Background and Aims Circulating proenkephalin (PENK) is a stable endogenous polypeptide with fast response to glomerular dysfunction and tubular damage. This study examined the predictive value of PENK for renal outcomes and mortality in patients with acute coronary syndrome (ACS). Methods Proenkephalin was measured in plasma in a prospective multicentre ACS cohort from Switzerland (n = 4787) and in validation cohorts from the UK (n = 1141), Czechia (n = 927), and Germany (n = 220). A biomarker-enhanced risk score (KID-ACS score) for simultaneous prediction of in-hospital acute kidney injury (AKI) and 30-day mortality was derived and externally validated. Results On multivariable adjustment for established risk factors, circulating PENK remained associated with in-hospital AKI [per log2 increase: adjusted odds ratio 1.53, 95% confidence interval (CI) 1.13-2.09, P = .007] and 30-day mortality (adjusted hazard ratio 2.73, 95% CI 1.85-4.02, P < .001). The KID-ACS score integrates PENK and showed an area under the receiver operating characteristic curve (AUC) of .72 (95% CI .68-.76) for in-hospital AKI and .91 (95% CI .87-.95) for 30-day mortality in the derivation cohort. Upon external validation, KID-ACS achieved similarly high performance for in-hospital AKI (Zurich: AUC .73, 95% CI .70-.77; Czechia: AUC .75, 95% CI .68-.81; Germany: AUC .71, 95% CI .55-.87) and 30-day mortality (UK: AUC .87, 95% CI .83-.91; Czechia: AUC .91, 95% CI .87-.94; Germany: AUC .96, 95% CI .92-1.00), outperforming the contrast-associated AKI score and the Global Registry of Acute Coronary Events 2.0 score, respectively. Conclusions Circulating PENK offers incremental value for predicting in-hospital AKI and mortality in ACS. The simple six-item KID-ACS risk score integrates PENK and provides a novel tool for simultaneous assessment of renal and mortality risk in patients with ACS.

Background Heart failure with preserved ejection fraction (HFpEF) is a heterogenous multi-system syndrome with limited efficacious treatment options. The prevalence of Type 2 diabetes (T2D) continues to rise and predisposes patients to HFpEF, and HFpEF remains one of the biggest challenges in cardiovascular medicine today. Novel therapeutic targets are required to meet this important clinical need. Deep phenotyping studies including -OMIC analyses can provide important pathogenic information to aid the identification of such targets. The aims of this study were to determine; 1) the impact of a low-energy diet on plasma sphingolipid/ceramide profiles in people with T2D compared to healthy controls and, 2) if the change in sphingolipid/ceramide profile is associated with reverse cardiovascular remodelling. Methods Post-hoc analysis of a randomised controlled trial (NCT02590822) including adults with T2D with no cardiovascular disease who completed a 12-week low-energy (∼810 kcal/day) meal-replacement plan (MRP) and matched healthy controls (HC). Echocardiography, cardiac MRI and a fasting blood for lipidomics were undertaken pre / post -intervention. Candidate biomarkers were identified from case–control comparison (fold change > 1.5 and statistical significance p < 0.05) and their response to the MRP reported. Association between change in biomarkers and change indices of cardiac remodelling were explored. Results Twenty-four people with T2D (15 males, age 51.1 ± 5.7 years), and 25 HC (15 male, 48.3 ± 6.6 years) were included. Subjects with T2D had increased left ventricular (LV) mass:volume ratio (0.84 ± 0.13 vs. 0.70 ± 0.08, p < 0.001), increased systolic function but impaired diastolic function compared to HC. Twelve long-chain polyunsaturated sphingolipids, including four ceramides, were downregulated in subjects with T2D at baseline. Three sphingomyelin species and all ceramides were inversely associated with LV mass:volume. There was a significant increase in all species and shift towards HC following the MRP, however, none of these changes were associated with reverse cardiac remodelling. Conclusion The lack of association between change in sphingolipids/ceramides and reverse cardiac remodelling following the MRP casts doubt on a causative role of sphingolipids/ceramides in the progression of heart failure in T2D. Trial registration NCT02590822.

Rationale: Heavy-labelled internal standards increasingly represent the gold standard for absolute quantitation in mass spectrometry (MS)-based bottom-up proteomics. The biggest drawbacks of using these standards are that they have high costs and lengthy lead times. Methods: We describe an efficient, low-cost optimised method to enable 'in-house' heavy labelling of synthetic tryptic peptides for absolute quantification using tandem LC-MS/MS mass spectrometry. Our methodology uses 18O water in a trypsin-catalysed oxygen exchange reaction at the carboxyl terminus with the overall aim of reducing the costs and lead time associated with sourcing heavy standards from commercial vendors. Results: Step-by-step instructions are provided on how to execute this protocol with high-throughput adaptations utilising a 96-well plate and a liquid-handling robot. Detailed notes on experimental setup, tips for troubleshooting and suggested improvements to maximise labelling efficiencies are highlighted to achieve the best results. Under optimum conditions, labelling efficiencies of peptides can reach from 95% to 100%. Conclusions: The application of the 'in-house' labelled standards in generating calibration curves to quantify endogenous peptide concentrations is just as effective as using the synthetically sourced standards while also having great cost reduction implications as well as saving time spent waiting for peptides to arrive. The protocol is highly adaptable and can be customized to fit the specific setup of any laboratory, maximizing achievable labelling efficiencies.

Background: Dysregulated epicardial adipose tissue (EAT) may contribute to the development of heart failure in Type 2 diabetes (T2D). This study aimed to evaluate the associations between EAT volume and composition with imaging markers of subclinical cardiac dysfunction in people with T2D and no prevalent cardiovascular disease. Methods: Prospective case-control study enrolling participants with and without T2D and no known cardiovascular disease. Two hundred and fifteen people with T2D (median age 63 years, 60 % male) and thirty-nine non-diabetics (median age 59 years, 62 % male) were included. Using computed tomography (CT), total EAT volume and mean CT attenuation, as well as, low attenuation (Hounsfield unit range -190 to -90) EAT volume were quantified by a deep learning method and volumes indexed to body surface area. Associations with cardiac magnetic resonance-derived left ventricular (LV) volumes and strain indices were assessed using linear regression. Results: T2D participants had higher LV mass/volume ratio (median 0.89 g/mL [0.82-0.99] vs 0.79 g/mL [0.75-0.89]) and lower global longitudinal strain (GLS; 16.1 +/- 2.3 % vs 17.2 +/- 2.2 %). Total indexed EAT volume correlated inversely with mean CT attenuation. Low attenuation indexed EAT volume was 2-fold higher (18.8 cm(3)/m(2) vs. 9.4 cm(3)/m(2), p < 0.001) in T2D and independently associated with LV mass/volume ratio (ss = 0.002, p = 0.01) and GLS (ss = -0.03, p = 0.03). Conclusions: Higher EAT volumes seen in T2D are associated with a lower mean CT attenuation. Low attenuation indexed EAT volume is independently, but only weakly, associated with markers of subclinical cardiac dysfunction in T2D.

Background Heart failure with preserved ejection fraction (HFpEF) and atrial fibrillation (AF) frequently co-exist. There is a limited understanding on whether this coexistence is associated with distinct alterations in myocardial remodelling and mechanics. We aimed to determine if patients with atrial fibrillation (AF) and heart failure with preserved ejection fraction (HFpEF) represent a distinct phenotype. Methods In this secondary analysis of adults with HFpEF (NCT03050593), participants were comprehensively phenotyped with stress cardiac MRI, echocardiography and plasma fibroinflammatory biomarkers, and were followed for the composite endpoint (HF hospitalisation or death) at a median of 8.5 years. Those with AF were compared to sinus rhythm (SR) and unsupervised cluster analysis was performed to explore possible phenotypes. Results 136 subjects were included (SR = 75, AF = 61). The AF group was older (76 ± 8 vs. 70 ± 10 years) with less diabetes (36% vs. 61%) compared to the SR group and had higher left atrial (LA) volumes (61 ± 30 vs. 39 ± 15 mL/m 2 , p < 0.001), lower LA ejection fraction (EF) (31 ± 15 vs. 51 ± 12%, p < 0.001), worse left ventricular (LV) systolic function (LVEF 63 ± 8 vs. 68 ± 8%, p = 0.002; global longitudinal strain 13.6 ± 2.9 vs. 14.7 ± 2.4%, p = 0.003) but higher LV peak early diastolic strain rates (0.73 ± 0.28 vs. 0.53 ± 0.17 1/s, p < 0.001). The AF group had higher levels of syndecan-1, matrix metalloproteinase-2, proBNP, angiopoietin-2 and pentraxin-3, but lower level of interleukin-8. No difference in clinical outcomes was observed between the groups. Three distinct clusters were identified with the poorest outcomes (Log-rank p = 0.029) in cluster 2 (hypertensive and fibroinflammatory) which had equal representation of SR and AF. Conclusions Presence of AF in HFpEF is associated with cardiac structural and functional changes together with altered expression of several fibro-inflammatory biomarkers. Distinct phenotypes exist in HFpEF which may have differing clinical outcomes.

During the COVID-19 pandemic, development of diagnostic tests was vital to chart the course and to reduce the impact of the infection. Continued testing and surveillance of vaccine escape will continue for years to come which presents an opportunity to integrate such testing into clinical biochemistry laboratories that form part of integrated healthcare testing. Here we describe a protocol for a targeted mass spectrometry based proteomic assay (COVIDCAP) developed to detect SARS-CoV-2 peptides from oro-nasopharyngeal swabs (ONP) and saliva. This uses novel SISCAPA antibodies bound to magnetic beads and subsequent analysis of captured and purified SARS-CoV-2 nucleocapsid (NCAP) peptides. The method involves immediate deactivation of the sample using an ethanolic solution. This simultaneously inactivates the virus and denatures viral proteins at sampling in contrast to the approach for RT-PCR testing, with benefits for the assay as well as for downstream processing. A plate-based preparation of the samples involving acetone precipitation followed by a short tryptic digestion and subsequent immunocapture allows LC-MS detection and quantification of peptides from the NCAP protein, in a 3-minute inject-to-inject assay with an LOD of 20 attomoles from starting sample. For 576 ONP swab samples taken as exemplars here, the sensitivity and specificity of this analysis is shown to be 97.0% and 96.6% respectively.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

Objectives: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. Methods: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. Results: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R-2 0.57, p-value 0.002). Conclusions: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Journal Article Corrected proof Differential implications of gut-related metabolites on outcomes between heart failure and myocardial infarction Get access Muhammad Zubair Israr, Muhammad Zubair Israr Department of Cardiovascular Sciences, University of Leicester and NIHR Leicester Biomedical Research Centre, Glenfield Hospital, Groby Road, Leicester LE3 9QP, UK https://orcid.org/0000-0003-1111-2780 Search for other works by this author on: Oxford Academic Google Scholar Andrea Salzano, Andrea Salzano Department of Cardiovascular Sciences, University of Leicester and NIHR Leicester Biomedical Research Centre, Glenfield Hospital, Groby Road, Leicester LE3 9QP, UKCardiac Unit, AORN A. Cardarelli, Naples, Italy https://orcid.org/0000-0003-2352-2103 Search for other works by this author on: Oxford Academic Google Scholar Sarir Sarmad, Sarir Sarmad Department of Cardiovascular Sciences, University of Leicester and NIHR Leicester Biomedical Research Centre, Glenfield Hospital, Groby Road, Leicester LE3 9QP, UK Search for other works by this author on: Oxford Academic Google Scholar Leong L Ng, Leong L Ng Department of Cardiovascular Sciences, University of Leicester and NIHR Leicester Biomedical Research Centre, Glenfield Hospital, Groby Road, Leicester LE3 9QP, UK Search for other works by this author on: Oxford Academic Google Scholar Toru Suzuki Toru Suzuki Department of Cardiovascular Sciences, University of Leicester and NIHR Leicester Biomedical Research Centre, Glenfield Hospital, Groby Road, Leicester LE3 9QP, UKThe Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo, 108-8639, Japan Corresponding author. Tel: +44 116 204 4741, Email: ts263@le.ac.uk https://orcid.org/0000-0002-8371-664X Search for other works by this author on: Oxford Academic Google Scholar European Journal of Preventive Cardiology, zwad305, https://doi.org/10.1093/eurjpc/zwad305 Published: 20 September 2023 Article history Received: 15 July 2023 Revision received: 31 August 2023 Accepted: 15 September 2023 Published: 20 September 2023 Corrected and typeset: 03 October 2023

AIMS:Iron deficiency (ID) is highly prevalent in patients with heart failure (HF) and associated with morbidity and poor prognosis, but pathophysiological mechanisms are unknown. We aimed to identify novel biological pathways affected by ID. METHODS AND RESULTS:We studied 881 patients with HF from the BIOSTAT-CHF cohort. ID was defined as a transferrin saturation <20%. Transcriptome profiling was performed in whole blood. Identified targets were validated in a human in vitro stem cell-derived cardiomyocyte ID model utilizing deferoxamine as iron chelator. ID was identified in 554 (62.9%) patients, and 89 differentially expressed genes between ID and non-ID were identified, of which 60 were up- and 29 were downregulated. Upregulated genes were overrepresented in pathways of erythrocyte development and homeostasis. Heme biosynthetic processes were confirmed as relatively upregulated in ID, while iron-sulfur cluster assembly was downregulated. Downregulated processes further included natural killer cell and lymphocyte mediated immunity. In agreement with patient data, cardiomyocyte iron depletion significantly induced the expression of two genes (SIAH2 and CLIC4), which could be normalized upon iron supplementation. Both SIAH2 and CLIC4 are associated with increased mortality in patients with HF (hazard ratio 2.40, 95% confidence interval 1.86-3.11, p < 0.001 hazard ratio 1.78, 95% confidence interval 1.53-2.07, p < 0.001, respectively). CONCLUSION:Iron deficiency is associated with the preservation of heme-related processes at the cost of iron-sulfur clusters. Immune processes are downregulated, uncovering another high energy demand system affected. SIAH2 and CLIC4 might be modifiable factors in the relation between ID and impaired prognosis.

Background Recent data suggest different causes of renal dysfunction between heart failure with reduced (HFrEF) versus preserved ejection fraction (HFpEF). We therefore studied a wide range of urinary markers reflecting different nephron segments in heart failure patients. Methods In 2070, in chronic heart failure patients, we measured several established and upcoming urinary markers reflecting different nephron segments. Results Mean age was 70 ± 12 years, 74% was male and 81% ( n = 1677) had HFrEF. Mean estimated glomerular filtration rate (eGFR) was lower in patients with HFpEF (56 ± 23 versus 63 ± 23 ml/min/1.73 m 2 , P = 0.001). Patients with HFpEF had significantly higher values of NGAL (58.1 [24.0–124.8] versus 28.1 [14.6–66.9] μg/gCr, P < 0.001) and KIM-1 (2.28 [1.49–4.37] versus 1.79 [0.85–3.49] μg/gCr, P = 0.001). These differences were more pronounced in patients with an eGFR > 60 ml/min/1.73m 2 . Conclusions HFpEF patients showed more evidence of tubular damage and/or dysfunction compared with HFrEF patients, in particular when glomerular function was preserved.

Aims: Heart failure (HF) is associated with cytokine activation and inflammation.Experimental evidence suggests that plasma interleukin-17 (IL-17) is associated with myocardial fibrosis and cardiac dysfunction in HF.IL-17D, a subtype of IL-17 originates from particular tissues such as the heart.However, there is very limited data on the IL-17 cytokine family in patients with HF.Therefore, we investigated the association between circulating IL-17D levels, clinical characteristics and outcome in a large cohort of patients with heart failure.Methods and results: Plasma IL-17D was measured in 2032 patients with HF from 11 European countries using a proximity extension assay.The primary outcome was a composite of HF hospitalization or all-cause mortality.Patients with higher plasma IL-17D concentrations were more likely to have atrial fibrillation (AF), renal dysfunction and heart failure with preserved ejection fraction (HFpEF) and had higher plasma N-terminal probrain natriuretic peptide (NT-proBNP) concentrations (all p < 0.001).IL-17D was not associated with interleukin-6 (IL-6) or C-reactive protein (CRP) concentrations.After adjustment for confounders in a multivariable Cox regression analysis, patients in the highest quartile of plasma IL-17D had a significantly increased risk of the composite outcome of HF hospitalization or all-cause mortality compared to patients in the lowest quartile [Hazard ratio (HR) 1.28, 95% confidence interval (CI) 1.05-1.57]. Conclusion:In patients with HF, elevated plasma IL-17D concentrations are associated with higher plasma NT-proBNP concentrations and a higher prevalence of AF and renal dysfunction.High IL-17D concentrations are independently associated with worse outcome.

Abstract Background Early assessment of renal and mortality risk in patients with acute coronary syndromes (ACS) is essential to guide treatment decisions according to international guidelines. Circulating proenkephalin (PENK) is a stable opioid metabolite with fast response to changes in kidney function. Purpose This study examined the predictive value of PENK for acute kidney injury (AKI) and mortality in patients presenting with acute coronary syndromes (ACS). Methods Plasma PENK levels were measured using a validated sandwich immunoassay in a prospective multicentre ACS cohort from Switzerland (n = 4 787) and in validation cohorts from the UK (n = 1 141) and Czechia (n = 920). A biomarker-enhanced risk score for simultaneous prediction of both in-hospital acute kidney injury (AKI) and 30-day death (KID-ACS score) with model coefficients adjusted to the outcome was derived and externally validated. In the development of the in-hospital AKI model, patients from one participating study centre were reserved for external validation (Swiss validation cohort). Results Circulating PENK ranked among the top predictors of renal outcomes and mortality in patients with ACS. Accounting for established risk factors and risk models, circulating PENK levels at presentation were independently associated with in-hospital AKI (per log2 increase: adjusted odds ratio [OR] 1.56, 95% CI 1.14 – 2.12, P = 0.005) and with 30-day mortality (per log2 increase: adjusted OR 2.69, 95% CI 1.89 – 3.84, P < 0.001). Dynamic changes in PENK levels within the first 12-24 hours after presentation were strongly and independently associated with in-hospital AKI (increase vs. decrease: adjusted OR 2.83, 95% CI 1.98 – 4.05, P < 0.001). The simple 6-item KID-ACS risk score for in-hospital AKI and 30-day mortality integrates PENK levels at presentation and showed an AUC of 0.72 (95% CI 0.68 – 0.76) for in-hospital AKI, and of 0.91 (95% CI 0.87 – 0.95) for 30-day mortality in the derivation cohort. Upon external validation, KID-ACS had similarly high performance for in-hospital AKI (Swiss validation cohort: AUC 0.73, 95% CI 0.70 – 0.77; Czech validation cohort: 0.74, 95% CI 0.68 – 0.80) and 30-day mortality (UK validation cohort: AUC of 0.87, 95% CI 0.82 – 0.91; Czech validation cohort: 0.91, 95% CI 0.87 – 0.94). The simple 6-item KID-ACS score was non-inferior to established risk models and significantly outperformed the CA-AKI risk score in predicting in-hospital AKI (Zurich cohort: delta AUC 0.11, 95% CI 0.07 – 0.15, P = 0.001) and the GRACE 2.0 in predicting 30-day mortality (UK cohort: delta AUC 0.05, 95% CI 0.01 – 0.09, P = 0.027). Conclusions Circulating PENK was identified as independent predictor of in-hospital AKI and 30-day mortality in patients with ACS. The simple 6-item KID-ACS risk score integrates circulating PENK levels and provides a novel tool for simultaneous assessment of renal and mortality risk in patients presenting with ACS.Independent Predictive Value PENKPerformance and validation of KID-ACS

In order for mass spectrometry to continue to grow as a platform for high-throughput clinical and translational research, careful consideration must be given to quality control by ensuring that the assay performs reproducibly and accurately and precisely. In particular, the throughput required for large cohort clinical validation in biomarker discovery and diagnostic screening has driven the growth of multiplexed targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assays paired with sample preparation and analysis in multiwell plates. However, large scale MS-based proteomics studies are often plagued by batch effects: sources of technical variation in the data, which can arise from a diverse array of sources such as sample preparation batches, different reagent lots, or indeed MS signal drift. These batch effects can confound the detection of true signal differences, resulting in incorrect conclusions being drawn about significant biological effects or lack thereof. Here, we present an intraplate batch effect termed the edge effect arising from temperature gradients in multiwell plates, commonly reported in preclinical cell culture studies but not yet reported in a clinical proteomics setting. We present methods herein to ameliorate the phenomenon including proper assessment of heating techniques for multiwell plates and incorporation of surrogate standards, which can normalize for intraplate variation.

OBJECTIVE:To characterise cardiac remodelling, exercise capacity and fibroinflammatory biomarkers in patients with aortic stenosis (AS) with and without diabetes, and assess the impact of diabetes on outcomes. METHODS:Patients with moderate or severe AS with and without diabetes underwent echocardiography, stress cardiovascular magnetic resonance (CMR), cardiopulmonary exercise testing and plasma biomarker analysis. Primary endpoint for survival analysis was a composite of cardiovascular mortality, myocardial infarction, hospitalisation with heart failure, syncope or arrhythmia. Secondary endpoint was all-cause death. RESULTS:Diabetes (n=56) and non-diabetes groups (n=198) were well matched for age, sex, ethnicity, blood pressure and severity of AS. The diabetes group had higher body mass index, lower estimated glomerular filtration rate and higher rates of hypertension, hyperlipidaemia and symptoms of AS. Biventricular volumes and systolic function were similar, but the diabetes group had higher extracellular volume fraction (25.9%±3.1% vs 24.8%±2.4%, p=0.020), lower myocardial perfusion reserve (2.02±0.75 vs 2.34±0.68, p=0.046) and lower percentage predicted peak oxygen consumption (68%±21% vs 77%±17%, p=0.002) compared with the non-diabetes group. Higher levels of renin (log10renin: 3.27±0.59 vs 2.82±0.69 pg/mL, p<0.001) were found in diabetes. Multivariable Cox regression analysis showed diabetes was not associated with cardiovascular outcomes, but was independently associated with all-cause mortality (HR 2.04, 95% CI 1.05 to 4.00; p=0.037). CONCLUSIONS:In patients with moderate-to-severe AS, diabetes is associated with reduced exercise capacity, increased diffuse myocardial fibrosis and microvascular dysfunction, but not cardiovascular events despite a small increase in mortality.

Objective: Non-adherence in hypertension can be as high as 30–45%. Chemical adherence testing (CAT) is the preferred method to assess non-adherence in European guidelines. New randomised controlled trials (RCTs), like the RADIANCE-HTN TRIO, have also started to implement CAT into their study framework to diagnoses non-adherence. Using high performance liquid chromatography (HPLC) in tandem mass spectrometry (MS/MS), existing quantitative urine CATs take > 40 minutes. These provide a yes/no answer. Recent guidance has suggested run-time be an area of development, along with the research of quantitative assays in plasma. Translated from the field of therapeutic drug monitoring, UltraFast (<1 minute) run times were explored using ultra performance short (UP)-LC columns with MS/MS for the detection and quantitation of nine cardiometabolic medications in plasma for use in CAT. UltraFast methods could increase clinical and research capacity while reducing cost. Design: An UltraFast quantitative CAT using UPLC-MS/MS was developed and optimised for the parallel detection of nine cardiometabolic medications in plasma. These medications were amlodipine, atenolol, atorvastatin, bisoprolol, edoxaban, enalapril, propranolol, ramipril, and saxagliptin. Methods: A mixed internal standard (IS; 10 ng/mL) was added to 100 μL of sample before the addition of 100 μL of water. The entire solution was added to pre-conditioned Oasis PRiME hydrophilic lipid balance (HLB) 96-well μElution solid phase extraction (SPE) plates. The loaded wells were washed (200 μL 5% methanol) and eluted (50 μL methanol/ acetonitrile) prior to UPLC-MS/MS analysis on a Waters Xevo TQ-XS triple quadrupole system. Sub 45 s run times were achieved using a Raptor Biphenyl Guard column cartridge (5 x 2.1 mm, 2.7 μm) as the UPLC column, used in reverse phase, and pumped at a 1.4 mL/min flow rate. Five replicate calibration curves were extracted over the range 0.01–500 ng/mL to assess model fits and detection limits. Results: All medications in plasma had lower limits of detection (LOD) between 0.01 ng/mL (atorvastatin) and 20 ng/mL (amlodipine). Optimum calibration curves were obtained. These were a mixture of quadratic and linear models. R2 ranged from 0.837 (enalapril) to 0.971 (atorvastatin). Clear chromatographic separation was achieved with the first eluted peak was atenolol, detected at 3 s. The last eluted peak was edoxaban at 22 s. Quantitation was possible over the range 0.01–500 ng/mL. Conclusion: A <45 second UPLC-MS/MS run time was achieved for a panel of nine cardiometabolic medications. LODs were well within the therapeutic range of interest, and quantitation was made possible over 0.01–500 ng/mL, the therapeutic range of interest. This method could be applied in clinical laboratories for same day adherence testing, or to large RCTs for their timely processing.

Background Mitral regurgitation (MR) frequently coexists with heart failure (HF). Objectives To better understand potential pathophysiological differences between patients with HF with or without moderate-severe MR, we compared differentially expressed circulating biomarkers between these two groups. Methods The Olink Proteomics® Multiplex Cardiovascular (CVD) -II, CVD-III, Immune Response and Oncology-II panels of 363 unique proteins from different pathophysiological domains were used to investigate the biomarker profiles of HF patients from index and validation cohorts of the BIOSTAT-CHF study stratified according to the presence of moderate-to-severe MR or no-mild MR. Results The index cohort included 888 patients (46%) with moderate-to-severe MR and 1029 (54%) with no-mild MR at baseline. The validation cohort included 522 patients (33%) with moderate-to-severe MR and 1076 (66%) with no-mild MR at baseline. Compared to patients with no-mild MR, those with moderate-to-severe MR had lower body mass index, higher comorbidity burden, signs and symptoms of more severe HF, lower systolic blood pressure, and larger left atrial and ventricular dimensions, in both cohorts. NT-proBNP, CA125, fibroblast growth factor 23 (FGF23) and growth hormone 1 (GH1) were up-regulated, whereas leptin (LEP) was down-regulated in patients with moderate-severe MR versus no-mild MR, in both index and validation cohorts. Conclusion Circulating biomarkers differently expressed in HF patients with moderate-severe MR versus no-mild MR were related to congestion, lipid and mineral metabolism and oxidative stress. These findings may be of value for the development of novel treatment targets in HF patients with MR.

Serum selenium levels have been associated with the incidence of heart failure (HF) and signs of the metabolic syndrome. In addition, notable differences have been reported between males and females in food intake and micronutrient metabolism, possibly explaining different health outcomes. Our objective was to elucidate sex-specific, cross-sectional phenotypic differences in the association of serum selenium concentrations with parameters of metabolic syndrome and HF. We investigated data from individuals from a community-based cohort (PREVEND; N = 4288) and heart failure cohort (BIOSTAT-CHF; N = 1994). In both populations, cross-sectional analyses were performed for potential interaction (p < 0.1) between sex and serum selenium with overlapping signs and clinical parameters of the metabolic syndrome and HF. Baseline selenium levels of the total cohort were similar between PREVEND (85.7 μg/L) and BIOSTAT-CHF (89.1 μg/L). Females with lower selenium levels had a higher BMI and increased prevalence of diabetes than females with higher selenium, in both PREVEND (pinteraction < 0.001; pinteraction = 0.040, resp.) and BIOSTAT-CHF (pinteraction = 0.021; pinteraction = 0.024, resp.), while opposite associations were observed for males. Additionally, in females, but not in males, lower selenium was associated with a higher prevalence of myocardial infarction (MI) in PREVEND (pinteraction = 0.021) and BIOSTAT-CHF (pinteraction = 0.084). Lower selenium was associated with a higher BMI and increased prevalence of diabetes in females, opposite to males, and was also associated with more MI in females. Interventional studies are needed to validate this observation.

Background Proenkephalin A 119-159 (PENK) is freely filtered in the glomerulus with plasma levels correlating with glomerular filtration rate. Therefore, PENK has been proposed as an early indicator of acute kidney injury (AKI) although its performance is dependent on the clinical setting. This meta-analysis aimed to investigate the correlation between PENK levels and the development of AKI. Methods We conducted a comprehensive search on the PubMed, Embase, Cochrane databases, the website ClinicalTrials.gov and Cnki.net until June 26, 2023. Summary receiver operating characteristic (SROC) curves were used to amalgamate the overall test performance. Diagnostic odds ratio (DOR) was employed to compare the diagnostic accuracy of PENK with other biomarkers. Quality of the evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) criteria. Results We incorporated 11 observational studies with 3969 patients with an incidence of AKI of 23.4% (929 out of 3969 patients) with the best optimal cutoff value of PENK for early detection of AKI being 57.3 pmol/L. The overall sensitivity and specificity of PENK in identifying AKI were 0.69 (95% CI 0.62–0.75) and 0.76 (95% CI 0.68–0.82), respectively. The combined positive likelihood ratio (LR) stood at 2.83 (95% CI 2.06–3.88), and the negative LR was 0.41 (95% CI 0.33–0.52). The SROC curve showcased pooled diagnostic accuracy of 0.77 (95% CI 0.73–0.81). Interestingly, patients with a history of hypertension or heart failure demonstrated a lower specificity of PENK in correlating the development of AKI. Conclusion Our results indicate that PENK possesses significant potential as a biomarker for the early detection of the development of AKI, using a cutoff point of 57.3 pmol/L for PENK.

Oxidative stress may be a key pathophysiological mediator in the development and progression of heart failure (HF). The role of serum-free thiol concentrations, as a marker of systemic oxidative stress, in HF remains largely unknown. The purpose of this study was to investigate associations between serum-free thiol concentrations and disease severity and clinical outcome in patients with new-onset or worsening HF. Serum-free thiol concentrations were determined by colorimetric detection in 3802 patients from the BIOlogy Study to TAilored Treatment in Chronic Heart Failure (BIOSTAT-CHF). Associations between free thiol concentrations and clinical characteristics and outcomes, including all-cause mortality, cardiovascular mortality, and a composite of HF hospitalization and all-cause mortality during a 2-years follow-up, were reported. Lower serum-free thiol concentrations were associated with more advanced HF, as indicated by worse NYHA class, higher plasma NT-proBNP (P < 0.001 for both) and with higher rates of all-cause mortality (hazard ratio (HR) per standard deviation (SD) decrease in free thiols: 1.253, 95