Quantification of choline in serum and plasma using a clinical nuclear magnetic resonance analyzer.

BACKGROUND: 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. METHODS: A non-negative deconvolution algorithm was developed to quantify choline. Assay performance was evaluated using CLSI guidelines. RESULTS: 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 was <7.1-20.0 µmol/L (serum) and <5.9-13.1 µmol/L (plasma). Linearity was demonstrated well beyond these intervals. No interference was observed for a number of substances tested. CONCLUSIONS: The newly developed, high-throughput NMR-based assay exhibited good performance characteristics enabling quantification of choline in serum and plasma for clinical use.

The extended lipid panel assay: a clinically-deployed high-throughput nuclear magnetic resonance method for the simultaneous measurement of lipids and Apolipoprotein B.

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.

Ketone Bodies Are Mildly Elevated in Subjects with Type 2 Diabetes Mellitus and Are Inversely Associated with Insulin Resistance as Measured by the Lipoprotein Insulin Resistance Index.

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 ß-hydroxybutyrate (ß-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: ß-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 ß-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 ß-HB, AcAc and acetone, respectively. In the IRAS, ketone bodies were elevated in subjects with T2DM versus non-diabetic individuals (p = 0.011 to ≤0.001). Age- and sex-adjusted multivariable linear regression analysis revealed that total ketone bodies and ß-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.