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European Heart Journal Advance Access originally published online on January 31, 2007
European Heart Journal 2007 28(4):484-491; doi:10.1093/eurheartj/ehl470
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© The European Society of Cardiology 2007. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Both vitamin B6 and total homocysteine plasma levels predict long-term atherothrombotic events in healthy subjects

Diego Vanuzzo1, Lorenza Pilotto1, Rossana Lombardi2, Guido Lazzerini3, Marisa Carluccio3, Silvia Diviacco4, Franco Quadrifoglio4, Giorgia Danek1, Dario Gregori6,7, Paolo Fioretti6, Marco Cattaneo2,5 and Raffaele De Caterina3,8,*

1 Cardiovascular Prevention Center, ASS 4 and Agenzia Regionale della Sanità del Friuli-Venezia Giulia, Udine, Italy
2 A. Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal Medicine, IRCCS Ospedale Maggiore, University of Milano, Milano, Italy
3 C.N.R. Institute of Clinical Physiology, Pisa, Italy
4 Department of Biomedical Sciences and Technologies, University of Udine, Udine, Italy
5 Unit of Hematology and Thrombosis, Ospedale San Paolo-DMCO University of Milan, Milan, Italy
6 IRCAB Foundation, Udine, Italy
7 Department of Public Health and Microbiology, University of Torino, Torino, Italy
8 Department of Cardiology, ‘G. d'Annunzio’ University, Chieti, C/o Ospedale S. Camillo de Lellis, Via Forlanini, 50-66100 Chieti, Italy

Received 7 April 2006; revised 13 November 2006; accepted 21 December 2006; online publish-ahead-of-print 31 January 2007.

* Corresponding author. Tel: +39 0871 41512; fax: +39 0871 553 461. E-mail address: rdecater{at}unich.it


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Aims The contribution of homocysteine and group B vitamins in determining cardiovascular risk is debated. We assessed the predictive value of total homocysteine (tHcy), vitamin B12, folate, and vitamin B6 on the long-term occurrence of coronary and cerebral atherothrombotic events in a nested case–control study.

Methods and results Within a cohort of 1021 healthy subjects (490 men and 531 women) recruited in 1987, 66 first-ever coronary and 43 first-ever cerebrovascular events were recorded at a 12-year follow-up (cases, n = 109). A total of 109 control subjects (remaining free from events) were matched with cases according to age, sex, smoking, hypertension, dyslipidaemia, and body mass index. Serum samples obtained in 1987 at baseline were used to measure tHcy, folate, and vitamins B12 and B6, as well as C-reactive protein plasma concentrations. We found a significant graded association between tHcy levels and the risk of coronary and cerebrovascular events [odds ratio (OR) for uppermost vs. lowermost quartile = 1.34, 95% CI 1.01–1.76)]. Folate and vitamin B12 did not significantly differ between cases and controls, but were negatively (P < 0.01) correlated with tHcy. Vitamin B6 did not correlate with tHcy levels, but differed significantly between cases and controls: for subjects in the uppermost quartile vs. the lowermost quartile of vitamin B6, OR = 0.69 (95% CI 0.49–0.98). For subjects in the lowermost quartile of vitamin B6 and the uppermost quartile of tHcy, OR = 17.50 (95% CI 1.97, 155.59). Cases and controls were not different as to C-reactive protein.

Conclusion tHcy and plasma vitamin B6 are long-term independent risk factors for coronary and cerebrovascular events.

Key Words: Vitamin B6 • Homocysteine • Coronary heart disease • Cerebrovascular disease • Atherothrombosis


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
The observation that severe hyperhomocysteinaemia is related to atherothrombotic vascular disease was first reported by McCully1 in 1969. Since then, many case–control and prospective studies have found that homocysteine is concentration-dependently related to cardiovascular risk, suggesting a causal role. A recent meta-analysis including 30 prospective or retrospective studies involving a total of 5073 coronary heart disease (CHD) events and 1113 stroke events has shown that after adjustment for known cardiovascular risk factors and regression dilution bias in the prospective studies, a 25% lower than usual homocysteine level [about 3 µmol/L (0.41 mg/L)] is associated with an 11% lower CHD risk [odds ratio (OR), 0.89; 95% confidence interval (CI), 0.83–0.96)] and 19% lower stroke risk (OR, 0.81; 95% CI, 0.69–0.95).2

Plasma homocysteine levels are known to be related to concentrations of B vitamins, specifically of folate, vitamin B12 and vitamin B6, all involved in homocysteine metabolism,3 and therefore one would expect some relationship of B vitamins status with the risk of cardiovascular events mediated by homocysteine plasma concentrations. However, a relationship between B vitamin status and cardiovascular disease has not been found in most studies.4–8 A partial exception has been vitamin B6, for which associations with vascular disease have been reported. Low circulating vitamin B6 concentrations were indeed identified as associated with vascular disease (CHD, stroke, and peripheral vascular disease) in two case–control studies, but this occurred independent of homocysteine levels,9,10 raising the possibility of some direct effect on vascular events. Two other case–control studies, however, did not report associations.6,7 In the Physicians' Health Study, where a relationship was found between baseline hyperhomocyst(e)inaemia and the risk of myocardial infarction and cardiac death at 5 years,11 further analysis of the risk of low folate and vitamin B6 at baseline for CHD at a 7.5-year follow-up showed trends for associations, but these were not statistically significant.12 In the ARIC Study, with a prospective case–cohort design evaluating the role of fasting homocysteine and B vitamins on the incidence of CHD over an average 3.3 years of follow-up, only vitamin B6 was associated, at multivariable analysis, with CHD incidence, with a relative risk for the highest vs. the lowest quintile of 0.28 (95% CI 0.1–0.7).8 Vitamin B6 significantly improved brachial artery flow-dependent dilation without changing homocysteine concentration in a small, randomized, prospective, double-blind trial.13 In the first randomized intervention trials so far reported, the combined administration of vitamin B6, vitamin B12, and folate decreased homocysteine without improving cardiovascular outcomes,14–16 but these studies did not clarify the possible benefit of the administration of some of these individual vitamins, and vitamin B6 in particular, independent from homocysteine lowering.

There is therefore a considerable uncertainty in the literature both on the relationship of group B vitamins with cardiovascular risk and on the possible independence of such relationship, if existing, from homocysteine levels. Such uncertainty is particular pertaining to prospective studies, potentially able to identify much needed new predictors of cardiovascular events.2,17

We therefore examined the role of homocysteine and other determinants of homocysteine metabolism, including folate, vitamin B12, and vitamin B6, in a prospective nested case–control study with a long follow-up. Since homocysteine may be related to risk factors such as smoking,18,19 hypertension,19–21 dyslipidaemia,19,22,23 hyperglycaemia,24 and high C-reactive protein,25 we matched also for these risk factors, besides age and sex, in identifying cases and control subjects.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Study cohort
The Martignacco Project is a population-based study of cardiovascular disease risk factors and a heart-health promotion program conducted in a North-Eastern Italian community, which started in 1977 and is still ongoing.26 It is the Italian section of the World Health Organization Comprehensive Cardiovascular Community Control Programmes (WHO-CCCCP), launched in 1976 after the presentation of the first 5 years of the Finnish North Karelia Project,27,28 with a detailed protocol.26

As an index cohort to evaluate trends for risk factors, mortality and incident cases of CHD and cerebrovascular disease, all men and women in the community aged 40–59 in 1977 (1302 subjects) were identified and invited to a first examination. The participation rate was 90%, resulting in the enrolment of 1171 subjects. Ten years later (1987) this cohort was re-examined (from here on referred to as ‘baseline examination’), and 1021 subjects (490 men and 531 women) were found ‘free of atherosclerotic cardiovascular diseases’ according to WHO—Cardiovascular Survey Methods—criteria,29 constituting the population of the present study (Table 1).


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  		Table 1 Characteristics of the original study population at the ‘baseline examination’a in 1987

 
At the 1987 recall, blood samples were obtained from the subjects between 8:00 a.m. and 9:30 a.m. after an overnight fast. Serum was separated within 60 min and stored at –80°C in four 1 mL aliquots, snap-frozen and maintained at –80°C, until the analysis. The storage facility had an alarm system with a technician available 24 h a day and back-up freezers to avoid accidental thawing.

The cohort of 1021 initially healthy subjects in this study was followed from the ‘baseline examination’ in 1987 up to 30 June 1999. The planned duration of the follow-up was 12 years in case of no cardiovascular events. Actual duration was [median (interquartile range)] 12.1 (11.5, 12.9) years.

Outcome measurements
Data on the first incident CHD or cerebrovascular events were obtained every 6 months during the follow-up period through the regional computerized health information system, including hospital discharge diagnoses and mortality registries, with each patient identified by a unique regional identification number. All new suspected acute CHD and stroke events were evaluated according to the WHO MONICA Project manual.30,31 CHD events were defined as diagnostic category 1 (in practice: ST-elevation acute myocardial infarction) or diagnostic category 2 (in practice: non-ST-elevation acute coronary syndromes), both requiring hospitalization. Cerebrovascular events were defined as MONICA category 1 (strokes) and WHO-CCCCP transient ischaemic attacks.30,31 All such events retrieved through hospital discharge diagnoses and mortality registries were validated reviewing hospital charts and interviewing relatives or the appropriate physicians in case of fatal out-of-hospital deaths. Peripheral arterial disease, a possibly logical outcome in this study, was not an endpoint of the MONICA project, and therefore no testing was performed to diagnose this condition. We, however, administered the London School of Hygiene questionnaire for peripheral arterial disease, and no cases of this conditions were recorded among cases or controls.

Matching criteria
Each of the case subjects was matched with one control subject, taken from the same cohort, for age, sex, smoking status, weight category, and the presence of hypertension, dyslipidaemia, and diabetes. According to the original WHO CCCCP Protocol,29 partly modified by the WHO MONICA Protocol,31 subjects were considered smokers if they regularly smoked at least one cigarette a day. Hypertension was here defined as a systolic blood pressure (mean of 2 readings) ≥140 mmHg or a diastolic blood pressure (mean of 2 readings) ≥90 mmHg, or as being on an anti-hypertensive drug treatment. Dyslipidaemia was defined by a total cholesterol ≥240 mg/dL or triglycerides ≥170 mg/dL or as being on a lipid-lowering drug. Such definition did not include low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol in keeping with the criteria in use at the 1977 start of the entire project. Diabetes was defined by a blood fasting glucose ≥126 mg/dL or being on either insulin or an oral antidiabetic drugs. For definition of weight classes, three weight categories were derived from assessment of the body mass index (kg/m2): normal weight: BMI < 25; overweight: 25 ≤ BMI < 30; obesity: BMI ≥ 30. For the medical history and the dietary questionnaire, the WHO Cardiovascular Survey Methods were adopted,29 after standardization and certification of the personnel (physicians and nurses) involved through the MONICA procedures.30 All initial biochemical assays were performed at the Department of Clinical Pathology of the S. Maria della Misericordia Hospital in Udine, Italy, a laboratory already in 1987 standardized according to the WHO Lipid Standardization Laboratory programme.32

All possible controls were identified for each case. If more than one control was available for the matching, this was chosen at random; if no strictly matching control was available, the nearest most similar one was selected, considering matching variables in the following order of importance: (i) sex; (ii) age; (iii) smoking status; (iv) dyslipidaemia; (v) hypertension; (vi) diabetes; (vii) weight category. After the matching, a number of data were recorded from the medical history, the dietary questionnaire, or biohumoral data, also pertaining to characteristics used for the matching, this time recorded and then analysed as continuous variables. This allowed an additional independent testing of the adequacy of the matching. In case of missing variables (always <10% of the data) for either a case or the corresponding control, the data of the corresponding control or case was dropped from calculations in order not to introduce biases in favour of cases or controls.

Other measurements
Total homocysteine (tHcy) levels (free and protein bound) were measured by a fluorescence polarized light immunoassay after reduction of the oxidized and protein-bound homocysteine to free homocysteine and subsequent enzymatic conversion to S-adenosyl-L-homocysteine (Abbott IMX, Abbott Laboratories, Abbott Park, IL, USA). Validation of the assay included a comparison with a previously validated high-performance liquid chromatography method with fluorimetric detection,33 yielding an intercept of 0.12, a slope of 0.980, and a correlation coefficient of 0.989 in a series of 114 samples in a range between 4.1 and 40.2 µmol/L. The between-run coefficient of variation for this assay was 4.2%.

Serum folates and vitamin B12 were measured by radioimmunoassay (ICN Biochemicals Inc., Irvine, CA, USA); the between-assay coefficients of variation were 9.2% for folate and 8.5% for vitamin B12. Pyridoxal-5'-phosphate (PLP), the coenzyme form of vitamin B6, was measured by the tyrosine decarboxylase method described by Shin-Buehring et al.34; the between-assay coefficient of variation was 11%.35 Plasma samples for PLP measurement were available from 102 cases and 100 controls only, with no plasma left over for this assay from seven cases and nine controls.

C-reactive protein (as C-reactive protein Latex, Cobas Integra 700, Roche Diagnostics, Basel, Switzerland, analytical range with linear response: 0.25–16 mg/dL) was measured to exclude acute-phase reactions and to monitor an inflammatory variable potentially related to outcomes.

Assays for each variable described here were performed simultaneously for cases and controls.

Statistical methods
Descriptive analysis is presented with percentages (number of cases) or median (first, third quartile), as appropriate. Pairwise relationhips for B6, B12, folates, and homocysteine are here reported using a scatter-plot matrix. Pairwise associations were estimated with non-parametric regression functions, fitted via local smoothers.36 In addition, Kendall's Tau-b correlation coefficient was estimated for each pair of variables because this procedure is robust in case of departure from normality for some variables. A conditional logistic regression model37 was used to model association of covariates with event status, with matching variables excluded from the models. Thus, a multivariable model was built. Variables related to outcome were entered in the model as-is, i.e. without transformations, cutting-off, or aggregation of levels, apart from homocysteine levels, which were log-transformed to stabilize model residuals. The model building strategy was established on the basis of the following steps: first, main effects were selected using a backward selection procedure38 and the score test.39 Secondly, all possible combinations between pairs of the main effects, as selected in the previous step, were evaluated and critically discussed from a biological point of view. Pairs resulted significant at the score test were finally retained for the model.

Final results are presented using OR along with 0.95 confidence intervals (CIs) and exact P-values.The entire analysis was performed using S-plus 2000 (Data Analysis Products Division, MathSoft, Seattle, WA, USA), with Harrel's and Therneaus's libraries.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 Conclusions
 Acknowledgements
 References
 
Characteristics of the study population at enrolment in 1987 are detailed in Table 1.

Throughout the follow-up period, there were 66 first-ever CHD cases (30 cases of acute myocardial infarction, of which 15 fatal, and 36 cases of unstable angina) and 43 first-ever cases of acute cerebrovascular episodes (including 29 strokes, of which 7 fatal, and 14 transient ischaemic attacks), for a total of 109 cases. The matching adequacy, by comparisons of continuous variables related to matching is illustrated in Table 2. There were no statistically significant differences as to any such variables. Levels of C-reactive protein turned out also to be similar between cases and controls (Table 2). There were also no statistically significant differences between cases and controls with regard to either categorical or continuous variables related to the use of medications potentially affecting cardiovascular outcomes, such as angiotensin-converting-enzyme (ACE)-inhibitors, beta-blockers, statins or aspirin, as well as to the percentage of people reporting alcohol drinking and the average alcohol intake, percentage of smokers and average number of cigarettes used, diabetes, anaemia potentially related to vitamin B deficiencies, renal dysfunction, lipid parameters, and percentage of smokers (Table 3). No subjects in the cohort reported, at a specific request, any use of vitamin supplements, either chronically or in the month preceding the evaluation. Medications that can alter vitamin B status and homocysteine levels (for vitamin B6: tricyclic antidepressants, mono-amino-oxidase inhibitors, isoniazid, birth control medications, erythropoietin, methotrexate, penicillamine, or theophylline; for vitamin B12: antacids, histamine receptor-2 antagonists, proton pump inhibitors, methotrexate, metformin, phenobarbital, or phenytoin) were also assumed in similar percentages of cases and controls. Calorie intake, evaluated by a 7-day recall questionnaire, as well as consumption of fruit and vegetables (semi-quantitative assessment) also showed similar dietary intakes (data not shown).


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  		Table 2 Characteristics of cases and controls related to matching variables and levels of C-reactive protein

 

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  		Table 3 Compared characteristics of cases and controls as of medications and other variables potentially affecting outcomes

 
Unadjusted associations between homocysteine and B vitamins with event status are shown in Table 4. Pairwise associations between each of these variables are shown in Figure 1. There were significant inverse correlations of vitamin B12 and folate with homocysteine, whereas no correlations were found between levels of vitamin B6 and homocysteine and between any of the vitamins assayed (Figure 1).


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  		Table 4 Homocysteine and B vitamins in cases and controls

 

Figure 1
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  		Figure 1 Scatter-plot matrix for homocysteine, vitamin B12, folates, and vitamin B6 representing the pairwise associations between each of these variables. Non-parametric regression analysis of the pairwise relationships are estimated and plotted as a line in each box. Triangles represent cases and squares represent controls. Kendall's Tau-b pairwise correlations are for Homocysteine vs. B6: 0.005 (NS), vs. B12: –0.161 (P < 0.05), vs. Folates: –0.33 (P < 0.05), and for Folates vs. B6: 0.025 (NS), vs. B12: 0.002 (NS), and finally for B12 vs. B6: 0.066 (NS).

 
At multivariable analysis, among all clinical and biochemical markers assessed, only homocysteine levels and vitamin B6 were found to be predictors of the risk of an event: the risk was found to increase by 34% with an increase of 4.2 µmol/L of homocysteine level, and by 45% for a, respective, decrease of 24.9 nmol/L in vitamin B6 level (Table 5). An interaction term between B6 and homocysteine was tested and resulted significant at the score test (P-value = 0.023). Thus, the combined risk of homocysteine and vitamin B6 (PLP) for cardiovascular events was studied. The risk of a cardiovascular event was highest when homocysteine was highest and vitamin B6 lowest (Table 6). As an example, for subjects in the lowermost quartile of vitamin B6 and the uppermost quartile of tHcy, OR = 17.50 (95% CI 1.97, 155.59).


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  		Table 5 Conditional multivariable regression model with interaction (regression coefficient –0.006026, P-value of interaction 0.023)

 

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  		Table 6 OR with 95% confidence intervals (in brackets) for homocysteine and vitamin B6 distribution expressed in quartiles (number of observations per cell in parentheses)

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Conclusions
 Acknowledgements
 References
 
In this prospective study, we found that both high levels of homocysteine and low levels of vitamin B6 are associated with an increased risk of subsequent acute CHD and cerebrovascular events, with an interaction between homocysteine and vitamin B6 in predicting such vascular events. This is the first report demonstrating the combined predictive value of these two factors in a prospective study. Only the Physicians' Health Study11,12 and the ARIC Study8 prospectively evaluated the prognostic significance of homocysteine and B vitamins, but only considering CHD events as the endpoints. In the Physicians' Health Study, the analysis, carried out for a mean 7.5-year follow-up, only showed trends for associations, without statistical significance.12 In the ARIC Study, conducted with a nested case–cohort design, homocysteine was associated with CHD incidence only at the univariable analysis, whereas low vitamin B6 values were associated with CHD incidence both at univariable and multivariable analysis.8 Here vitamin B6 showed an independent inverse correlation with CHD events, at a relative risk (RR) of 0.28 (95% CI 0.1–0.7) for the highest vs. the lowest quintile.8 The study follow-up in that study was short compared with ours (3.3 vs. 12 years). In a cross-sectional case–control study, Robinson et al.9 found an OR of 1.76 (95% CI 1.33–2.34) for vitamin B6 in the lowest vs. the highest quintile after adjustment for homocysteine, considering patients with vascular disease (stroke, peripheral vascular disease, and CHD) as cases. In our study, the OR for vitamin B6 in the III vs. the I quartile was 0.69 (95% CI 0.49, 0.98), consistent with these studies.

In a systematic meta-analysis, Ford et al.40 evaluated 57 publications relating homocysteine plasma concentrations with CHD and cerebrovascular disease, respectively. Ten nested case–control studies dealt with CHD and five with cerebrovascular disease. For a 5 µmol/L increase in homocysteine, these authors calculated an average OR of 1.23 for CHD and 1.58 for cerebrovascular disease in nested case–control studies. In a similar meta-analysis from case–control studies from 30 prospective or retrospective studies involving a total of 5073 CHD events and 1113 strokes, stronger associations were observed in retrospective studies of homocysteine measured in blood collected after the onset of disease than in prospective studies among individuals who had no history of vascular disease at blood collection. After adjustment for known cardiovascular risk factors and regression dilution bias in the prospective studies, a 3 µmol/L (0.41 mg/L) lower than usual homocysteine level was associated with an 11% (OR, 0.89; 95% CI, 0.83–0.96) lower CHD risk and 19% (OR, 0.81; 95% CI, 0.69–0.95) lower stroke risk.2 Considering that the difference between the highest and the lowest quartile is about 5 µmol/L in our study, the OR of 1.34 for the composite endpoint (CHD plus cerebrovascular events) is consistent with the OR reported in these meta-analyses. The long follow-up in our study, longer than in any previous study, may have led to an attenuation of the net effect of these analytes. Taking the regression dilution ratio of 0.53 at 12 years calculated by Clarke et al.41 into consideration, the risk of CHD or cerebrovascular disease associated with homocysteine was probably attenuated by one-half in our study.

Our study also provided an opportunity to assess the relative effect of C-reactive protein in relation to the other variables investigated. High-sensitivity C-reactive protein (hs-C-reactive protein) has recently gained widespread acceptance as a novel vascular risk factor among healthy subjects.42 In a prospective cohort of women, hs-C-reactive protein proved superior to homocysteine in predicting subsequent vascular events.43 Vitamin B6 was not, however, considered in that analysis. Recently, an association between low vitamin B6 plasma levels and C-reactive protein has also been reported.25 In the present study, we found plasma C-reactive protein levels not to be significantly different between cases and controls. This would indicate that, at least in our population, the predictive value of homocysteine and vitamin B6 is independent of C-reactive protein. That this result is due to our method of measurement, different from the high-sensitivity method used in other studies42,43 is unlikely, due to the fact that C-reactive protein was undetectable in only 4 of the 218 subjects studied here.

In agreement with the literature (reviewed in44), we found significant inverse relationships between folate or vitamin B12 and homocysteine plasma levels. However, we found no relationship between the status of folate or vitamin B12 and subsequent events. Plasma homocysteine levels, on the other hand, did not correlate with vitamin B6 status. Although this finding might in principle be the result of a Type II error (the inverse relationship between homocysteine and vitamin B6 being in general weaker than for folate or vitamin B129), it is noticeable that not even a trend for such a correlation was found in our study (Figure 1). Although this may reflect variable responses of homocysteine to vitamin B6 in different populations, these results further reinforce the concept that the relationship of B6 with cardiovascular events is largely independent of homocysteine. Explanations for a homocysteine-independent possibly causal relationship of vitamin B6 with vascular events have therefore to be sought in the numerous involvements of vitamin B6-PLP, as a co-enzyme, in more than 60 different enzymatic reactions in the body, with crucial roles in amino acid, carbohydrate, lipid, and nucleic acid metabolism.45,46

The main limitation of our study is the relatively small number of events: despite the long follow-up, only 109 new cases of CHD or cerebrovascular events were collected, increasing the possibility of both type I and type II errors and explaining the lack of a graded response in the analysis per quartiles. We a priori elected to combine CHD and cerebrovascular events, and not to consider, at variance from other studies,9 new diagnoses of peripheral vascular disease, which, however, did not occur in a sufficiently validated fashion in our cohort, because of their importance in the acute transition from cardiovascular health to disease, but any sub-analysis for these two separate categories, as well as of the few number of fatal events occurred, is precluded by the small numbers involved. Owing to the case–control methodology used in our design, only first-ever cases of CHD and cerebrovascular events were used in our study, with no collection on the total number (possibly more than one in the same patients) of events accrued.

Another possible limitation of this study is in the possibility that hidden variables, not considered here, might covariate with vitamin B6 and homocysteine and possibly play a real causal role in determining vascular events. This is a general limitation for association studies, which calls for further studies testing the cause–effect relationship. The long follow-up (12 years) in our study might have also led to an attenuation of some underlying relationships, but this—if anything—would make the probability of an overestimation of the effects of vitamin B6 and homocysteine unlikely.


   Conclusions
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
Acknowledgements
 References
 
In this prospective nested case–control study, both homocysteine and vitamin B6 levels at baseline were found to be independent and graded predictors of subsequent CHD and cerebrovascular events in an initially healthy middle-age population. These findings should stimulate more research on the predictive ability of this parameter. Despite the first reports on the protective role of vitamin B6 supplementation in vascular disease, which apparently dismiss the possibility of a therapeutic impact for the supplementation of this vitamin (along with folate and vitamin B12), with the regimens used, in cardiovascular disease,14–16 such studies cannot disprove the possibility that other non-measured biological variables are negatively affected by such treatments,47 and that a therapeutic potential may be restricted to some individual components of the vitamin cocktail so far used. In general, such findings warrant an improved understanding of the underlying biological reasons.


   Acknowledgements
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
References
 
This entire work was funded through research grants from the IRCAB Foundation Onlus—Udine (to D.V. and P.F.) and the Italian Ministry of the University and Scientific Research (M.I.U.R., ex-60%), and a grant to the Center of Excellence on Aging at the University of Chieti (to R.D.C.). S.D. was supported by a post-doctoral fellowship from the University of Udine. We thank Alessandro Perri, for laboratory measurements of vitamin B12 and folates, and Jacob Selhub for a revision of the original manuscript.

Conflict of interest: none declared.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 

  1. McCully KS. (1969) Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol 56:111–128.[ISI][Medline]
  2. The Homocysteine Studies Collaboration. (2002) Homocysteine and risk of ischaemic heart disease and stroke: a meta-analysis. JAMA 288:2015–2022.[Abstract/Free Full Text]
  3. Selhub J, Jacques PF, Wilson PW, Rush D, Rosenberg IH. (1993) Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 270:2693–2698.[Abstract]
  4. Miller J, Ribaya-Mercado J, Russel R. (1992) Effect of vitamin B-6 deficiency on fasting plasma homocysteine concentrations. Am J Clin Nutr 55:1154–1160.[Abstract/Free Full Text]
  5. Ubbink J, Vermaak W, van der Merwe A, Becker P. (1993) Vitamin B-12, vitamin B-6, and folate nutritional status in men with hyperhomocysteinemia. Am J Clin Nutr 57:47–53.[Abstract/Free Full Text]
  6. Verhoef P, Kok F, Knuyssen A, Schouten E, Witteman J, Grobbee D, Ueland P, Refsum H. (1997) Plasma total homocysteine, B vitamins, and risk of coronary atherosclerosis. Arterioscler Thromb Vasc Biol 17:989–995.[Abstract/Free Full Text]
  7. Siri P, Verhoef P, Kok F. (1998) Vitamins B6, B12, and folate: association with plasma total homocysteine and risk of coronary atherosclerosis. J Am Coll Nutr 17:435–441.[Abstract/Free Full Text]
  8. Folsom AR, Nieto FJ, McGovern PG, Tsai MY, Malinow MR, Eckfeldt JH, Hess DL, Davis CE. (1998) Prospective study of coronary heart disease incidence in relation to fasting total homocysteine, related genetic polymorphisms, and B vitamins: the Atherosclerosis Risk in Communities (ARIC) study. Circulation 98:204–210.
  9. Robinson K, Mayer EL, Miller DP, Green R, van Lente F, Gupta A, Kottke-Marchant K, Savon SR, Selhub J, Nissen SE, Kutner M, Topol EJ, Jacobsen DW. (1995) Hyperhomocysteinemia and low pyridoxal phosphate. Common and independent reversible risk factors for coronary artery disease. Circulation 92:2825–2830.
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