Pierre Allard

Distribution of Fasting Plasma Insulin, Free Fatty Acids, and Glucose Concentrations and of Homeostasis Model Assessment of Insulin Resistance in a Representative Sample of Quebec Children and Adolescents

BACKGROUND Plasma fasting insulin and the homeostasis model assessment of insulin resistance (HOMA-IR) are markers of IR, which, at least in part, mediates the relation of obesity to increased cardiovascular risk. Increased free fatty acids (FFAs) may be involved in the pathogenesis of IR. Our objectives were to describe the distributions of fasting plasma insulin, glucose, and FFAs and HOMA-IR in youth and to assess the relationship between FFAs and markers of IR. METHODS Fasting plasma insulin, glucose, and FFAs were measured in a representative sample of Quebec youth comprising 2244 individuals 9, 13, and 16 years of age. RESULTS In all age and sex groups, glucose exhibited remarkably tight distributions (median CV, 7.1%) in contrast to insulin, HOMA-IR, and FFAs (median CVs, 52%, 54% and 45%, respectively). For every percentile examined, 9-year-olds had lower insulin concentrations and HOMA-IR values than 13- and 16-year-olds. We observed strong correlations between insulin concentrations and HOMA-IR values, as well as close similarity in their rankings of individuals. The mean concentrations of glucose were higher in our population than in other Caucasian pediatric populations. No positive correlations were detected between FFAs and markers of IR. CONCLUSIONS We report some of the first data on the distributions of fasting plasma insulin, HOMA-IR, and FFAs from a representative sample of youth. HOMA-IR does not appear more informative than fasting insulin as a marker of IR. Our findings on higher mean glucose concentrations in this population require confirmation in other representative samples of youth to assess whether the North American distribution of glucose concentrations is shifting positively.

Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots

BACKGROUND Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS). METHODS We extracted 3/16-inch DBS punches with 300 microL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 microL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155-137) and 13C5-SUAC (m/z 160-142). Analysis time was 1.2 min. RESULTS SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13-81 micromol/L), with Tyr concentrations ranging from 65 to 293 micromol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 micromol/L. CONCLUSION The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.

Links between Fer tyrosine kinase expression levels and prostate cell proliferation.

In our cloning strategy to identify tyrosine kinases implicated in the regulation of prostate growth, the dog fer cDNA was obtained and shown to be highly homologous to known fer cDNAs. Using a polyclonal Fer antibody directed against a C-terminal peptide, we studied its associations with cortactin, beta-catenin and p120Cas in human prostate carcinoma PC-3 cells. In contrast to previous reports, no interactions were observed. To assess its functional role, fer cDNA constructs were transfected in PC-3 cells. Antisense clones exhibiting a marked diminution of Fer expression had a reduced growth rate (doubling time of 29 vs. 42 h) and were unable to form colonies in soft agar. In agreement with these results, Fer protein expression was linked to human prostatic proliferative diseases, with enhanced levels in extracts from cancer tissues as compared to those from normal and hyperplastic ones, and was also expressed in the human prostate carcinoma cell lines DU145 and LNCaP. In the dog model, Fer expression was up-regulated in dividing versus resting prostate epithelial cells in vitro, and also in vivo when basal cell hyperplasia and metaplasia was induced by estrogen after castration. Minimal effects were observed when renewing the luminal epithelium with androgens. Taken together, these results show that Fer expression is associated with prostate cell proliferation and enhanced in prostate cancer.

Biological variation of glycated haemoglobin in a paediatric population and its application to calculation of significant change between results

Background To determine precisely the probability that a change between two glycated haemoglobin A1c (HbA1c) results is significant and that clinical actions may be required, the biological variation of HbA1c must be known. However, it has not been evaluated in a paediatric population. We therefore determined the long-term biological variation of HbA1c in a paediatric population and used it to generate a probability curve for significant changes between two consecutive HbA1c measurements. Methods A group of 24 boys and 14 girls with cystic fibrosis (CF) but without diabetes or impaired glucose tolerance has been selected. HbA1c has been measured at least five times over five consecutive years for all subjects. We have used the Fraser and Harris method to calculate within-subject biological variation (CVI), which allowed the determination of the probability that a change is significant between results. Results As within-subject variances are equivalent for girls and boys (P > 0.1), both genders were merged for biological variation analysis. The CVI calculated for HbA1c was 4.8% and the between-subject variation (CVG) was 12.8%. Then, a probability curve based on the CVI found was generated and showed that a change of 14% between two consecutive HbA1c results corresponding to a probability of 95% was significant. Conclusions We determined for the first time the biological variation of HbA1c in a paediatric population, which is higher than the ones found for adult populations. The probability curves generated from these data could be invaluable tools for clinicians to balance HbA1c results with other clinical parameters.

Low citrulline in Leigh disease: still a biomarker of maternally inherited Leigh syndrome.

Two siblings presented with encephalopathy, lactic acidosis, and hypocitrullinemia. Muscle and liver biopsies were considered for respiratory chain studies, but because of hypocitrullinemia, molecular analysis for maternally inherited Leigh syndrome was first performed, revealing in both siblings the mitochondrial DNA T8993G mutation (95% heteroplasmy), allowing to avoid tissue biopsies. Hypocitrullinemia, an occasional finding in mitochondrial diseases, has been specifically associated with T8993G mutation. However, only few patients have been reported, and the prevalence of hypocitrullinemia in 8993 mitochondrial DNA mutations is unknown. In a small series of 16 Leigh syndrome patients, sensitivity and specificity of hypocitrullinemia (≤12 μmol/L) for 8993 mitochondrial DNA mutations were 66% and 85%, respectively. Although studies in larger cohorts are necessary, we suggest considering T8993G mutation early in the diagnostic evaluation of infantile mitochondrial diseases with hypocitrullinemia, which minimizes the need for invasive procedures associated with a small but nonnegligible risk of complications and incorrect diagnosis.

Disinfectant wipes containing hydrogen peroxide induce overestimation of glucose results obtained with Lifescan SureStep Flexx® glucose meter

OBJECTIVES Investigate the interference of Virox® disinfectant wipes containing hydrogen peroxide with Lifescan SureStep Flexx® glucose meter results. DESIGN AND METHODS Glucose measurements of control and whole blood samples were performed before and after cleaning with Virox® wipes to reveal the kinetics of this interference. RESULTS AND CONCLUSIONS After a period where the error code 2(b) is given by the device, an overestimation period of glucose results is always present at all levels tested for a duration of hours. As this interference cannot be detected by the operator, hydrogen peroxide containing wipes for sanitizing of the glucose oxidase based glucose meter must be forbidden.

Biosynthesis of glycosaminoglycans: associated disorders and biochemical tests

Glycosaminoglycans (GAG) are long, unbranched heteropolymers with repeating disaccharide units that make up the carbohydrate moiety of proteoglycans. Six distinct classes of GAGs are recognized. Their synthesis follows one of three biosynthetic pathways, depending on the type of oligosaccharide linker they contain. Chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin sulfate contain a common tetrasaccharide linker that is O-linked to specific serine residues in core proteins. Keratan sulfate can contain three different linkers, either N-linked to asparagine or O-linked to serine/threonine residues in core proteins. Finally, hyaluronic acid does not contain a linker and is not covalently attached to a core protein. Most inborn errors of GAG biosynthesis are reported in small numbers of patients. To date, in 20 diseases, convincing evidence for pathogenicity has been presented for mutations in a total of 16 genes encoding glycosyltransferases, sulfotransferases, epimerases or transporters. GAG synthesis defects should be suspected in patients with a combination of characteristic clinical features in more than one connective tissue compartment: bone and cartilage (short long bones with or without scoliosis), ligaments (joint laxity/dislocations), and subepithelial (skin, sclerae). Some produce distinct clinical syndromes. The commonest laboratory tests used for this group of diseases are analysis of GAGs, enzyme assays, and molecular testing. In principle, GAG analysis has potential as a general first-line diagnostic test for GAG biosynthesis disorders.

A Liver-Specific Defect of Acyl-CoA Degradation Produces Hyperammonemia, Hypoglycemia and a Distinct Hepatic Acyl-CoA Pattern

Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-14C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication.