Plasma Samples Research Articles

IGLON5 Frequency in Idiopathic REM Sleep Behavior Disorder: A Multicenter Study.

Idiopathic/isolated REM sleep behavior disorder (iRBD) has been strongly linked to neurodegenerative synucleinopathies such as Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. However, there have been increasing reports of RBD as a presenting feature of serious and treatable autoimmune syndromes, particularly IGLON5. This study's objective was to investigate the frequency of autoantibodies in a large cohort of participants with iRBD. Participants were enrolled in the North American Prodromal Synucleinopathy cohort with polysomnography-confirmed iRBD, free of parkinsonism and dementia. Plasma samples were systematically screened for the autoantibodies IGLON5, DPPX, LGI1, and CASPR2 using plasma IgG cell-based assay. Positive or equivocal results were confirmed by repeat testing, plus tissue-based indirect immunofluorescence assay for IGLON5. Of 339 samples analyzed, 3 participants (0.9%) had confirmed positive IGLON5 autoantibodies in the cell-based assay, which were confirmed by the tissue-based assay. An additional participant was positive for CASPR2 with low titer by cell-based assay only (of lower clinical certainty). These cases exhibited a variety of symptoms including dream enactment, cognitive decline, autonomic dysfunction, and motor symptoms. In 1 IGLON5 case and the CASPR2 case, evolution was suggestive of typical synucleinopathy, suggesting the possibility that findings were incidental. However, 2 participants with IGLON5 died before diagnosis was clinically suspected, with a final clinical picture highly suggestive of autoimmune disease. Our finding that nearly 1% of a large iRBD cohort may have a serious but potentially treatable autoantibody syndrome has important clinical implications. In particular, it raises the question of whether autoantibody testing for IGLON-5-IgG should be widely implemented for participants with iRBD, considering the difficulty in diagnosis of autoimmune diseases, their response to treatment, and the potential for rapid disease progression. However, any routine testing protocol will also have to consider costs and potential adverse effects of false-positive findings. NCT03623672.

Pharmacokinetic study of iptacopan and its two acyl glucuronide metabolites in monkey plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this study, a simple and sensitive liquid chromatography tandem mass spectrometric method was developed and validated for the determination of iptacopan and two acyl glucuronidation metabolites in monkey plasma. The plasma sample was precipitated with acetonitrile and then separated on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using 0.1% formic acid and 5 mM ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometry (MS) detection was performed in positive multiple reactions monitoring (MRM) mode with precursor-to-production transitions. The developed assay was validated over the range of 1-2000 ng/mL for three analytes with correlation coefficient (r) more than 0.99. The validation parameters including accuracy, precision, carryover effect, matrix effect, recovery, and stability were all within the acceptable limits. The validated method has been applied to investigate the pharmacokinetics of iptacopan and its two acyl glucuronidation metabolites in monkey plasma. After intravenous administration, iptacopan showed low clearance (2.75 mL/min/kg) in monkey plasma. After oral administration, the bioavailability was 55.43%. The exposure (AUC0-t) of direct acyl glucuronide (AG) of iptacopan accounts for 9.73% of the iptacopan plasma exposure. The AUC0-t of AG of dealkylated metabolite of iptacopan was present at a lower level, accounting for 0.5% of the iptacopan plasma exposure.

Population pharmacokinetics of fluconazole for prevention or treatment of invasive candidiasis in Chinese young infants.

The dosing of fluconazole for young infants remains empirical because of the limited pharmacokinetic (PK) data. We aimed to establish a population PK model and assess the systematic exposure-response of commonly used regimens of fluconazole in Chinese infants.We included infants with a postnatal age of less than 120 days and received intravenous fluconazole. Both scheduled and scavenged plasma samples were collected, and fluconzaole concentration was determined by a validated ultra-performance liquid chromatography-tandem mass spectrometry assay. Population PK analysis was conducted using Phoenix NLME, and then Monte Carlo simulation was conducted to predict the probability of target attainment (PTA) of empirically used regimens of both prophylactic and therapeutic purposes.Based on 304 plasma samples from 183 young infants, fluconazole concentration data was best described by a one-compartment model with first-order elimination. Gestational Age (GA), postnatal age (PNA), and body weight (BW) were included in the final model as CL = 0.02*(GA/214)2.77*(PNA/13)0.24*exp(nCL); V = 1.56*(BW/1435)0.90*exp(nV). Model validation revealed the final model had qualified stability and acceptable predictive properties. Monte Carlo simulation indicated that under the same minimum inhibitory concentration (MIC) value and administration regimen, PTA decreased with GA and PNA. The commonly used prophylactic regimens can meet the clinical need, while higher doses might be needed for treatment of invasive candidiasis.This population PK model of fluconazole discriminated the impact of GA and PNA on CL and BW on V. Dosing adjustment was needed according to the GA and PNA of infants to achieve targeted exposures.

Metabolomics reveals the potential metabolic mechanism of infliximab against DSS-induced acute and chronic ulcerative colitis.

Inflammatory bowel disease (IBD) is often accompanied by metabolic imbalance, and infliximab (IFX) can alleviate IBD symptoms, but its metabolic mechanisms remain unclear. To investigate the relationship between IBD, metabolism, and IFX, an acute and chronic ulcerative colitis (UC) model induced by dextran sulfate sodium (DSS) was established. Plasma samples were analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, followed by multivariate statistical analysis. The results showed that IFX could alleviate colonic shortening and reduce colonic pathological damage in acute and chronic mouse colitis, improve acute and chronic UC, and ameliorate metabolic disturbances. Among the 104 elevated metabolites and 170 decreased metabolites, these metabolites mainly belonged to amino acids, glucose, and purines. The changes in these metabolites were mainly associated with drug metabolism-other enzymes, riboflavin metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phosphonate and phosphinate metabolism, and phenylalanine metabolism. In summary, this study provides a valuable approach to explore the metabolic mechanisms of IFX in treating acute and chronic UC from a metabolomics perspective.

Surfactant-modified carbon nano-onion-β-cyclodextrin nanocomposite as an efficient sorbent in dispersive solid phase extraction of metoprolol and atenolol from plasma samples prior to HPLC–PDA analysis

Surfactant-modified carbon nano-onion-β-cyclodextrin nanocomposite as an efficient sorbent in dispersive solid phase extraction of metoprolol and atenolol from plasma samples prior to HPLC–PDA analysis

Impact of plasma from patients with severe aortic stenosis on valvular endothelial cells

Abstract Background The natural history of aortic stenosis (AS) encompasses a spectrum of pathophysiological stages, including endothelial dysfunction, inflammation, fibrosis and calcification. Accumulating data underscore the detrimental impact of the haemostatic system on AS, contributing to disease progression and the development of a prothrombotic phenotype within the valve. Understanding these mechanisms is pivotal for the exploration of novel therapeutic avenues, particularly in the context of transcatheter aortic valve replacement (TAVR). Following TAVR, the native diseased aortic valve persists, maintaining its pathological activity and thrombogenic effects on the bioprosthetic valve, potentially influencing its durability and patient outcomes. The precise effects of plasma and its various components on valvular endothelial cells (VECs) dysfunction remains inadequately explored. Purpose To evaluate the influence of plasma derived from individuals with severe AS on aortic VECs. Methods Plasma samples obtained from patients with severe AS undergoing TAVR (n=100) were collected immediately before the procedure, alongside plasma samples from healthy individuals. Factor (F)Xa activity within the plasma samples was quantified using fluorometer-based method. Porcine VECs were then exposed to either plasma (at 10% concentration) or isolated FXa (1 nM), with or without different pharmacological modulators. Protein expression levels were assessed by Western blot analyses. The level of reactive oxygen species (ROS) was evaluated by dihydroethidium staining. Results Exposure of VECs to plasma from severe AS patients promoted ROS formation compared to plasma from healthy individuals (1402 ± 438 AU vs 732 ± 137 AU, p=0.001) (Figure 1). The heightened oxidative stress elicited by plasma from severe AS patients was attenuated by empagliflozin, perindoprilat, losartan, neutralizing antibodies directed against IL-1, IL-6 and TNF-α. Plasma from severe AS patients showed elevated FXa activity compared to plasma from healthy individuals (179.4 ± 101.3 ng/mL vs 63.2 ± 87.2 ng/mL, p=0.02). Stimulation of VECs with isolated FXa resulted in a sustained formation of ROS, which was blunted by empagliflozin, perindoprilat, losartan, rivaroxaban, dabigatran and PAR-1, 2 and 4 antagonists. Quantification of protein expression in VECs stimulated by isolated FXa revealed an endothelial dysfunction phenotype characterized by increased expression of SGLT2, VCAM-1 and COX-1, and decreased expression of eNOS. Conclusions Plasma from patients with severe AS exerts a detrimental effect on aortic VECs, characterized by a pronounced oxidative stress response. This adverse impact is mediated by multiple mechanisms, including activation of the AT1R/NADPH oxidase/SGLT2 pathway, upregulation of pro-inflammatory cytokines, and activation of PAR. FXa plays a pivotal role by promoting oxidative stress in VECs and contributing to valvular endothelial dysfunction.Pro-oxidant state in VECs.

Evaluation of cfDNA fragmentation characteristics in plasma for the diagnosis of lung cancer: A prospective cohort study.

Lung cancer is one of the most prevalent cancers worldwide, yet only approximately 16% of patients are diagnosed in early stage, highlighting the urgent need for novel, highly accurate detection models. In our study, patients with suspected lung cancer or lung disease, as identified through radiographic imaging, along with healthy individuals, were consecutively recruited from Beijing Chest Hospital. Circulating free DNA (cfDNA) was extracted from plasma samples, and low-depth whole-genome sequencing was performed to identify fragmentomic features for model construction. A total of 265 participants were prospectively enrolled, comprising 124 lung cancer patients and 141 noncancer individuals. The model we developed was based on four cfDNA fragmentation characteristics, including 20-bp breakpoint nucleotides motif, fragmentation size coverage, fragmentation size distribution, and copy number variation. In an independent test cohort, the model achieved an area under the curve (AUC) of 0.861 (95% CI: 0.781-0.942) and demonstrated a sensitivity of 70% (95% CI: 53.5%-83.4%) at a specificity of 89.4% (95% CI: 76.9%-96.5%). Notably, the model was also effective in detecting early-stage cancer, with an AUC of 0.808 (95% CI: 0.69-0.925). In summary, our lung cancer detection model shows strong screening capabilities by leveraging four cfDNA fragmentation characteristics, exhibiting robust performance in early cancer diagnosis.

Circulating Amyloid beta 1-40 is independently associated with statin treatment and LDL cholesterol reduction: a prospective cohort study

Abstract Background Accumulating evidence suggests that circulating amyloid beta 1-40 (Aβ40), a pro-inflammatory and pro-atherogenic peptide, is associated with atherosclerotic cardiovascular disease (ASCVD), providing incremental prognostic value and risk stratification refinement in primary and secondary prevention settings. Existing evidence supports conflicting off-target effects of statins on plasma Aβ40. We aim to explore the impact of cardiovascular risk reduction through statin therapy on Aβ40 plasma levels in subjects with dyslipidemia and with/without ASCVD. Methods In this prospective study, patients with dyslipidemia who visited for the first time our lipid clinic for risk stratification and treatment plan assessment (n=146) were consecutively recruited. Αβ40 was measured in plasma samples by enzyme-linked immunosorbent assay at baseline and after a mean follow-up of 5.2 and 15 months. Patients in whom statin treatment was initiated or intensified in terms of dose or regimen (n= 68) were compared against patients not treated with statins or with unchanged statin intensity (n = 78). Statin intensity treatment was defined based on the European Society of Cardiology (ESC) guidelines. Results Increased Aβ40 plasma levels at baseline were associated with ASCVD presence, decreased glomerular filtration rate (GFR), and history of heart failure (p<0.05 for all). An increase in Aβ40 circulating levels between baseline and follow-up 1 was observed in patients in whom statin treatment was initiated or intensified (p=0.041). However, in patients not treated with statins or treated with unchanged statin intensity, an increase in Aβ40 circulating levels was observed between follow-up 1 and follow-up 2 (p=0.05, Figure 1A). These changes in Aβ40 were significantly different between the two groups during the short follow-up period (β=0.226, p for interaction =0.025) independently from age and changes in GFR, systolic blood pressure, and LDL-C. Additionally, patients who achieved a reduction in low-density lipoprotein cholesterol (LDL-C) ≥ 50% in follow-up 2 have unchanged Aβ40 levels during the long follow-up period, while an increase in Aβ40 was observed in the rest population (p=0.012, Figure 1B). These changes were significantly different between the two LDL-C groups independently of statin treatment and changes in high sensitive C-reactive protein (hs-CRP) (β= -0.231, p for interaction=0.035). Conclusion In a dyslipidemic population, statin initiation or intensification and LDL-C decrease showed a short-term increase and a long-term return to initial levels, respectively, in circulating Aβ40 levels. From a mechanistic point of view, our findings suggest that statins have an impact on Aβ40, possibly through an LDL-C-dependent and a non-LDL-C-dependent pathway. Further studies are needed to evaluate the utility of circulating Aβ40 as a novel biomarker in ASCVD risk reduction assessment.

Inhibition of ceramide de novo synthesis mitigates atherosclerosis

Abstract Background Atherosclerosis is the primary underlying cause of cardiovascular disease. Plasma ceramides are positively correlated with an increased risk of major adverse cardiovascular events and serve as an alternative biomarker for cardiovascular disease. Importantly, ceramide levels are notably higher within atherosclerotic plaques. Therefore, inhibiting the ceramide de novo synthesis pathway in the plaques may serve as a potential therapeutic intervention for atherosclerosis. Purpose We aim to assess the therapeutic efficacy of a nano-formulation of myriocin, a targeted inhibitor of serine-palmitoyl transferase, the key enzyme in ceramide de novo synthesis, in treating atherosclerosis. Methods Myriocin was encapsulated within the hydrophobic core of lipid-based nanoparticles specifically designed for this study. Male and female ApoE-/- mice, subjected to an 8-week high-fat diet regimen, received either myriocin nanoparticles (treatment group) or empty nanoparticles (control group) intravenously once a week for 4 weeks at a dose of 1.3 mg/kg body weight while still on the high-fat diet. At the study endpoint, whole aortas were harvested and subjected to Oil Red O staining to quantify the total lesion area. Histological staining was performed on serial sections of the aortic root to analyze the cellular composition within the plaques. Plasma samples were collected for total cholesterol measurement and lipidomic analysis. Results Upon intravenous administration, myriocin nanoparticles exhibited preferential accumulation in atherosclerotic plaques. Compared to control mice, those treated with myriocin nanoparticles exhibited a 58.7% reduction in total lesion area in male ApoE-/- mice and a 64.6% reduction in female ApoE-/- mice (p<0.0001). Consistently, the lesion areas in the aortic root were also reduced in the treated mice. Notably, myriocin nanoparticles enhanced collagen and alpha-smooth muscle content, while reducing macrophages and neutral lipids within the plaques, leading to a reduced plaque vulnerability index and preventing rupture events. TUNEL staining revealed reduced cellular apoptosis in the atherosclerotic plaques, resulting in a smaller necrotic area in the treated mice. In vitro experiments demonstrated that myriocin nanoparticles effectively inhibited lipopolysaccharide-induced inflammatory response and oxidative stress in bone marrow-derived macrophages. Furthermore, myriocin nanoparticles restored the gene expression of enzymes involved in the fatty acid beta-oxidation pathway, such as ACOX1 and CPT1A, in foam cells. Conclusion Using myriocin nanoparticles to inhibit excessive ceramide production due to inflammation reduces inflammation, and ROS production, while restoring fatty acid beta-oxidation in macrophages. This approach emerges as an effective strategy for targeting ceramide de novo synthesis within atherosclerotic plaques, thereby mitigating atherosclerosis progression and improving plaque stability.

Linking antibody against apolipoprotein A-1 to metabolic dysfunction-associated steatohepatitis in mice

Abstract Background Metabolic dysfunction-associated fatty liver disease (MASLD) is a common liver condition associated with increased cardiovascular disease (CVD) risk. Cytokeratin 18 (CK-18) is indicative of liver injury and used as a surrogate biomarker covering MASLD to cirrhosis phenotypes. Autoantibodies against apolipoprotein A-1 (AAA-1) are known CVD risk factors inducing MASLD in vitro, but their role in modulating steatohepatitis and fibrosis in vivo is still elusive. We investigated AAA-1's contribution to systemic low-grade inflammation, liver steatosis, and fibrosis using a MASLD mouse model and a validated passive immunization protocol (PIP). Methods 10 weeks-old male C57BL/6J mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) and immunized with AAA-1 (200ug) or control antibodies for ten days. At sacrifice, plasma samples were collected, and CK-18 levels, and proinflammatory cytokines were measured using the Mesoscale Discovery platform. Hematoxylin and Eosin and Sirius Red Fast Green staining were used to reveal liver steatosis and fibrosis, respectively. RNA were extracted from mouse liver samples and mRNA were then analyzed using a commercial fibrosis-specific genes panel (nCounter Human Fibrosis V2 Panel) and the nCounter Analysis System. Results Compared to control IgG recipients, CDAHFD mice injected with AAA-1 exhibited significantly elevated plasma levels of CK-18 (5.3 vs 2.1, p=0.031), IL-6 (13 vs 6.9, p=0.035), IL-10 (27.3 vs 9.8, p=0.007) and TNF-α (32.1 vs 24.2, p=0.032), and liver steatosis (93.4% vs 73.8%, p=0.007). Nanostring technology experiments revealed upregulation of several pro-fibrotic m-RNAs in liver tissue from AAA-1 immunized mice compared to the IgG immunized control group. These included Serum Amyloid A (Fold Change (FC) AAA-1 vs IgG : 2.5, p=0.02) , G-protein coupled receptors 65 (FC: 2.7, p=0.01), Periostin (FC: 2.6, p=0.001), Phospholipid transfer protein (FC: 2.2, p=0.0004), Vascular Cell Adhesion Molecule 1 (FC: 2.5, p=0.02) and Angiopoietin-like 4 (FC: 2.8, p=0.01). Histologically, the amount of liver fibrosis remained unaffected, probably due to the short time of the study. Conclusions Short AAA-1 PIP induced enhanced liver steatosis and inflammation accompanied by an elevation of the expression of several pro-fibrotic genes in CDAHFD mice, suggesting that AAA-1 may contribute to the transition from MASLD to MASH. Knowing whether increased exposure time to AAA-1 could lead to end stage disease (including cirrhosis and HCC), and if this could apply to humans as well warrant further investigations.

Early inflammation as a footprint of increased mortality risk in infants living with HIV from three African countries

In this work our aim was to identify early biomarkers in plasma samples associated with mortality in children with perinatal HIV treated early in life, to potentially inform early intervention targeting this vulnerable group. 20/215 children (9.3%) with perinatal HIV, enrolled within 3 months of age died prematurely within the first year of the study, despite early ART initiation. Using a propensity score, we selected 40 alive study participants having similar clinical and virological records compared to the deceased group. 13 HIV unexposed (HU) healthy children were additionally used as controls. Baseline plasma samples were analyzed using a targeted proteomic approach, and to assess pathogen-associated and damage-associated molecular patterns (PAMPs, DAMPs) levels. Data from deceased participants were compared to both control groups, with multivariate logistic regression models used to evaluate the association between mortality and plasma proteins. We developed a machine learning model to predict mortality risk, finding that IL-6 and CXCL11 not only were higher in deceased children than Matched-children with HIV (p < 0.001 and p = 0.0034) but also predictive of mortality (accuracy of 77%); levels of PAMPs were higher in deceased children (p = 0.0016). Thus, measuring early inflammatory biomarkers, particularly IL-6, could help mortality risk prediction and potentially guide targeted interventions.

Troponin T as a risk marker for recurrent cardiovascular events in the post-discharge setting one month after acute coronary syndrome: a TRACER substudy

Abstract Background Troponin T (TnT) is a protein found in cardiac myocytes that is released during myocardial injury, such as myocardial infarction (MI). TnT levels rapidly rise during an MI, remain elevated for several days, and are a key criterion for diagnosing MI. Additionally, circulating TnT levels may offer prognostic information later, particularly during the rehabilitation phase after an acute coronary syndrome (ACS). Purpose To examine the relationship between circulating TnT at one month after hospitalisation for ACS and subsequent cardiovascular (CV) events. Methods The TRACER trial randomly assigned 12,944 patients diagnosed with non-ST-segment elevation ACS (NSTE-ACS) to receive either vorapaxar or placebo. Plasma samples were obtained after one month, and high-sensitivity (hs)-TnT was measured among 5,313 patients using the Elecsys electrochemiluminescence immunoassays. In this sub-study, the primary composite endpoint was the one-year risk of CV mortality, MI, or ischaemic stroke. Associations with outcomes were evaluated using Cox regression models, both unadjusted and adjusted for clinical baseline characteristics. Results The median concentration of hs-TnT was 11 ng/L one month after hospitalisation for ACS. Higher levels of hs-TnT at one month were associated with older age, coronary artery bypass surgery during the index ACS, male sex, a history of heart failure, renal disease, and diabetes mellitus. The composite outcome of CV mortality, MI, or ischaemic stroke occurred in 381 patients (incidence rate 5.4 per 100 person-years). Hs-TnT at one month after ACS was independently associated with the risk of the composite CV endpoint (relative hazard ratio with 95% confidence interval comparing the third vs. the first sample quartile: 2.40 [1.80 – 3.20]) (see Figure 2). Similarly, hs-TnT, when added to a model including established risk factors, increased the concordance index of the multivariable model from 0.711 to 0.740 (p&amp;lt;0.001). The results were consistent across the different components of the composite endpoint. Conclusions The level of hs-TnT provides useful information on the risk of future CV events in patients when measured one month after an ACS. This information might be useful for optimising secondary preventive treatment following an ACS.Figure.Hs-TnT and composite CV outcome

Reference values for plasma and urine trace elements in a Swiss population-based cohort.

Trace elements (TEs) are ubiquitous. TE concentrations vary among individuals and countries, depending on factors such as living area, workplaces and diet. Deficit or excessive TEs concentrations have consequences on the proper functioning of human organism so their biomonitoring is important. The aim of this project was to provide reference values for TEs concentrations in the Swiss population. The 1,078 participants to the SKiPOGH cohort included in this study were aged 18-90 years. Their 24-h urine and/or plasma samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to determine 24 TEs concentrations: Ag, Al, As, Be, Bi, Cd, Co, Cr, Cu, Hg, I, Li, Mn, Mo, Ni, Pb, Pd, Pt, Sb, Se, Sn, Tl, V and Zn. Statistical tests were performed to evaluate the influence of covariates (sex, age, BMI, smoking) on these results. Reference intervals for the Swiss adult population were also defined. TEs concentrations were obtained for respectively 994 and 903 persons in plasma and urine matrices. It was possible to define percentiles of interest (P50 and P95) for almost all the TEs. Differences in TEs distribution between men and women were noticed in both matrices; age was also a cofactor. This first Swiss biomonitoring of a large TEs-panel offers reference values in plasma and in urine for the Swiss population. The results obtained in this study were generally in line with clinical recommendations and comparable to levels reported in other population-based surveys.

Comparison of multiple high sensitivity cardiac troponin assays for early low risk rule out of myocardial infarction: SCORECARD

Abstract Background/Introduction The use of a single low high-sensitivity cardiac troponin measurement to rule out myocardial infarction (MI) at presentation is advocated by international guidelines. Studies have not systematically compared high-sensitivity cardiac troponin (hs-cTn) I and T assays to determine which values should be used to rule out MI in patients with suspected acute coronary syndrome. Methods In a pooled analysis from three prospective observational cohort studies the lowest cTn concentration was determined that gave a negative predictive value (NPV) of ≥99.5% for MI or death at 30 days on presentation to the emergency department. EDTA or lithium heparin plasma or serum samples collected at presentation were measured using five hs-cTnI assays (Abbott ARCHITECT i2000, Beckman Coulter Access 2, Siemens Atellica IM, Ortho-Clinical Diagnostics VITROS 5600, LSI Medience PATHFAST) and one hs-cTnT assay (Roche Cobas e411). The diagnostic outcome of MI or death at 30 days was adjudicated according to the Fourth Universal Definition of MI. Optimal thresholds were determined to identify the highest proportion of patients that could be considered for discharge for a NPV of ≥99.5% for each assay. Results Among 941 patients, MI or death at 30 days occurred in 113 (12%). Optimized thresholds varied between assays ranging from &amp;lt;2 to &amp;lt;7 ng/L (Table) that identified between 22% and 60% of patients as low risk (0-1%) for MI or death at 30 days. Conclusions Our data identify differences in the effectiveness of thresholds between hs-cTnI and hs-cTnT assays for the same safety performance to rule out MI on presentation using a single measurement. In some cases, these thresholds differ from those used in clinical practice. Due to lack of assay standardization, optimal decision thresholds should be defined and validated for each high sensitivity cTn assay.

Evaluation of the biomarker-based ABC-AF-bleeding risk score and clinically based bleeding scores in 31,605 patients with atrial fibrillation treated with oral anticoagulation

Abstract Background International guidelines recommend a systematic evaluation of bleeding risk in patients with atrial fibrillation (AF) to guide oral anticoagulation. However, it remains challenging to accurately predict bleeding risk. The biomarker-based ABC-AF-bleeding risk score to predict bleeding in anticoagulated patients with AF has shown promise. Purpose To evaluate the performance of the biomarker-based ABC-AF-bleeding risk score and compare its performance with other bleeding risk scores in 31,605 patients randomized to direct oral anticoagulant or warfarin using individual patient data from three randomized clinical trials. Methods The COMBINE AF biomarker data set contains individual patient data from three pivotal randomized trials (ARISTOTLE, ENGAGE AF-TIMI 48, and RE-LY) comparing apixaban, edoxaban or dabigatran with warfarin, in patients with AF at increased risk of stroke. The ABC-AF-bleeding biomarkers were analyzed in plasma samples collected at baseline (hemoglobin, troponin T high-sensitivity, and GDF-15) using Roche Diagnostics Elecsys assays. The biomarker-based ABC-AF-bleeding risk score (Age, Biomarkers, Clinical history of prior bleeding) was calculated as previously published. The discrimination was assessed by Harrell’s c index and compared with three clinically based bleeding risk scores; HASBLED, ORBIT, and DOAC. Results During a median follow-up time of 2.0 years, a total of 1,572 ISTH major bleeding events occurred (incidence rate per 100 person-years of 2.79) including 593 gastrointestinal (1.04) and 270 intracranial (0.47) bleeding events. The biomarker-based ABC-AF-bleeding score was well calibrated for major bleeding in the total material (Figure). The c indices for major bleeding were 0.68 (95% confidence interval 0.67-0.70), for gastrointestinal bleeding 0.70 (0.68-0.72), and for intracranial bleeding 0.66 (0.63-0.69). The biomarker-based ABC-AF-bleeding risk score provided superior discrimination compared with the clinically based risk scores in the full cohort (Table). The ABC-AF-bleeding risk score also provided superior discrimination for major bleeding in clinically relevant subgroups based on age, sex, body mass index, coronary artery disease, diabetes, heart failure, kidney function, and irrespective of DOAC or VKA use. Conclusions In patients with AF treated with different types of OAC the biomarker-based ABC-AF-bleeding score provide better discrimination of the risk för major bleeding than risk scores based only on clinical factors. The results were consistent for different types of bleeding and across multiple subgroups. There was good calibration when comparing predicted vs observed rate of major bleeding. These findings support the utility of the biomarker-based ABC-AF-bleeding risk score for advancing precision medicine in patients with AF.Calibration of ABC-AF-bleeding scoreTable

Associations of omega-3 fatty acid subclasses with cardiovascular diseases in 500,000 individuals

Abstract Increased blood levels of omega-3 fatty acids have consistently been shown to be associated with reduced risk of cardiovascular diseases (CVD). However, large scale placebo-controlled trials of omega-3 supplementation have had discordant results: in 2018 REDUCE-IT reported a 25% risk reduction, whereas in 2020 STRENGTH reported no risk reduction. The two trials had both different placebos (mineral vs corn oil) and omega-3 formulations (EPA vs DHA+EPA). There is not yet a consensus on what explains these results. Here we describe the profiling of omega-3 fatty acid subclasses, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) with nuclear magnetic resonance (NMR), and test their associations with incident CVD in 500,000 individuals from UK Biobank. We utilized 500 MHz NMR spectrometers to analyze total omega-3 and its subclasses in EDTA plasma samples and employed biomarker-specific quantification algorithms to measure their absolute concentrations. In this study, we developed a novel algorithm to quantify EPA, complementing previously described algorithms for omega-3 and DHA. Correlation (r^2) of this novel NMR-based quantification and gas chromatography-based measurements was 0.77 for EPA. On average, DHA comprised 44.5% of the total omega-3 concentration, EPA accounted for 22.2%, and other subclasses constituted 34.3% in the cohort. We computed age and sex adjusted associations of omega-3, DHA, and EPA with 10-year risk of CVD among individuals who were not taking cholesterol lowering medication. We discovered protective associations: for DHA a hazard ratio of 0.85 (0.86-0.89, 95% confidence interval) per standard deviation versus 0.96 (0.95-0.98) for EPA, and 0.95 (0.93-0.97) for total omega-3 (Figure). Since DHA (used in the STRENGTH trial with a null result) has a significantly stronger epidemiological association than EPA, these results from a large-scale observational study do not support the hypothesis that the differential use of DHA and EPA in the REDUCE-IT and STRENGTH trials explains their divergent results.Figure

N-terminal prohormone of brain natriuretic peptide predicts recurrent cardiovascular events in patients with acute coronary syndrome both in the acute and post-discharge setting: a TRACER substudy

Abstract Background N-terminal prohormone of brain natriuretic peptide (NT-proBNP) is a circulating peptide released by the heart in response to increased wall tension and stretching, for example, occurring during myocardial and/or atrial dysfunction. NT-proBNP is a diagnostic and prognostic marker in patients with heart failure but might also have prognostic utility in the acute phase of patients with acute coronary syndrome (ACS) and at one month after ACS. Purpose To assess the prognostic value of NT-proBNP over time in patients with ACS both in the acute setting and when re-assessed after one month. Methods The TRACER study randomized 12,944 patients with non-ST-segment elevation ACS (NSTE-ACS) to treatment with vorapaxar vs. placebo. Plasma samples for biomarker analyses were obtained at randomization (median 21 hours after hospital admission) in 6,800 patients and at an outpatient visit after one month in 5,313 patients. NT-proBNP was analysed using the Elecsys electrochemiluminescence immunoassays method. The primary composite endpoint in this substudy was the one-year risk of cardiovascular (CV) mortality, myocardial infarction, or ischaemic stroke. Associations between continuous natural log-transformed NT-proBNP levels and outcomes were assessed using Cox regression models accounting for study treatment and established risk factors. Results The median concentration of NT-proBNP during hospitalization for ACS and after one month was 496 ng/L and 264 ng/L, respectively. Higher age, female sex, a history of heart failure, and lower body mass index were independently associated with higher levels of NT-proBNP among patients in the acute setting, with renal disease also being an important factor after one month. The composite outcome of CV mortality, MI, or ischaemic stroke occurred in 901 patients (incidence rate 10.1 per 100 person-years) included at baseline and in 381 patients (incidence rate 5.4 per 100 person-years) included at one month. Higher circulating levels of NT-proBNP were independently associated with a higher risk of the composite endpoint at baseline (adjusted hazard ratio with 95% confidence interval comparing the third vs. the first sample quartile: 1.25 [1.03 – 1.51]) and at one month (1.56 [1.15 – 2.11]) (Figure 1 and 2). Incorporating NT-proBNP at baseline into a model with other established risk factors increased the concordance index of the multivariable model from 0.656 to 0.660 (p &amp;lt; 0.001), and after one month, from 0.711 to 0.733 (p &amp;lt; 0.001). These findings remained consistent across the various components of the composite endpoint. Conclusions In patients with ACS, the level of NT-proBNP is an indicator of increased risk of CV outcomes both in the acute setting and, even more pronounced, one month after the index ACS. Measuring NT-proBNP provided incremental prognostic information beyond established risk factors, both in the acute and post-discharge settings.Fig 1.NT-proBNP quartile groupsFig 2.NT-proBNP and CV outcome

Circulating nucleases degrade high-molecular-weight cell-free DNA in coronary vessels during acute myocardial infarction

Abstract Introduction Extracellular cell-free DNA (cfDNA) originates from various biological processes, including cell turnover, apoptosis, necrosis, NETosis, and other forms of cell death. In healthy individuals, highly fragmented cfDNA is elevated in the bloodstream due to infection, cancer, inflammation and thrombosis. In thrombotic vascular diseases, neutrophils release elongated, high-molecular-weight DNA with bioactive and prothrombotic properties in a process termed NETosis. Circulating nucleases are thought to counteract pathogenic properties of NETs. However, attempts to connect total cfDNA concentration to nuclease activity have failed. Purpose We aimed to elucidate the in vivo connection between extracellular DNA and circulating nucleases in thrombotic vascular diseases by investigating DNA length as a potential novel biomarker in cardiovascular disease. Methods Citrated plasma from healthy volunteers (n=363) and from ST-elevation myocardial infarction (MI) coronary aspirates (n=59) and peripheral arterial sites (n=96) were analyzed for DNA concentration, length, and nuclease activity. Total DNA concentration was measured with a fluorescent DNA binding dye, and elongated DNA assessed using qPCR and Agilent bioanalyzer high-sensitivity DNA chip. Nuclease activity was measured by single radial enzyme-diffusion (SRED) assay. Cardiac MRI assessed infarct size acutely and six months after the event. Results Coronary aspirates exhibited an elevation in total DNA, with a significant rise in high-molecular weight cell-free DNA in addition to an increase in sub-nucleosome fragmentation (Figure 1). Peripheral and coronary plasma samples from individuals with myocardial infarction (MI) showed elevated levels of total and elongated cfDNA. When compared to healthy individuals, the integrity of cfDNA was lower in peripheral samples and higher in coronary aspirates. During myocardial infarction, circulating nuclease activity was elevated systemically but low at the coronary culprit lesion site. Elongated high-molecular-weight cfDNA accumulates primarily in coronary vessels but is also elevated systemically. In coronary aspirates, nuclease activity negatively correlated with elongated DNA and DNA integrity (rs=0.6255, p&amp;lt;0.0001). Conclusions Elongated DNA accumulates during MI at the culprit lesion site. Its inverse correlation with nuclease activity underscores the role of nucleases in cleaving elongated DNA to shorter, soluble fragments. This study establishes DNA length and integrity as novel biomarkers in thrombotic cardiovascular disease.Figure 1:Coronary cfDNA lengthFigure 2:Nuclease activity and cfDNA

Triple-hormone receptor agonist retatrutide significantly improves lipoprotein and apolipoprotein profiles in participants with obesity or overweight

Abstract Background In a phase 2 study in participants who have obesity or overweight but not type 2 diabetes (T2D) (n=338), retatrutide (RETA), an agonist of GIP, GLP-1 and glucagon receptors, reduced body weight, glycated hemoglobin, triglycerides (TG), LDL-C and VLDL-C. Purpose To better understand the effects of RETA on the serum lipid profile we analyzed changes in apolipoprotein levels and distribution and size of lipoprotein particles. Methods Adult participants with obesity (BMI ≥30 kg/m2), or overweight (BMI ≥ 27 kg/m2 and &amp;lt; 30 kg/m2) with a weight-related comorbidity, and without T2D were randomized to receive RETA 1, 4, 8, 12 mg or placebo for 48 weeks. Lipids panel (including calculated non-HDL-C), apolipoproteins (apo) and NMR lipoprotein profiles were measured at baseline, 24 and 48 weeks in fasting plasma samples. Data were analyzed using a mixed model for repeated measures after log transformation. Results RETA dose-dependently reduced non-HDL-C up to 22.2% and 26.9% and apoB up to 19.6% and 24.2% at 24 and 48 weeks, respectively (Figure). At 48 weeks, RETA reduced TG and apoC-III levels up to 40.6% and 38.0%, respectively. RETA greatly reduced the total number of TG-rich lipoprotein particles (TRLP) and all subfractions of TRLP (large, medium and small) at 48 weeks (Table). Since the reduction in large and medium TRLP was greater than the reduction in small TRLP, the average size of TRLP was smaller following treatment with RETA. RETA reduced the number of total and highly atherogenic small LDL particles (LDLP), while total and small HDL particles (HDLP) were only slightly reduced. The average size of HDLP increased following treatment with RETA. At 48 weeks, the NMR-derived lipoprotein insulin resistance score was dose-dependently decreased by 27.4%, 31.7% and 32.5% with 4 mg, 8 mg and 12 mg, respectively, following RETA treatment. Conclusions RETA treatment for 48 weeks dose-dependently reduced levels of non-HDL-C and apoB and the number of total and small LDL particles, suggesting an improvement in atherogenic lipoprotein profile and cardiovascular risk in subjects who have obesity or overweight but not T2D.

RGB Trichromatic Whiteness Assessment of Bio Analytical Chromatographic Tool Using Fluorescence for Quantitation of Semaglutide: Application to Pharmaceutical Preparations and Spiked Plasma.

Semaglutide (SEMG) is one of the most widely used and trending medications to treat type II diabetes and obesity.This work aimed to develop a liquid chromatography with spectroflourimetric detection (HPLC-flourimetry) analysis of SEMG in both its tablet dosage form and plasma. The power of fluorescence detection coupled with HPLC proved its capability as a bioanalytical tool to assay SEMG in plasma samples owing to its simplicity and sensitivity which reached below the Cmax of SEMG. Separation was done using a C18 column with mobile phase of acetonitrile and water acidified with orthophosphoric acid (pH 3.5) (1.41 × 10-5M) in isocratic mode in ratio 57:43 and 1mL/min flow rate after extraction using protein precipitation. Detection was carried out at λ excitation of 238nm and λ emission of 416 and 307nm for SEMG and the internal standard, respectively. Evaluation of greenness of the proposed method was done using AGREE (Analytical GREEnness Metric Approach), ComplexGAPI (Complementary Green Analytical Procedure Index) & the new algorithm RGB 12 model (Red-Green-Blue). They showed that these methods can be a greener alternative with acceptable sensitivity for analysis of SEMG. The developed seven min-assay was validated per ICH as well as FDA bio analytical methods' guidelines to prove its applicability for routine sample analysis and future pharmacokinetic studies.