V O L U M E 24, N O . 1 1 , N O V E M B E R 1 9 5 2
1839
would have been very large, the cylinder N&S very small. The cam and cylinder were then cut and assembled according to these plans. The performance of the slit drive was satidactmy. No final shaping necessary. The background curve is a function of the condition of the glower and the thermocouple. The slit drive has increased the value of the lithium fluoride monochromatar by permitting convenient operation of the unit and by presenting a nearly linear transmittance sesle. Figure 3 shows the lithium fluoride background curve. ACKNOWLEDGMENT
The authors wish to express their thanks to George Long, Takuzo Tsuchiys, and Peter Smiruov of the Mechanical Engi-
neering Department for preparing drawings of the design, and to E. G . Buday, Hilding Lindquist, and R. B. Hilton far building . .. the c:ams ana electrical systems.
.
.
LITERATURE CITED
(1) IcKimey, D. 5..and Friedel, R. A,, J . O p l i d Sac. Am., 38,22 (1948). (2)
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1 1 1 1 ,
A. O., presented before conference of Instrument Society of Amerioa. St. Louis, Mo.. September 12 to 16. 1949. (3) Shrew, 0. D.,and meether, M. R., ANAL. CHEM.,22, 836-7 D s y 18, 1951. Amepted August 19, 195.2. 1~ E C E ~ V EM tleries. Research Depsrtment, General Mills, Inc.
Paper 118. Journal
Determination of Cholesterol in Microgram Quantities of Tissue DANIEL J. CAVANAUGH'L AND DAVID GLICK Department of Physiological Chemistry, Medical Scho01, University of Minnesota, Minneapolis, Minn. T I S the purpose of this paper to describe a quantitative method
I for the determination of both free and total cholesterol in microgram quantities of tissue--e.g., microtome sections-w+th an accuracy equal to that of macroprocedures. Microtome sections are used so that a correlation of the histology with the cholesterol analyses can he made. The original method of Schoenheimer and Sperry (7), as modified by Sperry and Webb (a),was adapted to the micro scale required, and the solvent mixture of Nieft and Deuel(6) was employed for extraction of the cholesterol since it permitted extraction without heating.
stanaard aointion. n .024 o n9 . miP1.~O-PRm I_____._.. .... ~ ~ , t~. ...... .. phnieqt+Troi r__ microliter of glacial acetic acid. All steps were carried out in glass reaction tubes of about 25 mm. in length and 0.2 ml. in volume, which could he fitted with ~
tion (&I electric mkcrocentrifuge, Microchemical Specialties 50.~ Berkeley, Calif., %-as used). Stirring --as carried out .by .me-
EXPERIMENTAL
.
. "..
.
Preparation of Tissue Sections. The tissue was rapidly re. ,. .. 1 moveu nom %neanimal, neea 01 oiooa, connective ussue, ana fat, and then frozen either an a block of dry ice or in the freezing compartment of a refrigerator. No evidence of a difference in cholesterol distribution dependent on the freezing method was found: however, the former method was preferred since it should 1ead to less tissue change than the slower freezing procedure.
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Using stainless steel cylindrical tissue borers with diameter? of > I I"
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mediately frozen to the head of a freezing attachment on a rotary microtome. Careful alignment of the cylinders N&S necessary t o ensure that the plane of sectioning was perpendicular t o the axis of the cylinder. The cylinder diameters chosen depended on the cholesterol concentration and the section thickness. Sections were usually cut a t 30 microns. The sections to be analyzed far cholesterol were placed on illurninurn foil diRkR (4mm. in diameter) and dried t o constant
tended to stick to porcelain or glass'surfaces if dried on these miterials. Cholesterol Determination. REAGENTS A N D APPAR4TUS. Alcohol-ether solution, 3 to 2, volume for volume, made with ah8olute ethyl alcohol and anhydrous ethyl ether. Alcohol-acetone solution, 1 to 1 volume for volume, absolute ethyl alcohol and reagent grade acetone. Acetic acid, glacial, reagent grade. Thirty per cent acetic acid in distilled water. Digitonin, 0.5%, in 50% ethyl alcohol. Phenolphthalein, 0.2%, in 1t o 1alcohol-acetone solution. Potassium hydroxide, 50%, in distilled water. Color reagent, made by adding concentrated, reagent grade, sulfuric acid dropwise to ice-cold, reagent grade,,aceta anhydride in the proportion of 1 to 9, volume for volume, lust before using. This reagent must he freshly prepared, and should not he used aftera few hours. ~
1 Present address, Chemioal Department. Nuclear Instrument and Chemical Gorp., 223 West Erie St., Chicago 10. Ill.
F i g u r e 1.
Electric Heater for Small
Tuhes
ohanieal vibration or "buzzing," by touching the bottom of the vessel to a rapidly rotating flattened nail (I). Colorimetry N= carried out using a microscope colorimeter ( 5 ) with capillary glass cuvets having a length of 7 mm. and lumen of 1 mm. in diameter. A tung8ten ribbon filament light source was used with a Farrand interference filter having maximum transmission at 619 mN. The reaction vessels were heated electrically in an appazatus which was especially designed for this purpose, and which was capshle of maintaining temperatures constant within 1' C., Figures 1 and 2. The double pole, double throw, toggle switch of the heating apparatus permitted use of either one or both electric heating coils. A variable transformer (750 VA.) was used to regulate the temperature hy controlling the power Supply from the 115-volt house line t o the heater. Temperatures up to 290" C. (far a room temperature of 26" C.) could be obtained with one heating coil, and >360" C. with both coils. For the
1840
ANALYTICAL CHEMISTRY
cholesterol determinations temperatures of 30°, 60°, and 70" C. were used, and these were obtained with one heating coil supplied with approximatel 10,28 and 32 volts, respectively. PBOCBDURE. T i e weigbed dry sections were extracted in the reaction tubes with 40 microliters of the alcohol-ether by buzzing fairly vigorously for about 30 seconds, taking care not to get liquid in the upper part of the tubes, and then letting stand a t room temperature for about 30 minutes. Thirty minutes were found t o be in excess of the time necessary for complete extraction. In fact extension of the extraction period to 12 hours, an increase {n volume of the alcohol-ether, or disintegration of the section resulted in no further extraction. Even fresh tissue sections, which are more difficult to extract than the dried ones, were completely extracted within the 30-minute period. After a second brief buzzing, the desired aliquots (usually 12 microliters) of clear liquid were taken separately for free and total cholesterol determinations. Dried sections of the adrenal gland remained intact during extraction and it was rarely necessary to centrifuge them down. Fresh sections disintegrated and centrifugation of their extracts waa required before taking aliquots for analysis. The aliquots were evaporated to dryness a t room temperature or by heating carefully (70"C.) to avoid boiling, and the residues were taken Up in 20 microliters of acetone-alcohol. For the determination of free cholesterol, 4 microliters of digitonin solution were added to the acetone-alcohol solution, the mixture was gently buzzed, and then permitted to stand overnight a t room temperature.
6 rows hales eOCh
far switch
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sediment. This necessitated that the sediment be well packed by centrifugation, and that the aspiration be carried out very carefully with the aid of a magnifying lens. The recipitate was washed with 20 microlitem of ether and the centriggation was repeated. The ether-washed recipitates are prone t o poor packing and often require re-centriggation. The ether was removed by aspiration with a capillary pipet and the residue was brought to dryness by gentle heating. The residues were taken up in 7.5 microliters of glacial acetic acid and vigorously buzzed. The solutions were then mixed with 15 microliters of the color reagent and allowed to stand a t 30" C. for 30 minutes. The construction of the heating apparatus is such that the heated tubes are protected from light. At the end of the reaction period aliquots of the tube contents were pipetted into the microcuvets and the optical density was determined with the microscope-colorimeter, Blank measurements were made directly on the color reagent; combining it with glacial acetic acid had no appreciable effect on the optical density. Occasionally, the blank solutions gradually develop color after 30 minutes. This may be due to improperly cleaned cuvets, or to the fact that the color reagent is not fresh enough. Since a trace of water will spoil the color reagent, the cuvets should not be cleaned with water; wiping off excess grease, rinsing with the acetone-alcohol solution, draining on clean filter paper, and allowing to dry is the recommended procedure. The concentration of cholesterol was determined by comparison with a standard curve. It was found that the standard curves were linear over the concentration range tested, 0.008 to 0.3 microgram per microliter of final colored solution. Standards in glacial acetic acid solution were made in the usual manner-e.g. (2)-from a sample of commercial cholesterol which was compared analytically with a sample of highly purified cholesterol, kindly supplied by Richard von Xorff. A standard curve plotted from data obtained using the digitonide precipitate procedure (duplication of the procedure for free cholesterol) was identical with that obtained by simply allowing the glacial acetic solution of cholesterol to react with the color reagent. The validity of the over-all method was tested by recovery experiments in which cholesterol was added to brain and adrenal homogenates. In these experiments the total amount of tissue was adjusted to closely approximate that in a single microtome section. Both wet (corresponding to fresh sections) and dry preparations were used (Table I). In general, the method compared well with macro techniques which have been publishede.g. (2, 4 , 8). DISCUSSIOX
The volumes chosen in the determination were somewhat arbitrary, but they depended generally upon the following considerations: To minimize the photometric error the final colored solution should have a transmittancy in the range of about 30 to 60%.
U
Figure 2.
Diagram of Construction of Electric Heater for Small Tubes
For the determination of total cholesterol, the esters were hydrolyzed by adding 0.5 microliter of 50% potassium hydroxide and heating to 60" C . for thirty minutes. The tubes were cooled, and to each was added I microliter of the phenolphthalein solution ; then the solutions were made acid to. phenolphthalein with 30% acetic acid (usually 1 microliter of acid was required). The addition of solutions in volumes of less than 3 microliters waa conveniently carried out afith a microburet graduated in increments of 0.1 microliter. -4fter the acidification, the 8 0 1 ~ tions were treated with digitonin as previously described. The precipitated digitonide was centrifuged down at about 400 times gravity for 5 minutes and the supernatant aspirated from the tube with a fine capillary pipet. Great care was exercised t o withdraw the supernatant fluid without disturbing the
Table I. Homogenate Braina
Recovery of Added Cholesterol from Tissue Homogenates Microgram per Microliter Initial Added Calcd. Found 0,200 0.206 0.023 0.183
Adrenalb
0.028
AdrenalC
0.039
Adrenalc
0.051
0.023 0.023 0.167
0.167 0.010 0.010 0.011 0.011 0.005
0.206 0.206 0.196 n 195 0,049 0.049 0.062 0.062 0.056
0,200 0.201 0.194 0.190 0.049 0.049 0.062 0.066 0.061
Recovery, % ' 97
97 98 99
97 100 100 100 106 109
Concentrations given refer in all cases t o concentration in final colored reaction mixture. a Free cholesterol only determined. b Homogenate a n d added cholesterol brought t o dryness preceding extraction a n d only free cholesterol determined. Homogenates left in wet state and only total cholesterol determined.
1841
V O L U M E 2 4 , NO. 1 1 , N O V E M B E R 1 9 5 2 Under the conditions of the method described, this required that the cholesterol concentration of the final colored solution be in the range 0.08 to 0.25 microgram per microliter. A few experiments on a given tissue serve to determine the most convenient magnitudes for extraction and aliquot volumes which will enable one to attain final concentrations in the desired range. For the rabbit adrenal, the volumes given here were quite satisfactory. The adrenal work is being continued. The precision of the over-all method, checked by caloulation of the standard deviation in thirteen analyses of adrenal and brain homogenates, is 10.0036 microgram of chaleEtero1 per mjcroliter of final colored resction mixture. For the range 0.08 to 0.25 microgram per microliter, the corresponding range of error is 4.5
tc 1
Ern
LITERATURE CITED
(1) Beemy, O.,Lowry, 0. H., Brock, M. J., and Lopea, J. A., J . BW. C h m . . 166,177(1946). (2) Foldes, F. F..and Wilson, B. C., ANAL.CHEM.,22,1210 (1950). (3) ,Giiek,D., "Teohniquesof Risto-and Cytochemistry." New York, Interseienoe Publishers,Inc.. 1949. (4) Kiwdey, G. R., and Sohaffert. R. R., J . Bid. Chem., 180, 315 (1949).
( 5 ) Malrnstrom, B. G.. and Gliek,
D.,ANAL.CHEM.,23, 1699 (I!
RECEITBDfor review May 28, 1952. Accepted Auguat 11 , 1952. No.
,
XXIV in the series, Studies in Histochemistry. Aided by I grant from Division of Research Grants and lWlowships, Nations1 Institul.ea of Hedth, U. S. Public Health Service, Bethesda, Md.
An Improvement in the Vitamin 0 line 1 GLEN M. SHUE, LEO FRIEDMAN, A N D CHESTER D Division of Nutrition, Food and Drug Administration, Federal Security A(
N THE absence of reliahle chemical or physical chemical Ivariety method for the estimation of vitamin D potency in a wide of products, reliance must be placed on the biological a
assay. The U. S. Pharmacopeia method i8 based upon the cure of exnerimentallv induced rickets in rats
