- Email: [email protected]
Oxidation of various simple steroids by the cholesterol oxidase system
ARCHIVES
OF
BIOCHEMISTRY
Oxidation
AND
BIOPHYSICS
of Various
97,
Simple
Steroids
Oxidase EDMUND From
the Biochemistry
(1962)
~85-490
by the Cholesterol
System’
STEVENSON2
AND
EZRA
STAPLE3
Department, University of Pennsylvania Philadelphia, Pennsylvania
School
of Medicine,
Received November 8, 1961 Eight simple C24steroids with Ci4 in the 26 position were synthesized. These compounds, which differ from one another in the A-ring substituents, were used to study the substrate specificity of the rat liver cholesterol oxidase system. The rate of C’“0, formation was taken as a measure of oxidation of the substrates. In both the cholestane and coprostane series, the oxidation rates fall in the ascending order: 3,% sterol, 3a-sterol, 3-ketone. Members of coprostane series are oxidized faster than the corresponding members of the cholestane series. There is an initial lag period in the oxidation of most of the substrates tested, during which little C’“Ot is formed. Only coprostan-3-one-26-C’ and A’-cholesten-3-one-26-C? are oxidized without this initial lag period. Cholesterol-26-V can be enzymically converted to a CL 3-ketone which is neither A*- nor A5-cholestenone, but which appears to differ from them in the number and position of double bonds. A 3-ketone without a 5a-hydrogen is hypothesized as an intermediate in the degradation of the cholesterol side chain by the cholesterol oxidase system. INTRODUCTION
Studies in this and other laborator&
have
revealed that fortified mouse (1, 2) and rat (3) liver mitochondria preparations can convert the terminal methyl groups of the
cholesterol side chain to CO,. Activity of the preparations is considerably enhanced by a heat-stable cofactor found in the cytoplasmic
fraction
system carrying
of the cells. The
enzyme
out these reactions is called
1 Supported by a grant (H-1532-C) of the National Heart Institute of the National Institutes of Health. *From a Dissertation presented to the Faculty of the Graduate School of Arts and Sciences of the University of Pennsylvania in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Present address: Department of Zoology, Yale University, New Haven, Conn. 3Work done during the tenure of an Established Investigatorship of the American Heart Association, Inc. New York 10, New York.
the cholesterol oxidase system and has been suggested to be involved in bile acid formation (3). Several sterols other than cholesterol are attacked by the enzymes and have been suggested as possible intermediates (2, 4, 5), but nothing is known of the specificity of the system with regard to the A-ring of the substrate. We have prepared eight simple CZ7 steroids, all with Cl4 in the 26 position and differing from one another in the substituents on the A-ring. They are: cholesterol cholestan-3p-01 cholestan-3a-ol cholestan-3-one
A4-cholesten-3-one coprostan-3p-01 coprostan-h-01 coprostan-3-one
Each of these radioactive steroids was incubated with the rat liver cholesterol oxidase system, and the rate of C1402 formation was measured. In addition, the effect of a “pool” of unlabeled cholesterol on the oxidation 485
of the other
labeled
steroids
and the
486
STEVEXSON
effects of “pools” of the other unlabeled steroids on t,he oxidation of labeled cholesterol llave been studied. MATERIALS
AND
METHODS
In this work, alumina adsorption columns were used extensively to purify and identify the products of the synthetic and enzymic reactions. For the sake of simplicity of discussion, the various alumina colLmms can be divided into three types, according to the solvent used: type A, hexanebenzene, 4:l; iype B, hexane-benzene, 3:2; type C, benzene. In all cases, neut,ral alumina (Woelm), activity grade 3 was used. was prepared from 3p-hyCholesterol-26-C” droxy-5-norcholesten-25one (kindly supplied by Dr. A. I. Ryer, Schcring Corp., Bloomfield, N. J.) by the procedure rrported by Ryer et al. (6) and pnrified via the digitonide and by chromatography (type C column). It Ivas chromatographicallg pure in the system of Seller and Wettstein (7) and had a specific activit,y of 0.57 pc./mg. This sterol was us(d in t,he preparation of the other C’” steroids. Unlabeled cholesterol was recrystallized from 95% (xthanol and had an m.p. of 148-149”. Cholestan-3/j-01-26-C” was prepared from cholesteryl acetate-26-C” by the method of Ralls (8) and purified by the procedure of ilnderson and Xabenhaucr (9) and by rhromatography (type C c~olumn). It was chromatographirally pure in the system of Neher and Wettstcin (7). Unlabeled cholestan-3@-ol was prepared and purified in t,he same way. Cholestan-3-one-26-C” was prepared from cholestan-3@-ol-26-C” according to the method of Bruce (10) and purified on alumina (t-ppe A column). Unlabeled ketone was prepared and purified in the same way; its m.p. was 127-130”. Cholestan-3a-ol-26-C’4 was prepared by hydrogenation of cholestan-3-one-26-C14 in etherethanol-12 N HCl, 10:37: 1 over platinum oxide at room temperat,LLre and under slight pressure. After cholestan-3P-01-26-C” had been removed as the digitonide, cholestan-3a-01-26-C was pLlrified by chromatography (type A column). Unlabeled cholestan-3a-ol was prepared and purified in the same manner as the labeled stcrol. I’-Cholesten-3-one-26-C”, obtained by the Oppenauer oxidation of cholesterol-C’* (11) and purified by chromatography (type B rolumn), was chromatographically pure in the system of Neher and Wettstein (7). Unlabeled A’-c lolcsten-3-one was prepared and purified in the aam,: manner. Coprostan-3-one-26-C” was prepared from A*cholesten-3-one-26-C” as described by Grasshof (12) and purified on an slLlmina column (tye A). This material was not contaminated with unchanged a,fi-unsaturated ketone since its ultra-
AND
STAPLE
I-iolet absorption spectrum had no yak in the 240-mp region. Unlabeled coprostan-3-one prepared and purified in the same manner had :m m.p. of 60-62”. Coprostan-3p-ol-26-C>’ was prepared from coprostanone-26-C’& by the procedure of Gras&of (13) and purified both as the digitonide and on a type -4 alumina column. Unlabeled roprostan-3fl-ol prepared and purified in the same w-a?- had an m.p. of 97-98”. Coprostxn-3a-01-26-C” was prepared from coproStan-3-one-26-C? by catalytic hydrogenation as described by Ruzicka et c/l. (14). The small amount of 38 isomer that was formed at the same time was rcmo\.cxd a;; the digitonide, and the 3a isomer was purified b,v chromatography (type B column). Unlabeled coprostnn-3a-ol was prepared in the same way; it,s m.p. was 110.5-111.5” after recrystallization from 95% ethanol. A’-Cholesten-3-one was prepared by the oxidation of cholesterol dibromide as tlescribeci by Fieser (15). Its m.p. was 125-129” (rapid hea,ing), and its ultraviolet absorption spectrum showed no I)euk in the 240-mp region. The procedures for preparing the mitochondria and SF’ and for carrying out the incllbations have been described (2). In the studies on the rate of oxidation of any given steroid, the rate of CYO, formation from th:Lt steroid was compared with the rate of C”O, formation from c*holrsterol-26Cl*. In each de:e!mination, 20 flasks were used; as substrate (s),. five cont,aincd cholesterol-26-C’*, fire contained cholestrrol-26-C” plus a “pool” of the other, Lrnlnbcied steroid (0.1 mg./flnsk), fix-p contained the o!l.cr labPled with C” in the 26 position, steroid and five (‘1 :’ ::;n:.l the other, 26-labeled (0.1 mg.,/ plus a ((l)O.>. c,c Lmlabeled cholesterol &irate (s) were added as aqueous flask). These’ emulsions stat,. iz-d with a total of 3 mg. of Twcen 20 (-itlrs “(~a der Co.) as described earlirl
(2). ted in aqueous SaOH and conC”0, was cc verted to BaC’ : l3 Radioactivity was measured in a windowless, gas-flow counter and correct., :i for self-absorption. In order to compare ihc rates of oxidation of cholesterol-26-C’” and the other labeled steroids, equal concentrations of the snbstrates should be used. In practice, it is not always easy to achieve this. We corrected for the inequalities in the following way : We first established that within limits the amount of C”0, formed from cholesterol-26“SF, supcrnatant factor(a) in t,he sohble fraction of boiled extracts of liver; ATP, adenosine triphosphatt ; GSH, reduced glutathionine; AMP, adenosine monophosphate ; DPN, diphosphopyridine nuclrotide ; Tris, tris( hy-drometlLyl)uminomrth;mc.
OXIDATION C” is proportional to the substrate concentration. Figure 1 demonstrates that as long as less than cu. 55 pg. (30,000 counts/min.) of cholesterol-26-C’ per flask is used, a, linear relationship is maintained between C” substrate and C’“O, evolved. We then adjusted the counts/min. in the C”O, from cholesterol-26-C” to what they would have been had the concentration of the cholesterol-26-Cl4 been equal to the concentration of the other steroid. This was done by multiplying the counts/min. in the C401 from cholesterol-26-r? by the factor concentration of the other labeled steroid used concentration of cholesterol-26-Cl4 used ’ All of the experiments were repeated with different mitochondria preparations. The preparations varied considerably in their activities, so the final results are expressed as the per cent oxidation of the radioactive steroids relative to the maximum oxidation of radioactive cholesterol in the particular ekperiment, the latter having an assigned value of 100 (relative per cent oxidation). The relative per cent oxidation of any radioactive substrate was usually reproducible to cu. *IO%. In the search for ketosteroids formed enzymitally from cholesterol-26-C?, incubations carried out in the usual manner were stopped by the addition of 4 vol. of 95% ethanol. After the precipitated proteins had been removed, the supernatant fluids were evaporated to dryness and the ethersoluble portion of the residues chromatographed (type A and B columns). The eluted fractions (5 ml. each) were plated and the radfoactivities determined. RESULTS
487
OF STEROIDS
,’ 3 I’
When cholesterol-26-C?* i., AI‘d’ized by fortified rat liver mitochocd. a, there is a lapse of cu. i/s to Ii/i hr. b::are appreciable C1*Oa is formed. After this itial lag period the C?*O, formation r ,’ is linear for at Iiast 8 hr. TIP production of Cl402 from cholestan3p-ol-26-Cl4 proceeds both with a longer lag period and at a slower rate than from cholesterol-26-CY4. In addition, a “pool” of unlabeled cholesterol depresses cholestan3p-01-26-C?* oxidation considerably, while a “pool” of unlabeled cholestan-3p-ol does not depress cholesterol-26-C?* oxidation at all. These points are illustrated in Fig. 2; the data that we obtained when the other steroids were used as substrates are very briefly summarized in Table I. Cholestan3~01-26-Cl4 and cholesterol-26-Cl4 are oxidized similarly; a L’pool” of one unlabeled
750r-----l m 600 0 0 -0 g 450 .c f
. ./' .
300
a:
OV 0
I
60,000
45,000 30,000 15,000 C.P.M. Cholesterol - 26 - Cl4
used
pg:.5
used
I 0
100 ChOI.s+e::
- 26 -:I4
FIG. 1. Effect of cholesterol-26-r? concentration on C’“OZ formation by the cholesterol oxidase system. Incubation mixture consisted of 1 ml. of mitochondria suspension (‘VI of a rat liver), 5 ml. SF, 5 ml. of 0.25 M Tris-HCl buffer, pH 8.5, 25 mg. ATP, 15 mg. GSH, 8 mg. AMP, 5 mg. DPN, 10 mg. Mg(N0J2.6H20, and 22 mg. trisodium citrate dihydrate. Total volume, 12 ml. Cholesterol-26C” was added as Tween 20 suspension in buffer. Incubation time, 7% hr.
sterol has little effect on the oxidation of the other labeled sterol. Cholestan-3-one26-Cl4 is oxidized more rapidly than cholesterol-26-U*. Nowever, unlabeled cholesterol significantly depresses oxidation of the radioactive ketone, but unlabeled cholestan-3-one does not depress oxidation of the labeled sterol. Initial lag periods are found in the oxidations of both substrates. Both coprostan-3p-01-26-U* and cholesterol-26-U* are oxidized with lag periods that are alike, although the rate of oxidation of the former is less than that of the latter. Coprostan-3p-ol-26-Cl4 oxidation is depressed to about the same extent by a iipool” of unlabeled cholesterol as cholesterol-26-Cl4 oxidation is depressed by a “pool” of unlabeled coprostan-3p-01. The rate of oxidation of coprostan-3a-01-26-C?* is greater than t,hat of cholesterol-26-C’“. The presence of unlabeled coprostan-3a-ol depresses radioactive cholesterol oxidation more than Ihe presence of unlabeled cholesterol depresses radioactive coprostan-3a01 oxidation. Both labeled substrates are oxidized with about equal lag periods. M. W. Whitehouse (personal communication) has obtained similar results. Coprostan-3one-26-G4 oxidation is rapid and unaffected
488
STEVENSON
AND
STAPLE
Cholesterol- 26 -Cl4
f -f
I
0
2 Incubation
3
4
time,
of cholestan-3p-ol-C” FIG. 2. Oxidation steroids (0.1 mg./flask) and radioactive cubation mixture as in Fig. 1. TABLE
THE OXIDATION OF VARIOUS BY THE CHOLESTEROL OXIDASE SYSTEM
1?er cent depressionof oxidation of ‘ther, !abeled Labeled chostemd by lesterol by “pool” of “pool” of unlabeled other, unlacholesterol beledsteroid
. -
-
100
Cholestan3&ol Cholestan-3ol-01 Cholestan-3-one
40 95 170
35 10 20
0 10
Coprostan-SD-01 Coprostan-3a-01 Coprostan-3-one
75 175 235
40 10 0
40 25 20
A4-Cholesten-3one
330
20
50
-OL-26-C14
Cholesion-3b Cholesterol
-OL-26-Cl4
6
by the cholesterol oxidase system, Unlabeled sterols were added as Tween 20 suspensions. In-
I
Cholesterol
Choleston-3p
-
hours
COMPARISON OF STEROIDS
Final Radioactive substrate 1R.P.O.”
5
_-
0
-
a Final relative per cent oxidation (i.e., the oxidation of various radioactive steroids in 8 hr., compared to oxidation of radioactive cholesterol in the same t’ime by the same mitochondria preparation). The incubation mixture used is described in Fig. 1.
by a ‘ipool” of unlabeled cholesterol, although the oxidation of labeled cholesterol is depressed by a “pool” of unlabeled coprostanone. In addition, radioactive copro-
stanone is oxidized without an initial lag period. A4-Cholesten-3-one-26-Cl4 is also rapidly oxidized without an initial lag period. A “pool” of unlabeled cholesterol depresses oxidation of the radioactive ketone less than a ‘ipool” of unlabeled ketone depresses cholesterol-26-Cl4 oxidation. Although the cholesterol oxidase system attacks coprostan-3-one and A4-cholesten3-one more readily than cholesterol, we have been unable to show that it can form these ketones from radioactive cholesterol. However, a small amount of radioactive material that is chromatographically similar to A4cholesten-3-one can be formed. Figure 3 shows that this material is slightly more polar than carrier A4-cholesten-3-one on an alumina column (type A). The formation of this cholestenone-like material is unaffected by the presence of A4-cholesten-3one in the incubation medium, but is abolished when boiled mitochondria are used. The material forms a 2,4-dinitrophenylhydrazone which is chromatographically similar to, but not identical with A4-cholestenone-2,4-dinitrophenylhydrazone. The material cannot be A5-cholesten-3-one, since this compound is unstable under the incubation conditions. When an aqueous suspension of this ,B,y-unsaturated ketone is shaken at pH 8.5 and 38”, its spont,aneous isomerization to A4-cholestenone can be demonstrated by the increase in optical density at 240 mp (Fig. 4). The cholesten-
OXIDATION
489
OF STEROIDS
1300.
m
Radioactivity
1250 l
Optical
density
1200 5 .t e
-
I
200
$ a .g \ Ii 6
150
0.10
100
* E s N
0.50
50
z d d
0
-
15 Fraction
number
I
0
25
50
75 Vol.
eluted
100
125
150
in ML.
of radioactive cholestenone-like material formed FIG. 3. Adsorption chromatography from cholesterol-26-r?. Neutral alumina (7 g., activity grade III) used with hexane-bensene: 3:2 (fractions l-20) and benzene (fractions 21-26) as solvent. Column size: 1.1 X 7 cm.; 5-ml. fractions taken. First and last radioactive peaks are cholesterol esters and unchanged cholesterol, respectively. Fractions 9-16 contain cholestenone-like material. Fractions IO-15 contain carrier A4-cholesten-3-one.
one-like material, when hydrogenated over platinum oxide, is converted to cholestan3p-01 (identified by chromatography as the free sterol and as the acetate). DISCUSSION
The cholesterol oxidase system is obviously not specific for cholesterol; only one of the other steroids tested is attacked at a very much slower rate. Since the structures of these steroids are so varied, it is unlikely that they are all intermediates in cholesterol degradation.
If the oxidation rates of the cholestane series members are compared with the oxidation rates of the corresponding coprost.ane series members, it is seen that in each case the coprostane series members are oxidized more rapidly. Within either series, the oxidation rates in ascending order are: 3p-sterol, 3cx-sterol, 3-ketone. Of all the steroids tested, coprostanone and A4-cholestenone are the most rapidly oxidized. Unlike the other substrates, these two ketones are oxidized without initial lag periods. Cholesterol (and most of the other
490
STEVENSON
$ 0.08 cd ’ 6
/
0.06
/
AND
./ :
.‘.
t
0.00~ 0
:
I
I 2 Time
3 in
4
5
6
hours
FIG. 4. Isomerization of A’-cholesten-3-one at pH 8.5 and 38”. The ketone was dissolved in methanol containing 3 mg. of Tween 20. After evaporation of the methanol the A5-cholestenone-Tween 20 mixture was dispersed in 0.052 M Tris-HCl buffer and shaken in a water bath.
substrates that we have test,ed) undoubtedly must pass through a number of reaction stages before COZ is finally formed. Thus, this absence of lag periods might be taken to mean that these two ketones are structurally closer than cholesterol to that intermediate from which the terminal carbon is cleaved. Unlabeled iipoo1s” of these two ketones are fairly potent depressors of labeled cholesterol oxidation, but an unlabeled cholesterol LLpool” has relatively little effect on the oxidation of the labeled ketones. This makes it unlikely that the ketones and the sterol are attacked by different enzyme systems in the mitochondria. Coprostan-3-one and A4-cholesten-3-one both lack a %-hydrogen; in this respect they differ from the more slowly oxidized cholestan-3-one. It appears, then, that if a ketone is to be rapidly oxidized without an initial lag period, it is important that there not be a hydrogen in the a configuration on C-5. These findings suggest that somewhere along the pathway of cholesterol side-chain degradation by rat liver mitochondrial enzymes there is an intermediate that is a 3ketone without a 5a-hydrogen. Harold et al. (16) showed that the intact rat can convert cholesterol to acids similar to, but not identical with, the usual bile acids. They suggested that this conversion proceeds through A4-cholestenone, cholestanone, and cholestan-3p-01, in that order. It was not, established whether or not the
STAPLE
side chains of their acids had been cleaved. However, if cleavage did take place, the cholesterol oxidase system apparently was not involved, for in this system cholestan3,8-01 is attacked more slowly than either A4-cholestenone or cholestanone. The enzymically formed material that is chromatographically similar to A4-cholestenone must be a Czi 3-ketone, since it forms a 2,4-dinitrophenylhydrazone and can be converted to cholestan-3p-01. Its chromatographic behavior indicates that it probably has two or more double bonds. While we have no evidence at present to prove that this ketone is actually on the pathway of cholesterol degradation, the finding that it is formed strengthens the plausibility of the hypothesized 3-keto intermediate. REFERENCES 1. ANFINSEN, C. B., AND HORNING, M. G., J. Am. Chem. Sot. 75,151l (1953). 2. HORKING, M. G., FREDRICKSOS, D. S., AND ANFINSEN, C. B., Arch. Biochem. Biophys. 71, 266 (1957). 3. WHITEHOUSE, M. W., STAPLE, E., AND GURIN, S., J. Biol. Chem. 234,276 (1959). M. W., STAPLE, E., AR’D GURIS, 4. WHITEHOUSE, S., J. Bid. Chem. 236, 73 (1961). 5. BRIGCS, T., WHITEHOUSE, M. W., AXD STAPLE, E., J. Biol. Chem. 236, 688 (1961). 6. RYER, A. I., GEBERT, W. H., AKD MURRILL, N. M., J. Am. Chem. Sot. 72, 4247 (1950). 7. NEHER, R., AND WETTSTEIN, A., Helv. Chim. Acta 35, 276 (1952). 8. RALLS, J. O., in “Organic Syntheses, Coll.” (A. H. Blatt, ed.), Vol. II, p. 191. John Wiley and Sons, New York, 1943. 9. ANDERSON, R. J., AND NABEKHAUER, F. P., J. Am. Chem. Sac. 46,1957 (1924). 10. BRUCE, W. F., in “Organic Syntheses, Coil.” (A. H. Blatt, ed.), Vol. II, p. 139. John Wiley and Sons, New York, 1943. 11. OPPENAUER, R. V., in “Organic Syntheses, Coll.” (E. C. Horning, ed.), Vol. III, p. 207. John Wiley and Sons, New York, 1959. 12. GRASSHOF, H., 2. physiol. Chem. 223, 249 (1934). 13. GRASSHOF, H., 2. physiol. Chem. 225, 197 (1934). 14. RUZICKA, L., BRUNGER, H., EICHENBERGER, E., AND MEYER, J., Helv. Chim. Acta 17, 1407 (1934). 15. FIESER, L. F., Organic Syntheses, 35, 143 (1955). 16. HAROLD, F. M., CHAPMAN, D. D., AND CHAIKOFF, I. L., J. Biol. Chem. 224,609 (1957).
OF
BIOCHEMISTRY
Oxidation
AND
BIOPHYSICS
of Various
97,
Simple
Steroids
Oxidase EDMUND From
the Biochemistry
(1962)
~85-490
by the Cholesterol
System’
STEVENSON2
AND
EZRA
STAPLE3
Department, University of Pennsylvania Philadelphia, Pennsylvania
School
of Medicine,
Received November 8, 1961 Eight simple C24steroids with Ci4 in the 26 position were synthesized. These compounds, which differ from one another in the A-ring substituents, were used to study the substrate specificity of the rat liver cholesterol oxidase system. The rate of C’“0, formation was taken as a measure of oxidation of the substrates. In both the cholestane and coprostane series, the oxidation rates fall in the ascending order: 3,% sterol, 3a-sterol, 3-ketone. Members of coprostane series are oxidized faster than the corresponding members of the cholestane series. There is an initial lag period in the oxidation of most of the substrates tested, during which little C’“Ot is formed. Only coprostan-3-one-26-C’ and A’-cholesten-3-one-26-C? are oxidized without this initial lag period. Cholesterol-26-V can be enzymically converted to a CL 3-ketone which is neither A*- nor A5-cholestenone, but which appears to differ from them in the number and position of double bonds. A 3-ketone without a 5a-hydrogen is hypothesized as an intermediate in the degradation of the cholesterol side chain by the cholesterol oxidase system. INTRODUCTION
Studies in this and other laborator&
have
revealed that fortified mouse (1, 2) and rat (3) liver mitochondria preparations can convert the terminal methyl groups of the
cholesterol side chain to CO,. Activity of the preparations is considerably enhanced by a heat-stable cofactor found in the cytoplasmic
fraction
system carrying
of the cells. The
enzyme
out these reactions is called
1 Supported by a grant (H-1532-C) of the National Heart Institute of the National Institutes of Health. *From a Dissertation presented to the Faculty of the Graduate School of Arts and Sciences of the University of Pennsylvania in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Present address: Department of Zoology, Yale University, New Haven, Conn. 3Work done during the tenure of an Established Investigatorship of the American Heart Association, Inc. New York 10, New York.
the cholesterol oxidase system and has been suggested to be involved in bile acid formation (3). Several sterols other than cholesterol are attacked by the enzymes and have been suggested as possible intermediates (2, 4, 5), but nothing is known of the specificity of the system with regard to the A-ring of the substrate. We have prepared eight simple CZ7 steroids, all with Cl4 in the 26 position and differing from one another in the substituents on the A-ring. They are: cholesterol cholestan-3p-01 cholestan-3a-ol cholestan-3-one
A4-cholesten-3-one coprostan-3p-01 coprostan-h-01 coprostan-3-one
Each of these radioactive steroids was incubated with the rat liver cholesterol oxidase system, and the rate of C1402 formation was measured. In addition, the effect of a “pool” of unlabeled cholesterol on the oxidation 485
of the other
labeled
steroids
and the
486
STEVEXSON
effects of “pools” of the other unlabeled steroids on t,he oxidation of labeled cholesterol llave been studied. MATERIALS
AND
METHODS
In this work, alumina adsorption columns were used extensively to purify and identify the products of the synthetic and enzymic reactions. For the sake of simplicity of discussion, the various alumina colLmms can be divided into three types, according to the solvent used: type A, hexanebenzene, 4:l; iype B, hexane-benzene, 3:2; type C, benzene. In all cases, neut,ral alumina (Woelm), activity grade 3 was used. was prepared from 3p-hyCholesterol-26-C” droxy-5-norcholesten-25one (kindly supplied by Dr. A. I. Ryer, Schcring Corp., Bloomfield, N. J.) by the procedure rrported by Ryer et al. (6) and pnrified via the digitonide and by chromatography (type C column). It Ivas chromatographicallg pure in the system of Seller and Wettstein (7) and had a specific activit,y of 0.57 pc./mg. This sterol was us(d in t,he preparation of the other C’” steroids. Unlabeled cholesterol was recrystallized from 95% (xthanol and had an m.p. of 148-149”. Cholestan-3/j-01-26-C” was prepared from cholesteryl acetate-26-C” by the method of Ralls (8) and purified by the procedure of ilnderson and Xabenhaucr (9) and by rhromatography (type C c~olumn). It was chromatographirally pure in the system of Neher and Wettstcin (7). Unlabeled cholestan-3@-ol was prepared and purified in t,he same way. Cholestan-3-one-26-C” was prepared from cholestan-3@-ol-26-C” according to the method of Bruce (10) and purified on alumina (t-ppe A column). Unlabeled ketone was prepared and purified in the same way; its m.p. was 127-130”. Cholestan-3a-ol-26-C’4 was prepared by hydrogenation of cholestan-3-one-26-C14 in etherethanol-12 N HCl, 10:37: 1 over platinum oxide at room temperat,LLre and under slight pressure. After cholestan-3P-01-26-C” had been removed as the digitonide, cholestan-3a-01-26-C was pLlrified by chromatography (type A column). Unlabeled cholestan-3a-ol was prepared and purified in the same manner as the labeled stcrol. I’-Cholesten-3-one-26-C”, obtained by the Oppenauer oxidation of cholesterol-C’* (11) and purified by chromatography (type B rolumn), was chromatographically pure in the system of Neher and Wettstein (7). Unlabeled A’-c lolcsten-3-one was prepared and purified in the aam,: manner. Coprostan-3-one-26-C” was prepared from A*cholesten-3-one-26-C” as described by Grasshof (12) and purified on an slLlmina column (tye A). This material was not contaminated with unchanged a,fi-unsaturated ketone since its ultra-
AND
STAPLE
I-iolet absorption spectrum had no yak in the 240-mp region. Unlabeled coprostan-3-one prepared and purified in the same manner had :m m.p. of 60-62”. Coprostan-3p-ol-26-C>’ was prepared from coprostanone-26-C’& by the procedure of Gras&of (13) and purified both as the digitonide and on a type -4 alumina column. Unlabeled roprostan-3fl-ol prepared and purified in the same w-a?- had an m.p. of 97-98”. Coprostxn-3a-01-26-C” was prepared from coproStan-3-one-26-C? by catalytic hydrogenation as described by Ruzicka et c/l. (14). The small amount of 38 isomer that was formed at the same time was rcmo\.cxd a;; the digitonide, and the 3a isomer was purified b,v chromatography (type B column). Unlabeled coprostnn-3a-ol was prepared in the same way; it,s m.p. was 110.5-111.5” after recrystallization from 95% ethanol. A’-Cholesten-3-one was prepared by the oxidation of cholesterol dibromide as tlescribeci by Fieser (15). Its m.p. was 125-129” (rapid hea,ing), and its ultraviolet absorption spectrum showed no I)euk in the 240-mp region. The procedures for preparing the mitochondria and SF’ and for carrying out the incllbations have been described (2). In the studies on the rate of oxidation of any given steroid, the rate of CYO, formation from th:Lt steroid was compared with the rate of C”O, formation from c*holrsterol-26Cl*. In each de:e!mination, 20 flasks were used; as substrate (s),. five cont,aincd cholesterol-26-C’*, fire contained cholestrrol-26-C” plus a “pool” of the other, Lrnlnbcied steroid (0.1 mg./flnsk), fix-p contained the o!l.cr labPled with C” in the 26 position, steroid and five (‘1 :’ ::;n:.l the other, 26-labeled (0.1 mg.,/ plus a ((l)O.>. c,c Lmlabeled cholesterol &irate (s) were added as aqueous flask). These’ emulsions stat,. iz-d with a total of 3 mg. of Twcen 20 (-itlrs “(~a der Co.) as described earlirl
(2). ted in aqueous SaOH and conC”0, was cc verted to BaC’ : l3 Radioactivity was measured in a windowless, gas-flow counter and correct., :i for self-absorption. In order to compare ihc rates of oxidation of cholesterol-26-C’” and the other labeled steroids, equal concentrations of the snbstrates should be used. In practice, it is not always easy to achieve this. We corrected for the inequalities in the following way : We first established that within limits the amount of C”0, formed from cholesterol-26“SF, supcrnatant factor(a) in t,he sohble fraction of boiled extracts of liver; ATP, adenosine triphosphatt ; GSH, reduced glutathionine; AMP, adenosine monophosphate ; DPN, diphosphopyridine nuclrotide ; Tris, tris( hy-drometlLyl)uminomrth;mc.
OXIDATION C” is proportional to the substrate concentration. Figure 1 demonstrates that as long as less than cu. 55 pg. (30,000 counts/min.) of cholesterol-26-C’ per flask is used, a, linear relationship is maintained between C” substrate and C’“O, evolved. We then adjusted the counts/min. in the C”O, from cholesterol-26-C” to what they would have been had the concentration of the cholesterol-26-Cl4 been equal to the concentration of the other steroid. This was done by multiplying the counts/min. in the C401 from cholesterol-26-r? by the factor concentration of the other labeled steroid used concentration of cholesterol-26-Cl4 used ’ All of the experiments were repeated with different mitochondria preparations. The preparations varied considerably in their activities, so the final results are expressed as the per cent oxidation of the radioactive steroids relative to the maximum oxidation of radioactive cholesterol in the particular ekperiment, the latter having an assigned value of 100 (relative per cent oxidation). The relative per cent oxidation of any radioactive substrate was usually reproducible to cu. *IO%. In the search for ketosteroids formed enzymitally from cholesterol-26-C?, incubations carried out in the usual manner were stopped by the addition of 4 vol. of 95% ethanol. After the precipitated proteins had been removed, the supernatant fluids were evaporated to dryness and the ethersoluble portion of the residues chromatographed (type A and B columns). The eluted fractions (5 ml. each) were plated and the radfoactivities determined. RESULTS
487
OF STEROIDS
,’ 3 I’
When cholesterol-26-C?* i., AI‘d’ized by fortified rat liver mitochocd. a, there is a lapse of cu. i/s to Ii/i hr. b::are appreciable C1*Oa is formed. After this itial lag period the C?*O, formation r ,’ is linear for at Iiast 8 hr. TIP production of Cl402 from cholestan3p-ol-26-Cl4 proceeds both with a longer lag period and at a slower rate than from cholesterol-26-CY4. In addition, a “pool” of unlabeled cholesterol depresses cholestan3p-01-26-C?* oxidation considerably, while a “pool” of unlabeled cholestan-3p-ol does not depress cholesterol-26-C?* oxidation at all. These points are illustrated in Fig. 2; the data that we obtained when the other steroids were used as substrates are very briefly summarized in Table I. Cholestan3~01-26-Cl4 and cholesterol-26-Cl4 are oxidized similarly; a L’pool” of one unlabeled
750r-----l m 600 0 0 -0 g 450 .c f
. ./' .
300
a:
OV 0
I
60,000
45,000 30,000 15,000 C.P.M. Cholesterol - 26 - Cl4
used
pg:.5
used
I 0
100 ChOI.s+e::
- 26 -:I4
FIG. 1. Effect of cholesterol-26-r? concentration on C’“OZ formation by the cholesterol oxidase system. Incubation mixture consisted of 1 ml. of mitochondria suspension (‘VI of a rat liver), 5 ml. SF, 5 ml. of 0.25 M Tris-HCl buffer, pH 8.5, 25 mg. ATP, 15 mg. GSH, 8 mg. AMP, 5 mg. DPN, 10 mg. Mg(N0J2.6H20, and 22 mg. trisodium citrate dihydrate. Total volume, 12 ml. Cholesterol-26C” was added as Tween 20 suspension in buffer. Incubation time, 7% hr.
sterol has little effect on the oxidation of the other labeled sterol. Cholestan-3-one26-Cl4 is oxidized more rapidly than cholesterol-26-U*. Nowever, unlabeled cholesterol significantly depresses oxidation of the radioactive ketone, but unlabeled cholestan-3-one does not depress oxidation of the labeled sterol. Initial lag periods are found in the oxidations of both substrates. Both coprostan-3p-01-26-U* and cholesterol-26-U* are oxidized with lag periods that are alike, although the rate of oxidation of the former is less than that of the latter. Coprostan-3p-ol-26-Cl4 oxidation is depressed to about the same extent by a iipool” of unlabeled cholesterol as cholesterol-26-Cl4 oxidation is depressed by a “pool” of unlabeled coprostan-3p-01. The rate of oxidation of coprostan-3a-01-26-C?* is greater than t,hat of cholesterol-26-C’“. The presence of unlabeled coprostan-3a-ol depresses radioactive cholesterol oxidation more than Ihe presence of unlabeled cholesterol depresses radioactive coprostan-3a01 oxidation. Both labeled substrates are oxidized with about equal lag periods. M. W. Whitehouse (personal communication) has obtained similar results. Coprostan-3one-26-G4 oxidation is rapid and unaffected
488
STEVENSON
AND
STAPLE
Cholesterol- 26 -Cl4
f -f
I
0
2 Incubation
3
4
time,
of cholestan-3p-ol-C” FIG. 2. Oxidation steroids (0.1 mg./flask) and radioactive cubation mixture as in Fig. 1. TABLE
THE OXIDATION OF VARIOUS BY THE CHOLESTEROL OXIDASE SYSTEM
1?er cent depressionof oxidation of ‘ther, !abeled Labeled chostemd by lesterol by “pool” of “pool” of unlabeled other, unlacholesterol beledsteroid
. -
-
100
Cholestan3&ol Cholestan-3ol-01 Cholestan-3-one
40 95 170
35 10 20
0 10
Coprostan-SD-01 Coprostan-3a-01 Coprostan-3-one
75 175 235
40 10 0
40 25 20
A4-Cholesten-3one
330
20
50
-OL-26-C14
Cholesion-3b Cholesterol
-OL-26-Cl4
6
by the cholesterol oxidase system, Unlabeled sterols were added as Tween 20 suspensions. In-
I
Cholesterol
Choleston-3p
-
hours
COMPARISON OF STEROIDS
Final Radioactive substrate 1R.P.O.”
5
_-
0
-
a Final relative per cent oxidation (i.e., the oxidation of various radioactive steroids in 8 hr., compared to oxidation of radioactive cholesterol in the same t’ime by the same mitochondria preparation). The incubation mixture used is described in Fig. 1.
by a ‘ipool” of unlabeled cholesterol, although the oxidation of labeled cholesterol is depressed by a “pool” of unlabeled coprostanone. In addition, radioactive copro-
stanone is oxidized without an initial lag period. A4-Cholesten-3-one-26-Cl4 is also rapidly oxidized without an initial lag period. A “pool” of unlabeled cholesterol depresses oxidation of the radioactive ketone less than a ‘ipool” of unlabeled ketone depresses cholesterol-26-Cl4 oxidation. Although the cholesterol oxidase system attacks coprostan-3-one and A4-cholesten3-one more readily than cholesterol, we have been unable to show that it can form these ketones from radioactive cholesterol. However, a small amount of radioactive material that is chromatographically similar to A4cholesten-3-one can be formed. Figure 3 shows that this material is slightly more polar than carrier A4-cholesten-3-one on an alumina column (type A). The formation of this cholestenone-like material is unaffected by the presence of A4-cholesten-3one in the incubation medium, but is abolished when boiled mitochondria are used. The material forms a 2,4-dinitrophenylhydrazone which is chromatographically similar to, but not identical with A4-cholestenone-2,4-dinitrophenylhydrazone. The material cannot be A5-cholesten-3-one, since this compound is unstable under the incubation conditions. When an aqueous suspension of this ,B,y-unsaturated ketone is shaken at pH 8.5 and 38”, its spont,aneous isomerization to A4-cholestenone can be demonstrated by the increase in optical density at 240 mp (Fig. 4). The cholesten-
OXIDATION
489
OF STEROIDS
1300.
m
Radioactivity
1250 l
Optical
density
1200 5 .t e
-
I
200
$ a .g \ Ii 6
150
0.10
100
* E s N
0.50
50
z d d
0
-
15 Fraction
number
I
0
25
50
75 Vol.
eluted
100
125
150
in ML.
of radioactive cholestenone-like material formed FIG. 3. Adsorption chromatography from cholesterol-26-r?. Neutral alumina (7 g., activity grade III) used with hexane-bensene: 3:2 (fractions l-20) and benzene (fractions 21-26) as solvent. Column size: 1.1 X 7 cm.; 5-ml. fractions taken. First and last radioactive peaks are cholesterol esters and unchanged cholesterol, respectively. Fractions 9-16 contain cholestenone-like material. Fractions IO-15 contain carrier A4-cholesten-3-one.
one-like material, when hydrogenated over platinum oxide, is converted to cholestan3p-01 (identified by chromatography as the free sterol and as the acetate). DISCUSSION
The cholesterol oxidase system is obviously not specific for cholesterol; only one of the other steroids tested is attacked at a very much slower rate. Since the structures of these steroids are so varied, it is unlikely that they are all intermediates in cholesterol degradation.
If the oxidation rates of the cholestane series members are compared with the oxidation rates of the corresponding coprost.ane series members, it is seen that in each case the coprostane series members are oxidized more rapidly. Within either series, the oxidation rates in ascending order are: 3p-sterol, 3cx-sterol, 3-ketone. Of all the steroids tested, coprostanone and A4-cholestenone are the most rapidly oxidized. Unlike the other substrates, these two ketones are oxidized without initial lag periods. Cholesterol (and most of the other
490
STEVENSON
$ 0.08 cd ’ 6
/
0.06
/
AND
./ :
.‘.
t
0.00~ 0
:
I
I 2 Time
3 in
4
5
6
hours
FIG. 4. Isomerization of A’-cholesten-3-one at pH 8.5 and 38”. The ketone was dissolved in methanol containing 3 mg. of Tween 20. After evaporation of the methanol the A5-cholestenone-Tween 20 mixture was dispersed in 0.052 M Tris-HCl buffer and shaken in a water bath.
substrates that we have test,ed) undoubtedly must pass through a number of reaction stages before COZ is finally formed. Thus, this absence of lag periods might be taken to mean that these two ketones are structurally closer than cholesterol to that intermediate from which the terminal carbon is cleaved. Unlabeled iipoo1s” of these two ketones are fairly potent depressors of labeled cholesterol oxidation, but an unlabeled cholesterol LLpool” has relatively little effect on the oxidation of the labeled ketones. This makes it unlikely that the ketones and the sterol are attacked by different enzyme systems in the mitochondria. Coprostan-3-one and A4-cholesten-3-one both lack a %-hydrogen; in this respect they differ from the more slowly oxidized cholestan-3-one. It appears, then, that if a ketone is to be rapidly oxidized without an initial lag period, it is important that there not be a hydrogen in the a configuration on C-5. These findings suggest that somewhere along the pathway of cholesterol side-chain degradation by rat liver mitochondrial enzymes there is an intermediate that is a 3ketone without a 5a-hydrogen. Harold et al. (16) showed that the intact rat can convert cholesterol to acids similar to, but not identical with, the usual bile acids. They suggested that this conversion proceeds through A4-cholestenone, cholestanone, and cholestan-3p-01, in that order. It was not, established whether or not the
STAPLE
side chains of their acids had been cleaved. However, if cleavage did take place, the cholesterol oxidase system apparently was not involved, for in this system cholestan3,8-01 is attacked more slowly than either A4-cholestenone or cholestanone. The enzymically formed material that is chromatographically similar to A4-cholestenone must be a Czi 3-ketone, since it forms a 2,4-dinitrophenylhydrazone and can be converted to cholestan-3p-01. Its chromatographic behavior indicates that it probably has two or more double bonds. While we have no evidence at present to prove that this ketone is actually on the pathway of cholesterol degradation, the finding that it is formed strengthens the plausibility of the hypothesized 3-keto intermediate. REFERENCES 1. ANFINSEN, C. B., AND HORNING, M. G., J. Am. Chem. Sot. 75,151l (1953). 2. HORKING, M. G., FREDRICKSOS, D. S., AND ANFINSEN, C. B., Arch. Biochem. Biophys. 71, 266 (1957). 3. WHITEHOUSE, M. W., STAPLE, E., AND GURIN, S., J. Biol. Chem. 234,276 (1959). M. W., STAPLE, E., AR’D GURIS, 4. WHITEHOUSE, S., J. Bid. Chem. 236, 73 (1961). 5. BRIGCS, T., WHITEHOUSE, M. W., AXD STAPLE, E., J. Biol. Chem. 236, 688 (1961). 6. RYER, A. I., GEBERT, W. H., AKD MURRILL, N. M., J. Am. Chem. Sot. 72, 4247 (1950). 7. NEHER, R., AND WETTSTEIN, A., Helv. Chim. Acta 35, 276 (1952). 8. RALLS, J. O., in “Organic Syntheses, Coll.” (A. H. Blatt, ed.), Vol. II, p. 191. John Wiley and Sons, New York, 1943. 9. ANDERSON, R. J., AND NABEKHAUER, F. P., J. Am. Chem. Sac. 46,1957 (1924). 10. BRUCE, W. F., in “Organic Syntheses, Coil.” (A. H. Blatt, ed.), Vol. II, p. 139. John Wiley and Sons, New York, 1943. 11. OPPENAUER, R. V., in “Organic Syntheses, Coll.” (E. C. Horning, ed.), Vol. III, p. 207. John Wiley and Sons, New York, 1959. 12. GRASSHOF, H., 2. physiol. Chem. 223, 249 (1934). 13. GRASSHOF, H., 2. physiol. Chem. 225, 197 (1934). 14. RUZICKA, L., BRUNGER, H., EICHENBERGER, E., AND MEYER, J., Helv. Chim. Acta 17, 1407 (1934). 15. FIESER, L. F., Organic Syntheses, 35, 143 (1955). 16. HAROLD, F. M., CHAPMAN, D. D., AND CHAIKOFF, I. L., J. Biol. Chem. 224,609 (1957).