Investigation of the role of reactive oxygen species in bilirubin metabolism in the Gunn rat

Btl,

ELSEVIER

Biochi~ic~a et BiophysicaA~ta

Biochimica et Biophysica Acta 1243 (1995) 244-250

Investigation of the role of reactive oxygen species in bilirubin metabolism in the Gunn rat Minakshi Joshi

a,1,

Barbara H. Billing

a

Terence Hallinan

h,*

a Department of Medicine, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK b Department of Biochemistry, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK Received 6 April 1994

Abstract It has been previously established that the attenuation of hepatic lipid peroxidation by a fat-free diet is accompanied by a marked rise in plasma bilirubin in Gunn rats. Present in vitro studies confirmed that microsomal lipid peroxidation caused the concurrent degradation of added bilirubin but failed to show that microsomal superoxide, hydroxyl radical or hydrogen peroxide would degrade bilirubin. Moreover, although injection of vitamin E completely inhibited microsomal lipid peroxidation and bilirubin degradation it had no effect on plasma bilirubin. No evidence has therefore been obtained that in Gunn rats, in the absence of bilirubin glucuronidation, that reactive oxygen species provide a significant physiological pathway of bilirubin disposal. Keywords: Bilirubin; Reactive-Oxygen Species; Antioxidant; Gunn rat; Lipid-peroxidation

1. Introduction Previous studies have shown that in vitro there is a close association between enzymatic or non-enzymatic lipid peroxidation (LPO) and bilirubin degradation in Gunn rat liver microsomes [1]. This suggested that bilirubin catabolism might be an oxidative process dependent on lipid peroxidation. Moreover, in the Gunn rat the administration of a synthetic fat free diet strongly inhibited the hepatic microsomal LPO and bilirubin degradation and was accompanied by a marked increase in plasma bilirubin (BR). In contrast, when 25% corn oil was added to this diet the plasma BR decreased slightly while LPO and bilirubin degradation were similar to those on a stock diet [1]. It has not, however, been established whether there is a causal relationship between in vivo LPO activity and the level of plasma BR in the Gunn rat or indeed whether the co-oxidation of bilirubin during lipid peroxidation, which has been observed in vitro, is necessarily the mechanism responsible for the elimination of bilirubin in these animals.

* Corresponding author. Fax: +44 (071) 7949645. Present address - Dept. of Dermatology, Bowman Gray School of Medicine, Winston Salem, N. Carolina 27157 USA.

0304-4165/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 3 0 4 - 4 1 6 5 ( 9 4 ) 0 0 1 3 5 - 9

In order to examine this matter further, the effects of various reactive oxygen species on the degradation of bilirubin were studied. The dietary experiments were also extended to include the administration of large doses of the antioxidants vitamin E and C, to see whether they influenced the plasma BR.

2. Materials and methods 2.1. Materials

All chemicals (Analar grade) were purchased from either British Drug Houses or Sigma. Corn oil was obtained from Budgens and was shown by GLC to consist of 84% unsaturated fatty acids (29% oleic acid and 55% linoleic acid). Solkafloc (a wood cellulose product used as a source of dietary fibre) was purchased from Special Diet Services, Witham, Essex, UK. 2.2. Gunn rats

Unless otherwise stated the jaundiced male rats used in this study came from the original inbred strain of Gunn rats, which had been established at the Royal Free Hospital School of Medicine since 1962. Latterly it was observed

M. Joshi et al./ Biochimica et BiophysicaActa 1243 (1995)244-250 that the litter sizes in the colony had markedly decreased and fertility was low. A rebred colony was, therefore, created by crossing male Gunn rats from the original colony once with female Wistar rats (purchased from Bantin and Kingman, Hull). The female offspring (heterozygotes) were then crossed with male Gunn rats from the original colony and the jaundiced offspring were inbred for 3 or more generations. This rebred colony was used for some of the present experiments when synthetic fat free and 25% corn oil diets were fed and vitamin E or vitamin C were injected. 2.3. Dietary regimens Jaundiced male Gunn rats weighing 280-380 g were used in all experiments. They had free access to water and received ad libitum a stock diet ( S D S / B P purchased from Special Diet Services), which contained 2.6% lipid, 6.5% sucrose, 46% starch and 15% protein (w/w). In some studies they were fed for 7 days on powdered stock diet before receiving synthetic diets containing either 0% fat or 25% ( w / w ) corn oil with carbohydrate, in the form of corn starch or sucrose, contributing 69% or 44% of the diets, respectively. These diets contained 20% casein and balanced amounts of vitamins, minerals and Solkafloc [2]. The corn oil increased the dietary vitamin E to 92 m g / k g compared with 68 m g / k g in the stock diet. Blood samples (0.5 ml) were obtained under anaesthesia by cardiac puncture or via a tail vein. 2.4. Vitamin injections Vitamin E (a-tocopherol, 110 m g / k g ) was injected intraperitoneaUy (IP). It was dissolved in a vehicle [3] of ethanol-16% Tween 80 in 0.9% NaCI (1:9, v / v ) . In the vitamin C experiments, groups of Gunn rats were injected IP daily for 3 days with either saline (0.9% NaCI (w/v), n = 3) or vitamin C in saline in doses up to 750 m g / k g (n = 3). Three rats also received an IP injection of vitamin E on day 0 in addition to 3 doses of vitamin C (750 m g / k g ) on days 0, 1 and 2. Blood samples were taken on day 0 and at suitable time intervals and hepatic microsomes were prepared at the end of the experiment. In a second series of experiments, rats were injected for 3 days with doses of 180 or 375 mg vitamin C/kg. 2.5. Analytical methods Plasma bilirubin concentrations were assayed by a standard diazo method using caffeine benzoate, at a final concentration of 90 mM, as the accelerator [4]. Liver microsomes were generally prepared by a rapid calcium aggregation method [5] and stored at - 7 0 °. Microsomal protein was assayed by a modification of the Lowry method [6]. Assays were all conducted at 37° and

245

pH 7.4 in 40 mM (3-[N-morpholino] propanesulphonic acid) (Mops). 2.6. Hepatic microsomal bilirubin degradation and lipid peroxidation Bilirubin (BDH) dissolved in DMSO was diluted with sodium taurocholate in Mops to give a preparation containing 216 /xM bilirubin, 620 mM DMSO and 9.6 mM taurocholate. Microsomes (1.5-2 mg protein/ml, added last) were incubated aerobically in dim light in 40 mM Mops with 1.6 mM ADP, 18 /zM ferrous sulphate (dissolved in distilled water), 35 /xM unconjugated bilirnbin (100 mM DMSO, 1.55 mM taurocholate) and a NADPH generating system (0.18 mM NADP, 4.03 mM glucose-6-phosphate and 0.19 1 U / m l glucose-6-phosphate dehydrogenase) in order to initiate LPO and bilirubin degradation enzymatically. Non-enzymatic lipid peroxidation was initiated with 0.2 mM ascorbic acid instead of NADPH. Bilirubin degradation was assayed by measuring bilirubin disappearance from the incubation [7]. It was expressed as nmol bilirubin degraded/min per mg microsomal protein. Lipid peroxidation was determined by spectrophotometric measurement of the thiobarbituric acid reactive substances (TBARS) formed when samples were heated for 1 h at 80 ° with a TBA reagent containing 1 mM butylated hydroxytoluene [8]. It was expressed as nmol TBARS formed/min per mg protein. Unless otherwise stated LPO and bilirubin degradation were assayed in 10 min incubations. 2.7. Hydroxyl radical The method employed to generate and assay O H formation was essentially that of Cederbaum and Cohen [9]. DMSO was included in the bilirubin preparation and was therefore available to react with the 'OH to form methyl radicals which rearranged to formaldehyde. This was determined spectrophotometrically [10]. 2.8. Microsomal phospholipid unsaturated fatty acids Lipids were extracted from 4 mg microsomal protein suspended in Mops [11]. Phospholipids were then isolated using 3cc Bond Elut columns (Aminopropyl bonded phase) obtained from Analytichem International [12]. Methyl esters of the fatty acids were prepared and separated by GLC and quantitated so that the Double Bond Index (DBI, percentage mass multiplied by the number of double bonds in the fatty acid) could be compared for rats on different diets. This was calculated for the major unsaturated fatty acids (C16:1 palmitoleic; C18:1 oleic; C18:2 linoleic; C18:3 linolenic and C20:4 arachidonic) and was summated to give the total DBI for a particular sample.

M. Joshi et al./ Biochimica et BiophysicaActa 1243 (1995)244-250

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2.9. Statistics All results have been expressed as means _ SEM. Statistical analysis was performed using the unpaired t test.

3. R e s u l t s

3.1. The effect of reactive oxygen species on LPO and bilirubin degradation Superoxide Brodersen and Bartels [13] have reported that xanthine oxidase has the ability to oxidise bilirubin ' i n vitro'. Kaul et al. [14] found that the photodecomposition o f bilirubin w a s g r e a t l y a c c e l e r a t e d b y the p r e s e n c e of x a n t h i n e / x a n t h i n e oxidase but not if superoxide dismutase (SOD) was included. They also showed that an i.p. injection of xanthine increased the plasma clearance of an injected dose of bilirubin and postulated that the superoxide anion ( 0 2 ) might play a role in the degradation of bilirubin [15]. Studies were therefore performed to see whether superoxide was involved in a microsomal system. Data in Table 1 confirms that 0 2 generated by a x a n t h i n e / x a n t h i n e oxidase system degrades bilirubin in buffer; this was inhibited by Cu-Zn SOD. Degradation was also inhibited by the presence of either native or denatured microsomes, so it is unlikely that endogenous SOD caused this inhibition. Further evidence that 0 2 did not degrade bilirubin in the microsomal system comes from the observation that the addition of SOD to incubations of microTable 1 Effect of superoxide on bilirubin degradation (BRD) in the presence and absence of microsomes and superoxide dismutase Incubation conditions

n BRD (nmol min- 1)

BR + xanthine/xanthine oxidase BR + xanthine/xanthine oxidase + SOD BR + xanthine/xanthine oxidase + microsomes BR + xanthine/xanthine oxidase + microsomes+ 18 ~M FeSO4 / 1.6 mM ADP BR + xanthine/xanthine oxidase + denatured microsomes

4 0.40+ 0.04 2 0.04, 0.09

Standard enzymatic BRD system Standard enzymatic BRD system + SOD

2 0 4 0

2 0.10, 0.01 BRD (nmol min -1 mg prot- 1) 5 0.28+ 0.02 7 0.26+ 0.03

Bilirnbin (35 /.~M was incubated in Mops buffer pH 7.4 with 33 mM xanthine, 0.04 U/ml xanthine oxidase + microsomes (2 mg/ml): SOD (33 p.g/ml) was added as indicated. In the standard enzymatic BRD system, microsomes (1.5-2 mg protein/ml) were incubated aerobically for 10 min at 37° in 40 mM MOPS buffer pH 7.4 with 18 p.M FeSO4, 1.6 mM ADP, 35 uM unconjugated bilirubin and a NADPH generating system. Microsomes were denatured by heating the suspension for 5 min at 85° prior to addition of the other reagents The number of rats studied is given by n.

Table 2 Effect of hydroxyl radical (OH) on enzymatic microsomal BRD and lipid peroxidation (LPO).

Standard BRD assay Hydroxyl radical generating system

OH formation (nmol min- 1 mg prot- 1)

Microsomal activity (nmol min- 1 mg prot - 1) BRD

LPO

0.06 +_0.03 0.9 + 0.03

0.34 + 0.01 0.01 + 0.01

1.21 + 0.08 0.00

Bilirubin (35 p.M) was incubated with the standard enzymatic BRD/LPO system of ADP/Fe/NADPH as in Table 1. Alternatively it was incubated with an OH generating system consisting of microsomes (1.5-2 mg protein/ml) 0.1 mM EDTA, 18 p.M FeSO4, 1 mM azide to inhibit catalase and the standard NADPH generating system. Formation of OH was measured in both incubations. Results are means of 3-6 experiments somes with N A D P H / F e / A D P did not lessen their ability to degrade bilirubin (Table 1).

Hydrogen peroxide It has been reported that bilirubin can be oxidised by hydrogen peroxide ( 2 . 5 - 5 . 0 m M ) in aqueous solution in the presence of haem proteins [13,16] or F e - E D T A [16]. When 114 m M H 2 0 2 was added to native microsomes in the present study about 50% of the bilirubin was degraded. However, 11 m M H 2 0 2 and 1.1 m M H 2 0 2 failed to degrade bilirubin significantly. Moreover, the addition of catalase (2500 U / m l ) to a standard incubation of microsomes with bilirubin and N A D P H / F e / A D P did not inhibit the disappearance of bilirubin (data not shown). Since the physiological concentration of H 2 0 2 in the liver is reportedly only 1 /xM [17], it is unlikely that H 2 0 2 oxidises bilirubin in vivo.

Hydroxyl radical Hydroxyl radical was virtually undetectable in the standard assay of microsomes with bilirubin, an N A D P H generating system and F e / A D P , which gave optimal rates of bilirubin degradation and LPO (Table 2). In addition, hepatic m i c r o s o m e s , incubated with bilirubin and NADPH/Fe/EDTA/azide (conditions which optimise hydroxyl radical generation) formed 0.9 nmol OH m i n - 1 protein m g - 1 but caused no detectible LPO or degradation of bilirubin (Table 2). The absence of LPO in these incubations was expected since it is well established that O H , generated by N A D P H / F e / E D T A does not initiate microsomal LPO efficiently [18-20]. It is therefore unlikely that hydroxyl radicals degrade BR by a mechanism independent of LPO in an optimised "OH-system or in the standard assay.

3.2. The influence of fat free diet on lipid peroxidation and bilirubin metabolism Microsomal phospholipid composition When Gunn rats are fed a fat free diet their plasma BR is markedly increased [1,2]. In an attempt to understand

M. Joshi et al. / Biochimica et Biophysica Acta 1243 (1995) 244-250

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Table 3 Effect of fat free diets on microsomal LPO and plasma BR in rebred Gunn rats. Diet

n

Lipid peroxidation (nmol T B A R S / m g prot)

Plasma BR (% increase)

to

tl 0

t20

t40

Stock a Starch a Sucrose 14 days a 13 days b

4 6

1 + 0.1 1.3 + 0.1

53 + 1.8 36 + 5.7

57 + 2.5 54 + 4.9

63 -I- 3.3 64 _+ 3.7

276

3 3

1.4 5:0.1 0.8 5:0.02

30 5:10.4 12 5:5.2

48 5:5.2 25 5:6

56 5:3.5 64 5:5.2

102 * 148

Three groups of rats were studied after 13-14 days on stock diet or synthetic fat-free diets containing starch or sucrose as the source of carbohydrate. Microsomes in a were prepared by calcium aggregation and LPO was initiated with Fe/ADP//NADPH. In b, microsomes were sedimented at 105,000g and LPO was initiated with F e / A D P / 0 . 2 mM ascorbate. Bilirubin was not included in these incubations and the microsomal protein was lower at 0.2-0.4 mg/ml. * This value is low because the mean initial plasma BR (176 /zM) was very high in these rats.

how this diet influences LPO and plasma BR, the fatty acid composition of the liver microsomal phospholipids was compared with those from rats fed a stock diet. The DBI on the stock diet was 1 2 5 + 3 ( n = 7 ) and was significantly lowered by 2 weeks on a fat free diet to 8 6 + 6 ( n = 3 ; P < 0 . 0 5 ) . At the same time LPO, in agreement with other investigators [21,22] and bilirubin degradation became negligible. If the increase in plasma BR of 119 + 21% was due partly to the dietary abolition of bilirubin degradation, then it is theoretically possible that the decreased peroxidisability of the membrane lipids might be responsible for this. Different responses to diet in the rebred strain of Gunn rat On rebreeding the Gunn rats some strain-specific effects of diet on LPO and plasma BR levels became apparent. In the rebred strain, feeding for 14 days on a starch-based, fat-free diet increased the plasma BR by 270%. However, enzymatically initiated LPO was only modestly inhibited in 10 min assays and showed no inhibition in 20 or 40 min assays, which is at variance with our findings with the former sub-strain [1]. Similar results were obtained when sucrose was substituted for starch in the fat free diet, irrespective of whether LPO was initiated enzymatically or with ascorbate (Table 3). These observations suggest that the marked dietary increase in plasma BR could not be due to decreased LPO activity.

3.3. Effect of the antioxidants vitamin E and vitamin C on bilirubin metabolism in the Gunn rat

If LPO was important for determining plasma BR then it would be anticipated from studies on the original Gunn rat strain that the administration of antioxidants to inhibit LPO, would elevate the plasma BR. The following experiments were designed to test this hypothesis. Vitamin E In preliminary experiments, Vit E (110 mg/kg) was administered IP to the original strain of Gunn rats on the stock diet and blood was taken after 1 day (3 rats), 2 days (3 rats) and 3 days (2 rats). Lipid peroxidation and the degradation of bilirubin were abolished at each time, but plasma BR did not rise significantly. Similarly, plasma BR did not rise in 7 rats of the rebred strain 4 or 8 days after the Vit E injection, even when a second dose was given to 3 rats after 4 days [23]. The effect of Vit E was also examined in rats fed a 25% corn oil diet for 14 days. In these experiments controls received the Vit E vehicle instead of the vitamin. Since only 50% of the rebred rats responded to the high fat diet with a decrease in plasma BR [23], the study was repeated with the remaining Gunn rats from the original colony. (Table 4). In both groups, microsomal degradation of bilirubin and LPO were completely inhibited by Vit E but

Table 4 Effect of vitamin E on plasma BR and hepatic microsomal BRD and LPO in Gunn rats fed a 25% lipid diet Strain

Rebred Original

n

5 4 4 4

IP injection

Vitamin E Vehicle Vitamin E Vehicle

Plasma BR (/.tM)

Microsomal activity (nmol rain-1 mg prot-1)

Day 0

Day 3//4

Day 7

BRD

LPO

114 ___12 126 + 18 93 + 1 96 + 1

120 + 4 136 + 9 116,119 114,127

111 * ,132 * 116 * ,132 *

0 0.31 + 03 0 0.36 + 0.05

0.03 + 0.02 1.41 + 0.05 0 1.32 + 0.13

After 2 wk on a 25% corn oil diet (day 0) the rats were injected with either vitamin E (110 m g / k g ) or ethanol/tween/saline (vehicle). The majority of rats were killed on day 3 or 4 and plasma BR, BRD and LPO were assayed. * Four of the original strain of rats received a second dose of vitamin E or the vehicle on day 3 or 4 and were killed on day 7

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M. Josh± et al. / Biochimica et Biophysica Acta 1243 (1995) 244-250

Table 5 Effect of vitamin C on the plasma BR and microsomalBRD and LPO in the rebred strain of Gunn rats. Group Dailydose Decrease in n PlasmaBR (~M) Microsomal activity (nmol min -1 mg prot-1) body wt. (g) Initial Day 3 BRD LPO A B

0.9% NaC1 750 mg vit. C/kg 750 mg vit. C/kg + vit E * 180 mg vit.C/kg 375 mg vit.C/kg

3+ 6 42 ± 7 38_+6 7± 2 23 ± 6

3 3 2 4 8

159 ± 10 154 ± 25 150±9 178 ± 13 168 ± 12

150 ± 8 (-4%) 268 ± 17 (+ 7 4 % ) 236_+27(+51%) 178 ± 3 186 ± 10 ( + 13%)

0.37 ± 0.07 0.33,0.21 0.0.02 0.35 ± 0.06 0.37 ± 0.03

1.61 ± 0.07 1.42,1.3 0.07,0.05 1.24 _+0.19 1.26 ± 0.08

The rats were fed the stock diet. Group A rats were injected IP on days 0, 1 and 2 with 0.9% saline or 750 mg/kg vitamin C. : * Vit E (110 mg/kg) was injected on day 0. Blood was taken on day 0 and 24 h after the last injection. Rats were killed on day 8 for BRD and LPO determinations. Group B rats were injected on days 1, 2 and 3. Blood was taken on day 0 and 4 h after the last injection; the rats were then killed.

were not influenced by the vehicle. Although a slight rise in plasma BR occurred 3 / 4 days after the injection of Vit E, there was no significant increase in BR in 2 rats given a second injection. Since injection of the vehicle caused similar rises in plasma BR, it must be concluded that Vit E was therefore not responsible for these rises. The microsomal DBI values for rats on the corn oil diet were 156-160 and were not influenced by injected vitamin E [23]. Vitamin C

Because ascorbate has been reported to be the most effective aqueous phase ant±oxidant in plasma [24], its influence on plasma BR was investigated in the rebred strain of rats. Ascorbate has a short half life, so high doses were injected for 3 days. There were no significant changes in plasma BR in control rats injected with saline or those given 180 m g / k g ascorbate (Table 5). On an intermediate dose of 375 m g / k g , 4 of the 8 rats responded with an increase in plasma BR while the remainder showed no significant change. The highest dose of 750 m g / k g markedly increased the plasma BR in 2 of 3 animals. This increase in plasma BR was only slightly attenuated when a dose of vitamin E was also given on day 0. However, vitamin E completely inhibited the degradation of bilirubin by microsomes and peroxidation, in contrast to ascorbate which did not alter these activities. The rise in plasma BR might be due to high doses of ascorbate antagonising the oxidative destruction of bilirubin in an aqueous medium. However, the rats injected for 3 days with the highest doses of ascorbate lost considerable body weight suggesting that the changes in plasma BR could also reflect vitamin C toxicity.

4. Discussion Cuypers et al. [25] have previously reported that hepatic microsomes from Wistar rats are able to oxidise bilirubin in the presence of N A D P H independently of cytochrome P-450 and suggested that lipid peroxides might act as initiators of bilirubin degradation. Using N A D P H or ascorbate to initiate lipid peroxidation [1], we have confirmed their observations in Gunn rats and have attempted to

determine the nature of the reactive oxygen species responsible. Addition of excess C u / Z n superoxide dismutase or catalase to standard assays did not decrease the rate of bilirubin degradation. It is, therefore, unlikely that either superoxide or hydrogen peroxide are responsible for the oxidation of bilirubin by microsomes. When bilirubin was incubated with microsomes under conditions which were shown to maximise hydroxyl radical formation, no bilirubin degradation was detected, though O H was present. This could be due to the presence of 100 mM DMSO, which is a potent scavanger of hydroxyl radical, although this solvent did not prevent the oxidation of bilirubin under conditions favouring lipid peroxidation. Preliminary studies on the identity of the oxidised BR products have not detected propentdyopents, which are formed by hepatic mitochondria [26], or biliverdin, which accumulates during the oxidation of albumin-bound bilirubin by peroxyl radicals [27]. The identity of these products clearly requires further investigation. Since a previous study [1] had shown that plasma BR increased when microsomal LPO was inhibited by feeding Gunn rats with a synthetic fat free diet, it seemed possible that LPO might have an important influence upon bilirubin levels in vivo. However, when these studies were repeated with a rebred strain of Gunn rats, there was an even greater elevation of plasma BR although inhibition of LPO was only modest. The quantitative differences observed between the original and rebred strains favour the view that that hepatic LPO is unlikely to be an important factor governing BR metabolism in these rats in vivo. In order to probe any causal relationship between hepatic LPO and bilirubin metabolism in vivo, the influence of the ant±oxidants vitamin E and vitamin C was investigated. These studies likewise failed to support LPO as a major determinant of plasma BR. A single dose of vitamin E inhibited both microsomal LPO and bilirubin degradation for up to 8 days but did not cause any significant changes in plasma BR regardless of whether the rats were fed a standard or a 25% corn oil diet. Conversely, the administration of vitamin C in doses ranging from 180 to 750 m g / k g for 3 days, did not abolish LPO or bilirubin degradation in vitro. Elevations in plasma BR were seen

M. Joshi et al. / Biochimica et Biophysica Acta 1243 (1995) 244-250

with higher doses of vitamin C but these may reflect its toxicity since rats receiving 750 m g / k g i.p. lost 14 g / d a y body wt. It has been postulated that the increased plasma BR resulting from a fat free diet could be ascribed to decreased hepatic clearance of bilirubin due to changes in the hepatic sinusoidal membrane [28]. The present study confirms that the fatty acid composition of the hepatic microsomal phospholipids from Gunn rats changes in response to diet. Whether these alterations in membrane structure influence the clearance of bilirubin needs further study. Several mechanisms probably exist for the disposal of bilirubin in the Gunn rat. The intestine as well as the liver may be of importance [29,30] and mitochondrial bilirubin oxidases have been described in liver [26] and brain [13]. De Matteis et al. [16] have developed an assay for microsomal bilirubin degradation which is both oxygen and NADPH dependent but differs from that studied here in that EDTA is included in order to prevent LPO and F e / A D P is omitted. Their system is markedly stimulated by cytochrome P450A inducers which are known to decrease hyperbilirubinaemia in the Gunn rat [16,31,32] but do not appear to increase LPO [23]. It was of interest, therefore, to see whether P450 induction was associated with our observed dietary effects on plasma BR. However, no significant differences in microsomal cytochrome P450A (assayed as 7-ethoxyresorufin 0-deethylase activity) were found on the different diets; nevertheless the enzyme inducer fl-naphthoflavone did cause a 16.5% decrease in plasma BR [23]. The original and rebred strains of Gunn rat exhibited a similar degree of unconjugated hyperbilirubinaemia associated with the jaundice locus (jj). However feeding the rebred strain with different fat free diets generally caused an even larger increase in their plasma BR (64% increase by the original strain [1] compared with 102-276% increases by the new strain - Table 3). In addition the membrane peroxidisabilty responses of the two sub-strains to these diets, monitored by incubating their liver microsomes with F e / A D P and a reductant, differed markedly. Thus, microsomes of the original strain failed to peroxidise at all above the low zero time values in 10- or 20-min incubations and showed little LPO even at 60 min, [1]. In contrast, microsomes from the rebred strain incubated for 10 min showed 23-68% of the peroxidation on fat free diets as on stock diet and the two rates approximated at 20 min and were identical in 40 min incubations. This led us to suspect the hypothesis that dietary modulation of plasma BR was mediated by differences in membrane peroxidisability as was suggested by the double bond index results for microsomal phospholipid unsaturated fatty acids in the original strain. A further variation in the dietary bilirubinaemic response in the rebred strain was that on a 25% fat diet only 50% of the rats responded with a decrease in plasma bilirubin compared with 100% response in the original strain. These observations provide further evi-

249

dence that the genetic backgrounds of the Gunn rats produce quite different responses in parameters indirectly related to the jaundice phenotype [33]. Despite the several mechanisms proposed for the disposal of BR in the Gunn rat, none provides an adequate description of the alternate pathway for BR metabolism in untreated animals, which have not been administered enzyme-inducers or fed altered diets. Although many investigators consider that an oxidative pathway is involved, this study does not support a central role either for lipid peroxidation or for other reactive oxygen species in the disposition of bilirubin in Gunn rats.

Acknowledgements This study was supported by the Medical Research Council. The authors are grateful to Duncan Moore and Albert Shaw for the care of the Gunn rats. We would like to acknowledge helpful discussions with Professor R. Bonnett and Dr. F. De Matteis, who kindly provided facilities for the cytochrome P-450 determinations. Dr. D. Harry and Dr. J. Owen gave valuable advice for the analysis of the microsomal phospholipid fatty acid composition.

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