HISTOCHEMICAL STUDIES OF OXIDATION AND REDUCTION REACTIONS OF THE BILE PIGMENTS IN OBSTRUCTIVE ICTERUS, WITH SOME NOTES ON HEMATOIDIN

R. D. LILLIE ¹ and P. PIZZOLATO ¹

¹ Department of Pathology, Louisiana State University School of Medicine, and U.S. Veterans Administration Hospital, New Orleans, Louisiana

In parallel tests formol fixation preserves more hepatic bile pigment than chloroformmethanol. The latter extracts more yellow color into the solvent and a higher proportion of the remaining bile pigment is green. Titanous chloride-formol and bichromate-formol preserve about the same amount of pigment as formol; the color distribution between golden brown and green casts is altered only a little in the expected directions. Primary acetic silver and neutral silver nitrate fixations blacken bile casts in liver and kidney in a 4-hr fixation period; after formol fixation sections require ammoniacal or methenamine silver for a successful argentaffin reaction. Hepatic bile casts show a pK of 5 after formol and 6 after chloroform-methanol fixations, staining red-brown with azure eosin at lower pH levels and deep green blue at higher. A 4-hr methylation at 60°C produced strong oxyphilia in formol tissue at pH 6; saponification restored and enhanced the basophilia and also enhanced pH 6 basophilia in unmethylated sections. The last suggests liberation of the propionyl residues by hydrolysis of the glucuronide bonds. Staining with several red dyes effectively changed the color of green bile casts to brown; hence unstained controls are needed for evaluation of tests for oxidation of bilirubin to biliverdin. Green hepatic bile casts are reduced to yellow-brown by reducing agents: Na₂S₂O₄, Na₂S₂O₂, HC1 + Na₂S₂Q₅, FeSO₄. Use of reduced bile pigment may be needed to show convincingly the oxidative action of such agents as FeCl₂, K₂Cr₂O₇ and iodine. Bile casts in variously fixed livers show purplish to brownish red colors with the periodic acid-Schiff (glycol) reaction and also after saliva digestion. Saponification, to break the glucuronide bond with bilirubin propionyl residues, did not prevent the glycol reaction. If this reaction is due to glucuronic acid residues of conjugated bilirubin, they would appear to be otherwise bound to tissue, perhaps by an amide linkage to protein amino groups. Peptic digestion alone did not inhibit the glycol reaction; combination with saponification results in section loss. Previously sectioned paraffin blocks and cut paraffin sections on 2-3 months air oxidation present numbers of violet, purple and red bile casts. The same Gmelin colors are produced by 3-5-day oxidation at 60°C in 2.5% K₂Cr₂O₇ by 1-2 hr at 24°C in 0.1% CrO₃, and by short diammine silver baths followed by desilvering with iodine and thiosulfate, as well as by the dry Br,:CCl₄ and aqueous OSO₄ treatments previously reported (16). The Gmelin colors are not reduced by dithionite or thiosulfate.

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