Perspectives On Animal Research

Volume 1, Supplement

An Evaluation of Ten Randomly-Chosen Animal Models of Human Disease

Crigler-Najjar Syndrome (Rats)

(1)

Description of the Model:

Calabrese discussed the use of the Gunn rat, which has a hereditary hyperbilirubinemia, for toxicology studies with polychiorinated biphenols.(1) The use of the Gunn rat for toxicology research does not appear to be appropriate in a collection of animal models of human disease. Gunn rats have also been used for studies of kernicterus and as models for human Crigler-Najjar syndrome, type I(CNJ). Kernicterus is not a discrete disease, but a process with many etiologies. CNJ is a congenital metabolic disorder, and an earlier review in the Handbook Animal Models of Human Disease described the use of the Gunn rat for the study of CNJ(2) This paper, therefore, will address the relevance of the Gunn rat model for CNJ.

The Gunn rat, a mutant strain of the albino Wistar rat, was first observed in 1934, and it was characterized by C.K. Gunn in 1938.(3) Gunn noted that the rats have congenital jaundice that persists throughout life. They present clinically with acholuria, polyuria, kernicterus, subnormal growth rate, increased erythrocyte fragility, microcytosis, and reticulocytosis.(34) Gunn deduced that the jaundice is due to excessive serum bilirubin,(3) and subsequent investigation linked the hyperbilirubinemia to an enzymatic defect in the conjugation of bilirubin. These rats have an autosomal recessive deficiency of uridine diphosphate glucuronyliransferase (UDPGT) activity.(5) Patients with CNJ also have a defect in this enzyme. Because of a similar pathophysiology and because CNJ is rare, Cornelius and Arias advocated use of the Gunn rat animal model to study CNJ.(5)

Criterion I: Concordance between the Animal Model and the Human Disease

Clinical Presentation:

Like the Gunn rat, children with CNJ have jaundice, hyperbilirubinemia without bilirubinuria, and kernicterus. However, there are important differences. Cornelius and Arias observed, "Most patients die in infancy from kernicterus."(5) Those patients who do survive beyond the neonatal period eventually die from kernicterus later in life.(6) Teenagers may also develop CNJ with subsequent kernicterus, leading to an early death.(6) The clinical course in Gunn rats tends to be less severe. While all Gunn rats have degenerative brain lesions, only half have gross abnormalities of gait.(6) Serum bilirubin must exceed 12-l5mg/100ml before the Gunn rats suffer functional impairment, and they seldom die from kernicterus.(5-7) Thus, human and rat unconjugated hyperbilirubinemias are consequences of similar enzymatic defects, but clinically the disease causes more functional impairment and is more often fatal in humans. To a certain extent, the higher mortality in CNJ patients may be a consequence of the higher serum bilirubin levels in human patients. Bilirubin levels alone do not explain the lower mortality of Gunn rats because rats with significantly elevated serum bilirubin do not consistently die.

Another difference between the clinical presentation of the human and rat unconjugated hyperbilirubinemias is that Gunn rats develop papillary necrosis in the renal medulla.(6) Although CNJ patients and Gunn rats both have deposits of bilirubin in the kidney, only the Gunn rats have renal damage. Gunn rats have impaired sodium and water transport as a consequence of renal pathology. Therefore, Gunn rats, unlike CNJ patients, have difficulty concentrating urine and cannot tolerate water deprivation.(6)

Pathogenesis:

While both the Gunn rat and patients with CNJ syndrome have a deficiency of UDPGT, there are differences in enzymatic impairment. Onishi et al. examined UDP-glucuronyltransferase activity on 2-aminophenol and bilirubin in the livers of 70 human subjects. The malfunctioning UDPGT in Gunn rats does not act on bilirubin or 2-aminophenol.(8) However, Onishi et al. found that in CNJ patients, unlike Gunn rats, UDPGT activity on 2-aminophenol is normal. Thus, they recognized that the biochemical nature of the enzymatic deficiency in Gunn rats and CNJ patients is somewhat different. Onishi et al. concluded:

The Gunn rat is often recommended as a model of the Crigler-Najjar child, but our results suggest, contrary to earlier work, that the Crigler-Najjar liver, unlike the Gunn rat liver, contains normal UDP-glucuronyltransferase activity toward 2-aminophenol. This, together with our developmental results, indicates that human, rather than animal, material should be used to evaluate human pathophysiological conditions.(8)

Onishi and co-workers maintained that advanced techniques permitted detailed and valuable studies from autopsy liver specimens of CNJ patients. This, they concluded, should take the place of Gunn rats.(8) Later studies by Coughtrie et al. showed that the Gunn rat has deficiencies of at least two different UDPGT isoenzymes.(9) The UDPGT isoenzyme that acts on phenols, while deficient in Gunn rats, is functional in CNJ patients.

Both CNJ patients and Gunn rats eliminate bilirubin by alternative pathways from the usual conjugation route, but the Gunn rats have a more efficient mechanism. The Gunn rats catabolize most of the bilirubin to diazonegative polar bilirubin derivatives that are excreted in bile and urine.(6) In human patients, alternative pathways are invoked only when levels of serum bilirubin are greatly elevated. Patients with CNJ establish a higher bilirubin steady state, ranging between 14-45mg/100ml (normal 0.3-1.0). Virtually all bilirubin in CNJ patients is unconjugated.(5,6)

The Gunn rat has been used for the study of kernicterus due to CNJ and other diseases. While CNJ is rare, kernicterus is a major cause of death in premature infants. Stern and Brodersen asserted that unspecified rat strains "... and Gunn rats have been used more than any other animal, and much useful information has been obtained from them."(10) However, the Gunn rat has provided misinformation on a critical issue. Studies with Gunn rats indicated that hypercarbia opens the blood-brain barrier to bilirubin, but acidosis itself does not. However:

...in vitro studies of bilirubin-binding equilibria with rat and human serum albumins show that rat serum albumin binds bilirubin acid, not the anion, in contrast to the human protein which ... binds the anion, not the acid. Thus, it is understandable that acidosis as such does not induce precipitation of bilirubin acid in the rat, although it seems to do so in human babies.(10)

Thus, the rat model has misled investigators on the etiology of kenicterus in acidotic states. This is important because premature babies are prone to acidosis caused by sepsis. Stern and Brodersen commented:

The suggested applicability of these studies in man raises the crucial importance of the choice of animal models and the difficulty of directly translating findings from animal experiments to human infants.(10)

Criterion II: Citations

In 1957, both Axeirod et al.(11) and Carbone and Grodsky (12) found that the Gunn rat has a deficiency of UDPGT, the deficient enzyme in Crigler-Najjar syndrome. This established an important link between the animal model and the human disease. Later, Cornelius and Arias (5) did a detailed review of the Gunn rat as a model for CNJ. We located 102 articles in the Science Citation Index, 1965 to 1988, which cited at least one of these papers. (See Appendix A.) Of these papers, 15 were studies that involved human patients and/or tissues. Several of these clinical investigators attributed clinical importance to research with the Gunn rat.

1. Aziz and Siddiqui reported a case of CNJ that presented with clinical symptoms at age 22.(13) They did not credit the animal model with insights, but they did note similarities between CNJ and Gunn rats, including nonhemolytic unconjugated hyperbilirubinemia, UDPGT deficiency, and electron microscopic evidence of large areas of agranular endoplasmic reticulum in the liver.(13)

2. Robinson et al. studied bilirubin metabolism using a human CNJ patient.(14) Similar studies with Gunn rats preceded this research, and the authors noted that the animal research contributed to the protocol. The investigators found many important similarities between this patient with CNJ and Gunn rats, but there were also differences:

...phenobarbital markedly enhances early pigment formation in rats ... In our patient, on the other band, early labeled bilirubin formation from glycine-¹⁴C appeared to be unaffected by phenobarbital whereas that from ALA-3-5-³H was only moderately increased ... It is therefore impossible to be certain whether these findings reflect species variation between man and rat in the metabolic sources of early-labeled pigment or in the response of the microsomal heme enzymes to barbiturate, or whether they are explained by other mechanisms.(14)

This study did not describe CNJ per se, but rather used a CNJ patient to study bilirubin metabolism. The contribution of the Gunn rat to the understanding of normal bilirubin metabolism, a basic science issue, is beyond the scope of this study. The authors of this study, however, believe that the Gunn rat was valuable for that purpose.

3. Black and Billing studied UDPGT activity in patients with several types of liver disease. They mentioned that UDPGT is deficient in CNJ and in the Gunn rats, but they did not discuss the Gunn rat further.(15)

4. Arias et al. studied 16 jaundiced patients with UDPGT deficiency.(16) This important report yielded considerable insight into the characterization of CNJ. They cited research with the Gunn rat as follows:

Little unconjugated bilirubin is excreted in the bile and none in the urine in rats or human subjects with glucuronyl transferase deficiency. Unconjugated bilirubin accumulates in plasma and tissues, producing jaundice characterized by chronic, acholuric, nonhemolytic unconjugated hyperbilirubinemia.(16)

They also cited human tissue and Gunn rat tissue studies indicating that there was one UDPGT for ester and ether glucuronides. These investigators did not attribute any direct clinical contribution to the animal model.

5. Gardner and Konigsmark described a CNJ patient who developed neurological disease at 15 years of age.(17) The only reference to research with the Gunn rat was in support of the statement, "...the basic molecular abnormality was shown to be deficiency of glucuronyl transferase."(17) The role of the Gunn rat for defining the enzymatic deficiency in congenital nonhemolytic jaundice will be discussed below.

6. Black et al. described a method for determining bilirubin UDPGT activity from needle-biopsy specimens.(18) They mentioned that Gunn rat tissue was used to test this method prior to clinical use. It does not appear that the use of Gunn rat tissue was necessary, because investigators could have used liver tissue from human autopsies or liver biopsies from CNJ patients. However, Gunn rat liver tissue was probably more readily available. Black et al. also noted similarities between human and rat UDPGT.

7. Raia described a histochemic al staining technique to identify bilirubins by thin layer chromatography.(19) He mentioned that Gunn rat tissue was used to develop this technique. While liver tissue from CNJ patients would have been adequate, Gunn rat liver tissue was used first, presumably because it was more readily available. He noted that Gunn rat research has helped illustrate that several tissues can contain unconjugated bilirubin.

8. Stathakos et al. described a patient whose hyperbilirubinemia, it appeared, was due to peritoneal absorption of bilirubin.(20) They studied peritoneal absorption in Gunn and normal rats to test their hypothesis. The investigators did demonstrate bilirubin absorption through the peritoneum in rats, but the relevance of this to the human case is uncertain. Stathakos et al. noted that the patient appeared to have variable diversion of bile into the peritoneum and that pressure in the distended peritoneal cavity may have walled off the extravasating bile at its source.(20) Thus, it was unclear whether or not this patient had significant peritoneal absorption of bilirubin.

9. Bloomer et al. studied bilirubin metabolism in a patient with CNJ.(21) They noted that Gunn rat studies had demonstrated a deficiency of bilirubin glucuronyl transferase. This issue will be discussed below. They also used data from the Gunn rat to establish a mathematic model for bilirubin pools. However, in comparison with other possible models, they noted, "...all three pool models are equally compatible with the data."(21)

10. Black et al. evaluated bilirubin UDPGT activity and cytochrome P450 content in a surgical popu!ation(22) They cited the Gunn rat only in the following passage: "Thus, in the Crigler-Najjar syndrome (Types I and II) and also in the homozygous Gunn rat a very profound deficiency of bilirubin UDPGT results in severe unconjugated hyperbilinbinemia.(22)

11. Blaschke et al. described a patient with CNJ who developed neurologic damage at age 18(23) They discussed research with the Gunn rat extensively. They noted the UDPGT deficiency in Gunn rats, and they mentioned effects of drug metabolism on bilirubin turnover in humans and Gunn rats, They focused on therapeutic studies, including removal with albumin-conjugated agarose columns, phototherapy, and liver transplantation. These issues will be discussed in the next section.

12. Chowdhury et al. found that, although CNJ patients and Gunn rats lack UDPGT, they are able to convert bilirubin monoglucuronide to diglucuronide in vitro with bilirubin glucuronoside glucuronosyltransferase.(24) Like many previous reports, this study demonstrated a similarity between Gunn rats and CNJ patients, but it is unclear whether or not the animal research was necessary. Liver biopsy specimens for two CNJ patients were used, illustrating the availability of human clinical material. In addition to the in vitro studies, Chowdhury et al. reported correction of the hyperbilirubinemia by kidney transplantation in Gunn rats. However, for reasons that will be discussed below, kidney transplantation is not a viable treatment for human CNJ.

13. Wolkoff et al. led a discussion of CNJ, based upon a clinical case report.(25) The discussants, many of whom had done extensive research with Gunn rats, enthusiastically endorsed research with this model. For example, they stated that Gunn rats "...permitted major advances in understanding of bilirubin metabolism and transport, and kernicterus."(25) They also mentioned experimental therapeutic modalities. Liver transplantation had mixed results in Gunn rats. Removal of bilirubin with albumin-conjugated agarose gel was effective for Gunn rats, but there had been difficulties using human blood.

14. Onishi et al., as discussed above, found differences in the activity of UDPGT between CNJ patients and Gunn rats, and they advocated the use of human tissue for research.(8) They noted that much previous work on UDPGT activity had been with experimental animals, but they wrote:

...studies on human neonatal subjects have not been undertaken systematically. The main reasons for this may be as follows: (1) it has been widely assumed that human hepatic UDP-glucuronyltransferase began to be inactivated immediately after death; (2) since UDP-glucuronyltransferase is tightly bound to the microsomal membrane, the enzyme activity is easily affected by the storage conditions, presence of detergents, nucleotides, phospholipids and sonication conditions; (3) accurate measurement of the reaction products of bilirubin glucuronidation is difficult. With regard to problem (1), we observed that the enzyme activity remains almost unchanged (within 10%) until up to 12 hr after death. Moreover, with regard to problem (2), it was also confirmed that change of the activity in homogenate or microsomal fraction can be minimized by adding a detergent such as digitonin to the reaction mixture. Problem (3) was largely resolved by Heirwegh et al. However, they could not analyze further details in the case with very low UDP-glucuronyltransferase activity and low concentration of reaction products ... However, we have developed an excellent method involving the use of high-pressure liquid chromatography for the measurement of very low UDP-glucuronyltransferase activity towards bilirubin.(8)

15. Pett and Mowat reported the efficacy of different treatment modalities in seven patients with CNJ.(26) In their discussion of several promising therapies, they noted that liver transplantation has benefitted several patients. The role of the Gunn rat in the use of liver transplantation to treat CNJ will be discussed below.

In summary, clinical investigators have cited research with the Gunn rat quite frequently. Several studies noted basic science contributions in the understanding of bilirubin metabolism. Also, some credited the animal model with important clinical contributions. However, one might be skeptical of some claims because certain investigators, such as Arias and Chowdhury, also used the animal model. Because CNJ is such a rare disease, cases were generally referred to the few investigators who were familiar with the condition, and many of them also did research with the Gunn rat. The history of major developments in the understanding and treatment of CNJ is discussed in the next section.

Criterion III: Historical Impact

Crigler and Najjar first described CNJ in 1952. The main clinical features of the syndrome were characterized by observations of humans and by in vitro analysis.(6, 27-29) Investigators soon recognized similarities between the Gunn rat and CNJ patients, and identification of the underlying biochemical defects in the two conditions occurred nearly simultaneously. With the development in the late 1950s of modern enzymological techniques, researchers found that both CNJ patients and Gunn rats have a deficiency of UDPGT.(11, 29-32) A defect in bilirubin conjugation was first seen in a human patient in 1956;(29) shortly afterwards a similar abnormality was found in Gunn rats.(11,12)

The Gunn rat model appears to have had limited value in the therapy or prevention of CNJ. Several promising experimental approaches have not been useful clinically. In rats, removal of excess bilirubin has been successful using continuous extracorporeal perfusion through affinity chromatography containing immobilized albumin.(6) Wolkoff et al. noted, "While effective in reducing hyperbilirubinemia in Gunn rats, difficulties due to removal of formed elements are encountered with simian or human blood"(25) This treatment is not mentioned in Pett and Mowat's discussion of the treatment of CNJ (26) or in the 1987 edition of Nelson's Textbook of Pediatrics.(34) Therefore, it is not standard therapy in pediatric patients with CNJ.

Similarly, Gunn rats have been treated successfully by the oral administration of agar or cholestyramine, which binds intestinal stores of bilirubin and permits excretion by the gastrointestinal tract. However, this approach "...has not been consistently useful for the prolonged treatment of patients with Crigler-Najjar syndrome,"(6) and it is not mentioned by Pett and Mowat (26) or in Nelson's Textbook of Pediatrics.(34)

Kidney transplantation has been curative in rats, but it has not been helpful for human patients. Wolkoff et al. explained:

In the rat, bilirubin UDP-glucuronyltransferase is present in kidney and liver. Transplantation of kidney from a normal rat into a Gunn rat reduces hyperbilirubinemia. Enzyme activity in the human kidney is below the limit of detectability and, therefore, renal transplantation in Crigler-Najjar syndrome, type I, cannot be recommended.(6)

Liver transplantation was performed on Gunn rats prior to human patients, but the value of the Gunn rat for this advance is doubtful. It must have been obvious in the 1950s that liver transplantation would cure the deficiency of hepatic conjugation of bilirubin, but there were formidable anatomical, physiological, and inununological problems to overcome. When Crigler and Najjar first described CNJ in 1952, they concluded, "We are, therefore, left with what appears to be primarily a physiologic ... abnormality of the liver."(27) In 1986, Kaufman et al. performed the first successful liver transplant to treat CNJ.(35) They noted that, in 1976, Matas et al. demonstrated that transplantation of liver cells into the Gunn rat liver decreased plasma bilirubin.(36) Interestingly, Kaufman et al. also cited Blaschke et al., who wrote in 1974, "The ultimate approach to therapy in CNJ is likely to detive from advances in hepatic transplantation.(23) Ultimately, advances in surgical technique, medical support, and anti-rejection therapy were critical to the development of liver transplantation, and we found no evidence that research with the Gunn rat was responsible for the advances that made liver transplantation possible.

Some investigators reported conjugation of bilirubin in Gunn rats with transplanted cells and tissues. In 1970, Rugstad et al. injected rat hepatoma cells in the thighs of Gunn rats, and bilirubin levels increased.(37) In 1983, Chowdhury et al. implanted human hepatoma cells in athymic Gunn rats and reduced bilirubin levels.(38) However, Mukharjee and Crasner wrote in 1973, "...transplantation of a viable neoplastic tissue holds little promise of direct clinical application."(39) They transplanted normal rat hepatocytes into the livers of Gunn rats, and they documented lower bilirubin levels and increased UDPGT activity. They concluded, "Although this procedure holds promise for direct clinical application, extrapolation of data from animal experiments to humans must be cautiously evaluated."(39) Despite these encouraging laboratory results, we did not find evidence that this approach has been pursued clinically.

Phototherapy has proven successful in humans and Gunn rats for the long-term management of unconjugated hyperbilirubinemia. Exposure to high-intensity visible light induces catabolism of bilirubin to less toxic, diazonegative derivatives, which can be excreted in bile and urine.(6) This therapy was not derived from the Gunn rat model. In 1958, by measuring the effects of sunlight and artificial blue light on serum bilirubin concentrations in newborn infants, Cremer demonstrated that phototherapy had potential value in the prevention of hyperbilirubinemia.(40) Lucey et al. noted, "The decolorizing effect of sunlight and artificial light upon solutions of bilirubin has been known for many years. This observation prompted Cremer to first use phototherapy clinically"(41) in 1958. In 1968, Lucey et al. conducted the first controlled study of low-birthweight infants to test the effectiveness of phototherapy in the prophylaxis of hyperbilirubinemia. They found that, "...continuous phototherapy is effective in significantly modifying hyperbilirubinemia."(41) To date, the treatment of CJN syndrome "...usually requires exchange transfusions and phototherapy."(34)

The most recent comprehensive clinical review article we found was by Reichen, entitled "Familial unconjugated hyperbilirubinemia syndromes."(42) He devoted an entire section to the Gunn rat, and he noted the clinical and enzymatic similarities between CNJ and the Gunn rat. Although Reichen cited a 1971 paper by Ostrow with the Gunn rat as the basis for phototherapy, Cremer (40) and Lucey (41) had published previous human studies. Reichen also discussed interesting studies, first in the Gunn rat and later in human patients, which indicated that the UDPGT enzyme is normal, but that the microenvironment in the microsomal membrane is defective. The implications of these studies are not known at this time.

Pett and Mowat discussed the management of CNJ in 1987.(26) Six of 39 references were studies with Gunn rats. They cited studies by Matas et al.,(36) Rugstad et al.,(37) Chowdhury et al.,(38) and Mukharjee and Crasner,(39) which were discussed above. They also mentioned Axelrod et al.'s finding of UDPGT deficiency in Gunn rats,(11) and they cited work by Foiot et al. on kidney transplantation with Gunn rats.(43)

Conclusions:

Despite a similar genetic defect in Gunn rats and CNJ patients, certain differences may limit the applicability of the animal model. CNJ patients manifest higher serum bilirubin levels, and consequently they suffer much greater morbidity and mortality. It appears that the prominent features of CNJ were first noted in human patients, and later studies revealed analogous characteristics in Gunn rats. Although many therapeutic modalities have been tried in the experimental model, promising laboratory therapies have not been very useful clinically. Gunn rats might have played a role in our understanding of bilirubin metabolism. Because Gunn rat liver tissue is more available than liver from CNJ patients, Gunn rats may facilitate basic science research on bilirubin metabolism. However, Onisbi et al. contended that human liver tissue is often preferable for enzyme studies. Such basic science studies are outside the scope of this review of animal models of human diseases. Also, the Gunn rat might have been useful to test different techniques to measure unconjugated bilirubin. Although Gunn rats were not necessary, given the possible use of human tissues, Gunn rat tissues were probably more available. Thus, the issues are complex, and the exact contribution of the Gunn rat to our understanding of CNJ is not always clear, but it appears that the Gunn rat model has not been critical to important advances in the understanding, diagnosis, or treatment of CNJ.

References:

1. Calabrese, EJ: Hyperbilirubinemia, Model No. 157, in Jones TC, Hackel DB, Migaki G (eds): Handbook Animal Models of Human Disease Fasc 8. Washington DC, Registry of Comparative Pathology, Armed Forces Institute of Pathology, 1979.

2. Cornelius CE, Arias IM: Congenital hyperbilirubinemia, Model No. 26, in Jones TC, Hackel DB, Migaki G (eds): Handbook Animal Models of Human Disease Fasc 2. Washington DC, Registry of Comparative Pathology, Armed Forces Institute of Pathology,1972.

3. Gunn CK: Hereditary acholuric jaundice. J Hered 1938;29:137-139.

4. Gunn CK Hereditary acholuric jaundice in the rat. Canad Med Ass J. 1944;50:230-237.

5. Cornelius CE, Arias IM: Hereditary nonhemolytic unconjugated hyperbilirubinemia in Gunn rats. Am J Pathol 1972;69:369-372.

6. Wolkoff AW, Chowdhury JR, Arias IM: Hereditary jaundice and disorders of bilirubin metabolism, in, Stanbury JB, Wyngaarden JB, Fredrickson DS (eds): The Metabolic Basis of Inherited Disease, 5th Ed. New York, McGraw-Hill, 1983 pp 1399-1405.

7. Johnson L, Sarmiento F, Blanc WA, Day R: Kernicterus in rats with inherited deficiency of glucuronyltransferase. Am J Dis Child 1959;97:591-607.

8. Onishi S, Kawade N, Itoh S, Isobe K, Sugiyama S: Postnatal development of uridine phosphate glucuronyltransferase activity toward bilirubin and 2-aminophenol in human liver. Biochem J 1979;184:705-707.

9. Coughtrie MWH, Burchell B, Shepherd IM, Bend JR: Defective induction of phenol glucuronidation by 3-methyicholanthrene in Gunn rats is due to the absence of a specific UDP-glucuronosyltransferase isoenzyme. Molec Pharmacol 1987;31:585-591.

10. Stern L, Brodersen R: Kernicterus research and the basic sciences: a prospect for future development. Pediatrics 1987;79:154-155.

11. Axelrod J, Schmid R, Hamrnaker L: A biochemical lesion in congenital, non-obstructive, non haemolytic jaundice. Nature 1957;180:1426-1427.

12. Carbone JV, Grodsky GM: Constitutional nonhemolytic hyperbilirubinemia in the rat: Defect of bilirubin conjugation. P Soc Exp Biol Med 1957;94:461-463.

13. Aziz MA, Siddiqui AR: Congenital familial nonhemolytic hyperbilirubinemia in an adult with central nervous system derangement. Gastroenterol 1967;52:254-258.

14. Robinson SH, Lester R, Crigler JF, Tsong M: Early-labeled peak of bile pigment in man. N Eng J Med 1967;277:1323-1329.

15. Black M, Billing BH, Hepatic bilirubin UDP-glucuronyl transferase activity in liver disease and Gilbert’s syndrome. N Eng J Med 290;180:1266-1271.

16. Arias IM, Gartner LM, Cohen M, Ezzer JB, Levi AJ: Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency. Am J Med 1969;41:395-409.

17. Gardner WA, Konigsmark BW: Familial hemolytic jaundice: bilirubinosis and encephalopathy. Pediatrics 1969;43:365-376.

18. Black M, Billing BH, Heirwegh KPM: Determination of bilirubin UDP-glucuronyl transferase activity in needle-biopsy specimens of human liver. Clin Chim Acta 1970;29:27-35.

19. Raia S: Histochemical separation of conjugated and unconjugated bilirubin and its assessment by thin layer chromatography. J Histchem Cytochem 1970;18:153-158.

20. Stathakos P, Fanska R, Carbone JV, Grodsky GM: Jaundice associated with peritoneal absorption of bilirubin. Gastroenterol 1970;59:769-778.

21. Bloomer JR, Berk PD, Howe RB, Berlin NI: Bilirubin metabolism in congenital nonhemolytic jaundice. Pediat Res 1971;5:256-264.

22. Black M, Perrett RD, Carter AE: Hepatic bilirubin UDP-glucuronyl transferase activity and cytochrome P450 content in a surgical population, and the effects of preoperative drug therapy. J Lab Clin Med 1973;81:704-712

23. Blaschke TF, Berk PD, Scharschmidt BF, Guyther JR, Vergalla JM Waggoner JG: Crigler-Najjar syndrome: An unusual course with development of neurologic damage at age eighteen. Pediat Res 1974;8:573-590.

24. Chowdhury JR, Jansen PLM, Fischberg EB, Daniller A: Hepatic conversion of bilirubin monoglucuronide to diglucuronide in uridine diphosphate-glucuronyl transferase-deficient man and rat by bilirubin glucuronoside glucuronosyltransferase. J Clin Invest 1978;62:191-196.

25. Wolkoff AW, Chowdhury JR, Gartner LA, et al: Crigler-Najjar syndrome (type I) in an adult male. Gastroenterol 1979;76:840-848.

26. Pett S, Mowat AP: Crigler Najjar syndromes types I and II. Clinical experience-King’s College Hospital 1972-1978. Phenobarbitone, phototherapy and liver transplantation, Molec Aspecs Med 1987;9:473-482

27. Crigler Pi, Najjar VA Congenital familial nonhemolytic jaundice with kernicterus. Pediatrics 1952;10:169-180.

28. Childs B, Najjar VA: Familial nonhemolytic jaundice with kernicterus: Report of two cases without neurologic damage. Pedàurics 195418:369-377.

29. Schmid R: Direct-acting bilirubin, bilirubin glucuronide, in serum, bile, and urine. Science 1956;124:76-77.

30. Axelrod J, Schmid R, Hammaker L: Biochemical lesion in congenital non-obstructive nonhaemolytic jaundice. Nature 1957;180;1426-1427.

31. Arias IM, London IM: Bilirubin glucuronicle formation in vitro: Demonstration of a
defect in Gilbert's disease. Science 1957;126:563-564.

32. Carbone JV, Grodsky GM: Constitutional nonhemolytic hyperbilirubinemia in the rat: Defect of biirubin conjugation. Proc Soc Exp Biol Med 1957;94:461-463.

33. Peterson RE, Schmid R: A clinical syndrome associated with a defect in steroid glucuronide formation. J Clin Endocrinol 1957;17:1485-1488.

34. Behrman RE, Vaughn VC (eds): Nelson's Textbook of Pediatrics,13th Ed. Philadelphia, W.B. Saunders, 1987, pp 835-836.

35. Kaufman SS, Wood RP, Shaw BW, et al.: Orthotopic liver transplantation for Type I Crigler-Najjar syndrome. Hepatol 1986;6:1259-1262.

36. Matas AJ, Sutherland ER, Steffes MW, et al.: Hepatocellular transplantation for metabolic deficiency: Decrease in plasma bilirubin in Gunn rats. Science 1976;192:892-894.

37. Rugstad HE, Robinson SM, Yanoni C, Tasjia AH: Transfer of bilirubin UDP-glucuronyl transferase to enzyme deficient rats. Science 1970;170:553-555.

38. Chowdhury JR, Gatmaitan S, Shouval D, Chowdhury NR, Azias IM: The athymic Gunn rat: A new congeneic hybrid for testing billirubin UDP glucuronyl transferase activity of human tissues in vivo. Hepatol 1983;3:874A.

39. Mukharjee AB, Crasner J: Induction of an enzyme in genetically deficient rats after grafting of normal liver, Science 1973;182:68-69.

40. Cremer RJ, Perryman PW, Richards DH: Influences of light on hyperbilirubinemia of infants. Lancet 1958i:1094-1097.

41. Lucey J, Ferreiro M, Hewitt J: Prevention of hyperbilirubinemia of prematurity by phototherapy. Pediatrics 1968;41:1047-1054.

42. Reichen J: Familial unconjugated hyperbilirubinemia syndromes. Sem Liv Dis 1983;3:24-35.

43. Foliot A, Christoforov B, Petite JP, Etienne JP, Housset E, Dubois M: Bilirubin UDP glucuronyl transferase activity in Wistar rat kidney. J Physiol 1975;229340-343.