Hcy Metabolism Research Articles

Rate of T alleles and TT genotype at MTHFR 677C‐>T locus or C alleles and CC genotype at MTHFR 1298A‐>C locus among healthy subjects in Turkey: impact on homocysteine and folic acid status and reference intervals

Methylenetetrahydrofolate reductase (MTHFR) is important for folate and homocysteine (Hcy) metabolism. MTHFR 677C->T and 1298A->C MTHFR are two most common mutations which can affect folate and total homocysteine (tHcy) status. This study was designed to determine the rate of MTHFR 677C->T and 1298A->C mutations, and their influence on serum folate, Hcy and vitamin B12 status and the reference intervals in 402 healthy Turkish adults. The rate of MTHFR 677C->T or 1298A->C mutations was 50.7% or 54.7%, respectively. The MTHFR 677C->T mutation-specific reference intervals for serum folate and tHcy were characterized by marked shifts in their upper limits. In homozygote subjects for MTHFR 677C->T serum folate concentration was lower and serum tHcy concentration was higher than those in the wild genotype; all subjects had lower serum folate and 54% of the subjects had higher tHcy concentrations than the cutoff values of <or=10 nmol/L and >or=12 micromol/L, respectively. Serum vitamin B12 status was similar in all genotypes. Serum tHcy concentrations were inversely correlated with serum folate and vitamin B12 concentrations in all genotypes. These data show that the rate of MTHFR 677C->T and 1298A->C mutations is very high in Turks and serum folate and tHcy status are impaired by these mutations.

High-dose short-term folate administration modifies ambulatory blood pressure in postmenopausal women. A placebo-controlled study

In humans low intake or low levels of folate are related to elevated homocysteine (Hcy) or blood pressure (BP). This study was performed to test whether in healthy postmenopausal women a 3-week administration of folate (5-methyltetrahydrofolate: 5-MTHF) at the dose of 15 mg/day (n=15), was, in comparison to placebo (n=15), capable of modifying 24-h ambulatory BP, along with Hcy and insulin metabolism. Placebo did not modify any parameter. 5-MTHF significantly decreased nocturnal systolic (-4.48+/-1.8 mm Hg; P=0.029), diastolic (-5.33+/-1.3 mm Hg; P=0.001) and mean (-5.10+/-1.1 mm Hg; P=0.005) BP, in a way that was significantly different from that observed during placebo. 5-MTHF also reduced Hcy (11.77+/-1.15 vs 8.71+/-0.50 micromol/l; P=0.03), and insulin resistance, evaluated by HOMA-IR (2.58+/-0.04 vs 2.03+/-0.04; P=0.01). Whether maintained in the long term, 5-MTHF's cardiovascular and metabolic effect may contribute to primary cardiovascular prevention of postmenopausal women.

Maternal vitamin D status determines volumetric bone mineral density of the newborn

Introduction: Nutrition plays an important role in the acquisition and maintenance of skeletal integrity. There are some evidence that a well-balanced vegetarian diet may be consistent with good health and can potentially reduce the risk of several chronic diseases. On the other hand, people following a long-term vegetarian diet very often suffer from nutrient shortage like Ca, P, Fe, Zn, vitamin D or vitamin B12. The lack of vitamin B12 together with vitamin B6 and folate deficiency is close related to homocysteine (Hcy) metabolism. Hyperhomocysteinemia (HHcy) was found to be associated with increased bone turnover markers and increased fracture risk. Thus, Hcy, vitamin B12 and folate may be regarded as novel risk factors for micronutrient-deficiency-related osteoporosis. The aim of our study was to assess the possible impact of vegetarian diet on bone mass density in cohort of Slovak vegetarian women. Materials and methods: The study was performed on group of 141 women on long-term ovo-lacto-vegetarian diet and control group of 131 women on standard western diet. Standard biochemical analyses, plasma vitamin B12, folate and Hcy concentrations were measured. Bone mineral density (BMD) for lumbar spine (L1–L4), right femoral neck (FN), trochanter (Tr) and total of femur (ToFN) were measured with dual energy X-ray absorptiometry. Results: The mean Hcy levels were beyond the normal range in both groups (>12.0 μmol/l) but significantly higher in vegetarians (p<0.001). HHcy was found in 78% of vegetarians and in 48% of nonvegetarians. Vitamin B12 levels were significantly higher in nonvegetarians in both subgroups (p<0.05) and inversely correlated with Hcy (r=−0., p<0.001). Folate plasma levels were in normal range in all probands. Significant inverse correlations were found between Hcy and FN-BMD (r=−0.1887, p<0.003), Tr-BMD (r=−0.1725, p<0.01) and ToFN-BMD (r=−0.2053, p<0.001). No correlations were confirmed in nonvegetarians when they were evaluated separately. Conclusion: Our results indicate a strong association between plasma Hcy levels and decreased BMD in vegetarians. It suggests that vegetarian women can be at higher risk for low BMD than nonvegetarian women. This work was supported by Research and Development Support Agency under the contract No: APVT-21-010104 a APVT-21-017704. Conflict of interest: None declared.

Beneficial effects of danshensu, an active component of Salvia miltiorrhiza, on homocysteine metabolism via the trans‐sulphuration pathway in rats

Elevated plasma total homocysteine (tHcy) level has been established as an independent risk factor for cardiovascular diseases. Danshensu, an active ingredient of Salvia miltiorrhiza, shows wide cardiovascular benefit. However, in terms of its own methylation, danshensu could elevate tHcy level, which would act against its cardiovascular benefit, thus posing a 'therapeutic paradox'. As this paradox has not been fully assessed, we have evaluated the effects of danshensu on tHcy levels to uncover the underlying mechanisms. EXPERIMENT APPROACH: We evaluated the influence of danshensu on homocysteine (Hcy) metabolism in rats with normal tHcy levels and in rat models of elevated tHcy (single intravenous methionine loading model and a hyperhomocysteinemic model after 3 weeks methionine dosing, with and without 3 weeks of danshensu treatment). We also quantified some metabolic intermediates (S-adenosyl methionine, S-adenosyl-l-homocysteine, cysteine and glutathione) relevant to Hcy metabolism in rat liver and kidney. Acute treatment with a single dose of danshensu in rats with normal tHcy did not change plasma tHcy. In contrast, danshensu significantly lowered tHcy in rats with elevated tHcy. The relatively higher cysteine and glutathione levels after treatment with danshensu indicated that its tHcy-lowering effect was via increased activity of the trans-sulphuration pathway. Our results suggested that danshensu may act both acutely to increase trans-sulphuration and after chronic exposure to up-regulate the activity of the trans-sulphuration enzymes. The tHcy-lowering effect of danshensu is another cardiovascular benefit provided by S. miltiorrhiza and suggests a potential tHcy-lowering therapy.

Homocysteine and cognitive impairment in Parkinson's disease: A biochemical, neuroimaging, and genetic study

The role of the plasma level of homocysteine (Hcy), as a primary outcome, and the effect of silent cerebrovascular lesions and genetic variants related to Hcy metabolism, as secondary outcomes, in the cognitive decline and dementia in Parkinson's disease (PD) were studied. This case-control study focused on 89 PD patients of minimum 10 years of evolution and older than 60 years, who were neuropsychologically classified either as cognitively normal (n = 37), having mild cognitive impairment (Petersen criteria) (n = 22), or suffering from dementia (DSM-IV) (n = 30), compared with cognitively normal age-matched control subjects (n = 30). Plasma levels of Hcy, vitamins B12 and B6, folic acid, polymorphisms in genes related to Hcy metabolism (MTHFR, MTR, MTRR, and CBS) and silent cerebrovascular events were analyzed. Plasma levels of Hcy were increased in PD patients (P = 0.0001). There were no differences between the groups of patients. The brain vascular burden was similar among PD groups. There was no association between polymorphisms in the studied genes and the Hcy plasma levels or cognitive status in PD patients. We found no evidence for a direct relationship between Hcy plasma levels and cognitive impairment and dementia in PD. No indirect effect through cerebrovascular disease or genetic background was found either.

Effects of a Low-Protein Diet on Plasma Amino Acid and Homocysteine Levels and Oxidative Status in Rats

Background/Aims: Transmethylation reactions and antioxidant metabolism are linked by transsulfuration, where homocysteine (Hcy) is converted to cysteine and reduced glutathione (GSH). Low protein intake can modulate the balance of this metabolic reaction. The aim of the present investigation was to study the effect of a low-protein diet on Hcy metabolism by monitoring levels of the amino acids involved in these pathways, and relating these levels to GSH levels and lipid peroxidation in rats. Methods: Sixteen rats were divided into 2 groups: control (C; standard AIN-93 diet, 20% protein) and low-protein diet (LPD; 8% protein diet). Rats in both groups were placed on the diets for 28 days. Results: A significant reduction (p < 0.05) in plasma Hcy concentration was found in LPD rats (0.16 ± 0.04 μmol/mg protein) versus C rats (0.25 ± 0.03μmol/mg protein). Methionine levels were not significantly different between the 2 groups (C: 1.24 ± 0.22 μmol/mg protein; LPD: 1.03 ± 0.27 μmol/mg protein). A significant reduction (p < 0.05) in hepatic GSH concentrations (C: 44 ± 10 μmol/mg protein; LPD: 17.4 ± 4.3 μmol/mg protein) was accompanied by an increase in lipid peroxidation (C: 0.13 ± 0.01 μmol/mg protein; LPD: 0.17 ± 0.02 μmol/mg protein; r = –0.62, p < 0.01). Conclusion: Hcy levels were reduced under a low-protein diet, resulting in modulated methyl balance and reduced GSH formation leading to increased susceptibility of hepatic cells to oxidative events.

Characterization of homocysteine and methyl group metabolism in female non‐obese diabetic (NOD) mice

Methyl groups are critical for numerous biological reactions and proper regulation of methyl group and homocysteine (hcy) metabolism is essential for disease prevention. The hepatic protein glycine N‐methyltransferase (GNMT) is a key regulator of supply and utilization of methyl groups from the primary donor, S‐adenosylmethionine. Chemically‐induced type 1 diabetes has been shown to alter hcy and methyl group metabolism, resulting in a methyl deficient state, including DNA hypomethylation. The objective of this study was to characterize hcy and methyl group metabolism using a genetic model of diabetes, the non‐obese diabetic (NOD) mouse, which spontaneously develops autoimmune‐mediated type 1 diabetes. NOD mice and their controls (ICR), were sacrificed at 12, 18, and 24 wk of age for collection of blood and liver tissue. Plasma hcy concentrations in NOD mice were only 63% of the control values in 18 wk old ICR animals. NOD mice exhibited a 1.4‐fold increase in GNMT activity compared to ICR mice at 18 wk. Neither metabolic parameter was altered in 12 wk old animals. The NOD mouse, a genetic model for type 1 diabetes, exhibits similar perturbations in methyl group and hcy metabolism as the chemically‐induced rat model. This validation will be important in future studies directed at understanding the relation between diabetes, perturbation of methyl groups, and specific pathologies (Support: NIH HD057315‐02).

Folate‐independent remethylation of homocysteine is increased by dietary egg white protein

Proper maintenance and regulation of methyl group and homocysteine (hcy) metabolism is essential for optimization of health. Methyl deficiency and/or elevations in plasma hcy concentrations have been associated with numerous pathologies, such as vascular disease. It is important to identify dietary components that positively impact these metabolic pathways; thus, we examined dietary protein sources with varying sulfur amino acid content. Rats were fed either a casein or egg white protein (20%) diet for 2 wk after which half the rats in each group were fed folate‐deficient diets for 4 wk. Compared to casein, egg white protein resulted in: (1) elevated hepatic betaine‐hcy S‐methyltransferase activity, responsible for folate‐independent hcy remethylation; (2) increased hepatic activity of glycine N‐methyltransferase, a key methyl group regulatory protein; and (3) reduced plasma glutathione concentrations. Moreover, folate deficiency was without affect on plasma hcy levels in the egg white group. In a parallel study, soy protein did not differ from casein with respect to altering methyl group and hcy metabolism. In conclusion, dietary egg white protein appears to promote hcy remethylation at the expense of catabolism via transsulfuration. These findings may be attributed to the greater met‐cys content of egg white protein compared to casein and soy (Support: Amer. Egg Board, Egg Nutr. Center, IA Egg Council).

Genistein reduces plasma homocysteine concentrations in a folate deficient rat model

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine (Hcy) is an intermediate in methyl group metabolism, as S‐adenosylmethionine (SAM), the methyl group donor for most biological methylations, results in the formation of S‐adenosylhomocysteine and ultimately Hcy. Genistein, a 17β‐estradiol‐like isoflavone, has been shown to maintain normal methyl group metabolism in the agouti mouse model fed a methyl‐deficient diet. Although dietary genistein does not provide a methyl group, it may modulate the one‐carbon pool and influence DNA methylation in estrogen‐responsive tissues. To date, it is not known to what extent genistein directly influences Hcy and methyl group metabolism. In the present study, the effects of genistein supplementation on elevated Hcy during moderate folate deficiency were examined. Male Sprague Dawley rats (n=24) were fed ad‐lib control and folate deficient (FD) diets for 14 d, followed by supplementation of each diet with 300mg/kg diet genistein for an additional 24 d. Hcy concentrations in the genistein supplemented FD animals (18.2 + 0.9μM) were reduced (24%; P = 0.01) relative to concentration in unsupplemented, FD rats (24.0 + 2.2μM). Analysis of other hepatic enzymes (i.e. GNMT, PEMT) indicate that the Hcy‐lowering effect of genistein does not reside in reduced production, but rather in increased metabolism.

Evidence for enhanced transsulphuration (TS) as a mechanism underlying lower hyperhomocysteinemia in patients undergoing home nocturnal hemodialysis (HNHD)

ObjectivesElevated plasma total homocysteine (tHcy) &gt;12μmol/L, an independent risk factor for cardiovascular disease, is prevalent (&gt;85%) in patients undergoing standard hemodialysis (SHD). The current study was conducted to determine if lower tHcy associated with longer, more frequent dialysis (HNHD) is a function of dialysate HCY losses and/or enhanced HCY metabolism through the TS pathway (cysteine (Cys) and sulphate production).MethodsA cross‐sectional study including patients (n=33) on SHD (4 h, 3 days/wk), in‐centre nocturnal hemodialysis (INHD) (6‐8 h, 3 nights/wk) and HNHD (6‐8 h, 5‐7 nights/wk). Blood and dialysate were collected during a mid‐week dialysis session.ConclusionsThese data suggest that increased dialysate removal of Hcy alone does not explain lower plasma tHcy in HNHD. Rather, increased clearance of sulfate and cysteine may allow increased TS in patients on HNHD. Supported by Kidney Foundation of Canada

Genetic polymorphisms involved in folate metabolism and concentrations of methylmalonic acid and folate on plasma homocysteine and risk of coronary artery disease

Alterations in the enzymes involved in homocysteine (Hcy) metabolism or vitamin deficiency could play a role in coronary artery disease (CAD) development. This study investigated the influence of MTHFR and MTR gene polymorphisms, plasma folate and MMA on Hcy concentrations and CAD development. MMA and folate concentrations were also investigated according to the polymorphisms. Two hundred and eighty-three unrelated Caucasian individuals undergoing coronary angiography (175 with CAD and 108 non-CAD) were assessed in a case-control study. Plasma Hcy and MMA were measured by liquid chromatography/tandem mass spectrometry. Plasma folate was measured by competitive immunoassay. Dietary intake was evaluated using a nutritional questionnaire. Polymorphisms MTHFR and MTR were investigated by polymerase chain reaction (PCR) followed by enzyme digestion or allele-specific PCR. Hcy mean concentrations were higher in CAD patients compared to controls, but below statistical significance (P = 0.246). Increased MMA mean concentrations were frequently observed in the CAD group (P = 0.048). Individuals with MMA concentrations >0.5 micromol/l (vitamin B(12) deficiency) were found only in the CAD group (P = 0.004). A positive correlation between MMA and Hcy mean concentrations was observed in both groups, CAD (P = 0.001) and non-CAD (P = 0.020). MMA mean concentrations were significantly higher in patients with hyperhomocysteinemia in both groups, CAD and non-CAD (P = 0.0063 and P = 0.013, respectively). Folate mean concentration was significantly lower in carriers of the wild-type MTHFR 1298AA genotype (P = 0.010). Our results suggest a correlation between the MTHFR A1298C polymorphism and plasma folate concentration. Vitamin B(12) deficiency, reflected by increased MMA concentration, is an important risk factor for the development both of hyperhomocysteinemia and CAD.

Genetic or nutritional disorders in homocysteine or folate metabolism increase proteinN‐homocysteinylation in mice

Genetic disorders of homocysteine (Hcy) or folate metabolism or high-methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy-thiolactone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine beta-synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (N-Hcy-protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how N-Hcy-protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma N-Hcy-protein was elevated 10-fold in mice fed a high-methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton-coupled folate transporter (Pcft) gene resulted in a 10- to 30-fold increase in plasma or serum N-Hcy-protein levels. Liver N-Hcy-protein was elevated 3.4-fold in severely and 11-fold in extremely hyperhomocysteinemic Cbs-deficient mice, 3.6-fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr-deficient animals, suggesting that mice have a capacity to prevent accumulation of N-Hcy-protein in their organs. These findings provide evidence that N-Hcy-protein is an important metabolite associated with Hcy pathophysiology in the mouse.

Regulation of homocysteine homeostasis through the transcriptional coactivator PGC-1α

Plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. Hcy is a nonprotein amino acid derivative that is generated from the methionine cycle, which provides the methyl group for essentially all biological methylation reactions. Although plasma Hcy levels are elevated in patients with cardiovascular disease, the mechanisms that regulate Hcy homeostasis remain poorly defined. In this study, we found that the expression of key enzymes involved in Hcy metabolism is induced in the liver in response to fasting. This induction coincides with increased expression of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha, a transcriptional coactivator that regulates hepatic gluconeogenesis and mitochondrial function. PGC-1alpha stimulates the expression of genes involved in Hcy metabolism in cultured primary hepatocytes as well as in the liver. Adenoviral-mediated expression of PGC-1alpha in vivo leads to elevated plasma Hcy levels. In contrast, mice deficient in PGC-1alpha have lower plasma Hcy concentrations. These results define a novel role for the PGC-1alpha coactivator pathway in the regulation of Hcy homeostasis and suggest a potential pathogenic mechanism that contributes to hyperhomocysteinemia.

A Critical Evaluation of the Correlation Between Biomarkers of Folate and Vitamin B12 in Nutritional Homocysteinemia

Folate and vitamin are essential cofactors for homocysteine (Hcy) metabolism. Homocysteinemia has been related with cardiovascular and neurodegenerative disease. We examined the effect of folate and/or vitamin deficiency on biomarkers of one carbon metabolism in blood, liver and brain, and analyzed the correlation between vitamin biomarkers in mild and moderate homocysteinemia. In this study, Sprague-Dawley male rats (5 groups, n = 10) were fed folatesufficient diet (FS), folate-deficient diet (FD) with 0 or 3 g homocystine (FSH and FDH), and folate-/vitamin -deficient diet with 3 g homocystine (FDHCD) for 8 weeks. The FDH diet induced mild homocysteinemia (plasma Hcy 17.41 1.94 nmol/mL) and the FDHCD diet induced moderate homocysteinemia (plasma Hcy 44.13 2.65 nmol/mL), respectively. Although liver and brain folate levels were significantly lower compared with those values of rats fed FS or FSH (p 9.6 nmol/L) compared with rats fed FD and FDH (21.1 1.4 nmol/L, 22.0 2.2 nmol/L)(p deficiency. Plasma Hcy was correlated with hepatic folate (r = -0.641, p deficiency. This result suggests that dietary folate- and vitamin B12-deficiency may inhibit methylation in brain by increasing SAH rather than decreasing SAM level, which may be closely associated with impaired cognitive function in nutritional homocysteinemia.

Variabilidade genética MTHFR no desenvolvimento da doença arterial coronária

Increased homocysteine (Hcy) concentration is considered a risk factor for coronary artery disease (CAD). Genetic alterations of the metylenetetrahydrofolate reductase (MTHFR) enzyme could reduce its thermolability and alter the Hcy metabolism, contributing to development of atherosclerotic lesions. Objective of this study was to investigate the relation between MTHFR C677T and A1298C polymorphisms and presence, extension, and severity of CAD. One hundred seventy-five patients with CAD confirmed by angiography, and 108 individuals without CAD (control group) were evaluated. MTHFR C677T polymorphism was investigated by polymerase chain reaction (PCR) followed by enzyme digestion. The genotyping of the MTHFR A1298C polymorphism was performed by PCR allele-specific method. Frequency of the altered allele MTHFR 677C was 0.38 in the CAD group and 0.37 in the control group. Regarding the polymorphic allele MTHFR 1298C, frequency was 0.22 and 0.27, respectively. The genotype distribution MTHFR C677T and A1298C did not differ regarding number of affected vessels (P > 0.05). Also, relation between MTHFR C677T polymorphism and degree of arterial obstruction was not observed (P > 0.05), as well as the MTHFR A1298C polymorphism (P > 0.05). Results did not show association between MTHFR A1298C and MTHFR C677T polymorphisms and presence, extension or severity of CAD.

Plasma homocysteine levels in young male patients in the exacerbation and remission phase of schizophrenia

High levels of homocysteine (Hcy) were suggested to contribute to the pathogenesis of schizophrenia. Recent investigations have shown that treatment with folic acid, vitamin B-12 and pyridoxine are effective in reducing Hcy levels while concomitantly reducing the score of positive and negative symptoms in schizophrenic patients. In addition to the availability of nutrients (mainly folate, vitamins B6 and B12), plasma Hcy concentrations are dependent on complex metabolic regulation that could be disrupted in schizophrenia. This study was designed to test the influence of disease activity on plasma Hcy levels. Plasma Hcy concentrations were measured in male chronic schizophrenic patients with a predominantly positive (SCH (+)) or predominantly negative (SCH (−)) syndrome in schizophrenia immediately upon admission to the hospital (exacerbation phase) and one month later (remission phase). During this period patients received antipsychotic medications without vitamin therapy. The effects of age, duration of illness, folate and B12 concentrations, as well as smoking and coffee consumption habits on the observed changes were evaluated. Age- and sex-matched subjects were included in the control group. In the control group plasma Hcy concentration was 8.75 ± 1.84 μmol/L. In the exacerbation phase plasma Hcy concentrations were significantly increased both in SCH (+) (14.91 ± 6.19 μmol/L) and SCH (−) groups (12.8 ± 3.27 μmol/L). There was no difference in plasma Hcy concentrations between SCH (+) and SCH (−) patients. Serum folate and B12 concentrations were not significantly different in any of the investigated groups of subjects. The plasma Hcy concentrations could not be correlated with age, duration of illness, the score of positive symptoms or the concentration of folate and vitamin B12. A positive correlation was found between plasma Hcy level and score of negative symptoms in both groups of patients. No correlation was found between smoking or coffee consumption habits and plasma Hcy concentrations. All patients exhibited decreased plasma Hcy levels in the remission phase of the illness, with a mean decrease of 2.68 ± 1.57 μmol/L. Folate and B12 levels did not differ in the exacerbation and remission phases of the illness. The significant decrease of plasma Hcy levels, without changes in folate and vitamin B12 concentrations in the remission phase of schizophrenia, could indicate an influence of a pathogenetic process involved in schizophrenia on Hcy metabolism.

Mutations in cystathionine β‐synthase or methylenetetrahydrofolate reductase gene increaseN‐homocysteinylated protein levels in humans

Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also show that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients.

Homocysteine modulates the effect of simvastatin on expression of ApoA-I and NF- B/iNOS

Statins can ameliorate atherosclerosis by inhibition of cholesterol biosynthesis or by modulation of inflammation. In earlier work, we showed that homocysteine (Hcy) reduced synthesis of apolipoprotein A-I (ApoA-I). Our goal in this study was to determine whether Hcy could interfere with the ability of simvastatin to increase ApoA-I synthesis or to modify statin-dependent regulation of inflammatory factors. Human HepG2 hepatocarcinoma cells and murine RAW264.7 macrophages were treated with simvastatin, with and without Hcy, to examine the expression of ApoA-I and nuclear factor-kappaB (NF-kappaB) or the NF-kappaB target, inducible nitric-oxide synthase (iNOS), respectively. Mice with methylenetetrahydrofolate reductase (Mthfr) deficiency, an animal model of hyperhomocysteinemia, were administered simvastatin (in diets or by injection) for in vivo assessment of these interactions. In HepG2 cells, Hcy reduced the statin-dependent increases in ApoA-I protein, mRNA, and ApoA-I promoter activity. In RAW264.7 macrophages, simvastatin decreased, whereas Hcy increased, the expression of pro-inflammatory NF-kappaB protein; in the presence of both Hcy and simvastatin, the pro-inflammatory effect of Hcy prevailed. Hcy increased mRNA levels of iNOS in the macrophage line; the combination of Hcy and simvastatin resulted in a trend towards greater induction. In mouse studies, simvastatin decreased cholesterol levels, but levels of ApoA-I in Mthfr-deficient mice remained lower than those in Mthfr(+/+) mice. Simvastatin injection increased iNOS protein and mRNA levels in peripheral blood of hyperhomocysteinemic Mthfr-deficient mice, but not in Mthfr(+/+) mice. The drug also increased MTHFR protein in cells and mouse liver, an effect that was modified by Hcy. These findings provide a link between statins and folate-dependent Hcy metabolism, and suggest that Hcy interferes with some anti-atherogenic and anti-inflammatory properties of simvastatin. Our work may have clinical relevance for hyperhomocysteinemic individuals on statin therapy.

P4-181: Homocysteine, S-adenosylmethionine/s-adenosylhomocysteine ratio, glutathione and nitric oxide levels in TgCRDND8 mice fed with B vitamin–deficient diet

Increasing evidence indicates that oxidative and nitrosative stress, impaired glutathione (GSH) and homocysteine (Hcy) metabolism can interact in a vicious cycle, which is central to AD pathogenesis. Synthesis of GSH is regulated by cysteine, which is synthesized from Hcy via the transsulfuration pathway. Deficiencies of folate, vitamins B12 and B6 are important for S-adenosylmethionine (SAM)/Hcy metabolism alterations, leading to hyperhomocysteinemia and decrease of SAM/SAH ratio, known as “methylation potential” (MP). Nitric oxide (NO) is involved in physiological functions and in pathological processes leading to tissue damage due to its free radical nature. The production of NO is strongly related to Hcy metabolism, through the regulation of ADMA (asymmetric dymethyl arginine) levels. We already showed, in vitro, that SAM administration can regulate PS1 and BACE expression and β-amyloid production, and in vivo that inhibition of methylation reaction by diet induced hyperhomocysteinemia is able to up-regulate PS1 and BACE. Thus, it was intriguing to study the relationships between Hcy, SAM/SAH ratio, GSH/GSSG and NO levels to understand the link between aberration in redox homeostasis and methylation potential in AD models. We used transgenic mice that have an accelerated amyloid accumulation since they are a good model to study the metabolic alterations caused by B vitamins deprivations in AD. TgCRND8 mice together with wild type mice were fed with a deprived diet (without folate, vitamin B12 and B6) or with a control diet. Then, they were sacrificed to analyze Hcy, SAM, SAH, GSH and NO in plasma and tissues by Imx, HPLC and colorimetric assay kits. The deficient diet lead to a marked hyperhomocysteinemia, to a decrease in SAM/SAH ratio in plasma and brain, to an increase of GSH/GSSG ratio in TgCRND8 brain probably due to the attempt to compensate for the increased oxidative damage resulting from amyloid β. There is also a brain NO decrease in diet B in TgCRND8 mice, while there are no significant differences in plasma. Additional experiments with SAM administration in condition of hyperhomocysteinemia are actually in progress, to test the effect of SAM in GSH and NO production, and amyloid production.

Is there a relationship between homocysteine and vitiligo? A pilot study

Pigmentary dilution is observed in patients with homocystinuria. Therefore, it is possible that an increase of local homocysteine (Hcy) interferes with normal melanogenesis and plays a role in the pathogenesis of vitiligo. Vitamin B12 and folic acid, levels of which are decreased in vitiligo, are important cofactors in the metabolism of Hcy. Consequently, a nutritional deficiency in either of these two vitamins will result in an increase in homocysteine in the circulation, a finding that we expect to find in vitiligo. To determine the level of Hcy in the blood of patients with vitiligo as a first step in revealing if it has any relationship with the pathogenesis of vitiligo and consequently if this will have an impact on the treatment of vitiligo. Twenty-six patients of both sexes with vitiligo (age range 20-50 years, mean 31.4 +/- 8.09) and 26 age-matched healthy controls were included in the study. After excluding factors that may affect serum Hcy levels, blood samples from patients and controls were obtained for homocysteine determination by enzyme immunoassay. The mean serum level of Hcy was significantly higher in patients with vitiligo than in controls (21.61 +/- 13.28 vs. 13.1 +/- 4.88 micromol L(-1); P < 0.001). The Hcy level was significantly higher in male patients than in female patients (28.67 +/- 15.95 vs. 15.56 +/- 6.2 micromol L(-1); P < 0.001) and in male controls compared with female controls (15.07 +/- 4.61 vs. 12.05 +/- 4.82 micromol L(-1); P < 0.001). The homocysteine level was related to the activity of vitiligo and was significantly higher in patients with progressive disease than in controls (25.4 +/- 14.99 vs. 13.1 +/- 4.88 micromol L(-1); P < 0.001). No significant difference in Hcy levels was found between either untreated vitiligo patients (22.77 +/- 13.36 micromol L(-1)) or patients receiving ultraviolet therapy (20.45 +/- 13.73 micromol L(-1)) and the total patient group (21.62 +/- 13.28 micromol L(-1)). An elevated homocysteine level may be a precipitating factor for vitiligo in predisposed individuals. In view of the biological role of vitamin B(12) and folic acid in Hcy metabolism, we present our recommendations regarding the investigation and treatment of this common disease.