Uric acid analysis
Uric acid is a week acid that is distributed throughout the extracellular fluid compartment as sodium urate and is cleared from the plasma by glomerular filtration. Uric acid is the final breakdown product of purine (e.g. adenine, guanine) metabolism and is produced in the liver. Except for humans and higher apes, all mammals express uric acid oxidase, an enzyme responsible for conversion of uric acid to allantoin prior to excretion . Allantoin is a soluble water product; uric acid is onlysparingly soluble in aqueous media
Elevated levels of uric acid (hyperuricaemia) are most commonly associated with gout (a form of arthritis), Lesch-Nyhan syndrome, cardiovascular disease, diabetes, metabolic syndrome, increased breakdown of cell nuclei and renal disease. Patients on chemotherapy for proliferative diseases such as lymphoma, leukaemia or myeloma also often exhibit hyperuricaemia and levels must be monitored to avoid kidney damage. In gout patients, precipitation of uric acid in the joints leads to pain and inflammation and in many cases this can be directly linked to over-production of uric acid. These patients are also more susceptible to the formation of kidney stones. Plasma uric acid levels in such patients are usually high (> 0.36 mM) and can often reach 4.5 mM in urine. Over recent years there has been a significant effort to trace the link between raised serum uric acid concentration and cardiovascular disease and events, such as myocardial infarction. As insulin has a physiological action on renal tubules, causing reduced sodium and uric acid clearance, elevated serum uric acid is also a consistent feature of insulin resistance syndromes, which are also characterised by elevated plasma insulin level, blood glucose concentration and raised body mass index. It is not clear whether uric acid has a damaging or protective effect in these circumstances. However, a better understanding of the mechanisms underlying these associations is required.
A specific, non invasive, single component and highly sensitive assay for uric acid would certainly allow a clearer interpretation of the importance of elevated uric acid concentrations, and the specific urate-lowering treatment on cardiovascular disease. Lower serum uric acid values (hypouricaemia) are much less common but may result from under-production of uric acid from a reduction in uric acid excretion, as may occur in AIDS, diabetes mellitus and various malignant diseases. In conclusion, measurement of uric acid in urine and plasma is an important marker in clinical diagnosis and is essential in the treatment regime for both hyper- and hypouricaemia.
Measuring uric acid levels can be undertaken by common colorimetric methods, such as the reduction of phospho-tungstic acid to ‘tungsten blue’ by uric acid. Unfortunately, this method is subject to interference from other reducing agents and this combined with the problems encountered with these ‘popular’ colorimetric methods led to the development of enzymatic assays. Current in vitro clinical assays used to measure uric acid in urine and serum are predominantly based on the use of the uricase enzyme. Unfortunately, this enzymatic method also has its obvious downfalls, such as interference from ascorbate and bilirubin. The enzymes must be handled carefully to avoid being denaturated and the organic dyes are often light and temperature sensitive, requiring storage in the dark at low temperature. From a photophysical perspective, these organic dyes often suffer from overlapping absorption and emission spectra (Δλ < 15 nm) which further complicate their use, requiring high resolution spectrophotometers. In summary, it is clear that these enzymatic multi-component systems require relatively long and complex experimental procedures to be used on a daily basis. Therefore, the development of a suitable method for the rapid and accurate determination of uric acid concentration in serum or urine is of considerable interest.
Lanthanide ions afford considerable scope for the development of new chemical entities that can be used as analytical or imaging probes. The advantages of f-block ions have been demonstrated irrefutably: large excitation and emission maxima separation, intense, line-like and long-lived luminescence at a range of wavelengths spanning the visible and near infrared (NIR) regions allowing time-gated rejection of undesired signals arising from (short-lived) auto-fluorescence from bio-molecules.
The use of two different lanthanide(III)complexes of a common macrocyclic ligand, allows the analysis of the concentration of low molecular weight reductants, such as uric acid in diluted urine samples. Using a mixture of Eu and Tb complexes of the same macrocyclic ligand in a set ratio, measurement of the ratio of intensities of a Tb emission band (green) versus a dominant Eu emission band (red) is a direct function of the amount of urate present in solution, providing that interfering species contribute little to the overall quenching effect.
Prior to measuring uric acid concentrations in unknown urine samples, a calibration curve is required. This is defined by measuring the green/red intensity ratio as a function of added sodium urate concentration. The precision, which is an inherent feature of such a ratiometric assay, allows less than 1% variance in the measured intensity ratio for a given urate concentration. As the assay involves two complexes of the same ligand, other non-specific effects that may affect the observed emission intensity for a given sample occur to the same extent for the Eu and Tb complexes. Examples of such effects include sample to sample variation of protein, light scattering due to particulates and surface adhesion all that is eliminated by using our wn brand timegated small benchprint spectrophotometer. Extensive studies have been carried out to validate this unique method for urinalysis which involved co-measurements of samples using a commercial enzymatic uricase assay kit (Invitrogen®, Amplex Red™) that uses horseradish peroxidise. Agreement between the two methods was found to be less ±10%.
In summary, a simple, rapid non-enzymatic luminescence assay has been developed for uric acid. Application to diluted urine samples using equipment commonly found in analytical laboratories has been demonstrated and verified. The same methodology has been used in dilute (x100) lyophilised serum samples where the urate concentration was determined (350±10 μM) and validated with an enzymatic assay kit. These results confirm that this revolutionary ‘single component’ uric acid assay can be directly applied to common bio-fluids using commercially available instrumentation. The method is based on a reagent that can be prepared cheaply and quickly using a straightforward synthetic route. The fast response, short acquisition time and excellent photophysical and chemical properties suggest its use to determine uric acid concentration in low volume clinical samples, overcoming the limitations of the slow and rather elaborate current enzymatic assays..
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