Automatic fluorometric lactate determinations in human plasma samples

Abstract

Lactate is usually present in human blood at very low concentrations and its production is under hypoxic conditions. However, lactate is considered a signalling molecule promoting specific immune inflammatory responses since its levels increases according to the grade of inflammation. In this work, it was developed an automatic system based on sequential injection analysis (SIA) to determine the lactate concentrations in human samples. The reaction is based on the conversion of lactate in pyruvate by lactate dehydrogenase (LDH) in the presence of nicotinamide adenine dinucleotide (NAD+) that is converted in nicotinamide adenine dinucleotide reduced (NADH), a fluorescent compound. The effects of several parameters on the reactions were tested, such as reagents concentration, volumes, and aspiration order, reaction time and selected the best ones to get accuracy and quickness in the execution of the enzymatic assay. Finally, the methodology presented detection and quantification limits of 0.193 mM and 0.642 mM, allowing accurate and reliable quantifications of lactate in human plasma samples. This work confirmed the potential of the SIA system as a simple, versatile, robust, and rapid analytical tool for automating the determination of lactate levels.