to the fluorrscwiw is rcslntivoly independent of sample size (compare column 4 of Table I to Table 11); conscquontly, if one has thc facilitiw for scaling u p the extraction process to accomrnodatc 400gram tissue samples, the certainty of results in the 2-p.p.b. range may be increased significantly. It is of some interest to set up 95% confidence limits on the reported mean diethylstilbestrol content of samples as determined by these experiments (Table
tivity. The combined use of the split sample technique, incremrnt standard, and a pH changc offers in the above described method a suitable procedure for the correction of extraneous fluorescence and me:mircmcnt of diethylstilbestrol in biological samples. Applications of this mcthod to t h r drtermination of trace quantities of diethylstilbestrol in other biological material are being considered for future publication.
17).
ACKNOWLEDGMENT SUMMARY
Photochemical reactions offer a high degree of specificity as well as sensi-
The authors thank R. Q. Thompson and F. A. Smith for confirmatory data and technical contributions, M. M.
Marsh for consult:ttion, and H. L. Breunig for the design and st:Ltistical analysis. The authors rtpprcciatc the combined efforts of the Agricultural Products Assay Dcpartmcnt and Analytical Development Department in obtaining experimental data. LITERATURE CITED
(1) Cheng, E. W., Burroughs, W., J . Assoc. Ogic. Agr. Chemisk 38, 147 (1955). (2) Goodyesr, J. M . , Jenkinson, N. R., ANAL.CHEM.32, 1203 (1900). ( 3 ) Munscy, V. E., J . Assoc. O h . Agr. Chemists 41, 316 (1958).
RECEIVED for review Septembcr 27, 1960. Accepted February 1, 1961.
Spectrofluorometric Determination of Total Bile Acids in Bile SAMUEL J. LEVIN, J. LOGAN IRVIN,' and CHARLES G. JOHNSTON Deportment of Surgery, Wayne State University College of Medicine, Detroit, Mich The method of Minibeck has been modified to permit the accurate spectrofluorometric determination of total bile acids in bile. This procedure is applicable to the mixtures of bile acids found in the bile of human beings and various experimental animals.
T
HE BILE ACIDS of the bile of human beings and various experimental animals consist principally of mixtures of the glycine and taurine conjugates of mono-, di-, and trihydroxycholanic acids (7). The determination of total bile acids in bile has been a difficult procedure which usually has required the summation of separate analyscs for several different bile acids. Although it is important to have analytical procedures which are specific for the individual bile acids, there is a need for a method which will permit the rapid determination of total bile acids in a single analysis. The method of Doubilet ( 4 ) is of uncertain specificity and lacks sensitivity for the determination of milligram and microgram quantities of bile acids. Previously dcscribed spectrophotometric (6) and fluorornctric (IO) methods for bile acids yield low recoveries with the dihydroxycholanic acids, and they arc not suitable for the determination of total bile acids in unresolved mixtures due to marked differences in absorbance and fluorescence of various bile acids
Present address, Department of Biochemistry, University of North Carolina, School of Medicine, Chapel Hill, N. C. 856
ANALYTICAL CHEMISTRY
.
in concentrated sulfuric acid under the conditions previously described. In this paper thc method of Minibcck (IO) is modified to permit accurate fluorometric determination of total bile acids in mixtures of di- and trihydroxycholanic acids, and the method is applied in the determination of total bile acids in bile. EXPERIMENTAL
Sources and Purification of Bile Acids. Pure cholic a n d deoxycholic acids were obtained by repeated crystallization of commercial samples (Nutritional Biochemicals Corp.). Chenodeoxycholic acid was synthesized according to the method of Fieser and Rajagopalan (6). Glycocholic, glycodeoxycholic, glycochenodeoxycholic, and the sodium salts of taurocholic, taurodeoxycholic, and taurochenodcoxycholic acids were prepared according to the methods of Cortese et al. (1-3). Thc purity of all bile acid samples was confirmed by chromatography according to Sjovall (I.?) and by determination of characteristic physical constants and properties. Reagents. Peroxide-frce ethyl ether. Extract reagent grade ethyl ether with 2% ferrous sulfate solution, d r y over anhydrous calcium chloride, and distill. T h e ether will remain free of peroxides for several weeks if stored under nitrogen gas in a dark bottle. Some freshly opened samplrs of reagent grade ether are free of peroxides (starch-iodide test) and can be used without purification. Standard cholic acid. To prepare a stock standard solution of cholic acid,
dissolve 25.00 mg. of cholic acid in reagent grade absolute ethyl alcohol. and dilute the solution with this solvent to 25 ml. in a volumetric flask. Prepare working standards by suitable dilution of the stock standard with reagent grade absolute ethyl alcohol. When stored in a refrigewtor and protected from evaporation, the standard solutions can be kept for sweral months without change. Procedure. H Y D R O L YAND S I ~ EuTRACTION OF BILE SAMPLE. Mix 1.0 ml. of bile with 5 ml of absolute ethyl alcohol (reagent grade) in a test tube. Cover t h e tube with a glass bulb, and heat the contents at the boiling point for 5 minutes to denature protein. Cool the tube, and filter the contents into a 50-ml. Rockefeller tube. Wash the original tube and the residue on the filter paper twice with 5 ml. of hot absolute ethyl alcohol, and collect the washings in the Rockefeller tube. Attach the latter to a Rinco vacuum evaporator, and evaporate the contents to dryness. T o the residue add 5 ml of 1.25N sodium hydroxide, close the mouth of the tube with a cotton plug or a loosely fitting aluminum cap, and heat the tube in a n autoclave for 3 hours a t 15 pounds pressure. Cool thc tube and acidify the contents to p H 4.5 with approximately 3 ml. of 2.1N sulfuric acid. During this acidification, test the pH by touching small drops of the solution to Nitrazine paper with a pointcd glass rod. Transfer the solution quantitatively to a separatory funnel with several washings with distilled water such that the final volume in the separatory funncl is 15 ml. Extract the solution with three 15-ml. portions of peroxide-free ethyl ether. Combine the ether ex-
0~30r------1
i
0.251
.-In C
f 0.15-
Acid
C
--
.-2
0.10-
0
a"
Deoxychollc
0.05. 440
480
520
560
Wave Length (mu)
Figure 1. Fluorescence spectra of bile acids in 40% sulfuric acid after heating for 60 minutes a t 65" C. Exciting wave length, 436 rnpj concentration, 0.1 rng. bile acid in 10 rnl.
acid
tracts, and wash with two 10-ml. portions of distilled water. Evaporate the ether extract in a beaker, dissolve the residue in 10 ml. of 70% (by volume) ethyl alcohol, and transfer the solution quantitatively to a small hand-operated countercurrent extraction apparatus. Extract the alcoholic solution with three 10-ml. portions of n-hexane over 7Oy0 ethyl alcohol in this apparatus. Evaporate the alcoholic fractions to dryness in a beaker on a hot water bath. Dissolve the residue in absolute ethyl alcohol (reagent grade), transfer quantitatively to a 10ml. volumetric flask, and dilute to volume with absolute ethyl alcohol. This extract now contains the equivalent of 0.1 ml. of bile per ml. of extract. DETERMINATION OF TOTAL BILE ACIDS (TB.4). Dilute the alcoholic extract quantitatively with absolute ethyl alcohol to contain approximately 0.02 mg. of total bile acids per ml. This quantity usually is contained in the equivalent of 0.001 ml. of human hepatic bile or a smaller volume of gall bladder bile. Pipette 1.0- and 2.0ml. aliquots of the alcoholic solution into borosilicate test tubes and evaporate to dryness in a water bath with aliquots of a cholic acid standard solution equivalent to 0.01, 0.02, 0.03, 0.04, and 0.05 mg. of cholic acid in separate tubes. Be certain that the evaporation of the alcohol is complete in each tube. Set up an empty tube as a blank. Add 5.0 ml. of concentrated sulfuric acid (c.P.,96.5%) to each tube, and immediately place the tubes in a water bath regulated a t 65" 0.1" C. Maintain the tubes a t this temperature in the bath for 60 minutes, and stir the contents of the tubes intermittently with glass rods during the first 10 minutes of the period of heating. At the end of the 60-minute heating, remove the tubes from the bath and cool by immersing the tubes in running tap water. Transfer portions of the solutions to matched 10 X 75 mm. borosilicate test tubes, and measure the intensities of the fluorescence of these solutions with a Farrand Fluorometer,
*
Model A, which is adjusted so that the 0.05-mg. cholic acid standard gives a reading of 100 on the galvanometer scale. Use a combination of Corning glass filters Numbers 3389 and 5113 in the primary position in the fluorometer, and use Corning filter Number 3486 in the secondary position. Subtract the blank reading from each of the other intensity readings, and plot a graph of intensities us. concentration for the cholic acid standards. Calculate the quantities of bile acids in each of the unknown tubes by interpolation on the standard graph. With regard for the dilution factors, calculate the concentration of total bile acids in terms of milligrams of cholic acid equivalent per milliliter of bile. RESULTS AND DISCUSSION
The bile salts found in bile of man are the sodium salts of the glycine and taurine conjugates of the commonly occurring bile acids; cholic, deoxycholic, and chenodeoxycholic (7). The free bile acids are only rarely found in bile (14) and may, in fact, be artifacts. However, due to the strongly hydrophilic properties of the conjugates, in analytical procedures, the bile salts found in bile are usually hydrolyzed to give these free acids, which are readily extractable from aqueous solution by various organic solvents. All recent procedures for colorimetric determinations of individual bile acids include this hydrolysis step. Techniques vary from a 6-hour reflux with 3001, sodium hydroxide solution (IS) and 10 hours' autoclaving a t 15 pounds pressure with 3 N sodium hydroxide in 4Oy0 ethyl alcohol (8) to the milder conditions of a 3-hour hydrolysis at 15 pounds pressure with 5% sodium hydroxide (11). Our studies indicate that within limits there is increasing destruction or alteration of structure of the free bile acids when they are hydrolyzed with sodium or potassium hydroxide
solutions of decreasing strength. Autoclaving with distilled water not only fails to hydrolyze the conjugated bile acids but structurally alters the free bile acids so that recoveries are very poor. For example, losses with chenodeoxycholic acid are as much as 50%. On the other hand, it has already been indicated that strongly alkaliric solutions alter cholic acid in some manner (4, 11). The conditions of hydrolysis (11) adopted here gave maximum recoveries, as will be noted later. The most suitable concentration of sulfuric acid for fluorescent development with the bile acids was determined by a systematic study of fluorescence spectra of fluorescent solutions obtained by heating 0.1-mg. quantities of the bile acids in sulfuric acid concentrations ranging from 10% (by weight) to 96.5y0. Fluorescence spectra were obtained with a Farrand Spectrofluorometer, Model 104243, consisting of a xenon source and two UVIS grating monochromators for the primary and secondary radiation. Current output from the phototube was measured with an RCA ultrascnsitive direct current microammeter, Type WV-84A, range 0.01 to 100 pa. All spectra were obtained with the primary monochromator set for a wavc length of 436 mp. Silica cuvettes were used. The spectrofluorometric data are apparent values, and consequently the data may not be directly applicable for use with another fluorometer without correction. At low and intermediate concentrations of sulfuric acid the fluorescence spectra of the different bile acids differed greatly (for example, see Figure 1 for spectra in 40y0 sulfuric acid). Fluorescence spectra and intensities of fluorescence varied widely with the sulfuric acid concentration for any particular bile acid (for example, see spectra for cholic acid in Figure 2). Only in concentrated sulfuric acid (96.5%, specific gravity 1.84) were the fluorescence spectra of cholic, deoxycholic, and chenodeoxycholic acids sufficiently similar (Figure 3) to permit application in the determination of total bile acids in unresolved mixtures of these acids. The fluorescence of the bile acids was also more intense in relation to the blank fluorescence when the concentrated acid was used. As indicated in Figure 3, the fluorescence intensities of equal molar concentrations of cholic, deoxycholic, and chenodeoxycholic acids in concentrated sulfuric acid were identical at 510 mp and were practically coincident a t higher wave lengths, but they differed considerably at wave lengths shorter than 510 m p . Consequently, for routine analysis of mixtures of bile acids in the Farrand Fluorometer, Model A, it is essential to use a secondary filter which transmits radiation of 510 mp and higher VOL. 33, NO. 7, JUNE 1961
857
4
1
1.60
1.40
-
1.20
-
._ VI c a l
._0.80 0.60 1.00
0
.z 0.8
-
c
0
0.40
0.20
-
420
-
420
460
500 540 Wave Length (my)
500
540
580
Exciting wave length, 436 mp; concentration of cholic acid, 0.1 mg. per 10 rnl. acid; deoxycholic and chenodeoxycholic acids a t equivalent malar concentrations
Exciting wave length, 436 m p ; concentration, 0.1 mg. per 10 ml.
wave lengths but has a sharp cut-off of radiation below 510 mp, Corning glass color filter Number 3486 meets this requirement. The primary filter combination (Sunibers 51 13 and 3389) isohtes the exciting wave length of 436 mp, a strong line of the mercury emission spectrum. The temperature (65" C.) for fluorescence development n a s arbitrarily selected to permit use of a thernioregulated ~ a t wbath which also was enil~loyed a t this same teniperature in the determination of cholic acid by another method (8). The optimum duration of the reaction a t 65' C. in concentrated sulfuric acid was determined to be 60 minutes through experiments with single bile acids and with mixtures of cholic, deoxycholic, and chenodeoxycholic acids. This interval should be controlled with a precision of 1 2 minutes t o avoid errors when mixtures of the bile acids are determined against a cholic acid standard. The bile acids should not be permitted to stand longer than 10 minutes in concentrated sulfuric acid before commencement of heating in the water bath. Consequently, the number of tubes carried through the fluorescence development should not exceed 30 since the elapsed time between the addition of the sulfuric acid to the first and last tubes in a larger series of samples may cause an error in the results of the first samples in the series. After the reaction a t 65" C. has been terminated by cooling the solutions rapidly to room temperature, the intensities of fluorescence of unknown samples relative to simultaneously prepared standard solutions are constant for several hours. It is important to evaporate alcohol completely from standard and unknown samples before e
500
Figure 3. Fluorescence spectra of bile acids in 96.5% sulfuric acid after heating
Figure 2. Fluorescence spectra of cholic acid after heating 60 minutes a t 65" C. a t various concentrations of sulfuric acid
858
460
W a v e Length O f Fluorescence (mp)
ANALYTICAL CHEMISTRY
addition of concentrated sulfuric acid, since remaining alcohol in amounts greater than 0.1 ml. will cause serious errors in the determinations. The intensities of fluorescence are linear over the range 0.005 to 0.05 mg. of bile acid per 5 ml. of concentrated sulfuric acid when the Farrand fluorometer, Model A, is adjusted to give a galvanometer reading of 100 with the 0.05-mg. cholic acid standard. The galvanometer (Rubicon Co.) employed had a 100-mm. scale with a sensitivity of 0.0014 pa. per mm. With this setting of the instrument, the blank reading was in the range 0.5 to 1. Tntensities of standard solutions are reproducible to &I%. Under the conditions of this procedure cholic acid standards can be used in the determination of cholic, deoxycholic, or chenodeoxycholic, or mixtures of these bile acids. Final samples which have intensities of fluorescence greater than the highest cholic acid standard may be diluted to the proper range with concentrated sulfuric acid. Such diluted samples yield the same intensities of fluorescence as undiluted samples of identical final concentration. Certain naturally occurring constituents of bile interfere nith the determination of total bile acids if they are not removed before development of fluorescence. For example, while pure palmitic acid itself does not fluoresce under the conditions of this procedure, the fluorescence derived from a sample of bile acid is decreased by 1 to 3% by the presence of an equal weight of palmitic acid. On the other hand, cholesterol yields a highly fluorescent product when it is heated in concentrated sulfuric acid, the fluorescence intensity being approximately SO% of that given by an equal weight of
cholic acid. Therefore, it is imperative that all substances in bile samples which yield fluorescence or quench fluorescence be removed in the process of extraction of the bile acids for the fluorescence assay. The ether extraction of the acidified hydrolyzate, followed by the countercurrent extraction with hexane over 70% ethyl alcohol appears to remove interfering substances occurring in normal bile as indicated by the results of comparative analyses by different methods reported below. Infrared absorption spectra of the purified bile extracts were practically identical nith the infrared spectra of bile acids. Chromatographic analysis (12) of the final extracts of bile revealed only cholic, deoxycholic, and chenodeoxycholic acids. PEospholipides, cholesterol, and free fatty acids were virtually absent from the extracts after the countercurrent distribution. For example, even when cholesterol in the initial bile sample was present in a concentration 100 times that of the bile acids, the quantity of cholesterol remaining in the final extract was such that the error in the determination of the total bile acids was only 1 to 2%. Table I presents the recoveries obtained when known amounts of pure conjugated bile acids are determined by this method. Since it is known that the only bile acids present in significant amounts in the bile of man, the dog, and the rat are the glycine and taurine conjugates of cholic, deoxycholic, and chenodeoxycholic acids (7), these were the only bile acids subjected to individual analysis in this study. For the analyses reported in Table I, measured volumes of standard alcoholic solutions of the bile acids were evaporated in hydrolysis tubes, and the analytical procedure was continued exactly as described for bile samples. The quantities of the bile acids nere ey-
pressed as cholic acid equivalents in milligrams, viz., those quantities of cholic acid in milligrams which would be equivalent on a molar basis to the quantities of the bile acids in the sample. This was done since cholic acid was used as a standard in the fluorometric analyses. As stated previously, the conditions of hydrolysis used in the procedure appear to be optimal. However, some analytical loss in this step appears to be unavoidable. The conditions adopted are such t h a t reproducible analytical recoveries are obtained. While the free bile acids are not seriously affected by these conditions, analytical losses of the conjugates occur in varying degrees. These losses nere determined by photometric analyses designed for the individual bile acids: cholic ( 8 ) , deoxycholic ( I S ) , and chenodeoxycholic (9). Average recoveries after hydrolysis were as follows: the free bile acids, 98Y0 (range, 95 to 100); glycocholic, taurocholic, and glycodeoxycholic, 9570 (range 92 to 97) ; glycochenodeoxycholic, 90% (range, 87 to 92) ; taurodeoxycholic and taurochenodeoxycholic acids, 80% (range, 78 to 83). These average recoveries were used to calculate the quantities of bile acids present after hydrolysis (Table I, column 111). The results recorded in Table I show that even wide variations in the proportions of bile acids present in the sample do not appreciably alter the high degree of accuracy of the fluorometric procedure for determination of total bile acids. Table I1 illustrates the recoveries of free bile acids added to bile samples in the form of their sodium salts. The fluorometric procedure was used to determine the total bile acids in a sample of human bile (line 1). Then aqueous solutions of the sodium salts of various bile acids were added to other portions of this sample of bile, and the analyses were repeated exactly as described in the experimental procedure, above. Satisfactory analytical recoveries of the added bile acids were obtained. Table I11 records the results of analyses of a series of bile samples from three species of animals. I n these determinations, since the actual total bile acid concentrations were unknown, the total bile acid values determined by the fluorometric method were compared with the sum of the values obtained by separate photometric determinations (8, 9, I S ) of the three common bile acids. I n these determinations no corrections were made for losses during hydrolysis of the samples since all analyses were performed upon the same hydrolyzates. The sample of human hepatic bile was obtained by choledochostomy drainage following a cholecystectomy, and the sample was collected 14 days after the operation.
Table 1.
Analytical Recoveries of Pure Conjugated Bile Acids
I Added Bile Acids! (cholic acid equivalent in mg.) TC GDC TDC GCDC T C D c 1.04 1.04 0.52 0.52 0.52 1.04 1.00 0.52 0.52 1.00 0.52 1.04 0.52 1.04 2.00 0.10 1.04 0.10 1.04 0.10 1.04 0.52 2.08 0.52 2.08 1.00 0.52 2.08 1.00 0.52 2.08 0.52 2.08 0.50 0.52 2.08 0.50 0.52 2.08 0.10 0.10 2.00 0.10 0.10 0.50 0.10 2.08 0.10 1.04
GC 1.00 1.00 1.oo
2.00 2.00 2.00 1.00 1.oo
0.50 2.00 0.50
I1
Total Bile Acids (mg.) 3.08 2.04 2.56 2.04 2.56 3.56 3.14 3.14 3.14 3.60 3.60 3.60 3.60 3.10 3.10 3.10 2.20 2.20 2.68 1.64
IV IIIb Total Total Bile Acids by Fluorometric Bile Method Acids Per after cent Hydrolreysis (mg.) covery (mg.) 2.87 2.77 97 1.91 1.88 98 2.37 2.30 97 1.78 1.77 100 2.20 2.07 94 3125 100 3125 2.83 2.65 94 2.82 3.00 106 2.92 105 3.08 91 3.00 3.31 3.02 97 3.11 90 2.72 3.04 .~ 2.88 3.24 89 2.85 99 2.87 2.30 90 2.56 101 2.58 2.56 112 2.42 2.07 101 2.08 2.06 84 2.05 2.43 1.40 94 1.49
a GC = glycocholic acid; TC = taurocholic acid; GDC = glycodeoxycholic acid; TDC = taurodeoxycholic acid; GCDC = glycochenodeoxycholic acid; TCDC = taurochenodeoxycholic acid. Data of column I1 corrected for average losses known to occur in hydrolysis step.
Table
II.
Sample
Analytical Recoveries of Sodium Salts of Bile Acids Added to a Bile Sample
Added Salts" (mg./ml.) Deoxy- ChenodeCholic cholic oxycholic
...
5.0 10.0 5.0 5.0 20.0 10.0 5.0 a
...
2.5 5.0 5.0 5.0 5.0 10.0 10.0
...
1.25 2.5 5.0 7.5 2.5 2.25 7.5
Total Addition (mg./ml.)
...
8.75 17.5 15.0 17.5 27.5 22.25 22.5
TBAb Recovery Detnd. (per (mg./ml.) (mg./ml.) cent) - ... 12.4 ... 21.4 9.0 103 31.3 18.9 108 28.8 16.4 109 94 16.4 28.8 ~. ..~ 28.4 103 40.8 36.3 23.9 108 35.5 23.1 103 Av. 105
Cholic acid equivalent. TBA = total bile acids (cholic acid equivalent).
Table 111.
Analyses of Series of Samples of Sheep, Dog, and Human Bile
(Average of four determinations) ChenoDeoxy- deoxy- Sum of Cholic cholic cholic Bile Acida Acido Acid0 Acids (mg./ (mg./ (mg./ (mg./ ml.) ml.) ml.) ml.) 17.6 4.8 1.0 23.4 1.0 2.1 0.4 3.5 11.2 6.4 0.9 18.5 12.3 2.5 1.1 15.9 6.2 2.7 1.2 10.1 2.2 1.4 4.1 0.5 7.2 23.4 32.1 1.5 11.4 7.6 19.9 0.9 22.0 2.6 25.9 1.3 9.8 2.0 14.7 2.9 2.6 1.5 9.6 13.7 2.5 2.8 12.0 17.3
Recovery (per cent) 103 121 110 103 110 127 37-A 95 22-B 105 37-B 98 N-14 Human 97 P-5 97 Dog P-10 100 102 Z!Z 6% a Individual bile acids were determined by the following methods: cholic acid (8), deoxycholic acid (13), and chenodeoxychclic acid (9). Total bile acids (TBA) determined by the fluorometric method. Sample 22-A 25 26
Origin Sheep
TBAb Detd. (mg./ml.) 24.2 f 0 . 3 4.7 f 0 . 7 20.5 + 1 . 5 16.4 f 0 . 6 11.1 f 1 . 2 5.2 + 0 . 3 30.3 f 1 . 5 20.9 + 1 . 6 25.5 + 1 . 5 14.2 + 1 . 3 13.3 + 1 . 3 17.3 + 0 . 7 Av.
VOL. 33, NO. 7, JUNE 1961
859
The samples of shccp and dog bile were collected through permanent biliary fistulae established with a T-tube in the bile duct. All samples were analyzed shortly after collection. I n Figure 4 the results of the fluorometric determination of the concentration of total bile acids in 37 bile samples are compared with the summation of the concentrations of cholic, deoxycholic, and chenodeoxycholic acids determined on the same bile samples by specific analyses (8, 9, I S ) . The agreement is considered satisfactory with an average deviation of 8.57, between the results of the two procedures.
b Figure 4. Coricentrations of total bile acids determined in 37 bile samples b y fluorometric method (abscissa) Comparison with summation of cholic ( 8 ) , deoxycholic ( I 3), and chenodeoxycholic 19) acids determined by specific methods (ordinate)
LITERATURE CITED
(1) Cortcse, F., Bashour, J. T., J . Biol. Chem. 119, 177 (1937). (2) Cortese, F., Bauman, L., J . Am. Chem. SOC.57, 1393 (1935). (3) Cortese, F., Bauman, L., J . Biol. Chern. 113, 779 (1936). (4) Dmbilet, H., Ibid., 114,289 (1936). (5) Fieser, L. F., Rajagopalan, S., J. Am. Chem. SOC.72. 5530 (1950). ( 6 ) Siedman, M., Bye'rs, S.O., Michaelis, F., Am. J . Physiol. 164,786 (1951). (7) Haslewood, G. A., Wootton, V., Biochem. J . 47, 584 (1950). (8) Irvin, J. L., Johnston, C. G., Kopala, J., J. Biol.Chem. 153,439 (1944).
T o t a l B i l e A c i d C o n c e n t r a t i o n s b y Fluorometry ( m g . p e r ml. o f b i l e )
(9) Isaksson, B., Acta Chem. Scand. 8 , 889 (1954). (10) Minibeck. H.. Biochem. 2. 297, 29 . (i938). (11) Mosbach, E. H., Kalinski, H. J., Halpern, E., Kendall, F. E., Arch. Biochem. Biophp. 51,402 (1954). (12) Sjovall, J., Acta Chem. Scand. 8 , 339 (1954). (13) Szalkowski, C. R., Mader, W. J., ANAL.CHEM.24,1602 (1952). I
(14) Watanabe, Tu'., J. Biochem. (Tokyo) 46, 681 (1959).
.
De ns it o metric EvaIuatio n of
RECEIVEDfor review September 28, 1960. A4ccepted March 20, 1961. Research aided in part by funds and grants from the Detroit Receiving Hospital Research Corp., Parke, Davis & Co., and the National Institutes of Health, -4-659 and A-699.
Mic roeIe ct ropho retic Se rurn
Protein Patterns on Cellulose Acetate Membranes B. W. GRUNBAUM Department of Pediatrics, Universify of California Medical Center, San Francisco, Calif.
W. J. FESSEL The langley Porter Neuropsychiatric Institute, California Department of Mental Hygiene, San Francisco, Calif.
C. F. PlEL Departmenf of pediatrics, Universify of California Medical Center, Sarl Francisco Calif.
,A method is described for quantification of the serum protein separations achieved with a newly developed microelectrophoresis apparatus. Normal sera may show eight different protein fractions. Results of quantification of these fractions in 50 normal sera are given.
T
apparatus for microelectrophoresis described by Grunbaum and Kirk (8) was later found to be ideal for the use of cellulose acetate as the supporting medium (3). This paper presents a method of densitometric analysis of the microelectrophoretic separation using a slight modification of available apparatus. HE
MATERIALS A N D METHODS
Electrophoresis on cellulose acetate membranes was performed as described
860
ANALYTICAL CHEMISTRY
(3). From one to eight strips could be used simultaneously in each apparatus. The 50 supposedly normal sera from long-term prisoners, quantitative values for which are shown in Table I, were
Table 1. Quantification of Electrophoretic Analyses of Sera of 50 Prisoners
Number of
Albumin Postalbumin Alpha-1 Alpha-2 Beta-1 Beta-2 Total beta Gamma
Samples 50
Mean,
S.D. of
50.8
S.D. Mean 4.8 0.7
10 50 50 21 21
5.0 6.7 14.9 8.7 2.9
1.3 2.3 2.9 1.7 1.0
0.4 0.3 0.4 0.4 0.2
50 50
9.6 17.0
2.6 3.9
0.4 0.5
70
separated on strips 1 cm. wide, two strips at a time in each apparatus. Undiluted serum was applied a t the geometrical center of the membrane by an applicator made from a 26-gage hypodermic needle whose lumen had been completely exposed by grinding off one side. The applicator was made to fit the width of strip used. Each sample on the 1-cm.-wide strips was estimated to be about 0.25 ~ 1 . Electrophoresis was performed a t room temperature for 120 minutes at a constant voltage of 200 with a barbital buffer of p H 8.6 and ionic strength 0.075. After drying, the strip was stained for protein with Ponceau S, 0.2% in 37, trichloroacetic acid, and for lipoprotein and glycoprotein according to the method of Kohn ( 4 ) . The stained strips were quantified with the Spinco Analytrol apparatus, equipped with the B2 balancing cam, and using a neutral density filter in the rear holder and a blue interfering filter in both front and rear holders. The
