Fluorometric determination of D-lactate in biological fluids

Objective: D-lactic acid in the mammalian body is mainly of microbiological origin and is often located somewhere along the digestive tract. Surgical, extensive re-sectioning of the small bowel may be one of the risk factors for altered balance in the microbiological environment. Higher levels in the body may lead to D-lactate acidosis and neurotoxicity; consequently, the possibility of diagnosis of this condition is important. Several analytical procedures for D-lactate have been introduced, but it is absolutely mandatory to distinguish this metabolite from the much more abundant and naturally occurring stereoisomer L-lactate. If enzymatic analytical methods are used, it is consequently essential to eliminate the response from L-lactate and the ubiquitous enzyme L-lactate dehydrogenase (L-LDH) (and other oxido-reductases) which will interfere with the D-lactate determination heavily. Design and methods: The present paper introduces an enzymatic-fluorometric method for determination of D-lactate in biological matrices, including blood plasma, serum and urine. Macro molecules, including enzymes, were initially precipitated by ethanol and the supernatant used for analyses. Several plasma samples were analysed with and without standard addition of both L- and D-lactate in order to validate the assay. Results mid conclusions: The procedure effectively eliminates enzyme activities that may interfere with the D-lactate quantification, resulting in the situation that L-lactate in the sample does not interfere with the determination. Intra- and inter-assay precision, accuracy and recovery of the analyte were investigated and everything suggests that this method will be acceptable for analytical as well as descriptive purposes. The analytical procedure is suitable for a semi-automated large scale set-up in the laboratory.