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A reappraisal of xanthine dehydrogenase and oxidase in hypoxic reperfusion injury: the role of NADH as an electron donor.
Free Radic Res. 1998 Feb; 28(2):151-64.FR

Abstract

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.

Authors+Show Affiliations

Bone and Joint Research Unit, St Bartholomew's and the Royal London School of Medicine and Dentistry, University of London, UK. Z.ZHANG@MDS.QMW.AC.UKNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9645392

Citation

Zhang, Z, et al. "A Reappraisal of Xanthine Dehydrogenase and Oxidase in Hypoxic Reperfusion Injury: the Role of NADH as an Electron Donor." Free Radical Research, vol. 28, no. 2, 1998, pp. 151-64.
Zhang Z, Blake DR, Stevens CR, et al. A reappraisal of xanthine dehydrogenase and oxidase in hypoxic reperfusion injury: the role of NADH as an electron donor. Free Radic Res. 1998;28(2):151-64.
Zhang, Z., Blake, D. R., Stevens, C. R., Kanczler, J. M., Winyard, P. G., Symons, M. C., Benboubetra, M., & Harrison, R. (1998). A reappraisal of xanthine dehydrogenase and oxidase in hypoxic reperfusion injury: the role of NADH as an electron donor. Free Radical Research, 28(2), 151-64.
Zhang Z, et al. A Reappraisal of Xanthine Dehydrogenase and Oxidase in Hypoxic Reperfusion Injury: the Role of NADH as an Electron Donor. Free Radic Res. 1998;28(2):151-64. PubMed PMID: 9645392.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A reappraisal of xanthine dehydrogenase and oxidase in hypoxic reperfusion injury: the role of NADH as an electron donor. AU - Zhang,Z, AU - Blake,D R, AU - Stevens,C R, AU - Kanczler,J M, AU - Winyard,P G, AU - Symons,M C, AU - Benboubetra,M, AU - Harrison,R, PY - 1998/6/30/pubmed PY - 1998/6/30/medline PY - 1998/6/30/entrez SP - 151 EP - 64 JF - Free radical research JO - Free Radic Res VL - 28 IS - 2 N2 - Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS. SN - 1071-5762 UR - https://www.unboundmedicine.com/medline/citation/9645392/A_reappraisal_of_xanthine_dehydrogenase_and_oxidase_in_hypoxic_reperfusion_injury:_the_role_of_NADH_as_an_electron_donor_ L2 - https://www.tandfonline.com/doi/full/10.3109/10715769809065801 DB - PRIME DP - Unbound Medicine ER -