Lactic acid analysis
Lactic acid is a chemical compound that plays a significant role in several biochemical processes. The concentration of blood lactate in humans is usually 1-2 mM at rest, but can rise to over 20 mM during intense exertion. Lactic acid is naturally present in many foodstuffs and is formed by natural fermentation in products. It is also used in a wide range of food applications such as bakery products, beverages, meat products, confectionery, dairy products, salads, dressings and ready meals. Lactic acid in food products usually serves as either as a pH regulator, preservative or as a flavouring agent (E270). It also has several other important applications and roles in many aspects of our daily life, such as pharmaceutical and cleansing products or as a precursor for biodegradable polymers. Accumulation of lactic acid in the muscle, occurs only during short bouts of exercise of relatively high intensity, is often related to fatigue and muscle soreness. Lactic acid levels also increase in conditions such as heart failure, severe infection (sepsis), or shock. In each case, the flow of blood and oxygen throughout the body is lowered. Lactic acid levels are also elevated when the liver is severely damaged or diseased, because the liver normally breaks down lactic acid. Very high levels of lactic acid may cause a serious, sometimes life-threatening condition called lactic acidosis. Due to its key role not only in every day products but most importantly in the human body, the development of a single component, fast response and user friendly lactic acid sensor is paramount in order to undertand the role of (L)-lactic acid in sports science and medicine. The latter is of great importance for confirmation of the hypothesis of elevated lactic acid levels in malignant (cancerous) tissue samples and/or biological fluids.
Current commercially available (L-) lactic acid assays are based on enzymatic methods. This method of analysis has been found to be sensitive to certain interferents, such s pH, carbon dioxide, fatty acids, sample colour and protein. Thus whilst the method is accurate, it is based on a relatively insensitive measurement of absorbance at 340 nm. The need to treat certain samples prior to analysis makes it not very ‘user-friendly’. As with any UV absorbance based method, a reasonable amount of sample is required to execute a measurement. These undesired features prevents the assessment of any biological fluid with a small sample volume or low lactic acid concentration. Being a four component enzymatic assay kit, it also requires time and care to set up, and the kit requires cold storage and rather long data acquisition times (sometimes up to 30 min reaction time involving 3-4 readings).
Considering each of these issues, a single component, fast response L-lactate sensor is required. This should possess the following characteristics: tuneable affinity for lactate; insensitivity to common interferences found in typical bio-fluid samples; provide the facility for multiple readings, using emission techniques in the visible range of the spectrum. The single component L-lactate sensor developed by FScan Ltd. eliminates most of the problems assessed with the enzymatic lactate assay. The sensor is based on a europium-macrocyclic complex. The analysis utilises ratiometric detection, by plotting changes in the ratio of up to 4 different wavelengths as a function of lactic acid concentration. Therefore, establishment of a suitable calibration curve and appropriate dilution provides a fast method for the determination of lactate in various fluids or solutions. The precision of the measurement, which is an inherent feature of such a ratiometric assay, allows less than 1% variance in the measured intensity ratio for a given lactate concentration. It is also worth mentioning that one analysis in a typical sample volume of 10 μL requires 0.5 μg of compound with simple and fast sample preparation and a fast (1 min) acquisition time using our own brand Time-gated spectrophotometer), inexpensive disposable cuvettes and commercially available buffer solutions for dilution . This compound and the associated analytical method has been shown to be successful in the rapid determination of lactate concentration in a wide variety of biological samples (plasma, seminal and prostate fluid, urine and saliva), with confirmation of the accuracy of the analysis by comparison with the currently available enzymatic kit (Megazyme™ L-lactic acid assay kit) with an error of ± 0.25 mM (Fig. 1).
Figure 1: Comparison of results regarding lactic acid concentrations in various biofluid samples using LS01 Eu(III) based sensor compound and a commercially available enzymatic lactic acid assay kit.
In summary, a simple, rapid, non-enzymatic luminescence assay has been developed for lactic acid and its application demonstrated in a variety of diluted biological samples using equipment commonly found in high throughput screening laboratories. The method has been verified by comparison with a commonly used enzymatic assay kit. These encouraging results confirm this ‘single component’ lactic acid assay to be applicable to small sample volumes of common bio-fluids using commercially available instrumentation. The emissive metal complex has a relatively simple structure and is quick and cheap to produce by a straightforward synthetic route. The fast response, short acquisition time, excellent photophysical and chemical properties each favour its use to determine lactic acid concentration in low volume clinical samples, overcoming the disadvantages of the current enzymatic assay.
For more detailed information on our Lactate assay please visit our 'Literature' section.
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