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Enzyme induction with high doses of rifampicin in Wistar rats
Toxicology Letters, 17 (1983) 301-306 Elsevier
ENZYME INDUCTION IN WISTAR RATS (Rifampicin;
301
WITH HIGH DOSES OF RIFAMPICIN
enzyme induction; liver; rat)
A. PIRIOU, A. JACQUESON, J.M. WARNET and J.R. CLAUDE Laboratoire de Recherche de la D.A.S.S. de Paris, 27, rue Lacordaire, 75015 Paris, and Laboratoire Central de Biochimie et de Toxicologic, HGpitalJean Bernard, La Miletrie, 86021 Poitiers Cedex (France) (Received December 22nd, 1982) (Revision received February 14th, 1983) (Accepted February 15th, 1983)
SUMMARY Hepatic microsomal enzyme activities were determined in female Wistar rats after 1 and 8 days of oral administration of high doses of rifampicin (RFP) (400 mg/kg/day). After 8 days, the level of cytochrome P-450 doubled and the activities of NADPH-cytochrome c reductase and benzphetamine N-demethylase were significantly increased. The observed changes in enzymic activities are consistent with the possibility that RFP induces a special form of cytochrome P-450, responsible for the metabolism of the antibiotic (demethylation and reduction of rifampicin quinone). Considering the role of the endoplasmic reticulum in lipid metabolism, the inducing activity of RFP might also contribute to the observed accumulation of lipids in the liver.
INTRODUCTION
We previously showed [l, 21 that high oral doses of RFP resulted in an accumulation of lipids in the liver and a drop in circulating lipoprotein levels in .male and female Wistar rats. RFP intoxication in rats leads to complex metabolic modifications. The inhibition of DNA-dependent RNA polymerases observed in prokaryotes may also be noted in eukaryotes when the antibiotic is given at high doses. This inhibition could result in a defective synthesis of the protein moiety of lipoproteins, resulting in defective &lipoprotein excretion. In addition to a preferential augmentation of triglycerides when hepatic lipids accumulate, we also noted an accumulation of cholesterol. Although modest, this acAbbreviations: GGT, -r-glutamyl transferase; RFP, rifampicin; UDPGT, uridine diphosphoglucoronosyl transferase; VLDL, very low density lipoproteins. 0378-4274/83/0000-OOOO/$ 03.00 0 Elsevier Science Publishers
302
cumulation is not normally observed in fatty liver occurring in various intoxications. Other mechanisms must thus be considered. The involvement of the liver in lipid metabolism, especially the microsomal subcellular fraction, is known. RFP, like most exogenous substances, is metabolized primarily in the liver by direct metabolic transformation and by conjugations. The direct transformation reactions occur in the endoplasmic reticulum via cytochrome P-450 dependent enzymes. It has been shown in humans that rifampicin can induce cytochrome P-450 enzymes and cause disturbances in the metabolism of endogenous substances as well as in drugs. On the other hand, this effect is much less marked in animals. Existing observations, especially in rats, are contradictory [3, 41. The present work was performed to determine whether or not high doses of rifampicin could change the activities of certain enzymes bound to the endoplasmic reticulum. MATERIALS AND METHODS
Animals and experimental protocol Female Wistar rats weighing 200-220 g were used. RFP was suspended in tragacanth gum syrup and administered by gastric intubation at doses of 400 mg/kg/day. One group was dosed for 1 day and a second group for 8 days. Control and dosed rats were killed by cranial trauma and the liver was removed after perfusion; a 10% (w/v) liver homogenate was prepared. Liver microsomes were separated by differential sedimentation. Enzyme assays All enzyme activities were determined at 37°C and were expressed on a protein basis. (a) Total homogenate GGT was assayed by the kinetic method of Szasz [5], using Beckman reagents. UDPGT was assayed with the technique described by Cotte et al. [6]. (b) Microsomal fraction Cytochromes bs and P-450 were assayed with the method of Omura and Sato [7] by the difference spectrum obtained with a DW2 Aminco spectrophotometer equipped with the Midan T accessory. NADPH-cytochrome c reductase activity was determined as described by Maze1 [8]. p-Nitroanisol 0-demethylase was assayed with the technique of Zannoni [9]. Benzphetamine N-demethylase was assayed by determining the formaldehyde formed during the Nash reaction [lo]. Aniline hydroxylase was determined according to
303
Maze1 (81. Ethoxycoumarin-0-deethylase was assayed according to the method of Ullrich et al. [l 11. The formation of 7-hydroxycoumarin was followed by fluorescence in an Aminco Spectrofluorometer. The activities of various inhibitors were also studied: metyrapone, cy-naphthoflavone and tetrahydrofuran.
Protein The determination of protein was performed according to the method of Lowry et al. [12] using crystalline bovine albumin as standard.
Statistical analysis Each measurement was carried out at least in duplicate. The results obtained are expressed as means(m) and standard deviation (SD) and the significance of the differences was assayed by Student’s t-test.
TABLE I EFFECTS OF RIFAMPICIN (RFP) ON HEPATIC ENZYME ACTIVITIES IN FEMALE WlSTAR RATS AFTER 1 AND 8 DAYS OF TREATMENT AT 400 mg/kg Cytochrome levels expressed as nmol/mg, microsomal enzyme activities expressed as nmol/min/mg total homogenate enzyme activities expressed as IU/mg.
Cyt. bs cyt. P-450 NADPH-cyt.c
reductase
Aniline hydroxylase Benzphetamine N-demethylase p-Nitroanisole
0-demethylase
‘I-Ethoxycoumarin O-deethylase GGT UDPGT
m SD m SD m SD m SD m SD m SD m SD m SD m SD
and
Controls (N=6)
1 day RFP (N=5)
8 days RFP (N=5)
0.43 0.10 0.92 0.13 55 15 0.54 0.09 0.97 0.22 3.05 1.11 0.27 0.06 1.36 1.03 0.49 0.05
0.52 0.05 1.00 0.11 76 24 0.58 0.04 1.64** 0.33 2.44 0.40 0.30 0.08 1.41 1.08 0.44 0.11
0.62** 0.06 1.99*** 0.24 89++ 12 0.67 0.11 2.33** 0.35 3.36 0.89 0.34 0.08 5.84*** 2.12 1.67 1.24
N, number of animals. Significance of difference from controls: ‘P
304
RESULTS
The enzyme assays performed on the microsomal fractions of the rats are reported in Table I. Treatment with RFP at 400 mg/kg/day lead to an increase of cytochrome bs and P-450 activities, which was significant only after 8 days. The two-fold increase of cytochrome P-450 levels was accompanied by an increase of NADPH-cytochrome c reductase. Benzphetamine N-demethylase activity was significantly increased from the first day and to a greater extent after 8 days. By contrast, p-nitroanisole 0-demethylase and aniline hydroxylase were not modified. The activity of ethoxycoumarin Odeethylase was slightly but not significantly increased; the effects of the 3 added inhibitors was the same in all groups. The activities of GGT and UDPGT (Table I) did not vary after 1 day of treatment. After 8 days, however, there was a highly significant increase of GGT activity with a parallel increase of serum GGT (from 2.5 UI/I to 7.3 UM). The activity of UDPGT was also increased after 8 days, but this variation was not statistically significant because the variance was too high. DISCUSSION
Our data show that RFP, given orally to female Wistar rats at the dose of 400 mg/kg/day produces an enzyme-inducing effect. It seems that the action of RFP on enzyme activities takes place as early as the first day evidenced by an increase of benzphetamine-N-demethylase activity. This inducing effect increases gradually for 8 days. The benzphetamine N-demethylase activity as well as cytochrome bs, cytochrome P-450 and NADPH-cytochrome c reductase are then much higher. These results permit an explanation for the contradictions which exist between the work of Barone [3] and Otani [4]. The former observed no inducing effects in rats treated with RFP at 170 mg/kg/day for 10 days per OS. The latter administered the drug at 20 mg/kg/day for 5 days per OS; 2 days after the end of the treatment the rats were killed. Increased activities of NADPHcytochrome c reductase and benzphetamine N-demethylase were observed. The discrepancy is due to the fact that RFP causes a selective activation of cytochrome P-450-dependent enzymes in rat liver microsomes. The RFP-induced cytochrome P-450 seems able to demethylate particularly the substrates where the methyl group is bound to a nitrogen atom. On the contrary it does not appear especially active with regard to the other substrates; many other cytochrome P-450 dependent reactions are unaffected. This selective inducing effect leads us to consider two special points: (a) The metabolic process of RFP. RFP stimulates its own metabolism. This antibiotic is metabolised by microsomal enzymes to desacetylrifampicin. In addition to this
305
metabolite, large quantities of demethylrifampicin are produced in rats [ 131. The increased activity of NADPH-cytochrome c reductase may explain the fact that the antibiotic stimulates the hepatic reducing activity of quinone forms, especially rifampicin-quinone which is continually formed. This latter form might bind to the amino groups of proteins and nucleic acids which would explain its toxicity. Thus rifampicin exhibits a dual contradictory character because it acts successively as toxicant and as a modulator of its own toxicity. (b) The potential relationship between enzymic induction and the liver accumulation of lipids. The liver microsomal enzyme system may participate to the activation of fatty acids and stimulate the activity of phosphatidate phosphatase which converts CY,P-diglyceride phosphate to CY,/3-diglyceride and of hydroxymethyl-glutaryl CoA reductase [14], which are involved in the synthesis of cholesterol and also of VLDL. ACKNOWLEDGEMENTS
The authors thank Annie Guettier and Yvette Save for their technical assistance. REFERENCES 1 A. Piriou, J.M. Warnet, A. Jacqueson, J.R. Claude and R. Truhaut, Fatty liver induced by high doses of rifampicin in the rat: possible relation with an inhibition of RNA polymerases in eukariotic cells, Arch. Toxicol., Suppl. 2 (1979) 333-337. 2 A. Piriou, A. Jacqueson, M. Thevenin, J.M. Warnet and J.R. Claude, Study of the protective effect of an anabolic steroid, 1PNortestosterone phenylpropionate (IPNTPP) on the fatty liver induced by high doses of rifampicin in the rat, Arch. Toxicol., Suppl. 4 (1980) 331-334. 3 D. Barone, E. Beretta and L.T. Tenconi, Rifampicin and liver drug-metabolizing systems: studies in guinea-pigs, rats and mice, Acta Vitaminol. Enzymol., 26 (1972) 124-125. 4 G. Otani and H. Remmer, Participation of microsomal NAPDH-cytochrome c reductase in the metabolism of rifampicin. ,Arch. Pharmacol., 287 (1975) R76. 5 G.S. Szasz, A kinetic photometric method for serum gamma glutamyl transpeptidase, Clin. Chem., 15 (1969) 124-136. 6 J. Cotte, M. Mathieu, J.P. Andre, C. Collombel and L. Padis, Methode de dosage de l’activitt glucuronyltransferase htpatique applicable en biologie clinique, Enz. Biol. Clin., 8 (1%7) 387-399. 7 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes, J. Biol. Chem., 239 (1964) 2370-2385. 8 P. Maze], Experiments illustrating drug metabolism in vitro, in B.N. La Du, H.G. Mandel, and E.L. Way (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore, 1971, p. 546. 9 V.G. Zannoni, Experiments illustrating drug metabolism in vitro. Microsomal pnitroanisole Odemethylase, in B.N. La Du, H.G. Mandel and E.L. Way (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore 1971, pp. 566-569. 10 T. Nash, The calorimetric estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J., 55 (1953) 416-421. 11 V. Ullrich and P. Weber, The 0-dealkylation of ‘I-ethoxycoumarin by liver microsomes: a direct fluorimetric test, Z. Physiol. Chem., 353 (1972) 1171-1177.
306
12 O.H. phenol
Lowry,
N.J.
reagent,
13 T. Akimoto,
Rosebrough,
J. Biol. Chem.,
S. Hauser,
J. Antibiot., V. Lapar
liver of the rat, J. Lipid.
Res.,
Farr
and R.J.
Randall,
Protein
measurement
with the Folin
193 (1951) 265-275.
K. Ono and T. Nanpo,
picin in the rat, Jap. 14 S. Shefer,
A.L.
Absorption,
distribution,
metabolism
and excretion
of rifam-
23 (1970) 250-255. and E.H.
Mosbach,
13 (1972) 69-70.
HmG
CoA reductase
of intestinal
mucosa
and
ENZYME INDUCTION IN WISTAR RATS (Rifampicin;
301
WITH HIGH DOSES OF RIFAMPICIN
enzyme induction; liver; rat)
A. PIRIOU, A. JACQUESON, J.M. WARNET and J.R. CLAUDE Laboratoire de Recherche de la D.A.S.S. de Paris, 27, rue Lacordaire, 75015 Paris, and Laboratoire Central de Biochimie et de Toxicologic, HGpitalJean Bernard, La Miletrie, 86021 Poitiers Cedex (France) (Received December 22nd, 1982) (Revision received February 14th, 1983) (Accepted February 15th, 1983)
SUMMARY Hepatic microsomal enzyme activities were determined in female Wistar rats after 1 and 8 days of oral administration of high doses of rifampicin (RFP) (400 mg/kg/day). After 8 days, the level of cytochrome P-450 doubled and the activities of NADPH-cytochrome c reductase and benzphetamine N-demethylase were significantly increased. The observed changes in enzymic activities are consistent with the possibility that RFP induces a special form of cytochrome P-450, responsible for the metabolism of the antibiotic (demethylation and reduction of rifampicin quinone). Considering the role of the endoplasmic reticulum in lipid metabolism, the inducing activity of RFP might also contribute to the observed accumulation of lipids in the liver.
INTRODUCTION
We previously showed [l, 21 that high oral doses of RFP resulted in an accumulation of lipids in the liver and a drop in circulating lipoprotein levels in .male and female Wistar rats. RFP intoxication in rats leads to complex metabolic modifications. The inhibition of DNA-dependent RNA polymerases observed in prokaryotes may also be noted in eukaryotes when the antibiotic is given at high doses. This inhibition could result in a defective synthesis of the protein moiety of lipoproteins, resulting in defective &lipoprotein excretion. In addition to a preferential augmentation of triglycerides when hepatic lipids accumulate, we also noted an accumulation of cholesterol. Although modest, this acAbbreviations: GGT, -r-glutamyl transferase; RFP, rifampicin; UDPGT, uridine diphosphoglucoronosyl transferase; VLDL, very low density lipoproteins. 0378-4274/83/0000-OOOO/$ 03.00 0 Elsevier Science Publishers
302
cumulation is not normally observed in fatty liver occurring in various intoxications. Other mechanisms must thus be considered. The involvement of the liver in lipid metabolism, especially the microsomal subcellular fraction, is known. RFP, like most exogenous substances, is metabolized primarily in the liver by direct metabolic transformation and by conjugations. The direct transformation reactions occur in the endoplasmic reticulum via cytochrome P-450 dependent enzymes. It has been shown in humans that rifampicin can induce cytochrome P-450 enzymes and cause disturbances in the metabolism of endogenous substances as well as in drugs. On the other hand, this effect is much less marked in animals. Existing observations, especially in rats, are contradictory [3, 41. The present work was performed to determine whether or not high doses of rifampicin could change the activities of certain enzymes bound to the endoplasmic reticulum. MATERIALS AND METHODS
Animals and experimental protocol Female Wistar rats weighing 200-220 g were used. RFP was suspended in tragacanth gum syrup and administered by gastric intubation at doses of 400 mg/kg/day. One group was dosed for 1 day and a second group for 8 days. Control and dosed rats were killed by cranial trauma and the liver was removed after perfusion; a 10% (w/v) liver homogenate was prepared. Liver microsomes were separated by differential sedimentation. Enzyme assays All enzyme activities were determined at 37°C and were expressed on a protein basis. (a) Total homogenate GGT was assayed by the kinetic method of Szasz [5], using Beckman reagents. UDPGT was assayed with the technique described by Cotte et al. [6]. (b) Microsomal fraction Cytochromes bs and P-450 were assayed with the method of Omura and Sato [7] by the difference spectrum obtained with a DW2 Aminco spectrophotometer equipped with the Midan T accessory. NADPH-cytochrome c reductase activity was determined as described by Maze1 [8]. p-Nitroanisol 0-demethylase was assayed with the technique of Zannoni [9]. Benzphetamine N-demethylase was assayed by determining the formaldehyde formed during the Nash reaction [lo]. Aniline hydroxylase was determined according to
303
Maze1 (81. Ethoxycoumarin-0-deethylase was assayed according to the method of Ullrich et al. [l 11. The formation of 7-hydroxycoumarin was followed by fluorescence in an Aminco Spectrofluorometer. The activities of various inhibitors were also studied: metyrapone, cy-naphthoflavone and tetrahydrofuran.
Protein The determination of protein was performed according to the method of Lowry et al. [12] using crystalline bovine albumin as standard.
Statistical analysis Each measurement was carried out at least in duplicate. The results obtained are expressed as means(m) and standard deviation (SD) and the significance of the differences was assayed by Student’s t-test.
TABLE I EFFECTS OF RIFAMPICIN (RFP) ON HEPATIC ENZYME ACTIVITIES IN FEMALE WlSTAR RATS AFTER 1 AND 8 DAYS OF TREATMENT AT 400 mg/kg Cytochrome levels expressed as nmol/mg, microsomal enzyme activities expressed as nmol/min/mg total homogenate enzyme activities expressed as IU/mg.
Cyt. bs cyt. P-450 NADPH-cyt.c
reductase
Aniline hydroxylase Benzphetamine N-demethylase p-Nitroanisole
0-demethylase
‘I-Ethoxycoumarin O-deethylase GGT UDPGT
m SD m SD m SD m SD m SD m SD m SD m SD m SD
and
Controls (N=6)
1 day RFP (N=5)
8 days RFP (N=5)
0.43 0.10 0.92 0.13 55 15 0.54 0.09 0.97 0.22 3.05 1.11 0.27 0.06 1.36 1.03 0.49 0.05
0.52 0.05 1.00 0.11 76 24 0.58 0.04 1.64** 0.33 2.44 0.40 0.30 0.08 1.41 1.08 0.44 0.11
0.62** 0.06 1.99*** 0.24 89++ 12 0.67 0.11 2.33** 0.35 3.36 0.89 0.34 0.08 5.84*** 2.12 1.67 1.24
N, number of animals. Significance of difference from controls: ‘P
304
RESULTS
The enzyme assays performed on the microsomal fractions of the rats are reported in Table I. Treatment with RFP at 400 mg/kg/day lead to an increase of cytochrome bs and P-450 activities, which was significant only after 8 days. The two-fold increase of cytochrome P-450 levels was accompanied by an increase of NADPH-cytochrome c reductase. Benzphetamine N-demethylase activity was significantly increased from the first day and to a greater extent after 8 days. By contrast, p-nitroanisole 0-demethylase and aniline hydroxylase were not modified. The activity of ethoxycoumarin Odeethylase was slightly but not significantly increased; the effects of the 3 added inhibitors was the same in all groups. The activities of GGT and UDPGT (Table I) did not vary after 1 day of treatment. After 8 days, however, there was a highly significant increase of GGT activity with a parallel increase of serum GGT (from 2.5 UI/I to 7.3 UM). The activity of UDPGT was also increased after 8 days, but this variation was not statistically significant because the variance was too high. DISCUSSION
Our data show that RFP, given orally to female Wistar rats at the dose of 400 mg/kg/day produces an enzyme-inducing effect. It seems that the action of RFP on enzyme activities takes place as early as the first day evidenced by an increase of benzphetamine-N-demethylase activity. This inducing effect increases gradually for 8 days. The benzphetamine N-demethylase activity as well as cytochrome bs, cytochrome P-450 and NADPH-cytochrome c reductase are then much higher. These results permit an explanation for the contradictions which exist between the work of Barone [3] and Otani [4]. The former observed no inducing effects in rats treated with RFP at 170 mg/kg/day for 10 days per OS. The latter administered the drug at 20 mg/kg/day for 5 days per OS; 2 days after the end of the treatment the rats were killed. Increased activities of NADPHcytochrome c reductase and benzphetamine N-demethylase were observed. The discrepancy is due to the fact that RFP causes a selective activation of cytochrome P-450-dependent enzymes in rat liver microsomes. The RFP-induced cytochrome P-450 seems able to demethylate particularly the substrates where the methyl group is bound to a nitrogen atom. On the contrary it does not appear especially active with regard to the other substrates; many other cytochrome P-450 dependent reactions are unaffected. This selective inducing effect leads us to consider two special points: (a) The metabolic process of RFP. RFP stimulates its own metabolism. This antibiotic is metabolised by microsomal enzymes to desacetylrifampicin. In addition to this
305
metabolite, large quantities of demethylrifampicin are produced in rats [ 131. The increased activity of NADPH-cytochrome c reductase may explain the fact that the antibiotic stimulates the hepatic reducing activity of quinone forms, especially rifampicin-quinone which is continually formed. This latter form might bind to the amino groups of proteins and nucleic acids which would explain its toxicity. Thus rifampicin exhibits a dual contradictory character because it acts successively as toxicant and as a modulator of its own toxicity. (b) The potential relationship between enzymic induction and the liver accumulation of lipids. The liver microsomal enzyme system may participate to the activation of fatty acids and stimulate the activity of phosphatidate phosphatase which converts CY,P-diglyceride phosphate to CY,/3-diglyceride and of hydroxymethyl-glutaryl CoA reductase [14], which are involved in the synthesis of cholesterol and also of VLDL. ACKNOWLEDGEMENTS
The authors thank Annie Guettier and Yvette Save for their technical assistance. REFERENCES 1 A. Piriou, J.M. Warnet, A. Jacqueson, J.R. Claude and R. Truhaut, Fatty liver induced by high doses of rifampicin in the rat: possible relation with an inhibition of RNA polymerases in eukariotic cells, Arch. Toxicol., Suppl. 2 (1979) 333-337. 2 A. Piriou, A. Jacqueson, M. Thevenin, J.M. Warnet and J.R. Claude, Study of the protective effect of an anabolic steroid, 1PNortestosterone phenylpropionate (IPNTPP) on the fatty liver induced by high doses of rifampicin in the rat, Arch. Toxicol., Suppl. 4 (1980) 331-334. 3 D. Barone, E. Beretta and L.T. Tenconi, Rifampicin and liver drug-metabolizing systems: studies in guinea-pigs, rats and mice, Acta Vitaminol. Enzymol., 26 (1972) 124-125. 4 G. Otani and H. Remmer, Participation of microsomal NAPDH-cytochrome c reductase in the metabolism of rifampicin. ,Arch. Pharmacol., 287 (1975) R76. 5 G.S. Szasz, A kinetic photometric method for serum gamma glutamyl transpeptidase, Clin. Chem., 15 (1969) 124-136. 6 J. Cotte, M. Mathieu, J.P. Andre, C. Collombel and L. Padis, Methode de dosage de l’activitt glucuronyltransferase htpatique applicable en biologie clinique, Enz. Biol. Clin., 8 (1%7) 387-399. 7 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes, J. Biol. Chem., 239 (1964) 2370-2385. 8 P. Maze], Experiments illustrating drug metabolism in vitro, in B.N. La Du, H.G. Mandel, and E.L. Way (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore, 1971, p. 546. 9 V.G. Zannoni, Experiments illustrating drug metabolism in vitro. Microsomal pnitroanisole Odemethylase, in B.N. La Du, H.G. Mandel and E.L. Way (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore 1971, pp. 566-569. 10 T. Nash, The calorimetric estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J., 55 (1953) 416-421. 11 V. Ullrich and P. Weber, The 0-dealkylation of ‘I-ethoxycoumarin by liver microsomes: a direct fluorimetric test, Z. Physiol. Chem., 353 (1972) 1171-1177.
306
12 O.H. phenol
Lowry,
N.J.
reagent,
13 T. Akimoto,
Rosebrough,
J. Biol. Chem.,
S. Hauser,
J. Antibiot., V. Lapar
liver of the rat, J. Lipid.
Res.,
Farr
and R.J.
Randall,
Protein
measurement
with the Folin
193 (1951) 265-275.
K. Ono and T. Nanpo,
picin in the rat, Jap. 14 S. Shefer,
A.L.
Absorption,
distribution,
metabolism
and excretion
of rifam-
23 (1970) 250-255. and E.H.
Mosbach,
13 (1972) 69-70.
HmG
CoA reductase
of intestinal
mucosa
and