Jovan Evrovski

A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations

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.

Vitamin D-receptor genotypes as independent genetic predictors of decreased bone mineral density in primary biliary cirrhosis.

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.

Clinical chemistry and molecular biology of homocysteine metabolism: an update.

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.

Quantitation of sulfate and thiosulfate in clinical samples by ion chromatography

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.

The Clinical Chemistry of Inorganic Sulfate

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.

Pyridoxine improves endothelial function in cardiac transplant recipients

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.

Genetics University of Toronto Thrombophilia Study in Women (GUTTSI): genetic and other risk factors for venous thromboembolism in women.

BackgroundWomen may be at increased risk for venous thromboembolism (VTE) as compared with men. We studied the effects of genetic and biochemical markers of thrombophilia in women, in conjunction with other established risk factors for VTE.MethodThe present retrospective case-control study was conducted in a thrombosis treatment programme at a large Toronto hospital. The cases were 129 women aged 16-79 years with objectively confirmed VTE. Age-matched control individuals were women who were free of venous thrombosis. Neither cases nor control individuals had known cardiovascular disease. Participants were interviewed regarding personal risk factors for VTE, including smoking, history of malignancy, pregnancy, and oestrogen or oral contraceptive use. Blood specimens were analyzed for common single nucleotide polymorphisms of prothrombin, factor V and methylenetetrahydrofolate reductase (MTHFR; C677T, A1298C and T1317C), and the A66G polymorphism for methionine synthase reductase (MTRR).Fasting plasma homocysteine was also analyzed.ResultsWomen with VTE were significantly more likely than female control individuals to carry the prothrombin polymorphism and the factor V polymorphism, or to have fasting hyperhomocysteinaemia. Homozygosity for the C677T MTHFR gene was not a significant risk factor for VTE, or were the A1298C or T1317C MTHFR homozygous variants. Also, the A66G MTRR homozygous state did not confer an increased risk for VTE.ConclusionProthrombin and factor V polymorphisms increased the risk for VTE in women, independent from other established risk factors. Although hyperhomocysteinaemia also heightens this risk, common polymorphisms in two genes that are responsible for homocysteine remethylation do not. These findings are consistent with previous studies that included both men and women.

Simultaneous analysis of homocysteine and methionine in plasma.

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.

Calcium sensing receptor gene: analysis of polymorphism frequency

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.

Urinary thiosulfate determined by suppressed ion chromatography with conductimetric detection

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.