LC-MS/MS determination of dibasic amino acids for the diagnosis of cystinuria. Application in a family affected by a novel splice-acceptor site mutation in the SLC7A9 gene.

Cystinuria is an autosomal recessive disorder caused by defective transport of cystine and the dibasic amino acids ornithine, lysine and arginine across cell membranes. Poor solubility of cystine in urine leads to kidney stones and associated symptoms and complications. Mutations of genes SLC3A1 and SLC7A9 encoding for amino acid transport systems are responsible for different types of cystinuria. In this study we describe a new LC-MS/MS assay for these amino acids in urine. Moreover, we report a novel splice-acceptor site mutation in the SLC7A9 gene that we believe is the cause of the phenotype observed in four siblings from a first-cousin marriage. Into the wells of a 96-well microtitre plate, 10 microl of urine was mixed with 90 microl of a solution containing [(2)H4]cystine, [(2)H2]ornithine, [(13)C,(2)H4]arginine and [(2)H5]glutamine that was used as an internal standard for lysine. Chromatographic separation was achieved isocratically and detection was in the selected-reaction monitoring mode. The injection-to-injection time was 8 min. Calibration curves were linear up to 1000 micromol/L. Intra-day (n = 10) and inter-day (n = 6) variations (750 and 10 micromol/L) were less than 11.4%. Urine samples from healthy individuals (n = 135) were analysed and age-matched reference ranges were generated. The method was applied retrospectively and prospectively to analyse samples (n = 13) from nine cystinuria patients. The mutation reported here was not found in 100 controls with similar ethnicity to the studied family and is believed to have consequences for the transcribed mature RNA and protein structure and function.

Urological intervention in patients with cystinuria is decreased with medical compliance.

PURPOSE: Theoretically patients with cystinuria compliant with medical treatment should undergo fewer surgical procedures than those noncompliant with treatment. We describe a single urologist's experience (SYN) with the effects of medical management on the number of surgical interventions required in patients with cystinuria treated at our metabolic stone clinic (MSC). MATERIALS AND METHODS: The records of 20 patients with cystine stones seen at our MSC (mean followup of 42.5 months) were evaluated. The number of surgical interventions (endourology or open surgery) during followup were recorded as surgical events. Patients were placed into a compliant or noncompliant category based on their attendance record at scheduled appointments at our MSC and whether they adhered to the prescribed medical regimen by report. Patients were classified in an active disease category if recent imaging revealed a clinically significant stone burden greater than 3 mm. Statistical analysis of the number of surgical events for stone-free patients compliant with treatment versus noncompliant was conducted (Student's t test). RESULTS: Of the 20 patients 11 were categorized as compliant and 9 as noncompliant. Of the compliant patients the average number of surgical events was 1.0 per patient versus 4.0 in the noncompliant group (p <0.05). Of the 11 compliant patients 8 (73%) were stone-free compared to 3 (33%) in the noncompliant group. CONCLUSIONS: The majority of our patients with cystinuria compliant with treatment underwent a significantly fewer number of surgical procedures per year than those noncompliant with treatment. Active medical management in patients with cystinuria compliant with treatment decreases the incidence of surgical interventions.

Does carbamazepine-epoxide conjugate with glutathione? On an existing but almost forgotten possibility.

The anticonvulsant carbamazepine is widely used to treat affective disorders and behavioural disorders in non-epileptic children. We report an elevated plasma level of carbamazepine-10,11-epoxide in a cystinuric child after daily medication with 300 mg carbamazepine while the serum level of carbamazepine was in the therapeutic range. The concentrations of carbamazepine and its epoxide derivative were determined by HPLC. The formation of a glutathione conjugate of carbamazepine-10,11-epoxide is raised as a hypothesis.

High-performance liquid chromatography of 2-mercaptopropionylglycine and its metabolite 2-mercaptopropionic acid in plasma and urine after treatment with thiopronine.

2-Mercaptopropionic acid has been identified as a normal metabolite of 2-mercaptopropionylglycine (thiopronine) when this drug was given to humans and dogs. A high-performance liquid chromatographic method was developed to resolve the derivatives of these two thiols and thus enable simultaneous determination of the two compounds in plasma and urine.

Methods for assaying D-penicillamine in a clinical setting.

Penicillamine disulfides have been analysed by automatic amino acid analysis. Because the free thiol reacts poorly with ninhydrin, other detection methods are preferred, particularly high pressure liquid chromatography using an electrochemical detector or gas chromatography with a flame ionization detector. Pharmacokinetic studies have now been reported using these techniques. With patients on established penicillamine regimes, the concentration of free penicillamine in the plasma has been found to vary between 4 and 20 microM depending on dosage and time of administration. Disulfide concentration is higher than this by a factor of 3 or 4 and an even greater quantity is attached to plasma and tissue proteins.

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.

The renal clearance of amino acids in cystinuria.

The renal clearance of cystine, lysine, ornithine, arginine, and glycine has been compared with the simultaneously determined glomerular filtration rate in nine cystinuric patients. Five were studied before and after stabilization on penicillamine therapy, two were studied only while taking penicillamine, and two were studied only in the absence of penicillamine administration. The renal clearances of cysteine-penicillamine and of penicillamine disulfide were also determined in the patients who were taking the drug. Amino acids were determined by quantitative ion exchange chromatography, and the reliability of the method has been evaluated in terms of its reproducibility and of the recovery of known amounts of amino acids added to plasma and to urine. The plasma levels of cystine and of the basic amino acids were less than normal in all the patients. Cysteine-penicillamine and penicillamine disulfide were cleared by the kidney at rates similar to that of cystine. Two of the patients had glycine clearances that were considerably above the normal value. The renal clearance of cystine exceeded the glomerular filtration rate in six of the nine patients. The results form a continuum from values approximately equal to the glomerular filtration rate to values about twice this amount. The renal clearances of cystine and of the basic amino acids vary independently of one another in the disease. The significance of these results is discussed in terms of the nature of the renal tubular transport defect that underlies the disorder.