Simultaneous Determination of Lactate and 3-Hydroxybutyrate Enantiomers in Mouse Urine Using a Three-Dimensional Hplc System

The structures of lactate and 3-hydroxybutyrate (3HB) are similar, and both of them have a hydroxyl group, a carboxyl group, and a chiral carbon. Thus, there are two enantisomers each of lactate and 3HB. D-Lactate is the metabolite of methylglyoxal, and 3HB, the product of fatty acid metabolism, could be metabolized into methylglyoxal. D-Lactate is related diabetes mellitus, acidosis, encephalopathy, and renal damage, and the level of D-lactate in serum increased in the condition of disease or damaged tissue. D-3HB is the most of ketone bodies in human body, so it has been studied with diabetes mellitus. Recently, L-3HB was found in the serum of mice with aristolochic acid-induced nephropathy. According to the similarity of chemical structure, methylglyoxal-related metabolism, elevation in pathological state, it is important to develop a more efficient system to determine the enantiomers of lactate and 3HB. Furthermore, we could study the association among the change in enantiomers of lactate and 3HB and disease/damaged tissue. We have developed a two-dimensional high performance liquid chromatograph system (2D-HPLC) for the determination of lactate and 3HB enantiomers, however, the recovery in the urine was not enough. In this project, we are going to establish an off-line three-dimensional HPLC system, which is composed of 2D-HPLC and ion-exchange HPLC, to analyze the enantiomers of lactate and 3HB in the first year; we will set up an on-line three dimensional-high performance liquid chromatography (3D-HPLC) to separate them in the second year. Comparing to 2D-HPLC, 3D-HPLC might purify the samples and improve the efficiency of separation. Moreover, this method might be applied to study other samples which is trace or difficult to be detected.