ANALYTICAL CHEMISTRY
316 _
I_-
Table 111. Set NO.
1 2 3 4
6 6 7 8
9
10 11
-
~
Lead Taken
-5fQ.
MQ.
10.03
..
15.05 20.06 25.08
..
, . .
4.95 4.95 4.95 4.95 4.95
_
~
was dissolved in glacial acetic acid; this solvent offered no advantage over 95% ethyl alcohol. In determinations made E rofr o r in the absence of ammonium acetate, Test of .\veragt= Filtrates the precipitate was more difficult to re% move from the wall of the beaker. 0 N If the concentration of the hydro0 N oo color color -0.1 N o color chloric acid in the precipitating medium 0 N o color -0.2 Faint color was greater than 0.01 LV,a preliminary ... treatment was carried out, in which some 0 s o color +0.6 Faint color of the acid was neutralized by the +0.2 s o color -0.4 careful addition of dilute sodium hydro0 Faint gen carbonate solution. Alternatively, the sample was evaporated to dryness on a steam bath, the residue was dissolved in water, and the resulting solut,ion was filtered. Blank determinations were made with each increase in the amount of reagent added. When the sample weight of iridium was increased, the weight of 2-mercaptobenzothiazole was correspondingly increased. If the sample tveight of iridium was larger than 20 mg., there was increased difficulty in transferring and washing the bulky precipitate. In these cases the method offersno advantages over the hydrolytic precipitation of iridium. The results of the determination of iridium over a range of sample weights are shown in Table 111.
Determination of Iridium i n Presence of Lead w i t h 2-Mercaptobenzothiazole
Iridium Taken LO2
_
‘i ..
KO. of Detns.
Av. Iridium Recovered
Av. Deviation
.Mg.
4 3
4
2 1
...
1 2
3 7
2 3 2 4 3
5.02 10.03 15.03 20.05 25.04 -0.01 4.95 4.98 4.96 4.93 4.95
Mg.
h O.01 =tO.Ol *0.01
., .. ..
’
*0.02 *0.’02
*0.04
~~_______~
~
bumping. The sample was then boiled vigorously for 1 hour; during this time the iridium separated as a bulky orange precipitate. At the end of the hour (or before, if the volume of liquid in the sample became less than 20 ml.)] the cover glass and the aides of the beaker were washed down with a hot solution 2% in ammonium acetate and 2% in acetic acid. The final volume of the liquid was 50 to 70 ml. The sample n-as set aside overnight on a steam bath and then filtered through a Whatman No. 42,7-cm. filter paper and washed with 100 ml. or more of hot solution 2% in ammonium acetate and 2yo in acetic acid. After washing, the filter was dried under a heat lamp to remove the excess moisture and then transferred to a tared crucible. The filter was blackened in a muffle a t 350’ C., heated for 45 minutes a t 650’ to 700” C., cooled, reduced in hydrogen] cooled in hydrogen and nitrogen, and weighed. Saturated solutions of calcium nitrate were kept in the desiccator and in the balance case to maintain constant humidity. A blank, determined in a similar manner, was subtracted from the weight of the sample. The filtrates and washings were checked for the presence of iridium by evaporation to dryness and digesting with concentrated sulfuric and nitric acids on a steam bath. The liquid was transferred to a 50-ml. borosilicate glass Erlenmeyer flask and fumed strongly over a burner until colorless. After cooling, 1 or 2 drops of 7Oy0 perchloric acid were added and the contents were reheated until fumes of perchloric acid appeared. On cooling again, a mauve color indicated the presence of iridium, 0.01 mg. of iridium in 0.5 ml. of the solution could be detected. Qualitative observations showed that precipitation from an acetic acid solution is incomplete if much mineral acid is present. It was found that the optimum concentration of either hydrochloric or nitric acid for complete precipitation by the method outlined is 0.005 to 0.01 N . The length of time during Tvhich the sample is digested on the steam bath is also important. Low results were consistently obtained for a digestion time under 15 hours. A few tests were made in which the organic reagent
~~~
DETERMINATION OF IRIDIUM IN PRESENCE OF LEAD
Samples containing known quantities of iridium and lead as their chlorides were treated as described above for the determination of iridium by 2-mercaptobenzothiazole, A number of the recovered iridium samples were examined spectrographically for the presence of lead. Only set 11 showed trace contaminat,ion with lead above that int,roduced through the reagents. The results from single precipitation3 are recorded in Table 111. LITERATURE CITED
Currah, J. E., McBryde, TV. 4.E., Cruikshank, A. J., and Beamish, F. E., IND.ENG.CHEM.,ANAL.ED.,18, 120 (1946). (2) Gilchrist, R., J . Reseurch Nntl. Bur. Standards, 9, 547 (1932). (3) Ibid., 20, 745 (1938). (4) Ibid., 30,89-99 (1943). (5) Hill, M. A., and Beamish, F. E., ANAL.CHEM.,22, 590 (1950). (6) Holeer, H., and Zaussinger, E., 2. anal. Chem., 111,321 (1938). (7) Pollard, W. B., Bull. Inst. Mining Met., KO.497, 9 (1948). (8) Bchoeller, TV. R., and Powell, A. R., “Analysis of Minerals and Ores of the Rarer Elements,” p. 256, London, C. Griffin & (1)
Co., 1940. RECEIVEDJ u n e 2, 1950.
Cholesterol in Blood Serum Studies of Microestimation as the Pyridinium Cholesteryl Sulfate ALBERT E. SOBEL, JEROME GOODMAN, AND MONTE BL.4CT h e Jewish Hospital of Brooklyn a n d T h e Polytechnic I n s t i t u t e of Brooklyn, Brooklyn, N. Y .
T
HE most widely used method for the isolation of small amounts of cholesterol in naturally occurring substances prior to its estimation involves precipitation as the cholesterol digitonide (3). In 1936, Sobel, Drekter, and Natelson (’7) proposed the isolation of such cholesterol as the pyridinium cholesteryl sulfate salt. The method they used, however, was relatively cumbersome and required exacting technique, involving the use of the hygroscopic solid pyridine sulfur trioxide as the precipitating agent. The purpose of the present investigation was to develop a more practical procedure for the isolation of cholesterol as this sulfate
salt. It was found, after considerable investigation, that when small amounts of cholesterol (about 0.1 mg.) are dissolved in 0.5 ml. of carbon tetrachloride] and 0.1 ml. of pyridine and then 0.25 ml. of a saturated solution of cholorosulfonic acid in carbon tetrachloride are added, a t least 97% complete precipitation of the sulfate occurs in less than a half hour (see Table I), as determined by the colorimetric estimation of cholesterol remaining in solution. A longer time produced no increase in precipitation. The reaction involved may be written as
ROH
C5HsK + ClSOaH -AROS03HCsHJ + CbHaXHC1
V O L U M E 2 3 , NO. 3, M A R C H 1 9 5 1
517
This investigation was undertaken to provide a derivative other than the digitonide for the isolation and determination of small amounts of cholesterol and possibly other sterols. Small amounts of cholesterol may be quantitatively isolated as the pyridinium cholesteryl sulfate. Cholesterol in carbon tetrachloride and pyridine (5 to 1) is treated with chlorosulfonic acid. The precipitate is washed with petroleum ether and evaluated colorimetrically by the Liebermann-Burchard reaction. Values of total cholesterol in blood serum are similar to those
xherr It = steryl radical, and may be applicable to hydroxysterols other than cholesterol. The results obtained in the determination of cholesterol in blood with this new method weie compared with results obtained with Sobel and Mayer's ( 8 )variation of the Schocnheimer-Sperry (6) technique, both of which involve precipitation of cholesterol as the digitonide. This reference method of Sobel and ;\layer n a s modified so that, by reducing all volumes by half, smaller amounts of serum (0.1 ml.) could b(1 used, and a speetrophotometric method was devised which could be applied without change to the colorimetric determination of cholesterol in the form of the sulfate, the digitonidr, or uncombined cholesteiol.
obtained by digitonin. Depending upon the method of extraction, free cholesterol values are the same as or less than with digitonin. The new method not only provides a n alternative procedure for microestimation of cholesterol, but may serve to elucidate the various combinations in which cholesterol is present in the lipide extract of blood serum. The derivative may be useful in isolating a-sterols following removal of &sterols with digitonin. In large scale sterol isolation problems, it may obviate need for the more expensive digitonin.
precipitated as the digitonide as did those values based on the standards. This calibration curve, plotted in optical density against cholesterol in 1 ml. of acetic acid (up to 0.2 mg. of cholesterol), is a straight line in which 0.1 mg. of cholesterol gives an optical density reading of 0.50 when treated as described below. A sample of pyridinium cholesteryl sulfate of unknown purity was used to obtain a curve which similarly showed adherence to Beer's law. I n order to ascertain whether the digitonide and sulfate methods as used in this paper to precipitate cholesterol were suitable for the quantitative determination of precipitable cholesterol in blood serum, cholesterol in sera was determined both with and without the addition of known amounts of pure cholesterol. .I difference in blood cholesterol values corresponding to the added cholesterol is evidence that precipitable cholesterol has been quantitatively determined. As seen in Table 11, known amounts of cholesterol added to serum were quantitatively recovered as the pyridinium cholesteryl sulfate and cholesterol digitonide. To test the sulfate method further, the cholesterol in extracts of saponified ( 4 ) sera was determined as the pyridinium cholesteryl sulfate, the cholesterol digitonide, and by direct colorimetric evaluation of the lipide extract (see Table 111). The total cholesterol vdues obtained by the sulfate method were similar to the values obtained by the other two methods. Thus, the applicability of the sulfate method to total cholesterol estimation is indicated.
" '
0.39
4-
I
1
CHOLESTEROL
Table I.
25
30
35 TIME (rnin.)
40
Figure 1. Stability of Color Produced by action of 3 m l . of 25 to 1 acetic anhydride-sulfuric acid mixture on 1 m l . of acetic acid solution of cholesterol and pyridinium cholesteryl sulfate. Color developed in dark at 25' C., read at 625 mp o n Coleman spectrophotometer
The rate at which color developed after addition of color reagent, to a solutioii of pyridinium cholesteryl sulfate in acetic acid was found to be about the same as that with cholesterol or cholesterol digitonide in that solvent (set. Figure 1). Although it was verified that equivalent amounts of cholesterol and cholesterol dieitonide give the same intensity of color ( 6 ) ,t,he color developed on the pyridinium cholesteryl sulfate was observed to be somewhat less than that from a corresponding amount of cholesterol or the digitonide. In all cases, maximum absorption \vas a t 625 mp. All cholesterol values given in this paper were determined on the basis of cholesterol standards which were precipitated and treated in the same manner as the unknowns. However, a calibration curve for pure cholesterol (prepared by determining the color developed on standard solutions of cholesterol in acetic acid) gave essentially the same values for cholesterol whirl1 was
Precipitation of Cholesterol as Sulfate
Reaction time, min. Cholesterol precipitated,
(0.1 mg. used) r, 10 81.0 95.0
6%
15 96.7
20 97.0
30
97 2
Table 11. Typical Results in Recovery of Known Amounts of Cholesterol Added to Sera (Detei mined as cholesterol digitonide or pyridiniuni cholesteryl sulfate) Cholesterol Sample Cholesterol Cholesterol Cholesterol Cholesterol Found/ NO. Present Added Calculated Found Calcd y / O . l ml.
B 6 7 8
c
9 10
39.3 40.0 32.3 191 174
Y
50.0 50.0 50.0
200 200
Y
Y
%
89.0 90.0 82.3 391 374
88.7 88.5 84.0 391 380
99.7 98.4 102.0 100.0 101,B
correspondingly reduced. C. Cholesterol in saponified serum precipitated as pyridinium cholesteryl sulfate after extraction with petroleuni ether.
sia
ANALYTICAL CHEMISTRY Table 111.
Cholesterol in Saponified Sera
Determined colorimetrically a f t e r precipitation as pyridinium cholesteryl sulfate a n d cholesterol digitonide, and directly on lipide extract. Values in mg./100 m!. serum) Pyridinium Sample Cholesteryl Cholesterol Direct NO. Sulfate Digitonide Evaluation 164 164 158 185 185 192 192 192 188 268 266 265 277 270 278 271 278 283 284 287 281 377 380 8 3 84 843 833 83 5 9 Av.
316
319
318
The determination of “free” cholesterol as the sulfate is related to the basic problem of what free cholesterol is. In 1936, Drekter, Sobel, and Natelson ( 2 ) found that values for cholesterol in blood, determined as the pyridinium cholesteryl sulfate, mere from 18 to 37% of the values obtained as the digitonide, although cholesterol added to sera was quantitatively recovered, within experimental error, as the sulfate. They postulated that free unesterified cholesterol, precipitated by digitonin, consists of “loosely bound” and “unbound” cholesterol, only the latter being precipitable as the sulfate salt.
Table IV. Ratio of Cholesterol Determined as Pyridinium Cholesteryl Sulfate to Cholesterol from Same Extract Determined as Digitonide Chol. as Sulfate/Chol. Method of Extraction as Digitonide Drekter et al. 0 . 1 8 to 0 . 3 7 Petrolc 0 . 5 4 to 0 . 8 3 Alcoh~~amoea} 0.59 to 0.71 3 Alcohol-ether 4 10% H2SOeCClr 0 . 5 2 to 0.84 5 50% H2SOeCClr 0 . 3 8 to 0 . 9 0 Quantitative recovery of cholesterol added before extraction was obtained in all cases. All extractions were carried o u t a t room temperature. Give identical results with individual bloods. Other methods of extraction not compared on same bloods.
No. 1 2
Table S’. Cholesterol Determined as Sulfate after Extraction in Alcohol-Acetone Mixture Evaporated at High Temperature, and as Digitonide (Values given as y/O.l ml. of serum = mg. %) 1 2 3 4 Pyridiniurn cholesteryl sulfate, heating alcoholacetone extract 46.7 40.5 65.2 56.3 Cholesterol digitonide 46 2 4 3 . 7 6 1 . 8 5 4 . 5
Drekter, Sobel, and Natelson used three methods of extracting cholesterol from serum (two involving boiling alcohol-ether mixtures and the third involving extraction of serum dried on paper with a refluxing 3% solution of pyridine in alcohol), all of which gave essentially the same results. I t appears, however, from the authors’ data, that the ratio of unbound to free cholesterol depends on the method of extraction used (see Table IV). Although the results of these studies indicate a fairly wide range of unbound t o free cholesterol ratios, they suggested that relatively drastic methods of extracting cholesterol from serum might break the loosrly hound cholesterol combination which Drekter et al. postulated. Thus it might be possible to determine free cholesterol (digitonin-precipitable) by proper extraction followed by precipitation as the pyridinium cholesteryl sulfate. That such extraction methods are likely is indicated in Table V, which compares the cholesterol precipitated as the digitonide with that precipitated as the sulfate after extraction of the cholesterol by an alcohol-acetone mixture which is evaporated to dryness a t high temperature. The problem of the nature of loosely bound cholesterol will bear further investigation. In blood serum itself most of the
cholesterol is in some combination. Only 3% of the cholesterol can be extracted with the same amount of petroleum ether that will completely extract cholesterol from the same serum previously treated with alcohol (unpublished experiments). Thus, free cholesterol is not really free in the blood serum, but is defined as free only by the fact that it is precipitated by digitonin in the lipide extract. In making this lipide extract, lipide-protein combinations are broken. I t is possible that the combination of cholesterol with a compound (or compounds) that acts as a bridge to the protein still exists in the lipide extract. The possibility of such combination taking place during the process of lipide extraction is not excluded. Such combinations may be broken by digitonin as well as by evaporation of the acetone-alcohol extract a t high temperatures (see Table V). Bills ( 1 )points out the many combinations which cholesterol is known to enter into in addition to fatty acids and esters. That loosely bound cholesterol is some such combination and is broken down by digitonin is suggested by the results of an experiment in which cholesterol digitonide precipitates were dissolved in pyrithe digitonin was precipitated with ether, and the soluble dine (6), cholesterol was determined as the sulfate (see Table VI). The evidence that the sulfate method will quantitatively determine cholesterol as the free alcohol may be summarized as follows: Results on saponified sera are similar to results by two other methods (see Table 111). Known amounts of cholesterol added to sera as the free alcohol are quantitatively recovered with all types of extraction employed (see Table 11). Following the precipitation of free cholesterol as the digitonide, the cholesterol in the precipitate can be quantitatively determined as the sulfate after splitting the cholesterol digitonide (see Table VI). The estimation of serum cholesterol as the pyridinium cholesteryl sulfate should be of value: To study the significance of free versus loosely bound cholesterol in health and disease. To .gain further insight into the cholesterol fractions of the organism. As a procedure for estimation of total cholesterol in serum following saponification. As a procedure for free cholesterol estimation, when time is an important factor. The time required for evaporation of the alcohol-acetone extract is 1 hour and for precipitation 0.5 hour. The total of 1.5 hours is distinctly less than the time required for the digitonin precipitation alone. EXPERIMENTAL
Rea ents (all analytical grade). Carbon tetrachloride. If on adztion of redistilled chlorosulfonic acid the acid layer turns brown, the carbon tetrachloride should be washed with water, dried over anhydrous sodium sulfate, and redistilled. Pyridine, redistilled. Petroleumether, 30” to 60”. Standard solution of cholesterol in carbon tetrachloride, 20 mg. of cholesterol in 100 ml. (0.5 ml. of carbon tetrachloride contains 100 micrograms of cholesterol). Alcoholic potassium hydroxide solution, 0.5 N . Dilute 3 ml. of stock solution (10 grams of potassium h droxide in 20 ml. of water) to 50 ml. with aldehyde-free ethyl acohol. Alcohol-acetone mixture, 1 to 1 by volume of 9570 ethyl alcohol and acetone. Color-developing reagent (prepared freshly before use). Add concentrated sulfuric acid to ice-cold acetic anhydride in a 1 to 25 ratio and mix well. Saturated solution of chlorosulfonic acid in carbon tetrachloride. -4dd sufficient chlorosulfonic acid (redistilled in all-glass apparatus) to carbon tetrachloride so that, after shaking, a layer of chlorosulfonic acid remains on the bottom of the solution. This reagent is stable but should be discarded when a brown color develops to any appreciable extent. Shake shortly before using to ensure a saturated solution. Precipitation of Cholesterol as Pyridinium Cholesteryl Sulfate. To a solution of cholesterol in 0.5 ml. of carbon tetrachloride in a 15-ml. centrifuge tube 0.1 ml. of pyridine was added. The tube vas shaken to mix the pyridine and carbon tetrachloride lavers and to wash any pyridine from the tube walls. Then 0.25 ml. of a saturated solution of chlorosulfonic acid in carbon tetra
V O L U M E 23, NO. 3, M A R C H 1 9 5 1 chloride was added and the tube was shaken immediately to prevent caking of the precipitate which forms a t once. The tube was stoppered and left a t room temperature for 20 minutes, during which time it was shaken once or twice. Then 5 ml. of petroleum ether were added and the mixture was shaken and then centrifuged for 10 minutes at 2000 r.p.m. The supernatant liquid was discarded, the precipitate was washed by shaking with 4 ml. of petroleum ether, and the centrifuging and washing were repeated. The precipitate was dried in a stream of nitrogen and dissolved a t up to 60" C. in 1 ml. of acetic acid, which was added to wash the walls of the tube. If the acetic acid is not immediately added to the precipitate, the tube should be tightly stoppered to prevent absorption of moisture from the air. The precipitation mixture diluted to about 6 ml. contains 0.85 ml. of yridine and carbon tetrachloride, in which pyridinium choester 1 sulfate is soluble or slightly soluble. The use of more petroium ether might therefore increase the 97% precipitation of cholesterol which is obtained. Evaluation of Percentage of Cholesterol Precipitated as Sulfate. In a typical experiment 100 micrograms of cholesterol were precipitated in triplicate. The combined supernatant liquid and washings were evaporated to dryness a t 40" in a stream of nitrogen. The cholesterol found in the residue (determined colorimetrically) was 8.6 micrograms or 2.87 micrograms lost in each tube. Thus the cholesterol precipitated was 97.13 micrograms per 100 micrograms of sample. The validity of this method for evaluating losses was established by evaporating the supernatant fluid and washings of the reagents mixed in triplicate in the absence of cholesterol. Of 10.0 micrograms of cholesterol added to the resulting residue, 10.0 =t0.18 micrograms were recovered in replicate estimations. Cholesterol digitonide was precipitated by the method of Sobel and 3layer (8), using 0.1 ml. instead of 0.2 ml. of serum and reducing reagent volumes by one half.
P
COLORIMETRIC DETERMINATION
Ten minutes after the color-developing reagent was mixed, 3 ml. of the reagent were added to 1 ml. of acetic acid solution of pyridiniuni cholesteryl sulfate, cholesterol digitonide, or pure cholesterol. The color was allowed to develop a t 25" in the dark and after 35 * 5 minutes was read in a Coleman Universal spectrophotometer set a t 625 mp with a Coleman PC.4 filter in place, using horizontal cuvettes of 5-cm. light path and 2.8-ml. capacity (9). Readings of unknowns were compared with those of B standard cholesterol sample containing 100 micrograms of cholesterol, which was precipitated and treated in the same manner as the cholesterol in the unknowns. Color-developing reagent only was used as a blank, as the addition of acetic acid did not alter results. With recalibration of the system, chloroform may be substituted for acetic acid. Density readings made for this paper are the average of a t least two determinations. The precipitate of pyridinium cholesteryl sulfate fluoresces under ultraviolet light, so that a fluorometric method might replace the colorimetric method of determining cholesterol in this precipitate. SAPONIFICATION O F SERUM
To 0.2 ml. of blood serum in a 6-inch round-bottomed test tube were added 5 ml. of 0.5 N alcholic potassium hydroxide solution. The tube was shaken and placed in a 45 O oven for an hour. After saponification 5 ml. of petroleum ether and 3 ml. of water were added, and the mixture was shaken in the hand t o mix the la ers well. Then 0.5-ml. or 1.0-ml. ali uots of the petroleum etger were transferred into 15-ml. centrijuge tubes, evaporated to dryness, and taken up in 0.5 ml. of carbon tetrachloride, 2 ml. of 1 t o 1 alcohol-acetone mixture, or 1 ml. of acetic acid, and the cholesterol present was determined colorimetrically as the sulfate, digitonide, or directly without separation from other lipides. CHOLESTEROL EXTRACTION METHODS
Petroleum Ether Extraction (see Table IV). To 0.1 ml. of serum was added 0.1 ml. of alcohol and lipides were extracted with two 2-ml. portions of petroleum ether, which was then evaporated down. The extract was taken up in carbon tetrachloride and cholesterol was determined as the sulfate; and in alcohol-acetone cholesterol was determined as the digitonide. Alcohol-Acetone and Alcohol-Ether Extractions (see Table IV). A 1 to 1 alcohol-acetone (8) or 3 to 1 alcohol (8) ether extract was evaporated down a t room temperature and the extract was taken up in carbon tetrachloride for precipitation of the
519
sulfate. Precipitation of the digitonide was carried out in alcohol-acetone. Sulfuric Acid Extractions (10 and 50%, see Table IV). In a 3-inch test tube 1 ml. of 10% of 50% sulfuric acid and 1 ml. of carbon tetrachloride were added t o 0.1 ml. of serum and the mixture was shaken for 20 minutes and centrifuged a t 2500 r.p.m. for 20 minutes. The cholesterol in a 0.5-ml. aliquot of the carbon tetrachloride was determined as the sulfate and, after evaporation of the carbon tetrachloride, as the digitonide in alcohol-acetone. Heating Alcohol-Acetone Extract (see Table V). To 0.1 ml. of serum in a 15-ml. centrifuge tube or 3-inch test tube were added 2 ml. of alcohol-acetone mixture to precipitate protein. The tube was shaken and centrifuged a t 2000 r.p.m. for 10 minutes, after which the supernatant liquid was poured off into a 15-nd. centrifuge tube. The protein precipitate was washed with 1 ml. of alcohol-acetone mixture, and recentrifuged, and the washed liquid was added to the initial extract. The centrifuge tube containing the extract was placed in a hot water bath a t 60" and the bath was heated slowly, so that 45 minutes later the temperature was 100" C. The tubes were allowed to remain in the 100" bath for 15 minutes, after which they were removed and allowed to cool, and the dried extract was taken up in 0.5 ml. of carbon tetrachloride. Pyridinium cholesteryl sulfate was precipitated as described above.
Table VI.
Cholesterol in Sera
(Determined as sulfate from cholesterol recovered from digitonide precipitates, a n d directly as digitonide. Values given y/0.1 ml. of serum = mg. %) 1
2
?
Cholesterol digitonide 72.2 62.6 60.0 Cholesterol from digitonide determined as sulfate 76.2 59.4 58.60 a Value from same serum after extraction i n 50% HzSOd a n d precipitation as sulfate was 31.6 with recovery of cholesterol added before extraction of 97.1%.
Determination of Cholesterol in Digitonide Precipitates as Sulfate. To the dry cholesterol digitonide precipitate from 0.1 ml. of serum 0.3 ml. of pyridine was added. After solution of the digitonide, 10 ml. of diethyl ether were added. The precipitated digitonin was redissolved in 0.2 ml. of pyridine after centrifugation and removal of the ether, and then reprecipitated with 5 ml. of ether. The tube was centrifuged and the ether extracts were combined, evaporated down, and taken up in 0.5 ml. of carbon tetrachloride, and the cholesterol present was determined as the pyridinium cholesteryl sulfate. RECOMMENDED PROCEDURES FOR FREE AND TOTAL CHOLESTEROL
Total Cholesterol. To 0.05 to 0.1 ml. of blood serum are added 2.5 ml. of 0.5 N alcoholic potassium hydroxide solution. The contents of the tube are mixed and heated a t about 45' for 1 hour. This is followed by addition of 1.5 ml. of water and 2 ml. of petroleum ether, after which the tube is shaken for half a minute by hand. One milliliter of the petroleum ether layer is removed and evaporated to dryness. The residue is dissolved in 0.5 ml. of carbon tetrachloride, cholesterol is precipitated as the sulfate, and color is developed and evaluated as described above. Free Cholesterol. Free cholesterol is determined as described for cholesterol extraction methods, heating alcohol-acetone extract. LITERATURE CITED
(1) (2)
Bills, C. E., PhysioZ. Rev., 15, 1 (1935). Drekter, I. J., Sobel, A. E., and Natelson, S., J . B i d . Chem., 1 1 5 , 3 9 1 (1936).
Fieser, L. F., and Fieser, &I., "Natural Products Related to Phenanthrene," 3rd ed., p. 104, Now York, Reinhold Publishing Corp., 1949. (4) Kendall, F. E., Abell, L. L., Levy, B. B., Steele, J. M., and Brodie, B. B., Federation Proc., 8, 212 (1949). (5) Schoenheimer and Dam, 2.physiol. Chem., 215, 5 9 (1933). (6) Schoenheimer, R., and Sperry, W. M., J . Bid. Chem., 106, 7 4 9 (3)
(1 934). (7)
Sobel, A. E., Drekter, I. J., and Natelson, S., Ibid., 115, 381
(8) (9)
Sobel, A. E., and Mayer, A. hI.,Ibid., 157, 255 (1945). Sobel, A. E., and Snow, S. D., Ibid.,171, 617 (1947).
(1936).
RECEIVED M a y 22, 1950. Presented before the Division of Biological Chemistry a t the 116th Meeting of the AMERICAN CHEMICAL SOCIETY,Atlantic City, h'. J. Taken in part from theses submitted t o the Polytechnic Inatit u t e of Brooklyn b y Jerome Goodman (June 1949) a n d Monte Blau ( J u n e 1948) in partial fulfillment of the requirement for the B.S. degree.
