Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

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.

Is atrial fibrillation in HFpEF a distinct phenotype? Insights from multiparametric MRI and circulating biomarkers.

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/m2, 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.

Sexual dimorphism in selenium deficiency is associated with metabolic syndrome and prevalence of heart disease.

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

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.

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.

Fibro-inflammatory recovery and type 2 diabetes remission following a low calorie diet but not exercise training: A secondary analysis of the DIASTOLIC randomised controlled trial.

AIMS: To investigate the relationship between fibro-inflammatory biomarkers and cardiovascular structure/function in people with Type 2 Diabetes (T2D) compared to healthy controls and the effect of two lifestyle interventions in T2D. METHODS: Data were derived from the DIASTOLIC randomised controlled trial (RCT) and includes a comparison between those with T2D and the matched healthy volunteers recruited at baseline. Adults with T2D without cardiovascular disease (CVD) were randomized to a 12-week intervention either: (1) exercise training, (2) a low-energy (∼810 kcal/day) meal-replacement plan (MRP) or (3) standard care. Principal Component and Fisher's linear discriminant analysis were used to investigate the relationships between MRI acquired cardiovascular outcomes and fibro-inflammatory biomarkers in cases versus controls and pre- and post-intervention in T2D. RESULTS: At baseline, 83 people with T2D (mean age 50.5 ± 6.4; 58% male) and 36 healthy controls (mean age 48.6 ± 6.2; 53% male) were compared and 76 people with T2D completed the RCT for pre- post-analysis. Compared to healthy controls, subjects with T2D had adverse cardiovascular remodelling and a fibro-inflammatory profile (20 differentially expressed biomarkers). The 3D data visualisations showed almost complete separation between healthy controls and those with T2D, and a marked shift towards healthy controls following the MRP (15 biomarkers significantly changed) but not exercise training. CONCLUSIONS: Fibro-inflammatory pathways and cardiovascular structure/function are adversely altered before the onset of symptomatic CVD in middle-aged adults with T2D. The MRP improved the fibro-inflammatory profile of people with T2D towards a more healthy status. Long-term studies are required to assess whether these changes lead to continued reverse cardiac remodelling and prevent CVD.

Determination of N7-glycidamide guanine adducts in human blood DNA following exposure to dietary acrylamide using liquid chromatography/tandem mass spectrometry.

RATIONALE: Acrylamide is classified as a probable human carcinogen that is metabolised to glycidamide, which can covalently bind to DNA. The aim of this study was to investigate the formation of N7-glycidamide guanine (N7-GA-Gua) adducts in human blood DNA following exposure to acrylamide present in carbohydrate-rich foods as part of the normal human diet. METHODS: Lymphocyte DNA was extracted from blood samples obtained from healthy human volunteers. Following thermal depurination of the DNA samples, N7-GA-Gua adducts were quantified using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method incorporating a stable isotope labelled internal standard. Estimated dietary acrylamide intake was recorded by completion of food frequency questionnaires for the 24 hours prior to volunteer blood donation. RESULTS: An LC/MS/MS method was validated with a limit of detection of 0.25 fmol and a lower limit of quantitation of 0.50 fmol on column. N7-GA-Gua adducts were detected in human blood DNA with the levels ranging between 0.3 to 6.3 adducts per 108 nucleotides. The acrylamide intake was calculated from the food frequency questionnaires ranging between 20.0 and 78.6 µg. CONCLUSIONS: Identification and quantification of N7-GA-Gua adducts in the blood DNA of healthy volunteers suggests that dietary acrylamide exposure may lead to the formation of DNA adducts. This important finding warrants further investigation to ascertain a correlation between environmental/dietary acrylamide exposure and levels of DNA adducts.

Association of gut-related metabolites with outcome in acute heart failure.

BACKGROUND: Trimethylamine N-oxide (TMAO), a gut-related metabolite, is associated with heart failure (HF) outcomes. However, TMAO is the final product of a complex metabolic pathway (ie, choline/carnitine) that has never been entirely investigated in HF. The present study investigates a panel of metabolites involved in the TMAO-choline/carnitine metabolic pathway for their associations with outcome in acute HF patients. METHODS: In total, 806 plasma samples from acute HF patients were analyzed for TMAO, trimethyllysine, L-carnitine, acetyl-L-carnitine, γ-butyrobetaine, crotonobetaine, trimethylamine, betaine aldehyde, choline, and betaine using a developed liquid chromatography-tandem mass spectrometry method. Associations with outcome of all-cause mortality (death) and a composite of all-cause mortality and/or rehospitalization caused by HF (death/HF) at 30 days and 1 year were investigated. RESULTS: TMAO, trimethyllysine, L-carnitine, acetyl-L-carnitine, and γ-butyrobetaine were associated with death and death/HF at 30 days (short term; hazard ratio 1.30-1.49, P≤ .021) and at 1 year (long term; hazard ratio 1.15-1.25, P≤ .026) when adjusted for cardiac risk factors. L-carnitine and acetyl-L-carnitine were superior for short-term outcomes whereas TMAO was the superior metabolite for association with long-term outcomes. Furthermore, acetyl-L-carnitine and L-carnitine were superior for in-hospital mortality and improved risk stratification when combined with current clinical risk scores (ie, Acute Decompensated HEart Failure National REgistry, Organized Program To Initiate Lifesaving Treatment In Hospitalized Patients With Heart Failure, and Get With The Guidelines-Heart Failure; odds ratio (OR) ≥ 1.52, P≤ .020). CONCLUSIONS: Carnitine-related metabolites show associations with adverse outcomes in acute HF, in particular L-carnitine and acetyl-L-carnitine for short-term outcomes, and TMAO for long-term outcomes. Further studies are warranted to investigate the role and implications of carnitine metabolites including intervention in the pathogenesis of HF.

Short-term adverse remodeling progression in asymptomatic aortic stenosis.

OBJECTIVES: Aortic stenosis (AS) is characterised by a long and variable asymptomatic course. Our objective was to use cardiovascular magnetic resonance imaging (MRI) to assess progression of adverse remodeling in asymptomatic AS. METHODS: Participants from the PRIMID-AS study, a prospective, multi-centre observational study of asymptomatic patients with moderate to severe AS, who remained asymptomatic at 12 months, were invited to undergo a repeat cardiac MRI. RESULTS: Forty-three participants with moderate-severe AS (mean age 64.4 ± 14.8 years, 83.4% male, aortic valve area index 0.54 ± 0.15 cm2/m2) were included. There was small but significant increase in indexed left ventricular (LV) (90.7 ± 22.0 to 94.5 ± 23.1 ml/m2, p = 0.007) and left atrial volumes (52.9 ± 11.3 to 58.6 ± 13.6 ml/m2, p < 0.001), with a decrease in systolic (LV ejection fraction 57.9 ± 4.6 to 55.6 ± 4.1%, p = 0.001) and diastolic (longitudinal diastolic strain rate 1.06 ± 0.2 to 0.99 ± 0.2 1/s, p = 0.026) function, but no overall change in LV mass or mass/volume. Late gadolinium enhancement increased (2.02 to 4.26 g, p < 0.001) but markers of diffuse interstitial fibrosis did not change significantly (extracellular volume index 12.9 [11.4, 17.0] ml/m2 to 13.3 [11.1, 15.1] ml/m2, p = 0.689). There was also a significant increase in the levels of NT-proBNP (43.6 [13.45, 137.08] pg/ml to 53.4 [19.14, 202.20] pg/ml, p = 0.001). CONCLUSIONS: There is progression in cardiac remodeling with increasing scar burden even in asymptomatic AS. Given the lack of reversibility of LGE post-AVR and its association with long-term mortality post-AVR, this suggests the potential need for earlier intervention, before the accumulation of LGE, to improve the long-term outcomes in AS. KEY POINTS: • Current guidelines recommend waiting until symptom onset before valve replacement in severe AS. • MRI showed clear progression in cardiac remodeling over 12 months in asymptomatic patients with AS, with near doubling in LGE. • This highlights the need for potentially earlier intervention or better risk stratification in AS.

Circulating plasma concentrations of angiotensin-converting enzyme 2 in men and women with heart failure and effects of renin-angiotensin-aldosterone inhibitors.

AIMS: The current pandemic coronavirus SARS-CoV-2 infects a wide age group but predominantly elderly individuals, especially men and those with cardiovascular disease. Recent reports suggest an association with use of renin-angiotensin-aldosterone system (RAAS) inhibitors. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for coronaviruses. Higher ACE2 concentrations might lead to increased vulnerability to SARS-CoV-2 in patients on RAAS inhibitors. METHODS AND RESULTS: We measured ACE2 concentrations in 1485 men and 537 women with heart failure (index cohort). Results were validated in 1123 men and 575 women (validation cohort).The median age was 69 years for men and 75 years for women. The strongest predictor of elevated concentrations of ACE2 in both cohorts was male sex (estimate = 0.26, P < 0.001; and 0.19, P < 0.001, respectively). In the index cohort, use of ACE inhibitors, angiotensin receptor blockers (ARBs), or mineralocorticoid receptor antagonists (MRAs) was not an independent predictor of plasma ACE2. In the validation cohort, ACE inhibitor (estimate = -0.17, P = 0.002) and ARB use (estimate = -0.15, P = 0.03) were independent predictors of lower plasma ACE2, while use of an MRA (estimate = 0.11, P = 0.04) was an independent predictor of higher plasma ACE2 concentrations. CONCLUSION: In two independent cohorts of patients with heart failure, plasma concentrations of ACE2 were higher in men than in women, but use of neither an ACE inhibitor nor an ARB was associated with higher plasma ACE2 concentrations. These data might explain the higher incidence and fatality rate of COVID-19 in men, but do not support previous reports suggesting that ACE inhibitors or ARBs increase the vulnerability for COVID-19 through increased plasma ACE2 concentrations.

Identifying optimal doses of heart failure medications in men compared with women: a prospective, observational, cohort study.

BACKGROUND: Guideline-recommended doses of angiotensin-converting-enzyme (ACE) inhibitors or angiotensin-receptor blockers (ARBs), and ß blockers are similar for men and women with heart failure with reduced ejection fraction (HFrEF), even though there are known sex differences in pharmacokinetics of these drugs. We hypothesised that there might be sex differences in the optimal dose of ACE inhibitors or ARBs and ß blockers in patients with HFrEF. METHODS: We did a post-hoc analysis of BIOSTAT-CHF, a prospective study in 11 European countries of patients with heart failure in whom initiation and up-titration of ACE inhibitors or ARBs and ß blockers was encouraged by protocol. We included only patients with left ventricular ejection fraction less than 40%, and excluded those who died within the first 3 months. Primary outcome was a composite of time to all-cause mortality or hospitalisation for heart failure. Findings were validated in ASIAN-HF, an independent cohort of 3539 men and 961 women with HFrEF. FINDINGS: Among 1308 men and 402 women with HFrEF from BIOSTAT-CHF, women were older (74 [12] years vs 70 [12] years, p<0·0001) and had lower bodyweights (72 [16] kg vs 85 [18] kg, p<0·0001) and heights (162 [7] cm vs 174 [8] cm, p<0·0001) than did men, although body-mass index did not differ significantly. A similar number of men and women reached guideline-recommended target doses of ACE inhibitors or ARBs (99 [25%] vs 304 [23%], p=0·61) and ß blockers (57 [14%] vs 168 [13%], p=0·54). In men, the lowest hazards of death or hospitalisation for heart failure occurred at 100% of the recommended dose of ACE inhibitors or ARBs and ß blockers, but women showed approximately 30% lower risk at only 50% of the recommended doses, with no further decrease in risk at higher dose levels. These sex differences were still present after adjusting for clinical covariates, including age and body surface area. In the ASIAN-HF registry, similar patterns were observed for both ACE inhibitors or ARBs and ß blockers, with women having approximately 30% lower risk at 50% of the recommended doses, with no further benefit at higher dose levels. INTERPRETATION: This study suggests that women with HFrEF might need lower doses of ACE inhibitors or ARBs and ß blockers than men, and brings into question what the true optimal medical therapy is for women versus men. FUNDING: European Commission.

The measurement of KRAS G12 mutants using multiplexed selected reaction monitoring and ion mobility mass spectrometry.

RATIONALE: There is a considerable clinical demand to determine key mutations in genes involved with cancer which necessitates the deployment of highly specific and robust analytical methods. Multiplex liquid chromatography with selected reaction monitoring (LC/SRM) assays offer the ability to achieve quantitation down to levels expected to be present in clinical samples. Ion mobility mass spectrometry (IMS/MS) assays can provide increased peak capacity and hence separation in an extremely short time frame, and in addition provide physicochemical data regarding the collision cross-section of an analyte which can be used in conjunction with the m/z value of an ion to increase detection specificity. METHODS: For LC/SRM, unlabelled peptides and corresponding stable-isotope-labelled standards were spiked into digested human plasma and analysed using ultrahigh-performance liquid chromatography (UHPLC) coupled to a triple quadrupole mass spectrometer to enable the generation of analyte-specific calibration lines. Synthetic unlabelled peptides were infused into a Synapt G2 mass spectrometer for travelling wave ion mobility separation and TW CCSN2 values were derived from comparison with previously generated TW CCSN2 calibration values. RESULTS: Linear calibration lines (0.125 to 25 fmol/µL) were established for each of the KRAS peptides. UHPLC separated the peptides and hence enabled them to be split into different retention time functions/windows. This separation enabled detection of three or four transitions for each light and heavy peptide with at least 10 points per peak for accurate quantitation. All six KRAS G12 peptides were separated using IMS/MS, enabling precise TW CCSN2 values to be determined. Although some of the G12 peptides chromatographically co-eluted, all the peptides were distinguished by m/z, retention time and/or drift time. CONCLUSIONS: This study advocates that LC/SRM and IMS/MS could both be used to identify single amino acid substitutions in KRAS as an alternative to commonly used methods such as circulating tumour DNA analysis.

Plasma Tenascin-C: a prognostic biomarker in heart failure with preserved ejection fraction.

INTRODUCTION: Tenascin-C is a marker of interstitial fibrosis. We assessed whether plasma Tenascin-C differed between heart failure with preserved ejection fraction (HFpEF) and asymptomatic controls and related to clinical outcomes. MATERIALS AND METHODS: Prospective, observational study of 172 age- and sex-matched subjects (HFpEF n = 130; controls n = 42, age 73 ± 9, males 50%) who underwent phenotyping with 20 plasma biomarkers, echocardiography, cardiac MRI and 6-minute-walk-testing. The primary endpoint was the composite of all-cause death/HF hospitalisation. RESULTS: Tenascin-C was higher in HFpEF compared to controls (13.7 [10.8-17.3] vs (11.1 [8.9-12.9] ng/ml, p < 0.0001). Tenascin-C correlated positively with markers of clinical severity (NYHA, E/E', BNP) and plasma biomarkers reflecting interstitial fibrosis (ST-2, Galectin-3, GDF-15, TIMP-1, TIMP-4, MMP-2, MMP-3, MMP-7, MMP-8), cardiomyocyte stress (BNP, NTpro-ANP), inflammation (MPO, hs-CRP, TNFR-1, IL6) and renal dysfunction (urea, cystatin-C, NGAL); p < 0.05 for all. During follow-up (median 1428 days), there were 61 composite events (21 deaths, 40 HF hospitalizations). In multivariable Cox regression analysis, Tenascin-C (adjusted hazard ratio [HR] 1.755, 95% confidence interval [CI] 1.305-2.360; p < 0.0001) and indexed extracellular volume (HR 1.465, CI 1.019-2.106; p = 0.039) were independently associated with adverse outcomes. CONCLUSIONS: In HFpEF, plasma Tenascin-C is higher compared to age- and sex-matched controls and a strong predictor of adverse outcomes. Trial registration: ClinicalTrials.gov: NCT03050593.

Iron deficiency in worsening heart failure is associated with reduced estimated protein intake, fluid retention, inflammation, and antiplatelet use.

AIMS: Iron deficiency (ID) is common in heart failure (HF) patients and negatively impacts symptoms and prognosis. The aetiology of ID in HF is largely unknown. We studied determinants and the biomarker profile of ID in a large international HF cohort. METHODS AND RESULTS: We studied 2357 worsening HF patients from the BIOSTAT-CHF cohort. ID was defined as transferrin saturation <20%. Univariable and multivariable logistic regression models were constructed to identify determinants for ID. We measured 92 cardiovascular markers (Olink Cardiovascular III) to establish a biomarker profile of ID. The primary endpoint was the composite of all-cause mortality and first HF rehospitalization. Mean age (±standard deviation) of all patients was 69 ± 12.0 years, 26.1% were female and median N-terminal pro B-type natriuretic peptide levels (+interquartile range) were 4305 (2360-8329) ng/L. Iron deficiency was present in 1453 patients (61.6%), with highest prevalence in females (71.1% vs. 58.3%; P < 0.001). Independent determinants of ID were female sex, lower estimated protein intake, higher heart rate, presence of peripheral oedema and orthopnoea, chronic kidney disease, lower haemoglobin, higher C-reactive protein levels, lower serum albumin levels, and P2Y12 inhibitor use (all P < 0.05). None of these determinants were sex-specific. The biomarker profile of ID largely consisted of pro-inflammatory markers, including paraoxonase 3 (PON3) and tartrate-resistant acid phosphatase type 5. In multivariable Cox proportional hazard regression analyses, ID was associated to worse outcome, independently of predictors of ID (hazard ratio 1.25, 95% confidence interval 1.06-1.46; P = 0.007). CONCLUSION: Our data suggest that the aetiology of ID in worsening HF is complex, multifactorial and seems to consist of a combination of reduced iron uptake (malnutrition, fluid overload), impaired iron storage (inflammation, chronic kidney disease), and iron loss (antiplatelets).

Comparing biomarker profiles of patients with heart failure: atrial fibrillation vs. sinus rhythm and reduced vs. preserved ejection fraction.

Aims: The clinical correlates and consequences of atrial fibrillation (AF) might be different between heart failure with reduced vs. preserved ejection fraction (HFrEF vs. HFpEF). Biomarkers may provide insights into underlying pathophysiological mechanisms of AF in these different heart failure (HF) phenotypes. Methods and results: We performed a retrospective analysis of the BIOlogy Study to TAilored Treatment in Chronic Heart Failure (BIOSTAT-CHF), which was an observational cohort. We studied 2152 patients with HFrEF [ejection fraction (EF < 40%)], of which 1419 were in sinus rhythm (SR) and 733 had AF. Another 524 patients with HFpEF (EF ≥50%) were studied, of which 286 in SR and 238 with AF. For the comparison of biomarker profiles, 92 cardiovascular risk markers were measured (Proseek® Olink Cardiovascular III panel). The circulating risk marker pattern observed in HFrEF was different than the pattern in HFpEF: in HFrEF, AF was associated with higher levels of 77 of 92 (84%) risk markers compared to SR; whereas in HFpEF, many more markers were higher in SR than in AF. Over a median follow-up of 21 months, AF was associated with increased mortality risk [multivariable hazard ratio (HR) of 1.27; 95% confidence interval (CI) 1.09-1.48, P = 0.002]; there was no significant interaction between heart rhythm and EF group on outcome. Conclusion: In patients with HFrEF, the presence of AF was associated with a homogeneously elevated cardiovascular risk marker profile. In contrast, in patients with HFpEF, the presence of AF was associated with a more scattered risk marker profile, suggesting differences in underlying pathophysiological mechanisms of AF in these HF phenotypes.

B-type natriuretic peptide molecular forms for risk stratification and prediction of outcome after acute myocardial infarction.

BACKGROUND: B-type natriuretic peptide (BNP) is known to be a risk marker following acute myocardial infarction (MI). More recently, truncated molecular forms of the BNP molecule have been identified, with the association of these forms and outcome in acute MI not known. The present study investigated their use as risk stratifying biomarkers of this condition. METHODS: BNP molecular forms (BNP 5-32, BNP 4-32 and BNP 3-32) were measured in plasma from 1078 acute MI patients using immunocapture followed by MALDI-ToF-mass spectrometry. Associations of molecular forms with short-term and long-term adverse outcomes were assessed. RESULTS: BNP molecular forms were independent predictors of mortality/reinfarction, mortality/rehospitalization due to heart failure, and a composite of all events at 6 months, 1 year and 2 years and showed prognostic ability comparable with conventional BNP measurements (P<.001-0.026 vs. N-terminal [NT]-proBNP P<.001-0.020, respectively). Reclassification analyses showed BNP molecular forms successfully reclassified patient risk when used in addition to the GRACE clinical risk score (P≤.005). BNP 5-32 showed utility as a secondary risk stratification biomarker when used in combination with the GRACE score and NT-proBNP by successful down-classification of high-risk patients. CONCLUSIONS: BNP molecular forms were associated with poor prognosis at 6 months, 1 year and at 2 years in patients with acute MI. BNP 5-32 showed successful utility as a secondary marker in combination with NT-proBNP after GRACE scoring. This study suggests a potential role for BNP molecular forms in prognosis and risk stratification after acute MI.

Using matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) profiling in order to predict clinical outcomes of patients with heart failure.

BACKGROUND: Current risk prediction models in heart failure (HF) including clinical characteristics and biomarkers only have moderate predictive value. The aim of this study was to use matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) profiling to determine if a combination of peptides identified with MALDI-MS will better predict clinical outcomes of patients with HF. METHODS: A cohort of 100 patients with HF were recruited in the biomarker discovery phase (50 patients who died or had a HF hospital admission vs. 50 patients who did not have an event). The peptide extraction from plasma samples was performed using reversed phase C18. Then samples were analysed using MALDI-MS. A multiple peptide biomarker model was discovered that was able to predict clinical outcomes for patients with HF. Finally, this model was validated in an independent cohort with 100 patients with HF. RESULTS: After normalisation and alignment of all the processed spectra, a total of 11,389 peptides (m/z) were detected using MALDI-MS. A multiple biomarker model was developed from 14 plasma peptides that was able to predict clinical outcomes in HF patients with an area under the receiver operating characteristic curve (AUC) of 1.000 (p = 0.0005). This model was validated in an independent cohort with 100 HF patients that yielded an AUC of 0.817 (p = 0.0005) in the biomarker validation phase. Addition of this model to the BIOSTAT risk prediction model increased the predictive probability for clinical outcomes of HF from an AUC value of 0.643 to an AUC of 0.823 (p = 0.0021). Moreover, using the prediction model of fourteen peptides and the composite model of the multiple biomarker of fourteen peptides with the BIOSTAT risk prediction model achieved a better predictive probability of time-to-event in prediction of clinical events in patients with HF (p = 0.0005). CONCLUSIONS: The results obtained in this study suggest that a cluster of plasma peptides using MALDI-MS can reliably predict clinical outcomes in HF that may help enable precision medicine in HF.

Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy?

Homeostasis around vascular endothelium is a function of the equilibrium between the bioavailability of nitric oxide (NO) and oxidizing reactive oxygen species (ROS). Within the vascular endothelium, NO enhances vasodilatation, reduces platelet aggression and adhesion (anti-thrombotic), prevents smooth muscle proliferation, inhibits adhesion of leukocytes and expression of pro-inflammatory cytokines genes (anti-inflammatory), and counters the oxidation of low density lipoprotein (LDL) cholesterol. A shift in the equilibrium that favours NO deficiency and ROS formation leads to endothelial dysfunction and cardiovascular disease. The synthesis of NO is catalysed by nitric oxide synthase and co-factored by tetrahydrobiopterin (BH4), nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN). The focus of this review is on endothelial nitric oxide synthase (eNOS), although we recognize that the other nitric oxide synthases may contribute as well. Levels of homocysteine and the active metabolite of folate, 5-methyltetrahydrofolate (5-MTHF), play a determining role in circulating levels of nitric oxide. We review endothelial nitric oxide bioavailabilty in relation to endothelial dysfunction as well as the therapeutic strategies involving the nitric oxide synthesis pathway. Although folate supplementation improves endothelial function, results from large clinical trials and meta-analyses on palpable clinical endpoints have been inconsistent. There are however, encouraging results from animal and clinical studies of supplementation with the co-factor for nitric oxide synthesis, BH4, though its tendency to be oxidized to dihydrobiopterin (BH2) remains problematic. Understanding how to maintain a high ratio of BH4 to BH2 appears to be the key that will likely unlock the therapeutic potential of nitric oxide synthesis pathway.

Diagnostic and prognostic utility of cardiovascular magnetic resonance imaging in heart failure with preserved ejection fraction - implications for clinical trials.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a poorly characterized condition. We aimed to phenotype patients with HFpEF using multiparametric stress cardiovascular magnetic resonance imaging (CMR) and to assess the relationship to clinical outcomes. METHODS: One hundred and fifty four patients (51% male, mean age 72 ± 10 years) with a diagnosis of HFpEF underwent transthoracic echocardiography and CMR during a single study visit. The CMR protocol comprised cine, stress/rest perfusion and late gadolinium enhancement imaging on a 3T scanner. Follow-up outcome data (death and heart failure hospitalization) were captured after a minimum of 6 months. RESULTS: CMR detected previously undiagnosed pathology in 42 patients (27%), who had similar baseline characteristics to those without a new diagnosis. These diagnoses consisted of: coronary artery disease (n = 20, including 14 with 'silent' infarction), microvascular dysfunction (n = 11), probable or definite hypertrophic cardiomyopathy (n = 10) and constrictive pericarditis (n = 5). Four patients had dual pathology. During follow-up (median 623 days), patients with a new CMR diagnosis were at higher risk of adverse outcome for the composite endpoint (log rank test: p = 0.047). In multivariate Cox proportional hazards analysis, a new CMR diagnosis was the strongest independent predictor of adverse outcome (hazard ratio: 1.92; 95% CI: 1.07 to 3.45; p = 0.03). CONCLUSIONS: CMR diagnosed new significant pathology in 27% of patients with HFpEF. These patients were at increased risk of death and heart failure hospitalization. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03050593 . Retrospectively registered; Date of registration: February 06, 2017.