The Edge Effect in High-Throughput Proteomics: A Cautionary Tale

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

Plasma P‐selectin is a predictor of mortality in heart failure with preserved ejection fraction

AimsThe aim of the study was to assess the association of P‐selectin with outcomes in heart failure with preserved ejection fraction (HFpEF).Methods and resultsThis is a prospective, observational study of 130 HFpEF patients who underwent clinical profiling, blood sampling, 6 min walk testing, Minnesota Living with Heart Failure Questionnaire evaluation, echocardiography, cardiovascular magnetic resonance imaging, calculation of the Meta‐Analysis Global Group in Chronic Heart Failure (MAGGIC) risk scores, and blinded plasma P‐selectin measurement. Patients were followed up for the endpoint of all‐cause mortality. The HFpEF subgroup with higher P‐selectin levels [overall median 26 372, inter‐quartile range (19 360–34 889) pg/mL] was associated with lower age, higher heart rate, less prevalent atrial fibrillation, more frequent current smoking status, and lower right ventricular end‐diastolic volumes. During follow‐up (median 1428 days), there were 38 deaths. Following maximal sensitivity and specificity receiver operating characteristic curve analysis, P‐selectin levels above 35 506 pg/mL were associated with greater risk of all‐cause mortality [hazard ratio (HR) 2.700; 95% confidence interval (CI) 1.416–5.146; log‐rank P = 0.002]. Following multivariable Cox proportional hazards regression analysis and when added to MAGGIC scores, only P‐selectin (adjusted HR 1.707; 95% CI 1.099–2.650; P ConclusionsPlasma P‐selectin is an independent predictor of mortality and provides incremental prognostic information beyond MAGGIC scores in HFpEF.</div

Proenkephalin as a biomarker correlates with acute kidney injury: a systematic review with meta-analysis and trial sequential analysis

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

Fibroblast-growth-factor-23 in heart failure with preserved ejection fraction: relation to exercise capacity and outcomes

AimsThis study aimed to assess plasma fibroblast growth factor 23 (FGF23) in patients with heart failure with preserved ejection fraction (HFpEF) and its relation to inflammation, renal function, clinical and imaging characteristics, exercise capacity, and prognosis.Methods and resultsWe performed a prospective, observational study of 172 age‐matched and sex‐matched subjects (HFpEF n = 130; controls n = 42, age 73 ± 9, female 50%) who underwent plasma biomarker sampling, echocardiography, cardiac magnetic resonance imaging, and 6 min walk testing (6MWT). The primary endpoint was the composite of all‐cause death or HF hospitalization. FGF23 was higher in HFpEF compared with controls (62 [42–105] vs. 34 [22–41] pg/mL, P ConclusionsPlasma FGF23 is higher in HFpEF compared with age‐matched and sex‐matched controls and is strongly associated with exercise incapacity and prognosis. FGF23 correlates with plasma markers of inflammation and renal impairment.</div

Left atrial ejection fraction and outcomes in heart failure with preserved ejection fraction

The aim of this study was to determine whether left atrial ejection fraction (LAEF) quantified with cardiovascular magnetic resonance (CMR) was different between heart failure with preserved ejection fraction (HFpEF) and controls, and its relation to prognosis. As part of our single-centre, prospective, observational study, 188 subjects (HFpEF n = 140, controls n = 48) underwent phenotyping with contrast-enhanced CMR, transthoracic echocardiography, blood sampling and six-minute walk testing. LAEF was calculated using the biplane method. Atrial fibrillation (AF) was present in 43 (31%) of HFpEF subjects. Overall, LAEF (%) was lower in HFpEF patients inclusive of AF (32 ± 16) or those in sinus rhythm alone (41 ± 12) compared to controls (51 ± 11), p < 0.0001. LAEF correlated inversely with maximal and minimal left atrial volumes indexed (r = − 0.602, r = − 0.762), and plasma N-terminal pro-atrial natriuretic peptide (r = − 0.367); p < 0.0001. During median follow-up (1429 days), there were 67 composite events of all-cause death or hospitalization for heart failure (22 deaths, 45 HF hospitalizations) in HFpEF. Lower LAEF (below median) was associated with an increased risk of composite endpoints (Log-Rank: all p = 0.028; sinus p = 0.036). In multivariable Cox regression analysis, LAEF (adjusted hazard ratio [HR] 0.767, 95% confidence interval [CI] 0.591–0.996; p = 0.047) and indexed extracellular volume (HR 1.422, CI 1.015–1.992; p = 0.041) were the only parameters that remained significant when added to a base prognostic model comprising age, prior HF hospitalization, diastolic blood pressure, lung disease, NYHA, six-minute-walk-test-distance, haemoglobin, creatinine and B-type natriuretic peptide. CMR-derived LAEF is lower in HFpEF compared to healthy controls and is a strong prognostic biomarker

Daily remote ischaemic conditioning following acute myocardial infarction: a randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning (rIC) is a cardioprotective tool which has shown promise in preclinical and clinical trials in the context of acute ischaemia. Repeated rIC post myocardial infarction may provide additional benefits which have not previously been tested clinically. METHODS: The trial assessed the role of daily rIC in enhancing left ventricular ejection fraction (LVEF) recovery in patients with impaired LVEF (<45%) after ST segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (P-PCI). Patients were recruited from four UK hospitals and randomised to receive either 4 weeks of daily rIC or sham conditioning using the autoRIC Device (CellAegis) starting on day 3 post P-PCI. The primary endpoint was the improvement in LVEF over 4 months assessed by cardiac MRI (CMR). Seventy-three patients (38 cases, 35 controls) completed the study. RESULTS: The treatment and control groups were well matched at baseline including for mean LVEF (42.8% vs 44.3% respectively, p=0.952). There was no difference in the improvement in LVEF over 4 months between the treatment and control groups (4.8%±7.8% vs 4.6%±5.9% respectively, p=0.924). No differences were seen in the secondary outcome measures including changes in infarct size and left ventricular end-diastolic and systolic volumes, major adverse cardiac and cerebral event, mean Kansas City Cardiomyopathy Questionnaire score and change in N-terminal pro-brain natriuretic peptide levels. CONCLUSIONS: Daily rIC starting on day 3 and continued for 4 weeks following successful P-PCI for STEMI did not improve LVEF as assessed by CMR after 4 months when compared with a matched control group. TRIAL REGISTRATION NUMBER: NCT0166461

Surrogate markers of gut dysfunction are related to heart failure severity and outcome–from the BIOSTAT-CHF consortium

Background: The contribution of gut dysfunction to heart failure (HF) pathophysiology is not routinely assessed. We sought to investigate whether biomarkers of gut dysfunction would be useful in assessment of HF (eg, severity, adverse outcomes) and risk stratification. Methods: A panel of gut-related biomarkers including metabolites of the choline/carnitine- pathway (acetyl-L-carnitine, betaine, choline, γ-butyrobetaine, L-carnitine and trimethylamine-N-oxide [TMAO]) and the gut peptide, Trefoil factor-3 (TFF-3), were investigated in 1,783 patients with worsening HF enrolled in the systems BIOlogy Study to TAilored Treatment in Chronic Heart Failure (BIOSTAT-CHF) cohort and associations with HF severity and outcomes, and use in risk stratification were assessed. Results: Metabolites of the carnitine-TMAO pathway (acetyl-L-carnitine, γ-butyrobetaine, L-carnitine, and TMAO) and TFF-3 were associated with the composite outcome of HF hospitalization or all-cause mortality at 3 years (hazards ratio [HR] 2.04-2.93 [95% confidence interval {CI} 1.30-4.71] P≤.002). Combining the carnitine-TMAO metabolites with TFF-3, as a gut dysfunction panel, showed a graded association; a greater number of elevated markers was associated with higher New York Heart Association class (P<.001), higher plasma concentrations of B-type natriuretic peptide (P<.001), and worse outcome (HR 1.90-4.58 [95% CI 1.19-6.74] P≤ 0.008). Addition of gut dysfunction biomarkers to the contemporary BIOSTAT HF risk model also improved prediction for the aforementioned composite outcome (C-statistics P≤.011, NRI 13.5-21.1 [95% CI 2.7-31.9] P≤.014). Conclusions: A panel of biomarkers of gut dysfunction showed graded association with severity of HF and adverse outcomes. Biomarkers as surrogate markers are potentially useful for assessment of gut dysfunction to HF pathophysiology and in risk stratification

Bio-adrenomedullin as a marker of congestion in patients with new-onset and worsening heart failure.

BACKGROUND: Secretion of adrenomedullin (ADM) is stimulated by volume overload to maintain endothelial barrier function, and higher levels of biologically active (bio-) ADM in heart failure (HF) are a counteracting response to vascular leakage and tissue oedema. This study aimed to establish the value of plasma bio-ADM as a marker of congestion in patients with worsening HF. METHODS AND RESULTS: The association of plasma bio-ADM with clinical markers of congestion, as well as its prognostic value was studied in 2179 patients with new-onset or worsening HF enrolled in BIOSTAT-CHF. Data were validated in a separate cohort of 1703 patients. Patients with higher plasma bio-ADM levels were older, had more severe HF and more signs and symptoms of congestion (all P < 0.001). Amongst 20 biomarkers, bio-ADM was the strongest predictor of a clinical congestion score (r2  = 0.198). In multivariable regression analysis, higher bio-ADM was associated with higher body mass index, more oedema, and higher fibroblast growth factor 23. In hierarchical cluster analysis, bio-ADM clustered with oedema, orthopnoea, rales, hepatomegaly and jugular venous pressure. Higher bio-ADM was independently associated with impaired up-titration of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers after 3 months, but not of beta-blockers. Higher bio-ADM levels were independently associated with an increased risk of all-cause mortality and HF hospitalization (hazard ratio 1.16, 95% confidence interval 1.06-1.27, P = 0.002, per log increase). Analyses in the validation cohort yielded comparable findings. CONCLUSIONS: Plasma bio-ADM in patients with new-onset and worsening HF is associated with more severe HF and more oedema, orthopnoea, hepatomegaly and jugular venous pressure. We therefore postulate bio-ADM as a congestion marker, which might become useful to guide decongestive therapy

Proenkephalin and prognosis in heart failure with preserved ejection fraction: a GREAT network study.

BACKGROUND: Proenkephalin (PENK), a stable endogenous opioid biomarker related to renal function, has prognostic utility in acute and chronic heart failure. We investigated the prognostic utility of PENK in heart failure with preserved ejection fraction (HFpEF), and its relationship to renal function, Body Mass Index (BMI), and imaging measures of diastolic dysfunction. METHODS: In this multicentre study, PENK was measured in 522 HFpEF patients (ejection fraction > 50%, 253 male, mean age 76.13 ± 10.73 years) and compared to 47 age and sex-matched controls. The primary endpoint was 2-years composite of all-cause mortality and/or heart failure rehospitalisation (HF). A subset (n = 163) received detailed imaging studies. RESULTS: PENK levels were raised in HFpEF (median [interquartile range] 88.9 [62.1-132.0]) compared to normal controls (56.3 [47.9-70.5]). PENK was correlated to urea, eGFR, Body Mass Index and E/e' (rs 0.635, - 0.741, - 0.275, 0.476, respectively, p < 0.0005). During 2 years follow-up 144 patients died and 220 had death/HF endpoints. Multivariable Cox regression models showed PENK independently predicted 2 year death/HF [hazard ratio (for 1 SD increment of log-transformed biomarker) HR 1.45 [95% CI 1.12-1.88, p = 0.005]], even after adjustment for troponin (HR 1.59 [1.14-2.20, p = 0.006]), and Body Mass Index (HR 1.63 [1.13-2.33, p = 0.009]). PENK showed no interaction with ejection fraction status for prediction of poor outcomes. Net reclassification analyses showed PENK significantly improved classification of death/HF outcomes for multivariable models containing natriuretic peptide, troponin and Body Mass Index (p < 0.05 for all). CONCLUSIONS: In HFpEF, PENK levels are related to BMI, and measures of diastolic dysfunction and are prognostic for all-cause mortality and heart failure rehospitalisation

Multiomics Analysis Provides Novel Pathways Related to Progression of Heart Failure

Background: Despite major advances in pharmacological treatment for patients with heart failure, residual mortality remains high. This suggests that important pathways are not yet targeted by current heart failure therapies. Objectives: We sought integration of genetic, transcriptomic, and proteomic data in a large cohort of patients with heart failure to detect major pathways related to progression of heart failure leading to death. Methods: We used machine learning methodology based on stacked generalization framework and gradient boosting algorithms, using 54 clinical phenotypes, 403 circulating plasma proteins, 36,046 transcript expression levels in whole blood, and 6 million genomic markers to model all-cause mortality in 2,516 patients with heart failure from the BIOSTAT-CHF (Systems BIOlogy Study to TAilored Treatment in Chronic Heart Failure) study. Results were validated in an independent cohort of 1,738 patients. Results: The mean age of the patients was 70 years (Q1-Q3: 61-78 years), 27% were female, median N-terminal pro–B-type natriuretic peptide was 4,275 ng/L (Q1-Q3: 2,360-8,486 ng/L), and 7% had heart failure with preserved ejection fraction. During a median follow-up of 21 months, 657 (26%) of patients died. The 4 major pathways with a significant association to all-cause mortality were: 1) the PI3K/Akt pathway; 2) the MAPK pathway; 3) the Ras signaling pathway; and 4) epidermal growth factor receptor tyrosine kinase inhibitor resistance. Results were validated in an independent cohort of 1,738 patients. Conclusions: A systems biology approach integrating genomic, transcriptomic, and proteomic data identified 4 major pathways related to mortality. These pathways are related to decreased activation of the cardioprotective ERBB2 receptor, which can be modified by neuregulin