Janos M. Kanczler

A Reappraisal of Xanthine Dehydrogenase and Oxidase in Hypoxic Reperfusion Injury: the Role of NADH as an Electron Donor

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.

Antibacterial properties of xanthine oxidase in human milk

Formula-fed babies contract gastroenteritis more than breast-fed babies, which is of concern to mothers who cannot breastfeed or, as with HIV-infected mothers, are discouraged from breastfeeding. The ability of endogenous breastmilk xanthine oxidase to generate the antimicrobial radical nitric oxide has been measured and its influence on the growth of Escherichia coli and Salmonella enteritides examined. Breastmilk, but not formula feed, generated nitric oxide. Xanthine oxidase activity substantially inhibited the growth of both bacteria. An important natural antibiotic system is missing in formula feeds; the addition of xanthine oxidase may improve formula for use when breastfeeding is not a safe option.

Xanthine oxidase is a peroxynitrite synthase: newlyidentified roles for a very old enzyme

Xanthine oxidase (XO) was first identified in 1902 by the German scientist Schardinger in bovine milk by following the hydroxylation of hypoxanthine to xanthine. 1 Following this first discovery, the history of xanthine oxidase has followed the usual routes of investigation such as they were at the start of the 20th century. The electron donors and acceptors were identified and the constituents of the active sites were deduced. It turns out to be a classic, multicentred, redox enzyme. For many years the function of the enzyme in milk was questioned and various theories were proffered. These ideas ranged from being a carrier of iron and molybdenum to the infant, to purely an additional dietary protein, a notion still prevalent in the medical profession today. XO was thought not to have an enzymic role in milk. However, further thought on the matter brings to light the high, almost neutral pH of the neonatal gut in the first few weeks post partum. The acid pH, which develops later in life, acts as the primary defence to infective organisms via ingestion. In this time before the acid develops, other systems must be in place. The role of a radical generating enzyme may, therefore, be in the reduction of infective bacteria by the generation of superoxide and hydrogen peroxide. Unfortunately, the potency of these reactive oxygen species is dependent on a number of factors including detoxification enzymes in bacteria, particularly superoxide dismutases and catalase, and the availability of oxygen in the environment. In 1996, we began to study the nitrate reductase activity of XO, a largely ignored phenomenon, which culminated in the first description of nitrate and nitrite reductase activities with the formation of nitric oxide (NO) as the end product. 2‐4 In the light of these findings and our further studies, we have developed and tested the theory that XO-generated reactive molecules have antibacterial activities in the neonatal gut (see Fig. 1). It raises the possibility that, in areas where breast-feeding is not possible due to HIV infection in the mother, an alternative formula can be employed to reduce life-threatening diarrhoeal disease. This system acts as a low-cost, naturally occurring antibiotic for the reduction of infective bacteria. But first some background and comparative biochemistry.

Xanthine Oxidase Mediates Cytokine-induced, but not Hormone-induced Bone Resorption

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have been implicated as mediators of osteoclastic bone resorption. Xanthine oxidase (XO) a ubiquitous enzyme is widely known for its production of these ROS. We therefore evaluated the potential of XO as a source of ROS in cytokine- and hormone-induced bone resorption. XO activity in rat calvarial osteoblasts was found to be significantly elevated upon stimulation by the cytokines, TNF f and IL-1 g . These cytokines also caused a dose related increase in bone resorption of mouse calvariae, which was significantly inhibited by catalase (10 IU/ml). Allopurinol, the competitive inhibitor of XO, also caused a dose related (1-50 w M) inhibition of TNF f (20 ng/ml) and (0.01-10 w M) IL-1 g (50 IU/ml)-induced bone resorption, respectively. PTH- and 1,25-(OH)2 Vitamin D3-induced bone resorption could also be inhibited by catalase (100 IU/ml) but was unaffected by allopurinol, indicating that another mediator, other than XO, is required for hormone-induced bone resorption. These results demonstrate, that modulation of the redox balance in the bone microenvironment, which contains XO, can affect the bone resorbing process. Therefore, XO may play a pivotal role in cytokine-induced bone resorption and, if manipulated appropriately, could show a therapeutic benefit in inflammatory bone disorders such as RA.