Sulfate and Glucuronide Conjugates of Bilirubin in Experimental Liver Injury

SULFATE AND GLUCURONIDE CONJUGATES OF BILIRUBIN IN EXPERIMENTAL LIVER INJURY

Leslie J. Schoenfield, … , Jesse L. Bollman, Harry N. Hoffman II

J Clin Invest. 1962;41(1):133-140. https://doi.org/10.1172/JCI104455.

Research Article

Find the latest version: https://jci.me/104455/pdf Journal of Clinical Investigation Vol. 41, No. 1, 1962 SULFATE AND GLUCURONIDE CONJUGATES OF BILIRUBIN IN EXPERIMENTAL LIVER INJURY* By LESLIE J. SCHOENFIELD, JESSE L. BOLLMAN AND HARRY N. HOFFMAN, II (From the Mayo Clinic and Mayo Foundation, Rochester, Minn.) (Submitted for publication June 1, 1961; accepted September 7, 1961) Free, water-insoluble, indirect-reacting bilirubin 4-0 braided silk and the duct was cut between the ties is conjugated prior to excretion, predominantly for studies of short (acute) obstruction (24 hours) and of prolonged obstruction (6 to 10 days). In one group as an ester monoglucuronide and a diglucuronide, acute obstruction was accomplished after exposure to forming the water-soluble, direct-reacting pigments CCl4. This was done to study the added effect of car- I and II, respectively (1-6). The conjugation of bon tetrachloride injury on the plasma pigments inasmuch bilirubin with glucuronic acid, in the presence of as the exposure alone did not produce jaundiced plasma. uridine diphosphate glucuronic acid which serves Rat liver perfusion preparations (15) were utilized in the study of pigments in bile produced by the isolated as a glucuronyl donor, is brought about by the en- liver in response to acute CC14 injury. zyme, glucuronyl transferase. It has been shown After bile fistulas were prepared in the control groups that conjugated bilirubin also contains nonglu- and in the other groups in which CC14 was given later, curonide conjugates (7-13). 100 tec inorganic radioactive sulfate (S3O,) in normal Isselbacher and McCarthy (12), using S35-la- saline solution was injected intraperitoneally or into the isolated liver system. Bile was then collected for 5 hours beled sulfate, identified bilirubin sulfate chromato- on ice and in the dark. For study of the plasma pigments graphically and radioautographically in the bile the radioactive-labeled sulfate was administered immedi- of rats, cats, and humans and in the serum and ately after obstruction in the acute experiments and 24 urine of rats with ligated bile ducts. They ob- hours prior to the time that the animals were killed in served that the enzymatic biosynthesis of bilirubin chronic-obstruction experiments. sulfate in vitro involves active sulfate and requires Partition of the pigments. The proteins were precipi- tated from plasma with saturated (NH,) SO4 and ethanol adenosine triphosphate. These same authors spec- and from bile with ethanol alone. Siliconized kieselguhr ulated that the proportions of glucuronide and sul- was prepared according to the method of Howard and fate conjugates might be altered in certain disease Martin (16). Reversed-phase partition chromatography states. of the plasma and bile pigments was carried out by the was to method of Cole, Lathe and Billing (2), using the butanol- The present study designed investigate phosphate buffer, pH 6, solvent system for separation of the relative proportions of glucuronide and sul- the three pigments. The pigment bands were washed fate conjugates of bilirubin of laboratory animals from the kieselguhr with 5 ml of diazotized sulfanilic acid after hepatic injury. We also examined the rela- and separated by filtration. The relative amounts of pig- tive proportions of pigments I and II under these ments I and II were estimated by the 30-minute same conditions (14). direct van den Bergh reaction; the calculations were cor- rected for volume changes (17). When the diazotized pigments 1 from columns were dried and placed on paper MATERIALS AND METHODS for chromatography, several compounds were obtained Experimental conditions. Male Wistar rats, weighing from each, some of which coincided with the radioac- 250 to 300 g, were used. Each experimental group con- tivity. In order to purify the products the following tained 6 animals except those groups used for study of procedures taken from Schachter (19) were employed. bile from the isolated each of which contained 4 The azopigments from the columns were evaporated liver, 1.5 M animals. To accomplish acute injury, animals were ex- in vacuo at 300 C to a volume of 1.5 ml; 0.5 ml of posed in a closed chamber to vapors of carbon tetrachlo- glycine buffer, pH 2.0, was added, and the pigments were ride of constant concentration (25 mg CC1, per 50 L air) extracted to 8 ml of butanol saturated with glycine buf- for 7 Bile fistulas were fer. The azopigments were then placed in 4.5 ml of 2 M hours. prepared by cannulating 12 the common bile ducts with polyethylene catheters. The acetate buffer, pH 3, and ml of chloroform saturated common bile duct was ligated securely with two ties of 1 On diazotization bilirubin is split at the methene * Abridgment of thesis submitted by Dr. Schoenfield to bridge so that 1 mole of free bilirubin yields 2 moles of the Faculty of the Graduate School of the University of azopigment A; 1 mole of pigment I yields 1 mole of Minnesota in partial fulfillment of the requirements for azopigment A and 1 mole of azopigment B; 1 mole of the degree of Doctor of Philosophy in Medicine. pigment II yields 2 moles of azopigment B (18). 133 134 SCHOENFIELD, BOLLMAN AND HOFFMAN with acetate buffer. After shaking and centrifugation, making radioautographs, was directly proportional to the acetate layer containing azopigment B was evaporated the amount of radioactivity present. The area was to dryness. The azopigment A in the chloroform layer calculated for the entire strip and for that portion where was concentrated by transferring to 4.0 ml 0.3 N NaOH radioactivity coincided precisely with the azopigment and 16 ml petroleum ether. The NaOH layer containing band. In this manner the bilirubin sulfate could be re- azopigment A was shaken and centrifuged, then taken to lated to the total amount of sulfate as well as to the total dryness. The dried azopigments were dissolved in 50 to conjugated azopigment in the band. 100 gl of normal acetic acid in preparation for paper Radioautographs were made from the sheets on No- chromatography. Screen X-ray film after a 48-hour exposure and from the Paper chromatography. Ascending and two-dimen- strips on Eastman Linagraph ortho film after a 24-hour sional chromatography on Whatman no. 1 paper was exposure. carried out with two different solvent systems: 1) Inasmuch as another unidentified sulfate compound n-propionic acid, methyl ethyl ketone, and water (25: 75: (not bilirubin) was situated on the paper quite near the 30) (12), and 2) pyridine, ethyl acetate, and water (1: bilirubin, a perpendicular line was drawn between the 2:1) (20). Ten by 12-inch sheets were used for the two peaks on the curve of radioactivity in order to ap- two-dimensional paper chromatography and 1.5 by 22- proximate the area for the bilirubin sulfate. The azo- inch strips for the ascending paper chromatography. pigment bands were cut from the strips precisely, and the Since the amounts of pigment had been estimated after pigments were eluted from the paper with 0.05 N HCl. column chromatography, approximately equal quantities The optical density at 560 my was determined in a spec- of azopigment could be placed on each of the strips. Thus, trophotometer and compared with a standard curve 50 to 100 Al was generally used, depending on the con- prepared with crystalline bilirubin. The eluted pigment centration of the pigment. This served as a reference was rechromatographed with one or the other solvent for the estimation of the S35 incorporated as bilirubin sul- system. The radioactivity remained with the pigment fate in the various experiments. The chromatographic band when the chromatogram was scanned and radioau- systems were developed overnight, generally about 16 to tographs were made. 20 hours. As shown by Isselbacher and McCarthy (12), the The paper strips were then placed on a chromatogram eluted, labeled azopigment could not be hydrolyzed by a scanner, and a radioactivity curve was obtained. With the commercial arysulfatase (Mylase-P). Hydrolysis of use of a compensating polar planimeter, the amount of bilirubin sulfate could be carried out, however, in normal radioactivity present on the strips could be estimated by HCl at 1000 C for 90 minutes. The bilirubin glucuronide measuring the area under the curve of radioactivity. was hydrolyzed with 50 mg of bacterial j#-glucuronidase Preliminary work had demonstrated that the area under at 370 C for 24 hours at pH 6.2 with acetate buffer. both sharply peaked and broad curves, produced by Molar ratio of glucuronide to bilirubin in pigment II. placing known amounts of radioactivity on paper and This was studied in rats with bile fistulas and in isolated perfused rat livers before and after exposure to TABLE I CCl1. Simultaneous kieselguhr columns with 1 ml of deproteinized bile were used; one column was for esti- Reversed-phase partition chromatography of pigments I and II in bile and plasma of mation of the bilirubin content of the pigment II band as the rat after liver injury previously described, the other for the duplicate quan- titation of glucuronides in pigment II by the method of Per cent of conjugated pigment* Fishman and Green (21). The molar ratio of glucuronide Pigment I Pigment II to bilirubin in the pigment II band was calculated. Experiments Avg. Range Avg. Range RESULTS Bile Normal animals 14 9-18 86 82-91 Partition of pigments I and II in experimental After CCl4 exposuret 15 10-20 85 80-90 liver injury. The results of partition and esti- Plasma mation of pigments I and II as obtained from re- After obstruction of versed-phase column chromatography of bile or bile duct for 24 hours 33 29-38 67 62-71 plasma from six animals in each of the various After CCl4 exposure; experimental situations are shown in Table I. then obstruction The pigment patterns in the bile of the normal rat for 24 hours 58 49-68 42 32-51 and in the rat after exposure to CCl4 did not dif- After obstruction of bile duct for 6 to fer significantly. When the plasma pigments were 10 days 54 48-60 46 40-52 studied 24 hours after ligation of the common bile duct, pigment II predominated, but when acute * Six animals in each category. t Seven-hour exposure to 25 mg CCl4 per 50 L air. obstruction followed exposure to CC14 the per- CONJUGATES OF BILIRUBIN IN EXPERIMENTAL LIVER INJURY 135

FIG. 1. CHROMATOGRAPHIC AND RADIOAUTOGRAPHIC RESULTS OF f8-GLUCURONIDASE TREATMENT OF AZOPIGMENT CONJUGATED WITH SULFATE AND GLUCURONIDE FROM PIGMENT II OF RAT BILE AFTER EXPOSURE TO 25 MG OF CAR- BON TETRACHLORIDE PER 50 L AIR FOR 7 HOURS AND INJECTION OF S'O4. The solvent system used was n-propionic acid, methyl ethyl ketone, water (25: 75: 30). A is the unincubated control; B, its radioautograph. C shows the pigments after incubation with bacterial p-glucuronidase as described in the text; D, its radioautograph. It will be noted that a significant portion of the azopigment resists enzymatic hydrolysis and demon- strates radioactivity. By contrast, in normal bile, E, the hydrolyzed azopigment is the more conspicuous. centage of pigment I in the plasma increased and ment I. Rechromatography after elution of the usually slightly exceeded that of pigment II. The pigments did not alter their rate of movement on pigment pattern after prolonged obstruction did the paper. When the conjugated dipyrrol was not differ from that following combined CCI4 in- eluted from the paper chromatograms, incubated jury and acute obstruction. with ,/-glucuronidase and then rechromatographed, Pigment I. When the pigment I band from the all of the pigment migrated with an Rf of 0.5. kieselguhr column was diazotized and purified, Bilirubin sulfate in pigment II of bile and plasma. two pigments (A and B) resulted that could be When the pigment II band from the column was developed on paper chromatography with either diazotized, purified, and developed on paper chro- of the solvent systems. One pigment band mi- matography with either of the solvent systems, a grated with an Rf of about 0.5, which corresponds single band of azopigment with an Rf of about 0.3 to diazotized free dipyrrol; the other migrated with resulted. When this was rechromatographed, an Rf of about 0.3, corresponding to diazotized eluted from the paper, incubated with ,8-glucuroni- conjugated dipyrrol. No radioactivity was as- dase, and rechromatographed, two bands of azo- sociated with these azopigment bands from pig- pigment resulted. One pigment band migrated, 136 SCHOENFIELD, BOLLMAN AND HOFFMAN

with an Rf of 0.5 and was not radioactive. The Experiments with isolated liver (Table III) other migrated at an Rf of 0.3 and was radioac- showed a similar increase after CCI4 injury. The tive. When rats had been exposed to CCl4 the correlation of bilirubin-S3504 with the amount of azopigment band resistant to enzymatic hydrolysis nonbilirubin-S3504 may be open to question, al- with 83-glucuronidase was more significant than though the alignment of the calculations in Tables in normal controls, where the free azopigment band II and III gives credence to the method. It ap- was the more prominent (Figure 1). pears that the less S3504 available, the less is con- The work with two-dimensional chromatograms jugated with bilirubin. When strips with approx- likewise indicated a relative increase in the amount imately the same amount of total radioactivity are of bilirubin conjugated with sulfate after exposure compared, the differences in S35-bilirubin are sig- to CC14 as compared with the normal controls nificant. Omission of the two high values (nor- (Figure 2). mal rats, Table II) leaves all six CCl4-treated rats The results obtained by relating the area of ra- with higher S35-bilirubin values than have the other dioactivity associated with the azopigment to the four normal rats. total radioactivity on the strip are shown in Table The percentage of bilirubin sulfate in the total II. The amount of azopigment was constant in radioactive S3504 in the plasma after obstruction each experiment. After CCI4 injury the percentage of the common bile duct is given in Table III. of bilirubin sulfate in the total radioactivity in- Thus, the same amount of bilirubin in bile from creased. An average of 17.8 per cent was found control animals and normal isolated livers and in as compared with 12.6 per cent for the controls. the plasma after acute obstruction of the common

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- P-EA P-EA I FIG. 2. RADIOAUTOGRAPHY OF TWO-DIMENSIONAL CHROMATOGRAMS OF RAT BILE AZOPIGMENT AFTER INJECTION OF S5'O4. The azopigment was prepared from pigment II as described in the text. The chromatograms have been trimmed to contain the radioactivity and show the start (X) and the directions for the solvent development. The azopigment is outlined on the chromatogram. Both X-ray papers were in contact with the chromatogram for 48 hours. A. This is from the bile of normal rats. B. This is from rat bile after 7-hour exposure to CCL vapors (25 mg CCO4 per 50 L air). Note that with the same amount of azopigment and total radioactivity, there is a greater amount of radio- .activity associated with the azopigment after CC14. CONJUGATES OF BILIRUBIN IN EXPERIMENTAL LIVER INJURY 137 bile duct is conjugated with an essentially con- TABLE III stant percentage of total S3504. The correspond- Bilirubin sulfate (S3504) in pigment II in bile and plasma after liver injury and injection of radioactive sulfate; ing values increased significantly with CO4 injury isolated liver preparations and intact animals and after prolonged obstruction. Molar ratio of glutcuronide to bilirubin. The Bilirubin sulfate (S504), per cent of molar ratio of glucuronide to bilirubin in pigment total radioactivity * No. of II obtained from the bile of normal controls aver- Experiments animals Avg. Range aged 1.74. A decrease in the ratio to 1.17 was Bile, isolated liver preparation TABLE II Normal animals 4 12.2 11.4-13.4 Bilirubin sulfate (S3504) in pigment II in bile of the rat after After CC14 exposuret 4 17.4 17.0-18.6 exposure to carbon tetrachloride and injection of radioactive sulfate Plasma, intact animal After bile duct ob- Area of Bilirubin sulfate total (S304) struction for 24 radio- hours 6 13.3 11.7-13.9 active Total sulfate radio- After CC14 exposuret; (S3504) activity then bile duct ob- of strip Areat in strip struction for 24 sq cm sq cm % hours 6 19.8 19.2-20.4 Normal controls 26.1 3.2 12.3 After bile duct ob- 18.1 2.4 13.3 struction, 6 to 10 38.0 4.9 12.9 days 6 18.5 17.4-19.3 17.4 2.2 12.6 32.2 4.0 12.4 24.9 3.0 12.0 * The purified azopigment was developed on paper chromatography using n-propionic acid, methyl ethyl Average 26.1 3.3 12.6 ketone, and water (25:75:30). The amount of azopigment was constant in each of the experiments. The per cent After CC14* exposure 22.1 4.0 18.1 bilirubin sulfate of total radioactivity was calculated as 19.6 3.7 18.8 indicated in Table II. 24.3 4.2 17.3 t Seven-hour exposure to 25 mg CC14 in 50 L air. 18.9 3.3 17.5 23.2 4.0 17.2 24.0 4.3 17.9 fer from normal (14). It may be that the damaged Average 22.0 3.9 17.8 cells are unable to excrete conjugated pigment I into the bile. Two of us (L.J.S. and J.L.B.) and * Exposure to 25 mg CC14 in 50 L air for 7 hours. The others (22) have identified extrahepatic pigment purified azopigment was developed on paper chromatography using n-propionic acid, methyl ethyl ketone, and water (25:75: 30). Each strip contained the same amount TABLE IV of pigment. The strips were placed on a chromatogram scanner and the areas under the curve of radioactivity Molar ratio of glucuronide to bilirubin in pigment II * in were measured with a planimeter. bile of the rat after exposure to carbon tetrachloride tThe per cent error for repeated measurements of areas of 20 sq cm was 1 per cent. areas about less than Smaller Molar were brought to at least this dimension by recircumscribing ratio, the areas. The figures shown are the averages for three glucu- determinations. ronide/ Fistula bile Bilirubin Glucuronide bilirubin found after exposure to CO4 (Table IV). Similar mg/ 1O-7 mg/ 10-7 100 ml moles 100 ml moles results were obtained in four experiments using Normal controls 2.4 419 1.5 773 1.84 bile from the isolated liver. 2.0 349 1.2 618 1.77 2.8 489 1.6 824 1.69 3.2 559 1.8 927 1.66 COMMENT Average 2.6 454 1.5 786 1.74 After CC14 exposuret 3.0 524 1.2 618 1.18 The percentage of pigment I in bile of the 2.6 454 1.1 567 1.25 3.2 559 1.3 670 1.20 normal rat did not differ from that after CC14 ex- 3.4 594 1.2 618 1.04 posure. This is compatible with the report of Average 3.6 533 1.2 618 1.17 Hoffman, Whitcomb, Butt and Bollman who noted * Pigment II was obtained by reversed-phase partition chromatog- that the bile pigment pattern in duodenal drainage raphy using simultaneous columns for the determinations of bilirubin and glucuronide. of 12 patients with infectious hepatitis did not dif- t Seven-hour exposure to 25 mg carbon tetrachloride in 50 L air. 138 SCHOENFIELD, BOLLMAN AND HOFFMAN I and demonstrated its conversion to pigment II accounts for the similar results with pigment par- by isolated liver of the normal rat. Whether pig- titions in the two experimental conditions. Issel- ment I also is formed in the liver as an intermedi- bacher and McCarthy (29) observed a quantitative ate in the production of pigment II is not known. decrease in the liver cell microsomes isolated by Pigment I has not been found in bile from the iso- ultracentrifugation to account for the decreased lated rat liver. The significance of this is being hepatic glucuronide-conjugating capacity after in- studied further. traperitoneally injected CC14. This decreased ac- As Eberlein (23) pointed out, partition meth- tivity of glucuronyl transferase is in accord with ods supply more physiologic information than does our in vivo demonstration of a decrease in the the diazotization reaction. However, they do not molar ratio of glucuronide to bilirubin in pigment permit differentiation of primary parenchymal II of rat bile after acute exposure to CCl4. These hepatic disease from obstructive jaundice. Study experiments also afford evidence that nonglucuro- of the pigment patterns in plasma by column nide conjugation of bilirubin must have been in- chromatography did not provide a means of distin- creased. The work with paper chromatography guishing acute CCO4 injury followed by acute ob- showed that, relative to a constant amount of struction from prolonged obstruction. This prob- conjugated bilirubin and to the total output of ably results from the inevitable secondary pa- S3504 in the bile, the bilirubin sulfate (S35) in pig- renchymal damage of long-standing biliary ment II of rat bile increased after exposure to obstruction. CCl4 for 7 hours and increased equally after pro- No S3504-bilirubin was associated with pigment longed obstruction of the common bile duct. It I. All of the conjugated azopigment from pigment may be that other nonglucuronide conjugates are I was hydrolyzed by 3-glucuronidase to the free increased under these conditions. dipyrrol. It is possible, however, that more sen- Schachter's studies (19), showing a consider- sitive methods might reveal a small amount of sul- ably greater renal clearance of the diglucuronide fate or other conjugates of bilirubin in pigment I. than of the monoglucuronide, suggest that the rela- It has not been determined whether bilirubin tive proportions of pigments I and II in plasma sulfate exists as a monosulfate or disulfate or, in- may be related to differing renal thresholds for deed, whether a single bilirubin molecule may be these substances. That the isolated perfused rat conjugated with both sulfate and glucuronic acid. liver preparation did form less glucuronide and Hydrolysis of azopigment B (containing both sul- relatively more sulfate conjugate of bilirubin after fate andglucuronide conjugates) byfi-glucuronidase exposure to CC14 indicates that, under these con- indicated that at least some of the glucuronide ex- ditions, the differences obviously could not have ists without sulfate on the same molecule. Animal been functions of the renal thresholds. species differ greatly with regard to the extent of glucuronic acid conjugation and the relative SUMMARY AND CONCLUSIONS amounts of glucuronide and sulfate conjugates ex- Rats, including those with bile fistulas and iso- creted after administration of the same compound lated perfused liver systems, were utilized for the (24). study of sulfate and glucuronide conjugates of In vitro studies by Chojecki and Kern (25) bilirubin. The experimental conditions entailed showed a marked decrease of glucuronyl trans- exposure to carbon tetrachloride and ligation of ferase activity in rat liver homogenates 24 hours the common bile duct for short and long periods after intraperitoneal administration of CCl4. The followed by intraperitoneal injection of inorganic endoplasmic reticulum, from whence the microso- S3504. Pigments I and II of bile or plasma were mal fraction of ultracentrifugation is largely de- separated and estimated by the technic of reversed- rived (26), is apparently the earliest subcellular phase partition chromatography. The pigments structure to be pathologically altered in CCI4 were diazotized, purified further by chemical par- poisoning (27). It is likely that similar secondary tition, and placed on paper chromatographic sys- cytoplasmic organelle damage occurs with pro- tems for radioautography and analysis. longed biliary obstruction (28). This probably With acute obstruction to bile flow, pigment II CONJUGATES OF BILIRUBIN IN EXPERIMENTAL LIVER INJURY 139 was the predominant conjugated pigment in the 7. Arias, I. M. Recent advances in the metabolism of plasma. With added CC14 injury or with pro- bilirubin and their clinical implications. Med. Clin. N. Amer. 1960, 44, 607. longed biliary obstruction, the proportion of pig- 8. Jirsa, M., Vecerek, B., and Ledvina, M. Di- and ment I in the plasma increased. mono-taurobilirubin similar to a directly reacting Sulfate conjugate of bilirubin was not associ- form of bilirubin in serum. Nature (Lond.) 1956, ated with pigment I, but was found with the glu- 177, 895. curonide in pigment II. Incubation with fl-glu- 9. Billing, B. H. Bile pigments in jaundice in Advances in Clinical Chemistry, H. Sobotka and C. P. curonidase permitted the separation of glucuronide Stewart, Eds. New York, Academic Press, 1959, from sulfate when chromatographed on paper. vol. 2, pp. 267-299. After ligation of the common bile duct for a 10. Billing, B. H., Cole, P. G., and Lathe, G. H. The short period (24 hours), approximately the same excretion of bilirubin as a diglucuronide giving proportion of bilirubin sulfate appeared in the pig- the direct van den Bergh reaction. Biochem. J. II jaundiced plasma as is 1957, 65, 774. ment fraction of the 11. Watson, C. J. Color reaction of bilirubin with sul- ordinarily present in normal bile. With added furic acid: A direct diazo-reacting bilirubin sul- CC14 exposure or after prolonged obstruction, the fate. Science 1958, 128, 142. relative amount of serum bilirubin conjugated as 12. Isselbacher, K. J., and McCarthy, E. A. Studies on a sulfate increased. Carbon tetrachloride exposure bilirubin sulfate and other nonglucuronide con- also a increase in the sulfate conjugates of bilirubin. J. clin. Invest. 1959, 38, 645. caused relative 13. Bostr6m, H., and Vestermark, A. Ester sulphate jugate of bilirubin in the bile. 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Malloy, H. T., and Evelyn, K. A. The determination and glucuronide conjugates in response to CCl4 of bilirubin with the photoelectric colorimeter. injury are not functions of differing renal thresh- J. biol. Chem. 1937, 119, 481. olds for these substances. 18. Overbeek, J. T. G., Vink, C. L. J., and Deenstra, H. Kinetics of the formation of azobilirubin. Rec. Trav. chim. Pays-Bas 1955, 74, 85. REFERENCES 19. Schachter, D. Estimation of bilirubin mono- and di- 1. Cole, P. G., and Lathe, G. H. The separation of glucuronide in the plasma and urine of patients serum pigments giving the direct and indirect van with nonhemolytic jaundice. J. Lab. clin. Med. den Bergh reaction. J. clin. Path. 1953, 6, 99. 1959, 53, 557. 2. Cole, P. G., Lathe, G. H., and Billing, B. H. Separa- 20. Schmid, R. The identification of "direct-reacting" tion of bile pigments of serum, bile and urine. bilirubin as bilirubin glucuronide. J. biol. Chem. Biochem. J. 1954, 57, 514. 1957, 229, 881. 3. Billing, B. 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