Plasma Vitamin B12 Concentrations Research Articles

Hypoxia-induced megaloblastosis in vitamin B12-deficient rats.

In rats, in contrast with human subjects who develop megaloblastic anaemia due to vitamin B12 deficiency, haematological abnormalities with anaemia were not observed under normoxic conditions even though plasma vitamin B12 concentration was reduced to <15 % of a normal concentration by depleting dietary vitamin B12. To elucidate whether erythropoiesis was affected by vitamin B12 deficiency in rats, these vitamin B12-deficient rats were exposed to hypoxia (10.5 % O2) to stimulate erythropoiesis. In the vitamin B12-sufficient control rats, erythrocyte count was significantly (P<0.05) increased 1 week after starting the hypoxic exposure. However, the hypoxia-induced erythropoiesis was affected by vitamin B12 deficiency, and no significant increase in the erythrocyte count was observed even after 6-week exposure to hypoxia in the vitamin B12-deficient rats. In the vitamin B12-deficient rats in hypoxia, erythrocytes became abnormally enlarged, and haemoglobin concentration in peripheral blood was increased in proportion to the increase of mean corpuscular volume. However, the level of the increase in the haemoglobin concentration was significantly (P<0.05) lower in the vitamin B12-deficient rats compared with that in the -sufficient controls. In addition, in the vitamin B12-deficient rats, in contrast to the -sufficient rats, serum erythropoietin concentration was not normalized even after 6-week exposure to hypoxia. These results indicate that a megaloblastic anaemia-like symptom is induced when the vitamin B12-deficient rats are exposed to hypoxia.

Coronary heart disease mortality, plasma homocysteine, and B-vitamins: a prospective study

The results of prospective studies on the relations between the plasma concentration of total homocysteine (tHcy) and B-vitamins, on the one hand, and coronary heart disease (CHD) mortality, on the other hand, are inconclusive and scarce considering the relation with B-vitamins. We prospectively determined these relations in a case-cohort study. The full-cohort existed in ∼36,000 Dutch adults aged 20–59 years at baseline. The statistical analyses were done with a random sample from the cohort ( n=630) complemented with all subjects who died of CHD ( n=102) during a mean follow-up of 10.3 years. All subjects reported the absence of cardiovascular diseases (CVDs) at baseline. The plasma concentrations of tHcy, folate, PLP, and vitamin B12 were determined in samples obtained at baseline. Men with a tHcy concentration in the highest tertile (T3) compared with men in the lowest tertile (T1) had a relative risk (RR) of 1.14 for CHD (95% confidence interval (CI): 0.50, 2.61) after adjusting for age, study center, hypertension, HDL and total cholesterol, smoking, and creatinine. For women, this RR was 2.04 (95% CI: 0.48, 8.62). For each 5 μmol/l increase in tHcy, the RR of CHD was 1.03 (95% CI: 0.83–1.29) for men and women combined. In women only, high folate levels were associated with a statistically significant protection of fatal CHD (T3 versus T1; RR: 0.22, 95% CI: 0.06, 0.87). Plasma PLP (B6) and vitamin B12 concentrations were not associated with CHD risk. We conclude that elevated tHcy concentrations do not seem to be a risk factor for CHD mortality in these relatively young healthy Dutch subjects free of baseline CVD. Higher folate concentrations may be protective of CHD, but this needs confirmation.

This month in J Lab Clin Med: Issue highlights for January 2003

This month in J Lab Clin Med: Issue highlights for January 2003

Effect of fish oil on LDL oxidation and plasma homocysteine concentrations in health

Oxidation of low-density lipoprotein (LDL) and hyperhomocysteinemia are believed to play a role in therogenesis. Whether n-3 polyunsaturated fatty acids increase LDL susceptibility to oxidation or influence homocysteine (Hcy) metabolism has long been a subject of controversy. In this study, we evaluated the effect of 8 weeks of dietary supplementation with 6 g/day of fish oil (FO; 3 g of n-3 fatty acids) on plasma lipoproteins, in vitro LDL peroxidation, antioxidant status, and plasma Hcy concentrations in 16 normolipidemic subjects. FO rapidly and significantly (P <.01) decreased plasma total and very low density lipoprotein triglyceride concentrations and had no effect on LDL or high-density-lipoprotein cholesterol. The mean lag time before onset of Cu2+-induced LDL oxidation, as well as plasma and LDL α-tocopherol and β-carotene concentrations, was unchanged. However, changes in plasma aminothiol concentrations occurred during the study. Specifically, a progressive and significant increase in total Hcy plasma concentrations was observed (13.4% and 20% after 4 and 8 weeks, respectively; P <.01). Total glutathione concentrations were significantly higher after 8 weeks (P <.05). The tHcy increase was not associated with changes in plasma folate or vitamin B12 concentrations. However, concentrations of plasma nitric oxide metabolites (NOx = NO2 + NO3) were significantly higher than at baseline after 8 weeks of FO intake (74%; P <.01). Further, the changes in total Hcy and NOx plasma concentrations observed after 8 weeks of FO were found to be significantly correlated (r =.78, P <.001). With this study, we report for the first time the apparent interaction of n-3 fatty acids and nitric oxide on Hcy metabolism. (J Lab Clin Med 2003;141:41-9)

Plasma homocysteine and lipoprotein (a) levels as risk factors for atherosclerotic vascular disease in epileptic children taking anticonvulsants

Aim: To assess the effect of anticonvulsant treatment on plasma homocysteine level and lipoprotein (a) in epileptic children. Methods: Plasma total homocysteine, folate, vitamin B12 and lipoprotein (a) concentrations were measured in 111 epileptic children taking anticonvulsant drugs for longer than 12 mo. Forty‐six healthy, sex‐ and age‐matched children served as controls. Results: Patients and controls differed significantly in concentrations of homocysteine (p &gt; 0.05) and lipoprotein (a) (p &gt; 0.001). The number of patients with homocysteine concentrations of &gt;9 μM was significantly higher in the patient group than in the control group. A significant inverse relationship was found between vitamin B12 folate levels and plasma homocysteine levels in the patient group; 28.8% of the patient group had lipoprotein (a) concentrations above the cutoff value (30mg/dl) for increased risk of early atherosclerosis, whereas none of the control patients had concentrations above this value. Conclusion: These data indicate that prolonged anticonvulsant treatment could increase plasma homocysteine and lipoprotein (a) concentrations and that it may be useful to measure the levels routinely in order to prevent atherosclerosis in epileptic children taking anticonvulsant drugs.

Homocysteine levels in patients with Raynaud's phenomenon.

Patients with Raynaud's phenomenon (RP) have vasomotor dysregulation, mainly cause by dysfunction of the endothelium. Since homocysteine has been found to be damaging to endothelial cells, we investigated the concentrations of plasma homocysteine, folate and vitamin B12 in patients with primary or secondary RP compared to healthy individuals. We measured the concentrations of plasma fasting homocysteine, folate and vitamin B12 in a group of healthy individuals (n = 45) and in patients with primary (n = 26) or secondary RP (n = 42). Median homocysteine levels in healthy controls and in patients with primary RP, secondary RP were 7.9 (IQR 4.1 to 11.8) 9.8 (IQR 5.1 to 14.4), and 10.6 (6.0 to 15.3) mumol/L, respectively. Patients with primary and secondary RP had significantly higher homocysteine concentration compared to healthy controls (Kruskal Wallis p = 0.01). After matching for age and sex, patients with either primary or secondary RP showed significantly higher homocysteine levels (Wilcoxon p < 0.0001). No significant differences between the three groups were found concerning serum levels of vitamin B12 (p = 0.9) and serum folate levels (p = 0.2). These data demonstrate that patients with RP have higher plasma levels of homocysteine. No significant differences in folate and vitamin B12 levels were found between patients with primary RP, secondary RP, and healthy individuals. These data suggest that homocysteine may play a role in RP and may provide new clues in understanding of the vasomotor dysregulation.

Association of two MTHFR polymorphisms with total homocysteine plasma levels in dialysis patients

The effect of the combined 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C→T and 1298A→C genotype on total homocysteine (tHcy), folate, and vitamin B12 plasma levels was investigated in 983 subjects, including 415 hemodialysis patients, 179 peritoneal dialysis patients, and 389 healthy individuals. Mean tHcy plasma concentrations were 27.2 ± 15.8 μmol/L in hemodialysis patients, 25.4 ± 19.1 μmol/L in peritoneal dialysis patients, and 8.9 ± 3.5 μmol/L in healthy individuals. Hyperhomocysteinemia (tHcy > 15 μmol/L) was detected in 81.6% of patients and 2.6% of controls. Multiple stepwise regression analysis showed that the MTHFR 677C→T/1298A→C genotype (CC/AA, CC/AC, CC/CC, CT/AA, CT/AC, TT/AA), vitamin use, age, folate and vitamin B12 plasma level were significant predictors of tHcy plasma levels. Analysis of variance showed that this effect of MTHFR genotypes on tHcy level was caused by significantly greater tHcy levels in 677TT/1298AA hemodialysis and peritoneal dialysis patients versus other genotypes. Compound heterozygous controls (677CT/1298AC genotype) had significantly greater tHcy levels compared with 677CC/1298AA controls. There was no major effect of MTHFR polymorphisms on folate and vitamin B12 plasma concentrations. This study shows that the MTHFR 677TT/1298AA genotype, but not the 677CT/1298AC genotype, is a significant predictor of tHcy plasma levels in dialysis patients. © 2001 by the National Kidney Foundation, Inc.

Increased plasma homocysteine in liver cirrhosis

Background: Homocysteine (Hcy), is an atherogenic and thrombogenic risk factor which has also been proposed to be involved in hepatic fibrinogenesis. Hcy metabolism, depends on the cofactors folate, vit. B12, and the vit. B6 vitamer pyridoxalphosphate (PLP). Metabolism of these vitamins is frequently disturbed in cirrhotics, but little is known about plasma Hcy levels in these patients. Methods: Plasma levels of Hcy, methionine, serine, cysteine, PLP, vit. B12 and folate, and standard clinical/biochemical parameters of liver disease were measured in 43 postabsorptive patients with biopsy proven cirrhosis of different origin. Results: 74% of the patients had elevated plasma Hcy levels defined as >13.4 μmol/l (mean+2SD of healthy age matched controls). Increased plasma Hcy concentrations were seen in alcoholic as well as in non-alcoholic cirrhosis. Excluding patients with impaired renal function ( n=7), Hcy concentrations remained elevated in 69% of the patients. We found a high prevalence of pathological plasma vitamin concentrations of 33% for increased vit. B12 levels and 5% and 80% for decreased folate and vit. B6 levels, respectively. Mean plasma vitamin B12 concentrations increased, folate remained unchanged and PLP concentrations decreased with deteriorating liver function. Hcy concentrations were correlated with levels of creatinine ( r=0.44, P<0.01), serine ( r=−0.46, P<0.01), and cysteine ( r=0.38, P<0.05), but showed no association with parameters of liver function and with plasma levels of folate, vit. B12 und vit. B6. This was contrary to data obtained in healthy individuals. In a stepwise multiple regression serine and cysteine best explained the variance in Hcy levels. Conclusions: Elevated basal Hcy-plasma levels are frequently seen cirrhotic patients. Variations of Hcy concentration in liver cirrhosis are not explained by plasma levels of cofactors of Hcy metabolism.

Plasma folate, vitamin B(12), and homocyst(e)ine concentrations in preeclamptic and normotensive Peruvian women.

The authors measured maternal third trimester plasma folate, vitamin B(12), and homocyst(e)ine concentrations among 125 women with preeclampsia and 179 normotensive women in Lima, Peru (1997-1998), to determine whether these analytes were associated with the occurrence of preeclampsia. Logistic regression procedures were used to calculate maximum likelihood estimates of odds ratios and 95% confidence intervals. Relative to women in the upper quartile of the control distribution of maternal plasma folate concentrations, women with values in the lowest quartile experienced a 1.6-fold increased risk of preeclampsia (odds ratio = 1.6; 95% confidence interval: 0.8, 3.2). There was no evidence of an increased risk of preeclampsia associated with low plasma vitamin B(12) concentrations. The unadjusted relative risk of preeclampsia increased across successively higher quartiles of plasma homocyst(e)ine level (odds ratios were 1.0, 1.0, 1.5, and 2.9, respectively, with the lowest quartile used as the referent; p for linear trend = 0.0004). After adjustment for maternal age, parity, gestational age, use of prenatal vitamins, whether the pregnancy had been planned, and educational attainment, the relative risk between extreme quartiles was 4.0 (95% confidence interval: 1.8, 8.9). These findings are consistent with earlier reports suggesting that hyperhomocyst(e)inemia in pregnancy may be a risk factor for preeclampsia.

The effect of polymorphisms of MTHER gene and vitamin B on hyperhomocysteinemia.

The relationship between hyperhomocysteinemia and coronary artery disease (CAD) was investigated and the influence of environmental factors (Folate, VitB12) and genetic factors [N5, N10-methylenetetrahydrofolate reductase gene (MTHFR) or MTHFR gene mutation] on plasma homocysteine (Hcy) levels and the risk of CAD observed. Fifty-one CAD patients and 30 CAD-free subjects were recruited in the study. The polymorphisms of MTHFR gene were analyzed by PCR-RFLP and plasma total Hcy levels were measured by high performance liquid chromatography with fluorescence detection. Plasma folate and vitamin B12 concentrations were measured by an automated chemiluminescence method. It was found that mean total plasma Hcy concentrations were significantly higher in CAD patients than in CAD-free subjects (P < 0.01). The differences were also apparent among the three genotypes of MTHFR gene in CAD group (P < 0.05). There was no significant difference in the genotype distributions and allele frequencies between the two groups. A strong inverse correlation was found between folate or vitamin B12 and plasma Hcy levels according to MTHFR genotype (P < 0.01). It was concluded that hyperhomocysteinemia is a new independent risk factor for CAD. However, MTHFR gene mutation alone does not relate significantly to the morbidity of CAD since hyperhomocysteinemia and its influence on the risk of CAD are decided by both environmental and genetic factors. Supplementary treatment with vitamins B can effectively lower the plasma levels of Hcy, thus maybe reducing the risk of CAD.

Homocysteine Elevation in Sickle Cell Disease

Objective: Ischemic complications are common in patients with sickle cell disease. Hyperhomocysteinemia is a risk factor for arteriosclerosis and venous thrombosis, and given the propensity of patients with sickle cell disease to develop ischemic complications, we hypothesized that they might have elevated plasma homocysteine concentrations.Methods: Plasma concentrations of homocysteine, vitamin B12 and folate were measured in 49 adults with sickle cell disease and 16 normotensive Black controls. All subjects with sickle cell disease had been prescribed folic acid 1 mg by mouth daily.Results: The median plasma concentration of homocysteine of subjects with sickle cell disease was approximately 1.5-fold higher than that of controls (p=0.0008). This difference persisted, even when subjects with renal insufficiency were excluded. Plasma folate levels were 1.5-fold higher in subjects with sickle cell disease than in controls (p=0.0498). There was no significant difference in plasma vitamin B12 concentrations between the two groups. There was no difference in plasma homocysteine concentrations between transfused and non-transfused sickle cell subjects.Conclusions: Patients with sickle cell disease have elevated plasma concentrations of homocysteine in spite of elevated plasma folate levels and vitamin B12 concentrations similar to those observed in controls. Based on these data, we hypothesize that the concentration of folate required to normalize plasma homocysteine levels in patients with sickle cell disease may be higher than that of normal controls and that patients with sickle cell disease have a higher nutritional requirement for folic acid than the general population.

Effect of Chronic Alcohol Consumption on Total Plasma Homocysteine Level in Rats

Chronic alcoholism in humans is associated with the development of hyperhomocysteinemia, the mechanism of which remains unclear. Among the causes of hyperhomocysteinemia is depletion of folate, vitamin B12, or vitamin B6. Population-based studies indicate that folate is the strongest vitamin determinant of hyperhomocysteinemia and, in most settings, folate supplementation effectively lowers elevated homocysteine levels. However, it is not clear whether folate deficiency is the cause of alcohol-related hyperhomocysteinemia. In the present study, 10 male Sprague Dawley rats were fed ethanol-containing Lieber-DeCarli diets with 13 mg of folic acid per kilogram of diet. This represents a folate intake more than 20 times the basal requirement. Ethanol represented 36% of total energy, which yielded a concentration of 6.2% (vol/vol). The same number of rats were pair-fed with isocaloric control diets that contained an identical level of folate in which ethanol was entirely replaced by maltodextrin. At the end of 4 weeks, alcohol-fed rats did not show any significant reduction in plasma or hepatic folate concentrations, plasma pyridoxal-5'-phosphate concentration, or plasma vitamin B12 concentration. On the other hand, alcohol-fed rats were significantly hyperhomocysteinemic (17.24 +/- 4.63 micromol/liter,p < 0.01) compared to the nonalcohol group (10.73 +/- 2.76 micromol/liter). Alcohol-fed rats also had a significantly lower hepatic S-adenosylmethionine and higher hepatic S-adenosylhomocysteine levels. Chronic alcohol consumption produces hyperhomocysteinemia by a mechanism that is related to interference with one-carbon metabolism, and not through vitamin depletion.

Methylenetetrahydrofolate Reductase 677 C → T Polymorphism, Plasma Folate, Vitamin B12 Concentrations, and Risk of Preeclampsia among Black African Women from Zimbabwe

We conducted a case control study at Harare Maternity Hospital, Zimbabwe. We genotyped a total of 171 cases with preeclampsia or eclampsia and 185 normotensive control subjects for the methylenetetrahydrofolate reductase (MTHFR) 677 C → T genotype. The wild-type allele frequency among cases and controls was 91.2 and 91.3%, respectively. Only one subject (0.3%) was homozygous for the 677 C → T MTHFR genotype and this subject had preeclampsia. After adjustment for confounding factors, there was statistically no significant association between maternal MTHFR genotype and risk of preeclampsia (adjusted odds ratio = 1.0; 95% CI, 0.5–1.9). In addition, plasma homocyst(e)ine, vitamin B12, and folate concentrations were not statistically different between normotensive control subjects with wild-type genotype as compared with normotensive subjects who were heterozygous for the mutant allele. Conversely, there was a strong graded association between maternal plasma folate concentration and risk of preeclampsia. Women with plasma folate concentrations less than 5.7 nmol/L experienced a 10.4-fold increase in risk of preeclampsia. There was no clear pattern of preeclampsia risk and vitamin B12 concentrations.

Homocysteine, folate, vitamin B12, and cardiovascular risk in Indians, Malays, and Chinese in Singapore

OBJECTIVETo examine the hypothesis that the higher rates of coronary heart disease (CHD) in Indians (South Asians) compared with Malays and Chinese is partly attributable to differences in blood concentrations...

Transient lowering of plasma vitamin B12 concentrations in Ouessant sheep fed on a potassium-rich ration.

The hypothesis tested was that a high intake of potassium (K) interferes with cobalt (Co)-vitamin B12 metabolism in ruminants. Ouessant sheep were fed either a low- (8 g K/kg dry matter) or high-K diet (43 g K/kg dry matter) with an adequate amount of Co (124 micrograms Co/kg dry matter). Plasma vitamin B12 concentrations were measured. Sheep fed a low-K diet, but containing only 70 micrograms Co/kg dry matter, served as positive controls. Feeding the low-Co ration produced a significant decrease in plasma vitamin B12 concentrations. The high- versus low-K ration induced a significant decrease in plasma vitamin B12 during the first 6 weeks, but thereafter the values in the high-K group rose to those seen in the low-K group. The mechanism by which dietary K may interact with Co-vitamin B12 metabolism in sheep is unknown.

Micronucleus frequency in human lymphocytes is related to plasma vitamin B12 and homocysteine

In a series of studies, we have been able to confirm that the micronucleus index in cytokinesis-blocked lymphocytes is significantly negatively correlated with plasma vitamin B12 (B12) concentration and significantly positively correlated with plasma homocysteine (HC). Furthermore we have shown in a randomised double-blind placebo-controlled dietary intervention study that intake of 3.5 times the RDI of folic acid and B12 significantly reduces the micronucleus index only in those with above average levels of micronucleus frequency. Micronucleus frequency is minimised when plasma HC is below 7.5 μmol/l and plasma B12 is above 300 pmol/l. Therefore, it is important to take account of the effect of B12 and HC when using the micronucleus assay for human biomonitoring studies.

Peritoneal elimination of homocysteine moieties in continuous ambulatory peritoneal dialysis patients

The amount of total homocysteine eliminated by peritoneal dialysis and its relationship to peritoneal transport characteristics in continuous ambulatory peritoneal dialysis (CAPD) patients are unknown. The influence of total homocysteine, folate, and vitamin B12 plasma concentrations, serum albumin levels, age, sex, dialysate to plasma ratio (D/P) creatinine, D/D0 glucose, D/P albumin, dialysate effluent volume, and effluent albumin on the daily peritoneal excretion of total homocysteine was investigated in 39 CAPD patients. The relationship of D/P creatinine to D/P total homocysteine, D/P free homocysteine, and D/P protein-bound homocysteine was analyzed additionally in a subgroup of 25 patients. We observed a significant influence of plasma total homocysteine concentrations (P = 0.0001) of the daily dialysate effluent volume (P = 0.0221) and of the D/P creatinine (P = 0.0132) on peritoneal elimination of total homocysteine. The daily peritoneal excretion of total homocysteine was 38.94 +/- 20.82 mumol (5.27 +/- 2.81 mg). There was a positive linear association of the daily total homocysteine elimination with plasma total homocysteine concentrations (P = 0.0001). A significant linear correlation was observed between D/P creatinine and D/P total homocysteine (P = 0.0001), D/P free homocysteine (P = 0.0001), as well as D/P protein-bound homocysteine (P = 0.0001). The peritoneal elimination of total homocysteine primarily depends on the plasma total homocysteine concentration. Elevated total homocysteine plasma levels cannot be reduced efficiently by peritoneal dialysis.

Association between plasma homocysteine and peripheral arterial disease in older persons.

Plasma homocysteine is a risk factor for peripheral arterial disease. In a cross-sectional study design we investigated the association between plasma homocysteine, vitamin B12 and folate concentrations, and the prevalence of peripheral arterial disease in 158 men and 362 women, mean age 81 +/- 8 years. Univariate analysis showed that plasma homocysteine, vitamin B12 (inverse association) and folate (inverse association) concentrations and increased plasma homocysteine and low or indeterminate vitamin B12 concentrations were risk factors for peripheral arterial disease in older men and women. Stepwise multiple logistic regression analysis showed that significant independent risk factors for peripheral arterial disease were a greater value of plasma homocysteine (P = 0.0001; odds ratio = 1.125) and a low value of plasma folate (P = 0.0001; odds ratio = 0.709). High plasma concentrations of homocysteine and low plasma concentrations of folate are significant independent risk factors for peripheral arterial disease in older men and women.

Plasma Homocysteine, Folate and Vitamin B12 Concentrations in Coronary Artery Disease

Background:Elevated plasma homocysteine concentrations are an independent risk factor for vascular disease. The metabolism of homocysteine is closely related to vitamin B12 and folic acid. The purpose of this study is to evaluate the correlation between homocysteine and vitamin B12 as well as folic acid in patients with coronary artery disease. Methods:Sixteen patients, in whom coronary angiography revealed more than 50% of stenosis at least in one coronary vessel, were enrolled as the patient group, and sixteen hypertensive subjects were selected as the control group. Blood was collected in the peripheral vein to measure the concentrations of homocysteine, vitamin B12, and folate. We evaluated the correlation between homocysteine, vitamin B12 and/or folate in both groups. Results:Total plasma homocysteine concentrations were higher in the coronary artery disease group than in the control group. There were no significant differences in the folate and vitamin B12 levels between the coronary artery disease group and the control group. In the control group, the concentrations of homocysteine showed negative correlation with both vitamin B12 and folate concentrations, but similar trends were not observed in the coronary artery disease group. (Korean Circulation J 1998;28(4): 516-522)

Variability and determinants of total homocysteine concentrations in plasma in an elderly population

The variability of plasma total homocysteine (tHcy) was examined in 96 individuals over a 1-yr period. Blood tHcy concentrations varied from 7.1 micromol/L in the bottom quintile to 14.5 micromol/L in the top quintile. The mean tHcy was 10.4 micromol/L, the between-person SD was 2.5 micromol/L, and the within-person SD was 0.93 micromol/L. There was little seasonal variation, and the reliability coefficient was 0.88. Mean tHcy concentrations were inversely related to mean plasma folate (r = -0.36) and vitamin B12 (r = -0.35) concentrations. Median tHcy concentrations were approximately 1 micromol/L higher in men than in women and in older (70 to 74 years) than in younger (65 to 69 years) individuals and higher in those with the TT and CT genotypes for the methylenetetrahydrofolate reductase polymorphism than in those with the CC genotype (10.7 and 10.6 vs 9.6 micromol/L). Epidemiological studies based on single tHcy measurements may underestimate the magnitude of any risk associations with disease by 10-15%.