determination ofcholesterol bymeans ofphioroglucinol inacid solution

Determination of Cholesterol by Means

of Phioroglucinol in Acid Solution

Benjamin Sahagian* and Victor E. Levinet

Cholesterol is determined by taking advantage of the fact that the digitonin moietyof cholesteryl digitonide is a glycoside containing in its molecular structure onemolecule of xylose and four molecules of galactose, and therefore reacts with a sugar

reagent, such as phloroglucinol in acid solution, to form a stable color complex yield-ing quantitative results when measured spectrophotometrically. The method agrees

well with results obtained by the Schoenheimer and Sperry (8) procedure.

NI ANY REAGENTS employed in the colorimetric estimation of choles-

terol, the most popular being the Liebermann-Burchard reagent, react

directly with the sterol. Several methods, however, have been devised

which depend indirectly upon the analysis of the nonsteroid moiety of

a compound capable of combining with cholesterol. Engel and his as-

sociates (i) prepared cholesteryl hemidinitrophthalate by means of

3,5 dinitrophthalic anhydride. This compound reacts with primary

and secondary alcohols to yield alkali-soluble half esters. The half

esters of the anhydride of m-dinitrophthalic acid develop a character-

istic red color on treatment with alcoholic potassium hydroxide in the

absence of a ketone. This color can be measured quantitatively.

Recently, several methods have been developed depending upon the

precipitation of cholesterol with digitonin and estimating cholesterol

indirectly from the digitonin in union with the sterol. Rappaport and

Klapholz (2) determined cholesterol by the iodometric titration of the

hexoses liberated by the hydrolysis of excess digitonin. Michaelis et at.

From the Department of Biological Chemistry and Nutrition, The Creighton University

School of Medicine, Omaha, Neb.

The investigation reported was done as part of a project supported by a grant from the

U. S. Public Health Service.

Received for publication July 10, 1962.

5Present address: 3616 Mohawk St., Lineoln, Nab.

tDeceased.

116

Dlgltonin

+ 4 Galactose +Xylase

Digitogenin Carboli�drate moIety

(2g. 1,8, 1�,1 -triol)

Vol. 10, No. 2, 1964 DETERMINATION OF CHOLESTEROL 117

(3) described a method involving color formation by means of orcinol

and ferric chloride. Fechtmeir and Bregerman, (4) Webster et at., (5)

and Vahouney et at. (6) reported the determination of cholesterol by

treatment of its digitonide with anthrone, a compound which Drey-

wood (7) introduced as a color reagent for sugars.

Digitonin is a glycoside. The aglucon is in chemical union with 1

molecule of xylose and 4 molecules of galactose (Fig. 1). We have

Fig. 1. Structural formula of digitonin. Hydrolysis liberates digitogenin, four molecules

of galactose, aiid one molecule of xylose.

found cholesteryl digitonide to react quantitatively with resorcinol

and with phioroglucinol. We herein report the details of the method in

which free cholesterol or cholesterol freed from its esters by saponifi-

cation is precipitated as the digitonide and colorimetrically estimated

by means of phloroglucinol.

Experimental

We performed a series of preliminary experiments for the purpose

of investigating the reaction of the phenols, resorcinol, orcinol, and

phioroglucinol with xylose, galactose, and with xylose and galactose

(1:4), mixed in stoichiometric proportions, with digitonin, and with

cholesteryl digitonide in the presence of diluted sulfuric acid. Each

phenol was dissolved in 90% (v/v) acetic acid, 5 ml. containing 4 mg.

of the compound. The sugar solutions were prepared with distilled wa-

ter in concentrations of 1 mg./mI. The digitonin was dissolved in 50%

1)8 SAHAGIAN & LEVINE Clinical Chemistry

alcohol (equal volumes of alcohol and water) to the extent of 1 mg./ml.

Cholesteryl digitonide was precipitated from a cholesterol solution of

alcohol and ether (3:1), and the precipitate washed several times with

alcohol-ether mixture and finally dried in a desiccator.

The experiment was carried out in the following way: 1 ml. each of

xylose, galactose, xylose and galactose mixture, digitonin, and 1 mg.

cholesteryl digitonide was suspended in a glass-stoppered test tube.

To each of these was added 8 ml. of a cool previously prepared mixture

containing 5 ml. phenol solution and 3 ml. cool diluted sulfuric acid (7

vol. concentrated sulfuric acid and 3 vol. distilled water). Each reac-

tion mixture was heated in a water bath for 25 mm. The results are

presented in Table 1.

Table 1. CoLon REACTIONS OF SUGAES, DIOrroNIN, AND CHOLESTERYL DmIToNmE wI’rn

PHENOLS

Phenol Reaction

Xmosa

Resorcinol

Orcinol

Phloroglucinol

Deep red

Olive green changing to deeper green

Orange red

GALACTOSE

Resorcinol

Orcinol

Phloroglucinol

Deep red

Olive green changing to deeper green

Orange red

XYLOSE AND GALACTOSE

Besorcinol

Oreinol

Phloroglucinol

Cherry red

Green changing to deeper green

Orange red

DIOTTONIN

Resorcinol

Oreinol

Phloroglucinol

Orange red

Green changing to deeper green

Orange red

CHOLESTERYL DIGIT0NIDE

Resorcinol

Orcinol

Phloroglucinol

Cherry red

Red changing to deeper green

Orange red


MethodsReagents and Solutions

We employed the following reagents and solutions for the phioro-

glucinol method:

1. Alcohol-ether mixture 3 vol. alcohol and 1 vol. ether or alcohol

acetone mixture (equal volumes of alcohol and acetone)

2. Isopropanol-water-sodinm sulfate mixture After 30 ml.

isopropanol and 70 ml. distilled water are thoroughly mixed, anhy-

drous sodium sulfate is added a few grams at a time and the mixture

shaken thoroughly each time until saturation is obtained; undissolved

sodium sulfate is removed by decantation

3. Diluted sulfuric acid 7 vol. concentrated acid and 3 vol. dis-

tilled water

This mixture should be used only when cool. The diluted acid when

cool serves to minimize errors due to the volumetric measurement of

the viscous concentrated sulfuric acid. It also serves to minimize the

formation of extraneous color due to the dehydrating, condensing and

caramelizing effect of concentrated sulfuric acid upon sugars. It elimi-

nates the uncertainty of the color produced as a result of the heat de-

veloped upon mixing concentrated sulfuric acid with an aqueous solu-

tion. The diluted acid proves to be of sufficient concentration to pro-

duce distinct colors with resorcinol, orcinol, and phioroglucinol in the

presence of xylose, galactose, digitonin and cholesteryl digitonide.

Furthermore, since there is no necessity for maintaining anhydrous

conditions in this method as is the case for methods employing the

Liebermann-Burchard reagent, the use of a dilute acid offers a distinct

advantage.

4. Acetic acid solution 9 vol. glacial acetic and 1 vol. distilled

water

5. Phioroglucinol solution 100 mg. chemically pure compound

dissolved in 100 ml. 90% acetic acid (4)

This solution should not be prepared in greater quantities than 100

ml. and should be kept in the refrigerator in a brown glass-stoppered

bottle when not in use.

6. Phioroglucinol reagent 5 ml. phioroglucinol solution (5) and

3 ml. diluted sulfuric acid (3) freshly prepared before use

7. 50% Alcohol Equal volumes of absolute alcohol and distilled

water

8. Digitonin solution 1 gm. in 100 ml. of 50% alcohol (7).

120 SAHAGIAN & LEVINE Clinical Chemistry

This solution should be kept in the refrigerator in a glass-stoppered

brown bottle.

9. Dilute hydrochloric acid 5 ml. concentrated acid and 95 ml.

distilled water

10. 50% Potassium hydroxide solution

11. Phenolphthalein solution 1% solution in 95% neutral alcohol

12. Cholesterol standard stock solution 100 mg. chemically pure

cholesterol in 100 ml. alcohol-acetone (1:1)

This solvent is preferred to alcohol-ether (3:1) because of the dan-

ger of losing part of the ether by evaporation. This solution should be

kept in the refrigerator in a brown glass-stoppered bottle.

13. Working standard solutions These are prepared when needed

by dilution of the stock solution. According to the method, 0.2 ml. of

diluted serum used for the determination of cholesterol represents

0.02 ml. of the original serum. A dilution of 1 vol. of the stock solution

with 4 vol. of solvent yields a standard of which 0.2 ml. contains 40 1Lg.

cholesterol and corresponds to 200 mg. cholesterol in 100 ml. serum. A

dilution of 1 vol. of the stock solution with 3 vol. of solvent yields a

standard, of which 0.2 ml. contains 50 �g. cholesterol and corresponds

to 250 mg. cholesterol in 100 ml. serum. A dilution of 0.9 vol. of the

stock solution with 2.1 vol. of solvent gives a standard of which 0.2 ml.

contains 60 �g. cholesterol and corresponds to a serum content of 300

mg.% cholesterol. Diluting an equal volume of the stock solution with

an equal volume of solvent yields 0.2 ml. equivalent to 100 �g. choles-

terol and to 500 mg. cholesterol in 100 ml. serum.

A. Extraction of Cholesterol and its Esters from Blood

I. The alcohol ether mixture (9 ml.) or alcohol acetone mixture (9

ml.) is pipetted into a clean, dry glass-stoppered 15-mi. centrifuge tube.

2. Clear serum (1 ml.) is added to the above mixture.

3. The centrifuge tube is stoppered tight and shaken vigorously for

10 sec. The pressure is released by loosening the stopper. The tube is

shaken a second time and allowed to stand for 10 mm.

4. The tube is centrifuged for 15 mm. at 2500 rpm.

B. Precipitation of Free Cholesterol with Digitonin

1. The clear supernatant fluid (0.2 ml.) is drawn into a 15-ml. cen-

trifuge tube.

2. Digitonin solution (1 ml.) is now added, and the mixture stirred

thorou�hly with a glass rod.


3. The centrifuge tube is placed in a 40#{176}water bath for 2 hr.

4. At the end of this period, the tube is removed from the water

bath, cooled to room temperature, and centrifuged for 15 mm. at 2500

rpm.

5. The tube is carefully removed from the centrifuge without dis-

turbing the precipitate, and the supernatant liquid separated from the

precipitate by means of a fine capillary glass tube attached to a water

aspirator.

6. The precipitate is washed 3 successive times with 2 ml. isopro-

panol sodium sulfate solution. The tube is rotated gently by hand to

wash the sides with the solution. After each of three washings, tile

precipitate is thoroughly stirred with a glass rod, the wedged stopper

replaced, and the tube centrifuged for 15 mm. at 2500 rpm.

C. Hydrolysis of Cholesterol Esters and the Precipitation of Total Cholesterol

1. Alcohol ether or alcohol acetone extract (0.2 ml.) is drawn into a

clean, dry centrifuge tube.

2. The centrifuge tube is placed for 10 mm. in a water bath kept at

85#{176}to evaporate the solvent.

3. The residue is dissolved in 0.5 ml. absolute alcohol.

4. The solution is now treated with 2 drops of 50% potassium hy-

droxide to saponify cholesterol esters.

5. The centrifuge tube is placed for one half hour in a 60#{176}water

bath. At the end of this period the tube and its contents are cooled to

room temperature by placing the tube in a beaker of tap water.

6. The contents of the tube are first treated with one drop of phe-

nolphthalein solution and followed by one drop of concentrated hy-

drochloric acid and as many drops of 5% hydrochloric acid (Reagent

9) necessary to neutralize the alkali and to render the mixture slightly

acid.

7. The mixture is treated with 1 ml. digitonin solution to precipitate

the cholesterol, all of which now exists in the free state. The choles-

teryl digitonide is now subjected to treatment as per Section B, Steps

3-43, and subjected to further treatment as per Section D.

D. Development of the Color Complex of Cholesteryl Digitonide whenTreated with Phloroglucinol

1. The washed precipitate of cholesteryl digitonide in the centrifuge

tube is treated with 8 ml. freshly prepared phloroglucinol reagent (6).

The contents are mixed thoroughly with the glass rod which remains in

the tube during the washing and centrifugation procedures.


2. The centrifuge tube is placed for 25 mm. in the 85#{176}water bath.

3. At the end of this period, the tube is removed from the water bath

and cooled to room temperature by placing in a beaker of tap water.

4. The absorbance reading is compared with that of the standard

solutions of cholesterol which have been digitonized and treated with

the phloroglucinol reagent as described in A and D.

5. The blank consists of 8 ml. freshly prepared phloroglucinol re-

agent (5 ml. phloroglucinol solution and 3 ml. diluted sulfuric acid).

The color obtained with the phloroglucinol reagent is stable for at

least 2 hr.

Technic of Washing the Cholesteryl Digitonide Precipitate

The precipitate of cholesteryl digitonide comes down very slowly

as a fluffy substance. Heating to 40#{176}shortens the time of precipitation

and brings about the formation of fioccules of considerable size, which

pack down very nicely under centrifugation. Since digitonin itself

reacts with phloroglucinol in the presence of acid, it becomes neces-

sary to remove any excess from the precipitate. When the Liebermann-

Burchard reaction is employed as the color-forming reagent, it reacts

only with the cholesterol moiety of the cholesteryl digitonide and not

with digitonin itself, especially when pure digitonin is used. In order

to remove all traces of water, Schoenheimer and Sperry (8) and

Sperry and Webb (9) washed the cholesteryl digitonide precipitate

with a mixture of ether and acetone and finally with ether alone. In

the phloroglucinol procedure, moisture is not a critical factor. The

washing mixture is used only for the purpose of removing excess

digitonin.

The method of removing excess digitonin employed by Michaelis

et at. (3) involved the use of a mixture of 9 vol. of isopropyl alcohol and

1 vol. of distilled water. We tried this washing mixture but found par-

tial loss of precipitate by solution. Because we were dealing in the

phloroglucinol method with very minute quantities, in fact with micro-

chemical quantities of precipitate, peptization readily took place. We

tried a large number of solvents and mixtures of solvents to prevent

loss of precipitate. Our reagent No. 2 proved effective.

All precipitations are made in a 15-mi. centrifuge tube to which a

wedged one-hole rubber stopper has been carefully fitted. The upper

one-third is cut away at right angles to its longitudinal axis. This por-

tion is discarded and the lower two-thirds put into use. An arc Qf 5-6

mm. is cut into the stopper. It is fitted with a thin glass rod of such

P3 j� II ,7dlscard

cut here20 �ll U

Nn lone hole stopper

Fig. 2. Assembled apparatus

and parts employed in washing

cholesteryl digitonide precipi-

tate. Connect toaspirator

Thinglass rod

Capillarypipet

Assen*lodapparatus

Vol. 10, No. 2. 964 DETERMINATION OF CHOLESTEROL 123

length that it will protrude about 5 mm. out of the stopper when both

stopper and rod are placed in position in the centrifuge tube (Fig. 2).

The rod is left in the tube during centrifugation, thus obviating the

necessity of removing the glass rod each time the tube has to be cen-

ll�

trifuged. Accidental loss of the precipitate adhering to the glass rod is

thus avoided. A capillary pipet lowered into the centrifuge tube

through the wedge opening in the rubber stopper is used to remove the

supernatant fluid. An ordinary aspirator or water pump is quite satis-

factory in aiding in the removal of this fluid.

Care must be taken to keep the capillary tube above the precipitate.

Too great a negative pressure should not be employed to remove the

supernatant fluid.

The rubber stopper may be covered with aluminum foil.

Absorption Spectra

Aqueous solutions of xylose, of galactose, of a mixture of xylose and

galactose (1:4) in stoichiometric proportions, a solution of digitonin

555

555

550

010

060

ass

0

-C

am

am

555 �j

am

-C�0

C

555 Ms

alas

4as 510 500WAVELENGTH (MsP

0 440 4as 520 540 60) 640WAVElENGTH (M5J


in 50% alcohol, and cholesteryl digitonide were heated with phioro-glucinol reagent in the manner described in Section 1). Absorption

curves possessed a similar general character and there were some dif-

ferences in the location of the peak as noted below and in Fig. 3-5.

Complex Absorption peak (m/L)

Xylose and galactose-phloroglucinol

Digitonin-phioroglucinol

Cholesteryl-digitonide-phloroglucinol

Conformity with the Beer-Lambert Law

The cholesteryl digitonide phloroglucinol complex reacts in con-

formity with the Beer-Lambert law (Fig. 6). Duplicate samples, a set

from a solution of cholesterol in alcohol acetone (1:1) were placed in

15-mi. centrifuge tubes. The concentrations used were 20, 40, 60, 80,

and 100 pg., respectively. The cholesterol was precipitated with 1 ml.

of digitonin solution in 50% alcohol, and the procedure was continued

as described in Sections A, B, and D.

alas

Fig. 3 (left). Absorption spectrum of complex obtained by treating, in stochiometric pro-

portions, one part of xylose and four parts of galactose with phloroglucinol reagent. Fig. 4

(right). Absorption spectrum of complex obtained by treating digitonin with phloroglucinol

reagent.

550 M�

am

am

am0

C

am

am

0-Il

a’

Oil

‘40

/

vol. 10, No. 2, 1964 DETERMINATION OF CHOLESTEROL 125

20 40 60 � 160CONcENTRATION OF CHOUSIEROL 1

Comparison of the Phloroglucinol Method with the

Schoenheimer-Sperry Method

Solutions of cholesterol in glacial acetic acid were prepared for the

Schoenheimer-Sperry procedure (8). The green color developed with

the Liebermann-Burchard reagent as employed by Schoenheimer and

Sperry was measured at 650 mp� Specimens of blood for cholesterol

comparisons came from laboratory personnel and from patients in the

dispensary operated by the medical school.

A comparison of results (Table 2) indicates good agreement of the

Fig. 5. Absorption spectruM

of complex obtained by treating

cholesteryl digitonide with phlo-

roglucinol reagent.

0 5�Q �

WAVELENGTH(ffi�I

Fig. 6. Compliance with Beer-

Lambert law of cholesteryl digi-

tonide complex obtained by

treating cholesteryl digitonide

with phloroglucinol reagent.

Beckman DU spectrophotometer

was used. Similar results were

obtained with Coleman Junior

spectrophotometer using a 12 X

75-mm. cuvet, 5.5-ml. capacity.


Table 2. SEIEIJM CHOLESTEROL VALUES: COMPARATIVE DATA BY Two DIFFERENT METHODS

Schoenheimer-Sperry method Phloroglucinol method

(mg./lOO ml.) (mg.IlOO ml.)

Free Total Free Total

cholesterol cholesterol cholesterol cholesterol

1 80 238 81 240

2 75 234 76 233

3 73 222 76 225

4 94 331 96 335

5 78 212 80 212

6 114 279 111 279

7 62 198 62 197

8 73 251 75 253

9 60 239 62 241

10 81 257 82 260

11 82 252 80 251

12 50 188 54 191

13 55 203 53 206

14 72 191 74 195

15 64 215 63 214

16 49 209 50 210

17 57 212 61 212

18 93 271 95 270

19 57 213 60 215

20 71 251 73 253

21 64 243 65 244

22 50 194 52 196

23 52 165 50 164

Results are averages of replicates (two cases of duplicate samples).

Table 3. RECOVERIES BY THE PHLOROOLUCINOL METHOD OF CHOLESTEROL ADDED TO POOLED

SERUM

Free Total free Total Total free Total

cholesterol cholesterol cholesterol cholesterol cholesterol

So. added present present recovered recovered

1 0 64 232 - -

2 10 74 242 75 242

3 50 114 272 115 270

4 100 164 332 165 334

5 150 214 382 213 379

6 200 264 432 261 435

7 250 314 482 317 484

8 300 364 532 370 530

9 350 414 582 414 582

10 400 464 632 463 635

1n milligrams per 100 milliliters.Results are averages of replicates (two pairs of duplicate samples).


phioroglucinol procedure with those of the Schoenheimer-Sperry pro-

cedure.

Recovery of Cholesterol Added to Serum

To ascertain whether cholesterol added to serum can be recovered

quantitatively, we used a pooled sample of human serum which gave

an initial free cholesterol content of 64 mg./100 ml. and a total choles-

terol content of 232 mg./100 ml. To 1-ml. samples of serum, an exact

quantity of free cholesterol dissolved in alcohol-acetone (1 :1) was

added. The mixtures were diluted with alcohol-acetone to 10 ml. for

sera with a smaller content of cholesterol and to 25 ml. for those with

higher cholesterol content.

As indicated in Table 3, good recoveries were made in each case.

References

1. Engel, L. I., Patterson, H. R., Wilson, H., and Schinkerl, E. M., J. Biol. Chem. 47, 183

(1950).

2. Rappaport, F., and Klapholz, B., Biochem. Ztschr. 258, 467 (1933).

3. Michaelis, C. D., Fakayama, C., Chin, H. P., and Wheeler, P., Proc. Soc. Biol. 4- Exper.

lied. 98, 826 (1958).

4. Fechtmeir, T. V., and Bregerman, J., Am. J. Clin. Path. 23, 599 (1933).

5. Webster, W. W., Jr., Nichols, C. W., Jr, and Chaikoff, P., Proc. Soc. Biol. 4- Exper. Med.

98, 826 (1959).

6. Vahouney, G. V., Mayer, R. M., and Roe, J. A., Arch. Biochenz. Biophys. 86, 210 (1960).

7. Dreywood, B., lad. Eng. Chem. Anal. Ed. 18,499 (1946).

8. Schoenheimer, R., and Sperry, W. M., J. Biol. Chem. 106, 745 (1934).

9. Sperry, W. M., and Webb, M., J. Biol. Chem. 187, 97 (1950).

determination ofcholesterol bymeans ofphioroglucinol inacid solution

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