Tissue cholesterol preservationSolubility of cholesterol digitonide in ethanol.код для вставкиСкачать
BRIEF COMMUNICATION Tissue Cholesterol Preservation : Solubility of Cholesterol Digitonide in Ethanol ' PETER R. STERZING, JOSEPH V. SCALETTI AND LEONARD M. NAPOLITANO Departments of Anatomy and Microbiology, The University of N e w Mexico School of Medicine, Albuquerque, New Mexico 87106 ABSTRACT The solubility of purified cholesterol digitonide in absolute and aqueous ethanols was investigated. The results indicate that preservation of cholesterol (or other 3-B-hydroxysterols) in tissues prepared for electron and light microscopy by digitonin-containing fixatives may not be quantitative when ethanol and, in particular, absolute ethanol, is used for dehydration. The unique property of 3-B-hydroxysterols for form 1:l addition complexes with the saponin, digitonin (Windaus, '09), has recently been applied at the electron microscope level as a method for increased retention and/or ultrastructural localization of free sterols in various tissues (Okros, '68; Napolitano and Scallen, '69; Scallen and Dietert, '69; Williamson, '69). In the single report on the quantitative retention of cholesterol in tissue by this method (Scallen and Dietert, '69), acetone was the dehydration solvent of choice because its capacity to solubilize the digitonide in the test tube was lower than that of ethanol - 0.048 mg/ml (acetone) and 0.112 mg/ml (ethanol). Schonheimer and Dam ('33) reported the solubility of cholesterol digitonide in absolute ethanol as 0.9 mg/ml, after continuous agitation of solute in solvent for 24 hours. In our studies, we have begun to question the reported solubilities of the cholesterol-digitonin complex in ethanol owing to (1) the inability to retain better than 90% of the free sterol in rat sciatic nerve when a graded series of ethanol are used for dehydration, and (2) the observation that an increase in dehydration time results in a greater loss of sterol into the solvent (Napolitano et al., '69). The present report demonstrates that the solubility of the digitonide in absolute ethanol (commercial absolute ethanol dried over Linde 3A Molecular Sieve) is higher than previously reported, and is significant enough to warrant elimination of that solvent in ANAT. REC., 168: 569-572. tissue dehydration when maximum retention of free cholesterol is desired; i.e., the localization of cholesterol by light and electron microscopic autoradiography. MATERIALS AND METHODS Cholesterol (S.C.W.) and digitonin (purchased from Nutritional Biochemicals Corp., Cleveland, Ohio) were used for preparation of the digitonide and for analytical standards. The digitonide was prepared after the method of Sperry ('63). Absolute ethanol was the same as that used in our laboratory to dehydrate tissues for electron microscopy; i.e., ethanol (absolute) obtained from U. S. Industrial Chemicals Co., New Orleans, La., and dried over Linde 3A Molecular Sieve (Union Carbide Corp., San Francisco, Calif.) at least 24 hours prior to use. Aqueous ethanolic solutions (v/v) were made up immediately before experimentation using absolute alcohol that had remained capped after addition of Molecular Sieve. Quantities of the prepared (and dried) digitonide, usually about 40 mg, were weighed and placed in test tubes fitted with Teflon-lined screw caps. Ten (10.0) milliliters of solvent at room temperature were added, and the tubes were briefly agitated on a vortex agitator. After standing at room temperature for one hour, each tube was again agitated, and then centrifuged. Aliquots of the supernatants were taken, dried Received July 15, '70. Accepted Sept. 2, '70. 1 Supported i n part by U.S.P.H.S. grant 5 R01 AM 90432-06and 5 R01 CA 10239-04. 569 570 STERZING, SCALETTI AND NAPOLITANO under a stream of nitrogen, and analyzed colorimetrically for cholesterol by the FeCL procedure of Bowman and Wolfe ('62). Solubility calculations were determined relative to optical densities obtained for 100% solubility of the prepared digitonide in chloroform-methanol ( 2 : 1). Since digitonin has been shown to give a color reaction with FeCL-H2S04(Graham, '64), a standard curve was obtained with cholestrol, to which digitonin was added in an equimolar amount. RESULTS Table 1 shows the results of three experiments in which the solubility of cholesterol digitonide was determined in absolute ethanol, and in various aqueous ethanols. Close agreement was observed between experiments, except for solubility values in absolute ethanol. These apparently reflect the particle size (surface area) of the prepared digitonide. In experiment 1, the washed and dried precipitate was scraped into a weighing bottle without further grinding. In experiment 2, the precipitate was lightly ground before weighing, while in experiment 3 , a mortar and pestle was used to achieve a fine powder. The reported values for solubility in absolute ethanol correspond to a range of 45% dissolution in experiment 1, to 95% in experiment 3 . The difference in solvent action on the digitonide was visibly noticeable between absolute ethanol and absolute ethanol containing as little as 1% water. A definite turbidity was present in all aqueous ethanols, while the absolute ethanol rapidly cleared following agitation of solute in solTABLE 1 Solubilization of cholesterol digitonide in ethanol * ( m g / m l ) Experiment Ethanol 1 2 3 2.83 0.73 4.05 0.79 % Absolute 99 98 96 95 94 1.9 0.31 - 0.13 - 0.15 0.11 0.10 - 0.09 - 0.31 Absolute ethanol (see Methods) to which varying amounts of distilled water had been added. 1 vent. The turbidity in the former situations was clarified by centrifugation. As previously noted, solubilities were determined after subjection of solute to solvent for one hour at room temperature. Examination of test tubes at later times (up to 1 week) revealed that the digitonide eventually reached 100% solubility (approx. 4 mg/ml). In experiment 3, complete solubility was observed within two hours after aliquots were taken; in experiment 1, the solubility increased from 45% to 67% after standing 45 hours. DISCUSSION In preparation of tissue for electron microscopy, the preservation of lipids, especially cholesterol, is quite difficult due to the action of dehydrating solvents such as acetone and ethanol (Korn and Weisman, '66; Saunders et al., '68; Scallen and Dietert, '69; Moses et al., '69). Napolitano and Scallen ('69), and Scallen and Dietert ('69) have shown that by incorporating digitonin into preliminary fixation steps, the amount of cholesterol lost during dehydration steps is reduced. This is apparently due to the formation of a sparingly soluble complex between cholesterol (and other 3B-hydroxysterols) and digitonin, a property first demonstrated by Windaus ('09). Reports concerning the solubility of cholesterol digitonide in various solvents are sparse. For our purposes, those solvents utilized for electron microscope procedures are most pertinent. Windaus ('09) gives the solubility in 95% ethanol as 0.14 mg/ml; Schonheimer and Dam ( ' 3 3 ) report 0.2, 0.9, and 5.0 mg/ml for 96% ethanol, absolute ethanol, and methanol, respectively. Scallen and Dietert ('69) determined the solubility in acetone, 0.048 mg/ml; absolute ethanol, 0.112 mg/ml; and propylene oxide, 0.244 mg/ml. Our solubility values for 95% through 98% ethanol are in reasonably good agreement with those of Windaus ('09) and Schonheimer and Dam ( ' 3 3 ) , respectively. However, we observed a much higher solubility of the complex in absolute ethanol compared with other workers. The discrepancy might be explained by relating their lower values to the presence of small quantities of water. Furthermore, our data are compatible with recent studies on the TISSUE CHOLESTEROL PRESERVATION retention of cholesterol in rat sciatic nerve (unpublished observations). When nerve (100 mg), fixed in a buffered glutaraldehyde-digitonin solution and postfixed in Os04, is dehydrated through 60, 80 and 95% ethanol, 1% of the free sterol is lost. However, further dehydration with two changes (10 ml for 5 minutes each) of absolute ethanol yields a 7-15% loss. Extending the time for five hours (five 1hour changes in absolute ethanol) results in a 90% loss, a value comparable to direct extraction of the fixed tissue with chloroform-methanol (2 : 1). The solubility data presented here are defined for a single concentration, temperature, and time. Although maximal solubility in absolute ethanol was not determined, the values observed, when related to quantities of free sterol in mammalian tissues, become quite important in their quantitative preservation. Sciatic nerve exhibits a relatively high concentration of cholesterol per weight of tissue in the rat; i.e., 40% of the lipids present, or a value of 40 mg/g tissue (D'Hollander and Chevallier, '69). This corresponds to approximately 0.8 mg of cholesterol per nerve (- 20 mg) from a 100-150 g animal. The cholesterol content of rat liver is much lower - 1.6 mg/g tissue. Disregarding additional complexing of digitonides with other molecular constituents of cells, dehydration with absolute ethanol would be expected to remove considerable amounts of sterol. Extraction might even be expected when using an acetone series, considering the solubility figure of 0.048 mg/ml (Scallen and Dietert, '69). It is apparent, therefore, that in order to retain maximal quantities of free cholesterol in tissue prepared for electron microscopy by the digitonin method, dehydration through absolute ethanol should be avoided. In this regard, dehydration through 95% ethanol followed by the procedure of Idelman ('64) is recommended. In any case, such variables as choice of solvent, volume of solvent, and time of dehydration, relative to the weight and source of tissue used, should be clarified - 571 before conclusions based on quantitative preservation can be made. Such considerations become extremely important when the techniques of light and electron microscopic autoradiography are utilized for localization of cholesterol in tissues. LITERATURE CITED Bowman, R. E., and R. C. Wolf 1962 A rapid and specific ultramicro method for total serum cholesterol. Clin. Chem., 8: 302-309. D'Hollander, F., and F. Chevallier 1969 Estimation qualitative et quantitative des sterols libres et esterifies du rat in toto et de 23 de ses tissus ou organes. Biochim. Biophys. Acta, 176: 146-162. Graham, H. 0. 1964 Color reaction of veratrum alkaloids with sulfuric acid and sulfuric acid reagents. J. Pharm. Sci., 53: 86-91. Idelman, S . 1964 Modification de la technique de Luft en vue de la conservation des lipides en microscopie electronique. J. Micr., 3: 715718. Korn, E. D., and R. A. Weisman 1966 Loss of lipids during preparation of amoebae for electron microscopy. Biochim. Biophys. Acta, 116: 309-316. Moses, H. L., W. W. Davis, A. S. Rosenthal and L. 0. Green 1969 Adrenal cholesterol: localization by electron microscope autoradiography. Science, 163: 1203-1205. Napolitano, L. M., and T. J. Scallen 1969 Observations on the fine structure of peripheral nerve myelin. Anat. Rec., 163: 1-6. Napolitano, L. M., P. R. Sterzing and J. V. Scaletti 1969 Some observations on tissue fixed by glutaraldehyde-osmium tetroxide-digitonin mixtures. J. Cell Biol., 43: 96a. Okros, I. 1968 Digitonin reaction in electron microscopy. Histochemie, 13: 91-96. Saunders, D. R., J. Wilson and C. E. Rubin 1968 Loss of absorbed lipid during fixation and dehydration of jejunal mucosa. J. Cell Biol., 37: 183-187. Scallen, T. J., and S. E. Dietert 1969 The quantitative retention of cholesterol i n mouse liver prepared for electron microscopy by fixation i n a digitonin-containing aldehyde solution. J. Cell Biol., 40: 802-813. Schonheimer, R., and H. Dam 1933 Uber die spaltbarkeit und loslichkeit von sterindigitoniden. Hoppe-Seyler Z. Physiol. Chem., 215: 5963. Sperry, W. M. 1963 Quantitative isolation of sterols. J. Lipid Res., 4: 221-225. Williamson, J. R. 1969 Ultrastructural localization and distribution of free cholesterol (3-Bhydroxysterols) in tissues. J. Ultrastr. Res., 27: 118-125. Windaus, A. 1909 Uber die entgiftung der saponine durch cholesterin. Ber., 42: 238-246.