Alejandra Libia

Sonia García-Flores

Pedro Medina

Camille Martínez-Hernández

Jean-Marie Oger

Thierry Galano

Teresa Durand

Federico Casas-Pina

Ángel Ferreres

Gil-Izquierdo

García-Flores

Sonia Medina

Pedro Martínez-Hernández

Camille Oger

Jean-Marie Galano

Thierry Durand

Teresa Casas-Pina

Federico Ferreres

Ángel Gil- Izquierdo

Snapshot situation of oxidative degradation of the nervous system, kidney, and adrenal glands biomarkers-neuroprostane and dihomo-isoprostanesurinary biomarkers from infancy to elderly adults

Keywords: Oxidative stress Lipid peroxidation Neuroprostanes Dihomo-isoprostanes Age Urine biomarkers OS, (Oxidative stress), ROS, (Reactive oxygen species), PUFAs, (Polyunsaturated fatty

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Introduction

Biomarkers have been increasingly employed in empirical studies of human populations to understand physiological processes that change with age, diseases whose onset appears linked to age, and the aging process itself [START_REF] Crimmins | Biomarkers related to aging in human populations[END_REF]. The free radical/oxidative stress theory of aging is the most popular explanation of how aging occurs at a molecular level in aerobic biological organisms [START_REF] Valavanidis | The controversy for the validity of the free radical or oxidative stress theory of ageing: recent scientific evidence for oxidative damage at molecular level, animals and ageing populationbased studies[END_REF]. This theory of aging consisted of agerelated biochemical and physiological decline associated with cumulative oxidative damage to cellular components and tissues, promoting oxidative stress (OS) and leading to lesser longevity [START_REF] Harman | Aging: a theory based on free radical and radiation chemistry[END_REF]. Nowadays, this theory is controversial since there may be interventions independent of reactive oxygen species (ROS) that promote longevity without affecting ROS or OS [START_REF] Valavanidis | The controversy for the validity of the free radical or oxidative stress theory of ageing: recent scientific evidence for oxidative damage at molecular level, animals and ageing populationbased studies[END_REF][START_REF] Vina | The free radical theory of aging revisited: the cell signaling disruption theory of aging[END_REF]. Oxidative stress is widely accepted to be a perturbation in the balance of free radicals in a cell and the cell´s ability to cope with the change by means of its antioxidant defense mechanisms [START_REF] Venkataraman | Oxidative stress in aging-matters of the heart and mind[END_REF]. The balance between ROS production and antioxidant defenses determines the degree of OS according to Finkel and Holbrook [START_REF] Finkel | Oxidants, oxidative stress and the biology of ageing[END_REF]. Recently, it has been reported that mild stress stimulates endogenous defense systems, which will promote health, but if the stress becomes chronic or is too extensive, it induces cellular damage and/or aging and a shortening of lifespan [START_REF] Simm | Stress and biological aging: a double-edged sword[END_REF].

The brain and nervous system are prone to OS and are inadequately equipped with antioxidant defense system, which can lead to persistently increased levels of ROS and reactive nitrogen species reacting with the various target molecules (proteins, lipids, and DNA) [START_REF] Halliwell | Oxidative stress and neurodegeneration: where are we now?[END_REF]. The lipids, especially polyunsaturated fatty acids (PUFAs), are vulnerable to oxidation by both enzymatic and non-enzymatic process. In humans, the products of lipid peroxidation have been accepted as toxic mediators, but they are also known to exert diverse biological effects [START_REF] Niki | Biomarkers of lipid peroxidation in clinical material[END_REF]. Solberg et al. [START_REF] Solberg | Resuscitation with supplementary oxygen induces oxidative injury in the cerebral cortex[END_REF] mentioned that the determination of OS is complex and requires a quantification of the levels of free radicals or damaged biomolecules. The measurement of F 2 -isoprostanes (F 2 -IsoPs) by mass spectrometry has been extensively employed as a marker of oxidant stress and is widely considered to be the goldstandard index of lipid peroxidation in vivo. The measurement of free F 2 -IsoPs in plasma or urine can be utilized to assess the endogenous formation of IsoPs but not to reveal the organ in which they are formed. Unless determining the levels of IsoPs in the cerebrospinal fluid, which reflects the ongoing metabolic activity of the brain, provides a great opportunity to reveal the occurrence of OS and lipid peroxidation in the brain. However, there are now some IsoPs-like compounds that might be regarded as markers of lipid peroxidation of the nervous system [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF]. The F 2-dihomo-isoprostanes (F 2-dihomo-IsoPs), F 3 -neuroprostanes (F 3 -NeuroPs) and F 4 -neuroprostanes (F 4 -NeuroPs) are used to analyze the OS status of the nervous system in humans [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF][START_REF] Galano | and othersIsoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF][START_REF] Vigor | Non-enzymatic lipid oxidation products in biological systems: assessment of the metabolites from polyunsaturated fatty acids[END_REF]. These biomarkers are formed by a free radical nonenzymatic mechanism from adrenic acid (C22:4 n-6, AdA) [START_REF] Vanrollins | F2-dihomoisoprostanes arise from free radical attack on adrenic acid[END_REF], docosapentaenoic acid (C22:5 n-6, DPA) [START_REF] Auvinet | Total synthesis of 4-F3tneuroprostane and its 4-epimer[END_REF] and docosahexaenoic acid (C22:6 n-3, DHA) [START_REF] Roberts 2nd | Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid[END_REF] respectively. While DHA is 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 4 -NeuroPs provide a specific quantification of the OS suffered by neural membranes in vivo [START_REF] Roberts 2nd | The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid peroxidation[END_REF] and F 2 -dihomo-IsoPs are potential markers of free radical damage to myelin in the human brain [START_REF] Vanrollins | F2-dihomoisoprostanes arise from free radical attack on adrenic acid[END_REF]. Recent efforts have focused on the assessment of F 2-dihomo-IsoPs, F 3 -NeuroPs, and F 4 -NeuroPs as biomarkers in conditions associated with increased OS (particularly in disease conditions) and/or after dietary supplementation with antioxidants [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF][START_REF] Galano | and othersIsoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF][START_REF] Vigor | Non-enzymatic lipid oxidation products in biological systems: assessment of the metabolites from polyunsaturated fatty acids[END_REF][START_REF] De Felice | F(2)-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome (and others)[END_REF][START_REF] Gao | Formation of F-ring isoprostane-like compounds (F3-isoprostanes) in vivo from eicosapentaenoic acid[END_REF][START_REF] Medina | and othersDihomo-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]. Despite its increasing clinical us to the best of our knowledge the biological variation of these biomarkers in healthy people of different age has not been reported yet. The ability to quantify these compounds in non-invasive samples like urine could shed light on the changes in excretion values of products of lipid peroxidation across a wide age range and may be useful for comparing these values detected in healthy individuals with those obtained diseased individuals. Therefore, the aim of this cross-sectional study was to quantify biomarkers of lipid peroxidation in the nervous system (F 2 -dihomo-IsoPs, F 3 -NeuroPs, and F 4 -NeuroPs) in urine samples from healthy volunteers of different life stage (4-88 years), analyzing possible associations between their values and age intervals.

Materials and methods

Study population

This study was conducted in accordance with the Helsinki declaration. Approval was obtained from the Bioethics Committee of the University Hospital of Murcia. The participants were insured from the Hospital Virgen de la Arrixaca (Murcia, Spain) aged between 4 and 88 years of both genders (n=158). Age was reported at the time of the household interview as the age (years) at the last birthday. The assignment of the age ranges was based on social aging processes (childhood, adolescence, young adulthood, middle-aged adults, and elderly adults) according to Settersten and Mayer [START_REF] Settersten | The Measurement of Age, Age Structuring, and the Life Course[END_REF]. The age categories used in our statistical analyses were 4-12 years (childhood, n=20; mean: 8.20 ± 2.50 years), 13-17 years (adolescence, n=14; mean: 15.73 ± 1.43 years), 18-35 years (young adulthood, n=45; mean: 27.62 ± 4.97 years), 36-64 years (middle-aged adults, n=58; mean: 49.12 ± 9.03 years) and 65-88 years (old age, n=21; mean: 75.61 ± 6.62 years). All volunteers signed the informed consent document (18-88 years old). Volunteers under the age of 18 years had all referred to a doctor clinic for a routine check-up and a parent signed the informed consent document.

Regarding the exclusion criteria, individuals with chronic diseases, under drug treatments, and volunteers with overweight or obesity were excluded from the study. None of the subjects was cigarette smoker an alcoholic and pregnant. All the participants were submitted to clinical examination to confirm their health status. The health status of the participants was considered in the data analysis. The clinical parameters for determining the health status of the individuals are summarized in Supporting information 1.

Sample collection and preparation

A complete clinical analysis -consisting of hematology, chemistry, and urine chemical analysis -was performed in the volunteers. All samples (blood and urine) were collected, by a nurse at the University Hospital Virgen de la Arrixaca from the subjects early in the morning and under fasting conditions. Blood samples at rest were obtained by venipuncture and were placed in different tubes according to the analytical procedures. The samples were processed within 1 h of collection and stored at -80 °C for the analytical determinations. The hematological parameters were recorded using an automated hematological analyzer (Cell Dyn 3700 and 4000, Abbott, IL, USA) at the clinical analysis service of the University Hospital Virgen de la Arrixaca (Murcia, Spain). One-milliliter from the 24-h urine was used for analysis of the lipid peroxidation biomarkers. The metabolites were normalized as ng mg -1 creatinine and were assayed using the method described by Medina et al. [START_REF] Medina | and othersDihomo-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]. Clinical parameters results of our volunteers (mean ± standard deviations (SD)) are summarized in Table 1.

Chemicals and Standards

Six NeuroPs (4(RS)-4-F 4t -NeuroP, 4-F 4t -NeuroP, 10-epi-10-F 4t -NeuroP, 10-F 4t -NeuroP, 4-epi-4F 3t -NeuroP and 4-F 3t -NeuroP); as well as, four F 2-dihomo-IsoPs (ent-7-(R)-7-F 2t -dihomo-IsoP, ent-7-epi-7-F 2t -dihomo-IsoP, 17-F 2t -dihomo-IsoP, and 17-epi-17-F 2t -dihomo-IsoP) were analyzed in this experiment and 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. 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] 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]. β-glucuronidase, type H2 from Helix pomatia and BIS-TRIS (Bis-(2hydroxyethyl)-amino-tris (hydroxymethyl)-methane) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All LC-MS grade solvents were from J.T. Baker (Phillipsburg, NJ, USA). Strata X-AW (100 mg 3 mL -1 ) solid phase extraction cartridges were purchased from Phenomenex (Torrance, CA, USA).

UHPLC-QqQ-MS/MS analyses

The separation of NeuroPs and F 2 -dihomo-IsoPs in the urine samples was performed with an Ultra High-Performance Liquid Chromatography 6460-Triple Quadrupole-tandem Mass Spectrometry (Agilent Technologies, Waldbronn, Germany), using the set up previously described by Medina et al. [START_REF] Medina | and othersDihomo-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]. Data acquisition and processing were performed using Mass Hunter software version B.04.00 (Agilent Technologies, Waldron, Germany). The identification and quantification of NeuroPs and F 2 -dihomo-IsoPs were carried out using the authentic markers described by Medina et al. [START_REF] Medina | and othersDihomo-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

Quantitative data are presented as mean ± SEM (standard error of the mean) or SD (Table 2). Concerning the study population, women and men were analyzed together because no difference between them was detected according to the Student's t-test (data not shown). The Kolmogorov-Smirnov test and Shapiro-Wilk test were applied to assess the distribution of the data. Normality was not established, so nonparametric statistical tests were used for intergroup comparison. Comparison of non-normally distributed groups was carried out using the non-parametric Kruskal-Wallis test. In order to identify differences between the five groups, the Mann-Whitney U test was conducted with a Bonferroni correction, resulting in a significance level set at P < 0.005. Correlation between the variables (F 2 -dihomo-IsoPs) and age (years) was determined using Spearman´s correlation. The statistical analyses were made using the SPSS 21.0 software package (LEAD Technologies, Inc. Chicago, USA). The graphs were plotted using the Sigma Plot 12.0 software package (Systat Software, Inc. Sigma Plot for Windows).

Results

In this study four F 2 -dihomo-IsoPs stereoisomers (ent-7-(R)-7-F 2t -dihomo-IsoP, ent-7-epi-7-F 2t -dihomo-IsoP, 17-F 2t -dihomo-IsoP, 17-epi-17-F 2t -dihomo-IsoP) were detected in the volunteers' urine in all life-stages, and 4-F 3t -NeuroP (derived from n-6 DPA) was detected in the three groups of older volunteers (Table 2). We want to emphasize that the F 2 -dihomo-IsoPs were detected in the samples from all volunteers of the different life stages (from childhood to old age). The F 2 -dihomo-IsoPs (data for all volunteers) were correlated with age (4-88 years); the Spearman correlations were: 17-epi-17-F 2t -dihomo-IsoP, Rho=0.062, significant level (sig.)=0.448; 17-F 2t -dihomo-IsoP, Rho=0.940, sig.=0.253; ent-7-(R)-7-F 2t -dihomo-IsoP, Rho=0.264, sig.=0.001; ent-7-epi-7-F 2t -dihomo-IsoP, Rho=0.187, sig.=0.021; and total dihomo-IsoPs, Rho=0.197, sig.=0.015 (Fig. 1).

In order to provide a snapshot overview of the values of F 2 -dihomo-IsoPs in healthy volunteers, we have studied the most representative stages of human growth and development. To determine whether there were statistically significant differences between the groups, a nonparametric analysis of population medians (Kruskal-Wallis test, P < 0.05) was performed for each individual analyte (Table 2), as well for the total sum of the four F 2 -dihomo-IsoPs (Fig. 2). There was a statistically significant difference in 17-F 2t -dihomo-IsoP, ent-7- A) Dihomo-Isoprostane. Data presented as mean ± SEM (Standard error of the mean) ng mg -1 creatinine. Kruskal-Wallis test, χ2: Chi-square (degree of freedom), and P < 0.05 was significant. Abbreviations: W: women and M: men. B) Neuroprostane. Data presented as means ± SD (standard deviation) ng mg -1 creatinine. Abbreviations: n/d: not detected. No data on neuroprostanes were found with the only exception of 4-F 3t -NeuroP, which was found in a reduced number of volunteers within the experimental groups: young adults (a, detected in 3 women and 7 men), middle age adults (b, detected in 9 women and 6 men), and in elder volunteers (c, detected in 2 women and 6 men).

-(R)-F 4t -NeuroP n/d - n/d - n/d - n/d - n/d - - n-3 10-epi-10-F 4t -NeuroP n/d - n/d - n/d - n/d - n/d - - 10-F 4t -NeuroP n/d - n/d - n/d - n/d - n/d - - 4-F 4t -NeuroP n/d - n/d - n/d - n/d - n/d - - DPA n-6 4-epi-4F 3t -NeuroP n/d - n/d - n/d - n/d - n/d - - 4-F 3t
(R)-7-F 2t -IsoP, ent-7-epi-7-dihomo-IsoP, and total F 2 -dihomo-IsoPs when comparing the five groups. Thus, having analyzed the five groups, we used the results for the sum of these four F 2 -dihomo-IsoPs, since significant P-values were found for most of the biomarkers. The total F 2 -dihomo-IsoPs ranged from ~8.02 ng mg -1 creatinine (age group of 13-17 years old) to ~12.28 ng mg -1 creatinine (age group of 65-88 years old) (Fig. 2). The Mann-Whitney U test showed that among the first stage (childhood) and the later stages of life (middle-aged adults and elderly adults) the F 2 -dihomo-IsoPs values did not differ statistically; however, it should be noted that the elderly volunteers showed high values comparing to middle-aged. In the middle stage of life (adolescence and young adults) a significant decrease in the levelscompared to the first and last stages of life -was detected (P < 0.005) (Fig. 2).

Discussion

The measurement of OS biomarkers using a lipidomics approach has been useful for to compare values detected in a healthy population with those affected with different pathologies [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF][START_REF] Galano | and othersIsoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF][START_REF] Vigor | Non-enzymatic lipid oxidation products in biological systems: assessment of the metabolites from polyunsaturated fatty acids[END_REF][START_REF] De Felice | F(2)-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome (and others)[END_REF][START_REF] Gao | Formation of F-ring isoprostane-like compounds (F3-isoprostanes) in vivo from eicosapentaenoic acid[END_REF][START_REF] Medina | and othersDihomo-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]. To the best of our knowledge, this is the only study that evaluates F 2 -dihomo-IsoPs and F 4 -NeuroPs in a large population of healthy volunteers. The results obtained by correlation tests showed a small but positive association between the concomitant increases of urinary F 2 -dihomo-IsoPs (total sum) with the age (Fig. 1). Previously, in female volunteers (n=43), no statistically significant correlation (r=0.0841, P=0.40) between age and plasmatic values of F 2 -dihomo-IsoPs (ent-7(R)-F 2tdihomo-IsoP and 17-F 2t -dihomo-IsoP) was observed; the average age was 13.9 ± 6 years (range 1.5-32 years) and the values were 1.0 ± 0.11 pgmL -1 [START_REF] De Felice | F(2)-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome (and others)[END_REF]. Our study, with a wider age range and using data from women and men, showed that age may be related to changes in the excretion of F 2 -dihomo-IsoPs. In addition, individually, ent-7-epi-7-F 2t -dihomo-IsoP and ent-7-(R)-7-F 2t -dihomo-IsoP also exhibited a slight correlation with age. VanRollins et al. [START_REF] Vanrollins | F2-dihomoisoprostanes arise from free radical attack on adrenic acid[END_REF] mentioned that AdA is the most important PUFA in white matter and suggested the quantification of F 2 -dihomo-IsoPs as help to clarify the in vivo contributions of free radical to myelin and axons damage in white matter damage because of aging. Augmented levels of lipid peroxidation and myelin breakdown have been demonstrated in the myelin of older (normal) individuals, compared to younger. Age-related myelin alterations are ubiquitous and the correlations between their frequency and impairments of cognition occur because the conduction velocity along the affected nerve fibers is reduced so that the normal timing sequences within neuronal circuits breakdown [START_REF] Bartzokis | Age-related myelin breakdown: a developmental model of cognitive decline and Alzheimer's disease[END_REF][START_REF] Peters | Aging and the myelinated fibers in prefrontal cortex and corpus callosum of the monkey[END_REF]. Previous studies also showed an age-related increase in lipid peroxidation in humans and other animals [START_REF] Andriollo-Sanchez | Age-related oxidative stress and antioxidant parameters in middle-aged and older European subjects: the ZENITH study[END_REF][START_REF] Cui | Oxidative stress, mitochondrial dysfunction, and aging[END_REF][START_REF] Poon | Free radicals: key to brain aging and heme oxygenase as a cellular response to oxidative stress[END_REF][START_REF] Radak | Age-associated neurodegeneration and oxidative damage to lipids, proteins and DNA[END_REF][START_REF] Rizvi | Markers of oxidative stress in erythrocytes during aging in humans[END_REF]. Besides being a specific component of myelin, AdA also is present in several organs and tissues, including the kidney and the adrenal gland [START_REF] Vanrollins | F2-dihomoisoprostanes arise from free radical attack on adrenic acid[END_REF][START_REF] De Felice | F(2)-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome (and others)[END_REF]. A study in rats also showed evidence of age-related lipid peroxidation in adrenals [START_REF] Almeida | Age-related changes in lipid peroxidation products in rat adrenal gland[END_REF]. Therefore, our results also may reflect a positive correlation between non-enzymatic oxidation within the kidney and adrenal glands and advanced age.

The precise age of transition among the stages of life is heavily debated and this can vary from person to person [START_REF] Prenderville | Adding fuel to the fire: the impact of stress on the ageing brain[END_REF]. In order to provide a snapshot overview of the values of F 2 -dihomo-IsoPs in healthy volunteers, we have studied the most representative stages of human growth and development. The growth from childhood into adolescence has been associated with biological changes and could be influenced by the endocrinal and biochemical conditions generated in the pre-pubertal period. Our results are consistent with those obtained by Tamura et al. [START_REF] Tamura | Evaluation of a urinary multi-parameter biomarker set for oxidative stress in children, adolescents and young adults[END_REF] and Kaneko et al. [START_REF] Kaneko | Measurement of urinary 8-oxo-7,8-dihydro-2-deoxyguanosine in a novel point-ofcare testing device to assess oxidative stress in children[END_REF]. These authors studied urinary biomarkers produced under OS from lipids, proteins, DNA, and carbohydrates, and underlined that younger subjects (under the age of 10) were more vulnerable to oxidation than adolescent subjects, since they grow up rapidly, activation of the immune system and are probably exposed to high concentrations of ROS and nitric oxide. When we compared the F 2 -dihomo-IsoPs values of the young and middle-aged adults, higher OS values in the latter were apparent (Fig. 2). In humans, [START_REF] Harman | Free radical theory of aging: an update: increasing the functional life span[END_REF] according to the free radical theory of aging, the inborn aging process produces changes that increase in an exponential manner with age, becoming the major risk factor for disease and death in humans after the age of 28 years in developed countries. In our study, the volunteers aged from 36 to 88 years were divided into two groups -middle-aged (36-64 years) and elderly adults (65-88 years) -that did not differ significantly although, the values provided an ascending trend concomitantly with the age as is observed in Fig. 2.B. In the ZENITH study, European free-living, healthy, older adults (70-88 years) did not appear to be exposed to acute OS [START_REF] Andriollo-Sanchez | Age-related oxidative stress and antioxidant parameters in middle-aged and older European subjects: the ZENITH study[END_REF]. Our results showed a similar slow decline in the antioxidant status of elderly, healthy, free-living adults, and this may be one of the reasons why the values obtained for our group of older adults did not differ significantly from those of the middle-aged group. Currently, it is not clear which levels should be considered "normal" and which represent a serious imbalance between ROS generation and antioxidant defense. Some scientists reported that OS is an adaptation in the aging process and is not so harmful [START_REF] Valavanidis | The controversy for the validity of the free radical or oxidative stress theory of ageing: recent scientific evidence for oxidative damage at molecular level, animals and ageing populationbased studies[END_REF][START_REF] Vina | The free radical theory of aging revisited: the cell signaling disruption theory of aging[END_REF]. But, if the damage accumulates throughout the entire lifespan, as a by-product of normal cellular processes or a consequence of inefficient repair systems, this could lead to the diseases associated with the elderly [START_REF] Radak | Age-associated neurodegeneration and oxidative damage to lipids, proteins and DNA[END_REF][START_REF] Farooqui | Aging: an important factor for the pathogenesis of neurodegenerative diseases[END_REF]. On the other hand, Soares et al. [START_REF] Soares | How can age and lifestyle variables affect DNA damage, repair capacity and endogenous biomarkers of oxidative stress?[END_REF] highlighted the impact of the lifestyle (diet, environment, lifestyle among others factors of life-stage as physiological and/ or metabolic process) has an important role in the accumulation of oxidative damage or in the OS increase apart from growing older. The F 2 -dihomo-IsoPs have not been used previously as a biomarker of aging; they have been associated mainly with diseases in the elderly, adolescents, and children. Therefore, the normal values in our adults might be useful in subsequent comparisons evaluating OS progression in groups of older individuals.

Finally, regarding NeuroPs derived from DPA or DHA, these lipid metabolites were not detected in samples from healthy and sedentary volunteers. Only 4-F 3t -NeuroP, derived from omega 6-DPA, was detected, in a few volunteers in the age range from 18 to 65 years, who represents young adults, middle-aged adults, and elderly volunteers (Table 2). The 4-epimer of 4-F 3t -NeuroP was not detected in the urine samples, although its values may have been below the limits of detection (2.95 ng mL -1 ) and quantification (5.9 ng mL -1 ) [START_REF] Medina | and othersDihomo-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] since each is 4-fold higher than the corresponding limit found for 4-F 3t -NeuroP. The DPA-NeuroPs were synthesized with the aim of understanding their role in a ω3-depleted organism, as well as to broaden and deepen the study of neuronal OS [START_REF] Auvinet | Total synthesis of 4-F3tneuroprostane and its 4-epimer[END_REF]. In our study the metabolites of DHA n-3 peroxidation (4(RS)-4-F 4t -NeuroP, 4-F 4t -NeuroP, 10-F 4t -NeuroP, and 10-epi-10-F 4t -NeuroP) were not detected in the urine samples, suggesting that their values may also have been below the limit of detection. The physiological role of lipid peroxidation is not fully understood; some authors have shown that lipid peroxidation in various organs, including the brain, increases with aging and it is considered a risk factor for neurodegenerative diseases [START_REF] Niki | Biomarkers of lipid peroxidation in clinical material[END_REF][START_REF] Radak | Age-associated neurodegeneration and oxidative damage to lipids, proteins and DNA[END_REF]. Thus, further investigation is needed, regarding how or why age or the process of aging can influence the oxidative status of the central nervous system and whether the excretion of oxidative products derived from DPA n-6 and DHA n-3 is scarce in the healthy population, according to age.

Conclusions

A significant, positive correlation was found between the increase in age (4-88 years) and the values of ent-7-(R)-7-F 2t -dihomo-IsoP, ent-7-epi-7-F 2t -dihomo-IsoP, and total F 2 -dihomo-IsoPs. By dividing the participants into five groups according to social aging processes, we have been able to provide evidence for a decrease in total F 2 -dihomo-IsoPs during adolescence and the young-adult stage, when compared to childhood. The observation of a spurt in the total F 2 -dihomo-IsoPs values from the young adults to middle-aged adults is interesting, as is the fact that the elderly group did not show higher values than the middle-aged population. The findings of this work suggest that in healthy and sedentary the values of urinary F 4 -NeuroPs and F 3 -NeuroPs were not representative. We are conscious of the limitations

Fig. 1 .

 1 Fig. 1. Scatterplot showing: A) Ent-7-epi-7-F 2t -dihomo-IsoP, B) Ent-7-(R)-7-F 2tdihomo-IsoP, and C) the total sum of F 2 -dihomo-IsoPs (ng mg -1 creatinine in urine) versus age (years) in our volunteers. The Spearman rank correlation coefficient (Rho) and P-value are shown.

Fig. 2 .

 2 Fig. 2. The total F 2 -dihomo-IsoPs content in urine of volunteers (ent-7(R)-7-F 2t -dihomo-isoprostane, ent-7-epi-7-F 2t -dihomo-isoprostane, 17-F 2t -dihomo-isoprostane, and 17epi-17-F 2t -dihomo-isoprostane). The values are expressed as ng mg -1 creatinine and are categorized according to age range. A) Dot plot of the values from all volunteers, by life-stage. Significant P-values (≤0.005) from the Mann-Whitney U test are shown. B) Trend chart plot showing the averages of the values by life-stage.

Table 1

 1 Biochemical parameters of the volunteers categorized by their life-stage group (n=158).

			Childhood		Adolescence		Young adults		Middle-aged		Elderly
			(4-12 year)		(13-17 years)	(18-35 years)	(36-64 years)	(≥65 years)
	Clinical parameters		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
	Plasma Analysis	Glucose (mg dL -1 )	84.6	5.1	78.5	24.9	84.1	6.1	93.7	12.1	96.7	8.4
		Urea (mg dL -1 )	29.2	4.8	23.7	8.6	30.5	10.0	33.7	6.4	36.2	6.5
		Creatinine (serum) (mg dL -1 )	0.5	0.1	0.7	0.3	0.8	0.2	0.8	0.1	0.8	0.2
		Uric acid (mg dL -1 )	3.9	0.9	4.5	1.5	4.6	1.1	4.7	1.2	4.8	1.0
		Cholesterol (mg dL -1 )	170.4	24.3	126.0	45.0	172.7	74.4	173.2	22.3	173.3	23.8
		Total bilirubin (mg dL -1 )	0.5	0.3	0.5	0.2	0.5	0.2	0.5	0.2	0.5	0.2
		Triglyceride (mg dL -1 )	51.4	13.4	73.7	37.8	85.1	40.2	96.5	33.3	98.5	43.5
		Transaminase (ALT) (U/L)	17.8	3.9	11.7	4.4	20.7	9.0	20.9	5.6	19.1	6.4
		Transaminase (AST) (U/L)	25.7	4.2	18.0	6.2	20.0	5.5	20.9	5.6	20.6	4.2
	Blood count	Red blood cells (RBCs) (x 10 6 µL)	4.8	0.2	4.6	1.5	4.9	0.4	4.8	0.5	4.7	0.4
		Leukocytes (x10 3 µL)	6.4	1.2	6.7	2.3	7.5	1.6	6.9	1.2	6.3	1.2
		Haemoglobin (g dL -1 )	14.1	0.6	13.4	4.3	14.8	1.3	14.5	1.4	14.5	1.0
		Haematocrit (%)	41.1	1.8	40.3	13.1	44.2	4.2	43.6	1.4	43.7	3.0
		Platelet (x10 3 µL)	273.8	42.4	234.9	81.0	245.8	49.4	263.9	50.8	240.5	51.2
	Urine Analysis	Abnormal results and sediment	Negative		Negative		Negative		Negative		Negative

Data are represented as means ± SD. Childhood, n: 20; Adolescence, n: 14; Young adults, n: 45; middle-aged adults, n: 58; and elderly adults, n: 21.

Table 2

 2 Urinary F 4-neuroprostanes, F 3 -neuroprostanes, and F 2 -dihomo-isoprostanes (ng mg -1 creatinine) determined in the five study groups of the most representative life-stages in humans.

			Childhood	Adolescence	Young adults	Middle-aged	Elderly		Kruskal-Wallis
			(4-12 year)	(13-17 years)	(18-35 years)	(36-64 years)	(≥65 years)
		ng/mg creatinine	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	χ 2	Sig
	From	Number in age groups (W/M) 20 (9/11)	14 (7/7)		45 (23/22)	58 (31/27)		21 (10/11)
	A)	17-epi-17F 2t -dihomo-IsoP	2.14	0.18	1.63	0.22	1.68	0.12	1.74	0.11	1.93	0.18	8.37(4)		0.079
	AdA	17-F 2t -dihomo-IsoP	1.74	0.14	1.34	0.17	1.41	0.09	1.53	0.08	1.71	0.15	14.67(4)	0.005
	n-6
		ent-7-(R)-7-F 2t -dihomo-IsoP	4.45	0.28	3.05	0.34	3.48	0.19	4.56	0.17	5.26	0.28	21.85(4)	≤0.001
		ent-7-epi-7-F 2t -dihomo-IsoP	3.49	0.44	1.98	0.53	2.51	0.30	3.08	0.27	3.29	0.46	29.86(4)	≤0.001
			Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
	B) DHA	4

Acknowledgments

LAGF was awarded a pre-doctoral FPI fellowship (BES2012-060185) by the Spanish government. The authors are grateful to the Clinical Analysis Service of the University Hospital Virgen de la Arrixaca, Murcia (Spain) for its collaboration. We are grateful to Dr. David Walker (native English speaker) for his reviews of the English grammar and style of the current report.

Funding

This work was partially funded by the "Fundación Séneca de la Región de Murcia" Grupo de Excelencia 19900/GERM/15.

inherent in the performance of such analysis in healthy humans due to wide heterogeneity in the expression of OS biomarkers, as a consequence of aging. Nevertheless, this study in a matrix as human urine represents a powerful approach to advance our knowledge of the role of OS in the central nervous system across a wide age range and so it could serve as a baseline for future clinical studies in populations with different disorders.

Declarations of interest

The authors declare that they have no conflict of interest.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.redox.2017.01.008.


SSA 10 Pertuis charentais Freshwater allocation in the Charente river catchment SSA 10 Pertuis charentais Freshwater allocation in the Charente river catchment

Simulation results: oyster production

The policy issue concerns the quantitative management of the freshwater in the Charente river basin. This problem has been addressed by the regional plan for water management (SDAGE), which includes a "Water shortage Management Plan" (PGE) dedicated to the Charente river.

The SDAGE and the PGE have fixed the following: -the hierarchy of the freshwater uses = 1) good ecological status of the coastal ecosystems 2) drinking water for households 3) other uses: agriculture, shellfish farming… -Reachable Discharge Thresholds (RDT) at different control points, which are supposed to be sufficient to ensure the 2 first uses; the general objective of this management plan will be to make sure that the system is able to reach the RDTs during the summer at least 8/10 years.

The current debate aims at modifying the "authorised volumes of water" for each uses (consumptive uses: drinking water, irrigation) and improving the restriction rules.

Contact us: Jean.Prou@ifremer.fr, Remi.Mongruel@ifremer.fr & Francoise.Vernier@cemagref.fr

Extend simulation platform 4. Model calibration

The blocks and modules are organised in a user-friendly way. Graphical tools improve the visualisation of relevant indicators. Scenario, display and output blocks have been set to regroup all the model control panels.

Each module is linked to a corresponding database that records all model parameters and state variables. The model has been developped using Hierarchical and Custom blocks in ExtendSim ModL language. The above schematic presents the internal feedback loop which brings together the irrigation practices of farmers, their impacts on the river flow and the possible enforcement of irrigation restriction measures. After those measures have been implemented, irrigation is constrained and the water flow at monitoring station may increase again, leading to the suspension of the restriction measures.

At a wider scale, the system encompasses feedback loops which link the evolution of water uses and the inter-annual negotiation of the regulation framework. However, such policy changes are considered through scenarios.

The links between fresh water availability, coastal productivity and shellfish farming production are explored by the model. Outputs of the shellfish farming sub-model estimate additional costs (benefits) associated with the productivity losses (gains) due to freshwater relative scarcity, which depends on climatic changes and the joint evolution of rival anthropogenic uses.

According to the representation of the system which has been agreed upon by the participant group, the freshwater of the Charente river provides mainly "support services" for natural habitats and shellfish farming and "provisioning services" for households and agriculture.

The economic assessment of the ecosystem services will be based on the damage costs assessment method (costs of avoided damages, productivity losses and remediation costs).

  Bacher 3 , Johanna Ballé-Béganton 2 , Rémi Mongruel 2 , José A. Pérez Agúndez 2 , Françoise Vernier 4 , Paul Bordenave 4 , Jacqueline Candau 4 , François Delmas 4 , Yvon Mensencal 5 , Harold Réthoret 6 and Julien Neveu 7

Scenarios

The other modules (Hydrology, Coastal Productivity, Agriculture, Shellfish Farming) are also tested independently using existing datasets or outputs from existing models.

Example of the riverflow of watershed simulated during one year (blue) and comparison with the CycleauPE simulation (green).

The participant group (PG) of the SSA consists in regular de visu meetings between 6 local managers and a sub-group of 3 researchers from the SSA team. Each meeting is observed by a social scientist in order to analyse the exchanges between the managers and the researchers.

Recent meetings of the PG were dedicated to the building of scenarios, following a deliberation methodology based on transparent votes (work in progress). In a first step, the PG produced a list of 50 assumptions regarding the future of the system in three domains: trends (climate, demography), management options and changes in uses and practices. In a second step, the stakeholders voted on these assumptions in order to sort a hierarchy. In a third step, stakeholders will be asked to vote on combinations of assumptions using the deliberation matrix (in order to build complete scenarios).

Besides the existing decision support tools that they already use, the stakeholders expect from the SPICOSA experiment a scientific exploration of the complex and dynamic relationships between the three dimensions (ecology-economy-society) of the freshwater allocation problem.


Alexis Ferrand
email: alexis.ferrand@univ-lille1.fr

Regulation of health systems implies controls on the quality of care. Two kinds of controls exist:

1) Formal controls are organized by professional associations or by health organizations and insurances, or by the States .

Friedson (1963) has shown that the "processes of control in a company of equals" can be weak.

2) Indirect controls are exerted by patients as clients when -for example -they decide to consult such or such practitioner.

Patients'decisions and controls imply that they evaluate and judge various dimensions of the quality of cares. This is not simple for them 1) due to the asymmetry of knowledge about the technical content of the medical practice and to the fundamental uncertainty of medical "art" ;

2) due to the difficulty to test and to compare the qualities of several practitioners. The importance of discussion networks E.Freidson (1960) has suggested that people exchange information and form their opinion about practitioners and medical care through "lay referral systems", i.e. personal networks.

L. Karpic (1989) in a quite similar domain of the "economy of quality" supposes that justiciables look for a layer through "systems of interpersonal relations" because it is very difficult for them to judge the quality of services they can offer.

We have tried to test three hypothesis :

1) The existence of "lay refferal systems" as discussion networks about health issues ;

2) The effect of these networks on the formation of opinions on health care ;

3) The local variability of these networks ( in a kind of Wellman 's community perspective) -In each, two sub-samples : a) blue collars (men) and low wages clercks (wemen); b) professionals and managerial staff.

Networks : three names generators

1) Own health

It can happen that you talk about your own health with personal acquaintances, family members, colleagues, friends… For example you discuss a disease you recently caught, drugs you take, a diet you go on, with someone who is not one of your physicians… During the last six months, did it happen that you discuss about your health with someone…

2) A physician

It can happen that you talk about a physician, remedies he prescribes, or care he takes of you… During the last six months, did it happen that you discuss about a physician… More than one person out of two is unable to talk with someone about a physician or about three diseases (57% ; 55%).

3) Three diseases

Twice more people is unable to talk about a physician or about three diseases than about own health (55% vs 28%).

The existence of "lay refferal systems" is not generalized in the population.

The discrepancy between own health and the two other topics shows that a specific taboo exists when the question is to discuss medical practice or illness.

The effect of networks on the formation of opinions about quality of local treatments

We have asked the opinion of interviewed about the quality of treatments in the locality for the three illness : cancer, heart troubles, nervous breakdowns.

If interviewed answers "I don't know" for at least two diseases, then he/she is categorized as dubious.

Proportion of dubious people, by number of persons cited, by topic of discussion (cells percentages) Empirical evidences that people who discuss are more able to form an opinion fit well with interactionnist theory : the dyadic discussion process implies the expression of opinions.

Number of persons with who one discusses

In our results the discussion must implies Ego, plus two persons, i.e. a triad to decrease dramatically the proportion of dubious people.

That effect is independent from locality and social milieus : it is not an effect of cultural capital nor of local norms.

The proportion of each kind of ties in networks are the same : it is not an effect of the composition of networks.

We have to think of the triad as a specific structure in which opinions are formed :

-because triads allows confrontations and disputes ; -and / or because triads are the first "collective" structure, i.e. In Lens, more people can not discuss about a physician 60/54

In Lens, less people have large netwoks 13%/23%

In Lens : a stronger difference between social milieus

In lens -it is contradictory with a "cultural capital" hypothesisprofessionals are less able to discuss about a physician than working class milieu (70% vs 52%), when in Tourcoing there is no difference between social milieus (57% vs 51%, n.s.).

In fact, in Lens exists a stronger taboo among professionals.

XXI International Sunbelt Social Network Conference Budapest April 2001 8

Freidson:

  Client control and medical practice American Journal of Sociology, 1960,65 L.Karpic : L'économie de la qualité Revue Française de Sociologie 1989, XXX XXI International Sunbelt Social Network Conference Budapest April 2001 3 Samples -Two cities : Lens (37M) and Tourcoing (100M) ; 250 interviewed in each.

Finally

  I am going to ask you if you discuss about specific diseases… During the last six months, did it happen that you discuss one of theses diseases :a) cancer ; b) heart troubles ; c

  who have cited zero or one person with who they discusses about a physician are dubious, cann't form an opinion about the quality of local treatment. That proportion is five time lower (4%) when people have cited at least two persons. There is a strong effect of discussions networks on the possibility to have an opinion about quality of local treatments XXI International Sunbelt Social Network Conference Budapest April 2001 6

Difference between localities and social milieus

  .Ferrand The forming of opinions about quality of health care in local discussion networks Number of person with who it is possible to discuss of a physician

	Social milieu	Number of persons cited
	Locality A : LENS
		0	1	2 et +
	Professional	70	21	9
	Working class	52	32	16
	Chi2=8.4 Prob=0.01	60	27	13
	Locality B : TOURCOING
	Professional	57	23	20
	Working class	51	24	25
	Chi2= 1.1 Prob=0.55	54	23	23
	able to transform shared inter-individual uncertainty in collective
	opinion.

XXI International Sunbelt Social Network Conference Budapest April 2001 7 ADifferences between localities. In Lens : a stronger taboo

XXI International Sunbelt Social Network Conference

Budapest 25-29 April 2001 The forming of opinions about quality of health care in local discussion networks

Abstract

Alexis Ferrand*

The building up of opinions on the quality of care in local discussion networks Regulation of health systems implies controls on the quality of care. Apart of formal controls organized by professional associations or health organizations, patients, as clients, can play a role. But it needs that they evaluate and judge various dimensions of the quality of cares. And this is not simple for them due to the asymmetry of knowledge and legitimacy. We demonstrate that informal networks of discussion on health and cares exist -often, not always -and that they allows formation of opinion.

An empirical research compares in two cities networks of discussion about health among people. Items about opinions on the quality of care allow a description of references used by practitioners and by people to evaluate care. We examine the effects of belonging to various social milieus and to different local communities on the types of networks build and the types of opinions they convey. Doing so we propose new insights on the process by which quality of care is controlled by informal networks. * Sociologue, Université Lille 1, France, CNRS CLERSE (Communication is part of a research on « Local systems of health » with Philippe Lardé, Chargé de Recherche au CNRS, and directed by Geneviève Cresson , Université Lille 3)


Javier Marhuenda

Sonia Medina

Pedro Martínez-Hernández

Simón Arina

Pilar Zafrilla

Juana Mulero

Camille Oger

Jean-Marie Galano

Thierry Durand

Federico Ferreres

Angel Gil-Izquierdo
email: angelgil@cebas.csic.es

Melatonin and hydroxytyrosol protect against oxidative stress related to the central nervous system after the ingestion of three

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

Introduction

Lipid peroxidation products have been investigated in order to determine their use as biomarkers of the oxidative status in the human body. Linolenic acid (LNA), arachidonic acid (AA), adrenic acid (AdA), and docosahexanoic acid (DHA) are commonly-studied fatty acids [START_REF] Medina | A ultra-pressure liquid chromatography/triple quadrupole tandem mass spectrometry method for the analysis of 13 eicosanoids in human urine and quantitative 24 hour values in healthy volunteers in a controlled constant diet[END_REF][START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF] which can give rise to phytoprostanes (PhytoPs), [START_REF] Marhuenda | Dependency of Phytoprostane Fingerprints of Must and Wine on Viticulture and Enological Processes[END_REF] isoprostanes (IsoPs), 1,2 F 2 -dihomo-isoprostanes (F 2 -dihomo-IsoPs), and neuroprostanes (NeuroPs), [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF] respectively. IsoPs have been established as general oxidative stress biomarkers in humans. [START_REF] Medina | Assessment of oxidative stress markers and prostaglandins after chronic training of triathletes[END_REF] Moreover, F 2 -dihomo-IsoPs have been related to early brain damage in Rett syndrome, and to white matter injury, [START_REF] Durand | F2-Dihomo-isoprostanes and brain white matter damage in stage 1 Rett syndrome[END_REF] in particular, because of their specific location in neural membranes. [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF] The Mediterranean Diet (MD) has been shown to exert several benefits with regard to the maintenance of the redox balance in humans. Polyphenols have been thought to be mainly responsible for the antiradical effects of the foodstuffs included in the MD. [START_REF] Guerrero | Wine, resveratrol and health: A review[END_REF] Wine is a representative alcoholic component of the MD, considered responsible for the "French Paradox". [START_REF] Renaud | Wine, alcohol, platelets, and the French paradox for coronary heart disease[END_REF] In fact, ethanol exhibits antioxidant capacity in vivo. [START_REF] Renaud | Wine, alcohol, platelets, and the French paradox for coronary heart disease[END_REF] Therefore, alcohol could be partially responsible for the effects of red wine. Moreover, red wine polyphenols have demonstrated an in vivo and in vitro antioxidant capacity. [START_REF] Guerrero | Wine, resveratrol and health: A review[END_REF][START_REF] Bertelli | Grapes, wines, resveratrol, and heart health[END_REF] Nevertheless, despite these beneficial effects, polyphenols have very-low in vivo and in vitro bioavailability. [START_REF] Medina | A ultra-pressure liquid chromatography/triple quadrupole tandem mass spectrometry method for the analysis of 13 eicosanoids in human urine and quantitative 24 hour values in healthy volunteers in a controlled constant diet[END_REF][START_REF] Gil-Izquierdo | An in vitro method to simulate phenolic compound release from the food matrix in the gastrointestinal tract[END_REF] Aside from the more-deeply-studied polyphenols, melatonin (MEL), hydroxytyrosol (OHTyr), and their metabolites arising from the metabolic activity of the colonic microflora (such as homovanillic alcohol and homovanillic acid), among other known and/or unknown compounds, could be partly responsible for the antioxidant benefits of red wine. [START_REF] Fernández-Mar | Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review[END_REF][START_REF] Lafay | Bioavailability of phenolic acids[END_REF] In turn, MEL could be responsible for the health effects of the Mediterranean diet. [START_REF] Iriti | Melatonin in traditional Mediterranean diets[END_REF] Such bioactive compounds act as radical scavengers, regulating the oxidation status of organisms. [START_REF] Fernández-Mar | Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review[END_REF] Moreover, both compounds are able to go through physiological barriers, in particular the blood-brain barrier (BBB). Therefore, their neuroprotective capacity has been related previously. [START_REF] Miller | Melatonin Redox Activity. Its Potential Clinical Applications in Neurodegenerative Disorders[END_REF] The current research aims to elucidate, for the first time, the neuroprotection against oxidative stress provided by red wine, measured by urinary neuroprostanes and dihomoisoprostanes.

Materials and methods

Chemicals and reagents

HPLC-grade ACN and methanol were purchased from Scharlau Chemie (Barcelona, Spain). Ultrapure water (Milli-Q) was obtained from a Milli-Q Gradient A10 system (Millipore, Bedford, MA). F 4 -NeuroPs and F 3 -NeuroPs n-6 DPA including 4(RS)-F 4t -NeuroP, d 4 -4(RS)-F 4t -NeuroP, 10-epi-10-F 4t -NeuroP, d 4 -10-epi-10-F 4t -NeuroP, 10-F 4t -NeuroP, d 4 -10-F 4t -NeuroP, 4-F 4t -NeuroP, 4-epi-4-F 3t -NeuroP, and 4-F 3t -NeuroP and dihomo-IsoPs including ent-7(RS)-7-F 2t -Dihomo-IsoP, ent-7-epi-7-F 2t -Dihomo-IsoP, 17-F 2t -Dihomo-IsoP, and 17-epi-17-F 2t -Dihomo-IsoP were synthesized by Durand's research team at the Institut des Biomolecules Max Mousseron (IBMM) (Montpellier, France). [START_REF] García-Flores | Assessment oxidative stress biomarkers-neuroprostanes and dihomo-isoprostanes-in elite triathletes urine after two weeks of moderate altitude training[END_REF] β-Glucuronidase, type H2 from Helix pomatia, and BIS-TRIS (bis-(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane) were from Sigma-Aldrich (St Louis, MO, USA). The Strata X-AW, 100 mg 3 mL -1 SPE cartridges were from Phenomenex (Torrance, CA, USA).

Red wine and must samples in this clinical and nutritional trial

The description of red wines and their primary musts were detailed in our previous study. [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF] The red wines were provided by the Baigorri winery (Bodegas Baigorri S.A.U, Samaniego, Álava). Three different wines were selected in order to compare different vinification and aging procedures: "Baigorri carbonic maceration 2010", "Baigorri aged 2007" and "Baigorri high expression 2010". For their correct maintenance, they were stored at between 12 °C and 14 °C after bottling. The musts analyzed during the current assay were stored at -20 °C after the harvest of the grapes, so that the fermentation process did not begin. They were the original grape juices used for the winemaking procedure of each wine. This permitted determination of the differences between the wines that arose from the distinct winemaking processes, hence avoiding any differences in the grape variety or harvesting procedure from must to wine.

Study design

The present study was previously approved by the Bioethics Committee of the Catholic University San Antonio of Murcia (UCAM). The characteristics of the study have been described in our previous research. [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF] The study included a total of 18 (N = 18) healthy female volunteers, between 18 and 27 years old (characteristics were described in our previous research). [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF] Importantly, all individuals in this study were nonsmokers and during the study the women were not in their menstrual days. Informed and signed consent of each individual involved in the trial was obtained. The development of the study was double-blind, crossover and placebo-controlled, carried out in four stages. At the beginning of each stage, the volunteers (N = 18) were divided into two equal groups (N 1 = 9 and N 2 = 9) in order to facilitate the crossover of the study. The structure of the stages consisted of 48 hours of urine collection since the beginning of the treatment, followed by a 72 hours washout period. Finally, after the washout period the crossover was developed, collecting the urine of the volunteers along the following 48 hours. Afterwards, each stage was in turn separated by washout periods of 72 hours too. The graphical distribution of the different stages developed in the present study has been described in Fig. 1.

Urine samples were obtained equally in all the stages of the study (graphically represented in Fig. 1). At 0 hours, the first urine in the morning was obtained both before and after the crossover. Additionally, samples were collected from 0 to 48 hours in order to establish the in vivo effects of the intake of the different beverages. Importantly, the data obtained are expressed in µg per 24 h in order to facilitate the comparison with the previously reported data.

Sample preparation for neuroprostane and F 2 -dihomoisoprostane analysis

The extraction of NeuroPs and F 2 -dihomo-IsoPs from urine was carried out according to Medina et al. (2015). [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF] Calibration-standard stock solutions of NeuroPs and F 2 -dihomo-IsoPs were diluted with methanol : water (1 : 1, v : v) to obtain the appropriate working solutions. All solutions were stored at -80 °C. The concentrations of NeuroPs and F 2 -dihomo-IsoPs were calculated from standard curves prepared each day.

For the enzymatic hydrolysis, to remove the glucuronide and sulfate conjugates, 1 mL of urine was added to 250 µL of 0.1 M acetate buffer ( pH 4.9) and 55 µL of β-glucuronidase from Helix pomatia. The mixture was incubated at 37 °C in a thermal block for two hours. After this step, protein precipitation was carried out with 500 µL of methanol/chlorhydric acid (200 µM). Then, the samples were centrifuged at 10 000g for 5 min. [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF] One milliliter of the supernatant was subjected to solid phase extraction (SPE) using a Strata X-AW cartridge (100 mg/3 mL), according to Medina et al. (2015). [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF] Briefly, samples that had been diluted in 2 mL of methanol and 1.25 mL of BIS-TRIS buffer were passed through the previously-conditioned and equilibrated cartridges. The analytes were eluted with 1 mL of methanol and dried under vacuum using a SpeedVac concentrator (Savant SPD121P, Thermo Scientific, MA, USA). The dry extracts were reconstituted with the A : B (90 : 10, v : v) mobile phases and filtered through a 0.45 μm filter (Millipore, MA, USA). The samples were injected and analyzed by using UHPLC-QqQ-MS/MS.

UHPLC-QqQ-MS/MS analysis of neuroprostanes and F 2 -dihomo-isoprostanes

The NeuroPs/F 2 -dihomo-IsoPs were separated using a UHPLC system coupled to a 6460 triple quadrupole tandem mass spectrometer (Agilent Technologies, Waldbronn, Germany).

The urinary NeuroPs and F 2 -dihomo-IsoPs were extracted from the 24 h urines. [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF] Chromatographic separation was carried out on an ACQUITY BEH C 18 column (2.1 × 50 mm, 1.7 μm pore size) (Waters, MA, USA). The column temperatures were 6 °C (left) and 6 °C (right). Sample analysis was carried out as reported by Medina et al. (2015). The multiple reaction monitoring (MRM) mode was employed using the negative electrospray ionization (ESI) mode. The mobile phases A (water) and B (methanol) contained 0.01% acetic acid (v : v). 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), and (10.01; 60.00).

The injection volume was 20 μL in UHPLC-QqQ-MS/MS. The operating conditions for the MS were as follows: gas flow 8 L min -1 , nebulizer 30 psi, capillary voltage 4000 V, nozzle voltage 1000 V, gas temperature 325 °C, sheath gas temperature 350 °C, and jetstream gas flow 12 L min -1 . The time for a single analysis was 10.01 min. The MS parameters fragmentor (ion optics) and collision energy were optimized for each compound to generate the most abundant product ions. Data acquisition and processing were performed using MassHunter software version B.04.00 (Agilent Technologies, Walbronn, Germany). The urinary amounts of the NeuroPs/F 2 -dihomo-IsoPs were calculated from the area ratio of the ion peaks of the compounds to those of the corresponding standards (expressed as ng of analytes excreted over 24 h).

Analysis of melatonin and phenolics in wines and musts

The methods used for the characterization of the samples were described in our previous study. [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF] The different phenolic compounds detected in the samples of wine and must were identified by their UV-Vis spectra and chromatographic comparison with authentic commercial markers. [START_REF] Cantos | Postharvest UV-C-irradiated grapes as a potential source for producing stilbeneenriched red wines[END_REF][START_REF] Mulero | Antioxidant activity and phenolic compounds in organic red wine using different winemaking techniques[END_REF] For the determination of OHTyr and its metabolites, a UHPLC coupled to a 6460 QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany) was used, following the method designed by Domínguez-Perles et al. (2015). [START_REF] Domínguez-Perles | Gender Differences on Plasma and Urine metabolites from Sprague-dawley Rats after Oral Administration of Normal and High Dose of Hydroxytyrosol, Hydroxytyrosol Acetate, and DOPAC[END_REF] For the analysis of MEL and serotonin, the extraction procedure for the wines and musts was as previously described. [START_REF] Fernández-Pachón | Alcoholic fermentation induces melatonin synthesis in orange juice[END_REF] The separation and determination of MEL and serotonin were performed using a UHPLC coupled to a 6460 QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany) and a Waters Acquity UHPLC BEH C 18 1.7 µm 2.1 × 50 mm column.

Statistical analysis

The analysis of variance (ANOVA; Duncan) was applied to establish significant differences between the means obtained during the fermentation process. A probability value of p < 0.05 was adopted as the criteria for significant differences.

To determine the individual involvement of the different bioactive compounds and the total phenolic content of lipid peroxidation products, simple regression analysis (R 2 ) was performed. This statistical method allows one to know about the individual relationship of each of the bioactive compounds and TPC with the plasmatic concentration of lipid peroxidation products. However, since both wines and musts are matrices composed of various compounds of interest, it is essential to study all relationships simultaneously. Therefore, the results obtained after determining the simple regression were contrasted with multiple regression analysis (stepwise mode), in order to establish a real-based relationship. These analyses were performed with SPSS version 21 software (SPSS Inc., Chicago, IL, USA).

Results

Qualitative and quantitative analyses of melatonin and phenolics in wines and musts

The results related to the content of bioactive compounds of the different wine and must samples were reported in our previous study. [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF] As a resume, the results are detailed above with the aim to facilitate the comprehension of the present study. The concentrations of the different bioactive compounds are detailed in Table 1 describing the content of MEL, hydroxytyrosol (OHTyr) and their metabolites, and Table 2 showing the content of phenolic compounds.

Regarding MEL, the musts contained similar and very low amounts of this compound, compared to the wines. The MEL concentration of the carbonic maceration must (CMM) was 0.41 ± 0.03 ng mL -1 , aged must (AM) had 1.24 ± 0.13 ng mL -1 , and high expression must (HEM) had 0.03 ± 0.004 ng mL -1 . The MEL concentration found in carbonic maceration wine (CMW) (161.83 ± 7.14 ng mL -1 ) was higher than the other two wines ( p < 0.01): the aged wine (AW) (26.31 ± 1.35 ng mL -1 ) and the high expression wine (HEW) (19.63 ± 1.14 ng mL -1 ) had similar concentrations ( p > 0.05).

Red wine is one of the major dietary sources of OHTyr. The analysis of OHTyr and its metabolites yielded two metabolites related to the gut microflora, homovanillic acid (HV-Acid) and homovanillic alcohol (HV-Alc) (Table 1). The musts contained high amounts of HV-Acid, but lower contents of HV-Alc and a very low amount of OHTyr. HV-Alc followed a trend opposite to that of HV-Acid, its concentration increasing after winemaking for CMW and AW, but not for HEW (Table 1). The most important difference between the wines and musts was due to the increase in OHTyr after vinification ( p < 0.05). As a result, HEW showed the highest OHTyr concentration of all the beverages (35.11 ± 2.51 µg mL -1 ), followed by AW (29.65 ± 0.46 µg mL -1 ) and CMW (16.13 ± 2.98 µg mL -1 ) (Table 1).

The concentrations found for every phenolic compound from wines and musts are shown in Table 2. Anthocyanins were the major phenolic compounds found in wines and musts. Among all the five anthocyanin glucosides, malvidin-3-O-glucoside was the major anthocyanin both in wines and musts ( p < 0.05). Moreover, delphinidin-3-O-glucoside and petunidin-3-O-glucoside were also prominent from the rest of the anthocyanins. The individual content of hydroxycinnamic acid derivatives was divided into two groups. Caftaric acid and coutaric acid were the major compounds, mainly found in wines. On the contrary, p-coumaric acid and fertaric acid were minor compounds in wine, being undetectable in must. The individual analysis of the different flavonols identified revealed quercetin-3-O-rutinoside and kaempferol-3-O-glucoside as major flavonols. Finally, trans-resveratrol was the main stilbene, both in wines and musts.

Qualitative analysis of neuroprostanes and F 2 -dihomo-isoprostanes in 24 h urine from healthy volunteers

The NeuroPs and F 2 -dihomo-IsoPs analyzed are displayed in Table 3. In previous reports, the 4-series NeuroPs were selected to be the oxidative markers, due to their higher abundance in vivo. [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF][START_REF] Yin | Regiochemistry of neuroprostanes generated from the peroxidation of docosahexaenoic acid in vitro and in vivo[END_REF] The 7-and 17-series are the major F 2 -dihomo-IsoP metabolites; thereby, they were chosen as the reference for the other oxidative markers of neurodegeneration in human urine samples. [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF][START_REF] Vanrollins | F 2-Dihomo-isoprostanes arise from free radical attack on adrenic acid[END_REF] The NeuroPs and dihomo-IsoPs identified in the 24 h urine samples were as follows: 4(R)-F 4t -NeuroP; 10-epi-10-F 4t -NeuroP; 10-F 4t -NeuroP; 4-epi-4-F 3t -NeuroP; 4-F 3t -NeuroP; ent-7(R)-7-F 2t -Dihomo-IsoP; ent-7-epi-7-F 2t -Dihomo-IsoP; 17-F 2t -Dihomo-IsoP and 17-epi-17-F 2t -Dihomo-IsoP. These markers will help us to describe the neuroprotection capacity afforded by the intake of the different musts and wines in this study.

Quantitative assessment of total neuroprostanes and F 2dihomo-isoprostanes in 24 h urine from healthy volunteers

As can be seen in Fig. 2, the mean values varied between treatments. The baseline (control) 24 h urinary concentration of total-F 2 -dihomo-IsoPs was 6.14 ± 0.58 µg per 24 h, significantly higher ( p < 0.01) than that found after the intake of the different musts. The treatment with the AM triggered the highest urinary concentration of total-F 2 -dihomo-IsoPs (4.28 ± 0.59 µg per 24 h), followed by the CMM (3.97 ± 0.67 µg per 24 h) and the primary must of HEM (3.69 ± 0.76 µg per 24 h). There were no significant differences among these values ( p > 0.05). However, the intake of CMW, AW, or HEW caused the urinary total-F 2 -dihomo-IsoPs (2.53 ± 0.64, 2.96 ± 0.45 and 3.00 ± 0.53 µg per 24 h, respectively) to be lower than the baseline value (6.14 ± 0.58 µg per 24 h) ( p < 0.05). It is of note, as observed for the must intake, that no differences in total-F 2dihomo-IsoPs existed among the red wine treatments ( p > 0.05). Concerning the alcohol content, the group consuming the alcohol equivalent to a serving of wine (WR) also had a concentration of urinary total-F 2 -dihomo-IsoPs (4.95 ± 0.82 µg per 24 h), which was significantly lower than that of the baseline group (6.14 ± 0.58 µg per 24 h) ( p < 0.05). However, the ingestion of WR resulted in significantly higher urinary concentrations of these total-F 2 -dihomo-IsoPs ( p < 0.05) than the consumption of CMW, CMM, AW, HEW, or HEM.

The total amount of urinary NeuroPs found after the treatments varied widely (Fig. 2). The musts CMM and AM produced decreases ( p < 0.05) in the urinary excretion of total NeuroPs, compared to the baseline concentration (4.78 ± 0.72 µg per 24 h), while the intake of HEM exerted a particularly-marked protective effect ( p < 0.01) against the generation of these compounds (total NeuroP concentration: 2.81 ± 0.85 µg per 24 h). Regardless of the type, after the red wine intake the trend regarding the total NeuroP concentration was similar to that found after the ingestion of the different musts. Therefore, the three types of wine exerted similar DHA protection ( p > 0.05). Nevertheless, only after the HEW intake was significant ( p < 0.01) diminution of the total NeuroP level detected; of the three wines, HEW provided the lowest urinary concentration (3.17 ± 0.59 µg per 24 h). Alcohol intake, represented by the treatment with white rum (WR), did not exert neuroprotection. In fact, urinary excretion of total NeuroPs after WR intake was similar (4.64 ± 0.95 µg per 24 h) to the baseline level. HEW and HEM intake, among CMM, also produced lower total NeuroP excretion ( p < 0.05) than WR.

None of the three types of red wine exerted higher DHA protection against NeuroP production than their corresponding musts ( p < 0.05). However, the high expression must and wine (HEM and HEW) were able to reduce the urinary excretion of total NeuroPs, indicating the influence of the cultivation and harvesting of the grapes and of the winemaking process on the final DHA protection capacity of the resultant must and, consequently, of the finished wine. The vinification process did not seem to influence the antiradical properties of the final red wine when measured as the total NeuroP concentration in urine, according to the only-slight differences between each must and its corresponding finished wine ( p > 0.05).

Quantitative assessment of individual neuroprostanes and F 2 -dihomo-isoprostanes in 24 h urine from healthy volunteers

The disparity among the individual NeuroPs and F 2 -dihomo-IsoPs concerning their excretion after the intake of musts and wines was also detected (Table 3). The urinary concentrations of NeuroPs did not vary noticeably: in fact, NeuroPs such as 4-epi-4-F 3t , 4-F 3t , 10-epi-10-F 4t , and 10-F 4t did not differ significantly among the distinct types of must and wine ( p > 0.05).

Nevertheless, some findings can be highlighted. The main NeuroP found in urine was 4(R)-F 4t (baseline of 2.40 ± 0.19 µg per 24 h). Before must intake, the 4(R)-F 4t excretion was lower than the baseline for all types of musts, significantly so ( p < 0.05) in AM and HEM (1.92 ± 0.44 µg per 24 h for CMM; 1.66 ± 0.36 µg per 24 h for AM and 1.20 ± 0.26 µg per 24 h for HEM) (Table 3). The intake of all three types of wine also led to a reduction of the urine concentration of 4(R)-F 4t ( p < 0.05). The lowest value was found after HEW intake (1.27 ± 0.20 µg per 24 h). Besides, AW and CMW intake reduced the 4 (R)-F 4t concentration in urine to similar final values (1.27 ± 0.43 µg per 24 h for AW and 1.02 ± 0.17 µg per 24 h for HEW) (Table 3).

Regarding the individual variation of F 2 -dihomo-IsoPs, the concentrations in urine differed widely after the intake of the different musts. The baseline levels of 17-epi-17-F 2t and 17-F 2t in the urine were low (0.08 ± 0.02 µg per 24 h and 0.70 ± 0.10 µg per 24 h, respectively). After their administration to the healthy volunteers, CMM gave rise to urinary concentrations of both compounds that were similar to the baseline values ( p > 0.05) (0.04 ± 0.01 µg per 24 h and 0.44 ± 0.09 µg per 24 h, respectively), AM intake resulted in lower values ( p < 0.05) for 17-epi-17-F 2t (0.05 ± 0.02 µg per 24 h), and HEM intake produced a drop in the concentrations of 17-epi-17-F 2t (0.04 ± 0.012 µg per 24 h) and 17-F 2t (0.25 ± 0.09 µg per 24 h) (Table 3).

The highest baseline urinary concentrations were observed for ent-7(R)-7-F 2t (3.15 ± 0.31 µg per 24 h) and ent-7-epi-7-F 2t (2.21 ± 0.35 µg per 24 h). The musts caused similar decreases in the ent-7(R)-7-F 2t and ent-7-epi-7-F 2t levels ( p > 0.05). In fact, the ingestion of the musts significantly ( p < 0.05) diminished the concentration of ent-7(R)-7-F 2t (1.85 ± 0.35 µg per 24 h, 1.99 ± 0.18 µg per 24 h, 2.03 ± 0.43 µg per 24 h for MCM, AM and HEM intake, respectively). The urinary concentration of ent-7-epi-7-F 2t showed the same tendency, being lower after the intake of CMM (1.64 ± 0.22 µg per 24 h), AM (1.79 ± 0.32 µg per 24 h) and HEM (1.36 ± 0.22 µg per 24 h), compared to the baseline (Table 3). The wines also provoked decreases of the ent-7(R)-7-F 2t and ent-7-epi-7-F 2t levels ( p < 0.05). Thus, the ent-7(R)-7-F 2t excretion after CMW (1.30 ± 0.33 µg per 24 h), AW (1.66 ± 0.21 µg per 24 h), or HEW (1.41 ± 0.26 µg per 24 h) intake was markedly lower than the corresponding baseline value ( p < 0.05). The urinary excretion of ent-7-epi-7-F 2t was 0.89 ± 0.17 µg per 24 h, 1.03 ± 0.13 µg per 24 h and 1.27 ± 0.14 µg per 24 h as a consequence of the administration of CMW, AW, and HEW, respectively (Table 3).

The dietary intake of alcohol (WR) reduced the concentration of ent-7(R)-7-F 2t (to 2.4 ± 0.6 µg per 24 h), compared with the baseline ( p < 0.05). However, the urinary concentrations of the other F 2 -dihomo-IsoPs remained unchanged ( p > 0.05) (Table 3).

The winemaking processes did not influence the urinary concentration of individual NeuroPs: the differences observed between the musts and their respective wines were not significant ( p > 0.05). Conversely, differences did occur for two urinary F 2 -dihomo-IsoPs. The urinary concentration of ent-7(R)-7-F 2t was significantly higher ( p < 0.05) after CMM intake than after CMW intake and the urinary concentration of ent-7-epi-7-F 2t after the intake of AM was higher than after the intake of AW, highlighting the importance of the vinification process and the new compounds formed which boosted these effects on these markers.

Discussion

In recent years, polyphenols have been related to the beneficial effects on human health upon moderate consumption of red wine. [START_REF] Renaud | Wine, alcohol, platelets, and the French paradox for coronary heart disease[END_REF][START_REF] Basli | Wine polyphenols: Potential agents in neuroprotection[END_REF][START_REF] Renaud | Alcohol and platelet aggregation: The Caerphilly Prospective Heart Disease Study[END_REF] Among others, they have been attributed to antioxidant, [START_REF] Mulero | Antioxidant activity and phenolic compounds in organic red wine using different winemaking techniques[END_REF] anti-inflammatory 8 and anticancer properties. [START_REF] Artero | The impact of moderate wine consumption on health[END_REF][START_REF] Kampa | Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines[END_REF] Polyphenols exert neuroprotective effects; [START_REF] Basli | Wine polyphenols: Potential agents in neuroprotection[END_REF] especially, anthocyanins have also been reported to be able to trespass the blood brain barrier. [START_REF] Schaffer | Do polyphenols enter the brain and does it matter? Some theoretical and practical considerations[END_REF] Finally, they have antiatherogenic and anti-inflammatory effects and vasodilator capacity, and show cardioprotective properties. [START_REF] Bertelli | Grapes, wines, resveratrol, and heart health[END_REF] Despite the health effects of polyphenols on humans, the scientific literature shows a very low bioavailability. [START_REF] Bouayed | Bioaccessible and dialysable polyphenols in selected apple varieties following in vitro digestion vs. their native patterns[END_REF][START_REF] Bouayed | Total phenolics, flavonoids, anthocyanins and antioxidant activity following simulated gastro-intestinal digestion and dialysis of apple varieties: Bioaccessibility and potential uptake[END_REF][START_REF] Tagliazucchi | In vitro bio-accessibility and antioxidant activity of grape polyphenols[END_REF] Therefore, despite the antioxidant capacity of the polyphenols and the importance of these compounds in the wine and grape matrices, their low absorption appears to be a limiting factor when considering the intake of polyphenols as key factor in the antioxidant effects of red wine. In our study, the intake of unique polyphenols (OHTyr and total polyphenols)not including colon derivatives (only OHTyr microflora metabolites)has been approached in order to determine their preventive effect on lipid peroxidation. However, anthocyanins, being the major polyphenol in wine and must, have been demonstrated to be directly absorbed in the intestine. [START_REF] Talavéra | Anthocyanin metabolism in rats and their distribution to digestive area, kidney, and brain[END_REF] Moreover, other newly reported bioactive compounds in wine and must called phytoprostanes have shown healthy benefits regarding oxidative injury prevention and anti-inflammatory capacity. In fact, they have been reported to have absorption in the intestine although colonic metabolites have not yet been reported. [START_REF] Marhuenda | Dependency of Phytoprostane Fingerprints of Must and Wine on Viticulture and Enological Processes[END_REF][START_REF] Barden | Flaxseed oil supplementation increases plasma F1-phytoprostanes in healthy men[END_REF] Therefore, this work is a preliminary study that may help to understand the role of certain wine compounds in the oxidation of DHA, EPA and AdA.

Red wine has shown in vivo neuroprotective effects, mainly attributed to its polyphenolic composition and its antioxidant properties. [START_REF] Basli | Wine polyphenols: Potential agents in neuroprotection[END_REF][START_REF] Gómez-Serranillos | Study of red wine neuroprotection on astrocytes[END_REF] As mentioned above, MEL and OHTyr can act as local antioxidants due to their capacity to traverse physiological barriers. [START_REF] Fernández-Mar | Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review[END_REF][START_REF] Rodríguez-Naranjo | Melatonin is synthesised by yeast during alcoholic fermentation in wines[END_REF] To elucidate if MEL or OHTyr could be, at least partly, responsible for the observed neuroprotective effects of red wine, a regression study was carried out.

The results of regression analysis performed for the urinary excretion of total NeuroPs and total F 2 -dihomo-IsoPs are given in Table 4. As can be appreciated, the simple regression study shows that the excretion of NeuroPs is individually mainly explained by OHTyr intake (responsible for 15.5% of the observed variation). Meanwhile, the intake of MEL, HV-Acid, HV-Alc and TPC was responsible for 13.5%, 0.3%, 7.2% and 4.4% of the observed variation in the urinary excretion of NeuroPs, respectively. Finally, multiple regression analysis following the same trend was performed after simple regression, which showed little statistical weight for all compounds present in musts and wines. Only 23.6% of the observed change in the excretion of NeuroPs can be explained by the statistical model containing all the bioactive compounds and TPC simultaneously. This same model showed that the intake of OHTyr explains 16.5% of the observed change in the NeuroP variation, taking into account all the compounds included in the beverages. Therefore, it is clear that the excretion of NeuroPs must be influenced by various factors that cannot be determined in the present study. However, the intake of the various compounds (especially OHTyr) may be partially responsible for the decrease in the excretion of urinary NeuroPs. The simple regression in relation to the excretion of F 2dihomo-IsoPs showed a greater influence of the different compounds contained in the beverages than discussed above for the urinary excretion of NeuroPs. By conducting the study of simple regression, the urinary excretion of F 2 -dihomo-IsoPs is explained in 43.9% by the intake of MEL, in 20.7% by OHTyr, in 8% by HV-Acid, in 33.1% by HV-Alc and in 32.7% by TPC. Moreover, the study of multiple regression showed that the observed variation in the excretion of F 2 -dihomo-IsoPs is explained in 44.1% by the whole compounds studied simultaneously. Overall, the intake of MEL was the most significant describing the observed variation, explaining 43.9% of the observed decrease in the urinary excretion of F 2 -dihomo-IsoPs.

The scientific literature about neuroprostanes and F 2dihomo-isoprostanes is still limited to a few description and synthesis papers. The description of neuroprostanes and F 2 -dihomo-isoprostanes in human fluids is restricted to physiopathological conditions such as Rett syndrome, brain injury or Alzheimer disease among others. [START_REF] Signorini | F4-neuroprostanes mediate neurological severity in Rett syndrome[END_REF][START_REF] De Felice | F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome[END_REF][START_REF] Hsieh | Correlation of F4-neuroprostanes levels in cerebrospinal fluid with outcome of aneurysmal subarachnoid hemorrhage in humans[END_REF][START_REF] Kuo | Cerebrospinal fluid biomarkers for neuropsychological symptoms in early stage of lateonset Alzheimer's disease[END_REF][START_REF] Yen | Levels of F2-isoprostanes, F4-neuroprostanes, and total nitrate/nitrite in plasma and cerebrospinal fluid of patients with traumatic brain injury[END_REF] Some studies have reported the reduction of other lipid peroxidation biomarkers as isoprostanes. [START_REF] Schrieks | Effect of red wine consumption on biomarkers of oxidative stress[END_REF] However, it is not relevant in the present study due to the general location in the human body. Other studies reported a notable reduction in lipid peroxidation related to the central nervous system in young adult triathletes, showing similar values to those reported in the present study. García-Flores et al. reported that one serving of fruit juice rich in polyphenols leads to minor urinary excretion of neuroprostanes and dihomo-isoprostanes. [START_REF] García-Flores | Lipidomic approach in young adult triathletes: effect of supplementation with a poly-phenols-rich juice on neuroprostane and F 2-dihomoisoprostane markers[END_REF] They related those changes observed to the content of polyphenols of the fruit beverages. Therefore, the comparison of the physiological levels of the biomarkers with previous scientific literature is still limited in the case of a healthy population.

Therefore, all the bioactive compounds taken into account in this research study including TPC seem to exert neuroprotection. On one hand, MEL appears to exert its neuroprotective effect by the oxidative protection of AdA, measured by the formation of F 2 -dihomo-IsoPs. Meanwhile, OHTyr has proved to be responsible for the protection of DHA against oxidative species, determined by the measurement of the total production of NeuroPs.

According to the above statistical analysis, the differences observed among the wine and must treatments could be related to their MEL contents. The CMW, in which the concentration of MEL was the highest (161.83 ± 7.1 ng mL -1 ), was the only wine that gave a significantly greater decrease in total urinary F 2 -dihomo-IsoPs compared to its preceding must, CMM ( p = 0.015). Taking into account these results, the fact that MEL can act as an antioxidant and its ability to cross the BBB, it is evident that MEL in wine must be considered with neuro-protection capacity. [START_REF] Fernández-Mar | Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review[END_REF] In particular, the decrease in urinary F 2 -dihomo-IsoPs, compared to the baseline level, is representative of protection of the white brain matter against oxidative stress. [START_REF] Durand | F2-Dihomo-isoprostanes and brain white matter damage in stage 1 Rett syndrome[END_REF] The effects of wine on lipid peroxidation have been found to result in a decrease in the production of oxidized compounds. [START_REF] Schrieks | Effect of red wine consumption on biomarkers of oxidative stress[END_REF][START_REF] Pignatelli | Polyphenols synergistically inhibit oxidative stress in subjects given red and white wine[END_REF] Nonetheless, to the best of our knowledge, no reports about MEL or OHTyr and NeuroPs or F 2 -dihomo-IsoPs have been published. Therefore, this is the first in vivo assay of the relationship between the intake of these bioactive molecules as part of wine and neuroprotection.

There are few reports on the urinary excretion of F 4 -NeuroPs and F 2 -dihomo-IsoPs. However, similar results in urine were obtained using UHPLC-QqQ-MS/MS, [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF] reporting 3.30 ± 1.17 µg mL -1 for NeuroPs and 6.12 ± 1.51 µg mL -1 for F 2 -dihomo-IsoPs as mean values in a healthy population. Meanwhile, in epileptic patients there was greater degradation of neuronal membranes and myelin, giving mean values of 5.51 ± 1.86 µg mL -1 for NeuroPs and 7.81 ± 1.87 µg mL -1 for F 2 -dihomo-IsoPs. The urinary excretion of F 4 -NeuroPs and F 2 -dihomo-IsoPs was also studied in healthy subjects after the intake of a citrus-based juice, [START_REF] García-Flores | Assessment oxidative stress biomarkers-neuroprostanes and dihomo-isoprostanes-in elite triathletes urine after two weeks of moderate altitude training[END_REF] showing slightly higher values than those reported in the present study.

Additionally, NeuroPs and F 2 -dihomo-IsoPs have been described in other biological fluids such as plasma and cerebrospinal fluid. Other authors [START_REF] Signorini | F4-neuroprostanes mediate neurological severity in Rett syndrome[END_REF] (2011) [START_REF] De Felice | F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome[END_REF] found higher plasmatic levels of F 2 -dihomo-IsoPs in Rett syndrome patients. Finally, the cerebrospinal fluid content of F 4 -NeuroPs and F 2 -dihomo-IsoPs has also been reported, showing an increment of both biomarkers in many diseases such as brain injury, Alzheimer's disease, or aneurysmal subarachnoid hemorrhage. [START_REF] Hsieh | Correlation of F4-neuroprostanes levels in cerebrospinal fluid with outcome of aneurysmal subarachnoid hemorrhage in humans[END_REF][START_REF] Kuo | Cerebrospinal fluid biomarkers for neuropsychological symptoms in early stage of lateonset Alzheimer's disease[END_REF][START_REF] Yen | Levels of F2-isoprostanes, F4-neuroprostanes, and total nitrate/nitrite in plasma and cerebrospinal fluid of patients with traumatic brain injury[END_REF] As expected, the vinification processes enhanced the neuroprotective effect of the wines, with regard to total-F 2 -dihomo-IsoPs, in comparison with the musts. [START_REF] Rodríguez-Naranjo | Melatonin is synthesised by yeast during alcoholic fermentation in wines[END_REF] In general, unless not statistically significant ( p > 0.05), red wine intake resulted in a greater decrease of 24 h total F 2 -dihomo-IsoP excretion than the must intake did.

Conclusions

Red wine intake reduces oxidative injury in the central nervous system to a greater extent than must and ethanol intake, measured by the urinary excretion of neuroprostanes and F 2 -dihomo-isoprostanes. Melatonin may be the bioactive compound that better explains the increase in the percentage of protection against the oxidative attack of AdA, judged by the reduction in the formation of F 2 -dihomo-isoprostanes and their subsequent urinary excretion. Similarly, hydroxytyrosol may explain the increase in the protection of DHA and EPA from oxidative attack, measured by the reduction in the formation of F 3 -neuroprostanes and F 4 -neuroprostanes respectively.

Consequently, the intake of CMW containing high quantities of melatonin and hydroxytyrosol could develop an increased reduction of oxidative injury compared to aged wines (AW and HEW). However, only hydroxytyrosol metabolites have been approached in the present study. Therefore, further studies may help to understand the effect of other bioactive compounds such as polyphenol's colon derivatives and newly discovered phytoprostanes on reducing central nervous system lipid peroxidation.

Fig. 1

 1 Fig.1Graphical representation of the in vivo assay, divided into four stages. Stages 2, 3, and 4 were similar but the beverage drank changed between stages. A period of 72 hours was included between stages and before the crossover. MCW/MCM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must.

Fig. 2

 2 Fig.2Total neuroprostanes and F 2 -dihomo-isoprostanes. WR: white rum; MCW/MCM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must. The results are expressed as µg per 24 h ± SD.

  described a plasmatic increase of F 4 -NeuroPs in patients with Rett syndrome, compared to healthy subjects. Similarly, Durand et al. (2013) 5 and De Felice, Signorini, Durand, Oger, Guy, Bultel-Poncé et al.

Table 1

 1 Characterization of bioactive compounds of red wines and musts and the white rum ethanol content

				Hydroxytyrosol metabolites (µg mL -1 )
		Melatonin (ng mL -1 )	Ethanol (g per dose) a	Homovanillic acid	Homovanillic alcohol	Hydroxytyrosol
	WR	-	4.14	-	-	-
	CMW	161.83 ± 7.14 a	4.14	83.46 ± 13.59 c	189.23 ± 8.71 a	16.13 ± 2.98 b
	CMM	0.41 ± 0.039 d	-	130.76 ± 1.32 a	14.63 ± 0.17 c	0.53 ± 0.05 c
	AW	26.31 ± 1.35 b	4.14	74.38 ± 0.68 c	65.51 ± 1.31 b	29.65 ± 0.46 a
	AM	1.24 ± 0.13 c,d	-	112.41 ± 3.12 a,b	16.22 ± 1.02 c	0.96 ± 0.09 c
	HEW	19.63 ± 1.14 b	4.14	75.94 ± 7.29 c	51.56 ± 6.27 b	35.11 ± 2.51 a
	HEM	0.03 ± 0.004 d	-	101.04 ± 2.21 a,b	60.59 ± 1.31 b	0.54 ± 0.03 c

WR: white rum; MCW/MCM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must. a It is considered as the total dose ingested over 48 hours: 74 mL of white rum and 200 mL of wine and must.

Table 2

 2 Characterization of polyphenolic compounds of red wines and musts

		CMW	CMM	AW	AM	HEW	HEM
	Delphinidin-3-O-glucoside	55.97 ± 2.18 a	12.11 ± 0.56 c,d	39.81 ± 0.48 b	12.96 ± 1.10 c,d	11.36 ± 0.47 d	14.63 ± 1.14 c
	Cyanidin-3-O-glucoside	2.45 ± 0.05 b,c	4.03 ± 0.17 b	3.44 ± 0.09 b,c	3.69 ± 0.08 b	0.60 ± 0.04 c	7.29 ± 0.88 a
	Petunidin-3-O-glucoside	54.73 ± 1.26 a	9.13 ± 0.59 d	30.21 ± 0.27 b	9.12 ± 0.98 d	8.17 ± 0.20 d	13.85 ± 0.95 c
	Peonidin-3-O-glucoside	11.77 ± 1.04 b	7.36 ± 1.52 c	10.96 ± 0.35 b	7.38 ± 0.74 c	3.37 ± 0.04 d	14.66 ± 1.39 a
	Malvidin-3-O-glucoside	236.87 ± 10.00 a	35.80 ± 2.30 d	90.10 ± 5.09 b	38.53 ± 20.00 d	35.19 ± 0.61 c	74.16 ± 3.29 d
	Total anthocyanins	361.79 ± 11.27 a	68.43 ± 3.71 d	174.53 ± 5.98 b	71.68 ± 3.25 d	58.68 ± 0.74 c	124.59 ± 4.56 d
	Caftaric acid	45.09 ± 4.52 b,c	0.59 ± 0.02 d	48.81 ± 0.03 a,b	0.31 ± 0.02 d	55.70 ± 4.73 a	2.35 ± 0.12 d
	p-Coumaric acid	1.30 ± 0.07 c	ND	10.59 ± 0.73 b	ND	14.42 ± 1.04 a	ND
	Fertaric acid	0.76 ± 0.04 c	ND	2.40 ± 0.08 b	ND	5.20 ± 0.30 a	ND
	Coutaric acid	40.82 ± 1.75 a	0.39 ± 0.06 d	13.69 ± 0.32 c	0.28 ± 0.02 d	20.54 ± 0.71 b	1.13 ± 0.10 d
	Total hydroxycinnamic derivatives	87.96 ± 6.39 a	0.98 ± 0.08 c	75.50 ± 1.15 b	0.59 ± 0.04 c	95.85 ± 6.77 a	3.48 ± 0.22 c
	Myricetin-3-O-glucoside	3.96 ± 0.06 a	1.06 ± 0.04 c	4.21 ± 0.23 a	1.00 ± 0.04 c	2.84 ± 0.26 b	1.22 ± 0.05 c
	Quercetin-3-O-glucoside	2.33 ± 0.21 c	0.63 ± 0.10 d	3.36 ± 0.12 b	0.60 ± 0.09 d	6.88 ± 0.25 a	0.73 ± 0.12 d
	Quercetin-3-O-rutinoside	33.37 ± 1.88 b	2.15 ± 0.14 d	24.93 ± 3.32 c	2.03 ± 0.14 d	49.77 ± 5.24 a	2.49 ± 0.17 d
	Kaempferol-3-O-glucoside	10.19 ± 1.27 b	2.54 ± 0.13 d	6.49 ± 0.34 c	2.39 ± 0.12 d	18.82 ± 1.28 a	2.93 ± 0.15 d
	Myricetine	5.08 ± 0.16 a	1.83 ± 0.17 d	2.30 ± 0.14 c	1.73 ± 0.16 d	3.71 ± 0.18 b	2.12 ± 0.19 c,d
	Quercetine	3.23 ± 0.13 b	1.79 ± 0.24 c,d	1.25 ± 0.03 d	1.69 ± 0.23 c,d	4.82 ± 0.16 a	2.06 ± 0.28 c
	Total flavonols	58.15 ± 1.31 b	10.01 ± 0.32 c	42.54 ± 4.18 b	9.43 ± 0.30 c	86.84 ± 4.22 a	11.55 ± 0.36 c
	Piceatanol	0.26 ± 0.02 b	0.08 ± 0.01 c	0.20 ± 0.01 b	0.08 ± 0.01 c	0.57 ± 0.04 a	0.27 ± 0.03 b
	trans-Resveratrol	4.85 ± 1.03 a	0.84 ± 0.06 d	2.10 ± 0.15 c	0.79 ± 0.08 d	3.37 ± 0.25 b	0.82 ± 0.06 d
	Total stilbenes	5.12 ± 1.05 a	0.92 ± 0.07 c	2.30 ± 0.16 b	0.87 ± 0.09 c	3.94 ± 0.29 a	1.09 ± 0.09 b,c
	Total phenolic compounds (TPC)	513.02 ± 20.02 a	80.34 ± 4.18 c.d	294.87 ± 11.47 b	82.57 ± 3.68 c.d	245.31 ± 12.02 b	140.71 ± 5.23 c

WR: white rum; MCW/MCM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must. Concentration of polyphenolic compounds is expressed in µg mL -1 .

Food Funct.

This journal is © The Royal Society of Chemistry 2016

Table 3

 3 Urinary neuroprostanes (NeuroPs) and F 2 -dihomo-isoprostanes (F 2 -dihomo-IsoPs) after the treatments

			Control	WR	CMW	CMM	AW	AM	HEW	HEM
	NeuroPs	4(R)-F 4t	2.40 ± 0.19 1.95 ± 0.22 1.27 ± 0.2	1.92 ± 0.20 1.27 ± 0.43 1.66 ± 0.36 1.02 ± 0.17 1.20 ± 0.20
		4-epi-4-F 3t	0.67 ± 0.13 0.51 ± 0.11 0.41 ± 0.07 0.52 ± 0.19 0.48 ± 0.13 0.62 ± 0.21 0.37 ± 0.09 0.46 ± 0.08
		4-F 3t	0.26 ± 0.06 0.21 ± 0.05 0.18 ± 0.04 0.18 ± 0.04 0.19 ± 0.07 0.20 ± 0.07 0.19 ± 0.04 0.19 ± 0.06
		10-epi-10-F 4t	0.95 ± 0.24 1.12 ± 0.25 0.89 ± 0.18 0.73 ± 0.16 0.64 ± 0.28 0.86 ± 0.19 0.60 ± 0.17 0.56 ± 0.21
		10-F 4t	0.51 ± 0.11 0.85 ± 0.32 0.42 ± 0.09 0.36 ± 0.10 0.36 ± 0.20 0.39 ± 0.17 0.41 ± 0.17 0.37 ± 0.24
	F 2 -dihomo-IsoPs 17-epi-17-F 2t	0.08 ± 0.02 0.05 ± 0.01 0.04 ± 0.02 0.04 ± 0.01 0.03 ± 0.01 0.05 ± 0.02 0.04 ± 0.04 0.04 ± 0.02
		17-F 2t	0.70 ± 0.10 0.39 ± 0.09 0.31 ± 0.13 0.44 ± 0.09 0.25 ± 0.10 0.46 ± 0.08 0.28 ± 0.12 0.25 ± 0.09
		ent-7(R)-7-F 2t	3.15 ± 0.31 2.53 ± 0.33 1.30 ± 0.33 1.85 ± 0.35 1.66 ± 0.21 1.99 ± 0.18 1.41 ± 0.26 2.03 ± 0.43
		ent-7-epi-7-F 2t 2.21 ± 0.35 1.98 ± 0.39 0.89 ± 0.17 1.64 ± 0.22 1.03 ± 0.13 1.79 ± 0.32 1.27 ± 0.14 1.36 ± 0.22

Means are expressed in µg per 24 h. WR: white rum; MCW/MCM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must.

Table 4

 4 Determination coefficients derived from the study of simple regression and multiple regression of the urinary excretion of total neuroprostanes and F 2 -dihomo-isoprostanes Alc: homovanillic alcohol; TPC: total phenolic compounds. R 2 Tot.: coefficient of regression which expresses the statistical significance of all the compounds studied with respect to each biomarker. R 2 Select.: coefficient of determination of compounds selected by the statistical robustness model. All these values are treated as % of influence on the final concentration.

		Total NeuroPs	Total F 2 -dihomo-IsoPs
	Simple regression (R 2 )
	MEL	0.135	0.439
	OHTyr	0.155	0.207
	HV-Acid	0.003	0.080
	HV-Alc	0.072	0.331
	TPC	0.044	0.327
	Multiple regression
	R 2 Tot.	0.236	0.441
	R 2 Select.	0.165 (OHTyr)	0.439 (MEL)
	MEL: melatonin; OHTyr: hydroxytyrosol; HV-Acid: homovanillic acid;
	HV-

This journal is © The Royal Society of Chemistry 2016Food Funct.Published on 25 November 2016. Downloaded by University of Montpellier on 25/01/2017 15:00:04.View Article Online

Acknowledgements

This work has been partially funded by the "Fundación Séneca de la Región de Murcia" Grupo de Excelencia 19900/GERM/15. The authors are grateful to UCAM for the support bridged during the development of the assay for the study. Sonia Medina is grateful to the CICYT for a research contract (AGL2011-23690). Finally, the authors want to thank David Walker as an English Expert Reviewer and Jose Miguel Martínez-Sanz for providing information about melatonin intake in the Mediterranean diet.

Author contribution

Javier Marhuenda carried out the analytical processes and wrote and discussed the present paper. Sonia Medina supervised the analytical processes and developed the discussion of the paper. Pedro Martinez-Hernández and Simón Arina provided the beverages of the study and supervised the article. Pilar Zafrilla and Juana Mulero developed the randomized controlled trial. Camille Oger, Jean-Marie Galano and Thierry Durand provided the markers for the study and helped with the review of the manuscript. Federico Ferreres helped with the experimental procedures linked to UHPLC-QqQ-MS/MS. Ángel Gil-Izquierdo and Sonia Medina designed, supervised, and discussed this research work.

Conflict of interest

The authors declare that there is no conflict of interest associated with this manuscript.