Download Free radical scavenging and antioxidant effects of lactate ion: an in

J Appl Physiol
89: 169–175, 2000.
Free radical scavenging and antioxidant effects
of lactate ion: an in vitro study
CAROLE GROUSSARD,1 ISABELLE MOREL,2 MARTINE CHEVANNE,2 MICHEL MONNIER,1
JOSIANNE CILLARD,2 AND ARLETTE DELAMARCHE1
1
Laboratoire de Physiologie et de Biomécanique de l’Exercice Musculaire, UFRAPS Rennes 2,
UPRES A 1274, Campus la Harpe, CS 24414, 35044 Rennes Cedex; and 2Laboratoire de Biologie
Cellulaire et Végétale, Faculté de Pharmacie, Université de Rennes 1, 35043 Rennes Cedex, France
Received 15 June 1999; accepted in final form 9 March 2000
electron paramagnetic resonance experiments; linoleic acid
autoxidation; lactate; lipid peroxidation; oxidative stress
tion and lipid peroxidation, as evaluated by malondialdehyde (MDA), during a progressive incremental
exercise. However, Anbar and Neta (2), in tests of the
scavenging activity of various and numerous agents
toward the hydroxyl radical (䡠OH), noted that the lactate ion acted as a moderate 䡠OH scavenger at pH 9.
Given the substantial increase of lactate metabolism
during exercise, it seems of fundamental importance to
determine the exact effect of the lactate ion alone on
free radical production during exercise. In vivo, lactate
production during exercise can never be dissociated
from the concomitant acidosis, which is well known to
be a potent oxidative condition (28, 30). For this reason, we investigated the effect of the lactate ion in vitro
at concentrations usually found at rest or during exercise. Lactate concentrations ranged up to 60 mM because such high lactate levels can be found in muscle
(22, 29). Different methods were employed to test the
lactate effect on free radicals. Two methods were used
to determine its scavenging activity toward superoxide
anion (O2⫺䡠), 䡠OH, and lipid radical. We also investigated its cellular antilipoperoxidant effect by using
hepatocyte cultures. Lactate was dissolved in either
aqueous or plasma solution or culture medium at different concentrations.
MATERIALS AND METHODS
either in animals (1, 12) or
in humans (15, 19) have demonstrated that physical
exercise increases the production of reactive oxygen
species (ROS), thereby inducing oxidative stress. All
these studies have indicated that oxidative stress particularly appears during vigorous and exhaustive exercise, in which lactate production is nonnegligible. A
question thus arises: to what extent is lactate itself
responsible for free radical production during exercise?
The data on this subject are conflicting. In vitro studies
performed on kidney slices and homogenates have reported an oxidant effect of lactic acidosis (28, 30). In in
vivo experiments, Lovlin et al. (23) observed a significant relationship between plasma lactate concentraMANY EXPERIMENTS PERFORMED
Address for reprint requests and other correspondence: C. Groussard, Laboratoire de Physiologie et de Biomécanique de l’Exercice
Musculaire, UFRAPS Rennes 2, Ave. Charles Tillon, CS 24414,
35044 Rennes Cedex, France (E-mail: [email protected]).
http://www.jap.org
Three methods were used to evaluate the scavenging activity of lactate ion toward free radicals. Lactate ion was
dissolved in aqueous or plasma solution or culture at concentrations ranging from 1 to 60 mM, i.e., approximately the
concentrations usually found in plasma either at rest (1 mM)
or during exercise (10 and 15 mM) and in muscle during
exercise (30 and 60 mM) (22).
Experimental Procedure
First method: Electron paramagnetic resonance study of
the scavenging activity of lactate toward 䡠OH and O2⫺䡠. The
electron paramagnetic resonance (EPR) method allows direct
determination of the specific scavenging activity of lactate for
one radical species.
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8750-7587/00 $5.00 Copyright © 2000 the American Physiological Society
169
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Groussard, Carole, Isabelle Morel, Martine Chevanne, Michel Monnier, Josianne Cillard, and Arlette
Delamarche. Free radical scavenging and antioxidant effects of lactate ion: an in vitro study. J Appl Physiol 89:
169–175, 2000.—Divergent literature data are found concerning the effect of lactate on free radical production during
exercise. To clarify this point, we tested the pro- or antioxidant effect of lactate ion in vitro at different concentrations
using three methods: 1) electron paramagnetic resonance
(EPR) was used to study the scavenging ability of lactate
toward the superoxide aion (O2⫺䡠) and hydroxyl radical (䡠OH);
2) linoleic acid micelles were employed to investigate the
lipid radical scavenging capacity of lactate; and 3) primary
rat hepatocyte culture was used to study the inhibition of
membrane lipid peroxidation by lactate. EPR experiments
exhibited scavenging activities of lactate toward both O2⫺䡠
and 䡠OH; lactate was also able to inhibit lipid peroxidation of
hepatocyte culture. Both effects of lactate were concentration
dependent. However, no inhibition of lipid peroxidation by
lactate was observed in the micelle model. These results
suggested that lactate ion may prevent lipid peroxidation by
scavenging free radicals such as O2⫺䡠 and 䡠OH but not lipid
radicals. Thus lactate ion might be considered as a potential
antioxidant agent.
170
SCAVENGING AND ANTIOXIDANT EFFECTS OF LACTATE ION
acid concentration of 2.5 ⫻ 10⫺3 M and various final lactate
concentrations of 1, 10, 15, 30, and 60 mM, respectively.
Samples were placed in glass tubes and left in the dark under
air at 37°C. Controls without lactate were placed in the same
conditions. Linoleic acid autoxidation was determined by
conjugated diene measurement. Measures were performed
every 3 h except during the night by use of a Secomam
S1000PC spectrophotometer with a UV lamp set at 234 nm.
Third method: Effect of lactate on oxidative stress induced
in rat hepatocytes in culture. The cellular effect of lactate was
tested by using primary rat hepatocyte cultures as model. An
oxidative stress was induced in these cells by iron supplementation and was estimated by the extent of lipid peroxidation.
The reagents 1,1,3,3-tetramethoxypropane and sodium
lactate were obtained from Sigma Chemical. A ferric nitrilotriacetate solution (Fe-NTA) was prepared according to the
method of White and Jacobs (31). Briefly, 47 mg of nitrilotriacetic acid disodium salt (Sigma Chemical) and 20 mg of
ferric ammonium citrate (Merck) were dissolved in 10 ml of
sterile water to achieve a 10 mM solution of ferric iron.
Cell Isolation and Culture
Hepatocyte cultures were prepared according to the protocol of Guguen et al. (20). For experimental purposes, some
cultures were maintained for 4 h with Fe-NTA at a final iron
concentration of 100 ␮M (25), and other cultures were supplemented for 4 h with Fe-NTA (100 ␮M) and lactate (1, 10,
15, 30, or 60 mM at final concentrations) dissolved in culture
medium. Lipid peroxidation was estimated by the measure of
free MDA released in culture medium using the HPLC
method, according to the protocol of Morel et al. (25).
Free MDA Evaluation
HPLC procedure. MDA quantification was performed according to a method described previously (11). The HPLC
system (LDC-Milton Roy) was equipped with a spherogelTSK G1000 PW size exclusion column (7.5 mm ID ⫻ 30 cm;
Cluzeau, France). The eluant was composed of 0.1 M disodium phosphate buffer, pH 8, at a flow rate of 1 ml/min, at
ambient temperature. The absorbance was monitored at 267
nm, and the sensitivity was set at 0.05 absorbance units full
scale. The injections were performed by an automatic injector
(LDC Promis) set at a volume of 250 ␮l. The data were
recorded and integrated by a CI 3000 LDC integrator.
Preparation of free MDA standard. Ten microliters of
1,1,3,3-tetramethoxypropane were hydrolyzed in 10 ml of
0.1 N HCl for 5 min in boiling water. This solution was then
diluted 1,000 times in 0.01 M Na2HPO4 buffer, pH 7.45,
corresponding to a 6 ␮M MDA solution. The concentration of
MDA in samples was calculated by using a standard curve of
free MDA.
Preparation of the samples for HPLC analysis. Free MDA
was quantified from culture medium. Culture media were
collected, and hepatocytes were washed twice with 0.01 M
phosphate buffer, pH 7.45. They were resuspended in 1 ml of
the same buffer. The cells were lysed by use of an ultrasonic
homogenizer. Culture media were filtered through a 500-Da
membrane ultrafilter (Millipore, Yvelines, France) in a 10-ml
Amicon cell pressurized at 4 bars with nitrogen gas. The
filtrate was used for the HPLC procedure. All experiments
were performed at least on triplicate cultures. We determined protein content on cell homogenates according to the
Bradford reaction (5) using the Bio-Rad reagent, BSA as
standard, and a Cobas-Bio automatic analyzer.
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䡠OH was generated by decomposition of H2O2 (Merck,
Darmstadt, Germany) by ferrous ions (Fenton reaction) (7) in
the presence of the spin-trap 5,5-dimethylpyrroline-N-oxide
(DMPO) (Sigma Chemical, St Louis, MO) and with or without
sodium lactate (Sigma Chemical). The DMPO-䡠OH adduct
was formed and was then analyzed by EPR spectroscopy (9).
Briefly, 20 ␮l of lactate dissolved in water or plasma at
different concentrations (10, 100, 150, 300, and 600 mM), 30
␮l distilled water, 50 ␮l H2O2 (40 mM in aqueous solution),
10 ␮l DMPO (800 mM), and 90 ␮l iron sulfate (II) (1 mM)
(Prolabo, Paris, France) were added in that order to a small
glass tube. Final lactate concentrations were 1, 10, 15, 30,
and 60 mM. After 1 min of reaction, the sample was immediately analyzed by using a Bruker ECS 106 EPR spectrometer (France). Controls without lactate were prepared in the
same conditions as the samples. They contained only water
or plasma. The effect of lactate was estimated by the percentage of variation in the EPR signal intensity of the DMPO-䡠OH
adduct compared with that of the controls without lactate
(representing 100%).
To check whether lactate scavenged 䡠OH itself or reacted
with preformed DMPO-䡠OH adduct, postaddition of lactate at
the end of the Fenton reaction was tested (26). When the
amount of spin-adduct formed decreased during postaddition, this amount was taken into account and subtracted
from the previous results.
O2⫺䡠 was produced by a xanthine-xanthine oxidase system.
Xanthine oxidase (Sigma Chemical) catalyzes the oxidation
of xanthine (Sigma Chemical) in the presence of molecular
oxygen to yield uric acid and O2⫺䡠 as reaction products (18). In
the presence of the spin-trap DMPO, the O2䡠 leads to the
production of DMPO-䡠OOH adduct, which can then be analyzed by EPR spectroscopy (9). More precisely, 30 ␮l of
xanthine oxidase (0.8 U/ml of water) was added to a reaction
mixture containing 100 ␮l ethanol (1 M), 30 ␮l xanthine
aqueous solution (10 mM), 20 ␮l DMPO (800 mM), and 20 ␮l
of lactate dissolved in water or plasma at different initial
concentrations (10, 100, 150, 300, and 600 mM). Thus final
lactate concentrations were 1, 10, 15, 30, and 60 mM. The
EPR analysis was performed 2 min after the addition of
xanthine oxidase. As before, controls without lactate contained water or plasma alone. The effect of lactate was
estimated by the percentage of variation in the DMPO-䡠OOH
adduct compared with that of the controls without lactate
(representing 100%). In the xanthine-xanthine oxidase system, lactate cannot react with preformed DMPO-䡠OOH adduct. Thus postaddition experiments were not necessary.
However, the possible inhibition of xanthine oxidase by lactate was tested by measuring the concentration of uric acid
produced with and without lactate. Lactate did not modify
uric acid production by xanthine oxidase. Thus lactate did
not inhibit xanthine oxidase.
Second method: Effect of lactate ion on linoleic acid micelle
autoxidation. Fatty acid micelles are a good model to study
membrane lipid peroxidation. It is well known that propagation of lipid peroxidation involves the peroxyl radical, a lipid
radical (24). Thus, by scavenging this radical, lactate will
reduce lipid peroxidation. Linoleic acid (9,12-octadecadienoic
acid) was purchased (Koch Light; ⬎99% pure). This fatty acid
was dispersed with 0.5% Tween 20 (Merck) in 0.01 M phosphate-buffered aqueous solution (pH ⫽ 9) under nitrogen
atmosphere. Linoleic acid concentration was 10⫺2 M, and
this stock solution was stored at ⫹4°C. Lactate was dissolved
in phosphate buffer (pH ⫽ 7.4; 5 mM) at initial concentrations of 10, 100, 150, 300, and 600 mM, respectively, and
stored at ⫹4°C. Aliquots of each stock solution were adjusted
to pH 7.4 and mixed at time zero to achieve a final linoleic
SCAVENGING AND ANTIOXIDANT EFFECTS OF LACTATE ION
171
Each culture supplemented with both lactate and Fe-NTA
was compared with control cultures supplemented with FeNTA alone (representing 100%).
Statistical Analysis
The results are given as means ⫾ SE of our experiments.
We chose a nonparametric test to reveal significant differences between trials with and without lactate. The MannWhitney test was used instead of an ANOVA on ranks because the effect of lactate concentration for each experiment
had to be compared with its own control sample specific to the
experiment of that day. We have not provided the number of
assays performed because the number was never the same.
Nevertheless, there were at least five with a significant result
when we concluded. P ⬍ 0.01 was chosen as significance
level.
RESULTS
Scavenging of 䡠OH. Assuming that the signal intensity in the controls without lactate corresponds to 100%
DMPO-䡠OH formation, Fig. 1A shows that lactate addition to aqueous solution at final concentrations of 10,
15, 30, and 60 mM was associated with a decrease in
EPR signal. This decrease was due to a scavenging
activity of lactate toward the 䡠OH that was concentration dependent. In samples containing lactate in aqueous solution, the EPR signal values were, respectively,
64% (10 mM), 52.5% (15 mM), 46% (30 mM), and 38%
(60 mM). Figure 1B shows the effect of lactate dissolved in plasma. Because plasma alone exhibited a
scavenging activity toward 䡠OH, plasma without lactate was taken for reference 100%. Addition of lactate
dissolved in plasma reduced the EPR signal intensity
only at high concentration levels (30 and 60 mM). EPR
signal values were, respectively, 75% (30 mM) and
53.5% (60 mM) of the control value.
Scavenging of O2⫺䡠. As previously stated, controls
without lactate correspond to 100% DMPO-OOH䡠 adduct. Addition of lactate in aqueous solution caused a
decrease in DMPO-OOH䡠 adduct as shown in Fig. 2A,
indicating a scavenging effect of lactate (1, 10, 15, 30,
and 60 mM) toward superoxide radicals. EPR signal
intensities were, respectively, 57% (1 mM), 25.5% (10
mM), 23.5% (15 mM), 12.5% (30 mM), and 11.5% (60
mM) of control value. A scavenging effect was also
observed with lactate in plasma (Fig. 2B). EPR signal
intensities were, respectively, 86.5% (1 mM), 72% (10
mM), 54% (30 mM), 64% (15 mM), and 26% (60 mM).
DMPO-OOH䡠 adduct at 100% represented the plasma
control.
Lack of Lactate Effect to Inhibit Linoleic Acid
Autoxidation in Micelles
Conjugated dienes resulting from the spontaneous
autoxidation of linoleic acid (at pH 7.4 and temperature 37°C) were measured by their absorbance at 234
nm (Fig. 3). The level of conjugated dienes increased
rapidly to reach a maximum at 20 h and then remained
Fig. 1. Scavenging of hydroxyl radical (䡠OH) by lactate. Effect of
lactate concentrations on the electron paramagnetic resonance
(EPR) signal corresponding to the 䡠OH adduct with 5,5-dimethylpyrroline-N-oxide (DMPO). 䡠OH was generated by decomposition of
H2O2 by ferrous ion in presence or absence of lactate dissolved in
water (A) or in plasma (B) at different concentrations (1, 10, 15, 30,
and 60 mM). The 100% reference value corresponds to the level of
DMPO-䡠OH adduct produced in the corresponding control sample
without lactate. Results are expressed as means ⫾ SE. ** Values
significantly different (P ⬍ 0.01) from control without lactate.
constant. As shown in Fig. 3, addition of lactate at 1,
10, and 15 mM was without any effect on the time
course of the formation of conjugated dienes. Data for
lactate at 30 and 60 mM are not presented in Fig. 3, but
no inhibitory effect was observed even for these concentrations.
Antioxidant Effect of Lactate Toward Oxidative Stress
Induced in Hepatocyte Cultures
Addition of Fe-NTA to hepatocyte cultures for 4 h
induced a large increase in the level of free MDA
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Free Radical Scavenging Activity of Lactate
Toward 䡠OH and O2⫺䡠
172
SCAVENGING AND ANTIOXIDANT EFFECTS OF LACTATE ION
DISCUSSION
released in culture medium (25). MDA is commonly
used as a marker of lipid peroxidation. The level of
MDA in the iron-treated hepatocyte cultures was
taken as the reference of 100% free MDA production.
Addition of lactate reduced the amount of MDA, and
the decrease was concentration dependent, as shown
in Fig. 4. This antioxidant activity was significant
with 15, 30, and 60 mM lactate. MDA values expressed in percentage of controls (100%) were, respectively, 86% (15 mM), 58.5% (30 mM), and 49%
(60 mM).
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Fig. 2. Scavenging of superoxide anion (O2⫺䡠) by lactate. Effect of
lactate concentrations on the EPR signal corresponding to the O2⫺䡠
adduct with DMPO. O2⫺䡠 was generated by a xanthine-xanthine
oxidase system in presence or absence of lactate dissolved in water
(A) or in plasma (B) at different concentrations (1, 10, 15, 30, and 60
mM). The 100% reference value corresponds to the DMPO-䡠OH
adduct produced in the corresponding control sample without lactate. Results are expressed as means ⫾ SE. Values significantly
different (** P ⬍ 0.01, *** P ⬍ 0.001) from control without lactate.
The present study, using three different in vitro
models at physiological pH, provides evidence that the
lactate ion may act as a good antioxidant.
The first model consisted of EPR experiments to
determine the scavenging activity of lactate toward
free radicals such as 䡠OH and O2⫺䡠. Our data indicated
that EPR signals were reduced for both 䡠OH and O2⫺䡠 by
lactate in aqueous solution and in plasma. These results confirmed that lactate scavenged 䡠OH and O2⫺䡠.
Scavenging of 䡠OH by lactate was previously reported
by Anbar and Neta (2). For these authors, the rate
constant of reaction was calculated as 4.8 ⫻ 109 M⫺1 䡠
s⫺1 at pH 9, a value very close to the rate constant of
reaction evaluated in this study (6.9 ⫻ 109 M⫺1 䡠 s⫺1
when lactate was dissolved in aqueous solution and
3.8 ⫻ 109 M⫺1 䡠 s⫺1 when lactate was dissolved in
plasma). According to Herz et al. (21), this value demonstrates the powerful 䡠OH scavenging ability of lactate. Indeed, the rate constant of reaction of lactate
was greater than that of mannitol (which is commonly
referred to as an efficient 䡠OH scavenger), glucose, and
ethanol but less than that of catalase and ascorbic acid.
No information is currently available concerning the
scavenging activity of the well-known antioxidant
agents toward O2⫺䡠. To our knowledge, this study is also
the first report of the ability of lactate to scavenge O2⫺䡠.
Although O2⫺䡠 is considered to be a poorly reactive
radical, O2⫺䡠 formation is a major factor in oxygen
toxicity because it leads to the formation of toxic species such as H2O2 and 䡠OH. The free radical scavenging
effect of lactate was less marked in plasma than in
aqueous solution. In fact, plasma alone exerted an
antioxidant effect (data not shown) because of the
presence of well-known antioxidant plasma agents
such as vitamin C (55 ␮M), vitamin E (14–35 ␮M),
glucose (4.5 mM), and uric acid (0.25–0.45 ␮M) (17).
Thus lactate action at low concentrations (1–15 mM)
was partly masked by the effect of these antioxidants.
At high lactate levels such as 30 and 60 mM, lactate
action was increased. It should be noted that, because
the plasma concentration of these antioxidants varies
with the nutritional status, all experiments were performed with the same plasma sample.
The second model, based on linoleic acid autoxidation in micelles (10), showed that lactate did not inhibit
lipid peroxidation. No induction period was observed.
Indeed, in a micelle system, the autoxidation process
involves lipid radicals such as peroxyl radical (ROO䡠)
and alkoxyl radical (RO䡠) (24). Scavengers (e.g., tocopherol) of these radicals inhibit the autoxidation reaction.
Thus it can be deduced that lactate did not scavenge
lipid radicals.
The third model was a cellular model using hepatocytes in culture. Oxidative stress was induced in hepatocyte culture by addition of Fe-NTA. In this system,
lactate was able to inhibit lipid peroxidation. This
discrepancy between a cellular antilipoperoxidant effect of lactate and its ineffectiveness to prevent lipid
peroxidation in micelles could be explained by the
SCAVENGING AND ANTIOXIDANT EFFECTS OF LACTATE ION
173
Fig. 3. Effect of lactate on linoleic acid autoxidation.
Micelles of linoleic acid were autoxidized in the dark at
37°C. Linoleic acid autoxidation was estimated by the
absorbance of conjugated dienes at 234 nm. Linoleic
acid (2.5 ⫻ 10⫺3 M) without lactate (E), with 1 mM
lactate (䊐), with 10 mM lactate (‚), and with 15 mM
lactate (ƒ). Results obtained with lactate at 30 and 60
mM are not presented. No effect was noted at these
concentrations.
Fig. 4. Effect of lactate on lipid peroxidation induced in hepatocyte
cultures. Hepatocyte cultures were incubated for 4 h with ferric
nitrilotriacetate (100 ␮M) either alone or with lactate at different
concentrations (1, 10, 15, 30, and 60 mM). The malondialdehyde
(MDA) value of the control culture was taken as the reference 100%.
Results are expressed as means ⫾ SE, where 100% MDA recovery
corresponds to the MDA level in cultures supplemented with iron
alone. Values significantly different (* P ⬍ 0.05, *** P ⬍ 0.001) from
control without lactate.
lactate acted at the initiation step of lipid peroxidation
but not at the propagation step, which involves lipid
radicals. Such findings are not surprising because lactate is not liposoluble. Vitamin C, a well-known antioxidant agent, reacts rapidly with O2⫺䡠 and 䡠OH but not
with peroxyl radical. In contrast, the well-known liposoluble vitamin E is a powerful scavenger of lipid
radicals (8).
The antioxidant effect of lactate was concentration
dependent. These findings are important to further
our understanding of the effect of lactate on the free
radicals produced during exercise, because plasma
and muscular lactate levels increase with exercise
intensity. Plasma lactate concentration may rise
from 1 mM to 10 mM at maximal oxygen consumption and to 15 mM, possibly 30 mM, during supramaximal exercise (27). These concentrations were
therefore chosen for the study. Also, 30 and 60 mM
correspond to the lactate concentrations that could
be found in the active muscles after a very brief and
intensive exercise (22, 29). These high concentrations of lactate might protect cells from free radical
damage during exercise.
Some data in the literature have indicated the protective effect of lactate ion. Using 23Na and 31P NMR,
Yanagida et al. (32) showed that lactate can protect rat
heart from 䡠OH damage. In another NMR study of
biofluids, Herz et al. (21) concluded that consumption
of 䡠OH by lactate may serve to protect alternative
biofluid components against ROS-mediated damage in
vivo. Moreover, addition of lactate to ischemic reperfused hearts was found to prevent decline in the enzymatic defense system against oxygen toxicity (13).
The antioxidant effect of lactate thus seems undebatable. However, because a relationship was found
during maximal incremental exercise between plasma
MDA and lactate concentration, Lovlin et al. (23) in-
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property of lactate to scavenge radicals such as 䡠OH
and O2⫺䡠 but not lipid radicals. In hepatocytes, 䡠OH and
O2⫺䡠 are generated by the Fenton and Haber-Weiss
reactions (14). These radicals then initiate membrane
lipid peroxidation. Thus lactate, by eliminating 䡠OH
and O2⫺䡠, prevented lipid peroxidation. In other words,
174
SCAVENGING AND ANTIOXIDANT EFFECTS OF LACTATE ION
We thank C. Stott Carmeni for technical assistance and M. Benanni-Dosse for statistical assistance.
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criminated lactate as a promoting factor of exerciseinduced oxidative stress. In fact, lactate produced during exercise is always associated with acidosis, which
can act as a prooxidant agent (4, 6) by three mechanisms. First, during acidosis, increase in H⫹ concentration accelerates the rate of dismutation of O2⫺䡠 to
H2O2, which can react with Fe2⫹ and generate the
highly toxic 䡠OH. Second, during acidosis, increase in
H⫹ converts O2⫺䡠 to the more reactive and more lipid
soluble hydroperoxyl radical HO2. Third, another possible effect of tissue acidosis is to stimulate free radical
generation by increasing dissociation of protein-bound
iron, as originally suggested by Bernheim (4). Thus it
seems more likely that the oxidative stress noted in the
study by Lovlin et al. (23) was more in relationship
with acidosis than with lactate alone. Many previous
results support this hypothesis (6, 16), showing that
lactic acid and not lactate ion alone promotes lipoperoxidation.
The mechanisms underlying the antioxidant effect of
lactate have yet to be elucidated. Also using EPR
experiments, Anbar and Neta (2) reported a direct
scavenging activity of lactate toward 䡠OH. Nevertheless, an indirect antioxidant effect of lactate was also
assumed by Herz et al. (21). According to these authors, the consumption of 䡠OH by lactate generates
pyruvate, an antioxidant agent that is also able to
scavenge H2O2 and 䡠OH by its decomposition into acetate and CO2. Because hepatocyte culture is a biological model that contains the lactic dehydrogenase enzyme, the transformation of lactate into pyruvate was
possible. Thus this indirect mechanism cannot be totally excluded in the present study to explain the
reduction of MDA when lactate was present in culture
medium at 15, 30, and 60 mM.
This study nevertheless adds further information
about this subject. It clearly demonstrates for the first
time that lactate is also able to directly scavenge O2⫺䡠, a
potent oxygen reactive species that is produced in large
quantity during exercise (3).
In conclusion, the results of the present study contribute to the recent literature on the beneficial aspect
of lactate by suggesting that lactate ion can be considered as a potential antioxidant agent and by demonstrating that lactate ion, in vitro, is not prooxidant. We
showed that lactate was able to scavenge both 䡠OH and
O2⫺䡠, in vitro, with the effect toward O2⫺䡠 being predominant. No scavenging effect of lactate was observed
toward lipid radicals. This antioxidant capacity of lactate is very important regarding exercise performance.
By scavenging O2⫺䡠, the lactate ion may limit the high
exercise-induced increase in the production of ROS. By
scavenging both O2䡠 and 䡠OH, lactate may limit the
initiation step of lipid peroxidation and thus protect
cells against oxidative damage. The precise mechanisms remain to be elucidated.
SCAVENGING AND ANTIOXIDANT EFFECTS OF LACTATE ION
23. Lovlin R, Cottle W, Pyke I, Kavanagh M, and Belcastro
AN. Are indices of free radical damage related to exercise intensity? Eur J Appl Physiol 56: 313–316, 1987.
24. Mead JM. Free radical mechanisms of lipid damage and
consequences for cellular membranes. In: Free Radicals in
Biology, edited by Pryor WA. London: Academic Press, 1976,
p. 51–68.
25. Morel I, Lescoat G, Cillard J, Padeloup N, Bissot P, and
Cillard P. Kinetic evaluation of free malondialdehyde and enzyme leakage as indices of iron damage in rat hepatocyte cultures: involvement of free radicals. Biochem Pharmacol 39:
1647–1655, 1990.
26. Nagy IZS and Floyd RA. Hydroxyl free radical reactions with
amino acids and proteins studied by electron spin resonance
spectroscopy and spin trapping. Biochim Biophys Acta 790: 238–
250, 1984.
27. Osnes J and Hermansen L. Acid base balance after maximal
exercise of short duration. J Appl Physiol 3: 59–63, 1972.
175
28. Rehncrona S, Hauge HN, and Siesjö BK. Enhancement of
iron-catalysed free radical formation by acidosis in brain homogenates: differences in effect by lactic acid and CO2. J Cereb Blood
Flow Metab 9: 65–70, 1989.
29. Sahlin K and Ren JM. Relationship of contraction capacity to
metabolic changes during recovery from fatiguing contraction.
J Appl Physiol 67: 648–657, 1989.
30. Siesjo BK, Bendek G, Koide T, Westerberg E, and Wieloch
T. Influence of acidosis on lipid peroxidation in brain tissues in
vitro. J Cereb Blood Flow Metab 5: 253–258, 1985.
31. White GP and Jacobs A. Iron uptake by Chang liver cells from
transferrin, nitrilotriacetate and citrate complexes: the effect of
iron-loading and chelation with desferrioxamine. Biochim Biophys Acta 543: 217–225, 1978.
32. Yanagida S, Luo CS, Doyle M, Pohost GM, and Pike MM.
Nuclear magnetic resonance studies of cationic and energic alterations with oxidant stress in the perfused heart: modulation
with pyruvate and lactate. Circ Res 77: 773–783, 1995.
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