(10) Zimmerman, J., Hawley, D., Adam, R. N., unpublished data.
LITERATURE CITED
(1) Adams, R. X., Abstracts, American
Chemical Society Meeting, Symposium on Electrode Processes. New York. September 1963. (2) Bearman, R. J., J . Phys. Chem. 66, 2072 (1962).
T. A. MILLER BARBARA LAMB KEITHPRATER J. K. LEE RALPHN. ADAMS
(5) Sanborn, R.' H., Orlemann, E. F., Ibad., 77, 3726 (1955). (6) Wang, J. H., Ibid., 73, 510, 4181 (1951). (7) Ibid. , 76, 1528 (1954). (8) Wane. J. H.. Polestra. F. M.. Ibid.. . 76, 158'4.(1954j. (9) Yavorsky, P. M., Gorin, E., J . Am. Chem. SOC.84, 1071 (1962).
Department of Chemistry The University of Kansas Lawrence, Kan. RECEIVED for review November 8, 1963. Accepted November 27, 1963.
Tomatine and Digitonin as Precipitating Agents in the Estimation of Cholesterol SIR: The introduction of tomatine, a glycosidal alkaloid isolated from the leaves and stems of tomato plants, as a precipitating agent for the microdetermination of cholesterol by Kabara and coworkers (3) and the need for such a reagent in our work have prompted us to compare this compound with digitonin as a precipitant of cholesterol. Since the use of digitonin for cholesterol assay by Windaus (6), this reagent has been widely used, though it is well known that the glycoside is not specific for cholesterol (2, 5 ) . Kabara, McLaughlin, and Riegel (3) have reported t h a t tomatine is considerably more specific for the precipitation of cholesterol than digitonin. I n the present investigation digitonin and tomatine are compared as precipitants for cholesterol in radioactive tissues of rats.
Table
I.
Cholesterol digitonide
420
ANALYTICAL CHEMISTRY
RESULTS
The s'pecific activities of digitonides and tomatinides precipitated from the same tissues by this procedure are shown in Table I. The high and in-
At 30 min. 5 mg. of d.p.m./ ppt. mg.
8 . 1 3268 9 . 1 1737
tomatinide 5 . 6 542 Coproat anol digitonide 1 . 5 546 tomatinide . . . . . .
4.6 10.8 11.4 1.5
3703 3560 3774 1693
9:6 10.3
2519 1735
9.0 5.7
2793 439
10.4
684
1.9
425
2.3
310
2.8
208
0.7 2.3 1.4 12.3
697 2082 507 151
12.4
1388
......
0.5
289
7.3
313
9.3
2.0
119
6.0 3.8
101 19
...
. . . . . . . . . . . . 2.9 71
...
133
......
......
Precipitation by Tomatine and Digitonin of Carbon-1 4 Not Associated with Cholesterol
Radiocarbon added d./m.
Nonradioactive cholesterol used mg.
Digitonide 35 ,100 1.0 Tomatinide 35 ,100 1.0 Before and after recrystallization. (1
OF
At 24 hours Rat 1 2 3 4 mg. mg. mg. mg. of d.p.m./ of d.p.m./ of d.p.m./ of d.p.m./ ppt. mg. ppt. mg. ppt. mg. ppt. mg.
tomatinide 2 . 9 510 Cholesterol ester digitonide 1.1 1210
Table 11.
DISCUSSION
Specific Activities of Tomatinides and Digitonides of Cholesterol and Cholesterol Ester Derivatives from Rat Liver
EXPERIMENTAL
Eighteen milligrams of methioninem e t h ~ 1 - C (sp. ' ~ act. 1.8 mc. per mmole) contained in 4 grams of an adequate synthetic ration, were fed t o 4 adult rats b y stomach tube ( I ) . After 24 hours, these animals were sacrificed and livers were removed. One animal, R a t 5 , received 18mg. of methionine-methylC14 in aqueous solution and was sacrificed after 30 minutes. The quantities of radiocarbon absorbed by Rats 1 through 5 were 30.0, 20.2, 19.2, 19.3, and 8.2 million d.p.m., respectively. One gram of liver (cholesterol and cholesterol ester) or feces (coprostanol) was used for analyses. The procedure of Kabara et al. ( 3 ) was employed for the preparation of tomatinides, t h a t of Schoenheimer and Sperry (4) for the digitonides. To prepare cholesterol derivatives, 5 mg. of tomatine or 14 mg. of digitonin was added to 9 to 10 ml. of the filtered acetone-alcohol-
were washed with ether (digitonide) or acetone:ether, 1 : 2, (tomatinide) to constant specific activity. Eleven to 15 washings were required to accomplish this.
ether extract of the ground tissue. After the precipitation of free cholesterol, the extract was saponifled with potassium hydroxide to free the cholesterol esters and then acidified to the phenolphthalein end point prior t o the addition of tomatine or digitonin as described above. Both tomatinides and digitonides were allowed to precipitate overnight. After they had been plated on filter paper disks b y use of a microprecipitation apparatus, the derivatives
Cholesterol derivative precipitated Before After' wt . d.p.m. /mg. 4.5 4.9
164 180
21
26
consistent specific wtivities of the digitonide replicates discourage the use of digitonin for analysis of 24-hour radioactive tissues, anti perhaps also for colorimetric assays, whereas the significantly loner activitieb3 precipitated by tomatine (t = 4.96, p
