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Tissue cholesterol preservationSolubility of cholesterol digitonide in ethanol.

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Tissue Cholesterol Preservation : Solubility of
Cholesterol Digitonide in Ethanol '
Departments of Anatomy and Microbiology, The University of N e w
Mexico School of Medicine, Albuquerque, New Mexico 87106
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.
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.
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.
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 )
Absolute ethanol (see Methods) to which varying
amounts of distilled water had been added.
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.
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
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
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.
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:
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:
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:
Windaus, A. 1909 Uber die entgiftung der
saponine durch cholesterin. Ber., 42: 238-246.
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cholesterol, ethanol, tissue, preservationsolubility, digitonide
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