High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at -80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH+/toxin+, 53 GDH-/toxin-, and 124 GDH+/toxin- samples, of which 39 were CCNA+ and 85 were CCNA- Clarity had 96.2% negative agreement with GDH-/toxin- samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with GDH+/toxin-/CCNA- samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH+/toxin-/CCNA+ samples, 90.0% of GDH+/toxin-/CCNA+ (high-positive) samples, and 31.6% of GDH+/toxin-/CCNA+ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Effect of Health and Training on Ultrasensitive Cardiac Troponin in Marathon Runners.

PURPOSE: Cardiac troponin (cTn) is the gold standard biomarker for assessing cardiac damage. Previous studies have demonstrated increases in plasma cTn because of extreme exercise, including marathon running. We developed an easy-to-use, ultrasensitive assay for cardiac troponin I (cTnI) by combining single-molecule counting (SMC™) technology with dried blood spot (DBS) collection techniques and validated the assay on a cohort of marathon runners by correlating postmarathon cTnI elevations with training or risk variables. METHODS: An SMC-DBS method was developed for accurate and reproducible measurement of cTnI in fingerstick whole blood. Samples were collected from 42 runners both before and immediately after running a marathon. A similar collection was obtained from 22 non-running control individuals. Pre- and postrace questionnaires containing health and training variables were correlated with cTnI concentration. RESULTS: The assay quantified cTnI in all controls and marathon runners, both before and after the race. Prerace concentrations were significantly higher in marathon runners vs controls (median 3.1 vs 0.4 pg/mL; P < 0.0001). Immediate postmarathon concentrations were increased in 98% of runners (median elevation, 40.5 pg/mL; P < 0.001), including many above traditional cutoffs for acute myocardial infarction. Several health and training variables trended toward significant correlation with cTnI elevations. CONCLUSION: While further studies are needed to better understand the mechanisms and clinical implications of exercise-induced cTnI elevations, the present study suggests several variables that may be associated with such elevations and demonstrates a simple, cost-effective method for monitoring cTnI during exercise, managing chronic disease, and/or for assessing risk in large populations.

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay.

Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0 pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity+/CCCNA-) became CCCNA-, and 5/26 (19.2%) Clarity+/CCCNA- samples became CCCNA+, resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We evaluated the clinical performance of ultrasensitive single-molecule counting technology for detection of C. difficile toxins A and B. Stool specimens from 298 patients with suspected CDI were tested with the nucleic acid amplification test (NAAT; BD MAX Cdiff assay or Xpert C. difficile assay) and Singulex Clarity C. diff toxins A/B assay. Specimens with discordant results were tested with the cell cytotoxicity neutralization assay (CCNA), and the results were correlated with disease severity and outcome. There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity- and 4 NAAT-/Clarity+ samples, there were 26 CCNA- and 4 CCNA- samples, respectively. CDI relapse was more common in NAAT+/toxin+ patients than in NAAT+/toxin- and NAAT-/toxin- patients. The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was more than three times more common in NAAT+/toxin- than in NAAT+/toxin+ patients. The Clarity assay was superior to NAATs for the diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and the presence of toxins was associated with negative patient outcomes.

Laboratory comparison between cell cytotoxicity neutralization assay and ultrasensitive single molecule counting technology for detection of Clostridioides difficile toxins A and B, PCR, enzyme immunoassays, and multistep algorithms.

Diagnostic tests for Clostridioides difficile infection (CDI) lack either specificity (nucleic acid amplification tests) or sensitivity (enzyme immunoassays; EIAs). The performance of the Singulex Clarity® C. diff toxins A/B assay was compared to cell cytotoxicity neutralization assay. Testing was also performed using an EIA for glutamate dehydrogenase (GDH) and C. difficile toxins A and B (C. Diff Quik Chek Complete®), polymerase chain reaction (PCR) (BD MAX™ Cdiff Assay), and 2 multistep algorithms: algorithm 1 (discordant GDH/toxin results arbitrated by PCR) and algorithm 2 (PCR-positive samples tested with toxin EIA). The Clarity assay and PCR both had 97% sensitivity, while specificity was 100% for Clarity and 79% for PCR. Algorithm 1 yielded 41% discordant results, and both toxin EIA and algorithm 2 had 58% sensitivity. Median toxin concentrations, as measured by the Clarity C. difficile toxin assay, were 3590, 11.5, 0.4, and 0 pg/mL for GDH+/toxin+, GDH+/toxin-/PCR+, GDH+/toxin-/PCR-, and GDH-/toxin- samples, respectively (P < 0.001). The Clarity assay may offer a single-test solution for CDI.

Ultrasensitive quantification of cardiac troponin I by a Single Molecule Counting method: analytical validation and biological features.

AIM: To evaluate analytical and biological characteristics of the Singulex Clarity® cTnI assay, based upon Single Molecule Counting technology. METHODS: Assay's analytical sensitivity, precision, linearity, hook effect, cross-reactivity or interference by endogenous and exogenous substances, stability, 99th reference percentile [p99th] in EDTA plasma were evaluated in single or multi-site studies. RESULTS: Detection limit was 0.12 ng/L. Sensitivity was 0.14 ng/L at 20% CV (functional sensitivity) and 0.53 ng/L at 10% CV. Imprecision was 3.16%-10.0% in a multi-lot, single-site study, and 5.5%-12.0% in a single-lot, multi-site study; assay was linear from 0.08 to 25,000 ng/L. No hook effect was observed; any cross-reactivity/interference exceeded the 10%. Healthy subjects were recruited using clinical history, normal NT-proBNP and eGFR (n = 560) or plasma creatinine (n = 535) as inclusion criteria. cTnI was detectable in 96.8% of healthy subjects. The p99th were 8.01 (eGFR used) and 8.15 ng/L (plasma creatinine); both were measured with ≤5.7% CV. Median cTnI were significantly higher in older and male than in young and female subjects. CONCLUSIONS: The Singulex Clarity cTnI assay show analytical features and % detection in healthy subjects that improve the corresponding values of most of the existing high-sensitivity cTnI assays.

Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology.

Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

Fibroblast growth factor 23 and renal function among young and healthy individuals.

BACKGROUND: Fibroblast growth factor 23 (FGF-23), an osteocyte hormone involved in the regulation of phosphate metabolism, is associated with incident and progressive chronic kidney disease. We aimed to assess the association of FGF-23 with renal parameters, vascular function and phosphate metabolism in a large cohort of young and healthy individuals. METHODS: Healthy individuals aged 25-41 years were included in a prospective population-based study. Fasting venous blood and morning urinary samples were used to measure plasma creatinine, cystatin C, endothelin-1, phosphate and plasma FGF-23 as well as urinary creatinine and phosphate. Multivariable regression models were constructed to assess the relationship of FGF-23 with parameters of renal function, endothelin-1 and fractional phosphate excretion. RESULTS: The median age of 2077 participants was 37 years, 46% were males. The mean estimated glomerular filtration rate (eGFR - CKD-EPI creatinine-cystatin C equation) and fractional phosphate excretion were 110 mL/min/1.73 m2 and 8.7%, respectively. After multivariable adjustment, there was a significant inverse relationship of FGF-23 with eGFR (ß per 1 log-unit increase -3.81; 95% CI [-5.42; -2.20]; p<0.0001). Furthermore, we found a linear association between FGF-23 and endothelin-1 (ß per 1 log-unit increase 0.06; [0.01, 0.11]; p=0.01). In addition, we established a significant relationship of FGF-23 with fractional phosphate excretion (ß per 1 log-unit increase 0.62; [0.08, 1.16]; p=0.03). CONCLUSIONS: Increasing plasma FGF-23 levels are strongly associated with decreasing eGFR and increasing urinary phosphate excretion, suggesting an important role of FGF-23 in the regulation of kidney function in young and healthy adults.

High-sensitivity cardiac troponin I is a biomarker for occult coronary plaque burden and cardiovascular events in patients with rheumatoid arthritis.

Objectives: Patients with RA display greater occult coronary atherosclerosis burden and experience higher cardiovascular morbidity and mortality compared with controls. We here explored whether pro-inflammatory cytokines and high-sensitivity cardiac troponin I (hs-cTnI), a biomarker of myocardial injury, correlated with plaque burden and cardiovascular events (CVEs) in RA. Methods: We evaluated 150 patients with 64-slice coronary CT angiography. Coronary artery calcium, number of segments with plaque (segment involvement score), stenotic severity and plaque burden were assessed. Lesions were described as non-calcified, mixed or fully calcified. Blood levels of hs-cTnI and pro-inflammatory cytokines were assessed during coronary CT angiography. Subjects were followed over 60 (s.d. 26) months for both ischaemic [cardiac death, non-fatal myocardial infarction (MI), stroke, peripheral arterial ischaemia] and non-ischaemic (new-onset heart failure hospitalization) CVEs. Results: Plasma hs-cTnI correlated with all coronary plaque outcomes (P < 0.01). Elevated hs-cTnI (⩾1.5 pg/ml) further associated with significant calcification, extensive atherosclerosis, obstructive plaque and any advanced mixed or calcified plaques after adjustments for cardiac risk factors or Framingham D'Agostino scores (all P < 0.05). Eleven patients suffered a CVE (1.54/100 patient-years), eight ischaemic and three non-ischaemic. Elevated hs-cTnI predicted all CVE risk independent of demographics, cardiac risk factors and prednisone use (P = 0.03). Conversely, low hs-cTnI presaged a lower risk for both extensive atherosclerosis (P < 0.05) and incident CVEs (P = 0.037). Conclusion: Plasma hs-cTnI independently associated with occult coronary plaque burden, composition and long-term incident CVEs in patients with RA. Low hs-cTnI forecasted a lower risk for both extensive atherosclerosis as well as CVEs. hs-cTnI may therefore optimize cardiovascular risk stratification in RA.

Relationships of kidney injury molecule-1 with renal function and cardiovascular risk factors in the general population.

BACKGROUND: Kidney injury molecule-1 (KIM-1) has been associated with kidney damage in patients with preexisting renal disease. However, little is known about the relationships of KIM-1 with renal function and cardiovascular risk factors in healthy individuals from the general population. METHODS: Healthy individuals aged 25-41years were enrolled in a population-based study. Main exclusion criteria were a BMI >35kg/m2, preexisting kidney disease or established cardiovascular disease. KIM-1 was measured from frozen plasma samples using a high-sensitivity assay. Multivariable linear regression models were constructed to assess the relationships of KIM-1 with renal function and various cardiovascular risk factors. RESULTS: We included 2060 individuals (47% men, median (interquartile range) age: 37 (31-40) years) in this analysis. Median KIM-1 levels were 82.5 (IQR 59.4-112.7) pg/ml. We found no significant relationship of KIM-1 with creatinine (adjusted ß-coefficient (95% confidence interval) 0.0005 (-0.002; 0.003), p=0.61) and cystatin C (-0.02 (-0.21; 0.17), p=0.84). There were significant linear relationships of log-transformed KIM-1 with systolic blood pressure (adjusted ß-coefficient (95% confidence interval) 0.07 (0.04; 0.09), p<0.0001), diastolic blood pressure (0.04 (0.02; 0.07), p=0.001), low-density lipoprotein cholesterol (0.09 (0.06; 0.11), p<0.0001), high-density lipoprotein cholesterol (0.07 (0.05; 0.1), p<0.0001), high-sensitivity C-reactive protein (0.05 (0.03; 0.07), p<0.0001), age (0.09 (0.07; 0.11), p<0.0001), BMI (0.04 (0.01; 0.06), p=0.005) and current smoking (0.12 (0.07; 0.17), p<0.0001). CONCLUSION: Among healthy adults from the general population, plasma levels of KIM-1 were not associated with renal function but were independently related to multiple cardiovascular risk factors.

Low Plasma Testosterone Is Associated With Elevated Cardiovascular Disease Biomarkers.

BACKGROUND: The relation between testosterone (T) plasma concentration and cardiovascular (CV) risk is unclear, with evidence supporting increased risk in men with low and high T levels. Few studies have assessed CV risk as a function of plasma T levels using objective biomarkers. AIM: To determine the relation between T levels and high-sensitivity CV risk biomarkers. METHODS: Ten thousand forty-one male patients were identified in the database of a commercial clinical laboratory performing biomarker testing. Patients were grouped by total T concentration and associations with the following biomarkers were determined: cardiac troponin I (cTnI), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-17A, N-terminal pro-B-type natriuretic peptide (NTproBNP), high-density lipoprotein (HDL) cholesterol, high-sensitivity C-reactive protein (hs-CRP), hemoglobin A1c (HbA1c), and leptin. OUTCOMES: Association of CV risk markers with levels of T in men. RESULTS: The median age of the cohort was 58 years (interquartile range = 48-68), and the median plasma T level was 420 ng/dL (interquartile range = 304-565); T levels did not vary with patient age. An inverse relation between plasma T levels and CV risk was observed for 9 of 10 CV markers: cTnI, ET-1, IL-6, TNF-α, NTproBNP, HDL cholesterol, hs-CRP, HbA1c, and leptin. Even after adjusting for age, body mass index, HbA1c, hs-CRP, and HDL cholesterol levels, the CV markers IL-6, ET-1, NTproBNP, and leptin were significantly associated with a T level lower than 250 ng/dL. CLINICAL IMPLICATIONS: Men with low T levels could be at increased risk for increased CV disease as seen by increased CV risk markers. STRENGTH AND LIMITATIONS: This study was performed in a group of 10,041 men and is the first study to examine CV risk associated with circulating T levels using a large panel of 10 objective biomarkers. This study is limited by an absence of clinical data indicating whether men had pre-existing CV disease or other CV risk factors. CONCLUSION: Men with low plasma T levels exhibit increases in CV risk markers, consistent with a potential increased risk of CV disease. Pastuszak AW, Kohn TP, Estis J, Lipshultz LI. Low Plasma Testosterone Is Associated With Elevated Cardiovascular Disease Biomarkers. J Sex Med 2017;14:1095-1103.

Clinical utility of single molecule counting technology for quantification of KIM-1 in patients with heart failure and chronic kidney disease.

BACKGROUND: Acute kidney injury (AKI) is associated with high morbidity and mortality, and may lead to chronic kidney disease (CKD). Traditional serum biomarkers for acute and chronic renal dysfunction are insensitive and nonspecific. While urinary kidney injury molecule-1 (KIM-1) is a sensitive and specific measure of kidney tubular injury, it is difficult to obtain in acute settings. Thus, our objective was to develop a highly sensitive immunoassay for plasma KIM-1. METHODS: A novel plasma KIM-1 immunoassay was developed using Single Molecule Counting technology (SMC). It was clinically validated in: 120 healthy subjects to establish a preliminary reference range; 25 healthy subjects to assess biological variability; 200 patients with heart failure (CHF); and 60 patients from a CKD case control. RESULTS: SMC KIM-1 assay provided a limit of detection of 1.4pg/mL (reporting range from 2pg/mL to 1000pg/mL). Inter-assay precision was 9-15% CV. Median KIM-1 value in healthy subjects was 119pg/mL with a RR 95th percentile of 292pg/mL. KIM-1 demonstrated low weekly biological variability over 6weeks. KIM-1 was elevated in patients with CKD or CHF. Adjusted odds ratios for differentiating CHF or CKD from controls were 9.6 (95% CI 2.7-35.0) and 3.6 (95% CI 1.1-11.6), respectively. In CHF, KIM-1 values correlated inversely with eGFR (Spearman R=-0.32, p<0.0001). CONCLUSIONS: Plasma KIM-1 is quantifiable in healthy volunteers, elevated in CKD and CHF patients, and correlates with eGFR. Additional investigation is needed to determine if KIM-1 provides prognostic value for CKD and CHF patient outcomes.

Healthy lifestyle and glucagon-like peptide-1 in young and healthy adults: A population-based study.

A healthy lifestyle is associated with a lower risk of cardiovascular events and mortality, but underlying mechanisms are not fully understood. The aim of our study was to investigate the relationships between a healthy lifestyle and glucagon-like peptide-1 (GLP-1), an incretin hormone with both glycemic and cardiovascular properties. Healthy participants aged 25-41years without cardiovascular disease, diabetes or a body mass index (BMI) >35kg/m2 were enrolled in a population-based study. The following metrics were used to build a lifestyle score ranging from 0 to 7 (a higher score indicating a healthier lifestyle): blood pressure (BP) (<120/80mmHg), plasma levels of glycated hemoglobin (<5.7%), total cholesterol levels (<200mg/dl), BMI (<25kg/m2), not smoking cigarettes, moderate (≥150min/week) or vigorous (≥75min/week) physical activity and a healthy diet. Among 2133 participants median age was 36.7years and 53.3% were female. GLP-1 levels decreased significantly from 39.5 to 30.9ng/l (p<0.0001) across increasing lifestyle score categories. This linear relationship persisted in multivariable adjusted linear regression models (B for GLP-1 per 1-unit increase of the lifestyle score -0.06; 95% confidence intervals -0.07, -0.04; p<0.0001). Individual health metrics that were significantly associated with GLP-1 were a normal BMI (-0.07; -0.12, -0.03; p=0.001), low total cholesterol levels (-0.07; -0.12, -0.03; p=0.001), normal BP (-0.05; -0.10, -0.00; p=0.047) and not smoking (-0.06; -0.10, -0.01; p=0.01). A healthy lifestyle is strongly associated with lower GLP-1 levels in young and healthy adults.

Subclinical sleep apnoea and plasma levels of endothelin-1 among young and healthy adults.

OBJECTIVE: Obstructive sleep apnoea (OSA) is a risk factor for vascular disease and other adverse outcomes. These associations may be at least partly due to early endothelin-1 (ET-1)-mediated endothelial dysfunction (ED). Therefore, we assessed the relationships between subclinical sleep apnoea and plasma levels of ET-1. METHODS: We performed a population-based study among 1255 young and healthy adults aged 25-41 years. Cardiovascular disease, diabetes or a body mass index >35 kg/m2 were exclusion criteria. Plasma levels of ET-1 were measured using a high-sensitivity, single-molecule counting technology. The relationships between subclinical sleep apnoea (OSA indices: respiratory event index (REI), oxygen desaturation index (ODI), mean night-time blood oxygen saturation (SpO2)) and ET-1 levels were assessed by multivariable linear regression analysis. RESULTS: Median age of the cohort was 35 years. Median ET-1 levels were 2.9 (IQR 2.4-3.6) and 2.5 pg/mL (IQR 2.1-3.0) among patients with (n=105; 8%) and without subclinical sleep apnoea (REI 5-14), respectively. After multivariable adjustment, subclinical sleep apnoea remained significantly associated with plasma levels of ET-1 (ß=0.13 (95% CI 0.06 to 0.20) p=0.0002 for a REI 5-14; ß=0.10 (95% CI 0.03 to 0.16) p=0.003 for an ODI≥5). Every 1% decrease in mean night-time SpO2 increased ET-1 levels by 0.1 pg/mL, an association that remained significant after multivariable adjustment (ß=0.02 (95% CI 0.003 to 0.033) p=0.02). CONCLUSIONS: In this study of young and healthy adults, we found that participants with subclinical sleep apnoea had elevated plasma ET-1 levels, an association that was due to night-time hypoxaemia. Our results suggest that ED may already be an important consequence of subclinical sleep apnoea.

Plasma levels of endothelin-1 and renal function among young and healthy adults.

BACKGROUND: Endothelin-1 (ET-1), a vasoconstrictive and pro-inflammatory peptide, is associated with several cardiovascular risk factors and outcomes. We aimed to investigate the association of plasma ET-1 levels and renal function among young and healthy adults. METHODS: Individuals aged 25-41 years were enrolled in a population-based cohort study. Main exclusion criteria were established kidney disease, cardiovascular diseases, diabetes mellitus and a body mass index>35 kg/m2. Fasting venous plasma samples were used to measure creatinine, cystatin C and ET-1. The estimated glomerular filtration rate (eGFR) was calculated using the creatinine based chronic kidney disease epidemiology collaboration (CKD-EPI) formula. Multivariable regression models were constructed to assess interrelationships of plasma ET-1 with parameters of renal function. RESULTS: Median age of the 2139 participants was 37 years, 47% males. Median creatinine and eGFR were 67 µmol/L and 112 mL/min/1.73 m2, respectively. Using quartile one as the reference group, the ß-coefficients (95% confidence intervals [CIs]) for eGFR were 0.06 (- 1.22 to 1.35),-0.66 (- 1.95 to 0.62) and-1.70 (- 3.01 to-0.39) for quartiles 2-4 (p-for-trend=0.0056), respectively and ß-coefficients (95% CIs) for cystatin C were 0.002 (- 0.01 to 0.02), 0.02 (0.003-0.03) and 0.03 (0.01-0.04) for quartiles 2-4 (p-for-trend<0.0001), respectively. Using ET-1 as a continuous variable, the ß-coefficient (95% CI) for eGFR per 1-unit increase was-1.82 (- 3.19 to-0.44, p=0.0095) and 0.02 (0.01-0.04, p=0.0003) for cystatin C. Similar results were found between creatinine and ET-1 levels. CONCLUSIONS: ET-1 levels are strongly associated with parameters of renal function among young and healthy adults, suggesting an important role of ET-1 and endothelial function in the regulation of kidney function.

Urinary levels of novel kidney biomarkers and risk of true worsening renal function and mortality in patients with acute heart failure.

AIMS: Recent studies indicate the need to redefine worsening renal function (WRF) in acute heart failure (AHF), linking a rise in creatinine with clinical status to identify patients who develop 'true WRF'. We evaluated the usefulness of serial assessment of urinary levels of neutrophil gelatinase-associated lipocalin (uNGAL), kidney injury molecule-1 (uKIM-1), and cystatin C (uCysC) for prediction of 'true WRF'. METHODS AND RESULTS: In 132 patients with AHF, uNGAL, uKIM-1, and uCysC were measured using a highly sensitive immunoassay based on a single-molecule counting technology (Singulex, Alameda, CA, USA) at baseline, day 2, and day 3. Patients who developed WRF (a ≥0.3 mg/dL increase in serum creatinine or a >25% decrease in the estimated glomerular filtration rate from the baseline value) were differentiated into those 'true WRF' (presence of deterioration/no improvement in clinical status during hospitalization) vs. 'pseudo-WRF' (uneventful clinical course). 'True WRF' occurred in 13 (10%), 'pseudo-WRF' in 15 (11%), whereas the remaining 104 (79%) patients did not develop WRF. Patients with 'true WRF' were more often females, had higher levels of NT-proBNP, creatinine, and urea on admission, higher urine albumin to creatinine ratio at day 2, higher uNGAL at baseline, day 2, and day 3, and higher KIM-1 at day 2 (vs. pseudo-WRF vs. without WRF, all P < 0.05). Patients with pseudo-WRF did not differ from those without WRF. In the multivariable model, elevated uNGAL at all time points and uKIM-1 at day 2 remained independent predictors of 'true WRF'. CONCLUSION: Elevated levels of uNGAL and uKIM-1 may predict development of 'true WRF' in AHF.

Risk factors for premature ventricular contractions in young and healthy adults.

BACKGROUND: Premature ventricular contractions (PVCs) are associated with an increased risk of morbidity and mortality. Therefore, it was aimed to assess risk factors for the frequency of PVCs in young and healthy adults. METHODS: Our population-based study included 2048 healthy adults from the general population aged 25-41 years. PVC frequency was determined by 24-hour Holter ECG. We performed multivariable regression analysis using stepwise backward selection to identify factors independently associated with PVC frequency. RESULTS: Median age was 37 years, 953 (46.5%) were male. At least one PVC during the 24-hour monitoring period was observed in 69% of participants. Median number of detected PVCs was 2, the 95th percentile was 193. In multivariable regression analyses, we found 17 significant risk factors for PVC frequency. Low educational status (risk ratio (RR) 3.33; 95% CI 1.98 to 5.60), body height>median (1.58, 95% CI 1.11 to 2.24) and increasing levels of waist:hip ratio (2.15, 95% CI 1.77 to 2.61), N-terminal pro brain natriuretic peptide (1.52, 95% CI 1.30 to 1.76) and Sokolow-Lyon Index (1.38, 95% CI 1.15 to 1.66) (all p≤0.01) were associated with a higher PVC frequency. Physical activity (RR fourth vs first quartile 0.51, 95% CI 0.34 to 0.76) and increasing levels of haemoglobin (0.58, 95% CI 0.47 to 0.70) and glucagon-like peptide-1 (0.72, 95% CI 0.64 to 0.82) (all p<0.001) were related to a lower PVC frequency. CONCLUSIONS: PVC occurrence is common even in healthy low-risk individuals, and its frequency is associated with several covariates mainly related to cardiovascular risk factors, markers of cardiac structure and function and socioeconomic status.

Factors independently associated with cardiac troponin I levels in young and healthy adults from the general population.

BACKGROUND: Determinants of cardiomyocyte injury as quantified by high-sensitivity cardiac troponin I (cTnI) in young and healthy individuals, and sex-specific 99th percentiles are largely unknown. METHODS: Our study included 2077 adults from the general population aged 25-41 years without cardiovascular disease. cTnI was measured using a high-sensitivity assay. We performed stepwise backward linear regression analyses to identify variables independently associated with hs-cTnI levels, and calculated narrow-sense heritability from 1638-genotyped participants. RESULTS: Median age was 37 years. cTnI was quantifiable in all but 11 participants (99.5 %). Median (interquartile range) cTnI was significantly higher in men than in women [0.99 (0.71; 1.65) versus 0.47 (0.33; 0.71) ng/L, p < 0.0001]. The 99th percentile of cTnI was 15.79 ng/L in men and 5.11 ng/L in women. Out of 46 variables, 22 independent determinants for cTnI were identified. The strongest associations were observed with sex, age, systolic blood pressure, heart rate, left ventricular mass, N-terminal pro B-type natriuretic peptide, and creatine kinase (all p < 0.0001). The final model explained 36 % of the overall cTnI variability. Heritability of cTnI was estimated to be 29 % (p = 0.005), but became non-significant when the residuals of the multivariable model were used for analysis (5 %, p = 0.36). CONCLUSIONS: Sex, age, and systolic blood pressure belong to the strongest determinants of hs-cTnI in healthy adults. The 99th percentile was three times higher in men compared to women. Hence, sex-specific cut-off values may be preferable when applying hs-cTnI for screening purposes. Our results may also improve the interpretation of cTn levels in daily clinical practice.

Relationships of iron metabolism with insulin resistance and glucose levels in young and healthy adults.

AIMS: Several biomarkers within the iron metabolism pathway have been related to the occurrence of diabetes mellitus, but underlying mechanisms are unknown. The aim of our study was to investigate the differential relationships of iron metabolism with a broad range of diabetes markers in young and healthy adults. DESIGN: 2160 participants aged 25 to 41years were enrolled in a population-based study. Established cardiovascular disease, diabetes or a body mass index >35kg/m(2) were exclusion criteria. Multivariable linear regression models were built to assess the associations of ferritin and transferrin saturation (TSAT) with blood levels of glucagon-like peptide-1 (GLP-1), insulin, homeostatic model assessment-insulin resistance (HOMA-IR), fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c). RESULTS: Median (interquartile range) age was 37 (31, 40) years. In multivariable linear regression analyses, ß-coefficients (95% confidence intervals) per 1-SD increase in ferritin were 0.04 (0.02; 0.07, p=0.0008) for GLP-1, 0.06 (0.04; 0.08, p<0.0001) for insulin, 0.07 (0.04; 0.09, p<0.0001) for HOMA-IR, 0.004 (-0.00; 0.01, p=0.07) for FPG and -0.003 (-0.01; -0.00, p=0.07) for HbA1c. ß-coefficients (95% CI) per 1-SD increase in TSAT were -0.07 (-0.09; -0.05, p<0.0001) for GLP-1, -0.06 (-0.08; -0.04, p<0.0001) for insulin, -0.07(-0.09; -0.05, p<0.0001) for HOMA-IR, -0.01 (-0.01; -0.00, p<0.0001) for FPG and -0.01 (-0.01; -0.00, p=0.0004) for HbA1c. CONCLUSIONS: Markers of insulin resistance are strongly related with markers of iron metabolism in healthy subjects. These relationships were inconsistent and weaker for short-term and long-term glucose levels. These results may provide insights in the relationships between iron metabolism and diabetes occurrence.