MD Fabrice Lainé
email: fabrice.laine@chu-rennes.fr

PhD Marc Ruivard

PhD Véronique Loustaud-Ratti

PhD Fabrice Bonnet

MD Paul Calès

PhD Edouard Bardou-Jacquet

MD Sylvie Sacher-Huvelin

MD Xavier Causse

MD Christine Beusnel

study group* Alain Renault

PhD Eric Bellissant

MD Yves Deugnier

Institutions

Dominique Guyader

Romain Moirand

Caroline Jézéquel

Anne Guillygomarc'h

Pierre Brissot

Agnès Benses

Nolwenn Boissel

Marion Couegnat

Elise Thébault

Viviane Fortuna

Catherine Massart

Cécile Réminiac

Chloé Rousseau

Metabolic and hepatic effects of bloodletting in dysmetabolic iron overload syndrome: A randomized controlled study in 274 patients

Keywords:

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

Introduction

Insulin resistance-associated hepatic iron overload also coined as the dysmetabolic iron overload syndrome (DIOS) was first described in the late 90's [START_REF] Mendler | Insulin resistanceassociated hepatic iron overload[END_REF][START_REF] Moirand | A new syndrome of liver iron overload with normal transferrin saturation[END_REF] . It corresponds to mild increase of both liver and body iron stores [START_REF] Jezequel | Both hepatic and body iron stores are increased in dysmetabolic iron overload syndrome. A case-control study[END_REF] associated with various components of the metabolic syndrome in the absence of any identifiable cause of iron excess. It accounts for most cases of hyperferritinaemia referred to outpatient clinics and is much more frequent than genetic hemochromatosis [START_REF] Bozzini | Prevalence of body iron excess in the metabolic syndrome[END_REF] . The liver may be either normal or steatotic with non-alcoholic fatty liver injury (NAFLD) [START_REF] Turlin | Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients[END_REF] . DIOS shares the same cause as NAFLD, i.e. insulin-resistance, and about 10% of DIOS patients have type 2 diabetes [START_REF] Fernandez-Real | Mechanisms Linking Glucose Homeostasis and Iron Metabolism Toward the Onset and Progression of Type 2 Diabetes[END_REF] .

The role of iron excess in the development or the worsening of insulin resistance and, then, in the occurrence of diabetes and of NAFLD remains debated. Epidemiological studies have shown that elevated serum ferritin values were associated with a higher risk of developing type 2 diabetes [START_REF] Fumeron | Ferritin and transferrin are both predictive of the onset of hyperglycemia in men and women over 3 years: the data from an epidemiological study on the Insulin Resistance Syndrome (DESIR) study[END_REF] and were correlated with serum insulin and serum glucose levels [START_REF] Tuomainen | Body iron stores are associated with serum insulin and blood glucose concentrations. Population study in 1,013 eastern Finnish men[END_REF] , the best biological predictive factor of incident diabetes [START_REF] Balkau | Predicting diabetes: clinical, biological, and genetic approaches: data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF][START_REF] Bonora | Population-based incidence rates and risk factors for type 2 diabetes in white individuals: the Bruneck study[END_REF] . In early and small studies, achievement of low body iron stores by bloodletting [START_REF] Facchini | Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease[END_REF][START_REF] Fernandez-Real | Blood letting in high-ferritin type 2 diabetes: effects on vascular reactivity[END_REF][START_REF] Valenti | Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case-control study[END_REF] or low iron diet [START_REF] Hua | Low iron status and enhanced insulin sensitivity in lacto-ovo vegetarians[END_REF] was associated with decreased insulin resistance. This was not confirmed by a recent Australian study from Adams et al [START_REF] Adams | The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial[END_REF] who failed to find any improvement in both insulinresistance and steatosis in phlebotomized NAFLD patients. However, as stressed by Ryan et al [START_REF] Ryan | Does the death knell toll for phlebotomy in NAFLD?[END_REF] , only a few patients had increased hepatic iron in this study.

The aim of the present study was to assess, in a large population of DIOS patients with well-defined hepatic iron excess, whether iron depletion by sustained phlebotomies may improve insulin resistance and liver tests.

Patients and methods

Study design and patients

The SAIGNEES study was a multicentre, open-labelled, randomized, controlled phase 3 trial of oneyear maintenance of serum ferritin levels lower than 50µg/l by bloodletting associated with lifestyle and diet advices (LFDA) versus LFDA only in nondiabetic DIOS patients. Adult subjects with DIOS were recruited from January 2010 to May 2014 in 8 French hospitals (Angers, Clermont-Ferrand, La Roche Sur Yon, Limoges, Nantes, Orleans, Rennes and Saint-Malo). DIOS was diagnosed as follows: i) liver iron concentration, measured by magnetic resonance imaging [START_REF] Gandon | Non-invasive assessment of hepatic iron stores by MRI[END_REF] , higher than 50 µmol/g dry weight,

ii) presence of one of the following metabolic features at least: body mass index (BMI) >25 kg/m², waist size >80 cm in females and >93 cm in males, blood pressure higher than or equal to 130/85 mmHg or antihypertensive drug therapy, fasting blood glucose higher than 5.6 mmol/l but lower than 7 mmol/l, serum triglycerides levels higher than 1.7 mmol/l, serum high-density lipoprotein cholesterol levels lower than 1.29 mmol/l for females and lower than 1.03 mmol/l for males or cholesterol-lowering therapy and (iii) absence of the following conditions: diabetes mellitus, alcohol consumption >20g/day in females and >30g/day in males, homozygosity for the C282Y mutation of the HFE gene, aceruloplasminaemia, haematological disorder, acute or chronic inflammatory disease, viral hepatitis, auto-immune or cholestatic liver disease. The study (Clinical Trials.gov NCT01045525) was approved by the review board of the ethics committee of Rennes. All patients provided a written informed consent.

Randomisation and masking

Subjects were prospectively randomized in a 1:1 fashion to iron depletion by phlebotomy with lifestyle advice versus lifestyle advice only. Randomization was performed using computer software.

Stratification was made according to baseline values of fasting serum glucose (lower than 5.6 mmol/l or not). The study was open-labelled.

Procedures

Iron depletion by bloodletting

Subjects randomized to iron depletion underwent 2 phases of treatment. The initial phase consisted of bi-monthly phlebotomies (ranging from 350 ml to 450 ml according to body weight and sex) with the objective to achieve serum ferritin levels <50 µg/l with haemoglobin levels >11g/dl. Then, the maintenance phase consisted of bi-monthly 350 to 450 ml phlebotomy on the basis of serum ferritin measurement every two months in order to maintain serum ferritin lower than 50 µg/l and haemoglobin >11 g/dl.

Lifestyle advice

All subjects received written dietary recommendations promoting an healthy and varied diet, in the form of two books edited by the French National Program of Nutrition and Health (http://www.inpes.sante.fr) and were advised to have physical activity during at least 30 minutes per day. They were left free to consult a dietician.

Clinical assessment

It was performed at baseline and 12 months after randomisation and included medical history, medications, questionnaires about alcohol consumption, eating habits and physical activity, BMI, waist circumference, and systolic and diastolic blood pressure. Waist circumference was measured according to the recommendations of the NHANES III (http://www.cdc.gov). Blood pressure was measured on a subject lying down, at rest for at least 5 minutes. Three successive measurements spaced of 2 minutes were performed using automatic test equipment. The value used was the average of the last two measures.

Biochemical assessment

A fasting venipuncture and a 75 g OGTT were performed at baseline and at 12 months. Biochemical testing included blood cell count, aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), gamma-glutamyl transferase (GGT), HDL-and LDL-cholesterol, triglycerides, glucose, insulin, HbA1c, iron parameters (iron, transferrin, ferritin, and transferrin saturation), C Reactive protein and adiponectin. HOMA was calculated as (insulin T0 x glucose T0)/22.5, modified Matsuda index as 1000/ඥ[(glucose T0 x insulin T0) x (glucose T30 x insulin T30)], and insulinogenic index as the ratio of the increment of the insulin concentration over the first 30 minutes of the OGTT to the increment in blood glucose over the same period (insulin T0-T30/glucose T0-T30). Hepatic fibrosis was assessed using the Fib-4 score [START_REF] Mcpherson | Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease[END_REF] and steatosis using the Fatty Liver Index [START_REF] Bedogni | The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population[END_REF] .

Outcomes

The primary outcome was the mean value of fasting blood glucose one year after randomisation.

Secondary outcomes were 1) variations of metabolic features from baseline to end of treatment, 2) insulin resistance measured by homeostasis model of assessment of insulin resistance (HOMA), modified Matsuda index and insulinogenic index based upon a 75g oral glucose tolerance test (OGTT) with measurement of glycaemia and insulinemia at baseline and 30 and 120 minutes after glucose ingestion, one year after randomisation 3) serum adiponectin levels one year after randomisation, 4) serum C reactive protein levels one year after randomisation, 5) liver function tests and 6) FLI and Fib-4 scores.

Statistical analysis

An a priori power calculation indicated that a sample size of 270 subjects would provide 90% power to determine a difference in fasting blood glucose of 0.25 mmol/l, assuming a standard deviation of 0.61 mmol/l, a two-sided p value of 0.05 and a drop-out rate of 10%. Finally, standard deviation of fasting glycaemia was 0.71 at the end of the study; a posteriori power calculation indicated 80 % power, with a two-sided p value of 0.05 to determine a difference in fasting glycaemia of 0.25mmol/l. The statistical analysis, prospectively defined, was done by intention to treat (i.e., in all analyses, patients were grouped according to their original randomised treatment) with SAS statistical software (version 9.4; Cary, NC, USA). Continuous variables were expressed by mean and standard deviation and categorical variables were expressed by the number of patients and percentages. Baseline characteristics were compared between groups using Student's t-test for continuous variables and chi-squared test or Fisher's exact test for categorical variables. Student's t-tests were also performed between groups on the variation of continuous variables (between the baseline and the end of study).

The primary and secondary outcomes were evaluated first with Student's t-test and chi-square test, followed by analyses of variance with adjustment on the variation of weight. Regarding the insulinrelated parameters, analyses of variance with adjustment on the variation of weight and the insulin at baseline, were used. The same analysis was performed in subgroups of patients who lost weight.

All statistical tests had a significance level of 0.05.

Results

Patients groups and characteristics at baseline

Between Jan 8, 2010 and May 30, 2014, 274 patients were randomly assigned to receive venesections with LFDA (iron depletion group) and 128 to LFDA only (control group). Seven patients withdrew prior to the end of the study, one in the iron depletion group because he left abroad and 6 in the control arm (one suddenly died and 5 withdrew for personal decision). Finally, 267 patients were analysed, 145 in the iron depletion arm and 122 in the control arm (Figure 1). Baseline characteristics are summarized in Table 1. All patients were Caucasians and most were middle-aged males. Mean BMI was 28.5±3.6 kg/m 2 . Obesity was present in 31.1% of patients, increased waist circumference in 88%, hypertension in 54 %, elevated serum triglycerides in 55.1%, and hypercholesterolemia in 42.3%. Mean glycaemia was 5.61±0.61 mmol/l. Hyperglycaemia (fasting blood glucose higher than 5.6 mmol/l) was observed in 36.9% of patients. Metabolic syndrome, defined according to the International Diabetes Federation, was present in 59.5 %. Mean alcohol consumption was 90±80 g per week. Increase in serum ALT transaminase levels was present in 29.3 % of patients, but ALT levels were greater than 2 times the upper limit of normal in 7% only.

According to Fib-4 score, 3 patients had bridging hepatic fibrosis or cirrhosis. According to Fatty Liver Index, steatosis was present in 59.1% of patients, uncertain in 29.3% and absent in 11.6%. Liver biopsy was performed in 7 patients who all presented with steatosis. Fibrosis (METAVIR) was stage 0 in 1, stage 1 in 3 and stage 2 in 3. Allelic frequency of C282Y mutation of the HFE gene was 27.5% in the venesected group vs 27% in controls (p=0.93).

Iron removal

During the initial phase of treatment, a mean of 12±4 bi-monthly phlebotomies was performed in the iron depletion group. This corresponded to a mean of 4.9±1.6 l of blood removed, i.e. 2.45±0.8 g of iron. At the end of the initial phase, the mean value of serum ferritin levels was 62±37 µg/l. The threshold of 50 µg/l was achieved in 72 (50%) patients, whilst serum ferritin levels were comprised between 50 and 100 µg/l in 55 (38%) and greater than 100 µg/l in 17 patients (12%). During the maintenance phase, 91 (62%) of the iron-depleted patients had a mean of 2±1 phlebotomies. At the end of the study, the threshold of 50 µg/l was achieved in 51 (35%) patients, whilst serum ferritin levels were comprised between 50 and 100µg/L in 73 (50%) and greater than 100µg/L in 21 patients (15%). The main causes of non achievement of the goal of serum ferritin < 50 at the end of treatment were adverse events (43%) and poor adherence to phlebotomies (67%). There was no serious adverse event related to treatment. Dizziness, hypotension and vagal malaise related to irondepletion therapy were reported in 3, 2 and 5 patients, respectively. Anaemia, defined as haemoglobin level lower than 12 g/dl in females and lower than 13 g/dl in males, was observed in 6 patients. Fatigue was reported in 37 patients (25.3 %) of the iron-depleted group versus 3 patients (2.3 %) in the control group (p<0.0001).

Life style & diet

The percentage of patients who consulted a dietician was identical (12%) in both arms. Physical activity scores during sport and leisure did not significantly change during the study in the 2 groups.

Variation in alcohol consumption was not different in the 2 groups with a mean of -10 g per week.

Mean variation of BMI was +0.13±1.22 kg/m² in the iron depletion group versus -0.17±0.94 kg/m² in the control group; p=0•03. Mean percentage of variation of body weight was +0.5±4.3% in the iron depletion group vs -0.6±3.3% in the control group (p=0.03). Percentages of patients whose weight decreased, remained stable (±1 kg) or increased were 21%, 41% and 38% respectively in the iron depletion group vs 26%, 53% and 21% respectively in the control group (p=0.009).

Fasting blood glucose

There was no difference in end-of-treatment fasting blood glucose between the iron depletion and control groups (5.44±0.71 vs 5.49±0.70 respectively; p=0.57), even after adjustment for variation of weight (p=0.44). A stepwise multivariate analysis of the variation of glycaemia was performed. The following variables were included in the analysis: treatment group, presence of HTA, variations between baseline and end-of-study of weight, waist circumference, haemoglobin, baseline cholesterol, serum ferritin and liver enzymes values. Weight variations and -GT values were the only two variables independently and positively associated (p=0.01 and p=0.03 respectively) with the variation of glycaemia. As scheduled, analysis was performed in subgroups of patients according to baseline values of fasting glycaemia. One hundred and thirty-seven (51%) patients had glycaemia greater than or equal to 5.6 mmol/l at inclusion, of whom 74 were randomized in the iron depletion group and 63 in the control group. There was no difference in end-of-treatment fasting blood glucose between both groups (5.81±0.74 vs 5.83±0.77 respectively; p=0.91), even after adjustment for variation of weight (p=0.71). One hundred and thirty (49%) patients had glycaemia lower than 5.6 mmol/l at inclusion of whom 71 were randomized in the iron depletion group and 59 in the control group. There was no difference in end-of-treatment fasting blood glucose between the iron depletion and control groups (5.06±0.4 vs 5.13±0.4 respectively; p=0.27), even after adjustment for variation of weight (p=0. 24). Per protocol analysis comparing the 51 patients with serum ferritin levels <50µg/l one year after randomization with controls was performed. There was no difference in end-of-treatment fasting blood glucose between the iron depletion and control groups (5.45 ± 0.58 vs 5.49 ± 0.7; p=0.72) even after adjustment on weight variation (p=0.65).

Markers of insulin-resistance

There was no significant effect of iron depletion on mean serum values of triglycerides, HDL and LDL cholesterol, mean values of systolic and diastolic blood pressure and percentage of hypertensive patients (Table 2) even after adjustment on weight variation. Variations of insulin resistance indexes were compared between the two treatment groups without and with adjustment on variations of weight and adjustment on variations of both weight and baseline values of fasting insulin. Fasting insulinemia and HOMA index were significantly increased in the iron depletion group vs control group (Table 3). A stepwise multivariate analysis of the variation of HOMA was performed. The following variables were included in the analysis: treatment group, presence or absence of HTA, variations between baseline and end-of-study of weight, waist circumference, haemoglobin, baseline cholesterol, serum ferritin and liver enzymes values. Weight variations (p<0.001), -GT values (p<0.01), treatment group (p<0.01) and arterial hypertension (p=0.02) were significantly and positively associated with variation of HOMA. There was no difference in end-of-treatment adiponectin serum levels between iron depletion and control groups (6.2±4.3 vs 6.5±3.9 respectively; p=0.53) and in end-of-treatment C reactive protein serum levels between both groups (2.1±1.8 vs 2.4±3.1 respectively; p=0.24) even after adjustment for variation of weight (p=0.70 and p=0.20; respectively).

Liver tests and scores

As shown in table 2, end of study AST, ALT, and GGT did not differ between the iron-depleted and control groups. After adjustment for weight variation, serum ALT levels were significantly lower (p=0.03) in the iron-depleted group. Fib4 and FLI scores were not different according groups.

Subgroup analysis according to weight loss

Sixty-three patients lost more than 1 kg of body weight; 31 (21%) in the iron depletion group and 32 (26%) in the control group. Glycaemia, insulinemia, HOMA index, HDL and LDL-cholesterol, triglycerides and liver enzymes were or tended to be improved in these patients regardless of the treatment group (Table 4).

Discussion

The present study demonstrates that, in patients with DIOS, removal of iron excess by phlebotomies has no significant metabolic and hepatic effects. Even more, bloodletting may contribute to weight gain and to the worsening of some insulin-resistance-related features.

Blood glucose was chosen as primary end-point for the following reasons.

First, insulin resistance is the main determinant of both DIOS 1 and NAFLD [START_REF] Birkenfeld | Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes[END_REF] . Blood glucose is one of the best surrogate markers reflecting the degree of insulin resistance and the risk of forthcoming type 2 diabetes [START_REF] Balkau | Predicting diabetes: clinical, biological, and genetic approaches: data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF] . The epidemiological demonstration of an association between blood glucose and body iron stores concentrations was made by Finnish authors [START_REF] Tuomainen | Body iron stores are associated with serum insulin and blood glucose concentrations. Population study in 1,013 eastern Finnish men[END_REF] . Since then, a few clinical trials 11- 13,15,21-23 have reported conflicting results about the effects of phlebotomy therapy on blood glucose and markers of insulin resistance in patients with various metabolic abnormalities. In all but that conducted by Piperno et al [START_REF] Piperno | Effects of venesections and restricted diet in patients with the insulin-resistance hepatic iron overload syndrome[END_REF] , DIOS patients were mixed with NAFLD patients with no iron excess. In addition, most studies were not randomised [START_REF] Facchini | Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease[END_REF][START_REF] Valenti | Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case-control study[END_REF][START_REF] Piperno | Effects of venesections and restricted diet in patients with the insulin-resistance hepatic iron overload syndrome[END_REF] , included a limited number of patients [START_REF] Facchini | Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease[END_REF][START_REF] Adams | The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial[END_REF][START_REF] Laine | Effects of phlebotomy therapy on cytochrome P450 2e1 activity and oxidative stress markers in dysmetabolic iron overload syndrome: a randomized trial[END_REF][START_REF] Piperno | Effects of venesections and restricted diet in patients with the insulin-resistance hepatic iron overload syndrome[END_REF][START_REF] Valenti | A randomized trial of iron depletion in patients with nonalcoholic fatty liver disease and hyperferritinemia[END_REF][START_REF] Beaton | Phase II clinical trial of phlebotomy for non-alcoholic fatty liver disease[END_REF] , and mixed diabetics with non-diabetics [START_REF] Facchini | Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease[END_REF][START_REF] Adams | The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial[END_REF][START_REF] Laine | Effects of phlebotomy therapy on cytochrome P450 2e1 activity and oxidative stress markers in dysmetabolic iron overload syndrome: a randomized trial[END_REF][START_REF] Piperno | Effects of venesections and restricted diet in patients with the insulin-resistance hepatic iron overload syndrome[END_REF][START_REF] Beaton | Phase II clinical trial of phlebotomy for non-alcoholic fatty liver disease[END_REF] .

Second, Fernandez-Real et al reported a positive relationship between serum ferritin levels and insulin resistance and proposed serum ferritin as a component of the insulin resistance syndrome [START_REF] Fernandez-Real | Serum ferritin as a component of the insulin resistance syndrome[END_REF] .

In a controlled randomised trial conducted in 28 type 2 diabetics with elevated serum ferritin, they found that iron removal improved both insulin sensitivity and beta cell function but had no significant effect on blood glucose [START_REF] Fernandez-Real | Blood letting in high-ferritin type 2 diabetes: effects on vascular reactivity[END_REF] . Facchini et al, in a randomized study of 42 patients, mainly diabetics, showed that iron depletion was associated to decrease of both fasting and glucosestimulated plasma insulin concentrations in a subgroup of 17 hyperferritinemic subjects with nonbiopsy proven NAFLD [START_REF] Facchini | Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease[END_REF] . Piperno et al compared the respective effects of venesection therapy and diet restriction in 44 subjects with DIOS [START_REF] Piperno | Effects of venesections and restricted diet in patients with the insulin-resistance hepatic iron overload syndrome[END_REF] . They found that diet restriction only resulted in a significant decrease in blood glucose. Valenti et al, in a case-control study of 128 non-diabetic patients with NAFLD and either elevated serum ALT levels or increased serum ferritin levels or both, showed that serum insulin, serum glucose and HOMA improved in iron-depleted patients [START_REF] Valenti | Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case-control study[END_REF] . Finally, Adams et al, in a randomised controlled study, showed that iron depletion failed to improve blood glucose, HOMA and ISI in 61 patients with NAFLD and in a limited subgroup of 36 patients with hyperferritinemia [START_REF] Adams | The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial[END_REF] . Compared to these previous studies, we included a much larger sample of patients, which allowed for a satisfactory statistical power. Moreover, all of our subjects had hepatic iron excess demonstrated using a well-validated method [START_REF] Gandon | Non-invasive assessment of hepatic iron stores by MRI[END_REF] . Finally, our study was controlled and randomised with stratification on baseline serum glucose. As blood glucose value is associated in a continuous manner with the risk of diabetes [START_REF] Balkau | Predicting diabetes: clinical, biological, and genetic approaches: data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF] , the present data supports the conclusion that, in nondiabetic DIOS patients, iron depletion has no effect on the risk of developing diabetes, irrespective of the initial level of that risk.

Third, it would have not been realistic to propose liver biopsy as the primary end-point in our study because, in daily practice, it is rarely performed in DIOS patients. Indeed most of them have no increased serum transaminase levels -as illustrated by the present data -and the risk of severe fibrosis, which is very low in the absence of cofactors of liver disease, can be ruled out by noninvasive tests. Requiring histological end-points would have resulted in a selection bias and then in hardly conclusive results.

Our results raised concerns about putative deleterious effects of phlebotomy therapy in DIOS patients. Indeed, despite both groups had comparable baseline BMI and received similar lifestyle advices, BMI decreased by 0.5% in controls whilst it increased by 0.4% in venesected patients (p=0.03). This is in line with the observation of a trend to weight gain in venesected patients in the studies from Valenti et al [START_REF] Valenti | Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case-control study[END_REF] and from Adams et al [START_REF] Adams | The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial[END_REF] that did not reach statistical significance likely due to insufficient sample size. Weight gain could be partly related to looser motivation of venesected patients for lifestyle recommendations, because of the falsely reassuring feeling of being already treated.

Conflicting results about the effects of venesection therapy on HOMA index, a surrogate marker of insulin resistance, have been previously reported. Some authors found that HOMA index decreased in venesected patients [START_REF] Facchini | Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease[END_REF][START_REF] Fernandez-Real | Blood letting in high-ferritin type 2 diabetes: effects on vascular reactivity[END_REF][START_REF] Valenti | Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case-control study[END_REF] whilst others did not find any effect of iron removal [START_REF] Adams | The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial[END_REF][START_REF] Laine | Effects of phlebotomy therapy on cytochrome P450 2e1 activity and oxidative stress markers in dysmetabolic iron overload syndrome: a randomized trial[END_REF][START_REF] Beaton | Phase II clinical trial of phlebotomy for non-alcoholic fatty liver disease[END_REF] . In the present paper, we showed that HOMA index increased in venesected patients, mainly due to increase in serum insulin. This was significant at univariate analysis and confirmed at multivariate analysis which showed that increase in HOMA index was independently explained by both bloodletting and weight gain. Basal hypersecretion of insulin is viewed as an independent abnormality in the pathogenesis of type 2 diabetes and has been shown to predict diabetes development, independently of insulin resistance [START_REF] Weyer | A high fasting plasma insulin concentration predicts type 2 diabetes independent of insulin resistance: evidence for a pathogenic role of relative hyperinsulinemia[END_REF] . This increase in fasting insulin levels besides the improvement of hyperferritinaemia suggests the absence of benefit of iron depletion on the risk of type 2 diabetes. Nevertheless, the deleterious effect of venesection therapy per se is likely to remain weak compared to that induced by weight variation.

Our study demonstrates that weight variation is, in fact, the primary determinant of the evolution of not only HOMA index but also of metabolic features and liver tests in non-diabetic DIOS patients. This was well illustrated by the improvement of blood glucose, LDL and HDL cholesterol, triglycerides, ALAT and GGT in patients who had lost weight and by the absence of clinically significant effect of iron removal on these parameters in these patients (Table 4).

It could be discussed that iron depletion was insufficient to reach significant effects in the present study. However, previous studies had conflicting results independently of bloodletting scheme and endpoints of serum ferritin and serum haemoglobin. Moreover, analysis of the subgroup of our patients having achieved serum ferritin levels < 50 µg/l at one year failed to show any significant effect of venesection therapy on primary and secondary outcomes.

In addition, venesection therapy was not as well tolerated as expected [START_REF] Guillygomarc'h A | Venesection therapy of insulin resistance-associated hepatic iron overload[END_REF] . Indeed, a significant proportion of venesected patients experienced fatigue and mild malaises as previously reported by Piperno et al [START_REF] Piperno | Effects of venesections and restricted diet in patients with the insulin-resistance hepatic iron overload syndrome[END_REF] . This and the poor adherence to phlebotomy we observed render difficult to propose a more intensive scheme of bloodletting in routine practice.

Conclusion

In DIOS patients, iron depletion by phlebotomy has no significant effect on serum glucose level nor on liver biochemical tests and scores, may aggravate insulin-resistance, is associated with weight gain and is poorly tolerated. Weight loss alone is associated with improvement in most metabolic features including glycaemia and insulin resistance, and in serum ferritin and liver tests. Sustained modification of diet and life-style habits remains the first therapeutic intervention in these patients. *HOMA: homeostasis model assessment. † Insulinogenic index was calculated as as the ratio of the increment of the insulin concentration over the first 30 minutes of the OGTT to the increment in blood glucose over the same period (insulin T0-T30/glucose T0-T30). ‡ Modified Matsuda index was calculated as: 1000/ඥ[(glucose T0 x insulin T0) x (glucose T30 x insulin T30)]

Table 1 . Baseline characteristics of trial population.

 1

		Iron depleted (n=146)	Control (n=128)	P value
	Demographics
	Age (years)	58 (10)	56 (10)	0.09
	Male sex	119 (82%)	114 (89%)	0.08
	Iron parameters
	Ferritin (µg/L)	792 (290) 1	788 (290)	0.91
	Transferrin saturation (%)	37.4 (10.3) 3	38.4 (11.8)	0.43
	Haemoglobin (g/dL)	15.13 (1.2)	15.26 (1.0)	0.33
	MRI estimation of liver iron (µmol/g)	85 (32) 1	92 (39) 2	0.15
	Metabolic factors
	BMI (kg/m²)	28.67 (3.8)	28.41 (3.4)	0.55
	Waist circumference (cm)	102 (10) 2	101 (9) 3	0.56
	Hypertension*	105 (72%)	92 (73%) 2	0.84
	Triglyceride (mmol/L)	1.47 (0.7)	1.37 (0.6)	0.24
	HDL-cholesterol (mmol/L)	1.34 (0.4)	1.38 (0.4)	0.33
	Glucose (mmol/L)	5.57 (0.5)	5.65 (0.7)	0.27
	HbA1c (%)	5.56 (0.4) 1	5.57 (0.4)	0.92
	Insulin (µUI/mL)	11.81 (8.3)	10 (7.2) 1	0.06
	HOMA	2.97 (2.2)	2.58 (2.0) 1	0.13
	Insulin Sensitivity Index	107 (271.5) 1	89.1 (61.6) 3	0.47
	Liver tests & scores
	ALT (IU/L)	40 (21) 3	41 (22)	0.87
	AST (IU/L)	28 (11) 3	29 (10)	0.73
	Gamma GT (IU/L)	49 (40) 3	54 (41)	0.34
	Prothrombin (%)	101 (12) 1	103 (11) 1	0.34
	Fatty liver index	63.71 (23.49) 5	63.89 (22.97) 3	0.95
	FIB-4 score	1.34 (0.6) 3	1.32 (0.6)	0.73
	Data are n (%) or mean (SD). HOMA=homeostasis model assessment.

*Hypertension was defined as blood pressure > 140/90 mmHg or as receiving an anti-hypertensive drug. Numbers in subscript indicate the number of missing data.

Table 2 . End of study characteristics of trial population.

 2 Data are n (%) or mean (SD). HOMA=homeostasis model assessment. *Hypertension was defined as blood pressure > 140/90 mmHg or as receiving an anti-hypertensive drug. Numbers in subscript indicate the number of missing data.

					P value
		Iron depleted (n=145)	Control (n=122)	P value	(adjustment on the variation of
					weight)
	Iron parameters
	Ferritin (µg/L)	71 (48) 2	733 (277)	< 0.0001	< 0.0001
	Transferrin saturation (%)	26.4 (11.6) 6	37.3 (12.5) 1	< 0.0001	< 0.0001
	Hemoglobin (g/dL)	14.62 (1.2)	15.14 (1.1)	0.0004	0.0002
	Metabolic factors
	BMI (kg/m²)	28.76 (3.9)	28.12 (3.5) 1	0.17
	BMI change (%)	0.5 (4.3)	-0.6 (3.3) 1	0.03
	Waist circumference (cm)	101 (10) 2	101 (10) 1	0.86	0.72
	Hypertension*	103 (72 %) 3	93 (76%)	0.49	0.46
	Triglyceride (mmol/L)	1.3 (0.6) 2	1.37 (0.7)	0.36	0.24
	HDL-cholesterol (mmol/L)	1.39 (0.5) 2	1.36 (0.4)	0.63	0.56
	Glucose (mmol/L)	5.44 (0.7)	5.49 (0.7)	0.57	0.44
	HbA1c (%)	5.57 (0.6) 1	5.52 (0.4) 1	0.50	0.64
	Insulin (µUI/mL)	13.57 (10.5) 3	9.47 (6.3)	0.0002	0.0006
	HOMA	3.39 (3.0) 3	2.40 (1.9)	0.002	0.004
	Insulin Sensitivity Index	73.76 (57.3) 4	86.37 (52.2) 1	0.07	0.10
	Liver tests & scores
	ALT (IU/L)	33 (22) 1	37 (21)	0.10	0.03
	AST (IU/L)	27 (13) 1	27 (10)	0.81	0.54
	Gamma GT (IU/L)	54 (138) 1	49 (35)	0.72	0.91
	Prothrombin (%)	101 (11)	101(12)	0.75	0.80
	Fatty liver index	58.93 (24.6) 5	61.58 (22.9) 2	0.37	0.12
	FIB-4	1.35 (0.6) 1	1.30 (0.6)	0.51	0.47

Table 3 : Comparison at end-of-treatment of insulin and insulin resistance index between iron-depleted and control groups after adjustment on weight variation (left column) and after adjustment on weight variation and baseline insulin value (right column). Adjustment on weight variation Adjustment on weight variation and baseline insulin value

 3

		Iron-depleted	Controls	p value	Iron-depleted	Controls	p value
	Insulin (µUI/mL)	13.38	9.62	<0.01	12.57	10.64	<0.01
	HOMA*	3.33	2.44	<0.01	3.11	2.72	0.07
	Insulinogenic index †	24.82	17.14	0.13	24.54	17.48	0.17
	Modified Matsuda index ‡	74.62	85.84	0.10	77.98	80.49	0.66

Acknowledgments

The authors are indebted to Stuart Byrom, INSERM CIC1414, Rennes, for reviewing the English language.

Funding

French Ministry of Health -Programme Hospitalier de Recherche Clinique 2009.

Abbreviations

DIOS: dysmetabolic iron overload syndrome, NAFLD: non alholic fatty liver disease, LFDA: lifestyle and diet advice, AST: aspartate aminotransaminase, ALT: alanine aminotransaminase, GGT: gammaglutamyl transferase, MRI: magnetic resonance imaging, BMI: body mass index

Declaration of interests

All authors declare no competing interests.


domain: chim sdv

text:
A Hajjaji

M Elabidi

K Trabelsi

A A Assadi

B Bessais
email: brahim.bessais@crten.rnrt.tn

S Rtimi
email: s.rtimi@epfl.ch

Bacterial adhesion and inactivation on Ag decorated TiO2-nanotubes under visible light: Effect of the nanotubes geometry on the photocatalytic activity

Keywords: Ag decorated Titanium nanotubes, AgO/Ag2O, Nanotubes geometry, Crystallinity, Bacterial adhesion, Bacterial inactivation

Bacterial inactivation is seen to be related to the nanotubes anodization potential and the silverions decoration.

Escherichia coli (E. coli) is hazardous bacteria that can cause distinct diarrhea syndrome. it was recognized as pathogen because it can cause urinary tract infection, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura in human [START_REF] Zottola | The microbiology of foodborne disease outbreaks: an update[END_REF]. Thus, it is necessary to develop an effective material for the inactivation of E. coli.

Currently, the inactivation of bacteria is mainly through chemical products based on chloride as strong oxidant. Unfortunately, a big majority of these poducts produce carcinogenic disinfection by-products (DBPs) like trihalomethanes and haloacetic acids [START_REF] Gopal | Chlorination byproducts, their toxicodynamics and removal from drinking water[END_REF]. Oher products are based on bactericidal nanoparticles (mainly silver). These later products are showing limitations towards some germs. In addition, some pathogens can develop resistance to silver nanoparticles as recently reported [START_REF] Graves | Rapid evolution of silver nanoparticle resistance in Escherichia coli[END_REF]. World Health Organization (WHO) launched an alert towards the excessive use of antibiotics and their derivatives [START_REF] Tangcharoensathien | Complex determinants of inappropriate use of antibiotics[END_REF]. Antibiotics are today showed to become ineffective against many pathogens that are able to develop resistance; hence, many germs became multidrug resistant. Thus, it is necessity today to develop alternative ways to disinfect surfaces in hospital facilities since healthcare acquired infections are increasingly threatening our lives [START_REF] Rtimi | Indoor Light Enhanced Photocatalytic Ultra-Thin Films on Flexible Non-Heat Resistant Substrates Reducing Bacterial Infection Risks[END_REF][START_REF] Ballo | Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata[END_REF][START_REF] Mena | Grafted semiconductors on PE-films leading to bacterial inactivation: synthesis, characterization and mechanism[END_REF].

A C C E P T E D M A N U S C R I P T

Photocatalytic bacterial inactivation is found to be a promising alternative due to several advantages. The sustainable use of the photocatalytic material is one of the most unique properties of this method while conventional chemical root consumes disinfectants [START_REF] Ganguly | Antimicrobial activity of photocatalysts: Fundamentals, mechanisms, kinetics and recent advances[END_REF]. also this new method does not produce any toxic or carcinogenic product (DBPs). Additionally, Titanium dioxide (TiO2) is attracting great attention during the last decades because of its chemical stability, non-toxicity, high oxidative power and low fabrication cost [START_REF] Rtimi | Indoor Light Enhanced Photocatalytic Ultra-Thin Films on Flexible Non-Heat Resistant Substrates Reducing Bacterial Infection Risks[END_REF]. Titanium based photocatalysts have been used to inactivate bacteria (in the dark or under light irradiation) [START_REF] Nesic | New evidence for TiO2 uniform surfaces leading to complete bacterial reduction in the dark: Critical issues[END_REF][START_REF] Petronella | Uniform TiO2/In2O3 surface films effective in bacterial inactivation under visible light[END_REF], virus [START_REF] Guo | Virus removal and inactivation in a hybrid microfiltration-UV process with a photocatalytic membrane[END_REF] and even cancer cells/tumor [START_REF] Graves | Rapid evolution of silver nanoparticle resistance in Escherichia coli[END_REF][START_REF] Tangcharoensathien | Complex determinants of inappropriate use of antibiotics[END_REF][START_REF] Rtimi | Indoor Light Enhanced Photocatalytic Ultra-Thin Films on Flexible Non-Heat Resistant Substrates Reducing Bacterial Infection Risks[END_REF][START_REF] Ballo | Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata[END_REF]. However, the wide band gap energy (3.2 eV) and the high recombination rate of the photo-generated charges in TiO2 reduces its photocatalytic performance. To overcome this limitation, many strategies can be adopted like modifying TiO2 with anionic or cationic doping [START_REF] Kisch | Semiconductor Photocatalysis-Mechanistic and Synthetic Aspects[END_REF][START_REF] Milosevic | Synergistic Effect of Fluorinated and N Doped TiO2 Nanoparticles Leading to Different Microstructure and Enhanced Photocatalytic Bacterial Inactivation[END_REF], narrow-band semi-conductors coupling [START_REF] Rtimi | Coupling of narrow and wide band-gap semiconductors on uniform films active in bacterial disinfection under low intensity visible light: Implications of the interfacial charge transfer (IFCT)[END_REF] or metallic nanoparticles decoration [START_REF] Pearson | Decoration of TiO2 Nanotubes with Metal Nanoparticles Using Polyoxometalate as a UV-Switchable Reducing Agent for Enhanced Visible and Solar Light Photocatalysis[END_REF].

Although, it has been reported since years that anatase and rutile crystalline phases of TiO2 are more active than brookite form [START_REF] Fujishima | TiO2 Photocatalysis and Related Surface Phenomena[END_REF]. With respect to the photocatalytic activity of the polymorphs, although it is somewhat controversial, the anatase phase is generally regarded as being more active than rutile [START_REF] Landmann | The Electronic Structure and Optical Response of Rutile, Anatase and Brookite TiO2[END_REF][START_REF] Zhang | New Understanding of the Difference of Photocatalytic Activity among Anatase, Rutile and Brookite TiO2[END_REF]. An emerging field of interest in photocatalysis is the development of TiO2 nanotubes (TiO2-NTs) and their coupling with cations, metal-oxides and additional composites leading to a higher nanocomposite sensitization in the visible range [START_REF] Testino | Optimizing the Photocatalytic Properties of Hydrothermal TiO2 by the Control of Phase Composition and Particle Morphology. A Systematic Approach[END_REF][START_REF] Zhang | Synthesis of hierarchical TiO2 nanotube arrays assembled by anatase single crystal nanoparticles[END_REF][START_REF] Mangayayam | FeOx magnetization enhancing E. coli inactivation by orders of magnitude on Ag-TiO2 nanotubes under sunlight[END_REF]. Titanium anodization is a simple preparation leading to controllable nanotubes formation, chemical resistivity, high surface area (2-3 orders of magnitude higher than a flat surface) and therefore high loading capability. The large surface to volume ration of TiO2-NTs is related to the large internal and external surfaces along with the surface in their vertex and the interstitial surface.

In this study, TiO2-NTs were prepared by anodic oxidation. After optimization of the TiO2-NTs antibacterial activity and geometry, the most active NTs were decorated with different amounts of silver nanoparticles (Ag NPs) using the photo-reduction method. We focus on the effect of NTs geometry before and after the Ag-decoration and its influence on the charge separation and transport. Furthermore, this decoration affected the optical properties and photocatalytic performance against E. coli under low intensity solar simulated light. We also show that varying the anodization potential during the nanotubes growth influences their geometry and reactivity under visible light.

EXPERIMENTAL

A C C E P T E D

M A N U S C R I P T

Preparation of Ag-NPs/TiO2-NTs photo-electrodes

The anodization was performed under continuous stirring during 2 h at room temperature and at different potential starting from 20 V to 70 V. The electrolyte consisted of a mixture of 2 vol.% of water and ethylene glycol and 0.07 M of NH4F. The as-prepared TiO2-NTs were rinsed with water, air dried, and then annealed for 3 hours at 400°C (5°C/min). Subsequently, the prepared TiO2-NTs were decorated with Ag-NPs using the photo-reduction method. To this end, the TiO2-NTs were first immersed in a 0.1 M solution of AgNO3 for 24 hours, then rinsed with water and immersed again in methanol under UV radiation (254 nm) for 10 minutes. Finally, the Ag-NPs/TiO2-NTs samples were rinsed with water and dried in a vacuum oven for 1 h at 80°C.

Evaluation of the adhesion and inactivation of E. coli on Ag-NPs/TiO2-NTs under light and dark conditions

The antibacterial activity of the Ag-NPs/TiO2-NTs was performed taking Escherichia coli (E. coli K12 ATCC 23716; from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany) as a probe. The samples were sterilized by autoclaving at 121°C for 2 h. The 100 µL culture aliquots with an initial concentration of ~4 10 6 Colony-forming unit per milliliter (CFU mL -1 ) in NaCl/KCl (pH 7) were placed on the samples. The 100 µL of the E. coli inoculum was contacted with the Ag-NPs/TiO2-NTs uniform surface. The exposition was done at 23°C (+/-2°). The samples were then placed on Petri dishes provided with a lid to prevent evaporation. After each determination, the surface was transferred into a sterile 2 mL Eppendorf tube containing 900 µL autoclaved NaCl/KCl saline solution. This solution was subsequently mixed thoroughly using a Vortex for 2 min. Serial dilutions were made in NaCl/KCl solution. A 100 µL sample of each dilution was pipetted onto a nutrient agar plate and then spread over the surface of the plate using standard plating method. Agar plates were incubated lid down, at 37 °C for 24h before colonies were counted. Three independent assays were done for each sample.

The statistical analysis of the results were performed for the CFU values calculating the standard deviation values. The average values were compared by one-way analysis of variance and with the value of statistical significance. The one-way analysis of variance (one-way ANOVA) was used to compare the mean of the samples using the Fisher distribution. The response variable was approximated for the sample data obtained from the photocatalytic inactivation of test samples presenting the same distribution within the same sample (prepared at fixed anodization voltage).

A C C E P T E D M A N U S C R I P T

To verify that no attached/adsorbed bacteria remained on the surface, samples were incubated for 24 hours at 37°C on agar. No bacterial re-growth was observed.

The samples were irradiated in the cavity of an Atlas solar simulator (Atlas, GmbH, Hanau, Germany) with an overall power of 50 mW/cm 2 with light distribution wavelength distribution resembling solar irradiation emitting at wavelengths between 310 and 800 nm. The system contains an air-cooled Xenon lamp provided with filter to cut off wavelengths below 310 nm and above 800 nm. Samples were irradiated into covered glass petri dishes to avoid bacterial suspension evaporation due to the air-cooling. The material of the petri dishes does not cut the used light.

The adhesion of the E. coli on the TiO2-NTs and Ag/TiO2-NTs anodized surfaces was carried out by immersing the samples into 5 mL suspension of E. coli cell of a concentration of 4 10 6 CFU/ml. The tube was then shacked gently at 37°C for 4 h in dark [START_REF] Bonnefond | New evidence for hybrid acrylic/TiO2 films inducing bacterial inactivation under low intensity simulated sunlight[END_REF][START_REF] Seddiki | Evidence ofantibacterial activity on titanium surfaces through nanotextures[END_REF]. The non-adhered bacteria to the NTs were removed by washing the surface with phosphate buffer solution (pH 7.2).

The number of viable cells was determined after detachment of the adhered E. coli cells by ultrasonication (50 W) for 15 min. Non-adhered/weakly adhered bacteria on the surfaces were evaluated according to Hoffman [START_REF] Kadlec | A novel flotation technique for the separation of nonadherent microorganisms from a substrate[END_REF]. Bacterial adhesion experiments were carried out in the dark to avoid the photocatalytic action of the TiO2 and Ag/TiO2 surfaces.

2-3. Characterizations

The crystallinity and phase identification of the prepared samples were systematically investigated using X-ray diffraction (Philips X'PERT MPD, Cu Kα irradiation, λ=1.5406Å). The diffraction data were collected over the diffraction angle range of 20°-80° with a scanning step of 0.016°. The morphology and nanostructure of the Ag-NPs/TiO2-NTs samples were examined by scanning electronic microscope (SEM, Jeol JSM-6300) and FEI Tecnai G2 transmission electron microscopy (TEM) operating at 200 kV with a LaB6 filament. The local chemical analysis was performed using the energy dispersive X-ray spectroscopy (EDXS) system attached to the TEM.

The photoluminescence (PL) measurements on the Ag-NPs/TiO2-NTs samples were carried out by using a fluorescence spectrophotometer (Perkin Elmer LS55) equipped with a xenon lamp at an excitation wavelength of λ = 340 nm. The XPS measurements were performed using an AXIS NOVA (Kratos Analytical, Manchester, UK) photoelectron spectrometer with an achromatic Al Kα X-ray source at 400 W. The spectra were excited using Al Kα radiation (1486.6 eV). All XPS data were corrected from sample charging during XPS data acquisition, while using the C1s (284.6 eV) peak as a reference. The base pressure below 5 10 -9 mbar was maintained during the

A C C E P T E D M A N U S C R I P T measurement.
No argon sputtering was considered as to clean the coatings surface, neither ion or electron neutralizers were used during the measurement. The surface atomic concentration percentage for each element was determined from peak areas using the known sensitivity factors for each element [START_REF]Handbook of X-Ray Photoelectron Spectroscopy[END_REF][START_REF] Rivière | Handbook of Surface and Interface Analysis: Methods for Problem-Solving, 2 nd Edition[END_REF] on the surfaces. The spectrum background was subtracted according to Shirley [START_REF] Shirley | High-Resolution X-Ray photoemission spectrum of the valence bands of gold[END_REF] through the Shirley subtraction GL [START_REF] Pugazhenthiran | High surface area Ag-TiO2 nanotubes for solar/visible-light photocatalytic degradation of ceftiofur sodium[END_REF] program of the Kratos unit. The XPS spectra for the Ti and Ag-species were analyzed using CasaX-Vision 2 (Kratos Analytical, UK) and the peaks were assigned according to the NIST database. All measurements were done in the fixed analyzer transmission mode (FAT).

RESULTS AND DISCUSSIONS

X-ray diffraction (XRD) performed on the films showed Bragg reflections between 20 and 80°. Figure 1 shows the XRD spectra of the Ag-NPs/TiO2-NTs, which confirm that all samples, are crystallized in the anatase phase. The extra peaks observed at 2θ = 38.5°, 40.4°, 53.1°, 63°, 70.8° are due to the Ti metallic phase of the underlying substrate. The absence of silver related XRD peaks is mainly related to the very tiny amount of Ag NPs deposited on the TiO2-NTs.

Ag/TiO2 material have been extensively studied. Table 1 shows the diameter of TiO2-NTs expressed as mean value (± standard deviation) and the number of counted particles/NTs for the statistical analysis both for Ag-NPs and for TiO2-NTs. Crystallite size of TiO2-NTs are also estimated by the mean of XRD Scherrer equation as previously reported in detail [START_REF] Mangayayam | FeOx magnetization enhancing E. coli inactivation by orders of magnitude on Ag-TiO2 nanotubes under sunlight[END_REF]. Page et al., reported on Silver-titania nanocomposite as potent antimicrobial material and reported XRD peaks assigned to Ag2O at 2ɵ=31.5° [START_REF] Page | Titania and silver-titania composite films on glass -potent antimicrobial coatings[END_REF]. For our composite, this peak was not observed. This can be attributed to the few amounts of Ag that are likely below the XRD sensitivity threshold. It is readily seen that even at low anodization potentials, Ti crystallizes in anatase phase. This is due to the annealing step for 3 hours at 400°C as described in the experimental section. Recently, Mateus et al., observed anatase phase in TiO2 nanotubes prepared by different chemical solutions/precursors such as water/fluorhydric acid/ammonium fluoride/ethylene glycol (H2O/HF/NH4F/EG) as well as the conditions of anodization. The same authors showed by the mean of UV-Vis measurements that the band-gap varied with to the anodizing time and solution [START_REF] Mateus | Comparison of the Growth of TiO2 Nanotubes in Different Solutions[END_REF]. the NTs were not clearly seen. However, under 60 V anodization, a highly ordered TiO2 NTs, vertically aligned onto the Ti-substrate were formed as seen in Figure 2b. Figure 2c shows the diameter size distribution of TiO2 NTs anodized at 60 V with a mean diameter size of 97 nm. Figure 3d shows the evolution of mean diameter of TiO2-NTs as function of the applied anodization potentials. A maximum diameter size of 100 nm was reached for an anodization potential of 70 V.

The size distribution of Ag NPs was carried out using TEM on the Ag NPs decorated TiO2-NTs sample anodized at 60 V. Figure 3a (and the inset) shows a bare TiO2-NTs with typical outer diameter of 100 nm that confirm the SEM observation. The as-formed TiO2-NTs are seen in Figure 3b to be highly ordered and vertically aligned onto the underlying Ti-substrate. The observed nanotubes is 100 nm present a length is of ~15 µm (see the SEM cross-section view in the inset of Figure 3b). Figure 3c shows the TiO2-NTs after their Ag NPs decoration, the Ag NPs are attached on the external wall of the tube. In order to find the average size of Ag-NPs. It is normally difficult to efficiently deposit Ag-NPs in TiO2 nanotubes using the photo-reduction method. The TEM image were systematically analyzed as shown in Figure 3. The average size of Ag NPs decorating the TiO2-NTs is about 8 +/-2 nm.

Figure 4 shows the diffuse reflectance spectra of the anodized NTs. It is readily seen from Figure 4 that the spectral absorption of Ag-decorated samples are shifted to the visible region. The yellow zone of the figure shows the light emission of the used light source (solar simulated light, 310-800 nm). The UV-Vis absorbance spectra show that the absorption edges was bleu shifted with the increase of the NTs diameter, which can be explained by the increase of the crystallites average size. Although TiO2 is an indirect semiconductor and its energy gap is usually estimated by reflectance spectra, the difficulty to get accurate data (band-gaps) from DRS spectra in our case comes from the baseline/background correction before spectra acquisition. After anodization the substrate shows little colorization that is different from the raw material. This colorization limits to a large extend the possibility of using "non-anodized" material as baseline/background. Bandgaps (and intra-gap states) of the prepared NTs are described below by the mean of PL. nm) with a mono-modal peak at 390-450 nm or 2.7-3.1 eV. The PL spectra of Ag/TiO2-NTs show bimodal peaks. The first peak (390-450 nm) corresponds to TiO2-NTs; however, the second peak corresponds to the signal generated by Ag-NPs. This latter peak ranges from 460-650 nm corresponding to band energies 1.9-2.6 eV. The NTs defects were investigated through the shape and shifts in the intensity of the PL spectra correlated to electron/hole pairs and band-gap energies [START_REF] Pugazhenthiran | High surface area Ag-TiO2 nanotubes for solar/visible-light photocatalytic degradation of ceftiofur sodium[END_REF][START_REF] Pan | Engineering the intermediate band states in amorphous Ti 3+ -doped TiO2 for hybrid dyesensitized solar cell applications[END_REF]. The increase of the photoluminescence (PL) intensity of Ag/TiO2-NTs indicates the increase of the specific area when the nanotubes diameter increases. Figure 5b shows the bacterial inactivation mechanism on Ag-decorated TiO2-NTs. Ag NPs increase the separation of photogenerated charges and help in transferring the trapped electrons to the adsorbed O2. The Fermi level of Ag NPs is situated below the TiO2 conduction band. This promotes the interfacial charge transfer (IFCT) from TiO2-NTs to Ag-NPs [START_REF] Pugazhenthiran | High surface area Ag-TiO2 nanotubes for solar/visible-light photocatalytic degradation of ceftiofur sodium[END_REF] reducing consequently the charge recombination in TiO2.

XPS results showed the existence of Ti 4+ and Ti 3+ in the as-prepared samples. TiO2-NTs showed 89% Ti 4+ and 10% Ti 3+ as determined by XPS-peaks surface areas. Ag/TiO2-NTs showed slightly different proportions of 81% and 19%, respectively for Ti 4+ and Ti 3+ . Before their use for the antibacterial activity tests, the samples undergo the autoclaving step. After autoclaving, the samples showed slightly different surface properties (oxidative states) as detected by XPS. After autoclaving, the Ti 3+ content reached 22% for TiO2-NTs samples and up to 29% for Ag/TiO2-NTs samples. These changes in the oxidative states can be attributed to the molecular disorders at the NTs interface after autoclaving (H2Ovapor and pressure). After the bacterial inactivation time under light irradiation, the surface oxidative states were also followed. The Ti 3+ was seen to increase in all samples to 33 to 41% respectively for TiO2-NTs and Ag/TiO2-NTs. Ti-oxidative states (Ti +3 and Ti +4 ) have been reported to change between the as-prepared sample and the illuminated sample during the bacterial inactivation [START_REF] Rtimi | Innovative transparent non-scattering TiO2 bactericide thin films inducing increased E. coli cell wall fluidity[END_REF][START_REF] Rtimi | Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation[END_REF]. At these valences (+3 and +4), the ionic radii are located between 0.65 and 0.88 Å. In addition, Ag species used to decorate the TiO2-NTs exhibit oxidative states of +1 and +2. The ionic radii of these Ag-species were calculated to be 0.67-1.28 Å and 0.79-0.94 Å for Ag + and Ag 2+ , respectively [START_REF]Revised Effective Ionic Radii and Systematic Studies of Interatomic Distances in Halides and Chalcogenides[END_REF]. Ahrens previously calculated these radii from bond length -bond strength equations [START_REF] Ahrens | The use of ionization potentials Part 1. Ionic radii of the elements[END_REF]. The mechanism of bacterial inactivation on Ag/TiO2-NTs was reported in many studies before and will not be discussed in deep details in the present work.

A C C E P T E D

M A N U S C R I P T
Nonetheless, it is worth mentioning that this bacterial inactivation involves IFCT processes occurring between AgO/Ag2O and TiO2 [START_REF] Ubonchonlakate | Photocatalytic disinfection of P.aeruginosa bacterial Ag-doped TiO2 film[END_REF][START_REF] Rtimi | TiON and TiON-Ag sputtered surfaces leading to bacterial inactivation under indoor actinic light[END_REF][START_REF] Prakash | Noble metals-TiO2 nanocomposites: From fundamental mechanisms to photocatalysis, surface enhanced Raman scattering and antibacterial applications[END_REF].

Figure 6 shows E. coli inactivation on TiO2-NTs and Ag decorated TiO2-NTs under low intensity solar simulated light. The used light dose corresponds to the daylight dose during a cloudy day. It is readily seen in Figure 6 that TiO2-NTs by themselves reduced 1.6log E coli within 180 min. Ag decorated TiO2-NTs enhanced the bacterial inactivation. The fastest bacterial inactivation was seen to happen on Ag/TiO2-NTs anodized at 60 V and 70 V inactivating 99.99% E. coli within 90 min. It has been reported recently that bacteria can adhere to nanostructured TiO2 based surfaces due to Lifshitz-Van der Waals forces, hydrogen bonding, electrostatic interactions and Brownian motion interfacial forces [START_REF] Busscher | How Do Bacteria Know They Are on a Surface and Regulate Their Response to an Adhering State?[END_REF][START_REF] Tuson | Bacteria-surface interactions[END_REF]. This adhesion was also attributed to the cell wall proteins interaction with the surface [START_REF] Rabe | Understanding protein adsorption phenomena at solid surfaces[END_REF] and the surface wettability at the solid-liquid interface [START_REF] Kastantin | A bottom-up approach to understanding protein layer formation at solid-liquid interfaces[END_REF]. E. coli adhesion on the TiO2-NTs and Ag/TiO2-NTs in the dark showed that increasing the incubation time of bacteria with the surface increases the bacterial adhesion until a certain limit of ~10 3 CFU/ml/cm 2 . This load is weak compared to other surfaces able to harbor higher bacterial concentrations. This is due to: i) bacterial cell wall interaction with the polar groups on the NTssurface, ii) the wetting property of the NTs-surface (decorated with Ag or not), and iii) the surface potential "Eigenvalues" as recently reported [START_REF] Mena | Grafted semiconductors on PE-films leading to bacterial inactivation: synthesis, characterization and mechanism[END_REF]. It has been recently reported that the cell wall complexity controls the adhesion and invasion of biotic and abiotic surfaces [START_REF] Lamari | Assessment of the correlation among antibiotic resistance, adherence to abiotic and biotic surfaces, invasion and cytotoxicity of Pseudomonas aeruginosa isolated from diseased gilthead sea bream[END_REF]. Figure 6 shows that E. coli can adhere to TiO2-NTs in the dark. Under light, this bacterial adhesion facilitates the charge transfer between the photocatalyst and the bacterial cell wall leading to cell wall damage [START_REF] Baghriche | RF-plasma pretreatment of surfaces leading to TiO2 coatings with improved optical absorption and OH-radical production[END_REF]. The TiO2 photo-switchable surface wettability was extensively reported in the literature [START_REF] Rtimi | Innovative semi-transparent nanocomposite films presenting photo-switchable behavior and leading to a reduction of the risk of infection under sunlight[END_REF][START_REF] Ahmad | Visible-Light-Driven, Dye-Sensitized TiO2 Photo-Catalyst for Self-Cleaning Cotton Fabrics[END_REF] and is beyond the scope of the present study focusing more on the effect of the NTs geometry on the bacterial inactivation. Figure 6 shows also a slight bacterial inactivation in the dark on Ag/TiO2-NTs. This can be due to the diffusion of Ag-ions to the bacterial cytoplasm through the cell wall porins.

In addition, Figure 3d showed that the size distribution of Ag-NPs ranges from 1 to 12 nm. Nowack et al., [START_REF] Nowack | 120 Years of Nanosilver History: Implications for Policy Makers[END_REF] reviewed the use of nano-silver and the associated antibacterial effect during the last decades. Rtimi et al., reported that the dissolution of Ag-NPs releasing Ag-ions and attributed the antibacterial activity to an oligodynamic effect [START_REF] Rtimi | ZrNO-Ag co-sputtered surfaces leading to E. coli inactivation under actinic light: Evidence for the oligodynamic effect[END_REF][START_REF] Rtimi | Coupling of narrow and wide band-gap semiconductors on uniform films active in bacterial disinfection under low intensity visible light: Implications of the interfacial charge transfer (IFCT)[END_REF]. These ions are able to penetrate the bacterial cell envelop, reaching the cytoplasm and inactivating the cell machinery and [START_REF] Rtimi | Evidence for differentiated ionic and surface contact effects driving bacterial inactivation by way of genetically modified bacteria[END_REF][START_REF] Rtimi | First unambiguous evidence for distinct ionic and surface-contact effects during photocatalytic bacterial inactivation on Cu-Ag films: Kinetics, mechanism and energetics[END_REF]. The same study showed that porinless E. coli inactivation was six times longer than normal/wild E. coli (with porins). This study gave the evidence that Ag-ions are not the only agent leading to the bacterial inactivation but that surfacecontact effects play also an important role through ROS and/or charge transfer [START_REF] Rtimi | Evidence for differentiated ionic and surface contact effects driving bacterial inactivation by way of genetically modified bacteria[END_REF][START_REF] Rtimi | First unambiguous evidence for distinct ionic and surface-contact effects during photocatalytic bacterial inactivation on Cu-Ag films: Kinetics, mechanism and energetics[END_REF][START_REF] Ruales-Lonfat | Deleterious effect of homogeneous and heterogeneous near-neutral photo-Fenton system on Escherichia coli. Comparison with photo-catalytic action of TiO2 during cell envelope disruption[END_REF]. E. coli has a thinner peptidoglycan cell wall compared to the S. aureus and containing additional layers with a high structural complexity. The significant difference in the cell wall thickness/microstructure between these strains may lead to different interactions with TiO2-NTs and Ag/TiO2-NTs surfaces.

TiO2 was reported to damage the cell wall membrane through lipid-peroxidation [START_REF] Ruales-Lonfat | Deleterious effect of homogeneous and heterogeneous near-neutral photo-Fenton system on Escherichia coli. Comparison with photo-catalytic action of TiO2 during cell envelope disruption[END_REF] increasing the cell wall fluidity [START_REF] Rtimi | Innovative transparent non-scattering TiO2 bactericide thin films inducing increased E. coli cell wall fluidity[END_REF]. Nesic et al., reported recently on bacterial inactivation on TiO2 impregnated textiles in the dark [START_REF] Nesic | New evidence for TiO2 uniform surfaces leading to complete bacterial reduction in the dark: Critical issues[END_REF][START_REF] Rtimi | Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation[END_REF].

CONCLUSION

TiO2-NTs have been successfully prepared from the anodization of metallic Ti foils, and decorated them with Ag-NPs using the photochemical reduction method. By varying the anodization potential, we were able to increase the nanotubes diameter until reaching 100 nm for 70V. The increase of the anodization potential led to an increase in the crystallites sizes. It was also seen that PL spectra revealed band-gaps in the range of 2.7-3.1 eV for TiO2 and of 1.9-2.5 for AgxO. The reduced surface defects of TiO2 where seen to enhance the photocatalytic bacterial inactivation kinetics. The maximum photocatalytic performance of TiO2-NTs is obtained for NTs anodized at 60 and 70V. Bacterial adhesion was seen to be favorable in the dark. Under light irradiation, the bacterial inactivation was seen to be faster on larger NTs prepared at 60-70 V. The Ag/TiO2-NTs reported in this study have the potential to open new industrial segments in the rapidly developing health sector. Further research is required to translate the present findings into performing industrial products e.g. medical devices and implants.

Acknowledgment:

We thank the financial support from the Ministry of Higher Education, Scientific Research and Technology of Tunisia. We also thank the Swiss Federal Institute of Technology in Lausanne (EPFL). This article is dedicated to the retirement of Prof. H. Hofmann a key figure in the Nano-Bio-interactions at EPFL.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Figure 2

 2 Figure 2 Shows SEM top view images of Ag NPs decorated TiO2-NTs anodized respectively under potential 20 V (a) and 60 V (b). No tubular structures were seen when applying 20 V. At lower potentials, the fields assisted dissolution was not strong enough to remove the first anodic

Figure 5

 5 Figure 5 shows the typical PL spectra of TiO2-NTs and Ag decorated TiO2-NTs anodized at different potentials. When exited with UV light (see experimental) nano-crystalline TiO2 reveals a range of intra-bandgap defect states that emit at visible wavelengths as shown in the PL spectra.

  To probe the luminescent defect states of the anodized TiO2-NTs shown in this study, we used 340 nm excitation. The TiO2-NTs show a broad photo-luminescence in the visible (from 380 to 660

  thus leading to cell death. Recent reports used porinless bacteria to not allow the penetration of Agions through the bacterial cell wall porins

Figure 1 .

 1 Figure 1. XRD patterns of Ag-NPs decorated TiO2-NTs as a function of anodization potential.

Figure 2 .Figure 3 .

 23 Figure 2. SEM top view images of Ag-NPs decorated TiO2-NTs anodized respectively under : (a) potential 20 V, and (b) 60 V. (c) the variation of diameter of the TiO2-NTs as function of anodization potentials, and (d) the diameter size distribution of TiO2-NTs anodized at 60 V.

Figure 4 .

 4 Figure 4. Diffuse reflectivity spectra of TiO2-NTs decorated with Ag-NPs grown at different anodization potential. The yellow zone in the figure represents the emission range of the applied light (310-800 nm).

Figure 5 .

 5 Figure 5. a) Typical PL spectra of Ag-NPs decorated TiO2-NTs anodized at different potential; b) mechanism of bacterial inactivation on Ag decorated TiO2-NTs.

Figure 6 .

 6 Figure 6. Bacterial inactivation on TiO2-Nts and TiO2-NTs+Ag. Used light: solar simulated light (50 mW/cm 2 , 310-800 nm). For more details, see text. (Error bars: SD, n=5%).

Table 1 :

 1 TiO2-NTs diameter and Ag-NPs size expressed as mean value (± standard deviation).