Professional Documents
Culture Documents
antioxidants
ethanol
Hemidesmus indicus
INTRODUCTION
lipid peroxidation
liver
are probably the most susceptible target of free radical attack. The reaction of free radicals with the membrane lipid
components leads to lipid peroxidation. This process can
eventually cause increased membrane permeability and cell
death.7 To counteract these oxidants, cells have several
enzymatic antioxidants, including superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase (GPx),
and nonenzymatic antioxidants, including reduced glutathione (GSH), ascorbic acid, and -tocopherol, but their
levels are altered in alcoholics.
Hemidesmus indicus (Family Asclepiadaceae) is a widely
distributed medicinal plant in India. It has been used as a
traditional medicine in the treatment of biliousness, blood
diseases, diarrhea, respiratory disorders, skin diseases,
syphilis, fever, bronchitis, asthma, eye diseases, childhood
epilepsy, kidney and urinary disorders, loss of appetite,
burning sensation, and rheumatism.8 Jain and Singh9 reported that H. indicus is employed in traditional medicine
for gastric ailments. It is also used in the preparation of some
soft drinks. It mainly consists of essential oils and phytosterols, like hemidesmol, hemidesterol, and saponins. Although the protective effect of H. indicus against CCl4- and
peracetamol-induced hepatotoxicity in rats10 is known, the
effect of the extract on ethanol-induced hepatoxicity is un-
675
676
known. Because of its wide pharmacological actions and recent interest,11 the present study was undertaken to establish the hepatoprotective activity of the ethanolic root extract of H. indicus on an animal model of ethanol-induced
liver damage.
H. indicus
The plant material (H. indicus) was obtained from a local market, identified, and authenticated by the Department
of Botany, Annamalai University. The H. indicus roots were
cleaned, shade-dried, and disintegrated. Cold aqueous
ethanolic extract was prepared according to the methodology of the Indian Pharmacopoeia12 with a Soxhlet apparatus and screened for its antihepatotoxicity property. The
ethanolic extract (yield, 9.2%) was dark brown in color and
sticky in nature and gave positive results for flavonoids, terpenoids, tannins, coumarins, and glycoside and negative results for alkaloids, anthraquinones, lactones/ester, protein/amino acids, and saponins.13
Animals
AU
1
Study design
The animals were divided into four groups of 10 each.
Groups 1 and 2 received normal diets and isocaloric glucose
from 27% glucose solution (1 mL contains 1.08 cal) daily
by intragastric intubation. Liver cell damage was induced in
rats of groups 3 and 4 by administering 20% ethanol (5.0
g/kg of body weight/day)14 (1 mL contains 1.08 cal) as an
aqueous solution using an intragastric tube daily for 30 days.
At the end of this period the animals were treated as follows
for the next 30 days: Group 1 animals continued to receive
standard pellet diet and isocaloric glucose from 27% glucose solution in 1% carboxy methyl cellulose (CMC) and
served as controls. Group 2 animals continued to receive
standard pellet diet and isocaloric glucose from 27% glucose solution and were administered ethanolic root extract
of H. indicus (500 mg/kg of body weight/day)10,15 in 1%
CMC by intragastric intubation every day. Group 3 animals
continued to receive standard pellet diet, 20% ethanol, and
1% CMC daily. Group 4 animals continued to receive standard pellet diet, 20% ethanol, and ethanolic extract of H. indicus (500 mg/kg of body weight/day) in 1% CMC every
day. The total duration of the experiment was 60 days.
The animals were fasted overnight and then anesthetized
with an intramuscular injection of ketamine hydrochloride
(30 mg/kg of body weight), blood samples were collected
by retro-orbital puncture, and animals were sacrificed by
cervical decapitation. Blood samples were collected in heparinized test tubes and plain tubes and then centrifuged for
the separation of plasma and serum, respectively. The erythrocytes were washed with 0.9% NaCl three times, and distilled water was added for hemolysis. The hemolysate was
then used for estimating the total hemoglobin (Hb) content.
Liver tissues were immediately homogenized and used for
various biochemical estimations.
677
Statistical analysis
Data were analyzed by one-way analysis of variance followed by Duncans multiple range test (DMRT) using a
commercially available statistics software package (SPSS
for Windows, version 13.0, SPSS, Chicago, IL). Results
were presented as mean SD values. Values of P .05
were regarded as statistically significant.
RESULTS
Table 1 shows the average weight gain, food intake, and
liver/body weight ratios of control and experimental rats during the experimental period. The average food intake and
the average weight gained were significantly reduced in
ethanol-administered rats, and the liver/body weight ratio
was significantly higher compared to that of control animals.
Rats co-administered H. indicus along with ethanol from day
31 showed significantly greater weight gain and food intake
and decreased liver/body weight ratio compared to untreated
ethanol-fed rats.
TABLE 1.
EFFECT
OF
H.
INDICUS ON
BODY WEIGHT
AND
DISCUSSION
Chronic ethanol feeding caused significantly less weight
gain in rats compared to untreated controls. Wastage of energy during ethanol metabolism by the microsomal ethanol
oxidizing system (MEOS) (CYP2E1) can be one of the
causes for the decreased weight gain with ethanol supplementation.31 Other causes are mitochondrial insufficiency in
LIVER/BODY WEIGHT
OF
CONTROL
AND
ETHANOL-ADMINISTERED RATS
Day 1
Day 60
145.11 6.34
142.04 6.84
140.54 7.33
144.39 6.20
226.07 10.93
224.46 12.33
152.61 6.53
198.94 3.46
80.97
82.42 5.51c
12.07 0.96a
54.56 5.67b
9.18
9.27 0.25c
7.55 0.26a
8.71 0.22b
2.77 0.14a
2.81 0.23a
5.19 0.38c
3.18 0.07b
4.61c
0.25c
678
EFFECT
OF
H.
INDICUS ON
OF
CONTROL
AND
ETHANOL-ADMINISTERED RATS
Level (IU/L)
Group
AST
75.66
80.69 2.50a
127.91 7.13c
97.56 4.49b
3.39a
Control
H. indicus
Ethanol
Ethanol H. indicus
ALT
ALP
GGT
LDH
26.89
21.41 0.56a
58.11 1.85c
27.81 2.16b
102.77
111.80 14.09a
158.39 5.43b
108.64 23.15a
2.27
2.03 0.46a
7.33 1.23c
3.11 0.06b
120.17 3.08a
130.02 3.34b
332.95 8.55d
159.58 4.10c
1.28b
22.11a
0.66a
TABLE 3.
EFFECT
OF
Group
H.
INDICUS ON
CONTROL
OF
AND
ETHANOL-ADMINISTERED RATS
LOOH ( 10
5 mmol/mg of protein)
CD (nmol/mg of protein)
1.56 0.03a
1.46 0.04a
3.13 0.25c
1.77 0.08b
9.57 0.62b
7.77 0.51a
16.27 0.74c
10.00 0.78b
0.67 0.02b
0.56 0.02a
1.39 0.06d
0.75 0.03c
Control
H. indicus
Ethanol
Ethanol H. indicus
TBARS
LOOH
CD
1.52 0.06b
1.26 0.03a
3.08 0.23c
1.60 0.07b
6.87 0.11a
6.51 0.18a
13.25 1.02c
8.53 0.21b
6.07 0.10b
5.25 0.19a
10.73 0.96d
6.63 0.22c
TABLE 5.
OF
TBARS
LOOH
CD
0.73
0.65 0.02a
1.92 0.06d
0.82 0.04c
64.96
59.30 1.67a
90.47 3.25c
62.78 2.28ab
52.32 1.13b
43.73 1.78a
86.88 3.22c
59.90 2.26b
0.05b
4.13b
679
EFFECT
OF
AND
GSH
OF
SOD
CAT
GPx
2.60 0.06c
2.54 0.07c
1.59 0.08a
2.29 0.05b
3.54 0.32c
4.17 0.12d
1.76 0.03a
2.95 0.07b
22.70 1.43c
25.85 0.79d
13.15 0.49a
21.08 0.51b
35.22 0.61c
37.93 0.76d
15.06 0.71a
29.83 0.51b
Data are mean SD values for 10 rats in each group. Units for SOD, CAT, and GPx are amount of enzyme required for
50% inhibition of NBT reduction/minute, mol of hydrogen peroxide utilized/minute, and mol of glutathione utilized/minute,
respectively.
Values not sharing a common superscript letter within each column differ significantly at P .05 (DMRT).
TABLE 7.
EFFECT
OF
H.
INDICUS ON
ACTIVITIES
OF
AND
GSH
OF
NORMAL
AND
ETHANOL-ADMINISTERED RATS
SOD
CAT
GPx
2.03 0.06b
2.89 0.09c
1.29 0.37a
2.02 0.06b
154.21 2.56c
164.48 4.91d
96.75 2.89a
145.55 4.35b
14.37 0.42c
16.18 0.43d
7.55 0.20a
12.46 0.24b
3.61 0.19b
4.36 0.30c
1.52 0.14a
3.46 0.27b
Data are mean SD values for 10 rats in each group. Units for SOD, CAT, and GPx are amount of enzyme required for 50% inhibition of
NBT reduction/minute, mol of hydrogen peroxide utilized/minute, and mol of glutathione utilized/minute, respectively.
Values not sharing a common superscript letter within each column differ significantly at P .05 (DMRT).
TABLE 8.
EFFECT
OF
H.
INDICUS ON
ACTIVITIES
OF
AND
GSH
OF
NORMAL
AND
ETHANOL-ADMINISTERED RATS
SOD
CAT
GPx
5.75 0.15b
5.89 0.12b
2.07 0.19a
5.89 0.16b
75.31 1.72c
77.17 1.25c
52.34 2.67a
67.55 1.97b
13.22 1.06b
15.02 1.20c
6.00 0.48a
12.50 1.00b
17.64 0.37c
18.50 0.34d
10.02 0.39a
15.15 0.32b
Data are mean SD values for 10 rats in each group. Units for SOD, CAT, and GPx are amount of enzyme required for 50% inhibition of
NBT reduction/minute, mol of hydrogen peroxide utilized/minute, and mol of glutathione utilized/minute, respectively.
Values not sharing a common superscript letter within each column differ significantly at P .05 (DMRT).
TABLE 9.
EFFECT
OF
H.
INDICUS ON
NORMAL
Plasma (mg/dL)
Group
Control
H. indicus
Ethanol
Ethanol H. indicus
AND
VITAMIN E
OF
Vitamin C
Vitamin E
Vitamin C
Vitamin E
2.06 0.04c
2.31 0.08d
1.25 0.15
1.76 0.04b
2.68 0.17c
2.84 0.11d
1.28 0.17
2.16 0.05b
0.84 0.10b
1.02 0.11c
0.51 0.49a
0.82 0.73b
5.46 0.16c
5.96 0.35d
3.39 0.11a
5.10 0.22b
680
fatty acid oxidation secondary to chronic ethanol consumption and acetaldehyde toxicity.32 Significant improvement
in body weight gain was observed with H. indicus treatment,
which may be due to the inhibitory effect of H. indicus on
CYP2E1 in liver microsomes. The ratio between liver
weight and total body weight was significantly lower in
ethanol-fed rats supplemented with H. indicus extract compared to that of the unsupplemented ethanol-fed rats, which
may be because the extract increases the elimination of
ethanol directly from the intestines without absorption or
perhaps because H. indicus consumption prevents fat accumulation in the liver.
Liver damage after ethanol ingestion is a well-known phenomenon, and the obvious sign of hepatic injury is the leakage of cellular enzymes into plasma.33 The increased levels
of serum enzymes such as AST, ALT, ALP, GGT, and LDH
observed in ethanol-administered rats may indicate increased permeability, damage, and/or necrosis of hepatocytes.34 H. indicus extract supplementation showed a
marked hepatoprotective effect that is consistent with the results of previous researchers,10 and it is supported by the reversal of changes produced by ethanol. Moreover, the observed decrease in the activities of these enzymes shows that
H. indicus, to a certain extent, preserves the structural integrity of the liver from the toxic effects of ethanol.
Oxidation of polyunsaturated fatty acids (lipid peroxidation) of membranes is a common process in living organisms, since they are the target of oxygen-derived free radicals produced during mitochondrial electron transport.35
Increased lipid peroxidation associated with chronic ethanol
administration has often been used as an indicator of oxidative stress in both animal models and human clinical trials. Excess lipid peroxidation, as measured by formation of
TBARS, LOOH, and/or CD, has been found in most studies.36 In agreement with these findings, ethanol-administered rats showed increased blood and tissue levels of lipid
peroxidation markers such as TBARS, LOOH, and CD. The
increased peroxidation can result in changes in cellular metabolism of the hepatic and extrahepatic tissues. Products of
lipid peroxidation formed in the primary site reaching the
other organs and tissues via the bloodstream provoke lipid
peroxidation there and consequently cause cellular and tissue damage.37 Increased accumulation of lipid peroxidation
products in cells can result in cellular dehydration, whole
cell deformity, and cell death.38
Lipid peroxidation is an important cause of alcoholic liver
disease.36 Free radical generation and lipid peroxidation
products play a pivotal role in the mechanism by which
ethanol may exerts its toxic effects on the liver and other
extrahepatic tissues.36 The H. indicus co-administered rats
showed significantly lower levels of these lipid peroxidative
markers compared to ethanol-fed rats. H. indicus also exhibited its potent antioxidant activity by decreasing lipid peroxidation in control rats administered H. indicus extract
compared to normal controls. In this context, Mary et al.39
have also reported that H. indicus has an antioxidant property using in vitro studies. Decreased lipid peroxidation with
H. indicus extract administration suggests a decreased impact of reactive oxygen species (ROS) on lipid membranes,
and therefore increased protection against ethanol-induced
liver injury. Thus the inhibition of lipid peroxidation by H.
indicus may be one of the mechanisms by which H. indicus
exerts its protection against ethanol-mediated tissue injury.
Some studies have shown that treatment with silymarin protects the liver, probably through decreasing lipid peroxidation.40 H. indicus may have a similar mode of action.
Free radical scavenging enzymes such as SOD, CAT, and
GPx are the first line of defense against oxidative injury.
SOD scavenges excess superoxide anions and converts
them to H2O2. The primary role of CAT is to scavenge
H2O2 that has been generated by free radicals or by SOD
and convert it to water. GPx works in tandem with CAT to
scavenge excess H2O2 as well as other free radicals in response to oxidative stress. The equilibrium between these
enzymes is important for the effective removal of oxidative
stress in intracellular organelles. This antioxidant defense
system is significantly altered by ethanol administration.
Our results show decreased activities of SOD, CAT, and
GPx activities in chronic ethanol-administered rats. It has
been reported that ethanol impairs the antioxidant system
of the tissues in proportion to the amount of ethanol ingestion.41 Biphasic fluxes of these enzyme activities are
common; an increase or decrease may relate to the presence of excess ROS. The decreased activities of enzymatic
antioxidants observed in ethanol-administered rat erythrocytes and tissues may be a consequence of irreversible inactivation of enzyme proteins from increased free radical
production resulting from ethanol metabolism.42 Lowered
activities of these enzymes will result in the accumulation
of highly reactive free radicals, leading to deleterious effects such as loss of cell membrane integrity and membrane
function.43 There was a significant increase in the activities of these enzymes with H. indicus extract co-administration. H. indicus is reported to scavenge superoxide radicals and hydrogen peroxide.39 Because of these properties,
it was expected that H. indicus might decrease the workload of enzymatic antioxidants and reduce the free radicalmediated inactivation of enzyme proteins and thereby maintaining the activities of enzymatic antioxidants. Thus H.
indicus has the ability to increase the activity of the endogenous antioxidant enzymes.
The second line of defense consists of the nonenzymatic
scavengers, such as GSH, ascorbic acid, and -tocopherol,
which scavenge residual free radicals escaping decomposition by the antioxidant enzymes. Moreover, enzymatic antioxidants are inactivated by the excessive levels of free radicals, and hence the presence of nonenzymatic antioxidants
is presumably essential for the removal of these radicals.44
Glutathione is a major non-protein thiol in living organisms
that plays a central role in co-ordinating the antioxidant defense process in our body. Glutathione reacts directly with
ROS and electrophilic metabolites, protects essential thiol
groups from oxidation, and serves as a substrate for several
enzymes, including GPx.
We observed lower level of plasma, erythrocyte, and hepatic GSH in ethanol-fed rats, a consequence of increased
utilization to counter the increased oxidative stress. The
shortage of NADPH (due to the increased oxidation of
ethanol by the MEOS, which uses NADPH as a cofactor)
suppresses the reduction of oxidized glutathione by glutathione reductase and subsequently decreases glutathione
content. Generation of large quantities of acetaldehyde during ethanol metabolism ultimately will deplete cellular GSH
pools by forming S-conjugation with the
SH group. Perturbation in the GSH redox status not only can impair cell
defense against toxic compounds, but also result in increased
oxidative stress and oxidative injury.45
H. indicus co-administered rats exhibited significantly
improved GSH levels compared to ethanol-fed rats. The results obtained suggest that the maintenance of GSH by H.
indicus was mainly due to inactivation of ROS via its radical scavenging effects, sparing antioxidant enzymes such as
SOD46 and CAT. Restoration of GSH levels has been shown
to inhibit ethanol toxicity.47,48 Therefore, it was presumed
that the effects of H. indicus might be related to a normalization mechanism by maintaining adequate levels of GSH
for detoxification of xenobiotics.
Antioxidant systems other than GSH may also play a role
in preventing lipid peroxidation under experimental and clinical conditions. -Tocopherol and ascorbic acid are naturally
occurring free radical scavengers.49 Both ascorbic acid and tocopherol are known to be decreased in liver diseases, particularly in alcoholics.50 Under these conditions, thiol compounds,
such as GSH, might be involved in regenerating -tocopherol
from its radical form.51 The observed decrease in the levels of
-tocopherol and ascorbic acid may be due to their increased
utilization for scavenging ethanol- and/or oxygen-derived radicals. We have observed near normal levels of these antioxidants with H. indicus supplementation. The decrease in lipid
peroxidation with H. indicus treatment can be correlated with
the elevated levels of antioxidants. The ability of H. indicus to
enhance the levels of antioxidants along with its anti-lipid peroxidative activity suggest that this extract might be potentially
useful in countering free radical-mediated injuries involved in
the development of liver damage caused by alcohol abuse.
This study provides convincing evidence that H. indicus
treatment can effectively protect against oxidative injury in
alcoholics.
REFERENCES
1. Diehl AM: Liver disease in alcohol abusers: clinical perspective.
Alcohol 2002;27:711.
2. Lin CN, Chung MI, Gan KH: Novel antihepatotoxic principles of
Solanum incanum. Planta Med 1988;54:222.
3. Zima T, Fialova L, Mestek O, Janebova M, Crkovska J, Malbohan I, Stipek S, Mikulikova L, Popov P: Oxidative stress, metabolism of ethanol and alcohol-related diseases. J Biomed Sci
2001;1:5970.
4. Schlorff EC, Husann K, Somani SM: Dose- and time-dependent
effects of ethanol on plasma antioxidant system in rat. Alcohol
1999;17:97105.
681
5. Thurman RG, Handler JA: New perspectives in catalase-dependent ethanol metabolism. Drug Metab Rev 1989;20:679688.
6. Nordmann R, Ribiere C, Rouach H: Implication of free radical
mechanisms in ethanol-induced cellular injury. Free Radic Biol
Med 1992;12:219240.
7. Rakonczay Jr Z, Boros I, Jarmay K, Hegyi P, Lonovics J, Takacs
T: Ethanol administration generates oxidative stress in the pancreas and liver, but fails to induce heat-shock proteins in rats. J
Gastroenterol Hepatol 2003;18:858867.
8. Nadkarni AN: Indian Materia Medica, Vol. 1. Popular Book Depot, Bombay, 1989, p. 619.
9. Jain SP, Singh SC: Ethno-Medico-Botanical Survey of Ambikapur District, MP. Fourth International Congress of Ethnobiology.
NBRI, Lucknow, India, 1994.
10. Baheti JR, Goyal RK, Shah GB: Hepatoprotective activity of
Hemidesmus indicus R. br. in rats. Indian J Exp Biol 2006;
44:399402.
11. Sarasan V, Soniya EV, Nair GM: Regeneration of Indian sarasaparilla, Hemidesmus indicus R.Br., through organogenesis and somatic embryogenesis. Indian J Exp Biol 1994;32:284287.
12. Indian Pharmacopoeia, 2nd ed. Government of India, New Delhi,
1966, p. 57.
13. Verma PR, Joharapurkar AA, Chatpalliwar VA, Asnani AJ: Antinociceptive activity of alcoholic extract of Hemidesmus indicus
R.Br. in mice. J Ethnopharmacol 2005;102:298301.
14. Celec P, Jani P, Smrekova L, Mrlian A, Kudela M, Hodosy J,
Boor P, Kristova V, Jakubovsky J, Jezova D, Halcak L, Bozek P,
Slamova J, Ulicna O, Hojs(k D, Jurkovicova I: Effects of anabolic steroids and antioxidant vitamins on ethanol-induced tissue
injury. Life Sci 2003;74:419434.
15. Das S, Prakash R, Devaraj SN: Antidiarrhoeal effects of methanolic root extract of Hemidesmus indicus (Indian sarsaparilla)an
in vitro and in vivo study. Indian J Exp Biol 2003;41:363366.
16. Reitman S, Frankel S: A colorimetric method for the determination of serum glutamate oxaloacetic and glutamate pyruvic
transaminases. Am J Clin Pathol 1957;28:5663.
17. Kind PRN, King EJ: Estimation of plasma phosphatases by determination of hydrolyzed phenol with amino-antipyrine. J Clin
Pathol 1954;7:330322.
18. Rosalki SB, Rau D: Serum gamma-glutamyl transpeptidase activity in alcoholism. Clin Chim Acta 1972;39:4147.
19. King J: Practical Clinical Enzymology. D. Van Nostrand, London, 1965, pp. 8393.
20. Ohkawa H, Ohishi N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem
1979;95:351358.
21. Yagi K: Lipid peroxides and human disease. Chem Physiol Lipids
1978;45:337351.
22. Jiang ZY, Hunt JV, Wolff SP: Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxides in
low density lipoprotein. Anal Biochem 1992;202:384389.
23. Recknagel RO, Glende EA Jr: Spectrophotometric detection of
lipid conjugated dienes. Methods Enzymol 1984;105:331337.
24. Kakkar B, Das PN, Viswanathan A: Modified spectrophotometric assay of SOD. Indian J Biochem Biophys 1984;21:130132.
25. Sinha AK: Colorimetric assay of catalase. Anal Biochem 1972;
47:389394.
26. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG,
Hoekstra WG: Selenium: biochemical role as a component of glutathione peroxidase. Science 1973;179:588590.
682
41. Scott RB, Reddy KS, Husain K, Schlorff EC, Rybak LP, Somani SM: Dose response of ethanol on antioxidant defense system of liver, lungs and kidney in the rat. Pathophysiology
2000;7:2532.
42. Santiard D, Ribiere C, Nordmann R, Houee-Levin C: Inactivation
of Cu, Zn-superoxide dismutase by free radicals derived from
ethanol metabolism: a gamma radiolysis study. Free Radic Biol
Med 1995;19:121127.
43. Krishnakantha TP, Lokesh BR: Scavenging of superoxide anions
by spice principles. Indian J Biochem Biophys 1993;30:133134.
44. Allen RG: Oxygen-reactive species and antioxidant responses during development: the metabolic paradox by cellular differentiation. Proc Soc Exp Biol Med 1991;196:117129.
45. Reed DJ: Glutathione: toxicological implications. Annu Rev Pharmacol Toxicol 1990;30:603631.
46. Ravishankara MN, Shrivastava N, Padh H, Rajani M: Evaluation
of antioxidant properties of root bark of Hemidesmus indicus R.
Br. (Anantmul). Phytomedicine 2002;9:153160.
47. Garcia-Ruiz C, Morales A, Colell A, Ballesta A, Rodes J,
Kaplowitz N, Fernandez-Checa JC: Feeding S-adenosyl-Lmethionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes. Hepatology 1995;21:207214.
48. Iimuro Y, Bradford BU, Yamashina S, Rusyn I, Nakagami M,
Enomoto N, Kono H, Frey W, Forman D, Brenner D, Thurman
RG: The glutathione precursor L-2-oxothiazolidine-4-carboxylic
acid protects against liver injury due to chronic enteral ethanol
exposure in the rat. Hepatology 2000;31:391398.
49. Yu BP: Cellular defenses against damage from reactive oxygen
species. Physiol Rev 1994;74:139162.
50. Bjorneboe GA, Johnsen J, Bjorneboe A, Morland J, Drevon CA:
Effect of heavy alcohol consumption on serum concentration of
fat-soluble vitamins and selenium. Alcohol Alcohol 1987;1:
533537.
51. Wefers H, Sies H: The protection by ascorbate and glutathione
against microsomal lipid peroxidation is dependent on vitamin E.
Eur J Biochem 1988;174:353357.
NADANA SARAVANAN
AU1
One word in affiliation. Why different here?