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Introduction:

The liver injury due to acute and chronic ethanol

abuse has been proved to be dependent on its oxidative

effect at the cytosolic, peroxisomal and microsomal

levels.1 But despite extensive investigations, the

molecular mechanism leading to the hepatic damage

still needs to be clarified. Based on technologically

advanced procedures, it has been demonstrated that

a group of reactive species known as free radicals might

be taking a major role in the pathogenesis of tissue

changes during hepatic ethanol loading. A free radical

has been defined as a chemical species, capable of

independent existence that contains unpaired electrons.

They are energetically unstable, highly reactive and

short lived.2 Drugs including alcohol may exert toxic

effect by promoting free radical formation during their

metabolism and a decline of some of the antioxidant

defences like reduced glutathione, vitamin C, vitamin

E, vitamin A, etc. thereby increasing the ratio between

pro-oxidant and antioxidant reaction resulting to a

condition known as oxidative stress.3 Polyunsaturated

fatty acids within the cell membranes and lipoproteins

are particularly susceptible to oxidative attack often

as a result of metal ion dependent hydroxyl radical

formation. Long chains of lipid peroxides may be

formed causing serious disruption of cell membrane
Correspondence: Dr. A. R. Singh

E-mail: dr.drarsingh@rediffmail.com

Study of effect of ethanol on antioxidant - vitamin A and C in rat liver

A. R. Singh1, Sushil Kumar2, Roshan Takhelmayum2, J. N. Sihna3

1Asst. Prof, 2Lecturer, 3Prof and Head, Department of Biochemistry, College of Medical Sciences and Teaching Hospital,

Bharatpur, Nepal.

Abstract:

Objective: To see the effect of consumption of locally distilled alcohol (country liquor) continuously for

few months on hepatic vitamin A and C status in albino rats.

Materials and methods: The study was conducted in 36 male wistar strain albino rats for 3-4 months old

consisting six groups of six animals each.

Results: The first observation was weight gain among the series of alcoholic animals when compared to

the control and alcoholic fed animals supplemented with vitamin A and C, p-value by T-test between the

mean values of the initial weight and final weight was < 0.01 (0.006), significant.

Conclusion: It was found that the major effect on hepatic vitamin A and C contents were observed more

distinctly in mitochondrial fractions when compared with the rest fractions. Supplementation of vitamins

helped to protect loss of the vitamins which delayed the aging process at age 9-10 months in our study.

Key words: Albino rats, alcoholic, hepatic, vitamin A

Original Article, 29-34Journal of College of Medical Sciences-Nepal, 2010, Vol. 6, No. 1

29



function.4, 5 Proteins exposed to free radical attack may

fragment, cross link or aggregate. The consequences

include interference with ion channels, failure of cell

receptor, etc. Free radical damage to DNA may cause

destruction of bases, deoxyribose sugar and single or

double strand breaks6 and is implicated in mutagenesis,

carcinogenesis and even cell death.7 Antioxidants delay

and protect against oxidative damage produced by free

radicals. Vitamin A and C belong to nutrient

antioxidants. Vitamin A is a lipid soluble antioxidant

and membrane bound. It can suppress free radical

induced lipid peroxidation under conditions of low

partial pressure of oxygen in most tissues. Vitamin C,

a water soluble antioxidant which acts as free radical

scavenger could improve liver functions in alcoholic

patients8, 9 and that also maintains the vitamin E level.

It has been reported that both vitamins may behave as

pro-oxidants if aqueous phase antioxidant fall short.10

The present study was undertaken to see whether

the nutritional antioxidants like vitamin A and vitamin

C have got any definite role in checking liver injury in

alcoholics.

Materials and methods:

The study was carried out in the Department of

Biochemistry, Regional Institute of Medical Sciences

(RIMS), Imphal, Manipur. Albino rats (Wistar strain),

3-4 months old procured from National Institute of

Nutrition, Indian Council Of Medical Research

(ICMR), Hyderabad reared in the Central Animal

House, RIMS, Imphal were the animals used for the

study. Diet chart formulation was done according to

the method given for preparation of pellet diets as

published in LAIIS Centre, News (Nov, 1984). Ethical

clearance was obtained from the Institutional Ethical

Committee for conducting the animal experiment.

Thirty six male albino rats with average mean

weight of 165 gm were selected and divided into six

groups. The first group was given only normal diet and

served as Control No. 2. The second, third, fourth

and fifth were given alcohol over and above normal

diet. The sixth group was given both alcohol and nutrient

antioxidant along with normal diet. An additional group

(Control-1) of six albino rats (3-4 months old) with an

average mean weight of 165 gm were sacrificed at the

very beginning of the study for determining antioxidant

levels in various subcellular fractions. Determination

of antioxidant level in alcoholic group was done by

sacrificing the second, third, fourth and fifth groups after

one week, one month, three and six months

respectively. The first (Control No. 2, C
2
) and the sixth

groups were sacrificed after sixth months only to

determine the hepatic antioxidant levels.

All the chemicals and reagents used for the study

were of analytical grade and alcohol used for feeding

animals was collected from a local distiller. This sample

contains 37.95% alcohol as per analysis method given

by Department of Food and Technology and

Biochemical Engineering, Jadavpur University,

Calcutta. Four hundred I.U. of vitamin A (Retinol

procured from Eupharma Lab. Ltd. Mumbai), and 4

mg of vitamin C (Ascorbic acid from ABBOTT Lab.

India) were supplemented to the sixth group per day

per animal.

For collection of rat liver, the abdomen was cut

and tissue was dissected and then a homogenate was

prepared in 20% 0.25 M sucrose solution using Potter

Elvehjem type homogenizer. Differential centrifugation

of the homogenate was done in high speed refrigerated

centrifuge machine (Beckman’s Avanti-30) to separate

the various sub-cellular fractions. All the sub-cellular

fractions and 15000 X g, 1 hr supernatant were used

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30



for study of antioxidant levels in them. Methods of

Natelson S11 were used to estimate the levels of vitamin

A and vitamin C.

Results:

In table 1, the sixth group and the first group (C
2
)

show mean body weight of 201 and 204 gm

respectively showing a weight gain of 36 gm and 39

gm within a span of six months. The alcoholic group,

on the other hand showed a better rate of weight gain

showing the increase of 15 gm, 30 gm, 39 gm and 75

gm when recorded after one week, one month, three

months and six months respectively.

Table 1. Comparative body weight changes in different groups of animals.

Animal 

groups/ duration 

 

Initial weight in 

grams (mean) 

Final weight  in 

grams (mean) 

Weight changes 

in grams (mean) 

1 week ALC (2nd gr) 165 180 15 

1 month ALC (3rd gr) 165 195 30 

3 months ALC (4th gr) 165 204 39 

6 months ALC (5th gr) 165 240 75 

6 months ALC+AO (6th gr) 165 201 36 

6 months Control- C2 (1
st gr) 165 204 39 

p-value by T-test between mean values of initial weight

and final weight <0.01 (0.006), significant. ALC=

Alcoholic and AO= Antioxidant

Table 2 shows vitamin A distribution in all the subcellular

fractions though the nuclear fraction and light

mitochondrial fraction show slightly higher level. Effect

of alcohol loading in the level of vitamin A can be seen

in the subcellular fractions are all significant. Heavy

mitochondrial fraction shows the greatest fall (p

<0.001). Rearing the animal for six months showed

decrease in the level of vitamin A in nuclear fraction,

heavy and light mitochondrial fractions in the first

(Control 2, C
2
) and sixth groups (alcohol and

antioxidant).

Table 2: Comparative study of vitamin A concentration in various subcellular fractions of alcoholic rat liver.

Alcohol S.F. Control-1 

Mean±SD 1 week 

Mean±SD 

1 month 

Mean±SD 

3 months 

Mean±SD 

6 months 

Mean±SD 

Alcohol 

Antioxidant 6 

months 

Mean±SD 

Control C2 
Mean±SD 

H 68.80±5.00 71.35±0.74 66.75±1.68 60.20±2.62* 52.27±2.99* 76.62±2.98** 68.67±0.79 

N 18.36±0.99 18.35±0.63 14.45±1.73* 14.53±1.69* 13.83±1.47* 22.40±2.60* 17.00±1.81 

M1 16.67±0.47 17.14±0.38 12.76±1.39
** 12.50±1.41** 12.16±1.39** 20.69±2.59* 14.18±1.51* 

M2 18.28±0.34 18.11±0.43 14.27±1.74
** 14.26±1.74* 13.77±1.52** 17.05±1.02 17.09±0.74* 

Sup 15.31±0.67 15.88±1.03* 13.67±1.95 12.82±1.35* 12.73±1.29* 17.46±0.52** 15.17±0.85 

A. R. Singh et al. Study of effect of ethanol on antioxidant - vitamin A and C in rat liver

31



Values expressed as mg/G liver. *p <0.05 **p <0.001

S.F. = Subcellular fraction,                  H= Homogenate,

N= Nuclear fraction, M
1
= Heavy mitochondrial, M

2
=

Light mitochondrial, Sup= Supernatant.

The quantity of vitamin C recovered as sum of all

the fractions seems to be much higher than that of the

whole homogenate. The recovery is higher in the soluble

fraction than that of the whole homogenate. On alcohol

loading, vitamin C level decreases in the soluble fraction

and on antioxidant supplementation in the sixth group,

the level of vitamin C increases significantly in 15000

X g, 1 hr. supernatant. Sacrificing the animals after 6

months decreases in level of vitamin C in 15000 X g,

1 hr supernatant and the decrease is similar in both

alcoholic and non alcoholic groups, suggesting a

negligible role of alcohol in changing the hepatic vitamin

C level (Table 3). The changes must be simply because

of aging.

Table 3: Comparative study of vitamin C concentration in various subcellular fractions of alcoholic rat liver.

Alcohol S.F 

 

Control-1 

Mean±SD 1 week 

Mean±SD 

1 month 

Mean±SD 

3 months 

Mean±SD 

H 201.17±6.65 189.33±4.84* 189.33±4.84** 186.50±3.45* 1

N 44.95±2.44 44.70±2.51 43.75±0.76 43.75±0.76* 

M1 43.47±1.00 43.47±1.00 43.75±0.76 43.75±0.76 

M2 43.47±2.07 43.75±0.76 44.70±2.51 44.70±2.51 

Sup 220±10.73 206.33±4.97* 207.33±2.51 267.33±13.54* 2

Values expressed as mg/G liver. *p <0.05 **p <0.001 S.F. = Subcellular fraction, H= Homogenate, N= Nuclear

fraction, M
1
= Heavy mitochondrial, M

2
= Light mitochondrial, Sup= Supernatant.

Discussion:

In this study, we found that alcoholic animals had

a better weight gain when compared to control and

alcoholic groups supplemented with antioxidant

vitamins. Alcohol when given in reduced dose, instead

of causing any harmful hepatic changes, might have

simply stimulated the whole organ system thereby

increasing different vital functions leading to better

appetite and food intake. This may be one of the

possible reasons of getting higher weight gain in

alcoholic group.12 The rapid increase in weight may

not be a good sign of healthiness because most of

chronic alcoholics are always on higher side of

expected normal weight. The cause of the increased

body weight may be due to increased deposits of

hepatic lipids and also other adipose tissues. Alcohol

has extra calories of its own and if taken regularly

becomes an appetizer.

The fall in vitamin A content in the alcoholic liver

may be due to its utilisation in trying to control the

ethanol mediated free radical generation. The low

recovery from the subcellular fractions may be

explained on the basis of the loss of certain naturally

occurring protective antioxidant, after cell fractionation

which in turn leading to the mobilisation of more of the

vitamin A from the hepatic store in the alcoholic animals.

Due to the lack of ethanol mediated free radical

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32



generation in non alcoholic animals (control), naturally

occurring protective antioxidants may still be present

unaffected and thus recovery after cell fractionation

may still be maintained at 100%. The complete

recovery of vitamin from the subcellular fractions in

alcoholic animals supplemented with the antioxidant

vitamin shows the importance of vitamin A in

counteracting the generation of free radicals or

quenching the free radicals already generated.

One week ethanol feeding seems, to have no impact

on the antioxidant status of the animal. Only light

mitochondrial fraction shows just significantly

decreased level. Maria et al, 1982 reported the

changes in the structure of mitochondria in ethanol fed

rats.13 Further, one month of ethanol loading, affected

all the subcellular fractions. The homogenate however,

still maintain the vitamin level suggesting the presence

of naturally occurring protective antioxidant. The

sudden significant decrease of the vitamin level in the

subcellular fractions explains the loss of the said natural

antioxidant during the cell fractionation initiated by the

ethanol mediated free radicals. After three months the

alcoholic animals show the same pattern of changes as

that of one month already discussed. Here the only

change is that homogenate show a significant decrease

in its vitamin A content. After six months of ethanol

loading, the light mitochondrial fraction shows a further

fall in hepatic vitamin A content supporting the earlier

views that a drastic structural change may be related

to its increase fall of the vitamin.

When antioxidant vitamin is given along with the

alcohol for six months, the antioxidant status in the

hepatic subcellular fractions show a dramatic

improvement. All the subcellular fractions shows

significantly increased level of the vitamin except that

of light mitochondrial which has been considered as

the most sensitive fraction in terms of its capacity to

hold this vitamin because of its ethanol sensitive

structural changes, the extent of the decrease however,

is not significant. In short, a conclusion may be drawn

for the antioxidant vitamin A as having an important

role in checking the level of hepatic antioxidant levels

during ethanol loading thereby helping in the prevention

of ethanol mediated hepatic injury. Aging also seems

to affect the vitamin A status of two mitochondrial

fractions sparing rest of the fractions.

Vitamin C is mainly recovered from soluble fraction

and is not affected by the progress of alcohol loading.

The aging process seems to be the major factor in

changing vitamin levels in soluble fraction of the cells.

The study reaffirms that antioxidant supplementation

seems to be useful in maintaining the vitamin levels

affected by process of aging and alcohol.

Conclusion:

From all the findings, it is suggested that antioxidant

vitamin supplements will be beneficial to alcoholic

population but for it to be recommended, a thorough

trial study in human alcoholics with a well control dietary

chart and proper assessment of health status at different

stages of aging be very much needed.

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induced injury to liver and other tissues. N Engl J Med

1988; 319: 1639-50.

2. Halliwell B, Gutterrigde JMC. Oxygen toxicity, oxygen

radicals, transition metals and disease. Biochem J

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3. Bast A, Haenen GR, Doelman CJ. Oxidants and

antioxidants, State of art. Am J Med 1991; 2-13.

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4. Aust SD, Morehous LA, Thomas CE. Role of metal in

oxygen radical reactions. Free Radic Biol Med 1985;

1: 3-6.

5. Sevenian A, Hachstein P. Mechanisms and

consequences of lipid peroxidation in biological

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