None of the methods known for quantitative estimation of blile pigment has been found satisfactory for gallst one pigment estimations. van den Bergh's method' is unsatisfactory, as the unoxidized fractions of pigments are not included; the mlethod of Schmidt and Jones2 is objectionable for similar reasons ; Peterman and Cooley3 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 stabile. --I light filter is not necessary with this method Bile pigments are extracted from weighed amounts (10-20 nig) crf 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 ~Q Wdlered 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. Alicpots (usually 1-2 cc) if 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 951% alcohol. At least one cc of the alcohol should hie 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 kiiown strength of bilirubin solution in the triple mixture, which has been oxidized at the same time and in the same manner. A standard from a 3-5 mg % * This work was done in part under a grant from tlie Douglas Smith Founda-1 van den Bergh, Hymans A. A., Press Mcd., 1921, 88, 441.
NON-BACTERIAL CHOLECYSTITIS 89 and stiff and unbending. On microscopic examination the mucosa appeared normal or as nearly so as it usually does in human cholecystitis. The muscularis was moderately edematous and contained a considerable number of large and small mononuclear cells with a marked scarcity of pdymorphonuclear leucocytes. The reaction appeared to take place mostly in the serosa which was 8 to 10 times its normal thickness, being tremendously edematous, showing marked hyperemia and a number of punctate hemorrhages, together with the characteristic dilatation of lymphatics and moderate infiltration of round cells. The picture was similar but very much moxe marked than that produced by protein injection and resembled with a surprising accuracy that of acute human cholecystitis, differing markedly from the effects of bacterial injection.Considering the relatively slight increase in bile salt concentration beyond the 6-10% supposed to be normal for the human, these results may be looked upon as decidedly significant and further experiments are being undertaken to investigate the effect on this phenomenon of changes in the acid base equilibrium, variations of the osmotic pressure of its contents and other factors which might influence it.
PNon-Bacterial Cholecystitis.
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