W. J. Hayward Vermaak

Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum.

A rapid, isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of total homocysteine levels in human serum. Prior to reversed-phase HPLC analysis, the serum thiols were derivatized with SBD-F (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate), a thiolspecific fluorogenic probe which is commercially available. Retention of SBD-homocysteine was sensitive to pH, and a mobile phase pH of 2.1 ensured baseline separation of serum thiols within 6 min. The method is simple, sensitive, reproducible (between-run coefficient of variation of 6.6%) and very suitable for routine determination of serum homocysteine levels in a clinical pathology laboratory.

The effect of blood sample aging and food consumption on plasma total homocysteine levels

The stability of homocysteine in whole blood and plasma was investigated. Total homocysteine concentrations in whole blood increased rapidly to values in excess of 180% of the basal concentration if whole blood was left at ambient temperature. Sodium fluoride partially inhibited homocysteine accumulation, while refrigeration inhibited homocysteine accumulation for at least 4 h. Since intracellular concentrations of homocysteine were low, the results indicate continued metabolism of L-methionine to homocysteine after the blood sample had been obtained. In contrast to whole blood, homocysteine was stable in plasma, even at room temperature. Food consumption (normal breakfast) resulted in significantly lower plasma homocysteine concentrations, which returned to pre-prandial concentrations 8 h later. The results indicate that both blood sampling and food intake should be rigorously standardized in epidemiological studies to elucidate the possible role of elevated circulating homocysteine concentrations in premature vascular disease.

Homocysteine and ischaemic heart disease in the Caerphilly cohort

Elevated circulating total homocyst(e)ine concentrations are associated with a higher prevalence of ischaemic heart disease (IHD). We utilized data from the Caerphilly Prospective Cohort Study to assess the predictive power of the serum total homocyst(e)ine concentration for future IHD. Serum total homocyst(e)ine concentrations were measured in 2290 men in the Caerphilly cohort, a representative population sample of men aged 50-64 years. During a 5-year follow-up period, 56 men suffered fatal IHD, 77 had a non-fatal myocardial infarction, while 21 were found to have ECG evidence of myocardial infarction (MI) when examined at follow-up. The mean serum total homocyst(e)ine concentration in the total of 154 men who experienced an incident IHD event was 12.4 micromol/l, whereas the 2136 men who experienced no such event had a mean level of 11.7 micromol/l. The difference between these means, examined by logistic regression and standardising for the effects of differences in age, social class, smoking, BMI, diabetes, HDL-cholesterol and prevalent IHD is 0.47 micromol/l (95% CI = -0.13 to 1.11 micromol/l). The mean difference for the 56 men who died, and whose death was attributed to IHD, is 0.81 micromol/l (95% CI= -0.17 to 1.88 micromol/l) after correction for confounding factors. Vitamin nutritional status and alcohol intake were significant negative determinants of serum total homocyst(e)ine concentrations; the effect of alcohol is explained by the folic acid content of beer, which is the preferred alcoholic beverage in Caerphilly. It is concluded that the serum total homocyst(e)ine concentration is weakly predictive of IHD events, though in the present data adjustments for other factors attenuated the relationship and it became not statistically significant (P > 0.05).

The effect of Simvastatin on the plasma antioxidant concentrations in patients with hypercholesterolaemia

The aim of this study was to monitor the antioxidant status of patients with hypercholesterolaemia during treatment with Simvastatin. Forty-seven patients, of whom 25 had confirmed familial hypercholesterolaemia (FH), were treated with 10 or 20 mg of Simvastatin per day for 14 weeks. As expected, total cholesterol and LDL cholesterol concentrations decreased considerably, while HDL cholesterol concentrations increased during drug treatment. In neither FH nor non-FH patients were any significant changes observed for retinol status, while plasma vitamin C concentrations were also not adversely affected by the drug therapy. In both patient groups Simvastatin therapy led to a significant decrease in plasma alpha-tocopherol (P < 0.05) concentrations, however, the alpha-tocopherol/total cholesterol ratio increased by 9.1 (P < 0.01) and 12.1% (P < 0.01) in FH and non-FH patients, respectively, during the 14-week treatment period. The coenzyme Q10/total cholesterol ratio did not change significantly in non-FH patients, but was significantly lower (P < 0.05) than the baseline ratio after 4 and 14 weeks of Simvastatin treatment in FH patients. The alpha-tocopherol/total cholesterol ratio of FH patients remained consistently and significantly lower (P < 0.01) compared with non-FH patients, indicating that LDL from the former group may be more vulnerable to free radical-mediated damage and lipid peroxidation. Our results suggest that the significant decline in circulating alpha-tocopherol and coenzyme Q10 concentrations was mainly a function of the decrease in serum total cholesterol concentrations.

Folate status, homocysteine metabolism, and methylene tetrahydrofolate reductase genotype in rural South African blacks with a history of pregnancy complicated by neural tube defects.

The birth incidence of neural tube defects (NTDs) in South Africa is threefold to sixfold higher in rural compared with urban blacks. We investigated whether folate deficiency and aberrant homocysteine metabolism could explain the high NTD incidence in rural black populations. Plasma folate and total homocyst(e)ine (tHcy) concentrations were determined in apparently healthy rural black women (n = 107), rural black women with a history of pregnancy complicated by NTDs (n = 54), and urban blacks (n = 101). Methionine load tests were performed on the 54 women with a history of NTD-affected pregnancy and 54 controls matched for age and body mass. The presence of the 677C --> T mutation in the methylene tetrahydrofolate reductase (MTHFR) gene was investigated in both groups by a polymerase chain reaction (PCR) of genomic DNA and HinfI digestion of the PCR product. Apparently healthy urban black women (n = 101) had a lower (P < .001) plasma folate concentration compared with rural black women (n = 107). Women with a history of NTD-affected pregnancy did not differ significantly from controls with respect to plasma folate, fasting homocyst(e)ine, methionine, and the post-methionine load increase in plasma homocyst(e)ine. More than 50% of both of the latter groups had a post-methionine load increase in plasma tHcy less than the fifth percentile as observed in a healthy white control group. No homozygotes for the 677C --> T mutation in the MTHFR gene were found in black mothers with NTD-affected offspring or controls. It is concluded that black urbanization is characterized by a diminished folate status that is paradoxically associated with a lower NTD birth incidence. Homozygosity for the 677C --> T mutation in the gene coding for MTHFR does not constitute a genetic risk factor for NTDs in blacks. No aberrant homocysteine metabolism could be demonstrated in black women with NTD-affected pregnancies.

Antioxidant Vitamins and Coronary Artery Disease Risk in South African Males

Decreased antioxidant-vitamin nutritional status may increase lipid peroxidation and susceptibility of low-density lipoprotein (LDL) to oxidative modification. The aim of this study was to evaluate the vitamin nutritional status of coronary artery disease (CAD) patients and to assess the risk of CAD related to each individual antioxidant vitamin. The study was performed as a case-control study with 41 patients with angiographically demonstrated CAD and 41 apparently healthy age- and smoking status-matched controls. Plasma vitamin E, C and A concentrations were significantly decreased in CAD patients compared with controls (p < 0.001) after correcting for significant covariates. Per quartile decrease in vitamin A and E concentrations was associated with increased risk of CAD, even after adjusting for CAD risk factors, while per quartile decrease in vitamin C concentrations was not associated with significant CAD risk after adjusting for CAD risk factors. Decreased vitamin A and E concentrations are independently associated with increased risk of CAD independent from other CAD risk factors in white male South Africans and dietary intervention strategies are advocated.

Variability of post-methionine load plasma homocysteine assays.

BACKGROUND Numerous variations of the methionine load test are frequently used as dynamic function tests to assess homocysteine metabolism. Lack of standardization impedes inter-laboratory comparisons. Criteria based on biological variation are suggested to standardize the methionine load test. METHODS Weekly methionine load tests (n=5) with blood sampling at 0, 4, 6 and 8 h were performed on 15 young men. For both basal and post-methionine load homocysteine measurements, total variance (sigma(S)(2)), within-subject variance (sigma(I)(2)), between-subject variance (sigma(G)(2)) and analytical variance (sigma(A)(2)) were calculated from an appropriate analysis of variance (ANOVA). RESULTS Plasma homocysteine concentrations measured 6 h after methionine loading had analytical, within-subject and between-subject coefficients of variation of 5.2%, 17.5% and 9.7%, respectively. Measurements at 4 h had a higher within-subject coefficient of variation. Adjustment of post-methionine load homocysteine concentrations for basal levels resulted in considerable increases of all the measures of variation. CONCLUSIONS Adjustment of post-methionine load plasma homocysteine concentrations for basal levels does not improve the interpretation of changes in serial results due to the higher analytical and biological variance of adjusted concentrations. It is suggested that the methionine load test is standardized to a single, unadjusted homocysteine measurement at 6 h.

The Relationship between Vitamin B6 Metabolism, Asthma, and Theophylline Therapy

Asthma can be broadly defined as enhanced reactivity of the airways to specific and nonspecific stimuli, resulting in diffuse airways obstruction with clinical symptoms like dyspnea, cough, and a tight chest. Stimuli that may provoke asthmatic responses include environmental allergens, respiratory infection, certain drugs such as nonsteroidal anti-inflammatory drugs, and exercise.’ An important aspect of asthma treatment is the administration of bronchodilators of which theophylline is most commonly used.2 As will be reviewed in this paper, vitamin B, status and asthma have been studied by several researchers3^’; however, the effect of medication per se on vitamin B, status has not been investigated. Because it would be practically impossible to study vitamin B, status in asthmatics without the use of bronchodilators, we opted for the alternative: to study the effect of bronchodilators on vitamin B, metabolism in healthy volunteers. We therefore investigated the effect of theophylline on vitamin B, metabolism in healthy volunteers, and our results are summarized and reviewed in the second part of this paper.

Quantification of erythrocyte S-adenosyl-L-methionine levels and its application in enzyme studies

A highly selective high-performance liquid chromatographic method for the quantification of human erythrocyte S-adenosyl-L-methionine levels is described. A strong cation-exchange sorbent with propylsulphonic acid functional groups was used to extract S-adenosyl-L-methionine and S-adenosylethionine (internal standard) from erythrocytes. Quantification of erythrocyte S-adenosyl-L-methionine levels was achieved by using reversed-phase high-performance liquid chromatography and ultraviolet detection at 254 nm. This method was adapted to measure methionine-adenosyltransferase activity in erythrocytes, which enables us to study the possible role of altered methylation in different diseases.

Enzymes of Vitamin B6 Metabolism in Maternal and Cord Blood

The vitamin B, status of the neonate has been well documented,’ but the ability of the neonate to metabolize the different generic forms of vitamin B, has not been investigated. This is presumably due to ethical considerations, as studies on the kinetics of vitamin B, metabolism require multiple blood sampling. Alternatively, cord blood erythrocytes could be used to study the neonate’s potential to metabolize vitamin B, after parturition. Erythrocytes play an active role in human vitamin B, metabolism’ and can be regarded as a convenient cell system for studying neonatal capability to metabolize the different generic forms of vitamin B,. Directly after placental delivery, a 10-ml blood sample was collected from the mother and from the placental cord. EDTA was used as anticoagulant. Plasma pyridoxal-5’-phosphate (PLP) concentrations and erythrocyte activity levels of pyridoxal kinase (EC 2.7.1.39, pyridoxamine (pyridoxine)-5’-phosphate oxidase (PMPPNP) oxidase (EC 1.4.3.5), and PLP phosphatase were determined using highperformance liquid chr~matography.~ .~ TABLE 1 indicates that cord plasma PLP and cord erythrocyte PMP(PNP) oxidase levels were significantly elevated when compared to the respective maternal levels. Cord erythrocyte PLP phosphatase activities were significantly lower than maternal phosphatase levels. Linear regression between maternal and cord erythrocyte enzyme levels indicated that 64% of the variation in cord PMP(PNP) oxidase activity can be explained by maternal PMP(PNP) oxidase activity levels, while 32.5% of the variation in cord pyridoxal kinase activity levels is explained by maternal pyridoxal kinase activities. Erythrocyte enzyme activity levels indicate that the full-term neonate is fully capable of metabolizing the different generic forms of vitamin B,. The significantly higher PMP(PNP) oxidase activities in cord erythrocytes may relate to active transport of riboflavin across the placenta.’ Flavin mononucleotide is an essential cofactor for PMP(PNP) oxidase, and the activity of the enzyme is closely correlated with riboflavin nutritional status., The significance of the different PLP phosphatase activities in maternal and cord blood is still uncertain. It could either relate to a lower PLP turnover in the neonate, or increased PLP dephosphorylation in the maternal circulation to provide sufficient pyridoxal for placental transport.’ Nutritional and/or genetic determinants of PMP(PNP) oxidase and pyridoxal kinase activities are