Biomedical subjects
Publications and source records attributed to J P Colombo.
A second patient with N-acetylglutamate synthetase deficiency is described. The first symptoms were noted at 6 days of age. The course was lethal despite vigorous treatment. The diagnosis was established after death.
Quantitative analysis with dry reagent strips for the determination of clinical chemistry parameters and drug monitoring in serum, plasma and whole blood is expanding in small laboratories and in office laboratories. The systems predominantly used are Ektachem DT 60, the Seralyzer and the Reflotron. - The methods offered comprise substrates, enzymes, electrolytes, CO2 and drugs. The spectrum of feasible tests varies with the manufacturer. On the whole all the tests commonly used in small laboratories are covered. The instruments used for quantitative dry reagent strip chemistry are calibrated by the manufacturer. Calibration data are either inputted to the instrument with each method or already programmed in the apparatus. Thus calibration is outside the control of the user, a change of philosophy which does not correspond to present concepts of quality control in clinical chemistry. The analytical precision is satisfactory for all three instruments for the common parameters measured. A comparison between "dry-chemistry" determinations and the classical aqueous analyses show good accuracy as long as patient samples are used. This is, however, not true for all reference materials, presumably due to matrix effects. Our own evaluation of the Seralyzer showed a good correlation of potassium analysis compared with flame photometry; this was also true of gamma-glutamyltransferase using the Reflotron with patient samples. Internal quality control can only be performed with materials assessed by the instrument manufacturers, and the results will often differ for these materials when assayed with conventional methods or with other "dry-chemistry" methods. External quality control also has its limitations due to the fact that no sera are available which could be used universally.(ABSTRACT TRUNCATED AT 250 WORDS)
An enzymatic assay for creatine, depending on the creatine kinase reaction, has been modified for the determination of creatine in packed erythrocytes, using a centrifugal analyzer (COBAS BIO). The method is precise, sensitive and shows excellent accuracy in recovery experiments when compared to the diacetyl-alpha-naphthol method. The enzymatic red cell creatine correlates with the erythrocyte survival time determined with the radioactive chromium method. It can be used as a rapidly available parameter for the quantification of hemolytic processes.
In congenital urea cycle disorders, detoxification of ammonia is impaired, leading to hyperammonemia. Ammonia is the major component causing the acute neurological disturbances. It may influence the supply of substrate and its transport at the blood-brain barrier (BBB) which results in alterations in the synthesis and catabolism of neurotransmitters in the brain. In hyperammonemic rats, the uptake of tryptophan into the brain is increased with an augmented flux through the serotonin pathway. In the forebrain, glutamine as well as amino acids transported with the same L-carrier system, such as phenylalanine, tyrosine and tryptophan, are elevated. It is postulated that the increased transport of tryptophan at the BBB occurs in exchange with glutamine. Methionine sulfoximine (MSO) inhibits glutamine synthetase in the cerebral cortex. The activity drops from 5.85 +/- 0.38 to 1.07 +/- 0.37 mumol/min/g wet weight. Under MSO, the brain tryptophan uptake also decreased to 64.2 +/- 4.5% in hyperammonemic rats, to 54.1 +/- 8.0% in untreated hyperammonemic rats, whereas without MSO an increase of tryptophan uptake was observed. An effect of glutamine on tryptophan transport could also be demonstrated using brain microvessel preparations as a model for the BBB. Our findings indicate that preloading isolated microvessels with L-glutamine increases tryptophan uptake into the endothelia when L-glutamine is at concentrations found in brain homogenates under hyperammonemia. Since brain microvessels do not contain glutamine synthetase activity, enzymes from the gamma-glutamyl cycle may be involved in the glutamine-mediated tryptophan transport.(ABSTRACT TRUNCATED AT 250 WORDS)
Fractional tubular reabsorption (FTR) of free and acyl carnitine was measured in 15 patients with various selective tubular transport defects and in 19 patients with more generalized tubular dysfunction (Fanconi syndrome). FTR of free carnitine was normal in all patients with a selective tubulopathy, FTR of acyl carnitine was normal in most, and plasma carnitine levels were normal without exception. In these patients, there was no evidence for the existence of a defective renal transport mechanism shared by carnitine. In the patients with Fanconi syndrome, mean FTR of free and acyl carnitine was low; their plasma carnitine levels were lowered and correlated with the FTR. In individual patients, FTR of free and acyl carnitine also correlated with the severity of the disease. In the group of Fanconi syndrome patients, FTR of free and acyl carnitine correlated linearly with that of valine. We concluded that the lowering of plasma carnitine in the patients with Fanconi syndrome was caused by excessive loss of carnitine in urine. Its pathophysiological significance remained to be established.
Two patients with neonatal onset of hyperornithinemia-hyperammonemia-homocitrullinuria syndrome were studied at 4 and 2 1/2 yr of age, respectively. The aim of the investigation was to assess the effect of supplementing citrulline, arginine, or ornithine (2 mmol/kg per day) while on a protein-restricted diet. The peroral supplementation was carried out during 2 wk for each amino acid. While ammonia in plasma was not increased the supply of citrulline or arginine led to a reduction of plasma glutamine compared to ornithine supplement or to no supplement (control period). Plasmatic ornithine was raised in all instances. Homocitrulline excretion was lower with all additions compared to the control period. Adding citrulline to the diet (in contrast to supplementing arginine) did not lower tubular lysine reabsorption. A lowered creatine excretion was found which could be normalized by arginine or citrulline. The data are compatible with a product inhibition of arginino-glycine transamidinase suggesting that the enzyme is not located in the mitochondrial matrix in man. Citrulline supplement combined with a protein-restricted diet appears to allow a normal development. The additional finding of a factor VII and X deficiency in one of the patient and reports in the literature of this association in two other patients with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome suggest that the genetic defect leading to the syndrome might be located on chromosome 13.
A simple photometric procedure was developed for the determination of thiocyanate (SCN-) in plasma and saliva without deproteinisation or dialysis. Fe(III) ions form a red coloured complex with SCN- with a maximum absorbance at 460 nm. Mercury(II) nitrate is used to run a sample blank. A manual and an automated version (COBAS BIO) of the method is described. The method is linear up to 5000 mumol/l SCN-. The CV of the between-run precision is 2.8-8% for the manual and 2.6-6.6% for the automated method. The SCN- plasma concentration was 21-134 mumol/l in nonsmokers and 44-260 mumol/l in smokers. In mixed saliva, the concentration is much higher than in plasma: 1.57-5.5 mumol/l in smokers and 0.79-3.9 mumol/l in nonsmokers. Plasma, but not oral fluid SCN-, is a valuable parameter for studying smoking habits in population surveys. However, its use is limited. In our experience only heavy smokers can be distinguished from nonsmokers.
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The uptake of L-tryptophan into isolated porcine microvessels is increased by preincubation with L-glutamine as well as with D-glutamine. This could indicate that gamma-glutamyltranspeptidase is involved in the stimulation of uptake of large neutral amino acids into the brain observed in hyperammonemic conditions.
A protein load with an ensuing 20 hr fast was performed to assess the function of biotin dependent carboxylases in 3 girls with Rett syndrome. In plasma a moderate increase of ammonia and propionate was found. The fact that these discrete biochemical alterations were found in all of the patients supports earlier conclusions that further studies on biotin metabolism and carboxylase function should be performed, perhaps preferably in cerebrospinal fluid.
A patient with Rett syndrome (cerebral atrophy associated with hyperammonemia) was studied. Primary defects of urea cycle enzymes were excluded as causes of the disorder. The analysis of urinary organic acids showed a moderate increase of lactate, methylcitrate, tiglyglycine and 3-hydroxisovalerate, indicating an abnormality of multiple carboxylases. Biotin supplementation reversed the urinary abnormalities. In fibroblasts grown with a low biotin medium propionylCoA and 3-methylcrotonylCoA carboxylase activities were reduced. Holocarboxylase synthetase activity was normal (Vmax and Km). Surprisingly the biotinidase in fibroblasts was not decreased. The data indicate that some patients with Rett syndrome might suffer from a biotin-dependent defect of unknown nature.
The tryptophan uptake into forebrain and brainstem was measured in urease-injected rats and in controls after prior application of sodium benzoate or hippurate. Benzoate led to an increased brain uptake index of tryptophan. This effect was additive to the increase in tryptophan uptake effected by the provoked hyperammonemia. In contrast, hippurate did not alter tryptophan uptake across the blood-brain barrier. We conclude that benzoate should be monitored especially when applied intravenously to patients with hyperammonemic coma.
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Arginase from normal human mixed saliva was characterized. The enzyme was completely activated after a preincubation of 20 min at 55 degrees C and a Mn2+ final concentration of 5 mmol/l. The pH optimum was 9.6-9.8, and the Km for L-arginine was 4.2 +/- 0.7 mmol/l. In normal saliva only one form was found, which was chromatographically identical with the cationic form of arginase in liver and blood cells. Salivary arginase was completely precipitated by rabbit antiserum against human liver arginase. Arginase activity was not detectable in the saliva of patients suffering from argininaemia. Enzyme activities in the saliva of the heterozygous parents and the unaffected daughter were 0.08, 0.07 and 0.12 U/mg protein, respectively, whereas the activities in the saliva of 60 healthy adults and 8 children were 0.17 +/- 0.11 and 0.16 +/- 0.06 U/mg protein, respectively.
We evaluated the new Greiner G-400 selective multichannel analyzer. Thirty different tests can be run in combination profile and single-test mode at a rate between 150 and 300 tests per hour. The new photometer allows kinetic and equilibrium (endpoint) measurements. The G-400 analyzer demonstrated excellent precision, linearity, accuracy, ease of operation, and no carryover. Results for 16 different analytes as determined with the Hitachi 705, GSA II, and Corning 940 correlated favorably with those obtained with the G-400. The G-400 analyzer is suitable for both emergency and routine use.
Many, mostly congenital, hyperammonemic syndromes may occur in the first days of life. For the diagnosis of these disorders plasma ammonia levels physiologically present in neonates and early childhood should be known, preferably by the use of an enzymatic method. Full-term newborns had higher capillary ammonia concentrations on the first day of life (30-144 mumol/l) than on the fifth day (31-104 mumol/l). In the same individuals the arterial (38-89 mumol/l) are lower than the capillary concentrations (69-112 mumol/l). The venous concentrations in school children were not higher than in adults. The scatter of capillary values, however, is great. For diagnostic purposes of disorders of ammonia detoxication sampling of arterial or venous blood is preferable.
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Sparse-fur mice, 28 d of age with the x-chromosomal inherited defect of ornithine carbamoyltransferase, were used to investigate if tryptophan and the serotonin pathway in the brain are affected in this animal model which closely resembles the human inborn error of metabolism. Increased concentrations of tryptophan and 5-hydroxyindole acetic acid were found in forebrain and brainstem. Application of probenecid, which blocks the efflux of 5-hydroxyindole acetic acid from the brain, led to an augmented accumulation of this serotonin metabolite in the affected males. We conclude that the increased concentration of tryptophan in brain and the subsequent increased flux through the serotonin pathway are a consequence of hyperammonemia in this inherited defect of urea synthesis. Surprisingly, increased carbamoylphosphate synthetase was found in the liver of the male sparse-fur mice.