evaporated and diluted t o 1500 ml. so that the final p H was 1.5. This solution was passed through Dowex 50 x Q, 20- to 50-mesh cation exchange resin, 70 cm. deep and 4 em. in diameter, and the effluent was collected in a 4liter beaker. The column was aashed with acidu!ated water whose p H was adjusted to 1.5 with hydrochloric acid. The solution was evaporated to about 25 ml., transferred to a 150-ml. beaker and again evaporated in presence of 5 ml. of 2% iYaC1. The dry residue ivas treated with nitric acid-hydrogen peroxide mixture to destroy any organic matter coming through the column and the nitrates were then converted to chlorides by hydrochloric acid. The residue nas dissolved in 1 or 2 drops of €IC1 and about 30 ml. of water and the p H adjusted to 1.5. The solution was passed through a small cation exchange column, 4 cm. deep and 1 em. in diameter, to remove traces of base metals, and washed with 80 ml. of pI-1 1.5 water. The effluent was taken directly for the gravimetric determination of rhodium by the thiobarbituric acid method. For the microdetermination of rhodium by the stannous chloride procedure, the effluent from the small exchange column was evaporated to a very small volume and transferred into a 30-ml. borosilicate glass test tube. The absorbance measurements were made at a wave length of 470 mp (6). One
hundred to 500 pg. of Rh in 50 ml. mere determined with 1-em. path length while less than 100 pg. of Rh in 25 nil. were determined using a 5-cm. path length. RESULTS
Experimental data obtained for rhodium concentrations ranging from 10 pg. to 10 mg. are given in Table I. These results show essentially complete recovery of rhodium. I n the gravimetric analysis a positive error was occasionally noticed, due to traces of silica associated with the button. This error was eliminated by subjecting the residue t o dry chlorination and reprecipitating rhodium by thiobarbituric acid. Dry chlorination was carried out in the presence of excess sodium chloride in a borosilicate glass tube heated to about 650' C. for 6 to 7 hours. The chlorinated product was leached with dilute hydrochloric acid, the solution was filtered, and the rhodium in the filtrate determined gravimetrically as described above. This difficulty with silica was not experienced when determining rhodium spectrophotometrically. The slag and pot walls from the 10mg. rhodium sample were analyzed by the classical fire assay with lead as the
collector. No rhodium was thus detected. ACKNOWLEDGMENT
This work was supported by a grant from the National Research Council of Canada. The authors express their appreciation to Suseela B. Sant for the spectrophotometric analyses. LITERATURE CITED
( 2 ) Berman, S. S., V ~
Cruikshank, A. J., Bkamish, F. E.; IND.EKQ.CHEM.,ANAL. ED. 18, 120
(1946).
(4j Kavanagh, J. PII., Beamish, F. E., ANAL.CHEM.32,490 (1960). (5) Marks, A. G., Beamish, F. E., Ibid., 30, 1464 (1958). (6) Maynes, A. D., McBryde, W. A. E., Analyst 79,230 (1954). (7) Plummer, hl. E. V., Beamish, F. E., ANAL.CHEM.31,1141 (1959). (8) Plummer, hI. E. V., Lewis, C. L., Beamish, F. E., Ibid., 31,254 (1959). (9) Tertipis, G. G., Beamish, F. E., Zbid., 32, 486 (1960); cf. Zachariasen, H., Beamish, F. E., Talanta 4,44 (1960). (10) Truthe, W., 2. anorg. Chem. 154,413 (1926). (11) Westland, A. D., Beamish, F. E., Mikrochim.Acta 5, 625 (1957). \----,-
RECEIVED for review October 3, 1960. Accepted November 14, 1960.
Quantitative Microdetermination of Cholesterol Using Tomatine as Precipitating Agent JON J. KABARA, JAMES T. McLAUGHLIN, and CAROL A. RIEGEL Biochemistry Section, Department of Chemisfry, University of Detroit, Detroit, Mich.
b A routine quantitative method for isolating and assaying radioactive and nonradioactive cholesterol (free and ester) is described. The method involves the replacement of digitonin by the glycoside tomatine as a precipitating agent. The new reagent is more specific for cholesterol than is digitonin. While the glycoside does not possess absolute specificity, the sterol isolated from mice 15 minutes after methyllabeled tritium acetate administration is free of higher counting companions.
w
HILE a great deal of effort is being directed to the study of cholesterol metabolism, most of the analytical procedures used in these studies are not entirely specific for cholesterol. The most popular procedures, with appropriate modification, incorporate the use of digitonin as a precipitating agent as introduced by Kindaus (9) and use the Liebermann-Burchard color reaction
employed by Grigaut ( 2 ) . Because of the lack of specificity their use has been criticized even when combined into a single procedure. This short-coming becomes especially significant in experiments involving the isolation and assay of radioactive cholesterol. Precipitation with digitonin resulted in the isolation of various products along with the principal sterol so that after further purification (via the dibromide) much of the samples' original radioactivity was lost (8). Consequently it was necessary for each investigator to prove either that the cholesterol isolated a t the time interval soon after isotope injection (in vivo) or addition (in vitro) was free of higher counting companions or that the tedious dibromide purification method was a necessary additional step to ascertain purity. Since incorporation experiments involving radioactive precursors are most meaningful, during the early time inter-
vals when the concentration of higher counting companions is highest, a procedure was sought which would be relatively specific for cholesterol and sufficiently practical to permit the handling of large numbers of samples on a routine basis. Early success with tomatine, a glycosidal alkaloid isolated from the leaves and stems of tomato plants ( I ) as a specific precipitating agent was sufficiently encouraging for us to concentrate on its analytical use. As a modification of an earlier procedure (6) the present microtechnique using tomatine for either radioactive or nonradioactive cholesterol determinations represents a considerable increase in specificity and a concomitant decrease in work, time, and cost. APPARATUS AND REAGENTS
A Bausch & Lomb colorimeter was used with a 630-mp filter. VOL 33, NO. 2, FEBRUARY 1961
305
Table 1.
Time, Hours
Per Cent Cholesterol Precipitated by Various Concentrations of Digitonin and Tomatine Glycoside (10 Ml.) 2.0 Mg. (4: 1)a 1.5 Mg. (3:1)= 3.0 Mg. (6:1)0 Digitonin Tomatine Digitonin Tomatine Digitonin Tomatine 100 94 100 100
0.5 6.0 12.0 24.0
96 100 100 98
95 92 95 95
94 95 96 96
76 78 86 88
66 66 64 96
Ratio of glycoside (mg.): cholesterol (mg.).
A Packard (LaGrange, Ill.) liquid scintillation spectrophotometer Model 314 was used to assay carbon-14 and/or hydrogen-3 radioactive samples. Glass grinders were used to homogenize tissue samples: Ace Glass Co., Vineland, N. J. EXTRACTINGSOLVENT.A suitable solvent for cholesterol extraction and precipitation was a combination of acetone-alcohol-ether (4 :4: 1). The addition of ether to the usual acetonealcohol (1:l) system rendered the resulting precipitate more insoluble. TOMATINE REAGENT. A standard 15% stock solution was prepared by dissolving 1.0 gram of Tomatichol (Chemical Concentrates, Fort Washington, Pa. brand name of crystalline tomatine) in water, glacial acetic acid, and ethyl alcohol (44 : 1 :55 ml.) , COLORREAGENT.The LiebermannBurchard reagent was made with acetic anhydride and sulfuric acid in a ratio of ten to one. The acetic anhydride was cooled in an ice bath and sulfuric acid was added slowly so as not to cause a rise of temperature. Because of its instability, the reagent was used within 1 hour after being prepared. COU~TINGSOLVENT.The solvent system used for liquid scintillation counting consists of 1.0 ml. of glacial acetic acid containing cholesterol as the
Table 11. Influence of Time on Per Cent Cholesterol Precipitated by Tomatine Cholesterol Standard (10 Ml.) 0 . 5 Mg. 0.4 0.3 0.2
Precipitation Time, Hours 3 100
__
86
83 33 5
0.1
6 15 100 100 9n 97 ._ .. 90 98 88 95 55 85
Table 111. Per Cent Recovery of Free Sterol after Saponification of Cholesterol Oleate Time, 60 minutes; temperature, 50 i 5" C. 50%. KOH Concentration of Ester 111 Methanol 0.50 Mg. 0.10 Mg. 1.0 ml. 0.80 0.60 0.40 0.20
306
95 93 95 98 98
ANALYTICAL CHEMISTRY
29 41 78 90 90
tomatinide plus 2.0 ml. of ethyl acetate, and 15 ml. of toluene. The toluene solvent contained 3 grams per liter of 2,5-diphenyloxazole (PPO) and 150 mg. per liter of 1,4[bis-2-(5-phenyloxazolyl) ] benzene (POPOP). PROCEDURE
Extraction of Blood Cholesterol. Heparinized human or mouse blood was centrifuged and separated into plasma and red blood cells. The red blood cells were washed twice with equal volumes of physiological saline and centrifuged between washings. To avoid the usual clumping of the red blood cells upon the addition of the extracting solvent the cells are hemolyzed with an equal volume of distilled water before extraction. The cells hemolyzed in this manner can be handled with greater ease and extracted more completely. Boiling acetonealcohol-ether solution (15 to 20 ml.) was added to 1.0 ml. of plasma or red blood cells and extraction allowed to take place a t room temperature without further heating. While heating on a steam bath was unnecessary, it was important to add the solvent to the plasma or red blood cells to precipitate the protein in a finely divided state. The mixture was then filtered through Whatman No. 1 filter paper. If the solution was not clear a t this point, it was refiltered. Since the pH of the solution between 4.8 and 8.4 has no measurable effect on the precipitation reaction, no pH adjustment was necessary. Extraction of Tissue Cholesterol. Tissues surgically removed for analysis were rinsed in physiological saline, blotted dry, and quick-frozen in test tubes a t -70' C., and stored in a freezer chest until ready for analysis. Immediately prior to the analysis the weight of the frozen samples was recorded as wet weight. The tissues (0.5 to 1.0 gram) placed in 15 ml. of warm acetone-alcohol-ether (4:4:1) were ground with glass homogenizers until a fine suspension of the tissue protein was achieved. Extraction under these conditions was materially aided by the heating effect due to grinding. The mixture was filtered, the precipitated protein dried, weighed, and recorded as dry weight (lipidefree). Isolation of Free Cholesterol. The filtrates from the blood or tissue are evaporated to a more convenient volume (9 to 10 ml.) before precipita-
tion by tomatine. The glycoside concentration as well as the relative amount of cholesterol in the sample affect the rate and completeness of precipitation. On the basis of previous estimations as to the amount of free sterol in the filtrates approximately six times this amount of tomatine was added. For amounts of cholesterol 0.50 mg. or greater, quantitative precipitation takes place in less than 1 hour a t room temperature (Table I). When the total amount of sterol was less than 0.5 mg., quantitative recovery was possible only by increasing the time to 15 hours (Table 11). After precipitation was complete the tomatinide mixture was centrifuged. The supernatant fluid was then carefully decanted and the precipitate washed thoroughly by a jet stream of acetonealcohol-ether solution ejected from a syringe. The washed tomatinide was recentrifuged and rewashed with acetone-ether (1 : 2 ) , All the washings were combined with the first filtrate and used to ester-feed cholesterol. Isolation of Ester Cholesterol. When esterified cholesterol was determined, the combined washings as well as the filtrate from the free cholesterol were concentrated to 3 to 4 ml. Hydrolysis of the ester was acconiplished by adding 0.2 to 0.3 ml. of methanolic potassium hydroxide (50%) to the solution and then heating the tubes on a water bath a t 50' C. for 60 minutes. In our hands the saponification step was critical since too much alkali decreases the accuracy of the cholesterol assay (Table 111). After saponification the samples were removed from the water bath and allowed to cool to room temperature before acidifying to phenolphthalein with glacial acetic acid. The steps after acidification are similar to those given for isolating free cholesterol. Quantitative Measurement of Tomatinide. The tomatinide was dissolved in a known volume of glacial acetic acid by placing the sample tubes in a water bath (50' to 60' C.) for a few minutes. Since the upper limit of the standard curve was 0.500 mg. per ml., the amount of acetic acid added to each tube depended upon the amount precipitated. After the precipitate was dissolved, the solution was cooled to room temperature before taking a 1-ml. aliquot for colorimetric determination (4). In experiments involved with radioactive cholesterol another aliquot was assayed for radioactivity in a liquid scintillation spectrophotometer. Measurement of Radioactive Tomatinide. The problem of solubilizing the tomatinide in a solvent of reasonable efficiency for liquid scintillation counting was solved by using a mixture of glacial acetic acid, ethyl acetate, and toluene. Routinely the aliquot of acetic acid (1 ml.) was added to a 5-dram bottle and 2 ml. of ethyl acetate and 15 ml. of toluene containing 3 grams per liter of PPO and 150 mg. per liter of POPOP were then added, Both acetic acid and ethyl acetate lowered the counting efficiency
of the sample toluene system so that carbon-14 was counted a t 50 to 55% efficiency (background = 47 to 50 c.p.ni.) while hydrogen-3 compounds a t a background of 80 to 86 c.p.m. have an efficiency of 10 to 15%. The two isotopes could be assayed simultaneously a t somewhat lower efficiencies (carbon14, 30 to 33%; and tritium, 8 to 9%) by changes in the voltage settings (6). EXPERIMENTAL A N D DISCUSSION
In earlier attempts to work out a microprocedure for the isolation and liquid scintillation assay of radioactive cholesterol (5), it was recognized that the sterol isolated with the glycoside digitonin was not solely cholesterol. To find an agent more specific than digitonin, various types of compounds were screened for their ability to selectively precipitate cholesterol, beginning in 1957. An earlier report (7) as well as our initial success with tomatine was sufficiently encouraging to stimulate further work. Through the cooperation of the U. S. Department of Agriculture Eastern Regional Laboratories and Chemical Concentrates large quantities of crystalline tomatine (Tomatichol) were made available for extended studies. Tomatine has several desirable characteristics which make it the best precipitating reagent that we have tested. A solution of the glycoside can be prepared easily and kept for several months without signs of precipitation or cloudiness. Tomatine in contrast to several other glycosides studied, including digitonin, does not give any measurable color with the Liebermann-Burchard reagent. Table IV indicates the amount of color formed by some of the glycosides expressed in terms of milligrams of cholesterol. The most important quality of tomatine is its specificity for cholesterol. While this specificity is greater than that of digitonin, it is not the ultimate answer to the problem of an absolute analytical reagent. As can be seen from Table V, which includes only a representative number of the 40 steroids studied, 7-dehydrocholesterol and dihydrocholesterol were not precipitated by tomatine under our test conditions, while A?cholesten-38-01 was precipitated by both glycosides. The steroids omitted from the table were negative for both digitonin and tomatine and biologically unimportant. In a series of experiments to determine the radiochemical purity of cholesterol isolated shortly after intraperitoneal injection of methyl-labeled tritium ace-
tate (100 pc. per mouse), animals were injected with the radioactive metabolite and killed a t various time intervals. The livers were surgically removed, the cholesterol isolated and assayed by the procedure outlined above. The results (Table VI) of these in vivo experiments on mice indicate that only the sterol isolated 5 minutes after isotope injection and purified via a micro dibromide procedure (3) contained any high counting companions. Cholesterol isolated from mice 15 minutes after injection or later was free of any high counting companions, since the values found after diTable V.
Table IV. Color of Glycosides with Liebermann-Burchard Reagent
Per Cent Transmittancea (640 RIP) 95
Glycoside (10 Mg.) Tomatine Dieitonin 77 0.08 .. ._ Steollatus 41 0.27 Eichlamii 26 0.40 Benecki 20 0.49 Longispinus 15 0.57 Dioscin 0 1.20 Values obtained at time of maximum absorbance for cholesterol (20-25 minutes). 0
Specificity" for Various Steroids
Digitonin (10 mg.1 5+ 5 s
Steroid (0.1 Mg.) A5-Cholesten-3p-ol A7-Cholesten-3p-ol 5-Dihydroergosterol Dehydrocholesterol Calciferol A5.7-Cholestadien-38-01 Cholestan-3@-01-6-one
Tomatine (10 mg.) 5 s 5 s
'+ ..
4+
.. ..
3@,5,9,6p-Stigmastanetriol Methyl-3&5~dihydroxy-A~-etiocholenate
A5-Pregnene-3p,17@,21-triol-20-one,21 monoacetate A5-Pregnen-3p-ol,20-one
A~-Pregnene-3p,2l-diol-2O-one-2l-monoacetate A6-dI0pregnene-38~2 l-diol-20-one-21-monoacetate AS-dlopregnene-3@, 17~,2l-trio~-20-one-2l-monoacetate a
Expressed as Cholesterol (Mg.) 0.02
. .
..
2+
+ 4+
..
2+
.. . . ..
2+
+
..
2+
..
24 hours after addition of tomatine.
bromide purification differed less than 1070 of the original activity and are not considered significant. It would appear that for most in vivo studies the radioactive sterol isolated by tomatine needs no further purification. Tomat'ine has only relative specificity greater than digitonin. Under conditions other than those above each investigator must satisfy himself as to the purity of the isolated product. ACKNOWLEDGMENT
Gratitude expressed to Carl Djerassi and C. S. Brooks, and to Parke Davis & Co. for samples of glycosides and steroids. LITERATURE CITED
(1) Fontaine, T. D., Irving, G. W., Jr., Ma, M. A., Poole, J. B., Doolittle, S. P., Arch. Biochem. 18. 467 (1948). (2) Grigaut, A., Compt. rend. s&. biol. 68,
791 (1910). (3) Kabara,'J. J., Federation Proc.
14,
232
I 1 955). \ - - - - I .
(4) Kabara, J. J., J . Lab. Clin. Med. 44, 246 (1954). (5) Ibid., 50, 146 (1957). (6) Okita, G. T., Kabara, J. J., Richard-
Table VI. Specific Activity of Liver Cholesterol Isolated with Tomatine at Various Time Intervals
Time after Acetate-
Initial -2-H.3 Specific I.P. Injec- Activity, tion, Min. D.P.M./Mg. 5 2082 15 7180 30 13,532 60 5306
Specific Activity after
Dibromide, D.P.M./Mg. 1484 6976 14,800 5600
son, F., LeRoy, G. V., X'ucleonics 15, 111 (1957). (7) Schulz, G., Sander, H., Z. physiol. Chem. 308. 122 (1957). ( 8 ) Schwenk; E., kerthessen, N. T., Arch. Bzochem. Biophys. 40, 334 (1952). (9) Windaus, A., Ber. 42, 23&46 (1909). RECEIVED for review May 19, 1960. Accepted November 15, 1960. Work supported in part by grants from the Michigan Heart Association, Grant B-2235 from the National Institute of Neurological Disease and Blindness Public Health Service, and the Ciba Pharmaceutical Products, Inc. Meeting-in-Miniature, ACS, Detroit, Mich., 1960.
VOL 33, NO. 2, FEBRUARY 1961
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