ANALYTICAL BIOCHEMISTRY 33, 230-237 (180)

Micro Method for the Quantitative Determination

of A22-Sterols

F. B. MALLORY, K. A. FERGUSON, AND R. L. CONNER

Department of Chemistry and Department of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania 19010

Received July 11, 1969

Several methods have been described for the oxidation of A*‘-sterols and the subsequent determination of the side-chain cleavage products. Ozonolysis has long been a reliable method for the proof of side-chain unsaturation (1)) and a method for microgram quantities has been reported (2). However, this procedure involves the use of somewhat specialized apparatus, and is not entirely satisfactory for quantitative measurements because the products are relatively volatile aldehydes. Oxidation with osmium tetroxide (3, 4) has been performed on both A22- and A24-sterols, and gas-liquid chromatography (GLC) has been used for product identification. The osmium tetroxide method is lengthy, however. Hydroboration followed by GLC of the resultant steroid alcohol as the trimethylsilyl derivative has been used to characterize double bonds but is not a quantitative method (5).

In this laboratory, a study of sterol oxidation by the microorganism Tetrahymena pyriformis W created the need for a specific, quantitative method for analytical side-chain oxidation where small amounts (1 mg or less) of starting material would be sufficient.

The oxidation of unsaturated fatty acids to identifiable short-chain acidic products using a mixture of periodate and permanganate was reported by Lemieux and von Rudloff (6) and von Rudloff (7). Korn has described a method for permanganate-periodate oxidation of small amounts of unsaturated fatty acids, with qualitative and quantitative determination of the products by GLC (8).

This paper describes the application of this general method to the oxidation of a sterol containing a A22-unsaturation, in which only one low molecular weight product is detectable by GLC, and qualitative analysis may be performed on as little as 0.8 pg of A22-stero1. This oxidative method serves as an independent proof of side-chain structure, and can be used to supplement evidence obtained by chromatographic and spectroscopic analysis.

230

MI%3 METHOD FOR A”2-~~~~~~~ 231

MATERIALS AND METHODS

Tertiary butanol (Matheson, Coleman & Bell, special high-purity grade for estimation of corticosteroids) was distilled from permanganate. Reagent-grade diethyl ether (Merck) and purified benzene (J. T. Baker) were distilled before use.

Standard acids for retention time studies were used without further purification, except for acetic acid which was distilled. All acids showed single peaks when subjected to GLC. Isovaleric acid (3-methylbutyric acid), n-butyric acid, and propionic acid were obtained from Matheson, Coleman & Bell. Isobutyric acid was purchased from Paragon Testing Laboratories, Orange, New Jersey. n-Valerie acid and pivalic acid (2,2-dimethylpropionic acid) were obtained from Eastman Organic Chemicals. nn-2-Methylbutyric acid was prepared by hydrolysis of nn-2-methylbutyryl chloride (Eastman Organic Chemicals). K & K Laboratories graciously donated a sample of cY-methylisovaleric acid (2,3-dimethylbutyric acid). Lu-Ethylisovaleraldehyde (2-ethyl-3-methyl- butyraldehyde) was obtained from Distillation Products Industries and oxidized to cu-ethylisovaleric acid with permanganate.

Two of the sterols tested, 22-dehydrocholesterol and 7,22-bisdehydro- cholesterol (A5~7922 -cholestatrien-3/3-ol) , were isolated from cultures of Tetrahymena pyriformis W grown in a medium supplemented with cholesterol. The procedures for isolation and identification of these compounds from this biological source have been described (9). These sterols were analyzed as the acetates.

Ergosterol, stigmasterol, cholesterol, and 7-dehydrocholesterol were purchased from Sigma Chemical Company, and were further purified. Ergosterol and stigmasterol were each recrystallized from a 1:l (v:v) mixture of acetone/ethanol. 7-Dehydrocholesterol was recrystallized three times from acetone. Cholesterol was purified through the dibromide (10). Each sterol after purification gave a single peak on GLC analysis using a 6 ft glass column packed with 0.75% SE-52 on Diatoport S, W-100 mesh (F & M Scientific Co.). The column temperature was 235”, and the nitrogen carrier flow rate was 100 ml/min. The instrument was an F & M 400 biomedical gas chromatograph, equipped with a hydrogen flame detector and a disc integrat’or.

Lathosterol was prepared by selective hydrogenation of 7-dehydro- cholesterol in the presence of Raney nickel (11). The reduction of the As-double bond was shown to be complete by the disappearance from the ultraviolet spectrum of the characteristic absorption of the A517- conjugated diene (12). After recrystallization from methanol, the lathosterol sample showed only one peak on GLC.

Weighed quantities of sterols or sterol acetates were dissolved in

232 MALLORY, FERGUSON, AND CONNER

appropriate volumes of benzene, and the sterol content and purity were verified by quantitative GLC. Aliquots of the sterol solutions, amounting to from 1 to 2 pmoles, were pipetted into 10 ml Erlenmeyer flasks. The benzene was removed by gently warming under a nitrogen stream and 0.5 ml of tert-butanol was introduced to redissolve the sterol. The flasks were allowed to cool to room temperature and 2 pmoles of potassium carbonate (0.05 ml of a 0.04M solution) was added co each, followed by 28.7 pmoles of oxidant (0.3 ml of a solution 0.0023 M in potassium permanganate and 0.0933 M in sodium metaperiodate). One reagent blank and two flasks containing reagents plus a known amount of isovaleric acid (2.1 pmoles) were included in each experiment as controls. The flasks were tightly stoppered and allowed to stand overnight at 37”. The contents of each flask were then acidified with 0.1 ml of 5 N sulfuric acid. Excess oxidant was destroyed by dropwise addition of aqueous sodium bisulfite solution until the permanganate was decolorized. The reaction mixture was extracted twice with 2 ml portions of diethyl ether, and the two extracts from each flask were combined and dried with anhydrous sodium sulfate. The volume of each ether extract was then reduced under a nitrogen stream and, after transfer to a 1 ml volumetric tube, was further reduced to exactly 1 ml.

Aliquots of these ether extracts were analyzed on a 6 ft column packed with 10% diethylene glycol succinate polyester plus 2% phos- phoric acid on Diatoport S, 60-80 mesh (F B: M Scientific Co.) ; this column will be designated hereinafter as DGS. Determinations were carried out at 112-115”, with a nitrogen flow rate of 75 ml/min. A solution of isovaleric acid in benzene (9.1 PmolesJml) was used as a standard for quantitative analysis. Each sample was chromatographed alone and with added acetic acid, and the retention times were calculated relative to acetic acid. Ten different acids of low molecular weight were also chromatographed to determine the limits of separation under these conditions. These acids represent all the two- to five-carbon carboxylic acids (excluding unsaturated and cyclic acids) and the six- and seven-carbon acids to be expected from oxidation of the naturally occurring C?, and C?g sterols, respectively. These reference acids were acetic, propionic, isobutyric, isovaleric, n-Valerie, n-butyric, 2-methyIbutyric, pivalic, 2,3-dimethylbutyric, and 2-ethyl-3-methyl- butyric.

RESdLTS The relative retention times (RRT) of the various short-chain acids

(compared to acetic acid as a standard) are shown in Table 1. Analysis of the acids obtained from the oxidation of known A**-sterols and

MICRO METHOD FOR A??-STEROLS 233

TABLE 1 Retention Times of Some Carboxylic Acids, Relative to Acetic Acid,

on Diethylene Glycol Succinate GLC

Compound

Acetic acid Propionic acid Isobutyric acid n-Butyric acid Pivalic acid

(2,2-dimethylpropionic acid) 2-Methylbutyric acid Isovaleric acid

(3-methylbutyric acid) n-Valerie acid cY-Methylisovaleric acid

(2,3-dimethylbutyric acid) cu-Ethylisovaleric acid

(2-ethyl-2-methylbutyric acid)

Number of carbons

2 3 4 4 5

5 5

5 6

7

RRT

1.0 1.3 1.3 1.8 1.1

1.9 1.9

2.7 2.4

3.0

RRT, relative retention time.

sterol acetates (ergosterol, stigmasterol, 22-dehydrocholesterol acetate, and 7,22-bisdehydrocholesteryl acetate) revealed symmetrical peaks with RRT values corresponding to those expected of the product from each compound (Table 2). Sterols lacking A**-unsaturation (cholesterol, lathosterol and 7-dehydrocholesterol) failed to give rise to any products detectable by GLC. The method is thus specific for the side-chain unsaturation. In each case, the oxidized fragment related to the sterol nucleus is not sufficiently volatile (i.e., has too large an RRT value) to be detected with the DGS column under the conditions employed.

Many determinations were performed on known amounts of isovaleric acid in order to evaluate the error inherent in the method. Yields from sterol oxidations were then corrected to allow for the loss of isovaleric acid from controls (Table 2). Good yields were obtained from the C?, and C,, sterols, but stigmasterol oxidations were incomplete. When stigmasterol oxidations were allowed to remain at 37” for 4 days, the yields of cu-ethylisovaleric acid were increased, but did not reach the levels attained with C,, and CZ, sterols.

The practical limits of the method were explored with ergosterol. Using reagent volumes of 0.2, 0.12, and 0.02 ml of tert-butanol, oxidant and potassium carbonate solutions, respectively, qualitative identification of a-methylisovaleric acid by GLC was possible when only 2 nanomoles of sterol was present. Quantitative analyses could be performed on 20

234 MALLORY, FERGUSON, AND CONNER

TABLE 2 Yields of Acidic Products from Oxidation of Several Sterols and Sterol Acetates

Sterol

Number Corrected of Av. % %

analyses recovery recovery S.D. Range RRT on

GLC

Isovsleric acid 38 (control)

7,22-Bisdehydrocho- 9 lesteryl acetate

Ergosterol 34 Stigmasterol 7 Stigmasterol, 4 days 10 22-Dehydrocho- 1

lesteryl acetate Cholesterol 3 Lathosterol 2 7-Dehydrocholesterol 3

91 100 5.35

89 98 3.7

81 89 8.6 25 28 6.1 55 60 6.9 61 67 -

None - - None - None -

M-104 1.9

82-95 1.9

63-97 2.4 19-35 3.0 50-70 3.0

- 1.9

- - - -

-

nanomoles of sterol. The limits of the quantitative determinations are set by the volume of tert-butanol used, since the tert-butanol is extracted into the ether layer and remains after ether removal; the final volume is therefore approximately equal to the volume of tert-butanol originally introduced.

A sample of radioactive 7,22-bisdehydrocholesteryl acetate was obtained from experiments in which 26J4C-cholesterol was incubated with Tetrahymena pyriformis W (9). When the acetate was oxidized, “C-isovaleric acid was formed, and the corrected recovery was 89% as measured by GLC. After addition of carrier isovaleric acid, the p-bromophenacyl derivative was prepared (13) and the derivative was isolated. The radioactivity of the p-bromophenacyl isovalerate was determined by liquid scintillation counting; it was found that the derivative contained 78% of the radioactivity originally isolated in 7,22-bisdehydrocholesteryl acetate. The 89% recovery as determined by GLC and the 78% recovery of 14C label as the p-bromophenacyl derivative are consistent, indicating the accuracy of the oxidative procedure.

DISCUSSION

A difficulty encountered with this procedure is the presence of a peak with RRT 1.3, arising from an unknown reaction involving tert-butanol; this peak is present in reagent blanks, but aqueous controls do not contain this component. tert-Butanol itself is not retained on the DGS column. Consumption of oxidant, by tert-butanol

MICRO METHOD FOR A%TEROLS 235

was reported by von Rudloff (7), even after repeated purifications of the alcohol. However, this unknown peak with RRT 1.3 is sufficiently removed from the peaks of interest (the oxidation products isovaleric acid, a-methylisovaleric acid, and a-ethylisovaleric acid have RRT values of 1.9, 2.4, and 3.0, respectively) that blank corrections are easily performed.

A second contaminant peak encountered in early studies was eliminated by drying the ether layer lcefore injection of sample into the DGS column. A third undesirable peak from oxidation of ether is avoided by destruction of excess oxidant prior to ether extraction.

The ratio of organic solvent to water (0.5/0.35) is important, since the solubilities of both sterol and oxidant in the solvent mixture appear to be limited. It is therefore not feasible to simply increase the concentration of oxidant as might be desired in the case of stigmasterol oxidations,

The permanganate-periodate oxidative method described is advan- tageous in that it is comparatively rapid, requires only simple manipu- lations, and can be used to analyze minute quantities of sterol. The procedure may be used to indicate the amount of AZ2-sterol present in a sterol mixture, and it serves as a direct structural proof for the presence or ab’sence of A22-unsaturation in an unknown sterol. The method should only be applied to samples which have been sufficiently purified that other substances (e.g., carotenoids or other unsaturated materials) will not interfere by depleting the oxidant or contributing extraneous GLC peaks. If the oxidation mixture loses its permanganate color, too much sample (or too little oxidant) has been used. The oxidation of more than 1 mg of sterol is easily accomplished by increasing the reagent volumes while maintaining the critical solvent ratio.

The cleavage products from the side chains of C2,, &, and C,, sterols bearing a AZ2-unsaturation are readily identified by their characteristic RRT values on the DGS chromatographic column. Other four- and five-carbon acids are clearly separated from the three acids of biological origin (Table l), with the exception of 2-methylbutyric acid, which has the same RRT as isovaleric acid, and cochromatographs with it. Cochromatography of an authentic sample of cY-methylisovaleric acid with the product from ergosterol oxidation, and of an authentic sample of cu-ethylisovaleric acid with the product from stigmasterol oxidation, gave in each case one single symmetrical peak. Strong evidence for the position of the double bond at C-22 and the structure of the terminal portion of the side chain is therefore provided by the appearance of isovaltric, a-methylisovaleric, or a-cthylisovaleric acid

236 MALLORY, FERGUSON, AND CONNER

in the ether extract of the oxidation mixture. The recoveries for the series C,,, C,,, and C,, sterols (Table 2) indicate that the oxidation may be subject to steric hindrance by methyl and especially ethyl substituents at C-24.

Qualitative analyses of sterols with unsaturations at other positions may be possible with modifications of the chromatographic conditions. The oxidation of sterols with AZ4-unsaturation should produce acetone, but because of its high volatility a different method of extraction and a much lower GLC column temperature would be required for the analysis. 24-Ethylidene sterols should be oxidized to give acetic acid, which would be readily detected under the analytical conditions de- scribed; however, appreciable losses of acetic acid by evaporation during the extraction and volume reduction would be expected to preclude using the-method for quantitative purposes in these cases. Sterols having two side-chain double bonds of the type found in A5~7@,24(28)-ergostate- traen-3p-bl, for example, are expected to give 2-oxoisovaleric acid on oxidation, which may be detectable by GLC on a DGS column at higher temperatures than employed in the present study (judging from the behavior at 112” of pyruvic acid, RRT 5.3, relative to that of Fropionic acid, RRT 1.3) ; the applicability of our method for the analysis of this type of sterol is being investigated.

SUMMARY

A rapid and efficient method for the quantitative determination of sterols containing a A”-unsaturation has been developed. The technique consists of a controlled periodate-permanganate oxidation of the CZ7, C2g) or Cs9 sterol, extraction of the acidic fragments, and analysis by gas-liquid chromatography of the C,, Ce, or C, carboxylic acid cleavage product using a 10% diethylene glycol succinate + 2% phosphoric acid liquid phase. Oxidation of 22-dehydrocholesteryl acetate and 7,22-bisde- hydrocholesteryl acetate produced isovaleric acid, and oxidation of ergosterol and stigmasterol gave cY-methylisovaleric acid and a-ethyl- isovaleric acid, respectively. Except for the stigmasterol oxidation, the yields were high. As expected, no short-chain acidic products could be detected from cholesterol, 7-dehydrocholesterol, or lathosterol.

ACKNOWLEDGMENTS

We are grateful to the National Science Foundation (GB-4605 and GB-12133) and the Bryn Mawr College Fund for the Coordination of the Sciences for support of this work, K. A. F. was supported by a National Science Foundation Traineeship.

MICRO METHOD FOR A”“-STEROLS 237

REFERENCES

1. SHOPPEE, C. W., “Chemistry of the Steroids,” 2nd ed., p. 29. Butterworth, London, 1964.

2. BEROZA, M., AND B. A. BIERL, Anal. Chem. .39, 1131 (1967). 3. CLAYTON, R. B., AND K. BLOCH, J. Biol. Chem. 218, 305 (1956). 4. SMITH, F. R., AND E. D. KORN, J. Lipid Res. 9, 405 (1968). 5. KNIGHTS, B. A., J. Gas Chromatog. 2, 160 (1964). 6. LEMIEUX, R. U., AND E. VON RUDLOFF, Can. J. Chem. 33, 1701 (1955). 7. VON RUDLOFF, E., Can. J. Chem. 34, 1413 (1956); 43, 2660 (1965). 8. KORN, E. D., J. Biol. Chem. 238, 3584 (1963). 9. CONNER, R. L., F. B. MALLORY, J. R. LANDREY, AND C. W. L. IYENGAR, J. Biol.

Chem. 244, 2325 (1969). 10. FIESER, L. F., “Experiments in Organic Chemistry,” p. 67. Heath, Boston, 1957. 11. FRANTZ, I. D., A. G. DAVIDSON, E. DULIT, AND M. L. MOBBERLEY, J. Biol. Chem.

234, 2290 (1959). 12. DORFMAN, L., Chem. Rev. 53, 47 (1953). 13. VOGEL, A. I., “A Textbook of Practical Organic Chemistry,” 3rd ld., p. 362.

Longmans, Green, London, 1956.