Folic acid.

Folic acid is an essential nutrient from the B complex group of vitamins. Folate, as a cofactor, is involved in numerous intracellular reactions, and this is reflected in the various derivatives that have been isolated from biological sources. Folic acid is involved in single carbon transfer reactions and serves as a source of single carbon units in different oxidative states. The processes involved in the absorption, transport, and intracellular metabolism of this cofactor are complex. Much of folate is bound tightly to enzymes, indicating that there is not excess of this cofactor and that its cellular availability is protected as well as being strictly regulated. In animals, the liver controls the supply of folate through first pass metabolism, biliary secretion, enterohepatic recirculation, as well as through senescent erythrocyte recycling. Deficiencies of folate can occur for many reasons, including reduced intake, increased metabolism, and/or increased requirements as well as through genetic defects. The effects of folate deficiency include hyperhomocysteinemia, megaloblastic anemia, and mood disorders. Folate deficiency has also been implicated in disorders associated with neural tube defects. Supplementation of grain products such as cereals has been undertaken in several countries as a cost-effective means of reducing the prevelance of neural tube defects. Recently, common polymorphisms have been discovered in several genes associated with folate pathways that may play a role in diseases associated with folate deficiency, particularly mild folate deficiency.

Molecular diagnosis of hereditary thrombotic disorders.

Deep vein thrombosis (DVT) can be the result of coagulation pathway defects at the molecular level or damage to the vascular endothelium. Some of the acquired causes of DVT include malignancy, trauma, prolonged immobilization, and pregnancy (1). Thrombophilia can be owing in part to both acquired and inherited defects. The relative risk for thrombosis is increased by estrogen replacement therapy (2) and homocysteinemia (3). Homocysteine metabolism is influenced by the use of alcohol, anticonvulsant drugs, cyclosporine, methotrexate, inadequate dietary vitamin B(12), folate and pyridoxine intake, organ transplantation, and reduced creatinine clearance (4-8). Similar to venous thrombosis, homocysteinemia has genetic factors that influence susceptibility (9-11).

Neonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations.

Methylenetetrahydrofolate reductase (MTHFR) mutations are commonly associated with hyperhomocysteinemia, and, through their defects in homocysteine metabolism, they have been implicated as risk factors for neural tube defects and unexplained, recurrent embryo losses in early pregnancy. Folate sufficiency is thought to play an integral role in the phenotypic expression of MTHFR mutations. Samples of neonatal cord blood (n=119) and fetal tissue (n=161) were analyzed for MTHFR C677T and A1298C mutations to determine whether certain MTHFR genotype combinations were associated with decreased in utero viability. Mutation analysis revealed that all possible MTHFR genotype combinations were represented in the fetal group, demonstrating that 677T and 1298C alleles could occur in both cis and trans configurations. Combined 677CT/1298CC and 677TT/1298CC genotypes, which contain three and four mutant alleles, respectively, were not observed in the neonatal group (P=.0402). This suggests decreased viability among fetuses carrying these mutations and a possible selection disadvantage among fetuses with increased numbers of mutant MTHFR alleles. This is the first report that describes the existence of human MTHFR 677CT/1298CC and 677TT/1298CC genotypes and demonstrates their potential role in compromised fetal viability.

Prevalence of methylenetetrahydrofolate reductase mutations in patients with venous thrombosis.

BACKGROUND: The objectives of this study are to examine the prevalence of combined methylenetetrahydrofolate reductase (MTHFR) 677C-->T and 1298A-->C mutations in patients with venous thrombosis (VT) and healthy volunteers and to determine if these mutations are in Hardy-Weinberg equilibrium. METHODS AND RESULTS: Sixty-five patients with VT and 64 healthy volunteers were assessed for MTHFR 677T and 1298C alleles using polymerase chain reaction and restriction fragment length polymorphism. Observed MTHFR genotype frequencies were compared with expected genotype combinations, and their odds ratios were determined. MTHFR allele frequency did not differ between VT and control groups; however, differences were observed for MTHFR genotype distribution. MTHFR 677T and 1298C alleles occurred in cis in our population, and therefore mutation crossover has occurred. There was deviation from the Hardy-Weinberg equilibrium for combined MTHFR genotypes, although this may at least partly be attributable to linkage disequilibrium. MTHFR 677CT/1298CC and 677TT/1298CC genotypes (P<.05) were not observed in either group. CONCLUSIONS: The absence of MTHFR 677CT/1298CC and 677TT/1298CC genotypes in both groups suggests that certain MTHFR genotypes may carry a selective advantage. Our discovery of a substantial number of MTHFR mutations in cis configuration suggests that any MTHFR allele linkage disequilibrium present is incomplete.

Randomized trial of high-flux vs low-flux haemodialysis: effects on homocysteine and lipids.

BACKGROUND: Uncontrolled studies have found that high-flux haemodialysis favourably modifies homocysteine and lipid profiles. We sought to confirm these findings by carrying out a randomized prospective comparison of high-flux and low-flux polysulphone in chronic, stable dialysis patients. METHODS: Forty-eight patients were randomly assigned to either high or low-flux dialysis for 3 months. Serum levels of homocysteine, lipoprotein (a), and lipids were compared between the treatment groups at monthly intervals. RESULTS: All patient characteristics and laboratory variables were equally distributed between the groups at baseline. Over the study duration, we observed no differences between high- and low-flux treatment groups for the following outcomes: pre-dialysis homocysteine, lipoprotein (a), total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides (all P>0.05). Geometric mean (interquartile range) homocysteine at baseline was 20.0 (16.8-24.5) and 19.5 (15.3-22.0) micromol/l for the high-and low-flux groups respectively (P=0.80), and levels did not change significantly during the study. We did demonstrate a more pronounced intradialytic effect of high-flux dialysis on homocysteine levels, which fell during dialysis by 42%, compared to 32% with low-flux dialysis (P<0. 001). CONCLUSIONS: In this randomized controlled trial, the effects of high-flux and low-flux haemodialysis on homocysteine and lipid profiles were comparable. The greater intradialytic effect of high-flux dialysis on homocysteine did not translate into a significant difference in pre-dialysis levels after 3 months of study.

Occurrence of hyperhomocysteinaemia in cardiovascular, haematology and nephrology patients: contribution of folate deficiency.

We investigated the contribution of plasma folate deficiency to hyperhomocysteinaemia in selected patient groups. Based on our observations, we have determined a lower folate reference interval cut-off using homocysteine as a metabolic marker of folate deficiency. Four hundred and twenty-five consecutive plasma specimens from cardiology (n = 120), haematology (n = 190) and nephrology (n = 115) patients were analysed for homocysteine and plasma folate concentrations. Healthy volunteers were used as controls (n = 117). We observed elevated homocysteine values above our upper reference limit of 13 micromol/L in 20.1%, 28.4% and 74.8% of the cardiology, haematology and nephrology patients, respectively. All but 1.9% of the patients had plasma folate values greater than the lower reference interval limit (3.4 nmol/L) for our folate assay. The percentage of patients from cardiology and haematology clinics who were hyperhomocysteinaemic and had folate values > 15 nmol/L was 5.0% and 4.2%, respectively. In contrast, 58% of our nephrology patients with folate values > 15 nmol/L were hyperhomocysteinaemic. In all three groups, an inverse relationship was found between folate and homocysteine. The folate/homocysteine ratios in the patient groups were approximately one-third of the values observed in our control group. Folate deficiency appears to be the primary cause of hyperhomocysteinaemia in our cardiology and thrombosis patients. However, severe folate deficiency appears to be uncommon. The majority of our nephrology patients are hyperhomocysteinaemic without an apparent folate deficiency. We conclude that raising the lower reference interval cut-off for folate to 15 nmol/L would help to identify individuals at risk for hyperhomocysteinaemia in our non-uraemic patient population. Increasing folate supplementation to maintain a plasma concentration above 15 nmol/L in cardiac, thrombosis and renal patients would greatly reduce the occurrence of hyperhomocysteinaemia in these patients.

Non-fasting reference intervals for the Abbott IMx homocysteine and AxSYM plasma folate assays: influence of the methylenetetrahydrofolate reductase 677 C-->T mutation on homocysteine.

Plasma homocysteine comes under both genetic and nutritional control. B vitamins and particularly folate are important factors in homocysteine metabolism. We have obtained reference intervals for total plasma homocysteine and plasma folate. We have also determined the influence of methylenetetrahydrofolate reductase (MTHFR) genotype on plasma homocysteine concentrations in healthy individuals. Reference intervals for Abbott IMx homocysteine and AxSYM plasma folate assays were established using 116 volunteers recruited from hospital staff. Exclusion criteria included cardiac, hepatic or renal disorders, and use of over-the-counter prescription medications. An exception was the inclusion of three women using oral contraceptives and one woman receiving post-menopausal oestrogen supplementation. Methylenetetrahydrofolate reductase 677C-->T genotyping was performed on 101 of the volunteers to determine whether the MTHFR 677T allele influences homocysteine concentrations in healthy individuals. Reference intervals for homocysteine and folate were determined using the mean+/-2 standard deviations of the data. Folate/homocysteine ratios were sorted by MTHFR C677T genotype. Homocysteine correlated negatively with plasma folate. Mean male homocysteine concentrations were significantly higher (9.0 micromol/L; P<0.05) than the mean value (7.1 micromol/L) obtained for females. Mean homocysteine values were significantly higher in subjects who were homozygous for the MTHFR 677T allele when compared with the 677CC genotype (P<0.05). Ratios of folate/homocysteine were 20% and 7.4% lower in the male and female 677TT group than in the 677CC group, respectively. The mean homocysteine value of 43 volunteers who were taking multivitamins was not significantly different from that of 73 who were not vitamin supplemented. Conversely, the mean folate value was slightly greater, and statistically significant, in the group taking vitamin supplements. The mean folate values and reference intervals were not significantly different when grouped by sex or age. MTHFR 677C-->T mutations influenced homocysteine values observed in our study of healthy volunteers, even though we did not observe outright folate-deficient individuals. Our random homocysteine values were similar to the fasting homocysteine values obtained in other studies.

Evaluation of the Abbott IMx fluorescence polarization immunoassay and the bio-rad enzyme immunoassay for homocysteine: comparison with high-performance liquid chromatography.

We evaluated the precision, linearity and accuracy of the Abbott IMx and Bio-Rad (Axis) homocysteine assays. Both assays make use of S-adenosyl-homocysteine hydrolase and excess adenosine, to convert homocysteine to S-adenosylhomocysteine (SAH). A monoclonal anti-SAH antibody is then used to quantify SAH. The IMx assay measures the fluorescence polarization of a conjugated SAH analogue for the final analytical step, whereas the Bio-Rad method uses a microplate enzyme immunoassay (EIA) employing an anti-mouse antibody peroxidase conjugate. The Abbott procedure is completely automated whereas the Bio-Rad EIA is performed manually. Between-run coefficient of variation using commercial controls was 2.6% at 7 micromol/L, 2.5% at 13 micromol/L and 1.7% at 24 micromol/L for the Abbott method, and 19.7% at 6.4 micromol/L, 15.9% at 11.0 micromol/L and 14.5% at 23.4 micromol/L for the Bio-Rad method. Both assays correlated well with a high-performance liquid chromatography (HPLC) procedure for homocysteine: Bio-Rad EIA = 1.03HPLC + 1.0 micromol/L, r=0.98, s(y/x)=0.51; Abbott IMx = 1.02HPLC + 0.7 micromol/L, r=0.99, s(y/x) = 0.33. Both methods were linear up to 50 micromol/L homocysteine. The IMx assay had superior precision as well as the technological advantage of being completely automated. Both immunoassays exhibited greatly improved throughput compared with our existing HPLC method.

Genetic determinants of heritable venous thrombosis: genotyping methods for factor V(Leiden)A1691G, methylenetetrahydrofolate reductase C677T, prothrombin G20210A mutation, and algorithms for venous thrombosis investigations.

OBJECTIVES: To implement cost effective and clinically relevant thrombophilic genotyping and homocysteine analysis in our coagulation laboratory. METHODS: We describe genotyping assays for three of the genetic defects associated with hereditary thrombosis: factor V(Leiden) A1691G, methylenetetrahydrofolate reductase C677T, and prothrombin gene G20210A. A second confirmatory assay for factor V(Leiden) using allele specific oligonucleotide polymerase chain reaction is also presented. We suggest an algorithm for the rational integration of the traditional assays routinely used to investigate venous thrombosis with genotyping and plasma homocysteine measurements. RESULTS: These polymerase chain reaction based assays were designed to be performed under identical reaction conditions, permitting simultaneous setup, amplification, digestion, and analysis. CONCLUSIONS: The three genotyping assays presented are robust and relatively easy to perform. Use of an algorithm will ensure efficient resource utilization and minimize unnecessary testing.

Effect of multivitamins on plasma homocysteine and folate levels in patients on hemodialysis.

Hyperhomocysteinemia is a risk factor for cardiovascular disease in patients on hemodialysis. Causes include genetic enzyme deficiencies, chronic renal failure, and vitamin deficiencies. Homocysteine correlates negatively with folate status. In patients on hemodialysis, supraphysiologic doses of B vitamins and folate reduce homocysteine by 26-33%. No study has examined the effect of a standard multivitamin (Nephro-Vite Rx), containing B vitamins and 1 mg of folate, on erythrocyte-folate (RBC-folate) and homocysteine in patients on dialysis. We examined RBC-folate and homocysteine levels in 11 stable chronic patients on hemodialysis, mean duration of dialysis 9.8+/-4.1 months, who were not on vitamin or folate supplements, and repeated these levels after 3 weeks of once daily Nephro-Vite Rx dosage. Plasma homocysteine levels fell by 23.7% from 27.8+/-5.9 to 21.2+/-6.6 micromol/L (p = 0.007), whereas RBC-folate levels rose 60% from 631.2+/-208.3 to 1007.5+/-423.7 nmol/L (p = 0.001). The optimum dose of B vitamins and folate remains to be established, and a clinical benefit from lowering homocysteine has not yet been demonstrated. In summary, a standard multivitamin such as Nephro-Vite Rx reduces plasma homocysteine levels and increases RBC-folate levels in patients on hemodialysis. Our results may have implications for the modification of cardiovascular risk in these patients.

Establishing reference intervals for DPC's free testosterone radioimmunoassay.

OBJECTIVE: To establish reference intervals for serum free testosterone for DPC's Free Testosterone assay. METHODS: We used data from healthy subjects and patients to determine reference intervals by parametric and non-parametric methods after partitioning by sex and age. RESULTS: In males, there was a significant decrease in free testosterone concentrations with age. Reference intervals derived from a combination of 2075 "healthy" and patients' results gave similar values by parametric and nonparametric methods. However, the subgroups failed the test for Gaussian distribution. For each decade from 20 years on and > or = 60 years, the intervals based on 2.5th and 97.5th percentiles were: 24.1-94.8, 25.0-89.3, 23.4-81.7, 22.5-80.4, and 21.5-74.3 pmol/L respectively, in females, there was little change with age. The frequency distribution of 1915 patients was positively skewed, and showed a wider range than "healthy." Using square roots of values gave a Gaussian distribution. The central 95% intervals based on 187 "healthy" subjects were: 0.5-8.1 and 0.0-6.4 pmol/L for 20-59 and > or = 60 years, respectively. CONCLUSION: Developing reference intervals for free testosterone was complicated by the need to partition data by gender and age, difficulty in establishing disease in subjects and presence of physiological and other factors which can affect concentration in health and disease.