In vitro and ex vivo xanthine oxidoreductase activity in rat and guinea-pig hearts using hypoxanthine or xanthine as substrate.
Through oxyradical formation xanthine oxidoreductase (XOD) could play a role in the etiology of cardiac damage. Its measurement poses problems, due to little substrate specificity, self-inactivation and endogenous inhibitors. Perfusion of guinea-pig hearts with hypoxanthine gave rise to only little xanthine release; in contrast rat hearts showed vivid xanthine production. Therefore, xanthine breakdown was hypothesized to exceed its formation in guinea-pig hearts. The kinetics of both substrates for XOD in cardiac homogenates were therefore compared with those obtained in perfused hearts. Oxypurine contents and effluent catabolites were determined by HPLC. Regardless of substrate, Vmax values in homogenates were about 38 and 13 mU/g for rat and guinea-pig heart, respectively. Km values were in the 3-5 microM range; therefore the hypothesis concerning the low xanthine release in guinea-pig hearts must be rejected. Activities in hearts perfused with hypoxanthine (50 microM) were 40 and 18 mU/g for rat and guinea pig, respectively; perfusion with xanthine produced < 50% of the activities observed with hypoxanthine (p < 0.002). Intracellular xanthine concentration, estimated from sorbitol distribution space and myocardial xanthine content was negative in both species, contrasting intracellular hypoxanthine levels, which approached extracellular concentrations. This disparate distribution indicates that hypoxanthine transport across the cell membrane far exceeds that of xanthine. Consequently, hypoxanthine is preferable to xanthine as substrate in perfused hearts to estimate XOD activity in situ.
Janssen
M
M
Cardiochemical Laboratory, Erasmus University Rotterdam, The Netherlands.
Tavenier
M
M
Koster
J F
JF
de Jong
J W
JW
D000818:Animals; D006168:Guinea Pigs; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism*; D066298:In Vitro Techniques; D009206:Myocardium / Q000201:enzymology*; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D019820:Xanthine; D014968:Xanthine Dehydrogenase / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism*
D007042:Hypoxanthines; D014970:Xanthines; D019820:Xanthine; D019271:Hypoxanthine; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase