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The Formation of Bilirubin from Hemoglobin in Vivo

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The Formation of Bilirubin from Hemoglobin in Vivo THE FORMATION OF BILIRUBIN FROM HEMOGLOBIN IN VIVO J. Donald Ostrow, … , James H. Jandi, Rudi Schmid J Clin Invest. 1962;41(8):1628-1637. https://doi.org/10.1172/JCI104620. Research Article Find the latest version: https://jci.me/104620/pdf Journal of Clinical Investigation Vol. 41, No. 8, 1962 THE FORMATION OF BILIRUBIN FROM HEMOGLOBIN IN VIVO * By J. DONALD OSTROW,t JAMES H. JANDL AND RUDI SCHMID (From the Thorndikc Memorial Laboratory and the Second and Fourth [Harvard] Medical Services, Boston City Hospital, and the Departmcntt of Medicine, Harvard Medical School, Boston, Mass.) (Submitted for publication March 7, 1962; accepted March 30, 1962) About 80 to 85 per cent of the fecal bile pig- hepatic circulation of the bilirubin contained in mients are believed to be derived from the hemo- the administered bile (8). globin of senescent erythrocytes that are destroyed In the present investigation, these difficulties in the reticuloendothelial system (1). It is not have been surmounted by injecting C"4-labeled, known, however, whether the hemoglobin of se- antibody-sensitized red cells into rats with an ex- questered red cells is converted quantitatively to ternal bile fistula. This permitted both determina- bilirubin and eventually to urobilinogen, or is in tion of the excretory rate of bilirubin-C14 in the part metabolized to other products (2, 3). bile and estimation of the efficiency of conversion Earlier studies of this problem in man have re- of the injected hemoglobin to bilirubin, without vealed that the rate of fecal urobilinogen excretion regard for variations in endogenous bilirubin ex- falls short of the expected output as calculated cretion. Since the injected red cells were also la- from total red cell volume and mean erythrocyte beled with y-emitting Fe"s or Cr51, it was possible life span (4-6). This has been attributed to to follow their rate of removal from the circulation, incomplete intestinal conversion of bilirubin to and to determine the interval between sequestration urobilinogen (3, 4, 6, 7), destruction of urobili- of the cells and excretion of the resulting bilirubin- nogen in the large bowel (3, 4, 6), and loss of pig- C14 in the bile. ment through intestinal absorption (3, 4, 6, 8). At- Similar experiments were carried out with in- tempts to circumvent these difficulties by estimat- travenously injected solutions of hemoglobin la- ing bilirubin excretion through a T-tube in the beled with C14 and either Fe59 or Cr51. Urinary common bile duct have been unsatisfactory (9, loss of pigment was minimized or prevented by ad- 10) because this technique does not permit com- ministering only small amounts of hemoglobin or, plete bile collection. In animals with an external in some instances, by nephrectomizing the rats biliary fistula, this obstacle has been overcome, but prior to the experiment. the rate of endogenous bilirubin excretion was found to exhibit marked fluctuations (11, 12), MATERIALS AND METHODS which rendered difficult comparison of the ob- Preparation of red cells and hemioglobin labeled with served values with the endogenous production of C" and Fe59. Male Sprague-Dawley rats weighing ap- bilirubin calculated from hemoglobin turnover, or proximately 300 g were given, at 48-hour intervals, three with excess bilirubin formation after intravenous subcutaneous injections of 20 mg phenylhydrazine hydro- chloride in neutralized 2 per cent aqueous solution. Be- injection of a hemoglobin load. In animals with ginning three days after the last phenylhydrazine injec- chronic bile fistulae, these acute fluctuations in pig- tion, 0.2 mc glycine-2-C" was administered subcutaneously ment output tend to balance out, but the necessity twice daily to a total dose of 2.0 mc.1 Over the same of giving oral bile supplements to replace drainage period, a total dose of 100 Asc transferrin-bound Fe"Cl losses (11, 13) results in falsely high pigment ex- (14) was injected into a tail vein in three to five separate daily doses.2 Two weeks after the last isotope adminis- cretion due to intestinal absorption and entero- tration, the rats were exsanguinated with heparinized syringes, and the red cells were separated and washed * in Health Supported part by United States Public with cold isotonic saline. Service Grants A-1833 and RG-3507. This work was presented in part before the American Society for Clini- 1 Specific activity of glycine-2-C04, 17 to 20 mc per cal Investigation, Atlantic City, N. J., April 30, 1961. mmole; New England Nuclear Corp., Boston, Mass. t Postdoctoral Research Fellow, National Institutes of 2 Specific activity of Fe"Cl,, 3 to 10 ,tc per leg; Abbott Health, Bethesda, Md. Laboratories, Chicago, Ill. 1628 FORMATION OF BILIRUBIN FROM HEMOGLOBIN 1629 An aliquot of these red cells was sensitized in vitro or by incubation with heated rabbit serum containing "in- hemin (dpm perj~g) X 0.976 = bilirubini (dpm per Mg). complete" antibodies against rat erythrocytes (15) Af- ter these sensitized red cells were washed and resuspended Clearance of injectted labeled red cells or hemizoglobinl in isotonic saline, they showed slight agglutination in front the circilationi. In 16 male Sprague-Dawley rats, saline, but strong agglutination in an appropriate Coombs weighing 450 to 600 g, an external bile fistula was pro- serum (14) and in polyvinylpyrrolidone (16). On incu- duced (20) and the animals were then placed in loose re- bation for 2 hours at 370 C in a large volume of fresh straining cages. Bile flow was maintained in excess of rat serum, no hemolysis occurred. 0.7 ml per hour by appropriate hydration (21). After Solutions of labeled hemoglobin were prepared by collecting bile for 3 hours, the rats were briefly anesthe- lysing an aliquot of donor red cells in cold distilled wa- tized with ether and 1.6 to 2.0 ml of a suspension of the ter. After restoring isotonicity with 5 per cent saline, labeled red cells, or of the hemoglobin solution, xvas in- the stroma was removed by centrifugation in the cold for jected into the tail vein. From the total amount of he- 1 hour at 105,000 G. The clear supernatant was then ad- moglobin administered (Table I), the expected quantity justed to the desired hemoglobin concentration and to pH of bilirubin was calculated from the conversion ratio 1 g 7.8 by addition of appropriate volumes of isotonic saline hemoglobin = 34.5 mg bilirubin (22). and 0.15 M phosphate buffer, pH 8.1. The slightly alka- The rate of removal of the administered red cells or line pH was employed to obviate the slow, spontaneous hemoglobin from the circulation was determined by precipitation that occurred with rat hemoglobin in aque- following the gamma activity of serial blood samples col- ous solution. lected by incision of the tail. One-tenth ml blood was Isolation and radioassay of labeled heinin. From a pipetted into 1 ml water for determination of whole blood third aliquot of the C"-, Fe'-labeled red cells, hemin was radioactivity, and into 1 ml saline for measurement of isolated and crystallized (17). After recrystallization plasma radioactivity after removal of red cells by centrif- (18), the hemin was dissolved in pyridine and the con- ugation (15). The initial concentration of the inj ected in on centration estimated spectrophotometrically, with a stand- material the blood was estimated the basis of the predicted blood volume, which for male rats of this size ard curve prepared by dissolving weighed amounts of hemin in similar fashion. The pyridine solutions were was assumed to be 4.5 per cent of body weight (15). The rats were killed by cervical dislocation 22 hours then diluted with 1 M Hyamine-methanol 3 to yield a after the injection of the labeled heme pigment. The liver, final concentration of 10 Ag labeled hemin per ml. For 1 to 4 labeled hemin and 20 unlabeled car- spleen, kidneys, lungs, and right femur were removed, counting, Ag fig 5' rier hemin, dissolved in a total of 1 ml of 1 M Hyamine- blotted with damp gauze, weighed, and the Fe activity determined on specimens not exceeding 2 g in weight methanol, were mixed with 17 ml scintillator solution 4 (14). Total Fe" in the marrow was roughly estimated in a 20 ml low potassium glass counting vial.5 The vials by multiplying the femur radioactivity by 14.4 (23 ). were counted in a scintillation liquid spectrometers using Urine and bile Fe"' or Cr5' radioactivity were determined as the internal standard.7 After 6 to 8 toluene-C" months, on the total 22-hour specimens. had and the ac- most of the Fe" activity decayed, specific Because of the rapid reutilization and redistribution of of hemin a constant value attributable tivity the approached iron from the metabolized hemoglobin (24, 25), the or- to Absence of detectable emission due solely C". gamma gan content of Fe"' after 22 hours is not a valid meas- to remaining Fe"' was further ascertained in a well-type ure of the initial tissue distribution of injected heme. scintillation counter. Allowing for the difference in mo- Therefore six of the animals (Table I) were injected with lecular weights and for the loss of a labeled methene-bridge sensitized red cells or hemoglobin solution labeled with carbon atom (19), the C"-specific activity of the biliru- Cr5, which is released more slowly from the tissues (25, bin derived from the injected hemoglobin-C' was calcu- 26). C`-labeled red cells, prepared as described above, lated as follows: were labeled imt vitro with Na..Cr5"04 (15) prior to in- cubation with antiserum.9 Hemoglobin was prepared sp. act. of hemin X 584.7 X 7 = sp.
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