Sweaty organic acids: Lactic and pyruvic acids measured simultaneously in sweat by HPLC

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  • Published: Mar 19, 2012
  • Author: Steve Down
  • Channels: HPLC
thumbnail image: Sweaty organic acids: Lactic and pyruvic acids measured simultaneously in sweat by HPLC

Organic acids and exercise

The common contaminants found in commercially available diethyl ether and a plastic bag have been identified by Serbian scientists, in order to prevent them from being cited as native metabolites in studies on essential oils and plant extracts.

In the human body, pyruvic acid and lactic acid are two simple organic acids which are involved in the glycolysis pathway and their ratio in blood is used as an indicator of several conditions, including inborn errors of metabolism and cardiovascular diseases. It follows that there are established procedures for measuring the acids in plasma as well as in urine, which is the other common biological testing fluid.

In recent times, there has been a move towards less invasive methods of clinical testing, using fluids like saliva and sweat which are easier to collect, reduce the risk of infection and cause less stress to the patient. Several methods have been published for the simultaneous measurement of lactate and pyruvate in sweat but a team of Italian researchers has devised another one which they say is better.

It has already been proven that there is a good correlation between the lactate concentrations in blood and sweat, so a non-invasive approach using sweat could replace invasive methods like blood and urine.

Emilia Bramanti from the Institute of Chemical and Physical Processes, University of Pisa, with colleagues Simona Biagi, Silvia Ghimenti and Massimo Onor, developed an HPLC method which they claimed is simpler than the published procedures which are based on enzymatic assays, amperometric or electrochemiluminscent biosensors, or capillary electrophoresis.

Sweat solution

The team collected sweat on paper filters from eight volunteers who were accustomed to strenuous exercise, seven being kick boxers. The athletes were either at rest, had just completed a vigorous exercise regime, or had sweat induced by skin stimulation with the drug psilocarpine. The papers were simply extracted with water and injected directly into the HPLC system.

The sweat components were separated on a C18 column using a solution of methanol in phosphate buffer adjusted to pH 2.5. A UV detector operating at 220 nm was employed and each chromatographic run was completed within 20 minutes, including column washing and re-equilibration.

The chromatogram displayed peaks for pyruvate and lactate, which were confirmed by comparison with the chromatogram for a standard mixture of the two acids. Other compounds identified were acetic acid, uric acid and tyrosine.

The method performance was evaluated with the aid of standard solutions of the acids, leading to detection limits of 0.03 and 0.001 mM for lactate and pyruvate, respectively and good linear calibration curves. The recoveries, which were determined by spiking the filter papers, were 102 and 96%, respectively.

Lactate and pyruvate exercise indicators

The lactate levels in sweat varied markedly after physical exercise, indicating that this might be a useful parameter for estimating the physical condition. For some of the athletes, the levels increased during warm-up and after vigorous exercise, in agreement with published data. But lactate decreased in others, which the research team attributed to a dilution effect caused by a rise in sweating rate.

In order to be able to compare data from individuals, the results were examined as lactate mM/min, which is normalised with respect to sweating rate, and as the lactate/pyruvate ratio, to correct for individual variations like body mass, age and gender.

The plot of lactate rate followed a bell shape through the activities of rest, warm-up, and the exercise activities consisting of striking kick boxing and punch pads and boxing bags, and having ring matches. This pattern was consistent with the recognised metabolic pathways that are in play during exercise, following a switch from aerobic to anaerobic metabolism.

The trend of the lactate/pyruvate ratio was more complex and did not necessarily mirror the lactate rate. For most subjects, it rose during strenuous activity. This ratio is also a recognised parameter for monitoring the aerobic/anaerobic threshold.

It has long been known that lactate levels increase during exercise due to the switch to anaerobic metabolism, then fall sharply after reaching certain levels when exhaustion takes place. So, the new method could find a place as a tool for assessing the performance of athletes, as well as in clinical practice for metabolic disorders.

The simplicity and speed of the HPLC method, combined with non-invasive sweat collection and minimal sample preparation, should make it an attractive alternative for measuring the two metabolites in sweat, acting as a replacement for lactate/pyruvate monitoring in blood and urine.

Related Links

Biomedical Chromatography 2012 (Article in Press): "Simultaneous determination of lactate and pyruvate in human sweat using reversed-phase high-performance liquid chromatography: a noninvasive approach"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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