Comp. Biochem. Physiol., 1972, VoL 43113, pp. 929 to 933. Pergamon Press. Printed in Great Britain

BILIRUBIN URIDYLDIPHOSPHO-GLUCURONYL TRANSFERASE AND /~-GLUCURONIDASE ACTIVITY IN TISSUES OF HORSE (EQUUS CABALLUS) AND SHEEP

(OVIS ARIES)*

M. SAWKAT ANWER, ABDUS S. MIA t and RONALD G R O N W A L L

Department of Physiological Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66502

(Received 11 April 1972)

Abstract--1. Normal activities of bilirubin uridyldiphospho-glucuronyl trans- ferase (BUDPGT) (E.C. 2.4.1.17) and fl-glucuronidase (E.C. 3.2.1.31) were determined.

2. BUDPGT activity was elevated in two mutant strains of sheep with hyperbilirubinemia, suggesting no casual relationship to the hyperbilirubinemia.

3. Elevated fl-glucuronidase activity in mutant Corriedale sheep may reflect lysosomal retention related to hepatic pigmentation.

4. Plasma fl-glucuronidase activity decreased exponentially with age in normal neonatal lambs.

INTRODUCTION

CONJOGATION of bilirubin with glucuronic acid is considered essential for bilirubin excretion into bile. The reaction is catalyzed by the enzyme bilirubin uridyldi- phospho-glucuronyl transferase (BUDPGT), and UDP-glucuronic acid serves as the glucuronyl donor. The enzyme fi-glucuronidase will catalyze the cleavage of glucuronic acid from bilirubin diglucuronide (Billing & Lathe, 1956; Schmid, 1956). Deficiency of B U D P G T has been reported in Crigler-Najjar syndrome in man (Szabo et al., 1962) and in Gunn rats (Carbone & Grodsky, 1957), both being inherited defects characterized by marked elevation of plasma unconjugated bilirubin.

B U D P G T deficiency has also been reported in patients with inherited un- conjugated hyperbilirubinemia, known as Gilbert's syndrome (Black & Billing, 1969). fl-Glucuronidase activity has been found to be altered in certain physio- logical and pathological conditions (Fishman, 1955; Acocella et al., 1968), but its activity in spontaneous disorders of bilirubin metabolism has not been reported. Studies of the distribution of both B U D P G T and fi-glucuronidase in different tissues have been limited to laboratory animals with no reports on activities of these enzymes in different tissues under normal conditions in sheep and horse.

* Supported in part by U.S. Public Health Service Grant No. AM11384. Contribution No. 99, Department of Physiological Sciences, Kansas Agricultural Experiment Station.

t Present address: Agricultural University, Mymensingh, Bangladesh.

929

930 M. SAVCKAT ANWER, ABDUS S. MIA AND RONALD GRONWALL

In this study, BUDPGT and fl-glucuronidase activities were determined in horses and in normal and mutant hyperbilirubinemic sheep. Horses normally have higher plasma bilirubin levels than most domestic mammals or man, with about 90 per cent unconjugated. Evidence for a reduced hepatic bilirubin transport capacity in the horse has been reported by Gronwall & Mia (1972). Sheep normally have little bilirubin in their plasma, but two strains with inherited deficiencies in hepatic bilirubin transport have marked hyperbilirubinemia. A strain of South- down sheep has decreased ability to remove bilirubin from plasma and in many respects resembles Gilbert's syndrome in man (Cornelius & Gronwall, 1968; Mia et al., 1970a). A strain of Corriedale sheep has a reduced bilirubin excretory capacity and retain a black pigment in their liver. Thus they functionally resemble men with Dubin-Johnson syndrome (Cornelius et al., 1965; Mia et al., 1970b). Because young lambs, like young infants, have elevated plasma bilirubin during neonatal development (Cornelius et al., 1968), they were included in this study.

MATERIALS AND METHODS BUDPGT and ~-glucuronidase activities were determined in liver obtained by needle

biopsy from Southdown and Corriedale mutant sheep and normal sheep of approximately the same age. BUDPGT and fl-glucuronidase activity in various tissues, freshly collected from three slaughtered horses and sheep also were determined. Levels of plasma 8- glucuronidase activity of young normal lambs were followed from birth to 90 days of age.

BUDPGT activity was determined at pH 7"4 and optimal UDP glucuronic acid con- centration by the method of Black & Billing (1969). ]~-Glucuronidase activity was de- termined at the optimal pH, 4"5, using phenolphthalein fl-monoglucuronic acid (Sigma Chemical Co., St. Louis, Missouri) as substrate by the method of Fishman et al. (1948). Plasma bilirubin was determined by the method of Malloy & Evelyn (1937) as modified by Gronwall & Mia (1972).

RESULTS

BUDPGT and fl-glucuronidase activity in various tissues in normal sheep and horses are shown in Table I. BUDPGT activity was measurable only in liver and gall bladder mucosa of sheep. This result contrasts with that from a study in rats in which Grodsky & Carbone (1957) also found considerable activity in kidney. Hepatic BUDPGT activity per g of wet liver weight was not significantly different between horses and sheep.

Among the tissues studied, fl-glucuronidase activity was highest in liver. It was lower in most horse tissues than in sheep tissues (per g wet weight).

fl-Glucuronidase and BUDPGT activities in needle biopsy samples from normal and mutant sheep are shown in Table 2. BUDPGT activity was sig- nificantly higher in both mutant sheep strains while fi-glucuronidase was higher only in Corriedale mutant sheep.

fl-Glucuronidase activity in plasma of neonatal Iambs decreased exponentially with age (Fig. I). No BUDPGT activity was detected in plasma.

B U D P G T AND fl-OLUCURONIDASE ACTIVITY IN HORSE AND SHEEP TISSUES

TABLE 1--TlSSU~ ~NZYME ACTIVITIES

931

B U D P G T activity (/zM/g per hr)* fl-Glucuronidase activity (/zM/g per hr ) t

Tissues Horse (3) Sheep (3) Horse (3) Sheep (3)

Liver 0.92+0"23/; 0"82_+0"155 12"0+1"55 38"2+2"25 Kidney cortex - - Neg § 5"93 _+ 0-28 9-9 _+ 1-2 Kidney medulla - - Neg 2-23 + 0"23 5"7 + 1 "2 Heart muscle - - Neg 0"34 _+ 0"076 1"09 _+ 0"08 Lung - - Neg 3"17 _+ 0"41 2"45 + 0"58 Duodenum - - Neg 3"78 -+ 0"28 4"17 + 0"39 Ileum - - Neg - - 4.79 + 0-26 Gall bladder - - 0"48 + 0"06 - - 2"60 + 0.40

*/zM of bilirubin conjugated/g wet tissue per hr. t F M of phenolphthalein produeed/g wet tissue per hr. $ Mean + S.E. § Neg--negligible activity detected.

TABLE 2--ENzYM~ ACTIVITIES IN LIVER BIOPSY SPECIMENS

BUDPGT activity fl-Glucuronidase activity Adult sheep (~M/g per hr)* OzM/g per hr ) t

Normal (8) 0"88 + 0"07 § 33"3 + 3"3 § Southdown mutant (3) 1"29 + 0"151: 45"8 + 11"7 Corriedale mutant (4) 1.36 + 0"155 56.3 + 8"35

* FM of bilirubin conjugated/g wet tissue per hr. t" FM of phenolphthalein produced/g wet tissue per hr. $ Significantly different from normal at 5 per cent level by t-test. § Mean + S.E.

125,

>, tO0 ~ x , X X > 75 x

5C x x o

x ~ x x x x Adult X X ~ - - X 25 level

x I X X X X ~X

l I I I I I I I xlx I 0 I0 20 30 40 50 60 70 80 90 I00

Age , days FIG. 1. Plasma fl-glucuronidase activity (m/zM phenolphthalein produced per hr per ml) vs. age in normal lambs (seven lambs with four to five samples per lamb).

932 M. SAWKAT ANWER, ABDUS S. MIA AND RONALD GRONWALL

DISCUSSION BUDPGT deficiency has been reported to cause hyperbilirubinemia in man

and Gunn rats (Carbone & Grodsky, 1957; Szabo et al., 1962). Black & Billing (1969) have indicated that factors other than decreased transferase activity must help determine the degree of hyperbilirubinemia in Gilbert's syndrome as they obtained a negative correlation coefficient of 0.44 between transferase activity and serum unconjugated bilirubin in patients with that condition. The significantly higher level of BUDPGT in both sheep mutants with hyperbilirubinemia suggests that transferase activity was not the limiting factor causing hyperbilirubinemia.

Deconjugation of bilirubin by lysosomal fl-glucuronidase has been suggested in obstructive jaundice associated with increased plasma concentration of unconju- gated, as well as conjugated, bilirubin (Acocella et al., 1968). Intrahepatic de- conjugation of bilirubin diglucuronide also has been reported in normal and Gunn rats (Okolicsany et al., 1968). The higher fl-glucuronidase activity found in Corriedale mutant sheep does not reflect the plasma unconjugated bilirubin (10.16/~g/ml total plasma bilirubin with 70 per cent conjugated). Thus deconju- gation of bilirubin by fl-glucuronidase in that animal probably was not a significant factor.

As 80-95 per cent of fl-glucuronidase activity was found in the subcellular particles of rat liver (Walker, 1952) and lysosomal particles accumulated in the hepatocytes of Corriedale mutant sheep (Cornelius et al., 1965), the high level of the enzyme most likely resulted from accumulated lysosomal particles. The lysosomal enzyme may not, in fact, have access to bilirubin as it is transported through the cytoplasm under normal conditions. Mia et al. (1970b) suggested that marked hyperbilirubinemia of both conjugated and unconjugated bilirubin in mutant Corriedale sheep resulted from decreased transport of unconjugated and conjugated bilirubin from plasma to liver as well as from defective excretion of conjugated bilirubin.

The exponential decrease in plasma fl-glucuronidase of neonatal lambs could indicate a relationship of the enzyme with physiological icterus, although no direct evidence is provided. If hepatic activities of fi-glucuronidase paralleled plasma values, there would be a potential for decreasing excretion by deconjuga- tion. Further studies of this interesting correlation are justified.

SUMMARY Bilirubin uridyldiphospho-glucuronyl transferase (BUDPGT) and fl-glucu-

ronidase activities were determined in several tissues of normal sheep and in liver of normal horses. Hepatic levels of the two enzymes also were determined in mutant Southdown and Corriedale sheep with inherited hyperbilirubinemia. BUDPGT activity was elevated in both mutant strains of sheep, suggesting no causal relationship to hyperbilirubinemia, fl-Glucuronidase, elevated in mutant Corriedale sheep liver, may reflect lysosomal retention related to hepatic pig- mentation in that strain. Plasma fi-glucuronidase activity decreased exponentially

BUDPGT AND ~-GLUCURONIDASE ACTIVITY IN HORSE AND SHEEP TISSUES 933

with age in normal neonatal lambs, suggesting further studies of the relation of this enzyme to physiological icterus of the newborn.

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deconjugation of bilirubin glucuronide occur in obstructive jaundice ? Lancet I, 68-69. BILLING B. H. & LATHE G. H. (1956) The excretion of bilirubin as ester glucuronide,

giving direct Van den Bergh reaction. 13iochem..7. 63, 6P. BLACK M. & BILLING B. H. (1969) Hepatic bilirubin UDP-glucuronyl transferase activity in

liver disease and Gilbert's syndrome. New Eng. ft. IVied. 280, 1266-1271. CARBON~ J. V. & GRODSr:Y G. M. (1957) Constitutional nonhemolytic hyperbilirubinemia

in rats: defect of bilirubin conjugation. Proc. Soc. exp. Biol. Med. 94, 461-463. CORNELIUS C. E., ARIAS I. M. & OSBUaN B. I. (1965) Hepatic pigmentation with photo-

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CORNELIUS C. E. & GRONWALL R. R. (1968) Congenital photosensitivity and hyperbiliru- binemia in Southdown sheep in the United States. Am.ft . Vet. Res. 29, 291-295.

COa~LIUS C. E., OSBUaN B. I., GRONWALL R. R. & CARDINE'r G. H. (1968) Dubin- Johnson syndrome in immature sheep. Am. ft. Dig. Dis. 13, 1072-1076.

FISHMAN W. H. (1955) Beta glucuronidase. Adv. Enzymol. 16, 361-409. FISrtMAN W. H., SPRINGER B. & BRUNTEETI R. (1948) Application of an improved glu-

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GRODSKY G. M. & CARBONE J. V. (1957) The synthesis of bilirubin glucuronide by tissue homogenate, ft. biol. Chem. 226, 449-458.

GRONWALL R. R. & MIA A. S. (1972) Fasting hyperbilirubinernia in horses. Am.ft . Dig. Dis. 17, 473-476.

MALLOY H. T. & EVELYN K. A. (1937) The determination of bilirubin with the photo- electric colorimeter, ft. biol. Chem. 119, 481-490.

MIA A. S., GRONWALL R. R. & CORNELIUS C. E. (1970a) Bilirubin-14C turnover studies in normal and mutant Southdown sheep with congenital hyperbilirubinemia. Proc. Soc. exp. Biol. Med. 133, 955-959.

MIA A. S., GRONWALL R. R. & CORNELIUS C. E. (1970b) Unconjugated and conjugated bilirubin transport in normal and mutant Corriedale sheep with Dubin-Johnson syn- drome. Proc. Soc. exp. Biol. Med. 135, 33-37.

OKOLICSANY L., MAGNENAT P. & F ~ I J. (1968) Deconjugation of bilirubin glucuronide by the liver. Lancet 1, 1173-1174.

SCHMID R. (1956) Direct reacting bilirubin, bilirubin glucuronide in serum, bile and urine. Science 124, 76-77.

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Key Word lndex--Hyperbilirubinemia; bilirubin uridyldiphospho-glucuronyl trans- ferase; bilirubin conjugation;/3-glucuronidase ; horse; sheep ; neonatal hyperbilirubinernia; Equus caballus; Ovis aries.