A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

Zhongqin Li, Ruizhang Guan, Hongwei Liu

Fisheries College of Jimei University; Engineering Research Center of Eel Modern Industrial Technology, Ministry of Education, Xiamen, China.

DOI: 10.4236/ojmc.2013.31004                

Abstract

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Keywords

Xanthine Oxidase (XO); Enzyme Activity Assay; Reversed-Phase High Performance Liquid Chromatography (RP-HPLC)

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	R. Harrison, “Structure and Function of Xanthine Oxidoreductase [J],” Free Radical Biology & Medicine, Vol. 33, No. 6, 2002, pp. 774-797. doi:10.1016/S0891-5849(02)00956-5
[2]	S. A. Sanders, R. Eisenthal and R. Harrison, “NADH Oxidase Activity of Human Xanthine Oxidoreductase— Generation of Superoxide Anion [J],” European Journal of Biochemistry, Vol. 245, No. 3, 1997, pp. 541-548. doi:10.1111/j.1432-1033.1997.00541.x
[3]	F. Borges, E. Fernandes and F. Roleira, “Progress towards the Discovery of Xanthine Oxidase Inhibitors [J],” Current Medicinal Chemistry, Vol. 9, No. 2, 2002, pp. 195-217. doi:10.2174/0929867023371229
[4]	S. H. Giler, O. Sperling, S. Brosh, et al., “Serum Xanthine Oxidase in Jaundice [J],” Clinica Chimica Acta, Vol. 63, No. 1, 1975, pp. 37-40. doi:10.1016/0009-8981(75)90375-7
[5]	S. H. Giler, Y. Eshel and J. Pinkhas, et al., “Elevation of Serum Xanthine Oxidase Activity Following Halothane Anesthesia in Man [J],” Experientia, Vol. 33, No. 10, 1977, pp. 1356-1358. doi:10.1007/BF01920178
[6]	S. Tan, R. Radi and F. Gaudier, et al., “Physiological Levels of Uric Acid Inhibit Xanthine Oxidase in Human Plasma [J],” Pediatric Research, Vol. 34, No. 5, 1993, pp. 303-307. doi:10.1203/00006450-199309000-00013
[7]	A. H. Cory, T. C. Owen, J. A. Barltrop, et al., “Use of an aqueous Soluble Tetrazolium/Formazan Assay for Cell Growth Assays in Culture [J],” Cancer Communications, Vol. 3, No. 7, 1991, pp. 207-212.
[8]	R. Fried, “Xanthine Dehydrogenase [J],” Analytical Biochemistry, Vol. 16, No. 3, 1966, pp. 427-432. doi:10.1016/0003-2697(66)90224-7
[9]	G. G. Guilbault, D. N. Kramer and P. L. Cannon, “Electrochemical Determination of Organophosphorus Compounds and Analysis of Xamhine Oxidase and Sulfydryl Inhibitor [J],” Analytical Chemistry, Vol. 36, No. 7, 1964, pp. 606-614. doi:10.1021/ac60209a015
[10]	A. Weinstein, G. Mendes and G. Litwack, “Isotopic Method for Tyrosine Transaminase Activity [J],” Analytical Biochemistry, Vol. 21, No. 1, 1967, pp. 86-97. doi:10.1016/0003-2697(67)90086-3
[11]	T. Sasaoka, N. Kaneda and T. Nagatsu, “Highly Sensitive Assay for Xanthine Oxidase Activity by High-Performance Liquid Chromatography with Fluorescence Detection [J],” Journal of Chromatography B, Vol. 424, No. 2, 1988, pp. 392-397.
[12]	T. Yamamoto, Y. Moriwaki and S. Takahashi, et al., “Determination of Human Plasma Xanthine Oxidase Activity by High-Performance Liquid Chromatography [J],” Journal of Chromatography B, Vol. 681, No. 2, 1996, pp. 395-400. doi:10.1016/0378-4347(96)00071-0
[13]	Y. Xia and J. L. Zweier, “Substrate Control of Free Radical Generation from Xanthine Oxidase in the Postischemic Heart [J],” Journal of Biological Chemistry, Vol. 270, No. 32, 1995, pp. 18797-18803. doi:10.1074/jbc.270.32.18797
[14]	Y. Xia, G. Khatchikian and J. L. Zweier, “Adenosine Deaminase Inhibition Prevents Free Radical-Mediated Injury in the Postischemic Heart [J],” Journal of Biological Chemistry, Vol. 271, No. 17, 1996, pp. 10096-10102. doi:10.1074/jbc.271.17.10096
[15]	G. Buonocore, S. Perrone and M. Longini, et al., “Total Hydroperoxide and Advanced Oxidation Protein Products in Preterm Hypoxic Babies [J],” Pediatric Research, Vol. 47, No. 2, 2000, pp. 221-224. doi:10.1203/00006450-200002000-00012
[16]	U. Al-Khalidi, S. Nasrallah, A. K. Khachadurian, et al., “A Sensitive Method for the Determination of Xanthine Oxidase Activity [J],” Clinica Chimica Acta, Vol. 11, No. 1, 1965, pp. 72-77. doi:10.1016/0009-8981(65)90091-4
[17]	A. McHale, H. Grimes and M. P. Coughlan, “Human Serum Xanthine Oxidase: Fluorometric Assay Applicable to the Investigation of Liver Disorders [J],” International Journal of Biochemistry, Vol. 10, No. 4, 1979, pp. 317-319. doi:10.1016/0020-711X(79)90096-X
[18]	J. S. Beckman, D. A. Parks, J. D. Pearson, et al., “A Sensitive Fluorometric Assay for Measuring Xanthine Dehydrogenase and Oxidase in Tissues [J],” Free Radical Biology and Medicine, Vol. 6, No. 6, 1989, pp. 607-615. doi:10.1016/0891-5849(89)90068-3
[19]	M. G. Battelli, A. Abbondanza, S. Musiani, et al., “Determination of Xanthine Oxidase in Human Serum by a Competitive Enzyme-Linked Immunosorbent Assay [J],” Clinica Chimica Acta, Vol. 281, No. 1-2, 1999, pp. 147-158. doi:10.1016/S0009-8981(98)00220-4
[20]	A. Atlante, D. Valenti, S. Gagliardi, et al., “A Sensitive Method to Assay The Xanthine Oxidase Activity in Primary Cultures of Cerebellar Granule Cells [J],” Brain Research Protocols, Vol. 6, No. 1-2, 2000, pp. 1-5. doi:10.1016/S1385-299X(00)00030-1