According to the authors, because there was no reduc- tion in CYP3A4 mRNA, it is unlikely that the decrease in CYP3A4 protein involves decreased gene transcription or reduced stability of mRNA. Most likely, the data reflect either accelerated protein degradation or reduced mRNA translation. Because the effects are rapid (observed within 4 hours), the researchers speculate that a component of grapefruit juice damages CYP3A4 through suicide inhibi- tion, resulting in accelerated degradation of the enzyme. They also surmise that the observed down-regulation of intestinal cytochrome P450 represents the first example of a dietary component that causes a decrease in an intesti- nal xenobiotic-metabolizing enzyme in humans. The re- sults also suggest that intact cell systems are required to identify the active ingredients in grapefruit juice.

The researchers speculate that in the future these natural and presumably safe substances, when identified, may be added to oral formulations of drugs that are cyto- chrome P450 CYP3A4 substrates to improve the reliability of medications and reduce their costs by lowering the

Another question for the future exists: Is grapefruit juice a source of risk in patients?’Because the Lown et al. study demonstrated effects with 8 ounces of grapefruit juice three times daily, and other studies used 8 ounces of double-strength grapefruit juice, the clinical importance of potential grapefruit juice-drug interactions may be lim- ited, but deserves further study.

The flavonoids in grapefruit juice are likely the re- sponsible agent(s). The grapefruit flavonoids are among

effective dose. 4

the five major flavonoids commonly ingested in fruits and vegetables. These polyphenols may have antiestrogenic properties and are under investigation as anticarcinogenic agents, particularly in the colon. They may affect the im- mune system as well as drug resistance. Because they are found not only in grapefruit juice but also in grapes, grape juice, and strawberries, we may become increasingly aware of other fruit-drug interactions in the future.

1.

2.

3.

4.

5.

6.

7.

Bailey DG, Spence JD, Munoz C, Arnold JMO. Inter- action of citrus juices with felodipine and nifedipine. Lancet 1991 ;337:268-9 Bailey DG, Malcolm J , Arnold JMO, Spence JD. Grape- fruit juice and drugs: how significant is the interac- tion? Clin Pharmacokinet 1994;26:91-8 Miniscalco A, Lundahl J , Regardh CG, et al. Inhibi- tion of dihydropyridine metabolism in rat and hu- man liver microsomes by flavonoids found in grape- fruit juice. J Pharrnacol Exp Ther 1992;261:1195-9 Ducharme MP, Provenzano R, Debourne-Smith M, Edwards DJ. Trough concentrations of cyclosporine in blood following administration with grapefruit juice. Br J Clin Pharmacol 1993;36:457-9 Bailey DG, Arnold JMO, Bend JR, et al. Grapefruit juice-felodipine interaction: reproducibility and char- acterization with the extended-release drug formu- lation. Br J Clin Pharmacol 1995;40:35-40 Lown KS, Bailey DG, Fontana RJ, et al. Grapefruit juice increases felodipine oral availability in humans by decreasing intestinal CYP3A protein expression. J Clin Invsst 1997;99:2545-53 Abernethy DR. Grapefruits and drugs: when is sta- tistically significant clinically significant? [Editorial] J Clin Invest 1997;99:2297-8

Micronutrient Supplementation and Infection in Institutionalized Elders

A randomized, double-blind, placebo-controlled clinical trial was conducted to determine the effects of a low-dose (< 2 times the Recommended Dietary Allowance) micronutrient supplement containing trace minerals (zinc and selenium) and1 or vitaminslprovitamins (vitamin C, a-tocopherol, and @carotene) on the incidence of respiratory and urogenital infections in institutionalized elderly. After 2 years, there was a significant decrease in the mean number of infections in elders given trace elements (p c 0.07) but not vitamins.

Advancing age is associated with increased susceptibil- ity to many illnesses, including infectious disease. In a study of short-term hospital stays, hospitalization for in- fectious and parasitic disease was three times more com-

This review was prepared by Mary Ann Johnson, Ph.D., and Kimberly H. Porter, M.S. , Department of Foods and Nutrition, University of Georgia, Athens, GA 30602, USA.

mon in adults 65 years old and over compared with those 45-64 years old.’ In long-term care facilities, prevalence rates for infections range from five to 33 infections per 100 residents per month and incidence rates range from 11 to 20 per 100 residents per month.2 The most common infec- tions among residents of long-term care facilities are uri- nary tract infections, pneumonia, and skidsoft tissue in- fectiom2

Aging alters both humoral and cell-mediated immu- r~ity.~ However, cell-mediated immunity is disproportionally a f f e~ ted .~ Across species, immune parameters that decline include thymic tissue mass, antibody production, delayed hypersensitivity tests (DHST), and T cell responsiveness to polyclonal activator^.^ Immune parameters that increase include heterogeneity of immune response between indi- viduals, monoclonal immunoglobulin production, autoan- tibody production, and a higher proportion of memory T cell subsets with a compensatory decrease in the number of naive T cells.5 Overall, T cells in many elderly individu- als are anergic and calcium resistant.6

400 Nutrition Reviews, Vol. 55, No. 11

There is interest in the potential for micronutrient supplements to enhance immune function and decrease the incidence of infections in the e l d e r l ~ . ~ About 40% of Americans use nutritional supplements, and elderly people are as likely as younger adults to use supplement^.^ De- spite the high prevalence of supplement use among the elderly, poor micronutrient status is common in older adults.s Factors that contribute to nutritional disorders in the elderly include economic, social, environmental, men- tal, physiologic, and medical problem^.^ Evidence is emerg- ing that the Recommended Dietary Allowances (RDA) for some micronutrients may be too low.8 It also has been suggested that nutritional recommendations must be de- veloped to prevent frank nutritional deficiencies as well as chronic diseases.'O The role of nutrition and micronutri- ent supplementation in preventing or minimizing the de- cline in immunity with aging has not been fully explored.

Evidence supporting the positive influence of micro- nutrients on immune function in the elderly has been col- lected from a small number of cross-sectional studies, depletiodrepletion studies, clinical trials of singk nutri- ents, and clinical trials of combinations of micro nutrient^.^ Cross-sectional studies have not consistently yielded strong evidence that micronutrient status is related to im- mune f ~ n c t i o n . ~ Differences in socioeconomic status, di- etary practices, and health behaviors between users and nonusers of nutritional supplements1IJ2may make it diffi- cult to identify specific effects of supplement use on im- mune function in cross-sectional studies. Intervention tri- als are needed to provide more definitive evidence for a role of micronutrients in immune f ~ n c t i o n . ~ Certain indi- ces of immune function in elderly people were positively influenced during intervention trials with single supple- ments of high doses of vitamin E (a-tocopherol, 800 mg/ day),13 pyridoxine (50 mg/day),I4 and p-carotene (1 5-60 mg/day).15 However, the immune system of older adults was shown to be adversely influenced by high doses of other micronutrients, such as zinc (100-150 rng/day).l6J7

Because many essential nutrients are believed to in- fluence the immune system, there is also interest in the effects of multimicronutrient supplements on immune func- tion. However, there are very few published placebo-con- trolled trials of the effects of multimicronutrient supple- ments on the immune function of elderly p e ~ p l e . I ~ - ~ ~ These micronutrient intervention trials have been conducted in elders who were living independently,17-20 homebound,21 or hospitalized.22 The duration of these studies ranged from 1 4 months2s22 to 1 year.I7-l9 The multimicronutrient supplements contained as few as three nutrients,z2 but usually contained 18-24 different n~tr ients . '~-~l Outcome measures of immune function included laboratory indi- ces,17,19,21*22 delayed hypersensitivity t e ~ t s , l ~ J ~ % ~ ~ and func- tional outcomes such as incidence of infection^.^^.^^ The multimicronutrient supplements influenced immune func- tion in some17J8J9~22 but not all studies.z0.21

It is possible that multimicronutrient supplements will have the greatest influence on immune function during long-term supplementation of elders in poor health. This hypothesis was not directly tested until the recent study of Girodon et al.,23 who examined the influence of multimicronutrient supplements during a 2-year period on the incidence of infections in geriatric patients at a long- term care facility in France. The micronutrient supplement contained moderate amounts of five nutrients or provita- mins that in previous studies showed some potential for enhancing immune function (p-carotene, a-tocopherol, vitamin C, zinc, and selenium). The investigators focused on functional endpoints of immunocompetence related to common health problems of elderly people residing in long- term care facilities: urogenital and respiratory infections.2

an average age of 84*8 years (range 65-102 years; 25% men, 75% women), and 56 subjects were followed for 2 full years. Meals were prepared in a central kitchen and ther- mally processed. Exclusion criteria included history of cancer or taking vitamin or mineral supplements. Subjects were also excluded if they were taking medications that might interfere with nutrition status or immunocompetence; however, no details were given concerning the disorders that constituted a medical condition affecting nutrition or immune status. Subjects had age-related diseases (such as osteoarthniis or residual stroke), and 22% had heart failure, 23% had hypertension, 4% had bronchitis, and 4% had prostate problems.

The study was a double-blind, placebo-controlled, randomized block design. Participants were stratified by age and sex and randomly assigned to one of four treat- ments: (1) placebo (calcium phosphate and cellulose), (2) trace elements (20 mg zinc, 100 pg selenium), (3) vitamins/ provitamins (120 mg vitamin C, 6 pg p-carotene, 15 mg a- tocopherol), or (4) trace elements plus vitamins. Zinc was provided as zinc sulfate and selenium as selenite. Supple- ments were specifically formulated for the study, and there were no visible differences between the supplements and the placebo. The nutrient contents were moderate and less than two times the U.S. and French RDAs (Table 1).

Compliance was assessed by nursing staff, pill counts, and serum levels of micronutrients. Fasting blood samples were used to assess micronutrient status at baseline and after 6 months, 1 year, and 2 years of supplementation. In the total sample at baseline, 6 1 % had low zinc (< 10.7 pmoV L, < 70 pg/dL), 68% had low selenium (< 0.86 pmol/L, < 6.8 pg/dL), 75% had low vitamin C (< 3.5 pg/mL, < 20 pmol/L), 0% had low a-tocopherol (< 4 mg/L, < 9.3 pmol/L), and 15% had low p-carotene (< 161 pa, < 0.3 pmol/L). Serum concentrations of the nutrients increased in the supple- ment groups but not in the placebo group. For example, in subjects receiving both trace elements and vitamins, low serum concentrations of zinc, selenium, vitamin C, and p- carotene were 40%, lo%, lo%, and 5%, respectively, after

Girodon and colleagues23 enrolled 8 1 subjects with'

Nutrition Reviews, Vol. 55, No. I 1 401

Table 1 Multimicronutrient Supplement Compared with the US. and French Recommended Dietary Al I owances

Nutrient Supplement Women Men French RDA (Elderly)

Selenium, pg 100 55 70 70

U.S. RDA (> 51 years)

Zinc, mg 20 12 15 12

Vitamin C, mg 120 60 60 80 Vitamin E, mg 15 8 10 12

- - &Carotene 6 -

6 months. In each respective supplement group, serum selenium, vitamin C, and p-carotene increased through- out the 2-year period; however, serum zinc and a-toco- pherol concentrations increased above baseline at 6 months and 1 year but returned to baseline at 2 years. The changes in serum a-tocopherol were somewhat variable in the vitamin-supplemented group, but increased above baseline after 2 years when expressed per milligram of cho- lesterol.

Respiratory and symptomatic urogenital infections were recorded. Diagnosis of urogenital or respiratory in- fections was made by the same physician and was based on clinical, biologic, and radiologic tests. Urogenital in- fections were documented if a specific bacteria exceeded 1 06/mL of urine and leukocytes exceeded 1 OS/mL of urine.

Number of infectious events and mortality are shown in Table 2. Mortality was similar among the four groups. A 2 x 2 factorial analysis of variance suggested a significant decrease in the mean number of infectious events associ- ated with taking the trace element supplement 07 < 0.01) but not the vitamin supplement; the interaction term was not significant. The influence of trace elements alone ap- peared particularly striking. Compared with the placebo group, those given trace elements alone had two to four times fewer infections during the course of the study.

This study has several strengths, including examina- tion of a high-risk population, monitoring of blood levels of micronutrients, and outcome measures that included two common infectious problems in elders in long-term

care.2 The primary limitation of the study is the presenta- tion and statistical treatment of the data. The number of infectious events during 2 years was not corrected for loss of subjects who died, duration of infections was not reported, and the effectiveness of the supplements in those with the poorest micronutrient status was not examhed. A more appropriate statistical treatment of the data would include estimation of the percentage of subjects who ex- perienced one or more infections, as well as information on the duration and severity of infectious episodes.

The infection history of the participants was not re- ported; it is important to document that the history of respiratory and urogenital infections was the same in all four treatment groups. Although cognitive status was not reported, it is possible that poor cognition might impair an elder’s ability to report an infection. Recording only symp- tomatic infections may have underestimated the number of infections. A high percentage of participants had low serum folate (70%), but folate was not included in the vitamin supplement. Information on food intake was col- lected but not reported. As noted by the authors, the high prevalence of poor folate and vitamin C status may be partially attributed to the thermal processing of the food, which could decrease the amounts of heat-sensitive vita- mins. Despite limitations in the design and statistical analy- ses of the study, multimicronutrient supplementation of elders residing in long-term care facilities appears to have potential for decreasing the incidence of respiratory and urogenital infections.

Table 2. Infectious Events and Mortality in Subjects Supplemented with Trace Elements and/or Vitamins for 2 Years

Trace Elements Placebo Trace Elements Vitamins +Vitamins

n 20 20 20 21

Total deaths 7 6 5 7

Deaths from infections 2 0 0 1

Infections, year 1 16 7 10 14

Infections, year 2 19 5 14 9

Total infections 35 12 24 23

Mean number of infections 1.75 0.60 1.20 1.09 M D ) (1.48) (0.99) (1.43) (1.09)

Note: The effect of the trace element supplement on mean number of infections was significant (p < 0.01).

402 Nutrition Reviews, Vol. 55, No. 11

The only other studies of the influence of multimicro- nutrient supplements on infections were conducted in in- dependently living elderly people. Chandra19 conducted a 1-year study of 96 people 65 years old and older. Com- pared with those given placebo, those given a multimicro- nutrient supplement had fewer days of illness owing to infections (4817 versus 2315 dayslyear, p = 0.002) and fewer days during which antibiotics were used (1 8rk4 ver- sus 3215,p= 0.004). Chavance et a1.20 studied 218 people 60 years old and older for 4 months and reported no differ- ences in the incidence of infections between those given placebo and those given multimicronutrient supplements. It is difficult to account for the differences in the out- comes of these two studies. Both investigators used multinutrient supplements that contained either 18 nutri- e n t ~ ~ ~ or 21 nutrients20 at levels that ranged from nutrition- ally insignificant to three to four times the RDA. However, the types and amounts of individual nutrients differed between the two studies. Chavance and colleagues2" used a questionnaire to assess the incidence of infections, whereas ChandraL9 apparently used a physician's assess- ment and laboratory tests to diagnose infections. ?he in- hibitory effect of multimicronutrient supplements on in- fections in Chandra's study19 might be related to the long duration (1 year) of the study and inclusion of p-carotene (1 6 mg) and selenium (20 pg) in the supplement, while the lack of effects in Chavance et al.'s studyZo might be related to the short duration (4 months) and lack of p-carotene and selenium in the supplement. Differences in the amounts and/or bioavailability of other micronutrients in the supplement or differences in the baseline nutritional status of the two samples of elders also may have ac- counted for the different outcomes. Baseline nutritional status was reported in both studies but are not easily compared, because one study reported the percentage of participants with low blood values of certain nutrientsLq whereas the other reported mean blood values of nutri- ents and dietary intakes of selected nutrients.20

Bogden and Louria3 suggest several directions for future research concerning micronutrients and immunity in the elderly (Table 3). They recommend examination of dietary modification alone, single micronutrients, antioxi- dant micronutrients, and multivitamidminerals, as well as characterization of host factors, such as gender, race, health status, or physical activity, that might influence the interactions of micronutrients with immune function. In- tervention trials in hospital or long-term care settings will provide valuable insight into the potential of multimicro- nutrient supplements to improve immunocompetence in elders at high risk for infections. Although somewhat dif- ficult to define in high-risk elders, the characteristics of the sample must be very well defined. Studies of healthy elders could follow the recommended protocols for exami- nation of immune function in these population^.^^ Indices of cellular immunity, such as the DHST reaction, lympho-

Table 3. Research on Micronutrients, Immunity9 and Aaina Well-defined inclusion and exclusion criteria for

Studies of frail as well as healthy elders.

Long-term placebo-controlled double-blind clinical

Prospective epidemiologic studies.

Clinically relevant outcomes (infectious disease

Humoral immunity (e.g., antibody responses to

Cellular immunity (e.g., DHST responses).

Identify host-specific factors that may influence the

Identify the molecular and cellular mechanisms for

research participants.

trials (> 1-2 years).

incidence, duration, and severity).

vaccination).

interactions of micronutrients and immune function.

effects of micronutrients on immunity.

Adapted from reference 3.

proliferative response to polyclonal activators, and anti- sheep red blood cell responseS should be included along with functional endpoints of immunocompetence. Addi- tional research in well-controlled human studies is needed, as is a continued focus on the molecular and cellular mecha- nisms that underlie the influence of micronutrients on im- mune functio;.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

Graves EJ, Owings MF. 1995 Summary: National Hospital Discharge Survey. DHHS Publication No. (PHS) 97-1250 7-0737 (Advance Data from Vital and Health Statistics, No 291). Hyattsville, MD: National Center for Health Statistics, 1997 Yoshikawa TT, Norman DC. Approach to fever and infection in the nursing home. J Am Geriatr SOC

Bogden JD, Louria DB. Micronutrients and immu- nity in older people. In: Bendich A, Deckelbaum RJ, eds. Preventive nutrition: the comprehensive guide for health professionals. Totowa, NJ: Humana Press Inc, 1997;317-36 Meydani SN, Hayek M, Coleman L. Influence of vita- mins E and B6 on immune response. Ann NY Acad Sci 1992;669: 125-39 Makinodan T. Patterns of age-related immunologic changes. Nutr Rev 1995;53:S27-S34 Miller RA. Aging and immune function: cellular and biochemical analyses. Exp Gerontol 1994;29:21- 35 Block G, Cox C, Madans J, et al. Vitamin supple- ment use, by demographic characteristics. Am J Epidemiol 1988;127:297-309 Russell RM. New views on the RDAs for older adults. J Am Diet Assoc 1997;97:515-8 Davies L. Risk-factors for malnutrition. In: Horwitz A, Macfadyen DM, Munro H, et al, eds. Nutrition in the elderly. Oxford: Oxford University Press, 1989;153- 66 Anon. Revisiting dietary allowances and require-

1996;44:74-82

Nutrition Reviews, Vol. 55, No. 11 403

ments. Nutr Rev 1996;8:246-7 11. Daniel TD, Houston DK, Johnson MA. Vitamin and

mineral supplement use among the elderly. In: Vellas BJ, Sachet P, Baumgartner RJ, eds. Nutritional In- tervention in the elderly. Facts Res Gerontol (suppl)

12. Houston DK, Daniel TD, Johnson MA, Poon LW. Health and dietary characteristics of supplement users. Int J Vitam Nutr Res 1997;67:183-91

13. Meydani SN, Barklund MP, Liu S, et al. Vitamin E supplementation enhances cell-medicated immu- nity in healthy elderly subjects. Am J Clin Nutr

14. Talbott MC, Miller LT, Kerkvliet NI. Pyridoxine supple- mentation: effect on lymphocyte responses in eld- erly persons. Am J Clin Nutr 1987;46:659-64

15. Watson RR, Prabhala RH, Plezia PM, Alberts DS. Effect of beta-carotene on lymphocyte subpopula- tions in elderly humans: evidence for a dose-re- sponse relationship. Am J Clin Nutr 1991 ;53:90-4

16. Chandra RK. Excessive intake of zinc impairs im- mune response. JAMA 1984;252:1443-6

17. Bogden JD, Oleske JM, Lavenhar MA, et al. Effects of one year of supplementation with zinc and other micronutrients on cellular immunity in the dderly. J Am Coll Nutr 1990;9:214-25

18. Bogden JD, Bendich A, Kemp FW, et al. Daily micro-

1995; 163-79

1990;52:557-63

nutrient supplements enhance delayed hypersen- sitivity skin test responses in older people. Am J Clin Nutr 1994;60:437-47

19. Chandra RK. Effect of vitamin and trace-element supplementation on immune responses and infec- tion in elderly subjects. Lancet 1992;340:1124-7

20. Chavance M, Herbeth B, Lemoine A, Bao-Piong Z. Does multivitamin supplementation prevent infec- tions in healthy elderly subjects? a controlled trial. Int J Vitam Nutr Res 1993;63:11-6

21. Bunker VW, Stansfield MF, Deacon-Smith R, et al, Dietary supplementation and immunocompetence in housebound elderly subjects. Br J Med Sci

22. Penn ND, Purkins L, Kelleher J, et al. The effect of dietary supplementation with vitamins A, C, and E on cell-mediated immune function in elderly long- stay patients: a randomized controlled trial. Age Age- i n g 1 9 9 1 ; 2 0 : 1 6 9-74

23. Girodon F, Lombard M, Galan P, et al. Effect of micro- nutrient supplementation on infection in institution- alized elderly subjects: a controlled trial. Ann Nutr Metab 1997;41:98-107

24. Legthart GJ, Corberand JX, Fournier C, et al. Admis- sion criteria for immunogerontological studies in man. Mech Ageing Dev 1984;28:47-55

1994;51:128-35

*

lsoprostanes as Oxidant Stress Markers in Coronary Reperfusion

The measurement of urinary levels of 8-epi-PGFZa provides a noninvasive in vivo index of free radical generation and a relatively reliable index for the determination of oxidative stress and administration of antioxidant nutrients and drugs.

It has been suggested that oxidative stress and free radi- cal-mediated lipid peroxidation may play a fundamental role in several disease states, including cancer, cardiovas- cular disease, chronic inflammatory diseases, toxicity, and reperfusion injury, as well as in the degenerative processes associated with aging.' Our current understanding of the contribution of free radicals to oxidative stress conditions and disease states has emerged mostly from animal stud- ies in which tissues from target organs are readily avail- able for measurement of free radical generation. However, direct measurement of free radical generation in vivo has proven to be problematic.

The detection and measurement of lipid peroxidation has been most frequently used to support the involve- ment of free radical reactions in pathophysiologic condi- tions. Furthermore, these indices have been the basis for

This review was prepared by Mohsen Meydani, D.V.M., Ph.D., Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 0211 1, USA.

the development and use of antioxidant drugs or dietary supplements to prevent oxidative injury. A wide range of methods have been developed to measure the rate of this process; however, each has its limitations and pitfalls and is often not applicable to all circumstances, especially when applied to human samples.2 The thiobarbituric acid (TBA) assay measures an aldehydic breakdown product of lipid hydroperoxides (malodialdehyde) and is the most frequently used assay. TBA reacts with a variety of other biologic compounds, such as prostaglandins, thrombox- anes, carbohydrates, and sialic acids. TBA and other com- monly used assays, such as diene conjugation, are nei- ther specific nor sensitive enough to monitor small changes in free radical status and lipid peroxidation in vivo. There- fore, development of a more specific and sensitive assay to detect and better represent in vivo rates of lipid peroxidation as an index of free radical generation and oxidative stress is desirable.

In recent years, the measurement of isoeicosanoids, the products of arachidonic acid formed by free radical- catalyzed reactions, appears to be a promising assay that might be specific and sensitive enough to detect in vivo lipid peroxidation. Production of isoeicosanoids is not dependent on cyclooxygenase (COX) activity and, thus, is not inhibited by COX inhibitors such as aspirin, but might be inhibited by antioxidants. Morrow et aL3 reported the detection of one class of these compounds in vivo,

404 Nutrition Reviews, Vol. 55, No. I1