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Determination of dihydrocholesterol in serum
ANALYTICAL
BIOCHEMISTRY
Determination
10,
435-443
of
(1965)
Dihydrocholesterol
D. P. CHATTOPADHYAY
AND
in
Serum
E. H. MOSBACH
From the Department of Laboratory Diagnosis, Public Health Research Institute of the City of New York, Inc., and the Bureau of Laboratories, New York City Department of Health, New York, New York Received
July
9, 1964
The saturated sterol dihydrocholesterol (5a-cholestan-3/3-ol) accompanies cholesterol in the nonsaponifiable fraction of all mammalian tissuesstudied. The proportion of this stanol in tissue sterol fractions ranges from about 0.3% in human gallstones to more than 10% in rabbit and guinea-pig adrenals (1, 2). The high percentage of dihydrocholesterol found in adrenals suggestsan important function of this sterol which is as yet unknown. Studies on the metabolism of dihydrocholesterol required a method for its determination in serum. The procedure for the analysis of tissues previously developed in this laboratory (1) was time consuming and required excessively large amounts of serum. Therefore, a method was devised suitable for the determination of dihydrocholesterol in 0.2 to 1.0 ml of serum. The method is based on the finding that the serum sterols (consisting predominantly of cholest.erol and dihydrocholesterol) react quantitatively with acetic-l-C?’ anhydride and that the acetates can be separated completely and rapidly by thin-layer chromatography on AgNO,-silica gel plates (3-5). From the ratio of the radioactivities of the acetates, the rat,io of dihydrocholesterol to cholesterol in the samples can be calculated. By means of this procedure the percentage of dihydrocholesterol (0.1-1.0%/o) usually encountered in the serum total sterols can be determined with fair accuracy. MATERIALS
Acetic-l-W anhydride, New England Nuclear Corporation, Boston, Mass. (2.0 mc diluted to 1.0 ml with unlabeled acetic anhydride, Fisher Scientific Co., .#A-10). Pyridine, Fisher Scientific Co., #P-384, dried over KOH and redistilled. Digitonin, Hoffmann-La Roche, Nutley, N. J. Methylene chloride, Fisher Scientific Co,, #D-35. 435
436
CHATTOPADHYAY
AND
MOSBACH
Cholesterol, USP, purified twice via the dibromide (6). Dihydrocholesterol, supplied through the courtesy of Schering Corp., purified by the procedure described in “Organic Syntheses,” Coll. Vol. 2, p. 191 (1943). Dihydrocholesterol acetate prepared as described in “Organic Syntheses,” Coll. Vol. 2, p. 191 (1943). Silica gel G, #8076, Research Specialties Co., Richmond, Calif. 2,7-Dichlorofluorescein, spray reagent, Brinkmann Instruments, Inc., ,#9677. Silver nitrate, Fisher Scientific Co., ,#S-181. Capillary pipets, Harshaw Scientific Co., #H-55698. Scintillation liquid, 6 gm 2,5-diphenyloxazole (Fisher Scientific Co., .#6775) in 1000 ml toluene (Fisher Scientific Co., #T-324). Chloroform, Fisher Scientific Co., #C-298. METHOD
Extraction
of Sterol Fractiolz from Semm
The nonsaponifiable fraction of serum was obtained by a modification of the technique of Abel1 et al. (7) as follows: 0.2 to 1.0 ml serum was mixed with 5.0 ml alcoholic KOH solution (6 ml 33% KOH in 94 ml absolute ethanol) in 25-ml glass-stoppered centrifuge tubes. The tubes were heated at 60 + 1°C for 30 min and then allowed to cool to room temperature. n-Hexane (10.0 ml) and 5.0 ml of distilled water were added to each tube and the contents mixed thoroughly on a Vortex mixer. The tubes were then centrifuged at 2000 rpm for 5 min. An 8.0-ml aliquot of each hexane extract was pipetted into a 12-ml glass-stoppered centrifuge tube and the solvent was evaporated to dryness at 60°C under a stream of air. Determination
of Total Sterol Concentration
A separate sample of serum was processed as described above and the sterols present in the nonsaponifiable fraction were determined gravimetrically as the digitonides (1) ; in some samples cholesterol was determined calorimetrically by the method of Abel1 et al. (7). Acetylation
of Sterol Mixture
To each tube containing the dried nonsaponifiable fraction there was added 0.1 ml redistilled pyridine and 20 pl acetic-l-c” anhydride. This proportion of reagents was chosen to achieve quantitative acetylation and to use as little of the labeled acetic anhydride as possible. The tubes were stoppered, stirred on a Vortex mixer, and kept in a boiling water
DIHYDROCHOLESTEROL
DETERMINATION
437
bath for 1 hr. After cooling to room temperature, 3 ml methanol was added to each tube and the methanol was evaporated under a stream of air at 6O’C. The addition and evaporation of methanol was repeated. A third evaporation was performed with 4 ml of a methanol: benzene mixture (1: 1, v/v). These evaporations removed most of the pyridine and unreacted acetic anhydride. The acetylated sterols were dissolved in 0.5 ml chloroform containing 2.0 mg unlabeled carrier dihydrocholesterol acetate (the latter makes it possible to detect the dihydrocholesterol acetate on the thin-layer plate). A standard sample consisting of 1.99 mg cholesterol and 0.010 mg dihydrocholesterol was run with each set of unknowns. Preparation
of Plates
Glass plates (20 X 20 cm) were coat,ed with silver nitrate-silica gel G (40: 100 w/w) with a glass rod as described by Lees and De Muria (8). The layers were 0.17 mm thick. The adsorbent slurry was prepared with 37.5 gm silica gel G and a solution of 15 gm AgNO, in 75 ml distilled water. The plates were dried in air overnight and were activated for 30 min at 120°C. They were stored over anhydrous CaSOa. Plates older than 5 days were discarded. Chromatography
Aliquots (30 ~1) of the chloroform solutions of radioactive acetylated samples were applied on the thin-layer plates at 2.5-cm intervals. One spot on each plate was reserved for a 30-~1 aliquot of unlabeled dihydrocholesterol acetate (4.0 mg/ml chloroform) to serve as the “plate blank.” The plate was then developed in a standard chromatographic tank saturated with fresh methylene chloride. When the solvent had migrated to a height of about 15 cm, the plate was removed and air-dried under a hood. The plate was sprayed lightly and gently with 2,7-dichlorofluorescein, dried in air, and inspected under long-wave ultraviolet light (3660 A). As a rule only spots corresponding to cholesterol acetate and dihydrocholesterol acetate were observed, with RI values averaging 0.31 and 0.51, respectively. The position of the spots was marked with a teasing needle. Radioactivity
Determination
To recover the radioactive material from a spot, a straight capillary pipet was cut in half and the circular constriction of the upper part was plugged with a small piece of cotton (9). The two halves were connected with a short piece of rubber tubing. The entire spot was then aspirated as quantitatively as possible into the cotton plug. Any traces of silica gel which still remained on the plate were loosened by scraping with a micro-
438
CHATTOPADHYAY
AND
MOSBACH
spatula and likewise aspirated into the pipet. The rubber tubing was detached and the cotton containing the adsorbent was pushed into a scintillation vial; the two halves were rinsed twice with 1 ml of scintillation liquid. An additional 13 ml of scintillation liquid was added to the vial and the contents were mixed thoroughly in a Vortex mixer. The radioactivity was then measured in a Nuclear-Chicago model 702 scintillation counter equipped with an automatic turntable. Radioactivity data were not corrected for the difference in molecular weight between dihydrocholesterol and cholesterol. This difference of about 0.5% would be within the experimental error of the method. To minimize the error introduced by statistical fluctuation of the count rate, all samples were counted to a total of at least 2000 counts. The background count was taken from the unlabeled dihydrocholesterol acetate spot (“plate blank”). RESULTS
Known binary mixtures of cholesterol and dihydrocholesterol ranging in dihydrocholesterol content from 0. to 4.76% of the total sterol were analyzed. The recovery of dihydrocholesterol from the sterol mixtures was found to vary from 96.2 to 110.0%. The sample of cholesterol used in these experiments had been purified twice (6) and still contained 0.050% dihydrocholesterol when analyzed by the present procedure. This figure was subtracted from the calculated percentage of dihydrocholesterol listed in Table 1. In addition, a standard sample containing 0.498% dihydro-
ANALYSES
OF KNOWN
TABLE 1 MIXTURES OF CHOLESTEROL
%
4.76 2.44 1.96 1.48 0.99 0.74 0.50 0.25 0.10 0.00 = Dihydrocholesterol. 6 The cholesterol
mg
DHC.0 me
Total stero1, m3
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
0.100 0.050 0.040 0.030 0.020 0.015 0.010 0.005 0.002 0.000
2.100 2.050 2.040 2.030 2.020 2.015 2.010 2.005 2.002 2.000
used in this
AND DIHYDROCHOLESTEROL
D
C
IA3
present in mixture,
BINARY
study
still
contained
4.63-0.05b 2.60-0.05 2.10-0.05 1.60-0.05 1.04-0.05 0.81-0.05 0.55-0.05 0.32-0.05 0.16-0.05 0.050b
0.050%
= = = = = = = = =
4.58 2.55 2.05 1.55 0.99 0.76 0.50 0.27 0.11
dihydrocholesterol.
96.2 104.5 104.6 104.7 100.0 102.7 100.0 108.0 110.0
DIHYDROCHOLESTEROL
439
DETERMINATION
cholesterol and 99.502oJo cholesterol was analyzed 16 times, and the results obtained averaged 0.482% dihydrocholesterol with a standard error of the mean of 0.016. It can be seen that there is good agreement between the calculated composition of the samples and the analytical results.
EFFECT
TABLE 2 OF SAMPLE SIZE ON ANALYSES OF KNOWN OF CHOLESTEROL-DIHYDROCHOLESTEROL
BINARY
MIXTURES
Totalt;k;;;~dded DHC present in mixture, %
4.76
2.44
0.99
0.00
a Dihydrocholesterol. b The cholesterol
Radioactivity. Weight, Irg
42.0 84.0
41.0 82.0 123.0
40.4 80.8 121.2
80.0 160.0
sample
volre’
10 20
10 20 30
10 20 30
20 40
used
cpm
Cholesterol
DHC-
5,527 10,863
260 518
4,772 10,856 15,869
53 117 192
6,904 13,200
contained
31 52
0.0420/,
DHC recovered. %
4.49 4.55 4.52 - 0.042b = 4.478
129 293 424
4,926 10,789 816,558
DHC found, %
2.63 2.63 2.60 2.62 - 0.042 = 2.578 1.06 1.07 1.14 1.09 - 0.042 = 1.048
94.08
105.65
105.85
0.045 0.039 0.042b
dihydrocholesterol
on the average.
The experiments listed in Table 2 were carried out in order to determine the effect of sample size on the accuracy of the determination. The data show that the total sterol applied to each spot can be varied from 40 to 120 pg without incurring significant errors. It should be pointed out that the lower limit of the sample size is set by the radioactivity of the dihydrocholesterol acetate. If the radioactivity is too low, unfavorable counting statistics will be encountered. If the sample size exceeds 200 pg, overloading of the plate may cause trailing of the sample and prevent an accurate analysis.
440
CHATTOPADHYAY
AND
TABLE REPLICATE
ANALYSES
OF POOLED Number of determinations
Specier,
Monkey Dog Man a Standard
MONKEY,
3 DOG,
AND
HUMAN
Dihydrocholesterol in total sterol,
5 5 5 error
MOSBACH
0.963 0.356 0.738
SERUM
%
SAMPLES Range
f O.O1la f 0.009 f 0.008
0.935-0.988 0.339-0.389 0.719-0.769
of the mean.
Table 3 summarizes analyses of pooled serum from 3 sources, analyzed in quintuplicate. The data indicate that, on the average, there may be an error of &50/o. The maximum error observed in the analyses listed in Table 3 was +8.5%. Recovery experiments were carried out with monkey serum and hypercholesterolemic dog serum and the results are listed in Table 4. Recoveries ranging from 102.1 to 107.7% were observed.
RECOVERY
EXPERIMENT
selvm sample
Total sterol, mg/~
Monkey
1.673
Dog
2.96
DHC
USING added, Irg
0 20 40 60 80 0 20 40 60 80
TABLE SERUM DHC
present, La
17.2 38.2 58.6 78.5 102.7 14.2 35.6 55.6 78.8 100.3
4 SAMPLES DHC
FROM
MONKEY
Added recovered, w
AND DOG Per centage of added DHC recovered
21.0 41.4 61.3 85.5 -
105.0 103.5 102.2 106.9 -
21.4 41.4 64.6 86.1
107.0 103.5 107.7 107.6
Table 5 shows the results of dihydrocholesterol determinations in the serum of some common species of laboratory animals and in samples of pooled serum from germ-free mice. All the animals had been maintained on standard laboratory chow diets. Dihydrocholesterol in the serum sterols of the animals ranged from 0.122% in the rat to 0.854% in the monkey. DISCUSSION
The accuracy of the method described in this paper depends largely on two factors: First, the resolution of the sterol acetates on the thin-layer plates must be adequate to prevent any contamination of the dihydro-
DIHYDROCHOLESTEROL
TABLE DIHYDROCHOLESTEROL
Species
Monkey Dog= Dog Rabbit Rat Guinea pig Chicken Mouse Germ-free mouse
Number animals
5 3 3 5 5 5 5 6d 12d
CONTENT
of
Average 8emlIn cholesterol concn., rng/lOO ml
167 1450 182 39.1 70.3 40.3 132 c e
441
DETERMINATION
5
OF SERUM
Average serum DHC” concn., mg/loo ml
1.42 5.60 0.471 0.186 0.086 0.155 0.296 e e
IN
LABORATORY
Per cent of DHC’ in serum tots1 sterols
0.854 0.378 0.259 0.475 0.1% 0.353 0.244 0.485 0.290
S.E.M.b
0.04s 0.058 0.017 0.026 0.066 B e
ANIMALS
Range
0.716-0.967 0.363-0.391 0.211-0.304 0.327-0.645 0.115-0.131 0.303-0.450 0.141-0.257 e e
a Dihydrocholesterol. b Standard error of the mean. c Cholesterol + thiouracil treated. d Pooled sample. e Not determined.
cholesterol acetate with the much more highly labeled cholesterol acetat,e or the percentage of dihydrocholesterol found will be too high. Second, the specific activity of the labeled acetic anhydride must be high enough to give satisfactory counting rates for the dihydrocholesterol acetate. Statistical and practical consideration lead to the conclusion that the counting rate of the dihydrocholesterol acetate sample should be 2 to 4 times greater than the background counting rate. If the two conditions are met satisfactorily it seems possible to achieve accurate determinations of dihydrocholesterol (+5%). The dihydrocholesterol content of serum is calculated by assuming that the stanol and cholesterol are the only sterols present in the serum. The occurrence of other sterols, whose acetates have Rf values identical to that of dihydrocholesterol acetate would interfere with the procedure described here. It is more likely, however, that other trace sterols in serum would either be unsaturated or have additional oxygen functions so that they would not migrate at the same rate as dihydrocholesterol acetate. In this connection it is apparent that the serum of animals containing appreciable amounts of noncholesterol sterol (e.g., desmosterol following triparanol administration) might also be analyzed by this procedure providing the Rf values of the sterols differ sufficiently from those of cholesterol and dihydrocholesterol. The dihydrocholesterol content of the serum of some common labora-
442
CHATTOPADHYAY
AND
MOSBACH
tory animals ranged from 0.122% in the rat to 0.854% in the monkey. In each species the serum concentration of dihydrocholesterol varied over a relatively narrow range. Not enough data are available at the present time to decide whether serum dihydrocholesterol concentrations are subject to the same type of dietary and pharmacological manipulation as has been demonstrated in the case of serum cholesterol. In connection with Table 5, two findings are of interest: First, the dihydrocholesterol content of rabbit and guinea pig serum was of the order of 0.5%, although certain other organs of these species, particularly liver and adrenal, contained a much larger percentage of dihydrocholesterol, ranging from 1 to 5% in liver to 10 to 15% in adrenals (1, 2). Second, the serum of germ-free mice contained appreciable concentrations of dihydrocholesterol. This is in accord with the results of previous studies that dihydrocholesterol is not exclusively derived via the action of intestinal microorganisms on cholesterol, but is produced by animal tissues in viva and in vitro (10). However, since the food of the germ-free mice was not available for analysis, the possibility was not excluded that the tissue dihydrocholesterol in these animals was of exogenous origin. SUMMARY
(1) An isotope derivative procedure was developed for the determination of dihydrocholesterol (5a-cholestan-3p-01) in 0.2 to 1.0 ml of serum. The method is based on the finding that the serum sterols, consisting predominantly of cholest,erol and dihydrocholesterol, react quantitatively with acetic-l-V anhydride and that the acetates are separated completely and rapidly by thin-layer chromatography on AgNO,-silicic acid plates. The ratio of the radioactivities of the acetates will then correspond to the molar ratio of cholesterol to dihydrocholesterol in the sample. (2) The feasibility of the method was tested by analyses of known cholesterol and dihydrocholesterol mixtures and by recovery experiments. The reproducibility of the procedure was checked by replicate analyses of serum from different species of animals. (3) Serum dihydrocholesterol concentrations were determined in 7 species of laboratory animals maintained on a stock diet. The concentration of dihydrocholesterol ranged from 0.093 mg per 100 ml in the rat to 1.43 mg per 100 ml in the monkey. Chicken, guinea pig, dog, and mouse exhibited intermediate values. 1. MOSBACH, (1903). 2. WERBIN,
E. H., BLUM,
REFERENCES J., ARROYO, E., AND MILCH,
S., And.
H., CHAIKOFF, I. L., AND IMADA, M. R., J. Biol. Chem. 3. DE VRIES, B., Chem. Id. (London) 1962,1049.
Biochem. 237,
5, 158
2073 (1962).
DIHTDROCHOLESTEROL
DETERMINATION
443
4. BARRETT, C. B., DALLAS, M. S. J., AND PADLEY, F. B., Chem. Znd. (London) 1962, 1050. 5. AVIGAN, J., GOODMAN, DEW. S., AND STEINBERG, D., J. Lipid Res. 4, 100 (1963). 6. “Organic Syntheses,” Coll. Vol. IV, p. 193. Wiley, New York, 1963. 7. ABELL, L. L., LEVY, B. B., BRODY, B. B., AND KENDALL, F. E., J. Biol. Chem. 195, 357 (1952). 8. LEES, T. M., AND DE MURIA, P. J., J. Chromatog. 8, 168 (1962). 9. MOT-TIER, M., Mitt. Gebiete Lebensm. Hyg. 49, 454 (19581, cited in Bobbitt, J. M., “Thin-Layer Chromatography,” p. 113. Reinhold, New York, 1963. 10. SHEFER, S., MILCH, S., AND MOSBACH, E. H., J. Biol. Chem. 239, 1731 (1964).
BIOCHEMISTRY
Determination
10,
435-443
of
(1965)
Dihydrocholesterol
D. P. CHATTOPADHYAY
AND
in
Serum
E. H. MOSBACH
From the Department of Laboratory Diagnosis, Public Health Research Institute of the City of New York, Inc., and the Bureau of Laboratories, New York City Department of Health, New York, New York Received
July
9, 1964
The saturated sterol dihydrocholesterol (5a-cholestan-3/3-ol) accompanies cholesterol in the nonsaponifiable fraction of all mammalian tissuesstudied. The proportion of this stanol in tissue sterol fractions ranges from about 0.3% in human gallstones to more than 10% in rabbit and guinea-pig adrenals (1, 2). The high percentage of dihydrocholesterol found in adrenals suggestsan important function of this sterol which is as yet unknown. Studies on the metabolism of dihydrocholesterol required a method for its determination in serum. The procedure for the analysis of tissues previously developed in this laboratory (1) was time consuming and required excessively large amounts of serum. Therefore, a method was devised suitable for the determination of dihydrocholesterol in 0.2 to 1.0 ml of serum. The method is based on the finding that the serum sterols (consisting predominantly of cholest.erol and dihydrocholesterol) react quantitatively with acetic-l-C?’ anhydride and that the acetates can be separated completely and rapidly by thin-layer chromatography on AgNO,-silica gel plates (3-5). From the ratio of the radioactivities of the acetates, the rat,io of dihydrocholesterol to cholesterol in the samples can be calculated. By means of this procedure the percentage of dihydrocholesterol (0.1-1.0%/o) usually encountered in the serum total sterols can be determined with fair accuracy. MATERIALS
Acetic-l-W anhydride, New England Nuclear Corporation, Boston, Mass. (2.0 mc diluted to 1.0 ml with unlabeled acetic anhydride, Fisher Scientific Co., .#A-10). Pyridine, Fisher Scientific Co., #P-384, dried over KOH and redistilled. Digitonin, Hoffmann-La Roche, Nutley, N. J. Methylene chloride, Fisher Scientific Co,, #D-35. 435
436
CHATTOPADHYAY
AND
MOSBACH
Cholesterol, USP, purified twice via the dibromide (6). Dihydrocholesterol, supplied through the courtesy of Schering Corp., purified by the procedure described in “Organic Syntheses,” Coll. Vol. 2, p. 191 (1943). Dihydrocholesterol acetate prepared as described in “Organic Syntheses,” Coll. Vol. 2, p. 191 (1943). Silica gel G, #8076, Research Specialties Co., Richmond, Calif. 2,7-Dichlorofluorescein, spray reagent, Brinkmann Instruments, Inc., ,#9677. Silver nitrate, Fisher Scientific Co., ,#S-181. Capillary pipets, Harshaw Scientific Co., #H-55698. Scintillation liquid, 6 gm 2,5-diphenyloxazole (Fisher Scientific Co., .#6775) in 1000 ml toluene (Fisher Scientific Co., #T-324). Chloroform, Fisher Scientific Co., #C-298. METHOD
Extraction
of Sterol Fractiolz from Semm
The nonsaponifiable fraction of serum was obtained by a modification of the technique of Abel1 et al. (7) as follows: 0.2 to 1.0 ml serum was mixed with 5.0 ml alcoholic KOH solution (6 ml 33% KOH in 94 ml absolute ethanol) in 25-ml glass-stoppered centrifuge tubes. The tubes were heated at 60 + 1°C for 30 min and then allowed to cool to room temperature. n-Hexane (10.0 ml) and 5.0 ml of distilled water were added to each tube and the contents mixed thoroughly on a Vortex mixer. The tubes were then centrifuged at 2000 rpm for 5 min. An 8.0-ml aliquot of each hexane extract was pipetted into a 12-ml glass-stoppered centrifuge tube and the solvent was evaporated to dryness at 60°C under a stream of air. Determination
of Total Sterol Concentration
A separate sample of serum was processed as described above and the sterols present in the nonsaponifiable fraction were determined gravimetrically as the digitonides (1) ; in some samples cholesterol was determined calorimetrically by the method of Abel1 et al. (7). Acetylation
of Sterol Mixture
To each tube containing the dried nonsaponifiable fraction there was added 0.1 ml redistilled pyridine and 20 pl acetic-l-c” anhydride. This proportion of reagents was chosen to achieve quantitative acetylation and to use as little of the labeled acetic anhydride as possible. The tubes were stoppered, stirred on a Vortex mixer, and kept in a boiling water
DIHYDROCHOLESTEROL
DETERMINATION
437
bath for 1 hr. After cooling to room temperature, 3 ml methanol was added to each tube and the methanol was evaporated under a stream of air at 6O’C. The addition and evaporation of methanol was repeated. A third evaporation was performed with 4 ml of a methanol: benzene mixture (1: 1, v/v). These evaporations removed most of the pyridine and unreacted acetic anhydride. The acetylated sterols were dissolved in 0.5 ml chloroform containing 2.0 mg unlabeled carrier dihydrocholesterol acetate (the latter makes it possible to detect the dihydrocholesterol acetate on the thin-layer plate). A standard sample consisting of 1.99 mg cholesterol and 0.010 mg dihydrocholesterol was run with each set of unknowns. Preparation
of Plates
Glass plates (20 X 20 cm) were coat,ed with silver nitrate-silica gel G (40: 100 w/w) with a glass rod as described by Lees and De Muria (8). The layers were 0.17 mm thick. The adsorbent slurry was prepared with 37.5 gm silica gel G and a solution of 15 gm AgNO, in 75 ml distilled water. The plates were dried in air overnight and were activated for 30 min at 120°C. They were stored over anhydrous CaSOa. Plates older than 5 days were discarded. Chromatography
Aliquots (30 ~1) of the chloroform solutions of radioactive acetylated samples were applied on the thin-layer plates at 2.5-cm intervals. One spot on each plate was reserved for a 30-~1 aliquot of unlabeled dihydrocholesterol acetate (4.0 mg/ml chloroform) to serve as the “plate blank.” The plate was then developed in a standard chromatographic tank saturated with fresh methylene chloride. When the solvent had migrated to a height of about 15 cm, the plate was removed and air-dried under a hood. The plate was sprayed lightly and gently with 2,7-dichlorofluorescein, dried in air, and inspected under long-wave ultraviolet light (3660 A). As a rule only spots corresponding to cholesterol acetate and dihydrocholesterol acetate were observed, with RI values averaging 0.31 and 0.51, respectively. The position of the spots was marked with a teasing needle. Radioactivity
Determination
To recover the radioactive material from a spot, a straight capillary pipet was cut in half and the circular constriction of the upper part was plugged with a small piece of cotton (9). The two halves were connected with a short piece of rubber tubing. The entire spot was then aspirated as quantitatively as possible into the cotton plug. Any traces of silica gel which still remained on the plate were loosened by scraping with a micro-
438
CHATTOPADHYAY
AND
MOSBACH
spatula and likewise aspirated into the pipet. The rubber tubing was detached and the cotton containing the adsorbent was pushed into a scintillation vial; the two halves were rinsed twice with 1 ml of scintillation liquid. An additional 13 ml of scintillation liquid was added to the vial and the contents were mixed thoroughly in a Vortex mixer. The radioactivity was then measured in a Nuclear-Chicago model 702 scintillation counter equipped with an automatic turntable. Radioactivity data were not corrected for the difference in molecular weight between dihydrocholesterol and cholesterol. This difference of about 0.5% would be within the experimental error of the method. To minimize the error introduced by statistical fluctuation of the count rate, all samples were counted to a total of at least 2000 counts. The background count was taken from the unlabeled dihydrocholesterol acetate spot (“plate blank”). RESULTS
Known binary mixtures of cholesterol and dihydrocholesterol ranging in dihydrocholesterol content from 0. to 4.76% of the total sterol were analyzed. The recovery of dihydrocholesterol from the sterol mixtures was found to vary from 96.2 to 110.0%. The sample of cholesterol used in these experiments had been purified twice (6) and still contained 0.050% dihydrocholesterol when analyzed by the present procedure. This figure was subtracted from the calculated percentage of dihydrocholesterol listed in Table 1. In addition, a standard sample containing 0.498% dihydro-
ANALYSES
OF KNOWN
TABLE 1 MIXTURES OF CHOLESTEROL
%
4.76 2.44 1.96 1.48 0.99 0.74 0.50 0.25 0.10 0.00 = Dihydrocholesterol. 6 The cholesterol
mg
DHC.0 me
Total stero1, m3
2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
0.100 0.050 0.040 0.030 0.020 0.015 0.010 0.005 0.002 0.000
2.100 2.050 2.040 2.030 2.020 2.015 2.010 2.005 2.002 2.000
used in this
AND DIHYDROCHOLESTEROL
D
C
IA3
present in mixture,
BINARY
study
still
contained
4.63-0.05b 2.60-0.05 2.10-0.05 1.60-0.05 1.04-0.05 0.81-0.05 0.55-0.05 0.32-0.05 0.16-0.05 0.050b
0.050%
= = = = = = = = =
4.58 2.55 2.05 1.55 0.99 0.76 0.50 0.27 0.11
dihydrocholesterol.
96.2 104.5 104.6 104.7 100.0 102.7 100.0 108.0 110.0
DIHYDROCHOLESTEROL
439
DETERMINATION
cholesterol and 99.502oJo cholesterol was analyzed 16 times, and the results obtained averaged 0.482% dihydrocholesterol with a standard error of the mean of 0.016. It can be seen that there is good agreement between the calculated composition of the samples and the analytical results.
EFFECT
TABLE 2 OF SAMPLE SIZE ON ANALYSES OF KNOWN OF CHOLESTEROL-DIHYDROCHOLESTEROL
BINARY
MIXTURES
Totalt;k;;;~dded DHC present in mixture, %
4.76
2.44
0.99
0.00
a Dihydrocholesterol. b The cholesterol
Radioactivity. Weight, Irg
42.0 84.0
41.0 82.0 123.0
40.4 80.8 121.2
80.0 160.0
sample
volre’
10 20
10 20 30
10 20 30
20 40
used
cpm
Cholesterol
DHC-
5,527 10,863
260 518
4,772 10,856 15,869
53 117 192
6,904 13,200
contained
31 52
0.0420/,
DHC recovered. %
4.49 4.55 4.52 - 0.042b = 4.478
129 293 424
4,926 10,789 816,558
DHC found, %
2.63 2.63 2.60 2.62 - 0.042 = 2.578 1.06 1.07 1.14 1.09 - 0.042 = 1.048
94.08
105.65
105.85
0.045 0.039 0.042b
dihydrocholesterol
on the average.
The experiments listed in Table 2 were carried out in order to determine the effect of sample size on the accuracy of the determination. The data show that the total sterol applied to each spot can be varied from 40 to 120 pg without incurring significant errors. It should be pointed out that the lower limit of the sample size is set by the radioactivity of the dihydrocholesterol acetate. If the radioactivity is too low, unfavorable counting statistics will be encountered. If the sample size exceeds 200 pg, overloading of the plate may cause trailing of the sample and prevent an accurate analysis.
440
CHATTOPADHYAY
AND
TABLE REPLICATE
ANALYSES
OF POOLED Number of determinations
Specier,
Monkey Dog Man a Standard
MONKEY,
3 DOG,
AND
HUMAN
Dihydrocholesterol in total sterol,
5 5 5 error
MOSBACH
0.963 0.356 0.738
SERUM
%
SAMPLES Range
f O.O1la f 0.009 f 0.008
0.935-0.988 0.339-0.389 0.719-0.769
of the mean.
Table 3 summarizes analyses of pooled serum from 3 sources, analyzed in quintuplicate. The data indicate that, on the average, there may be an error of &50/o. The maximum error observed in the analyses listed in Table 3 was +8.5%. Recovery experiments were carried out with monkey serum and hypercholesterolemic dog serum and the results are listed in Table 4. Recoveries ranging from 102.1 to 107.7% were observed.
RECOVERY
EXPERIMENT
selvm sample
Total sterol, mg/~
Monkey
1.673
Dog
2.96
DHC
USING added, Irg
0 20 40 60 80 0 20 40 60 80
TABLE SERUM DHC
present, La
17.2 38.2 58.6 78.5 102.7 14.2 35.6 55.6 78.8 100.3
4 SAMPLES DHC
FROM
MONKEY
Added recovered, w
AND DOG Per centage of added DHC recovered
21.0 41.4 61.3 85.5 -
105.0 103.5 102.2 106.9 -
21.4 41.4 64.6 86.1
107.0 103.5 107.7 107.6
Table 5 shows the results of dihydrocholesterol determinations in the serum of some common species of laboratory animals and in samples of pooled serum from germ-free mice. All the animals had been maintained on standard laboratory chow diets. Dihydrocholesterol in the serum sterols of the animals ranged from 0.122% in the rat to 0.854% in the monkey. DISCUSSION
The accuracy of the method described in this paper depends largely on two factors: First, the resolution of the sterol acetates on the thin-layer plates must be adequate to prevent any contamination of the dihydro-
DIHYDROCHOLESTEROL
TABLE DIHYDROCHOLESTEROL
Species
Monkey Dog= Dog Rabbit Rat Guinea pig Chicken Mouse Germ-free mouse
Number animals
5 3 3 5 5 5 5 6d 12d
CONTENT
of
Average 8emlIn cholesterol concn., rng/lOO ml
167 1450 182 39.1 70.3 40.3 132 c e
441
DETERMINATION
5
OF SERUM
Average serum DHC” concn., mg/loo ml
1.42 5.60 0.471 0.186 0.086 0.155 0.296 e e
IN
LABORATORY
Per cent of DHC’ in serum tots1 sterols
0.854 0.378 0.259 0.475 0.1% 0.353 0.244 0.485 0.290
S.E.M.b
0.04s 0.058 0.017 0.026 0.066 B e
ANIMALS
Range
0.716-0.967 0.363-0.391 0.211-0.304 0.327-0.645 0.115-0.131 0.303-0.450 0.141-0.257 e e
a Dihydrocholesterol. b Standard error of the mean. c Cholesterol + thiouracil treated. d Pooled sample. e Not determined.
cholesterol acetate with the much more highly labeled cholesterol acetat,e or the percentage of dihydrocholesterol found will be too high. Second, the specific activity of the labeled acetic anhydride must be high enough to give satisfactory counting rates for the dihydrocholesterol acetate. Statistical and practical consideration lead to the conclusion that the counting rate of the dihydrocholesterol acetate sample should be 2 to 4 times greater than the background counting rate. If the two conditions are met satisfactorily it seems possible to achieve accurate determinations of dihydrocholesterol (+5%). The dihydrocholesterol content of serum is calculated by assuming that the stanol and cholesterol are the only sterols present in the serum. The occurrence of other sterols, whose acetates have Rf values identical to that of dihydrocholesterol acetate would interfere with the procedure described here. It is more likely, however, that other trace sterols in serum would either be unsaturated or have additional oxygen functions so that they would not migrate at the same rate as dihydrocholesterol acetate. In this connection it is apparent that the serum of animals containing appreciable amounts of noncholesterol sterol (e.g., desmosterol following triparanol administration) might also be analyzed by this procedure providing the Rf values of the sterols differ sufficiently from those of cholesterol and dihydrocholesterol. The dihydrocholesterol content of the serum of some common labora-
442
CHATTOPADHYAY
AND
MOSBACH
tory animals ranged from 0.122% in the rat to 0.854% in the monkey. In each species the serum concentration of dihydrocholesterol varied over a relatively narrow range. Not enough data are available at the present time to decide whether serum dihydrocholesterol concentrations are subject to the same type of dietary and pharmacological manipulation as has been demonstrated in the case of serum cholesterol. In connection with Table 5, two findings are of interest: First, the dihydrocholesterol content of rabbit and guinea pig serum was of the order of 0.5%, although certain other organs of these species, particularly liver and adrenal, contained a much larger percentage of dihydrocholesterol, ranging from 1 to 5% in liver to 10 to 15% in adrenals (1, 2). Second, the serum of germ-free mice contained appreciable concentrations of dihydrocholesterol. This is in accord with the results of previous studies that dihydrocholesterol is not exclusively derived via the action of intestinal microorganisms on cholesterol, but is produced by animal tissues in viva and in vitro (10). However, since the food of the germ-free mice was not available for analysis, the possibility was not excluded that the tissue dihydrocholesterol in these animals was of exogenous origin. SUMMARY
(1) An isotope derivative procedure was developed for the determination of dihydrocholesterol (5a-cholestan-3p-01) in 0.2 to 1.0 ml of serum. The method is based on the finding that the serum sterols, consisting predominantly of cholest,erol and dihydrocholesterol, react quantitatively with acetic-l-V anhydride and that the acetates are separated completely and rapidly by thin-layer chromatography on AgNO,-silicic acid plates. The ratio of the radioactivities of the acetates will then correspond to the molar ratio of cholesterol to dihydrocholesterol in the sample. (2) The feasibility of the method was tested by analyses of known cholesterol and dihydrocholesterol mixtures and by recovery experiments. The reproducibility of the procedure was checked by replicate analyses of serum from different species of animals. (3) Serum dihydrocholesterol concentrations were determined in 7 species of laboratory animals maintained on a stock diet. The concentration of dihydrocholesterol ranged from 0.093 mg per 100 ml in the rat to 1.43 mg per 100 ml in the monkey. Chicken, guinea pig, dog, and mouse exhibited intermediate values. 1. MOSBACH, (1903). 2. WERBIN,
E. H., BLUM,
REFERENCES J., ARROYO, E., AND MILCH,
S., And.
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Biochem. 237,
5, 158
2073 (1962).
DIHTDROCHOLESTEROL
DETERMINATION
443
4. BARRETT, C. B., DALLAS, M. S. J., AND PADLEY, F. B., Chem. Znd. (London) 1962, 1050. 5. AVIGAN, J., GOODMAN, DEW. S., AND STEINBERG, D., J. Lipid Res. 4, 100 (1963). 6. “Organic Syntheses,” Coll. Vol. IV, p. 193. Wiley, New York, 1963. 7. ABELL, L. L., LEVY, B. B., BRODY, B. B., AND KENDALL, F. E., J. Biol. Chem. 195, 357 (1952). 8. LEES, T. M., AND DE MURIA, P. J., J. Chromatog. 8, 168 (1962). 9. MOT-TIER, M., Mitt. Gebiete Lebensm. Hyg. 49, 454 (19581, cited in Bobbitt, J. M., “Thin-Layer Chromatography,” p. 113. Reinhold, New York, 1963. 10. SHEFER, S., MILCH, S., AND MOSBACH, E. H., J. Biol. Chem. 239, 1731 (1964).