Congenital Jaundice in Rats, Due to a Defect in Glucuronide Formation Rudi Schmid, … , Lydia Hammaker, Richard L. Swarm J Clin Invest. 1958;37(8):1123-1130. https://doi.org/10.1172/JCI103702. Research Article Find the latest version: https://jci.me/103702/pdf CONGENITAL JAUNDICE IN RATS, DUE TO A DEFECT IN GLUCURONIDE FORMATION 1 By RUDI SCHMID,2 JULIUS AXELROD, LYDIA HAMMAKER,2 AND RICHARD L. SWARM (From the National Institutes of Health, United States Public Health Service, Bethesda, Md.; the Thorndike Memorial Laboratory, and the Second and Fourth (Harvard) Medical Services, Boston City Hospital; and the Department of Medicine, Harvard Medical School, Boston, Mass.) (Submitted for publication March 4, 1958; accepted April 24, 1958) It has recently been reported that bilirubin is permitted the separate collection of urine and feces (11) excreted in the bile as a water-soluble glucuronide and were fed Purina laboratory chow. The conjugation of bilirubin is believed to Blood was removed from the tail or the subclavian veins (1-3). for the following determinations: hemoglobin concentra- occur mainly in the liver (4, 5). In vitro, forma- tion (12), reticulocyte count (13), one minute direct- tion of bilirubin glucuronide has been demon- reacting and total serum bilirubin (14), and paper chro- strated with a system containing liver microsomes matographic analysis of the serum bilirubin azo deriva- as the enzyme source and uridine diphosphate glu- tives (15). The urine was examined for bilirubin with the Harrison test (16) and by paper chromatographic curonic acid as the glucuronic acid donor (5, 6). analysis (15). Fecal urobilinogen excretion was esti- Since conjugation of bilirubin appears to be es- mated in stool samples collected over four consecutive sential for its excretion in the bile, a defect in the days, and the values were expressed in mg. per day per glucuronide forming mechanism would result in 100 Gm. body weight (17). impaired pigment excretion and, hence, in reten- o-Aminobenzoic acid and menthol were dissolved in 50 per cent ethanol and were administered by stomach tube tion of free bilirubin in the blood. in doses of 25 mg. per 100 Gm. body weight. During the In preliminary communications (7, 8), such de- 24 hour period immediately following the drug adminis- fects in glucuronide formation have been reported tration, the total urinary glucuronides were estimated in congenital nonhemolytic nonobstructive jaun- (18) and expressed as glucuronic acid. For comparison, dice in man (9) and in hereditary nonhemolytic glucuronide excretion was determined during similar con- trol periods when the rats were given only 50 per cent jaundice in rats (10). The present report is con- ethanol. Aqueous sodium o-aminobenzoate was injected cerned with the details of the murine studies, while intraperitoneally (100 mg. salt per 100 Gm. body weight) the human syndrome will be considered in a sepa- and urinary glucuronide excretion was estimated over rate communication. a seven hour period immediately following the adminis- tration of the compound. The values obtained were com- pared with those found after injection of an equivalent MATERIALS AND METHODS amount of sodium chloride at the same hour of the the difference in the the preceding day. In all instances, Nonjaundiced Wistar rats, known to be carriers of amount of glucuronic acid excreted during the test pe- jaundice trait, were donated by Dr. W. E. Castle at the riod and the control periods was expressed in per cent University of California in Berkeley. Litters obtained of the dose of o-aminobenzoic acid and of menthol ad- by mating pairs of these animals 3 regularly included one ministered. Sodium benzoate was injected intraperitone- or more offspring with jaundice. Nonjaundiced litter ally (50 mg. benzoic acid per 100 Gm. body weight) and mates and normal Wistar and Sprague-Dawley rats the urinary excretion of hippurate was estimated over a served as controls. Only male animals of at least 200 12 hour period, following the injection of the compound. Gm. body weight were used for the experiments. The The hippuric acid was extracted and crystallized (19), rats were housed in specially built glass cages which and the amino nitrogen of the crystals was determined by the micro-Kjeldahl method (20). Results were ex- 1 Presented at the annual meeting of the American ad- Atlantic N. pressed in per cent of the dose of benzoic acid Society for Clinical Investigation, City, J., ministered. May, 1957. rats were de- 2 Present address: Thorndike Memorial Laboratory, For estimation of enzymatic activity, the Boston City Hospital, Boston, Mass. capitated and the livers were immediately chilled and ho- 3 The authors are grateful to Mr. Samuel M. Poiley, mogenized with cold isotonic KCl. Microsomes were pre- Animal Production Section, National Institutes of Health, pared as described previously (21). Uridine diphosphate for his help and cooperation. (UDP) glucose dehydrogenase activity in the liver was 1123 -1124 RUDI SCHMID, JULIUS AXELROD, LYDIA HAMMAKER, AND RICHARD L. SWARM TABLE I taining 0.05 ml. of 1 N sodium hydroxide. For intra- Serum bilirubin concentration in Wistar rats venous injection, this solution was mixed with normal rat serum to give a final concentration of 1 mg. bilirubin per ml. As source of bilirubin glucuronide, serum was One minute collected from patients with complete biliary obstruction, direct-reacting Total bilirubin or sterile bile was obtained from patients with a T-tube Rats No. Mean S.D.* Mean S.D. in the common bile duct. Before these specimens were mg.% mg.% injected, their approximate concentration of conjugated Jaundiced 14 0.21 0.05 8.09 2.85 bilirubin was estimated by the direct van den Bergh Nonjaundiced method (14) and by paper chromatography (15). litter mates 5 <0.1 <0.1 Bromsulphalein (0.5 mg. per 100 Gm. body weight) was Normal controls 5 <0.1 <0.1 injected intravenously and the concentration of the dye in serum and bile was determined at different time in- * Standard deviation. tervals (23). CholografinO (iodopamide) was injected intravenously (0.1 ml. per 100 Gm. body weight) and assayed by the method of Strominger, Maxwell, Axel- the excretion of the contrast medium into the duodenum rod, and Kalckar (21). The rate of formation of o-ami- was followed by serial X-rays. nophenyl glucosiduronic acid (22) was determined by in- cubating microsomes obtained from one Gm. of liver with 0.2 micromole of o-aminophenol and 0.3 micromole RESULTS of UDP glucuronic acid which was prepared enzymati- A. Hematological and pigmentary studies. cally (21). In the kidney, glucuronide formation was estimated by incubating tissue slices in Krebs-Ringer bi- Hematological studies, performed on 10 jaun- carbonate with o-aminophenol and UDP glucuronic acid diced rats, yielded a mean hemoglobin concentra- (22). tion of 13.2 Gm. per cent (S.D., 1.5 Gm. per cent) For histological examination frozen sections were and a mean reticulocyte count of 0.69 per cent used, as well as paraffin sections of material that had been fixed in formalin or in Zenker's solution. The golden (S.D., 0.41 per cent). These values are within material found at the tip of the renal papilla of jaundiced normal limits (24). The values for serum bili- animals was freed under a dissecting microscope and was rubin concentration are summarized in Table I. dissolved in 0.2 ml. of chloroform. The azo derivatives On paper chromatographic analysis, the sera of prepared from this material were studied by paper chro- jaundiced rats exhibited only the azo derivatives of matography (15). bilirubin. In nonjaundiced For collection of bile, the rats were anesthetized with free, nonconjugated ether, the peritoneal cavity was opened by a midline in- litter mates and in normal control rats the plasma cision, and the common bile duct was ligated in its distal was colorless and bilirubin azo derivatives were portion. The dilated proximal part of the duct was then not demonstrable on paper chromatography. Bili- intubated with a polyethylene tube (outside diameter, rubin could not be detected in the urine of any of 0.024 inch), which was anchored with a silk ligature. Bile flow at the rate of 0.8 to 1.0 ml. per hour was main- the three groups of rats. tained by subcutaneous injection of 1.0 ml. normal saline In the bile, the bilirubin concentration of five per hour per 100 Gm. body weight. Aliquots of the bile jaundiced rats was 3.32 mg. per cent (S.D., 1.04 were removed at different time intervals for estimation of mg. per cent). On paper chromatographic analy- bilirubin concentration (14) and for paper chromato- sis, azo derivatives of bilirubin glucuronide could graphic analysis of the bilirubin azo derivatives (15). not be demonstrated, but there were small amounts Crystalline bilirubin (Armour and Company) was dis- solved in 0.2 ml. of 2 per cent sodium carbonate, con- of other azo derivatives, which exhibited Rf's simi- lar to that of the azo derivatives of crystalline bili- TABLE II rubin. In addition, these bile specimens contained Fecal urobilinogen excretion in Wistar rats some yellow pigments which gave a negative van den Bergh reaction. In normal control rats and Urobilinogen in nonjaundiced litter mates, virtually all of the Rats No. Mean S.D. pigment in the bile was present as bilirubin glu- mg./day/100 Gm. curonide. body weight As shown in Table II, fecal urobilinogen excre- Jaundiced 3 0.009 0.004 Nonjaundiced tion was much smaller in jaundiced rats than in litter mates 3 0.039 0.013 nonjaundiced animals. Patency of the biliary ex- Normal controls 4 0.077 0.024 cretory apparatus was demonstrated by the ob- CONGENITAL DEFECT IN BILIRUBIN GLUCURONIDE FORMATION 1125 TABLE III Urinary excretion of glucuronic acid after administration of o-aminobenzoic acid and of menthol to Wistar rats Glucuronic acid excreted in % of dose administered* Dose given Mode of ad- Rats No.