A High-Throughput NMR Method for Lipoprotein-X Quantification.

Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin-cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort (n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

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.

Creatine and creatinine quantified using nuclear magnetic resonance: A method validation study and clinical associations between circulating creatine and fatigue in kidney transplant recipients.

BACKGROUND: A potential contributor to fatigue in kidney transplant recipients (KTR) may be impaired creatine homeostasis. We developed and validated a high-throughput NMR assay allowing for simultaneous measurement of circulating creatine and creatinine, and determined plasma creatine and estimated intramuscular creatine concentrations in KTRs, delineated their determinants and explored their associations with self-reported fatigue. METHODS: An NMR assay was developed and validated for measurement of circulating creatinine and creatine concentrations. Plasma creatine and creatinine concentrations were measured in 618 KTR. Fatigue was assessed using the checklist individual strength. Associations of creatine parameters with fatigue was assessed using linear mixed effect models. RESULTS: The NMR-based assay had good sensitivity, precision and demonstrated linearity across a large range of values. Among KTR, the mean age was 56 ± 13 years, 62% were men and eGFR was 54 ± 18 ml/min/1.73 m2. Plasma creatine concentration was 27 [19-39] µmol/L. Estimated intramuscular creatine concentration was 27 ± 7 mmol/kg. Higher plasma creatine concentration and higher estimated intramuscular creatine concentration were independently associated with a lower total fatigue score and less motivation problems. CONCLUSION: An NMR method for measurement of circulating creatine and creatinine which offers the potential for accurate and efficient quantification was developed. The found associations suggest that improving creatine status may play a beneficial role in mitigating fatigue.

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.

Vantera Mediated Quantification of Urine Citrate and Creatinine: A New Technology to Assess Risk of Nephrolithiasis.

Urine citrate is often used to identify patients at risk of recurrent nephrolithiasis or kidney stones. A high-throughput assay was developed to measure urine citrate and creatinine on the Vantera® Clinical Analyzer, a nuclear magnetic resonance (NMR) instrument designed for the clinical laboratory. Assay performance was evaluated and comparisons between the NMR and chemistry results were conducted. Linearity was demonstrated over a wide range of concentrations for citrate (6 and 2040 mg/L) and creatinine (2.8 and 1308 mg/dL). Intra-and inter-assay precision (%CV) ranged from 0.9 to 3.7% for citrate and 0.4 to 2.1% for creatinine. The correlation coefficients for the comparison between NMR and chemistry results were 0.98 (Y = 1.00X + 5.0) for citrate and 0.96 (Y = 0.968X + 0.97) for creatinine. The reference intervals for both analytes were confirmed. Ten endogenous and exogenous substances were tested and none were found to interfere with the assay results. In conclusion, the newly developed high-throughput NMR assay exhibited robust performance and generated results comparable to the currently utilized chemistry tests, thereby providing an alternative means to simultaneously quantify urine citrate and creatinine for clinical and research use. Furthermore, the NMR assay does not exhibit the same interference limitations as the chemistry tests and it enables multiplexing with other urine metabolite assays which saves time and costs.

Nuclear Magnetic Resonance-Measured Ionized Magnesium Is Inversely Associated with Type 2 Diabetes in the Insulin Resistance Atherosclerosis Study.

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.

High-throughput nuclear magnetic resonance measurement of citrate in serum and plasma in the clinical laboratory.

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 â€‹= â€‹553), and in the general population (n â€‹= â€‹133,576). RESULTS: The LOQ for the assay was determined to be 1.48 â€‹mg/dL. Linearity was demonstrated over a wide range of concentrations (1.40-4.46 â€‹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 <1% bias between serum and plasma samples, except for heparin plasma (3% bias). Stability was demonstrated up to 8 days at room temperature and longer at lower temperatures. In a cohort of apparently healthy adults, the reference interval was <1.48-2.97 â€‹mg/dL. Slightly higher values were observed in the general population. CONCLUSIONS: The newly developed NMR-based assay exhibits analytical characteristics that allow the accurate quantification of clinically relevant citrate concentrations. The assay provides a simple and fast means to analyze samples for research and clinical studies.

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.

Characterization of LP-Z Lipoprotein Particles and Quantification in Subjects with Liver Disease Using a Newly Developed NMR-Based Assay.

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.

A novel NMR-based assay to measure circulating concentrations of branched-chain amino acids: Elevation in subjects with type 2 diabetes mellitus and association with carotid intima media thickness.

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 (R2 = 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.

GlycA, a marker of acute phase glycoproteins, and the risk of incident type 2 diabetes mellitus: PREVEND study.

BACKGROUND: GlycA is a recently developed glycoprotein biomarker of systemic inflammation that may be predictive of incident type 2 diabetes mellitus (T2DM). METHODS: Analytical performance of the GlycA test, measured on the Vantera® Clinical Analyzer, was evaluated. To test its prospective association with T2DM, GlycA was measured in 4524 individuals from the PREVEND study and a survival analysis was performed with a mean follow-up period of 7.3y. RESULTS: Imprecision for the GlycA test ranged from 1.3-2.3% and linearity was established between 150 and 1588µmol/l. During the follow-up period, 220 new T2DM cases were ascertained. In analyses adjusted for relevant covariates, GlycA was associated with incident T2DM; hazard ratio (HR) for the highest vs. lowest quartile 1.77 [95% Confidence Interval (CI): 1.10-2.86, P=0.01], whereas the association of high sensitivity C-reactive protein (hsCRP) with T2DM was not significant. GlycA remained associated with incident T2DM after additional adjustment for hsCRP; HR 1.71 [1.00-2.92, P=0.04]. A multivariable adjusted analysis of dichotomized subgroups showed that the hazard for incident T2DM was highest in the subgroup with high GlycA and low hsCRP (P=0.03). CONCLUSIONS: The performance characteristics of the GlycA test reveal that it is suitable for clinical applications, including assessment of the risk of future T2DM.

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.

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.