Basic research
The effects of palmvitee on d-aminolevulinic acid-induced hyperbilirubinaemia in suckling rats

Introduction
Jaundice or hyperbilirubinaemia is a common phenomenon that occurs during the first days of postnatal life and it affects nearly two thirds of human newborns. It is often attributed to physiological immaturity of bilirubin metabolism, leading to excessive quantities of unbound bilirubin that can cross the blood brain barrier, damage the neuronal tissue and finally can increase the risk of neonatal death due to kernicterus [1]. Enhanced erythrocyte destruction rate contributes about 75% of bilirubin metabolism [2] in neonates and this leads to a higher production of bilirubin. The increased production of bilirubin exceeding its elimination activity, plays an important role in the mechanism of neonatal hyperbilirubinaemia [3]. The major factor regulating the elimination of bilirubin is the rate of its glucuronidation in the liver which is catalyzed by UDP-glucuronyltransferase (UGT) [4]. Bilirubin is taken up by the liver and conjugated with glucuronides forming water-soluble and more easily excreted bilirubin [5]. Therefore, a low activity of UGT will increase the level of unconjugated bilirubin, which is lipid soluble and neurotoxic [3]. Rifampicin was reported to successfully treat unconjugated hyperbilirubinaemia in patients with Gilbert syndrome, a syndrome caused by a deficiency in UGT activity via the induction of its isoenzyme, UGT1A1 [6].
Phototherapy, an accepted modality for neonatal hyperbilirubinaemia management has been related to increased oxidative stress. In full term jaundiced neonates, plasma lipid peroxidation was elevated after the therapy [7, 8]. Therefore, the therapy can expose the infants to potent oxidative stress, and alternative treatment should be sought.
a-Tocopherol afforded a protective effect against hyperbilirubinaemia in a few studies. It decreased serum total bilirubin and the duration of phototherapy in the preterm [9] and full term babies [10]. The protective effect of the a-tocopherol could be due to its antioxidative property as well as its stabilizing effect on the erythrocytes [11]. Palmvitee, a vitamin E extract from palm oil, contains both tocopherol and tocotrienol. Similar to tocopherol, tocotrienol has also been shown to possess high antioxidative activity [12] and somewhat better than the former [13]. Abdul-Razzak et al. [14] had demonstrated that low level of plasma vitamins C and E were associated with significant hyperbilirubinaemia in full term neonates due to increased oxidative stress and intern red blood haemolysis. The red blood cells of preterm neonates were more prone to lipid peroxidation than that of the full term neonates [15].
Our previous study [16] has shown that maternal supplementation of the palmvitee throughout pregnancy reduced hyperbilirubinaemia in rat neonates. Therefore, in this study, we investigated the effects of the palmvitee administration in suckling rats on hyperbilirubinaemia.

Material and methods
Animals, reagents and diet
Fifteen-day pregnant female Wistar were supplied by the Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia. The pups born later were housed together with their littermates and individual mothers in polyethylene cages sized 45 × 28 × 20 cm during the course of the treatment. The dams were given free access to a commercial rat chow (Gold Coin Ltd., Malaysia) and water.
All chemicals and enzymes were of highest grade obtainable from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. The palmvitee used in this study was prepared by Malaysian Palm Oil Board according to Gapor et al. [17], comprising 21% a-tocopherol, 17% a-tocotrienol, 4% g-tocopherol, 33% g-tocotrienol and 24% d-tocotrienol. Olive oil being the edible oil with the lowest content of vitamin E [18] was used as vehicle. The rat chow contained about 25.11 mg/kg total vitamin E and its composition is as follows (mg/kg food): a-tocopherol acetate 10.2, a-tocopherol 5.43, g-tocopherol 0.8, a-tocotrienol 2.69, g-tocotrienol 4.54 and d-tocotrienol, 1.38.

Experimental design
Sixty suckling rats were divided into two groups. They were given 30 mg/kg body palmvitee intraperitoneally once daily or vehicle (control group) starting from day 1 postnatal for 14 days. At day 14 postnatal, each group was subdivided into another two groups; one group was administered d-aminolevulinic acid (ALA, 500 µmol/kg body weight) intraperitoneally in three separate doses, 24, 20 and 16 h prior to sacrifice, following a procedure described by Drummond and Kappas [19] and another group was given an equivalent volume of normal saline. The final volume of each injection was 0.1 ml. The dose of the ALA and the age of the sucklings used in this study were based on our previous study [16]. Twenty-four hours after the first dose of ALA, the neonates were sacrificed. Neonatal blood via cardiac puncture and livers were taken under diethyl ether anesthesia. The plasma and livers were stored at –70°C until subsequent analyses. Pups with lysed blood were excluded because that would affect the bilirubin mea-surement.
The experimental procedure and animal handling in this study were approved by the Universiti Kebangsaan Malaysia Animal Care and Use and Medical Research Ethics Committee, which conforms to Malaysian Ministry of Science, Technology and Innovations guidelines.

Biochemical parameter measurements
Plasma total bilirubin was determined by means of a commercially available kit using an automated analyzer Cobas Mira S (Roche Diagnostic, Switzerland). The hepatic microsomes were prepared following a standard procedure [20] prior to the measurement of UDP-glucuronyltransferase (UGT) activity [21]. Malondialdehyde measured as thiobarbituric acid reactive substance (TBARS) content [22] and protein concentration [23] were determined in the livers of the neonates following the established methods. Hepatic vitamin E was extracted using the method of Podda et al. [24] with some modifications [25] and analysed using an analytical high performance liquid chromatography (HPLC; Waters Corp., Milford MA, USA).

Statistical analysis
Results are expressed as mean ± standard error of mean. Data were statistically compared using one way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test using GraphPad Prism 2.1 software (1997; GraphPad Software Inc., San Diego, CA, USA). A p value < 0.05 was considered to indicate a significant difference between the groups.

Results

Effect of palmvitee on plasma total bilirubin
ALA administration increased plasma total bilirubin in rat neonates (Figure 1). Palmvitee pretreatment (30 mg/kg body weight) for 14 days, prevented the increase significantly (p < 0.05).

Effect of palmvitee on hepatic UGT
Figure 2 shows that ALA administration did not affect the activity of UDP-glucuronyltransferase (UGT) significantly in the control group. However, in the neonates given palmvitee, the enzyme activity in the ALA-treated group was significantly lower than the saline-treated group (p < 0.05).


In Figure 3, the hepatic lipid peroxidation measured as thiobarbituric reactive acid substance (TBARS) was not affected by the ALA administration. Howver, in the palmvitee pretreatment groups, the hepatic TBARS content was reduced significantly (p < 0.05).

Effect of palmvitee administration on hepatic vitamin E content
Only a- and g-tocopherols were detected in the livers of the rat neonates from the control group. ALA however, did not affect neonatal hepatic vitamin E content. Palmvitee pretreatment for 14 days increased all the tocopherol and tocotrienol isomers in the neonatal liver (Table I).

Discussion
In this study, as expected, d-aminolevulinic acid (ALA) administration increased the plasma total bilirubin, and palmvitee pretreatment prevented the increase. This finding is in agreement with our previous study which showed that maternal administration of palmvitee reduced the parameter in the rat neonates [16]. a-Tocopherol has been shown to reduce plasma total bilirubin in preterm and term hyperbilirubinaemic newborns [9, 10]. It is believed that the low levels of the antioxidant vitamins (C and E) may predispose them to in-creased oxidative stress and lead to hyper-bilirubinaemia [14, 26].
ALA administration was used as a hyper-bilirubinaemia model, by increasing haem synthesis and finally bilirubin production in ani-mal models dose-dependently. The induction of hyperbilirubinaemia in rat neonates should be done between the age of 7 and 21 days due to inconsistencies in haem metabolism before the former age and maturation of hepatic glucuronidation after the latter age [19]. In our study, ALA administration did not affect the activity of UDP-glucuronyltransferase (UGT) in the control group. However, in the palmvitee-treated neonates, the significant reduction of the enzyme was observed in the ALA-treated group compared to the saline-treated group. In our previous study [16], the maternal administration of palmvitee at 50 and 200 mg/kg body weight decreased the hepatic UGT activity in the rat neonates. It is possibly that palmvitee competes with the bilirubin for the binding site in the enzyme, produces conformational changes that impede the binding of the substrate or depletes the hepatic UDP-glucuronic acid, the same hypotheses made on silymarin which was demonstrated to inhibit UGT1A1 [27, 28], the isoenzyme of UGT that metabolizes bilirubin. This isoenzyme is detectable from day 22 of gestation and gradually increases after birth in rats [29]. We could not explain why the inhibition of the enzyme was apparent in the palmvitee-treated group given ALA. These will need further investigation. However, we can suggest the use of phenobarbitone as a countermeasure should the palmvitee be used clinically in the treatment of neonatal jaundice and the reduction of UGT by the vitamin become apparent. Phenobarbitone is used clinically to treat neonatal jaundice [30] by inducing the UGT1A1 activity [31]. Nonetheless, the interaction that may occur between palmvitee and phenobarbitone requires another study.
Diethyl ether anesthesia was reported to reduce bilirubin glucuronidation which finally affects the UGT activity [32]. In our preliminary study, we found that the anesthestic did not influence the UGT activity sgnificantly (data not shown), could be due to a shorter duration of exposure in our study i.e. less than a minute, than reported by Dills and Klaassen [32] (30 min to 4 h).
Our finding suggests that the decrease in plasma total bilirubin by palmvitee was not mediated by the increase in the hepatic bilirubin glucuronidation activity. a-Tocopherol was reported to decrease haem oxygenase activity, the rate-limiting enzyme in haem synthesis [33] which was found to be elevated after ALA administration [19, 34]. It could be that the palmvitee acts via the same mechanism. It is noteworthy to mention that this current study was a preliminary study. It will need further investigation regarding the effects of palmvitee on haem oxygenase and other enzymes involved in the bilirubin metabolism. On the other hand, Yin Zhi Huang, a traditional Chinese herbal medicine which has been used in neonatal jaundice treatment was shown to enhance bilirubin clearance and induce the expression of UGT [35].
Hepatic TBARS was not increased following ALA administration. However, the increase in hepatic lipid peroxidation was demonstrated following ALA administration at 40 mg/kg body weight for 10 days [36]. The dissimilarity could be due to the differ-ence in ALA exposure. Plasma lipid peroxidation was also shown to be increase in babies with hyperbilirubinaemia compared to the healthy controls [7, 37]. Palmvitee pretreatment reduced this parameter significantly and this is afforded by its antioxidant property. In our previous study [16], ALA and maternal palmvitee pretreatment did not affect hepatic TBARS. The discrepancy could be due to the difference in dosage of palmvitee used and exposure timing. In the present study, 100% of the palmvitee administered received by the sucklings, but it was reported that in vitamin E placental transfer, only 20 to 30% those of the mother reached the fetus [38].
In the suckling rats pretreated with palmvitee, there was an increase in both hepatic tocopherol and tocotrienol contents, indicating that both types of vitamin E were taken up by the liver. ALA did not have any significant effect on the hepatic vitamin content. In the control groups, only tocopherol isomers were detected. More than 90% of the total vitamin E detected in the control groups was in the form of a-tocopherol, but in the groups given palmvitee, the hepatic vitamin E distribution were similar to the composition of the palmvitee used i.e. the highest being the g-tocotrienol followed by a-tocopherol, d-tocotrienol, a-tocotrienol and g-tocopherol. In adult rats fed a combination of a-tocopherol and a-tocotrienol, the major isomer detected was a-tocopherol (more than 90%) [39]. A possible explanation could that a-tocopherol transfer protein, the protein which is responsible for biodiscriminational uptake of a-tocopherol into tissues [40] is poorly expressed in the neonatal rat liver. The activity of the protein results in high concentration of a-tocopherol in various tissues compared to the tocotrienols. However, in our previous study [16], in maternally palmvitee-pretreated neonates, the major vitamin E isomer detected was a-tocopherol, while d-tocotrienol was not detected at all. The hepatic protein of a-tocopherol transfer protein expression was very low immediately after birth and only increased steadily during the two weeks of life before weaning [41], but it is mainly expressed in the mouse uteri [42].
In conclusion, the results obtained showed a promising therapeutic effect of palmvitee. The protective effect of the palmvitee in reducing plasma total bilirubin is possibly neither through its antioxidative mechanism nor by enhancing the activity of UGT. The exact mechanism of palmvitee would be pursued further. Its concomitant use with other drugs which undergo glucuronidation, also needs further investigations.

Acknowledgments
The authors wish to thank Mr Arizi Aziz (UKM) for his technical assistance. This work was supported by a grant from Universiti Kebangsaan Malaysia (UKM F66/99).

References
1. Shah Z, Chawla A, Patkar D, Pungaonkar S. MRI in kernicterus. Australas Rad 2003; 47: 55-7.
2. Shapiro SM. Bilirubin toxicity in the developing nervous system. Pediatr Neurol 2003; 29: 410-21.
3. Drummond GS, Kappas A. Chemoprevention of severe neonatal hyperbilirubinaemia. Semin Perinatol 2004; 28: 365-8.
4. Mackenzie PI, Owens IS, Burchell B, et al. The UDP-glycosyltransferase gene superfamily: Recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 1997; 7: 255-69.
5. Dennery PA. Pharmacological interventions for the treatment of neonatal jaundice. Semin Neonatol 2002; 7: 111-9.
6. Ellis E, Wagner M, Lammert F, et al. Successful treatment of severe unconjugated hyperbilirubinaemia via induction of UGT1A1 by rifampicin. J Hepatol 2006; 44: 243-45.
7. Dahiya K, Tiwari AD, Shankar V, Kharb S, Dhankhar R. Antioxidant status in neonatal jaundice before and after phototherapy. Indian J Clin Biochem 2006; 21: 157-60.
8. Aycicek A, Erei O. Total oxidant/antioxidant status in jaundiced newborns before and after phototherapy. J Pediatria (Rio J) 2007; 83: 319-22.
9. Gross SJ. Vitamin E and neonatal hyperbilirubinaemia. Pediatrics 1979; 64: 321-23.
10. Pourarian S, Khalili HA. Effect of vitamin E on neonatal jaundice (G6PD vs. Normal) Iranian J Med Sci 2000; 25 (3-4). [Cited 12 June 2003]. Available from URL: http//www.sums.ac.ir/0034/pourarian0034.html.
11. Urano S, Inomori Y, Sugawara T, et al. Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behaviour. J Biol Chem 1992; 267: 18365-70.
12. Asmadi AY, Adam A, Wan Ngah WZ, et al. Tocotrienols and alpha-tocopherol reduced acute and chronic lung lipid peroxidation induced by paraquat in rats. Pakistan J Nutr 2005; 4: 97-100.
13. Maniam S, Mohamed N, Shuid AN, Soelaiman IN. Palm tocotrienol exerted better antioxidant activities in bone than alpha-tocopherol. Basic Clin Pharmacol Toxicol 2008; 103: 55-60.
14. Abdul-Razzak KK, Nusier MK, Obediat AD, Salim AM. Antioxidant vitamins and hyperbilirubinaemiain neonates. GMS Ger Med Sci 2007; 5: Doc03.
15. Agil A, Fraile R, Acuňa-Castroviejo. Changes in plasma susceptibility to lipid peroxidation and vitamin C in preterm C in preterm and full-term neonates. Arch Med Sci 2008; 4: 324-8.
16. Kamisah Y, Norhayati MY, Mazliadiyana M, Zakri B, Zaliza-wati DS, Asmadi AY. Maternal palmvitee administration reduced plasma total bilirubin and uridine diphosphate glucuronyltransferase activity in hyperbilirubinaemic rat neonates. Arch Med Sci 2008; 4: 32-9.
17. Gapor MT, Leong WL, Ong ASH, Kawada T, Watanabe H, Tsuchiya N. Production of high concentration tocopherols and tocotrienols from palm oil byproducts. US Patent No. 5,190,618. 2 March 1993; Malaysian Patent No. MY-110779-A.
18. Cottrell RC. Introduction: nutritional aspects of palm oil. Am J Clin Nutr 1991; 53: 989S-1009S.
19. Drummond GS, Kappas A. An experimental model of postnatal jaundice in the suckling rat: Suppression of induced hyperbilirubinaemia by Sn-protoporphyrin. J Clin Invest 1984; 74: 142-9.
20. Kamimura S, Gaal K, Britton RS, Bacon BR, Triadafilopoulos G, Tsukamoto H. Increased 4-hydroxynonenal levels in experimental alcoholic liver disease: association of lipid peroxidation with liver fibrogenesis. Hepatology 1992; 16: 448-53.
21. Temple AR, Done AK, Clement MS. Studies of glucuronidation: the measurement of p-nitrophenyl glucuronide. J Lab Clin Med 1971; 77: 1015-9.
22. Ledwozyw A, Michalak J, Stepian A, Kadziolka A. A relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986; 155: 275-85.
23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin Phenol reagent. J Biol Chem 1951; 193: 265-75.
24. Podda M, Weber C, Traber MG, Packer L. Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols and ubiquinones. J Lipid Res 1996; 37: 893-901.
25. Kamisah Y, Norhayati MY, Zakri B, et al. Comparative distribution of tocotrienols in livers of suckling and adult rats. Pakistan J Nutr 2006; 5: 539-43.
26. Turgut M, Basaran O, Cekmen M, Karatas F, Kurt A, Aygun AD. Oxidant and antioxidant levels in preterm newborns with idiopathic hyperbilirubinaemia. J Paediatr Child Health 2004; 40: 633-7.
27. D’Andrea V, Perez LM, Pozzi EJ. Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide. Life Sci 2005; 77: 683-92.
28. Chrungoo VJ, Reen RK, Singh K, Singh J. Effects of silymarin on UDP-glucuronic acid and glucuronidation activity in the rat isolated hepatocytes and liver in relation to D-galactosamine toxicity. Indian J Exp Biol 1997; 35: 256-63.
29. Bustamante N, Cantarino MH, Arahuetes RM, Cubero FJ, Ortiz A. Evolution of the activity of UGT1A1 throughout the development and adult life in a rat. Life Sci 2006; 78: 1688-95.
30. Murki S, Dutta S, Narang A, Sarkar U, Garewal G. A randomized, triple-blind, placebo-controlled trial of prophylactic oral phenobarbital to reduce the need for phototherapy in G6PD-deficient neonates. J Perinatol 2005; 25: 325-30.
31. Mirkov S, Komoroski BJ, Ramírez J, et al. Effects of green tea compounds on irinotecan metabolism. Drug Metab Dispos 2007; 35: 228-33.
32. Dills RL, Klaassen CD. Decreased glucuronidation of bilirubin by diethyl ether anesthesia. Biochem Pharmacol 1984; 33: 2813-4.
33. Ossola JO, Groppa MD, Tomaro ML. Relationship between oxidative stress and haem oxygenase induction by copper sulfate. Arch Biochem Biophys 1997; 337: 332-7.
34. Anderson KE, Drummond GS, Freddara U, Sardana MK, Sassa S. Porphyrogenic effects and induction of haem oxygenase in vivo by delta-aminolevulinic acid. Biochem Biophys Acta 1981; 676: 289-99.
35. Elferink RO. Yin Zhi Huang and other plant-derived preparations: where herbal and molecular medicine meets. J Hepatol 2004; 41: 691-5.
36. Noriega GO, Tomaro MI, del Batlle AM. Bilirubin is highly effective in preventing in vivo delta-aminolevulinic acid-induced oxidative cell damage. Biochim Biophys Acta 2003; 1638: 173-8.
37. Yigit S, Yurdakok M, Kilinc K, Oran O, Erdem G, Tekinalp G. Serum malondialdehyde concentration in babies with hyperbilirubinaemia. Arch Dis Child Fetal Neonatal Ed 1999; 80: F235-7.
38. McEvoy GK (ed). American hospital formulary service – Drug information 1993, Bethesda, MD: American Society of Hospital Pharmacists, Inc. 1993 (Plus Supplements); 2314.
39. Ikeda S, Tohyama T, Yoshimura H, Hamamura K, Yamashita K. Dietary alpha-tocopherol decreases alpha-tocotrienol but not g-tocotrienol concentration in rats. J Nutr 2003; 133: 428-34.
40. Blatt DH, Leonard SW, Traber MG. Vitamin E kinetics and the function of tocopherol regulatory proteins. Nutrition 2001; 17: 799-805.
41. Kim H, Arai H, Arita M, et al. Age-related changes of alpha-tocopherol transfer protein expression in rat liver. J Nutr Sci Vitaminol 1996; 42: 11-8.
42. Kaempf-Rotzoll DE, Igarashi K, Aoki J, et al. alpha-Tocopherol transfer protein is specifically localized at the implantation site of pregnant mouse uterus. Biol Reprod 2002; 67: 599-604.