ABSTRACT
We here present a conceptually novel reaction-based ELISA principle (ReactELISA) for quantitation of the carbon nucleophilic lipid metabolite acetoacetate. Key to the assay is the utilization of a highly chemoselective Friedländer reaction that captures and simultaneously stabilizes the nucleophilic metabolite directly in the biological matrix. By developing a bifunctional biotinylated capture probe, the Friedländer-acetoacetate adduct can be trapped and purified directly in streptavidin coated wells. Finally, we outline the selection and refinement of a highly selective recombinant antibody for specific adduct quantitation. The setup is very robust and, as we demonstrate via miniaturization for microplate format, amenable for screening of compounds or interventions that alter lipid metabolism in liver cell cultures. The assay-principle should be extendable to quantitation of other nucleophilic or electrophilic and perhaps even more reactive metabolites provided suitable capture probes and antibodies.
Subject(s)
Acetoacetates/blood , Enzyme-Linked Immunosorbent Assay/methods , Hepatocytes/metabolism , Lipid Metabolism , Acetoacetates/chemistry , Acetophenones/chemical synthesis , Acetophenones/chemistry , Aniline Compounds/chemical synthesis , Aniline Compounds/chemistry , Animals , Antibodies, Monoclonal/immunology , Biotin/analogs & derivatives , Biotin/chemical synthesis , Biotin/immunology , Humans , MiceABSTRACT
OBJECTIVES: For the quantification of ß-hydroxybutyrate (BHB) and ß-hydroxy-ß-methylbutyrate (HMB) in human whole blood, a method using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was developed, which does not require chemical modification of the analytes. DESIGN AND METHODS: Samples were deproteinised by a mixture of methanol and acetonitrile, and the extracts were cleaned-up using both polymeric strong cation exchange and strong anion exchange sorbents. The analytes and their structural isomers were separated using a column with a zwitterionic stationary phase. Isotope dilution of both analytes was used for quantitative analysis. RESULTS: Separation of BHB from isobaric interferences was achieved through chromatography. The relative intra-laboratory reproducibility standard deviations were better than 10% for blood samples at concentration levels of 10-20µM BHB and 1µM HMB and better than 5% at concentration levels 10 times higher. The mean true extraction recoveries were close to 100%. The trueness expressed as the relative bias of test results was within ±5% at concentration levels of 10-1000µM BHB and 1-20µM HMB. The lower limits of quantification were estimated to be 3µM for BHB and 0.4µM for HMB. CONCLUSIONS: A simple and highly sensitive and selective HILIC-MS/MS method was developed that is suitable for the quantification of BHB and HMB in whole blood.