1859
V O L U M E 2 4 , NO. 11, NOVEMBER 1 9 5 2 unsuitable end points, irrespective of solvent ratios. However, Fritz ( 4 ) has successfully titrated caffeine as a base potentiometrically.
tion of the perchlorate salt of theobromine, materially aiding in the viewing of the correct end point. LITER4TCRE CITED
IhTERFEREYCES
Inteifering substances include other bases and water. Most common tablet excipients are not basic and hence do not interfere. DISCUSSION
Theobromine is amphoteric in nature. Recently, Fritz and Vespe (.5) proposed to determine it as an acid. Hoaevei, many tablets containing theobromine also contain other a c t h e ingredients, some of them acidic in character. The proposed procedure circumvents this difficulty. The addition of carhon tetrachloride promotes the precipita-
(1) Conant, J. B., Hall, S . F., aiid Werner, T . H., J . A m . Chent Soc., 49, 3047, 3062 (1927); 50, 2367 (1928); 52, 4-130. 5 1 1 5
(1930). S..-%s%L. CHEY..22. 578 (1950). . . i3j Ibid.,'p. 1028. (4)Ibid., p. 1029. (5) Fritz, J. S.,and Vespe, I-.,.J. A m , Pharni. Assoc.. Sci. Ed., 41, 197 (1952). (6) Sadeau, G. F., aiid Branchen, L. E., J . Am. C h e w SOC., 57, 1336 (1935). ( i )Pifer, C. W., and IT'cllish, E. G., ASAL. CHEM.,24, 300 (1952). (8) Wileon, H. h-.,J . SOC.C h o n . I d . (London), 67, 237 ilO4S). 12) Fritz. J.
RECEIVED for review M a y 29, 1952. Accepted August 2 5 , 1952.
Quantitative Estimation of Total Bilirubin in Serum J4I)IES J. QUIGLEY Diriuion of Laboratories and Research, Y e w Y o r k S t a t e Department of Health, 'ilbany, N. Y .
iM -4s'-
workers hare endeavored t o find a method for estimating the total bilirubin content of serum in a medium in which the protein remains soluble. M o s t of the earlier textbook methods required the addition of ammonium sulfate for precipitation of protein followed by 95% alcohol for solution of the bilirubin. Malloy and Evelyn (6) describe a method, employing a photoelectric colorimeter, for the estimation of bilirubin in serum in which the precipitation of protein and consequent loss of bilirubin has been eliminated. This is accomplished by diluting the serum 1 t o 10 with water and adding absolute methanol t o a final concentration of 50%. A4dlerand Strauss (1, 3) found that caffein, urea, and certain other substances facilitate the coupling reaction between the diazo reagent and biliruliin. Powell ( 7 ) noted that a mixture of sodium benzoate and urea aided in t h e coupling of the diazo reagent and bilirubin and also that the proteins remained soluble. I n his study he used a KlettSummerson photoelectric colorimeter which employs color filters. However, he suggested that the method could be adapted to other photoelectric instruments. Maclay ( 4 ) extended Powell's work for use with a Coleman spectrophotometer and in the preparation of bilirubin standards. However, t h e use of large volumes of reagents makes t h e method cumbersome. The following procedure is an extension and simplification of Powell's and Maclay's methods. REAGENTS
Solution A. One gram of sulfanilic acid (Baker's c.P.) is dissolved in a siiiall amount of water containing 15 ml. of concentrated hydrochloric acid and diluted t o 1 liter xvith distilled water. Solution B. Five-tenths gram of sodium nitrite (1Ierck reagent) is dissolved in 100 m]. of water. Freshly prepared. Solution C. A mixture is also freshly prepared by adding 0.3 ml. of solution B to 10 ml. of solution A. Sodium Benzoate-Urea Solution. Fifty grams of sodium benzoate (llallinckrodt, V.S.P.) are dissolved in 200 ml. of water, warmed if necessary. Fift,v grams of urea are dissolved separately in 200 ml. of water. The two solutions are mixed, diluted t o 1000 ml. with water, and filtered if necessary. The final solution should be Tvater clear. For the diazo blank, solution A is used. Sodium Carbonate Solution. Anhydrous sodium carbonate, 0.75 gram, is dissolved in 100 ml. of water. PROCEDURE
Coleman's spectrophotometer (Model 6A) was used for the measurements in this study. 9 solution containing 10 nig. of bilirubin (Pfansteil) in 100 ml. of redistilled chloroforn~ was prepared as a stock solution. The working solution, W , prepared as needed, was 10 ml. of the stock solution diluted t o
50 ml. with chloroform. \\lien not in use, these solutions were kept cold in the dark. Into six test tubes (19 h). 150 nini.) are added respectively 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ml. of the working solution, V'. This is carefully evaporated, one tube a t a time, with constant shaking in a beaker of hot ivater (80" to 90" C.) until all the chloroform has evolved. Chloroform is poisonous but not flammable. Then 1 ml. of t,he sodium carbonate solution is added to each tube, which is lvarmed to effect complete solution of the residue. The tuhes are cooled in water. Then 1 ml. of a Seitz-filtered serum pool, 1 ml. of solution C, and 7 ml. of sodium benzoate-urea solution are added and well mixed, and 10 to 15 minutes are allowed for full development of color. The final pH should be about 6.0 to 6.5. I n the Coleman spectrophotonieter using 12 X i 5 mm. cuvettes and a wave length of 530 nipfound to be maximal-the percentage transmittance or optical density is read. -4serum reagent blank is made from the same solutions in the same volumes except that solution .\ is substituted for solution C. Five different serum pools, containing negligible amounts of bilirubin were used, as it is imprartiralile to attempt to obtain bilirubin-free serum. While this study was originally aimed a t attaining a rapid, roughly quantitative estimation of bilirubin in serum, the results obtained were more precise than expected. Table I gives the observed transmittance values of the serum pools and of seruin with added bilirubin. Table I1 records optical density data on the same samples. There is a statistically significant trend Is, towards an increasing ratio of 3 to C as C increases-that Beer's law is inadequate t o describe the relationship. However, by the use of either transmittance or optical density data, a curve or table could be prepared suitable for the routine estimation of bilirubin in serum. For the transmittance TITO semilogarithmic paper is used, whereas, for optical density, 0 , the conventional cross-section paper is more suitable.
Table 1. Transmittance T Observed for Given Bilirichin Content Bilirubin Added. Ca
Experiinent Number 1
7
4
5
0.81
0.82
o
0.80 0 73 0.64
3
Transmittance
Blank 0.85 0 85 0.85 o ( T ~ )0 . 8 4 0 825 o 82 1 0 77 0.743 a 0.655 0 . 6 5 5 0.635 3 0.50 0.49 0.49 6 0.37 0.37 0.36 8 0.27 0 26 0 27 10 0.19 0.20 0.19 a C is bilirubin added in milligrams of serum was used.
0.78 ... 63 0.475 0.25
0.48 0.34 0.26
Mean
T ITO
0:8i3 0.738 0.643 0.487 0.360 0.262 0.192
1'000 0.920 o 791
0.599 0 343 0.322 0 236
0.19 0.19 per 100 nil. of serum: but only 1
111
1860
ANALYTICAL CHEMISTRY SUMMARY AND CONCLUSION
Table C 1 2 4
6 8 10
11.
Corresponding Optical x = 108 X D“
-Values of 1 2
2
Density
for Expt. No.
3 4 38 . . . 42 , . . 93 108 100 111 225 226 224 215 356 348 358 . . . 493 501 482 494 646 615 635 613
5 40 97 222 372 488 624
.tb
f/CC
G/Cd
40.0 101.8 222.4 358.6 491.6 626.6
40.00 50.09 55.60 59.75 61.45 62.66
1.155 1.684 0.491 0.832 0.396 0.622
Observed, 95% Confidence Interval0 35.34 44.66 45.41 54.77 54.24 56.96 57.10 62.40 60.35 62.55 60.93 64.39
D = -log(T/To). b li: = mean z.
C = bilirubin content. d s d C is estimated standard deviation of li:/C for value of C given for tabular row. e Based on R. A. Fisher’s t - distributioD in the usual way (I).
In estimating the bilirubin content of a submitted serum sample three tubes should be employed: the serum reagent blank, the serum sample, and a tube containing 0.04 mg. of bilirubin as a control on the reactivity of reagents. If the serum sample is hemolyzed, the serum reagent blank should cancel the effect of the hemolysis. If the sample contains a large amount of bilirubin. the blank and sample can be diluted with sodium benzoate-uiea solution.
The method described for the total bilirubin content of serum is simple, rapid, and precise. I n a series of 20 routine serum samples examined for total bilirubin, only one developed the typical rose-mauve color of azobilirubin. It was thus possible to eliminate 19 of the samples from further consideration with the consequent saving of time. According to Peters and Van Slyke ( 6 ) , the serum of normal individuals contains between 0.1 and 0.25 mg. of bilirubin per 100 ml. of serum. This paper is concerned only with the total bilirubin content of serum. LITERATURE CITED
(1) ddler, A , , and Strauss, L., Klin. Wochschr., 1, 2285 (1922). (2) Fisher, R. A., “Statistical Methods for Research Workers,” p. 120, New York, Hafner, 1950. (3) Goodson, W.H., and Sheard, C. S., J . Lab. Clin. Med., 26, 423
(1940-41). (4) LIaclay, E., Am. J . M e d . Technol., 17, 267 (1951). ( 5 ) Malloy, H. T., and Evelyn, K. A., J . Bid. Chem., 119, 4S1 (1937). (6) Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemistry,” Vol. 2, p. 917, Baltimore, Williams and Wilkins. 1932. (7) Powell, IT. N., Am. J . Clin. Path., 14, 55 (1944). RECEIVED for review July 18, 1952. Accepted hugust 28. 1932.
Table for Calculation of
“5
Concentration
By Mass Spectrometric Isotope Analysis LYNN D. ABBOT”, JR., AND MARY J. DODSON D e p a r t m e n t of Biochemistry, Medical College of Virginia, R i c h m o n d , Va.
TABLE has been found to be very useful in saving the time of L4 calculation involved in the analysis of nitrogen samples
for “5 concentration by the isotope-ratio mass spectrometer. Concentration of N15 was calculated a t intervals of current
~ ~
~-
ratios
(129) by the formula:
(128)
Conversion Table
Atom
z29
Ratio - to Atom yo 128
N16
For interpolation, a d d 0.0005 t o a t o m % E15 for each 0.00001 increase in ratio, a n d round off t o 3 figures. Atom Atom Atom Atom Ratio % N1S Ratio % N15 Ratio % “5 Ratio % Si3 0.00720 0.3587 0.00930 0.4627 0.01280 0.6357 0.01700 0.8432 0.00725 0.3611 0.0093B 0.4653 0.01290 0.6410 0.01710 0.8475 0.00730 0.3636 0.00940 0.4677 0.01300 0.6460 0.01720 0.8525 0.00735 0.3662 0.00945 0.4704 0.01310 0.6506 0.01730 0.8576 0.00740 0.3686 0.00950 0.4728 0.01320 0.6557 0.01740 0.8628 0.00745 0.3710 0.00955 0.4753 0.01330 0.6605 0.01750 0.8673 0.00750 0.3735 0.00960 0.4779 0.01340 0.6653 0.01760 0.8726 0.00755 0,3761 0.00965 0.4801 0.01350 0.6707 0.01770 0.8772 0.00760 0.3785 0.00970 0.4826 0.01360 0.6752 0.01780 0.8819 0.00765 0.3811 0.00975 0.4852 0.01370 0.6803 0.01790 0.8873 0.00770 0.3836 0.00980 0.4876 0.01380 0,6854 0.01800 0.8921 0.00775 0.3859 0,00985 0.4902 0.01390 0.6901 0.01810 0.8969 0.00780 0.3885 0.00990 0.4926 0.01400 0.6949 0.01820 0.9017 0.00785 0.3909 0.00995 0.4951 0.01410 0.7003 0.01830 0.9066 0.00790 0.3934 0.01000 0.4975 0.01420 0.7052 0.01840 0.9116 0.00795 0.3959 0.01010 0.5025 0.01430 0.7097 0.01850 0.9166 0.00800 0.3984 0,01020 0.5074 0.01440 0.7148 0.01860 0.9217 0.00805 0.4010 0.01030 0.5123 0.01450 0.7199 0.01870 0.9259 0.00810 0.4034 0.01040 0.5173 0.01460 0.7246 0.01880 0.9311 0.00815 0.4088 0.01050 0.5225 0.01470 0.7294 0.01890 0.9363 0.00820 0.4083 0.01060 0.5272 0.01480 0.7348 0.01900 0.9408 0.00825 0.4108 0.01070 0.5322 0.01490 0.7397 0.01910 0.9461 0.00830 0.4132 0.01080 0.5371 0,01500 0.7446 0.01920 0.9506 0.00835 0.4158 0.01090 0.5420 0.01510 0.7491 0,01930 0.9560 0.00840 0.4182 0.01100 0.5470 0.01620 0.7542 0.01940 0.9606 0.00845 0.4207 0.01110 0.5519 0.01530 0.7593 0,01950 0 9653 0.00850 0.4232 0,01120 0 5568 0.01540 0.7639 0.01960 0.9709 0.00855 0.4257 0.01130 0.5618 0.01550 0.7692 0.01970 0.9756 0.00860 0.4281 0.01140 0.5669 0.01560 0.7740 0.01980 0.9804 0.00865 0.4307 0 01150 0.5718 0.01570 0.7788 0,01990 0.9852 0.00870 0.4331 0.01160 0 5767 0.01580 0.7837 0.02000 0,9901 0.00875 0.4355 0 01170 0.5817 0.01590 0.7887 0.02010 0.9950 0.00880 0.4380 0.01180 0.5865 0.01600 0.7937 0.02020 1.0000 0.00885 0.4405 0.01190 0.5914 0.01610 0.7987 0.00890 0.4431 0.01200 0,5963 0.01620 0.8032 0.00895 0.4454 0,01210 0.6013 0.01630 0.8084 0.00900 0.4480 0.01220 0 6064 0.01640 0.8130 0.00906 0.4505 0.01230 0.6113 0.01650 0.8183 0.00910 0,4529 0.01240 0.6161 0.01660 0.8231 0.00915 0.4551 0.01250 0.6211 0.01670 0.8278 O.M)920 0.4579 0.01260 0.6262 0.01680 0.8333 0.00925 0.4604 0.01270 0 6309 0.01690 0.8382
Note:
yoS I 5
=
100 -
2R
+1
(1”)( 8 ) n-here R = ~
(129
Zz8 = ion current of mass 28 ZZ9 = ion current of mass 29 Tables of reciprocals ( 1 ) n’ere used, and it was found that interpolation as indicated was satisfactory for the authors’ purposes. Ratios less than 0.00720 occasionally may be obtained. In these cases extrapolation may be made readily and accurately by subtracting 0.0005 from the atom % “5 for each 0.00001 decrease in ratio. I t is evident that the table can be expanded or modified further to suit individual needs. For convenience two sheets of data may be combined back t o back with transparent plastic covers to provide a protected table for ready reference. Such covers are obtainable from American Kleer Vu Plastic, Inc., 57 Thames St., Brooklyn, N. Y. LITERATURE CITED
(1) Fisher, R. A., and Yates, F., “Statistical Tables for Biological, Agricultural and Medical Re-
search,” 2nd ed., London, Oliver and Boyd, 1943. ( 2 ) Rittenberg, D., “Preparation of Gas Samples for Mass Spectrographic Isotope Analysis,” in Wilson, D. W., el al., “Preparation and Measurement of Isotopic Tracers,” Ann Arbor, Mich., J. W. Edwards, 1948. RECEIVED for review J u n e 30, 1952. Accepted Beptember 8, 1952.
