Investigation of vitamin B6 inadequacy, induced by exposure to the anti-B6 factor 1-amino D-proline, on plasma lipophilic metabolites of rats: a metabolomics approach.

PURPOSE: Vitamin B6 status in the body is affected by several factors including dietary supply of the antivitamin B6 factor, 1-amino D-proline (1ADP), which is present in flaxseed. Owing to the prevalence of moderate B6 deficiency in the general population, a co-occurrence of 1ADP may lead to a further deterioration of B6 status. To this end, we applied a nontargeted metabolomics approach to identify potential plasma lipophilic biomarkers of deleterious effect of 1ADP on moderately vitamin B6-deficient rats using a high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. METHODS: Twenty-four rats were fed with a semi-purified diet containing pyridoxine·HCl (PN·HCl) either 7 mg/kg diet (optimal B6) or 0.7 mg/kg diet (moderate B6). The rats were divided into four treatments (n = 6), and one treatment in each B6 diet group was also fed ad libitum with 10 mg/kg diet of synthetic 1ADP. After 5 weeks of study, plasma was collected from the rats and lipophilic metabolites were extracted using acetonitrile as a solvent for analysis. RESULTS: Ten potential plasma lipophilic biomarkers were identified out of >2500 detected entities, which showed significant differences between the treatments. Plasma glycocholic acid, glycoursodeoxycholic acid, murocholic acid, N-docosahexaenoyl GABA, N-arachidonoyl GABA, lumula, nandrolone and orthothymotinic acid concentrations were significantly elevated, while plasma cystamine and 3-methyleneoxindole concentrations were significantly reduced as a result of either low B6 status or 1ADP or their interaction. CONCLUSION: Changes in these metabolites revealed a potential defect in pathways linked with the biosynthesis and metabolism of bile acid components, N-acyl amino acids, analgesic androgens, anti-inflammatory and neuroprotective molecules. We also noted that the changes in these biomarkers can be alleviated by the application of adequate vitamin B6.

Plasma homocysteine is adversely associated with glomerular filtration rate in asymptomatic black and white young adults: the Bogalusa heart study.

That plasma homocysteine is elevated markedly in renal dysfunction is well recognized. But whether the increased homocysteine is an independent correlate of glomerular filtration rate, a marker of renal function, in asymptomatic younger individuals is not clear. The aim of this study was to determine the association between plasma homocysteine and renal function in a biracial (black-white) community-based cohort of asymptomatic young adults. Plasma homocysteine along with cardiovascular disease risk factor variables were measured in 805 white and 330 black subjects, ages 24-44 years, enrolled in the Bogalusa Heart Study. Modification of Diet in Renal Disease Study equation was used to calculate the estimated glomerular filtration rate (eGFR) from serum creatinine level. Males versus females showed higher homocysteine levels (8.83 +/- 3.16 vs. 7.35 +/- 2.84 micromol/L, p < 0.0001) and lower eGFR (99.1 +/- 17.6 vs. 102.5 +/- 21.0 mL/min/1.73 m(2), p = 0.024). Whites versus blacks had lower eGFR (97.3 +/- 18.0 vs. 110.0 +/- 20.6 mL/min/1.73 m(2), p < 0.0001). In a multivariate regression analysis that included age, race, sex, body mass index, blood pressure, lipoprotein variables, insulin resistance index and homocysteine, white race, age and homocysteine, in that order, were independently and negatively associated with eGFR. The odds ratio (95% confidence interval) of individuals in the homocysteine quintiles II, III, IV and V vs. I for having the risk of impaired eGFR defined as <10th percentile was 2.28 (0.95-5.50, p = 0.065), 2.97 (1.24-7.12, p = 0.015), 3.32 (1.45-7.60, p = 0.005) and 6.99 (3.06-15.94, p < 0.0001), respectively. Homocysteine is an independent correlate of renal function in asymptomatic black and white young adults.

No association between global leukocyte DNA methylation and homocysteine levels in schizophrenia patients.

Meta-analysis recently suggested that a 5 muM increase in homocysteine is associated with a 70% higher risk for schizophrenia. Elevated homocysteine is reported to alter macromolecule methylation. We studied whether elevated plasma homocysteine levels in schizophrenia are associated with altered leukocyte global DNA methylation. DNA was extracted from peripheral blood leukocytes of 28 schizophrenia patients vs. 26 matched healthy controls. Percent of global genome DNA methylation was measured using the cytosine-extension method. Homocysteine levels were higher in schizophrenia patients than in controls. No difference in global DNA methylation between schizophrenia patients and control subjects was found (74.0%+/-14.8 vs. 69.4+/-22.0, p=0.31). A significant interaction between diagnosis and smoking on DNA methylation was obtained (F=6.8, df=1,47, p=0.032). Although leukocytes may be a useful cell model to evaluate epigenetic changes such as global DNA methylation in brain, future studies should compare global DNA methylation in peripheral tissue vs. brain in laboratory animals.

Methylenetetrahydrofolate reductase gene polymorphism and its association with coronary artery disease.

CONTEXT AND OBJECTIVE: Obstructive coronary artery disease (CAD) is characterized by the deposition of atherosclerotic plaque on the coronary artery wall. Its manifestations depend on interactions between environmental and genetic risk factors. The aim of this work was to analyze the frequency of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in patients with CAD and its association with plasma homocysteine levels. Risk factors for CAD were also evaluated. DESIGN AND SETTING: Retrospective with blind quantitative analysis, at Hospital de Base, Faculdade de Medicina de São José do Rio Preto. METHODS: One hundred and twenty-seven individuals were studied. All completed a questionnaire to analyze risk factors for CAD. MTHFR polymorphism was investigated by restriction fragment length analysis and correlated with the number of affected arteries and degree of arterial obstruction determined by coronary cineangiography, and with plasma homocysteine levels measured by liquid chromatography/sequential mass spectrometry. RESULTS: Smoking (p = 0.02) and high-density lipoprotein cholesterol (p = 0.01) were associated with CAD. The C allele was the most prevalent in patients (0.61) and controls (0.66). There was no correlation between MTHFR/C677T polymorphism and plasma homocysteine levels. However, in patients with the TT genotype there was a correlation with the prevalence of coronary obstruction greater than 95% (p = 0.02) and the presence of two affected arteries (p = 0.04). CONCLUSIONS: The TT genotype is associated with coronary artery obstruction greater than 95% and the presence of two affected arteries. This confirms the relationship between genetic variants in specific patient subgroups and cardiovascular diseases.

MTHFR 677T is a strong determinant of the degree of hearing loss among Polish males with postlingual sensorineural hearing impairment.

Hearing impairment (HI) is the most common sensory handicap. Congenital HI often has a genetic basis, whereas the etiology of nonsyndromic postlingual HI (npHI) usually remains unidentified. Our purpose was to test whether the MTHFR C677T (rs1801133) polymorphism affecting folate metabolism is associated with the occurrence or severity of npHI. We studied rs1801133 genotypes in 647 npHI patients (age <40, sudden sensorineural loss excluded, HI characterized as mean of better ear hearing thresholds for 0.5-8 kHz) and 3273 adult controls from the background population. Genotype distribution among patients and controls was similar, but among male cases (n = 302) we found a dose-dependent correlation of MTHFR 677T with the degree of HI (mean thresholds in dB: 38.8, 44.9, and 53.3, for CC, CT, and TT genotypes, respectively; p = 0.0013, p(cor.) = 0.017). Among male patients rs1801133 TT significantly increased the risk of severe/profound HI (odds ratio = 4.88, p = 0.001). Among controls the known effect of MTHFR 677T on plasma total homocysteine was more pronounced in men than in women (p<0.00004 for genotype-sex interaction) suggesting that in Poland folate deficiency is more prevalent in males. In conclusion, we report a novel strong effect of MTHFR 677T among males with npHI. The functional significance of rs1801133 suggests that these patients may benefit from folate supplementation-an intervention which is simple, cheap, and devoid of side effects.

A novel serum metabolomics-based diagnostic approach for colorectal cancer.

BACKGROUND: To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer. METHODOLOGY/PRINCIPAL FINDINGS: We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0-2 colorectal cancer (82.8%). CONCLUSIONS/SIGNIFICANCE: Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer.

The role of physical activity level and B-vitamin status on blood homocysteine levels.

UNLABELLED: Exercise increases methionine metabolism, which also increases its amino acid metabolic intermediate, homocysteine (Hcy). High Hcy levels increase cardiovascular disease (CVD) risk, whereas B-vitamins (folate, vitamins B6, and B12) can reduce Hcy. Research exploring the relationship between exercise and Hcy is equivocal. PURPOSE: To determine whether plasma Hcy values, independent of plasma B-vitamin concentrations, are higher in active (HighPA; > 420 min x wk(-1)) than less active (LowPA; < or = 420 min x wk(-1)) males (M = 38) and females (F = 38). METHODS: Subjects were healthy, young (26 +/- 5 yr), used no B-vitamin supplements in last 30 d, and reported being physically active for the last 5 yr. Physical activity (PA) groups were based on moderate- to high-intensity PA (min x wk(-1)) using 7-d PA records. Dietary intakes of B-vitamins were assessed using 7-d weighed food records. The differences of Hcy between PA and gender were examined using ANCOVA, with plasma B-vitamins as covariates. RESULTS: Mean PA was 220 min x wk(-1) for LowPA (n = 36; VO2max = 42.8 +/- 8.8 mL x kg(-1) x min(-1)) and 652 min x wk(-1) for HighPA (n = 40; VO2max = 54.2 +/- 9.7 mL x kg(-1) x min(-1)). Hcy (micromol x L(-1)) was not different between PA levels (LowPA = 7.5 +/- 1.6; HighPA = 7.7 +/- 1.6, P = 0.36) or sex (M = 7.8 +/- 1.7; F = 7.4 +/- 1.1; P = 0.13). Plasma folate was the only significant covariate (P<0.001). However, secondary analysis revealed that Hcy levels were significantly higher in the most active and fit (ExHighPA; range = 758-1085 min x wk(-1); n = 11; > 90% VO2max) compared with the sedentary ones (ExLowPA; range = 9-130 min x wk(-1); n = 9; < 70% VO2max; 8.6 +/- 1.8 vs 6.7 +/- 1.5 micromol x L(-1); P = 0.007, respectively). CONCLUSION: Hcy, independent of plasma B-vitamin levels, was not different between PA levels in nonsupplementing young adults, unless PA was high (> 758 min x wk(-1)).

Hyperhomocysteinemia and low plasma folate as risk factors for central retinal vein occlusion: a case-control study in a Chinese population.

PURPOSE: To determine whether hyperhomocysteinemia and low plasma folate are risk factors for central retinal vein occlusion (CRVO) in the Chinese population. METHODS: A matched case-control study was conducted between July 2004 and May 2005. The study cohort consisted of 64 individuals that had been diagnosed to have CRVO and 64 normal controls (matched for age, gender, hypertension, smoking and drinking habits). None of the cases or controls had a history of diabetes, glaucoma, medication or any other vascular events that might minimize the influence on plasma homocysteine levels. A cross-sectional analysis among the 64 cases was performed to compare the prevalence of hyperhomocysteinemia and low plasma folate among subjects with and without ischemia and subjects with age above 45 and below 45 years. Plasma homocysteine level was measured by means of high-performance liquid chromatography and plasma folate concentration by radioimmunoassay. RESULTS: The CRVO patients had a significantly higher homocysteine level (13.83+/-1.71 micromol/l) than the normal controls (8.05+/-0.58 micromol/l; p=0.003). The plasma folate levels were significantly lower in CRVO patients than in controls (5.62+/-0.39 ng/dl vs 7.23+/-0.60 ng/dl; p=0.032). A 1 micromol/l increase of plasma homocysteine level was associated with an odds ratio of 1.368. Hyperhomocysteinemia was defined as a homocysteine level of >14.97 micromol/l and was seen in 11 patients in the ischemic group, significantly more often than in the non-ischemic group (5 patients; p=0.030). CONCLUSIONS: The results suggest that hyperhomocysteinemia and low plasma folate are independent risk factors for CRVO and are associated with the development of CRVO in the Chinese population.

Treatment of YAC128 mice and their wild-type littermates with cystamine does not lead to its accumulation in plasma or brain: implications for the treatment of Huntington disease.

Cystamine is beneficial to Huntington disease (HD) transgenic mice. To elucidate the mechanism, cystamine metabolites were determined in brain and plasma of cystamine-treated mice. A major route for cystamine metabolism is thought to be: cystamine --> cysteamine --> hypotaurine --> taurine. Here we describe an HPLC system with coulometric detection that can rapidly measure underivatized cystamine, cysteamine and hypotaurine, as well as cysteine and glutathione in the same deproteinized tissue sample. A method is also described for the coulometric estimation of taurine as its isoindole-sulfonate derivative. Using this new methodology we showed that cystamine and cysteamine are undetectable (< or = 0.2 nmol/100 mg protein) in the brains of 3-month-old HD transgenic (YAC128) mice (or their wild-type littermates) treated daily for 2 weeks with cystamine (225 mg/kg) in their drinking water. No significant changes were observed in brain glutathione and taurine but significant increases were observed in brain cysteine. Cystamine and cysteamine were not detected in the plasma of YAC128 mice treated daily with cystamine between the ages of 4 and 12 or 7 and 12 months. These findings suggest that cystamine is not directly involved in mitigating HD but that increased brain cysteine or uncharacterized sulfur metabolites may be responsible.

Disulphide reduction in glutathione-deficient erythrocytes from a patient with pyroglutamic acidemia.

Erythrocytes from a patient with pyroglutamic acidemia, containing about 5% of the normal content of glutathione, were able to reduce cystamine at a relatively high rate (about 50% that of normal cells) at low concentrations of the disulphide. Compared to normal cells, the reduction of disulphide by the patient's cells were highly sensitive to inhibition by high concentrations of the disulphide. This inhibition was noted whether glucose or inosine were used as carbohydrate substrate, and was also present when hemolysates were used and NADPH was added directly to the reaction mixture. The "disulphide poisoning" is assumed to be due to the formation of mixed disulphides at the expense of oxidized glutathione available for reduction by glutathione reductase. The glutathione/glutathione reductase system is probably the only disulphide reducing system of importance in human erythrocytes.

Replacement therapy for vitamin B12 deficiency: comparison between the sublingual and oral route.

AIMS: To compare the efficacy of sublingual and oral administration of 500 micro g of cobalamin in subjects with cobalamin deficiency. MATERIALS AND RESULTS: Thirty subjects with low serum concentrations of cobalamin participated in the study. Subjects were randomly allocated to receive one tablet daily of 500 micro g cobalamin sublingually or orally, or two tablets daily of a vitamin B complex. Serum cobalamin concentrations before treatment were 94 +/- 30 pmol l-1, 108 +/- 17 pmol l-1 and 98 +/- 14 pmol l-1 in the sublingual B12, oral B12 and oral B-complex groups, respectively. After 4 weeks, concentrations rose to 288 +/- 74 pmol l-1, 286 +/- 87 pmol l-1 and 293 +/- 78 pmol l-1, respectively. The increase in each group across time was statistically significant (P = 0.0001, differences [95% confidence intervals] 194.2 (114.5, 273.9), 178.3 (104.2, 252.4), and 195.1 (135.0, 255.2) pmol l-1, respectively). There was no significant difference in concentrations between the treatment groups. CONCLUSION: A dose of 500 micro g of cobalamin given either sublingually or orally is effective in correcting cobalamin deficiency.

[Quantitative determination and reversible modification of sulfhydryl groups in low and high molecular weight compounds using a biradical spin marker]. / Kolichestvennoe opredelenie i obratimaia modifikatsiia sul'fgidril'nykhgrupp nizko- i vysokomolekuliarnykh soedinenii s ispol'zovaniem biradikal'noi spinovoi metki.

A new method for quantitation of sulfhydryl groups of low and high molecular weight compounds is proposed. The method is based on the use of a biradical spin label carrying a disulfide bond, RS-SR, where R is the imidazoline radical. It was found that this biradical is involved in the reaction of thiol-disulfide exchange with thiols; the EPR spectra of the original biradical and monoradical products differ essentially. This circumstance made it possible to determine the bimolecular rate constant for the biradical interaction with cysteamine, cysteine, glutathione and human serum albumin. The method was used for measuring glutathione and cysteine levels in murine and rat blood and for assaying the insect acetylcholine esterase activity and reversible inhibition of NADPH-cytochrome P-450 reductase. The method is marked for a high sensitivity (10(-6)-10(-7) M) towards sulfhydryl groups and allows the determination of thiol groups in coloured and nontransparent solutions.

Methylenetetrahydrofolate reductase gene polymorphism and its association with coronary artery disease

CONTEXT AND OBJECTIVE: Obstructive coronary artery disease (CAD) is characterized by the deposition of atherosclerotic plaque on the coronary artery wall. Its manifestations depend on interactions between environmental and genetic risk factors. The aim of this work was to analyze the frequency of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in patients with CAD and its association with plasma homocysteine levels. Risk factors for CAD were also evaluated. DESIGN AND SETTING: Retrospective with blind quantitative analysis, at Hospital de Base, Faculdade de Medicina de São José do Rio Preto. METHODS: One hundred and twenty-seven individuals were studied. All completed a questionnaire to analyze risk factors for CAD. MTHFR polymorphism was investigated by restriction fragment length analysis and correlated with the number of affected arteries and degree of arterial obstruction determined by coronary cineangiography, and with plasma homocysteine levels measured by liquid chromatography/sequential mass spectrometry. RESULTS: Smoking (p = 0.02) and high-density lipoprotein cholesterol (p = 0.01) were associated with CAD. The C allele was the most prevalent in patients (0.61) and controls (0.66). There was no correlation between MTHFR/C677T polymorphism and plasma homocysteine levels. However, in patients with the TT genotype there was a correlation with the prevalence of coronary obstruction greater than 95 percent (p = 0.02) and the presence of two affected arteries (p = 0.04). CONCLUSIONS: The TT genotype is associated with coronary artery obstruction greater than 95 percent and the presence of two affected arteries. This confirms the relationship between genetic variants in specific patient subgroups and cardiovascular diseases.

CONTEXTO E OBJETIVO: A doença arterial coronariana (DAC) caracteriza-se pelo depósito de placa aterosclerótica na parede arterial coronária. Sua manifestação é dependente da interação entre fatores de risco ambientais e genéticos. O objetivo deste trabalho é analisar a freqüência do polimorfismo MTHFR/C677T em pacientes com doença arterial coronária e sua associação com o nível de Hcy plasmática. Fatores de risco para DAC também foram avaliados. TIPO DE ESTUDO: Retrospectivo com análise cega quantitativa, no Hospital de Base, Faculdade de Medicina de São José do Rio Preto. MÉTODOS: Foram estudados 127 indivíduos. Todos responderam a um questionário para análise dos fatores de risco para DAC. O polimorfismo MTHFR/C677T, investigado por análise de comprimento de fragmentos de restrição, foi correlacionado com número de artérias afetadas e grau de obstrução arterial, determinadas pela cinangiocoronariografia, e com o nível de Hcy plasmática. RESULTADOS: Tabagismo (p = 0,02) and HDLc (p = 0,01) foram associados com DAC. O alelo C foi o mais prevalente em pacientes (0,61) e controles (0,66; p = 0,49). O polimorfismo MTHFR/C677T não apresentou associação com níveis de Hcy plasmática. Entretanto, nos pacientes com genótipo TT observou-se a prevalência de obstrução coronariana acima de 95 por cento (p = 0,02) e a presença de duas artérias lesadas (p = 0,04). CONCLUSÕES: Associou-se o genótipo TT com o grau de obstrução arterial coronária acima de 95 por cento e a presença de duas artérias lesadas; confirma-se, assim, a relação de variantes genéticas em subgrupos específicos de pacientes com doenças cardiovasculares.