Lipoprotein Research Articles

A computational biology approach to identify potential protein biomarkers and drug targets for sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the loss of upper and lower motor neurons. The sporadic ALS (sALS) is a multigenic disorder and the complex mechanisms underlying its onset are still not fully delineated. Despite the recent scientific advancements, certain aspects of ALS pathogenic targets need to be yet clarified. The aim of the presented study is to identify potential genetic biomarkers and drug targets for sALS, by analysing gene expression profiles, presented in the publicly available GSE68605 dataset, of motor neurons cells obtained from sALS patients. We used different computational approaches including differential expression analysis, protein network mapping, candidate protein biomarker (CPB) identification, elucidation of the role of functional modules, and molecular docking analysis. The resultant top ten up- and downregulated genes were further used to construct protein-protein interaction network (PPIN). The PPIN analysis resulted in identifying four CPBs (namely RIOK2, AKT1, CTNNB1, and TNF) that commonly overlapped with one another in network parameters (degree, bottleneck and maximum neighbourhood component). The RIOK2 protein emerged as a potential mediator of top five functional modules that are associated with RNA binding, lipoprotein particle receptor binding in pre-ribosome, and interferon, cytokine-mediated signaling pathway. Furthermore, molecular docking analysis revealed that cyclosporine exhibited the highest binding affinity (−8.6 kJ/mol) with RIOK2, and surpassed the FDA-approved ALS drugs, such as riluzole and edaravone. This suggested that cyclosporine may serve as a promising candidate for targeting RIOK2 downregulation observed in sALS patients. In order to validate our computational results, it is suggested that in vitro and in vivo studies may be conducted in future to provide a more detailed understanding of ALS diagnosis, prognosis, and therapeutic intervention.

Lipoprotein Characteristics and Incident Coronary Heart Disease: Prospective Cohort of Nearly 90 000 Individuals in UK Biobank.

Background Associations of coronary heart disease (CHD) with plasma lipids are well described, but the associations with characteristics of lipoproteins (which transport lipids) remain unclear. Methods and Results UK Biobank is a prospective study of 0.5 million adults. Analyses were restricted to 89 422 participants with plasma lipoprotein and apolipoprotein measures from Nightingale nuclear magnetic resonance spectroscopy and without CHD at baseline. CHD risk was positively associated with concentrations of very-low-density lipoproteins, intermediate-density lipoproteins, and low-density lipoproteins (LDL), and inversely associated with high-density lipoproteins. Hazard ratios (99% CIs) per SD were 1.22 (1.17-1.28), 1.16 (1.11-1.21), 1.20 (1.15-1.25), and 0.90 (0.86-0.95), respectively. Larger subclasses of very-low-density lipoproteins were less strongly associated with CHD risk, but associations did not materially vary by size of LDL or high-density lipoprotein. Given lipoprotein particle concentrations, lipid composition (including cholesterol) was not strongly related to CHD risk, except for triglyceride in LDL particles. Apolipoprotein B was highly correlated with LDL concentration (r=0.99), but after adjustment for apolipoprotein B, concentrations of very-low-density lipoprotein and high-density lipoprotein particles remained strongly related to CHD risk. Conclusions This large-scale study reliably quantifies the associations of nuclear magnetic resonance-defined lipoprotein characteristics with CHD risk. CHD risk was most strongly related to particle concentrations, and separate measurements of lipoprotein concentrations may be of greater value than the measurement by apolipoprotein B, which was largely determined by LDL concentration alone. Furthermore, there was strong evidence of positive association with mean triglyceride molecules per LDL particle but little evidence of associations with total triglycerides or other lipid and lipoprotein fractions after accounting for lipoprotein concentrations.

FRI099 Advanced Lipoprotein Analysis In Patients With Cushing’s Syndrome

Abstract Disclosure: R.T. Varghese: None. R. McGlotten: None. M. Sampson: None. A. Prakash: None. R. Shamburek: None. A.T. Remaley: None. R. Muniyappa: None. L.K. Nieman: None. Introduction: Cushing syndrome (CS) patients have a higher mortality rate due to increased cardiovascular disease (CVD). Analysis of lipoprotein particles with nuclear magnetic resonance (NMR) spectroscopy provides additional insights into CVD risk beyond the standard lipid panel. The goal of the study was to better characterize the lipid abnormalities in patients with CS by using high-resolution separation of lipoprotein particles. Methods: We measured plasma lipids (Cholesterol, TC; calculated low density lipoprotein, LDL; triglycerides, TG; high density lipoprotein, HDL) by the traditional enzymatic assay and lipoprotein particles by NMR spectroscopy. Measures of inflammation and insulin resistance were also derived from NMR spectra. Values are shown as mean ± SEM or median [IQR]. Results: After initial evaluation, 42 subjects (mean age 47.4 ± 2.5 years; 28 females; weight 85.0 ± 3.1 kg) had Cushing's disease (n=23), ectopic ACTH secretion (n=17), or adrenal adenoma (n=2). Ten patients were on chronic statin therapy, and 14 subjects had type 2 diabetes mellitus (T2DM). The control group comprised 45 subjects without T2DM [mean age 45.4 ± 2.1 years and weight (83.5 ± 2.7) kg]. None in the control group were on statin therapy. Compared to controls, CS patients had significantly higher systolic (135 ±2 vs. 119 ±2 mm Hg, p<0.001) and diastolic blood pressure (79 ±2 vs. 71 ±1 mm Hg, p<0.001), HbA1c (6.1 [5.4-7.5] vs. 5.4 [5.0-5.7], p<0.001), plasma glucose (103 [90-137] vs 94 [89-100] mg/dL, p=0.003), and insulin (17[12-26] vs 11[7-15] uU/mL, p=0.01) levels. Compared to controls, patients with CS had significantly higher triglyceride concentration (116 [84 -146] vs 87 [62-120] mg/dL, p=0.02) and lower LDL concentration (90 [69 -113] vs 108 [90 -125] mg/dL, p=0.04). However, total cholesterol and HDL cholesterol concentrations were not different. LDL particle size in CS was smaller (20.5[20.0-21.0] vs 21.0 [20.7-21.2] nm, p=0.01) and VLDL particle size was larger (49.8 ± 0.9 vs 45.7 ± 1.0 nm, p=0.002) than in controls. No difference was noted between groups in HDL particle number or size or in LDL and VLDL particle number. In the group with CS, a marker of inflammation, GlycA (Glycan N-acetylglucosamine) (494.5 ± 18.2 vs. 382.5 ± 9.0 µmol/L, p<0.001) and Lipoprotein Insulin Resistance score (LPIR) (46.4 ± 3.1 vs. 36.2 ± 3.1, p=0.02) were significantly higher. There were no group differences in branch-chained amino acids (BCCA) levels. These differences remained even when patients with type 2 diabetes and those on statin therapy were excluded. Conclusions: These data suggest that the higher inflammatory and insulin resistance state, dysglycemia, hypertension, and altered lipoprotein profiles may contribute to the increased CV risk in patients with Cushing’s syndrome. Early detection and treatment for these abnormalities may attenuate CV risk. Presentation: Friday, June 16, 2023

THU335 Hyperinsulinemia Is Associated With An Atherogenic Lipoprotein Profile In Humans With Selective Insulin Resistance And An Atheroprotective Profile In Humans With Non-selective Insulin Resistance

Abstract Disclosure: D. Walzer: None. M. Lightbourne: None. M. Walter: None. Y. Dai: None. R.D. Shamburek: None. R.J. Brown: None. Insulin resistance (IR), a major risk factor for cardiovascular disease (CVD), leads to hyperglycemia and dyslipidemia. In obesity-associated IR, dyslipidemia attributed to increased stimulation of hepatic lipogenesis by insulin, whereas IR in glucoregulatory pathways leads to hyperglycemia. This dichotomy in insulin signaling pathways is termed selective IR. Lipodystrophy syndromes (LD) are rare conditions of severe, selective IR in which, like obesity, hyperinsulinemia with excess insulin receptor signaling is thought to cause high triglycerides (TG) & low HDL. By contrast, in the rare condition of insulin receptor pathogenic variants (INSR) (nonselective IR) lack of insulin signaling through its receptor despite hyperinsulinemia leads to low TG and high HDL. Rodents with hepatic INSR k/o have an atherogenic lipid profile, but the lipoprotein profile in humans with INSR is unknown. Factors influencing atherogenicity of lipoprotein particles, including cholesteryl ester transfer protein (CETP) activity and adiponectin, have not been explored in these human models of severe selective vs. non-selective IR. We characterized atherogenicity of lipoprotein particles and factors influencing these particles in subjects with LD matched 2:1 with subjects with INSR based on age and sex. All subjects had fasting lipoprotein profiles by NMR spectroscopy, apolipoproteins AI (ApoI) and B (ApoB), total adiponectin by ELISA, and CETP activity by fluorometric assay. Subjects with LD (N=24, 75% female; 8 generalized, 16 partial) had a more atherogenic lipoprotein profile vs INSR (N=12, 50% female; 6 homozygous, 6 heterozygous), with 45-fold higher large TRLP, 20-fold higher medium TRLP, 15-fold higher very small TRLP (representing IDL), 3-fold higher small LDLP, 4-fold lower large HDLP, 1.7—fold higher ApoB, and 1.4—fold lower ApoAI (p <0.05 for all). CETP activity was comparable in INSR and LD. Adiponectin was 5.9- fold lower in LD vs INSR (p <0.0001). Among all subjects, adiponectin was positively associated with HDL-C, large HDLP, HDL size, and ApoA-I (p=0.001), and negatively correlated with TRLP number & size, small LDLP, and very small TRLP (p<0.0001). Selective IR in LD is associated with an atherogenic lipoprotein particle profile. By contrast, non-selective IR in INSR is associated with an atheroprotective profile, which differs from rodent models. These divergent lipid phenotypes are not explained by different CETP activity. Although preclinical studies suggest that adiponectin promotes a favorable lipoprotein profile consistent with our observed associations, a causal link in humans is not supported by Mendelian randomization studies or rare ADIPOQ gene deletions. Overall, these findings suggest a key role of selective insulin resistance in the development of an atherogenic lipid profile. Further studies are needed to see if this profile leads to increased CVD risk. Presentation: Thursday, June 15, 2023

FRI181 Increased LDL Particle And Apolipoprotein B Concentrations In Patients With Nonfunctioning And Cortisol Secreting Adrenal Adenomas

Abstract Disclosure: R. Sandooja: None. A. Kittithaworn: None. P. Dogra: None. D. Gruppen: None. E. Atkinson: None. S. Achenbach: None. K. Yu: None. M. Connelly: None. V. Simha: None. R.P. Dullaart: None. I. Bancos: None. INTRODUCTION: Based on the spectrum of cortisol excess, adrenal adenomas are classified as adenomas with Cushing syndrome (CS), adenomas with mild autonomous cortisol secretion (MACS), and non-functioning adenomas (NFA). Patients with adrenal adenomas demonstrate a high prevalence of cardiovascular morbidity and mortality proportional to the degree of hypercortisolism. Evidence on whether dyslipidemia is a contributing cardiovascular risk factor in patients with NFA and MACS is discrepant. We aimed to characterize (apo) lipoprotein abnormalities across the spectrum of cortisol excess in patients with adenomas as compared to referent subjects. METHODS: Study Design: Single center cross sectional study between 01/2015 - 05/2021.Patients: Adults with NFA, MACS, or CS were consecutively enrolled. MACS was diagnosed based on post-dexamethasone cortisol >1.8 mcg/dL. Referent subjects were recruited from local population of patients with available abdominal imaging and no adrenal disorders. All subjects provided fasting ethylenediaminetetraacetic acid (EDTA) venous samples. Measurements: Nuclear magnetic resonance (NMR) spectroscopy measurements included total cholesterol (TC), low-density lipoprotein (LDL) and LDL particle concentrations, high-density lipoprotein (HDL) subclasses, apolipoprotein B (apo B), triglycerides (TG) and TG-rich lipoprotein concentrations. RESULTS: Participants included 101 patients with CS (median age 44 years, IQR 31-58, 85% women), 158 patients with MACS (median age 62 years, IQR 51-70, 62% women), 166 patients with NFA (median age 60 years, IQR 53--66, 63% women) and 136 referent subjects (median age 68 years, IQR 57-77, 52% women). Patients had higher BMI (32 vs 27 kg/m2), higher prevalence of hypertension (72% vs 46%) and diabetes mellitus (27% vs 9%) as compared to reference subjects. Statin therapy was used in 32% of patients and referent subjects. After adjusting for age, sex, BMI, smoking, diabetes, hypertension, and statin therapy, as compared to referent subjects, we found inconsistent increase in TC, LDL cholesterol and TG across CS, MACS and NFA and no consistent differences in LDL size, HDL variables, TG and TG-rich lipoproteins. However, when compared to referent subjects, LDL particle concentrations- CS(OR 1.78, 95% CI 1.33-2.39), MACS (OR 1.31, 95% CI 1.03-1.67), and NFA (OR 1.43, 95% CI 1.12-1.82) and Apo B levels were higher in all patient categories: CS (OR 2.26, 95% CI 1.57-3.24), MACS (OR 1.36 95% CI 1.00-1.85 ), and NFA (OR 1.54 95% CI 1.13-2.09). These differences were particularly evident in patients not on statin therapy. CONCLUSIONS: We found that LDL particle and Apo B concentrations were elevated in all patient groups, including those with MACS and NFA. In comparison to conventional lipid measurements, LDL particle and Apo B may serve as a better biomarker of the future cardiovascular risk in patients with adrenal adenomas. Presentation: Friday, June 16, 2023

Interaction of energy and sulfur microbial diet and smoking status with polygenic variants associated with lipoprotein metabolism.

Hypo-high-density lipoprotein cholesterolemia (hypo-HDL-C) contributes to the development of cardiovascular diseases. The hypothesis that the polygenic variants associated with hypo-HDL-C interact with lifestyle factors was examined in 58,701 middle-aged Korean adults who participated in the Korean Genome and Epidemiology Study (KoGES). Participants were categorized into the Low-HDL (case; n = 16,980) and Normal-HDL (n = 41,721) groups. The participants in the Low-HDL group were selected using the guideline-based cutoffs for hypo-HDL-C (<40 mg/dL for men and < 50 mg/dL for women) and included those taking medication for dyslipidemia. The genes associated with hypo-HDL-C were determined through a genome-wide association study (GWAS) in a city hospital-based cohort, and the results were validated in the Ansan/Anung study. The genetic variants for the single nucleotide polymorphism (SNP)-SNP interaction were selected using a generalized multifactor dimensionality reduction analysis, and the polygenic risk score (PRS) generated was evaluated for interaction with lifestyle parameters. The participants with hypo-HDL-C showed a 1.45 and 1.36-fold higher association with myocardial infarction and stroke, respectively. The High-PRS with four SNPs, namely ZPR1_rs3741297, CETP_rs708272, BUD13_rs180327, and ALDH1A2_rs588136, and that with the 11q23.3 haplotype were positively associated with hypo-HDL-C by about 3 times, which was a 2.4-fold higher association than the PRS of 24 SNP with p < 5×10-8. The risk alleles of CETP_rs708272 and ALDH1A2_rs588136 were linked to increased expression in the heart and decreased in the brain, respectively. The selected SNPs were linked to the reverse cholesterol transport pathway, triglyceride-rich lipoprotein particle remodeling pathway, cholesterol storage, and macrophage-derived foam cell differentiation regulation. The PRS of the 4-SNP model interacted with energy intake and smoking status, while that of the haplotype interacted with a glycemic index of the diet, sulfur microbial diet, and smoking status. Adults with a genetic risk for hypo-HDL-C need to modulate their diet and smoking status to reduce their risk.

Particle profiling of EV-lipoprotein mixtures by AFM nanomechanical imaging.

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.

Lipid carriers for mRNA delivery.

Messenger RNA (mRNA) is the template for protein biosynthesis and is emerging as an essential active molecule to combat various diseases, including viral infection and cancer. Especially, mRNA-based vaccines, as a new type of vaccine, have played a leading role in fighting against the current global pandemic of COVID-19. However, the inherent drawbacks, including large size, negative charge, and instability, hinder its use as a therapeutic agent. Lipid carriers are distinguishable and promising vehicles for mRNA delivery, owning the capacity to encapsulate and deliver negatively charged drugs to the targeted tissues and release cargoes at the desired time. Here, we first summarized the structure and properties of different lipid carriers, such as liposomes, liposome-like nanoparticles, solid lipid nanoparticles, lipid-polymer hybrid nanoparticles, nanoemulsions, exosomes and lipoprotein particles, and their applications in delivering mRNA. Then, the development of lipid-based formulations as vaccine delivery systems was discussed and highlighted. Recent advancements in the mRNA vaccine of COVID-19 were emphasized. Finally, we described our future vision and perspectives in this field.

Lipoprotein (a) in atherosclerosis: A bibliometric and visualization analysis

Abstract: Lipoprotein (a) [Lp (a)] is a complex polymorphic lipoprotein consisting of one low-density lipoprotein particle with one molecule of apolipoprotein B100 and another apolipoprotein (a) linked by a disulfide bond. In recent years, due to its causal role in premature atherosclerotic cardiovascular diseases and calcified aortic stenosis, Lp (a) has attracted more and more attention. Our study aimed to illustrate the trend of Lp (a) research in atherosclerosis (AS) through bibliometric analysis. The Science Citation Index-Expanded was used to locate Lp (a) and AS studies published between December 1, 2012 and December 1, 2022. VOSviewer and CiteSpace bibliometric software packages were used to analyze literature information. LP (a) has seen an overall increase in annual publications. The United States had the highest number of publications worldwide, with 192 publications. The University of California, San Diego, has contributed significantly to Lp (a) with 29 publications and led research collaboration. In the past few decades, there has been close collaboration between countries or regions, institutions, and authors. In addition, the European Heart Journal was the most cited, followed by the Journal of Lipid Research and AS with 2033, 1096, and 806 citations, respectively. Recent studies were on genes and lipid-lowering therapies. Our study comprehensively evaluated the research status and trends of Lp (a) in AS worldwide for the first time and provided a valuable reference for clinical researchers.

Women with gestational diabetes mellitus, controlled for plasma glucose level, exhibit maternal and fetal dyslipidaemia that may warrant treatment

AimsTo compare maternal and fetal cord plasma and lipoprotein triglyceride (TG) concentrations in women with Gestational Diabetes Mellitus (GDM), with hyperglycaemia and hypertriglyceridaemia, and healthy women. MethodsFasted maternal blood at 28.6 ± 3.4 (T1) and 36.2 ± 1.0 (T2) [mean ± S.D] weeks of gestation, and cord blood were collected. Plasma lipoprotein fractions underwent compositional analysis. ResultsPlasma glucose did not differ between GDM and healthy women. T1 maternal plasma TG (2.60 ± 0.89 mmol/l versus 1.71 ± 0.69 mmol/l) and plasma apolipoprotein B (1.30 ± 0.48 g/l versus 0.75 ± 0.40 g/l) were higher in GDM compared to healthy. Maternal plasma TG increased over gestation in both groups. T1 plasma VLDL total protein (38 ± 15 mg/dl versus 25 ± 11 mg/dl), total cholesterol (TC) (30 ± 14 mg/dl versus 16 ± 13 mg/dl) and phospholipid (PL) (43 ± 17 mg/dl versus 26 ± 16 mg/dl) were higher in GDM than healthy, and similarly for IDL, suggesting increased lipoprotein particle number. T1 VLDL-TG enrichment was higher in healthy and increased over gestation in GDM women but decreased in healthy. IDL-TG enrichment (TG/TC) increased over gestation in women with GDM and decreased in healthy. Cord blood VLDL, IDL and LDL from GDM had a two-fold higher TG enrichment than healthy pregnancy. ConclusionIncreased maternal lipoprotein number, but not TG enrichment, in GDM mothers may explain TG enrichment of cord lipoproteins.

Serum Activities of Paraoxonase 1 (PON1) in Predicting Liver Damage Among Patients Diagnosed With Hepatocellular Carcinoma: A Case-Control Study.

Introduction Hepatocellular carcinoma (HCC)is one of the most common cancers in the world and unless diagnosed timely has limited options for treatment. Paraoxonase(PON) isa glycosylated proteinthat has been implicated in antioxidant and other biochemical functions.Paraoxonase 1 (PON1)is an esterase associated with high-density lipoprotein (HDL) particles. The present study was carried out to assess the PON1 activity and compare it with the standard liver function tests (LFTs) in assessing the predictability of liver damage among patients diagnosed with HCC. Methods This case-control study was carried out in the Department of Biochemistry attached to Great Eastern Medical School and Hospital, Srikakulam, Andhra Pradesh. Serum PON1 activities and LFTs like total bilirubin, direct bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total protein, and albumin were estimated in 30 patients diagnosed with HCC and 30 healthy persons. All the parameters were estimated using standard biochemical methods.The data was analyzed using GraphPad Prism version 6.0 (GraphPad Software, Inc).A probability (p) value <0.05 was considered to be statistically significant. Receiver operating characteristic curve (ROC) analysis was performed to assess the area under the curve (AUC) for accuracy, sensitivity, specificity, and diagnostic efficiency. Results The serum activities of PON1 had identical sensitivity (70%) to albumin (70%) and were superior to other tested parameters. Additionally, PON1 activities showed lower specificity (86.67%) than the other tested parameters. ROC analysis showed increased diagnostic efficacy (DE) of PON1 (DE=78.3%; p<0.0001) when compared with total bilirubin (DE=76.6%; p=0.0039), direct bilirubin (DE=74.9%; p=0.04), ALT (DE=73.30%; p=0.0006), and total protein (DE=71.6%; p=0.0005). However, the DE of PON1 was comparable with AST (DE=81.60%; p<0.0001), ALP (DE=79.9%; p<0.0001), and albumin (DE=83.30%, p<0.0001). Conclusions Serum activities of PON1 could be used as a diagnostic marker for assessing liver damage among HCC patients.

A-202 Establishing of Lipid-Apolipoprotein Connectivity Map in Hyperglycemia

Abstract Background Hyperglycemia, a condition characterized by a significantly increased glucose level in the blood, can lead to insulin deficiency and pre-diabetes. Lack of insulin production also interferes with lipid metabolism and increases the risk for cardiovascular disease. Thus, diagnosis and treatment of hyperglycemic patients can benefit from advanced proteomics and lipidomics testing approaches. Methods Disease-focused serum samples from 50 specimens were collected and evaluated, including 29 specimens with a glucose level&amp;gt;180 mg/dL and 21 normal lipidemic specimens. The sample proteomics analysis was conducted with a Perfinity IDP workstation (Shimadzu Scientific, Columbia, MD, USA) using on-line protein digestion with an immobilized enzyme reactor (IMER) directly coupled to a HALO-C18 column (Advanced Materials Technology, Wilmington, DE, USA). Lipidomics data were collected with the Lipidyzer platform (AB SCIEX, Framingham, MA, USA). Statistical analysis was conducted using JMP software (SAS Institute, USA) and custom scripts. Results Using targeted lipidomics and proteomics platforms we identified 20 proteins and 575 lipid species across 10 lipid subclasses. We identified specific lipids and lipoproteins dysregulated in hyperglycemic specimens as compared to the normolipidemic samples and linked them to major metabolic pathways. Lipoprotein particles related-proteins apos A1/B/C2/C3/E and lecithin–cholesterol acyltransferase (LCAT) were significantly dysregulated and associated with changes in HDL-mediated lipid transport, very-low-density lipoprotein particle remodeling, acylglycerol homeostasis, and triglyceride homeostasis. Conclusion The conducted integrated analysis of lipid and protein concentrations in hyperglycemia specimens to provide new insights into molecular changes associated with hyperglycemia that may inform new diagnostic and therapeutic strategies. The network analysis can allow improved categorization of patients that can lead to more effective and individualized therapeutic intervention approaches. This study provides new experimental and informatics approaches to better assess underlying metabolic conditions and related diseases, including cardiovascular disease, diabetes, and metabolic syndrome. Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the CDC.

A-207 Analytical Validation of Helena SPIFE Touch to Determine ApoB-Containing Lipoproteins Lp(a)-P, LDL-P and VLDL-P

Abstract Background Elevated Lp(a) is an independent risk factor for atherosclerotic (ASCVD) events and associates with aortic stenosis. Accurate immunochemical measurement of Lp(a) is complicated by heterogeneity of Lp(a) molecular size. The objective of this study was to validate the analytical performance of an electrophoretic assay for measurement of ApoB-containing lipoprotein particles including Lp(a)-P, LDL-P, and VLDL-P and to compare results to other standard clinical methods. Method Lp(a)-P, LDL-P, and VLDL-P were quantified by electrophoretic serum separation followed by immuno-staining with an ApoB antibody using the SPIFE Touch (Helena Laboratories, Beaumont, TX). Stability was evaluated by comparison to results at time zero (n = 10). Precision was assessed by 5 measurements twice daily for five days. Limit of quantitation (LoQ) was determined by 20 replicate measurements. Linearity was assessed by mixing high and low concentrations at various ratios. Accuracy was assessed by split sample comparison (n = 126) with Helena Laboratories. Lp(a)-P and LDL-P results were compared to nuclear magnetic resonance (NMR) using a Bruker Ascend 600 NMR (Bruker, Billerica, MA) with AXINON software (numares, AG, Regensberg, Germany) (n = 114). Lp(a)-P is included with LDL-P by NMR. Therefore, the SPIFE Lp(a)-P + LDL-P was compared to NMR LDL-P. SPIFE Lp(a)-P was compared directly to Roche cobas c501 Lp(a) immunoassay calibrated to molar units (Roche Diagnostics, Indianapolis, IN) (n = 126). Results The analytical performance of Lp(a)-P, LDL-P, and VLDL-P measured on the SPIFE Touch met all acceptance criteria. Bias to the reference method was &amp;lt;20% with R2 &amp;gt; 0.98. All analytes were stable 14 days refrigerated and 14 days frozen. Lipoprotein particle measurements on SPIFE Touch correlated to those obtained by both NMR and Roche assays. Conclusion The analytical performance of the SPIFE Touch for measuring the concentration of ApoB-containing lipoproteins is acceptable. Further studies are needed to determine clinical implications and utility compared to NMR and Roche assays.

A-216 Measuring Inflammation and Cardiovascular Markers at Benchtop NMR using Diffusion and Relaxation Edited Experiments

Abstract Background Metabolic phenotyping is an established tool in systems medicine that captures a profile of one’s individual health status and reflects the interaction between genes and external stressors. It uses analytical platforms such as NMR or MS to acquire molecular profiles, and modelling to extract actionable knowledge. Applying these techniques to a cohort of Australians infected by SARS-CoV-2 revealed a strong alteration in regions of 1D NMR spectra associated with lipoproteins and glycoproteins, and referred as Supramolecular Phospholipid Composite SPC (δ = 3.2 ppm) and Glyc (δ = 2.07 ppm). The latter is an established marker of inflammation. These results were later confirmed using larger cohorts from Spain (n = 525) and the UK (n = 1022). The urgent need for very rapid testing, at the early stage of the pandemic, prompted the development of bespoke NMR experiments able to measure this lipoproteins/glycoproteins signature without requiring complex modelling. Methods Physico-chemical properties of lipoprotein particles, such as diffusion, transverse and longitudinal relaxation rates, differ from low molecular weight metabolites. Therefore, an edited experiment JEDI (PGPE) was designed that combines diffusion, relaxation and scalar coupling editing blocks to produce a lipoprotein profile devoid of chemical noise (overlapping peaks) and where peaks give quantitative results. Results The SPC peak was further broken down into 3 sub-regions that are related to the main subfraction HDL and LDL (choline headgroups of phospholipids). Interestingly the ratio of SPC3/SPC2 highly correlates with the Apo-B100/Apo-A1 ratio, an established cardiovascular risk marker that is increased in patients with COVID19. Longitudinal data suggest this latter remains elevated even 30 days after onset in some patients. Furthermore, the edited experiment performs equally well at low field using a benchtop NMR. Conclusion Translation of a spectral signature relevant for monitoring COVID patients from high to low field NMR is possible using sophisticated editing techniques.

Retardation of Aβ42 fibril formation by apolipoprotein A-I and recombinant HDL particles

The double nucleation mechanism of amyloid β (Aβ) peptide aggregation is retained from buffer to cerebrospinal fluid (CSF) but with reduced rate of all microscopic processes. Here, we used a bottom-up approach to identify retarding factors in CSF. We investigated the Aβ42 fibril formation as a function of time in the absence and presence of apolipoprotein A-I (ApoA-I), recombinant high-density lipoprotein (rHDL) particles, or lipid vesicles. A retardation was observed in the presence of ApoA-I or rHDL particles, most pronounced with ApoA-I, but not with lipid vesicles. Global kinetic analysis implies that rHDL interferes with secondary nucleation. The effect of ApoA-I could best be described as an interference with secondary and to a smaller extent primary nucleation. Using surface plasmon resonance and microfluidics diffusional sizing analyses, we find that both rHDL and ApoA-I interact with Aβ42 fibrils but not Aβ42 monomer, thus the effect on kinetics seems to involve interference with the catalytic surface for secondary nucleation. The Aβ42 fibrils were imaged using cryogenic-electron microscopy and found to be longer when formed in the presence of ApoA-I or rHDL, compared to formation in buffer. A retarding effect, as observed in CSF, could be replicated using a simpler system, from key components present in CSF but purified from a CSF-free host. However, the effect of CSF is stronger implying the presence of additional retarding factors.

Ti4+ functionalized β-cyclodextrin covalent organic framework as a new immobilized metal ion affinity chromatography platform for selective capture of phosphorylated peptides and exosomes.

A Ti4+ functionalized β-cyclodextrin covalent organic framework nanoparticle (named as β-CD-COF@Ti4+) was synthesized using a one-pot method successfully realizingthe enrichment of phosphorylated peptides and exosomes based on the immobilized metal ion affinity chromatography strategy. The functionalized β-CD-COF@Ti4+ exhibited superior performance on the enrichment of phosphopeptides, including high selectivity (1:1000), low detection limit (0.5 fmol), and loading capacity for phosphopeptides (100 mg·g-1). After treatment with β-CD-COF@Ti4+, 9 phosphopeptides from defatted milk, 29 phosphopeptides related to 23 phosphoproteins from normal group serum, and 24 phosphopeptides related to 22 phosphoproteins from the serum of uremia patients were captured. Through the analysis of Gene Ontology, the captured phosphoprotein is closely related to kidney disease, including lipoprotein metabolism, very-low-density lipoprotein particle, high-density lipoprotein particle, and lipid binding activity process. Furthermore, western blot verification showed that this nanoparticle could successfully capture exosomes from human serum. This study demonstrates great prospects for the enrichment of phosphopeptides and exosomes from actual bio-samples.

Immune capture and protein profiling of small extracellular vesicles from human plasma.

Extracellular vesicles (EVs), the key players in inter-cellular communication, are produced by all cell types and are present in all body fluids. Analysis of the proteome content is an important approach in structural and functional studies of these vesicles. EVs circulating in human plasma are heterogeneous in size, cellular origin, and functions. This heterogeneity and the potential presence of contamination with plasma components such as lipoprotein particles and soluble plasma proteins represent a challenge in profiling the proteome of EV subsets by mass spectrometry. An immunocapture strategy prior to mass spectrometry may be used to isolate a homogeneous subpopulation of small EVs (sEV) with a specific endocytic origin from plasma or other biofluids. Immunocapture selectively separates EV subpopulations in biofluids based on the presence of a unique protein carried on the vesicle surface. The advantages and disadvantages of EV immune capture as a preparative step for mass spectrometry are discussed.

Cardiometabolic risk factor clustering in persons with spinal cord injury: A principal component analysis approach

Context/Objective To identify cardiometabolic (CM) measurements that cluster to confer increased cardiovascular disease (CVD) risk using principal component analysis (PCA) in a cohort of chronic spinal cord injury (SCI) and healthy non-SCI individuals. Approach A cross-sectional study was performed in ninety-eight non-ambulatory men with chronic SCI and fifty-one healthy non-SCI individuals (ambulatory comparison group). Fasting blood samples were obtained for the following CM biomarkers: lipid, lipoprotein particle, fasting glucose and insulin concentrations, leptin, adiponectin, and markers of inflammation. Total and central adiposity [total body fat (TBF) percent and visceral adipose tissue (VAT) percent, respectively] were obtained by dual x-ray absorptiometry (DXA). A PCA was used to identify the CM outcome measurements that cluster to confer CVD risk in SCI and non-SCI cohorts. Results Using PCA, six factor-components (FC) were extracted, explaining 77% and 82% of the total variance in the SCI and non-SCI cohorts, respectively. In both groups, FC-1 was primarily composed of lipoprotein particle concentration variables. TBF and VAT were included in FC-2 in the SCI group, but not the non-SCI group. In the SCI cohort, logistic regression analysis results revealed that for every unit increase in the FC-1 standardized score generated from the statistical software during the PCA, there is a 216% increased risk of MetS (P = 0.001), a 209% increased risk of a 10-yr. FRS ≥ 10% (P = 0.001), and a 92% increase in the risk of HOMA2-IR ≥ 2.05 (P = 0.01). Conclusion Application of PCA identified 6-FC models for the SCI and non-SCI groups. The clustering of variables into the respective models varied considerably between the cohorts, indicating that CM outcomes may play a differential role on their conferring CVD-risk in individuals with chronic SCI.

Nuclear Magnetic Resonance–Based Metabolomics and Risk of CKD

Chronic kidney disease (CKD) leads to lipid and metabolic abnormalities, but a comprehensive investigation of lipids, lipoprotein particles, and circulating metabolites associated with the risk of CKD has been lacking. We examined the associations of nuclear magnetic resonance (NMR)-based metabolomics data with CKD risk in the UK Biobank study. Observational cohort study. A total of 91,532 participants in the UK Biobank Study without CKD and not receiving lipid-lowering therapy. Levels of metabolites including lipid concentration and composition within 14 lipoprotein subclasses, as well as other metabolic biomarkers were quantified via NMR spectroscopy. Incident CKD identified using ICD codes in any primary care data, hospital admission records, or death register records. Cox proportional hazards regression models were used to estimate hazard ratios and 95% confidence intervals. We identified 2,269 CKD cases over a median follow-up period of 13.1 years via linkage with the electronic health records. After adjusting for covariates and correcting for multiple testing, 90 of 142 biomarkers were significantly associated with incident CKD. In general, higher concentrations of very-low-density lipoprotein (VLDL) particles were associated with a higher risk of CKD whereas higher concentrations of high-density lipoprotein (HDL) particles were associated with a lower risk of CKD. Higher concentrations of cholesterol, phospholipids, and total lipids within VLDL were associated with a higher risk of CKD, whereas within HDL they were associated with a lower risk of CKD. Further, higher triglyceride levels within all lipoprotein subclasses, including all HDL particles, were associated with greater risk of CKD. We also identified that several amino acids, fatty acids, and inflammatory biomarkers were associated with risk of CKD. Potential underreporting of CKD cases because of case identification via electronic health records. Our findings highlight multiple known and novel pathways linking circulating metabolites to the risk of CKD. The relationship between individual lipoprotein particle subclasses and lipid-related traits and risk of chronic kidney disease (CKD) in general population is unclear. Using data from 91,532 participants in the UK Biobank, we evaluated the associations of metabolites measured using nuclear magnetic resonance testing with the risk of CKD. We identified that 90 out of 142 lipid biomarkers were significantly associated with incident CKD. We found that very-low-density lipoproteins, high-density lipoproteins, the lipid concentration and composition within these lipoproteins, triglycerides within all the lipoprotein subclasses, fatty acids, amino acids, and inflammation biomarkers were associated with CKD risk. These findings advance our knowledge about mechanistic pathways that may contribute to the development of CKD.

Present and Future of Dyslipidaemia Treatment-A Review.

One of the greatest burdens on the healthcare systems of modern civilization is cardiovascular diseases (CVDs). Therefore, the medical community is looking for ways to reduce the incidence of CVDs. Simple lifestyle changes from an unhealthy to a healthy lifestyle are the cornerstone of prevention, but other risk factors for cardiovascular disease are also being currently targeted, most notably dyslipidaemia. It is well known that lowering serum lipid levels, and in particular lowering elevated LDL-cholesterol, leads to a reduction in major cardiovascular events. Although the focus to date has been on LDL-cholesterol levels and lowering them with statin therapy, this is often not enough because of increased concentrations of other lipoprotein particles in the serum and residual cardiovascular risk. Since lowering LDL-cholesterol levels is successful in most cases, there has been a recent focus on lowering residual cardiovascular risk. In recent years, new therapeutic options have emerged that target triglyceride-rich lipoproteins, lipoprotein (a) and apolipoproteins C and B. The effects of these drugs on serious adverse cardiovascular events are not yet known, but recent studies with some of these drugs have shown significant results in lowering total lipid levels. The aim of this review is to present the current therapeutic options for the treatment of dyslipidaemia and to describe the newly approved drugs as well as the drugs that are still in development. Although at this stage we cannot say with certainty whether these agents will be approved and widely used, it is safe to say that our views on the treatment of dyslipidaemia are certainly changing.