Molecular characterization of MPS IIIA, MPS IIIB and MPS IIIC in Tunisian patients.

Sanfilippo syndrome (mucopolysaccharidosis type III, MPS III) is a progressive disorder in which patients are characterized by severe central nervous system degeneration together with mild somatic disease. MPS III results from a deficiency in one of the four enzymes involved in the heparan sulfate degradation, with sulfamidase (SGSH), α-N-acetylglucosaminidase (NAGLU), acetyl-coenzyme A: α-glucosaminide N-acetyltransferase (HGSNAT), and N-acetylglucosamine-6-sulfatase (GNS) being deficient respectively in MPS IIIA, MPS IIIB, MPS IIIC and MPS IIID. Mutation screening using PCR reaction/sequencing analysis on genomic DNA fragments was performed in seven Tunisian index cases with MPS IIIA, three with MPS IIIB and two with MPS IIIC. QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) analysis was developed for the detection of genomic deletions and duplications in the SGSH gene. These approaches allowed the identification of 11 mutations, 8 of them were novel including a mutation involving the start codon (p.Met1?), one small duplication (p.Leu11AlafsX22), one small deletion (p.Val361SerfsX52) and a large deletion of exon 1 to exon 5 in the SGSH gene, one missense mutation (p.Pro604Leu) and one nonsense mutation (p.Tyr558X) in the NAGLU gene and, finally, one missense mutation (p.Trp627Cys) and one nonsense mutation (p.Trp403X) in the HGSNAT gene.

Two-year stability of NT-proBNP in frozen samples using the Roche Elecsys system.

BACKGROUND: The in vitro stability of N-terminal (NT)-proBNP (brain natriuretic peptide) at room temperature and at 4 degrees C is excellent and has been well studied. However, less is known concerning its stability after a long-term frozen storage. This notion could be of major interest in the context of clinical evaluations. METHODS: NT-proBNP was measured on 97 heparinized samples before and after a two-year frozen storage (-20 degrees C) using the Roche Elecsys system. RESULTS: There is a slight but significant decrease of NT-proBNP concentration after frozen storage. However, this decrease is <10% for more than 90% of the samples and the maximum decrease is 16%. Moreover, values on frozen samples are well correlated with values on fresh samples (r = 0.99) using the following equation: NT-proBNP(stored sample) = 0.93 NT-proBNP(fresh sample) + 81. A similar equation was found in lower concentrations (NT-proBNP < or = 2000 ng/L). CONCLUSIONS: These data show that NT-proBNP can be stored for at least two years at -20 degrees C before measurement without a substantial loss of immunoreactivity.

[Evaluation of NT-proBNP as an early biological marker of cardiac dysfunction in septic shock]. / Evaluation du NT-proBNP comme marqueur biologique de la dysfonction cardiaque du choc septique.

OBJECTIVE: To evaluate the NT-proBNP as a biological diagnosis marker of the myocardial dysfunction in septic shock. STUDY DESIGN: Non-randomized prospective clinical study with written assent. The analysis of the data obtained was retrospective. PATIENTS AND METHODS: All the patients with septic shock in the beginning of evolution (less than 24h) were included. Patients with cardiac insufficiency, insufficient respiratory function and chronic renal insufficiency as well as cirrhotic patients were excluded. Among patients in shock, a NT-proBNP concentration measurement and a cardiac echography by transthoracic way were carried out at inclusion. The rates of NT-proBNP were compared with the data of the echography. RESULTS: Thirty-three patients in septic shock were included. On the whole of the collective, whether or not there is a cardiac dysfunction, the rates of NT-proBNP are not significantly different (11,306+/-16,196 pg/ml versus 10,697+/-12,346 pg/ml). By eliminating the patients with severe renal failure, we show that the NT-proBNP is non-significantly increased in the event of right and/or left ventricular failure (5751+/-4180 pg/ml versus 1,256+/-999 pg/ml). CONCLUSION: The NT-proBNP can help to detect the cardiogenic share sometimes implied in the haemodynamic failure of the septic shock. However, because of the influence of the renal insufficiency and the respiratory, cardiologic and hepatic comorbidities on its secretion, its use cannot be recommended for patients in septic shock.

[NT-proBNP measurement on Immulite 2500 (DPC): analytical performance and comparison with Roche Diagnostics and Dade-Behring NT-proBNP immunoassays]. / Dosage du NT-proBNP sur l'Immulite 2500 (DPC): évaluation analytique et comparaison avec les trousses Roche-Diagnostics et Dade-Behring.

The measurement of the N-terminal part of the proBNP (NT-proBNP) may be used to assess the secretion of the B-type natriuretic peptide (BNP), a marker of heart failure. In this study, we have evaluated the NT-proBNP immunoassay proposed by DPC Company for the Immulite 2500 analyzer and compared the results with those obtained with the two other immunoassays respectively commercialized by Roche Diagnostics (Elecsys 2010 analyzer) and Dade-Behring (Dimension RXL). The obtained results show very good general performance of the DPC's technique with a CV inferior to 8% for the values superior to 40 ng/L. The within run CVs are 3.1, 3.5 and 3.5% and the between run CVs are 3.8, 4.7 and 4.8% for the NT-proBNP levels of 151, 1601 and 5255 ng/L, respectively. We found a very good correlation between DPC's and Roche Diagnostics's assays (regression analysis: y = 0.88 x + 25.2 ; r = 0.998) and DPC's and Dade-Behring assays (regression analysis: y = 0.93 x + 16.4 ; r = 0.997). Although a small bias appeared between these assays, similar cut-points may be used to exclude both heart failure in ambulatory patients and cardiac origin in acute dyspnea.

[Macromolecular complex of cardiac troponin I: differences of reactivity with DPC and Dade-Behring assays]. / Complexe macromoléculaire de troponine I cardiaque: différence de réactivité des trousses de dosage DPC et Dade-Behring.

We have recently identified a macromolecular 440-kDa cardiac troponin I (cTnI) complex after successful percutaneous transluminal coronary angioplasty (PTCA) (Clin Chem 2003; 49: 505-7). The aim of the work was to confirm the existence of such a complex by using another cTnI assay (Dimension RXL, Dade-Behring). We have first studied the correlation between the two assays by using heparinized samples [cTnI(Immulite) = 2.00 cTnI(Dimension) - 0.01 (n = 176; r = 0,987)]. Then, cTnI taken 120 minutes after PTCA for two patients was measured with the two assays after fractionation by FPLC. The obtained results confirmed the existence of the 440-kDa cTnI complex and showed that the reactivity between the assays (DPC/Dade-Behring ratio) depended on the nature of the complex: the ratio increased from 0.7 (440-kDa cTnI complex) to 3 (80-kDa cTnI complex) therefore suggesting caution in the comparison between the different cTnI assays in the context of reperfusion therapy.

[Prescription, assay and interpretation of cardiac troponins tests: guidelines from SFBC-CNBC troponin working group]. / Recommandations sur la prescription, le dosage et l'interprétation des troponines cardiaques.

Troponin (I or T) has become the gold-standard marker in acute coronary syndromes during the last few years, as confirmed by a national survey realized within french clinical chemists, cardiologists and emergency practitioners. The importance of this marker and the heterogeneousness of circulating forms of troponin after myocardial necrosis fully justify international studies about standardization of this assay, which is a central bulk to reach a global market coherence. Checking analytical problems, although necessary, must be absolutely associated with an informed clinical interpretation. The knowledge of the crucial thresholds of each assay, the kinetic curves and the specificity limits of troponin assays allow the best use of their potential in diagnosis and prognosis together with an optimal patient care in very different clinical settings, in addition to others clinical and technical arguments. The quality improvement through successive generations of assay kits must nowadays persuade the physicians never to ignore a significant and valid troponin increase, which mainly reveals a cardiac injury, whatever its origin.

[Brain natriuretic peptide: physiological, biological and clinical aspects]. / Le BNP: aspects physiologiques, biologiques et cliniques.

Brain natriuretic peptide is one member of the natriuretic peptide family, including also ANP, CNP, DNP and urodilatin. In human, brain natriuretic peptide is mainly secreted by the cardiac ventricles. BNP is synthetized as pre-proBNP form, secondary cleaved in proBNP, itself equimolarly cleaved in BNP and NT-proBNP. The biological action of BNP is mediated by the NPR-A receptor. This peptide is eliminated from the systemic circulation by a neutral endopeptidase and by a clearance receptor (NPR-C). The BNP and NT-proBNP concentrations are measured using automated rapid immunoassay techniques. Plasma concentrations of the two peptides physiologically increase with age and are found to be higher in women than in men. The action of BNP against fluid expansion is explained by its vascular (vasodilatation), renal (diuretic and natriuretic) and cerebral activities. The measurement of these two peptides contributes to the diagnosis of heart failure. These peptides are prognostic markers both in heart failure and in acute coronary syndromes. In renal insufficiency, the interpretation of the increase in these two peptide concentrations may be difficult, particularly with the NT-proBNP which is mainly excreted by the kidneys.

[Cardiac troponin I and CK-MB mass after cardiac surgery with cardiopulmonary bypass]. / Troponine Ic et CK-MB masse en chirurgie cardiaque sous circulation extracorporelle.

Cardiac troponin I (cTnI) assay is used in the diagnosis of myocardial infarction after cardiac surgery. Variations in the cut-off value have been reported even with the same assay method. The aim of this work is to investigate the release profile of cTnI and CK-MB mass after cardiac surgery and to determine the cut-off value of cTnI and CK-MB mass allowing the diagnosis of perioperative myocardial infarction. In patients without postoperative cardiac complication, the cTnI peak was observed 24 hours after surgery both in coronary artery bypass grafting and in valve replacement. Moreover, the amount of cTnI released within the three hours after surgery is 2.5 fold higher in valve replacement than in coronary artery bypass grafting. The CK-MB peak was observed 3 hours after surgery in the two surgical procedures. In these patients, cTnI and CK-MB concentrations increased with the cross clamp time duration. In patients with postoperative myocardial infarction, the cTnI and CK-MB peaks were observed 24 hours after surgery. Diagnosis of perioperative myocardial infarction can be performed with a sensitivity of 100% at 24 hours with cut-off values of 32 and 7 microg/L for CK-MB and cTnI, respectively, both with Stratus (Dade Behring) and Immulite (DPC) analysers.

[Cardiac troponin I and T: specific biomarkers of cardiomyocyte]. / Les troponines I et T cardiaques : des marqueurs spécifiques du cardiomyocyte.

PURPOSE: Cardiac troponin I and troponin T have replaced creatine kinase MB (CK-MB) for the diagnosis of cardiomyocyte necrosis. Cardiac specificity of these new markers leads to a change in our practice. CURRENT KNOWLEDGE AND KEY POINTS: Following necrosis, intracellular proteins are released into blood. This easy concept overlaps a biological complexity since troponins are released as complexes leading to various cut-off values depending on the assay used, as least for cardiac troponin I. The increase in both specificity and analytical sensitivity of these markers reached to propose a new definition of myocardial infarction. The diagnosis of acute coronary syndrome is a clinical based diagnosis, the use of troponin contributing to their classification. Finally, pathological processes leading to cardiac injury may induce an increase in the cardiac troponin level. FUTURE PROSPECTS AND PROJECTS: Troponin standardization is a challenge for the near future leading to better follow-up of patients and comparison between cohorts.

[Plasma determination of homocysteine on CPC Immulite 2000: comparison with determination on IMX Abbott]. / Dosage plasmatique de l'homocystéine sur l'Immulite 2000 DPC: comparaison avec le dosage sur l'IMX Abbott.

Plasma total homocysteine is a parameter frequently included in the biological investigation of arterious or venous thrombotic diseases. Different techniques, chromatographic, enzymatic, and immunochemical, are used in the measurement of homocysteine. Among immunochemical methods, some are conceived to function on multiparametric automates, leading to a greater accessibility of the test for most of the clinical laboratories. In this study, we have evaluated a new immunoassay proposed by the DPC Company for the Immulite 2000 analyzer (chemiluminescence) and compared its performance against the Abbott's immunoassay on IMx (fluorescence polarization). The results obtained show very good general performance of the DPC's technique. Linearity is also excellent. The within run CVs are 9.9, 7.0 and 5.4% and the between run CVs are 8.2, 3.9 and 4.3% respectively for homocysteine levels of 4.2, 13.9 and 27.7 pmol/L. We found a very good correlation between DPC's and Abbott's methods (regression analysis:y=0.948 x + 0.05; r=0.895). The mean of differences between both methods is -0.55 micromol/L. On the whole, the DPC technique appeared in our experience as easily exchangeable, from the analytical point of view, with Abbott's technique.

Influence of glutamine on cytokine production by human gut in vitro.

BACKGROUND: glutamine modulates cytokine production by immune cells in vitro and protects the gut from experimental enterocolitis, but data on the effect of glutamine on cytokine production in human gut are lacking. AIM: to assess the effect of glutamine pre-treatment in vivo and in vitro on cytokine production by intestinal mucosa. METHODS: nine fasted volunteers received either enteral glutamine or saline over 6 h in a cross-over design. Duodenal biopsies were cultured for 24 h with or without glutamine. Cytokine content of culture media was analysed by ELISA, and the expression of cytokine mRNA in biopsies was assessed by semi-quantitative RT-PCR. RESULTS: glutamine given in vivo and in vitro significantly decreased IL-6 [1.4 (0.8-8.5) vs 8.9 (1.0-43.9)] and IL-8 production [5.8 (0-51.4) vs. 53.0 (2.5-114.6), pg/mg wet tissue], median (range), both P< or =0.01, in comparison to no glutamine experiments. Glutamine did not influence IL-4 production. IL-1beta, IL-10 and TNF-alpha were not detectable in culture media. The expression of any cytokine mRNA was not influenced by glutamine. CONCLUSIONS: glutamine reduces pro-inflammatory cytokine production by human intestinal mucosa, probably by a post-transcriptional pathway. Glutamine could be useful to modulate inflammatory conditions with imbalanced cytokine production.

AMP-activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes.

The effect of AMP-activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.

Synthesis of interleukin 1beta and interleukin 6 by stimulated rat peritoneal macrophages: modulation by glutamine.

Synthesis and secretion of IL-1beta and IL-6 were compared in LPS-stimulated rat peritoneal macrophages, and the effect of glutamine studied. LPS induced a parallel increase in mRNA and synthesis of IL-1beta and IL-6. IL-1beta accumulated mainly in the cytosol and IL-6 in the culture medium. Glutamine addition increased the synthesis of both cytokines, but the overall production (intra-+extracellular) of IL-1beta increased two-fold, although that of IL-6 increased only 1.3-fold. The influence of glutamine is discussed.

Effects of enteral glutamine on gut mucosal protein synthesis in healthy humans receiving glucocorticoids.

In hypercatabolic patients, the beneficial effects of glutamine on gut mucosa could be partly due to a stimulation of protein synthesis. The fractional synthesis rate (FSR) of gut mucosal protein was measured in four groups of healthy volunteers treated with glucocorticoids for 2 days. Two groups were studied in the postabsorptive state while receiving glutamine or a nitrogen equivalent (control) and two groups in the fed state with or without glutamine, using a 5-h intravenous infusion of [(13)C]leucine, [(2)H(5)]phenylalanine, and cortisone. After nutrient and tracer infusion, duodenal biopsies were taken. In the postabsorptive state, FSR of gut mucosal protein were 87 and 76%/day in the control group and 130% (P = 0.058 vs. control) and 104% (P = 0.17 vs. control)/day in the glutamine group, with leucine and phenylalanine as tracers, respectively. During feeding, FSR did not increase and no significant difference was observed between glutamine and control groups. Overall, FSR of the four groups were two- to threefold higher than those obtained previously in healthy humans, suggesting that glucocorticoids may increase gut mucosal protein synthesis. However, in this situation, a moderate enteral glutamine supply failed to demonstrate a significant effect on gut mucosal protein synthesis in the postabsorptive state and during feeding.