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Radioassay of cholesterol-C14 digitonide
88
SHORT
Radioassay
COMMUNICATIONS
of Cholesterol-Cl4
Digitonide
’
In the course of an investigation involving the isolation and assay of a large number of samples of cholesterol-U4 as the digitonide, it was deemed advisable to investigate all methods available for solubilization of the cholesterol-digitonin complex in order to ascertain which was most suited to rapid and efficient radioassay. Cholesterol digitonide has been assayed by liquid scintillation spectrometry after solution in H,P04-p-dioxane (1) or methanol (2) or by sealing the solid in plastic which is immersed in the solution of liquid scintillator (3). Since cholesterol digitonide can be split by heating with pyridine (4), xylene (5)) benzene (6), or pyridine: acetic anhydride 1: 1 (7)-all materials which are completely miscible in common liquid scintillation solvent systems-we compared them for efficiency and ease in radioassay of cholesterol-C4 digitonide. The digitonide was prepared by the method of Sperry and Webb (8). Assays were carried out in a Packard TriCarb Liquid Scintillation Spectrometer using a solution of 0.6% PPO and 0.02% dimethyl POPOP in toluene. The quenching effects of a number of potential solvents were studied. Pyridine lowered the counting efficiency too much to be of value; Hyamine and acetic anhydride could be of potential utility if small (less than 1 ml) volumes were used. In the volume range under consideration (l-2 ml) glacial acetic acid, methanol, and ethanol gave comparatively little quenching. The findings with pyridine, methanol, and ethanol are in accord with those of other authors (9, 10). Cholesterol, cholesterol dibromide, cholesterol digit.onide, and digitonin did not give appreciable quenching. To test the efficiency of the solvent systems under study, triplicate samples of a standard amount of digitonide (0.8 mg) were treated with one of the following: 0.5 ml Hyamine, 2 ml each of ethanol, methanol, boiling pyridine, boiling acetic acid, boiling acetic anhydride, and boiling acetic anhydride-pyridine (1: 1) or 4 ml each of boiling xylene, benzene, or toluene. Complete solution was effected only with Hyamine, methanol, acetic acid, pyridine and acetic anhydride-pyridine (1: 1). After 2 nun the solution was transferred to the scintillation counting vial and the residue washed with ether, which was added to the solution to be assayed. While all the solvents used will split the digitonide on prolonged heating, only those listed above gave complete solution under our ‘This National
work was supported (in part) by grants H-3299 and H-5299 from the Institutes of Health, U. S. Public Health Service.
SHORT
89
COMMUNICATIONS
conditions of treatment. In every case in which a residue remained, it was dissolved in methanol and the resulting solution accounted for the counts not recovered in initial treatment. Samples were also prepared using phosphoric acid-p-dioxane (1:99). Since Funt and Hetherington (11) have shown that some insoluble materials can be dispersed for counting with the aid of gel formation, samples of cholesterol digitonide were suspended in the scintillation solution containing either 2.4% Cab0-Sil or 2.5% Thixcin R. Recovery of radioactivity is summarized in Table 1. Corrections have been made to account for quenching effects TABLE RECOVERY
OF CHOLESTEROL-Cl4
Solvents Original solution Acetic anhydride-pyridine Phosphoric acid-p-dioxane Hyamine Methanol Et,hanol Aretic anhydride Thixcin R Toluene Acetic acid Pyridine Cab-O-N Sylene Benzene
1 FROM CHOLESTEROL Dpm recovered~
(1: 1) (1:99)
19,514 19,302 19,152 18,882 18,721 18,609 18,416 14,531 13,203 11,489 10,972 10,200 6,314 3,518
DIGITONIDE Recovery
(%,I
99 98 97 96 95 94 74 68 59 56 5’2 32 18
a Treatment for 2 min with 2-4 ml of solvent (except for Hyamine, 0.5 ml used). b Average of triplicate determinations. Corrected by internal standardization.
of the various solvents. The extent of solubilization of digitonide by the various treatments is reflected in the recovered radioactivity. It is evident that with ethanol and methanol one can achieve the most rapid quantitative assay of cholesterol from labeled cholesterol digitonide. In a study of the solubility of cholesterol and its digitonide, Schoenheimer and Dam (4) found that 1 ml of methanol could dissolve 6 mg of cholesterol and 5 mg of the digitonide, while 1 ml of ethanol could dissolve 19 mg of cholesterol but only 0.7 mg of the digitonide. In our study, 0.8 mg of digitonide was used. One milliliter of Hyamine will dissolve as much as 100 mg of cholesterol digitonide. From these considerations methanol or Hyamine would appear to be best suited for direct assay of cholesterol digitonide. The radioassay of cholesterol-CP digitonide by gas flow technique
90
SHORT
COMMUNICATIONS
was also investigated using methanol as the solvent for solution and plating. A Tracerlab CompuMatic II gas flow counter was used. Construction of self-absorption curves for cholesterol-W and cholesterolC?” digitonide was carried out, with results shown in Fig. 1. Infinite SELF-ABSORPTION CURVES CHOLESTEROL 0l0lT0Nl0E
moo -
Y
CHOLESTEROL
2400 *200
-
2000 IS00 -
0
10
FIG.
1. Self-absorption
25
20
SAMPLE
30
THICKNESS,
35
45
40
50
MG/CM’
curves for cholesterol-C’”
and cholesterol-C”
digitonide.
thickness for cholesterol was reached at 30 mg/cmz and for the digitonide at 20 mg/cm*. Plating of duplicate aliquots of cholesterol-C4 digitonide from methanol gave an average recovery of 3443 cpm as compared with 12,684 cpm for aliquots assayed by liquid scintillation count’ing. Thus it would appear that the plating and flow counting method is practical for the assay of cholesterol-C I4 digitonide when no liquid scintillation spectrometer is available or when it is desired to recover the sample for subsequent chemical manipulations. ACKNOWLEDGMENT We are indebted to Hoffmann-La Roche Co., Nutley, New Jersey, and to Merck and Co., Rahway, New Jersey, for generous gifts of the digitonin used in this study. REFERENCES 1. KABARA, J. J., J. Lab. C&n. Med. 50, 146 (1957). 2. WILSON, J. D., AND SIPERSTEIN, M., Am. J. Physiol. 3. GRIMES, E. T., Anal. Chem. 34, 1175 (1962). 4. SCHOENHEIMER, R., AND DAM, H., 2. physiol. Chem. 5. WINDAUS,
A.,
2.
physiol.
Chem.
65,
110
(1910).
196, 215,
596
(1959).
59
(1933).
91
SHORT COMMUNICATIONS 6. KELSEY, 7. WINDAUS, 8.
9. 10. 11.
F. E., J. Biol. Chem. 127, 15 (1939). A., Z. physiol. Chem. 101, 276 (1918). SPERRI., W. M., AND WEBB, M., J. Biol. Chem. 187, 97 (1950). KERR, V. N., HAYES, F. N., AND OTT, D. G., Intern. J. Appl. Radiation 1, 2846 (1957). STITCH, S. R., Biochem. J. 73, 287 (1959). FUNT, H. L., AND HETHERINGTON, A., Science 131, 1608 (1960).
Isotopes
IRWIN L. SHAPIRO DAVID KRITCHEVSKY The Wistar Philadelphia, Received
Institute of Anatomy Pennsylvania September 11, 1962
and
Biology
SHORT
Radioassay
COMMUNICATIONS
of Cholesterol-Cl4
Digitonide
’
In the course of an investigation involving the isolation and assay of a large number of samples of cholesterol-U4 as the digitonide, it was deemed advisable to investigate all methods available for solubilization of the cholesterol-digitonin complex in order to ascertain which was most suited to rapid and efficient radioassay. Cholesterol digitonide has been assayed by liquid scintillation spectrometry after solution in H,P04-p-dioxane (1) or methanol (2) or by sealing the solid in plastic which is immersed in the solution of liquid scintillator (3). Since cholesterol digitonide can be split by heating with pyridine (4), xylene (5)) benzene (6), or pyridine: acetic anhydride 1: 1 (7)-all materials which are completely miscible in common liquid scintillation solvent systems-we compared them for efficiency and ease in radioassay of cholesterol-C4 digitonide. The digitonide was prepared by the method of Sperry and Webb (8). Assays were carried out in a Packard TriCarb Liquid Scintillation Spectrometer using a solution of 0.6% PPO and 0.02% dimethyl POPOP in toluene. The quenching effects of a number of potential solvents were studied. Pyridine lowered the counting efficiency too much to be of value; Hyamine and acetic anhydride could be of potential utility if small (less than 1 ml) volumes were used. In the volume range under consideration (l-2 ml) glacial acetic acid, methanol, and ethanol gave comparatively little quenching. The findings with pyridine, methanol, and ethanol are in accord with those of other authors (9, 10). Cholesterol, cholesterol dibromide, cholesterol digit.onide, and digitonin did not give appreciable quenching. To test the efficiency of the solvent systems under study, triplicate samples of a standard amount of digitonide (0.8 mg) were treated with one of the following: 0.5 ml Hyamine, 2 ml each of ethanol, methanol, boiling pyridine, boiling acetic acid, boiling acetic anhydride, and boiling acetic anhydride-pyridine (1: 1) or 4 ml each of boiling xylene, benzene, or toluene. Complete solution was effected only with Hyamine, methanol, acetic acid, pyridine and acetic anhydride-pyridine (1: 1). After 2 nun the solution was transferred to the scintillation counting vial and the residue washed with ether, which was added to the solution to be assayed. While all the solvents used will split the digitonide on prolonged heating, only those listed above gave complete solution under our ‘This National
work was supported (in part) by grants H-3299 and H-5299 from the Institutes of Health, U. S. Public Health Service.
SHORT
89
COMMUNICATIONS
conditions of treatment. In every case in which a residue remained, it was dissolved in methanol and the resulting solution accounted for the counts not recovered in initial treatment. Samples were also prepared using phosphoric acid-p-dioxane (1:99). Since Funt and Hetherington (11) have shown that some insoluble materials can be dispersed for counting with the aid of gel formation, samples of cholesterol digitonide were suspended in the scintillation solution containing either 2.4% Cab0-Sil or 2.5% Thixcin R. Recovery of radioactivity is summarized in Table 1. Corrections have been made to account for quenching effects TABLE RECOVERY
OF CHOLESTEROL-Cl4
Solvents Original solution Acetic anhydride-pyridine Phosphoric acid-p-dioxane Hyamine Methanol Et,hanol Aretic anhydride Thixcin R Toluene Acetic acid Pyridine Cab-O-N Sylene Benzene
1 FROM CHOLESTEROL Dpm recovered~
(1: 1) (1:99)
19,514 19,302 19,152 18,882 18,721 18,609 18,416 14,531 13,203 11,489 10,972 10,200 6,314 3,518
DIGITONIDE Recovery
(%,I
99 98 97 96 95 94 74 68 59 56 5’2 32 18
a Treatment for 2 min with 2-4 ml of solvent (except for Hyamine, 0.5 ml used). b Average of triplicate determinations. Corrected by internal standardization.
of the various solvents. The extent of solubilization of digitonide by the various treatments is reflected in the recovered radioactivity. It is evident that with ethanol and methanol one can achieve the most rapid quantitative assay of cholesterol from labeled cholesterol digitonide. In a study of the solubility of cholesterol and its digitonide, Schoenheimer and Dam (4) found that 1 ml of methanol could dissolve 6 mg of cholesterol and 5 mg of the digitonide, while 1 ml of ethanol could dissolve 19 mg of cholesterol but only 0.7 mg of the digitonide. In our study, 0.8 mg of digitonide was used. One milliliter of Hyamine will dissolve as much as 100 mg of cholesterol digitonide. From these considerations methanol or Hyamine would appear to be best suited for direct assay of cholesterol digitonide. The radioassay of cholesterol-CP digitonide by gas flow technique
90
SHORT
COMMUNICATIONS
was also investigated using methanol as the solvent for solution and plating. A Tracerlab CompuMatic II gas flow counter was used. Construction of self-absorption curves for cholesterol-W and cholesterolC?” digitonide was carried out, with results shown in Fig. 1. Infinite SELF-ABSORPTION CURVES CHOLESTEROL 0l0lT0Nl0E
moo -
Y
CHOLESTEROL
2400 *200
-
2000 IS00 -
0
10
FIG.
1. Self-absorption
25
20
SAMPLE
30
THICKNESS,
35
45
40
50
MG/CM’
curves for cholesterol-C’”
and cholesterol-C”
digitonide.
thickness for cholesterol was reached at 30 mg/cmz and for the digitonide at 20 mg/cm*. Plating of duplicate aliquots of cholesterol-C4 digitonide from methanol gave an average recovery of 3443 cpm as compared with 12,684 cpm for aliquots assayed by liquid scintillation count’ing. Thus it would appear that the plating and flow counting method is practical for the assay of cholesterol-C I4 digitonide when no liquid scintillation spectrometer is available or when it is desired to recover the sample for subsequent chemical manipulations. ACKNOWLEDGMENT We are indebted to Hoffmann-La Roche Co., Nutley, New Jersey, and to Merck and Co., Rahway, New Jersey, for generous gifts of the digitonin used in this study. REFERENCES 1. KABARA, J. J., J. Lab. C&n. Med. 50, 146 (1957). 2. WILSON, J. D., AND SIPERSTEIN, M., Am. J. Physiol. 3. GRIMES, E. T., Anal. Chem. 34, 1175 (1962). 4. SCHOENHEIMER, R., AND DAM, H., 2. physiol. Chem. 5. WINDAUS,
A.,
2.
physiol.
Chem.
65,
110
(1910).
196, 215,
596
(1959).
59
(1933).
91
SHORT COMMUNICATIONS 6. KELSEY, 7. WINDAUS, 8.
9. 10. 11.
F. E., J. Biol. Chem. 127, 15 (1939). A., Z. physiol. Chem. 101, 276 (1918). SPERRI., W. M., AND WEBB, M., J. Biol. Chem. 187, 97 (1950). KERR, V. N., HAYES, F. N., AND OTT, D. G., Intern. J. Appl. Radiation 1, 2846 (1957). STITCH, S. R., Biochem. J. 73, 287 (1959). FUNT, H. L., AND HETHERINGTON, A., Science 131, 1608 (1960).
Isotopes
IRWIN L. SHAPIRO DAVID KRITCHEVSKY The Wistar Philadelphia, Received
Institute of Anatomy Pennsylvania September 11, 1962
and
Biology