By David Glick
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R. Blinks, Proc. Null. Acud. Sci. ,46,327 (1960). 71. J. Myers and C. S. French, J. Gen. , 43, 723 (1960). 72. J. Myers and J. Graham, J. , 38, 1 (19&3). 73. W. Cockburn, C. W. Baldry, and D. A. Walker, Biochim. Bwphys. Acta, 131, 594 (1967). 74. M. Rorth and P. K. Jensen, Biochini. Biophys. Acta, 139, 171 (1967). Methods of Biochemical Analysis, VolumeI 7 Edited by David Glick Copyright 6 1969 John Wiley & Sons, Inc. METHODS OF BIOCHEMICAL ANALYSIS VOLUME 17 Separation and Determination of Bile Pigments .
Sulfanilic acid solution. 0 g of sulfanilic acid, 15 ml of conc. hydrochloric acid, water to 1 liter. 3. Sodium nitrite solution. 5 g in 100 ml of water. Keep in a brown bottle for no more than 2 weeks. 4. 25 ml of reagent 3; use presently. 5. Ascorbic acid solution. 2 g in 5 ml of water. Keep in a brown bottle and use during 1 day. 6. Fehling 11 solution. 100 g of sodium hydroxide, 350 g of potassium sodium tartrate, water to 1 liter. Procedure. 5 ml of reagent 4. Mix and stand for 10 min. 5 ml of reagent 6.
The composition of pigment I corresponds to bilirubin monoglucuronide or a complex of bilirubin and bilirubin diglucuronide (27). Rechromatography of pigment I or I1 again gives three zones. This method has been used by Billing (24) and Baikie (28) for determination of the three fractions from icteric sera. Schoenfield, Foulk, and Bollman (29) have compared the results of reverse-phase chromatography with those obtained by phase partition according to Eberlein, by 34 R. BRODERSEN AND J. JACOBSEN the classic diazo method of Malloy and Evelyn, and by the method of Schachter (Section 111-2), and have found little correlation.