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Food Safety and Toxicology
TOXICITY OF NATURAL DEEP EUTECTIC SOLVENT (NaDES) BETAINE:GLYCEROL IN RATS Melha Benlebna, Mariana RUESGAS-RAMON, Beatrice Bonafos, Gilles Fouret, Françcois Casas, Charles Coudray, Erwann DURAND, maria-cruz Figueroa, and Christine Feillet-Coudray J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01746 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018
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Journal of Agricultural and Food Chemistry
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TOXICITY
OF
NATURAL
2
BETAINE:GLYCEROL IN RATS
DEEP
EUTECTIC
SOLVENT
(NaDES)
Melha BENLEBNA£§, Mariana RUESGAS-RAMON$§, Beatrice BONAFOS£, Gilles FOURET£, François CASAS£, Charles COUDRAY£, Erwann DURAND¤,ll, Maria Cruz FIGUEROA-ESPINOZA$*, Christine FEILLET-COUDRAY£*
£
DMEM, INRA, Univ Montpellier, Montpellier, France
$
IATE, Montpellier SupAgro, INRA, CIRAD, Univ Montpellier, Montpellier, France
¤
CIRAD, UMR IATE, Montpellier F-34398, France
ll
IATE, Univ Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France
Running title: NaDES toxicity in rats
*Corresponding authors: Dr. Christine FEILLET-COUDRAY, DMEM, INRA, Univ Montpellier, Montpellier, France Tel: 33 4 99 61 26 87, Fax: 33 4 67 54 56 94, Email:
[email protected] Dr. Maria Cruz FIGUEROA-ESPINOZA, IATE, Montpellier SupAgro, INRA, CIRAD, Univ Montpellier, Montpellier, France, Email:
[email protected] §both authors contributed equally to this work
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ABSTRACT
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The natural deep eutectic solvents (NaDES) are new natural solvents in green chemistry that
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in some cases have been shown to allow better extraction of plant bioactive molecules
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compared to conventional solvents and higher phenolic compounds absorption in rodents.
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However, there is a serious lack of information regarding their in vivo safety. The purpose of
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this study was to verify the safety of a NaDES (glycerol:betaine (mole ratio 2:1) + 10 % (v/v)
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of water) extract from green coffee beans, rich in polyphenols.
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Twelve 6-weeks-old male Wistar rats were randomized into two groups of 6 animals each and
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twice daily gavaged for 14 days either with 3 ml water or with 3 ml phenolic NaDES extract.
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Oral administration of phenolic NaDES extract induced mortality in 2 rats. In addition, it
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induced excessive water consumption, reduced dietary intake and weight loss, hepatomegaly,
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plasma oxidative stress associated with high blood lipid levels. In conclusion, this work
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demonstrated the toxicity of oral administration of the selected NaDES, under a short-term
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condition. This occurs despite the fact that this NaDES extract contains polyphenols, whose
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beneficial effects have been shown. Therefore, complementary work is needed to find the best
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dose and formulation of NaDES that are safe for the environment, animals and ultimately for
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humans.
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KEYWORDS
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NaDES, toxicity, phenolic compounds, glycerol, betaine, chlorogenic acids
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
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When molecules abundantly present in nature (e.g., sugars, polyols, amino acids, urea,
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carboxylic acids, vitamins), are combined in a well-defined mole ratio, these compounds may
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form a liquid phase which has been defined as Natural Deep Eutectic Solvents (NaDES)
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The presence of an extensive hydrogen bond network between each component and, to a
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lesser extent, van der Waals and electrostatic interactions, are responsible of these liquid
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phase formation 3. Since they are formed with molecules coming from the nature, most of the
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NaDES are biodegradables, cheap, and present a lower toxicity in comparison with
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conventional chemical liquids (organic solvents and ionic liquids) 4. Many of the NaDES are
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formed with choline chloride (ChCl) paired with urea, ethylene glycol, or glycerol 3, but
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organic acid, sugars and other molecules can also be used 1. Besides their potential role in
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living organisms (protection from harsh environmental conditions or biotransformation of
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poor-water soluble substances) the physicochemical characteristics of NaDES (liquids over a
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very large temperature range, non-volatile with excellent thermal stability) opened interesting
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perspectives for bioactive molecules extraction from plants, particularly phenolic compounds
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5
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Concomitantly, it has been demonstrated that an oral administration of phenolic compounds
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dissolved in NaDES lead to higher phenolic compounds absorption in rodents 6. Moreover,
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formulation of phenolic compounds with NaDES was shown to improve the in vitro
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antioxidant activity of phenolic molecules in fibroblast cells 7. Although the NaDES have an
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important potential for application in the food or pharmaceutical industry (high economic and
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environmental interest, improve the bioactive molecules extraction, promote the absorption of
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molecules), there is still a lack of information regarding their in vivo safety.
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Chlorogenic acids (CGA) refer to a related polyphenol family of esters, including
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hydroxycinnamic acids (caffeic acid, ferulic acid and p-coumaric acid) with quinic acid. CGA
1,2
.
.
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are natural antioxidants present in coffee beans. The 5-O-caffeoyl quinic acid (5-CQA), often
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called chlorogenic acid, is the commonest individual CGA
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interesting biological activities 10.
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While a few studies report the effect of NaDES administration on mice and rats 11,12 no study
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reports the effect of a polyphenolic NaDES extract supplementation.
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The purpose of this study was to prepare a NaDES extract rich in CGAs (extracted from green
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Arabica coffee beans, GCB) and to evaluate for the first time the effects of this formulation by
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oral administration in rats, in order to evaluate if NaDES can be used for oral bioactive
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molecules or therapeutic agents delivery.
8,9
. CGA possess several
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MATERIALS AND METHODS
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Reagents and plant material
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Chlorogenic acid (5-CQA), lactic acid, glycerol, caffeine reagent plus, 1,4 butanediol, choline
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chloride (ChCl), betaine > 98% (perchloric acid titration) , acetic acid ≥ 99.9%, methanol
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CHROMASOLV® ≥ 99.9%, sodium carbonate anhydride, and water CHROMASOLV® Plus
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grade HPLC were purchased from Sigma-Aldrich (Saint Louis, USA). Dry GCB (Coffea
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arabica) came from the Huatusco municipality (Veracruz, Mexico), and were harvested in
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2016. GCB were milled in a grinding mill IKA-WERKE M20 (Wilmington, USA), then it
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was sieved in particle size to 630 µm, and then stored at -20 °C before experiments
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NaDES preparation and phenolic compound extraction
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The NaDES was prepared using the heat stirring extraction (HSE) method. Constituents,
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according the mole ratio described in table 1, were placed in closed glass flasks, heated
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(60°C) and stirred (250 rpm) in an orbital shaker incubator (STUART Scientific S150,
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Legallais, Montferrier-sur-Lez, France) until a colorless liquid was formed (approximatively 2 4 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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h). Then 10 % (v/v) of water was added and the hydrated NaDES was heated and stirred
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(60°C, 250 rpm) for another 10 min to get a perfect phase mixing.
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An extract rich in CGA was obtained from GCB using NaDES as solvent. The extraction was
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performed under the following conditions: 60°C, 250 rpm, 3 h, in sealed flasks protected from
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light using 0.4 g of ground GCB/mL of NaDES. For the control, the NaDES was replaced by
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ethanol in water (EtOH 70 %, v/v). Finally, the total phenolic compounds concentration in
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both extracts (NaDES and EtOH 70 %) was quantified by HPLC and expressed as mg of
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chlorogenic acid (5-CQA) equivalents (CAE) per mL of NaDES, or per mL of EtOH 70 %.
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Mono and di-esters of CGA are the main phenolic compounds in the GCB extract. The
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NaDES4, obtained after GCB extraction, was diluted to reach a final concentration in phenolic
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compound of ~10 mg CAE/mL prior rats feeding. Thus, a dilution (1:3 with free-NaDES) of
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the raw NaDES4 extract was performed.
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HPLC analysis
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For phenolic compounds analysis in the NaDES and EtOH 70% extracts, each extract was
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diluted 400-fold in water in duplicate. The solution was then filtered through a 0.45 µm
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cellulose filter (Minisart Legallais, Montferrier-sur-Lez, France), and analyzed by HPLC. The
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analysis was performed with a Shimadzu LC-20AD equipped with a DAD SPO-M20A,
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column oven CTO-10ASVP (Shimadzu, Noisel, France) and a Kinetex 5 µm C-18 column
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(100 Å, 4.6×250 mm, Phenomenex, Le Pecq, France). The elution solvents were (A) water
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(0.1 % acetic acid, v/v) and (B) methanol (0.1 % acetic acid, v/v). The gradient elution
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program was performed as follows (time (min), solvent B (%)): (3, 5), (13, 5), (23, 100), (33,
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100), (34, 0), (40, 0). The flow rate, injection volume, and detection wavelength were 1.0
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mL/min, 20 µL, respectively. The wavelengths used were 327 nm and 280 nm, to detect the
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CGA and the caffeine, respectively. The results were expressed in g of CAE or caffeine
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equivalents per 100 g of dry weight GCB (g CAE/ 100 g d.w.). In both cases, a calibration
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curve of the standard molecules (namely 5-CQA and caffeine) was used to assess the
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quantification in the extracts.
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Animals and diets
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Twelve 6-weeks old male Wistar rats (210 ± 6 g/rat) (Charles River, L’Arbresle, France) were
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housed (2 per cage) under conditions of constant temperature (20-22°C), humidity (45-50 %)
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and a standard dark cycle (20.00-08.00 hours). The rats were randomized into two groups of 6
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animals each and fed by gavage for 14 days at 9h00 and at 17h00 either with 3 mL of water or
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with 3 mL of NaDES4 extract (containing 28.01 mg of phenolic compound expressed in CAE
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and 5.89 mg of caffeine). Rats were fed with a standard pellet diet (UAR A2) and given free
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access to food and tap water. Rat body weight, food consumption, and drinking were
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measured every two days. Our institution guidelines for the care and use of laboratory animals
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were observed and all experimental procedures were approved by the national ethical
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committee in France (Reference 2016080911004286).
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Sampling
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Nine hours-fasted rats (23h00–08h00) were anesthetized with pentobarbital (40 mg/kg body
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weight) (Ceva Santé Animale, Libourne, France) blood was drawn from the abdominal artery
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with slightly heparinized syringes, to prevent possible coagulation during the blood sampling,
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and distributed into dry and heparinized tubes. Blood tubes were centrifuged at 1000 g for 10
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min at 4°C, plasma or serum were collected and stored at –80°C until analysis. Beforehand,
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for GSH analysis, 200 µL aliquots of heparinized blood sample were mixed with 1800 µL of
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ice-cold 6 % (w/v) meta-phosphoric acid, incubated for 30 min on ice, centrifuged for 10 min
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at 1000xg at 4°C and supernatants were collected and stored until analysis. The liver was
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quickly removed, rinsed, weighted, and frozen in liquid nitrogen, then kept at -80°C.
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Gastrocnemius and soleus muscles, heart, kidney, spleen, and adipose tissue were also
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removed, rinsed and weighted.
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Plasma/serum routine biochemical analyses
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The following analyses were performed using commercial kits: plasma levels of glucose
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(Sobioda, MontBonnot Saint-Martin, France), uric acid (Biolabo, Maizy, France), urea
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(Elitech, Puteaux, France); serum levels of creatinine, total cholesterol and triglycerides
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(Sobioda, MontBonnot Saint-Martin, France), free fatty acids (Wako, Oxoid, Dardilly,
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France), glycerol (Biosentec, Auzeville-Tolosane, France), aspartate aminotransferase
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(ASAT), and alanine aminotransferase (ALAT) activity (Elitech, Puteaux, France). Plasma
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and liver supernatant protein levels were measured by the Bradford technique 13.
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Blood and liver oxidative stress measurement
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Lipid peroxidation levels represented by the thiobarbituric acid-reactive substances (TBARS)
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were measured in plasma and in liver homogenates according to the method of Sunderman et
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al.
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groups (-SH)
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advanced glycation end products (AGE) were measured in plasma by fluorescence according
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to the method of Munch et al. 17.
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Liver homogenates were prepared on ice in a ratio of 1 g of wet tissue for 9 mL phosphate
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buffer (50 mM, pH 7) using a Polytron homogenizer (VWR, Fontenay sous Bois cedex,
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France). Homogenates were divided into three parts: part 1 was used directly for the
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assessment of lipid oxidation using the TBARS assay; part 2 was mixed with identical volume
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of ice-cold 12 % (w/v) meta-phosphoric acid, incubated for 30 min on ice, centrifuged for 10
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. Protein oxidation was assessed in plasma and liver by measurement of sulfhydryl 15
and advanced oxidation protein product (AOPP)
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. Glucoxidation or
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min at 1000xg at 4°C, then supernatants were collected and stored until analysis of total GSH;
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part 3 was centrifuged at 1000xg for 10 min at 4°C, supernatant was collected and stored at -
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80°C until analysis of other oxidative and anti-oxidative stress markers.
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Lipids content in liver
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The liver samples were homogenized in NaCl (9 g/L) and Triton X-100 (0.1 %), using an
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Ultra Turax homogenizer. Hepatic levels of triglycerides, free fatty acids (Wako, Oxoid,
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Dardilly, France) and total cholesterol (Sobioda, MontBonnot Saint-Martin, France) were
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quantified directly in the liver homogenate using enzymatic colorimetric methods.
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Glycogen content
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The liver glycogen content was determined from a slice of the main lobe of the liver,
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according to Burcelin et al 18. Briefly, about 50 mg of the liver were dissolved in NaOH, 1 M
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(ratio 1/6) at 55 °C for 1 h, neutralized with HCl (1 M) and spun down. A sample of the
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supernatant was incubated in the presence of amyloglucosidase (Sigma) for 2 h at 37 °C in a
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shaking incubator (750 rpm). After centrifugation, the glucose present in the supernatant was
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assayed by a glucose oxidase method (Sobioda, MontBonnot Saint-Martin, France).
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Statistical analysis
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Results were expressed as means ± SD (standard deviation), with n=5. Statistical analyses
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were based on student t test. The limit of statistical significance was set at p
