A PHOTOMETRIC METHOD FOR THE DETERMINATION OF CHOLATES IN BILE AND BLOOD* BY J. LOGAN IRVIN,t

Reinhold and Wilson (1) have described a modification of the method of Gregory and Pascoe (2) which permits the determination of cholic acid in pure solutions and in bile with an accuracy approaching the maximum that can be attained with the visual color comparator. By this procedure chelates (glycocholate, taurocholate, and unconjugated cholate) can be determined with specificity even in the presence of dihydroxyand monohydroxycholanates. Modifications of this method have been described (3-6), but a critical evaluation of essential factors in the method is desirable. Procedures for the determination of cholate involving other color reactions (7-9) do not appear to offer advantages in specificity or in the accuracy of analytical recoveries. There is much uncertainty regarding the question of the presence of bile salts in the peripheral circulation of normal animals (10). Employing modifications of the method of Gregory and Pascoe, Josephson (3) reported that the normal concentration of cholate in blood is 0.5 to 2 mg. per 100 ml., and Schmidt (5) stated that the concentration is less than 2 mg. per 100 ml. By the use of another method, Perlzweig and Barron (11) were unable to demonstrate the presence of bile salts in normal blood. This was in agreement with the report of Herzfeld and Haemmerlei (12). Greene and Aldrich (13) and Aldrich and Bledsoe (14), by the use of a modified Pettenkofer reaction, found normal concentrations of bile salts in blood ranging from 3 to 6 mg. per 100 ml. With a method which was stated to be specific for the bile acid group of compounds but not for any particular bile acid, Tashiro (15) reported normal concentrations of 60 to 100 mg. per 100 ml. of blood. These analytical uncertainties are emphasized by reports of unsuccessful attempts to isolate bile acids from blood obtained from the peripheral circulation of normal animals (10). In this paper the method of Reinhold and Wilson is adapted for the photoelectric filter photometer. Certain modifications in the conditions for the color reaction are employed in order to secure the maximum increase