Caroline Genoux Ense

Philippe Larroude
email: larroude@hmg.inpg.fr

Lucile Poulleau

Thomas Pochetat

Comparison of sediment transport formulae during a flood wave in the river: an application of Telemac teaching

Comparison of sediment transport formulae during a flood wave in the river: an application of Telemac teaching

Caroline Genoux, Lucile Poulleau, Thomas Pochetat, Philippe

INTRODUCTION

The Telemac chain of code is use since several years to teach the numerical approach in a project. The project (propose to the student) is during 10 weeks in the second year of the engineering school ENSE 3 . The student work only a half day a week on the subject. The idea of this practical lecture is to teach to them how conduct a project with a numerical approach.

During the two last years, we propose to our student a comparison between numerical simulation with Telemac and data from a physical model. The experimental took place at the CNR in Lyon and the model represent a study on a possible creation of a natural dam.

The study site is located in the department of Isere. Since several years, the State gives a very detailed attention to these risks of falls of ground: they could create a natural dam which would block the bed of Romanche, creating a water reserve to the upstream. With the origin, the assumption was that the accumulation of water would end up causing the rupture of the dam, thus creating a wave of destroying immersion; more recently, studies showed that this scenario was not realistic: in fact, the dam would be eroded gradually, creating intermittent overflow rates in the downstream part of the valley.

These modifications led to the following formulation of the subject of this workshop of engineering: "Study of progressive erosion and sedimentary transport on the top of the natural dam of Séchilienne".

In 2008, CNR (National Company of the Rhone) carried out various tests on a small-scale model of Séchilienne, in particular two tests numbered 17 and 21 (see [START_REF] Cnr | Rapport DPFI-Labo[END_REF]).

The objective general of our work is the study of test 21 and the comparison between the results and the data of CNR obtained with their model. We will carry out comparisons between various existing formulas of erosion and the data.

II. LOCALIZATION AND PRESENTATION

Séchilienne is a commune of Isere, localized at 30 kilometers in the south-east of Grenoble, on the road connecting Grenoble and Briançon. It belongs to the canton of Vizille and is located in the valley of the Romanche river.

Séchilienne is known for its active zone of landslide located on the Southern slope of right bank of the valley of the Romanche, at the southern end of the mountain chain of Belledonne (see Fig. 1).

This site, called "Ruins of Séchilienne", knew many falls of blocks, in particular during years 1726, 1762, 1794, 1833 and 1906. The scree cone of the Ruins is visible from the secondary road, which serves Bourg d'Oisans.

III. PRESENTATION OF THE STUDY

A. Presentation of former studies

As indicated in the introduction, the scenarios considered up to now were very pessimistic. At the time of a rainy event of strong intensity, an important landslide of the mountain would lead to the complete obstruction of the valley and the creation of a dam of several tens meters. That would lead to the formation of a lake of approximately 20 million m 3 to the upstream and the road would be cut. According to former studies, this dam will resist at the rising waters due to flooding and would be broken only when passing the peak of the hydrograph, i.e. at the most critical moment. This rupture would involve an overflow rate with the downstream, forming a wave of immersion which would reach the cities and industrial facilities located downstream from the dam. All these scenarios were deduced from digital models. On these various conclusions, several parades were installing. Many measuring apparatus were also installed in order to monitor closely the evolution of the phenomenon (contours exact of the zone of landslide, kinematics and movement of the zone…). These measurements make it possible to generate alarms in real time.

B. General presentation of the study of CNR

These data were use by CNR, which enabled it to make a more thorough study of this landslide through the construction of a small-scale physical model. The purpose of this model, on the scale 1/60e, was to study the speed of erosion of a dam by overflow of water of Romanche and to provide complementary data (flow in downstream, flow upstream, dimension of water on the level of reserve, quantity of transported materials …)

This scientific tool made it possible to model a very complicated physical process that the mathematical models could not entirely represent. Several scenarios were considered according to the characteristics below:

• The height of the dam formed by the landslide of the 3 million m 3 ;

• The materials (size, nature) constituting the dam;

• Flow of Romanche;

• Test of a second material constituting the dan different from the first testing;

• Tests relating to a 18 m height dam and to a second landslide falling into water reserve formed by a first landslide of 6 m height.

This study by the CNR on this reduced model gives the behavior of the dam formed in bottom of valley of Romanche. The principal conclusions are as follows:

• A 6 m height dam, corresponding to the landslide of a volume of 3 million cubic meters (able to occur in the 10 next years) does not present a risk of brutal rupture because of a progressive erosion. Moreover, a simultaneous hundred-year flood would increase the downstream flow only of 10%.

• A second landslide falling into water reserve formed by a first landslide does not present a real risk, neither for the downstream, nor for the upstream.

• A long term landslide (in 50 years) forming a 18 m height dam for a volume from 5 to 6 million cubic meters would present a more important risk for the downstream with an increase of the flow in the case of a hundred-year flood, in the order of 20%.

C. Presentation of our study

Thanks to the data of CNR [START_REF] Cnr | Rapport DPFI-Labo[END_REF], we could recover the bathymetry of Romanche. We tried to set up a digital model which will make it possible to have the results of simulation to compare with thus obtained on the small-scale physical model. We had to model the different flood that might occur, the various transformations undergone by the bottom of the river (erosion, "un-paving" ...). This will calculate the over-flow in each case and to allow predictions on new models. We have two tests on the site of Séchilienne made by CNR: test 17 which was treated last year and testing 21, we will fully address this year. Both tests were carried out for a dam of 6 m above the bed of the Romanche. The same size of material was used for both tests. The results of last year on over-rates are consistent with the observations of the CNR, so we considered that the hydraulic model was correct. We have therefore chosen to focus primarily on modeling the erosion of the river bottom.

1) Test 17:

During test 17, the landslide occurs out flood of the Romanche River, forming a dam in the Romanche valley. The flow of Romanche gradually fills the reserve created by the dam and flows on this and then a flood occurs later. In this scenario, the passage of a hundred-year flood which was studied. The students of last year already modeled this entire test.

We initially tried to improve modeling the un-paving and erosion by using zones polygonal rather than rectangular (what was used previously). Then, we modeled erosion thanks to various formulas to try to take into account the phenomenon observed by CNR.

2) Test 21: During test 21, the landslide occurs for one period of the flood. In the first time, we thought that the test of CNR consisted in making fall the landslide at a given time at the maximum of the flood. However, after having looked at the provided documents more attentively, we realized that CNR used same bathymetry as in test 17.

In this case, the landslide is established in the model (what corresponds to the bathymetry of case 17), then a constant flow (corresponding to the maximum of flood) is introduced very quickly into the model. In this scenario, it is the maximum flow of the hundred-year flood which was studied. We thus modeled this scenario, by using various formulas of erosion as for test 17 (because we have same granulometry). We could thus compare our results with those of CNR. Bathymetry being identical to the preceding case, the study of this test especially allowed us to model erosion more easily.

IV. STANDARD MODELING FOR THIS PROJECT

The system TELEMAC is a whole of software of numerical simulation applied to the flows on free face, at sea or in river, in two or three dimensions.

The applicability goes from the local study of impact of the construction of works (bridges, ears, mole…) until the calculation of the current due to the waves or tides while passing by the reproduction of the flood, rupture of dams and the transport of sediments.

All the software of the system uses powerful algorithms based on the finite element method. The field of calculation is discretized with not structured grids using triangular elements, which makes it possible in particular to refine the grid in the zones of particular interest. System TELEMAC proposes a complete data processing sequence with software of simulation, "preprocessors" and "post-processors".

The pre and post-processors are the elements of the chain which make it possible to prepare and manage a calculation, to display the results. These tools are common to all the modules of calculation, which ensures the homogeneity of the unit.

After having traced contours external and the interior lines of the field, it is necessary to define the size of the mesh. We choose here to create constant meshes. During the project, we tested several sizes of grids: 20m, 10m, 5m, 2m in the aim of evaluating the influence of the grid in calculations. We could observe that the precision of calculation increased when the mesh decreased. However, the computing times then were increased considerably. We thus found a compromise by choosing for the majority of the cases the grid of 10m.

For our study, the border upstream is with flow imposed and the border downstream on imposed height. For the side borders, they correspond to the banks and are thus regarded as solid walls.

Fig. 2 shows the hydrograph of the hundred-year flood used by CNR and that which we used in our simulations.

After having controlled the result, we use Blue Kenue to treat the data so extracting information to be analyzed and compare with the results of CNR. Thus, we could extract from the profiles height of water and bathymetry at various moments with the same sections transversely as those used by CNR. Fig. 3 represents an example of evolution of the bottom in function of time on a transversely given section obtained with Blue Kenue. V. MODELING EROSION Sediment transport can be considered as a matter production, which will be moved by the river and then sediment again: it is also called transit sediment or sediment transport. This is a complex phenomenon involving a large number of meteorological, hydrological, geological parameters, etc.

There are two types of moving materials:

• The bed load, for materials of large size, which corresponds to a transport on the bed in shifts.

• Suspension, that is to say, the finer sediment transport by the flow. This is increased by the force of floods, the slope of the river bed and the narrowness of the channel. There are two main phases in the sediment transport: the phase of erosion and the deposition phase. For our study we only studied the phenomenon of erosion because it is the cause of the overflow downstream (see [START_REF] Smart | Sediment transport on steep slopes[END_REF] and [START_REF] Sogreah | Etude générale sur les seuils de correction torrentielle et les plages de dépôts (Couvert[END_REF]).

In our project, we are dealing with fluvial erosion, and more precisely to regressive erosion, observed by the CNR in the various tests. Regressive erosion is a mechanism for widening stream that comes after the lowering of the bed downstream. It begins with the digging at baseline also called fixed point before heading upstream (the base level of a river corresponds to the lowest level at which a river can erode bed.).

Regressive erosion is a mechanism for digging of rivers that comes after the lowering of the bed downstream. It begins with downstream at the basic level also called fixed point before rising towards upstream (the basic level of a river corresponds to the lowest level to which a river can erode bed.). This type of erosion is much faster than the traditional erosion which, it, acts in the downward direction.

This phenomenon occurs when the slope is higher than the slope of balance. This increase in slope led to an increase in the erosive power of the river: the transport capacity becomes higher than the contributions, this difference involving an erosion of the bed and banks.

Erosion is propagated then upstream to restore the initial slope of balance. In our case, the landslide is done on only one side of Romanche, which generates a nonsymmetrical deposit. An angular part called "breach" or "gap" by CNR is formed, and it is on this level that the erosion occurs mainly, which digs the dam starting from this point.

Many formulas are proposed to model solid transport by bed load in the rivers. They use various variables, in particular data concerning the granulometry, the width of the bed, the hydraulic diameter and the slope. In this case, it was not found yet of sedimentary formula of sediment transport and erosion compatible with the bathymetry (strong slope).

Within the framework of our study with the Sisyphe code, we used several different formulas, in order to compare the numerical results with the data and to find the most adapted one:

• The formula of Bijker is normally used in the cases of bed load and suspension. It is made of two components: bed load, which is entirely empirical, and suspension, which takes as a starting point the preceding component, and which is drawn from the theory of Einstein.

• The formula of Rickenmann (see [START_REF] Rickenmann | Bedload transport capacity of slurry flows at steep slopes, 249[END_REF]) which makes it possible to obtain the bed load starting from the liquid flow of the river.

• Einstein-Brown formula [START_REF] Einstein | The bed-load function for sediment transportation in open channel flows[END_REF].

• Grass formula [START_REF] Grass | Sediments transport by waves and currents[END_REF]. It is build by using the morphodynamic equation of Exner.

• Empirical formula.

In addition to these theoretical formulas, we tried to propose our own formula in order to represent erosion. We tried various simple formulas, by choosing relations of proportionality between erosion and the flow per linear meter, since the flow is one of the principal parameters influencing erosion. We thus programmed relations of the form:

n Aq E = ( 1
)
where A is a constant and n varying from 1 to 3. E represents the sea bed evolution at each time step. For n=3, erosion observed was really too strong, even with low values of A. Finally, we chose A=1×10-7 and n=1, which we integrated in a standard file FORTRAN for our simulations only with Telemac2d module (without the coupling with Sisyphe module.

In our study cases, we chose to look at the evolution of the bed on 3 profiles, corresponding to profiles 15, 16 and 17 of the study of CNR (see Fig. 4).

VI. RESULTS

A. Comparison with test 17 data (hundred-year flood)

For this modeling, the tests are made during three days of real time in order to make entirely the hydrograph hundredyear flood. We did not model the "un-paving" (abrupt setbacks of part of the dam) observed by CNR, because we observe some divergence of our results compared to the observations on the small-scale model. On the grid of 10m, we need approximately 18 hours to carry out the simulation on a PC. All the profiles show for the results are taken looking in the upstream direction. The profiles of CNR's data are truncated to facilitate the comparison.

1) Profile 15: with Bijker (see Fig. 6), until t = 2h, we see that our curves are smoothed in contrast to those of the CNR (see Fig. 5). However, the numerical values coincide. For the curves representing the time above 17h, there is too much erosion, the theoretical result moves away significantly from the experimental result. With Einstein formula (see Fig. 7), we obtain similar results to those obtained for Bijker, except that the results are good up to t = 5h40. There is still a significant erosion of the profiles at the final time. With the empirical formulae (see Fig. 8), the shapes of the curves are quite consistent with those of the CNR, but erosion is not large enough.

2) Profile 16: With Bijker (see Fig. 10), the results for short times are not very good (see fig. 9): the shape is moderate and the erosion is not large enough. However, the profile obtained for the curve t = 18h is closer to the data, but the following simulations are incorrect (the erosion is too great). With Einstein (see Fig. 11), the results at short times are not correct. On the other hand at very long times (t = 29h), the behavior and the values are correct. With the empirical formula (see Fig. 12), the behavior is correct for the all times, but on the other hand the erosion is for each time step too weak.

3) Profile 17: with Bijker (see Fig. 14), the behavior is quite correct. In contrast the values of erosion are not very good at long times (about one meter gap between simulate and experimental test (see Fig. 13)). With Einstein (see Fig. 15), the curves at short times are incoherent because we observe identical profile from t = 2h to t=18h. On the other hand, the shape of the last curve is good, but erosion is too weak. With the empirical formula (see Fig. 16): The results are wrong. The shape is correct but the erosion is quasi non-existent.

B. Comparison with test 21 data (ten-year flood)

For this modeling, the tests are made during 15h to simulate the maximum of the ten-year flood (Q = 300m 3 /s). We either did not model the "un-paving" observed by CNR. On the grid of 10m, we need approximately 6 hours to carry out the simulation on a PC.

1) Profile 15: with Bijker (see Fig. 17), the results are good for very short times. On the other hand, it seems according to the figure of CNR that an "un-paving" occurs after 30mn so our results after thi time are wrong because of the gap create by the "un-paving" on the river bed. With a translation of our curves, the shape seems to be correct.

With Einstein (see Fig. 17), the depths of erosion are correct along the entire simulation in time. On the other hand, the digging of the channel is done over a width much more reduced than in the results of CNR. That can be due to that the "un-paving" occurred after 30mn changes the shape of the breach in the dam.

With Grass formula (see Fig. 17): There still, the phenomenon of "un-paving" is not modeled, which distorts interpretation. If not, the profiles are overall good, with a broader channel which corresponds well to the results provided by CNR.

1) Profile 16: with Bijker (see Fig. 18), the depths of erosion are not good. But temporal data of the CNR are missing on their graph, so the interpretations are less easy. The shape is relatively correct but the erosion is too weak (only 2m in 15 hours).

With Einstein (see Fig. 18), the shapes and the values are good. But, we observe on the profile of CNR at t = 11h40 an increase of the river bed elevation, which can correspond to a deposit of sediment which is difficult to simulate.

With Grass model (see Fig. 18), the results are not very realistic. Erosion is progressive but does not correspond at all to the results observed on the small-scale model. 3) Profile 17: with Bijker and Grass models, erosion is nonexistent in this part upstream of the dam, which is in clear disagreement with experimental data from the CNR. With Einstein, there is an erosion that has generally the same shape as the experimental profiles, but the values are not very good (too much erosion before t = 6h not enough after).

VII. CONCLUSIONS

This project of Workshop of Engineering on the topic of the Ruins of Séchilienne allowed us to extend our knowledge as regards numerical modeling of flow in river and to control the bases of the software Matisse, Telemac and Bluekenue. We are now able, starting from data of bathymetry of a river bed and hydrological data, to model a floof and to extract from the results information necessary to their good comprehension.

This study was the occasion for us to measure the difficulty in following objectives: many data-processing problems forced us to reorganize the aim to achieve. In addition, of new elements of comprehension reached us during the project and led us to adapt our study.

For test 17, the simulation which approaches more the tests carried out by CNR is the one using the formula of Bijker. In addition, the phenomenon of "un-paving" is not taken into account in our study, which can explain why erosions obtained numerically are often too weak. Our empirical formulae seem correct but remain to be improved. It would be necessary to continue to make evolve/move the coefficients to obtain erosion a little more important and to better follow the results of CNR.

For test 21, the simulation which approaches more of the tests carried out by CNR is the one using the formula of Einstein.

We decided this year to look further into the modeling of erosion; we hope that our results will make it possible to make progress for the study in the future projects of Workshop of Engineering. Modeling must still be improved, in particular with the "un-paving" phenomenon.

Figure 1 .

 1 Figure 1. Localisation of the Ruins of Séchilienne.

Figure 2 .

 2 Figure 2. Hydrographs of CNR (purple) and of our study (pink) for the hundred-year flood (m 3 /s) over time (h).

Figure 3 .

 3 Figure 3. Erosion of the bottom of section 16 during the passage of the hundred-year flood (m 3 /s) over time (h).

Figure 4 .

 4 Figure 4. Simulation domain with the position of the Dam and the profiles 15, 16 and 17.

Figure 5 .

 5 Figure 5. Test Case 17: profile 15 data of the CNR.

Figure 6 .

 6 Figure 6. Test Case 17: profile 15 simulated with Bijker formulae.

Figure 7 .

 7 Figure 7. Test Case 17: profile 15 simulated with Einstein formulae.

Figure 8 .

 8 Figure 8. Test Case 17: profile 15 simulated with Empirical formulae.

Figure 9 .

 9 Figure 9. Test Case 17: profile 16 data of the CNR.

Figure 10 .

 10 Figure 10. Test Case 17: profile 16 simulated with Bijker formulae.

Figure 11 .

 11 Figure 11. Test Case 17: profile 16 simulated with Einstein formulae.

Figure 12 .

 12 Figure 12. Test Case 17: profile 16 simulated with Empirical formulae.

Figure 13 .

 13 Figure 13. Test Case 17: profile 16 data of the CNR.

Figure 14 .

 14 Figure 14. Test Case 17: profile 17 simulated with Bijker formulae.

Figure 15 Figure 16

 1516 Figure 15 Test Case 17: profile 17 simulated with Einstein formulae

Figure 17 .

 17 Figure 17. Test Case 21: profile 15, the CNR data and simulated data with Bijker, Einstein and Grass formulae.

Figure 18 .

 18 Figure 18. Test Case 21: profile 15, the CNR data and simulated data with Bijker, Einstein and Grass formulae.

ACKNOWLEDGEMENT

Thank to the CNR and Sylvain Reynaud for the data. We also acknowledge Daniel Verdeil and Olivier Manin from the Symbhi who authorized the CNR to give us the data and the results of the experiment. This work is the continuation of the work of the previous year by Amoroso Lauranne, Lambert Marine, De Bortoli Romain and Saidon Simon.


Marie Michèle Boulet

David Cheillan

Mathilde Di Filippo

Taïssia Lelekov-Boissard

Stéphanie Charline Buisson

Julie-Anne Nazare

Jessica Tressou

Marie-Caroline Michalski

Ph.D Catherine Calzada

Stéphanie Lambert-Porcheron

Philippe Moulin

Boulet

Page

Postprandial Triglyceride-Rich Lipoproteins from Type 2 Diabetic Women Stimulate Platelet Activation Regardless of the Fat Source in the Meal

Keywords: arachidonic acid, CM: chylomicrons, HCS: hazelnut cocoa spread, FA: fatty acids, LPL: lipoprotein lipase, PP: postprandial, PPP: platelet-poor plasma, PRP: platelet-rich plasma, SFA: saturated fatty acid, TC: total cholesterol, TG: triglycerides, TGRL: triglyceride-rich lipoproteins, TxB fatty acids, platelets, postprandial, triglyceride-rich lipoproteins, type 2 diabetes

INTRODUCTION

Type 2 diabetic (T2D) patients have a higher risk of cardiovascular diseases, including ischemic coronary artery events, atherosclerosis and atherothrombosis [START_REF] Einarson | Prevalence of cardiovascular disease in type 2 diabetes: a systematic literature review of scientific evidence from across the world in 2007-2017[END_REF]. Epidemiologic studies also showed that women with T2D exhibit a worse cardiovascular risk profile [START_REF] Kautzky-Willer | Sex-specific differences in metabolic control, cardiovascular risk, and interventions in patients with type 2 diabetes mellitus[END_REF] and have greater increase in cardiovascular morbidity and mortality compared to men [START_REF] Huxley | Excess risk of fatal coronary heart disease associated with diabetes in men and women: meta-analysis of 37 prospective cohort studies[END_REF].

Platelets from T2D individuals have been shown to be hyperactive, thus more easily prone to agonist-triggered aggregation in the event of plaque rupture [START_REF] Santilli | Platelets and diabetes mellitus[END_REF]. Moreover, it is well documented that increased concentration of plasma triglycerides (TG), carried in the bloodstream by "triglyceride-rich lipoproteins" (TGRL) that are chylomicrons (CM), VLDL and their remnants, is an important risk factor in T2D [START_REF] Taskinen | New insights into the pathophysiology of dyslipidemia in type 2 diabetes[END_REF]. Our group previously showed that large TGRL from fasting T2D patients enhanced in vitro platelet activation and aggregation [START_REF] Boulet | Large triglyceride-rich lipoproteins from fasting patients with type 2 diabetes activate platelets[END_REF].

It is now well acknowledged that postprandial (PP) TG concentrations are involved in cardiovascular risk. The concept of the atherogenicity of CM and VLDL was first introduced by Zilversmit 40 years ago [START_REF] Zilversmit | Atherogenesis: a postprandial phenomenon[END_REF]. Non-fasting TG levels were subsequently associated with the incidence of various cardiovascular events [START_REF] Iso | Serum triglycerides and risk of coronary heart disease among Japanese men and women[END_REF][START_REF] Nordestgaard | Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women[END_REF][START_REF] Freiberg | Nonfasting triglycerides and risk of ischemic stroke in the general population[END_REF][START_REF] Egeland | Non-fasting triglycerides predict incident acute myocardial infarction among those with favourable HDL-cholesterol: Cohort Norway[END_REF][START_REF] Eberly | Relation of triglyceride levels, fasting and nonfasting, to fatal and nonfatal coronary heart disease[END_REF], independently of other risk factors [START_REF] Bansal | Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women[END_REF][START_REF] Sloop | Interstitial fluid lipoproteins[END_REF].

TGRL remnants are likely to be the main contributors of an enhanced atherosclerosis development [START_REF] Sloop | Interstitial fluid lipoproteins[END_REF][START_REF] Nakano | Detection of apolipoproteins B-48 and B-100 carrying particles in lipoprotein fractions extracted from human aortic atherosclerotic plaques in sudden cardiac death cases[END_REF]. However, regarding atherothrombosis, the contribution of TGRL is not clearly established. The few studies on this topic were performed mostly in non-diabetic hyperlipidemic and normolipidemic individuals and had major differences in methodology.

They showed that CM from both groups can have an inhibitory effect on platelet functions [START_REF] Aviram | Platelet activation by plasma lipoproteins[END_REF], while VLDL from healthy subjects [START_REF] Englyst | A novel role for CD36 in VLDL-enhanced platelet activation[END_REF] and remnants from healthy and T2D individuals as well as patients with lipoprotein lipase (LPL) deficiency [START_REF] Knofler | Remnant-like lipoproteins stimulate whole blood platelet aggregation in vitro[END_REF] would have an enhancing effect. No study had been performed on the potential implication of PP TGRL from T2D

Boulet et al. 2020, Page 5

patients in atherothrombosis, and it is relevant to study, especially in this population at higher risk for cardiovascular complications.

Because the CM fraction of TGRL in the PP state is mainly constituted of dietary TG, the fatty acid (FA) composition of the ingested lipid influences their fatty acid composition in healthy individuals [START_REF] Bysted | Effect of dietary fatty acids on the postprandial fatty acid composition of triacylglycerol-rich lipoproteins in healthy male subjects[END_REF]. It is also known that there are exchanges of FA from plasma and lipoproteins to phosphatidylcholine in platelet membranes [START_REF] Guillot | Increasing intakes of the longchain ω-3 docosahexaenoic acid: effects on platelet functions and redox status in healthy men[END_REF][START_REF] Skeaff | Dietary-Induced Changes in Fatty Acid Composition of Human Plasma, Platelet, and Erythrocyte Lipids Follow a Similar Time Course[END_REF]. Chain length of the saturated fatty acids (SFA) can also impact platelet aggregation. Studies in humans suggest deleterious stimulating effects of dietary long chain SFA on platelet function [START_REF] Renaud | Influence of long-term diet modification on platelet function and composition in Moselle farmers[END_REF][START_REF] Renaud | Effect of smoking and dietary saturated fats on platelet functions in Scottish farmers[END_REF][START_REF] Jakubowski | Modification of human platelet function by a diet enriched in saturated or polyunsaturated fat[END_REF]. Also, it was shown that the PP state was associated with increased platelet activation in T2D patients following the consumption of a carbohydrate-rich meal and related to PP insulin [START_REF] Razmara | Food intake enhances thromboxane receptor-mediated platelet activation in type 2 diabetic patients but not in healthy subjects[END_REF][START_REF] Spectre | Postprandial platelet activation is related to postprandial plasma insulin rather than glucose in patients with type 2 diabetes[END_REF].

However the specific effect of TGRL secreted following the consumption of fats with different SFA composition on platelet aggregation has never been investigated in T2D women.

In this context, we hypothesized that PP TGRL from T2D women would have a modified FA composition following a realistic fat intake via a mixed meal from different food sources (dairy vs vegetal) and that this would change their effect on platelet activation and aggregation.

Boulet et al. 2020, Page 6

EXPERIMENTAL SECTION

Study participants

Thirty T2D women were recruited at the Department of Endocrinology and Metabolic Diseases, Cardiovascular Hospital, Lyon Bron (France) and randomized in one of the two groups. To be included, the subjects required to be women aged between 50 and 80 years old, with a post-menopausal status, a stable weight and a BMI ≥27 and ≤40 kg/m 2 , an HbA 1c ≥6,5 and ≤10% (≥48 and ≤86 mmol/mol) and no history of cardiovascular events or major hypertriglyceridemia. The anti-diabetic and lipid lowering medication was not discontinued for ethical reasons: 28 (93%) had metformin, 18 (60%) had sulfonylurea, 11 (37%) had glucagon-like peptide 1 analog, 8 (27%) had gliptin and 1 (3%) had Acarbose; none was treated with insulin injection. In addition, 17 (57%) had statins and 1 (0.03%) had a combined therapy of statins and ezetimibe. Table 1 shows study participants characteristics. They were postmenopausal women with grade 1 obesity (mean BMI of 34.1±1.0), and long duration of T2D (12.9±1.4 years). They had low serum LDL cholesterol due to statin treatment and none was treated with fibrates. They had a mild increase in serum TG concentration (33.3% had plasma TG above 2 mmol/L).

Study design

The study is a randomized interventional parallel trial entitled The Composition of Triglyceride Rich Lipoproteins and Platelet Activation in Type 2 Diabetes (COMPLETE, clinical trial registration number at ClinicalTrials.gov: NCT03561571). The trial was approved by the Scientific Ethics Committee Ile de France VII and conducted in accordance with the principles of the Helsinki declaration. Informed written consent was obtained from all subjects. The subjects performed the trial from October 2018 to May 2019. A minimum sample size of 10 T2D women per group was necessary to detect a 30% difference in TxB 2

Boulet et al. 2020, Page 7

concentrations with an alpha level of 0.05 and 80% power, 15 women were recruited in each group to increase the statistical power. The primary outcome was the difference in TxB 2 concentrations in platelets incubated with fasting and PP TGRL. Secondary outcomes were the difference in the aggregation of platelets incubated with fasting and PP TGRL, the difference in TxB 2 concentrations in platelets incubated with TGRL collected after the consumption of a fat of different FA composition; and the difference in FA composition between fasting and PP TGRL from each group.

The interventions were randomized according to a block randomization performed by a biostatistician associated to the study using the R software (The R Foundation, Miami, FL).

The block randomization (7 blocks of 4 and 1 block of 2 patients) was generated so that both treatments could be tested on platelets from the same donor in each block. The outcome assessors were blinded to the group assignment of the patients. After a 12 hour overnight fast, the first blood draw was made and the subjects were asked to eat the whole test breakfast according to their randomization group. A second blood sample was collected 4 hours after the ingestion of the breakfast. Blood sampling and test breakfast administration were done as part of the annual T2D follow-up at the outpatient clinic.

Test breakfasts composition

Both experimental breakfasts contained 20g of fats, one containing a majority of vegetal oil (mostly refined palm oil) within a commercial hazelnut-cocoa spread (HCS) and the other with butter. The FA composition of both fats was assessed by gas chromatography from 30 mg of butter and 70 mg of HCS with the same method as for the TGRL. Lipids were extracted with chloroform/methanol (2:1) according to the Folch method [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF]. The HCS mostly contained palmitic (16:0) and oleic (18:1 n-9) acids. Butter contained a broader range of SFA

Boulet et al. 2020, Page 8

(Table 2). Both breakfasts were designed to be similar in carbohydrates, proteins and caloric contents. The butter breakfast was composed of plain crackers (30g), commercial butter (22.5g, 82% fat), jam (30g), orange juice (200 ml) and plain coffee or tea (200 ml) for a total of 440 calories, with 51.7%, 40.9% and 4.5% of energy as carbohydrates (56.9g), lipids (20g) and proteins (5g), respectively. The HCS breakfast contained plain crackers (30g), commercial HCS (60g) and plain coffee or tea (200 ml) providing 439 calories with 51.1%, 40.9% and 6.5% of energy as carbohydrates (56.1g), lipids (20g) and proteins (7.1g), respectively. All subjects were given a maximum of 20 minutes to eat the breakfast.

Plasma biochemistry

Blood was collected into EDTA tubes and plasma was immediately isolated by centrifugation (1500 × g, 10 min, at 4 °C) and stored in aliquots at -20 °C. Measurement of ApoB-48 concentrations was performed by ELISA according to the manufacturer's instruction (SHIBAYAGI, Shibukawa, Gunma Prefecture, Japan). Total (TC), HDL cholesterol (HDL-c), TG, total ApoB and glucose were enzymatically determined using commercial kits (ABBOTT Diagnostics) on an Architect C16000 autoanalyser (ABBOTT Diagnostics, Illinois, USA).

LDL cholesterol was calculated using Friedewald equation (LDL-c = TC -HDL-c -0.45*TG). HbA 1c was determined by capillary electrophoresis using a Sebia Capillarys 3 (Sebia). Insulin was determined by immunoassay on an Architect I2000 autoanalyser. ApoB-100 was calculated by subtraction of ApoB-48 from total ApoB.

Isolation and characterization of TGRL fractions

In order to collect TGRL fractions, 250µL of plasma was carefully deposited under a layer of distilled water (850µL) followed by ultracentrifugation at 9777 × g for 1 hour at 12°C using a

Boulet et al. 2020, Page 9

Beckman Coulter Optima TLX ultracentrifuge and a Beckman TLA 100.2 rotor. Top 100 µL of the floating layer corresponding to TGRL with density <1.000 g/ml was collected and stored at 4°C for no longer than 2 days or stored at -20°C for lipid analysis. Protein concentration of the fractions was measured using a modified Lowry method [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF]. TG concentrations were obtained on an Architect C16000 autoanalyser. The hydrodynamic diameter of TGRL was measured by dynamic light scattering at 25°C with a ZetaSizer NanoS (Malvern, UK) using 1.0658 as viscosity and 1.445 as refractive index and 1.330 as refractive index of the aqueous phase [START_REF] Vors | Modulating absorption and postprandial handling of dietary fatty acids by structuring fat in the meal: a randomized crossover clinical trial[END_REF].

TGRL fatty acid composition by gas chromatography

Total lipids were extracted twice from TGRL (200µL + 300µL H 2 O) with ethanol:chloroform (1:3:6, respectively) after addition of 1,2,3-triheptadecanoyl-sn-glycerol as internal standard and BHT (50µM). FA were transmethylated with BF 3 /methanol (1.3 M, 10% in methanol) at 100°C for 1h30. FA methyl esters were extracted twice with isooctane and separated by gas chromatography using a gas chromatograph (HP6890 G1530) equipped with a SP 2380 capillary column (0.25μm, 30m × 0.25mm, Supelco, Bellefonte, PA, USA) and a flame ionization detector.

Platelet isolation

Venous blood was obtained from the local blood bank (Etablissement Français du Sang) on citrate-phosphate-dextrose anticoagulant (19.6 mmol/L citric acid, 89.4 mmol/L sodium citrate, 16.1 mmol/L NaH 2 PO 4 , 128.7 mmol/L dextrose; pH 5.6) from healthy volunteers who had not ingested any aspirin or anti-inflammatory drugs in the previous 10 days. Platelet-rich plasma (PRP) was prepared by centrifugation of the blood at 200 × g for 17 minutes at 20°C.

Boulet et al. 2020, Page 10

PRP was collected, acidified to pH 6.4 with 0.15 M citric acid and centrifuged at 900 × g for 12 minutes at 20°C. Platelet-poor plasma (PPP) was removed and platelets were suspended in a Tyrode-HEPES buffer (pH 7.35) [START_REF] Lagarde | A simple and efficient method for platelet isolation from their plasma[END_REF]. The mean concentration for all platelet suspensions was 239 ± 24 G/L (min: 141 G/L, max: 354 G/L).

Platelet aggregation experiments

Aggregation was measured in isolated platelets in a Chrono-log dual-channel aggregometer (Coulter, Margency, France) according to the method of Born [START_REF] Born | Aggregation of blood platelets by adenosine diphosphate and its reversal[END_REF]. Platelet suspensions were preincubated for 5 minutes at 37°C in the presence or absence of different preparations of TGRL (25 µg proteins/mL) and then stimulated with subthreshold concentrations of collagen (0.13±0.02 µg/mL) (type I from equine tendons, Diagnostica Stago, Asnières sur Seine, France) with continuous stirring at 1000 rpm. The subthreshold concentrations of collagen were defined as the concentrations of collagen that induced approximately less than a 10-20% increase in light transmission. The extent of platelet aggregation was expressed as percentage of change in light transmission 4 minutes after the addition of collagen [START_REF] Colas | LDL from obese patients with the metabolic syndrome show increased lipid peroxidation and activate platelets[END_REF].

Platelet thromboxane B 2 measurement

Freshly isolated platelet suspensions were incubated at 37°C for 30 minutes in the absence or presence of TGRL (25 µg proteins/mL) while being slowly agitated. Platelets were incubated at 37°C for an hour with non aggregating concentrations of collagen (0.5 µg/mL) or thrombin (0.05 U/mL) while being slowly agitated before being immediately stored at -80°C until TxB 2 measurement. Following 3 cycles of thawing and freezing of samples for platelets lysis, TxB 2 concentrations were determined by enzyme immunoassays according to the manufacturer's recommendations (Enzo Life Sciences Inc., Villeurbanne, France). Platelet proteins

Boulet et al. 2020, Page 11

concentrations were measured using the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] in order to express TxB 2 results.

Statistical analysis

Data were expressed as means ± SEM. Normality of data distribution was assessed using d'Agostino-Pearson normality test. Paired Student's t-test or Wilcoxon test was used to assess differences between platelets incubated with TGRL and stimulated with an agonist compared to platelets stimulated with an agonist. Differences between platelets stimulated with an agonist compared to platelets alone were performed to ensure platelet functionality in response to agonists using Student's t-test or Wilcoxon test as well. Differences between T240 and T0 in each group were assessed using paired Student's t-test or Wilcoxon test and differences between the two groups for the means of variation deltas (T240-T0) were determined using unpaired Student's t-test or Mann-Whitney test. Differences were considered significant at p<0.05 (2 sided test). All statistical analyses were performed using GraphPad Prism 8.0 (Graphpad Software, San Diego, CA).

Boulet et al. 2020, Page 12

RESULTS

Postprandial modification of plasma and triglyceride-rich lipoproteins

At the plasma level, consumption of both breakfasts increased ApoB-48 (HCS: +78%, Butter: +67%, p<0.001 for both) and TG (HCS: +33%, Butter: +30%, p≤0.0001 for both) concentrations similarly (Figure 1A and B) at 4 hours after 20g fat meal. TGRL isolated from plasma of fasting T2D patients had a mean diameter of 146±5 nm and a mean TG and total FA concentrations of 0.07±0.01 mmol/L and 512±6.5 nmol/mg protein, respectively. TGRL collected after the breakfasts had an increased diameter, TG and FA content 171±4 nm, 0.12±0.02 mmol/L and 907±14 nmol/mg protein, respectively. TGRL mean concentration of TG was significantly increased in the PP state. As shown in Figure 2A, there was a 200% and 71% increase in TGRL-TG after the HCS and butter breakfasts, respectively (p<0.01 for both groups). The type of fat does not impact TGRL-TG concentrations in PP plasma. Following the HCS breakfast, mean TGRL diameter was 10% higher, while it was increased by 23% after the butter breakfast (Figure 2B) (p<0.01 for both groups). This increase in diameter after the butter breakfast was significantly higher than after the HCS breakfast (Figure 2B, p<0.01).

As shown in Table 3, the main FA found in TGRL were palmitic (16:0), oleic (18:1 n-9) and linoleic (18:2 n-6) acids and their molar percents were not different between both groups in the fasting state. Ingestion of the HCS breakfast increased the proportion of oleic acid, the main MUFA, with decreased proportions of SFA and PUFA. After the butter breakfast, molar percentages of palmitic acid, the main SFA, and of medium chain SFA were significantly elevated, except for stearic acid (18:0), and proportions of PUFA were significantly decreased. As shown in Figure 3, there was a significant PP increase in TGRL SFA molar

Boulet et al. 2020, Page 13

percent (T240-T0) following the butter breakfast compared to the HCS one, while the MUFA molar percent increased in TGRL after the HCS breakfast compared to the butter breakfast.

Effect of fasting and postprandial TGRL on platelet aggregation

As shown in Figure 4, TGRL from fasting T2D patients increased collagen-stimulated platelet aggregation (HCS group: +62%, butter group: +86%, p<0.01 for both) compared with collagen-stimulated platelets alone. When platelets were pre-incubated with TGRL isolated in the PP state, collagen-stimulated aggregation increased similarly in both groups (HCS group: +86%, butter group: +129%, p<0.01 for both). When platelets pre-incubated with PP TGRL are compared to platelets pre-incubated with fasting TGRL, the difference was not significant.

Effect of fasting and postprandial TGRL on platelet thromboxane B 2 concentration

Concentrations of TxB 2 in collagen and thrombin-stimulated platelets alone or pre-incubated with TGRL from T2D patients before and after the test breakfasts are shown in Figure 5.

When platelets were stimulated with non-aggregating concentrations of collagen, TGRL isolated in the fasting state in both groups significantly increased TxB 2 concentration similarly (HCS group: +28%, butter group: +31%, p<0.05 for both) compared to collagenstimulated platelets. TGRL isolated after the breakfasts did not further increase platelet TxB 2 concentration compared to TGRL collected in the fasting state (HCS group: +29%, p=0.07, butter group: +23%, p=NS vs control, Figure 5A). When platelets were pre-incubated with fasting and PP TGRL and stimulated with thrombin, there was an increase of TxB 2 platelet concentration (HCS group at T0: +54%, p=0.06, HCS group at T240: +54%, p=0.02, butter group at T0: +34%, p=0.007, butter group at T240: +55%, p=0.001 vs control) (Figure 5B).

We also tested the effect of a higher concentration of PP TGRL (50 µg/mL) on platelets, since TGRL concentration is elevated in the PP compared to the fasting state. The incubation of platelets with 50 µg/mL of PP TGRL significantly increased TxB 2 concentration compared to 25 µg/mL PP TGRL and fasting TGRL. This effect was not different between the HCS and butter group (Figure 6).

Boulet et al. 2020, Page 15

DISCUSSION

The association of non-fasting TG concentrations with the risk of ischemic cardiovascular events, independently of traditional risk factors, has highlighted the need of specific studies in order to understand the implication of PP TGRL on platelet function. Our randomized controlled study establishes that TGRL secreted in the PP state have a stimulatory effect on platelet aggregation. This finding extends our previous observation of the enhancing effect of large TGRL from fasting T2D patients on platelet aggregation [START_REF] Boulet | Large triglyceride-rich lipoproteins from fasting patients with type 2 diabetes activate platelets[END_REF]. This effect of PP TGRL was not dependent on their FA composition.

The concentrations of plasma TG and ApoB-48 increased in T2D patients after the ingestion of both realistic breakfasts in a similar way. This is in accordance with the observations of Sciarrillo et al. who showed that plasma TG levels in lean and obese subjects were increased in a similar manner after a meal containing 61% of calories (51-84g) from different fats (butter, coconut oil, olive oil and canola oil) [START_REF] Sciarrillo | Postprandial Lipemic Responses to Various Sources of Saturated and Monounsaturated Fat in Adults[END_REF]. Our results support the fact that PP TG metabolism is altered in the T2D population, because a similar lipemic response in healthy individuals would necessitate around 30-40g of lipids [START_REF] Dubois | Effects of graded amounts (0-50 g) of dietary fat on postprandial lipemia and lipoproteins in normolipidemic adults[END_REF].

Residual fasting TGRL have a mean diameter of 146 nm, which is much larger than what is reported in healthy individuals. For example, Mekki et al. reported a mean diameter of ~91 nm for fasting TGRL in healthy individuals [START_REF] Mekki | Butter differs from olive oil and sunflower oil in its effects on postprandial lipemia and triacylglycerol-rich lipoproteins after single mixed meals in healthy young men[END_REF]. Interestingly, despite a similar amount of fat (20 g), we observed a greater diameter of PP TGRL 4 hours after the ingestion of the butter breakfast (183 nm) compared to the PP TGRL after the HCS breakfast (159 nm), and compared to the mean diameter reported for PP TGRL 4 hours after the consumption of a meal containing 40g of butter (~120 nm). In addition, our study shows that ingestion of a moderate amount of lipids (20g) leads to modification of the FA composition of TGRL 4 hours after the intake in T2D women. FA composition of PP TGRL reflects the main FA

Boulet et al. 2020, Page 16

composition of each fat consumed. TGRL secreted after the HCS breakfast, containing 45% of oleic acid, were enriched in MUFA (particularly oleic acid, 18:1 n-9). Those collected after the butter breakfast, which contained 79% of SFA (mainly palmitic acid, 16:0) and were accordingly rich in SFA (53% of total FA), especially palmitic acid (16:0).

The increased size of TGRL collected after the butter breakfast is intriguing and the difference in FA composition of TGRL after both breakfasts could be due to the positional distribution of fatty acids on the glycerol backbone of the TG molecules. In butter, palmitic acid (16:0) is mostly in the sn-2 position, while it is on the external sn-1 and 3 positions in TG from palm oil [START_REF] Michalski | Multiscale structures of lipids in foods as parameters affecting fatty acid bioavailability and lipid metabolism[END_REF]. Our results are in line with studies on animal models demonstrating that the FA position at sn-2, particularly for palmitic acid (16:0), has a determining effect on its absorption and subsequent incorporation in TG [START_REF] Renaud | The positional distribution of fatty acids in palm oil and lard influences their biologic effects in rats[END_REF][START_REF] Innis | Formula containing randomized fats with palmitic acid (16:0) in the 2-position increases 16:0 in the 2-position of plasma and chylomicron triglycerides in formula-fed piglets to levels approaching those of piglets fed sow's milk[END_REF]. The FA in the sn-2 position of the ingested TG is favorably incorporated in the CM [START_REF] Pufal | Effect of dietary triacylglycerol structure on lipoprotein metabolism: A comparison of the effects of dioleoylpalmitoylglycerol in which palmitate is esterified to the 2-or 1(3)-position of the glycerol[END_REF] and the presence of palmitic acid in the sn-2 position is likely to increase TG absorption [START_REF] Karupaiah | Effects of stereospecific positioning of fatty acids in triacylglycerol structures in native and randomized fats: a review of their nutritional implications[END_REF]. However, in our case the palmitic acid content of both fats was similar and this may have minimized the effect of stereospecificity on TG absorption. Also, although the PP TGRL is significantly higher by 24 nm after the butter breakfast, it may not be enough of an increase to have a physiological impact on TGRL lipolysis.

Our results on the effect of TGRL from T2D patients on platelets show that PP TGRL significantly stimulate in vitro platelet aggregation. To the best of our knowledge, no study has already investigated the specific effect of PP TGRL from T2D patients on isolated platelets. For example, Aviram et al. tested the effect of different isolated lipoproteins fractions from healthy or hyperlipoproteinemic men after two meals containing 50g of fat either rich in SFA (25g) or PUFA (38g) on platelet aggregation. They observed that 3h after both meals, CM decreased collagen-stimulated aggregation while VLDL enhanced it in both groups [START_REF] Aviram | Postprandial plasma lipoproteins in normal and hypertriglyceridaemic subjects and their in vitro effect on platelet activity: differences between saturated and polyunsaturated fats[END_REF]. An original aspect of our study is that we isolated and tested both fractions from

Boulet et al. 2020, Page 17

the fasting and PP state, which are different in composition. Indeed, the presence of large TGrich particles in the basal state suggest that these are remnants of an inadequate CM and VLDL lipolysis [START_REF] Panarotto | Insulin resistance affects the regulation of lipoprotein lipase in the postprandial period and in an adipose tissue-specific manner[END_REF], while the largest TGRL fractions isolated in the PP state contain CM and are representative of the heterogeneous mix of particles following a meal. We also show for the first time in vitro that an increase in TGRL, in physiological concentration, in the PP state has an enhancing effect on platelets TxB 2 concentration after a meal containing a realistic amount of fat rich in SFA. The increase in platelet activation and aggregation was similar after both fats, even though the TGRL FA composition was modified in PP. It is possible that an acute intervention of one breakfast containing a moderate quantity of lipids is not enough to cause a sufficient change in TGRL composition leading to a significant effect of TGRL FA on TGRL-induced platelet activation. A longer dietary intervention with different lipid amount and sources could induce more pronounced effects. Delgado-Lista et al studied in 20 healthy young men the effects of a chronic 28 days intake of a controlled diet followed by 3 tests meals containing 1g/kg of lipids of different FA composition [44]. A lipidrich meal caused an increase of plasma tissue-type plasminogen activator, d-dimers and TxB 2 in the PP irrespective of the type of fat consumed, but not in the fasting state.

Our results show that fasting and PP TGRL also increase platelet TxB 2 concentration with differences in the extent of TGRL-induced stimulation according to the agonist used. We aimed to use physiological agonists that would not interfere with the lipid composition of TGRL and platelets (eg: arachidonic acid), and that would activate platelets by different signalling pathways. Tested collagen concentrations induce a moderate stimulation while used thrombin concentrations provoke strong platelet activation. The platelets used are from healthy donors, so it is possible that the effect of fasting and PP TGRL would be greater on platelets from T2D patients, since they are already hyperactive in the basal state.

Boulet et al. 2020, Page 18

Overall, we acknowledge some limitations to our study. The test fats were comprised in mixed meals so we cannot exclude that other components of the meals could have had an effect on TGRL composition and their effect on platelets, for example compounds from coffee or tea. Also, we could not obtain dietary data from the patients, which would have been useful to evaluate the usual fat intake, but randomization reduces the risk of bias in this case.

Finally, our trial included only women, because of their higher risk for cardiovascular morbidity and mortality compared to diabetic men, so our results would need to be confirmed in T2D men.

In conclusion, this trial shows that 20g of lipids is sufficient to modify the FA composition of TGRL 4 hours after the ingestion in T2D patients. We confirm that fasting TGRL from T2D patients activate platelets and further demonstrate that PP TGRL have a similar enhancing effect on platelet aggregation and TxB 2 formation, independently of their FA composition.

This study also highlights the importance of TG management in the diabetic population for prevention of cardiovascular complications, as well as dietary counseling regarding fat intake.

Our randomized trial shows that a realistic acute intake of either a commercial hazelnut cocoa spread or butter has a similar detrimental effect on TGRL-induced platelet activation compared with TGRL from fasting patients with T2D.

[44] Delgado-Lista, J., Lopez-Miranda, J., Cortes, B., Perez-Martinez, P., et al., Chronic dietary fat intake modifies the postprandial response of hemostatic markers to a single fatty test meal. Am J Clin Nutr 2008, 87, 317-322.

Characteristics

FIGURE 1 .

 1 FIGURE 1. Increase in plasma ApoB-48 and triglyceride concentrations following the

FIGURE 2 .FIGURE 3 .

 23 FIGURE 2. Increase of TGRL triglyceride concentration and diameter following the test

FIGURE 4 . 33 FIGURE 5 . 2 ( A )

 43352A FIGURE 4. Collagen-stimulated platelet aggregation is increased by fasting and

FIGURE 6 .

 6 FIGURE 6. Platelet activation by postprandial TGRL is concentration dependant, but

TABLE 2 .

 2 Fatty acid composition of the fats in the test breakfasts

		Type 2	Type 2 diabetes	Type 2 diabetes
		diabetes	HCS group	Butter group	p
		(n=30)	(n=15)	(n=15)
	Age (years)	65.5 ± 1.4	62.9 ± 2.3	68.1 ± 1.4	NS
	BMI (kg/m 2 )	34.1 ± 1.0	34.4 ± 1.7	33.9 ± 1.2	NS
	Duration of diabetes (years)	12.9* ± 1.4	12.5 ± 1.7	13.2 ‡ ± 2.4	NS
	Glucose homeostasis
	HbA 1c (%)	7.8 ± 0.2	7.7 ± 0.3	7.8 ± 0.3	NS
	Glycemia (mmol/L)	9.0 ± 0.7	8.8 ± 1.0	9.3 ± 0.9	NS
	Insulin (mU/L)	12.3* ± 1.3	11.2 ± 1.4	13.4 ‡± 2.3	NS
	HOMA-IR	5.0* ± 0.7	4.1 ± 0.4	5.9 ‡± 1.4	NS
	Plasma lipids (mmol/L)
	Triglycerides	1.69 ± 0.1	1.53 ± 0.1	1.85 ± 0.3	NS
	Total cholesterol	4.55 ± 0.2	4.49 ± 0.2	4.62 ± 0.2	NS
	HDL cholesterol	1.24 ± 0.1	1.27 ± 0.1	1.21 ± 0.1	NS
	LDL cholesterol	2.56 ± 0.1	2.53 ± 0.2	2.60 ± 0.2	NS
	Plasma apolipoproteins B (g/L)
	Total ApoB	0.89 †± 0.04	0.86 ‡ ± 0.1	0.92 ‡ ± 0.1	NS
	ApoB-100	0.88 † ± 0.04	0.85 ‡ ± 0.05	0.91 ‡ ± 0.1	NS
	ApoB-48	0.006 † ± 0.001	0.005 ± 0.001	0.006 § ± 0.001	NS
	Antidiabetic and lipid lowering-medication (n(%))
	Metformin	28 (93)	14 (93)	14 (93)	-
	Sulfonylurea	18 (60)	8 (53)	10 (67)	-

ACKNOWLEDGEMENTS

We thank the nurses and the medical team of the department of Endocrinology, Metabolic Disease, Diabetes and Nutrition at Louis Pradel hospital for their essential collaboration and the dieticians at Centre de Recherche en Nutrition Humaine Rhône-Alpes for designing the test breakfasts.

FUNDING

This work was supported by Inserm and promoted by Hospices Civils de Lyon. MMB received a doctoral contract from the Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation and a grant from the Nouvelle Société Francophone d'Athérosclérose. CC thanks the CNRS.

Data are presented as means ± SEM. Other minor FA are not listed. HCS: hazelnut cocoa spread, NS: not significant, SFA: saturated fatty acids.

CONFLICT OF INTEREST STATTEMENT


Prince N Amaniampong

Thang Quang

Karine Trinh

De Oliveira Vigier

Quang Duy

Ngoc Han Dao

Yingqiao Tran

Matthew Wang

François Sherburne

Jérôme

] Quang

Thang Trinh

Karine De

Oliveira Vigier

♠ ] Duy

Quang Dao

# ] Ngoc

Han Tran

Yingqiao Wang

Matthew P Sherburne

François Jérôme

Synergistic Effect of High-Frequency Ultrasound with Cupric Oxide Catalyst Resulting in a Selectivity Switch in Glucose Oxidation under Argon

Keywords: Oxidation, catalysis, ultrasound, glucuronic acid, DFT, Copper oxide

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

INTRODUCTION

Glucuronic acid is an important chemical both for social and industrial reasons, for example it plays crucial role in the detoxification of drugs and toxins. Glucuronic acid is a promising intermediate in the fledgling, but fast growing, renewable chemical industry, allowing the synthesis of chemicals with improved performances. For instance, glucuronic acid is a key precursor in the synthesis of proteoglycans, glycoglycerolipids, ascorbic acid, surfactants, and monomers such as glucaric and adipic acids used in the fabrication of renewably-sourced polymers. [START_REF] Michal | Biochemical pathways: an atlas of biochemistry and molecular biology[END_REF] The selective oxidation of glucose to glucuronic acid (oxidation at the C6 position) remains a formidable challenge in the field of catalysis. Indeed, most of catalysts, including noble and non-noble metal catalysts in combination with oxygen or hydrogen peroxide, oxidize glucose at the anomeric position (oxidation at the C1 position), leading to the formation of gluconic acid instead of glucuronic acid (Scheme 1). [START_REF] Amaniampong | Titania-Supported Gold Nanoparticles as Efficient Catalysts for the Oxidation of Cellobiose to Organic Acids in Aqueous Medium[END_REF] To circumvent this problem, the anomeric position of glucose is protected by Fisher glycosylation [START_REF] Fel'dman | Methods of obtaining D-glucuronic acid from D-glucose. II. Synthesis of glucuronides and their conversion into D-glucuronic acid[END_REF] , or by acetalization [START_REF] Mehltretter | A Practical Synthesis of D-Glucuronic Acid through the Catalytic Oxidation of 1, 2-Isopropylidene-D-glucose[END_REF] , or by reaction with phosphate groups 51 prior the oxidation of the C6 position, followed by deprotection of the C1 position. Wojcieszak and coworkers [START_REF] Wojcieszak | Selective oxidation of glucose to glucuronic acid by cesiumpromoted gold nanoparticle catalyst[END_REF] reported one of the rare examples of selective oxidation of unprotected glucose to glucuronic acid (53% yield), using a hydrazine neutralized basic cesiumpromoted gold nanoparticles as a catalyst. However, the reaction mechanism and the origin for this unexpected selectivity were not reported on. In other work reporting on the catalytic oxidation of glucose, low selectivity to glucuronic acid (< 5%) was always observed. [START_REF] Marioli | Electrochemical characterization of carbohydrate oxidation at copper electrodes[END_REF] In the current state of the art, glucuronic acid is produced by fermentation routes which produces glucuronate salt, thus requiring an extra acidification step (Scheme 1). 1b The development of alternative catalytic technologies capable of selectively oxidizing unprotected glucose to glucuronic acid is an important scientific challenge, which is addressed in this work.

When water is subjected to an ultrasonic irradiation at a high frequency (100-800 kHz), small size gaseous cavitation bubbles are formed (< 10 µm), which contain sufficient Scheme 1. Contribution of this work to the state of the art amount of energy to dissociate water to H• and •OH radicals. 8 On implosion of the cavitation bubbles, radicals are propelled into the bulk solution where they react with organic solutes. The selectivity of the resulting radical reactions is quite difficult to control and, for this reason, high frequency ultrasound (HFUS) is mainly used for the total free oxidation of aqueous pollutants. [START_REF] Mahamuni | Advanced oxidation processes (AOPs) involving ultrasound for waste water treatment: a review with emphasis on cost estimation[END_REF] The ability to control the selectivity of these radical oxidative reactions is highly desirable to the implementation of this technology to the synthesis of a variety of chemicals, such as glucuronic acid, but it remains an elusive task. One solution consists of the introduction of a catalyst during the ultrasonic irradiation. This strategy, coined sonocatalysis, has been previously explored, with the focus being mostly on low frequency ultrasound (<20 kHz), i.e. the formation of radicals was very low and mainly physical effects occur. In this case, the turbulent flow and shock waves produced by the implosion of cavitation bubbles improve the dispersion of catalyst/reactant, mass transfer and, sometimes, prevent the catalyst from coking, resulting in improved reaction rates. [START_REF] Suslick | Applications of ultrasound to materials chemistry[END_REF] For instance, this strategy has been explored to accelerate the iron-catalyzed oxidation rate of glucose with hydrogen peroxide but, as expected, it yields gluconic acid as the main reaction product [START_REF] Rinsant | Efficient and Selective Oxidation of D-Glucose into Gluconic acid under Low-Frequency Ultrasonic Irradiation[END_REF] . To the best of our knowledge, controlling the selectivity of radical reactions induced by HFUS with the help of solid catalysts has not been reported yet. To optimize the reaction selectivity, in situ produced radicals by HFUS should interact faster with the catalyst surface than with organic solutes present in the bulk solution. In a heterogeneous solution, the formation of cavitation bubbles occurs preferentially on the particle surface via heterogeneous nucleation. [START_REF] Shchukin | Ultrasonic cavitation at solid surfaces[END_REF] In contrast to homogeneous solution, implosion of cavitation bubbles on a solid surface thus generates high-speed jets of liquid directed towards the surface. It occurred to us that this physical behavior could be a means to greatly increase selective transfer of radicals produced inside the cavitation bubble to a solid catalyst surface, and thus allowing better control of the reaction selectivity.

Recently, we reported the catalyst-free oxidation of glucose induced by HFUS. [START_REF] Amaniampong | Selective and Catalyst-free Oxidation of D-Glucose to D-Glucuronic acid induced by High-Frequency Ultrasound[END_REF] Surprisingly, the selectivity of the reaction was significantly impacted by the nature of the gaseous atmosphere. Under Ar, gluconic acid was formed as a major product while under O2 glucuronic acid was formed in a significant amount. Our efforts to rationalize this difference of selectivity, and inspired by our previous investigations in catalytic oxidation, [START_REF] Amaniampong | Unraveling the mechanism of the oxidation of glycerol to dicarboxylic acids over a sonochemically synthesized copper oxide catalyst[END_REF] led us to discover a synergistic effect between CuO and HFUS on the glucose oxidation. While CuO and HFUS are able to independently oxidize glucose to gluconic acid under argon, the combination of CuO with HFUS led to a complete reverse of the reaction selectivity, with glucuronic acid being formed as the major product. Through a combined experimental-theoretical approach, we rationalize here the synergistic effect between CuO and HFUS on glucose oxidation. In particular, we show that, under ultrasonic irradiation of water at a high frequency (550 kHz), the surface lattice oxygen of a CuO catalyst traps H• radicals stemming from the sonolysis of water, eliminating the participatory role of H• in the ring opening of glucose and favoring the selective oxidation of glucose to glucuronic acid. We also highlight that the particle size of CuO is an important parameter governing an optimal transfer of radicals from the cavitation bubbles to the catalyst surface.

RESULTS AND DISCUSSION

In line with our previous report, under argon, the catalyst-free ultrasonic irradiation of an aqueous solution of glucose at high frequency (550KHz, 40°C) led to the formation of gluconic acid (55% yield) as a major product. [START_REF] Nørskov | Rate Constants[END_REF] DFT calculations performed at a M05-2X/6-311++G(d,p) level of theory [START_REF] Zhao | Design of Density Functionals by Combining the Method of Constraint Satisfaction with Parametrization for Thermochemistry, Thermochemical Kinetics, and Noncovalent Interactions[END_REF] using the Gaussian DFT code 16 confirmed the preferential oxidation of the anomeric position (calculated on the glucopyranose form predominantly existing in water). The generation of H• and •OH radicals, as a consequence of cavitation bubbles collapse, favors the ring opening of glucose and follows a widely accepted concerted mechanism: (1) abstraction of the H atom at the anomeric position by the •OH radical (Ea = 2.6 kJ/mol, TS1a in Figure 1), forming water and (2) a very energetically favorable addition of H• radical on the oxygen atom of the ring (∆E = -524.0 kJ/mol, Int1b to Int2b in Figure 1), which induce the ring-opening. The dehydrogenation on the C6 position of glucose is also a feasible reaction, but with a higher activation energy barrier (Ea = 14 kJ/mol [START_REF] Amaniampong | Selective and Catalyst-free Oxidation of D-Glucose to D-Glucuronic acid induced by High-Frequency Ultrasound[END_REF] ) than the ring opening path. Interestingly, without assistance of H• radicals, the ring opening of glucose becomes significantly less favorable energetically (Ea = 116.2 kJ/mol, TS2a in Figure 1) than the oxidation on the C6 position of glucose. Hence, we hypothesize that if H• radicals are completely suppressed (i.e. only •OH radicals remain) during the sonolysis of water, then glucose should be selectively oxidized to glucuronic acid.

Previous studies have reported that •OH radicals bound to CuO surface sites Cu3, while H• radicals adsorb strongly at under-coordinated lattice oxygen sites on the surface of CuO. 14a, 17 Adsorbed H• radicals have a diffusion barrier of 0.53 eV [START_REF] Song | A DFT Study and Microkinetic Simulation of Propylene Partial Oxidation on CuO (111) and CuO (100) Surfaces[END_REF] allowing migration and the reaction with lattice oxygen to form H2O with a reaction energy of -0.82 eV. 17b This process then results in generating an oxygen vacancy at the CuO surface, 2f, 14a, 19 and a surface of CuO covered by •OH radicals, as reported by Bhola et al. 17a and Song et al. [START_REF] Song | A DFT Study and Microkinetic Simulation of Propylene Partial Oxidation on CuO (111) and CuO (100) Surfaces[END_REF] From these result, a reasoned approach suggested that the association of CuO catalyst with HFUS should be a viable strategy to in situ trap the radical H•, which would inhibit the ring-opening of glucose, and thus would shift the selectivity of the oxidation reaction towards glucuronic acid.

Unless otherwise noted, CuO used in this work was prepared according to our previously reported methodology 14a (see ESI for more details on CuO characterization). In agreement with DFT calculations, when CuO was introduced during the ultrasonic irradiation of an aqueous solution of glucose under argon (550 kHz, 80°C), the selectivity of the reaction was changed and glucuronic acid was now obtained as a major product, with 66% yield. A similar trend was observed when other hexoses such as mannose and galactose were tested under the same experimental conditions. In these cases, the corresponding uronic acids were obtained with 70% and 64% yield from mannose and galactose, respectively. CuO catalytic material can be up to 10 wt % without affecting the reaction selectivity, suggesting that CuO can be used in a catalytic amount and was therefore in situ re-oxidized under HFUS conditions (discussed later in the text). The in situ regeneration of CuO was confirmed by XRD analysis, with XRD patterns of the spent CuO being rigorously studied and showed similar patterns to those of the fresh CuO (Figures S1a andS1b).

To obtain additional data, the catalytic reaction was performed under silent conditions (i.e. 80°C, under Ar, without HFUS). Under argon, CuO reacted with glucose in a stoichiometric way, leading to the formation of gluconic acid instead of glucuronic acid. This oxidation under silent conditions occurs through the insertion of surface lattice oxygen from CuO into the glucose, resulting in a partial reduction of CuO to CunOn-1, as confirmed by XRD analysis (Figure S1c) (i.e. Mars-Krevelen-type mechanism). On the basis of the gluconic acid yield, we determined that about 93% of the oxygen of CuO was consumed by glucose in this case (a value in line with a previous work of Amaniampoing et al. 14b ) The oxidation mechanism of glucose to gluconic acid over bare CuO is discussed later in the manuscript. When this partly reduced CuO was then treated by HFUS (50 mL of water, 80°C, 550 KHz), XRD analysis unambiguously confirmed that CuO was in situ re-oxidized under HFUS conditions (Figure S4). DFT calculations we performed confirm that the re-oxidation of CuO by •OH radicals is a favorable energetic reaction (E = -66 kJ/mol). To rationalize this synergistic effect between HFUS and CuO, we performed calculations with the periodic planewave implementation of DFT using VASP (Vienna ab initio simulation package). [START_REF] Kresse | Efficiency of ab-initio total energy calculations for metals and semiconductors using a planewave basis set[END_REF] Under HFUS conditions, the surface of CuO is covered by •OH radicals. Without HFUS, the water splitting of H2O on Cu (111) of CuO is energetically non-favorable (132 kJ/mol) 2f and, in this case, bare CuO surface was computed.

First the oxidation of glucose to gluconic acid was investigated, with the ring-opening of glucose being the key step [START_REF] Trinh | Adsorption and Reactivity of Cellulosic Aldoses on Transition Metals[END_REF] , energy profiles are shown in Fig. 2. The first step of the glucose ring-opening is the dehydrogenation of the hydroxyl attached to the C1 position (TS1 in Figure 2). [START_REF] Trinh | Adsorption and Reactivity of Cellulosic Aldoses on Transition Metals[END_REF][START_REF] Mushrif | Insights into the Cr(iii) catalyzed isomerization mechanism of glucose to fructose in the presence of water using ab initio molecular dynamics[END_REF] This step is feasible on both bare CuO surface (barrier of 56 kJ/mol, TS1-1) and on CuO surface covered by •OH radicals (HFUS, Ea = 74 kJ/mol, TS1-2). However, the second step (hydrogen transfer to the ring, TS2 in Figure 2), has very different behavior with or without HFUS. On bare CuO surface, the barrier for this step is 47 kJ/mol (TS2-1). Under HFUS conditions (with high coverage of surface by the hydroxyl group), the barrier to abstract the H• atom from a surface hydroxyl group and transfer back to the ring has an extremely high barrier of 209 kJ/mol (TS2-3, Figure 2), due to the endothermic energy of this process. If the hydrogen is abstracted from a surface adsorbed water molecule, the barrier is lower but still very high at 121 kJ/mol (TS2-2, Figure 2), which is 74 kJ/mol higher than the corresponding step on bare CuO surface. These high barriers therefore, largely inhibit the ring-opening of glucose on CuO surface under HFUS conditions. [START_REF] Nørskov | Rate Constants[END_REF] Then we investigated the oxidation of glucose to glucuronic acid, on both the bare CuO surface and under HFUS conditions (with the presence of surface HO• group on CuO), Figure 3 Color code is the same as the color code used in Figure 2.

On bare CuO surface, the subsequent oxidation of "glucose aldehyde" requires an activation of the formyl O=C-H bond which possess a very high barrier of 153 kJ/mol (TS1-3, Figure 3), making the formation of glucuronic acid very unlikely through this mechanism. Under HFUS conditions, the presence of high coverage surface HO• groups on CuO opens an alternative pathway for glucose oxidation. In this alternative pathway, glucose is oxidized via the incorporation of surface HO• into glucose aldehyde with a barrier of only 35 kJ/mol (TS2-3, Fig. 3). This type of reaction has also been proposed and validated for alcohol oxidation on transition metals catalysts. [START_REF] Zope | Reactivity of the Gold/Water Interface During Selective Oxidation Catalysis[END_REF] After, the incorporation of surface HO• group, the formed molecule (Figure 3) undergoes the C-H abstraction either by surface lattice oxygen of CuO (barrier of 57 kJ/mol, TS2-4, Figure 3) or facilitated by adsorbed HO• group on CuO (slightly higher barrier of 66 kJ/mol), generating glucuronic acid as the final product. To gain insight into the reaction mechanism, the kinetic profile of the reaction was then recorded by plotting the yield of the reaction products as a function of the reaction time (Figure 4). Using 10 wt% of CuO under HFUS, glucuronic acid was formed as a major product (66% yield after 6 h of reaction). Fructose, commonly formed by isomerization of glucose in water, was also detected at the initial stage of the reaction, with a maximum yield of 8% at 28% conversion. The isomerization glucose/fructose is an equilibrated reaction and fructose is reconverted back to glucose and then glucuronic acid, as illustrated in the kinetic profile by a drop of the fructose yield at conversion higher than 28%. To support this claim, fructose was used as the starting material, affording glucuronic acid with 70% yield. In this case, formation of glucose was also initially with a maximum yield of 5% at 50% conversion of fructose (see SI for detailed kinetics).

Gluconic acid was also detected as a co-product, albeit in a low amount (yield < 10%), Figure 4. The side formation of gluconic acid may result from diffusion of H• and •OH radicals into the bulk solution, leading to unwanted oxidation of glucose to gluconic acid. When the catalyst loading was varied from 5 to 60 wt%, the formation of gluconic acid was decreased but not completely inhibited, suggesting that the transfer of radicals produced by cavitation bubbles to the CuO surface is not optimal (Figure S5). The heterogeneous nucleation of cavitation bubbles on a material surface is affected by the particle size. 12b A particle with large surface area (lower particle size) favors efficient nucleation and growth of cavitation bubble, owing to enhanced cavitation bubble-solid particles contact angles 12b . In this context, the present HFUS reactor was employed to prepare CuO, hereafter named CuOHFUS, using our previously reported strategy (sonication of an alkaline solution of CuNO3). 14a Using the same reactor and ultrasonic frequency for the catalyst preparation and the oxidation of glucose is, in our views, a good strategy to optimize the particle size of CuO to that of the cavitation bubbles, thus to maximize the transfer of radicals to the CuO surface. The optimized particle sizes of CuOHFUS (3.5-4.7 μm, 30 m 2 /g) are approximately four order of magnitude lower than those of CuO (10-24 μm, 18 m 2 /g) while the shape remained similar as confirmed by SEM and TEM analysis (Figure S7). Pleasingly, over CuOHFUS, formation of gluconic acid was completely suppressed and glucuronic acid was formed with a maximum yield of 88% (95% selectivity at 93% conversion), confirming that, in this case, radicals are selectively transfer to the CuO surface and not into the bulk solution, as above observed (Figure 5). At 80°C, the other detected product was formic acid stemming from the over-oxidation of glucose. The formation of formic acid is however significantly more important over CuO than over CuOHFUS, a result in line with less efficient cavitation bubble/CuO interactions in the former case. It is noteworthy that, in the presence of CuOHFUS, formic acid was only detected at a conversion higher than 80%, but its formation remained rather low (< 10% yield at 93% conv. of glucose, Figure 5b).

As a control reaction, tert-butanol, a known radical scavenger in sonochemistry, was added during the reaction [START_REF] Tauber | Sonolysis of tert-butyl alcohol in aqueous solution[END_REF] . As expected, in this case, radicals were trapped and glucuronic acid was no longer formed. Instead, we observed the classical and stoichiometric oxidation of glucose to gluconic acid (21 % yield) by CuO, illustrating the synergistic effect between CuO and HFUS in the selective oxidation of glucose to glucuronic acid.

If the popular low frequency (<20 kHz) are known to damage catalyst, mainly by abrasion resulting from intense shock waves, this phenomenon is much less likely to occur at HFUS for which cavitation bubbles are much smaller (4 m at 550 kHz vs 170 m at 20 kHz), 8,[START_REF] Suslick | Ultrasound: its chemical, physical, and biological effects[END_REF] thus considerably limiting the undesirable effects of shock waves on catalyst stability. To assess the stability of CuOHFUS during the ultrasonic irradiation at a high frequency, the CuOHFUS was filtered out at the end of the reaction and reused as collected without any further purification.

As anticipated, the CuOHFUS catalyst was found to be stable and was successfully recycled at least five times, without any appreciable decrease of the glucuronic acid yield, confirming that abrasion of CuOHFUS was drastically limited under HFUS. SEM analysis of the spent CuOHFUS catalyst showed no drastic change in morphology in comparison to the fresh catalysts (Fig. S7c)

CONCLUSION

We have provided theoretical and experimental evidence for an alternative reaction pathway provided by synergistic cavitation-copper oxide catalyst interactions. That is, H• and •OH radicals generated during cavitation bubble implosions in water are diffused onto the surface of the CuO catalyst, and selectively promote the oxidation of glucose to glucuronic acid under argon, in contrast to the gluconic acid formation in homogeneous bulk liquid solution. Through a combined experimental-theoretical approach, we showed that H• radical formed in situ by sonolysis of water are trapped by the surface lattice oxygen of CuO, making the ring opening of glucose energetically non-favorable. Instead, the reaction takes an alternative pathway oriented to the selective oxidation of glucose to glucuronic acid, a valuable chemicals whose synthesis remains a formidable scientific challenge in the field of catalysis. These results, providing first-hand insight into the participatory role of H• in the overall oxidation mechanism, also open a scientific line of thought on the unexplained role of the atmosphere (O2 vs Ar) previously observed on the catalyst-free oxidation of glucose assisted by ultrasound. [START_REF] Tauber | Sonolysis of tert-butyl alcohol in aqueous solution[END_REF] This aspect is the topic of current investigations in our groups.

From a more general point of view, this work demonstrates the general concept that the insertion of a solid catalyst during ultrasonic irradiation of organic solutes at a high frequency allows in situ radical reactions to be finely controlled. It also points towards a path to optimize the size of the catalyst for an ultrasonic frequency which minimizes the damage to the catalyst resulting in reusable catalyst. [START_REF] Koda | A standard method to calibrate sonochemical efficiency of an individual reaction system[END_REF] . The ultrasound probe was immersed directly in the reaction medium and a minichiller cooler (Huber) was used to control the reaction temperature at 25 o C. On completion of sonication at the desired time, the dark brown precipitates were washed thoroughly with distilled water and dried in an oven at 60 o C overnight.

EXPERIMENTAL SECTION

Preparation of Copper (II) Oxide Nanoleaves (CuO NLs) under High frequency ultrasound irradiation.

All chemical reagents were used without further purification. In a typical synthesis method, 40 mL of 0.25 M NaOH aqueous solution was added to 10 mL of 0.5 M Cu(NO3)2 aqueous solution and a sky-blue suspension was obtained. This suspension was subsequently subjected to an ultrasonic irradiation at high-frequency (550 kHz) and at a controlled temperature of 40 °C (standby power P0 = 13.9 W, nominal electric power of the generator Pelec = 70.4 W), with an acoustic power in water of Pacous.vol = 0.44 W.mL -1 determined by calorimetry [START_REF] Koda | A standard method to calibrate sonochemical efficiency of an individual reaction system[END_REF] . A minichiller cooler (Huber) was used to control the reaction temperature at 25 o C. On completion of sonication at the desired time, the dark brown precipitates were washed thoroughly with distilled water and dried in an oven at 60 o C overnight.

Sample characterization.

The as-synthesized CuO morphology was studied by SEM (JEOL JSM 6700F field emission), TEM and HR-TEM (JEOLJEM-2100F). In each run, approximately 50 mg of the catalyst was pretreated at 300 °C under a flow of He (30 mL min -1 ), and then heated to 700 °C with a ramp of 10 °C min -1 in the stream of 5 vol% H2/Ar (40 mL min -1 ). Surface area analysis was determined by nitrogen physisorption on a Micromeritics TrisStar apparatus. The specific area was calculated using the Brunauer-Emmett-Teller (BET) equation. Crystallographic analysis for the tested were performed by means of XRD measurements in 2ϴ mode on a Bruker AXS D8diffractometer with CuKɑ (= 0.154056 Å) radiation at 40 kV and20 mA. XPS was performed on a Thermo Escalab 250 spectrometer. The binding energy was calibrated using C1s (284.6 eV) as a reference.

Carbohydrates oxidation. Oxidation of glucose (or fructose, mannose, galactose) was carried out in a 250 mL high-frequency ultrasonic reactor (SinapTec Ultrasonic Technology, NextGen Lab 1000). Typically, 2.0 g of glucose in 100 mL of distilled water was subjected to highfrequency ultrasound irradiation at a controlled temperature of 25 o C and a frequency of 550 kHz (standby power P0 = 13.9 W, nominal electric power of the generator Pelec = 46.1 W), with an acoustic power in water of Pacous.vol = 0.36 W.mL -1 determined by calorimetry using the procedure described in the literature. [START_REF] Koda | A standard method to calibrate sonochemical efficiency of an individual reaction system[END_REF] Product analysis. Glucose and fructose were analyzed using a Shimadzu HPLC equipped with a pump system (LC-20AD), an autosampler SIL-10A, a controller CBM 20A, a refractive index detector from Waters and a Zorbax NH2-column (250 ¥ 4.6 mm). A mixture acetonitrile/water (20:80) was used as a mobile phase (0.4 mL min -1 ). Glucuronic acid and other acid products were detected and quantified from the HPLC analysis by using the Varian Pro Star HPLC equipped with an ICE-COREGEL 107H column 300 × 7.8 mm from Transgenomic, a UV/Vis detector (Varian Pro Star, 210 nm) and a refractive index detector (Varian 356-LC). A H2SO4 aqueous solution was used as the eluent with 0.4 mL.min -1 flow rate. External calibration of liquid chromatography was performed using standards of glucose, fructose, gluconic, formic and glucuronic acid was quantified by the difference between the two HPLC analyses.

Density Functional Theory (DFT) details.

The roles of OH  , H3O  radicals on the ring-opening reaction of glucose by DFT calculations using Gaussian 16 RevA.03 package 16 in the vacuum at the M05-2X/6-311++G(d,p) level of theory. [START_REF] Zhao | Design of Density Functionals by Combining the Method of Constraint Satisfaction with Parametrization for Thermochemistry, Thermochemical Kinetics, and Noncovalent Interactions[END_REF] The M05-2X functional combined with the 6-311++G(d,p) basis set was proved to provide the best accuracy of the radical-molecule reaction including the H-atom transfer especially the barrier-less reaction with reactive radical like OH  . [START_REF] Galano | Kinetics of radicalmolecule reactions in aqueous solution: A benchmark study of the performance of density functional methods[END_REF] All the intermediate compounds (Int) and transition states (TS) were identified by the number of imaginary frequencies (0 or 1, respectively). The intrinsic coordinate (IRCs) calculations were also performed to verify if the TS were well connected with the intermediate compounds. All the relative energies on the potential energy profiles are calculated in compared with the initial reactant, i.e. glucose.

All first-principles calculations for adsorptions and reactions on CuO(111) surface are performed based on periodic boundary conditions and plane-wave pseudopotential implementation of DFT using the Vienna ab-initio simulation package (VASP) developed at the Fakultät für Physik of the Universität Wien. 20a, 20b Projector augmented wave (PAW) method [START_REF] Blöchl | Projector augmented-wave method[END_REF] employed with Perdew-Burke-Ernzerhof (PBE) exchange-correlation functional [START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF] is used to describe the interaction between valence electrons and ions with a plane wave cut-off energy of 450 eV. We used a k-points sampling of 3×3×1 with Monkhorst-pack scheme for integration over the Brillouin zone in reciprocal space and spin polarization is turned on for all simula-tions. The Generalized Gradient Approximation (GGA) and the Hubbard correction U = 4.5 eV within the GGA+U scheme was used to correct the electron delocalization that occurs in strongly correlated systems such as transition metal oxides. 20c, 31 The optimized lattice parameters for CuO with GGA+U method is a = 4.5597 Å; b = 3.6059 Å; c = 5.1782 Å; β = 96.3385 o , which also matches very well with reported experimental data. 2f CuO catalyst is modeled by using the structure of p(4×2) slab of the most stable CuO(111) surface with 4 layers and the vacuum thickness of 15 Å above the topmost layer was employed to avoid interactions between repeted slabs. 20c The bulk-like magnetic configuration was reported to be the most stable arrangement for CuO(111) surface, and therefore, was used for all calculations in this study. [START_REF] Hu | Effects on Electronic Properties of Molecule Adsorption on CuO Surfaces and Nanowires[END_REF] Geometries were fully relaxed using the conjugategradient algorithm until the energy changes by less than 0.1 kJ/mol. Transition states were located using the Climbing-Nudged Elastic Band (Cl-NEB) method, [START_REF] Henkelman | A climbing image nudged elastic band method for finding saddle points and minimum energy paths[END_REF] and frequency calculations confirmed the nature of the transition states with only one imaginary vibrational frequency.

ASSOCIATED CONTENT

Supporting information is available free of charge via the internet at http://pubs.acs.org. It contains CuO characterization before and after reaction, effect of catalyst loading, kinetic profile from fructose, characterization of the crude glucuronic acid and additional informations on DFT

Figure 1 .

 1 Figure 1. Potential energy profiles for glucose ring-opening reaction in vacuum calculated at the M05-2X/6-311++G(d,p) level of theory facilitated by HO  , HO  / H3O  radicals (H  + H2O). Label of structure: Int: intermediate compounds; TS: transition state.

Figure 2 .

 2 Figure 2. The energy profile for glucose ring-opening process under different conditions. Label of structure: Int1: adsorbed glucose; Int2: Product of first step OH activation; Int3: adsorbed open-chain glucose; TS1, TS2: transition states of step 1 and step 2, respectively. Color code: Large peach and red balls represent Copper (Cu) and Oxygen (O) atoms of the CuO substrate; and small white, grey, and red balls represent Hydrogen (H), Carbon (C), and Oxygen (O) atoms of the adsorbates, respectively.

  . The initial OH activation and the subsequent CH dehydrogenation steps from the -CH2OH group of glucose forming "glucose-dialdehyde" on bare CuO surface have barriers of 45 kJ/mol (TS1-1) and 83 kJ/mol (TS1-2), respectively, and the reaction is facilitated by the under-coordinated oxygen of CuO(111) surface. These two steps are even more favorable with the surface HO• group present on the CuO surface under HFUS conditions, with the CH dehydrogenation having a relatively low barrier of Ea = 58 kJ/mol, TS2-2.Micro-kinetic modeling of glucose ring-opening on bare CuO surface and under HFUS conditions (see SI for detailed discussion) confirmed that the rate of glucose open-chain formation was reduced to negligible values on CuO with high coverage of surface •OH (assuming the initial coverage of surface OH is 0.7 mono-layer (ML) under HFUS conditions).

Figure 3 .

 3 Figure 3. Overall reaction network for the glucose oxidation on CuO surface under HFUS conditions. Two pathways are illustrated. The pathway where reactions are facilitated by surface lattice oxygen of CuO is presented on the left side and activation barriers are indicated by red color. The pathway where reactions are facilitated by surface OH* group generated under HFUS is presented on the right side and activation barriers are indicated by green color. The bold blue arrows are highlighted the more preferred pathway. Color code is the same as the color code used in Figure 2.

Figure 4 .

 4 Figure 4. Kinetic profile of the reaction (10 wt% CuO, 80°C, 550 kHz, glucose concentration 20g.L -1 , Pacoust. = 0.36 W.mL - 1 ).

Figure 5 .

 5 Figure 5. Plot of the selectivity as a function of the conversion of glucose (a) CuO and (b) CuOHFUS (10 wt% copper oxide, 80°C, 550 kHz, glucose concentration 20g.L -1 , Pacoust. = 0.36 W.mL -1 ).

Figure 6 .

 6 Figure 6. Recyling of CuOHFUS (80°C, 550 kHz, 10 wt% CuOH-FUS, glucose concentration 20g.L -1 , Pacoust. = 0.36 W.mL -1 ).

Table of Contents artwork OH OH OH H 2 O (vap) H • • OH + CuO H 2 O Implosion on CuO surface Cu n O n-1

 of

H 2 O liquid 2 2 HIGH FREQUENCY ULTRASOUND

	H	•
			•	•	•
	H 2 O	• HO

ACKNOWLEDGMENT PNA, KV and FJ are grateful to the CNRS, the University of Poitiers, the Région Nouvelle Aquitaine for financial support. The International Consortium on Eco-conception and Renewable Resources (FR CNRS INCREASE 3707) and the chair "TECHNOGREEN" are also acknowledged for their funding. QTT and MPS acknowledge the financial support by the National Research Foundation (NRF), Prime Minister's Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) program. The computational work for this research is performed with resources of the National Supercomputing Centre, Singapore (NSCC). DQD is also indebted to the Centre de Ressources Informa-tiques (CRI) of the University of Lille and the Centre Régional Informatique et d'Applications Numériques de Normandie (CRIANN) for providing computer time of the theoretical calculations.

Funding Sources

This work was funded by the CNRS, the University of Poitiers, the Région Nouvelle-Aquitaine, the National Research Foundation (NRF),

AUTHOR INFORMATION

Corresponding Author * francois.jerome@univ-poitiers.fr (experimental part) * mpsherb@berkeley.edu (theoretical part)

Notes

The authors declare no competing financial interest.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

ABBREVIATIONS

HFUS, High frequency ultrasound; DFT, Density functional theory; XRD, X-Ray diffraction; XPS, X-ray photoelectron spectrometry; TS, Transition state.