THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 10,Issue of May 25, pp. 3623%3630,*1973

Printed in U.S.A.

Differences between Germ-free and Conventional Rats in Liver Microsomal Metabolism of Steroids*

(Received for publication, December 26, 1972)

KURT EINARSSON, JAN-&E GUSTAFSSON, AND BENGTE. GUSTAFSSON

From the Department of Medicine, XeraJimerlasarettet, Department of Chemistq and Department of Germ-fyee Research, Karolinska Institute, Stockholm, Sweden

SUMMARY

The metabolism of some steroids has been investigated in liver microsomal preparations from male germ-free and conventional rats. Hydroxylation of 4-[4-14C]androstene- 3,17-dione in positions 60 and 16~~ is about 2 and 1.5 times more efficient in germ-free than in conventional rats. The same is true for 6/3- and Zol-hydroxylation of 4-[4J4C]preg- nene-3,20-dione (about 1.6 and 1.2 times larger, respectively, in germ-free animals), for 1%hydroxylation of 5a-[4J4C]- androstane-3a, 17P-diol (about 1.6 times larger in germ-free rats), and for 6P-hydroxylation of lithocholic acid (about 2 times larger in germ-free rats). All these differences are statistically significant (significance level, p < 0.05). Sim- ilar differences (although not statistically significant) are also found for 16or-hydroxylation of 4-pregnene-3,20-dione and for 2/3- and 2a-hydroxylation of Sac-androstane-30c, 17p- diol. The amount of cytochrome P-450 in germ-free animals is 2.53 f 0.45 nmoles per mg of protein as compared to 1.72 i 0.04 nmoles per mg of protein in conventional animals (p < 0.005). %-Reduction of 4-androstene-3,17-dione, 4-pregnene-3,20-dione and of 7~[6/3-~H]hydroxy-4-cho- lesten-3.one tends to be lower in germ-free than in con- ventional rats.

In contrast to these results, 12cz-hydroxylation of 7~ hydroxy-4-cholesten-3-one and 7or-hydroxylation of [4J4C]- cholesterol are larger in conventional than in germ-free rats. These findings are in accordance with the slower cholesterol and bile acid turnover in germ-free compared to conventional rats.

The metabolism of steroid hormones and bile acids in vivo is widely different in germ-free rats compared to conventional rats (1, 2). The biological half-lives of steroids have been found to be shorter in conventional than in germ-free animals (3)) plasma levels of some steroids are higher in germ-free than in conven- tional rats,’ and the patterns of steroids in feces and urine

* This work was supported by grants from Ekhagastiftelsen and from the Swedish Medical Research Council (Proiects 16X- 206 and 13X-2819).

I

1 T. Nomura, M. Saito, 0. Nakaaki, and K. Kageyama (1972) The Fourth Znfernational Symposium on Germ-free Research, Xew Orleans, Louisiana, April 16 to 22, Abstract 59.

from germ-free and conventional rats are quite different (2). Several of these differences are due to the direct participation of the intestinal microflora in the metabolism of steroids (4, 5). Comparatively little is known, however, about the influence of the intestinal bacteria upon steroid-metabolizing enzyme levels in the tissues of the host. The hydroxylase system present in the microsomal fraction of liver is inducible by various drugs such as phenobarbital (6) and steroids such as 16ar-cganopreg- nenolone (7), and it is possible that the microsomal enzyme activities could be influenced by endogenous plasma steroids or by absorbed intestinal bacterial steroidal or nonsteroidal metabo- lites. An investigation concerning the metabolism of steroids in the microsomal fraction of livers from germfree and conven- tional rats was therefore considered of interest. In the present paper we have studied the metabolism of some steroid hormones (androstenedione, progesterone, and 5a-androstane-?a , 17fi-diol), sterols (cholesterol and 7cr-hydroxy-4-cholesten&one), and one bile acid (lithocholic acid).

MATERIALS AND METHOD'

Reference Compounds-Several of the reference compounds used in this investigation were generous gifts from colleagues: Dr. J. Babcock (4.androstene-3,17-dione, 4-pregnene-3,20-dione, 17fi-hydroxy-4-androsten-3-one, 200(- and 20@-hydroxy-4-preg- nen-a-one, and SD- and 16a-hydroxy-4-pregnene-3,20-dione), Dr. M. Ehrenstein (6fi-hydroxy-4-androstene-3,17-dione), Dr. D. K. Fukushima (3a, l%dihydroxy-5a-androstan-17.one), Dr. W. Staib (7ol-hydroxy-4-androstene-3,17-dione), Dr. J. Ufer (5oc-androstane-3,17- dione, and 3a- and 3@-hydroxy- SOL- androstan-17.one), Dr. M. Wolff (Sa-androstane-2fi, 3cu, 17fi- triol), Dr. P. N. Rao (2a-hydroxy-4-pregnene-3,20-dione). 16a-Hydroxy-4-androstene-3,17-dione was obtained from USP Steroid Reference Collection (New York). Sa-Androstane- 3ol(and 3/3) ,17P-diol and 17&hydroxy-5oc-androstari-3-one were purchased from Sigma Chemical Company (St. Louis, Missouri). 3a-Hydroxy-5ar-pregnan-20.one and 3&hydroxy-5a-pregnan-20- one were purchased from Ikapharm (Ramat-Gan, P.O.B. 31, Israel).

Sodium borohydride reduction of 30(, 18-dihSdros!--5cr~andro- stair-17.one yielded 5ol-androstane-3ol, 17/3,1%triol. 2a-Hy- droxy-4-pregnene-3,20-dione was hydrogenated with palladium on charcoal as catalyst (8). The resulting mixture of tetra- hydro compounds was analyzed by gas chromatography-mass spectrometry after formation of silyl ethers. Two peaks were

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seen in the gas chromatogram, representing the 3p,5a! isomer (tn = 1.36 on SE-30) and a mixture of the 3/3,50(; 3cr, 5a and ICY ,5fi isomers (tB = 1.00) of 2,3-dihydroxy-pregnan-20-one.

Radioactive Steroids-4-[4-14C]Androstene-3, 17-dione (specific radioactivity, 1.2 PCi per mg), 4-[4-14C]pregnene-3, 20.dione (specific radioactivity, 1.6 &i per mg), and [4-14C]cholesterol (specific radioactivity, 145 PCi per mg) were obtained from the Radiochemical Centre, Smersham, England. Prior to use, the labeled cholesterol was purified by chromatography on a column with aluminium oxide, grade III (Woelm, Eschwege, Germany). 5a-[4-%]Androstane-3c, l’ifl-diol was prepared by incubation of 4-[4-r4C]androstene-3,17-dione (specific radioactivity, 210 PC1 per mg) with minced testicular tissue from 4-week-old Sprague- Dawley rats and purified as described elsewhere.2 Prior to incubation the prepared Sa-[4-%]androstane-3oL, 17fi-diol was diluted with unlabeled 5ar-androstane-3a, 17fi-diol (the purity of which was assayed by gas-liquid chromatography) to yield a specific activity of 3.0 $.Zi per mg. 7a-[6fi-3H]Hydroxy-4-cho- lesten-3-one (specific radioactivity, 6.7 PCi per mg) was prepared as described by Bjorkhem (9). [24-i4C]Lithocholic acid (sys- tematic name Sa-hydroxy-5P-cholanoic acid) (specific radio- activity 4.0 PCi per mg) was purchased from New England Nuclear Corporation, Boston, Massachusetts.

Animals and Preparation of flomogenates-For the incubation studies three germ-free and three conventional rats of the Sprague-Dawley strain were used. For the determination of concentration of cytochrome P-450 six germ-free and six con- ventional rats of the Long-Evans strain were used. The germ- free animals were reared according to the technique of Gustafsson (10, 11) and fed a standard diet ad lib&m (11). This diet is semisynthetic with 10% arachis oil as source of fat. Control animals of the same strain were reared outside the germ-free isolators on the same sterilized diet. Male rats weighing about 300 g were used. The germ-free rats were killed by a blow on the head immediately after they had been taken out of the germ- free isolators; the conventional animals were killed in the same way. The liver was excised immediately and chilled on ice. Liver homogenates, 20% (w/v) were prepared in a modified Bucher medium (la), pII 7.4, with a Potter-Elvehjem homoge- nizer equipped with a loosely fitting Teflon pestle. The ho- mogenate was centrifuged at 20,000 X g for 15 min. The micro- somal fraction was obtained by centrifuging the 20,000 x g supernatant fluid at 105,000 x g for 70 min. The microsomal fraction was suspended in the homogenizing mediurn in a volume corresponding to that of the 20,000 x g supernatant fluid, from which it had been isolated, and was homogenized with a loosely fitting pestle. The protein concentration of the 20,000 x g supernatant fluid and of the microsomal fraction was determined according to Lowry et al. (13).

Incubations with 4-[/,-14C]Androsfene-S,l?-dione, 4-[4-W]Preg- nene-S ,20-dione, and ~01-[4-‘1C]Bndrostnne-Sa,17p-diol-4-[4- 1%]8ndrostene-3,17-dione (300 pg) , dissolved in 50 ~1 of acetone, was added to a mixture of 0.3 ml of microsomal fraction and 2.7 ml of Bucher medium fortified with 3 pmoles of NADPH. 4-[4-rhC]Pregnene-3,20-dione (250 pg), dissolved in 50 ~1 of ace- tone, was added to a mixture of 0.5 ml of microsomal fraction and 2.5 ml of Bucher medium fortified with 3 pmoles of NADPH. 5a(-[4-i%]Androstane-3cr, 17P-diol (200 pg), dissolved in 50 ~1 of acetone, was added to a mixture of 2.5 ml of microsomal fraction, 1.5 ml of Bucher medium, 0.03 pmole of MnC12, 3 pmoles of NADP, 12.5 pmoles of isocitrate, and 10 ~1 of isocitric dehydro-

0 2 A. Berg and J.-A. Gustafsson, manuscript in preparation.

genase solution. NADP, NADPH, nn-isocitric acid (trisodium salt), and isocitric dehydrogenase (Type IV) were obtained from Sigma Chemical Co., St. Louis, MO.

Incubations were carried out for 10 min at 37”. The condi- tions during which the incubations were performed had been tested and, with the use of the conditions described, the ob- tained conversions were linear with respect to time and enzyme concentration. The incubations were terminated by the addi- tion of 20 volumes of chloroform-methanol (2: 1, v/v). The precipitate was filtered off, and 0.2 volume of a solution of sodium chloride (0.9%, w/v) was added. The chloroform phase was collected, and the solvent was evaporated. The residue was dissolved in 0.5 ml of chloroform-methanol (2:1, v/v) and applied on precoated silica gel plates (250 p, E. Merck, Darm- stadt, Germany), The extracts from the incubations with 4- [4-14C]androstene-3, 17-dione were developed once in the solvent system chloroform-ethyl acetate, 4:l (v/v), and the extracts from the incubations with 4-[4-r4C]pregnene-3 ,20-dione once in the solvent system benzene-ethyl acetate, 3:l (v/v). The extracts from the incubations with 5oc-[4-i4C]androstane-3a, 17/3- diol were developed five times in the solvent system ethyl acetate- cyclohexane, 3:2 (v/v). The chromatoplates were subjected to autoradiography with an exposing time of 10 days. The radio- active zones on the thin layer chromatographic plates were determined exactly from the x-ray film and were scraped off, eluted with methanol and measured for radioactivity.

Incubations with [/t-14C]Cholesterol--[4-14C]Cholesterol, 7 pg in 50 ~1 of acetone, was incubated for 20 min with a mixture of 3 ml of 20,000 x g supernatant fluid. The incubation was terminated as described above, and the obtained extract was analyzed as described previously (14).

Incubations with 7oc-[6flP-3H]Hydroxy-4-cholesten-3-one-7ar-[6fl- 3H]Hydroxy-4-cholesten-3-one (50 pg), dissolved in 50 ~1 of acetone, was added to a mixture of 2 ml of microsomal fraction and 1 ml of Bucher medium fortified with 3 pmoles of NADPH. Incubations were carried out for 12 min at 37”. The incubations were terminated as described above, and the incubation extracts were analyzed as described in a previous paper (15).

Incubations with [d.J-~4C]LithochoZic Acid-Microsomal fraction (1 ml) was diluted with 1 ml of Bucher medium, and 2 pmoles of NADPH were added. [24-14C]Lithocholic acid, 50 pg, was added to the incubation mixture dissolved in 50 ~1 of ethanol. The incubation was carried out for 20 min and was terminated by the addition of an equal volume of 95% aqueous ethanol (v/v). After filtration the mixture was acidified with 0.2 M

HCl and extracted with ether. The ether extract was washed with water until neutral and the solvent was evaporated. The residue together with suitable reference compounds as internal standards was subjected to thin layer chromatography with phase system S 11 as solvent (16). The compounds were located by exposing the chromatoplates to iodine vapor. The iodine vapor was allowed to evaporate at room temperature, and the appropriate zones of the chromatograms were scraped off, eluted with methanol, and measured for radioactivity.

Measurement of Cytochrame P-450-Cytochrome P-450 was assayed in an Aminco-Chance dual wave length split beam scanning spectrophotometer according to the method by Omura and Sato (17). Each cuvette was bubbled with CO for 1 min, and the sample cuvette was then reduced with Nn&04 (18).

Gas Chromatography-A1fass Specfrometry Analysis oj Metabolites of 4-[.&14C]8ndrostene-3, 1 7-dione, 4-[4J4C]Pregn,ene-S , Ddione, and 5a-[4-14C]Androsfane-ba ,17fi-dio2-The methanol extracts of the radioactive zones pooled from three incubations from the

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thin layer plates were taken to dryness, and all material left after radioactivity measurements was (trimethyl)silylated and analyzed by gas chromatography-mass spectrometry (LKB 9000 instrument) with the use of a 1.5% SE-30 column. Mass spectra were recorded on magnetic tape with the incremental mode of operation and were then treated in an IBM 1800 computer (19). A compound was considered identified only if it had the same mass spectrum and gas-liquid chromatogra,phic behavior as the reference compound.

Radioactivity Measurements-Radioactivity was assayed in a Packard liquid scintillation spectrometer, model 3003.

Statistical Analysis-Student’s t test was used and the signifi- cance level was set at 0.05.

RESULTS

No steroids were identified in zone 1. The metabolites iden- tified in zones 2 to 9 are summarized in Table I. Zones 2 to 4 contained hydroxylated metabolites of 4-androstene-3,17-dione: 7oc-(zone 2), 6/%(zone 3), and lba-(zone 4) hydroxy-4-androstene- 3,17-dione. Reduced metabolites were found in zones 5 to 7: 17/3-hydroxy-4-androsten-3-one (testosterone, zone 5), 3p-hy- droxy-5or-androstan-17-one (zone 6)) and 3/3-hydroxy-4-andro- sten-17-one (zone 7). Zone 8 contained the substrate (4-andro- stene-3,17-dione) and zone 9 the reduction product 5cr-andro- stane-3,17-dione. The mass spectrometric characteristics of the silylated compounds are also indicated in Table I, and references are given to publications where these mass spectra are discussed in more detail.

Incubations of .J-[~-14C]Androstene-S,1?‘-dione with Xicrosomal Table II summarizes the quantitative data on microsomal Fraction Fortified with NADPH-After incubation of 4-[4-14C]an- metabolism of 4-androstene-3,17-dione. It can be seen that drostene-3,17-dione with the microsomal fraction of livers from microsomal preparations from germ-free rats are significantly germ-free and conventional male rats there were total conversions more active in hydroxylating 4-androstene-3,17-dione in posi-

-9

-a -7 -6

-5

-4 -3 -2 -1

of the substrate of about 10%. Labeled products were seen in the chromatographic zones 1 to 9 shown in Fig. 1.

FIG. 1 (left). Thin layer chromatogram of extract of incubation of 4-[4-Wlandrostene-3,17-dione with the microsomal fraction of liver homogenate from male germ-free rats fortified with NADPH. The solvent system used was chloroform-ethyl acetate, 4:l (v/v). The following compounds were identified in the radioactive zones : zone 2, 7a+hydroxy4-androstene-3,17-dione; zone 3, Gp-hydroxy- 4-androstene-3,17-dione; zone 4, 16a-hydroxy-4-androstene-3,17- dione; zone 5, 17p-hydroxy-4-androsten-3-one (testosterone) ; zone 6, 3p-hydroxy-5a-androstan-17-one; zone 7, 3p-hydroxy-4-andro- sten-17-one; zone 8, 4-androstene-3,17-dione; zone 9, 5ol-andro- stane3,17-dione.

FIG. 2 (center). Thin layer chromatogram of extract of incu- bation of 4-[4-Wlpregnene-3,20-dione with the microsomal frac- tion of liver homogenate from male germ-free rats fortified with NADPH. The solvent system used was benzene-ethyl acetate, 3: 1 (v/v). The following compounds were identified in the radio- active zones : zone 2, 16cx-hydroxy-4-pregnene-3,20-dione; zone 3,

-4 ” : \, _ -2

-3 -2 “1 ‘\

-1

2~,3~-dihydroxy-5~+pregnan-20-one; zone 4, 6p-hydroxy-4-preg nene-3,20-dione; zone 5, 2001- and 20@-hydroxy-4-pregnen-3-one; zone 6, 2a-hydroxy-4-pregnene-3,20-dione; zone 7, 3&hydroxy-5or- pregnan-20-one; zone 8, 4-pregnene-3,20-dione; zone 9, 5a+preg- nane-3,20-dione.

FIG. 3 (right). Thin layer chromatogram of extract of incu- bation of 5ol-[4-W]androstane-3~,17@-diol with the microsomal fraction of liver homogenate from male germ-free rats fortified with an NADPH-regenerating system. The solvent system used was ethyl acetate-cyclohexane, 3:2 (v/v) (5 developments). The following compounds were identified in the radioactive zones: zone 9, 5a-androstane-3cy,7or,17p-triol; zone 3, 5ol-androstane-2p,- 3cr,l7@triol; zone 4, &-androstane-2or,3a:,17p-triol; zone 5, 5a-an- drostane-3p, 17p, Wtriol; zone 6, 5a-androstane-3a, 17p, Wtriol; zone 7, 5a-androstane-3a, 17p-diol and 5a-androstane-3p, 17p-diol; zone 8, 3a-hydroxy-5a-androstan-17-one and 17p-hydroxy-5a-an- drostan-3-one; zone 9,5or-androstane-3,17-dione.

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TABLE I

Gas chromatographic and mass spectrometric characteristics of isolated metabolites of ~-[4-14C]anclrostene-3,17-clione

Compound is&ted

7or-Hydroxy-4.androstene-3,17-dione. ......... Bp-Hydroxy-4-androstene-3,17-dione ...........

16or-Hydroxy-4-androstene-3,17-dione. ........ 17&Hydroxy-4-androsten&one (testosterone). 3p-Hydroxy-5a-androstan-17.one. ............. 3@-Hydroxy-4-androsten-17.one. .............. 4-Androstene-3,17-dione. ..................... 5a-Androstane-3,17-dione .....................

Thin layer chromato-

graphic zone

-

O.G9 0.62

374 (M),b 359 (M - 15, base peak) 374 (M), 359 (M - 15, base peak), 318

(M - 56) 0.84 374 (M), 303 (n/r - 71, base peak) 0.63 360 (M), 129 (base peak) 0.50 362 (M), 347 (M - 15, base peak) 0.48 360 (M), 142 (base peak) 0.54 286 (n/r, base peak) 0.45 288 (M, base peak)

a tR, relative retention time (5ol-cholestane = 1.00). b $1, molecular ion.

TABLE II

Metabolism of .$-[4-‘T]androstene-3,lrdione in liver microsomes from germ-free and conventional rats

The conversions are calculated from the amounts of radio- activity in t’he different zones of the thin layer chromatograms. The values listed are the means f S.D. of experiments with three male rats.

Compound I GFQ I cb I *

nmoles metabolite jormed/mg protein/10 min

7a-Hydroxy-4.androstene- 3,17-dione. ._.............. 4.09 f 0.23 4.07 f 0.30NSc

G@-Hydroxy-4-androstene- 3,17-dione.................20.07 Y!Z 2.6210.88 & 1.14<0.01

16a-Hydroxy-4-androstene- 3,17-dione.................40.47 f 4.5026.88 f 2.80!<0.02

Testosterone. _. 11.00 f 0.92 9.41 i: 3p-Hydroxy-5a-androstan-17-

0.601<0.10

one....................... 9.07 f 1.0511.22 =t 1.62 <0.20 3p-Hydroxy-4-androsten-17-

one....................... 4.93 f 0.56 4.05 f 0.35 <O.lO 5a-Androstane-3,17-dione..... 8.93 f 2.3212.03 f 0.66 <O.lO

a GF, germ-free rats. b C, conventional rats. e NS, no significance.

Characteristic ma;;jytrometric peaks

tions 60 and 16a thau are corresponding preparations from con-

ventional rats. Furthermore, conventional rats seem to be

more active in 5a-reduction of 4-androstene-3,17-dione (about 1.3 times, as judged by the increased formation of BP-hydrosy-

5a-androstan-17-one and 5or-androstane-3,17-dione), even if no statistically significant difference is observed.

Incubations of 4-[4-W]Pregnene-S , SO-done with Microsomal

Fraction Fortified with NADPH-Incubation of 4-[4-W]preg- nene-3,20-dione with the microsomal fraction of livers from germ-free and conventional rats resulted in a total conversion of

the substrate of about 10 to 15%. Labeled products were seen in the chromatographic zones 1 to 9 shown in Fig. 2.

Zone 1 did not give any peaks when analyzed by gas-liquid chromatography after silyla,tion. I-Iydroxylated metabolites were present in zones 2 to 4 and 6 (see Table III) : 16cr-hydroxy- 4-pregnene-3,20-dione (zone 2)) 2cr, 3cu-dihydroxy-5cr-pregnan- 20.one (tentative identification, cf. Ref. 24) (zone 3)) GP-hy- droxy+pregnene-3,20-dione (zone 4)) and 2a-hydroxy-4-preg- nene-3,20-dione (zone 6). The other zones (except zone 8 that contained unmetabolized 4-pregnene-3,20-dione) contained re- duced metabolites: 2Ocr- and 20&hydroxy-4-pregnen-3-one (zone 5)) 3fl-hydroxy-5cu-pregnaIl-20-one (zone 7)) and 5ol-pregnane- 3,20-dione (zone 9).

As can be seen from Table IV, which summarizes the quanti-

tative results on 4-pregnene-3,20-dione metabolism, germ-free

Compound isolated Thin layer chromato-

graphic zone

16cu-Hydroxy-4-pregnene-3,20-dione.. 2 2a,3~Dihydroxy-5a-pregnan-20-one. 3

Gp-Hydroxy-4-pregnene-3,20-dione. 4 20~ and 20p-hydroxy-4-pregnen-3-one. 5 2a-Hydroxy-4.pregnene-3,20-dione. 6 3p-Hydroxy-5a-pregnan-20-one. . 7 4-Pregnene-3,20-dione.. . 8 5a-Pregnane-3,20-dione . . 9

Reference

(20) (20)

(20)

(21) (22)

TABLE III

Gas chromatographic and mass spectrometric characteristics of isolated metabolites of 4-[4-14C]pregnene-3,20-dione

1.25 402 (iM),h 186,‘172 (base peak), 159, 157, 109, 96 23 1.02 478(M), 463 (M - 15), 299(M - [90 + 891, base peak), 24

143, 142, 129 1.01 402(M), 387(M - 15), 346(M - 56, base peak) 1.18, l.llc 388(M), lli(base peak) 1.34 402(M), 387(M - 15, base peak) 0.80 390(M), 375(M - 15, base peak) 0.83 314 (M, base peak) 0.75 316 (M, base peak)

25

26 7

Characteristic mass spectrometric peaks (m/e) Reference

* tz, relative retention time (5ol-cholestane = 1.00). b M, molecular ion. c Respect.ively.

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rats are significantly more efficient in liver microsomal 6P-hy- drosylation and 2a-hydrosylation (p < 0.025, as judged by the sum of formed amounts of 2or, 3oc-tlil~ydros~-5a-l)regllall-20-one and 2~-lz)-clrox~-4-pregllene-3,20-dione). Also 16c&ydroxyla- tion seems to occur more readily in microsomal preparations from germ-free rats even if the differences between germ-free and coc ventional rats was not statistically significant kth respect to this reaction. The formation of 5a-pregnane-3,20-dione was significantly higher in control rats compared to germ-free rats (p < 0.05). I-Iolvever, when the amounts of all 5ar-reduced products formed (20(,3cr-dihydroxy-5cu-pregnan-20-one, 3fi-hy- drosy-5a-~~rcgnau-2O-oi~e, and 5a-pregnane-3,20-dione) were added, the 5a-reductase activity was found to be 1.4 times higher

TABLE IV

ilfelabolism of .I-[4-‘4C]pregnene-5,20-dione tin liver microsomes from germfree and conventional rats

The conversions are calculated from the amounts of radio- activity in the different zones of the thin layer chromatograms. The values listed are the means =t S.D. of experiments with two germ-free male and three conventional male rats.

Compound

16or-Hydroxy+-pregnene- 3,20-dione,

2a,3a-Dihydroxy-5a-preg- nan-20-one,

GP-Hydroxy&pregnene- 3,20-dione.

2001- and 20fl-hydroxy-4.preg- nen-3-one. .

2cr-HydroxyA-pregnene- 3,20-dione .

3&Hydroxy-5a-pregnan-20- one

5a-Pregnane-3,20-dione..

LL GF, germ-free rats. * C, conventional rats c XS, no significance.

GFa I

Cb P

?zmoles metabolite form?d/mg protein/10 ntilt

11.43 f 1.13 8.78 + 1.30 <O.lO

5.17 f 0.47 5.25 f 0.18 NSc

10.68 f 0.53 6.70 f 0.07 <0.005

4.65 rt 0.57 4.94 + 0.64 NS

9.25 f 0.86 6.12 zk 0.57 <0.025

4.04 f 0.60 4.27 f 0.21 NS 7.44 f 1.3313.GO f 1.73 <0.05

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in control rats compared to germ-free rats, but this difference was not significant statistically.

Incubations oj 5or-[4-‘4C]Androstane-3a ,17’p-dial with Micro-

somol Fraction Forti$ed wiih NADPH-After incubation of 501- [4-14C]androstane-3a, 17p-diol with the microsomal fraction of livers from germ-free and conventional male rats the substrate was converted to about 50 to 607;. Labeled products n-ere seen in t.hc chromatographic zones 1 to 9 shown in Fig. 3.

Xo metabolitcs were found in zone 1. Hydroxylated metabo- lites were found in zones 2 to 6 : 5cu-andr-ostane-3a, 7a!, 17@-trio1 (zone 2)) Sol-androstane-2P, 30(, 17@-trio1 (zone 3), 5ol-androstane- ICY, 30(, 170.trio1 (zone 4)) Scu-androstane-3/3,17p, 1%trio1 (zone 51, and 5cr-androstane-3cr, 17p, l&trio1 (zone 6). Zone 7 con- tained 5a-androstane-Ya, 17fl-diol (substrate) but also small amounts of 5a-androstane-3P,l7&diol. Zone 8 contained a mixture of about 50% of 3a-hydroxy-5cr-androstan-17.one and 50y0 of 17fi-hydroxy-5a-androstan&one. Zone 9 only con- tained 5cu-androstanc-3,17-dionr. The gas-liquid chromato- graphic and mass spectrometric data obtained during the iden- tification of these compounds are summarized in Table V.

As is obvious from Table VI microsomal preparations from germ-free rats were more efficient in carrying out 2fi-, 2~, and 1%hydroxylation than corresponding preparations from conven- tional rats. In the case of 2p- and 2u-hydroxylation only trends were observed (p < O.lO), but for IS-hydroxylation (measured as the sum of formed 5a-androstane-3cr,17fl, E-trio1 and 5~ androstane-3@, 17fl,l%triol) the difference found was statistically significant (p < 0.05). Germ-free rats were also more efficient than conventional rats in microsomal oxidation of 5c+androstane- 3a(, 17fi-diol to 5oc-androstane-3,17-dione (p < 0.05).

Incubations cf [p”C]Cholesterol with 20,000 x g Supernatant Fluid--The pattern of polar products formed from cholesterol in the presence of t,his enzyme fraction was the same for germ-free and for conventional rats of the Sprague-Dawley strain. The main 7a-hydroxylatcd metabolites formed have previously been identified as 5-cholestene-Sfi , 7ar-diol, 7a-hydroxy-4-cholesten- 3-oue, and 7cr, 12a-dihydroxy-4-cholestell-3-6ne (31). The amounts of these three products were calculated from the amounts of radioactivity in the zones of the thin layer chromato- grams corresponding to these products (Fig. 4). As seen in

TABLE V

Gas chromatographic and mass spectrometric characteristics of isolated metabolites of 5ol-[4-‘4C]androstane-3ff, 17&diol

Compound isolated

5a-Androstane-3a,7a, 170.triol. 5~-Androstane-2fl,3~,17@-triol_

5a-Androstane-2a,3a, l’ip-triol.. .

Bcu-Androstane-3p,17b,18-triol. . . 5cu-Androstane-3cu,17@,18-triol_ Sa-Androstane-3a, 170.diol + 5cu-Andro-

stane-3p,17p-diol. 3or-Hydroxy5ol.androstan-17.one + 17p-

Hydroxy-5ti-androstan-3-one. .

5or-Androstane-3,17-dione. .

I

Thin layer chromato-

:raphic zone fP

(SE-30)

0.61 0.76

0.79

0.91 0.72

0.50, 0.62c

0.41, 0.53

0.45

Characteristic mass spectrometric peaks (m/e)

524(M),b 393(M - 131, base peak) 524(M), 509(M - 15), 255(M - [2 X 90 + 891, base

peak), 143, 142, 129 524(M), 509(M - 15), 255(M - [2 X 90 + 891, base

peak), 143, 142, 129 524 (M), 217 (base peak), 191, 129 524 (M), 217 (base peak), 191, 129

436 (M), 129 (base peak) (for both compounds)

362 (M), 272 (M - 90, base peak) and 362 (M), 129 (base peak), respectively

288 (M, base peak)

-- --

Reference

(28) (29)

(10)

(30) (30)

(21)

(21)

Q f~, relative retention time (5or-cholestane = 1.00). * M, molecular ion. c Respectively.

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TABLE VI

Metabolism of 6cr-[~-14C]androstane-Scu,iYp-diol in liver microsomes

from germ-free and conventional rats The conversions are calculated from the amounts of radio-

activity in the different zones of the t,hin layer chromatograms. The values listed are the means & S.D. of experiments with three male rats.

5cu-Androstane-3ol,701,17fl- trial.......................

5ol-Androstane-2p,3Lu,17p- trial.......................

5ol-Androstane-2ol,3or,17P- triol. . . . . . . . . .

5a-Androstane-3p, 17p, B-trio1 + 5a-Androstane-3a, 17p, 18. trio1 . . . . . . . . . . . .

3a-Hydroxy-5a-androstan-17- one + 17fl-Hydroxy-5or-an- drostan-3-one. . . .

5ol-Androstane-3,17-dione..

I

-I

1

GF” I

c* B

nnroles ntetabolite formed/mg protein/10 min

2.84 f 0.09 2.59 f 0.56NSc

3.33 f 0.32 2.13 f 0.76 <O.lO

19.00 f 1.3714.4G f 3.39<0.10

4.56 f O.G4 2.92 f 0.57 <0.05

10.84 f 1.97 8.6G f 1.04 NS 1.23 + 0.12 0.84 f 0.17 <0.05

a GF, germ-free rats. b C, conventional rats. c NS, no significance.

TABLE VII

Metabolism of [4-W]chdesterol, Yoc-[6~-3H]hydroxy-.-cholesten-3- one and [24-14C]lithocholic acid

The values listed are the means f S.D. of experiments with

T three male rats of the Sprague-Dawley strain.

Substrate Reaction GFQ I

C”

nmoles metebolite .foiormea/ nzg j?rotein

Cholesterol 7a-Hydroxyla- 0.026+0.0070.036f0.0~ tion

7a-Hydroxy-4- 12a-Hydroxyla- 2.26~kO.CG 3.44f0.34 cholesten- lation a-one

5cr-Reduction 0.75f0.15 0.87f0.13 Lithocholic Gp-Hydroxyla- 12.02~t2.06 G.18f1.22

acid lation

a GF, germ-free rats. * C, conventional rats. c NS, no significance.

-.

1

P

NSc

<O.Ol

NS <0.02

Table VII the extent of 7ol-hydroxylation of cholesterol was about 30% less in incubations with livers from germ-free rats compared to incubations with livers from conventional rats of the Sprague-Dawley strain. In a second experiment where male rats of the Long-Evans strain were used the percentage of ‘icu-hydroxylation of [4-Wlcholesterol was 0.63 f 0.11 (n = 5) for germ-free animals and 0.80 f 0.20 for conventional animals (n = 6) (p < 0.20). Thus, also rats of the Long-Evans strain showed the same principal difference in microsomal cholesterol ‘icu-hydroxylase activity as rats of the Sprague-Dawley strain.

Incubations of 7ol-[6P-3H]Hydroxy-Q-Cholesten-S-one with Micro- somal Fraction Forti$ed with NADPH-l’he main metabolites formed from 7oc-hydroxy-4-cholesten-3-one in the presence of this enzyme fraction have previously been identified as 7a, 12or-dihy- droxy-4-cholesten-3-one, 7cr-hydroxy-5oc-cholestan-3-one, and

2

1

1.299

START

c

3.330

- - - - - - START - - ~ - - - START l-----i

- - - - - - 954 2112

2

1

2.341 :::1 1.556

54.923

FIG. 4 (left). Thin layer chromatogram of extract of incubation of [4-W!]cholesterol with the 20,000 X g supernatant fluid fraction of liver homogenate from male germ-free rats. The solvent system used was benzene-ethyl acetate, 2:3 (v/v). The numbers on the chromatogram represent counts per min. Reference com- pounds were: (1) 7cu, 12or-dihydroxy-4-cholesten-3-one; (2) 5- cholestene-3p,7a-diol; (3) 5-cholestene-3fi,7p-diol; (4) 7a-hy- droxy-4-cholesten-3-one; (5) cholesterol.

FIG. 5 (center). Thin layer chromatogram of extract of incu- bation of 7or-[6P-3H]hydroxy-4-cholesten-3-one with the micro- somal fraction of liver homogenate from male germ-free rats fortified with NADPH. The solvent system used was benzene- ethyl acetate, 1:l (v/v). The numbers on the chromatogram represent counts per min. Reference compounds were: (1) 7a,120~-dihydroxy-4-cholesten-3-one; (2) 5a-cholestane-3@,7a-diol; (3) 7a-hydroxy-4-cholesten-3-one; (4) 7a-hydroxy-5a-cholestan-3- one.

FIG. 6 (right). Thin layer chromatogram of extract of incu- bation of [24-‘%llithocholic acid with the microsomal fraction of liver homdgenate from male germ-free rats fortified with NADPH. The solvent system used was trimethylpentane-ethyl acetate- acetic acid, 5:5:1 (by volume). The numbers on the chromato- gram represent counts per min. Reference compounds were: (1) 3or,6p-dihydroxy-5p-cholnnoic acid; (2) chenodeoxycholic acid; (3) lithocholic acid.

Scr-cholestane-3@,7c+diol (15, 31) (Fig. 5). The extent of 12a-hydroxylation was found to bc larger in conventional than in germ-free rats (p < 0.01) (Table VII). The 5a-reduction

(as calculated from the sum of 7cr-hydroxy-5cr-cholestan-3-one and Sa-cholestane-3P ,7cu-diol) secmcd to be more efficient in conventional than in germ-free animals although the difference was small and not statistically significant.

Incubation of [~4-14C]Lit/zocholic Acid with Microsomal Fraction

Forti$ed with NADPH-The main metabolite formed from lithocholic acid in this cellular fraction is 30(, 6/%dihydroxy-SD- cholanoic acid as has previously been shown (32) (Fig. 6). The extent of 6&hydroxylation II-as found to be about 2 times larger in germ-free rats than in conventional rats (p < 0.02) (Table VII).

Measurement of Cytochrome P-450 Concentration--The amount of cytochrome P-450 in germ-free rats is 2.53 f 0.45 nmolcs per mg of protein as compared to 1.72 f 0.04 nmoles per mg of protein in conventional rats. This difference is statistically significant (p < 0.005).

DISCUSSIOX

All animals used in the present study were of the same strain and sex and had received the same diet during their lives. There- fore, the differences found between germ-free and conventional

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rats are most probably due to the presence of an indigenous microflora in the latter group of animals.

The microsomal hydroxylating activity (with 4-androstene- 3,17-dione, 4-pregnene-3,20-dione, 5oc-androstane-3oc, 17fi-diol, and lit.hocholic acid as substrates) is found to be higher in germ- free than in conventional rats. Statistically significant differ- ences are found for 66. and 16oc-hydroxylation of 4-androstene- 3,17-dione (about 2 and 1.5 times higher enzyme activities, respectively, in germ-free than in couventional rats), for 6@- and 2cy-hydroxylation of 4-pregnene-3,20 dione (about 1.6 and 1.2 times higher, respectively, in germ-free rats), for E-hydroxyla- tion of 5ol-androstane-3oc,l7P-diol (about 1.6 times higher in germ-free rats), and for 6&hydroxylation of lithocholic acid (about 2 times higher in germ-free rats). Similar differences, although not statistically significant, are also found for 16a- hydroxylation of 4-pregnene-3,20-dione and for Zp- and 2cr- hydroxylation of 5a-androstane-3oc, lib-diol. Furthermore, the concentration of cytochrome P-450 is about 1.5 times higher in microsomes from germ-free rats than in microsomes from con- ventional rats (p < 0.005).

As mentioned above, plasma levels of steroid hormones have been reported to be higher in germ-free than in conventional rats (4). Several data also indicate that in the germ-free rat there is a greater reabsorption of bile acids from the gastroin- testinal tract and a greater recirculat’ion than in the conventional rat (33). Certain naturally occurring and synthetic steroid compounds are known to cause proliferation of the endoplasmic reticulum in hepatocytes and to stimulate microsomal drug hydroxylation (34-36). A possible explanation for the higher microsomal hydroxylase activity with respect to steroid hormones found in germ-free compared with conventional rats in the present study could therefore be induction by circulating steroids. On the other hand certain drugs are known to depress the activity of the liver microsomal hydroxylase system (37), and it is pos- sible that a similar depressive effect could be exerted in conven- tional rats by intestinal bacterial products of a steroid or non- steroid nature.

The results presented in this investigation are at variance with the results reported by Short and Davis (38). These authors studied the hepatic microsomnl metabolism of hexobarbital, zoxazolamine, neoprontosil, and p-nitroanisole in germ-free and conventional rats but observed no significant differences between the animals. It may be that the differences observed in our study are not evident with all substrates. Another factor of importance is the diet which is known to influence hepatic micro- somal enzyme activities. Short and Davis compared germ-free animals to conventional rats given nonautoclaved diet, whereas in the present investigation both the conventional and the germ-free animals were fed the same sterilized diet. It is pos- sible that essential nutritional factors absent from the diet of germ-free rats but present in the diet of conventional rats in the experiment of Short and Davis could have abolished any differ- ences in microsomal metabolism evident when germ-free and conventional rats are fed the same diet.

Another interesting metabolic difference between germ-free and conventional rats is seen in microsomal reduction. 5or-Re- duction of 4-pregnene-3,20-dione and 4-androstene-3,17-dione is higher in conventional compared to germ-free rats. The regu- lation of the activity of microsomal 5ol-reductase activity is incompletely known, and it is therefore difficult to evaluate the biological significance of this finding. Interestingly, a high microsomal hydroxylase activity seems to be combined with a low 5a-reductase activity; male rats have generally a high micro-

somal hydroxylase activity but a low 5ol-reductase activity and the reverse is true for female rats (39). Administration of 16cr- cyanopregnenolone to female rats results in increased liver micro- somal hydroxylation but decreased 5oc-reduction (7). The re- sults obtained with germ-free and conventional rats thus agree well with this general tendency. Germ-free rats have a more efficient microsomal hydroxylation of steroid hormones but a less efficient 5a-reduction when compared to conventional rats.

In contrast to the results obtained with steroid hormones as substrates 7a-hydroxy-4-cholesten-3-one was more efficiently 12cy-hydroxylated in conventional than in germ-free rats, and the same was true for 7oc-hydroxylation of cholesterol, although in this case the difference was not statistically significant. The hydroxylating systems responsible for these two reactions have been found to differ from the common drug- and steroid hormone- metabolizing enzyme systems in response to phenobarbital ad- ministration and carbon monoxide (40) and to 16ac-cyanopreg- nenolone (7), and the findings in the present study further emphasize that the hydroxylating systems participating in 12c+ hydroxylation of 7cr-hydroxy-4-cholesten&one and 7cr-hydroxyl- ation of cholesterol are different from the common microsomal drug-metabolizing enzyme system.

Kellogg and Wostmann have demonstrated that germ-free rats fed a cholesterol-containing semipurified diet accumulate liver cholesterol concentrations three times higher than do similar conventional rats (33, 41). The intestinal microflora thus seem to protect its host from accumulating high tissue cholesterol levels when the host is consuming a cholesterol-con- taining diet (33). The 7cu-hydroxylation of cholesterol has been suggested to be the rate-limiting step in the conversion of cho- lesterol to bile acids (42). Our findings indicate that the 701- hydroxylase activity is higher in conventional than in germ-free rats, and this may thus help to explain the smaller susceptibility of the conventional rats to dietary cholesterol. Furthermore, the fecal excretion of bile acids in conventional rats is two times larger than in germ-free rats (43). This effect of the microflora may be partially mediated via an increased cholesterol 7cr-hy- droxylase activity in the liver.

As mentioned above, several data indicate that in the germ-free rat there is a greater reabsorption of bile acids from the gastro- intestinal tract and a greater recirculation than in the conven- tional rat (33). This would lead to a more extensive feedback inhibition of liver bile acid biosynthesis in the germ-free rat and therefore to a diminished 7oc-hydroxylation of cholesterol. The findings in the present investigation indicating a lower cholesterol 7cu-hydroxylase activity in germ-free than in conventional rats are in agreement with this hypothesis.

Shefer et al. found that when cholic acid was infused into intact rats the de no~lo bile acid synthesis decreased markedly (44). The decrease in bile acid biosynthesis was much more pronounced for cholic .acid than for P-muricholic acid, and after long term cholic acid infusion, P-muricholic acid became the predominant bile acid biosynthesized in the conventional rat. Similar mech- anisms may be operating in the germ-free rat since approximately 61oj, of the total fecal bile acids are in the form of P-muricholic acid in these animals (33). A diminished excretion of cholic acid could be partially mediated via a decreased 12a-hydroxylase activity, and the present findings of a less efficient microsomal 12cr-hydroxylation in germ-free compared to conventional rats are in agreement with this view. ,4 more extensive formation of P-muricholic acid in germ-free than in conventional rats is also in agreement with our finding of a higher Go-hydroxylase

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Kurt Einarsson, Jan-Åke Gustafsson and Bengt E. GustafssonMetabolism of Steroids

Differences between Germ-free and Conventional Rats in Liver Microsomal

1973, 248:3623-3630.J. Biol. Chem. 

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