Uricase methods

The two predominant analyses of uric acid are the phosphotungstic acid (PTA) method and the uricase method. In the PTA method, urate reduces PTA to form a blue product. In the uricase analysis method, urate is oxidized by uricase oxidoreductase. 

Ingebretsen OC, Borgen J, Farstad M. Uric acid determination Reversed-phase liquid chromatography with ultraviolet detection compared with kinetic and equilibrium adaptations of the uricase method. Clin Chem 1982 28 496-8. 

This integration strategy for enzyme substrate assay gives the linear response from about 1.5 imol/L up to 60 imol/L uric acid at 40 U/L uricase (Fig.4, unpublished), and shows resistance to the action of xanthine at 30. imol/L in reaction solutions (this level of xanthine always caused negative interference with all available kits commercialized for serum uric acid assay). Therefore, the integration strategy for uric acid assay is clearly superior to any other uricase method reported. 

His diet was essentially the same during the three studies. %-glycine C60 atan % excess), 100 mg/kg body weight was given with 250 ml milk. Thereafter, urine was collected daily for one we, using toluene as preservative. Uric acid was determined using the uricase method. Uric acid was isolated and crystallized. Isotope enrichment was analyzed by standard mass spectroneter techniques as previously described CD. ... 

The detailed plan of the study was set out clearly in writing so that all subjects would follow the same protocol. This plan (Fig. 1) began with two control days during which the person took the normal unrestricted diet, including their usual alcohol. During this time, two consecutive 24 hour urine collections were examined for volume, urate concentration and creatinine concentration. Urate was measured by an automated uricase method based on that of Liddle et alii (1959) (2) which gave a molar absorption of urate within 15 of published values. Blood was collected for urate... 

Blood samples were taken Monday, Wednesday and Friday in the morning before breakfast. Urine samples were collected during 2k hours daily. To check for errors in the urine collection endogenous creatinine in blood and urine were determined. Uric acid was measured by our modification [ ZtJLLNER, I963 ] of the uricase method. 

Standard inulin clearances were obtained. After four adequate 20 minute clearance periods 3.0 g of pyrazinamide were given orally. Fifteen to 20 minute clearance periods were collected for the next two hours. Uric acid was measured by the uricase method (4) and inulin by a modification of the Fjelbo autoanalyser technique (5). 

In 50 adult patients with sickle cell anemia, mean serum uric acid by the uricase method was 6.2 0.3 mg%. Twenty of these 50 patients or 40% were hyperuricemic as defined by a serum uric acid greater than 6.5 mg%. Mean urinary acid/creatinine ratio was increased to. 56 . 06 in 20 sickle cell anemia patients compared to. 41 . 04 in 12 normal adults (p <. 05). 

Uric acid was determined by uricase method (3), total oxypurines were determined by the method of Klinenberg (4). 

Modified procedure (ACU kit) The plasma sample is preincubated with uricase (urate oxidase) and then quantified in the ACW assay. The method requires 10 pL of plasma. It is also possible to determine the uric acid (UA) portion in ACW UA = ACW - ACU. 

Phosphotungstic acid (PTA), uricase, and HPLC-based methods have been described for measuring uric acid. ... 

Although many methods for the quantitation of uric acid are described in the literature, the most popular methods today employ the uricase-mediated reaction however, the specificity of this reaction may be compromised by the choice of detector reaction, owing to either an interfering enzyme or a molecule that competes in the final color generation step. Today reactions that generate a visible end product are preferred because of the higher color yield however, care should be taken that interference caused by ascorbate, bilirubin, and unspecified interferants in plasma from patients with kidney failure is minimized. 

Meites S, Thompson C, Roach RW. Two ultramicroscale methods for plasma uric acid analysis with uricase. Clin Chem 1974 20 790-3. 

Methods have been devised for the determination of these compounds in the presence of uric acid through the use of uricase. The uricase is destroyed and the xanthine and hypoxanthine determined enzymatically. In the absence of uric acid (e.g., patients with xanthinuria), addition of xanthine oxidase converts the hypoxanthine and xanthine to uric acid and any method for uric acid determination could then be employed. The determination of hypoxanthine and xanthine by any method is complicated if the patient has received allopurinol, since allopurinol does not separate well from xanthine on ion-exchange resins and both allopurinol and alloxanthine are inhibitors of xanthine oxidase. Carefully... 

K8. Klein, F., and Lafeber, G. J. M., Improvements of the uricase-U.V. method for the determination of uric acid in serum and urine. Clin. Chim. Acta 14, 708-710 (1966). 

In view of the low physiological uric acid concentrations, in the assay of uric acid electrochemical interferences by other anodically oxidizable substances become particularly troublesome. Kulys et al. (1983) coimmobilized HRP with uricase in order to eliminate these interferences. This approach permits uric acid measurement to be performed at a potential of 0 V vs SCE. However, the autoxidation of ferrocyanide used as HRP substrate is a serious drawback of this method. 

The Glukometer (see Table 23) has been equipped with a uricase membrane and employed for uric acid assay in serum. Satisfactory agreement with the uricase-catalase reference method was obtained the deviation of the mean value was as low as +2.4 pmol/1. The reagent costs of the method amount to only one tenth of those required for the manual photometric one. 

The UA-300 analyzer of Fuji Electric (Japan) uses a uricase membrane fixed to a hydrogen peroxide selective layer (Osawa et al., 1981). Only 20 pi of blood serum is required and a sample throughput of 50-60/h at a CV of 3% is achieved. The correlation with the uricase-catalase method is reflected by the following equation ... 

Manual or automated methods involving uricase enzyme were subsequently developed for use with cereal products (Farn and Smith, 1963b Sen and Smith, 1966). Laessig et al. (1972) used an autoanalyzer, an instrument deployed for determining uric acid in blood, for uric acid analysis in... 

Uric acid is usually determined by measuring its ultraviolet absorption at 292 nm. However, the amount of uric acid in blood is small and the absorption is not specific. So, after the measurement, the uric acid is destroyed by adding the enzyme uricase. The absorbance is measured again. The difference in absorbance is due to the uric acid present. Since only uric acid wiU be decomposed by uricase, the method becomes specific. A similar procedure for the colorimetric determination of uric acid involves the oxidation of uric acid with molybdate to form molybdenum blue, a molybdenum(V) compound. In principle, uric acid could be determined as glucose was, but impurities in uricase preparations usually rapidly destroy the very small amount of hydrogen peroxide produced. 

Y. Nishikawa, F. Morishita, and T. Kojima, Determination of Urate by a FIA Method Using a Uricase-Immobilized Open-Tubular Reactor [in Japanese]. Bunseki Kagaku, 35 (1986) 575. 

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