Alteration in Antioxidant Defense System and Oxidative Stress in blood for Non Alcoholic Fatty Liver Disease

Received 12 Aug 2017, Revised 28 Aug 2017, Accepted 18 Sep 2017, Online 30 Oct 2017 AbstractAntioxidants neutralize free radicals or reactive oxygen species which cause oxidative stress. Oxidative stress is a major contributor in the pathogenesis of Non-alcoholic fatty liver disease (NAFLD). Present study is aimed to know level of antioxidant defense system and oxidative stress in controls and NAFLD participants. The cross-sectional study was carried out at tertiary centre of gastroenterologist unit at Anand. Experimental participants were divided into two groups: Control (n= 52, Age26.7±1.23 years, 39 M/ 13 F) and NAFLD (n= 48, Age41.71±1.71, 43 M/5 F). All participants underwent anthropometric assessment, clinical examination, biochemical parameters, antioxidant parameters and lipid peroxidation marker or oxidative stress marker (MDAMalondialdehyde). Results of our study indicate significant increase in markers of metabolic syndrome (obesity, dyslipidaemia) and liver enzymes in NAFLD compared to controls. Total antioxidant activity (Gallic acid equivalent (GAE (p=0.0008)) and Trolox equivalent (TE (p<0.0001)) and antioxidant enzymes such as superoxide dismutase (SOD (p=0.0023)), Glutathione Peroxidase (GSHPx (p<0.0001)), Glutathione-s-transferase (GST (p<0.0001)) and Catalase (CAT (p<0.0001)) were significantly decreased in NAFLD compared to controls. Lipid peroxidation marker or oxidative stress marker (MDA) was significantly increased (p<0.0001) in NAFLD compared to controls. Our study concludes that NAFLD participants have increased oxidative stress and decreased antioxidant levels compared to controls. Study strongly indicates that antioxidant enzymes namely GST and CAT can be used as surrogate markers of oxidative stress for NAFLD participants.


INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) is defined as steatosis affecting >5% of hepatocytes in the absence of excessive significant alcohol consumption.The histological spectrum of NAFLD includes non-alcoholic fatty liver (NAFL; steatosis without hepatocellular injury), steatohepatitis (NASH; steatosis with inflammation and hepatocyte ballooning degeneration), fibrosis and ultimately cirrhosis [1].NAFLD has become the most common cause of chronic liver disease worldwide in the last three decades and a major cause of liver-related morbidity and mortality.
Clinical burden of NAFLD is not only confined to liverrelated morbidity and mortality, but there is a growing evidence that NAFLD is a multi-system disease, affecting several extra-hepatic organs like kidney, lung and heart etc. [2,3].
NAFLD can occur at all ages, the highest prevalence is at the age of 40-50 years [4].The majority (56%-79%) of NAFLD participants are overweight or obese (BMI (body mass index) > 25 kg/m 2 ), only one third have the metabolic syndrome.Sometimes lean participants (BMI < 25 kg/m 2 ) also have metabolic risk factors for NAFLD [5].Prevalence of NAFLD is inadequately known due to absence of signs, symptoms and lacks sensitive and specific diagnostic tools [4,6].Most individuals with NAFLD are usually asymptomatic, however, sometimes fatigue, discomfort in the right upper quadrant of the abdomen is observed [5][6][7][8].In India, prevalence of NAFLD is around 9% to 32% of general population with higher prevalence (57-74%) in those with overweight/obesity and with diabetes/ pre-diabetes [4].In western countries, the prevalence of NAFLD varies between 20 to 30%, rising up to 70-90% in obese individuals [7,9,10].The pathogenesis of NAFLD is a very complicated process and model proposed in 2010 known as "multiple parallel hits" which includes obesity, insulin resistance and oxidative stress as a causative factors for onset of NAFLD [11].
Insulin resistance has a central role in both steatosis and in its progression to more advanced forms of the disease as non-alcoholic steatohepatitis (NASH) makes it the main pathogenic mechanism of NAFLD [12].Oxidative stress, result of excess amount of ROS production regarded as one of the pathological mechanisms that results in initiation and progression of various liver diseases such as alcoholic liver diseases and non-alcoholic fatty liver disease to fibrosis, cirrhosis and Hepatocellular Carcinoma [13].
Liver enzymes namely SGPT, SGOT, GGT are reported as a surrogate marker for liver disease, but they are non-specific because up to 78% of the NAFLD individuals possess normal values for liver enzymes and do not reliably correlate with liver histology [5][6][7].Even though Ultrasonography is a commonly used method for prognosis of NAFLD with sensitivity of 85%, it cannot detect stages of fibrosis and inflammation [6].Liver biopsy is useful invasive method for detection of stages of NAFLD progression from steatosis, NASH to cirrhosis and it is known as "gold standard method" for detection of fibrosis, but has low accuracy and is assessed with complications [6,14].Review studies suggest that approximately 30% of the general population have radiological evidence of steatosis, 8% have raised transaminases due to NAFLD and remaining sufferers go unrecognised [1].The human body is equipped with a variety of highly sophisticated and complex antioxidant protection system to protect the cells and organ systems of the body against reactive oxygen species (ROS) [15].Antioxidant activity is known to reflect the altered redox balance of affected fluids, tissues or organs in several pathological processes [16].Increased antioxidant status can reduce the risk for chronic diseases including liver diseases, cardiovascular diseases, cancer, neurodegenerative diseases, and immune dysfunction [17].Enzymatic Antioxidants have ability to catalyse free radical quenching reactions, convert free radicals into non-reactive species [15,17].They are important in preventing lipid peroxidation and maintaining the structure and function of biologic membranes [18].Reactive oxygen species present in excessive amounts may cause extreme damage to biomolecules such as lipids, proteins, and DNA [17]."The imbalance between oxidants and antioxidants in favour of the oxidants, potentially leading to damage", termed as "oxidative stress" [19].Oxidative stress is among the major causative factors in induction of many chronic and degenerative diseases including atherosclerosis, ischemic heart disease, aging, diabetes mellitus, cancer, immunosuppression, neurodegenerative diseases, male infertility, liver diseases, vitiligo and others [13,19,20].Oxidative stress is a process to explain progression to hepatocellular damage, inflammation and fibrosis [21].Lipid peroxidation caused by oxidative stress is implicated in the pathogenesis of several hepatic disorders in human [22].Damage to cells caused by free radicals is believed to play a central role in disease progression [15].Oxidative stress due to augmented generation of reactive oxygen species (ROS) can induce lipid peroxidation leading to inflammation and fibrogenesis through the activation of stellate cells [23,24].Fibrosis as a precursor of cirrhosis is a pivotal pathological process in the evolution of all chronic liver diseases to cirrhosis [25].Hepatic fibrosis is fibrous scarring of the liver.Hepatic fibrosis itself causes no symptoms but can lead to the end-stage cirrhosis [26].NAFLD condition may alter the level of antioxidants because of oxidative stress which are undertaken for the investigation.Review studies do not show sequential and integrated work for total antioxidant activity, antioxidant enzyme system and lipid peroxidation marker from human blood samples for NAFLD participants.Literature reviewed cannot suggest any parameter which gives clear idea regarding onset or stages of fatty liver disease.Many researchers studied selected parameters of antioxidants and only limited information is available to conclude the presence of liver disease which cannot be considered as a strong evaluation parameter.Therefore, present study is aimed to analyse the level of plasma /serum antioxidants status and oxidative stress marker (MDA) in NAFLD condition.Systematic study includes taking informed consent from selected participants followed by blood collection and lab analysis of selected parameters.Parameters selected were antioxidant activity, antioxidant enzymes (SOD, GR, GST, GSHPx, CAT) and lipid peroxidation marker (MDA).Investigation of our study point out that as oxidative stress increases, total antioxidant activity and antioxidant enzymes decreases which is specific for selected parameters.Present study helps to diagnose the fatty liver condition at the very early stage of NAFLD using analytical blood values for selected parameters.Our study recommends that similar research can be carried out for both the genders and at different geographical condition.Our study concludes that GST and CAT can be used as surrogate markers for NAFLD participants.

II. RELATED WORK
Many researchers have separately worked on selected total antioxidant activities [27,28] or selected antioxidant enzymes [18, 28 -32] or oxidative stress marker [28,29] from miscellaneous mammalian plasma /serum samples.Very little sequential work is found on total antioxidant activity, selected antioxidant enzymes (SOD, Catalase, GSHPx, GR and GST) and oxidative stress marker (MDA) from the human plasma /serum sample in NAFLD condition compared to the controls which is limited for only selected parameters.Therefore, present study was planned to analyse total antioxidant activity, all sequential antioxidant enzymes and lipid peroxidation marker from blood samples of each, i.e. controls and NAFLD participants.

Study Design:
All participants underwent anthropometric assessment, clinical examination, biochemical parameters, antioxidant parameters and oxidative stress marker.

Exclusion Criteria:
Pregnant and lactating women, Alcoholic, Smokers/ tobacco users, Exposure to radiation, Exposure to ozone therapy or hyperbaric oxygen therapy, Exposure to heavy metals or ayurvedic medicine, Infection within 30 days, Cancer, Cardiac, gastro-intestinal and brain ischemic disease, Currently not using antioxidant drugs which affect the oxidative stress and antioxidant status were excluded.

Informed Consent:
Explain interest of the study to the participants and informed consent were taken.

Anthropometric assessment and clinical parameters and biochemical parameters
Anthropometric assessment includes the record for age, height and weight (Height was measured using height board which is fixed to the wall and weight using high quality electronic digital scale).BMI was calculated using formula BMI= [body weight (kg)]/ [(height) (m)

Antioxidant parameters and oxidative stress marker
Total antioxidant activity (Gallic acid equivalent and Trolox equivalent) [34] and enzymatic antioxidants (SOD [35], GSHPx [36], GR [37], CAT [38] and GST [39] were analysed using standard protocol in our laboratory after cross-checking the standards and samples.U.V. Spectrophotometer (Systronic-model no.117) was used for investigation of all the above parameters.MDA was analysed from plasma by standard method [40].

Statistical analysis
Data are expressed as mean ± S.E. and percentages.Statistical analysis was conducted using z-test and t-test [41].

Ethical approval:
The institutional ethics committee approved the study protocol.

Anthropometric assessment, clinical parameters and biochemical parameters
In this cross-sectional study, controls and NAFLD participants were studied for anthropometric assessment, clinical parameters and biochemical parameters.Values are presented in table: 1.The prevalence of NAFLD increases with the rising incidence of obesity.In our study 50% and 29% of the NAFLD participants were obese and overweight respectively.Population and hospital-based studies from the West India also reported around 10-24% of general population and 57-74% of obese individuals suffering from NAFLD [5].Our study indicates presence of metabolic syndrome such as high BMI, dyslipidaemia or/and elevated liver enzyme levels (SGPT, SGOT and GGT) compared to controls.Results of our study were in agreement with the study carried out on human participants suffering from NAFLD condition.[42,43].Almost 90% of NAFLD participants have more than one characteristic of metabolic syndrome [12].

Antioxidant parameters and oxidative stress marker
Antioxidant parameters such as total antioxidant activity (Gallic acid equivalent and Trolox equivalent) and antioxidant enzymes (SOD, GSHPx, GST and CAT) were decreased in NAFLD participants compared to controls.Antioxidant parameters such as total antioxidant activity (Gallic acid equivalent and Trolox equivalent), antioxidant enzymes (SOD, GSHPx, GR, GST and CAT) decreases may be due to defences against free radicalmediated injury includes enzymatic deactivation and direct reaction with free radicals [18].Result of our study for total antioxidant activity was significantly decreased in NAFLD compared to controls [Gallic acid equivalent (46% vs. 34%) and Trolex equivalent (67% vs. 24%)] depicted in figure: 1.Compared to reference value [44,45] total antioxidant status was found lower for controls, which further decreases (GAE (P=0.0008),TE (P<0.0001))among NAFLD participants.Previous study carried out for NAFLD participants also reveal decrease in total antioxidant activity [28].Antioxidant enzymes such as SOD, GSHPx, GST and CAT decreased in NAFLD compared to controls are as presented in figure: 2. Decreased activity of SOD is important factor for pathogenesis of NASH [46].Our study revealed significant lower value for SOD in controls compared to reported value, which further significantly decreased (p=0.0023) in NAFLD participants.SOD is considered as the first line of defence against oxygen derived free radicals, converts superoxide anion into H 2 O 2 , forming as neutral products O 2 and H 2 O. SOD scavenges superoxide and inhibits the formation of Peroxynitrite, thereby suppressing the resulting injury.Therefore, it acts as a protective mechanism against tissue injury.SOD level decreases with increase in injury due to higher the utilization of SOD in defence mechanism [24].GSHPx catalyses reductive destruction of hydrogen and lipid hydro peroxides, using glutathione as an electron donor [18].Study carried out by few researchers found decrease in SOD and GSHPx activity which is in accordance with our results [28,16].Initial level of SOD and GSHPx is lower in controls may be due to lower intake of antioxidant containing foods.Decrease in the activity of glutathione peroxidase may be due to exhaustion or inactivation of the enzyme by reactive oxygen species, since oxidative damage to hemoglobin and cell membrane has been reported to reduce the activity of glutathione peroxidase [47].
Finding of our study indicates non-significant alteration in GR activity for NAFLD participants compared to controls [18].
Our study indicates a significant (p<0.0001)decrease in the activity of catalase in NAFLD participants compared to controls may be due to less availability of NADPH [29,47].
The function of GST is to detoxify foreign compounds.GST in plasma provides an exceptionally sensitive index of hepatocellular damage.Serum GST may be a more sensitive marker of hepatocellular damage than transaminases [48].
Our study also indicates significant decrease (p<0.0001) in GST activity for NAFLD participants compared to controls (100% vs. 0%).Study carried out on liver tissue of wistar rats suffering from NASH condition also showed decrease in GST activity [49].GST (4.02-6.46 vs. 0.005-1.78)and CAT (0.24-0.98 vs. 0.002-0.1)values in controls vs. NAFLD are highly significant (p<0.0001)so GST and CAT were considered as most sensible parameters to know oxidative stress in NAFLD.Both enzymes can be used as surrogate marker to know oxidative stress in NAFLD.
Oxidative stress due to increased ROS production has a role in the pathogenesis of NAFLD [46].Liver is continuously exposed to ROS and is protected from oxidative injury by a range of antioxidant pathways [18].

V. CONCLUSION AND FUTURE SCOPE
Study concludes that NAFLD participants indicates the presence of metabolic syndrome such as obesity, dyslipidaemia or/and elevated liver enzyme levels (SGPT, SGOT and GGT) compared to controls.Total antioxidant activity decreases in NAFLD condition which can be supported by values obtained for various antioxidant enzymes.Maximum decreased in values were obtained for CAT followed by GST, GSHPx and SOD whereas GR shows non-significant difference.Even though SGPT and SGOT do not drastically vary, antioxidant enzymes namely GST and CAT drastically changes compared to controls which can be used as surrogate markers of oxidative stress in NAFLD participants.Result of our study strengthens by the increase value obtained for oxidative stress marker which is considered as an important factor for pathogenesis of NAFLD.
The results of this study can be used to establish GST and CAT as markers for recognition of onset of NAFLD condition by planning and implementing future study after evaluating more samples from both the genders and at different geographical condition.

LIMITATIONS
Study is limited for non-alcoholic fatty liver condition.
Grouping of the NAFLD condition on the basis of other disease/disorder conditions such as obesity, diabetes mellitus or any other associated condition to conclude lower and upper limits for all above parameters for both the genders is not included in present study.

DISCLOSURE STATEMENT
The authors have nothing to disclose.

CONFLICT OF INTEREST STATEMENT
The authors have no conflict of interest regarding the subject of the study.