Comparison of the EMIT test with high-performance liquid chromatography for the determination of phenobarbital in serum.

The new enzyme immunoassay technique (EMIT) is compared to high-performance liquid chromatography used for determining phenobarbital in serum. After resuming the methods of both techniques, we compare the results of 116 sera collected from patients receiving treatment for epilepsy. The equation of the correlation curve is: HPLC = 0.98 EMIT + 0.12. The reliability characteristics are comparable. The enzyme immunoassay technique is less specific but more practicable.

Diagnosis of alcaptonuria: rapid analysis of homogentisic acid by HPLC.

The clinical and biochemical features of five cases of alcaptonuria were reported. The concentration of homogentisic acid was determined in urine and also in plasma using a rapid, sensitive and specific HPLC method. In all five cases, the concentrations of homogentisic acid were elevated in urine rising up to 46.5 mmol/24 h. In plasma, homogentisic acid levels ranged from 33 to 38 mumols/l. In healthy individuals, homogentisic acid was not detectable in plasma and urine. The method described represents a very useful and suitable analytical tool for the diagnosis of alcaptonuria.

Hypoxanthine and xanthine concentrations determined by high performance liquid chromatography in biological fluids from patients with xanthinuria.

In the present paper, we report the biochemical features of six cases of xanthinuria. For these studies, the concentrations of hypoxanthine and xanthine have been measured in urine, plasma and also erythrocyte samples by a rapid, sensitive high performance liquid chromatographic (HPLC) method. The analyses of plasma and erythrocyte samples require a very sensitive method relative to physiological concentrations and rigorous sampling conditions in order to achieve accurate results. In the cases reported in the literature, total oxypurine levels (hypoxanthine + xanthine) have been generally measured in plasma and urine by an enzymatic spectrophotometric method. In our studies, using HPLC, we found that xanthine is the major oxypurine compound in plasma and urine samples from patients with xanthinuria. In erythrocytes, a biological sample which has not been analysed up to now, we found that xanthine is present at high concentrations whereas it is not detectable in erythrocytes from healthy subjects.

Comparison of capillary electrophoretic and liquid chromatographic determination of hypoxanthine and xanthine for the diagnosis of xanthinuria.

A capillary electrophoretic (CE) method for the determination of hypoxanthine and xanthine in urine was developed to diagnose xanthinuria. The linearity was excellent up to 200 mumol l-1 for the two compounds and the limit of quantitation was 2 mumol l-1. A comparison o the results obtained using CE was made with those obtained by the high-performance liquid chromatographic (HPLC) technique described previously. With regard to specificity, sensitivity and reproducibility, the results are similar but CE is more rapid than HPLC.

Capillary electrophoretic analysis of DNA restriction fragments and PCR products for polymorphism and mutation studies in cystic fibrosis and Gaucher's disease.

Two methods are described for the analysis of DNA restriction fragments and PCR products in studies on polymorphism and mutation in cystic fibrosis and Guacher's disease, based on capillary electrophoresis. In one CE system, a Beckman kit for producing a chemical gel (polymerized within the capillary) is used for single-stranded DNA fragments from 10 to 300 bases in size. Its performance was demonstrated on the separation of a mixture of polydeoxyadenylic acids p(dA)40-60 at 30 degrees C. Electrokinetic injection was used (5-7 kV for 5-20 s), the applied field being 300 V cm-1 for an effective length of 7, 20 or 30 cm and 100 microns i.d., with Tris-borate buffer containing urea. Typical electropherograms are presented for the analysis of CF mutation delta F508 in PCR products from homozygous and heterozygous individuals, illustrating the resolution of two complementary single strands (95b and 95b) of a DNA fragment. DNA fragments differing in size by only one base could also be resolved, as shown for the 105b and 106b fragments obtained from a heterozygote for 3905 insT CF mutation, with a run time of ca-45 min. If discrimination were only required between fragments differing by two or more bases, run times could be reduced by 6 when using a capillary length of only 7 cm x 100 microns i.d.(ABSTRACT TRUNCATED AT 250 WORDS)

[Polyethylene glycol-adenosine deaminase: a new adenosine deaminase deficiency therapy. Value of deoxyadenosine triphosphate determination for therapeutic monitoring]. / Le polyéthylène glycol-adénosine désaminase: nouveau traitement du déficit en adénosine désaminase. Intérêt de la détermination du désoxyadénosine triphosphate dans la surveillance thérapeutique.

The effect of polyethylene glycol-adenosine deaminase (PEG-ADA) therapy on biochemical, immunological and clinical abnormalities in an ADA-deficit child with severe combined immunodeficiency has been studied. Following PEG-ADA therapy, total lymphocytes, lymphocyte subsets (CD3, CD4 and CD8) and the response of lymphocytes to non specific mitogens increase significantly. The improvement of immunological functions is closely related to a decrease of erythrocyte deoxyadenosine triphosphate (dATP) concentrations. This study shows that PEG-ADA therapy is sufficiently effective to reduce and to maintain erythrocyte dATP levels at values compatible with normal immune functions. PEG-ADA represents an important progress for the treatment of ADA deficiency associated with severe combined immunodeficiency disease.

High-performance liquid chromatographic determination of thiopurine metabolites of azathioprine in biological fluids.

A selective and sensitive reversed-phase liquid chromatographic method for the analysis of thiopurine bases, nucleosides and nucleotides in biological samples was developed. A simple and rapid sample treatment procedure using perchloric acid deproteinization with dithiothreitol for the analysis of thiopurine bases and nucleosides is presented. The addition of dithiothreitol during sample collection and treatment improves recoveries. This procedure also allows the determination of thiopurine nucleotides by hydrolysis to their free bases after heating of the perchloric acid extract. The method was applied to the analysis of thiopurine metabolites in plasma and erythrocytes from lung-transplant patients under azathioprine therapy.

Liquid-chromatographic measurement of purine nucleotides in blood cells.

In this anion-exchange "high-performance" liquid-chromatographic method of analysis for purine nucleotides, the nucleotides are separated with high efficiency and selectivity on a weak anion exchanger (Hypersil APS 2, 3-micron particle size) by elution with a gradient of eluent pH and concentration. Applying this method to analysis for these compounds in human blood cells, we determined them in a patient with adenosine deaminase deficiency who was treated with a bone-marrow transplantation, finding that the transplantation did not entirely correct the patient's abnormalities of purine metabolism.

Liquid-chromatographic study of purine metabolism abnormalities in purine nucleoside phosphorylase deficiency.

Using HPLC methods, we measured the concentrations of nucleosides and nucleotides for a patient with no purine nucleoside phosphorylase (PNP; EC 2.4.2.1) enzymatic activity. Concentrations of inosine and guanosine were abnormally high in urine and plasma, whereas guanosine diphosphate (GDP) and guanosine triphosphate (GTP) concentrations in erythrocytes were depleted. The unusual presence of deoxyribonucleosides (deoxyinosine and deoxyguanosine) and deoxyribonucleotides (dGDP and dGTP) was also notable. Thus, HPLC represents an accurate and useful tool for the study of purine metabolic disorders.

Hypoxanthine and xanthine levels determined by high-performance liquid chromatography in plasma, erythrocyte, and urine samples from healthy subjects: the problem of hypoxanthine level evolution as a function of time.

The levels of hypoxanthine and xanthine are determined in plasma, erythrocyte, and urine samples by a reverse-phase high-performance liquid chromatographic (HPLC) method. The hypoxanthine concentration increases in erythrocyte and plasma samples when whole blood is stored at room temperature between sampling and centrifugation. Furthermore, the hypoxanthine concentration increases in erythrocyte samples when they are kept apart at room temperature before analysis, whereas the plasma hypoxanthine level remains constant. This result proves an endogenous formation of hypoxanthine in erythrocytes with time, at room temperature. These studies show the necessity of rigorous conditions for the collection, transport, and treatment of blood samples. In order to achieve accurate results, the blood must be centrifuged immediately after collection. The erythrocyte and plasma samples must be stored frozen until deproteinization and HPLC analysis. Under these conditions, the concentrations of hypoxanthine and xanthine in plasma are 2.5 +/- 1 and 1.4 +/- 0.7 microM, respectively. In erythrocyte samples, hypoxanthine concentration reaches 8.0 +/- 6.2 microM.

High-performance liquid chromatographic determination of hypoxanthine and xanthine in biological fluids.

A rapid and selective reversed-phase high-performance liquid chromatographic method for the simultaneous determination of hypoxanthine and xanthine in biological fluids was developed. The identification of hypoxanthine and xanthine was confirmed by xanthine oxidase reaction. This method was applied to the investigation of purine metabolism in subjects with xanthine oxidase deficiency or gout. Hypoxanthine concentrations three to ten times higher than those determined in plasma were found in erythrocyte samples from normal subjects and from patients with xanthine oxidase deficiency or hyperuricemia under allopurinol therapy.

Homogentisic acid determined in biological fluids by HPLC.

In this rapid, specific, and sensitive high-performance liquid-chromatographic method of analysis for homogentisic acid in biological fluids, homogentisic acid is separated on a column of Nucleosil CN. This method, which we applied to the diagnosis of three cases of alcaptonuria, represents a suitable analytical tool for the diagnosis of alcaptonuria.

[Hyponatremia and theophylline in very low birthweight premature infants]. / Hyponatrémie et théophylline chez le prématuré de très petit poids de naissance.

A retrospective study of the occurrence of hyponatremia (i.e. less than 135 mmol/l) was carried out in 17 control preterm neonates and 25 infants treated with theophylline. These infants had a birthweight lower than 1,301 g and a gestational age less than 36 weeks. Despite a similar sodium and water intake in both groups, hyponatremia was detected at the age of 7-8 days in 37% infants treated with theophylline and none of the control infants. Their mean weight loss was 11% with theophylline and 5% in the controls. These data suggest that theophylline may play a role in the occurrence of hyponatremia of very low birthweight infants.

Metabolism of theophylline to caffeine in premature newborn infants.

Plasma concentrations of theophylline and caffeine in seven premature neonates receiving theophylline orally for treatment of apnea at age one to 9 days were measured by high performance liquid chromatography, ultraviolet spectrophotometry, and mass spectrometry. Plasma concentrations of caffeine increased from 1.8 mg/l (range = 0.1 to 3.7) at day one to 3.7 mg/l (1.3 to 8.0) seven days after initiation of theophylline therapy. Similarly, plasma concentrations of theophylline were 4.6 mg/l (1.5 to 7.5) and 11.0 mg/l (4.0 to 19.0) on days one and 7 of theophylline therapy, respectively. In contrast, four normal adult volunteers given theophylline orally for eight to ten days had plasma theophylline concentrations ranging from 3 to 14 mg/l but no measurable caffeine. This indicates that caffeine is a biotransformation product of theophylline in premature neonates and that the metabolic pathway followed by theophylline in premature infants includes a methylation reaction producing caffeine, whereas in adults, the major metabolic pathway involves oxidative reactions (demethylation and oxidation). Some pharmacologic effects attributed to theophylline during chronic therapy for apnea may in part be due to caffeine. Routine monitoring during theophylline therapy in premature neonates with apnea should include plasma concentrations of both theophylline and caffeine in order to assess the total methylxanthine load.

[Use of thiopental in man. Determination of this drug and its metabolites in plasma and urine by liquid phase chromatography and mass spectrometry]. / Utilisation du thiopental chez l'homme. Détermination de ce médicament et de ses métabolites dans le plasma et les urines par chromatographie en phase liquide et spectrométrie de masse.

Thiopental is an anaesthetic drug which is currently used for cerebral resuscitation. In this last indication the drug is administrated at high doses over a period of several days. Under clinical trials thiopental and two metabolites namely 5 ethyl-5 (1' methyl-3' hydroxy-butyl) 2 thiobarbituric acid and 5 ethyl-5 (1' methyl-3' carboxy-propyl) 2 thiobarbituric acid have been determined by high performance liquid chromatography and mass spectrometry. In human plasma high concentrations of thiopental and 5 ethyl-5 (1' methyl-3' hydroxy-butyl) 2 thiobarbituric acid and a lower concentration of 5 ethyl-5 (1' methyl-3' carboxy-propyl) 2 thiobarbituric acid have been found. In urine samples these metabolites are excreted in large and approximately equal quantities, whereas small amounts of thiopental were recovered.