Xanthine oxidase homogenization

In 1975, the Life Sciences Research Office of the Federation of American Societies for Experimental Biology (Carr et al. 1975), upon an extensive review of the available evidence, concluded that it was doubtful whether XO in homogenized cow s milk was a causal or risk factor for heart disease. More recently, Clifford et al (1983) and Deeth (1983), in critical reviews of the homogenized cow s milk XO hypothesis, have arrived at a similar, if not more definitive, conclusion. As stated by Clifford et al. (1983), experimental evidence has failed to substantiate, and in many cases has refuted, the hypothesis that homogenized bovine milk xanthine oxidase intake or plasmalogen depletion are causal factors in the development of atherosclerosis. And, according to Deeth (1983), there appears to be no unequivocal evidence that the absorbed enzyme has any pathological effects that may contribute to development of atherosclerotic heart disease. ... 

Clark, A. J. and Pratt, D. E. 1976. Xanthine oxidase activity in rat serum after administration of homogenized bovine cream preparation. Life Sci. 19, 887-892. 

Clifford, A. J., Ho, C. Y. and Swenerton, H. 1983. Homogenized bovine milk xanthine oxidase A critique of the hypothesis relating to plasmalogen depletion and cardiovascular disease. Am J. Clin. Nutr. 38, 327-332. 

Nathans, G. R. and Hade, E. P. K. 1978. Bovine milk xanthine oxidase. Purification by ultrafiltration and conventional methods which omit addition of proteases. Some criteria for homogeneity of native xanthine oxidase. Biochim. Biophys. Acta 526, 328-344. 

In addition to liver aldehyde dehydrogenase, a number of other enzymes present in the soluble fraction of liver homogenates will oxidize aldehydes and certain N-heterocyclic compounds. Among these are aldehyde oxidase and xanthine oxidase (see below), both flavoprotein enzymes containing molybdenum. These enzymes catalyze the oxidation of aldehydes formed by the deamination of endogenous amines by amine oxidases. 

Fig. 10. Glycated Cu,Zn-SOD in streptozotocin-induced diabetic rat lens. Pooled lenses from normal and streptozotocin-induced diabetic rats [9 weeks after intravenous injection of streptozotocin (STZ)] were homogenized, and Cu,Zn-SOD was extracted by chloroform/ethanol and subjected to a boronate affinity column. Upper panel, normal rat lens lower panel, diabetic rat lens. The SOD activity and immunoreactive Cu,Zn-SOD were measured using the xanthine/xanthine oxidase method and ELISA, respectively.

Whole brain homogenates have been found to contain very low levels or to be devoid of either of the molybdenum hydroxylases [92, 107, 108, 120]. However, more specific assays for xanthine oxidase gave values of 2-20 nmol xanthine transformed/mg per h for homogenates of cortex or whole brain from mouse, rat, guinea-pig, rabbit and cow [46, 121], Less activity was detected in the cerebellum [121] and, again, the cranial capillary endothelial cells were found to be enriched in xanthine oxidase [46], Brain hypoxanthine concentrations are reported to rise during ischaemia due to increased ATP breakdown, and Betz [46] proposed that brain capillaries may be susceptible to damage... 

Martin also has a word about the harm from the use of homogenized milk, as per the work of cardiologist Kurt Oster, M.D., of Fairfield University. Thus, natural milk contains xanthine oxidase, an oxidizing enzyme, in large particles, which, consequently, do not get into the bloodstream. Homogenization reduces the particle size, allowing entry into the bloodstream, which Oster claims is harmful to the vascular system. 

Livers are thawed, weighed, homogenized with Tris-HCl (5mM containing 2-mM EDTA, pH 7.4), and centrifuged (1000g, 10min, 4°C), and MDA and SOD in the supernatant are immediately analyzed. MDA in liver tissue is determined by the thiobarbituric acid method [72]. The assay for total SOD is based on its ability to inhibit the oxidation of oxyamine by the xanthine-xanthine oxidase system. The absorbance of the red nitrite produced by the oxidation of oxyamine is determined at 550 nm. 

The inhibition of lipid peroxidation by ciimari-zine seems to be independent of the oxidant system used to induce the peroxidation, as was verified when using xanthine oxidase and iron (Janero et al. 1988), as well as simple exposure to air (for rat liver homogenates Fernandes et al. 1991), copper (for human plasma and erythrocytes Fernandes et al. 1991) or H2O2 (for human erythrocytes Fernandes et al. 1991). 

Xanthine oxidase, found in liver and in milk, has been purified from chicken and calf liver and has been crystallized from milk. The crystalline enzyme (mol wt 290,000) is homogeneous in the ultracentrifuge and on electrophoresis. Each protein molecule contains two molecules of FAD. The enzyme also contains iron and molybdenum. The role of xanthine oxidase is generally assumed to be to oxidize xanthine. However, an almost complete deficiency of xanthine oxidase in xanthinuria has been described, and no symptoms were observed except for the formation of xanthine stones. 

Xanthine oxidase activity of the duodenal mucosa obtained by gastrofiberscopy was determined by the following procedures. Tissue specimens weighing 5-6 mg were homogenized in pyrophosphate buffer solution (pH 8.0, 0.06M) and centrifuged at 10,000 g for 40 minutes. 

Figure 2. SDS-PAGE showing a single protein band (XO) of purified xanthine oxidase coincident with an immunoprecipitate. Forty yg of purified enzyme (A) and 60 yg of an immunoprecipitate (B) prepared from antiserum and crude liver homogenate were applied to gels. Gels were stained with Coomassie blue. Immunoglobulin heavy (HC) and light (LC) are seen.

Figure 3. Ouchterlony immunodiffusion, (C) Crude liver homogenate. (1,3,5) Antiseriam from a single rabbit. (2,4) Control Sera. Single immunoprecipitin band was stained for xanthine oxidase activity.

Figure 4. Standard curve showing parallelism between purified xanthine oxidase and serial dilutions of crude liver homogenate.

In an in vitro experiment with liver homogenate (Dunn et al. 1981b), mercury volatilization activity was enhanced by the addition of ethanol but disappeared on heating. The highest mercury volatilization activity was found in the cytosol fraction. However, the actual entity responsible for the reduction of mercury to its volatile form is still unclear. Ogata et al. (1987) reported a higher rate of mercury volatilization in vitro in the presence of a superoxide anion-producing system such as xanthine/xanthine oxidase. 

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