Benjamin Dieplinger

A Variant Associated with Nicotine Dependence, Lung Cancer and Peripheral Arterial Disease

Smoking is a leading cause of preventable death, causing about 5 million premature deaths worldwide each year. Evidence for genetic influence on smoking behaviour and nicotine dependence (ND) has prompted a search for susceptibility genes. Furthermore, assessing the impact of sequence variants on smoking-related diseases is important to public health. Smoking is the major risk factor for lung cancer (LC) and is one of the main risk factors for peripheral arterial disease (PAD). Here we identify a common variant in the nicotinic acetylcholine receptor gene cluster on chromosome 15q24 with an effect on smoking quantity, ND and the risk of two smoking-related diseases in populations of European descent. The variant has an effect on the number of cigarettes smoked per day in our sample of smokers. The same variant was associated with ND in a previous genome-wide association study that used low-quantity smokers as controls, and with a similar approach we observe a highly significant association with ND. A comparison of cases of LC and PAD with population controls each showed that the variant confers risk of LC and PAD. The findings provide a case study of a gene–environment interaction, highlighting the role of nicotine addiction in the pathology of other serious diseases.

Analytical and clinical evaluation of a novel high-sensitivity assay for measurement of soluble ST2 in human plasma — The Presage™ ST2 assay

BACKGROUND The protein ST2 is a member of the interleukin-1 receptor family. Blood concentrations of the soluble isoform of ST2 (sST2) are increased in inflammatory diseases and in heart disease and are considered a prognostic marker in both. The aim of this study was the analytical and clinical evaluation of the novel Presage ST2 assay for the determination of sST2 in human plasma. METHODS We evaluated precision and linearity of the assay, analyte stability, and biological variability, determined reference values, performed a method comparison with an established ELISA, and quantified sST2 concentrations in various diseases. RESULTS Within-run and total coefficients of variation were <2.5% and <4.0%. The method was linear across the whole measurement range of the assay. The analyte was stable for 48 h at room temperature, for 7 days at 4 degrees C, and for at least 2 months at -20 degrees C and -80 degrees C. The reference change value for healthy individuals was 30%. Age-independent reference values were 3-28 U/mL in males, and 2-16 U/mL in females. The method comparison revealed a high proportional bias. sST2 plasma concentrations were increased modestly in heart failure and moderately in pneumonia and chronic obstructive pulmonary disease. Patients with sepsis exhibited highly elevated sST2 values. In patients with chronic renal disease, however, there was no difference compared to healthy individuals. CONCLUSION The Presage ST2 assay meets the needs of quality specifications of laboratory medicine. The results of the clinical assay evaluation are novel with respect to sST2 in various diseases and should initiate further studies.

Increased Plasma Concentrations of Soluble ST2 are Predictive for 1-Year Mortality in Patients with Acute Destabilized Heart Failure

BACKGROUND The soluble isoform of the interleukin-1 receptor family member ST2 (sST2) has been implicated in heart failure. The aim of the present study was to evaluate the capability of sST2 as a prognostic marker in patients with acute destabilized heart failure. METHODS sST2 plasma concentrations were obtained in 137 patients with acute destabilized heart failure attending the emergency department of a tertiary care hospital. The endpoint was defined as all-cause mortality, and the study participants were followed up for 365 days. RESULTS Of the 137 patients enrolled, 41 died and 96 survived during follow-up. At baseline the median sST2 plasma concentration was significantly higher in the patients who died than in those who survived (870 vs 342 ng/L, P <0.001). Kaplan-Meier curve analyses demonstrated that the risk ratios for mortality were 2.45 (95% CI, 0.88-6.31; P = 0.086) and 6.63 (95% CI, 2.55-10.89; P <0.001) in the second tercile (sST2, 300-700 ng/L; 11 deaths vs 34 survivors) and third tercile (sST2, >700 ng/L; 25 deaths vs 21 survivors) of sST2 plasma concentrations compared with the first tercile (sST2, < or =300 ng/L; 5 deaths vs 41 survivors). In multivariable Cox proportional-hazards regression analyses, an sST2 plasma concentration in the upper tercile was a strong and independent predictor of all-cause mortality. CONCLUSIONS Increased sST2 concentrations determined in plasma samples drawn from patients with acute destabilized heart failure at their initial presentation indicate increased risk of future mortality. Increased sST2 plasma concentrations are independently and strongly associated with one-year all-cause mortality in these patients.

Long-term stability of endogenous B-type natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) in frozen plasma samples.

Abstract The aim of the present study was to assess the long-term stability of endogenous B-type natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) in plasma samples stored at –20°C without addition of protease inhibitors (e.g., aprotinin). Stability of BNP and NT-proBNP was tested in 60 EDTA plasma samples with BNP values between 30 and 420 pg/ml. Initial BNP and NT-proBNP plasma concentrations were determined within four hours after blood collection using the AxSYM BNP and the Elecsys NT-proBNP assays. Subsequently, all samples were stored at –20°C and were thawed for the second BNP and NT-proBNP determination on the two instruments after one day, 30 days, 60 days, 90 days and 120 days, respectively. Mean recovery (i.e., residual immunoreactivity) of BNP and NT-proBNP expressed in percent of the initial value for the given time interval of storage was calculated. Mean recovery of BNP was less than 70% after one day of storage at –20°C and decreased to less than 50% after two to four months of storage (e.g., recovery of endogenous BNP after three months of storage at –20°C ranging from 0% to 71%). In contrast, mean recovery of NT-proBNP was generally greater than 90%, irrespective of the duration of storage at –20°C (e.g., recovery of endogenous NT-proBNP after three months of storage at –20°C ranging from 91% to 112%). In conclusion, the determination of endogenous BNP with the AxSYM assay using frozen plasma samples may not be valid under the conditions tested. In contrast, NT-proBNP as measured by the Elecsys assay may be stored at –20°C for at least four months without a relevant loss of the immunoreactive analyte.

Proteomic Profiling Identifies Afamin as a Potential Biomarker for Ovarian Cancer

Purpose: To discover and validate serum glycoprotein biomarkers in ovarian cancer using proteomic-based approaches. Experimental Design: Serum samples from a “discovery set” of 20 patients with ovarian cancer or benign ovarian cysts or healthy volunteers were compared by fluorescence two-dimensional differential in-gel electrophoresis and parallel lectin-based two-dimensional profiling. Validation of a candidate biomarker was carried out with Western blotting and immunoassay (n = 424). Results: Twenty-six proteins that changed significantly were identified by mass spectrometric sequencing. One of these, confirmed by Western blotting, was afamin, a vitamin E binding protein, with two isoforms decreasing in patients with ovarian cancer. Validation using cross-sectional samples from 303 individuals (healthy controls and patients with benign, borderline, or malignant ovarian conditions and other cancers) assayed by ELISA showed significantly decreased total afamin concentrations in patients with ovarian cancer compared with healthy controls (P = 0.002) and patients with benign disease (P = 0.046). However, the receiver operating characteristic areas for total afamin for the comparison of ovarian cancer with healthy controls or benign controls were only 0.67 and 0.60, respectively, with comparable figures for CA-125 being 0.92 and 0.88 although corresponding figures for a subgroup of samples analyzed by isoelectric focusing for afamin isoform 2 were 0.85 and 0.79. Analysis of a further 121 samples collected prospectively from 9 patients pretreatment through to relapse indicated complementarity of afamin with CA-125, including two cases in whom CA-125 was noninformative. Conclusions: Afamin shows potential complementarity with CA-125 in longitudinal monitoring of patients with ovarian cancer, justifying prospective larger-scale investigation. Changes in specific isoforms may provide further information.

Evaluation of novel biomarkers for the diagnosis of acute destabilised heart failure in patients with shortness of breath

Objective: The evaluation of novel biomarkers for the diagnosis of acute destabilised heart failure (HF). Design: Prospectively conducted study on diagnostic accuracy. Setting: Emergency department of a tertiary care hospital. Patients: 251 consecutive patients presenting to the emergency department with dyspnoea as the chief complaint. Main outcome measures: Index tests were plasma concentrations of 10 biomarkers (BNP, MR-proANP, MR-proADM, copeptin, CT-proET-1, ST2, adiponectin, chromogranin A, proguanylin and prouroguanylin). The reference standard was the diagnosis of acute destabilised HF, which was based on the Framingham score for HF plus echocardiographic evidence of systolic or diastolic dysfunction. Results: Median plasma concentrations of all 10 biomarkers were higher in patients with dyspnoea attributable to acute destabilised HF (n = 137) than in patients with dyspnoea attributable to other reasons (n = 114). Applying receiver operating characteristic curve (ROC) analyses, areas under the curve (AUCs) for BNP (0.92) and MR-proANP (0.88) were significantly higher than the AUCs of the other eight biomarkers (MR-proADM, 0.75; adiponectin, 0.73; CT-proET-1, 0.72; proguanylin, 0.68; ST2, 0.67; prouroguanylin, 0.62; copeptin, 0.62; and chromogranin A, 0.56). In multivariate logistic regression analysis only increased BNP and MR-proANP concentrations remained independent markers for the diagnosis of HF. Both markers alone or in combination added similar diagnostic information besides all clinical information available in the emergency department. Conclusions: The data showed that BNP and MR-proANP were the only independent diagnostic markers of HF. Both markers provided similar diagnostic information and were clinically useful as an aid in the diagnosis of acute destabilised HF in an emergency setting.

Prognostic value of established and novel biomarkers in patients with shortness of breath attending an emergency department.

OBJECTIVES Acute dyspnea is a common cause for emergency department visits. The aim of this study was to evaluate the prognostic value of established and novel biomarkers in patients with acute dyspnea. DESIGN AND METHODS We measured 10 biomarkers [B-type natriuretic peptide (BNP), midregional pro-A-type natriuretic peptide (MR-proANP), midregional-proadrenomedullin (MR-proADM), copeptin, C-terminal endothelin-1 precursor fragment (CT-proET-1), soluble ST2 (sST2), chromogranin A (CgA), adiponectin, proguanylin, and prouroguanylin] in 251 consecutive patients with acute dyspnea presenting to the emergency department of a tertiary care hospital. Outcome measure was all-cause mortality at 1 year. RESULTS At baseline decedents (n=62) had significantly higher median plasma concentrations of all 10 biomarkers than survivors (n=189). Applying univariate Cox proportional-hazard regression analyses, all biomarkers were significant outcome predictors displaying risk ratios (RR) from 1.4 to 2.4 (per 1 SD increase in log transformed values). In multivariate Cox proportional-hazard regression analysis, however, only MR-proANP (RR 1.6; 95% CI, 1.1-2.2; p=0.008), sST2 (RR 1.7; 95% CI, 1.3-2.3; p<0.001), and CgA (RR 1.5; 95% CI, 1.2-1.9, p<0.001) were independently associated with 1-year mortality. We provide a possible explanation for the complementary prognostic value of those three biomarkers in our cohort, where coincidence of heart failure and inflammatory pulmonary disease was common and also related to worse outcome. CONCLUSIONS Our evaluation of biomarkers in patients with acute dyspnea suggests that MR-proANP, sST2, and CgA are strong, independent and complementary outcome predictors. MR-proANP is considered a specific marker of cardiac stretch, sST2 might reflect both inflammation and cardiac stretch, and CgA obviously indicates neuroendocrine activation in various diseases.

Pro-A-type natriuretic peptide and pro- adrenomedullin predict progression of chronic kidney disease: the MMKD Study

A-type natriuretic peptide (ANP) and adrenomedullin (ADM) are potent hypotensive, diuretic, and natriuretic peptides involved in maintaining cardiovascular and renal homeostasis. We conducted a prospective 7-year study of 177 nondiabetic patients with primary chronic kidney disease to see if ANP and ADM plasma concentrations predict the progression of their disease, using novel sandwich immunoassays covering the midregional epitopes of the stable prohormones (MRproANP and MR-proADM). Progression of chronic kidney disease was defined as doubling of baseline serum creatinine and/or terminal renal failure, which occurred in 65 patients. Analysis of the receiver operating characteristic curve for the prediction of renal endpoints showed similar areas under the curve for the glomerular filtration rate (GFR) (0.838), MR-proANP (0.810), and MRproADM (0.876), respectively, as did the Kaplan-Meier curve analyses of the patients stratified according to the median of the respective markers. In separate multiple Cox-proportional hazard regression analyses, increased plasma concentrations of both peptides were each strongly predictive of the progression of chronic kidney disease after adjustments for age, gender, GFR, proteinuria and amino-terminal pro-B-type natriuretic peptide. Our study suggests that MR-proANP and MR-proADM are useful new markers of progression of primary nondiabetic chronic kidney disease.

Delayed In Vivo Catabolism of Intermediate-Density Lipoprotein and Low-Density Lipoprotein in Hemodialysis Patients as Potential Cause of Premature Atherosclerosis

Objective—Premature cardiovascular disease is the leading cause of death in patients with end-stage renal disease treated by hemodialysis (HD). Low-density lipoprotein (LDL) levels are not generally increased in HD patients, but their LDL metabolism is still poorly understood. We therefore investigated the in vivo metabolism of apoB-containing lipoproteins in two different ethnic populations of HD patients and controls. Methods and Results—We performed stable isotope kinetic studies using a primed constant infusion of deuterated leucine in 12 HD patients and 13 healthy controls. Tracer/tracee ratio of apoB was determined by means of gas chromatography/mass spectrometry, and the modeling program SAAMII was used to estimate the fractional catabolic rate (FCR) of apoB. Mean LDL-apoB plasma concentrations were almost identical in both groups (HD: 95±30 mg/dL, controls: 91±40 mg/dL), whereas LDL-apoB FCR was 50% lower in HD patients as compared with controls (0.22±0.12 days−1 versus 0.46±0.20 days−1, P=0.001) with concomitantly decreased production rates of LDL. Compared with controls, intermediate-density lipoprotein (IDL)-apoB FCR was 65% lower (2.87±1.02 days−1 versus 8.89±4.94 days−1, P=0.014), accompanied by 1.5-fold higher IDL-apoB levels in HD. Very low-density lipoprotein metabolism was similar in both study groups. Conclusions—In vivo catabolism of LDL and IDL is severely impaired in HD patients but misleadingly masked by normal plasma cholesterol levels. The resulting markedly prolonged residence times of both IDL and LDL particles might thus significantly contribute to the well-documented high risk for premature cardiovascular disease in HD patients.

Utility of the PFA-100 Instrument and the Novel Multiplate Analyzer for the Assessment of Aspirin and Clopidogrel Effects on Platelet Function in Patients With Cardiovascular Disease

This study evaluated the utility of the PFA-100 and the Multiplate analyzer for the assessment of aspirin and clopidogrel effects on platelet function in patients with cardiovascular disease. Platelet function was determined with the PFA-100 using collagen+epinephrine (CEPI) and collagen+adenosine-5’-diphosphate (CADP) cartridges, and with whole blood impedance aggregometry using the Multiplate ASPI and ADP+PG tests (aggregation triggered with arachidonic acid and ADP+ prostaglandin E1, respectively). Four study groups were identified from the 154 patients enrolled: patients without antiplatelet therapy, patients with 100 mg aspirin daily but without clopidogrel treatment, patients with 75 mg clopidogrel daily but without aspirin treatment, and patients with both 100 mg aspirin daily plus 75 mg clopidogrel daily. It was found that the PFA-100 instrument is useful for detection of aspirin but not for detection of a clopidogrel effect, while the Multiplate analyzer is useful for specific detection of both aspirin and clopidogrel effects on platelet function.