Administration of CORM-2 inhibits diabetic neuropathy but does not reduce dyslipidemia in diabetic mice.

The antinociceptive effects of the carbon monoxide-releasing molecule tricarbonyldichlororuthenium (II) dimer (CORM-2) during chronic pain are well documented, but most of its possible side-effects remain poorly understood. In this work, we examine the impact of CORM-2 treatment on the lipoprotein profile and two main atheroprotective functions attributed to high-density lipoprotein (HDL) in a mouse model of type 1 diabetes while analyzing the effect of this drug on diabetic neuropathy. Streptozotocin (Stz)-induced diabetic mice treated with CORM-2 (Stz-CORM-2) or vehicle (Stz-vehicle) were used to evaluate the effect of this drug on the modulation of painful diabetic neuropathy using nociceptive behavioral tests. Plasma and tissue samples were used for chemical and functional analyses, as appropriate. Two main antiatherogenic properties of HDL, i.e., the ability of HDL to protect low-density lipoprotein (LDL) from oxidation and to promote reverse cholesterol transport from macrophages to the liver and feces in vivo (m-RCT), were also assessed. Stz-induced diabetic mice displayed hyperglycemia, dyslipidemia and pain hypersensitivity. The administration of 10 mg/kg CORM-2 during five consecutive days inhibited allodynia and hyperalgesia and significantly ameliorated spinal cord markers (Cybb and Bdkrb1expression) of neuropathic pain in Stz mice, but it did not reduce the combined dyslipidemia shown in Stz-treated mice. Its administration to Stz-treated mice led to a significant increase in the plasma levels of cholesterol (∼ 1.4-fold vs. Ctrl, ∼ 1.3- fold vs. Stz-vehicle; p < 0.05) and was attributed to significant elevations in both non-HDL (∼ 1.8-fold vs. Ctrl; ∼ 1.6-fold vs. Stz-vehicle; p < 0.05) and HDL cholesterol (∼ 1.3-fold vs. Ctrl, ∼ 1.2-fold vs. Stz-vehicle; p < 0.05). The increased HDL in plasma was not accompanied by a commensurate elevation in m-RCT in Stz-CORM-2 compared to Stz-vehicle mice; instead, it was worsened as revealed by decreased [3H]-tracer trafficking into the feces in vivo. Furthermore, the HDL-mediated protection against LDL oxidation ex vivo shown by the HDL isolated from Stz-CORM-2 mice did not differ from that obtained in Stz-vehicle mice. In conclusion, the antinociceptive effects produced by a high dose of CORM-2 were accompanied by antioxidative effects but were without favorable effects on the dyslipidemia manifested in diabetic mice.

Valor diagnóstico del cociente de cadenas ligeras libres de inmunoglobulinas en suero en las gammapatías monoclonales de significado incierto / Diagnostic value of serum immunoglobulin free light chains ratio in monoclonal gammopathies of undetermined significance

Introducción. La medida de la concentración de las cadenas ligeras libres de inmunoglobulinas en suero proporciona información clínicamente relevante en el diagnóstico, pronóstico y monitorización de pacientes con mieloma múltiple (MM) y con gammapatías monoclonales de significado incierto (GMSI). En este trabajo se ha calculado el valor predictivo negativo (VPN) del cociente kappa/lambda libres (kappa/lambda) en un suero incluido dentro del rango diagnóstico (0,26-1,65), además de establecer unos nuevos valores discriminantes con un VPN del 100% en el grupo de pacientes estudiado. Material y métodos. La medida de la concentración de cadenas ligeras libres en suero se realizó por nefelometría en 157 muestras de pacientes diagnosticados de MM o GMSI y se calculó el cociente kappa/lambda libres. Resultados. El área bajo la curva de rendimiento diagnóstico para el cociente kappa/lambda libres fue de 0,885 en los pacientes con gammapatías con isotipo kappa y 0,879 en pacientes con gammapatías con isotipo lambda. El VPN para un cociente kappa/lambda libres entre 0,26 y 1,65 fue del 92%. Utilizando un intervalo de 0,36-1,0 se alcanzó un VPN del 100%. Conclusiones. La modificación del rango diagnóstico de 0,26-1,65 del cociente kappa/lambda libres por el comprendido entre 0,36 y 1,0, podría ser útil para obviar la realización de un aspirado de médula ósea en pacientes con criterios clínicos y analíticos de GMSI (AU)

Background. Measurement of immunoglobulin free light chains provides relevant clinical information for the diagnosis, prognosis and monitoring of multiple myeloma (MM) and monoclonal gammopathies of undetermined significance (MGUS). We evaluated the negative predictive value (NPV) of a serum free kappa to lambda (kappa/Lambda) ratio included within the described reference range (0.26-1.65) and also set cut-off points for the ratio in order to ensure a 100% NPV. Methods. Serum concentration of free light chains was measured by nephelometry in 157 individuals diagnosed as having MM or MGUS, and the free kappa/Lambda ratio was calculated. Results. The area under the curve of the free kappa/lambda ratio was 0.885 in the kappa type gammopathies subgroup, and 0.879 for the lambda subgroup. The NPV for a free kappa/lambda ratio between 0.26 and 1.65 was 92%. Using cut-off points of 0.36 and 1.0 for the ratio, achieved a 100% NPV. Conclusions. A change from the cut-off point 0.26-1.65 to 0.36-1.0 for the free kappa/lambda ratio could be useful in order to avoid performing a bone marrow aspirate in patients with MGUS clinical and laboratory criteria (AU)

Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry.

Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum requires optimized, reproducible methods for handling and deposition of protein samples. Data acquisition in MALDI MS is also a critical issue, since heterogeneity of sample deposits leads to attenuation of ion signals in MALDI MS. In order to improve the robustness and reproducibility of MALDI MS for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard to chromatographic properties; reversed phase (C8, C18, SDB-XC), ion-exchange (anion, weak cation, mixed-phase (SDB-RPS)) and magnetic beads were tested with regard to chromatographic properties; reversed phase (C8) or affinity chromatography (Cu-IMAC). The reproducibility of each sample preparation method was determined by enumeration and analysis of protein signals that were detected in at least six out of nine spectra obtained by three triplicate analyses of one serum sample.A candidate for best overall performance as evaluated by the number of peaks generated and the reproducibility of mass spectra was found among the tested methods. Up to 418 reproducible peaks were detected in one cancer serum sample. These protein peaks can be part of a possible diagnostic profile, suggesting that this sample preparation method and data acquisition approach is suitable for large-scale analysis of serum samples for protein profiling.

Análisis del proteoma hepático de ratones transgénicos de apo A-II humana: identificación de proteínas potencialmente implicadas en la regulación del metabolismo de triglicéridos y la respuesta a la insulina / Analysis of the liver proteome of human apolipoprotein A-II transgenic mice: identification of proteins potentially involved in the regulation of triglyceride metabolism and insulin response

Introducción. La función de la apolipoproteína A-II (apo A-II) en el metabolismo lipoproteico y su relación con la arteriosclerosis es poco conocida. Los estudios realizados en humanos y ratones modificados genéticamente han demostrado un efecto directo de la apo A-II en el metabolismo de los triglicéridos y los ácidos grasos libres (AGL) y la sensibilidad a la insulina. El objetivo principal de este estudio es la identificación de proteínas diferencialmente expresadas en el hígado de ratones transgénicos de apo A-II humana (h) y su potencial relación con el metabolismo de los triglicéridos y la glucosa. Material y métodos. Se realizaron estudios metabólicos de las lipoproteínas ricas en triglicéridos, la betaoxidación hepática y la prueba de tolerancia a la glucosa en ratones transgénicos de apo A-IIh y en controles en situación de ayunas y tras una carga oral de aceite de oliva. Los cambios en el perfil de expresión proteica se analizaron mediante el análisis comparativo de geles bidimensionales y la identificación de proteínas mediante espectrometría de masas MALDI-TOF. Resultados. Los ratones transgénicos de apo A-IIh presentaban un incremento del colesterol y los triglicéridos de las lipoproteínas que contienen apo B, hipertrigliceridemia aumentada tras la carga oral de ácido oleico, así como un aclaramiento acelerado de la glucosa tras la prueba de sobrecarga de glucosa. Estos cambios estaban asociados a una reducción en el catabolismo de las lipoproteínas ricas en triglicéridos y la tasa de betaoxidación hepática, pero sin cambios significativos en la actividad de la lipoproteína lipasa. De las más de 1.000 manchas resueltas en el rango pH 3 a 10, se identificaron 55 alteraciones significativas en los ratones transgénicos en comparación con los ratones controles, 16 de las cuales estaban relacionadas directamente con el metabolismo de los AGL y los carbohidratos. Conclusiones. La sobreexpresión apo A-IIh en ratones transgénicos induce hipertrigliceridemia posprandial debido, al menos en parte, a un defecto en el catabolismo de las lipoproteínas ricas en triglicéridos. La aproximación proteómica nos ha permitido detectar y caracterizar diferencias en el proteoma hepático de los ratones transgénicos de apo A-IIh y establecer proteínas potencialmente involucradas en el metabolismo de los AGL. Se requieren más estudios bioquímicos y moleculares para investigar el significado funcional de los cambios encontrados (AU)

Introduction. The role of apolipoprotein A-II (apo A-II) in lipoprotein metabolism and its relationship with atherosclerosis is poorly understood. Several studies both in humans and genetically modified mice have shown a direct effect of apo A-II on triglyceride and free fatty acid (FFA) metabolism and on insulin sensitivity. The aim of this study was to identify the proteins differentially expressed in the livers of human apo A-II transgenic mice and their potential relationship with triglyceride and glucose metabolism. Matherial and methods. Metabolic studies of triglyceride-rich lipoproteins, liver beta-oxidation and the glucose tolerance test were conducted in C57BL/6 control mice and human apo A-II transgenic mice in both fasting and postprandial states. The changes in protein expression patterns were determined by analysis of two-dimensional polyacrylamide gel electrophoresis while protein identification was performed by mass spectrometry (MALDI-TOF). Results. Human apo A-II transgenic mice showed an increase in cholesterol and triglycerides from apo B-containing lipoproteins, a higher response after oral fat test with oleic acid and higher glucose clearance after the glucose test. These changes were associated with a reduction in the clearance of triglyceride-rich lipoproteins and in the rate of liver beta-oxidation, but there were no significant changes in lipoprotein lipase activity. Within the pH 3-10 range, 55 out of more than 1,000 spots were identified to be significantly altered in human apo A-II transgenic mice compared with control mice, and 16 of these spots were directly related with FFA and carbohydrate metabolism. Conclusions. Overexpression of human apo A-II in transgenic mice induces postprandial hypertriglyceridemia. This finding is at least partly due to reduced catabolism of triglyceride-rich lipoproteins. We have been able to detect and characterize differences in the liver proteome of human apo A-II transgenic mice and to identify proteins potentially related to FFA metabolism. Further biochemical and molecular studies are required to investigate the functional significance of the changes found (AU)

NT-ProBNP reduction percentage during admission for acutely decompensated heart failure predicts long-term cardiovascular mortality.

BACKGROUND: N-terminal brain natriuretic peptide (NT-proBNP) improves emergency room diagnosis of acutely decompensated heart failure. Less evidence is available on the usefulness of NT-proBNP as a prognostic marker after hospitalization for acute heart failure. The percentage of NT-proBNP reduction during admission and its prognostic significance were studied. METHODS AND RESULTS: This was a prospective study of 74 patients in the emergency department who were diagnosed with acute heart failure and who had follow-up evaluation for 6 and 12 months after admission. Plasma NT-proBNP concentrations were measured on admission, at 24 hours, at day 7, and at 6 and 12 months. Eighteen patients died during the 12-month follow-up; 12 deaths were from cardiovascular causes. NT-proBNP concentrations were significantly higher in the emergency department and at 24 hours than those concentrations that were found at day 7 and beyond (P < .001). During admission, the NT-proBNP concentration fell a mean of 15% in patients who died of cardiovascular causes during the 1-year follow-up evaluation, in 75% in those patients who died of non-cardiovascular causes, and in 50% in survivors (P = .004). The area under the receiver operator characteristic curve for NT-proBNP reduction percentage to predict cardiovascular death was 0.78 (95% CI, 0.66-0.90; P = .002). A 30% NT-proBNP reduction percentage cutoff value had 75% accuracy for the identification of high-risk patients and was the only variable that was associated with cardiovascular death in multivariate analysis (odds ratio, 4.4; 95% CI, 1.12-17.4; P = .03). CONCLUSION: NT-proBNP reduction percentage during admission for acutely decompensated heart failure appeared to be the best predictor of cardiovascular death during the follow-up period. A <30% NT-proBNP reduction percentage identified a subgroup of high-risk patients.

N-terminal pro-brain natriuretic peptide reflects pulmonary capillary leakage in patients with acute dyspnea.

Natriuretic peptides have proved useful in the diagnosis of heart failure in patients presenting to the emergency department with shortness of breath. Dyspnea and orthopnea in heart failure are clinical expressions of pulmonary capillary congestion and leakage, which may be assessed by the percentage of pulmonary hemosiderin-laden macrophages (HLM) in induced sputum. We found a significant difference in the percentage of HLM present in sputum among patients with acute heart failure, patients with noncardiac dyspnea with ventricular dysfunction, and patients without heart failure (p = 0.008). N-terminal pro-brain natriuretic peptide (N-BNP) concentrations were also different among these 3 patient groups (p = 0.006). N-BNP concentrations were positively associated with the percentage of HLM in patients with acute dyspnea (r = 0.6; p < 0.0001). N-BNP, in addition to being a ventricular dysfunction marker, may reflect the severity of pulmonary capillary congestion and leakage in patients with acute shortness of breath.

Evaluation of two nonisotopic immunoassays for determination of glutamic acid decarboxylase and tyrosine phosphatase autoantibodies in serum.

BACKGROUND: Autoantibodies for the 65-kDa form of glutamic acid decarboxylase (GAD65) and protein tyrosine phosphatase-like protein (IA-2) are measured for risk prediction and diagnosis of autoimmune diabetes mellitus. There is a lack of adequate nonisotopic alternatives to the most widely used method for both autoantibodies, which is a radiobinding assay (RBA). METHODS: We compared two commercially available immunoassays, an ELISA and a time-resolved immunofluorometric assay (TR-IFMA), with RBA. RESULTS: We found excellent agreement between the RBA and ELISA for measurement of GAD65 autoantibodies (GADAs); they showed comparable analytical precision in the cutoff range and achieved similar diagnostic specificity. The ELISA identified more GADA-positive individuals among patients with new-onset type 1 diabetes than did the RBA [89% (95% confidence interval, 78-95%), vs 71% (58-82%); P <0.03]. For IA-2 autoantibodies (IA-2As), only the TR-IFMA achieved analytical performance and diagnostic accuracy comparable to that of the RBA. These results with the GADA ELISA and IA-2A TR-IFMA were consistent with those obtained blindly in the Diabetes Antibody Standardization Program 2003. The performance of the GADA TR-IFMA and IA-2A ELISA was unsatisfactory, and these tests were not subjected to clinical evaluation. CONCLUSIONS: The GADA ELISA and IA-2A TR-IFMA behave comparably with RBA and are thus suitable for use in the clinical laboratory.

Non-HDL cholesterol and apolipoprotein B in the dyslipidemic classification of type 2 diabetic patients.

OBJECTIVE: To compare non-HDL cholesterol (HDLc) and apolipoprotein B (apoB) in the identification of nonconventional high-risk dyslipidemic phenotypes in type 2 diabetic patients. RESEARCH DESIGN AND METHODS: Total cholesterol and triglycerides, HDLc, LDL cholesterol, non-HDLc, apolipoprotein B (apoB), and LDL size were determined in 122 type 2 diabetic patients (68% male, aged 59.6 +/- 9.7 years, and HbA(1c) 7.5% [range 5.2-16.0]). They were then classified as normo- and hypertriglyceridemic if their triglyceride concentrations were below/above 2.25 mmol/l, as normo/hyper-non-HDLc if non-HDLc concentrations were below/above 4.13 mmol/l, and as normo- and hyperapoB if apoB concentrations were below/above 0.97 g/l. Both classifications were compared (concordance assessed with the kappa index), and low HDLc and LDL phenotype B were identified in each category. RESULTS: A total of 26 patients were hypertriglyceridemic and 96 were normotriglyceridemic. All hypertriglyceridemic subjects had increased non-HDLc, whereas 24 had increased apoB (kappa= 0.95). In the normotriglyceridemic group, 44 had increased non-HDLc, 68 had increased apoB, and 25 of the 52 patients with normal non-HDLc had increased apoB (kappa= 0.587). Low HDLc and LDL phenotype B were similarly distributed into the equivalent categories. CONCLUSIONS: Non-HDLc and apoB are equivalent risk markers in hypertriglyceridemic patients, but apoB identifies additional patients with high-risk dyslipidemic phenotypes in normotriglyceridemic type 2 diabetic patients.

The effect of VLDL particles on the accuracy of a direct LDL-cholesterol method in type 2 diabetic patients.

OBJECTIVE: To assess the accuracy of the direct method LDL-c Plus, in type 2 diabetic patients.LDL-c Plus was measured in 64 consecutive samples of type 2 diabetic patients and compared with betaquantification (BQ), Friedewald's and an alternative formula. METHODS: LDL-c Plus was also measured in the VLDL (d<1.006 Kg/L) fraction of these samples and in total serum and the VLDL fraction of a phenotype III patient, before and after diluting it with saline or VLDL from normolipidemic subjects. RESULTS: LDL-c Plus showed a significant, constant bias (-8.5 +/- 5.6%) against BQ which correlated with VLDL-cholesterol/total triglyceride ratio (r = 0.760, p < 0.0005); bias decreased to zero when the ratio increased. In the VLDL fraction of the diabetic patients and the phenotype III patient LDL-c Plus measured 20.7 +/- 11.6% and 56.2% of the cholesterol, respectively. Dilution with saline did not alter the latter percentage, whereas dilution with normolipidemic VLDL reduced it showing that LDL-c Plus recognized cholesterol-enriched particles in the d<1.006 Kg/L. Friedewald's formula also showed a significant, constant bias (-3.1 +/- 6.4%) against BQ, whereas the alternative formula did not (0.5 +/- 6.1%). Both calculations classified patients better than LDL-c Plus did at NCEP cut-off points. CONCLUSIONS: In type 2 diabetic patients, LDL-c Plus underestimates LDL-c but measures cholesterol associated to IDL particles in the d<1.006 Kg/L fraction. Although LDLc-Plus might be a better cardiovascular risk estimator when well standardized, at the moment, it does not seem to be superior to calculations.