HPLC-MS/MS method for simultaneous analysis of plasma 2-hydroxybutyrate and 2-hydroxyisobutyrate: Development and clinical significance

Clin Chim Acta. 2025 Jan 15:565:120023. doi: 10.1016/j.cca.2024.120023. Epub 2024 Oct 28.

Abstract

Recent studies have identified relationships between diabetes mellitus and short-chain fatty acids, including 2-hydroxybutyrate (2-HB) and 2-hydroxyisobutyrate (2-HiB); 2-HB has been associated to the early stages of insulin resistance, while 2-HiB with the risk and progression of complications of Type 1 diabetes. Their metabolism and pathophysiological role in humans are not fully clarified. The possible association between 2-HB and 2-HiB and diabetes mellitus was investigated with a novel mass spectrometry-based assay, capable of discriminating plasma 2-HiB and 2-HB from their HB isomers. Accuracy and precision (RSD%) were always in the range 99-102% and 0.7-3.5%, respectively. The study involved samples from subjects with normal glucose tolerance (NGT) and Type 2 diabetes (T2D), originally included in a multicenter study investigating mechanisms involved in atherothrombosis. NGT subjects exhibited concentrations of 2-HB and 2-HiB of 61 (36) and 3.1 (1.9) µmol/L, median (interquartile range), respectively, that were significantly lower than those of the T2D patients, whose values were 74 (4.0) and 3.8 (2.9) µmol/L, respectively. The pattern of association of these molecules with clinical and metabolic variables is partially different: both compounds were directly related to male sex, BMI, HbA1c, and plasma glucose, 2-HiB also with age, systolic blood pressure, and HDL-cholesterol. Furthermore, they correlate with free fatty acids, glycerol, and triglyceride concentrations, but the latter correlation was negative for 2-HB and positive for 2-HiB. Results confirmed the clinical significance of 2-HB and 2-HiB, in differential association with metabolic features of T2D.

Keywords: 2-hydroxybutyrate; 2-hydroxyisobutyrate; HPLC; Mass spectrometry.

MeSH terms

  • Adult
  • Chromatography, High Pressure Liquid
  • Clinical Relevance
  • Diabetes Mellitus, Type 2* / blood
  • Female
  • Humans
  • Hydroxybutyrates* / blood
  • Liquid Chromatography-Mass Spectrometry
  • Male
  • Middle Aged
  • Tandem Mass Spectrometry*

Substances

  • 2-hydroxybutyric acid
  • Hydroxybutyrates