umgang mit homocystein 2004

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O. StangerW. HerrmannK. PietrzikB. FowlerJ. GeiselJ. DierkesM. Weger

Clinical use and rational managementof homocysteine, folic acid,and B vitamins in cardiovascularand thrombotic diseases

Z Kardiol 93:439–453 (2004)DOI 10.1007/s00392-004-0075-3

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Received: 12 May 2003Accepted: 17 December 2003

Prof. Dr. med. Olaf Stanger ())Universitätsklinik für HerzchirurgiePrivate Medizinische Universität (PMU)Landeskliniken SalzburgMüllner Hauptstraße 485020 Salzburg, AustriaTel.: 0043-662/4482-57512Fax: 0043-662 /82 8318E-Mail: [email protected]

W. Herrmann · J. GeiselZentrallabor, Bldg 40Universitätskliniken des SaarlandesHomburg, Germany

K. PietrzikInstitut für ErnährungswissenschaftenRheinische Friedrichs-Wilhelm-UniversitätBonn, Germany

B. FowlerUniversitäts-Kinderspital beider BaselBasel, Switzerland

J. DierkesInstitut für Klinische Chemie

Otto-von-Guericke-Universität MagdeburgMagdeburg, Germany

M. WegerUniversitäts-AugenklinikLKH GrazGraz, Austriaon behalf of the D.A.CH.-LIGAHomocystein e.V.(German, Austrian, and SwissHomocysteine Society www.dach.liga-homocystein.org)

Über den rationellen klinischenUmgang mit Homocystein, Folsäureund B-Vitaminen bei kardio-

vaskulären und thrombotischenErkrankungen

n Zusammenfassung Etwa dieHälfte aller Todesfälle sind auf Herz-Kreislauf-Erkrankungen

bzw. deren Komplikationen zu-rückzuführen. Volkswirtschaftund Gesundheitswesen werdenzusätzlich durch gewaltige Kostenfür Arbeitsausfälle, Folgeerkran-kungen und -behandlungen bela-stet, besonders unter dem Aspekteiner raschen Zunahme ältererBevölkerungsschichten. Nachdemdie konventionellen Risikofakto-ren einen Teil der Fälle nicht er-klären können, wird dem „neuen“Risikofaktor Homocystein großesInteresse entgegen gebracht. Ho-mocystein ist ein schwefelhaltigesIntermediärprodukt im Stoff-wechsel der essentiellen Amino-säure Methionin. Defizite der Vi-tamine Folsäure, Vitamin B12 undB6 sowie eingeschränkte Enzym-aktivitäten führen durch Abbau-hemmung zur intrazellulärenKonzentrationserhöhung von Ho-mocystein. Zahlreiche retrospek-tive und prospektive Studien fin-den übereinstimmend eine unab-hängige Beziehung zwischen be-reits leicht erhöhtem Homocys-tein und kardiovaskulären Er-krankungen sowie der Gesamt-mortalität. Eine Risikoerhöhungist ab einem Homocysteinwertvon etwa 9 lmol/l in einer linea-ren Dosis-Wirkungsbeziehungohne Schwellenwert darstellbar.Die Hyperhomocysteinämie als

unabhängiger Risikofaktor fürHerz-Kreislauf-Erkrankungenwird für etwa 10% des Gesamtri-

sikos verantwortlich gemacht. Er-höhte Konzentrationen (moderateHyperhomocysteinämie,>12 lmol/l) gelten als zelltoxischund werden bei 5–10% der Allge-meinbevölkerung und bei bis zu40% der Patienten mit Gefäßer-krankungen gemessen. Zusätzli-che Risikofaktoren (Rauchen, ar-terieller Hypertonus, Diabetesund Hyperlipidämie) können dasGesamtrisiko additiv oder durchInteraktion mit Homocystein syn-ergistisch und überproportionalerhöhen. Bei Hyperhomocystein-ämie kommt es neben Verände-rungen der Gefäßmorphologie zueinem Verlust der antithromboti-schen Endothelfunktion und zurInduktion eines prokoagulatori-schen Milieus. Den meisten derbekannten Schädigungen liegenHomocystein-vermittelte oxidativeStressbelastungen zugrunde.Zahlreiche Wirkstoffe, Medika-mente, Erkrankungen und Le-bensstilfaktoren beeinflussen denHomocystein-Stoffwechsel, zu-meist als direkte oder indirekteAntagonisten von Kofaktoren undEnzymaktivitäten. Als häufigsteUrsache erhöhter Homocystein-werte gilt der Folsäuremangel.Die ausreichende Versorgung mitmindestens 400 lg Folat/Tag istauch bei ausgewogener Ernäh-

REVIEW



440 Zeitschrift für Kardiologie, Band 93, Heft 6 (2004)© Steinkopff Verlag 2004

rung schwierig und besonders fürRisikogruppen häufig nicht reali-sierbar. Aufgrund der bereits vor-liegenden Erkenntnisse wird zu-nehmend die Bestimmung undBehandlung erhöhter Homocys-teinkonzentrationen bei Hochrisi-

kogruppen und besonders vonPatienten mit manifesten Gefäß-erkrankungen gefordert. In bei-den Fällen sollte zunächst eineHomocysteinbestimmung durch-geführt werden (Ausgangswert).Außer bei Manifestationen richtetsich das weitere Vorgehen nachdem Befund (Grafik). In Überein-stimmung mit anderen Arbeits-und Konsensusgruppen ist alsTherapieziel ein Homocysteins-piegel < 10 lmol/l anzustreben.

Durch Senkung erhöhter Homo-cysteinspiegel könnten, basierendauf verschiedenen Berechnungs-grundlagen, theoretisch bis zu25% der kardiovaskulären Ereig-nisse vermieden werden. Auf Grund der billigen, potentiell ef-fektiven und nebenwirkungsfreienTherapiemöglichkeit besteht einaußerordentlich günstiger Kosten-Nutzen-Quotient. Vor einer mög-lichen Empfehlung für die gene-relle Bestimmung und Behand-lung erhöhter Homocysteinwertebei Gesunden müssen erst die Er-gebnisse derzeit laufender kon-trolliert-randomisierter Interven-tionsstudien bekannt sein.

n Schlüsselwörter Homocystein –Hyperhomocysteinämie –Vitamin B12 – Folat – Therapie

n Summary About half of alldeaths are due to cardiovasculardisease and its complications. Theeconomic burden on society andthe healthcare system from cardi-ovascular disability, complications,and treatments is huge and be-coming larger in the rapidly agingpopulations of developed coun-

tries. As conventional risk factorsfail to account for part of the cases,homocysteine, a “new” risk factor,is being viewed with mountinginterest.

Homocysteine is a sulfur-con-taining intermediate product in

the normal metabolism of methionine, an essential aminoacid. Folic acid, vitamin B12, andvitamin B6 deficiency and reducedenzyme activities inhibit thebreakdown of homocysteine, thusincreasing the intracellular homo-cysteine concentration. Numerousretrospective and prospectivestudies have consistently found anindependent relationship betweenmild hyperhomocysteinemia andcardiovascular disease or all-cause

mortality. Starting at a plasmahomocysteine concentration of approximately 10 lmol/l, the riskincrease follows a linear dose-re-sponse relationship with no spe-cific threshold level. Hyperhomo-cysteinemia as an independentrisk factor for cardiovascular dis-ease is thought to be responsiblefor about 10 percent of total risk.Elevated plasma homocysteinelevels (>12 lmol/l; moderate hy-perhomocysteinemia) are consid-ered cytotoxic and are found in 5to 10 percent of the general popu-lation and in up to 40 percent of patients with vascular disease.Additional risk factors (smoking,arterial hypertension, diabetes,and hyperlipidemia) may addi-tively or, by interacting withhomocysteine, synergistically (andhence overproportionally) in-crease overall risk. Hyperhomo-cysteinemia is associated with al-terations in vascular morphology,loss of endothelial antithromboticfunction, and induction of a pro-coagulant environment. Mostknown forms of damage or injury are due to homocysteine-mediatedoxidative stresses. Especially when

acting as direct or indirect antag-onists of cofactors and enzymeactivities, numerous agents, drugs,diseases, and life style factors havean impact on homocysteine meta-bolism. Folic acid deficiency isconsidered the most common

cause of hyperhomocysteinemia.An adequate intake of at least400 lg of folate per day is difficultto maintain even with a balanceddiet, and high-risk groups oftenfind it impossible to meet thesefolate requirements. Based on theavailable evidence, there is an in-creasing call for the diagnosis andtreatment of elevated homocys-teine levels in high-risk indivi-duals in general and patients withmanifest vascular disease in par-

ticular. Subjects of both popula-tions should first have a baselinehomocysteine assay. Except wheremanifestations are already present,intervention, if any, should beguided by the severity of hyper-homocysteinemia. Consistent withother working parties and con-sensus groups, we recommend atarget plasma homocysteine levelof <10 lmol/l. Based on variouscalculation models, reduction of elevated plasma homocysteineconcentrations may theoretically prevent up to 25 percent of car-diovascular events. Supplementa-tion is inexpensive, potentially effective, and devoid of adverseeffects and, therefore, has an ex-ceptionally favorable benefit/riskratio. The results of ongoing ran-domized controlled interventiontrials must be available beforescreening for and treatment of hyperhomocysteinemia can be re-commended for the apparently healthy general population.

n Key words Homocysteine –hyperhomocysteinemia –vitamin B12 – folate – therapy



Introduction

Each year about 4 million Europeans die from cardi-ovascular disease and its complications (CAD, PAOD,myocardial infarction, stroke, venous thrombosis).In the three D.A.CH. countries (Germany, Austria,Switzerland), there were 443498 cardiovascular

deaths in 2001 (www.statistik.at, www.destatis.de,www.statistik.admin.ch), accounting for 46 percent of all deaths there [51, 85]. The economic burden onsociety and the healthcare system from cardiovascu-lar disability, complications, and treatments is hugeand becoming larger in the rapidly aging popula-tions of developed countries [27, 50, 55, 85]. Athero-sclerosis is today considered a chronic condition thatprogresses in bouts rather than as a continuous pro-cess [84]. Atherosclerosis is often detectable at ayouthful age and therefore amenable to early, effi-cient prophylaxis [7, 8]. There is therefore an in-creasing call for starting risk factor identification at

age 20, and absolute individual risk should beknown when a person turns 40 [46, 101].Hyperhomocysteinemia as an independent risk

factor for cardiovascular disease is thought to be re-sponsible for about 10 percent of total risk [9, 36].Based on various calculation models, reduction of elevated plasma homocysteine concentrations may prevent up to 25 percent of cardiovascular events[65, 105, 107]. Based on the available evidence, thereis an increasing call for the diagnosis and treatmentof elevated homocysteine levels in high-risk popula-tions [20, 21, 28, 36, 55, 109]. The results of ongoingrandomized controlled intervention trials must be

available before screening for and treatment of hy-perhomocysteinemia can be recommended for theapparently healthy general population [14]. Apartfrom its significance as an independent risk factorof additional prognostic value, homocysteine is asensitive diagnostic indicator of folate, vitamin B12,and vitamin B6 deficiencies [15, 69, 103]. The deter-

mination of the plasma homocysteine concentrationis also useful for documenting response to vitaminsupplementation.

The purpose of this consensus paper is to provideorientation on how to handle the risk factor homo-cysteine in terms of its diagnostic and clinical rolein atherothrombotic conditions and the need fortherapeutic intervention.

Metabolism and pathobiochemistry

Homocysteine is a sulfur-containing intermediateproduct in the normal metabolism of methionine, anessential amino acid (Fig. 1). “Activated” S-adenosyl-methionine (SAM) is the most important methyl do-nor in numerous biological reactions (DNA, pro-teins, neurotransmitters, hormones, phospholipids)[13]. Acquiring a methyl group from 5-methyltetra-hydrofolate (5-methyl-THF), homocysteine is re-methylated to methionine. This reaction is catalyzedby the enzyme methionine synthase, and vitamin B12

is required as a cofactor. Alternatively, homocysteinecan, by condensation with serine and via cystathio-nine, be irreversibly broken down to cysteine and

441O. Stanger et al.Management of homocysteine in atherothrombotic disease

Fig. 1 Homocysteine metabolism(THF tetrahydrofolate, A methyl trans-ferases, B 5,10-methylenetetrahydro-folate reductase)



glutathione (transsulfuration). The activities of bothenzymes involved in this metabolic pathway, i.e.,cystathionine beta-synthase (CBS) and gamma-cys-tathionase, depend on the cofactor vitamin B6.

In addition to their functions as cofactors for theenzymes involved in homocysteine metabolism, vita-mins B12, B6, and folic acid have yet other important,

independent properties [5, 11, 82, 106]. Folic acidand vitamin B6 deficiencies are independent risk fac-tors for cardiovascular disease. Apart from being in-volved in the development of hyperhomocysteine-mia, folate deficiency is associated with hypomethy-lation, DNA damage (chromosome strand breaks),or impaired cell proliferation with an increased riskof malignant disease [48, 96]. As vitamin B12 acts asa cofactor for methionine synthase and is involvedin folate metabolism, vitamin B12 deficiency may,even with adequate folate intake, lead to reduced re-methylation as well as to hypomethylation [91]. Thisresults in elevated plasma homocysteine levels and

functional folate deficiency despite (seemingly) ade-quate plasma folic acid concentrations (because fo-late is “trapped” as methyltetrahydrofolate).

Folic acid, vitamin B12, and vitamin B6 deficien-cies and reduced enzyme activities inhibit the break-down of homocysteine, thus increasing the concen-tration of intracellular homocysteine [26, 44, 90].Being cytotoxic, homocysteine is increasingly ex-ported from the cell to become detectable in plasma.

Homocysteine is present in plasma (serum) invarious forms in different proportions. The free, re-duced form accounts for less than 2 percent, whilemost homocysteine in plasma is present in the oxi-dized form bound to albumin or as the disulfide[78]. Only minute amounts of homocysteine arefound in the urine of healthy subjects. The term“homocystinuria” should therefore be reserved forinborn errors of metabolism characterized by ex-tremely elevated plasma homocysteine levels andsubstantially increased excretion of homocysteine inthe urine.

Homocysteine as a risk factor

Numerous retrospective and prospective studies haveconsistently found an independent relationship be-tween mild hyperhomocysteinemia (fasting or afteroral methionine loading) and cardiovascular diseaseor all-cause mortality [9, 20, 26, 68, 95, 105, 108]. Start-ing at a plasma homocysteine concentration of ap-proximately 10 lmol/l, an associated risk increase fol-lows a linear dose-response relationship with no spe-cific threshold level [7, 9, 59, 69, 96, 109]. Practically allessential criteria for a causal association [40] between

cardiovascular events and elevated homocysteine con-centrations are considered met [9, 107]. The impor-tance of homocysteine as a risk factor is approximately equivalent to that of smoking or hyperlipidemia [9,36]; relative risk is at least 1.3 to 1.7 for a 5 lmol/l in-crease in plasma homocysteine [26, 105] and is furtherincreased in preexisting vascular disease. Meta-ana-

lyses have calculated that homocysteine is responsiblefor at least 10 percent of the total risk for atherothrom-botic vascular disease [36, 65, 107].

Evidence from epidemiological studies suggests anincreased risk for venous thrombosis with elevatedhomocysteine concentrations [22, 52, 107]. In meta-analyses, the odds ratio for venous thrombosis wascalculated to be 1.6 (1.1–2.2) for a 5 lmol/l increasein plasma homocysteine and to be 2.5 (1.8–3.5) re-spectively, for fasting plasma homocysteine levelsabove the 95th percentile compared with the controlgroup [22, 107]. Furthermore, hyperhomocysteinemiamay increase the risk associated with some inherited

disorders. In a 10-year prospective study, the riskfor idiopathic thrombosis increased in carriers of fac-tor V leiden mutation from 4-fold to 22-fold whensubjects were hyperhomocysteinemic [80]. Recentstudies suggested an independent association betweenlow folate and vitamin B12 status and the risk of ve-nous thromboembolism [72, 77].

Additional risk factors (smoking, arterial hyper-tension, diabetes, and hyperlipidemia) may addi-tively or, by interacting with homocysteine, synergis-tically (and hence overproportionally) increase over-all risk [3, 26, 30, 36, 73]. Meta-analyses have calcu-lated that a 3 to 5 lmol/l reduction in plasma homo-cysteine may reduce the incidence of venous throm-bosis, stroke, and CAD mortality by up to 25 percent[105, 107].

Causes of hyperhomocysteinemia

n Age and gender

Plasma homocysteine increases with age, and young-er men normally have higher levels than women of the same age. In people around age 40, the genderdifference is approximately 2 lmol/l and can be ex-plained by the effect of estrogen in women becausethis difference disappears rapidly after menopause.The age-related increase in plasma homocysteinecan be explained, at least in part, by the physiologicdecline in renal function with age. Plasma homocys-teine levels show an essentially linear increase up toage 60–65 but a much faster rise thereafter, increas-ing by approximately 10 percent or 1 lmol/l per de-cade [20, 26].




n Genetic factors

The enzyme 5,10-methylenetetrahydrofolate reduc-tase (MTHFR) irreversibly reduces 5,10-methylene-THF to 5-methyl-THF. About 5 to 15 percent of thegeneral population in Germany, Austria, and Swit-zerland are homozygous carriers of a thermolabile

variant of MTHFR that is due to a point mutation atnucleotide position 677 (MTHFR 677C?T) [32].MTHFR activity is reduced by approximately 70 per-cent in affected individuals. Carriers of the mutationare therefore particularly sensitive to folate deficien-cy, experiencing an increase in plasma homocysteineby approximately 25 percent (or about 2.6 lmol/l)[44]. Recent meta-analyses including sufficiently large numbers of cases have found an associated 16to 23 percent risk increase for homozygotes, ex-plained by the plasma homocysteine increase or fo-late deficiency [47, 49, 63, 107]. Approximately 1 per-cent of the general population are heterozygotes for

mutations in the CBS gene. Carriers of this mutationshow elevated homocysteine concentrations afteroral methionine loading and also have an increasedrisk for vascular disease [109]. Other mutations witha possible impact on homocysteine metabolism(methionine synthase [53], methionine synthase re-ductase [111], etc.) are very rare, and their clinicalsignificance is all but unexplored.

n Vitamin deficiency

Vitamin deficiency is by far the leading cause of hy-perhomocysteinemia, and it may be due to inade-quate intake, reduced absorption from the gastroin-testinal tract, increased consumption, and (drug) in-teractions. Individuals who do not eat a balanceddiet (e.g., vegetarians), elderly people, pregnantwomen, patients with renal disease, malabsorption(inflammatory bowel disease) or malignant diseaseare at risk for clinically significant vitamin deficien-cy. In addition, alcohol abuse and use of certaindrugs (see Table 1) may lead to vitamin deficiency.Folate deficiency is the most common vitamin defi-ciency in Europe, partly because of a lack of freshfruits and vegetables. Good dietary sources of folatesinclude green vegetables, cereals, fruits, yeast, andliver (with reservations). However, up to 90 percentof folates may be lost during processing of cerealsand other foods [89]. Folates are also lost becausefolic acid is sensitive to heat, storage, and light. Anumber of professional associations recommend fiveservings of fruits and vegetables a day (600–700 g),but most people find it all but impossible to comply with this recommendation. An average daily intake

of approximately 400 lg of dietary folate equivalents(DFE) would optimize all folate-dependent metabolicparameters (e.g., homocysteine). However, the aver-age daily dietary folate intake in Germany, Austria,and Switzerland is currently clearly below 300 lg(197 to 235 lg for men and 168 to 214 lg for wom-en) [19] so that a large proportion of the general

population fails to attain the required natural dietary folate intake [4].Vitamin B12 intake usually exceeds requirements.

High-risk populations may still experience problems.Vitamin B12 deficiency in elderly people is frequently due to inadequate absorption resulting from an age-related decrease in gastric acid secretion or a slightincrease in (gastric) pH, or to intrinsic factor defi-ciency, and may affect as many as 30 to 40 percentof the elderly population [58, 90]. As vitamin B12

can only be synthesized by bacteria, animal foods(fish, meat, eggs, dairy products) are the only goodsources of vitamin B12 [38]. Unlike folate, cobalamin

is a relatively stable vitamin and almost all of it isleft intact by food processing.Meat, dairy products, wholemeal cereals, potatoes,

fruits, and vegetables are particularly rich in vita-min B6 [37]. No representative surveys of vitamin B6

intake in Germany, Austria, and Switzerland are avail-able. Data from the Framingham Heart Study show asignificant increase in plasma homocysteine levelsfor vitamin B6 intakes of less than approximately 1.4 mg/day [34]. Vitamin B6 shows greater stability than folic acid: Not more than 10 to 50 percent of vi-tamin B6 is lost during storage and cooking [37].

n Other causes of changes in plasma homocysteine

Numerous agents, drugs, diseases, and life style fac-tors have an impact on homocysteine metabolism,especially when acting as direct or indirect antago-nists of cofactors and enzyme activities but also as aconsequence of disulfide exchange reactions, impair-ment of absorption, and enzyme induction [26, 70].Most of the resultant clinically significant changesmay therefore be important to interpreting the over-all clinical picture. Moreover, plasma homocysteinelevels are a useful indicator of the efficiency of sometreatments (Table 1).

Mechanisms of homocysteine-mediatedvascular damage (atherothrombosis)

Homocysteine metabolism in cardiovascular cells re-lies exclusively on folate and vitamin B12 dependentremethylation since no transsulfuration has to date




been demonstrated in endothelial cells of humanblood vessels [12]. Because of the absence of irrever-sible breakdown of homocysteine to cysteine, homo-cysteine synthesis may rapidly exceed cell export, re-sulting in specific cell injury to the point of celldeath. Compared with other organ systems, the car-diovascular system is therefore particularly sensitiveto elevated homocysteine levels [12]. Hyperhomocys-teinemia may alter vascular morphology, stimulateinflammation, activate the endothelium and theblood clotting cascade, and inhibit fibrinolysis. As aresult, hyperhomocysteinemia is associated with lossof endothelial antithrombotic function and inductionof a procoagulant environment [26, 98]. Most knownforms of damage or injury (Table 2) are due to

homocysteine-mediated oxidative stresses. Chief among these are changes in the intracellular redoxpotential, interference with the NO system, and acti-vation of transcription factors with stimulation of gene expression [99]. Numerous mechanisms aresupported by in vivo studies and models of diet-in-duced folate deficiency and physiologic homocys-teine elevation.


Table 1 Causes of plasma homocys-teine (Hcy) changes Cause Hcy Mechanism

DrugsTheophylline : Vitamin B6 antagonist; inhibits pyridoxal kinaseNitrous Oxide (N2O) : Oxidation of cobalt, cobalamin and MS inactivationLipid Lowering Drugs

Fibrates : PPARa activation? Renal function?Niacin : Vitamin B6 antagonist; inhibits pyridoxal kinase

Colestipol/cholestyramine : Impairment of folic acid and cobalamin absorptionAntifolatesMethotrexate : Inhibits dihydrofolate reductase, folic acid antagonistTrimethoprim : Inhibits dihydrofolate reductase

HormonesPostmenopausal HRT ; Estrogen effectOral contraceptives : (?) Interference with folic acid?

(relevance still unclear)Antiepileptic drugs : Folic acid antagonism, enzyme modulationMetformin : Inhibition of vitamin B12 absorption,

binding of Ca2+

Omeprazole : Impairment of vitamin B12 absorptionMesna ; Disulfide exchangeLevodopa : Levodopa is a substrate for SAM-dependent methylationD-Penicillamine ; Disulfide exchangeN-Acetylcysteine ; Disulfide exchangeAntiestrogens

Tamoxifen ; Partial estrogen antagonist,enzyme induction?

Raloxifene ; Enzyme induction?Aminoglutethimide : Enzyme induction?

Cyclosporin A : Renal function?Sulfasalazine : Inhibits dihydrofolate reductase and folate absorptionIsoniazid : Vitamin B6 antagonist through complex formation

(Hyper)proliferative ConditionsPsoriasis : Cell proliferationAcute lymphocytic leukemia : Cell proliferationRheumatoid arthritis : Cell proliferation

Thyroid DisordersHypothyroidism : Enzyme inductionHyperthyroidism ; Enzyme induction

Renal Impairment :: Impaired remethylationSmoking : Interference with vitamin B6, B12, and folate; redox

Coffee/Caffeine : Vitamin B6 antagonist (caffeine), methyl group requirements :

Alcohol _ Interference with v itamin B6, B12, and folate; enzyme inhibition



Methods and sample handling

n Analytical methods

A variety of methods are available for the quantita-tive determination of homocysteine in plasma [104].Only a minor part of total homocysteine in plasmais present as a free, reduced form. The rest (&98%)forms disulfides with homocysteine, cysteine or al-bumin. Available methods for the determination of homocysteine measure total plasma homocysteine(tHcy), i.e., the sum of free and bound homocys-teine, after a reduction step. Sulphydryl compoundssuch as dithiothreitol, mercaptoethanol, tri-n-butyl-phosphine and others are used as reducing agents[104]. Quantitative shifts between the two fractionswill therefore not show up in the reported concen-tration.

Most common assay techniques are based on high-pressure liquid chromatography (HPLC) and immu-nologic methods. In addition, the stable isotope dilu-tion assay that uses gas chromatography-mass spec-trometry (GCMS) depends on the simultaneous useof a deuterated homocysteine as an internal standard[94]. After a reduction step, homocysteine is extractedon a disposable anion-exchange chromatography col-umn. The extract is then dried, derivatized with a tert-

butyldimethylsilyl agent and finally separated andquantified by GCMS. This method, although highly precise, is expensive and relatively time consuming.HPLC can be applied using pre- or post-column deri-vatization with photometric, fluorometric or electro-chemical detection [1, 60]. These assays have been im-proved to offer a shorter run time, and a higher pre-cision. Immunoassays initially reduce homocysteinewhich is then converted into S-adenosylhomocysteineusing S-adenosylhomocysteine hydrolase. The quanti-fication step depends on using monoclonal antibodiesagainst S-adenosylhomocysteine combined with dif-ferent approaches for detection [67, 92]. All of theseassay methods show good concordance in patientpopulations but may show considerable within-sub-

ject differences.

n Quality control

While there is sufficient concordance in differentiat-ing between normohomocysteinemia and hyperho-mocysteinemia, among-method variations are stillunsatisfactory [60]. The mean among-laboratory andamong-method variations ranged from 12.5 to 18%in a recent study that compared 6 different methodsin 9 laboratories [23]. International standardization(development of a plasma standard) would be


Table 2 Atherogenic effects of homocysteine (selection) Vascular Architecture Oxidative Stress :

Endothelial damage : Production of peroxinitrite, H2O2, etc. :VSMC proliferation : Antioxidative enzymes (SOD, GPx) :Collagen synthesis, fibrosis of media : Lipid peroxidation :Constrictive remodeling : Chemotaxis, Leukocyte Adhesion :Foam cell formation : Leukocyte adhesion :(Proliferative) fibrous plaques : sICAM-1, VCAM-1 :Cell Structure Damage : Chemotaxis (IL-8, MCP-1), vWF :Mitochondrial damage : Clotting Activation :ER stress : Tissue factor :Metalloproteinases : Inactivation of protein C :Elastolysis : Thrombin (thrombin-antithrombin complex) :HSP-70 expression ; D-Dimer :Endothelial Dysfunction : Fibrinolysis ;NO System :; Heparin sulfate ;NO bioavailability ; Annexin II ;ADMA : Thrombomodulin ;Transcription Factors PAI-1, t-PA antigen :Activation of NF-:B, SREBP, PKC : Prothrombin fragment F1+2 :Gene expression :; Inactivation of Factor Va ;HMG-CoA reductase : Platelet Aggregation :Lipid biosynthesis : Fibronectin (function) ;Inactivation of PPARa and c : COX, production of TXA2 and TXB2 :

VSMC vascular smooth muscle cell, ER endoplasmic reticulum, HSP heat shock protein, NO nitric oxide, ADMA asym-metric dimethylarginine, SREBP sterol regulatory element binding protein, PKC protein kinase C, PPAR peroxisomeproliferator-activated receptor, H 2O 2 hydrogen peroxide, SOD superoxide dismutase, GPx glutathione peroxidase, sICAMsoluble intercellular adhesion molecule, VCAM vascular cell adhesion molecule, IL interleukin, MCP monocyte chemo-tactic protein, vWF von Willebrand Factor, PAI-1 plasminogen activator inhibitor 1, t-PA tissue plasminogen activator,COX cyclooxygenase, TXA 2 thromboxane A2



needed to improve between-laboratory comparability and to increase the quality of assay results. Partici-pation in an interlaboratory (roundrobin) testingprogram for external quality assurance (e.g., in a Eu-ropean Research Network for evaluation and im-provement of screening, diagnosis and treatment of Inherited Metabolic Disorders (ERNDIM) quality as-

surance scheme; www.erndimqa.nl) would thereforebe desirable and useful.

n Sample preparation

A fasting blood sample collected into an EDTA tubeshould be used for the measurement of plasmahomocysteine. The blood sample should be centri-fuged immediately after collection to separate theplasma. If immediate centrifugation is impracticable,the blood sample can be stored on ice for not morethan one hour. Failure to immediately centrifuge and

separate the plasma from the blood cells causes arapid increase in plasma homocysteine (by as muchas 10% per hour) as a function of temperature andtime, giving false high readings [79]. After centrifu-gation, homocysteine is stable in plasma (for24 hours at room temperature, for up to one week inthe refrigerator (48C), or for several months whendeep-frozen (–20 8C)).

Serum samples should not be used because serumcannot be separated by centrifugation before theblood sample has coagulated completely. Collectiontubes anticoagulated with substances other thanEDTA have been used on various occasions to in-crease the time to centrifugation. However, as thecomparability of those assay results with the read-ings obtained with the method described here isquite limited, the practice of immediate plasma sepa-ration by centrifugation or brief storage on iceshould be followed if at all possible for the sake of better comparability of results.

n Intraindividual variability

Intraindividual variability of homocysteine is very low. Repeat measurements after 6 to 18 months inhealthy volunteers show good reproducibility of baseline levels with nonsignificant intraindividualvariations of as little as 0.85 to 1.2 lmol/l [18, 33].Despite the low variability of homocysteine assays,repeat measurements can improve the diagnostic re-liability within the range where a decision to treat ornot to treat is to be made. One-time measurements,on the other hand, tend to underestimate actual riskby approximately 10 to 15 percent because of the as-sociated misestimate of the true set-point [17].

Without appropriate correction, risk is underesti-mated by approximately 20 percent after 2 years andapproximately 50 percent after 10 years [17]. This re-gression dilution increases with time, and a correc-tion formula should be used for appropriate risk es-timation in prospective clinical trials [17]. This alsoexplains why various prospective studies have tended

to underestimate relative risk compared with retro-spective studies [16].

n Oral methionine loading

Plasma homocysteine levels are measured before and4 or 6 hours after oral methionine loading (100 mgof methionine per kg of body weight). The valuemeasured after methionine loading mainly reflectsCBS activity or vitamin B6 availability. The fastingplasma homocysteine concentration determinedwithout methionine loading, on the other hand, is

not a good indicator of vitamin B6

deficiency [103].Subjects with a fasting plasma homocysteine be-tween 12 and 15 lmol/l often have an abnormal oralmethionine loading test (>38 lmol/l) [36]. Oralmethionine loading can identify more subjects withhyperhomocysteinemia than determination of fastingplasma homocysteine alone [36]. However, there arecurrently no generally accepted criteria for interpret-ing methionine loading test results so that this testcan as yet not be recommended for use as a routinediagnostic tool; its use should rather be reserved for(clinical) research studies.

High-risk populationsand plasma homocysteine risk ranges

n High-risk populations and profiles

Moderate hyperhomocysteinemia (plasma homocys-teine concentration > 12 lmol/l) is found in 5 to10 percent of the general population and in up to40 percent of patients with vascular disease [26, 79,90]. Hyperhomocysteinemia is associated with an in-creased risk for atherothrombotic diseases. The de-termination of plasma homocysteine should there-fore be part of the individual risk profile for patientswith cardiovascular disease. Synergistic interactionsof homocysteine with additional risk factors (smok-ing, arterial hypertension, diabetes, hyperlipidemia)produce an overproportional increase in total risk;the identification of subjects/patients at high risk of vascular disease is therefore of particular importance[21, 36, 93]. These target populations are likely toderive particular benefit from homocysteine-lower-




ing treatments. Once diagnosed, patients with dia-betes mellitus or metabolic syndrome should betreated like those with vascular manifestations. Indi-viduals with a family history of cardiovascular dis-ease/events are very likely to suffer manifestations of vascular disease at some point in their lives. Early screening for hyperhomocysteinemia is recom-mended for close relatives of patients in such high-risk populations. About 50 percent of men over age40 and about 33 percent of women over age 40 willdevelop CAD [56]. This is why the plasma homocys-teine concentration should be known also in appar-ently healthy individuals at age 50 at the latest.Other target populations for plasma homocysteinescreening include people at (increased) risk for de-veloping (atherothrombotic) vascular complicationsand vitamin deficiencies (Table 3).

n Plasma homocysteine risk ranges

It is not helpful to specify reference ranges in theusual sense because plasma homocysteine levels be-low 10 lmol/l are already associated with a gradedincrease in risk or manifestations of cardiovasculardisease (dose-response relationship) [9, 25, 69]: Each1 lmol/l increment in plasma homocysteine concen-tration is associated with a 6 to 7 percent risk in-crease [8].

However, differentiated prophylactic and thera-peutic risk ranges for cardiovascular disease can bedefined for clinical practice. For the sake of simplifi-cation, plasma homocysteine levels >12 lmol/l and< 30 lmol/l are traditionally referred to as “moderatehyperhomocysteinemia” (commonly found in people

with vitamin deficiency); the range from 30 to100 lmol/l has been termed “intermediate hyperho-mocysteinemia” (often found in individuals withhomozygous enzyme defects as well as in patientswith chronic kidney disease); and plasma homocys-teine concentrations > 100 lmol/l are traditionally defined as “severe hyperhomocysteinemia” (typically found in individuals with severe congenital disordersor homocystinuria) [43, 45] (Table 4).

Goals of intervention

n prophylaxis

While there is clearly an overall need to improve fo-lic acid intake by the general population, there iscurrently no cogent evidence from cardiovascularoutcome studies that would justify the definition of general guidelines for vitamin supplementation inapparently healthy subjects and low-risk individuals.Vitamin supplementation continues to be a recom-mendable option for prophylaxis. Dosages for pro-phylaxis are given in Fig. 2 (low-dose supplementa-tion: folic acid, 0.2 to 0.8 mg/day; vitamin B12, 3 to100 lg/day; vitamin B6, 2 to 25 mg/day). Also, every-body is recommended to eat a diet rich in vitamins.

Although the results of ongoing intervention stud-ies of the impact of B vitamin supplementation onmortality reduction are not yet available, plasmahomocysteine reduction (through dietary supple-mentation) should also be considered in apparently healthy individuals at increased risk and especially in patients with vascular disease because secondary endpoint evidence from previous intervention stud-ies suggests potential benefit.

n Treatment

Consistent with other working parties and consensusgroups, we recommend a target plasma homocys-teine level of <10 lmol/l for patients with manifestvascular disease and high-risk individuals [4, 20, 34,


Table 3 Plasma homocysteine assay target populations based on risk

Manifest VascularDisease

Populations at Risk forCardiovascular Disease

Populations at Riskfor Vitamin Deficiency

Coronary artery disease Family history of CVD Elderly people

Myocardial infarction Arterial hypertension Vegetarians

Carotid artery

atherosclerosis

Smoking habit Inflammator y

gastrointestinalconditions (gastritis,malabsorption)

Peripheral arterialocclusive disease

Hyperlip idemia Preeclampsia

Cerebral arteryatherosclerosis

Renal insufficiency Kidney disease

Stroke Diabetes Alcohol abuse

Venous thrombosis Metabolic syndrome Unbalanced diet

Pulmonary arteryembolism

Drugs (see Table 1)

Table 4 Classification of plasma homocysteine levels by need to treat

>12 to 30 lmol/l Moderatehyperhomocysteinemia

Intervention required forall (apparently healthyindividuals and patients)

10 to 12 lmol/l Tolerable(in healthy subjects)

Need to treat patients atincreased risk

< 10 lmol/l Safe No need to treat (target

level of intervention)



35, 64, 71, 75] (Fig. 2). Renal impairment and thy-roid dysfunction as well as vitamin deficiency should be ruled out as the cause of hyperhomocys-teinemia in individuals with plasma homocysteinelevels > 12 lmol/l. The factors listed in Table 1should always be considered when interpreting find-ings. Thus, a significant reduction in plasma homo-cysteine can often be achieved by merely switchingmedications, making a dosage adjustment, or start-ing treatment for hypothyroidism.

n Folate requirements and therapeutic options

An adequate intake of at least 400 lg of folate perday is difficult to maintain even with a balanceddiet, and high-risk groups often find it impossible tomeet these folate requirements [4, 19]. As the recom-mendation to eat a healthy diet has little or limitedimpact on elevated homocysteine levels, (folate)-for-tified foods and/or vitamin supplements are rationaland therefore recommended [4, 58]. Maintaining atotal folate intake of 600 to 650 lg/day, say, by sup-plementing 400 lg/day, may help lower elevatedhomocysteine levels; this is usually easy to achievewith fortified foods and/or vitamin supplements[19].

The bioavailability of dietary folates is 55 percent.The RDAs recommended by German, Austrian, andSwiss nutrition societies are based on a polygluta-mate to monoglutamate ratio of 60 : 40 and a bio-availability of 20 percent for polyglutamates and

100 percent for monoglutamates. The extent of ab-sorption of synthetic folic acid added to foods is 90to 95 percent and that of folic acid in supplement ta-blets is almost 100 percent. As the bioavailability of synthetic folic acid is about twice that of naturally occurring folates, RDAs are given in terms of dietary folate equivalents (DFE): 1 lg of DFE is equivalent to1 lg of dietary folate or 0.5 lg of synthetic folic acid[2, 54, 58].

n Recommendations for vitamin supplementation

The absolute and relative reductions in plasmahomocysteine that can be achieved with vitaminsupplementation depend on the baseline homocys-teine concentration and are greater for higher base-line levels. Supplementation with 0.2 to 5 mg of folicacid per day is expected to lower plasma homocys-teine by 16 to 39 percent (the average reduction fora standardized baseline concentration of 12 lmol/l isapproximately 25%) [42]. Additional supplementa-tion with vitamin B12 is recommended to avoid rela-tive folate deficiency, i.e., to support folate utilization(because folate is “trapped” as methyltetrahydrofo-late in relative vitamin B12 deficiency) [91]. Vita-min B12 supplementation is also recommended forprevention of neurodegenerative damage, a particu-lar problem especially among elderly people. Basedon these considerations, (long-term) supplementa-tion of folic acid alone is discouraged. Instead, folatesupplementation should always be combined with vi-


Fig. 2 Decision tree for the diagnosis andprophylaxis/treatment of hyperhomocysteinemia(does not apply to patients with renal impair-ment)



tamin B12 supplementation. Vitamin B6 has little im-pact on fasting plasma homocysteine levels, but it isan important cofactor in catabolic transsulfurationand, therefore, should be supplemented as well. Body (folate) stores are quite limited. Vitamin supplemen-tation therefore needs to be administered chroni-cally. Once folate (+vitamin B12+ vitamin B6) supple-

mentation is stopped, plasma homocysteine is boundto rise again.

n Supplementationin moderate hyperhomocysteinemia

If plasma homocysteine determination suggestsmoderate hyperhomocysteinemia, a repeat measure-ment after 4 to 6 weeks may be useful to confirmthe diagnosis. Once (moderate) hyperhomocysteine-mia is established, vitamin supplementation shouldbe started, supplementing 0.2 to 0.8 mg of folic acid,

3 to 100l

g of vitamin B12

(elderly people should re-ceive at least 100 lg because of malabsorption), andideally also 2 to 25 mg of vitamin B6. If this supple-mentation regimen lowers plasma homocysteine to< 10 lmol/l within 4 weeks, repeat measurements of plasma homocysteine should be obtained first every 6 months and later on once a year. If response (i.e.,plasma homocysteine reduction) is still inadequate,the dosage of folic acid should be increased to, say,1 to 5 mg of folic acid per day (while supplementa-tion with vitamin B12 and vitamin B6 can be contin-ued unchanged for some time). Repeat determina-tions of plasma homocysteine should be performedat 4-week intervals.

n Possible other causesof increased vitamin requirements

If plasma homocysteine fails to be adequately low-ered despite adequate vitamin supplementation, thepatient should be evaluated for vitamin deficiency,renal impairment, and thyroid dysfunction. It shouldbe borne in mind that a (low) “normal” vitamin B12

level does not rule out intracellular vitamin B12 defi-ciency. Serum methylmalonic acid and holotransco-balamin II levels are more reliable markers of vita-min B12 deficiency than is the serum vitamin B12

concentration [39, 86]. Mutations of the genes en-coding the enzymes involved in homocysteine meta-bolism may also result in increased vitamin require-ments. The best known example is MTHFR677C?T polymorphism. Other possible causes of hyperhomocysteinemia are listed in Table 1. The de-termination of specific metabolites, includingmethylmalonic acid, 2-methylcitric acid, cystathio-

nine, cysteine, and glutathione, may provide addi-tional information about the type of disorder presentin a particular patient.

n Supplementation in patientswith renal dysfunction and enzyme deficiency

Patients with overt renal failure (insufficiency, dialy-sis) may require very large vitamin doses (including3 g of betaine/day) and still fail to achieve normal plas-ma homocysteine levels. Patients with no renal impair-ment whose plasma homocysteine is >30 lmol/l may have some form of congenital enzyme deficiency whose prevalence has been underestimated in the past.If pharmacologic doses of, say, 1 to 5 mg of folic acid,1 mg of vitamin B12, and > 20 mg of vitamin B6 fail toachieve normalization of plasma homocysteine, thepatient should be referred to a specialist for furtherevaluation.

n Safety

The toxicity of folic acid is extremely low even afterprolonged use of high doses. Thus, 10 mg/day admi-nistered for 5 years has been tolerated without ad-verse reactions [10]. Higher doses have, in isolatedinstances, been associated with gastrointestinalsymptoms, insomnia, irritability, excitation, and de-pression. Because of the theoretical risk of maskingmegaloblastic anemia and causing irreversible neu-rologic disorders, high doses of folic acid should notbe administered alone without ruling out underlyingvitamin B12 deficiency beforehand, especially in el-derly people [10]. This is why the United StatesFood and Nutrition Board (FNB) has defined a toler-able upper intake level (UL) of 1 mg/day of folicacid, which is considered safe even with life-longsupplementation. Vitamin B12 has for decades beenused for the treatment of pernicious anemia, mainly by the parenteral (intravenous or intramuscular)route. In this indication, patients receive standardsingle doses of several hundred micrograms, oftenfor the rest of their lives. Based on this extensivetherapeutic experience, vitamin B12 (cyanocobalaminand hydroxocobalamin) can be considered nontoxic.The FNB, therefore, has not specified a UL for vita-min B12. Vitamin B6 has for many years been used inthe treatment of a number of conditions, and evenvery high doses are usually well tolerated. A UL of 100 mg/day would not be expected to be associatedwith side effects even with life-long use [6]. Vita-min B6 supplementation in mild hyperhomocysteine-mia typically only involves supplementation with 2




to 20 mg, and there is rarely a need to use doses of 100 mg and above.

Cost/benefit assessment

The accepted response-to-injury model of the devel-

opment of atherosclerosis inherently includes theconcept of reversibility through control of theagent(s) that cause(s) the injury [41, 50, 84]. Thereis clearly much greater prevention potential althoughthe prevalence of cardiovascular disease is bound toincrease dramatically as a consequence of the steadi-ly increasing life expectancy [28, 29, 41, 51, 85]. Ef-fective reduction of elevated plasma homocysteinelevels by 3 to 5 lmol/l through vitamin supplementa-tion might reduce the relative risk for cardiovasculardisease by approximately 10 percent in the generalpopulation and by as much as 25 percent in high-risk groups [58, 105]. This prevention potential is

clearly supported by epidemiological data [31, 57,81, 113] and numerous favorable study results al-ready available for secondary endpoints, includingimprovement in endothelial function in healthy indi-viduals [112] and patients [24, 97, 99, 110], slowingof progression of (carotid artery) atherosclerosis[75], and substantial reduction of the rate of coro-nary restenosis following percutaneous transluminalcoronary angioplasty (PTCA) [87, 88]. More indirectevidence of the efficacy of plasma homocysteine re-duction is provided by the observation that, in thefirst year after the introduction of food fortificationwith folic acid (140 [lg]/100 g) in the United States,there were 26696 fewer deaths from myocardial in-

farction and stroke (compared with 1997) [61]. Amost recent report [H. Lange, personal communica-tion, ACC Chicago, 2003] discourages vitamin sup-plementation following coronary stent implantationfor the time being.

Such an inexpensive and potentially effective in-tervention is rarely available for reducing morbidity,

mortality, and associated costs [65, 100]. For all ap-proaches to improving vitamin intake, variable yetinvariably conservative calculation models have dem-onstrated a very favorable cost/benefit ratio [65, 66,83, 100, 102]. Cost-effectiveness analyses are greatly dependent upon the defined baseline risk. The cur-rently most efficient approach is therefore to screenand treat high-risk groups [66, 100]. Maximum cost-effectiveness has also been calculated for screeningall men over age 45 (and women over age 55) withno known vascular disease and treatment of indivi-duals with plasma homocysteine levels > 10 lmol/l[65, 100]. Most models do not take account of syner-

gistic savings although diseases and their treatment/prevention should, in fact, not be treated as isolatedentities [50, 65, 100]. Thus, the reduction of CAD in-cidence would also include a reduced risk of cost-in-tensive conditions of old age, such as senile demen-tia and stroke, which account for about 30 percent of healthcare costs in over 85-year-olds. In addition tothe cited cardiovascular disease prevention potential,improved folate and vitamin B12 intake/supplementa-tion is likely to have preventive effects on congenitalmalformations/birth defects, malignant disease, per-nicious anemia, depression, and Alzheimer’s disease.Further position papers are planned to address theseissues.


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