Validated method for quantitation of biomarkers for benzene and its alkylated analogues in urine.

A validated gas chromatography-mass spectrometric method for the analysis of the metabolites of benzene and its alkylated analogues in urine is reported. A number of metabolites, as required by authorities for biomonitoring of industrial exposure to aromatic vapour, were analysed simultaneously with preservation of quantitative information concerning positional isomers. The use of this method replaces a combination of analytical methods required for the analysis of all these metabolites. Urine samples were subjected to acidic deconjugation followed by a derivatization step. Phenol, ortho-, meta-, para-cresol, mandelic acid, and ortho-, meta-, para-methylhippuric acid were analysed as their corresponding ethoxycarbonyl derivatives, with single ion monitoring. The mass-to-charge ratios (m/z) of the ions used for quantitation by single ion monitoring of the metabolites were: phenol, 94 m/z; cresols, 108 m/z; mandelic acid, 206 m/z; hippuric acid, 105 m/z; methylhippuric acids, 119 m/z. The mass-to-charge ratios for the internal standards were: [(2)H(6)]phenol, 99 m/z; p-chlorophenol, 128 m/z and 3-chloro-4-hydroxyphenyl acetic acid, 214 m/z. The limits of detection for phenol and the cresols were below 0.4 micromol/l and below 0.05 micromol/l for mandelic acid and the hippuric acids. Within-run precision for mandelic acid was 6.2%, for hippuric acid was 7.32% and was below 5% for the rest of the analytes.

Gas chromatographic-mass spectrometric method for quantitation of phenylalanine and tyrosine in serum.

A validated gas chromatographic-mass spectrometric method for quantitation of phenylalanine and tyrosine in serum is described. Quantitation of phenylalanine and tyrosine with a non-labelled non-endogenous internal standard, L-2-chlorophenylalanine, compared favourably with isotope dilution mass spectrometric quantitation. The 95% reference ranges for phenylalanine. tyrosine and the phenylalanine-tyrosine molar ratio in neonate cord blood serum were determined by isotope dilution mass spectrometry and were found to be 77.1-144.7, 33.3-109.3 micromol/l and 1.1-3.0, respectively.

Does a single vitamin B-supplementation induce functional vitamin B-deficiency?

In a pilot study we measured the effect of three different combinations of the vitamins B6, folate and B12 on the serum concentrations of homocysteine, cystathionine and methylmalonic acid in five healthy young men without hyperhomocysteinemia. The results indicate that there are still undescribed interactions between vitamin B6 and folate, suggesting that these two vitamins should be given together to avoid depletion of the one not given. With regard to the well known metabolic pathways of methionine and cysteine, this confirms the hypothesis that a combined supplementation with the vitamins B6 and folate (and B12) is superior to folate alone in order to lower homocysteine.

Homocysteine and ischaemic stroke in men: the Caerphilly study.

OBJECTIVE: To assess the risk of ischaemic stroke associated with total serum homocyst(e)ine (tHcy) concentration. DESIGN: Cohort study. SETTING: Caerphilly, South Wales PARTICIPANTS: 2254 men age 50 to 64 years recruited between 1984 and 1988. RESULTS: 107 men developed ischaemic stroke and mean follow up time was 10.2 years. There was no significant difference in mean serum total homocyst(e)ine levels between stroke cases (12.2 micromol 95% CI 11.6 to 13.1) and non-cases (11.7 micromol 95% CI 11.5 to 11.9) (p=0.14). There was no significant risk for a standard deviation increase in homocyst(e)ine (adjusted hazard ratio = 1.1, 95% CI 0.9 to 1.4). An interaction was observed between homocyst(e)ine and age at entry (p=0.003). The adjusted odds ratio comparing the top quintile of homocyst(e)ine with the rest was 2.5 (95% CI 1.0 to 6.2) for strokes occurring under 65 years and 0.5 (95% CI 0.2 to 1.3) at 65 years or older (p value for interaction =0.02). Risk also differed by blood pressure status. The adjusted hazard ratio for a standard deviation increase in homocyst(e)ine was 0.8, (95% CI 0.6 to 1.2) for normotensive men and 1.3 (95% CI 1.1 to 1.7) for hypertensive men (p value for interaction =0.01). CONCLUSIONS: Overall, there is no significant relation between homocyst(e)ine and ischaemic stroke in this cohort. However, its aetiological importance may be greater for premature ischaemic strokes (<65 years) and in hypertensive men.

Homocysteine and coronary heart disease in the Caerphilly cohort: a 10 year follow up.

OBJECTIVE: Prospective assessment of the risk of coronary heart disease associated with total serum homocyst(e)ine (homocysteine) concentration. DESIGN: Nested case-control study. SETTING: Caerphilly and surrounding villages in south Wales, UK. PARTICIPANTS: 2290 men who participated in phase II of the study in 1984. After a mean follow up of 10 years, 312 men developed coronary heart disease and were compared with 1248 randomly selected, age frequency matched controls. MAIN OUTCOME MEASURE: Acute myocardial infarction or death from coronary heart disease. RESULTS: The geometric mean serum homocysteine concentration was higher in cases (12.2 micromol/l, 95% confidence interval (CI) 11.8 to 12.6 micromol/l) than in controls (11.8 micromol/l, 95% CI 11.3 to 12.5 micromol/l) (p = 0.09). There was a graded increase in the odds ratio of coronary heart disease across quintiles of the homocysteine concentration distribution compared with the first (p = 0.04), which was attenuated when adjusted for confounding variables (p = 0.4). There was a small but non-significant increase in the adjusted odds ratio of coronary heart disease per standard deviation change in the log distribution of homocysteine concentration (OR = 1.07 (95% CI.93 to 1.24), p = 0.34). Comparing the top quintile of the homocysteine concentration with the remaining 80%, the adjusted odds ratio of coronary heart disease was 1.03 (95% CI 0.73 to 1.45) (p = 0.8) and comparing the top 5% with the remaining 95% it was 1.05 (95% CI 0.56 to 1.95) (p = 0.9). CONCLUSIONS: These findings do not support the hypothesis that a raised homocysteine concentration is a strong independent risk factor for coronary heart disease. Randomised controlled trials of homocysteine lowering treatment such as folic acid are needed before generalizing the early positive results of observational studies.

Assay methods for the measurement of total homocyst(e)ine in plasma.

Analytical methods to measure plasma total homocyst(e)ine (tHcy) concentrations are reviewed. High-pressure liquid chromatography (HPLC) with fluorometric detection is the most widely used method to determine plasma tHcy concentrations. Both monobromobimane and ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F) are popular thiol-specific fluorogenic agents suitable for tHcy analysis. Monobromobimane has the advantage that it is highly reactive towards thiols, but its hydrolysis products are also fluorogenic, thus necessitating complex chromatography to obtain satisfactory separation between the compounds of interest and interferents. SBD-F does not show fluorescence, thus allowing isocratic separation of SBD-F derivatized thiols. SBD-thiol adducts are light sensitive and require protection against light to ensure reliable results. HPLC with electrochemical detection is also often used and has the advantage that no derivatization of thiols is required prior to detection. A recently reported liquid chromatography electrospray tandem mass spectrometric assay has the potential to become the reference method for plasma tHcy analysis. Other methods to measure plasma tHcy concentrations include gas-liquid chromatography, capillary electrophoresis, and immunoassays. Fluorescence polarization immunoassay compares well with gas chromatography-mass spectrometry and may become the method of choice in routine diagnostic clinical chemistry laboratories. Instability of tHcy in whole blood as well as postprandial and orthostatic variation are preanalytical factors that should be accounted for in plasma tHcy analysis. Between-method and between-laboratory variations in serum tHcy analysis are not yet satisfactory; certified reference material and standardization of the plasma tHcy assay will be essential to reduce between-laboratory bias.

Effect of magnesium supplementation on the fractional intestinal absorption of 45CaCl2 in women with a low erythrocyte magnesium concentration.

The cosupplementation of magnesium with calcium has been suggested to be beneficial in the prevention of osteoporosis. We investigated the effect of magnesium supplementation on parameters of bone resorption and fractional 45Ca absorption. Twenty apparently healthy women with a mean age of 39.2 +/- 9.2 years and an erythrocyte magnesium concentration less than 1.97 mmol/L were recruited into a controlled magnesium supplementation trial. During weeks 1 to 4, they received a daily control preparation, potassium/sodium citrate malate (PSCM). During weeks 5 to 8, the subjects received magnesium citrate malate (MCM) equivalent to 250 mg magnesium per day. During the fourth and eighth weeks, blood was collected for measurement of the serum intact parathyroid hormone (PTH) concentration and serum and erythrocyte magnesium concentration. Urine was collected for measurement of calcium, magnesium, creatinine, and deoxypyridinoline excretion. On the final day of each treatment period, 5 microCi45CaCl2 was administered orally, and the isotope was traced in the blood and urine over 7 hours. Urinary calcium, 45Ca, and deoxypyridinoline excretion, as well as serum intact PTH levels, showed no statistically significant changes as a result of magnesium supplementation. However, urinary magnesium excretion increased by 31.1% (P < .005) while fractional 45Ca absorption decreased by 23.5% (P < .001) as a result of magnesium supplementation. It is concluded that magnesium supplementation does not result in changes in bone resorption, while the fractional intestinal absorption of 45Ca appears to decrease.

Analytical quality of near-patient blood cholesterol and glucose determinations.

BACKGROUND: Screening for diabetes and hypercholesterolemia is widely advocated, and extra-laboratory testing could play a major role in cost-effective population screening. We wished to assess the analytical quality and interchangeability of capillary blood cholesterol and glucose assays as performed on near-patient devices in pharmacies in Pretoria, South Africa. METHODS: Accuracy of near-patient and laboratory analyzers was assessed by analyses of human-serum-based reference material. To assess interchangeability in routine use, six volunteers visited each of 12 randomly selected pharmacies consecutively during a 3-week period to have their fasting blood glucose and cholesterol concentrations determined. For comparison purposes, a similar procedure was followed to evaluate the eight clinical chemistry laboratories servicing Pretoria and surroundings. RESULTS: The analytical performances in our laboratory of a single point-of-care instrument and of a laboratory analyzer compared well. Nevertheless, between-pharmacy analytical variation was larger than between-laboratory variation (11% vs 6.1% for cholesterol; 10% vs 7.6% for glucose). For glucose measurements, near-patient testing in pharmacies demonstrated a bias of -48.1% to 16.2%, whereas bias for laboratory measurements was -1.0% to 7.4%. Cholesterol assays showed a bias of -5.6% to 16.6% in pharmacies compared with -10.6% to 3.7% in laboratories. The percentage of closeness to the homeostatic set point for a single glucose and cholesterol determination done in any pharmacy was 24.6% and 23.6%, respectively. The corresponding values for laboratories were 16.9% and 15.6%, respectively. CONCLUSIONS: Although modern point-of-care instruments allow high-quality blood analyses under ideal conditions, performance goals often are not achieved in practice as indicated by a higher uncertainty of cholesterol and glucose blood results when determined in pharmacies. Nonuniformity of calibration procedures, deficiencies in training, a lack of internal quality control, and the absence of an external quality assessment program may all contribute to the current state of affairs.

Nutritional status influences plasma fibrinogen concentration: evidence from the THUSA survey.

Nutritional status and risk factors for chronic diseases, including plasma fibrinogen and its determinants, of Africans in the Northwest Province of South Africa, have been studied in a cross-sectional survey. A representative sample of 1854 "apparently healthy" African men and women volunteers aged 15 years and older was recruited from 37 randomly selected sites throughout the Province and stratified for level of urbanisation. Information was collected using validated and culture-sensitive questionnaires. Fasting blood samples were drawn, and all measurements were done with standardised methodology using appropriate equipment, procedures, and controls. Fibrinogen concentration was measured in citrated plasma with the method of Clauss, using the ACL200 automated system and the international fibrinogen standard. The results revealed a population with a high mean plasma fibrinogen (3.17+/-1.10 g/L for HIV-negative men and 3. 64+/-1.12 g/L for HIV-negative women). Factors known to influence plasma fibrinogen, such as age, gender, smoking habit, and physical activity, were also observed in this population. Young rural men and women had the lowest fibrinogen level. Nasal snuff taking and HIV infection did not influence fibrinogen concentration. Multivariate analyses revealed that lower plasma fibrinogen was associated with low to normal body mass index in women, and with dietary intakes compatible with prudent dietary guidelines in men and women (low intakes of animal protein; trans fatty acids and higher intakes of plant protein; dietary fibre, vitamin E, and iron, and a high dietary P/S ratio). Subjects in the higher quartiles of plasma fibrinogen had significantly lower iron, vitamin E, and vitamin B6 (women) status. Increases in fibrinogen were associated with significant increases in serum lipids. Both under- and overnutrition seem to be associated with high plasma fibrinogen. It is concluded that overall nutritional status, possibly in addition to specific nutrients (and foods), influences plasma fibrinogen.

Effect of consumption of red wine, spirits, and beer on serum homocysteine.

Serum homocysteine increases after moderate consumption of red wine and spirits, not after moderate consumption of beer. Vitamin B6 in beer seems to prevent the alcohol-induced rise in serum homocysteine.

Erythrocyte magnesium concentration as an index of magnesium status: a perspective from a magnesium supplementation study.

We investigated the utility of erythrocyte magnesium concentrations as an index of magnesium status. Calculated from the analytical and biological variation, the critical difference, i.e. the difference between results from serial specimens before they can be said to be statistically different, was 22.4% (P<0.05). Magnesium supplementation of 250 mg/day for 3 weeks in 20 women with an erythrocyte magnesium concentration less than the 15th percentile of a population sample (n=219), resulted in an increase in erythrocyte magnesium concentration of only 1.6%. We therefore conclude that sequential erythrocyte magnesium measurements are not useful for the monitoring of individual changes in magnesium intake and status.

Comparison of three different plasma homocysteine assays with gas chromatography-mass spectrometry.

BACKGROUND: Various methods are available to measure plasma total homocyst(e)ine (tHcy) concentrations, but whether plasma tHcy assays may be used interchangeably is not known. METHODS: Results from three different methods [HPLC with fluorescence detection, enzyme immunoassay (EIA), and fluorescence polarization immunoassay (FPIA)] to determine fasting (n = 163) and post-methionine load (n = 80) plasma tHcy concentrations were compared with those obtained by gas chromatography-mass spectrometry (GC-MS). Difference plots on non-transformed and log-transformed data were used to assess the agreement between HPLC and GC-MS, EIA and GC-MS, and FPIA and GC-MS. RESULTS: The closest agreement between methods was observed between GC-MS and FPIA for fasting tHcy concentrations, with 95% of the FPIA values between 19% above and 24% below the corresponding GC-MS results. Post-methionine load tHcy concentrations measured by EIA showed the least agreement with GC-MS, with 95% of values measured by EIA ranging between 52% above and 16% below the GC-MS values. With respect to GC-MS, the above-mentioned methods showed a negative bias for fasting tHcy concentrations, but a positive bias for both immunoassays for post-methionine load tHcy concentrations. CONCLUSIONS: The agreement among methods is insufficient to allow them to be used interchangeably. The intermethod differences emphasize the need for standardization of plasma tHcy assays.

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.

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).

Should all elderly people receive folate supplements?

Although it was initially thought that folate deficiencies are uncommon in the elderly, the use of the plasma total homocysteine concentration as a metabolic marker of folate status is changing this attitude. Plasma homocysteine measurements in epidemiological studies suggest that subclinical folate deficiencies are common in various populations, including the elderly. The clinical consequences of a compromised folate status may include increased coronary heart disease and cancer risk, and an association between folate deficiency and neuropsychiatric illness has been reported. Although a suboptimal folate status is associated with at least 2 major chronic diseases, it would be premature to recommend folate supplements to all elderly people. We still lack evidence that a suboptimal folate status is causally involved in the pathogenesis of the above mentioned disorders. Furthermore, cancer and the cardiovascular diseases are chronic conditions and it may be too late to use folate supplementation as preventative measures in elderly persons. However, folate supplementation should be considered in elderly people with elevated plasma total homocysteine concentrations and proven cardiovascular disease, in elderly patients treated with drugs known to induce a folate deficiency, and in those who experience neuropsychiatric disorders. Cyanocobalamin (vitamin B12) deficiencies should be excluded before folate supplementation is commenced; if in doubt, it may be safer to supplement folate and vitamin B12 together. The daily folate supplement should be at least 0.5 mg/day and it should never be used as a surrogate for a diet rich in fruit and vegetables.

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.

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.