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.

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.

Erythroblastopenia associated with methylmalonic aciduria. Case report and in vitro studies.

Various cytopenias, including neutropenia, thrombocytopenia and pancytopenia, have been reported in association with inborn errors of branched amino acid metabolism. We report here on a case of anemia associated with erythroblastopenia-that is less frequent in this context-in a neonate with methylmalonic aciduria. We used a semisolid erythroid culture system to investigate the effect on in vitro erythropoiesis of organic acids found in excess in this patient: methylmalonic (MMA) and propionic (PA) acids. First, the addition of 10% serum of the patient to a normal bone marrow progenitor culture suppressed the erythrocyte colony-forming unit growth in comparison to a pool of normal serum, while the addition of a 1:1 mixture of normal and patient serum resulted in an intermediate inhibition. MMA, when added to culture medium, resulted in a moderate inhibition of erythropoiesis only at a higher concentration than observed in our patient or reported in other cases. Conversely, PA showed an inhibitory effect at a concentration commonly observed in methylmalonic aciduria. The same effect was observed when the cells were in the presence of PA only for 72 h and then secondly plated in semisolid culture. Neither MMA nor PA showed any effect on the cell number and viability after a 3-, 4- or 7-day incubation except at the highest concentration tested.

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.

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.

Plasma and urinary cortisol levels after dexamethasone in affective disorders.

The validity of the dexamethasone suppression test was evaluated for the differential diagnosis of primary and secondary depressions. Forty hospitalized psychiatric patients (14 primary depressed, 15 secondary depressed and 11 non-depressed controls) were studied. The Research Diagnostic Criteria of Spitzer et al. (1978) were used to classify these patients. Eight out of the 14 primary depressed patients had an abnormally high plasma cortisol at 4 p.m. after dexamethasone. Only 2 out of the 15 secondary depressed patients and none of the 11 controls had an abnormal response to dexamethasone. Based on these results, the dexamethasone suppression test has a sensitivity of 57%, a specificity of 87% and a predictive value of 80%. The determination of urinary free cortisol excretion does not improve the performance of the test.

Reinvestigation of disulfiram-like biological activity of Coprinus atramentarius (Bull. ex Fr.) Fr. extracts.

A ninhydrin-positive aqueous fraction isolated from Coprinus atramentarius (Bull. ex Fr.) Fr. showed significant disulfiram-like activity in mice. The isolation of the fraction from the mushroom crude extract and a convenient pharmacological test for following the physiological activity in each fraction are described.

Intravenous loading with arginine-hydrochloride and ornithine-aspartate in siblings of two families, presenting a familial neurological syndrome associated with cystinuria.

1. Arginine-hydrochloride and ornithine-aspartate solutions have been infused intravenously to children of two families. Three children of the WOL. family are affected with hyperargininemia and hyperammonemia, due to a lack of arginase. They present a secondary cystine-lysinuria. The three WIL. siblings are suffering from muscular hypotonia, dwarfism, incomplete renal tubular acidosis and primary cystinuria. 2. The aim was to verify how and to what extent the artificial rise of one serum amino acid could influence the serum concentrations and the urinary losses of the other amino acids. The results found for the serum have been submitted to a statistical analysis of variance. 3. The variations observed for the amino acids of the urea cycle can be interpreted as being the reflections of known metabolic pathways. 4. Additional remarks are made on a paradox in the lysinemia-lysinuria relation after arginine infusion, with a simultaneous rise of this essential amino acid in serum and urine.