Erik Gerlo

Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12

In patients with mitochondrial encephalomyopathies an increasing number of causative gene defects have been detected. The number of identified pathogenic mitochondrial DNA mutations has largely increased over the past 15 years. Recently, much attention has turned to the investigation of nuclear oxidative phosphorylation (OXPHOS) gene defects. Within the OXPHOS defects, complex V deficiency is rarely found and, so far, these defects have only been attributed to mutations in the mitochondrial MTATP6 gene. Mutation analysis of the complete coding regions at the cDNA level of the nuclear ATP11, ATP12, ATPα, ATPβ and ATPγ genes and the mitochondrial MTATP6 and MTAT8 genes was undertaken in two unrelated patients. Blue Native polyacrylamide gel electrophoresis followed by catalytic staining had already documented their complex V decreased activity. Extensive molecular analysis of five nuclear and two mitochondrial genes revealed a mutation in the ATP12 assembly gene in one patient. This mutation is believed to be the cause of the impaired complex V activity. To our knowledge, this is the first report of a pathogenic mutation in a human nuclear encoded ATPase assembly gene.

Acrodermatitis enteropathica-like cutaneous lesions in organic aciduria

Cutaneous lesions resembling acrodermatitis enteropathica were present in two infants with methylmalonic acidemia and in one infant with propionic acidemia. All three infants were being fed a low-protein diet limited in branched-chain amino acids when the skin lesions developed. A deficiency in plasma levels of essential amino acids, particularly isoleucine, was confirmed. Supplementation of the diet with isoleucine in one of the patients led to a prompt improvement of the skin lesions. We conclude that dietary deficiencies associated with the treatment of organic aciduria should be added to the causes of acrodermatitis enteropathica-like cutaneous lesions.

Pyruvate-dehydrogenase Deficiency - Clinical and Biochemical-diagnosis

A female neonate with pyruvate dehydrogenase (PDH) deficiency is presented with clinical, radiologic, biochemical, neuropathologic, and molecular genetic data. She was dysmorphic, with a high forehead, lowset ears, thin upper lip, upturned nose, and rhizomelic limbs. Cranial MRI revealed severe cortical atrophy, ventricular dilatation, and corpus callosum agenesis. Pyruvate and lactate levels were increased in CSF and blood. Urinary organic acid profile was compatible with PDH deficiency. PDH activity was normal in fibroblasts, lymphocytes, and muscle. The PDH E1-alpha gene was sequenced and a single base mutation was found within the regulatory phosphorylation site in exon 10. It is postulated that this mutation causes a cerebral form of PDH deficiency. Tissue-specific expression of the disease could be explained by differential X chromosome inactivation because the PDH E1-alpha gene is located on this chromosome. Dysmorphism with severe cerebral malformations in female patients merits a metabolic evaluation, including determination of lactate and pyruvate levels in CSF.

High-performance liquid chromatographic assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid

A procedure is described for the concurrent assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid in physiological fluids using high-performance liquid chromatography with electrochemical detection. The column packing is an octadecyl-bonded silica. A single mobile phase containing 1-octanesulphonate is used for the assay of catecholamines and for the assay of the acidic metabolites. An efficient sample preparation scheme is presented for the isolation of the catecholamines and their acidic metabolites from the same sample aliquot. Catecholamines are extracted by ion exchange on small columns and adsorption on alumina, using dihydroxybenzylamine as an internal standard. Vanillylmandelic acid and homovanillic acid are recovered from the combined loading and washing effluents of the ion-exchange column by a solvent extraction procedure. Recovery of catecholamines averages 67%. The limit of detection for individual catecholamines is ca. 30 pg. Recoveries of vanillylmandelic acid and homovanillic acid average 77% and 87%, respectively. The use of the same mobile phase for the concurrent assay of catecholamines and their acidic metabolites considerably increases the throughput of samples in the chromatographic system by eliminating the time-consuming column-equilibration periods.

How should HbA1c measurements be reported

In 1968, amidst the turmoil of student protests, a more discreet event—the reporting of increased minor Hb fractions in erythrocyte lysates from diabetic patients [1]—set the stage for yet another revolution, this time in our understanding of the pathogenesis of chronic complications of hyperglycaemia and in the management of diabetic patients. Since then, non-enzymatic glycation of proteins has been identified both as the reaction responsible for this increase in minor Hb bands [2], and as the first step in one of the key biochemical mechanisms underlying the development of chronic complications of diabetes [3]. Incidentally, the reaction of reducing sugars with amino groups was already long known to the food industry, when it was first identified, to the benefit of all of us, as the starting point of a complex series of reactions implicated in the formation of flavours and colours in, for example, beer, confectionery products and roasts (Fig. 1) [4]. Glycated Hb levels provide a time-integrated measure of glycaemia over the lifespan of the red blood cell (about 120 days), and therefore represent an index of glycaemic control for that period. They are used to assess the risk of developing chronic complications of diabetes and the quality of diabetes care [5]. It became progressively clear that non-enzymatic glycation reactions can occur to a significant degree between any free amino group on a protein (N-terminal or intracatenar groups from dibasic amino acids) on the one hand, and a free aldehyde or keto group from a variety of (phospho)hexoses or pentoses on the other, provided that the half-life of the protein is sufficiently long (Fig. 2) [5]. Glucose, as it happens, appears to be the least reactive aldohexose in this respect. Some have speculated that this property may have conferred a selective advantage to glucose, and that it was for this reason that it emerged as the universal metabolic fuel rather than one of the other hexoses or pentoses involved in carbohydrate metabolism [6]. Glycation apart, long-lived proteins such as Hb may undergo various other non-enzymatic post-translational modifications, such as carbamylation by urea or acetylation by acetylsalicylic acid.

Free amino acid distribution inside the first trimester human gestational sac

The trophoblast functions of nutrient transport and protein synthesis generate high concentrations of amino acids in the placenta and in fetal blood during the second half of pregnancy, but little is known about these metabolic processes in embryonic and early fetal periods. The aim of this study is to compare the distribution of amino acids inside the first trimester gestational sac. Free amino acid concentrations were measured in homogenates of placental villi, in samples of coelomic and amniotic fluid, and in the maternal serum from 17 normal pregnancies between 7 and 11 weeks of gestation. Significant positive relationships between maternal serum and placental tissue were found for 10 amino acids, indicating that active amino acid transport and accumulation by the human syncytiotrophoblast occurs as early as 7 weeks of gestation. The transplacental flux of most amino acid transport from maternal blood to the exocoelomic cavity was against a concentration gradient. The highest placental amino acid concentrations were found for taurine, glutamic acid, glycine and alanine. The amniotic fluid contained lower mean concentration of all amino acids than coelomic fluid and maternal serum. The concentration distribution of individual amino acids in coelomic and amniotic fluid were related indicating a passive transfer through the amniotic membrane. A coelomic-maternal gradient was observed in 19 out of 24 amino acids measured and positive correlations were found between maternal serum and coelomic fluid for concentrations of alpha-aminobutyric acid, tyrosine and histidine, suggesting that these amino acids are only partially retained and/or transferred more rapidly by the early placenta.

Evaluation of an immunoturbidimetric assay for haemoglobin A1c on a Cobas® Mira S analyser

We evaluated a homogenous immunoturbidimetric assay for haemoglobin A1c (Tina-quant Haemog bin A1c, Boehringer Mannheim, GmbH, Mannheim, Germany) adapted to a Cobas Mira S analyser (F. Hoffmann-La Roche & Co., Basel, Switzerland) and not requiring sample pretreatment. Between-day CVs determined over a 6 week period were 5.9% and 5.3% for mean haemoglobin A1c values of 5.4% and 17.0% of total haemoglobin respectively. The imprecision was higher than with an in-house ion-exchange high performance liquid chromatographic method. Bilirubin, triacylglycerols and the labile fraction of haemoglobin A1c did not interfere with the assay of haemoglobin A1c. Fetal haemoglobin is not recognized by the antibodies used. Results correlated well with those obtained by high performance liquid chromatography. In conclusion, the Tina-quant assay is not prone to common interferences and allows the rapid and automated determination of haemoglobin A1c on open photometric analysers.

Determination of glycerol in plasma by an automated enzymatic spectrophotometric procedure

Abstract The plasma concentration of glycerol, the backbone of triglycerides and the end product of triacylglycerol breakdown, is considered to reflect lipolysis in adipose tissue. We evaluated an automated enzymatic procedure for the measurement of glycerol in plasma. The assay was linear up to 250μmol/l. The detection limit was 8μmol/l. Recovery averaged 94% from spiked plasma and 104% from diluted plasma. An extensive precision study performed according to the guidelines of the National Committee for Clinical Laboratory Standards showed within-run coefficients of variation between 14.8% and 2.6% for concentrations ranging from 28μmol/l to 164 μmol/l. The reference range for fasting healthy adult men was 14–69 μmol/l. Glycerol levels were significantly correlated with free fatty acid levels. This automated enzymatic method is rapid and reliable, and provides greater sensitivity or convenience than previously described procedures.

Irreversible inactivation of arginyl-tRNA ligase by periodate-oxidized tRNA

Aminoacyl-tRNA ligases play a crucial role in protein synthesis. Each enzyme esterifies one of the amino acids to the corresponding tRNAin the presence of ATP and Mg*‘. Periodate-oxidized tRNA is often used as a deadend inhibitor in kinetic studies in this class of enzymes [l-5]. With the arginyl-tRNA ligase from Escherichia coli K12 we observed apparent mixed-type non-competitive inhibition with tRNA as the variable substrate. We also demonstrated an irreversible inactivation of the enzyme upon preincubation with periodate-oxidized tRNA. This finding indicates that special attention has to be paid to the interpretation of inhibition patterns obtained with this inhibitor.

Preliminary performance evaluation of blood gas analyzers.

Abstract Background: We evaluated the imprecision and bias of three instruments for the determination of blood gases, pH and ionized calcium (Ca2+) in human arterial blood samples, in comparison with the performance of an established methodology. Methods: The ABL 735, Omni S and Rapidpoint 405 blood gas analyzers were evaluated and compared to the ABL 620 analyzer. Imprecision was determined according to the NCCLS EP10-A2 evaluation protocol. The NCCLS EP9-A2 evaluation protocol was used to determine bias relative to the ABL 620 system. Experimental data were compared against preset quality specifications. Results: The three new instruments showed excellent imprecision for the measurement of pH, but only the ABL 620 met the preset imprecision goals for all analytes tested. All new instruments showed good correlation with the comparative instrument. The slope of the regression equation was significantly different from 1.0 in six out of the 12 comparisons, indicating systematic differences between the instruments. Nevertheless, the predicted bias values relative to the comparative instrument did not exceed the preset quality specifications for two out of the three new instruments. Conclusions: Preliminary evaluation using the NCCLS evaluation protocols EP10-A2 and EP9-A2, may provide valuable information on performance characteristics of blood gas analyzers. Clin Chem Lab Med 2006;44:1030–4.