Abstract
None of the methods known for quantitative estimation of bile pigment has been found satisfactory for gallstone pigment estimations. van den Bergh's method 1 is unsatisfactory, as the unoxidized fractions of pigments are not included; the method of Schmidt and Jones 2 is objectionable for similar reasons; Peterman and Cooley 3 carried oxidation with peroxide to a blue end-point and used a light filter when making the colorimetric reading.
The method offered here uses perchloric acid for oxidation, as the steps are better controlled and the end-products are more stable. A light filter is not necessary with this method. Bile pigments are extracted from weighed amounts (10-20 mg) of dry, well powdered stones, by refluxing with 5 to 8 cc of a mixture of equal parts of chloroform, ethyl alcohol, and glacial acetic acid, until the solvent drops colorless from the tip of the filter cone holding the powdered stone. Previous to this, cholesterol, and calcium and phosphorus are removed by washing first with warm ether and then with hydrochloric acid. Excess temperature and prolonged extraction are avoided to diminish the oxidation as far as possible. The extract is cooled to room temperature and made to a known volume, using the triple mixture. Aliquots (usually 1-2 cc) of this extract, as the intensity of the solution indicates, are measured into small test tubes and made up to a volume of 3 cc with 95% alcohol. At least one cc of the alcohol should be used.
Oxidation to a blue color is accomplished by the addition of 0.8 cc of 72% perchloric acid. Colorimetric comparison is made after 10 minutes against an aliquot from a known strength of bilirubin solution in the triple mixture, which has been oxidized at the same time and in the same manner.
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