our work to improve the resolution of the nuclides listed in Table I. ACKNOWLEDGMENT
The author is indebted to L. C. Bate, J. F. Emery, C. F. Goeking, G. W. Leddicotte,l W. T. Mullins, and R. R. Rickard of the Oak Ridge National Laboratory for their interest while this method was being developed and evaluated. (IC-min count1
LLTERATURE CITED
ENERGY, MeV
Figure 12.
water and then extracting all elements, except iron, from HBOs solutions with tri-n-octylphosphine oxide (TOPO) (9). The gamma activities observed after chemical separation of one sample are shown in Figure I;!. The limits of detection that can be achieved with the irradiation periods used in the nondestructive and chemical procedures have also been applied in the analysis of graphite by completely nondestructive techniques. The absence of an appreciable amount of any impurity other than chromium resulted in the production of very low levels of gamma activity so that all nuclides with gamma energies above 0.3 m.e.v. could be readily identified. Consequently, limits of detection of 54 elements were established by com-
High purity iron
parison of the gross spectra with those in the catalog of standards. Although designed for the analysis of ultrapure materials, this method has been modified and extended by the inclusion of additional chemical separations to remove intensely radioactive nuclides so that alloys could be analyzed. These supplementary procedures emphasized rapid extractions with CC1, (Br, I), tri-n-octylphosphine oxide (As, Sb, Re, Tc, Au, Mo, Sc, Ga, Zr, Th, U, Fe, and Cr) (Q), TTA (Sc, Zr, U, Th, and rare earth elements), and di(2-ethylhexyl)phosphoric acid (rare earth elements). Meinke (3)has shown that many of the short-lived nuclides in Table I can be separated rapidly by chemical means; however, such techniques have not been applied as yet in
( 1 ) Albert, P., Pure Appl. Chem. 1, 111 (1960). (2) Anders, 0. U., Beamer, W. H., ANAL. CHEM.33.226 (1961). (3) Kusaka; Y.,' Meinke, W. W., iiatl.
Acad. Sei.-Natl. Research Council, Nucl. Sei. Ser. NAS-NS-3104 (1961). (4) Leddicotte, G. W., ANAL.CHEY.34, 144R (1962).
(5) Mullins, W. T., Oak Ridge National Laboratory, private communication, October 1962. (6) Reynolds, S. A., Mullins, W. T., Intern. J . Appl. Radiation Isotopes 14, 421 (1963).
(7) Samsahl, K., Aktiebolageb Atomenergi, Stockholm, Sweden, Rept. AE-82 (1961). ( s j I b i d : , 1962. (9) White, J. C., Ross, W. J., Natl. Acad. Sci.-Natl. Research Council, Nuel. Sci. Ser. NAS-NS-3102 (1961).
Present address: Union Carbide Nuclear Go., P.O. Box 324, Tuxedo, N. Y. RECEIVEDfor review July 3, 1963. Accepted February 6, 1964. Oak Ridge National Laboratory is operated by Union Carbide Nuclear Go. for the U. S. Atomic Energy Commission.
Spectrophotometric Determination of Nanogram Amounts (of Total Cholesterol in Microgram Quantities; of Tissue or Microliter Volumes of Serum DAVID GLICK, BERNARD F. FELL1, and KNUD-ERIK SJQ)LIN2 Division of Histochemistry, Department of Pathology, Stanford University Medical School, Palo Alto, Calif.
b To achieve the refinement indicated in the title, by spectrophotometric analysis with the ferric chloride-sulfuric acid reagent, volumes of solutions were reduced to a submilliliter scale and handled with micro equipment. No loss in precision or accuracy, compared to analysis at the usuiil milliliter level, was incurred. The procedure given incorporates improvements from a number of laboratories, and it provides 0.1 ml. of solution for measurement, which can be reduced to 0.01 ml. with appropriate equipment indicated. Precision of 2.570 relative standard deviation was obtained with tissue samples, and 1.370 with blood serum.
Recovery of cholesterol added to adrenal homogenate was 102%. This method permits determinations on histologically defined biological material such as microtome sections of tissue as well as on drop samples of blood or other fluids.
A
SPECTROPHOTOMETRIC METHOD for the determination of free and total cholesterol in microgram quantities of tissue--e.g., microtome sections-microadapted from modifications of the original Schoenheimer-Sperry method employing sulfuric acid-acetic andescribed hydride color reagent-was
by Cavanaugh and Glick (3). This method was used by Glick and Ochs (7) in a study of the quantitative histological distribution of cholesterol in the adrenal gland. Subsequently, Glick and Lopez (6, Vol. 11, pp. 2537) incorporated the sulfuric acid-ferric chloride color reagent of Zlatkis, Zak, and Boyle @ I ) , with modifications by Brown et al. ( 2 ) , and Zak and Epstein (20),into the procedure of Cavanaugh and Glick to utilize the greater senl Present address, The Rowett Research Institute, Bucksburn, Aberdeen, Scotland. Present address, Department of Pathology, Sundby Hospital, Copenhagen, Denmark.
VOL 36, NO. 6, MAY 1964
1 119
titivity, stability, and simplicity of the ferric chloride method. Substitution of ethanol for glacial acetic acid was shown by Bowman and Wolf (1) to increase the specificity of the color reaction and to obviate interferences caused by traces of impurities even in the purest commercial glacial acetic acid. Earlier, Figueroa and Pike (6) had described an adaptation of the ferric chloride method using glacial acetic acid to the analysis of 5-pl. samples of serum. The present method includes utilization of the ferric chloride-sulfuric acid reaction in ethanolic medium, and the microadaptation exploits techniques indigenous to this laboratory.
(\I
\ L
+ 1.0 -$ 0 0 T j 0.5 t 0 t
S
0.0
Apparatus. Glass reaction tubes, 6 X 50, 6 X 30, and 6 X 35 mm. are used. The 6- X 35-mm. size are ground-glass stoppered. Mixing of liquids is effected by vibration or “buzzing” the tubes-e.g., with a Vortex Jr. Mixer (Scientific Industries), a LabLine Super Mixer (LabLine Instruments), or a home-made rubber-stopper type (6, Vol. I, p. 89, Figure 53). Lowry-Bessey cuvettes with a Beckman spectrophotometer (Model DU) are used for 100-pl. volumes, while capillary cuvettes may be employed for 10 pl. (6, Vol. 11,pp. 12-2 5). Constriction pipets are used for submilliliter volumes. Tubes are centrifuged in a Misco (Microchemical Specialties) microcentrifuge with Powerstat (Superior Electric) variable transformer speed control. Reagents. Absolute Ethanol. 2.5% FeCls.GHzO (reagent grade) in 86%. Hap04 (reagent grade). Solution is stable. Color Reagent. 0.8 ml. of the F e C k H3P04 solution made up to 10 ml. with concentrated H,SOa (Du Pont, reagent grade). Use as long as clear (6 to 8 weeks). Blank Solution. Before use mix equal volumes of absolute ethanol and color reagent. Cholesterol Standard. Cholesterol, c.P.,recrystallized from absolute ethanol and desiccated. Stock solution, 1 mg. of cholesterol per ml. of absolute ethanol. Store in refrigerator. Working standards, 2, 4, 6, 8, 10 pg. % in absolute ethanol.
0.2
0.6 0.8 mm. from surfaca
0.4
No. of
samples
Liver, 4 circular sections, 4mm. X 16microns
ANALYTICAL CHEMISTRY
9
1.0
1.2
Figure 1. Distribution of total cholesterol in a rat adrenal gland shown by analysis of microtome sections of tissue (1.5 mm. in diameter, 16 microns thick, 0.028-pl. volume). Histological zones are indicated by G, glomerulosa; F, fascicutala; R, reticularis; M, medulla; and sections containing material from two zones are designated by two letters
Preparation of Sample. Tissue. Place tissue-e.g., microtome section -into bottom of a reaction tube (6 X 30 mm.) and add 75 pl. of absolute ethanol, Seal tube with Parafilm and mix every 15 minutes for 1 hour to extract cholesterol and its esters. Centrifuge 5 minutes a t about 9000 X g (70-volt setting on Powerstat), and use the clear supernatant fluid for analysis. For homogenatese.g., a 20-mg. rat adrenal per ml. of watel-use 10-p1. sample and 250 p1. of absolute ethanol in a 6- X 50-mm. tube. Serum. Pipet 10 pl. of serum into bottom of a reaction tube (6 X 50 mm.), add 250 ~ lof. absolute ethanol, and mix well. Centrifuge for 5 to 10 minutes a t about 9000 X g and use the clear supernatant fluid for analysis. Procedure. Pipet 50 pl. of alcoholic extract into a 6- X 35-mm. tube with ground-glass mouth and add 50 p1. of color reagent. Mix well and stopper. Let stand a t room temperature for 1 hour and transfer liquid to microcuvette. Measure absorbance at 558 mp. (peak a t 550 to 560) against blank solution. (With Beckman DU spectrophotometer, 0.1-mm. slit and sensitivity setting 4 are used.)
Effect of Drying on Extractability of Cholesterol from Rat Adrenal and Liver
Tissue
1 120
f: 1.5
@
T
EXPERIMENTAL
Table 1.
?
0 .4-
Fresh Mean concn., pg./lOO pl.
No. of
Dried Mean concn., pg./lOO pl.
extract
samples
1.14 1.30
10 10
extract 1.06 1.20
1.70
2
1.75
1.46
9
1.47
RESULTS
Color Development. I n a test of the effect of time of standing on the color to be measured, absorbance increased a t room temperature up to about 45 minutes after mixing extract and color reagent, and then i t became constant. Accordingly, the mixtures were allowed to stand 1 hour before measurement. Extraction. To establish if extraction of cholesterol and its esters from tissue with absolute ethanol is influenced by drying, tissue homogenates and microtome sections were analyzed both fresh and after drying to constant weight. (The tissue material was dried overnight in vacuo over silica gel a t room temperature.) Table I shows that the extraction is equally effective on either the fresh or dried material. Standards. A linear relationship between absorbance and concentration of cholesterol in the alcoholic extract obtained up to a t least 1.1 absorbance, equivalent to 0.1 fig. of cholesterol per pl. Analyses were confined to 0.015 to 0.060 pg. per p l . ] corresponding to 0.165 to 0.715 absorbance. Recovery and Precision. Cholesterol, 0.37 pg. in 10 pl. of absolute ethanol, was added to 10 pl. of rat adrenal homogenate (20 mg. per ml. of water), which was allowed to stand overnight a t 4’ C. for the larger particles to settle. The cholesterol was recovered to 102y0 as the mean of 10 experiments, Table 11. In these experiments, the 2 0 4 . volume was extracted with 250 p1. of absolute ethanol and 50 p1. of the extract was used. For comparable analysis of the cholesterol alone, 10 pl, of water was substituted for the homogenate, and conversely the homogenate alone was analyzed by
substituting 10 pl. of absolute alcohol for the cholesterol solution. Ten separate analyses of an extract of rat serum and 10 analyses of a r:tt adrenal homogenate had relative standard deviations of 1.3% and 2.5y0, respectively, for corresponding mean absorbances of 0.219 and 0.270. Distribution in Adrenal. To demonstrate the applicability of the method t o analysis of micro samples of tissue, the following experiment was repeated 9 times. A f-esh rat adrenal gland was frozen with solid carbon dioxide, a cylinder of the tissue was bored out through the entire gland, and serial microtome sections (1.5 mm. in diameter, 16 microns thick, 0.028-pl. volume) were cut in a cryostat at - 15’ C. The technique of the operation followed that dexribed by Glick (6, Vol. I? pp. 24-33) using the equip ment designed by Grunbaum, Geary, and Glick (8). Two successive sections were taken first for analysis, the next one was taken for histological examination after staining with toluidine blue, the following two for analysis, and so on from the outer capsule to the middle of the medulla. Each pair used for an analysis was placed in a reaction tube in the cryostat and, after all of the sections were cut, the tubes were removed from the cryostat; 75 pl, of absolute ethanol were added to each tube and they were sea ed with Parafilm to stand during extraction. The histological distribution of total cholesterol in the rat :idrenal is shown in a typical experiment (Figure 1). Analysis of cholesterol n whole adrenals by the criginal methoc’ of Zlatkis, Zak, and Boyle ($1) had been described by Knobil et al. (13). DISCUSSION
After this work wao completed, the report of Momose et a,!. (15) appeared, in which it was shown that the higher cholesterol values caused by impurities in the sulfuric acid used in the fprric chloride method can be eliminated by use of a stable cnlcr reagent consisting of 8.0 grams of FeCla.6T&0 in a mixture of 200 nil. of perchloi,ic acid and 600 ml. of 85% phpsphcric acid. This reagent can be incorporated into the method given here, but with the Du Pont reagent grade sulfuric acid specified by Bowman and Wolf ( I ) , which was used in this work, the dif-
Table II. Recovery of Cholesterol Added to Rat Adrenal Homogenate
Mean concn., No. of Sample analyses Homogenate 10 Cholesterol, added Total, theor Total, f o u n l 10
bilirubin in serum by using isopropanolpetroleum ether for the extraction, followed by addition of sodium hydroxide to separate bilirubin.
PP./
100 pl. extract 2.10 3.70 5.80 5.90
ficulties encountered with other brands do not occur. Fortunately, either fresh or dried biological samples can be employed since it is often necessary to determine the dry weight of the micro sample and this can be performed prior to cholesterol analysis by direct weighing on a quartz fiber balance or another of suitable range (6, Vol. I, pp. 111-120). Microtome sections can be placed on previously weighed disks of aluminium foil up to 4 mm. in diameter and freeze dried or dried a t room temperature in vacuo over silica gel in a desiccator. After the foil with the dried sections is weighed, it can be placed directly in the tube for extraction. Drying the serum samples on filter paper has been used for some time in cases where this procedure offers special convenience for storage, and Searcy, Bergquist, and Jung (17) employed dried samples for determination on 10-p1. volumes of serum by direct elution from the paper with a so’ution of glacial acetic acid saturated with ferrous sulfate, and then by addition of sulfuric acid to develop a salmon-pink color. Separation of free cholesterol from its esters can be carried out on the analytical scale described in this paper by the digitonide procedure used by Cavanaugh and Glick (6, Vol. 11, pp. 254-7). The use of tomatine to precipitate the cholesterol (10-12, 16) could also be employed, although it has not been used by the authors. Other possibilities of adaptation to the micro scale are separations of cholesterol and its esters on paper (Q), silicic acid (4, 18), or alumina (19). Leffler and McDougald (14) modified the ferric chloride method to eliminate interference from
LITERATURE CITED
(1) Bowman, R. E., Wolf, R. C., Clin. Chem. 8,302 (1962). (2) Brown, H. H., Zlatkis, A., Zak, B., Boyle, A. J., ANAL. CHEY. 26, 397 119541. (3) Cavanaugh, D. J., Glick, D., Zbid., 24,1839 (1952). (4) Creech, B. G., Sewell, B. W., Anal. Riochem. 3,119 (1962). ( 5 ) Fieueroa. I. S.. Pike, R. L., Ibid.,
1, 163 (1960). ’ (6) Glick, D., “Quantitative Chemical Techniques of Histo- and Cytochernis~
try,” Vol. I, 11, Interscience, Xew York, 1963. ( 7 ) Glick, D., Ochs, hf. J., Endocrinology 56,285 (1955).
( 8 ) Grunbaum, B. W., Geary, J. R,., Jr., Glick, D., J . Histochem. Cytochem. 4 , 558 (1956). (9) Hansen, P. W., Dam, H., Acta Chem. Scand. 11,1658 (1957). (10) Huang, T. C., Wefler, V., Raftery, A., ANAL.CHEY.35,1757 (1963). (11) Kabara, J. J., in “Methods of Biochemical Analysis,” D. Glick, ed., Vol. 10, pp. 263-318, Interscience, Xew York, 1962. (12). Kabara, J. J., McLaughlin, J. T., Rieeel. C. A.. ANAL. CHEM.33. 305 (1961): (13) Knobil, E., Hagney, M. G., Wilder, E. J., Briggs, F. N., Proc. SOC.Exptl. B i d Med. 87,48 (1954). (14) Leffler, H. E., McDougald, C. H., Am. J . Clin. Pnthol. 39, 311 (1963). (15) Momose, T., Ueda, Y., Yamamoto,
K., Masumura, T., Ohta, K., ANAL.
CHEM.35,1751 (1963). (16) Rinehard, R. K., . Delaney, S. E., Sheppard, H., J . Lqpid Res. 3, 383 (1962). (17) Searcy, R . L., Rergquist, L. M., Jung, R . C., Ibid., 1,349 (1960). (18) Shin, Y. 6., Anal. Biochem. 5, 369 (1963) -,. (19) Webster, D., Clin. Chim. Acta 8 , 19 (1963). (20) Zak, B., Epstein, E., Ibid., 6 , 72 (1961). (21) Zlatkis, A., Zak, B., Royle, A. J., J.Lab. Clin. &fed.41,486 (1953). \ - -
RECEIVED for review January 6, 1964. Accepted February 17, 1964. Work mpported by research grants RG9227 and HEO6716, Sational Institutes of Health, U. S. Public Health Service. D. G. supported by Public Health Service research career program award ( S o . 5K6-AM-18,513) from the Xational Institute of hrthritis and Metabolic Diseases. B. F. is a W. K. Kellogg Foundation Fellow.
VOL. 36, NO. 6, MAY 1964
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