Clinical Science (2000) 99, 315–320 (Printed in Great Britain)
S-Adenosylmethionine prevents hepatic
tocopherol depletion in carbon
tetrachloride-injured rats
Ramo! n DEULOFEU*, Albert PARE! S†, Mireia RUBIO†, Marta GASSO! †, Juan ROMA! N†,
Ame! rica GIME! NEZ†, Gregorio VARELA-MOREIRAS‡, Joan CABALLERIA†,
Antonio M. BALLESTA*, Jose! M. MATO‡ and Joan RODE! S†
*Laboratory of Biochemistry, Hospital Clı! nic i Provincial, University of Barcelona, Barcelona, Spain, †Alcohol and Liver Units,
Hospital Clı! nic i Provincial, University of Barcelona, Barcelona, Spain, and ‡Department of Medicine, Division of Hepatology
and Gene Therapy, University of Navarra, Pamplona, Spain
A
B
S
T
R
A
C
T
In various experimental models, S-adenosylmethionine (SAMe) has been shown to reduce liver
injury by preventing depletion of glutathione, one of the antioxidant systems that plays a critical
role in defence against oxidative stress. On the other hand, α-tocopherol may be decreased in
liver diseases, and treatment with this vitamin reduces liver injury in CCl4-treated rats. Since
there is a close relationship among the different antioxidant systems (mainly glutathione, αtocopherol and ascorbic acid), we have assessed whether, as well as restoring hepatic glutathione
content, SAMe has any effect on liver α-tocopherol and ascorbic acid levels in CCl4-injured rats.
Four groups of seven male Wistar rats treated for 9 weeks were studied : rats induced to cirrhosis
with CCl4, rats induced to cirrhosis plus SAMe administration (10 mg:kg−1:day−1) and
their respective controls. Liver samples were obtained for measuring levels of glutathione,
α-tocopherol, ascorbic acid and thiobarbituric acid-reactive substances (TBARS), and
hydroxyproline concentration as an index of collagen content. The hydroxyproline content
was higher in CCl4-injured rats than in the control group (4.4p1.8 and 1.1p0.3 µmol/g
respectively ; P 0.05). In CCl4-injured rats, SAMe administration decreased collagen content
(2.7p1.0 µmol/g ; P 0.05) and TBARS, and corrected glutathione depletion. α-Tocopherol was
significantly lower in CCl4-injured rats than in controls (17.3p4.9 and 23.0p4.0 µmol/g
respectively ; P 0.05). By contrast, α-tocopherol levels were similar (23.8p5.1 µmol/g) in
CCl4-injured rats receiving SAMe and in controls. In CCl4-injured rats, liver ascorbic acid was
decreased in comparison with controls (4.9p1.8 and 8.2p1.0 µmol/g respectively ; P 0.05),
levels which were not replenished by SAMe (4.6p0.4 µmol/g). In conclusion, SAMe not only
decreases fibrosis and protects against hepatic glutathione depletion, but has a further
antioxidant effect of preventing α-tocopherol depletion in CCl4-injured rats.
INTRODUCTION
S-Adenosylmethionine (SAMe), a cellular metabolite
resulting from the activation of methionine by SAMe
synthetase, is the principal methylating agent in a variety
of transmethylation reactions [1,2] and plays a key role in
cellular sulphur metabolism, as well as being a precursor
of glutathione (GSH) [3]. In various experimental models
SAMe has been shown to decrease liver injury by
preventing depletion of reduced GSH [4–7], one of the
Key words : ascorbic acid, CCl , glutathione, lipid peroxidation, vitamin C, vitamin E.
%
Abbreviations : SAMe, S-adenosylmethionine ; TBARS, thiobarbituric acid-reactive substances.
Correspondence : Dr Albert Pare! s, Alcohol and Liver Units, Hospital Clı! nic i Provincial, C\. Villarroel 170, 08036-Barcelona, Spain
(e-mail pares!medicina.ub.es).
# 2000 The Biochemical Society and the Medical Research Society
315
316
R. Deulofeu and others
antioxidant systems that plays a critical role in oxidative
stress. SAMe is also effective in some human liver diseases
[8–11], and this drug restores depleted GSH levels in
patients with alcoholic liver disease [8]. These effects of
SAMe on liver injury may result in decreased lipid
peroxidation after exposure to a number of noxious
agents, including carbon tetrachloride (CCl ). Indeed,
%
increased free radical production and lipid peroxidation
have been proposed as major cellular mechanisms involved in CCl -induced hepatotoxicity [12,13].
%
Antioxidant systems other than GSH may also play a
role in preventing lipid peroxidation under experimental
and clinical conditions. In this respect, α-tocopherol
(vitamin E) may be decreased in liver diseases [14–18],
particularly in alcoholics [15,16], and treatment with this
vitamin reduces liver injury in CCl -treated rats [19].
%
Ascorbic acid (vitamin C) is also a powerful antioxidant
[20]. Both α-tocopherol and ascorbic acid are naturally
occurring free-radical scavengers [21] which protect cell
membranes against toxic agents such as CCl and alcohol.
%
They are active in different media : while tocopherol
is located in the lipid membrane environment, ascorbate is
hydrophilic. Moreover, ascorbic acid can restore the
antioxidant properties of oxidized vitamin E, suggesting
that a major function of vitamin C is to recycle the
vitamin E radical [21]. In this respect, an in vivo sparing
action of vitamin C has been demonstrated [22]. GSH
also maintains tissue ascorbic acid levels [23].
Given the close relationship among these antioxidant
systems (GSH, α-tocopherol and ascorbic acid), we have
assessed whether, as well as correcting hepatic GSH
levels, SAMe has any effect on the liver content of αtocopherol and ascorbic acid in CCl -injured rats.
%
MATERIALS AND METHODS
Chemicals
Vitamin E (α-tocopherol), tocopheryl acetate, ascorbic
acid, o-phthaldehyde and hydroxyproline were purchased from Sigma Chemical Co. (St. Louis, MO,
U.S.A.). Ascorbate oxidase spatula and reduced glutathione were from Boehringer Mannheim G.m.b.H.
(Mannheim, Germany). SAMe, in the stable form of
sulphate p-toluenesulphonate, was kindly provided by
Europharma (Madrid, Spain). Tocopherol and tocopheryl acetate standards were adjusted for concentration
by spectrophotometry, and the method was initially
calibrated by means of the standard reference material
SRM 968b from the National Institute for Standards and
Technology (Gaithersburg, MD, U.S.A.). All other
reagents were of analytical grade or better.
Animals and experimental design
The study was performed in 28 male Wistar rats, of body
weight 250p3.5 g, which were fed a standard diet ad
# 2000 The Biochemical Society and the Medical Research Society
libitum (Purina Chow A03 ; Panlab, Barcelona, Spain).
Animals were housed in metabolic stainless steel cages in
a room with a controlled 12 h light\dark cycle at 22 mC.
Cirrhosis was induced in two groups of seven rats by
intraperitoneal injection of 0.5 ml of CCl , diluted 1 : 1
%
(v\v) in vegetal oil, twice a week for 9 weeks. One group
of these rats also received SAMe (intramuscular ;
10 mg\kg body weight ; daily) during the study. Two
additional groups of seven rats, a control group and
another which received SAMe only, were also studied.
The animals received humane care in compliance with
our institution’s criteria for the care and use of laboratory
animals.
At the end of the study, rats were killed by cardiac
puncture and exsanguination. The liver was removed
immediately and prepared for analytical measurements
or frozen immediately in liquid nitrogen and stored at
k80 mC for further determinations. Some samples were
fixed in 4 % formaldehyde and embedded in paraffin for
histological analysis. Other liver samples were processed
for measurement of levels of α-tocopherol, ascorbic
acid, GSH and thiobarbituric acid-reactive substances
(TBARS) (as a measure of lipid peroxidation), and of
hydroxyproline content as a measure of liver collagen.
Plasma samples were also collected from each animal.
Biochemical determinations
Liver α-tocopherol concentrations were measured by
HPLC after tissue homogenization in cold acetone
according to the method of Shearer [24]. Total ascorbic
acid, including ascorbic acid and dehydroascorbic acid,
was measured by HPLC using the method of Speek et al.
[25] after o-phenylenediamine derivatization to allow
fluorimetric detection. The total GSH concentration
was determined by the method of Hissin and Hill as
described previously [26], and the TBARS concentration
in liver homogenates was measured by HPLC after
deproteinization of tissue homogenates with phosphoric
acid according to the method of Bird et al. [27]. The
hydroxyproline concentration in liver homogenates was
measured by Pico-Tag HPLC (Waters, Milford, MA,
U.S.A.) using phenylisothiocyanate to produce the
phenylthiocarbamyl amino acids after overnight tissue
hydrolysis in 6 mol\l HCl [28]. All liver measurements
were expressed per g of wet liver tissue. HPLC analyses
were performed on a Waters Chromatographic system
(Waters, Milford, MA, U.S.A.) equipped with two M510
pumps, a gradient AGL 560 controller, a WISP 712
automatic sampler, and a M440 fixed-wavelength detector or a M480 fluorescence detector. The chromatograms were recorded and calculated using the computerbased program Baseline (Waters). Standard liver function
tests, including aspartate aminotransferase, alanine
aminotransferase and alkaline phosphatase activities,
were measured as an index of hepatic cellular necrosis,
Effects of S-adenosylmethionine on vitamin E in CCl4-injured rats
using a DAX-72 analyser (Bayer Corp., Barcelona,
Spain).
Histological analysis
Sections of 4 µm thickness were stained with
haematoxylin\eosin and Masson’s trichrome, coded for
blind reading and classified as normal liver, fibrosis and
cirrhosis. Fibrosis was graded from 0–3 as follows : 0, no
fibrosis ; 1, sinusoidal or perivenular fibrosis ; 2, fibrotic
septa between portal tracts ; 3, cirrhosis.
Statistical analysis
Results are expressed as meanspS.E.M. One-way
ANOVA was used to assess differences between groups,
and the Student–Newman–Keuls test was used for
multiple comparisons. Fisher’s exact test was used to
analyse differences in categorical variables. Correlations
between continuous variables were evaluated by linear
regression analysis. A P value of 0.05 was considered
significant.
RESULTS
After 9 weeks of treatment, all CCl -injured rats had
%
developed cirrhosis, while this was only observed in two
out of seven CCl -injured rats treated with SAMe (P l
%
0.02). The remaining CCl -injured rats receiving SAMe
%
had different degrees of liver damage and fibrosis, but not
Figure 1 Effects of SAMe on liver fibrosis, measured as
hepatic hydroxyproline content, in CCl4-injured and control
rats
F l 13.4, P
0.0001 ; *P
0.05 compared with other groups.
Figure 2 Effects of SAMe on hepatic α-tocopherol content in
CCl4-injured and control rats
F l 3.04, P l 0.04 ; *P
0.05 compared with other groups.
cirrhosis (sinusoidal or perivenular fibrosis in two cases,
and septal fibrosis in three cases). Liver histology was
normal in the control groups. These results were consistent with the hepatic hydroxyproline levels, which
were significantly increased in CCl -injured rats in
%
comparison with control rats. SAMe treatment significantly decreased hepatic hydroxyproline levels in
CCl -injured rats (Figure 1). Aspartate aminotransferase,
%
alanine aminotransferase and alkaline phosphatase levels
were also increased in CCl -injured rats, effects which
%
were partially prevented by SAMe (Table 1).
The hepatic GSH concentration was significantly
diminished in CCl -injured rats with respect to controls,
%
but normal levels were observed in rats receiving CCl
%
and SAMe (Table 1). Hepatic TBARS were increased in
CCl -injured rats, effects which were neutralized by
%
SAMe (Table 1). Moreover, the hydroxyproline content
was correlated inversely with GSH concentration (r l
k0.75, P 0.001), and directly with TBARS levels
(r l 0.63, P l 0.001).
The liver α-tocopherol concentration was significantly
lower in rats induced to cirrhosis than in controls
(17.3p4.9 and 23.0p4.0 µmol\g respectively ; P 0.05).
By contrast, the liver α-tocopherol level was similar in
CCl -injured rats treated with SAMe (23.8p5.1 µmol\g)
%
and in control rats. SAMe administration was not
associated with increased α-tocopherol levels in noninjured rats (Figure 2). Furthermore, the α-tocopherol
level was correlated directly with GSH content (r l 0.38,
Table 1 Effects of SAMe on biochemical parameters of liver disease and on hepatic glutathione
and TBARS levels in CCl4-injured and control rats
AST, aspartate aminotransferase ; ALT, alanine aminotransferase ; AP, alkaline phosphatase. Significance of differences : *P
0.05 compared with all other groups ; †P 0.05 compared with control and controljSAMe groups.
Parameter
Control
Control j SAMe
CCl4
CCl4jSAMe
F; P
AST (units/l)
ALT (units/l)
AP (units/l)
Glutathione (µmol/g)
TBARS (nmol/g)
46p5
29p4
252p24
5.9p0.6
36.4p12.9
69p7
41p4
270p16
6.5p0.9
23.5p6.3
2351p876* 932p140
1179p506† 557p86
540p98*
370p37
4.2p0.4* 5.8p0.5
60.7p7.5* 35.1p5.9
5.9 ;
4.9 ;
5.8 ;
15.9 ;
18.5 ;
0.01
0.01
0.01
0.001
0.001
# 2000 The Biochemical Society and the Medical Research Society
317
318
R. Deulofeu and others
Figure 3 Effects of SAMe on hepatic ascorbic acid content in
CCl4-injured and control rats
F l 38.6, P
0.001 ; *P
0.05 compared with other groups.
P 0.05) and inversely with TBARS levels (r l k0.41, P
l 0.04). The liver ascorbic acid concentration was
also decreased in CCl -injured rats (4.9p1.8 µmol\g)
%
in comparison with control rats (8.2p1.0 µmol\g ;
P 0.05), levels which were not corrected by SAMe
(4.6p0.4 µmol\g). In control rats SAMe administration significantly increased ascorbic acid levels
(12.8p0.9 µmol\g) (Figure 3). Ascorbic acid tissue concentrations were correlated directly with GSH (r l 0.62,
P l 0.001) and inversely with TBARS (r l k0.57, P
0.01) and hydroxyproline (r l k0.47, P 0.02).
DISCUSSION
The results of the present study indicate that, in CCl %
injured rats, as well as decreasing liver fibrosis and lipid
peroxidation and increasing hepatic GSH levels (as
reported in other studies [29,30]), SAMe administration
prevents hepatic vitamin E depletion without having any
effect on liver vitamin C content. Therefore, in rats
induced to cirrhosis, SAMe administration results in
normal levels of major known natural antioxidants, such
as GSH and vitamin E, which are commonly diminished
in acute and chronic liver damage generated by various
toxic agents. By contrast, SAMe had no effect on ascorbic
acid levels in injured animals, although it increased the
levels of this vitamin in control rats, thus suggesting that
this agent may enhance the antioxidant reserves under
non-toxic conditions. Indeed, apart from the beneficial
effects of SAMe in CCl -injured rats, this agent also had
%
some effect in normal rats, since the lipid peroxidation
levels (measured as TBARS) were significantly lower and
vitamin C levels significantly higher, with a trend of
higher GSH levels, in normal rats treated with SAMe.
These effects of SAMe on the antioxidant systems in
the liver may explain in part the favourable effects on
liver fibrosis, since, in addition to the well known
association between GSH and lipid peroxidation, close
correlations have been found between both vitamin E
and vitamin C and TBARS, thus indicating that the
protective effect on liver fibrosis is secondary to the
# 2000 The Biochemical Society and the Medical Research Society
reduced lipid peroxidation. In this respect, α-tocopherol
can inhibit stimulation of collagen synthesis by fibroblasts through decreasing lipid peroxidation [31]. On the
other hand, low GSH [8,32] and vitamin E levels have
been found in chronic liver diseases, particularly in
cirrhosis of different aetiologies [14–18]. Low blood
vitamin C levels have also been found associated with
liver diseases [33–35]. Moreover, vitamin E-deficient
animals are more susceptible to oxidative damage [36]. In
CCl -induced liver damage, several studies have demon%
strated that a high hepatic vitamin E content may prevent
the acute liver damage induced by the toxin [19,37]. In
addition, several clinical and experimental studies suggest
that a vitamin E intake greater than the recommended
dietary allowance may be of benefit in the prevention or
treatment of several pathological conditions [38]. It is
also known that, following dietary supplementation, the
liver content of vitamin E increases only in hepatocytes
and not in non-parenchymal cells [39]. The major role of
this vitamin involves its ability to protect cell membranes
from damage mediated by lipid peroxidation. This
concept is supported by a large number of studies
showing that α-tocopherol, as well as its synthetic
derivatives, is able to prevent the onset of cell damage
consequent to the induction of oxidative stress
[19,40–42]. In fact, because of its lipophilic properties,
vitamin E is expected to be highly effective in protecting
against membrane lipid peroxidation by reacting with
lipid peroxyl and alkoxyl radicals [43].
Ascorbic acid serves as both an antioxidant and a prooxidant. In fact, excess amounts of vitamin C may act as
a pro-oxidant in the presence of metals such as iron and
copper by generating cofactors of activated oxygen
radicals during the promotion of lipid peroxidation.
Indeed, ascorbate is related to the promotion of collagen
synthesis in some experimental models. As an antioxidant, vitamin C exerts a sparing effect on the
antioxidant action of vitamin E and selenium. GSH acts
as an antioxidant by serving as a substrate for several
enzymes that reduce hydrogen peroxide and organic
peroxides, and by mediating the reduction of dehydroascorbate [44,45] and the oxidized forms of α-tocopherol.
Thus GSH deficiency would be expected to produce
effects that also result from deficiency of ascorbate and
α-tocopherol. Furthermore, GSH deficiency decreases
tissue ascorbate. On the other hand, ascorbate protects
against GSH deficiency and spares GSH in various rat
models. Likewise, ascorbic acid, which alone is a moderately effective antioxidant, may interact with vitamin E,
thus enhancing the antioxidant activity of the latter. The
lack of an effect of SAMe on ascorbate levels in CCl %
injured rats could be explained in terms that ascorbate is
used for regenerating α-tocopherol. In this respect, SAMe
administration was associated with increased ascorbate
levels in healthy rats.
Our findings confirm that SAMe may lead to an
Effects of S-adenosylmethionine on vitamin E in CCl4-injured rats
increase in GSH levels, which could prevent inactivation
of SAMe synthetase and inhibit lipid peroxidation, and
consequently attenuate the development of liver fibrosis
and cirrhosis. Moreover, SAMe prevents hepatic vitamin
E depletion in this model of CCl -induced liver damage,
%
thus supporting the view that SAMe not only increases
GSH levels, but also spares α-tocopherol, in the livers of
rats induced to liver injury by CCl intoxication. This
%
may be an additional means of preventing lipid peroxidation and liver fibrosis, and provides a rationale for
using SAMe as a treatment for chronic liver disease,
particularly in alcoholic liver disease which is characterized by low hepatic levels of antioxidants such as
vitamin E and GSH.
ACKNOWLEDGMENTS
This work was supported in part by grants FIS 95\0485
and CICYT SAF 98-009.
REFERENCES
1 Mato, J. M. (1986) Regulation of phospholipid methylation
and its effect on membrane protein. In Progress in
Protein-Lipid Interactions, vol. 2 (Watts, A. and de Pont,
J. H., eds.), pp. 267–282, Elsevier Scientific Publishing,
New York
2 Giulidori, P., Galli-Kienle, M., Catto, E. and
Stramentinoli, G. (1984) Transmethylation,
transsulphuration and aminopropylation reactions of
S-adenosyl-L-methionine in vivo. J. Biol. Chem. 259,
4205–4211
3 Friedel, H. A., Goa, K. L. and Benfield, P. (1989)
S-adenosyl-L-methionine : a review of its pharmacological
properties and therapeutic potential in liver dysfunction
and affective disorders in relation to its physiological role
in cell metabolism. Drugs 38, 389–416
4 Lieber, C. S., Casini, A., DeCarli, L. M., Kim, C. and
Lorre, N. (1990) S-adenosyl-L-methionine attenuates
alcohol-induced liver injury in the baboon. Hepatology 11,
165–172
5 Stramentinoli, G., Gualano, M. and Ideo, G. (1978)
Protective role of s-adenosyl-L-methionine on liver injury
induced by d-galactosamine in rats. Biochem Pharmacol.
27, 1431–1433
6 Tribble, D. I., Aw, T. Y. and Jones, D. P. (1987) The
pathophysiological significance of lipid peroxidation in
oxidative cell injury. Hepatology 7, 377–387
7 Jover, R., Ponsoda, X., Fabra, R., Trullenque, R., Go! mezLecho! n, M. J. and Castell, J. V. (1992) S-adenosyl-Lmethionine prevents intracellular glutathione depletion by
GSH-depleting drugs in rat and human hepatocytes. Drug
Invest. 4 (suppl. 4), 46–53
8 Vendemiale, G., Altomare, E., Trizio, T. et al. (1989)
Effects of oral S-adenosyl-L-methionine on hepatic
glutathione in patients with liver diseases. Scand. J.
Gastroenterol. 24, 407–415
9 Frezza, M., Surrenti, C., Manzillo, G., Fiaccadori, F.,
Bortolini, M. and DiPadova, C. (1990) Oral S-adenosyl-Lmethionine in the symptomatic treatment of intrahepatic
cholestasis : a double blind, placebo-controlled study.
Gastroenterology 99, 211–215
10 Ribalta, J., Reyes, H. and Gonzalez, M. C. (1991)
S-adenosyl-L-methionine in the treatment of patients with
intrahepatic cholestasis of pregnancy : a randomized,
double blind placebo-controlled study with negative
results. Hepatology 13, 1084–1089
11 Mato, J. M., Ca! mara, J., Ferna! ndez, J. et al. (1999)
S-adenosylmethionine in the treatment of alcoholic liver
cirrhosis : results from a multicentric, placebo-controlled,
randomized, double-blind clinical trial. J. Hepatol. 30,
1081–1089
12 Recknagel, R. O. and Ghoshal, A. K. (1966)
Lipoperoxidation as a vector in carbon tetrachloride
hepatotoxicity. Lab. Invest. 15, 135–146
13 Recknagel, R. O. and Glende, E. A. (1973) Carbon
tetrachloride hepatotoxicity : an example of lethal cleavage.
CRC Crit. Rev. Toxicol. 2, 263–297
14 Tanner, A. R., Bantock, I., Hinks, U. et al. (1986)
Depressed selenium and vitamin E levels in alcoholic
population : possible relationships to hepatic injury
through increased lipid peroxidation. Dig. Dis. Sci. 31,
1307–1312
15 Bjorneboe, G. E. A., Johnsen, J., Bjorneboe, A. et al.
(1987) Effect of heavy alcohol consumption on serum
concentration of fat soluble vitamins and selenium.
Alcohol Alcohol. (suppl.) 1, 533–537
16 Bell, H., Bjorneboe, A., Eidsvoll, B. et al. (1992) Reduced
concentration of hepatic α-tocopherol in patients with
alcoholic liver cirrhosis. Alcohol Alcohol. 27, 39–46
17 Leo, A. M., Rosman, A. S. and Lieber, C. S. (1993)
Differential depletion of carotenoids and tocopherol in
liver disease. Hepatology 17, 977–986
18 Sokol, R. J., Balistreri, W. F., Hoofnagle, J. H. and Jones,
E. A. (1985) Vitamin E deficiency in adults with chronic
liver disease. Am. J. Clin. Nutr. 41, 66–72
19 Parola, M., Leonardizzi, G., Biasi, F. et al. (1992) Vitamin
E dietary supplementation protects against carbon
tetrachloride-induced chronic liver damage and cirrhosis.
Hepatology 16, 1014–1021
20 Frei, B., Stocker, R., England, L. and Ames, B. N. (1990)
Ascorbate : the most effective antioxidant in blood plasma.
Adv. Exp. Med. Biol. 264, 155–163
21 Yu, B. P. (1994) Cellular defenses against damage from
reactive oxygen species. Physiol. Rev. 74, 139–162
22 Bendich, A., Machlin, L. J. and Scandurra, O. (1986) The
antioxidant role of vitamin C. Adv. Free Radical Biol.
Med. 2, 419–444
23 Martensson, J. and Meister, A. (1991) Glutathione
deficiency decreases tissue ascorbate levels in newborn
rats : ascorbate spares glutathione and protects. Proc. Natl.
Acad. Sci. U.S.A. 88, 4656–4660
24 Shearer, M. J. (1986) Vitamins : Vitamin E. In HPLC of
Small Molecules (Lim, C. K., ed.), pp. 177–182, IRL Press,
Oxford
25 Speek, A. J., Shrijver, J. and Scheuers, W. P. H. (1984)
Measurements of total ascorbic acid in biological fluids.
J. Chromatogr. 305, 53–59
26 Hissin, P. J. and Hill, R. (1976) A fluorimetric method for
determination of oxidized and reduced glutathione in
tissues. Anal. Biochem. 74, 214–226
27 Bird, R. P., Hung, S., Hadley, M. and Draper, H. H.
(1983) Determination of malonaldehyde in biological
materials by high-pressure liquid chromatography. Anal.
Biochem. 128, 240–244
28 Heinriksin, R. L. and Meredith, S. C. (1984) Amino acid
analysis by reverse phase High-Performance Liquid
Chromatography : precolumn derivatization with
phenylisothiocyanate. Anal. Biochem. 136, 65–69
29 Corrales, F., Gime! nez, A., Alvarez, L. et al. (1992)
S-adenosylmethionine treatment prevents carbon
tetrachloride induced S-adenosylmethionine synthetase
inactivation and attenuates liver injury. Hepatology 16,
1022–1027
30 Gasso, M., Rubio, M., Varela, G. et al. (1996) Effects of
S-adenosylmethionine on lipid peroxidation and liver
fibrogenesis in carbon tetrachloride-induced cirrhosis.
J. Hepatol. 25, 200–205
31 Houglum, K., Brenner, D. A. and Chojkier, M. (1991)
α-Tocopherol inhibits collagen alpha (1) gene expression in
cultured human fibroblasts : modulation of constitutive
gene expression by lipid peroxidation. J. Clin. Invest. 87,
2230–2235
32 Shaw, S., Rubin, K. P. and Lieber, C. S. (1983) Depressed
hepatic glutathione and increased diene conjugates in
# 2000 The Biochemical Society and the Medical Research Society
319
320
R. Deulofeu and others
33
34
35
36
37
38
39
alcoholic liver disease. Evidence of lipid peroxidation. Dig.
Dis. Sci. 28, 585–589
Beattie, A. D. and Sherlock, S. (1976) Ascorbic acid
deficiency in liver disease. Gut 17, 571–575
Sinclair, P. R., Gorman, N., Shedlofsky, S. I. et al. (1997)
Ascorbic acid deficiency in porphyria cutanea tarda. J.
Lab. Clin. Med. 130, 197–201
Yamanoto, Y., Yamashita, S., Fujisawa, A., Kokura, S. and
Yoshikawa, T. (1998) Oxidative stress in patients with
hepatitis, cirrhosis, and hepatoma evaluated by plasma
antioxidants. Biochem. Biophys. Res. Commun. 247,
166–170
Maellaro, E., Casini, A. F., Del Bello, B. and Comporti, M.
(1990) Lipid peroxidation and antioxidant systems in the
liver injury produced by glutathione depleting agents.
Biochem. Pharmacol. 39, 1513–1521
Cheeseman, K. H., Davies, M. J., Emery, S., Maddix, S. P.
and Slater, T. F. (1987) Effects of alpha-tocopherol on
carbon tetrachloride metabolism in rat liver microsomes.
Free. Radical Res. Commun. 3, 325–330
Traber, M. G. (1996) Vitamin E in humans : demand and
delivery. Annu. Rev. Nutr. 16, 321–347
Drevon, C. A. (1991) Absorption, transport and
metabolism of vitamin E. Free. Radical Res. Commun. 14,
229–246
40 Fariss, M. W. (1990) Oxygen toxicity : unique
cytoprotective properties of vitamin E succinate in
hepatocytes. Free. Radical Biol. Med. 9, 333–343
41 Sharma, B. K., Bacon, B. R., Britton, R. S. et al. (1990)
Prevention of hepatocyte injury and lipid peroxidation by
iron chelators and alpha-tocopherol in isolated iron-loaded
hepatocytes. Hepatology 12, 31–39
42 Carini, R., Poli, G., Dianzani, M. U., Maddid, S. P., Slater,
T. F. and Cheeseman, K. H. (1990) Comparative
evaluation of the antioxidant activity of alpha-tocopherol,
alpha-tocopherol polyethylene glycol 1000 succinate and
alpha-tocopherol succinate in isolated hepatocytes and
liver microsomal suspensions. Biochem. Pharmacol. 39,
1597–1601
43 Ross, D. and Moldeus, P. (1991) Antioxidant defense
systems and oxidative stress. In Membrane Lipid
Oxidation (Vigo-Pelfrey, C., ed.), pp. 151–173, CRC Press,
Boca Raton
44 Burns, J. J., Rivers, J. M. and Machlin, L. J. (1987) Safety
of high levels of vitamin C ingestion. Ann. N.Y. Acad. Sci.
589, 498–506
45 Rose, R. C. (1989) Renal metabolism of the oxidized form
of ascorbic acid (dehydro-L-ascorbic acid). Am. J. Physiol.
256, F52–F56
Received 11 May 2000; accepted 22 June 2000
# 2000 The Biochemical Society and the Medical Research Society