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Int. J. Pharm. Med. & Bio. Sc. 2014 Roopa Rani Bhandary and Sukanya Shetty, 2014

ASSESSMENT OF ANTIOXIDANT STATUS

IN PATIENTS WITH NON-ALCOHOLIC

FATTY LIVER DISEASE

Roopa Rani Bhandary1*and Sukanya Shetty1

Research Paper

Background: Non-Alcoholic Fatty Liver Disease (NAFLD) is an increasing global health concern,with an estimated prevalence of 20% to 30% in Western countries and 15% in Asian countries.Oxidative stress is considered a major contributor as the “second hit” in the pathogenesis ofNAFLD. Objectives: We aimed to compare serum total antioxidant, MDA and Nitric Oxide levelsin patients with NAFLD and normal individuals. Methods and Materials: Patients with NAFLD (n=100) were compared to control subjects (n=100). Lipid peroxidation (Malondialdehyde) levelwas estimated using Thiobarbituric acid (TBA) method. Total antioxidant level was estimatedusing Phosphomolybdenum method. Nitric-oxide level was estimated using Griess-reagentmethod. Results are presented as mean + standard deviation value. Results: The mean serumlevel of total antioxidant in NAFLD individuals was found to be decreased when compared tonormal individuals, whereas the levels of MDA and nitric oxide was found to be significantlyincreased compared normal individuals. Conclusion: This study may provide clinicians to drawdifferent baselines for total antioxidant, Lipid peroxidation and Nitric oxide levels in patients withNon-Alcoholic Fatty Liver Diseases.

Keywords: Non-Alcoholic Fatty Liver Diseases, Oxidative stress, Malondialdehyde, Nitricoxide

*Corresponding Author: Roopa Rani Bhandary � rooparbhandary@gmail.com

INTRODUCTION

Non-Alcoholic Fatty Liver Disease (NAFLD) is an

increasing global health concern, with an

estimated prevalence of 20%-30% in Western

countries and 15% in Asian countries (Bellentani

et al., 2010).

Non-Alcoholic fatty liver disease is an

increasingly recognized condition that may

progress to end-stage liver disease. The

ISSN 2278 – 5221 www.ijpmbs.com

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Int. J. Pharm. Med. & Bio. Sc. 2014

1 Department of Biochemistry, K S Hegde Medical Academy, Nitte University, Deralakatte, Mangalore 575018.

pathological picture resembles that of alcohol-

induced liver injury, but it occurs in patients who

do not abuse alcohol. A variety of terms have been

used to describe this entity, including fatty-liver

hepatitis, nonalcoholic Laennec’s disease,

diabetes hepatitis, alcohol-like liver disease, and

nonalcoholic steatohepatitis. Nonalcoholic fatty

liver disease is becoming the preferred term, and

it refers to a wide spectrum of liver damage,

ranging from simple steatosis to steatohepatitis,

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Int. J. Pharm. Med. & Bio. Sc. 2014 Roopa Rani Bhandary and Sukanya Shetty, 2014

advanced fibrosis, and cirrhosis. Steatohepatitis

(nonalcoholic steatohepatitis) represents only a

stage within the spectrum of nonalcoholic fatty

liver disease (Paul et al., 2002; Elmar Aigner et

al., 2010; Guha et al., 2006; Michael Charlton,

2004).

Oxidative stress is known to play an important

role in the onset of NAFLD. When pro-oxidant

pathways generate more reactive species than

can be consumed by antioxidant pathways (e.g.,

via protein disulfide isomerase or reduced

glutathione peroxidase), oxidative stress occurs,

with resulting accumulation of reactive oxygen

species, chiefly superoxide and hydroxyl radicals

plus hydrogen peroxide). Oxidative stress is

considered a major contributor as the “second

hit” in the pathogenesis of NAFLD and NASH,

justifying the study of several antioxidants in

NAFLD treatment (Shadid and Jensen, 2003).

Given the increased mortality rates in patients

with NAFLD, the rising rates of obesity, diabetes,

and metabolic syndrome in this population, finding

an effective therapy is of utmost importance. In

this study, our aim is to determine the oxidative

stress markers in patients with NAFLD.

MATERIALS AND METHODS

Groups

Control: 100 healthy patients Serum wascollected from K S Hegde Hospital, Mangalore.

Study: 100 Patients with Non-Alcoholic Fatty LiverDiseases serum was collected from K S Hegde

Hospital, Mangalore.

Inclusion Criteria: Non fatty liver diseasesubjects who were diagnosed by USG.

Exclusion Criteria: Alcoholic fatty liver subjects.

• Patients taking lipid lowering drugs.

• Patients suffering from hepatitis (ALT > 350).

Blood Sampling: 2 mL of blood collected in aplain bottle, centrifuged to separate serum.

Oxidative Stress Markers: Lipid peroxidation(Malondialdehyde) level was estimated using

Thiobarbituric acid (TBA) method (Prieto et al.,

1999). Total antioxidant level was estimated using

Phosphomolybdenum method (Buege and Aust,

1978). Nitric-oxide level was estimated usingGriess-reagent method (Wink et al., 1995).

STATISTICAL ANALYSIS

Results are presented as mean + standard

deviation value. Student ’t’ test was used to

correlate between total antioxidant level and

control groups. A ‘p’ value of 0.05 or less was

considered significant.

RESULTS

The mean serum level of total antioxidant in

NAFLD individuals was found to be decreased

when compared to normal individuals, whereas

the levels of MDA and nitric oxide was found to

be significantly increased compared to normal

individuals. Serum total antioxidant level in case

of NAFLD was 146.05±24.89 to that of normal

individuals 185.15±39.91. Showed in Table 1 and

Figure 1.

The Serum Malondialdehyde level in case of

NAFLD was 3.67±0.40 and that of control group

0.85±0.28. The Table 1 and Figure 2 shows the

comparison values of MDA in case NAFLD and

normal group.

The serum Nitric oxide level was increased in

case of NAFLD patients 70.20±5.47 when

compared to that of normal individuals 48.93±6.67.

Values are shown in Table 1 and Figure 3.

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Int. J. Pharm. Med. & Bio. Sc. 2014 Roopa Rani Bhandary and Sukanya Shetty, 2014

DISCUSSION

The prevalence of NAFLD is continuously rising

and represents a growing clinical problem. NAFLD

is an increasingly recognized form of chronic liver

condition affecting both children and adults within

the wide spectrum of fatty liver diseases. Its

incidence and prevalence are increasing,

paralleling the increase in obesity and diabetes

mellitus. It is well-known that lipid peroxidation andoxidative stress play significant roles in the

pathogenesis of various diseases including

chronic liver diseases. NAFLD is present in 10%

to 24% of the general population in various

countries (Elmar Aigner et al., 2010).

Increased oxidative stress is considered a key

trigger in the pathogenesis of human NAFLD and

one of the enzymes counteracting oxidative

stress, copper/zinc (Cu/Zn) Superoxide

Dismutase (SOD) depends on adequate copper

availability, suggesting a potential link between

copper availability and impaired antioxidant

defence in NAFLD. (Elmar Aigner et al., 2012;

Baquial and Sorenson, 1995).

Living organisms have evolved different

molecules that speed up termination by catching

free radicals and therefore protect the cell

membrane. One important such antioxidant is

vitamin E. Other anti-oxidants made within the

body include the enzymes

Table 1: Comparison of Serum Total antioxidant, Nitric Oxide, MDA Levels in Normal and NAFLD

Parameters Normal NAFLD ‘P’ Value

Total antioxidant capacity (μg/mL) 185.15±39.91 146.05±24.89

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Int. J. Pharm. Med. & Bio. Sc. 2014 Roopa Rani Bhandary and Sukanya Shetty, 2014

Superoxide dismutase, catalase and

peroxidase. In addition, end products of lipid

peroxidation may be mutagenic and carcinogenic.

For instance, the end product malondialdehyde

reacts with deoxyadenosine and deoxyguanosine

in DNA, forming DNA adducts to them, primarily

M1G (Fracasso et al., 2002).

Free radicals are electrically charged

molecules, i.e., they have an unpaired electron,

which causes them to seek out and capture

electrons from other substances in order to

neutralize themselves. Antioxidants are capable

of stabilizing, or deactivating, free radicals before

they attack cells. Antioxidants are absolutely

critical for maintaining optimal cellular and

systemic health and well-being. Hence body

maintains complex system of enzymatic

antioxidants such as catalase, SOD,

peroxidases, etc., and non enzymatic

antioxidants such as Vitamin C, E and glutathione,

etc. Oxidative stress occurs as a result of

increased oxidative metabolism. An inadequate

intake of antioxidant nutrients may compromise

antioxidant potential, thus compounding overall

oxidative stress .Conditions associated with

oxidative damage include heart disease, cancer,

pulmonary disorders, ageing, etc. (Mataix et al.,

1998; Srikrishna and Suresh, 2009).

An antioxidant is a molecule capable of

inhibiting the oxidation of other molecules.

Oxidation is a chemical reaction that transfers

electrons from a substance to an oxidizing agent.

Oxidation reactions can produce free radicals. In

turn, these radicals can start chain reactions that

damage cells. Antioxidants terminate these chain

reactions by removing free radical intermediates,

and inhibit other oxidation reactions. They do this

by being oxidized themselves, so antioxidants are

often reducing agents such as thiols, ascorbic

acid or polyphenols. Although oxidation reactions

are crucial for life, they can also be damaging;

hence, plants and animals maintain complex

systems of multiple types of antioxidants, such

as glutathione, vitamin C, and vitamin E as well

as enzymes such as catalase, superoxide

dismutase and various peroxidase. Low levels of

antioxidants, or inhibition of the antioxidant

enzymes, cause oxidative stress and may

damage or kill cells.

Lipid peroxidation refers to the oxidative

degradation of lipids. It is the process whereby

free radicals “steal” electrons from the lipids in

cell membranes, resulting in cell damage. This

process proceeds by a free radical chain reaction

mechanism. It most often affects polyunsaturated

fatty acids, because they contain multiple double

bonds in between which lie methylene -CH2-

groups that possess especially reactive

hydrogens. As with any radical reaction the

reaction consists of three major steps: initiation,

propagation and termination.

NO is an important signaling molecule in the

body of mammals, including humans and is

important intermediate in the chemical industry.

(Hou et al., 1999) NO is an important messenger

molecule involved in physiological and

pathological processes within the mammalian

body both beneficial and detrimental. Appropriate

levels of NO production are important in

protecting an organ such as the liver from

ischemic damage. Chronic expression of NO is

associated with various carcinomas and

inflammatory conditions including juvenile

diabetes, multiple sclerosis, arthritis and ulcerative

colitis.

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Int. J. Pharm. Med. & Bio. Sc. 2014 Roopa Rani Bhandary and Sukanya Shetty, 2014

In tumor biology, nitric oxide has a complex

array of concentration dependent action, including

both inhibitory and promoting effect. It is thought

that the levels of nitric oxide found in many human

cancer lead to enhanced angiogenesis and tumor

dissemination. A high salt intake was

demonstrated to attenuate NO production,

although bioavailability remains unregulated.

(Osanai et al., 2002)

In the present study we observed that the

serum level of total antioxidant in NAFLD

individuals was found to be decreased when

compared to normal individuals, whereas the

levels of MDA and nitric oxide was found to be

significantly increased compared normal

individuals.

CONCLUSION

This study may provide clinicians to draw different

baselines for total antioxidant, Lipid peroxidation

and Nitric oxide levels in patients with Non-

Alcoholic Fatty Liver Diseases.

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