Claude Bachmann

Endogenous synthesis and transport of creatine in the rat brain: an in situ hybridization study

Creatine is synthesized from arginine by L-arginine:glycine amidinotransferase (AGAT) and S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT) and can be taken up by cells by creatine transporters (CRT). While creatine is mainly synthesized by the liver and the kidney, most of other tissues, including the brain, also express AGAT and GAMT. There is evidence that the permeability of the blood-brain barrier (BBB) for creatine is limited, suggesting that the brain is dependent on its own creatine synthesis. In order to better understand creatine synthesis and transport in the central nervous system (CNS), we studied the regional distribution of cells expressing AGAT, GAMT and the creatine transporter CRT1 in the adult rat brain by non-radioisotopic in situ hybridization. AGAT and GAMT presented an ubiquitous neuronal and glial expression, whereas CRT1 was present in neurons and oligodendrocytes throughout the brain, but not in astrocytes. This indicates that all cells in the CNS can synthesize creatine from arginine. The absence of expression of CRT1 in astrocytes and particularly in those contacting capillary endothelial cells (BBB) reinforces the idea that under normal conditions the creatine used by the brain is synthesized mainly in the CNS. Furthermore, the expression of CRT1 by neurons and oligodendrocytes indicates that creatine trafficking is possible in those brain areas of main creatine consumption.

Urinary phosphate/creatinine, calcium/creatinine, and magnesium/creatinine ratios in a healthy pediatric population

OBJECTIVE To determine reference values for urinary phosphate/creatinine (Cr) concentration ratios and to complete reference values for urinary calcium/creatinine and magnesium/creatinine ratios in the second morning urine sample of healthy infants, children, and adolescents. DESIGN Urinary P/Cr, Ca/Cr, and Mg/Cr ratios were determined from the second morning urine sample. Two urine samples were obtained 1 week apart from most subjects to assess reproducibility. SETTING Kindergartens and schools of Lausanne, Switzerland. PARTICIPANTS A total of 410 healthy children aged 1 month to 17 years (197 girls and 213 boys) participated in the study. RESULTS The 5th and 95th percentiles were estimated from 664 urine samples. There were no differences related to sex. A nonlinear regression in terms of age was used to smooth the estimated percentiles yielding reference curves from which critical values may be obtained for any given age. The 95th percentile for urinary Ca/Cr and Mg/Cr agreed with previously reported values in children older than 7 years. The upper limit of the three solute/creatinine ratios decreased significantly with age: for urinary P/Cr from 19.0 mol/mol at 1 month to 2.7 at 14 years; for urinary Ca/Cr from 2.2 to 0.7 mol/mol, and for urinary Mg/Cr from 2.2 to 0.6 mol/mol. Lower limits varied little. Interindividual and intraindividual variations decreased with age. CONCLUSIONS Urinary P/Cr, Ca/Cr, and Mg/Cr ratios vary strongly with age. We provide reference values, expressed both in SI and in mass units, for urinary P/Cr, Ca/Cr, and Mg/Cr in children aged one month to 17 years.

Ammonium-Induced Impairment of Axonal Growth Is Prevented through Glial Creatine

Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cotreatment protected axons from ammonium toxic effects, although this did not restore high-energy phosphates. The protection by creatine was glial cell-dependent. Our findings suggest that the means to efficiently sustain CNS creatine concentration in hyperammonemic neonates and infants should be assessed to prevent impairment of axonogenesis and irreversible brain damage.

Progressive neuropathy and recurrent myoglobinuria in a child with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency

From the Department of Metabolism, Bambino GesO Childrens Hospital, lstituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy, the Department of Pediatrics, University of Padua, Padua, Italy, the Department of Clinical Chemistry, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland, and the Division of Endocrinology and Diabetes, Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania

Assay of D-lactate in urine of infants and children with reference values taking into account data below detection limit

Accumulation of D-lactic acid produced by intestinal bacteria such as streptococci and lactobacilli has been extensively studied in ruminants [1-4]. In humans an increased production of D-lactate by intestinal bacteria under pathological conditions such as the short bowel syndrome can cause metabolic acidosis [5-8]. Since the lactate assays routinely used only measure L-lactate we developed a sensitive method of D-lactate quantification and established reference values in spot urines of infants and children (0 to 4 years of age). The enzymatic method with fluorimetric quantification of NADH is linear up to 2 mmol/l. It has a detection limit of 3.4 micromol/l. Among structurally related organic acids an interference was found only for L-lactate and DL-2-hydroxybutyrate at concentrations which are way beyond their physiological excretion. One hundred and sixty five spot urines of healthy Swiss (S), Austrian (A), German (G) and Chilean (CHI) infants aged from 0 to 4 years were analyzed. The distribution of the data is close to a lognormal one. Values below the detection limit were simulated and age groups were formed. In all populations D-lactate excretion was found highest during the first year of life; it declines with age during infancy and remains stable from 2.5 to 4 years of age. We show that D-lactate is excreted physiologically by healthy infants and children below 4 years of age and present reference values for D-lactate excretion which show some differences between the populations tested.

Microheterogeneity of serum glycoproteins and their liver precursors in patients with carbohydrate-deficient glycoprotein syndrome type I: Apparent deficiencies in clusterin and serum amyloid P

Serum and liver protein patterns were studied, respectively, in 5 patients (serum) and 1 patient (liver) with carbohydrate-deficient glycoprotein syndrome (CDGS) type I by high-resolution two-dimensional electrophoresis (2-DE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pattern of serum glycoproteins in all 5 patients presented abnormal trains of isoforms with decreased mass (delta molecular weight 3000) and all showed a cathodal shift. Two-dimensional electrophoresis and SDS-PAGE mass analysis of transferrin, alpha1 -antitrypsin, haptoglobin beta-chain, and alpha1-acid glycoprotein after neuraminidase and N-glycosidase F treatments demonstrated that the additional trains of the isoforms found in CDGS type I contain homologous species of isoforms. Some of them still showed charge differences, and all still contained glycans except for transferrin, with some unusual nonglycosylated isoforms. In addition, deficiencies in clusterin and serum amyloid P, not described so far, have been found in all 5 patients. The two-dimensional pattern of immunodetected precursors of serum proteins in liver cells from 1 patient with CDGS showed abnormal low-mass precursors and the absence of the precursors normally found in controls. These results suggest that these abnormal precursors accumulate during the early oligosaccharide processing of the nascent protein-bound oligosaccharides and that glycoprotein precursors undergo an altered intracellular transport while the post-translational processing along the normal pathway is still apparently functioning in patients with CDGS.

Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycaemic conditions

The neuronal effects of glucose deficiency on amino acid metabolism was studied on three‐dimensional cultures of rat telencephalon neurones. Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone‐specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult. Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose‐deficiency. Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased. Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage. Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage. Both lactate and glutamine were readily oxidized in these neuronal cultures. The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions. This results in increased ammonia production and neuronal damage.

Isovaleric acidemia: Identification of isovalerate, isovalerylglycine, and 3-hydroxyisovalerate in urine of a patient previously reported as having butyric and hexanoic acidemia

Urine from one of the original patients with butyric and hexanoic aidemia was examined for the possible excrection of glycine conjugates of butyryl-CoA and hexanoyl-CoA. Thin-layer chromatography of acylglycines revealed a distinct spot with an Rf values imilar to that of isovalerylglycine. Gas liquid chromatography of a distillate of urine showed a peak with the same retention time as the isovaleric acid and a second peak with a retention time longer than that of hexanoic acid. Furthermore, gas liquid chromatography of the trimethylsilyl derivative of an extract of urine showed peaks of isovalerylglycine and β-hydroxyisovaleric acid. The structure of these compounds was established by mass spectrometry. These findings indicate that the case reported actually had isovaleric acidemia.

Organic acids in the second morning urine in a healthy Swiss paediatric population.

Abstract Organic acid analysis is used for the early detection/exclusion and for the follow-up of inherited disorders of amino acid and organic acid metabolism. Urinary organic acid concentrations in 417 healthy Caucasian children (1 day to 17 years of age) were determined after liquid solid extraction, as their trimethylsilyl derivatives, by gas chromatography and mass spectrometry. Concentrations of most of the organic acids adjusted for creatinine tend to decrease with age. No differences were found between gender except for the Krebs cycle intermediates in the older age groups. In neonates, the immaturity of the neonatal kidney led to a much larger variation of organic acid levels when related to creatinine. The low number of subjects (n = 36−52) per age class resulted in large 95% confidence intervals of the percentiles used for decision. This must be taken into account when using the data for exclusion or diagnosis of disorders.

Clinical chemistry variables in normal elderly and healthy ambulatory populations : comparison with reference values

Laboratory values of the most commonly assayed clinical chemistry variables were determined in selected elderly and healthy ambulatory populations. The upper and lower limits (2.5 and 97.5 fractiles) were compared with the adult reference values in use in university hospitals of Switzerland. The results suggest that conventional adult reference values can be used for most variables in the elderly and that these values are also useful in an ambulatory population.