EFFECTS OF TREATMENT WITH MELATONIN AND TRYPTOPHAN ON LIVER ENZYMES, PARAMETRS OF FAT METABOLISM AND PLASMA LEVELS OF CYTOKINES IN PATIENTS WITH NON-ALCOHOLIC FATTY LIVER
DISEASE - 14 MONTHS FOLLOW UP

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic hepatic pathology (1). Its prevalence in the developed countries is estimated at 1/3 of the population (2). The clinical spectrum of NAFLD involves simple hepatic steatosis, non-alcoholic steatohepatitis (NASH) and cirrhosis with all the features of portal hypertension (3-5). NAFLD may remain stable for a long time; however, in about 32–37% of patients, histological progression is likely to be observed within 3–6 years whereas in 12% of the affected cirrhosis may develop within 8–10 years (4-6). Advanced fibrosis accompanying NASH inevitably progresses to cirrhosis and consequently to hepatocellular carcinoma. Therefore, identification of advanced fibrosis in patients with NAFLD is of crucial importance.

In recent years the pathophysiology of NAFLD has been extensively studied. Amongst the numerous pathogenetic factors, oxidative stress and apoptosis of hepatocytes are involved in the progression of disease, particularly in transformation of NASH to cirrhosis (7-11). Day and James (12) have suggested the 'double-hit' theory, according to which the 'first hit' induces fat accumulation in the liver whereas the 'second hit' involves oxidative stress resulting in lipid peroxidation, stellate cell activation and fibrogenesis. The dysfunction of mitochondria are likely to play the pivotal role in induction of both "hits" as the mitochondria are involved in β-oxidation of free fatty acids and are the main cellular source of reactive oxygen metabolites (13). In patients with NASH, the activity of the mitochondrial respiratory chain (MRC) is markedly reduced (14). Despite advances in understanding of pathomechanism of NAFLD and numerous studies on novel therapeutic options, the standards of pharmacological treatment have not been established. Some new effective treatment methods for NAFLD are still being searched for, including those that could effectively reduce both, the oxidative stress, apoptosis of hepatocytes and finally inhibiting progression of steatosis into fibrosis.

Melatonin (N-acetyl-5-methoxytryptamine) produced by the pineal gland is considered an endogenous substance exerting the antioxidative, anti-inflammatory and anti-apoptotic activity (15, 16). Melatonin is a scavenger of reactive oxygen metabolites (17); therefore, its use in NAFLD could be recommended, considering the pathogenetic mechanisms involved in the development of NAFLD, especially NASH. Melatonin is produced from its precursor L-tryptophan in human body. L-tryptophan cannot be synthesized by the organism, and therefore is provided from daily diet and ingested due to digestion and intestinal absorption. Tryptophan is an important source for the few other compounds such as serotonin, niacin and auxin. Serotonin in turn, can be converted to melatonin via N-acetyltransferase (NAT) and 5-hydroxyindole-O-methyltransferase (HIOMT) activities.

The aim of our present study was: (1) to determine the effects of tryptophan and melatonin on the selected biochemical parameters in patients with NAFLD, and (2) to evaluate the effects of daily treatment with tryptophan and melatonin on selected parameters of liver tissue in patients with NAFLD. The results presented in this study are the continuation of the study published previously (18) in which melatonin or its precursor L-tryptophan given for shorter period of time (four weeks) was found to be highly effective in reducing the proinflammatory cytokines in patients with NAFLD.

MATERIAL AND METHODS

The protocol of the study concept and all procedures were approved by the local Ethics Committee in Lublin (no. 23/2008).

Patients

The study encompassed 74 patients with NAFLD, including 51 males aged 22–56 years and 23 females aged 26-47 years. The diagnosis of NAFLD was based on morphological testing of liver biopsy; 56 patients were diagnosed with simple hepatic steatosis and the remaining 18 patients with NASH. Microscopic evaluation of liver biopsy specimens was based on the current recommendations (19). The histopathological diagnosis of adult NASH was based on the presence of certain microscopic features accepted in 2005 by NASH Clinical Research Network (NASH CRN) (19, 20). Briefly, these histopathological criteria for NASH diagnosis include: steatosis of hepatocytes, hepatocyte ballooning, lobular inflammation and fibrosis in advanced stage. In liver biopsies diagnosed as simple hepatic steatosis, microvesicular accumulation of fat droplets (macro- or microvesicular) within hepatocytes was observed. This accumulation of fat was not associated with degenerative changes within their cytoplasm or lobular inflammation. The concomitant conditions included arterial hypertension (50 patients), diabetes mellitus (34 patients including 22 requiring insulin therapy and 12 receiving oral anti-diabetic drugs), glucose intolerance (3 patients). Forty-two patients showed dysfunctional fat metabolism: hypercholesterolemia was diagnosed in 34, hypertriglyceridemia in 22 and elevated LDL cholesterol levels in 18 persons. None of the enrolled patients received cholesterol-lowering agents before and during the study.

Procedures

On inclusion, the following biochemical parameters were determined in all patients: alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGTP), bilirubin, lipid profile (total cholesterol, LDL and HDL, cholesterol, triglycerides), IL-1, IL-6 cytokines, TNF-α and melatonin.

For routine laboratory assays standard automated techniques were used (Alab Sp. z o.o. Lublin, Poland). Cytokines and TNF-α were examined by ELISA test (Cytokine Immunoassays, R&D System, USA). Plasma melatonin concentration was determined using RIA-technique as described in details previously (21). Blood samples were collected in EDTA-coated polypropylene tubes and centrifugated at 1700 g for 20 minutes at 4°C. Than plasma was stored at –60°C. Melatonin was determined using Human MT RIA kit (DRG Diagnostics GmbH, Marburg, Germany).

The patients were randomly assigned to three groups. The characteristics of patients are presented in Table 1.

Table 1. Characteristics of patients and study grups: I (tryptophan), II (melatonin), III (placebo).

n=number of patients

Group I received 300 mg of phospholipids, 3 times a day - glycerol esters of cholinephosphoric acid and unsaturated fatty acids (linolic, linoleic, oleic) in the form of preparation Essentiale (Rhone-Poulenc Rorer GmBH, Germany) in the dose of 3 × 1 tablet/day and Tryptophan (Ardeydorm, Ardeypharm, Germany) 2 × 500 mg/day over the period of 14 months.

Group II received 300 mg of phospholipids, 3 times a day - glycerol esters of cholinephosphoric acid and unsaturated fatty acids (linolic, linoleic, oleic) in the form of Essentiale (Rhone-Poulenc Rorer GmBH, Germany) and Melatonin (Lekam, Zakroczym, Poland) 2 × 5 mg/day over 14 months.

Group III received 300 mg of phospholipids, 3 times a day - glycerol esters of cholinephosphoric acid and unsaturated fatty acids (linolic, linoleic, oleic) in the form of Essentiale (Rhone-Poulenc Rorer GmBH, Germany) done over the period of 14 months.

None of the patients enrolled for the study was treated with any preparations containing antioxidative compounds, vitamins or diet supplements before and during the study. After 14 months, the above-mentioned biochemical parameters were re-determined in each group of patients.

After 14 months treatment period the liver biopsy which was optional was performed in patients who agreed for this procedure. Percutaneous liver biopsy was performed by Tru-Cut automatic needle 16 G (COOK Quick-Core® Biopsy-Needle, Cook Medical Inc., USA) with specimen size obtained at least 2 cm long with presence of no fewer than 11 complete portal tracts. Liver biopsy specimens were fixed in 10% neutral buffered formalin and were processed to paraffin blocks. Four µm thick slides were cut on the microtome and stained with haematoxylin and eosin (H+E), Masson Trichrome, silver stain and PAS. Slides were then examined by one pathologist under the light microscope OLYMPUS CX 45.

All patients gave their informed written consent to be recruited for the study.

Statistical analysis

All results were expressed as a mean ± standard de viation. Numeric data were analyzed by the t-student test. Statistical significance was assumed at p<0.05. All calculations were performed using STATISTICA PL software.

RESULTS

No side effects of melatonin and tryptophan were observed; for instance none of patients complained on excessive sleepiness and/or dizzines.

As shown in Table 2 after the 14-month treatment period, GGPT activity and the plasma levels of triglycerides and LDL-cholesterol were significantly reduced in group I and II. The remaining lipidogram parameters did not differ between these groups. The level of melatonin after the therapy with melatonin and tryptophan was significantly elevated in group I and II compared with that recorded in group III. Moreover, statistically significantly lower plasma levels of IL-1, IL-6 and TNF-α were observed in patients receiving melatonin and tryptophan after 14-months therapy (Table 3). The remaining biochemical parameters did not show significant differences after the 14-month treatment period compared with respective values observed at the initiation of the study.

Table 2. Comparison of plasma levels of liver enzymes and biochemical metabolic parameters in the study groups I, II and III after.

The values are given as mean ± standard deviation. NS- differences not statistically significant. Asterisk (*) indicates statistically significant (p<0.05) decrease in plasma levels of GGTP, triglycerides and LDL-cholesterol measured after 14 month of treatment with tryptophan or melatonin compared to the baseline level (recorded at the start of the study).
Except values of GGTP, triglycerides and LDL cholesterol the differences between the other measured parameters in groups I, II and III were not statistically significant.

Table 3. Comparison of plasma levels of pro-inflammatory cytokines and melatonin in the study groups I, II and III after 14 months of treatment.

The values are given as mean ± standard deviation. Asterisk (*) indicates statistically significant decrease (p<0.05) below the values recorded at the start of the treatment. Cross (+) indicates statistically significant (p<0.05) increase above the values recorded at the start of the treatment.

In group III receiving Essentiale, the differences in all the biochemical parameters studied were not statistically significant at the start of study and at follow up 14 months later.

In the nine patients of all groups the liver biopsy was performed after the end of 14-months treatment period. Three of the patients were from group I, 4 out of 9 from group II and remaining 2 were from group III, respectively.

In patients from group I and II the histological examination of liver showed NASH, (Figs. 1 and 2) After treatment in all of them, the features of steatosis were observed (Figs. 3 and 4). NASH was not found in any of them. Results of liver biopsy were not changed in group III. Figs. 1-4 present microscopic examples of NASH. Macrovesicular steatosis and lobular inflammation are most prominent on Fig. 1, while Fig. 2 shows scattered fatty change in hepatocytes associated with ballooning of hepatocytes and focal lobular inflammation.

Fig. 1. Liver biopsy. NASH (non-alcoholic steatohepatitis). Macrovesicular steatosis, ballooned hepatocytes, focal intralobular inflammation and scattered necrotic hepatocytes. H+E × 400.

Fig. 2. Liver biopsy NASH (non-alcoholic steatohepatitis). Macrovesicular steatosis, ballooned hepatocytes, focal intralobular inflammation and scattered necrotic hepatocytes. Single apoptotic hepatocyte present. H+E × 400.

Oedema within the cytoplasm of hepatocytes forming ballooning change are also well seen on the Fig. 2 and Fig. 4. The first one shows also foci of lobular inflammation. Minor microscopic changes are shown on Fig. 3. Fig. 4 shows also some microvesicular steatosis.

Fig. 3. Liver biopsy. NASH (non-alcoholic steatohepatitis). Focal macrovesicular steatosis. Liver cells show mild oedema of their cytoplasm and glycogenated nuclei. H+E × 400.

Fig. 4. Liver biopsy. NASH (non-alcoholic steatohepatitis). Focal macrovesicular steatosis. Liver cells show mild edema of their cytoplasm and enlarged nuclei. Very focal mononuclear inflammatory cells present within the lobule. H+E × 400.

DISCUSSION

Our present study which follows up of short term observation published before (18), demonstrated the decreased plasma levels of GGTP, triglycerides and LDL cholesterol after the 14-month therapy with melatonin and its precursor tryptophan. Moreover, the plasma levels of the pro-inflammatory cytokines IL-1, IL-6 and TNF-α, were found to be attenuated in patients treated with melatonin and tryptophan. The level of total cholesterol, HDL-cholesterol and activity of aminotransferases, ALT, AST and ALP and GGTP did not show statistically significant differences after 14 months of treatment with melatonin or tryptophan.

The literature data on this issue are scarce. The similar research was conducted by Gonciarz et al., yet their observation period was shorter, i.e. 4, 8, 12 and 24 weeks of treatment with melatonin (21, 22). According to their study, the mean levels of aminotransferases ALT and AST after the 24-week treatment with melatonin were 42% and 33%, respectively (p<0.05), compared with the baseline levels in patients with NAFLD (22). These findings differ from our results, as our study did not show statistically significant differences in the activity of aminotransferases throughout the period of treatment with melatonin. As far as triglycerides are concerned, our data reported reduced levels of triglycerides whereas the results published by Gonciarz et al. (22) showed no changes in their levels. This discrepancy is likely to result from the longer period of treatment with melatonin in our study, namely 14 months versus 24 weeks, respectively. On the other hand, the results concerning GGTP activity (22) are comparable in both studies. Both, our results and those reported by Gonciarz et al. demonstrated reduced activity of GGTP in patients who underwent the therapy with melatonin and tryptophan (22).

GGTP was considered as surrogate marker of NAFLD and NASH and is positively associated with cardiovascular events (23). Some researches believe that triglycerides can play the same role. Cardiovascular disease is increased in NAFLD and represents the main cause of death in these patients (24). It is found that NAFLD is a predictor of cardiovascular events (25). Longitudinal increase of GGTP level is observed in patients with cardiovascular disease and NAFLD (25). The oxidative stress and the dysfunction of mitochondria are linked with disorders in β-oxidation of free fatty acids. According to the literature data, melatonin can improve these processes and restore the proper balance in mitochondrial metabolism. Therefore, the decrease of GGTP and triglycerides levels observed after long term treatment with melatonin and tryptophan can reflect the beneficial effect of this indoleamine and its precursor on the elements of metabolic syndrome and particularly cardiovascular events (23-25).

Melatonin and its precursor tryptophan could be useful in NAFLD because melatonin is an antioxidative agent and the oxidative stress is considered as one of the factors involved in the pathogenesis of NAFLD, especially NASH (9-11, 26, 27). Recent studies showed promising results of antioxidants e.g. vitamin C, E and betadine in prevention of NASH (28-31).

Some animal studies revealed the hypocholesterolemic and lipid peroxidation inhibitory effect of melatonin (32). The serum levels of cholesterol and triglycerides were reduced in mice with experimental diet-induced hypercholesterolemia in mice (33). This hypocholesterolemic effect of melatonin may be associated with enhanced catabolism of cholesterol to bile acids, inhibition of cholesterol synthesis and activity of LDL receptors as well as direct effects on the adipose tissue exerted via the specific MT1 and MT2 receptors (34, 35). It is worth to undeline that LDL can support the atherogenic effect of NAFLD. According to Pan et al., (36) the effect of melatonin on liver lipometabolism in rats was dose-dependent. Melatonin in the doses of 5 and 10 mg/kg reduced high cholesterol levels in rats fed with high-fat diet whereas only the high dose, i.e. 10 mg/kg, additionally decreased the liver triglyceride content (36). This dose reduced hepatic steatosis, serum levels of ALT, AST and oxidative stress parameters. The protective effect of melatonin in hepatic steatosis in rats could be attributed to its antioxidative action. In the other animal study melatonin alone or administered in combination with pioglitazone or pentoxifylline reduced the insulin resistance index, total cholesterol and triglycerides, activities of liver enzymes and the increased hepatic reduced glutathione level in rats with NAFLD. Data in this study indicate that melatonin can be used as promising adjunct therapy in the clinical management of NAFLD (37).

According the available knowledge regarding the pathogenesis of NAFLD, melatonin is considered as antiinflammatory factor. Our findings demonstrated reduced levels of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α after the use of melatonin and tryptophan in patients with NAFLD. TNF-α is considered an important pro-inflammatory cytokine produced predominantly by the immune cells of the liver in subjects with NASH. IL-6, a multifunctional cytokine promotes insulin resistance (38). These cytokines are involved in the transformation of hepatic stellate cells (HSCs) into myofibroblasts, which contribute to the progression of liver fibrosis. Serum levels of IL-6 in patients with NASH is associated with liver fibrosis (39). These data suggest that cytokines may play an important role in liver fibrosis in NAFLD patients, and the inhibition of proinflammatory cytokines appears as a primary target for the treatment of liver fibrosis.

The role of oxidative and nitrosative stress in the pathogenesis of NAFLD has been implicated (14, 40). Furthermore, the involvement of increased levels of TNF-α and of cytokines in induction of oxidative and nitrosative stress in obese patients was emphasized. Obesity may lead to impaired mitochondrial respiratory chain (MRC) activity and dysfunction of mitochondria (41-43). The protective impact of melatonin on MRC activity was demonstrated in in vitro and in vivo studies (44, 45).

Likewise, we confirmed that melatonin improved the metabolic parameters of NAFLD and attenuated the plasma levels of TNF-α and other proinflammatory cytokines IL-1, IL-6, which indicates the antioxidative and anti-inflammatory properties of melatonin in patients with NAFLD. Our present study confirms the original data of Gonciarz et al. (22) who demonstrated the beneficial effect of 12-24 weeks course of melatonin on the liver enzymes, namely AST and GGT in NASH patients. In their study (22), amelioration of liver enzymes was accompanied by the 6-7 folds increase in plasma malatonin levels at 24th week of treatment with melatonin. It is of interest that plasma cholesterol, triglycerides and glucose concentrations as well as plasma alkaline phosphatase were unchanged as compared to control group plasma levels during this prolonged study period (22).

In another study, Grigorescu et al., (46) proposed the non-invasive biochemical markers that might be useful in distinguishing between NASH and simple steatosis. According to this analysis (46) the novel pathophysiological-based panel of biomarkers combining total cytokeratin-18 (M65 antigen), IL-6 and adiponectin could efficiently predict NASH, being useful in differentiation between NASH and simple steatosis. This is of great importance since the incidence of NAFLD has increased in recent years and in some patients an unexpected evolution starting from fatty liver towards cirrhosis and hepatocellular carcinoma (HCC) has been observed (47).

For the first time we observed that melatonin and tryptophan reduced inflammation in liver tissue in patients with NASH who underwent the liver biopsy, since there is lack of a similar evidence in the literature. Undoubtely, a certain limitation of the present study is the lack of histopathological evaluation of the liver after 14 months of melatonin and tryptophan use in all examined patients. To assess in detail the beneficial effect of melatonin in the development of NAFLD, the results of morphological parameters in liver biosies determined in large cohort of patients would be helpful, but these studies await further research.

Our study demonstrates that melatonin and tryptophan substantially attenuate the levels of pro-inflammatory cytokines and improve some parameters of fat metabolism in patients with NAFLD. Thus, melatonin seems to be worth considering for the therapy of NAFLD, particularly in patients with impaired fat metabolism accompanied by hypertriglyceridemia and hyper-LDL cholesterolemia. The mechanism of this beneficial effect of melatonin and tryptophan in NASH patients may depend on amelioration of the inflammation in liver and antioxidative properties of this indoleamine.

Conflict of interests: None declared.

REFERENCES

1. McCullough AJ. The epidemiology and risk factors of NASH. In: Fatty Liver Disease. NASH and Related Disorders. GC Farrell, J GeorgeJ, P Hall P (eds.), Malden, Blackwell Publ Ltd, 2005; pp. 23-37.
2. Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old questions and new insights. Science 2011; 332: 1519-1523.
3. Bellentani S, Scaglioni F, Marino M, Bedogni G. Epidemiology of non-alcoholic fatty liver disease. Dig Dis 2010; 28: 155-161.
4. Starley B, Calcagno Ch, Harrison S. Non-alcoholic fatty liver disease and hepatocellular carcinoma: a weight connection. Hepatology 2010; 51: 1820-1832.
5. Ascha M, Hanouneh I, Lopez R, Tamimi T, Feldstein A, Zein N. The incidence and risk factors of hepatocellular carcinoma in patients with non-alcoholic steatohepatitis. Hepatology 2010; 51: 1972-1978.
6. Harrison SA, Oliver D, Arnold HL, Gogia S, Neuschwander-Tetri BA. Development and validation of a simple NALFD clinical scoring system for identifying patient without advanced disease. Gut 2008; 57: 1441-1447.
7. Dowman J, Tomlinson J, Newsome P. Pathogenesis of non-alcoholic fatty liver disease. QJM 2010; 103: 71-83.
8. Bian Z, Ma X. Liver fibrogenesis in non-alcoholic steatohepatitis. Front Physiol 2012; 3: 248.
9. Larter C, Chitturi S, Heydel D, Farrell GC. A fresh look at NASH pathogenesis. Part 1: the metabolic movers. J Gastroenterol Hepatol 2010; 25: 672-690.
10. Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: present concepts. J Gastroenterol Hepatol 2011; 26(Suppl. 1): 173-179.
11. Ono M, Okamoto N, Saibara T. The latest idea in NAFLD/NASH pathogenesis. Clin J Gastroenterol 2010; 3: 263-270.
12. Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology 1998; 114: 842-845.
13. Begriche K, Igoudjil A, Pessayre D, Fromenty B. Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it. Mitochondrion 2006; 6: 1-28.
14. Perez-Carreras M, Del Hoyo P, Martin MA, et al. Defective hepatic mitochondrial respiratory chain in patients with non-alcoholic steatohepatitis. Hepatology 2003; 38: 999-1007.
15. Tan DX, Manchester LC, Hardeland R, et al. Melatonin: a hormone, a tissue factor, an autocoid, a paracoid, and an antioxidant vitamin. J Pineal Res 2003; 34: 75-78.
16. Solis Herruzo JA, Solis Munoz P. Melatonin and oxidative stress. Rev Esp Enferm Dig 2009; 101: 453-459.
17. Reiter RJ, Tan DX, Osuna C, Gitto T. Actions of melatonin in the reduction of oxidative stress. A Review. J Biomed Sci 2000; 7: 444-458.
18. Cichoz-Lach H, Celinski K, Konturek SJ, Konturek SJ, Slomka M. The effects of L-tryptophan and melatonin on selected biochemical parameters in patient with steatohepatitis. J Physiol Pharmacol 2010; 61: 577-580.
19. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for non-alcoholic fatty liver disease. Hepatology 2005; 41: 1313-1321.
20. Hubscher SG. Histological assessment of non-alcoholic fatty liver disease. Histopathology 2006; 49: 450-465.
21. Gonciarz M, Gonciarz Z, Bielanski W, et al. The pilot study of 3-month course of melatonin treatment of patients with non-alcoholic steatohepatitis: effect on plasma levels of liver enzymes, lipids and melatonin. J Physiol Pharmacol 2010; 61: 705-710.
22. Gonciarz M, Gonciarz Z, Bielanski W, et al. The effects of long-term melatonin treatment on plasma liver enzyme levels and plasma concentrations of lipids and melatonin in patients with non-alcoholic steatohepatitis: a pilot study. J Physiol Pharmacol 2012; 63: 35-40.
23. Ghouri N, Preiss D, Sattar N. Liver enzymes, non-alcoholic liver disease and incidentcardiovascular disease: a narrative review and clinical perspective data. Hepatology 2010; 52: 1156-1161.
24. Lu H, Liu H, Hu F, Zou L, Luo S, Sun L. Independent association between non-alcoholic fatty liver disease and cardiovascular disease: a systematic review and meta-analysis. Int J Endocrinol 2013; 2013: 124958.
25. Choi SY, Kim D, Kang JH, Choi SY, Kim D, Kang JH. Non-alcoholic fatty liver disease as a risk factor of cardiovascular disease: relation of non-alcoholic fatty liver disease to carotid atherosclerosis. Korean J Hepatol 2008; 14: 77-88.
26. Zhao F, Liu ZQ, Wu D. Antioxidative effect of melatonin on DNA and erythrocytes against free-radical-induced oxidation. Chem Phys Lipids 2008; 151: 77-84.
27. McCarty MF. Full-spectrum antioxidant therapy featuring astaxanthin coupled with lipoprivic strategies and salsalate for management of non-alcoholic fatty liver disease. Med Hypotheses 2011; 77: 550-556.
28. Arendt B, Allard J. Effect of atorvastatin, vitamin E and C on non-alcoholic fatty liver disease is the combination required? Am J Gastroenterol 2011; 106: 78-80.
29. Sanyal AJ, Chalsani N, Kowdley KV, et al. Pioglitazone, vitamin E or placebo for non-alcoholic steatohepatitis. N Eng J Med 2010; 362: 1675-1685.
30. Nobili V, Manco M, Devito R, et al. Lifestyle intervention and antioxidant therapy in children with non-alcoholic fatty liver disease: a randomized, controlled trial. Hepatology 2008; 48: 119-128.
31. Dufour J. Vitamin E for non-alcoholic steatohepatitis: ready for prime time? Hepatology 2010; 52: 789-792.
32. Hoyos M, Guerrero JM, Perez-Cano R, et al. Serum cholesterol and lipid peroxidation are decreased by melatonin in diet-induced hypercholesterolemic rats. J Pineal Res 2000; 28: 150-155.
33. Sener G, Balkan J, Cevikbas U, Keyer-Uysal M, Uysal M. Melatonin reduces cholesterol accumulation and prooxidant state induced by high cholesterol diet in the plasma, the liver and probably in the aorta of C57BL/6J mice. J Pineal Res 2004; 36: 212-216.
34. Brydon L, Petit L, Delagrange P, Strosberg AD, Jockers R. Functional expression of MT2 (Mel 1b) melatonin receptors in human PAZ 6 adipocytes. Endocrinology 2001; 142: 4264-4271.
35. Alonso-Vale MI, Andreotti S, Peres SB, et al. Melatonin enhances leptin expression by rat adipocytes in the presence of insulin. Am J Physiol Endocrinol Metab 2005; 288: E805-E812.
36. Pan M, Song YL, Xu JM, Gan HZ. Melatonin ameliorates non-alcoholic fatty liver induced by high-fat diet in rats. J Pineal Res 2006; 41: 79-84.
37. Zaitone S, Hassan N, El-Orabi N, El-Awady el-S. Pentoxifylline and melatonin in combination with pioglitazone ameliorate experimental non-alcoholic fatty liver disease. Eur J Pharmacol 2011; 662: 70-77.
38. Tarrats N, Moles A, Morales A, Garcia-Ruiz C, Fernandez-Checa J C, Mari M. Critical role of tumor necrosis factor receptor 1, but not 2, in hepatic stellate cell proliferation, extracellular matrix remodeling, and liver fibrogenesis. Hepatology 2011; 54: 319-327.
39. Lemoine M, Ratziu V, Kim M, et al. Serum adipokine levels predictive of liver injury in non-alcoholic fatty liver disease. Liver Int 2009; 29: 1431-1438.
40. Koek GH, Liedorp PR, Bast A. The role of oxidative stress in non-alcoholic steatohepatitis. Clin Chim Acta 2011; 412: 1297-1305.
41. Garcia Ruiz I, Rodriguez-Juan C, Diaz-Sanjuan T, et al. Uric acid and anti-TNF antibody improve mitochondrial dysfunction in ob/ob mice. Hepatology 2006; 44: 581-591.
42. Paradies G, Petrosilo G, Paradies V, Reiter R, Ruggiero F. Melatonin, cardiolipin and mitochondrial bioenergetics in health and disease. J Pineal Res 2010; 48: 297-310.
43. Garcia-Ruiz I, Fernandez-Moreira D, Solis-Munoz P, et al. Mitochondrial complex I subunits are decreased in murine non-alcoholic fatty liver disease: implication of peroxynitrite. J Proteome Res 2010; 9: 2450-2459.
44. Peyrot F, Houee-Levin C, Ducrocq C. Melatonin nitrosation promoted by NO●2; comparison with the peroxynitrite reaction. Free Radic Res 2006; 40: 910-920.
45. Solis-Munoz P, Solis-Herruzo JA, Fernandez-Moreira D, et al. Melatonin improves mitochondrial respiratory chain activity and liver morphology in ob/ob mice. J Pineal Res 2011; 51: 113-123.
46. Grigorescu M, Crisan D, Radu C, Grigorescu MD, Sparchez Z, Serban A. A novel pathophysiological-based panel of biomarkers for the diagnosis on non-alcoholic steatohepatitis. J Physiol Pharmacol 2012; 63: 347-353.
47. Fierbinteanu-Braticevici C, Negreanu L, Tarantino G. Is fatty liver always benign and should not consequently be treated? J Physiol Pharmacol 2013; 64: 3-9.