A Simplified Spectrophotometric Method for Determination of Total a n d Esterified Cholesterol with Tomatine SIR: The classical method of cholesterol and cholesterol eater determinat.ion is by precipitation of free cholesterol by digitonin in acid medium and of total cholesterol after saponificat'ion. The amount of precipitated digitonide is then determined by the LiebermannBurchard color react..on aft'er mixing with the acetic snhya ride-sulfuric acid reagent ( I , 3 ) . TKOproblems are involved. One, digitonin is not a specific cholesterd precipitant and digitonin itself gives a color reaction; therefore, excess digit'oniri must be removed by many washings n-hich is time consuming. Second, the color reagent is not
1 niimber of reports of rapid total cholesterol :may indictated t'hat if certain prot>eins and bilirubin can be excluded from the blood sample, extraction of fat and precipitstion is not necessnry (4, 6.8'). These methods failed t o separate free from esterified cholesterol. Kabara et al. (A) found a glycoside tomatine which is m3re specific than digit,oninin choleat,erol precipitation and also is negligible in giving a LiebermannBurchard color reaction. However, repeated centrifugations and washings were still required. TT-e formulated a color reagent, wh..ch is stable for 2 n-eek; at rnoni teinperature and 4 weeks in the refrigerator and a solvent which is able t o precipitate protein and biliruliin but, able to dissolve t h e cholesterol and cholesterol ester. Combining these methods. we present, a total and wterified cholesterol assay procedure nThich eliminates the ordinary repeated washings a,nd centrifugations and shortens the entire procediwe from a day to an hour.
Inc., Cleveland. Ohio) is dissolved in a 100-ml. mixture of 95y0 ethyl alcoholwater-glacial acetic acid (20:79.5:0.5). Procedure. Add 15 ml. of fat solvent a t room temperature t o a test tube containing 0.5 t o 1.0 ml. of unknown serum, other body fluid, or homogenized tiqsue. Rubber stoppers are used for all t h e tubes t o prevent evaporation. Invert t h e mixture slowly and completely 30 times a n d centrifuge i t for 5 minutes at 1500 r.p.m. Transfer 2 ml. of t h e supernatant to a 50-ml. beaker for total cholesterol, and another 10 ml. of i t to a 15-ml. centrifuge tube. Add 1 ml. of tomatine reagent to the centrifuge tube, mix, and let stand for 10 minutes. Centrifuge the tube and transfer 2 ml. of its supernatant to another 50-ml. beaker for the cholesterol ester. Dry the beakers on a steam bath. After drying, add 0.5 ml. of glacial acetic acid t o each beaker, followed by 5.0 ml. of color reagent. Let the solutions stand for 20 minutes and read against the stable color reagent (as the blank) at 610 mp with a spectrophotometer. A standard is also run with the unknown throughout the procedure. Since the results previously reported showed that the present reagents for LiebermannBurchard follow Beer's law, the unknown cholesterol values are obtained b y Beer's law uiing the cholesterol standard (4).
Table I.
Reagents. C O L O RREAGEXT.A miyture contdining 307, glacial acetic acid. 6 0 7 acetic anhydride, and 10% qnlfuric :iciil 1)r volume if prepared in an ice water b t t h . Anhydrous sodium suliate if added to the miuture to ;i coilcentration of 2%. It i. stored i n the refriserator a n d used n ithout ~varniing F ~ SOLYEYT T 937, ethyl alcoholacetone-ether 16:3: 1) 17)- volume. CHOLE ~ T E R O L STAXDARD.Cholesterol acetate (144 mg., assayed equivalent to 130 mg cholesterol) and 70 mg. of cholestwol are di\wlved in a 100-ml. mixture of 9 5 7 ethyl alcohol and ether (98 :2 ) . TOLITISF:R E A G E N T . T n o grams of tomatine manufactured by Chemical Concentrate-, Fort \Vashington. Pa., and alw l n Sutritional Biochemical,
Extraction. T h e common f a t extraction solvents used by Bloor ( I ) , Schoenheimer and Sperry (9), and Kabara, McLaughlin, and Riegel (6) are combinations of ethyl alcohol, acetone, and ether. Our results from experiments with various combinations of alcohol, acetone, and ether show t h a t the greater the quantity of ether, the better the extraction (Table I). However, the difference is only hundredths of the actual value obtained. Since we attempted t o save time b y avoiding the adjustment of volume due to evaporation, Ive used the lower percentage of ether. The second reason for choosing the 6 : 3: 1 is due to some precipitating effect5 observed in bolvents 1 and 3. K h e n C.P. cholesterol acetate was added ieparately to these solvents along with tomatine, immediate precipitates were observed in solvents 1 and 3. No precipitation TT a< observed in these two solvents containing no cholesterol ester or iolvent. 2 and 4 containing cholesterol acetate (Table I). The 6.3:1 sohent was also tested to show its incapability of dissolving C.P. bilirubin, rrhich is one of the most important interference materials in blood for the Lieberinaiin-Rurchard reaction.
Cholesterol Extraction Ability of Various Solvent Combinations
Solvent composition Alcohol Acetone Ether 1 1 1 2
1 4 3
4 6
EXPERIME"4TAL
RESULTS AND DISCUSSION
0
Cholesterol extracted, yo Precipitation (unknown of cholesterol serum j acetatea
-
100 99 98 9s
1 1 1
-
Precipitate observed when using tomatine manufactured by Chemical Concentrates.
Table 11.
Cholesterol Esters of Human Serum Determined a t Different Tomatine Precipitation Times
Precipitation time, min. Ob
5
10 20 30 60 12 hrs.
Unknon-n serum, mg. % Kormal (approx. 0.2 mg. free LOX (approx. 0.1 mg. f X cholesterol j cholesterol) __ 1
2
3
4
5
6"
205 143 143 137 143 141 139
198 125 119 119 114 110 114
220 155 155 154 155 154 150
98
90
105
78
63
91
76 70
67 63
93 67
This was done using tomatine manufactured by Sutritional Biochemical, Inc. Represents total cholesterol; other values represent cholesterol esters after precipitation. 5
I,
VOL. 35, NO. 11, OCTOBER 1963
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Precipitation of Free Cholesterol by Tomatine. Precipitation of free cholesterol b y digitonin and tomatine requires hours for completion. T h e present tomatine solution, which is nearly saturated, will precipitate free cholesterol immediately. We observed no difference among precipitation times varying from 5 t o 60 minutes (Table 11). Kabara, McLaughlin, and Riegel(5) observed that a low level of cholesterol requires a longer period of precipitation. This was seldom noticed in the present trial. I n case no immediate precipitation was observed, as in sample 6 in Table 11,this solution was left t o stand overnight. The failure of immediate precipitation mas mainly due to improper preparation of the tomatine reagent and incomplete mixing of tomatine reagent before using. Variation of Replicates. T o study the reproducibility of t h e present method, 100 unknown serum samples Tyere determined in duplicate. We used Bennett and Franklin’s method (2) to determine the standard deviation is. D. = 4.%d2/2kwhere d represents difference between duplicates and k, the number of such differences obtained). Values obtained were 229.2 f 3.9 and 158.5 + 4.4 for the total and esterified cholesterol, respectively. Comparison with Other Methods. T h e present method was compared
with various methods. One human serum was used as t h e testing material. T h e results are shown in Table 111. The present method gave significantly smaller values for both total and esterified cholesterol than Bloor’s method. The data also show that the present method gave a slightly higher value than the Schoenheimer and Sperry method. Therefore, the present cholesterol values are between those of Bloor and Schoenheimer and Sperry. Practical Findings. It has been shown t h a t in humans, t h e female has consistently lower serum cholesterol t h a n t h e corresponding male until the sixth decade ( 7 ) . This difference among infants, however, is not definite, because the extremely lorn infant serum cholesterol value is rather hard to detect. We used the present method to compare the cholesterol levels among a number of day-old white infants. The infant serum cholesterol is less than 100; therefore, it was necessary to use 0.5 ml. (or 100 mg.) of cholesterol standard to ensure that the absorbance produced remained within the best range on the spectrophotometer. The results in Table IV shorn a significant difference of cholesterol values between male and female infants according to a t-test (10) for group comparison. Cnder normal conditions, human serum cholesterol rarely rises higher than
Table Ill. Cholesterol Determinations of Unknown Serum in Mg. Different Methods So. samples
Total Cholesterol cholesterol ester 10 10
Method Schoenheimer
Bloora
Present
170 f 7
156 =t 4
153 f 9
144 =k 6
113 3= 2
103 f 7
78 f 16
20
% by
Huang ( 4 )
78 f 17
4 Significantly higher than those of present method according to t-test for group comparison ( l a ) .
Table IV. Total and Esterified Cholesterol in Various Samples Total No. cholesterol, Cholesterol ester, mg. yo Samples assayed hlethod mg. 7% Serum from day old: 12 Present 85 3= 12” 60 =t 6 boy girl 12 Present 66f 6 55 f 9 Serum from rabbit 4 Present 1058 334 4 Huang ( 4 ) 1119 Rabbit liver homogenate 4 Present 1625 1374 4 Boiling 1420 1210 a
Significant difference from female according tmot-test for group comparison (IO).
1758
ANALYTICAL CHEMISTRY
400 mg.%. The latter can Le determined easily by the present method providing that a higher cholesterol standard of (2 ml. or 400 rng.%) is used to ensure that the absorbance produced remains within the best range on the spectrophotometer. However, in esperimental work, especially in animal? or organ assay, t h e cholesterol level quite often rises to more than 1000 mg.Yc. The present method was also tested for its applicability. Four sets of rabbit serum with abnormally high cholesterol values were tested. One milliliter of serum was extracted and total and esterified cholesterol were assayed as described. It was found that their absorbance values were too high for the spectrophotometer. Since we still had enough supernatant left from total and cholesterol esters, we redid the assays by using 0.5 ml. of filtrate instead of 2 ml. We obtained the values of 1058, comparable to 1119 with the previous method (Table IV), (4). This shows that even when one obtains unreadable values, the present method can be used to obtain true values from the material available. Tissue Cholesterol. Four samples of high cholesterol rabbit liver were analyzed individually. After homogenization in a Potter-Elvehjem homogenizer (Kontes Glass Co., Vineland, N. J.), 0.2 ml. of homogenate was used. The present method was compared t o the boiling method of Schoenheimer and Sperry. The results show that the present method gave slightly higher values than the boiling method (Table IV) . LITERATURE CITED
(1) Bloor, W. R., J . Biol. Chenz. 24, 227 (1916). (2) Bennett, C. A., ? a n k h , ?;. L., “Statistical Analysis, p. 168, Wiley, New York, 1954. (3) Burchard, H., Chem. Zenfr. 61 ( I ) , 25 (1890). (4) Huang, T. C., Chen, C. C., Refler, V., Raftery, A,, ANAL. CHEM.33, 1405 (1961). (5) Kabara, J. J., Mclaughlin, J. T., Riegel, C. A., Ibid., 33, 305 (1961). (6) Klungsoyr, L., Haukenes, E., Closs K., Clin. Chem. Acta 3, 514 (1960).
(7) Lawry, E. Y . , Mann, G. V., Peterson, A., Wysocki, A. P., Connell, R. O., Stare, F. J., Am. J. dled. 22, 605 (1957). (8) Pearson, S., Stern, S., McGavack, T. H., ANAL.CHEM.25,813 (1953). (9) Schoenheimer, D., Sperry, W . W., J . Biol. Chem. 106, 745 (1934). (10) SEedecor, G. W., “Statistical blet,h-
ods, Iowa State College Press, hmes, Iowa, 1956. T. C. HUANQ VERNAWEFLER ALANRAFTERY Research Department Timken Mercy Hospital Canton, Ohio Presented in part a t the Division of Biological Chemistry, 141st Meeting, ACS, Washington, D. C., March 1962. This work was partially support’ed by U. S. P. H. (H5171-4).
