Franck Ceppa

Analytical performance of the Albumin Cobalt Binding (ACB®) test on the Cobas MIRA® Plus analyzer

Abstract Recently a new biological marker, Ischemia Modified Albumin (IMA®), measured by the Albumin Cobalt Binding (ACB®) test, was introduced for detection of myocardial ischemia. During ischemia, the metal binding capacity of albumin for certain transition metals like cobalt is reduced. The precise mechanism of action for producing IMA is not known but appears to be related to the production of reactive oxygen species that modify the metal binding sites. The ACB test is a quantitative assay that detects IMA by measuring the cobalt binding capacity of albumin in human serum. We evaluated the analytical characteristics of the ACB test, and reagent and specimen stability, using the Cobas MIRA® Plus instrument. Coefficients of variation for within-run and between-run assays were <4%. No significant interference was observed for concentrations of triglycerides and hemoglobin up to 7 mmol/l and 3.8 g/l, respectively. No interference was apparent with bilirubin. Measures from paired samples of heparinized plasma and serum were not equivalent. The assay is validated for commercial use with serum, therefore our study reported results for serum specimens only. All assays were completed within 5 hours after blood withdrawal. The one-sided upper 95th percentile, calculated for the ACB test in 150 healthy subjects, was 87.00 U/ml. There was no observed difference between men and women or with age. We conclude that the ACB test adapted on the Cobas MIRA Plus analyzer is satisfactory, but strict attention to sample handling procedures is necessary to maintain stability of the analyte.

High-performance liquid chromatographic determination of cotinine in urine in isocratic mode.

A simple procedure for the determination of cotinine, major metabolite of nicotine in urine, is described. The assay involved a liquid-liquid extraction with dichloromethane in alkaline environment. The extract was dried at ambient temperature under a gentle stream of nitrogen. The residue was dissolved in 300 microl of mobile phase and 30 microl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile phase (10-9%, v/v methanol and acetonitrile, respectively in potassium dihydrogenphosphate buffer adjusted to pH 3.4) at a flow rate of 1 ml/min on a C8 Symmetry cartridge column (5 microm, 150 mm x 3.9 mm, Waters) at 25 degrees C. The eluate was detected at 260 nm. Internal standard was 2-phenylimidazole. Sensitive and specific, this technique was performed to test urine of diabetic patients (smokers and non-smokers) admitted in an endocrinology service. Urinary cotinine seems to be a better marker of smoking status than thiocyanates.

Characterization of a Novel BCHE “Silent” Allele: Point Mutation (p.Val204Asp) Causes Loss of Activity and Prolonged Apnea with Suxamethonium

Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a “silent” phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 µM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.

Analytical performances of the Diazyme ADA assay on the Cobas® 6000 system

OBJECTIVE To evaluate the analytical performance of the Diazyme ADA assay on the Cobas® 6000 system for pleural fluid samples analysis. DESIGN AND METHODS Imprecision, linearity, calibration curve stability, interference, and correlation studies were completed. RESULTS The Diazyme ADA assay demonstrated excellent precision (CV<4%) over the analytical measurement range (0.5-117 U/L). Bilirubin above 50 μmol/L and haemoglobin above 177 μmol/L interfered with the test, inducing a negative and a positive interference respectively. The Diazyme ADA assay correlated well with the Giusti method (r(2)=0.93) but exhibited a negative bias (~ -30%). CONCLUSIONS The Diazyme ADA assay on the Cobas® 6000 system represents a rapid, accurate, precise and reliable method for determination of ADA activity in pleural fluid samples.

Military Community: A Privileged Site for Clinical Research: Epidemiological Study of Metabolic Syndrome Risk Factors in the Military Environment

The metabolic syndrome is considered to be an important public health problem. The Epidemiological Study of Metabolic Syndrome Risk Factors in the Military Environment is a prospective epidemiological study that is designed to identify clinical and laboratory parameters of metabolic syndrome and cardiovascular risk factors with an initial 1-year cross-sectional study followed by a 10-year follow-up and patient care. One hundred eight-five (9%) of 2,045 military personnel subjects presented at least three of the five National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) criteria. They were significantly older (42.2 +/- 8.5 years) than the other subjects (37.3 +/- 8.7 years, p < 0.001), had a higher body mass index (BMI) (29.5 +/- 3.4 vs. 24.8 +/- 2.9, p < 0.001), and a greater body weight at age 20 (75.4 +/- 11 vs. 70.4 +/- 8.5 kg, p < 0.001). Smoking, little physical activity, and family histories of diabetes and arterial hypertension were more frequent in these subjects. Total plasma cholesterol and C-reactive protein were higher. Plasma insulin and BMI (r = 0.456, p < 0.0001) and plasma insulin and waist circumference (r = 0.446, p < 0.0001) were well correlated. Plasma insulin and homeostasis model assessment increased with the number of metabolic syndrome criteria. These results demonstrate a strong association with insulin resistance. Men with several risk factors require specific care especially for hypertension and dyslipidemia that will be evaluated during the follow-up period. Genotyping of subjects having metabolic syndrome vs. controls for genes, presumably involved should enlarge the area of exploration of this syndrome.

Hyponatremia with consciousness disturbance associated with esomeprazole

tion. Zolpidem had been prescribed by several physicians and obtained from other sources until the intake was abruptly discontinued by the patient due to supply problems. Within 24 hours, 2 epileptic seizures were observed by relatives. A further generalized convulsion of about 6 minutes occurred in the emergency department and was terminated by administration of intravenous diazepam 10 mg. Her substance use history was significant for intermittent use of barbiturates and diazepam during adolescence due to an anxiety disorder (not further specified). Experiences of illegal drugs/alcohol or previous detoxification treatments were not reported (actual toxicology without abnormal findings). Six years previously, the patient had developed severe facial pain after excessive treatment of the skin with various external cleansing lotions of unknown composition. Presently, a symmetrical burning pain, including all sensory branches of the trigeminal nerve, with an intensity of 8 out of 10 on a visual analog rating scale (VAS) and persistence during the most part of the day was described. Trigger points were not mentioned, but there was increased sensitivity to cold and touch. Extensive examinations, including electrophysiologic and imaging techniques, were within the normal range. The symptomatology was considered painful polyneuropathy of neurotoxic genesis on the basis of excessive dermal treatment. Various treatment efforts in the past, including tricylic and other antidepressants, non-opioidergic and opioidergic analgesics, memantine, glucocorticoids, anticonvulsive agents, α-lipoic acid, lidocaine, benzodiazepines, and blockade of the superior cervical ganglion, were ineffective. However, the patient noted substantial pain relief (VAS score 1) with zolpidem therapy, which had been prescribed as temporary sleep medication starting with 10 mg/day. Over the past 2 years, the dose of zolpidem was gradually escalated by the patient to reach optimal pain relief. Detoxification was accomplished using a standardized 10-day tapering protocol of oxazepam (starting with 20 mg for 4 days) in combination with carbamazepine (starting with 200 mg for 3 days), which was successfully completed.1,2 However, the pain reappeared and did not respond to any further treatment efforts. Discussion. Our case further corroborates the assumption of a definite abuse and dependence potential for zolpidem and a possible loss of benzodiazepine-1 receptor specificity at high doses, with the majority of reported patients having a history of former drug or alcohol abuse and/or other psychiatric conditions.3 Furthermore, this case illustrates an exceptional off-label effectivity of high-dose zolpidem in a patient with therapy-resistant facial pain, although this regimen holds high medical risks. Remarkably, 2 reports of temporary improvements of catatonia and aphasia after a single dose of zolpidem indicate a mode of action apart from hypnotic properties.4,5 Thus, further investigations of the pharmacodynamic properties of zolpidem might be useful. Nevertheless, zolpidem should be prescribed with the awareness of a definite dependence potential.

Rapid Identification of Atypical Variant of Plasma Butyrylcholinesterase by PCR

Abstract Human butyrylcholinesterase is the enzyme responsible of mivacurium and succinylcholine metabolism, which may be significantly impaired when mutation Asp70Gly is found in patients. We describe a simple PCR method for the detection of this variant. Thirteen out of sixteen patients tested after prolonged apnea were positive for the presence of this mutation (50.0% homozygotes and 31.3% heterozygotes), suggesting that this test contributes to the explanation of some clinical events and to their prevention in relatives of these patients.

Pancréatite aiguë survenant après la prise de celecoxib

Resume Les atteintes pancreatites induites par le celecoxib, un inhibiteur de Cox-2 de premiere generation, sont reputees comme etant rares. Nous rapportons le cas d’une pancreatite aigue severe chez une femme apres un traitement de 3 mois par le celecoxib. Le bilan etiologique de la pancreatite est reste negatif. L’imputabilite du celecoxib a ete consideree comme vraisemblable. Cette observation et quelques cas rapportes dans la litterature suggerent de rechercher une pancreatite en cas de douleurs abdominales lors de la prise d’anti-inflammatoires selectifs de COX-2.

Déficit génétique en butyrylcholinestérase

Butyrylcholinesterase (EC 3.1.1.8; BChE) is a sister enzyme of acetylcholinesterase. Though BChE lacks obvious physiological functions, it is of toxicological and pharmacological importance in detoxifying or catabolising ester-containing drugs. Furthermore, individuals deficient in BChE appear asymptomatic, apart from a heightened sensitivity to the muscle relaxants suxamethonium and mivacurium, two BChE substrates used as myorelaxant. Although many acquired conditions may affect BChE activity, BChE deficiency is mainly due to mutations in the BCHE gene (OMIM 177400). Currently, more than 70 natural mutations have been documented in human BCHE. They have an adverse effect on BChE activity by affecting the catalytic functioning or the protein expression. However, the atypical variant (rs1799807) is the most frequently involved in prolonged apnea.

Pleural adenosine deaminase determination: an inter-laboratory comparison is required.

We read with interest the article of Keng et al. about the interest of various biomarkers for diagnosing tuberculous pleurisy (TB). In accordance with previous studies, the authors concluded that pleural adenosine deaminase (ADA) determination is a useful tool. However, they reported an optimal cut-off value significantly lower than those commonly mentioned in the literature (15.5 IU/L versus 30 or 40 IU/L) and especially in another study performed in the same country (cut-off: 30 IU/L). The authors hypothesized that this difference may be related to either the study population (i.e., TB patients older than those of the other Taiwanese study), or to the methodology used for pleural ADA determination. As underlined in the article, the laboratory methodologies, such as Giusti or non-Giusti methods, have a significant impact on the pleural ADA determination. Recently, we have reported a lack of transferability between pleural ADA assays. The two studies performed in Taiwan used the same commercial kit (Adenosine Deaminase Assay Kit, Diazyme Laboratories, San Diego, CA, USA). This kit can be adapted to various analytical systems. For example, in our daily practice, we have adapted it on the Cobas 6000 system (Roche Diagnostics, GmbH, Mannheim, Germany) with a cut-off value of 30 IU/L. It cannot be excluded that the analytical system on which the kit is adapted can impact the inter-studies comparisons. To confirm or infirm this hypothesis, it would be interesting to perform an interlaboratory comparison to determine the potential impact of the analytical systems on ADA determination with the Diazyme Laboratories’ kit. Based on the results obtained, specific cut-off values could be proposed depending on the analytical system or on the age of the patients.