Cholesterol depletion perturbs calcium handling by rat submandibular glands.

The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption.

Assessment of an electron-impact GC-MS method for organic acids and glycine conjugates in amniotic fluid.

We assessed the reliability of a method designed for common electron-impact GC-MS systems to determine in a single run most organic acids and glycine conjugates of clinical interest in amniotic fluid. Suitable sensitivity was achieved by dividing the selected-ion chromatogram into 12 time segments during which the monitoring dwelt on specific ions. Twelve metabolites were simultaneously quantified in amniotic fluid, with performances ranging from very good to clinically acceptable. The total coefficient of variation was 2.5-14.1% and the detection limit was well below the lower value of the physiological range. For five other metabolites, the precision was lower and/or the detection limit was near the physiological range. The method was clinically assessed by the prenatal detection of three cases of tyrosinaemia type I and one case of propionic acidaemia. Analytical and clinical evaluation of the method showed that GC-MS with electron-impact fragmentation can be an informative analytical approach for low-level organic acids in physiological fluids. Apart from the case of glycine conjugates, the method shows a fair reliability for amniotic fluid analysis, which might warrant its use for prenatal diagnosis of organic acidurias. However, this method cannot replace procedures using isotopic internal standards, nor GC-MS based on chemical ionization fragmentation, which remain confirmatory analytical methods of choice.

Statistics of the Comet assay: a key to discriminate between genotoxic effects.

The alkaline Comet assay is a widely used single cell gel electrophoresis technique for the quantification of DNA strand breaks, crosslinks and alkali-labile sites induced by a series of physical and chemical agents. DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. Breaks are quantified from geometric and fluorescence measurements by image analysis of comet-shaped DNA, often reported parameters being tail DNA and tail moment. Although a variety of statistical approaches have been used in the literature, most of these do not take into account the distribution patterns of comet data. In order to investigate a methodology for statistically demonstrating a comet effect, two different experiments, a reproducibility study and a trend analysis, were undertaken on a murine lymphoma cell line (P388D1) photodynamically stressed after induction of porphyrins with delta-aminolaevulinic acid. This treatment results in significant heterogeneity of DNA damage, producing values ranging from 0 to 100% tail DNA in the same sample. The comparison of distribution curves for stressed and non-stressed samples shows that none of the application conditions are verified, either for parametric tests (which require normal distributions), or non-parametric tests (which assume essentially similar distributions). Meaningful statistics (median and 75th percentile) were consequently extracted from repeated experiments and found suitable for comparing stress conditions in an ANOVA and in a trend analysis; the 75th percentile is theoretically more sensitive but tends to more rapidly saturate at extensive stress levels. We conclude that a trend analysis of median comet metrics from repeated experiments at different stress levels is certainly an efficient way to statistically demonstrate a genotoxic effect. Whether the considered comet parameter is tail DNA or tail moment had no influence on the conclusions of our experiments, which were carried up to stress levels leading to a median 70% tail DNA.

Growth failure, encephalopathy, and endocrine dysfunctions in two siblings, one with 5-oxoprolinase deficiency.

UNLABELLED: Two female siblings, born to consanguineous parents, presented with a similar phenotype characterized by severe growth and developmental failure, dysmorphic features, thyroid and gonadal dysfunction, autistic traits and hand stereotypes resembling Rett syndrome. In the elder patient, analysis of urinary organic acids disclosed a very high excretion of 5-oxoproline (4.2 to 8.1 mol/mol creatinine) and enzyme assays of leucocyte extracts revealed a profound deficiency of 5-oxoprolinase. However, normal urinary organic acid profiles were found in the younger child. In view of their distinct dysmorphic features and severe growth deficiency, these siblings cannot be considered as Rett Syndrome variants. The Dubowitz and carbohydrate-deficient glycoprotein syndromes were also excluded clinically and biochemically respectively. We conclude that these patients suffer from a hitherto undescribed autosomal recessive disorder, unrelated to the 5-oxoprolinase deficiency of the elder sib. CONCLUSION: The present findings give evidence that 5-oxoprolinase deficiency is not associated with a distinct morbid phenotype.

GC-MS profiling of urinary organic acids evaluated as a quantitative method.

We assessed the quantitative performances of a classical method for profiling urinary organic acids: ethyl acetate extraction/oxime-trimethylsilyl derivatization/GC-MS. Twenty-seven acids were quantified on the basis of specific ions in both scan and selected-ion monitoring modes. We found that the tuning of the mass detector severely affects the calibration factors, being critical to achieve quantitative results, and we propose a practical procedure for reproducible tuning. Of seven compounds tested, tropic acid was retained as the internal standard suitable for most of the acids of clinical interest; a second internal standard, 2-ketocaproic acid, was used in quantifying keto-acids. The within-day and total relative standard deviations (CVs), estimated from scan-mode analyses of urine, ranged from 2.6% to 12.7% and from 4.2% to 11.8%, respectively. Curvilinear relationships between analytical response and concentration were observed for most of the acids investigated.

Gas chromatographic profiling and determination of urinary acylcarnitines.

A method is described for the routine profiling and determination in urine of most of the acylcarnitines clinically relevant for the diagnosis of organic acidurias. The procedure, which does not require expensive apparatus, involves extraction of the acylcarnitines on strong cation-exchange disposable columns, mild alkaline hydrolysis and gas chromatography of the liberated monocarboxylic acids. The different steps were optimized in order to increase the analytical performance. No significant interferences were encountered, the limit of detection (signal-to-noise ratio = 3:1) ranged from 0.1 to 4 mg/l and the between-day coefficient of variation from 3.6 to 17.7%, depending on the acyl species. The rapidity of the method results from the application of a single solid-phase extraction on disposable columns. The acyl moieties are chromatographed underivatized in order to permit the identification of short-, medium- and long-chain acylcarnitines. The method was assessed by analysing fourteen urine specimens from patients presenting an organic aciduria.

Congenital absence of insulin cells in a neonate with diabetes mellitus and mutase-deficient methylmalonic acidaemia.

We report on a female neonate with diabetes mellitus and methylmalonic acidaemia, who died at age 16 days. Using immunocytochemistry, electron microscopy and in situ hybridisation, we were unable to demonstrate any insulin cells in the pancreatic islets. Methylmalonic acidaemia was caused by a methylmalonyl coenzyme A mutase apoenzyme defect. The metabolic crisis of the methylmalonic acidaemia aggravated the diabetes and may explain the failure of insulin therapy. Our results suggest that the infant suffered from a congenital absence of beta cells associated with a genetically transmitted mutase apoenzyme defect.

Comparison of two extraction procedures for urinary organic acids prior to gas chromatography-mass spectrometry.

We have compared a new isolation procedure for urinary organic acids using strong anion-exchange columns with a solvent partition (ethyl acetate) method. Urinary samples from two healthy children and from nine children with organic acidurias were analysed by both procedures. Although the solid-phase extraction is more efficient for polyhydroxy acids, some polar acids, and some glycine derivatives, clinically important compounds such as oxalic, methylcitric, pyruvic, glyoxylic and 2-ketoglutaric acids, are not recovered or are only poorly recovered. However, both procedures may be used as a routine method for the diagnosis of the organic acidurias included in this study.

Oxcarbazepine disposition: preliminary observations in patients.

We describe a preliminary retrospective study based on the concentration of two hydroxylated metabolites of oxcarbazepine (OCZ), a new anticonvulsant substance, measured in the plasma of 15 patients with epilepsy. Their ages ranged from 8 to 68 years, 6 of them also received phenobarbital and/or phenytoin as co-medication. The concentration of 10-hydroxy-10,11-dihydrocarbamazepine (HCBZ) or of trans-10,11-dihydroxy-10,11-dihydrocarbamazepine (DHCBZ), the metabolites measured, are significantly correlated with the dose of OCZ (p less than 0.05 and p less than 0.01, respectively). DHCBZ concentrations, standardized to a constant OCZ dose or to a constant HCBZ concentration, are significantly higher during co-medication (p less than 0.01 and p less than 0.05, respectively); HCBZ levels are unaffected. These results confirm that enzyme-inducing drugs, although accelerating the oxidation HCBZ, do not induce its formation. Since HCBZ is the active metabolite, such drug interaction seems unlikely to alter OCZ pharmacological activity.

Cross-reactivity assessment of oxcarbazepine and its metabolites in the EMIT assay of carbamazepine plasma levels.

To study the possible cross-reactivity of trans-10, 11-dihydroxy-10, 11-dihydro-carbamazepine (DHCBZ), the diol metabolite of carbamazepine (CBZ), of oxcarbazepine (OCZ), and of its metabolites in the CBZ-enzyme multiplied immunoassay technique (EMIT), this technique was used to analyze sera spiked with CBZ, OCZ, DHCBZ, and 10-hydroxy-10, 11-dihydro-carbamazepine (HCBZ). OCZ and, to a lesser extent, HCBZ cross-reacted with the CBZ-EMIT reagents. However, from a clinical point of view, only HCBZ could significantly interfere in the quantitation of CBZ levels in the plasma of patients taking both CBZ and OCZ. There was no interference by DHCBZ.

Therapeutic drug monitoring: a comprehensive and critical review of analytical methods for anticonvulsive drugs.

In the last 20 years, there has been considerable improvement in the determination of anticonvulsive drugs in body fluids. Gas-liquid chromatography, high-performance liquid chromatography and immunological methods (radio-, enzyme-, fluoro-, and nephelo-immunoassays) have progressively supplanted spectrophotometry and thin-layer chromatography. As the number of publications shows, these methods have included a great variety of procedures. At present, gas-liquid chromatography and enzyme-immunoassays are routinely performed, awaiting a wider spread of liquid chromatography and other immunological techniques. This paper is a comprehensive review of the analytical literature on the determination of phenobarbitone, primidone, phenytoin, carbamazepine, valproic acid, ethosuximide, clonazepam, and some of their metabolites in physiological fluids. Gas-liquid chromatographic methods are more particularly reviewed. In order to facilitate the choice between these procedures, a critical selection of the techniques is given and recommendations are made. Emphasis is laid on technical problems encountered with the assays, as well as the need for a rigorous analytical assessment and internal and external quality controls. This review is completed by considerations on the determination of the free drug fraction and on sample collection and storage.

Dose-dependency of the ratio between carbamazepine serum level and dosage in patients with epilepsy.

Carbamazepine serum levels have been determined by gas-liquid chromatography in 24 children and 26 adults with epilepsy on chronic carbamazepine treatment. A significant correlation has been found between carbamazepine steady-state levels and doses per kilogram body weight in both children (p less than 0.01) and adults (p less than 0.05). This relationship is characterized by a significant decline in the level/dose ratio with the doses for adults (p less than 0.001) and, to a lesser extent, for children (p less than 0.05). These results are consistent with a dose-dependent bioavailability.