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Aberration of heme and hemoprotein in aged female rats
Mechanisms of Ageing and Development, 38 (1987) 189-197 Elsevier Scientific Publishers Ireland Ltd.
189
ABERRATION OF HEME AND HEMOPROTEIN IN AGED FEMALE RATS
MILAD S. BITAR and BERNARD H. SHAPIRO University of Pennsylvania, Department of Animal Biology, Laboratories of Biochemistry, School of Veterinary Medicine, Philadelphia, PA 19104-6048 (U.S.A.) (Received October 14th, 1986)
SUMMARY The magnitude and duration of drug action is determined partially by the activity of the drug metabolizing enzyme systems in the liver. The pharmacological effectiveness of many drugs is altered during the aging process. In this study, the regulation of heme metabolism and hemoprotein content was examined in livers of aged female rats. The activities of hexobarbital hydroxylase and aniline hydroxylase, indicators of m o n o oxygenase function, were decreased in aged rats by 31% and 24%, respectively, as compared to values in young rats. This was accompanied by a proportional decrease in the level of cytochrome P-450 (26%). Additionally, the activity of 8-aminolevulinic acid syhthetase (ALA-S), the rate-limiting enzyme in heme synthesis, and the microsomal concentration of heme were also decreased by 33% and 26%, respectively, in these animals. In contrast, the basal activity of microsomal heme oxygenase (MHO), the rate-limiting enzyme in heme degradation, and the percent heme saturation of tryptophan pyrrolase (TPO), a sensitive indicator of changes in the availability of heme in the "regulatory" heme pool, were increased by (87%) and (31%), respectively, in the aged rats. The serum concentration of bilirubin, an indicator of erythrocyte breakdown and/or liver function was likewise increased in these animals. In view of these findings, we suggest that the high activity of MHO and the low level of ALA-S may be a significant causative factor for the decreased microsomal concentration of heme, cytochrome-P-450 and its dependent monooxygenase activities in senescent female rats.
Key words: Heme metabolism; Cytochrome P-450; Aging
Address all correspondence to." Miiad S. Bitar, University of Pennsylvania, Department of Animal Biology, Laboratories of Biochemistry, School of Veterinary Medicine, Philadelphia, PA 19104-6048, U.S.A. 0047-6374/87/$03.50 Printed and Published in Ireland
© 1987 Elsevier Scientific Publishers Ireland Ltd.
190 INTRODUCTION The ability to respond to xenobiotics is altered during the aging process. Early investigations of drug metabolism in aged rats have demonstrated that the altered response to chemicals could be due, in part, to decreases in hepatic biotransformation of drugs, as was observed in vitro and in vivo experiments [1-2]. In this regard, it has been reported that the level of cytochrome P-450 and its dependent monooxygenase activities are decreased in liver microsomes from aged rats [3-5]. Furthermore, assessments of drug metabolism of antipyrine, aminopyrine and propranolol were faster in younger subjects than in the elderly [6-8]. These authors suggested that the activities of drug metabolizing enzymes may be decreased in humans, as in the case of aged rats. Most of the studies concerning drug biotransformation were conducted in aged male rats. Because sex can influence hepatic disposition of xenobiotics [9], data on age-related changes in pharmacokinetics in one sex do not necessarily apply to the other. Since alteration in hepatic biotransformation of xenobiotics during the aging process could have an important pharmacokinetic and toxicological implication in the elderly, it is desirable, therefore, to learn more about the underlying biochemical mechanisms of these age-related changes in cytochrome P-450 and its dependent monooxygenase activity. In the light of previous studies showing that heme plays a cardinal role in the regulation of cytochrome P-450 [10-14] and of the lack of information concerning heme metabolism during aging, we have examined the relationship between heme biosynthetic and degradative capabilities and monooxygenase activities in senescent female rats. MATERIALSAND METHODS Animals Female Fisher-344 rats were obtained from a colony maintained under barrier husbandry conditions by Charles River Breeding Laboratories (Wilmington, MA). In order to study a representative sample of each age group, all surviving animals that were received were used, with the exception of a few that were obviously debilitated or had large tumor masses that contributed substantially to their body weight, and thus could possibly alter drug metabolism. Animals were maintained under standardized conditions of 12 h light/12 h dark and temperature (22°C) on a diet of laboratory chow and water for the duration of the study. They were housed 2 per cage on hardwood bedding chips in clear plastic cages. Their ages are designated as follows: young, 3 - 4 months old; and old or senescent, 3 0 - 3 2 months old. Tissue Preparation Livers were perfused with ice-cold 0.9% saline, then removed, weighed and minced into small pieces and homogenized in three volumes of cold 0.15 M KCI containing 50 mM Tris-HC1 (pH 7.4). Microsomes were isolated as described previously [15].
191
Heme Synthesis ( 8-Aminolevulinic Acid Synthetase, ALA-S] ALA-S activity was measured by the method of Sassa et al. [16] as modified by Bitar and Weiner [ 17].
Heme Degradation (Microsomal Heme Oxygenase, MHO) MHO activity was measured by the method of Maines and Kappas [18] as modified by Bitar and Weiner [17]. This assay measures the amount of bilirubin formed from biliverdin, using the 100 000 g supernatant solution from young animals as the source of biliverdin reductase.
Heme and Hemopro teins ( Cy t ochrome P-450 and bs and Drug Metabolizing Enzymes] Cytochrome P-450 was determined from the reduced carbon monoxide difference spectrum according to the method of Omura and Sato [19]. Cytochrome b5 was measured as described previously [ 19]. The activity of aniline hydroxylase was measured by the formation of p-aminophenol (PAP) according to the procedure of Mieyal and Blumer [20] using approximately 2 mg of microsomal protein and 0.45 mM aniline in a total volume of 1 ml. The activity of hexobarbital hydroxylase was measured by the method of Kupfer and Rosenfeld [21] as modified by Shapiro and Szczotka [22].
Tryptophan Pyrrolase ( TPO) The activity of TPO was determined in liver cytosol [17] either in the absence (holoenzyme activity) or in the presence (total enzyme activity) of added methemoglobin. The percent heme saturation for TPO was expressed as the ratio of holoenzyme to total enzyme ×100. Berne
The microsomal concentration of heme was determined by the pyridine hemochromogen method of Falk [23]. Microsomal pellets were suspended in enough 0.05 M TrisHC1 buffer (pH 7.5) to result in 2.3 mg of protein/ml. The microsomal heme content was calculated from the difference in absorbance between 557 and 575 nm and an extinction coefficient of 32.2 mM cm -1.
Bilirub in Serum bilirubin concentration was measured according to a previously published procedure [24].
Protein Protein was measured by the method of Lowry et al [25] using bovine serum albumin as a standard.
Statistics Data were expressed as the mean + S.E.M. The two-tailed Student t-test was used to distinguish significant differences between treatment and control groups.
192 TABLE I HEME METABOLISM IN AGED RATS
8-aminolevulinic acid synthetase (nmol ALA/mg protein/h) Microsomal heme oxygenase (nmol bilirubin/mg protein/h) Microsomal heme (nmol/mg protein) Percent heine saturation of tryptophan pyrrolase Serum bilirubin (mg/100 ml) Total Unconjugated
Young (3-4 months)
Aged (30-32 months)
0.21 _+0.03 a
0.14 + 0.02*
2.30 _+0.20
4.30 + 0.42*
1.38 _+0.12
1.02 _+0.09*
48.34 _+2.27
63.30 + 2.76*
0.87 +_0.07 0.72 + 0.08
2.81 +_0.39* 2.37 + 0.40*
a Values are the mean + S.E.M. for at least 4 animals. * P < 0.05 when compared to young animals.
RESULTS
B o d y and liver weights were increased as a f u n c t i o n o f age. However, the relative ratio o f liver w e i g h t / b o d y weight (mg/g, 43.7 + 2.6 young, 44.6 -+ 1.0 aged) as well as microsomal
protein
unaltered
(mg
protein/g
liver, 9 . 8 7 - + 0 . 4 4
young,
9.82 + 0 . 5
aged) remained
in the aged female rats. Alterations in heine m e t a b o l i s m in aged rats are
depicted in Table I. Microsomal c o n c e n t r a t i o n o f h e m e and the activity o f ALA-S in aged rats were reduced by 26% and 33%, respectively, as c o m p a r e d to y o u n g values. In TABLE II MICROSOMAL COMPONENTS OF THE HEPATIC MONOOXYGENASE SYSTEM IN AGED FEMALE RATS
Aniline hydroxylase (nmol/mg protein/min) Hexobarbital hydroxylase (nmol/mg protein/min) Cytochrome P-450 (nmol/mg protein) Cytochrome b (nmol/mg protein)
Young (3-4 Months)
Aged (30-32 Months)
1.10 +_0.10 a
0.84 + 0.06*
1.20 + 0.11
0.83 + 0.09*
0.51 + 0.03
0.38 + 0.03*
0.22 -4 0.02
0.24 + 0.03
a Values are the mean -+ S.E.M. for at least 4 animals. * P < 0.05 when compared to young animals.
193 contrast, marked elevations in the activity of MHO (87%), percent heme saturation of TPO (32%), and serum bilirubin concentration (322% and 329% for total and unconjugated, respectively) were observed in these animals. Changes in the microsomal components of the hepatic monooxygenase system in aged rats are summarized in Table II. In these studies, hexobarbital and aniline were chosen as prototypes for type I and type II substrates. In aged rats, the activities of cytochrome P-450 dependent enzymes, hexobarbital hydroxylase and aniline hydroxylase were decreased by 31% and 24%, respectively, as compared to young values. Moreover, there was a consistent correlation between decreased microsomal content of cytochrome P-450 (26%) and the inhibition of the activities of drug metabolizing enzymes. It is noteworthy that in aged rats the level of cytochrome b5 remained unaltered despite the decrease in the microsomal concentration of the closely related hemoprotein, cytochrome P-450. DISCUSSION The results of the present study document specific age-related decreases in the levels of hepatic microsomal cytochrome P-450, and certain associated enzymatic activities, such as aniline hydroxylase and hexobarbital hydroxylase in female rats. Additionally, microsomal contents of heme and the activities of the key enzymes involved in Its synthesis (ALA-S) and degradation (MHO) were decreased and increased, respectively, in these animals. These findings are in accordance with previously published data on heme and hemoprotein in aged male rats [26-28]. In a similar vein, a decrease in the level of cytochrome P-450 and its dependent monooxygenase activity (e.g. hexobarbital hydroxylase, aminopyrine-N-demethylase and 7-ethoxycoumarin O-deethylase) has also been reported in 28-month-old female rats [29]. Contrary to these findings, others have indicated that the drug metabolizing enzyme system in female rats is not affected by aging [30]. Our findings of a low level of heme, cytochrome P-450 and its dependent monooxygenase activities during the senescent period may be due to the low ALA-S activity which in turn limited heme biosynthesis. Furthermore, with heme catabolism in aged rats proceeding at a higher rate than in young rats, the microsomal content of heme as well as cytochrome P-450 would be lower, since the level of this hemoprotein is influenced by the synthesis and catabolism of heme [ 10-14]. Indeed, our data support this contention. This inverse relationship between increased MHO and diminished ALA-S activity together with impaired cytochrome P-450 dependent monooxygenase activity is not unique to aged animals, since similar results have been reported in tumor bearing female rats [31] and in newborn rats [32-34]. Additionally it is consistent with the contention that a decreased concentration of cytochrome P-450 is usually accomplished by an induced MHO activity [35]. Little information, however, is available concerning mechanisms responsible for this modification in heine metabolism in aged rats. A possibility to be entertained is the enlargement of the "regulatory heine pool" by intra and/or extra hepatic heme. In this vein, it is generally accepted that in rat liver there is an intracellular "heme pool", fed by
194 both the endogenously synthesized heme, as well as by the preformed heme, e.g. the cytochrome P-450 heme and heme derived from extra hepatic tissues [36-38]. The "free" heine of this pool has been reported to regulate the activities of ALA-S and MHO through two separate feedback mechanisms operating in opposite directions, negative feedback at the level of ALA-S and positive feedback at the level of MHO [36,39,40]. In this context, the administration of heme or bacterial endotoxin, an agent which causes a release of heme from cytochrome P-450, has been shown to produce enlargement of the hepatic heme pool, and as a consequence, a stimulation of MHO, repression of ALA-S, decreased level of cytochrome P-450 and accumulation of heme in tryptophan pyrrolase [36,41-43]. The latter enzyme is a cytosolic hemoprotein which catalyzes the oxidative cleavage of L-tryptophan to N-formylkynurenine during the first and rate-limiting step of the kynurenine-nicotinic acid pathway of tryptophan degradation [44]. In rat liver tryptophan pyrrolase exists in two forms, the active heine-containing holoenzyme and the inactive heme-free apoenzyme [45]. The relative changes in the holo- and apoenzyme activities has been suggested to reflect the availability of "free" heme of the intracellular "regulatory" heme pool [46-49]. Based on the above regulatory mechanism of heme metabolism, it is possible that during the aging process, high rates of erythrocyte bretakdown may take place. This would be expected to enhance the uptake of heine into the liver with concurrent enlargement of the hepatic heme pool, and thus more "free" heme is available both to induce the activity of MHO as well as to repress ALA-S activity. This regulatory action could account for the decrease in cytochrome P-450 and its dependent monooxygenase activities. Indeed, our findings of increased heme saturation of tryptophan pyrrolase supports the contention of an enlargement of the hepatic heme pool as a function of age, since this enzyme has been reported to serve as a sensitive indicator of changes in the availability of heme in the "regulatory" heme pool [46-48]. Additionally, the increased serum concentration of bilirubin, both total as well as unconjugated, in these animals in conjunction with past reports showing that erythrocytes derived from aged animals exhibit a number of abnormalities, including decreased half-life, decreased enzymatic protection to oxidative damage and reduced hemoglobin content [49,50], give credence to the second tenet of our hypothesis of an increase in erythrocyte breakdown during the aging process. In this regard, the increase in plasma bilirubin concentration has been reported to be proportional to the rate of hemolysis, i.e. inversely related to erythrocyte life span [13]. Alternatively, an age-related enlargement in the hepatic heme pool could exist as a result of increases in the rate of dissociation of heme from cytochrome P-450 and/or decreases in the rate of assembly of this hemoprotein. In summary, we propose that an expansion in the hepatic heme pool of aged animals, possibly due to increased erythrocyte breakdown, results in a perturbation of heine metabolism as indicated by increases and decreases in the activities of MHO and ALA-S, respectively. This reciprocal action on heine regulatory enzymes, therefore, causes the reduction in the microsomal level of cytochrome P-450 and certain associated enzymatic activities.
195 ACKNOWLEDGEMENTS The technical assistance o f M. Peter Sorensen is gratefully acknowledged. This study was partially s u p p o r t e d b y N I H grant HD-16358. REFERENCES 1 R. Kate and A. Takanaka, Effect of phenobarbital on electron transport system, oxidation and reduction of drugs in liver microsomes of rats of different ages. J. Biochem. (Tokyo), 63 (1968) 406 -408. 2 R. Kate and A. Takanaka, Metabolism of drugs in old rats. I. Activities of NADPH-linked electron transport and drug-metabolizing enzyme systems in liver microsomes of old rats. Jpn. J. Pharma. col., 18 (1968) 381-388. 3 D.N. McMartin, J.A. O'Connor, Jr., MJ. Fasco and L.S. Kaminsky, Influence of aging and induction on rat liver and kidney microsomal mixed function oxidase systems. Toxicol. Appl. Pharmacol., 54 (1980) 411-419. 4 D.L. Schmucker and R.K. Wang, Age-related changes in liver drug-metabolizing enzymes. Exp. Gerontoi., 15 (!980) 321-329. 5 J. Kao and P. Hudson, Induction of the hepatic cytochrome P-450 dependent monooxygenase system in young and geriatric rats. Biochertt PharmacoL, 29 (1980) 1191-1194. 6 K. O'Malley, J. Crooks, E. Duke and LH. Stevenson, Effect of age and sex on human drug metabolism. Br. Med. J., 3 (1971) 607-609. 7 C.M. Castleden and C.F. George, The effect of aging on the hepatic clearance of propranolol. Br. J. Clin. Pharmacol., 7 (1979) 49-53. 8 R.E. lrvine, J. Grove, P.A. Toseland and J.R. Trounce, The effect of age on the hydroxylation of amylobarbital sodium in man. Br. J. Clin. Pharmacol, I (1974) 41-45. 9 R. Kate, Sex-related differences in drug metabolism. DrugMetab, Rev., 3 (1974) 1-32. 10 H.S. Murty, P.I. Caasi, S.K. Brooks and P.P. Nair, Biosynthesis of heine in the vitamin E-deficient rat. J. BioL Chem., 245 (1970) 5498-5505. 11 O. Wada, Y. Yano, G. Urata and K. Nakao, Behavior of hepatic microsomal cytochromes after treatment of mice with drugs known to disturb porphyrin metabolism in liver. Biochem. Pharmacol., ] 7 (1968) 596-603. -12 J.R. Paterniti, Jr., C.I. Lin and D.S. Beattie, 6-Aminolevulinic acid synthase: Regulation of activity in various tissues of the aging rat. Arch. Biochem. Biophy~, 191 (1978) 792-797. 13 D.M. Bissell, Formation and elimination of bilirubin. Gastroenterology, 69 (1975) 519-538. 14 H.C. Krasny and D.J. Holbrook, Jr., Effect of cadmium on microsomal hemoproteins and heme oxygenase in rat liver. Mol. PharmacoL, 13 (1979) 759-765. 15 M. Bitar and M. Weiner, Diabetes-induced m'etabolic alterations in heine synthesis and degradation and various heme-containing enzymes in female rats. Diabetes, 33 (1984) 37-44. 16 S. Sassa, A. Kappas, S.E. Bernstein and A.P. Alvares, Heine biosynthesis and drug metabolism in mice with hereditary hemolytic anemia. J. Biol. Chem., 254 (1979) 729-735. 17 M. Bitar and M. Weiner, Heine and hemoproteins in streptozotocin diabetic female rats. Biochem. PharmacoL, 32 (1983) 1921-1928. 18 M.D. Maines and A. Kapps, Cobalt stimulation of heme degradation in the liver. J. Biol. Chem., 250 (1975) 4171-4177. 19 T. Omura and R. Sate, The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J. Biol. Chem., 239 (1964) 2370-2378. 20 J.1. Mieyal and J.L. Blumer, Acceleration of the autoox!dation of human oxyhemoglobin by aniline and its relation to hemoglobin-catalyzed aniline hydroxylation. J. Biol. Chem., 251 (1976) 3442-3446. 21 D. Kupfer and J. Rosenfled, A sensitive radioactive assay for hexobarbital hydroxylase in hepatic microsomes. Drug Metab. Dispos., 1 (1973)763-765.
196 22 B.H. Shapiro and S.M. Szczotka, Androgenic repression of hexobarbitone metabolism and action in Crl: CD-I(ICR)BR mice. Br. J. Pharmacol., 81 (1984) 4 9 - 5 4 . 23 J.E. Falk, Haems. I. Determination as pyridine haemochromes. In Porphyrins and Metalloporphy. rins, Elsevier, Amsterdam, New York, 1964, pp. 181-188. 24 M. Waiters and H. Gerarde, An ultramieromethod for the determination of conjugated and total bilirubin in serum or plasma. Microchem. J., 15 (1970) 2 3 1 - 2 4 3 . 25 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent. J. Biol. Chem., 193 (1951) 2 6 5 - 2 7 5 . 26 M. Bitar and M. Weiner, Modification of age-induced changes in heme and hemoproteins by testosterone in male rats. Mech. Ageing Dev., 23 (1983) 285-296. 27 J.R. Paterniti, Jr., C.I. Lin and D.S. Beattie, Regulation of heme metabolism during senescence: activity of several heine-containing enzymes and heme oxygenase in the liver and kidney of aging rats. Mech. Ageing Dev., 12 (1980) 8 1 - 9 1 . 28 A.W. Scotto, R.W. Rinehart, D.S. Beattie, Aging-related decreases in hepatic mitochondrial and cytosolic 6-aminolevulinic acid synthases during experimental porphyria. Arch. Biochem, Biophys., 222 (1983) 150-157. 29 S. Fujita, T. Uesugi, H. Kitagawa, T. Suzuki and K. Kitani, Hepatic microsomal monooxygenase and azoreductase activities in aging Fischer-344 rats. Importance of sex difference for aging studies. In K. Kitani (ed.), Liver and Aging, Elsevier Biomedical Press, Amsterdam, 1982, pp. 55 - 7 4 . 30 T. Kamataki, K. Maeda, M. Shimada, K. Kitani, T. Nagai and R. Kato, Age-related alteration in the activities of drug-metabolizing enzymes and contents of sex-specific forms of cytochrome P450 in liver microsomes from male and female rats. J. Pharmacol. Exp. Ther., 233 (1985) 222-228. 31 W.T. Beck, M.L. Dedmon and M.A. Ouellette, Biochemical basis for impaired drug metabolism in tumor-bearing rats. Biochem. Pharmacol., 31 (1982) 1535-1543. 32 M.M. Thaler, D.L. Gemes and A.F. Bakken, Enzymatic conversion ofheme to bilirubin in normal and starved fetuses and newborn rats. Pediatr. Res., 6 (1972) 197-201. 33 M.D. Maines and A. Kappas, Study of the developmental pattern of heme catabolism in liver and the effects of cobalt on cytochrome P450 and the rate of heme oxidation during the neonatal period. J. Exp. Med., 141 (1975) 1400-1410. 34 M.D. Maines and A. Kappas, Prematurely evoked synthesis and induction of 6-aminolevulinate synthetase in neonatal liver. J. Biol. Chem., 253 (1978) 2321-2326. 35 M.D. Maines, New development in the regulation of heme metabolism and their implications. CRC Crit. Rev. Toxicol., 12 (1984) 241-314. 36 D.M. Bisseli and L.E. Hammaker, Cytochrome P450 heme and the regulation of 6-aminolevulinic acid synthetase in the liver. Arch. Biochem. Biophys., 1 76 (1976) 103-112. 37 U. Muller-Eberhard and S.H. Vincent, Concepts of heme distribution within hepatocytes. Biochem. Pharmacol. , 34 (1985) 719-725. 38 G.C. Farrell, J.L. Gollan and R. Schmid, Efflux of bilirubin into plasma following hepatic degradation of exogenous heme. Proc. Soc. Exp. Biol. Med., 163 (1980) 5 0 4 - 5 0 9 . 39 S. Granick, The induction in vitro of the synthesis of 6-aminolevulinic acid synthetase in chemical porphyria: A response to certain drugs, sex hormones and foreign chemicals. J. Biol. Chem., 241 (1966) 1359-1375. 40 R. Schmid and A.F. McDonagh, The enzymatic formation of bilirubin. Ann. N. Y. Acad. Sei., 244 (1975) 5 3 3 - 5 5 2 . 41 A.D. Waxman, A. Collins and D.P. Tschudy. Oscillations of hepatic delta-aminolevulinic acid synthetase produced in vivo by heme. Biochem. Biophys, R es. Commun., 24 (1966) 6 7 5 - 6 8 3 . 42 D.M. Bissell and L.E. Hammaker, Effect of endotoxin on tryptophan pyrrolase and g-aminolevulinate synthetase. Evidence for an endogenous regulatory haem fraction in rat liver. Biochem. J., 166 (1977) 301-304. 43 D.M. Bissell and L.E. Hammaker, Cytochrome P-450 heine and the regulation of hepatic heme oxygenase activity. Arch. Biochem. Biophys., 176 (1976) 9 1 - 1 0 2 . 44 W.E. Knox and M.M. Piras, Tryptophan pyrrolase of liver. III. Conjugation in vivo during cofactor induction by tryptophan analogus. J. Biol. Chem., 242 (1967) 2959-2965.
197 45 P. Feigelson and O. Greengard, A microsomal iron-porphyrin activator of rat liver tryptophan pyrroiase, d. Biol. Chem., 236 (1961) 153-157. 46 A.A.-B. Badawy, Tryptophan pyrroiase, the regulatory free heine and hepatic porphyria. Biochem. J., 172 (1978) 487-494. 47 A.A.-B. Badawy, Central role of tryptophan pyrrolase in heine metabolism. Biochem. Soe. Trans., 7 (1979) 575-583. 48 D.A. Litman and M.A. Correia, Elevated brain tryptophan and enhanced 5-hydroxytryptamine turnover in acute hepatic heme deficiency clinical implications. J. Pharmacol. Exp. Ther., 232 (1985) 337-345. 49 G.A. Glass and D. Gershon, Enzymatic changes in rat erythrocytes with increasing cell donor age: loss of superoxide dismutase activity associated with increases in catalytically defective forms. Biochem. Biophys. Res. Commun., 103 (1981) 1245-1253. 50 G.A. Glass and D. Gershon, Decreased enzymic protection and increased sensitivity to oxidative damage in erythrocytes as a function of cell donor aging. Biochem. J., 218 (1984) 531-537.
189
ABERRATION OF HEME AND HEMOPROTEIN IN AGED FEMALE RATS
MILAD S. BITAR and BERNARD H. SHAPIRO University of Pennsylvania, Department of Animal Biology, Laboratories of Biochemistry, School of Veterinary Medicine, Philadelphia, PA 19104-6048 (U.S.A.) (Received October 14th, 1986)
SUMMARY The magnitude and duration of drug action is determined partially by the activity of the drug metabolizing enzyme systems in the liver. The pharmacological effectiveness of many drugs is altered during the aging process. In this study, the regulation of heme metabolism and hemoprotein content was examined in livers of aged female rats. The activities of hexobarbital hydroxylase and aniline hydroxylase, indicators of m o n o oxygenase function, were decreased in aged rats by 31% and 24%, respectively, as compared to values in young rats. This was accompanied by a proportional decrease in the level of cytochrome P-450 (26%). Additionally, the activity of 8-aminolevulinic acid syhthetase (ALA-S), the rate-limiting enzyme in heme synthesis, and the microsomal concentration of heme were also decreased by 33% and 26%, respectively, in these animals. In contrast, the basal activity of microsomal heme oxygenase (MHO), the rate-limiting enzyme in heme degradation, and the percent heme saturation of tryptophan pyrrolase (TPO), a sensitive indicator of changes in the availability of heme in the "regulatory" heme pool, were increased by (87%) and (31%), respectively, in the aged rats. The serum concentration of bilirubin, an indicator of erythrocyte breakdown and/or liver function was likewise increased in these animals. In view of these findings, we suggest that the high activity of MHO and the low level of ALA-S may be a significant causative factor for the decreased microsomal concentration of heme, cytochrome-P-450 and its dependent monooxygenase activities in senescent female rats.
Key words: Heme metabolism; Cytochrome P-450; Aging
Address all correspondence to." Miiad S. Bitar, University of Pennsylvania, Department of Animal Biology, Laboratories of Biochemistry, School of Veterinary Medicine, Philadelphia, PA 19104-6048, U.S.A. 0047-6374/87/$03.50 Printed and Published in Ireland
© 1987 Elsevier Scientific Publishers Ireland Ltd.
190 INTRODUCTION The ability to respond to xenobiotics is altered during the aging process. Early investigations of drug metabolism in aged rats have demonstrated that the altered response to chemicals could be due, in part, to decreases in hepatic biotransformation of drugs, as was observed in vitro and in vivo experiments [1-2]. In this regard, it has been reported that the level of cytochrome P-450 and its dependent monooxygenase activities are decreased in liver microsomes from aged rats [3-5]. Furthermore, assessments of drug metabolism of antipyrine, aminopyrine and propranolol were faster in younger subjects than in the elderly [6-8]. These authors suggested that the activities of drug metabolizing enzymes may be decreased in humans, as in the case of aged rats. Most of the studies concerning drug biotransformation were conducted in aged male rats. Because sex can influence hepatic disposition of xenobiotics [9], data on age-related changes in pharmacokinetics in one sex do not necessarily apply to the other. Since alteration in hepatic biotransformation of xenobiotics during the aging process could have an important pharmacokinetic and toxicological implication in the elderly, it is desirable, therefore, to learn more about the underlying biochemical mechanisms of these age-related changes in cytochrome P-450 and its dependent monooxygenase activity. In the light of previous studies showing that heme plays a cardinal role in the regulation of cytochrome P-450 [10-14] and of the lack of information concerning heme metabolism during aging, we have examined the relationship between heme biosynthetic and degradative capabilities and monooxygenase activities in senescent female rats. MATERIALSAND METHODS Animals Female Fisher-344 rats were obtained from a colony maintained under barrier husbandry conditions by Charles River Breeding Laboratories (Wilmington, MA). In order to study a representative sample of each age group, all surviving animals that were received were used, with the exception of a few that were obviously debilitated or had large tumor masses that contributed substantially to their body weight, and thus could possibly alter drug metabolism. Animals were maintained under standardized conditions of 12 h light/12 h dark and temperature (22°C) on a diet of laboratory chow and water for the duration of the study. They were housed 2 per cage on hardwood bedding chips in clear plastic cages. Their ages are designated as follows: young, 3 - 4 months old; and old or senescent, 3 0 - 3 2 months old. Tissue Preparation Livers were perfused with ice-cold 0.9% saline, then removed, weighed and minced into small pieces and homogenized in three volumes of cold 0.15 M KCI containing 50 mM Tris-HC1 (pH 7.4). Microsomes were isolated as described previously [15].
191
Heme Synthesis ( 8-Aminolevulinic Acid Synthetase, ALA-S] ALA-S activity was measured by the method of Sassa et al. [16] as modified by Bitar and Weiner [ 17].
Heme Degradation (Microsomal Heme Oxygenase, MHO) MHO activity was measured by the method of Maines and Kappas [18] as modified by Bitar and Weiner [17]. This assay measures the amount of bilirubin formed from biliverdin, using the 100 000 g supernatant solution from young animals as the source of biliverdin reductase.
Heme and Hemopro teins ( Cy t ochrome P-450 and bs and Drug Metabolizing Enzymes] Cytochrome P-450 was determined from the reduced carbon monoxide difference spectrum according to the method of Omura and Sato [19]. Cytochrome b5 was measured as described previously [ 19]. The activity of aniline hydroxylase was measured by the formation of p-aminophenol (PAP) according to the procedure of Mieyal and Blumer [20] using approximately 2 mg of microsomal protein and 0.45 mM aniline in a total volume of 1 ml. The activity of hexobarbital hydroxylase was measured by the method of Kupfer and Rosenfeld [21] as modified by Shapiro and Szczotka [22].
Tryptophan Pyrrolase ( TPO) The activity of TPO was determined in liver cytosol [17] either in the absence (holoenzyme activity) or in the presence (total enzyme activity) of added methemoglobin. The percent heme saturation for TPO was expressed as the ratio of holoenzyme to total enzyme ×100. Berne
The microsomal concentration of heme was determined by the pyridine hemochromogen method of Falk [23]. Microsomal pellets were suspended in enough 0.05 M TrisHC1 buffer (pH 7.5) to result in 2.3 mg of protein/ml. The microsomal heme content was calculated from the difference in absorbance between 557 and 575 nm and an extinction coefficient of 32.2 mM cm -1.
Bilirub in Serum bilirubin concentration was measured according to a previously published procedure [24].
Protein Protein was measured by the method of Lowry et al [25] using bovine serum albumin as a standard.
Statistics Data were expressed as the mean + S.E.M. The two-tailed Student t-test was used to distinguish significant differences between treatment and control groups.
192 TABLE I HEME METABOLISM IN AGED RATS
8-aminolevulinic acid synthetase (nmol ALA/mg protein/h) Microsomal heme oxygenase (nmol bilirubin/mg protein/h) Microsomal heme (nmol/mg protein) Percent heine saturation of tryptophan pyrrolase Serum bilirubin (mg/100 ml) Total Unconjugated
Young (3-4 months)
Aged (30-32 months)
0.21 _+0.03 a
0.14 + 0.02*
2.30 _+0.20
4.30 + 0.42*
1.38 _+0.12
1.02 _+0.09*
48.34 _+2.27
63.30 + 2.76*
0.87 +_0.07 0.72 + 0.08
2.81 +_0.39* 2.37 + 0.40*
a Values are the mean + S.E.M. for at least 4 animals. * P < 0.05 when compared to young animals.
RESULTS
B o d y and liver weights were increased as a f u n c t i o n o f age. However, the relative ratio o f liver w e i g h t / b o d y weight (mg/g, 43.7 + 2.6 young, 44.6 -+ 1.0 aged) as well as microsomal
protein
unaltered
(mg
protein/g
liver, 9 . 8 7 - + 0 . 4 4
young,
9.82 + 0 . 5
aged) remained
in the aged female rats. Alterations in heine m e t a b o l i s m in aged rats are
depicted in Table I. Microsomal c o n c e n t r a t i o n o f h e m e and the activity o f ALA-S in aged rats were reduced by 26% and 33%, respectively, as c o m p a r e d to y o u n g values. In TABLE II MICROSOMAL COMPONENTS OF THE HEPATIC MONOOXYGENASE SYSTEM IN AGED FEMALE RATS
Aniline hydroxylase (nmol/mg protein/min) Hexobarbital hydroxylase (nmol/mg protein/min) Cytochrome P-450 (nmol/mg protein) Cytochrome b (nmol/mg protein)
Young (3-4 Months)
Aged (30-32 Months)
1.10 +_0.10 a
0.84 + 0.06*
1.20 + 0.11
0.83 + 0.09*
0.51 + 0.03
0.38 + 0.03*
0.22 -4 0.02
0.24 + 0.03
a Values are the mean -+ S.E.M. for at least 4 animals. * P < 0.05 when compared to young animals.
193 contrast, marked elevations in the activity of MHO (87%), percent heme saturation of TPO (32%), and serum bilirubin concentration (322% and 329% for total and unconjugated, respectively) were observed in these animals. Changes in the microsomal components of the hepatic monooxygenase system in aged rats are summarized in Table II. In these studies, hexobarbital and aniline were chosen as prototypes for type I and type II substrates. In aged rats, the activities of cytochrome P-450 dependent enzymes, hexobarbital hydroxylase and aniline hydroxylase were decreased by 31% and 24%, respectively, as compared to young values. Moreover, there was a consistent correlation between decreased microsomal content of cytochrome P-450 (26%) and the inhibition of the activities of drug metabolizing enzymes. It is noteworthy that in aged rats the level of cytochrome b5 remained unaltered despite the decrease in the microsomal concentration of the closely related hemoprotein, cytochrome P-450. DISCUSSION The results of the present study document specific age-related decreases in the levels of hepatic microsomal cytochrome P-450, and certain associated enzymatic activities, such as aniline hydroxylase and hexobarbital hydroxylase in female rats. Additionally, microsomal contents of heme and the activities of the key enzymes involved in Its synthesis (ALA-S) and degradation (MHO) were decreased and increased, respectively, in these animals. These findings are in accordance with previously published data on heme and hemoprotein in aged male rats [26-28]. In a similar vein, a decrease in the level of cytochrome P-450 and its dependent monooxygenase activity (e.g. hexobarbital hydroxylase, aminopyrine-N-demethylase and 7-ethoxycoumarin O-deethylase) has also been reported in 28-month-old female rats [29]. Contrary to these findings, others have indicated that the drug metabolizing enzyme system in female rats is not affected by aging [30]. Our findings of a low level of heme, cytochrome P-450 and its dependent monooxygenase activities during the senescent period may be due to the low ALA-S activity which in turn limited heme biosynthesis. Furthermore, with heme catabolism in aged rats proceeding at a higher rate than in young rats, the microsomal content of heme as well as cytochrome P-450 would be lower, since the level of this hemoprotein is influenced by the synthesis and catabolism of heme [ 10-14]. Indeed, our data support this contention. This inverse relationship between increased MHO and diminished ALA-S activity together with impaired cytochrome P-450 dependent monooxygenase activity is not unique to aged animals, since similar results have been reported in tumor bearing female rats [31] and in newborn rats [32-34]. Additionally it is consistent with the contention that a decreased concentration of cytochrome P-450 is usually accomplished by an induced MHO activity [35]. Little information, however, is available concerning mechanisms responsible for this modification in heine metabolism in aged rats. A possibility to be entertained is the enlargement of the "regulatory heine pool" by intra and/or extra hepatic heme. In this vein, it is generally accepted that in rat liver there is an intracellular "heme pool", fed by
194 both the endogenously synthesized heme, as well as by the preformed heme, e.g. the cytochrome P-450 heme and heme derived from extra hepatic tissues [36-38]. The "free" heine of this pool has been reported to regulate the activities of ALA-S and MHO through two separate feedback mechanisms operating in opposite directions, negative feedback at the level of ALA-S and positive feedback at the level of MHO [36,39,40]. In this context, the administration of heme or bacterial endotoxin, an agent which causes a release of heme from cytochrome P-450, has been shown to produce enlargement of the hepatic heme pool, and as a consequence, a stimulation of MHO, repression of ALA-S, decreased level of cytochrome P-450 and accumulation of heme in tryptophan pyrrolase [36,41-43]. The latter enzyme is a cytosolic hemoprotein which catalyzes the oxidative cleavage of L-tryptophan to N-formylkynurenine during the first and rate-limiting step of the kynurenine-nicotinic acid pathway of tryptophan degradation [44]. In rat liver tryptophan pyrrolase exists in two forms, the active heine-containing holoenzyme and the inactive heme-free apoenzyme [45]. The relative changes in the holo- and apoenzyme activities has been suggested to reflect the availability of "free" heme of the intracellular "regulatory" heme pool [46-49]. Based on the above regulatory mechanism of heme metabolism, it is possible that during the aging process, high rates of erythrocyte bretakdown may take place. This would be expected to enhance the uptake of heine into the liver with concurrent enlargement of the hepatic heme pool, and thus more "free" heme is available both to induce the activity of MHO as well as to repress ALA-S activity. This regulatory action could account for the decrease in cytochrome P-450 and its dependent monooxygenase activities. Indeed, our findings of increased heme saturation of tryptophan pyrrolase supports the contention of an enlargement of the hepatic heme pool as a function of age, since this enzyme has been reported to serve as a sensitive indicator of changes in the availability of heme in the "regulatory" heme pool [46-48]. Additionally, the increased serum concentration of bilirubin, both total as well as unconjugated, in these animals in conjunction with past reports showing that erythrocytes derived from aged animals exhibit a number of abnormalities, including decreased half-life, decreased enzymatic protection to oxidative damage and reduced hemoglobin content [49,50], give credence to the second tenet of our hypothesis of an increase in erythrocyte breakdown during the aging process. In this regard, the increase in plasma bilirubin concentration has been reported to be proportional to the rate of hemolysis, i.e. inversely related to erythrocyte life span [13]. Alternatively, an age-related enlargement in the hepatic heme pool could exist as a result of increases in the rate of dissociation of heme from cytochrome P-450 and/or decreases in the rate of assembly of this hemoprotein. In summary, we propose that an expansion in the hepatic heme pool of aged animals, possibly due to increased erythrocyte breakdown, results in a perturbation of heine metabolism as indicated by increases and decreases in the activities of MHO and ALA-S, respectively. This reciprocal action on heine regulatory enzymes, therefore, causes the reduction in the microsomal level of cytochrome P-450 and certain associated enzymatic activities.
195 ACKNOWLEDGEMENTS The technical assistance o f M. Peter Sorensen is gratefully acknowledged. This study was partially s u p p o r t e d b y N I H grant HD-16358. REFERENCES 1 R. Kate and A. Takanaka, Effect of phenobarbital on electron transport system, oxidation and reduction of drugs in liver microsomes of rats of different ages. J. Biochem. (Tokyo), 63 (1968) 406 -408. 2 R. Kate and A. Takanaka, Metabolism of drugs in old rats. I. Activities of NADPH-linked electron transport and drug-metabolizing enzyme systems in liver microsomes of old rats. Jpn. J. Pharma. col., 18 (1968) 381-388. 3 D.N. McMartin, J.A. O'Connor, Jr., MJ. Fasco and L.S. Kaminsky, Influence of aging and induction on rat liver and kidney microsomal mixed function oxidase systems. Toxicol. Appl. Pharmacol., 54 (1980) 411-419. 4 D.L. Schmucker and R.K. Wang, Age-related changes in liver drug-metabolizing enzymes. Exp. Gerontoi., 15 (!980) 321-329. 5 J. Kao and P. Hudson, Induction of the hepatic cytochrome P-450 dependent monooxygenase system in young and geriatric rats. Biochertt PharmacoL, 29 (1980) 1191-1194. 6 K. O'Malley, J. Crooks, E. Duke and LH. Stevenson, Effect of age and sex on human drug metabolism. Br. Med. J., 3 (1971) 607-609. 7 C.M. Castleden and C.F. George, The effect of aging on the hepatic clearance of propranolol. Br. J. Clin. Pharmacol., 7 (1979) 49-53. 8 R.E. lrvine, J. Grove, P.A. Toseland and J.R. Trounce, The effect of age on the hydroxylation of amylobarbital sodium in man. Br. J. Clin. Pharmacol, I (1974) 41-45. 9 R. Kate, Sex-related differences in drug metabolism. DrugMetab, Rev., 3 (1974) 1-32. 10 H.S. Murty, P.I. Caasi, S.K. Brooks and P.P. Nair, Biosynthesis of heine in the vitamin E-deficient rat. J. BioL Chem., 245 (1970) 5498-5505. 11 O. Wada, Y. Yano, G. Urata and K. Nakao, Behavior of hepatic microsomal cytochromes after treatment of mice with drugs known to disturb porphyrin metabolism in liver. Biochem. Pharmacol., ] 7 (1968) 596-603. -12 J.R. Paterniti, Jr., C.I. Lin and D.S. Beattie, 6-Aminolevulinic acid synthase: Regulation of activity in various tissues of the aging rat. Arch. Biochem. Biophy~, 191 (1978) 792-797. 13 D.M. Bissell, Formation and elimination of bilirubin. Gastroenterology, 69 (1975) 519-538. 14 H.C. Krasny and D.J. Holbrook, Jr., Effect of cadmium on microsomal hemoproteins and heme oxygenase in rat liver. Mol. PharmacoL, 13 (1979) 759-765. 15 M. Bitar and M. Weiner, Diabetes-induced m'etabolic alterations in heine synthesis and degradation and various heme-containing enzymes in female rats. Diabetes, 33 (1984) 37-44. 16 S. Sassa, A. Kappas, S.E. Bernstein and A.P. Alvares, Heine biosynthesis and drug metabolism in mice with hereditary hemolytic anemia. J. Biol. Chem., 254 (1979) 729-735. 17 M. Bitar and M. Weiner, Heine and hemoproteins in streptozotocin diabetic female rats. Biochem. PharmacoL, 32 (1983) 1921-1928. 18 M.D. Maines and A. Kapps, Cobalt stimulation of heme degradation in the liver. J. Biol. Chem., 250 (1975) 4171-4177. 19 T. Omura and R. Sate, The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J. Biol. Chem., 239 (1964) 2370-2378. 20 J.1. Mieyal and J.L. Blumer, Acceleration of the autoox!dation of human oxyhemoglobin by aniline and its relation to hemoglobin-catalyzed aniline hydroxylation. J. Biol. Chem., 251 (1976) 3442-3446. 21 D. Kupfer and J. Rosenfled, A sensitive radioactive assay for hexobarbital hydroxylase in hepatic microsomes. Drug Metab. Dispos., 1 (1973)763-765.
196 22 B.H. Shapiro and S.M. Szczotka, Androgenic repression of hexobarbitone metabolism and action in Crl: CD-I(ICR)BR mice. Br. J. Pharmacol., 81 (1984) 4 9 - 5 4 . 23 J.E. Falk, Haems. I. Determination as pyridine haemochromes. In Porphyrins and Metalloporphy. rins, Elsevier, Amsterdam, New York, 1964, pp. 181-188. 24 M. Waiters and H. Gerarde, An ultramieromethod for the determination of conjugated and total bilirubin in serum or plasma. Microchem. J., 15 (1970) 2 3 1 - 2 4 3 . 25 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent. J. Biol. Chem., 193 (1951) 2 6 5 - 2 7 5 . 26 M. Bitar and M. Weiner, Modification of age-induced changes in heme and hemoproteins by testosterone in male rats. Mech. Ageing Dev., 23 (1983) 285-296. 27 J.R. Paterniti, Jr., C.I. Lin and D.S. Beattie, Regulation of heme metabolism during senescence: activity of several heine-containing enzymes and heme oxygenase in the liver and kidney of aging rats. Mech. Ageing Dev., 12 (1980) 8 1 - 9 1 . 28 A.W. Scotto, R.W. Rinehart, D.S. Beattie, Aging-related decreases in hepatic mitochondrial and cytosolic 6-aminolevulinic acid synthases during experimental porphyria. Arch. Biochem, Biophys., 222 (1983) 150-157. 29 S. Fujita, T. Uesugi, H. Kitagawa, T. Suzuki and K. Kitani, Hepatic microsomal monooxygenase and azoreductase activities in aging Fischer-344 rats. Importance of sex difference for aging studies. In K. Kitani (ed.), Liver and Aging, Elsevier Biomedical Press, Amsterdam, 1982, pp. 55 - 7 4 . 30 T. Kamataki, K. Maeda, M. Shimada, K. Kitani, T. Nagai and R. Kato, Age-related alteration in the activities of drug-metabolizing enzymes and contents of sex-specific forms of cytochrome P450 in liver microsomes from male and female rats. J. Pharmacol. Exp. Ther., 233 (1985) 222-228. 31 W.T. Beck, M.L. Dedmon and M.A. Ouellette, Biochemical basis for impaired drug metabolism in tumor-bearing rats. Biochem. Pharmacol., 31 (1982) 1535-1543. 32 M.M. Thaler, D.L. Gemes and A.F. Bakken, Enzymatic conversion ofheme to bilirubin in normal and starved fetuses and newborn rats. Pediatr. Res., 6 (1972) 197-201. 33 M.D. Maines and A. Kappas, Study of the developmental pattern of heme catabolism in liver and the effects of cobalt on cytochrome P450 and the rate of heme oxidation during the neonatal period. J. Exp. Med., 141 (1975) 1400-1410. 34 M.D. Maines and A. Kappas, Prematurely evoked synthesis and induction of 6-aminolevulinate synthetase in neonatal liver. J. Biol. Chem., 253 (1978) 2321-2326. 35 M.D. Maines, New development in the regulation of heme metabolism and their implications. CRC Crit. Rev. Toxicol., 12 (1984) 241-314. 36 D.M. Bisseli and L.E. Hammaker, Cytochrome P450 heme and the regulation of 6-aminolevulinic acid synthetase in the liver. Arch. Biochem. Biophys., 1 76 (1976) 103-112. 37 U. Muller-Eberhard and S.H. Vincent, Concepts of heme distribution within hepatocytes. Biochem. Pharmacol. , 34 (1985) 719-725. 38 G.C. Farrell, J.L. Gollan and R. Schmid, Efflux of bilirubin into plasma following hepatic degradation of exogenous heme. Proc. Soc. Exp. Biol. Med., 163 (1980) 5 0 4 - 5 0 9 . 39 S. Granick, The induction in vitro of the synthesis of 6-aminolevulinic acid synthetase in chemical porphyria: A response to certain drugs, sex hormones and foreign chemicals. J. Biol. Chem., 241 (1966) 1359-1375. 40 R. Schmid and A.F. McDonagh, The enzymatic formation of bilirubin. Ann. N. Y. Acad. Sei., 244 (1975) 5 3 3 - 5 5 2 . 41 A.D. Waxman, A. Collins and D.P. Tschudy. Oscillations of hepatic delta-aminolevulinic acid synthetase produced in vivo by heme. Biochem. Biophys, R es. Commun., 24 (1966) 6 7 5 - 6 8 3 . 42 D.M. Bissell and L.E. Hammaker, Effect of endotoxin on tryptophan pyrrolase and g-aminolevulinate synthetase. Evidence for an endogenous regulatory haem fraction in rat liver. Biochem. J., 166 (1977) 301-304. 43 D.M. Bissell and L.E. Hammaker, Cytochrome P-450 heine and the regulation of hepatic heme oxygenase activity. Arch. Biochem. Biophys., 176 (1976) 9 1 - 1 0 2 . 44 W.E. Knox and M.M. Piras, Tryptophan pyrrolase of liver. III. Conjugation in vivo during cofactor induction by tryptophan analogus. J. Biol. Chem., 242 (1967) 2959-2965.
197 45 P. Feigelson and O. Greengard, A microsomal iron-porphyrin activator of rat liver tryptophan pyrroiase, d. Biol. Chem., 236 (1961) 153-157. 46 A.A.-B. Badawy, Tryptophan pyrroiase, the regulatory free heine and hepatic porphyria. Biochem. J., 172 (1978) 487-494. 47 A.A.-B. Badawy, Central role of tryptophan pyrrolase in heine metabolism. Biochem. Soe. Trans., 7 (1979) 575-583. 48 D.A. Litman and M.A. Correia, Elevated brain tryptophan and enhanced 5-hydroxytryptamine turnover in acute hepatic heme deficiency clinical implications. J. Pharmacol. Exp. Ther., 232 (1985) 337-345. 49 G.A. Glass and D. Gershon, Enzymatic changes in rat erythrocytes with increasing cell donor age: loss of superoxide dismutase activity associated with increases in catalytically defective forms. Biochem. Biophys. Res. Commun., 103 (1981) 1245-1253. 50 G.A. Glass and D. Gershon, Decreased enzymic protection and increased sensitivity to oxidative damage in erythrocytes as a function of cell donor aging. Biochem. J., 218 (1984) 531-537.