Libia Alejandra

Sonia Medina

Camille Oger

Jean-Marie Galano

Thierry Durand

Roberto Cejuela

José Miguel Martínez-Sanz

Federico Ferreres

Ángel Gil-Izquierdo

Alejandra Libia

García-Flores

Lipidomic approach in young adult triathletes: effect of supplementation with a polyphenols-rich juice on neuroprostane and F2 -dihomo-isoprostane markers

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Exercise-induced reactive oxygen species (ROS) production could be an important signaling pathway to induce biological adaptations to training. [START_REF] Peternelj | Antioxidant supplementation during exercise training: beneficial or detrimental?[END_REF][START_REF] Mankowski | Dietary Antioxidants as Modifiers of Physiologic Adaptations to Exercise[END_REF] In addition, regarding the effect of exercise on the brain, regular and moderate aerobic exercise appears to promote the antioxidant capacity, but anaerobic or high-intensity exercise, aerobic-exhausted exercise, or the combination of both types of training could worsen the antioxidant response. [START_REF] Camiletti-Moiron | Does exercise reduce brain oxidative stress? A systematic review[END_REF] The literature shows that polyphenols (abundant in plants and plant-derived foods such as fruits and vegetables) can provide protection against exercise-induced muscle damage and oxidative stress (OS) thanks to their antioxidant and antiinflammatory properties. [START_REF] Malaguti | Polyphenols in exercise performance and prevention of exercise-induced muscle damage[END_REF][START_REF] Del Rio | Dietary ( poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases[END_REF] There has also been growing reco-gnition of the possible beneficial influence of polyphenols on the development and health of brain structure and function, [START_REF] Gomez-Pinilla | Natural mood foods: the actions of polyphenols against psychiatric and cognitive disorders[END_REF][START_REF] Schaffer | Do polyphenols enter the brain and does it matter? Some theoretical and practical considerations[END_REF] as well as their positive effects, which involve a decrease in oxidative/inflammation damage in the nervous system. [START_REF] Gomez-Pinilla | The combined effects of exercise and foods in preventing neurological and cognitive disorders[END_REF][START_REF] Myburgh | Polyphenol supplementation: benefits for exercise performance or oxidative stress?[END_REF] The use of antioxidant supplementation is common in athletes, primarily to prevent overproduction of ROS and their deleterious impact on cells and tissues through lipid and protein protection. There is evidence that beverages, such as fruit juice, containing a diversity of polyphenol compounds can have a favorable impact on human health. [START_REF] Del Rio | Dietary ( poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases[END_REF][START_REF] Myburgh | Polyphenol supplementation: benefits for exercise performance or oxidative stress?[END_REF] It has been mentioned that fruit juices can provide a blend of polyphenols in a single serving of the drink that cannot be obtained from a portion of fruit. [START_REF] Gil-Izquierdo | Influence of Industrial Processing on Orange Juice Flavanone Solubility and Transformation to Chalcones under Gastrointestinal Conditions[END_REF][START_REF] Manach | Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice[END_REF] For example, the combination of Aronia (Aronia melanocarpa) with citrus juices has provided synergistic effects of flavanones plus anthocyanins, among other bioactive compounds. [START_REF] Habauzit | Differential effects of two citrus flavanones on bone quality in senescent male rats in relation to their bioavailability and metabolism[END_REF] Black chokeberry (Aronia melanocarpa) contains high amounts of polyphenol compounds that are bioavailable and show health-promoting properties for humans by different mechanisms. [START_REF] Medina | Physical activity increases the bioavailability of flavanones after dietary aronia-citrus juice intake in triathletes[END_REF] Among them, the intake of this berry may be beneficial against OS, in both humans and animals. [START_REF] Denev | Bioavailability and antioxidant activity of black chokeberry (Aronia melanocarpa) polyphenols: in vitro and in vivo evidences and possible mechanisms of action: A review[END_REF] In addition, citrus flavonoids have antioxidant and antiinflammatory bioactivities. Previous in vitro and in vivo studies showed that these flavonoids exert neuroprotection at high a Dept. of Food Science and Technology. CEBAS-CSIC. Campus de Espinardo 25, 30100 Espinardo, Murcia, Spain. E-mail: smescudero@cebas.csic.es, angelgil@cebas.csic.es; Fax: +34 968396213; Tel: +34 968396363, 6253 and low doses. [START_REF] Hwang | Neuroprotective effects of citrus flavonoids[END_REF] Supplementation with the polyphenols-rich juice used in this study-aronia-citrus juice (ACJ)-may provide health protection to triathletes (200 mL per day), according to previously published results. [START_REF] Medina | Physical activity increases the bioavailability of flavanones after dietary aronia-citrus juice intake in triathletes[END_REF] In fact, the bioavailability of flavanones (eriodictyol and hesperetin) in the triathletes was augmented after the ACJ intake (during 2 weeks) by the physical exercise compared to sedentary volunteers. Besides, the intake of this ACJ, in conjunction with adequate training, was able to influence the plasmatic and urinary values of OS biomarkers (15-F 2t -IsoP; also termed 8-iso-prostaglandin-F 2α urinary biomarker, as well as the biomarkers guanosine-3′,5′-cyclic monophosphate and 8-hydroxyguanine analyzed in plasma samples). [START_REF] Garcia-Flores | DNA catabolites in triathletes: effects of supplementation with an aronia-citrus juice ( polyphenols-rich juice)[END_REF] Llorach et al. recently published a metabolomic study in healthy volunteers after regular ACJ intake (250 mL per day) for 16 weeks and found an association with markers of intake of the components of juice: proline betaine, ferulic acid, and two unknown mercapturate derivatives. [START_REF] Llorach | Discovery of human urinary biomarkers of aronia-citrus juice intake by HPLC-q-TOF-based metabolomic approach[END_REF] Regarding lipid oxidation markers, F 2 -dihomo-isoprostanes (F 2 -dihomo-IsoPs) and F 4 -neuroprostanes (F 4 -NeuroPs) are formed by a free radical, non-enzymatic mechanism from adrenic acid (AdA, C22:4 n-6) [START_REF] Vanrollins | F2-dihomo-isoprostanes arise from free radical attack on adrenic acid[END_REF][START_REF] Galano | Isoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF] and docosahexaenoic acid (DHA, C22:6 n-3), [START_REF] Roberts 2nd | Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid[END_REF] respectively. F 4 -NeuroPs originate from DHA, an essential constituent of nervous tissue, highly enriched in neurons and highly prone to oxidation. [START_REF] Nourooz-Zadeh | F4-isoprostanes as specific marker of docosahexaenoic acid peroxidation in Alzheimer's disease[END_REF] F 2dihomo-IsoPs are specific markers generated from AdA and are potential markers of free radical damage to myelin in the human brain. [START_REF] Vanrollins | F2-dihomo-isoprostanes arise from free radical attack on adrenic acid[END_REF] Currently, researchers tend to focus more on the assessment of these biomarkers in disease conditions and their increase in different biological fluids. [START_REF] Galano | Isoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF][START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF][START_REF] De Felice | F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome[END_REF][START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF] Besides, no attention has been paid to the investigation of these central nervous system (CNS) degradation markers and their relationship with physical exercise, to the ability of nutrition with functional foods enriched in polyphenols to attenuate to this type of OS generation, or to the elucidation of potential pathways of the OS biomarkers with exercise adaptation and/or the effect of functional foods on the CNS.

Based on the foregoing statements, the aim of this work was to evaluate urinary biomarkers of OS associated with the CNS, namely four F 4 -NeuroPs and four F 2 -dihomo-IsoPs, and whether supplementation of the diet with one serving (200 mL per day) of ACJ for 45 days could produce changes in these OS biomarkers. In this study, the identification was carried out by UHPLC-QqQ-MS/MS thanks to its superior advantages to techniques used in other studies to distinguish the regioisomers and diastereomers of the metabolites in samples. [START_REF] Roberts 2nd | Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid[END_REF] This is the first study to investigate these CNS degradation markers in relation to physical exercise, as well as the influence of nutrition with functional foods enriched in polyphenols.

Materials and methods

Physical characteristics of participants

The anthropometric measurements were performed according to the International Society for the Advancement of Kinanthropometry (ISAK: http://www.isakonline.com), in all cases by the same internationally certified anthropometrist (level 2 ISAK) to minimize the technical error of the measurement. The body composition was determined by GREC Kineanthropometric consensus, using a model that consists of: total fat by Withers' formula, [START_REF] Withers | Relative body fat and anthropometric prediction of body density of male athletes[END_REF] lean weight by a previous procedure, [START_REF] Lee | Total-body skeletal muscle mass: development and cross-validation of anthropometric prediction models[END_REF] and residual mass by the difference in weight (Table 1).

Dietary intake of participants

The diet was kept constant to avoid any interference with urinary analysis (Table 2). The calculation of the dietary parameters and caloric intake was accurately designed and overviewed during the experimental intervention by nutritionists and specific software was used for the calculation. The data were calculated using the software available on the website (http://www.easydiet.es), with the additional assistance of the Spanish and USDA databases (http://www.bedca.net/ and http://www.nal.usda.gov/fnic/foodcomp/search/). The dietary assessment and planning for our volunteers were estimated based on their energy needs, [START_REF]A Report of the Panel on Macronutrients; Subcommittees on Upper Reference Levels of Nutrients and Interpretation and Uses of Dietary Reference Intakes; Standing Committee on the Scientific Evaluation of Dietary Reference Intakes; Food and Nutrition Board[END_REF] on their energy expenditure, [START_REF] Ainsworth | Compendium of physical activities: an update of activity codes and MET intensities[END_REF] and on different recommendations for triathletes [START_REF] Jeukendrup | Nutritional considerations in triathlon[END_REF] as well as sports men/women. [START_REF] Martínez-Sanz | Necesidades energéticas, hídricas y nutricionales en el deporte[END_REF] The dietary fulfillment was individually conducted for each elite triathlete by the University of Alicante nutritionists (Chief responsible for the dietary control: Dr José Miguel Martínez-Sanz). Dietary information was obtained via 24 h recall, [START_REF] Salvador | Entrevista dietética: herramientas útiles para la recogida de datos[END_REF] in which they described in detail all foods and drinks consumed 24 hours prior to each provision of urine.

2.2.1 Aronia-citrus juice and placebo beverage. The juice composition was based on a mixture of citrus juice (95%) with 5% Aronia melanocarpa juice, based on a drink model developed before. [START_REF] Gonzalez-Molina | Aronia-enriched lemon juice: a new highly antioxidant beverage[END_REF] The composition was developed in the industry at pilot scale with organoleptically acceptable criteria, to mimic the flavonoids composition of the original beverage. The supplementation with this natural fruit juice has been used in other studies, as aforesaid in the introduction, the daily dose being around 200 mL (ref. 13 and 17) in healthy subjects. The nutrient content and caloric supply of the ACJ are summarized in Table 3, as well as the contents of fruit flavanones, flavones, and anthocyanins. The results are expressed as milligrams per serving of juice. One serving of juice corresponds to 240 mL according to the US FDA (US Food and Drug Administration), but in this study it was adjusted to 200 mL to adapt to the caloric requirements of the triathletes (represented only 2.6% of the calories of the diet).

The placebo beverage was a mixture of water, authorized red dye, flavoring, and sweetener, with sensory characteristics very similar to those described for the ACJ. This placebo drink has been used in two other previous studies. [START_REF] Llorach | Discovery of human urinary biomarkers of aronia-citrus juice intake by HPLC-q-TOF-based metabolomic approach[END_REF][START_REF] Bernabé | Effects of a citrus based juice on biomarkers of oxidative stress in metabolic syndrome patients[END_REF]

Training load

Triathlon is a sport where three exercises (swimming, cycling, and running) are performed in a continuous way, these three are being the most common exercises among human forms of locomotion. [START_REF] Cejuela Anta | Training load quantification in triathlon[END_REF] The quantification of training programs was addressed to evaluate their effects on physiological adaptation and subsequent performance. [START_REF] Borresen | The quantification of training load, the training response and the effect on performance[END_REF] The training load quantification was performed using the objective load scale (ECOs); to learn more about this scale, refer to the papers below. [START_REF] Cejuela Anta | Training load quantification in triathlon[END_REF][START_REF] Saugy | Comparison of "Live High-Train Low" in normobaric versus hypobaric hypoxia[END_REF] The training loads developed by triathletes in the present trial were similar to those found in other studies. [START_REF] Medina | Physical activity increases the bioavailability of flavanones after dietary aronia-citrus juice intake in triathletes[END_REF][START_REF] Lucia | Physiological characteristics of the best Eritrean runners-exceptional running economy[END_REF][START_REF] Medina | Assessment of oxidative stress markers and prostaglandins after chronic training of triathletes[END_REF] The values of daily and weekly trainings have been summarized to assess the ECOs of each volunteer, depending on their physical characteristics and the intensity of the training program (the ECOs data presented are the average of the individual ECOs of the triathletes; Fig. 1). Briefly, and from a general point of view, the intensity was exponentially-not linearly-considered, with the aim of leveling off the total training stress for a given performance level. The volume was quantified by time and this allowed better comparison of different performance levels and terrain conditions ( pavement, uneven laps). [START_REF] Cejuela Anta | Training load quantification in triathlon[END_REF]

Study design

Sixteen Caucasian triathletes (6 training women and 10 training men), aged 19-21 years from the University of Alicante (Spain) agreed to participate in the project. The recruitment started on 28 th -29 th October 2010 and was completed on 24 th -25 th March 2011. The volunteers were non-smokers, had stable food habits, and did not receive any medication (including the specific absence of the acute administration of anti-inflammatory drugs) during the experimental procedure. The study was approved by the Bioethics Committee of the University Hospital of Murcia, in accordance with the principles of the Declaration of Helsinki, and all participants signed written informed consent.

This was a randomized, double-blind, placebo-controlled, and crossover study (Fig. 1). Before the supplementation with ACJ, two urine-sampling periods (as controls) were used: the first was a control baseline (C-B) with minimal training loads (ECOs) and the second control (control-training: C-T) started with an increase in ECOs; both lasted 15 days. Both groups consumed ACJ or placebo for 45 days (200 mL of beverage). Ten days were utilized as the washout period without drink intake, while maintaining the training and the control diet. Subsequently, the intervention protocol was repeated, swapping the two groups according to the corresponding drink intake and maintaining their ECOs. The drink intake was 15 minutes after their training finished, to improve the bioavailability of ACJ. [START_REF] Medina | Physical activity increases the bioavailability of flavanones after dietary aronia-citrus juice intake in triathletes[END_REF] After the crossover period, the control post-treatment (CP-T) was started for the last 15 days of study without supplementation and with decreased ECOs (active recovery phase) with the objective of analyzing the post-training adaptations. Twenty-four-hour urine samples were collected at the end of each period (as shown in Fig. 1). To learn more about the study design, refer to the previously published paper. [START_REF] Garcia-Flores | DNA catabolites in triathletes: effects of supplementation with an aronia-citrus juice ( polyphenols-rich juice)[END_REF]

Sample collection and preparation

Twenty-four-hour urine samples were collected on the last day of each stage. They were collected in sterile and clear polystyrene pots with screw caps and were protected from light. One milliliter of the urine excreted over 24 hours was analyzed and used for the absolute calculation of the amounts of F 4 -NeuroPs and F 2 -dihomo-IsoPs excreted by all volunteers. All F 4 -NeuroPs and F 2 -dihomo-IsoPs were assayed using a previously described method. [START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF]

Chemicals and standards

Four F 4 -NeuroPs (4(RS)-4-F 4t -NeuroP, 4-F 4t -NeuroP, 10-epi-10-F 4t -NeuroP, and 10-F 4t -NeuroP) as well as four F 2 -dihomo-IsoPs (ent-7(R)-7-F 2t -dihomo-IsoP, ent-7(S)-7-F 2t -dihomo-IsoP, 17-F 2tdihomo-IsoP, and 17-epi-17-F 2t -dihomo-IsoP) were utilized in this experiment. Three deuterated internal standards (d 4 -4(RS)-F 4t -NeuroP, d 4 -10-epi-10-F 4t -NeuroP, and d 4 -10-F 4t -NeuroP) were used for the quality control of the analyses (Fig. 2). All standards were synthesized using our published strategies. [START_REF] Guy | Oxygenated metabolites of n-3 polyunsaturated fatty acids as potential oxidative stress biomarkers: total synthesis of 8-F3t-IsoP, 10-F4t-NeuroP and [D4]-10-F4t-NeuroP[END_REF][START_REF] Durand | Total syntheses of iso-, neuro-and phytoprostanes: new insight in lipid chemistry[END_REF][START_REF] Oger | The handy use of Brown's P2-Ni catalyst for a skipped diyne deuteration: application to the synthesis of a [D4]-labeled F4t-neuroprostane[END_REF][START_REF] Oger | Total Synthesis of Isoprostanes Derived from adrenic acid and EPA[END_REF] All our compounds were up to 99% pure and the structures were confirmed by microanalyses, HRMS (high resolution mass spectrometry) and full NMR (1H, 13C, HMQC). The β-glucuronidase, type H2, from Helix pomatia and bis-tris (bis-(2-hydroxyethyl)-amino-tris (hydroxymethyl)-methane) used were purchased from Sigma-Aldrich (St Louis, MO, USA). All LC-MS grade solvents were from J.T. Baker (Phillipsburg, NJ, USA). The Strata X-AW SPE cartridges (100 mg per 3 mL) were obtained from Phenomenex (Torrance, CA, USA).

UHPLC-QqQ-MS/MS analyses

The separation of F 4 -NeuroPs and F 2 -dihomo-IsoPs in the urine samples was performed by ultra high pressure liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS; Agilent Technologies, Waldbronn, Germany), using the set-up described in ref. 22. Chromatographic separation was carried out on an ACQUITY BEH C 18 column (2.150 mm, 1.7 μm pore size) (Waters, MA, USA). The column temperatures were 6 °C (left) and 6 °C (right). The MRM was performed using the negative electrospray ionization (ESI) mode and the dwell time was 25 ms for all MRM transitions. The mobile phases were: (A) H 2 O containing 0.01% acetic acid (v/v) and (B) MeOH containing 0.01% acetic acid (v/v). The injection volume was 20 μL. The analysis time for each sample was 10.01 min. The flow rate was 0.2 mL min -1 , using a linear gradient scheme: (t; %B): (0.0; 60.00), (7.00; 70.00), (7.01; 90.00), (10.00; 90.00), (10.01; 60.00). The MS parameters fragmentor (ion optics) and collision energy were optimized for each compound. Data acquisition and processing were performed using Mass Hunter software version B.04.00 (Agilent Technologies, Waldbronn, Germany). The identification and quantification of F 4 -NeuroPs and F 2dihomo-IsoPs were carried out using the authentic markers previously described. [START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF]

Statistical analyses

Specific differences between the amounts of F 4 -NeuroPs and F 2 -dihomo-IsoPs excreted (ng per 24 h) in the different stages were analyzed by Friedman's non-parametric repeated measures analysis of variance (ANOVA), since the normality and/or equal variance tests failed. When a significant difference was found in the ANOVA, a pair-wise comparison was performed using the Wilcoxon signed rank test with Bonferroni correction. A posteriori, sample size was calculated using the value r, calculated by r = Z/√N, in which Z is the Z-score that SPSS produces, and N is the size of the study on which Z is based. An r value of 0.1, 0.3, or 0.5 was considered to show a small, moderate, or large effect, respectively. [START_REF] Field | How to Design and Report Experiments[END_REF] The data are shown as mean ± SD, as well as the quartiles (upper values 75%, median 50%, and lower values 25%), of the F 4 -NeuroPs and F 2 -dihomo-IsoPs excreted throughout the study. Because the crossover period data of the two phases did not differ, data from both groups were pooled into one placebo or ACJ treatment. The statistical analyses were carried out using the SPSS 23.0 software package (LEAD Technologies Inc. Chicago, USA). The graphs were plotted using the Sigma Plot 12.0 software package (Systat Software, Inc. SigmaPlot for Windows).

Results and discussion

In a previous study realized in our group, we observed that urinary levels of the F 4 -NeuroPs and F 2 -dihomo-IsoPs remained constant during a short triathlon training period (2 weeks) at sea level. [START_REF] Garcia-Flores | Assessment of oxidative stress biomarkers -neuroprostanes and dihomo-isoprostanes -in the urine of elite triathletes after two weeks of moderate-altitude training[END_REF] This study analyzed the same eight biomarkers in urine, but the present trial had a longer period (145 days) allowing us to analyze the chronic effects of exercise, as well as of the supplementation of our polyphenols-rich juice (200 mL) in the diet after training. As was mentioned in the introduction, during chronic training, an increase in OS products was detected and this increase can then disrupt the balance of the OS status. [START_REF] Peternelj | Antioxidant supplementation during exercise training: beneficial or detrimental?[END_REF][START_REF] Mankowski | Dietary Antioxidants as Modifiers of Physiologic Adaptations to Exercise[END_REF][START_REF] Camiletti-Moiron | Does exercise reduce brain oxidative stress? A systematic review[END_REF] In athletes, an option for balancing their OS status is to strictly follow an appropriate diet in which fruit is included thanks to its antioxidant and healthpromoting properties. [START_REF] Jeukendrup | Nutritional considerations in triathlon[END_REF][START_REF] Martínez-Sanz | Necesidades energéticas, hídricas y nutricionales en el deporte[END_REF] In addition, beverages in the world of sport are among the best food products since they can provide benefits for voluntary fluid intake, rapid fluid absorption, improvement of performance and enhanced rehydration. [START_REF] Murray | Sports Drinks, Basic science and practical Aspects[END_REF] The excretion values of the lipid peroxidation products from CNS were used to compare them through of the five stages of our clinical trial. Only six biomarkers were quantified (Table 4). Identification was confirmed according to their molecular mass, the characteristic MS/MS fragmentation product ions, and the retention time relative to the corresponding standard. The measured ions were the product ions at m/z 152.9 (10-epi-10-F 4t -NeuroP and 10-F 4t -NeuroP), m/z 337.1 (17-F 2t -dihomo-IsoP and 17-epi-17-F 2t -dihomo-IsoP), and m/z 362.2 (ent-7(R)-7-F 2t -dihomo-IsoP, ent-7(S)-7-F 2t -dihomo-IsoP) derived from the precursor ions m/z 377.1 (for NeuroPs) and m/z 381.1 (F 2t -dihomo-IsoP).

Our volunteers did not show representative differences through the experimental study, according to the working Group of Kinanthropometrics procedure (Table 1). The majority of our triathletes ranged from 19 to 21 years old (Table 1), belonging to the young adult period in accordance with the human life-stages. According to our current knowledge, [START_REF] García-Flores | Snapshot situation of degradation of the nervous system biomarkers -neuroprostane and dihomoisoprostanes-from infancy to elderly[END_REF] this life-stage is ideal for quantification of these specific markers for DHA and AdA peroxidation (F 4 -NeuroPs and F 2 -dihomo-IsoPs), since in sedentary and healthy young adults we detected low amounts of oxidative damage biomarkers. Thereby, the evaluation in this group indicated a realistic demonstration of the effects due to triathlon training and supplementation of our ACJ in the diet on lipid peroxidation from CNS.

The information that follows below may open new avenues for research of the possible roles of polyphenols and other bioactive compounds from a polyphenols-rich beverage-ACJ-on oxidative damage to lipids, essential constituent of nervous tissue (conceived in a chronic triathlon training context using the objective load scale (ECOs)), thanks to the properties of the phenolic compounds to scavenge free radicals in vivo or to activate redox antioxidant pathways in the human body. [START_REF] Garcia-Flores | DNA catabolites in triathletes: effects of supplementation with an aronia-citrus juice ( polyphenols-rich juice)[END_REF][START_REF] Bernabé | Effects of a citrus based juice on biomarkers of oxidative stress in metabolic syndrome patients[END_REF] It must be taken into account that the biomarkers used in this study are oxidative products deriving from the radical attack on adrenic acid (AdA, C22:4 n-6) or docosahexaenoic acid (DHA, C22:6 n-3) and are good prognostic markers for the evolution of oxidative stress linked to the CNS, [START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF][START_REF] Garcia-Flores | Assessment of oxidative stress biomarkers -neuroprostanes and dihomo-isoprostanes -in the urine of elite triathletes after two weeks of moderate-altitude training[END_REF] like isoprostanes or DNA oxidation catabolites are at the systemic level. [START_REF] Garcia-Flores | DNA catabolites in triathletes: effects of supplementation with an aronia-citrus juice ( polyphenols-rich juice)[END_REF][START_REF] Bernabé | Effects of a citrus based juice on biomarkers of oxidative stress in metabolic syndrome patients[END_REF][START_REF] Medina | Assessment of oxidative stress markers and prostaglandins after chronic training of triathletes[END_REF]

F 2 -dihomo-isoprostanes

The F 2 -dihomo-IsoPs are specific markers for free radicalinduced AdA peroxidation, being potential markers of free radical damage to myelin in the human brain. [START_REF] Vanrollins | F2-dihomo-isoprostanes arise from free radical attack on adrenic acid[END_REF] For example, in cerebrospinal fluid, the F 2 -dihomo-IsoPs levels were associated with some neuropsychological symptoms of Alzheimer's disease. [START_REF] Kuo | Cerebrospinal fluid biomarkers for neuropsychological symptoms in early stage of late-onset Alzheimer's disease[END_REF] De Felice et al. published [START_REF] De Felice | F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome[END_REF] that the plasma F 2dihomo-IsoPs were involved in the pathogenesis of Rett syndrome. In this assay, the urinary biomarkers derived from AdA were detected in all samples during the whole period of the study, and ranged from ∼1787 to ∼4813 ng per 24 h (Table 4). The two F 2 -dihomo-IsoP metabolites of the 17-series showed significant changes (Table 4); the values decreased with the increase of ECOs training and continued to decline during the ACJ intake. Particularly, 17-epi-17-F 2t -dihomo-IsoP differed significantly in the C-B stage compared to in the C-T (Z = -2.783, P = 0.005, r = 0.695), placebo (Z = -3.124, P = 0.002, r = 0.781), and ACJ stages (Z = -3.408, P = 0.001, r = 0.852). The excretion of 17-F 2t -dihomo-IsoP reached its highest value in the C-B stage. The Bonferroni correction of the results from the Wilcoxon test gave P < 0.005, showing that the C-B stage value was statistically higher than those from placebo (Z = -3.124, P = 0.002, r = 0.781), ACJ (Z = -3.067, P = 0.002, r = 0.766), and CP-T stages (Z = -3.181, P = 0.001, r = 0.795) (Fig. 3). Therefore, our results demonstrated that the F 2 -dihomo-IsoPs values showed significant changes owing to the increase or decrease of the training loads, as well as the influence depending on the time (acute or chronic). The OS elicits different responses depending on the type of the organ tissue and its endogenous antioxidant levels upon acute and chronic exercise. [START_REF] Camiletti-Moiron | Does exercise reduce brain oxidative stress? A systematic review[END_REF] In fact, regular aerobic, moderate training or physical activity programs could increase the resistance against OS to promote antioxidant capacity in the brain. [START_REF] Camiletti-Moiron | Does exercise reduce brain oxidative stress? A systematic review[END_REF] Highlighting also that our athletes have no influence according to their range age, since a study found that there were no significant differences in levels of ent-7(R)-7-F 2t -dihomo-IsoP, ent-7-epi-7-F 2tdihomo-IsoP, 17-F 2t -dihomo-IsoP, and 17-epi-17-F 2t -dihomo- IsoP in sedentary and healthy volunteers between the ages of 13 and 35 years. [START_REF] García-Flores | Snapshot situation of degradation of the nervous system biomarkers -neuroprostane and dihomoisoprostanes-from infancy to elderly[END_REF] Otherwise, the Friedman test showed a significant difference in the ent-7-(R)-7-F 2t -dihomo-IsoP values (Table 4), and also a significant increase in the CP-T stage compared with in the C-T stage. In the CP-T stage, the training load was decreased around 50% after 115 days with high load training (1008 ± 105 ECOs). Post hoc analysis with the Wilcoxon signedrank test showed that values were higher in the CP-T stage (Fig. 3), although only the C-T stage (Z = -3.389, P = 0.001, r = 0.847) differed significantly with the Bonferroni correction (P < 0.005). This result indicates that an acute decrease of training loads after a chronic exercise programme may stimulate an adaptation response where this oxidative product deriving from radical attack on AdA (ent-7(RS)-7-F 2t -dihomo-IsoP) could play a role in this adaptation post-training, although typically the F 2 -dihomo-IsoPs provide a relatively selective insight into oxidative damage to myelin since they are the oxidative products deriving from radical attack on AdA. These markers are also considered to reflect cerebral white matter injury; [START_REF] Durand | F(2)-Dihomo-isoprostanes and brain white matter damage in stage 1 Rett syndrome[END_REF] however, we should also remember that AdA is present in other organs, like the kidney and adrenal glands. [START_REF] Vanrollins | F2-dihomo-isoprostanes arise from free radical attack on adrenic acid[END_REF][START_REF] De Felice | Systemic oxidative stress in classic Rett syndrome[END_REF] Thereby, physical exercise effects on OS from kidney and adrenal glands could also reflect similar results. Besides, a previous study reflected that the urinary levels of F 2 -IsoP decreased with chronic exercise in most of the cases and chronic exercise may rarely result in increased urine F 2 -IsoP levels, [START_REF] Durand | F(2)-Dihomo-isoprostanes and brain white matter damage in stage 1 Rett syndrome[END_REF] while some studies have supported no changes. Our results are consistent with the three changes that were mentioned by Nikolaidis et al. [START_REF] Nikolaidis | F(2)-isoprostane formation, measurement and interpretation: the role of exercise[END_REF] in their review, since any change in the ent-7-epi-7-F 2tdihomo-IsoP values was also observed [START_REF] Vanrollins | F2-dihomo-isoprostanes arise from free radical attack on adrenic acid[END_REF] remaining at constant levels throughout the study with no statistical differences.

Regarding the possible role of the compounds from our juice on the lipid peroxidation from AdA (whatever the current physiological origin: brain white matter, adrenal gland or kidney), the 17-epi-17-F 2t -dihomo-IsoP in the ACJ stage was significantly lower than the CP-T stage values (Z = -3.013, P = 0.003, r = 0.753; Fig. 3). From our point of view, this significant difference perhaps is due to over-activation of the steroid biosynthesis pathway in the particular case of citrus juices, [START_REF] Medina | Non-targeted metabolomic approach reveals urinary metabolites linked to steroid bio-synthesis pathway after ingestion of citrus juice[END_REF] since this pathway is mainly located in the adrenal glands and gonads as well as within the nervous system. There is evidence of neurotrophic and neuroprotective effects on the CNS involving a steroid mechanism, for example, the progesterone has been linked with a decreased of the amount of LPP. [START_REF] Schumacher | Steroid synthesis and metabolism in the nervous system: trophic and protective effects[END_REF] A steroid conjugate from progesterone (17-hydroxyprogesterone) was identified as a significant metabolite after the citrus juice intake, [START_REF] Medina | Non-targeted metabolomic approach reveals urinary metabolites linked to steroid bio-synthesis pathway after ingestion of citrus juice[END_REF] suggesting a possible role in OS status. Another explanation is that due to food biomarkers discovered after the ingestion of ACJ in healthy volunteers, proline betaine, ferulic acid, and two mercapturate derivatives, [START_REF] Llorach | Discovery of human urinary biomarkers of aronia-citrus juice intake by HPLC-q-TOF-based metabolomic approach[END_REF] they may be related with the decrease of 17-epi-17-F 2t -dihomo-IsoP levels in combination with the training sessions. For example, the proline betaine (specific and sensitive marker of citrus fruit intake) had a lowering effect on plasma homocysteine concentration in healthy volunteers. [START_REF] Atkinson | Effects of orange juice and proline betaine on glycine betaine and homocysteine in healthy male subjects[END_REF] Lowering plasma homocysteine levels has been related with lowered OS, conversely if this amino acid increases its levels it can lead to prooxidative activity, agerelated cognitive impairment, and neurodegenerative and cerebrovascular disease. [START_REF] Hainsworth | Homocysteine, hyperhomocysteinemia and vascular contributions to cognitive impairment and dementia (VCID)[END_REF] In addition, ferulic acid also provides protection against lipid peroxidation and prevents attacks to the membrane, acting as a potential antioxidant due to its structural characteristics, the presence of electron donating groups on the benzene ring and its carboxylic acid group. [START_REF] Srinivasan | Ferulic acid: Therapeutic potential through its antioxidant property[END_REF] In biological models, the ferulic acid showed a role as an inhibitor or disaggregating agent of amyloid structure, suggesting a positive effect in the first steps to trigger Alzheimer's disease. [START_REF] Sgarbossa | Ferulic Acid: A hope for Alzheimer's disease therapy from plants[END_REF] Alzheimer's disease has been related with the increase of F 2 -dihomo-IsoPs levels. [START_REF] Vanrollins | F2-dihomo-isoprostanes arise from free radical attack on adrenic acid[END_REF] On the other hand, it is noteworthy that ACJ, besides its phytochemicals, provides other compounds such as vitamins and minerals that appear to have or help antioxidative activities providing health benefits. The vitamin C from the mixture (from citrus to Aronia) is a representative compound. [START_REF] Gonzalez-Molina | Aronia-enriched lemon juice: a new highly antioxidant beverage[END_REF] Ascorbic acid (vitamin C) is an electron donor and reducing agent, so it prevents the oxidation of biomolecules. [START_REF] Sawicka-Glazer | Vitamin C: A new auxiliary treatment of epilepsy?[END_REF] Ascorbic acid is accumulated in adrenal glands and the central nervous system, indicative of the importance of ascorbate function in the CNS, even with low plasma levels. [START_REF] Halliwell | Oxidative stress and neurodegeneration: where are we now?[END_REF] Besides its function as a reactive oxygen species scavenger, it also helps to restore other substances with antioxidant properties, such as alpha-tocopherol (vitamin E) or glutathione (antioxidant in plants). [START_REF] Sawicka-Glazer | Vitamin C: A new auxiliary treatment of epilepsy?[END_REF] There is no conclusive data regarding the anti-oxidative effects related to mineral intake from Aronia and/or citrus, although orange juice consumption has been shown to enhance the absorption of minerals (iron, aluminum, calcium, zinc, and selenium) from the diet. [START_REF] Franke | Orange juice and cancer chemoprevention[END_REF] Besides, we found that in animal models the hesperidin intake (a monomethylated flavanone found abundantly in oranges) due to its antioxidant and anti-inflammatory properties showed protective effects on bone mineral density. [START_REF] Habauzit | Increased bioavailability of hesperetin-7-glucoside compared with hesperidin results in more efficient prevention of bone loss in adult ovariectomised rats[END_REF] The minerals in vivo are involved in the production of free radicals, since they can accelerate or delay the oxidative stress and neurodegeneration occurring in the CNS. [START_REF] Halliwell | Oxidative stress and neurodegeneration: where are we now?[END_REF] Therefore, minerals and vitamins from our ACJ were maybe involved in the lipid peroxidation pathways for this result.

Nonetheless, further research is needed on the correlation of potential beneficial effects of polyphenols-rich dietary supplements and their particular mechanisms of action of each compound on its own or in conjunction with others on the markers of central nervous system degradation in athletes, although some experimental studies have indicated positive biological effects of polyphenols-rich dietary supplements in athletes. [START_REF] Del Rio | Dietary ( poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases[END_REF][START_REF] Myburgh | Polyphenol supplementation: benefits for exercise performance or oxidative stress?[END_REF][START_REF] Medina | Physical activity increases the bioavailability of flavanones after dietary aronia-citrus juice intake in triathletes[END_REF][START_REF] Lafay | Grape extract improves antioxidant status and physical performance in elite male athletes[END_REF][START_REF] Pilaczynska-Szczesniak | The influence of chokeberry juice supplementation on the reduction of oxidative stress resulting from an incremental rowing ergometer exercise[END_REF] Thus, we are developing further research to clarify the positive influence that the intake of functional fruit juices and polyphenols could have in athletes. [START_REF] Garcia-Flores | DNA catabolites in triathletes: effects of supplementation with an aronia-citrus juice ( polyphenols-rich juice)[END_REF]

F 4 -neuroprostanes

The F 4 -NeuroPs originate from the free radical-catalysed peroxidation of DHA-an essential constituent of nervous tissue that is highly enriched in neurons and highly susceptible to oxidation. [START_REF] Nourooz-Zadeh | F4-isoprostanes as specific marker of docosahexaenoic acid peroxidation in Alzheimer's disease[END_REF] Looking at our findings, we note a possible effect of ACJ at the neuronal level, since 10-epi-10-F 4t -NeuroP and 10-F 4t -NeuroP were not detected during the intake period compared to in the placebo stage. In the C-T stage, two F 4 -NeuroPs (10-epi-10-F 4t -NeuroP (Z = -2.845, P = 0.004, r = 0.711) and 10-F 4t -NeuroP (Z = -2.499, P = 0.012, r = 0.624)) showed a decrease before the crossover intake of the beverages ( placebo or ACJ) (Fig. 3). The 10-F 4t -NeuroP values continued to decline significantly in the placebo stage (Z = -3.130, P = 0.002, r = 0.782) (Fig. 3). During the ACJ and CP-T stages, these F 4 -NeuroPs were not detected (Table 4). The decline of the excretion of the NeuroPs in our study could partially be attributed to the ingestion of bioactive compounds found in our polyphenols-rich juice. There is evidence showing that citrus fruits intake could alter the OS of the CNS [START_REF] Schaffer | Do polyphenols enter the brain and does it matter? Some theoretical and practical considerations[END_REF] and, particularly, that polyphenols may alter brain function at three locations: outside the CNS (for instance, by improving cerebral blood flow or by modulating signaling pathways from peripheral organs to the brain), at the blood-brain barrier (e.g., by altering multi-drug-resistant protein-dependent influx and efflux mechanisms of various biomolecules), and inside the CNS (e.g., by directly modifying the activity of neurons and glial cells). In addition, citrus fruits, which are rich and abundant sources of hesperidin and other polyphenols, are promising for the development of general foodbased neuroprotection and "brain foods". [START_REF] Hwang | Neuroprotective effects of citrus flavonoids[END_REF] A recent review gathered evidence about the neuroprotective actions of the flavonoids mentioned that may influence the survival cascade and transcription factors by modulating the redox potential of neurons and glia. In vivo activities of flavonoids in the brain remain to be elucidated, but have shown potential functions against oxidative damage, 63 as has been shown in this study.

The health effects of polyphenols depend on the amount consumed and their bioavailability. The bioavailability is a key aspect to exert antioxidant activity in humans, since many polyphenols have a scarce bioavailability and are extensively metabolized. [START_REF] Rao | Effects of acute graded exercise on human colonic motility[END_REF] According to our previous study, the bioavailability of flavanones from ACJ intake increased in the triathletes, suggesting that over-activation of the microbiota and intestinal motility were caused by physical exercise, helping to increase the bioavailability of the compounds in the ACJ. [START_REF] Medina | Physical activity increases the bioavailability of flavanones after dietary aronia-citrus juice intake in triathletes[END_REF] The results obtained in this study with ACJ supplementation (one serving, 200 mL), which was adjusted to the normal diet of our athletes (the intake always being around 15 minutes after training for 45 days) suggest an effect of the ACJ due to the combination with the physical exercise. This could be based on the physiological changes that may re-establish colonic motility after exercise, when blood flow is restored, allowing maximum exposure and absorption of nutrients including polyphenols and thus, the increase in the flavonoids bioavailability. [START_REF] Rao | Effects of acute graded exercise on human colonic motility[END_REF] In support of the above affirmation, Gomez-Pinilla 8 mentioned that the combination of polyphenols intake and physical activity can deliver more beneficial effects than intervention alone or the mixed effects of exercise. For example, a study in athletes showed that the increase of the intake of anthocyanins can limit exercise-induced oxidative damage to red blood cells, most probably by enhancing the endogenous antioxidant defense system. These athletes consumed 150 mL of chokeberry juice daily-providing 23 mg per 100 mL antho-cyanin-for a period of one month. [START_REF] Pilaczynska-Szczesniak | The influence of chokeberry juice supplementation on the reduction of oxidative stress resulting from an incremental rowing ergometer exercise[END_REF] Other nutritional interventions in athletes also showed the protective effect against OS induced by the consumption of polyphenols from grape extract (400 mg per day). [START_REF] Lafay | Grape extract improves antioxidant status and physical performance in elite male athletes[END_REF] Furthermore, berry extracts could have effects associated with their ability to maintain metabolic homeostasis, thus protecting membranes from lipid peroxidation and affecting synaptic plasticity. [START_REF] Gomez-Pinilla | Brain foods: the effects of nutrients on brain function[END_REF] In vitro and animal models have proved the beneficial effects of polyphenols on exercise-induced OS, muscle damage and exercise performance, but in human studies further research is required for the better assessment of their benefits. [START_REF] Malaguti | Polyphenols in exercise performance and prevention of exercise-induced muscle damage[END_REF] Currently, the mechanisms by which physical exercise exerts its effects in the brain remain largely unknown, although researchers have provided promising evidence about physical exercise-induced outcomes for several prevalent neurological and psychiatric conditions (CNS). [START_REF] Morgan | Effects of physical exercise on central nervous system functions: a review of brain region specific adaptations[END_REF] The reduction of oxidative stress has provided possible evidence to suggest the positive effects on CNS health. [START_REF] Camiletti-Moiron | Does exercise reduce brain oxidative stress? A systematic review[END_REF][START_REF] Morgan | Effects of physical exercise on central nervous system functions: a review of brain region specific adaptations[END_REF] Thus, our study provides evidence of the effect of the intake of ACJ (rich in polyphenols) during a training period with regard to the decrease of the NeuroPs values, suggesting a potential positive effect on the nervous system during training.

Another interesting point besides the apparent absence of 10-epi-10-F 4t -NeuroP and 10-F 4t -NeuroP in the ACJ stage was the significant changes in the values of these NeuroPs during the stages in which they were detected (C-B, C-T, and placebo stages) (Table 4). The excretion of these metabolites tended to decrease, as we could observe for 10-F 4t -NeuroP during the study, but in the placebo stage 10-epi-10-F 4t -NeuroP exhibited a significant increase (Z = -2.543, P = 0.011, r = 0.635) compared with in the C-T stage, but returned to previous values in the C-B stage. This behavior of the stereoisomers can depend on different mechanisms, but the precise roles of these isomers in vivo have not been elucidated yet. In the urine analysis of the systemic neuroprostane-like compounds (isoprostane, IsoPs) formed in vivo via the non-enzymatic, free radicalinitiated peroxidation of polyunsaturated fatty acids, it is important to consider that these molecules are not only excreted in the original form since they are extensively metabolized in the liver, producing a biotransformation of the metabolites. [START_REF] Milne | Measurement of F2-isoprostanes and isofurans using gas chromatography-mass spectrometry[END_REF] For example, in a study of smokers, all IsoPs are equally increased by any source of OS (e.g., smoking), but some are more efficiently metabolized so that their determined concentrations appear less affected by variations in oxidant levels. [START_REF] Yan | Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS[END_REF] This would mean that highly metabolized IsoPs appear less correlated with smoking than less-metabolized IsoPs. Another possibility was that exposure to different types of oxidants may affect the mechanisms that create IsoPs, thereby affecting their distribution. In our study, the closest relationship was between chronic physical exercise and the metabolite 10-epi-10-F 4t -NeuroP.

Finally, two F 4t -NeuroPs (4-(RS)-4-F 4t -NeuroP and 4-F 4t -NeuroP) were analyzed in this study, but they were below the limit of detection/quantification. Therefore, these data are not shown. In previous work, 4-(RS)-F 4t -NeuroP and 4F 4t -NeuroP were also not detected. [START_REF] Medina | Dihomo-isoprostanes-nonenzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography-triple quadrupole-tandem mass spectrometry method[END_REF] In addition, another mediator of oxidative stress from omega-3 fatty acid, docosapentaenoic acid (4-F 3t NeuroP), was only detected in 22.22% of the 45 young adults volunteers. [START_REF] García-Flores | Snapshot situation of degradation of the nervous system biomarkers -neuroprostane and dihomoisoprostanes-from infancy to elderly[END_REF] Thus, the latest data continue to support the idea that the NeuroPs do not appear to be specific biomarkers in healthy or sedentary volunteers.

Conclusions

The F 4 -NeuroPs 10-epi-10-F 4t -NeuroP and 10-F 4t -NeuroP were not detected after the consumption of ACJ. The changes in the excretion values of these biomarkers suggest health benefits that could be attributed to the ingestion of bioactive compounds that include partial co-responsibility of flavonoids and others phenolic found in ACJ on the oxidative status neuronal membrane. The changes in the excretion of 17-epi-17-F 2tdihomo-IsoP show a positive connection between physical exercise and ACJ intake, suggesting that the combination of polyphenols intake and physical activity can deliver beneficial effects on the neuromotor system. The physical exercise by itself was also able to exert different responses depending on the increase (17-F 2t -dihomo-IsoP) or the decrease (ent-7-(RS)-7-F 2t -dihomo-IsoP) of the training loads. Thus, the chronic intake of one serving of ACJ rich in polyphenols (200 mL, adjusted to the diet) and adequate training influenced the OS of the CNS in young adult triathletes, which will help to elucidate novel interactions and mechanisms between excretion of lipid peroxidation metabolites, supplementation of polyphenols-rich juice in the diet and physical exercise during a training season. These actions and mechanisms may be linked to the properties of polyphenols to scavenge free radicals in vivo themselves or to activate redox antioxidant pathways in the human body.

Fig. 1

 1 Fig. 1 Study design. This crossover study was randomized, double-blind, and placebo-controlled. Sixteen athletes (n = 16), randomly divided into two groups (n = 8), were assigned supplementation with either 200 mL of ACJ (aronia-citrus juice) or 200 mL of placebo. After 45 days of supplementation and a 10 day washing-out period, the beverages were reversed. Urine samples were collected on the last day at the end of each stage. The training load was quantified by the Objective Load Scale (ECOs).

Fig. 2

 2 Fig. 2 Chemical structures of F 4 -NeuroPs, F 2 -dihomo-IsoPs, and deuterated internal standards. A: F 4 -NeuroPs, B: F 2 -dihomo-IsoPs.

  This journal is © The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 4343-4355 | 4347 Published on 30 August 2016. Downloaded by University of Montpellier on 13/10/2016 17:47:38.

Fig. 3

 3 Fig. 3 Box plots with quartiles (upper values 75%, median 50%, and lower values 25%) of the (A) F 2 -dihomo-IsoPs and (B) F 4 -NeuroPs in 24 h -1 urine throughout the study (ng per 24 h). • Outlier data are shown. *: shows a significant difference compared to the C-B stage, §: shows a significant difference compared to the ACJ stage and ‡: shows a significant difference compared to the C-T stage. Significant P-values are shown according to post hoc analysis with Wilcoxon signed-rank tests (with a Bonferroni correction P < 0.005, for F 2 -dihomo-IsoPs and P < 0.016, for F 4 -NeuroPs). Abbreviations: C-B; Control Baseline, C-T; Control Training, ACJ; Aronia-Citrus Juice, CP-T; Control Post-Treatment.

  This journal is © The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 4343-4355 | 4349 Published on 30 August 2016. Downloaded by University of Montpellier on 13/10/2016 17:47:38.

  This journal is © The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 4343-4355 | 4351 Published on 30 August 2016. Downloaded by University of Montpellier on 13/10/2016 17:47:38.

Table 3

 3 Nutritional and phenolic composition of aronia-citrus juice

	ACJ	200 mL
	Energy intake (kcal)	76
	Proteins (g)	0.9
	Carbohydrate (g)	18
	Fat (g)	0.06
	Phenolic compounds a
	Total flavonoids (mg)	129.31 ± 1.79
	Hydroxycinnamic acids (mg)	68.82 ± 0.6
	The values are means ± standard deviation (n = 3, expressed as mg per
	200 mL of juice). a For more detailed analysis of the phenolic com-
	pounds from this juice, see ref. 16.

Table 2

 2 Dietary parameters and caloric intake of the triathletes during the study

		Male triathletes	Female triathletes
	Energy intake (kcal) Carbohydrate (g d -1 ) Dietary fiber (g d -1 ) Sugars (g d -1 ) Proteins (g d -1 ) Total lipids (g d -1 ) SFA a (g d -1 ) MUFA b (g d -1 ) PUFA c (g d -1 ) Vitamin C (mg d -1 ) Vitamin A (µg d -1 ) Vitamin E (mg d -1 ) Vitamin D (mg d -1 ) Iron (mg d -1 ) Selenium (mg d -1 )	2820.0 ± 241.2 326.1 ± 63.5 27.3 ± 7.4 121.3 ± 33.9 133.7 ± 12.9 113.7 ± 13.3 33.5 ± 6.5 56.5 ± 5.5 16.9 ± 2.7 178.9 ± 71.9 2970.0 ± 913.9 21.0 ± 5.6 988. ± 47.5 20.9 ± 2.4 149.8 ± 21.5	2072.6 ± 223.4 211.3 ± 43.9 15.5 ± 4.4 80.5 ± 18.3 83.5 ± 9.0 107.1 ± 14.4 29.6 ± 4.4 56.6 ± 7.5 15.9 ± 6.7 135.0 ± 60.4 1427.4 ± 573.1 13.9 ± 3.4 751.6 ± 163.0 14.9 ± 2.6 103.0 ± 17.4
	Dietary parameters and caloric intake of the triathletes during the
	study.

a Saturated fatty acids. b Monounsaturated fatty acids. c Polyunsaturated fatty acids.

Table 1

 1 Physical and metabolic characteristics and training loads of the elite triathletes Published on 30 August 2016. Downloaded by University of Montpellier on 13/10/2016 17:47:38.

	Food & Function					Paper
		Stages of study
	Physical characteristics	CB	CT	Placebo	ACJ	CP-T
	Male (n = 10)
	Age (y)	19.0 ± 1.7	19.0 ± 1.7	19.0 ± 1.7	19.4 ± 1.3	19.6 ± 1.3
	Weight (kg)	69.0 ± 6.2	69.0 ± 6.4	70.7 ± 6.9	71.2 ± 4.6	72.2 ± 6.8
	Height (m) BMI a (kg m -2 )	1.8 ± 0.1 22.2 ± 1.0	1.8 ± 0.1 22.2 ± 1.0	1.8 ± 0.1 21.7 ± 1.4	1.8 ± 0.1 21.6 ± 1.3	1.8 ± 0.1 21.8 ± 1.7
	Total fat (kg)	9.2 ± 2.8	8.8 ± 2.6	8.0 ± 1.7	6.4 ± 2.8	6.8 ± 1.2
	Lean weight (kg)	31.4 ± 2.1	30.5 ± 2.7	31.6 ± 3.0	33.8 ± 3.2	32.4 ± 2.4
	Subscapular skinfold (mm)	9.6 ± 3.0	9.5 ± 2.1	9.1 ± 1.7	8.6 ± 2.0	8.6 ± 1.8
	Triceps skinfold (mm)	8.9 ± 3.0	9.7 ± 2.6	8.7 ± 2.1	7.4 ± 2.4	7.3 ± 1.5
	Biceps skinfold (mm)	5.4 ± 2.4	4.7 ± 1.5	4.1 ± 0.6	4.5 ± 1.5	3.7 ± 0.4
	Iliac crest skinfold (mm)	12.0 ± 2.6	13.1 ± 4.1	12.5 ± 4.2	11.2 ± 3.4	9.6 ± 2.5
	Supraspinale skinfold (mm)	9.0 ± 2.6	8.9 ± 2.8	8.7 ± 2.5	7.6 ± 1.9	6.7 ± 1.4
	Abdominal skinfold (mm)	16.4 ± 8.0	15.5 ± 6.8	14.5 ± 5.9	11.8 ± 5.2	10.0 ± 3.7
	Front thigh skinfold (mm)	14.9 ± 4.4	14.0 ± 4.4	11.5 ± 2.3	10.1 ± 2.9	10.0 ± 2.5
	Medial calf skinfold (mm)	9.0 ± 3.0	9.5 ± 3.1	8.2 ± 2.1	7.2 ± 2.3	7.3 ± 1.8
	Training loads ECOs	37.5 ± 5.5	1008 ± 105	923 ± 119	923 ± 119	552 ± 45
	Female (n = 6)
	Age (y)	21.0 ± 3.0	21.0 ± 3.0	21.08 ± 3.0	21.0 ± 3.0	21.0 ± 3.0
	Weight (kg)	54.8 ± 12.2	54.8 ± 11.6	56.2 ± 4.8	54.4 ± 5.0	53.1 ± 2.9
	Height (m) BMI a (kg m -2 )	1.6 ± 0.1 21.2 ± 4.1	1.6 ± 0.1 21.2 ± 4.1	1.6 ± 0.1 20.7 ± 1.3	1.6 ± 0.1 21.6 ± 2.4	1.6 ± 0.1 20.5 ± 1.6
	Total fat (kg)	8.7 ± 4.1	8.9 ± 4.7	9.2 ± 0.9	7.5 ± 1.2	7.3 ± 1.4
	Lean weight (kg)	20.8 ± 3.6	20.6 ± 2.7	20.8 ± 2.4	19.4 ± 2.8	20.9 ± 2.0
	Subscapular skinfold (mm)	12.7 ± 6.7	13.4 ± 8.2	11.7 ± 2.5	10.7 ± 1.9	9.9 ± 2.8
	Triceps skinfold (mm)	16.3 ± 2.3	18.4 ± 3.8	19.3 ± 5.4	16.1 ± 4.6	17.4 ± 4.6
	Biceps skinfold (mm)	10.3 ± 2.8	9.8 ± 3.2	7.2 ± 0.4	5.7 ± 1.0	5.7 ± 1.3
	Iliac crest skinfold (mm)	19.7 ± 4.5	17.1 ± 6.9	20.9 ± 4.5	17.3 ± 3.7	13.7 ± 4.3
	Supraspinale skinfold (mm)	14.3 ± 6.5	14.4 ± 6.9	15.0 ± 1.0	12.8 ± 2.1	11.6 ± 2.5
	Abdominal skinfold (mm)	23.1 ± 5.9	23.6 ± 6.9	24.5 ± 4.7	21.3 ± 4.1	17.9 ± 4.6
	Front thigh skinfold (mm)	27.2 ± 5.2	26.4 ± 5.0	25.8 ± 3.6	23.8 ± 12.5	26.0 ± 5.4
	Medial calf skinfold (mm)	14.8 ± 3.8	13.9 ± 3.0	15.7 ± 2.1	12.5 ± 1.8	14.4 ± 2.9
	Training loads ECOs	37.5 ± 5.5	1008 ± 105	923 ± 119	923 ± 119	552 ± 45

a Body mass index. CB; Control Baseline, CT; Control Training, ACJ; Aronia-Citrus Juice, CP-T; Control Post-Treatment. This journal is © The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 4343-4355 | 4345

Table 4

 4 Urinary F 4 -neuroprostanes and F 2 -dihomo-isoprostane (ng per 24 h) a determined throughout the assay The data are shown as means ± SD. n.d.: not detected. a The volume of urine excreted by the volunteers was 1212.42 ± 716.50 ml per 24 h, on average, in all the periods. b Average of the two urine collections in the crossover period (Placebo/ACJ). C-B; Control Baseline, C-T; Control Training, ACJ; Aronia-Citrus Juice, CP-T; Control Post-Treatment.

						Stage of study
								Placebo b
	From Analyte a (ng per 24 h)	X 2	df Sig	C-B (n = 16)	C-T (n = 16)	(n = 16)	ACJ b (n = 16)	CP-T (n = 16)
	Ω3	Neuronal membrane degradation
	DHA 10-epi-10-F 4t -NeuroP	11.37 2	0.003 4930.3 ± 1844.4 2953.2 ± 1176.3 4135.4 ± 1005.0 n.d.	n.d.
		10-F 4t -NeuroP	20.93 2	0.000 2711.6 ± 294.5	1909.9 ± 116.7	891.6 ± 372.7	n.d.	n.d.
	Ω6	Neuromotor system degradation
	AdA	17-epi-17-F 2t -dihomo-IsoP	27.14 4	0.000 2689.4 ± 487.5	2018.6 ± 507.0	2016.6 ± 330.4	1787.0 ± 328.6	2319.9 ± 444.9
		17-F 2t -dihomo-IsoP	24.48 4	0.000 3604.4 ± 628.4	2677.7 ± 444.7	2842.8 ± 316.7	2559.1 ± 504.4	2607.1 ± 450.9
		Ent-7(R)-7-F 2t -dihomo-IsoP 22.56 4	0.000 4045.3 ± 763.5	3551.1 ± 534.2	3914.9 ± 444.2	4070.2 ± 599.5	4639.7 ± 612.8
		Ent-7-epi-7-F 2t -dihomo-	8.80 4	0.066 4179.0 ± 815.7	4020.6 ± 1115.9 4216.3 ± 629.4	4813.23 ± 1040.9 4255.0 ± 834.2
		IsoP

This journal is © The Royal Society of Chemistry 2016

This journal is © The Royal Society of Chemistry 2016 Published on 30 August 2016. Downloaded by University of Montpellier on 13/10/2016 17:47:38.View Article Online

Acknowledgements

This study was supported by the project AGL2011-23690 (CICYT) (Spanish Ministry of Economy and Competitiveness). This work has been partially funded by the "Fundación Séneca de la Región de Murcia" Grupo de Excelencia 19900/GERM/15. LAGF was granted a pre-doctoral FPI fellowship (BES2012-060185) by the Spanish government. The authors are grateful to the University of Alicante for its collaboration. We are grateful to Dr David Walker (native English speaker) and Dr Pablo Rodríguez, for their reviews of the English grammar and style of the current report.

Notes and references

Conflict of interest

The authors declare that they have no conflict of interest.


F P Duval

S Quellec

A Trémier

C Druilhe

F Mariette

Non-Destructive Quantification of Water Gradient in Sludge Composting with Magnetic Resonance Imaging

Keywords: NMR, MRI, relaxation, compost, biodegradability, moisture content, bulking agent, sludge

Sludge from a slaughterhouse wastewater plant, and mixtures of bulking agent (crushed wood pallet) and sludge were studied by Nuclear Magnetic Resonance (NMR). The NMR spin-spin relaxation (T 2 ) and spin-lattice relaxation (T 1 ) signals for sludge, wet crushed wood pallet and mixtures of sludge and bulking agent were decomposed into three relaxation time components. Each relaxation time component was explained by a non-homogeneous water distribution on a microscopic length scale and by the porosity of the material. For all samples, the T 2 relaxation time value of each component was directly related to the dry matter content.

The addition of wet crushed wood to sludge induced a decrease in the relaxation time, explained by water transfer between the sludge and the wood.

Magnetic Resonance Imaging (MRI) and respirometric measurements were performed on sludge and wood mixtures. MR images of the mixtures were successfully obtained at different biodegradation states. Based on specific NMR measurements in an identified area located in the MRI cells, the results showed that grey levels of MR images reflected dry matter content. This preliminary study showed that MRI would be a powerful tool to measure water distribution in sludge and bulking agent mixtures and highlights the potential of this technique to increase the understanding of sludge composting.

Introduction

Treatment of wastewater is an important issue in western countries and research is currently being undertaken to investigate the elimination or re-use of residual sludge at better cost, including composting of wastewater sludge. In order to do so, sludge needs to be mixed with a bulking agent which creates a porous matrix. Proportions of sludge and bulking agent are chosen so that sludge covers the bulking agent with a thin layer, and then micro-organisms in the sludge are sufficiently aerated to consume the available biodegradable organic matter.

During sludge composting treatment, the active biodegradation takes place in three to four weeks, followed by several months of maturation [START_REF] Gupta | Stabilization of primary sewage sludge during vermicomposting[END_REF]. Selection of an initial waste recipe with optimal physical and biochemical parameters is essential to create an environment conducive to high microbial growth and activity, and then to shorten and improve the biodegradation in the active phase [START_REF] Haug | The practical handbook of composting engineering[END_REF][START_REF] Agnew | The physical properties of compost[END_REF]. Some of the important physical parameters for this waste recipe are water content, air porosity and specific surface area.

Research on composting is often focused on overall understanding of the biodegradation phenomena, i.e. on a macro-scale. All the parameters of biodegradation such as oxygen consumption, bacteria activity, porosity and moisture content are averaged, and little is known regarding the influence of local behaviour of the physical parameters on overall biodegradation. Since solid wastes are very heterogeneous, better understanding of water content, porosity and specific surface spatial distribution is needed, and Magnetic Resonance Imaging (MRI) could help to fill this gap. Indeed, most of the common techniques to measure physical parameters can not be applied along the composting process because they are invasive or destructive. Moisture measurements need sampling and drying. Classic porosity measurement implies to fill the porous volume with a liquid [START_REF] Annan | Evaluation of techniques for measuring air filled porosity in composts of municipal biosolids and wood chips[END_REF], which is destructive for the sample. Pycnometry is a non destructive method but results directly depend on the applied pressure leading to the measurement of total air space (TAS) or only free air space (FAS) [START_REF] Su | Comparison of free air space test methods[END_REF]. Measurement of the specific surface is not easier. The direct measurement technique through gaseous absorption (BET) used by Palmowski et al. [START_REF] Palmowski | Anaerobic degradation of organic materials-significance of the substrate surface area[END_REF] can only be applied after drying the waste. On the contrary, MRI is nondestructive and non-invasive and thus makes it possible to follow the evolution of samples over time. MRI is well known for its medical applications, but it has been used in other fields including the wood industry [START_REF] Muller | Detection of incipient fungal attack in wood using magnetic resonance parameter mapping[END_REF], study of bacterial chemotaxis [START_REF] Sherwood | Analysis of bacterial random motility in a porous medium using magnetic resonance imaging and immunomagnetic labelling[END_REF][START_REF] Olson | Quantification of bacterial chemotaxis in porous media using magnetic resonance imaging[END_REF], chemical engineering [START_REF] Mantle | Dynamic MRI in chemical process and reaction engineering[END_REF], civil engineering [START_REF] Faure | An MRI-SPI and NMR relaxation study of dryinghydration coupling effect on microstructure of cement-based materials at early age[END_REF], fluid mechanics [START_REF] Bonn | Some applications of magnetic resonance imaging in fluid mechanics: Complex flows and complex fluids[END_REF], pharmaceutical science [START_REF] Richardson | Pharmaceutical applications of magnetic resonance imaging (MRI)[END_REF] and food science [START_REF] Hills | Magnetic resonance imaging in food science[END_REF][START_REF] Butz | Recent developments in noninvasive techniques for fresh fruit and vegetable internal quality analysis[END_REF]. In an MRI experiment, the volume in which the signal is collected is called a voxel, and its typical order of magnitude is the cubic millimetre. Voxel intensity is a function of local proton density and of their respective spin-lattice (T 1 ) and spin-spin (T 2 ) relaxation times. Proton density is proportional to the amount of material containing protons.

In most materials, water is the main constituent related to proton density. Consequently, the grey level intensity of a voxel may be related to water content or air content (i.e. porosity).

Indeed when the voxel size is larger than the pore size the porosity is directly related to the intensity of the MRI signal: the greater the quantity of gas, the lower the signal. This method has been used in food science to quantify the distribution of porosity during proving of dough [START_REF] Grenier | Assessment by MRI of local porosity in dough during proving. Theoretical considerations and experimental validation using a spin-echo sequence[END_REF][START_REF] Lucas | MRI quantification of ice gradients in dough during freezing or thawing processes[END_REF]. In the extreme case where the pore volume is much larger than the voxel size, the pore size can be measured. For example, air cavities have been detected and quantified in food products such as cheese [START_REF] Mariette | NMR relaxometry and MRI techniques: a powerful association in food science[END_REF][START_REF] Mariette | NMR imaging of dairy products[END_REF]. In addition to the porous fraction, the specific surface area can be estimated if the resolution of the MR images is good enough.

Spin-lattice (T 1 ) and spin-spin (T 2 ) relaxation times are related to water content and also to material microstructure and to molecular structure [START_REF] Brownstein | Importance of classical diffusion in NMR studies of water in biological cells[END_REF][START_REF] Belton | The effects of diffusive exchange in heterogeneous systems on N.M.R line shapes and relaxation processes[END_REF]Hills, 1989b;a;[START_REF] Denisov | Hydrogen exchange rates in proteins from water H-1 transverse magnetic relaxation[END_REF]. Several authors have used the T 2 and T 1 relaxation times as tracers of water content for the investigation of draining processes [START_REF] Mariette | NMR imaging of dairy products[END_REF] and rehydration processes [START_REF] Ziegler | Moisture migration in starch molding operations as observed by magnetic resonance imaging[END_REF].

The aim of this study was to evaluate the potential of the MRI technique in sludge composting to quantify the water distribution. . The first part of this report focuses on the description of the nuclear magnetic resonance (NMR) signals in sludge and in mixtures of sludge and bulking agent and their sensitivity to water content. The second part describes the preliminary results of MRI images from sludge and bulking agent mixture. The potential for determination of water content and measurement of local porosity by MRI is discussed.

Materials and Methods

Sludge and bulking agent

The sludge came from the biological activated sludge treatment of the wastewater from a slaughter-house. Iron chloride and a polymeric flocculent (PRAESTOL K133) were added to the sludge to induce flocculation and, after a decantation step, the sludge was dewatered using a press band. The collected sludge was stored frozen at -20°C in 1kg batches. Sludge was unfrozen before each experiment, initially in a refrigerator at 4°C and then at room temperature.

The dry matter (DM, measured by drying at 105°C until constant weight) in the sludge was 13.6% of total weight, with approximately 80% organic matter to DM. The exact quantity of living bacteria present in the sludge was unknown. Flocs of bacteria were observed using optical microscopy (data not shown). Sludges with higher dry matter content were prepared using a low pressure oven (34 Mb and 30°C). Mixtures of sludge and bulking agent were prepared by hand mixing the sludge with crushed wood pallet as bulking agent. The size of the wood chips considered was 8 to 12 mm. Wood was stored after drying at 80°C. Different samples were then studied by NMR, i.e. the sludge without bulking agent, mixtures of wood and sludge, and a rehydrated wood sample (DM 45%). Since there is water transfer between sludge and dried wood after mixing, the saturated wood sample was prepared to mimic the contribution of the re-hydrated wood fraction in the mixture.

A combined experiment using MRI and respirometry was set up. Two large respirometric cells were prepared with 4.5 kg of sludge (wet) mixed with 2.5 kg of wood (dry mass ratio 1/4.1 g DM /g DM ). The moisture content in both cells was approximately 56%. MRI measurements were performed at day 1, day 3 and day 27 of biodegradation. Samples were taken from these cells for NMR measurement and moisture analysis at 27 days.

Respirometry

Respirometry is a useful tool to assess microbial activity [START_REF] Iannotti | Oxygen Respirometry to Assess Stability and Maturity of Composted Municipal Solid-Waste[END_REF][START_REF] Adani | Determination of biological stability by oxygen uptake on municipal solid waste and derived products[END_REF][START_REF] Gea | Monitoring the biological activity of the composting process: Oxygen uptake rate (OUR), respirometric index (RI), and respiratory quotient (RQ)[END_REF]. The respirometric apparatus used in this study consisted of a 10 L airtight cylindrical reactor made of glass. Three to four kilos of precisely weighed material were introduced into the respirometric reactor on a 3 mm round mesh grid placed 70 mm above the cell bottom. An entering air flow rate of 65 L/h (compressed air) was applied throughout the experiments.

Homogeneity of aeration of the entire sample was provided by means of the rapid recirculation of the exhaust gas in the cell (360 L/h). The entering and exhaust air streams were monitored for O 2 content using a paramagnetic O 2 gas analyser (MAHAIK Technology, Germany).

The apparatus was monitored for the main environmental factors influencing microbial activity, i.e. temperature and moisture. As shown on Figure 1 the temperature of samples was held constant by placing each respirometric cell in a water-bath at 40°C, which is optimal for biodegradation activity [START_REF] Tremier | A respirometric method for characterising the organic composition and biodegradation kinetics and the temperature influence on the biodegradation kinetics, for a mixture of sludge and bulking agent to be co-composted[END_REF]. The inlet air was preheated to 40°C using a copper tubular coil with a diameter of 10 mm, thickness of 2 mm and length of 2 m, submerged in the water-bath. The sample temperatures were monitored by means of a Pt100 temperature probe (OMNI Instrument, UK) inserted in the centre of the sample. For the moisture control, the inflowing air was saturated by bubbling through two water-filled glass bottles immersed in the water-bath. Moisture condensing in the exhaust air was collected in beakers above the water-bath to prevent its return into the sample cell and its entrance into the gas analyser.

NMR measurements

All NMR measurements were performed with a 10 MHz Minispec Mq10 (Bruker, Wissembourg, France). Typical 90° and 180° pulse durations were 14 µs and 32 µs, respectively, with an 8 dB pulse attenuation. The temperature was fixed at 20°C. Spin-Spin relaxation time (T 2 ) measurements were performed using a Carr Purcell Meiboom and Gill (CPMG) sequence [START_REF] Meiboom | Compensation for pulse imperfections in Carr-Purcell NMR experiments[END_REF]. Three thousand consecutive echoes were recorded with a time spacing of 0.1 ms between the 90° and the first 180° pulse. Recycle delay and number of scans were 2 s and 8 s, respectively. Spin-Lattice relaxation time (T 1 ) measurements were performed using a saturation recovery (SR) sequence. Times between the two 90° pulses were varied one hundred times from 5 ms to 2000 ms. The NMR signal was acquired 28 µs after the second 90° pulse in order to detect only the longest relaxation components, i.e, the water proton relaxation. Recycle delay and number of scans were 2 s and 8 s, respectively.

Calculation of NMR parameters

Relaxation curves were fitted using two different methods, the Maximum Entropy Method (MEM) [START_REF] Mariette | Continuous relaxation time distribution decomposition by MEM[END_REF], which provides a continuous distribution of relaxation components, and the Levenberg-Marquardt algorithm, which allows discrete solution for the fitting (Table Curve, Jandel) [START_REF] Marquardt | An algorithm for least squares estimations of nonlinear parameters[END_REF]. Moreover, no assumptions regarding the number of relaxation times are necessary with MEM compared to the Levenberg-Marquardt algorithm method.

For the spin-spin relaxation decay signal, the equation used for fitting according to the Levenberg-Marquardt method was:

( )

c e I e I e I t S T t T t T t CPMG + + + = - - - 23 22 21 3 2 1
Eq.1 where S(t) CPMG is the intensity of the total relaxation signal (a.u.), t is the time of the relaxation process (ms), T 2i the spin-spin relaxation time of the component i (ms) and I i the associated signal (a.u.). A constant c was added into the model in order to take into account change in the decay baseline. Spin-Lattice relaxation curves were fitted with the Levenberg-Marquardt algorithm with the following equation:

( ) where S(t) SR is the intensity of the total relaxation signal (a.u.), t is the time of the relaxation process (ms), T 1i the spin-lattice relaxation time of the component i (ms) and I i the associated signal (a.u.). A constant α was introduced to correct for imperfections in the 90° pulse.

) 1 ( ) 1 ( ) 1 (

MRI measurements

A 1.5 Tesla whole body MRI scanner (Magneton Avento Siemens, Erlangen, Germany) was used. The MR images were acquired with the standard spin echo sequence. Forty-five slices of 2 mm were recorded, the distance between the centres of two consecutive slices being 4 mm. The field of view was 220 mm x 220 mm for 256 x 256 pixel sampling. Echo time (T E ), repetition time (T R ) and pixel bandwidth were 8 ms, 1000 ms and 395 Hz/pixel, respectively.

The grey level intensity S ES (a.u) within a voxel with a standard spin-echo sequence was given by the equation:

2 1 1 T T T T ES E R e e I S - - × ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - = β Eq.3
with I the intensity related to the relaxation times T 1 and T 2 of the protons in the voxel. β is a parameter introduced to correct for imperfections in the 90° pulse. This equation assumed a single proton component with a single spin-spin relaxation and a single spin-lattice relaxation.

In the case of multi-exponential behaviour, the grey level intensity would be a combination of the different relaxing components for both T 1 and T 2 .

Results and discussion

NMR

Figure 2 shows typical CPMG (a) and SR (b) signals for two wood and sludge mixtures with dry matter of 24% and 70%. CPMG signal intensities decreased throughout the acquisition time t, according to equation 1. An increase in signal intensity was obtained for SR as expected from equation 2. The effects of water content on NMR parameters can be described from the two CPMG signals. Firstly, since the initial signal intensity was proportional to the sample proton content, and because water is one of the most abundant protonic molecules in our system, this explains the reduction in signal intensity from 3.7 to 2.2 a.u. observed between the two samples. Secondly, the water relaxation time is highly sensitive to water content. Indeed, T 2 relaxation was directly related to molecular mobility, i.e., the slower the molecular mobility the shorter the relaxation time [START_REF] Belton | Can Nuclear Magnetic Resonance Give Useful Information About the State of Water in Foodstuffs?[END_REF]. The relaxation time of a small molecule is thus always higher than the relaxation time of a large molecule. Moreover, the exchange between water protons and exchangeable protons from the molecule contained in the dry matter has to be taken into account for water. This exchange, known as chemical exchange, is one of the most efficient mechanisms of water relaxation [START_REF] Belton | The effects of diffusive exchange in heterogeneous systems on N.M.R line shapes and relaxation processes[END_REF]Hills, 1989b;a;[START_REF] Belton | Can Nuclear Magnetic Resonance Give Useful Information About the State of Water in Foodstuffs?[END_REF][START_REF] Denisov | Hydrogen exchange rates in proteins from water H-1 transverse magnetic relaxation[END_REF]. Consequently, water relaxation is always sensitive to water content and its value decreases with water content, explaining the more rapid decrease in the CPMG signal observed for the driest sample. The same mechanisms are also involved in the T 1 relaxation from the Saturation Recovery NMR sequence. The intensity measured at 2 s was lower for the driest sample and the recovery of the signal (shorter T 1 ) was faster for the driest sample.

The spin-spin relaxation distributions (T 2 ) from MEM obtained from fitting the CPMG curve are presented in Figure 3. For all samples, two or three peaks were extracted from the signal according to the sample dry matter. Two mechanisms can be proposed to explain such multiexponential relaxation behaviour. The first is based on the assumption of the chemical composition of the sample. For example, in the case of the water and fat mixture in an emulsion, a specific exponential can be attributed to each phase, i.e. water and fat [START_REF] Duval | Practical implications of the phasecompositional assessment of lipid-based food products by time-domain NMR[END_REF]. However, since our samples were mainly composed of water, this hypothesis did not hold. The second mechanism is based on the sample microstructure which may induce nonhomogeneous water distribution on a microscopic length scale. Indeed, when water exchange by diffusion between different compartments is slow compared to the relaxation time value, the averaging effect is not sufficient to yield a single relaxation time value, and several peaks are observed for the water relaxation. This multi-exponential relaxation for water has already been reported for many samples which exhibited restriction to the water diffusion. For example three peaks have been reported from the relaxation decay curve of vegetable tissues. These peaks were attributed to water in the vacuole, in the cytoplasm and in the cell wall, respectively [START_REF] Snaar | Probing water compartments and membrane permeability in plant cells by H 1 NMR relaxation measurements[END_REF][START_REF] Van Dusschoten | Extracting diffusion constants from echo-time-dependent PFG NMR data using relaxation-time information[END_REF][START_REF] Duval | NMR study of tomato pericarp tissue by spin-spin relaxation and water self-diffusion[END_REF][START_REF] Musse | An investigation of the structural aspects of the tomato fruit by means of quantitative nuclear resonance imaging[END_REF]. This mechanism therefore seems the most relevant to explain the multi-exponential behaviour of the relaxation.

For the sludge, depending on the dry matter, two or three peaks were extracted from the signal ( Figure 3-a). Their T 2 values covered a range from 5 to 60 ms. The sludge was composed of flocculated bacteria which induced heterogeneity in the water distribution. Indeed, water was always present in bacteria, in and around the flocs. Since water protons are thus in three different compartments, it is tempting to associate each relaxation time with a water compartment. Granular sludges have already been studied by NMR [START_REF] Lens | Use of H 1 NMR to study transport processes in sulfidogenic granular sludge[END_REF][START_REF] Lens | H 1 NMR characterisation of the diffusional properties of methanogenic granular sludge[END_REF], and a relaxing agent was used to lower the proton relaxation times in the solution. Relaxation times of 10-15 ms and 30 ms were attributed to methanogenic [START_REF] Lens | H 1 NMR characterisation of the diffusional properties of methanogenic granular sludge[END_REF] and sulfidogenic bacteria [START_REF] Lens | Use of H 1 NMR to study transport processes in sulfidogenic granular sludge[END_REF], respectively. Water in the granular matrix had a relaxation time of 40-100 ms [START_REF] Lens | H 1 NMR characterisation of the diffusional properties of methanogenic granular sludge[END_REF]. According to these results, the first peak could be related to the bacteria and the second to the water around the bacteria (Figure 3-a). However, a water proton from the solution could well contribute to the first or the second peak. Since no peak was found with a value close to the free water value (1.5-3 s), this means that there were some molecules in the sludge solution that lowered the overall relaxation of the proton of this compartment.

The polymer added for the flocculation could explain the decrease in the water relaxation time, but the effect of the iron chloride (used to dehydrate the sludge) has also to be taken into account.

Wood relaxation is well documented in the literature [START_REF] Hsi | Nuclear magnetic resonance relaxation study of water absorbed on milled northern white-cedar[END_REF][START_REF] Menon | An NMR Determination of the physiological water distribution in wood during drying[END_REF][START_REF] Araujo | Proton Magnetic-Resonance Techniques for Characterization of Water in Wood -Application to White Spruce[END_REF][START_REF] Araujo | Behaviour of Solid Wood and Bound Water as a Function of Moisture Content a Proton Magnetic Resonance Study[END_REF][START_REF] Hartley | Absolute Moisture Content Determination of Aspen Wood Below the Fiber Saturation Point Using Pulsed NMR[END_REF][START_REF] Xu | Proton spin-lattice relaxation in wood -T-1 related to local specific gravity using a fast-exchange model[END_REF]. Three main relaxation times were found related to water in cell wall fibrils (fast T 2 ), water in the ray and late wood tracheid lumens (medium T 2 ) and water in early wood tracheid lumens (fast T 2 ). Above the fiber saturation point (FSP), wood relaxation times are relatively constant over a wide range of water content [START_REF] Menon | An NMR Determination of the physiological water distribution in wood during drying[END_REF]. Below FSB, the relaxation times are a function of water content. The wood used in this study had a similar relaxation time distribution (Figure 3-b) as that reported for Western Red Cedar sapwood [START_REF] Menon | An NMR Determination of the physiological water distribution in wood during drying[END_REF]. At a water content of 55%, the mean relaxation times were 10 ms, 35 ms and 115 ms. The last two peaks were broad. Wood relaxation is sensitive to cell size and wood species. Since the wood used in this study was heterogeneous in origin, this would explain the peak shapes.

In a wood and sludge mixture there was water equilibrium between the two. After mixing, the sludge tended to loose water and the wood tended to gain water. Three typical relaxation distributions of wood and sludge mixtures are presented in Figure 3-c. The dry matter mixtures were 24%, 50% and 71%, respectively. Two or three peaks could be seen, the first being narrow (in the range of 0-10 ms), and the second peak broad, the range becoming larger with the increase in water content. Eventually, two broad peaks could be seen for the wettest samples. For the driest samples, the wood FSP was most probably not reached since no relaxation times were found above 40 ms. When the samples became wetter, the wood water content must have been above the FSP. Water filled the wood cells and then the relaxation time distribution could become broader. One important result in this study was that most of the T 2 relaxation distributions for wood and sludge were in the same range. Thus it was not possible to distinguish specific NMR signals from the sludge and the wood in the woodsludge relaxation distribution.

Using the Levenberg-Marquardt algorithm to fit the T 2 relaxation decay curves, the smallest residues were obtained with a three component fitting equation (equation 1). The three T 2 values were consistent with the T 2 distribution discussed above. Relaxation rates (R 2 = 1/T2) for the three components are plotted on Figure 4 for sludge samples (a) and for sludge and wood mixtures (b). For all samples, a constant decrease was observed with the increase in water mass over dry matter mass. Moreover, the variation in relaxation rate was the same for the three components. This demonstrates that all fractions were in equilibrium and were affected by the dehydration process in the same way, despite the micro heterogeneity in the water distribution in the sample. It should be noted that the same variation was observed for sludge dehydrated by air drying and by exchange with dry wood. These results confirm that the main effect on the water relaxation rate in the two samples was the dry matter content.

Spin-lattice relaxation times (T 1 ) were measured for wood and sludge mixtures. As for the spin-spin relaxation curves, the spin-lattice relaxation curves were fitted with MEM and the Levenberg-Marquardt algorithm (equation 2). The MEM distributions were composed of two or three peaks (Figure 5-a), the first peak being narrow (range 5-20 ms), the other peaks being broad (ranging from 15 ms to 250 ms). The Levenberg-Marquardt algorithm had the smallest residues for the three components. The difference between the two fitting methods obtained for some mixtures (two components from MEM and three from the discrete method) is explained by the wide distribution of the second peak. Indeed, this effect is known to induce discrepancies between the fitting methods [START_REF] Mariette | Continuous relaxation time distribution decomposition by MEM[END_REF]. The second peak obtained by MEM was decomposed into two discrete T 1 values with the discrete method. As for R 2 , a linear relationship in a log-log plot was observed between the spin-lattice relaxation rate R 1 and the water mass over dry matter mass (Figure 5-b). Moreover, this relationship was obtained for the three components. Only the T 2 sensitivity to water content will be considered for the following MRI measurement.

MRI

Two experimental cells were scanned with the whole body MRI scanner at different periods of biodegradation. In the mean time, the cells were monitored for biodegradation through respirometry measurements (Figure 6). Indeed, oxygen consumption is a good indicator of sludge biodegradation. The respirometric profiles obtained, that were similar for the two cells, are typical. The oxygen uptake rate increased quickly due to biodegradation of the most easily biodegradable substrate. Then, when all this matter had been degraded, a break in the exponential growth was observed. The oxygen consumption rate then decreased until all the biodegradable substrates had completely disappeared. During this decrease, the slowly biodegradable organic matter was hydrolysed before being consumed by micro-organisms.

After 27 days, consumption rate returned to ground state and respirometric study could then be stopped [START_REF] Tremier | A respirometric method for characterising the organic composition and biodegradation kinetics and the temperature influence on the biodegradation kinetics, for a mixture of sludge and bulking agent to be co-composted[END_REF][START_REF] Berthe | Coupling a respirometer and a pycnometer, to study the biodegradability of solid organic wastes during composting[END_REF].

Both respirometric cells were thus scanned at D0 before biodegradation, then cell 1 at D3 after the biodegradation peak, and finally cell 2 at D27 when biodegradation was low and constant. Examples of MR images at D0, D3 and D27 are presented in Figure 7. Images (a) and (b) and images (c) and (d) were taken at the same location in cell 1 and in cell 2, respectively. At D0, cell 1 (Figure 7-a) and cell 2 (Figure 7-c) images were similar: no differences between the MR images for the two cells could be seen. The wet pieces of wood covered with sludge were clearly visible. The black areas were the air space between the bulking agents. The contrast in the image will allow to implement image segmentation method to compute the inter porosity and the specific surface area. For larger pieces of wood, the edges of the wood tended to be more intense than the centres, showing that the water had not yet fully hydrated the centres of the wood. NMR relaxation experiments showed that this process required at least 10h (data not shown). After 3 days, the edge effect was less visible for cell 1 and the wood chips were even better defined ( Figure 7-b). MR images of cell 2 taken after 27 days were highly contrasted. The MR image (Figure 7-d) showed two distinct areas. Very low signal intensity was detected at the bottom of the image, compared with a high level signal intensity at the top. This was due to the fact that cell 2 became highly heterogeneous in water content in 27 days. The low intensity area had dry matter content of around 60-70%. The intensity of the mixture signal was too low to be fully detected with MRI. The other area had a dry matter content of around 25%, which demonstrated water transfer in the cell. At D3, the water content in cell 1 also began to be inhomogeneous, but to a lesser extent. The average dry matter content was 44%, two percent less than at D0. The contrast between D3 and D27 was thus due to the difference in dry matter content. MRI images were obtained with a spin echo sequence, the grey level intensities being a function of both relaxation times (T 1 , T 2 ) and the amount of water (see eq.3). Since the repetition time, T R , was 1 s and the largest spin-lattice relaxation time (T 1 ) found for the wettest wood and sludge mixture was 250 ms, then in equation 3,

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - - 1 1 T T R e β ~ 1
. Thus, the MR images were not T 1 weighted. Equation 3 can be rewritten for the three components as: Eq.4

Since the intensity of each component I i and the three T 2 relaxation times were strongly dependent on the water content of the sample, the global signal intensity should consequently also be directly related to water content in the mixture. This direct relationship was checked by taking samples from cell 1 at D3 and from cell 2 at D27 at different heights in the cells where MR images showed inhomogeneity. NMR and moisture content measurements were performed on these samples. Signal intensities at 8 ms were extracted from the NMR CPMG experiments and plotted against dry matter (Figure 8). As expected, the intensities decreased with dry mater content. Despite the biological activity during the 27 days of storage, no biodegradation effect was observed. The results provide evidence that the effect of biological activity on the NMR signal of wood and sludge mixtures is negligible with regard to dry matter content. The decay was non-linear. Indeed, with an 8 ms echo time, the multiexponential behaviour introduced a weighting from the different components. We demonstrated that the grey levels of the MRI images were directly related to dry matter content when the water content was above 40-30%. Below this value the signal was too low to be detected with this MRI protocol.

MRI has been used previously to monitor wood decay by fungus [START_REF] Muller | Detection of incipient fungal attack in wood using magnetic resonance parameter mapping[END_REF]: indeed biological activity means a local increase in water content, which leads to a local change in relaxation times and intensities. In this experiment, respirometry showed that biodegradation occurred and both the NMR measurements and the MR images showed changes in the dry matter content. MR images gave 3D representation of the heterogeneity of dry matter content in the cells after 27 days of biodegradation ( Figure 9). Heterogeneity was both vertical and horizontal. Biodegradation may contribute to the heterogeneity of the dry matter, but the main cause seems to be related to the cell design. The dried area is above the tube where air flows into the cell. The air rate must be too fast and then has a drying effect. The humid area is located under the tube where air flows out of the cell (Figure 2). Even with the water trap, some water seems to condense in this tube and then drops to the wood and sludge mixture.

Thus, these MR images cannot be taken as proof of biodegradation activity. Nevertheless, they will be used to improve the design of the respiratory cell.

Conclusion

We have shown that the NMR water relaxation for wood and sludge exhibited a multiexponential relaxation time behaviour which can be explained by the microstructure of the matrix. Moreover, a high dry matter content effect on the relaxation was observed for sludge samples and for sludge and wood mixtures. This effect was the same whatever the relaxation time component, which confirmed that the dry matter content had the main effect on the NMR parameters. Wood and sludge mixtures were scanned successfully by MRI using a spin echo sequence. The grey level intensities were correlated with the dry matter content, thus demonstrating the feasibility of MRI to quantify the water distribution in respiratory cells.

Moreover, image resolution was good enough for image analysis. Since voxel size was much smaller than pore volume, porosity and specific surface area should be available.

Figure 1 :

 1 Figure 1 : Diagram of respirometric apparatus

Figure 3 :

 3 Figure 3 : Spin-spin relaxation time (T 2 ) distributions. Adjustments were made with the MEM

Figure 4 :

 4 Figure 4 : Three component spin-spin relaxation rates (1/T 2 =R 2 ) for (a) sludge samples and

Figure 5 :

 5 Figure 5 : Spin-lattice relaxation time (T 1 ) distributions for mixtures of sludge and wood with

Figure 6 :

 6 Figure 6 : Oxygen uptake rate over time for the two respirometric cells used in the combined

Figure 7 :

 7 Figure 7 : MRI images of respirometric cells 1 and 2 taken with a spin echo sequence (T E = 8

Figure 8 :

 8 Figure 8 : Spin-spin relaxation intensities normalised by sample mass extracted from CPMG

Figure 9 :

 9 Figure 9 : MRI images of respirometric cell 2 at D27, taken with a spin echo sequence (TE =

  Figure 4

  Figure 7

Figure 9

 9 Figure 9

CemOA : archive ouverte d'Irstea / Cemagref

Acknowledgments 388 389 The authors thank Mylene Launay and Mireille Cambert for their technical assistance.