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OBJECTIVE: The present study aims to evaluate the performance of the Diabetes Risk Index (DRI), a metabolomic index based on lipoprotein particles and branched chain amino acids, on the incidence of newly developed hypertension in a large community dwelling cohort.METHODS: The DRI was calculated by combining 6 lipoprotein parameters [sizes of very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), concentrations of large VLDL, small LDL, and large HDL particles], and the concentrations of valine and leucine. DRI scores were estimated in 4169 participants from the PREVEND prospective cohort. Cox proportional hazards regression was used to evaluate the association of DRI scores with incident hypertension.RESULTS: During a median follow-up of 8.6 years, 924 new hypertension cases were ascertained. In analyses adjusted for age and sex, there was a significant association between DRI and incident hypertension with a hazard ratio (HR) per 1 SD increase of 1.45 (95% CI 1.36,1.54; p < 0.001). After additional adjustment for traditional risk factors, the HR remained significant (HRadj 1.21, 95% CI 1.10, 1.33, p CONCLUSION: Higher DRI scores were associated with an increased risk of incident hypertension. Such association was independent of traditional clinical risk factors for hypertension.

Choline, a gut microbiome metabolite, is associated with cardiovascular risk and other chronic illnesses. The aim was to develop a high-throughput nuclear magnetic resonance (NMR)-based assay to measure choline on the Vantera® Clinical Analyzer.A non-negative deconvolution algorithm was developed to quantify choline. Assay performance was evaluated using CLSI guidelines.Deming regression analysis comparing choline concentrations by NMR and mass spectrometry (n = 28) exhibited a correlation coefficient of 0.998 (intercept = -9.216, slope = 1.057). The LOQ were determined to be 7.1 µmol/L in serum and 5.9 µmol/L in plasma. The coefficients of variation (%CV) for intra- and inter-assay precision ranged from 6.2 to 14.8% (serum) and 5.4-11.3% (plasma). Choline concentrations were lower in EDTA plasma by as much as 38% compared to serum, however, choline was less stable in serum compared to plasma. In a population of apparently healthy adults, the reference interval was7.1-20.0 µmol/L (serum) and5.9-13.1 µmol/L (plasma). Linearity was demonstrated well beyond these intervals. No interference was observed for a number of substances tested.The newly developed, high-throughput NMR-based assay exhibited good performance characteristics enabling quantification of choline in serum and plasma for clinical use.

Background & aims Lipoprotein Z (LP-Z) is an abnormal free cholesterol (FC)-enriched low density lipoprotein (LDL)-like particle discovered from patients with cholestatic liver disease. This study aims to define the diagnostic value of LP-Z in alcoholic hepatitis (AH) and interrogate the biology behind its formation. Approach & results We measured serum levels of LP-Z using nuclear magnetic resonance spectroscopy, a well-established clinical assay. Serum levels of LP-Z were significantly elevated in four AH cohorts compared to control groups, including heavy drinkers and cirrhosis. We defined a Z-index, calculated by the ratio of LP-Z to total apolipoprotein B-containing lipoproteins, representing the degree of deviation from normal very low density lipoprotein (VLDL) metabolism. A high Z-index was associated with 90-day mortality independent from the Model for End-Stage Liver Disease and provided added prognosticative value. Both a Z-index ≤0.6 and a decline of Z-index by ≥ 0.1 in two weeks predicted 90-day survival. RNAseq analyses of liver tissues demonstrated an inverse association in the expression of enzymes responsible for the extrahepatic conversion of VLDL to LDL and AH disease severity, which was further confirmed by the measurement of serum enzyme activity. To evaluate whether the FC in LP-Z could contribute to the pathogenesis of AH, we found significantly altered FC levels in liver explant of AH patients. Furthermore, FC in reconstituted LP-Z particles caused direct toxicity to human hepatocytes in a concentration-dependent manner, supporting a pathogenic role of FC in LP-Z. Conclusions Impaired lipoprotein metabolism in AH leads to the accumulation of LP-Z in the circulation, which is hepatotoxic from excessive FC. A Z-index ≤ 0.6 predicts 90-day survival independent from conventional biomarkers for disease prognostication.

BACKGROUND: Early assessment of insulin resistance may be a way of identifying patients at risk as well as monitoring treatments that increase insulin sensitivity and reduce the risk of developing type II diabetes mellitus (T2DM).OBJECTIVE: The objective of the study was to evaluate the ability of the Lipoprotein Insulin Resistance Index (LP-IR) to predict incident T2DM in a large cohort.METHODS: LP-IR scores were calculated using 6 lipoprotein particle concentrations and sizes measured by nuclear magnetic resonance spectroscopy. In total, 5977 nondiabetic men and women were included. Cox proportional hazards regression was used to evaluate the association of LP-IR scores with incident T2DM.RESULTS: LP-IR scores were closely associated with insulin resistance, assessed by homeostatic model assessment of insulin resistance (r = 0.51; P CONCLUSION: LP-IR may be a convenient way to assess insulin resistance and T2DM risk, as well as to monitor preventative treatments. (C) 2018 National Lipid Association. All rights reserved.

Objectives: Despite reports highlighting citrate association with different diseases, serum citrate is scarcely used for diagnosis. Existing methods to quantify citrate are limited by their complexity and practicality of implementation. A simple and rapid NMR-based method to measure circulating citrate is described here, and its analytical performance evaluated. Design: and Methods: Citrate was quantified from NMR spectra using a non-negative linear least squares deconvolution algorithm. The analytical characteristics of the assay were evaluated using CLSI guidelines. To determine if the assay has adequate sensitivity to measure clinically relevant concentrations of citrate, the assay was used to quantify citrate in apparently healthy adults (n ​= ​553), and in the general population (n ​= ​133,576). Results: The LOQ for the assay was determined to be 1.48 ​mg/dL. Linearity was demonstrated over a wide range of concentrations (1.40–4.46 ​mg/dL). Coefficients of variation (%CV) for intra- and inter-assay precision ranged from 5.8–9.3 and 5.2–9.6%, respectively. Substances tested did not elicit interference with assay results. Specimen type comparison revealed

Background In the failing heart, energy metabolism is shifted towards increased ketone body oxidation. Nevertheless, the association of beta-hydroxybutyrate (beta-OHB) with development of heart failure (HF) remains unclear. We investigated the association between plasma beta-OHB and the risk of HF in a prospective population-based cohort. Design Plasma beta-OHB concentrations were measured in 6134 participants of the PREVEND study. Risk of incident HF with reduced (HFrEF) or preserved (HFpEF) ejection fraction was estimated using multivariable-adjusted Cox regression models. Results During median follow-up for 8.2 years, 227 subjects were diagnosed with HF (137 with HFrEF; 90 with HFpEF). Cox regression analyses revealed a significant association of higher beta-OHB concentrations with incident HF (HR per 1 standard deviation increase, 1.40 (95% CI: 1.21-1.63; P

The aims were to optimize a nuclear magnetic resonance (NMR)-based assay for quantifying ionized or free magnesium and investigate its association with type 2 diabetes (T2D). A high-throughput, ionized magnesium assay was optimized and evaluated. Plasma magnesium was quantified, and associations with T2D were ascertained in Insulin Resistance Atherosclerosis Study (IRAS) participants. Coefficients of variation for the ionized magnesium assay ranged from 0.7–1.5% for intra-assay and 4.2–4.7% for inter-assay precision. In IRAS (n = 1342), ionized magnesium was significantly lower in subjects with prediabetes and T2D than in normoglycemic subjects, and lower in participants with T2D than those with prediabetes (p < 0.0001). Cross-sectional regression analyses revealed that magnesium was associated with T2D at baseline in models adjusted for multiple clinical risk factors (p = 0.032). This association appeared to be modified by sex, in such a way that the associations were present in women (OR = 0.54 (95% CI 0.37–0.79), p = 0.0015) and not in men (OR = 0.98 (95% CI 0.71–1.35), p = 0.90). Longitudinal regression analyses revealed an inverse association between magnesium and future T2D in the total population (p = 0.035) that was attenuated by LP-IR (p = 0.22). No interactions were detected between magnesium and age, race, BMI, glucose, insulin, triglycerides, or LPIR for the prospective association with future T2D. However, a significant interaction between magnesium and sex was present, now with a trend for an association in men (OR = 0.75 (95% CI 0.55–1.02), p = 0.065 and absence of an association in women (OR = 1.01 (0.76–1.33), p = 0.97). Conclusions: lower ionized magnesium, as measured by an NMR-based assay optimized for accuracy and precision, was associated cross-sectionally with T2D at baseline and longitudinally with incident T2D in IRAS.

Background Standard lipid panel assays employing chemical/enzymatic methods measure total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), from which are calculated estimates of low-density lipoprotein cholesterol (LDL-C). These lipid measures are used universally to guide management of atherosclerotic cardiovascular disease risk. Apolipoprotein B (apoB) is generally acknowledged to be superior to LDL-C for lipid-lowering therapeutic decision-making, but apoB immunoassays are performed relatively infrequently due to the added analytic cost. The aim of this study was to develop and validate the performance of a rapid, high-throughput, reagent-less assay producing an “Extended Lipid Panel” (ELP) that includes apoB, using the Vantera® nuclear magnetic resonance (NMR) analyzer platform already deployed clinically for lipoprotein particle and other testing. Methods Partial least squares regression models, using as input a defined region of proton NMR spectra of plasma or serum, were created to simultaneously quantify TC, TG, HDL-C, and apoB. Large training sets (n > ~ 1000) of patient sera analyzed independently for lipids and apoB by chemical methods were employed to ensure prediction models reflect the wide lipid compositional diversity of the population. The analytical performance of the NMR ELP assay was comprehensively evaluated. Results Excellent agreement was demonstrated between chemically-measured and ELP assay values of TC, TG, HDL-C and apoB with correlation coefficients ranging from 0.980 to 0.997. Within-run precision studies measured using low, medium, and high level serum pools gave coefficients of variation for the 4 analytes ranging from 1.0 to 3.8% for the low, 1.0 to 1.7% for the medium, and 0.9 to 1.3% for the high pools. Corresponding values for within-lab precision over 20 days were 1.4 to 3.6%, 1.2 to 2.3%, and 1.0 to 1.9%, respectively. Independent testing at three sites over 5 days produced highly consistent assay results. No major interference was observed from 38 endogenous or exogenous substances tested. Conclusions Extensive assay performance evaluations validate that the NMR ELP assay is efficient, robust, and substantially equivalent to standard chemistry assays for the clinical measurement of lipids and apoB. Routine reporting of apoB alongside standard lipid measures could facilitate more widespread utilization of apoB for clinical decision-making.

BackgroundLipoprotein Insulin Resistance Index (LP-IR) and Diabetes Risk Index are novel spectroscopic multimarkers of insulin resistance and type 2 diabetes risk. As the Studies of a Targeted Risk Reduction Intervention through Defined Exercise (STRRIDE) randomized trials have previously demonstrated the ability of exercise training to improve traditional markers of insulin action, the aim of this study was to examine the effects of exercise amount, intensity, and mode on LP-IR and the Diabetes Risk Index.MethodsA total of 503 adults with dyslipidemia [STRRIDE I (n = 194), STRRIDE AT/RT (n = 139)] or prediabetes [STRRIDE-PD (n = 170)] were randomized to control or one of 10 exercise interventions, ranging from doses of 8–23 kcal/kg/week; intensities of 50–75% V̇O2peak; and durations of 6–8 months. Two groups included resistance training and one included dietary intervention (7% weight loss goal). Fasting plasma samples were obtained at baseline and 16–24 h after the final exercise bout. LP-IR, the Diabetes Risk Index, and concentrations of the branched chain amino acids valine and leucine were determined using nuclear magnetic resonance spectroscopy. LP-IR and the Diabetes Risk Index scores range from 0–100 and 1–100, respectively (greater scores indicate greater risk). Paired t-tests determined significance within groups (p < 0.05).ResultsAfter training, six exercise groups significantly improved LP-IR (ranging from −4.4 ± 8.2 to −12.4 ± 14.1), and four exercise groups significantly improved the Diabetes Risk Index (ranging from −2.8 ± 8.2 to −8.3 ± 10.4). The most beneficial interventions for both LP-IR and the Diabetes Risk Index were low amount/moderate intensity aerobic, aerobic plus resistance, and aerobic plus diet.SummaryMultiple exercise interventions improved LP-IR and the Diabetes Risk Index. In those with dyslipidemia, adding resistance to aerobic training elicited a synergistic effect on insulin resistance and type 2 diabetes risk. In individuals with prediabetes, combining a dietary intervention and weight loss with aerobic training resulted in the most robust type 2 diabetes risk improvement.

Background: Lipoprotein particles with abnormal compositions, such as lipoprotein X (LP-X) and lipoprotein Z (LP-Z), have been described in cases of obstructive jaundice and cholestasis. The study objectives were to: (1) develop an NMR-based assay for quantification of plasma/serum LP-Z particles, (2) evaluate the assay performance, (3) isolate LP-Z particles and characterize them by lipidomic and proteomic analysis, and (4) quantify LP-Z in subjects with various liver diseases. Methods: Assay performance was assessed for linearity, sensitivity, and precision. Mass spectroscopy was used to characterize the protein and lipid content of isolated LP-Z particles. Results: The assay showed good linearity and precision (2.5–6.3%). Lipid analyses revealed that LP-Z particles exhibit lower cholesteryl esters and higher free cholesterol, bile acids, acylcarnitines, diacylglycerides, dihexosylceramides, lysophosphatidylcholines, phosphatidylcholines, triacylglycerides, and fatty acids than low-density lipoprotein (LDL) particles. A proteomic analysis revealed that LP-Z have one copy of apolipoprotein B per particle such as LDL, but less apolipoprotein (apo)A-I, apoC3, apoA-IV and apoC2 and more complement C3. LP-Z were not detected in healthy volunteers or subjects with primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, or type 2 diabetes. LP-Z were detected in some, but not all, subjects with hypertriglyceridemia, and were high in some subjects with alcoholic liver disease. Conclusions: LP-Z differ significantly in their lipid and protein content from LDL. Further studies are needed to fully understand the pathophysiological reason for the enhanced presence of LP-Z particles in patients with cholestasis and alcoholic liver disease.

Objective: Evaluate the ability of a newly developed diabetes risk score, the Diabetes Risk Index (DRI), to predict incident type 2 diabetes mellitus (T2D) in a large adult population. Methods: The DRI was developed by combining the Lipoprotein Insulin Resistance Index (LP-IR), calculated from 6 lipoprotein subspecies and size parameters, and the branched chain amino acids, valine and leucine, all of which have been shown previously to be associated with future T2D. DRI scores were calculated in a total of 6134 nondiabetic men and women in the Prevention of Renal and Vascular End-Stage Disease (PREVEND) Study. Cox proportional hazards regression was used to evaluate the association of DRI scores with incident T2D. Results: During a median follow-up of 8.5 years, 306 new T2D cases were ascertained. In analyses adjusted for age and sex, there was a significant association between DRI scores and incident T2D with the hazard ratio (HR) for the highest versus lowest quartile being 12.07 (95% confidence interval: 6.97&ndash, 20.89, p &lt, 0.001). After additional adjustment for body mass index (BMI), family history of T2D, alcohol consumption, diastolic blood pressure, total cholesterol, triglycerides, HDL cholesterol and HOMA-IR, the HR was attenuated but remained significant (HR 3.20 (1.73&ndash, 5.95), p = 0.001). Similar results were obtained when DRI was analyzed as HR per 1 SD increase (HR 1.37 (1.14&ndash, 1.65), p &lt, 0.001). The Kaplan&ndash, Meier plot demonstrated that patients in the highest quartile of DRI scores presented at higher risk (p-value for log-rank test &lt, 0.001). Conclusions: Higher DRI scores are associated with an increased risk of T2D. The association is independent of clinical risk factors for T2D including HOMA-IR, BMI and conventional lipids.

Objective: Examine the effects of amount, intensity, and mode of exercise training on type 2 diabetes (T2D) risk. Methods: A total of 531 men and women with dyslipidemia [STRRIDE I (n=222), STRRIDE AT/RT (n=142) or prediabetes [STRRIDE-PD (n=167)] were randomized to control group or one of 10 exercise interventions, ranging from doses of 8-22 kcal/kg/week; intensities of 50-75% V?O2peak; and duration of 6-9 months. Two groups included resistance training and one included dietary intervention (weight loss goal of 7%). Fasting plasma samples were obtained at baseline and 16-24 h after last exercise bout. The Diabetes Risk Index (DRI) and its components—Lipoprotein Insulin Resistance Index (LP-IR) and branched chain amino acids (BCAA) valine and leucine—were determined by NMR spectroscopy. DRI scores range from 1-100, the latter representing those at greatest T2D risk. Paired t-tests determined significance within groups (p Results: The inactive control group did not significantly change DRI score. After training, four of ten exercise groups significantly improved DRI scores, ranging from -2.8 ± 8.2 to -8.3 ± 10.4. In STRRIDE AT/RT, only the aerobic-plus-resistance group significantly improved DRI. In STRRIDE-PD, the moderate-intensity-plus-diet group had a greater DRI change than the other exercise-only moderate intensity groups. Reductions in DRI appeared to be driven largely by decreases in insulin resistance (LP-IR) but not dysmetabolism (BCAA), except for the moderate-intensity-plus-diet group, which exhibited a reduction in both. Conclusions: Multiple exercise interventions led to improvements in DRI scores. Results provide compelling evidence that adding resistance to aerobic training elicits a synergistic effect on insulin resistance and T2D risk. In individuals with prediabetes, incorporating dietary intervention with aerobic training results in the most robust improvement in T2D risk. Disclosure L.M. Ross: None. C.A. Slentz: None. I.Y. Shalaurova: None. J.D. Otvos: Employee; Self; LabCorp. M.A. Connelly: Employee; Self; LabCorp. C.W. Bales: None. J.A. Houmard: None. W.E. Kraus: None. Funding National Institutes of Health (R01HL057354, R01DK081559, T32HL007101)

Introduction: Lipoprotein Insulin Resistance Index (LP-IR) is a novel spectroscopic multimarker linked to future diabetes risk. We recently assessed changes in LP-IR across the three STRRIDE trials, where on average, STRRIDE exercise interventions improved LP-IR. In the present study, we sought to determine if there were effects of gender, race, and glucose tolerance on LP-IR responses across the STRRIDE trials. Methods: A total of 461 adults with dyslipidemia (STRRIDE I and STRRIDE AT/RT) or prediabetes (STRRIDE-PD) were randomized to one of 7 exercise interventions, ranging from doses of 8-22 kcal/kg/week (KKW); intensities of 50-75% VO 2peak ; and durations of 6-9 months. Six groups included aerobic exercise, two groups included resistance training, and one group included dietary intervention (weight loss goal of 7%). Fasting blood samples were obtained at both baseline and 16-24 h after the final exercise bout. In STRRIDE-PD only (n=165), subjects completed oral glucose tolerance tests and were categorized into normal (NGT) and impaired glucose tolerance (IGT) groups at baseline. NMR spectroscopy was performed at LabCorp to determine LP-IR score (comprised of six lipoprotein subclass and size parameters). LP-IR score ranges from 0 (most insulin sensitive) to 100 (most insulin resistant). Irrespective of intervention group, we assessed change in LP-IR in three stratified analyses: by gender, race, and baseline glucose tolerance category. Paired t-tests determined whether the post- minus pre- intervention change scores within each group were significant (p Results: At baseline, women had lower LP-IR scores compared to men (47.8 ± 22.3 vs 62.6 ± 21.5; p Conclusion: There were significant baseline differences in LP-IR among gender, racial, and glucose tolerance groups. However, after adjusting for these baseline differences, there were similar beneficial responses to exercise in this marker of insulin resistance.

Background: Quantifying mildly elevated ketone bodies is clinically and pathophysiologically relevant, especially in the context of disease states as well as for monitoring of various diets and exercise regimens. As an alternative assay for measuring ketone bodies in the clinical laboratory, a nuclear magnetic resonance (NMR) spectroscopy-based test was developed for quantification of &beta, hydroxybutyrate (&beta, HB), acetoacetate (AcAc) and acetone. Methods: The ketone body assay was evaluated for precision, linearity and stability and method comparisons were performed. In addition, plasma ketone bodies were measured in the Insulin Resistance Atherosclerosis Study (IRAS, n = 1198, 373 type 2 diabetes mellitus (T2DM) subjects). Results: &beta, HB and AcAc quantified using NMR and mass spectrometry and acetone quantified using NMR and gas chromatography/mass spectrometry were highly correlated (R2 = 0.996, 0.994, and 0.994 for &beta, HB, AcAc, acetone, respectively). Coefficients of variation (%CVs) for intra- and inter-assay precision ranged from 1.3% to 9.3%, 3.1% to 7.7%, and 3.8% to 9.1%, for &beta, HB, AcAc and acetone, respectively. In the IRAS, ketone bodies were elevated in subjects with T2DM versus non-diabetic individuals (p = 0.011 to &le, 0.001). Age- and sex-adjusted multivariable linear regression analysis revealed that total ketone bodies and &beta, HB were associated directly with free fatty acids (FFAs) and T2DM and inversely with triglycerides and insulin resistance as measured by the Lipoprotein Insulin Resistance Index. Conclusions: Concentrations of the three main ketone bodies can be determined by NMR with good clinical performance, are elevated in T2DM and are inversely associated with triglycerides and insulin resistance.

Nonalcoholic fatty liver disease (NAFLD) is a complex disease dictated by both genetic and environmental factors. While insulin resistance (IR) is a key pathogenic driver, two common genetic variants in patatin‐like phospholipase domain containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) also impart significant risk for disease progression. Traditional approaches to NAFLD risk stratification rely on biomarkers of fibrosis, an end result of disease progression. We hypothesized that by combining genetics and a novel measurement for IR we could predict disease progression by the NAFLD activity score (NAS) and histologic presence of significant fibrosis. A total of 177 patients with biopsy‐proven NAFLD were enrolled in this cross‐sectional study. PNPLA3 I148M and TM6SF2 E167K genotypes were determined by TaqMan assays. The enhanced lipoprotein IR index (eLP‐IR) was calculated from serum biomarkers using nuclear magnetic resonance (NMR) spectroscopy. Multivariate regression models were used to study the relationships between genetics, IR, and histologic features of NAFLD. In the multivariate analysis, the eLP‐IR was strongly associated with histologic features of NAFLD activity and hepatic fibrosis (P < 0.001 to 0.02) after adjustment for potential confounders. PNPLA3 148M and TM6SF2 E167K genotypes were significantly associated with steatosis (P = 0.003 and P = 0.02, respectively). A combination of the eLP‐IR and genetic score was able to predict the presence of NAS ≥3 with an area under the receiver operating characteristic curve (AUROC) of 0.74. Adding age to this model predicted stages 3‐4 liver fibrosis with an AUROC of 0.82. Conclusion: This proof‐of‐concept study supports the hypothesis that genetics and IR are major determinants of NAFLD severity and demonstrates the feasibility of a new risk stratification paradigm using exclusively pathogenic factors.

Objectives Plasma branched-chain amino acid (BCAA) levels, measured on nuclear magnetic resonance (NMR) metabolomics research platforms or by mass spectrometry, have been shown to be associated with type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). We developed a new test for quantification of BCAA on a clinical NMR analyzer and used this test to determine the clinical correlates of BCAA in 2 independent cohorts. Design and methods The performance of the NMR-based BCAA assay was evaluated. A method comparison study was performed with mass spectrometry (LC-MS/MS). Plasma BCAA were measured in the Insulin Resistance Atherosclerosis Study (IRAS, n = 1209; 376 T2DM subjects) and in a Groningen cohort (n = 123; 67 T2DM subjects). In addition, carotid intima media thickness (cIMT) was measured successfully in 119 subjects from the Groningen cohort. Results NMR-based BCAA assay results were linear over a range of concentrations. Coefficients of variation for inter- and intra-assay precision ranged from 1.8–6.0, 1.7–5.4, 4.4–9.1, and 8.8–21.3%, for total BCAA, valine , leucine , and isoleucine , respectively. BCAA quantified from the same samples using NMR and LC-MS/MS were highly correlated (R 2 = 0.97, 0.95 and 0.90 for valine, leucine and isoleucine). In both cohorts total and individual BCAA were elevated in T2DM (P = 0.01 to ≤0.001). Moreover, cIMT was associated with BCAA independent of age, sex, T2DM and metabolic syndrome (MetS) categorization or alternatively of individual MetS components. Conclusions BCAA levels, measured by NMR in the clinical laboratory, are elevated in T2DM and may be associated with cIMT, a proxy of subclinical atherosclerosis.

BACKGROUND: Trimethylamine-N-oxide (TMAO), an atherogenic metabolite species, has emerged as a possible new risk factor for cardiovascular disease. Animal studies have shown that circulating TMAO levels are regulated by genetic and environmental factors. However, large-scale human studies have failed to replicate the observed genetic associations, and epigenetic factors such as DNA methylation have never been examined in relation to TMAO levels. METHODS AND RESULTS: We used data from the family-based Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) to investigate the heritable determinants of plasma TMAO in humans. TMAO was not associated with other plasma markers of cardiovascular disease, e.g. lipids or inflammatory cytokines. We first estimated TMAO heritability at 27%, indicating a moderate genetic influence. We used 1000 Genomes imputed data (n=626) to estimate genome-wide associations with TMAO levels, adjusting for age, sex, family relationships, and study site. The genome-wide study yielded one significant hit at the genome-wide level, located in an intergenic region on chromosome 4. We subsequently quantified epigenome-wide DNA methylation using the Illumina Infinium array on CD4+ T-cells. We tested for association of methylation loci with circulating TMAO (n=847), adjusting for age, sex, family relationships, and study site as the genome-wide study plus principal components capturing CD4+ T-cell purity. Upon adjusting for multiple testing, none of the epigenetic findings were statistically significant. CONCLUSIONS: Our findings contribute to the growing body of evidence suggesting that neither genetic nor epigenetic factors play a critical role in establishing circulating TMAO levels in humans. This work was funded by the American Heart Association (14CRP18060003, PI: Aslibekyan) and the National Institutes of Health (R01HL104135, PI: Arnett). Sí

Background: Gut microbiota-related metabolites, trimethylamine-N-oxide (TMAO), choline, and betaine, have been shown to be associated with cardiovascular disease (CVD) risk. Moreover, lower plasma betaine concentrations have been reported in subjects with type 2 diabetes mellitus (T2DM). However, few studies have explored the association of betaine with incident T2DM, especially in the general population. The goals of this study were to evaluate the performance of a newly developed betaine assay and to prospectively explore the potential clinical associations of betaine and future risk of T2DM in a large population-based cohort. Methods: We developed a high-throughput, nuclear magnetic resonance (NMR) spectroscopy procedure for acquiring spectra that allow for the accurate quantification of plasma/serum betaine and TMAO. Assay performance for betaine quantification was assessed and Cox proportional hazards regression was employed to evaluate the association of betaine with incident T2DM in 4336 participants in the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study. Results: Betaine assay results were linear (y = 1.02X − 3.75) over a wide range of concentrations (26.0−1135 µM). The limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ) were 6.4, 8.9, and 13.2 µM, respectively. Coefficients of variation for intra- and inter-assay precision ranged from 1.5−4.3% and 2.5−5.5%, respectively. Deming regression analysis of results produced by NMR and liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS) revealed an R2 value of 0.94 (Y = 1.08x − 1.89) and a small bias for higher values by NMR. The reference interval, in a cohort of apparently healthy adult participants (n = 501), was determined to be 23.8 to 74.7 µM (mean of 42.9 ± 12.6 µM). In the PREVEND study (n = 4336, excluding subjects with T2DM at baseline), higher betaine was associated with older age and lower body mass index, total cholesterol, triglycerides, and hsCRP. During a median follow-up of 7.3 (interquartile range (IQR), 5.9−7.7) years, 224 new T2DM cases were ascertained. Cox proportional hazards regression models revealed that the highest tertile of betaine was associated with a lower incidence of T2DM. Hazard ratio (HR) for the crude model was 0.61 (95% CI: 0.44−0.85, p = 0.004). The association remained significant even after adjusting for multiple clinical covariates and T2DM risk factors, including fasting glucose. HR for the fully-adjusted model was 0.50 (95% CI: 0.32−0.80, p = 0.003). Conclusions: The newly developed NMR-based betaine assay exhibits performance characteristics that are consistent with usage in the clinical laboratory. Betaine levels may be useful for assessing the risk of future T2DM.

Introduction: Metabolic Vulnerability Index (MVX) is a novel spectroscopic multimarker of mortality risk. MVX reflects components of nutrition and systemic inflammation status and is hypothesized to enhance disease and mortality risk stratification. As Studies Targeting Risk Reduction Interventions through Defined Exercise (STRRIDE) has previously demonstrated the ability of aerobic exercise training to improve traditional markers of cardiometabolic health, we hypothesized that aerobic exercise intervention also would result in beneficial changes in the components of MVX. Methods: Participants (n=222, previously sedentary, overweight men and women with mild to moderate dyslipidemia from STRRIDE) were randomized to either a 6-month control group or to one of three supervised aerobic exercise groups for 8-months: 1) low amount moderate intensity (LM): 14 kcal/kg/wk at 40-55% VO 2peak ; 2) low amount vigorous intensity (LV): 14 kcal/kg/wk at 65-80% VO 2peak ; 3) high amount vigorous intensity (HV): 21 kcal/kg/wk at 65-80% VO 2peak . Blood samples were obtained after an overnight fast at both baseline and study completion (16-24 hr after the final training bout for exercisers). Nuclear magnetic resonance spectroscopy was performed on the Vantera ® clinical analyzer at LabCorp to determine MVX score. MVX has two components: inflammation index [INFX; combined concentrations of GlycA and small HDL particle subspecies (diameters < 9 nm)] and metabolic malnutrition index (MALNX; combined concentrations of total branched chain amino acids and ketone bodies). MVX, INFX, and MALNX are all scaled from 1 (lowest risk) to 100 (highest risk). Paired t-tests determined whether the post- minus pre- intervention change scores within each group were significant (p Results: The inactive control group did not exhibit significant changes in any of the multimarkers. After 8-months of exercise training, the LM group experienced significant decreases in all three scores: INFX (-2.1 ± 5.2), MALNX (-3.1 ± 11.3), and MVX (-3.9 ± 9.1). The LV group significantly decreased their INFX score (-1.5 ± 3.8), yet significantly increased MALNX (2.2 ± 6.8). The HV group significantly decreased their INFX (-1.6 ± 4.8) and MVX scores (-1.9 ± 5.6). For the LM group, the beneficial changes in MALNX and MVX were significantly greater than the LV group. Conclusion: Aerobic exercise improved components of the novel risk-prediction multimarker MVX, with the LM group displaying the best responses across all three scores. As ongoing research investigates the clinical utility of these mulitmarkers, this study provides evidence for the ability of exercise intervention to beneficially change MVX score.

Background: Low circulating magnesium (Mg) is associated with an increased risk of developing type 2 diabetes mellitus (T2DM). We aimed to study the performance of a nuclear magnetic resonance (NMR)-based assay that quantifies ionized Mg in EDTA plasma samples and prospectively investigate the association of Mg with the risk of T2DM. Methods: The analytic performance of an NMR-based assay for measuring plasma Mg was evaluated. We studied 5747 subjects free of T2DM at baseline in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study. Results: Passing&ndash, Bablok regression analysis, comparing NMR-measured ionized Mg with total Mg measured by the Roche colorimetric assay, produced a correlation of r = 0.90, with a slope of 1.08 (95% CI: 1.00&ndash, 1.13) and an intercept of 0.02 (95% CI: &minus, 0.02&ndash, 0.08). During a median follow-up period of 11.2 (IQR: 7.7&ndash, 12.0) years, 289 (5.0%) participants developed T2DM. The association of NMR-measured ionized Mg with T2DM risk was modified by sex (Pinteraction = 0.007). In women, we found an inverse association between Mg and the risk of developing T2DM, independent of adjustment for potential confounders (HR: 1.80, 95% CI: 1.20&ndash, 2.70). In men, we found no association between Mg and the risk of developing T2DM (HR: 0.90, 95%: 0.67&ndash, 1.21). Conclusion: Lower NMR-measured plasma ionized Mg was independently associated with a higher risk of developing T2DM in women, but not in men.

BACKGROUNDNuclear magnetic resonance (NMR) spectra of serum obtained under quantitative conditions for lipoprotein particle analyses contain additional signals that could potentially serve as useful clinical biomarkers. One of these signals that we named GlycA originates from a subset of glycan N-acetylglucosamine residues on enzymatically glycosylated acute-phase proteins. We hypothesized that the amplitude of the GlycA signal might provide a unique and convenient measure of systemic inflammation.METHODSWe developed a spectral deconvolution algorithm to quantify GlycA signal amplitudes from automated NMR LipoProfile® test spectra and assessed analytic precision and biological variability. Spectra of acute-phase glycoproteins and serum fractions were analyzed to probe the origins of the GlycA signal. GlycA concentrations obtained from archived NMR LipoProfile spectra of baseline plasma from 5537 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) were used to assess associations with demographic and laboratory parameters including measures of inflammation.RESULTSMajor acute-phase protein contributors to the serum GlycA signal are α1-acid glycoprotein, haptoglobin, α1-antitrypsin, α1-antichymotrypsin, and transferrin. GlycA concentrations were correlated with high-sensitivity C-reactive protein (hsCRP) (r = 0.56), fibrinogen (r = 0.46), and interleukin-6 (IL-6) (r = 0.35) (all P < 0.0001). Analytic imprecision was low (intra- and interassay CVs 1.9% and 2.6%, respectively) and intraindividual variability, assessed weekly for 5 weeks in 23 healthy volunteers, was 4.3%, lower than for hsCRP (29.2%), cholesterol (5.7%), and triglycerides (18.0%).CONCLUSIONSGlycA is a unique inflammatory biomarker with analytic and clinical attributes that may complement or provide advantages over existing clinical markers of systemic inflammation.

Objectives Nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to the measurement of high-density lipoprotein (HDL) particles, providing particle concentrations for total HDL particle number (HDL-P), HDL subclasses (small, medium, large) and weighted, average HDL size for many years. Key clinical studies have demonstrated that NMR-measured HDL-P was more strongly associated with measures of coronary artery disease and a better predictor of incident cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C). Recently, an NMR-based clinical analyzer, the Vantera®, was developed to allow lipoprotein measurements to be performed in the routine, clinical laboratory setting. The aim of this study was to evaluate and report the performance characteristics for HDL-P quantified on the Vantera® Clinical Analyzer. Design and methods Assay performance was evaluated according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In order to ensure that quantification of HDL-P on the Vantera® Clinical Analyzer was similar to the well-characterized HDL-P assay on the NMR profiler, a method comparison was performed. Results The within-run and within-lab imprecision ranged from 2.0% to 3.9%. Linearity was established within the range of 10.0 to 65.0 μmol/L. The reference intervals were different between men (22.0 to 46.0 μmol/L) and women (26.7 to 52.9 μmol/L). HDL-P concentrations between two NMR platforms, Vantera® Clinical Analyzer and NMR Profiler, demonstrated excellent correlation ( R 2 = 0.98). Conclusions The performance characteristics, as well as the primary tube sampling procedure for specimen analysis on the Vantera® Clinical Analyzer, suggest that the HDL-P assay is suitable for routine clinical applications.

Background GlycA is a novel spectroscopic marker of systemic inflammation with low intra-individual variability and other attributes favoring its clinical use in patients with chronic inflammatory and autoimmune diseases. GlycA is unique in its composite nature, reflecting both increased glycan complexity and circulating acute phase protein levels during local and systemic inflammation. Recent studies of GlycA from cross-sectional, observational and interventional studies have been highly informative, demonstrating that GlycA is elevated in acute and chronic inflammation, predicts death in healthy individuals and is associated with disease severity in patients with chronic inflammatory diseases such as rheumatoid arthritis, psoriasis and lupus. Moreover, following treatment with biological therapy in psoriasis, reduction in skin disease severity was accompanied by a decrease in GlycA levels and improvement in vascular inflammation. Conclusions Collectively, these findings suggest GlycA is a marker that tracks systemic inflammation and subclinical vascular inflammation. However, larger prospective studies and randomized trials are necessary in order to assess the impact of novel therapies on GlycA in patients with chronic inflammatory conditions, which may be concomitant with cardiovascular benefits.

Background: Lipoprotein particle sizes and concentrations are characteristically altered in patients with insulin resistance (IR) or type 2 diabetes mellitus (T2DM). This study assessed the ability of an IR score, based on nuclear magnetic resonance (NMR)-derived lipoprotein information, to detect IR in otherwise healthy individuals.Lipoprotein subclass and size information were evaluated for strength of association with IR, as measured by homeostasis model assessment of insulin resistance (HOMA-IR) in the Multi-Ethnic Study of Atherosclerosis (MESA). To increase the likelihood of identifying subjects with IR, six lipoprotein measures were combined into a single algorithm. The resulting assay [Lipoprotein Insulin Resistance Index (LP-IR)] was developed using HOMA-IR in 4972 nondiabetic subjects from MESA and verified independently using glucose disposal rates (GDRs) measured during hyperinsulinemic-euglycemic clamps in 56 insulin-sensitive, 46 insulin-resistant, and 46 untreated subjects with T2DM.LP-IR exhibited stronger associations with HOMA-IR (r=0.51) and GDR (r=-0.53) than each of the individual lipoprotein parameters as well as the triglycerides/high-density lipoprotein cholesterol (TGs/HDL-C) ratio (r=0.41 and -0.44, respectively). In MESA, associations between the LP-IR score and HOMA-IR were strong in men (r=0.51), women (r=0.52), European Americans (r=0.58), African Americans (r=0.48), Chinese Americans (r=0.49), and Hispanic Americans (r=0.45). When LP-IR was categorized by HOMA-IR and either body mass index (BMI) or fasting plasma glucose (FPG), subgroups were revealed whose LP-IR scores were high (≥ 50), despite having normal BMIs (24 kg/m(2)) or FPG (100 mg/dL).LP-IR scores had strong associations with multiple measures, HOMA-IR, and GDR, the former being more reflective of hepatic and the latter of peripheral insulin sensitivity, and may represent a simple means to identify individuals with IR.

The amount of cholesterol per low-density lipoprotein (LDL) particle is variable and related in part to particle size, with smaller particles carrying less cholesterol. This variability causes concentrations of LDL cholesterol (LDL-C) and LDL particles (LDL-P) to be discordant in many individuals.LDL-P measured by nuclear magnetic resonance spectroscopy, calculated LDL-C, and carotid intima-media thickness (IMT) were assessed at baseline in the Multi-Ethnic Study of Atherosclerosis, a community-based cohort of 6814 persons free of clinical cardiovascular disease (CVD) at entry and followed for CVD events (n = 319 during 5.5-year follow-up). Discordance, defined as values of LDL-P and LDL-C differing by ≥ 12 percentile units to give equal-sized concordant and discordant subgroups, was related to CVD events and to carotid IMT in models predicting outcomes for a 1 SD difference in LDL-C or LDL-P, adjusted for age, gender, and race.LDL-C and LDL-P were associated with incident CVD overall: hazard ratios (HR 1.20, 95% CI [CI] 1.08-1.34; and 1.32, 95% CI 1.19-1.47, respectively, but for those with discordant levels, only LDL-P was associated with incident CVD (HR 1.45, 95% CI 1.19-1.78; LDL-C HR 1.07, 95% CI 0.88-1.30). IMT also tracked with LDL-P rather than LDL-C, ie, adjusted mean IMT of 958, 932, and 917 microm in the LDL-PLDL-C discordant, concordant, and LDL-PLDL-C discordant subgroups, respectively, with the difference persisting after adjustment for LDL-C (P = .002) but not LDL-P (P = .60).For individuals with discordant LDL-C and LDL-P levels, the LDL-attributable atherosclerotic risk is better indicated by LDL-P.

Great advances are being made at the mechanistic level in the understanding of the structural and functional diversity of high-density lipoprotein (HDL). HDL particle subspecies of different sizes are now known to differ in the protein and lipid cargo they transport, conferring on them the ability to perform different functions that in aggregate would be expected to provide protection against the development of atherosclerosis and its downstream clinical consequences. Exacerbating what is already a very complex system is the finding that inflammation, via alteration of the proteomic and lipidomic composition of HDL subspecies, can modulate at least some of their functional activities. In contrast to the progress being made at the mechanistic level, HDL epidemiologic research has lagged behind, largely because the simple HDL biomarkers used (mainly just HDL cholesterol) lack the needed complexity. To address this deficiency, analyses will need to use multiple HDL subspecies and be conducted in such a way as to eliminate potential sources of confounding. To help account for the modulating influence of inflammation, effective use must also be made of inflammatory biomarkers including searching systematically for HDL-inflammation interactions. Using nuclear magnetic resonance (NMR)-measured HDL subclass data and a novel NMR-derived inflammatory biomarker, GlycA, we offer a case study example of the type of analytic approach considered necessary to advance HDL epidemiologic understanding.

Metabolomic studies have shown several serum or plasma biomarkers are raised in subjects with insulin resistance and type 2 diabetes (T2D). In addition, several of these biomarkers, including various lipoprotein parameters, markers of inflammation and branched chain amino acids have been shown to be associated with new-onset T2D. A recently developed clinical Nuclear Magnetic Resonance (NMR) instrument the Vantera® Clinical Analyzer has the ability to measure these biomarkers in a single serum sample. Combining these NMRmeasured biomarkers of future diabetes risk into a single algorithm called the Diabetes Risk Index or DRI, allows better diabetes prediction than any one of the individual biomarkers. This review summarizes the data suggesting that DRI may be a simple, inexpensive way to alert patients of their heightened risk of developing diabetes before they become prediabetic, allowing time for effective lifestyle modification to theoretically prevent, not just delay, onset of the disease.

Background The Vantera Clinical Analyzer was developed to enable fully-automated, high-throughput nuclear magnetic resonance (NMR) spectroscopy measurements in a clinical laboratory setting. NMR-measured low-density lipoprotein particle number (LDL-P) has been shown to be more strongly associated with cardiovascular disease outcomes than LDL cholesterol (LDL-C) in individuals for whom these alternate measures of LDL are discordant. Objective The aim of this study was to assess the analytical performance of the LDL-P assay on the Vantera Clinical Analyzer as per Clinical Laboratory Standards Institute (CLSI) guidelines. Results Sensitivity and linearity were established within the range of 300–3500 nmol/L. For serum pools containing low, medium and high levels of LDL-P, the inter-assay, intra-assay precision and repeatability gave coefficients of variation (CVs) between 2.6 and 5.8%. The reference interval was determined to be 457–2282 nmol/L and the assay was compatible with multiple specimen collection tubes. Of 30 substances tested, only 2 exhibited the potential for assay interference. Moreover, the LDL-P results from samples run on two NMR platforms, Vantera Clinical Analyzer and NMR Profiler, showed excellent correlation (R 2 = 0.96). Conclusions The performance characteristics suggest that the LDL-P assay is suitable for routine testing in the clinical laboratory on the Vantera Clinical Analyzer, the first automated NMR platform that supports NMR-based clinical assays.

Background— Changes in conventional lipid risk factors with gemfibrozil treatment only partially explain the reductions in coronary heart disease (CHD) events experienced by men in the Veterans Affairs High-Density Lipoprotein Intervention Trial (VA-HIT). We examined whether measurement of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particle subclasses provides additional information relative to CHD risk reduction. Methods and Results— This is a prospective nested case-control study of 364 men with a new CHD event (nonfatal myocardial infarction or cardiac death) during a 5.1-year (median) follow-up and 697 age-matched controls. Nuclear magnetic resonance (NMR) spectroscopy was used to quantify levels of LDL and HDL particle subclasses and mean particle sizes in plasma obtained at baseline and after 7 months of treatment with gemfibrozil or placebo. Odds ratios for a 1-SD increment of each lipoprotein variable were calculated with adjusted logistic regression models. Gemfibrozil treatment increased LDL size and lowered numbers of LDL particles (−5%) while raising numbers of HDL particles (10%) and small HDL subclass particles (21%). Concentrations of these LDL and HDL particles achieved with gemfibrozil were significant, independent predictors of new CHD events. For total LDL and HDL particles, odds ratios predicting CHD benefit were 1.28 (95% CI, 1.12 to 1.47) and 0.71 (95% CI, 0.61 to 0.81), respectively. Mean LDL and HDL particle sizes were not associated with CHD events. Conclusions— The effects of gemfibrozil on NMR-measured LDL and HDL particle subclasses, which are not reflected by conventional lipoprotein cholesterol measures, help to explain the demonstrated benefit of this therapy in patients with low HDL cholesterol.

Background: The sex differential in coronary heart disease (CHD) risk, which is not explained by male/female differences in lipid and lipoprotein concentrations, narrows with age. We examined whether this differential CHD risk might, in part, be attributable to the sizes of lipoprotein particles or concentrations of lipoprotein subclasses.Methods: We analyzed frozen plasma samples from 1574 men and 1692 women from exam cycle 4 (1988–1990) of the Framingham Offspring Study. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the subclass concentrations and mean sizes of VLDL, LDL, and HDL particles. Concentrations of lipids and apolipoproteins were measured by standard chemical methods.Results: In addition to the expected sex differences in concentrations of triglycerides, LDL-cholesterol, and HDL-cholesterol, women also had a lower-risk subclass profile consisting of larger LDL (0.4 nm) and HDL (0.5 nm) particles. The sex difference was most pronounced for HDL, with women having a twofold higher (8 vs 4 μmol/L) concentration of large HDL particles than men. Furthermore, similar to the narrowing of the sex difference in CHD risk with age, the observed male/female difference in HDL particle size also decreased with age. Although lipoprotein particle sizes were highly correlated with lipid and lipoprotein concentrations, the sex differences in the mean sizes of lipoprotein particles persisted (P Conclusions: Women have a less atherogenic subclass profile than men, even after accounting for differences in lipid concentrations.

Lipoprotein subclass analyses may facilitate coronary heart disease (CHD) risk stratification and provide insight into the cardioprotective benefits of statins (3-hydroxymethylglutaryl-coenzyme A reductase inhibitors). This study evaluated the influence of pravastatin on lipoprotein subclass profiles to determine whether subjects with predominantly large LDL (LDL size >20.5 nm) or small LDL (LDL size < or =20.5 nm) at baseline differ in responsiveness to drug treatment. Frozen plasma specimens were analyzed from a subset of participants in the Pravastatin Limitation of Atherosclerosis in the Coronaries (PLAC-I) trial at baseline and after treatment for 6 months with pravastatin (n=154) or placebo (n=138). Lipids were measured by standard chemical methods and lipoprotein subclasses by nuclear magnetic resonance (NMR) spectroscopy. Pravastatin-induced changes in lipid levels were similar in subjects with large or small LDL at baseline. Levels of the most abundant LDL subclass were preferentially lowered by pravastatin, resulting in an increase in average LDL size for those with a predominance of small LDL. High-risk CHD subjects with small LDL particles gain at least as much pharmacological benefit from pravastatin as those with large LDL, as evidenced by reductions in the numbers of total and small LDL particles, and increases in average LDL and HDL particle size.

Background Glycosylation patterns of serum proteins, such as α1-acid glycoprotein, are modified during an acute phase reaction. The response of acute Kawasaki disease (KD) patients to IVIG treatment has been linked to sialic acid levels on native IgG, suggesting that protein glycosylation patterns vary during the immune response in acute KD. Additionally, the distribution and function of lipoprotein particles are altered during inflammation. Therefore, the aim of this study was to explore the potential for GlycA, a marker of protein glycosylation, and the lipoprotein particle profile to distinguish pediatric patients with acute KD from those with other febrile illnesses. Methods Nuclear magnetic resonance was used to quantify GlycA and lipoprotein particle classes and subclasses in pediatric subjects with acute KD (n = 75), post-treatment subacute (n = 36) and convalescent (n = 63) KD, as well as febrile controls (n = 48), and age-similar healthy controls (n = 48). Results GlycA was elevated in acute KD subjects compared to febrile controls with bacterial or viral infections, IVIG-treated subacute and convalescent KD subjects, and healthy children (P