[The N-terminal pro-brain natriuretic peptide (NT-proBNP) assay with the Stratus CS analyzer]. / Evaluation des performances analytiques du dosage du NT-proBNP sur analyseur semi-automatisé Stratus CS.

BACKGROUND: NT-proBNP is an efficient biomarker for the evaluation, management and prognosis of patients with heart failure. METHODS: We evaluated the analytical performance of the NT-proBNP immunoenzymatic assay with the Stratus CS semi-automated analyzer in two hospital laboratories. The characteristics assessed included imprecision, functional sensitivity, linearity/recovery, interferences study, high-dose hook effect and a comparison of Acute Care(TM) pPBNP (on Stratus)CS) versus PBNP (on Dimension HM) results on patient heparinized plasma samples. RESULTS: Total imprecision reached < 5% coefficient of variation at NT-proBNP concentrations of 186-19,649 ng/L; recovery values for diluted samples were between 89.0 and 110.0 %; functional sensitivity reached 21 ng/L; there was no high-dose hook effect at concentrations up to 400,000 ng/L; hemaoglobin affected negatively but <10% the NT-proBNP assay, while bilirubin and triglycerides did not affected it more than 5%; Stratus CS results were strongly correlated with Dimension HM results (R(2)=1,00). CONCLUSION: The Stratus CS Acute Care pPBNP assay demonstrated excellent analytical performance which agreed with the Dimension HM PBNP assay. This analyzer is therefore suitable for use by low NT-proBNP test volume laboratories, and also by Emergency departments and Intensive care units.

[Evaluation of the analytical performance characteristics of NT-proBNP immunoassay on dimension RxL-HM (Dade Behring)]. / Evaluation des performances analytiques du dosage du NT-proBNP sur automate d'immunoanalyse Dimension RxL-HM (Dade Behring).

BACKGROUND: Plasma B-type natriuretic peptide (BNP) and N terminal prohormone (NT-proBNP) are promising markers for the diagnosis, prognosis and follow up of heart failure. Elevated levels have been associated with adverse long-term outcome in patients with acute coronary syndromes. METHOD: The analytical performance of a new automated NT-proBNP assay on Dimension-HM system (Dade Behring) and comparison with the results obtained with the Roche Diagnostics NT-proBNP assays using the Elecsys instrument have been evaluated according to the Valtec protocol. RESULTS: Total imprecision (CV) was below 9% for NT-proBNP concentrations between 100 and 10,000 pg/mL using quality control samples and pooled patients' plasma. Analytical sensitivity was found 9.8 pg/mL the day of calibration and 10.1 pg/mL ten days later. Dilution linearity showed overall recovery between 98.4 and 104.7%. No hook effect was observed for NT-proBNP concentration up to 96,000 pg/mL. The interference of turbidity was below 4%. Hemoglobin interfered negatively with assay upper 240 micromol/L but less than 10%. A precision profile demonstrated a total imprecision of 10% CV at a NT-proBNP concentration of 70 pg/mL and 20% at 30 pg/mL. Dimension results highly correlated (r = 0.99) with Roche Elecsys NT-proBNP.

Cardiac troponin I: its contribution to the diagnosis of perioperative myocardial infarction and various complications of cardiac surgery.

OBJECTIVE: To study the value of assaying cardiac troponin I (cTnI) for the early diagnosis of perioperative myocardial infarction (PMI) and various complications of cardiac surgery. DESIGN: A prospective observational clinical study. SETTING: Biochemical laboratory, anesthesia, and cardiac surgery department of Hôpital Broussais. PATIENTS: Two hundred and sixty consecutive patients undergoing cardiac surgery. INTERVENTIONS: All patients underwent coronary artery bypass grafting and/or valvular surgery under extracorporeal circulation. Per-operative and postoperative follow-up consisted of electrocardiogram, echocardiography (mainly by the transesophageal approach), and serial determinations of biochemical markers such as creatinine kinase-MB isoenzyme (CK-MB) and cTnI. PMI, new ST segment changes, and ventricular arrhythmias were considered postoperative adverse cardiac outcome. MEASUREMENTS AND MAIN RESULTS: CTnI was measured before cardiopulmonary bypass (T0) and 12 and 24 hrs after (T12, T24). CK-MB was measured on arrival in the intensive care unit and on the first postoperative day (D1). Patients were divided into three groups according to the type of surgery: coronary artery bypass graft (CABG), valvular surgery (VS), or both procedures. The plasma CK-MB and cTnI concentrations were high in all patients after extracorporeal circulation because of aortic clamping or cardioplegia. The CK-MB and cTnI values were higher in the VS group than in the CABG group. Values peaked at T12 and fell by T24, except when PMI occurred. Eight patients developed a PMI. Patients with PMI had significantly higher cTnI levels at T12 and T24, and higher CK-MB values at D1 than patients without PMI. Cutoff values of cTnI for diagnosing PMI were >19 microg/L at T12 with 100% sensitivity and 73% specificity, and >36 microg/L at T24, with 100% sensitivity and 93% specificity. Lower cTnI values were highly suggestive of the absence of PMI after CABG and/or VS. Other complications such as ST segment changes, ventricular arrhythmias and cardiac failure were indicated by high cTnI levels at T12 and T24. Myocardial protective measures were associated with a nonsignificant increase in cTnI values. CONCLUSIONS: CTnI is more sensitive and specific than CK-MB for diagnosing PMI and other forms of heart failure after cardiac surgery.

Homocysteine-induced decrease in endothelin-1 production is initiated at the extracellular level and involves oxidative products.

The increased cardiovascular risk associated with hyperhomocysteinemia has been partly related to homocysteine (Hcy)-induced endothelial cell dysfunction. However, the intra or extracellular starting point of the interaction between Hcy and endothelial cells, leading to cellular dysfunction, has not yet been identified. We investigated the effects of both intracellular and extracellular Hcy accumulation on endothelin-1 (ET-1) synthesis by cultured human endothelial cells. Incubation of cultures with methionine (1.0 mmol x L(-1)) for 2 h induced a slight increase in cellular Hcy content but no change in ET-1 production. Incubation of cells with Hcy (0.2 mmol x L(-1)) led to a significant fall in ET-1 generation, accompanied by a significant increase in cellular Hcy content. Addition of the amino-acid transport system L substrate 2-amino-2-norbornane carboxylic acid had no effect on the Hcy-induced decrease in ET-1 production but significantly inhibited the Hcy-induced increase in the cellular Hcy content. Incubation of cells with a lower Hcy concentration (0.05 mmol x L(-1)) also reduced ET-1 production without increasing the cellular Hcy content. Co-incubation with extracellular free-radical inhibitors (superoxide dismutase, catalase and mannitol) markedly reduced the effect of Hcy on ET-1 production. Thus, it is extracellular Hcy accumulation that triggers the decrease in ET-1 production by endothelial cells through oxidative products.

Factors associated with increased serum levels of cardiac troponins T and I in chronic haemodialysis patients: Chronic Haemodialysis And New Cardiac Markers Evaluation (CHANCE) study.

BACKGROUND: Serum concentrations of the cardiac troponins (cTn) T and I, specific markers of myocardial injury, are frequently elevated in haemodialysis patients. The clinical relevance of this is unclear. The aim of this study was to investigate factors associated with increased serum levels of cTn in haemodialysis patients. METHODS: We included in this cross-sectional study 258 chronic haemodialysis patients (150 men, age 60+/-15 years) without acute coronary symptoms. Clinical data, echocardiographic hypertrophy, biochemical status, and haemodialysis regimen were evaluated for each patient. Pre-dialysis serum cTnT (Elecsys, Roche), cTnI (Stratus and RXL, Dade-Berhing), and CK-MB (Stratus, Dade-Berhing) concentrations were determined. Logistic regression was the principal method of analysis. RESULTS: Pre-dialysis levels of cTnT >0.1 ng/ml (n=48, 18.6% of patients) were associated with age (P<0.001), diabetes (P<0.005), history of ischaemic heart disease (P<0.05), and left ventricular hypertrophy (P<0.05). In multivariate analysis, age odds ratio ((OR) 1.04), diabetes (OR 4.9), and indexed left ventricular mass (OR 1.01) were found to be independently associated with cTnT concentration above the threshold. Only six patients had cTnI-Stratus levels >0.6 ng/ml. cTnI-RXL levels >0.3 ng/ml (n=13, 5.0%) were associated with age (P=0.05) and hypercholesterolaemia (P<0.05). Only age (OR 1.06) remained associated in multivariate analysis. CONCLUSION: Elevated baseline serum levels of cardiac troponins were associated with cardiovascular risk factors, history of ischaemic heart disease and left ventricular hypertrophy in asymptomatic chronic haemodialysis patients.

Cellular therapy reverses myocardial dysfunction.

OBJECTIVES: Cellular cardiomyoplasty refers to the implantation of autologous skeletal muscle cells into the myocardium to reinforce its structure and function. In this study a reproducible method for the creation of a myocardial lesion was developed. The functional benefit of cell implantation was evaluated by 2-dimensional echocardiography for global contraction and color kinesis echocardiography, which allows the precise assessment of the regional contraction. METHODS: A left ventricular intramyocardial injection with snake cardiotoxin was carried out on a sheep model to induce a well-delineated transmural lesion. Three weeks later, the lesion was assessed by echocardiography. Thereafter, autologous skeletal muscle cells or culture media (control) were injected into the lesion. Two months after cell implantation, the myocardial contraction was again evaluated by echocardiography and the implanted cells were analyzed by a fast myosin heavy chain antibody. RESULTS: 1. The snake cardiotoxin produced a well-delineated transmural lesion in all animals. 2. Echocardiographic studies showed a significant improvement in global and regional left ventricular function in cell-treated sheep. 3. Histologic analyses demonstrated satellite cell survival at the periphery of the lesions. CONCLUSION: Satellite cells implanted in a cardiotoxin-induced myocardial lesion survived for a 2-month period and were associated with a significant functional improvement of both local and global contraction.

Homocysteine decreases endothelin-1 production by cultured human endothelial cells.

Hyperhomocysteinemia is believed to be responsible for the development of vascular disease via several mechanisms, including the impairment of endothelial-cell functionality. In-vitro studies have demonstrated that homocysteine decreases the production or bioavailability of vasodilator autacoids, such as prostacyclin and NO. Here, we show that the treatment of human endothelial cells with noncytotoxic homocysteine concentrations leads to a dose-dependent decrease in both the secretion of the vasoconstrictor agent endothelin-1 (ET-1) and the level of its mRNA. Homocysteine had an inhibitory effect at pathophysiological (0.1 and 0.5 mmol.L(-1)) and pharmacological noncytotoxic (1.0 and 2.0 mmol.L(-1)) concentrations. Mean percentage variation from control for ET-1 production was -36. 2 +/- 18.9% for 0.5 mmol.L(-1) homocysteine and -41.5 +/- 26.8% for 1.0 mmol.L(-1) homocysteine, after incubation for 8 h. Mean percentage variation from control for steady-state mRNA was -17.3 +/- 7.1% for 0.5 mmol.L(-1) homocysteine and -46.0 +/- 10.1 for 1.0 mmol.L(-1) homocysteine, after an incubation time of 2 h. ET-1 production was also reduced by incubation with various other thiol compounds containing free thiol groups, but not by incubation with thiol compounds with no free thiol group. Co-incubation of cells with homocysteine and the sulfhydryl inhibitor N-ethylmaleimide prevented the effect of homocysteine on ET-1 production, confirming a sulfhydryl-dependent mechanism. Based on the reciprocal feedback mechanism controlling the synthesis of vasoactive mediators, these preliminary data suggest a mechanism by which homocysteine may selectively impair endothelium-dependent vasodilation by primary inhibition of ET-1 production.

Opposite effects of plasma homocysteine and the methylenetetrahydrofolate reductase C677T mutation on carotid artery geometry in asymptomatic adults.

Studies of symptomatic patients have identified hyperhomocysteinemia as an independent risk factor for vascular disease. In case-control studies, a point mutation (C677T) in the gene encoding 5,10-methylenetetrahydrofolate reductase (MTHFR) has also been linked to an increased risk of vascular disease through its effect on homocysteinemia. Our aim was to extend these observations to asymptomatic subjects by studying the influence of both homocysteinemia and its mutation on carotid artery geometry. We examined 144 subjects free of atherosclerotic lesions. Fasting homocysteinemia was measured by high-performance liquid chromatography with fluorometric detection. MTHFR genotype was analyzed by polymerase chain reaction followed by HinfI digestion. Carotid artery geometry was characterized by internal diameter and intima-media thickness, as assessed by a high-resolution echo-tracking system. Subjects in the upper homocysteine tertile had a greater carotid internal diameter than did subjects in the middle and lower tertiles (6516+/-770 versus 6206+/-641 and 5985+/-558 microm, respectively; P<0.001). Subjects homozygous for the mutation had a smaller carotid artery internal diameter than did subjects heterozygous or homozygous for the wild-type allele (5846+/-785 versus 6345+/-673 and 6199+/-671 microm, respectively; P<0.05). Homocysteinemia was not significantly increased in subjects homozygous for the mutation. In multivariate regression analysis, homocysteinemia was independently and positively associated with lumen diameter (P=0.0008) and wall thickness (P=0.020). Conversely, homozygosity for the mutation was negatively associated with internal diameter (P=0.009). These preliminary data suggest that mildly elevated homocysteinemia and homozygosity for the MTHFR C677T mutation are associated with opposite preclinical modifications of carotid artery geometry. If confirmed, these results may have important implications for new treatment strategies for vascular disease before the onset of clinical manifestations.

Endothelin and cardiovascular remodelling in end-stage renal disease.

BACKGROUND: Plasma endothelin (ET) is elevated in end-stage renal disease (ESRD), but the origin and consequences of this increase remain unclear. In the present study we analysed the relationships between plasma ET levels and cardiovascular alterations in ESRD. METHODS AND RESULTS: Common carotid artery (CCA) intima-media thickness (IMT) and diameter, atherosclerotic plaque occurrence, and left ventricular (LV) geometry and function were determined by ultrasound imaging in 76 haemodialysis patients and in 57 age-, sex-, and blood pressure-matched controls. Arterial stiffness was evaluated via carotid-femoral pulse wave velocity (CF-PWV), forearm post-ischaemic vasodilation was measured by venous plethysmography, and plasma ET levels were determined using a specific immunoenzymoassay. Compared with controls, ESRD patients had elevated plasma ET levels (1.6 +/- 1.4 vs 4.6 +/- 3.8 pg/ml; P < 0.001), increased LV mass (P < 0.001), increased CCA-IMT (P < 0.001), a higher prevalence of atherosclerotic plaques (P < 0.001) and increased CF-PWV (P < 0.01). Plasma ET levels correlated positively with LV outflow velocity integral (r = 0.57; P < 0.0001), stroke index (P < 0.01), and baseline forearm blood flow (P < 0.001) which were all significantly higher in ESRD patients than in controls (P < 0.01). After adjustment for age, blood pressure, haemoglobin levels, gender and body dimensions, plasma ET levels were significantly correlated to LV mass (r = 0.46; P < 0.001), CCA-IMT and CCA intima-media cross-sectional area (r = 0.41; P < 0.001), and CF-PWV (P < 0.05). Post-ischaemic forearm vasodilation was decreased in ESRD (85 +/- 31 vs 119 +/- 28%; P < 0.001) and there was a negative correlation between post-ischaemic flow recovery and ET levels (r = -0.49; P < 0.001). In ESRD patients, plasma ET levels were positively and independently correlated with the prevalence of CCA atherosclerotic plaque (P < 0.01). CONCLUSIONS: These results indicate that the increased plasma ET levels in ESRD patients are associated with left ventricular hypertrophy and arterial intima-media thickening, suggesting that increased ET concentrations in ESRD patients may be of pathophysiological significance in the process of cardiovascular remodelling.

Increased serum concentration of IgA2 subclass and IgA2/IgA1 ratio: specific markers of chronic alcoholic abuse?

Enhanced serum IgA concentrations are common in alcoholic liver cirrhosis, but functional differences between IgA subclasses and their relation with interleukin-6 (IL-6) have not been described. Distinct immunoregulatory mechanisms may exist that selectively affect one subclass. This possibility prompted us to investigate the distribution of IgA1 and IgA2 subclasses in the serum of 25 heavy alcohol drinkers (alcohol: 80 to 200 g per day) without clinical disorders, in comparison with 35 patients affected by alcoholic liver cirrhosis, 29 viral hepatitis patients and 33 social drinkers as a control group. Mean (+/- SD) IgA2 concentration (0.56 +/- 0.31 g/l) was significantly increased (p < 0.01) in heavy alcohol drinkers, with an IgA2/IgA1 ratio of 0.33 +/- 0.12, while the mean total IgA concentration was similar to the control group. Mean IgA1 and IgA2 concentrations were significantly increased (p < 0.001) in alcoholic liver cirrhosis patients (6.13 +/- 4.52 g/l and 1.83 +/- 1.93 g/l respectively, with an IgA2/IgA1 ratio of 0.32 +/- 0.19) and viral hepatitis patients (3.66 +/- 2.59 g/l and 0.69 +/- 0.67 g/l respectively, with an IgA2/IgA1 ratio of 0.21 +/- 0.14) High serum IL-6 concentrations (34 +/- 33 ng/l) were correlated with elevated IgA1 and IgA2 concentrations only in patients with alcoholic liver cirrhosis. IgA2 subclass and IgA2/IgA1 ratio could therefore be used as markers of chronic alcohol abuse directly related to the extent and duration of the alcohol abuse and the effectiveness of alcohol withdrawal.

Characteristics of ten charge-differing subfractions isolated from human native low-density lipoproteins (LDL). No evidence of peroxidative modifications.

Native plasma low-density lipoproteins (LDL) were fractionated into ten subfractions with increasingly negative charges (LDL-1, the least electronegative, to LDL-10) using an anion-exchange column coupled to a fast protein-liquid chromatography system. Prior to fractionation, contaminating Lp(a) and apo A-I-containing lipoproteins were removed from LDL preparations by immunoaffinity chromatography. No significant difference in thiobarbituric acid-reactive substances, vitamin E or free aminogroup was found among subfractions, and no peptide with a higher molecular weight than apo B was observed on SDS-PAGE. We observed a gradual increase in cholesterol esters and a concomitant decrease in triglycerides from LDL-1 to LDL-7, and a reverse tendency from LDL-8 to LDL-10 (P < 0.01). Free cholesterol increased linearly from LDL-1 to LDL-10 (P < 0.01). LDL-1 to -3 had a homogeneous density profile, while other more electronegative subfractions showed a bimodal distribution with a second, minor peak of slightly higher density. A gradual increase in apolipoprotein C-III content related to LDL electronegativity was observed (P < 0.001). Apolipoprotein E content was also increased in the last two subfractions (P < 0.01). LDL subfractions displayed a similar binding fate on human fibroblasts, with the exception of the most electronegative subfractions [LDL-(9 + 10)], which bound more actively to apo B/E receptors (P < 0.05). This study shows that charge heterogeneity of native LDL is not related to lipid peroxidation or derivatization of free aminogroups of apolipoprotein B. In contrast, the enrichment of LDL in apolipoproteins other than apo B may explain, in part, the difference in their particle charge.

[Multicenter study of sialic acid deficient transferrin determined by two chromatographic techniques]. / Etude multicentrique du dosage de la transferrine déficiente en acide sialique par deux techniques chromatographiques.

Serum carbohydrate-deficient-transferrin (CDT) was measured by a micro anion-exchange chromatography/enzyme immunoassay. Results obtained on 245 sera analyzed in four laboratories were compared. Moreover, one laboratory used a commercial kit with ready-to-use microcolumns and a radioimmunoassay for measuring eluted CDT. Imprecision was judged to be satisfactory. Within-assay coefficients of variation ranged from 5 to 10%, between-assay coefficients of variation ranged from 9 to 18%. Between-laboratory results were compared for 110 sera from control subjects (daily alcohol intake < 40 g), for 57 sera from chronic ethylic subjects and for 78 sera from patients suffering from non-alcoholic liver diseases. There was a large between-laboratory variation, suggesting that the method is difficult to standardize and that results are not transferable. Results of enzyme and radioimmunoassays were compared on 325 sera. The best correlation was obtained in the groups of ethylic subjects and those with non-alcoholic hepatic diseases. Finally the performance of the CDT-test was evaluated by calculating sensitivity and specificity. With both methods specificity was very high (> 85%) but sensitivity was poor (< 50%).

[Hemodialysis and ferritinemia]. / Hémodialyse et ferritinémie.

Iron-deficiency is a common phenomenon in chronic renal diseases and haemodialysis patients, a treatment with iron or transfusions is always provided in an early preventive way; yet, an overload may appear. Serum ferritin, in spite of analytic variability, remains at the present time a good witness to appreciate patients' iron stores. Authors report the results obtained with four commercial reagents in healthy population and in haemodialysed children or adults. The comparison of results for this parameter shows that haemodialysed sera, with or without treatment, have the same behaviour with reagents as those of healthy subjects.

Monitoring of heart allograft rejection by simultaneous measurement of serum beta 2-microglobulin and urinary neopterin.

In patients with heart transplant, the combined determination of serum beta 2-microglobulin and urinary neopterin, as rejection marker, prevented the interference by renal function and cyclosporin therapy. Unfortunately, the simultaneous measurement of these two parameters cannot distinguish between a rejection episode and the early stage of viral infection.

[Normal serum concentration of retinol transport protein (RBP) measured by an immunonephelometric method]. / Concentration sérique usuelle de la protéine transporteuse du rétinol (RBP) mesurée par une méthode immunonéphélémétrique .

The authors give serum retinol binding protein (RBP) normal values, established by immunonephelometry, for two healthy populations in their hospital laboratory. The mean values for men's and women's group are significantly different. They note for women's group with estroprogestative drugs a higher mean value. Relations between retinol and RBP metabolism are known. RBP values give a good estimation of retinal reserves in patients.

[A manual method for the direct determination of serum iron using a new chromogen: chromazurol B]. / Méthode manuelle de dosage direct du fer sérique par un nouveau chromogène: le chromazurol B.

The authors describe a manual method for the direct assay of iron in human serum using the ammonium salt of Chromazurol B. This new chromogen reacts with ferric or ferrous ions and cetyltrimethylammonium bromide giving a purple ternary complex. Reliability and practicability of the method are studied. The results correlate very well either with those of the SFBC's method or with a direct ferrozine-guanidine kit. The reagents are very cheap, the method is quickly performed; no significant interference could be observed in case of high concentrations of added bilirubin, copper or hemoglobin.

[Diagnosis of AL amyloidosis in the absence of detectable serum or urinary monoclonal component]. / Diagnostic d'une amylose AL en l'absence de composant monoclonal sérique ou urinaire détectable.

A case of massive amyloidosis without detectable monoclonal Ig component either in serum or in urines on classical electrophoresis and without obvious plasma cell proliferation is reported. Immunohistochemical study of renal and bone marrow biopsies showed that amyloid deposits were specifically stained with the anti-lambda antiserum and that 92% of the plasma cells were also lambda positive. Immunofixation following electrophoretic analysis of serum and urinary proteins exhibited a monoclonal lambda light chain at an advanced stage of the disease. Simple but not routinely used techniques are therefore of great interest to characterize apparently idiopathic amyloidosis which could have therapeutic implications.