DETERMINATION OF FREE AND TOTAL CHOLESTEROL BY DIRECT CHLOROFORM EXTRACTION*

BY GEORGE R. KINGSLEY AND ROSCOE R. SCHAFFERT

(From the Clinical Biochemistry Laboratory, General Medical and Surgical Hospital, Veterans Administration Center, Los Angeles)

(Received for publication, October 30, 1948)

Since the Liebermann-Burchard reaction was adopted by Grigaut (1) for the quantitative determination of serum total cholesterol, many modified procedures of the original method have been made. The various pro- cedures have differed in extraction method and in the reagents used in the color development. The separation of cholesterol from its esters with digitonin has had limited application because of the time and labor required in the involved and delicate technique. The errors which are encountered in the Liebermann-Burchard color reaction of different cho- lesterol and cholesterol ester methods have been described by Sperry (2).

Because of the different methods of extraction in use, the wide range of accepted normal values for total cholesterol, the difficulty of perform- ing cholesterol ester determinations, and the instability of color produced in the Liebermann-Burchard reaction, an investigation of the possible improvement in the method of determination of cholesterol and its ester was undertaken.

The method reported has been satisfactorily used over the past 2 years for the determination of several hundred serum cholesterols and choles- terol esters.

Reagents- Method

Suljuric acid, concentrated (c.P. or A. C. S.). Acetic anhydride (c.P. or A. C. S.). Magnesium sulfate, anhydrous. Dry U. S. P. magnesium sulfate at

150-200”. Discard caked or melted crystals. Heat slowly, to avoid melting or caking of crystals.

Acetic anhydride-sulfuric acid reagent. Mix cautiously 4 parts of cold acetic anhydride and 1 part of cold sulfuric acid (O-5”) in a stoppered

* Jean Weiser of this laboratory aided in the investigation of this method. Albert De Lalla (T/4) and Leon Shew (T/5) of the 193rd (United States) General Hospital, gave technical assistance in a preliminary investigation of this method.

Published with the permission of the Chief Medical Director, Department of Medicine and Surgery, Veterans Administration, who assumes no responsibility for the opinions expressed or conclusions drawn by the authors.

316

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316 DETERMINATION OF CHOLESTEROI~

Erlenmeyer flask placed in powdered ice. Use immediately after mix- ing.

Chloroform (c.P. or A. C. S.). Chloroform-digitonin reagent, 0.1 per cent. Add 0.4 gm. of digitonin

to 400 ml. of reagent grade chloroform. Mix 5 minutes in a Waring blendor, cool, and mix again for 5 minutes. If a high speed blendor is not available, shake the mixt,ure in a Kahn shaker for 30 minutes and store at 37” for 1 week before using. Shake the mixture each time before use.

Stock cholesterol standard. Dissolve 100 mg. of ash-free cholesterol (m.p. 149”) in chloroform and dilute to 100 ml. at the temperature of the refrigerator where it is stored.

Working cholesterol standard. Dilute 10 ml. of stock standard to 100 ml. with redistilled chloroform in the same manner as the stock standard and store in the refrigerator.

Xtandardization-Add 1, 2, 3, 4, 5 ml., of working cholesterol stand- ard to photometer cuvett’es and dilute each to 5 ml. with chloroform. Add 2 ml. of ice-cold fresh acetic anhydride-sulfuric acid reagent, mix, and read the color as directed in “Procedure.”

Procedure

Total Cholesterol-Add 0.2 ml. of serum to 10 ml. of chloroform in a 15 X 150 mm. hollow glass stopper test-tube. Stopper tightly. (Use chloroform-washed corks if glass-stoppered t,est,-tubes are not available.) In the same manner add 0.2 ml. of distilled water to 10 ml. of chloroform for the blank determination, and also to 2 ml. of working cholesterol standard and 8 ml. of chloroform for standard determinations. Shake for 5 minutes in a Kahn shaker. Add 1.5 gm. of anhydrous magnesium sulfate and stopper. Mix immediately t,o avoid ca.king of sulfate and again shake 5 minutes in a Kahn shaker. Add 0.5 gm. of fullers’ earth and stopper. Mix vigorously 10 seconds by hand and centrifuge imme- diately for 10 minutes at 3000 R.P.M. Pipette 5 ml. of the clear super- natant chloroform extract into a photometer cuvette and add 2 ml. of freshly prepared acetic anhydride-sulfuric acid reagent. Mix by twirl- ing; do not invert. After mixing, read the color developed at 4 to 7 minute intervals to obtain the maximum reading if the temperature of the reaction mixture is 25”, at 10 to 15 minute intervals at 20°, or at other time intervals as indicated by the temperature (see Fig. 2). Read in the photometer against a chloroform blank set at 100 per cent transmis- sion wit.h 625 mp light transmission. 1Jse the maximum reading for calculation.

Ch.olesterol Ester-Add 0.2 ml. of serum and 0.2 ml. of distilled water

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G. R. EINCIBLEY AND R. R. SCHAFFERT 317

for a blank, separately, to two 10 ml. portions of chloroform-digitonin reagent; stopper tightly. Shake for 5 minutes in a Kahn shaker. Add 1.5 gm. of anhydrous magnesium sulfate and stopper. Mix immediately to avoid caking of the sulfate and mix in a Kahn shaker for 5 minutes. Add 0.2 ml. of acetic anhydride and stopper. Mix vigorously by hand 10 seconds, add 0.5 gm. of fullers’ earth, and stopper. Mix 10 seconds by hand, and centrifuge 10 minutes at 3000 R.P.M. Continue as directed in the total cholesterol procedure.

Calculations

General Formula-Concentration in mg. per 100 ml. = K’(2 - log per cent T).

Total Cholesterol, Ester and Reagent BZunk*-Substitute per cent T in the above formula.

Cholesterol Ester Correction-(Cholesterol ester minus blank) - (choles- terol ester minus blank) X O.8.3

Corrected Total Cholesterol-(Total cholesterol minus blank) - (cho- lesterol ester correction).

Corrected Cholesterol Ester-(Cholesterol ester minus blank) X 0.8.

EXPERIMENTAL

SimpliJication of Method of Chobsterol Extraction

In most cholesterol methods hot alcohol-ether or alcohol-acetone is used to extract cholesterol from serum. These solvents must be removed by evaporation before the cholesterol is redissolved in chloroform or acetic anhydride for color development. This procedure involves possible loss of cholesterol by oxidation from overheating, or retardation of color development by water remaining after incomplete drying of the residue from previous extractions. Preliminary extraction with additional sol- vents other than chloroform increases the possibility of mechanical loss of cholesterol.

When serum is shaken with chloroform, very little coagulation of pro- tein occurs for several minutes. A fine emulsion is formed which is ideal for extraction of the cholesterol. The use of alcohol, acetone, and heat coagulat,es the serum proteins. The addition of 0.3 ml. of 10 per cent sulfuric acid, 0.3 ml. of 95 per cent ethyl alcohol, or 3 gm. of anhydrous magnesium sulfate to 0.5 ml. of serum before extraction with chloroform

1 K = 688 at 20" for Coleman speotrophotometers, models 6 and 14, with No. 6-394B cuvettes.

2 Reagent blank = 97 to 99 per cent T with Coleman spectrophotometers. 30.8 = relative color correction for the ester when read against the cholesterol

standard.

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318 DETERMINATION OF CHOLESTEROL

prevented the extraction of 11, 66, and 86 per cent, respectively, of the cholesterol present. If the serum is heated on a water bath 10 to 20 minutes at 100” before extraction, 83 per cent of the cholesterol is not extractable with chloroform.

Direct chloroform extraction is also advantageous, as this extract can be used directly for color development once it is free of water and serum proteins. The water is easily removed by the additicn of anhydrous magnesium sulfate, which also precipitates the serum proteins after the cholesterol has entered the chloroform solvent phase. The use of chloro-

TABLE I

Comparison of Color Intensity Developed by Reagents Specijed by Various Cholesterol Methods

Temperature 19’; light transmission 625 rnp; total volume of reaction mixture 7 ml.; cholesterol 0.5 mg.

Method

Time of maxi-

c%%- t$$T Acetic Chloro- LiKht &y;o”, tion,

HdO4 dride acid form absorp-

tion de- velop- merit

---~-- vol. ool. vol. 001.

per ten4 per cent per ccnl per cent per czn6 7&*

Kingsley-Schaffert........................ 5.7 22.9 0.0 71.4 81.0 10 Myers-Wardeli (6). . . . . . . . . . . . . . . . . . . . . . . . 1.6 16.4 0.0 82.0 71.4 45 Macheboeuf -Delsal cholesterol method (5) 0.7 71.0 0.0 28.3 62.5 100

“ I‘ ester method (5)............................ 0.7 49.6 0.0 49.6 65.5 100

Srhoenheimer-Sperry (4). . . . . . . . . . . . . . . . . . 3.3 64.4 32.2 0.0 50.0 85 Bloor (3). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 28.2 0.0 70.4 76.5 15

form for the direct extraction of serum cholesterol avoids the use of other solvents, heat for extraction and drying, and the loss of cholesterol by protein precipitation at the time of extraction. Mechanical loss is also decreased, as direct extraction with chloroform eliminates additional steps in the method.

Determination of Optimum Proportion of Reagents for Development of Max- imum Color and Color Xtabilitg in Liebermann-Burchard Reaction

The method of Bloor (3) and its modifications for the determination of cholesterol are widely used. In this method chloroform is used as a cholesterol solvent in the final color development. In the Schoenheimer and Sperry (4) method glacial acetic acid is used for the final solution of

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Q. R. KINBSLEY ANTI R. R. YCHAFFERT 319

cholesterol digitonide. This modification was made to stabilize the color development. However, as shown in Table I, much less color is de- veloped, and the color must be measured at exact temperatures and time intervals. Macheboeuf and Delsal (5) investigated the effect of varia- tion of the proportion of reagents in the Liebermann-Burchard reaction upon speed and intensity of color development and concluded that maxi- mum color with free cholesterol was obtained with 2 ml. of chloroform, 5 ml. of acetic anhydride, and 2 drops of N sulfuric acid in 30 minutes

WAVELENGTH-MILLlt4ICftON5

400 500 600 700

Fra. 1. Spectral transmittance of the color developed by 2 ml. of acetic anhydride sulfuric acid reagent (4:l) added to 0.6 mg. of cholexterol in 6 ml. of chloroform at 0”. Transmittance 80 minutes after color development.

at 25”. The maximum color for cholesterol esters was obtained with 3.5 ml. of chloroform, 3.5 ml. of acetic anhydride, and 2 drops of N sul- furic acid at 25”. Macheboeuf and Delsal also noted that changes in temperature gave different rates of color development as greater color intensity was obtained at higher temperatures.

After testing all possible combinations of reagents, the ratio of reagents as specified in the procedure for the new method was found to give greater maximum color than was yielded by other methods at 19”, as shown in Table I. Light transmission of 625 rnF was used to make these readings, as maximum light absorption for the color developed by the Liebermann- Burchard reaction occurred at this wave-length (see Fig. 1).

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320 DETERMIN.%TIOK OF CHOLESTEROL

E.fect of Tenqxrature, Time, Light, Cholesterol Conwntration, and Water upon Xta.bility md Intmsity of Color Developmnt

The color developed with chole&erol in chloroform solution by the Liebermann-Burchard rea,ction wit,h the magems specified in the new metShod reached approximately t,he sa,me maximum intensity, rega,rdless of the bemperature, as shown in Fig. 2. So correction was made for the change in volume of the reaction mixture at t,he lower or higher tempers- tures. -4t 0” the color reached ma.ximum development at. 70 minutes and remained stable for the following 80 minutes a,t t#his l,empcrature. At 10” the color was stable at the 30 to 45 minute interval after the start of color development. At 20” maximum color was ma.intained at the 10 to 15 minutSe interval after the st,art of the color development and at, 30” a,t the 3

TIME MINUTES

FIG. 2. Effect of temperature on the stability and rate of color development in the Liebermann-Burchard reaction when 2 ml. of acetic anhydride-sulfuric acid re- agent (4:l) are added to 0.5 mg. of cholesterol in 5 ml. of chloroform.

t,o 6 minute interval. The effect of temperature on the intensit,y, stabilit,y, and rate of color development of cholesterol acetate in the Liebermann- Burchard reaction was the same as that on cholesterol, as equal, maximum color density of cholesterol acetate standards was reached in the same time intervals from O-30”. Maximum color density of chloroform extracts of total cholesterol and cholesterol ester of serum also was reached in the same time intervals at 030” as those shown in Fig. 2 for cholesterol. These data indicate that at 20” maximum color development of both cholesterol and its ester will occur between 10 and 15 minutes, which gives ample time for making readings in the spectrophotometer. This is a distinct advantage over other methods, in which color readings must be made at time intervals of 1 minute or less.

The per cent transmission at 625 rnp produced by different concentra- tions of cholesterol plotted on a semilog scale, as shown in Fig. 3, gave a straight line function from 0 to 500 mg. of cholesterol per 100 ml. of se- rum. The constant for this curve, including the dilution factors for 0.2

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G. R. KINGSLEY AND R. R. SCHAFFERT 321

ml. of serum and 5 ml. of the 10 ml. of chloroform solvent used, as out- lined in the method was 692 at 0” and 688 at 20”, for substitution in the formula, cholesterol concentration per 100 ml. of serum = K(2 - log per cent T).

All determinations were carried out in the absence of direct sunlight. Indirect light had no effect on the color development.

Water could be tolerated up to the addition of 0.02 ml. to the reaction mixture with less than 4 per cent loss in color development. Small

100 200 300 400 500

FIG. 3. Relation between cholesterol concentration and per cent transmittance. The curve represents cholesterol standardization in mg. equivalence per 100 ml. of serum for 5 ml. of the 10 ml. chloroform extract of 0.2 ml. of serum. 2 ml. of acetic anhydride-sulfuric acid reagent (4:l) added to 5 ml. of chloroform containing 0.06 to 0.5 mg. of cholesterol at 0”. Transmittance 30 minutes after color development.

amounts of water had no effect on the time of maximum color develop- ment.

Simpli$cation of Cholesterol Ester Def.errnination

The determination of cholesterol esters with digitonin by the older methods is a long tedious procedure, which must be carried out with meticulous care. The cholesterol ester determination was incorporated in the simplified direct chloroform extraction to obtain a short, simple accurate method for cholesterol fractionation. This possibility became apparent when it was found that sufficient digitonin* was soluble or in

‘Digitonin preparations from The British Drug Houses, Ltd., Merck and Com- pany, Inc., S. B. Penick and Company, Hoffmann-La Roche, Inc., were found to be equally satisfactory.

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322 DETERMINATION OF CHOLESTEROL

suspension in 10 ml. of chloroform to precipitate up to 0.5 mp. of added cholesterol. If the cholesterol concentration is greater than 250 mg. per cent, less serum should be used. A chloroform-digitonin reagent was prepared which was used to extract the cholesterol ester and simultane- ously precipitate the free cholesterol directly from the serum as it was extracted. Anhydrous magnesium sulfate and acetic anhydride were added and the cholesterol-digit,onin filtered off with the proteins. No overnight standing is required for the complete precipitation or absorp- tion of the cholesterol digitonide. Only cholesterol est,er remains in the filtrate, which is determined in the same manner as the total cholesterol. The addition of 0.2 ml. of acetic anhydride to the chloroform-digitonin extract of 0.2 ml. of serum, dried over anhydrous magnesium sulfate, was found necessary for the complete precipitation or absorption of the cho- lesterol digitonide with the protein precipitate. The addition of less than 0.1 ml. or more than 0.3 ml. of acetic anhydride did not give satisfactory separation of the digitonide. The additionof 0.2 ml. of acetic anhydrid: to the dry chloroform extract of 0.2 ml. of serum had no effect on the to- tal cholesterol determination.

Determination of Relative Color Density Developed in Liebermann-Burchard Reaction by Cholesterol and Cholesterol Ester

The ratio of color density developed by the naturally occurring serum cholesterol esters to that of cholesterol was determined by comparing the total cholesterol values obtained on samples of hydrolyzed and unhydro- lyzed serum. 0.2 ml. of serum was incubated at 40” with 0.2 ml. of 30 per cent KOH for 30 minutes to convert all cholesterol esters to choles- terol. Hydrolysis was complete at 30 minutes, as a series of hydrolyses at 0, 10, 20, 30, and 60 minutes indicated 98 per cent hydrolysis at 10 minutes and no increase from 20 to 60 minutes. Serum hydrolyzed for 0 minute gave values identical to those from serum to which no hydrox- ide had been added. The hydrolyzed serum was neutralized (phenol- phthalein) with acetic anhydride, 10 ml. of chloroform were added, and the remainder of the procedure carried out as directed in the total cholesterol method. The unhydrolyzed serum developed greater color than the hydrolyzed, the difference indicating the proportionately greater amount of color produced by the cholesberol esters. In twenty-one hydrolyzed and unhydrolyzed serums, presented in Table II, the ratio of color pro- duced by hydrolyzed to unhydrolyzed specimens is quite constant, rang- ing from 82 to 88 per cent (average 84 per cent). The range of 179 to 68 per cent cholesterol ester of the serums used could cause an additional difference of 2 per cent to the expected 5 per cent experimental error in the ratio of unhydrolyzed to hydrolyzed serum cholesterol. In order to make a correction for the greater color produced by cholesterol ester when

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cf. R. KINGSLEY AND R. R. SCHAFFERT 323

cholesterol standards are used with unhydrolyzed serums, the difference between unhydrolyzed and hydrolyzed serums is subtracted from the color value obtained with serum from which cholesterol has been pre- cipitated with digitonin to obtain a corrected cholesterol ester. A cor-

TABLE II

Ratio of Color Developed by Cholesterol and Cholesterol Ester in Serum of Fasting Norvnal Subjects and Hospital Patients

Sample No.

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21

__-

- Total chulesterol

hydz&ed’ q. per IO3 ml

307 129 375 307 365 316 164 365 181 303 184 170 164 178 224 214 230 355 298 114 224

-__

Hydrolyzed*

q. )M JOJ ml

270 106 321 268 316 263 137 312 149 254 154 140 137 149 193 175 193 290 244 95

190 ---

1

‘. m

-

Jncorrected’

g. $% 103 ml

251 98

286 233 278 247 125 294 140 240 148 135 128 140 174 168 183 294 240 84

166 ~__-.

Cholesterol ester

Corrected

g. per 103 ml

214 75

232 194 229 194 98

241 108 191 118 105 101 111 143 129 145 229 186 65

132 ,----.-

Average...............................................

* Average of duplicate determinations.

0.796 I I

!.

--

Ratio of corrected to uncorrected

0.85 0.77 0.81 0.83 0.82 0.79 0.79 0.82 0.77 0.80 0.80 0.78 0.79 0.79 0.82 0.77 0.79 0.78 0.78 0.77 0.80

-

-

In total cholesterol

per cent

79.0 71.0 72.0 72.4 72.1 73.7 71.5 77.4 72.4 75.1 76.5 75.0 73.6 74.1 74.1 73.6 75.0 78.7 76.4 68.4 69.4

rection averaging 0.8 is obtained with twenty-one serums (Table II) from a ratio of the corrected to the uncorrected cholesterol ester. As shown above, this correction would also be approximately the same at all temperatures from 030” if optimum time at a specific temperature is allowed for maximum color development.

Other laboratories should determine their om correction factor for cholesterol ester, as their reagents, conditions, and technique of analyses may vary from those of the authors.

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324 DETERMINATION OF CHOLESTEROL

The correct cholesterol ester is calculated as follows: cholesterol ester (as read against the cholesterol standard) - (the unhydrolyaed total cholesterol minus the hydrolyzed total cholesterol) = the corrected cho- lesterol ester.

TABLE III RecoverzJ of Cholesterol and Cholesterol Acetate Added to 0.8 Ml. Samples of Pooled

Serums

Sample No. Original I

Added Recovered Recovery - ___-.-

Cholesterol in serum --

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

w. 0.060 0.077 0.163 0.117 0.163 0.149 0.111 0.163 0.133 0.111 0.163 0.102 0.102 0.102 0.113 0.102 0.102

-i- w. 0.04 0.08 0.12 0.16 0.16 0.20 0.24 0.28 0.32 0.36 0.36 0.48 0.61 1.00 1.00 2.00 3.00

j- I , ,

I

-

T.

0.040 0.078 0.124 0.152 0.156 0.196 0.228 0.288 0.312 0.344 0.364 0.474 0.600 1.008 1.040 2.046 3.126

__--_- Average...................................................

Cholesterol ester in serum

18 0.340 0.252 0.246 97.8 19 0.340 0.504 0.486 96.2 20 0.340 0.630 0.602 95.5 21 0.340 1.260 0.121 96.0 22 0.340 2.520 2.530 100.0 23 0.340 3.780 3.860 102.2

per cent 100.0 97.5

103.3 95.0 97.5 98.0 95.0

102.7 97.5 95.5

101.0 98.5 98.3

100.8 104.0 102.3 104.2

- 99.5

---

Average . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97.9

Determination of Accuracy of Method by Recovery Experiments 0.04 to 3.8 mg. of cholesterol and cholesterol acetate was added to 0.2

ml. samples of fresh pooled serum and the per cent recoveries determined by comparison with the cholesterol and cholestSerol ester originally present in the serum. The recoveries, shown in Ta.ble III, are all within 5 per cent of the expected 100 per cent recovery.

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G. R. KINGSLEY AA% R. R. SCHAFFERT 325

TABLE IV

Partition of Serum Cholesterol in Fasting Normal Adults*

The results are given in mg. per 100 ml. --

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

SC2

--

M. “ I‘ “ ‘I ‘I “ ‘I “ ‘I “ “ “ “ “ I‘ “ I‘ “ ‘C “ “ “ ‘C ‘I “ “ U ‘I “ ‘6

F. ‘C ‘I “ 1‘ ** ‘I “ “ “ I‘

T- I Total cholesterol

pecimen I

257 172 218 221 177 206 160 161 250 173 215 240 185 195 213 222 248 231 197 240 251 182 246 218 236 192 187 217 226 232 233 212 189 168 177 202 172 207 156 242 262 205

mimen 11 --.- Average

265 261 172 172 218 218 215 218 183 180 212 209 165 163 165 163 250 250 179 176 209 212

212 212 189 189 164 161 179 178 196 199 178 175 207 207 161 159 242 242 249 256 211 208

specimen 1

201 135 165 169 132 156 118 119 189 128 164 170 136 140 155 172 178 168 141 168 190 143 191 166 178 143 149 168 179 186 186 156 136 116 133 158 133 162 116 196 186 166

3 --

-

Ester

pecimen I

204 203 131 133 160 163 169 169 136 134 156 156 122 120 121 120 195 192 128 128 160 162

156 156 141 139 116 116 133 133 153 156 133 133 156 159 118 117 196 196 182 184 171 169

---- Average

Per cent

77.8 77.5 74.7 77.5 74.4 74.6 73.7 73.7 76.8 72.8 76.5 70.8 73.5 71.8 72.7 77.5 71.8 72.6 71.5 70.0 75.7 78.5 77.5 76.0 75.4 74.5 80.2 77.4 79.3 80.1 79.9 73.6 73.5 72.0 74.8 78.3 76.0 76.7 73.6 81.0 72.0 81.0

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826 DETERMINATION OF CIIOLESTEROL

TABLE IV-Concluded

Total cholesterol

Specimen I 15 specimen I:

43 44 45 46 47 48 49 50

228 202 162 173 195 183 245 244

Average

220 224 202 202 157 160 176 175 195 195 178 181

- Ester

Per cent Specimen I Specimen II Average ____~--

168 168 168 75.0 146 146 146 72.4 129 133 131 81.8 138 146 142 81.1 158 158 158 81.0 137 133 135 74.6 181 73.8 178 i3.0

* Determinations on 20 to 50 year-old individuals 12 hours after evening meal.

TABLE V

Comparison of Direct Chloroform Extraction Mefhod with Schoenheimer-Sperry Method for Determination oj Total Cholesterol and Ester6 on Serum of Hospital Patients

%P -

1 2 3 4 5 6 7 8 9

10 11 12 13 14

Direct chlorolorm extraction

Cholesterol-digitonin

Toral - _- Ester

mg. per mg. per 100 ml. NO ml.

226 150 188 136 170 107 213 143 183 130 245 173 156 91 109 70 306 228 188 136 161 119 231 165 116 63 164 84

Cholesterol -

-

1

-

7-

- Water-digitonin

Cholesterol _ Ester

66 72 63 68 71 71 58 64 74 72 74 71 54 51

-

Total

mg. per 100 ml.

225 186 178 219 187 244 157 105 307 188 160 230

- -

-

Ester

mg. per JUIJ nz.

155 146

99 153 138 178

90 67

221 136 123 170

- .-

-I- Ester Total

!Jer ccn;

69 78 56 70 74 73 57 64 72 72 77 74

7”; E 5 2:

238 164 192 136 167 105 216 159 184 132 245 178 157 88 105 67 302 220 187 134 158 117 230 166 119 61 159 88

Schoenheimer-Sperry (4)

Cholesterol

-

--

-

Ester

--

-

i

-

Ester

per cent

69 71 63 73 72 73 56 64 73 72 75 72 51 55

Determination of Normal Range of Serum Chdesterol and Cholesterol Esters

Determinations of serum total cholesterol and cholesterol ester were carried out in duplicate on 50 fasting normal adults (Table IV). Single and average duplicate determinations for both sexes were within the range of 159 to 261 mg. of total cholesterol, and 116 to 203 mg. of cholesterol ester per 100 ml. of serum. The per cent cholesterol ester ranged in narrow limits, from 70.0 to 81.8 per cent. Duplicate determinations of

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G. It. KINGSLEY AND R. R. SCE.4FFERT 327

cholesterol and cholesterol ester agreed to within 5.0 per cent or less. These values are in close agreement with those of Sobel and Mayer (i’), who found normal serum total cholesterol to range between 160 and 267 and cholesterol ester between 118 and 202 mg. per 100 ml. of serum. The per cent ester levels were within the narrow limits found by Schoenheimer and Sperry (4) and more recently by Clarke and Marney (8) and Sobel and Mayer (7).

Comparison of New Method with Meth.od oj Schoenheimer and Sperry (4) for Determination of Cholesterol and Cholesterol Ester

The procedure, as directed under “Method,” was compared with that of Schoenheimer and Sperry. An additional comparison for ester de- termination was made hy use of a saturated solution of digitonin in water (0.5 to 0.8 per cent), with a slight modification of the procedure described for the chloroform-digitonin reagent, which is as follows: 0.2 ml. of serum and 10 ml. of chloroform are shaken 5 minutes and 0.2 ml. of digitonin in water (5 per cent) added. The mixture is shaken again for 5 minutes. 1.5 gm. of anhydrous magnesium sulfate is added and the mixture shaken for 5 minutes. 0.3 ml. acetic anhydride is added, shaken 5 minutes, and the remainder of the determination continued as directed in the cho- lesterol ester procedure with the chloroform-digitonin reagent. A com- parison of these two methods with that of Schoenheimer and Sperry is shown in Table V, in which the determinations were made upon serum of fasting hospital patients.

SUMMARY

1. A simple, rapid, accurate method for the determination of serum cholesterol and cholesterol esters by direct extraction with chloroform has been presented, in which evaporation, drying, and ester hydrolysis are not required and a cholesterol fractionation is completed within 1 to 2 hours. It has been assumed that only cholesterol and its esters in chloroform extracts of serum are responsible for the development of color in the Liebermann-Burchard reaction.

2. The optimum proportion of rea.gents in the Liebermann-Burchard reaction has been determined in order to obtain maximum and equal rate of color development for both cholesterol and cholesterol esters in the same interval of time.

3. Approximately the same maximum color intensity is obtained by both cholesterol and cholesterol ester at different temperatures, as the temperature affects only the rate of the reaction. The time for the meas- urement of maximum color is determined by the temperature of the reaction.

4. The accuracy of the method has been confirmed by recovery experi-

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328 BETERMISATION OF CHOLESTEROL

ments, by close agreement of duplicate determinations, and by com- parison with the method of Schoenheimer and Sperry.

5. Total serum cholesterol in 50 normal adults ranged from 159 to 260 mg. per 100 ml. and cholesterol ester from 70 to 82 per cent as de- termined by the new method.

BIBLIOGRAPHY

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George R. Kingsley and Roscoe R. SchaffertCHLOROFORM EXTRACTION

TOTAL CHOLESTEROL BY DIRECT DETERMINATION OF FREE AND

1949, 180:315-328.J. Biol. Chem. 

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