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

Abstract

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 β-hydroxybutyrate (β-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: β-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 β-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 β-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 β-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.

Association of beta‐hydroxybutyrate with development of heart failure: Sex differences in a Dutch population cohort

“…Higher concentrations of β-OHB were found in participants with T2D, as expected. 19,24 Remarkably, the prospective association of β-OHB with HF in women was independent of prevalent T2D.…”

“…Plasma samples were prepared on board the instrument and automatically delivered to the flow probe in the NMR spectrometer's magnetic field. Data acquisition on the Vantera and spectra data processing have been reported in greater detail elsewhere 19 . Leucine was measured by NMR spectroscopy as reported 20 …”

“…Data acquisition on the Vantera and spectra data processing have been reported in greater detail elsewhere. 19 Leucine was measured by NMR spectroscopy as reported. 20 Total cholesterol, triglycerides, insulin, serum creatinine and serum cystatin C were measured using standard protocols.…”

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

“…Multiplexing the citrate assay with additional analytes into a panel would be readily feasible because it uses the same acquisition and processing parameters as that of the NMR LipoProfile® test. The spectrum used by the NMR LipoProfile ® test provides clinically relevant analytes/markers such as lipoprotein (LDL and HDL) particle numbers, lipoprotein subclasses, the Lipoprotein Insulin Resistance Index (LP-IR), the GlycA marker of systemic inflammation, and concentrations of ketone bodies and branched chain amino acids [ 15 , 16 , 18 , 27 , [29] , [30] , [31] ]. Citrate, in addition to these analytes/markers, could help improve risk assessment for a disease such as NAFLD or mortality.…”

“…These methods involve removal of serum/plasma proteins by precipitation prior to sample analysis and throughput of several minutes (sample preparation plus run time) per sample. NMR spectroscopy has been shown to provide an automated, high-throughput platform to interrogate serum/plasma samples with minimal sample preparation (i.e., only mixing phosphate buffer with serum/plasma) [ [14] , [15] , [16] , [17] , [18] ]. Here, we report a simple and rapid (1.5 ​min per sample) NMR-based method to quantify circulating citrate, and extensively validate its analytical performance.…”