Simultaneous electrochemical monitoring of metabolites related to the xanthine oxidase pathway using a grinded carbon electrode.

Using a mechanically grinded pyrolytic graphite electrode in edge orientation, a sensitive electrochemical method was developed for simultaneous determination of uric acid (UA), xanthine (XAN), hypoxanthine (HYP) (products of purine catabolism in human), allopurinol (ALO), and oxypurinol (OXY) (a drug used in treatment of purine catabolism disorders and its metabolite, respectively). It is demonstrated that differential pulse voltammetry in connection with this electrode can serve as a simple and efficient tool for monitoring transformation of purine catabolites (HYP --> XAN --> UA) catalyzed by xanthine oxidase (XO) as well as inhibition of this pathway by ALO being enzymatically converted to OXY. Our protocol is based on direct electrochemical measurement of oxidation peaks for each of the substances during in vitro reactions in a single detection step by the same electrode system. In addition, we show that the proposed electrochemical technique can be applied to parallel detection of metabolites involved in the XO pathway excreted in urine without any pretreatment of the clinical samples.

A cyclic N7,C-8 guanine adduct of N-nitrosopyrrolidine (NPYR): formation in nucleic acids and excretion in the urine of NPYR-treated rats.

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen that is present in the diet and tobacco smoke and may form endogenously in humans. Biomarkers to assess NPYR exposure and metabolic activation in humans are needed. The cyclic N7,C-8 guanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purin-4(3H)-one (8), which is formed in tissues of rats treated with NPYR, is one potential candidate for such a biomarker. In this study, we evaluated the formation of this and other NPYR adducts in reactions of alpha-acetoxyNPYR with dGuo, Guo, DNA, and RNA and determined the extent of urinary excretion of adduct 8 in rats treated with NPYR. alpha-AcetoxyNPYR, a stable precursor to the major product of NPYR metabolic activation, was allowed to react with dGuo, Guo, DNA, or RNA at 37 degrees C, pH 7. The most striking observation was that the cyclic N7,C-8 guanine adduct 8 was formed 9 times more extensively in the reaction with Guo than with dGuo. It was also formed 2.5 times more extensively in RNA than in DNA. In rats treated with NPYR, levels of the cyclic N7,C-8 guanine adduct 8 were 2 times as high in RNA than in DNA. Rats treated with [14C]adduct 8 excreted 51% of this adduct unchanged in urine. Rats treated with [3,4-3H]NPYR excreted 0.00004% of the dose as adduct 8. The major differences in product formation in reactions of alpha-acetoxyNPYR with dGuo versus Guo are unusual for alkylating agents; potential mechanisms are discussed. The higher levels of adduct 8 in RNA than in DNA suggest that RNA may be superior as a source of adduct 8 as a biomarker.

Effect of glucagon on renal excretion of oxypurinol and purine bases.

OBJECTIVE: To investigate whether glucagon increases the urinary excretion of oxypurinol and purine bases. METHODS: We administered 1 mg glucagon intravenously to 5 healthy subjects taking 300 mg allopurinol orally, and determined plasma concentrations and urinary excretion of oxypurinol and purine bases. RESULTS: Glucagon increased the urinary excretion and fractional clearances of uric acid, xanthine, and oxypurinol, together with an increase in creatinine clearance, while it decreased plasma concentrations of xanthine and hypoxanthine. CONCLUSION: Glucagon-induced increases in urinary excretion of uric acid, xanthine, and oxypurinol were attributable to increases in the fractional clearances of uric acid, xanthine, and oxypurinol in addition to an increase in glomerular filtration rate. It is suggested that glucagon affects the renal common transport pathway of uric acid, xanthine, and oxypurinol by stimulating the release of a liver derived renal vasodilator.

Direct injection/h.p.l.c. methods for the analysis of drugs in biological samples.

1. Direct injection h.p.l.c. methods for zaprinast, and pantoprazole and its sulphone metabolite were developed. 2. Optimal recovery of pantoprazole and its sulphone metabolite was effected by the absence of transfer losses and the effective adjustment of sample pH on-line. 3. Acetonitrile reduced the recovery of pantoprazole and its sulphone metabolite at acetonitrile concentrations greater than 5% in serum. 4. Direct injection h.p.l.c. methods minimize sample handling losses, reduce human contact with biological samples and are sufficiently accurate and reproducible to be used to support pharmacodynamic and toxicokinetic studies.

Purine metabolism during strenuous muscular exercise in man.

This study was designed to examine the influence of exercise on purine metabolism in man. In 15 men, the plasma uric acid concentration increased from 6.9 to 8.5 mg/dl following a 5000-m race and from 6.2 to 7.9 mg/dl in 11 men following a 42-km marathon. During a progressive exercise test on a cycle ergometer, the plasma uric acid ocnentration did not change significantly in 11 subjects. However, the plasma oxypurines increased from 19 micrM at rest to 50 microM at exhaustion and the urinary excretion of oxypurines increased from 140 to 400 mumol/g creatinine. Intracellular ATP decreased from 5.17 to 2.91 mumol/g and ADP and AMP increased from 0.85 to 1.29 and from 0.12 to 0.15 mumol/g wet weight, respectively. These observations suggest that there is an accelerated degradation of purine nucleotides to the precursors of uric acid in skeletal muscle during vigorous exercise.

Renal urate transport during variations in urate synthesis in the rat.

In order to determine the effect of intrarenal synthesis of urate upon the urinary urate excretion in the rat, we effected large changes in urate synthesis by increasing it with allopurinol. Hypoxanthine infusion increased plasma urate rapidly and also increased the urinary urate excretion and its renal clearance. However, when the plasma urate was maintained constant, hypoxanthine had no effect upon renal urate transport. Conversely, allpurinol infusion rapidly diminished the plasma urate, urinary urate excretion and its renal clearance. Again, the maintenance of a constant plasma urate concentration prevented any change in urate transport during allopurinol. The urinary degradative purine metabolic pattern was altered predictably by hypoxanthine and allopurinol. Assuming than any putative intrarenal component of urate synthesis would be affected predictably and consistently by hypoxanthine and allopurinol, these results suggest that changes in intrarenal urate synthesis are not an important determinant of urate excretion in the rat.