Acid freezes uricolysis in addition to glycolysis: Utility of fluoride-EDTA-citrate tube in the measurement of uric acid for patients administered rasburicase

Background In samples from patients administered rasburicase, ex vivo uricolysis leads to spuriously low uric acid results. The manufacturer’s recommendation of storing the sample in ice-water until analysis, however, does not fully arrest uricolysis. Since uricase activity is affected by pH and metal chelators, we assessed uricolysis inhibition in sodium fluoride-ethylenediaminetetraacetic acid (EDTA)-citrate sample tube (FC Mix tube, Greiner) used primarily for plasma glucose. Method A serum pool was spiked with rasburicase and uric acid measured at 15, 45, 90, 150, 240 and 1080 min in a lithium heparin tube in ice-water, plain tube at room temperature (RT), EDTA tube at RT, FC Mix tube in ice-water, FC Mix tube at RT and FC Mix tube at RT prepared by dissolving FC Mix in serum. Results The rate of urate decay was lowest in the FC Mix tube independent of temperature, then lithium heparin tube in ice-water, then EDTA tube at RT and highest in the plain tube at RT. Uric acid concentrations in the prepared FC Mix tube at RT and heparin tube in ice-water were, respectively, 98.2% and 93.8% of control values at 90 min, 97.1% and 89.3% of control values at 4 h, and remained higher in the prepared FC Mix tube at all time points. Conclusion NaF-EDTA-citrate mixture largely arrested rasburicase mediated ex vivo uricolysis without the need for sample cooling. We propose that sample tubes containing NaF-EDTA-citrate be used for the measurement of uric acid in patients administered rasburicase.

Selection of uric acid oxidizing-Lactobacillus plantarum isolates based on their genetic determinant and uricase kinetics

Hyperuricemia is a condition characterized by abnormally elevated levels of uric acid in the blood. It has been a leading morbidity disease. Microbial uricase can be used to oxidize uric acid into allantoin and hydrogen peroxide in the presence of oxygen and therefore has the potential to play an essential role in reducing uric acid in the people suffering from degenerative disease of hyperuricemia. The present study aims to select uric acid oxidizing-Lactobacillus plantarum isolates based on their genetic determinant and uricase kinetics. A collection of Lactobacillus plantarum isolates were grown on a selective differential medium followed by measuring their uricase activity spectrophotometrically. Specific primers for detection of uricase gene were designed. The uricase coding gene (uox) was then detected in all of the selected isolates by using a qPCR method employing the designed specific primers. The uricase kinetics was determined by the Lineweaver-Burk method. Results showed that all isolates had uricase activity and 4 potential isolates were selected based on their superior uricase activity. The uox gene was detected in all of the selected isolates. The kinetics analysis, however, revealed that only the L. plantarum K-Mar-A2 show strongest substrate affinity and was considered a potential candidate to be developed as a source of therapeutic agent for hyperuricemia.

Pharmacokinetics of Intravenous Uricase-PEG 20, a Drug for Managing Hyperuricemia in Tumor Lysis Syndrome.

Abstract 3086 Poster Board III-23 Background Tumor lysis syndrome can result in hyperuricemia and the precipitation of monosodium urate crystals in the renal tubules, leading to renal failure and dialysis. The enzyme uricase, found in most mammals but not humans, metabolizes uric acid into the highly soluble allantoin. Rasburicase (Elitek/Fasturtec, Sanofi Aventis), an unmodified recombinant uricase, is used to treat and prevent hyperuricemia associated with tumor lysis syndrome. However, it requires multiple doses and is highly immunogenic. Uricase-PEG 20 is a pegylated recombinant uricase, designed to have an extended half life and reduced immunogenicity compared with unmodified uricase, that has been administered intramuscularly to gout patients in clinical studies. However, it has not previously been studied as an intravenous agent. Methods Intravenous toxicology studies, including pharmacokinetic studies, were performed with Uricase-PEG 20 in rats and cynomolgus monkeys. Uricase-PEG 20 was administered to rats at 1, 4 and 10 mg/kg and uricase enzyme activity was assessed at 1, 6, 24, 72 and 144 hrs after dosing. In monkeys, Uricase-PEG 20 was administered at 0.5, 1 and 5 mg/kg and uricase activity was assessed at 15 min and 1, 4, 24, 72 and 144 hrs after administration. Results In rats, the half-life for Uricase-PEG 20 ranged from 44.2 to 104.5 hrs (median, 70.7 hrs); the volume of distribution varied from 38.8 to 129.0 mL/kg (median 94.6), and the clearance varied from 0.70 to 1.48 mL/hr/kg. In monkeys, the half-life ranged from 184 to 214 hrs (median 194.5 hrs), the volume of distribution ranged from 17.2 to 25.2 mL/kg (median 19.9 mL/kg), and the clearance ranged from 0.063 to 0.102 mL/hr/kg. In both species, measures of exposure including Cmax and AUClast increased in a dose-proportional manner. The median half-lives of 70.7 hrs in rats and 194.5 hrs in cynomolgus monkeys is significantly longer than the half-life reported for rasburicase in similar models (<2 hrs in rats and <4 hrs in baboon monkeys). Conclusions The long half-life of Uricase-PEG 20 compared to that of unmodified uricase suggests that Uricase-PEG 20 may require less frequent dosing and thus have better clinical utility than unmodified uricase. Uricase-PEG20 will be used at intravenous doses ranging from 0.05 to 0.4 mg/kg in initial human studies. Disclosures Zhang: EnzymeRx: Employment. Fiorino:EnzymeRx: Employment.

Regulation of expression of theRhizopusoryzaeuricase and urease enzymes

The regulation of intracellular urease and uricase activities was examined in Rhizopus oryzae. Urease activity (2.4 U/mg protein) was present in R. oryzae mycelium grown in minimal medium containing NH4Cl as sole nitrogen source. This activity increased threefold under nitrogen derepression conditions, but no induction by urea was detected. Control of urease activity in R. oryzae differs from that found in Neurospora crassa but resembles the situation in Aspergillus nidulans. No uricase activity was detected in R. oryzae mycelium grown in minimal medium containing NH4Cl as sole nitrogen source. Uricase activity was increased 10- to 40-fold under derepression conditions and was induced by exogenous uric acid (60- to 78-fold). Control of the R. oryzae uricase differs from that found in N. crassa and A. nidulans. This is the first analysis of the regulation of enzymes from the purine catabolic pathway in any member of the Zygomycetes.Key words: Rhizopus oryzae, uricase, urease, nitrogen metabolism.