Relationship of Uric Acid with Superoxide Dismutase (Sod) in Induced Hyperuricemic Rat Model

doi:10.4236/pp.2012.34054

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Pharmacology & Pharmacy, 2012, 3, 404-408

http://dx.doi.org/10.4236/pp.2012.34054 Published Online October 2012 (http://www.SciRP.org/journal/pp) 1

Relationship of Uric Acid with Superoxide Dismutase (Sod)

in Induced Hyperuricemic Rat Model

Shiza Batool1, Iftikhar Ahmed2*, Muhammad Sarwar3, Hafeez ul Hassan4

1Biochemistry Department, Aziz Fatima Medical and Dental College, Faisalabad, Pakistan; 2Biochemistry Department, Baqai Medi-

cal University, Karachi, Pakistan; 3Chemical Pathology Department, Al Jouf University, Al-Jawf, KSA; 4Physiology Department,

Baqai Medical Unive rsity, Karachi, Pa ki st an.

Email: *[email protected], *[email protected]

Received July 16th, 2012; revised August 18th, 2012; accepted September 12th, 2012

ABSTRACT

Increase uric acid levels have been found in oxidative stress. Urate radicals do not react with oxygen to form another

peroxy radical, thus increasing the efficacy of uric acid as an antioxidant. Therefore, this study is designed to measure

the level of uric acids and find out the relationship of uric acid with superoxide dismutase in induced hyperuricemic

model. Forty male albino rats with an average weight of 180 ± 2 g were selected. The rats were grouped. The animals

were fed on standard diet and given tap water ad libitum until treatment. Albino rats were divided into four groups.

Group A (10)—con trol giv en only stand ard diet, gr oup B (10) fed on 60% fructose w ith standard diet, group C (10) fed

on fructose, standard diet and intraperitonially oxonic acid 250 mg/kg and group D (10) only on injection intraperotoni-

ally oxonic acid 250 mg/kg. At the end of study 10 mL of blood was drawn from heart of rats. Then blood was esti-

mated for superoxide dismutase and uric acids done by kit methods randox-manual/Rx monza UA230/UA 233. Results:

In Group C superoxide dismutase was found to be 32% (244 ± 2.23 mg/dL) more than control. In the same group the

uric acid concentration was highly significantly correlated with control. Conclusion: The uric acid concentration in-

creases when we take fructose up to 60% in our diet. It also increases superoxide dismutase concentration. More than

this value may have inverse effect on the uric acid level and its role as an antioxidant may become inversed.

Keywords: Uric Acid; Superoxide Dismutase; Albino Rats; Fructose Induced Hyperuricemia

1. Introduction

Free radical can be defined as any molecular species ca-

pable of independent existence that contains an unpaired

electron in an atomic orbital [1]. They are capable of tr ig-

gering chain reactions which can damage the different

cell constituents.

The most important free radicals in many disease

states are oxygen derivatives, particularly superoxide and

the hydroxyl radical. Superoxide is formed from several

molecules by oxidation including adrenaline, flavine nu-

cleotides, thiol compounds and glucose. Superoxide is

also produce during important biological reactions in-

cluding electron transport chain in mitochondria [2].

In order to check free radicals formation to avoid oxi-

dative stress, body has different anti-oxidant defense

systems. An antioxidant can be defined as: “any sub-

stance that when present in low concentrations compared

to that of an oxidisable substrate significantly delays or

inhibits the oxidatio n of that substrate. The physiolog ical

role of antioxidants, as this definition suggests, is to pre-

vent damage to cellular components arising as a conse-

quence of chemical reactions involving free radicals [3].

Superoxide dismutase is believed to serve as first line of

defense against toxicity of superoxide radicals. Also

takes part in cell signaling regarding to reactive oxygen

species levels [4]. Supero xide is one of the main reactive

oxygen species in the cell and as such, SOD serves a key

antioxidant role. The physiological importance of SODs

is illustrated by the severe pathologies evident in mice

genetically engineered to lack these enzymes. Mice lack-

ing SOD2 die several days after birth, amid massive oxi-

dative stress [5] Mice lacking SOD1 develop a wide range

of pathologies, includ ing hepatocellular carcinoma [6] an

acceleration of age-related muscle mass loss [7], an ear-

lier incidence of cataracts and a reduced lifespan. Mice

lacking SOD3 do not show any obvious defects and ex-

hibit a normal lifespan, though they are more sensitive to

hyperoxic injury [8].

Uric acid now is not considered as merely a metabolic

waste. It ha s been proposed that increase in life span ob-

served in human evolution to some extent might be due

to protective action of uric acid [9]. Increase uric acid

*Corresponding a uthor.

Relationship of Uric Acid with Superoxide Dismutase (Sod) in Induced Hyperuricemic Rat Model 405

levels have been found in oxidative stress and ischemia

which might be compensatory mechanism of protection

against free radicals [10]. Urate radicals do not react with

oxygen to form another peroxy radical which is seen with

the ascorbic acid, thus increasing the efficacy of uric acid

as an antioxidant [11]. Uric acid cause inactivation of

Nitric oxide and peroxynitrite radicals [12,13]. Another

important function of urate is found in its ability to form

chelating agents with transition metals ions like iron and

copper thus scavenging them. We carried out a study to

assess the relationship of uric acid with superoxide dis-

mutase in an induced hyperuricemic rats.

2. Methodology

Locally bred fort y (40) male Albino ra ts with an averag e

weight of 180 ± 20 g were purchased. The rats were

grouped and housed in environmentally controlled room

(ambient temperature 24˚C ± 2˚C and relative humidity

of 55% ± 5%) in the animal house and acclimatized for

07 days. The animals were fed standard diet and given

tap water ad libitum until treatment. The protocols for

experimentation was approved and performed in strict

accordance with the Guide for the care and use of labo-

ratory animals (Institute of Laboratory Animal Resources

on Life Sciences, US National Research Council, 1996)

and the Institutional Animal Ethical Committee (IAEC)

of Baqai Medical University, Karachi. Pakistan. The

cage size was 8" × 18" × 10" to keep a group of 5 ani-

mals in the cage to prevent from cannibalism.

Sodium Tungstate 10%, 2/3N sulphuric acid, 10% so-

dium bicarbonate, LiCO3, 40% Formaline, Acetic acid,

Fructose, Oxonic acid. Spectrosol grade reagents and

acids from B.D.H., Poole, UK, were employed. All puri-

fied enzymes, coenzymes, substrates, standards and buff-

ers will be purchased from Sigma Chemicals Company,

USA. All other chemicals were of analytical grade and

will be procured from SRL and Qualigens, USA.

All animals housed in standard conditions were ini-

tially fed standard diet and allowed adaptation of one

(1) week. Albino rats were divided in four (4) groups: A,

B, C & D.

Group A: Ten (10) male albino rats as Control were

kept as control and were fed standard diet and water ad

libitum for 10 weeks. Group B: Ten (10) male albino rats

[F] were fed 60% fructose mixed in standard diet and

water ad libitum for 10 weeks. Group C: Ten (10) male

albino rats [FO] were fed 60% fructose mixed in standard

diet and water ad libitum for 10 weeks. They were also

injected intraperitonealy oxonic acid 250 mg/kg every

third day for 10 weeks. Group D: Ten (10) male albino

rats [O] were injected intraperitonealy oxonic acid 250

mg/kg every third day for 10 weeks. They were fed

standard diet and water ad libitum for 10 weeks. Body

weights were measured at the commencement and at the

end of study. The amount of diet was measured before

giving and then subtracted from the amount of food left

over daily. At the end of study, rats were dissected in a

nearby room separate from experiment area. Approxi-

mately 10 mls of blood was drawn from heart using dis-

posable syringe. 8 mls of blood was transferred in he pa rin -

ized tube, mixed and centrifuged to separate plasma and

divided in two epindorf cups for estimation of uric acid

and SOD done by kit methods by randox-manual /Rx

monza UA230/UA 233.

3. Results

3.1. Graph 1

It demonstrates the comparison of mean plasma uric acid

levels of Control with rest of the groups. Mean plasma

level of uric acid of Control is found to be 1.97 mg/dL

(±0.09). Group “F” (fructose) showed mean plasma uric

acid of 3.15 mg/dL (±0.17). This reflects that uric acid

was raised to 37% in rats which were exposed to diet

comprising 60% Fructose than control. On comparing

both groups i.e., Control with Group “F” (highly signifi-

cant statistical correlation (P < 0.001) was observed.

The mean plasma uric acid levels of Group “O”

SERUM URIC ACID

GROUPS CONTROL GROUP = F GROUP = O GROUP = F + O

M.V. 1.97 3.15 3.63 4.43

S.D 0.3 0.55 0.63 0.43.

Graph 1. Comparison of serum uric acid levels of all study groups.

Relationship of Uric Acid with Superoxide Dismutase (Sod) in Induced Hyperuricemic Rat Model

406

(oxonic acid) was 3.63 mg/dL (±0.22) which is 45%

higher than Control. The probability calculated was high l y

significant (P < 0.0 01 ) w hen both groups were eval uat ed .

While comparing Group “F + O” (Fructose + Oxonic

acid) with Control, highly sign ificant correlation was ob-

served (P < 0.001). It was due to high mean plasma se-

rum uric acid level of Group “F + O” which was 4.41

mg/dL (±0.14). The combinatio n of fructose with uricase

inhibitor, Oxonic acid raises uric acid to 55% from con-

trol and this level is highest of all these groups.

3.2. Graph 2

It shows the comparison of mean plasma SOD levels of

Control with rest of the groups. Mean plasma level of

SOD of Control was found to be 165.15 mg/dL (±2.65).

Group “F” (Fructose) showed mean plasma SOD level of

176.65 mg/dL (±2.60) reflecting SOD levels were raised

to 6.5% in rats which were exposed to diet comprising

60% Fructose. On comparing both groups i.e., Control

with Group “F” significant statistical correlation (P <

0.01) was observed. The mean plasma SOD levels of

Group “O” (oxonic acid) has been measured to 181.2

mg/dL (±3.52) which is again 8% more than Control.

Therefore, significant (P < 0.01) correlation was ob-

served on comparison of both groups.

While comparing Group “F + O” (Fructose + Oxonic

acid) with Control, highly significant correlation was

observed (P < 0.001). It was due to high mean plasma

SOD levels of Group “F + O” which was 244 mg/dL

(±2.23) which is high est of all these groups. In this gro up

SOD was 32% more than control which might be due to

antioxidant action of uric acid .

4. Discussion

One of the important features of this study was the

method by which hyperuricemia have been induced in

animal model. The group B was given fructose, group D

was treated with “oxonic acid” and group C was offered

both fructose and oxonic acid (G = Fructose + Oxonic

acid). The principle hyperuricemic factor in this study

was fructose as it is extensively used in beverages and

food. Its a rather controversial factor as number of stud-

ies both animals and human, are in the favour that fruc-

tose can induce hyperuricemia [14] but many studies

have opposed this hypothesis [15] and even mixed re-

sponse has been shown [16]. Present investigation has

tried to verify this theory. Very few studies have used

this combined model of fructose plus oxonic acid. In

order to make conditions similar to human, uricase in-

hibitor oxonic acid was incorporated to ab olish the effect

of this enzyme in rats. Also these different regimens

were used to establish the extent of hyperuricemia caused

by fructose.

Superoxide dismutase levels were found to be raised in

all three groups in comparison to control. The highest

level was obser ved in gro up C of 244 mg/dL as sh own in

graph 1. They were 32% more than the control. This was

in agreement with H. Ulrich Hink and Nalini Santanam

et al. The possible explanation can be drawn from num-

ber of studies showing that superoxide dismutase during

catalyzing dismutation of 2 to H2O2 can form copper

bound hydroxyl radical from hydrogen peroxide H2O2

[17]. Hydroxyl radical when gets bounded to SOD, then

it can attack adjacent histidine residue which is attached

to copper resulting in inactivation of both SOD1 and

SOD3 [18]. This might be prevented when small anions

or reductants including Uric acid are co incubated [19] as

shown in graph 1 the comparison of mean plasma SOD

levels of Control with rest of the groups. Mean plasma

level of SOD of Control was found to be 165.15 mg/dL

(±2.65). Group B (Fructose) showed mean plasma SOD

level of 176.65 mg/dL (±2.60) reflecting SOD levels

were raised to 6.5% in rats which were exposed to diet

comprising 60% Fructose. On comparing both groups, i.e.

Control with Group B significant statistical correlation (P <

0.01) was observed.

O

SUPEROXIDE DISMUTASE

GROUPS CONTROL GROUP = F GROUP = O GROUP = F + O

M.V. 165.15 176.65 181.25 244

S.D 8.38 8.23 9.96 6.69

Graph 2. Comparison of serum SOD levels of all study groups.

Relationship of Uric Acid with Superoxide Dismutase (Sod) in Induced Hyperuricemic Rat Model 407

The mean plasma SOD levels of Group D (oxonic acid)

has been measured to 181.2 mg/dL (±3.52) which is

again 8% more than Control. Therefore, significant (P <

0.01) correlation was observed on comparison of both

groups.

While comparing Group C (Fructose + Oxonic acid)

with Control, highly significant co rrelation was observed

(P < 0.001). It was due to high mean plasma SOD levels

of Group “F + O” which was 244 mg/dL (±2.23) which

is highest of all these groups. In this group SOD was

32% more than control which might be due to antioxi-

dant action of uric acid.

It has been suggested that in group C there was signi-

ficant increase in the concen tration of uric acid. This was

in accordance with some studies done on human that

high dietary intake of fructose contributes significantly to

hyperuricemia [20]. In a large study in the United States,

consumption of four or more sugar-sweetened soft drinks

per day gave an odds ratio of 1.82 for hyperuricemia [21].

Increased pr oduction of uric acid is th e result of interfer-

ence, by a product of fructose metabolism, in purine me-

tabolism. This interference has a dual action, both in-

creasing the conversion of ATP to inosine and increasing

the synthesis of purine [22] http://en.wikipedia.org/wiki/

Hyperuricemia-cite_note-pmid8213607-14. Fructose also

inhibits the excretion of uric acid, apparently by compet-

ing with uric acid for access to the transport protein

SLC2A9 [23]. The effect of fructose in reducing excre-

tion of uric acid is increased in people with a hereditary

(genetic) predisposition toward hyperuricemia and/or

gout [22] .

Starvation causes the body to metabolize its own

(purine-rich) tissues for energy. Thus, like a high purine

diet, starvation increases the amount of purine converted

to uric acid. A very low calorie diet without carbohydrate

can induce extreme hyperuricemia; including some car-

bohydrate (and redu cing the protein) reduces th e level of

hyperuricemia [24]. Starvation also impairs the ability of

the kidney to excrete uric acid, due to competition for

transport between uric acid and ketones [25]. Many

studies are controversial to our results all were done on

human some supported that high doses of fructose (200

g/day for 2 weeks) raise the blood pressure and cause the

features of metabolic syndrome. Some suggested that

lowering of the uric acid level prevents the increase in

mean arterial blood pressure. Excessive intake of fruc-

tose may have a role in the current epidemics of obesity

and diabetes [26]. Some authors also suggested that in-

creased dietary fructose was not associated with increase

uric acid level [27].

5. Conclusion

The uric acid concentration does increase when we take

fructose up to 60% in our diet, e.g., beverages soft drinks.

It also increases superoxide dismutase concentration. It

has been concluded that more than this value of fructose

may have inverse effect on the uric acid level and its role

as an antioxidant may become inversed. Therefore, it is

suggested from our study that further work need to be

done on the effect of fructose on uric acid levels in hu-

man.

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