JBC-1942-Bloch K-625-36-胆固醇合成代谢

ON THE UTILIZATION OF ACETIC ACID FOR CHOLESTEROL FORMATION* BY KONRAD BLOCH AND D. RITTENBERG (From the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York) (Received for publication, August 14, 1942) The information at present available as to the metabolism of cholesterol and its relationship to the other biologically important sterols is meager. Balance experiments (1) have demonstrated that cholesterol can be syn- thesized and destroyed in the animal but have given no convincing evidence as to the nature of specific precursors of cholesterol. In general, balance experiments fail to differentiate between two alterna- tives, direct utilization of the dietary component or stimulation of a specific process of metabolism. It cannot, for example, be decided whether the increase of cholesterol observed in some experiments after the feeding of fatty substances is the result of conversion of these substances to cholesterol or whether it merely reflects an increased metabolic activity of the or- ganism. An experiment (2) on the rate of formation of cholesterol, carried out with the aid of deuterium, indicated that about half of all the hydrogen atoms of the newly formed cholesterol was derived from the hydrogen of water; the other half must have originated from the carbon-bound hydrogen atoms of some dietary constituent. This evidence eliminated large mole- cules as immediate precursors, and suggested that “cholesterol . . . is formed by the coupling of smaller molecules, possibly those which have been postulated to be intermediates in the fat and carbohydrate metab- olism” (2). The recent observation (3) that the ingestion of deuterio acetic acid, CD&OOH, leads to the formation of deuterio cholesterol supports this view. On the other hand, deuterio cholesterol is not formed after a,& dideuterio propionic acid and deuterio succinic acid are fed. cr,P-Di- deuterio butyric acid and 0, y-dideuterio butyric acid were only slightly effective. The deuterium concentrations in the total cholesterol of the experimental animals after the feeding of deuterio acetate for 8 days arc given in Table I. Positive evidence for the utilization of acetate for choles- terol formation was found for mice, growing rats, and adult rats. In each case the concentration of deuterium in the cholesterol was over 3 times that in the body water. The figures in the seventh column give the proportion * This work was carried out bvith the aid of grants from the Rockefeller Foundat,ion and the Josiah Macy, Jr., Foundation. 625 at BEIJING UNIV LIBRARY, on June 12, 2012 w w w .jbc.org D ow nloaded from 626 ACETIC ACID IN CHOLESTEROL FORMATION of all the hydrogen in the cholesterol which was derived from the dietary acetate during the experimental period. These values must of necessity be small, if only for the reason that the rate of synthesis of cholesterol is slow. In considering possible steps in the transformation of acetate to sterol, a mechanism must be postulated in which the acetate molecules are reduc- tively coupled to form larger units. Intermediates containing only labile hydrogen, i.e. readily exchangeable hydrogen, can be ruled out. For example, a pathway via oxalacetic acid cannot be considered, as it would result in the elimination of deuterium by enolization. We have tested some compounds into which acetic acid is known to be converted in viva in order to determine whether any of these reactions is involved in sterol synthesis. TABLE I Feeding of Deuterio Acetic Acid Experiment Deuterio acetic acid Deuterium in Hydrogen in Animals used fed per day cholesterol D:uterium per 100 gm. body weight Sodium Is&ted ,,,“,“g’;& f&%?ds acetate Body water cholesterol m. atom per atom per atom per atom per cent erce5s cent ezce3s cent ezcess per cent cent ezcess A Adult mice 372 9.9 0.04 0.13 1.3 0.02 B Young rats 82 68.0 0.08 0.27 0.4 0.04 C Adult ” 97 27.6 0.05 0.21* 0.8 * In this experiment the cholesterol was converted to cholesteryl chloride, which was analyzed. Yeast dehydrogenases rapidly convert acetate to succinate (4). As the results obtained on feeding deuterio succinate were negative, the pathway from acetate to cholesterol does not go through succinic acid. A conceivable explanation for the failure of succinate to generate deuterio cholesterol might be that the succinic-fumaric-malic equilibrium causes a rapid loss of deuterium to the body fluids and therefore renders detection of cholesterol formation impossible. However, this cannot be the reason. Assuming that acetate were converted to succinate and that the latter lost deuterium by exchange, then deuterio acetate should not be more effective in forming deuterio cholesterol than deuterio succinate. Succinate does not represent an intermediate step. Little is known of acetate metabolism in normal animals. It has ap- parently been believed, but not proved, that acetic acid is directly burned to carbon dioxide and water at a rapid rate, Under certain condi- tions, such as in fasting animals (5) or in liver slices (6), formation of aceto- at BEIJING UNIV LIBRARY, on June 12, 2012 w w w .jbc.org D ow nloaded from K. BLOCH AND D. RITTENBERG 627 acetic acid and P-hydroxybutyric acid from acetic acid has been demon- strated and ascribed to condensation of acetic acid with either pyruvic acid (7) or another molecule of acetic acid (8). Morehouse (9) has isolated deuterio ,8-hydroxybutyric acid from the urine of fasting rats which had been given ~,y-dideuterio butyric acid, but found normal fi-hydroxybutyric acid after 01 ,/%dideuterio butyric acid was fed. This demonstrates that, while the CY- and P-hydrogen atoms are rapidly replaced, the y-hydrogen atoms are relatively stable. P,r-Di- deuterio butyric acid can be expected to give rise to deuterio acetoacetic acid in normal animals also. When its sodium salt was fed to rats, the isotopic content of the cholesterol was only slightly higher than that of the body fluids, in contrast to a comparable feeding of deuterio acetate, when the cholesterol formed contained 3 to 4 times more deuterium than the sur- rounding body water. In view of the known relationship of butyric, aceto- acetic, and P-hydroxybutyric acids these compounds may be ruled out as intermediates in sterol formation from acetic acid. In another experiment our animals received a corresponding amount of a! ,p-dideuterio butyric acid. This compound was expected to exchange its deuterium by enolization after being oxidized to acetoacetic acid, and the cholesterol isolated should not contain significant concentrations of deuterium. The concentration of deuterium in the isolated cholesterol was slightly higher than that of the body fluids. Proplonic acid was tested as a possible cholesterol precursor, because of its close biological relationship to pyruvic acid. Sodium cr,fi-dideuterio propionate was fed to rats as a source of deuterio pyruvic acid, but failed to produce a cholesterol with a significant deuterium content. If this fail- ure were due to removal of deuterium by enolization, the same loss of isotope should occur with acetate if it were converted to cholesterol via pyruvate. Our experimental results indicate that propionic acid and pre- sumably pyruvic acid are not intermediates in the synthesis of cholesterol from acetate. We have attempted, by chemical degradation of the deuterio cholesterol, to obtain some more detailed information on the mechanism of sterol syn- thesis. Unfortunately the yields of cholesterol fragments which could be obtained by oxidative breakdown of the available deuterio cholesterol sam- ples would be too small to permit deuterium analysis. The only reaction found to be suitable for our purpose was the thermal degradation of choles- teryl chloride described by Mauthner (10). When this compound is heated to about 300” in a nitogen atmosphere, hydrochloric acid is eliminated; at about 400” fission occurs between carbon atoms 17 and 20, yielding iso- octane, isooctene, and a high boiling oil to which the structure shown in the accompanying formula has been assigned by Bergmann and Bergmann (11). at BEIJING UNIV LIBRARY, on June 12, 2012 w w w .jbc.org D ow nloaded from 628 ACETIC ACID IN CHOLESTEROL FORMATION CJ% / ‘3% il-I /v By converting the deuterio cholesterol isolated from our animals into cholesteryl chloride and subjecting the latter to the thermal degradation, we have obtained two fractions, representing the side chain and the sterol nucleus respectively. The concentrations of deuterium in the isooctane- isooctene mixture and in the hydrocarbon ClgH30, which may be regarded as the primary degradation products, are in good agreement with that in the cholesteryl chloride. The occurrence of side reactions leading to a loss of fractions with high deuterium concentrations is therefore unlikely. Small errors in the calculation may arise from two sources. The hydro- chloric acid split off in the initial phase of the degradation was not analyzed. However, unless the deuterium concentration at carbon atom 2 or 4 of the sterol nucleus was very much higher than that at other carbon atoms the analytical figure for the nucleus could not have been influenced pro- foundly. Secondly, the high boiling hydrocarbon CIgH30 contains only one double bond instead of the two or three expected. The drastic treatment of the cholesteryl chloride evidently involves hydrogen shifts, which, how- ever, should not greatly change the isotopic concentration of the frag- ments unless the deuterium concentration at any particular carbon is of a different order of magnitude from that at others. From the analytical values obtained for the two breakdown products, it becomes evident that acetate had been utilized for the biosynthesis of the side chain as well as the steroid nucleus. Indeed the deuterium concentra- tion found in the side chain exceeds that of the nucleus by approximately 50 per cent. The difference may arise from the fact that the methyl groups of the cholesterol at positions 18, 19,21,26, and 27 may originate directly from the acetic acid with the retention of all 3 hydrogen atoms, while the other carbon atoms probably retain only 1 or 2 of the original hydrogen atoms. Since the methyl groups account for 50 per cent of all the hydrogen in the side chain, and for only 20 per cent of the total hydrogen in the nucleus, more of the deuterium could be expected to be incorporated into the side chain.1 1 The foregoing considerations ignore the possibility that intramolecular hydrogen shift had occurred between the side chain and the nucleus during the pyrolysis. This, however, cannot be excluded at present. As the deuterium content of the isooctane is found to be higher than that of the ring hydrocarbon, the isotope must at BEIJING UNIV LIBRARY, on June 12, 2012 w w w .jbc.org D ow nloaded from K. BLOCH AND D. RWFENBERG 629 EXPERIMENTAL Preparation of Deuterio Compounds Deuterio Acetic Acid-Deuterio malonic acid was prepared by exchange with heavy water and decarboxylated to deuterio acetic acid (12). For feeding experiments the sodium salt was used. cu,&Dideuterio propionic acid was prepared from methyl acrylate as previously described (13). The sodium salt of the acid contained 34.4 atom per cent excess deuterium. a,~-Dideuterio butyric acid was prepared by hydrogenating ethyl cro- tonate with deuterium gas. The sodium butyrate contained 23.1 atom per cent excess deuterium. p ,r-Dideuterio butyric acid was similarly prepared from vinylacetic acid. The sodium salt of the 0, y-dideuterio butyric acid contained 16.0 atom per cent excess deuterium. As the 0,~ double bond in vinylacetic acid is known to shift easily towards the or,/3 position (14), it was felt necessary to prove that the isotope was actually located at the p- and y-carbon atoms. To 0.398 gm. of the sodium butyrate (3.57 mM) in water, 10.34 gm. (117.5 mM) of ordinary butyric acid and an excess of sulfuric acid were added. The butyric acid mixture was extracted from the aqueous solution by ether and the ether dried and distilled off. The butyric acid thus obtained was brominated according to the method of Fischer (15). The a-bromo- butyric acid obtained distilled at 120-122”, 19 mm. It contained 0.49 atom per cent excess deuterium or 16.6 per cent when calculated for the undiluted acid. This compares with a value of 16.0 per cent for the deuterium analy- sis of the sodium p,y-dideuterio butyrate. As the sodium butyrate and the a-bromobut,yric acid both contain 7 hydrogen atoms, the agreement between the two values demonstrates that no deuterium had been lost as the result of the bromination; i.e., no shift of double bonds from the 0,~ to the oc,p positions had occurred during the catalytic hydrogenation of vinylacetic acid. Isolation of Cholesterol-Cholesterol was obtained in the usual manner from the unsaponifiable fractions of the total pooled carcasses. The samples were converted into the dibromides, debrominated (16), and re- crystallized. The melting points of the purified cholesterol samples ranged from 147-149”. Cholesterol Degradation-l.3 gm. of crude sterol obtained from Experi- ment C, Table I, were converted into cholesteryl chloride by dissolving in have been present in the cholesterol side chain prior to the degradation. The same, however, does not hold necessarily for the sterol nucleus. The amounts of deuterio cholesterol available at present do not permit as yet the carrying out of reactions from which the deuterium content at individual carbon atoms could be determined. at BEIJING UNIV LIBRARY, on June 12, 2012 w w w .jbc.org D ow nloaded from 630 ACETIC ACID IN CHOLESTEROL FORMATION chloroform and adding 0.6 gm. of freshly distilled thionyl chloride. The mixture was refluxed for 1 hour, and the solvent and excess reagent were distilled off. The dark brown residue failed to crystallize. Purification was achieved by passing a petroleum ether solution of the crude product through a column of activated alumina. The colorless petroleum ether filtrate was evaporated and the residue crystallized from a small volume of acetone. There was obtained 0.75 gm. of cholesteryl chloride, m.p. g&95”, containing 0.21 atom per cent excess deuterium. C2,H&1. Calculated, C 80.1, H 11.1; found, C 79.7, H 11.4 For the thermal degradation, 0.65 gm. of the deuterio cholesteryl chloride, diluted with an equal amount of non-isotopic cholesteryl chloride, was slowly heated to 300’ in an atmosphere of nitrogen. Hydrochloric acid was evolved. The temperature was kept constant until the HCI evolution ceased (4 hours). The bath temperature was then slowly raised to about 400”. At this temperature a volatile product distilled over very slowly and was collected in a trap cooled by dry ice. About 250 mg. of a mobile distillate were obtained; it was redistilled into a second trap. The liquid was presumed to be a mixture of isooctane and isooctene. Its boiling range was 115-120”; i.e., roughly the same range as that observed by Bergmann and Bergmann (11). For deuterium analysis the liquid was volatilized directly into the combustion furnace by a slow oxygen stream. The re- sulting water contained 0.128 atom per cent excess deuterium. Since the deuterio cholesteryl chloride had been diluted l:l, the actual value for the isooctane-isooctene mixture is 0.256 atom per cent excess deuterium. The residue which remained after the pyrolysis was heated over a free flame, and a viscous yellow oil distilled over between 380400”. An at- tempt to obtain a crystalline hydrocarbon from this fraction was unsuccess- ful. 0.50 gm. of this high boiling oil was dissolved in petroleum ether and the solution passed through a column of activated alumina. Only 10 per cent of the fraction was adsorbed. The combined filtrate and petroleum ether washings were free of pigments. The colorless oil remaining after removal of the solvent had the following composition. C19Hz0. Calculated, C 88.4, H 11.6; found, C 88.3, H 11.7 [cJ E = +30.7” (2% in benzene) Bergmann and Bergmann reported C 87.9, H 11.9, [(II]~ = i-31.4”. The hydrocarbon contained 0.089 atom per cent excess deuterium, or 0.178per cent calculated for the undiluted starting material. On the basis of data obtained by Bergmann and Bergmann, we assume that the composition of the volatile hydrocarbon corresponds closely to that of isooctane, GHM. The average deuterium content of the hydrogen at BEIJING UNIV LIBRARY, on June 12, 2012 w w w .jbc.org D ow nloaded from K. BLOCH AND D. RITTENBERG 631 in a compound composed of the fragments CgHlg and C19H30, as calculated from their isotope content, would be (18 X 0.26 + 30 X 0.18)/48 = 0.21 per cent. This compares well with the value 0.21 per cent found for the deuterium content of the hydrogen in the cholesteryl chloride which had been used as a starting material. The catalytic hydrogenation of the hydrocarbon C19H3~ with platinum in acetic acid consumed only one-third of the amount of hydrogen required for one double bond. The resistance to hydrogenation is in agreement with the view of Bergmann and Bergmann that the double bond in the hydro- carbon C19H3~ is located at quaternary carbon atoms. Animal Experiments Feeding of Deuterio Acetic Acid. Experiment A-Seven adult mice received the following diet (Stock Diet I): 50 per cent casein, 20 per cent Wesson oil, 14 per cent salt mixture (17), 16 per cent yeast. The high salt content of the diet resulted in a large water consumption and urine excre- tion, thereby favoring the “washing out” of heavy water from the body fluids. In addition to the stock diet, each mouse received 110 mg. of sodium deuterio acetate (9.9 atom per cent excess deuterium) per day for 8 days. At the end of the experimental period, each mouse had gained an average of 4 gm. The animals were killed, body water was distilled from the tissues, and total fatty acids and cholesterol were isolated from the animal carcasses after removal of the gastrointestinal tracts. Experiment B-Two growing rats, weighing 102 and 110 gm. respectively, were given the carbohydrate-free Stock Diet I and in addition 137 mg. of sodium deuterio acetate (68 atom per cent excess deuterium) per animal per day over a period of 8 days. Each animal gained about 30 gm. during the feeding period. Experiment C-Four adult rats having a combined weight of 1113 gm. received the following Stock Diet II: 86 per cent casein, 3 per cent Wesson oil, 5 per cent yeast, 5 per cent salt mixture (17), 1 per cent cod liver oil. During the 8 day feeding period, each animal received 400 mg. of sodium deuterio acetate (27.6 atom per cent excess deuterium) per day. At the end of the experimental period, the combined weight of the animals was 1196 gm. A total of 1.4 gm. of crude cholesterol was obtained from the animal carcasses. The results of the deuterium analysis of body water, fatty acids, and cholesterol are given in Table I. The data suggest that variation of dietary composition has had little effect on the formation of deuterio cholesterol. During the experimental period, the feces were collected from the animals of Experiment C, Table I. They were ground up with anhydrous sodium sulfate, extracted with ether and acetone, and the sterols precipitated from at BEIJING UNIV LIBRARY, on June 12, 2012 w w w .jbc.org D ow nloaded from 632 ACETIC ACID IN CHOLESTEROL FORMATION the unsaponifiable fraction by digitonin. If we assume that the fecal sterols were derived from the body sterols and that the deuterium con- cent