This Article Abstract Full Text (PDF) Erratum (v99,p983) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Robinson, K. Articles by Graham, I. Search for Related Content PubMed PubMed Citation Articles by Robinson, K. Articles by Graham, I. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB FOLIC ACID PYRIDOXINE HYDROCHLORIDE Medline Plus Health Information Peripheral Vascular Diseases

(Circulation. 1998;97:437-443.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Low Circulating Folate and Vitamin B₆ Concentrations

Risk Factors for Stroke, Peripheral Vascular Disease, and Coronary Artery Disease

Killian Robinson, MD; Kristopher Arheart, EdD; Helga Refsum, PhD; Lars Brattström, MD, PhD; Godfried Boers, MD; Per Ueland, PhD; Paolo Rubba, MD; Roberto Palma-Reis, MD; Raymond Meleady, MRCPI; Leslie Daly, PhD; Jacqueline Witteman, MD; Ian Graham, FRCPI; ; for the European COMAC Group¹

From the Departments of Cardiology (K.R.) and Biostatistics and Epidemiology (K.A.), The Cleveland Clinic Foundation, Cleveland, Ohio; Department of Clinical Biology, Division of Pharmacology (H.R., P.U.), University of Bergen, Norway; Department of Medicine (L.B.), County Hospital, Kalmar, Sweden; Department of Endocrinology (G.B.), Katholieke Universiteit, Nijmegen, Netherlands; Facolta di Medicina e Chirurgia (P.R.), Universita degli Studi di Napoli, Federico II, Naples, Italy; Servico de Medicina (R.P.-R.), Hospital de S. Francisco Xavier Lisbon, Portugal; Department of Cardiology, Adelaide Hospital, Trinity College, Dublin and the Department of Epidemiology, Royal College of Surgeons in Ireland (R.M., I.G.); Department of Public Health Medicine and Epidemiology (L.D.), University College Dublin, Ireland; and Department of Epidemiology (J.W.), Erasmus University Medical School, Rotterdam, Netherlands.

Correspondence to Killian Robinson, MD, Desk F15, Department of Cardiology, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail robinsk{at}ccsmtp.ccf.org

	Abstract

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Background—A high plasma homocysteine concentration is a risk factor for atherosclerosis, and circulating concentrations of homocysteine are related to levels of folate and vitamin B₆. This study was performed to explore the interrelationships between homocysteine, B vitamins, and vascular diseases and to evaluate the role of these vitamins as risk factors for atherosclerosis.

Methods—In a multicenter case-control study in Europe, 750 patients with documented vascular disease and 800 control subjects frequency-matched for age and sex were compared. Plasma levels of total homocysteine (before and after methionine loading) were determined, as were those of red cell folate, vitamin B₁₂, and vitamin B₆.

Results—In a conditional logistic regression model, homocysteine concentrations greater than the 80th percentile for control subjects either fasting (12.1 µmol/L) or after a methionine load (38.0 µmol/L) were associated with an elevated risk of vascular disease independent of all traditional risk factors. In addition, concentrations of red cell folate below the lowest 10th percentile (<513 nmol/L) and concentrations of vitamin B₆ below the lowest 20th percentile (<23.3 nmol/L) for control subjects were also associated with increased risk. This risk was independent of conventional risk factors and for folate was explained in part by increased homocysteine levels. In contrast, the relationship between vitamin B₆ and atherosclerosis was independent of homocysteine levels both before and after methionine loading.

Conclusions—Lower levels of folate and vitamin B₆ confer an increased risk of atherosclerosis. Clinical trials are now required to evaluate the effect of treatment with these vitamins in the primary and secondary prevention of vascular diseases.

Key Words: atherosclerosis • cerebrovascular disorders • coronary disease • peripheral vascular disease • risk factors

	Introduction

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
An increased plasma homocysteine concentration is associated with premature arterial disease^{1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16} and may reflect deficiency states of folate, vitamin B₁₂, or vitamin B₆^{17 18 19} or of certain essential enzymes.^{20 21 22 23 24 25} The relationship between these B vitamins and vascular diseases, however, remains poorly defined. The present study demonstrates that lower circulating levels of folate and vitamin B₆ are often seen in patients with atherosclerosis and confer an increased and independent risk of cardiovascular disease.

	Methods

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Case Subjects
Patients with clinical evidence of coronary artery disease, peripheral vascular disease, or cerebrovascular disease confirmed by standard

diagnostic techniques were included. The inclusion and exclusion criteria have been reported extensively elsewhere.¹⁵ Briefly, 750 case subjects with vascular disease and 800 control subjects younger than 60 years of age, of both sexes, were recruited at 19 centers in nine European countries. Case subjects had defined clinical and objective investigational evidence of vascular disease. Newly or recently diagnosed case subjects were recruited wherever possible, and 69% were recruited within 1 year of diagnosis. Exclusion criteria for both case and control subjects included nonatherosclerotic vascular disease, cardiomyopathy, diabetes mellitus, pregnancy, recent (within 3 months) systemic illness, and psychiatric illness. Conditions thought to influence homocysteine concentrations, such as renal or thyroid disease, anticonvulsant therapy, and recent (<3 months) exposure to nitrous oxide, also served as exclusion criteria.

Control Subjects
Control subjects were clinically healthy and free of overt disease. Where possible, subjects were recruited from a geographic background similar to that of case subjects. Community-based control subjects from random population samples, family practice registers, and occupational registers were considered ideal sources. Just less than half of these subjects came from community samples, one third were recruited from employee health insurance registers, and one sixth were hospital employees. Two percent of control subjects were hospital patients. Control subjects recruited from the three main sources were similar in terms of the major variables studied and plasma total homocysteine (tHcy) levels.

Risk Factors for Vascular Disease
Age, sex, smoking habits, blood pressure, lipid concentrations, weight, and both drug and vitamin usage were documented in all subjects and are shown in Table 1.

View this table: [in this window] [in a new window]   Table 1. Clinical Data in 750 Case Subjects With Vascular Disease and 800 Control Subjects

Methionine-Loading Test
A methionine-loading test was performed on all subjects in standard fashion.²⁶ Blood was drawn into tubes containing EDTA for measurement of fasting tHcy. An oral dose of 0.1 g/kg L-methionine was administered, and blood was drawn again 6 hours later for the postload measurement. We refer to the difference between these two concentrations as the increase in tHcy.

Laboratory Measurements
Homocysteine Assay
Total plasma homocysteine was measured by use of a previously described method involving reduction with sodium borohydride, derivatization with monobromobimane, high-performance liquid chromatography (HPLC) separation, and fluorescence detection.²⁷ Blinded analyses were performed on all samples that were reanalyzed twice on two separate days. A maximum of 10% difference between the two results, ie, 5% difference from the mean, was allowed. If this was exceeded, the analyses were repeated for a third time. The average of these analyses is presented.

Vitamin Concentrations and Other Assays
Measurements of red cell folate, vitamin B₁₂, vitamin B₆ (measured as pyridoxal 5'-phosphate), and creatinine were performed centrally at Mimelab-AB, Soraker, Sweden. Vitamin B₁₂ and folate concentrations were measured by use of a radioimmunoassay technique,²⁸ and pyridoxal 5'-phosphate was measured by enzymatic photometry with HPLC separation.²⁹

Definitions
Traditional Risk Factors
Smokers were defined as those currently smoking any tobacco (at the time of diagnosis for case subjects and at the time of the methionine-loading test for control subjects). Hypertension was considered present if at the time of the methionine-loading test a systolic blood pressure 160 mm Hg or a diastolic pressure of 95 mm Hg was observed or if treatment for high blood pressure was administered. For both systolic and diastolic blood pressures, the mean of four values was used (two obtained before and two after the administration of methionine). Hypercholesterolemia was considered present if subjects were taking lipid-lowering drug treatment or had a serum cholesterol 6.5 mmol/L (251.4 mg/dL).

Homocysteine Concentrations
For categorical analyses, high tHcy concentrations were defined as levels greater than the 80th percentile for control subjects in both the fasting (12 µmol/L) and the post–methionine-loading state (38 µmol/L). The 80th percentile for control subjects was also used to define an abnormally high increase after methionine loading (27 µmol/L).

Vitamin Deficiencies and Low Vitamin Status
Folate deficiency was defined as a red cell folate concentration <372 nmol/L, which is similar to widely used reference ranges.³⁰ Low folate status was defined as a concentration below the 10th percentile for control subjects (513 nmol/L). Concentrations of fasting tHcy below this level of folate were higher than those in the upper decile of folate concentration (see Figure). Because this difference persisted when adjusted for deficiencies of both vitamin B₁₂ and vitamin B₆, we inferred a functional folate deficiency at and below this level. Vitamin B₁₂ deficiency was defined as a plasma concentration <125 pmol/L.³⁰ Low vitamin B₁₂ status was defined arbitrarily as a value below the 10th percentile for control subjects (139.5 pmol/L).

View larger version (29K): [in this window] [in a new window]   Figure 1. Geometric mean and 95% CI bars of fasting and postload homocysteine concentrations in case and control subjects defined by decile cutpoints of folic acid, vitamin B12, and vitamin B6. Decile cutpoints are based on control samples only. Vitamin values on the x axis are the group mean of case and control subjects combined. Case subjects with deficiencies in vitamins other than the one being graphed have been eliminated from the analysis. *Homocysteine level differs significantly (P<.05) between case and control subjects in the relevant group. +Case or control homocysteine level is significantly different (P<.05) from the level observed in the group above the highest vitamin decile.

Definitions of vitamin B₆ deficiency are not uniform,^{30 31} and values <30³⁰ or <20 nmol/L³¹ may indicate deficiency. In the present study, frank deficiency was defined as <20 nmol/L. Because this was almost identical to the 10th percentile for control subjects (20.8 nmol/L), low vitamin B₆ status was defined as less than the 20th percentile for control subjects (23.3 nmol/L).

Diagnostic Criteria for Vascular Disease
The following criteria were used for the diagnosis of vascular diseases:

1. Coronary heart disease: clinical evidence of angina or myocardial infarction plus a 2-fold rise in cardiac enzymes with evolutionary ST-T changes or pathological Q waves alone or angiographic evidence of 70% stenosis of a major coronary artery.

2. Cerebrovascular disease: clinical evidence of stroke or transient ischemic attack plus carotid stenosis 50% on Doppler or angiography or unequivocal atherosclerotic plaque on angiography or computed tomographic evidence of cerebral infarction without demonstrable source of embolism.

3. Peripheral vascular disease: clinical evidence of intermittent claudication or clearly diminished foot pulses plus obstruction of one major peripheral artery on angiography or Doppler ankle-arm index <0.9.

Statistical Methods
Sample size considerations for this study have been presented elsewhere.¹⁵ Data are presented as mean±SE or percents. When necessary, log transformation was used for skewed variables, and these data are presented as geometric means and 95% CIs. We compared risk factors between case and control subjects using a t test or ² test as appropriate. We examined the relationship among tHcy and vitamin concentrations using Pearson correlations. Conditional logistic regression stratified by center, age, and gender was used to investigate models of the risk of coronary artery disease; odds ratios with 95% CIs are reported for these analyses. Differences in tHcy among vitamin deciles were evaluated with ANOVA. A two-sided 5% level of significance is considered significant for all statistical tests; exact probability values are reported down to P<.001.

	Results

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Concentrations of tHcy
Geometric means for fasting, postload, and increase in tHcy values and for the vitamins are shown in Table 2 according to gender and case status. Overall, fasting tHcy values were higher in case subjects than in control subjects in both men and women. Age and weight adjustment had little effect on the values shown in the tables or on significance levels (data not shown). After the methionine-loading test, tHcy values were higher in case subjects than in control subjects, both in men and women. These concentrations also remained high when adjusted for age and weight (data not shown). The increase in tHcy after methionine loading was significantly greater in case subjects than in control subjects in both sexes but was more marked in women than in men. These concentrations also remained high when adjusted for age and weight (data not shown).

View this table: [in this window] [in a new window]   Table 2. Total Homocysteine Concentrations and Vitamin Levels According to Gender and Case Status

Vitamins
Folate concentrations were higher in men than in women. Within men as a group, however, folate levels were lower in case subjects than in control subjects (819.0±1.0 versus 876.2±1.0 nmol/L; P=.005; see Table 2). Mean vitamin B₁₂ concentrations were similar in both case and control subjects. Vitamin B₆ concentrations were lower in case subjects than in control subjects.

Correlations Between Vitamins and tHcy
The correlations between tHcy and the three vitamins are shown in Table 3 and the Figure.

View this table: [in this window] [in a new window]   Table 3. Correlations Between Plasma Total Homocysteine and Vitamin Levels in Case and Control Subjects

Fasting tHcy correlated negatively with folate. Postload values correlated negatively with folate in male case subjects and in female control subjects. In contrast, values for the increase in tHcy did not correlate with folate. Fasting, postload, and increases in tHcy values correlated negatively with vitamin B₁₂ (see Table 3) in both case and control subjects. The majority of these correlations were significant. Fasting, postload, and increases in tHcy values correlated negatively with vitamin B₆ (see Table 3), and the majority of these correlations were significant. Across the range of vitamin B₆ concentrations, postload tHcy levels were greater in case than control subjects (see Figure).

Vitamin Deficiencies
Prevalences of vitamin deficiencies (defined by use of conventional definitions) and values for the lower 10th and 20th percentiles are shown in Table 4. When a definition of folate deficiency of 372 nmol/L was used, folate deficiency was seen in 2% of control subjects and 4% of case subjects (P=.048). Low folate status, corresponding to the 10th percentile for control subjects (<513 nmol/L), was seen in 15% of case subjects (P=.002). Prevalences of deficiency of vitamin B₁₂ (<125 pmol/L) and low vitamin B₁₂ status were no different in case subjects than in control subjects. Deficiency (<20 nmol/L) was seen in 21% of case subjects (P<.001). This concentration was almost identical to the 10th percentile for control subjects (20.75 nmol/L; see Table 4). Low vitamin B₆ status (less than the 20th percentile for control subjects, or 23.2 nmol/L) was seen in 35% of case subjects (P<.001).

View this table: [in this window] [in a new window]   Table 4. Prevalence of Low Vitamin Status and Conventionally Defined Vitamin Deficiencies in Case and Control Subjects

Relationships Between Homocysteine, Vitamins, and Vascular Disease
Variables included in the conditional logistic regression models for vascular diseases included hypertension, smoking, hypercholesterolemia, creatinine, and the concentrations of fasting tHcy, postload tHcy, increase in tHcy, folate, vitamin B₁₂, and vitamin B₆. The results for these analyses are shown in Table 5.

View this table: [in this window] [in a new window]   Table 5. Adjusted Odds Ratio of Vascular Disease in Subjects With High Total Homocysteine or Low Vitamin Levels Relative to Subjects With Normal tHcy or Vitamin Levels

Homocysteine
Odds ratios for vascular disease for tHcy have already been reported, adjusted for conventional risk factors.¹⁶ High fasting, increase, and postload tHcy concentrations were significant risk factors for vascular disease after adjustment was made for traditional risk factors and vitamins (see Table 5).

Vitamins
When a conventional definition (<372 nmol/L) was used, folate deficiency was not associated with an increased odds ratio of vascular disease (1.12; CI, 0.52 to 2.41; P=.77; data not shown in Table 5). A level of folate below the lowest decile (513.0 nmol/L) conferred an odds ratio of 1.50 (CI, 1.03 to 2.20; P=.045; see Table 5) for vascular disease, adjusted for traditional risk factors. When adjusted for fasting tHcy but not the increase or postload values, this was no longer significant.

Neither vitamin B₁₂ deficiency (data not shown) nor low vitamin B₁₂ status was associated with a significant likelihood of vascular disease (see Table 5). An increased odds ratio of vascular disease was seen both with vitamin B₆ deficiency (not shown in Table 5) and low vitamin B₆ status (odds ratio, 1.84; CI, 1.39 to 2.42; P<.001). The risk associated with low vitamin B₆ status persisted when adjusted for the concentrations of tHcy (fasting, postload, or increase; see Table 5).

	Discussion

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Increases in plasma concentrations of homocysteine are common in patients with stroke, coronary disease, and peripheral vascular disease and confer an independent risk of atherosclerosis.^{1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16} In the present study, important links between homocysteine, low vitamin concentrations, and vascular disease risk were seen. The causes of hyperhomocysteinemia in these patients are poorly understood, although reduced activity of cystathionine ß-synthase^{2 4} or methylenetetrahydrofolate reductase,^{24 25} which are essential for the metabolism of homocysteine, could play a role. More importantly, however, concentrations of homocysteine rise as the levels of folate, vitamin B₁₂, and vitamin B₆ fall,^{21 22} and high homocysteine concentrations are often seen with deficiency of these vitamins.^{17 18 19}

In this investigation, homocysteine levels were higher in men, although the postload increase was greater in case subjects, with a consequently greater value in total homocysteine level. The gender difference may be because of the fact that more homocysteine is formed in men than in women in conjunction with creatine-creatinine synthesis.³² It is also possible that there are gender differences in the transsulfuration and remethylation of homocysteine, with more efficient remethylation in women and more efficient transsulfuration in men. Men may therefore have a higher folate requirement. Indeed, in the present study, folate levels were lower in women than in men, and case-control differences were only apparent in men.

In the present study, homocysteine correlated negatively with all three vitamins, although the rise in homocysteine was steepest with lower vitamin levels. When a standard definition (372 nmol/L) was used, folate deficiency was not associated with an increased risk of vascular disease. Low folate status, however, was associated with an increased risk of vascular disease. This risk was reduced by the inclusion of fasting homocysteine in the model, implying that the increased risk of vascular disease accompanying lower folate levels may be explained by the higher circulating homocysteine concentrations. These findings are consistent with those of Pancharuniti et al,¹² who showed an association between lower folate levels and angiographic evidence of 50% occlusion of one or more major coronary arteries in white males younger than 50 years of age. Recently, Morrison et al³³ reported a higher 15-year coronary mortality rate in patients with lower folate concentrations. In their study, however, no data were available on homocysteine levels. Our findings are consistent with the observation that low functional levels of folate and other B vitamins, including vitamin B₆, that are prevalent in the general population¹⁹ are also commonplace in patients with atherosclerosis. Because it is possible to lower homocysteine levels with folic acid, such treatment may reduce the risk of atherosclerosis.¹⁶

Concentrations of vitamin B₆ were lower in case subjects than in control subjects, and deficiency was common (>20%). These findings are unlikely to be a consequence of vascular disease because although vitamin B₆ levels may fall after myocardial infarction,^{34 35} concentrations return to baseline levels after 3 to 4 days.³⁶ Confounding disorders associated with reduced vitamin B₆ levels, such as cancer, renal disease, diabetes, or alcoholism,³¹ also could not have been responsible because such patients had been excluded from the present study.¹⁵ Control subjects had also been selected carefully, and values for random population control subjects were similar to those seen in control subjects recruited from other sources. The large sample size permitted the exploration of a number of models of vitamin B₆ deficiency and low vitamin B₆ status that confirmed the increased relative risk of vascular disease with lower vitamin concentrations. Risk fell with rising vitamin B₆ concentrations and was independent of traditional risk factors. Adjustment for fasting, postload, and increase in homocysteine concentrations did not abolish this effect. High homocysteine concentrations often follow a methionine load^{1 2 3 4 16 21 22} and have been ascribed to cystathionine ß-synthase deficiency.^{2 4} In such patients, however, deficiency of vitamin B₆ may be a more satisfactory explanation, because the loading test may be abnormal in such case subjects³¹ and the gene frequency for cystathionine ß-synthase deficiency is low.^{20 36} Other studies have also pointed to an increase in coronary artery disease risk with lower vitamin B₆ concentrations.^{14 33} In the study of Selhub et al,¹³ a relationship between lower vitamin B₆ levels and carotid disease was seen that diminished when adjusted for homocysteine. In other studies,^{37 38} arterial lesions have been seen in animals given pyridoxine-deficient diets. The mechanism for the vascular damage is unclear, although vitamin B₆ may alter platelet function,^{39 40} cholesterol concentrations,⁴¹ and antithrombin III activity⁴¹ as well as homocysteine concentrations.^{2 3 42 43 44}

In summary, low concentrations of folate and vitamin B₆ are often associated with high homocysteine concentrations. Lower levels of both these vitamins confer an increased risk of vascular disease. This risk may be mediated through homocysteine in the case of folate but not in the case of vitamin B₆. Such vitamin levels are commonplace in the population and include many individuals now thought to have vitamin concentrations in a normal range. The abnormalities may be readily reversed by folic acid either alone^{45 46} or in combination with vitamins B₁₂ and B₆.^{44 47} Intervention studies are now required to test the effects of such treatment on the primary and secondary prevention of vascular disease.

	Appendix 1

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 
Other Investigators in the European Concerted Action Project
Isabella Higgins (Department of Cardiology, Adelaide Hospital, Trinity College, Dublin, Ireland); Armando Sales Lúis (Servico de Medicina, Hospital de S. Francisco Xavier Lisbon, Portugal); Richard Sheahan (Division of Cardiology, University of Texas Medical Branch at Galveston); Bo Israelsson (Department of Medicine, Malmo General Hospital, Malmo, Sweden); Dorothy McMaster and Alun Evans (Department of Medicine, The Queen's University of Belfast, Northern Ireland); Petra Verhoef (Department of Public Health and Epidemiology, Agricultural University, Wageningen, Netherlands); Cuno Uiterwaal (Department of Epidemiology and Biostatistics, Erasmus University Medical School, Rotterdam, Netherlands); Generoso Andria (Facolta di Medicina e Chirurgia, Universita degli Studi di Napoli, Federico II, Naples, Italy); Hélene Bellet (Laboratoire de Medicine Experimentale, Institut de Biologie, Montpelier, France); Claude Wautrecht (Service de Pathologie Vasculaire, Clinique Médicale, University Libre de Bruxelles, Belgium); Harald de Valk (Department of Internal Medicine, University Hospital, Utrecht, Netherlands); Françoise Parrot Roulaud (Département Chromatographie, Hôpital Pellegrin, Bordeaux, France); Kok Soon Tan (Department of Cardiology, Toa Payoh Hospital, Singapore); Danielle Garçon (Laboratoire de Biochimie, Faculté de Pharmacie, Marseille, France); Maria José Medrano (Instituto de Salud "Carlos III", Centro Nacional de Epidemiologia, Madrid, Spain); Mirande Candito (Laboratoire de Biochimie, Hôpital Pasteur, Nice, France).

	Acknowledgments

 
We acknowledge with gratitude the funding for this work partially supplied by the following research organizations: Irish Heart Foundation; The Irish Health Research Board; The Norwegian Council on Cardiovascular Diseases and the Norwegian Research Council; The Netherlands Organization for Scientific Research; The Foundation for Metabolic Research of Utrecht; The Northern Ireland Chest Heart and Stroke Association; Instituto Nacional de Investigacao Sanitarias de la Seguridad Social; FIS No. 92/0914E, Spain; Progetto strategico "Farmaci per malattie orfane" nell'ambito del sottoprogetto Omocistinuria, CNF Roma, Consiglio Nazionale delle Ricerche, Rome, Italy; S.A. Roche, Brussels; The Swedish Heart and Lung Foundation; The Swedish Medical Research Council; and several other bodies.

	Footnotes

 
¹ For a complete list of investigators and their affiliations, please see the "Appendix."

Received June 5, 1997; revision received November 17, 1997; accepted November 17, 1997.

	References

Top Abstract Introduction Methods Results Discussion Appendix 1 References

 

1. Wilcken DEL, Wilcken B. The pathogenesis of coronary artery disease: a possible role for methionine metabolism. J Clin Invest. 1976;57:1079–1082.
   
2. Boers GHJ, Smals AGH, Trijbels FJM, Fowler B, Bakkeren JAJM, Schoonderwaldt HC, Kleijer WJ, Kloppenborg PWC. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med. 1985;313:709–715.[Abstract]
   
3. Brattström L, Israelsson B, Norrving B, Bergqvist D, Thörne J, Hultberg B, Hamfelt A. Impaired homocysteine metabolism in early-onset cerebral and peripheral occlusive arterial disease: effects of pyridoxine and folic acid treatment. Atherosclerosis. 1990;81:51–60.[Medline] [Order article via Infotrieve]
   
4. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med. 1991;324:1149–1155.[Abstract]
   
5. Malinow MR, Kang SS, Taylor LM, Wong PWK, Coull B, Inahara T, Mukerjee D, Sexton G, Upson B. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation. 1989;79:1180–1188.[Abstract/Free Full Text]
   
6. Taylor LM Jr, DeFrang RD, Harris J Jr, Porter JM. The association of elevated plasma homocyst(e)ine with progression of symptomatic peripheral arterial disease. J Vasc Surg. 1991;13:128–136.[Medline] [Order article via Infotrieve]
   
7. Arnesen E, Refsum H, Bonaa KH, Ueland PM, Forde OH, Nordrehaug JE. Serum total homocysteine and coronary heart disease. Int J Epidemiol. 1995;24:704–709.[Abstract/Free Full Text]
   
8. Brattström L, Lindgren A, Israelsson B, Malinow MR, Norrving B, Upson B, Hamfelt A. Hyperhomocysteinaemia in stroke: prevalence, cause and relationships to type of stroke and stroke risk factors. Eur J Clin Invest. 1992;22:214–221.[Medline] [Order article via Infotrieve]
   
9. Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, Ullmann D, Tishler PV, Hennekens CH. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA. 1992;268:877–881.[Abstract]
   
10. Dudman NPB, Wilcken DEL, Wang J, Lynch JF, Macey D, Lundberg P. Disordered methionine/homocysteine metabolism in premature vascular disease: its occurrence, cofactor therapy, and enzymology. Arterioscler Thromb. 1993;13:1253–1260.[Abstract/Free Full Text]
    
11. Wu LL, Wu J, Hunt SC, James BC, Vincent GM, Williams RR, Hopkins PN. Plasma homocysteine as a risk factor for early familial coronary artery disease. Clin Chem. 1994;40:552–561.[Abstract/Free Full Text]
    
12. Pancharuniti N, Lewis CA, Sauberlich HE, Perkins LL, Go RCP, Alvarez JO, Macaluso M, Acton RT, Copeland RB, Cousins AL, Gore TB, Cornwell PE, Roseman JM. Plasma homocyst(e)ine, folate, and vitamin B12 concentrations and risk for early-onset coronary artery disease. Am J Clin Nutr. 1994;59:940–948.[Abstract/Free Full Text]
    
13. Selhub J, Jacques PF, Bostom AG, D'Agostino RB, Wilson PWF, Belanger AJ, O'Leary DH, Wolf PA, Schaefer EJ, Rosenberg IH. Association between plasma homocysteine concentrations and extracranial carotid artery stenosis. N Engl J Med. 1995;332:286–291.[Abstract/Free Full Text]
    
14. Robinson K, Mayer EL, Miller D, Green R, van Lente F, Gupta A, Kottke-Marchant K, Savon S, Selhub J, Nissen SE, Kutner M, Topol EJ, Jacobsen DW. Hyperhomocysteinemia and low pyridoxal phosphate: common and independent reversible risk factors for coronary artery disease. Circulation. 1995;92:2825–2830.[Abstract/Free Full Text]
    
15. Graham IM, Daly LE, Refsum HM, Robinson K, Brattström L, Ueland P, Palma-Reis R, Boers G, Sheahan R, Israelsson B, Uiterwaal CS, Meleady R. Plasma homocysteine as a risk factor for vascular disease: the European Concerted Action Project. JAMA. 1997;277:1775–1781.[Abstract]
    
16. Boushey CJ, Beresford SA, Omenn GC, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes. JAMA. 1995;274:1049–1057.[Abstract]
    
17. Stabler SP, Marcell PD, Podell ER, Allen RH, Savage DG, Lindenbaum J. Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry. J Clin Invest. 1988;81:466–474.
    
18. Brattström LE, Israelsson B, Lindgärde F, Hultberg B. Higher total plasma homocysteine in vitamin B12 deficiency than in heterozygosity for homocystinuria due to cystathionine ß-synthase deficiency. Metabolism. 1988;37:175–178.[Medline] [Order article via Infotrieve]
    
19. Selhub J, Jacques PF, Wilson PWF, Rush D, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA. 1993;270:2693–2698.[Abstract]
    
20. Mudd SH, Levy HL, Skovby F. Disorders of transsulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 7th ed. New York, NY: McGraw Hill; 1995:1279–1327.
    
21. Ueland PM, Refsum H, Brattström L. Plasma homocysteine and cardiovascular disease. In: Francis RB Jr, ed. Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function. New York, NY: Marcel Dekker Inc; 1992:183–236.
    
22. Mayer EM, Jacobsen DW, Robinson K. Homocysteine and coronary atherosclerosis. J Am Coll Cardiol. 1996;27:517–527.[Abstract]
    
23. Rosenblatt DS. Inherited disorders of folate transport and metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 7th ed. New York, NY: McGraw-Hill; 1995:3111–3128.
    
24. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJH, den Heijer M, Kluijtmans LAJ. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10:111–113.[Medline] [Order article via Infotrieve]
    
25. Kang S-S, Wong PWK, Susmano A, Sora J, Norusis M, Ruggie N. Thermolabile methylenetetrahydrofolate reductase: an inherited risk factor for coronary artery disease. Am J Hum Genet. 1991;48:536–545.[Medline] [Order article via Infotrieve]
    
26. Boers G. Refinement of the methionine loading test. In: Robinson K, ed. Homocysteinaemia and Vascular Disease. Proceedings of an EC COMAC Epidemiology Expert Group Workshop. Luxembourg, Belgium: Commissioners of the European Communities; 1990:61–66.
    
27. Fiskerstrand T, Refsum H, Kvalheim G, Ueland PM. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin Chem. 1993;39:263–271.[Abstract]
    
28. Chen IW, Silberstein EB, Maxon HR, Volle CP, Sohnlein BH. Semiautomated system for simultaneous assays of serum vitamin B12 and folic acid in serum evaluated. Clin Chem. 1982;28:2161–2165.[Abstract/Free Full Text]
    
29. Hamfelt A. A simplified method for determination of pyridoxal phosphate in biological samples. Ups J Med Sci. 1986;91:105–109.[Medline] [Order article via Infotrieve]
    
30. Tietz NW, ed. Clinical Guide to Laboratory Tests. 3rd ed. Philadelphia, Pa: WB Saunders Co; 1995.
    
31. Leklem JJ. Vitamin B6. In: Shils ME, Olson JA, Shike M, eds. Modern Nutrition in Health, Disease. 8th ed. Philadelphia, Pa: Lea & Febiger; 1994:383–394.
    
32. Brattström L, Lindgren A, Israelson B, Andersson A, Hultberg B. Homocysteine and cysteine: determinants of plasma levels in middle-aged and elderly subjects. J Int Med. 1994;236:633–641.[Medline] [Order article via Infotrieve]
    
33. Morrison HI, Schaubel D, Desmeules M, Wigle DT. Serum folate and risk of fatal coronary heart disease. JAMA. 1996;275:1893–1896.[Abstract]
    
34. Kok FJ, Schrijver J, Hofman A, Witteman JC, Kruyssen DA, Remme WJ, Valkenburg HA. Low vitamin B6 status in patients with acute myocardial infarction. Am J Cardiol. 1989;63:513–516.[Medline] [Order article via Infotrieve]
    
35. Vermaak WJH, Barnard HC, Potgieter GM, Theron H du T. Vitamin B6 and coronary artery disease: epidemiological observations and case studies. Atherosclerosis. 1987;63:235–238.[Medline] [Order article via Infotrieve]
    
36. Vermaak WJ, Barnard HC, Van Dalen EM, Potgieter GM, Van Jaarsveld H, Myburgh SJ. Compartmentalization of pyridoxal-5'-phosphate during the acute phase of myocardial infarction. Klin Wochenschr. 1988;66:428–433.[Medline] [Order article via Infotrieve]
    
37. Rinehart JF, Greenberg LD. Arteriosclerotic lesions in pyridoxine-deficient monkeys. Am J Pathol. 1949;25:481–491.[Medline] [Order article via Infotrieve]
    
38. Smolin LA, Crenshaw TD, Kurtycz D, Benevenga NJ. Homocyst(e)ine accumulation in pigs fed diets deficient in vitamin B-6: relationship to atherosclerosis. J Nutr. 1983;113:2122–2133.
    
39. Krishnamurthi S, Kakkar VV. Studies on the effect of platelet inhibitors on platelet adhesion to collagen and collagen-induced human platelet activation. Thromb Haemost. 1985;53:337–342.[Medline] [Order article via Infotrieve]
    
40. Subbarao K, Kuchibhotla J, Kakkar VV. Pyridoxal 5'-phosphate: a new physiological inhibitor of blood coagulation and platelet function. Biochem Pharmacol. 1979;28:531–534.[Medline] [Order article via Infotrieve]
    
41. Brattström L, Stavenow L, Galvard H, Nilsson-Ehle P, Berntorp E, Jerntorp P, Elmstahl S, Pessah-Rasmussen H. Pyridoxine reduces cholesterol and low-density lipoprotein and increases antithrombin III activity in 80-year-old men with low plasma pyridoxal 5-phosphate. Scand J Clin Lab Invest. 1990;50:873–877.[Medline] [Order article via Infotrieve]
    
42. Franken DG, Boers GHJ, Blom HJ, Trijbels FJM, Kloppenborg PWC. Treatment of mild hyperhomocysteinemia in vascular disease patients. Arterioscler Thromb. 1994;14:465–470.[Abstract/Free Full Text]
    
43. Ryan M, Robinson K, Clarke R, Refsum R, Ueland P, Graham I. Vitamin B6 and folate reduce homocysteine concentrations in coronary artery disease. Ir J Med Sci. 1993;162:197A. Abstract.
    
44. Naurath HJ, Joosten E, Reizler R, Stabler SP, Allen RH, Lindenbaum J. Effects of vitamin B12, folate, and vitamin B6 supplements in elderly people with normal serum vitamin concentrations. Lancet. 1995;346:85–89.[Medline] [Order article via Infotrieve]
    
45. Brattström LE, Israelsson B, Jeppsson J-O, Hultberg BL. Folic acid: an innocuous means to reduce plasma homocysteine. Scand J Clin Lab Invest. 1988;48:215–221.[Medline] [Order article via Infotrieve]
    
46. Landgren F, Israelsson B, Lindgren A, Hultberg B, Andersson A, Brattström L. Plasma homocysteine in acute myocardial infarction: homocysteine-lowering effect of folic acid. J Intern Med. 1995;237:381–388.[Medline] [Order article via Infotrieve]
    
47. Ubbink JB, Vermaak WJH, van der Merwe A, Becker PJ. Vitamin B-12, vitamin B-6, and folate nutritional status in men with hyperhomocysteinemia. Am J Clin Nutr. 1993;57:47–53.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				M. Ebbing, O. Bleie, P. M. Ueland, J. E. Nordrehaug, D. W. Nilsen, S. E. Vollset, H. Refsum, E. K. Ringdal Pedersen, and O. Nygard Mortality and Cardiovascular Events in Patients Treated With Homocysteine-Lowering B Vitamins After Coronary Angiography: A Randomized Controlled Trial JAMA, August 20, 2008; 300(7): 795 - 804. [Abstract] [Full Text] [PDF]

				C. Weikert, J. Dierkes, K. Hoffmann, K. Berger, D. Drogan, K. Klipstein-Grobusch, J. Spranger, M. Mohlig, C. Luley, and H. Boeing B Vitamin Plasma Levels and the Risk of Ischemic Stroke and Transient Ischemic Attack in a German Cohort Stroke, November 1, 2007; 38(11): 2912 - 2918. [Abstract] [Full Text] [PDF]

				J. Dierkes, C. Weikert, K. Klipstein-Grobusch, S. Westphal, C. Luley, M. Mohlig, J. Spranger, and H. Boeing Plasma pyridoxal-5-phosphate and future risk of myocardial infarction in the European Prospective Investigation into Cancer and Nutrition Potsdam cohort Am. J. Clinical Nutrition, July 1, 2007; 86(1): 214 - 220. [Abstract] [Full Text] [PDF]

				C. P. Lima, S. R. Davis, A. D. Mackey, J. B. Scheer, J. Williamson, and J. F. Gregory III Vitamin B-6 Deficiency Suppresses the Hepatic Transsulfuration Pathway but Increases Glutathione Concentration in Rats Fed AIN-76A or AIN-93G Diets J. Nutr., August 1, 2006; 136(8): 2141 - 2147. [Abstract] [Full Text] [PDF]

				K. E. MacKenzie, E. J. Wiltshire, R. Gent, C. Hirte, L. Piotto, and J. J. Couper Folate and Vitamin B6 Rapidly Normalize Endothelial Dysfunction In Children With Type 1 Diabetes Mellitus Pediatrics, July 1, 2006; 118(1): 242 - 253. [Abstract] [Full Text] [PDF]

				K. H. Bonaa, I. Njolstad, P. M. Ueland, H. Schirmer, A. Tverdal, T. Steigen, H. Wang, J. E. Nordrehaug, E. Arnesen, K. Rasmussen, et al. Homocysteine Lowering and Cardiovascular Events after Acute Myocardial Infarction N. Engl. J. Med., April 13, 2006; 354(15): 1578 - 1588. [Abstract] [Full Text] [PDF]

				O. Midttun, S. Hustad, E. Solheim, J. Schneede, and P. M. Ueland Multianalyte Quantification of Vitamin B6 and B2 Species in the Nanomolar Range in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry Clin. Chem., July 1, 2005; 51(7): 1206 - 1216. [Abstract] [Full Text] [PDF]

				J. J. Carrero, E. Lopez-Huertas, L. M. Salmeron, L. Baro, and E. Ros Daily Supplementation with (n-3) PUFAs, Oleic Acid, Folic Acid, and Vitamins B-6 and E Increases Pain-Free Walking Distance and Improves Risk Factors in Men with Peripheral Vascular Disease J. Nutr., June 1, 2005; 135(6): 1393 - 1399. [Abstract] [Full Text] [PDF]

				A.M.W. Spijkerman, Y.M. Smulders, P.J. Kostense, R.M.A. Henry, A. Becker, T. Teerlink, C. Jakobs, J.M. Dekker, G. Nijpels, R.J. Heine, et al. S-Adenosylmethionine and 5-Methyltetrahydrofolate Are Associated With Endothelial Function After Controlling for Confounding by Homocysteine: The Hoorn Study Arterioscler. Thromb. Vasc. Biol., April 1, 2005; 25(4): 778 - 784. [Abstract] [Full Text] [PDF]

				S. R Davis, J. B Scheer, E. P Quinlivan, B. S Coats, P. W Stacpoole, and J. F Gregory III Dietary vitamin B-6 restriction does not alter rates of homocysteine remethylation or synthesis in healthy young women and men Am. J. Clinical Nutrition, March 1, 2005; 81(3): 648 - 655. [Abstract] [Full Text] [PDF]

				J. Dierkes, K. Hoffmann, K. Klipstein-Grobusch, C. Weikert, H. Boeing, B.-C. Zyriax, E. Windler, and J. Kratzsch Low plasma pyridoxal-5'phosphate and cardiovascular disease risk in women: results from the Coronary Risk Factors for Atherosclerosis in Women Study Am. J. Clinical Nutrition, March 1, 2005; 81(3): 725 - 727. [Full Text] [PDF]

				B. Smolkova, M. Dusinska, K. Raslova, M. Barancokova, A. Kazimirova, A. Horska, V. Spustova, and A. Collins Folate levels determine effect of antioxidant supplementation on micronuclei in subjects with cardiovascular risk Mutagenesis, November 1, 2004; 19(6): 469 - 476. [Abstract] [Full Text] [PDF]

				O. Bleie, H. Refsum, P. M. Ueland, S. E. Vollset, A. B. Guttormsen, E. Nexo, J. Schneede, J. E. Nordrehaug, and O. Nygard Changes in basal and postmethionine load concentrations of total homocysteine and cystathionine after B vitamin intervention Am. J. Clinical Nutrition, September 1, 2004; 80(3): 641 - 648. [Abstract] [Full Text] [PDF]

				C. Cantu, E. Alonso, A. Jara, L. Martinez, C. Rios, M. d. l. A. Fernandez, I. Garcia, and F. Barinagarrementeria Hyperhomocysteinemia, Low Folate and Vitamin B12 Concentrations, and Methylene Tetrahydrofolate Reductase Mutation in Cerebral Venous Thrombosis Stroke, August 1, 2004; 35(8): 1790 - 1794. [Abstract] [Full Text] [PDF]

				R. H. Samson, Z. Yungst, and D. P. Showalter Homocysteine, a Risk Factor for Carotid Atherosclerosis, Is Not a Risk Factor for Early Recurrent Carotid Stenosis Following Carotid Endarterectomy Vascular and Endovascular Surgery, July 1, 2004; 38(4): 345 - 348. [Abstract] [PDF]

				C. A. Perry, S. A. Renna, E. Khitun, M. Ortiz, D. J. Moriarty, and M. A. Caudill Ethnicity and Race Influence the Folate Status Response to Controlled Folate Intakes in Young Women J. Nutr., July 1, 2004; 134(7): 1786 - 1792. [Abstract] [Full Text]