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UNLABELLED: The risk of cardio vascular disease (CVD) doubles after menopause. Plasma homocysteine (hCy) is a risk factor which is influenced by vitamins B12,B6 and folate. The present study was conducted to examine the relationship of plasma hCy to the three vitamins and other contributing variables in early natural menopause. METHODS: Participants were healthy, non smoking Caucasian women 3 to 5 years postmenopausal (n = 26) or premenopausal between 30 and 45 y(n = 30). Anthropometric data, dietary records and plasma concentrations of hCy, vitamin B6, vitamin B12 and folate were obtained. RESULTS: The nutritional status of vitamins B6, B12 and folate as measured by dietary intake and blood concentrations was adequate in both groups. Mean fasting plasma total (t) hCy concentration of postmenopausal group was 2-fold higher than the value found for control group (P < 0.0001) without oral methionine loading. The difference between the two groups remained highly significant after adjustment for confounding variables by multivariate analysis, suggesting that the effect of estrogen deficiency was direct. CONCLUSION: In addition to the loss of the protective effects of estrogen on their cardiovascular physiology and lipid metabolism, postmenopausal women are exposed to higher plasma hCy concentrations and deleterious cardiovascular effects. The exact mechanism is not known but does not seem to be related to coenzyme deficiency.

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BACKGROUND: N-acetylcysteine is a novel antioxidant that has been reported to reduce plasma homocysteine concentrations and improve endothelial function. Cardiac transplant recipients have a high incidence of coronary endothelial dysfunction and hyperhomocysteinemia, both of which may lead to the development of transplantation coronary artery disease. It was hypothesized that N-acetylcysteine would reduce plasma homocysteine concentrations and improve brachial endothelial function in cardiac transplant recipients. PATIENTS AND METHODS: A cohort of stable cardiac transplant recipients was recruited from the outpatient clinic at the Toronto General Hospital, Toronto, Ontario. Brachial artery endothelial functions were studied according to standard techniques to determine flow-mediated dilation of the brachial artery. Plasma homocysteine concentrations were assayed using high performance liquid chromatography with electrochemical detection and pulsed integrated amperometry. After baseline testing, patients were treated in an unblinded fashion with N-acetylcysteine 500 mg/day. After 10 weeks of therapy, patients returned for follow-up endothelial function and homocysteine testing. RESULTS: Thirty-one patients were initially enrolled. Two patients withdrew due to excessive gastrointestinal upset. Two patients did not return for follow-up testing. The remaining 27 patients tolerated the treatment well. At baseline, 85% of the patients had hyperhomocysteinemia (greater than 15 mol/L) with a mean plasma concentration of 18.6 4.7 mol/L. No changes in homocysteine concentrations were seen at follow-up. At baseline, the average flow-mediated dilation was only 4.7 6.3%. No changes were seen at follow-up. CONCLUSIONS: Hyperhomocysteinemia and brachial endothelial dysfunction are common in stable cardiac transplant recipients and are unaffected by supplementation with N-acetylcysteine.

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BACKGROUND: In cardiac transplant recipients, long-term survival may be limited by transplant coronary artery disease (TxCAD). Hyperhomocysteinemia (Hhcy) has been associated with vascular disease and is common in transplant recipients. The objective of this study was to determine the relationship between fasting homocysteine (Hcy) concentrations and TxCAD in a cohort of cardiac transplant recipients. METHODS: Forty-eight patients more than 5 yr after transplant were recruited from a cohort of 72 consecutive patients with in-depth analysis of homocysteine levels from the Cardiac Transplant Clinic. Early morning fasting blood was obtained, and the plasma separated and frozen within 30 min. Hcy concentrations were determined by high-performance liquid chromatography (HPLC) with pulsed integrated amperometry. Coronary angiograms were reviewed in a blinded fashion. TxCAD was diagnosed, using the most recent angiogram, when a >25% lesion was present anywhere in the coronary tree. RESULTS: Forty-eight patients transplanted between 1985 and 1994 were studied. The mean Hcy concentration for the cohort was 23.5+/-5.0 micromol/L, all patients had homocysteine levels above the upper range of normal (5-15 micromol/L). Hcy concentrations were significantly higher in patients with angiographic evidence of TxCAD: 25.0+/-5.9 vs. 21.9+/-3.4 micromol/L, p=0.03. This effect persisted when covariates were taken into account using logistic regression analysis. CONCLUSIONS: Hhcy is associated with TxCAD. Prospective studies are required to confirm this association and to assess the efficacy of Hcy-lowering therapy in this patient population.

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BACKGROUND: Endothelial dysfunction is common in cardiac transplant recipients and predicts the development of transplant coronary artery disease. Hyperhomocysteinemia is associated with endothelial dysfunction in the general population, is common in transplant recipients, and has been associated with transplant coronary artery disease. Thus therapy that decreases homocysteine concentrations might also improve endothelial function and decrease the risk of transplant coronary artery disease. Folate and pyridoxine are important cofactors in distinct aspects of homocysteine metabolism. The purpose of this study was to determine whether folate or pyridoxine supplementation improves endothelial function in cardiac transplant recipients. METHODS AND RESULTS: This was a double-blind, randomized, placebo-controlled trial. We assigned 31 transplant recipients to either pyridoxine (n = 11:100 mg/day), folate (n = 12:5 mg/day), or placebo (n = 8) for 10 weeks. Fasting and post-methionine-load (methionine 100 mg/kg orally) homocysteine concentrations were determined. Brachial artery flow-mediated dilatation was used as a measure of endothelial function. At follow-up, we noted no significant changes in homocysteine concentrations in any of the groups. However, pyridoxine supplementation was associated with a significant improvement in endothelial function (2.8 +/- 6.7 to 6.9 +/- 6.3, p = 0.05). No significant changes were seen in patients treated with folate or placebo. CONCLUSIONS: Pyridoxine, but not folate supplementation, significantly improves endothelial function in cardiac transplant recipients.

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The new isocratic cation exchange method separates up to eight different amino thiols. The separated sample components are detected electrochemically using a gold electrode and the integrated pulsed amperometry. The eluent composition is, for example, 0.15 M sodium perchlorate, 0.02 M perchloric acid and 5% acetonitrile. The report describes the optimization of chromatographic parameters such as column diameter and eluent composition. Quantitative performance is discussed for eight different amino thiols using standards. Also presented is a long term quantitative study for homocysteine and methionine in plasma samples. The preparation of plasma samples is simpler than with the previously reported version of the method. Only a reduction step is required, and neither column switching nor derivatization are necessary.

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Although inorganic sulfate is an essential and ubiquitous anion in human biology, it is infrequently assayed in clinical chemistry today. Serum sulfate is difficult to measure accurately without resorting to physicochemical methods, such as ion chromatography, although many other techniques have been described. It is strongly influenced by a variety of physiological factors, including age, diet, pregnancy, and drug ingestion. Urinary excretion is the principal mechanism of disposal for the excess sulfate produced by sulfur amino acid oxidation, and the kidney is the primary site of regulation. In renal failure, sulfoesters accumulate and hypersulfatemia contributes directly to the unmeasured anion gap characteristic of the condition. In contrast, sulfate in urine is readily assayed by a number of means, particularly nephelometry after precipitation as a barium salt. Sulfate is most commonly assayed today as part of the clinical workup for nephrolithiasis, because sulfate is a major contributor to the ionic strength of urine and alters the equilibrium constants governing saturation and precipitation of calcium salts. Total sulfate deficiency has hitherto not been described, although genetic defects in sulfate transporters have been associated recently with congenital osteochondrodystrophies that may be lethal. New insights into sulfate transport and its hormonal regulation may lead to new clinical applications of sulfate analysis in the future.

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BACKGROUND: Elevation of plasma homocyst(e)ine level is an independent risk factor for arterial and venous thrombosis. We studied the degree to which hyperhomocyst(e)inemia contributes to the development of venous thromboembolism, using a retrospective case-control study design. METHODS: Cases were individuals with objectively confirmed venous thromboembolism and no history of atherosclerosis seen at the Toronto Hospital Thrombosis Clinic, Toronto, Ontario, between January 1, 1996, and July 31, 1998. Three controls were matched for every case according to sex and age within 5 years and were derived from a large community cohort. All subjects underwent assessment for fasting plasma homocyst(e)ine levels. Hyperhomocyst(e)inemia was defined as a fasting total homocyst(e)ine concentration above the 95th percentile control value. RESULTS: Seventy cases and 210 matched controls were included. Men and women were equally represented, and most were younger than 60 years. Among cases with venous thromboembolism, the mean (+/- SD) plasma homocyst(e)ine level was significantly higher than in controls (13.0 +/- 6.9 micromol/L vs 9.0 +/- 4.8 micromol/L, respectively; P<.001). Sixteen (23%) of 70 cases had hyperhomocyst(e)inemia compared with 10 (5%) of 210 controls (odds ratio, 5.9; 95% confidence interval [CI], 2.5-13.8). Among subjects aged 60 years or younger, the odds ratio was 4.9 (95% CI, 1.4-16.4), while for those aged 60 years or older, it was 7.3 (95% CI, 2.2-24.0). Even with the exclusion of cases showing abnormal renal function or low serum vitamin B12 or folate levels, the odds ratio remained significantly elevated at 3.3 (95% CI, 1.1-10.0). CONCLUSIONS: We found that fasting hyperhomocyst(e)inemia is a significant risk factor for venous thromboembolic disease in patients at a thrombosis clinic. Given the magnitude of effect and consistency across these studies, it is likely that homocyst(e)ine plays a causative role in the development of venous thrombosis, and it should be considered in the workup for venous thromboembolism.

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BACKGROUND & AIMS: Hepatic osteodystrophy is a complication of primary biliary cirrhosis (PBC). Allelic polymorphisms of the vitamin D receptor (VDR) gene are related to bone mineral density (BMD) in normal cohorts and those with primary osteoporosis. We sought to establish the prevalence of reduced bone mass in PBC, correlate BMD with VDR gene polymorphisms, and identify risk factors for the development of hepatic osteodystrophy. METHODS: Seventy-two female patients with PBC were evaluated prospectively. Clinical information, BMD assessment, disease severity, and osteoporosis risk factors were documented, and multivariate regression modeling was performed. RESULTS: Twenty-four percent of the patients were osteoporotic at the lumbar spine and 32% at the femur. Severe bone loss (z score <-2.0) occurs 4 times more frequently in patients with PBC compared with controls. Body weight (P = 0.003) and postmenopausal status (P = 0.012) correlated independently with BMD. VDR genotype (P = 0.01) correlated with lower BMD at the spine only. CONCLUSIONS: Osteoporosis is a common complication of PBC. VDR genotype predicts lower BMD in patients with PBC. Studies are warranted to investigate the mechanism(s) by which VDR as well as other candidate genes may contribute to the development of hepatic osteodystrophy in PBC.

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BACKGROUND: The regulation of extracellular calcium concentration by parathyroid hormone is mediated by a calcium-sensing, G-protein-coupled cell-surface receptor (CASR). Mutations of the CASR gene alter the set-point for extracellular ionised calcium [Ca2+]o and cause familial hypercalcaemia or hypocalcaemia. The CASR missense polymorphism, A986S, is common in the general population and is, therefore, a prime candidate as a genetic determinant of extracellular calcium concentration. METHODS: We genotyped the CASR A986S variant (S allele frequency of 16.3%) in 163 healthy adult women and tested samples of their serum for total calcium, albumin, total protein, creatinine, phosphate, pH, and parathyroid hormone. A prospectively generated, random subset of 84 of these women provided a whole blood sample for assay of [Ca2+]o. FINDINGS: The A986S genotype showed no association with total serum concentration of calcium, until corrected for albumin. In a multivariate regression model, biochemical and genetic variables accounted for 74% of the total variation in calcium. The significant predictors of serum calcium were: albumin (p<0.001), phosphate (p=0.02), parathyroid hormone (p=0.007), pH (p=0.001), and A986S genotype (p=0.009). Fasting whole-blood [Ca2+]o also showed an independent positive association with the 986S variant (p=0.013). INTERPRETATION: The CASR A986S variant has a significant effect on extracellular calcium. The CASR A986S polymorphism is a likely candidate locus for genetic predisposition to various bone and mineral disorders in which extracellular calcium concentrations have a prominent part.

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Increased circulating total homocysteine (tHcy) has been implicated as an independent risk factor for atherosclerotic disease. In cardiac transplant patients, accelerated coronary atherosclerosis is an important cause of late allograft failure; however, studies of tHcy in this at-risk group are limited. We sampled a cohort of 72 subjects 3.95+/-3.14 (mean +/- SD) years after transplantation and found that all had tHcy concentrations above our upper reference limit (15.0 micromol/L). The mean tHcy in the transplant group (25.4+/-7.1 micromol/L) was significantly greater than in our reference group (9.0+/-4.3 micromol/L; n = 457; P <0.001). We also examined the effect of age, gender, time since transplant, serum folate and cobalamin, total protein, urate, creatinine, albumin, and trough whole blood cyclosporine concentrations. In a multiple linear regression model, only creatinine (mean 144+/-52 micromol/L; P = 0.021) and trough cyclosporine concentrations (191+/-163 microg/L; P = 0.015) were independent positive predictors of tHcy, whereas serum folate (8.35+/-7.43 nmol/L; P = 0.018) and time since transplant (P = 0.049) were significant negative predictors. We conclude that hyperhomocysteinemia is a common characteristic of cardiac transplant recipients. Our analysis suggests that folate and renal glomerular dysfunction are important contributory factors; however, whole blood cyclosporine concentrations may also predict the degree of hyperhomocysteinemia in this population and therefore influence interpretation of any apparent response to treatment.

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OBJECTIVE: To summarize recent developments in our understanding of homocysteine as a clinically relevant and independent predictor of vaso-occlusive disease (including atherosclerosis and thromboembolism), as an early indicator of folate or cobalamin deficiency, and as a key factor in the pathogenesis of neural tube defects. METHODS AND RESULTS: To determine total homocysteine, plasma or serum must be separated shortly after collection and subjected to chemical reduction. Reference intervals should take into account the prevalence of physiological hyperhomocystinemia. A common cause of hyperhomocystinemia is a genetic predisposition caused by a polymorphic substitution in the methylenetetrahydrofolate reductase (MTHFR) gene, which can be readily detected by molecular means. CONCLUSION: Determination of homocysteine and MTHFR testing should be limited to laboratories with relevant expertise and ability to maintain the high degree of precision required for reliable interpretation. Assays should be offered in selected cases with clinical features or laboratory findings suggestive of hyperhomocystinemia, since treatment is simple and may be highly effective.

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The role that the Calcium Sensing Receptor (CASR) plays in extracellular calcium regulation had been ascertained through studies of inactivating as well as activating mutations of CASR gene in a number of multiplex families. We have extended these observations to a polymorphism analysis of the intercellular domain of CASR in a cohort of healthy young women. The results demonstrate significant allelic polymorphism as a result of nonconservative changes at two specific sites. Further studies will be required to determine what, if any, relationship this may have to CASR phenotype.

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For assay of serum sulfate, quantitation by ion conductimetry after separation by anion-exchange chromatography is the method of choice. In comparison to classical barium precipitation methods, chromatographic methods demonstrate increased precision, specificity and sensitivity, and they may be superior to spectrophotometric methods that rely on organic cation precipitation of sulfate. The increased sensitivity and specificity, as well as the inherent capacity of chromatographic methods for simultaneous determination of other anions, has led to its increasing use in the determination of excreted sulfate in clinical profiles of urinary anion composition. Ion chromatography can also be used to quantitate free sulfate in other clinical samples, including cerebrospinal fluid, sweat, saliva, breast milk and human tissues. Finally, ion chromatography shows promise as a more precise and sensitive method for measurement of total acid-labile sulfoesters and thiosulfate.

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Thiosulfate is a naturally occurring product of sulfur metabolism. Assays of urinary thiosulfate have been based on the reaction with cyanide to form thiocyanate. However, matrix interferences and background variation in endogenous thiocyanate excretion place serious constraints on this method for determination of physiological amounts of thiosulfate in urine. We describe a column-switching ion chromatographic separation for urinary thiosulfate that allows for sensitive and accurate detection by ion conductimetry. In 20 adult volunteers, we found a lower urinary thiosulfate (8.50 +/- 7.39 mumol/24 h, mean +/- S.D.) than others have described, although the upward skew of the results (median, 6.90; range, 0.84-32 mumol/24 h) was similar. However, we have not observed any of the interferences and the sensitivity of our technique (< 0.2 mumol/24 h) allows for detection of thiosulfate in all control samples. This sort of methodological improvement will be essential for any study of physiological thiosulfate metabolism.

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