A liquid chromatographic-tandem mass spectrometric method for the determination of two selective thymidylate synthase inhibitors, BGC945 and BGC638, in mouse plasma

A LC-tandem mass spectrometry method to quantify the quinazoline-based thymidylate synthase inhibitors BGC945 and BGC638 in mouse plasma was developed. BGC945 and BGC638 were extracted from mouse plasma using protein precipitation with acetonitrile. Chromatography was performed on a Fluophase RP 5 microm, 100 mmx2.0mm i.d. column using a gradient of ammonium acetate and acetonitrile as a mobile phase with a flow rate of 0.2 mLmin(-1). The injection volume for each sample was 20 microL with a total run time of 7.5 min. This method was validated in the range 25-4000 nM (r2=0.99). The analytical assay performance showed that the method was accurate (mean intra- and inter-day assay R.E. were below 12% and 11%, respectively), reproducible (mean intra- and inter-day R.S.D. were less than 13% and 5% for all quality control levels, respectively) and sensitive (lower limit of quantification was 25 nM) in the range studied. This validated method has been used to define the first pharmacokinetic report of BGC945 and BGC638 in mice.     

Effect of postmenopausal hormone replacement therapy on lipoprotein and homocysteine levels in Chinese women.

Epidemiological studies have revealed that postmenopausal estrogen replacement therapy results in a marked reduction in the risk for cardiovascular diseases. In the present study, we evaluated plasma lipoprotein profile as well as homocysteine levels in 145 postmenopausal and premenopausal Chinese women living in Hong Kong. We also investigated the effect of hormone-replacement therapy (HRT) with estrogen or estrogen combined with progestin on plasma lipoprotein profile and homocysteine concentrations in those individuals. Postmenopausal women displayed significantly higher plasma levels of total cholesterol, LDL-cholesterol and apoB as well as higher plasma homocysteine levels than that of premenopausal women. HRT with either estrogen (17beta-estradiol or conjugated equine estrogen) alone or estrogen combined with progestin for 3.5-4.5 years significantly improved the lipoprotein profile in postmenopausal women by decreasing the levels of total cholesterol (12-20% reduction), LDL-cholesterol (26-29% reduction) and apoB (21-25% reduction). In women treated with 17beta-estradiol or conjugated equine estrogens their plasma levels of apoAl were significantly elevated (18% elevation) as compared to non-users. HRT also reduced plasma concentrations of homocysteine (13-15% reduction). In conclusion, we found that long-term HRT was associated with improvement in plasma lipoprotein profile and a reduction in homocysteine concentration in postmenopausal women. These results support the notion that the improvement of lipoprotein profile and a reduction in homocysteine concentration may contribute to the beneficial effect of HRT on cardiovascular risk.     

Mechanisms for the formation of protein-bound homocysteine in human plasma

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Greater than 70% of homocysteine in circulation is protein-bound. An in vitro model system using human plasma has been developed to study mechanisms of protein-bound homocysteine formation and establish the equilibrium binding capacities of plasma for homocysteine. Addition of homocysteine to plasma caused an initial rapid displacement of cysteine and a subsequent increase in protein-bound homocysteine. This rapid reaction was followed by a slower oxygen-dependent reaction forming additional protein-bound homocysteine. To determine the equilibrium binding capacity of plasma proteins for homocysteine, plasma was treated with 0.5-10 mM dl-homocysteine for 4 h at 37 degrees C under aerobic conditions. Under these conditions the equilibrium binding capacity was 4.88 +/- 0.51 and 4.74 +/- 0.68 micromol/g protein for male (n = 10) and female (n = 10) donors, respectively. The mechanism of protein-bound homocysteine formation involves both thiol-disulfide exchange and thiol oxidation reactions. We conclude that plasma proteins have a high capacity for binding homocysteine in vitro.     

Effect of folate deficiency on placental DNA methylation in hyperhomocysteinemic rats

We report that the maternal folate status can influence folate-mediated one-carbon metabolism and DNA methylation in the placenta. Thirty-six female Sprague-Dawley rats were divided into the following three dietary groups: folate-supplemented (FS; 8 mg/kg folic acid, n=12), homocystine- and folate-supplemented (HFS; 0.3% homocystine and 8 mg/kg folic acid, n=12) and homocystine-supplemented and folate-deficient (HFD; 0.3% homocystine and no folic acid, n=12). The animals were fed their experimental diets from 4 weeks prior to mating until Day 20 of pregnancy (n=7-9 per group). The HFS diet increased the plasma homocysteine and placental DNA methylation but did not affect plasma folate, vitamin B-12, S-adenosyl methionine (SAM) or S-adenosyl homocysteine (SAH) levels, or the SAM/SAH ratio in the liver and placenta compared with the FS diet. The HFD diet induced severely low plasma folate concentrations, with plasma homocysteine levels increasing up to 100 micromol/L, and increased hepatic SAH and decreased placental SAM levels and SAM/SAH ratio in both tissues, with a concomitant decrease in placental DNA methylation. Placental DNA methylation was significantly correlated with placental (gamma=0.819), hepatic (gamma=0.7) and plasma (gamma=0.752) folate levels; plasma homocysteine level (gamma=-0.688); hepatic SAH level (gamma=-0.662) and hepatic SAM/SAH ratio (gamma=0.494). These results suggest that the maternal folate status in hyperhomocysteinemic rats influences the homeostasis of folate-mediated one-carbon metabolism and the methyl pool, which would, in turn, affect placental DNA methylation by altering the methylation potential of the liver.     

Differences in plasma homocysteine levels between Zucker fatty and Zucker diabetic fatty rats following 3 weeks oral administration of organic vanadium compounds

PURPOSE: Recently, our laboratory group has reported that rats with Type 1 diabetes have decreased plasma homocysteine and cysteine levels compared to non-diabetic controls and that organic vanadium treatment increased plasma homocysteine concentrations to non-diabetic concentrations. However, to date, no studies have been done investigating the effects of organic vanadium compounds on plasma homocysteine and its metabolites in Type 2 diabetic animal model. These studies examined the effect of organic vanadium compounds [bis(maltolato)oxovanadium(IV) and bis(ethylmaltolato)oxovanadium(IV); BMOV and BEOV] administered orally on plasma concentrations of homocysteine and its metabolites (cysteine and cysteinylglycine) in lean, Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rats. ZF rats are a model of pre-diabetic Type 2 diabetes characterized by hyperinsulinemia and normoglycemia. The ZDF rat is a model of Type 2 diabetes characterized by relative hypoinsulinemia and hyperglycemia. METHODS: Zucker lean and ZF rats received BMOV in the drinking water at a dose of 0.19 +/- 0.02 mmol/kg/day. Lean and ZDF rats received BEOV by oral gavage daily at dose of 0.1 mmol/kg. The treatment period for both studies was 21 days. At termination, animals were fasted overnight (approximately 16 h) and blood samples were collected by cardiac puncture for determination of plasma glucose, insulin and homocysteine levels. Plasma homocysteine and its metabolites levels were determined using high-pressure liquid chromatography. Plasma glucose was determined using a Glucose Analyzer 2. Plasma insulin levels were determined by radioimmunoassay. Plasma triglycerides were determined by an enzymatic assay methodology. RESULTS: ZF (n = 4) and ZDF (n = 10) rats had significantly lower plasma homocysteine as compared to their respective lean groups (ZF 0.78 +/- 0.1 micromol/L vs. Zucker lean 2.19 +/- 0.7 micromol/L; ZDF 1.71 +/- 0.2 micromol/L vs. Zucker lean 3.02 +/- 0.3 micromol/L; p < 0.05). BMOV treatment in ZF rats restored plasma homocysteine levels to those observed in lean untreated rats (ZF treated: 2.04 +/- 0.2 micromol/L; lean 2.19 +/- 0.7 micromol/L). There was a modest effect of BMOV treatment on plasma glucose levels in ZF rats. BEOV treatment significantly decreased the elevated plasma glucose levels in the ZDF rats (lean 7.9 +/- 0.1 mmol/L; lean + vanadium 7.7 +/- 0.2 mmol/L; ZDF 29.9 +/- 0.4 mmol/L; ZDF + vanadium 17.4 +/- 0.3 mmol/L, p < 0.05). Organic vanadium treatment reduced cysteine levels in both ZF and ZDF rats. No differences in total plasma cysteinylglycine concentrations were observed. CONCLUSION: Plasma homocysteine levels are significantly reduced in a pre-diabetic model of Type 2 diabetes, which was restored to lean levels upon vanadium treatment; however, this restoration of plasma homocysteine levels was not seen in ZDF Type 2 diabetic rats following vanadium treatment. In the latter case vanadium treatment may not have totally overcome the insulin resistance seen in these animals.     

The determination of homocysteine-thiolactone in human plasma

The thioester homocysteine-thiolactone, a reactive metabolite of homocysteine, has been implicated in human cardiovascular disease. However, data on the levels of homocysteine-thiolactone in humans are limited, mostly due to a lack of facile and reliable assays. Here we describe a sensitive assay for the determination of plasma homocysteine-thiolactone and demonstrate its utility with a cohort of 60 healthy human subjects. Plasma homocysteine-thiolactone is first separated from macromolecules by ultrafiltration and then selectively extracted with chloroform/methanol. Further purification of plasma homocysteine-thiolactone is achieved by high-performance liquid chromatography on a cation exchange microbore column. The detection and quantification is by monitoring fluorescence after postcolumn derivatization with o-phthaldialdehyde. The limit of detection is 0.36 nM. Using this assay, homocysteine-thiolactone concentrations in plasma from normal healthy human subjects (n=60) were found to vary from zero to 34.8 nM, with an average of 2.82+/-6.13 nM. In 29 of the 60 human plasma samples analyzed, homocysteine-thiolactone levels were below the detection limit. Homocysteine-thiolactone represented from 0 to 0.28%, on average 0.023+/-0.05%, of plasma total homocysteine.     

Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection

A rapid and sensitive method for quantification of homocysteine total forms and glutathione levels in blood plasma via HPLC was developed. Dithiotreitol as a water soluble agent has been used as a reductant for both protein and nonprotein disulphides. Dithiotreitol reacts with the mixed disulphides under 60 degrees C treatment within 10 min. Reduced aminothiols and homocystein were easily derivated with 5,5'-dithiobis-(2-nitrobenzoic acid) and the resultant ultraviolet absorbance within 330 nm was detected by the HPLC method. The concentration of total plasma homocysteine was significantly higher in groups of patients: with the end stage of renal disease: 45.5+/-40.9 micromol/l (n=79), with cerebral vascular disorders 12.3+/-7.0 micromol/l (n=65), and with coronary atherosclerosis 15.4+/-10.9 micromol/l (n=15) than that in healthy subjects (6.2+/-1.74 micromol/l, n=20). Some major advantages of the method include: simultaneous measurement of both total homocysteine and total glutathione, no loss of oxidized form during processing of blood plasma for aminothiols measurement, use of protein-bound aminothiols solution as a calibrator.     

Hyperhomocysteinemia and recurrent carotid stenosis

Background

Hyperhomocysteinemia has been identified as a potential risk for atherosclerotic disease in epidemiologic studies. This study investigates the impact of elevated serum homocysteine on restenosis after carotid endarterectomy (CEA).

Methods

In a retrospective study, we compared fasting plasma homocysteine levels of 51 patients who developed restenosis during an eight year period after CEA with 45 patients who did not develop restenosis. Restenosis was defined as at least 50% stenosis and was assessed by applying a routine duplex scan follow up investigation. Patients with restenosis were divided into a group with early restenosis (between 3 and 18 months postoperative, a total of 39 patients) and late restenosis (19 and more months; a total of 12 patients).

Results

The groups were controlled for age, sex, and risk factors such as diabetes, nicotine abuse, weight, hypertension, and hyperlipidemia. Patients with restenosis had a significant lower mean homocysteine level (9.11 μmol/L; range: 3.23 μmol/L to 26.49 μmol/L) compared to patients without restenosis (11.01 μmol/L; range: 5.09 μmol/L to 23.29 μmol/L; p = 0.03). Mean homocysteine level in patients with early restenosis was 8.88 μmol/L (range: 3.23–26.49 μmol/L) and 9.86 μmol/L (range 4.44–19.06 μmol/L) in late restenosis (p = 0.50).

Conclusion

The finding suggests that high plasma homocysteine concentrations do not play a significant role in the development of restenosis following CEA.     

A rapid and sensitive micro-assay for the enzymatic determination of plasma and lipoprotein cholesterol

A rapid and inexpensive micro-assay for determining cholesterol in plasma and isolated lipoprotein fractions has been established which utilizes a commercially available enzymatic reagent with semi-automated instruments and microtiter plates. The assay is sensitive, precise, and easy to perform. The color development is linear from 0.4 to 20 micrograms cholesterol/well, with sample volumes of 2 to 100 microliters. Inter- and intra-assay variability yielded coefficients of variation (CV) of 2.75% (n = 51) and 1.09% (n = 32), respectively. The concentrations of total plasma and lipoprotein cholesterol (d greater than 1.006 g/ml) obtained with this method were compared with those analyzed in a lipid laboratory standardized to the Centers for Disease Control. The correlation coefficients between the two methods were 0.976 and 0.964, respectively. For total high density lipoprotein (HDL) and the HDL3 subfraction, inter-assay variability was 4.12% and 6.33% (n = 27), respectively; the intra-assay variability was 2.79% and 4.19% (n = 12).     

Pancreastatin-like immunoreactivity in human carcinoid disease

Pancreastatin-like immunoreactivity has been demonstrated in human carcinoid tumors by immunohistochemistry and radioimmunoassay, employing antisera raised to a synthetic C-terminal fragment of porcine pancreastatin. Immunohistochemistry revealed intense immunoreactivity in all tumors. By radioimmunoassay, high concentrations of pancreastatin-like immunoreactivity were measured in carcinoid tumors arising from the fore-gut (mean +/- S.D. and range: 369 +/- 955 and 9.4-3670 pmol g-1, respectively, n = 14), mid-gut (mean +/- S.D. and range: 1354 +/- 1538 and 337-3978 pmol g-1, respectively, n = 5) and in metastases associated with mid-gut tumors (mean +/- S.D. and range: 684 +/- 739 and 31-2255 pmol g-1, respectively, n = 7), compared to corresponding normal tissues (less than 1.4 pmol g-1). Individuals with hepatic metastases and carcinoid syndrome had elevated circulating levels of pancreastatin-like immunoreactivity (mean +/- S.D. and range: 770 +/- 1249 and 42-4120 pmol l-1; n = 12), significantly above the normal, fasting range (mean +/- S.D. and range: 14.9 +/- 7.5 and 4-37.5 pmol l-1, respectively, n = 42). However, patients with non-metastatic carcinoid tumors (n = 4), who had been clinically cured after primary tumor resection, had plasma levels within the normal range. Chromatographic analysis of extracts of primary lung and ileal tumors, hepatic metastases from ileal tumors and plasma from individuals with carcinoid syndrome revealed molecular heterogeneity of pancreastatin-like immunoreactivity.     

Homocysteine is positively associated with cytokine IL-18 plasma levels in coronary artery bypass surgery patients

Homocysteine, cytokines (IL-18, IL-6, IL-8) are involved in vascular inflammation and coronary artery disease. Homocysteine influences endothelial IL-6 and IL-8 cytokine expression and release, however, an association between homocysteine and IL-18 has not been previously investigated in endothelial/smooth muscle cells and or in coronary artery disease. We report in 9 coronary artery bypass surgery (CABG) patients a positive correlation r = 0.86 between homocysteine and IL-18 plasma levels (p < 0.05). Plasma IL-18 levels are significantly higher in those patients with elevated homocysteine compared to those with normal levels (p < 0.02; 153 +/- 19 pg/ml versus 116 +/- 14 pg/ml respectively). Our in vitro cell culture studies suggest that the source of IL-18 in CABG patients with elevated homocysteine is not from vascular smooth muscle or endothelial cells.     

Prospective evaluation of folic acid supplementation on plasma homocysteine concentrations during weight reduction: a randomized, double-blinded, placebo-controlled study in obese women

Elevated total homocysteine concentrations and obesity are both associated with an increased risk of cardiovascular disease. However, previous studies of weight reduction on serum homocysteine concentrations have obtained inconsistent reports. We investigated the effect of folic acid supplementation on serum homocysteine concentrations via a randomized, double-blinded, placebo-controlled study. Seventy-four obese women [age (mean +/- SEM) 41 +/- 1 years; body mass index, 29.6 +/- 0.5 kgs/m2] completed a 12 weeks weight reduction program with dietary advice and light exercise. They were also randomized to take either folic acid supplementation (5 mg daily, n = 36) or placebo (n = 38) groups. This program led to a weight reduction of 7.7% and 8.9% of initial weight for folic acid supplementation and placebo groups, respectively. Serum folate concentrations increased for 3 folds (p < 0.001) in the folic acid group. In the folic acid group, there was a trend of lower fasting serum homocysteine concentrations (7.6 +/- 0.2 vs. 7.3 +/- 0.3 micromol/L), but it did not reach statistical significance (p = 0.170). However, we found that serum homocysteine concentrations decreased significantly in those with higher baseline homocysteine concentrations (8.7 +/- 1.3 vs. 7.8 +/- 1.5 micromol/L, p = 0.004), while it did not change in those with lower baseline homocysteine concentrations (6.6 +/- 0.6 vs. 6.8 +/- 1.2 micromol/L, p = 0.334). Reduction of serum homocysteine concentrations did not correlate with elevation of serum folate concentrations (p = 0.646) in obese women with higher baseline homocysteine concentrations. In conclusion, serum homocysteine concentrations can be maintained in obese women during mild to moderate weight loss. Folic acid supplementation decreased serum homocysteine concentrations in those women who had higher serum homocysteine concentrations before participating in the weight reduction program.     

Homocysteine levels in acromegaly patients

Acromegaly is associated with a two to three-fold increase in mortality related predominantly to cardiovascular disease. The excess mortality is associated most closely with higher levels of growth hormone (GH). Survival in acromegaly may be normalized to a control age-matched rate by controlling GH levels; in particular, GH levels less than 2.5 ng/mL are associated with survival rates equal to those of the general population. Hyperhomocysteinemia has also been recognized as a risk factor for cardiovascular disease, yet there are limited data on the prevalence of hyperhomocysteinemia in patients with acromegaly. Eighteen acromegaly patients (7 male, 11 female, mean age 42.8 +/- 11.0 years) in our endocrine clinic consented to having the following tests performed: complete blood count (CBC), thyroid hormones, folic acid, vitamin B12, plasma homocysteine levels, uric acid, fibrinogen, CRP, fasting glucose, insulin, C-peptide, total serum cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, GH, insulin-like growth factor-1 (IGF-1) and GH levels after an oral glucose tolerance test (OGTT). By history, fourteen had macroadenomas and four had microadenomas; eight had hypertension; two had glucose intolerance, and four had diabetes. Fifteen had had transsphenoidal or transfrontal surgery: two had been cured, but 13 others were taking long-acting octreotide. Five patients had undergone radiotherapy and the acromegaly in two was treated primarily with long-acting octreotide. CBC, thyroid hormone, folic acid, and vit B12 levels were normal in all patients. We divided the patients into two groups according to mean GH levels after an OGTT: Group 1 (GH<2.5 ng/mL, n=10), and Group 2 (GH<2.5 ng/mL, n=8). Comparison of the two groups using Mann-Whitney U testing revealed statistically significant lower levels in Group 1 of the following parameters: GH (1.91 +/- 0.90 vs. 8.58 +/- 5.55 ng/mL, p=0.002), IGF-1 (338.30 +/- 217.90 vs. 509.60 +/- 293.58 ng/dL, p=0.06), GH after an OGTT (1.42 +/- 0.81 vs. 9.01 +/- 4.53 ng/mL, p=0.001), plasma homocysteine (12.85 +/- 4.47 vs. 18.20 +/- 4.99 micromol/L, p=0.05), total cholesterol (164.0 +/- 20.81 vs. 188.0 +/- 22.26 mg/dL, p=0.05) and LDL cholesterol (81.0 +/- 9.64 vs. 116.70 +/- 13.03 mg/dl, p=0.01). Differences between the other parameters were not significantly different. Acromegaly patients with high GH levels after an OGTT have much higher levels of homocysteine than patients with lower GH levels. The role of elevated homocysteine levels as an independent cardiovascular risk factor in the mortality of acromegaly patients should be determined in future studies.     

Red blood cell folate vitamer distribution in healthy subjects is determined by the methylenetetrahydrofolate reductase C677T polymorphism and by the total folate status

BACKGROUND: Red blood cells (RBCs) represent a storage pool for folate. In contrast to plasma, RBC folate can appear in different biochemical isoforms. So far, only the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype has been identified as a determinant of RBC folate vitamer distribution. OBJECTIVE: The purpose of this study is to identify clinical and biochemical determinants of RBC folate vitamer distribution in healthy subjects. DESIGN: In an observational study, 109 subjects, aged 18 to 65 years, were studied. Red blood cell folate vitamers were analyzed using a liquid chromatography-tandem mass spectrometry method. Other variables recorded included vitamin B(2), B(6) and B(12) status, homocysteine, plasma and RBC S-adenosylhomocysteine and S-adenosylmethionine, renal function and the MTHFR C677T polymorphism. RESULTS: The MTHFR C677T genotype was the dominant determinant of nonmethylfolate accumulation. The median (range) nonmethylfolate/total folate ratio was 0.58% (0-12.2%) in the MTHFR CC group (n=55), 0.99% (0-14.3%) in the CT group (n=39) and 30.3% (5.7-73.3%) in the TT genotype group (n=15), P<.001. The 95th percentile for the nonmethylfolate/total folate ratio was 2.8% for the CC group, 9.1% for the CT group and 73.3% for the TT group. In the CC and CT genotype subjects, the T-allele and total folate status were positively and independently correlated with nonmethylfolate accumulation, but the degree of nonmethylfolate accumulation in these subjects was usually minor compared with those with the TT genotype. None of the other studied variables was associated with nonmethylfolate accumulation. CONCLUSIONS: The MTHFR C677T genotype is the dominant determinant of nonmethylfolate accumulation in RBCs. In addition, high total folate status may contribute to minor to moderate nonmethylfolate accumulation in MTHFR CC and CT subjects.     

The relationship between micronuclei in human lymphocytes and selected micronutrients in vegetarians and non-vegetarians

A vegetarian diet results in higher intake of vitamins and micronutrients, which - although providing antioxidant defence - may lead to deficiency in other micronutrients involved in DNA metabolism and stability (such as vitamins belonging to the B group). The principal difference among various vegetarian diets is the extent to which animal products are avoided. We have performed a pilot study to determine the relationship between the micronucleus frequency in lymphocytes and diet, and we compared the levels of Vitamins C and E, beta-carotene, B(12), folic acid, homocysteine and total antioxidant capacity in healthy vegetarians and non-vegetarians. The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among the vegetarians were 13 lacto-ovo-vegetarians with average duration of vegetarian diet 10.8 years (ranging from 5 to 26 years) and 11 lacto-vegetarians with average duration of vegetarian diet 8.2 years (ranging from 3 to 15 years). Homocysteine, Vitamins C and E and beta-carotene levels in plasma were assayed by HPLC, and serum folate and Vitamin B(12) were determined with Elecsys Immunoassay tests. The total antioxidant capacity of plasma was estimated by measuring the ferric-reducing activity in a spectrophotometric assay. Micronuclei were measured in cytokinesis-blocked lymphocytes. Vegetarians had significantly higher levels of Vitamin C and beta-carotene (but not Vitamin E) in plasma compared with non-vegetarians (P<0.001). There were no significant differences in serum levels of folic acid and Vitamin B(12) between the monitored groups. Levels of folic acid in vegetarians correlated with length of vegetarianism (r=0.62, P=0.001, N=24). Vegetarians had elevated levels of homocysteine compared with non-vegetarians (P=0.007), as did vegetarian women compared with non-vegetarian women (P=0.031). We did not find any differences in total antioxidant capacity or in micronucleus frequency between the groups. Micronuclei correlated with age (r=0.62, P<0.001, N=48), women having higher frequencies than men. Multifactorial regression analysis showed significant effects of age, sex and total antioxidant capacity on micronucleus frequency (N=48, P<0.001).     

MTHFR Gene Polymorphism and Its Relationship with Plasma Homocysteine and Folate in a North Indian Population

There is a paucity of data on the prevalence of elevated homocysteine and its relation with plasma folate and the methylenetetrahydrofolate reductase (MTHFR) gene in the population of North India. This study evaluates MTHFR gene polymorphism and its relationship with plasma homocysteine and folate levels in a healthy North Indian population. The age of the 200 subjects included in this study was in the range 18–73 (mean 39.4) years. The plasma homocysteine level was elevated in 56.5%, and the plasma folate level was low in 49.5% of the subjects. Heterozygous MTHFR gene polymorphism (CT) was present in 15.5%, and homozygous (TT) in 3.5% of the subjects. Age, diet, and MTHFR gene polymorphisms were related to homocysteine level. All the subjects with the TT and 79% with the CT genotype had a high level of plasma homocysteine, whereas 51% with the CC genotype had a high homocysteine level. After adjustment for the effect of covariates, however, homocysteine was not related to MTHFR gene polymorphism.     

Effects of hemodialysis, dialyser type and iron infusion on oxidative stress in uremic patients

Uremic patients undergoing hemodialysis (HD) are considered to face an elevated risk for atherosclerosis and cancer. This has been attributed in part to an increased oxidative stress. In this pilot study, oxidative cell damage in blood of HD-patients was compared to those of controls: total DNA damage (basic and specific oxidative DNA damage), modulation of glutathione levels (total and oxidized glutathione) and of lipid peroxidation were monitored via the Comet assay (with and without FPG), a kinetic photometric assay and HPLC quantification of plasma malondialdehyde (MDA), respectively. In some samples, leukocytes were analysed for malondialdehyde-deoxyguanosine-adducts (M1dG) with an immunoslot blot technique. HD-patients (n=21) showed a significant increase of total DNA damage (p<10(-12)), compared to controls (n=12). In a subset of patients and controls, GSSG levels and M1dG, however, only increased slightly, while tGSH and MDA levels did not differ. The influence of different low flux HD-membranes was tested in a pilot study with nine patients consecutively dialysed on three membrane types for four weeks each. In addition to the individual disposition of the patient, the dialyser membrane had a significant impact on oxidative stress. Total DNA damage was found to be almost identical for polysulfone and vitamin E coated cellulosic membranes, whereas a slight, but significant increase was observed with cellulose-diacetate (p<0.001). In patients receiving iron infusion during HD, MDA-formation (n=11) and total DNA damage (n=10) were additionally increased (p<0.005). Our results show an increased oxidative damage in HD-patients, compared to healthy volunteers. Significant influences were found for the dialyser membrane type and iron infusion.     

Tracer-derived total and folate-dependent homocysteine remethylation and synthesis rates in humans indicate that serine is the main one-carbon donor

Hyperhomocysteinemia in humans is associated with genetic variants of several enzymes of folate and one-carbon metabolism and deficiencies of folate and vitamins B12 and B6. In each case, hyperhomocysteinemia might be caused by diminished folate-dependent homocysteine remethylation, but this has not been confirmed in vivo. Because published stable isotopic tracer approaches cannot distinguish folate-dependent from folate-independent remethylation, we developed a dual-tracer procedure in which a [U-13C5]-methionine tracer is used in conjunction with a [3-13C]serine tracer to simultaneously measure rates of total and folate-dependent homocysteine remethylation. In young female subjects, plasma [U-13C4]homocysteine enrichment, a surrogate measure of intracellular [U-13C5]methionine enrichment, reached approximately 90% of the plasma [U-13C5]methionine enrichment. Methionine-methyl and -carboxyl group fluxes were in the range of previous reports (approximately 25 and approximately 17 micromol.kg(-1).h(-1), respectively). However, the rate of overall homocysteine remethylation (approximately 8 micromol.kg(-1).h(-1)) was twice that of previous reports, which suggests a larger role for homocysteine remethylation in methionine metabolism than previously thought. By use of estimates of intracellular [3-13C]serine enrichment based on a conservative correction of plasma [3-13C]serine enrichment, serine was calculated to contribute approximately 100% of the methyl groups used for total body homocysteine remethylation under the conditions of this protocol. This contribution represented only a small fraction (approximately 2.8%) of total serine flux. Our dual-tracer procedure is well suited to measure the effects of nutrient deficiencies, genetic polymorphisms, and other metabolic perturbations on homocysteine synthesis and total and folate-dependent homocysteine remethylation.     

Elevated lipid peroxidation products and depleted transferrin levels in the plasma of kidney transplant recipients

Lipid peroxidation products were measured in the plasma of 24 kidney transplant patients and 12 healthy volunteers (controls) by: (1) 2-thiobarbituric acid assay and (2) the intensity of fluorescence products of malonaldehyde cross-linked proteins. Plasma levels of creatinine, ceruloplasmin, transferrin, prealbumin, albumin and total protein were also measured. Elevated lipid peroxidation products and lowered transferrin levels were observed in transplant patients compared to controls. Ceruloplasmin levels were slightly but significantly elevated in recent transplant recipients (less than 6 months, n = 12, Group A) while no difference was observed between older transplant recipients (greater than 6 months, n = 12, Group B) and controls. Serum, creatinine levels were also slightly but significantly elevated in both groups of patients compared to controls. Serum prealbumin, albumin and total protein levels in both groups of transplant recipients were not different from controls or reference range values.     

Methylenetetrahydrofolate reductase gene C677T polymorphism,homocysteine, vitamin B12, and DNA damage in coronary artery disease

Elevated levels of plasma homocysteine (Hcy), a risk factor for coronary artery disease (CAD), can result from genetic errors, e.g., the methylenetetrahydrofolate reductase (MTHFR) polymorphism, or nutritional deficiencies, e.g., in vitamin B12 and folate. The mechanism by which Hcy induces atherosclerosis is not fully understood. Recently, Hcy has also been observed to induce DNA damage. In this study, we have investigated whether DNA damage is related to the C677T variant in the MTHFR gene and to plasma levels of Hcy, B12, and folate in patients with CAD. Patients ( n=46) with angiographically proven CAD were studied by using the micronucleus (MN) test, an accepted method for evaluating genetic instability. TT patients had plasma Hcy levels higher than those with the CT or CC genotypes (27.8+/-5.2 vs 13.7+/-2.2 and 12.9+/-1.9 micro mol/l, respectively; P=0.02). Patients with multi-vessel disease had higher plasma Hcy levels (11.6+/-1.2, 22.0+/-4.7, 19.3+/-3.9 micromol/l for one-, two- and three-vessel disease, respectively; P=0.05). The MN index increased with the number of affected vessels (8.4+/-0.7, 11.1+/-2.0, 14.2+/-1.7 for one-, two-, and three-vessels disease, respectively; P=0.02) and was significantly higher in subjects with the TT genotype compared with the CC or CT genotypes (15.7+/-2.4 vs 8.9+/-1.7 and 9.9+/-0.8; P=0.02). The MN index was also correlated negatively with plasma B12 concentration ( r=-0.343; P=0.019) and positively with plasma Hcy ( r=0.429, P=0.005). These data indicate that the MN index is associated with the severity of CAD and is related to the MTHFR polymorphism, suggesting an interesting link between coronary atherosclerosis and genetic instability in humans.