Glutathione Decreased Parenteral Nutrition–Induced Hepatocyte Injury in Infant Rabbits
From the * Department of Pediatric Surgery and Correspondence: Wei Cai, Department of Pediatric Surgery and Clinical Nutrition Center, Xinhua Hospital and Shanghai Children's Medical Center, School of Medicine, Shanghai Jiaotong University, 1665 Kongjiang Road, Shanghai, China. Electronic mail may be sent to caiw204{at}yahoo.com.cn. Background: This study was designed to explore the mechanisms in parenteral nutrition (PN)-associated hepatic dysfunction, and the possible effectiveness of glutathione (GSH) to alleviate this injury. Methods: Thirty 1-week-old New Zealand rabbits were divided into 3 groups: 10 in the control group (maternal fed); 10 in the PN group (PN for 10 days); and 10 in the GSH + PN group (PN plus glutathione for 10 days). At the end of the study, blood biochemistry analysis and liver histologic examination were performed by light and electronic microscope; malondialdehyde (MDA) content of liver tissues and apoptotic hepatocytes were also measured. Results: Direct bilirubin and bile acid in the PN group were significantly higher than that in the control group and in the GSH + PN group (p < .05, for both). In the PN group, there were some cholestatic or steatotic changes. In the GSH + PN group, histologic changes were reduced compared with the PN group. The electron microscopy appearances were in agreement with the histologic findings. MDA value was higher in the PN group than in the control group and in the GSH + PN group (p < .05, respectively). Terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assays showed that the rate of apoptotic hepatocytes in the PN group was the highest and the control group was the lowest among 3 groups (comparison between groups, p < .01, individually.) Conclusions: The study showed that PN can induce hepatic dysfunction in infant rabbits. GSH can effectively reduce this injury. The study implies that oxidative stress and apoptosis contribute to PN-associated hepatic dysfunction. Parenteral nutrition (PN) has been widely used for >30 years. In that time, the prognosis has been improved in patients with gastrointestinal failure or other diseases. However, hepatic dysfunction is a major complication of PN. The cause of PN-associated hepatic dysfunction is still under extensive investigation. Recent studies showed that oxidative stress plays an important role in PN-associated hepatic dysfunction.1–3 In patients requiring PN, both recurrent catheter-related sepsis and bacterial translocation across the intestinal mucosal barrier are major complications. They pose a considerable oxidative challenge, particularly to the liver. Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine and is the most important intracellular antioxidant in mammalian tissues. It protects cells against oxidative damage caused by free radicals through the GSH peroxidase reaction and the detoxification of electrophilic metabolites by multiple GSH transferases. GSH concentration is particularly high in the liver, which is the major site of GSH synthesis and detoxification.4 However, hepatic GSH concentration is decreased during PN.2,5,6 This might be caused by increased GSH consumption, increased oxidation, and conjugation-depleted intracellular GSH stores. On the other hand, oxidative stress induces or accompanies apoptosis,7 and antioxidants can reduce hepatocyte apoptosis by preventing the generation of oxidant stress.8 Furthermore, studies by Albright et al9 showed that reduction of GSH is associated with a higher degree of apoptosis. According to these findings, the present study was conducted to explore the relationship among oxidative stress, apoptosis, and PN-associated hepatic dysfunction. We hypothesize that GSH supplementation to parenteral feeding can protect hepatic function by ameliorating oxidative damage to the liver.
Thirty New Zealand white rabbits (specific pathogen free, age 6–8 days, body weight 80–110 g) were obtained from the animal experiment center of the Chinese Academy of Science. The rabbits were divided into 3 groups: control group (n = 10, maternal fed); PN group (n = 10, PN for 10 days); and GSH + PN group (n = 10), the PN infusion plus GSH (20 mg/kg/d; reduced GSH: TAD, product of Foscama Pharmacy Co, Ferentino, Italy). Immediately following 24 hours' adaptation to the environment, experiments were started. After anesthesia with Shumianxin (a complex of ketamine and haloperidol), all of the rabbits in the PN groups had a Silastic catheter inserted into the superior vena cava through the right jugular vein. The catheter was fixed to the skin of the midscapular region with a swivel spring, which allowed free movement for the caged rabbits. The rabbits were housed in individual cages under controlled conditions (12-hour light and dark cycles and temperature of 30°C–32°C, humidity 60%–70%). The rabbits were initially infused with saline (5% dextrose in 0.9% normal saline) at a rate of 0.5–1 mL/h for 6–8 hours, then this was changed to PN solution with an infusion pump (Graseby 3100, GBR) for 10 days. The PN solution consisted of amino acids, dextrose, fat emulsion, electrolytes, vitamins, and trace elements (Table I). Rabbits in the PN group received the PN at the calorie density of 877 kJ/kg/d (210 kcal/kg/d).10
After 10 days of PN infusion, 3 mL of blood was collected from the inferior vena cava for biochemistry, and euthanasia immediately was performed by an overdose anesthesia. The right lobe of the liver was taken. About 3 mm3 liver tissue was fixed immediately using 5% glutaraldehyde for electron microscopy. Histologic examination was performed using standard H&E staining. According to the Loff et al11 criteria (Table II), an experienced pathologist was invited to score the pathologic changes in every slide in a singleblinded fashion.
The remaining liver tissue was frozen immediately in liquid nitrogen until processing: MDA (malondialdehyde, an oxidative parameter) content in liver tissues was measured by standard TBARS (thiobarbituric acid reactive substances) method (MDA Kit from Jiancheng Bioengineering Institute, NanJing, China). Paraffin sections were used for TUNEL assays (TDT-FragEL Kit; Oncogene, Cambridge, MA) to detect apoptotic hepatocytes. Axioplan 2 imaging system and KS400 version 3.0 software were used to take photos and count the rate of apoptotic positive cells (apoptotic cells/total cells) for every slide. Data were entered into a database and analyzed using SPSS software 13.0; summaries were expressed as mean ± SD. Data analysis was performed using one-way ANOVA (analysis of variance) or nonparametric test and a p value < .05 was considered statistically significant.
Liver Function Test Serum biochemical parameters are shown in Table III. The level of direct bilirubin and bile acid in the PN group was significantly higher than that seen in the control group and in the GSH + PN group (p < .05, individually). Alanine aminotransferase (ALT) level was elevated in the GSH-PN group. However, the differences in other parameters (aspartate aminotransferase [AST],  -glutamyl transpeptidase [-GT], alkaline
phosphatase [AKP], total bilirubin [TBi]) between the groups were not
statistically significant.
Histology
The electron microscopy appearances showed normal mitochondria and nuclei, and dense microvilli in microbile ducts in the control group. However, in the PN group, there was a disappearance of microvilli in the microbile ducts and swollen mitochondria. Apoptotic change (including karyorrhexis and apoptotic body) were found in some specimens. In the GSH-PN group, rare microvilli and mildly dilated microbile ducts were detected (Figure 2), indicating a reduced extent of damage.
The Liver Content of MDA and Apoptosis Index The MDA value in the PN group was higher than that in the control group (2.04 ± 0.44 nmol/mgpro vs 1.35 ± 0.29 nmol/mgpro; p < .05) and in the GSH-PN group (2.04 ± 0.44 nmol/mgpro vs 1.37 ± 0.24 nmol/mgpro; p < .05; Table V). TUNEL assays showed that the apoptosis index (apoptotic hepatocytes positive rate) in the PN group was the highest among the 3 groups (which were control group 0.92 ± 0.85, PN group 44.59 ± 6.68, and GSH-PN group 4.14 ± 2.76, individually). The PN group value was compared with the control group or the GSH-PN group, which showed statistical significance, respectively (Table VI; Figure 3).
Progressive hepatobiliary disease is potentially a life-threatening complication in patients receiving long-term PN, especially in the neonates.12 Numerous factors including "gut starvation," sepsis, endotoxin, and toxicity of PN components have been considered as etiology.13,14 More recent studies have suggested that a combination of oxidative stress and GSH depletion could be an important factor in the pathogenesis of PN-induced hepatic damage.2,5,6 Sokol demonstrated that hepatic GSH levels decreased to 16% of control values after 5 days of PN in weanling rats.2 GSH is a low-molecular-weight thiol that plays a critical role in the cellular defense against oxidative stress in mammalian cells. It removes oxygen-derived free radicals, inhibits lipid peroxidation, and maintains cells' normal structure and function.4 Prolonged PN decreases the GSH levels in blood and liver because of oxidative stress. Recent studies have shown that PN induces epithelial cell apoptosis associated with decreased Bcl-2 mRNA expression.15 Additionally, PN with lipid increased the expression of Fas and both the proapoptotic factor Bad and the antiapoptotic factor Bcl-xl. These changes may contribute to PN-induced hepatocyte injury (apoptosis), or they may suppress hepatocyte regeneration.16 Diminished GSH levels have been seen to be association with the early stages of apoptosis.8 The ability to decrease intracellular oxidative and apoptotic damage has been shown to correlate directly with GSH level. Dzakovic et al17 reported that targeted trophic feeding of precursors for GSH synthesis (glutamine, cysteine, and glycine) during PN almost doubled hepatic GSH concentration and prevented liver oxidative damage. The Schauer et al18 studies showed that IV GSH administration during reperfusion to ischemic livers prevented reperfusion injury in rats. However, little literature has investigated whether IV GSH prevents PN-associated hepatic dysfunction directly.
The present study showed that in infant rabbits, continuous PN for 10 days results in severe liver injury, including increased bile acid and direct bilirubin levels. Histologic and electron microscopy manifestations confirm this cholestatic injury. In the PN group, MDA values and apoptosis index were higher than those in the control group, demonstrating that oxidative damage and apoptosis may play important roles in PN-associated liver injury. On the other hand, bile acids, direct bilirubin levels, and histologic score in the GSH-PN group are lower than those in the PN group. Significantly, the electron microscopy manifestations displayed similar results. At the same time, MDA values and apoptosis index in the GSH-PN group were lower than those in the PN group. Inconsistently, ALT level increased in the GSH-PN group. Routine laboratory tests are of little relevance in the beginning of PN-induced hepatobiliary dysfunction in humans. Several investigators stress that often there is a discrepancy between severe histologic damage and moderate or absent pathologic findings in routine laboratory investigations.19,20 Loff et al21 also found that within the first 4 weeks of PN, biochemical changes in infants and rabbits are inconsistent. The examination of bile acid may draw a more precise and early picture of the damage.22 In the Loff et al21 and Hata et al10 rabbit models of PN, they found that mild to moderate periportal inflammation and foci necrosis occurred not only in the PN group but also in the control group. Our experiments showed similar results, and severe histologic changes such as cholestasis or fibrosis occurred only in the PN groups. The pathologic changes in the control group may be caused by anesthesia, experiment procedure, or other endogenous factors. The statistical difference in histologic score between the 3 groups excluded these factors and showed the meaning of the intervention's (PN or GSH) effect. On the other hand, there are no normal ranges of biochemical parameters in infant rabbits. Therefore, preferable biochemical parameters that demonstrated PN-associated hepatobiliary dysfunction, especially in animal model, should be further studied.
In conclusion, our experiments showed that IV GSH administration during PN can alleviate liver injury and decrease hepatocyte oxidative damage and apoptosis. We concluded that IV exogenous GSH dosage supplement can decrease PN-associated liver injury in infant rabbits. This effect may be related to the nature of GSH that defends against oxidative stress and antiapoptosis. Further studies should be performed to explain the individual or interactive pathways of oxidative and apoptotic damage in this PN-associated hepatic dysfunction, and related clinical trials are needed to assess GSH effectiveness in human beings. The authors thank Professor Aifen Fu for her perfect work of histologic scoring. We would also like to thank the Experimental Center of Xinhua Hospital and Shanghai Children's Medical Center for their assistance in the study. Received for publication July 16, 2006. Accepted for publication December 4, 2006.
Journal of Parenteral and Enteral Nutrition, Vol. 31, No. 3,
199-204 (2007)
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MATERIALS AND METHODS
RESULTS
