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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Background:

Non-alcoholic steatohepatitis is a distinct entity, characterized by fatty change, lobular inflammation and fibrosis of the liver. Some cases of non-alcoholic steatohepatitis progress to cirrhosis, but it is not easy to distinguish this disease from non-alcoholic fatty liver by non-invasive examinations. No proven therapy for non-alcoholic steatohepatitis exists. Transforming growth factor-β1 is implicated in the development of liver fibrosis, and is inhibited by α-tocopherol (vitamin E) in the liver. Therefore, in this study, the significance of the measurement of the level of plasma transforming growth factor-β1 and the effect of α-tocopherol on the clinical course of non-alcoholic steatohepatitis were investigated.

Methods:

Twelve patients with non-alcoholic steatohepatitis and 10 patients with non-alcoholic fatty liver, with a diagnosis confirmed by liver biopsy, were studied. None of the patients had a history of alcohol abuse, habitual medicine or malignant or inflammatory diseases. All patients were negative for hepatitis B, C and G virus. Patients were given dietary instruction for 6 months, and then α-tocopherol (300 mg/day) was given for 1 year. Blood chemistries, measurement of plasma transforming growth factor-β1 level and liver biopsies were undertaken before and after the 1-year α-tocopherol treatment.

Results:

The serum alanine transaminase level decreased in non-alcoholic fatty liver patients, but not in non-alcoholic steatohepatitis patients, after 6 months of dietary therapy. Although the serum alanine transaminase level in non-alcoholic steatohepatitis patients was reduced during the 1-year α-tocopherol treatment, α-tocopherol had no effect on the serum alanine transaminase level in non-alcoholic fatty liver patients. The histological findings, such as steatosis, inflammation and fibrosis, of the non-alcoholic steatohepatitis patients were improved after α-tocopherol treatment. The plasma transforming growth factor-β1 level in non-alcoholic steatohepatitis patients was significantly elevated compared with that in non-alcoholic fatty liver patients and healthy controls, and decreased, accompanied by an improvement in serum alanine transaminase level, with α-tocopherol treatment.

Conclusions:

lOur data suggest that the measurement of the level of plasma transforming growth factor-β1 represents a possible method of distinguishing between non-alcoholic steatohepatitis and non-alcoholic fatty liver. Long-term α-tocopherol treatment may be safe and effective for non-alcoholic steatohepatitis. A randomized, controlled, double-blind trial is needed to confirm the full potential of α-tocopherol in the management of non-alcoholic steatohepatitis.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Non-alcoholic steatohepatitis (NASH) was first described as a distinct entity in 1980.1 Patients lacking a history of significant alcohol consumption developed liver disease that was histologically indistinguishable from alcoholic liver damage. Although non-alcoholic fatty liver (NAFL) is a reversible condition, in some patients with NASH the liver damage can progress to cirrhosis.2, 3 It is difficult to distinguish between NASH and NAFL by non-invasive examinations, such as computed tomography and ultrasonography. The only definitive diagnosis of NASH is by biopsy.4 However, it is ethically and practically impossible to perform liver biopsies for all patients with clinically diagnosed NAFL. The pathogenesis of NASH remains elusive, but most patients are obese, diabetic or have hyperlipidaemia.1–4 A large percentage of patients are asymptomatic and are discovered only after liver biochemistry is incidentally noted to be abnormal.5 The clinical features of NASH are very similar to those of NAFL, such as a ‘bright liver’ on ultrasonography.6, 7 A non-invasive method of differentiating between NASH and NAFL has not been established.

No known treatment for NASH exists.8 Weight reduction is frequently advocated, but difficult to evaluate, because these patients seldom achieve or maintain sustained weight loss; some patients have shown a marked worsening of the disease in association with weight loss.9, 10 A close relation between transforming growth factor-β1 (TGF-β1) and hepatic fibrosis and a pivotal role of TGF-β1 in hepatic fibrogenesis and hepatocyte programmed death have been reported.11–13 Vitamin E, α-tocopherol, known to have antioxidant properties and to suppress peroxidation,14 has been reported to inhibit hepatic TGF-β1 gene expression and protect against liver fibrosis in an animal model.15, 16 Very recently, the efficacy of α-tocopherol in paediatric NASH was reported in a preliminary study.17

In this study, the significance of the measurement of the level of plasma TGF-β1 in the diagnosis of NASH and the effect of α-tocopherol treatment in adult patients with NASH were investigated.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Twelve NASH patients and 10 NAFL patients were included in this study (Table 1). None of the patients had any malignancy or inflammatory disease. Non-alcoholic steatohepatitis was diagnosed by liver biopsy findings, showing hepatic steatosis, lobular inflammation and fibrosis, 1 month prior to study entry. Patients were excluded if younger than 18 years or if pregnancy testing was positive. A detailed alcohol history was taken by at least two physicians and confirmed by at least one close relative who lived with the patient. All patients consumed less than 40 g of alcohol per week. None of the patients had used medications known to precipitate steatohepatitis (e.g. prednisone), and there was no history of treatment with lipid-reducing agents, ursodeoxycholic acid or vitamin supplements in the 6 months prior to study entry. Protocols were approved by the Ethical Committee of Asahikawa Medical College for Clinical Study and informed consent was obtained from all patients.

Table 1.   Patient characteristics Thumbnail image of

Laboratory evaluation included serum liver tests total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT), γ-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and total serum bilirubin), hepatitis B serology (hepatitis B surface antigen, antibody to hepatitis B surface antigen, antibody to hepatitis B core antigen), antibody to hepatitis C virus, hepatitis C RNA polymerase chain reaction, hepatitis G RNA polymerase chain reaction, autoantibodies (antinuclear antibody, anti-smooth muscle antibody and anti-mitochondrial antibody), iron profile (serum iron, transferrin saturation and ferrin) and ceruloplasmin. All NASH and NAFL patients were negative for hepatitis B serological tests, antibody to hepatitis C virus, hepatitis C virus RNA, hepatitis G virus RNA or autoantibodies. Iron profile and serum ceruloplasmin levels were normal in all patients. Serum electrolytes, urea, creatinine, fasting glucose, complete blood count, cholesterol and triglyceride levels were also obtained.

All patients were given dietary instruction, and recorded their weight weekly during the study. The total calculated calories consumed per day was 30 kcal/kg body weight. After 6 months of dietary therapy, α-tocopherol (Eisai Pharmaceutical Co., Tokyo, Japan; 100 mg three times a day for a total of 300 mg/day; 100 mg is equivalent to 100 IU) was given to all patients after each meal for a total duration of 1 year. Serum biochemistry, lipid profile and measurement of body weight were performed at entry and every month. Liver biopsies were performed after 1 year of α-tocopherol treatment in nine of the 12 NASH patients. An experienced hepatopathologist (K.V.) evaluated all liver biopsy specimens stained with haematoxylin and eosin. Hepatic steatosis, inflammation and fibrosis were graded in a blind fashion for the specimens obtained before and after α-tocopherol treatment. Inflammation and fibrosis were scored according to the scoring system of Knodell et al.,18 and steatosis was scored by the percentage of fatty degeneration according to the following criteria: 3, over 60%; 2, 31–60%; 1, 1–30%; 0, no fatty degeneration. To avoid observer bias, quantification of histological evaluations was verified by another independent pathologist unaware of the treatment.

The plasma TGF-β1 level was measured at entry, after dietary therapy and after α-tocopherol treatment. Blood samples were taken and centrifuged. Plasma was stored at −80 °C until TGF-β1 assay. The plasma TGF-β1 level was determined by enzyme-linked immunosorbent assay (Quantikine Kit, R & S Systems, Minneapolis, MN, USA) after extraction by HCl–ethanol (1:1) solution according to the method of Danielpour et al.19 The plasma TGF-β1 levels of 10 healthy subjects were also measured as a control group. At the time of blood sampling, it was confirmed that the patients did not have any acute inflammation, such as influenza.

All data were expressed as the mean ± s.e. Student’s t-test was used to compare the patient groups at entry if normally distributed; otherwise, the Wilcoxon rank sum test was used. Analysis of variance (ANOVA) with Fisher’s LSD was used to compare the plasma TGF-β1 levels of healthy subjects and the NASH and NAFL groups at entry. Repeated ANOVA was used to compare the data before and after each treatment.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Patient characteristics

All NASH and NAFL patients demonstrated an impaired glucose tolerance curve, and were hyperlipidaemic and over their ideal body weight (body mass index was over 23). The age, gender, body weight, body mass index and serum cholesterol, triglyceride, GGT and ALT levels were not significantly different between the NASH and NAFL groups (Tables 1 and 2). The serum ALP level in NASH patients was higher than that in NAFL patients, but the difference was not statistically significant. The serum AST level in the NASH group was significantly higher than that in the NAFL group (Table 2).

Table 2.   Biochemical data of patients with non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver (NAFL) before and after diet and α-tocopherol treatments Thumbnail image of

Effect of dietary therapy

The body weight in all NASH and NAFL patients was significantly reduced after the 6-month dietary therapy (Table 2). In the NASH group, serum AST, ALT, GGT and ALP levels were slightly reduced after the 6-month dietary therapy; however, these changes were not statistically significant. On the other hand, the serum AST, ALT and GGT levels in NAFL patients were significantly reduced by dietary therapy.

Effect of α-tocopherol treatment

None of the NASH and NAFL patients complained of side-effects during the 1-year α-tocopherol treatment. The body weight in the NASH and NAFL patients did not change during α-tocopherol treatment (Table 2). Although the serum AST, ALT and GGT levels in NAFL patients did not change after α-tocopherol treatment, those in the NASH patients were significantly reduced. Liver biopsies after α-tocopherol treatment were performed in nine of the 12 NASH patients. In six NASH patients, steatosis was improved, and, in five patients, inflammation and fibrosis were improved. None of the NASH patients had histological worsening after α-tocopherol treatment (Table 3).

Table 3.   Histological changes after α-tocopherol treatment Thumbnail image of

Plasma TGF-β1 level

Although the plasma TGF-β1 level in NAFL patients was not different from that in healthy subjects, the plasma TGF-β1 level in NASH patients was significantly higher than that in NAFL patients and healthy subjects (Figure 1). The plasma TGF-β1 level in NASH patients was unchanged after the 6-month dietary therapy, but was significantly reduced after the 1-year α-tocopherol treatment (Figure 2).

image

Figure 1.  Plasma transforming growth factor-β1 (TGF-β1) level in non-alcoholic steatohepatitis (NASH) patients, non-alcoholic fatty liver (NAFL) patients and healthy subjects at baseline.

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image

Figure 2.  Changes in plasma transforming growth factor-β1 (TGF-β1) level of non-alcoholic steatohepatitis (NASH) patients after dietary therapy and 1-year α-tocopherol treatment.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The clinical features of NASH are very similar to those of NAFL, such as a ‘bright liver’ on ultrasonography, but some cases of NASH can progress to cirrhosis.6, 7 Routine biochemical tests are not helpful for either the diagnosis or exclusion of steatohepatitis.2–7 Therefore, liver biopsies are needed to confirm the diagnosis of NASH.4 Because liver biopsies cannot be performed for all NAFL patients, a diagnostic test is needed to differentiate between NASH and NAFL.

Transforming growth factor-β1 is a multifunctional and ubiquitous peptide involved in many areas of cell biology.20 It was first identified as a factor promoting cell growth in transformed fibroblasts, but it rapidly became obvious that TGF-β regulates many other cell functions, including wound healing and programmed cell death.20 In the liver, TGF-β1 is the most abundant isoform and its concentration is about 10-fold higher than that of TGF-β2 and TGF-β3.21 Transforming growth factor-β1 messenger RNA and extracellular matrix deposits have been detected in the liver tissue of many hepatic fibrosis models.11, 22 In humans, an increase in TGF-β1 has been found in chronic active hepatitis, and correlates with the histological activity index and collagen messenger RNA production.23, 24 An increase in TGF-β1 gene expression has been found in fibrotic alcoholic liver diseases and active cirrhosis of various origins.25 Moreover, there is evidence for a causative role of TGF-β1 in fibrogenesis from in vitro and in vivo studies. Kupffer cells and stellate cells secrete TGF-β1, and TGF-β1 accelerates the transformation of resting stellate cells to myofibroblasts.26 Transforming growth factor-β1 also increases the production of many extracellular matrix proteins by fibroblasts and stellate cultured cells.27 Type I collagen production and cell proliferation are mediated by TGF-β.12 From these facts, TGF-β1 is suggested to play an important role in hepatic fibrosis, and TGF-β1 production is enhanced in the process of hepatic fibrosis.20

Until now, nothing was known about the relationship between NASH and TGF-β1. In this study, it was found that the plasma TGF-β1 level in NAFL patients was no different from that in healthy subjects, but the plasma TGF-β1 level in NASH patients was significantly higher than that in NAFL patients or healthy subjects. The elevated plasma TGF-β1 level in NASH patients was reduced, accompanied by an improvement in other liver indices, with 1-year α-tocopherol treatment. Although there was no overlap between the plasma TGF-β1 levels of NASH and NAFL patients, a further study with a larger sample size is needed to confirm the clinical significance of measuring the plasma TGF-β1 level in NASH. In this study, we used an enzyme-linked immunosorbent assay to measure the plasma TGF-β1 level. The accuracy of this assay has already been confirmed,28 but it is costly and time consuming. These problems need to be solved before clinical application.

The efficacy of α-tocopherol treatment in adult NASH patients was also investigated in this study, as the efficacy of treatment in paediatric NASH patients has recently been reported.17 Although weight reduction is most commonly recommended and steatosis is usually improved, fibrosis in the liver sometimes worsens even after dietary therapy.9 In fact, weight reduction by dietary therapy improved the liver biochemistry in NAFL patients, but did not significantly improve that in NASH patients in this study. Ursodeoxycholic acid, used for the treatment of patients with primary biliary cirrhosis29 and chronic hepatitis,30 has been given to NASH patients in a pilot study.31 Vitamin E, α-tocopherol, known to have antioxidant effects and to suppress peroxidation,14 has been reported to inhibit hepatic TGF-β1 gene expression,15 and to protect against liver fibrosis in an animal model.16 In this study, we observed a significant improvement in serum ALT, AST, ALP and GGT levels and histological grade of hepatic inflammation and fibrosis after 1 year of α-tocopherol treatment at a dose of 300 mg/day. The dose of α-tocopherol in our study (300 IU/day) was relatively low compared with that in the previous study.17 However, 400 IU/day of α-tocopherol was effective in the study reported by Lavine,17 whose subjects had a higher average body weight than those in our study. α-Tocopherol appears to be safe as no side-effects were noted during the 1 year of treatment in this study. We believe that α-tocopherol has a beneficial therapeutic effect in patients with NASH. The full potential of α-tocopherol in the management of NASH will be more apparent after a large placebo-controlled trial with long-term follow-up has been carried out.

In conclusion, the measurement of the plasma TGF-β1 level may be useful in differentiating between NASH and NAFL, and is also a good indicator of the efficacy of treatment for NASH. α-Tocopherol may have a beneficial therapeutic effect in adult patients with NASH.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This work was supported in part by a Grant-in-Aid for Scientific Research from the Promotion of Science (No. 12670512) and a grant from the Smoking Research Foundation.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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