Isabelle Legroux-Gérot

Jean Vignau

Odile Viltart

Pierre Hardouin

Christophe Chauveau

Bernard Cortet

Adipokines and bone status in a cohort of anorexic patients

Keywords: anorexia nervosa, adipokines, adiponectin, Pref-1, leptin, bone mineral density

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Introduction

The pathophysiological mechanisms involved in bone metabolism impairment in anorexia nervosa (AN) are still largely unknown. Various mechanisms have been proposed and are probably interrelated, including oestrogen deficiency, disturbances in the growth hormone (GH)-insulin growth factor-1 (IGF1) axis, hypercorticism, and vitamin D deficiency [START_REF] Legroux-Gérot | Evaluation of bone loss and mechanisms in anorexia nervosa[END_REF][START_REF] Munoz | Anorexia nervosa in female adolescents : endocrine and bone mineral density disturbances[END_REF][START_REF] Jacoangeli | Osteoporosis and anorexia nervosa : relative role of endocrine alterations and malnutrition[END_REF][START_REF] Misra | Alterations in growth hormone secretory dynamics in adolescent girls with anorexia nervosa and effects on bone metabolism[END_REF].

More recently, several studies have highlighted the role of fat tissue -as an endocrine organ in its own right -in controlling bone metabolism via leptin -and more generally adipokines -as well as factors involved in adipocyte/osteoblast differentiation, such as Preadipocyte factor-1(Pref-1) [START_REF] Is | Anorexia nervosa, osteoporosis and circulating leptin: the missing link[END_REF][START_REF] Biver | Influence of adipokines and ghrelin on bone mineral density and fracture risk : a systematic review and metaanalysis[END_REF][START_REF] Kajimura | Adiponectin regulates bone mass via opposite central and peripheral mechanisms through FoxO1[END_REF][START_REF] Aronis | Preadipocyte factor-1 levels are higher in women with hypothalamic amenorrhea and are associated with none mineral content and bone mineral density through a mechanism independant of leptin[END_REF]. Leptin is secreted primarily by adipocytes in subcutaneous fat -and to a lesser degree in visceral fat -and its serum level correlates with fat mass [START_REF] Spiegelman | Obesity and the regulation of energy balance[END_REF].

In addition to inhibiting appetite, leptin also regulates bone mass through a dual mechanism producing, on one hand, an inhibitory effect by stimulating the sympathetic nervous system, and on the other hand, a stimulatory effect by binding directly to receptors on osteoblasts [START_REF] Ducy | Leptin inhibits bone formation through a hypothalamic relay : a central control of bone mass[END_REF][START_REF] Karsenty | Convergence between bone and energy homeostases : leptin regulation of bone mass[END_REF]. Adiponectin regulates energy homeostasis through the modulation of glucose and fatty acid metabolism in peripheral tissues. It circulates in the bloodstream in the form of oligomers of different sizes. Low levels of circulating adiponectin have been reported in obesity and type 2 diabetes [START_REF] Weyer | Hypoadiponectinemia in obesity and type 2 diabetes : close association with insulin resistance and hyperinsulinemia[END_REF][START_REF] Nakashima | Decreased total and high molecular weight adiponectin are independent risk factors for the development of type 2 diabetes in Japanese-Americans[END_REF]. In osteoporosis, several studies have mentioned its association with low bone mass [START_REF] Biver | Influence of adipokines and ghrelin on bone mineral density and fracture risk : a systematic review and metaanalysis[END_REF][START_REF] Jurimae | Adiponectin is associated with bone mineral density in perimenopausal women[END_REF][START_REF] Napoli | Adiponectin and bone mass density : The InCHIANTI study[END_REF]. Serum adiponectin levels seem to correlate inversely with fat mass; in situations of energy deficit, bone marrow adipose tissue may be a major source of circulating adiponectin [START_REF] Cawthorn | Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction[END_REF]. This could explain the higher serum adiponectin levels reported by some authors in anorexic patients with osteoporosis, in whom histological and MRI analyses have indicated an increase in marrow adiposity [START_REF] Delporte | Hyperadiponectinaemia in anorexia nervosa[END_REF][START_REF] Pannacciulli | Anorexia nervosa is characterized by increased adiponectin plasma levels and reduced nonxidative glucose metabolism[END_REF][START_REF] Fazeli | Marrow fat and bone-New perspectives[END_REF][START_REF] Abella | Bone marrow changes in anorexia nervosa are correlated with the amount of weight loss and not with other clinical findings[END_REF]. The increase in marrow adiposity reported in anorexic patients with osteoporosis probably plays a role in bone loss [START_REF] Abella | Bone marrow changes in anorexia nervosa are correlated with the amount of weight loss and not with other clinical findings[END_REF][START_REF] Ecklund | Bone marrow changes in adolescent girls with anorexia nervosa[END_REF][START_REF] Hardouin | L'adiposité médullaire osseuse[END_REF]. The physical proximity of adipocyte and osteoblast cells in bone marrow has led to the hypothesis that a dialogue exists between these cells. Further, adiponectin receptors have been reported on osteoblast cells, and serum adiponectin contributes to low bone mass by promoting osteoclast activity [START_REF] Luo | Adiponectin stimulates RANKL and inhibits OPG expression in human osteoblats through the MAPK signaling pathway[END_REF]. Moreover, as osteoblast and adipocyte cells come from the same cell line, the decrease in osteoblast differentiation would tend to induce precursors to take the adipocyte pathway.

Previous studies have shown conflicting results for patients with anorexia nervosa (18,17,24,25,26,). One explanation should be that adiponectin level is related to the severity of the disease and previous studies did include heterogeneous population. Moreover most of the previous studies focused on total adiponectin and not on high molecular weight (HMW) adiponectin.

Also very few studies did assess both leptin and adiponectin in patients with AN and their relative role for explaining bone involvement remain controversial [START_REF] Maïmoun | Evidence of a link between resting energy expenditure and bone remodelling, glucose homeostasis and adipokine variations in adolescent girls with anorexia nervosa[END_REF].

Pref-1 is secreted primarily by preadipocytes but also by many tissues like placenta, pituitary gland and adrenal glands. It belongs to the Epithelial Growth factor (EGF) family of proteins and regulates bone-marrow osteoblast and adipocyte differentiation. Plasma Pref-1 was found to be high in anorexic patients, and to correlate negatively with blood leptin and whole body bone mineral density (BMD), but positively with marrow adiposity in L4 lumbar vertebrae [START_REF] Fazeli | Marrow fat and preadipocyte factor-1 levels decrease with recovery in women with anorexia nervosa[END_REF].

The present study aimed 1-To explore the relationship between leptin, adiponectin, Pref-1 levels and phenotypes of patients with anorexia nervosa according to their BMI and BMD status (recovery, slight decrease in BMD and BMI, slight decrease in BMD and large decrease in BMI, large decrease in BMD and slight decrease in BMI and finally large decrease in both BMI and BMD) 2-To explore the additive values of these 3 previous parameters compared to other more classical parameters who play a role in the pathophysiology of AN or in bone involvement (estrogens, IGF-1 and markers of bone remodelling).

3-To assess the role of both leptin, adiponectin and Pref-1for explaining the variability of BMD in the whole cohort and particularly in women with osteoporosis.

Methods

Patients

The study involved 80 anorexia nervosa patients. Mean BMI was 16.8 ± 2.4 kg/m 2 . All patients were being followed up in the Department of Psychiatry (CHR-U de Lille) and were referred to the Department of Rheumatology for a bone-health assessment. The assessment was approved by the local Ethics Committee, and all patients gave their written informed consent to participate (No. 2012 A01009 34).

We recorded patients' weight and height, and determined, by interview, their risk factors for osteoporosis as well as the types of treatment they had been receiving. According to their weight and bone status, we divided our population into 5 groups: Group 1: Normal BMD and BMI and regular menstrual cycle Group 2: Osteopenia (-2 < Z-score < -1) and BMI >17 kg/m 2 Group 3: Osteopenia and BMI≤17 kg/m 2 Group 4: Osteoporosis (Z-score ≤ -2) and BMI >17 kg/m2 kg/m 2 Group 5: Osteoporosis and BMI≤17 kg/m 2 Patients assigned to Group 1 had had one episode of anorexia nervosa but were considered "cured", having regained weight and a regular menstrual cycle. This group served as the "control" group.

For assessing the relationship between BMD (raw data and z-scores) and adiponectin metabolism, we divided adiponectin levels (total adiponectin, HMW adiponectin and % HMW) in tertiles and analysed in the whole anorexic population, the distribution of adiponectin levels in tertiles against BMD and Z score.

Biological data

Plasma concentrations of leptin, total adiponectin, high molecular weight (HMW) adiponectin, and Pref-1 were measured. Blood and urinary calcium and phosphate levels were also determined (calcemia, phosphatemia, alkaline phosphatases, 24-hour calciuria), and 25hydroxyvitamin D3 -25(OH)D3 -and parathyroid hormone (PTH), FSH, LH, estradiol, cortisol, somatomedin and bone remodelling markers Plasma leptin concentrations were measured by radio immunologic assay (normal values: 3.7 to 11.1 ng/ml). Plasma concentrations of total adiponectin (µg/ml), high molecular weight adiponectin (HMW adiponectin µg/ml), and Pref-1 (ng/ml) were determined by ELISA (Quantikine Immunoassay kits, R&D Systems Inc., Minneapolis, USA). The ratio of HMW adiponectin to total adiponectin -HMW (percentage) -was calculated. Bone remodelling markers -osteocalcin and bone alkaline phosphatase, for bone formation, and serum crosslaps or CTX and for bone resorption -were assessed. Osteocalcin total was measured by radioimmunoassay (Cis-Bio International, Gif-sur-Yvette; normal values: 10.4-45.6 ng/ml). Bone alkaline phosphatase were measured using a human-specific immunoradiometric method (Hybritech, Inc., Dan Diego, CA, USA; normal values: 2.9-14.5µg/l). Serum CTX was measured by immuno-enzyme assay (ELISA) (serum crossLaps One Step, Osteometer Biotech, Herlev; normal values: 232-5115 pmol/l). IGF-I was measured by chemiluminescence (normal values : 114-451 ng/ml).

All biological samples were collected from fasting patients between 8 a.m. and 10 a.m. at inclusion.

Measurement of BMD, body fat and fat-free soft tissues

Lumbar-spine and hip bone mineral densities were determined by dual-energy X-ray absorptiometry using a Discovery device (Hologic Inc., Bedford MA, USA). The coefficient of variation was 1% for lumbar spine measurements and 2% for hip measurements. Given the mean age of our population, results were expressed as Z-scores, and we used the WHO criterion for osteoporosis, i.e. Z-score < -2.

We also performed a total body composition analysis, measuring lean mass, fat mass, and percentage fat.

Statistical analysis

Results were expressed as mean ± standard deviation. Groups were compared using ANOVA or the Kruskal-Wallis test. When significant differences were found, the results were compared 2-by-2 using Student's t-test for independent samples, or the Mann-Whitney U test.

Lastly, stepwise multiple regression analysis was used to evaluate the factors affecting the variance in BMD. A p value <0.20 was retained for selecting the variables included in the stepwise analysis.

Role of the funding source

This study was conducted through a grant obtained from the Nord Pas de Calais District (ARCIR 2009 : actions de recherches concertées d'initiative régionale)

Results

Clinical characteristics

Our study involved 80 female patients, aged 23.8 ± 4.7 years, diagnosed with anorexia nervosa (AN) according to DSM-IV criteria. The mean duration of anorexia was 5.5 ± 4 years. Mean age of disease onset was 18.2 ± 4.4 years. Mean duration of amenorrhea was 3.4 ± 3.8 years, with a mean age of onset of 19.6 ± 4.4 years. Of the 80 patients, 56 (70%) were found to have pure restrictive anorexia while 24 (30%) exhibited a mixed form of the disorder (bulimia and anorexia). Mean weight was 46.1 ± 7.3kg. Mean BMI was 16.8 ± 2.4 kg/m 2 .

The clinical data for the various groups are shown in Table I. There was no significant difference in the mean age of patients across the various groups. Mean age of disease onset was significantly lower in Group 2 compared to Groups 1, 3 and 5. There was no difference in disease duration. BMI was obviously significantly lower in Groups 3 and 5 compared to Group 1, Group 2 and Group 4.

Biological analysis of plasma samples

The results are shown in Figure 1.

There was no significant difference in total and HMW adiponectin plasma concentrations between the 5 groups. HMW (percentage) was significantly higher in Group 5 compared to Group 1. No significant difference was observed for Pref-1. Leptin was significantly lower in Groups 3 and 5 compared to the other groups; in Groups 3 and 5 versus 1 and 2, and in Groups 3 and 5 versus 4. Plasma concentrations of IGF-I were also significantly lower in Groups 3 and 5 compared to Group 2 (p=0.008 and p=0.01 respectively). No difference was observed for the other parameters (serum calcium and phosphate, bone remodelling markers, thyroid function, hormone levels).

When we divided the adiponectin levels in tertiles and analysed the distribution against BMD and Z score, we found significant results with total adiponectin and HMW adiponectin but not with %HMW. The total adiponectin levels were higher in the lower tertile for spine (p=0.01) and femoral neck BMD (p=0.002). The results were similar, when we expressed the results as Z scores for all the sites: spine (p=0.02), hip (p=0.01), femoral neck (p=0.03) and whole body (p=0.01). The HMW adiponectin levels were higher in the lower tertile for spine (p=0.01), femoral neck (p=0.001) and whole body BMD (p=0.02). The results were similar for Z scores for all the sites: spine (p=0.01), hip (p=0.003), femoral neck (p=0.01) and whole body (p=0.02) (Figure 2). The same analyses were done with % HMW but the results were not significant.

Bone mineral density and body composition

In our population of 80 patients, 35 (43.75%) had osteoporosis at least at one site and 30 (37.5%) had osteopenia (Z-scores between -1 and -2). BMD was found to be normal in 15 patients (18.75%).

The results for the various groups are shown in Table 2. As expected, BMD at all 3 sites was significantly lower in the osteoporosis groups -Groups 4 and 5 -than in Groups 1, 2 and 3.

Regarding body-composition analysis results, percentage fat and fat mass were found to be significantly lower in Groups 3 and 5 compared to Groups 1, 2 and 4 (p<0.0001). A significant drop in lean mass was also observed in Group 5 compared to Groups 1, 2 and 4 (p=0.002), and in Group 3 compared to Group 1 (p=0.001) and Groups 2 and 4 (p=0.01).

Linear regression analysis

We sought correlations between adiponectin levels and the other biological parameters and BMD results in the entire study population (Table 3), in Groups 4 and 5 -patients with osteoporosis -in Groups 3 and 5 (patients with BMI≤17 kg/m 2 )(Table 4).

In the entire study population

No correlation was found between adipokines and BMI. Negative correlations were found between weight and HMW adiponectin and weight and HMW (percentage). Total adiponectin correlated negatively with fat mass and IGF-I. HMW adiponectin was correlated negatively with percentage fat, fat mass, lean mass and IGF-I. HMW (percentage) correlated positively with PTH and blood estradiol. No correlation was found between the various adipokine forms and insulin. Pref-1 correlated positively with HMW (percentage) (r=0.24; p<0.05), but not with total adiponectin or HMW adiponectin.

Positive correlations were found between fat mass and BMI (r=0.81; p<0.0001), IGF-1 (r=0.46; p<0.0001), insulin (r=0.58; p<0.0001) and leptin. Additionally, percentage fat correlated positively with BMI (r=0.75; p<0.0001), IGF-1 (r=0.46; p<0.0001), insulin (r=0.54; p<0.0001), and leptin. There was a positive correlation between lean mass and BMI (r=0.64; p<0.0001) and leptin, but not with IGF-1 or insulin.

No correlation was found between spine BMD and the biological parameters. Femoral neck BMD correlated negatively with duration of amenorrhea (r=-0.37; p<0.01), total adiponectin, and HMW adiponectin, but positively with BMI (r=0.32; p<0.01). Total hip BMD correlated negatively with duration of amenorrhea (r=-0.35; p<0.01), total adiponectin (r=-0.31; p<0.01), and HMW adiponectin, but positively with BMI (r=0.38; p<0.001). Whole body BMD also correlated negatively with total adiponectin. No correlation was found between BMD at any of the sites and Pref-1 or leptin. Markers of bone remodelling were not correlated with both BMD and other biological variables.

In Groups 4 and 5 (patients with osteoporosis)

Spine BMD correlated positively with weight (r=0. [START_REF] Zgheib | Long term physiological alterations and recovery in a mouse model of separation associated with time-restricted feeding : a tool to study anorexia nervosa related consequences[END_REF] For femoral neck BMD, the 2 explanatory factors were BMI and total adiponectin (adjusted R-squared=0.14, p=0.002). For total hip, the variance in BMD was explained by 3 factors: leptin, BMI, and total adiponectin (adjusted R-squared = 0.27, p<0.0001).

In the patients with osteoporosis -Groups 4 and 5 -the factors explaining the variance in spine BMD were leptin, BMI and total adiponectin (adjusted R-squared=0.35, p=0.001). For femoral neck, the factors explaining the variance in BMD were BMI and total adiponectin (adjusted R-squared=0.2, p=0.01), and for total hip the 3 explanatory factors were leptin, BMI, and total adiponectin, which explained a significant part of the variance at this site (adjusted R-squared=0.47, p<0.0001). For whole body BMD, leptin, BMI, and total adiponectin (adjusted R-squared=0.33, p= 0.002) were also found to be explanatory factors.

In patients with BMI≤17 kg/m 2 , the factors explaining the variance in spine BMD were BMI and total adiponectin (adjusted R-squared=0.21, p=0.004). This was also the case for femoral neck and total hip BMDs (adjusted R-squared=0.22, p=0.003 and 0.27, p=0.001 respectively).

For whole body BMD, the factors explaining the variance in BMD were BMI and total adiponectin (adjusted R-squared=0.21, p=0.004).

Discussion

Our study involved a sizeable cohort of anorexia nervosa patients, allowing us to form subgroups based on BMI and bone status. Group results were compared with Group 1, which comprised patients who had a history of anorexia nervosa but could be considered "cured".

No difference was found in total serum adiponectin, but a significant increase in HMW (percentage) adiponectin was observed in Group 5 -the group of patients with osteoporosis and BMI ≤17 kg/m 2 -compared to Group 1. Moreover, the total adiponectin levels and HMW adiponectin were higher in the lower tertile for spine and femoral neck BMD. The results were similar, when we expressed the results as Z score for all the sites: spine, hip, femoral neck and whole body. In anorexia nervosa patients, adiponectin results are contradictory. Whereas earlier studies [START_REF] Amitani | The role of adiponectin multimers in anorexia nervosa[END_REF][START_REF] Tagami | Adiponectin in anorexia nervosa and bulimia nervosa[END_REF] reported lower or unchanged adiponectin levels in anorexia nervosa patients compared to controls, a more recent study conducted by Terra et al (26) compared 28 patients to 33 controls and reported significantly higher concentrations of circulating total and HMW adiponectin in patients. The patients were older, with a mean age of 27.4 ± 1.4 ans. In a study involving 11 anorexia nervosa patients (mean age 16.4 years) compared to 26 controls, Pannacciulli et al (18) also found significantly higher adiponectin levels in their patient group. Similarly, in a study conducted by Delporte et al [START_REF] Delporte | Hyperadiponectinaemia in anorexia nervosa[END_REF] involving 26 anorexia nervosa patients versus 24 controls, adiponectin levels were found to be 30% higher in the patient group while leptin levels were lower. They also reported negative correlations between adiponectin and BMI and adiponectin and fat mass. The most recent data seem to argue in favour of an increase in HMW adiponectin -and indeed total adiponectin -in anorexia nervosa patients with osteoporosis. In our study, we found no difference in total adiponectin, but in the absence of a real control group, comparisons with data from the literature are difficult.

We sought correlations between adipokines and other metabolic parameters and found that total adiponectin correlated negatively with fat mass. HMW adiponectin was also found to correlate negatively with percentage fat, fat mass and lean mass. In the study by Terra et al [START_REF] Terra | Adipocytokine levels in women with anorexia nervosa. Relationship with weight restoration and disease duration[END_REF], the authors also reported a negative correlation between total adiponectin and percentage fat, but also BMI and disease duration after adjusting for age. Several authors have also mentioned negative correlations between adiponectin and BMI [START_REF] Terra | Adipocytokine levels in women with anorexia nervosa. Relationship with weight restoration and disease duration[END_REF][START_REF] Amitani | The role of adiponectin multimers in anorexia nervosa[END_REF], contrary to our findings. These data tend to confirm a negative correlation between total adiponectin and fat mass.

Thus, anorexia nervosa patients with low BMIs were able to produce adiponectin despite having little peripheral fat, which suggests that the adiponectin is not being secreted by subcutaneous or visceral adipocytes.

Several authors have reported an increase in circulating adiponectin levels during caloric restrictions in animals and man [START_REF] Pannacciulli | Anorexia nervosa is characterized by increased adiponectin plasma levels and reduced nonxidative glucose metabolism[END_REF][START_REF] Tagami | Adiponectin in anorexia nervosa and bulimia nervosa[END_REF][START_REF] Dolezalova | Changes of endocrine function of adipose tissue in anorexia nervosa : comparison of circulating levels versus subcutaneous mRNA expression[END_REF], despite the loss of visceral and subcutaneous fat.

A strong relationship between resting energy expenditure and adipokines was found in a recent study which explored both leptin and adiponectin in AN patients [START_REF] Cawthorn | Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction[END_REF]. The authors demonstrated a positive correlation with resting energy expenditure and markers of bone formation, leptin and a negative correlation with bone resorption markers but no correlation with BMD. In the present study we did not find relationships between the level of bone remodelling markers and both biological variables (including adipokines) and BMD whereas we found a significant negative correlation between adiponectin levels and hip BMD as Maïmoun et al found [START_REF] Maïmoun | Evidence of a link between resting energy expenditure and bone remodelling, glucose homeostasis and adipokine variations in adolescent girls with anorexia nervosa[END_REF]. Also Maïmoun et al [START_REF] Maïmoun | Evidence of a link between resting energy expenditure and bone remodelling, glucose homeostasis and adipokine variations in adolescent girls with anorexia nervosa[END_REF] measured only total adiponectin whereas we measured different type of adiponectin according to their molecular weight. Finally they did not perform multivariate analysis perhaps owing to the sample size which was smaller compared with our study (50 AN patients against 80 in our own study). Cawthorn et al [START_REF] Cawthorn | Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction[END_REF] compared adiponectin secretion in peripheral fat and marrow fat in mice and rabbits. They found that marrow fat secreted more adiponectin than peripheral fat, and that mice with no marrow fat did exhibit an increase in serum adiponectin levels in response to caloric restrictions but this increase is blunted compared to wild-type control mice . These results clearly demonstrate that the increase in marrow fat observed during caloric restrictions contributes to increasing serum adiponectin levels, and several recent studies have reported an increase in marrow fat, evaluated by MRI, in anorexia nervosa patients with osteoporosis [START_REF] Bredella | Increased bone marrow fat in anorexia nervosa[END_REF][START_REF] Ecklund | Bone marrow changes in adolescent girls with anorexia nervosa[END_REF]. However, the role of adiponectin in bone metabolism remains unclear, and other animal studies failed to confirm these results [START_REF] Zgheib | Long term physiological alterations and recovery in a mouse model of separation associated with time-restricted feeding : a tool to study anorexia nervosa related consequences[END_REF]. Lastly, data from studies in man are lacking or insufficient. Unlike some studies, we did not find a correlation between adiponectin and spine BMD in our population. In a study comparing 17 anorexia nervosa patients to 19 controls, Misra et al [START_REF] Misra | Relationships between serum adipokines, insulin levels, and bone density in girls with anorexie nervosa[END_REF] found an inverse correlation between spine BMD, as well as femoral neck and whole body BMD, and adiponectin. In our study we also found an inverse correlation between femoral neck, total hip BMD and total and HMW adiponectin ; however spine BMD correlated inversely with total and HMW adiponectin in only anorexia nervosa patients with osteoporosis. These correlations were also found in patients with BMI≤17 kg/m2. Contrary to other authors, we found no significant difference in serum Pref-1 level between the various groups in our study, but we did not have a group of healthy controls without a history of anorexia nervosa. We found no correlation between serum Pref-1 levels and fat mass, percentage fat and lean mass results from BMD examinations. For the various adiponectin forms, we only found a positive correlation between Pref-1 and HMW (percentage, r=0.24; p=0.03). However, when the two groups of patients with BMI≤17 kg/m2 were considered together, a moderate correlation was observed between spine BMD and Pref-1 (r=0.32; p=0.04). Pref-1 seems to be an interesting candidate since it regulates marrow osteoblast and adipocyte differentiation. Aronis et al [START_REF] Aronis | Preadipocyte factor-1 levels are higher in women with hypothalamic amenorrhea and are associated with none mineral content and bone mineral density through a mechanism independant of leptin[END_REF] reported an increase in serum Pref-1 in 18 anorexia nervosa patients compared with healthy subjects. They also reported negative correlations between Pref-1 and BMD, and positive correlations with marrow fat, in both anorexia nervosa patients and healthy subjects. Other authors, such as Fazelli et al [START_REF] Fazeli | Preadipocyte factor-1 is associated with marrow adiposity and bone mineral density in women with anorexia nervosa[END_REF], also reported an increase in Pref-1 levels in anorexia nervosa patients compared to healthy subjects, as well as an inverse correlation between Pref-1 and percentage fat. Pref-1 is one of many factors involved in adipocyte differentiation and its role may vary depending on the nutritional status of the patient. The role of serum Pref-1 in peripheral fat deposits is unknown and further studies will be needed to determine its effect on marrow fat compared to visceral and subcutaneous fat.

We also found that serum leptin levels were significantly lower in Groups 3 and 5 (with BMI≤17 kg/m2) compared to the other groups. In the literature, several authors reported low leptin levels in anorexia nervosa patients [START_REF] Misra | Secretory dynamics of leptin in adolescant girls with anorexia nervosa and healthy adolescents[END_REF][START_REF] Lear | Body fat, caloric intake and plasma leptin levels in women with anorexia nervosa[END_REF][START_REF] Haluzik | Serum leptin levels in patients with anorexia nervosa before and after partial refeeding, relationships to serum lipids and biochemical nutritional parameters[END_REF]. We previously evaluated serum leptin levels in 103 anorexia nervosa patients grouped according to bone status and found low levels in patients with osteoporosis [START_REF] Is | Anorexia nervosa, osteoporosis and circulating leptin: the missing link[END_REF]. In the previous study when multivariate analyses were performed, the independent factors that could best explain the variance in spine BMD were found to be duration of amenorrhea and leptin level, which accounted for 28% of the variance. On the other hand in the previous study, leptin did not explain the variance in hip BMD. Similarly in the present study, leptin and BMI were found to explain 10% of the variance in spine BMD. However, when patients with osteoporosis were considered alone, 35% of the variance in spine BMD was explained by 3 factors: leptin, BMI and total adiponectin. For femoral neck BMD, 14% of the variance was explained by BMI and total adiponectin. In anorexia nervosa patients with osteoporosis, the figure increased to 20% with the same factors. For total hip, 27% of the variance in BMD was explained by 3 factorsleptin, BMI, and total adiponectin, and in anorexia nervosa patients with osteoporosis, the figure increased to 47% with the same factors. These findings suggest that adipokines seem to particularly influence BMD for patients with the most severe disease (low BMI and or low BMD).

Moreover apart from IGF-I level, other parameters measured in this present study (estradiol, FSH, LH, cortisol, markers of bone turn over) did not seem to play a role for explaining phenotypes of anorexia nervosa patients according to BMD and BMI. In the same manner these parameters did not explain the variability of BMD.

There are certain limitations to our study. We did not have a real control group, but we compared our patients with those in Group 1, who were anorexia nervosa patients who were considered "cured" and who had normal BMIs and BMDs. Therefore this Group 1 could be considered as a surrogate for a control group. We divided our population into several groups according to bone status and BMI to reflect different disease profiles. While this approach may seem logical, it necessarily results in fewer subjects in each group. Lastly, since our study was a cross-sectional study, no longitudinal data is available. However, compared to data in the literature, our study group was sizeable, and many parameters were analysed in detail.

This study argues in favour of adipokine involvement as an explanatory factor of bone status impairment in anorexia nervosa. The variance in BMD in anorexia nervosa patients with osteoporosis was explained predominantly by 3 factors: BMI, leptin, and adiponectin. To the extent that these 3 factors are, by definition, independent -which is the principle underlying step-wise linear regression analysis -our hypothesis is that BMI is a reflection of lean mass, leptin a reflection of subcutaneous and visceral fat, and adiponectin a possible reflection of marrow fat.

Conflits d'intérêts

Isabelle Legroux-Gérot, Jean Vignau, Odile Viltart, Pierre Hardouin, Christophe Chauveau Bernard Cortet declare that they have no conflict of interest.

  ; p<0.05), and BMI (r=0.35; p<0.05), but negatively with total adiponectin and HMW adiponectin. Femoral neck BMD also correlated positively with weight (r=0.43; p=0.01) and with fat mass (r=0.35; p<0.05), and negatively with total adiponectin. Total hip BMD correlated positively with weight (r=0.64; p<0.0001), BMI (r=0.54; p=0.001), percentage fat (r=0.41; p=0.01), fat mass (r=0.51; p=0.01), and lean mass (r=0.54; p<0.001), and negatively with total adiponectin and HMW adiponectin. Whole body BMD correlated positively with weight (r=0.37; p<0.05), BMI (r=0.33; p<0.05), and lean mass (r=0.38; p<0.05), and negatively with total adiponectin and HMW adiponectin. Markers of bone remodelling were not correlated with both BMD and other biological variables.3.4.3. In Groups 3 and 5 (patients with BMI≤17 kg/m 2 )Spine BMD correlated negatively with total adiponectin, HMW adiponectin, and Pref-1.Femoral neck BMD correlated positively with weight (r=0.32; p<0.05) and BMI (r=0.34; p<0.05), and negatively with total adiponectin. Total hip BMD correlated positively with weight (r=0.35; p<0.05) and BMI (r=0.34; p<0.05), and negatively with total adiponectin and HMW adiponectin. Markers of bone remodelling were not correlated with both BMD and other biological variables.Multivariate analysis performed on the entire study population revealed that 2 factorsleptin and BMI (adjusted R-squared = 0.1, p=0.008) -explained the variance in spine BMD.

Figure 1 :Figure 2 :

 12 Figure 1: Biological description of the population in the 5 groups * Groups 3 and 5 vs. Groups 1 and 2, p=0.0002; Groups 3 and 5 vs. Group 4, p=0.003 ° Group 5 vs. Group 1, p=0.04

4 :

 4 Correlations between adipokines and bone parameters in osteoporosis or BMI<17.

Table 1 :

 1 Clinical description of the population in the 5 groups

		Group 1	Group 2	Group 3	Group 4	Group 5
		(n=15)	(n=9)	(n=21)	(n=15)	(n=20)
	Age years	25.0 ± 4.4	22.9 ± 3.9	24.7 ± 4.7	21.9 ± 3.9	23.9 ± 5.7
	Age onset AN years	19.3 ± 3.4	15.3 ± 3.8*	19.1± 5.2	16.7 ± 3.2	19 ± 4.4
	Age onset AMN	21.5 ± 4.2	17.5 ± 4	20.6 ± 4.9	18 ± 3.6	19.5 ± 4.4
	years
	Duration AN years	5.8 ± 3.7	7.5 ± 5.2	5.1 ± 4.2	5.2 ± 3.1	4.9 ± 4.1
	Duration AMN	1.9 ± 2.9	3.4 ± 3.4	3.4 ± 4.7	2.4 ± 1.6	4.6 ± 4.2
	years
	Weight (kg)	51.8 ± 4.2	53.2 ± 7.2	41.3 ± 4.5**	50.1 ± 6.2	40.5 ± 4.1**
	Height (cm)	168 ± 5.8	164.6 ± 6.0	164.4 ± 5.3	165.8 ± 4.8	164.7 ± 5.9
	BMI kg/m 2	18.5 ± 1.7	19.5 ± 1.9	15.3 ± 1.5**	18.3 ± 1.6	14.9 ± 1.2**
	** BMI and weight are significantly lower in Groups 3 and 5 compared to Group 1 (p<0.0001)
	*Mean age of disease onset is significantly lower in Group 2 compared to Group 1,3 and 5 (p=0,003)

Table 2 :

 2 Densitometric description and total body analysis results of the population in the 5 groups

		Group 1	Group 2	Group 3	Group 4	Group 5
		(n=15)	(n=9)	(n=21)	(n=15)	(n=20)
	Spine BMD	0.95 ± 0.24	0.85 ± 0.05	0.89 ± 0.06	0.78 ± 0.06*	0.73± 0.08*
	Spine Z-score	-0.05 ± 1.01	-1.54 ± 0.49	-1.11 ± 0.6	-2.67 ± 0.68*	-2.68 ± 0.81*
	Hip BMD	0.93 ± 0.09	0.8 ± 0.06	0.82 ± 0.07	0.78 ± 0.09*	0.66 ± 0.09*
	Hip Z-score	-0.16 ± 0.77	-1.21 ± 0.49	-0.99 ± 0.59	-1.46 ± 0.77*	-2.3 ± 0.64*
	Neck BMD	0.82 ± 0.1	0.69 ± 0.04	0.72 ± 0.07	0.66 ± 0.08*	0.6 ± 0.09*
	Neck Z-score	-0.35 ± 0.99	-1.48 ± 0.38	-1.17 ± 0.56	-1.87 ± 0.68*	-2.25 ± 0.73*
	% fat	26.8 ± 5.3	29.5 ± 7.8	19.2 ± 3.8 #	27.2 ± 6.8	19.5 ± 4.5 #
	Fat mass (g)	13899 ± 3587	16205 ± 6810	7927 ± 1924 #	13610 ± 4308	7831 ± 2274
						#
	Lean mass (g)	35163 ± 3299	34796 ± 3026	31119 ± 3934	34104 ±	30245 ±
				#	4733	2211 #
	*BMD significantly lower Groups 4 and 5 vs. Groups 1,2 and 3
	# Values significantly lower Groups 3 and 5 vs. Groups 1, 2 and 4

Table 3 :

 3 Correlations between adipokines and other parameters in the entire study population*BMD

		Clinical				Biological			Bone
		data				data
	Adipokines	Weight	Fat mass	% fat	Lean	IGF-1	PTH	Blood	Whole	Spine	Femoral	Total
					mass			estradiol	Body	BMD	Neck	Hip
									BMD		BMD	BMD
	Leptin	ns	r = 0.77
		*p<0,05; # p≤0,01; ° p<0,0001


Emmanuelle Lipka
email: emmanuelle.lipka@univ-lille.fr

Anca-Elena Dascalu

Yasmine Messara

Eka Tsutsqiridze

Tivadar Farkas

Bezhan Chankvetadze

Separation of enantiomers of native amino acids with polysaccharide-based chiral columns in supercritical fluid chromatography

Keywords: Separation of enantiomers, Polysaccharide-based chiral stationary phases, Native aminoacids, Mobile phase composition, Extra column band-broadening

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

Introduction

Although polysaccharide derivatives represent the group of most universal chiral selectors for liquid phase separation of enantiomers [START_REF] Shen | Efficient separation of enantiomers using stereoregular chiral polymers[END_REF][START_REF] Chankvetadze | Recent developments on polysaccharide-based chiral stationary phases for liquid-phase separation of enantiomers[END_REF][START_REF] Leek | Strategy for largescale isolation of enantiomers in drug discovery[END_REF], there are certain groups of chiral analytes towards which these chiral selectors have been reported to exhibit inadequate chiral recognition ability. Small nonaromatic carboxylic acids and most native amino acids belong to this category of chiral compounds. Therefore, these analytes are commonly converted into various derivatives prior to separation of their enantiomers on polysaccharide-based chiral columns [START_REF] Chankvetadze | Enantiomer elution order reversal of FMOC-isoleucine by variation of mobile phase temperature and composition[END_REF][START_REF] López-Ram-De-Víu | High-performance liquid chromatographic enantioseparation of unusual amino acid derivatives with axial chirality on polysaccharide-based chiral stationary phases[END_REF][START_REF] Jin | Comparative studies between covalently immobilized and coated chiral stationary phases based on polysaccharide derivatives for enantiomer separation of N-protected alpha-amino acids and their ester derivatives[END_REF][START_REF] Jin | Comparison of several polysaccharide-derived chiral stationary phases for the enantiomer 12 separation of N-fluorenylmethoxycarbonyl alpha-amino acids by HPLC[END_REF][START_REF] Lee | Enantioseparation of Nfluorenylmethoxycarbonyl alpha-amino acids on polysaccharide-derived chiral stationary phases by reverse mode liquid chromatography[END_REF][START_REF] Khater | Thermodynamic enantioseparation behavior of phenylthiohydantoin-amino acid derivatives in supercritical fluid chromatography on polysaccharide chiral stationary phases[END_REF][START_REF] West | Chiral separation of phospine-containing α-amino acid derivatives using two complementary cellulosic stationary phases in supercritical fluid chromatography[END_REF]. Although derivatization of analytes provides certain advantages from the viewpoint of separation (improved peak shape, additional intermolecular interactions) and detection, it also has disadvantages such as an additional synthetic step prior to separation, possibility of incomplete derivatization, interference of residual derivatizing reagent in the separation process, additional costs, etc. In addition, if the analyte is required in its intact but enantiomerically pure form after the separation, recovering the analyte from its derivative used in the separation process in its initial form will also require an additional step which may be burdened by additional shortcomings.

Since many amino acids do not possess strong UV-chromophores in their structures, in earlier studies their derivatization was performed both, for improving their enantiorecognition and also for improving their detectability with UV/VIS or fluorescence detectors. However, with the successful detection of native amino acids by alternative techniques such as mass spectrometry (MS), refractive index (RI), evaporative light scattering (ELS), corona charged aerosol (CA) detectors or based on label-free fluorescent detection, the derivatization of amino acids with the sole purpose of improving their detectability becomes unnecessary. Thus, in some cases the separation of native amino acids may offer certain advantages over the separation of their derivatives.

Several efficient chiral selectors are available for the separation of enantiomers of native amino acids. Actually, the history of enantioselective high-performance liquid chromatography (HPLC) started by the successful separation of enantiomers of native amino acids reported by Davankov and Rogozhin in 1971 [START_REF] Rogozhin | Ligand chromatography on asymmetric complexforming sorbents as a new method for resolution of racemates[END_REF]. Today, the most useful chiral selectors for the separation of enantiomers are cation-and anion-exchangers, as well as zwitterionic chiral selectors developed by Lindner and co-workers [START_REF] Ilisz | Liquid chromatographic enantiomer separations applying chiral ion-exchangers based on Cinchona alkaloids[END_REF][START_REF] Ianni | Quinine-based zwitterionic chiral stationary phase as a complementary tool for peptide analysis: Mobile phase effects on enantio-and stereoselectivity of underivatized oligopeptides[END_REF][START_REF] Hoffmann | Synergistic effects on enantioselectivity of zwitterionic chiral stationary phases for separations of chiral acids, bases, and amino acids by HPLC[END_REF],

macrocyclic antiobiotic-and cyclofructane-based materials proposed by Armstrong and co-workers [START_REF] Orosz | Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene skeleton on macrocyclic glycopeptide-based chiral stationary phases[END_REF][START_REF] Padivitage | Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography[END_REF][START_REF] Armstrong | Chiral stationary phases for HPLC[END_REF], crown ether-based [START_REF] Hyun | Liquid chromatographic enantioseparations on crown ether-based chiral stationary phases[END_REF] and few other chiral stationary phases [19,[START_REF] Sajewicz | HPLC-DAD evidence of the oscillatory chiral conversion of phenylglycine[END_REF]. In early 2000s Ye and co-authors have published the article reporting a separation of underivatized amino acid enantiomers on polysaccharide-based chiral column, Chiralpak AD in HPLC [START_REF] Ye | Enantioseparation of amino acids on a polysaccharide-based chiral stationary phase[END_REF]. Later, Stringham reported separation of enantiomers of underivatized proline and 4 aromatic amino acids, baclofen, 2-phenylglycine, phenylalanine and tyrosine on the same chiral column in supercritical fluid chromatography (SFC) [START_REF] Stringham | Chiral separation of amines in subcritical fluid chromatography using polysaccharide stationary phases and acidic additives[END_REF].

Separation of enantiomers of underivatized amino acids under SFC conditions on few polysaccharide-based chiral columns was also reported in the conference presentation by Yan and Riley [START_REF] Yan | Presentation at PittCon 2017[END_REF].

In this manuscript our results are reported on the evaluation of several coated and covalently immobilized polysaccharide-based chiral selectors for the separation of enantiomers of 14 native amino acids, namely valine, norvaline, threonine, phenylalanine, leucine, isoleucine, norleucine, lysine, alanine, histidine, serine, carnitine, oxoproline (pyroglutamic acid) and aspartic acid (Fig. 1a) in sub/supercritical fluid chromatography (SFC).

Experimental part

Materials

All studied amino acids, trimethylamine and n-butylamine were purchased from Sigma-Aldrich (Steinheim, Germany) and Acros Organics (Illkirch, France) respectively.

Sample solutions

Screening solutions of racemic amino acids were prepared at 3 mg/mL concentration in methanol/H 2 O 50/50 (v/v) mixture, except for aspartic acid which was prepared in 1 mg/mL concentration. The solutions were degassed by an ultrasonic bath and filtered through a 0.45 µm PTFE syringe-filter (15 mm diameter) prior to be used.

Supercritical fluid chromatography apparatus

The SFC system used was an SFC-PICLAB hybrid 10-20 apparatus (PIC Solution, Avignon, France). The pump head used for pumping the (Table 1). Some typical examples are shown in Fig. 2. The high enantiomer resolving ability of cellulose tris(3,5-dichlorophenylcarbamate) has been known for many years [START_REF] Okamoto | Chromatographic resolution: XI. Controlled chiral recognition of cellulose triphenylcarbamate derivatives supported on silica gel[END_REF][START_REF] Chankvetadze | Extremely high enantiomer recognition in HPLC separation of racemic 2-(benzylsulfinyl)benzamide using cellulose tris(3,5-dichlorophenylcarbamate) as a chiral stationary phase[END_REF][START_REF] Chankvetadze | HPLC Enantioseparation with cellulose tris(3,5-dichlorophenylcarbamate) in aqueous methanol as a mobile phase[END_REF][START_REF] Chankvetadze | Enantioseparations using cellulose tris(3,5-dichlorophenylcarbamate) in high-performance liquid chromatography in common size and capillary columns: potential for screening of chiral compounds[END_REF][START_REF] Gallinella | Direct separation of the enantiomers of oxaliplatin on a cellulose-based chiral stationary phase in hydrophilic interaction liquid chromatography mode[END_REF] and has been observed also for rather hydrophilic non-aromatic compounds, such as chiral metal complexes [START_REF] Chankvetadze | Enantioseparations using cellulose tris(3,5-dichlorophenylcarbamate) in high-performance liquid chromatography in common size and capillary columns: potential for screening of chiral compounds[END_REF][START_REF] Gallinella | Direct separation of the enantiomers of oxaliplatin on a cellulose-based chiral stationary phase in hydrophilic interaction liquid chromatography mode[END_REF]. Considering this experience, the results observed in the present study for non-aromatic amino acids do not seem surprising.

Effect of mobile phase optimization

Type of co-solvent

The mobile phase was CO 2 /modifier mixtures; firstly 40% of either methanol or ethanol were tested. As it can be seen from Fig. S1, for the enantiomers of norvaline and leucine, ethanol was preferable modifier. Thus, ethanol has been used in further studies.

Use of mobile phase additive

In liquid-phase separations of enantiomers with polysaccharide-based chiral columns commonly basic additives are used for the separations of basic compounds and acidic additives for acids. The mixture of a basic and an acidic additive has also been used [START_REF] Klerck | Enantioselectivity of polysaccharide-based chiral stationary phases insupercritical fluid chromatography using methanol-containing carbondioxide mobile phases[END_REF][START_REF] Klerck | updating a genericapproach in sub-or supercritical fluid chromatography for theenantioseparation of pharmaceuticals[END_REF][START_REF] Mosiashvili | On the effect of basic and acidic additives on the separation of the enantiomers of some basic drugs with polysaccharide-based chiral selectors and polar organic phases[END_REF]. In SFC, due to the acidic nature of carbon dioxide [START_REF] West | Unravelling the effects of mobile phase additive in supercritical fluid chromatography. Part I: polarity and acidity of the mobile phase[END_REF] especially in the presence of alcohols [START_REF] Grand-Guillaume Perrenoud | Analysis of basic compounds by supercritical fluid chromatography: attempts to improve peak shape and maintain mass spectrometry compatibilty[END_REF], for the acidic solutes the addition of an acidic additive in the mobile phase is not as common as in HPLC. Triethylamine (TEA) in the amount 0.3% (v/v) was used as the mobile phase additive in initial experiments. As shown in Fig. S2a the additive enabled good baseline separation of enantiomers of norvaline, but broad peaks were observed. Similar results were obtained for enantiomers of valine. For enantiomers of leucine only partial separation was observed. Further adjustment of the ethanol content and increasing the concentration of TEA in the mobile phase following the recently published work by Speybrouck et al. [START_REF] Speybrouck | The effect of high concentration additive on chiral separations in supercritical fluid chromatography[END_REF] enabled to improve peak shapes and resolution further (Figs. S2b-f). Thus, except for phenylalanine and histidine, for which 0.3% of TEA additive was used, 3% TEA allowed a good compromise between peak shape, analysis time and peak resolution and was therefore chosen.

In order of further improving peak shapes, EtOH-water mixtures in different ratios was applied as mobile phase component. The use of water in the mobile phase can suppress peak tailing by counteracting the limited solubility of amino acids in CO 2 -based mobile phases. The use of 2 or 5% water in MeOH with chiral polysaccharide-based stationary phases has been reported earlier [START_REF] Liu | Extending the range of supercritical fluid chromatography by use of water-rich modifiers[END_REF]. Thus, a mobile phase of 20% (2% H 2 O and 3% TEA in EtOH)/CO 2 was tested but did not improve resolution significantly.

Lastly, whenever analysis times were too long, the flow-rate was increased from 3 mL/min to 4 mL/min for isoleucine, phenylalanine and carnitine and to 6 mL/min in case of histidine. The best results obtained for the ten amino-acids are summarized in Table 1.

3.3

Evaluation of the potential contribution of ELSD to extra-column bandbroadening ELSD is a useful universal detector for non-aromatic amino acids having week UV-absorbance. However, this detector may generate additional extra-column bandbroadening. In order to study this phenomenon, the separation of phenylalanine enantiomers on Lux i-Cellulose-5 was monitored with sequentially connected UV-VIS and ELS detectors in a rather wide flow-rate range (from 2 to 8 mL/min). Selected chromatograms are shown in Fig. 3 for medium and high flow-rates. As it can be seen from these overlayed chromatograms, ELSD does not contribute significantly to extracolumn band-broadening, at least not in this particular case.

Conclusions

As this study illustrates polysaccharide-based chiral columns may successfully be used for the separation of enantiomers of underivatized nonaromatic amino acids in SFC.

As expected, the nature of the chiral selector and the composition of the mobile-phase play key roles for differentiation of enantiomers. Among studied chiral columns Lux i- norleucine on Lux i-Cellulose-5. Separation conditions were as shown in Table 1.

Fig. 3 Chromatograms of phenylalanine enantiomers with the flow rates of the mobile phase (a) 8 mL/min and (b) 4 mL/min when detected with DAD and ELSD. Other separation conditions were as shown in Table 1.

Supplementary Figures:

Figure S2

Separation of enantiomers of norvaline on Lux i-Cellulose-5, with 20%

EtOH and a) 0.3 %; b) 1.0% ; c) 2.0%; d) 3.0% ; e) 5.0% and f) 10% of TEA additive, at 4 mL/min, 40°C, 15 MPa, T ELSD 60°C, gain 8.

  HPLC grade methanol (MeOH) and ethanol (EtOH) were purchased from Carlo-Erba Reagents (Val-de-Reuil, France). Carbon dioxide (CO 2 ) with purity of 99.995% was purchased from Linde (Saint Priest, France). Chiral columns, such as Chiralpak AS based on amylose tris((S)-α-phenylethylcarbamate) as chiral selector, Chiralcel OD-H and Chiralpak IB based on cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, Chiralpak AD-H and Chiralpak IA based on amylose tris(3,5-dimethylphenylcarbamate) as chiral selector were purchased from Chiral Technologies Europe (Illkirch, France). Lux Cellulose-2 based on cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector, Lux Amylose-2 based on amylose tris(5-chloro-2-methylphenylcarbamate) as chiral selector and Lux i-Cellulose-5 based on cellulose (3,5-dichlorophenylcarbamate) as chiral selector were provided by Phenomenex (Le Pecq, France). The structures of all chiral selectors part of the studied columns are shown in Fig. 1b. All columns were 4.6 x 250 mm in size and packed with 5 micrometer particles.

  CO 2 was cooled to -8° C by a cryostat (model Minichiller, Huber, Offenburg, Germany). The injection valve was provided with a 19.6 µL sample loop. The system also included a Smartline 2600 diode array detector (DAD) (Knauer, Berlin, Germany). Detection wavelength was set at 210 nm. A Sedex 85 evaporative light scattering detector (ELSD) (Sedere, Alfortville, France), was connected in-line with the DAD and right before the backpressure regulator using a 0.064 mm ID × 160 cm long peek tubing. The pressure of the nebulizer gas (N 2 ) was set at 3.7 bar. After optimization, the drift tube temperature was equal to 60° C and the gain was set at 8. The system was controlled, and the data acquired and processed with the SFC PicLab Analytic Online v.3.1.2 and Offline v.3.2.0 software (PIC Solution), respectively. During the separation optimization, the mobile phase composition was varied between 20-40 % methanol or ethanol as modifiers in CO 2 . The used flow rate was 3 mL/min if not indicated otherwise. All analyses were run in isocratic mode. The column oven temperature was 40°C and the outlet pressure was maintained at 15 MPa for all experiments. The dead time was marked as the first disturbance of the baseline after injection, caused by the solvent peak. Retention times were average values of two replicate measurements. Screening of polysaccharide-based chiral columns for the separation of enantiomers of underivatized amino acids SFC is well established technique for separation of enantiomers[START_REF] West | Enantioselective separations with supercritical fluids[END_REF][START_REF] Speybrouck | Preparative supercritical fluid chromatography: A powerful tool for chiral separations[END_REF][START_REF] Klerck | Exploratory data analysis as a tool for similarity assessment and clustering of chiral polysaccharide-based systems used to separate pharmaceuticals in supercritical fluid chromatography[END_REF][START_REF] West | Enantioseparation of novel chiral sulfoxides on chlorinated polysaccharide stationary phases in supercritical fluid chromatography[END_REF]. Due to lower viscosity of and fast analyte diffusion in supercritical fluids this technique offers the possibility of operating at higher flow rates (fast separations) as well as generating higher plate numbers compared to HPLC. In addition, in preparative scale separations removal of the mobile phase is easier and less costly compared to other separation modes. This technique has been explored for the separation of derivatized amino acids also with polysaccharide-based chiral columns[START_REF] Khater | Thermodynamic enantioseparation behavior of phenylthiohydantoin-amino acid derivatives in supercritical fluid chromatography on polysaccharide chiral stationary phases[END_REF][START_REF] West | Chiral separation of phospine-containing α-amino acid derivatives using two complementary cellulosic stationary phases in supercritical fluid chromatography[END_REF]. However, to the best of our knowledge only one journal publication describes the separation of 5 underivatized amino acids, among which 4 are aromatic, with polysaccharide-based chiral columns in SFC[START_REF] Stringham | Chiral separation of amines in subcritical fluid chromatography using polysaccharide stationary phases and acidic additives[END_REF].All analytes were injected on the eight columns with either 40% methanol or ethanol as co-solvent. The separation was performed at 40°C. The results on enantioseparation of the 14 amino acids on 8 polysaccharide-based chiral columns with coated or covalently immobilized chiral selectors showed that some of the studied columns did not provide separation of any pair of enantiomers under this study. In particular, coated-type columns such as Chiralcel OD, Chiralpak AD and Chiralpak AS belong to this category. In addition, no separation of enantiomers was observed on the covalently immobilized version of Chiralcel OD and Chiralpak AD columns, Chiralpak IB and Chiralpak IA, respectively. Lux Cellulose-2, having a chlorinated chiral selector provided baseline separation for the enantiomers of phenylalanine, valine, isoleucine and partial resolution of enantiomers of leucine (Data not shown). Lux Amylose-2 provided partial resolution of enantiomers of phenylalanine (Data not shown). The best enantiomer resolving ability among studied chiral columns was provided with Lux i-Cellulose-5 based on cellulose-(3,5-dichlorophenylcarbamate) as chiral selector in immobilized form

Cellulose- 5 Fig. 1 Fig. 2

 512 Fig. 1 Structure of (a) studied amino acids and (b) chiral selectors part of studied chiral

  Figure S1Chromatograms of Leucine and Norvaline with either MeOH or EtOH as

Table 1 :

 1 Chromatographic parameters of the separation of underivatized amino acids under optimized conditions by SFC with ELS detection on Lux i-Celllulose-5. Other experimental conditions were: The oven temperature 40°C, pressure 150 bar; T ELSD = 60°C; gain = 8.

	Amino acid	Conditions	tr 1 (min)	tr 2 (min)	k 1	k 2	α	Rs
	Leucine	20% EtOH+TEA 3%, 3mL/min a)	11.44	16.05	6.15	9.00	1.49	1.63
	Isoleucine	20% EtOH+TEA 3%, 4mL/min	10.51	15.43	5.57	8.64	1.55	1.61
		20% EtOH+TEA 3%,
	Norleucine	3mL/min	12.23	19.98	7.09	12.23	1.72	2.17
	Phenylalanine	20% EtOH+TEA 0.3%, 4mL/min	14.62	20.46	11.72	16.79	1.43	1.59
	Alanine	20% EtOH+TEA 3%, 3mL/min	16.08	23.76	10.08	15.38	1.50	1.53
	Valine	20% EtOH+TEA 3%, 3mL/min	10.55	16.90	5.65	9.65	1.71	2.24
	Norvaline	20% EtOH+TEA 3%, 3mL/min	12.90	19.59	6.77	10.80	1.60	1.83
	Serine	20% EtOH+TEA 3%, 3mL/min	13.12	15.25	6.41	7.62	1.19	1.51
	Carnitine	40% EtOH+TEA 3%, 4mL/min	11.12	16.21	10.34	15.54	1.50	1.95
		30% EtOH+TEA 0.3%,
	Histidine	6mL/min	14.37	21.47	16.96	26.17	1.25	1.99
	a)

Acknowledgement

Anca-Elena Dascalu is grateful to the Fondation de la Catho for her PhD-scholarship. Dr. Alina Ghinet is recognized for her gift of some amino acids used in this study.