STUDIES ON THE UTILIZATION OF ACETIC ACID FOR THE BIOLOGICAL SYNTHESIS OF CHOLESTEROL*?

BY HENRY N. LITTLE AND KONRAD BLOCH

(From the Department of Biochemistry and the Institute of Radiobiology and Biophysics, University of Chicago, Chicago)

(Received for publication, July 29, 1949)

Previous work (1, 2) has demonstrated that acetic acid is an important source of carbon and hydrogen in the total synthesis of steroids by the ani- mal body, and that this compound is superior in this respect to various other organic molecules. Experiments with deuterioacetate led to the con- clusion that 2-carbon compounds participate in the synthesis of both the nucleus and of the side chain of cholesterol and that, of the carbon and hydrogen atoms in the steroid molecule, at least half is derived from those of acetate. The present investigation was undertaken in order to examine the role of acetic acid in greater detail. To this end cholesterol synthesis has been studied with the aid of acetic acid labeled in either the carboxyl or methyl group, or acetic acid labeled by Cl3 as well as C4. In this way the relative utilization of the 2 carbon atoms of acetic acid for the synthesis of cholesterol as a whole, as well as for the aliphatic and cyclic moieties of the molecule, could be determined. In a few cases the source of individual carbon atoms of cholesterol has been identified. By degradation of isotopic cholesterol the origin of the isopropyl group in the sterol side chain, of the angular methyl groups, and of 2 carbon atoms in the ring system has been established.

The labeled cholesterol used in the present experiments was obtained by synthesis in vitro in surviving rat liver (3). Under these conditions the isotopic substrate is utilized with greater economy than in intact animals. In order to secure the relatively large quantities of steroid which were nec- essary for degradation studies, the biosynthesis was carried out with ace- tate labeled by Cl4 of high specific activity. The resulting cholesterol could then be diluted considerably by normal material.

DISCUSSION

Although the earlier work with deuterioacetate and with acetic acid la- beled in the carboxyl group has shown qualitatively that both carbon atoms

* Presented in part at the meeting of the American Society of Biological Chemists at Detroit, April, 1949.

t Supported by a grant-in-aid from the Life Insurance Medical Research Fund and by the Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago.

33

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34 ACETIC ACID UTILIZATION

of this compound participate in cholesterol formation, it was unknown whether they were utilized equally in the synthetic process. Since cho- lesterol has an odd number of carbon atoms, it is clear that, unless other precursors exist, the carboxyl and methyl carbon atoms of acetic acid can- not appear in the sterol molecule in equal numbers. Also, the presence in cholesterol of branched structures, such as the isopropyl group of the ali- phatic chain, suggests that decarboxylations may occur at an intermediate stage of the synthesis. To what extent the 2 carbon atoms of acetic acid are utilized may be determined with the aid of acetic acid in which 1 of the carbon atoms is labeled by Cl3 and the other by C14. The ratio of t,he 2

TABLE I

Utilization of Methyl and Carboxyl Carbon Atoms of Acetic Acid for Cholesterol

PWXrSOI

Experiment I. C13H&1400H. Cl3 9.26 atom y0 excess ; Cl’, 26&l c.p:m.

Experiment II. C14HKF00H. CY, 9.9 atom y. excess; C14, 103,OCO c.p.m.

Synthesis

-

C’”

atom per ccltt czccss

0.200

0.307

Cholesterol

C”

3900

Relative isotope con- centration’ due to

2.16

3.78

1.64

3.09

If

-

Methyl carbon grboxyl

1.32

1.22

* Relative isotope concentration = atom o/o excess Cl3 or counts per min. Cl4 in cholesterol

atom 70 excess Cl3 or counts per min. CL4 in precursor

carbon isotopes in cholesterol compared to the isotope ratio in the precur- sor will indicate the relative proportion of methyl and carboxyl carbon atoms of acetic acid in the synthetic product. The data in Table I indi- cate that the ratio of methyl carbon to carboxyl carbon in cholesterol is significantly greater than 1 and hence that methyl carbons predominate. If it is assumed that all carbon atoms of the sterol are furnished by acetate, then the observed ratio of 1.27, the average value of Experiments I and II, indicates that of the total of 27 carbon atoms in the sterol molecule 15 are derived from methyl groups and 12 from carboxyl groups, respectively, of acetic acid. This deduction will be valid only if acetic acid is the sole carbon source for cholesterol and if a given carbon atom of cholesterol is derived from either a carboxyl or methyl carbon atom of acetic acid but

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H. N. LITTLE AND K. BLOCH 35

not from both. Furthermore the carbon atoms of acetic acid which serve as the immediate source should have the same isotope concentration re- gardless of their subsequent location in cholesterol. As is shown later, the isotope concentrations observed for individual carbon atoms of cho- lesterol are on the whole consistent with these assumptions.

The calculation of the methyl carbon to carboxyl carbon ratio required four isotope analyses. Since the error of each determination is 5 per cent, the probable error of this ratio is about 10 per cent. Nevertheless, the close agreement of the data from two different experiments suggests that the ratio of 1.27 has real significance.l An independent check of the ac- curacy of the analytical values is provided by the isotope ratios in the saturated fatty acids which were isolated from the same experiments. In accord with previous findings, which indicated that the higher fatty acids are formed by multiple condensation of 2-carbon units (4), the ratio of methyl carbon to carboxyl carbon in the saturated fatty acids in two ex- periments was found to be 0.98 and 1.08.

The value of 1.27 for the ratio of methyl carbon to carboxyl carbon shows that in the course of cholesterol formation from acetate decarboxylations take place. There is no evidence to indicate at which stage of the con- densation process carboxyl groups are removed. However, it may be mentioned here that, as shown later in this report, the isopropyl portion of the cholesterol side chain is composed of two methyl groups and one carboxyl group of acetic acid, and hence may result from the condensation of 2 molecules of acetate to a 4-carbon compound and its subsequent de- carboxylation to a 3-carbon unit.

The participation of acetic acid in the synthesis of both the isooctyl and cyclic moieties of cholesterol had been established with the aid of deuterium as a tracer (1). The quantitative significance of the results obtained on thermolysis of deuteriocholesteryl chloride was open to question, since the drastic conditions necessary for the splitting of the cholesterol molecule could have resulted in intramolecular hydrogen shifts. We have therefore repeated the degradation with cholesterol derived from Cl”-acetate and have determined the Cl4 content of the isooctane-isooctene mixture and of the hydrocarbon &Ha, fractions which represent the side chain and the nucleus of cholesterol. The results obtained (Table II) with choleste- rol synthesized from carbon-labeled acetic acid confirm our earlier con- clusion that the carbon atoms of acetic acid are utilized in the formation of both the aliphatic and the cyclic portion of cholesterol. The isotope concentration of the nucleus compared to that of the side chain was found to depend on the position of the label in the precursor. In the case of

1 If the ratio methyl carbon to carboxyl carbon were 16:ll or 14:13, the isotope ratios should be 1.45 and 1.08 respectively.

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36 ACETIC ACID UTILIZATION

cholesterol derived from deuterioacetate, the lower isotope concentration in the nucleus may merely be due to the fact that hydrogen bound to methyl groups accounts for more than half of all the hydrogen atoms in the isooctyl side chain but only for 20 per cent of all the hydrogen atoms in the ring structure. It may be assumed that, during the condensation process, the deuterium in those methyl groups of acetic acid which still appear as methyl groups in cholesterol is less likely to be lost or replaced by ordinary hydrogen than the deuterium of methyl groups which become part of the ring structure. The isotope concentrations in the two portions of the sterol did not differ significantly when acetic acid labeled by Cl4 in the methyl group was the precursor. On the basis of these data methyl groups of acetic acid appear to supply approximately the same fraction of the carbon atoms in the side chain and in the nucleus of cholesterol. On

TABLE II Incorporation of Labeled Acetic Acid into Cholesterol and Degradation Products

Atom per cent excess D or counts per min. CY in

Deuterioacetic acid*. .................. Acetic acid-2-W. .....................

“ “ ...................... “ acid-l-U’. ..................... “ “ ......................

0.21 71

93 39

NUCkUS Isoocty1 KhIxao) side chain

(-4) 0)

0.18 0.26 76 74

400 424 98 72 45 36

-

-

(-4) m

0.69 1.03 0.94 1.36 1.25

* Data taken from Bloch and Rittenberg (1).

the other hand, in cholesterol formed from CHJY400H, the radioactivity was significantly higher in the nucleus, indicating that the carboxyl group of acetic acid contributes a greater share of the ring carbon atoms than of those in the side chain. The observation that the isotope distribution in the two moieties of cholesterol depends on the position of the label in the acetic acid used could not be explained readily if the 2 carbon atoms of acetic acid participated equally in cholesterol synthesis. However, it is consistent with the above finding that a greater number of methyl than carboxyl carbon atoms of acetic acid is employed in steroid synthesis. From the data in Tables I and II, it follows that the ratio of methyl carbon to carboxyl carbon, which is 1.27 for the total cholesterol molecule, must be greater than this value for the side chain and smaller than 1.27 in the cholesterol nucleus. The ratios which are in nearest agreement with the experimental dat.a are 5: 3 or 1.67 for the isooct,yl side chain and 10: 9 or 1.1 for t.he cholestero! nucleus. On the basis of this distribution of

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H. N. LITTLE AND K. BLOCH 37

methyl and carboxyl carbons in the nucleus and side chain, the calculated ratio (A): (B) for acetic acid-2-Cl4 is 0.84 and for acetic acid-l-Cl4 1.26. Isotope analyses of greater precision are needed to establish more com- pletely the accuracy of these ratios.

Isotope Concentration of Individual Carbon Atoms in Cholesterol-By iso- tope analysis of the angular methyl groups and of the isopropyl moiety of the isooctyl side chain, the origin of some individual carbon atoms of the cholesterol molecule has been determined. The choice of these carbon atoms was prompted by the relative ease of their isolation and by our

TABLE III Distribution of Cl4 in Isopropyl Group of Cholesterol Side Chain

Compound analyzed CH8CWOH

-

Cholestane ........................... Acetone ............................... Iodoform (Czs, C23 .................... Carbonyl carbon of acetone ((32,). .....

Cholesterol formed from -

Found Calculated

c.p.m.

93 62 20

146%

c.p.n.

70*t ot

209t

C”H8COoH

Found

c.p.m.

71 72

166 W

Calculated

c.p.m.

85:s 1285

05

* Calculated on the assumption that the methyl and carboxyl carbon atoms of acetic acid are present in cholesterol in a ratio of 15:12.

t Calculated from the C** content of cholestane for 1 isotopic carbon (C,,) per molecule of acetone.

$ Calculated from the observed values for acetone and iodoform. $ Calculated from the 04 content of cholestane for 2 isotopic carbon atoms

(C&, Gr) per molecule of acetone. The predicted value of iodoform is 168 if the Cl4 concentration of CS and Gr is calculated from the observed value for acetone (72 counts) from which the iodoform was obtained.

interest in the biological synthesis of branched chain structures. Choles- terol synthesized from acetic acid-l-U4 and from acetic acid-2-C4 waz used for this purpose.

The formation of acetone and allocholanic acid on chromic acid oxida- tion of cholestane (5) constitutes the classical proof for the presence of a terminal isopropyl group in the aliphatic side chain of cholesterol. Hence, we have assumed that the acetone obtained by oxidation of cholestane is derived exclusively from carbon atoms 25, 26, and 27. The isotope con- centrations were determined in acetone and in the iodoform derived from it, which represents C&c and C&T. The isotope content of the carbonyl atom of acetone (C,,) was calculated by difference. It is clear from the dat,a in Table III that acetic acid had been utilized in the synthesis of the isopro-

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38 ACETIC ACID UTILIZATION

pyl group of cholesterol. When methyl-labeled acetic acid was the precur- sor of the steroid, the 04 content of the iodoform fraction (CM and C&) accounted for nearly all the Cl4 in the acetone. On the other hand, in cholesterol synthesized from carboxyl-labeled acetic acid, the methyl group of acetone had a very low radioactivity, showing that in this case the carbonyl group of acetone (CZ6) contained most of the isotope. Carbon atoms 26 and 27 of cholesterol therefore originate from the methyl groups of acetic acid, while carbon atom 25 is derived from the carboxyl carbon of acetate (Fig. 1). It should be pointed out that in both experiments the Cl4 content of the “labeled” carbon atoms was about 15 to 20 per cent lower than was expected from the isotope concentration of the steroid and that those positions which one would expect to be unlabeled contained a small but significant radioactivity. These discrepancies may be attrib-

C,H3 JJ

,G, CH b3-& C 25

D H\27

CH3

FIG. 1

uted either to shortcomings of the degradative and analytical procedures or perhaps to a biological redistribution of labeled carbon during the bio- synthesis. It is worthy of note that the formation of the isopropyl group in the side chain of cholesterol from the elements of acetic acid is the only known instance of the synthesis of a branched chain structure in the animal organism. All other constituents of the animal body which have branched carbon chains are either essential amino acids or vitamins.

Origin of Carbon Atoms 10, 17, 18, and IQ-In the oxidation of organic compounds by a mixture of chromic and sulfuric acids, methyl groups and their adjoining carbon atoms are converted to acetic acid. This procedure has been made the basis for an analytical determination of carbon-bound methyl groups (6). We have applied this procedure to the nuclear hydrocarbon G9HS0 which is obtained by thermolysis of cholesteryl chloride. If it is assumed that this hydrocarbon has the structural formula proposed by Bergmann and Bergmann (7), chromic acid oxidation should yield a maximum of 2 moles of acetic acid (degradation)2 from the methyl carbon atoms 18 and 19 and their adjoining carbon atoms (CM and Cl,) (Fig. 2). The observed yield of acetic acid (degradation) was 1.3 to 1.5 moles. The acid was found to contain isotopic carbon both when methyl-

f The acetic acid resulting from the degradation is designated as acetic acid (degradation) in order to distinguish it from the acetic acid which serves as a pre- cursor in steroid synthesis and which is designated acetic acid (precursor).

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H. N. LITTLE AND K. BLOCH 39

labeled and carboxyl-labeled acetic acid had been the cholesterol precursor. The acetic acid obtained on chromic acid oxidation of the cholesterol nu- cleus is derived from 4 carbon atoms of the steroid. Therefore, the isotope concentrations observed for the methyl groups of acetic acid (degradation) reflect the average Cl4 content of carbon atoms 18 and 19, and those given for the carboxyl group of acetic acid3 (degradation) represent the average

- 2cH3cooH

!lK% %-c17

FIG. 2

TABLE IV Isotope Concentration in Angular Methyl GTOUPS and C;o and Crr of Cholesterol

Compound analyzed

1. Cholesterol ............................ 2. Acetic acid by oxidation of CLH30 ...... 3. C02(C10 + Crr) from (2) ............... 4. Iodoform from methyl carbons

(Cl8 + Cd of (2) ....................

Cholesterol synthesized from

-

CHS?OOH (Experiment I)

c.p.m. C.#.rn. 93 64 lost

120 128

2 0

eik- B’

c.p.m.

=t 128

0

C’HaCOOH (Experiment II)

Calcu- Found la$n

_____ c.p.m. c.p.m.

71 76 64t 40 0

105 152

c.g.m.

W 50

101 L

* The two methods of calculation (A and B) are explained in the text. t Calculated on the assumption that the methyl and carboxyl carbon atoms of

acetic acid are present in cholesterol in a ratio of 15:12. The calculated values in lines (3) and (4) are based on the observed Cl4 content of the acetic acid (line(a)).

concentration of carbon atoms 10 and 17 (Table IV). In cholesterol for which CH3CY400H had been the precursor, the angular methyl groups contained no C14. On the other hand, the isotope concentration was high at carbon atoms 18 and 19 in cholesterol which had been synthesized from CYH&OOH, demonstrating that methyl groups of acetic acid are the source of the angular methyl groups of the steroid molecule. In Experi- ment I (Table IV), the isotope concentration of the acetic acid obtained

8 It is assumed that the acetic acid (degradation) is formed in equal amounts from carbon atoms 18 and 10, and 19 and 17 respectively.

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40 ACETIC ACID UTILIZATION

by degradation was entirely accounted for by the Cl4 content of its car- boxy1 group, and therefore either carbon atom 10 or 17, or both, must have been derived from carboxyl groups of the precursor acetic acid. It will be noted that in Experiment II (Table IV) the CO2 fraction also had a sig- nificant radioactivity, which suggests that this fraction (Cl0 and Cl,) had originated in part from methyl groups of acetic acid. It may further be seen that in Experiment I (Table IV) the acetic acid (degradation) had an appreciably lower isotope content and in Experiment II a higher isotope concentration than would have been expected if methyl groups of acetic acid (precursor) had been the source of carbon atoms 18 and 19 only, and if carboxyl groups of acetic acid had supplied both carbon atoms 10 and 17 (Calculation A). It should be recalled here that the methyl carbon 19 changes its position during the thermolysis of cholesteryl chloride and be- comes attached to C1, (7). While one might expect both angular methyl groups in cholesterol to be attached to carbons which were originally car- boxy1 carbons of acetic acid, this need not be the case for Cl9 after it has migrated to Cl?. Since, as indicated in Experiment II (Table IV), the mixture of Cl0 and Cl7 contained a significant isotope concentration, 1 of these 2 carbon atoms may have been derived from methyl groups of acetic acid, and it seems likely that Cl, was the labeled one in this case. With these considerations in mind, we have calculated (Calculation B) the ex- pected isotope concentrations of carbon atoms 10, 17, 18, and 19 and have assumed that, in Experiment I, of the 2 molecules of acetic acid formed on degradation, 1 contains no isotope and the other 04 in the carboxyl group (Clo), while in Experiment II 1 molecule of acetic acid is doubly labeled (Cl7 and C19) and the other singly (CM). The experimental data for the most part are in satisfactory agreement with this assumption.

Admittedly the procedures which have been employed in order to deter- mine the isotope concentration of the angular methyl groups are not un- equivocal. We have therefore carried out an independent degradation experiment to substantiate the above results. When cholesterol is heated in the presence of palladium-charcoal, about 4 moles of hydrogen and 1 mole of methane are evolved per mole of cholesterol (8, 9). According to Ruzicka et al. (9), the aromatization of Rings A and B of cholesterol causes the elimination of the angular methyl group (Cl,) in the form of methane. When this reaction was applied to cholesterol which had been synthesized from CH3U400H, the methane produced had a low isotope concentration, but when cholesterol synthesized from CY4H3COOH was subjected to de- hydrogenation, the methane contained 73 to 81 per cent of the expected radioactivity (Table V). These results taken together with those ob- served on chromic acid oxidation of the cholesterol nucleus (CleH& strongly indicate that the angular carbon atoms 18 and 19 have their origin in methyl groups of acetic acid. The source of carbon atoms 10

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H. N. LITTLE AND K. BLOCH 41

and 17 is less certain, although it appears from the analytical data that one of them is derived from a carboxyl and the other from a methyl group of acetic acid. We conclude tentatively that Cl0 is furnished by a carboxyl carbon of acetic acid and (317 by a methyl group.

Previous considerations of the quantitative significance of acetic acid as a sterol precursor were based on experiments with intact rats (1,2). These animals received deuterioacetate or U3-acetate for a relatively short time, and it was computed that under these conditions acetate furnished about, half of the carbon and hydrogen atoms of cholesterol. Since the calcula- tion of these values included an estimate of the half life time of cholesterol and of the dilution of dietary isotopic acetate by acetic acid from endoge- nous sources, quantities which cannot be determined very accurately, the

TABLE V

Radioactivity of Methane (Cl*) Formed by Dehydrogenation of Cholesterol

Cholesterol synthesized from

Cholesterol. . . Methane Sample l*. . . .

‘I “ 2...................... “ I‘ 3 . . . . “ I‘ 4......................

CHsCWOH C”‘IWDOH (Experiment I) (Experiment II)

c.p.m. C.).rn.

92 59 13 (207)T 86 (106)t 23 77 32 80 43 83

* See “Experimental ” for explanation of Samples 1 to 4. t The values in parentheses are the calculated isotope concentrations of a single

carbon if derived from the labeled carbon of the precursor, corrected for the ratio of 15:12 for the ratio of methyl carbon to carboxyl carbon in cholesterol.

values given were considered to be approximate only. The results reported here on the isotope concentration of individual carbon atoms of the steroid molecule furnish independent evidence that 2-carbon compounds are the principal source of carbon in the biosynthesis of cholesterol. If acetic acid or an equivalent 2-carbon compound was the sole precursor, then any single carbon atom in the sterol which is synthesized from acetic acid la- beled in one position only should contain either about twice4 the isotope concentration of the total molecule or no excess isotope at all. On the other hand, if, for example, acetic acid supplied only half of all the carbon atoms, then any individual carbon of cholesterol should contain either

4 Since the methyl and carboxyl carbon atoms of acetic acid a,re not used equally in the synthetic process but in a ratio of 1.27, the isotope concentration of an indi- vidual carbon atom should be either 1.8 or 2.5 times that of the total steroid mole- cule.

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42 ACETIC ACID UTILIZATION

about 4 times the isotope concentration of the total molecule or no excess isotope at all. Since the radioactivities at those carbon atoms which were analyzed individually were found to contain very little radioactivity when acetic acid labeled in one position was the precursor and contained close t*o the theoretical radioactivity in the case of the acetic acid labeled at the other carbon atom, it is probable that this will also be the case for the re- mainder of carbon atoms in the sterol molecule. The close agreement between postulated and observed isotope distribution is perhaps the most convincing evidence that a 2-carbon compound is the principal if not the sole building block of cholesterol.

EXPERIMENTAL

Preparation of Labeled Acetic Acid-Acetic acid-l-C14 was prepared from Cl402 and methyl magnesium bromide.

Acetic acid-l-Cl3 was obtained from acetonitrile, as described by Wein- house et al. (10).

Acetic Acz&~-C~~-~O mu of methyl alcohol containing CL4 were mixed with 15 ml. of hydriodic acid (sp. gr. 1.7) and a trace of red phosphorus, and heated under a reflux for 1 hour. A slow stream of nitrogen was passed through the reaction flask and the methyl iodide was collected in a trap cooled by dry ice. The methyl iodide (1.05 ml.) was transferred to a solution containing 1.01 gm. of sodium cyanide in 1.8 ml. of water and the mixture heated under a reflux for 6 hours.6 Acetonitrile was distilled from the reaction mixture with several additions of water to the reaction flask. The distillate containing the acetonitrile was hydrolyzed by heating under a reflux with 10 per cent sodium hydroxide for 24 hours. For purification the solution was heated for an additional half hour in the presence of potas- sium permanganate. Acetic acid was obtained by stea,m distillation of t<he acidified solution. The yield based on methyl alcohol was 66 per cent.

Acetic Acid-2-C13-CJ3-methyl iodide6 was allowed to react with KCN and converted to acetic acid as described for the preparation of acetic acid-2-U4.

Preparation of Labeled Cholesterol-The isotopic cholesterol used in the present experiments was obtained by incubation of rat liver slices in the presence of isotopic acetate as described before (3). Tissue from young male rats of the Sprague-Dawley strain (60 to 100 gm. in weight) was em- ployed, since it had been noted (3) that the incorporation of isotopic car- bon into cholesterol was considerably faster in younger than in older ani- mals. In a typical experiment, 1.5 gm. of liver slices were incubated in 12 ml. of Krebs’ phosphate buffer, pH 7.4, which contained 3 mg. of appro-

6 This procedure was worked out by I. Zabin in this laboratory. 6 Furnished as a contribution by the American Cancer Society on recommenda-

tion of the Committee on Growth of the National Research Council.

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H. N. LITTLE AND R. BLOCK 33

priately labeled acetic acid. The flasks were shaken at 37” for 6 hours. The gas phase was pure oxygen which was replaced every 2 hours. Cho- lesterol was isolated by way of cholesterol digitonide in the usual manner. Under our experimental conditions, the isotope concentration or specific activity in the isolated cholesterol varied from 2 to 4 per cent of that of the acetate used. This permitted a several hundred fold dilution of the CY4-containing sterol by non-isotopic cholesterol, thus providing adequate amounts of marked sterol for degradation studies. In the experiments in which the utilization of the 2 carbon atoms of acetic acid for cholesterol synthesis was determined, appropriate mixtures of the differently labeled acetates were added to the incubation medium. Their isotope concen- trations are given in Tables I to VI.

Degradation of Cholesterol into Nucleus and Side Chain-Cholesteryl chloride prepared from cholesterol was pyrolyzed as described before (1). It was assumed that the low boiling fraction was a mixture of isooctane and isooctene and represented the isooctyl side chain of cholesterol. This fraction was analyzed for isotope content after redistillation. The mate- rial remaining after removal of the volatile hydrocarbon was distilled in vacua and yielded as the main fraction a hydrocarbon distilling at 185- 230” at 1 mm. of Hg. The elementary composition and optical rotation of this material corresponded to those for the hydrocarbon C19H3,, de- scribed by Bergmann and Bergmann (7).

G&I. Calculated, C 88.4, H 11.6; found, C 88.3, H 11.7

The hydrocarbon obtained from two different runs had [tr]: = +30.3” and +33.5” (2 per cent in benzene) respectively. This hydrocarbon rep- resents the intact steroid skeleton and is referred to in this paper as the cholesterol nucleus.

Oxidation of Hydrocarbon C19H30 by Chromic Acid-A mixture of 1 mM of hydrocarbon C 19H30, 7.5 gm. of chromium trioxide, and 6.0 ml. of sulfuric acid was heated under a reflux for 3 hours. Under these con- ditions methyl groups and their adjacent carbon atoms are converted to acetic acid (6). The reaction mixture was then subjected to steam dis- tillation, the acid in the distillate determined by titration, and acetic acid isolated as the silver salt. The yield of acetic acid from the oxidation of different samples varied from 1.30 to 1.47 moles per mole of hydrocarbon C 19H30. The isotope concentration of the carboxyl carbon of this acetic acid (degradation) was determined either by decarboxylation of silver acetate with bromine in carbon tetrachloride (11) or by pyrolysis of lithium acetate at 350400” to yield acetone and lithium carbonate (12). Analy- sis of iodoform obtained from this acetone yielded the isotope concentra- tion of the methyl group of acetic acid (degradation). In order to test the reliability of the procedures for the degradation of acetic acid, control

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44 ACETIC ACID UTILIZATION

experiments were carried out with a sample of acetic acid-2-Cl4 of known specific activity. The results given in Table VI show that the degrada- tion products have the expected isotope concentrations, except for iodo- form which had a specific activity about 10 per cent lower than that cal- culated. No corrections have been made for this dilution in the values reported in Tables III and IV.

Dehydrogenation of Cholesterol by Palladium-Charcoal-O.40 gm. of iso- topic cholesterol was intimately mixed with 0.15 gm. of 10 per cent palladium-charcoal and heated, after initial evacuation of the system, to 330-350” for 24 hours (8, 9). A gas trap cooled by liquid nitrogen was connected to the vacuum line in order to condense any hydrocarbons

TABLE VI Degradation of Acetic Acid-S-Cl’

CPH&OOH ........................ CH&OCHa ......................... CHIl ............................... co**. .............................. co,t ...............................

- Found

c.p.m.

2040 2680 3740

20 0

cakuhtea

c.p.m.

2720 4080

0 0

* By thermolysis of lithium acetate. t By decarboxylation of silver acetate with bromine

formed. After gas evolution had ceased, nitrogen was admitted to the system until atmospheric pressure had been reached. Nitrogen gas was

passed slowly through the gas trap which was kept at -180”, and the exit gases were swept directly into a micro combustion furnace. Oxygen was fed into the combustion tube from a side arm. The combustion gases were passed through a Ba(OH)z solution in order to precipitate the car- bon dioxide as BaC03. The baryta traps were changed every 20 min- utes and the Cl* content of the BaC03 samples measured separately (Samples 1 to 4, Table V). Since methane is the only hydrocarbon which has a sufficiently high vapor pressure at the temperature of liquid nitro- gen, it is assumed that the gas removed is mainly methane. However, the varying isotope concentration of the different samples indicates that at least in Experiment I, Table V, the gas contained a second component.

Degradation of Cholesterol to Allocholanic Acid and Acetone-Cholesterol was converted to cholestane by way of dihydrocholesterol and cholesta- none (13). The hydraaone of cholestanone was reduced to cholestane by the Wolff-Kishner method as described by Dutcher and Wintersteiner (14). The over-all yield from cholesterol was 52 to 57 per cent.

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H. N. LITTLE AND K. BLOCH 45

Oxidation of cholestane by chromic acid according to Windaus and Eeukirchen (5) yields allocholanic acid and acetone. For the degradation of isotopic cholestane, 3 gm. of the steroid were suspended in 125 ml. of glacial acetic acid and 6.5 gm. of CrOs dissolved in 25 ml. of 80 per cent acetic acid were added in t,he course of 30 minutes. The reaction mixture was heated on the steam bath for 6 hours. Acetone was removed from the oxidation mixture by a slow stream of nitrogen and precipitated as the mercury complex (15) by passage through a boiling solution of mer- curic sulfate. After completion of the oxidation, water was added to the flask and the reaction mixture was distilled to obtain the remaining ace- tone. The yield of acetone, calculated from the weight of the mercury- acetone complex, ranged from 10 to 20 per cent. Allocholanic acid was isolated according to the directions of Windaus and Neukirchen (5). The yield of crude potassium salt was about 10 per cent of the cholestane used. Purification of the allocholanic acid by recrystallization so diminished the yield as to prevent further use of this material for degradation studies. Attempts to increase the recovery by use of the procedure described by Fieser (16) for the oxidation of isoalkylnaphthoquinones were unsuccess- ful.

The Cl4 content of the acetone was determined by combustion of the mercury-acetone complex. For isotope analysis of the methyl groups of acetone, the mercury-acetone complex was decomposed by hydrochloric acid and the acetone converted to iodoform after distillation. The iso- tope content of the carbonyl group of acetone was calculated by difference.

Isotope Analyses-For CL4 analysis all the samples were burned in a micro combustion apparatus, except the samples of mercury-acetone and iodoform, which were converted to carbon dioxide by the wet combustion procedure of Van Slyke and Folch (17). CO2 was precipitated as barium carbonate and counted in a flow gas counter. Samples were counted for a sufficient length of time to insure less than 5 per cent probable error. The CL2 values are given as counts of Cl4 per minute of BaC03 samples, cor- rected for infinite thickness. Cl3 was determined by mass spectrometric analysis.

SUMMARY

1. The biological synthesis of cholesterol has been investigated with the aid of acetic acid-l-C’*, acetic acid-2-CY4, and with acetic acid labeled by both Cl3 and CY4.

2. The utilization of the 2 carbon atoms of acetic acid as a carbon source for the total molecule and for the cholesterol nucleus and side chain has been determined.

3. Methyl groups of acetic acid have been identified as sources of carbon

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46 ACETIC ACID UTILIZATION

atoms 18, 19, 26, 27, and presumably 17 in cholesterol. Carbon atom 25 and proabably carbon atom 10 are furnished by carboxyl groups of acetic acid.

BIBLIOGRAPHY

1. Bloch, K., and Rittenberg, D., J. Biol. Chem., 146, 625 (1942). 2. Bloch, K., and Rittenberg, D., J. Biol. Chem., 169, 45 (1945). 3. Bloch, K., Borek, E., and Rittenberg, D., J. Biol. Chem., 162, 441 (1946). 4. Rittenberg, D., and Bloch, K., J. Biol. Chem., MO, 417 (1945). 5. Windaus, A., and Neukirchen, K., Ber. &em. Ges., 62. 1915 (1919). 6. Kuhn, R., and L’Orsa, F., 2. cmgew. Chem., 44, 847 (1931). 7. Bergmann, E., and Bergmann, F., J. Chem. Sot., 1019 (1939). 8. Diels, O., Gaedke, W., and Koerding, I?., Ann. Chem., 469, 1 (1927). 9. Ruzicka, L., Furter, M., and Thomann, G., HeZu. chim. acta, 16, 812 (1933).

10. Weinhouse, S., Medes, G., and Floyd, N. F., J. BioZ. Chem., 156, 411 (1945). 11. Hunsdiecker, H., and Hunsdiecker, C., Ber. them. Ges., 76, 291 (1942). 12. Kroenig, W., 2. angew. Chem., 87, 667 (1924). 13. Bruce, W. F., Org. Syntheses, Coll. 2, 139 (1943). 14. Dutcher, J. D., and Wintersteiner, O., J. Am. Chem. Sot., 61, 1992 (1939). 16. Van Slyke, D. D., J. BioZ. Chem., 88, 415 (1929). 16. Fieser, L. F., J. Am. Chem. Sot., 70, 3237 (1948). 17. Van Slyke, D. D., and Folch, J., J. BioZ. Chem., 136, 509 (1940).

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Henry N. Little and Konrad BlochSYNTHESIS OF CHOLESTEROL

ACETIC ACID FOR THE BIOLOGICAL STUDIES ON THE UTILIZATION OF

1950, 183:33-46.J. Biol. Chem. 

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