Research Note: Antioxidant Activity of Japanese Quail Liver Cytosol in the Absence and Presence of Reduced Glutathione

Research Note: Antioxidant Activity of Japanese Quail Liver Cytosol in the Absence and Presence of Reduced Glutathione

RESEARCH NOTES Research Note: Antioxidant Activity of Japanese Quail Liver Cytosol in the Absence and Presence of Reduced Glutathione M. E. SPURLOCK1 ...

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RESEARCH NOTES Research Note: Antioxidant Activity of Japanese Quail Liver Cytosol in the Absence and Presence of Reduced Glutathione M. E. SPURLOCK1 and J. E. SAVAGE2 Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211 (Received for publication July 5, 1991)

1992 Poultry Science 71:928^931

and nonenzymatic biological reactions (Wefers and Sies, 1983), as a consequence Fatty liver hemorrhagic syndrome of xenobiotic metabolism (Taylor et ah, (FLHS) is a metabolic abnormality of 1988) and as products of oxidative metablaying chickens (see Butler, 1976 for a olism (Chow, 1979). Oxygen metabolism detailed review) and Japanese quail imposes on respiring cells the need to (Mateo, 1980; Rhoades, 1985; Spurlock and maintain complex antioxidative mechaSavage, 1988). Studies conducted in the nisms to protect against the deleterious authors' laboratory have shown the inci- effects of oxygen (Combs, 1981). dence of FLHS increased in laying hens Gibson et al. (1985) have identified a and Japanese quail fed diets containing cytosolic protein in rat liver that protects marginal or deficient sulfur amino acid rat liver microsomal lipids from peroxida(SAA.) concentrations (Mateo, 1980; tion. The 15-kDa protein requires Rhodes, 1985; Spurlock, 1989). Given the glutathione (GSH) or related thiols, such increased metabolic demands placed on as cysteine, mercaptoethanol, and the liver during egg production, the dithiothreitol, to sustain activity. In their authors have hypothesized that FLHS in vitro system the thiols alone were totally occurs in hens fed SAA-deficient diets as a inactive, suggesting a catalytic function for consequence of oxidative damage to the the protein. Treatment of the protein with liver vascular and parenchymal tissue. iodoacetate abolished activity, leading to The increased use of oxygen during egg the speculation that the protein itself production and the related increased pro- contains sulfhydryl groups that exert anduction of oxygen free radicals may con- tioxidant activity temporarily. tribute to the development of FLHS. Free The postulated oxidative damage that radicals are generated in both enzymatic occurs in FLHS could result from lack of reducing potential, i.e., lack of GSH, or from lack of a protein such as described by Gibson et al. (1985). The objective of the Present address: Department of Animal Science, present study was to determine whether Purdue University, West Lafayette, IN 47907. To whom correspondence should be addressed. or not Japanese quail liver contains a INTRODUCTION

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ABSTRACT The antioxidant activity of Japanese quail liver cytosol was assessed in the presence of .1,1.0, or 10.0 mM reduced glutathione (GSH) using an in vitro assay system that utilized quail liver microsomes as the lipid substrate. The formation of malonaldehyde (MA), an indicator of lipid peroxidation, was reduced (P<.01) by 1.0 and 10.0 mM GSH, even in the absence of cytosolic protein. Cytosolic protein at a level of 1 mg/mL of assay medium did not further reduce MA formation at any of the GSH concentrations tested. Increasing the cytosolic protein concentration to 6 mg/mL decreased (P<.01) MA formation, even in the absence of added GSH. (Key words: Japanese quail, glutathione, liver cytosol, microsomes, oxidation)

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protein that, in conjunction with GSH, protects against peroxidation.

MATERIALS AND METHODS

Glutathione

Cytosolic protein

Malonaldehyde2

(ng/mg microsomal (mM) protein) .1 44 ± 9 + 41 ± 4 1.0 13 ± 4 + 18 ± 7 10.0 11 ± 5 + 14 ± 5 The incubation system contained .4 mM adenosine diphosphate, 12 \lM FefJH), and .66 mM ascorbic acid. The presence of cytosolic protein (1 mg/mL) is indicated by a +. 2 Adding cytosolic protein did not reduce (P>.05) malonaldehyde production at any glutathione concentration.

of GSH to regenerate the antioxidant activity of cytosolic protein. The basal incubation solution contained .4 m M adenosine diphosphate, 12 uM Fe(III), and .66 mM ascorbic acid. Solutions were prepared in .15 M potassium phosphate buffer, p H 7.5. The concentrations of GSH and cytosolic protein used are indicated in Table 1. All incubation reagents were prepared just prior to use. The assay solution was incubated in a water bath at 37 C for 1 h. An equal volume of 1% thiobarbituric acid (TBA) was added following removal of the microsomes by centrifugation. The solution was mixed with a vortex mixer, heated in a boiling water bath for 30 min, and filtered through a .2-(i methanolresistant filter. Malonaldehyde (MA) was quantified as the TBA complex as described by Bird et al. (1983). Data are reported as nanograms of MA per milligram of microsomal protein. The data were analyzed using the Student's * test (Snedecor and Cochran, 1980).

RESULTS Initially, the antioxidant activity of cytosolic protein (1 m g / m L assay volume) was assessed in the absence and presence of 10 m M GSH. Cytosolic protein alone

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Eight mature Japanese quail hens were selected randomly for each experiment from the breeder colony maintained by the authors' laboratory. Two quail livers were used for microsomal membrane preparation and six for preparation of cytosol. Liver microsomes were prepared as described by Flohe and Zimmerman (1970). All centrifugations were performed at 4 C. Following the final centrifugation, the supernatant fraction was discarded, the tube covered, and the microsomal pellet stored at -20 C until used. Immediately prior to use, the microsomes were resuspended in 2 to 3 mL of the potassium phosphate buffer and heated to 100 C for 10 min to inactivate the factor, which inhibits lipid peroxidation in the presence of GSH (Gibson et al, 1985). Microsomal protein was assayed prior to use according to the bicinchoninic acid (BCA) method (Smith et al, 1985). Liver cytosol was prepared by the method of McCay et al. (1976). All sedimentations w e r e carried out in refrigerated centrifuges maintained at 4 C. Following the final centrifugation, the supernatant fraction was collected, care being taken not to contaminate the cytosol with lipid suspended on top of the aqueous fraction, placed in clean tubes, and kept on ice. The cytosol was treated for 30 min with .25 mM (final concentration) 2-mercaptoethanol to reduce potential protein sulfhydryls (Gibson et al, 1985). The cytosol (3 to 4 mL) was placed in dialysis tubing (molecular weight cutoff = 8 kDa) and dialyzed overnight against approximately 2 L of the Tris-HCl buffer. The dialysis was carried out in a cold room at 4 C. Cytosolic protein was measured using the aforementioned BCA method. The antioxidant activity of the cytosol was measured using the incubation system of Gibson et al. (1985). However, smaller amounts of cytosolic protein (1 to 6 m g / m L assay volume) were tested so that emphasis was placed on the capacity

TABLE 1. Production of malonaldehyde (x ± SEM) in file absence and presence of cytosolic protein at different glutathione concentrations1

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DISCUSSION The ability of GSH to quench peroxidation of quail microsome lipid in the absence of cytosolic protein was surprising and complicated efforts to determine whether an antioxidant cytosolic protein exists in quail liver. Gibson et al. (1985) reported that GSH alone had no antioxidant effect. The independent antioxidant activity exerted by GSH in the present study indicates that the thiol reacted directly to prevent peroxidation or that the microsomal system was contaminated with the protein, although the latter seems highly unlikely. Microsomes are vesicles formed from endoplasmic reticulum following disruption (Gutteridge, 1987) and could contain proteins common to the cytosol. However, presence of enzymatic activity seems unlikely when one considers the pre-experimental heat treatment applied to the microsomal preparation. In agreement with present observations, a direct scavenging of radicals by GSH has been reported by Wefers and Sies (1983). It is possible that the difference between the present observations and those of Gibson et al. (1985) are due to the different sources of microsomes. It is also possible that the antioxidant activity of GSH is related to the postexperimental heat treatment applied to facilitate the formation of the MA-TBA chromogen. A substantial portion of the MA complexed with TBA may form during the

heating process rather than in experimental incubation period (Gutteridge, 1987). The authors attempted to minimize MA formation during the heating process by removing the microsomes prior to heating. However, if all lipid substrate was not removed, the antioxidant activity of GSH may be indicative of the thiol reducing available iron or quenching other oxidants during the heating process. Consequently, oxidation of residual lipid would be lessened and MA formation decreased. If the latter is true, perhaps one way to minimize MA formation during the heating process, provided GSH is not a part of the experimental design, is to add the thiol prior to heating. Consistent with the results of Gibson et al. (1985), a substantial reduction in MA formation occurred when the cytosolic protein concentration was increased to 6 mg/mL. It is possible that the reduction in MA associated with the higher concentration of cytosolic protein was because of endogenous GSH. Analysis of dialyzed cytosol indicated that approximately 70% of the GSH was removed by dialysis. Although autoxidation of GSH to the disulfide proceeds rapidly in alkaline solutions, perhaps enough GSH remained to regenerate the antioxidant activity of cytosolic protein, which became a p p a r e n t when its concentration was increased. However, the nature of the protein identified by Gibson et al. (1985) is such that its maintenance of antioxidant activity relies on an electron donor. As stated by the authors, their experiments demonstrate that GSH has the property of preserving the inhibitory activity of the cytosolic factor and that this property is particularly apparent w h e n the concentration of cytosol in the system is relatively low. Hence, it seems likely that an effect of GSH would have been more apparent at the low (1 mg) concentration of cytosolic protein, at which the antioxidant activity of the cytosol itself was minimal. Using the assay system described, a protein similar to the one described by Gibson et al. (1985) could not be demonstrated in cytosol prepared from Japanese quail liver. However, the existence of such a protein cannot be ruled out. Based on the variation observed, the power to

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did not decrease MA formation. However, 10 mM GSH prevented the formation of measurable amounts of MA, even in the absence of cytosolic protein (data not shown). The results of another experiment are presented in Table 1. Glutathione alone reduced (P<.01) MA formation when added to the assay system at 1.0 and 10 mM. Adding 1 m g / m L cytosolic protein did not further decrease MA formation at any of the GSH concentrations tested. Finally, in the absence of added GSH, increasing the concentration of cytosolic protein from 1 to 6 m g / m L resulted in a decrease (P<.01) in MA formation (69 ± 11 versus 24 ± 6 ng M A / m g microsomal protein, x ± SEM).

RESEARCH NOTE

ACKNOWLEDGMENTS The authors wish to express their thanks to BioKyowa, Inc. (Chesterfield, M O 63017) for generously providing GSH and to Boyd C D e l l for allowing us to use his laboratory and equipment. REFERENCES Bird, R. P., S. O. Hung, M. Hadley, and H. H. Draper, 1983. Determination of malonaldehyde in biological materials by high-pressure liquid chromatography. Anal. Biochem. 128:240-244. Butler, E. J., 1976. Fatty liver diseases in the domestic fowl: A review. Avian Pathol. 5:1-14. Chow, C. K., 1979. Nutritional influence on cellular

antioxidant defense systems. Am. J. Clin. Nutr. 32:1066-1081. Combs, G. F., Jr., 1981. Influences of dietary vitamin E and selenium on the oxidant defense system of the chick. Poultry Sci. 60:2098-2105. Flohe, L., and R. Zimmerman, 1970. The role of GSH peroxidase in protecting the membrane of rat liver mitochondria. J. Biol. Chem. 243:2288-2295. Gibson, D. D., J. Hawrylko, and P. B. McCay, 1985. GSH-dependent inhibition of lipid peroxidation: Properties of a potent cytosofic system which protects cell membranes. Lipids 20:704-711. Gutteridge, J.M.C., 1987. Intracellular production of oxygen-derived free radicals. Pages 9-19 in: Oxygen Radicals and Tissue Injury. Proceedings of the Brook Lodge Symposium. Augusta, MX Mateo, C. D., 1980. Influence of dietary modification on the incidence of fatty liver hemorrhagic syndrome in Japanese quail and laying hens. Ph.D. dissertation, University of Missouri, Columbia, MO. McCay, P. B., D. D. Gibson, K. Fong, and K. R. Hornbrook, 1976. Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes. Biochim. Biophys. Acta 431: 459-468. Rhoades, J. F., 1985. Dietary modifications and fatty liver hemorrhagic syndrome in laying hens and Japanese quail. M.S. thesis, University of Missouri, Columbia, MO. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk, 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85. Snedecor, G. W., and W. G. Cochran, 1980. Statistical Methods. 7th ed. Iowa State University Press, Ames, IA. Spurlock, M. E., 1989. The effects of cellular antioxidants on the incidence of fatty liver hemorrhagic syndrome. Ph.D. dissertation, University of Missouri, Columbia, MO. Spurlock, M. E., and J. E. Savage, 1988. Effects of glutathione supplementation and dietary protein on plasma and liver glutathione and incidence of fatty liver hemorrhagic syndrome. Poultry Sci. 67(Suppl. l):159.(Abstr.) Taylor, C. G., W. J. Bettger, and T. M. Bray, 1988. Effect of dietary zinc or copper deficiency on the primary free radical defense system in the rat. J. Nutr. 118:613-621. Wefers, H., and H. Sies, 1983. Oxidation of glutathione by the superoxide radical to the disulfide and the sulfonate yielding singlet oxygen. Eur. J. Biochem. 13729-36.

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detect a 3-ng difference (the difference observed at .1 m M GSH) between treatment means was approximately .36. However, the power to detect differences as large as those attributed to GSH was considerably greater (>.99). Species variation in cytosolic concentrations of the various antioxidant proteins is one possible explanation for the different results obtained by the present authors as compared with those of Gibson et al. (1985). In the present study, cytosolic protein at concentrations as low as 6 m g / m L decreased MA formation, whereas Gibson et al. (1985) reported that concentrations as high as 12 m g / m L did not. This difference may be indicative of species variation or subtle differences in the execution of the protocol, i.e., dialysis procedures. The key difference in the present results, as compared with those of Gibson et al. (1985), is that GSH alone decreased oxidation greatly. It seems that future studies will have to be conducted in such a w a y that the independent activity of GSH can be distinguished clearly from that of any GSH-dependent protein contained in the cytosol.

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