EFFECTS OF LYOPHILIZATION AND STORAGE OF RAT LIVER MICROSOMES ON ACTIVITY OF ANILINE HYDROXYLASE, CONTENTS OF CYTOCHROME b<SUB>5</SUB> AND CYTOCHROME P-450 AND ANILINE-INDUCED P-450 DIFFERENCE SPECTRUM

Effects of lyophilization and storage of rat liver 9,000&times;g supernatant frac tion or microsomes on activity of aniline hydroxylase, contents of cyt. P-450 and cyt. b5 and aniline-induced P-450 difference spectrum were studied. The lyophiliza tion of rat liver 9,000&times;g supernatant fraction or microsomes produced about 20% decrease in the activity of aniline hydroxylase. Aniline hydroxylase was stably stored for 6 months if lyophilized 9,000&times;g supernatant fraction of rat liver had been stored in an atmosphere of nitrogen or in a low pressure of air. Activity of aniline hydro xylase and contents of cyt. P-450 and cyt. b5 were not reduced during aerobical storage for a week. When lyophilized microsomes were stored for 6 months anaerobically, aniline hydroxylase, cyt. P-450 and cyt. b5 were more stable as compared to aerobical storage. However, activity of aniline hydroxylase was reduced approx. 40% by the storage. The reduction of aniline hydroxylase activity was more significant than that of contents of cyt. P-450 and cyt. b5. Reduction of either activity of aniline hydroxylase or contents of cyt. P-450 and cyt. b5 was more marked when lyophilized microsomes were stored with glycerol. Aniline hydroxylase in lyophilized 9,000&times;g supernatant fraction from phenobarbital treated rabbit was stably stored, and aer obically little activity was lost during storage for 7 months. There have been reports on the stability of drug metabolizing enzymes in liver micro somes (1-6). Gram and Fouts (6) reported that there was a marked species difference in stability of drug metabolizing enzymes between mice, rats and rabbits using various substrates, and that enzymes in rat liver were in most cases more unstable than those in rabbit liver. In addition, Levin et al. (5) reported that liver microsomes kept in a pellet form tended to lose activities less than those in a suspension or lyophilized form. In some experiments, investigators have used microsomes kept in a pellet form. Moreover, Ichikawa and Yamano (7) demonstrated that liver microsomal cyt. P-450 was stably stored aerobically by addition of glycerol even at 16°C. It was of interest for the authors to investigate the effects of lyophilization and storage of microsomes for long period on the liver microsomal enzymes, as these effects have not yet been established. For this purpose, the authors compared the stabilities of aniline hydro xylase, cyt. P-450, cyt. b5 and aniline-induced P-450 difference spectrum using rat liver 9,000 x g supernatant fraction or microsomal fraction lyophilized and stored in various environments. Animals: Male rats of Wistar strain weighing 100 to 150 g and male albino rabbits weighing about 2.7 kg were used. The animals were fasted for 18 hr prior to sacrifice but were given water ad libitum. Phenobarbital pretreatment comprised five consecutive daily s.c. injections of phenobarbital sodium (100 mg/kg in the rabbit), and the last in jection was given approx. 48 hr prior to sacrifice. In the rat, 8 to 10 livers were pooled and used. Preparation of liver microsonres: The animals were stunned by a blow on the head, and decapitated. The livers were immediately perfused with 1.15% KCl solution from inferior vena cava to portal vein. This procedure was carried out at room temp., since cold KCI solution could contract the blood capillaries in the liver and all blood cannot be quickly removed. The perfusion from inferior vena cava to portal vein also improved with a rapid removal of the blood. If one employs this method to remove blood from liver, the process can be completed in only one or two min. After perfusion, the livers were quickly removed and placed in 1.15% KCI solution on ice. All remaining procedures were carried out at a temp. under 4°C. After pouring off the KCl solution through a layer of paper, the livers were minced with scissors, then 20 % homogenate of the livers in 1.15 KCI solution was prepared with a Potter-Elvehjem homogenizer which had a teflon pestle. The homogenate was centrifuged at 9,000.,g for 20 min in a TOMINAGA refrigirated centrifuge, Model S-62. The supernatant fluid was re-centrifuged at 105,000 xg for 60 min in a HITACHI ultracentrifuge, Model 65 P. All g values were calculated for the center of the centrifuge tube. The resulting microsomal pellet was suspended in 0.1 M phosphate buffer (pH 7.5) with a Potter-Elvehjem homogenizer having a teflon pestle. Protein was assayed by a method of Lowry et al. (8). Lyophilization and storage of 9,000 x g supernatant fraction, microsonres and soluble fraction: The 9,000 xg supernatant fraction, microsomes and soluble fraction were frozen in a deep freezer for 2 to 3.4 hr, then lyophilized in a KYOWA lyophilizer, Model RL 300S. The final atmospheric pressure in the sample chamber was below 80 1zHg. Dis placement of the gas phase in a vessel was made by the vacuum-flash method. When storage of the preparations lasted for 6 months, the gas phase was displaced by the vacuum flash method, then stored in a refrigerator at -20°C. At the end of 3 months, the vessel was re-flashed with the gas, re-sealed and returned to the refrigerator. Moreover, in the case of storage under low atmospheric pressure of air, the air in the vessel was removed by an aspirator to 24 mmHg of pressure, and the vessel was placed in a deep freezer. At 3 months, the air was removed again and the vessel was placed in the deep freezer. In the case of addition of glycerol, 0.7 ml of glycerol was added into a vessel containing lyo philized microsonres being equivalent to 3 ml of 9,000 xg supernatant fluid. At time intervals after lyophilization, re-distilled water was added in the lyophilized samples, then homogenized with a homogenizer and resultant microsomal suspensions were used for the enzyme assays and spectrophotometric measurements. Assay of aniline hydroxylase activity: Incubations were carried out at 37°C aerobic ally for 30 min. Incubation media contained glucose-6-phosphate (20.0 ,umoles), NADP (0.4 pmole), MgCl2 (37.5 tvioles), nicotinamide (75.0 ,umoles), aniline (5 ,umoles), 1.4 ml of 0.2 M Na-K phosphate buffer (pH 7.4) and 3.0 ml of enzyme preparation in a final vol ume of 5.0 ml. Aromatic hydroxylation of aniline was determined by measuring p-amino phenol according to the method of Kato and Gillette (9). Measurement of difference spectra of microsomal cyt. P-450: Difference spectra were recorded using a HITACHI recording spectrophotometer, Model EPS-3T, with an inte grating sphere accessory. The carbon monoxide-induced difference spectrum of dithio nite-reduced microsomes was obtained by the method essentially as described by Omura and Sato (10). Cyt. P-450 and cyt. P-420 were determined from CO difference spectra of dithionite-reduced samples using a value of 91 cm-'mM-' for the molar extinction in crement between 450 and 490 m/t for cyt. P-450, and a value of 110 cni 'mM-' between 420 and 490 m/2 for cyt. P-420 (11). Cyt. P-450 difference spectrum induced by aniline was obtained using oxidized microsomes. A 0.2 ml of substrate solution was added to 3.8 ml of the microsomal suspension. With each addition to the sample cuvette, a cor responding volume of water was added to the reference cuvette. Cyt. b5 content in micro somes was determined as described by Omura and Sato (10). The difference in absorb ance between 424 and 409 m/.e was measured, and an extinction coefficient of 185 cm 'mM-' was used to convert the data from absorbancy to lntcmoles of cyt. b5. RESULTS 1. Effect of storage of lyophilized 9,000 x g supernatant fraction for 6 months on activity of aniline hydroxylase in the rat The lyophilization of 9,000 xg supernatant fraction produced about 20 % loss of the activity. During storage of lyophilized samples for 6 months, varying loss of the activity was seen with the conditions. When they were stored in a low pressure of air or in an atmosphere of nitrogen, they did not lose their activity during a 6 months storage, where as they lost about 20% of their activity by storage in the atmosphere of air. Accord ingly, the best result for storage of aniline hydroxylase was obtained if lyophilized 9,000 xg supernatant fraction was stored anaerobically. TABLE 1. Effects of six months storage of lyophilized 9,000 x g supernatant fraction on activity of aniline hydroxylase in rats *Microsomal protein **Range in duplication 2. Effects of storage of lyophilized inicrosoines and soluble fraction for I week in air on activity of aniline hydroxylase and contents of cyt. P-450 and cyt. b5 in the rat As shown in Table 2, about 20% loss of the activity of aniline hydroxylase was seen with lyophilization. With storage of lyophilized microsomes and soluble fraction for 1 week in air, further loss of the activity of aniline hydroxylase did not occur. Similar effect of lyophilization and storage was observed in content of cyt. P-450, whereas, unexpectedly, content of cyt. b5 was apparently increased by the storage (Table 3). TABLE 2. Effects of one week storage of lyophilized microsornes and soluble fraction on the activity of aniline hydroxylase in the rat. * Soluble fraction stored at 8'C aerobically for 1 week without lyophilization. ** Range in duplication TABLE 3. Effects of storage for 1 week of lyophilized microsomes on the contents of cyt. P-450 and cyt. b5. 3. Effects of conditions of storage for 6 months of lyophilized inicrosonies and soluble frac tion on activity of aniline hydroxylase in the rat Effects of long term storage (6 months) of microsomes and soluble fraction of rat liver on activity of aniline hydroxylase are shown in Table 4 to Table 7. The activities just after lyophilization were 84.816.1 % (S.E., n=6) of those in fresh sam ples. The activity of aniline hydroxylase appeared to be dependent not only on the ac tivity of microsomal enzyme but also on the enzymes in the soluble fraction. This fact may suggest that enzymes localized in soluble fraction such as glucose-6-phosphate de hydrogenase lose the activities if the soluble fraction is stored in aerobic conditions. Micro somes stored in an atmosphere of low pressure of air or in nitrogen lost relatively small activity of aniline hydroxylase, but about 60 to 70 % of the activity was lost when they were kept in an atmosphere of oxygen or of air. Practically most of the activity was lost when lyophilized microsomes were stored with glycerol, ethylene glycol or propyrene glycol. TABLE 4. Effects of storage for 6 months of lyophilized microsomes (in air) and soluble fraction on the activity of aniline hydroxylase. *Range in duplication TABLE 5. Effects of storage for 6 months of lyophilized microsomes (in low pressure of air) and soluble fraction nn the activity of aniline hydroxylase_ *Microsomal protein , **Range in duplication TABLE 6. Effects of storage for 6 months of lyophilized microsomes (in N2) and soluble fraction on the activity of aniline hydroxylase. *Range in duplication TABLE 7. Effects of storage for 6 months of lyophilized microsomes (in 02) and soluble fraction on the activity of aniline hydroxylase. * Range in duplication TABLE 8. Effects of six months storage of lyophilized microsomes on the content of cyt. P-450. TABLE 9. Effect of storage for 6 months of lyophilized microsomes on the content of cyt. b5. TABLE 10. Effect of storage for 6 months of lyophilized microsomes on the aniline P-450 binding difference spectrum. Aniline : 1 mM *OD peak-trough correlated with baseline;' protein concentration . 4. Effects of various conditions of storage for 6 months of lyophilized microsomes on cyt. P-450 content, cyt. b, content and aniline-induced difference spectrum of cyt. P-450 In microsomes stored in an atmosphere of low pressure of air and in an atmosphere of nitrogen, cyt. P-450, cyt. b, and aniline-P-450 binding spectrum tended to be more stably stored than those kept in other conditions. It was found that microsomes stored with glycerol were kept less stably than those stored without glycerol. In the case of glyc erol, cyt. P-420 appeared instead of cyt. P-450, and less cyt. b, and aniline-P-450 binding spectrum remained. FIG. 1. Periodical changes in the activity of aniline hydroxylase in lyophilized 9,000 x g supernatant fraction of phenobarbital treated rabbit liver. 5. Effects of storage of 9,000 x g supernatant fraction on the activity of aniline hydroxylase in the rabbit In preliminary experiments, microsomes and soluble fraction from rabbit liver were lyophilized and stored in an atmosphere of air, after which the periodical change in the ac tivity of aniline hydroxylase was observed. The lyophilization produced about 20 loss of activity, but little loss of the activity was observed during storage for 120 days (data not shown). To investigate the stability of aniline hydroxylase in the 9,000 xg super natant fraction, 9,000-g supernatant fraction from phenobarbital treated rabbits was employed, since the enzyme from phenobarbital treated rabbits was assumed to be un stable compared to that from control rabbits (Fig. 1). Unexpectedly, the activity of aniline hydroxylase in 9,000 xg supernatant fraction from phenobarbital treated rabbits was apparently increased by the lyophilization and the ac tivity decreased slowly during storage for 210 days, however, the activity at 210 days was not lower than that before lyophilization. Thus, it can be concluded that drug meta bolizing enzymes of rabbit liver are more stable than those of rat liver from the findings presented here and as described by Gram and Fouts (6). DISCUSSION In the rat liver, 9,000 xg supernatant fraction was more stably stored than micro somes. Recently, the authors found that heat labile factor in the soluble fraction inhibits formation of lipid peroxides of liver microsomes and then stabilizes microsomal N-de methylase of ethylmorphine (12). If the inactivation of aniline hydroxylase is caused by the lipid peroxidation in the microsomes, the stability of 9,000>,g supernatant fraction may be depending upon the inhibitory factor of lipid peroxidation in the soluble fraction. In most cases, disappearance of aniline hydroxylase during storage of microsomes was more than that of magnitude of aniline-induced P-450 difference spectrum and cyt. P 450 content. This finding is supported by a finding that activity of ethylmorphine N demethylase is rapidly lost without significant loss of cyt. P-450 content (at most 10"/) when microsomes are aged at 45`C aerobically for 30 min (12). Since activity of aniline hydroxylase was not recovered even when fresh soluble fraction was added, the loss of the activity of aniline hydroxylase during storage might be accompanied either with in activation of NADPH-cyt.c reductase or with change in membrane structure of endo plasmic reticulum. Recent observations of Wills (13) and the authors (14) that NADPH linked lipid peroxidation in liver microsomes produced an inactivation of drug metaboliz ing enzymes, and another one by Gram and Fouts (15) that significant lipid peroxidation was seen in the rat but negligible peroxidation occurred in the rabbit, may suggest that drug metabolizing enzymes in the rabbit liver are more stable than those in the rat. As previously described (14), addition of EDTA, a potent inhibitor of lipid peroxidation (16), prevented inactivation of ethyl morphine N-demethylase which occurs during incubation. Accordingly, the fact that rabbit liver was stably stored is confirmed as being due to lack of lipid peroxidase in rabbit liver, however, the reason why apparent activation of aniline hydroxylation is observed with the storage of lyophilized 9,000 xg supernatant fraction of rabbit liver is yet to be determined. Acknowledgement: The authors thank Miss Yuko Hanawa for technical assistance. 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