E Ponthier

H Domínguez

M.D Torres
email: matorres@uvigo.es

The microwave assisted extraction sway on the features of antioxidant compounds and gelling biopolymers from Mastocarpus stellatus

Keywords:

Microwave assisted water extraction of hybrid carrageenan and antioxidant compounds from Mastocarpus stellatus red alga was first proposed to recover biopolymers with specific rheological properties, saving time, avoiding chemicals and enhancing extraction yields when compared with conventional treatments. The hybrid carrageenans, precipitated from the liquid fractions recovered after microwave assisted water extraction were analyzed by Fourier transform infrared attenuated total reflectance, high-performance size-exclusion chromatography and rheology. The corresponding soluble extracts were also characterized by total phenolic content, antioxidant capacity, proteins, sulfate and carbohydrate content. The highest phenolic, antioxidant capacity and sulfate content were observed at 190 °C, without notable effect of heating time between 3 and 6 min. In contrast, a clear impact of both microwave heating temperature and time on the molecular weight distribution of hybrid carrageenans as well as on the viscoelastic behavior of formulated hydrogels was identified, being the strongest hydrogels achieved at 170 °C for 6 min. It should be highlighted that all proposed hydrogels exhibited stable mechanical properties in the absence of syneresis for a month of cold storage, except those made with biopolymers obtained with the mildest extraction conditions (3 min at 70 °C).

Introduction

In the last decade, there has been a growing interest in the macroalgae because they are rich sources of unique bioactive and biopolymerbased compounds with great potential for the food and non-food sector [START_REF] Campo | Carrageenans: biological properties, chemical modifications and structural analysis -a review[END_REF][START_REF] Torres | Coeliacs cannot live by gluten-free bread alone -every once in awhile they need antioxidants[END_REF]. Macroalgae are generally classified in the following three groups, Chlorophyta (green algae), Phaeophyta (brown algae) and Rhodophyta (red algae) [START_REF] Adl | The revised classification of eukaryotes[END_REF][START_REF] Dawes | Chapter 4 macroalgae systematics[END_REF]. Namely, the Rhodophyta are rich in bioactive compounds such as proteins, carotenoids, phenolics or vitamins [START_REF] Torres | Seaweed biorefinery[END_REF] as well as an important source of gelling biopolymers like carrageenan or agar, which represents about 20% on dry basis [START_REF] Wells | Algae as nutritional and functional food sources: revisiting our understanding[END_REF].

Carragenophyte seaweeds produce different carrageenan types like kappa-, iota-or lamba-and are widely distributed by the western and eastern coasts of the North to mid-Atlantic [START_REF] Wells | Algae as nutritional and functional food sources: revisiting our understanding[END_REF]. These polysaccharides are sulfated galactans which are produced mainly by Gigartinales species as their main cell-wall material [START_REF] Pereira | Portuguese carrageenophytes: carrageenan composition and geographic distribution of eight species (Gigartinales, Rhodophyta)[END_REF]. Carrageenans can have gelling, thickening and emulsifying properties [START_REF] Torres | Rheology of κ/ι-hybrid carrageenan from Mastocarpus stellatus: critical parameters for the gel formation[END_REF], representing a huge growing market of several millions dollars [START_REF] Bixler | A decade of change in the seaweed hydrocolloids industry[END_REF][START_REF] Boulho | Antiherpetic (HSV-1) activity of carrageenans from the red seaweed Solieria chordalis (Rhodophyta Gigartinales) extracted by microwave-assisted extraction (MAE)[END_REF]. This relevant fact can be explained taking into account that carrageenan is a natural polymer and the world is more and more concerned about sustainability issues [START_REF] Moreira | Gelling characteristics and rheology of kappa/ iota-hybrid carrageenans extracted from Mastocarpus stellatus dried at different temperatures[END_REF]. A number of factors can affect the chemical structure of the carrageenan and thus the gel properties such as alga culture conditions, season when collected, algae health or disease state, maturity degree, post-harvest storage or processing conditions. The extraction method of these biopolymers and the involved conditions with parameters such as temperature, pH or time can also dramatically impact the biopolymer properties and consequently the mechanical features of the gel [START_REF] Azevedo | Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis[END_REF].

Model red seaweeds as Mastocarpus stellatus, which is an important biomass on the Atlantic coast of Spain and Portugal, can be a suitable alternative for the carrageenan production [START_REF] Moreira | Gelling characteristics and rheology of kappa/ iota-hybrid carrageenans extracted from Mastocarpus stellatus dried at different temperatures[END_REF]. This seaweed is considered as a model because it shows a wide geographical distribution with limited variability in the chemical structure of extracted kappaiota carrageenan attributable to the collection site conditions [START_REF] Pereira | Portuguese carrageenophytes: carrageenan composition and geographic distribution of eight species (Gigartinales, Rhodophyta)[END_REF]. Moreover, it is a promising candidate for integrated multitrophic aquaculture and thus a future sustainable resource for the production of hybrid carrageenan in Europe [START_REF] Azevedo | Tailoring kappa/iota-hybrid carrageenan from Mastocarpus stellatus with desired gel quality through pre-extraction alkali treatment[END_REF][START_REF] Pereira | A comparative analysis of carrageenans produced by underutilized versus industrially utilized macroalgae (Gigartinales Rhodophyta)[END_REF]. Namely, this seaweed produce kappa/iota-hybrid carrageenans, which are natural polyelectrolyte copolymers made of blocks of kappa-(sequences of alternating 3-linked β-D-galactopyranose with a sulphate group on the fourth carbon of the sugar ring and 4-linked α-D-anhydrogalactopyranose) and blocks of iota-(sequences of alternating 3-linked β-D-galactopyranose with a sulphate group on the fourth carbon of the sugar ring and 4-linked α-D-T anhydrogalactopyranose bearing a sulphate group on the second carbon of the anhydro ring). These biopolymers commonly show enhanced mechanical properties when compared with individual kappa or iota carrageenan, exhibiting intermediate gelling properties [START_REF] Moreira | Drying kinetics of biofilms obtained from chestnut starch and carrageenan with and without glycerol[END_REF][START_REF] Domingues | On the bioremediation efficiency of Mastocarpus stellatus (Stackhouse) Guiry, in an integrated multi-trophic aquaculture system[END_REF]. Namely, kappa carrageenan can provide hard, strong or brittle gels, whereas iota carrageenan commonly forms soft and weak gels. Thus, the hybridization allows the formation of a strong elastic gel [START_REF] Moreira | Drying kinetics of biofilms obtained from chestnut starch and carrageenan with and without glycerol[END_REF]. The kappa/iota hybrid carrageenan gels can have many applications in different industries [START_REF] Domingues | On the bioremediation efficiency of Mastocarpus stellatus (Stackhouse) Guiry, in an integrated multi-trophic aquaculture system[END_REF]. It could be used in the dairy industry for cakes, in cosmetic for creams or lotions, in the pharmaceutical industry for semi-synthetic antibiotic production, in chemicals for paints or shoe polish, in experimental medicine for the testing of anti-inflammatory drugs or in biotechnology to immobilize cells/enzymes, among many others [START_REF] Abdul Khalil | Seaweed based sustainable films and composites for food and pharmaceutical applications: a review[END_REF][START_REF] Arufe | Air drying modelling of Mastocarpus stellatus seaweed a source of hybrid carrageenan[END_REF][START_REF] Pereira | Seaweeds as source of bioactive substances and skin care therapy-cosmeceuticals, algotheraphy, and thalassotherapy[END_REF].

Microwave assisted extraction (MAE) is a relatively new, green and scalable process for the recovery of high valuable natural compounds [START_REF] Boulho | Antiherpetic (HSV-1) activity of carrageenans from the red seaweed Solieria chordalis (Rhodophyta Gigartinales) extracted by microwave-assisted extraction (MAE)[END_REF][START_REF] Pérez | Microwave hydrodiffusion and gravity processing of Sargassum muticum[END_REF][START_REF] Vázquez-Delfín | Microwave-assisted extraction of the carrageenan from Hypnea musciformis (Cystocloniaceae, Rhodophyta)[END_REF][START_REF] López-Hortas | Valorisation of edible brown seaweeds by the recovery of bioactive compounds from aqueous phase using MHG to develop innovative hydrogels[END_REF], that could also be an attractive alternative for the recovery of hybrid carrageenans or antioxidants from macroalgae [START_REF] Flórez-Fernández | Microwave hydrogravity pretreatment of Sargassum muticum before solvent extraction of antioxidant and antiobesity compounds[END_REF][START_REF] Barral-Martínez | Tailoring hybrid carrageenans from Mastocarpus stellatus red seaweed using microwave hydrodiffusion and gravity[END_REF]. MAE allows a fast and uniform extraction, compact equipment, fast start-up, short time, less solvent and energy, which can be explained by the effect of microwaves on molecules by ionic conduction and dipole rotation [START_REF] Soria | Microwave-assisted extraction of polysaccharide[END_REF][START_REF] Ch | Antioxidant Activity of Sulfated Seaweeds Polysaccharides by Novel Assisted Extraction[END_REF]. Some authors [START_REF] Navarro | Microwave-assisted alkaline modification of red seaweed galactans[END_REF] studied the microwaveassisted alkaline modification of red seaweed galactans to enhance their gelation behavior, accelerating the reaction several times with respect to that performed with conventional heating [START_REF] Arufe | Air drying modelling of Mastocarpus stellatus seaweed a source of hybrid carrageenan[END_REF][START_REF] Flórez-Fernández | Microwave hydrogravity pretreatment of Sargassum muticum before solvent extraction of antioxidant and antiobesity compounds[END_REF]. Later works indicated the integrity of the polysaccharides treated by MAE was not affected by this procedure [START_REF] Navarro | Microwave-assisted desulfation of sulfated polysaccharides[END_REF]. However, a thermal degradation of some components can occur [START_REF] Magnusson | A comparative assessment of microwave assisted (MAE) and conventional solid-liquid (SLE) techniques for the extraction of phloroglucinol from seaweeds[END_REF][START_REF] Razzaghi | Operational efficiencies of six microwave based extraction methods for orange peel oil[END_REF][START_REF] Llompart | Microwave-assisted Extraction[END_REF].

In this context, the main objective of this novel work is to study the impact of microwave assisted extraction on the properties of the hybrid carrageenans and antioxidant compounds recovered from Mastocarpus stellatus for the integral valorization of this model red seaweed. For this purpose, the composition of the soluble extracts and the physical characterization of the gelling biopolymers obtained at different microwave processing conditions, as well as the rheological characterization of the corresponding gels were analyzed.

Materials and methods

Raw materials

Dehydrated Mastocarpus stellatus algae of ecological production (moisture content of 11.54 ± 0.25%, w.b. and ash content of 17.83 ± 0.19%) were provided by Porto Muíños company (A Coruña, Spain). Algae were stored in closed dark plastic bags at room temperature before and after being milled until to be processed. Fundamental chemical properties of the raw material were reported in a parallel work [START_REF] Barral-Martínez | Tailoring hybrid carrageenans from Mastocarpus stellatus red seaweed using microwave hydrodiffusion and gravity[END_REF].

Microwave assisted extraction

Milled algae (average particle size < 200 μm) were processed by Microwave Assisted Extraction (MAE) using a Microwave reactor Monowave 450 with autosampler Mas 24 (Anton Paar, Austria) for the recovery of high valuable compounds. Particularly, dried milled algae and distilled water were added in the microwave vials at a solid liquid ratio of 1:30 (w:w). Preliminary trials were performed to choose the operation conditions taking into account the outcomes achieved in previous works with seaweeds [START_REF] Flórez-Fernández | Recovery of bioactive and gelling extracts from edible brown seaweed Laminaria ochroleuca by non-isothermal autohydrolysis[END_REF]. The first set of samples were heated for a microwave time (t MAE ) of 3 min at a wide range of temperatures: 70, 90, 130, 150, 170, 190 °C and the second set of samples were heated for t MAE of 6 min at the same temperatures. Then, both were cooled until 55 °C. Subsequently, the liquid fraction was separated from the solid one by vacuum filtration. The samples were kept in the freezer until further use.

One volume of the liquid fraction obtained by MAE was precipitated with one volume of ethanol (96%, Sigma-Aldrich, USA). The precipitate was separated by filtration if necessary. Then, the recovered biopolymer was dried in an oven at 40 °C. The dried carrageenan was weighed to obtain the yield (g hybrid carrageenan/g dried alga powder) [START_REF] Azevedo | Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis[END_REF].

Composition analysis of the soluble extracts 2.3.1. Total phenolic compounds

Total phenolic content of the soluble extracts obtained by MAE was determined by the Folin-Ciocalteau method [START_REF] Singleton | Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents[END_REF]. The results were given as grams of Gallic Acid Equivalents (GAE). This is a spectrophotometric method in which it was necessary to mix 0.25 mL of soluble extracts or gallic acid (CAS no. 149-91-7, Sigma-Aldrich, USA) used as reference, 1.875 mL of distilled water, 0.175 mL of Folin reagent (CAS no. 7647-01-0, Panreac Química, Spain): water (1:1, v/v) . Note here that standard deviations were lower than 1% in all cases (n = 3). and 0.25 mL of 10% sodium carbonate solution (CAS no. 497-19-8, Sigma-Aldrich, USA). After leaving the test tubes for 60 min without light, the absorbance was measured at 765 nm.

Antioxidant capacity

Antioxidant characteristics of the soluble extracts were obtained by the ABTS (2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging method [START_REF] Re | Antioxidant activity applying an improved ABTS radical cation decolorization assay[END_REF]. The used unit was Trolox Equivalents Activity Antioxidant (TEAC). This is a spectrophotometric method in which it was necessary to add 1 mL of diluted ABTS (CAS no. 30931-67-0, Sigma-Aldrich, USA) (until the absorbance was 0.7-0.72) to 10 μL of soluble extracts or Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (CAS no. 53188-07-1, Sigma-Aldrich, USA). The test tubes had to be in bath at 30 °C. Six minutes after the addition of diluted ABTS reagent, the absorbance was measured at 734 nm.

Protein content

Protein content of the soluble extracts was obtained by the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. This is a spectrophotometric method which consists in the addition of 790 μL of distilled water and 200 μL of Bradford reagent (CAS 6104-58-1, Sigma-Aldrich, USA) to 10 μL of soluble extracts or Bovine Serum Albumin (BSA) (CAS no. 9048-46-8, Sigma-Aldrich, USA). After an incubation of 5 min at the room temperature, the absorbance was measured at 595 nm.

Sulfate groups

Free sulfate content of the soluble extracts was determined by gelatin-barium chloride method [START_REF] Dodgson | Determination of inorganic sulphate in studies on the enzymic and non-enzymic hydrolysis of carbohydrate and other sulphate esters[END_REF]. This is a spectrophotometric method which consists in mixing 0.2 mL of soluble extracts, 3.8 mL of 4% trichloroacetic acid (TCA) (CAS no. 76-03-9, Sigma-Aldrich, USA) and 1 mL of gelatin -barium chloride reagent. This reagent was prepared by adding 0.5 g of gelatin powder (CAS no. 9000-70-8, Sigma-Aldrich, USA) in 100 mL of hot water (60-70 °C) and let at 4 °C for at least 6 h, before adding 0.5 g of BaCl₂ (CAS no. 10361-37-2, Sigma Aldrich, USA). After leaving the test tubes for 15 min at room temperature, the absorbance was measured at 500 nm.

Monosaccharide analysis

Liquid samples obtained by MAE were filtered through 0.45 μm membranes, neutralized with barium carbonate (CAS no. 513-77-9, Thermo Fisher Scientific, Belgium) before HPLC analysis of carbohydrate compounds. These measurements were conducted on a 1100 series Hewlett-Packard chromatograph (IR detector) operating with a 300 × 7.8 mm CARBOsep CHO 682 column (Transgenomic, Glasgow, U.K.) at 80 °C using Milli Q water as mobile phase (0.4 mL/min). Other carbohydrate components were determined by HPLC using 300 × 7.8 mm Aminex HPX87H column (BioRad, Hercules, CA) operating at 60 °C with 0.003 M H 2 SO 4 (CAS no. 7664-93-9, Sigma-Aldrich, USA) at 0.6 mL/min as mobile phase. The content of oligosaccharides was determined after quantitative posthydrolysis (8% sulfuric acid at 121 °C, 20 min). Glucose, xylose, and galactose (Sigma Aldrich, Missouri, USA) were used as standards.

Physical characterization of the extracted biopolymer 2.4.1. Fourier transform infrared attenuated Total reflectance

The carrageenan fractions were determined by Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR). The measurements were conducted on a Nicolet 6700 spectrometer from 500 to 1500 cm -1 at room temperature for the qualitative characterization of the extracted carrageenan, as detailed elsewhere [START_REF] Pereira | Identification of selected seaweed polysaccharides (phycocolloids) by vibrational spectroscopy (FTIR-ATR and FT-Raman)[END_REF].

High-performance size-exclusion chromatography

The molar mass distribution of extracted carrageenan was analyzed by high-performance size-exclusion chromatography (HPSEC) using two 300 × 7.8 mm TSKGel G3000PWXL and G2500PWXL columns in series (Tosoh Bioscience, Stuttgart, Germany), a 40 × 6 mm PWXguard column at 70 °C with a refractive index detector. Milli Q water (0.4 mL/min) was used as a mobile phase. Dextrans (Fluka Chemie AG, Buchs, Switzerland) with molar masses between 1000 and 80,000 g/ mol were used as standards.

Rheology of the developed hydrogels

Aqueous hybrid carrageenan solutions were made at 1.0 w/w (0.5% at the highest yields) and a fixed ionic strength (0.1 mol/L potassium chloride (CAS no. 7447-40-7, Sigma-Aldrich, USA). The used conditions were chosen based on the results previously reported for this alga using conventional extraction procedures [START_REF] Azevedo | Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis[END_REF]. Note here that the potassium chloride solutions were prepared by dissolving potassium chloride powder in distilled water. Hybrid carrageenan solutions were prepared by dissolving biopolymers at 70-80 °C under strong stirring until the complete dissolution of carrageenan. Then, the solutions remained in the fridge at 5 °C for at least 24 h to allow the full maturation of the gels.

The rheological behavior of above prepared hydrogels was determined on a stress-controlled rheometer (MCR 302, Anton Paar, Graz, Austria) using parallel plates (25 mm diameter and 1 mm gap). To prevent evaporation, the samples were covered with paraffin oil. First, the samples were kept during 5 min at 25 °C to allow temperature equilibration. Then, frequency sweep tests were conducted from 0.1 to 10 Hz, at 10 Pa and at 25 °C.

Statistical analysis

All measurements were performed at least in triplicate. One-factor analysis of variance was employed to the statistical analysis of the experimental data using PASW Statistics v.22 software (IBM SPSS Statistics, New York, USA). A Scheffé test was carried out to distinguish means with a 95% of confidence (p < 0.05).

Results and discussion

Microwave assisted processing

The influence of the heating time and temperature during the microwave assisted water extraction of Mastocarpus stellatus was studied following the scheme presented in Fig. 1. After extraction, the solid phase was discarded, and the extract was analyzed for carrageenan and other high valuable compounds. Fig. 2 shows the heating and cooling profiles during water extraction in the microwave assisted process. Note here that the required times are significantly shorter than in a conventionally heated equipment as found in an ongoing work for Mastocarpus stellatus and other marine biomass [START_REF] Ruiz | Hydrothermal processing, as an alternative for upgrading agriculture residues and marine biomass according to the biorefinery concept: a review[END_REF]. Fast microwave treatments with satisfactory results were also previously reported for the alkaline modification of red seaweed galactans using a sharp domestic microwave [START_REF] Navarro | Microwave-assisted alkaline modification of red seaweed galactans[END_REF]. It should be noteworthy that microwave extraction times of 3-6 min can provide thermal profiles comparable to those attained in conventionally heated equipment.

Composition of microwave-treated soluble extracts

The impact of different microwave-assisted treatments on the antioxidant features of some sources of marine biomass has been reported ) [START_REF] Navarro | Microwave-assisted alkaline modification of red seaweed galactans[END_REF]. This eco-friendly treatment not only can accelerate the extraction process, but also promote the recovery of fractions rich in phenolic or sulfate compounds selecting the adequate processing conditions [START_REF] Soria | Microwave-assisted extraction of polysaccharide[END_REF][START_REF] Navarro | Microwave-assisted desulfation of sulfated polysaccharides[END_REF][START_REF] Santos | The quest for phenolic compounds from macroalgae: a review of extraction and identification methodologies[END_REF]. Fig. 3 shows that there is no influence of the heating time between 3 and 6 min at different temperatures on the extract composition. The highest phenolic and sulfate content were observed at 190 °C, but the protein content was similar between 150 and 190 °C. The magnitudes of above components were consistent with those previously reported for carrageenophyte red seaweeds using MAE-assisted treatments [START_REF] Navarro | Microwave-assisted desulfation of sulfated polysaccharides[END_REF] or conventional alkaline/acid treatments [START_REF] Gómez-Ordóñez | Dietary fibre and physicochemical properties of several edible seaweeds from the northwestern Spanish coast[END_REF][START_REF] Gómez-Ordóñez | Bioactivity of sulfates polysaccharides from the edible red seaweed Mastocarpus stellatus[END_REF][START_REF] Vega | Antioxidant activity of extracts from marine macroalgae, wild-collected and cultivated, in an integrated multi-trophic aquaculture system[END_REF]. A dramatic increase in the ABTS radical scavenging capacity was observed at 190 °C, more marked than the rise in the phenolic content. Probably this activity was due not only to the phenolic content, but also to other components. Similar behaviors highlighting a high increase in yields using optimized microwave aqueous extraction conditions (algae: solvent ratio 1:30, 160 °C, 3 min) were previously reported for other marine biomass [START_REF] Soria | Microwave-assisted extraction of polysaccharide[END_REF]. Higher total phenolic content and antioxidant activity values using this green technique when compared with conventional liquid-solid extractions was also reported in a comprehensive recent review for different macroalgae [START_REF] Santos | The quest for phenolic compounds from macroalgae: a review of extraction and identification methodologies[END_REF]. Higher bioactive compounds than those reported for Mastocarpus stellatus and other similar red seaweeds using conventional techniques (sulfate, 0.6-3.9 g/ 100 g, protein, 0.1-3.4 g/100 g, 4.9 μmol Trolox equivalents/g) was also reported [START_REF] Gómez-Ordóñez | Bioactivity of sulfates polysaccharides from the edible red seaweed Mastocarpus stellatus[END_REF][START_REF] Øverland | Marine macroalgae as sources of protein and bioactive compounds in feed for monogastric animals[END_REF].

Overall, the extracts obtained at 190 °C mainly contained 12% phenolics, 23% protein and 51% carbohydrates. Concerning the latter compounds, fundamental content for MAE soluble extracts treated at tested temperature and time conditions are summarized in Table 1. Again, no statistical impact of microwave heating time on carbohydrates compositions was found. However, a clear impact of microwave heating temperature was observed, with the highest carbohydrate content (around 66%) at 170 °C. The obtained carbohydrates are in well harmony with those previously reported for red seaweeds [START_REF] Cotas | A comprehensive review of the nutraceutical and therapeutic applications of red seaweeds (Rhodophyta)[END_REF][START_REF] Rodrigues | Impact of enzyme-and ultrasound-assisted extraction methods on biological properties of red, brown, and green seaweeds from the central west coast of Portugal[END_REF]. Glucose, xylose and galactose contents increased with rising heating Mechanical spectra (G': elastic modulus, G": viscous modulus) of representative hydrogels formulated with extracted hybrid carrageenan (1%, w/w). Note here that the visualization scale of y-axis of both plots is different. Note here that in all cases deviations were lower than symbol sizes.

temperature up to 170 °C, whereas their content significantly dropped at the highest tested temperature, which suggests some polysaccharides degradation at the highest processing temperatures [START_REF] Soria | Microwave-assisted extraction of polysaccharide[END_REF][START_REF] Ch | Antioxidant Activity of Sulfated Seaweeds Polysaccharides by Novel Assisted Extraction[END_REF]. It should be highlighted that the carbohydrates recovered in soluble extracts treated at 170 °C are even higher than those obtained from other similar red seaweeds treated using enzyme-or ultrasound-assisted extraction methods [START_REF] Sánchez-Camargo | Considerations on the use of enzyme-assisted extraction in combination with pressurized liquids to recover bioactive compounds from algae[END_REF]. Note here that acetic acid significantly decreased over the tested temperature range; whereas formic acid exhibited the reverse trend.

Physical properties of the extracted biopolymers

The hybrid carrageenan separated from the liquid extracts was also characterized. Fig. 4 presents the yield of the biopolymers obtained after microwave treatment of Mastocarpus stellatus red seaweed. The extraction time was influential at temperatures under 150 °C, but less important at higher temperatures. This behavior can be related with the solubilization temperatures of hybrid carrageenans, which is around 90 °C for conventional extraction procedures [START_REF] Azevedo | Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis[END_REF]. Moreover, the extraction yield showed a maximum of 60% at 150 °C, more than double the yield attained at 190 °C. Even though, the hybrid carrageenan extraction yields were improved using microwave assisted extraction when compared with long optimized conventional procedures, except at the milder conditions (70 °C) where similar magnitudes were identified. Namely, yields from conventional carrageenan extraction treatments for these type of carragenophyte seaweeds are usually around 20% [START_REF] Bixler | A decade of change in the seaweed hydrocolloids industry[END_REF][START_REF] Boulho | Antiherpetic (HSV-1) activity of carrageenans from the red seaweed Solieria chordalis (Rhodophyta Gigartinales) extracted by microwave-assisted extraction (MAE)[END_REF].

Fig. 5 shows the corresponding FTIR-ATR spectra of extracted hybrid carrageenans, mainly made for kappa and iota disaccharide units in the backbone. In general, data in plot shows that the extraction time did not notably influenced the FTIR-ATR profiles, although some attenuation in the intensities were observed at 190 °C for 3 min and 70-90 °C for 6 min. In all cases, the characteristic iota (at 805 cm -1 ) and kappa (at 845 cm -1 ) bands corresponding to 3,6-anhydro-galactose-2-sulfate and galactose-4-sulfate, respectively, were clearly identified [START_REF] Gómez-Ordóñez | Dietary fibre and physicochemical properties of several edible seaweeds from the northwestern Spanish coast[END_REF]. The existence of 3,6-anhydro-galactose was corroborated by a relatively strong IR absorption band at 930 cm -1 . Extracted carrageenans also featured a wide band at 1240 cm -1 ascribed to the S]O stretching vibration of the sulfated groups [START_REF] Pereira | Identification of selected seaweed polysaccharides (phycocolloids) by vibrational spectroscopy (FTIR-ATR and FT-Raman)[END_REF]. Consequently, the obtained FTIR profiles for Mastocarpus stellatus extracts are consistent with those described for kappa/iota-hybrid carrageenans from a number of red seaweeds [START_REF] Azevedo | Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis[END_REF][START_REF] Re | Antioxidant activity applying an improved ABTS radical cation decolorization assay[END_REF]. In order to estimate the kappa/iota carrageenan hybridization, the intensity ratio of IR absorption bands at 805 and 845 cm -1 was calculated as previously described [START_REF] Gómez-Ordóñez | Bioactivity of sulfates polysaccharides from the edible red seaweed Mastocarpus stellatus[END_REF]. For all samples, the estimated ratios presented values between 0.29 and 0.35, showing the prevalence of kappa against iota carrageenan nature. This behavior is consistent with the outcomes previously found for carrageenans from Mastocarpus stellatus using conventional alkali treatments with values of hybridization around 0.31 [START_REF] Gómez-Ordóñez | Bioactivity of sulfates polysaccharides from the edible red seaweed Mastocarpus stellatus[END_REF][START_REF] Hilliou | Hybrid carrageenans: isolation, chemical structure and gel properties[END_REF].

The molecular weight distribution in the hybrid carrageenan extracted at 3 and 6 min is presented in Fig. 6. No peaks in the studied interval were observed for the extracts obtained at 70 °C. The extracts obtained at higher temperatures showed peaks under 1 kDa and higher than 80 kDa. The extracts obtained after 3 min at 90 and at 130 °C showed two peaks higher than 80 kDa, but at higher temperatures or at 6 min heating time, only one peak was observed. The signals of these peaks increased with the extraction temperature and were maximal in the extracts obtained at 130 and 150 °C when the extraction time was 3 min and at 170 °C for the extracts produced after 6 min. The intensity of the signals was three times higher for the extracts obtained at 6 min. The molecular weights of extracted hybrid carrageenans are consistent with those previously reported for other edible carrageenophyte red seaweeds [START_REF] Azevedo | Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis[END_REF]. The average molecular weights of those hybrid carrageenans recovered at 170 °C for 6 min are comparable with equivalent commercial kappa and iota-carrageenans [START_REF] Azevedo | Tailoring kappa/iota-hybrid carrageenan from Mastocarpus stellatus with desired gel quality through pre-extraction alkali treatment[END_REF]. To sum up, the range of molecular weight distributions obtained in extracted hybrid carrageenans is a critically relevant feature in order to achieve biopolymers with a wide range of mechanical properties [START_REF] Campo | Carrageenans: biological properties, chemical modifications and structural analysis -a review[END_REF][START_REF] Hilliou | Effect of extraction parameters on the chemical structure and gel properties of kappa/iota-hybrid carrageenans obtained from Mastocarpus stellatus[END_REF].

Mechanical characterization of formulated hybrid carrageenan hydrogels

Fig. 7 shows the viscoelastic behavior at 25 °C of representative kappa/iota-hybrid carrageenans (1%, w/w) prepared in KCl (0.1 mol/ L). Most systems presented elastic and viscous moduli independent of frequency with the elastic modulus larger than the viscous one over the tested frequency, which is indicative of gel materials [START_REF] Hilliou | Effect of extraction parameters on the chemical structure and gel properties of kappa/iota-hybrid carrageenans obtained from Mastocarpus stellatus[END_REF]. Note here that only very weak gel properties were identified for those systems formulated with hybrid carrageenans extracted at the milder microwave conditions (70 °C for 3 min), which is consistent with the molecular weight distribution profiles aforementioned. Another developed hydrogels here featured intermediate strength according the elastic and viscous moduli values. Similar viscoelastic tendencies were observed for those systems formed at lower carrageenan content (0.5%, w/w), although with lower values (around half) of both moduli and then weaker gelling characteristics. The achieved viscoelastic magnitudes are in the range of those previously reported for other hybrid carrageenans extracted using conventional procedures [START_REF] Azevedo | Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis[END_REF][START_REF] Hilliou | Hybrid carrageenans: isolation, chemical structure and gel properties[END_REF][START_REF] Pereira | Identification of selected seaweed polysaccharides (phycocolloids) by vibrational spectroscopy (FTIR-ATR and FT-Raman)[END_REF].

The impact of microwave heating temperature and time on mechanical properties of formulated hydrogels can be clearly observed in Fig. 8. In general, G' gel (1Hz) increased with increasing microwave temperature and time up to 170 °C, which involves the enhancement of the gel strength. A higher effect of both parameters was identified at higher biopolymer content. Note here that no gels were achieved with hybrid carrageenans at 190 °C independently of extraction time at selected ionic strengths, may be related with the small signals observed in HPSEC profiles. This suggests that higher biopolymer content or ionic strength should be tried to obtain hydrogels for this carrageenan [START_REF] Azevedo | Tailoring kappa/iota-hybrid carrageenan from Mastocarpus stellatus with desired gel quality through pre-extraction alkali treatment[END_REF]. Overall, analyzing jointly the carrageenan extraction performance and the mechanical properties of the corresponding hydrogels, it seems that hybrid carrageenans treated between 150 and 170 °C for 6 min presented the most adequate biopolymer features.

Conclusions

Microwave assisted water extraction of hybrid carrageenans, proteins, carbohydrates and antioxidant compounds from Mastocarpus stellatus red alga was tested using an environmentally friendly process with enhanced extraction yields. A noticeable effect of microwave temperature was observed on the recovered soluble extracts properties, achieving the maximum values of proteins at 150 °C, of carbohydrates at 170 °C and of antioxidants at 190 °C. Concerning the gelling biopolymer fractions, the most adequate microwave treatment to obtain the highest yields is 150 °C for 6 min. This microwave heating temperature should be increased up to 170 °C whenever it is sought to promote the strength of the hydrogels. Hybrid carrageenan-based hydrogels with stable viscoelastic properties and intermediate strength can be developed with the proposed microwave processing conditions. Another important industrial benefit is the absence of syneresis of the above hydrogels. Overall, it should be noteworthy that this novel work provided a set of kappa/iota -hybrid carrageenans with potential applications in food and non-food sectors to develop functional hydrogels, resulting from algae processed to different microwave heating temperatures for times, without jeopardizing the mechanical characteristics of the corresponding hydrogels.

CRediT authorship contribution statement

All the authors have read, approved, and made substantial contributions to the manuscript. HD and MDT conceived the study, made the drafting of the manuscript and the critical revision. EP and MDT made the experimental tests.

Fig. 1 .Fig. 2 .

 12 Fig. 1. General scheme of Mastocarpus stellatus treatment by microwave assisted extraction. MW: Microwave, RSL: Solid liquid ratio, FTIR: Fourier transform infrared attenuated total reflectance, HPSEC: high-performance size-exclusion chromatography.

Fig. 3 .

 3 Fig. 3. Influence of microwave assisted extraction (MAE) temperature and heating time on a) total phenolic content, b) Trolox equivalents activity antioxidant (TEAC) value, c) protein content, and d) sulfate content of the extracts. Symbols: time of 3 min (blue bars), time of 6 min (orange bars). Data values were presented as average ± standard deviations (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 .

 4 Fig. 4. Influence of microwave assisted extraction (MAE) temperature and heating time on the yield of extracted carrageenan. Symbols: time of 3 min (blue bars), time of 6 min (orange bars). Data values were presented as average ± standard deviations (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .

 5 Fig. 5. Fourier transform infrared attenuated total reflectance spectra of hybrid carrageenan obtained by (a) 3 min and (b) 6 min of microwave assisted extraction (MAE) heating time. Note here that standard deviations were lower than 1% in all cases (n = 3).

Fig. 6 .

 6 Fig. 6. High-performance size-exclusion chromatography elution profiles of hybrid carrageenan obtained by (a) 3 min and (b) 6 min of microwave assisted extraction (MAE) heating time. Note here that standard deviations were lower than 1% in all cases (n = 3).

  Fig. 7.Mechanical spectra (G': elastic modulus, G": viscous modulus) of representative hydrogels formulated with extracted hybrid carrageenan (1%, w/w). Note here that the visualization scale of y-axis of both plots is different. Note here that in all cases deviations were lower than symbol sizes.

Fig. 8 .

 8 Fig. 8. Overview of the elastic modulus (G') at 1 Hz for all tested systems prepared at 1% of biopolymer content. Inset represents the effect microwave assisted extraction (MAE) temperatures (x-axis) in the elastic behavior at 1 Hz (y-axis) for gelled samples made at 0.5%. Symbols: time of 3 min (blue bars), time of 6 min (orange bars). Data values were presented as average ± standard deviations (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1

 1 Carbohydrate

		Composition	70 °C	90 °C	130 °C	150 °C	170 °C	190 °C
	Time, 3 min	Glucose (%)	4.1 ± 0.1 e	4.9 ± 0.3 d	6.2 ± 0.2 c	6.8 ± 0.1 b	7.3 ± 0.1 a	5.8 ± 0.1 c
		Xylose (%)	1.5 ± 0.1 d	2.0 ± 0.1 c	2.6 ± 0.2 b	2.8 ± 0.1 b	3.2 ± 0.1 a	1.7 ± 0.4 c,d
		Galactose (%)	19.7 ± 0.4 e	22.4 ± 0.8 d	29.2 ± 1.1 c	30.6 ± 0.6 b	32.8 ± 0.5 a	23.9 ± 1.2 c
		Acetic acid (%)	31.6 ± 0.5 a	30.9 ± 0.9 a	29.1 ± 0.2 b	20.1 ± 0.8 c	15.5 ± 0.6 d	13.8 ± 0.5 e
		Formic acid (%)	3.9 ± 0.2 d	4.1 ± 0.1 d	4.6 ± 0.2 c	5.5 ± 0.3 b	6.0 ± 0.2 a	6.1 ± 0.1 a
	Time, 6 min	Glucose (%)	4.5 ± 0.5 e	5.1 ± 0.4 d	6.0 ± 0.4 c	6.9 ± 0.2 b	7.5 ± 0.2 a	5.9 ± 0.3 c
		Xylose (%)	1.7 ± 0.3 d	2.1 ± 0.1 c	2.8 ± 0.3 b	2.9 ± 0.1 b	3.4 ± 0.2 a	1.9 ± 0.2 c,d
		Galactose (%)	19.9 ± 0.3 e	22.0 ± 0.5 d	29.5 ± 0.9 c	31.5 ± 0.5 b	33.3 ± 0.7 a	22.5 ± 1.5 c
		Acetic acid (%)	31.9 ± 0.3 a	31.4 ± 0.5 a	29.6 ± 0.4 b	20.9 ± 0.4 c	16.0 ± 0.2 d	14.1 ± 1.0 e
		Formic acid (%)	3.5 ± 0.1 e	4.3 ± 0.2 d	4.9 ± 0.4 c	5.7 ± 0.3 b	6.2 ± 0.3 a	6.7 ± 0.6 a
	Data values are presented as average ± standard deviations (n = 3). Data values in a raw with different superscript letters are significantly different at the p ≤ 0.05
	level.

content for soluble extracts recovered from microwave assisted extraction (MAE) at different microwave heating temperatures (70-190 °C) and times (3 and 6 min).

Acknowledgements

Authors thank the financial support to the Ministry of Science, Innovation and Universities of Spain (RTI2018-096376-B-I00). Financial support from the Xunta de Galicia (Centro singular de investigación de Galicia accreditation 2019-2022) and the European Union (European Regional Development Fund -ERDF), is grate fully acknowledged (Ref. ED431G2019/06). M.D.T. thanks Spanish Ministry of Science, Innovation and Universities for her postdoctoral grant (IJCI-2016-27535 and RYC2018-024454-I).

Informed consent, human/animal rights

No conflicts, informed consent, human or animal rights applicable.

Declaration of competing interest

The authors declare no conflict of interest.

Declaration of authors

All authors agree to authorship and submission of the manuscript for peer review.


Florin Huţu

Vincent Lechappé

Guillaume Villemaud

Michaël Di Loreto

Study of Distributed Beamforming for Wireless Power Transfer in Presence of RF Impairments

For an equivalent performance (sensitivity, communication range, computational capability, etc.), the energy efficiency of connected devices is continuously increasing. Providing the energy for such objects by the means of electromagnetic waves (wireless power transfer) becomes a feasible option in various scenarios, especially those in which the use of conventional energy resources (e.g. batteries) is unfeasible.This paper tackles a particular method of wireless power transfer, which is the distributed beamforming, i. e. the use of several sources, spatially distributed and which are coordinating together in order to maximize the power of the electromagnetic field in a particular location. Through simulation results, this paper highlights the robustness of this approach to different nonidealities, i.e. frequency and phase synthesis, phase noise and channel characteristics. A first experimental setup is also presented.

Introduction

All studies predicted an exponential increase of the number of connected devices (50 billions in 2020 [START_REF]More Data, Less Energy, Making Network Standby More Efficient in Billions of Connected Devices[END_REF]) in a multitude of applications related to agriculture, industry, health monitoring, etc. This huge amount of connected devices gives an idea of how important is to find new ways to decrease their environmental impact.

In many Internet of Things (IoT) applications, the use of classical energy source is inappropriate. One can cite the case of sensors buried in concrete that are monitoring the mechanical efforts inside the walls. The sensor life span will be strictly related to the battery lifetime. For these scenarios, the solution to transmit the entire or a part of the energy through electromagnetic waves may be taken into account. Indeed, this solution is feasible because of the increasing efficiency of the electronic devices. Some studies (e.g. [START_REF] Koomey | plications of Historical Trends in the Electrical Efficiency of Computing[END_REF]) are predicting that in the next decade the energy needed for a computational task will decrease by a factor 10000 compared to the current levels.

Wireless power transfer has already been proposed as a method to energize connected objects. Generally, the research is focusing at the receiver side, in order to increase the efficiency of the RF power rectifier since here is the main limitation. This efficiency is varying with respect to the received input power. To the best of our knowledge, with the current technology, in order to have a rectifier's efficiency greater than 25%, the received signal should be superior to -20 dBm [START_REF] Paz | A survey on low power RF rectifiers efficiency for low cost energy harvesting applications[END_REF].

In order to increase the rectifier's efficiency, some recent works are focusing on the use of multi-carrier signals [START_REF] Rousseau | On the use of Vector Fitting and State-Space Modeling to Maximize the DC Power Collected by a Wireless Power Transfer System[END_REF]. Indeed, such signals are renowned for having a high PAPR (Peak to Average Power Ratio) and to increase the DC voltage level at the rectifier's output [START_REF] Boaventura | Boosting the Efficiency: Unconventional Waveform Design for Efficient Wireless Power Transfer[END_REF]. Some other works are proposing a systemic view on maximizing the efficiency of the WPT by taking into account the power source, the effects of the propagation channel and the characteristics of the rectifier [START_REF] Clerckx | Fundamentals of Wireless Information and Power Transfer: From RF Energy Harvester Models to Signal and System Designs[END_REF].

Wireless power transfer by using time reversal technology was proposed in [START_REF] Ku | Power Waveforming: Wireless Power Transfer Beyond Time Reversal[END_REF]. This technique is based on spatial-temporal focusing effect. After a channel probing, made by sending an impulse signal, the transceiver will return a time-reversed, conjugate and amplified replica of the received impulse. These replicas will combine at the exact spot of the initial pulse, bringing there a relatively high amount of electromagnetic field energy.

Distributed beamforming has already been proposed as a way to maximize the wireless power transfer and some theoretical solutions are proposed [START_REF] Goguri | Maximizing wireless power transfer using distributed beamforming[END_REF]. Here, the authors are formulating the problem of maximizing the power delivered by a distributed antenna array to a receiver by taking into account the channel effects. A fixed-point algorithm is giving the optimum power allocations in a finite set of frequencies.

In distributed beamforming, a network of individual wireless nodes having single antennas are cooperating in order to generate synchronized signals. The considered sources may be Access Points (AP) in Wireless Local Area Networks (WLAN) or cellular base stations. The initial phase of the emitted signals are chosen such way that, in a desired spot, the phase coherence occurs. Consequently, the amplitude of the electromagnetic field is locally maximized. The main contribution of this paper is to study the impact of the RF impairments on the amount of energy transferred by employing distributed beamforming. More precisely, by taking as reference an ideal scenario, the power decrease in presence of frequency and phase synthesis errors, in presence of sources phase noise and in presence of a realistic propagation channel is studied.

In Section 2, the distributed beamforming for wireless power transfer purpose is formalized. Then, some simulation results tackle the impact of different beam-forming synthesis errors on the received power level. Section 3 presents an experimental setup built in order to validate the distributed beamforming scenarios. Conclusion and future directions are given in Section 4.

Problem statement and simulation results

Consider N identical sources randomly distributed in a Cartesian plane. These sources are coordinating together in order to obtain phase coherent signals at the origin of the coordinate plane in order to perform wireless power transfer efficiently. Each source is defined by its coordinates (x i , y i ) where i ∈ [1, N]. Consider also that each source is able to generate a signal at a frequency f 0 and with an identical power level P. At the center of the coordinate system, a receiver having low energy resources is placed.

ϕ i = 2π λ 0 x i 2 + y i 2 (1)
where λ 0 is the wavelength of the emitted waveform.

In the case of the phase coherence, the power level collected by the receiver increases with the number of sources. In the particular case in which N sources are placed at the same distance around the receiver, the gain related to the power level received from one source can be expressed as:

[G] dB = 20 • log 10 (N) . (2)
Indeed, suppose that only one source is emitting. The received power level can be written as:

[P R ] dBm = 10 • log 10 A 2 R ( 3
)
where A is the amplitude of the received sinusoidal waveform and R is the load presented by the receiver.

Consider now that all N sources are emitting sinusoidal waveforms at the same frequency so that the signals arrive in phase coherence at the receiver level. In this case, the received power level become:

[P RN ] dBm = 10 • log 10 (N • A) 2 R . ( 4
)
If the gain in terms of power G is defined as :

G P RN P R , (5)
then, the result given in equation ( 2) is straightforward.

As an example, from Figure 2, which is the graphical representation of equation ( 2), one can remark that when the four sources are emitting, the gain in terms of received power is 12 dB. In order to verify this theoretical gain in a more realistic scenario, a simulator based on Keysight's ADS software was implemented. In this simulator, each source is placed at a certain distance from the sink and is able to deliver a CW (continuous wave) signal. Between the different sources and the sink, free space path loss propagation channel was considered as a first step.

If the scenario given in Table 1 is simulated, the received signals have an identical phase of 150.8 degrees and the received power level is -33.04 dBm. In this particular case, the emitted power levels are of -20 dBm and antenna gains are 4.1 dBi. Cables connecting different antennas and having an insertion loss of 0.85 dB were also taken into account.

Moreover, the simulator is able to test the impact on the received power of the frequency and phase synthesis deviation. Figure 3 and shows the sensitivity of the distributed beamforming strategy to frequency variations. Figure 4 presents the impact of the phase deviation on the received power level. More precisely, the phase of one emitter is modified with respect to the desired value, while the other three phases are remaining at their ideal values. The received power level is insensitive on the phase synthesis since power diminutions of 1 dB are obtained for errors ∆ϕ greater than ±60 degrees, with respect to the ideal phase ϕ i .

These simulation results are showing the consequence of the synchronization between the different sources which seems to be more important than the one of phase synthesis.

Another parameter which was suspected to have an influence on the distributed beamforming accuracy was the local oscillator's phase noise. Indeed, real oscillators are presenting random variations of their instantaneous Figure 4: The received power level variation with respect to the deviation of the desired phases ϕ i phase which may corrupt the phase accuracy at the receiver side. In order to test this influence, the phase noise of the NI PXIe-5644R RF vector signal transceiver was considered as reference. This transceiver has good performance in terms of phase noise and it is expected that the different sources used for distributed beamforming will present poor performance. The VST's phase noise levels are given in Table 2.

Freq.

[kHz] 10 -1 10 0 10 5 presents the variation of the received power level when the phase noise floor is increased with respect to the values given in Table 3. A parametric simulation was performed while increasing the power level of the phase noise with a quantity P N . All the four sources are considered to have identical phase noise levels. As can be remarked, a phase noise of -38 dBc/Hz @ 100 Hz will decrease the received power level by 1 dB compared to the ideal value. One can say that the local oscillator's Figure 5: The received power variation with respect to the increase of the phase noise level phase noise has a small impact on the accuracy of the distributed beamforming performance since this value is beyond the ones announced for most of the commercial radio front-ends. Moreover, this reinforces the remark that distributed beamforming is robust in terms of phase synthesis. The propagation channel plays also an important role in the accuracy of the phase at the ceiver side. In a realistic distributed beamforming scenario, multipath phenomena between the emitters and the receiver are unavoidable. Propagation channels having multiple paths (Rice channels) were considered between each emitter and the receiver.

The Rician factor α ∈ [0 • • • 1] was considered as in [START_REF] Abdi | On the estimation of the K parameter for the Rice fading distribution[END_REF], being the ratio between the amplitude of the LOS (line of sight) power level and the power of the NLOS (non line of sight) version. Moreover, the NLOS path was considered to be delayed by a factor τ ∈ [0 • • • T ], where T = 1/ f 0 is the period of the emitted signals.

As can be remarked from Figure 6, a multi path channel is decreasing the received power level only if the NLOS version of the emitted signal is closed to quadrature (i. e. τ ≈ T /2). This degradation is more important when the rician factor α is high (i. e. power level of the NLOS received signal closed to the LOS power level).

Experimental setup

A distributed beamforming scenario was implemented in FIT/CorteXlab testbed [START_REF]villemaud@insa-lyon[END_REF], a quasi-anechoic environment. A National Instruments' PXI chassis containing four 5645 VSTs is the core of the experimental setup. The four VST (Vector Signal Transceiver) were used as CW generators and a spectrum analyzer was employed in order to measure the received power level. Experiments were conducted at 868 MHz and, as can be seen from Figure 7, four omnidirectional antennas have been connected to the VST's RF output. The antennas have a gain of 4.1 dBi, the coaxial cables have the same phase shift at 868 MHz and the emitted power level was set to -20 dBm, identical for the four VST. One receiving omnidirectional antenna has been placed at the center of a Cartesian coordinate system. The synchronization of the four VST and their phase coherence were tested beforehand.

More precisely, the phase coherence was assured by sharing the local oscillator of one of the VSTs to the other three. A calibration procedure was implemented in order to cancel phase offset brought by the coaxial cables connecting the VTS's local oscillators inputs and outputs.

The scenario presented in Table 1 was implemented and the received power level was approximately -34.5 dBm, close to the simulated one. The difference between the simulated and measured received powers may come from the antenna gains uncertainty.

Conclusion

This article presented the distributed beamforming as a solution for wireless power transfer. The impact of the RF chain non idealities on the received power level is also tackled. Moreover, the paper presented an experimental setup able to implement distributed beamforming scenarios. Future work will focus on implementation of algorithms able to take into account the impact of the propagation channel since it is the most unpredictable part of the distributed beamforming setup.

Figure 1 :

 1 Figure 1: Scenario of wireless power transfer using geographically distributes RF sourcesThis receiver is power supplied by the electromagnetic field produced by the N sources. As stated, the power of the received electromagnetic field is maximized if the arriving signals are in phase coherence. In order to obtain the phase coherence at the receiver level, the initial phase of the emitted signals should be :

Figure 2 :

 2 Figure 2: The gain in terms of received power

Figure 3 :

 3 Figure 3: The received power level variation with respect to the CFO (Central Frequency Offset)

Figure 6 :

 6 Figure 6: Influence of the propagation channel on the received power level

Figure 7 :

 7 Figure 7: Measurement setup deployed in an quasianechoic chamber

Table 1 :

 1 presents the variation of the received power level if one of the emitters among the four has a Coordinates and the initial phases of the CW sources frequency deviation ∆ f with respect to the central frequency f 0 =868 MHz. As can be remarked, compared to the maximum power level (-33 dBm), the received power level is about 1 dB lower for deviations of the emitted frequency around ± 25 kHz with respect to the ideal value. This value corresponds to a variation of the synthesized frequency of approximately 28.8 ppm (parts per million)

	Antenna x (meters) y (meters) ϕ (degrees)
	1	-0.75	0.6
	2	0.75		68.2
	3	-0.65	-0.75	33.4
	4	0.85	-0.85	-108.2

Table 2 :

 2 NI's 5644 VST's phase noiseFigure

	1	10 2	10 3

Acknowledgment

This work was supported by the "Institut National des Sciences Appliquées" Lyon, France under the BQR 2019-2021 program.


C Blanc-Bisson
email: cblancbisson@free.fr

F L Velayoudom-Cephise

A Cougnard-Gregoire

C Helmer

K Rajaobelina

C Delcourt

L Alexandre

L Blanco

K Mohammedi

M Monlun

V Rigalleau

Skin autofluorescence predicts major adverse cardiovascular events in patients with type 1 diabetes: a 7-year follow-up study

Keywords: Skin autofluorescence, Macroangiopathy, Advanced glycation end-products, Type 1 diabetes

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

Skin autofluorescence predicts major adverse cardiovascular events in patients

Introduction

Despite the decline of cardiovascular disease in patients with type 1 diabetes (T1D) [START_REF] Rawshani | Mortality and cardiovascular disease in type 1 and type 2 diabetes[END_REF], it remains by far the first cause of mortality, as recently reported in patients with long duration of diabetes [START_REF] Tinsley | Association of glycemic control with reduced risk for large-vessel disease after more than 50 years of type 1 diabetes[END_REF], and a major concern even in children who often present features of subclinical cardiovascular disease [START_REF] Gourgari | Modifiable risk factors for cardiovascular disease in children with type 1 diabetes: can early intervention prevent future cardiovascular events?[END_REF]. The cardiovascular risk of subjects with T1D is influenced by modifiable conventional risk factors such as arterial hypertension, dyslipidemia, and smoking, [START_REF] De Ferranti | Type 1 diabetes mellitus and cardiovascular disease: a scientific statement from the American Heart Association and American Diabetes Association[END_REF], and is strongly higher in the presence of diabetic nephropathy [START_REF] Tuomilehto | Incidence of cardiovascular disease in Type 1 (insulin-dependent) diabetic subjects with and without diabetic nephropathy in Finland[END_REF], but hyperglycaemia by itself is thought to play an important role.

Long-term hyperglycaemia promotes cardiovascular mortality, with a gradual increase of hazard ratios for death among patients with T1D compared to non diabetic subjects according to the HbA1C levels [START_REF] Lind | Glycemic control and excess mortality in type 1 diabetes[END_REF]. However a risk persists for well-controlled subjects with HbA1C ≤ 6.9%.

The effect of intensive glucose control on cardiovascular events was significant only 11 years after the intensive glucose control intervention in the Diabetes Control and Complications Trial, owing to a "glucose memory" effect [START_REF] Nathan | Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes[END_REF]. A recent detailed analysis of cardiovascular risk factors of the DCCT-EDIC participants emphasized the importance of the mean time-weighted HbA1C [START_REF] Diabetes | Complications Trial/Epidemiology of Diabetes I, Complications Research G. Risk factors for cardiovascular disease in type 1 diabetes[END_REF].

Advanced glycation end-products (AGEs) generated from excess glucose have deleterious effects on endothelial cells [START_REF] Brownlee | Biochemistry and molecular cell biology of diabetic complications[END_REF]. They are thought to contribute to the memory of hyperglycaemia [START_REF] El-Osta | Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia[END_REF]. The skin concentrations of AGEs have been related to the progression of intimamedia thickness in the DCCT-EDIC study [START_REF] Monnier | Skin collagen advanced glycation endproducts (AGEs) and the long-term progression of sub-clinical cardiovascular disease in type 1 diabetes[END_REF]. Due to their fluorescent properties, the accumulation of AGEs in tissues can be simply estimated by measuring the skin autofluorescence (SAF) [START_REF] Meerwaldt | Simple non-invasive assessment of advanced glycation endproduct accumulation[END_REF]. SAF predicted later cardiovascular events in patients with type 2 diabetes [START_REF] Lutgers | Skin autofluorescence provides additional information to the UK Prospective Diabetes Study (UKPDS) risk score for the estimation of cardiovascular prognosis in type 2 diabetes mellitus[END_REF]. We have related it to later impaired renal function [START_REF] Velayoudom-Cephise | Skin autofluorescence predicts cardio-renal outcome in type 1 diabetes: a longitudinal study[END_REF] and neuropathy [START_REF] Rajaobelina | Skin autofluorescence and peripheral neuropathy four years later in type 1 diabetes[END_REF] in patients with T1D. However, the relation between SAF and later cardiovascular events has not been reported yet in T1D. We aimed to test the association between SAF and future occurrence of major adverse cardiovascular event (MACE) in patients with T1D followed-up over 7 years.

Subjects and methods

Design and patients

Two hundred and thirty-two patients with T1D were included consecutively in 2009 after consent during annual visit in University Hospital of Bordeaux. The only exclusion criterion was ultraviolet reflectance < 10% that precluded the measurement of SAF. The patients with skin phototypes V and VI were therefore not included. The following data were recorded at inclusion on the day of SAF measurement: age, sex, body mass index, duration of diabetes, treatment by Continuous Subcutaneous Insulin Infusion, arterial hypertension (blood pressure ≥ 140/90 mmHg and/or treatment with antihypertensive drug), treatment by statins, previous macrovascular events, and smoking history. The biological data recorded included HbA1c levels, blood lipids, the Albumin Excretion Rates (AER), and serum creatinine to estimate the Glomerular Filtration Rates calculated with the CKD-EPI formula [START_REF] Sprenger | Skin advanced glycation end products in HIV infection are increased and predictive of development of cardiovascular events[END_REF]. Participants were prospectively followed-up until the occurrence of new cases of major adverse cardiovascular events (MACE) till March 2017. Number of patients was mentioned when data were missing.

Skin autofluorescence (SAF)

The accumulation of AGEs was evaluated at inclusion [START_REF] Meerwaldt | Simple non-invasive assessment of advanced glycation endproduct accumulation[END_REF], using the AGE-Reader (diagnOptics BV, Groningen, The Netherlands). The device illuminated 1 cm 2 of the forearm skin. SAF values were calculated by dividing the mean emitted light intensity (excitation light source ranging from 300 to 420 nm) by the mean reflected excitation light intensity from the skin (over 300-420 nm). The patients with Fitzpatrick phototypes V and VI were not included due to their skin pigmentation, which had ultraviolet reflectance of < 10%. The results were the mean of three consecutive measurements of SAF and were expressed in arbitrary units (AU).

Outcomes

MACE were defined as myocardial infarction, stroke, lower limb amputation, or a revascularization procedure for coronary, carotid or lower limb arteries. Every year, patients came in visit at hospital, MACE was collected by physician if occurred since previous visit and recorded in the informatic medical folder of each patient. If we had no recent news we called practitioner to ask if MACE or death occurred.

Statistical methods

The results are presented as mean ± SD for continuous variables, or median (IQR) for those with skewed distribution. Categorial variables are presented as numbers (percentages). A Cox model with delayed entry (taking age as the scale-time) was used to investigate the association between SAF and MACE. R2 of the model has been calculated by the following formula: R2 = 1e -(LRT/n) , where LRT is the likelihood-ratio statistic. Log-linearity of the model has been tested using SAF as a continuous variable and in quartile. Risk proportionality has been verified both by testing the interaction between SAF and time and by using Schoenfeld residuals. In a first step, analyses were performed to test the association between each co-variable and MACE in order to select variables for the multivariate analysis. Then, all the variables associated with MACE with a p value ≤ 0.10 were retained in the multivariate Cox model. All statistical analyses were performed using SAS version 9.3 (SAS Institute Inc, Cary, NC) and results were considered significant when p < 0.05. Follow-up was analysed using Kaplan-Meier curve.

Results

Characteristics of the population at inclusion

Participants were mainly men (59.5%), 51.5 ± 16.7 years old, with a 21.5 ± 13.6 years diabetes duration. Their BMI was 25.0 ± 4.1 kg/m 2 , HbA1c 7.6 ± 1.1%, eGFR was 86.0 ± 26.3 ml/min/1.73 m 2 . Among them, 25.1% were smokers, 45.3% had arterial hypertension, 15.9% had elevated AER (≥ 30 mg/24H), and 9.9% had a history of previous macrovascular event. Their SAF was 2.13 ± 0.58 AU. The scatterplot for SAF over age is presented as a Additional file 1: Figure S1.

Subjects with new major adverse cardiovascular events during the follow-up

Twenty-two patients presented MACE during a median (IQR) duration of follow-up of 7.8 [7.6-8.0] years: 6 myocardial infarctions, 1 gangrene and 15 revascularization procedures. As compared to subjects without MACE, they were significantly more men, 10 years older, with 9 more years of diabetes duration, but similar HbA1c (Table 1). The lipid profiles did not significantly differ, but subjects with MACE were more likely to be treated by statins in comparison with subjects without MACE. Arterial hypertension, abnormal eGFR (< 60 ml/ min/1.73 m 2 ) and abnormal (> 30 mg/24 h) AER were significantly more frequent.

SAF and new major adverse cardiovascular events (Table 2, Fig. 1)

At inclusion, the SAF was 2.63 ± 0.73 AU in patients who presented MACE during the follow-up, vs 2.08 ± 0.54 for the others (p = 0.002). In addition to SAF, gender, duration of diabetes, BMI, hypertension, use of statins, smoking, renal parameters, and previous MACE were associated with MACE during the follow-up at a p value ≤ 0.10 and were retained in the multivariate analysis. SAF high level in 2009 was associated with significant increased risk of MACE during the 7 next years: HR = 4.13 [1.30;13.07] for increasing of +1 SAF AU (p = 0.02). Elevated hazard ratio for sex, MACE at baseline, BMI, statins treatment, abnormal eGFR, albumin excretion rates, hypertension and diabetes duration were not significantly associated with MACE. Using Kaplan-Meier curve, the follow-up of patients categorized in two groups according to SAF median (< 2.0 vs ≥ 2.0) showed that patients with high SAF more frequently developed MACE than those with SAF lower than median (p = 0.001). R2 of the model was estimated at 19%.

Discussion

Higher SAF measured in 2009 was associated with increased risk of later cardiovascular events (HR: 4.13, CI 95%: 1.30-13.07) after adjusting for diabetes duration, arterial hypertension, treatment by statins, renal parameters and previous cardiovascular events. SAF therefore appears as a significant predictor of cardiovascular events in Type 1 Diabetes. Participants with DT1 who experienced a cardiovascular event during the 7 years followup were older, with a 9 years longer diabetes duration, but similar HbA1C as compared to the others. They had more arterial hypertension, more dyslipidemia according to similar blood lipids despite twice more treated by statins, more frequent abnormal albumin excretion and estimated glomerular filtration rates, and more previous cardiovascular events. Their SAF were + 30% higher.

SAF to predict MACE

The prediction of cardiovascular events by SAF has already been reported in other diseases, as HIV infection [START_REF] Sprenger | Skin advanced glycation end products in HIV infection are increased and predictive of development of cardiovascular events[END_REF] and Chronic Kidney Diseases although significance was lost (p = 0.09) after multivariate adjustments [START_REF] Fraser | Skin autofluorescence and all-cause mortality in stage 3 CKD[END_REF]. In type 2 diabetes, Lutgers et al. have shown that a high SAF (upper than median) was related to the 3 years cardiovascular prognosis, adjusted to the UKPDS risk score [START_REF] Lutgers | Skin autofluorescence provides additional information to the UK Prospective Diabetes Study (UKPDS) risk score for the estimation of cardiovascular prognosis in type 2 diabetes mellitus[END_REF]. For Type 1 Diabetes, cross-sectional studies have reported relationship between SAF or skin intrinsic fluorescence and coronary calcifications [START_REF] Conway | Skin fluorescence correlates strongly with coronary artery calcification severity in type 1 diabetes[END_REF], and clinical history of coronary heart disease [START_REF] Conway | Skin intrinsic fluorescence is associated with coronary artery disease in individuals with long duration of type 1 diabetes[END_REF]. But no prospective study had shown that SAF could predict cardiovascular events. In our previous analysis on the same cohort with a shorter follow-up of only 4 years [START_REF] Velayoudom-Cephise | Skin autofluorescence predicts cardio-renal outcome in type 1 diabetes: a longitudinal study[END_REF], SAF predicted later eGFR impairement and later MACE: ). However, this previous analysis was based on lower cases of cardio-vascular events. Three years later, the relation between SAF keeps significant after this adjustment: OR 4.13 (1.30-13.07).

The longer follow-up with higher numbers of events now allows to reach significance for the prediction of macrovascular events in our patients independently from classical risk factors.

The role of AGEs

The main mechanism underlying this prediction is the deleterious effect of AGEs on the vascular system [START_REF] Brownlee | Biochemistry and molecular cell biology of diabetic complications[END_REF]. Previous studies found that serum levels of advanced glycation end-products-modified LDL [START_REF] Lopes-Virella | Levels of oxidized LDL and advanced glycation end products-modified LDL in circulating immune complexes are strongly associated with increased levels of carotid intima-media thickness and its progression in type 1 diabetes[END_REF], and Methylglyoxal [START_REF] Hanssen | Higher plasma methylglyoxal levels are associated with incident cardiovascular disease in individuals with type 1 diabetes: a 12-year follow-up study[END_REF] were related to later cardiovascular events or to the progression of atherosclerosis as reflected by the carotid intima-media thickness, in type 1 diabetes. AGEs are generated inside peripheral cells before they are liberated in the extracellular space [START_REF] Brownlee | Biochemistry and molecular cell biology of diabetic complications[END_REF]. Their accumulation in tissues as estimated by SAF is expected to better reflect their influence, than their circulating levels. SAF has been related to cardiac tissue glycation [START_REF] Hofmann | Relationship between cardiac tissue glycation and skin autofluorescence in patients with coronary artery disease[END_REF], inappropriate left ventricular mass and diastolic dysfunction [START_REF] Wang | Skin autofluorescence is associated with inappropriate left ventricular mass and diastolic dysfunction in subjects at risk for cardiovascular disease[END_REF], long wavelenght-1 and sublinical markers as the intima-media thickness [START_REF] Sell | Skin collagen fluorophore LW-1 versus skin fluorescence as markers for the long-term progression of subclinical macrovascular disease in type 1 diabetes[END_REF] and it is increased in patients with an abdominal aortic aneurysm [START_REF] Boersema | Skin accumulation of advanced glycation end products is increased in patients with an abdominal aortic aneurysm[END_REF]. The skin concentrations of AGEs have been related to coronary calcifications, left ventricular masses and progression of intima-media thickness in the DCCT-EDIC study [START_REF] Monnier | Skin collagen advanced glycation endproducts (AGEs) and the long-term progression of sub-clinical cardiovascular disease in type 1 diabetes[END_REF]. SAF was related to the age of the participants (ß = + 0.397; p < 0.001) which is very similar to reports from other groups in participants with T1D [START_REF] Cho | Higher skin autofluorescence in young people with Type 1 diabetes and microvascular complications[END_REF][START_REF] Sugisawa | Skin autofluorescence reflects integration of past long-term glycemic control in patients with type 1 diabetes[END_REF][START_REF] Duda-Sobczak | Association between self-reported physical activity and skin autofluorescence, a marker of tissue accumulation of advanced glycation end products in adults with type 1 diabetes: a cross-sectional study[END_REF]. Although an indirect estimation of the accumulation of AGEs, SAF has some advantages on skin biopsies: low cost, simplicity, noninvasiveness, and repeatability.

SAF and metabolic memory

The SAF may predict macrovascular events because it is a marker of the metabolic memory of hyperglycaemia, which seems critical for macroangiopathy in T1D. The cardiovascular benefits of the intensive glucose control during the DCCT came apparent only later, 11 years after the end of its active phase [START_REF] Nathan | Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes[END_REF], which led to propose the concept of glucose memory. This concept is also supported by the long-term results of the UKPDS for type 2 diabetes. A recent analysis of the risk factors for cardiovascular disease in the DCCT-EDIC participants has emphasized the major role of their mean time-weighted HbA1C, second only to age [START_REF] Diabetes | Complications Trial/Epidemiology of Diabetes I, Complications Research G. Risk factors for cardiovascular disease in type 1 diabetes[END_REF]. Previous studies have reported that SAF relates to the HbA1C of the previous years, better than to the present HbA1C in T1D [START_REF] Sugisawa | Skin autofluorescence reflects integration of past long-term glycemic control in patients with type 1 diabetes[END_REF][START_REF] Genevieve | Skin autofluorescence is associated with past glycaemic control and complications in type 1 diabetes mellitus[END_REF][START_REF] Araszkiewicz | Skin autofluorescence is associated with carotid intimamedia thickness, diabetic microangiopathy, and long-lasting metabolic control in type 1 diabetic patients. Results from Poznan Prospective Study[END_REF][START_REF] Banser | Advanced glycation end products, measured in skin, vs. HbA1c in children with type 1 diabetes mellitus[END_REF]. In elderly participants of the 3-City cohort study from the general population, we have shown that SAF relates to glycaemia and HbA1C measured ten years before, rather than to their values measured at the same time as SAF [START_REF] Rajaobelina | Autofluorescence of skin advanced glycation end products: marker of metabolic memory in elderly population[END_REF]. Most of the associations between skin intrinsic fluorescence and T1D complications lost significance after adjustment to the mean HbA1C over time for the DCCT participants [START_REF] Orchard | The association of skin intrinsic fluorescence with type 1 diabetes complications in the DCCT/EDIC study[END_REF]. The association between skin fluorescence and coronary arterial calcifications was however still significant after this adjustment, arguing for an additional value of the fluorescence measurement. But no prospective study had shown that SAF could predict cardiovascular events. In our previous analysis on the same cohort with a shorter follow-up of only 4 years [START_REF] Velayoudom-Cephise | Skin autofluorescence predicts cardio-renal outcome in type 1 diabetes: a longitudinal study[END_REF], SAF predicted later eGFR impairement and later MACE: OR 4.84 (1.31-17.89), but the relation with later MACE did not reach significance with adjustment for previous MACE: OR 2.98 (0.77-11.47). Three years later, the relation between SAF keeps significant after this adjustment: OR 4.13 (1.30-13.07). In the real life, the history of HbA1C is often not complete for many T1D patients, and death from cardiovascular causes are thrice more frequent even for the best controlled (HbA1C ≤ 6.9%) according to the Swedish register [START_REF] Lind | Glycemic control and excess mortality in type 1 diabetes[END_REF]. This underlines the interest of a metabolic memory marker that predicts cardiac events.

Others factors

Hyperglycaemia-related mechanisms may not be the only explanation for the relationship between SAF and macrovascular diseases. SAF increases with age, the strongest risk factor for cardiovascular events in T1D [START_REF] Diabetes | Complications Trial/Epidemiology of Diabetes I, Complications Research G. Risk factors for cardiovascular disease in type 1 diabetes[END_REF]. SAF has recently been related to the components of the metabolic syndrome [START_REF] Van Waateringe | Skin autofluorescence, a non-invasive biomarker for advanced glycation end products, is associated with the metabolic syndrome and its individual components[END_REF], especially low HDL-cholesterol and arterial hypertension [START_REF] Botros | Advanced glycation end-products (AGEs) and associations with cardio-metabolic, lifestyle, and dietary factors in a general population: the NQplus study[END_REF], that increasingly mediate the effect of glucose control on cardiovascular risk in aging T1D [START_REF] Tuomilehto | Incidence of cardiovascular disease in Type 1 (insulin-dependent) diabetic subjects with and without diabetic nephropathy in Finland[END_REF]. The progression of SAF in our patients with T1D highly depends on their glomerular filtration rates [START_REF] Rajaobelina | Skin autofluorescence and peripheral neuropathy four years later in type 1 diabetes[END_REF], as expected because renal insufficiency reduces the clearance of AGEs. This effect probably worsens as SAF predicts the impairment of GFR in T1D [START_REF] Velayoudom-Cephise | Skin autofluorescence predicts cardio-renal outcome in type 1 diabetes: a longitudinal study[END_REF]. Diabetic nephropathy is a well-known, major contributor to cardiovascular disease in T1D [START_REF] Jensen | Coronary heart disease in young type 1 (insulin-dependent) diabetic patients with and without diabetic nephropathy: incidence and risk factors[END_REF]. But the relation between SAF and cardiovascular events reported in our study, was still significant after adjusting for age, arterial hypertension, blood lipids and renal parameters.

Limitations

Some other hypothesis can however not be excluded on the basis of our study, which can be considered as limitations. We have recently shown that SAF quickly rises during acute renal failure [START_REF] Lavielle | Skin autofluorescence in acute kidney injury[END_REF]. That is a powerful predictor of major cardiovascular events in type 2 diabetes [START_REF] Monseu | Acute kidney injury predicts major adverse outcomes in diabetes: synergic impact with low glomerular filtration rate and albuminuria[END_REF], but to our knowledge this has not been reported for T1D. We also found that SAF progressed less in our patients whose T1D was treated by continuous subcutaneous insulin infusion CSII [START_REF] Rajaobelina | Skin autofluorescence and peripheral neuropathy four years later in type 1 diabetes[END_REF], which has been related to less cardiovascular mortality [START_REF] Steineck | Insulin pump therapy, multiple daily injections, and cardiovascular mortality in 18,168 people with type 1 diabetes: observational study[END_REF]. This suggests that glycaemic variability may play a role in the accumulation of AGEs: even transient pikes of hyperglycaemia may have deleterious effects on vascular cells [START_REF] El-Osta | Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia[END_REF]. Long-term glycemic variability has been related to a twice-higher risk of cardiovascular events in T1D [START_REF] Gorst | Long-term glycemic variability and risk of adverse outcomes: a systematic review and meta-analysis[END_REF]. Skin autofluorescence is however not influenced by short-term glycemic variations [START_REF] Noordzij | Skin autofluorescence and glycemic variability[END_REF]. Finally, some reports have related SAF to physical activity indexes: aerobic exercise capacity, muscle strength [START_REF] Momma | Skin advanced glycation end product accumulation and muscle strength among adult men[END_REF], and regular performance of physical activities [START_REF] Klenovics | Reference values of skin autofluorescence as an estimation of tissue accumulation of advanced glycation end products in a general Slovak population[END_REF][START_REF] Pilleron | Accumulation of advanced glycation end products evaluated by skin autofluorescence and incident frailty in older adults from the Bordeaux Three-City cohort[END_REF], which has been related to less cardiovascular events in the FinnDiane cohort [START_REF] Tikkanen-Dolenc | Frequent and intensive physical activity reduces risk of cardiovascular events in type 1 diabetes[END_REF]. Some practical limitations must also be mentioned. According to French law, we did not categorize the participants according to their ethnicity, but some subjects were excluded due to their skin pigmentation as already mentioned. The measurement of SAF were performed on intact skin, but we can not rule out that some subjects might have applied some body lotions locally. Draelos et al. applied a topical AGE inhibitor (blueberry extract) on the skin of 20 women with diabetes during three months, and they did not report any change of their SAF on this time interval [START_REF] Draelos | An evaluation of the effect of a topical product containing C-xyloside and blueberry extract on the appearance of type II diabetic skin[END_REF]. An important proportion of the variability of SAF is not explained by age and usual covariates, some studies have suggested that genetic [START_REF] Barat | Advanced glycation end products in children with type 1 diabetes: family matters?[END_REF] or dietary factors may play a role, as diet is a source of AGEs [START_REF] Uribarri | Advanced glycation end products in foods and a practical guide to their reduction in the diet[END_REF]. Smoking has been related to SAF [START_REF] Botros | Advanced glycation end-products (AGEs) and associations with cardio-metabolic, lifestyle, and dietary factors in a general population: the NQplus study[END_REF] and the relation between SAF and MACE was adjusted for tobacco use in our study, but we did not collect detailed dietary informations, so we can not exclude the role of some specific diet components, like coffee [START_REF] Botros | Advanced glycation end-products (AGEs) and associations with cardio-metabolic, lifestyle, and dietary factors in a general population: the NQplus study[END_REF].

Conclusion

SAF seems to be a good mean to predict MACE in T1D.

Further works are now required to explore these hypothesis with more long term follow-up because fortunately only a few MACE occurred, a multicentric study with more patients is required to find strategies to slower the progression of SAF in T1D.

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  with type 1 diabetes: a 7-year follow-up study C. Blanc-Bisson, F. L. Velayoudom-Cephise, Audrey Cougnard-Gregoire, Catherine Helmer, Kalina Rajaobelina, Cécile Delcourt, L. Alexandre, L. Blanco, K. Mohammedi, M. Monlun, et al. To cite this version: C. Blanc-Bisson, F. L. Velayoudom-Cephise, Audrey Cougnard-Gregoire, Catherine Helmer, Kalina Rajaobelina, et al.. Skin autofluorescence predicts major adverse cardiovascular events in patients with type 1 diabetes: a 7-year follow-up study: Cardiovasc Diabetol. Cardiovascular Diabetology, In press, 17 (1), pp.82. 10.1186/s12933-018-0718-8. hal-02977826

OR 4 .

 4 84(1.31-17.89), but the relation with later MACE did not reach significance with adjustment for previous MACE: OR 2.98 (0.77-11.47

Fig. 1

 1 Fig. 1 MACE during follow-up according to SAF median in patients with SAF < 2.0 and patients with SAF 2.0. p = 0.001

Table 1 Characteristics of participants at baseline according to the incidence of major adverse cardiovascular event over the 7-year follow-up

 1

Data presented in N (%) and mean ± standard deviation p value: COX test for qualitative variables and quantitative

variables Major cardiovascular event No (N = 210) Yes (N = 22) p

	Age (years) Sex men	50.54 ± 16.76 60.56 ± 13.30 121 (57.6) 17 (77.2)	0.007 0.07
	BMI (kg/m 2 ) Diabetes duration (years) HbA1c (mmol/ml) in 2009 (%) 7.6 ± 1.0 24.7 ± 3.7 20.6 ± 12.8 Insulin pump 40 (19.0)	27.1 ± 6.3 29.6 ± 17.8 7.5 ± 1.0 5 (22.7)	0.09 0.03 0.74 0.77
	Smoking habits (n = 219) HDL (g/l) (n = 222) LDL (g/l) (n =t221) Triglycerides (g/l) (n = 205) Statin at baseline (n = 222) Hypertension	51 (25.8) 0.64 ± 0.19 1.03 ± 0.29 0.89 ± 0.63 45 (22.5) 87 (41.43)	4 (18.1) 0.58 ± 0.17 1.04 ± 0.33 1.11 ± 0.53 13 (59.0) 18 (81.82)	0.43 0.17 0.88 0.13 0.0002 0.0003
	Previous history of MACE	13 (6.19)	10 (45.45)	< 0.0001
	eGFR (mL/min) (n = 225) ≥ 60 < 60	179 (88.18) 24 (11.82)	13 (59.09) 9 (40.91)	0.001
	Albumin excretion rate (mg/24 h) (n = 227) < 30 179 (86.89)	12 (57.14)	0.002
	≥ 30 SAF (AU)	27 (13.11) 2.08 (0.54)	9 (42.86) 2.63 (0.73)	0.002

Table 2 Risk of major adverse cardiovascular event over the follow-up according to SAF at baseline

 2

	Cox multivariate analysis (n = 205, 21 events)

Acknowledgements

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Funding

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Availability of data and materials

Yes.

Abbreviations

AER: albumin excretion rate; AGE: advanced glycation end-products; AU: arbitrary unit; BMI : body mass index; CSII: continuous subcutaneous insulin infusion; DCCT : diabetes control and complications trial; eGFR: estimated glomerular filtration rates; HbA1c: glycated haemoglobin; HIV: human immunodeficiency virus; HR: hazard ratio; IQR: interquartile range; MACE : major adverse cardiovascular event; SAF: skin autofluorescence; SD: standard deviation; T1D: type 1 diabetes; UKPDS: United Kindom Prospective Diabetes Study.

Authors' contributions

Writting: CBB and VR, Patients' inclusion and follow-up: FLC, LB, LA, MM, KM, data analyses: ACG, CH, CD, Review before submission: all authors. All authors read and approved the final manuscript Author details 1 Nutrition Diabetology Unit, CHU Bordeaux, Haut-Levêque Hospital, Avenue Magellan, 33600 Pessac Cedex, France. 2 INSERM, Bordeaux Population Health Research Center, Team LEHA, UMR 1219, Univ Bordeaux, 33000 Bordeaux, France.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Additional file

Additional file 1. Scatterplot for SAF over age.

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