Mohammad Azam Mansoor

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma.

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 microM dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (less than 2 pmol) to detect the various forms of the four thiol compounds in human plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.

Determination of reduced, oxidized, and protein-bound glutathione in human plasma with precolumn derivatization with monobromobimane and liquid chromatography

This assay measures reduced (GSH), oxidized (GSSG, GSSR), and protein-bound (glutathione-protein mixed disulfides, ProSSG) glutathione in human plasma. Oxidized glutathione and ProSSG are converted to GSH in the presence of NaBH4, and, after precolumn derivatization with monobromobimane, GSH is quantitated by reversed-phase liquid chromatography and fluorescence detection. The NaBH4 concentration is optimized so that total recovery of oxidized glutathione is obtained and no interference with the formation/stability of the GSH-bimane adduct occurs. The presence of 50 microM dithioerythritol prevents reduced recovery at low concentrations of GSH, and the standard curve for GSH is linear over a wide concentration range and is super-imposed upon that obtained with GSSG. Selective determination of oxidized glutathione exploits the fact that N-ethylmaleimide (NEM) blocks free sulfhydryl groups and excess NEM is inactivated by the subsequent addition of NaBH4. To measure total glutathione including the protein-bound forms, the protein is solubilized with dimethyl sulfoxide, which is compatible with the other reagents and slightly increases the yield of the fluorescent GSH derivative. The assay is characterized by a sensitivity (less than 2 pmol) sufficiently high to detect the various forms of glutathione in plasma, by an analytical recovery of GSH and GSSG close to 100%, and by a within-day precision corresponding to a coefficient of variation of 7%. The assay was used to determine the dynamic relationships among various glutathione species in human plasma.

Plasma total homocysteine in a representative sample of 972 British men and women aged 65 and over.

Objectives: To provide a reference range for plasma total homocysteine (tHcy), an independent risk factor for vascular disease, and to explore relationships with nutritional indices for people aged 65 y and over, in the UK National Diet and Nutrition Survey (NDNS). Design: The survey procedures described in the National Diet and Nutrition Survey Report (1997) included a health-and-lifestyle interview, a four-day weighed diet record, anthropometric and blood pressure measurements and a fasting blood sample for biochemical indices, including tHcy. Setting: Eighty randomly selected postcode sectors from mainland Britain during 1995–1996. Subjects: Of 2060 people interviewed, 1527 were visited by the nurse, 1276 gave a blood sample and 972 had tHcy measured. About 80% were in their own homes and the remainder were in nursing homes or similar institutions. Results: Significant cross-sectional relationships, both univariate and multivariate were found between tHcy and index concentrations of folate and vitamin B12 (P<0.0001), and between tHcy and plasma creatinine, urea, calcium, zinc, α1-antichymotrypsin, lutein and cysteine (P=0.013 to <0.0001). Dietary nutrient analyses showed an association with folate intake. tHcy was also correlated with age and with domicile (free-living or institution), with history of vascular disease and with use of four classes of drugs, two of which are prescribed for vascular diseases. There was a north-south gradient in tHcy (P=0.005), and also in food choices, blood micronutrient indices and vascular disease prevalence. Conclusions: The concentrations of tHcy found in this study provide a reference range for people aged 65 y and over, in mainland Britain. tHcy is a valuable functional index of micronutrient status and intakes for British people aged 65 y and over, which can assist the development of health-promotion strategies. Sponsorship: This study was part of the National Diet and Nutrition Survey of people aged 65 y and over, which was funded jointly by the Ministry of Agriculture, Fisheries and Food and the Department of Health, and conducted by Social Community Planning and Research in conjunction with the MRC Dunn Nutrition Unit. The authors acknowledge financial support for the study from the Department of Health, the European Commission and British Heart Foundation.

Plasma pyridoxal phosphate and pyridoxic acid and their relationship to plasma homocysteine in a representative sample of British men and women aged 65 years and over

Concentrations of pyridoxal phosphate and pyridoxic acid were measured in fasting plasma samples from British men and women aged 65 years and over, participating in a National Diet and Nutrition Survey during 1994-5, selected to be representative of the population of mainland Britain. In this population, the concentration of pyridoxal phosphate declined, whereas pyridoxic acid rose, with increasing age and frailty; however, both status indicators were strongly and directly (with a positive coefficient) correlated with estimates of vitamin B6 intake. This was little affected by the inclusion of food energy and protein intakes in the model. Forty-eight percent of the participants living in the community and 75% of those living in institutions had plasma pyridoxal phosphate concentrations below a range considered normal from other studies. In a univariate regression model, plasma pyridoxal phosphate concentrations were inversely correlated with plasma homocysteine concentrations, consistent with the hypothesis that vitamin B6 status may influence plasma homocysteine levels, and hence vascular disease risk. However, this relationship was partly attenuated in a multiple regression model including age, sex, domicile and biochemical status indices, including those of folate and vitamin B12. There was evidence that plasma pyridoxal phosphate was sensitive to metabolic conditions associated with inflammation and the acute-phase reaction, and that plasma pyridoxic acid was sensitive to renal function. Thus, neither index is an ideal predictor of vitamin B6 status in older people, unless these confounding factors are allowed for. Since poor vitamin B6 status may have health implications, e.g. for immune function, cognition, and for essential intermediary metabolic pathways in older people, it needs to be investigated as a possible public health problem.

Redox status and protein binding of plasma homocysteine and other aminothiols in patients with homocystinuria.

Elevations of homocyst(e)ine levels in the blood of patients with homocystinuria may explain the high cardiovascular morbidity. We determined levels of reduced, oxidized, and protein-bound homocyst(e)ine, cyst(e)ine, and cyst(e)inylglycine in plasma from eight patients with homocystinuria. The technique used involved trapping of reduced thiols by collecting blood directly into tubes containing sulfhydryl-reactive reagents. All patients had high levels of homocysteine (range, 1.9 to 91.2 mumol/L), and among the aminothiols investigated, this species showed the most drastic elevation compared with trace levels (< 0.4 mumol/L) found in healthy subjects. The ratio between free homocysteine and total homocyst(e)ine (reduced to total ratio) was above normal and positively correlated to the reduced to total ratio for cyst(e)ine, suggesting that an equilibrium exists between these species through sulfhydryl disulfide exchange. The other homocyst(e)ine species (oxidized and protein-bound) were also markedly increased in patients with homocystinuria. Plasma cysteine and cysteinylglycine levels were moderately increased, whereas plasma concentrations of protein-bound cyst(e)ine, protein-bound cyst(e)inylglycine, and free cystine were below normal. Homocysteine in particular and other homocyst(e)ine species are markedly increased in plasma of homocystinurics, and these changes are associated with pronounced alterations in the level and the redox status of other aminothiols. This should be taken into account when considering homocyst(e)ine as an atherogenic agent, and the role of various homocyst(e)ine species in the pathogenesis of homocystinuria.

Correlates of plasma homocysteine, cysteine and cysteinyl-glycine in respondents in the British National Diet and Nutrition Survey of young people aged 4-18 years, and a comparison with the survey of people aged 65 years and over.

Plasma total homocysteine (tHcy), cysteine and cysteinyl-glycine were measured in a representative sample of 922 young people aged 4-18 years, participating in the National Diet and Nutrition Survey in mainland Britain in 1997. Both tHcy and cysteine increased markedly with age; cysteinyl-glycine less so. Neither tHcy nor cysteine differed between genders; cysteinyl-glycine was higher in males. tHcy concentrations were lowest in the winter; cysteine and cysteinyl-glycine varied only slightly with season. In respondents aged >15 years, tHcy was higher in smokers, but in respondents aged 7-11 years, tHcy was higher in those whose mothers smoked. tHcy was inversely correlated with serum folate, serum vitamin B12 and vitamin B6 status, but neither cysteine nor cysteinyl-glycine shared these relationships. The relationships between tHcy and B-vitamin status indices ran parallel with those of the 65 years and over survey, but at much lower tHcy concentrations for any given B-vitamin concentration. Age-adjusted tHcy was not correlated with anthropometric indices, blood pressure, haematology, plasma creatinine, urea or cholesterol, but was directly correlated with fasting triacylglycerol. We conclude that disease-risk indices, like tHcy and perhaps cysteine, if established during early life, may be modulated by diet and lifestyle, thereby providing an opportunity for public health intervention.

Hyperhomocysteinemia in patients operated for lower extremity ischaemia below the age of 50—effect of smoking and extent of disease

Moderate hyperhomocysteinemia may be a risk factor for atherosclerotic peripheral vascular disease (PVD). In order to develop PVD at an early age risk factors are more strongly expressed and hyperhomocysteinemia may be one such factor. Homocysteine is derived from methionine and is metabolised by cystathionine-synthase to cystathionine or remethylated to methionine. Cystathionine-synthase activity is dependent on vitamin B6 while the remethylation of homocysteine is dependent on vitamin B12 and folate. The present study analyses homocysteine in patients operated on for lower extremity ischaemia before the age of 50. Homocysteine before and after loading with methionine, vitamin B6, B12 and folate were measured at follow-up. The patients were compared to age- and sex-matched controls. Significantly more patients than controls had hyperhomocysteinemia, 16/58 vs. 4/65, defined as fasting total homocysteine above 18.6 mumol/l. Loading with methionine did not further discriminate between patients and controls. Smoking patients had higher levels of homocysteine than non-smoking patients or smoking and non-smoking controls. Smoking patients also had lower levels of vitamin B6. When comparing patients with suprainguinal, infrainguinal and multilevel disease the highest homocysteine levels were seen in the latter group. Also, in this group smoking patients had higher homocysteine levels. Multivariate analysis revealed that homocysteine was associated with low levels of vitamin B12, folate and smoking. Smoking therefore seems to be connected to increased homocysteine levels in patients with early development of atherosclerosis, partly explained by decreased levels of B6, B12 and folate.

Reduction in Homocysteine by n-3 Polyunsaturated Fatty Acids after 1 Year in a Randomised Double-Blind Study following an Acute Myocardial Infarction: No Effect on Endothelial Adhesion Properties

We hypothesized that n–3 polyunsaturated fatty acids (n–3 PUFAs) as compared to corn oil administered for 1 year following an acute myocardial infarction (MI) may reduce plasma total homocysteine (p-tHcy), ultrasensitive C-reactive protein (µCRP), and the adhesive properties of the endothelium, expressed as soluble E-selectin (sE-selectin) and soluble intercellular adhesion molecule-1 (sICAM-1). In a prospective, randomised, double-blind study, 300 acute MI patients were allocated to highly concentrated n–3 PUFAs (n = 150) or corn oil (n = 150). After 1 year on treatment there was an intergroup difference in p-tHcy in favour of the n–3 group (n = 118), p = 0.022. However, sE-selectin, sICAM-1 and µCRP were unaffected by the treatment. In conclusion, reduction of p-tHcy by long-term n–3 PUFAs treatment was not associated with demonstrable effects on markers of endothelial adhesion properties.

The effect of B-vitamins on hyperhomocysteinemia in patients on antiepileptic drugs.

Patients on antiepileptic drugs (AEDs) may have elevated levels of plasma total homocysteine (p-tHcy). The aim of this study was to assess the effect of B-vitamin supplementation on the levels of p-tHcy and markers of endothelial activation and lipid peroxidation. A total of 33 adult patients on AEDs were identified with either fasting (Group 1, n=23) or post methionine load (PML) (Group 2, n=10) hyperhomocysteinemia. Subjects were supplemented with B-vitamins for 30 days: folic acid 0.4 mg, pyridoxine 120 mg and riboflavin 75 mg per day. After supplementation, serum folate and pyridoxal phosphate had increased, while fasting and PML p-tHcy had decreased (P<0.0001) by 36 and 26%, respectively. Prior to supplementation, the Group 1 patients had elevated levels of P-selectin and von Willebrand factor (vWF) (P=0.05 and 0.03, respectively). After supplementation, the levels of intercellular cell adhesion molecules had decreased (P=0.01) and E-selectin decreased nonsignificantly (P=0.07). However, the levels of vascular cell adhesion molecules had increased (P<0.0001), while lipid peroxidation were unchanged. In conclusion, the combined supplementation with folic acid, pyridoxine and riboflavin reduced fasting and PML hyperhomocysteinemia in patients on AEDs. Patients with fasting hyperhomocysteinemia had elevated levels of P-selectin and vWF, which may indicate an increased risk of cardiovascular disease. Furthermore, B-vitamin supplementation influenced endothelial activation, although the clinical implication is uncertain.

Folate, Related B Vitamins, and Homocysteine in Childhood and Adolescence: Potential Implications for Disease Risk in Later Life

OBJECTIVES. Folate and the metabolically related B vitamins are an important priority throughout life, but few studies have examined their status through childhood and adolescence. The aims of the current study were to investigate age, gender, and lifestyle factors as determinants of folate, related B-vitamin status, and homocysteine concentrations among British children and adolescents and to propose age-specific reference ranges for these biomarkers, which, at present, are unavailable. PARTICIPANTS AND METHODS. Data from the National Dietary and Nutritional Survey of 2127 young people aged 4 to 18 years were accessed to provide a representative sample of British children. All of the subjects who provided a blood sample for homocysteine concentrations were included in the current study (n = 840). Of these, laboratory biomarkers of folate (serum and red cell folate: n = 832 and 774, respectively), vitamin B12 (n = 828), vitamin B6 (n = 770), and riboflavin (n = 839) were also examined. RESULTS. The biomarker status of all 4 of the relevant B vitamins decreased significantly with age. Correspondingly, homocysteine concentrations progressively increased, with median values of 5.6, 6.3, and 7.9 μmol/L for children aged 4 to 10 years, 11 to 14 years, and 15 to 18 years, respectively, and were higher in boys compared with girls (15–18 years only). Independent of age and gender, fortified breakfast cereal intake (consumed by 89% of the sample) was associated with significantly higher B-vitamin status and lower homocysteine concentrations. CONCLUSIONS. It is not generally appreciated that the well-established progressive increase in homocysteine from 4 to 18 years reflects decreases in the biomarker status of all 4 metabolically related B vitamins. We suggest age-specific laboratory reference ranges for homocysteine and related B-vitamin concentrations for potential use within a pediatric setting.