Fasting tHcy Research Articles

High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri- n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65±2.3 and 8.9±2.4 μmol/l, while post-methionine load gave tHcy levels of 19.9±5.5 and 26.8±5.5 μmol/l, for women and men, respectively ( n=40).

Vitamins B6, B12, and Folate: Association with Plasma Total Homocysteine and Risk of Coronary Atherosclerosis

Objectives: To investigate the association of status of vitamins B6, B12 and folate with plasma fasting total homocysteine (tHcy) and with risk of coronary atherosclerosis; and to establish whether associations between vitamins and risk of coronary atherosclerosis are mediated by tHcy.Methods: The study population consisted of 131 patients with angiographically-defined severe coronary atherosclerosis and 88 referents with no or minor coronary stenosis. Previous analyses in this study population have shown that fasting tHcy is an independent risk factor for coronary atherosclerosis. In the present analyses, using multiple linear regression, we estimated differences in tHcy concentrations between subjects in the lowest and highest quartiles of concentrations of each of the vitamins, adjusting for age, gender, total:HDL cholesterol ratio, smoking habits, alcohol intake, blood pressure, serum creatinine, body mass index and the two other vitamins. We used logistic regression analysis conditional on the set of potential confounders described above to study the association between vitamin concentration and risk of coronary atherosclerosis. By comparing these estimated odds ratios (ORs) with those that were additionally adjusted for fasting tHcy, we determined whether the vitamins exerted their effects on disease risk via homocysteine metabolism.Results: Cases who were in the upper quartile of serum vitamin B12 and erythrocyte folate concentrations showed statistically significantly lower tHcy concentrations (−4.00 and −4.71 μmol/L, respectively) than those in the lowest quartile. Referents in the upper quartile of plasma B6 showed significantly lower tHcy concentrations (−2.36 μmol/L) than referents in the lowest quartile. Subjects in the lowest quartile of vitamin B12 concentration had higher risk of coronary atherosclerosis (OR: 2.91; 95% CI: 1.10, 7.71) compared to those in the highest quartile. The ORs and 95% CIs for low B6 and low folate were 0.86 (95% CI: 0.33, 2.22) and 0.58 (95% CI: 0.23, 1.48), respectively. Additional adjustment for fasting tHcy weakened associations, although data indicated that low vitamin B12 concentration is a risk factor for coronary atherosclerosis, independently of tHcy.Conclusion: The presently accepted view that vitamin B6 mainly affects tHcy after methionine loading, and not fasting tHcy, is contradicted by our findings in referents. Low vitamin B12 concentrations were associated with an increased risk of coronary atherosclerosis, partly independently of tHcy. Although low folate status was a strong determinant of elevated tHcy concentrations, it was not associated with increased risk of coronary atherosclerosis.

Fasting and post—methionine load homocyst(e)ine values are correlated with microalbuminuria and could contribute to worsening vascular damage in non—insulin-dependent diabetes mellitus patients

The study aim was to assess the relationship between homocyst(e)inemia and microalbuminuria in non—insulin-dependent diabetes mellitus (NIDDM) patients. The study was performed on 33 NIDDM patients (16 males and 17 females), and 16 healthy control subjects (seven males and nine females). Plasma fasting and post—methionine load homocyst(e)ine (tHcy), together with other parameters that could modify tHcy levels, were assessed. There were no significant differences between NIDDM patients with controls for fasting tHcy (8.12 ± 3.17 v 7.19 ± 2.40 μmol/L) and post—methionine load tHcy (26.51 ± 11.50 v 25.06 ± 10.76 μmol/L). Moreover, there was a significant correlation between urinary albumin excretion (IUAE) and fasting tHcy ( r = .340, P = .05) and post—methionine load tHCy ( r = .502, P = .004) in NIDDM patients. Fasting tHCy was correlated both with post—methionine load tHcy ( r = .429, P = .01) and with vitamin B 12 ( r = .349, P = .04) in NIDDM patients. Microalbuminuric NIDDM patients had higher fasting tHCy (9.05 ± 3.83 μmol/L) than normoalbuminurics (7.12 ± 1.95 μmol/L). In addition, NIDDM patients with complications presented higher fasting tHcy values than the group without complications (9.61 ± 3.34 v 6.53 ± 2.09 μmol/L, Kolmogorov-Smirnov two-sample test for nonparametric data [KS] = 1.794, P = .003), without any other significant differences in the parameters considered. tHcy could be an important risk factor worsening the prognosis in NIDDM patients, especially microalbuminuric patients. Microalbuminuric NIDDM patients could be particularly prone to hyperhomocyst(e)inemia, probably due to endothelial or renal dysfunction with a reduction in the scavenging of tHcy.

Methylenetetrahydrofolate Reductase Polymorphism Affects the Change in Homocysteine and Folate Concentrations Resulting from Low Dose Folic Acid Supplementation in Women with Unexplained Recurrent Miscarriages

To determine the effects of daily supplementation of 0.5 mg folic acid on homocysteine and folate concentrations, we investigated 49 women with a history of unexplained recurrent miscarriages. A methionine loading test (including the vitamin concentrations of concern) was used preceding and after 2 mo of folic acid intake. Subsequently, these effects were studied after stratification for C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphism. Folic acid supplementation (for 2 mo) reduced the median fasting and delta (after-load minus fasting) total plasma homocysteine (tHcy) concentrations 27% (P < 0.001) and 14% (P < 0.05), respectively. Median serum and red cell folate concentrations increased 275 and 70%, respectively (P < 0.01). The homocysteine-lowering effect was most marked in women with the highest tHcy concentrations at baseline. All MTHFR-genotypes (homozygous T/T, n = 8; heterozygous T/C, n = 23; wild type C/C, n = 18) had a different response to the supplementation. After 2 mo, homozygous women showed the greatest decline in median fasting (−41%; P < 0.01) tHcy concentrations, but the lowest absolute increase in serum folate concentration (+26 nmol/L; P < 0.05). In conclusion, 2 mo of daily supplementation of 0.5 mg folic acid in women with a history of unexplained recurrent miscarriages caused, in general, substantially reduced tHcy concentrations. This effect was most distinct in women with the highest tHcy concentrations at baseline and in women homozygous for the 677 C→T mutation of the MTHFR-gene.

Methylenetetrahydrofolate Reductase C677T Mutation, Plasma Homocysteine, and Folate in Subjects From Northern Italy With or Without Angiographically Documented Severe Coronary Atherosclerotic Disease: Evidence for an Important Genetic-Environmental Interaction

Moderate elevation of plasma total homocysteine (tHcy) is a strong and independent risk factor for coronary artery disease (CAD). It can result from genetic or nutrient-related disturbances in the transsulfuration or remethylation pathways for Hcy metabolism. A point mutation (C677T; Ala-to-Val) in the gene encoding the 5,10-methylenetetrahydrofolate reductase (MTHFR) has been recently reported to render the enzyme thermolabile and less active. Studies on the role of this mutation as a risk factor for CAD have given conflicting results. We studied a total of 415 subjects, 278 with angiographically documented multivessel CAD and 137 with angiographically documented normal coronary arteries. The overall frequency of the MTHFR V/V homozygous genotype was 15.7% (with 52.5% heterozygous and 31.8% normal). Subgroup analysis showed no significant differences between CAD and CAD-free subjects. A genotype/phenotype correlation study showed a marked effect of folate on the association between MTHFR genotypes and tHcy. Among individuals with folate levels below the median (11.5 nmol/L), fasting tHcy was significantly increased not only in V/V homozygotes (by 59%) but also, at intermediate values, in A/V heterozygotes (by 21% on average). Conversely, the mutation resulted neutral with respect to tHcy levels in subjects with adequate folate levels. We conclude that, in our population, the MTHFR C677T mutation is rather common, but it does not appear to be associated per se to CAD. A genetic-environmental interaction may contribute to the vascular risk by elevating tHcy when folate status is low.

Methylenetetrahydrofolate Reductase C677T Mutation, Plasma Homocysteine, and Folate in Subjects From Northern Italy With or Without Angiographically Documented Severe Coronary Atherosclerotic Disease: Evidence for an Important Genetic-Environmental Interaction

AbstractModerate elevation of plasma total homocysteine (tHcy) is a strong and independent risk factor for coronary artery disease (CAD). It can result from genetic or nutrient-related disturbances in the transsulfuration or remethylation pathways for Hcy metabolism. A point mutation (C677T; Ala-to-Val) in the gene encoding the 5,10-methylenetetrahydrofolate reductase (MTHFR) has been recently reported to render the enzyme thermolabile and less active. Studies on the role of this mutation as a risk factor for CAD have given conflicting results. We studied a total of 415 subjects, 278 with angiographically documented multivessel CAD and 137 with angiographically documented normal coronary arteries. The overall frequency of the MTHFR V/V homozygous genotype was 15.7% (with 52.5% heterozygous and 31.8% normal). Subgroup analysis showed no significant differences between CAD and CAD-free subjects. A genotype/phenotype correlation study showed a marked effect of folate on the association between MTHFR genotypes and tHcy. Among individuals with folate levels below the median (11.5 nmol/L), fasting tHcy was significantly increased not only in V/V homozygotes (by 59%) but also, at intermediate values, in A/V heterozygotes (by 21% on average). Conversely, the mutation resulted neutral with respect to tHcy levels in subjects with adequate folate levels. We conclude that, in our population, the MTHFR C677T mutation is rather common, but it does not appear to be associated per se to CAD. A genetic-environmental interaction may contribute to the vascular risk by elevating tHcy when folate status is low.

Elevated Fasting Total Plasma Homocysteine Levels and Cardiovascular Disease Outcomes in Maintenance Dialysis Patients

There is an excess prevalence of hyperhomocysteinemia in dialysis-dependent end-stage renal disease (ESRD) patients. Cross-sectional studies of the relationship between elevated total homocysteine (tHcy) levels and prevalent cardiovascular disease (CVD) in this patient population suffer from severe methodologic limitations. No prospective investigations examining the association between tHcy levels and the subsequent development of arteriosclerotic CVD outcomes among maintenance dialysis patients have been reported. To assess whether elevated plasma tHcy is an independent risk factor for incident CVD in dialysis-dependent ESRD patients, we studied 73 maintenance peritoneal dialysis or hemodialysis patients who received a baseline examination between March and December 1994, with follow-up through April 1, 1996. We determined the incidence of nonfatal and fatal CVD events, which included all validated coronary heart disease, cerebrovascular disease, and abdominal aortic/lower-extremity arterial disease outcomes. After a median follow-up of 17.0 months, 16 individuals experienced at least one arteriosclerotic CVD event. Cox proportional-hazards regression analyses, unadjusted and individually adjusted for creatinine, albumin, and total cholesterol levels, total/HDL cholesterol ratio, dialysis adequacy/residual renal function, baseline CVD, and the established CVD risk factors (ie, age, sex, smoking, hypertension, diabetes/glucose intolerance, and dyslipidemia) revealed that tHcy levels in the upper quartile (> or = 27.0 mumol/L) versus the lower three quartiles (< 27.0 mumol/L) were associated with relative risk estimates (hazards ratios, with 95% confidence intervals for the occurrence of (pooled) nonfatal and fatal CVD ranging from 3.0 to 4.4; 95% confidence intervals (1.1-8.1) to (1.6-12.2). We conclude that the markedly elevated fasting tHcy levels found in dialysis-dependent ESRD patients may contribute independently to their excess incidence of fatal and nonfatal CVD outcomes.

Excess prevalence of fasting and postmethionine-loading hyperhomocysteinemia in stable renal transplant recipients.

Hyperhomocysteinemia, either fasting or after methionine loading, may contribute to the increased incidence of cardiovascular disease events experienced by renal transplant recipients. Limited data are available on fasting homocysteine (Hcy) levels, and none on postmethionine-loading Hcy levels, in these patients. We assessed the prevalence and potential determinants of fasting and postmethionine-loading hyperhomocysteinemia in 29 stable renal transplant recipients and 58 age- and sex-matched, population-based controls free of renal disease with serum creatinine levels of 1.5 mg/dL or less. Total (t) plasma Hcy was determined fasting and 2 hours after methionine loading, along with fasting determinations of the B-vitamin cofactors/substrates for Hcy metabolism, ie, pyridoxal 5'-phosphate, B-12, and folate and serum creatinine. Geometric mean fasting (18.1 versus 9.8 microM, P < .001) and postmethionine-loading increase (22.0 versus 15.2, P = .001) in tHcy levels were significantly greater in the renal transplant recipients, as were the prevalence odds (with 95% confidence intervals) for fasting [14.8 (3.4-64.7)], postmethionine loading [6.9 (1.5-32.8)], combined fasting and postmethionine-loading [18.0 (2.3-142.1)] hyperhomocysteinemia, and inadequate circulating folate [4.2 (1.1-16.5)] or pyridoxal 5'-phosphate [3.2 (0.9-11.0) status. Correlation analyses suggested important potential relationships between creatinine and both fasting (+0.64, P < .001) and postmethionine-load increase (+0.38, P = .045) in tHcy, folate and fasting (-0.41, P = .025) tHcy, and pyridoxal 5'-phosphate and postmethionine-loading increase (-0.33, P = .091) in tHcy. We conclude that there is an excess prevalence of fasting and postmethionine-loading hyperhomocysteinemia in stable renal transplant recipients. Renal function is related to both fasting and postmethionine loading-hyperhomocysteinemia, inadequate folate status is associated with fasting hyperhomocysteinemia, and inadequate vitamin B-6 status may be related to postmethionine-loading hyperhomocysteinemia in this patient population.

Kinetic basis of hyperhomocysteinemia in patients with chronic renal failure

We investigated the elimination of total homocysteine (tHcy) from plasma after peroral homocysteine (Hcy) loading in eight patients with chronic renal failure. Data on bioavailability and distribution volume were obtained from two patients and two healthy controls by performing both intravenous and peroral Hcy loading. Response to high-dose folic acid was studied in six cases. Mean (SD) basal plasma tHcy was 27.4 (11.0) microM at inclusion. The half-life and the area under the curve were about four times higher, and clearance was reduced to 29.8% compared to controls. High-dose folic acid had no influence on half-life for tHcy, but the basal tHcy level declined by 26.8%. The reduction in tHcy was particularly pronounced in three patients with low-normal serum folate, and the enhanced methionine response to Hcy loading after folic acid suggested improved Hcy remethylation in tissues. In conclusion, patients with renal failure had markedly reduced clearance of tHcy from plasma, which probably accounts for their hyperhomocysteinemia. High-dose folic acid reduces fasting tHcy by improving tissue Hcy remethylation without affecting the low renal clearance of tHcy.

The 677C→T mutation in the methylenetetrahydrofolate reductase gene: associations with plasma total homocysteine levels and risk of coronary atherosclerotic disease

Homozygosity for a 677C→T mutation at the locus that codes for 5,10-methylenetetrahydrofolate reductase (MTHFR), a folate-dependent crucial enzyme in homocysteine metabolism, may render the enzyme thermolabile and less active and has been associated with increased levels of plasma total homocysteine (tHcy). We assessed whether this mutation was associated with increased risk of coronary atherosclerosis and plasma levels of tHcy and furthermore studied whether folate status would modify the associations. Data were collected from subjects with substantial coronary atherosclerosis (≥90% occlusion in one and ≥40% occlusion in a second coronary artery, referred to as cases, n=131) or virtually no coronary narrowing (referred to as coronary controls, n=87) and from a population-based control group (n=100), all residing in the Rotterdam area, The Netherlands. Both males and females, aged 25–65 years were studied. The frequency of homozygosity for the mutation (+/+) in cases (10.0%) did not significantly differ statistically from that observed in coronary controls (11.5%, P=0.71), population-based controls (7.0%, P=0.43), or combined control groups (9.1%, P=0.80). In the overall group (as well as in the three subgroups), plasma tHcy levels, fasting and to a lesser extent after a methionine-loading test, were higher in +/+ subjects than in homozygous normal subjects (−/−), whereas heterozygous subjects (+/−) had intermediate levels (Ptrend=0.001). The +/+ subjects with erythrocyte folate levels <790 nmol/l (population median) had a 77% (95% CI, 27–144%) higher geometric mean fasting tHcy (21.4, μmol/l) than those with higher erythrocyte folate (12.1 μmol/l). The odds ratio (OR) of coronary atherosclerosis for +/+ subjects, with +/− and −/− subjects as the reference group, in analyses with combined control groups, was 1.1 (95% CI, 0.5–2.4). The ORs were 2.2 (95% CI, 0.7–6.8) and 0.6 (95% CI, 0.2–1.7) among subjects with low and high folate levels, respectively. Our study indicates that homozygosity for the 677C→T MTHFR mutation, especially in combination with low folate status, predisposes to high plasma levels of fasting tHcy. However, homozygosity for this mutation, whether or not in combination with low folate status, was not associated with increased risk of coronary artery disease.

Plasma total homocysteine, B vitamins, and risk of coronary atherosclerosis.

Epidemiological research has shown that elevated plasma total homocysteine (tHcy) is a risk factor for atherosclerotic disease. In the present case-control study, we investigated whether fasting or postmethionine-loading tHcy was a stronger predictor of risk of severe coronary atherosclerosis. Furthermore, we studied levels of B vitamins, which are involved in homocysteine metabolism. Subjects were recruited from men and women, aged 25 to 65 years, who underwent coronary angiography between June 1992 and June 1994 in a hospital in Rotterdam, The Netherlands. Cases (n=131) were defined as those with > or =90% occlusion in one and > or =40% occlusion in a second coronary artery, while control subjects (n=88) had <50% occlusion in only one coronary vessel. In addition, a population-based control group free from clinical cardiovascular disease (n=101) was studied. Coronary patients were studied at least 2.5 months after angiography or other acute illness, such as myocardial infarction. After adjusting for age and sex differences between the groups, cases had 9% (P=.01) higher geometric mean fasting and 7% (P=.04) higher geometric mean postload tHcy than the combined control groups. Despite higher levels of tHcy for cases, their geometric mean levels of red cell folate and pyridoxal 5'-phosphate were higher than for control subjects, whereas plasma vitamin B12 was only slightly lower in cases. The frequency distribution of tHcy values in cases was slightly shifted toward the right, across the entire range, compared with the distribution in the combined control group. This was somewhat more obvious for fasting than postload tHcy levels. The odds ratio (OR) for severe coronary atherosclerosis (case status) for each 1 SD increase in fasting tHcy (5 micromol/L) was 1.3 (95% confidence interval [CI], 1.0-1.6), similar to the OR for each 1 SD increase (12 micromol/L) in postmethionine-loading tHcy (1.3 [95 CI, 1.0-1.7]), after adjustment for sex, age, and other potential confounders. Furthermore, there was a significant linear trend of increasing fasting tHcy with increasing number of occluded arteries (P=.01), correcting for sex, age, and other potential confounders. Our data show a positive association between plasma tHcy and risk of severe coronary atherosclerosis, of similar strength for fasting and postload tHcy levels. The data suggest that the association exists over a wide range of tHcy levels, without a clear cutoff point below which there is no increased risk.

The effect of supraphysiologic doses of testosterone on fasting total homocysteine levels in normal men

Elevated total homocysteine (tHcy) levels are associated with increased risk for atherosclerotic cardiovascular disease. tHcy levels are higher in men than in women, and estrogen replacement therapy may reduce tHcy levels in postmenopausal women. The effect of androgenic hormones on tHcy levels in men has not been examined. The present study determined the effect of supraphysiologic doses of testosterone, with or without its aromatization to estradiol, on fasting tHcy levels in 14 normal male weightlifters aged 19–42 years. Subjects received testosterone enanthate (200 mg/week intramuscularly), the aromatase inhibitor, testolactone (1 g/day orally), or both drugs together in a crossover design. Each treatment lasted 3 weeks and each treatment was separated by a 4-week washout. Both testosterone regimens increased serum testosterone levels, whereas estradiol increased only during testosterone alone. Mean tHcy levels were not significantly altered when testosterone was given alone or together with testolactone. Testolactone did not significantly influence tHcy levels. We conclude that short-term, high-dose testosterone administration does not affect fasting tHcy levels in normal men.