PRECURSORS OF SQUALENE AND CHOLESTEROL*

BY FRANK DITURI,t JOSEPH L. RABINOWITZ,$ ROY P. HULLIN,Q AND SAMUEL GURIN

(From the Department of Biochemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania)

(Received for publication, June 17, 1957)

The nature of the “biological isoprene unit” believed to be the precursor of cholesterol, rubber, and other terpenoid substances has been the subject of considerable investigation (l-20). Until recently, no single 5- or 6- carbon compound has been found to be consistently superior as a precursor to any other or even to acetate. The discovery by Tavormina et al. (21) that 2-Cr4-3,5-dihydroxy-3-methylvaleric acid (mevalonic acid (MVA))’ is readily incorporated into cholesterol has focused attention on this 6-carbon branched chain acid.

This laboratory has attempted to gain some insight into the nature of such precursors by investigating the pattern of labeling observed in the squalene derived from them. Two compounds have been investigated, 3’-C4-3-hydroxy-3-methylglutaric acid (HMG) and 2-C”-mevalonic acid. In a preliminary communication (22), we have reported that, when 2-C”- mevalonic acid2 is incubated with rat liver homogenates in the presence of carrier squalene, the recovered squalene is found to be radioactive. When degraded by ozonolysis with subsequent degradation of the products of ozonolysis, the distribution of labeling found in the squalene suggests that either the mevalonic acid is decarboxylated subsequent to polymeri- zation or that, if it is decarboxylated before polymerization, the resulting 5-carbon compound is dealt with in an asymmetric manner. Further data verifying this finding are presented.

Work in several laboratories (7,11) has demonstrated that liver is capable of synthesizing HMG from acetate, and Rudney (7, 10) has shown that

* Supported in part by grants from the National Heart Institute of the National Institutes of Health, the American Heart Association, and the American Cancer Society.

t Scholar in Cancer Research of the American Cancer Society. $ Present address, Department of Biochemistry, Baylor University, Houston,

Texas. 0 Visiting Research Fellow of the Mental Health Research Fund of Great Britain. r Abbreviations used in this paper are as follows: MVA, mevalonic acid; HMG,

3-hydroxy-3-methylglutaric acid; CoA, coenzyme A; bMG, 3-methylglutaconic acid; ATP, adenosine triphosphate; DPN, diphosphopyridine nucleotide.

2 The authors express their appreciation to Merck Sharp and Dohme for a generous supply of labeled and unlabeled mevalonic acid.

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826 SQUALESE .4ND CHOLESTEROL

the synt.hesis involves acetyl CoA and acetoacetyl CoA in the microsomal fraction of rat liver and in a yeast preparation. It has also been established that HMGCoA can be formed in heart muscle by means of fixation of carbon dioxide by P-hydroxyisovaleryl CoA (23, 24). In all these studies, the identity of the HhlG has been carefully established. CL4-HMG, pre- pared synthetically and purified by crystallization, has been reported to be incorporated into cholesterol (1 l), squalene (20), and farnesenic acid (20) by homogenates and soluble enzyme systems from rat liver. It was therefore surprising to find that such synthetic C”-HMG, when further purified by chromatography, can no longer be incorporated into squalene or cholesterol by in V&O systems. Chromatography revealed two radio- active contaminants, one of which appears to be partly responsible for the biological activity previously reported from this laboratory (11-14, 18, 20). The identity of this substance has not been established.

Previously reported work on the incorporation of C14-HMG into farne- senic acid (20) has been repeated with C”-mevalonic acid as a substrate. The recovered farnesenic acid was found to be radioactive. Biosynthetic C4-farnesenic acid was prepared and shown to be incorporated into choles- terol by rat liver homogenates.

EXPERIMENTAL

The homogenates used in these studies were prepared by homogenizing minced rat liver in 2.5 volumes of cold 0.1 M potassium phosphate buffer, pH 7.0, which contained nicotinamide, 0.03 M, magnesium chloride, 0.005 M, and potassium bicarbonate, 0.025 M. The cellular debris was removed by centrifugation at 1000 X q for 10 minutes.

Soluble enzyme systems were prepared in phosphate buffer containing nicotinamide and magnesium chloride, as previously described (20).

The isolation, purification, and radioassay of squalene, cholesterol, and farnesenic acid have been described previously (20). An additional pro- cedure used in this study involved the incubation of homogenates with squalene emulsified in 2 per cent gelatin solution. The preparation of the emulsion has been previously described (20).

Chromatography of Squalene-Squalene purified by chromatography on alumina (20) was found to contain small amounts of an impurity which did not cleave in the usual manner upon ozonolysis. The squalene was further purified by chromatography on silicic acid.

15 gm. of silicic acid, Mallinckrodt, 200 mesh, were suspended in petro- leum ether (boiling range 60-110”) and packed under pressure to give a column 20 mm. in diameter and 150 mm. long. Squalene (75 to 100 mg.) was dissolved in a small amount of petroleum ether and put on the column and 15 ml. of petroleum ether were added above the silicic acid. The top

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DITURI, RABINOWITZ, HULLIN, AND GURIN 827

of the column was fitted with a 300 ml. mixing bulb, and a magnetic stirrer was suspended beside the bulb to hold in position a stirring bar on the inside of the bulb. The bulb was filled with petroleum ether and another bulb was connected above it. The upper bulb was filled with a solution of 10 per cent benzene in petroleum ether. Stirring was started in the mixing bulb, and 5 ml. fractions were collected at a rate of 0.5 to 1.0 ml. per minute.

With use of this gradient elution method, the impurity present in the squalene came out at the solvent front and was found to contain no radio- activity. The squalene was eluted as a single peak in Fractions 18 to 25. The specific activity of the individual fractions was determined and found to be the same within the limits of experimental error.

Degradation of SquaZene-Squalene (30 to 100 mg.), chromatographed on silicic acid, was dissolved in 100 ml. of ethyl acetate and ozonized with 3 per cent ozone at O-1”. Rupture of a length of rubber tubing was used to indicate the presence of excess ozone. The process was repeated three times to insure completeness of ozonization. To the solution of ozonide there were added 5 ml. of 30 per cent hydrogen peroxide and 1 ml. of 2 per cent sulfuric acid. The mixture was shaken for 1 hour at room tem- perature and then allowed to stand overnight. 10 ml. of water, 25 ml. of acetic acid, and 5 ml. of 30 per cent hydrogen peroxide were added and the mixture was refluxed for 3 hours.

The resulting mixture was neutralized to pH 5 by addition of dilute sodium hydroxide, and the acetone fraction was removed by steam distilla- tion and collected in Deniges reagent. The recovered acetone was con- verted to iodoform and acetic acid, and the acetic acid further was de- graded to give carbon dioxide and methylamine (14).

Levulinic acid was isolated and purified by the method of Cornforth and PopjBk (25). It was treated with hypoiodite and carbon atom 5 was re- covered as iodoform (14). The remaining succinic acid was degraded by the method of Phares and Long (26) to give carbon dioxide and ethylene- diamine.

The original succinic acid produced from squalene by ozonization was recovered and purified by the method of Cornforth and PopjiLk (25) and degraded by the method of Phares and Long (26).

Samples of the original squalene and each of the degradation products were converted to carbon dioxide by wet oxidation (27). The carbon dioxide was counted as barium carbonate in a windowless gas flow counter; a minimum of 3000 counts was obtained on each sample. All counts were corrected to infinite thinness.

Chromatography of S-Hydroxy-S-methylglutaric Acid (HMG)-HMG was chromatographed on silicic acid by the method of Adamson and Greenberg (28) and was found in approximately the same fractions that they reported.

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828 SQUALENE AND CHOLESTEROL

In addition, HMG was found to be readily purified by chromatography on Dowex 1-formate form by the gradient elution technique of Busch et al. (29). HMG appears in the fractions in which the normality of the formic acid in the eluting solution is 0.8 to 0.9 N. Crystalline HMG can be ob- tained from the eluates by vacuum distillation or lyophilization. HMG recovered by this method had the correct melting point, did not depress the melting point of an authentic sample, had the same RF as an authentic sample when chromatographed on paper (11)) and behaved as expected when chromatographed on silicic acid (28). 3-Methylglutaconic acid can also be separated by this method with the appearance of two peaks (prob- ably cis and trans forms) at 0.4 to 0.5 N formic acid. Dimethylacrylic acid appears with the first trace of formic acid in the eluting solution.

Since titrimetric methods cannot be used for estimation of the eluted acids, advantage was taken of the color reaction given by anhydrides of dibasic acids with pyridine (30). Small samples of each fraction were evaporated to dryness on a steam bath and treated with 0.2 ml. of 30 per cent acetic anhydride in pyridine. After heating in a boiling water bath for 30 minutes, the color intensity could be estimated by eye, or, after dilution, to 10 ml. with carbon tetrachloride, in a filter photometer at 420 rnp. As little as 0.025 mg. of HMG or 3-methylglutaconic acid could be detected by this method.

Results

Incorporation of Mevalonic Acid into Squakne- When 2-Cl*-mevalonic acid along with emulsified squalene was incubated with homogenates of rat liver, the recovered squalene was found to be radioactive (Table I). Such squalene preparations were degraded as described above, and the specific activities of the resulting fragments determined. Experiment 1 (Table I) demonstrates that approximately 85 per cent of the Cl* incor- porated into squalene is found in the terminal methyl groups and in carbon 3 of levulinic acid (24). In a duplicate experiment (Experiment 2), yield- ing squalene of considerably higher specific activity, over 95 per cent of the isotopic carbon was recovered in the same carbon atoms. Although carbon atoms 2 and 3 of levulinic acid have been counted together, it is clear that the radioactivity was predominantly confined to carbon 3. Were this not the case (Fig. l), the succinic acid fragment representing the four central carbons of squalene would have contained much more radioactivity. If the carbon atoms representing carbon 2 of levulinic acid had been appreciably labeled, then labeling would have been expected in the methylene carbons of the succinic acid moiety derived from the center of squalene. This was not the case. Furthermore, if carbon 2 of levulinic acid were labeled, this would presuppose the formation of a 5-

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DITURI, RABINOWITZ, HULLIN, AND GURIN 829

carbon intermediate labeled in a terminal position as well as at the branched methyl carbon atom. The close correspondence in the radioactivity of

TABLE I

Specific Activity of Biosynthetic C%S’qualene and Its Degradation Products

Compound

Squalene Acetone

Carbonyl carbon CHII

Succinic acid -COOH -CHs-

Levulinic acid CHIa (carbon 5) -COOH (carbon atoms 1 and 4) -CH2- ( “ ‘I 2 “ 3)

- Specilic activity

Experiment 1 Experiment 2

c.p.m. pa nib. c c.p.m. pn **. c

150* 715 396 790

10 60 360 1966

70 35 30 30 80 50

150 1996 17 13 28 16

310 2160

* This sample of barium carbonate was contaminated, and the specific activity was estimated from direct counting of the original squalene.

OH I

HOOC-C*H&+CHg-CHsOH I CHa

8 C*Hp---CT -%H-CHp-C*Hg-Cl+

CHI: I ’

CHo I

CH-C&-C*Hn--Cl PCH-CHr- 1 ! C&

Squalene

FIG. 1. Distribution of isotopic carbon in biosynthetic squalene

the iodoform (representing the 2 methyl carbon atoms of acetone) and carbon 2 and 3 of the levulinic acid suggests also that only one of the two methyl groups appearing ultimately in the acetone fraction was appreciably labeled.

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830 SQUALENE AND CHOLESTEROL

PuriJication of S’-P4-HMG-Labeled HMG was synthesized by the method of Klosterman and Smith (31) and apparently pure material was obtained by recrystallization (m.p. 105-106”). When incubated with homogenates and soluble enzyme systems of rat liver, this material was found to contribute Cl4 to cholesterol (ll), squalene (20), farnesenic acid (20)) and 3-hydroxyisovaleric acid (14). After chromatographic purifica- tion by the two methods described above, the recovered material had the same specific activity, but was no longer incorporated into squalene or cholesterol.

TABLE II Incorporation of S’4WHMG and Chromatographic

Fractions into Squalene

Each flask contained 3.0 ml. of soluble enzyme system, 0.3 mg. of ATP, 0.3 mg. of DPN, 1 mg. of squalene in 0.1 ml. of emulsion, and radioactive substrate; final volume, 3.4 ml. Substrate in Experiment 1 was 5 mg. of crystallized 3’-CWHMG or the equivalent fraction obtained by the chromatography of 5 mg. of the HMG on a silicic acid column; in Experiment 2, the substrate was 1 mg. of 3’-Cl’-HMG or the equivalent fraction obtained by chromatography of 1 mg. on Dowex 1-formate form. Gas phase, 95 per cent 02-5 per cent CO*; temperature, 34”; time, 2 hours. At the end of the incubation periods, 4 mg. of squalene carrier were added to each flask.

Experi- ment No.

-

Substrate

Crystallized HMG Fractions 21-30 (probably @MG) HMG fractions Methanol eluates Crystallized HMG Column fractions

0.2 N formic acid 0.9 “ “ “ (HMG)

-

1

_-

Total radioactivity added

c&m.

2.5 X 10’ 2.0 x 106 2.0 x 106 2.0 x 106 5 x 106

4 x 10’ 4 x 106

-i Recovered squalene

Specific Substrate activity 1corporation

p.m. pn mg

400 0 0

80 150

30 0

pn celu

0.08 0 0 0.25 0.15

0.38 0

A cruder synthetic preparation of high specific activity (10” c.p.m. per mg. of C) was crystallized once from ether-petroleum ether. This prepara- tion did not have a sharp melting point and appeared grossly yellow, but radioactivity was incorporated into squalene when it was incubated with soluble enzyme systems (Table II). When chromatographed on silicic acid, three radioactive components were obtained. The first was a titrat- able peak conforming to that found with 3-methylglutaconic acid; the specific activity of this material was the same as that of the HMG. It was not incorporated into squalene (Table II). The second and largest peak, representing 80 per cent of the weight and radioactivity of the start- ing material, was demonstrated to be HMG by criteria of melting point,

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DITURI, RABINOWITZ, HULLIN, AND GURIN 831

mixed melting point, and chromatography in other systems. This was not incorporated into squulene. After elution of the HMG, the column was further cluted with methanol and another radioactive sample obtained. This material, representing 5 to 8 per cent of the original radioactivity, was incorporated into squalene (Table II).

Chromatography of the same material on Dowex 1-formate gave only two radioactive peaks, the expected one for HMG (0.9 N formic acid) and a peak which was eluted at 0.2 N formic acid. The HMG was not incor- porated into squalene. The less polar peak, representing about 10 per cent of the original radioactivity, was incorporated into squalene (Table II).

TABLE III

Incorporation of Isotopic Substrates into Cholesterol by Rat Liver Homogenates

Each flask contained 4.5 ml. of liver homogenate, 0.5 mg. of ATP, 0.5 mg. of DPN, and 1 mg. of isotopic substrate as indicated below; final volume, 5.0 ml.; gas phase, 95 per cent 02-5 per cent CO,; temperature, 34”; time, 2 hours. At the end of the incubation, carrier cholesterol was added to give a total of 1 mg. per flask.

Substrate

Recovered cholesterol

Total activity I Substrate

incorporation

I-W-Acetate 2-Cl’-Mevalonate 3’-CWHMG Crystalline (m.p. 105-107”) Chromatographed (m.p. 108-109”) 4-Cl’-Dimethylacrylate

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

3.3 x 10’ 256 1.0 x 10’ 830

4.0 x 106 54 2.0 x 106 0 4.0 x 106 20

per ccn1

0.08 8.3

0.015 0 0.0008

The radioactive impurity present in CY4-HMG was stable up to 2 years when the crystalline Cl”-HMG was stored in a desiccator. It was also stable in aqueous solution at a range from pH 4 to 7 when stored in the frozen state. When heated to 50” or higher for more than a few minutes, the impurity was completely inactivated. In addition, exposure to strong alkali or strong acid appeared to inactivate it rapidly.

The identity of the impurity has not been established. The amounts present are small, and it is inactivated to a considerable extent in the process of isolation by chromatography (Table 11). In view of chromatographic evidence and comparison with known labeled compounds in the biosyn- thesis of squalene and cholesterol, the following have been eliminated as possibilities: acetate, dimethylacrylic acid, 3-methylglutaconic acid, 3- hydroxyisovaleric acid, and mevalonic acid.

Since most of our experiments dealt with the conversion of HMG to

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832 SQUALENE AND CHOLESTEROL

squalene by aqueous enzyme systems, the incorporation of labeled HMG into cholesterol was reinvestigated in homogenates. Crystalline HMG (m.p. 10.5107”), chromatographed HMG (m.p. 108-log”), acetate, meva- ionic acid, and dimethylacrylic acid (all labeled with CY4) were tested as substrates for cholesterol synthesis (Table III). It was observed that the chromatographically pure HMG is no longer incorporated into choles- terol. Mevalonic acid-204 is obviously far superior as a substrate to all the other substances tested.

TABLE IV

Incorporation of Mevalonic Acid into Squalene and Fatnesenic Acid by Soluble Enzyme System of Rat Liver

Each flask contained supernatant enzyme system as indicated below, DPN, 0.1 mg. per ml., squalene, 1.0 mg. in gelatin emulsion, and potassium farnesenate as indicated. At the end of the incubation, carrier squalene was added to all flasks to give a total of 5 mg. per 3.0 ml. of enzyme system; flasks containing farnesenic acid received additional farnesenic acid to make a total of 20 mg.

Exper men1 No.

Conditions

Enzyme, 3 ml., and 2-C“- mevalonic acid, 2 pmoles (10’ c.p.m. per pmole)

Same as above plus 2 mg. far- nesenic acid

Enzyme, 6 ml., and 2-C”- mevalonic acid, 1.7 pmoles (5 X 10” c.p.m. per pmole)

Same as above, plus 1 mg. farnesenic acid

Recovered squalene Recovered farms&c acid

Specific activity

c.p.m. QW w.

500

50

500

120

-

1

_

-

Total vztivity

250

5ooo

1200

iubstrate Specific incorP activity ration --

pm& c.p.m. QW w.

0.025

0.0025 300

0.100

0.025 850

Tota, Qubstrate activity UlCOWC-

ration --

6,000 0.06

17,000 0.33

Upon incubation of the soluble enzyme system with 2-C”-mevalonic acid in the presence of farnesenic acid, a reduction in the amount of radio- active substrate incorporated into squalene (Table IV) was observed. Similarly, the addition of carrier farnesenic acid to homogenates reduced the incorporation of labeled mevalonic acid into cholesterol. When the carrier farnesenic acid was recovered and purified by chromatography on a reversed phase column with partitioning between hexane and 80 per cent methanol-water (20), it was found to contain significant radioactivity. The recovered farnesenic acid was further chromatographed on the re- versed phase column, this time partitioning between hexane and 70 per

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DITURI, RABINOWITZ, HULLIN, AND GURIN 833

cent methanol-water. There was no change in specific activity of the recovered farnesenic acid, and the specific activity of serial fractions re- mained constant within the limits of error of the method. After repeated chromatography, the farnesenic acid was diluted with additional carrier and the benzylthiouronium salt prepared (20) and recrystallized (m.p. 123-124”). There was no change in specific activity.

Biosynthetic C!“-farnesenic acid, isolated from homogenates and purified as described, was then incubated with liver homogenates. On recovery of cholesterol, significant activity was found (Table V). The cholesterol digitonide was converted to cholesterol dibromide (32) and recrystallized

TABLE V Incorporation of Labeled Substrates into Cholesterol by Homogenates of Rat Liver Each flask contained 5.0 ml. of homogenate, 1.0 mg. of ATP, and 1.0 mg. of DPN.

Substrates used were W-farnesenic acid (3509 c.p.m. per mg.), 2-Cl’-mevalonic acid (5500 c.p.m. per mg.), and I-W-acetic acid (3500 c.p.m. per mg.). Substrates were added as potassium salts. Final volume, 5.3 to 5.4 ml. Conditions and addition of carrier cholesterol as in Table III.

Substrate added

Amount

I-W-Acetate 2-CY-MVA Cl’-Famesenate 2-C”-MVA CY-Famesenate 2-C”-MVA Cl’-Farnesenate

m. 1.0 1.0 1.0 2.0 2.0 2.0 2.0

Total activity

c.p.m.

3,500 5,500 3,500

11,000 7,000

11,000 7,000

T Recovered cboksterol

Total Substrate in- activity corporation

c.p.n. pn ten;

5 0.14 250 4.5 47 1.3

210 1.9 97 1.4

318 2.9 95 1.3

(m.p. 114L115”) (33) ; no change in specific activity of the cholesterol was observed.

DISCUSSION

The hypothesis that liver tissue is capable of condensing active aceto- acetate and acetyl CoA to form HMG (11) has been confirmed by subse- quent studies. Cl’-acetate is readily incorporated into this 6-carbon branched chain acid (8, 11). Rudney has demonstrated that a microsomal fraction of rat liver can form HMGCoA from acetoacetyl CoA and acetyl CoA (7). His recent report (10) that the enzyme system from yeast can be freed from thiolase provides strong evidence that acetoacetate reacts as an intact 4-carbon unit in the biosynthesis of HMG.

This biosynthetic process is quite distinct from the reaction studied by

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834 SQUALENE AND CHOLESTEROL

Bachhawat et al. (23,24), who have reported that HMGCoA can be formed by a carbon dioxide fixation reaction with isovaleryl CoA. The subsequent cleavage of the product to yield acetoacetate and acetyl CoA has not been found to be reversible. This reaction mechanism as well as the condensa- tion reaction studied by Rudney, therefore, represent the only known mech- anisms for the biosynthesis of HMG.

CY4-HMGCoA prepared synthetically by the method described by Bach- hawat et al. (24) was tested as a precursor of squalene and was not incor- porated.* It is now clear that free HMG likewise is not an effective pre- cursor of squalene or cholesterol in in vitro systems.

The close chemical relationship between mevalonic acid and HMG sug- gests that HMGCoA or some other active form of the acid must somehow be involved in cholesterol synthesis. It is possible that the form of HMG- CoA isolated by Rudney may be isomeric with that obtained by Bachhawat et al. It will be of interest to learn whether Rudney’s compound is an effective precursor.

The fact that the carboxyl carbon of mevalonic acid is almost completely lost during the biosynthesis of cholesterol (34) suggests that the condensa- tion occurs between carbon atom 5 of one molecule and the active methyl- ene group (carbon 2) of another condensing molecule. The results of the chemical degradation of radioactive squalene confirm the decarboxylation and are consistent with the concept outlined. Similar findings have been recently reported by Cornforth et al. (35).

?Vhether decarboxylation occurs before polymerization or later is not clear. If decarboxylation occurs first, then the resulting 5-carbon inter- mediate is either asymmetric or is treated asymmetrically by the enzyme system.

The recovery of radioactive farnesenic acid from in vitro systems and the incorporation of biosynthetic C!“-farnesenic acid into cholesterol sug- gest that an intermediate containing the farnesyl structure must be con- sidered. A recent report (36) states that synthetic CY4-farnesenic acid is not incorporated into cholesterol by the whole animal. As was the case with squalene (37, 39, this may be a matter of synthesizing the correct isomer.

SUMMARY

Homogenates of rat liver have been shown to incorporate 2-CY4-mevalonic acid into squalene and cholesterol. Biosynthetic CY4-squalene was ozo- nized and the radioactivity of the cleavage products determined. The results suggest that mevalonic acid is incorporated directly into squalene with little randomization of isotope before polymerization. The evidence

* F. Dituri, unpublished.

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DITURI, RABINOWITZ, HULLIN, AND GURIN 835

also suggests that condensation occurs between carbon atom 5 of one mole- cule and carbon 2 of a second molecule, with decarboxylation possibly taking place during or after polymerization.

Chromatographic purification of synthetic 3’-CY4-3-hydroxy-3-methyl- glutaric acid yields a preparation which is not incorporated into squalene or cholesterol by in vitro systems. The previously reported biological activity may be ascribed to an unidentified impurity.

It has been demonstrated that mevalonic acid is incorporated into farne- senic acid by soluble enzyme systems of rat liver, and that biosynthetic CY4-farnesenic acid is incorporated into cholesterol by rat liver homogenates.

The authors express their appreciation for the technical assistance of Jessie V. B. Warms and Katherine P. Janeway.

BIBLIOGRAPHY

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Hullin and Samuel GurinFrank Dituri, Joseph L. Rabinowitz, Roy P.

CHOLESTEROLPRECURSORS OF SQUALENE AND

1957, 229:825-836.J. Biol. Chem. 

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