Harald Sourij

Multicentric Validation of Proteomic Biomarkers in Urine Specific for Diabetic Nephropathy

Background Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration ≥5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82). Methodology/Principal Findings Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patients subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients. Conclusions These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin.

Short-term effects of high-dose oral vitamin D3 in critically ill vitamin D deficient patients: a randomized, double-blind, placebo-controlled pilot study

IntroductionVitamin D deficiency is encountered frequently in critically ill patients and might be harmful. Current nutrition guidelines recommend very low vitamin D doses. The objective of this trial was to evaluate the safety and efficacy of a single oral high-dose vitamin D3 supplementation in an intensive care setting over a one-week observation period.MethodsThis was a randomized, double-blind, placebo-controlled pilot study in a medical ICU at a tertiary care university center in Graz, Austria. Twenty-five patients (mean age 62 ± 16yrs) with vitamin D deficiency [25-hydroxyvitamin D (25(OH)D) ≤20 ng/ml] and an expected stay in the ICU >48 hours were included and randomly received either 540,000 IU (corresponding to 13.5 mg) of cholecalciferol (VITD) dissolved in 45 ml herbal oil or matched placebo (PBO) orally or via feeding tube.ResultsThe mean serum 25(OH)D increase in the intervention group was 25 ng/ml (range 1-47 ng/ml). The highest 25(OH)D level reached was 64 ng/ml, while two patients showed a small (7 ng/ml) or no response (1 ng/ml). Hypercalcemia or hypercalciuria did not occur in any patient. From day 0 to day 7, total serum calcium levels increased by 0.10 (PBO) and 0.15 mmol/L (VITD; P < 0.05 for both), while ionized calcium levels increased by 0.11 (PBO) and 0.05 mmol/L (VITD; P < 0.05 for both). Parathyroid hormone levels decreased by 19 and 28 pg/ml (PBO and VITD, ns) over the seven days, while 1,25(OH)D showed a transient significant increase in the VITD group only.ConclusionsThis pilot study shows that a single oral ultra-high dose of cholecalciferol corrects vitamin D deficiency within 2 days in most patients without causing adverse effects like hypercalcemia or hypercalciuria. Further research is needed to confirm our results and establish whether vitamin D supplementation can affect the clinical outcome of vitamin D deficient critically ill patients.EudraCT Number2009-012080-34German Clinical Trials Register (DRKS)DRKS00000750

The influence of probiotic supplementation on gut permeability in patients with metabolic syndrome: an open label, randomized pilot study

Background/objectives:Obesity and metabolic disorders are linked to inflammation via gut microbiota and/or gut permeability. Gut-derived endotoxin triggers inflammation leading to metabolic syndrome (MetS) and contributing to oxidative stress. We intended to investigate the effect of Lactobacillus casei Shirota on gut permeability, presence of endotoxin and neutrophil function in MetS.Subjects/methods:Patients with MetS were randomized to receive 3 × 6.5 × 109 CFU L. casei Shirota (probiotic group) or not for 3 months. Gut permeability was assessed by a differential sugar absorption method and by determination of diaminooxidase serum levels, endotoxin by an adapted limulus amoebocyte lysate assay, neutrophil function and toll-like receptor (TLR) expression by flow cytometry and ELISA was used to detect lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14) levels.Results:Twenty-eight patients and 10 healthy controls were included. Gut permeability was significantly increased in MetS compared with controls but did not differ between patient groups. None of the patients were positive for endotoxin. LBP and sCD14 levels were not significantly different from healthy controls. High-sensitive C-reactive protein and LBP levels slightly but significantly increased after 3 months within the probiotics group. Neutrophil function and TLR expression did not differ from healthy controls or within the patient groups.Conclusions:Gut permeability of MetS patients was increased significantly compared with healthy controls. L. casei Shirota administration in the MetS patients did not have any influence on any parameter tested possibly due to too-short study duration or underdosing of L. casei Shirota.

Chronic TNF-a neutralization does not improve insulin resistance or endothelial function in "healthy" men with metabolic syndrome

The possible contribution of tumor necrosis factor-α (TNF-α) to the development of obesity-associated insulin resistance in humans is still controversial. Our study investigated the effect of TNF-α neutralization on insulin resistance in healthy, obese and insulin resistant men. We performed a prospective, randomized, double-blind placebo-controlled trial in nine young, healthy obese male subjects with metabolic syndrome and insulin resistance. Volunteers received three infusions (wks 0, 2 and 6) of infliximab or placebo. Insulin resistance was measured at baseline and after 70 d by homeostatic model assessment (HOMA) index as well as by minimal model analysis of an intravenous glucose tolerance test. Endothelial function was accessed before and after intervention by flow mediated dilation. Infliximab improved the inflammatory status as indicated by reduced high sensitivity C-reactive protein (hsCRP) and fibrinogen levels (2.77 ± 0.6 to 1.8 ± 0.5 µg/L, and 3.42 ± 0.18 to 3.18 ± 0.28 g/L; (day 0 and day 70, P = 0.020 and 0.037 respectively), but did not improve insulin resistance (HOMA index and intravenous glucose-tolerance test [ivGGT]) or endothelial function. Despite improvements in inflammatory status, chronic TNF-α neutralization does not improve insulin resistance or endothelial function in seemingly healthy, but obese, insulin-resistant volunteers. This study severely questions the proposal that TNF-α is a causative link between adiposity and insulin resistance.

Key role of postchallenge hyperglycemia for the presence and extent of coronary atherosclerosis: An angiographic study

BACKGROUND The associations between impaired glucose tolerance (IGT) and postchallenge diabetes with the presence and extent of angiographically characterized coronary atherosclerosis are unclear. MATERIALS AND METHODS We enrolled 1040 consecutive Caucasian patients undergoing coronary angiography for the evaluation of coronary artery disease (CAD). An oral 75-g glucose tolerance test was performed in patients without previously diagnosed diabetes. RESULTS From our patients, 394 had normal glucose tolerance (NGT), 190 impaired glucose tolerance (IGT), 90 isolated postchallenge diabetes (postchallenge glucose >or=200 mg/dl), and 366 type 2 diabetes previously established or newly diagnosed on the basis of fasting glucose (conventional diabetes). Coronary atherosclerosis was more frequent in patients with IGT, isolated postchallenge diabetes, or conventional diabetes when compared to NGT subjects (87.9, 95.6, 89.1 versus 80.7%; p=0.030, 0.001, 0.043, respectively). The prevalence of significant coronary stenoses >or=50%, compared to NGT subjects (57.4%), was similar in IGT patients (59.5%; p=0.628), but significantly higher in patients with isolated postchallenge diabetes (77.8%; p=0.001) or conventional diabetes (68.0%; p=0.002). Also the number of significant stenoses compared to NGT subjects was similar in IGT patients, but significantly higher in those with isolated postchallenge or conventional diabetes. These results were confirmed after multivariate adjustment. CONCLUSIONS Abnormal glucose tolerance is strongly and independently associated with angiographically characterized coronary atherosclerosis. In IGT, non-significant coronary atherosclerosis is more frequent than in NGT; the prevalence and number of significant stenoses increases when postchallenge diabetes evolves.

Arginine bioavailability ratios are associated with cardiovascular mortality in patients referred to coronary angiography

OBJECTIVES Arginine is the only source for nitric oxide (NO) synthesis. The bioavailability of NO plays a pivotal role in endothelial function and consequently in cardiovascular disease. The aim of the current study is to investigate the association of arginine bioavailability ratios with markers of endothelial function and cardiovascular mortality in patients referred to coronary angiography. METHODS We investigated 2236 patients recruited within the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study that were followed up for a median of 7.7 years. Arginine, ornithine and citrulline were chromatographically determined after precolumn-derivatisation followed by postcolumn continuous reaction with ninhydrin. Global arginine bioavailability (GABR) was calculated by arginine divided by the sum of ornithine plus citrulline. RESULTS We observed a significant rise in cardiovascular mortality with decreasing GABR and arginine to ornithine ratio quartiles. The adjusted Cox proportional HRs for GABR were 1.27 (0.88-1.83), 1.27 (0.89-1.80) and 1.75 (1.24-2.45) for the 3rd, the 2nd and the 1st quartile respectively in comparison to the 4th quartile. The HRs for the quartiles of the arginine to ornithine ratio were 1.83 (1.25-2.67), 2.17 (1.50-3.20) and 2.02 (1.39-2.92) respectively. Patients with type 2 diabetes mellitus had a significantly lower GABR than persons without diabetes (0.88 ± 0.23 vs. 0.94 ± 0.24, p<0.001). GABR was found to be inversely correlated with endothelial markers as VCAM-1 (r=-0.301, p<0.001) or ICAM-1 (r=-0.136, p<0.001). CONCLUSIONS GABR and the arginine to ornithine ratio are associated with markers of endothelial dysfunction and increased risk of cardiovascular mortality. Further studies are warranted to elucidate the pathobiology and clinical relevance of the arginine bioavailability ratios in cardio-metabolic diseases.

Low-dose atorvastatin improves dyslipidemia and vascular function in patients with primary biliary cirrhosis after one year of treatment.

OBJECTIVE Primary biliary cirrhosis (PBC) is frequently associated with hypercholesterolemia and with an increased cardiovascular morbidity and mortality. Statins lower serum cholesterol levels and may thus improve the cardiovascular risk in PBC patients. The aim of our study was to prospectively examine the efficacy of low-dose atorvastatin on cholestasis as well as cardiovascular risk markers such as dyslipidemia and vascular function in patients with PBC. METHODS Nineteen patients with early-stage (biopsy proven and AMA positive) PBC and low-density lipoprotein cholesterol (LDL-C) above 130mg/dL were included in this single-center study and treated with atorvastatin 10mg per day for one year. RESULTS Concentrations of total cholesterol, LDL-C, LDL triglycerides, oxLDL, IgG and sVCAM-1 decreased significantly after 48 weeks of atorvastatin treatment. Flow-mediated dilation (FMD) of the brachial artery as an indicator of vascular function significantly increased, while carotid artery intima-media thickness and vascular wall stiffness did not progress under treatment. No statistical differences in liver enzymes were observed except a transient increase of alkaline phosphatase. CONCLUSION Treatment with low-dose atorvastatin is safe in early-stage PBC, effectively reduces total cholesterol, LDL-C, LDL triglycerides, oxLDL and sVCAM-1 and improves vascular function as reflected by FMD, without affecting cholestasis progression. Therefore, statin therapy should be considered in PBC patients with additional risk factors for cardiovascular disease.

Post-challenge hyperglycaemia is strongly associated with future macrovascular events and total mortality in angiographied coronary patients

AIMS The prevalence of post-challenge hyperglycaemia in coronary patients is high. Until now, it is unclear whether post-challenge hyperglycaemia is associated with an increased risk for future macrovascular events in this clinically important patient population. METHODS AND RESULTS We enrolled 1040 patients undergoing coronary angiography for the evaluation of suspected or established coronary artery disease. In patients without previously established diabetes mellitus, an oral glucose tolerance test (oGTT) was performed. Prospectively, mortality and macrovascular events were recorded over a mean follow-up period of 3.8 years. From our patients, 394 had normal glucose tolerance (NGT), 280 post-challenge hyperglycaemia (this subgroup includes both impaired glucose tolerance and post-challenge diabetes) and 366 had conventional diabetes. The incidence of macrovascular events was significantly higher in patients with post-challenge hyperglycaemia as well as in patients with conventional diabetes than in subjects with NGT (23.6 and 29.5% vs. 18.5%; P = 0.013 and P < 0.001, respectively). Adjusted hazard ratios were 1.46 (95% CI 1.03-2.07, P = 0.033) for patients with post-challenge hyperglycaemia and 1.73 (1.25-2.37, P = 0.001) for patients with conventional diabetes. CONCLUSION Post-challenge hyperglycaemia is associated with future macrovascular events in patients undergoing coronary angiography for the evaluation of stable coronary artery disease (CAD). Oral glucose tolerance tests in this high-risk population thus identify patients with a particularly unfavourable prognosis.

Short communication: Effect of supplementation with Lactobacillus casei Shirota on insulin sensitivity, β-cell function, and markers of endothelial function and inflammation in subjects with metabolic syndrome—A pilot study

Based on animal studies, intake of probiotic bacteria was suggested to improve insulin sensitivity by reducing endotoxinemia and inflammation. The objective of this study was to determine the effects of supplementation with the probiotic strain Lactobacillus casei Shirota (LcS) over 12 wk on insulin sensitivity, β-cell function, inflammation, and endothelial dysfunction parameters in subjects with metabolic syndrome. In a randomized-controlled study, 30 subjects with metabolic syndrome either received Lactobacillus casei Shirota 3 times daily for 12 wk or served as controls with standard medical therapy. Fasting blood samples were taken and a 75-g oral glucose tolerance test was performed to derive indices for insulin sensitivity and β-cell function. In addition, parameters to assess endothelial function and inflammation markers were determined. Even though the insulin sensitivity index significantly improved after 3 mo of probiotic supplementation (0.058±0.021 vs. 0.038±0.025), the change was not significantly different compared with the control group. No improvements were seen in additional indices of insulin sensitivity (quantitative insulin sensitivity check index, insulin sensitivity by oral glucose tolerance test, and homeostasis model assessment for insulin resistance) and β-cell function (first and second phase insulin secretion, and homeostasis model assessment for β-cell function). Probiotic supplementation resulted in a significant reduction in soluble vascular cell adhesion molecule-1 (sVCAM-1) level (1,614±343 vs. 1,418±265 ng/mL). No significant changes in parameters used to assess low-grade inflammation or endothelial dysfunction were observed. Intake of LcS for 12 wk in subjects with metabolic syndrome did not improve insulin sensitivity, β-cell function, endothelial function, or inflammation markers in this trial.

Graz Endocrine Causes of Hypertension (GECOH) study: a diagnostic accuracy study of aldosterone to active renin ratio in screening for primary aldosteronism

BackgroundPrimary aldosteronism (PA) affects approximately 5 to 10% of all patients with arterial hypertension and is associated with an excess rate of cardiovascular complications that can be significantly reduced by a targeted treatment. There exists a general consensus that the aldosterone to renin ratio should be used as a screening tool but valid data about the accuracy of the aldosterone to renin ratio in screening for PA are sparse. In the Graz endocrine causes of hypertension (GECOH) study we aim to prospectively evaluate diagnostic procedures for PA.Methods and designIn this single center, diagnostic accuracy study we will enrol 400 patients that are routinely referred to our tertiary care center for screening for endocrine hypertension. We will determine the aldosterone to active renin ratio (AARR) as a screening test. In addition, all study participants will have a second determination of the AARR and will undergo a saline infusion test (SIT) as a confirmatory test. PA will be diagnosed in patients with at least one AARR of ≥ 5.7 ng/dL/ng/L (including an aldosterone concentration of ≥ 9 ng/dL) who have an aldosterone level of ≥ 10 ng/dL after the saline infusion test. As a primary outcome we will calculate the receiver operating characteristic curve of the AARR in diagnosing PA. Secondary outcomes include the test characteristics of the saline infusion test involving a comparison with 24 hours urine aldosterone levels and the accuracy of the aldosterone to renin activity ratio in diagnosing PA. In addition we will evaluate whether the use of beta-blockers significantly alters the accuracy of the AARR and we will validate our laboratory methods for aldosterone and renin.ConclusionScreening for PA with subsequent targeted treatment is of great potential benefit for hypertensive patients. In the GECOH study we will evaluate a standardised procedure for screening and diagnosing of this disease.