Advances in the metabolic profiling of acidic compounds in children's urines achieved by comprehensive two-dimensional gas chromatography.

The main objective of this work was to evaluate a comprehensive two-dimensional gas chromatographic (GCxGC) coupled to quadrupole mass spectrometry (qMS) method in the field of biomarker candidates' discovery. To this purpose we developed a GCxGC-qMS method suitable for the separation of organic acids and other classes of compounds with silylable polar hydrogen such as sugars, amino-acids, and vitamins. As compared to those obtained by a widely used 1D-GC method, the urinary chromatographic profiles performed by the proposed 2D-GC method exhibit higher resolution and sensitivity, leading to the detection of up to 92 additional compounds in some urine samples including some well-known biomarkers. In order to validate the proposed method we focused on three metabolites of interest with various functional groups and polarities including CH3-malonic acid (MMA: biomarker of methylmalonic acidemia), 3-hydroxy-3-methyl-glutaric acid (3-OHMGA: biomarker of 3-hydroxy-3-methylglutaric acidemia), and phenylpiruvic acid (PhPA: marker of phenylketonuria). While these three metabolites can be considered as representative of organic acids classically determined by 1D-GC, they cannot be representative of new detected metabolites. Thus, we also focused on quinolic acid (QUIN), taken as an example of biomarker not detected at basal levels with the classical 1D GC-qMS method. In order to obtain sufficient recoveries for all tested compounds, we developed a sample preparation protocol including a step of urea removal followed by two extraction steps using two solvents of different polarity and selectivity. Recoveries with the proposed method reached more than 80% for all targeted compounds and the linearity was satisfactory up to 50µmol/L. The CVs of the within-run and within-laboratory precisions were less than 8% for all tested compounds. The limits of quantification (LOQs) were 0.6µmol/L for MMA, 0.4µmol/L for 3-OHMGA, 0.7µmol/L for PhPA, and 1µmol/L for QUIN. The LOQs of these metabolites obtained by a classical GC-MS method under the same chromatographic conditions were 5µmol/L for MMA, 4µmol/L for 3-OHMGA, 6µmol/L for PhPA while QUIN was below the limit of detection. As compared to 1D-GC, these results highlight the enhanced detectability of urine metabolites by the 2D-GC technique. Our results also show that for each new detected compound it is necessary to develop and validate an appropriate sample preparation procedure.

[Validation and study of the transferability of the selected technique by SFBC for the determination of creatinine in plasma]. / Validation et étude de la transférabilité de la technique sélectionnée par la SFBC pour le dosage de la créatinine dans le plasma.

A selected method for the determination of creatinine in plasma, using the reaction with alkaline picrate without prior pretreatment has been proposed by the Commission 'Validation de techniques' in the SFBC (Société Française de biologie clinique). The transferability step was conducted in seven laboratories, equipped with different automatic analyzers, using analytical procedures derived from the recommended method. Its goal was to test whether the original analytical performances could be maintained and consistent results obtained. The validation step was designed to evaluate the linearity limits of the analytical range, the detection limit, to assess accuracy as compared to a high performance liquid chromatography and to investigate the effect of the main interferents. Linearity limits are 15 and 2000 mumol/L. The detection limit is 3 to 8 mumol/L according to the analytical systems. The selected method can fulfil the set imprecision goals: intralaboratory CV minus than 2% (within-run), minus than 4% (run-to-run), interlaboratory CV minus than 5% (for 100 mumol/L creatinine). Inaccuracy evaluated for the chosen control sera is 1 to 15% as compared to the chromatographic method, according to the sera and to the analytical systems. The results obtained with the selected method are more consistent with the HPLC than are those obtained with an alkaline picrate method without SDS or with an enzymatic method. No interference could be demonstrated for acetoacetate (up to 8 mmol/L), hemoglobin (up to 210 mumol/L), unconjugated bilirubin (up to 250 mumol/L), glucose (up to 30 mmol/L), IgG (up to 45 g/L), albumin (up to 60 g/L). The effect of cephalosporins depends on the molecule. The reagents are stable for at least 6 months when stored in closed vials at +20 degrees C. The alkaline reagent is stable 30 days at +4 degrees C. Reference limits (0.025 and 0.975 fractiles) have been established for healthy adults. They are respectively 73 to 126 mumol/L for men and 59 to 100 mumol/L for females.

[Method selected for the determination of creatinine in plasma or serum. Choice of optimal conditions of measurement]. / Technique sélectionnée pour le dosage de la créatinine dans le plasma ou le sérum, Choix des conditions optimales de mesure.

The method selected by the SFBC (Société française de biologie clinique) is derived from the colorimetric reaction of creatinine with alkaline picrate, measured kinetically, without any pretreatment step. The key parameters of the reaction determining the quality of the results are studied, with special regard to samples including known interferents. The aims of the study were to gain an optimal analytical sensitivity and to reduce main interferences (acetoacetate, bilirubine, glucose, protein) which plague the Jaffé reaction, through a comprehensive study of the reagents, of their concentrations and of the analytical procedures. The selected concentrations (in the test) are: 150 mmol/L sodium hydroxide, 10 mmol/L picric acid and 2 g/L sodium dodecyl sulfate. Ten millilitres of a BRIJ solution (30% volvol) are added to the reagent. The operating procedures are as follow: sample ratio 0.07 to 0.08; wavelength 505 to 510 nm; temperature 37 degrees C; incubation of the specimen with the alkaline reagent 5 mn (at least), before starting the reaction with picric acid. A seric calibrator is recommended. The first measurement is taken 20 to 40 s after starting the reaction. Total measurement time is 120 to 150 seconds.

Sucrase-isomaltase deficiency: changing pattern over two decades.

Twenty-two cases of sucrase-isomaltase deficiency (SID) were observed over a period of 20 years. Since 1977 delay of introduction of sucrose and its decrease in infants' diets have modified the symptomatology. In general, onset of diarrhea has not taken place immediately but 15 days to 2 months after introduction of sucrose. Out of 12 cases with dehydration, five occurred 3 to 7 months after the beginning of sucrose diet. Hypotrophy was not constant (11 of 22 cases), thus diagnosis was delayed in 17 of 22 cases. A yellow complexion due to rising carotene levels in the blood is a striking feature. Because of falsely positive sucrose load tests (four out of 14 nonSID infants) and failure of the hydrogene breath test (one out of five studied cases), disaccharidase determination remains the key to diagnosis. Despite the genetic difference symptoms seem to depend on infant feeding practices.

Method of measurement of pancreatic elastase II activity and postnatal development of proteases in human duodenal juice and bovine and porcine pancreatic tissue.

A specific method for pancreatic elastase II activity analysis was developed. True elastase II activity could be discriminated from that of elastase I and chymotrypsin. The postnatal development of four pancreatic proteases in the duodenal juice of children and in the pancreatic homogenates of calves and piglets was measured. The study was carried out on patients without (14 children) and with (5 children) pancreatic insufficiency. Calves and piglets were either milk-fed or weaned until slaughter at different ages. Profiles of enzyme development were globally similar in milk-fed piglets and calves, while in children without pancreatic insufficiency, no significant change was observed between 4 and 168 months. In children with pancreatic insufficiency, enzyme activity was low. In animals, elastase II and chymotrypsin activities were maximal at birth, decreased with age, and probably were associated with the digestion of milk protein. In contrast, elastase I and trypsin activities increased markedly after weaning in connection with the intake of solid food.