Automatic fluorometric lactate determination in human plasma samples

Abstract

Lactate is usually present in human blood at very low concentrations and its production occurs under hypoxic conditions. However, lactate is considered to be a signalling molecule promoting specific immune inflammatory responses since its level increases according to the grade of inflammation. In this work, we 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 to pyruvate by lactate dehydrogenase (LDH) in the presence of nicotinamide adenine dinucleotide (NAD⁺) that is converted to reduced nicotinamide adenine dinucleotide (NADH), a fluorescent compound. The effects of several parameters on the reactions were tested, such as reagent concentration, volume, aspiration order, and reaction time, and the best ones were selected to maximise 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, respectively, allowing accurate and reliable quantification 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.