MSc Sophie Guillotin

PhD Alexandra Vallet

PhD Sylvie Lorthois

PharmD Philippe Cestac

MD Eric Schmidt

PharmD Nicolas Delcourt
email: delcourt.n@chu-toulouse.fr

Affiliations

Association between Homocysteine, Frailty and Biomechanical Response of the CNS in NPH-suspected Patients

Keywords: biochemical parameters, biomechanic, brain, aging

Frailty is a geriatric syndrome that combines physiological decline, disruptions of homeostatic mechanisms across multiple physiologic systems and thus, strong vulnerability to further pathological stress. Previously, we provided the first evidence that increased risk of poor health outcomes, as quantified by a frailty index, is associated with an alteration of the central nervous system (CNS) biomechanical response to blood pulsatility. In this study, we explored correlation between fourteen biological parameters, the CNS elastance coefficient and frailty index. We included 60 adults (52-92 years) suspected of normal pressure hydrocephalus (NPH) and presenting with markers of multiple co-existing brain pathologies, including Parkinson disease (PD), Alzheimer disease (AD) and vascular dementia. We showed that the homocysteine (Hcy) level was independently and positively associated with both frailty index and the CNS elastance coefficient (adjusted R² of 10% and 6%). We also demonstrated that creatinine clearance and folate level were independently associated with Hcy level. Based on previous literature results describing the involvement of Hcy in endothelial dysfunction, glial activation and neurodegeneration, we discuss how Hcy could contribute to the altered biomechanical response of the CNS and frailty.

Introduction

Frailty is a clinical syndrome characterized by a decrease of the physiological reserve capacities with failure of homeostatic mechanisms and vulnerability to stressors [START_REF] Fried | Frailty in older adults: evidence for a phenotype[END_REF][START_REF] Clegg | Frailty in elderly people[END_REF]. Frailty is also a marker of risk for mortality and adverse events, including disabilities, falls, hospitalizations and institutionalizations [START_REF] Fried | Frailty in older adults: evidence for a phenotype[END_REF]. Notably, age is considered as a determinant of frailty but does not alone explain this syndrome. Its clinical expression is indeed modulated by comorbidities and psychological, social, economic and behavioral factors [START_REF] Fried | The physical frailty syndrome as a transition from homeostatic symphony to cacophony[END_REF].

With regard to pathologies of the central nervous system (CNS), it is well known that frailty is associated with an increased risk of cognitive decline and dementia [START_REF] Avila-Funes | Is Frailty a Prodromal Stage of Vascular Dementia? Results From the Three-City Study[END_REF][START_REF] Buchman | Brain Pathology Contributes to Simultaneous Change in Physical Frailty and Cognition in Old Age[END_REF][START_REF] Solfrizzi | Frailty syndrome and the risk of vascular dementia: The Italian Longitudinal Study on Aging[END_REF]. Interestingly, few studies also suggested that neurodegenerative or neurovascular changes are substrates of the frailty phenotype. Notably, it was shown that the presence of macro infarcts, Alzheimer disease (AD), Lewy body pathology, and nigral neuronal loss were associated with a more rapid progression of frailty [START_REF] Buchman | Association of brain pathology with the progression of frailty in older adults[END_REF]. In the same manner, reduced total brain or gray matter volume and increased cortical brain infarcts could be part of the structural substrate of the frailty phenotype.

Frailty is indeed associated with the presence, degree, and some specific localizations of cerebral atrophy in a population of cognitive-decline patients [START_REF] Gallucci | Associations between the Frailty Index and Brain Atrophy: The Treviso Dementia (TREDEM) Registry[END_REF]. Frailty is also associated with arterial stiffness [START_REF] Orkaby | Cross-Sectional Association of Frailty and Arterial Stiffness in Community-Dwelling Older Adults: The Framingham Heart Study[END_REF], failure of homeostatic mechanisms and vulnerability to stressors [START_REF] Hale | Frailty, inequality and resilience[END_REF].

The research of circulating frailty biomarkers that reflect the physiological state of individuals and the underlying mechanisms related to homeostatic changes throughout an individual lifespan remains a challenge. Among them, inflammatory, nutritional or metabolic risk markers are of the most important targets due to their potential modifiable characteristics [START_REF] Gale | Inflammatory markers and incident frailty in men and women: the English Longitudinal Study of Ageing[END_REF][START_REF] Baylis | Understanding how we age: insights into inflammaging[END_REF][START_REF] Walker | Midlife Systemic Inflammation Is Associated With Frailty in Later Life: The ARIC Study[END_REF][START_REF] Artaza-Artabe | The relationship between nutrition and frailty: Effects of protein intake, nutritional supplementation, vitamin D and exercise on muscle metabolism in the elderly. A systematic review[END_REF][START_REF] Yannakoulia | Frailty and nutrition: From epidemiological and clinical evidence to potential mechanisms[END_REF][START_REF] Tessier | An Update on Protein, Leucine, Omega-3 Fatty Acids, and Vitamin D in the Prevention and Treatment of Sarcopenia and Functional Decline[END_REF][START_REF] Vatic | Inflammatory biomarkers of frailty[END_REF][START_REF] Lu | Biological and Neuroimaging Markers as Predictors of 5-Year Incident Frailty in Older Adults: A Secondary Analysis of the MAPT Study[END_REF]. In parallel, several pathophysiological mechanisms have been proposed to result in frailty, such as chronic, low-grade inflammation related to aging [START_REF] Álvarez-Sánchez | Homocysteine and C-Reactive Protein Levels Are Associated With Frailty in Older Spaniards: The Toledo Study for Healthy Aging[END_REF]. Homocysteine (Hcy), known to be related to inflammation and oxidative stress, is associated with frailty but also with physical and cognitive decline [START_REF] Álvarez-Sánchez | Homocysteine and C-Reactive Protein Levels Are Associated With Frailty in Older Spaniards: The Toledo Study for Healthy Aging[END_REF][START_REF] Vidoni | Relationship between Homocysteine and Muscle Strength Decline: The Baltimore Longitudinal Study of Aging[END_REF][START_REF] Wong | Homocysteine, frailty, and all-cause mortality in older men: the health in men study[END_REF], and patients with hyperhomcysteinemia (HHcy) (>15 µmol/L) are at higher risk for decline of both physical and cognitive functions [START_REF] Smith | Homocysteine, B Vitamins, and Cognitive Impairment[END_REF].

In line with the demonstration that frailty is associated with arterial stiffness [START_REF] Orkaby | Cross-Sectional Association of Frailty and Arterial Stiffness in Community-Dwelling Older Adults: The Framingham Heart Study[END_REF], we recently provided the first evidence that frailty index (FI) is also associated with the CNS elastance coefficient [START_REF] Vallet | Biomechanical response of the CNS is associated with frailty in NPH-suspected patients[END_REF]. An increased CNS elastance coefficient induces larger intracranial pressure rises for given intracranial blood volume change. Such an abnormal transmission of mechanical stresses from blood to intracranial tissues at each cardiac cycle could lead to brain damage.

Similar to frailty but based on the physical behavior of the CNS rather than on biomedical markers, the CNS elastance coefficient could be considered as an index reflecting a decrease in mechanical reserve capacities with failure of homeostatic mechanisms and vulnerability to stressors.

We seek for plasma parameters statistically correlated to both FI and the CNS elastance coefficient in an observational cohort of patients suspected of normal pressure hydrocephalus (NPH) and presenting markers of multiple co-existing pathologies, including AD, Parkinson disease (PD) and vascular dementia. We integrate several plasma parameters divided into biological profiles: renal profile, metabolic profile, lipid profile and inflammatory markers. These plasma parameters make it possible to describe the body as a whole by integrating risk factors related to frailty, vascular and cognitive risks. Then, based on previous results from the literature, we discuss several hypothesis underlying Hcy based molecular mechanisms by which increased CNS elastance coefficient at the macroscopic scale could possibly contribute to frailty.

Methods

Study design and participants

One hundred patients suspected of NPH (46 women and 54 men aged 52-92 years) were prospectively included in the PROLYPHIC cohort between 2013 and 2015 at Toulouse University Hospital. The suspicion of NPH was based on established guidelines, i.e. the presence of (1) clinical symptoms of gait or balance disturbance, cognitive impairment or urinary disorders, (2) neuroimaging evidence of ventriculomegaly (i.e. Evans Index > 0.3) and

(3) narrow callosal angle, temporal horn enlargement or periventricular signal changes [START_REF] Relkin | Diagnosing Idiopathic Normal-pressure Hydrocephalus[END_REF].

The study was approved by the competent authority and registered at ClinicalTrials.gov under no NCT02016352. The local ethics committee approved the study. All patients gave their written informed consent.

Clinical characterization of the population

NPH is an important differential diagnosis of neurodegenerative diseases [START_REF] Skalický | Normal pressure hydrocephalus-an overview of pathophysiological mechanisms and diagnostic procedures[END_REF]. It is important to define these different diseases within our cohort. The increased risk of AD was examined based on the ratio between cerebrospinal fluid (CSF) tau and amyloid beta 42 (Aβ42) peptides using a cut-off of 0.215 [START_REF] Mo | A multinational study distinguishing Alzheimer's and healthy patients using cerebrospinal fluid tau/Aβ42 cutoff with concordance to amyloid positron emission tomography imaging[END_REF]. Presynaptic dopamine deficiency, which is a characteristic of PD, was examined visually by an expert neurologist using [123I]FP-CIT DATSPECT. The presence of white matter lesions, which is a characteristic of increased risk of vascular dementia, was ascertained on FLAIR MRI images using the Fazekas scale with a cut-off of ≥ grade 2 [START_REF] Fazekas | MR signal abnormalities at 1.5 T in Alzheimer's dementia and normal aging[END_REF].

Finally, abnormal CSF dynamics was quantified via the CSF outflow resistance R 0 [START_REF] Marmarou | Diagnosis and management of idiopathic normalpressure hydrocephalus: a prospective study in 151 patients[END_REF] derived from the lumbar infusion test [START_REF] Vallet | Biomechanical response of the CNS is associated with frailty in NPH-suspected patients[END_REF]. A threshold of R 0 >12 mmHg/mL/min was chosen as a marker of altered CSF dynamics and increased risk of NPH. Indeed, the symptoms mentioned above are not specific to NPH and may be associated with other neurodegenerative diseases.

That is why the infusion test is recommended in the clinical practice in order to characterize CSF dynamic alterations by a quantitative measurement of the resistance to CSF outflow R 0 .

Although the above in vivo markers do not, by themselves, corroborate the clinical diagnosis of neurodegenerative diseases, they do flag an increased risk of having or developing them. For each marker, the prevalence of patients at risk was calculated only in patients with available data.

Data collection

Comorbidities and lifestyles were recorded using a registration questionnaire for admission: tobacco, alcohol, diabetes mellitus, high blood pressure and dyslipidemia as qualitative variables. Questioning about alcohol and tobacco consumption were based on the current use.

Lipid profile with Very Low Density Lipoprotein (VLDL), Low Density Lipoprotein (LDL), High Density Lipoprotein (HDL), Apolipoprotein A1 and B, triglycerids and total cholesterol, inflammatory profile with C reactive protein (CRP) and Hcy, renal profile with creatinine, uric acid, blood urea nitrogen (BUN), and metabolic profile with cobalamin and folates were measured by the hospital laboratory according to routine procedures. The creatinine clearance was estimated with the Cockroft-Gault equation.

Frailty assessment

Frailty was quantified by means of a FI based on the deficit accumulation approach, which has been widely validated across many settings and populations [START_REF] Searle | A standard procedure for creating a frailty index[END_REF][START_REF] Rockwood | Frailty Defined by Deficit Accumulation and Geriatric Medicine Defined by Frailty[END_REF]. The FI was defined as the ratio, ranging from 0 (not frail) to 1 (very frail), between the number of health deficits presented by the individual and the total number of deficits considered. A total of 40 variables were selected to measure the deficits in (1) executive functioning, (2) mentation, behaviour and mood, (3) gait, (4) rigidity and agility of extremities, (5) blood laboratory tests of markers related to kidney function, metabolic function and inflammatory conditions, which are of particular importance in adults over 50 years old (31), (6) urinary symptoms, [START_REF] Buchman | Association of brain pathology with the progression of frailty in older adults[END_REF] nutrition and (8) history including medication intake and the presence of previously diagnosed hypertension or diabetes mellitus. Supplementary table 1 presents the variables and thresholds for deficit status. Following the guidelines [START_REF] Searle | A standard procedure for creating a frailty index[END_REF] for creating a FI, we did not include variables that had more than 5% missing data and less than 1% of participants meeting criteria for the deficit.

Patients presenting more than 10% of missing data on the 40 health deficits considered were excluded.

CNS mechanical response assessment

Infusion tests were performed in patients according to standard infusion test protocol [START_REF] Katzman | A simple constant-infusion manometric test for measurement of CSF absorption. I. Rationale and method[END_REF].

Lumbar CSF pressure, a surrogate marker of intracranial pressure (ICP), was measured; then a constant rate lumbar saline injection was performed in order to gauge CNS mechanical properties. Patients also underwent phase contrast magnetic resonance imaging (PCMRI) (see below) in order to gauge intracranial fluids dynamics. The biomechanical response of the CNS was characterized by the CNS elastance coefficient inferred in vivo from PCMRI and ICP monitoring during lumbar infusion test. This latter parameter characterizes the biomechanical response of the CNS to stress and measures its ability to adapt to a change of volume. It results from a complex interplay between the global stiffness of the brain parenchyma (homogeneous to a Young modulus E [Pa]), and the volume V [ml] of the compressible volumes within the CNS, and dimensionally scales as E/(V.p), where p is still to be identified but must be homogeneous to a pressure or stress [Pa] (for details, see [START_REF] Vallet | Biomechanical response of the CNS is associated with frailty in NPH-suspected patients[END_REF]).

PCMRI protocol and image analysis

The 2D PCMRI was used as a non-invasive technique to quantify intracranial blood flows as reported elsewhere [START_REF] Balédent | Cerebrospinal fluid dynamics and relation with blood flow: a magnetic resonance study with semiautomated cerebrospinal fluid segmentation[END_REF]. Patients who could not undergo PCMRI were excluded from the study.

All other participants were imaged in a 3.0 T magnetic resonance (MR) scanner (Philips Achieva, Philips, Best, Netherlands) with a commercial 32-channel head coil. Retrospective cardiac-gated PCMRI sequences were performed with a plethysmograph set on the subject's finger to synchronize the acquired images with the subject's heartbeat. The selected segment was perpendicular to the intracranial internal carotids, basilar artery, straight sinus and sagittal sinus vessels. The acquisitions were made with an encoding velocity of 80 cm/s and 32 time points were sampled for each cardiac cycle. Preprocessing steps, including velocity aliasing correction, noise reduction and vessel segmentation, were performed by a person blinded to the biomechanical data and using the dedicated Flow Analysis software, version 2018-12-12. Data identified by an independent reviewer as non-reliable due to poor image or segmentation quality were excluded from the study. The instantaneous intracranial arterial flow Qa(t) was defined as the sum of blood flows in the intracranial internal carotid and basilar arteries. The instantaneous intracranial sinus flow Qs(t) was defined as the sum of blood flows in the straight and sagittal sinuses. It was assumed that during the whole cardiac cycle, the blood volume entering the cranial cavity through arteries was equal to the blood volume exiting it through veins. However, the straight and sagittal sinuses are not the only exit routes for blood. A correction factor was therefore defined to take the undetected blood volume exiting the cranial cavity into account:

αcorr = ∫ T0Qa(t)dt ∕ ∫ T0Qs(t)dt ,
where T is the cardiac cycle period. The total instantaneous venous flow was then estimated as Qv(t) = _corrQs(t). The instantaneous blood volume change was V(t) = ∫ t0Qa(t)dt -∫ t0Qv(t)dt . The maximal blood volume change was defined as ΔV =

max{V(t)}t∈[0,T] -min{V(t)}t∈[0,T] .

Statistical analysis

Descriptive analysis of quantitative parameters was performed with mean ± standard deviation (SD) and qualitative parameters with percentage.

The associations between quantitative independent and dependent variables were assessed by univariate and multivariate linear regressions. In univariate regression analysis, the associations were evaluated and any independent variate that displayed p-value < 0.05 in a given univariate analysis model was considered as a potential independent variable. In multivariate regression analysis, the independent variable selection was based on the preliminary univariate analysis.

Three models were tested with Hcy as the dependent variable. For each model and its variables, β-estimate, regression coefficients, adjusted regression coefficients, and p-values were reported. Multicollinearity was checked using the variance inflation factor.

The associations between qualitative independent variables and Hcy as the dependent variable were assessed by logistic regressions. To illustrate the logistic correlation, a forest plot was built from odds ratio.

To ascertain normality, continuous variables were inspected through histograms and Lilliefors tests.

Statistical analyses were performed using R version 4.0.2.

Results

Clinical and biochemical characteristics of the study population

After careful evaluation, 40 patients were excluded from the initial cohort of 100 patients, for low quality of PCMRI data, FI reliability criterion or missing data [START_REF] Vallet | Biomechanical response of the CNS is associated with frailty in NPH-suspected patients[END_REF]. This resulted in a final analytical sample of 60 patients, with statistical characteristics of demographic variables, FI and biomechanical parameters (Table 1). Concerning markers of neurodegenerative pathologies, 57% of patients had abnormal CSF dynamics and increased risk of NPH, 75% had increased risk of AD, 25% (of 53 patients) had increased risk of PD, and 62% (of 53 patients) had increased risk of vascular dementia. In addition, among 52 patients without missing markers, 4% presented no risks of any of the four diseases considered, 19% presented risks of one disease, 38% presented risks of two diseases, 35% presented risks of three diseases, and 4% presented risks of the four diseases (data not shown). In this cohort, there were 48.3% of women. Concerning environmental factors, among the patients, 24.6% and 26.3% respectively smoked and drunk alcohol. Regarding vascular risk factors, 43.3% were hypertensive, 28.3% diabetic and 28.3% had dyslipidemia. The CSF outflow resistance R 0 was observed to be higher than average due to the specific cohort suspected of NPH. For the metabolism profile, folates and cobalamins levels were in normal ranges. Despite the prevalence of dyslipidemia, the mean of different parameters in the lipid profile were in the norms. Moreover, the renal profile is relatively conserved for this cohort with multiple comorbidities. Finally, the inflammatory markers were also in normal ranges.

Hcy plasma level is independently associated with frailty index, the CNS elastance coefficient and age

Table 2 displays univariate regression analysis between all measured biochemical parameters and two factors, the CNS elastance coefficient and FI. CRP, Hcy and folates were associated with FI (p-value < 0.05) (Table 2). Hcy, LDL, total cholesterol and apolipoprotein B were associated with the CNS elastance coefficient (p < 0.05). Among all measured biochemical parameters, Hcy plasma level was the sole to be correlated with both the CNS elastance coefficient (Adjusted R² 6% and p-value <0.05) and FI (Adjusted R² 12% and p-value <0.05).

These associations are represented in Figure 1, where an increase in Hcy is associated with an increase in both the CNS elastance coefficient and FI. An increase in Hcy is also associated with an increase in age (Adjusted R² 23% and p-value <0.001).

Hcy plasma level is not associated with comorbidities and lifestyles

The association between Hcy plasma level with qualitative lifestyle parameters and comorbidities was measured by logistic regression analysis (Supplementary Figure 1). In this cohort of patients with NPH, Hcy plasma level was not different between males and females (OR = 0.989; 95% CI: 0.881 -1.105). Moreover, with the high frequency of cardiovascular risk factors in this cohort, links between high blood pressure (OR = 0.888; 95% CI: 0.755 -1.01), diabetes mellitus (OR = 0.96; 95% CI: 0.831 -1.082) and presence of dyslipidemia (OR = 0.968 ; 95% CI: 0.835 -1.094) with Hcy plasma level were measured and no link was found.

Concerning lifestyles with alcohol (OR = 1.057; 95% CI: 0.928 -1.2) and tobacco (OR = 1.091; 95% CI: 0.965 -1.246), Hcy plasma level was also independent of these two features. Finally, the Hcy plasma level was also independent on comorbidity risks, including increased risk of AD (OR = 1.11; 95% CI: 0.961-1.34), of PD (OR = 1.08; 95% CI: 0.936-1.246), of vascular dementia (OR = 1; 95% CI: 0.891-1.132) and of NPH (OR = 0.971; 95% CI: 0.866-1.088). Moreover, Supplementary table 2 displays the univariate regression analysis between R 0 with CRP, FI and the CNS elastance coefficient. No linear correlation was measured with R 0 (p-value > 0.05). No parameters depend on R 0 despite the patients' suspicion of NPH. We checked that the main counfounder related to our NPH suspected population, R 0 , is not such a confounder variable.

Hcy plasma level is independently associated with creatinine clearance and folate

Associations between Hcy plasma level and plasmatic biochemical parameters were measured by Pearson univariate linear regression (Supplementary Table 3 andTable 4). Concerning the serum lipid profile, no correlation was found (p-value > 0.05). For the metabolic profile, our data showed a link between folate level (p-value < 0.05) and Hcy plasma level contrary to cobalamin level (p-value > 0.05). In the renal profile, only creatinine clearance was correlated with homocysteine plasma level (p-value <0.001). Finally, for the inflammatory profile, Hcy and CRP were not associated (p-value > 0.05). Then, only folates levels (figure 2A) and creatinine clearance (figure 2B) were associated with Hcy plasma level. As shown in figure 2C, three models of multivariate regression analysis were performed from the significant biochemical parameters in figure 2A

Discussion

Our cohort is a convenient model of pathological brain aging with a wide range of degrees of frailty, suitable to assess biomechanical and biological parameters. Indeed, the NPH suspicion provides access to various parameters (CSF sample, ICP measure, PCMRI, …), especially in relation to neurodegenerative diseases. Moreover, with 43% of patients having an unconfirmed diagnosis of NPH, we are in the context of non-specific disease cohort. This fact is reinforced by the lack of correlation between R 0 and all parameters tested.

Here, we took a step back and focused on a potential biological parameter that might link frailty and CNS biomechanics in a group of patients presenting markers of multiple co-existing pathologies and suspected of NPH. Among the various biochemical parameters tested, Hcy was the sole parameter independently associated with both FI and biomechanical response of the CNS. Thus, our data suggest that the importance of Hcy may be a parameter involved in the shift from robust brain aging to pathological brain aging (Figure 3).

The first part of our work aimed to identify a potential plasma parameter associated with frailty. Among all the tested plasmatic biological parameters, we showed that FI is associated with Hcy, folates and CRP (Table 2). Our data are in concordance with the general view that clinical frailty is associated with inflammation [START_REF] Vatic | Inflammatory biomarkers of frailty[END_REF], as suggested from previous cross-sectional studies [START_REF] Álvarez-Sánchez | Homocysteine and C-Reactive Protein Levels Are Associated With Frailty in Older Spaniards: The Toledo Study for Healthy Aging[END_REF][START_REF] Wong | Homocysteine, frailty, and all-cause mortality in older men: the health in men study[END_REF] and meta-analysis [START_REF] Soysal | Inflammation and frailty in the elderly: A systematic review and meta-analysis[END_REF][START_REF] Mailliez | Circulating biomarkers characterizing physical frailty: CRP, hemoglobin, albumin, 25OHD and free testosterone as best biomarkers. Results of a meta-analysis[END_REF]. Among inflammatory parameters, CRP, hsCRP, interleukin-6 (IL-6) and Tumor necrosis factor-α (TNF-α) were shown to be significantly higher in frail subjects. Nevertheless, a recent study including 1394 adults aged 70 years and more showed that Hcy and CRP levels were not able to predict frailty for a period of 5 years, in an adjusted model [START_REF] Lu | Biological and Neuroimaging Markers as Predictors of 5-Year Incident Frailty in Older Adults: A Secondary Analysis of the MAPT Study[END_REF], and several studies observe different results on a possible correlation between CRP and frailty [START_REF] Gale | Inflammatory markers and incident frailty in men and women: the English Longitudinal Study of Ageing[END_REF][START_REF] Walker | Midlife Systemic Inflammation Is Associated With Frailty in Later Life: The ARIC Study[END_REF]. Differences in study designs, such as the use of different frailty scores or techniques to measure parameters, the recruitment of only men or women or differences in participants' ages, might explain these differences. Thus, further investigations would be needed to complete these latter studies in terms of biomarkers such as interleukins.

Then, we showed that impaired creatinine clearance, a marker of renal dysfunction, and folate are independently associated with Hcy. Concerning the association with folates, these results are in accordance with previous literature showing that a lack of folate can lead to HHcy [START_REF] Ganguly | Role of homocysteine in the development of cardiovascular disease[END_REF] and may be related to cognitive impairment [START_REF] Mccaddon | Assessing the association between homocysteine and cognition: reflections on Bradford Hill, meta-analyses, and causality[END_REF]. Deficiencies in vitamin B12 and folates are nutritional deficiencies that contribute to variations in Hcy metabolism, as these molecules are involved in the de novo synthesis of the methionine methyl group. Interestingly, it was demonstrated that folate and vitamin B12 supplementation reduce the level of Hcy in the serum of patients with mild cognitive impairment and improve cognitive function [START_REF] Jiang | The effect of folate and VitB12 in the treatment of MCI patients with hyperhomocysteinemia[END_REF]. Moreover, we found that Hcy is independently associated with creatinine clearance. These results are of interest as HHcy has been recognized to be associated with chronic kidney disease (CKD) [START_REF] Iacobazzi | Hyperhomocysteinemia: Related genetic diseases and congenital defects, abnormal DNA methylation and newborn screening issues[END_REF].

It was also recently demonstrated that Hcy is positively and independently associated with serum creatinine levels in male patients with hypertension [START_REF] Yang | Homocysteine level is positively and independently associated with serum creatinine and urea nitrogen levels in old male patients with hypertension[END_REF]. Interestingly, a recent metaanalysis confirmed that frail individuals had higher plasmatic creatinine levels than robust individuals [START_REF] Mailliez | Circulating biomarkers characterizing physical frailty: CRP, hemoglobin, albumin, 25OHD and free testosterone as best biomarkers. Results of a meta-analysis[END_REF].

Finally, we aimed to determine whether some biochemical parameters measured in plasma could be associated with the CNS elastance coefficient. In a recent work, we found a significant association between the CNS elastance coefficient and FI [START_REF] Vallet | Biomechanical response of the CNS is associated with frailty in NPH-suspected patients[END_REF]. Noteworthy, Phillip et al. already suggested that aging was not only accompanied by major biochemical changes, but also biomechanical alterations, including increased stiffness, at macroscopic and microscopic length scales [START_REF] Phillip | The Mechanobiology of Aging[END_REF]. Consistently, as illustrated in Table 2, we showed that the CNS elastance coefficient is associated with biochemical parameters, including Hcy, LDL-Cholesterol, total-Cholesterol and Apolipoprotein B, but not CRP. Thus, we demonstrated that Hcy is independently associated with both FI and CNS biomechanics, as quantified by the CNS elastance coefficient.

Then, by integrating all the biochemical and biomechanical parameters, we proposed a representative model of the variability of Hcy (adjusted R² 56%) that is composed of the CNS elastance coefficient, age, FI, creatinine clearance and folate level (Figure 2C). Interestingly, this model does not include CRP, a parameter that is only associated with FI in our cohort.

These results suggest that whereas low-grade inflammation could be associated with frailty, only Hcy seems to be linked with both renal and brain disorders. While these correlations are not sufficient to prove a causal relationship between Hcy, FI and the CNS elastance coefficient, they nevertheless raise the question of the possible underlying pathways biological plausibility by which Hcy could result in increased vulnerability of these organs. In the brain, Hcy is linked to neurodegeneration, notably by modulating PP2A activity and tau phosphorylation, and to neuroinflammation by inducing inflammatory cytokine such as TNF-α and IL-1β [START_REF] Zhang | Homocysteine induces tau phosphorylation by inactivating protein phosphatase 2A in rat hippocampus[END_REF][START_REF] Moretti | Homocysteine in Neurology: A Possible Contributing Factor to Small Vessel Disease[END_REF]. The neurotoxicity of Hcy was also shown to be mediated by the NMDA and metabotropic glutamate receptors [START_REF] Lipton | Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor[END_REF][START_REF] Ziemińska | Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones[END_REF][START_REF] Yeganeh | Neuroprotective Effects of NMDA and Group I Metabotropic Glutamate Receptor Antagonists Against Neurodegeneration Induced by Homocysteine in Rat Hippocampus: In Vivo Study[END_REF]. It is also well known as a cardiovascular risk factor, in particular involved in kidney microvascular dysfunction.

It can thus be hypothesized that Hcy induces damage of small blood vessels. Thus, high Hcy levels is now studied as a possible contributing factor to microvascular dysfunction and cerebral small vessel disease (43) by modulating inflammation, oxidative stress, immune activation and endothelial dysfunctions [START_REF] Moretti | Homocysteine in Neurology: A Possible Contributing Factor to Small Vessel Disease[END_REF][START_REF] Hainsworth | Homocysteine, hyperhomocysteinemia and vascular contributions to cognitive impairment and dementia (VCID)[END_REF][START_REF] Nowroozpoor | Is microvascular dysfunction a systemic disorder with common biomarkers found in the heart, brain, and kidneys? -A scoping review[END_REF]. Notably, HHcy was well described to induce oxidative damage by ROS accumulation [START_REF] Moretti | Homocysteine in Neurology: A Possible Contributing Factor to Small Vessel Disease[END_REF]. Indeed, longer HHcy exposure induces a downregulation of endothelial nitric oxide synthase (eNOS), leading to the decrease in L-citrulline and nitrite formation, and then to oxidative damages [START_REF] Jiang | Hyperhomocystinemia Impairs Endothelial Function and eNOS Activity via PKC Activation[END_REF]. Another mechanism that contributes to vascular inflammation mediated by Hcy concerns the homocysteinylation process [START_REF] Jakubowski | Homocysteine Modification in Protein Structure/Function and Human Disease[END_REF]. Nhomocysteinylation is a post-translational modification that alters protein structure, function, causes protein aggregation, as well as protein misfolding, and induces apoptosis [START_REF] Jakubowski | Homocysteine Modification in Protein Structure/Function and Human Disease[END_REF].

Interestingly, it was also shown that treatment of microvascular endothelial cells derived from mouse brains with Hcy leads to the decrease of growth factor such as VEGF, FGFα and leptin, and increase of the matrix metalloproteinase MMP-9 [START_REF] Shastry | Homocysteine induces metalloproteinase and shedding of ?-1 integrin in microvessel endothelial cells[END_REF]. Finally, in this same study, by modulating β1 integrin abundance at the plasma membrane, Hcy also disturbed the extracellular matrix integrity in the intimal layers around endothelial cells, and potentially their biomechanical properties.

Moreover, microvascular dysfunction is a leading cause for renal dysfunction, which may thus explain the association between Hcy level and creatinine clearance. It was indeed previously shown that impaired kidney function, as measured by decreased glomerular filtration rate, is related to subclinical markers of cerebral small vessel disease, independent of cardiovascular risk factors [START_REF] Ikram | Kidney Function Is Related to Cerebral Small Vessel Disease[END_REF]. From a biomechanical point of view, we can hypothesize that the consequences of increased Hcy described above, such as the loss of neural cells, misfolded protein aggregation, microvascular dysfunction or vessel wall alteration could modify the mechanical properties of both the brain parenchyma and the cerebro-vascular system, resulting in an increased CNS elastance coefficient [START_REF] Vallet | Biomechanical response of the CNS is associated with frailty in NPH-suspected patients[END_REF].

All together, we hypothesize from the statistically significant results that pathological brain aging, pathological kidney aging and frailty share common underlying biological processes, which could involve microvascular dysfunction. Consistently, CRP, which is not associated with the CNS elastance coefficient nor creatinine clearance, was not previously found to play a role neither in brain nor in renal microvascular dysfunctions.

The included patients were suspected of NPH. However, 43% of patients had normal CSF dynamics, demonstrating that about half of our population was not hydrocephalic. Moreover, 75% had increased risk of AD, 25% of PD and 62% of vascular dementia, demonstrating that our population was wider than NPH. Hence, the associations between Hcy, FI and the CNS elastance coefficient seems not to be specific to NPH and the other neurological diseases and makes acceptable the absence of a non NPH control group [START_REF] Vallet | Biomechanical response of the CNS is associated with frailty in NPH-suspected patients[END_REF][START_REF] Cesari | The cognitive impairment of frail older persons[END_REF].

Despite convincing results, the study showed some limitations. First, our study did not provide information on the relevance of our approach in the preclinical stage as all patients presented clinical symptoms. Second as the number of cases is small, it will be necessary to confirm our results in a larger cohort. Third, in the statistical study, the cross-sectional design of the study did not allow resolving the directionality of the association between Hcy, FI and the CNS elastance coefficient. Fourth, concerning the biochemical exploration of plasma, we did not analyze other inflammatory parameters such as IL-6 and TNF-α, we cannot conclude on a link between the inflammasome and the biomechanical response of CNS in NPH suspected patients.

Then, further investigations are needed to argue in favor of a causality according to Bradford Hill criteria [START_REF] Hill | The Environment and Disease: Association or Causation?[END_REF].

In conclusion, our exploratory study demonstrated that Hcy level is positively associated with FI and the CNS elastance coefficient, providing a potential biological link between pathological brain aging and a biomechanical response of the CNS. The biological plausibility at the origin of the different correlations described above are reported (Figure 3). In a recent perspective article, Fried and Coll. raised the question of whether there is a shared biological driver of multi system dysregulation in parallel in physical frailty [START_REF] Fried | The physical frailty syndrome as a transition from homeostatic symphony to cacophony[END_REF]. Our results reinforce the interest to work on the role of Hcy at the mechanistic level in order to understand whether Hcy could be one of these drivers. Notably it will be interesting to explore how modulation of Hcy homeostasis supports a possible causality in the frailty-related increase in the CNS elastance coefficient.

Future works could use global approaches such as OMICs or experimental models in order to generate comprehensive data that help to understand the biological mechanisms underlying the transition from normal to pathological aging of the brain.

  and 2B, the biomechanical response and the clinical response. The model A associated Hcy plasma level with the CNS elastance coefficient (pvalue < 0.05). The model B added on top the folates and the creatinine clearance (p-value <0.001). Finally, the model C added on the top the age and the FI. The model C was the most representative model of the variability of Hcy (p-value < 0.001), with an adjusted R² of 56%.

Figure 1 :Figure 2 :

 12 Figure 1: (A) Linear relations between FI and homocysteine (R² = 0.1233; p-value = 0.014*), between (B) the CNSa elastance coefficient and homocysteine (R² = 0.0773; p-value = 0.044*) and (C) between age and homocysteine (R² = 0.2435; p-value = 1.734 E-04***). R² and p-value lead to describe the Pearson univariate regression analysis. Linear regressions (blue lines) and 95% confidence intervals (grey bands) are indicated.Homocysteine level is expressed in logarithmic form to respect the normality condition.aCNS: Central Nervous Systemp-value < 0.05: * ; p-value < 0.01: **; p-value < 0.001: *** 33x63mm (300 x 300 DPI)

Figure 3 :

 3 Figure 3: Known associations between molecular and cellular roles of homocysteine and their interplay with the biomechanical response of the central nervous system (CNS) and frailty index 1 vascular inflammation/endothelial dysfunction; 2 neurotoxicity; 3 glial activation; 4 endoplasmic reticulum / oxidative stress; 5 blood pulsatility; <----------> correlation

Table 1 :

 1 Characteristics of subjects. Continuous variables were presented with mean, standard deviation and normal range and categorical data were presented as the percentage of deficit.

		Quantitative parameters		Qualitative parameters
			Mean
		Parameter (unit)	(Standard	Normal range	Parameter	% score = 1	Score
			deviation)
		Age (years)	74.22 (6.58)	-	Sex	48.3	0: man / 1: woman
	Clinical features
		Frailty index	0.324 (0.142)	-	Tobacco	24.6	0: no / 1: yes
		Creatinine clearance (mL/min)	75.93 (25.38)	[90-140]	Alcohol	26.3	0: no / 1: yes
		Plasmatic creatinine (μM)	77.83 (20.91)	[60-115] M c / [45-105] W d	High blood pressure	43.3	0: no / 1: yes
		Blood urea nitrogen (mM)	6.85 (2.59)	[2.5-7.5]	Diabetes mellitus	28.3	0: no / 1: yes
		Uric acid (μM)	313.03 (10.21)	[180-420] M c / [150-360] W d	Dyslipidemia	28.3	0: no / 1: yes
		Apolipoprotein A1 (g/L)	1.42 (0.34)	[1.1-2] M c /[1.2-2.22] W d	Alzheimer disease	75	0: no/ 1: yes
		Apolipoprotein B (g/L)	0.96 (0.22)	[0.55-1.35] M c / [0.55-1.25] W d	Parkinson disease	25	0: no/ 1:yes
		Very Low Density Lipoprotein (mM)	0.61 (0.26)	[0.1-1.7]	Vascular dementia	62	0: no/ 1:yes
	Biochemical parameters	Low Density Lipoprotein (mM)	2.83 (0.92)	<4.1	Normal pressure hydrocephalus	57	0: no/1: yes
		High Density Lipoprotein (mM)	1.43 (0.51)	>1.0
		Total cholesterol (mM)	4.87 (1.04)	[4.1-5.2]
		Triglycerides (mM)	1.37 (0.64)	[0.4-1.7]
		Plasmatic homocysteine (μM)	13.61 (5) <15
		C reactive protein (mg/L)	3.01 (3.77)	<5
		Cobalamins (pg/mL)	402.02 (163.81)	[200-900]
		Folates (ng/mL)	8.6 (12.87)	[5-15]
	Biomechanical	CNS a elastance coefficient (1/mL)	0.511 (0.129)	-
	parameters
		CSF b outflow resistance (R0)	14.92	-

Table 2 :

 2 Biochemical parameters associated with the CNS elastance coefficient and frailty index. Parameters associated were described by Pearson univariate correlation with R²,

	adjusted R² and p-value
	p-value < 0.05: *

Table 1 :

 1 Variables used in the construction of the frailty index a UPDRS: Unified Parkinson's Disease Rating Scale / b SEGA: short emergency geriatric assessment / c IPSS: International Prostate Score Symptom / d MNA: Mini Nutritional Assessment

	Supplementary data
	Numb er	Parameters	Conditions for deficit
	Executives functions
	1	Dependence for dressing
	2	Dependence for hygiene
	3	Handwriting	UPDRS a item score >=2
	4	Speech
	5	Transferring
	6	Chewing and swallowing
	7	Doing hobbies and other activities
	Mood and cognition
	8	Cognitive impairment	UPDRS a item score >=2
	9	Depressed mood	SEGA b item > 1
	10	Apathy	UPDRS a item score >=2
	11	Self-reported health compared to others	SEGA b item > 1
	12	Night-time sleep problems	UPDRS a item score >=2
	13	Anxious mood
	Gait
	14	Walking and balance	UPDRS a item score >=2
	15	Freezing	UPDRS a item score >=2
	16	Gait
	17	Global spontaneity of movement
	Rigidity and agility of extremities
	18	Tapping fingers
	19	Tapping toes	UPDRS a item score >2 left + right
	20	Rigidity upper extremities
	21	Rigidity lower extremities
	22	Hand movements

Journal of Gerontology: Biological Sciences

Acknowledgements

We would like to acknowledge Pr. Pascal Swider, Institute of Fluid Mechanics of Toulouse for helpful discussions.

Funding This work was supported by a grant from the Clinical Research Hospital Program from the French Ministry of Health (PHRC 2011-A01091-40), by the Occitania Region research funding (RPBIO 2015 n°14054344), by the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013).

Conflict of interest

The authors declare no conflict of interest.


W Liu

F Morfin

K Provost

M Bahri

W Baaziz

O Ersen

L Piccolo

C Zlotea

Unveiling the Ir single atoms as selective active species for the partial hydrogenation of butadiene by operando XAS

Keywords: Ir-single atom catalyst, operando XAS, butadiene partial hydrogenation, EXAFS, XANES

Single-atom catalysts represent an intense topic of research due to their interesting catalytic properties for a wide range of reactions. Clarifying the nature of the active sites of single-atom catalysts under realistic working conditions is of paramount importance for the design of performant materials. We have prepared an Ir single-atoms catalyst supported on a nitrogen-rich carbon substrate that proven a substantial activity toward hydrogenation of butadiene with nearly 100% selectivity to butenes even at full conversion. We evidence here, by quantitative operando X-ray absorption spectroscopy, that the initial Ir single atoms are coordinated with four light atoms i.e., Ir-X4 (X = C/N/O) with an oxidation state of +3.2. During the pre-treatment under hydrogen flow at 250 °C the Ir atoms loses one neighbour (possibly oxygen) and partially reduces to an oxidation state around +2.0. We clearly demonstrate that Ir-X3 (X = C/N/O) are the active species with very good stability under reactive conditions. Moreover, Ir single atoms remain isolated under reducing atmosphere at a temperature as high as 400 °C.

Introduction

Heterogeneous catalysis is the most important industrial process for producing large-scale chemicals and energy products nowadays. However, the design of catalysts with high efficiency, selectivity and stability remains a major challenge. [START_REF] Li | [END_REF]2 Recently, single-atom catalysts (SACs), which contain isolated atoms dispersed on a support, have gained considerable attention due to their maximized atomic utilization and unique electronic properties. [3][4][5][6][7][8] Because of the changes in the active-site structure in comparison with metal nanoparticles or nanoclusters, SACs are expected to have distinct behaviours in catalysis, such as high selectivity and stability. [9][10][11] Since the first practical report of SACs (Pt/FeOx) by Qiao et al 12 in 2011, significant progress has been made in this field. [13][14][15][16] SACs have shown superior catalytic performance (activity and/or selectivity) for several industrial relevant reactions. [17][18][19] Selective hydrogenation reactions play a key role in the field of sustainable chemistry. The over-hydrogenation of the desired product remains a major issue for these reactions. 20 Among them, refining of C4 cuts from steam cracking by catalytic selective hydrogenation is an important industrial process. Butene products, 1-butene and 2-butenes, are intermediates used in industry for polymerization and alkylation processes, respectively. [START_REF] Derrien | Studies in Surface Science and Catalysis[END_REF][START_REF] Yang | [END_REF][23] The presence of 1,3-butadiene (0.3% to 6%) in C4 species is considered as a poison which causes unwanted side reactions and deactivates the catalysts. 24 Commonly, catalysts based on Pd and Pt are widely used due to their high activity [25][26][27][28][29] but their selectivity to butene is limited at high butadiene conversion. [START_REF] Wang | Selective Hydrogenation of Butadiene over Non-noble Bimetallic Catalysts[END_REF] Over the last few years, researchers have reported the selective hydrogenation of 1,3butadiene on SACs which showed promising catalytic performance. For example, Lucci et al. [START_REF] Lucci | [END_REF] reported that low concentration Pt single atoms supported on a Cu surface showed 90% selectivity to butenes at full conversion and the catalyst remained stable over 2 days at 160 °C.

Through a DFT study, Lv et al. 32 showed that Pt single atoms adsorbed on Cu surface [Pt1/Cu(1 1 1)] has a higher selectivity toward 1-butene than a clustered Pt-content catalyst [Pt4-line/Cu(1 1 1)]. Yan et al. 33 compared an atomically dispersed Pd1/graphene catalyst with 3 different Pd carbon-supported nanocatalysts. The SAC showed a high activity by retaining 100% butenes selectivity up to 95 % conversion while the selectivity of Pd nanocatalysts dropped sharply at high 1,3-butadiene conversion. Furthermore, Zhang et al. 34 found that a very low concentration (0.08 wt.%) of isolated Au 3+ on a ZrO2 support is highly selective in the hydrogenation of 1,3-butadiene to butene isomers. However, Ir single atom catalysts have never been reported for this reaction.

A fine description of the active species of single-atom catalysts and their local and electronic structures are very scarcely identified, whatever the reaction targeted. Only very recently, Fang et al. 35 proposed the evolution of Pt single-atoms into a near-free state as active species in the electrocatalytic hydrogen evolution reaction. They revealed that during the reaction, the single Pt atom tends to dynamically release from the nitrogen-carbon substrate, with the structure less coordinated to the support and the electronic properties close to zero valence.

In this work, we report the synthesis and characterization of Ir single-atoms dispersed on a nitrogen-rich carbon substrate and their catalytic activity for the partial hydrogenation of butadiene. To reveal the active species of the Ir SAC and their local and electronic structures under working conditions, we have carried out operando XAS experiments. For the sake of comparison, Ir nanocatalysts have been also investigated under the same conditions.

Experimental methods

Catalyst preparation

We have prepared Ir single-atom catalyst (Ir-SAC) supported on a nitrogen-doped carbon starting from a commercial porous activated carbon (AC with a specific surface area of 1400 m 2 /g purchased from STREM Chemicals) as initial substrate. The initial AC contains small amount of impurities, mainly aluminosilicates, that are noticeable in the XRD patterns as sharp peaks. The Ir-SAC was prepared by a cascade anchoring strategy adapted from Zhao et al. 36 The Ir atoms were dispersed on a activated carbon in a nitrogen-rich carbonaceous medium by the liquid impregnation of the pristine AC with IrCl3•xH2O precursor in aqueous solution in the presence of a chelating agent (ethylenediaminetetraacetic acid: EDTA) and a source of nitrogen (melamine) followed by pyrolysis under Ar flow at 800 °C.

For the sake of comparison, we have prepared Ir nanocatalysts (Ir-NC) in the form of well dispersed nanoparticles by a simple method based on the liquid-phase impregnation of the same AC support with the IrCl3•xH2O precursor in aqueous solution, followed by reduction under 5%H2/Ar flow at 800 °C, as described earlier. 37

Synthesis of Ir-SAC

First, 400 mg of EDTA (Sigma-Aldrich), used here as chelating agent, was dispersed in 10 mL distilled water and NH3•H2O was added to adjust the solution p-H to dissolve the EDTA. Then, 0.0213 mmol of IrCl3•xH2O (Alfa Aesar) and 200 mg of AC were added to the solution and the mixture was stirred and evaporated at 60 °C. The metal ions are thus chelated by EDTA then anchored onto the carbon support by solvent evaporation. Moreover, the excessive chelating agent bound to the support surface will physically isolate the metal complex. The recovered solid was dried at 70 °C overnight. During the second step, the dried carbon-based powder was hand ground with 1000 mg of melamine (Sigma-Aldrich), as a nitrogen source for the subsequent step.

The final stage of the synthesis consists in the pyrolysis of the obtained mixture at 800 °C under Ar flow for 2 h in a furnace (ramp 3 °C/min, 200 mL/min). During this step, the chelated metal complex can react with carbon nitrogen species CNx (such as C3N4 etc.) decomposed from melamine at high temperature (> 600 °C) and subsequently metal-Nx moieties are formed, thus preventing metal atoms from aggregation. The Ir single atoms are finally anchored in a nitrogenrich carbon substrate. The as-prepared Ir-SAC powder was recovered and used without any further treatment.

The elemental analysis of the Ir-SAC sample was determined by ICP-OES and CHNS techniques.

The metal amount is 1.0 wt.% for Ir-SAC, while N concentration reaches 14.1 wt.% after the synthesis which is higher than 0.5 wt.% found in the initial AC. Additional results from the elemental analysis of the as-prepared samples are shown in table SI1.

Synthesis of Ir-NC

For the Ir nanocatalyst preparation, 300 mg of AC and 0.065 mmol of IrCl3•xH2O were dispersed in 10 mL distilled water and the solution was stirred and evaporated at 60 °C. The mixture was then dried at 70 °C overnight. The dried powder was reduced under 5% H2/Ar (0.5 L/min) at 800 °C for 2 h. The as-prepared powder material was further used without any treatment. The metal amount is 3.7 wt.% for Ir-NC, as determined by ICP-OES.

Physicochemical characterizations

The structural properties of the samples were characterized by powder X-ray diffraction (XRD) using a D8 Advance Bruker diffractometer (Cu Kα radiation  = 1.5406 Å, Bragg-Brentano geometry).

The samples were characterized by Transmission Electron Microscopy (TEM) with a 200 kV FEG microscope (FEI Tecnai F20 equipped with a Gatan Energy Imaging Filter, resolution 0.24 nm).

A small amount of sample was ultrasonicated in ethanol solvent, then small drops of suspension were placed on carbon grids. The Ir nanoparticles size distribution and the average size were determined by statistical analyses of several TEM images.

The Ir-SAC specimen was analysed by high-angle annular dark-field imaging in scanning transmission electron microscopy (HAADF-STEM) using a probe corrected JEOL 2100F electron microscope operating at 200 kV. This imaging technique was used to characterize the as-prepared Ir-SAC sample as well as to assess the single atoms stability against coalescence after treatment at high temperature. The thermal treatments were carried out by heating the sample at 250 °C and 400 °C with 10°C/min under 5%H2/Ar flow (200 ml/min) in an external resistive furnace.

Catalytic test

The hydrogenation of 1,3-butadiene on Ir-SAC and Ir-NC samples was carried out under atmospheric pressure in a continuous flow fixed-bed stainless steel reactor. The reactor was placed in a ceramic furnace with temperature control. The temperature of the catalyst was recorded via a thermocouple inserted in the catalytic bed. Before the reaction, 40 mg of catalyst was mixed with 100 mg of α-Al2O3 (8 m 2 /g, purity 99.95% from Alfa-Aesar) and activated in situ with hydrogen (70 ml/min) by heating from room temperature to 250 °C at a heating rate of 4 °C/min and remained at 250 °C for 1 h. Then the reactor was cooled down to 25 °C under H2.

The feed flow rate was set to 100 ml/min. The reactive gases (C2H2:H2:He) were mixed using mass flow controllers (Brooks and Vögtlin) with a composition of 2:10:88%. The temperature was heated to 200 °C with a ramp of 1 °C/min and remained at 200 °C for 20 minutes under reactive mixture. The feed and effluent gases were analysed online using Shimadzu GC-2014 gas chromatograph equipped with a Supelco alumina sulphate plot coupled with an FID detector.

Operando XAS

To check the local and electronic structures of Ir-SAC and Ir-NC samples during the hydrogenation of butadiene, operando X-ray absorption spectroscopy (XAS) experiments coupled with mass spectroscopy were carried on the ROCK beam line 38 at the SOLEIL synchrotron. The Xray absorption spectra at the L3 edge of Ir (11.215 eV) were measured and metal Ir foil was used as reference. The powder samples were placed inside a Lytle-type cell, which was connected to a gas distribution system that controls the gas flow composition at atmospheric pressure. The experiment followed as close as possible the laboratory reaction conditions. Prior to each experiment, the samples were pre-treated to remove contaminants at the surface by heating to 250 °C (4 °C/min) under continuous H2/He flow (15 ml/min H2 and 15 ml/min He) followed by a dwell at 250 °C for 1 h and then cooled down to 25 °C. Afterward, the catalysts were exposed to the reactive gas mixture (35 mL/min): 0.5% C4H6 + 2.5% H2 + 97% He and the temperature was raised to 200 °C with a ramp of 1 °C/min. The temperature was maintained at 200 °C for 1 h under reaction conditions. The XAS measurements were coupled with continuous mass spectrometry (MS) to identify the reaction products.

All XANES spectra were calibrated in energy and normalized. The XAS data treatment and EXAFS refinement were performed with the MAX program package (E0 = 11215 eV for Ir L3 edge, Fourier Transform 4-15 Å -1 ). 39,40 EXAFS fitting was performed on the first sphere filtered spectra.

Theoretical phases and amplitudes were computed with FEFF8 based on Ir-X4 structure. The refined parameters are the coordination number (N), the Debye-Waller factor (σ 2 ) and the nearest neighbour distance (R). The energy shift ΔE0 was first refined for the Ir reference and then fixed for the subsequent refinements. The goodness of fit was evaluated using the quality factor (QF).

Results and discussions

XRD patterns of the as-prepared Ir-SAC and Ir-NC samples showed only the contribution from the starting activated carbon (AC) support, as shown in Figure 1A. Moreover, no diffraction peaks from the face centred cubic (fcc) lattice of bulk Ir metal were observed in both samples. This suggests that Ir is well dispersed on the carbon host either as small clusters/nanoparticles with too short coherence length to diffract X-rays 41 or as single atoms.

Figure 1. (A) XRD patterns of the starting activated carbon (AC) support together with the as-prepared Ir-SAC and Ir-NC samples; The angular positions of the expected diffraction peaks (111) and (200) of fcc bulk Ir metal are indicated by dashed lines. (B) HAADF-STEM image of Ir-SAC and (C) TEM image of Ir-NC with an average size of 1.3 nm (size histogram in inset).

To check the dispersion of Ir in both as-prepared samples, TEM/STEM characterizations were carried out (Figures 1B and1C). HAADF-STEM images of the Ir-SAC sample clearly shows isolated bright spots homogeneously dispersed on the support (figure 1B). We hypothesize that these spots are Ir single atoms, as this will be confirmed later by XAS. In the light of the above XRD and STEM-HAADF investigations and the chemical properties of the as-prepared Ir-SAC (1.0 wt.% of Ir and ~ 14 wt.% of N), this sample consists of Ir single-atoms on a nitrogen-rich carbon substrate, as also proposed by Zhao et al. 36 On the contrary, Ir nanoparticles can be clearly visualized on the carbon support for the Ir-NC sample (Figure 1C). The mean size of Ir nanoparticles is 1.3 (±0.5) nm, in agreement with the literature for similar synthetic conditions. 42 Additional HAADF-STEM and TEM images for the asprepared samples are shown in figure SI1. The conversion and selectivity of Ir-SAC and Ir-NC during the hydrogenation of butadiene reaction are shown in Figure 2. Butadiene conversion is a slightly higher for Ir-NC, however Ir-SAC shows an impressive nearly 100% selectivity to butenes in the whole temperature range (25-200 °C). The conversion increases steadily with the temperature and reaches 80% at 200 °C. Ir-NC behaves differently with high selectivity at low temperature and low conversion (below 30%).

However, when the conversion exceeds 30% a sudden drop of the selectivity occurs to almost full hydrogenation to butane at high temperature. As shown in Figure 3A, the Ir L3-edge XANES spectra are characterized by a white line, which corresponds to transitions from the occupied 2p3/2 core electron level to empty 5d states, thus probing the local density of unoccupied final states in the system. 43 It is obvious from Figure 3 that the white line of Ir-SAC is comparable to that of IrO2 and more intense than the one of Ir-NC.

The white line of the latter catalyst is close to the one of Ir bulk metal. The increase of the intensity of the white line from Ir-NC to Ir-SAC indicates a larger unoccupancy of 5d states in the Ir-SAC which can be explained by the hybridization between Ir and C/N/O atoms from the support.

The above-mentioned white line features can be also understood in terms of oxidation state of the catalyst. The XANES spectra can be exploited to determine the oxidation state more precisely. Hambley et. al proposed a simple mathematical method to calculate the oxidation state using the following equation 44 :

𝑂𝑆 𝑠𝑎𝑚𝑝𝑙𝑒 = [( 𝑎 𝑏 ) 𝑠𝑎𝑚𝑝𝑙𝑒 -( 𝑎 𝑏 ) 𝐼𝑟 𝑏𝑢𝑙𝑘 ] / [( 𝑎 𝑏 ) 𝐼𝑟𝑂 2 -( 𝑎 𝑏 ) 𝐼𝑟 𝑏𝑢𝑙𝑘 ] × 4,
where a is the intensity of the white line and b is the gap of the adsorption spectra as shown in Figure SI2. Thus, the oxidation state of Ir in Ir-SAC and Ir-NC were calculated accordingly and compared to those of well-known references: 0 and +4 for Ir metal and IrO2, respectively (Figure SI2). Nevertheless, it is worth noting that no Ir-Ir scattering peak (around 2.6 Å) is visible in the asprepared Ir-SAC sample, which clearly confirms the atomic dispersion of Ir, in good agreement with HAADF-STEM investigations (Figure 1B).

The XANES spectra of Ir-SAC during the pre-treatment at 250 °C (under H2/He flow) and under operando conditions at 200 °C (under reactive gas mixture) are illustrated in Figure 4A andB, respectively. The intensity of the white line smoothly decreases during the pre-treatment heating and stabilizes when reaching 250 °C (Figure 4A). Under reactive conditions, only a very slight variation of the white line intensity can be noticed with increasing and stabilisation of the temperature at 200 °C (Figure 4B). To monitor the change of the electronic properties during pretreatment and under operando conditions we applied Humbley's approach for all XANES data and the calculated oxidation of state is given in Table 1. To reveal the local structure of Ir-SAC during pre-treatment and under reactive conditions, we performed EXAFS refinements based on a structural model where iridium atom is coordinated to four in-plane light atoms Ir-NxCyOz (x + y +z = 4), as previously proposed in the literature. 46,47 The EXAFS fitting results such as, the coordination number (N), the Debye-Waller factor (σ²) and the nearest neighbour Ir-X distance (R) are listed in Table 1. Typical refinements results are plotted in Figure SI3.

The evolution of the coordination number, the nearest neighbour RIr-X distance and the oxidation state during hydrogenation of butadiene are plotted in Figure 5. It is obvious that the pre-treatment under H2/He gas mixture at 250 °C strongly affects the single-atom catalyst state whereas only minor changes can be observed under reactive conditions. Therefore, the following discussion will firstly focus on the initial state and the pre-treatment effect on the Ir-SAC and secondly on the catalyst under working conditions.

Pre-treatment cooling

H2/He 34.1 the coordination number (N), the Debye-Waller factor (σ 2 ), the nearest Ir-X (X = N,C,O) distance (R) and the quality of the fit (QF). The values of oxidation state (OS) from XANES analysis are also given.

Introduction of reactive gas mixture

Initially, the Ir atoms are surrounded by four light atoms (N/C/O) and the valence state is +3.2 (Table 1), which is similar to Ir 3+ in iridium (III) porphyrin and phthalocyanine complexes. [48][49][50] During the pre-treatment under H2 gas at 250°C, the coordination number reduces from 4.0 (0.1) to 2.9 (0.1), as illustrated in Figure 5A. This three-fold configuration has been already reported for Cu, Mn and Co single atom catalysts supported on carbon nitride based materials, [51][52][53] as well as Ir (II) complexes ( R N4)Ir(COD) 2+ , where Ir was found to be coordinated with three nitrogen atoms. 54 Moreover, Fang et al. 35 found that Pt in Pt1/N-C SAC shows a diminution of the coordination number (from 4 to 2) and the oxidation state (from + 1.89 to + 1.12) under electroreduction conditions, as evidenced by operando XAS. 35 The authors hypothesized that the high catalytic activity of the electrochemical hydrogen evolution reaction was mainly due to this evolution of the electronic properties and of the local arrangement of the atoms.

The coordination number reduction is accompanied by the decrease of the RIr-x distance from 2.016 to 1.996 Å (Figure 5B) during the pre-treatment. A first explanation would be that the decoordination allows a stronger interaction between the remaining neighbours and therefore smaller RIr-x distances as for C-C bond whose length is reduce form 1.54 Å to 1.34 Å and 1.20 Å for the single, double and triple C-C bond respectively. A second explanation is based on the decoordination of one longer Ir-X distance amongst the initial four neighbouring atoms. Previous literature reported smaller bond lengths of M-N as compared to M-O in metal SACs. For example, the distances Cu-N and Cu-O were found to be 1.91 Å and 2.01 Å, respectively, in a Cu SAC supported on carbon. 55 In a ZnNx/C catalysts, the distances Zn-N and Zn-O are 2.00 Å and 2.14 Å, respectively. 15 Furthermore, the Ir-N bond length in an Iridium Pincer complex, (NCN)Ir(CH2CH3)Cl(OH2), is 2.051 Å which is significantly shorter than 2.259 Å for Ir-O. [START_REF] Wingard | PhD thesis Accessing Intermediate and High Oxidation States with Tungsten and Iridium Pincer Complexes[END_REF] Therefore, we hypothesize that RIr-N is lower than RIr-O in Ir-SAC supported on nitrogen doped carbon and consequently, this catalyst might lose one oxygen neighbour under reductive atmosphere. Assuming that the initial configuration is Ir-NxCyOz (x + y + z = 4), we hypothesize that it evolves to Ir-NxCyOz (x + y + z = 3) during the reduction pre-treatment. Moreover, a diminution of the oxidation state from +3.2 to +2.0 is also noticed during pre-treatment pointing out to a partial reduction under these conditions (Figure 5C). Therefore, these three parameters (coordination number, RIr-x and oxidation state) are varying concomitantly pointing out that the reduction pretreatment might imply the loss of one nearest neighbour around Ir atoms (possibly oxygen) accompanied by a decrease of both the average distance RIr-X and the oxidation state.

The coordination number, the RIr-x distance and the oxidation state slightly increase with the introduction of the reactive gas mixture at 25 °C. They continue to slightly raise during the temperature ramp up to around 80 °C which might be an artefact due to the gradual arrival of butadiene in the reactor. This is followed by a smooth decrease at higher temperature up to 200 °C that might be the evidence of the adsorption of carbonaceous reactants and products in the vicinity of Ir single-atoms. The decrease at high temperature is consistent with a classical decrease of the reactant/products surface coverage when the temperature increases. At steady reactive conditions at 200 °C and during cooling to 25 °C, these three parameters are stable, within the error bar proving that Ir SAC is exceptionally stable during the hydrogenation reaction.

It is interestingly to notice the matching evolutions of the coordination number, the nearest neighbour distance, and the oxidation state, irrespective of the gaseous environment and temperature. This is surprising since these values are extracted from different parts of the XAS spectrum: the coordination number and the nearest neighbour distance are determined from the EXAFS fitting requiring a structure input and the oxidation state is calculated from the XANES data without structural model. This relationship among two different sets of data confirms our robust data treatment and supports our following conclusion: Ir atoms surrounded by three light elements (N, C, O) are the main reactive species in the hydrogenation of butadiene.

It is obvious from these experiments that the geometric and the electronic state of the asprepared Ir-SAC is different from the pre-treated (before reaction) and final (after reaction) states.

This variation is mainly reflected by the reduction of the intensity of both the white line in the XANES spectra and the Fourier Transform modulus of the k²-weighed EXAFS signals from the initial state to the catalyst before and after reaction (Figure 6). Moreover, it is important to notice that Ir-SAC is stable during reaction without coalescence and formation of metallic aggregates since no additional peak at 2.6 Å (typical for Ir-Ir nearest distance) is observed after reaction (Figure 6B). The outstanding thermal stability of Ir single atoms will be addressed separately in a next paragraph. Moreover, no significant change of Ir-NC could be noticed when introducing the reactive gas mixture as well as during reaction. Selected XANES, EXAFS spectra and corresponding fits for Ir-NC are displayed in Figure SI4.

The gaseous product distributions during operando XAS experiments of both Ir-SAC and Ir-NC were detected by mass spectroscopy (Figure SI5). Ir-SAC is highly active and selective to butene isomers while butane is the principal product for Ir-NC. The result is consistent with laboratory catalytic tests, confirming that operando XAS experiments were conducted in real working conditions.

To further confirm the thermal stability of Ir-SAC, we have carried out ex situ STEM-HAADF imaging on the Ir-SAC powder treated under 5%H2/Ar flow at 250 °C and 400 °C. Figure SI6 clearly shows isolated bright spots homogeneously dispersed on the support, irrespective of the temperature. This confirms that Ir single atoms are stable after the thermal treatment under reducing gas, even at 400 °C. This result is particularly meaningful since the stability of singleatoms toward coalescence is a real issue, especially in reducing atmosphere [START_REF] Dessal | [END_REF] . A thorough catalytic study complemented by detailed physicochemical characterizations will be addressed in a future report.

Conclusion

We have prepared Ir single atoms well dispersed on a nitrogen-doped carbon support and carried out catalytic tests for the partial hydrogenation of butadiene. The Ir-SAC proves a substantial activity toward hydrogenation of butadiene with nearly 100% selectivity to butenes even at full conversion. In comparison, Ir nanoparticles dispersed on a carbon support behave

Acknowledgments

Fabrice Couturas and Stéphanie Belin are acknowledged for help during the XAS experiments on ROCK beam line at Soleil. WL and CZ would like to thank Julie Bourgon for TEM measurements.

The authors acknowledge the French National Research Agency (ANR) for financial support (contract Ultracat ANR-17-CE06-0008-02). This work was supported by a public grant overseen by

Figure 2 .Figure 3 .

 23 Figure 2. Conversion and selectivity to butenes of Ir-SAC (A) and Ir-NC (B) in the hydrogenation of butadiene as a function of temperature.

  has an oxidation state of +3.2(0.1) in Ir-SAC, while this value is close to 0 in Ir-NC i.e., comparable to Ir bulk sample. The FT modulus of the k²-weighed EXAFS signals of the as-prepared samples are compared to those of bulk Ir and IrO2 references in Figure3B. Ir bulk shows typical peaks of the fcc metal with the dominant one at approximately 2.58 Å (Ir-Ir nearest distances) whereas, IrO2 displays a main peak at around 1.62 Å corresponding to the Ir-O nearest neighbours of the tetragonal structure of the oxide, in good agreement with previous results45 .The FT modulus of the k²-weighed EXAFS signal of Ir-SAC is clearly different as compared to those of the metal and oxide references and features a single peak at around 1.6 Å with low intensity. This unique and less intense peak can be ascribed to the backscattering from the nearest Ir neighbouring light elements such as, N, C or O. However, EXAFS cannot distinguish between light elements close to each other such as, C, N or O because of their similar scattering amplitudes.

Figure 4 .

 4 Figure 4. XANES spectra at the Ir L3-edge of the Ir-SAC during (A) the pre-treatment under H2/He flow and (B) under operando conditions.

Figure 5 .

 5 Figure 5. Variation of coordination number (A), the nearest neighbour distance Ir-X with X = C, N or O (B) and the Ir oxidation state (C) of Ir-SAC in operando conditions.

Figure 6 .

 6 Figure 6. XANES spectra at the Ir L3-edge (A) and the modulus of the Fourier transforms of the k²weighed EAXFS signals (B) of the Ir-SAC initial (black), after pre-treatment under H2/He flow (blue) and after reaction (red) at room temperature.

  differently with low selectivity to butenes while the conversion exceeds 30%. By means of operando XAS experiments, we demonstrated that the reduction pre-treatment under H2 at 250 °C involves the loss of one nearest neighbour (possibly oxygen) around Ir atoms accompanied by a decrease of the RIr-X distance as well as the oxidation state from +3.2 to +2.0. This evolution of the electronic and atomic structure of Ir-SAC might be the reason for the high catalytic selectivity in the hydrogenation of butadiene uncovering that the active species are Ir atoms surrounded by three light elements (N, C, O). Moreover, the Ir single atoms are stable at high temperature (up to 400 °C) without any coalescence into metallic clusters or nanoparticles. These results are of paramount importance for in-depth understanding of the underlying reaction mechanisms in the fast-growing filed of the single-atom catalysis.

Table 1 .

 1 EXAFS refinements result for Ir-SAC under operando conditions (hydrogenation of butadiene) :

		C4H6/H2/He	24.9	3.0 (1)	0.0049 (5)	1.997 (1)	0.06	2.0
			24.8	3 (1)	0.0048 (4)	1.998 (1)	0.05	2.0
			64.7	3.1 (1)	0.0051 (5)	2.001 (1)	0.06	2.1
	Reaction ramp	C4H6/H2/He	106.5	3.1 (1)	0.0053 (4)	2.001 (1)	0.05	2.1
			147.7	3.1 (1)	0.0054 (4)	1.999 (1)	0.05	2.0
			198.6	3.0 (1)	0.0055 (4)	1.996 (1)	0.05	1.9
			200	3.0 (1)	0.0054 (4)	1.996 (1)	0.05	1.9
	Reaction at 200 °C	C4H6/H2/He	200	2.9 (1)	0.0051 (5)	1.997 (1)	0.06	1.9
			200	3.0 (1)	0.0053 (4)	1.996 (1)	0.05	1.9
	Final state	He	33.4	3.1 (1)	0.0053 (4)	1.996 (1)	0.05	2.0

Conditions