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).
.
Dens it o metric EvaIuat io 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 Serurn
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
electrophoresis strips were carried in the Spinco Microanalyzer attachment which wm modified to carry a directdrive d.c. motor with a speed variable from 1 to 10 r.p.m. For most purposes a speed of 3 r.p.m. is ideal; but slower speeds may be used for greater sensitivity and the recording of fine lines. Experiments showed that a slit 0.5 mni. wide and 4 mm. long was ideal for scanning the strips in this system. Good results were also obtained by using a pinpoint source of light which was concentrated via a biconvex lens, focal length 2.2 cm. This arrangement allowed a strip 3 mm. wide to be scanned and integrated. RESULTS
Normal sera were separated by this technique into the five classical fractions plus, in many instances, prealbnmin, postalbumin, and &globulin. The demonstration of prealbumin required the use of larger amounts (0.5 PI. or more) of serum with short periods of separation. Not every normal serum gave each of these fractions. Figure 1 shows a record of an abnormal serum in which eight zones are clearly demonstrated. One of the zones in that sernm is a split eI fraction; this was not seen~ in any normal sera. In Table I are shown the results of analyses of sera from 50 persons. They are comparable to analyses of normal sera compiled from the literature by Ehrmantraut ( I ) . Results given here differ only in showing a higher mean value for wglobulin, which may be the more correct value, since the method used gave exceptionally clear separation of a,-globulin (Figure 1). Evidence shows that postalbumin, when nnseparated, is covered by the al-globulin. The rather wide scatter of values for the a,-globulin may, therefore, be explained by the fact that postalbumin separated in only 10of the 50 sera. It is, of course, desirable that each laboratory determine its own set of normal ranges. Figure 2 shorn% an example of a densitometric evaluation of a serum separated simultaneously on two strips and stained for lipoprotein and glycoprotein.
Figure 1. Electrophoresis strip (insert) and graph of on abnormal serum showing eight distinct zones
CONCLUSION
w i t h the ability to quantify the microelectrophoretic separations, the .authors believe the previously described . teehGqne of microelectrophoresis on cellulose acetate has reached the point where it is more rapid, discriminative, and versatile than other available methods. ACKNOWLEDGMENT
The authors are very grateful tc Carter C. Collins, Director of the R e search and Development Laboratory at the University of California Medical Center, for invaluable technical advice
and criticism, and to John Koch and Luther Dong for very competent techniea1 help. The normal sera from Alcatraz Federal Penitentiary were kindly provided by the Irwin Memorial Blood Bank, San Francisco. LITERATURE CITED
CHEM. 32, 5G4 (1960).
fig&;
( 3 a ~ ~ ~ ~ ~ ~ A (4) Kohn, J., Queen Maw's Hosmtal. London, England, prix tion, October 1960.
RECEIVEQ for review N o Acceoted March 21, 15 or6d in part by a grsnc Irom licit: u. U. h d i o Health Service, Institute of Arthritis and Metabolic Diseases (Grant A-3774), the Medical Research Gilbert Fund and in part by a grant from the California. State Oepartment of Mental Hygiene. VOL. 33, NO. 7, JUNE 1961
861
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B
