1829
V O L U M E 27, NO. 11, N O V E M B E R 1 9 5 5 the end point. The addition of standard perchloric acid to the solvent corrects the small negative blank. REAGENTS
Perchloric acid, 0.05-\-. Dissolve ca. 4.2 ml. of i 2 y 0 perchloric acid in 1 liter of dioxane and standardize against potassium acid phthalate according t o the method of Seaman and Allen ( 7 ) . Mercuric acetate solution. Dissolve ca. 6 grams of mercuric acetate in 100 ml. of hot glacial acetic acid ( 5 ) . Xitromethane, C.P. Fisher Scientific Go. Formic acid, 98%, Fisher Scientific Go. 1,3-Di-o-tolylguanidine,0.I-\-. Dissolve 24 grams of reagent in 100 ml. of methanol and 900 ml. of methylchloroform. Standardize potentiometrically against 20-ml. portions of standard 0.1-V aqueous hydrochloric acid or 40 ml. of the 0.05.V perchloric acid in dioxane. Methanol is used as sample solvent. Dimethylformamide, E . I. du Pont de Nemours &- Co. PROCEDURE
Sample weights are Iiased on an expected base content of the polymer of 5yG. Determination of Total Base. Accurately weigh 0.3 to 0.5 giam of the polvmer into a tared 150-ml. beaker and stir with 20 ml. of nitromethane until the material is well dispersed. Add 2 ml. of 9Syo formic acid, and heat until the suspension dissolves completely. Dilute with 50 ml. of cold nitromethane and allow the solution to come to room temperature. If the amine or quaternary salts present contain halide ion, add 2 ml. of 6 % mercuric acetate solution.
Introduce the glass and calomel electrodes into solution, set the pH meter to mv., and titrate the solut,ion potentiometrically with Ptandard 0.05.V perchloric acid. A microburet should be used. The end point of the titration is the maximum value of ( a E / A V ) . Appropriate plots may be made. A blank must also be titrated and the titer of the sample corrected for the solvent titration. Determination of Heterocyclic Amine Salt. Dissolve a rveighed 0.5-gram sample of polymer in 70 ml. of dimethylformamide. TJsing a glass and calomel electrode combination titrate the solution potent,iometrically with the standardized solut.ion of 1,3-di-o-tolylguanidine. The end point is det'ermined by the maximum value of ( a E / A V ) . The negative solvent blank is detcrrnined by tit,rating 70 nil. of dimet,hylformamide with standard perchloric acid and adding this correction t o the sample tit,er.
+
LITERATURE CITED
(1) Davis, 11.AI., and Hetaer, H. B., J . Research .\-afZ. Bur. Standa,ds, 48, 381 (1952).
(2) Fritz, J. S.,"Acid-Base Titrations in Sonaqueous Media," G. Frederick Smith Chemical Co. Columbus. Ohio. 1952. (3) Fritz, J. S.,and Fulda, 11.O., h s a ~ CHEX.. . 25, 1837 (1953). (4) Leussing, D. L., unpublished data. (5) Pifer, C. W., and Wollish. E. G., -%N\L. CHEY..24, 300 (1952). (6) Pifer, C . W., Wollish, E. G., and Schmall. I1 , J . Am Phaiin. Assoc., 42, 509 (1953). (7) Seaman, W., and .Illen, E., . i s 4 ~ CHEM., . 23, 592 (1951). RECEIVED for review May 18, 1955.
Accepted July 23, 1933
Fluorometric Determination of 0.1 to 10 Micrograms of Cholesterol R. WAYNE ALBERS
and
OLIVER H. LOWRY
Department of Pharmacology, Washington University School o f M e d i c i n e , St. Louis, M o .
A fluoronietric method is described for the nieasurement of as littleas 0.1 y of cholesterol in animal tissues. The simplicity of the procedure and the stabilitj of the fluorescence facilitate the measurement of a large number of samples. The effect on the reaction of other substances likely to be present in lipide extracts of animal tissues has been studied. No substances have been encountered which seriously affect the results. Free and esterified cholesterol produce nearly the same final fluorescence on an equimolar basis.
I
C O N S E C T I O S with n quantitative histochemical study of brain ( 6 ) a method vas needed for measuring as little as 0.1 y of cholesterol, or 1000 times less than the amount required for example. The reliability of the in the Sperry method (9), colorimetric Liebermann-Burchard reaction prompted an investigation of its fluorescence possibilities. A simple procedure resulted, which is based upon the measurement of a stable fluorescent product and which has the required sensitivity when used x i t h a photomultiplier-type fluorometer ( 5 ) . Glick (3)has recently adapted the colorimetric Sperry method t o the measurement of as little as 0.2 7 of cholesterol. Severtheless, the fluorometric method proposed may have certain advantages including greater ultimate sensitivity. Chen has reported a fluorometric method for hydroxy steroids ( 2 ) ,but no details have been published. ?;
METHOD
Reagents and Equipment. ..icetic anhydride, analytical grade, is used without further purification. Sulfuric acid, analytical grade. If the sulfuric acid contributes to the reagent blank, it may be purified by heating with 5o/c by
volume of 70yoperchloric acid until the solution hecomes colorless and fuming subsides. 1,1,2-Trichloroethane. The solvent obtained from Distillat,ion Products Industries (No. T 8 5 l ) is suitable after furt,her purification. The fraction boiling a t 111-113° C. is washed four times with 0.1 volume of concentrated sulfuric acid, twice x i t h water, and is finally dried over anhydrous sodium suliat,r. Redistilled absolute et.hyl alcohol. St'andard cholesterol solutions are prep:ired in trichloroethane a t concentrations of 0.02 to 0.4 mg. per ml. Solutions of 0.01 to 0.1 mg. % safranine 0 (Sational .hiline Co.) or rhodanine B (Distillation Products Industries) in 0.01S hydrochloric acid. These are not essential, but may be convenient t o control instrument, settings during fluorescence measurements. Lang-Levy pipets ( 6 , 7 ) are used throughout,. For the initial extraction the tip must be rather slender ( 7 ) . Fluorometer tubes are selected from 7 X 70 mm. serological tubes (Iiimble, S o . 45060-Sl81, -4.S. .\lot: CJ., St. Louis). Tolerances are kept within 1 or 2y0for 170th inner and outer diameter. The fluorometer tubes need to he very carefully cleaned before use. The folloning procpdure is recommended: two rinses with a det,ergent, two with water follon-rd by heat,ing in half concentrated nitric acid a t 100" C. for 43 minutes, then three rinses n-ith distilled witer The Farrand fluorometer is suitahle for me:isuring the fluorescence after adaptation to hold tubes of 7 miii. in diameter with 15O-pl. volumes ( 7 ) . Centrifuge evaporator (Figure 1 ), The motor, Jf, 280 r.p.m., Type KCI-23, Bodine Electric Co.. 2254 R'est Ohio St., Chicago, is suspended from desiccator plate, P , and is fitted with a 9-inch Lurite rotor, R. The rotor is d d l e d nitli 50 holes 8 nim. in half-inch strap of metal, S , permits diameter a t a 45" angle. easy removal of the assembly, which is mounted in a 10-inch vacuum desiccator. The electrical leads are stripped bare a t the point of emergence and sealed in with a De Khotinsky type of cement. The low speed motor for the small centrifuge constructed within the vacuum desiccator provides centrifugal force to prevent boiling or creeping of t.he solvent. up the vialls of the tubes during evaporation. K i t h the vacuum from a water aspirator and a little heat from an infrared lamp: the samples (alcohol) A\
1830
ANALYTICAL CHEMISTRY
evaporate to dryness within 15 minutes; however, with higher boiling solvents a better vacuum is required. If desired, the tubes may he loosely capped with aluminum foil during the operation. Procedure. The description given he1017 is applicable to 1 to 25 y of frozen-dried sections of brain or other tissue containing 0.1 to 2 y of cholesterol. -4general description of analvtical tools and techniques suitable for measurements at this scale has been given ( 7 ) .
appropriate selection of tube s k e and reagent volumes. For example, 2 to 20 y of cholesterol may he extracted with 0.1-nil. volume of ethyl alcohol in 7 X 70-mm. tubes and the fluorescence developed a t a volume of 1 ml. in standard 3-ml. fluorometer tubes. DISCUSSIOY 4 Y D RESULTS
The strong fluorescence of the reaction products of cholesterol in concentrated sulfuric acid (Salkomki reaction) was investigated in preliminary experiments. ;ilthough the fluorescence was found to he much more stable than the color, i t was difficult to achieve precision. Various modifications of the method of Trappe (10) were explored. Strong fluorescence could he ohtained, but the blank fluorescence could not be reduced to a workable level. The Liebermann-Burchard reaction in chloroform yielded more reproducible results; however, a higher boiling solvent than chloroform was necessary for use on a micro scale
..
>
L1 .
c
F i g u r e 1.
BRAIN IP IDES
C e n t r i f u g e eiaporator
’1 0
Extraction.
I n tubes of 2-mm. bore and 40-mm. lengt,h
( ? ) > the samples are extracted directly n-ith 15 to 20 pl. of re-
distilled absolute ethyl alcohol. With the thin porous frozendried tissue sections til-o short ext,ractions a t room t,emperature have proved adequak. Ext,ract,ion is accomplished by mixing with a “buzzer” ( 7 ) , follon-ed by centrifugat’ion and removal of iiiost, of the solvent \Tith a pipet. The ext’racts are transferred dirertly to fluorometer tubes which are kept in an ice bath during the extraction procedure to prevent the solvent from creeping up the n-a119 of the tubes. i K h e n analyzing wet samples or when it is dcsirable to measure various acid-soluble constituents or protein in the same sample (S),tlie extraction procedure is modified as follo~m: T h e tissue samples are extracted with 10 pl. of 5 % ti.icahloroacetic arid. They are buzzed and allowed to stand in t,hr a(4d for a t le:& 10 minutes), then centrifuged for 15 minutes at 3000 r.p.m. The centrifuge is allowed to coast to a halt. Of t8hr supernatant fluid 8 pl. are removed and saved for other an:tlyseP. The rePidue is then extracted once with 12 to 15 pl. of 0.l.V potassium acetate in ethyl alcohol and twice wit,h absolute ethyl alcohol. The three alcoholic extract,s are combined for anal>-sis.) The extract,s are evaporated to dryness with precautions to kcvp the residue in the hottom of the tube. This may be acwmplished with 50 samples a t once in the centrifuge device described, or with one sample a t a t,ime by heating in a water bath n-hilr directing a gentle stream of nit,rogen into the tube from a glass vapillary; Development and Measurement of Fluorescence. The dried liiii(k~residues are disaolvpd in I50 ~ 1 of, a fresh 5 to 1 mixture of t~ic~tiloroethane and acetic anhydride. About 20 minutes with tn-o or three mixings arc’ allowed for complete solution of the saniple and to permit any moisture present to react with the acetic. anhydride. Six microlit,ers of concentrated sulfuric acid :ire added to each sample with prompt and thorough mixing. The suniples are capped xvit,h aluminum foil and read in the fluorometer after standing at room temperature for 1 to 2 hours. Stxidards are prepared by adding 0.1 to 2 y of cholesterol in 5 p l , of trichloroethane to fluorometer tubes and carrying them t,hrough the analytical procedure, An appropriate small volume correction is applied before calculating the unknown samples. I t may he necessary to calculate from a curve, since fluorescence is not strictly proportional to concentration. The A546 mp mercury line isolated with Corning filters KO,4010 and 5120 is used as the exciting source. The secondary filt,er is the Corning N o . 2121, a cutoff filter with 377@t,ransmittance a t about A590 mp. All samples may be read against one of the standards or against a fluorescent dye solution (safranine 0 or rhodanine B). T h r procedure may be readily adapted to larger samples by
F i g u r e 2.
5
-
1.0
15...- ..- . .....
MINUTES
R a t e of fluorescence d e v e l o p m e n t
Trichloroethane proved to he the liest of a long series of solvent3 tested. The time course of the reaction is influenced by the solvent. Thus, with trichloroethane fluorescence reaches a peal; value in 5 to 10 minutes, then falls off slightly t o a steady value after itbout 45 minutes (Figure 2 ) , whereas in tetrachloroethane a slow development of fluorescence continues for over 3 hours. The fluorescence intensity is recorded as per cent of the filial fluorexence obtained with cholesterol. Cholesterol and choleiterol stearate are compared on :in equimolar basis. Brain cholesterol, which is chiefly nonesterified, is plotted relittive to free cholesterol. Adrenal cholesterol, which is chiefly esterified, is plotted relative t,o cholesterol Ptearate. I n the presence of brain lipides a higher early peak value is reached (Figure 2). This is ascrihed to the effect of other lipidep on the course of the reaction rather than to the presence of other steroids. In spite of the fact that the kinetics of the fluorescence development with free and esterified cholesterol are somewhat different (Figure 2), the final fluorescence intensity is only 37@ less with the ester. The composition of the resgent is such t h a t the components must he reasonably anhydrous to avoid turbidity. Beyond this moisture has not been found to be a critical factor. The fluorescence intensity increises markedly n-ith increasing acid concentration. Increasing amounts of acetic anhydride decrease the fluorescence somewhat. The reagent chosen was that n-hich produced the highest ratio of cholesterol fluorescence to reagent blank fluorescence. The choice of filters was dictated by the same criterion. The loss of sensitivity entailed b y measuring only the red fluorescence is compensated by a greater specificity.
1831
V O L U M E 2 7 , N O . 11, N O V E M B E R 1 9 5 5 T h e visible fluorescence excited by the A546 mp line is redorange, whereas that resulting from A365 mp excitation is bluewhite. When the concentration of cholesterol in the reagent is high enough t o produce a visible color, the color sequence is from pink t o rose t o blue nithin 30 seconds; the phase from blue to green requires about 10 minutes, whereas t h a t from green to yellow occurs slowly over 2 or 3 hours. The development of fluorescence nearly parallels that of the yelloL5 color as measured by the A420 mp absorption, although the yellow color continues to develop slowly after the fluorescence has attained a stable value. Quenching becomes marked a t concentrations greater than 100 per nil., but the fluorescence-concentration curve is practically linear up t o 25 -r per nil. Validation of Method. The coefficient of variation with the proposed procedure is about 2y0 down to a few tenths of a microgram of cholesterol (Tables I a n d 111). Even with 0.1 -/ of cholesterol, the coefficient of variation is only about 5% (Table I). Rabbit brain and rat adrenal samples were analyzed with and mitholit amounts of added cholesterol (Ch) as follows:
-,
A.
Table 111. Fluorescence of Other Steroids Relath e to That of Cholesterol
brain, 1 . 5 8 y Ch brain, 0 . 3 7 7 y Ch C. adrenal, 0 . 4 0 y Ch D. adrenal 0 . 2 0 y Ch E. adrenal: 0 . 1 0 Y Ch F. no tissue, 0 . 2 0 y C h 88.4 y 20.8 y J . 10 y 2.55 y 1.28 Y
B.
Primary filtera
Recovery of cholesterol added t o brain homogenate is satisfactory (Table I).
Table I. Cholesterol Found, Tissue After addition alone A.
1.91 1.88 1.92 1.91
3.38 3.53 3.43 3.37
B.
0.314 0.319 0.322
C.
0.440 0.435 0.446 0.438
Table 11.
Precision and Recovery Calcd. Total D.
0.237 0.215 0.206 0.220
0.401 0.398 0.386 0.411
0.420 0.420 0.420 0,420
0.728 0.G95 0 703
3.48 3.48 3.48 3.48 0.695 0.695 0.695
E.
0.116
0.839 0.846 0.835 0.845
0.840 0.840 0.840 0.840
F.
0.224 0.212 0.213 0.201 0.197 0.197 0.210 0.195
0.210 0.210 0.210 0.210 0.200 0,200 0.200 0,200
0.102 0.113 0.110
...
... .
..
...
Comparison of Fluorometric and Colorimetric Methods
Grams of Cholesterol per Kg. of Protein Rabbits, Differ.\Ionths Fluor. Color ence 8 244 231 +13 8 248 245 3 8 254 261 - 7 .ige of
+
II
Cholesterol Found, y Tissue after Calcd. addition Total alone
A ,
Grams of Cholesterol .kge of per K g . of Protein Rabbits DifferMonths' Fluor. Color ence 11 11 14
261 263
2QQ
significant difference was found between the values obtained by the two procedures (Table 11). A number of steroids were examined for interference (Table 111). Many steroids fluoresce in the reagent, but there fluorescence differs both quantitatively and qualitatively. With the filter combination recommended most of the steroids tested produced considerably less fluorescence than cholesterol. Of those tested only Ab-pregnenolone and A7-cholestenol were equal t o cholesterol. The fluorescence with other filter combinations may be of interest (Table 111). The fluorescences are recorded as per cent of t h a t obtained with the same weight of cholesterol and TTith the same filter combination. The filter combination in the first column is that recommended in the proposed procedure. D a t a should be considered only approximate. The secondarj filter was the Corning No. 2424 in all cases. Since in most unfractionated lipide extracts the amount of cholesterol present is greater than other steroids by several orders of magnitude, interference would not be a serious problem.
264 2R4 2
-
-
3 1
27
+
5120
5113
Exciting wave length 546 m u 436 mp Cholesterol 100 100 Dihydrocholesterol 30 28 AWholestenol 135 158 Cortisone 0 20 Pregnandiol 1 8 Dihydroepiandrostane 10 70 Alloprenanolone 12 45 Androsterone 22 105 26 45 Estroneb Progesterone 35 135 Desoxycorticosterone 66 470 A6-Pregnenolone 115 49 5 Corning filter numbers. b Estrone fluorescence faded rapidly on irradiation.
5860 365 inp 100 22 187 0 3
RO
28 123 16 120 250 210
Purified samples of phosphatidyl choline, phosphatidyl ethanolamine, and sphingomyelin do not interfere when present in amounts 10 times greater than the cholesterol. Cholic acid produces about one tenth the fluorescence of an equal weight of cholesterol. The substitution of fluorometry for colorimetry in the measurement of cholesterol has several advantages. Not only is it easy to obtain high sensitivity without sacrifice of precision, but the readings are relatively stable for some time, which is not true of present colorimetric methods. I n addition, free and esterified cholesterol give almost the same readings (Figure 2). No attempt has been made to attain maximal sensitivity. Merely reducing volumes tenfold-a completely feasible changewould increase the useful sensitivity 10 times (since the reagent blank is the present limiting factor).
a Standard error of difference. Coefficient of variation for fluorometric determinations waa 2.0%; for colorimetric determinations i t was 1.1%.
.A comparison was made between the proposed fluorometric procedure and a modification of the colorimetric method of Sperry (9) by means of parallel determinations of the total cholesterol in lipide extracts of 13 rabbit brains of three age groups. For the colorimetric procedure an amount of the dried residue of the lipide extract containing about 50 y of cholesterol was dissolved in 0.25 ml. of glacial acetic acid; 0.50 ml. of acetic anhydride \ias then added t o each sample and mixed, followed by the addition of 25 pl. of concentrated sulfuric acid with immediate and thorough mixing. The reaction was timed from the addition of sulfuric acid and read a t 620 mp between 27 and 37 minutes. The fluorometric analyses were performed on samples containing 4 t o 8 y in a final volume of 175 p1. Protein was determined on the extracted residues by a colorimetric method (10). No
4010
REFERENCES
Bessey, 0. A., Lowry, 0. H., and Brock, LI. J., J . Biol. C h e m , 164,321 (1946). Chen, C . , Federation Proc., 10,2 5 (1951). Glick, D., ANAL.CHEM.,24, 1839 (1952). Levy, LI., Compt. rend. trav. lab. Cadsberg, St%. chim., 21, 101 (1936).
Lowry, 0.H., J . Biol. Chem,, 173, 677 (1948). Lowry, 0. H., Roberts, N. R., Leiner, K. Y., Wu, AI. L., Farr, A. L., and Albers, R. W., Ibid., 207, 39 (1954). Lowry, 0. H.. Roberts, N. R., Leiner, K. Y., Wu, hl. L., Farr, A. L.,Ibid., 207, 1 (1954). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., Ibid., 193, 265 (1951). Sperry, W. hf., Am. J . Clin. Pathol., Tech. Suppl., 2, 91 (1938). Trappe, W., Hoppe-Seyler's 2. physiol. Chem., 273, 177 (1942). R E C E I V E D for review April 28, 1954. Accepted August 4, 1955. Supported in part by a grant from American Cancer Society, through the Committee on Growth of the Kational Research Council. R. Wayne Albers, a predoctoral fellow of the National Cancer Institute of the C . S. Public Health Service.
