Liver International 2005: 25: 946–953 Printed in Denmark. All rights reserved

Copyright r Blackwell Munksgaard 2005

Clinical Studies

DOI: 10.1111/j.1478-3231.2005.01126.x

Increase in liver antioxidant enzyme activities in non-alcoholic fatty liver disease

Perlemuter G, Davit-Spraul A, Cosson C, Conti M, Bigorgne A, Paradis V, Corre M-P, Prat L, Kuoch V, Basdevant A, Pelletier G, Oppert J-M, Buffet C. Increase in liver antioxidant enzyme activities in non-alcoholic fatty liver disease. Liver International 2005: 25: 946–953. r Blackwell Munksgaard 2005 Abstract: Aims: Steatosis may increase oxidative stress, which is counteracted by cellular enzymatic (cytosolic and mitochondrial superoxide dismutases (Cu/Zn-SOD and Mn-SOD), glutathione peroxidase (GPx), catalase) and non-enzymatic antioxidant systems. We aimed to determine, in patients with non-alcoholic fatty liver disease (NAFLD), the level of antioxidant defenses (1) in liver biopsies, to demonstrate the existence of oxidative stress; (2) in erythrocytes and plasma, to determine whether their antioxidant defenses reflect liver oxidative stress. Methods: Erythrocyte and plasma antioxidant defenses were prospectively studied in two groups of 16 patients: patients with NAFLD and controls. Liver biopsies were performed in eight NAFLD patients; liver antioxidant enzyme activities were measured and compared with those in 12 control livers used for transplantation. Results: Cu/Zn-SOD, GPx and catalase activities were significantly higher in NAFLD livers than in controls whereas no significant differences were observed in Mn-SOD activity, and thiobarbituric acid-reactive substance (TBARS) concentration. No differences were observed in erythrocyte antioxidant enzyme activities (GPx, catalase, Cu/Zn-SOD), erythrocyte TBARS concentration, and plasma a-tocopherol concentration. Conclusions: Liver antioxidant enzyme activities were high in patients with NAFLD, reflecting an oxidative stress possibly involved in inflammation and fibrogenesis. However, erythrocyte and plasma antioxidant defenses did not reflect intrahepatic peroxidation.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease in which the liver displays histological features similar to those induced by excessive alcohol intake in the absence of significant alcohol consumption (1). Non-alcoholic steatoAbbreviations: a-T, a-tocopherol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; GPx, glutathione peroxidase; Hb, hemoglobin; NAFLD, nonalcoholic fatty liver disease; N, upper limit of normal value; NASH, non-alcoholic steatohepatitis; ROS, reactive oxygen species; SOD, superoxide dismutase; Cu/Zn-SOD, copper/ zinc-dependent SOD; Mn-SOD, manganese-dependent SOD; TBARS, thiobarbituric acid-reactive substances

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Gabriel Perlemuter1,2, Anne Davit-Spraul3, Claudine Cosson3, Marc Conti3, Ame´lie Bigorgne2, Vale´rie Paradis4, Marie-Pierre Corre3, Lydie Prat5, Viceth Kuoch6, Arnaud Basdevant5, Gilles Pelletier7, Jean-Michel Oppert5 and Catherine Buffet7 1 Service d’he´pato-gastroente´rologie, Hoˆpital Antoine Be´cle`re; AP-HP, 157, rue de la Porte de Trivaux, 92141 Clamart Cedex, France, 2INSERM AVENIR, Institut Paris-Sud sur les Cytokines (IFR 13), 32, rue des Carnets, 92140 Clamart, France, 3Service de biochimie 1, Hoˆpital Biceˆtre; AP-HP, Kremlin Biceˆtre, France, 4Service d’anatomo-pathologie, Hoˆpital Beaujon; AP-HP, Clichy, France, 5Service de nutrition, Hoˆpital Hoˆtel-Dieu; AP-HP, Paris, France, 6Service de radiologie and 7Service des maladies du foie et de l’appareil digestif, Hoˆpital Biceˆtre; AP-HP, Kremlin Biceˆtre, France

Key words: catalase – fatty liver – glutathione peroxidase – non-alcoholic fatty liver disease – obesity – oxidative stress – superoxide dismutase Gabriel Perlemuter, Service d’he´pato-gastroente´rologie, Hoˆpital Antoine Be´cle`re, 157, rue de la Porte de Trivaux, 92141 Clamart Cedex, France. Tel: 133-1-45-37-43-69 Fax: 133-1-40-94-06-56 E-mail: [email protected] Received 19 July 2004, accepted 14 February 2005

hepatitis (NASH), defined as steatosis together with necroinflammatory activity, is one stage in the spectrum of NAFLD (2), which ranges from benign hepatic steatosis to cirrhosis and liver failure (3). Although the prevalence of NAFLD is unclear, it seems to be currently the most prevalent form of liver disease in the US population (4) and probably in other western countries. There is no distinct clinical or biochemical abnormality that can be used for the accurate diagnosis of NAFLD (5). However, ultrasound scans can identify hepatic steatosis and have been advocated as a non-invasive diagnostic test for

Oxidative stress in non-alcoholic fatty liver fatty liver disease (6, 7). The most common laboratory findings are high plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (1, 8–11), with the increase in ALT greater than that in AST (1, 8). Metabolic abnormalities, such as dyslipidemia (i.e. hypertriglyceridemia and hypercholesterolemia) and hyperglycemia because of insulin resistance, are also associated with NAFLD (12–14). Several predictors of liver fibrosis have been identified in patients with NAFLD, including body mass index (BMI; equal to weight divided by height squared)  28 kg/m2, age450 years, ALT4 twice the upper limit of normal values (N), AST/ALT ratio 41, and type 2 diabetes (5, 15). This subgroup of patients with NAFLD stands to benefit most from liver biopsy and investigational therapies (5, 15). The pathogenesis of NAFLD remains unclear. Several mechanisms may play a role, including insulin resistance, amino acid imbalance, and endotoxemia (reviewed in (16, 17)). Each of these processes can shift metabolism towards lipogenesis rather than lipolysis, leading to fat accumulation in the liver. Reactive oxygen species (ROS) may trigger the peroxidation of polyunsaturated fatty acids (18). Thus, the fats accumulating in the liver may be a target for lipid peroxidation. The aldehyde end products of lipid peroxidation (i.e. malondialdehyde and 4-hydroxynonenal) are known to be proinflammatory mediators and can activate stellate cells, which in turn synthesize collagen, leading to liver fibrosis (19). Evidence has been obtained suggesting that a state of oxidative stress exists in non-alcoholic murine models of liver diseases (20–25). Cellular antioxidant defenses against such oxidative stress involve antioxidant enzymes such as glutathione peroxidase (GPx), catalase and two forms of superoxide dismutase, a cytosolic copper/zincdependent SOD (Cu/Zn-SOD), and a mitochondrial manganese-dependent SOD (Mn-SOD) (26–28). Erythrocytes, which are potential target cells for peroxidative damage, exhibit oxidative stress in patients with nephrotic syndrome (29) and with type 1 diabetes (30). The unusually high susceptibility of erythrocyte lipids to peroxidation is thought to reflect similar abnormalities in organs and tissues including the liver. As lipid peroxidation might play a major role in the pathogenesis of NAFLD, the aim of this study was to determine the level of antioxidant defenses: (i) in the liver of NAFLD patients, to demonstrate the existence of oxidative stress, and (ii) in erythrocytes and plasma, to investigate whether cells much more readily accessible than

hepatocytes, such erythrocytes, could reflect the liver antioxidant status.

Patients and methods

Patients

This prospective study included 16 patients with clinically suspected NAFLD and 16 control subjects. For both groups, consecutive eligible patients were included at Biceˆtre Hospital between June 2000 and December 2001. The NAFLD patients fulfilled the following criteria: ALT activity greater than the upper limit of normal values, BMI  25 kg/m2, and a hepatorenal difference in echo intensities  7.0 dB. Exclusion criteria were daily alcohol consumption 420 g, positive testing for hepatitis B virus surface antigen (AgHBs) and hepatitis C virus (HCV) antibodies, positive testing for antinuclear, antismooth muscle actin, anti-liver kidney microsome, and anti-mitochondrial antibodies, consumption of corticosteroids, amiodarone, tamoxifene or cyclins, evidence of genetic hemochromatosis (i.e. transferrin saturation 445% or presence of the C282Y mutation in the homozygous state), creatininemia 4120 mmol/l, weight changes 410% of body weight in the 3 months preceeding the study, vitamin A, C or E supplement consumption, abnormal TSH value, evidence of cardiac failure. Control subjects had BMI o25 kg/m2 and normal liver function tests. Informed written consent was obtained from each patient after detailed explanation of the protocol. Methods

Evaluation of study subjects BMI was calculated as weight (in kg) divided by height (in meters) squared. A threshold value of 25 kg/m2 was used, based on the current WHO classification for weight status (in both sexes, overweight was defined as BMI 25–29.9 kg/m2, and obesity as BMI  30 kg/m2) (31). Alcohol and vitamin intakes were assessed during interviews, by a dietician and a senior physician. Blood samples were collected on the day of inclusion in the protocol. Plasma AST, ALT, glucose and triglyceride concentrations were measured by automated techniques (Hitachi 747, Roche diagnostics, Meylan, France). HbA1c was measured by low-pressure liquid chromatography separation of hemoglobin (Hb) fractions, with a reference value of 3.9–6.2% on a 745 Glycomat apparatus (Bayer Diagnostics, Puteaux, France). 947

Perlemuter et al. Liver sample collection Liver biopsy was performed in the eight patients who had with high risk factors for fibrosis: ALT activity greater than 2 N and BMI 428 kg/m2. The biopsy tissue was divided into two samples. One sample was used for histological assessment. The other sample, weighing 5–10 mg, was immediately frozen and stored in liquid nitrogen until used for biochemical studies. Twelve control liver samples were obtained from transplant donors (age range, 5–42 years). These liver samples were obtained from normal human livers and surgically reduced for pediatric transplantation. These healthy liver samples were perfused at 4 1C in situ with Belzer’s liquid used for organ preservation. Within 15 min of surgical removal, blocks of these liver tissues were frozen and stored in liquid nitrogen until use. Sampling was carried out in accordance with French ethical and legal regulations. Some of these liver sample blocks had already been used as controls for determination of the glucuronidation of drugs by hepatic microsomes (32). They were histologically documented and did not exhibit any features of steatosis or fibrosis. Histological assessment Fixed paraffin-embedded liver samples from NAFLD patients were reviewed by a single pathologist (V. P.) blind to clinical and laboratory data. The histological variables commonly described in NAFLD (i.e. steatosis, necroinflammatory grade, fibrosis stage) were analyzed according to Brunt’s classification (33). Macrovacuolar steatosis grade was assessed by determining the percentage of hepatocytes containing fat droplets. Biochemical studies Blood and liver biopsy preparations: Venous blood samples were collected into vacutainers containing EDTA, which were centrifuged at 3000g for 10 min at 4 1C. Plasma and buffy coats were removed and erythrocytes were washed three times with 0.9% NaCl solution. Aliquots of washed erythrocytes and plasma were stored at 80 1C until analysis. For erythrocyte assays, the erythrocytes were subjected to hemolysis by thawing and diluted (1:10) in 10 mM phosphate buffer pH 7.8. Frozen liver samples were homogenized in 10 mM phosphate buffer pH 7.8 containing 1 mM EDTA, and then subjected to sonic disruption on ice for 30 s in pulsed mode. Antioxidant enzyme assays: Superoxide dismutase activities (Cu/Zn-SOD and Mn-SOD) were measured with a commercially available kit (Ransod, Randox Lab., Montpellier, France) based on 948

the method developed by McCord and Fridovich (34). Total SOD activity was measured at pH 7.8, and Cu/Zn-SOD at pH 10.2. Mn-SOD activity was calculated as the difference between total SOD and Cu/Zn-SOD activity. GPx activity was determined with a commercial kit (Ransel, Randox Lab.) using a method based on that developed by Paglia and Valentine (35). Catalase activity was determined for diluted samples, using a spectrophotometric method, as previously described (36). Hb concentration in the 1:10 diluted hemolysate was determined by the cyanmethemoglobin method, using Sigma Diagnostic reagents (Sigma, Saint Quentin Fallavier, France). The protein content of liver homogenates was determined according to the Bradford procedure. Specific enzyme activities were expressed in U/g Hb for erythrocytes or in U/mg protein for liver biopsies. Thiobarbituric acid-reactive substances (TBARS): The extent of lipid peroxidation was evaluated by determining TBARS, as previously described (37). a-tocopherol assay: Plasma a-tocopherol (a-T) concentration was determined by reverse-phase HPLC (38) with ultraviolet detection at 292 nm. As there is a metabolic relationship between plasma a-T concentration and plasma lipid concentration (39), a-T concentrations were expressed as a ratio of a-T/total lipids (total cholesterol1triglycerides). Statistical analysis Characteristics of study subjects, erythrocyte data and liver biopsy data were compared by the nonparametric Mann–Whitney variance analysis test; the level of significance was set at Po0.05. Results

Study population

Thirty-two consecutive patients fulfilling the inclusion criteria were enrolled in this study. The clinical and laboratory data for these patients are shown in Table 1. The groups were similar in terms of sex ratio and age. Blood glucose, HbA1c and triglycerides concentrations were higher in patients with NAFLD than in control patients. Histological findings

Steatosis was observed in all eight liver biopsies performed in NAFLD patients. Moderate steatosis (440% of hepatocytes) was observed in four patients. Pure steatosis without inflammation was observed in three patients. The remaining five

Oxidative stress in non-alcoholic fatty liver Table 1. Characteristics of study subjects Usual laboratory values Sex M/F Age (years) BMI (kg/m2) AST (IU/l) ALT (IU/l) Glucose (mmol/l) HbA1c (%) Triglycerides (mmol/l)

Mo40 Fo35 Mo45 Fo35 3.8–5.8 o6.2 0.6–1.7

Controls subjects (n 5 16)

NAFLD patients (n 5 16)

P

7/9 46.0  8.4 23.8  5.2 24  7

5/11 51  14.1 33.4  9.2 46  17

NS o0.007 o0.004

18  8

100  68.1

o0.0001

6.2  1.5 5.8  0.9 1.44  0.3

o0.0001 o0.004 o0.003

4.9  0.3 4.9  0.4 0.93  0.3

Values are means  SD. NS, not statistically significant; NAFLD, nonalcoholic fatty liver disease; BMI, body mass index; AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Cu/Zn-SOD

A 30

patients had features of NASH with inflammation and fibrosis. The necroinflammatory grade was one in three patients and two in two patients. There was no fibrosis in the three patients with pure steatosis. In the five patients with NASH, fibrosis was seen in all: stage 1 was seen in two patients and stage 2 in 3 patients. Antioxidant status in liver biopsies

Figure 1 shows the Cu/Zn-SOD (A), Mn-SOD (B), catalase (C) and GPx (D) activities in the livers of patients with NAFLD (n 5 8) and in control livers (n 5 12). Cu/Zn-SOD activity was significantly higher (by 60%; Po0.0003) in NAFLD livers than in control livers (Fig. 1A). No significant difference in Mn-SOD activity was observed between these two groups (Fig. 1B).

Mn-SOD

B * 30

U/mg prot

U/mg prot

26 22 18

20

10 14 10 Control liver Catalase

C

Control liver

100 80

*

1300

mU/mg prot

120

NAFLD liver GPx

D *

140

U/mg prot

0

NAFLD liver

1100 900 700

60 500

40 0

300 Control liver

NAFLD liver

Control liver

NAFLD liver

Fig. 1. Antioxidant enzyme activities in liver. Cu/Zn-SOD, Mn-SOD, catalase, and GPx activities in the livers of eight non-alcoholic fatty liver disease (NAFLD) patients were compared with those in control livers (12 samples). (A) Significantly higher levels of Cu/ZnSOD activity in the livers of NAFLD patients than in control livers (160%; Po0.0003); (B) No statistically significant difference in Mn-SOD activity between the livers of NAFLD patients and control livers; (C) Significantly higher levels of catalase activity in the livers of NAFLD patients than in control livers (1130%; Po0.0007); (D) Significantly higher levels of GPx activity in the livers of NAFLD patients than in control livers (176%; Po0.0007)). Graphs show median  percentiles; comparisons were made using the non-parametric Mann–Whitney variance analysis test (statistically significant difference from control is indicated by an asterisk). SOD, superoxide dismutase; Cu/Zn-SOD, copper/zinc-dependent SOD; Mn-SOD, manganese-dependent SOD; GPx, glutathione peroxidase.

949

Perlemuter et al. Discussion

TBARS

pmol/mg prot

35

25

15

5 Control liver

NAFLD liver

Fig. 2. Thiobarbituric acid-reactive substance (TBARS) concentration in the liver. No significant difference in liver lipid peroxidation, as assessed by TBARS concentration, was observed between the livers of eight NAFLD patients and control livers (12 samples). Graph shows median  percentiles; comparisons were made using the non-parametric Mann–Whitney variance analysis test.

Catalase (1130%, Po0.0007; Fig. 1C) and GPx (176%, Po0.0007; Fig. 1D) activities were also significantly higher in the livers of NAFLD patients than in control livers. Although the number of samples was to small to draw any conclusion, there was no difference in the level of antioxidant enzyme activities between livers with pure steatosis and livers with steatohepatitis. TBARS concentrations did not differ significantly between the livers of NAFLD patients and control livers (Fig. 2). Antioxidant status in plasma and erythrocytes

No significant difference was observed in a-T concentrations standardized according to total lipid levels between patients with NAFLD (2.08  0.51 mg/mmol) and control subjects (1.95  0.33 mg/mmol). No significant difference in Cu/Zn-SOD (Fig. 3A), catalase (Fig. 3B) or GPx (Fig. 3C) activity was observed between the erythrocytes of NAFLD patients and control subjects. Moreover, within the eight patients who had liver biopsy, we did not observe any correlation between hepatic and erythrocyte levels of antioxidants. The erythrocyte TBARS concentrations of the NAFLD population did not differ significantly from that of control subjects (Fig. 4). 950

NAFLD is characterized by fat accumulation in hepatocytes, providing a potential substrate for lipid peroxidation. Antioxidant enzyme activities (SOD, GPx and catalase) were found to be markedly higher in the livers of patients with NAFLD than in the control livers. These findings provide evidence for the occurrence of oxidative stress in the livers of patients with NAFLD. Oxidative stress also occurs in other situations of hepatocyte fat accumulation, such as experimental alcoholic liver, with a positive correlation between lipid peroxidation and liver injury (40). ROS are thought to play a major role in the development of steatohepatitis lesions (41). Mitochondrial ROS, which may oxidize polyunsaturated fatty acids from hepatic fat deposits, have been shown to play a particularly important role in animal models (21, 42). Our results show that only cytosolic defenses (Cu/Zn-SOD, GPx and catalase activities) were increased in the liver of NAFLD patients. Up-regulation of the same antioxidant enzymes in the liver has been reported in murine models of NASH (43). Interestingly, no increase in Mn-SOD activity, located in mitochondria, was observed in the liver of patients with NAFLD. This lack of increase in MnSOD activity may reflect low levels of ROS production in mitochondria. However, mitochondria are thought to be the main source of ROS in the cell and mitochondrial ROS production increases markedly if the electron flow in the mitochondrial respiratory chain is impaired. Recent reports have shown that a defective hepatic mitochondrial respiratory chain in patients with NASH may increase ROS production and decrease the synthesis of enzymes encoded by mitochondrial DNA (44). Thus, mitochondrial ROS production may increase Mn-SOD activity whereas mitochondrial alterations may decrease the synthesis of enzymes encoded by mitochondrial DNA, in turn decreasing Mn-SOD activity. These two opposite effects may cancel each other out at early stages of NAFLD, thereby accounting for the lack of difference in Mn-SOD activity between control subjects and patients with NAFLD. At advanced stages of NAFLD, the down-regulation of genes encoding antioxidant enzymes would impair both cytosolic and mitochondrial defenses against oxidative stress. Indeed, a recent study of hepatic gene expression in patients with NASH showed that genes encoding proteins involved in the dismutation of ROS (i.e. Cu/Zn-SOD, catalase and GPx) displayed significant down-regulation in subjects with cirrhosis secondary to NASH (45). A down-regulation of

Oxidative stress in non-alcoholic fatty liver Cu/Zn-SOD

A

320

1300

280 KU/g Hb

1200 U/g Hb

Catalase

B

1400

1100

240 200

1000 160 900 120 Control subjects C

NAFLD patients

Control subjects

NAFLD patients

GPx

90

U/g Hb

70

50

30

Control subjects

NAFLD patients

Fig. 3. Antioxidant enzyme activities in erythrocytes. Cu/Zn-SOD, catalase, and GPx activities were determined in the erythrocytes of patients with non-alcoholic fatty liver disease (n 5 16), and of control subjects (n 5 16). No statistically significant difference was found between the two groups for Cu/Zn-SOD (A), catalase (B) and GPx (C) activities. Results are normalized on the basis of erythrocyte hemoglobin concentration. Graphs show median  percentiles; comparisons were made using the non-parametric Mann– Whitney variance analysis test. SOD, superoxide dismutase; Cu/Zn-SOD, copper/zinc-dependent SOD; Mn-SOD, manganesedependent SOD; GPx, glutathione peroxidase.

SOD and catalase activities has also been shown in NAFLD patients (46). This would be expected to result in an increase in the amount of lipid peroxidation products and the pathological progression of liver lesions. In this context, it has been shown that the progression of NAFLD from pure steatosis to fibrosis and cirrhosis is associated with an increase in the amount of lipid peroxidation products (47, 48). The lack of increase in the amount of peroxidation products in our study is consistent with the low-level or mild necroinflammatory activity and the absence of extensive fibrosis on liver biopsy. We also investigated whether the activity of antioxidant enzymes in erythrocytes reflected oxidative stress in the liver. No significant difference in Cu/Zn-SOD, catalase, and GPx erythrocyte activities was observed between patients with NAFLD and the control group. The plasma concentration of the liposoluble antioxidant a-T standardized according to total lipid levels did not differ significantly between groups. There-

fore, antioxidant status in erythrocytes and plasma does not appear to reflect the oxidative status of the liver in patients with low-level or mild pathological liver lesions. In conclusion, despite the limited number of liver biopsies performed in the present study, our data provide evidence for a state of oxidative stress with an efficient adaptive antioxidant response in the livers of patients with NAFLD. The failure of this compensatory antioxidant response may be involved in the inflammation and fibrogenesis observed during the progression of NAFLD. As oxidative stress can be detected early in the course of NAFLD, antioxidant treatment is potentially of major value for preventing progression towards fibrosis and cirrhosis in NAFLD patients. Acknowledgements This study was supported by INSERM, AP-HP, and French foundation for medical research. A. B. was supported by grants from INSERM and re´gion Ile-de-France.

951

Perlemuter et al. TBARS

3.5

13.

µmol/g Hb

3

2.5

14.

2

15.

1.5

16. 17.

1

18. 0.5 Control subjects

NAFLD patients

Fig. 4. Thiobarbituric acid-reactive substance (TBARS) concentration in erythrocytes. No significant difference in erythrocyte lipid peroxidation, as assessed by TBARS, was observed between patients with non-alcoholic fatty liver disease (n 5 16) and control subjects (n 5 16). Graphs show median  percentile; comparisons were made using the non-parametric Mann–Whitney variance analysis test.

19.

20.

21.

22.

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