Results Action ofÆOH/O2Æ-free radicals A 450 lmolÆL1 solution of metformin in 10 mmolÆL1 sodium phosphate buffer, pH 7 was irradiated at doses ranging from 52 to 627 Gy, with a dose rate
Trang 1Radical-induced oxidation of metformin
H Khouri1, F Collin1, D Bonnefont-Rousselot2,3, A Legrand2, D Jore1and M Garde`s-Albert1
1 Laboratoire de Chimie Physique UMR 8601-CNRS, Universite´ Paris 5, France; 2 Laboratoire de Biochimie Me´tabolique et Clinique, Faculte´ de Pharmacie, Paris 5, France; 3 Laboratoire de Biochimie B, Hoˆpital de la Salpeˆtrie`re (AP-HP), Paris, France
Metformin (1,1-dimethylbiguanide) is an
antihyperglycae-mic drug used to normalize glucose concentrations in type 2
diabetes Furthermore, antioxidant benefits have been
reported in diabetic patients treated with metformin This
work was aimed at studying the scavenging capacity of this
drug against reactive oxygen species (ROS) likeÆOH and
O2Æ-free radicals ROS were produced by gamma
radio-lysis of water The irradiated solutions of metformin were
analyzed by UV/visible absorption spectrophotometry It
has been shown that hydroxyl free radicals react with
met-formin in a concentration-dependent way The maximum
scavenging activity was obtained for concentrations of metformin‡ 200 lmolÆL)1, under our experimental condi-tions An estimated value of 107LÆmol)1Æs)1 has been determined for the second order rate constant k(ÆOH + metformin) Superoxide free radicals and hydro-gen peroxide do not initiate any oxidation on metformin in our in vitro experiments
Keywords: metformin; hydroxyl radical; antioxidant; radio-lysis
Metformin (MTF) (1,1-dimethylbiguanide, see structure in
Fig 1) is one of the most used oral antihyperglycaemic
agents It normalizes plasma glucose concentration without
any stimulation of insulin production It has been
demon-strated that elevated glucose levels induce oxidative stress
in diabetes, i.e an imbalance between the production of
oxidant species, particularly radical species, and the
anti-oxidant defences [1] This might partly explain the elevated
risk factors for diabetic patients to develop cardiovascular
complications [2,3] This imbalance can be detected by
oxidative stress markers such as those of lipid peroxidation
and protein oxidation
Previous in vivo and in vitro studies have demonstrated
several antioxidant properties of metformin such as the
inhibition of the formation of advanced glycation end
products (AGEs) [4,5] that are thought to be responsible
for further diabetic complications, and the decrease in
the formation of methylglyoxal, one of the precursors of
AGEs [6]
Metformin improves liver antioxidant potential in rats
fed a high-fructose diet [7] It has been observed that the
administration of metformin in diabetic patients ameliorates
the antioxidant status This was shown by a decrease in lipid
peroxidation [monitored by determining the production of
thiobarbituric acid reactive substances (TBARS)] [8,9], a
decrease in lipid peroxidation markers in both LDL and HDL [10], an increase in reduced glutathione (GSH) blood concentration (usually low in diabetic patients) [11] and in antioxidant enzyme activities (such as catalase and CuZn superoxide dismutase) [11]
Furthermore, clinical benefits against vascular complica-tions have been obtained, and protective effects against diabetic complications have been observed with metformin monotherapy [12] Patients with type 2 diabetes receiving either metformin alone or accompanied by another treat-ment reduced by 40% the risk of developing further vascular complications compared to those receiving other treatments [12–14]
In order to improve the knowledge of MTF antioxidant mode of action, this work focused on the direct antioxidant properties of metformin in vitro against different oxygen-derived free radical species generated in aqueous solution
by gamma radiolysis Gamma radiolysis of water is a well known method that has many advantages, such as the homogeneous production of known quantities of free radicals (as superoxide anion O2Æ or hydroxyl radical
Æ
OH), as well as the possibility to selectively produce one specific radical to be studied at a time [15–18] Free radicals thus generated have been used to initiate one electron oxidation reaction(s) on metformin dissolved in water In a previous work, we have identified the oxida-tion end-products ofÆOH-induced oxidation of metformin [19] Four products have been characterized (Fig 1): methylbiguanide (MBG), a dimer of MTF (diMTF), a hydroperoxide of MTF (MTFOOH) and 4-amino-2-imino-1-methyl-1,2-dihydro-1,3,5-triazine (4,2,1-AIMT) The generation of these oxidation end-products was shown to be dependent on the experimental conditions: MTFOOH is only produced under aerated conditions, while diMTF occurs only in nonaerated solutions, saturated with nitrogen protoxide The two other products, MBG and 4,2,1-AIMT, have been found in both aerated
Correspondence to H Khouri or F Collin, Laboratoire de Chimie
Physique, CNRS UMR 8601 universite´s Paris 5, 45 rue des
Saints-Pe`res, 75270 Paris Cedex 06, France Fax: + 33 1 42862213,
Tel.: + 33 1 42862173, E-mail: hania.khouri@univ-paris5.fr or
fabrice.collin@univ-paris5.fr
Abbreviations: 4,2,1-AIMT,
4-amino-2-imino-1-methyl-1,2-dihydro-1,3,5-triazine; AGE, advanced glycation end products; GSH,
gluta-thione; MBG, methylbiguanide; MTF, metformin; ROS, reactive
oxygen species; TBARS, thiobarbituric acid reactive substance.
(Received 17 August 2004, accepted 14 October 2004)
Trang 2and nonaerated irradiated solutions of metformin
How-ever, only two radiation doses (50 and 300 Gy) and only
one concentration of metformin (200 lmolÆL)1) have been
studied [19] In order to specify theÆOH-induced oxidation
mechanism of metformin, we present in this paper the
effect of several radiation doses (from 52 to 627 Gy) on
different metformin concentrations (from 4 to 500 lmolÆ
L)1) UV/visible differential absorption spectra (where the
reference is non-irradiated solutions) have been recorded as
a function of the radiation dose
In addition, we have determined the initial slope of the
curves [Dabsorbancek¼ f(radiation dose)] which is
propor-tional to the radiolytic yield (initial slope¼ GÆDekÆl, were G
is the radiolytic yield, Dekthe differential molar extinction
coefficient and l the optical path-length Kinetic data have
been obtained from the dilution curves {i.e GÆDekÆl¼
f([metformin])}, allowing us to discuss the possible
compe-tition of hydroxyl radicals between metformin and
radio-lytically generated hydrogen peroxide
Materials and methods
Chemicals
All chemicals were purchased from Sigma (St Louis,
MO, USA) except when mentioned Metformin solutions
(4–500 lmolÆL)1) were prepared in 10 mmolÆL)1phosphate
buffer NaH2PO4Æ2H2O (purchased from Prolabo,
Manche-ster, UK) at pH 7 Ultra pure water (Maxima Ultra-pure
Water, ELGA, resistivity 18.2 MW) was used to prepare the
solutions Irradiations were carried out in test tubes that
have been previously cleaned with hot TFD4 detergent
(Franklab S.A., France), rinsed thoroughly with ultra pure
water, and then heated at 400C for 4 h to avoid any
pollution by remaining organic compounds
Gamma radiolysis Radiolysis corresponds to the chemical transformations of a solvent due to the absorption of ionizing radiations, which allows, within a few nanoseconds, the production of a homogeneous solution of free radicals In addition, this method allows selective generation of particular radicals from the solvent, and thus it is possible to study their action towards the dissolved entities Radiolytically generated free radicals are independent of the nature and of the concen-tration of the dissolved compound as long as its concentra-tion remains lower than or equal to 10 mmolÆL)1[20] Gamma radiolysis was carried out by using an IBL 637 irradiator (CIS Biointernational, Gif-sur-Yvette, France) of
137Cs source, whose activity was 222 TBq (6000 Ci) In our experiments the dose rate was 10.45 GyÆmin)1 The dosimetry was determined by Fricke’s method [21], namely radio-oxidation of 1 mmolÆL)1of iron(II) sulfate solution in 0.4 molÆL)1 sulfuric acid (under an aerated atmosphere) taking kmax (Fe3+)¼ 304 nm, e(304 nm)¼ 2204 LÆmol)1Æ
cm)1) at 25C, and a radiolytic yield of G(Fe3+)¼ 1.62 lmolÆJ)1 Different radiation doses, ranging from 52
to 627 Gy, were delivered to 5 mL of the solution depend-ing on the time of the exposure to the c-ray source: the longer the time of the exposure, the higher the radiation dose For each experimental set, 5 mL of non-irradiated solution was taken as a control
Water radiolysis by c-rays generates the free radical species e–aq, ÆOH, ÆH, and the molecular species H2 and
H2O2 Under aerated conditions (oxygen concentration is about 0.2 mmolÆL)1), hydroxyl and superoxide radicals (resulting from the scavenging of e–aqandÆH species) were simultaneously produced with radiolytic yields (G-values expressed in molÆJ)1) of 0.28 and 0.34 lmolÆJ)1, respectively
In order to select only hydroxyl radicals, radiolysis was
Fig 1 Structures of the protonated form of metformin (1,1-dimethylbiguanide) and of the oxidation products generated from ÆOH attacks
on metformin, according to [19].
Trang 3carried out in a nonaerated medium saturated with nitrogen
protoxide (N2O) N2O scavenges hydrated electrons and
converts them into hydroxyl radicals: as a result,ÆOH is
produced with a final G-value of 0.56 lmolÆJ)1, that is twice
as high as the G-value in an aerated medium [20] To
selectively obtain superoxide anions, sodium formate
(Pro-labo) was added to the solution at a concentration of
0.1 molÆL)1in order to convert all radicals (ÆOH,ÆH and
e–aq) into O2Æ–radicals with a final G-value of 0.62 lmolÆJ)1
[20]
Analysis
Detection of the oxidation products was achieved by
spectrophotometric measurements with an UV/visible
spec-trophotometer (Beckman DU 70) Samples were scanned
from 200 nm to 300 nm At pH 7, metformin, like all
biguanides, is present in its mono-protonated form (Fig 1)
Therefore, the possibility of resonance gives to biguanides a
characteristic absorption band at about 230 nm [22] Beer–
Lambert law was applicable on metformin within the
studied range of concentrations (4–500 lmolÆL)1), and the
molar extinction coefficient at 232 nm was found to be
12 300 ± 490 LÆmol)1Æcm)1)
Results
Action ofÆOH/O2Æ-free radicals
A 450 lmolÆL)1 solution of metformin (in 10 mmolÆL)1 sodium phosphate buffer, pH 7) was irradiated at doses ranging from 52 to 627 Gy, with a dose rate of 10.45 GyÆmin)1 The absolute absorption spectra (refer-ence¼ phosphate buffer, 10 mmolÆL)1, pH 7) are presen-ted in Fig 2A, as a function of the radiation dose The non-irradiated solution shows a main absorption band at
232 nm corresponding to the absorption of metformin [22]
As the radiation dose increased, the absorption at this wavelength decreased (illustrating the consumption of metformin) and two new bands were detected at 208 nm (intensified) and 258 nm, probably due to the generation of oxidation products Differential absorption spectra (refer-ence¼ non-irradiated metformin solution) allows us to better show the same phenomenon (Fig 2B) The arrows in
Fig 2 UV/visible absorption spectra of
metformin (450 lmolÆL)1) as a function of the
radiation dose (52–627 Gy) in aerated medium.
(A) Absolute absorption spectra
(refer-ence, phosphate buffer, 10 mmolÆL)1, pH 7).
(B) Differential absorption spectra
(refer-ence, non-irradiated metformin solution).
Optical path-length: l ¼ 0.2 cm, dose rate:
I ¼ 10.45 GyÆmin)1 The arrows indicate the
decrease (disappearance) and the increase
(appearance) in the absorbance values as a
function of the enhancing radiation dose.
Trang 4Fig 2 indicate the variations in the absorption intensity of
every characterized band as a function of the increasing
radiation dose Differential spectra highlight the previous
observation: at 232 nm, the differential absorbance is
decreasing (consumption of metformin), while it increases
at 208 and 258 nm (formation of oxidation products)
The differential absorbances at 232 nm and 258 nm have
been reported as a function of the radiation dose (Fig 3) At
232 nm, the differential absorbances decrease confirming
the consumption of metformin as a function of the radiation
dose (Fig 3A) At 208 nm (not shown) and 258 nm
(Fig 3B), the differential absorbance increases with the
radiation dose, indicating the simultaneous formation of
one or more oxidation products However, the 258-nm
absorption band has been selected for this study, as
non-irradiated metformin solution does not absorb at all at this
wavelength; 258 nm is a characteristic wavelength of
aromatic structure In fact, Collin et al [19] have identified
4,2,1-AIMT as one oxidation product of metformin
(Fig 1), which might be considered as the compound that
absorbs at this particular wavelength The other oxidation
products identified (methylbiguanide and metformin
peroxide) seem to share the same spectral characteristics
as metformin since their chemical structures are very close Similar analyses have been replicated for several metfor-min initial concentrations (4–500 lmolÆL)1) Initial slopes
of the curves [DAbsk¼ f(dose)], corresponding to GÆDekÆl (where G is the radiolytic yield, Dekthe molar extinction coefficient and l the optical path-length) at 232 and 258 nm, respectively, have been reported as a function of the initial concentration of metformin (Fig 4A,B) These dilution curves give the evolution of GÆDek (corrected for optical path-length l¼ 1 cm) with the initial concentration of metformin Both dilution curves at 232 nm and 258 nm exhibit the same profile, namely increasing values of GÆDek
at low metformin concentration (from 4 to 200 lmolÆL)1) followed by plateau values of GÆDek at high metformin concentration (200–500 lmolÆL)1) Hence, these dilution curves exhibit two key areas At the plateau, the value G.Dek
at 232 nm or 258 nm reaches a steady state, meaning that all free radicals (ÆOH/O2Æ) produced by water radiolysis have reacted with metformin independently of its initial concentration (200–500 lmolÆL)1) The second key area is characterized by GÆDekvalues that decrease as concentra-tions of metformin decrease (200–4 lmolÆL)1) This latter phenomenon might be due to a competition between
Fig 3 Differential absorbances as a function of the radiation dose.
[Metformin] ¼ 450 lmolÆL)1, [phosphate buffer] ¼ 10 mmolÆL)1,
pH 7, aerated medium Reference ¼ non-irradiated metformin
solu-tions (A) 232 nm, (B) 258 nm Optical path-length: l ¼ 0.2 cm, dose
rate: I ¼ 10.45 GyÆmin)1 Uncertainties (RSD) have been calculated
as being equal to 4%, at the 95% confidence level (2 r, n ¼ 3).
Fig 4 Dilution curves of metformin (GÆDe k as a function of the initial concentration of metformin), [phosphate buffer] = 10 mmolÆL)1, pH 7, aerated medium (A) 232 nm, (B) 258 nm – values are corrected for an optical path-length of 1 cm Uncertainties (RSD) have been calculated
as being equal to 4%, at the 95% confidence level (2 r, n ¼ 3).
Trang 5metformin and either phosphate buffer or hydrogen
peroxide radiolytically generated, towards the action of
Æ
OH/O2Æradicals
To verify this assumption, the effect of various phosphate
buffer concentrations (0.05, 0.5 and 5 mmolÆL)1) at pH 7
was studied in the presence of 50 lmolÆL)1of metformin
After irradiation, solutions were analyzed by absorption
spectrophotometry at 232 nm Any change was observed in
the consumption of metformin, indicating that phosphate
buffer did not compete at all with metformin towards
ÆOH/O2Æ-free radicals oxidation (data not shown)
When hydrogen peroxide was added to the metformin
solutions, it was been first verified that there was no
detectable effect of H2O2 as an initiator of metformin
oxidation in the absence of irradiation However, under
irradiation, there was a noticeable effect of the
concentra-tion of H2O2 (0.05, 0.5 and 5 mmolÆL)1) added to
metformin (50 lmolÆL)1) as shown in Fig 5 These
metformin–H2O2 solutions were irradiated from 52 to
520 Gy and analyzed at 232 nm by absorption
spectro-photometry The consumption of metformin was gradually
decreased by increasing the concentration of H2O2 from
0.05 to 5 mmolÆL)1 regardless of the radiation dose At
5 mmolÆL)1H2O2, it can be seen in Fig 5 that metformin
was not consumed as the radiation dose increased, i.e
metformin no longer reacted with the radiolytically
gener-ated free radicals
Action of O2Æradicals
In order to study the effect of superoxide radicals as
initiators of metformin oxidation, metformin solutions at
different concentrations ranging from 50 to 100 lmolÆL)1
were irradiated in the presence of sodium formate
(0.1 molÆL)1) Under these conditions (0.1 molÆL)1 of
sodium formate), O2Æradicals are the only radical species
produced by water radiolysis with a formation yield of
0.62 lmolÆJ)1, as described in Materials and methods
Metformin consumption was measured by absorption
spectrophotometry at 232 nm Under our experimental
conditions, no detected effect of O2Æ radicals on the
initiation of metformin oxidation has been observed This
phenomenon implies that superoxide radicals would mainly dismutate in such conditions (k¼ 6 · 105LÆmol)1Æs)1at
pH 7 [23])
Action ofÆOH radicals
In order to study the action ofÆOH radicals on the initiation
of metformin oxidation in nonaerated medium, different solutions of metformin (4–500 lmolÆL)1) were saturated with nitrogen protoxide (N2O) Under these conditions,
ÆOH radicals are the main radical species produced from water radiolysis with a radiolytic yield of 5.6· 10)7molÆJ)1 (see Materials and methods) The apparition of metformin oxidation product(s) was followed by absorption spectro-photometry at 258 nm
In Fig 6, for a metformin concentration of 500 lmolÆ
L)1, differential absorbances at 232 nm (Fig 6A) and
258 nm (Fig 6B) have been reported as a function of the radiation dose (from 52 to 627 Gy) The formation of oxidized product(s) exhibit the same profile as under aerated conditions (Fig 3A,B), confirming that ÆOH radicals are responsible for the initiation of metformin oxidation Several metformin concentrations were studied under the same experimental conditions (nonaerated and N2 O-satur-ated medium) The initial slope of the curves [DAbsk¼ f(radiation dose)] allowed us to determine the GÆDekvalues (corrected for an optical path-length l¼ 1 cm) Dilution curves {GÆDek¼ f([MTF])} were plotted on Fig 7 It can
be observed that GÆDek values increase with metformin initial concentration up to 200 lmolÆL)1and plateau values are reached for metformin initial concentrations superior to
200 lmolÆL)1 At 232 and 258 nm (Fig 7A,B, respectively),
it can be noted that GÆDekplateau values (13 ± 2· 10)4 and 6.5 ± 0.5 · 10)4, respectively) are twice as high as those obtained under aerated medium (6.5 ± 0.3· 10)4, Fig 4A and 3.2 ± 0.2· 10)4, Fig 4B) These observa-tions can be explained by the fact thatÆOH radicals have a formation yield under N2O atmosphere (0.56 lmolÆJ)1) twice as high as those ofÆOH radicals formed under aerated medium (0.28 lmolÆJ)1) However, the exact G-values of metformin oxidation products formation are not actually known
Fig 5 Differential absorbance at 232 nm as a
function of the radiation dose for metformin
solutions (50 lmolÆL)1) with or without H 2 O 2
(0.05, 0.5 and 5 mmolÆL)1) [phosphate
buf-fer] ¼ 10 mmolÆL)1, pH 7, aerated medium,
optical path-length: l ¼ 1 cm, dose rate:
I ¼ 10.45 GyÆmin)1 (Reference,
non-irradiated metformin solution) Uncertainties
(RSD) have been calculated as being equal to
4%, at the 95% confidence level (2 r, n ¼ 3).
Trang 6According to our experimental results, it seems that neither
superoxide radicals nor hydrogen peroxide react with
metformin, but that ÆOH radicals are the only species
initiating metformin oxidation Knowing that ÆOH-free
radicals can abstract one electron (charge transfer) or one H
atom, or add to a double bond, we may assume thatÆ
OH-free radicals can abstract an H atom from the CH3groups
and/or from the N–H between the N(CH3)2 and NH2
Hydroxyl radicals can also add to the C¼NH double bonds
(giving nitrogen-centred free radicals) It can be noted that,
because of the conjugation of the nitrogen electron pair [of
NH2, NH and N(CH3)2] with the C¼NH double bonds, the
charge transfer process ofÆOH abstracting an electron from
the nitrogen electron pair seems rather unfavourable
Scheme 1 summarizes the radical-induced oxidation of
metformin MTFÆ symbolizes the ÆOH-induced radical of
metformin Once metformin radicals are produced, they
might undergo various reactions leading to different
oxida-tion products [19] In the presence of oxygen, metformin
radical may react with oxygen molecules leading to peroxy
radicals which could be reduced (maybe by superoxide
radicals) to give metformin hydroperoxide (MTFOOH),
whereas in the absence of oxygen, metformin radicals would
tend to dimerize (diMTF) The occurrence of these latter compounds is oxygen dependent [19] Another two oxida-tion end-products have been observed by Collin et al., i.e MBG and 4,2,1-AIMT [19] whose mechanisms of forma-tion are unknown In order to specify the different steps
of the proposed mechanism, additional results would be necessary, mainly the quantification of the oxidation products
The observed progressive inhibition of metformin oxida-tion, in the presence of added hydrogen peroxide, would come from the reaction ofÆOH radicals with H2O2 An estimated value of the second order rate constant of k(ÆOH + MTF) could be determined, by comparing the initial rates ofÆOH radical with hydrogen peroxide [relation (1)] or with metformin [relation (2)]
vðÆOHþ H2O2Þ ¼ kðÆOHþ H2O2Þ ½ÆOH ½H2O20
ð1Þ
vðÆOHþ MTFÞ ¼ kðÆOHþ MTFÞ ½ÆOH ½MTF0
ð2Þ
It is well known that the rate constant ofÆOH radicals with
HO is close to 107LÆmol)1Æs)1 [24] For the highest
Fig 6 Differential absorbances as a function of the radiation dose.
[Metformin] ¼ 500 lmolÆL)1, [phosphate buffer] ¼ 10 mmolÆL)1,
pH 7, N 2 O-saturated solutions Reference, non-irradiated metformin
solution (A) 232 nm, (B) 258 nm Optical path-length: l ¼ 0.2 cm,
dose rate: I ¼ 10.45 GyÆmin)1 Uncertainties (RSD) have been
cal-culated as being equal to 17% (A) and 8% (B), at the 95% confidence
level (2 r, n ¼ 3).
Fig 7 Dilution curves of metformin (GÆDe k as a function of the initial concentration of metformin), [phosphate buffer] = 10 mmolÆL)1, pH 7,
N 2 O-saturated solutions (A) 232 nm, (B) 258 nm – values are correc-ted for an optical path-length of 1 cm Uncertainties (RSD) have been calculated as being equal to 17% (A) and 8% (B), at the 95% con-fidence level (2 r, n ¼ 3).
Trang 7hydrogen peroxide concentration (5 mmolÆL)1), a
quasi-total inhibition of metformin (50 lmolÆL)1) oxidation
(Fig 5) has been observed, involving a reaction rate of it
ÆOH radicals with H2O2at least 10 times higher than those
ofÆOH radicals with metformin [relation 3]
vðÆOHþ H2O2Þ > 10 vðÆOHþ MTFÞ ð3Þ
From relations 1–3, it can be deduced that the second order
rate constant [k(ÆOH + metformin)] is lower than 108
LÆmol)1Æs)1) Therefore, this rate constant is likely of the
same order of magnitude ( 107LÆmol)1Æs)1) than that of
hydrogen peroxide withÆOH radicals It is worth
mention-ing that this value is rather weak for a reaction involvmention-ing
hydroxyl radicals whose k-values are usually diffusion
controlled, and approximately equal to 109)1010
LÆmol)1Æs)1[24] Accordingly, metformin exhibits a
relat-ively weak radical scavenging capacity againstÆOH radicals
in vitro
In the radiolysis solutions, H2O2 could come from
different pathways: (i) fromÆOH radical recombination (in
the spurs) giving H2O2with a G-value of 0.7· 10)7molÆJ)1
(this production being independent of the presence of
metformin); (ii) from O2Æ (in equilibrium with HOÆ2)
radical dismutation (in homogeneous phase) leading to
H2O2 with a G-value of (3.4· 10)7)/2 molÆJ)1, i.e
(Ge–aq+ GH)/2, in the case where O2Æ– radicals do not
react neither with metformin nor with the metformin
radical, and (iii) from O2Æradical oxidation of metformin
radical giving H2O2with a G-value of 3.4· 10)7molÆJ)1
H2O2 concentration in the radiolysis solution is
propor-tional to G(H2O2) and to the radiation dose ([H2O2]¼
G(H2O2)· dose) For example, at 50 Gy (which is a dose
where G-value can be determined), the following H2O2
concentration can be calculated: 3.5 lmolÆL)1 [pathway
(i)], 12 lmolÆL)1 [pathway (i) + (ii)] or 20.5 lmolÆL)1
[pathway (i) + (iii)] Such H2O2concentrations are similar
to the lowest concentrations of metformin (from 4 to
50 lmolÆL)1) Hence, the hypothesis of a competition of
ÆOH radicals between H2O2 and metformin is plausible
providing that the rate constants [k(ÆOH + H2O2) and
k(ÆOH + metformin)] be of the same order of magnitude
[i.e. 107LÆmol)1Æs)1] In agreement with these
consider-ations, it can be proposed that the decrease of GÆDekvalues
at low metformin concentration (4–200 lmolÆL)1) (Figs 4
and 7) would come from the competition ofÆOH radicals
between metformin and radiolytically generated hydrogen peroxide
Conclusion
We have investigated the antioxidant properties of metfor-min againstÆOH and O2Æ-free radicals produced by water gamma radiolysis Metformin aqueous solutions (from 4 to
500 lmolÆL)1) were analyzed by UV/visible absorption spectroscopy We have shown that metformin does not scavenge O2Æ radicals, but is able to react with ÆOH radicals However, under our experimental conditions, the
Æ
OH-induced oxidation of metformin depended on its initial concentration because of the possible competitive reaction ofÆOH radicals with radiolytically generated H2O2 Moreover, we have determined an estimated value of
107LÆmol)1Æs)1) for the second order rate constant of the reaction ofÆOH radicals with metformin
Our results obtained with an in vitro model allow assuming that metformin, at a molecular level, is not a very good scavenger of reactive oxygen species Consequently, it seems that metformin would certainly exert its in vivo antioxidant activity by different pathways other than the simple free radical scavenging action, such as increasing the antioxidant enzyme activities [8,11,25], decreasing the markers of lipid peroxidation [10,11] and inhibiting the formation of AGEs [4,5]
Acknowledgements
Authors show gratitude towards Dr N Wiernsperger (LIPHA S.A., Lyon, France) for his support to this work As well our thanks to
Dr Averbeck of the Institut Curie – Paris for c irradiation facilities.
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