j5阿托伐他汀对非酒精性脂肪肝氧磷酶1、丙二醛、谷胱甘肽过氧化物酶的效果影响

a ef pt Article history: Received 24 August 2010 Accepted 2 April 2011 Keywords: Non-alcoholic fatty liver diseases Paroxonase-1 activity Antioxidant Lipid peroxidation the liver, mainly triglycerides [4]. The mechanisms leading to lipid accumulation are not com- pletely understood, but it could possibly result from insulin resis- tance [5,6], and decreased disposal of fatty acids due to impaired mitochondrial b-oxidation or deficient production of very low den- sity lipoprotein [7]. The pro-inflammatory and profibrogenic antioxidant defences. It protects cells from damage by free radicals, specifically lipid peroxides and hydrogen peroxide, reducing them to alcohols and water, respectively, which can then be detoxified further and removed from the body or, in the case of water, used as needed. Deficiency of GP is associated with accelerated ageing and degenerative diseases [10]. In light of these data, this study was designed to: (1) investigate the relationship between hepatic antioxidant PON1 activity, lipid peroxidation and antioxidant enzymes in patients with NAFLD; ⇑ Corresponding author. Mobile: +966553420886. Arab Journal of Gastroenterology 12 (2011) 80–85 Contents lists availab G w.e E-mail address: wsmohamed1@yahoo.com (W. Samy). Introduction Non-alcoholic fatty liver disease (NAFLD) is an increasingly diagnosed condition that may progress to end-stage liver disease, affecting approximately 30% of Western populations. The histo- logic spectrum of NAFLD includes simple steatosis, which has a be- nign prognosis, and non-alcoholic steatohepatitis (NASH), a more aggressive form of liver injury that may progress to cirrhosis and its complications [1]. NAFLD has histological features of alcohol-in- duced liver disease that occurs in individuals who do not consume alcohol [2,3]. Fatty liver occurs due to the accumulation of lipid in properties of lipid peroxidation end products, such as malondialde- hyde (MDA), may account for all of the typical histological features observed in this disorder [2]. Paraoxonase 1 (PON1) is a calcium- dependent esterase closely associated with high-density lipopro- tein cholesterol (HDL) that has been reported to confer antioxidant properties by decreasing the accumulation of lipid peroxidation products [8]. The liver plays a key role in the synthesis of serum PON1 that is able to hydrolyse a number of substrates, such as paraoxon, phenyl acetate, lipid peroxides, cholesterol esters and hydroperoxides [9]. Glutathione peroxidase (GP) is one of the body’s most potent Atorvastatin 1687-1979/$ - see front matter � 2011 Arab Journal o doi:10.1016/j.ajg.2011.04.008 Background and study aims: The prevalence of non-alcoholic fatty liver disease (NAFLD) appears to be increasing. The aim of the present study was to investigate the relationship between hepatic antioxidant paraoxonase 1 (PON1) activity, lipid peroxidation and antioxidant enzymes in patients with NAFLD and the effect of atorvastatin. Patients and methods: This study was conducted on 50 patients with NAFLD and 20 normal subjects matched for age and sex. All of them were subjected to the following investigations: abdominal ultraso- nography, serum PON1 activity level, liver function tests, serum lipid profile, fasting and postprandial blood glucose and serum levels of malondialdehyde (MDA) and glutathione peroxidase (GP). NAFLD patients were further randomly classified into two groups (25 patients each), groups Ia and Ib. Only group Ia received atorvastatin 40 mg tablet for 8 months. Results: Obesity, dyslipidaemia and impaired glucose tolerance were prevalent in group I. There was a significant decrease in serum PON1 activity with a significant increase in MDA and GP activity (i.e., there is a significant increase in lipid peroxidation rate) in group I compared with group II. After atorvastatin therapy, there was a significant increase in serum PNO1 activity and significant decrease in serum MDA levels. Conclusion: Patients with NAFLD show enhanced oxidative stress which may lead to non-alcoholic ste- atohepatitis (NASH). Reduced PON1 activity and increased MDA could be considered a biochemical mar- ker for lipid peroxidation, which require follow-up in patients with NAFLD. Atorvastatin may have a role in prevention of, or delay, the transformation of liver steatosis into NASH. � 2011 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved. a r t i c l e i n f o a b s t r a c t Original Article Paraoxonase-1 activity, malondialdehyde non-alcoholic fatty liver disease and the Waleed Samy a,⇑, Mohammed A. Hassanian b aDepartment of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt bDepartment of Clinical Biochemistry, Faculty of Medicine, Tanta University, Tanta, Egy Arab Journal of journal homepage: ww f Gastroenterology. Published by El nd glutathione peroxidase in fect of atorvastatin le at ScienceDirect astroenterology lsev ier .com/ locate/a jg sevier B.V. All rights reserved. Hepatic ultrasonography scanning was performed on all partic- ipants by the same well-experienced sonographer using a Siemens Liver function tests (alanine aminotransferase (ALT), aspartate curve [14]. al o Sonoline Omnia instrument with a 5-MHz transducer (Siemens Medical Solutions, Mountain View, CA, USA). Control cases had normal ultrasonographic findings. A fatty liver was diagnosed by the presence of at least two of three abnormal findings: diffusely (2) investigate the role of PON1 and MDA as a biochemical marker of lipid peroxidation and follow-up patients with NAFLD; and (3) re-evaluate these parameters after a short course of atorvastatin therapy as an antihyperlipidaemic agent. Patients and methods This study was conducted at the Internal Medicine Department and Outpatient Clinic at Tanta University Hospital. It was conducted in two stages: � Stage one: analytic cross-sectional study and � Stage two: randomised clinical trial (patients with NAFLD). This study was conducted on 70 subjects classified into two groups. Group I included 50 patients with NAFLD (28 females and 22 males with an age range of 35–62 years). Patients were admitted or referred to the Internal Medicine Department and Outpatient Clinic at Tanta University Hospital because of elevated aminotrans- ferases or a bright liver on abdominal ultrasound, which were acci- dentally discovered. The diagnostic evaluation of our patients with NAFLD was based on criteria used by others [11]. This group of pa- tients with abnormal aminotransferases’ concentration, who otherwise had normal clinical, biochemical, virological and radio- logical profiles, was considered as having NAFLD. Exclusion criteria included: � Schistosomiasis (excluded by negative indirect haemaggluti- nation). � Viral hepatitis (excluded by negative hepatitis B surface antigen, antibody to hepatitis B core antigen and antibody to hepatitis C virus). � Auto-immune hepatitis (auto-immune serology including antimitochondrial antibody, antinuclear antibody and anti- smooth muscle antibody were negative). � Haemochromatosis and Wilson’s disease (fasting serum iron, transferrin saturation, ferritin levels and coeruloplasmin levels were normal). � Intake of oestrogen hormones or corticosteroid therapy. � Alcoholism. Group II included 20 healthy volunteers matched for age and sex (nine females and 11 males within the age range of 30– 58 years) as controls. The studied groups were subjected to: � Proper history taking and thorough clinical examination. � Body mass index (BMI) was calculated as weight (in kilograms) divided by the square of height (in metres). Overweight was defined as BMI between 25 and 29.9 kg m�2, and obesity was defined as BMIP 30 kg m�2. Furthermore, obesity was classi- fied into class 1 obesity with BMI between 30 and 34.9 kg m�2, class 2 obesity with BMI between 35 and 39.9 kg m�2 and extreme obesity with BMIP 40 kg m�2 [12]. W. Samy, M.A. Hassanian / Arab Journ increased echogenicity (bright) liver with liver echogenicity great- er than kidney, with vascular blurring and deep attenuation of ultrasound signal [13]. Serum GP activity level was determined spectrophotometrically from a lysate of the washed packed erythrocyte fraction by using a test reagent kit (Calbiochem–Novabiochem Corp., San Diego, CA, USA). Absorbance was measured at 340 nm. Results were ex- pressed as milliunits of GPx activity per milligram of haemoglobin (Hb). Hb was assayed by the commercial cyanmethemoglobin method (Sigma Diagnostics, St. Louis, MO, USA) [15]. NAFLD patients were further randomly allocated into two groups (each 25 patients): group Ia, which received atorvastatin 40 mg tablet at night for 8 months and followed up by liver en- zymes (ALT and AST) to detect any rise in their level, and, group Ib, which did not receive atorvastatin therapy. All patients com- pleted the follow-up till the end of the study. The same chemical parameters were repeated after the trial in both groups. The re- search members were blinded to the case and controls. Statistical methods Values were expressed as mean ± SD. The statistical analysis of the results was carried out using Statistical Package for Social Sci- ences (SPSS), version 10.0 for Microsoft Windows XP. All variables were tested for normality of distribution. Independent samples t- test (t value) was applied to detect the difference between two arithmetic means. Paired-samples t-test was used for comparison between after and before parametric values. Pearson’s correlation coefficient was used for parametric results, while non-parametric results were correlated using Spearman’s rank correlation. The la- belled independent frequencies or proportions between the differ- ent groups were estimated using chi-square test (v2 value). A stepwise multiple regression analysis was done to find the inde- pendent variables associated with NAFLD. Results were considered statistically significant when p value was less than 0.05. Results Tables 1 and 2 summarise the basic characteristics of the study population. There was no significant difference between the stud- ied groups regarding age and sex. There was a significant increase �2 aminotransferase (AST), serum albumin, total protein, total biliru- bin, direct bilirubin and prothrombin time), lipid profile (serum tri- glycerides (TGs), total cholesterol level (TC), HDL, low density lipoprotein-cholesterol level (LDL)) fasting and postprandial blood glucose were done in all subjects. PON1 activity towards paraoxon (O,O-diethyl-O-p-nitrophenyl phosphate, Sigma) was determined by measuring the initial rate of substrate hydrolysis to p-nitrophenol, the absorbance of which was monitored in the assay mixture (950 ll containing 1 mM paraoxon;1 mM CaCl2 and 100 mM Tris/HCl buffer, pH 8.0, incu- bated at 37 �C for 3 min). A total of 50-ll serum was added to this mixture and the change in absorbance at 405 nm was immediately recorded spectrophotometrically. Enzyme activity was calculated from the molar extinction coefficient (405) of p-nitrophenol (18,053 M�1 cm�1), and expressed in U ml�1, where 1 U of enzyme hydrolyses 1 nmol of paraoxon min�1 [9]. Serum MDA level was derived from a pre-constructed standard All subjects received the same dietary protein intake at least 1 day before the study. After an overnight fast and bed rest, blood samples were collected. After centrifugation (3000g;10 min), the sera were separated and stored at �20 �C for further assay of the studied parameters. f Gastroenterology 12 (2011) 80–85 81 in BMI in group I (26.78 ± 3.92 kg m ) as compared with group II (23.72 ± 3.34 kg m�2). According to BMI, in group I, 80% (40 pa- tients) were categorised as obese and 20% (10 patients) as Table 3 PON1 MDA and GP in the studied groups. Parameter Group I Group II p PON1 (U ml�1) 39.66 ± 12.38 28.33 ± 15.93 0.002* MDA (nmol ml�1) 2.91 ± 0.17 3.03 ± 0.23 0.023* GP (U l�1) 554.25 ± 133.17 642.36 ± 112.33 0.003* * Statistically significant. Table 4 Correlation of PNO1 activity with the different parameters in the studied groups. Parameter Group I (r1) Group II (r2) PNO1 activity BMI (kg m�2) �0.033 �0.264 FBG �0.692** �0.722** TC (mg dl�1) �0.571** �0.824** TG (mg dl�1) �0.296 �0.757** HDL (mg dl�1) 0.822** 0.944** �1 ** ** al of Gastroenterology 12 (2011) 80–85 overweight; the obese patients were further categorised as class I obesity 55% (22 patients) and as class II obesity 45% (18 patients), while in group II seven were obese (28%), nine were overweight Table 1 Comparison between the different studied groups. Parameter Group I N = 25 Group II N = 50 p Age (years) 44.1 ± 7.31 46.6 ± 5.56 0.603 Gender (female/male) 28/22 9/11 0.043a BMI 26.78 ± 3.92 23.72 ± 3.34 0.001* Blood pressure (normal/ hypertensive) 13/50 – 0.05⁄,a a v2 (Scheffe test). * Statistically significant. Table 2 Biochemical tests in the studied groups. Laboratory test Group I Group II p-value ALT (U l�1) 17.65 ± 4.54 25.44 ± 15.6 0.017* AST (U l�1) 15.85 ± 7.36 20.64 ± 6.69 0.006* S. Albumin (g dl�1) 4.43 ± 0.22 4.36 ± 0.31 0.316 Total Protein (g dl�1) 7.18 ± 0.19 7.20 ± 0.23 0.708 Total Bilirubin (umol l�1) 10.28 ± 2.28 11.3 ± 2.1 0.057 PT (s) 12.67 ± 0.2 12.78 ± 0.31 0.111 FBS (mg dl�1) 79.78 ± 19.17 90.86 ± 10.21 0.001* PPBS (mg dl�1) 159.73 ± 52.62 183.11 ± 23.9 0.009* TC (mg dl�1) 174.75 ± 25.85 190.15 ± 13.32 0.001* TG (mg dl�1) 122.91 ± 44.14 138.23 ± 18.42 0.037* HDL (mg dl�1) 41.77 ± 8.1 39.24 ± 4.09 0.754 LDL (mg dl�1) 111.80 ± 11.7 121.47 ± 12.28 0.001* * Significant p < 0.05. 82 W. Samy, M.A. Hassanian / Arab Journ (35%) and nine were of average body weight (35%). None of the subjects enrolled in the study had morbid obesity. There was a sig- nificant increase in fasting blood glucose levels in group I com- pared with group II (90.86 ± 10.21 mg dl�1) versus (79.78 ± 19.17 mg dl�1) (p > 0.001). In addition, there was a signif- icant increase in postprandial blood glucose levels in group I com- pared with group II (183.11 ± 23.9 mg dl�1) versus (159.73 ± 52.62 mg dl�1) (p > 0.009). Thirteen patients in group I had hypertension, and seven of them showed the criteria of meta- bolic syndrome diagnosed by a recently modified Adult Treatment Panel III definition [16]. Regarding lipid profile, there was a significant increase of TC (190.15 ± 13.32 mg dl�1), TG (138.23 ± 18.42 mg dl�1) and LDL (121.47 ± 12.28 mg dl�1) in group I compared with group II (174.75 ± 25.85 mg dl�1), (122.91 ± 44.14 mg dl�1) and (111.80 ± 11.7 mg dl�1), respectively. More than two-thirds (68%) of patients showed TC over 200 mg dl�1 and 56% of patients showed TG equal to or over 150 mg dl�1. In addition, there was a significant decrease in serum HDL levels in group I when compared with group II. As regards liver function tests AST and ALT, showed a significant increase in group I: (20.64 ± 6.69 U l�1) and (25.44 ± 15.6 U l�1), as compared with group II: (15.85 ± 7.36 U l�1) and (17.65 ± 4.54 U l�1), respectively, and the AST/ALT ratio was 0.85 (less than 1). Serum total bilirubin (11.3 ± 2.1 umol l�1), serum albumin (4.36 ± 0.31 g dl�1), total protein (7.20 ± 0.23 g dl�1) and pro- thrombin time (12.78 ± 0.31 s) were within normal expected range in patients with NAFLD. Serum MDA levels were significantly in- creased (3.03 ± 0.23 nmol ml�1) in group I as compared with group II (2.91 ± 0.17 nmol ml�1). In addition, there was a significant in- crease in GP activity (642.36 ± 112.33 U l�1) in group I as compared with group II (554.25 ± 133.17 U l�1). Serum PON1 activity showed a significant decrease (28.33 ± 15.93 U l�1) in group I when compared with group II (39.66 ± 12.38 U l�1) (Table 3). When PON1 activity was correlated to the studied parameters (Table 4), it showed a significant nega- tive correlation with glucose levels in all the studied groups (Figs. 1 and 2). Regarding lipid profile parameters, there was a significant negative correlation of PON activity with TC levels of both groups and a significant negative correlation with TG levels of group I. PON1 activity showed significant positive correlation with HDL levels and significant negative correlation with LDL levels in both groups (Figs. 3–6). PON1 activity showed significant negative cor- relation with GP in group I. PON1 activity revealed non-significant correlation with either MDA or BMI in both the studied groups. After stepwise linear regression analysis, low serum PON1 levels, increased serum levels of MDA, TC and GP were the only indepen- LDL (mg dl ) �0.682 �0.893 GR (U ml�1) �0.272 �0.0.539* MDA (nmol ml�1) �0.325 �0.041 * Significant at p < 0.05. ** Significant at p < 0.01. dent predictor of hepatic steatosis scores in patients with NAFLD. After a trial of atorvastatin therapy (40 mg day�1 for 8 months), there was a significant increase in serum PON1 activity and a sig- nificant decrease of serum MDA levels. In addition, total choles- terol and LDL-cholesterol levels were significantly reduced with a significant increase in HDL-cholesterol levels. There was a r = - 0.692 , p<0.01 0 20 40 60 80 100 120 0 20 40 60 80 PON1 (U/ml) G lu co se (m g/ dl ) Fig. 1. Correlation between PON1 and glucose in control group. r = -0.722 , p<0.01 0 50 100 150 200 250 0 20 40 60 PON1 (U/ml) G lu co se (m g/ dl ) Fig. 2. Correlation between PON1 and glucose in NASH group. r= 0.822 , p<0.01 0 10 20 30 40 50 60 70 0 20 40 60 80 PON1 (U/ml) H D L (m g/ dl ) Fig. 3. Correlation between PON1 and HDL in control group. r = 0.944 , p<0.05 0 5 10 15 20 25 30 35 40 45 50 0 20 40 60 PON1 (U/ml) H D L (m g/ dl ) Fig. 4. Correlation between PON1 and HDL in NASH group. W. Samy, M.A. Hassanian / Arab Journal o r= - 0.682 , p<0.01 120 f Gastroenterology 12 (2011) 80–85 83 non-significant decrease in serum TG and GP (Table 5). A further comparison of chemical parameters in group Ia after atorvastatin trial and group Ib revealed a significant increase in serum PON1 activity and HDL-cholesterol levels with a significant decrease in serumMDA levels, serum total cholesterol, serum TG and LDL-cho- lesterol levels (Table 6). 0 20 40 60 80 100 0 20 40 60 80 PON1 (U/ml) LD L (m g/ dl ) Fig. 5. Correlation between PON1 and LDL in control group. r = -0.893 , p<0.01 0 20 40 60 80 100 120 140 160 180 0 20 40 60 PON1 (U/ml) LD L (m g/ dl ) Fig. 6. Correlation between PON1 and LDL in NASH group. Table 5 Clinical parameters before and after atorvastatin therapy in group Ia. Before atorvastatin therapy After atorvastatin therapy p-value PON1 (U ml�1) 28.33 ± 15.93 36.84 ± 10.13 0.028* MDA (nmol ml�1) 3.03 ± 0.23 2.77 ± 0.41 0.008* GP (U l�1) 642.36 ± 112.33 614.21 ± 102.76 0.281 TC (mg dl�1) 190.15 ± 13.32 182.22 ± 10.26 0.022* TG (mg dl�1) 138.23 ± 18.42 130.38 ± 15.31 0.107 HDL (mg dl�1) 39.24 ± 4.07 41.54 ± 2.04 0.014* LDL (mg dl�1) 122.73 ± 13.28 114.17 ± 10.38 0.014* * Statistically significant. tients with NAFLD and as predictors of developing NASH in these al o Discussion The present study demonstrated that obesity, impaired glucose tolerance and dyslipidaemia are associated with NAFLD and may have a role in its pathogenesis. The percentage of obese subjects was 80% in group I, while it represented 30% in group II, indicating that those patients might have had higher body fat or higher sub- cutaneous fat than control subjects. This observation was reported by many workers and established as the most important factors in developing NAFLD [17,18]. These metabolic disorders lead to in- creased circulating levels of free unsaturated fatty acids with en- hanced concentration in the liver leading to hepatic steatosis (fatty liver), which is considered the first hit to the liver [19,20]. Oxidation of free fatty acids results in the autopropagative pro- cess of lipid peroxidation. To assess whether there are relation- ships between oxidative stress and the development of NAFLD, w