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Haem biosynthesis in the unconjugated hyperbilirubinaemias: Observations in the gunn rat model
CllnBzochem, Vol 22, pp. 177-179. 1989 Printed m Canada All rights reserved
0009-9120/89 $3 00 * O0 Copyright © 1989 The Canadian Socmty of Chn]cal Chemists
Haem Biosynthesis in the Unconjugated Hyperbilirubinaemias: Observations in the Gunn Rat Model ANNE M. GRAHAM, KENNETH E. L. McCOLL, and MICHAEL R. MOORE University Department of Medicine, Western Infirmary, Glasgow G11 6NT, Scotland Disturbances of the enzymes of haem biosynthesis have been reportedin patients with unconjugated hyperbihrubinaemia. We have examined the excretton of haem precursors in the Gunn rat which suffers from severe unconjugated hyperbiltrubinaemia. In urine, levels of aminolaevuhnic actd and total porphyrin were significantly reduced when compared to controls. In feces both coproporphyrm~and protoporphynn levels were reduced in Gunn rats, the former being more affected. Blood porphyrm levels in control and Gunn rats were srmilar.
KEY WORDS: bilirubin; Gilbert's syndrome; Gunn rats; haem biosynthesis; porphyrin.
Introduction ilbert's syndrome was first described in 1907 (1) and is characterised by mild chronic variable unG conjugated hyperbiluribinaemia without the presence of overt haemolysis (2,3). The prevalence of the disease is thought to be between 2-5% of the United States and Western European populations (4,5). The increased level of plasma bilirubin is due to abnormal bilirubin clearance (6). Compartmental analysis suggests defects in both hepatic uptake and conjugation of bilirubin (7). There is evidence that Gilbert's syndrome is a heterogenous condition both in respect to daily bilirubin turnover rate and morphology. Electron micrographs show intracellular differences (8) and this has been supported by subcellular biochemical analysis (9). The reasons behind the abnormalities in bilirubin clearance have still to be determined. In vitro assays of the bilirubin conjugating enzyme, bilirubin UDP-glucuronyl transferase shows reduced activity in Gilbert's syndrome when compared with control patients (10). Unfortunately, little correlation exists between the severity of the enzyme defect and the extent to which serum bilirubin levels are raised (10). This tends to suggest that the reduced level of bilirubin UDP glucuronyl transferase is only one factor contributing to the unconjugated hyperbilirubinaemia. We have recently observed that patients with Gil-
Manuscript received December 1, 1987; accepted July 2, 1988. CLINICAL BIOCHEMISTRY, VOLUME 22, JUNE 1989
bert's syndrome also have distrubances of the enzymes of haem biosynthesis with reduced activity of the penultimate enzyme of the pathway, protoporphyrinogen oxidase (Proto-Oxidase) and increased activity of the initial and rate controlling enzyme of the pathway, delta-aminolaevulinic acid (ALA) synthase (11). This pattern of enzyme disturbance is similar to that seen in patients with variegate porphyria, who, unlike those with Gilbert's syndrome have increased excretion of porphyrins (12). The Gunn rat, a homozygous mutant of the Wistar strain (13), suffers from congenital unconjugated hyperbilirubinaemia. These rats have a nervous disposition and often unsteady gait, which may be due to brain damage, possibly in a fashion similar to kernicterus (20). The high levels of serum bilirubin (14,15) make the Gunn rat a suitable model for further investigations of the relationship between unconjugated hyperbilirubinaemia and disturbance of haem and porphyrin metabolism. In this paper the excretion patterns of haem precursors and serum bilirubin levels of Gunn rats were compared with control rats. The merits of the pure Wistar and heterozygous Gunn-Wistar cross, as controls, were also considered.
Methods Groups of Gunn rats, heterozygous Gunn-Wistar cross rats and Wistar rats, weighing between 150-250 g (aged 4 weeks) and heterozygous Gunn-Wistar cross rats weighing approximately 400 g (aged 8 weeks) were studied. They were confined to individual metabolic cages for a 24-h period to permit separate collections of urine and feces. Since porphyrins are sensitive to light, the collections were kept in the dark. All rats used were male, and had free access to standard diet and tap water. Urinary porphyrins and their precursors were measured by column chromatography (BIO-RAD). Feces were softened to a slurry with water and porphyrins extracted and measured, as already described for human porphyrin excretion (16). Blood protoporphyrin levels were measured using a haematofluorimeter. As there is some evidence that bilirubin interferes with the haematofluorometric assay (17), blood porphyrin 177
GRAHAM, McCOLL, AND MOORE
TABLE 1 Blood Levels of Porphyrins and Bilirubin (Mean -+ SD) Blood Porphyrin I~evel
Type of Rat
Total Serum Bilirubin (~mol/L)
Extraction (nmol/L)
Haematofluorimeter (nmol/L)
Wistar Number Cross Number Gunn Number
3.1 ± 2.5 9 2.9 ± 2.0 8 55.1 -+ 4.7 7*
670 ± 308 7 612 ± 218 10 723 ± 289 9
706 -+ 92 7 596 ± 50 10 1062 ± 206 9*
heterozygous Gunn-Wistar cross rat urine revealed a difference in the pattern of excretion. In the heterozygous Gunn-Wistar rat approximately 80% of porphyrin excreted was coproporphyrin while in the Gunn rat, coproporphyrin accounted for only 45% of total urinary porphyrin.
Discussion
*p < 0.001 by Mann-Whitney U test, Gunn vs Cross or Wistar. was also measured after extraction from blood (16). Total serum bilirubin was measured by autoanalyser. High Performance Liquid Chromatography (HPLC) was applied to pooled urine by a variation of the method of Seubert and Seubert (16).
Results The level of serum bilirubin in Gunn rats was markedly raised (55.1 -+ 4.7 ~mol/L) (Mean -+ S.D.) when compared to the heterozygous Gunn-Wistar cross (2.9 +_ 2.0) and Wistar rats (3.1 _+ 2.5) (Table 1). Significantly higher blood porphyrin levels were recorded in the Gunn rat as compared with the other two strains when the results were calculated using a haematofluorimeter (Table 1). However, blood porphyrin levels were similar in the three groups of animals when measured following extraction. The patterns of excretion were different in all three types of rat (Table 2). Using Wistar rats as controls, u r i n a r y ALA excretion, total u r i n a r y porphyrin excretion and fecal protoporphyrin and coproporphyrin were reduced in the Gunn rat. PBG excretion was normal. Using the heterozygous Gunn Wistar cross as control, there was also reduced total u r i n a r y porphyrin and ALA excretion in the Gunn rat. Fecal porphyrin excretion was lower but this did not reach statistical significance. U r i n a r y PBG excretion was similar in the two animals. Qualitative HPLC analysis of pooled Gunn rat and
The Gunn rats studied in our experiments have bilirubin levels in excess of normal and so provide a model to study the relationship between hyperbilirubinaemia and h a e m biosynthesis. Reported average total serum bilirubin levels in Gunn rats are approximately 100 ~mol/L, whilst heterozygous Gunn rats have normal serum bilirubin levels (15). In our colony, bilirubin levels were half those previously reported, but remained in the region of 10 times the levels in Wistar and heterozygous Gunn-Wistar cross rats. In this study, heterozygous Gunn-Wistar cross rats were regarded as the best control animal since they are genetically closest to Gunn rats. As a consequence of the unconjugated hyperbilirubinaemia, Gunn rats are smaller t h a n heterozygotes of the same age. It is, therefore, impossible to match animals by age and weight. As preliminary studies had shown no difference in porphyrin excretion in 4-week-old and 8-week-old heterozygous Gunn-Wistar cross rats, we elected to study animals matched for weight rather t h a n age. When blood porphyrins were measured by haematofluorimetry, higher values were found in the Gunn rats t h a n in the heterozygous Gunn-Wistar or Wistar rats. When the assay was repeated after extraction from whole blood there were no significant differences between the groups of animals. The high blood porphyrin values using the haematofluorimeter can be explained by interference by bilirubin, as previously reported (17). The excretion studies show that Gunn rats excrete less porphyrin in urine and feces than either heterozygous Gunn-Wistar cross rats or Wistar rats. Analysis of the pattern of u r i n a r y excretion shows that the reduction is principally in coproporphyrin, which is the major porphyrin excreted. The reason for the reduced
TABLE 2
Daily Excretion of Porphyrins and Precursors in Rats (nmol/24 h) Urine
Feces
Type of Rat
ALA
PBG
Total Porphyrin
Copro
Proto
Wistar Number Cross Number Gunn Number
191 ± 41.7 9 61.3 ± 13.2 6* 46.4 ± 32.1 6*tt
32.5 ± 29.3 9 39.6 ± 20.8 4 43.7 ± 50.5 5
4.5 -+ 3.7 9 8.0 -+ 4.3 9** 1.1 ± 1.0 9***t
236 -+ 150 6 163 ± 26.9 6 41.8 ± 14.4 6*
426 -+ 504 6 290 ± 82.1 6 157 ± 69.5 6**
Significance: Wistar *p < 0.001 -v**p < 0.05 Gunn ***p < 0.01
Cross t p < 0.001 -vGunn t t p < 0.05
All statistics computed by the Mann-Whitney U Test. 178
CLINICAL BIOCHEMISTRY, VOLUME 22, JUNE 1989
HAEM BIOSYNTHESIS IN THE HYPERBILIRUBINAEMIAS porphyrin excretion is unclear and several possibilities must be considered. It is known t h a t bilirubin can crystallise out in the kidney of the G u n n r a t (21). This m a y result in mild renal i m p a i r m e n t which could account for decreased u r i n a r y excretion of porphyrin. However, the fact t h a t fecal excretion of porphyrin is also decreased suggests the results are due to reduced porphyrin synthesis. It has previously been reported t h a t porphyrin excretion in Wistar rats follows a bimodal distribution (18). The r a n g e of values previously obtained are from 0.31 to 63.1 nmol/24 h. F r o m this it would a p p e a r t h a t all the Wistar r a t s used in our experiments are from the lower end of this distribution. In our experiments, values for G u n n r a t u r i n a r y total porphyrin excretion (1.1 _+ 1.0) are significantly lower t h a n those of Wistar rats (4.5 -+ 3.7), but these results are still within the range previously reported for Wistar rats (18). If the Gunn is a m u t a n t strain of a Wistar r a t at the low end of the bimodal distribution, t h e n the a b n o r m a l strain would all h a v e a low porphyrin excretion. HPLC analysis shows t h a t coproporphyrin, the p r e d o m i n a n t p'orphyrin excreted in urine, is reduced. U n f o r t u n a t e l y the pattern of u r i n a r y excretion was not determined in the previous study d e m o n s t r a t i n g the bimodal distribution in Wistar rats (18), and we are unable to say w h e t h e r the pattern in those with low excretion is similar to that seen in our homozygous G u n n rats. It is interesting to compare our findings of decreased porphyrin excretion in G u n n rats with hyperbilirubinaemias in humans. In the mild unconjugated hyperbilirubinaemia of Gilbert's syndrome and the severe unconjugated h y p e r b i l i r u b i n a e m i a of Crigler-Najjar syndrome no a b n o r m a l i t y of porphyrin excretion has been reported. However, in the conjugated hyperbilirubinaemias of Rotor's syndrome (19) and Dubin-Johnson syndrome there is increased coproporphyrin excretion, p r i m a r i l y of the isomer 1 series (22). Thus, the Gunn rat, which has severe unconjugated hyperbilirubinaemia and reduced porphyrin excretion, is not an ideal model for a n y of these h u m a n conditions. References
1. Gilbert A, Lereboullet P, Herscher M. Les trois cholemies congenitales. Bull Soc Med Hop Paris 1907; 24: 1203. 2. Arias IM. Chronic unconjugated hyperbilirubinaemia without overt signs of hemolysis in adolescents and adults. J Clin Invest 1962; 41: 2233-45. 3. Foulk WT, Butt HR, Owen CA Jr, Whitcomb FF Jr, Mason HL. Constitutional hepatic dysfunction (Gilbert's disease): its natural history and related syndromes. Medicine 1959; 38: 25-46.
CLINICAL BIOCHEMISTRY, VOLUME 22, JUNE 1989
4. Bailey A, Robinson D, Dawson AM. Does Gilbert's disease exist? The Lancet 1977; i: 931-3. 5. Franken FH. Konstitutionelle oder post hepatikische hyperbilirubinamie? Dtsch Med Wschr 1970; 95: 844-6. 6. Berk PD, Bloomer JR, Howe RB, Berlin NI. Constitutional hepatic dysfunction (Gilbert's syndrome). Amer J Med 1970; 49: 296-305. 7. Zeneroli ML, Piagi V, Cremonini C, Gozzi C, Ventura E. Sources of bile pigment overproduction in Gilbert's syndrome: studies with non-radioactive bilirubin kinetics and with 5 (3,5-3H) aminolaevulinic acid and (214C) glycine. Clin Sci 1982; 62: 643-9. 8. Dawson J, Carr-Locke DL, Talbot IC, Rosenthal FD. Gilbert's syndrome: evidence of morphological heterogeneity. Gut 1979; 20: 848-53. 9. Dawson J, Seymour CA, Peters TJ. Gilbert's syndrome: analytical subcellular fractionation of liver biopsy specimens. Enzyme activities, organelle pathology and evidence for subpopulations of the syndrome. Clin Sci 1979; 57: 491-7. 10. Black M, Billing BH. Hepatic bilirubin UDP Glucuronyl transferase activity in liver disease and Gilbert's syndrome. New Engl J Med 1969; 280: 1266-71. 11. McColl KEL, Thompson GG, E1 Omar E, Moore MR, Goldberg A. Porphyrin metabolism and haem biosynthesis in Gilbert's syndrome. Gut 1987; 28: 125-30. 12. Mustajoki P. Variegate porphyria. Quart J Med 1980; 49: 191-203. 13. Gunn CH. Hereditary acholuric jaundice. J Hered 1938; 29: 137-9. 14. Carbone JV, Grodsky GM. Constitutional non-haemolytic hyperbilirubinaemia in the rat: defect of bilirubin conjugation. Proc Soc Exp Biol Med 1957; 94: 461-3. 15. Johnson L, Sarmiento F, Blanc WA, Day R. Kernicterus in rats with an inherited deficiency of glucuronyl transferase. Amer Med Assoc J of Dis in Children 1959; 97: 91-108. 16. Moore MR. Laboratory investigation of disturbances of porphyrin metabolism. Assoc Clin Path Broadsheet 1983; 109: 1-16. British Medical Association, London. 17. Buhrmann E, Mentzer WC, Lubin BH. The influence of plasma bilirubin on zinc protoporphyrin measurement by a haematofluorimeter. J Lab Clin Med 1978; 91: 710-6. 18. Gartzke Von J, Burck D. Renale ausscheidung von gesamtporphyrinen und hippursaure bei der ratte. J Clin Chem Clin Biochem 1986; 24: 637-9. 19. Berk PD. Pathophysiology, diagnosis and treatment ofthe hereditary hyperbilirubinaemias. In: Williams R, Maddrey WC, eds. Liver. Pp. 1-51. London: Butterworths, 1986. 20. Walker, PC. Neonatal bilirubin toxicity. Chn Pharmacokinetics 1987; 13: 26-50. 21. Schmid R, Axelrod J, Hammaker L, Swarm RL. Congenital jaundice in rats due to a defect in glucuronide formation. J Clin Invest 1958; 37: 1123-30. 22. Cohen C, Kirsch RE, Moore MR. Porphobilinogen deaminase and the synthesis of porphyrin isomers in the Dubin-Johnson syndrome. S Afr Med J 1986; 70: 36-9.
179
0009-9120/89 $3 00 * O0 Copyright © 1989 The Canadian Socmty of Chn]cal Chemists
Haem Biosynthesis in the Unconjugated Hyperbilirubinaemias: Observations in the Gunn Rat Model ANNE M. GRAHAM, KENNETH E. L. McCOLL, and MICHAEL R. MOORE University Department of Medicine, Western Infirmary, Glasgow G11 6NT, Scotland Disturbances of the enzymes of haem biosynthesis have been reportedin patients with unconjugated hyperbihrubinaemia. We have examined the excretton of haem precursors in the Gunn rat which suffers from severe unconjugated hyperbiltrubinaemia. In urine, levels of aminolaevuhnic actd and total porphyrin were significantly reduced when compared to controls. In feces both coproporphyrm~and protoporphynn levels were reduced in Gunn rats, the former being more affected. Blood porphyrm levels in control and Gunn rats were srmilar.
KEY WORDS: bilirubin; Gilbert's syndrome; Gunn rats; haem biosynthesis; porphyrin.
Introduction ilbert's syndrome was first described in 1907 (1) and is characterised by mild chronic variable unG conjugated hyperbiluribinaemia without the presence of overt haemolysis (2,3). The prevalence of the disease is thought to be between 2-5% of the United States and Western European populations (4,5). The increased level of plasma bilirubin is due to abnormal bilirubin clearance (6). Compartmental analysis suggests defects in both hepatic uptake and conjugation of bilirubin (7). There is evidence that Gilbert's syndrome is a heterogenous condition both in respect to daily bilirubin turnover rate and morphology. Electron micrographs show intracellular differences (8) and this has been supported by subcellular biochemical analysis (9). The reasons behind the abnormalities in bilirubin clearance have still to be determined. In vitro assays of the bilirubin conjugating enzyme, bilirubin UDP-glucuronyl transferase shows reduced activity in Gilbert's syndrome when compared with control patients (10). Unfortunately, little correlation exists between the severity of the enzyme defect and the extent to which serum bilirubin levels are raised (10). This tends to suggest that the reduced level of bilirubin UDP glucuronyl transferase is only one factor contributing to the unconjugated hyperbilirubinaemia. We have recently observed that patients with Gil-
Manuscript received December 1, 1987; accepted July 2, 1988. CLINICAL BIOCHEMISTRY, VOLUME 22, JUNE 1989
bert's syndrome also have distrubances of the enzymes of haem biosynthesis with reduced activity of the penultimate enzyme of the pathway, protoporphyrinogen oxidase (Proto-Oxidase) and increased activity of the initial and rate controlling enzyme of the pathway, delta-aminolaevulinic acid (ALA) synthase (11). This pattern of enzyme disturbance is similar to that seen in patients with variegate porphyria, who, unlike those with Gilbert's syndrome have increased excretion of porphyrins (12). The Gunn rat, a homozygous mutant of the Wistar strain (13), suffers from congenital unconjugated hyperbilirubinaemia. These rats have a nervous disposition and often unsteady gait, which may be due to brain damage, possibly in a fashion similar to kernicterus (20). The high levels of serum bilirubin (14,15) make the Gunn rat a suitable model for further investigations of the relationship between unconjugated hyperbilirubinaemia and disturbance of haem and porphyrin metabolism. In this paper the excretion patterns of haem precursors and serum bilirubin levels of Gunn rats were compared with control rats. The merits of the pure Wistar and heterozygous Gunn-Wistar cross, as controls, were also considered.
Methods Groups of Gunn rats, heterozygous Gunn-Wistar cross rats and Wistar rats, weighing between 150-250 g (aged 4 weeks) and heterozygous Gunn-Wistar cross rats weighing approximately 400 g (aged 8 weeks) were studied. They were confined to individual metabolic cages for a 24-h period to permit separate collections of urine and feces. Since porphyrins are sensitive to light, the collections were kept in the dark. All rats used were male, and had free access to standard diet and tap water. Urinary porphyrins and their precursors were measured by column chromatography (BIO-RAD). Feces were softened to a slurry with water and porphyrins extracted and measured, as already described for human porphyrin excretion (16). Blood protoporphyrin levels were measured using a haematofluorimeter. As there is some evidence that bilirubin interferes with the haematofluorometric assay (17), blood porphyrin 177
GRAHAM, McCOLL, AND MOORE
TABLE 1 Blood Levels of Porphyrins and Bilirubin (Mean -+ SD) Blood Porphyrin I~evel
Type of Rat
Total Serum Bilirubin (~mol/L)
Extraction (nmol/L)
Haematofluorimeter (nmol/L)
Wistar Number Cross Number Gunn Number
3.1 ± 2.5 9 2.9 ± 2.0 8 55.1 -+ 4.7 7*
670 ± 308 7 612 ± 218 10 723 ± 289 9
706 -+ 92 7 596 ± 50 10 1062 ± 206 9*
heterozygous Gunn-Wistar cross rat urine revealed a difference in the pattern of excretion. In the heterozygous Gunn-Wistar rat approximately 80% of porphyrin excreted was coproporphyrin while in the Gunn rat, coproporphyrin accounted for only 45% of total urinary porphyrin.
Discussion
*p < 0.001 by Mann-Whitney U test, Gunn vs Cross or Wistar. was also measured after extraction from blood (16). Total serum bilirubin was measured by autoanalyser. High Performance Liquid Chromatography (HPLC) was applied to pooled urine by a variation of the method of Seubert and Seubert (16).
Results The level of serum bilirubin in Gunn rats was markedly raised (55.1 -+ 4.7 ~mol/L) (Mean -+ S.D.) when compared to the heterozygous Gunn-Wistar cross (2.9 +_ 2.0) and Wistar rats (3.1 _+ 2.5) (Table 1). Significantly higher blood porphyrin levels were recorded in the Gunn rat as compared with the other two strains when the results were calculated using a haematofluorimeter (Table 1). However, blood porphyrin levels were similar in the three groups of animals when measured following extraction. The patterns of excretion were different in all three types of rat (Table 2). Using Wistar rats as controls, u r i n a r y ALA excretion, total u r i n a r y porphyrin excretion and fecal protoporphyrin and coproporphyrin were reduced in the Gunn rat. PBG excretion was normal. Using the heterozygous Gunn Wistar cross as control, there was also reduced total u r i n a r y porphyrin and ALA excretion in the Gunn rat. Fecal porphyrin excretion was lower but this did not reach statistical significance. U r i n a r y PBG excretion was similar in the two animals. Qualitative HPLC analysis of pooled Gunn rat and
The Gunn rats studied in our experiments have bilirubin levels in excess of normal and so provide a model to study the relationship between hyperbilirubinaemia and h a e m biosynthesis. Reported average total serum bilirubin levels in Gunn rats are approximately 100 ~mol/L, whilst heterozygous Gunn rats have normal serum bilirubin levels (15). In our colony, bilirubin levels were half those previously reported, but remained in the region of 10 times the levels in Wistar and heterozygous Gunn-Wistar cross rats. In this study, heterozygous Gunn-Wistar cross rats were regarded as the best control animal since they are genetically closest to Gunn rats. As a consequence of the unconjugated hyperbilirubinaemia, Gunn rats are smaller t h a n heterozygotes of the same age. It is, therefore, impossible to match animals by age and weight. As preliminary studies had shown no difference in porphyrin excretion in 4-week-old and 8-week-old heterozygous Gunn-Wistar cross rats, we elected to study animals matched for weight rather t h a n age. When blood porphyrins were measured by haematofluorimetry, higher values were found in the Gunn rats t h a n in the heterozygous Gunn-Wistar or Wistar rats. When the assay was repeated after extraction from whole blood there were no significant differences between the groups of animals. The high blood porphyrin values using the haematofluorimeter can be explained by interference by bilirubin, as previously reported (17). The excretion studies show that Gunn rats excrete less porphyrin in urine and feces than either heterozygous Gunn-Wistar cross rats or Wistar rats. Analysis of the pattern of u r i n a r y excretion shows that the reduction is principally in coproporphyrin, which is the major porphyrin excreted. The reason for the reduced
TABLE 2
Daily Excretion of Porphyrins and Precursors in Rats (nmol/24 h) Urine
Feces
Type of Rat
ALA
PBG
Total Porphyrin
Copro
Proto
Wistar Number Cross Number Gunn Number
191 ± 41.7 9 61.3 ± 13.2 6* 46.4 ± 32.1 6*tt
32.5 ± 29.3 9 39.6 ± 20.8 4 43.7 ± 50.5 5
4.5 -+ 3.7 9 8.0 -+ 4.3 9** 1.1 ± 1.0 9***t
236 -+ 150 6 163 ± 26.9 6 41.8 ± 14.4 6*
426 -+ 504 6 290 ± 82.1 6 157 ± 69.5 6**
Significance: Wistar *p < 0.001 -v**p < 0.05 Gunn ***p < 0.01
Cross t p < 0.001 -vGunn t t p < 0.05
All statistics computed by the Mann-Whitney U Test. 178
CLINICAL BIOCHEMISTRY, VOLUME 22, JUNE 1989
HAEM BIOSYNTHESIS IN THE HYPERBILIRUBINAEMIAS porphyrin excretion is unclear and several possibilities must be considered. It is known t h a t bilirubin can crystallise out in the kidney of the G u n n r a t (21). This m a y result in mild renal i m p a i r m e n t which could account for decreased u r i n a r y excretion of porphyrin. However, the fact t h a t fecal excretion of porphyrin is also decreased suggests the results are due to reduced porphyrin synthesis. It has previously been reported t h a t porphyrin excretion in Wistar rats follows a bimodal distribution (18). The r a n g e of values previously obtained are from 0.31 to 63.1 nmol/24 h. F r o m this it would a p p e a r t h a t all the Wistar r a t s used in our experiments are from the lower end of this distribution. In our experiments, values for G u n n r a t u r i n a r y total porphyrin excretion (1.1 _+ 1.0) are significantly lower t h a n those of Wistar rats (4.5 -+ 3.7), but these results are still within the range previously reported for Wistar rats (18). If the Gunn is a m u t a n t strain of a Wistar r a t at the low end of the bimodal distribution, t h e n the a b n o r m a l strain would all h a v e a low porphyrin excretion. HPLC analysis shows t h a t coproporphyrin, the p r e d o m i n a n t p'orphyrin excreted in urine, is reduced. U n f o r t u n a t e l y the pattern of u r i n a r y excretion was not determined in the previous study d e m o n s t r a t i n g the bimodal distribution in Wistar rats (18), and we are unable to say w h e t h e r the pattern in those with low excretion is similar to that seen in our homozygous G u n n rats. It is interesting to compare our findings of decreased porphyrin excretion in G u n n rats with hyperbilirubinaemias in humans. In the mild unconjugated hyperbilirubinaemia of Gilbert's syndrome and the severe unconjugated h y p e r b i l i r u b i n a e m i a of Crigler-Najjar syndrome no a b n o r m a l i t y of porphyrin excretion has been reported. However, in the conjugated hyperbilirubinaemias of Rotor's syndrome (19) and Dubin-Johnson syndrome there is increased coproporphyrin excretion, p r i m a r i l y of the isomer 1 series (22). Thus, the Gunn rat, which has severe unconjugated hyperbilirubinaemia and reduced porphyrin excretion, is not an ideal model for a n y of these h u m a n conditions. References
1. Gilbert A, Lereboullet P, Herscher M. Les trois cholemies congenitales. Bull Soc Med Hop Paris 1907; 24: 1203. 2. Arias IM. Chronic unconjugated hyperbilirubinaemia without overt signs of hemolysis in adolescents and adults. J Clin Invest 1962; 41: 2233-45. 3. Foulk WT, Butt HR, Owen CA Jr, Whitcomb FF Jr, Mason HL. Constitutional hepatic dysfunction (Gilbert's disease): its natural history and related syndromes. Medicine 1959; 38: 25-46.
CLINICAL BIOCHEMISTRY, VOLUME 22, JUNE 1989
4. Bailey A, Robinson D, Dawson AM. Does Gilbert's disease exist? The Lancet 1977; i: 931-3. 5. Franken FH. Konstitutionelle oder post hepatikische hyperbilirubinamie? Dtsch Med Wschr 1970; 95: 844-6. 6. Berk PD, Bloomer JR, Howe RB, Berlin NI. Constitutional hepatic dysfunction (Gilbert's syndrome). Amer J Med 1970; 49: 296-305. 7. Zeneroli ML, Piagi V, Cremonini C, Gozzi C, Ventura E. Sources of bile pigment overproduction in Gilbert's syndrome: studies with non-radioactive bilirubin kinetics and with 5 (3,5-3H) aminolaevulinic acid and (214C) glycine. Clin Sci 1982; 62: 643-9. 8. Dawson J, Carr-Locke DL, Talbot IC, Rosenthal FD. Gilbert's syndrome: evidence of morphological heterogeneity. Gut 1979; 20: 848-53. 9. Dawson J, Seymour CA, Peters TJ. Gilbert's syndrome: analytical subcellular fractionation of liver biopsy specimens. Enzyme activities, organelle pathology and evidence for subpopulations of the syndrome. Clin Sci 1979; 57: 491-7. 10. Black M, Billing BH. Hepatic bilirubin UDP Glucuronyl transferase activity in liver disease and Gilbert's syndrome. New Engl J Med 1969; 280: 1266-71. 11. McColl KEL, Thompson GG, E1 Omar E, Moore MR, Goldberg A. Porphyrin metabolism and haem biosynthesis in Gilbert's syndrome. Gut 1987; 28: 125-30. 12. Mustajoki P. Variegate porphyria. Quart J Med 1980; 49: 191-203. 13. Gunn CH. Hereditary acholuric jaundice. J Hered 1938; 29: 137-9. 14. Carbone JV, Grodsky GM. Constitutional non-haemolytic hyperbilirubinaemia in the rat: defect of bilirubin conjugation. Proc Soc Exp Biol Med 1957; 94: 461-3. 15. Johnson L, Sarmiento F, Blanc WA, Day R. Kernicterus in rats with an inherited deficiency of glucuronyl transferase. Amer Med Assoc J of Dis in Children 1959; 97: 91-108. 16. Moore MR. Laboratory investigation of disturbances of porphyrin metabolism. Assoc Clin Path Broadsheet 1983; 109: 1-16. British Medical Association, London. 17. Buhrmann E, Mentzer WC, Lubin BH. The influence of plasma bilirubin on zinc protoporphyrin measurement by a haematofluorimeter. J Lab Clin Med 1978; 91: 710-6. 18. Gartzke Von J, Burck D. Renale ausscheidung von gesamtporphyrinen und hippursaure bei der ratte. J Clin Chem Clin Biochem 1986; 24: 637-9. 19. Berk PD. Pathophysiology, diagnosis and treatment ofthe hereditary hyperbilirubinaemias. In: Williams R, Maddrey WC, eds. Liver. Pp. 1-51. London: Butterworths, 1986. 20. Walker, PC. Neonatal bilirubin toxicity. Chn Pharmacokinetics 1987; 13: 26-50. 21. Schmid R, Axelrod J, Hammaker L, Swarm RL. Congenital jaundice in rats due to a defect in glucuronide formation. J Clin Invest 1958; 37: 1123-30. 22. Cohen C, Kirsch RE, Moore MR. Porphobilinogen deaminase and the synthesis of porphyrin isomers in the Dubin-Johnson syndrome. S Afr Med J 1986; 70: 36-9.
179