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Microsomal ethanol-oxidizing system (MEOS): Purification and properties of a rat liver system free of catalase and alcohol dehydrogenase
BIOCHEMICAL
Vol. 49, No. 5,1972
AND BIOPHYSKAL
RESEARCH COMMUNICATIONS
MICROSOMAL ETHANOL-OXIDIZING SYSTEM (MEOS): PURIFICATION AND PROPERTIES OF A RAT LIVER SYSTEM FREE OF CATALASE AND ALCOHOL DEHYDROGENASE s R. Teschke*, Y. Hasumura, J.-G. Joly’, H. Ishii , and C.S. Lieber. lepartment of Medicine, Mt. Sinai School of Medicine (CUNY) and Section of Liver Xsease and Nutrition, Veterans Administration Hospital, Bronx, New York 19468. Received
October
6,1972
Summary: A separation of the microsomal ethanol-oxidizing system (MECS) from alcohol dehydrogenase (ADH) and catalase in rat liver microsomes is described. After solubilization of microsomes , ADH and catalase were eluted by DEAE-cellulose chromatography with the hemoglobin containing fraction, whereas MEOS activity was recovered separately. The oxidation of ethanol to acetaldehyde required 02 and NADPH, and was partially inhibited by CO. The fractions exhibiting MEOS activity contained cytochrome P-450, NADPH-cytochrome c reductase and phospholipids. These three components also increased in total microsomes after chronic ethanol consumption and may play a role in the associated enhancementof MEOS activity. The observation that chronic ethanol consumption produces proliferation of the hepatic smooth endoplasmic reticulum (1,2) suggestedthat, in addition to its oxidation by ADH in the cytosol, ethanol may be metabolized by the microsomal fraction.
This
was substantiated by the description of a microsomal ethanol-oxidizing system (MEOS) (3). MEOS was differentiated from catalase and ADH primarily by the use of inhibitors (3,4), but these experiments have been the subject of controversy (5-9). The present study describes the physical separation of MEOS from ADH and catalase activity by column chromatography and reveals the effect of chronic ethanol feeding on three components in the MEOS fraction, namely cytochrome P-450, NADPH-cytochrome c reductase and phospholipids. Materials and Methods Female Swarrue Dawlev rats (Charles River, C. D. ) of a starting bodv weight *Recipient of a grant from the Deutsche Forschungsgemeinschaft . ‘Present address: Centre de Recherches Cliniques , Hopital Saint-Luc Montreal 129, P.Q., Canada. * Present address: Department of Internal Medicine, School of Medicine, Keio University, Shinijuku-Ku, Tokyo, Japan Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Vol. 49, No. 5, 1972
Of
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
130-16Ogwere fed an alcohol containing liquid diet for 3-4 weeks (10). The prepam-
tion and solubilization of microsomes were achieved by a modification of the procedure of LU and Coon (11). Fresh livers were perfused with ice-cold 0.15 M KCl, pooled and homogenized in 4 volumes of 0.25 M sucrose. After the centrifugation of the homogenateat 10,000 x g for 15 min. , the supernatant layer was spun again at 10,000 x g for 15 min. The supernatant layer was then centrifuged at 105,000 x g for 2 hours. The pellet was suspendedwith 0.25 M sucrose to give a microsomal protein concentration (12) of 30 mg/ml.
To approximately 23 ml of such a suspensionthe following
were added: 14.5 ml glycerol, 4. ‘7ml 1 M KC1, 4.7 ml 1 M potassium citrate (in 0 .l M phosphate buffer pH 7.4) and 0.05 ml of 0.1 M dithlothreitol.
This mixture was
sonlcated with the microtip of a Sonifier Cell Disruptor (Ultrasonics Incorp. ) at full output for 6 periods of 20 sec. each. After the addition of 2.7 ml 10%deoxycholate (w/v) the mixture was stirred for 20 min. and passedthrough glasswool. The filtrate was diluted with 2 volumes of distilled water and put onto a DEAE-cellulose column (2.5 x 50 cm). The column was then washed and eluted (ll). ADH (13)and catalase (14)activities were determined respectively in 0.5 and 0 .l ml samples of the column eluates . Catalase units were calculated according to LUck Q4). Absence of catalase activity in the fractions containing MECS activity was also verified with the oxygen electrode technique (15). Cytochrome P-450 content was measured in microsomes (16) and column eluates (11). NADPH-cytochrome c reductase was determined with 0.5 ml of eluate (11). Phospholipids were estimated by their phosphorous content (17)after extraction according to Folch et al (18). Eluates to be used for MECS assay were dialyzed overnight against 0 .l M phosphate buffer @H 7.0). Ethanol oxidation to acetaldehyde was determined according to a modification of the procedure of Lieber and DeCarli (3) and ME06 activity was expressed in units corresponding to the nanomolesof acetaldehyde produced per min. Two ml aliquots Of
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Vol. 49, No. 5,1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dialyzed column eluates were preincubated with 50 mM ethanol (in 0.5 ml 0.1 M phosphate buffer, pH 7.4) for 5 min. at 37’C.
The reaction was started by adding NADPH,
EDTA and Iv&C12(in 0.5 ml 001 M phosphate buffer, pH 7.4) at final concentrations of 0.4, 1 and 5 mM respectively.
For each determination, at least 6 flasks were used
with duplicate incubations for 0, 5 and 10 min. ; MEOS activity was linear over the period tested. The identity of the product with acetaldehyde was confirmed by cornpalative absorption spectra and by measurement of the retention time by gas liquid chromatography (3). In the studies of the effects of gasses,NADPH was replaced by a NADPH generating system with 0.4 mM NAD@ , 8 mM sodium isocitrate, and 2 g per liter of isocitrate dehydrogenase (crude type I, Sigma Chemical Company). The NADPH generating system was preincubated with the column eluates for 5 min. at 37’C, the flasks were flushed with the gas for another 5 min. and the reaction was started by addition of ethanol. In the studies of the effects of gassesand cofactors, all column eluates containing cytochrome P-450 were dialyzed and pooled prior to the MEOS assay. The mean of each group was compared with the corresponding control value, and the difference was tested by the Student t-test for groups. Cytochrome P-450 (16), NADPH-cytochrome c reductase (19)) phospholipids(l7) and MEOS activity (3) were also determined in washed microsomes (3) of 18 rats pairfed either an alcohol or a carbohydrates containing control diet (10). The values were corrected for preparative losses of microsomes (20). Each individual result was compared with its corresponding control, and the mean of the individual differences was tested by the Student t-test for pairs. Results During the elution procedure, ADH and catalase appeared in the hemoglobin containing fraction,
whereas MEOS activity was recovered separately with the linear
KC1 gradient (fig. 1). Chromatographic separation of MEOS activity from that of cata-
1189
BIOCHEMICAL
Vol. 49, No. 5, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3
2
I
150
300
450
600
ELUTION
VOLUME
750
900
1050
0
(ml)
Fig. 1 Separation of MEOS from catalase and ADH activities by DEAE-cellulose column chromatography after solubilization of microsomes obtained from rats fed an ethanol containing liquid diet for 3 weeks.
lase and ADH was consistently achieved in twelve different preparations. The fractions containing MEOS activity were free of ADH as established by enzymatic assay. Absence of catalase in these fractions was demonstrated spectrophotometrically by the failure of Hz02 disappearance Q4) and by lack of O2 liberation (15). Recovery of MEOS activity and cytochrome P-450 content in the eluates varied with each individual column and averaged 50%of the amount originally present in the microsomes. The cytochrome P-450 recovery was comparable to that achieved by others (11). Substantial MEOS activity was observed only in the presence of NADPH or the NADPH generating system. With 0.4 mM NADP’ or NAD’, the activities were less than 10%and, with 0.4 mM NADH , the activity was less than 25% of that obtained with 0.4 mM NADPH. In MEOS containing eluates obtained from five different microsomal preparations , replacement of air by N2 markedly diminished MEOS activity (9.18 5 0.49 units/ml vs. 1.59 50.72; p
1190
Vol. 49, No. 5,1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
34% (6.092 0.49 units/ml; p
1191
Vol. 49, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNKATIONS
The biochemical nature of MECS and the mechanism of its increase in activity after chronic ethanol consumption are still unknown. MECS activity is 02 and NADPH dependentand is inhibited by CO. It thereby shares properties with microsomal drug detoxifying enzymes, though some pecularities of MEGS have been described such as insensitivity to SKF 525A (3). In the present experiments, MEGS activity could be demonstrated in fractions which contained cytochrome P-450, NADPH-cytochrome 2 reductase and phospholipids. An apparent correlation between the activity of MEGS and NADPH-cytochrome 2 reductase was found, when phospholipids and sufficient amounts of cytocbrome P-450 were present (fig. 1). These three components also increased in activity in total microsomes after chronic ethanol consumption, and this coincided with an enhancementof MECS activity.
In addition, using a reconstituted
microsomal enzyme system, Lu et al Ql, 21-23)have shownthe role of hepatic microsomal cytochrome P-450, NADPH-cytochrome 2 reductase and phospholipids in the hydroxylation of various drugs. It is conceivable that cytochrome P-450, NADPHcytochrome 2 reductase and phospholipids play a comparable function in ethanol oxidation in microsomes, though the role of some other as yet unidentified mechanisms has not been ruled out. For instance, cytochrome P-450 has been shownto exhibit significant peroxidative activity (24). It is conceivable therefore that cytochrome P-450 (or a componentwith similar properties) may promote the oxidation of ethanol by the latter mechanism. Acknowledgement: We are grateful to Dr. A. Y. H. Lu for his generous advice and to Miss L. M. DeCarli for her encouragementthroughout this work. We also thank Miss N. Lowe, Mrs. E . Jayatilleke and Mr. J. Per1 for their expert technical assistance. This work was supported in part by USPHSgrants MH 15558, AM 12511and the Veterans Administration. References 1. 2. 3. 4.
Iseri, O.A., Gottlieb, L.S. and Lieber, C.S., Fed. Proc. 23:579 (1964). Iseri, O.A., Lieber, C.S. and Gottlieb, L.S., Amer. J. Path. 48:535 (l966). Lieber, C .S. and DeCarli, L. M., J. Biol. Chem. 245:2505 Q970). Lieber, C.S. Rubin, E. and DeCarli, L. M., Biochem . Biophys . Res. Commun. 40:858 (1970). 1192
Vol. 49, No. 5, 1972
5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Isselbacher, K. J. and Carter, E.A., Biochem. Biophys . Res. Commun. 39: 530 Q970). Khanna, J.M. and Kalant, H., Biochem. Pharmacol. 19:2033 (1970). Carter, E.A., and Isselbacher, K. J., Ann. N.Y. Acad. Sci. 179:282(1971). Thurman, R.G., Ley, H.G. and Scholz, R., Europ. J. Biochem. 25:420 (1972). Mezey, E., Potter, J. J. and Reed, W.D., Clin. Res. 20:461 0972). DeCarli, L.M. and Lieber, C.S., J. Nutr. 91:331(1967). Lu, A.Y.H. and Coon, M. 3.) J. Biol. Chem. 243: 13310968). Lowry, O.H., Rosebrough, N. J., Farr, A. and Randall, R. J., J. Biol. Chem. 193:265(1951). Bonnichsen, R.K. and Brinck, N.G. , in Methods in Enzymology I, ed. by S. P. Colowick and N.O. Kaplan, Academic Press, New York, p. 495 (1955). Ltick, H. , in Methods of Enzymatic Analysis, ed. by H. U. Bergmeyer, Academic Press, New York, p. 885 (1963). Goldstein, D.B., Analyt. Biochem. 24:431 (1968). Gmura, T., and Sato, R., J. Biol. Chem. 239:2370 (1964). Bartlett, G.R., J. Biol. Chem. 234: 466 (1959). Folch, J., Lees, M., and Sloane-Stanley, G.H., J. Biol. Chem. 226:497 Q957). Masters, B.S.S., Williams, C. M. and Kamin, H. , in Methods of Enzymology X, ed. by S.P.Colowickand N.O. Kaplan, Academic Press, New York,p. 565 (1967). Greim, H., Naunyn-Schmied. Arch. Pharmakol. 266:261(1970). Lu, A. Y.H. , Strobel, H.W. and Coon, M. J., Biochem. Biophys. Res. Commun. 36:545 (1969). Lu, A.Y.H., Kuntzman, R., West, S. and Conney, A.H., Biochem. Biophys. Res. Commun. 42:1200(1971). Lu, A.Y.H., Kuntzman, R. ) West, S., Jacobson, M. and Conney, A.H. J. Biol. Chem. 247:1727(1972). Hrycay, E.G., and GBrien, P. J., Arch. Biochem. Biophys. 147:14(1971).
1193
Vol. 49, No. 5,1972
AND BIOPHYSKAL
RESEARCH COMMUNICATIONS
MICROSOMAL ETHANOL-OXIDIZING SYSTEM (MEOS): PURIFICATION AND PROPERTIES OF A RAT LIVER SYSTEM FREE OF CATALASE AND ALCOHOL DEHYDROGENASE s R. Teschke*, Y. Hasumura, J.-G. Joly’, H. Ishii , and C.S. Lieber. lepartment of Medicine, Mt. Sinai School of Medicine (CUNY) and Section of Liver Xsease and Nutrition, Veterans Administration Hospital, Bronx, New York 19468. Received
October
6,1972
Summary: A separation of the microsomal ethanol-oxidizing system (MECS) from alcohol dehydrogenase (ADH) and catalase in rat liver microsomes is described. After solubilization of microsomes , ADH and catalase were eluted by DEAE-cellulose chromatography with the hemoglobin containing fraction, whereas MEOS activity was recovered separately. The oxidation of ethanol to acetaldehyde required 02 and NADPH, and was partially inhibited by CO. The fractions exhibiting MEOS activity contained cytochrome P-450, NADPH-cytochrome c reductase and phospholipids. These three components also increased in total microsomes after chronic ethanol consumption and may play a role in the associated enhancementof MEOS activity. The observation that chronic ethanol consumption produces proliferation of the hepatic smooth endoplasmic reticulum (1,2) suggestedthat, in addition to its oxidation by ADH in the cytosol, ethanol may be metabolized by the microsomal fraction.
This
was substantiated by the description of a microsomal ethanol-oxidizing system (MEOS) (3). MEOS was differentiated from catalase and ADH primarily by the use of inhibitors (3,4), but these experiments have been the subject of controversy (5-9). The present study describes the physical separation of MEOS from ADH and catalase activity by column chromatography and reveals the effect of chronic ethanol feeding on three components in the MEOS fraction, namely cytochrome P-450, NADPH-cytochrome c reductase and phospholipids. Materials and Methods Female Swarrue Dawlev rats (Charles River, C. D. ) of a starting bodv weight *Recipient of a grant from the Deutsche Forschungsgemeinschaft . ‘Present address: Centre de Recherches Cliniques , Hopital Saint-Luc Montreal 129, P.Q., Canada. * Present address: Department of Internal Medicine, School of Medicine, Keio University, Shinijuku-Ku, Tokyo, Japan Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
1187
Vol. 49, No. 5, 1972
Of
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
130-16Ogwere fed an alcohol containing liquid diet for 3-4 weeks (10). The prepam-
tion and solubilization of microsomes were achieved by a modification of the procedure of LU and Coon (11). Fresh livers were perfused with ice-cold 0.15 M KCl, pooled and homogenized in 4 volumes of 0.25 M sucrose. After the centrifugation of the homogenateat 10,000 x g for 15 min. , the supernatant layer was spun again at 10,000 x g for 15 min. The supernatant layer was then centrifuged at 105,000 x g for 2 hours. The pellet was suspendedwith 0.25 M sucrose to give a microsomal protein concentration (12) of 30 mg/ml.
To approximately 23 ml of such a suspensionthe following
were added: 14.5 ml glycerol, 4. ‘7ml 1 M KC1, 4.7 ml 1 M potassium citrate (in 0 .l M phosphate buffer pH 7.4) and 0.05 ml of 0.1 M dithlothreitol.
This mixture was
sonlcated with the microtip of a Sonifier Cell Disruptor (Ultrasonics Incorp. ) at full output for 6 periods of 20 sec. each. After the addition of 2.7 ml 10%deoxycholate (w/v) the mixture was stirred for 20 min. and passedthrough glasswool. The filtrate was diluted with 2 volumes of distilled water and put onto a DEAE-cellulose column (2.5 x 50 cm). The column was then washed and eluted (ll). ADH (13)and catalase (14)activities were determined respectively in 0.5 and 0 .l ml samples of the column eluates . Catalase units were calculated according to LUck Q4). Absence of catalase activity in the fractions containing MECS activity was also verified with the oxygen electrode technique (15). Cytochrome P-450 content was measured in microsomes (16) and column eluates (11). NADPH-cytochrome c reductase was determined with 0.5 ml of eluate (11). Phospholipids were estimated by their phosphorous content (17)after extraction according to Folch et al (18). Eluates to be used for MECS assay were dialyzed overnight against 0 .l M phosphate buffer @H 7.0). Ethanol oxidation to acetaldehyde was determined according to a modification of the procedure of Lieber and DeCarli (3) and ME06 activity was expressed in units corresponding to the nanomolesof acetaldehyde produced per min. Two ml aliquots Of
1188
Vol. 49, No. 5,1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dialyzed column eluates were preincubated with 50 mM ethanol (in 0.5 ml 0.1 M phosphate buffer, pH 7.4) for 5 min. at 37’C.
The reaction was started by adding NADPH,
EDTA and Iv&C12(in 0.5 ml 001 M phosphate buffer, pH 7.4) at final concentrations of 0.4, 1 and 5 mM respectively.
For each determination, at least 6 flasks were used
with duplicate incubations for 0, 5 and 10 min. ; MEOS activity was linear over the period tested. The identity of the product with acetaldehyde was confirmed by cornpalative absorption spectra and by measurement of the retention time by gas liquid chromatography (3). In the studies of the effects of gasses,NADPH was replaced by a NADPH generating system with 0.4 mM NAD@ , 8 mM sodium isocitrate, and 2 g per liter of isocitrate dehydrogenase (crude type I, Sigma Chemical Company). The NADPH generating system was preincubated with the column eluates for 5 min. at 37’C, the flasks were flushed with the gas for another 5 min. and the reaction was started by addition of ethanol. In the studies of the effects of gassesand cofactors, all column eluates containing cytochrome P-450 were dialyzed and pooled prior to the MEOS assay. The mean of each group was compared with the corresponding control value, and the difference was tested by the Student t-test for groups. Cytochrome P-450 (16), NADPH-cytochrome c reductase (19)) phospholipids(l7) and MEOS activity (3) were also determined in washed microsomes (3) of 18 rats pairfed either an alcohol or a carbohydrates containing control diet (10). The values were corrected for preparative losses of microsomes (20). Each individual result was compared with its corresponding control, and the mean of the individual differences was tested by the Student t-test for pairs. Results During the elution procedure, ADH and catalase appeared in the hemoglobin containing fraction,
whereas MEOS activity was recovered separately with the linear
KC1 gradient (fig. 1). Chromatographic separation of MEOS activity from that of cata-
1189
BIOCHEMICAL
Vol. 49, No. 5, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3
2
I
150
300
450
600
ELUTION
VOLUME
750
900
1050
0
(ml)
Fig. 1 Separation of MEOS from catalase and ADH activities by DEAE-cellulose column chromatography after solubilization of microsomes obtained from rats fed an ethanol containing liquid diet for 3 weeks.
lase and ADH was consistently achieved in twelve different preparations. The fractions containing MEOS activity were free of ADH as established by enzymatic assay. Absence of catalase in these fractions was demonstrated spectrophotometrically by the failure of Hz02 disappearance Q4) and by lack of O2 liberation (15). Recovery of MEOS activity and cytochrome P-450 content in the eluates varied with each individual column and averaged 50%of the amount originally present in the microsomes. The cytochrome P-450 recovery was comparable to that achieved by others (11). Substantial MEOS activity was observed only in the presence of NADPH or the NADPH generating system. With 0.4 mM NADP’ or NAD’, the activities were less than 10%and, with 0.4 mM NADH , the activity was less than 25% of that obtained with 0.4 mM NADPH. In MEOS containing eluates obtained from five different microsomal preparations , replacement of air by N2 markedly diminished MEOS activity (9.18 5 0.49 units/ml vs. 1.59 50.72; p
1190
Vol. 49, No. 5,1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
34% (6.092 0.49 units/ml; p
1191
Vol. 49, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNKATIONS
The biochemical nature of MECS and the mechanism of its increase in activity after chronic ethanol consumption are still unknown. MECS activity is 02 and NADPH dependentand is inhibited by CO. It thereby shares properties with microsomal drug detoxifying enzymes, though some pecularities of MEGS have been described such as insensitivity to SKF 525A (3). In the present experiments, MEGS activity could be demonstrated in fractions which contained cytochrome P-450, NADPH-cytochrome 2 reductase and phospholipids. An apparent correlation between the activity of MEGS and NADPH-cytochrome 2 reductase was found, when phospholipids and sufficient amounts of cytocbrome P-450 were present (fig. 1). These three components also increased in activity in total microsomes after chronic ethanol consumption, and this coincided with an enhancementof MECS activity.
In addition, using a reconstituted
microsomal enzyme system, Lu et al Ql, 21-23)have shownthe role of hepatic microsomal cytochrome P-450, NADPH-cytochrome 2 reductase and phospholipids in the hydroxylation of various drugs. It is conceivable that cytochrome P-450, NADPHcytochrome 2 reductase and phospholipids play a comparable function in ethanol oxidation in microsomes, though the role of some other as yet unidentified mechanisms has not been ruled out. For instance, cytochrome P-450 has been shownto exhibit significant peroxidative activity (24). It is conceivable therefore that cytochrome P-450 (or a componentwith similar properties) may promote the oxidation of ethanol by the latter mechanism. Acknowledgement: We are grateful to Dr. A. Y. H. Lu for his generous advice and to Miss L. M. DeCarli for her encouragementthroughout this work. We also thank Miss N. Lowe, Mrs. E . Jayatilleke and Mr. J. Per1 for their expert technical assistance. This work was supported in part by USPHSgrants MH 15558, AM 12511and the Veterans Administration. References 1. 2. 3. 4.
Iseri, O.A., Gottlieb, L.S. and Lieber, C.S., Fed. Proc. 23:579 (1964). Iseri, O.A., Lieber, C.S. and Gottlieb, L.S., Amer. J. Path. 48:535 (l966). Lieber, C .S. and DeCarli, L. M., J. Biol. Chem. 245:2505 Q970). Lieber, C.S. Rubin, E. and DeCarli, L. M., Biochem . Biophys . Res. Commun. 40:858 (1970). 1192
Vol. 49, No. 5, 1972
5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Isselbacher, K. J. and Carter, E.A., Biochem. Biophys . Res. Commun. 39: 530 Q970). Khanna, J.M. and Kalant, H., Biochem. Pharmacol. 19:2033 (1970). Carter, E.A., and Isselbacher, K. J., Ann. N.Y. Acad. Sci. 179:282(1971). Thurman, R.G., Ley, H.G. and Scholz, R., Europ. J. Biochem. 25:420 (1972). Mezey, E., Potter, J. J. and Reed, W.D., Clin. Res. 20:461 0972). DeCarli, L.M. and Lieber, C.S., J. Nutr. 91:331(1967). Lu, A.Y.H. and Coon, M. 3.) J. Biol. Chem. 243: 13310968). Lowry, O.H., Rosebrough, N. J., Farr, A. and Randall, R. J., J. Biol. Chem. 193:265(1951). Bonnichsen, R.K. and Brinck, N.G. , in Methods in Enzymology I, ed. by S. P. Colowick and N.O. Kaplan, Academic Press, New York, p. 495 (1955). Ltick, H. , in Methods of Enzymatic Analysis, ed. by H. U. Bergmeyer, Academic Press, New York, p. 885 (1963). Goldstein, D.B., Analyt. Biochem. 24:431 (1968). Gmura, T., and Sato, R., J. Biol. Chem. 239:2370 (1964). Bartlett, G.R., J. Biol. Chem. 234: 466 (1959). Folch, J., Lees, M., and Sloane-Stanley, G.H., J. Biol. Chem. 226:497 Q957). Masters, B.S.S., Williams, C. M. and Kamin, H. , in Methods of Enzymology X, ed. by S.P.Colowickand N.O. Kaplan, Academic Press, New York,p. 565 (1967). Greim, H., Naunyn-Schmied. Arch. Pharmakol. 266:261(1970). Lu, A. Y.H. , Strobel, H.W. and Coon, M. J., Biochem. Biophys. Res. Commun. 36:545 (1969). Lu, A.Y.H., Kuntzman, R., West, S. and Conney, A.H., Biochem. Biophys. Res. Commun. 42:1200(1971). Lu, A.Y.H., Kuntzman, R. ) West, S., Jacobson, M. and Conney, A.H. J. Biol. Chem. 247:1727(1972). Hrycay, E.G., and GBrien, P. J., Arch. Biochem. Biophys. 147:14(1971).
1193