Elias J. Jeyarajah

Relation of Lipoprotein Subclasses as Measured by Proton Nuclear Magnetic Resonance Spectroscopy to Coronary Artery Disease

Although each of the major lipoprotein fractions is composed of various subclasses that may differ in atherogenicity, the importance of this heterogeneity has been difficult to ascertain owing to the labor-intensive nature of subclass measurement methods. We have recently developed a procedure, using proton nuclear magnetic resonance (NMR) spectroscopy, to simultaneously quantify levels of subclasses of very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins; subclass distributions determined with this method agree well with those derived by gradient gel electrophoresis. The objective of the current study of 158 men was to examine whether NMR-derived lipoprotein subclass levels improve the prediction of arteriographically documented coronary artery disease (CAD) when levels of lipids and lipoproteins are known. We found that a global measure of CAD severity was positively associated with levels of large VLDL and small HDL particles and inversely associated with intermediate size HDL particles; these associations were independent of age and standard lipid measurements. At comparable lipid and lipoprotein levels, for example, men with relatively high (higher than the median) levels of either small HDL or large VLDL particles were three to four times more likely to have extensive CAD than were the other men; the 27 men with high levels of both large VLDL and small HDL were 15 times more likely to have extensive CAD than were men with low levels. In contrast, adjustment for levels of triglycerides or HDL cholesterol greatly reduced the relation of small LDL particles to CAD. These findings suggest that large VLDL and small HDL particles may play important roles in the development of occlusive disease and that their measurement, which is not possible with routine lipid testing, may lead to more accurate risk assessment.

NMR measurement of LDL particle number using the Vantera® Clinical Analyzer

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.

Incubation of MDCO-216 (ApoA-IMilano/POPC) with Human Serum Potentiates ABCA1-Mediated Cholesterol Efflux Capacity, Generates New Prebeta-1 HDL, and Causes an Increase in HDL Size

MDCO-216 is a complex of dimeric ApoA-IMilano and palmitoyl oleoyl phosphatidylcholine (POPC), previously shown to reduce atherosclerotic plaque burden. Here we studied the effect of incubation of human plasma or serum with MDCO-216 on cholesterol efflux capacity from J774 cells, on prebeta-1 high density lipoprotein (prebeta-1 HDL) and on HDL size assessed by proton nuclear magnetic resonance (1H-NMR). MDCO-216 incubated in buffer containing 4% human serum albumin stimulated both ABCA1-mediated efflux and ABCA1-independent cholesterol efflux from J774 macrophages. When incubated with human serum a dose- and time-dependent synergistic increase of the ABCA1-mediated efflux capacity were observed. Using a commercially available ELISA for prebeta-1 HDL, MDCO-216 as such was poorly detected (12–15% of nominal amount of protein). Prebeta-1 HDL was rapidly lost when human plasma alone is incubated at 37°C. In contrast, incubation of human plasma with MDCO-216 at 37°C produced a large amount of new prebeta-1 HDL. Native 2D electrophoresis followed by immunoblotting with an apoA-I antibody, which also detects ApoA-I Milano, confirmed the increase in prebeta-1 HDL upon incubation at 37°C. With the increase of prebeta-1 HDL, the concomitant disappearance of the small alpha-3 and alpha-4 HDL and MDCO-216 and an increase in the large alpha-1 and alpha-2 HDL were observed. Immunoblotting with Mab 17F3 specific for ApoA-I Milano showed the appearance of ApoA-I Milano in alpha-1 and alpha-2, but not in prebeta-1 HDL. 1H-NMR analysis of plasma incubated with MDCO-216 confirmed rapid disappearance of small-sized HDL particles and increase of medium- and large-sized HDL particles accompanied with a decrease in total HDL particle number. In conclusion, incubation of human plasma or serum with MDCO-216 strongly enhanced ABCA1-mediated cholesterol efflux, caused a strong increase of prebeta-1 HDL, and drastically changed the distribution of HDL subpopulations. Overall, the results are in line with the hypothesis that MDCO-216 fuses with small alpha-migrating HDL particles forming larger particles containing both apoA-I WT and ApoA-I Milano, meanwhile liberating the endogenous wild-type apoA-I which enriches prebeta-1 HDL subpopulation.

HDL particle number measured on the Vantera®, the first clinical NMR analyzer.

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.

High-Density Lipoprotein Subfractions and Cholesterol Efflux Capacities After Infusion of MDCO-216 (Apolipoprotein A-IMilano/Palmitoyl-Oleoyl-Phosphatidylcholine) in Healthy Volunteers and Stable Coronary Artery Disease Patients

Objective— To determine effects of single ascending doses of MDCO-216 on high-density lipoprotein (HDL) subfractions in relation to changes in cholesterol efflux capacity in healthy volunteers and in patients with stable angina pectoris. Approach and Results— Doses of 5- (in volunteers only), 10-, 20-, 30-, and 40-mg/kg MDCO-216 were infused during 2 hours, and plasma and serum were collected during 30 days. Plasma levels of HDL subfractions were assessed by 2-dimensional gel electrophoresis, immunoblotting, and image analysis. Lipoprotein particle concentrations and sizes were also assessed by proton nuclear magnetic resonance (1H-NMR). There was a rapid dose-dependent increase of total apolipoprotein A-I (apoA-I) in pre-&bgr;1, &agr;-1, and &agr;-2 HDL levels and decrease in &agr;-3 and &agr;-4 HDL. Using a selective antibody apoA-IMilano was detected in the large &agr;-1 and &agr;-2 HDL on all doses and at each time point. ApoA-IMilano was also detected at the &agr;-4 position but only at high doses. 1H-NMR analysis similarly showed a rapid and dose-dependent shift from small- to large-sized HDL particles. The increase of basal and ATP-binding cassette transporter A1–mediated efflux capacities reported previously correlated strongly and independently with the increase in pre-&bgr;1-HDL and &agr;-1 HDL, but not with that in &agr;-2 HDL. Conclusions— On infusion, MDCO-216 rapidly eliminates small HDL and leads to formation of &agr;-1 and &agr;-2 HDL containing both wild-type apoA-I and apoA-IMilano. In this process, endogenous apoA-I is liberated appearing as pre-&bgr;1-HDL. In addition to pre-&bgr;1-HDL, the newly formed &agr;-1 HDL particle containing apoA-I Milano may have a direct effect on cholesterol efflux capacity.

TMAO is Associated with Mortality: Impact of Modestly Impaired Renal Function

Trimethylamine-N-Oxide (TMAO) is a microbiome-related metabolite that is cleared by the kidney and linked to renal function. We explored the relationship between TMAO and all-cause mortality, and determined whether this association was modified by renal function. A prospective study was performed among PREVEND participants to examine associations of plasma TMAO with all-cause mortality. After median follow-up of 8.3 years in 5,469 participants, 322 subjects died. TMAO was positively associated with age, body mass index, type 2 diabetes mellitus and inversely with estimated glomerular filtration rate (eGFRcreatcysC)(all P < 0.001). Subjects in the highest versus lowest TMAO quartile had a crude 1.86-fold higher mortality risk (Ptrend < 0.001). After adjustment for several risk factors, TMAO remained associated with all-cause mortality [HR:1.36 (95% CI, 0.97–1.91),Ptrend = 0.016]. This association was lost after further adjustment for urinary albumin excretion and eGFR [HR:1.15 (95% CI, 0.81–1.64),Ptrend = 0.22]. The association of TMAO with mortality was modified by eGFR in crude and age- and sex-adjusted analyses (interaction P = 0.002). When participants were stratified by renal function (eGFR < vs. ≥90 mL/min/1.73 m2), TMAO was associated with all-cause mortality only in subjects with eGFR <90 mL/min/1.73 m2 [adjusted HR:1.18 (95% CI, 1.02–1.36),P = 0.023]. In conclusion, TMAO is associated with all-cause mortality, particularly in subjects with eGFR <90 mL/min/1.73 m2.

Genome- and CD4+ T-cell methylome-wide association study of circulating trimethylamine-N-oxide in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN)

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.

Plasma phospholipid transfer protein activity is inversely associated with betaine in diabetic and non-diabetic subjects.

BackgroundThe choline metabolite, betaine, plays a role in lipid metabolism, and may predict the development of cardiovascular disease and type 2 diabetes mellitus (T2DM). Phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) require phosphatidylcholine as substrate, raising the possibility that there is an intricate relationship of these protein factors with choline metabolism. Here we determined the relationships of PLTP and LCAT activity with betaine in subjects with and without T2DM.MethodsPlasma betaine (nuclear magnetic resonance spectroscopy), PLTP activity (liposome-vesicle HDL system), LCAT activity (exogenous substrate assay) and (apo)lipoproteins were measured in 65 type 2 diabetic (T2DM) and in 55 non-diabetic subjects.ResultsPLTP and LCAT activity were elevated in T2DM (p < 0.05), whereas the difference in betaine was not significant. In age-, sex- and diabetes status-controlled correlation analysis, betaine was inversely correlated with triglycerides and positively with HDL cholesterol (p < 0.05 to 0.01). PLTP and LCAT activity were positively correlated with triglycerides and inversely with HDL cholesterol (p < 0.05 to 0.001). PLTP (r = −0.245, p = 0.006) and LCAT activity (r = −0.195, p = 0.035) were correlated inversely with betaine. The inverse association of PLTP activity with betaine remained significant after additional adjustment for body mass index and lipoprotein variables (β = −0.179, p = 0.034), whereas its association with LCAT activity lost significance (β = −0.056, p = 0.44).ConclusionsBetaine may influence lipoprotein metabolism via an effect on PLTP activity.

NMR quantification of trimethylamine-N-oxide in human serum and plasma in the clinical laboratory setting

BACKGROUND AND OBJECTIVES Trimethylamine-N-oxide (TMAO) produced by gut microbiota metabolism of dietary choline and carnitine has been shown to be associated with increased risk of cardiovascular disease (CVD) and to provide incremental clinical prognostic utility beyond traditional risk factors for assessing a patients CVD risk. The aim of this study was to develop an automated nuclear magnetic resonance (NMR) spectroscopy assay for quantification of TMAO concentration in serum and plasma using a high-throughput NMR clinical analyzer. METHODS Key steps in assay development included: (i) shifting the TMAO analyte peak to a less crowded region of the spectrum with a pH buffer/reagent, (ii) attenuating the broad protein background signal in the spectrum and (iii) using a non-negative least squares algorithm for peak deconvolution. Assay performance was evaluated according to Clinical and Laboratory Standards Institute guidelines. A method comparison study was performed to compare TMAO concentrations quantified by NMR and mass spectrometry (MS). RESULTS The within-run and within-lab imprecision ranged from 4.3 to 14.5%. Under the acquisition method employed, the NMR assay had a limit of blank, detection and quantitation of 1.6, 3.0 and 3.3μM, respectively. Linearity was demonstrated within the reportable range of 3.3 to 3000μM. TMAO measurements using the NMR assay, which involves minimal sample preparation, compared well with values obtained with the MS-based assay (R2=0.98). CONCLUSIONS The NMR based assay provides a simple and accurate measurement of circulating TMAO levels amenable to the high-throughput demands of the clinical chemistry laboratory. Moreover, assay performance enables the levels of TMAO to be quantified in serum or plasma at clinically actionable concentrations for the assessment of cardiovascular disease risks and individualized dietary monitoring.