micronutrientes

Nutrição no Exercício Físico e Desporto Pedro Carrera Bastos, 2013

VITAMINAS E MINERAIS

COMPOSIÇÃO DO CORPO HUMANO Elementos gasosos Proporção no corpo

humano Oxigénio 65%

Hidrogénio 10% Nitrogénio 3%

Subtotal 78% Minerais Carbono 18.5% Cálcio 1.2%

Fósforo 1.0% Potássio, Enxofre, Sódio, Cloro,

Magnésio 1.2%

Vitaminas e outros nutrientes 0.1% Total 100%

Colgan, M. Sports nutrition guide – Minerals, vitamins & antioxidants for athletes. Apple Publications, 2002

FONTES DE NUTRIENTES

Nutrientes Fontes Oxigénio Ar e água Hidrogénio Ar e água Carbono Base estrutural de todos os

alimentos Nitrogénio Proteínas Restantes elementos Distribuição heterogénea em

vários alimentos


VITAMINAS E OUTROS NUTRIENTES

Vitaminas hidrossolúveis

Vitaminas Lipossolúveis

Outros micronutrientes

Vitamina B1 Vitamina A Colina Vitamina B2 Vitamina D Betaína Vitamina B3 Vitamina E PABA

Vitamina B5 Vitamina K Fitonutrientes Vitamina B6

Vitamina B12 Ácido Fólico

Biotina Vitamina C


MINERAIS Macrominerais Microminerais

Cálcio Ferro Fósforo Zinco Potássio Cobre Enxofre Iodo Sódio Crómio Cloro Selénio

Magnésio Manganês Molibdénio

Sílicio Boro

Vanádio Flúor

Cobalto Arsénico, Estanho e Níquel


Stipanuk. MH. Biochenical, Physiological, Molecular aspects of Human Nutrition. Saunders, 2006

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Suberythemal UV irradiationAn amount of UV irradiation that is not able to induce any detectable redness in the skin over a period of 24 hours after exposure.

Contact hypersensitivity responseA form of delayed-type hypersensitivity (type IV), in which T cells respond to antigens that are introduced through skin contact. This step requires dendritic cell mobilization from the skin to the draining lymph nodes to prime the antigen-specific T cells.

has also been shown to suppress human immune responses against tumour-associated, self and experi-mental antigens2. An involvement of multiple comple-mentary pathways may be dictated by an evolutionary advantage not to respond to antigens of commensal organisms in the skin, damaged skin cells or nuclear antigens of sunburnt cells.

Of sunlight reaching the earth’s surface, the UVB wavelengths (290–315 nm) are generally considered to be the most potent at regulating the immune system. The contribution of UVA wavelengths (315–400 nm) to both UV-induced carcinogenesis3 and UV-mediated regulation of the immune system4 is controversial. In some studies, UVA radiation suppressed immune responses4, whereas in others UVA radiation modu-lated the regulatory effects of UVB radiation5. As

solar UV radiation predominantly comprises UVA wavelengths, there is a need for further studies using solar-simulated sources of UV radiation that allow wavelength interactions.

It is also important to consider the effects of dif-ferent doses of UV radiation on immune func-tion. Suberythemal UV irradiation was found to inhibit local immune responses to antigens applied to the UV-irradiated sites6. Suberythemal doses of UV radia-tion have also been shown to suppress systemic immune responses in both mice and humans7, but it is generally believed that erythemal doses of UV radiation are more successful at achieving systemic immunoregulation. The immune indices used also dictate the sensitivity with which UV-induced immuno regulation can be detected. For example, the contact hypersensitivity response

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Hart PH, et al. Nature Rev Immun 2011;11:584–596

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REVIEW

Fracture Prevention WithVitamin D SupplementationA Meta-analysis of Randomized Controlled TrialsHeike A. Bischoff-Ferrari, MD, MPHWalter C. Willett, DrPHJohn B. Wong, MDEdward Giovannucci, ScDThomas Dietrich, MPHBess Dawson-Hughes, MD

FRACTURES CONTRIBUTE SIGNIFI-cantly to morbidity and mortal-ity of older persons. Hip frac-tures increase exponentially

with age so that by the ninth decade oflife, an estimated 1 in every 3 womenand 1 in every 6 men will have sus-tained a hip fracture.1 With the agingof the population, the number of hipfractures is projected to increase world-wide.2 The consequences of hip frac-tures are severe: 50% of older personshave permanent functional disabili-ties, 15% to 25% require long-termnursing home care, and 10% to 20% diewithin 1 year.3-6 Besides the personalburden, hip fractures account for sub-stantial health care expenses3,7 with an-nual costs in the United States pro-jected to increase from $7.2 billion in1990 to $16 billion in 2020.7

Given the high prevalence, severity,and cost of osteoporotic fractures, pre-vention strategies that are effective, lowin cost, and well-tolerated are needed.One promising prevention strategy maybe oral vitamin D supplementation. Sev-eral randomized controlled trials(RCTs) have examined vitamin Dsupplements for fracture prevention,but the results were conflicting. Thegoal of our analysis was to determine

Author Affiliations: Department of Nutrition, Har-vard School of Public Health (Drs Bischoff-Ferrari, Wil-lett, and Giovannucci); Division of Rheumatology, Im-munology, and Allergy, The Robert B. Brigham Arthritisand Musculoskeletal Diseases Clinical Research Cen-ter, and Division of Aging, Brigham and Women’s Hos-pital (Dr Bischoff-Ferrari); Department of Epidemiol-ogy and Channing Laboratory, Brigham and Women’sHospital (Drs Willett and Giovannucci); Departmentof Medicine, Tufts-New England Medical Center

(Dr Wong); Department of Health Policy and HealthServices Research, Boston University Goldman Schoolof Dental Medicine (Mr Dietrich); and Jean Mayer USDepartment of Agriculture Human Nutrition Re-search Center on Aging, Tufts University (Dr Dawson-Hughes), Boston, Mass.Corresponding Author: Heike A. Bischoff-Ferrari, MD,MPH, Department of Nutrition, Harvard School of Pub-lic Health, 651 Huntington Ave, Boston, MA 02115([email protected]).

Context The role and dose of oral vitamin D supplementation in nonvertebral frac-ture prevention have not been well established.

Objective To estimate the effectiveness of vitamin D supplementation in prevent-ing hip and nonvertebral fractures in older persons.

Data Sources A systematic review of English and non-English articles using MEDLINEand the Cochrane Controlled Trials Register (1960-2005), and EMBASE (1991-2005).Additional studies were identified by contacting clinical experts and searching bibliog-raphies and abstracts presented at the American Society for Bone and Mineral Research(1995-2004). Search terms included randomized controlled trial (RCT), controlled clini-cal trial, random allocation, double-blind method, cholecalciferol, ergocalciferol, 25-hydroxyvitamin D, fractures, humans, elderly, falls, and bone density.

Study Selection Only double-blind RCTs of oral vitamin D supplementation (cho-lecalciferol, ergocalciferol) with or without calcium supplementation vs calcium supple-mentation or placebo in older persons (!60 years) that examined hip or nonvertebralfractures were included.

Data Extraction Independent extraction of articles by 2 authors using predefineddata fields, including study quality indicators.

Data Synthesis All pooled analyses were based on random-effects models. Five RCTsfor hip fracture (n=9294) and 7 RCTs for nonvertebral fracture risk (n=9820) met ourinclusion criteria. All trials used cholecalciferol. Heterogeneity among studies for both hipand nonvertebral fracture prevention was observed, which disappeared after pooling RCTswith low-dose (400 IU/d) and higher-dose vitamin D (700-800 IU/d), separately. A vi-tamin D dose of 700 to 800 IU/d reduced the relative risk (RR) of hip fracture by 26% (3RCTs with 5572 persons; pooled RR, 0.74; 95% confidence interval [CI], 0.61-0.88) andany nonvertebral fracture by 23% (5 RCTs with 6098 persons; pooled RR, 0.77; 95%CI, 0.68-0.87) vs calcium or placebo. No significant benefit was observed for RCTs with400 IU/d vitamin D (2 RCTs with 3722 persons; pooled RR for hip fracture, 1.15; 95%CI, 0.88-1.50; and pooled RR for any nonvertebral fracture, 1.03; 95% CI, 0.86-1.24).

Conclusions Oral vitamin D supplementation between 700 to 800 IU/d appears toreduce the risk of hip and any nonvertebral fractures in ambulatory or institutional-ized elderly persons. An oral vitamin D dose of 400 IU/d is not sufficient for fractureprevention.JAMA. 2005;293:2257-2264 www.jama.com

©2005 American Medical Association. All rights reserved. (Reprinted) JAMA, May 11, 2005—Vol 293, No. 18 2257

on April 4, 2008 www.jama.comDownloaded from

groups, the pooled RR was 0.74 (95%CI, 0.61-0.88), suggesting that 700 to800 IU/d vitamin D reduces hip frac-ture risk by 26% (FIGURE 2). Thepooled risk difference was 2% (95% CI,1%-4%; P!.001), so the NNT was 45(95% CI, 28-114) for a treatmentduration of 24 to 60 months. For 2trials,13,16 which included 3722 indi-viduals and a vitamin D dose of 400IU/d, the pooled RR was 1.15 (95% CI,0.88-1.50), suggesting that 400 IU/d vi-tamin D supplementation does not re-duce hip fracture risk.

We also examined the achieved levelof serum 25-hydroxyvitamin D in re-lation to reduction in hip fracture risk(FIGURE 3). A greater reduction in hipfractures was observed with higherachieved 25-hydroxyvitamin D levels

in the treatment group (meta-regression P=.02).

When we included the 2 trials eachwith only 1 hip fracture report in a sen-sitivity analysis, the correspondingpooled results were as follows for 7 trialswith 9820 individuals and hip frac-ture by vitamin D supplementation be-tween 400 to 800 IU/d (RR, 0.87; 95%CI, 0.70-1.09), hip fracture by vita-min D supplementation between 700to 800 IU/d (RR, 0.73; 95% CI, 0.61-0.88), and hip fracture by vitamin Dsupplementation of 400 IU/d (RR, 1.15;95% CI, 0.88-1.50).

Any Nonvertebral FractureThe pooled RR for any vitamin D dosepreventing nonvertebral fractures was0.83 (95% CI, 0.70-0.98). However,

variation between studies was morethan expected indicating heterogene-ity (Q test P=.07).

After stratifying trials by vitamin Ddose, there was homogeneity (Q testP=.41 for high-dose trials and P=.36 forlow-dose trials). For 5 trials,12,14,15,17,18

which included 6098 individuals and avitamin D dose of 700 to 800 IU/d, thepooled RR was 0.77 (95% CI, 0.68-0.87), suggesting that 700 to 800 IU/dvitamin D supplementation reducesnonvertebral fracture risk by 23%(Figure 2). The pooled risk differencewas 4% (95% CI, 2%-5%), P=.02); there-fore, the NNT was 27 (95% CI, 19-49)for a treatment duration of 12 to 60months. For 2 trials,13,16 which in-cluded 3722 individuals and a vitaminD dose of 400 IU/d, the pooled RR was

Figure 2. Forest Plots Comparing the Risk of Hip and Nonvertebral Fractures Between Vitamin D (700-800 IU/d and 400 IU/d) and ControlGroups

Hip Fracture

Favors Vitamin D

Nonvertebral Fracture

Favors Control Favors Vitamin D Favors Control

Source

1.00.2Relative Risk (95% CI) Relative Risk (95% CI)

1.00.2 0.50.5 5.05.0

1.00.2 1.00.2 0.50.5 5.05.0

Meyer et al,16 2002

Pooled

Source

Pooled

Vitamin D 700-800 IU/d Vitamin D 700-800 IU/d

Vitamin D 400 IU/d Vitamin D 400 IU/d

Pooled Pooled

Chapuy et al,17 2002


Trivedi et al,18 2003

Lips et al,13 1996 Lips et al,13 1996

Meyer et al,16 2002


Dawson-Hughes et al,14 1997



Pfeifer et al,15 2000

Squares represent relative risks (RRs) and size of squares is proportional to the size of the trials. Error bars represent 95% confidence intervals (CIs). Trials are sorted bytrial duration ranging from 24 to 60 months for hip fracture and 12 to 60 months for nonvertebral fracture. For 3 trials with hip fractures,12,17,18 which included 5572individuals with a vitamin D dose of 700 to 800 IU/d, the pooled RR was 0.74 (95% CI, 0.61-0.88; Q test P=.74). For 5 trials with nonvertebral fractures,12,14,15,17,18

which included 6098 individuals with a vitamin D dose of 700 to 800 IU/d, the pooled RR was 0.77 (95% CI, 0.68-0.87; Q test P=.41). For the 2 trials,13,16 with avitamin D dose of 400 IU/d, trial duration ranged from 24 months to 36 to 41 months.

FRACTURE PREVENTION WITH VITAMIN D SUPPLEMENTATION



groups, the pooled RR was 0.74 (95%CI, 0.61-0.88), suggesting that 700 to800 IU/d vitamin D reduces hip frac-ture risk by 26% (FIGURE 2). Thepooled risk difference was 2% (95% CI,1%-4%; P!.001), so the NNT was 45(95% CI, 28-114) for a treatmentduration of 24 to 60 months. For 2trials,13,16 which included 3722 indi-viduals and a vitamin D dose of 400IU/d, the pooled RR was 1.15 (95% CI,0.88-1.50), suggesting that 400 IU/d vi-tamin D supplementation does not re-duce hip fracture risk.

We also examined the achieved levelof serum 25-hydroxyvitamin D in re-lation to reduction in hip fracture risk(FIGURE 3). A greater reduction in hipfractures was observed with higherachieved 25-hydroxyvitamin D levels

in the treatment group (meta-regression P=.02).

When we included the 2 trials eachwith only 1 hip fracture report in a sen-sitivity analysis, the correspondingpooled results were as follows for 7 trialswith 9820 individuals and hip frac-ture by vitamin D supplementation be-tween 400 to 800 IU/d (RR, 0.87; 95%CI, 0.70-1.09), hip fracture by vita-min D supplementation between 700to 800 IU/d (RR, 0.73; 95% CI, 0.61-0.88), and hip fracture by vitamin Dsupplementation of 400 IU/d (RR, 1.15;95% CI, 0.88-1.50).

Any Nonvertebral FractureThe pooled RR for any vitamin D dosepreventing nonvertebral fractures was0.83 (95% CI, 0.70-0.98). However,

variation between studies was morethan expected indicating heterogene-ity (Q test P=.07).

After stratifying trials by vitamin Ddose, there was homogeneity (Q testP=.41 for high-dose trials and P=.36 forlow-dose trials). For 5 trials,12,14,15,17,18

which included 6098 individuals and avitamin D dose of 700 to 800 IU/d, thepooled RR was 0.77 (95% CI, 0.68-0.87), suggesting that 700 to 800 IU/dvitamin D supplementation reducesnonvertebral fracture risk by 23%(Figure 2). The pooled risk differencewas 4% (95% CI, 2%-5%), P=.02); there-fore, the NNT was 27 (95% CI, 19-49)for a treatment duration of 12 to 60months. For 2 trials,13,16 which in-cluded 3722 individuals and a vitaminD dose of 400 IU/d, the pooled RR was

Figure 2. Forest Plots Comparing the Risk of Hip and Nonvertebral Fractures Between Vitamin D (700-800 IU/d and 400 IU/d) and ControlGroups

Hip Fracture

Favors Vitamin D

Nonvertebral Fracture

Favors Control Favors Vitamin D Favors Control

Source

1.00.2Relative Risk (95% CI) Relative Risk (95% CI)

1.00.2 0.50.5 5.05.0

1.00.2 1.00.2 0.50.5 5.05.0

Meyer et al,16 2002

Pooled

Source

Pooled

Vitamin D 700-800 IU/d Vitamin D 700-800 IU/d

Vitamin D 400 IU/d Vitamin D 400 IU/d

Pooled Pooled




Lips et al,13 1996 Lips et al,13 1996

Meyer et al,16 2002


Dawson-Hughes et al,14 1997



Pfeifer et al,15 2000

Squares represent relative risks (RRs) and size of squares is proportional to the size of the trials. Error bars represent 95% confidence intervals (CIs). Trials are sorted bytrial duration ranging from 24 to 60 months for hip fracture and 12 to 60 months for nonvertebral fracture. For 3 trials with hip fractures,12,17,18 which included 5572individuals with a vitamin D dose of 700 to 800 IU/d, the pooled RR was 0.74 (95% CI, 0.61-0.88; Q test P=.74). For 5 trials with nonvertebral fractures,12,14,15,17,18

which included 6098 individuals with a vitamin D dose of 700 to 800 IU/d, the pooled RR was 0.77 (95% CI, 0.68-0.87; Q test P=.41). For the 2 trials,13,16 with avitamin D dose of 400 IU/d, trial duration ranged from 24 months to 36 to 41 months.

FRACTURE PREVENTION WITH VITAMIN D SUPPLEMENTATION



JAMA. 2005;293:2257-‐2264

The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006

a vitamin D deficiency–like state that was resistant to physiologic doses of vitamin D (Figure 4). There is only one documented case of 25-OHase–deficiency rickets (62). The most likely reason why more cases are not reported is that there are at least 4 different enzymes that have the ability to convert vitamin D to 25(OH)D (63).

Since vitamin D undergoes its final activation in the kidneys, several studies of individuals with “vitamin D–resistant” diseases causing rickets in which patients were evaluated for a defect in the metabolism of 25(OH)D to 1,25(OH)2D have been reported. Pseu-dovitamin D–deficiency rickets (also known as hereditary, vitamin D–dependent rickets type 1), a rare hereditary disorder, was found to be associated with very low or undetectable levels of 1,25(OH)2D in the circulation (64). These children responded to orally adminis-tered 1,25(OH)2D3 (64). The cloning of the renal 1-OHase enzyme led to the identification of a multitude of point mutations of the CYP27B1 gene, which result in either a poorly functional 1-OHase or the complete absence of 1-OHase activity (65).

Several investigators reported children with severe rickets who often had alopecia and extremely elevated levels of 1,25(OH)2D

(65, 66). Some children with this disease, vitamin D–resistant rick-ets (hereditary, vitamin D–dependent rickets type 2), responded to pharmacologic doses of vitamin D or 1,25(OH)2D3, while oth-ers did not (66, 67). Point mutations in the VDR gene are respon-sible for the vitamin D resistance. Chen et al. (68) reported a new form of vitamin D resistance, hereditary vitamin D–dependent rickets type 3, caused by the abnormal expression of a hormone response element–binding protein (HRBP) that binds to the VDRE and therefore prevents the 1,25(OH)2D-VDR-RXR complex from binding to its responsive element. This patient had normal VDR expression and was completely resistant to 1,25(OH)2D3 action. Children with these vitamin D–resistance syndromes often suffer from severe bone deformities and more marked hypocalcemia than children with vitamin D–deficiency rickets. Treatment depends on the cause and severity of the vitamin D resistance. Children have responded to pharmacologic doses of vitamin D, physiologic and pharmacologic doses of 1,25(OH)2D3 and its analog 1α-hydroxyvi-tamin D3, as well as intravenous infusions of calcium and phos-phorus (30, 64, 66–69).

×Holick MF. J Clin Invest. 2006 Aug;116(8):2062-‐72

Mayo Clin Proc, December 2003, Vol 78 Musculoskeletal Pain and Severe Hypovitaminosis D 1463

Mayo Clin Proc. 2003;78:1463-1470 1463 © 2003 Mayo Foundation for Medical Education and Research

Original Article

Prevalence of Severe Hypovitaminosis D in PatientsWith Persistent, Nonspecific Musculoskeletal Pain

GREGORY A. PLOTNIKOFF, MD, MTS, AND JOANNA M. QUIGLEY, BA

From the Departments of Internal Medicine and Pediatrics, Univer-sity of Minnesota Medical School, Minneapolis, Minn, and Depart-ment of Oriental Medicine, Keio University Medical School, Tokyo,Japan (G.A.P.); and Center for Spirituality and Healing, University ofMinnesota Academic Health Center, Minneapolis, Minn (G.A.P.,J.M.Q.).

Address reprint requests and correspondence to Gregory A. Plot-nikoff, MD, MTS, Department of Oriental Medicine, Keio UniversitySchool of Medicine, 35 Shinano-Machi, Shinjuku-Ku, Tokyo 160-8582, Japan (e-mail: [email protected]).

• Objective: To determine the prevalence of hypovita-minosis D in primary care outpatients with persistent,nonspecific musculoskeletal pain syndromes refractory tostandard therapies.

• Patients and Methods: In this cross-sectional study,150 patients presented consecutively between February2000 and June 2002 with persistent, nonspecific muscu-loskeletal pain to the Community University Health CareCenter, a university-affiliated inner city primary careclinic in Minneapolis, Minn (45 north). Immigrant (n=83)and nonimmigrant (n=67) persons of both sexes, aged 10 to65 years, from 6 broad ethnic groups were screened forvitamin D status. Serum 25-hydroxyvitamin D levels weredetermined by radioimmunoassay.

• Results: Of the African American, East African, His-panic, and American Indian patients, 100% had deficientlevels of vitamin D (≤20 ng/mL). Of all patients, 93% (140/150) had deficient levels of vitamin D (mean, 12.08 ng/mL;95% confidence interval, 11.18-12.99 ng/mL). Nonimmi-grants had vitamin D levels as deficient as immigrants(P=.48). Levels of vitamin D in men were as deficient as inwomen (P=.42). Of all patients, 28% (42/150) had severelydeficient vitamin D levels (≤8 ng/mL), including 55% of

ANOVA = analysis of variance; CI = confidence interval;PTH = parathyroid hormone

whom were younger than 30 years. Five patients, 4 of whomwere aged 35 years or younger, had vitamin D serum levelsbelow the level of detection. The severity of deficiency wasdisproportionate by age for young women (P<.001), by sexfor East African patients (P<.001), and by race for AfricanAmerican patients (P=.006). Season was not a significantfactor in determining vitamin D serum levels (P=.06).

• Conclusion: All patients with persistent, nonspecificmusculoskeletal pain are at high risk for the consequencesof unrecognized and untreated severe hypovitaminosis D.This risk extends to those considered at low risk for vita-min D deficiency: nonelderly, nonhousebound, or nonimmi-grant persons of either sex. Nonimmigrant women ofchildbearing age with such pain appear to be at greatestrisk for misdiagnosis or delayed diagnosis. Because osteo-malacia is a known cause of persistent, nonspecific muscu-loskeletal pain, screening all outpatients with such pain forhypovitaminosis D should be standard practice in clinicalcare.

Mayo Clin Proc. 2003;78:1463-1470

Prevalence surveys suggest that 9% to 20% of adults inthe United States experience chronic pain.1-4 Of these,

89% have some degree of long-term or short-term disabil-ity,5 and nearly all have substantially reduced health-related quality of life.6 Direct and indirect costs related totheir chronic pain are estimated at $50 billion annually.7

Of the many types of chronic pain, nonspecific or idio-pathic musculoskeletal pain, such as noninflammatoryarthritis, nonarticular rheumatism, and nonradicular lowback pain, is seen frequently in medical and chiropracticclinics. Despite the prevalence, severity, and burdens ofsuch pain, precise diagnosis and effective treatment areoften elusive.

Chronic nonspecific musculoskeletal pain is one conse-quence of hypovitaminosis D. For the past 30 years, pub-lished reports from Europe have documented persistent,nonspecific musculoskeletal pain in immigrant patientssecondary to severe hypovitaminosis D.8-19 This study ex-tends the European studies to include patients in the UnitedStates presumed to be vitamin D–sufficient, namely youngambulatory outpatients.20,21

For editorial comment, see page 1457.

PATIENTS AND METHODSStudy Population

Six broad categories of ethnic groups were divided intoimmigrant and nonimmigrant populations. The immigrantethnic groups considered in this study are East African(primarily Somalian), Hispanic (primarily Mexican), andSoutheast Asian (primarily Hmong, Cambodian, or Lao-tian). The Southeast Asian patients began translocating toMinnesota in 1975, most of whom arrived from refugeecamps. The East African and Hispanic patients began arriv-ing in Minnesota in 1995. The nonimmigrant ethnic groups

Mayo Clin Proc, December 2003, Vol 78 Musculoskeletal Pain and Severe Hypovitaminosis D 1463

Mayo Clin Proc. 2003;78:1463-1470 1463 © 2003 Mayo Foundation for Medical Education and Research

Original Article

Prevalence of Severe Hypovitaminosis D in PatientsWith Persistent, Nonspecific Musculoskeletal Pain

GREGORY A. PLOTNIKOFF, MD, MTS, AND JOANNA M. QUIGLEY, BA

From the Departments of Internal Medicine and Pediatrics, Univer-sity of Minnesota Medical School, Minneapolis, Minn, and Depart-ment of Oriental Medicine, Keio University Medical School, Tokyo,Japan (G.A.P.); and Center for Spirituality and Healing, University ofMinnesota Academic Health Center, Minneapolis, Minn (G.A.P.,J.M.Q.).

Address reprint requests and correspondence to Gregory A. Plot-nikoff, MD, MTS, Department of Oriental Medicine, Keio UniversitySchool of Medicine, 35 Shinano-Machi, Shinjuku-Ku, Tokyo 160-8582, Japan (e-mail: [email protected]).

• Objective: To determine the prevalence of hypovita-minosis D in primary care outpatients with persistent,nonspecific musculoskeletal pain syndromes refractory tostandard therapies.

• Patients and Methods: In this cross-sectional study,150 patients presented consecutively between February2000 and June 2002 with persistent, nonspecific muscu-loskeletal pain to the Community University Health CareCenter, a university-affiliated inner city primary careclinic in Minneapolis, Minn (45 north). Immigrant (n=83)and nonimmigrant (n=67) persons of both sexes, aged 10 to65 years, from 6 broad ethnic groups were screened forvitamin D status. Serum 25-hydroxyvitamin D levels weredetermined by radioimmunoassay.

• Results: Of the African American, East African, His-panic, and American Indian patients, 100% had deficientlevels of vitamin D (≤20 ng/mL). Of all patients, 93% (140/150) had deficient levels of vitamin D (mean, 12.08 ng/mL;95% confidence interval, 11.18-12.99 ng/mL). Nonimmi-grants had vitamin D levels as deficient as immigrants(P=.48). Levels of vitamin D in men were as deficient as inwomen (P=.42). Of all patients, 28% (42/150) had severelydeficient vitamin D levels (≤8 ng/mL), including 55% of

ANOVA = analysis of variance; CI = confidence interval;PTH = parathyroid hormone

whom were younger than 30 years. Five patients, 4 of whomwere aged 35 years or younger, had vitamin D serum levelsbelow the level of detection. The severity of deficiency wasdisproportionate by age for young women (P<.001), by sexfor East African patients (P<.001), and by race for AfricanAmerican patients (P=.006). Season was not a significantfactor in determining vitamin D serum levels (P=.06).

• Conclusion: All patients with persistent, nonspecificmusculoskeletal pain are at high risk for the consequencesof unrecognized and untreated severe hypovitaminosis D.This risk extends to those considered at low risk for vita-min D deficiency: nonelderly, nonhousebound, or nonimmi-grant persons of either sex. Nonimmigrant women ofchildbearing age with such pain appear to be at greatestrisk for misdiagnosis or delayed diagnosis. Because osteo-malacia is a known cause of persistent, nonspecific muscu-loskeletal pain, screening all outpatients with such pain forhypovitaminosis D should be standard practice in clinicalcare.

Mayo Clin Proc. 2003;78:1463-1470

Prevalence surveys suggest that 9% to 20% of adults inthe United States experience chronic pain.1-4 Of these,

89% have some degree of long-term or short-term disabil-ity,5 and nearly all have substantially reduced health-related quality of life.6 Direct and indirect costs related totheir chronic pain are estimated at $50 billion annually.7

Of the many types of chronic pain, nonspecific or idio-pathic musculoskeletal pain, such as noninflammatoryarthritis, nonarticular rheumatism, and nonradicular lowback pain, is seen frequently in medical and chiropracticclinics. Despite the prevalence, severity, and burdens ofsuch pain, precise diagnosis and effective treatment areoften elusive.

Chronic nonspecific musculoskeletal pain is one conse-quence of hypovitaminosis D. For the past 30 years, pub-lished reports from Europe have documented persistent,nonspecific musculoskeletal pain in immigrant patientssecondary to severe hypovitaminosis D.8-19 This study ex-tends the European studies to include patients in the UnitedStates presumed to be vitamin D–sufficient, namely youngambulatory outpatients.20,21

For editorial comment, see page 1457.

PATIENTS AND METHODSStudy Population

Six broad categories of ethnic groups were divided intoimmigrant and nonimmigrant populations. The immigrantethnic groups considered in this study are East African(primarily Somalian), Hispanic (primarily Mexican), andSoutheast Asian (primarily Hmong, Cambodian, or Lao-tian). The Southeast Asian patients began translocating toMinnesota in 1975, most of whom arrived from refugeecamps. The East African and Hispanic patients began arriv-ing in Minnesota in 1995. The nonimmigrant ethnic groups

ORIGINAL ARTICLE

Hypovitaminosis D in female patients with chroniclow back pain

Ahmed Lotfi & Ahmed M. Abdel-Nasser &

Ahmed Hamdy & Ahmed A. Omran &

Mahmoud A. El-Rehany

Received: 7 October 2006 /Revised: 4 March 2007 /Accepted: 5 March 2007 / Published online: 22 March 2007# Clinical Rheumatology 2007

Abstract Chronic low back pain (LBP) is an extremelycommon problem in practice, where it is often labeledidiopathic. No sufficient studies have been conducted toanalyze the contribution of hypovitaminosis D to the eti-ology of chronic LBP in populations wherein vitamin Ddeficiency is endemic. The present study was, therefore,carried out to examine hypovitaminosis D and its determi-nants in female patients with chronic LBP during thechildbearing period. Sixty female patients complaining ofLBP lasting more than 3 months were clinically studiedrheumatologically and neurologically. Questionnaires andindices quantifying risk factors associated with vitamin Ddeficiency were utilized. Biochemical assays of serum cal-cium, phosphorus, alkaline phosphatase (ALP), parathor-mone (PTH), and 25-hydroxyvitamin D (25 OHD) wereperformed and compared to those of 20 matched healthycontrols. The determinants of vitamin D levels in patientswere examined by stepwise regression. Patients with LBP

had significantly lower 25 OHD levels (p<0.05) andsignificantly higher PTH (p<0.05) and ALP (p<0.001)than controls, although there were no significant groupdifferences in calcium and phosphorus. Hypovitaminosis D(25 OHD < 40 ng/ml) was found in 49/60 patients (81%)and 12/20 (60%) of controls, with an odds ratio of 2.97.Although many risk factors related to sun exposure,clothing, diet, and pregnancy were significantly correlatedwith vitamin D levels in patients, only limited duration ofsun exposure, contributing 55% to the variance of 25 OHD,limited areas of skin exposed (13%), and increased numberof pregnancies (2%), were significant determinants ofvitamin D levels in patients. Despite the sunny climate,hypovitaminosis D is prevalent among Egyptian women inthe childbearing period, especially those presenting withchronic LBP, where it is associated with hyperphosphatasiaand hyperparathyroidism, without alterations in serumcalcium. The major determinant of hypovitaminosis D inour patients is limited sun exposure.

Keywords Chronic low back pain . Hypovitaminosis D .

Osteomalacia . 25(OH) D

Introduction

About two thirds of adults suffer from low back pain (LBP)at some time. It is second only to upper respiratoryproblems as a symptom-related reason for visiting aphysician [1, 2]. Most population-based surveys of LBPreport a point prevalence of 15–30%, an annual incidenceof 50%, and a lifetime prevalence of 60–80% [3].Experimental studies suggest that LBP may originate frommany spinal structures, including ligaments, facet joints, the

Clin Rheumatol (2007) 26:1895–1901DOI 10.1007/s10067-007-0603-4

A. Lotfi :A. M. Abdel-Nasser (*) :A. HamdyRheumatology and Rehabilitation Department,Faculty of Medicine, Minia University,13 Botros Ghally Street,11341 Heliopolis, Cairo, Egypte-mail: [email protected]

A. A. OmranClinical Pathology Department,Faculty of Medicine, El-Minia University,Heliopolis, Cairo, Egypt

M. A. El-RehanyBiochemistry Department,Faculty of Medicine, El-Minia University,Heliopolis, Cairo, Egypt

ORIGINAL ARTICLE

Association between nonspecific skeletal pain and vitaminD deficiency

Behzad HEIDARI,1 Javad Shokri SHIRVANI,1 Alireza FIROUZJAHI,2 Parnaz HEIDARI3 and

Karim O. HAJIAN-TILAKI4

1Deparment of Medicine, Division of Rheumatology, 2Deparment of Pathology and Laboratory Medicine, Rouhani Hospital, BabolUniversity of Medical Sciences, Babol, 3Faculty of Medicine, Islamic Azad University, Tehran and 4Department of Social Medicine,Babol University of Medical Sciences, Babol, Iran

AbstractBackground: Deficiency of vitamin D has been reported in patients with many types of musculoskeletal pain.The present study was designed to determine the association between serum 25-hydroxyvitamin D [25-(OH)D] deficiency and nonspecific skeletal pain.

Methods: A total of 276 patients with nonspecific skeletal pain at different regions of the skeletal systemdiagnosed as leg pain, widespread pain, arthralgia, rib pain, back pain and fibromyalgia were compared with202 matched controls with regard to mean serum 25-(OH)D level and 25-(OH)D deficiency. Serum 25-(OH)D was measured by enzyme-linked immunosorbent assay method and levels < 20 ng/mL were consid-ered as deficient. Nonparametric one-way analysis of variance, Kruskal Wallis and Wilcoxon tests were usedfor group comparisons. Multiple logistic regression analysis with calculation of adjusted odds ratio (OR) and95% confidence interval (95% CI) were performed to determine associations.

Results: In patients with nonspecific skeletal pain the mean 25-(OH)D was significantly lower (P = 0.0001)and the proportion of 25-(OH)D deficiency was significantly higher (63.4% vs. 36.1%, P = 0.0001) comparedwith controls. There was a significantly positive association between 25-(OH)D deficiency and skeletal pain(OR = 2.94, 95% CI = 1.01–4.3, P = 0.0001). The strength of association varied across the groups with stron-gest association observed with leg pain (OR = 7.4; 95% CI = 3.9–13.9, P = 0.0001) followed by arthralgia(OR = 3.9, 95% CI = 2.1–7.1, P = 0.0001) and widespread pain (OR = 2.8, 95% CI = 1.1–6.6, P = 0.020) butno association with back pain and fibromyalgia. There was a greater positive associations in women com-pared with men (OR = 2.1, 95% CI = 1.1–4.3, P = 0.001).

Conclusion: The results of this study indicate a positive association of vitamin D deficiency with a variety ofnonspecific bone pain, particularly in women. More studies with larger samples are required to confirm thesefindings. Increasing serum vitamin D to sufficient levels and longitudinal follow-up of patients may providefurther evidence in relation to vitamin D deficiency and skeletal pain.

Key words: arthralgia, leg pain, musculoskeletal pain, vitamin D deficiency, widespread pain.

INTRODUCTIONVitamin D deficiency constitutes a largely unrecognizedepidemic worldwide.1–8 It is common in patientswith many types of musculoskeletal symptoms,

Correspondence: Dr. Behzad Heidari, Department of medicine,Shaheed Beheshti Hospital, Babol 1325, Iran.Email: [email protected]

International Journal of Rheumatic Diseases 2010; 13: 340–346

ª2010 Asia Pacific League of Associations for Rheumatology and Blackwell Publishing Asia Pty Ltd

Holick MF. J Clin Invest. 2006 Aug;116(8):2062-‐72

RECEPTORES EM VÁRIAS CÉLULAS

The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006

study in hypertensive adults exposed to simulated sunlight 3 times a week for 3 months resulted in an increase in their 25(OH)D levels by more than 150% and a significant (6 mmHg) reduction in both systolic and diastolic blood pressure (113).

Circulating levels of 1,25(OH)2D are very low or undetectable in patients with chronic kidney disease (114). It has been assumed that the kidneys are the sole source of 1,25(OH)2D. However, just as most tissues and cells in the body have a VDR, so too do these tissues and cells possess the ability to express CYP27B1. Thus, the skin, prostate, breast, colon, lung, brain, and placenta not only express the VDR but also have the capacity to produce 1,25(OH)2D (22, 24, 115, 116). It is now recognized that 1,25(OH)2D helps con-trol the expression of more than 200 genes (20–22, 24, 117). It is thought that 1,25(OH)2D maintains cellular health by acting as a sentinel for preventing malignancy (24, 95, 96) (Figure 5).

Activated macrophages also express CYP27B1 and thus pro-duce 1,25(OH)2D. This is the mechanism by which patients with

chronic granulomatous diseases such as sarcoidosis and tubercu-losis develop a disorder in calcium metabolism that causes hyper-calcuria and hypercalcemia (1, 22, 24). Why macrophages produce 1,25(OH)2D was unknown until Liu et al. (118) reported that activation of TLRs with LPS resulted in the upregulation of the expression of not only VDR but also the CYP27B1 gene. The local production of 1,25(OH)2D induced the expression of the antimi-crobial peptide cathelicidin (LL-37), which is thought to be a key factor in the innate immune response when TLR is activated by an infective agent such as Mycobacterium tuberculosis (Figure 5). This remarkable observation explains why patients with TB often do better when placed in a solarium and exposed to sunlight or taken to higher altitudes where the vitamin D3 production in the skin is more efficient (14). This also is the likely reason why African Americans, who are often vitamin D deficient, and children with vitamin D deficiency have increased susceptibility to TB infec-tion (118). This also may explain why it was widely reported that children with rickets often are more prone to infectious diseases, including the common cold virus (12–14, 119).

Catelicidina

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n engl j med 357;3 www.nejm.org july 19, 2007272

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AUTHOR PLEASE NOTE:Figure has been redrawn and type has been reset

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Decreased reninDecreased

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Enhances p21 and p27Inhibits angiogenesisInduces apoptosis

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Figure 2. Metabolism of 25-Hydroxyvitamin D to 1,25-Dihydroxyvitamin D for Nonskeletal Functions.

When a macrophage or monocyte is stimulated through its toll-like receptor 2/1 (TLR2/1) by an infectious agent such as Mycobacterium tuberculosis or its lipopolysaccharide, the signal up-regulates the expression of vitamin D re-ceptor (VDR) and 25-hydroxyvitamin D-1!-hydroxylase (1-OHase). A 25-hydroxyvitamin D [25(OH)D] level of 30 ng per milliliter (75 nmol per liter) or higher provides adequate substrate for 1-OHase to convert 25(OH)D to its active form, 1,25 dihydroxyvitamin D [1,25(OH)2D]. 1,25(OH)2D travels to the nucleus, where it increases the expression of cathelicidin, a peptide capable of promoting innate immunity and inducing the destruction of infectious agents such as M. tuberculosis. It is also likely that the 1,25(OH)2D produced in monocytes or macrophages is released to act locally on activated T lymphocytes, which regulate cytokine synthesis, and activated B lymphocytes, which regu-late immunoglobulin synthesis. When the 25(OH)D level is approximately 30 ng per milliliter, the risk of many com-mon cancers is reduced. It is believed that the local production of 1,25(OH)2D in the breast, colon, prostate, and other tissues regulates a variety of genes that control proliferation, including p21 and p27, as well as genes that in-hibit angiogenesis and induce differentiation and apoptosis. Once 1,25(OH)2D completes the task of maintaining normal cellular proliferation and differentiation, it induces expression of the enzyme 25-hydroxyvitamin D-24-hy-droxylase (24-OHase), which enhances the catabolism of 1,25(OH)2D to the biologically inert calcitroic acid. Thus, locally produced 1,25(OH)2D does not enter the circulation and has no influence on calcium metabolism. The para-thyroid glands have 1-OHase activity, and the local production of 1,25(OH)2D inhibits the expression and synthesis of parathyroid hormone. The 1,25(OH)2D produced in the kidney enters the circulation and can down-regulate renin production in the kidney and stimulate insulin secretion in the beta islet cells of the pancreas.

Holick M. NEJM 2007;357:266-81.

Cannell JJ, Zasloff M, Garland CF, Scragg R, Giovannucci E. Virol J. 2008 Feb 25;5:29.

Virology Journal 2008, 5:29 http://www.virologyj.com/content/5/1/29

Page 4 of 12(page number not for citation purposes)

bial activity against bacteria, fungi, and viruses [29]. Ingeneral, they act by rapidly and irreversibly damaging thelipoprotein membranes of microbial targets, includingenveloped viruses, like influenza [30]. Other AMPs, suchas human beta-defensin 3, inhibit influenza haemaggluti-nin A mediated fusion by binding to haemagglutinin Aassociated carbohydrates via a lectin-like interaction [31].

AMPs protect mucosal epithelial surfaces by creating ahostile antimicrobial shield. The epithelia secrete themconstitutively into the thin layer of fluid that lies abovethe apical surface of the epithelium but below the viscousmucous layer. To effectively access the epithelium amicrobe, such as influenza, must penetrate the mucousbarrier and then survive damage inflicted by the AMPspresent in the fluid that is in immediate contact with theepithelial surface. Should this constitutive barrier bebreached, the binding of microbes to the epithelium and/or local tissue injury rapidly provokes the expression ofhigh concentrations of specific inducible AMPs such ashuman beta-defensin 2 and cathelicidin, that provide a"back-up" antimicrobial shield. These inducible AMPsalso act as chemo-attractants for macrophages and neu-trophils that are present in the immediate vicinity of thesite of the microbial breach [28-30]. In addition, catheli-cidin plays a role in epithelial repair by triggering epithe-lial growth and angiogenesis [32].

The crucial role of vitamin D in the innate immune systemwas discovered only very recently [33,34]. Both epithelialcells and macrophages increase expression of the antimi-

crobial cathelicidin upon exposure to microbes, anexpression that is dependent upon the presence of vita-min D. Pathogenic microbes, much like the commensalsthat inhabit the upper airway, stimulate the production ofa hydroxylase that converts 25(OH)D to 1,25(OH)2D, aseco-steroid hormone. This in turn rapidly activates a suiteof genes involved in defense [35].

In the macrophage, the presence of vitamin D alsoappears to suppress the pro-inflammatory cytokines,Interferon J, TNFD, and IL12, and down regulate the cel-lular expression of several PAMP receptors. In the epider-mis, vitamin D induces additional PAMP receptors,enabling keratinocytes to recognize and respond tomicrobes [36]. Thus, vitamin D appears to both enhancethe local capacity of the epithelium to produce endog-enous antibiotics and – at the same time – dampen certainarms of the adaptive immune response, especially thoseresponsible for the signs and symptoms of acute inflam-mation, such as the cytokine storms operative when influ-enza kills quickly.

Of particular note is that not all animals appear to dependon vitamin D for their innate immune circuitry. The cathe-licidin genes of mouse, rat, and dog, lack a vitamin Dreceptor-binding site, and do not require vitamin D forexpression [34]. Therefore, one cannot extrapolate therole vitamin D plays in human infections from studies ofsuch animals.

Plasma levels of vitamin 25(OH)D in African Americans,known to be lower than white skinned individuals, areinadequate to fully stimulate the vitamin D dependentantimicrobial circuits operative within the innateimmune system. However, the addition of 25(OH)Drestored the dependent circuits and greatly enhancedexpression of AMPs [37]. High concentrations of melaninin dark-skinned individuals shield the keratinocytes fromthe ultraviolet radiation required to generate vitamin D inskin [38]. In addition, the production of vitamin D in skindiminishes with aging [39]. Therefore, relative – but easilycorrectable – deficiencies in innate immunity probablyexist in many dark-skinned and aged individuals, espe-cially during the winter.

Because humans obtain most vitamin D from sun expo-sure and not from diet, a varying percentage of the popu-lation is vitamin D deficient, at any time, during anyseason, at any latitude, although the percentage is higherin the winter, in the aged, in the obese, in the sun-deprived, in the dark-skinned, and in more poleward pop-ulations [40,41]. However, seasonal variation of vitaminD levels even occur around the equator [42] and wide-spread vitamin D deficiency can occur at equatorial lati-tudes [43], probably due to sun avoidance [44], rainy

Incidence of reported cold/influenza symptoms according to seasonFigure 2Incidence of reported cold/influenza symptoms according to season. The 104 subjects in the placebo group (light shade) reported cold and flu symptoms year around with the most symptoms in the winter. While on 800 IU per day (intermediate shade) the 104 test subjects were as likely to get sick in the summer as the winter. Only one of the 104 test subjects had cold/influenza symptoms during the final year of the trial, when they took 2,000 IU of vitamin D per day (dark shading). Adapted from: Aloia JF, Li-Ng M: Epi-demic influenza and vitamin D. Epidemiol Infect 2007; 135: 1095–1096. (Reproduced with permission, Cambridge Uni-versity Press).

25

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Autumn

VITAMINA D E GRIPE

N = 104 Placebo vs 104 Vit D

than those whose mothers did not (OR 0.67, 95% CI 0.53–0.86) [9], and a case–control study by Stene and colleaguesfound that the risk of diabetes in children of mothers whotook cod liver oil during pregnancy was lower than inmothers who did not (OR 0.30, 95% CI 0.12–0.75) [10].Neither study measured serum 25(OH)D in the participants.

In a cohort study in Colorado, intake of vitamin Dduring the third trimester of pregnancy was assessed in themothers of 233 children. The children were then followed

for an average of 4 years [11]. Maternal intake of vitamin Dfrom food had a protective effect against the appearance ofislet cell autoantibodies (multiple-adjusted HR 0.37, 95%CI 0.17–0.78). Such antibodies are associated with thedevelopment of type 1 diabetes [12]. Another study reportedlower incidence rates of type 1 diabetes in association withhigher ultraviolet radiation in Australia [13].

Some findings from previous research appear to beinconsistent with the hypothesis regarding an overall

61

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82

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Sao Paulo, Brazil

Santiago, Chile

Bogota,Columbia

87 88

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BarbadosCuba

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US Virgin IslandsNew South Wales, Australia97

Canterbury, New Zealand

–60 –50 –40 –30 –20 –10 0 10 20 30 40 50 60 70

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44

Fig. 1 Age-standardised incidence rates of type 1 diabetes per100,000 boys <14 years of age, by latitude, in 51 regions worldwide,2002. Data points are shown by dots; names shown adjacent to thedots denote location, where space allows. Where space was limited,numerical codes (below) designate location. Source: data from WHODiaMond [3]. Lux., Luxembourg. Numerical codes for areas: 2. Beja,Tunisia; 3. Gafsa, Tunisia; 4. Kairoan, Tunisia; 5. Monastir, Tunisia; 7.Mauritius; 8. Wuhan, China; 9. Sichuan, China; 10. Huhehot, China;16. Nanjing, China; 17. Jinan, China; 21. Harbin, China; 23.Changsha, China; 25. Hainan, China; 29. Hong Kong, China; 31.Israel; 32. Chiba, Japan; 33. Hokkaido, Japan; 34. Okinawa, Japan;36. Novosibirsk, Russia; 38. Antwerp, Belgium; 39. Varna, Bulgaria;40. Denmark; 43. France; 44. Baden, Germany; 45. Attica, Greece;48. Sicily, Italy; 49. Pavia, Italy; 50. Marche, Italy; 52. Lazio, Italy;59. Krakow, Poland; 61. Algarve, Portugal; 62. Coimbra, Portugal; 63.

Madeira Island, Portugal; 64. Portalegre, Portugal; 65. Bucharest,Romania; 67. Slovakia; 68. Catalonia, Spain; 71. Leicestershire, UK;72. Northern Ireland, UK; 77. Allegheny, PA, USA; 80. Avellaneda,Argentina; 82. Corrientes, Argentina; 87. Paraguay; 88. Lima, Peru;90. Caracas, Venezuela; 97. Auckland, New Zealand. Data points notlabelled because of space constraints (latitude in degrees, rate per100,000): 11. Dalian, China (39, 1.1); 12. Guilin, China (24, 0.6); 13.Beijing, China (40, 0.7); 14. Shanghai, China (32. 0.7); 15. ChangChun, China (44, 0.6); 18. Jilin, China (43, 0.4); 19. Shenyang, China(42, 0.4); 20. Lanzhou, China (36, 0.5); 22. Nanning, China (23, 0.3);24. Zhengzhou, China (35, 0.2); 26. Tie Ling, China (42, 0.2); 27.Zunyi, China (28, 0.1); 28. Wulumuqi, China (44, 0.9); 35. Karachi,Pakistan (25, 0.5); 37. Austria (48, 9.8); 46. Hungary (47, 8.7); 51.Turin, Italy (45, 11.9); 53. Lombardia, Italy (46, 7.6); 66. Slovenia(46, 6.8); 79. Chicago, IL, USA (42, 10.2). R2=0.25, p<0.001

Diabetologia (2008) 51:1391–1398 1393

ORIGINAL CONTRIBUTION

Serum 25-Hydroxyvitamin D Levelsand Risk of Multiple SclerosisKassandra L. Munger, MScLynn I. Levin, PhD, MPHBruce W. Hollis, PhDNoel S. Howard, MDAlberto Ascherio, MD, DrPH

MULTIPLE SCLEROSIS (MS) ISamong the most commonneurological diseases inyoung adults, affecting

350 000 individuals in the United Statesand 2 million worldwide.1 Prevailingthought is thatMSisanautoimmunedis-order whereby an unknown agent oragents triggersaTcell–mediated inflam-matoryattack, causingdemyelinationofcentral nervous system tissue.2

A striking feature of the global dis-tribution of MS is a multifold increasein incidence with increasing latitude,both north and south of the equator.3

Genetic predisposition contributes tothis variation,4 but the change in MSrisk with migration among people ofcommon ancestry5 strongly supports arole for environmental factors. One po-tential factor may be vitamin D,6-9 a po-tent immunomodulator that in its hor-monal form can prevent experimentalautoimmune encephalomyelitis (EAE),an animal model of MS.10 Because foodprovides little vitamin D, the majorsource for most people is through skinexposure to sunlight.11 At latitudes of42° or more (eg, Boston, Mass), in win-ter most UV-B radiation is absorbed bythe atmosphere, and even prolongedsun exposure is insufficient to gener-ate vitamin D.12 As a result, seasonal vi-tamin D deficiency is common.11

AprotectiveeffectofvitaminDonMSis supported by the reduced MS risk as-

sociated with sun exposure13,14 and useofvitaminDsupplements,15 butevidenceremainsinconclusive.Inthepresentstudy,we examined prospectively for the firsttime whether high blood levels of 25-hydroxyvitamin D, a good marker of vi-tamin D availability to tissues,11 predicta lower risk of MS.

METHODSThis study has been approved by theinstitutional review boards of theHarvard School of Public Health andthe Walter Reed Army Institute of

Research, both of which waived theneed for informed consent to use

See also Patient Page.

Author Affiliations: Departments of Nutrition (Ms Mu-nger and Dr Ascherio) and Epidemiology (Dr Asche-rio), Harvard School of Public Health, and ChanningLaboratory, Department of Medicine, Brigham andWomen’s Hospital and Harvard Medical School(Dr Ascherio), Boston, Mass; Division of PreventiveMedicine, Walter Reed Army Institute of Research,Silver Spring, Md (Dr Levin); Departments of Pediat-rics, Biochemistry, and Molecular Biology, Medical Uni-versity of South Carolina, Charleston (Dr Hollis); andDepartment of the Navy, Secretary of the Navy Coun-cil of Review Boards, Washington, DC (Dr Howard).Corresponding Author: Alberto Ascherio, MD, DrPH,Harvard School of Public Health, 655 Huntington Ave,Third Floor, Boston, MA 02115 ([email protected]).

Context Epidemiological and experimental evidence suggests that high levels of vi-tamin D, a potent immunomodulator, may decrease the risk of multiple sclerosis. Thereare no prospective studies addressing this hypothesis.

Objective To examine whether levels of 25-hydroxyvitamin D are associated withrisk of multiple sclerosis.

Design, Setting, and Participants Prospective, nested case-control study amongmore than 7 million US military personnel who have serum samples stored in the De-partment of Defense Serum Repository. Multiple sclerosis cases were identified throughArmy and Navy physical disability databases for 1992 through 2004, and diagnoseswere confirmed by medical record review. Each case (n=257) was matched to 2 con-trols by age, sex, race/ethnicity, and dates of blood collection. Vitamin D status wasestimated by averaging 25-hydroxyvitamin D levels of 2 or more serum samples col-lected before the date of initial multiple sclerosis symptoms.

Main Outcome Measures Odds ratios of multiple sclerosis associated with con-tinuous or categorical levels (quantiles or a priori–defined categories) of serum 25-hydroxyvitamin D within each racial/ethnic group.

Results Among whites (148 cases, 296 controls), the risk of multiple sclerosis signifi-cantly decreased with increasing levels of 25-hydroxyvitamin D (odds ratio [OR] for a50-nmol/L increase in 25-hydroxyvitamin D, 0.59; 95% confidence interval, 0.36-0.97).In categorical analyses using the lowest quintile (!63.3 nmol/L) as the reference, the ORsfor each subsequent quintile were 0.57, 0.57, 0.74, and 0.38 (P=.02 for trend acrossquintiles). Only the OR for the highest quintile, corresponding to 25-hydroxyvitamin Dlevels higher than 99.1 nmol/L, was significantly different from 1.00 (OR, 0.38; 95% con-fidence interval, 0.19-0.75; P=.006). The inverse relation with multiple sclerosis risk wasparticularly strong for 25-hydroxyvitamin D levels measured before age 20 years. Amongblacks and Hispanics (109 cases, 218 controls), who had lower 25-hydroxyvitamin D lev-els than whites, no significant associations between vitamin D and multiple sclerosis riskwere found.

Conclusion The results of our study suggest that high circulating levels of vitamin Dare associated with a lower risk of multiple sclerosis.JAMA. 2006;296:2832-2838 www.jama.com

2832 JAMA, December 20, 2006—Vol 296, No. 23 (Reprinted) ©2006 American Medical Association. All rights reserved.

at University of Calgary, on December 19, 2006 www.jama.comDownloaded from

ORIGINAL CONTRIBUTION

Serum 25-Hydroxyvitamin D Levelsand Risk of Multiple SclerosisKassandra L. Munger, MScLynn I. Levin, PhD, MPHBruce W. Hollis, PhDNoel S. Howard, MDAlberto Ascherio, MD, DrPH

MULTIPLE SCLEROSIS (MS) ISamong the most commonneurological diseases inyoung adults, affecting

350 000 individuals in the United Statesand 2 million worldwide.1 Prevailingthought is thatMSisanautoimmunedis-order whereby an unknown agent oragents triggersaTcell–mediated inflam-matoryattack, causingdemyelinationofcentral nervous system tissue.2

A striking feature of the global dis-tribution of MS is a multifold increasein incidence with increasing latitude,both north and south of the equator.3

Genetic predisposition contributes tothis variation,4 but the change in MSrisk with migration among people ofcommon ancestry5 strongly supports arole for environmental factors. One po-tential factor may be vitamin D,6-9 a po-tent immunomodulator that in its hor-monal form can prevent experimentalautoimmune encephalomyelitis (EAE),an animal model of MS.10 Because foodprovides little vitamin D, the majorsource for most people is through skinexposure to sunlight.11 At latitudes of42° or more (eg, Boston, Mass), in win-ter most UV-B radiation is absorbed bythe atmosphere, and even prolongedsun exposure is insufficient to gener-ate vitamin D.12 As a result, seasonal vi-tamin D deficiency is common.11

AprotectiveeffectofvitaminDonMSis supported by the reduced MS risk as-

sociated with sun exposure13,14 and useofvitaminDsupplements,15 butevidenceremainsinconclusive.Inthepresentstudy,we examined prospectively for the firsttime whether high blood levels of 25-hydroxyvitamin D, a good marker of vi-tamin D availability to tissues,11 predicta lower risk of MS.

METHODSThis study has been approved by theinstitutional review boards of theHarvard School of Public Health andthe Walter Reed Army Institute of

Research, both of which waived theneed for informed consent to use

See also Patient Page.

Author Affiliations: Departments of Nutrition (Ms Mu-nger and Dr Ascherio) and Epidemiology (Dr Asche-rio), Harvard School of Public Health, and ChanningLaboratory, Department of Medicine, Brigham andWomen’s Hospital and Harvard Medical School(Dr Ascherio), Boston, Mass; Division of PreventiveMedicine, Walter Reed Army Institute of Research,Silver Spring, Md (Dr Levin); Departments of Pediat-rics, Biochemistry, and Molecular Biology, Medical Uni-versity of South Carolina, Charleston (Dr Hollis); andDepartment of the Navy, Secretary of the Navy Coun-cil of Review Boards, Washington, DC (Dr Howard).Corresponding Author: Alberto Ascherio, MD, DrPH,Harvard School of Public Health, 655 Huntington Ave,Third Floor, Boston, MA 02115 ([email protected]).

Context Epidemiological and experimental evidence suggests that high levels of vi-tamin D, a potent immunomodulator, may decrease the risk of multiple sclerosis. Thereare no prospective studies addressing this hypothesis.

Objective To examine whether levels of 25-hydroxyvitamin D are associated withrisk of multiple sclerosis.

Design, Setting, and Participants Prospective, nested case-control study amongmore than 7 million US military personnel who have serum samples stored in the De-partment of Defense Serum Repository. Multiple sclerosis cases were identified throughArmy and Navy physical disability databases for 1992 through 2004, and diagnoseswere confirmed by medical record review. Each case (n=257) was matched to 2 con-trols by age, sex, race/ethnicity, and dates of blood collection. Vitamin D status wasestimated by averaging 25-hydroxyvitamin D levels of 2 or more serum samples col-lected before the date of initial multiple sclerosis symptoms.

Main Outcome Measures Odds ratios of multiple sclerosis associated with con-tinuous or categorical levels (quantiles or a priori–defined categories) of serum 25-hydroxyvitamin D within each racial/ethnic group.

Results Among whites (148 cases, 296 controls), the risk of multiple sclerosis signifi-cantly decreased with increasing levels of 25-hydroxyvitamin D (odds ratio [OR] for a50-nmol/L increase in 25-hydroxyvitamin D, 0.59; 95% confidence interval, 0.36-0.97).In categorical analyses using the lowest quintile (!63.3 nmol/L) as the reference, the ORsfor each subsequent quintile were 0.57, 0.57, 0.74, and 0.38 (P=.02 for trend acrossquintiles). Only the OR for the highest quintile, corresponding to 25-hydroxyvitamin Dlevels higher than 99.1 nmol/L, was significantly different from 1.00 (OR, 0.38; 95% con-fidence interval, 0.19-0.75; P=.006). The inverse relation with multiple sclerosis risk wasparticularly strong for 25-hydroxyvitamin D levels measured before age 20 years. Amongblacks and Hispanics (109 cases, 218 controls), who had lower 25-hydroxyvitamin D lev-els than whites, no significant associations between vitamin D and multiple sclerosis riskwere found.

Conclusion The results of our study suggest that high circulating levels of vitamin Dare associated with a lower risk of multiple sclerosis.JAMA. 2006;296:2832-2838 www.jama.com

2832 JAMA, December 20, 2006—Vol 296, No. 23 (Reprinted) ©2006 American Medical Association. All rights reserved.


25-hydroxyvitamin D levels of less than75nmol/Las the reference (69casesand114controls) therewasanonsignificantreduction in risk among those with25-hydroxyvitaminDlevelsof75 to lessthan 100 nmol/L (62 cases and 124 con-trols; OR, 0.83; 95% CI, 0.54-1.29;P=.41) and a significant 51% reductionamong those with 25-hydroxyvitaminDlevelsof100nmol/Lorhigher(17casesand58controls;OR,0.49;95%CI,0.27-0.91; P=.02).

Adolescence appears to be a crucialexposure period for MS.5 Therefore, wefurther examined whether serum 25-hydroxyvitamin D concentrations be-fore age 20 years predict MS risk. Oneof 39 cases and 16 of 76 controls (2 ofthe 78 matched controls were 20 yearsold at time of blood collection and wereexcluded) had 25-hydroxyvitamin Dlevels of 100 nmol/L or higher, result-ing in an OR of 0.09 (95% CI, 0.01-0.75; P=.03) compared with levels lessthan 100 nmol/L.

We also were concerned that our re-sults could reflect an effect of MS on 25-hydroxyvitamin D levels rather than aneffect of 25-hydroxyvitamin D levels onMS risk. Multiple sclerosis could affect25-hydroxyvitamin D levels either bysome as yet unknown effect on vita-min D metabolism or, more likely, bychanges in behavior—because heatcommonly exacerbates MS symp-

toms, individuals with MS tend to avoidsun exposure and, thus, may have lower25-hydroxyvitamin D levels thanhealthy individuals.29 If heat intoler-ance and sun avoidance preceded theneurological symptoms recognized asthe first onset of MS, higher serum lev-els of 25-hydroxyvitamin D would spu-riously appear to be protective. To ad-dress this possibility, we examined thetemporal relationship between serum25-hydroxyvitamin D concentrations

and the date of onset of MS symptomsamong white cases. Average 25-hydroxyvitamin D levels among indi-viduals who developed MS were stableduring the years preceding symptomonset (P= .42 for trend) but signifi-cantly decreased after onset of symp-toms (P=.002). Mean 25-hydroxyvita-min D levels were 71.8 nmol/L morethan 6 years before symptom onset (51cases), 71.6 nmol/L between 4 and 6years (51 cases), 73.5 nmol/L between

Table. Selected Characteristics of Multiple Sclerosis Cases and Matched Controls*

CharacteristicsCases

(n = 257)Controls(n = 514)

Male 174 (68) 348 (68)Race

White, non-Hispanic 148 (57.6) 296 (57.6)Black, non-Hispanic 77 (30) 154 (30)Hispanic/other 32 (12.5) 64 (12.5)

Latitude of residence at entry into the military†‡Northern 42 (16.3) 104 (20.2)Middle 97 (37.7) 156 (30.4)Southern 101 (39.3) 205 (39.9)Outside continental United States 3 (1.2) 6 (1.2)

UV index of residence at entry into the military†!5 10 (3.9) 32 (6.2)5 to !6 124 (48.3) 221 (43.0)"6 106 (41.3) 212 (41.3)

No. of serum samples available2 81 (32) 172 (33.5)3 176 (68) 342 (66.5)

Age at first serum sample collection, mean (SD) [range], y 23.3 (5.3) [16-40] 23.3 (5.3) [17-41]*Data are expressed as No. (%) unless otherwise indicated.†Does not total to 100% because of missing information on place of residence at entry into the military.‡See “Methods” section of text for definitions of northern, middle, and southern latitudes.

Figure. Odds Ratios of MS by Quantile of Serum 25-Hydroxyvitamin D Among Whites and Blacks

P = .02 for Trend P = .90 for Trend

CasesControls

Quintile of 25-Hydroxyvitamin D, nmol/L

Whites

Odd

s R

atio

15.2-63.2 63.3-75.3 75.4-84.8 84.9-99.1 99.2-152.9

41 29 27 33 1856 60 63 57 60

3.0

1

0.1

CasesControls

Tertile of 25-Hydroxyvitamin D, nmol/L

Blacks

Odd

s R

atio

10.4-36.6 36.8-49.7 50.1-97.9

24 30 2351 56 47

3.0

11.0 1.0

2.22 2.17

1.05

0.51

1.15

0.59

1.07

0.57 0.57

0.30 0.30

1.071.36

0.74 0.75

0.38

0.19

0.40

0.1

Error bars indicate 95% confidence intervals.

SERUM 25-HYDROXYVITAMIN D LEVELS AND RISK OF MULTIPLE SCLEROSIS

©2006 American Medical Association. All rights reserved. (Reprinted) JAMA, December 20, 2006—Vol 296, No. 23 2835


RISCO RELATIVO DE QUEDAS AJUSTADO PARA A IDADE EM 124 IDOSOS

NÍVEL IDEAL DE 25OHD PARA ÓPTIMA FUNÇÃO MUSCULAR NOS MEMBROS INFERIORES

attenuates the ability of 1a,25(OH)2D3 to inhibit cellgrowth and induce 24-OHase [6].

Antiproliferative actionsThe precise pathways through which 1a,25(OH)2D trans-duces signals in prostate cells are less well understood.Recent studies indicate that 1a,25(OH)2D might actthrough different pathways to inhibit cell proliferation indifferent cell types. For example, LNCaP cells, which areandrogen-sensitive prostate-cancer cells, accumulate inthe G0–G1 phase of the cell cycle after treatment with1a,25(OH)2D3 [8,14] but no such accumulation is observedin ALVA-31 and PC-3 cells, even though the growth of bothis inhibited by 1a,25(OH)2D3 [14]. 1a,25(OH)2D3-inducedcell-cycle arrest of LNCaP cells in the G0–G1 phaseinvolves decreased phosphorylation of the retinoblastomagene-encoded protein (Rb). This is followed by a reductionin the activity of the E2F transcription factor, which leadsto increased activity of p21waf1, the CDK inhibitor, anddecreased CDK2 activity. 1a,25(OH)2D3-induced arrest ofLNCaP cells in G0 might also require functional p53 [15].In p53-negative PC-3 cells and a line of LNCaP cells (calledLN-56) in which p53 function is impaired by stabletransfection with the genetic suppressor element 56,1a,25(OH)2D3 does not cause G0 arrest. This allowsthese cells to quickly regain normal growth capabilitieswhen 1a,25(OH)2D3 is withdrawn from the media [15].Although abrogating Rb function with the SV40 large-Tantigen compromises the ability of 1a,25(OH)2D3 to inhibit

the growth of prostate-cancer cells [7], 1a,25(OH)2D3 doesinhibit prostate-cell growth in a Gg/T-15 transgenic mouseline that contains the human fetal globin promoter linkedto the SV40 large-T antigen [16]. Moreover, the growth ofDU145 cells, which lack functional Rb and have high levelsof 24-OHase, is inhibited by 1a,25(OH)2D3 in the presenceof Liarozole [an inhibitor of 24-OHase that prevents the24-hydroxylation of 1a,25(OH)2D3 and so prolongs thehalf-life of 1a,25(OH)2D3 [7]. These findings indicate that1a,25(OH)2D3-mediated growth inhibition in DU145 cellsand in cell lines generated using the SV40 large-T antigenmight be mediated by an alternative mechanism.

Apoptotic actionsUnder some experimental conditions 1a,25(OH)2D alsoinduces apoptosis in LNCaP cells [8,15,17,18]. Using theterminal deoxynucleotide transferase-mediated dUTPnick end labeling (TUNEL) assay followed by flowcytometric analysis to quantify DNA fragmentation,Blutt et al. [18] have observed apoptosis after treatingLNCaP cells with 1a,25(OH)2D3. This is accompanied bydownregulation of two antiapoptotic proteins, Bcl2 andBclXL, and is prevented by overexpression of the gene thatencodes Bcl2. Other antiapoptotic proteins (Mcl-1, BAG1L,XIAP, cIAP1 and cIAP2) are also downregulated by1a,25(OH)2D3 in LNCaP cells but proapoptotic Bax andBak are unaltered [17]. This downregulation leads to theactivation of caspase-3 and caspase-9, the apical proteasesin the mitochondrial pathway for apoptosis [17]. Neitherapoptosis nor changes in synthesis of pro-apoptotic proteinhave been observed in DU145 cells treated with1a,25(OH)2D3. Thus, both growth arrest and apoptosisare involved in growth regulation of LNCaP cells inresponse to 1a,25(OH)2D3.

Interaction between vitamin D and other hormones1a,25(OH)2D does not act alone in regulating prostate cellproliferation. RARs and ARs are involved in regulating thegrowth of some cancer cell lines [7]. Weigel and associates[8] have demonstrated that 1a,25(OH)2D3 and 9-cis RA, aligand of RXR, act synergistically to inhibit the growth ofLNCaP cells and cause cells to accumulate in G0. Thisappears to be dependent on functional p53 [15]. Zhao et al.showed that 1a,25(OH)2D3 and 9-cis RA increase theexpression of mRNA that encodes the androgen receptor(AR) and act synergistically to inhibit LNCaP cell growth[19]. Because both actions are prevented by the pure ARantagonist, Casodex, they concluded that growth inhi-bition of LNCaP cells by 1a,25(OH)2D3 and/or 9-cis RA ismediated by an AR-dependent mechanism and precededby the induction of AR gene expression. To re-examine therole of androgens in the antiproliferative effects of1a,25(OH)2D3 in prostate cancer cells, Yang et al. [20]have utilized two androgen-independent cell models ofprostate cancer, ALVA-AR and LNCaP-104R1, that con-tain functional ARs and VDRs. They found that neithergrowth of ALVA-AR nor of control ALVA-NEO cells isinhibited substantially by 1a,25(OH)2D3 either in thepresence or absence of androgen, which indicates that theresistance of ALVA-AR to 1a,25(OH)2D3-mediated growthinhibition is not caused by lack of AR. They also found that

Fig. 2. Mechanism of vitamin D3 activity. Both 25-hydroxyvitamin D3 [25(OH)D3]and 1a,25-dihydroxyvitamin D3 [1a,25(OH)2D3] are transported into prostate cells.25(OH)D3 is then converted to 1a,25(OH)2D3 by 25(OH)D-1a-hydroxylase(1a-OHase) in mitochondria. Binding of 1a,25(OH)2D3 to the vitamin D receptor(VDR) causes the VDR to heterodimerize with the retinoid X receptor (RXR). TheVDR–RXR heterodimer binds to specific vitamin D-response elements in the pro-moter region of vitamin D3-responsive genes and induces gene transcription. Thegene products include proteins involved in apoptosis (Bcl-2, Bcl-XL, Mcl-1, BAG1L,XIAP, clAP1 and clAP2), differentiation [prostate specific antigen (PSA) and theandrogen receptor (AR)], and cell-cycle regulation, including cyclin-dependentkinase (CDK), CDK inhibitors (p21 and p27), tumor suppression (p53 and E-Cad-herin), and cell proliferation-associated nuclear antigen (Ki 67).

TRENDS in Endocrinology & Metabolism

1α,25(OH)D3

Nucleus

Gene transcription

Mitochondria

25(OH)D31α,25(OH)2D3

RXR

ApoptosisBcl-2, Bcl-XL, Mcl-1BAG1L, XIAP, cIAP1

cIAP2

DifferentiationPSA, AR

Cell-cycle arrestCDK2, p21, p27, p53,

Ki67, E-Cadherin

VDR

Prostate cell

1α-OHase25(OH)D3 1α,25(OH)2D3

Review TRENDS in Endocrinology and Metabolism Vol.14 No.9 November 2003 425

http://tem.trends.com

Vitamin D and prostate cancerprevention and treatmentTai C. Chen and Michael F. Holick

Vitamin D, Skin and Bone Research Laboratory, Section of Endocrinology, Diabetes and Nutrition, Department of Medicine, BostonUniversity School of Medicine, Boston, MA 02118, USA

Human prostate cells contain receptors for 1a,25-dihy-droxyvitamin D, the active form of vitamin D. Prostatecancer cells respond to vitamin D3 with increases indifferentiation and apoptosis, and decreases in prolifer-ation, invasiveness and metastasis. These findingsstrongly support the use of vitamin D-based therapiesfor prostate cancer and/or as a second-line therapy ifandrogen deprivation fails. The association betweeneither decreased sun exposure or vitamin D deficiencyand the increased risk of prostate cancer at an earlierage, and with a more aggressive progression, indicatesthat adequate vitamin D nutrition should be a priorityfor men of all ages. Here we summarize recent advancesin epidemiological and biochemical studies of the endo-crine and autocrine systems associated with vitamin Dand their implications for prostate cancer and in theevaluation of vitamin D3 and its analogs in preventingand/or treating prostate cancer.

Vitamin D2 (ergocalciferol) is derived from fungi andplants, whereas vitamin D3 (cholecalciferol) is produced inthe skin. Both forms (referred to here as vitamin D) arehydroxylated to create the active hormone. The firsthydroxylation step, which forms 25(OH)D (see Glossary),occurs in the liver. 25(OH)D is then further hydroxylatedat the 1a-position by 25(OH)D-1a-hydroxylase (1a-OHase,also known as CYP27B1) in the kidney to form 1a,25(OH)2D,the active form of vitamin D (Fig. 1) [1]. The cDNAs thatencode 1a-OHase in mice, rats and humans have beencloned [2,3], and 1a,25(OH)2D is now known to playimportant roles in the regulation of .60 genes, includingthose associated with calcium homeostasis, immuneresponses, blood pressure control, cell proliferation,differentiation and apoptosis. [1,3,4].

Prostate cancer is the most commonly diagnosed andthe secondmost fatal cancer in Americanmen. The inversecorrelation !P , 0:0001" between the mortality rate ofprostate cancer and exposure to ultraviolet radiation(UVR) in the US population, as well as the greater riskof prostate cancer in Afro-Caribbeanmen indicate that oneprecipitating factor for prostate cancermight be vitamin Dinsufficiency [5]. The biochemical evidence to support arole for vitamin D in prostate cancer includes thedemonstration of VDR and the antiproliferative, apoptotic

and prodifferentiation activities of 1a,25(OH)2D and itsanalogs in prostate cells in vitro and in vivo [6–8].

Here we summarize recent findings of: (1) the associ-ation between vitamin D deficiency, UVR exposure and therisk of prostate cancer; (2) the mechanism of 1a,25(OH)2Daction; (3) the identification of 1a-OHase in the prostateand its implications; (4) the evaluation of antiproliferativeactivity of 1a,25(OH)2D3 and its analogs in prostate cells inculture, in animal models and in clinical trials; and (5) thecontroversy that surrounds the association between VDRpolymorphism and the risk of prostate cancer.

Vitamin D deficiency, UV exposure and the risk ofprostate cancerAn association between vitamin D deficiency and prostatecancer was reported by Ahonen et al. in a 13-year follow-upof 19 000middle-agedmen in theHelsinkiHeart Study [9].In this study, 149 cases of prostate cancer were identifiedand matched to 566 sample controls. The study showedthat low circulating levels of 25(OH)D (,40 nmol l21 or16 ng ml21) were associated with an increased risk ofsubsequent earlier onset and more aggressive progressionof prostate cancer, especially before the age of 52.

UVR exposure has a significant protective effect inprostate cancer [10,11]. Luscombe et al. [10] showed thatcancer patients with the lowest quartile of sun exposuredeveloped cancer at a median age of 67.7 years comparedwith 72.1 years in patients in other quartiles. Although themechanism of this association is unclear, it is likely thatincreased cutaneous synthesis of vitamin D3 increases thecirculating levels of 25(OH)D3 and the subsequentformation of 1a,25(OH)2D3 in the prostate by prostatic1a-OHase [12]. 1a,25(OH)2D3 then interacts with VDR in

Glossary

1a,25(OH)2D3: 1a,25-dihydroxyvitamin D3

25(OH)D3: 25-hydroxyvitamin D3

EB1089: Seocalcitol, 1a,25-dihydroxy-22,24-diene-24,26,27-trihomovitamin D3

CDK: cyclin-dependent kinaseCKI: cyclin-dependent kinase inhibitorE2F: early gene 2 factorIGFBP: insulin-like growth factor binding proteinp21waf1: cyclin-dependent kinase inhibitor p21Cip1/Waf1p27: cyclin-dependent kinase inhibitor p27Kip1p53: p53 tumor suppressorRFLP: restriction fragment length polymorphismVDR: vitamin D receptorVDRE: vitamin D response element

Corresponding authors: T.C. Chen ([email protected]),M.F. Holick ([email protected]).

Review TRENDS in Endocrinology and Metabolism Vol.14 No.9 November 2003 423

http://tem.trends.com 1043-2760/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2003.09.004

of aggressive disease. Both studies indicate that high serum levelsof 25(OH)D can be problematic and thus, the general commu-nication of the result in this study should be done with caution.The plausibility of an effect of 25(OH)D exists. 25(OH)D can act

as a substrate for the 1,a-hydroxylation and production of1,25(OH)2D in the prostate or 25(OH)D might bind itself to theVDR in the prostate cells and act by itself (Schwartz, 2005). Severalexperimental studies have demonstrated the anti-invasive andanti-metastatic effect of vitamin D on prostate cells, through itspromotion of differentiation and apoptosis and its inhibiting effecton angiogenesis and proliferation (see Dunlap et al, 2003). Ourresults are consistent with the ecological studies showing thelowest death risk among patients diagnosed in seasons with highultraviolet radiation exposure (Robsahm et al, 2004; Lim et al,2006). These results from ecological studies are not in accordancewith an influence of the prostate cancer disease on the level ofvitamin D. The results in the present study are also consistent withan inverse relationship between plasma 25(OH)D levels and

mortality that have been reported for early-stage lung cancer andfor colorectal cancer (Zhou et al, 2007; Ng et al, 2008).It has been hypothesised that vitamin D can amplify the effect of

cancer treatment; a synergistic effect that has been observed inboth experimental and clinical studies (Dunlap et al, 2003; Deebet al, 2007). Furthermore, results from experimental studies alsoindicate an interaction between sexual hormones and vitamin Dmetabolism (Nyomba et al, 1987; Sarem and Pedersen, 1988;Bolland et al, 2007). The androgen testosterone is synthesised fromcholesterol and has a similar structure as vitamin D, and actsthrough nuclear receptors that belong to the same chemical familyas VDR. Thus, a possible explanation for the observed associationbetween 25(OH)D levels and mortality in group I (Table 2) and forthe patients on hormone therapy in groups I and II combined(Table 3) might be that 25(OH)D amplifies the therapeutic effectsof lowering androgen levels and/or activity and hence improve thecancer prognosis. Among the patients treated without hormonesonly seven patients died of prostate cancer. This makes itimpossible to make an appropriate survival analysis for othertreatments than hormone therapy. The different results betweengroup I and II (Table 2, model III), might be due to a difference inthe stage of disease. In group II only 19 deaths occurred duringfollow-up, which indicate the need for longer follow-up or newlarger studies.To conclude, this study shows a strong association between

25(OH)D levels and cause-specific mortality in patients with

Table 2 The estimated relationship between serum calcidiol and deathfrom prostate cancer for all patients and patient group I and II separately

VariablesModel I RR(95%CI)

Model II RR(95%CI)

Model III RR(95%CI)

Group I+II (n! 160)Calcidiol (nmol l"1)Low (o50) 1.00 (ref) 1.00 (ref) 1.00 (ref)Medium (50–80) 0.48 (0.24–0.97) 0.41 (0.20–0.85) 0.33 (0.14–0.77)High (480) 0.34 (0.15–0.77) 0.22 (0.09–0.53) 0.16 (0.05–0.43)

Group status 0.05 (0.03–0.10) 0.05 (0.03–0.10)

Age (1 year) 1.00 (0.96–1.03) 1.00 (0.95–1.03)

Differentiation gradea

High 1.00 (ref)Moderate 1.56 (0.42–5.06)Low 7.01 (1.91–25.7)

Functional statusGood 1.00 (ref)Less good 1.22 (1.00–1.50)

Group I (n! 37)Calcidiol (nmol l"1)Low (o50) 1.00 (ref) 1.00 (ref) 1.00 (ref)Medium (50–80) 0.31 (0.13–0.73) 0.30 (0.12–0.73) 0.51 (0.14–1.78)High (480) 0.26 (0.10–0.68) 0.25 (0.09–0.69) 0.41(0.11–1.54)

Age (1 year) 0.99 (0.95–1.04) 0.99 (0.94–1.04)


Group II (n! 123)Calcidiol (nmol l"1)Low (o50) Medium 1.00 (ref) 1.00 (ref) 1.00 (ref)(50–80) 0.73 (0.24-2.24) 1.14 (0.24–5.36) 1.05 (0.21–5.13)High (480) 0.57 (0.17–1.91) 0.62 (0.11–3.57) 0.59 (0.10–3.47)

Age (1 year) 0.92 (0.85–1.00) 0.93 (0.86–1.00)




aDifferentiation grade of tumour tissue; WHO three-grade system.

Table 3 The estimated relationship between serum calcidiol and deathfrom prostate cancer among patients receiving hormone therapy (n! 97)

VariablesModel I

RR (95%CI)Model II

RR (95%CI)Model III

RR (95%CI)

Calcidiol (nmol l"1)Low 1.00 (ref) 1.00 (ref) 1.00 (ref)Medium 0.39 (0.19–0.81) 0.35 (0.17–0.73) 0.18 (0.07–0.46)High 0.29 (0.12–0.68) 0.20 (0.08–0.50) 0.09 (0.03–0.27)

Group status 0.08 (0.04–0.16) 0.06 (0.02–0.13)

Age (1 year) 1.00 (0.94–1.03)




aDifferentiation grade of tumour tissue; WHO three-grade system.

Table 4 The estimated relationship between serum calcidiol and deathfrom all causes (n! 160)

VariablesModel I

RR (95%CI)Model II

RR (95%CI)Model III

RR (95%CI)

Calcidiol (nmol l"1)Low 1.00 (ref) 1.00 (ref) 1.00 (ref)Medium 0.41 (0.21–0.79) 0.37 (0.19–0.73) 0.40 (0.20–0.78)High 0.35 (0.17–0.2) 0.23 (0.11–0.51) 0.24 (0.11–0.53)

Group status 0.08 (0.05–0.14) 0.08 (0.05–0.14)

Age (1 year) 1.00 (0.97–1.04)


Vitamin D and cancer prognosisS Tretli et al

453

British Journal of Cancer (2009) 100(3), 450 – 454& 2009 Cancer Research UK

ClinicalStudies

(< 50) (50-‐80)

(> 80)

Association between serum 25(OH)D and death from prostatecancer

S Tretli1,2, E Hernes1,3, JP Berg4,5, UE Hestvik1 and TE Robsahm*,1

1The Cancer Registry of Norway, Institute of Population-based Cancer Research, Oslo, Norway; 2Department of Community Medicine and GeneralPractice, NTNU, Trondheim, Norway; 3The Norwegian Radium Hospital, Montebello, Norway; 4Hormone Laboratory, Aker University Hospital, Oslo,Norway; 5Faculty Division Ulleval University Hospital, University of Oslo, Oslo, Norway

Based on observations that for certain cancers, mortality varies according to sun exposure, vitamin D has been proposed to influenceon disease progression. This study aims to investigate whether serum levels of 25(OH)D are associated with prognosis in patientswith prostate cancer. In total, 160 patients with a serum sample in the JANUS serum bank were included. For 123 patients a pre-treatment serum sample was taken, whereas 37 of the patients had received hormone therapy prior to the blood collection. Theserum level of 25(OH)D was classified as low (o 50 nmol l!1), medium (50–80 nmol l!1) or high (480 nmol l!1). A Coxproportional hazard regression model was used to assess the association between serum 25(OH)D and cancer mortality. Duringfollow-up, 61 deaths occurred, of whom 52 died of prostate cancer. The median time of follow-up was 44.0 months (range, 1.2–154.6). Serum 25(OH)D at medium or high levels were significantly related to better prognosis (RR 0.33; 95% CI 0.14–0.77, RR 0.16;95% CI 0.05–0.43) compared with the low level. Analysis restricted to patients receiving hormone therapy gave a strongerassociation. The serum level of 25(OH)D may be involved in disease progression and is a potential marker of prognosis in patientswith prostate cancer.British Journal of Cancer (2009) 100, 450–454. doi:10.1038/sj.bjc.6604865 www.bjcancer.comPublished online 20 January 2009& 2009 Cancer Research UK

Keywords: 25(OH)D; serum; prostate cancer; prognosis; mortality

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The prehormone vitamin D is well known for its important role incalcium regulation and mineralisation of the bone. However,during the last two decades accumulating evidence suggests thatvitamin D also influences several other biological processes. Asearly as 1941, an inverse association between ultraviolet radiationand cancer mortality was suggested (Apperly, 1941). By 1980, thehypothesis that vitamin D deficiency resulting from insufficientsun exposure increased mortality from colon cancer was proposed(Garland and Garland, 1980). The mortality rates for cancer of thebreast, prostate, lung, skin and lymphoma have subsequently beenshown to vary according to variation in sun exposure (John et al,1999; Freedman et al, 2002; Grant, 2002; Robsahm et al, 2004;Berwick et al, 2005; Porojnicu et al, 2005, 2007). Results fromrecent studies indicate that vitamin D also might influence cancerincidence (see Giovannucci, 2005) through mechanisms thatinfluence cancer development and progression and less likely asa part of the cancer initiation. Vitamin D deficiency is suggested tobe a risk factor for prostate cancer (Schwartz and Hulka, 1990;Luscombe et al, 2001). The effect of vitamin D on cancer processeshas repeatedly been demonstrated in experimental studies (seeHolick, 2006) as it regulates cell cycle processes such asproliferation, apoptosis and angiogenesis in different tissues.

Although several factors influence the level of circulatingvitamin D, skin exposure to sunlight is the most important factor.The main dietary contributors include fatty fish, cod liver oil, eggs,and vitamin D fortified dairy products. The biologically mostactive form of vitamin D, calcitriol (1,25(OH)2D), is formed fromcalcidiol (25(OH)D) in the kidney and is kept at a virtuallyconstant level in serum by parathyroid hormone. However, localproduction of 1,25(OH)2D occurs in different tissues includingprostate cells (Schwartz et al, 1998), where it regulates keyprocesses as cell differentiation and proliferation (Hansen et al,2001; Omdahl et al, 2002). 1,25(OH)2D acts by binding to nuclearvitamin D receptors (VDR), and regulates gene transcription. Theamount of vitamin D available in the body is closely associated tothe concentration of the vitamin D metabolite, 25(OH)D, in theblood. Thus, measurement of circulating 25(OH)D is the bestmethod to estimate vitamin D availability in the body (Freedmanet al, 2007).During the winter months at northern latitudes, there is an

insufficient amount of UVB in the sunlight to generate vitamin Dproduction in the skin. In Norway, the daily maximum UV-indicesvary from zero during the winter months (November–February)to 4.5–6.5 (possible range 0–10) in the mid-summer (Johnsenet al, 2002). Similarly, the level of 25(OH)D vary throughout theyear, with a mean in the Norwegian population of about50 nmol l!1 during the winter to about 70–80 nmol l!1 during thesummer (Moan and Porojnicu, 2006). By using season of diagnosisas an indicator for the level of vitamin D, we previously haveinvestigated a possible relationship between vitamin D and cancer-specific survival. Patients who were diagnosed with cancer of thebreast, colon, prostate, lung or lymphoma during summer or

Received 23 September 2008; revised 2 December 2008; accepted 5December 2008; published online 20 January 2009

*Correspondence: Dr TE Robsahm, The Cancer Registry of Norway,Institute of Population-based Cancer Research, PB 5313, Majorstua, Oslo0304, Norway; E-mail: [email protected]

British Journal of Cancer (2009) 100, 450 – 454& 2009 Cancer Research UK All rights reserved 0007 – 0920/09 $32.00

www.bjcancer.com

Clin

icalStudies

by quantiles of serum 25(OH)D provided a clear linear dose-response gradient (61) (Fig. 4). A serum 25(OH)D levelgreater than 38 ng/mL (95 nmol/L) (top quintile) was asso-ciated with an odds ratio of 0.45 (95% CI 0.28–0.69), cor-responding to 55% lower risk of colorectal cancer comparedto individuals with 25(OH)D of less than 16 ng/mL (40nmol/L) (bottom quintile) (61).

Prostate Cancer

Observational studies of the inverse association of prediag-nostic serum 25(OH)D with prostate cancer were recentlyreviewed by Giovannucci (62). The geographic epidemi-ology of prostate cancer is not as clearly linked with solarirradiance levels as it is for cancer of the breast, colon, ovary,endometrium and kidney.

Recent observational studies of the incidence of prostatecancer have had promising, although mixed, results. A studyby Li and colleagues of the Physicians’ Health Study cohort

(10) found that physicians whose 25(OH)D and1,25(OH)2D levels were both below the median,25(OH)D of 28 ng/mL (70 nmol/L) and 1,25(OH)2D of32 pg/mL (77 pmol/L) had twice the incidence of aggressiveprostate cancer (odds ratio 2.1, 95%CI 1.2–3.4, p! 0.05) asmen whose levels were above the median.

In a nested case-control study of 90 Kaiser Foundationcases and 91 controls matched on age, race, and day of serumstorage, the estimated relative risk of prostate cancer was0.41 (not significant) in men in the top quartile of serumvitamin D metabolites, specifically, 25(OH)D greater than28 ng/mL (70 nmol/L) and 1,25(OH)2D greater than 39pg/mL (94 pmol/L). The risk of aggressive prostate cancer(palpable mass or Gleason score 7–10) in men in the topquartiles of serum 25(OH)D and 1,25(OH)2D wasextremely low (relative risk 0.03, not significant) in menolder than 57 years of age (the median age of the cohort)(9). These effects were not present in younger men or for

0.2

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FIGURE 1. Relative risk of breast cancer mortality, by baselineserum 25(OH)D concentration, divided at the median,NHANES III cohort, 1988–2000. (Source: Drawn from data inFreedman et al. [56].)

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58 reduction in breastcancer risk associated with38 ng/ml serum 25(OH)D

FIGURE 2. Pooled odds ratio for breast cancer, according toserum 25(OH)D concentration, meta-analysis, 2008. (Sources:Bertone-Johnson et al. [5], Lowe et al. [4], Garland et al. [57].)(Graphic: E. D. Gorman.)

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FIGURE 3. Relative risk of colon cancer mortality, by baselineserum 25(OH)D concentration, in tertiles, NHANES cohort,1988-2000. (Source: Drawn from data in Freedman et al. [56].)

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55 reduction in coloncancer risk associated with38 ng/ml serum 25(OH)D

FIGURE 4. Pooled odds ratio for colorectal cancer, according toserum 25(OH)D concentration, meta-analysis, 2007. (Source,meta-analysis of six studies: Gorham et al. [61].) (Graphic: E. D.Gorham, S. B. Mohr.)

Garland et al. AEP Vol. 19, No. 7VITAMIN D FOR CANCER PREVENTION July 2009: 468–483

470


Prostate Cancer





0.2

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Rel

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skFIGURE 3. Relative risk of colon cancer mortality, by baselineserum 25(OH)D concentration, in tertiles, NHANES cohort,1988-2000. (Source: Drawn from data in Freedman et al. [56].)

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Rel

ativ

e ri

skFIGURE 3. Relative risk of colon cancer mortality, by baselineserum 25(OH)D concentration, in tertiles, NHANES cohort,1988-2000. (Source: Drawn from data in Freedman et al. [56].)

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13

Why the minimum desirable serum 25-hydroxyvitamin Dlevel should be 75 nmol/L (30 ng/ml)

Reinhold Vieth, Ph.D., F.C.A.C.B., Professor a,b,c,*aDepartment of Nutritional Sciences, University of Toronto, CanadabDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Canadac Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave, Toronto, Ontario, Canada M5G 1X5

Keywords:dietary guidelinesoptimal vitamin D nutritional statushealthdisease prevention

The Institutes of Medicine (IOM) recently revised the recom-mended dietary allowances (RDA) for vitamin D, to maintainserum 25-hydroxyvitamin D (25(OH)D) at or above 50 nmol/L, tosustain bone density, calcium absorption, and to minimize risk ofosteomalacia and rickets. However there are compelling reasonswhy 25(OH)D should preferably exceed 75 nmol/L: (A) Scrutiny ofactual data specified by the IOM relating 25(OH)D to bone densityand osteomalacia shows the desirable minimum 25(OH)D to be75 nmol/L (30 ng/mL). (B) Humans are primates, optimizedthrough evolution to inhabit tropical latitudes, with serum 25(OH)D over 100 nmol/L. (C) Epidemiologic relationships show healthbenefits if 25(OH)D levels exceed 70 nmol/L; these include fewerfalls, better tooth attachment, less colorectal cancer, improveddepression and wellbeing. Some studies of populations at high-latitude relate higher 25(OH)D to risk of prostate cancer, pancre-atic cancer or mortality. Those relationships are attributable to thedynamic fluctuations in 25(OH)D specific to high latitudes, andwhich can be corrected by maintaining 25(OH)D at steady, highlevels throughout the year, the way they are in the tropics. (D)There are now many clinical trials that show benefits and/or noadversity with doses of vitamin D that raise serum 25(OH)D tolevels beyond 75 nmol/L. Together, the evidence makes it veryunlikely that further research will change the conclusion that riskof disease with serum 25(OH)D higher than 75 nmol/L is lowerthan the risk of disease if the serum 25(OH)D is approximately53 nmol/L.

! 2011 Elsevier Ltd. All rights reserved.

* Tel.: !1 416 586 5920; Fax: !1 416 586 8628.

Contents lists available at ScienceDirect

Best Practice & Research ClinicalEndocrinology & Metabolism

journal homepage: www.elsevier .com/locate/beem

1521-690X/$ – see front matter ! 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.beem.2011.06.009

Best Practice & Research Clinical Endocrinology & Metabolism 25 (2011) 681–691

RELAÇÃO ENTRE INGESTÃO CÁLCIO E NÍVEIS SÉRICOS DE 25OHD

©2008 by American Society for Nutrition Heaney R P Am J Clin Nutr 2008;88:541S-544S

Ingestão de 300 mg Cálcio

[Dermato-Endocrinology 1:4, 207-214; July/August 2009]; ©2009 Landes Bioscience

Emerging scientific evidence strongly supports the beneficial role of vitamin D in reducing the risk of incidence and death from many chronic and infectious diseases. This study esti-mates increases in melanoma and nonmelanoma skin cancer mortality rates and decreases in chronic and infectious disease mortality rates in the US from the standpoint of approximately doubling population doses of solar UVB to increase mean serum 25-hydroxyvitamin D levels from 16 ng/mL for black Americans and 25 ng/mL for white Americans to 45 ng/mL. The primary benefits are expected to come from reductions in cancer and cardiovascular diseases. Although a few thousand excess deaths per year might occur from melanoma and skin cancer, the avoided premature death rate could be near 400,000/year, with most of the avoided deaths coming late in life. While oral sources of vitamin D could be used instead of UVB or when UVB irradiance is not available, public health policies do not yet recommend the 3,000–4,000 IU/day required to raise serum 25-hydroxyvitamin D levels to the levels required for optimal health, which would be required before vitamin D fortification levels in food can be raised. Until then, moderate solar UVB irra-diance remains an import source, and the health benefits greatly outweigh the risks.

Introduction

Solar UVB (290–315 nm) irradiance correlates with reduced risk of about 14 types of cancer.1-4 It is hypothesized to explain the latitudinal variation of multiple sclerosis,5 the seasonality of epidemic influenza,6 the epidemiology of septicemia,7 and case fatality rates during the 1918–1919 influenza pandemic.8 The beneficial effect of ultraviolet irradiance (UVR) arises from production of vitamin D. Serum 25-hydroxyvitamin D [25(OH)D] level also inversely correlates with incidence and/or mortality

rates of other diseases such as type 2 diabetes mellitus,9,10 coronary heart disease (CHD)11 and congestive heart failure.12

Let us put vitamin D production into the context of human history on Earth. The human species originated in the eastern portion of tropical Africa. Skin pigmentation in that region was very dark to protect against the adverse effects of solar UVR, primarily free radical production and DNA damage leading to melanoma and other skin cancer.13 Because UVB doses were high and clothes were not worn, sufficient UVB penetrated the epidermis to produce adequate vitamin D. As people migrated poleward from the tropics, skin pigmentation lightened to become very pale in northern Europe because those with dark skin had lower survival rates because of rickets and both chronic and infec-tious diseases.14

One underlying reason for concern about skin cancer and mela-noma today is that many people with skin that has adapted for life at high latitudes are now living at lower latitudes, where their skin pigmentation does not afford adequate protection against the adverse effects of solar UV. Conversely, many with dark skin have moved poleward and have chronically low serum 25(OH)D levels and, as a result, higher disease rate.15,16

This report will estimate the health benefits and risks of increasing solar UVB irradiance and oral intake of vitamin D to increase mean serum 25-hydroxyvitamin D levels from 16 ng/mL for black Americans and 25 ng/mL for white Americans17,18 to 45 ng/mL, a level that seems to be in the range required for optimal health,19,20 which requires the production from UVB irradiance or oral intake of about 3,600 IU/day.21,22 The indices used for this study are mortality rates for diseases affected by either vitamin D or UVR leading to death. Although incidence and prevalence rates and the economic burden could also be used, they should yield similar results.

Results

The index used to estimate the changes in health due to increased serum 25(OH)D levels is mortality rates of UV- and vitamin D-sensitive diseases. It is assumed that raising mean serum 25(OH)D levels for white Americans from 25 ng/mL to 45 ng/mL would take 2–2.5 times the current solar UVB irradi-

*Correspondence to: William B. Grant; Sunlight, Nutrition and Health Research Center (SUNARC); P.O. Box 641603; San Francisco, CA 94164-1603 USA; Tel.: 415.409.1980; Email: [email protected]

Submitted: 08/17/09; Accepted: 08/19/09

Previously published online as a Dermato-Endocrinology E-publication: http://www.landesbioscience.com/journals/dermatoendocrinology/article/9841

Review

In defense of the sunAn estimate of changes in mortality rates in the United States if mean serum 25-hydroxyvitamin D levels were raised to 45 ng/mL by solar ultraviolet-B irradiance

William B. Grant

Sunlight, Nutrition and Health Research Center (SUNARC); San Francisco, CA USA

Key words: cancer, cardiovascular diseases, melanoma, respiratory infections, skin cancer, vitamin D, ultraviolet-B

www.landesbioscience.com Dermato-Endocrinology 207

review article

T h e n e w e ng l a nd j o u r na l o f m e dic i n e

n engl j med 357;3 www.nejm.org july 19, 2007266

Medical Progress

Vitamin D DeficiencyMichael F. Holick, M.D., Ph.D.

From the Department of Medicine, Sec-tion of Endocrinology, Nutrition, and Di-abetes, the Vitamin D, Skin, and Bone Research Laboratory, Boston University Medical Center, Boston. Address reprint requests to Dr. Holick at Boston University School of Medicine, 715 Albany St., M-1013, Boston, MA 02118, or at [email protected].

N Engl J Med 2007;357:266-81.Copyright © 2007 Massachusetts Medical Society.

Once foods were fortified with vitamin d and rickets appeared to have been conquered, many health care professionals thought the major health problems resulting from vitamin D deficiency had been resolved. How-

ever, rickets can be considered the tip of the vitamin D–deficiency iceberg. In fact, vitamin D deficiency remains common in children and adults. In utero and during childhood, vitamin D deficiency can cause growth retardation and skeletal deformi-ties and may increase the risk of hip fracture later in life. Vitamin D deficiency in adults can precipitate or exacerbate osteopenia and osteoporosis, cause osteomalacia and muscle weakness, and increase the risk of fracture.

The discovery that most tissues and cells in the body have a vitamin D receptor and that several possess the enzymatic machinery to convert the primary circulating form of vitamin D, 25-hydroxyvitamin D, to the active form, 1,25-dihydroxyvitamin D, has provided new insights into the function of this vitamin. Of great interest is the role it can play in decreasing the risk of many chronic illnesses, including common can-cers, autoimmune diseases, infectious diseases, and cardiovascular disease. In this review I consider the nature of vitamin D deficiency, discuss its role in skeletal and nonskeletal health, and suggest strategies for its prevention and treatment.

Sources a nd Me ta bol ism of V i ta min D

Humans get vitamin D from exposure to sunlight, from their diet, and from dietary supplements.1-4 A diet high in oily fish prevents vitamin D deficiency.3 Solar ultravio-let B radiation (wavelength, 290 to 315 nm) penetrates the skin and converts 7-dehy-drocholesterol to previtamin D3, which is rapidly converted to vitamin D3 (Fig. 1).1 Because any excess previtamin D3 or vitamin D3 is destroyed by sunlight (Fig. 1), ex-cessive exposure to sunlight does not cause vitamin D3 intoxication.2

Few foods naturally contain or are fortified with vitamin D. The “D” represents D2 or D3 (Fig. 1). Vitamin D2 is manufactured through the ultraviolet irradiation of ergosterol from yeast, and vitamin D3 through the ultraviolet irradiation of 7-dehy-drocholesterol from lanolin. Both are used in over-the-counter vitamin D supplements, but the form available by prescription in the United States is vitamin D2.

Vitamin D from the skin and diet is metabolized in the liver to 25-hydroxyvitamin D (Fig. 1), which is used to determine a patient’s vitamin D status1-4; 25-hydroxyvi-tamin D is metabolized in the kidneys by the enzyme 25-hydroxyvitamin D-1!-hydroxylase (CYP27B1) to its active form, 1,25-dihydroxyvitamin D.1-4 The renal pro-duction of 1,25-dihydroxyvitamin D is tightly regulated by plasma parathyroid hormone levels and serum calcium and phosphorus levels.1-4 Fibroblast growth fac-tor 23, secreted from the bone, causes the sodium–phosphate cotransporter to be internalized by the cells of the kidney and small intestine and also suppresses 1,25-dihydroxyvitamin D synthesis.5 The efficiency of the absorption of renal calcium and of intestinal calcium and phosphorus is increased in the presence of 1,25-dihy-

Holick M. NEJM 2007;357:266-81.

medical progress

n engl j med 357;3 www.nejm.org july 19, 2007 269

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AuthorFig #TitleME

DEArtist

Issue date

COLOR FIGURE

Draft 13Holick

KMK

Vitamin D Deficiency

7/19/07

Koopman

Skin

Solar UVB radiationPrevitamin D3

Heat

Vitamin D

Vitamin D3

Inactive photoproducts

Vitamin D2

Diet

Vitamin D-25-hydroxylase

Liver

25(OH)D

1-OHase

Phophorus, calcium, FGF-23, and other factors +/–

Preosteoclast

RANKLRANK

OsteoblastParathyroid

hormone

Fat cell

Parathyroid glands

Osteoclast

Blood calcium and phosphorus

Ca2+ and HPO42!

AbsorptionCalcification

Intestine

Calcitroic acid

Bile

Excreted

24-OHase1,25(OH)2D

TRPV6

>150 ng/ml

(major circulating metabolite)

7-Dehydrocholesterol

Chylomicrons

Solar UVB radiation

Kidneys

Ca2+ and HPO42!

Reference range20–100 ng/ml

1,25(OH)2D

Intoxication

<20 ng/ml

Deficiency Preferred range30–60 ng/ml

CaBP

_

_

(290–315 nm)

Circulation

Circulation

Bone

VDR–RXR

VDR–RXR

Calcium

Solar UVB radiation

+

+ Calcium AbsorptionCalcium Resorption

Vitamin D3

CH3

HO

CH2

HO

CH2

PORQUE EXISTE DEFICIÊNCIA

Webb AR, Kline L, Holick MF. J Clin Endocrinol Metab. 1988 Aug;67(2):373-8.

Figure 1. The potential for synthesis of previtamin D3 in lightly pigmented human skin computed from annual average UVMED. The highest annualvalues for UVMED are shown in light violet, with incrementally lower values in dark violet, then in light to dark shades of blue, orange, green and gray(64 classes). White denotes areas for which no UVMED data exist. Mercator projection. In the tropics, the zone of adequate UV radiation throughoutthe year (Zone 1) is delimited by bold black lines. Light stippling indicates Zone 2, in which there is not su!cient UV radiation during at least one monthof the year to produce previtamin D3 in human skin. Zone 3, in which there is not su!cient UV radiation for previtamin D3 synthesis on average forthe whole year, is indicated by heavy stippling.

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RADIAÇÃO UV E VITAMINA D

Jablonski NG, Chaplin G. J Hum Evol. 2000 Jul;39(1):57-106

Vitamina D todo o ano

Défice de Vitamina D 1 ou + meses/ano




Sunlight, UV-Radiation, Vitamin D and Skin Cancer 5

Figure 3. Influence of season, time of day in July and latitude on the synthesis of previtamin D3 in Boston (42°N)-o-, Edmonton (52°N)-n-, Bergen (60°) - ^ - . The hour is the end of the one hour exposure time in July. Holick copyright 2007 with permission.

cells in the kidneys. l,25(OH)2D is responsible for the maintenance of calcium homeostasis and bone health by increasing the efficiency of intestinal calcium absorption, stimulating osteoblast function and increase bone calcium resorption. It also enhances the tubular resorption of calcium in the kidneys (Fig. 5).

1,25(OH)2D is such a potent regulator of calcium metabolism that in order to control its own actions, it induces its own destruction by enhancing the expression of the 25-hydroxyvitamin D-24-hydroxylase (CYP24)?^-^^ CYP24 causes oxidation on carbons 24 and 23 leading to the formation of a C23 acid known as calcitroic acid. This water soluble inactive metabohte is excreted in the bile (Fig. 5).

Role of Vitamin D in the Prevention of Chronic Diseases Most tissues and cells in the body including brain, skin, breast, prostate, colon and activated

T and B lymphocytes possess a VDR. ' ^ It is now recognized that l,25(OH)2D is one of the most potent hormones for regulating cell growth and maturation. It is estimated that more than 200 genes are either directly or indirectly influenced by l,25(OH)2D.^^

There have been numerous studies that have implicated living at higher latitudes and being at increased risk of vitamin D deficiency with many serious and chronic and deadly diseases including cancers of the colon, prostate and breast, autoimmune diseases including multiple sclerosis, type I diabetes and rheumatoid arthritis, infectious diseases including tuberculosis and influenza and hypertension and heart disease. ' '

What has been perplexing is the fact that exposure to sunlight results in an increase of circulating levels of 25(OH)D but not l,25(OH)2D. The reason is that parathyroid hormone, calcium and phosphorus and fiberblast growth factor 23 tightly control the production of l,25(OH)2D in the kidneys ^ (Fig. 5). Since 25(OH)D is incapable of altering vitamin D responsive gene expression at physiologic concentrations, there needed to be another explanation for the sunlight-vitamin D health connection.

It has been recognized for more than 30 years that activated macrophages, placenta and skin expressed the l-OHase." ^ ^ In the late 1990 s, there were numerous reports of various cell culture systems that expressed the 1-OHase that were capable of converting 25(OH)D3 to l,25(OH)2D3 including colon, prostate, breast and lung cell cultures. " " It was also observed that normal prostate cells obtained from prostate biopsies and both normal and colon cancer cells obtained at the time of surgery expressed the 1-OHase and had the capacity to make l,25(OH)2D. ^^^ These observa-tions have led to the hypothesis that by raising blood levels of 25 (OH)D, there is enough substrate

Holick MF. Adv Exp Med Biol. 2008;624:1-15.

REVIEW ARTICLEEpidemic influenza and vitamin D

J. J. CANNELL 1*, R. VIETH 2, J. C. UMHAU 3, M. F. HOLICK 4, W. B. GRANT 5,S. MADRONICH 6, C. F. GARLAND 7

AND E. GIOVANNUCCI 8

1 Atascadero State Hospital, 10333 El Camino Real, Atascadero, CA, USA2 Mount Sinai Hospital, Pathology and Laboratory Medicine, Department of Medicine, Toronto, Ontario,Canada3 Laboratory of Clinical and Translational Studies, National Institute on Alcohol Abuse and Alcoholism,National Institutes of Health, Bethesda, MD4 Departments of Medicine and Physiology, Boston University School of Medicine, Boston, MA, USA5 SUNARC, San Francisco, CA, USA6 Atmospheric Chemistry Division, National Center for Atmospheric Research, Boulder, CO, USA7 Department of Family and Preventive Medicine, University of California San Diego, La Jolla, CA, USA8 Departments of Nutrition and Epidemiology, Harvard School of Public Health, Boston, MA, USA

(Accepted 5 August 2006, first published online 7 September 2006)

SUMMARY

In 1981, R. Edgar Hope-Simpson proposed that a ‘seasonal stimulus ’ intimately associated withsolar radiation explained the remarkable seasonality of epidemic influenza. Solar radiation triggers

robust seasonal vitamin D production in the skin; vitamin D deficiency is common in the winter,and activated vitamin D, 1,25(OH)2D, a steroid hormone, has profound e!ects on human

immunity. 1,25(OH)2D acts as an immune system modulator, preventing excessive expressionof inflammatory cytokines and increasing the ‘oxidative burst ’ potential of macrophages. Perhapsmost importantly, it dramatically stimulates the expression of potent anti-microbial peptides,

which exist in neutrophils, monocytes, natural killer cells, and in epithelial cells lining therespiratory tract where they play a major role in protecting the lung from infection. Volunteers

inoculated with live attenuated influenza virus are more likely to develop fever and serologicalevidence of an immune response in the winter. Vitamin D deficiency predisposes children to

respiratory infections. Ultraviolet radiation (either from artificial sources or from sunlight) reducesthe incidence of viral respiratory infections, as does cod liver oil (which contains vitamin D). An

interventional study showed that vitamin D reduces the incidence of respiratory infections inchildren. We conclude that vitamin D, or lack of it, may be Hope-Simpson’s ‘seasonal stimulus ’.

INTRODUCTION

Whoever wishes to investigate medicine properly shouldproceed thus: in the first place to consider the seasons of theyear …

Hippocrates(circa 400 B.C.)

… the characteristic microbe of a disease might be asymptom instead of a cause.

George Bernard Shaw(Preface on Doctors, The Doctor’s Dilemma, 1911)

Perhaps the most mysterious feature of epidemic

influenza is its remarkable and recurrent seasonality –wintertime surfeit and summertime scarcity – a

feature first explored in detail by R. Edgar Hope-Simpson, the British general practitioner and

* Author for correspondence: Dr J. J. Cannell, Atascadero StateHospital, 10333 El Camino Real, Atascadero, CA 93422, USA.(Email : [email protected])

Epidemiol. Infect. (2006), 134, 1129–1140. f 2006 Cambridge University Pressdoi:10.1017/S0950268806007175 Printed in the United Kingdom

REVIEW ARTICLEEpidemic influenza and vitamin D

J. J. CANNELL 1*, R. VIETH 2, J. C. UMHAU 3, M. F. HOLICK 4, W. B. GRANT 5,S. MADRONICH 6, C. F. GARLAND 7

AND E. GIOVANNUCCI 8

1 Atascadero State Hospital, 10333 El Camino Real, Atascadero, CA, USA2 Mount Sinai Hospital, Pathology and Laboratory Medicine, Department of Medicine, Toronto, Ontario,Canada3 Laboratory of Clinical and Translational Studies, National Institute on Alcohol Abuse and Alcoholism,National Institutes of Health, Bethesda, MD4 Departments of Medicine and Physiology, Boston University School of Medicine, Boston, MA, USA5 SUNARC, San Francisco, CA, USA6 Atmospheric Chemistry Division, National Center for Atmospheric Research, Boulder, CO, USA7 Department of Family and Preventive Medicine, University of California San Diego, La Jolla, CA, USA8 Departments of Nutrition and Epidemiology, Harvard School of Public Health, Boston, MA, USA

(Accepted 5 August 2006, first published online 7 September 2006)

SUMMARY

In 1981, R. Edgar Hope-Simpson proposed that a ‘seasonal stimulus ’ intimately associated withsolar radiation explained the remarkable seasonality of epidemic influenza. Solar radiation triggers

robust seasonal vitamin D production in the skin; vitamin D deficiency is common in the winter,and activated vitamin D, 1,25(OH)2D, a steroid hormone, has profound e!ects on human

immunity. 1,25(OH)2D acts as an immune system modulator, preventing excessive expressionof inflammatory cytokines and increasing the ‘oxidative burst ’ potential of macrophages. Perhapsmost importantly, it dramatically stimulates the expression of potent anti-microbial peptides,

which exist in neutrophils, monocytes, natural killer cells, and in epithelial cells lining therespiratory tract where they play a major role in protecting the lung from infection. Volunteers

inoculated with live attenuated influenza virus are more likely to develop fever and serologicalevidence of an immune response in the winter. Vitamin D deficiency predisposes children to

respiratory infections. Ultraviolet radiation (either from artificial sources or from sunlight) reducesthe incidence of viral respiratory infections, as does cod liver oil (which contains vitamin D). An

interventional study showed that vitamin D reduces the incidence of respiratory infections inchildren. We conclude that vitamin D, or lack of it, may be Hope-Simpson’s ‘seasonal stimulus ’.

INTRODUCTION

Whoever wishes to investigate medicine properly shouldproceed thus: in the first place to consider the seasons of theyear …

Hippocrates(circa 400 B.C.)

… the characteristic microbe of a disease might be asymptom instead of a cause.

George Bernard Shaw(Preface on Doctors, The Doctor’s Dilemma, 1911)

Perhaps the most mysterious feature of epidemic

influenza is its remarkable and recurrent seasonality –wintertime surfeit and summertime scarcity – a

feature first explored in detail by R. Edgar Hope-Simpson, the British general practitioner and

* Author for correspondence: Dr J. J. Cannell, Atascadero StateHospital, 10333 El Camino Real, Atascadero, CA 93422, USA.(Email : [email protected])

Epidemiol. Infect. (2006), 134, 1129–1140. f 2006 Cambridge University Pressdoi:10.1017/S0950268806007175 Printed in the United Kingdom

cause significant summertime illness despite being anew antigenic variant in a non-immune population

(Fig. 2). However, clinical case rates increased inintensity as the sun became progressively lower in

the sky each day (autumn), waiting until the wintersolstice of 1968 before the first community outbreaks

appeared. Influenza case rates peaked for severalmonths but waned as the sun rose higher in the sky

each day (spring). Predictably, influenza virtuallyceased following the summer solstice. Clinical caserates for Hong Kong influenza increased from

September 1969, only to explode again in the dayspreceding the winter solstice, even though a much

higher proportion of the British population had virus-specific antibodies at the beginning of the lethal

second wave than they did at the beginning of itsless lethal first wave.

Hope-Simpson also observed that influenza out-breaks in the tropics, where solar UV radiation is less

seasonal, are also much less seasonal, but are gener-ally more severe when solar radiation is impaired (therainy season) – observations recently confirmed [15].

This intimate association with sunlight led the natu-ralist in Hope-Simpson to see influenza as a winter

‘crop, and, as with other crops, some years are goodinfluenza years, and other years produce a poor crop

of influenza cases ’ [3, p. 92].Influenza is but one of several respiratory viral

pathogens that show a distinct predilection forinfecting us in the wintertime. Noah found that inEngland and Wales respiratory syncytial virus and

parainfluenza 1 and 2 display marked wintertimeexcess [16]. More than 200 viruses cause the common

cold, which, as the name implies, also shows a distinctwintertime excess [17]. However, in clinical practice,

and in much published research, specific identifi-cation of respiratory viral infections is frequently

absent. With full appreciation of its inherent limi-tations, we will use the term viral respiratory infection

in this review, unless the literature cited was morespecific.

The seasonal stimulus?

As Hope-Simpson pointed out, solar radiation maybe a!ecting the ‘virus, the human host, or theirinteraction …’. That is, he theorized that humans

might have a physiological system directly dependenton solar radiation that improves innate immunity

around the summer solstice but impairs it in thewinter. There is a seasonal steroid hormone system

with profound e!ects on human immunity whose

substrate levels reach their nadir during influenzaseason but peak when influenza is rare (Fig. 3) [18, 19].

Cholecalciferol (vitamin D) is a prehormonenormally made in the skin during sunny months

when UVB radiation triggers the conversion of 7-dehydrocholesterol in the skin into vitamin D [20].

The liver converts vitamin D into 25-hydroxyvitaminD [25(OH)D] and then cells all over the bodyconvert 25(OH)D to 1,25-dihydroxyvitamin D

[1,25(OH)2D] – a potent steroid hormone. Locallyproduced 1,25(OH)2D performs autocrine and para-

crine functions in a wide variety of tissues, includingthe immune system. Local tissue levels of 1,25(OH)2D

are dependent on available serum substrate[25(OH)D]. Dangers of vitamin D deficiency may

include more than just low 25(OH)D levels. Vieth hasproposed that progressively falling serum levels of25(OH)D (as occurs in the autumn), may trigger

intracellular deficiencies of 1,25(OH)2D, despiteapparently adequate serum levels of 25(OH)D and

1,25(OH)2D [21].Many distinctive features of the biology, physi-

ology, and epidemiology of vitamin D point to itas a likely candidate for Hope-Simpson’s ‘seasonal

stimulus ’.

(1) Vitamin D has profound and multiple e!ects on

human immunity [22, 23].(2) Inadequate vitamin D nutrition is endemic among

the elderly in the winter [24–26].

(3) Serum levels of 25(OH)D are low in manypeople of all ages who live at temperate latitudes,

especially in the winter [20].(4) Humans acquire most of their vitamin D

from casual sun exposure, and to a degree that

35

30

25

20

15

10Aug. Sep. Oct.Nov.Dec. Jan. Feb.

Month

25(O

H)D

(ng/

ml)

Mar.Apr. MayJune JulyAug.

Fig. 3. Seasonal variation of 25(OH)D levels in a popu-lation-based sample of inhabitants of a small southernGerman town, aged 50–80 years. (Reproduced/amendedwith kind permission of Springer Science and BusinessMedia, Scharla, S.H., 1998.)

Epidemic influenza and vitamin D 1131

When analysing the data according to BMI, a non-significantand progressive decrease of 25(OH)D concentrations withincreasing BMI is observed, the lowest levels being observedin obese adolescents (equivalent to BMI .30 kg/m2;Table 3). The highest mean levels were for boys in the under-weight group and for girls in the optimal weight group (66·6(SD 28·9) and 61·1 (SD 23·5) nmol/l, respectively). Most ofthe adolescents had optimal weight status (BMI 20–25 kg/m2).Table 4 shows 25(OH)D levels by study centre, for the

whole group split by sex. The highest levels were obtainedin Rome, Athens, Vienna and Zaragoza, and the lowestlevels were found in Dortmund, Heraklion and Ghent forthe whole group, where the sampling procedure went onfor most of the academic year. In none of the cities weremean levels above the proposed cut-off of 75 nmol/l. Girlshad higher mean levels in all cities except for Athens,Pecs and Lille. Deficient levels (,50 nmol/l) were highest in

Dortmund (62·9% of the population) and Ghent (53·3%),and lowest in Athens (25·7%) and Rome (26·4%) (datanot shown).

Discussion

Since the publication of the results of the SENECA (Survey inEurope on Nutrition and the Elderly; a Concerted Action)study(38), where unexpectedly only 3·5% of the analysedEuropean elderly presented optimum 25(OH)D levels(.60 nmol/l), public health authorities have been concernedabout the widespread 25(OH)D deficiency in the Europeanpopulation. To the best of our knowledge, the data obtainedin the framework of the HELENA study are the first to aim atestablishing descriptive 25(OH)D status in adolescents at aEuropean level. According to the Institute of Medicine report2011, vitamin D intake for bone health should correspond to

Table 2. 25-Hydroxycholecalciferol concentrations by age and sex in European adolescents (nmol/l)

(Mean values, standard deviations, number of participants and percentiles)

n Mean SD P2·5 P5 P10 P25 P50 P75 P90 P95 P97·5

Total (n 1006) 1006 58·8 23·1 20·9 24·9 31·6 43·5 57·0 71·3 87·8 99·1 112·9Males (n 470)† 470 57·4 22·7 21·6 24·3 32 42·6 56·0 69·1 86·7 96·4 107·4

Age 13 (years) 124 56·9 22·8 24·2 27·0 32·5 41·8 55·8 66·6 78·3 101·5 132·0Age 14 (years) 124 55·6 20·6 22·5 24·9 31·7 42·3 53·5 65·9 81·9 91·2 96·4Age 15 (years) 122 58·3 21·3 20·7 24·4 33·6 44·8 56·2 71·3 89·0 94·9 102·0Age 16 (years) 99 59·3 26·5 20·4 22·6 24·9 43·0 59·6 70·8 92·3 107·6 121·9

Females (n 536)† 536 60·0 23·4 20·9 25·5 31·0 44·8 57·9 74·3 88·7 103·0 115·9Age 13 (years) 133 52·1* 19·2 17·0 20·9 25·6 40·6 50·8 64·0 78·0 85·8 96·8Age 14 (years) 140 62·5* 22·3 20·9 27·8 33·6 49·1 62·0 77·9 90·2 99·2 111·3Age 15 (years) 143 59·6* 20·8 21·3 26·0 36·2 46·6 56·8 71·5 90·2 97·2 106·0Age 16 (years) 120 66·2* 28·9 25·6 26·9 27·4 46·4 61·8 80·3 109·3 120·8 133·5

*Mean values were significantly different between the 13 years age group and the rest of the age groups (P,0·05).† Four age groups: 13 years, age between 12·5 and 13·99 years; 14 years, age between 14 and 14·99 years; 15 years, age between 15 and 15·99 years; 16 years,

age between 16 and 17·49 years.

40

30

15·0

27·4

38·8

18·9

Sufficient(>75 nmol/l)

Insufficient(50–75 nmol/l)

Deficient(27·5–50 nmol/l)

Severe deficiency(<27·5 nmol/l)

25(OH)D status groups

20

Sub

ject

s (%

)

10

0

Fig. 1. 25-Hydroxycholecalciferol (25(OH)D) status classification.

M. Gonzalez-Gross et al.4

British

JournalofNutrition

Vitamin D status among adolescents in Europe: the Healthy Lifestylein Europe by Nutrition in Adolescence study

Marcela Gonzalez-Gross1,2*†, Jara Valtuena1,3†, Christina Breidenassel2, Luis A. Moreno4,5,Marika Ferrari3, Matilde Kersting6, Stefaan De Henauw7, Frederic Gottrand8, Elena Azzini3,Kurt Widhalm9, Anthony Kafatos10, Yannis Manios11 and Peter Stehle2 on behalf of theHELENA Study Group1Department of Health and Human Performance, Faculty of Physical Activity and Sport Sciences (INEF), UniversidadPolitecnica de Madrid, C/Martın Fierro, 7, 28040 Madrid, Spain2Institut fur Ernahrungs- und Lebensmittelwissenschaften – Humanernahrung, Rheinische Friedrich-Wilhelms Universitat,Bonn, Germany3National Research Institute on Food and Nutrition, Rome, Italy4Growth, Exercise, Nutrition and Development (GENUD) Research Group, Universidad de Zaragoza, Zaragoza, Spain5Escuela Universitaria de Ciencias de la Salud, Universidad de Zaragoza, Zaragoza, Spain6Research Institute of Child Nutrition Dortmund, Rheinische Friedrich-Wilhelms Universitat, Bonn, Germany7Department of Public Health, Ghent University, Ghent, Belgium8Inserm U995, IFR114, University Lille 2, Lille, France9Department of Paediatrics, Medical University of Vienna, Vienna, Austria10Preventive Medicine and Nutrition Clinic, University of Crete School of Medicine, Iraclion, Crete, Greece11Department of Nutrition and Dietetics, Harokopio University, Athens, Greece

(Received 12 November 2010 – Revised 23 May 2011 – Accepted 31 May 2011)

AbstractAn adequate vitamin D status is essential during childhood and adolescence, for its important role in cell growth, skeletal structure anddevelopment. It also reduces the risk of conditions such as CVD, osteoporosis, diabetes mellitus, infections and autoimmune disease.As comparable data on the European level are lacking, assessment of vitamin D concentrations was included in the Healthy Lifestyle inEurope by Nutrition in Adolescence (HELENA) study. Fasting blood samples were obtained from a subsample of 1006 adolescents (470males; 46·8%) with an age range of 12·5–17·5 years, selected in the ten HELENA cities in the nine European countries participating inthis cross-sectional study, and analysed for 25-hydroxycholecalciferol (25(OH)D) by ELISA using EDTA plasma. As specific referencevalues for adolescents are missing, percentile distribution were computed by age and sex. Median 25(OH)D levels for the whole popu-lation were 57·1 nmol/l (5th percentile 24·3 nmol/l, 95th percentile 99·05 nmol/l). Vitamin D status was classified into four groups accordingto international guidelines (sufficiency/optimal levels $75 nmol/l; insufficiency 50–75 nmol/l; deficiency 27·5–49·99 nmol/l and severedeficiency ,27·5 nmol/l). About 80% of the sample had suboptimal levels (39% had insufficient, 27% deficient and 15% severely deficientlevels). Vitamin D concentrations increased with age (P,0·01) and tended to decrease according to BMI. Geographical differences werealso identified. Our study results indicate that vitamin D deficiency is a highly prevalent condition in European adolescents and should bea matter of concern for public health authorities.

Key words: Adolescents: Vitamin D: Prevention: Europe

Adolescents are considered as a risk group for malnutritionbecause of their increasing needs of nutrients and energyfor adequate growth and development that vary withage(1–3). Specifically different levels of vitamin D deficiencyat these early ages could be considered a risk factor for

osteomalacia(4–6), impaired cognitive function and concen-tration problems(7), hyperactivity(8) and immune systemdeficiency(4). Inadequate vitamin D levels have also beenrelated to other diseases such as diabetes, multiple sclerosisand cancer(9–11). One of the most important applications

† Equal authorship.

*Corresponding author: Professor M. Gonzalez-Gross, email [email protected]

Abbreviations: 25(OH)D, 25-hydroxycholecalciferol; HELENA, Healthy Lifestyle in Europe by Nutrition in Adolescence.

British Journal of Nutrition, page 1 of 10 doi:10.1017/S0007114511003527q The Authors 2011

British

JournalofNutrition

DEFICIÊNCIA DE VITAMINA D EM ATLETAS Modalidades 25OHD3

ng/ml Referências

Mulheres Finlandesas (corredoras e ginastas)

67%: < 15

Lehtonen-Veromaa M, et al. Eur J Clin Nutr. 1999;53(9):746–51.

Ginástica (Alemanha)

77%: < 35 37%: < 10

Willis KS, Peterson NJ, Larson-Meyer DE. Int J Sport Nutr Exerc Metab. 2008;18:204–24.

Ginástica (EUA - Mulheres)

83%: < 30 33%: < 20

Lovell G. Clin J Sport Med. 2008;18(2):159–61.

Ciclistas franceses (treino de 16 h/dia)

Média: 32

Maïmoun L, et al. Int J Sports Med. 2006;27(2):105–11.

Cannell JJ, et al. Med Sci Sports Exerc. 2009 May;41(5):1102-10

PERFORMANCE AUMENTA NOS MESES DE VERÃO

USO DE RADIAÇÃO UVB EM ATLETAS MELHORA PERFORMANCE

Once formed, previtamin D3, which is entrapped within theplasma membrane lipid bilayer, rapidly undergoes rearrange-ment of its double bonds to form the more thermodynamicallystable vitamin D3. During this transformation process, vitaminD3 is ejected from the plasma membrane into the extracellularspace (14). The vitamin D-binding protein in the dermal capillarybed has an affinity for vitamin D3 (14–16) and draws it into thecirculation.

Prolonged sun exposure does not result in the production ofexcess quantities of vitamin D3 to cause intoxication. The reasonfor this is that, during sun exposure, the previtamin D3 that isformed and the thermal isomerization product vitamin D3 thatdoes not escape into the circulation absorb solar UV radiation andisomerize to several photoproducts that are thought to have littleactivity on calcium metabolism (2, 3, 14, 15) (Figure 3).

FACTORS THAT ALTER THE CUTANEOUSPRODUCTION OF VITAMIN D3

Anything that either influences the number of solar UVB pho-tons that penetrate the skin or alters the amount of7-dehydrocholesterol in the skin influences the cutaneous pro-duction of vitamin D3. The amount of 7-dehydrocholesterol inthe epidermis is relatively constant until later in life, when itbegins to decline (17, 18). A person 70 years of age exposed to thesame amount of sunlight as a 20-year-old person makes !25% ofthe vitamin D3 that the 20-year-old person can make (Figure 4).

Melanin evolved as an effective natural sunscreen. Because itefficiently absorbs UVB photons, people with increased skin

FIGURE 3. Schematic diagram of cutaneous production of vitamin D andits metabolism and regulation for calcium homeostasis and cellular growth.During exposure to sunlight, 7-dehydrocholesterol (7-DHC) in the skin ab-sorbs solar UVB radiation and is converted to previtamin D3 (preD3). Onceformed, previtamin D3 undergoes thermally induced transformation to vita-min D3. Additional exposure to sunlight converts previtamin D3 and vitaminD3 to biologically inert photoproducts. Vitamin D originating from the dietor from the skin enters the circulation and is metabolized to 25(OH)D3 in theliver by vitamin D 25-hydroxylase (25-OHase). 25(OH)D3 reenters the cir-culation and is converted to 1,25(OH)2D3 in the kidney by 25(OH)D3 1!-hydroxylase (1-OHase). A variety of factors, including serum phosphorus(Pi) and PTH, regulate the renal production of 1,25(OH)2D. 1,25(OH)2Dregulates calcium metabolism through interactions with its major target tis-sues, ie, bone and intestine. 1,25(OH)2D3 also induces its own destruction byenhancing the expression of 25(OH)D 24-hydroxylase (24-OHase).25(OH)D is metabolized in other tissues for regulation of cellular growth.

FIGURE 4. A: Circulating concentrations of vitamin D3 after a singleexposure to 1 MED of simulated sunlight, with a sunscreen (SPF 8) or atopical placebo cream. B: Circulating concentrations of vitamin D in re-sponse to whole-body exposure to 1 MED among healthy young and elderlysubjects. Reproduced with permission from reference 3.

1680S HOLICK

Holick M. Am J Clin Nutr 2004;80(suppl):1678S– 88S.

ENVELHECIMENTO

UVB

The evolution of light pigmentation at high latitudes has longbeen related to the significance of production of vitamin D in theskin under conditions of reduced sunlight (19, 46). Vitamin D3 ismade in the skin whenUVR penetrates the skin and is absorbed by7-dehydrocholesterol (7-DHC) in the epidermis and dermis to formprevitamin D3. This reaction only occurs in the presence of wave-lengths of 290–310 nm in the UVB range, with peak conversionoccurring at 295–297 nm. Photosynthesis of vitamin D3 in the skindepends upon the solar zenith angle, which changes with season,latitude, and time of day, and is further controlled by the amount ofpigment and thickness of the skin (47, 48). The importance ofvitaminD3 as a selective force in the evolution of skin pigmentationis related to the manifold effects of the vitamin on fitness asreviewed inearlierpapers (7, 49).The vitaminDendocrine system isinvolved in the regulation ofmany independent biological processesincluding bone metabolism, the innate immune response, cell pro-liferation, anddifferentiation (50, 51).The roles of vitaminD3 in theregulation of intestinal calcium absorption, and in bone formationand remodeling, havebeenknown fordecades, but only recently hasthe importanceof vitaminD3 in the establishment andmaintenanceof innate immunity, and in the normal functioning of the pancreas,brain, andheart, been recognized (51, 52).Reductionof fertility dueto vitamin D3 deficiencies is greatest in cases of nutritional rickets,but is also significant because of increased prevalence of bacterialand viral infections and increased risk of autoimmune diseases suchas multiple sclerosis and Type 1 diabetes (53). Natural selection topromote continued vitamin D production through loss of con-stitutive pigmentation under conditions reduced UVR was strong,and its independent action on hominin populations dispersing tolow UVR habitats was inferred before genetic evidence demon-strating positive selection for depigmentation became known (7).Generally low andhighly seasonally variable levels forUVBcreateda selective environment favoring the capture of UVB photonsrequired for vitamin D3 photosynthesis through loss of melaninpigmentation. Genetic verification of three independent occur-rences of evolutionof depigmented skin in hominin populations hasbeen documented in the lineages leading to modern northernEuropeans and modern east Asians (54, 55) and in Homo nean-derthalensis (56). It is significant that the genetic and physiologicalmechanisms causing light-skinned phenotypes in each group weredifferent from one another. Regulatory mechanisms involve thecontrol of the formation of melanosomes (the organelles in whichmelanins are produced and stored) (54, 55), and the production ofthe different types and mixtures of melanins. The mechanismswhereby similar phenotypic ends have been reached by differentgenetic means have been reviewed recently (57). One of the mostinteresting questions remaining to be answered about the physiol-ogy of vitamin D is humans concerns the nature of variation in thevitamin D receptor (VDR), specifically whether the pattern andnature of polymorphisms in the VDR is related to UVB levels and/or length of habitation under specific UVB regimes.

Geographic Variation in UV RadiationMounting genetic evidence demonstrating the role of naturalselection in establishing and maintaining darkly and lightly pig-mented cutaneous phenotypes near the equator and poles,respectively, prompts a closer look at the nature of the primeselective agent, UVR. Differences in the strength, seasonal dis-tribution, and bioactivity of UVA and UVB have been recognizedfor a long time (58, 59), but the relevance of these variables to theevolution of human skin pigmentation has not been fully appre-ciated. The dispersals of hominins out of Africa that occurredabout 1.9 Ma and 80 ka, respectively, involved the movement ofpeople out of highly UVR-rich environments into habitats thatwere much more mixed with respect to the seasonal pattern,intensity, and wavelength mixture of UVR. Although two distinctforms of hominin (early Homo and Homo sapiens, respectively)were involved in these dispersals, neither form made or used

clothing or used other portable methods of sun protection. Thus,apart from the time they spent in the shade, their bodies weresubjected to the full force of UVR wherever they went.The Earth’s surface receives much less UVB than UVA because

most UVB reaching the Earth is scattered and absorbed by oxygen,ozone, and water molecules in the atmosphere. Because of this andthe geometry of sunlight reaching different places in different sea-sons, UVB is much more variable in its intensity and distributionthan UVA. Levels UVB are highest near the equator in more aridregions, and in high altitude areas such as the Tibetan Plateau andthe Altiplano (Fig. 1A). North or south of about 46°, levels of UVBare insufficient to initiate cutaneous production of previtamin D3for much of the year (7, 17). The pattern of distribution of UVB ismost strongly influenced by latitude because of atmospheric scat-tering and absorption. Africa receives high and uniform amounts,whereas northern Eurasia receives negligible amounts. The coef-ficient of variation (CoV) for UVB (Fig. 1B) is strongly associatedwith its seasonal nature outside of the tropics, and is lowest in theequatorial zone and highest in northern Eurasia and North Amer-ica.Humidity andmonsoonprecipitation reduce averageUVB, andtheCoV ishigher relative to themean level inmoisture-rich regions.Levels of UVA (Fig. 2A) are considerably higher than those for

UVB. The latitudinal bands of UVA distribution are wider thanthose of UVB and higher levels of UVA exist toward the poles.UVA at 380 nm is about fifteen times more plentiful than UVB at305 nm, with Western Europe receiving an average 283–570 Jm!2

ofUVAcompared to only 20–40 Jm!2 ofUVB.Equatorial regionsreceived slightly less UVA than tropical and subtropical areas.Albedo from lighter-colored ground and, especially, from snow

Fig. 1. (A) Annual mean UVB (305 nm). Intensity is indicated by gradationsfrom dark to light varying from 1 to 135 Jm!2 in 10 steps with oceans partiallygrayed-out. (B) Annual CoV for UVB (305 nm). Gradations of dark to lightvarying from 10 to 300 in 10 steps, with oceans area partially grayed-out.

8964 | www.pnas.org/cgi/doi/10.1073/pnas.0914628107 Jablonski and Chaplin

Jablonski NG, Chaplin G. Proc Natl Acad Sci U S A. 2010 May 11;107 Suppl 2:8962-8

Holick M. Am J Clin Nutr 2004;80(suppl):1678S– 88S.!

Pele muito pigmentada

Pele muito pigmentada

Pele pouco pigmentada

54 mJ/cm2

320 mJ/cm2

MEDO DO SOL

PROTECTOR SOLAR

Holick M. Am J Clin Nutr 2004;80(suppl):1678S– 88S.!

ü  SPF de 8 diminui a capacidade de produzir Vitamina D3 em 95%

ü  SPF de 15 diminui a capacidade de produzir Vitamina D3 em 98%

Endocrinol Nutr. 2011;58(6):267—273

ENDOCRINOLOGÍA Y NUTRICIÓN

www.elsevier.es/endo

ORIGINAL

Elevada prevalencia de hipovitaminosis D en los estudiantes demedicina de Gran Canaria, Islas Canarias (Espana)

Esther González-Padillaa, Adela Soria Lópezb, Elisa González-Rodrígueza,Sabrina García-Santanaa, Ana Mirallave-Pescadora, María del Val Groba Marcoa,Pedro Saavedrac, José Manuel Quesada Gómezd y Manuel Sosa Henríqueza,e,!

a Grupo de Investigación en Osteoporosis y Metabolismo Mineral, Universidad de Las Palmas de Gran Canaria, Gran Canaria,Espanab Servicio de Bioquímica Clínica, Hospital Universitario Insular, Las Palmas de Gran Canaria, Gran Canaria, Espanac Departamento de Matemáticas, Universidad de Las Palmas de Gran Canaria, Gran Canaria, Espanad Unidad de Investigación, Iniciativas y Desarrollo, Sanyres y Unidad de Metabolismo Mineral, Servicio de Endocrinología, HospitalUniversitario Reina Sofía, RETICEF, Córdoba. Espanae Unidad Metabólica Ósea, Hospital Universitario Insular, Gran Canaria, Espana

Recibido el 3 de agosto de 2010; aceptado el 9 de marzo de 2011Disponible en Internet el 8 de mayo de 2011

PALABRAS CLAVEVitamina D;Deficiencia;Insuficiencia;Estudiantes;Exposición solar;Islas Canarias

ResumenFundamento: Se ha descrito la existencia de deficiencia de vitamina D tanto en la poblacióngeneral como en un gran número de enfermedades. Sin embargo, se han publicado pocos estu-dios realizados en población joven y sana en Espana. Teóricamente no debería encontrarsedeficiencia de vitamina D entre los estudiantes de Medicina de la Universidad de Las Palmas deGran Canaria, porque disponen de todos los medios para evitarla.Objetivo: Estimar la prevalencia de deficiencia de vitamina D en una población de estudiantesde Medicina de ambos sexos de la Universidad de Las Palmas de Gran Canaria.Método: Se estudiaron 103 alumnos de Medicina de ambos sexos de la Universidad de Las Palmasde Gran Canaria. A todos se les realizó un cuestionario y una exploración física. Se determinóla vitamina D 25-hidroxicolecalciferol (25-HCC), la hormona paratiroidea, varios marcadoresbioquímicos de remodelado óseo y un estudio bioquímico general. Se estimó la densidad mineralósea por absorciometría radiológica dual en la columna lumbar y en la extremidad proximal delfémur. Asimismo, se midieron los parámetros ultrasonográficos en el calcáneo.Resultados: Sólo el 38,8% de los estudiantes de Medicina (el 42,1% de los varones y el 44,9%de las mujeres) presentaron niveles de 25-HCC superiores a 30 ng/dl tal y como se recomienda

! Autor para correspondencia.Correo electrónico: [email protected] (M. Sosa Henríquez).

1575-0922/$ – see front matter © 2010 SEEN. Publicado por Elsevier España, S.L. Todos los derechos reservados.doi:10.1016/j.endonu.2011.03.002

Documento descargado de http://www.elsevier.es el 19/08/2011. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.

Elevada prevalencia de hipovitaminosis D en los estudiantes de medicina de Gran Canaria, Islas Canarias (Espana) 271

Tabla 5 Valores de densidad mineral ósea, medidos en la columna lumbar (L2-L4) y en la extremidad proximal del fémur

DXA Todos los alumnos (103) Varones (31) Mujeres (72) Valor de p

Columna lumbar (L2-L4) (g/cm2) 1,026 ± 0,116 1,025 ± 0,999 1,026 ± 0,124 0,968T-score —0,1 ± 0,8 —0,1 ± 1,2 0,935

Cuello femoral (g/cm2) 0,886 ± 0,127 0,936 ± 0,124 0,863 ± 0,122 0,007T-score 0,1 ± 1,1 0,2 ± 1,1 0,557

Total de cadera (g/cm2) 0,988 ± 0,126 1,053 ± 0,115 0,959 ± 0,121 0,001T-score 0,2 ± 0,8 0,4 ± 1,2 0,223

Trocánter (g/cm2) 0,743 ± 0,104 0,789 ± 0,960 0,722 ± 0,101 0,003T-score 0,1 ± 0,8 0,4 ± 1,2 0,189

Intertrocánter (g/cm2) 1,138 ± 0,151 1,213 ± 0,134 1,104 ± 0,147 0,001T-score 0,1 ± 0,8 0,4 ± 1,3 0,218

Tabla 6 Valores de ultrasonidos medidos en el calcáneo

QUS Todos los alumnos Varones Mujeres Valor de p

BUA (dB/MHz) 76,8 (13,7) 80,3 (12,8) 75,1 (14) 0,084T-score —0,2 ± 0,7 —0,2 ± 0,9 0,733

SOS (m/s) 1.561,8 (24,5) 1.560,4 (25,7) 1.562,5 (24,1) 0,692T-score —0,2 ± 0,8 —0,1 ± 0,8 0,529

QUI 102,7 (18) 102,8 (15,7) 102,6 (19,1) 0,960T-score —0,1 ± 0,8 —0,1 ± 1 0,514

Tabla 7 Prevalencia de insuficiencia y deficiencia de vitamina D (%)

Todos los estudiantes Varones Mujeres Valor de p

Deficiencia (25-HCC < 30 ng/dl) 32,6 48,3 26,1 0,048Insuficiencia (25-HCC < 20 ng/dl) 28,6 27,6 29 0,927Deficiencia e insuficiencia conjuntas 61,2 75,9 55,1 0,081Valores óptimos (> 30 ng/ml) 38,8 24,1 44,9 0,081

que fueron estadísticamente superiores en todos los lugaresanatómicos de la extremidad proximal del fémur.

La tabla 6 muestra los parámetros ultrasonográficosmedidos en el calcáneo. No se observaron diferencias esta-dísticamente significativas entre ambos sexos en ninguno delos parámetros estudiados: BUA, SOS y QUI.

Finalmente, en la tabla 7 se muestra la prevalenciade insuficiencia y deficiencia de vitamina D en la pobla-ción estudiada. El 48,3% de los varones y el 26,1% de lasmujeres mostraron unos niveles de 25-HCC por debajo de30 ng/ml y el 27,6% de los varones y el 29% de las mujerespresentaron unos niveles de 25-HCC inferiores a 20 ng/ml.Valorados conjuntamente, el 75,9% de los varones y el 55,1%de las mujeres presentaron insuficiencia o deficiencia devitamina D y sólo el 24,1% de los varones y el 44,9% delas mujeres mostraron unos valores de 25-HCC superioresa 30 ng/ml.

Discusión

En condiciones normales, la vitamina D es sintetizada porla piel, que convierte el 7-dihidrocolesterol en previta-mina D3, que es rápidamente convertida en vitamina D3. Lavitamina D de la piel y la dieta es metabolizada en el hígado a

25-HCC que es el metabolito utilizado para valorar el estadode vitamina D en el organismo18,19.

Aunque no existe un consenso sobre cuáles son los nivelesóptimos de 25-HCC, hoy en día se acepta la clasificación dedeficiencia de vitamina D cuando los valores de 25-HCC soninferiores a 20 ng/ml20 e insuficiencia cuando las cifras de25-HCC están por debajo de 30 ng/ml3,21.

Las personas que viven cerca del Ecuador, expuestas ala luz solar sin protección, habitualmente tienen unos nive-les altos de 25-HCC, por encima de 30 ng/ml3. Sin embargo,incluso en las áreas más soleadas, la deficiencia de vita-mina D es un hallazgo común, dado que es práctica habitualesconder la piel del sol. En estudios realizados en ArabiaSaudí, Emiratos Árabes Unidos, Australia, India y Líbano,entre el 30 y el 50% de los ninos y adultos tienen unos nivelesde 25-HCC por debajo de 20 ng/ml22—25.

La deficiencia de vitamina D está considerada como unaverdadera pandemia. Su principal causa es la falta de con-cienciación de que la exposición al sol es la principal fuentede vitamina D para la mayor parte de los seres humanos11 yentre las posibles consecuencias de su déficit se encuen-tran: osteoporosis, osteomalacia, algunas enfermedadesautoinmunes, enfermedades cardiovasculares, enfermeda-des infecciosas y algunas formas de cáncer3,4,11.

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Gran Canária: 28º N

Low Vitamin D Status despite Abundant Sun Exposure

N. Binkley, R. Novotny, D. Krueger, T. Kawahara, Y. G. Daida, G. Lensmeyer, B. W. Hollis, andM. K. Drezner

University of Wisconsin Osteoporosis Clinical Research Program (N.B., D.K., T.K., M.K.D.), Madison, Wisconsin 53705;Human Nutrition, Food and Animal Sciences (R.N., Y.G.D.), University of Hawaii at Manoa, Honolulu, Hawaii 96822;Laboratory Medicine (G.L.), University of Wisconsin, Madison, Wisconsin 53792; and Medical University of South Carolina(B.W.H.), Charleston, South Carolina 29425

Context: Lack of sun exposure is widely accepted as the primary causeof epidemic low vitamin D status worldwide. However, some individualswith seemingly adequate UV exposure have been reported to have lowserum 25-hydroxyvitamin D [25(OH)D] concentration, results thatmight have been confounded by imprecision of the assays used.

Objective: The aim was to document the 25(OH)D status of healthyindividuals with habitually high sun exposure.

Setting: This study was conducted in a convenience sample of adultsin Honolulu, Hawaii (latitude 21°).

Participants: The study population consisted of 93 adults (30 womenand 63 men) with a mean (SEM) age and body mass index of 24.0 yr (0.7)and 23.6 kg/m2 (0.4), respectively. Their self-reported sun exposure was28.9 (1.5) h/wk, yielding a calculated sun exposure index of 11.1 (0.7).

Main Outcome Measures: Serum 25(OH)D concentration was mea-sured using a precise HPLC assay. Low vitamin D status was definedas a circulating 25(OH)D concentration less than 30 ng/ml.

Results: Mean serum 25(OH)D concentration was 31.6 ng/ml. Usinga cutpoint of 30 ng/ml, 51% of this population had low vitamin Dstatus. The highest 25(OH)D concentration was 62 ng/ml.

Conclusions: These data suggest that variable responsiveness toUVB radiation is evident among individuals, causing some to havelow vitamin D status despite abundant sun exposure. In addition,because the maximal 25(OH)D concentration produced by natural UVexposure appears to be approximately 60 ng/ml, it seems prudent touse this value as an upper limit when prescribing vitamin Dsupplementation. (J Clin Endocrinol Metab 92: 2130–2135, 2007)

LOW VITAMIN D status1 is extremely common (1– 4),and may contribute to the development of osteopo-

rosis and osteomalacia/rickets, as well as increase the riskfor falls (5, 6). Moreover, low vitamin D status may playa role in nonmusculoskeletal diseases, including a varietyof cancers, multiple sclerosis, infection, hypertension, anddiabetes mellitus (7, 8). Although it is widely accepted thatvitamin D status is determined by the measurement of thecirculating concentration of 25-hydroxyvitamin D[25(OH)D] (9), the cutoff value to define low vitamin Dstatus and a definition for success of vitamin D repletiontherapy remain controversial (10, 11). This is partially dueto the variability of vitamin D concentration by geograph-ical location and differences in assay methodology (12–16).Despite this controversy, clinicians often endeavor to cor-rect vitamin D deficiency by prescribing high-dose vita-min D (17). However, the goal for such therapy is unclearand could include achieving a serum 25(OH)D levelgreater than an accepted cutpoint (e.g. 30 ng/ml) or, al-ternatively, the upper limit of normal, a value that variesbetween laboratories (18).

The high prevalence of low vitamin D status is assumedto result from inadequate sun exposure. Because highly sun-exposed individuals likely possess normal vitamin D statusfrom an evolutionary standpoint, the use of such individualsto define normal 25(OH)D status has been proposed (19).This argument is based on the view that contemporary hu-mans are genetically adapted to the environment of ourancestors and that the profound lifestyle changes that haveoccurred over the past approximately 10,000 yr (importantlyincluding reduced sun exposure) have been much too rapidfor the human genome to adjust (20, 21). The current studywas designed to assess whether, in fact, people living at a lowlatitude with high amounts of sun exposure have adequatevitamin D status, as expected, and to identify a target valueof 25(OH)D for use in vitamin D therapy.

Subjects and MethodsSubjects and study design

Subjects older than 18 yr were recruited approximately equally fromthe University of Hawaii at Manoa (UH) and from patrons of the A’alaPark Board Shop, Honolulu, Hawaii (latitude 21° north), in late March2005. The A’ala Park Board Shop is a skateboard shop frequented byyoung adults. Recruitment was performed by posted notice at the BoardShop and on the UH campus; volunteers were reimbursed for studyparticipation. Volunteers were required to have self-reported sun ex-posure of 3 or more hours per day on 5 or more days per week for atleast the preceding 3 months, and not to be currently taking phenobar-bital, phenytoin, or prednisone. A total of 93 subjects (63 male and 30female) participated.

The University of Wisconsin Health Sciences Institutional ReviewBoard and the Committee on Human Studies at the UH reviewed and

First Published Online April 3, 2007Abbreviations: BMI, Body mass index; CV, coefficient of variation;

25(OH)D, 25-hydroxyvitamin D; UH, University of Hawaii at Manoa.1 We have chosen to use the terminology “low vitamin D status” for

a syndrome that others have variously described as “vitamin D insuf-ficiency,” “vitamin D inadequacy,” or “hypovitaminosis D.”JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the en-docrine community.

0021-972X/07/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 92(6):2130–2135Printed in U.S.A. Copyright © 2007 by The Endocrine Society

doi: 10.1210/jc.2006-2250

2130

The Journal of Clinical Endocrinology & Metabolism 92(6):2130–2135

Serum 25(OH)D and PTH

Using the HPLC assay for serum 25(OH)D and applyinga widely recommended cutpoint of 30 ng/ml (10), 51% (47of 93) of these subjects had low vitamin D status (Fig. 2). Thehighest serum 25(OH)D concentration observed was 62 ng/ml. No correlation between serum whole PTH and 25(OH)Dconcentration was observed (Fig. 3). Moreover, there was nocorrelation between serum 25(OH)D measured by HPLC andage, lightest or darkest skin color, delta skin color, hours/week of sun exposure without sunscreen, sun index, totalhours of sun exposure/week, or BMI (data not shown). Spe-cifically, delta skin color was not correlated with either PTH(P ! 0.10; r2 ! 0.03) or serum 25(OH)D (P ! 0.18; r2 ! 0.02).

In an effort to evaluate determinants of serum 25(OH)Dstatus, the quartile of individuals (n ! 23) with the lowestcirculating levels of 25(OH)D was compared with the re-maining cohort. The serum 25(OH)D in the lowest quartile(20.7 " 0.7 ng/ml) was significantly lower (P # 0.0001) thanin the rest of the population (35.2 " 1.1 ng/ml). In accord,PTH was higher (P # 0.01) in the lowest quartile (15.9 " 1.4pg/ml) compared with the remainder of the subjects (12.7 "0.5 pg/ml). In addition, the lowest quartile compared withthe remaining population demonstrated a significantly lower(P # 0.05) sun exposure score (7.2 " 0.8 vs. 12.3 " 0.9) anddelta skin color (11.6 " 0.7 vs. 13.8 " 0.4) than the remainderof the subjects. Age, BMI, vitamin D supplement intake,serum calcium, alkaline phosphatase, and creatinine did notdiffer between groups.

Serum 25(OH)D as measured by reverse-phase HPLC andRIA was highly correlated (r2 ! 0.76; Fig. 4). However, asystematic bias was present with 25(OH)D values deter-mined by RIA being approximately 6.8 ng/ml higher than byHPLC. Thus, if the Diasorin RIA had been used to determinethe prevalence of low vitamin D status (using a cutpoint of30 ng/ml), fewer individuals would have been classified as“low.” However, even using the RIA, 25% of this populationwould be classified as having low vitamin D status. Finally,

although these assays were highly correlated, greater scatterat higher 25(OH)D values was observed with the RIA, as hasbeen previously reported (18). This greater scatter at highervalues slightly increases the mean bias noted previously;however, even when limiting the analysis to the 47 individ-

FIG. 4. Comparison of 25(OH)D as measured by HPLC and RIA.Although the correlation between these assays is good, a positive biasof 6.8 ng/ml is present using the Diasorin RIA in comparison to theHPLC assay used in this study.

FIG. 2. Low vitamin D status in highly sun-exposed subjects. Whenan accepted cutpoint of 30 ng/ml is used to define low vitamin D status,51% of these subjects (open bars) are low.

FIG. 3. PTH and 25(OH)D. Specific 1–84 PTH or whole PTH (“CAPPTH”) was not related to serum 25(OH)D status measured by HPLCin this cohort.

2132 J Clin Endocrinol Metab, June 2007, 92(6):2130–2135 Binkley et al. • Low Vitamin D despite Sun Exposure

Hawai: 21º N

Journal of the National Cancer Institute, Vol. 97, No. 3, February 2, 2005 ARTICLES 197

each melanoma site, patients with elastosis had better survival than those without. For melanomas of the head and neck, the hazard ratio of death for patients with solar elastosis compared with those without solar elastosis was 0.44 (95% CI = 0.12 to 1.65); for melanomas on the trunk, the hazard ratio was 0.34 (95% CI = 0.16 to 0.79); and for melanomas on the extremities, the hazard ratio was 0.64 (95% CI = 0.24 to 1.70).

We examined the association between screening and survival from melanoma. Screening for melanoma was inferred from skin awareness, skin self-examination, and physician skin examina-

tion. Compared with individuals who did not report skin awareness, individuals who did were at statistically signi cantly lower risk of death from melanoma ( Table 1 , P <.001). However, reported skin self-examination and physician skin examination were not statistically signi cantly associated with risk of death from melanoma ( P = .28 for each).

When melanoma-speci c mortality was analyzed in a multivariable setting, including all the variables from the univari ate analysis and accounting for the competing risk of death from other causes, solar elastosis and skin awareness remained statistically signi cant predictors of survival, as did anatomic site, Breslow thickness, and mitotic index ( Table 2 ). In addition, the point estimates of the hazard ratios in the mul-tivariable model for intermittent sun exposure (HR = 0.6, 95% CI = 0.3 to 1.1) and ever severely sunburned (HR = 0.6, 95% CI = 0.4 to 1.1) changed little from their univariate values ( Table 1 ), but the con dence intervals widened and P values rose, both to 0.12. In a multivariable model that included only the 80% of subjects for whom nevus counts were available and containing nevus count as a covariate, the hazard ratio of nevus count in association with death from the 95% con dence inter-val for melanoma increased slightly to 1.0 to 1.7 over its crude value. The point estimates of the hazard ratios for the other variables in this model were changed little from those shown in Table 2 .

D ISCUSSION

The results of this population-based study of survival from melanoma suggest that some factors associated with high levels of sun exposure, such as solar elastosis and, to a lesser extent, sunburns and intermittent sun exposure, are inversely associated with death from melanoma. The association between survival and solar elastosis was not explained by confounding with early detection or screening behaviors, represented by skin awareness, skin self examination, and physician examination, or by con-founding by social class, represented by educational level, all of which were also inversely associated with death from melanoma in univariate analyses. The inverse associations of solar elastosis and skin awareness with death from melanoma were also inde-pendent of its strong associations with melanoma site, lesion thickness, and mitoses.

One limitation of this study was the crude evaluation of sunscreen use. Moreover, the study was conducted during the 1980s, when few individuals would have used sunscreen regu-larly during most of their life. Thus, the weak and nonstatistically signi cant reductions in the risk of melanoma death may or may not be relevant to any contribution sunscreen use might make to an outcome of melanoma.

Our study was also limited by the use of the simple qualitative variables in a questionnaire assessment of early detection behaviors. Skin awareness, however, was a strong and indepen-dent predictor of survival of patients with melanoma and is a plausible indicator of likelihood of detecting melanoma early. Our study also lacked complete information on number of nevi, which could be confounded with sun exposure. However, analyses that included number of nevi as a covariate based on the 80% data set that had nurse-assessed numbers of nevi found no difference in point estimates of the solar exposure variables or other independent variables from those without adjustment of nevi from the full data set.

Table 1. Predictors of risk of death from melanoma in a population-based study of residents from Connecticut

No. of Hazard ratio Total melanoma (95% con dence Variable no. * deaths interval) P value

Demographic variables Sex Male 272 33 1.0 (referent) .33 Female 256 25 0.8 (0.5 to 1.3)

Age at diagnosis 528 58 1.2 (1.0 to 1.4) .08 10-year increase

Education Up to high school 202 28 1.0 (referent) .11 Greater than 326 30 0.7 (0.4 to 1.1) high school

Sun exposure variables Ever severely sunburned No 173 27 1.0 (referent) .02 Yes 353 31 0.5 (0.3 to 0.9)

Intermittent sun exposure index Low 189 27 1.0 (referent) .04 High 328 28 0.6 (0.3 to 1.0)

Solar elastosis Absent 254 36 1.0 (referent) .02 Present 268 21 0.5 (0.3 to 0.9)

Screening variables Skin self-examination No 458 53 1.0 (referent) .28 Yes 70 5 0.6 (0.2 to 1.5)

Skin awareness No 225 37 1.0 (referent) <.001 Yes 303 21 0.4 (0.2 to 0.7)

Physician skin examination No 318 39 1.0 (referent) .28 Yes 210 19 0.7 (0.4 to 1.3)

Clinical variables Site of melanoma Head and neck 36 10 1.0 (referent) .001 Trunk 284 30 0.4 (0.2 to 0.8) Extremity 208 15 0.3 (0.2 to 0.7)

Breslow thickness 1-mm increase 528 58 1.4 (1.3 to 1.5) <.001

Ulceration Absent 443 35 1.0 (referent) <.001 Present 75 21 4.2 (2.4 to 7.2)

Mitoses None 208 3 1.0 (referent) <.001 Any 307 54 7.6 (2.5 to 23.1)

* Numbers may vary because of missing data for some variables.

J Natl Cancer Inst 2005;97:195–9

FONTES DE VITAMINA D

Alimentos Vit. D (UI)

Óleo de Fígado de Peixe (10 g) 1360 Sardinha (105 g) 500 Atum (105 g) 402 Salmão Cozinhado (105 g) 360 Ovo (Unidade) 20 Fígado de Vitela cozinhado (105 g) 15

Ozkan B. J Clin Res Pediatr Endocrinol. 2010 Dec;2(4):137-‐43

result of fortification of frequently consumed commercial bev-erages (not shown) (8).

For children, even with small amounts of TF being consumed,diets of Dene/Yukon children containing TF had significantlymore protein, iron, zinc, copper, magnesium, phosphorus, potas-sium, vitamin E, riboflavin, and vitamin B-6. There were noother significant differences in micronutrient contents of chil-dren’s diets comparing days with and without TF.

DiscussionCurrent attention has been called to the contribution of dietarydiversity to dietary quality. It has been shown that increasingdiversity in either individual food items or in food groups leadsto a corresponding increase in dietary quality in North Americandiets (18). Consideration of the number of animal food speciesor food groups correlates with dietary diversity because dietdiversity appears to be a prerequisite to dietary quality. Animalfoods must be considered carefully. All ASF are not of equalnutritional quality, as is evident in the comparison of lean Arcticwildlife mammal tissues with processed meats such as frank-furters and lunch meats, which are high in saturated fat.Furthermore, 1 serving of 1 type of ASF provides more nutrientvalue (more nutrients, greater density) than any 1 plant food;therefore, animal food or food group should not be given equal‘‘weight’’ in diversity scores. It remains to be understood how tobest include meat and other ASF in dietary diversity calculationsand correlations with nutrient quality.

Our work with Arctic food systems and diets reveals that adiversity of animal and plant species are known and used, al-though many fewer species are used frequently. Nevertheless,fewer than 2 dozen animal and fish species make significantcontributions to Arctic dietary quality.

ASF, both TF and MF, are important sources of energy indiets of Arctic adults and children. Larger amounts of traditionalmeats and fish (combined) were eaten than of purchased meats.Adults’ and children’s diets invariably included MF, and theymay have contained portions of TF, usually as meat or fish.When traditional meats or fish were included, daily intakes weresignificantly higher in percentage of energy as protein and manymicronutrients, including vitamin D, vitamin E, riboflavin,vitamin B-6, iron, zinc, copper, magnesium, manganese, phos-phorus, potassium, and selenium than when MF were con-sumed. For children, daily diets containing these foods had

significantly more iron, zinc, copper, magnesium, phosphorus,potassium, vitamin E, riboflavin, and vitamin B-6 than did recalldays with no TF. It is very clear that traditional ASF areextremely important for dietary quality in Arctic populationswhen MF are the major source of energy.

AcknowledgmentsWe thank Rula Soueida and Tomoko Nakano for contributionsto the original data sets, and Liz Ansell for assistance with themanuscript. We continue to be appreciative of the originalparticipants, interviewers, and data managers who contributedto this work.

Literature Cited

1. Murphy SP, Allen LH. Nutritional importance of animal source foods.J Nutr. 2003;133(11 Suppl 2)S3932–5.

2. Gittlesohn J, Vastine AE. Sociocultural and household factors impactingonthe selection, allocation and consumption of animal source foods: currentknowledge and application. J Nutr. 2003;133(11 Suppl 2)S4036–41.

3. Allen LH. Interventions for micronutrient deficiency control in devel-oping countries: past, present and future. J Nutr. 2003;133(11 Suppl2)S3875–8.

4. Morrison NE, Receveur O, Kuhnlein HV, Appavoo M, Soueida R,Pierrot P. Contemporary Sahtu Dene/Metis use of traditional andmarket food. Ecol Food Nutr. 1995;34:197–210.

5. Fediuk K, Hidiroglou N, Madere R, Kuhnlein HV. Vitamin C in Inuittraditional food and women’s diets. J Food Compos Anal. 2002;15:221–35.

6. Kuhnlein HV, Chan HM, Leggee D, Barthet V. Macronutrient, mineraland fatty acid composition of Canadian Arctic traditional food. J FoodCompos Anal. 2002;15:545–66.

7. Kuhnlein HV, Barthet V, Farren A, Falahi E, Leggee D, Receveur O,Berti P. Vitamins A, D, and E in Canadian Arctic traditional food andadult diets. J Food Compos Anal. 2006;19:495–506.

8. Kuhnlein HV, Receveur O, Soueida R, Egeland GM. Arctic IndigenousPeoples experience the nutrition transition with changing dietarypatterns and obesity. J Nutr. 2004;134:1447–53.

9. Receveur O, Boulay M, Kuhnlein HV. Decreasing traditionalfood use affects diet quality for adult Dene/Metis in 16 communities ofthe Canadian Northwest Territories. J Nutr. 1997;127:2179–86.

10. Nakano T, Fediuk K, Kassi N, Kuhnlein HV. Food use of Dene/Metis and Yukon children. Int J Circumpolar Health. 2005;64:137–46.

11. Nakano T, Fediuk K, Kassi N, Egeland GM, Kuhnlein HV. Dietarynutrients and anthropometry of Dene/Metis and Yukon children. Int JCircumpolar Health. 2005;64:147–56.

TABLE 4 Micronutrient intake on days with and without TF for Yukon, Dene/Metis, and Inuit adults and Yukon and Dene children

Adults1 Children1

Yukon Dene/Metis Inuit Yukon and Dene

NutrientDays with

TF (n ! 410)Days withoutTF (n ! 387)

Days withTF (n ! 661)

Days withoutTF (n ! 346)





Iron, mg 23.3 6 0.6a 14.1 6 0.7b 26.5 6 0.9a 15.6 6 1.3b 37.4 6 1.1a 15.0 6 1.4b 16 6 0.6a 14 6 0.7b

Zinc, mg 27.7 6 0.9a 13.1 6 1.1b 23.8 6 1.0a 15.4 6 1.3b 21.5 6 0.5a 9.5 6 0.6b 11 6 0.5a 8.6 6 0.6b

Copper, mg 1655 6 46a 1163 6 53b 2025 6 89a 1439 6 122b 2076 6 44a 1041 6 58b 1293 6 41a 1066 6 48b

Phosphorus, mg 1602 6 35a 1155 6 40b 1759 6 31a 1224 6 43b 1663 6 27a 947 6 36b 1044 6 32a 924 6 37b

Potassium, mg 3520 6 76a 2608 6 87b 3516 6 63a 2561 6 86b 2997 6 53a 1999 6 0b 2291 6 86a 2043 6 99b

Magnesium, mg 297 6 6a 240 6 7b 305 6 5a 237 6 7b 597 6 31a 280 6 40b 203 6 5.7a 182 6 6.6b

Manganese, mg 3.7 6 0.1a 3.3 6 0.1b 3.6 6 0.1a 3.3 6 0.1b 3.3 6 0.1a 2.7 6 0.1b 2.4 6 0.1 2.1 6 0.2Riboflavin, mg 2.2 6 0.1a 1.5 6 0.1b 2.5 6 0.1a 1.6 6 0.1b 2.9 6 0.1a 1.3 6 0.1b 1.6 6 0.07a 1.2 6 0.08b

Vitamin B-6, mg 3.3 6 0.1a 1.7 6 0.1b 3.7 6 0.1a 1.9 6 0.1b 4.0 6 0.1a 1.4 6 0.1b 1.9 6 0.08a 1.6 6 0.09b

Vitamin D, mg 7.3 6 0.6a 2.1 6 0.7b 7.9 6 0.9a 3.5 6 1.3b 25.1 6 1.3a 8.6 6 1.7b 3.2 6 0.4 2.5 6 0.5Vitamin E, mg 4.8 6 0.1a 3.5 6 0.2b 6.5 6 0.4a 3.9 6 0.5b 5.4 6 0.2a 3.1 6 0.3b 3.5 6 0.2a 3.1 6 0.2b

1 Significant differences in nutrient intakes with TF and without TF are represented by the presence of different superscripts (a, b). Absence of superscripts means results are not

significantly different. Adults, P , 0.01; children P , 0.05.

Arctic indigenous peoples and animal source food 1113

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The Journal of Nutrition

Symposium: Food-Based Approaches to Combating Micronutrient Deficiencies in Children of Developing Countries

Local Cultural Animal Food Contributes HighLevels of Nutrients for Arctic CanadianIndigenous Adults and Children1,2

Harriet V. Kuhnlein3,4* and Olivier Receveur5

3Centre for Indigenous Peoples’ Nutrition and Environment (CINE) and 4School of Dietetics and Human Nutrition, McGill University,Ste. Anne de Bellevue H9X3V9, Canada and 5Department of Nutrition, University of Montreal, Montreal J3C3KS, Canada

Abstract

Food systems of Canadian Arctic Indigenous Peoples contain many species of traditional animal and plant food, but the

extent of use today is limited because purchased food displaces much of the traditional species from the diet. Frequency

and 24-h dietary interviews of Arctic adults and children were used to investigate these trends. The most frequently

consumed Arctic foods were derived from animals and fish. In adults these foods contributed 6–40% of daily energy of

adults. Children ate much less, 0.4–15% of energy, and .40% of their total energy was contributed by ‘‘sweet’’ and ‘‘fat’’

food sources. Nevertheless, for adults and children, even a single portion of local animal or fish food resulted in increased

(P , 0.05) levels of energy, protein, vitamin D, vitamin E, riboflavin, vitamin B-6, iron, zinc, copper, magnesium,

manganese, phosphorus, and potassium; although children had similar results for these nutrients, they did not reach

significance for energy, vitamin D, or manganese. Because market foods are the major source of energy in the Arctic,

traditional animal-source foods are extremely important to ensure high dietary quality of both adults and children. J. Nutr.

137: 1110–1114, 2007.

Animal-source foods (ASF)6 are viewed as essential in mosthuman societies because of their high nutrient content. In food-based approaches to ameliorating multiple micronutrient defi-ciencies, particularly in developing countries, the use of ASF incombination with plant foods is seen as effective and efficacious(1) provided that cultural and household-level constraints areaccommodated (2). Successful efforts to increase intake of ASFhave been documented, yet concerns have been expressed aboutthe amount of saturated fat and additional energy from ASF or

their derivatives and their possible contribution to the nutritiontransition in developing societies (2,3). In Canadian Arctic foodsystems there are abundant animals and fish in addition topurchased foods. Studies of Yukon First Nations, Dene/Metis,and Inuit cultural groups demonstrate extensive knowledge ofdiverse food sources, unique foods with exceptional nutrientquality, and unique patterns of food use incorporating varyinglevels of local cultural food with purchased market food (MF)(4–9). In this article, we present data on dietary food sources foradults of these 3 groups and selected samples of Dene (10,11)and Inuit children.

Methods of data collectionDietary data were collected during 2 seasons in 3 major dietarysurveys of adults and a study of Dene children residing in Yukonand Northwest Territories (NWT) communities as describedearlier (8–11). For this article, data from 24-h food intake re-calls and frequency interviews, comprised of lists of traditionalfood (TF) species, were derived from these studies. Communitymeetings were conducted to create complete food lists of speciescurrently known and used and to develop lists of species andtheir consumed parts, which were incorporated into frequencyinterviews for each cultural group. Daily intake from 24-h recalldata was dichotomized into the TF and MF. Recall data wereused to calculate mean intakes of food groups as percentage oftotal energy. TF groups consisted of land mammals, sea mam-mals, fish, birds, and plants. For MF, food groups represent themain categories (grains, meats, fruits and vegetables, dairy, meatalternatives). ‘‘Mixed dishes’’ included primarily savory foods

1 Presented as part of the symposium ‘‘Food-Based Approaches to CombatingMicronutrient Deficiencies in Children of Developing Countries’’ given at the2006 Experimental Biology meeting on April 3, 2006, San Francisco, CA.Previously presented in part at the 18th International Congress of Nutrition,September 2005, Durban, South Africa, abstract page 11. The symposium wassponsored by the American Society for Nutrition and supported in part by aneducational grant from Merck & Company. The supplement is the responsibilityof the Guest Editors to whom the Editor of The Journal of Nutrition has delegatedsupervision of both technical conformity to the published regulations of TheJournal of Nutrition and general oversight of the scientific merit of each article.The opinions expressed in this publication are those of the authors and are notattributable to the sponsors or the publisher, Editor, or Editorial Board of TheJournal of Nutrition. Guest Editors for the symposium publication are CharlotteG. Neumann and Suzanne P. Murphy.2 Supported by the Canadian Institutes of Health Research (CIHR), Institute ofAboriginal Peoples’ Health (IAPH) and Institute of Nutrition, Metabolism andDiabetes (INMD).6 Abbreviations used: ASF, animal-source foods; CINE, Centre for IndigenousPeoples’ Nutrition and Environment; MF, market food; NWT, NorthwestTerritories; TF, traditional food.* To whom correspondence should be addressed. E-mail: [email protected].

1110 0022-3166/07 $8.00 ª 2007 American Society for Nutrition.

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TF – Alimentos Tradicionais 1 mcg Vit D = 40 UI

Kuhnlein HV, Receveur O. J Nutr. 2007 Apr;137(4):1110-4!

Óleo de Fígado de Peixe: 8400 IU"

INGESTÃO PROLONGADA DE

100 IU/DIA DE VITAMINA D3 RESULTA NUM AUMENTO DE 25(OH)D DE

2,5 NMOL/L (1 NG/ML)

Heaney RP. Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 635–641

DNA synthesis and, therefore, cellular growth (33). A humancolon cancer cell line that expressed the vitamin D receptor(VDR) HT-29 responded to 1,25(OH)2D3 by a dosage-depen-dent inhibition of cellular growth and induction of differentia-tion. A wide variety of other tumor cell lines including leuke-mia; melanoma; and lung, breast, and prostate cancer cells havebeen shown to respond to the antiproliferative and prodiffer-entiating activity of 1,25(OH)2D3 (33–38). In addition,1,25(OH)2D3 induced apoptosis and has been demonstrated tobe antiangiogenic both in vivo and in vitro (33,39).

It is well established that the kidneys are the major source of1,25(OH)2D that is present in the circulation and is responsiblefor regulating the efficiency of intestinal calcium absorptionand mobilizing calcium stores from the skeleton (11). In ne-phrectomized patients, circulating levels of 1,25(OH)2D areundetectable. Thus, it had been assumed that only the kidneyswere capable of producing 1,25(OH)2D. It was recognized thatkeratinocytes in skin expressed the 25-hydroxyvitamin D-1-hydroxylase (CYP27B1; 1-OHase) (40); however, it was notuntil 1995, when it was observed that human prostate cells thatwere recovered from prostate biopsies not only expressed1-OHase but also converted 25(OH)D3 to 1,25(OH)2D3, that itwas realized that the body had other resources to produce1,25(OH)2D in an autocrine manner (41). It has been reportedthat many tissues and cells in the body, including activatedmacrophages, colon, prostate, breast, and brain, among others,have the ability to express 1-OHase and make 1,25(OH)2D3

(11,37). With this new revelation, it was appreciated that therewas another function for vitamin D that was unrelated to

calcium metabolism, and it was important not only for regu-lating cellular proliferation and differentiation but also for awide variety of other biologic processes, including regulation ofimmune function, modulating vascular tone, and influencingrenin and insulin synthesis, among other functions (11). Thus,although increasing vitamin D intake or sun exposure will notlead to an increase in the renal production of 1,25(OH)2D, it isbelieved that raising blood levels of 25(OH)D !30 ng/ml pro-vides an adequate amount of substrate for the nonrenal con-version of 25(OH)D to 1,25(OH)2D in many of these organs andcells. Once 1,25(OH)2D carries out its autocrine function withinthe cell, it then induces its own destruction by markedly in-creasing the expression of the 25-hydroxyvitamin D-24 hydrox-ylase (CYP24R; 24-OHase) (11,33).

Clinical Uses of the AntiproliferativeActivity of 1,25(OH)2D3 and Analogs

Epidermal cells have a VDR, and their proliferation is inhib-ited by 1,25(OH)2D3 (2,11). This observation led to the conceptthat 1,25(OH)2D3 could be used to treat the hyperproliferativeskin disorder psoriasis. Topical application of 1,25(OH)2D3 wasfound to be very effective for treating psoriasis with no unto-ward toxicity (42). 1,25(OH)2D3 and several of its analogs arenow one of the first-line treatments for psoriasis (11).

The observation that 1,25(OH)2D3 was effective in inducingleukemic cells to differentiate in vitro led to a clinical trial inwhich patients with preleukemia were given 1,25(OH)2D3. Ini-tially, patients responded favorably to the treatment but overtime developed severe toxicity, including hypercalcemia, andultimately died from their aggressive leukemia (43).

1,25(OH)2D3 has been given to men with metastatic prostatecancer. When given daily, it caused significant hypercalcemia,thus limiting its usefulness (44); however, when men weretreated with docetaxel and 45 mg of 1,25(OH)2D3 three times aweek, there seemed to be improvement in overall survivalwithout toxicity (44).

Numerous analogs of 1,25(OH)2D3 have been made, some ofwhich have potent antiproliferative activity while having min-imum calcemic activity (12,33). In animal models, some of theseanalogs, including novel analogs that have two side armsknown as gemini analogs, have been found to be very effectivein inhibiting colon tumor growth in mice while having mini-mum calcemic activity (33). To date, no active analogs of vita-min D have been proved to be efficacious for the treatment ofany human cancer by themselves.

Role of Vitamin D Nutrition in TumorPromotion and Progression

There is overwhelming scientific evidence suggesting thatmaintenance of an adequate vitamin D status is important forthe prevention of a wide variety of deadly cancers (7–11). Wooet al. (45) reported that men who had metastatic prostate cancerand received 2000 IU/d vitamin D had as much as a 50%reduction in prostate-specific antigen levels after 21 mo. Tang-pricha et al. (46) investigated the role of vitamin D sufficiencyand deficiency on colon tumor growth. They observed that

Figure 2. Comparison of the percentage increase in serum25(OH)D levels of healthy adults who were in a bathing suitand exposed to suberythemal doses (0.5 MED) of ultraviolet Bradiation once a week for 3 mo with healthy adults who re-ceived either 1000 IU of vitamin D2 or 1000 IU of vitamin D3

daily during the winter and early spring for a period of 11 wk.Fifty percent increase represented approximately 10 ng/mlfrom baseline 18 " 3 to 28 " 4 ng/ml. Skin type is based on theFitzpatrick scale: Type II always burns, sometimes tans; type IIIalways burns, always tans; type IV sometimes burns, alwaystans; type V never burns, always tans. Data are means " SEM.Reprinted with permission, copyright Michael F. Holick, 2008.

1550 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: 1548–1554, 2008

Holick MF. Clin J Am Soc Nephrol. 2008 Sep;3(5):1548-‐54

EXPOSIÇÃO SOLAR!

Todo o corpo (1 MED): 10 000 – 25 000 UI ! (20 000-50 000 UI em suplemento)"

Braços e pernas (0,25 – 0,5 MED): 2 000 – 4 000 UI!!

Mãos e cara: 200 UI!

Holick M. The Vitamin D Solution. 2010. Hudson Street Press!

EDITORIAL 1109

will be photochemically produced by an exposure of skin to sun-light. The two principal determinants are the quantity (intensity)and quality (appropriate wavelength) of the ultraviolet (UV) B irradiation reaching the 7-dehydrocholesterol deep in the stra-tum basale and stratum spinosum. 7-Dehydrocholesterol absorbsUV light most efficiently over the wavelengths of 270–290 nmand thus only UV light in this wavelength range has the capabil-ity to produce vitamin D3. There have been many studies show-ing the influence of season and latitude on the cutaneous photo-chemical synthesis of vitamin D3 (10, 11); maximal vitamin D3production occurs in summer months and, depending on latitude,little or no vitamin D3 may be generated in winter months.

A third factor, of course, is the actual concentration of 7-dehydrocholesterol in these two strata. Under normal physio-logic circumstances in humans there are ample quantities of 7-dehydrocholesterol available in the stratum spinosum and

stratum basale (! 25–50 !g/cm2 of skin) but in some species,such as domestic cats, this is an important consideration. Morris (12) reported that kittens exposed to UV-B are unable to photo-chemically produce vitamin D3. The concentration of 7-dehydrocholesterol in cat skin, in contrast with skin in ham-sters, rats, pigs, and sheep, was virtually undetectable. Thus, itmay be that for domestic cats vitamin D3 is an essential dietarytrace nutrient, ie, a vitamin.

The fourth determinant of vitamin D3 production is the con-centration of melanin in the skin. Melanin, which absorbs UV-Bin the 290–320 nm range, functions as a light filter and thereforedetermines the proportion of the incident UV-B that is actuallyable to penetrate the outer three strata and arrive at the stratumbasale and stratum spinosum (13). Appreciation of this factallowed Loomis (14) to propose that the evolution of the world’sracial distribution by latitude was due to regulation of vitamin D

FIGURE 1. Photochemical pathway occurring in the skin that describes the production of vitamin D3 (cholecalciferol) from 7-dehydrocholesterol.A brief description of the cellular anatomy of skin is provided in the text. The starting point is the irradiation of a provitamin D, which contains themandatory "5,7-conjugated double bonds; in the skin the highest concentrations of 7-dehydrocholesterol are present in the stratum spinosum and stra-tum basale. After absorption of a quantum of light from sunlight [ultraviolet (UV)-B], the activated molecule can return to its ground state and gener-ate at least six distinct products. The four steroids that do not have a broken 9,10 carbon bond (the starting 7-dehydrocholesterol and the products pyro-calciferol, lumisterol, and isopyrocalciferol) represent four diastereoisomers with either an #- or $-orientation of the methyl group on C-10 and thehydrogen on C-9. The three secosteroid products all have a broken 9,10 carbon-carbon bond with a differing position of the three conjugated doublebonds; they include previtamin D3, vitamin D3, and tachysterol. In the skin the principal irradiation product produced is previtamin D3, which thenundergoes a 1,7-sigmatropic hydrogen transfer from C-19 to C-9, yielding the final vitamin D3. Under normal sunlight exposure, irrespective of theextent of skin pigmentation, the amount of previtamin D3 produced is not greater than !15% of the available substrate 7-dehydrocholesterol (11). Fur-ther UV exposure simply causes the photoisomerization of previtamin D3 to lumisterol and tachysterol, which are both biologically inert. The newlyproduced vitamin D3 that is formed in the skin is removed by binding to the plasma transport protein, the vitamin D–binding protein (DBP), present inthe capillary bed of the dermis. DBP–vitamin D3 then enters the general circulatory system where it ultimately can be metabolized in the liver to 25-hydroxyvitamin D3 [25(OH)D3, or calcidiol] and then in the kidney to 1#,25-dihydroxyvitamin D3 [1#,25(OH)2D3, or calcitriol].

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Norman AW. Am J Clin Nutr 1998;67:1108–10

TIPOS DE PEL TIPO! DESCRIÇÃO!Tipo I! Queima-se sempre, nunca se bronzeia, pele muito

clara com cabelo louro ou ruivo"

Tipo II! Queima-se rápido, bronzeia-se com dificultade, pele clara"

Tipo III! Queima-se ocasionalmente, bronzeia-se gradualmente (origen mediterrânica e do médio oriente)"

Tipo IV! Muito raro queimar-se, bronzeia-se sempre (Médio Oriente, Índia, Paquistão)"

Tipo V! África, Sudeste asiático, alguns nativos de India e Paquistão"

Tipo VI! África, Tamils de Ásia (Índia, Sri Lanka), Nativos de Papua-Nova Guiné, Aborígenes Australianos"

Holick M. The Vitamin D Solution. 2010. Hudson Street Press!

PENÍNSULA IBÉRICA

8-11 H / 15 – 18 H

Tipo de Pele

Nov-Fev Mar-Maio Jun-Ag Sep-Oct

Tipo 1 15-20 min 10-15 min 15-20 min

Tipo II 20-30 min 15-20 min 20-30 min

Tipo III 30-40 min 20-30 min 30-40 min

Tipo IV 40-60 min 30-40 min 40-60 min

Tipo V e VI 60-75 min 40-60 min 60-75 min

Holick M. The Vitamin D Solution. 2010. Hudson Street Press

Tipo de Pele

Nov-Fev Mar-Maio Jun-Ag Sep-Oct

Tipo 1 10-15 min 2-8 min 10-15 min

Tipo II 15-20 min 5-10 min 15-20 min

Tipo III 20-30 min 15-20 min 20-30 min

Tipo IV 30-40 min 20-25 min 30-40 min

Tipo V e VI 40-60 min 25-35 min 40-60 min

Holick M. The Vitamin D Solution. 2010. Hudson Street Press

PENÍNSULA IBÉRICA

11 - 15 H

65

DEFICIÊNCIA EM VITAMINA D

Holick MF, Chen TC. Am J Clin Nutr. 2008 Apr;87(4):1080S-6S

Ossos e dentes: 99% Sangue e tecidos moles: 1%

DISTRIBUIÇÃO NO CORPO HUMANO

Mataix J. Nutrición y Alimentación Humana – Tomo I: Nutrientes y Alimentos. Ergon, 2002.

FUNÇÕES

o  Mineralização óssea e dentária;

o  Vasoconstrição e vasodilatação;

o  Transmissão de impulsos nervosos;

o  Contracção muscular;

o  Secreção e sensbilidade à Insulina;

o  Lipólise;

o  Ritmo cardíaco, Pressão Arterial e coagulação


Stipanuk. MH. Biochemical, Physiological, Molecular aspects of Human Nutrition. Saunders, 2006

ü  Infância: 60%

ü Antes da puberdade: 28%

ü  Puberdade: 34%

ü Adolescência e idade adulta: 25 a 30%

ü Gravidez: Aumenta

ü  Envelhecimento: Diminui 0.21% por ano

ü Menopausa: Diminui 2%

ABSORÇÃO


Shils M.E., Shike M., Ross A.C. et al. Modern Nutrition in Health and Disease. Lippincott Williams & Wilkins, US; 10Rev Ed edition, 2005


ü Gordura

ü  Fósforo

ü  Sódio, potássio, estrôncio y Magnésio

ü Ácido Fítico

ü Mais potente: Ácido Oxálico

ü  Ácido Hidroclorídrico

ü  Ácido Ascórbico

ü  Ácido Cítrico

ü  Glicina e Lisina

ü  Fibra solúvel

ü  Lactose (crianças)


Institute for Functional Medicine. Clinical Nutrition – A functional approach. IFM, 2004


ABSORÇÃO DE CÁLCIO

ABSORÇÃO ACTIVA DE CÁLCIO

Heaney R P Am J Clin Nutr 2008;88:541S-544S ©2008 by American Society for Nutrition

Sem Transporte Accvo: ü  Absorção = 0 à Ingestão = 1100 mg ü  Absorção = 200 mg à Ingestão = 3000 mg

Heaney RP, Weaver CM, Recker RR. Am J Clin Nutr. 1988 Apr;47(4):707-9.

% ABSORÇÃO DE Ca N= 13 (Cross-over) 200 mg Ca (marcado com 45Ca) por alimento

0%

5%

10%

15%

20%

25%

30%

Espinafre Leite

Heaney RP, Weaver CM, Fitzsimmons ML. Am J Clin Nutr. 1991 Mar;53(3):745-7.

% ABSORÇÃO DE Ca N= 16 (Cross-over) 100 mg Ca (marcado com 45Ca) por alimento

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

Soja alta Fitato Leite Soja baixa Fitato

ü Cafeína??

ü Álcool

ü  Excesso de Sódio

ü  Excesso de Proteína

ü Aspartame

ü Glucose

ü Hiperinsulinemia

ü Carga Ácida >0

EXCREÇÃO DE CÁLCIO


Institute for Functional Medicine. Clinical Nutrition – A functional approach. IFM, 2004


EQUILÍBRIO ÁCIDO-BASE

PH ARTERIAL

pH H+

(nanoeq/L) PCO2

mmHg HCO3- (mEq/L)

Sangue Arterial 7,37-7,43 37-43 36-44 22-26

Rose BD, Post TW. Clinical Physiology of Acid-base and Electrolyte Disorders. McGraw-Hill, 2001.

pH = 7,62 + log HCO3- /PCO2

(Henderson-‐Hasselbach)

PRODUÇÃO ÁCIDA ENDÓGENA LÍQUIDA

PAEL

Sebastian A, et al. Am J Clin Nutr. 2002 Dec;76(6):1308-16

Produção de Ácido Sulfúrico + Produção de Ácidos orgânicos - Produção de

Bicarbonato

PAEL

ü  Produção de Ácido sulfúrico resulta do metabolismo dos aminoácidos sulfurados metionina e cisteína.

ü  Produção de Ácidos orgânicos resulta da combustão incompleta dos hidratos de carbono e dos ácidos gordos.

ü  Produção de Bicarbonato (PB) = ∑ catiões inorganicos - ∑ aniões inorganicos

Corrigido para a taxa de absorção intestinal

PB = 0.95 Na+ + 0.8 K+ + 0.25 Ca2+ + 0.32 Mg2+ - 0.95 Cl - 0.63 Pi

Sebastian A, et al. Am J Clin Nutr. 2002 Dec;76(6):1308-16

PRAL DE ALGUNS GRUPOS DE ALIMENTOS

Peixe 14,6 Carne 12,4 Aves 7,8 Ovo 7,3

Marisco 7,3 Queijo 3,3 Leite 1,3

Cereais 1,1

Oleaginosas -1,1

Fruta -5,2

Tubérculos -5,4

Cogumelos -11,2

Raízes (cenoura, nabo) -17,1

Tomate -17,5

Hortaliças -23,4

ALCALINIZANTES ACIDIFICANTES

PRAL(mEq/100 kcal)

Leguminosas -0,4

PAEL (mEq/100 kcal)

PRAL (mEq/100 kcal)

NEUTROS Para cálculo da PAEL da dieta, há que somar 32.9 mEq/d que é a produção ácida orgânica basal independente da dieta.

Frassetto L.A. et al. J Nephrol. 2006 Mar-Apr;19 Suppl 9:S33-40.

NaCl determina ~ 50% da PAEL

CLORO

Frassetto LA, Morris RC Jr, Sebastian A. Am J Physiol Renal Physiol. 2007 Aug;293(2):F521-5

Jajoo R, et al. J Am Coll Nutr. 2006 Jun;25(3):224-30.

ü  40 H e M > 50 anos

ü  Duração 60 dias

ü  2 Grupos:

GRUPO 1: Elevada ingestão de Fruta e Vegetais

GRUPO 2: Fruta e Vegetais substituídas por Cereais

ü  RESULTADOS:


GRUPO 2:

•  PTH

•  Excreção UCa

•  Excreção urinária de N-Telopéptido


RECOMENDAÇÕES DE CÁLCIO

Etapas da vida Dose adequada mg

Limite mg

0-6 meses 210 N.D. 7-12 meses 270 N.D.

1-3 anos 500 2500 4-8 anos 800 2500

9-18 anos 1300 2500 19-50 anos 1000 2500 > 50 anos 1200 2500

Gravidez e Amamentação Até 18 anos 1300 2500 > 18 anos 1000 2500

National Academy of Sciences. Dietary Reference Intakes: National Academy Press; 1 edition (Dec 2006)

FONTES DE CALCIO NÃO LÁCTEAS

Alimento Dose Cálcio (mg)

Nº de doses necessárias para = Cálcio absorvido a partir de 225 ml de leite

Leite 225 ml 300 1

Couve chinesa * ½ chávena 239 1,3

Repolho chinês * 55 g 79 2,3

Feijão branco ½ chávena 113 3,9

Brócolo * ½ chávena 35 4,5

Ruibarbo ½ chávena 174 9,5

Feijão vermelho ½ chávena 41 9,7

Espinafre ½ chávena 115 16,3

Institute for Functional Medicine. Clinical Nutrition – A functional approach. IFM, 2004 Shils M.E., Shike M., Ross A.C. et al. Modern Nutrition in Health and Disease. Lippincott Williams & Wilkins, US; 10Rev Ed edition, 2005

* Não acumulam oxalatos como mecanismo para desentoxicar excesso de cálcio, de modo a prevenir a morte celular

Heaney RP, Weaver CM. Am J Clin Nutr. 1990 Apr;51(4):656-7

N= 11 (Cross-over) 300 mg Ca (marcado com 47Ca) por alimento

% ABSORÇÃO DE Ca

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

Leite Couve

HORTÍCOLAS PORTUGUESES COMO FONTES DE CÁLCIO

Alimento Ca (mg)

Crú Cozido

Couve-Galega 286 264 Grelo de Couve 147 131 Grelo de Nabo 131 106

Couve-Portuguesa 76 71 Brócolo 67 56

Couve-Lombarda 51 46 Couve-Branca 50 45

Couve-De-Bruxelas 26 20 Couve-Flor 21 19

Instituto Ricardo Jorge, 2006

DEFICIÊNCIA E TOXICIDADE

SESSÃO DE BASQUETEBOL (2 H)

à PERDA DE Ca (SUOR) DE 400 MG.

.

Kiesges RC, et al. JAMA. 1996 Jul 17;276(3):226-30

DEFICIÊNCIA

o Cãibras,

o Espasmos musculares,

o Hipertensão,

o Osteopenia,

o Osteomalacia.




TOXICIDADE

> 2500 mg/dia:

ü Cálculos renais ü Perturba o equilíbrio de Fe, Mg e Zn

A partir de 5 gramas/dia pode afectar a função renal.




RESEARCH

Effect of calcium supplements on risk of myocardialinfarction and cardiovascular events: meta-analysis

Mark J Bolland, senior research fellow,1 Alison Avenell, clinical senior lecturer,2 John A Baron, professor,3

Andrew Grey, associate professor,1 Graeme SMacLennan, senior research fellow,2 Greg D Gamble, researchfellow,1 Ian R Reid, professor1

ABSTRACTObjective To investigate whether calcium supplements

increase the risk of cardiovascular events.

Design Patient level and trial level meta-analyses.

Data sourcesMedline, Embase, and Cochrane Central

Register of Controlled Trials (1966-March 2010),

reference lists of meta-analyses of calcium supplements,

and two clinical trial registries. Initial searches were

carried out in November 2007, with electronic database

searches repeated in March 2010.

Study selection Eligible studies were randomised,

placebo controlled trials of calcium supplements

(!500 mg/day), with 100 or more participants of mean

agemore than 40 years and study durationmore than one

year. The lead authors of eligible trials supplied data.

Cardiovascular outcomeswere obtained fromself reports,

hospital admissions, and death certificates.

Results 15 trials were eligible for inclusion, five with

patient level data (8151 participants, median follow-up

3.6 years, interquartile range 2.7-4.3 years) and 11 with

trial level data (11921 participants, mean duration

4.0 years). In the five studies contributing patient level

data, 143 people allocated to calcium had a myocardial

infarction compared with 111 allocated to placebo

(hazard ratio 1.31, 95% confidence interval 1.02 to 1.67,

P=0.035). Non-significant increases occurred in the

incidence of stroke (1.20, 0.96 to 1.50, P=0.11), thecomposite end point of myocardial infarction, stroke, or

sudden death (1.18, 1.00 to 1.39, P=0.057), and death

(1.09, 0.96 to 1.23, P=0.18). The meta-analysis of trial

level data showed similar results: 296 people had a

myocardial infarction (166 allocated to calcium, 130 to

placebo), with an increased incidence of myocardial

infarction in those allocated to calcium (pooled relative

risk 1.27, 95% confidence interval 1.01 to 1.59,

P=0.038).Conclusions Calcium supplements (without

coadministered vitamin D) are associated with an

increased risk of myocardial infarction. As calcium

supplements are widely used these modest increases in

risk of cardiovascular disease might translate into a large

burden of disease in the population. A reassessment of

the role of calcium supplements in the management of

osteoporosis is warranted.

INTRODUCTION

Osteoporosis is a major cause of morbidity and mor-tality in older people.1 Calcium supplements margin-ally reduce the risk of fracture,23 and most guidelinesrecommend adequate calcium intake as an integralpart of the prevention or treatment of osteoporosis.4 5

Consequently, calcium supplements are commonlyused by people over the age of 50. Observational stu-dies suggest that high calcium intake might protectagainst vascular disease,6-8 and the findings are consis-tent with those of interventional studies of calcium sup-plements that show improvement in somevascular riskfactors.9-11 In contrast, calcium supplements acceleratevascular calcification and increasemortality in patientswith renal failure, in both dialysis and predialysispopulations.12-14 Furthermore, a five year randomisedcontrolled trial of calcium supplements in healthyolder women, in which cardiovascular events wereprespecified as secondary end points, recentlyreported possible increases in rates of myocardialinfarction and cardiovascular events in women allo-cated to calcium.15 16 We carried out a meta-analysisof cardiovascular events in randomised trials of cal-cium supplements.

METHODS

InNovember 2007we searchedMedline, Embase, andthe Cochrane Central Register of Controlled Trials forrandomised placebo controlled trials of calcium sup-plements, using the terms “calcium”, “randomisedcontrolled trial”, and “placebo” as text words, and cor-respondingMeSH terms (full details are available fromthe authors). We searched for studies in the referencelists of meta-analyses published between 1990 and2007 of the effect of calcium supplements on bone den-sity, fracture, colorectal neoplasia, and blood pressure,and in two clinical trial registries (ClinicalTrials.govand Australian New Zealand Clinical Trials Registry).No language restrictions were applied. In March 2010we updated the searches of the electronic databases(Medline: January 1966-March 2010, Embase: Janu-ary 1980-March 2010, Central Register of ControlledTrials: first quarter 2010).

1Department of Medicine, Facultyof Medical and Health Sciences,University of Auckland, Private Bag92 019, Auckland 1142, NewZealand2Health Services Research Unit,University of Aberdeen3Department of Medicine, andDepartment of Community andFamily Medicine, DartmouthMedical School, NH, USA

Correspondence to: I R [email protected]

Cite this as: BMJ 2010;341:c3691doi:10.1136/bmj.c3691

BMJ | ONLINE FIRST | bmj.com page 1 of 9Cardiovascular events were not primary study endpoints

A potential limitation of our meta-analysis is that cardio-vascular outcomes were not the primary endpoints of any ofthe contributing studies (although they were pre-specifiedsecondary endpoints in two of the Auckland studies [15,17]), and consequently, data on these outcomes were notgathered in a standardized manner. The events wereobtained variously from self-reports, hospital admissions,death certificates, or adjudication of medical records. TheRECORD study was the single largest contributor ofparticipants, and diagnoses were based on self-reports ofhospital admissions, and data from death certificates. Thelatter were the source for about 60% of MI, and 46% ofstrokes in that study. Two key facts address these concerns.First, the magnitude of the increased risk of MI withcalcium was consistent across the trials irrespective of themethod of event ascertainment. Secondly, the likelihood ofdifferential misclassification or misreporting of cardiovas-cular events by treatment allocation is small, because thedata came from blinded, placebo-controlled trials. The useof variable methods for collecting adverse event reports willintroduce noise, not bias, and would tend to obscure a trueeffect rather than introduce a false one. This contention issupported by the results of the adjudication process of self-reported events from the Auckland Calcium Study, whichchanged the numbers of events in the two groups but notthe asymmetry of their distribution [15]. It has also beensuggested that failure of randomization may have producedan uneven distribution of risk factors for cardiovasculardisease. This is unlikely across the number and size ofstudies included in the meta-analysis, and is not supportedby examination of the baseline data from the calcium andplacebo groups.

Adverse effect restricted to subgroups

Others have suggested that the effects might be related totrial- or participant-related characteristics, such as gender ortype of calcium supplement used. However, there was nointeraction between age, gender, baseline vitamin D status,or type of supplement used and risk of MI, stroke, or thecomposite endpoint with calcium (Fig. 2). When the cohortwas divided into two groups by baseline dietary calciumintake (above and below the median), there was aninteraction between dietary calcium and the risk of MI(but not stroke or the composite endpoint) with calcium.But when the cohort was divided by quintile of baselinedietary calcium intake, there was no evidence of a dose–response relationship between baseline dietary calcium andthe risk of MI with calcium, and in four of the five groups,the hazard ratios exceeded 1 and were similar to the hazardratio for the entire cohort (Fig. 2).

No effect on mortality

Some have questioned the internal consistency of the data sincethe increase in risk of MI is not accompanied by increasedmortality. About 10–20% of individuals having an MI die as aresult of that event, so the 30% increase in MI found withcalcium use should result in a 3–6% increase in mortality. Infact, there is a 9% increase in mortality with calcium in thepatient-level analysis and a 7% increase in the trial-levelanalysis, consistent with expectations. These results were notstatistically significant, but the study did not have the power todetect differences of this magnitude. Hormone therapy androsiglitazone are other examples of interventions that increaserisk of MI to a similar degree as calcium supplements withoutevidence for a significant increase in mortality [18, 19]

Myocardial infarction

0 1 2 3 4 50

1

2

3

4

5

6

Calcium

Placebo

HR 1.31, 95%CI 1.02-1.67, P=0.035

No. at risk40974054

38703865

35393588

26702728

12941320

373388

CalciumPlacebo

Years

Cum

ulat

ive

inci

denc

e (%

)

Total

Reid 2008

Lappe 2007

Reid 2006

Prince 2006

Grant 2005 Vit D

Grant 2005

Baron 1999

Favours calcium

Study

0.5 1 21.50.8

Relative Risk of Myocardial Infarction[95% Confidence Interval]

1.27 [1.01-1.59] P=0.038Test for heterogeneity: I! = 0%, P = 0.96

Weight(%)

17

29

13

1

1

26

13

1.2 3Favours placebo

Fig. 1 Effect of calcium supplements on risk of myocardial infarctionfrom an individual patient meta-analysis of five trials (8,151participants), and a trial-level analysis of 11 studies (11,921 subjects).

Significant increases in risk were shown for both analyses. Figurefrom Bolland et al. [16], used with permission

Osteoporos Int (2011) 22:1649–1658 1651

. .

ü  7 estudos prospectivos, 170

991 mulheres, 2954 fracturas da anca.

ü  5 estudos prospectivos, 68 606 Homens, 214 fracturas da anca.

ü  5 intervenções (5666 Mulheres + 1074 Homens), 814 fracturas não-vertebrais.

ü  4 intervenções c/ resultados separados para fracturas da anca (6504 pessoas, 139 fracturas da anca).

Bischoff-Ferrari HA, Dawson-Hughes B, Baron JA, et al. Am J Clin Nutr. 2007 Dec;86(6):1780-90

100 Bischoff-Ferrari HA, Dawson-Hughes B, Baron JA, et al. Am J Clin Nutr. 2007 Dec;86(6):1780-900

Total n: 252,841

ü Estudos Prospectivos NÃO MOSTRAM ASSOCIAÇÃO ENTRE INGESTÃO DE CA E FRACTURAS DA ANCA em Homens e Mulheres

ü RCTs observam um LIGEIRO AUMENTO DO RISCO DE FRACTURAS DA ANCA com a SUPLEMENTAÇÃO DE CA

. .

101 Bischoff-Ferrari HA, Dawson-Hughes B, Baron JA, et al. Am J Clin Nutr. 2007 Dec;86(6):1780-900

Total n: 252,841

ü Estudos Prospectivos NÃO MOSTRAM ASSOCIAÇÃO ENTRE INGESTÃO DE CA E FRACTURAS DA ANCA em Homens e Mulheres

ü RCTs observam um LIGEIRO AUMENTO DO RISCO DE FRACTURAS DA ANCA com a SUPLEMENTAÇÃO DE CA

. .

Reid IR, Bolland MJ, Grey A. Osteoporos Int. 2008 Aug;19(8):1119-23.

Bischoff-Ferrari HA et al. J Bone Mineral Res 2011; online Oct 2010, DOI 10.1002/jbmr.279!

Pooled analysis for categories of milk intake and hip fracture risk in women from prospective cohort studies (6 studies, 195 102 women, 3574 fractures). !

RR/+1glass"

LEITE E FRACTURAS"

SEM ASSOCIAÇÃO EM ADULTOS

MAGNÉSIO ü Distribuição:

o Ossos e dentes: 60 a 65%

o Músculo: 27%

o Outras células: 6 a 7%

o  Líquido extracelular: menos de 1%




37

Mg-ATP and Mg-DNA complexes:Mg2+involved in all enzymes using ATP and all enzymes in DNA and RNA synthesis

Mg-DNA complexes: Mg2+>Co2+> Ni2+> Mn 2+> Zn2+>Cd2+>Cu2+

Mg-ATP complexes: triphosphates are normally found with Mg2+. If the Mg2+content of a system is

low the enzymes using triphosphates as substrates cease function

Magnesium biochemical functions

– Regulation enzyme function (>300)

– All energy requiring metabolic processes

– Membrane and channel functions

– Calcium antagonist action

– Structural functionsBarbagallo, Curr Pharm Design 2010

Magnesium and excitability

–Conduction of nerve pulses

–Muscle contraction

–Heart rhythmBarbagallo, Curr Pharm Design 2010

Magnesium is needed for glutathione synthesis

low magnesium is associated with dramatic increases in free radical generation as well as glutathione depletion

Mg deficiency causes low RBC glutathione in hamsters

Weglicki, Ann NY Acad Sci 1994

Magnesium food sources

– Vegetables (notably dark leavy vegetables)– Fruits (bananas, dried apricots, and

avocados) – Nuts (almonds and cashews) – Peas and beans (legumes), seeds – Soy products (soy flour and tofu) – Whole grains (brown rice and millet)

Mg está envolvido em todos os enzimas que usam ATP e todos os enzimas envolvidos na síntese de ADN e RNA

(1) gamma-Glutamylcysteine synthetase; (2) glutathione synthetase; (3) phytochelatine synthase; (4) glutathione S-transferase (GST).

Mg

113

W-6 Ácido Linoleico

18:2 w6

W-3 Ácido Alfa Linolénico

18:3 w3

Enzima Delta-6-

-desaturase Ácido Gama Linoleico

18:3 w6

Enzima Elongase

Ácido Estearidónico

18:4 w3

Ácido Dihomogama

Linoleico 20:3 w6

Ácido Eicosatetraenoico

20:4 w3 Enzima Delta-5-

-desaturase

EPA 20:5 w3

Ácido Araquidónico

20:4 w6

Bastos P. An Nutr Esp Func 2007; 7(36): 17-24

DHA 22:6 w3

Delta-6- -desaturase

Mg

Santos DA, Matias CN, Monteiro CP, Silva AM, Rocha PM, Minderico CS, Bettencourt Sardinha L, Laires MJ. Magnes Res. 2011 Dec;24(4):215-9

DOSES DE MAGNÉSIO FOOD AND NUTRITION BOARD

Homens RDA/AI* (mg)

Limite (mg) suplemento

Mulheres RDA/AI* (mg) Limite (mg) suplemento

0-6 meses 30* N.D. 0-6 meses 30* N.D.

7-12 meses 75* N.D. 7-12 meses 75* N.D.

1-3 anos 80 65 1-3 anos 80 65

4-8 anos 130 110 4-8 anos 130 110

9-13 anos 240 350 9-13 anos 240 350

14-18 anos 410 350 14-18 anos 360 350

19-30 anos 400 350 19-30 anos 310 350

> 30 anos 420 350 > 30 anos 320 350

Gravidez

Sugestão de Seelig

(Am J Clin Nutr 1964, 14:342-90)

6 mg/kg peso

Até 18 anos 400 350

19-30 anos 350 350

31-50 anos 360 350

Lactação Até 18 anos 360 350

19-30 anos 310 350

31-50 anos 320 350

FONTES DE MAGNÉSIO

Hands ES. Nutrients in Food. Lippincott Williams & Wilkins; 2003.

Alimentos Dose (g) Magnésio (mg) Semente de sésamo 30 102 Semente de abóbora 30 159 Amêndoa 30 91 Avelã 30 85 Caju 30 72 Castanha do Pará 30 71 Noz 30 50 Pistachio 30 47 Semente de girassol 30 36 Macadâmia 30 34 Banana 120 35 Melancia 250 26 Kiwi 75 23 Abacate 50 20 Manga 200 17 Morango 150 16 Papaia 140 14 Sumo de Laranja natural 250 27 Sumo de Uva 250 25

Alimentos Dose (g) Magnésio (mg) Feijão Preto cozido 150 70 LenYlha cozida 100 36 Ervilha cozida 100 29 Pão Integral 60 51 Arroz Integral cozido 100 43 Arroz Branco cozido 100 12 Aveia cozida 100 24 Farelo de Aveia 10 22 Beterraba fresca cozida 100 68 Alcachofra cozida 100 60 Couve Cozida 100 23 Brócolo cozido 100 22 Pescada 100 29 Atum 100 28 Sardinha 100 25 Linguado 100 25 Gambas 100 42 Leite 250 29 Iogurte 125 22

ü Gordura

ü Fitatos e Oxalatos

ü Cálcio e Fósforo

ü Ácidos biliares

ü Ingestão proteica < 30 grs/día

ü Ingestão de Cálcio >2 grs juntamente con Mg

ü Ingestão elevada de Zn (> 100 mg)

ü Hipocloridria

PDR, 2001 Mataix, 2002

Linus Pauling Institute, 2003 IFM, 2004

ABSORÇÃO

o Lactose

o FOS

o Boro

ABSORÇÃO


Linus Pauling Institute, 2003 IFM, 2004

o Excesso de Na, Ca,

açúcar, cafeína e álcool

o Diuréticos

o Diabetes

o Dieta com PAEL > 0


Linus Pauling Ins;tute, 2003 IFM, 2004

J. Nutr. 2006; 136:2374-‐2377

EXCREÇÃO DE MAGNÉSIO

SINAIS DE DEFICIÊNCIA

Cãibras Diminuição dos níveis de Ca e K

Espasmos musculares Alteração do perfil lipídico

Fadiga RI

Insónia Arritmia cardíaca

Irritabilidade e Sintomas Depressão Aumento PA com Stress

Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ. Modern Nutrition in Health and Disease. Lippincott Williams & Wilkins; 2005. Gibney MJ, et al. Introduction to Human Nutrition (Nutrition Society Textbook). 2nd ed. Wiley-Blackwell; 2009.

Nutriente % H 14-18 a

% H 19-30 a

% H 31-50 a

% M 14-18 a

% M 19-30 a

% M 31-50 a

Vit E 97 89 90 97 97 97 Magnésio 78 55 61 91 64 65 Vit C 26 37 40 42 40 41 Zinco 4 6 4 26 13 11 Folato 4 6 < 3 19 14 16 Ferro < 3 < 3 < 3 16 15 17

DEFICIÊNCIAS DE MICRONUTRIENTES (POPULAÇÃO GERAL - EUA)

USDA What we Eat in America (NHANES 2001-‐2002) Sept. 2005

Disponível em: hmp://www.ars.usda.gov/SP2UserFiles/Place/12355000/pdf/usualintaketables2001-‐02.pdf

DEFICIÊNCIAS DE MAGNÉSIO EM ATLETAS

Modalidades % DDR Referências

Basquetebol (Mulheres)

66 Hickson JF Jr, Schrader J, Trischler LC. J Am Diet Assoc 1986;86: 251–3

Ginástica (Mulheres)

66 Hickson JF Jr, Schrader J, Trischler LC. J Am Diet Assoc 1986;86: 251–3

Futebol Americano 69 Hickson JF, et al. Nutr Res 1987;7:27–34.

Futebol 90 Hickson JF, et al. Nutr Rep Int 1986;34:85–91

Atletismo (Mulheres com amenorreia)

53 Zierath J, Kaiserauer S, Snyder AC.

Med Sci Sports Exerc 1986; 18(suppl):S55–6

Atletismo (Mulheres sem amenorreia)

89 Zierath J, Kaiserauer S, Snyder AC.

Med Sci Sports Exerc 1986; 18(suppl):S55–6

Triatlo (Homens) 91 Worme JD, et al. Am J Clin Nutr 1990;51:690–7

Adaptado de Lukaski HC. Am J Clin Nutr. 2000 Aug;72(2 Suppl):585S-‐93S

TOXICIDADE

o Diarreia (pode surgir a partir de 600 mg/dia);

o Hipotensão que pode levar a tonturas, letargia, confusão e alteração do ritmo cardíaco

o Grave: debilidade, dificultade em respirar e paragem cardíaca.

ü Atenção a quem tem Insuficiência Renal

ü Doses de Suplementos: Comum entre 100 e 1000 mg/dia

PDR, 2001,

Colgan, 2002, IFM, 2004, Linus Pauling Institute, 2003

Seelig e Rosanoff 2003

VITAMINA K

Morley SL. Management of acquired coagulopathy in acute paediatrics. Arch Dis Child Educ Pract Ed. 2011 Apr;96(2):49-60.

Best practice

Arch Dis Child Educ Pract Ed 2011;96:49–60. doi:10.1136/adc.2007.13574950

The clotting cascade contains a number of anticoagulants that help to prevent excessive clot formation. Protein C is activated by throm-bin into activated protein C (aPC) and forms a complex with thrombin, thrombomodulin and protein S. This complex downgrades activated factors VIIIa and Va making them inactive. Antithrombin (AT) III is a constantly active enzyme that can inactivate all the factors depen-dent on vitamin K. It has low activity but in the presence of heparin (which acts as a catalyst) its activity increases 1000-fold. In the circulatory system the heparin role is probably fulfi lled by naturally occurring heparin-like compounds (glycosaminoglycans) on the surface of endothe-lium. The low molecular weight heparins have a preferential role in inactivation of factor Xa rather than the wider panel inhibited by conven-tional heparin. Tissue factor pathway inhibitor limits the downstream effects of tissue factor by inhibiting factor VIIa (when bound to tissue factor) and also Xa and thrombin.

up these pathways are found in plasma, almost all the reactions in the cascade occur on the surface of platelets, endothelial cells or other blood cells. Normal endothelium cannot support coagulation because it carries neutral phospholipids on its sur-face. Disruption of endothelial cell function leads to loss of neutral phospholipids and exposure of negatively charged phospholipids that do support coagulation. Negatively charged phospholipids are also released by activated platelets. Calcium is an important cofactor for many coagulation fac-tor reactions. Fibrinolysis is initiated through the clotting pathways (via thrombin) and is triggered by release of tissue plasminogen activator from damaged endothelial calls.

Vitamin K is an essential factor to a hepatic γ-glutamyl carboxylase that adds a carboxyl group to glutamic acid residues on factors II, VII, IX, X, protein S and protein C. If vitamin K is not available proteins formed in vitamin K absence are produced and are unable to contribute to coagulation.

Figure 1 Pathways of the human coagulation system.

03_edpract135749.indd 5003_edpract135749.indd 50 2/24/2011 2:31:59 AM2/24/2011 2:31:59 AM

group.bmj.com on October 13, 2012 - Published by ep.bmj.comDownloaded from

Vit K

Vit K

Vit K Vit K

144 Port J Nephrol Hypert 2008; 22(2): 143-148

CMYKP

which this contributes to the daily intake is still unknown (Suttie, 1995).

The discovery of vitamin K goes back to the 1930s, with the observation that chickens on a fat-free diet developed haemorrhages (Dam H., 1934). Shortly thereafter, the clotting factor prothrombin was the first coagulation factor to be discovered as being decreased in animals with haemorrhagic syn-drome. Administration of vitamin K was then shown to cure bleeding complications in patients with hepatic and biliary diseases (Suttie, 1978).

The individual denomination of the several isoforms of menaquinones is dependent on the number of isoprenoid residues in the side chain. In human food we find the isoforms MK -4 to MK -10, and here we find those having 7, 8 or 9 isoprenoid groups in the side chain are most common. Due to the increased hydro-phobic properties of longer side chains, the various isoforms exert different half -lives. This results in a half-life time of MK -4 of 1 hour, and a half -life time of MK -7 of around 3 days. The kinetic of the half -life of MK -7 is biphasic; within the first 1.5h it lasts 6 -8h, where-as in a second phase the half -life is around 50h, sug-gesting initial redistribution and tissue uptake followed by the incorporation of vitamin K2 in lipoproteins and

release by the liver (Schurgers, 2000). This difference in bioavailability is important for the supply of vitamin K2 towards extra -hepatic tissues such as the arterial vessel wall.

In mammalians, vitamin K serves as a redox part-ner in cellular metabolism pathways of gamma-glutamyl carboxylations, and a recycling mechanism helps to reduce the daily requirement of vitamin K. The vitamin K cycle can be effectively inhibited by coumarins like warfarin, primarily known as potent coagulation inhibitors.

Both vitamin K1 and vitamin K2 catalyse the gamma -glutamyl carboxylation of all vitamin K -depen-dent proteins, including coagulation factors, osteo-calcin, and MGP. Buitenhuis et al. (1989) showed that K2 vitamins have a lower Km for the enzyme gamma -glutamyl carboxylase, demonstrating a pref-erence for K2 vitamins as cofactor. Additionally, while vitamin K1 predominantly accumulates in the liver, it is most important in the catalysation of the J -glutamyl carboxylation of coagulation factors. However, vitamin K2 has a more wide -spread tissue distribution and is thus also involved in the car-boxylation of osteocalcin and MGP. Osteocalcin (OC), also called bone Gla -protein (BGP), is exclusively

Thilo Krueger, Vincent M. Brandenburg, Leon J. Schurgers, Jürgen Floege

Figure 1

The Vitamin-K-Cycle (from Stafford, 2005)

Nefro - 22-2.indd 144Nefro - 22-2.indd 144 17-04-2008 11:00:4817-04-2008 11:00:48

Krueger T. Port J Nephrol Hypert 2008; 22(2): 143-148

DOSES DE VITAMINA K FOOD AND NUTRITION BOARD

Homens AI (mcg) Limite (mcg) Mulheres AI (mcg)

Limite (mcg)

0-12 meses 2.0-2.5 Nd 0-12 meses 2.0-2.5 Nd

1-3 anos 30 Nd 1-3 anos 30 Nd

4-8 anos 55 Nd 4-8 anos 55 Nd

9-13 anos 60 Nd 9-13 anos 60 Nd

14-18 anos 75 Nd 14-18 anos 75 Nd

>18 anos 120 Nd >18 anos 90 Nd

Gravidez (<18 anos) 75 Nd

Gravidez (>18 anos) 90 Nd

Lactação (<18 anos) 75 Nd

Lactação (>18 anos) 90 Nd

Alimentos Vitamina K1 (mcg) Couve Galega cozida (130 g) 1146 Espinafre cozido (180 g) 888 Couve-de-Bruxelas cozida (155 g) 300 Brócolo cozido (156) 220 Cebola crua (100 g) 207

USDA Naconal Nutrient Database for Standard Reference, Release 20

CALCIFICAÇÃO DA AORTA

MORTALIDADE CARDIOVASCULAR

Arch Intern Med. 2006;166:1256-‐1261

power of bias tests remains low to de-tect bias and funnel plot asymmetry(Egger test: Phip= .09; Pvertebral= .61;Pnonvertebral= .08). Visual inspectionof funnel plots showed no evidenceof bias (data not shown).24 In termsof reporting quality, only 2 stud-ies25,30 reported that they had useda method of concealing the alloca-tion mechanism. Attrition, anothersource of potential bias, ranged from0% to as high as 30%.

ADVERSE EVENTS

No study reported any serious ad-verse events associated with vita-min K. However, minor gastrointes-tinal problems were reported bysome authors.

COMMENT

In this systematic review and meta-analysis, we have shown that supple-mentation with phytonadione andmenaquinone, particularly menaqui-none-4, is associated with increasedBMD and reduced fracture inci-dence. The reduction in fracture in-cidence is particularly striking, withan approximate 80% reduction in hipfractures. Our findings should be

treated cautiously, however, be-cause the studies were not primarilydesigned to show a fracture effect.Another reason for caution is that theeffect on fractures is much larger thanwith other treatments, such as bis-phosphonates. Therefore, it is pos-sible that such a large effect is due tochance or some other unidentified

reason. In addition, all the studieswith fracture outcomes were under-taken in Japan, and there may be di-etary differences that could mean thatthese findings are not applicable else-where. The quality of many of thetrials was not high. Few trials, nonewith fracture outcomes, reported howthe randomization process was con-

Table 2. Trial Outcomes

Source

No. of Randomized Patients(Attrition Rate, %)

No. of Fractures/Total No. of Patients*

Difference in BMD, %(95% CI)Control Intervention Control Intervention

Braam et al,25 2003 61 (13) 66 (12) NA NA 1.3 (hip) (0.10 to 3.41);0.9 (spine) (!0.70 to 2.23)

Ishida and Kawai,26 2004 66 (6) 68 (3) 17/66 (spine);1/66 (hip);3/66 (nonspine)

9/66 (spine);0/66 (hip);0/66 (nonspine)

1.4 (radius)

Iwamoto et al,27 1999 35 (0) 17 (0) NA NA 2.0 (spine)Iwamoto et al,28 2000 49 (0) 43 (0) NA NA 1.34 (spine)Iwamoto et al,29 2001 24 (0) 23 (0) 6/24 (spine) 2/23 (spine) 1.6 (radius)Braam et al,30 2003 !42 (30†) !37 (30†) NA NA !0.1 (spine)

!1.3 (hip)Nishiguchi et al,31 2001 15 (1) 15 (2) NA NA 3.8 (spine)Sasaki et al,32 2005 10 (0) 10 (0) 1/10 (spine) 0/10 (spine) Not possible to calculateSato et al,33 1998 60 (6) 60 (4) 1/54 (hip) 0/54 (hip) 1.8 (metacarpals)Sato et al,34 2002 60 (4) 60 (6) 8/56 (hip);

10/56 (nonspine)1/54 (hip);

1/54 (nonspine)5.2 (metacarpals)

Sato et al,35 2005 100 (12) 100 (10) 15/88 (hip);22/88 (nonspine)

2/90 (hip)3/90 (nonspine)

7.5 (metacarpals)

Shiraki et al,36 2000 121 (22) 120 (29) 30/121 (spine);2/121 (hip);5/121 (nonspine)

13/120 (spine);0/120 (hip);1/120 (nonspine)

2.8 (spine)

Somekawa et al,37 1999 52 (0) 52 (0) NA NA 1.1 (spine)

Abbreviations: BMD, bone mineral density; CI, confidence interval; NA, not applicable.*Indicates number of patients who underwent follow-up for outcome.†This is an estimated attrition rate.

Sato et al,33 1998Shiraki et al,36 2000Sato et al,34 2002Ishida and Kawai,26 2004Sato et al,35 2005

Subtotal

Hip

0.05 0.1 0.2 0.5

Favors Vitamin K Favors Control

1

OR2 5 10 20

Sasaki et al,32 2005Shiraki et al,36 2000Iwamoto et al,29 2001Ishida and Kawai,26 2004

Subtotal

Vertebral

Sato et al,33 1998Shiraki et al,36 2000Sato et al,34 2002

Sato et al,35 2005Ishida and Kawai,26 2004

Subtotal

0.36 (0.02 to 5.90)0.26 (0.03 to 2.55)0.19 (0.05 to 0.75)0.37 (0.02 to 5.90)0.22 (0.08 to 0.59)0.23 (0.12 to 0.47)

0.36 (0.02 to 5.90)0.26 (0.05 to 1.30)0.17 (0.05 to 0.58)0.22 (0.03 to 1.56)0.18 (0.08 to 0.41)0.19 (0.11 to 0.35)

0.35 (0.02 to 6.00)0.39 (0.20 to 0.75)0.32 (0.07 to 1.46)0.47 (0.20 to 1.10)0.40 (0.25 to 0.65)

6.39.4

26.46.3

51.6100.0

4.513.423.0

8.950.2

100.0

2.954.410.432.3

100.0

All Nonvertebral

Study%

WeightOR

(95% Cl)

Figure 2. Meta-analysis of treatment effects on fractures. Peto odds ratios (ORs) with 95% confidenceintervals (CIs).

(REPRINTED) ARCH INTERN MED/ VOL 166, JUNE 26, 2006 WWW.ARCHINTERNMED.COM1259

©2006 American Medical Association. All rights reserved.

Alimentos (100 g) Vitamina K2 (mcg) Natto 1103 Paté de fígado de Ganso 369 Queijos envelhecidos 76,3 Queijos gordos 56 Gema de ovo 15-32 Coxa de Ganso 21 Manteiga 15 Fígado de Galinha 14 Coxa de Galinha 8,5 Bife de vaca 8,1

Elder SJ, et al. J Agric Food Chem. 2006; 54: 463-‐467

Schurgers LJ, Vermeer C. Haemostasis. 2000; 30: 298-‐307.

VITAMINA K1 E OSTEOCALCINA

Binkley NC. Am J Clin Nutr. 2002 Nov;76(5):1055-‐60.

Echocardiography was performed in all patients using acommercially available ultrasound system (GE Vingmed,Vivid 7, Horten, Norway) by 1 experienced echocardiogra-pher. Aortic valve sclerosis was defined as a focal area ofincreased echogenicity and thickening of the aortic valveleaflets with a transaortic flow velocity of !2.0 m/s ontransthoracic echocardiography, using the criteria of Otto etal.7 Patients with a Doppler peak aortic velocity between 2and 3 m/s had mild aortic stenosis, those with a peakvelocity between 3.1 and 3.9 m/s had moderate aortic ste-nosis, and those with a peak velocity of !4 m/s had severeaortic stenosis.

The cardiovascular risk factors were assessed from thepatient charts, including nicotine abuse, hypertension (useof antihypertensive medication or blood pressure at rest"140/90 mm Hg), diabetes mellitus (use of insulin or oralantidiabetic agents or fasting serum glucose "130 mg/dl),hypercholesterolemia (total fasting serum cholesterol "200mg/dl or use of cholesterol-lowering medication), and obe-sity (body mass index "25 kg/m2). Additionally, the pres-ence of coronary artery disease (coronary artery stenosis"50% assessed by cardiac catheterization or known myo-cardial infarction) was evaluated.

Continuous variables are expressed as means # SDs.Student’s t test was used for comparisons of continuousvariables. Categorical data are presented as frequencies andwere compared by chi-square analysis. Multiple linear re-gression analysis was used to evaluate the patient contribu-tions of age, gender, risk factors, and OAC treatment forthe amount of cardiac valvular and coronary calcium. A pvalue !0.05 was considered statistically significant. Statis-tical analysis was performed using the Statistical Package ofSocial Sciences, version 10.0 (SPSS, Inc., Chicago, Illi-nois).

Patient characteristics are listed in Table 1. The indica-

tion for OAC therapy was chronic atrial fibrillation in 16patients (70%), cardiomyopathy with a low ejection fractionin 3 patients (13%), recurrent deep vein thrombosis in 2patients (9%), pulmonary embolism in 1 patient (4%), andischemic stroke in 1 patient (4%). No correlation betweenthe duration of OAC treatment and the amount of coronary(p $ 0.35) or valvular (p $ 0.31) calcium was observed.The median valvular Agatston score was 1,064 (range 0 to6,535), and the median coronary Agatston score was 474(range 0 to 4,621).

No differences were found in the demographic and clin-ical characteristics between patients on long-term OACtreatment and non–anticoagulated patients (Table 1). Inaddition, the severity of aortic valve disease did not differsignificantly between the 2 groups (Table 2). Patients withlong-term OAC treatment had significantly higher aorticvalve and coronary calcium scores than did non–anticoagu-lated patients (Table 3). Fifteen patients were excluded fromthe assessment of the coronary calcification score becauseof coronary artery bypass grafts. Multiple linear regressionanalysis revealed that OAC treatment was the only indepen-dent predictor of the valvular calcium score (p !0.001), andmale gender (p $ 0.045), arterial hypertension (p $ 0.049),and OAC treatment (p $ 0.011) were independent predic-tors of the coronary calcium score.

The results of this observational study in patients with

Table 1Demographic and clinical characteristics of study population

Variable Oral Anticoagulants p Value

Yes(n $ 23)

No(n $ 63)

Age (yrs) 71.5 # 6.4 71.2 # 8.3 0.86Men 18 (78%) 35 (56%) 0.08Body mass index (kg/m2) 28.2 # 4.0 27.5 # 4.5 0.50C-reactive protein (mg/L) 15.8 # 13.1 15.2 # 19.0 0.89Calcium (mg/dl) 2.36 # 0.1 2.38 # 0.1 0.54Creatinine (mg/dl) 1.40 # 0.5 1.24 # 0.4 0.18Low-density lipoprotein (mg/dl) 118 # 28 143 # 65 0.07International normalized ratio 2.43 # 0.57 1.01 # 0.07 0.0001Hypertension 16 (70%) 52 (83%) 0.23Hypercholesterolemia 14 (61%) 43 (68%) 0.61Smoker 13 (57%) 23 (37%) 0.14Diabetes mellitus 5 (22%) 14 (22%) 1.00Obesity 15 (65%) 35 (56%) 0.47Coronary artery disease 15 (65%) 37 (59%) 0.63Myocardial infarction 6 (26%) 19 (30%) 0.79

Data are presented as means # SDs of numbers (percentages).

Table 2Echocardiographic characteristics of study population

Variable Oral Anticoagulants p Value

Yes(n $ 23)

No(n $ 63)

Aortic sclerosis 4 (17%) 13 (21%) 0.74Mild aortic stenosis* 5 (22%) 24 (38%) 0.16Moderate aortic stenosis† 11 (48%) 20 (32%) 0.17Severe aortic stenosis‡ 3 (13%) 6 (10%) 0.64Peak to peak transvalvular

gradient (mm Hg)45 # 20 39 # 24 0.28

Mean transvalvular gradient(mm Hg)

24 # 12 21 # 14 0.37

Peak aortic velocity (m/s) 3.14 # 0.72 2.93 # 0.85 0.31

Data are presented as numbers (percentages) or means # SDs.* Peak aortic velocity between 2 and 3 m/s.† Peak aortic velocity between 3.1 and 3.9 m/s.‡ Peak aortic velocity !4 m/s.

Table 3Valvular and coronary calcium scores assessed by multislice spiralcomputed tomography stratified by anticoagulation status

Variable Oral Anticoagulants pValue

Yes (n $ 23) No (n $ 63)

Valvular Agatstonscore

2,409.9 # 1,758.5 1,070.1 # 1,084.6 0.002

Coronary Agatstonscore

1,561.3 # 1,140.5 738.2 # 977.5 0.024

748 The American Journal of Cardiology (www.AJConline.org)

Koos R, et al. Am J Cardiol. 2005 Sep 15;96(6):747-‐9.

Reduced Bone Mineral Density Is Associated withBreast Arterial Calcification

Jhansi Reddy, John P. Bilezikian, Suzanne J. Smith, and Lori Mosca

Department of Obstetrics, Gynecology, and Reproductive Biology (J.R.), Brigham and Women’s Hospital, Boston, Massachusetts 02115;Departments of Medicine (J.P.B., L.M.), Pharmacology (J.P.B.), and Radiology (S.J.S.), Columbia University College of Physicians andSurgeons, New York, New York 10032

Background: Arterial calcification, a marker of atherosclerosis, results from a complex process ofbiomineralization resembling bone formation. Breast arterial calcification (BAC) has been associ-ated with angiographic and clinical cardiovascular disease. The purpose of this study was to de-termine the association between reduced bone mineral density (BMD) and BAC, which may sharea common pathophysiology.

Methods: We conducted a retrospective study of 228 women (55% Hispanic, mean age 64 !10 yr)who had both mammography and BMD evaluation at Columbia University Medical Center from2001–2003. Each mammogram was reviewed for the presence of BAC using standardized methods.BMD was measured using dual-energy x-ray absorptiometry and categorized as normal, low bonedensity (osteopenia), or osteoporosis as defined by the World Health Organization. Univariate andmultivariate logistic regression analyses were performed to evaluate the association betweenreduced BMD and BAC.

Results: The prevalence of BAC, low bone density (osteopenia), and osteoporosis was 39, 42, and29%, respectively. Women with BAC were significantly more likely to be older, Hispanic, andpostmenopausal and have osteoporosis as compared with women without BAC. In age-adjustedanalyses, women with BAC were more likely to have reduced BMD (odds ratio 3.0, P " 0.01) ascompared with women without BAC. Furthermore, osteoporosis was strongly associated with thepresence of BAC (odds ratio 3.5, P " 0.01).

Conclusion: These data suggest that osteoporosis and arterial calcification are strongly and inde-pendently correlated. Reduced BMD may identify women at risk of vascular disease. (J Clin Endo-crinol Metab 93: 208–211, 2008)

Cardiovascular disease (CVD) and osteoporosis are signifi-cant causes of morbidity and mortality among older

women and may coexist in many women (1). Recent research andseveral prospective studies have documented an association be-tween reduced bone mineral density (BMD) and clinical CVD,including acute stroke, angiographically documented coronaryartery disease, and increased cardiovascular mortality (2–5). Inaddition, reduced BMD has been independently associated withsurrogate markers of subclinical vascular disease including aor-tic artery calcification, coronary artery calcium score, and ca-rotid artery atherosclerosis. This suggests that results of BMD

testing may identify women at risk of future clinical cardiovas-cular events (6–9).

Breast arterial calcification (BAC) is the deposition of calciumwithin the media of arteries supplying breast tissue. Readily iden-tifiable on routine screening mammography, its presence hasbeen associated with numerous cardiovascular risk factors in-cluding diabetes and hypertension (10–12). In a recently pub-lished study of a large multiethnic cohort, women with BAC hadsignificantly increased risk of coronary heart disease, ischemicstroke, and heart failure over a median follow-up of 25 yr (11).Accumulating data suggest that the mammogram may be an

0021-972X/08/$15.00/0

Printed in U.S.A.

Copyright © 2008 by The Endocrine Society

doi: 10.1210/jc.2007-0693 Received March 27, 2007. Accepted October 22, 2007.

First Published Online October 30, 2007

Abbreviations: BAC, Breast arterial calcification; BMD, bone mineral density; CVD, cardio-vascular disease; DXA, dual-energy x-ray absorptiometry.

O R I G I N A L A R T I C L E

E n d o c r i n e C a r e — B r i e f R e p o r t

208 jcem.endojournals.org J Clin Endocrinol Metab. January 2008, 93(1):208–211

by on August 16, 2009 jcem.endojournals.orgDownloaded from

sclerosis measured on carotid artery ultrasound and reducedbone density.

Several studies have suggested that BAC is an independentpredictor of cardiovascular mortality (10–12). Kemmeren et al.(12) concluded that BAC represented a significant risk factor forcardiovascular mortality in women over 50 yr based on datagathered from a large breast cancer screening trial of 12,239women conducted in The Netherlands. In a 25-yr prospectivestudy of 12,761 ethnically diverse women, Iribarren et al. (11)reported that after adjustment for multiple potential confound-ers, BAC was associated with a 1.32-fold increased risk of cor-onary heart disease, a 1.41-fold increased risk of ischemic stroke,and 1.52-fold increased risk of heart failure. In our study, His-panic race/ethnicity was found to be a significant independentpredictor of BAC. Reddy et al. (14) were the first to report thatHispanic women had a significantly higher prevalence of BACcompared with whites of similar age. This finding is in line withthe high prevalence of cardiovascular risk factors among His-panics. If BAC is a marker for future CVD, then our data suggestreduced BMD may also be a marker, suggesting a sharedpathophysiology.

Basic research and animal studies have shown arterial calci-fication is the result of a biologically active and highly regulatedprocess of calcium deposition that resembles processes seen inthe metabolism of bone tissue itself (16–20). Molecular studiesindicate that factors and specific proteins involved in osteogen-esis arealso expressed in calcified atherosclerotic vascular lesions(19). For example, mice deficient in a !-carboxylated matrixprotein, osteocalcin, develop extensive vascular calcification re-sulting in premature death from CVD (19). In addition, osteo-protegerin, an inhibitor of bone resorption, may also play a crit-ical role. Mice deficient in osteoprotegerin develop severeosteoporosis and medial arterial calcification of the aorta (20).Whether the association between osteoporosis and arterial cal-cification is causal has yet to be determined.

There are limitations to our study. Using a cross-sectionaldesign, subjects were selected in a retrospective manner frompatients who underwent both routine screening mammographyand BMD evaluation from 2001–2003. At present, universalscreening for osteoporosis in women under the age of 65 yr is notrecommended. Thus, women are referred for BMD evaluationbased on the presence of certain risk factors. This may representa selection bias, potentially accounting for the higher prevalenceof low bone density (osteopenia) and osteoporosis in our study

cohort than the general population. Thus, our results may not begeneralized to the population at large. Demographic and clinicaldata were ascertained from medical records and by self-report ofa physician’s diagnosis of the condition. The study design pre-cluded the systematic determination of some clinical data, inparticular the diagnosis of diabetes mellitus and hypertension in23 study participants. Objective measurements of bone densityand BAC, however, were applied to all participants. Our studywas limited to white and Hispanic woman; therefore, the resultsmay not be generalized to other ethnicities. Nevertheless, thesignificance of the correlation may well help to identify womenwho may need further evaluation.

The paradigm of CVD as a categorical diagnosis has beenreplaced by the growing recognition that there is a continuum ofrisk. It is important to identify women in preclinical stages ofvascular disease so optimal preventive treatments can be offered.The possibility that routine screening mammography can also beused to identify women at increased risk for osteoporosis andCVD is appealing, but future research still needs to further ex-plore the relationship between vascular calcification and boneloss.

Our data, thus, add to a growing body of evidence that linksarterial calcification, a measure of CVD, to bone loss in olderwomen. Strategies for the early identification of women at riskfor CVD may also be considered for testing to slow the progres-sion of osteoporosis. In the future, effective secondary preventiveand treatment interventions may substantially reduce the risk ofthese two very common diseases in older women.

Acknowledgments

We are grateful to the staff of the Preventive Cardiology Program, Co-lumbia University, New York, NY, for their support and editorialassistance.

Address all correspondence and requests for reprints to: Dr. LoriMosca, Preventive Cardiology Program, New York Presbyterian Hos-pital, 622 W 168th Street, PH 10-203B, New York, New York 10032.E-mail: [email protected] (copy to: [email protected]).

This work was generously supported by the American Heart Asso-ciation Student Scholarship in Cardiovascular Disease and Stroke (toJ.R.), the Doris Duke Clinical Research Fellowship for medical students(to J.R.), and the National Heart Lung Blood Institute (to L.M.).

Disclosure Statement: The authors have nothing to disclose.

References

1. McFarlane SI, Ranganath M, Shin JJ, Bahtiyar G, Sowers JR 2004 Osteopo-rosis and cardiovascular disease, brittle bones and boned arteries, is there alink? Endocrine 23:1–10

2. Jørgensen L, Engstad T, Jacobsen B 2001 Bone mineral density in acute strokepatients, low bone mineral density may predict first stroke in women. Stroke32:47–51

3. von der Recke P, Hansen MA, Hassager C 1999 The association between lowbone mass at menopause and cardiovascular mortality. Am J Med 106:273–278

4. Kiel DP, Kauppila LI, Cupples LA, Hannan MT, O’Donnell CJ, Wilson PW2001 Bone loss and the progression of abdominal aortic calcification over a 25year period: the Framingham Heart Study. Calcif Tissue Int 68:271–276

TABLE 2. Multivariate adjusted odds ratios (OR) and 95%confidence intervals (CI) for predictors of BAC

Variable OR 95% CI

Age (10 yr) 1.5 1.0–2.2Hispanic 3.1 1.5–6.3Menopause 4.1 0.8–21Diabetes mellitusa 1.6 0.7–3.7Hypertensiona 0.9 0.5–1.7Bone density

Low bone density (osteopenia) 2.7 1.1–6.8Osteoporosis 4.4 1.6–12

a Total n ! 205 due to missing data on 23 patients.

210 Reddy et al. Bone Density and Arterial Calcification J Clin Endocrinol Metab, January 2008, 93(1):208–211

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Reddy J, et al. J Clin Endocrinol Metab 93: 208–211, 2008

Bolland MJ, et al. BMJ. 2008 Feb 2;336(7638):262-‐6

PORQUÊ?? Talvez Déficit Vit. K

DOSES DE POTÁSSIO FOOD AND NUTRITION BOARD

Fases da vida AI grs

Limite

0-6 meses 0.4 N.D.

7-12 meses 0.7 N.D.

1-3 anos 3.0 N.D

4-8 anos 3.8 N.D

9-13 anos 4.5 N.D

> 13 anos 4.7 N.D

Gravidez 4.7 N.D

Lactação 5.1 N.D

FONTES DE POTÁSSIO LPI, 2004

Alimentos Dose Qtd de Potássio mg

Banana 1 média 467 Batata cozida com casca 1 média 721

Sumo de ameixa 180 ml 530 Sumo de laranja 180 ml 354

Laranja 1 média 237

Sumo de tomate 180 ml 400

Tomate 1 médio 273

Passas ½ chávena 598 Alcachofra cozida 1 média 425

Espinafres cozidos ½ chávena 419

Amêndoas 30 g 211

CONCENTRAÇÕES DE ELECTRÓLITOS NO SORO E SUOR, E CONCENTRAÇÕES DE HC E ELECTRÓLITOS EM ALGUMAS

BEBIDAS COMUNS.

Plasma 140 4,5 2,5 1,5-‐2,1 110 300 -‐ Suor 60-‐80 4,5 1,5 3,3 40-‐90 170-‐220 -‐ Coca-‐Cola 3,0 -‐ -‐ -‐ 1,0 650 107 Gatorade 23,0 3,0 -‐ -‐ 14,0 280 62 Sumo Fruta 0,5 58,0 -‐ -‐ -‐ 690 118 Pepsi Cola 1,7 VesYgios -‐ -‐ Vesbgios 568 81 Àgua Vesbgios Vesbgios -‐ -‐ Vesbgios 10-‐20 -‐

Na+ K+ Ca2+ Mg2+ Cl-‐ Osmolalidade CHO (mEq.L-‐1) (mEq.L-‐1) (mEq.L-‐1) (mEq.L-‐1) (mEq.L-‐1) (mOsm.L-‐1 ) (g.L-‐1 )

McArdle W, Katch F, Katch V. Sports & Exercise Nutricon, 2nd Edicon. Lippincom Williams & Wilkins, 2005

PERDA DE FERRO:

ü  SUOR: 0.4 A 0.6 MG/HORA

ü HEMÓLISE: 0.25 a 0.75 mg

ü HEMORRAGIA GASTROINTESTINAL, ACIDOSE E PEROXIDAÇÃO DAS MEMBRANAS CELULARES POR RL: 0.5-1 MG/DIA


SHILS, M.E. et al. Modern Nutrition in Health and Disease. 10 ed. Lippincott Williams & Wilkins, 2005.

NECESSIDADES DE ATLETAS EM TREINO INTENSO:

ü HOMENS = 3,25 MG (PERDAS) + 1 MG (FUNÇÕES BÁSICAS) = 4,25MG/DIA

ü MULHERES = 3,25 MG (PERDAS) + 1 MG (FUNÇÕES BÁSICAS) + 0.5MG (MENSTRUAÇÃO) = 4,75MG/DIA

ABSORÇÃO: ENTRE 1 A 25%


SHILS, M.E. et al. Modern Nutrition in Health and Disease. 10 ed. Lippincott Williams & Wilkins, 2005.

144

FONTES DE FERRO

PERDA DE Zn:

ü SUOR: 3 A 12 MG/DIA

ü UTILIZAÇÃO PELA SOD


DEFICIÊNCIAS DE ZINCO EM ATLETAS

Modalidades % DDR Referências

Atletismo (Mulheres)

86 Deuster PA, et al. Am J Clin Nutr 1989;49:1295–301

Maratonistas (Mulheres)

73 Lukaski HC. Am J Clin Nutr. 2000 Aug;72(2 Suppl):585S-93S

Velocistas (Mulheres)

81 Lukaski HC. Am J Clin Nutr. 2000 Aug;72(2 Suppl):585S-93S

Triatlo (Mulheres)

88 Worme JD, et al. Am J Clin Nutr 1990;51:690–7

Triatlo (Homens) 91 Worme JD, et al. Am J Clin Nutr 1990;51:690–7

Adaptado de Lukaski HC. Am J Clin Nutr. 2000 Aug;72(2 Suppl):585S-‐93S

DOSES DE ZINCO FOOD AND NUTRITION BOARD

Homens RDA/AI* (mg) Limite (mg) Mulheres RDA/AI* (mg) Limite (mg)

0-‐6 meses 2* 4 0-‐6 meses 2* 4

7-‐12 meses 3 5 7-‐12 meses 3 5

1-‐3 anos 3 7 1-‐3 anos 3 7

4-‐8 anos 5 12 4-‐8 anos 5 12

9-‐13 anos 8 23 9-‐13 anos 8 23

14-‐18 anos 11 34 14-‐18 anos 9 34

> 18 anos 11 40 > 18 anos 8 40

Gravidez

Até 18 anos 12 34

19-‐50 anos 11 40

Lactação

Até 18 anos 13 34

19-‐50 12 40

FONTES DE ZINCO

Alimentos Dose Qtd de Zinco (mg)

Ostras 6 médias (cozidas) 43,4

Caranguejo 90 g (cozido) 4,6

Bife de vaca 90 g (cozido) 5,8

Carne de porco 90 g (cozido) 2,2

Galinha (coxa e asa) 90 g (cozido) 2,4

Perú (coxa e asa) 90 g (cozido) 3,5

Queijo 30 g 0,9 Leite 240 ml 1,0

Amêndoas 30 g 1,0

Amendoins 30 g 0,9

Feijão cozido ½ chávena 1,8

Grão de bico ½ chávena 1,3

FITATOS

DIMINUI A ABSORÇÃO DE:

ü  Ferro ü  Zinco ü  Cálcio ü  Magnésio

Cordain L. World Rev Nutr Diet 1999; 84:19-‐73. Bohn T, et al. Am J Clin Nutr. 2004 Mar;79(3):418-‐23.

A biodisponibilidade de minerais em amostras chinesas de vários tipos de

arroz é <4%.

DOSES DE COBRE FOOD AND NUTRITION BOARD

Fases da vida RDA/AI* (mcg) Limite (mcg) 0-‐6 meses 200* N.D.

7-‐12 meses 220* N.D.

1-‐3 anos 340 1,000

4-‐8 anos 440 3,000

9-‐13 anos 700 5,000

14-‐18 anos 890 8,000

> 18 anos 900 10,000

Gravidez

Até 18 anos 1000 8,000

> 18 anos 1000 10,000

Lactação

Até 18 anos 1300 8,000

> 18 anos 1300 10,000

FONTES DE COBRE

Alimentos 100 grs Qtd de Cobre mg

Carne 0,2-‐1,1 Lombo de porco 0,4

Frango 0,2

Mariscos 0,3-‐13,7 Amêijoa 0,3

Lacccínios 0,02-‐0,4 Leite inteiro 0,02

Leite desnatado 0,2

Leguminosas 0,14-‐1,2 Ervilhas 1,2

LenYlhas 0,1

Frutos secos 0,8-‐2,4 Amêndoas 1,4 Amendoins 0,8

Nozes 1,3

Alimentos 100 grs Qtd de Cobre mg

Cereais 0,02-‐0,4 Pão branco 0,02

Pão de centeio 0,1

Trigo inteiro 0,2

Verduras e hortaliças 0,04-‐0,3 Cenouras 0,3

Espargos 0,04

Couve galega 0,1

Frutas 0,07-‐0,14 Banana 0,1

Maçã 0,1

Gorduras 0,1-‐0,3 Azeite 0,3

Manteiga 0,4

Condimentos 0,3-‐2,4 Alho 0,3

Pimentão 0,6

DOSES DE SELÉNIO FOOD AND NUTRITION BOARD

Fases da vida RDA/AI* (mcg)

Limite (mcg)

0-‐6 meses 15* 45 7-‐12 meses 20* 60 1-‐3 anos 20 90 4-‐8 anos 30 150 9-‐13 anos 40 280

> 14 55 400 Gravidez 60 400 Lactação 70 400

FONTES DE SELÉNIO Alimentos Dose Qtd de Selenio (mcg)

Castanha do Pará 30 g (6 a 8 unidades) 839 Camarão pequeno

90 g (10 a 12 unidades)

34

Caranguejo 90 g 40 Salmão 90 g 40

Arroz integral cozido

1 chávena 19

Peito de galinha 90 g 20 Bife de porco 90 g 33 Bife de vaca 90 g 17 Pão de trigo integral

2 faYas 15

Leite 240 ml 5 Nozes 30 g 5

VITAMINA B1

DOSE DIÁRIA RECOMENDADA DE TIAMINA FOOD AND NUTRITION BOARD

Homens RDA/AI* (mg)

Limite (mg) Mulheres RDA/AI* (mg)

Limite (mg)

0-12 meses 0.2-0.3* Nd 0-12 meses 0.2-0.3* Nd

1-8 anos 0.5-0.6 Nd 1-8 anos 0.5-0.6 Nd

9-13 anos 0.9 Nd 9-13 anos 0.9 Nd

> 14 anos 1.2 nd 14-18 anos 1.0 Nd

< 18 anos 1.1 Nd

Gravidez 1.4 Nd

Lactação 1.4 Nd

Cereais Tiamina (mg/100g)

Gérmen de Trigo 2

Farelo de Trigo 0,72

Trigo Integral 0,4-0,5

Centeio Integral 0,43

Millet 0,73

Trigo Sarraceno 0,6

Aveia 0,6

Farelo de Arroz 2,3

Arroz Integral 0,5

Arroz polido 0,03

Arroz Selvagem 0,45 Leguminosas Tiamina (mg/100g)

Farinha de soja 0,85

Feijão Vermelho 0,84

Ervilha 0,74

Lentilhas 0,37

Grão 0,31

Rebentos de soja 0,23

Oleaginosas Tiamina (mg/100g)

Sem. Girassol 1,96

Pinhão 1,28

Amendoim 1,14

Castanha do Pará 0,96

Pistácio 0,67

Avelã 0,46

Caju 0,43

Noz 0,33

Amêndoa 0,24

Sem. de abóbora 0,24

Sem. Sésamo 0,18

Carne, Leite e Levedura

Tiamina (mg/100g)

Levedura de Cerveja 15,61

Bife de Porco 0,93

Coração de Borrego 0,45

Fígado de Borrego 0,4

Fígado de Vaca 0,25

Bife de Vaca 0,3

Bife de Borrego 0,2

Leite de Vaca 0,04

Frango 0,1

Liska D, Quinn S, Lukaczer D, et al. Clinical Nutrition: A Functional Approach. Second Edition. IFM; 2004. Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ. Modern Nutrition in Health and Disease. Lippincott Williams & Wilkins; 2005.

FONTES DE TIAMINA

FACTORES QUE AFECTAM ESTADO DE TIAMINA


Tiaminases (Degradam a

Tiamina)

Alimentos

Acção

Como eliminar

Tiaminase 1

Algumas plantas, Peixe Crú, marisco Crú,

Clostridium thiaminolyticus

Actuam durante o armazenamento dos alimentos ou

durante a passagem pelo

tracto gastrointestinal

Temperatura Tiaminase 2 Candida aneurinolytica

FACTORES QUE AFECTAM ESTADO DE TIAMINA


1.  Compostos anti-tiamina, que são termoestáveis: Tanino (chá)

2.  Sulfitos destroem a Tiamina

3.  Perda significativa na água da cozedura

4.  Temperatura destrói Tiamina (incluindo pasteurização do Leite)

5.  Exposição às luz solar (p.e. alimentos numa montra expostos à luz solar)

6.  Ingestão crónica e excessiva de Álcool (reduz absorção intestinal e transporte celular de Tiamina e a sua conversão em co-factor)

7.  Envenenamento por Arsénico (Bloqueia uso celular da Tiamina)

Tiamina é co-factor

DEFICIÊNCIA

Beriberi “Seco” (Neuropatia periférica) Beriberi “Molhado” (Neuropatia Periférica, Edema,

Taquicardia, Cardiomegalia, Insuficiência Cardíaca)

Beriberi Infantil (Vómitios, Taquicardia, Convulsões e Morte) Estomatite Angular (Irritação e fissuras nos cantos dos

lábios)

Encefalopatia Alcoólica

Lesões neurológicas


VITAMINA B6

Homens RDA/AI* (mg)

Limite (mg) Mulheres RDA/AI* (mg)

Limite (mg)

0-12 meses

0.1-0.3* Nd 0-12 meses 0.1-0.3* Nd

1-8 anos 0.5-0.6 30-40 1-8 anos 0.5-0.6 30-40

9-13 anos 1.0 60 9-13 anos 1.0 60

14-50 anos

1.3 80-100 14-18 anos 1.2 80

> 50 anos 1.7 100 19-50 anos 1.3 100

> 50 anos 1.5 100

Gravidez 1.9 80-100

Lactação 2.0 80-100

DOSE DIÁRIA RECOMENDADA DE VITAMINA B6 FOOD AND NUTRITION BOARD

FONTES DE VITAMINA B6

BIODISPONIBILIDADE DA B6 EM CEREAIS É BAIXA

ü  Pode chegar a ser de 25-20% ü Nos alimentos de origem animal: biodisponibilidade de quase

100% ü Baixo status de vitamina B6 em vegetarianos

Cordain L. World Rev Nutr Diet 1999; 84:19-‐73.

PLP

SÍNTESE DE HEME

PLP

SÍNTESE DE CARNITINA

PLP

GLICOGENÓLISE

Giardina G et al. PNAS 2011;108:20514-20519

©2011 by National Academy of Sciences

DOPAMINA, SEROTONINA, GABA E HISTAMINA

SINTOMAS DE DEFICIÊNCIA

Dermatite

Irritabilidade

Convulsões

Anemia microcitica (Diminuição da Hemoglobina)

Diminuição da Proliferação Linfocitária

Hiperhomocisteinemia

Neuropatia Periférica


EXCESSO

Neuropatias (> 500 mg, mas casos isolados com dosagens > 100 mg)

Inibição da Lactação na Mulher (> 150 mg)


HOMOCISTEÍNA

FOLATO

Homens RDA/AI* (mcg)

Limite (mcg) Mulheres RDA/AI* (mcg)

Limite (mcg)

0-12 meses

65-80* Nd 0-12 meses 65-80* Nd

1-8 anos 150-200 300 1-8 anos 150-200 300

9-18 anos 300-400 600-800 9-13 anos 300-400 600-800

Adultos 400 1000 Adultos 400 1000

Gravidez (<18 anos) 600 800

Gravidez ( >18 anos) 600 1000

Lactação (> 18 anos) 500 800

Lactação (> 18 anos) 500 1000

DOSE DIÁRIA RECOMENDADA DE FOLATO FOOD AND NUTRITION BOARD

184

FONTES DIETÉTICAS DE FOLATO

Miller AL, Kelley GS. Altern Med Rev. 1996;1(4):220-‐235

FOLATO

536 Pietrziket al.

The importance of periconceptual folie acid supplementa-tion in reducing the risk of neural tube defects and other con-genital malformations is generally accepted. Furthermore,evidence is accumulating to support a possible role of folie acidin the reduction in risk of other diseases, including dementiaand certain types of cancer, making folie acid the subject ofintense discussion by the medical community and by the public.

Despite the important functions of the vitamin folie acid, itschemical nature has seldom been the focus of scientific dis-cussion. Folie acid is an oxidized synthetic form of the vitamin,which does not exist in nature except in fortified foods, sup-plements and Pharmaceuticals. Folie acid itself is not active as acoenzyme, and it undergoes several metabolic steps to be acti-vated. The advantage of folie acid over natural folates has beenits greater stability and lower cost, which make it particularlysuitable for use in vitamin supplements, Pharmaceuticals andfortified foods with a sufficient shelf-life. In addition, until re-cently, the natural forms of folate (reduced derivatives) haveonly been available as mixed diastereoisomers, of which onlyhalf possess biological activity.

The predominant active form of folate in the body is L-5-methyltetrahydrofolate (L-5-methyl-THF), which accounts forapproximately 98% of folates in human plasma. L-5-methyl-THF is also the predominant active metabolite after intake offolie acid. Because of its limited stability, pharmaceutical use ofL-5-methyl-THF was not feasible until its stable calcium saltwas developed (Metafolin®; Merck Eprova AG, Schaffhausen,Switzerland), Since then, folie acid has been replaced by thecalcium salt of L-5-methyl-THF in several vitamin supplements

and Pharmaceuticals to enable direct use of the predominantnatural form of the vitamin.

The objective of this review is to provide evidence that L-5-methyl-THF is as effective as folie acid in improving folatestatus. Differences in the metabolic pathways and the modeof action are discussed, with particular emphasis on geneticpolymorphisms and the potential to mask vitamin B,; defi-ciency symptoms. In this review, L-5-methyl-THF refers tothe biologically active diastereoisomer, also known as 6S-5-methyl-THF,

1. Clinical Pharmacokinetics

1.1 Absorption

Folie acid (pteroylmonoglutamate) is absorbed as such,whereas food folates (polyglutamate derivatives) are hydro-lysed to monoglutamates in the gut by a brush border hydrolaseprior to absorption. Both forms are absorbed in the proximalsmall intestine via the proton coupled folate transporter(PCFT), a saturable transporter that transports oxidized andreduced folates with similar efficiency.''' In addition to sharinga common transport mechanism, most dietary folates and folieacid that is added to the diet share a common metabolic fate, asthey are metabolized to L-5-methyl-THF during their passageacross the intestinal mucosa (figure 1). Folie acid is reducedto dihydrofolate (DHF) and then to tetrahydrofolate (THF)by DHF reductase (DHFR) in the mucosal cell, and it is

Folie acid

Polyglutamates

Food folates

Monoglutamates

L-5-methyl-THF

Folie acid>200 ng

DHF

THF

*\ 5,10-methyiene-THF

*\ L-5-methyl-THF

Folk: acid

L-5-methyl-THF

Fig. 1. Intestinal folate absorption (from ttie brush border membrane ttirough the mucosal cell via the portal vein into the peripheral circulation)DHF = dihydrofolate; THF=tetrahydrofolate. , _

© 2010 Adis Data information BV. All rights reserved. Clin Piiarmacoidnet 2010; 49 (B)

5-‐MTHFR

Pietrzik K, Bailey L, Shane B. Clin Pharmacokinet. 2010 Aug;49(8):535-‐48.

ÁCIDO FÓLICO ≠ FOLATO

Meta-analysis of cancer risk in folic acid supplementation trials

Joseph E. Baggott a, Robert A. Oster b, Tsunenobu Tamura a,*a Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USAb Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA

1. Introduction

Several research groups have conducted large prospectiveclinical trials to evaluate the effect of the supplementation of B-vitamins including folic acid (pteroylglutamic acid) to evaluate itseffect on the risk of occlusive vascular disease and neoplasm inboth men and women [1–6]. Some of these were performedbecause a higher occlusive vascular disease incidence wasassociated with higher homocysteine concentration in thecirculation, which can be lowered by supplementation of folicacid and vitamins B-12 and B-6. Based on cancer incidences inthese trials, researchers commented on the possibility that folicacid supplementation increases the incidence of various cancers[7–9]. It is possible that increased folate intake would reducecancer incidence, because of the role of folate coenzymes in purinebiosynthesis de novo and thymidylate synthesis. Therefore,increased pools of purine and thymidine nucleotides could beutilized to repair DNA damage before initiation and earlypromotion of cancer. However, folic acid supplementation mayincrease cancer incidence. The increased supply of nucleotides,required for replication of mutated cells, could stimulate formationof occult foci of cancer [7]. We performed a meta-analysis toevaluate the risk of cancer secondary to folic acid supplementation,and discuss studies suggesting a procarcinogenic effect of folicacid.

2. Methods

A weighted meta-analysis of six trials that involved folic acidsupplementation in men and women was performed. Weidentified a total of 1124 articles by a PubMed search using keywords, ‘‘folic acid,’’ limiting only in ‘‘randomized-controlled trial,’’‘‘humans,’’ and ‘‘English’’ (access date: June 11, 2009). Of thesearticles, 370 involved the use of folate supplements withanticancer drugs and were eliminated, leaving 754. Of these,343 were cross-sectional or metabolic studies, case-controlledinvestigations, methodological articles, reviews and letters to theeditor, leaving 411. Of these, 303 studies lasted for less thanone year (63; 68; 70; 73; and 29 trials lasted for <1; 2–3; 3–6; and6–12 months, respectively). The durations were 12 months ormore for the remaining108 prospective trials involving folatesupplements without the concomitant use of anticancer drugs. Ofthese, 43 trials involved multi-nutrient only (vitamins B-12 and B-6, iron, etc.) supplementation including folate without anappropriate placebo, leaving 65. Of these, 59 included no dataon cancer morbidity or no placebo control for a valid randomizedtrial. Finally, only six of these reports were identified asprospective trials of folic acid supplementation (including othernutrients) that had an appropriate placebo control group andincluded cancer morbidity of the subjects [1–5,10]. The subjects infive of these six trials were both female and male adults. In five ofsix trials, the mean body-mass index (BMI) was < 30 kg/m2 (range26.3–27.5), although subjects in one study involved females onlywith a mean BMI of 30.6 kg/m2 [10]; therefore, the prostate cancerincidence was not possible, although it was significantly increased

Cancer Epidemiology xxx (2011) xxx–xxx

* Corresponding author. Tel.: +1 205 934 7478; fax: +1 205 934 7049.E-mail address: [email protected] (T. Tamura).

A R T I C L E I N F O

Article history:Received 5 November 2010Received in revised form 29 April 2011Accepted 6 May 2011Available online xxx

Keywords:Folic acidCancer riskSupplementation trialsMeta-analysis

A B S T R A C T

Several reports suggest that folate has a procarcinogenic effect. Folate has a unique role because itscoenzymes are needed for de novo purine and thymine nucleotide biosynthesis. Antifolates, such asmethotrexate, are used in cancer treatment. Using a meta-analysis weighted for the duration of folic acid(pteroylglutamic acid) supplementation, we analyzed the cancer incidence of six previously publishedlarge prospective folic acid-supplementation trials in men and women. These articles were carefullyselected from over 1100 identified using PubMed search. Our analyses suggest that cancer incidenceswere higher in the folic acid-supplemented groups than the non-folic acid-supplemented groups(relative risk = 1.21 [95% confidence interval: 1.05–1.39]). Folic acid-supplementation trials should beperformed with careful monitoring of cancer incidence. Solid monitoring systems to detect side effects,including increase in cancer risk, should be established before the initiation of folic acid supplementationtrials.


G Model

CANEP-315; No. of Pages 4

Please cite this article in press as: Baggott JE, et al. Meta-analysis of cancer risk in folic acid supplementation trials. Cancer Epidemiology(2011), doi:10.1016/j.canep.2011.05.003


Cancer EpidemiologyThe International Journal of Cancer Epidemiology, Detection, and Prevention

jou r nal h o mep age: w ww.c an cer ep idem io log y.n et

1877-7821/$ – see front matter ! 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.canep.2011.05.003

Meta-analysis of cancer risk in folic acid supplementation trials

Joseph E. Baggott a, Robert A. Oster b, Tsunenobu Tamura a,*a Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USAb Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA

1. Introduction

Several research groups have conducted large prospectiveclinical trials to evaluate the effect of the supplementation of B-vitamins including folic acid (pteroylglutamic acid) to evaluate itseffect on the risk of occlusive vascular disease and neoplasm inboth men and women [1–6]. Some of these were performedbecause a higher occlusive vascular disease incidence wasassociated with higher homocysteine concentration in thecirculation, which can be lowered by supplementation of folicacid and vitamins B-12 and B-6. Based on cancer incidences inthese trials, researchers commented on the possibility that folicacid supplementation increases the incidence of various cancers[7–9]. It is possible that increased folate intake would reducecancer incidence, because of the role of folate coenzymes in purinebiosynthesis de novo and thymidylate synthesis. Therefore,increased pools of purine and thymidine nucleotides could beutilized to repair DNA damage before initiation and earlypromotion of cancer. However, folic acid supplementation mayincrease cancer incidence. The increased supply of nucleotides,required for replication of mutated cells, could stimulate formationof occult foci of cancer [7]. We performed a meta-analysis toevaluate the risk of cancer secondary to folic acid supplementation,and discuss studies suggesting a procarcinogenic effect of folicacid.

2. Methods

A weighted meta-analysis of six trials that involved folic acidsupplementation in men and women was performed. Weidentified a total of 1124 articles by a PubMed search using keywords, ‘‘folic acid,’’ limiting only in ‘‘randomized-controlled trial,’’‘‘humans,’’ and ‘‘English’’ (access date: June 11, 2009). Of thesearticles, 370 involved the use of folate supplements withanticancer drugs and were eliminated, leaving 754. Of these,343 were cross-sectional or metabolic studies, case-controlledinvestigations, methodological articles, reviews and letters to theeditor, leaving 411. Of these, 303 studies lasted for less thanone year (63; 68; 70; 73; and 29 trials lasted for <1; 2–3; 3–6; and6–12 months, respectively). The durations were 12 months ormore for the remaining108 prospective trials involving folatesupplements without the concomitant use of anticancer drugs. Ofthese, 43 trials involved multi-nutrient only (vitamins B-12 and B-6, iron, etc.) supplementation including folate without anappropriate placebo, leaving 65. Of these, 59 included no dataon cancer morbidity or no placebo control for a valid randomizedtrial. Finally, only six of these reports were identified asprospective trials of folic acid supplementation (including othernutrients) that had an appropriate placebo control group andincluded cancer morbidity of the subjects [1–5,10]. The subjects infive of these six trials were both female and male adults. In five ofsix trials, the mean body-mass index (BMI) was < 30 kg/m2 (range26.3–27.5), although subjects in one study involved females onlywith a mean BMI of 30.6 kg/m2 [10]; therefore, the prostate cancerincidence was not possible, although it was significantly increased

Cancer Epidemiology xxx (2011) xxx–xxx

* Corresponding author. Tel.: +1 205 934 7478; fax: +1 205 934 7049.E-mail address: [email protected] (T. Tamura).

A R T I C L E I N F O

Article history:Received 5 November 2010Received in revised form 29 April 2011Accepted 6 May 2011Available online xxx

Keywords:Folic acidCancer riskSupplementation trialsMeta-analysis

A B S T R A C T

Several reports suggest that folate has a procarcinogenic effect. Folate has a unique role because itscoenzymes are needed for de novo purine and thymine nucleotide biosynthesis. Antifolates, such asmethotrexate, are used in cancer treatment. Using a meta-analysis weighted for the duration of folic acid(pteroylglutamic acid) supplementation, we analyzed the cancer incidence of six previously publishedlarge prospective folic acid-supplementation trials in men and women. These articles were carefullyselected from over 1100 identified using PubMed search. Our analyses suggest that cancer incidenceswere higher in the folic acid-supplemented groups than the non-folic acid-supplemented groups(relative risk = 1.21 [95% confidence interval: 1.05–1.39]). Folic acid-supplementation trials should beperformed with careful monitoring of cancer incidence. Solid monitoring systems to detect side effects,including increase in cancer risk, should be established before the initiation of folic acid supplementationtrials.


G Model




Cancer EpidemiologyThe International Journal of Cancer Epidemiology, Detection, and Prevention

jou r nal h o mep age: w ww.c an cer ep idem io log y.n et

1877-7821/$ – see front matter ! 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.canep.2011.05.003

by folic acid supplementation in two trials [3,11]. Furthermore, inthis trial [10], the median plasma folate concentrations in the folicacid supplemented and placebo groups were 88 and 35 nmol/l,respectively, which were higher than corresponding means in theother five trials (range: 43–78 and 8–30 nmol/l, respectively) [1–5]. Therefore, this trial of only females, who were mostly obesewith folate concentrations above those previously considered tobe normal, was excluded from our analysis. Since the initialPubMed search, we searched on a nearly daily basis using the keyword ‘‘folate’’ to update our search. In June of 2010; we added onenewly published trial [6]. We did not use unpublished or non-peerreviewed data; because it was impossible for us to verify theaccuracy of the data [12]. Finally, we performed a meta-analysisusing the data presented in a total of six trials.

A weighted meta-analysis of the trial-specific effects wasperformed. The following information from the trials was includedin this analysis: the number of study subjects per supplement

group, a description of the daily supplements (which containedfolic acid or a placebo), the number of cancer cases per supplementgroup, and the duration of the study in years. The study durationserved as the weighting factor. The incidence of all skin cancer wasexcluded, where possible, from the analysis either by theinvestigators [2,4,6] or by us [1] as shown in Table 1, althoughtypes of skin cancer were unknown. All results are expressed aspercentage increase in risk of cancer, 95% confidence intervals(95% CIs) associated with the cancer risk (assuming a null risk of1), and the weights as a percentage of the overall total. The overallrisk ratio was compared to a null risk ratio of 1.0 using the Mantel–Haenszel test. Statistical analyses were performed using Stata(version 11.1; StataCorp LP, College Station, TX). Incidences werenot adjusted for dropouts or deaths, and these were assumed tobe the same in folic acid supplemented and non-folic acidsupplemented groups.

3. Results

Investigators used daily supplementation of 0.5–2.5 mg of folicacid with or without vitamins B-12 and B-6 and aspirin (Table 1).Only one of these six trials showed a significant increase in cancerincidences (P = 0.02) [3], although all trials had risk ratios over 1.0.For each study, a relative risk is shown in Fig. 1. A weighted meta-analysis with the overall risk ratio of 1.21, and its corresponding95% CI of 1.05–1.39 are shown in Fig. 1.

4. Discussion

A risk ratio of 1.21 (95% CI, 1.03–1.41) was recently reported byEbbing et al. [13] for a combination of two large trials that lastedfor six years. This is similar to our risk ratio of 1.21 (95% CI, 1.05–1.39). In contrast, our findings differ from those of Clarke et al. [12]who did not include the trials by Cole et al. [3] and Logan et al. [5];however, we find no substantial reason for elimination of thesetrials. We realize that subjects in these trials had a history ofcolorectal adenoma [3,5]. However, eliminating the enrolment ofsubjects with any risk factors (e.g. family history, smoking and etc.)would make trials nearly impossible, because such a selectionwould lead to a small sample size and impair meaningful datainterpretation. Furthermore, Clarke et al. [12] did not weight forduration of the trials, and reported that the risk ratio was 1.05 (95%CI, 0.98–1.13). Since the progression of cancer may be slow, thelonger the trial, the more likely that occult foci of cancer will growto a clinically detectable mass.

Fig. 1. Weighted meta-analysis of cancer risk during folic acid supplementationtrials. The solid diamonds are risk ratios with 95% confidence intervals (95% CI)displayed by the horizontal lines. The vertical-broken line is for the overall cancerrisk ratio of 1.21, and the open diamond represents the 95% CI (1.05–1.39,P = 0.008). Percentage weights of each trial were 17.7, 11.7, 24.8, 11.4, 10.6 and 23.8,and relative risk ratios (95% CI) were 1.08 (0.94–1.24), 1.22 (0.88–1.68), 1.55 (1.04–2.31), 1.24 (0.91–1.69), 1.08 (0.51–2.26) and 1.06 (0.96–1.18) for the HOPE [1],Bønaa et al. [2], Cole et al. [3], Ebbing et al. [4] and Logan et al. [5], and SEARCH [6],respectively.

Table 1Folic acid supplementation trials and cancer risk.

Trials No of subjects (% male)a Duration (years) Daily supplement (mg/day) Cancer cases

HOPE [1] 2758 (71.1) 5 Folic acid (2.5) + vitamin B-6 (50) + vitamin B-12 (1.0) 353b

2764 (72.4) 5 Placebo 328b

Bønaa et al. [2] 1872 (73.7) 3.3 Folic acid (0.8) + vitamin B-6 (40 or 0) + vitamin B-12 (0.4) 79c

1877 (74.1) 3.3 Vitamin B-6 (40) or placebo 65c

Cole et al. [3] 516 (64.0) 7d Folic acid (1.0) 57505 (63.6) 7d placebo 36

Ebbing et al. [4] 1540 (80.8) 3.2 Folic acid (0.8) + vitamin B-6 (40 or 0) + vitamin B-12 (0.4) 85c

1550 (78.3) 3.2 vitamin B-6 (40) or placebo 69c

Logan et al. [5] 470 (56.2) 3 Folic acid (0.5) ! aspirin (300) 14469 (57.6) 3 Placebo ! aspirin (300) 13

SEARCH [6] 6033 (83.0) 6.7 Folic acid (2) ! vitamin B-12 (1) 678c

6031 (83.0) 6.7 Placebo 639c

a These are estimates and are not corrected for dropouts or deaths.b We eliminated melanoma.c The authors excluded basal-cell carcinoma.d The duration was 6–8 years; seven years represents the average duration.

J.E. Baggott et al. / Cancer Epidemiology xxx (2011) xxx–xxx2

G Model



ingestão ≅ 254mcg/día ≅ 254mcg/día ≅ 254mcg/día

Ashfield-‐Wam PAL et al. Am J Clin Nutr 2002;76:180-‐6.

GENOTIPO MTHFR TT

1654 Arnfinn Hykkerud Steindal et a/.

80 A*, = 360 nm A,, 280 nm

Y W + s s40

p

!i

C

a,

4 20 E

0 . 250 300 350 400 450 500

Wavelength [nm]

Figure 6. Fluorescence excitation and fluorescence emission spectra of 5 pM 5MTHF exposed to UVB irradiation for 0,30,60,90, 120 and 150 min. A,, is 360 nm for the excitation spectra and he, is 280 nm for the emission spectra; pH 7.4.

Figure 6 shows the evolution of the fluorescence excitation and emission spectra after 30,60, 120 and 150 min of UVB exposure. When solutions of SMTHF are exposed to UVB radiation, the fluorescence of the solutions increases. This means that at least one of the photoproducts of SMTHF fluoresces stronger than SMTHF itself. The peak at 284 nm is blueshifted to 277 nm.

The fluorescence yield for PGA is around 20 times higher than that of SMTHF (cf. Fig. 5 ) . A two-step process may explain the observed kinetics: SMTHF is first oxidized to SMDHF and then the bond between atom 9 and 10 is broken.

Reaction kinetics

Figure 7 shows the fluorescence emission intensity of irradiated SMTHF (5 pM) at 360 nm as a function of exposure time. The shape of the graph is sigmoidal.

When FA was UV exposed it was found that the degradation rate was concentration dependent (7). The photodegradation rate of SMTHF is not concentration depended and, therefore, follows first order kinetics. The rate constant was 9.2 X lop3 m i d . This rate

- ,

0 50 100 150 200

Exposure time [min]

0.12

0.10 s 0.08 i

4 0.06

0.04

1

Figure 7. The fluorescence emission intensity at 360 nm (hex = 280 nm) and the absorption at 290 and 250 nm as a function of exposure time for 5 pM SMTHF, pH 7.4. Data are from three different experiments with mean and standard deviation given. The line for fluorescence intensity at 360 nm as a function of exposure time is sigmoidal (P < 0.0001 far all parameters) and the fitting was made with Sigmaplot 2001 version 7.101 (SPSS Inc., Chicago).

I lJV 1 poaH

Unknown molecule + H a

Figure 8. A possible scheme of the photodegradation of SMTHF.

constant is five times larger than that of an unirradiated solution (1.8 x min-I).

In Fig. 8 a scheme of SMTHF photodegradation is proposed. The unknown molecule is probably a reduced form of a pterin. 5MTHF is first oxidized to SMDHF, and then the molecule is cleaved into PGA and an unknown pterin.

5h4THF absorbs UV radiation, mostly in the UVB region. This means that the UV penetrating the epidermis and reaching the human blood, mainly UVA (17), probably does not degrade SMTHF significantly by direct absorption. It is more likely that SMTHF in the human body is oxidized by radical oxygen species produced by naturally photosensitizers (flavins, porphyrins, bilirubin, etc.) after UVA and near-UV exposure, SMTHF is a strong antioxidant, comparable to ascorbic acid (l8,19). The oxidized form of SMTHF, SMDHF, is probably not reentering the folate pool (20), and therefore the first step of folate degradation is sufficient to reduce the folate concentrations in humans. Further studies have to be performed to be able to conclude whether folates are degraded in humans and the implications for human health.

Acknowledgements-A.H.S. was supported by the Research Foundation of the Norwegian Radium Hospital and A. Juzeniene was supported by the Norwegian Cancer Society.

REFERENCES 1. Lucock, M. (2000) Folk acid nutritional biochemistry, molecular

biology, and role in disease processes. Mol. Genet. Metah. 71, 121-138.

2. MRC Vitamin Study Research Group (1991) Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet

3. Rimm, E. B., W. C. Willett, F. B. Hu, L. Sampson, G. A. Colditz, J. E. Manson, C. Hennekens and M. J. Stampfer (1998) Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA 279, 359-364.

4. McCullough, M. L. and E. L. Giovannucci (2004) Diet and cancer prevention. Oncogene 23, 6349-6364.

338, 131-137.

Photochemistry and Photobiology, 2006, 82: 1651-1 655

Photodegradation of 5-methyltetrahydrofolate: Biophysical Aspects Arnfinn Hykkerud Steindal*' ?', Asta Juzeniene', Anders Johnsson' and Johan Moan'13 'Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

2Department of Physics, Norwegian University of Science and Technology - NTNU, N-7491 Trondheim, Norway 3Department of Physics, University of Oslo, N-0316 Oslo, Norway

Received 09 June 2006; accepted 24 July 2006; published online 31 July 2006 DOI: 10.1562/2006-06-09-RA-915

ABSTRACT 5-methyltetrahydrofolate (SMTHF) absorbs UV radiation and has an absorption coefficient of 24250 f 1170 M-' cm-' at 290 nm. It has a weak fluorescence emission in the wavelength region around 360 nm. Our data demonstrated induction of 5methyldihydrofolate by exposure to UVB and, after con- tinues irradiation, p-aminobenzoyl-L-glutamic acid was found. The photodegradation of SMTHF follows a first order kinetic with a degradation rate constant of 9.2 X min-' under our conditions (fluence rate of 2.15 mW crn-', exposure wavelengths from 280 to 350 nm). Our results indicate that a direct degradation of SMTHF by UV exposure in humans in vivo is rather unlikely. 5MTHF mainly absorbs, and is degraded by, UVB and UVC, radiation that does not penetrate the earth's atmosphere and the human skin well.

INTRODUCTION Folate is an important vitamin for human health (1). Folate deficiency, or impairment of the folate metabolism in an organism, leads to several diseases, including megaloblastic anemia (1) and complications arising in pregnancy, such as neural tube defects (2). Folate deficiency may also increase the risk of developing cardiovascular diseases (3) and cancer (4).

It has been suggested that exposure to large doses of solar radiation may lead to folate deficiency, and that sun-induced folate degradation may play a key role in evolution of human skin color. This hypothesis was first proposed by Branda and Eaton (5) and further developed by Jablonski and Chaplin (6). No definite conclusion about the possibility of folate photodegradation in vivo has been drawn yet. As a first step in the elucidation of this problem, investigations of the photophysics and photochemistry of folate in simple model systems would be of great value.

The photodegradation of folic acid (FA), a synthetic form of the folate, has been thoroughly studied (7,s). 5-methyltetrahydrofolate (SMTHF) belongs to the naturally occurring folates. In the present work we have investigated the photodegradation kinetic of SMTHF by absorption and fluoresence measurements. The chemical structure of SMTHF is shown in Fig. 1. It consists of three groups: a pteridine residue, p-aminobenzolate and glutamk acid.

*Corresponding author e-mail: [email protected]

0 2006 American Society for Photobiology 0031-8655/06 (Arnfinn Hykkerud Steindal)

MATERIALS AND METHODS Chemicals. (6R,S)-5-methyl-5,6,7,8-tetrahydrofolate calcium salt and (6R,S)- 5-methyl-5,6-dihydrofolate ammonium salt was purchased from Schircks Laboratories (JoM, Switzerland), while p-aminobenzoyl-L-glutamic acid was purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco's phosphate buffered saline (PBS) was purchased from PAA Laboratories GmbH (Paschmg, Austria). The solutions were freshly made in PBS before each experiment. Because of the instability of the compounds, the solutions were always kept on ice before use.

UVIVis absorption and fluorescence measurements. Absorption spectm were registered with a Perkin-Elmer Lambda 40 UVPis spectrometer (Shelton, CT). Fluorescence emission and excitation spectra were recorded by means of a Perkin-Elmer LS5OB luminescence spectrometer equipped with a Hamamatsu R-928 photomultiplier (Iwata, Japan). All measurements were performed in standard 10 mm quartz cuvettes.

Photoolysis. Solutions of 5MTHF were exposed to UV radiation in closed quartz cuvettes (1 mL). The radiation source was a broad-band UVB lamp with five Philips TL 2OW/12 RS UVB tubes (Amsterdam). The fluence rate of the lamp was measured after each experiment with an UVB detector (PMA2106) connected to a photometer (PMA2200), both from Solar Light Co. (Philadelphia, PA). The intensity at the sample position was 2.15 mW an-*, and the radiation was emitted mainly in the wavelength region 280-350 nm with a maximum at 3 12 nm. The temperatures of the samples were approximately 25 to 30°C. All experiments were performed in constant dim light in order to avoid external, uncontrolled light exposure.

One point that must be stressed is the fact that our UV source contains radiation that is not present in the solar radiation (9). In the starting phase of our project we used a narrow-band UVB lamp (Phillips TL 20W/01 RS, 312 nm peak). The results were the same as for the broad-band UVB lamp, but the process was slower because 5MTHF absorbs less at 312 nm than at shorter wavelengths. SMTHF is rather unstable in vibo, so we decided to use the broad-band UVB lamp.

RESULTS AND DISCUSSION Absorption measurements

The absorption spectra of different concentrations of 5MTHF are shown in Fig. 2. SMTHF absorbs radiation in the UV region and has an absorption peak at 290 nm. At wavelengths longer than 340 nm, the absorption is weak. The insert shows a linear plot of the absorbance at 290 nm as a function of concentration. The linear relationship between absorbance and concentration indicates that the absorbance of SMTHF follows the Beer-Lambert law, thus aggregation plays no major role.

The absorption coefficient, E , for SMTHF was found to be 24 250 % 1170 M-' cm-' at 290 nm. This is lower than 30 800 M-' cm-I, found by Donaldson and Keresztesy (10) and 29000 M-' cm-' found by h a b e e et ul. (1 I), and the reason for this can be that SMTHF is a hygroscopic substance. The powder of 5MTHF may absorb water from the air and increase its weight. This may lead to

1651

Photochemistry and Photobiology, 2006, 82: 1651-1 655

Photodegradation of 5-methyltetrahydrofolate: Biophysical Aspects Arnfinn Hykkerud Steindal*' ?', Asta Juzeniene', Anders Johnsson' and Johan Moan'13 'Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

2Department of Physics, Norwegian University of Science and Technology - NTNU, N-7491 Trondheim, Norway 3Department of Physics, University of Oslo, N-0316 Oslo, Norway

Received 09 June 2006; accepted 24 July 2006; published online 31 July 2006 DOI: 10.1562/2006-06-09-RA-915

ABSTRACT 5-methyltetrahydrofolate (SMTHF) absorbs UV radiation and has an absorption coefficient of 24250 f 1170 M-' cm-' at 290 nm. It has a weak fluorescence emission in the wavelength region around 360 nm. Our data demonstrated induction of 5methyldihydrofolate by exposure to UVB and, after con- tinues irradiation, p-aminobenzoyl-L-glutamic acid was found. The photodegradation of SMTHF follows a first order kinetic with a degradation rate constant of 9.2 X min-' under our conditions (fluence rate of 2.15 mW crn-', exposure wavelengths from 280 to 350 nm). Our results indicate that a direct degradation of SMTHF by UV exposure in humans in vivo is rather unlikely. 5MTHF mainly absorbs, and is degraded by, UVB and UVC, radiation that does not penetrate the earth's atmosphere and the human skin well.

INTRODUCTION Folate is an important vitamin for human health (1). Folate deficiency, or impairment of the folate metabolism in an organism, leads to several diseases, including megaloblastic anemia (1) and complications arising in pregnancy, such as neural tube defects (2). Folate deficiency may also increase the risk of developing cardiovascular diseases (3) and cancer (4).

It has been suggested that exposure to large doses of solar radiation may lead to folate deficiency, and that sun-induced folate degradation may play a key role in evolution of human skin color. This hypothesis was first proposed by Branda and Eaton (5) and further developed by Jablonski and Chaplin (6). No definite conclusion about the possibility of folate photodegradation in vivo has been drawn yet. As a first step in the elucidation of this problem, investigations of the photophysics and photochemistry of folate in simple model systems would be of great value.

The photodegradation of folic acid (FA), a synthetic form of the folate, has been thoroughly studied (7,s). 5-methyltetrahydrofolate (SMTHF) belongs to the naturally occurring folates. In the present work we have investigated the photodegradation kinetic of SMTHF by absorption and fluoresence measurements. The chemical structure of SMTHF is shown in Fig. 1. It consists of three groups: a pteridine residue, p-aminobenzolate and glutamk acid.

*Corresponding author e-mail: [email protected]

0 2006 American Society for Photobiology 0031-8655/06 (Arnfinn Hykkerud Steindal)

MATERIALS AND METHODS Chemicals. (6R,S)-5-methyl-5,6,7,8-tetrahydrofolate calcium salt and (6R,S)- 5-methyl-5,6-dihydrofolate ammonium salt was purchased from Schircks Laboratories (JoM, Switzerland), while p-aminobenzoyl-L-glutamic acid was purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco's phosphate buffered saline (PBS) was purchased from PAA Laboratories GmbH (Paschmg, Austria). The solutions were freshly made in PBS before each experiment. Because of the instability of the compounds, the solutions were always kept on ice before use.

UVIVis absorption and fluorescence measurements. Absorption spectm were registered with a Perkin-Elmer Lambda 40 UVPis spectrometer (Shelton, CT). Fluorescence emission and excitation spectra were recorded by means of a Perkin-Elmer LS5OB luminescence spectrometer equipped with a Hamamatsu R-928 photomultiplier (Iwata, Japan). All measurements were performed in standard 10 mm quartz cuvettes.

Photoolysis. Solutions of 5MTHF were exposed to UV radiation in closed quartz cuvettes (1 mL). The radiation source was a broad-band UVB lamp with five Philips TL 2OW/12 RS UVB tubes (Amsterdam). The fluence rate of the lamp was measured after each experiment with an UVB detector (PMA2106) connected to a photometer (PMA2200), both from Solar Light Co. (Philadelphia, PA). The intensity at the sample position was 2.15 mW an-*, and the radiation was emitted mainly in the wavelength region 280-350 nm with a maximum at 3 12 nm. The temperatures of the samples were approximately 25 to 30°C. All experiments were performed in constant dim light in order to avoid external, uncontrolled light exposure.

One point that must be stressed is the fact that our UV source contains radiation that is not present in the solar radiation (9). In the starting phase of our project we used a narrow-band UVB lamp (Phillips TL 20W/01 RS, 312 nm peak). The results were the same as for the broad-band UVB lamp, but the process was slower because 5MTHF absorbs less at 312 nm than at shorter wavelengths. SMTHF is rather unstable in vibo, so we decided to use the broad-band UVB lamp.

RESULTS AND DISCUSSION Absorption measurements

The absorption spectra of different concentrations of 5MTHF are shown in Fig. 2. SMTHF absorbs radiation in the UV region and has an absorption peak at 290 nm. At wavelengths longer than 340 nm, the absorption is weak. The insert shows a linear plot of the absorbance at 290 nm as a function of concentration. The linear relationship between absorbance and concentration indicates that the absorbance of SMTHF follows the Beer-Lambert law, thus aggregation plays no major role.

The absorption coefficient, E , for SMTHF was found to be 24 250 % 1170 M-' cm-' at 290 nm. This is lower than 30 800 M-' cm-I, found by Donaldson and Keresztesy (10) and 29000 M-' cm-' found by h a b e e et ul. (1 I), and the reason for this can be that SMTHF is a hygroscopic substance. The powder of 5MTHF may absorb water from the air and increase its weight. This may lead to

1651

ü  5MTHF absorve UVB, sendo oxidado a 5MDHF

ü  5MDHF não volta a entrar no ciclo de folato

ü  UVA, através de ROS produzidos por fotosensibilizadores (ex: flavinas, porfirinas, bilirrubina, etc), oxida 5MTHF

The effect of different cooking methods on folate retention invarious foods that are amongst the major contributors to folate intake

in the UK diet

Derek J. McKillop, Kristina Pentieva*, Donna Daly, Joseph M. McPartlin, Joan Hughes, J. J. Strain,John M. Scott and Helene McNulty

Northern Ireland Centre for Food and Health (NICHE), University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK

(Received 14 January 2002 – Revised 04 July 2002 – Accepted 15 August 2002)

Folate intake is strongly influenced by various methods of cooking that can degrade the naturalforms of the vitamin in foods. The aim of the present study was to determine the effect ofdifferent cooking methods on folate retention in various foods that contribute to folate intakein the UK diet. Typical purchasing and cooking practices of representative food folate sourceswere determined from a questionnaire survey of local shoppers (n 100). Total folate was deter-mined by microbiological assay (Lactobacillus casei NCIMB 10463) following thermal extrac-tion and tri-enzyme (a-amylase, protease and conjugase) treatment in raw foods and aftertypical methods of cooking. Boiling for typical time periods resulted in only 49% retentionof folate in spinach (191·8 and 94·4mg/100 g for raw and boiled spinach respectively;P,0·005), and only 44% in broccoli (177·1 and 77·0mg/100 g for raw and boiled broccolirespectively, P,0·0001). Steaming of spinach or broccoli, in contrast, resulted in no significantdecrease in folate content, even for the maximum steaming periods of 4·5min (spinach) and15·0min (broccoli). Prolonged grilling of beef for the maximum period of 16·0min did notresult in a significant decrease in folate content (54·3 and 51·5mg/100 g for raw and grilledbeef respectively). Compared with raw values, boiling of whole potatoes (skin and flesh) for60·0min did not result in a significant change in folate content (125·1 and 102·8mg/100 gfor raw and boiled potato respectively), nor was there any effect on folate retention whetheror not skin was retained during boiling. These current results show that the retention offolate in various foods is highly dependent both on the food in question and the method of cook-ing. Thus, public health efforts to increase folate intake in order to improve folate status shouldincorporate practical advice on cooking.

Food folate retention: Cooking methods: Food folates: Dietary folate intake

Optimal folate status may have a role in the prevention ofcardiovascular disease via plasma homocysteine-lowering(Boushey et al. 1995), and possibly in the prevention ofcertain cancers (Branda & Blickenderfer, 1993; Kim et al.1997; Jacob et al. 1998; Choi & Mason, 2000). However,the most compelling evidence for the benefit of optimalfolate status is its link with the prevention of neural tubedefects (Medical Research Council Vitamin StudyResearch Group, 1991; Czeizel & Dudas, 1992; Bottoet al. 1999). The national committees tasked with publichealth in the UK, USA and Australia (Department ofHealth, 1992; Public Health Service Centers for DiseaseControl and Prevention, 1992; National Health and Medi-cal Research Council, 1993) have recommended an extra

400mg folic acid/d in addition to normal dietary folateintake to prevent the occurrence of neural tube defects.However, more recent studies suggest that an additionalintake of 200mg folic acid/d may be optimal both for theprevention of neural tube defect occurrence (Daly et al.1997) and for the lowering of plasma homocysteine(Ward et al. 1997). This level could potentially be achievedby doubling the current dietary folate intake of about190mg/d in the UK (Ball, 1998) through the increasedconsumption of folate-rich foods.Natural folates are a group of water-soluble compounds

with similar biological activity to the synthetic vitamin,folic acid. Unlike folic acid, which is a fully oxidisedmolecule, natural folates are reduced at the 5, 6, 7 and 8

*Corresponding author: Dr Kristina Pentieva, fax +44 2870 324965, email [email protected]: Ches, 2-(N-cyclohexylamino)ethanesulfonic acid; Hepes, N-(2-hydroxyethyl)piperazine-N0-(2-ethanesulfonic acid).

British Journal of Nutrition (2002), 88, 681–688 DOI: 10.1079/BJN2002733q The Authors 2002

Table 1. Folate retention in major food folate sources following typical cooking procedures†

(Mean values with their standard errors for duplicate samples in three independent experiments)

Raw food§ Cooked food§

Food Cooking method Cooking duration (min)‡ Folate (mg/100g) SEM Folate (mg/100g) SEM

Spinach Boiled 3·5 191·8 5·8 94·4*** 13·3Spinach Steamed 3·0 189·5 9·0 218·5 22·6Broccoli Boiled 10·0 177·1 8·5 77·0**** 3·6Broccoli Steamed 10·0 172·0 9·4 156·2 14·5Potato Boiled 60·0 125·1 11·7 102·8 15·0Beef Grilled 11·0 54·3 4·8 50·6 5·8

Mean values were significantly different from those of the raw food (paired t test: ***P,0·005, ****P,0·0001.† ‘Typical’ cooking procedures were established from the results of a consumer questionnaire (for details see p. 682).‡ Times shown reflect the duration required to cook the food item (determined from preliminary experiments, i.e. not ‘undercooked’or ‘overcooked’; for details, see p. 683).

§ To avoid differences in moisture content as a result of different cooking procedures, spinach, broccoli and beef were weighed rawbefore cooking and values therefore given per 100 g raw weight. Values for potatoes relate to whole potatoes (skin and flesh)and are given per 100 g raw or cooked weight as appropriate (for details of procedures see p. 682).

Fig. 2. The effect of duration and method of cooking on folate retention in: (a), spinach;(b), broccoli. V, Boiled; B, steamed. For details of samples and procedures, see p. 682.Values are means for six samples, with their standard errors shown by vertical bars.

Effect of cooking on food folate retention 685

ESPINAFRE

COZIDO EM ÁGUA

COZIDO AO VAPOR

The effect of different cooking methods on folate retention invarious foods that are amongst the major contributors to folate intake

in the UK diet

Derek J. McKillop, Kristina Pentieva*, Donna Daly, Joseph M. McPartlin, Joan Hughes, J. J. Strain,John M. Scott and Helene McNulty

Northern Ireland Centre for Food and Health (NICHE), University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK

(Received 14 January 2002 – Revised 04 July 2002 – Accepted 15 August 2002)

Folate intake is strongly influenced by various methods of cooking that can degrade the naturalforms of the vitamin in foods. The aim of the present study was to determine the effect ofdifferent cooking methods on folate retention in various foods that contribute to folate intakein the UK diet. Typical purchasing and cooking practices of representative food folate sourceswere determined from a questionnaire survey of local shoppers (n 100). Total folate was deter-mined by microbiological assay (Lactobacillus casei NCIMB 10463) following thermal extrac-tion and tri-enzyme (a-amylase, protease and conjugase) treatment in raw foods and aftertypical methods of cooking. Boiling for typical time periods resulted in only 49% retentionof folate in spinach (191·8 and 94·4mg/100 g for raw and boiled spinach respectively;P,0·005), and only 44% in broccoli (177·1 and 77·0mg/100 g for raw and boiled broccolirespectively, P,0·0001). Steaming of spinach or broccoli, in contrast, resulted in no significantdecrease in folate content, even for the maximum steaming periods of 4·5min (spinach) and15·0min (broccoli). Prolonged grilling of beef for the maximum period of 16·0min did notresult in a significant decrease in folate content (54·3 and 51·5mg/100 g for raw and grilledbeef respectively). Compared with raw values, boiling of whole potatoes (skin and flesh) for60·0min did not result in a significant change in folate content (125·1 and 102·8mg/100 gfor raw and boiled potato respectively), nor was there any effect on folate retention whetheror not skin was retained during boiling. These current results show that the retention offolate in various foods is highly dependent both on the food in question and the method of cook-ing. Thus, public health efforts to increase folate intake in order to improve folate status shouldincorporate practical advice on cooking.

Food folate retention: Cooking methods: Food folates: Dietary folate intake

Optimal folate status may have a role in the prevention ofcardiovascular disease via plasma homocysteine-lowering(Boushey et al. 1995), and possibly in the prevention ofcertain cancers (Branda & Blickenderfer, 1993; Kim et al.1997; Jacob et al. 1998; Choi & Mason, 2000). However,the most compelling evidence for the benefit of optimalfolate status is its link with the prevention of neural tubedefects (Medical Research Council Vitamin StudyResearch Group, 1991; Czeizel & Dudas, 1992; Bottoet al. 1999). The national committees tasked with publichealth in the UK, USA and Australia (Department ofHealth, 1992; Public Health Service Centers for DiseaseControl and Prevention, 1992; National Health and Medi-cal Research Council, 1993) have recommended an extra

400mg folic acid/d in addition to normal dietary folateintake to prevent the occurrence of neural tube defects.However, more recent studies suggest that an additionalintake of 200mg folic acid/d may be optimal both for theprevention of neural tube defect occurrence (Daly et al.1997) and for the lowering of plasma homocysteine(Ward et al. 1997). This level could potentially be achievedby doubling the current dietary folate intake of about190mg/d in the UK (Ball, 1998) through the increasedconsumption of folate-rich foods.Natural folates are a group of water-soluble compounds

with similar biological activity to the synthetic vitamin,folic acid. Unlike folic acid, which is a fully oxidisedmolecule, natural folates are reduced at the 5, 6, 7 and 8

*Corresponding author: Dr Kristina Pentieva, fax +44 2870 324965, email [email protected]: Ches, 2-(N-cyclohexylamino)ethanesulfonic acid; Hepes, N-(2-hydroxyethyl)piperazine-N0-(2-ethanesulfonic acid).


BRÓCOLO

Table 1. Folate retention in major food folate sources following typical cooking procedures†

(Mean values with their standard errors for duplicate samples in three independent experiments)

Raw food§ Cooked food§

Food Cooking method Cooking duration (min)‡ Folate (mg/100g) SEM Folate (mg/100g) SEM

Spinach Boiled 3·5 191·8 5·8 94·4*** 13·3Spinach Steamed 3·0 189·5 9·0 218·5 22·6Broccoli Boiled 10·0 177·1 8·5 77·0**** 3·6Broccoli Steamed 10·0 172·0 9·4 156·2 14·5Potato Boiled 60·0 125·1 11·7 102·8 15·0Beef Grilled 11·0 54·3 4·8 50·6 5·8

Mean values were significantly different from those of the raw food (paired t test: ***P,0·005, ****P,0·0001.† ‘Typical’ cooking procedures were established from the results of a consumer questionnaire (for details see p. 682).‡ Times shown reflect the duration required to cook the food item (determined from preliminary experiments, i.e. not ‘undercooked’or ‘overcooked’; for details, see p. 683).

§ To avoid differences in moisture content as a result of different cooking procedures, spinach, broccoli and beef were weighed rawbefore cooking and values therefore given per 100 g raw weight. Values for potatoes relate to whole potatoes (skin and flesh)and are given per 100 g raw or cooked weight as appropriate (for details of procedures see p. 682).

Fig. 2. The effect of duration and method of cooking on folate retention in: (a), spinach;(b), broccoli. V, Boiled; B, steamed. For details of samples and procedures, see p. 682.Values are means for six samples, with their standard errors shown by vertical bars.

Effect of cooking on food folate retention 685

COZIDO EM ÁGUA

COZIDO AO VAPOR

VITAMINA B12

Homens RDA/AI* (mcg)

Limite (mcg) Mulheres RDA/AI*

(mcg)

Limite (mcg)

0-12 meses

0.4-0.5* Nd 0-12 meses 0.4-0.5* Nd

1-8 anos 0.9-1.2 Nd 1-8 anos 0.9-1.2 Nd

9-13 anos 1.8 Nd 9-13 anos 1.8 Nd

> 14 anos 2.4 Nd > 14 anos 2.4 Nd

Gravidez 2.6 Nd

Lactação 2.8 Nd

DOSE DIÁRIA RECOMENDADA DE VITAMINA B12 FOOD AND NUTRITION BOARD

195


TABLE 2Vitamin B-12 and homocysteine status among vegetarians, vegans, and omnivores: summary of studies from differentcountries

Study (reference) Plasma vitamin B-12 Plasma homocysteine

pmol/L lmol/LMajchrzak et al, 2006, Austria (10)

Vegetarians (n ! 36)1 238.5 6 99.1 14.0 6 5.4Vegans (n ! 42)1 203.2 6 101.5 16.5 6 8.2Omnivores (n ! 40)1 251.5 6 83.0 12.2 6 5.6

Koebnick et al, 2005, Germany (11)Vegetarians (n ! 38)2 143.2 (121.2–175.9) 17.1 (13.1–20.2)Vegans (n ! 39)2 126.2 (87.8–182.3) 18.5 (13.5–28.9)Omnivores (n ! 109) 2 174.5 (142.2–249.8) 14.7 (11.9–18.3)

Herrmann et al, 2005, Germany (13)Vegetarians (n ! 66)3 192 (127–450) 10.6 (6.4–27.7)Vegans (n ! 29)3 148 (99–314) 12.8 (5.9–57.1)Omnivores (n ! 79)3 287 (190–471) 8.8 (5.5–16.1)

Waldmann et al, 2004, Germany (12)Moderate vegans (n ! 45)3 185 (97.6–689) 12.3 (4.6–23.6)Strict vegans (n ! 86)3 122 (71.2–276) 13.4 (6.0–82.5)

Huang et al, 2003, Taiwan (14)Vegetarians (n ! 37)4 191.8 (164.0, 220.0) 13.2 (10.6, 15.7)Omnivores (n ! 32)4 310.9 (278.2, 343.6) 9.8 (9.1, 10.6)

Su et al, 2005, Taiwan (15)Vegetarians (n ! 57)1 265.2 6 179.3 11.0 6 3.3Omnivores (n ! 61)1 380.3 6 199.4 9.0 6 2.1

Hung et al, 2002, Taiwan (16)Vegetarians (n ! 45)1 207.7 6 127.1 11.20 6 4.27Omnivores (n ! 45)1 403.5 6 138.9 8.64 6 2.06

Bissoli et al, 2002, Italy (17)Vegetarians (n ! 14)1 163.8 6 57.1 17.4 6 11.1Vegans (n ! 31)1 155 6 73.6 26.9 6 24.1

Kazimırova et al, 2006, Slovak Republic (18)Vegetarians (n ! 24)5 209.79 6 27.52 Not reportedOmnivores (n ! 24)5 229.33 6 15.01 Not reported

Koebnick et al, 2004, Germany (19)Vegetarian (n ! 60)6,7 159.6 (127–176) 6.7 (5.7–7.5)Omnivores (n ! 108)6,7 218.6 (169–249) 6.2 (5.7–6.7)

Ambroszkiewicz et al, 2006, Poland (20)Vegetarians (n ! 32)1,8 404.9 6 106.6 6.1 6 1.2

Karabudak et al, 2008, Turkey (21)Vegetarian (n ! 26)1 200.5 6 137.3 12.6 6 5.97Omnivores (n ! 26)1 269.1 6 234.2 10.8 6 3.72

Haddad et al, 1999, United States (22)Vegans (n ! 25)1 312 6 125 7.9 6 1.5Omnivores (n ! 20)1 313 6 99 8.0 6 1.9

Hokin et al, 1999, Australia (23)Vegetarians (n ! 245)7 199 (58–538) Not reportedOmnivores (n ! 53)7 292 (134–721) Not reported

Leung et al, 2001, China (24)Vegetarians (n ! 51)7,9 389 (313–437) Not reported

Refsum et al, 2001, India (26)Vegetarian (n ! 78)3 124 (66–625) 22 (9.6–48)Omnivores (n ! 126)3 161 (62–492) 19.4 (9.7–45.7)

1 All values are means 6 SDs.2 All values are medians; 25th to 75th percentiles in parentheses.3 All values are medians, 5th to 95th percentiles in parentheses.4 All values are means; 95% CIs in parentheses.5 All values are means 6 SEMs.6 Subjects were women at various stages of pregnancy.7 All values are means; range in parentheses.8 Subjects were children, including 5 vegans (values originally in pg/ml: 548.6 6 144.4).9 Subjects were children aged 4–14 y.

1696S ELMADFA AND SINGER

at Lund University Libraries on October 3, 2011

www.ajcn.orgDownloaded from

Elmadfa I, Singer I. Am J Clin Nutr. 2009 May;89(5):1693S-‐1698S

DEFICIÊNCIA DE VITAMINA B12

EM VEGETARIANOS

778 www.japi.org © JAPI • VOL. 54 • OCTOBER 2006

vitamin B12-concentration (68% rural, 51% slumresidents, 81% urban middle-class). Median red cellfolate concentration was in the normal range in the threegroups (>283 nmol/L), and low folate concemThere wasno significant relationship between plasma vitamin B12and red cell folate concentrations. Plasma vitamin B12concentration was inversely related to plasma tHcyconcentration (r= -0.41, p<0.001), an association that wasindependent of red cell folate concentration. Red cellfolate concentration was inversely related to plasma tHcyconcentration (r= -0.18, p<0.001). Adjusted for age andplace of residence, low vitamin B12 concentrationscontributed 28.4% to the risk of hyperhomocysteinemia(population attributable risk) while low folateconcentrations contributed only 2.2%.

Plasma vitamin B12 concentration decreased andplasma tHcy increased with increasing age (p< 0.01 andp<0.05 respectively). Plasma vitamin B12, tHcy and redcell folate concentrations were not related to body sizemeasurements. Compared to Hindu men, Muslim menhad higher plasma vitamin B12 (148 vs 107 pmol/L),lower plasma tHcy (15.8 vs 19.5 µmol/L) and lower redcell folate concentrations (423.7 vs 505.3 nmol/L) (p<0.01, all), of which difference between plasma vitaminB12 and plasma tHcy became non-significant afteradjusting for non-vegetarian food intake. Highereducation and income were associated with lowerplasma vitamin B12 and higher tHcy concentrations(p<0.05). These relations were not independent of intakeof non-vegetarian foods. Smoking and alcohol habitswere not related to plasma vitamin B12 or tHcy or to redcell folate concentrations.Associations with diet and gastrointestinal factors

Only 3 men took vitamin supplements, nonecontaining vitamin B12. Daily energy intakes of thesemen were lower than those recommended by the Indian

Council of Medical Research (2425 kcals/day) in all 3groups, and were not related to plasma vitamin B12 ortHcy and red cell folate concentrations. Protein intakeswere comparable to the ICMR recommendation (60g/day). Higher protein intake was associated with higherred cell folate concentration (p<0.05) independent of ageand place of residence.

Food frequency data was available on 424 men. Noneof these men were vegan. Forty one percent rural, 11%slum residents and 44% urban middle-class men werelacto-vegetarians. Non-vegetarian foods were eaten morefrequently by Muslims than Hindus (p<0.001), by thosewho were less educated or poorer (p<0.001), and by theslum residents compared to urban middle class(p<0.001). The portion size of non-vegetarian foods wasusually small (~100 g cooked). Most men ate chicken,fish and eggs and very few ate red meat. There was aprogressive and graded relation between frequency ofconsumption of non-vegetarian foods and plasmavitamin B12 (r= 0.27, p<0.001) and tHcy concentrations(r= -0.26, p<0.001). On univariate analysis (age adjusted)lacto-vegetarians had a 4.3 (95%CI, 2.4, 7.8) times higherrisk of low vitamin B12 concentrations and 4.3 (95%CI,2.4, 7.6) times higher risk of hyperhomocysteinemiacompared to those who ate non-vegetarian foods on atleast alternate days (data not shown). Ninety-three menhad non-vegetarian food frequently (Table 1).Approximately half of the men who ate non-veg foodfrequently had low vitamin B12 concentration andhyperhomocysteinemia. Nine men had hyper-homocysteinemia despite frequent non-vegetarian foodintake, normal circulating vitamin B12, normal folateand plasma creatinine concentrations. Intake ofvegetables, milk and coffee was not related to plasmavitamin B12 and tHcy or red cell folate concentrations.

Plasma vitamin B12 and tHcy and red cell folateconcentrations were not related to gastrointestinal

Table 2 : Circulating vitamin B12, total homocysteine and folate concentrations and haematological parameters and inrural and urban men. The CRISIS study

Rural(n=149) Slums(n=142) Urban middle-class(n=150)

Plasma vitamin B12 (pmol/L) 119 (73, 171) 145 (90, 241) ** 89 (58, 133) ++

<150 pmol/L (%) 68 51 * 81 *, +++

Total Hcy (µmol/L) 14.6 (12.0, 22.6) 14.2 (11.4, 19.7) 23.7 (15.3, 40.7) ***, +++

>15 µmol/L (%) 48 47 79 **, ++

Red cell folate (nmol/L) 522 (424, 647) 461 (360, 585) * 525 (406, 707) ++

<283 nmol/L (%) 7 12 5 ++

Haematological parametersHaemoglobin (g/L) 140 (134,147) 143 (137,149) 141 (132, 146)Anemia (< 135 g/L) (%) 25 19 31Mean corpuscular volume (fL) 85.7 (82.3, 89.0) 85.1 (81.8, 89.7) 85.3 (82.2,89.3)Macrocytosis (>100 fL) (%) 1 2 1Microcytosis (<80 fL) (%) 15 14 15Thrombocytopenia (<140 * 109/L) (%) 4 4 2Leucopenia (<4.5*109/L) (%) 18 5 * 5 *

Plasma ferritin(<45.0 pmol/L) (%) 18 17 21

Median (interquartile range) or percentages. *p<0.05, **p<0.01, ***p<0.001, different from rural and adjusted for age+ p<0.05, ++p<0.01, +++p<0.001, different from urban slums and adjusted for age.

© JAPI • VOL. 54 • OCTOBER 2006 www.japi.org 775

Original Article

Vitamin B12 Deficiency and Hyperhomocysteinemia inRural and Urban IndiansCS Yajnik*, Swapna S Deshpande*, Himangi G Lubree*, SS Naik*, DS Bhat*Bhagyashree S Uradey*, Jyoti A Deshpande*, Sonali S Rege*, Helga Refsum**,JS Yudkin***

AbstractBackground : Low vitamin B12 concentration in South Asian Indians is common, but the exact prevalence isnot known.Aim : To investigate prevalence and associations of low vitamin B12 concentration and hyperhomocysteinemiain rural and urban Indian men living in and around Pune, Maharashtra.Method : We studied 441 middle-aged men (149 rural, 142 slum and 150 urban middle-class residents, meanage 39 y). Data on lifestyle, socio-economic status, nutrition and medical history were obtained. Circulatingconcentrations of vitamin B12, folate, ferritin, total homocysteine (tHcy), and haematological indices, andcardiovascular risk variables were measured.Results : Median plasma B12 concentration was low (110 pmol/L): Overall, 67% of men had low vitamin B12concentration (<150 pmol/L) and 58% had hyperhomocysteinemia (>15 µmol/L). Of the urban middle class,81% had low vitamin B12 concentration and 79% had hyperhomocysteinemia. Low vitamin B12 concentrationcontributed 28% to the risk of hyperhomocysteinemia (population attributable risk) while low red cell folatecontributed 2%. Vegetarians had 4.4 times (95%CI 2.1, 9.4) higher risk of low vitamin B12 concentrations and3.0 times (95%CI 1.4, 6.5) higher risk of hyperhomocysteinemia compared to those who ate non-vegetarianfoods frequently. Urban middle-class residence was an additional independent risk factor ofhyperhomocysteinemia (odds ratio 7.6 (95%CI 2.5, 22.6), compared to rural men). Low vitamin B12concentration was related to lower blood haemoglobin concentration and higher mean corpuscular volume,but macrocytic anemia was rare.Conclusion : Low vitamin B12 concentration and hyperhomocysteinemia are common in Indian men,particularly in vegetarians and urban middle class residents. Further studies are needed to confirm thesefindings in other parts of India. ©

INTRODUCTION

Elevated circulating total homocysteine (tHcy)concentration is a risk factor for cardiovascular

disease1,2 and elevated tHcy or low folate and vitaminB12 concentrations are a risk factor for birth defects,poor pregnancy outcomes and neurocognitiveperformance.3-5 Indians in India6,7 as well as thosemigrated abroad8, 9 have high circulating tHcy

*Diabetes Unit, King Edward Memorial Hospital and ResearchCentre, Pune, India. **Institute of Basic Medical Sciences,Department of Nutrition, University of Oslo and Institute ofMedicine, Section of Pharmacology, University of Bergen,Norway. ***Diabetes and Cardiovascular Disease AcademicUnit, University College, London, UK.Received : 7.2.2006; Revised : 12.7.2006;Re-revised : 16.8.2006; Accepted : 25.8.2006

concentration compared to other ethnic groups. InCaucasian populations not eating folic acid fortifiedfood, hyperhomocysteinemia is usually explained by lowblood folate concentrations.10 In contrast, hyper-homocysteinemia in Indians living in India is moreattributable to low concentrations of vitamin B12.4

Eventhough low circulating vitamin B12concentration have been recognised in Indians for a longtime,11 there is little appreciation of this amongst Indianmedical professionals and policy makers. This may bedue to a number of reasons: 1) vitamin B12 and tHcy arenot routinely measured in clinical practice, 2) despitelow circulating vitamin B12 concentrations, specifichematological and neurological manifestationsconsistent with vitamin B12 deficiency are rare, and 3)the majority of previous reports are clinic based andtherefore may not represent community prevalence.

Yajnik CS, et al. J Assoc Physicians India. 2006 Oct;54:775-‐82

Applied nutritional investigation

Vegetarianism produces subclinical malnutrition, hyperhomocysteinemiaand atherogenesis

Yves Ingenbleek M.D. a, Kilmer S. McCully M.D. b,c,*a Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur, Strasbourg, Franceb Pathology and Laboratory Medicine Service, VA Boston Healthcare System, West Roxbury, Massachusetts, USAcDepartment of Pathology, Harvard Medical School, Boston, Massachusetts, USA

a r t i c l e i n f o

Article history:Received 30 December 2010Accepted 27 April 2011

Keywords:Protein malnutritionLean body massTransthyretinHomocysteineSulfur deficiencyCardiovascular disease

a b s t r a c t

Objective: To explain why vegetarian subjects develop morbidity and mortality from cardiovasculardiseases unrelated to vitamin B status and Framingham criteria.Methods: A study of 24 rural male subjects 18 to 30 y old and 15 urban male controls was con-ducted in the Sahel region of Chad. Food consumption was determined from a dietary question-naire, and overall health status was assessed by body weight, body mass index, serum albumin,plasma transthyretin, urinary nitrogen, and creatinine. Plasma lipids, vitamins B6, B9 and B12,homocysteine, and related sulfur amino acids were measured as selected cardiovascular diseaserisk factors.Results: Body weight, body mass index, blood, and urinary markers of protein status were signif-icantly lower, with an estimated 10% decrease of lean body mass in the study group comparedwith urban controls. Neither lipid fractions nor plasma levels of vitamins B6, B9, and B12 weresignificantly different between the two groups. Although the mean consumption of sulfur aminoacids (10.4 mg$kg!1$d!1) by rural subjects was significantly below the recommended dietaryallowances (13 mg$kg!1$d!1), plasma methionine values were similar in the two groups. Incontrast, homocysteine concentration was significantly increased (18.6 mmol/L, P < 0.001), and thelevels of cysteine and glutathione were significantly decreased in the study group, demonstratinginhibition of the trans-sulfuration pathway. The strong negative correlation (r " !0.71) betweentransthyretin and homocysteine implicated lean body mass as a critical determinant ofhyperhomocysteinemia.Conclusion: The low dietary intake of protein and sulfur amino acids by a plant-eating populationleads to subclinical protein malnutrition, explaining the origin of hyperhomocysteinemia and theincreased vulnerability of these vegetarian subjects to cardiovascular diseases.

! 2011 Elsevier Inc. All rights reserved.

Introduction

According to World Health Organization estimates [1], 16.7million deaths are attributable to cardiovascular disease (CVD)each year, accounting for 30% of all deaths worldwide andconstituting an international pandemic [2]. More than half ofthese deaths occur in developing countries where vegetariandiets consisting of several varieties of legumes and starchy foodshave prevailed for millennia. The beneficial health effects of suchregimens in the prevention of chronic disorders such as CVD,

diabetes, and cancer have been documented by many investi-gators [3,4], explaining their growing popularity in Westernizedcountries during the past several decades. It is estimated thatabout 2% to 5% of individuals living in developed countries haveadopted vegetarianism as a lifestyle choice [5,6].

Plant-based regimens, however, do not optimally fulfill thenutritional requirements of population groups living in devel-oping countries, as illustrated by the increasing incidence ofhyperhomocysteinemia (HHcy) states [7,8], now recognized asa critical factor in occurrence of CVD and stroke [9]. HHcy wasoriginally associated with the pathogenesis of atherosclerosis bythe study of the vascular pathology of children with inheriteddisorders of methionine (Met) metabolism [10]. Dietary insuffi-ciency or malabsorption of any of three water-soluble B vitamins

This work was funded by University Louis Pasteur, Strasbourg, France.* Corresponding author. Tel.: #857-203-5990; fax: #857-203-5623.

E-mail address: [email protected] (K. S. McCully).

0899-9007/$ - see front matter ! 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.nut.2011.04.009


Nutrition

journal homepage: www.nutr i t ionjrnl .com

Please cite this article in press as: Ingenbleek Y, McCully KS, Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia andatherogenesis, Nutrition (2011), doi:10.1016/j.nut.2011.04.009

Nutrition xxx (2011) 1–6

Ingenbleek Y, McCully KS. Nutricon. 2012 Feb;28(2):148-‐53.

transthyretin (TTR; P < 0.001) compared with the control group.Despite an apparently satisfactory state of health, plasma TTRvalues of all rural subjects were !240 mg/L (Fig. 1), indicatingthat an element of protein malnutrition was present in allmembers of the study group. The lower excretion of urinarynitrogen and creatinine by the study group reflected lowerprotein consumption and decreased lean body mass (LBM) andmuscle mass, respectively, compared with the control group.

There was no difference in serum levels of triacylglycerols,cholesterol, LDL cholesterol, or HDL cholesterol between the twogroups (Table 2). There was no difference in plasma levels ofvitamins B6 or B9 between the two groups, and the lower plasmalevel of vitamin B12 of the study group (174 pmol/L) was ofborderline significance (P " 0.067) compared with the controlgroup (269 pmol/L). In the study group, only 4 of 24 participantshad plasma vitamin B12 levels below the lower limit of normal

(!140 pmol/L). A previous study undertaken in the same Cha-dian area [17] in a larger group of 60 rural participants diddemonstrate a weak inverse correlation between B12 and Hcyconcentrations in the 20 subjects most severely protein depleted,as identified by the lowest TTR plasma values. It is thereforelikely that the HHcy status of some of our rural subjects in thepresent survey might have resulted from combined B12 andprotein deficiencies. The correlation of B12 deficiency with HHcycould well reach statistical significance if a larger group ofsubjects were studied. Analysis of plasma SAAs showed nodifference in concentration ofMet between the study and controlgroups (Table 2). In contrast, plasma levels of Hcy were signifi-cantly higher (18.6 mmol/L, P < 0.001) in the study group than inthe control group (mean average 10.8 mmol/L, with no value#11.7 mmol/L). Plasma levels of Cys (P < 0.001) and GSH (P <0.01) were significantly lower in the study group compared withthe control group.

The Spearman correlation coefficients among plasma Hcy,nutritional indicators, and dietary variables (Table 3) indicated

Table 2Health and plasma indices

Indices Study group Control group P

(n " 24) (n " 15)

Overall health statusHeight (m) 1.68 $ 0.3 1.73 $ 0.3 NSWeight (kg) 61.7 $ 3.6 69.3 $ 1.9 <0.05Body mass index (kg/m2) 19.3 $ 0.8 22.1 $ 0.6 <0.05Serum albumin (g/L) 3.8 $ 0.6 4.3 $ 0.4 <0.05Plasma transthyretin (mg/L) 178.1 $ 27.5 292.9 $ 10.8 <0.001Urinary nitrogen (g/d) 9.6 $ 0.9 11.2 $ 0.9 <0.05Urinary creatinine (g/d) 1.18 $ 0.37 1.43 $ 0.26 <0.05

Serum lipidsTriacylglycerols (mmol/L) 0.85 $ 0.55 1.08 $ 0.41 NSTotal cholesterol (mmol/L) 3.98 $ 0.59 4.31 $ 0.92 NSHDL cholesterol (mmol/L) 1.21 $ 0.41 1.49 $ 0.30 NSLDL cholesterol (mmol/L) 2.27 $ 0.32 2.12 $ 0.25 NS

Plasma vitaminsPyridoxine (B6, nmol/L) 41.9 $ 7.4 52.4 $ 13.8 NSFolate (B9, nmol/L) 16.9 $ 2.7 14.4 $ 2.6 NSCobalamin (B12, pmol/L) 174 $ 28 269 $ 36 <0.06

Plasma SAAsMethionine (mmol/L) 29.3 $ 3 28.0 $ 4 NSHomocysteine (mmol/L) 18.6 $ 2.9 10.8 $ 0.7 <0.001Cysteine (mmol/L) 14.5 $ 8.3 43.4 $ 4.5 <0.001Glutathione (mmol/L) 2.7 $ 0.7 4.3 $ 0.7 <0.01Taurine (mmol/L) 32 $ 6 58 $ 9 <0.01

HDL, high-density lipoprotein; LDL, low-density lipoprotein; SAA sulfur aminoacidParameters of general health status and serum lipids, plasma vitamins, andplasma SAAs were determined after 1 wk of observation

Fig. 1. Relation of plasma homocysteine and transthyretin. Regression line analysisplots total plasma homocysteine and plasma transthyretin values in participantswith protein malnutrition (diamonds) and in controls (circles). Decreased plasmatransthyretin values are negatively correlated (r " %0.71) with increased plasmahomocysteine values in the study group, indicating that a decrease of lean bodymass is a critical determinant of hyperhomocysteinemia.

Table 3Spearman correlation coefficients between total homocysteine and nutritionalindicators and significance

Nutritional indicator Study group Control group

(n " 24) (n " 15)

r P r P

Body mass index %0.139 NS 0.047 NSSerum albumin %0.273 NS 0.029 NSPlasma transthyretin %0.709 <0.001 %0.275 0.004Urinary nitrogen %0.357 0.003 0.058 NSUrinary creatinine %0.285 0.004 0.067 NSTriacylglycerols 0.215 NS 0.086 NSTotal cholesterol 0.188 NS 0.127 NSHDL cholesterol %0.315 NS %0.075 NSLDL cholesterol 0.209 NS 0.286 NSPyridoxine (B6) 0.189 NS 0.279 NSFolate (B9) 0.237 NS 0.189 NSCobalamin (B12) %0.175 0.061 %0.067 NSMethionine 0.260 NS %0.023 NSCysteine %0.668 <0.001 0.215 NSGlutathione %0.319 0.013 0.181 NSTaurine %0.182 0.043 0.098 NS

HDL, high-density lipoprotein; LDL, low-density lipoprotein

Table 1Characteristics of food consumption

Nutrient classes Study group Control group P

(n " 24) (n " 15)

Energy (MJ/d) 7.33 (6.1–8.4) 7.22 (6.4–8.9) NSFiber (g/d) 73.4 (62–97) 49.4 (38–73) <0.01Carbohydrate (g/d) 265.3 (174–389) 220.5 (160–291) <0.05Total energy (%) 63.1 54.1

Fat (g/d) 52.1 (34–83) 62.4 (48–84) <0.05Total energy (%) 25.7 32.6

Protein (g/d) 49.7 (24–51) 63.4 (29–59) <0.01Total energy (%) 11.2 13.3

SAA (mg$kg%1$d%1) 10.4 (9–12) 14.6 (11–21) <0.001

SAA, sulfur amino acidIntakes of dietary nutrients were calculated from the analysis of a 1-wk dietaryquestionnaire for rural vegetarian participants in the study group and urbanomnivorous participants in the control group. Intakes of dietary SAAs werecalculated from tables of composition of dietary proteins

Y. Ingenbleek, K. S. McCully / Nutrition xxx (2011) 1–6 3

Please cite this article in press as: Ingenbleek Y, McCully KS, Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia andatherogenesis, Nutrition (2011), doi:10.1016/j.nut.2011.04.009

Alimento mcg/100 g!Brócolos, Espargos, Rebentos de feijão Mung >0,1

Folhas de Chá verde 0,1-‐0,5

Folhas de Chá Vermelho 0,7

Folhas de chá preto 0,3-‐1,2

Tempeh 0,7-‐8

Natto 0,1-‐1,5

Nori 32-‐78

Alga Nostoc (Ishikurage) 99 (apenas 12% é accva)

Watanabe F. Exp Biol Med (Maywood). 2007 Nov;232(10):1266-‐74!

FONTES DE B12 BIODISPONÍVEL PARA VEGANS

VITAMINA B2

Homens RDA/AI* (mg)

Limite (mg) Mulheres RDA/AI*

(mg)

Limite

(mg) 0-12

meses 0.3-0.4* Nd 0-12 meses 0.3-0.4

* Nd

1-8 anos 0.5-0.6 Nd 1-8 anos 0.5-0.6 Nd

9-13 anos

0.9 Nd 9-13 anos 0.9 Nd

> 14 anos 1.3 Nd 14-18 anos 1.0 Nd

< 18 anos 1.1 Nd

Gravidez 1.4 Nd

Lactação 1.6 Nd

DOSE DIÁRIA RECOMENDADA DE RIBOFLAVNA FOOD AND NUTRITION BOARD

Cereais e Leguminosas

Tiamina (mg/100g)

Gérmen de Trigo 0,68

Farelo de Trigo 0,35

Centeio Integral 0,22

Millet 0,38

Arroz Selvagem 0,73

Farelo de Arroz 0,25

Farinha de soja 0,35

Ervilha 0,29

Lentilha 0,22

Feijão Vermelho 0,21

Feijão Preto 0,21

Oleaginosas e Hortaliças

Tiamina (mg/100g)

Amêndoa 0,92

Caju 0,25

Pinhão 0,23

Sem. Girassol 0,23

Cogumelos 0,46

Pimento vermelho 0,36

Couve 0,26

Salsa 0,26

Brócolo 0,23

Rábano 0,22

Ameixa 0,22

Carne, Leite e Levedura

Tiamina (mg/100g)

Levedura de Cerveja 4,28

Fígado de Borrego 3,28

Fígado de Porco 3,03

Fígado de Vitela 2,72

Fígado de Galinha 2,49

Gema de Ovo 0,44

Ovo 0,3

Salmão 0,23

Bife de Porco 0,22

Leite 0,18

Liska D, Quinn S, Lukaczer D, et al. Clinical Nutrition: A Functional Approach. Second Edition. IFM; 2004.

FONTES DE RIBOFLAVINA

Reservas duram 2-6 Semanas

CONVERSÃO DA RIBOFLAVINA EM COENZIMAS OCORRE NO CITOPLASMA DE VÁRIAS CÉLULAS

Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ. Modern Nutrition in Health and Disease. Lippincott Williams & Wilkins; 2005.

Marriage B, Clandinin MT, Glerum DM. Nutritional cofactor treatment in mitochondrial disorders. J Am Diet Assoc. 2003 Aug;103(8):1029-38

VIT B2

VITAMINA B3

Homens RDA/AI* (mg)

Limite (mg) Mulheres RDA/AI*

(mg)

Limite

(mg) 0-12

meses 2-4* Nd 0-12 meses 2-4* Nd

1-8 anos 6-8 10-15 1-8 anos 6-8 10-15

9-18 anos 12-16 20-30 9-18 anos 12-16 20-30

Adultos 16 35 Adultos 14 35

Gravidez 18 30-35

Lactação 17 30-35

DOSE DIÁRIA RECOMENDADA DE NIACINA FOOD AND NUTRITION BOARD

210


60 g

1 g

B6, B2 Ferro, Cobre

Marriage B, Clandinin MT, Glerum DM. Nutritional cofactor treatment in mitochondrial disorders. J Am Diet Assoc. 2003 Aug;103(8):1029-38

EXCESSO

Calor e rubor causado pela liberação de prostaglandinas durante a formação do ácido nicotinúrico (deriva da conjugada da niacina com a glicina) à Mais comum com Niacina de Libertação rápida

Hepatotoxicidade à Mais comum com Niacina de Libertação lenta (a partir de 2 gramas)

Diarreia, Náusea, Dispepsia, Dor Abdominal

Resistência à Insulina Atraso no Crescimento em crianças à Mais

comum com Nicotinamida


Antioxidants prevent health-promoting effectsof physical exercise in humansMichael Ristowa,b,1,2, Kim Zarsea,2, Andreas Oberbachc,2, Nora Klotingc, Marc Birringera, Michael Kiehntopfd,Michael Stumvollc, C. Ronald Kahne, and Matthias Bluherc,2

aDepartment of Human Nutrition, Institute of Nutrition, University of Jena, Jena D-07743, Germany; bGerman Institute of Human Nutrition,Potsdam-Rehbrucke D-14558, Germany; cDepartment of Medicine, University of Leipzig, Leipzig D-04103, Germany; dInstitute of Clinical Chemistry andLaboratory Medicine, University of Jena, Jena D-07743, Germany; and eResearch Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215

Contributed by C. Ronald Kahn, March 31, 2009 (sent for review March 14, 2009)

Exercise promotes longevity and ameliorates type 2 diabetesmellitus and insulin resistance. However, exercise also increasesmitochondrial formation of presumably harmful reactive oxygenspecies (ROS). Antioxidants are widely used as supplements butwhether they affect the health-promoting effects of exercise isunknown. We evaluated the effects of a combination of vitamin C(1000 mg/day) and vitamin E (400 IU/day) on insulin sensitivity asmeasured by glucose infusion rates (GIR) during a hyperinsuline-mic, euglycemic clamp in previously untrained (n ! 19) and pre-trained (n ! 20) healthy young men. Before and after a 4 weekintervention of physical exercise, GIR was determined, and musclebiopsies for gene expression analyses as well as plasma sampleswere obtained to compare changes over baseline and potentialinfluences of vitamins on exercise effects. Exercise increased pa-rameters of insulin sensitivity (GIR and plasma adiponectin) only inthe absence of antioxidants in both previously untrained (P <0.001) and pretrained (P < 0.001) individuals. This was paralleledby increased expression of ROS-sensitive transcriptional regulatorsof insulin sensitivity and ROS defense capacity, peroxisome-proliferator-activated receptor gamma (PPAR"), and PPAR" coac-tivators PGC1# and PGC1$ only in the absence of antioxidants (P <0.001 for all). Molecular mediators of endogenous ROS defense(superoxide dismutases 1 and 2; glutathione peroxidase) were alsoinduced by exercise, and this effect too was blocked by antioxidantsupplementation. Consistent with the concept of mitohormesis,exercise-induced oxidative stress ameliorates insulin resistanceand causes an adaptive response promoting endogenous antiox-idant defense capacity. Supplementation with antioxidants maypreclude these health-promoting effects of exercise in humans.

aging ! hormesis ! insulin resistance ! oxidative stress !reactive oxygen species

Type 2 diabetes mellitus is increasing worldwide at epidemicrates and is associated with both microvascular and macro-

vascular complications (1). Type 2 diabetes mellitus is caused bya combination of insulin resistance involving a number ofperipheral tissues, including skeletal muscle (2, 3), and aninadequate !-cell response despite normal or even increasedamounts of circulating insulin.

Physical exercise exerts numerous favorable effects on generalhealth (4) and specifically has been shown to improve glucosemetabolism in the insulin-resistant state (5). This effect may beindependent of exercise-related changes in body mass (6). More-over, physical exercise has been shown to be effective in pre-venting type 2 diabetes in high risk individuals (7, 8) and may beeven more effective than the most widely used anti-diabetic drug,metformin (9).

These beneficial effects of physical exercise on insulin resis-tance involve multiple mechanisms, including enhanced expres-sion of glucose transporters and translocation of glucose trans-porters to the plasma membrane independent of insulin (10).Exercise, as well as weight loss, has been linked to activation ofmitochondrial metabolism, and reduced mitochondrial metab-

olism has been functionally connected with type 2 diabetes (11).Mitochondria, however, are also the main source of reactiveoxygen species (ROS), which are inevitable by-products ofoxidative glucose metabolism. Muscle is also known to generatefree radicals, especially during contraction and physical exercise(12). It has been suggested that ROS may mediate some health-promoting effects, at least in nonprimate model systems (13–17).

We here evaluated the possibility that ROS are required forthe insulin-sensitizing capabilities of physical exercise in healthyhumans and that commonly used antioxidants, such as vitaminC and vitamin E, may abrogate the health-promoting effects ofboth physical exercise and oxidative stress in humans.

ResultsBaseline Characteristics. Of the 40 individuals included in thepresent study, 20 were known to be previously trained, and 20were previously untrained. Study subject characteristics in thepreinterventional state are given in Table 1. No significantdifferences in age, height, body mass index, fat free mass, or VO2maximum were observed within the groups (Table 1) and nosignificant differences in age, height and body mass index wereobserved between untrained and pretrained groups. Not sur-prisingly, pretrained individuals had a significantly higher fatfree mass (P ! 0.03) and VO2 maximum (P " 0.001).

Half of the previously untrained and previously trained groupswere randomly assigned to either antioxidant supplementationas described in Methods or to no supplementation (creating 4groups of 10 each) (supporting information (SI) Fig. S1). Allsubjects underwent a 4 week exercise training program irrespec-tive of antioxidant supplementation and previous training status.One untrained individual withdrew during the study for personalreasons unrelated to the experimental protocol.

Induction of Oxidative Stress by Short-Term Exercise. It is well-established that physical exercise increases ROS formation inskeletal muscle (12); however, it is not known if the health-promoting effects of exercise are partly due to this effect. Toreplicate the ROS-inducing capacity of exercise in our specificexperimental set-up, we subjected previously untrained individ-uals to 3 days of exercise with muscle biopsy before (Fig. S1,‘‘pre’’) and after this short-term intervention (Fig. S1, ‘‘early’’).We measured concentrations of thiobarbituric acid-reactive

Author contributions: M.R., M. Birringer, M.S., C.R.K., and M. Bluher designed research;M.R., K.Z., A.O., N.K., M. Birringer, M.K., M.S., and M. Bluher performed research; K.Z. andM.S. analyzed data; and M.R., M.S., C.R.K., and M. Bluher wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: The study design described in this paper has been deposited at Clinical-Trials.gov (registration no. NCT00638560).1To whom correspondence should be addressed. E-mail: [email protected]., K.Z., A.O., and M. Bluher contributed equally to this work.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903485106/DCSupplemental.

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intervention and antioxidant supplementation on expression ofthese genes (P ! 0.21 to 0.89 for interaction of vitamins bypretraining-status by ANOVA).

Taken together, these findings indicate that physical exerciseinduces several molecular regulators of insulin sensitivity irre-spective of previous training status and that this induction iswidely inhibited by antioxidant supplementation.

Molecular Promotion of Muscle Antioxidant Defense Following Phys-ical Exercise Is Abrogated by Antioxidants. The transcriptionalcoactivators PGC1! and PGC1" have not only been linked toincreased insulin sensitivity but have also been shown to induceexpression of several enzymes known to be involved into detox-ification of reactive oxygen species (ROS), including superoxidedismutase 2 (SOD2), glutathione peroxidase 1 (GPx1) andpossibly other enzymes of similar biochemical function (20).Accordingly, in the present study, physical exercise resulted in astrongly increased expression of SOD1 (Fig. 2 G and H, Left pairof bars, P " 0.05 to P " 0.001), SOD2 (Fig. 2 I and J, Left pairof bars, P " 0.05 to P " 0.001), and GPx1 (Fig. 2 K and L, Leftpair of bars, P " 0.001) in previously untrained and previouslytrained, antioxidant naïve individuals, whereas pretreatmentwith antioxidants prevented this induction (Fig. 2 G, I, and K,Right pair of bars, P ! 0.92, P ! 0.06, and P ! 0.10, respectively).Similar while less pronounced effects were observed for catalase(CAT) (P ! 0.045 and P ! 0.13, not depicted). When comparingthe relative expression of these enzyme-encoding mRNAs in thepostinterventional state in the presence and absence of antioxi-dants, exercise increased expression to a much lesser, if any,extent in antioxidant-supplemented individuals whether previ-ously untrained or previously trained (Fig. 2 G-L, shaded bars,P " 0.05 to P " 0.001). As observed for PPAR#, PGC1!, andPGC1", we found a strong effect of antioxidant supplementationto block exercise training-induced expression of antioxidantenzyme mRNAs for the entire sample of previously trained anduntrained individuals (n ! 39) (Fig. S4 D–F) (SOD1: P " 0.001;SOD2: P ! 0.01; GPx1: P " 0.001; and CAT: P ! 0.81, all forANOVA). In some of these cases, previous training status hadan impact on the effects of exercise intervention and antioxidantsupplementation on 2 of the expression levels (SOD1: P ! 0.003;SOD2: P ! 0.32; GPx1: P ! 0.046; and CAT: P ! 0.83, all forinteraction of vitamins by pretraining-status by ANOVA). How-ever, this effect was restricted to SOD1 and GPx1, while, notably,the mitochondrially active SOD2 appeared to be unaffected byprevious training status.

Taken together, physical exercise induces numerous molecularregulators of insulin sensitivity and antioxidant defense, most ofwhich are almost completely inhibited by antioxidant pretreat-ment in healthy young men (Fig. 3).

DiscussionBased on the evidence derived from the current study, we herepropose an essential role for exercise-induced ROS formation inpromoting insulin sensitivity in humans. This induction appearsto involve the ROS-dependent transcriptional coactivatorsPGC1! and PGC1", and the transcription factor PPAR# andtheir targets SOD1, SOD2, GPx1, and, to a reduced extent, CAT.Most importantly, these changes in gene expression and theincrease in insulin sensitivity following physical exercise arealmost completely abrogated by daily ingestion of the commonlyused antioxidants vitamin C and vitamin E. Thus, antioxidantsupplementation blocks many of the beneficial effects of exerciseon metabolism.

This direct molecular link between exercise-dependent for-mation of ROS, activation of PGC1!, PGC1" and PPAR# on theone hand and increased insulin sensitivity on the other hand,strongly suggest that oxidative stress can be instrumental inpreventing type 2-diabetes. The transcriptional coactivator

PGC1! has been previously linked to type 2 diabetes in humans(21, 22). This protein has also been shown to be inducible byvarious oxidative stressors (20), as well as physical exercise inrodents, notably in a vitamin C sensitive manner (17). Given itssynergistic potency in coactivating the transcription factorPPAR# to promote insulin sensitivity, the previously establishedrole of PGC1! as a ROS sensor in neurons that in turn inducesROS defense (20), as well as in rodent muscle (17), suggests thatactivation of PGC1! and possibly PGC1" may be importantfactors in promoting insulin sensitivity by both exercise and ROSin skeletal muscle. Moreover, in addition to the increase ininsulin sensitivity following exercise-induced ROS formation, wealso observe an induction of all relevant ROS defense enzymeexpression levels, namely SOD1 and SOD2, GPx1, and, to areduced extent, CAT. Notably, some of these enzymes have beenpreviously linked to transcriptional promotion by PGC1! (20),and, at least for SOD2, exercise has been shown to induce itsexpression in rodents (23).

Nevertheless, the published evidence is ambiguous with anumber of studies suggesting that exposure to ROS may promoteinsulin resistance (24, 25) whereas others find the opposite (14).In the present study, we find that increased ROS formationefficiently counteracts insulin resistance. Previously publishedfindings in nonprimate models also support this interpretation(13–17). One possible explanation for the apparent conflictbetween the different studies may be that those studies suggest-ing an inverse relation between ROS and insulin sensitivity wereobtained in models of continuous exposure to increased levels ofROS (24, 25), whereas our current findings and those of otherstudies (23, 26) may reflect transient increases in ROS duringlimited periods of physical exercise only.

This notion is further supported by the fact that most negativeeffects of antioxidant supplements observed in the current studyoccur irrespective of previous training status. While most effectsappear quantitatively more pronounced within the previouslyuntrained study group (i.e., Left arms of Figs. 1, 2, and S1), thedata do not support the assumption that antioxidant supplementintake is less detrimental in previously trained subjects.ANOVA, which considered covariables as stated in Methods,indicates that most effects of antioxidants are similar in bothpretrained and untrained individuals (Fig. S3 and Fig. S4) and

Fig. 3. Mitohormesis links physical exercise and subsequent formation ofreactive oxygen species to insulin sensitivity and antioxidant defense. Physicalexercise exerts ameliorating effects on insulin resistance by increasing mito-chondrial formation of reactive oxygen species in skeletal muscle to induceexpression of PGC1!, PGC1", and PPAR# as inducers of insulin sensitivity, aswell as superoxide dismutases 1 and 2 and glutathione peroxidase 1, keyenzymes of ROS defense. Notably, by blocking exercise-dependent formationof reactive oxygen species due to ingestion of antioxidant supplements,health promoting effects of physical exercise are abolished, and physicalexercise fails to promote insulin sensitivity and antioxidant defense in thepresence of vitamin C and vitamin E.

8668 ! www.pnas.org"cgi"doi"10.1073"pnas.0903485106 Ristow et al.

Antioxidants prevent health-promoting effectsof physical exercise in humansMichael Ristowa,b,1,2, Kim Zarsea,2, Andreas Oberbachc,2, Nora Klotingc, Marc Birringera, Michael Kiehntopfd,Michael Stumvollc, C. Ronald Kahne, and Matthias Bluherc,2

aDepartment of Human Nutrition, Institute of Nutrition, University of Jena, Jena D-07743, Germany; bGerman Institute of Human Nutrition,Potsdam-Rehbrucke D-14558, Germany; cDepartment of Medicine, University of Leipzig, Leipzig D-04103, Germany; dInstitute of Clinical Chemistry andLaboratory Medicine, University of Jena, Jena D-07743, Germany; and eResearch Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215

Contributed by C. Ronald Kahn, March 31, 2009 (sent for review March 14, 2009)

Exercise promotes longevity and ameliorates type 2 diabetesmellitus and insulin resistance. However, exercise also increasesmitochondrial formation of presumably harmful reactive oxygenspecies (ROS). Antioxidants are widely used as supplements butwhether they affect the health-promoting effects of exercise isunknown. We evaluated the effects of a combination of vitamin C(1000 mg/day) and vitamin E (400 IU/day) on insulin sensitivity asmeasured by glucose infusion rates (GIR) during a hyperinsuline-mic, euglycemic clamp in previously untrained (n ! 19) and pre-trained (n ! 20) healthy young men. Before and after a 4 weekintervention of physical exercise, GIR was determined, and musclebiopsies for gene expression analyses as well as plasma sampleswere obtained to compare changes over baseline and potentialinfluences of vitamins on exercise effects. Exercise increased pa-rameters of insulin sensitivity (GIR and plasma adiponectin) only inthe absence of antioxidants in both previously untrained (P <0.001) and pretrained (P < 0.001) individuals. This was paralleledby increased expression of ROS-sensitive transcriptional regulatorsof insulin sensitivity and ROS defense capacity, peroxisome-proliferator-activated receptor gamma (PPAR"), and PPAR" coac-tivators PGC1# and PGC1$ only in the absence of antioxidants (P <0.001 for all). Molecular mediators of endogenous ROS defense(superoxide dismutases 1 and 2; glutathione peroxidase) were alsoinduced by exercise, and this effect too was blocked by antioxidantsupplementation. Consistent with the concept of mitohormesis,exercise-induced oxidative stress ameliorates insulin resistanceand causes an adaptive response promoting endogenous antiox-idant defense capacity. Supplementation with antioxidants maypreclude these health-promoting effects of exercise in humans.

aging ! hormesis ! insulin resistance ! oxidative stress !reactive oxygen species

Type 2 diabetes mellitus is increasing worldwide at epidemicrates and is associated with both microvascular and macro-

vascular complications (1). Type 2 diabetes mellitus is caused bya combination of insulin resistance involving a number ofperipheral tissues, including skeletal muscle (2, 3), and aninadequate !-cell response despite normal or even increasedamounts of circulating insulin.

Physical exercise exerts numerous favorable effects on generalhealth (4) and specifically has been shown to improve glucosemetabolism in the insulin-resistant state (5). This effect may beindependent of exercise-related changes in body mass (6). More-over, physical exercise has been shown to be effective in pre-venting type 2 diabetes in high risk individuals (7, 8) and may beeven more effective than the most widely used anti-diabetic drug,metformin (9).

These beneficial effects of physical exercise on insulin resis-tance involve multiple mechanisms, including enhanced expres-sion of glucose transporters and translocation of glucose trans-porters to the plasma membrane independent of insulin (10).Exercise, as well as weight loss, has been linked to activation ofmitochondrial metabolism, and reduced mitochondrial metab-

olism has been functionally connected with type 2 diabetes (11).Mitochondria, however, are also the main source of reactiveoxygen species (ROS), which are inevitable by-products ofoxidative glucose metabolism. Muscle is also known to generatefree radicals, especially during contraction and physical exercise(12). It has been suggested that ROS may mediate some health-promoting effects, at least in nonprimate model systems (13–17).

We here evaluated the possibility that ROS are required forthe insulin-sensitizing capabilities of physical exercise in healthyhumans and that commonly used antioxidants, such as vitaminC and vitamin E, may abrogate the health-promoting effects ofboth physical exercise and oxidative stress in humans.

ResultsBaseline Characteristics. Of the 40 individuals included in thepresent study, 20 were known to be previously trained, and 20were previously untrained. Study subject characteristics in thepreinterventional state are given in Table 1. No significantdifferences in age, height, body mass index, fat free mass, or VO2maximum were observed within the groups (Table 1) and nosignificant differences in age, height and body mass index wereobserved between untrained and pretrained groups. Not sur-prisingly, pretrained individuals had a significantly higher fatfree mass (P ! 0.03) and VO2 maximum (P " 0.001).

Half of the previously untrained and previously trained groupswere randomly assigned to either antioxidant supplementationas described in Methods or to no supplementation (creating 4groups of 10 each) (supporting information (SI) Fig. S1). Allsubjects underwent a 4 week exercise training program irrespec-tive of antioxidant supplementation and previous training status.One untrained individual withdrew during the study for personalreasons unrelated to the experimental protocol.

Induction of Oxidative Stress by Short-Term Exercise. It is well-established that physical exercise increases ROS formation inskeletal muscle (12); however, it is not known if the health-promoting effects of exercise are partly due to this effect. Toreplicate the ROS-inducing capacity of exercise in our specificexperimental set-up, we subjected previously untrained individ-uals to 3 days of exercise with muscle biopsy before (Fig. S1,‘‘pre’’) and after this short-term intervention (Fig. S1, ‘‘early’’).We measured concentrations of thiobarbituric acid-reactive

Author contributions: M.R., M. Birringer, M.S., C.R.K., and M. Bluher designed research;M.R., K.Z., A.O., N.K., M. Birringer, M.K., M.S., and M. Bluher performed research; K.Z. andM.S. analyzed data; and M.R., M.S., C.R.K., and M. Bluher wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: The study design described in this paper has been deposited at Clinical-Trials.gov (registration no. NCT00638560).1To whom correspondence should be addressed. E-mail: [email protected]., K.Z., A.O., and M. Bluher contributed equally to this work.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903485106/DCSupplemental.

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substances (TBARS), a well-established marker of overall oxi-dative stress reflecting oxidized lipids and thus ROS formationin mammals, within skeletal muscle of these previously untrainedindividuals in the presence or absence of antioxidant treatment.As expected (12), we observed a more than 2-fold increase inoxidative stress, as reflected by TBARS levels, following physicalexercise in the absence of antioxidants (Fig. S2, Left pair of bars,P ! 0.008). By contrast, those individuals taking antioxidantsupplements showed no significant increase in muscle TBARSlevels after exercise (Fig. S2, Right pair of bars, P ! 0.19) resultingin significantly reduced TBARS formation after 3 days ofexercise in comparison to untreated individuals (Fig. S2, shadedbars, P ! 0.03). Thus, consistent with previous findings (12),these observations suggest that short-term physical exerciseinduces skeletal muscle ROS formation and that antioxidantsupplements reduce this formation, at least during the first 3 days.

Antioxidants Prevent Increase of Insulin Sensitivity Following PhysicalExercise. Glucose infusion rates. Physical training has been shown toameliorate insulin resistance and to improve glucose metabolism(5). Thus, both previously untrained and previously trainedindividuals were subjected to training with twenty 85 minsessions of defined physical exercise on 5 days per week (with orwithout addition of antioxidant supplementation) with measure-ment of insulin sensitivity by glucose infusion rates (GIR) duringa hyperinsulinemic euglycemic clamp (18). As expected (5),nonsupplemented individuals showed a significant increase inGIR, i.e., increased insulin sensitivity after 4 weeks of exerciseirrespective of previous training status (previously untrained:Fig. 1A, Left pair of bars, P " 0.001; and previously trained: Fig.1B, Left pair of bars, P " 0.001), confirming previous findingsthat physical exercise induces an increase in insulin sensitivity. Bycontrast, neither previously untrained individuals and pretrainedindividuals who received antioxidants exhibited significantchanges in GIR following exercise (Fig. 1 A, Right pair of bars,P ! 0.07, and B, Right pair of bars, P ! 0.89). Thus physicalexercise induced an increase in insulin sensitivity only in theabsence of antioxidants. The same impaired effect of exercise onGIRs seen in the antioxidant-treated group was also apparent inthe previously untrained individuals when the supplement-treated individuals were compared to nonsupplemented indi-viduals (Fig. 1 A, shaded bars, P ! 0.003). Finally, analysis of all39 individuals, irrespective of straining status, demonstrates ahighly significant (Fig. S3A, P " 0.001 for ANOVA) effect ofantioxidant supplementation on the blockage of exercise-induced improvement of GIR. Interestingly, inclusion of thebaseline training status in the statistical analyses revealed thatthe effect of exercise on GIR was independent of the pretrainingstatus of the study participants (P ! 0.58 for interaction term ofpretraining status by ANOVA), indicating that antioxidants canabolish the insulin sensitizing effects of exercise in both un-trained and previously well-trained subjects. We additionally

measured serum concentrations of TBARS. When comparingthese to GIRs in the postinterventional state in all 39 individuals,we observed a significant correlation with TBARS serum levels(Pearson’s r ! 0.353, P " 0.05); this suggests that TBARS serumlevels, at least to some extent, correlate with insulin sensitivityirrespective of antioxidant supplementation and previous train-ing status, whereas no significant effect of antioxidant supple-mentation on postinterventional TBARS levels was observed(P ! 0.73 for ANOVA, and P ! 0.43 for interaction of vitaminsby pretraining status by ANOVA).Additional plasma markers of insulin sensitivity. Plasma concentrationsof the adipocyte-derived secretory protein adiponectin havebeen shown to be positively correlated with insulin sensitivity inhumans and inversely correlated with type 2 diabetes risk (19).We observe an increase in circulating adiponectin levels follow-ing physical exercise in both previously untrained individuals(Fig. 1C, Left pair of bars, P " 0.001) and pretrained individuals(Fig. 1D, Left pair of bars, P " 0.001). In contrast, previously

Table 1. Baseline characteristics of study subjects

Previously untrained study group Pretrained study group

No supplements Vitamin C/Vitamin E

P-Value

No supplements Vitamin C/Vitamin E

P-ValueMean SD Mean SD Mean SD Mean SD

Number/sex 10/male n.a. 10/male n.a. n.a. 10/male n.a. 10/male n.a. n.a.Age, years 26.70 4.34 27.44 3.02 0.69 25.40 2.15 26.00 1.95 0.54Height, cm 179.20 4.98 182.32 8.83 0.38 182.96 3.87 177.91 7.16 0.08Body Mass Index, kg/m2 24.37 2.53 24.19 1.84 0.87 24.33 1.31 23.33 1.36 0.13Fat free mass, kg 60.64 5.88 66.20 7.28 0.10 69.71 3.85 65.84 5.31 0.09VO2 max, ml/min!kg#1 45.85 5.46 45.21 7.03 0.84 54.42 4.90 54.31 5.10 0.96

n.a. ! not applicable.

Fig. 1. Antioxidants prevent exercise-dependent induction of insulin sensi-tivity. (A) Glucose infusion rates (GIR) during euglycemic hyperinsulinemicclamps in previously untrained individuals before (white bars) and after(shaded bars) physical exercise over 4 weeks. (Left pair of bars) Individuals nottaking any medication or placebo; (Right pair of bars) individuals taking bothvitamin C (1000 mg/day) as well as vitamin E (400 IU/day). Bars depict means,error bars show standard error means (applies to all subsequent panels andfigures). Significances (applies to all subsequent panels and Fig. 3): * indicates0.01 " P " 0.05 comparing data before and after 4 weeks of exercise, #indicates 0.01 " P " 0.05 comparing ‘‘no suppl.’’ with ‘‘Vit.C/Vit.E’’ groupsafter intervention, ** indicates 0.001 ! P ! 0.01 comparing data before andafter 4 weeks of exercise, ## indicates 0.001 ! P ! 0.01 comparing ‘‘no suppl.’’with ‘‘Vit.C/Vit.E’’ groups after intervention, *** indicates P " 0.001 compar-ing data before and after 4 weeks of exercise, ### indicates P " 0.001comparing ‘‘no suppl.’’ with ‘‘Vit.C/Vit.E’’ groups after intervention. (B) Thesame set of data derived from a physically pretrained group of individuals. (C)Plasma adiponectin levels in the previously untrained and previously trained(D) state.

8666 ! www.pnas.org"cgi"doi"10.1073"pnas.0903485106 Ristow et al.

Oral administration of vitamin C decreases muscle mitochondrialbiogenesis and hampers training-induced adaptations in enduranceperformance1–3

Mari-Carmen Gomez-Cabrera, Elena Domenech, Marco Romagnoli, Alessandro Arduini, Consuelo Borras,Federico V Pallardo, Juan Sastre, and Jose Vina

ABSTRACTBackground: Exercise practitioners often take vitamin C supple-ments because intense muscular contractile activity can result inoxidative stress, as indicated by altered muscle and blood glutathi-one concentrations and increases in protein, DNA, and lipid peroxi-dation. There is, however, considerable debate regarding the bene-ficial health effects of vitamin C supplementation.Objective: This study was designed to study the effect of vitamin Con training efficiency in rats and in humans.Design: The human study was double-blind and randomized. Four-teen men (27–36 y old) were trained for 8 wk. Five of the men weresupplemented daily with an oral dose of 1 g vitamin C. In the animalstudy, 24 male Wistar rats were exercised under 2 different protocolsfor 3 and 6 wk. Twelve of the rats were treated with a daily dose ofvitamin C (0.24 mg/cm2 body surface area).Results: The administration of vitamin C significantly (P ! 0.014)hampered endurance capacity. The adverse effects of vitamin C mayresult from its capacity to reduce the exercise-induced expression ofkey transcription factors involved in mitochondrial biogenesis.These factors are peroxisome proliferator–activated receptor co-activator 1, nuclear respiratory factor 1, and mitochondrial transcrip-tion factor A. Vitamin C also prevented the exercise-induced ex-pression of cytochrome C (a marker of mitochondrial content) and ofthe antioxidant enzymes superoxide dismutase and glutathione per-oxidase.Conclusion: Vitamin C supplementation decreases trainingefficiency because it prevents some cellular adaptations toexercise. Am J Clin Nutr 2008;87:142–9.

KEY WORDS Free radicals, VO2max, antioxidant enzymes,antioxidant supplements, exercise, exhaustion, vitamins, gene ex-pression, hormesis, reactive oxygen species

INTRODUCTION

Acute physical exercise induces augmented generation of re-active oxygen species (ROS) in muscle and in other organs (1–3).Because of that, it has been generally accepted over the past 20 ythat increasing the concentrations of antioxidants within a mus-cle cell should provide greater protection against these oxidizingagents and should reduce fatigue (4–7). However, the functionalsignificance of exercise-induced oxidative stress is open to dis-cussion. Results from several laboratories indicate that ROS aresignals that serve to up-regulate the expression of a number of

genes (8, 9). Thus, ROS can exert favorable effects and can beinvolved in the process of training adaptation. Up-regulation ofendogenous antioxidant systems in response to regular trainingexerts beneficial effects in the prevention of chronic diseaseprocesses (10) and has also been related to longevity in flies (11)and mice (12).

The maximal capacity to take up, transport and utilize oxygenduring exercise is VO2max (13). Endurance is defined as the timelimit of a person’s or animal’s ability to maintain a specific powerlevel during a running protocol (14). Large-scale epidemiologicstudies of humans with and without cardiovascular disease showthat low aerobic exercise capacity is an stronger predictor ofmortality than are other established risk factors, such as diabetes,smoking, hypertension, or chronic obstructive pulmonary dis-ease (15–18). These observations are consistent with the role ofimpaired regulation of mitochondrial function as an importantmechanism for low aerobic capacity (19). The relations amongVO2max, muscle oxidative capacity, endurance capacity, andmaximal aerobic workload capacity have been discussed foryears (20). Davies et al (21) concluded that muscle oxidativecapacity (ie, the mitochondrial content of muscle) was a majordeterminant of endurance capacity, whereas VO2max was onlyindirectly related to endurance capacity but was directly relatedto exercise intensity. In eukaryotic cells, mitochondrial biogen-esis requires gene products from 2 physically separated ge-nomes—one contained within the organelle and the other con-tained within the nucleus. Peroxisome proliferator–activatedreceptor co-activator 1 (PGC-1) is a recently identified coacti-vator of nuclear receptors. It powerfully induces mRNA expres-sion for important nuclear transcription factors such as nuclearrespiratory factor 1 (NRF-1) and mitochondrial transcription

1 From the Department of Physiology, Faculty of Medicine, University ofValencia, Valencia, Spain (M-CG-C, ED, AA, FVP, JS, and JV); the CatholicUniversity of Valencia, Valencia, Spain (CB); and the Polytechnic Univer-sity of Valencia, Valencia, Spain (MR).

2 Supported by grants no. SAF 2004-03755 (to JV) and GV06/289 (toM-CG-C) and by la Red Tematica de investigacion cooperativa en enveje-cimiento y fragilidad (RETICEF) from the Instituto de Salud Carlos III(ISCIII2006-RED13-027).

3 Reprints not available. Address correspondence to J Vina, Department ofPhysiology, Faculty of Medicine, Blasco Ibanez, 15, Valencia, Spain 46010.E-mail: [email protected].

Received May 27, 2007.Accepted for publication September 5, 2007.

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Ascorbic acid supplementation does not attenuate post-exercise musclesoreness following muscle-damaging exercise but may delay the recoveryprocess

Graeme L. Close1*, Tony Ashton2, Tim Cable1, Dominic Doran1, Chris Holloway1, Frank McArdle2

and Don P. M. MacLaren1

1Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Henry Cotton Campus, 15–21

Webster Street, Liverpool L3 2ET, UK2School of Clinical Sciences, Division of Metabolic and Cellular Medicine, University of Liverpool, Liverpool L69 3GA, UK

(Received 7 October 2005 – Revised 15 December 2005 – Accepted 19 December 2005)

Exercise involving lengthening muscle actions, such as downhill running, results in delayed onset muscle soreness (DOMS), which may be attribu-table to reactive oxygen species (ROS). Although exercise causes oxidative stress, any link between ROS and DOMS remains speculative. There isemerging evidence to suggest that ROS play an important physiological role, assisting in the recovery process and protecting the cell from futuredamage; however, this has not been fully established. Despite this uncertainty as to the precise role of ROS, attempts to prevent post-exercise ROSproduction through antioxidant intervention are still common. The study investigated the effects of ascorbic acid supplementation on ROS pro-duction and DOMS following downhill running. Subjects were assigned to two groups. The ascorbic acid group (group AA) received 1 g ascorbicacid 2 h pre-, and for 14 d post-downhill running, whilst the placebo group (Pl group) received a placebo. Blood samples were drawn pre-sup-plement, pre- and post-exercise, and then 1, 2, 3, 4, 7 and 14 d post-exercise for analysis of ascorbate, malonaldehyde and total glutathione.DOMS was assessed using a visual analogue scale and pressure algometry. Muscle function was assessed using isokinetic dynamometry.Plasma ascorbate was elevated throughout in group AA compared with the Pl group. Downhill running resulted in DOMS in both groups.Muscle function was impaired post-exercise in both groups, although a delayed recovery was noted in group AA. Malonaldehyde increased 4 dpost-exercise in the Pl group only. Ascorbic acid supplementation attenuates ROS production following downhill running, without affectingDOMS. Furthermore, ascorbic acid supplementation may inhibit the recovery of muscle function.

Reactive oxygen species: Exercise: Eccentric muscle torque: Antioxidants: Muscle damage

Unaccustomed or excessive exercise often results in the sen-sation of muscular discomfort and pain that is characterisedby its delay in onset and has been termed ‘delayed onsetmuscle soreness’ (DOMS) (Newham et al. 1983). DOMS isoften first noticed 24 h post-exercise and presents as a dullache similar to that of a bruise. Despite extensive researchinto DOMS, the underlying causes and methods of preventionremain unresolved (Close et al. 2005).We have previously demonstrated that 30min of downhill

running at a sub-maximal intensity results in muscle damageand a significant increase in reactive oxygen species (ROS)production and subsequent lipid peroxidation in the days fol-lowing the exercise (Close et al. 2004). This increase inlipid peroxidation was speculated to be due to phagocyte-derived superoxide (O2

z2) production resulting in the sub-sequent formation of the more potent hydroxyl radical(zOH). However, it is still unclear if this production of ROSin the days following lengthening muscle contractions is apathological process that amplifies the sensations of DOMS,

or is an essential physiological process assisting in the recov-ery from the initial trauma. The reason for this is that merelythe presence of ROS cannot determine if their production isinvolved in the observed pathology. The only way that ROScan be confirmed to be involved in the pathology is throughspecific intervention (Jackson, 1999). Despite this uncertaintyas to the exact role of ROS following contraction-induceddamage, it is common practice for athletes to use antioxidanttherapy to prevent post-exercise ROS production and, further-more, there is still extensive research investigating ways toprevent DOMS and muscle damage using antioxidant sup-plementation (Jakeman & Maxwell, 1993; Goldfarb, 1999;Thompson et al. 2001a).

Ascorbic acid is a water-soluble, dietary antioxidant presentin the cytosolic compartment of the cell and the extracellularfluid, and is known to be a powerful inhibitor of lipid peroxi-dation (Powers et al. 2004), in conjunction with a-tocopherol(Evans, 2000). The spatial arrangement of ascorbic acidallows it to scavenge aqueous-phase a-tocopherol radicals

*Corresponding author: Dr Graeme L. Close, fax !44 151 706 5802, email [email protected]

Abbreviations: DOMS, delayed onset muscle soreness; group AA, ascorbic acid group; MDA, malonaldehyde; Pl group, placebo group; ROS, reactive oxygen

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ASCORBIC ACID SUPPLEMENTATION ATTENUATES ROS PRODUCTION FOLLOWING DOWNHILL RUNNING, WITHOUT AFFECTING DOMS.

FURTHERMORE, ASCORBIC ACID SUPPLEMENTATION MAY INHIBIT THE RECOVERY OF MUSCLE FUNCTION.

[68,69]. We have reported that a single bout of exercise, whichcaused oxidative damage to skeletal muscle [18], liver, andkidney [53], did not cause damage to the brain [70]. Further, theactivities of antioxidant enzymes (Cu,Zn-SOD, Mn-SOD,catalase (CAT), GPX) were not significantly altered by anexercise session. A similar phenomenon has been reported afterexercise training. Treadmill running did not alter the activitiesof SOD, CAT, or GPX in the brain of rats. However, exercisedrats with diabetes have shown decreased Cu,Zn-SOD and GPXactivities [71].

The first study, which described a causative relationshipbetween the accumulation of oxidative damage to brainproteins, RCD, and certain cognitive functions, was an age-related study [72], and their results have been confirmed byother laboratories [73]. Oxidative damage has been associatedwith poor physiological function of the brain [72–74]. We havealso shown that regular exercise training attenuated the age-related accumulation of RCD in the brain, increased the activityof the proteasome complex, and improved brain function [75].Chronic exercise training, using the rat model, did not causesignificant alteration of lipid peroxidation levels in the brain. Onthe other hand, the supplementation of vitamin C elevated theoxidative damage of lipids [76]. We have subjected rats tomoderate, very hard, and overtraining and found, even withvery hard training and overtraining, beneficial effects on brainfunction and lowered accumulation of RCD [77]. The content of8-OHdG was not significantly altered by the overtrainingprotocol, and activity of OGG1 was also not changed in thecrude cell extract [77]. We recently evaluated the activity of theDNA damage/repair enzyme of OGG1, in the nucleus andmitochondria of trained and detrained rats, and did not detectany significant alterations [78].

Brain-derived neurotrophic factor (BDNF) is one of the mostversatile, important neurotrophic factors in the brain. It plays acurricular role in the learning process, including memory,locomotion, behaviors, and a wide range of stress responses[79,80]. It has been suggested that BDNF regulates braindevelopment, neuroplasticity, neurogenesis, neurite outgrowth,synaptic plasticity, and cell survival [81,82]. The expression andprotein content of BDNF have been shown to be up-regulatedby exercise and oxidative stress [60]. We have measured ROSlevels by electron spin resonance and found that in some brainregions, exercise training increases the levels of ROS, althoughthe level of oxidative damage does not increase ([78,83];Z. Szabo, unpublished; S. Siamilis, unpublished), and a cor-relation was found between ROS level and BDNF concentrationin the spinal cord (S. Siamilis et al., unpublished). In addition,exercise results in angiogenesis in the brain by increasing thelevel of vascular endothelial growth factor (VEGF) in thehippocampus [84] and it seems that ROS are the mediators ofVEGF expression [85].

The activity of some redox-sensitive transcription factorswas investigated in exercise studies and the findings revealedthat CREB, synapsin, and MAPK activity increased in the brainwith exercise, and oxidative challenge alone also regulatedthese [79,80], suggesting that exercise-induced changes inredox homeostasis could be, at least in some part, mediated by

ROS. The observation that exercise training attenuates theoxidative stress-related damage in brain is in accordance withthis statement [62,66,74,86].

Data relating to the effects of exercise on brain indicate thataccumulation of oxidative damage impairs brain function, andexercise, under certain conditions, can attenuate the accumula-tion of damage, causing improved brain function. Moreover,ROS could play a role in the induction of neurotrophins, whichmight be important for neurotrophin-caused neurogenesis.

Conclusion

The available data strongly indicate that regular exerciseplays a preventive role against lifestyle-dependent diseases andthe molecular mechanism behind this favorable effect could belinked to redox homeostasis, a free radical-related adaptivemechanism. The adaptive mechanism is initiated by transcriptionfactors, resulting in increased activities of the antioxidantenzymes, and more effective repair and housekeeping by theDNA repair enzymes and proteasome complex. The molecularadaptation then leads to an improved physiological function andenhanced resistance to oxidative stress. Most importantly, theexercise-induced oxidative challenge-associated adaptation issystemic. These beneficial consequences of regular exercise are insharp contrast to the effects of exhaustive exercise on unpreparedtissues that results in, apparently, harmful outcomes (Fig. 1).These consequences of exercise fit well with the concept ofhormesis [87].

On the other hand, physical inactivity leads to impairment inphysiological functions and reduces the whole body resistanceto oxidative stress. Moreover, it seems that physical inactivitythrough molecular pathways could facilitate the incidence of

Fig. 1. The redox homeostasis-associated changes as a result of single bout ofexercise and regular exercise compared to physical inactivity. Sedentarylifestyle, of human beings or experimental animals, is often regarded as acontrol, but this most probably should be considered as a nonphysiological,physically inactive condition. One can consider that regular exercise is a normalpart of everyday life and it is phylogenetically conserved in evolution; henceinactivity has very serious consequences, which are reflected in redoxhomeostasis. The numbers represent selected references that support thehypothesis. RONS, reactive oxygen and nitrogen species.

156 Z. Radak et al. / Free Radical Biology & Medicine 44 (2008) 153–159

Radak Z, Chung HY, Goto S. Free Radic Biol Med. 2008 Jan 15;44(2):153-‐9.

most well-described age-related disorders (Booth and Lees, 2007; Booth et al., 2002; Feher and Lengyel, 2006; Taylorand Poston, 2006; Thompson, 2006; Wendel-Vos et al., 2004). Muscular activity results in increased capillarizationand better oxygen supply to different regions of the brain, and, naturally, the increased metabolic activity of neuronsresults in increased oxygen uptake, which probably is associated with increased activity of antioxidant and oxidativedamage repairing enzymes (Cotman and Berchtold, 2002; Fabel et al., 2003; Radak et al., 2001a, 2006). Moreover,physical activity results in up-regulation of neutrophins, which not only enhance brain function but play a critical rolein cell survival, and increase resistance against a variety of stressors (Mattson and Wan, 2005; Mattson et al., 2004;Mattson, 2005).

A causative relationship has been shown between the accumulation of carbonyl groups in amino acid residues, dueto the interaction with ROS, and specific brain functions (Carney et al., 1991; Radak et al., 2001b).We have shown thatregular exercise can increase the activity of proteasome complex, which is responsible for the degradation ofcarbonylated and other damaged proteins. A recent study by Lazarov et al. (2005) has shown that physical activityreduced the b-amyloid content in the brain of transgenic mice and this is due to the increased activity of neprilysin.

The systemic effects of exercise can also be observed in the liver, in which single bouts of exercise significantlyincrease the level of ROS and cause oxidative damage to lipids (Davies et al., 1982; Radak et al., 1995, 1996).Furthermore, we have demonstrated that regular exercise decreases the ROS concentration in liver, and attenuates theage-associated increase in ROS and the associated oxidative damage (Radak et al., 2004a). In addition, the DNAbinding of NF-kB, which is one of the most potent inflammatory transcription factors, is modified by aging, andregular exercise and has a rejuvenating effect in the context of NF-kB.

Therefore, the available information suggests regular exercise at moderate intensity can retard the aging processand ameliorate the insidious onset of age-associated diseases.

8. Conclusion

The response of biological systems to stressors can be described by a U-shaped curve. Physical exercise also evokesthis hormesis curve-response by the organism. The two end-points of the hormesis curve are inactivity andovertraining, and both of these result in decreased physiological function (Fig. 1). Normal and positively adaptedfunction of the organism can be achieved with regular moderate exercise bouts. The effects of exercise on the immunesystem, free radicals, muscle function, vascular function and aging appear to fit the hormesis curve.

Acknowledgement

The present work was supported by Hungarian grants: ETT 38388 awarded to Z. Radak.

Z. Radak et al. / Ageing Research Reviews 7 (2008) 34–42 39

Fig. 1. Typical hormesis curve and the effects of exercise. Moderate exercise increases the physiological function of different organs, increases the

rate of prevention against diseases and improves quality of life. Physical inactivity and strenuous exercise and overtraining increases the risk of

diseases and decreases physiological function.

Abstract Exercise increases oxygen consumption andcauses a disturbance of intracellular pro-oxidant-antioxi-dant homeostasis. Few data are available as to the cumu-lative effects of exercise on the antioxidant defenses ofthe neutrophil. We studied the effects of 90 days’ supple-mentation with placebo or an antioxidant cocktail of vi-tamin E (500 mg/day) and β-carotene (30 mg/day) andthe last 15 days also with vitamin C (1 g/day) on sports-men’s basal neutrophil antioxidant defenses. We ana-lyzed the activities of catalase, glutathione peroxidase,glutathione reductase and the activities and levels of su-peroxide dismutase, glutathione and glutathione disul-fide in neutrophils purified from antecubital vein bloodof sportsmen before and after diet supplementation. Plas-ma vitamin E, β-carotene and vitamin C concentrationsin the antioxidant-supplemented group were approxi-mately 1.6, 10, and 1.2 times higher respectively thanthose of the placebo group. The antioxidant-supplement-ed group presented a significantly higher glutathioneversus glutathione disulfide ratio in neutrophils (about20%) than the placebo one. Antioxidant supplementationenhances the antioxidant enzyme activity of superoxidedismutase and catalase in neutrophils.

Keywords Antioxidants · Exercise · Glutathione · Neutrophil · Oxidative stress

Introduction

Exercise increases oxygen consumption and causes adisturbance of intracellular pro-oxidant-antioxidant ho-meostasis [21]. The mitochondrial electron transportchain [26], polymorphonuclear neutrophils [27], andxanthine oxidase [29] have been identified as majorsources of intracellular reactive oxygen species (ROS)and free radical generation during exercise. The cellularantioxidant defense systems have demonstrated great ad-aptation to acute and chronic exercise [32]. However, ex-treme physical exercise causes oxidative damage to well-trained athletes, as indicated by an increase in plasmalevels of malondialdehyde [22, 28] and conjugated di-enes [28] or by an increase in urine 8-hydroxydeoxygua-nosine (8-OhdG) after an ultra-marathon [41] and as alsoindicated by an increase in erythrocyte catalase activityafter a duathlon competition [45]: in these situations ofstrenuous exercise the antioxidant defenses are over-whelmed [28]. Recent studies point to negative effects ofoxidative stress in sportsmen on the functionality of dif-ferent cells of the immune system [38]. Epidemiologicalreports suggest a higher risk of upper respiratory tract in-fection in endurance athletes due to prolonged exercise,which leads to a transient yet significant perturbation ofimmune and host defenses [39]. Some studies haveshown that diet supplementation with vitamin C reducesthe risk of upper respiratory tract infection [39].

The phagocytic cell of the immune system, which isthe cornerstone in host defense as the resistance mecha-nism to upper respiratory tract infections, is mediated byneutrophil action. Oxygen-dependent antimicrobialmechanisms are set in motion when neutrophils undergoa “respiratory burst” and produce toxic oxygen species[31]. These toxic oxygen species produced by neutro-phils could also be toxic to the neutrophil itself and con-tribute to the overall oxidative stress situation. Neutro-phils are protected against ROS by antioxidant enzymessuch as superoxide dismutase (SOD), catalase, glutathi-one peroxidase, and glutathione reductase. Althoughmany studies have focused on the adaptations of neutro-

P. Tauler · A. Aguiló · J.A. Tur · A. Pons (✉)Laboratori de Ciències de l’Activitat Física and Departament deBiologia Fonamental i Ciències de la Salut, Edifici Guillem Colom. Facultat de Ciències, Universitat de les Illes Balears, Campus Universitari, Crta, Valldemossa km 7.5, 07071, Palma de Mallorca, Balears, Spaine-mail: [email protected].: +34-971-173171, Fax: +34-971-173184E. FuentespinaLaboratori del Carme, Hospital Son Dureta, INSALUD, Plaça del Carme, 07071, Palma de Mallorca, Balears, Spain

Pflügers Arch - Eur J Physiol (2002) 443:791–797DOI 10.1007/s00424-001-0770-0

O R I G I N A L A RT I C L E

P. Tauler · A. Aguiló · E. Fuentespina · J. A. TurA. Pons

Diet supplementation with vitamin E, vitamin C and β-carotene cocktail enhances basal neutrophil antioxidant enzymes in athletes

Received: 14 June 2001 / Accepted: 10 October 2001 / Published online: 31 January 2002© Springer-Verlag 2002

dant or placebo intake, or by the training/competition pe-riod.

The changes observed in the total glutathione levelsin neutrophils over the 3 months were different in place-bo and in the antioxidant diet-supplemented group, asevidenced by the statistically significant interaction be-tween the two ANOVA factors (Table 2). The 3 monthsof training/competition decreased the total glutathionecontent of neutrophils about 17% in the placebo group,whereas the antioxidant supplementation maintained theinitial basal levels. In the end, the total glutathione con-tent of neutrophils in the supplemented group was 25%higher than in the placebo group. The antioxidant sup-plementation for 3 months significantly influenced theneutrophil GSH levels and the GSH/GSSG ratio, where-as the training/competition period significantly influ-enced the GSSG levels in neutrophils. No statisticallysignificant differences were observed in the initial valuesof GSH, GSSG, and the ratio GSH/GSSG between theplacebo and the antioxidant-supplemented group. How-ever, after the 3 months, the antioxidant diet supplemen-tation group presented significantly higher levels of GSHand higher values of GSH/GSSG than the placebo group.No differences were observed in the GSSG levels be-tween groups after the 3 months; however, the placebogroup increased this parameter about 20% whereas theantioxidant-supplemented group maintained the initialvalues.

Discussion

Plasma vitamin levels of sportsmen at the beginning ofthe study were within the normal range of well-nour-ished people [20], and we think that the intake of these

antioxidant nutrients with food was enough to cover therecommended daily allowance (RDA). The observed in-crease in vitamin E is less compared to that described insimilar experiences with well-nourished adults [48] andsimilar to others with sportsmen [43], probably becausesportsmen should take a higher amount of vitamin E thannormal adults in order to protect themselves against theoxidative stress associated with physical activity. In fact,daily diet supplementation with 1000 IU of vitamin E for10 days significantly decreases breath pentane excretion(an index of lipid peroxidation) [24], and vitamin E dietsupplementation also decreases the malondialdehyde se-rum level [15, 43].

β-Carotene is the supplemented antioxidant that in-creased most in the plasma after 3 months of supplemen-tation. The liposoluble nature of this compound is re-sponsible for its accumulation in the body. The very highlevels of plasma β-carotene in the supplemented groupare partly accounted for by the synergistic effects be-tween vitamin E and β-carotene, because vitamin E pre-vents β-carotene oxidation [18]. On the other hand, plas-ma vitamin A levels were similar in all groups and situa-tions studied, in accordance with the existence of ho-meostatic mechanisms for regulating vitamin A synthesisfrom its precursor, β-carotene [44]. The intake of β-caro-tene supplements could be beneficial against the intakeof vitamin A supplements in order to avoid the toxic ef-fects of an excess of vitamin A.

The increase of plasma vitamin C levels in the antiox-idant-supplemented group was the lowest of those pro-duced by the antioxidants supplemented. The hydrosolu-ble nature of this vitamin and the existence of homeo-static mechanisms for regulating vitamin C plasma levels[7, 42] could be responsible for this low plasma increasewith a vitamin C intake 16 times higher than the RDA.

795

Table 2 Basal neutrophil glutathione of sportsmen before and after supplementation with antioxidants. The results are the mean ±s.e.m.of ten subjects in the placebo group and ten subjects in the antioxidant-supplemented group

Initial Final ANOVA

Placebo Supplemented Placebo Supplemented G×T G T

Total glutathionenmol/ml of blood 2.34±0.08a 2.41±0.09a 2.06±0.20b 2.36±0.16a * * *nmol/109neutrophils 961±33a 991±37a 793±33b 989±41a * * *

GSHnmol/ml of blood 2.18±0.12 2.20±0.14 1.99±0.12 2.22±0.10# *nmol/109 neutrophils 887±33 900±37 700±39 901±48# *

GSSGnmol/ml of blood 0.10±0.01 0.10±0.01 0.13±0.01& 0.11±0.01a *nmol/109 neutrophils 44.1±3.3 45.0±2.7 52.7±1.3& 46.9±1.6a *

GSH/GSSG 20.2±1.9 20.5±2.3 13.8±1.8 19.4±2.0# *

* Significant effects of factor G, T or the interaction G×T (two-wayANOVA). Factor G represents the diet antioxidant supplementa-tion; factor T represents the training and competition period; G×Trepresents the interaction between the two factors. Different lettersindicate significant different values (ANOVA one-way test,P<0.05) when a significant G×T interaction is observed

# Significant differences between placebo and supplementedgroups (Student’s t-test for unpaired data, P<0.05)& Significant differences between initial and final values (Stu-dent’s t-test for unpaired data, P<0.05)

Alimento Ascorbato (mg) Acerola 1300 Pimientos Vermelhos 369 Goiaba 242 Couve 186 Pimentos verdes 128 Brócolos 113 Couve de Bruxelas 102 Couve-Flor 78 Morango 59 Papaia 56 Espinafre 51 Laranja 50 Toranja 38 Manga 35 Tangerina 31

COUVES DE BRUXELAS CONGELADAS DURANTE 6 MESES APRESENTAM MENOS 14 A 32% DE VITAMINA C EM RELAÇÃO AO MOMENTO DA COLHEITA.

Kmiecik W, Lisiewska Z. Rocz Panstw Zakl Hig. 1989;40(3):215-‐22.

S8 D. RUMM-KREUTER and I. DEMMEL

peroxidase between 80 and 100 degrees celsius [(Hottenroth (8), Zobel (20)].The amount of vitamin losses due to cooking depends on several factors, for

example, the type of food, the variety of food, the way of cutting, preparation, duration and method of cooking. Due to the high degree of variability a direct comparison of the results of the numerous investigations is difficult [Klein (9, 10), Warthesen (18), Augustin (1), Somogyi (14)]. Even a slight alteration could lead to different results.

DISCUSSION OF DIFFERENT RESULTS

Many investigators are engaged in the Ascorbic acid losses due to cooking. Blumenthal (3) compared for example the Ascorbic acid content in fresh Spinach when boiled (with water), steamed (without adding water) and cooked in a pressure cooker at approximately 120 degrees celsius.

The results show a distinctly higher Ascorbic acid content after the steaming method as opposed to pressure cooking or boiling. Pressure cooking and boiling produced similar ascorbic acid results.

Tests carried out by Meier-Ploeger (12), Somogyi (14) and Tempelhoff (17) confirm that after the steaming method without adding water the most significant Ascorbic acid content was found to be present. Pressure-cooking, however, in these tests showed better results than boiling.

Bielig (2), Herrmann (7) and Szoke (16) investigated the influence on Ascorbic acid content when it is affected by different pressure levels. They determined that after low pressures the Ascorbic acid content was greater than after high pressures.

Of course, cooking duration and the type of foodstuff play an important role as shown by some of our unpublished tests. Products, which need a long cooking period, like cabbage and bean soup, showed a higher Ascorbic acid content after pressure cooking than after steaming. On the other hand, products, which require only a short cooking period, like spinach and kohlrabi in slices, contained a higher concentration of Ascorbic acid after steaming rather than after pressure cooking (Rumm-Kreuter, 1986).

Further interesting cooking methods are stirfrying and microwave cooking.

Table 1. Ascorbic acid contents (mg/100g) in fresh spinach and in spinach after different cooking methods.

Blumenthal (1980).

J. Nutr. Sci. Vitaminol.

J. Nutr. Sci. Vitaminol., 36, S7-S15, 1990

Comparison of Vitamin Losses in Vegetables Due to Various Cooking Methods

D. RUMM-KREUTER and I. DEMMEL

Alfa Institut Eltville (FDG)

Summary Preparing vegetables with heat the contents of their constituents will change to a various extend. Particularly the water-soluble and the heat-sensitive vitamins are affected. At an early stage the vitamin C losses were investigated, because of vitamin C's indicating function for oxidations and leaching-out processes (1, 2, 7, 11-13, 15, 17). The degree of vitamin losses is influenced by various factors, for example the type of food, variety of vegetables, the way of cutting, preparation, duration and method of cooking. The influence of the various cooking methods with regard to the losses of certain water-soluble vitamins will be discussed.Key Words cooking methods, ascorbic acid, folic acid, thiamine, riboflavin, niacin, pyridoxine

Foods are prepared in order to become edible and enjoyable. The choice of the cooking method depends on the individual or cultural dietary habits.

By preparing food with heat, not only alterations in carbohydrates, fats and proteins, but also alterations in the vitamins and minerals take place to a various extent.

Particularly the water soluble and heat sensitive vitamins are affected. Therefore the main subject of this report is the reduction in the content of vitamins like Ascorbic acid, Thiamine, Riboflavin, Niacin, Pyridoxine and Folic acid caused by the preparation of food with heat.

The degree of vitamin losses is influenced by the vitamin's sensitivity to factors like light, oxygen, water and temperature.

According to Paulus (13) the losses are higher, the longer the period of time when the different factors can affect the food and the greater the surface of the food.

Ascorbic acid is often used as an indicating vitamin, because it is very sensitive to heat and to water. The Ascorbic acid losses during the cooking process are caused by the lixiviation and the destruction by oxidation. According to Weise (19) the enzymatic activity of the oxidases reaches the highest level at 40 degrees celsius. Higher temperatures destroy the enzymes. The irreversible inactivation of phenoloxidase takes place between 70 and 80 degrees celsius, the inactivation of

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Yuan et al. / J Zhejiang Univ Sci B 2009 10(8):580-588

583

stir-frying (16% and 24%, respectively) treatments. In contrast, steaming did not cause any significant loss of vitamin C, compared with the raw sample (Fig.3).

The cooking methods of microwaving, steaming, and stir-frying mostly kept the content of total caro-tenoids after cooking (Fig.3), while boiling and stir-frying/boiling caused the loss of total carotenoids by 13% and 28%, respectively (P<0.05) (Fig.3). Effect of cooking on total and individual glucosi-nolates

The contents of total and individual glucosi-nolates were quantitatively determined in broccoli cooked by different methods. The main aliphatic glucosinolates in broccoli were glucoraphanin, fol-lowed by glucoiberin, while the main indole glucosi-nolates were glucobrassicin and neoglucobrassicin (Table 1). The glucosinolate profile of broccoli in our study was consistent with the previous reports (Jia et al., 2009; Schreiner et al., 2006).

The contents of total aliphatic and indole glu-cosinolates in broccoli after different cooking treat-ments are presented in Fig.4. Total aliphatic glu-cosinolates were significantly decreased by 55%, 54%, 60%, and 41%, respectively in stir-fried, stir-fried/boiled, microwaved, and boiled broccoli (P<0.05). However, the contents of total aliphatic glucosinolates remained almost unchanged in steamed broccoli (Fig.4).

0

1

2

3

4 0 1 2 3 4 5 6

Fig.2 The contents of total soluble proteins and soluble sugars in broccoli cooked by different methods Each value is mean±SD of three replicate samples. Values not sharing a common letter are significantly different at P<0.05. Cooking methods: 1. Raw; 2. Boiled; 3. Streamed; 4. Microwaved; 5. Stir-fried; 6. Stir-fried/boiled

1 2 3 4 5 6

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Fig.4 The contents of total aliphatic and indole glucosi-nolates in broccoli cooked by different methods Each value is mean±SD of three replicate samples. Values not sharing a common letter are significantly different at P<0.05. DW: dry weight. Cooking methods: 1. Raw; 2. Boiled; 3. Streamed; 4. Microwaved; 5. Stir-fried; 6. Stir-fried/boiled

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Fig.3 The contents of vitamin C and total carotenoids in broccoli cooked by different methods Each value is mean±SD of three replicate samples. Values not sharing a common letter are significantly different at P<0.05. Cooking methods: 1. Raw; 2. Boiled; 3. Streamed; 4. Microwaved; 5. Stir-fried; 6. Stir-fried/boiled

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Yuan et al. / J Zhejiang Univ Sci B 2009 10(8):580-588 580

Effects of different cooking methods on health-promoting compounds of broccoli*

Gao-feng YUAN1, Bo SUN1, Jing YUAN1, Qiao-mei WANG†‡1,2

(1Department of Horticulture, Zhejiang University, Hangzhou 310029, China) (2Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310029, China)

†E-mail: [email protected]

Received Feb. 24, 2009; Revision accepted Apr. 20, 2009; Crosschecked July 14, 2009

Abstract: The effects of five domestic cooking methods, including steaming, microwaving, boiling, stir-frying, and stir-frying followed by boiling (stir-frying/boiling), on the nutrients and health-promoting compounds of broccoli were investigated. The results show that all cooking treatments, except steaming, caused significant losses of chlorophyll and vitamin C and significant decreases of total soluble proteins and soluble sugars. Total aliphatic and indole glucosinolates were significantly modified by all cooking treatments but not by steaming. In general, the steaming led to the lowest loss of total glucosinolates, while stir-frying and stir-frying/boiling presented the highest loss. Stir-frying and stir-frying/boiling, the two most popular methods for most homemade dishes in China, cause great losses of chlorophyll, soluble protein, soluble sugar, vitamin C, and glucosinolates, but the steaming method appears the best in retention of the nutrients in cooking broccoli. Key words: Broccoli, Cooking, Glucosinolates, Vitamin C, Chlorophyll, Soluble sugar doi:10.1631/jzus.B0920051 Document code: A CLC number: S635

INTRODUCTION

Broccoli (Brassica oleracea var. italica) con-tains high levels of vitamins, antioxidants, and anti-carcinogenic compounds and has been described as a vegetable with high nutritional value. Glucosinolates, a diverse class of sulfur- and nitrogen-containing secondary metabolites, are found in Brassica vegeta-bles including broccoli. These compounds have gained renewed interest in recent years due to the chemoprotective properties of their major hydrolysis products, isothiocyanates. Glucosinolates are chemi-cally stable until they come in contact with the deg-radation enzyme myrosinase (ȕ-thioglucoside glu-cohydrolase, EC 3.2.1.147), which is stored in dif-

ferent compartments of the plant cells to separate from glucosinolates. When plant tissues are damaged, myrosinase rapidly hydrolyzes the glucosinolates to glucose and other unstable intermediates, which spontaneously rearrange to a variety of biologically active products, including isothiocyanates, thiocy-anates, epithionitriles, or nitriles depending on chemical conditions (Jia et al., 2009). The hydrolysis products vary depending largely upon the level and activity of myrosinase, presence of specifier protein, e.g., epithiospecifier protein, and hydrolysis condi-tions, e.g., pH, metal ions and temperature, and these can be influenced by species, cultivar, and cooking time and conditions (Verkerk et al., 2008). Epidemi-ological studies and experimental researches with cell and animal models have shown that isothiocyanates have the health-promoting effects, e.g., cancer pro-tection (Traka and Mithen, 2009).

Most vegetables are commonly cooked before being consumed. In general, vegetables are prepared at home on the basis of convenience and taste

Journal of Zhejiang University SCIENCE B ISSN 1673-1581 (Print); ISSN 1862-1783 (Online) www.zju.edu.cn/jzus; www.springerlink.com E-mail: [email protected]

‡ Corresponding author * Project supported by the High-Tech R&D Program (863) of China (No. 2008AA10Z111), the National Natural Science Foundation of China (No. 30320974), the Fok Ying Tong Education Foundation of China (No. 104034), and the Program for New Century Excellent Talents in the University of China (No. NCET-05-0516)

Yuan et al. J Zhejiang Univ Sci B 2009 10(8):580-‐588

Crudo

Cozido

Vapor

Frito Cozido y frito

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1245

Research ArticleReceived: 21 September 2009 Revised: 8 January 2010 Accepted: 20 February 2010 Published online in Wiley Interscience:

(www.interscience.wiley.com) DOI 10.1002/jsfa.3967

Vitamin C, total phenolics and antioxidativeactivity in tip-cut green beans (Phaseolusvulgaris) and swede rods (Brassica napus var.napobrassica) processed by methods usedin cateringPernille Baardseth,a! Frøydis Bjerke,b Berit K Martinsena and Grete Skredea

Abstract

BACKGROUND: Retention of nutrients in vegetables during blanching/freezing, cooking and warm-holding is crucial in thepreparation of both standard and therapeutic diets. In the present study, conventional cooking in water, and cooking by pouchtechnology (boil-in-bag, sous vide) were compared in their ability to retain vitamin C, total phenolics and antioxidative activity(DPPH and FRAP) in industrially blanched/frozen tip-cut green beans and swede rods.

RESULTS: After conventional cooking, 50.4% total ascorbic acid, 76.7% total phenolics, 55.7% DPPH and 59.0% FRAP wererecovered in the drained beans. After boil-in-bag cooking, significantly (P < 0.05) higher recoveries were obtained, i.e. 80.5%total ascorbic acid, 89.2% total phenolics, 94.8% DPPH and 92.9% FRAP. Recoveries after sous vide cooking were comparable tothose of boil-in-bag cooking. By conventional cooking, 13.5–42.8% of the nutrients leaked into the cooking water; by sous videabout 10% leaked to the exuded liquid, while no leakage occurred by boil-in-bag cooking. Warm-holding beans after cookingreduced recoveries in all components. Recoveries in swede rods were comparable but overall slightly lower.

CONCLUSION: Industrially blanched/frozen vegetables should preferably be cooked by pouch technology, rather thanconventional cooking in water. Including cooking water or exuded liquid into the final dish will increase the level ofnutrients in a meal. Warm-holding of vegetables after cooking should be avoided.c! 2010 Society of Chemical Industry

Keywords: green beans; swede; cooking; boil-in-bag; sous vide; warm-holding; vitamin C; total phenolics; DPPH; FRAP

INTRODUCTIONConsumption of vegetables rich in nutrients and phytochemicalsis today recommended as a means to ensure a health-beneficialdiet.1 – 3 Many vegetables are consumed fresh, but others areprocessed to various extents in the catering and foodserviceindustries or in the private home prior to consumption. Processingmay influence nutrients or phytochemicals positively by releaseof compounds and increase in bioavailability4 or negatively byphysical losses and chemical degradation.5,6 Thus, to ensurethe nutritional quality of diets for healthy persons as wellas for therapeutic use, it is crucial to know how the variousprocessing steps contribute to the levels of nutrients at the timeof consumption.7 – 9 This is emphasised by the fact that manyhospitalised patients today have inadequate intakes of nutrients,such as water-soluble vitamins.8

Common processing steps for vegetables include blanching,freezing, cooking and, occasionally, warm-holding. Blanching andsubsequent freezing facilitate distribution and further processingof vegetables year-round, independent of season and placeof growing.5 Various technologies are available for cookingin catering and foodservice kitchens as well as in-home, i.e.

conventional cooking in water,6,10 – 17 baking in oven,10 heating bymicrowave,6,10,11,13 – 15,17,18 steam,7,11,13,15 stir-frying,6,10,12,14,16,17

cook–chill,8,19 or the more recent technologies of sous vide20

and boil-in-bag21 cooking. Both intentional19,22 and unintentionalwarm-holding after cooking are also practised in many cases. Thepouch technologies, boil-in-bag and sous vide, will most likelyplay an important role in culinary treatment of blanched/frozenvegetables in catering, professional kitchens and in the retailmarket in the future. Boil-in-bag involves one heating step butrequires available freezing and heating capacities close to wherethe food is to be served. Sous vide requires two heating steps as wellas cooling and heating facilities. The initial heating can, however,be performed efficiently at locations separate from where the food

" Correspondence to: Pernille Baardseth, Nofima Mat AS, Osloveien 1, NO-1430Aas, Norway. E-mail: [email protected]

a Nofima Mat AS, Osloveien 1, NO-1430 Aas, Norway

b Animalia – Meat and Poultry Research Centre, PB 396 Økern, NO-0513 Oslo,Norway

J Sci Food Agric 2010; 90: 1245–1255 www.soci.org c! 2010 Society of Chemical Industry

PERDE-‐SE 13 – 42 % DA VITAMINA C E COMPOSTOS FENÓLICOS NA ÁGUA DA COZEDURA

160 Am J Clin Nutr 2003;77:160–6. Printed in USA. © 2003 American Society for Clinical Nutrition

Smoking and exposure to environmental tobacco smoke decreasesome plasma antioxidants and increase !-tocopherol in vivo afteradjustment for dietary antioxidant intakes1–3

Marion Dietrich, Gladys Block, Edward P Norkus, Mark Hudes, Maret G Traber, Carroll E Cross, and Lester Packer

ABSTRACTBackground: Free radicals in cigarette smoke may cause oxida-tive damage to macromolecules, contributing to cardiovasculardiseases and cancer. Decreased plasma antioxidant concentrationsmay indicate cigarette smoke–related oxidative stress.Objective: We compared the effects on plasma antioxidant con-centrations in cotinine-confirmed active and passive smokers withthose in nonsmokers, independent of differences in dietary intakesand other covariates.Design: Plasma samples from 83 smokers, 40 passive smokers,and 36 nonsmokers were analyzed for total ascorbic acid, "- and!-tocopherols, 5 carotenoids, retinol, and cotinine. Groups werecompared by using analysis of variance with adjustment for sex,age, race, body mass index, alcohol intake, triacylglycerol con-centration, fruit and vegetable intakes, and dietary antioxidants.Results: After adjustment for dietary antioxidant intakes and othercovariates, smokers and passive smokers had significantly lowerplasma #-carotene concentrations than did nonsmokers (0.15,0.17, and 0.24 $mol/L, respectively) and significantly higher!-tocopherol concentrations (7.8, 7.8, and 6.5 $mol/L, respec-tively). Smokers had significantly lower plasma ascorbic acid and#-cryptoxanthin concentrations than did nonsmokers and passivesmokers (ascorbic acid: 43.6, 54.5, and 54.6 $mol/L, respectively;#-cryptoxanthin: 0.12, 0.16, and 0.16 $mol/L, respectively) andsignificantly lower concentrations of lutein and zeaxanthin thandid nonsmokers (0.33 compared with 0.41 $mol/L). The P valuesfor all the differences described above were < 0.05. No significantdifferences in plasma concentrations of "-tocopherol, "-carotene,total carotenoids, lycopene, or retinol were observed.Conclusions: These results indicate that cigarette smokers andnonsmokers exposed to cigarette smoke have a significantly lowerplasma antioxidant status than do unexposed nonsmokers, inde-pendent of differences in dietary antioxidant intakes. Furtherresearch is required to explain why plasma !-tocopherol concen-trations were significantly higher in smokers and passive smok-ers than in nonsmokers. Am J Clin Nutr 2003;77:160–6.

KEY WORDS Hydrophilic antioxidants, lipophilic antioxidants,!-tocopherol, smokers, passive smokers, environmental tobaccosmoke, body mass index, dietary micronutrients

INTRODUCTIONActive and passive smokers are exposed to reactive free radi-

cals that are present in cigarette smoke (CS) (1). Because free

1 From the School of Public Health (MD and GB) and the Department ofNutritional Sciences (MH), University of California, Berkeley; the Departmentof Biomedical Research, Our Lady of Mercy Medical Center, Bronx, NY(EPN); the University of California, Davis, School of Medicine, Sacramento(MGT and CEC); the Department of Nutrition and Food Management, LinusPauling Institute, Oregon State University, Corvallis, (MGT); and the Depart-ment of Molecular Pharmacology and Toxicology, University of Southern Cal-ifornia, Los Angeles (LP).

2 Supported by the University of California Tobacco-Related DiseaseResearch Program (6RT-0008 and 7RT-0160), NIH grants P30 ESO01896 and1R03 CA96412-01, and the German Academic Exchange Service, DAAD, Post-doctoral Fellowship (to MD).

3 Address reprint requests to G Block or M Dietrich, School of PublicHealth, 140 Warren Hall, University of California, Berkeley, CA 94720-7360.E-mail: [email protected] or [email protected].

Received August 15, 2001.Accepted for publication March 14, 2002.

radicals cause oxidative damage to macromolecules such as lipids,proteins, and DNA, they are believed to be involved in the patho-genesis of cardiovascular diseases and cancer (2–5). Free radicalsin CS deplete some plasma antioxidants in vitro (4, 6), and severalstudies found lower plasma antioxidant concentrations in smok-ers in vivo (7–13). Less information is available on the effect ofCS exposure on plasma antioxidant concentrations in passivesmokers (14–18).

It has been difficult to determine whether differences inplasma antioxidants between smokers and nonsmokers are actu-ally due to the effect of CS exposure or are due instead to dif-ferences in dietary antioxidant intakes or in other covariates [eg,body mass index (BMI; in kg/m2)]. Epidemiologic studiesshowed that cigarette smokers consume fewer fruits and vegeta-bles than do nonsmokers (19–23). In addition, cigarette smok-ers consume fewer vitamin supplements than do nonsmokers(24–26). The dietary habits of passive smokers were found to beintermediate between those of smokers and nonsmokers (27).Few of the existing studies of the effect of smoking on plasmaantioxidant status adjusted for dietary or supplement intake (8,9), and only one study on passive smokers did so (17). As aresult, the in vivo effect of smoking or passive smoking onplasma antioxidant status remains unclear.

In this study, we confirmed active smoking, passive smoking, ornonsmoking status with plasma cotinine measures, excluded cur-rent or recent vitamin supplement users, and adjusted for dietaryantioxidant intakes and other covariates. This permitted us to

CIGARETTE SMOKE AND PLASMA ANTIOXIDANTS 163

TABLE 2Fruit and vegetable intakes, dietary antioxidant intakes, and crude plasma antioxidant concentrations in the 159 subjects by study group1

Nonsmokers Passive smokers Smokers(n = 36) (n = 40) (n = 83) P2

Dietary intakesFruit and fruit juice (servings/d) 1.3b (1.1, 1.5) 1.2b (0.9, 1.6) 0.7a (0.6, 0.8) <0.0001Vegetables (servings/d) 2.5 (2.1, 2.9) 2.5 (1.9, 3.0) 2.2 (1.8, 2.5) 0.421Ascorbic acid (mg/d) 86.5b (73.0, 103.5) 89.1b (70.1, 113.3) 63.4a (54.6, 73.7) 0.008!-Tocopherol (!-TE/d) 8.8 (7.5, 10.2) 9.5 (7.5, 11.8) 8.0 (7.0, 9.1) 0.32"-Tocopherol (mg/day) 18.6 (15.1, 22.9) 23.3 (17.7, 30.7) 20.3 (17.5, 23.5) 0.38!-Carotene (#g/d) 478b (365, 620) 344a,b (257, 464) 293a (240, 361) 0.032$-Carotene (#g/d) 2515 (2039, 3103) 1998 (1541, 2566) 1826 (1556, 2143) 0.10$-Cryptoxanthin (#g/d) 69 (49, 98) 71 (53, 95) 47 (37, 59) 0.052Lycopene (#g/d) 4629 (3533, 6063) 3328 (2465, 4537) 3197 (2670, 3866) 0.10Lutein (#g/d) 1002 (804, 1249) 750 (578, 973) 706 (590, 846) 0.08Retinol (#g/d) 392 (330, 464) 358 (284, 450) 351 (305, 408) 0.70

Plasma antioxidants!-Tocopherol (#mol/L) 30.0 (27.4, 32.3) 29.3 (26.7, 31.8) 30.0 (28.3, 31.6) 0.89"-Tocopherol (#mol/L) 6.0a (5.1, 6.9) 7.8b (7.0, 8.6) 7.9b (7.1, 8.8) 0.006Total ascorbic acid (#mol/L) 60.8b (54.5, 67.6) 59.1b (51.6, 67.0) 40.3a (35.2, 45.4) 0.0001!-Carotene (#mol/L) 0.064b (0.047, 0.089) 0.039a,b (0.028, 0.053) 0.036a (0.019, 0.044) 0.011$-Carotene (#mol/L) 0.25b (0.20, 0.31) 0.15a (0.11, 0.19) 0.16a (0.13, 0.19) 0.005Total carotenoids (#mol/L)3 1.84b (1.60, 2.10) 1.52a, b (1.32, 1.73) 1.51a (1.37, 1.65) 0.04$-Cryptoxanthin (#mol/L) 0.16b (0.13, 0.21) 0.15b (0.12, 0.19) 0.11a (0.09, 0.12) 0.002Lutein and zeaxanthin (#mol/L) 0.40b (0.34, 0.46) 0.33a, b (0.28, 0.38) 0.32a (0.29, 0.35) 0.04Lycopene (#mol/L) 0.73 (0.64, 0.83) 0.67 (0.58, 0.76) 0.71 (0.63, 0.79) 0.68Retinol (#mol/L) 2.17a,b (2.02, 2.33) 2.06a (1.89, 2.26) 2.33b (2.19, 2.45) 0.04

1 x–; 95% CI in parentheses (arithmetic means for fruit and vegetable intake and plasma total ascorbic acid and geometric means for all other plasmaantioxidants and for all dietary antioxidants). Values in the same row with different superscript letters are significantly different at a 5% procedure-wise errorrate. !-TE, !-tocopherol equivalent.

2 ANOVA.3 Composite assay total of all identified and unidentified peaks during the carotene analysis.

have concentrations that were intermediate between those of thesmokers and nonsmokers, but in neither case were the concentra-tions in the passive smoking group significantly different fromthose in either of the other groups.

In summary, after adjustment for race, age, sex, BMI, alco-hol intake, fruit and vegetable intakes, the respective dietaryantioxidant, and triacylgycerol (for lipid-soluble antioxidantsonly), the smokers had significantly lower plasma concentra-tions of $-carotene, total ascorbic acid, $-cryptoxanthin, andlutein and zeaxanthin than did the nonsmokers. The passive

smokers had significantly lower plasma $-carotene concentra-tions than did the nonsmokers. The smokers and passive smok-ers had higher plasma "-tocopherol concentrations than did thenonsmokers. All of these comparisons were significant with theuse of Tukey-Kramer’s correction with a 5% procedure-wiseerror rate.

The importance of other covariates on plasma antioxidant con-centrations is also notable. BMI was highly significant for severalcarotenoids (data not shown), consistent with earlier findings onthe importance of body weight to plasma ascorbic acid status (40).

TABLE 3Plasma antioxidant concentrations in the 159 subjects by study group after adjustment for race, age, sex, body mass index, alcohol intake, fruit andvegetable intake, the respective dietary antioxidant, and triacylglycerol concentration1

Nonsmokers Passive smokers Smokers(n = 36) (n = 40) (n = 83) P2

!-Tocopherol (#mol/L) 30.0 30.4 29.7 0.933"-Tocopherol (#mol/L) 6.5a 7.8b 7.8b 0.032Total ascorbic acid (#mol/L) 54.5b 54.6b 43.6a 0.014!-Carotene (#mol/L) 0.05 0.04 0.04 0.210$-Carotene (#mol/L) 0.24b 0.15a 0.17a 0.026Total carotenoids (#mol/L) 1.80 1.60 1.53 0.141$-Cryptoxanthin (#mol/L) 0.16b 0.16b 0.12a 0.034Lutein and zeaxanthin (#mol/L) 0.41b 0.36 a,b 0.33a 0.047Lycopene (#mol/L) 0.71 0.70 0.71 0.977Retinol (#mol/L) 2.15 2.15 2.26 0.549

1 Adjustment for triacylglycerol concentration was performed for lipid-soluble antioxidants only. Values in the same row with different superscript lettersare significantly different at a 5% procedure-wise error rate.

2 Overall general linear model.

Dietrich M, et al. Am J Clin Nutr. 2003 Jan;77(1):160-‐6.

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