A. Cadenhead

Radioisotope studies of purine metabolism during administration of guanine and allopurinol in the pig

Abstract In pigs pre-fed guanine, some 33 per cent of [8-14C]guanine administered orally appeared in the urine in 24 hr, principally in the form of allantoin. Little incorporation (less than 1 per cent) of radioactivity into body tissues occurred and only 5 per cent of the radioactivity could be found in the faeces. When allopurinol was added to the guanine diet the pattern of excretion of [8-14C]-guanine changed considerably. Only 11 per cent of the radioactivity was recovered from the urine in 24 hr while 83 per cent appeared in the faeces in 3 days. Again, less than 1 per cent of the radioactivity was found in the tissues at slaughter. Intravenous administration of [8-14C]guanine to a pig on the above mixture resulted in the rapid incorporation of approximately 50 per cent of the radioactivity into body tissues with a slow subsequent daily excretion of approximately 2 per cent of this radioactivity in faeces and urine. The finding of 13 per cent of the radioactivity in the faeces is considered evidence of purine excretion into the gut. The recovery of urinary radioactivity (34 per cent of dose) principally in xanthine, but also in hypoxanthine, showed the existence of a rapid additional route of guanine catabolism via hypoxanthine. Experimental evidence is also presented to demonstrate the existence of a reciprocal relationship between urinary [14C]hypoxanthine and allopurinol riboside excretion suggesting competitive inhibition of allopurinol riboside formation by hypoxanthine in vivo. In the allopurinol treated pig, orally administered [6-14C]allopurinol was rapidly absorbed and almost totally excreted in the urine in 24 hr (90 per cent). The remainder of the radioactivity (approximately 7 per cent) was excreted in the faeces in 3 days and no radioactivity could be detected in tissue nucleic acids or in tissues to any extent (less than 0.01 per cent of the dose). The significance of these results in relation to the metabolic studies is discussed.

Metabolic studies of purine metabolism in the pig during the oral administration of guanine and allopurinol.

Abstract (1) Allopurinol and guanine, both separately and together, were fed over varying periods to diiferent groups of large white/landrace cross pigs. Although allantoin and not uric acid represented the end point of purine metabolism in this species, total urinary purine excretion approximated that of a man of equivalent weight on a purine-free diet. No abnormality in the handling of guanine was found, up to 50 per cent of an exogenous load being absorbed, metabolized and rapidly excreted in the urine as allantoin. (2) Allopurinol alone, in increasing dosage, was capable of total saturation of the enzyme xanthine oxidase at high dosage levels, but produced only slight reduction in total purine excretion at any dosage. Allopurinol riboside was the principal urinary metabolite, increasing with increasing allopurinol dosage, even at dosages above enzyme saturation levels, when oxipurinol excretion had levelled off. Xanthine replaced allantoin as the principal urinary purine metabolite during allopurinol therapy but, despite saturation of the xanthine oxidase, allantoin excretion did not fall to zero and hypoxanthine excretion was not increased. Allopurinol also had an effect on pyrimidine excretion in the pig as indicated by increased urinary orotic acid and orotidine levels, an effect not eliminated when guanine was given together with allopurinol. (3) Combined therapy with allopurinol and guanine produced three additional effects to those when allopurinol was given alone, (a) Allopurinol reduced substantially the considerable increase in total purine excretion resulting from guanine alone, a finding difficult to explain on the basis of feedback inhibition of de novo purine synthesis. (b) Urinary hypoxanthine excretion increased and at the same time allopurinol riboside excretion decreased substantially, suggesting competitive inhibition of allopurinol riboside formation as mediated by the enzyme purine nucleoside phosphorylase. (c) Xanthine was again the principal urinary metabolite, but at levels in excess of its solubility, so that coprecipitation with oxipurinol occurred in the renal tubules causing a considerable degree of renal dysfunction. Crystals were not found in any other tissue, including muscle, so that no evidence of guanine gout was noted, nor any other abnormality of purine metabolism which could be related to leg weakness in pigs.

Metabolism of intravenous adenine in the pig.

1. Adenine administered either parenterally or orally is less toxic to the pig than to other species; doses of 100 mg/kg are rapidly catabolised and excreted largely as soluble purine end-products in the urine. 2. The low toxicity is explained by the excretion of less than 1% of the dose as 2,8-dihydroxyadenine. 3. These results suggest that adenine dosages which give rise to kidney damage must be above a threshold-like level which varies in the different mammalian species, and is higher in the pig than in the rat, dog, rabbit or man.

A comparative study of purine metabolism by human and pig erythrocytes in vitro

Abstract Comparative rates of riboside and ribotide formation have been studied in pig and human intact and haemolysed erythrocytes in vitro by means of [ 14 C]labelled bases and electrophoretic separation of metabolites. In a physiological buffer system, ribosides were the major metabolites of hypoxanthine, guanine and xanthine in human intact cells, but pig erythrocytes showed little formation of metabolites in this system. When ribose-1-phosphate was added to a medium employing ribose in place of glucose, nucleoside formation by the pig erythrocytes exceeded that of the human cells, but the order of specificity, Gu > Hx > X > Allopurinol was common to both and no adenosine was formed by either. In either buffer system in both human and pig intact erythrocytes nucleotide formation was exceedingly low and allopurinol ribotide was not detectable. When intact cells were replaced by an equivalent volume of haemolysed cells, however nucleotides formed the major metabolites in both species in the presence of PRPP. In the pig erythrocyte PRPP and ribose-1-phosphate levels were exceedingly low and likewise the pig cell was capable of synthesizing these compounds at only minimal rates compared with humans. These results are discussed in relation to studies concerning the metabolic fate of allopurinol in pigs.

Valine oxidation: the synthesis and evaluation of l -[3-H]valine as a tracer in vivo

The suitability of L-[3-3H]valine for measuring valine oxidation was studied by comparing its oxidation rate with that of L-[1-14C]valine in rats and pigs. L-[3-3H]valine was synthesized by removal of the tritium on carbon-2 of L-[2,3-3H]valine by acetylation. The acetyl group was removed enzymatically using pig renal acylase 1 (EC 3.5.1.14) and the product was purified by ion-exchange and paper chromatography. For the first rat experiment L-[3-3H]valine was synthesized in our laboratory; for the subsequent experiments it was produced by Amersham International plc. In the first experiment in rats the two tracers were given by injection and 14CO2 was collected for 2 h. The oxidation of tritiated valine was significantly higher than that of L-[1-14C]valine. In a second experiment there was no difference. This was probably due to the higher purity of the labelled valine which, for the second experiment, was shown by nuclear magnetic resonance to contain only one tritium atom. In a study with pigs in which the two tracers were given by continuous infusion there was no significant difference between them in flux or oxidation. The results of this experiment were used to evaluate a model to estimate amino acid requirements. With pigs given a methionine-limiting diet a reduction in methionine intake, by reducing protein accretion, increased valine oxidation by the same proportion.

The Pig as an Animal Model for Purine Metabolic Studies

Small laboratory rodents have many disadvantages for the study of purine metabolism in relation to human disease. They normally pass their purine metabolites in much smaller volumes of urine both in relation to bodyweight and filtration rate (1); in addition, the distribution of enzymes of purine catabolism in these species differs from man (2,3). The pig in contrast has a kidney which structurally and functionally resembles human kidneys closely (4,5), and the distribution of xanthine oxidase and guanase is similar to man (2,3). Because of these advantages, and the finding of “guanine gout” in pigs (6), we studied purine metabolism in this animal (7). The pig has proved a much better model.

Effects of allopurinol and oxonic acid on pyrimidine metabolism in the pig.

Pigs given allopurinol showed an increase in urinary orotic acid and orotidine excretion from a mean of 5 mg to a mean of 50 mg/24 hours. Although the dose of allopurinol was constant the levels of orotic acid and orotidine fell gradually from their initial peak. When the drug was stopped levels returned rapidly to normal. These results are similar to those obtained in rat and man (1), where this effect has been attributed to inhibition of the enzyme orotidylic decarboxylase (2).

The Effect of Allopurinol on Oral Purine Absorption and Excretion in the Pig

Allopurinol therapy in hyperuricaemic man has been shown to be advantageous from two points of view. Firstly, it reduces urinary uric acid excretion and increases the excretion of the precursor purines xanthine and, to a lesser extent, hypoxanthine. In addition, total urinary purine excretion (the sum of these three) may be reduced by as much as 50% during allopurinol therapy (1). This latter effect has been attributed to the formation of nucleotides of either hypoxanthine (1) or allopurinol itself (2), which in turn exert a “feed back” inhibitary effect on the first enzyme of de novo purine synthesis.