A new method for the determination of dihydrocholesterol in tissues

ANALYTICAL

BIOCHEMISTRY

A New

5, 158-169 (1963)

Method

for the

Dihydrocholesterol E. H. MOSBACH,

J. BLUM,

Determination

of

in Tissues E. ARROYO,

AND

S. MILCH

From the Division of Laboratory Diagnosis, Public Health Research Institute of the City of New York, Inc., and the Bureau of Laboratories, New York City Health Department, New York, New York Received June 25, 1962

INTRODUCTION Dihydrocholesterol (cholestan-S/3-01) has been found in the nonsaponifiable fraction of all mammalian tissues studied so far (1). The biological origin and functions of this sterol are not known but it has been suggested that dihydrocholesterol is involved in the pathogenesis of atherosclerosis and cholelithiasis. This conclusion is based upon reports claiming that relatively large concentrations of dihydrocholesterol were present in atherosclerotic lesions and cholesterol gallstones. For example, Schoenheimer (2) found 1.2-1.9s dihydrocholesterol in the sterol fraction isolated from human gallstones and 5.1-5.3s dihydrocholesterol in the sterols of human atherosclerotic aortas. McArthur (3) found 11.7-12.9s dihydrocholesterol in the nonsaponifiable matter obtained from human atherosclerotic aortas. The results of animal experiments support the assumption that dihydrocholesterol may play a role in the etiology of cholelithiasis and atherosclerosis. It is known, for example, that the feeding of dihydrocholesterol to rabbits and mice will produce cholecystitis and cholelithiasis (4), and this sterol has further been shown to be atherogenic in rabbits and chicks (5, 6). Studies of the metabolism of dihydrocholesterol in mammalian tissues required a method for the quantitative determination of this sterol in the presence of a large excess of cholesterol, since these two sterols always seem to occur together. The bromination method of Schoenheimer (2) was found to be unsatisfactory when the dihydrocholesterol content of the sterol fraction was less than 3% (which is usually the case in most tissues). The present report describes a new method for the determination of dihvdrocholesterol. This method is based upon the finding that oxidation of cholesterol-dihydrocholesterol mixtures with performic acid (7) converts cholesterol to cholestane-3/3,&,6@triol leaving the saturated

DIHYDROCHOLESTEROL

DETERMINATION

159

sterol, dihydrocholesterol, unchanged. The oxidation mixture is resolved into its components by chromatography on silicic acid columns (8). Dihydrocholesterol in the column effluent is determined as the digitonide, either gravimetrically or calorimetrically by the anthrone reaction (9). By means of this procedure as little as 0.2 mg of dihydrocholesterol can be determined with good accuracy even if a loo-fold excess of cholesterol is present. MATERIALS

Fmmic acid, Fisher Scientific Company, #A-118. Di1ut.e with water to make an 85% solution (v/v). n-Hexune, spectroanalyzed, Fisher Scientific Company. #H-334. Hydrogen peroxide, 30% solution, Becco Chemical Division, Food Machinery and Chemical Corporation, Buffalo 7, N. Y. Ethyl ether, anhydrous, absolute. Silicic acid, Bio-Rad, California Corporation for Biochemical Research, Los Angeles 63, Calif. Digitonin, Hoffman LaRoche, Nutley, N. ,J. Cho,lesteroE,free of dihydrocholesterol, prepared from cholesterol, USP, by the bromination procedure of Schoenheimer (2). Dihydrocholesterol, free of cholesterol, prepared by the procedure described in “Organic Syntheses,” Coll, Vol. 2, p. 191 (1943). Cholesterol-4-W, Nuclear-Chicago. Purified by chromatography on silicic acid (8). METHOD

1. Extraction

of Sterol Fraction from Tissues

(a) Serum. The serum was heated with 5 vol of 5% (w/v) KOH in 95% ethanol at 60 & 1°C for 1 hr. An equal volume of water was added and the solution was extracted 3 times with r/c its voIume of n-hexane. An aliquot of this solution was taken for determination of total sterol concentration by digitonin precipitation. After evaporation of the solvent (60°C water bath, under a stream of nitrogen) this nonsaponifiable fraction was used directly for the oxidation step (step 2). (b) Other Tissues. The tissues were hydrolyzed with 5 or 10% KOH (w/v) in 95% ethanol using at least 5 ml of KOH solution per gram of tissues. In the case of cholesterol gallstones 25 ml of alcoholic KOH per gram of stone was required to achieve complete solution. In all cases the tissues were refluxed for 3 hr, and after cooling to room temperature an equal volume of water was added. The nonsaponifiable fraction was obtained by extracting the solution 3 times with vc its volume of n-

160

MOSBACH,

BLUM,

ARFlOYO,

AND

MILCH

hexane. An aliquot of the hexane extract was used for the determination of total sterol by digitonin precipitation. In most cases the nonsaponifiable fraction of tissues was used directly for the oxidation step. However, if the nonsaponifiable material was highly colored or contained oily material it was first purified, either via the digitonide or by silicic acid chromatography. $2. Cixidation

The dry sterol mixture, or nonsaponifiable fraction, was placed in an Erlenmeyer flask (a 50-ml flask for samples containing up t.o 25 mg sterol, and a 125-ml flask for samples up to 100 mg of sterol), containing a Teflon-covered stirring bar. A solution of cholesterol-4-V4, previously purified by silicic acid chromatography (8), (0.05 PC; 1 pg/ml of benzene, 30,009 cpm/ml under our conditions of radioactivity assay) was added to serve as an indicator for completeness of oxidation. The benzene was evaporated at 60°C under a stream of nitrogen. This radioactive cholesterol must be purified by silicic acid chromatography; otherwise trace impurities will appear in the dihydrocholesterol fraction. Radioactive contamination of this fraction will make it impossible to obtain an accurate measure of the amount of unoxidized cholesterol. Following evaporation of the benzene, formic acid (85%, v/v) was added in the proportion of 1 ml of acid per milligram of sterol. The minimum volume of formic acid used was 10 ml. The flask was placed on a thermostatically controlled hot plate-magnetic stirrer combination. The surface temperature of the hot plate had previously been adjusted to 170°C (thermometer). The reaction mixture was stirred until all of the sample had dissolved, and stirring was continued 1 min longer, for a total period of 6-10 min, depending upon sample size. These conditions were chosen so as to avoid excessively prolonged heating periods yet assure complete dissolution of the sterols and allow the formylation reaction to proceed at 90-95°C for 2-3 min. This is the most critical step in the procedure since overheating will lead to lowering of the oxidation efficiency. The sample flask was then transferred to an unheated magnetic stirrer, a thermometer was inserted into the solution, and when the temperature reached 75 + 5’C a 30y0 solution of hydrogen peroxide (1 ml of peroxide per 20 ml of formic acid solution) was added dropwise while stirring. After addition of the peroxide solution was completed (about 1 min) stirring was continued for 30 min. (The temperature at which the peroxide solution is added is not as critical as the formylation temperature. It is necessary, however, to keep within the 10” range to avoid

DIHYDROCHOLESTEROL

DETERMINATION

161

either destruction of the dihydrocholesterol which takes place above 8O”C, or poor oxidation efficiency observed at low temperatures due to incomplete solution of the sterols.) After the sample had come to room temperature (usually at the end of the 30-min oxidation period) a volume of water equal to that of the formic acid solution was added, plus an extra 25 ml. This aqueous solution was transferred to a separatory funnel and extracted twice with l/z its volume of n-hexane. (Some of the n-hexane is used to rinse the thermometer.) The sample was then further extracted with 3 portions of 20% peroxide-free ethyl ether in n-hexane (v/v), again using I/ the volume of the formic acid solution. All five hexane and hexane-ether extracts were combined and washed with 10 ml of water, twice with 10% (w/v) NaHCOs solution, and 3 times with 10 ml of water. The organic solvents were evaporated on a 60°C water bath under nitrogen, and the sample was then dried in vucuo at room temperature for at least 1 hr. The mixture of sterol formates obtained is unstable in solution and should be used for the next step (silicic acid chromatography) as soon as possible. Storage for 24 hr at room temperature was found to be permissible both for the dry samples or for hexane solutions. S. Silicic

Acid Chromatography

The mixture of dihydrocholesterol formate, cholestane-3,8,5a,6@triol 3,6-diformate and any cholest,erol formate which remained unoxidized was chromatographed on silicic acid, using columns 20 cm in length and 15 mm in diameter, at 25°C. The columns were prepared and conditioned as described by Hirsch and Ahrens (8). The sample was put on the column in 2-3 ml of n-hexane, and the flask containing the sample was rinsed 3 times with 2-ml portions of n-hexane; these washings were used for rinsing the column. Elution was carried out with 1% anhydrous ethyl ether in n-hexane. Dihydrocholesterol formate (plus traces of cholesterol formate, if present) was eluted first, requiring approximately 50 ml of eluent. Trio1 formate was eluted with 4 or S$% ethyl ether in n-hexane or, more rapidly, with methanol. In order to obtain reproducible results each lot of silicic acid must be tested with known compounds since the retention volumes vary from batch to batch. The effluent was collected in 5-ml fractions and the solvent was evaporated under nitrogen at 60°C. The fractions containing a visible residue of dihydrocholesterol formate (2-3 tubes) were selected and rinsed with absolute ethanol. The rinses were combined and made up to a volume of 5.0 ml. The tubes containing cholestanetriol diformate were combined in a similar manner, and the

162

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dLUM,

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AND

MILCH

weight of the diformate (divided by 1.233) was used as a measure of the initial weight of cholesterol. Recoveries of cholesterol should range from 9P99%. 4. Determination

of Dihydrocholesterol

as the Digitwide

(a) Gravimetric Method. If the dihydrocholesterol content of the sample was 0.5 mg or greater, the sterol was determined gravimetrically as the digitonide. The solution containing dihydrocholesterol formate usually will have a volume of 5.0 ml; for samples of high dihydrocholesterol content a 5-ml aliquot should be taken so that the sterol content of the sample will not exceed 1 mg/ml. The dihydrocholesterol formate first must be hydrolyzed as follows: 0.3 ml of 33% KOH (w/v) solution in water is added to 5 ml of the dihydrocholesterol formate solution, and this alkaline solution is then heated in a 60°C water bath for 1 hr. The solution is cooled to room temperature and 1 drop of a 0.5% solution of phenolphthalein in 95% ethanol is added. Glacial acetic acid is then added dropwise to neutrality, followed by an additional drop of glacial acetic acid. An equal volume of a 1% solution of digitonin in 80% ethanol (v/v) is then added, and quantitative precipitation of dihydrocholesterol digitonide is found to be complete after the sample has stood for 30 min at room temperature. The digitonide is suctionfiltered on a Whatman No. 42 tared filter paper, 2.4 cm in diameter, using a Millipore No. XX 1002500 filtration apparatus. The filter paper must be equilibrated in room air for 30 min before weighing. In the present experiment a Mettler No. H-16 balance having a precision of +O.Ol mg was used. The digitonide was washed first with 3-5 ml portions of boiling water until the wash water no longer foamed (this is essential for removing all traces of potassium acetate and excess digitonin which otherwise will produce erroneously high weights of the digitonide). Washing was completed with &ml portions of 80% alcohol, acetone, and ethyl ether. The digitonin precipitate on the filter paper was then dried at 60°C for 1 hr in vacua. The digitonide was allowed to equilibrate at room temperature for 0.5 hr before weighing. The weight of the digitonide was multiplied by the factor of 0.239 to obtain the weight of dihydrocholesterol in the sample. The digitonide was next counted in a Nuclear-Chicago D-47 flow counter to determine the amount of unoxidized cholesterol. If the radioactivity of the sample, corrected for background and self-absorption, was 3 cpm or less the oxidation of the sample was assumed to be complete. If the radioactivity was greater than 3 cpm this was assumed to be due to unoxidized cholesterol and the weight of dihydrocholesterol was corrected accordingly.

DIHYDROCHOLESTEROL

DETERMINATION

163

(b) Anthrone Method. When the total dihydrocholesterol content of the sample was less than 0.5 mg the sterol was determined colorimetritally by the anthrone color reaction. As before (Section .Ja) the dihydrocholesterol formate fraction was made up to a volume of 5.0 ml, but only 4.0 ml was used for digitonin precipitation, using a 0.5% digitonin SO~Ution in 50% aqueous alcohol. The remaining 1.0 ml was used for radiaactivity determination by liquid scintillation counting (Nuclear-Chicago Model 702). The determination of the sterol digitonide by the anthrone method was carried out exactly as described by Vahouny et al (9). RESULTS

The validity of this method was established by (I) analysis of known binary mixtures of cholesterol and dihydrocholesterol, (2) replication experiments, (3) recovery experiments, and (4) infrared spectrophotometry of dihydrocholesterol samples isolated from tissues. Table 1 provides a summary of 67 analyses of known binary mixtures of cholesterol and dihydrocholesterol, ranging in dihydrocholesterol content from 0-1000/o. When care was taken during the formylation step oxidation efficiencies of 99.9 to 100% could be obtained with samples containing 0.5 to 2.0% dihydrocholesterol, thus making it possible to achieve good accuracy. As will be seen below, samples with this low dihydrocholesterol content are typical of most biological sterol mixtures with the exception of certain rabbit tissues. Table 1 further indicates that the cholestanetriol weights served as a fair measure of the original choIestero1 content of the sterol mixture, although, as pointed out above, this information is usually available from the original determination of total sterol content. When the weight of dihydrocholesterol in the sample to be analyzed is low, ranging from 0.2-0.4 mg, the anthrone method has proved useful for the determination of this st,erol. The data listed in Table 2 demonstrate that good agreement with the theoretical values could be obtained even without the use of labeled cholesterol as an indicator of oxidation efficiency. This is feasible since very careful attention to the formylation procedure results in negligibly small corrections for unoxidized cholesterol. This possibility of achieving consistent and high oxidation efficiencies is also demonstrated in the reproducibility study shown in Table 3. It can be seen that the procedure gives good agreement among duplicate or triplicate determinations even when the amount of dihydrocholesterol is quite small. Table 4 lists the results of 8 recovery experiments in which known amounts of dihydrocholestero1 were added to the nonsaponifiable extracts

2

124 10 7

9 5

8 4

6

0.00

0.5 1.0 2.0 5.0

10.0 20.0

50.0 80.0

100.0

2.00

25.00 40.00

5.00 10.00

0.50 1.00 2.00 2.50

0.00

DHC (ms)

a DHC = dihydrocholesterol. bu = standard deviation.

No. of samples

DHC(%I

2.00

50.00 10.00

50.00 50.00

100.00 100.00 100.00 50.00

100.00

Total sterol (mg)

ANALYSIS

24.78 38.62 1.965

4.67 9.81

0.488 0.978 1.914 2.41

0.00

DHC recovered kw)

OF KNOWN

0.80 1.42 0.052

0.12 0.27

0.020 0.040 0.095 0.06

-

d

99.1 96.6 98.3

93.6 98.1

97.6 97.8 95.7 96.2

-

DHC recovered (%)

TABLE 1 CHOLESTEROL-DIHYDROCHOLESTEROL (GRAVIMETRIC METHOD)

96.3 99.1 -

97.5 96.6

96.9 95.8 94.0 97.1

-

Cholestanetriol recovered (%)

MIXTURES

3.52 -

1.67 3.03

0.98 3.03 9.01 1.14

-

cd

99.80

98.62 99.04

99.89 99.33

99.97 99.85 99.89 99.74

100.00

Oxidation (%)

0.11

2.07 0.65

0.14 0.39

0.02 0.39 0.09 0.23

0.00

d

z z P 8

3

ti 55

2

i?

is 0m tij Fi m

DIHYDROCHOLESTEROL

TABLE ANALYSIS

DHCQ (%I

0.5

1.0

2.0

100.0

a DHC

OF KNOWN

165

DETERMINATION

2

DIHYDROCHOLESTEROL-CHOLESTEROL (ANTHRONE METHOD)

MIXTURES

DHC recovered hg)

DHC recovered (%I

40.0 40.0 60.0 80.0

0.194 0.182 0.294 0.389

97.0 91.0 98.0 97.3

95.8

0.20 0.20 0.30 0.40

20.0 20.0 30.0 40.0

0.186 0.198 0.287 0.387

93.0 99.0 95.6 96.7

96.1

0.20 0.20 0.30 0.40

10.0 10.0 20.0

0.175 0.199 0.285 0.384

87.5 99.5 95.0 96.0

94.5

0.20 0.20 0.30 0.40

0.20 0.20 0.30 0.40

0.189 0.177 0.284 0.377

94.5 88.5 94.7 94.2

93.0

DHC (w)

Total sterol bwf

0.20 0.20 0.30 0.40

15.0

Av. DHC recovery (%)

= dihydrocholesterol. TABLE REPRODUCIBILITY

3 STUDY

Total stem1 (mg)

DHCa fmd

DHC (%I

Human gallstone

100.42 100.47 100.90

0.454 0.460 0.471

0.452 0.458 0.467

99.98 99.98 99.97

Intima of human thoracic aorta

112.25 112.25 109.00

0.225 0.234 0.237

0.200 0.208 0.218

99.96 99.95 99.97

62.7 62.5

0.220 0.220

0.350 0.352

99.98 99.98

Sample

Human serum pool

Oxidation (%)

6 DHC = dihydrocholesterol.

of the tissue studied. It is apparent that, regardless of the biological origin of the sample, 96-1040/o recovery could be obtained. Table 5 summarizes the results obtained when the method was applied to various mammalian tissues and to human cholesterol gallstones.

166

MOSBACH,

BLUM,

ARROYO,

TABLE RECOVERY

Tissue

Rabbit

liver

Gallstone

AND

MILCH

4

EXPERIMENTS

Total sterol b&x)

DHC. h)

DHC (%)

116.3 91.2 103.9

4.04 3.82 3.60

3.47 4.19 3.47

3.00 3.00 2.00

3.04 3.12 2.07

101.3 103.9 103.3

100.5

0.468

0.465

2.00

2.45

96.8

DHC added h3)

DHC recovered (mo)

DHC recovered

es?)

Human

aorta

175.1 99.99

0.683 0.645

0.390 0.648

0.994 0.994

1.012 0.985

101.8 99.1

Human

serum

54.95 53.40

0.481 0.467

0.876 0.876

1.490 1.00

1.550 1.04

103.8 104.0

Q DHC

= dihydrocholesterol. TABLE DIHYDROCHOLESTEROL No.

Tissue

Rabbit Liver Small intestine Adrenal Serum Kidney Human gallstones Intima, human aorta Media, human aorta Human serum Hypercholesterolemic Pool A Pool B Human red cells, pool Dog serum, pool Q DHC

of

samples

5 CONTENT

Total sterol

(mg/gm)

2.00 1.65 51.50 0.289 3.11 998 33.8 4.79 8.57 2.34 2.51 1.60 1.75

OF TISSUES DHCa (70)

4.25 5.28 8.24 2.08 1.97 0.475 0.341 0.530 1.030 0.88 0.35 0.32 0.563

R(a+y 0

1.39-4.65 1.54-7.53 5.76-10.72 1.68-2.48 0.327-0.599 0.158-0.646 0.422-0.639 -

= dihydrocholesterol.

Dihydrocholesterol samples isolated from rabbit liver, rabbit small intestine, and rabbit adrenals by the method described were subjected to infrared spectroscopy. The spectra obtained were identical with the spectrum of pure dihydrocholesterol, indicating that the isolated material was at least 95% pure and that the removal of cholesterol was practically complete. Table 6 illustrates experiments comparing the results of the present

DIHYDROCHOLESTEROL

COMPARISON

167

DETERMIh’ATIOh-

TABLE OF SCHOENHEIMER

6 AND

OXIDATION

METHODS Per cent

Sample Cholesterol

Sterol A

fraction

gallstone

of human

Method used5

Sample wt. h3)

DHC” wt. (mg)

contsminatiorl with cholesterols

B 0

300.0 300.0

13.6 0.98

63.9 0.02

1~ 0

106.5 99.4

1.43 0.38

ti4.i 0.00

B 0

113.1 94.3

4.40 0.23

77.0 0.00

intima

B

0 B, bromination; 0, oxidation. b DHC = dihydrocholesterol. c Determined wiQh labeled cholesterol.

method with those obtained by the Schoenheimer procedure. It is apparent that the dibromination method gives high results due to the fact that a considerable quantity of the cholesterol and perhaps of other sterols escape dibromination and appear in the dihydrocholesterol fraction. Inspection of this table suggests that some of the high dihydrocholesterol concentrations reported in the past may be due, at least in part, t,o this factor. DISCUSSION

The method described in this report is based upon a method for the preparation of cholestane-3P,%,6/3-triol from cholesterol published by Fieser and Rajagopalan (7). In order to adapt the performic acid oxidation employed by these workers to the determination of dihydrocholesterol on a milligram scale the oxidation had to be carried out at elevated temperatures since the solubility of dihydrocholesterol-cholesterol mixtures in formic acid solutions is low at room temperature, resulting in relatively poor oxidation efficiencies. It was surprising, therefore, that the critical step in this procedure is not the peroxidation itself but rather the formylation step which precedes the oxidation. Apparently the formylation reaction goes to completion only at temperatures close to the temperature at which these sterols undergo decomposition in 85% formic acid. The present procedure requires at least 0.2 mg of dihydrocholesterol for accurate measurement. Therefore, for samples containing approximately 1% dihydrocholesterol the weight of total sterol in the sample

168

MOSBACH,

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ARROYO,

AND

MILCH

must be at least 20 mg. This constitutes a serious limitation of the method since, for example, an entire rat liver (containing 2 mg of sterol per gram wet weight) or 10 ml of human serum (having a cholesterol concentration of 200 mg per 100 ml) are required for a single analysis. It should be possible, however, to adapt the procedure to much smaller samples by employing gas chromatography to analyze the components of the oxidized mixture (10). The biological data presented in Table 5 are of interest since they serve to correct a widely held opinion that gallstones and atheromata usually are of high dihydrocholesterol content. The results of the present study indicate that the sterol fractions obtained from these materials have a surprisingly low concentration of dihydrocholesterol. Other human tissues also may prove to have a low dihydrocholesterol content since the results of the serum analyses showed a range of dihydrocholesterol in the total sterol fraction ranging from 0.351.03%. The latter value was obtained with a sample of hypercholesterolemic serum having a total cholesterol concentration of 857 mg per 100 ml. The relatively high proportion of dihydrocholesterol in rabbit tissues was likewise unexpected. This finding suggests that the rabbit will be a useful species for further studies dealing with the metabolism of dihydrocholesterol. SUMMARY

1. A new method has been developed for the analysis of dihydrocholesterol in the sterol fraction of mammalian tissues and in gallstones. 2. The method is based on the finding that cholesterol-dihydrocholesterol mixtures can be oxidized with performic acid, which converts the cholesterol quantitatively to cholestane-3/3,5a,6j3-triol, leaving the dihydrocholesterol unchanged. The oxidation products can then be separated by chromatography on silicic acid, and the amount of dihydrocholesterol can be measured by digitonin precipitation either gravimetrically or by the anthrone method. 3. The feasibility of this method has been demonstrated by analysis of known cholesterol-dihydrocholesterol mixtures, by recovery experiments, and by infrared spectrophotometry of the isolated dihydrocholesterol. 4. It has been shown that previous concepts of the high dihydrocholesterol content of gallstones and atheromata may be in error since these materials frequently contain less than 0.5% dihydrocholesterol. 5. It has further been shown that rabbit tissues, particularly the intestinal wall and adrenals, may contain as much as 5-10s of dihydrocholesterol in the tot,al sterol mixture obtained from these tissues.

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DETERMINATION

169

REFERENCES 1. BLADON, P., in “Cholesterol” (R. P. Cook, ed.), pp. 21-22.. Academic Press, New York, 1958. 2. SCHOENHEIMER, R., VON BEHRING, H., AND HUMMEL, R., 2. physiol. Chem. 192, 93 (1930). 3. MCARTHUR, C. S., Biochem. J. 36, 559 (1942). 4. MOSBACH, E. H., AND BEVANS, M., Am. J. Path. 37, 631 (1960). 5. NICHOLS, C. W., JR., SIPERSTEIN, M. D., AND CHAIKOFF, I. L., Proc. Sot. Expll. Biol. und Med. 83, 756 (1953). 6. NICHOLS, C. W., JR., LINDSAY, S., AND CHAIKOFF, I. L., Proc. Sot. Ezpptl. Biol. nrLd Med. 89, 609 (1955). 7. FIESER, L. F., AND RAJAGOPALAN, S., J. Am. Chem. Sot. 71, 3938 (1949). 8. HII~SCH, J., AND AHRENS, E. H., JR., J. Biol. Chem. 233, 311 (1958). 9. VAHOUNY, G. V., MAI~R, R. M., ROE, J. H., AND TREADWELL, C. R., Arch. Biochew Biophys. 86, 210 (1960). 0. I~LANKENHORN, D. H., personal communication,