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[26] Enzymatic degradation of the cholestane side chain in the biosynthesis of bile acids
562
ENZYME SYSTEMS: BILE ACID FORMATION
[26]
The 3a-hydroxysteroid dehydrogenase fraction obtained from the Sephadex G-75 column, 0.05-0.1 ml, is diluted to 4 ml with 0.1 M TrisHC1 buffer, pH 7.4; 1 mg of N A D P H is added and the mixture is incubated for 20 minutes with 50 ~g of labeled 7a-hydroxy-5fl-cholestan3-one, added to the incubation mixture dissolved in 0.1 ml of acetone. The chloroform extract of the incubation mixture is chromatographed with benzene-ethyl acetate 1:1 as solvent (cf. Table I).
[ 2 6 ] E n z y m a t i c D e g r a d a t i o n of t h e C h o l e s t a n e S i d e C h a i n in t h e B i o s y n t h e s i s o f B i l e A c i d s B y EZRA STAPLE~
Work on the cleavage of the side chain of cholestanes to form bile acids may be considered to begin with the work of Anfinsen and Horning. 1~ These investigators found that a homogenate of mouse liver and, later, mitoehondrial fractions of this homogenate were capable of yielding radioactive carbon dioxide from incubation with cholesterol-26-14C. Later this enzymatic reaction was studied with rat liver mitochondria by Whitehouse, Staple, and Gurin, 2,~ and with liver mitoehondria of other species. 4 This reaction has been used to study the influence of various factors on the cleavage process. Following suggestions first made by BergstrSm, 5 5fi-cholestane-3~,7~, 12~-triol and various derivatives were studied. This work led to the discovery by Danielsson 6 tha~ this triol can be enzymatically hydroxylated in the 26-position by liver mitoehondrial preparations. This was confirmed by Suld, S~aple, and Gurin. 7 Further, both of these groups of investigators found that 3%7a,12a-trihydroxy-Sfl-cholestan-26-oie acid is also produced. Suld et al. 7 also found that production of the 26-oic acid from the 26-hydroxylated compound is dependent on the presence of NAD ÷. A partial replacement of NAD ÷ by N A D P + in a crude system is claimed by Deceased. 1~C. B. Aniinsen and M. G. Homing, J. Am. Chem. Soc. 75, 1511 (1963). *M. W. Whitehouse, E. Staple, and S. Gurin, J. Biol. Chem. 234, 276 (1959). M. W. Whitehouse, E. Staple, and S. Gurin, J. Biol. Chem. 236, 68 (1961). *M. W. Whitehouse, M. C. Cottrell, T. Briggs, and E. Staple, Arch. Biochem. Biophys. 9@, 305 (1962). 5S. Bergstr6m, Ciba Found. Syrup. Biosyn. Terpenes Sterols. Little, Brown, Boston, Massachusetts, 1959. *H. Danielsson, Acta Chem. ~cand. 14, 348 (1960). It. M. Suld, E. Staple, and S. Gurin, J. Biol. Chem. 237, 338 (1962).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
563
Dean and Whitehouses for this reaction. This dehydrogenase system has been purified and studied by Herman and Staple9 and further by Masui, Herman, and Staple 1° to indicate that this is a two-step dehydrogenation reaction, in which the enzyme for the first step can be separated by enzymatic purification techniques. They also have confirmed the obligatory requirement for NAD ÷ as hydrogen acceptor. As an extension of their work with 3~,7~,12~-trihydroxy-5fi-cholestane-26-oic acid, Suld et al. ~ found that the side chain was cleaved between carbon atoms 24 and 25 of the side chain to yield cholic acid and propionie acid (propionyl-CoA). These authors proposed a thiolytie cleavage similar to that which occurs in the fl-oxidation of fatty acids. Consistent with this hypothesis, Masui and Staple 11 have recently reported the formation of 3%7~,12a,24~-tetrahydroxy-5fl-cholestan-26-oic acid from 3~,7%12~-trihydroxy-5fi-cholestan-26-oic acid and the conversion of the former compound to cholic acid. In sum, the enzyme reactions for the cleavage of the cholestane side chain to form the cholanic acids appears to parallel those for u-oxidation of hydrocarbons and fl-oxidation of fatty acids. The enzyme reactions described below give evidence for some of these steps. I. Preparation of Substrate Materials A. Cholesterol-26-14C This material can be synthesized according to methods previously reported 12-14 and as given below. It may also be obtained commercially from several sources. Seventy-two milligrams (0.5 millimole) of methyl iodide-~4C (2 mC per millimole) is diluted with 4.5 millimoles of unlabeled methyl iodide and added to 146 mg of magnesium (for Grignard reactions) (6 millimoles) in 4 ml of anhydrous ether. The reaction is initiated by cautious, gentle warming and proceeds for 1 hour. Then 715 mg (1.67 millimoles) of 3fl-acetoxy-27-norcholest-5-en-25-one in 5 ml of dry benzene is added slowly with stirring and the mixture is refluxed for 3 hours. It is then allowed to stand overnight. The complex is decomposed by addition of 2 ml of ice water and 4 ml of 50% acetic acid. The mixture is then steams p. D. G. Dean and M. W. Whitehouse, Biochem J. 98, 410 (1966). 9R. H. Herman and E. Staple, Federation Proc. ~4, 661 (1965). 1oT. Masui, R. Herman, and E. Staple, Biochim. Biophys. Acta 117, 260 (1966). ,1T. Masui and E. Staple, J. Biol. Chem. 9.41, 3889 (1966). J~A. I. Ryer, W. H. Gebert, and N. M. Murrilt, J. Am. Chem. Soc. 7g, 4247 (1950). ~ W. G. Dauben and H. L. Bradlow, J. Am. Chem. Soc. 72~ 4248 (1950). 1~M. G. ttorning, D. S. Fredrickson, and C. B. Anfinsen, Arch. Biochem. Biophys. 71, 266 (1957).
564
ENZYME SYSTEMS: BILE A.CID FOHMA.TION
[26]
distilled until no more water-insoluble distillate is obtained. The reaction mixture is extracted with ether, and the ether extract is washed with NaHCOa solution. The washings are made alkaline and extracted with ether, and the two ether extracts are combined and dried over MgS04. The M g S Q is then filtered off and the ether is evaporated, leaving 743 mg of residue of crude cholest-5-ene-3fl,25-diol-26-14C. The residue is dissolved in 5 ml of anhydrous pyridine, and 1.2 ml (12.7 millimole) of acetic anhydride is added. The mixture is refluxed for 7 minutes and then added dropwise to 50 ml of water. The water is extracted with ether, the ether is washed with 2 N H2S04, and the acid wash is extracted again with ether. The combined ether extracts are dried over MgS0~, after addition of a little dry NaHCO~. After evaporation of the ether, 806 mg of yellowish residue of crude 3fl-acetoxy-eholest-5en-25-ol-26-14C remains. The crude acetate is dissolved in 20 ml of anhydrous pyridine and 0.85 ml (9.3 millimole) of freshly distilled phosphorus oxychloride is added. A red color, deepening to black, forms on refluxing for 30 minutes. The solution is poured into 50 ml of ice water, diluted with more water, and extracted with ether. The ether extracts are combined and washed with 2 N H2S04 to remove the pyridine. The extract is then washed with 5% NaHC03 solution. The acidified washings are extracted with ether, and the ether extract is washed with NaHCOs solution. The ether extracts are combined and evaporated. The orange residue of mixed 26-1~C labeled 24and 25-dehydrocholesteryl acetates weighs 665 rag. It is hydrolyzed by refluxing for 1 hour in 10 ml of 2 N ethanolic KOH. This solution is diluted with 50 ml of water and extracted several times with ether. The ether extracts are washed repeatedly with water and the water washes are combined and extracted once with ether. The combined ether extracts are dried overnight over MgSO~ and filtered into a 100-ml hydrogenation flask. The solvent is evaporated, leaving 580 mg of orange residue containing the crude 24- and 25-dehydrocholesterols. The mixture is dissolved in 20 ml of absolute ethanol and hydrogenated at atmospheric pressure using 50 mg of 10% palladium-on-charcoal as catalyst. The process is stopped after uptake of 23 ml (1.0 millimole) of hydrogen per millimole of steroid. The hydrogenation requires about 30 minutes. The solution of crude cholesterol is filtered, the solvent is evaporated, and the residue is chromatographed on a 30-g column of neutral alumina (Woelm, grade 1). The crude cholesterol is placed on the column, a minimal quantity of 45% (v/v) ether in petroleum ether (b.p. 30-60 °) being used; the column is then eluted with 2.3 liters of the same solvent followed by 1.5 liters of 65% ether in petroleum ether. The fractions con-
[26]
BILE ACIDS: SIDE-CHAIN" DEGRADATION
565
raining the principal radioactive peak are combined and evaporated. Approximately 509 mg of crude cholesterol-26-14C is obtained at this point. The partially purified product is dissolved in 60 ml of 90% aqueous ethanol and to this solution is added a solution containing 1.7 g of digitonin in 200 ml of 90% aqueous ethanol. After it has stood for 1 hour at room temperature, the digitonide is kept at 7 ° for 60 hours. It is collected by eentrifugation and washed with cold 90% aqueous ethanol, cold ether-acetone (2:1), and cold ether, then dried. The cholesterol is recovered by decomposition of the digitonide dissolved in 12 ml of pyridine. Ether, 120 ml, is added and the digitonin is allowed to precipitate several hours at room temperature. The precipitate is centrifuged and washed with ether, and the ether solutions are combined. The precipitated digitonin is redissolved in 85% aqueous ethanol and the above precipitation procedure is repeated; additional cholesterol is obtained in the ether solution. On evaporation of the ether solution, 326 mg (0.84 millimole) cholesterol-26-~4C, specific activity 0.57 /~C/mg is obtained. The overall yield from 3fl-acetoxy-27-norcholest-5-en-25-one is 51%. On the basis of methyl iodide-l~C the yield is 35%. B. 5fl-Cholestane-3a,7a,12a-triol-26,27-14C
The synthesis is carried out, as previously reported, 1~ as follows: Triformylcholyl chloride is prepared from cholic acid by the method of Cortese and Bauman26 Using benzene-petroleum ether (b.p. 60-80 °) as solvent, triformylcholic acid can be crystallized with m.p. 209-211 °. The acid is converted to the acid chloride by treatment with oxalyl chloride. 2-Propanol-l,3-1~C (specific activity 1.0 mC per millimole) is diluted 20-fold with unlabeled 2-propanol and then converted to 2-bromopropane-l,3-14C by reaction with phosphorus tribromide. A Grignard reagent is prepared from 2.40 g of 2-bromopropane-l,3-~4C (50 ~C per millimole) and 600 mg of magnesium (for Grignard reactions) in 25 ml of anhydrous ether. The solution is cooled in an ice bath and stirred. To this 7.5 g of powdered anhydrous cadmium bromide (dried for 3 hours at 120 °) is added. The preparation is stirred for 30 minutes, the ice bath is removed, and a solution of 1.50 g of triformylcholyl chloride in 8 ml of benzene is added. Stirring is continued for 30 minutes after this addition, and the reaction mixture is heated to reflux for 1 hour then allowed to stand overnight at room temperature. Ice water and then 3 N HC1 solution are added to the reaction mixture. Sufficient acid should be ~'E. Staple and M. W. Whitehouse, J. Org. Chem. 9.4, 433 (1959). ~eF. Cortese and L. Bauman, J. Am. Chem. Soc. 57, 1393 (1935).
566
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
added to dissolve the initial precipitate. The benzenc cther layer is separated and washed with water until the washings are neutral. The washed solvent layer is then evaporated to dryness and the residual gum is further dried in vacuo. The residue is heated for 1 hour with 10 ml of 5% methanolic KOH. This reaction mixture is then diluted with 100 ml of water and extracted with ether. The ether extract is evaporated to dryness and the residue of crude 24-keto-5fl-cholestane-3a,7a,12a4riol26,27-14C is subjected to silicic acid column chromatography. The column, 2.0 cm in diameter, contains 50 g of silicic acid (Merck) and is prepared with benzene. It is eluted with benzene first and then with ether-benzene (1:2). The ketone is eluted by the latter solvent. After evaporation of this eluate, 698 mg of 24-keto-5fl-cholestane-3~,7~,12~-triol-26,27-~4C, m.p. 143-145 °, is obtained. This product may be recrystallized from acetone if desired. To 500 mg of 24-keto-5fl-cholestane-3a,7a,12a-triol-26,27-~C dissolved in 1 ml of ethanol is added 1 ml of hydrazine hydrate (99%). The mixture is swirled for a few minutes to give a homogeneous solution. Ten milliliters of triethylene glycol and 1 g of KOH are added to this solution, and the mixture is heated under reflux for 30 minutes. Then the reflux condenser is removed and the mixture is heated at 180-200 ° for 2 hours. At the end of this heating period, the reaction mixture is allowed to cool in a stream of nitrogen gas and it is then poured into 50 ml of water. The precipitated compound is filtered, washed with water, and dried in vacuo. This crude product is crystallized from acetone; 269 mg 5fl-cholestane3~,7a,12a-triol-26,27-1~C, m.p. 184--185°, is obtained.
C. 3a,7a,12a-Trihydroxy-Sfl-cholestan-26-oic Acid The D-isomer of this acid is best obtained from the bile of Alligator mississippiensis. Bile from other crocodilia may be used in place of A. mississippiensis bile, which is now difficult to obtain. It is necessary, however, thoroughly to identify and characterize the isolated trihydroxycoprostanic acid in each case. There may be some racemization about carbon atom 25 during the hydrolysis procedure although no definitive information on this has yet been obtained. It may also be obtained synthetically, 1~ but with great difficulty and in low yield. A procedure for isolation of the acid from A. mississippiensis bile is given below. Bile from A. mississippiensis is mixed with 2-3 volumes of ethanol to precipitate the mucin and other proteins. The precipitate is removed by centrifugation and again washed with ethanol. The combined alcoholic solutions are evaporated to dryness giving x grams of gummy residual ~7R. J. Bridgwater, Biochem. J. 64, 593 (1956).
[26]
ruby. ACIDS: SIDE-CHAIN DEGRADATION
567
"crude bile salts." This residue is taken up in water, the pH adjusted to 8-9 and 0.5 x g of decolorizing charcoal is added. The mixture is evaporated on a steam bath with occasional stirring. The residue is transferred to a Soxhlet extractor, and extraction with absolute ethanol is continued until fresh extracts are colorless. Evaporation of the alcohol gives a gummy, occasionally brittle, residue, "purified bile salts." In the case where the quantity of "crude bile salts" is small (less than 100 rag) the charcoal purification step can be omitted. Hydrolysis of the bile salts is carried out in 2.5 N NaOH in an autoclave at a pressure of 20 psi (125 ° for 16-18 hours). The alkaline hydrolyzate is diluted with water, saturated with NaC1, and acidified to pH 1 with HC1. The precipitated bile acids are extracted with three portions of ethyl acetate, and the combined extract is washed three times with water. The combined water washings are extracted once with ethyl acetate. The ethyl acetate extracts are combined and evaporated to dryness, and the crude bile acids so obtained are further purified by partition column chromatography. The chromatographic column system used by Mosbach, Zomzely, and Kendall is is recommended. Four milliliters of 70% acetic acid on 5 g of Celite 545 (Johns-Manville Corp.) is the stationary phase, and petroleum ether (b.p. 30-60 °) is the first moving phase. The packed column measures 0.9 cm X 18 cm. The sample to be separated is dissolved in 3--4 drops of glacial acetic acid, mixed thoroughly with 0.2-0.3 g of Celite, slurried in petroleum ether, and added to the column. Elution is effected with (1) 45 ml of petroleum ether, (2) 90 ml of 40% isopropy] ether in petroleum ether, and (3) 100 ml of 60% isopropyl ether in petroleum ether. The 3%7~,12~-trihydroxy-5fl-cholestan-26-oic acid is eluted with solvent mixture (2). Material of m.p. 176-178 ° is readily obtained by isolation from the bile of A. mississippiensis. The acid obtained in this way can be tritiated by the Wilzbach method? 9 Several commercial laboratories offer this tritiation service. The acid which has been through this procedure must be carefully purified by several chromatographic systems. D. 5fl-Cholestane-3a,Ta,12a,26-tetraol This compound is prepared synthetically in high yield from 3a,7a,12atrihydroxy-5fi-eholestan-26-oic acid by reduction with lithium aluminum hydride as follows: The methyl ester of the trihydroxy acid is prepared by treatment of 1SE. H. Mosbach, C. Zomzely, and F. E. Kendall, Arch. Biochem. Biophys. 48, 95 (1954). ~9K. E. Wilzbaeh, J. Am. Chem. Soc. 79, 1013 (1957).
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ENZYME SYSTEMS: :BILE ACID FORMATION
[26]
an ether solution of the acid with an excess of freshly generated diazomethane solution in ether. The mixture is allowed to stand until all the diazomethane decomposes, then the ether is evaporated. The ester in the residue, after removal of the ether, represents a yield of 99-[-% of the ester. The ester (50 rag) is dissolved in 10 ml of anhydrous benzene and added slowly to the lithium aluminum hydride (25 mg) dissolved in 15 ml of ether. After the addition, and after the reaction has subsided, the mixture is heated under reflux for 30 minutes. The reaction mixture is then cooled, and ethanol (95%) is then added cautiously, dropwise, to decompose the excess of the hydride. When no further gas evolution occurs after addition of ethanol, the mixture is added to cold water (50 ml) and sufficient 2 N H~S04 is added to dissolve the precipitate. The e~herbenzene layer is washed with water until neutral. The original water layer is extracted with ether, and this ether extract is washed with water until neutral. The ether extract is combined with the original etherbenzene layer, and the solvents are removed by evaporation. The residue consists of practically pure 5fl-cholestane-3a,7a,12a,26-tetraol, in a yield of 98-]-%. After crystallization from methanol, the tetraol melts at 203-204%
E. 3a,7a,12a,24~-Tetrahydroxy-5fl-cholestan-26-olc Acid-24-14C This compound is prepared from cholic acid-24-14C by a modified Reformatsky reaction 2° as follows. 3a,7a,12a-Trihydroxycholan-24-al-14C (0.10 mC, 95 rag) is synthesized from cholic acid-24-1~C (activity 0.50 mC) (500 mg) by the method of Yashima21 This method utilizes the oxidation of homocholane-3a,7a,12a,24,25-pentaol by lead tetraacetate to give cholanyl aldehyde directly. The aldehyde-14C is dissolved in 10 ml of dry toluene and 3 ml of ethyl dl-2-bromopropionate containing granulated zinc and a small amount of both iodine and powdered copper as catalysts. The reaction mixture is then poured into a mixture of crushed ice and excess 10% H2SO,. This is then extracted with ethyl acetate. The ethyl acetate extract is washed with dilute Na2S~03 to remove the iodine and then with water. The solvent layer is then evaporated to dryness. The residue is saponified with 5% of methanolic KOH solution for 1 hour on a water bath. After removal of the methanol, the residual aqueous solution is extracted with ethyl acetate to remove 3a,Ta,12a-trihydroxycholan24-al-24-~4C. This alkaline solution is then acidified with 10% HCI solution and extracted with ethyl acetate. The extract is washed with water ,oy. Inai, Y. Tanaka, S. Betsukl, and T. Kazuno, I. Biochem. (Tokyo) 56, 591 (1964). ~IH. Yashima, J. Biochem. (Tokyo) 54, 47 (1963).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
569
to neutrality and then the solvent is evaporated. A reaction product (activity 0.03 mC) (31 rag) is obtained. On further purification by thin layer chromatography on silica gel G (Brinkmann Instruments) using benzenc-isopropanol-acctic acid 30:I0:1 as developing solvent, a mixture of the isomers of 3~,7~,]2~,24~-tetrahydroxy-5fl-cholestan-26-oic acid-24-14C, with R / s of 0.31 and 0.34 and m.p. 98-102 ° is obtained. For further separation of the stereoisomers of this acid, see Masui and Staple21 II. Enzymatic Transformation A. Formation of 1~CO~ from Cholest-5-en-3fl-ol-26-14C Principle. This sequence of enzyme reactions in liver mitochondria involves the cleavage of the terminal side-chain carbon atoms of cholesterol and their oxidation to carbon dioxide. By comparison with 26- or 2Z-unlabeled cholesterol, the origin of these carbon atoms in the side chain can be identified. In the light of later findings7 the formation of carbon dioxide from the terminal fragment is due to the oxidation of propionic acid which is cleaved from the side chain. This represents, therefore, a portion of nonsteroid metabolism in this overall reaction. Although the cholanoic acids formed in this reaction have been reported by Fredrickson and Ono22 to represent "unnatural" forms, a recent report differs.2s The yield of carbon dioxide from carbon atom 26 of cholesterol-26-14C is variable and is dependent on the preparation of mitochondria and their source. The yield may vary from a few percent to more than 30% in very favorable cases2 ,24 The yield from other possible steroid intermediates is given by Stevenson and Staple ~5 and also by Dean and Whitehouse.8 The overall reaction is given by Eq. (1).
Ho*
--~"
Substituted C*O2 + eholanicacids
(1)
D. S. Fredrickson and K. Ono, Biochim. Biophys. Acta 22, 183 (1965). :3K. A. Mitropoulos and N. D. Myant, Biochem. J. 99, 51P (1967). ~4D. Kritchevsky, in "Metabolism of Lipids as Related to Atherosclerosis" (F. A. Kummerow, ed.), pp. 106-128. Thomas, Springfield, Illinois, 1965. BE. Stevenson and E. Staple, Arch. Biochem. Biophys. 97, 485 (1962).
570
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
Procedure. White, male, Wistar strain rats weighing 150-160 g are sacrificed by decapitation, the livers are excised rapidly and transferred to a beaker containing 10T sucrose (w/v) at 0 °. The livers are then minced with a scissors and homogenized in cold aqueous sucrose solution (10~'o) using 3 volumes of sucrose solution per volume of minced liver. The homogenizer is a loose-fitting (1.5 mm clearance) glass PotterElvehjem homogenizer. The homogenate is then centrifuged at 600 g for 10 minutes for removal of unbroken cells, nuclei, and cellular debris. The resultant, supernatant portion from the previous centrifugation is then centrifuged in an ultracentrifuge (Spinco Model L preparative ultracentrifuge) for 12 minutes at 8500 g to separate the mitochondrial fraction. The mitochondrial suspension obtained from the first centrifugation is resuspended in l0 T aqueous sucrose solution and recentrifuged at 8500 g for 12 minutes to wash the mitochondria and remove adhering mierosomes. The supernatant fraction from this second centrifugation is discarded. The mitoehondria are resuspended in 10% sucrose solution so that a concentration of mitoehondria equivalent to 1 g of original wet weight of liver tissue is obtained. Dispersions of cholesterol-26-~4C are most conveniently and consistently prepared by dissolving the cholesterol (usually 0.05 rag) in a small amount of methanol containing 1.0 to 4.0 mg Tween 20 (Atlas Powder Co.) and removal of the methanol in a stream of nitrogen at 40-50 °. When the methanol has been entirely removed, the still warm solution of cholesterol in Tween 20 is diluted with the desired quantity of buffer solution at room temperature. This procedure yields a visually transparent dispersion of cholesterol which is consistently reproducible. Other methods of dispersion have been used. These include serum albumin stabilized emulsions (cf. Anfinsen and Homing 1) and lipoprotein solutions, but the Tween 20 dispersion has been found to be reliably consistent, provided however that quantities of Tween 20 are held sufficiently low so as to have no deleterious effect on the enzymes involved. Boiled soluble factor (SF), which is added to the incubation, is deproteinized, heat-stable fraction obtained by heating for 5 minutes at 90 ° the supernatant fraction from the first ultracentrifugation at 8500 g mentioned above. After heating, the solution containing particulate coagulum is filtered or centrifuged at low speed and the resultant supernatant fraction is used in the incubations. A typical incubation mixture contains 1 ml of mitochondrial suspension (equivalent to 1 g wet weight of liver) ; 1 ml of a solution containing ATP (25 rag) and AMP (8 rag) ; NAD ÷ (5 mg) ; reduced glutathione (15
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
571
mg), sodium citrate monohydrate (30 rag), M g ( N 0 ~ ) ' 6 H20 (10 rag), potassium penicillin G (2000 units), and streptomycin sulfate (1 mg); 5 ml of cholesterol-26-14C dispersion in 0.25 M tris(hydroxymethyl)aminomethane HC1, pH 8.5, and 5 ml of boiled supernatant fluid prepared as described above. The incubations are carried out in 125-ml stoppered Erlenmeyer flasks provided with a center well which contains 2.0 ml of 2.5 N sodium hydroxide solution to trap the carbon dioxide evolved during the incubation. The flasks are shaken at 37 ° in air for suitable periods of time (up to 18 hours). After the incubation periods are completed, 2.5 ml of 25% aqueous triehloroacetic acid are added to each flask, and the flasks are promptly restoppered and shaken for 3 hours to displace 14C02 from the incubation medium. The pH after addition of trichloroaeetic acid should not be more than 2.5. This point should be tested and additional acid be added if necessary. The acidified incubation mixture is shaken, then the contents of the center well are removed with a pipette and added to 2.5 ml of 2 N NH~C1 solution and 1 ml of 1.5 M BaC12 solution. The precipitated BaC03 is filtered, washed carefully, dried, weighed, and then counted, either directly in a planchet G-M counter, or dispersed in a thixotropie gel -"6 containing appropriate phosphors and counted in a liquid scintillation counter. B. Formation of 5fl-Cholestane-3~,7a,12a,26-tetraol-25,27-~4C from 5fi-Cholestane-3~,7a,12a-triol-25,27-14C Principle. This reaction involves the enzymatic hydroxylation of 5flcholestane-3a,7%12~-triol. Both Danielsson 6 and Suld et al. 7 have demonstrated that this hydroxylation can take place in mouse or rat liver mitochondria. Yields of 10-35% have been obtained for the conversion of triol to tetraol using this preparation. The mechanism and stereochemistry of this reaction are yet to be elucidated. It is indicated in Eq. (2) :
OH
(2) H
H
~ B. L. Funt and A. Hetherington, Science 125, 986 (1957).
572
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
Procedure. Washed mitochondria from white, male, Wistar strain rats (150-160 g) are obtained as mentioned in Section II,A. A dispersion of 0.05 mg substrate, 5fl-cholestane-3a,Ta,12a-triol-26,27-~4C, is prepared in 5 ml of 0.25 M tris (hydroxymethyl)aminomethane buffer, pH 8.5, with 4 mg Tween 20 as described for cholesterol-26-14C in Section II,A. The dispersion is cooled to room temperature and to it is added 1 ml of a solution containing ATP (25 rag), GSH (15 mg), MgC12.6 H20 (8 mg), trisodium citrate dihydrate (22 rag). This mixture is added to each incubation flask. A suspension of washed mitochondria in 10% sucrose solution equivalent to 4 g of liver tissue (wet weight) along with 105,000 g supernatant fluid (obtained from the same liver homogenate above) equivalent to 1.5 g liver tissue (wet weight) is added next to each incubation flask. The final volume is made up, as necessary, with 10% sucrose solution, to 12.5 ml. The incubations are conducted aerobically at 37 ° for 2.5 hours. At the end of this period, the incubation is terminated by the addition of 40 m! of 95% ethanol. The precipitated proteins and other insoluble substances are removed by eentrifugation, and the supernatant ethanolic solution is evaporated to dryness in vacuo at 25-30 °. The residue, suspended in water (3-4 ml) is acidified with 10 N H~S04 to pH 1-2, mixed with Celite 535 (Johns-Manville Corp.) which has been previously purified by treatment with water, methanol, and ether and then air dried. The residueCelite mixture (2 g of Celite per milliliter of residue suspension)is packed firmly into a 2 em diameter column and eluted with 150 ml of ether. (For isolation of crystalline tetraol the incubation size is increased by 10-fold over that given above and the ether eluates from twenty of these large-scale incubations are combined for paper chromatography.) The ether eluate is evaporated and this residue is then chromatographed on paper using 70% aqueous acetic acid as the stationary phase and isopropyl ether-heptane (60:40) as described by SjSvalF 7 and Suld et al. 7 Whatman No. 1 paper is used, and the chromatography carried out by the descending technique for periods of 3-10 hours. After development, the paper is dried and may be preliminarily scanned in a suitable scanning radioactivity-detection device or the tetraol spot may be visualized by other techniques. The radioactive peak corresponding to the 5fl-cholestane-3a,7a,12a,26tetraol spot is marked on the paper and cut out, and the material is eluted from the paper with methanol. The tetraol may be further purified by the use of reversed-phase column chromatographyY s Hydrophobic Hyflo Supercel (Johns-Manville Corp.) is prepared by treatment with
~J. SjSvall, Acta Chem. Seand. 6, 1552 (1952). ~S. BergstrSm, R. J. Brldgwater, and U. Gloor, Acta Chem. Scand. 11, 836 (1957).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
573
dimethyldichlorosilane as described by BergstrSm and SjSvall. 29 The stationary phase is ehloroform-heptane (90:10). Four milliliters of this solvent mixture is added to 4.5 g of treated Hyflo Supercel. The moving phase is 50% aqueous methanol. The tetraol is usually eluted by 50-75 ml of the moving phase. Crystalline tetraol, m.p. 203-204 ° is obtained. C. Formation of 3a,7a,12a-Trihydroxy-Sfl-Cholestan-26-oic Acid25,27-14C from 5fl-Cholestane-3a,7a,12a,25-tetraol-26,27-14C Principle. These enzymatic reactions involve the sequence, in which 5fi-cholestane-3a,Ta,12a,26-tetraol is converted to 3a,7a,12a-trihydroxy5fl-eholestan-26-oie acid. Yields of 20-30% of the acid from enzymatic oxidation of the tetraol by the preparation given here have been reported. A further elaboration of these reactions is given in Section II,D. The reaction is described by Eq. (3).
HO" ~ ( ~ H
'OH
H
Procedure. The complete system as described in procedure of Section II,B is used here. In addition, NAD ÷ (5 mg) is added to each incubation mixture. In place of the substrate added above (Section II,B), 0.5 mg of 5fl-eholestane-3a,7a,12a,26-tetraol-26,27-1~C is used. The dispersion in Tween 20 is prepared as previously described. The incubation is carried out at 37 ° , in air, for 1.5 hours and terminated by addition of 90% ethanol as previously described. The centrifuged ethanolic supernatant is evaporated in vacuo and mixed with Celite 535 as described in procedure of Section II,B. The ether eluate from this chromatography is evaporated and the residue is subjected to paper chromatography, as described in Section II,B, for 10 hours. The 3a,7a,12atrihydroxy-5fl-cholestan-26-oie acid-26,27-14C peak is eluted from the paper with methanol and subjected to partition column chromatography according to the procedure of Mosbach, Zomzely, and Kendall. 1~ Celite 545, 4 g, is treated with 5 ml of 70% aqueous acetic acid and packed in a column (1.0 cm X 18 cm). One hundred milliliters of petroleum ether (b.p. 30-60 °) is passed through the column. The sample, dis~S. BergstrSm and J. SjSvall. Acta Chem. Scand. 5~ 1267 (1950).
574
[26]
ENZYME SYSTEMS: BILE ACID FORMATION
solved in two drops of glacial acetic acid mixed with 0.2 g Celite 545, is placed on top of the colunm. The column is eluted with 45 ml of petroleum ether and then with 150 ml petroleum ether-isopropyl ether (60:40) mixture. The 3~,7~,12a-trihydroxy-5fl-cholestan-26-oic acid-26,27-1~C can be obtained by evaporation of the solvent of elu~ion. Crystalline acid, m.p. 172-173 ° can be obtained from ether solution. D. Formation of 3~,7~,12~-Trihydroxy-5fi-Cholestan-26-oic Acid-SH and 3a, Ta,12a-Trihydroxy-5fl-cholestan-26-al-3H from 5fl-Cholestane-3a,7a,12a,26-tetraol-~H Principle. The enzyme preparation, which is soluble and partially purified as described, has two component activities: (1) a 26-hydroxyl dehydrogenase which produces the 26-aldehyde, and (2) an aldehyde oxidase which yields the 26-acid. NAD ÷ is the hydrogen receptor in both these steps although another report indicates a utilization of N A D P in an analogous oxidation, s Conversions of tetraol to aldehyde in 45% yield and of aldehyde to acid in 5-20% yield have been obtained with the preparation given here. The latter yield of 20% was obtained after addition of ammonium sulfate to the enzyme preparation. The crude system before precipitation with (NH4)2S04 forms the acid from the tetraol in 50% yield. These two activities have been observed in the same preparation. After further purification only the first, or alcohol dehydrogenase, activity is observed. Whether both activities are present in the same enzyme or whether each is present in a separate enzyme remains to be determined. The reactions are indicated in Eq. (4).
HO
HO~~I~IV~'OH
I
~
~0
NAD
HO~
H
~
1~ H
"OH (4)
HO~ ~ ' / J ~ / " H
"OH
[26]
BILE &CIDS: SIDE-CHAIN DEGRA.DATION
575
Procedure. Rat livers (30 g) from male, Wistar strain rats, 150-160 g weight, are homogenized in 10% sucrose solution (75 ml) and then centrifuged at 500 g for 12 minutes. The supernatant layer after removal of nuclei is centrifuged at 105,000 g for 1 hour. (:NH4)2S04 is added to the I05,000 g supernatant fraction. The precipitate obtained at between 40 and 65% of the saturation concentration of (:NH4)2S04 at pH 7.4 is collected after this mixture has been held at 4 ° for 1/2 hour, then centrifuged at 8500 g for 20 minutes. The precipitate (400 mg protein) is dissolved in 20 ml of 0.02 M Tris buffer (pH 8.5). The protein solution is kept at 5960 ° for 10 minutes, then cooled rapidly to 4 ° and centrifuged at 8500 g for 10 minutes. This heat treatment serves to inactivate the 3-hydroxysteroid dehydrogenase, which interferes with the reaction observed. The precipitate from this treatment is removed and discarded after centrifugation at 8500 g for 10 minutes. The supernatant fraction is treated again with sufficient (NH4)2S0~ to attain 40-65% of saturation concentration. The precipitate (120 mg of protein) obtained in this case is dissolved in 0.02 M Tris buffer, pH 8.5 (30 ml) and dialyzed against the same buffer, overnight, at 4 ° . The resultant dialyzed preparation is the enzyme preparation used in these incubations. The incubation is carried out with a volume of solution equivalent to 1.2 mg of protein, 0.5 mg substrate, 5fl-cholestane-3%7%12%26-tetraol26-SH in 0.01 ml of methanol, NAD ÷ (2 mg), 0.25 M Tris buffer, pH 9.5, to give a final volume of 3 ml. The resultant mixture is incubated, in air, at 37 ° for ~/2 hour. The incubation is terminated by heating on a water bath for 5 minutes. The incubation mixture is then acidified with dilute HC1 and extracted with ether. The resultant extract is chromatographed on thin-layer plates coated with silica gel G using ethyl acetate-acetone (70:30) as developing solvent. The three compounds are separated quite well: the most polar spot is 3a,Ta,12~-trihydroxy-5fi-cholestan-26-oic acid-~H, R~ = 0.04; the intermediate spot corresponds to 5fl-cholestane-3%Ta,12a,26-tetraol-~H, RI = 0.31; and the least polar spot is 3%7a,12a-trihydroxy-5fl-cholestan-26al-3H, R I = 0.54. The aldehyde may be converted to the corresponding acid by an aqueous ethanolic Ag20 suspension, and the acid identified by comparison with the authentic compound. The spots may also be located by radioactive scanning of the plate, followed by elution of the spot with methanol and determining its radioactivity in a liquid scintillation counter. This enzyme preparation produces aldehyde predominantly. If a cruder preparation is used 9 the acid is practically the only product observed. The dehydrogenase preparation described here may also be used with N A D H in a reverse conversion of the aldehyde to tetraol. 1°
576
ENZYME
[26]
SYSTEMS: BILE ACID F O R M A T I O N
E. Formation of Propionic Acid-l,3-1~C from
5fl-Cholestane-3a,7a,12a-triol-26,27-'4C Principle. The enzymatic transformations carried out here involve the formation of propionic acid-l,3-14C and cho]ic acid from 5fl-cholestane-
3a,7a,12a-triol-26,27-:*C. This involves the formation of the 3a,Ta,12a,26-
HO~ ~ ' ~ 1 ~ H
H
~o ~
~o
"OH
~o ~
.~o
oH(scorn
HO ~ ~
Iv H
HO ~ ~
"OH
~o ~
~H
I~ H
~o
tI
"OH
~o
~o ~C~oH~Co~
tt
c~
~_C I "OH(SCoA) "-" \CH.
c "o~(scoA) ~
H O ~ ~ H
C'-oH(sco~
HO"
~/I H
~/ "OH
0
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
577
tetraol, 3a,7a,12a-trihydroxy-26-aldehyde, and the 3a,7a,12a-triol-26-oic acid. This latter acid is believed to be cleaved by a series of reactions, closely resembling the fl-oxidation of fatty acids, to yield propionic acid and cholic acid. This reaction has been observed to occur in a mitochondrial fraction of liver homogenate to which a supernatant fraction, centrifuged at 105,000 g for 1 hour, has been added. Yields of propionic acid of 20-34% have been obtained with the preparation given here. The postulated reaction sequence is outlined in Eq. (5). Procedure. The incubation mixture and additions are the same as for the formation of 5fl-cholestane-3a,7a,12a,26-tetraol (see procedure of Section II,C) except for the addition of 1.5 mg of potassium propionate to each flask containing 12.5 ml of incubation mixture. The incubation is carried out aerobically, with mechanical shaking at 37 ° for 1.5 hours. The reaction is terminated by heating for 1-2 minutes on a boiling water bath. The resultant, precipitated proteins are removed by centrifugation and the precipitate is washed thoroughly. The supernatant fluid and washings are treated with 2 mg of potassium propionate and 4 N H2SO4 to yield a pH of 1-2. The solution is steam-distilled until 150-200 ml of distillate is collected. The steam distillate is neutralized with 0.I N NaOH solution, using phenolphthalein as indicator. An aliquot of the neutralized steam-distillate may be evaporated on a planchet and the radioactivity measured by use of a suitable G-M counter. (Frecautions to avoid the loss of radioactive propionic acid when evaporating the propionate solution should be taken by keeping the pH of the evaporating solution at 10 or above.) The remainder of the steam distillate can be ehromatographed, as below, to isolate pure propionic acid for further G-M or liquid scintillation counting and derivative formation. The neutralized steam distillate is evaporated to dryness in vacuo and then chromatographed on a Celite 535 column according to the procedure of Swim and Utter? ° The stationary phase contains 7.5 ml of 0.2 N H2SO4 mixed with 15 g of Celite 535; the moving phase contains chloroform saturated with 0.2 N H~SO4. The eluate is collected in 10-ml portions to which 5 ml of C02-free water is added. The acidity is then titrated with 0.01 N NaOH solution using phenol red as indicator. Aliquots of these fractions can be taken for further counting and the neutralized purified propionic acid can be used also for further solid derivative formation. F. Formation of Propionyl-l,3-14C-CoA from 5fl-Cholestane-3a,7a,12 a,26-tetraol-26,27 -~C Principle. The formation of propionyl-l,3-~4C-CoA from 5fi-cholestane-3a,Ta,12a,26-tetraol-26,27-~4C is observed in a microsomal fraction It. E. Swim and M. F. Utter, see Vol. IV, p. 584.
578
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
from a rat liver homogenate to which is added a supernatant fraction from this same liver homogenate, spun at 105,000 g for 1 hour. Twenty to thirty percent of the theoretical yield of propionyl-CoA frmn the cleavage of the side chain of the tetraol has been obtained with this preparation. This reaction sequence is included in Eq. (5). Procedure. The microsomes as separated by differential centrifugation of the 8500 g supernatant fluid at 105,000 g for 1 hour are used for this incubation. The microsomes equivalent to 1.25 g of rat liver (wet weight) are used for this incubation. The 105,000 g-1 hour supernatant fluid equivalent to 0.5 g of rat liver (wet weight) is also added to the above microsomal preparation. Other rat liver and homogenization specifications are as previously described in procedure of Section II,A. To microsomes and supernatant fluid in a 50-ml flask is added a dispersion of 0.05 mg of 5fl-cholestane-3a,7a,12a,26-tetraol-26,27-~4C prepared with 1.5 mg Tween 20 as previously described (Section II,A) in 1.5 ml of 0.25 M phosphate buffer, pH 8.0, containing potassium propionate (0.5 mg), ATP (7.3 mg), NAD ÷ (1.3 mg), GSH (3.7 rag), nicotinamide (8.8 rag), MgC12.6 H20 (2.0 rag), trisodium citrate dihydrate (5.5 rag) and Na4t)207 (11.8 rag). The total volume is 3.5 ml. The mixture is allowed to incubate at 37 ° for 10 minutes, at which time 5 mg of unlabeled, freshly prepared propionyl-CoA is added to each flask. The mixtures are allowed to incubate at 37 ° for 10 minutes longer for a total incubation time of 20 minutes. The incubation mixture can then be worked up for isolation of propionyl hydroxamate or for isolation of propionyl-CoA directly. The isolation of propionyl hydroxamate is by the method of Stadtman and Barker21 After the incubation is terminated, the pH is adjusted to 5.5-6.0 with 1 N HC1. Four milliliters of hydroxylamine reagent, prepared by mixing equal volumes of 3.5 N N a 0 H solution and 28% hydroxylamine hydrochloride solution, is added to each flask. After 10 minutes at room temperature, 100 ml ethanol is added and the resultant precipitate is removed by centrifugation. The supernatant solution is evaporated in vacuo to dryness and the dry residue is extracted with three 2-ml portions of absolute ethanol and the combined ethanolic extracts are evaporated in a stream of nitrogen to a volume of 0.3 ml. The precipitated solid is removed by eentrifugation and the supernatant solution is subiected to descending paper chromatography using the octanol-formic acid-water (75:25:75) system 27 for 5-6 hours. The developed chromatograms are dried overnight at room temperature; a narrow lengthwise strip is cut out of the chromatogram and dipped into an acid-ferric chloride ~ E. R. S t a d t m a n and H. A. Barker, ]. Biol. Chem. 184, 769 (1950).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
579
ethanolic solution prepared as described by Thompson2 ~ The area of the chromatogram corresponding to the colored area of the indicating strip is then eluted with ethanol and the eluate is evaporated under nitrogen to a volume of 0.3 ml. This solution is in turn rechromatographed on paper using the solvent system n-butanol-acetic acid-water (4:1:5). Again, a narrow lengthwise strip is cut out and stained with ethanolic ferric chloride solution. The area of the ehromatogram corresponding to the stained area of the strip is eluted with ethanol, and the propionyl hydroxamate remaining after evaporation of the ethanol is assayed for radioactivity. For isolation of propionyl-CoA a modification of the procedure of Lynen, Reichert, and Rueff~ is used. After the incubation is terminated by treatment with 1.2 g of (NH4)~S04, the mixture is extracted with three 1-g portions of phenol. The combined phenol extracts are mixed with an equal volume of ether, and the resultant solution is extracted with five 1-ml portions of water. The aqueous extracts are combined and reextraeted with 2 ml of ether. The aqueous layer is evaporated in vacuo (20 °) to a volume of 0.3 ml. This solution is SlJotted on paper (Whatman No. 1) and ehromatographed using i~opropanol-pyridine-water (1:I:1) as solvent24 After development the paper is air-dried. A lengthwise strip is cut from the chromatogram and dipped into nitroprusside reagent ~ made as follows: Dissolve 1.5 g of sodium nitroprusside in 5 ml of 2 N H~SO~. Add 95 ml of absolute methanol and then 10 ml of 28% NH~OH solution. Filter off the white precipitate and use the clear orange filtrate. Then spray the strip with methanolic NaOH solution. The propionyl-CoA area ean then be visualized. Cut out the area on the original chromatogram corresponding to this spot and then spray with hydroxylamine reagent solution (previously described) ; the resulting propionyl hydroxamate is eluted from the paper with ethanol and can be ehromatographed by either of the systems mentioned in the preceding paragraph. The eluted propionyl hydroxamate can also be assayed for radioactivity. G. Formation of 3a,7a,12a,24~-Tetrahydroxy-Sfl-cholestan-26-oic Acid-~H from 3a,7a,12a-Trihydroxy-5fi-cholestan-26-oic Acid-~H Principle. The reaction involved here is the enzymatic formation of the fi-hydroxy acid from 3~,7%12~-trihydroxy-5fl-cholestan-26-oic acid in a rat liver mitochondrial preparation to which 105,000 g supernatant
3~A. R. Thompson, Australian J. Sci. Res. Ser. B4, 180 (1951). ~F. Lynen, E. Reichert, and L. Rueff, Ann. Chem. 574, 1 (1951). ~ E. R. Stadtman, see Vol. III, p. 940. 3~G. Toennies and J. J. Kolb, Anal. Chem. 23, 823 (1951).
580
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
fraction has been added. The intermediate formation of the 24,25-dehydro acid and subsequent hydration of this dehydro acid by an enzyme similar to the enoyl-CoA hydratase of fatty acid metabolism are probable steps in the conversion. The resultant 24-hydroxy (or p-hydroxy) acid is produced by a stereo-specific enzymatic reaction, the actual stereochemistry of which has not yet been established. It appears likely, by analogy with the report of Masui and Staple 3~ on the separation of the stereoisomers of 5fl-cholestane-3a,7a,12a,24-tetraols, that the configuration of the hydroxyl group at carbon atom 24 is alpha. A yield of 30% of the tetrahydroxy acid has been obtained with the preparation given here. This sequence is included in Eq. (5). Procedure. A 40% rat liver homogenate from male Wistar rats (150160 g) is prepared by homogenizing the tissue in 10% sucrose solution with a loose-fitting (1.5 mm clearance) Potter-Elvehjem glass homogenizer. The homogenate is centrifuged at 600 g for 10 minutes to remove unbroken cells, nuclei, and large cell debris. The mitochondrial portion is isolated by centrifugation of this supernatant fraction at 8500 g for 12 minutes. The supernatant layer from this centrifugation is centrifuged at 105,000 g for 1 hour to give the supernatant fraction used below. The mitochondria are suspended in 10% sucrose to yield a suspension equivalent to 4 g of rat liver (wet weight) in 1 ml. In addition, 2 ml of 105,000 g supernatant fraction prepared as above, is also taken for each incubation. The incubation medium contains in addition to the above mitoehondrial and supernatant fractions, 3a,7a,12a-trihydroxy-Sfl-cholestane-26oic acid-~H (0.10 rag, 5 X 104 cpm) in 0.2 ml of methanol; ATP (25 rag), MgC12 (8 rag), FAD (1 mg), and CoA (2 rag) ; 0.25M Tris buffer (5 ml), pH 8.5. The volume is made up to 10 ml with 10% sucrose solution. The incubations are carried out in air at 37 ° for 1 hour. At the end of this time the incubation is terminated by the addition of ethanol (20 ml). The precipitated protein is removed by centrifugation and then the precipitate is washed with ethanol. The combined supernatant fractions are evaporated in vacuo until most of the ethanol is removed. The aqueous residue is then acidified to pH 1 with diluted HC1 and extracted with ethyl acetate. The ethyl acetate solution is then spotted on a thin-layer plate, 0.2 mm silica gel G. A spot of authentic 3a,Ta,12a,24~-tetrahydroxy5fl-cholestan-26-oic acid is also placed on the same plate. The ehromatogram can be developed with various solvent systems: (1) benzeneisopropanol-acetic acid (30:10:1 by volume) ; (2) benzene-isopropanolacetic acid (55:25:2 by volume); (3) ether-petroleum ether-methanolacetic acid (70:30:8:1 by volume); (4) ethyl acetate-acetone (70:30 by 3~T. Masui and E. Staple, Steroids 9, 443 (1967).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
581
volume). The steroid compounds on the plates can be detected in various ways (see the article by Lisboa~7). Iodine vapor has the advantage that the spots can be marked after they become visible and then the iodine may be allowed to disappear on standing for some time in air. The marked spots may be scraped off the plate and eluted with methanol and the eluted steroid assayed for radioactivity. Alternatively, the plates, appropriately marked with the positions of substrate and product standard, may be scanned in a radiochromatogram scanner. The enzyme has been found to be stereospeeific and if synthetic 3a,7a,12a,24~-tetrahydroxy-5flcholestan-26-oic acid is used as standard, radioactivity will be detected in only one (the more polar) of the two spots obtained, using solvent system (4) above. FI. Formation of Cholic Acid-24-14C from 3a,7a,12a,24~-tetrahydroxy5fl-cholestan-26-oic Acid-24-~4C
Principle. The conversion of the 24-hydroxy acid to cholic acid demonstrated here occurs in the supernatant fraction of rat liver homogenate (spun at 105,000 g for 1 hour). It appears to involve a 24-hydroxyacyl-CoA dehydrogenase and thiolase. The reaction in this crude system has been found to require NAD ÷ or NADP ÷, CoA, and ATP. These requirements are consistent with those required for similar conversions of fatty acids. A yield of 15% of cholic acid from the tetrahydroxy acid has been obtained with this preparation. The reaction sequence is shown in the last part of Eq. (5). Procedure. Two milliliters of 105,000 g supernatant fluid obtained from the 40% rat liver homogenate in 10% sucrose obtained in procedure of Section II,G above is used as the enzyme preparation. To this is added 3a,7a,12a,24~-tetrahydroxy-5fi-cholestan-26-oie acid-24-14C (0.01 mg, 1 X 104 cpm) prepared as described in Section I,E in 0.2 ml of methanol, ATP (28 rag), MgC12 (8 rag), NAD ÷ (5 mg), CoA (2 mg), GSH (15 rag), 0.25 M Tris buffer, pH 9.0 (5 ml). Final volume is made up to 10 ml with ]0% sucrose solution. The incubation is carried out in air at 37 ° for 1 hour. At the end of this time ethanol (20 ml) is added to terminate the incubation. The precipitated protein is removed by centrifugation, washed with ethanol, and recentrifuged. The combined extracts are evaporated in vacuo to remove the ethanol. The aqueous residue is acidified with diluted HC1 to pH 1, and the steroid acids are extracted with ethyl acetate. This extract is spotted on thin-layer plates (0.2 ram, silica gel G), and the chromatogram is developed with the solvent system, benzene-isopropyl alcohol-acetic acid, 30: I0:I. Authentic cbolie acid, Rr ~ 0.26, and 3a,7a,12a,24~-tetraa, B. P. Lisboa, this volume [1].
582
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
hydroxy-Sfl-cholestan-26-oic acid, R / = 0.54 are spotted on the plate as standards. The plate is exposed to iodine vapor. The area of the plate containing the radioactive sample and which is opposite the standard spot is scraped from the plate and eluted with methanol; the eluate is counted in a suitable counter to determine its radioactivity. Conversely, the plate may be scanned for radioactivity in a suitable thin-layer plate radiochromatogram scanner, and the radioactivity opposite the standard spots can be measured from the curve.
ENZYME SYSTEMS: BILE ACID FORMATION
[26]
The 3a-hydroxysteroid dehydrogenase fraction obtained from the Sephadex G-75 column, 0.05-0.1 ml, is diluted to 4 ml with 0.1 M TrisHC1 buffer, pH 7.4; 1 mg of N A D P H is added and the mixture is incubated for 20 minutes with 50 ~g of labeled 7a-hydroxy-5fl-cholestan3-one, added to the incubation mixture dissolved in 0.1 ml of acetone. The chloroform extract of the incubation mixture is chromatographed with benzene-ethyl acetate 1:1 as solvent (cf. Table I).
[ 2 6 ] E n z y m a t i c D e g r a d a t i o n of t h e C h o l e s t a n e S i d e C h a i n in t h e B i o s y n t h e s i s o f B i l e A c i d s B y EZRA STAPLE~
Work on the cleavage of the side chain of cholestanes to form bile acids may be considered to begin with the work of Anfinsen and Horning. 1~ These investigators found that a homogenate of mouse liver and, later, mitoehondrial fractions of this homogenate were capable of yielding radioactive carbon dioxide from incubation with cholesterol-26-14C. Later this enzymatic reaction was studied with rat liver mitochondria by Whitehouse, Staple, and Gurin, 2,~ and with liver mitoehondria of other species. 4 This reaction has been used to study the influence of various factors on the cleavage process. Following suggestions first made by BergstrSm, 5 5fi-cholestane-3~,7~, 12~-triol and various derivatives were studied. This work led to the discovery by Danielsson 6 tha~ this triol can be enzymatically hydroxylated in the 26-position by liver mitoehondrial preparations. This was confirmed by Suld, S~aple, and Gurin. 7 Further, both of these groups of investigators found that 3%7a,12a-trihydroxy-Sfl-cholestan-26-oie acid is also produced. Suld et al. 7 also found that production of the 26-oic acid from the 26-hydroxylated compound is dependent on the presence of NAD ÷. A partial replacement of NAD ÷ by N A D P + in a crude system is claimed by Deceased. 1~C. B. Aniinsen and M. G. Homing, J. Am. Chem. Soc. 75, 1511 (1963). *M. W. Whitehouse, E. Staple, and S. Gurin, J. Biol. Chem. 234, 276 (1959). M. W. Whitehouse, E. Staple, and S. Gurin, J. Biol. Chem. 236, 68 (1961). *M. W. Whitehouse, M. C. Cottrell, T. Briggs, and E. Staple, Arch. Biochem. Biophys. 9@, 305 (1962). 5S. Bergstr6m, Ciba Found. Syrup. Biosyn. Terpenes Sterols. Little, Brown, Boston, Massachusetts, 1959. *H. Danielsson, Acta Chem. ~cand. 14, 348 (1960). It. M. Suld, E. Staple, and S. Gurin, J. Biol. Chem. 237, 338 (1962).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
563
Dean and Whitehouses for this reaction. This dehydrogenase system has been purified and studied by Herman and Staple9 and further by Masui, Herman, and Staple 1° to indicate that this is a two-step dehydrogenation reaction, in which the enzyme for the first step can be separated by enzymatic purification techniques. They also have confirmed the obligatory requirement for NAD ÷ as hydrogen acceptor. As an extension of their work with 3~,7~,12~-trihydroxy-5fi-cholestane-26-oic acid, Suld et al. ~ found that the side chain was cleaved between carbon atoms 24 and 25 of the side chain to yield cholic acid and propionie acid (propionyl-CoA). These authors proposed a thiolytie cleavage similar to that which occurs in the fl-oxidation of fatty acids. Consistent with this hypothesis, Masui and Staple 11 have recently reported the formation of 3%7~,12a,24~-tetrahydroxy-5fl-cholestan-26-oic acid from 3~,7%12~-trihydroxy-5fi-cholestan-26-oic acid and the conversion of the former compound to cholic acid. In sum, the enzyme reactions for the cleavage of the cholestane side chain to form the cholanic acids appears to parallel those for u-oxidation of hydrocarbons and fl-oxidation of fatty acids. The enzyme reactions described below give evidence for some of these steps. I. Preparation of Substrate Materials A. Cholesterol-26-14C This material can be synthesized according to methods previously reported 12-14 and as given below. It may also be obtained commercially from several sources. Seventy-two milligrams (0.5 millimole) of methyl iodide-~4C (2 mC per millimole) is diluted with 4.5 millimoles of unlabeled methyl iodide and added to 146 mg of magnesium (for Grignard reactions) (6 millimoles) in 4 ml of anhydrous ether. The reaction is initiated by cautious, gentle warming and proceeds for 1 hour. Then 715 mg (1.67 millimoles) of 3fl-acetoxy-27-norcholest-5-en-25-one in 5 ml of dry benzene is added slowly with stirring and the mixture is refluxed for 3 hours. It is then allowed to stand overnight. The complex is decomposed by addition of 2 ml of ice water and 4 ml of 50% acetic acid. The mixture is then steams p. D. G. Dean and M. W. Whitehouse, Biochem J. 98, 410 (1966). 9R. H. Herman and E. Staple, Federation Proc. ~4, 661 (1965). 1oT. Masui, R. Herman, and E. Staple, Biochim. Biophys. Acta 117, 260 (1966). ,1T. Masui and E. Staple, J. Biol. Chem. 9.41, 3889 (1966). J~A. I. Ryer, W. H. Gebert, and N. M. Murrilt, J. Am. Chem. Soc. 7g, 4247 (1950). ~ W. G. Dauben and H. L. Bradlow, J. Am. Chem. Soc. 72~ 4248 (1950). 1~M. G. ttorning, D. S. Fredrickson, and C. B. Anfinsen, Arch. Biochem. Biophys. 71, 266 (1957).
564
ENZYME SYSTEMS: BILE A.CID FOHMA.TION
[26]
distilled until no more water-insoluble distillate is obtained. The reaction mixture is extracted with ether, and the ether extract is washed with NaHCOa solution. The washings are made alkaline and extracted with ether, and the two ether extracts are combined and dried over MgS04. The M g S Q is then filtered off and the ether is evaporated, leaving 743 mg of residue of crude cholest-5-ene-3fl,25-diol-26-14C. The residue is dissolved in 5 ml of anhydrous pyridine, and 1.2 ml (12.7 millimole) of acetic anhydride is added. The mixture is refluxed for 7 minutes and then added dropwise to 50 ml of water. The water is extracted with ether, the ether is washed with 2 N H2S04, and the acid wash is extracted again with ether. The combined ether extracts are dried over MgS0~, after addition of a little dry NaHCO~. After evaporation of the ether, 806 mg of yellowish residue of crude 3fl-acetoxy-eholest-5en-25-ol-26-14C remains. The crude acetate is dissolved in 20 ml of anhydrous pyridine and 0.85 ml (9.3 millimole) of freshly distilled phosphorus oxychloride is added. A red color, deepening to black, forms on refluxing for 30 minutes. The solution is poured into 50 ml of ice water, diluted with more water, and extracted with ether. The ether extracts are combined and washed with 2 N H2S04 to remove the pyridine. The extract is then washed with 5% NaHC03 solution. The acidified washings are extracted with ether, and the ether extract is washed with NaHCOs solution. The ether extracts are combined and evaporated. The orange residue of mixed 26-1~C labeled 24and 25-dehydrocholesteryl acetates weighs 665 rag. It is hydrolyzed by refluxing for 1 hour in 10 ml of 2 N ethanolic KOH. This solution is diluted with 50 ml of water and extracted several times with ether. The ether extracts are washed repeatedly with water and the water washes are combined and extracted once with ether. The combined ether extracts are dried overnight over MgSO~ and filtered into a 100-ml hydrogenation flask. The solvent is evaporated, leaving 580 mg of orange residue containing the crude 24- and 25-dehydrocholesterols. The mixture is dissolved in 20 ml of absolute ethanol and hydrogenated at atmospheric pressure using 50 mg of 10% palladium-on-charcoal as catalyst. The process is stopped after uptake of 23 ml (1.0 millimole) of hydrogen per millimole of steroid. The hydrogenation requires about 30 minutes. The solution of crude cholesterol is filtered, the solvent is evaporated, and the residue is chromatographed on a 30-g column of neutral alumina (Woelm, grade 1). The crude cholesterol is placed on the column, a minimal quantity of 45% (v/v) ether in petroleum ether (b.p. 30-60 °) being used; the column is then eluted with 2.3 liters of the same solvent followed by 1.5 liters of 65% ether in petroleum ether. The fractions con-
[26]
BILE ACIDS: SIDE-CHAIN" DEGRADATION
565
raining the principal radioactive peak are combined and evaporated. Approximately 509 mg of crude cholesterol-26-14C is obtained at this point. The partially purified product is dissolved in 60 ml of 90% aqueous ethanol and to this solution is added a solution containing 1.7 g of digitonin in 200 ml of 90% aqueous ethanol. After it has stood for 1 hour at room temperature, the digitonide is kept at 7 ° for 60 hours. It is collected by eentrifugation and washed with cold 90% aqueous ethanol, cold ether-acetone (2:1), and cold ether, then dried. The cholesterol is recovered by decomposition of the digitonide dissolved in 12 ml of pyridine. Ether, 120 ml, is added and the digitonin is allowed to precipitate several hours at room temperature. The precipitate is centrifuged and washed with ether, and the ether solutions are combined. The precipitated digitonin is redissolved in 85% aqueous ethanol and the above precipitation procedure is repeated; additional cholesterol is obtained in the ether solution. On evaporation of the ether solution, 326 mg (0.84 millimole) cholesterol-26-~4C, specific activity 0.57 /~C/mg is obtained. The overall yield from 3fl-acetoxy-27-norcholest-5-en-25-one is 51%. On the basis of methyl iodide-l~C the yield is 35%. B. 5fl-Cholestane-3a,7a,12a-triol-26,27-14C
The synthesis is carried out, as previously reported, 1~ as follows: Triformylcholyl chloride is prepared from cholic acid by the method of Cortese and Bauman26 Using benzene-petroleum ether (b.p. 60-80 °) as solvent, triformylcholic acid can be crystallized with m.p. 209-211 °. The acid is converted to the acid chloride by treatment with oxalyl chloride. 2-Propanol-l,3-1~C (specific activity 1.0 mC per millimole) is diluted 20-fold with unlabeled 2-propanol and then converted to 2-bromopropane-l,3-14C by reaction with phosphorus tribromide. A Grignard reagent is prepared from 2.40 g of 2-bromopropane-l,3-~4C (50 ~C per millimole) and 600 mg of magnesium (for Grignard reactions) in 25 ml of anhydrous ether. The solution is cooled in an ice bath and stirred. To this 7.5 g of powdered anhydrous cadmium bromide (dried for 3 hours at 120 °) is added. The preparation is stirred for 30 minutes, the ice bath is removed, and a solution of 1.50 g of triformylcholyl chloride in 8 ml of benzene is added. Stirring is continued for 30 minutes after this addition, and the reaction mixture is heated to reflux for 1 hour then allowed to stand overnight at room temperature. Ice water and then 3 N HC1 solution are added to the reaction mixture. Sufficient acid should be ~'E. Staple and M. W. Whitehouse, J. Org. Chem. 9.4, 433 (1959). ~eF. Cortese and L. Bauman, J. Am. Chem. Soc. 57, 1393 (1935).
566
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
added to dissolve the initial precipitate. The benzenc cther layer is separated and washed with water until the washings are neutral. The washed solvent layer is then evaporated to dryness and the residual gum is further dried in vacuo. The residue is heated for 1 hour with 10 ml of 5% methanolic KOH. This reaction mixture is then diluted with 100 ml of water and extracted with ether. The ether extract is evaporated to dryness and the residue of crude 24-keto-5fl-cholestane-3a,7a,12a4riol26,27-14C is subjected to silicic acid column chromatography. The column, 2.0 cm in diameter, contains 50 g of silicic acid (Merck) and is prepared with benzene. It is eluted with benzene first and then with ether-benzene (1:2). The ketone is eluted by the latter solvent. After evaporation of this eluate, 698 mg of 24-keto-5fl-cholestane-3~,7~,12~-triol-26,27-~4C, m.p. 143-145 °, is obtained. This product may be recrystallized from acetone if desired. To 500 mg of 24-keto-5fl-cholestane-3a,7a,12a-triol-26,27-~C dissolved in 1 ml of ethanol is added 1 ml of hydrazine hydrate (99%). The mixture is swirled for a few minutes to give a homogeneous solution. Ten milliliters of triethylene glycol and 1 g of KOH are added to this solution, and the mixture is heated under reflux for 30 minutes. Then the reflux condenser is removed and the mixture is heated at 180-200 ° for 2 hours. At the end of this heating period, the reaction mixture is allowed to cool in a stream of nitrogen gas and it is then poured into 50 ml of water. The precipitated compound is filtered, washed with water, and dried in vacuo. This crude product is crystallized from acetone; 269 mg 5fl-cholestane3~,7a,12a-triol-26,27-1~C, m.p. 184--185°, is obtained.
C. 3a,7a,12a-Trihydroxy-Sfl-cholestan-26-oic Acid The D-isomer of this acid is best obtained from the bile of Alligator mississippiensis. Bile from other crocodilia may be used in place of A. mississippiensis bile, which is now difficult to obtain. It is necessary, however, thoroughly to identify and characterize the isolated trihydroxycoprostanic acid in each case. There may be some racemization about carbon atom 25 during the hydrolysis procedure although no definitive information on this has yet been obtained. It may also be obtained synthetically, 1~ but with great difficulty and in low yield. A procedure for isolation of the acid from A. mississippiensis bile is given below. Bile from A. mississippiensis is mixed with 2-3 volumes of ethanol to precipitate the mucin and other proteins. The precipitate is removed by centrifugation and again washed with ethanol. The combined alcoholic solutions are evaporated to dryness giving x grams of gummy residual ~7R. J. Bridgwater, Biochem. J. 64, 593 (1956).
[26]
ruby. ACIDS: SIDE-CHAIN DEGRADATION
567
"crude bile salts." This residue is taken up in water, the pH adjusted to 8-9 and 0.5 x g of decolorizing charcoal is added. The mixture is evaporated on a steam bath with occasional stirring. The residue is transferred to a Soxhlet extractor, and extraction with absolute ethanol is continued until fresh extracts are colorless. Evaporation of the alcohol gives a gummy, occasionally brittle, residue, "purified bile salts." In the case where the quantity of "crude bile salts" is small (less than 100 rag) the charcoal purification step can be omitted. Hydrolysis of the bile salts is carried out in 2.5 N NaOH in an autoclave at a pressure of 20 psi (125 ° for 16-18 hours). The alkaline hydrolyzate is diluted with water, saturated with NaC1, and acidified to pH 1 with HC1. The precipitated bile acids are extracted with three portions of ethyl acetate, and the combined extract is washed three times with water. The combined water washings are extracted once with ethyl acetate. The ethyl acetate extracts are combined and evaporated to dryness, and the crude bile acids so obtained are further purified by partition column chromatography. The chromatographic column system used by Mosbach, Zomzely, and Kendall is is recommended. Four milliliters of 70% acetic acid on 5 g of Celite 545 (Johns-Manville Corp.) is the stationary phase, and petroleum ether (b.p. 30-60 °) is the first moving phase. The packed column measures 0.9 cm X 18 cm. The sample to be separated is dissolved in 3--4 drops of glacial acetic acid, mixed thoroughly with 0.2-0.3 g of Celite, slurried in petroleum ether, and added to the column. Elution is effected with (1) 45 ml of petroleum ether, (2) 90 ml of 40% isopropy] ether in petroleum ether, and (3) 100 ml of 60% isopropyl ether in petroleum ether. The 3%7~,12~-trihydroxy-5fl-cholestan-26-oic acid is eluted with solvent mixture (2). Material of m.p. 176-178 ° is readily obtained by isolation from the bile of A. mississippiensis. The acid obtained in this way can be tritiated by the Wilzbach method? 9 Several commercial laboratories offer this tritiation service. The acid which has been through this procedure must be carefully purified by several chromatographic systems. D. 5fl-Cholestane-3a,Ta,12a,26-tetraol This compound is prepared synthetically in high yield from 3a,7a,12atrihydroxy-5fi-eholestan-26-oic acid by reduction with lithium aluminum hydride as follows: The methyl ester of the trihydroxy acid is prepared by treatment of 1SE. H. Mosbach, C. Zomzely, and F. E. Kendall, Arch. Biochem. Biophys. 48, 95 (1954). ~9K. E. Wilzbaeh, J. Am. Chem. Soc. 79, 1013 (1957).
568
ENZYME SYSTEMS: :BILE ACID FORMATION
[26]
an ether solution of the acid with an excess of freshly generated diazomethane solution in ether. The mixture is allowed to stand until all the diazomethane decomposes, then the ether is evaporated. The ester in the residue, after removal of the ether, represents a yield of 99-[-% of the ester. The ester (50 rag) is dissolved in 10 ml of anhydrous benzene and added slowly to the lithium aluminum hydride (25 mg) dissolved in 15 ml of ether. After the addition, and after the reaction has subsided, the mixture is heated under reflux for 30 minutes. The reaction mixture is then cooled, and ethanol (95%) is then added cautiously, dropwise, to decompose the excess of the hydride. When no further gas evolution occurs after addition of ethanol, the mixture is added to cold water (50 ml) and sufficient 2 N H~S04 is added to dissolve the precipitate. The e~herbenzene layer is washed with water until neutral. The original water layer is extracted with ether, and this ether extract is washed with water until neutral. The ether extract is combined with the original etherbenzene layer, and the solvents are removed by evaporation. The residue consists of practically pure 5fl-cholestane-3a,7a,12a,26-tetraol, in a yield of 98-]-%. After crystallization from methanol, the tetraol melts at 203-204%
E. 3a,7a,12a,24~-Tetrahydroxy-5fl-cholestan-26-olc Acid-24-14C This compound is prepared from cholic acid-24-14C by a modified Reformatsky reaction 2° as follows. 3a,7a,12a-Trihydroxycholan-24-al-14C (0.10 mC, 95 rag) is synthesized from cholic acid-24-1~C (activity 0.50 mC) (500 mg) by the method of Yashima21 This method utilizes the oxidation of homocholane-3a,7a,12a,24,25-pentaol by lead tetraacetate to give cholanyl aldehyde directly. The aldehyde-14C is dissolved in 10 ml of dry toluene and 3 ml of ethyl dl-2-bromopropionate containing granulated zinc and a small amount of both iodine and powdered copper as catalysts. The reaction mixture is then poured into a mixture of crushed ice and excess 10% H2SO,. This is then extracted with ethyl acetate. The ethyl acetate extract is washed with dilute Na2S~03 to remove the iodine and then with water. The solvent layer is then evaporated to dryness. The residue is saponified with 5% of methanolic KOH solution for 1 hour on a water bath. After removal of the methanol, the residual aqueous solution is extracted with ethyl acetate to remove 3a,Ta,12a-trihydroxycholan24-al-24-~4C. This alkaline solution is then acidified with 10% HCI solution and extracted with ethyl acetate. The extract is washed with water ,oy. Inai, Y. Tanaka, S. Betsukl, and T. Kazuno, I. Biochem. (Tokyo) 56, 591 (1964). ~IH. Yashima, J. Biochem. (Tokyo) 54, 47 (1963).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
569
to neutrality and then the solvent is evaporated. A reaction product (activity 0.03 mC) (31 rag) is obtained. On further purification by thin layer chromatography on silica gel G (Brinkmann Instruments) using benzenc-isopropanol-acctic acid 30:I0:1 as developing solvent, a mixture of the isomers of 3~,7~,]2~,24~-tetrahydroxy-5fl-cholestan-26-oic acid-24-14C, with R / s of 0.31 and 0.34 and m.p. 98-102 ° is obtained. For further separation of the stereoisomers of this acid, see Masui and Staple21 II. Enzymatic Transformation A. Formation of 1~CO~ from Cholest-5-en-3fl-ol-26-14C Principle. This sequence of enzyme reactions in liver mitochondria involves the cleavage of the terminal side-chain carbon atoms of cholesterol and their oxidation to carbon dioxide. By comparison with 26- or 2Z-unlabeled cholesterol, the origin of these carbon atoms in the side chain can be identified. In the light of later findings7 the formation of carbon dioxide from the terminal fragment is due to the oxidation of propionic acid which is cleaved from the side chain. This represents, therefore, a portion of nonsteroid metabolism in this overall reaction. Although the cholanoic acids formed in this reaction have been reported by Fredrickson and Ono22 to represent "unnatural" forms, a recent report differs.2s The yield of carbon dioxide from carbon atom 26 of cholesterol-26-14C is variable and is dependent on the preparation of mitochondria and their source. The yield may vary from a few percent to more than 30% in very favorable cases2 ,24 The yield from other possible steroid intermediates is given by Stevenson and Staple ~5 and also by Dean and Whitehouse.8 The overall reaction is given by Eq. (1).
Ho*
--~"
Substituted C*O2 + eholanicacids
(1)
D. S. Fredrickson and K. Ono, Biochim. Biophys. Acta 22, 183 (1965). :3K. A. Mitropoulos and N. D. Myant, Biochem. J. 99, 51P (1967). ~4D. Kritchevsky, in "Metabolism of Lipids as Related to Atherosclerosis" (F. A. Kummerow, ed.), pp. 106-128. Thomas, Springfield, Illinois, 1965. BE. Stevenson and E. Staple, Arch. Biochem. Biophys. 97, 485 (1962).
570
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
Procedure. White, male, Wistar strain rats weighing 150-160 g are sacrificed by decapitation, the livers are excised rapidly and transferred to a beaker containing 10T sucrose (w/v) at 0 °. The livers are then minced with a scissors and homogenized in cold aqueous sucrose solution (10~'o) using 3 volumes of sucrose solution per volume of minced liver. The homogenizer is a loose-fitting (1.5 mm clearance) glass PotterElvehjem homogenizer. The homogenate is then centrifuged at 600 g for 10 minutes for removal of unbroken cells, nuclei, and cellular debris. The resultant, supernatant portion from the previous centrifugation is then centrifuged in an ultracentrifuge (Spinco Model L preparative ultracentrifuge) for 12 minutes at 8500 g to separate the mitochondrial fraction. The mitochondrial suspension obtained from the first centrifugation is resuspended in l0 T aqueous sucrose solution and recentrifuged at 8500 g for 12 minutes to wash the mitochondria and remove adhering mierosomes. The supernatant fraction from this second centrifugation is discarded. The mitoehondria are resuspended in 10% sucrose solution so that a concentration of mitoehondria equivalent to 1 g of original wet weight of liver tissue is obtained. Dispersions of cholesterol-26-~4C are most conveniently and consistently prepared by dissolving the cholesterol (usually 0.05 rag) in a small amount of methanol containing 1.0 to 4.0 mg Tween 20 (Atlas Powder Co.) and removal of the methanol in a stream of nitrogen at 40-50 °. When the methanol has been entirely removed, the still warm solution of cholesterol in Tween 20 is diluted with the desired quantity of buffer solution at room temperature. This procedure yields a visually transparent dispersion of cholesterol which is consistently reproducible. Other methods of dispersion have been used. These include serum albumin stabilized emulsions (cf. Anfinsen and Homing 1) and lipoprotein solutions, but the Tween 20 dispersion has been found to be reliably consistent, provided however that quantities of Tween 20 are held sufficiently low so as to have no deleterious effect on the enzymes involved. Boiled soluble factor (SF), which is added to the incubation, is deproteinized, heat-stable fraction obtained by heating for 5 minutes at 90 ° the supernatant fraction from the first ultracentrifugation at 8500 g mentioned above. After heating, the solution containing particulate coagulum is filtered or centrifuged at low speed and the resultant supernatant fraction is used in the incubations. A typical incubation mixture contains 1 ml of mitochondrial suspension (equivalent to 1 g wet weight of liver) ; 1 ml of a solution containing ATP (25 rag) and AMP (8 rag) ; NAD ÷ (5 mg) ; reduced glutathione (15
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
571
mg), sodium citrate monohydrate (30 rag), M g ( N 0 ~ ) ' 6 H20 (10 rag), potassium penicillin G (2000 units), and streptomycin sulfate (1 mg); 5 ml of cholesterol-26-14C dispersion in 0.25 M tris(hydroxymethyl)aminomethane HC1, pH 8.5, and 5 ml of boiled supernatant fluid prepared as described above. The incubations are carried out in 125-ml stoppered Erlenmeyer flasks provided with a center well which contains 2.0 ml of 2.5 N sodium hydroxide solution to trap the carbon dioxide evolved during the incubation. The flasks are shaken at 37 ° in air for suitable periods of time (up to 18 hours). After the incubation periods are completed, 2.5 ml of 25% aqueous triehloroacetic acid are added to each flask, and the flasks are promptly restoppered and shaken for 3 hours to displace 14C02 from the incubation medium. The pH after addition of trichloroaeetic acid should not be more than 2.5. This point should be tested and additional acid be added if necessary. The acidified incubation mixture is shaken, then the contents of the center well are removed with a pipette and added to 2.5 ml of 2 N NH~C1 solution and 1 ml of 1.5 M BaC12 solution. The precipitated BaC03 is filtered, washed carefully, dried, weighed, and then counted, either directly in a planchet G-M counter, or dispersed in a thixotropie gel -"6 containing appropriate phosphors and counted in a liquid scintillation counter. B. Formation of 5fl-Cholestane-3~,7a,12a,26-tetraol-25,27-~4C from 5fi-Cholestane-3~,7a,12a-triol-25,27-14C Principle. This reaction involves the enzymatic hydroxylation of 5flcholestane-3a,7%12~-triol. Both Danielsson 6 and Suld et al. 7 have demonstrated that this hydroxylation can take place in mouse or rat liver mitochondria. Yields of 10-35% have been obtained for the conversion of triol to tetraol using this preparation. The mechanism and stereochemistry of this reaction are yet to be elucidated. It is indicated in Eq. (2) :
OH
(2) H
H
~ B. L. Funt and A. Hetherington, Science 125, 986 (1957).
572
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
Procedure. Washed mitochondria from white, male, Wistar strain rats (150-160 g) are obtained as mentioned in Section II,A. A dispersion of 0.05 mg substrate, 5fl-cholestane-3a,Ta,12a-triol-26,27-~4C, is prepared in 5 ml of 0.25 M tris (hydroxymethyl)aminomethane buffer, pH 8.5, with 4 mg Tween 20 as described for cholesterol-26-14C in Section II,A. The dispersion is cooled to room temperature and to it is added 1 ml of a solution containing ATP (25 rag), GSH (15 mg), MgC12.6 H20 (8 mg), trisodium citrate dihydrate (22 rag). This mixture is added to each incubation flask. A suspension of washed mitochondria in 10% sucrose solution equivalent to 4 g of liver tissue (wet weight) along with 105,000 g supernatant fluid (obtained from the same liver homogenate above) equivalent to 1.5 g liver tissue (wet weight) is added next to each incubation flask. The final volume is made up, as necessary, with 10% sucrose solution, to 12.5 ml. The incubations are conducted aerobically at 37 ° for 2.5 hours. At the end of this period, the incubation is terminated by the addition of 40 m! of 95% ethanol. The precipitated proteins and other insoluble substances are removed by eentrifugation, and the supernatant ethanolic solution is evaporated to dryness in vacuo at 25-30 °. The residue, suspended in water (3-4 ml) is acidified with 10 N H~S04 to pH 1-2, mixed with Celite 535 (Johns-Manville Corp.) which has been previously purified by treatment with water, methanol, and ether and then air dried. The residueCelite mixture (2 g of Celite per milliliter of residue suspension)is packed firmly into a 2 em diameter column and eluted with 150 ml of ether. (For isolation of crystalline tetraol the incubation size is increased by 10-fold over that given above and the ether eluates from twenty of these large-scale incubations are combined for paper chromatography.) The ether eluate is evaporated and this residue is then chromatographed on paper using 70% aqueous acetic acid as the stationary phase and isopropyl ether-heptane (60:40) as described by SjSvalF 7 and Suld et al. 7 Whatman No. 1 paper is used, and the chromatography carried out by the descending technique for periods of 3-10 hours. After development, the paper is dried and may be preliminarily scanned in a suitable scanning radioactivity-detection device or the tetraol spot may be visualized by other techniques. The radioactive peak corresponding to the 5fl-cholestane-3a,7a,12a,26tetraol spot is marked on the paper and cut out, and the material is eluted from the paper with methanol. The tetraol may be further purified by the use of reversed-phase column chromatographyY s Hydrophobic Hyflo Supercel (Johns-Manville Corp.) is prepared by treatment with
~J. SjSvall, Acta Chem. Seand. 6, 1552 (1952). ~S. BergstrSm, R. J. Brldgwater, and U. Gloor, Acta Chem. Scand. 11, 836 (1957).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
573
dimethyldichlorosilane as described by BergstrSm and SjSvall. 29 The stationary phase is ehloroform-heptane (90:10). Four milliliters of this solvent mixture is added to 4.5 g of treated Hyflo Supercel. The moving phase is 50% aqueous methanol. The tetraol is usually eluted by 50-75 ml of the moving phase. Crystalline tetraol, m.p. 203-204 ° is obtained. C. Formation of 3a,7a,12a-Trihydroxy-Sfl-Cholestan-26-oic Acid25,27-14C from 5fl-Cholestane-3a,7a,12a,25-tetraol-26,27-14C Principle. These enzymatic reactions involve the sequence, in which 5fi-cholestane-3a,Ta,12a,26-tetraol is converted to 3a,7a,12a-trihydroxy5fl-eholestan-26-oie acid. Yields of 20-30% of the acid from enzymatic oxidation of the tetraol by the preparation given here have been reported. A further elaboration of these reactions is given in Section II,D. The reaction is described by Eq. (3).
HO" ~ ( ~ H
'OH
H
Procedure. The complete system as described in procedure of Section II,B is used here. In addition, NAD ÷ (5 mg) is added to each incubation mixture. In place of the substrate added above (Section II,B), 0.5 mg of 5fl-eholestane-3a,7a,12a,26-tetraol-26,27-1~C is used. The dispersion in Tween 20 is prepared as previously described. The incubation is carried out at 37 ° , in air, for 1.5 hours and terminated by addition of 90% ethanol as previously described. The centrifuged ethanolic supernatant is evaporated in vacuo and mixed with Celite 535 as described in procedure of Section II,B. The ether eluate from this chromatography is evaporated and the residue is subjected to paper chromatography, as described in Section II,B, for 10 hours. The 3a,7a,12atrihydroxy-5fl-cholestan-26-oie acid-26,27-14C peak is eluted from the paper with methanol and subjected to partition column chromatography according to the procedure of Mosbach, Zomzely, and Kendall. 1~ Celite 545, 4 g, is treated with 5 ml of 70% aqueous acetic acid and packed in a column (1.0 cm X 18 cm). One hundred milliliters of petroleum ether (b.p. 30-60 °) is passed through the column. The sample, dis~S. BergstrSm and J. SjSvall. Acta Chem. Scand. 5~ 1267 (1950).
574
[26]
ENZYME SYSTEMS: BILE ACID FORMATION
solved in two drops of glacial acetic acid mixed with 0.2 g Celite 545, is placed on top of the colunm. The column is eluted with 45 ml of petroleum ether and then with 150 ml petroleum ether-isopropyl ether (60:40) mixture. The 3~,7~,12a-trihydroxy-5fl-cholestan-26-oic acid-26,27-1~C can be obtained by evaporation of the solvent of elu~ion. Crystalline acid, m.p. 172-173 ° can be obtained from ether solution. D. Formation of 3~,7~,12~-Trihydroxy-5fi-Cholestan-26-oic Acid-SH and 3a, Ta,12a-Trihydroxy-5fl-cholestan-26-al-3H from 5fl-Cholestane-3a,7a,12a,26-tetraol-~H Principle. The enzyme preparation, which is soluble and partially purified as described, has two component activities: (1) a 26-hydroxyl dehydrogenase which produces the 26-aldehyde, and (2) an aldehyde oxidase which yields the 26-acid. NAD ÷ is the hydrogen receptor in both these steps although another report indicates a utilization of N A D P in an analogous oxidation, s Conversions of tetraol to aldehyde in 45% yield and of aldehyde to acid in 5-20% yield have been obtained with the preparation given here. The latter yield of 20% was obtained after addition of ammonium sulfate to the enzyme preparation. The crude system before precipitation with (NH4)2S04 forms the acid from the tetraol in 50% yield. These two activities have been observed in the same preparation. After further purification only the first, or alcohol dehydrogenase, activity is observed. Whether both activities are present in the same enzyme or whether each is present in a separate enzyme remains to be determined. The reactions are indicated in Eq. (4).
HO
HO~~I~IV~'OH
I
~
~0
NAD
HO~
H
~
1~ H
"OH (4)
HO~ ~ ' / J ~ / " H
"OH
[26]
BILE &CIDS: SIDE-CHAIN DEGRA.DATION
575
Procedure. Rat livers (30 g) from male, Wistar strain rats, 150-160 g weight, are homogenized in 10% sucrose solution (75 ml) and then centrifuged at 500 g for 12 minutes. The supernatant layer after removal of nuclei is centrifuged at 105,000 g for 1 hour. (:NH4)2S04 is added to the I05,000 g supernatant fraction. The precipitate obtained at between 40 and 65% of the saturation concentration of (:NH4)2S04 at pH 7.4 is collected after this mixture has been held at 4 ° for 1/2 hour, then centrifuged at 8500 g for 20 minutes. The precipitate (400 mg protein) is dissolved in 20 ml of 0.02 M Tris buffer (pH 8.5). The protein solution is kept at 5960 ° for 10 minutes, then cooled rapidly to 4 ° and centrifuged at 8500 g for 10 minutes. This heat treatment serves to inactivate the 3-hydroxysteroid dehydrogenase, which interferes with the reaction observed. The precipitate from this treatment is removed and discarded after centrifugation at 8500 g for 10 minutes. The supernatant fraction is treated again with sufficient (NH4)2S0~ to attain 40-65% of saturation concentration. The precipitate (120 mg of protein) obtained in this case is dissolved in 0.02 M Tris buffer, pH 8.5 (30 ml) and dialyzed against the same buffer, overnight, at 4 ° . The resultant dialyzed preparation is the enzyme preparation used in these incubations. The incubation is carried out with a volume of solution equivalent to 1.2 mg of protein, 0.5 mg substrate, 5fl-cholestane-3%7%12%26-tetraol26-SH in 0.01 ml of methanol, NAD ÷ (2 mg), 0.25 M Tris buffer, pH 9.5, to give a final volume of 3 ml. The resultant mixture is incubated, in air, at 37 ° for ~/2 hour. The incubation is terminated by heating on a water bath for 5 minutes. The incubation mixture is then acidified with dilute HC1 and extracted with ether. The resultant extract is chromatographed on thin-layer plates coated with silica gel G using ethyl acetate-acetone (70:30) as developing solvent. The three compounds are separated quite well: the most polar spot is 3a,Ta,12~-trihydroxy-5fi-cholestan-26-oic acid-~H, R~ = 0.04; the intermediate spot corresponds to 5fl-cholestane-3%Ta,12a,26-tetraol-~H, RI = 0.31; and the least polar spot is 3%7a,12a-trihydroxy-5fl-cholestan-26al-3H, R I = 0.54. The aldehyde may be converted to the corresponding acid by an aqueous ethanolic Ag20 suspension, and the acid identified by comparison with the authentic compound. The spots may also be located by radioactive scanning of the plate, followed by elution of the spot with methanol and determining its radioactivity in a liquid scintillation counter. This enzyme preparation produces aldehyde predominantly. If a cruder preparation is used 9 the acid is practically the only product observed. The dehydrogenase preparation described here may also be used with N A D H in a reverse conversion of the aldehyde to tetraol. 1°
576
ENZYME
[26]
SYSTEMS: BILE ACID F O R M A T I O N
E. Formation of Propionic Acid-l,3-1~C from
5fl-Cholestane-3a,7a,12a-triol-26,27-'4C Principle. The enzymatic transformations carried out here involve the formation of propionic acid-l,3-14C and cho]ic acid from 5fl-cholestane-
3a,7a,12a-triol-26,27-:*C. This involves the formation of the 3a,Ta,12a,26-
HO~ ~ ' ~ 1 ~ H
H
~o ~
~o
"OH
~o ~
.~o
oH(scorn
HO ~ ~
Iv H
HO ~ ~
"OH
~o ~
~H
I~ H
~o
tI
"OH
~o
~o ~C~oH~Co~
tt
c~
~_C I "OH(SCoA) "-" \CH.
c "o~(scoA) ~
H O ~ ~ H
C'-oH(sco~
HO"
~/I H
~/ "OH
0
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
577
tetraol, 3a,7a,12a-trihydroxy-26-aldehyde, and the 3a,7a,12a-triol-26-oic acid. This latter acid is believed to be cleaved by a series of reactions, closely resembling the fl-oxidation of fatty acids, to yield propionic acid and cholic acid. This reaction has been observed to occur in a mitochondrial fraction of liver homogenate to which a supernatant fraction, centrifuged at 105,000 g for 1 hour, has been added. Yields of propionic acid of 20-34% have been obtained with the preparation given here. The postulated reaction sequence is outlined in Eq. (5). Procedure. The incubation mixture and additions are the same as for the formation of 5fl-cholestane-3a,7a,12a,26-tetraol (see procedure of Section II,C) except for the addition of 1.5 mg of potassium propionate to each flask containing 12.5 ml of incubation mixture. The incubation is carried out aerobically, with mechanical shaking at 37 ° for 1.5 hours. The reaction is terminated by heating for 1-2 minutes on a boiling water bath. The resultant, precipitated proteins are removed by centrifugation and the precipitate is washed thoroughly. The supernatant fluid and washings are treated with 2 mg of potassium propionate and 4 N H2SO4 to yield a pH of 1-2. The solution is steam-distilled until 150-200 ml of distillate is collected. The steam distillate is neutralized with 0.I N NaOH solution, using phenolphthalein as indicator. An aliquot of the neutralized steam-distillate may be evaporated on a planchet and the radioactivity measured by use of a suitable G-M counter. (Frecautions to avoid the loss of radioactive propionic acid when evaporating the propionate solution should be taken by keeping the pH of the evaporating solution at 10 or above.) The remainder of the steam distillate can be ehromatographed, as below, to isolate pure propionic acid for further G-M or liquid scintillation counting and derivative formation. The neutralized steam distillate is evaporated to dryness in vacuo and then chromatographed on a Celite 535 column according to the procedure of Swim and Utter? ° The stationary phase contains 7.5 ml of 0.2 N H2SO4 mixed with 15 g of Celite 535; the moving phase contains chloroform saturated with 0.2 N H~SO4. The eluate is collected in 10-ml portions to which 5 ml of C02-free water is added. The acidity is then titrated with 0.01 N NaOH solution using phenol red as indicator. Aliquots of these fractions can be taken for further counting and the neutralized purified propionic acid can be used also for further solid derivative formation. F. Formation of Propionyl-l,3-14C-CoA from 5fl-Cholestane-3a,7a,12 a,26-tetraol-26,27 -~C Principle. The formation of propionyl-l,3-~4C-CoA from 5fi-cholestane-3a,Ta,12a,26-tetraol-26,27-~4C is observed in a microsomal fraction It. E. Swim and M. F. Utter, see Vol. IV, p. 584.
578
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
from a rat liver homogenate to which is added a supernatant fraction from this same liver homogenate, spun at 105,000 g for 1 hour. Twenty to thirty percent of the theoretical yield of propionyl-CoA frmn the cleavage of the side chain of the tetraol has been obtained with this preparation. This reaction sequence is included in Eq. (5). Procedure. The microsomes as separated by differential centrifugation of the 8500 g supernatant fluid at 105,000 g for 1 hour are used for this incubation. The microsomes equivalent to 1.25 g of rat liver (wet weight) are used for this incubation. The 105,000 g-1 hour supernatant fluid equivalent to 0.5 g of rat liver (wet weight) is also added to the above microsomal preparation. Other rat liver and homogenization specifications are as previously described in procedure of Section II,A. To microsomes and supernatant fluid in a 50-ml flask is added a dispersion of 0.05 mg of 5fl-cholestane-3a,7a,12a,26-tetraol-26,27-~4C prepared with 1.5 mg Tween 20 as previously described (Section II,A) in 1.5 ml of 0.25 M phosphate buffer, pH 8.0, containing potassium propionate (0.5 mg), ATP (7.3 mg), NAD ÷ (1.3 mg), GSH (3.7 rag), nicotinamide (8.8 rag), MgC12.6 H20 (2.0 rag), trisodium citrate dihydrate (5.5 rag) and Na4t)207 (11.8 rag). The total volume is 3.5 ml. The mixture is allowed to incubate at 37 ° for 10 minutes, at which time 5 mg of unlabeled, freshly prepared propionyl-CoA is added to each flask. The mixtures are allowed to incubate at 37 ° for 10 minutes longer for a total incubation time of 20 minutes. The incubation mixture can then be worked up for isolation of propionyl hydroxamate or for isolation of propionyl-CoA directly. The isolation of propionyl hydroxamate is by the method of Stadtman and Barker21 After the incubation is terminated, the pH is adjusted to 5.5-6.0 with 1 N HC1. Four milliliters of hydroxylamine reagent, prepared by mixing equal volumes of 3.5 N N a 0 H solution and 28% hydroxylamine hydrochloride solution, is added to each flask. After 10 minutes at room temperature, 100 ml ethanol is added and the resultant precipitate is removed by centrifugation. The supernatant solution is evaporated in vacuo to dryness and the dry residue is extracted with three 2-ml portions of absolute ethanol and the combined ethanolic extracts are evaporated in a stream of nitrogen to a volume of 0.3 ml. The precipitated solid is removed by eentrifugation and the supernatant solution is subiected to descending paper chromatography using the octanol-formic acid-water (75:25:75) system 27 for 5-6 hours. The developed chromatograms are dried overnight at room temperature; a narrow lengthwise strip is cut out of the chromatogram and dipped into an acid-ferric chloride ~ E. R. S t a d t m a n and H. A. Barker, ]. Biol. Chem. 184, 769 (1950).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
579
ethanolic solution prepared as described by Thompson2 ~ The area of the chromatogram corresponding to the colored area of the indicating strip is then eluted with ethanol and the eluate is evaporated under nitrogen to a volume of 0.3 ml. This solution is in turn rechromatographed on paper using the solvent system n-butanol-acetic acid-water (4:1:5). Again, a narrow lengthwise strip is cut out and stained with ethanolic ferric chloride solution. The area of the ehromatogram corresponding to the stained area of the strip is eluted with ethanol, and the propionyl hydroxamate remaining after evaporation of the ethanol is assayed for radioactivity. For isolation of propionyl-CoA a modification of the procedure of Lynen, Reichert, and Rueff~ is used. After the incubation is terminated by treatment with 1.2 g of (NH4)~S04, the mixture is extracted with three 1-g portions of phenol. The combined phenol extracts are mixed with an equal volume of ether, and the resultant solution is extracted with five 1-ml portions of water. The aqueous extracts are combined and reextraeted with 2 ml of ether. The aqueous layer is evaporated in vacuo (20 °) to a volume of 0.3 ml. This solution is SlJotted on paper (Whatman No. 1) and ehromatographed using i~opropanol-pyridine-water (1:I:1) as solvent24 After development the paper is air-dried. A lengthwise strip is cut from the chromatogram and dipped into nitroprusside reagent ~ made as follows: Dissolve 1.5 g of sodium nitroprusside in 5 ml of 2 N H~SO~. Add 95 ml of absolute methanol and then 10 ml of 28% NH~OH solution. Filter off the white precipitate and use the clear orange filtrate. Then spray the strip with methanolic NaOH solution. The propionyl-CoA area ean then be visualized. Cut out the area on the original chromatogram corresponding to this spot and then spray with hydroxylamine reagent solution (previously described) ; the resulting propionyl hydroxamate is eluted from the paper with ethanol and can be ehromatographed by either of the systems mentioned in the preceding paragraph. The eluted propionyl hydroxamate can also be assayed for radioactivity. G. Formation of 3a,7a,12a,24~-Tetrahydroxy-Sfl-cholestan-26-oic Acid-~H from 3a,7a,12a-Trihydroxy-5fi-cholestan-26-oic Acid-~H Principle. The reaction involved here is the enzymatic formation of the fi-hydroxy acid from 3~,7%12~-trihydroxy-5fl-cholestan-26-oic acid in a rat liver mitochondrial preparation to which 105,000 g supernatant
3~A. R. Thompson, Australian J. Sci. Res. Ser. B4, 180 (1951). ~F. Lynen, E. Reichert, and L. Rueff, Ann. Chem. 574, 1 (1951). ~ E. R. Stadtman, see Vol. III, p. 940. 3~G. Toennies and J. J. Kolb, Anal. Chem. 23, 823 (1951).
580
ENZYME SYSTEMS: BILE ACID FORMATION
[25]
fraction has been added. The intermediate formation of the 24,25-dehydro acid and subsequent hydration of this dehydro acid by an enzyme similar to the enoyl-CoA hydratase of fatty acid metabolism are probable steps in the conversion. The resultant 24-hydroxy (or p-hydroxy) acid is produced by a stereo-specific enzymatic reaction, the actual stereochemistry of which has not yet been established. It appears likely, by analogy with the report of Masui and Staple 3~ on the separation of the stereoisomers of 5fl-cholestane-3a,7a,12a,24-tetraols, that the configuration of the hydroxyl group at carbon atom 24 is alpha. A yield of 30% of the tetrahydroxy acid has been obtained with the preparation given here. This sequence is included in Eq. (5). Procedure. A 40% rat liver homogenate from male Wistar rats (150160 g) is prepared by homogenizing the tissue in 10% sucrose solution with a loose-fitting (1.5 mm clearance) Potter-Elvehjem glass homogenizer. The homogenate is centrifuged at 600 g for 10 minutes to remove unbroken cells, nuclei, and large cell debris. The mitochondrial portion is isolated by centrifugation of this supernatant fraction at 8500 g for 12 minutes. The supernatant layer from this centrifugation is centrifuged at 105,000 g for 1 hour to give the supernatant fraction used below. The mitochondria are suspended in 10% sucrose to yield a suspension equivalent to 4 g of rat liver (wet weight) in 1 ml. In addition, 2 ml of 105,000 g supernatant fraction prepared as above, is also taken for each incubation. The incubation medium contains in addition to the above mitoehondrial and supernatant fractions, 3a,7a,12a-trihydroxy-Sfl-cholestane-26oic acid-~H (0.10 rag, 5 X 104 cpm) in 0.2 ml of methanol; ATP (25 rag), MgC12 (8 rag), FAD (1 mg), and CoA (2 rag) ; 0.25M Tris buffer (5 ml), pH 8.5. The volume is made up to 10 ml with 10% sucrose solution. The incubations are carried out in air at 37 ° for 1 hour. At the end of this time the incubation is terminated by the addition of ethanol (20 ml). The precipitated protein is removed by centrifugation and then the precipitate is washed with ethanol. The combined supernatant fractions are evaporated in vacuo until most of the ethanol is removed. The aqueous residue is then acidified to pH 1 with diluted HC1 and extracted with ethyl acetate. The ethyl acetate solution is then spotted on a thin-layer plate, 0.2 mm silica gel G. A spot of authentic 3a,Ta,12a,24~-tetrahydroxy5fl-cholestan-26-oic acid is also placed on the same plate. The ehromatogram can be developed with various solvent systems: (1) benzeneisopropanol-acetic acid (30:10:1 by volume) ; (2) benzene-isopropanolacetic acid (55:25:2 by volume); (3) ether-petroleum ether-methanolacetic acid (70:30:8:1 by volume); (4) ethyl acetate-acetone (70:30 by 3~T. Masui and E. Staple, Steroids 9, 443 (1967).
[26]
BILE ACIDS: SIDE-CHAIN DEGRADATION
581
volume). The steroid compounds on the plates can be detected in various ways (see the article by Lisboa~7). Iodine vapor has the advantage that the spots can be marked after they become visible and then the iodine may be allowed to disappear on standing for some time in air. The marked spots may be scraped off the plate and eluted with methanol and the eluted steroid assayed for radioactivity. Alternatively, the plates, appropriately marked with the positions of substrate and product standard, may be scanned in a radiochromatogram scanner. The enzyme has been found to be stereospeeific and if synthetic 3a,7a,12a,24~-tetrahydroxy-5flcholestan-26-oic acid is used as standard, radioactivity will be detected in only one (the more polar) of the two spots obtained, using solvent system (4) above. FI. Formation of Cholic Acid-24-14C from 3a,7a,12a,24~-tetrahydroxy5fl-cholestan-26-oic Acid-24-~4C
Principle. The conversion of the 24-hydroxy acid to cholic acid demonstrated here occurs in the supernatant fraction of rat liver homogenate (spun at 105,000 g for 1 hour). It appears to involve a 24-hydroxyacyl-CoA dehydrogenase and thiolase. The reaction in this crude system has been found to require NAD ÷ or NADP ÷, CoA, and ATP. These requirements are consistent with those required for similar conversions of fatty acids. A yield of 15% of cholic acid from the tetrahydroxy acid has been obtained with this preparation. The reaction sequence is shown in the last part of Eq. (5). Procedure. Two milliliters of 105,000 g supernatant fluid obtained from the 40% rat liver homogenate in 10% sucrose obtained in procedure of Section II,G above is used as the enzyme preparation. To this is added 3a,7a,12a,24~-tetrahydroxy-5fi-cholestan-26-oie acid-24-14C (0.01 mg, 1 X 104 cpm) prepared as described in Section I,E in 0.2 ml of methanol, ATP (28 rag), MgC12 (8 rag), NAD ÷ (5 mg), CoA (2 mg), GSH (15 rag), 0.25 M Tris buffer, pH 9.0 (5 ml). Final volume is made up to 10 ml with ]0% sucrose solution. The incubation is carried out in air at 37 ° for 1 hour. At the end of this time ethanol (20 ml) is added to terminate the incubation. The precipitated protein is removed by centrifugation, washed with ethanol, and recentrifuged. The combined extracts are evaporated in vacuo to remove the ethanol. The aqueous residue is acidified with diluted HC1 to pH 1, and the steroid acids are extracted with ethyl acetate. This extract is spotted on thin-layer plates (0.2 ram, silica gel G), and the chromatogram is developed with the solvent system, benzene-isopropyl alcohol-acetic acid, 30: I0:I. Authentic cbolie acid, Rr ~ 0.26, and 3a,7a,12a,24~-tetraa, B. P. Lisboa, this volume [1].
582
ENZYME SYSTEMS: BILE ACID FORMATION
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hydroxy-Sfl-cholestan-26-oic acid, R / = 0.54 are spotted on the plate as standards. The plate is exposed to iodine vapor. The area of the plate containing the radioactive sample and which is opposite the standard spot is scraped from the plate and eluted with methanol; the eluate is counted in a suitable counter to determine its radioactivity. Conversely, the plate may be scanned for radioactivity in a suitable thin-layer plate radiochromatogram scanner, and the radioactivity opposite the standard spots can be measured from the curve.