Julie Mardon

Veronique Habauzit

Anna Trzeciakiewicz

M.-J Davicco

Patrice Lebecque

Sylvie Mercier

Jean-Claude Tressol

Marie-Noëlle Horcajada

Christian Demigné

Veronique Coxam

Véronique Habauzit

Marie-Jeanne Davicco

Véronique Coxam

Long-Term Intake of a High-Protein Diet with or without Potassium Citrate Modulates Acid-Base Metabolism, but Not Bone Status, in Male Rats 1

Keywords: BMD, bone mineral density, DPD, deoxypyridinoline, HP, high protein diet

Introduction

Compared to "optimum" nutritional requirements, which are genetically determined, Western diets are characterized by an excessive protein intake, frequently associated with insufficient fruit and vegetable consumption. It has been suggested that this mismatch could lead to a number of pathological conditions [START_REF] Eaton | Paleolithic vs. modern diets--selected pathophysiological implications[END_REF].

In particular, an imbalanced protein-potassium ratio results in low-grade metabolic acidosis [START_REF] Frassetto | Estimation of net endogenous noncarbonic acid production in humans from diet potassium and protein contents[END_REF]. Protein-rich diets are considered acidogenic due to the excess of sulphuric anions produced by the catabolism of sulphur amino acids. Moreover, a low fruit and vegetable intake results in decreased mineral and organic anion provision, including potassium citrate and malate. Thus, the availability of alkaline organic salts potentially is insufficient to neutralize the endogenous acid production associated with excessive protein intake.

To counteract the excess dietary acid load, the homeostatic systems excrete increased amounts of urinary calcium and phosphorus [START_REF] Lemann | The importance of renal net acid excretion as a determinant of fasting urinary calcium excretion[END_REF][START_REF] Alpern | The clinical spectrum of chronic metabolic acidosis: homeostatic mechanisms produce significant morbidity[END_REF]. Hypercalciuria can have multiple pathological consequences [START_REF]Metabolic and clinical consequences of metabolic acidosis[END_REF], including nephrolithiasis, and it may contribute to the pathogenesis of osteoporosis. Acidosis promotes continual release of alkaline calcium salts from the skeleton, to maintain the acid-base balance [START_REF] Green | The role of bone in the regulation of systemic acid-base balance[END_REF][START_REF] Barzel | The skeleton as an ion exchange system: implications for the role of acid-base imbalance in the genesis of osteoporosis[END_REF], and this may result in bone loss in the long-term. Indeed, metabolic acidosis is associated with lower bone mineral density in women [START_REF] New | Lower estimates of net endogenous non-carbonic acid production are positively associated with indexes of bone health in premenopausal and perimenopausal women[END_REF] and rats [START_REF] Won | Bone histomorphometric changes due to differences in calcium intake under metabolic acidosis in rats[END_REF]. In an in vitro model for metabolic acidosis, significant bone calcium efflux was observed [START_REF] Bushinsky | Net calcium efflux from live bone during chronic metabolic, but not respiratory, acidosis[END_REF] and cell function was impaired as evidenced by stimulation of osteoclastic bone resorption and inhibition of osteoblastic formation [START_REF] Arnett | Acid-base regulation of bone metabolism[END_REF][START_REF] Krieger | Acidosis inhibits osteoblastic and stimulates osteoclastic activity in vitro[END_REF].

Similarly, high protein intake and the associated acidosis are considered potentially detrimental for bone health in humans [START_REF] Feskanich | Protein consumption and bone fractures in women[END_REF][START_REF] Zwart | Amino acid supplementation alters bone metabolism during simulated weightlessness[END_REF] and rats [START_REF] Amanzadeh | Effect of high protein diet on stone-forming propensity and bone loss in rats[END_REF]. However, in contrast to the wellcharacterized effects of dietary protein on urinary calcium excretion, the effect on bone status needs further insight. Actually, the impact of dietary protein on bone remains somewhat controversial as several reports have emphasized that high protein intake benefits bone health [START_REF] Promislow | Protein consumption and bone mineral density in the elderly : the Rancho Bernardo Study[END_REF][START_REF] Kerstetter | The impact of dietary protein on calcium absorption and kinetic measures of bone turnover in women[END_REF][START_REF] Dawson-Hughes | Effect of dietary protein supplements on calcium excretion in healthy older men and women[END_REF]. Indeed, protein intake may favourably influence bone metabolism by stimulating the anabolic action of IGF-1 (Insulin-like Growth Factor 1) on bone [START_REF] Rizzoli | Dietary protein and bone health[END_REF].

Fruit and vegetable intake provides potassium salts, especially potassium citrate.

Previous investigations have reported beneficial effects of dietary potassium and potassiumrich foods (fruits and vegetables) on bone health [START_REF] New | Dietary influences on bone mass and bone metabolism: further evidence of a positive link between fruit and vegetable consumption and bone health?[END_REF][START_REF] Tucker | Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women[END_REF][START_REF] Marangella | Effects of potassium citrate supplementation on bone metabolism[END_REF][START_REF] Sellmeyer | Potassium citrate prevents increased urine calcium excretion and bone resorption induced by a high sodium chloride diet[END_REF]. Hypothesizing that a high-protein diet could induce osteopenia, potassium citrate intake would neutralize the diet-induced metabolic acidosis, and may be associated with a higher bone mass [START_REF] Macdonald | Low dietary potassium intakes and high dietary estimates of net endogenous acid production are associated with low bone mineral density in premenopausal women and increased markers of bone resorption in postmenopausal women[END_REF]. Thus, further investigations with more long-term outcomes would be needed to completely define the role of high protein intake and acid-base balance in age-related bone loss. The goal of this study was to carefully identify the long-term effects of protein content in the diet on bone status and to test if alkaline-forming supplements had favourable effects on bone health. Using rats as a model, the study was designed to address two specific questions: i) Does a long-term high-protein diet (HP: 26%) have beneficial or detrimental effects on bone status when compared to a normal-protein diet (NP: 13%).

ii) Does potassium citrate supplementation (Kcit) modulate the bone response to dietary protein intake.

Materials and methods

Animals and experimental design

The study was conducted in accordance with the regional Ethics Committee for animal experiments, in France. One hundred and twenty male Wistar rats (2 mo old, mean weight 316 g) were purchased from INRA (Institut National de la Recherche Agronomique, Clermont-Ferrand/Theix, France). The rats were housed individually in plastic cages at 25°C, in 12/12 h light/dark cycles and had free access to water. Body weight and food intake were measured weekly. Male rats were chosen in order to eliminate the potential confounding effect of hormonal fluctuations.

At the beginning of the study, the rats were randomly assigned to four groups (n=30).

Two groups received diets containing either a normal protein content (13 % (g/100g)) (NP) or a high protein content (26 %) (HP). The other two groups received identical diets supplemented with potassium citrate (3.60 %) (NPKcit and HPKcit) (Table 1). All diets were purchased from INRA (Jouy en Josas, France). All rats were fed a measured amount of diet, corresponding to 90-95 % of the mean ad libitum intake of the HP groups, which had the lowest food intake. The pair-feeding allowed us to energy match the food intake, providing healthy, non-obese control animals for the study [START_REF] Pugh | Controlling caloric consumption: protocols for rodents and rhesus monkeys[END_REF]. All diets contained the same amount of lipid, fiber, vitamins and minerals, excepting potassium (0.36 % vs. 1.66 % in supplemented groups). Thus, all rats consumed similar amounts of dietary calcium (0.25 %) and phosphorus (0.20 %) (Ca/P ratio=1.25). At 9, 16 and 21 months of age, 10 rats from each group were killed. One week before the end of the study, the rodents were transferred to individual metabolic cages specially designed for rats. After a three-day adaptation period, food intake was controlled daily, and urine and faeces were collected for three consecutive days. At the end of the experiment, the rats were food-deprived for 12 h then killed by injection of a lethal dose of anaesthetic (Imalgen 1000, Merial, Lyon, France; 0,75mL/kg bw and Vetranquil 1%, Ceva santé animale, Libourne, France; 0,25mL/kg bw). Blood samples were collected from the abdominal aorta and frozen at -20°C until analysis. Femurs were cleaned from adjacent tissues. Left femurs were harvested in saline solution (9 g NaCl/L) and frozen (-20°C) until mechanical testing.

Right femurs were kept in 80 % alcohol until bone mineral density was measured.

Biochemical analyses

Minerals. Urine samples were appropriately diluted in lanthanum oxide solution.

Urinary calcium and magnesium concentrations were determined by atomic absorption spectrophotometry (AA800, Perkin Elmer, Norwalk, CT, USA). Sodium and potassium concentrations were measured by atomic emission spectrophotometry. 200 mg of dried samples (food and faeces) were dried-ashed (10 h at 500°C) and extracted until discoloration at 130°C in HNO3-H2O2 (2:1) (Suprapur; Merck, Darmstadt, Germany). For calcium determination by atomic absorption spectroscopy, the final dilution was performed with lanthanum oxide. Calcium retention (µmol / 24 h), defined as [calcium intake -(urinary calcium excretion + faecal calcium excretion)] was calculated.

Organic anions. Urine was diluted 400-fold with milli-Q water. Chloride, phosphate, sulphate and citrate concentrations were determined by ionic chromatography (DX320, Dionex, Sunnydale, CA, USA). The anions were separated on a 4 × 250 mm AS 11 column / AG 11 precolumn (flow rate 1 mL/min). An EG40 eluent generator controlled the elution using an OH -gradient (0.5 to 35 mmol/L in 20 min) and the conductimetry detector was preceded by an ASRS self-regenerating suppressor.

IGF-1. Serum insulin-like growth factor I concentrations were measured using a twosite immunoenzymometric assay (OCTEIA Rat/Mouse IGF-1 kit, IDS, Paris, France). The sensitivity of the assay was 82 µg/L. Intra-and interassay variations were 5.7 % and 10.7 %, respectively.

PTH. Plasma parathyroid hormone concentrations were determined using an immunoradiometric assay kit (Immunotopics, Inc., San Clemente, CA, USA). The sensitivity was 1.0 ng/L. The intra-and interassay variations were 4.1 % and 4.5 %, respectively. Bone biomarkers. Plasma osteocalcin (OC) concentrations were measured by an immunoradiometric assay (Immunotopics, Inc., San Clemente, CA, USA). The sensitivity was 0.01 µg/L. The intra-and interassay variations were 2.0 % and 4.5 %, respectively. The urinary deoxypyridinoline (DPD) concentration (nmol/L) was determined using a radioimmunoassay (Pyrilinks-D RIA kit, Metra Biosystems, Mountain View, CA, USA). The sensitivity was 2 nmol/L. The intra-and interassay variations were 4 and 6%, respectively. DPD excretion rates (nmol/24 h) were calculated using the prior 24-hour urine volumes.

Physical measurements

Bone mineral density (BMD). BMD was assessed by dual-energy X-ray absorptiometry (DEXA), using a Hologic QDR-4500 A X-ray bone densitometer (Hologic, Massy, France). Total femoral BMD (T-BMD), metaphyseal BMD (M-BMD) and diaphyseal BMD (D-BMD) were determined. For M-BMD and D-BMD measurements, scans were cut and analyzed as follow: the first cut of the femur was performed at the upper quarter, and the next cut at the lower quarter. D-BMD corresponded to the density of the second and the third quarter of the femur. M-BMD was calculated as the mean of the upper and the lower quarters of the femur [START_REF] Omi | The effect of ovariectomy on bone metabolism in rats[END_REF].

Mechanical testing of femurs. Femoral length and mean diaphyseal diameter were measured with a precision calliper (Mitutoyo, Shropshire, UK). Because of the irregular shape of the femoral diaphysis, the femoral diameter used in the calculation was the mean of the greatest and smallest femoral diaphyseal diameters. The femoral failure load was determined using a 3-point bending test [START_REF] Turner | Basic biomechanical measurements of bone: a tutorial[END_REF], with a Universal Testing Machine (Instron 4501, Instron, Canton, MA, USA). The two lower supports were separated by a 20 mm distance and an upper crosshead roller (diameter, 6 mm) was applied in front of the middle of the bone at a speed of 0.5 mm/min until failure, to guarantee that 85-90 % of the bone flexure was due to bending. Load (Newtons) at rupture was recorded.

Statistical analyses

Results are expressed as mean ± standard error of mean (SEM) and were analyzed with XLSTAT (Addinsoft, Paris, France). The sample size was based on a previous study of the effect of protein intake on BMD in 8 months-old male rats (28) followed during 7 months, which used 7 rats per group. Given the duration of the present study, we used 10 rats per group. The normality of the data was verified. All variables were subjected to a three-way analysis of variance (ANOVA), testing for any difference among the groups. Thus, the main effects assessed were protein intake (NP / HP), potassium citrate supplementation (Kcit), age (9, 16 and 21 mo) and their interactions. If a result was statistically significant (P<0.05), the Student-Newman-Keuls multiple comparison test was used to determine specific differences between means. Linear regressions were performed to study internal correlations among variables and the Pearson test was carried out to assess their significance.

Results

Body weight and dietary intake. The body weight of all rats increased between 9 and 16 mo of age (P=0.01) (Table 2). At 21 months of age, body weight appeared to decrease (non significant). There was no difference in body weight between the four groups, whatever the level of protein or the supplementation of potassium citrate in the diet. The mean dietary intake did not vary among the four groups, due to the pair-feeding, and it was 22.1 ± 0.9 g, 22.4 ± 1.2 g and 21.0 ± 1.3 g at 9, 16 and 21 months of age, respectively. Urinary cation excretion. After 9 months, the urine pH of rats adapted to the NP diet was slightly acidic, whereas it was markedly lower in rats adapted to the HP diet (P<0.0001) (Table 2). The urine pH of rats fed the diets supplemented with Kcit was around 8.8 regardless of dietary protein intake. With age, all groups showed urine acidification: from 9 to 21 months of age, the decline in pH was -0.73 in the NP group and -0.57 in the HP group. The age-related decline in urine pH was more pronounced when the rats were fed the Kcitsupplemented diets (-0.87 and -1.94 with NPKcit and HPKcit, respectively). As a result, the urine was still alkaline in the NPKcit group, but marginally acidic in the HPKcit group. There was no difference in urine sodium excretion between the groups, and age had little effect on the efficiency of renal sodium excretion. The rate of potassium excretion was dependent on the dietary potassium level (P<0.0001) and was not affected by the dietary protein level (Table 2). A decline in renal potassium excretion was observed in the 21 months old rats adapted to the NP and HP diets (P=0.039), and it was much less pronounced in the rats adapted to the Kcit-supplemented diets. Urinary calcium (Table 3) and magnesium (Table 2) excretion displayed similar features: (i) an increase in rats fed the HP diet when compared to those fed the NP diet (P=0.009 and P=0.089 respectively), (ii) a blunting of this protein effect when Kcit was included and (iii) increased calcium (P<0.082) and magnesium (P=0.001) concentrations in the urine of older rats.

Calcium retention. Calcium intake was lower in all rats at 21 mo compared to 9 and 16 mo (P<0.0001). In parallel, fecal calcium excretion increased significantly from 16 mo of age (P<0.0001). Fecal calcium excretion was also higher in the HP group compared to the other groups at 9, 16 and 21 mo of age (P=0.027). The urinary calcium excretion represented less than 1% of total calcium excretion. Calcium retention decreased during the experimental period (Table 3). This decrease was significant between 9 and 21 mo of age (P<0.0001). At 21 mo of age, the HP group showed a lower calcium retention rate than the NP, NPKcit and HPKcit groups (P=0.044), whereas no differences were detected at the earlier stages. Plasma IGF-1. Plasma IGF-1 concentrations were lower after 21 mo (P<0.0001), compared to 9 and 16 mo old rats (data not shown). No differences were observed between the groups at any age.

Plasma PTH. Plasma PTH concentrations increased at 16 and 21 mo (P=0.001) compared to 9 mo of age. There were no significant variations between the four groups (data not shown) at any age.

Discussion

A relatively high dietary protein intake can generate a substantial fixed acidity, which inconsistently has been reported to adversely affect bone health [START_REF] Feskanich | Protein consumption and bone fractures in women[END_REF][START_REF] Zwart | Amino acid supplementation alters bone metabolism during simulated weightlessness[END_REF][START_REF] Amanzadeh | Effect of high protein diet on stone-forming propensity and bone loss in rats[END_REF]. Potassium citrate (Kcit) could possibly be used to neutralize this acid load. We investigated the effect of two levels of dietary protein (13% or 26%), in the presence or absence of Kcit supplementation, on acid-base metabolism and bone status. Male Wistar rats were used as an experimental model for age-related bone loss (29) and were monitored until 21 months of age.

Characterization of high-protein diet-induced low-grade metabolic acidosis

The high-protein diet (HP) elicited an acid load, which was characterized by a decreased urine pH when compared to the NP diet group at all ages (Table 2). The HP diet was associated with significant calciuria (Table 3). Hypercalciuria is correlated with net acid excretion [START_REF] Lemann | Relationship between urinary calcium and net acid excretion as determined by dietary protein and potassium: a review[END_REF] and is a well-known consequence of high-protein diet ingestion [START_REF] Allen | Calcium metabolism, intestinal calcium-binding protein, and bone growth of rats fed high protein diets[END_REF][START_REF] El & Swaminathan | The effect of high protein and high salt intake for 4 months on calcium and hydroxyproline excretion in normal and oophorectomized rats[END_REF]. In the kidney, it has been attributed to an increased glomerular filtration rate and a decreased tubular calcium reabsorption, which neutralizes SO4 2-generated by catabolism of sulphur amino acids (4). The changes in magnesium excretion were similar (30% increase between the NP and HP groups). This trend is consistent with previous short-term studies and suggests that magnesium, in addition to calcium, compensates for metabolic acidosis in the long-term [START_REF] Sabboh | Effect of potassium salts in rats adapted to an acidogenic high-sulfur amino acid diet[END_REF].

Increased urinary sulphate and phosphate excretion was observed with the HP diet (Fig 1).

The increase in sulphate elimination resulted from a greater sulphur amino acid intake [START_REF] Nakamura | A study on the estimation of sulfur-containing amino acid metabolism by the determination of urinary sulfate and taurine[END_REF].

The higher phosphate excretion rate could be due to the presence of more organic phosphorus in the HP diet [START_REF] Sabboh | Effects of plant food potassium salts (citrate, galacturonate or tartrate) on acid-base status and digestive fermentations in rats[END_REF], but impaired tubular reabsorption of phosphate ( 15) is another possibility.

Hypocitraturia was another major factor associated with dietary protein excess (Fig 1A) [START_REF] Breslau | Relationship of animal protein-rich diet to kidney stone formation and calcium metabolism[END_REF]. This is consistent with reports of renal acidosis resulting in decreased proximal tubular reabsorption of citrate and impaired citrate transport [START_REF] Brennan | Citrate transport in rabbit nephron[END_REF][START_REF] Labow | Mechanisms governing the expression of the enzymes of glutamine metabolism--glutaminase and glutamine synthetase[END_REF].

Potassium citrate supplementation affects acid-base metabolism

In contrast to western diets, the NPKcit and HPKcit diets provided a relative excess of organic anions (Kcit: 35.95 g/kg) compared to sulphur amino acids (around 6.9 g/kg in the NP diets and 13.8 g/kg in the HP diets). The protein-potassium ratio is a reliable predictor of dietnet-acid-load and these two diets were previously shown to prevent metabolic acidosis [START_REF] Frassetto | Estimation of net endogenous noncarbonic acid production in humans from diet potassium and protein contents[END_REF]. Indeed, potassium citrate supplementation significantly increased urine pH, consistent with previous work [START_REF] Marangella | Effects of potassium citrate supplementation on bone metabolism[END_REF][START_REF] He | Fortnightly review: Beneficial effects of potassium[END_REF]. The administration of Kcit resulted in increased urinary potassium (Table 2) and citrate excretion (Fig 1) in both the NPKcit and HPKcit group. However, the 26% protein diet limited the effect of the Kcit supplementation on citraturia.

The Kcit neutralizing effect was further confirmed by lower urinary calcium and magnesium excretion (Table 2, Table 3). The potassium citrate probably exerted its effects by neutralizing endogenous acidity by KHCO3 -generation, glutamine sparing [START_REF] Demigne | Protective effects of high dietary potassium: nutritional and metabolic aspects[END_REF][START_REF] Remer | Potential renal acid load of foods and its influence on urine pH[END_REF] and modulation of hepatic urea synthesis for removal of bicarbonate [START_REF] Haussinger | Liver regulation of acid-base balance[END_REF]. Furthermore, potassium influences the expression of the proximal tubular Na/sulphate-cotransporter. Although Kcit supplementation may have restored SO4 2-reabsorption through a reversal of transporter down-regulation by acidosis, it increased sulphate excretion (Fig 1C). This was consistent with data from Sabboh et al. [START_REF] Sabboh | Effects of plant food potassium salts (citrate, galacturonate or tartrate) on acid-base status and digestive fermentations in rats[END_REF] who suggested that sulphate reabsorption could not match increased generation of SO4 2-. Moreover, it seems the capacity of the kidney to excrete sulphate and phosphate was maintained during aging with Kcit supplementation, whereas it was altered after 21 mo in the NP and HP rats.

High-protein diet and potassium citrate supplementation had no effect on bone quality

The sole change reported on bone status was a marked age-related bone loss at the end of the experimental period (21 mo of age) (Fig 2). Bone density changes were consistent with the observed biomarker variations and resulted in decreased bone strength (Fig. 2B). These alterations may be related to the decreased IGF-1 secretion, IGF-1 being a bone anabolic hormone. The lower plasma IGF-1 levels probably result age-induced perturbation of the hypothalamo-adenohypophysial-somatotrophic axis [START_REF] Sonntag | Effects of aging and dietary restriction on tissue protein synthesis: relationship to plasma insulin-like growth factor-1[END_REF][START_REF] Thissen | Nutritional regulation of the insulin-like growth factors[END_REF]. Secondary hyperparathyroidism has been reported in old rats and was suggested to contribute to bone alteration. In parallel, these changes were associated with impaired ability of the kidney to excrete the dietary acid load with aging, and characterized by acidification of excreted urine and increased loss of urinary magnesium and calcium.

The femoral BMD was not affected by protein intake or Kcit supplementation (Fig 2A). Indeed, no significant differences were recorded among the groups at any of the time points. Corresponding femoral biomechanical properties remained unchanged as well (Fig 2B). These results are consistent with previous observations in rats by Whiting & Draper (45), Calvo et al. [START_REF] Calvo | Effect of protein-induced calciuria on calcium metabolism and bone status in adult rats[END_REF], Goulding & Campbell (47), showing no change in femoral bone mass or macro-mineral composition in response to dietary protein excess (30%, 35% and 60% excess, respectively), despite hypercalciuria. In contrast, using histomorphometric analysis, Amanzadeh et al. [START_REF] Amanzadeh | Effect of high protein diet on stone-forming propensity and bone loss in rats[END_REF] observed bone loss in rats fed a 48% casein diet during 59 days and displaying hypercalciuria and hypocitraturia.

The physical bone measurements were consistent with the bone biomarker levels, which did not vary between the four groups (Fig 2C andFig 2D). Yet, metabolic acidosis has been demonstrated to stimulate osteoclastic resorption and inhibit osteoblastic collagen synthesis in vitro [START_REF] Krieger | Acidosis inhibits osteoblastic and stimulates osteoclastic activity in vitro[END_REF][START_REF] Arnett | Regulation of bone cell function by acid-base balance[END_REF]. As far as hydroxyproline can be considered as a relevant indicator of bone resorption, high protein intake was associated with increased resorption levels [START_REF] El & Swaminathan | The effect of high protein and high salt intake for 4 months on calcium and hydroxyproline excretion in normal and oophorectomized rats[END_REF][START_REF] Goulding | Effects of oral loads of sodium chloride on bone composition in growing rats consuming ample dietary calcium[END_REF][START_REF] Schuette | Renal acid, urinary cyclic AMP, and hydroxyproline excretion as affected by level of protein, sulfur amino acid, and phosphorus intake[END_REF]. However, Creedon & Cashman (50) and Shapses et al. [START_REF] Shapses | Short-term changes in calcium but not protein intake alter the rate of bone resorption in healthy subjects as assessed by urinary pyridinium cross-link excretion[END_REF] reported no effect of high-protein diets on urinary excretion of collagen pyridinium crosslinks in rats and human subjects. In this study, the absence of variation in bone metabolism can be attributed to stable circulating PTH levels, a potent stimulator of bone resorption. Thus, all examined bone parameters concur to conclude that long-term low-grade metabolic acidosis, induced by a slightly hyperproteic diet (26% casein), does not affect the skeleton. However, it would be interesting to repeat the analysis with female rats that are subject to greater hormonal variations. Actually, it appears that the effects of dietary protein on bone status depend on multiple, interacting parameters [START_REF] Sebastian | Dietary protein content and the diet's net acid load: opposing effects on bone health[END_REF], including additional dietary factors. Protein and calcium, in particular, seem to act synergistically on human bone [START_REF] Heaney | Protein and calcium: antagonists or synergists?[END_REF][START_REF]Calcium and protein in bone health[END_REF].

An adequate calcium supply, as in the present study, may reduce or offset the negative effects of protein on bone mass. Here, calcium retention was not significantly affected by the highprotein (26%) diet or citrate salt intake at 9 and 16 mo of age (Table 3), although calcium fecal excretion was increased. Nevertheless, calcium retention was altered in the HP rats compared to the NP rats after 21 mo. This was a result of both higher fecal and urinary calcium excretion. Calcium retention did not vary between the NPKcit and the HPKcit groups. In summary, in the older rats the HP diet had deleterious effects on calcium retention and Kcit supplementation could prevent this impairment. The impact of high-protein intake on calcium retention is controversial since Creedon and Kashman [START_REF] Creedon | The effect of high salt and high protein intake on calcium metabolism, bone composition and bone resorption in the rat[END_REF] found no effect of this parameter in growing rats. Femoral BMD was unchanged in this study at 21 mo, but it is conceivable that bone density would be altered at later time points. These results remain to be confirmed in humans, given the differences suspected in intestinal calcium absorption.

The role of dietary protein in bone health is currently debated. Some authors argue that high-protein diets diminish bone health [START_REF] Feskanich | Protein consumption and bone fractures in women[END_REF][START_REF] Sellmeyer | A high ratio of dietary animal to vegetable protein increases the rate of bone loss and the risk of fracture in postmenopausal women. Study of Osteoporotic Fractures Research Group[END_REF], whereas others speculate that it is favourable for the skeleton [START_REF] Kerstetter | The impact of dietary protein on calcium absorption and kinetic measures of bone turnover in women[END_REF][START_REF] Hannan | Effect of dietary protein on bone loss in elderly men and women: the Framingham Osteoporosis Study[END_REF]. Considering that dietary protein supplies the amino acid building blocks for the bone matrix and stimulates the IGF-1 system, it may have a magnitudedependent anabolic effect on bone. We did not observe any differences in plasma IGF-1 levels between the four groups. However, IGF-1 is an osteotrophic factor and it was previously shown to be regulated by nutritional status, especially dietary protein intake [START_REF] Thissen | Nutritional regulation of the insulin-like growth factors[END_REF].

Inappropriate protein intake (2.5 % casein diet) impairs the IGF-1 system and has been suggested to lead to decreased bone mineral mass and fragility in rats [START_REF] Ammann | Protein undernutritioninduced bone loss is associated with decreased IGF-I levels and estrogen deficiency[END_REF]. The absence of variation in IGF-1 levels in this study is consistent with the BMD values, and probably reflects that our diets were less restrictive, particularly with respect to protein content.

In conclusion, the high-protein diet induced a low-grade metabolic acidosis, which persisted over the entire duration of the experiment. Potassium citrate supplementation had alkalinizing effects and neutralized the increased acid load. Neither beneficial nor detrimental effects associated with high-protein intake or potassium citrate supplementation was reported on bone status or metabolism under our experimental conditions. This could be explained by an adequate calcium supply because calcium intake may offset the effects of a proteingenerated acid load on bone mass.

  Urinary anion excretion. As expected, rats fed potassium citrate excreted larger amounts of citrate in the urine (around 12-to 16-fold more) than those who where not supplemented (P<0.0001) (Fig 1A). At 9, 16 and 21 mo of age, citrate excretion was modulated by the protein level in the diet (P<0.0001): it was lower in the HP group than in the NP group and, at 9 mo, it was lower in the HPKcit group than in the NPKcit group. Similar trends were observed at 16 and 21 mo of age (P=0.021). Urinary phosphate (Fig 1B)and sulphate (Fig 1C) excretion was increased in the groups fed the high-protein diets (HP and HPKcit) (P<0.0001), compared with those on the normal-protein diets (NP and NPKcit). At 21 mo, both phosphate and sulphate excretion was significantly lower in the HP group than in the HPKcit group (P=0.001). Chloride excretion did not vary between the groups (data not shown). Bone mineral density. Total BMD (T-BMD) did not vary between 9 and 16 mo (Fig 2A). M-BMD and D-BMD data are not represented since similar patterns were observed between T-BMD and both M-BMD (R=0.930, P<0.0001) and D-BMD (R=0.929, P<0.0001) values respectively. T-BMD (P=0.004), M-BMD (P=0.004) and D-BMD (P=0.026) were reduced at 21 mo compared to 9 and 16 mo. The level of protein intake and potassium citrate supplementation had no effect. Biomechanical properties. Femurs from 21 mo old rats had lower resistance to fracture (Fig 2B) than those from 9 and 16 mo old rats (P<0.0001). The biomechanical data were positively correlated with the BMD data (R=0.709, P<0.0001). No significant differences were recorded between the four groups at any age. Bone biomarkers. Plasma OC was reduced in all groups at 16 and 21 mo (P<0.0001) compared to 9 mo (Fig 2C). The urinary DPD excretion rate was similar at 9 and 16 mo of age then decreased at 21 mo (P<0.0001) (Fig 2D). Both bone biomarkers were unaffected by diet protein content and potassium citrate supplementation. Osteocalcin was positively correlated with calcium retention (R=0.660, P=0.014).

Table 1 .

 1 Diet compositions.

	Component (g/100 g)	NP	HP	NPKcit	HPKcit
	Casein	13.0	26.0	13.0	26.0
	Corn starch	57.7	47.1	54.8	44.2
	Sucrose	14.4	11.8	13.7	11.1
	Groundnut and rape oils	6.0	6.0	6.0	6.0
	Cellulose	4.0	4.0	4.0	4.0
	Potassium citrate	0	0	3.60	3.60
	L-cystin	0.19	0.39	0.19	0.39
	Choline bitartrate	0.25	0.25	0.25	0.25
	Mineral mix 1	3.50	3.50	3.50	3.50
	Vitamin mix 2	1.00	1.00	1.00	1.00
	1 Expressed in g/kg of mixture: CaCO3, 178.5; KH2PO4, 250; K3C6H5O7, 28.00; NaCl,
	74; K2SO4, 46.60; MgO, 24.00; FeC6H8O7: 6.06; ZnCO3: 1.65; Na2SiO3•9H2O, 1.45;
	MnCO3, 0.63; CuCO3, 0.30; CrKO8S2•12H2O, 0.275; H3BO3, 0.0815; FNa, 0.0635;
	NiCO3, 0.0318; LiCl, 0.0174; Na2SeO4, 0.01025; KIO3, 0.010; (NH4)6Mo7O24•4H2O,
	0.00795; NH4VO3, 0.0066.
	2 Expressed in g/kg of mixture : nicotinic acid, 3.000; calcium pantothenate, 1.600;
	pyridoxine-HCL, 0.700; thiamine-HCL, 0.600; riboflavin, 0.6000; folic acid, 0.200;
	biotin, 0.020; cyanocobalamin, 2.500; all-rac-α-tocopheryl acetate (500 IU/g), 5.000;
	all-trans-retinyl palmitate (500000 IU/g), 0.800; cholecalciferol (400000 IU/g), 0.083;
	phylloquinone, 0.075.

Table 2 .

 2 Body weight, urine pH, urinary potassium, sodium and magnesium excretion in rats fed a normal protein diet, a high protein diet, a normal protein potassium citrate supplemented diet and a high protein potassium citrate supplemented diet for 9, 16 and 21 months. -d : significance of difference between NP, HP, NPKcit and HPKcit groups at a given age (Means without a common letter differ, P≤0.05).

			NP	HP	NPKcit	HPKcit	Statistical significance, P values :
		9 mo	640.8 ± 20.0	651.2 ± 14.9	642.8 ± 16.2	653.9 ± 15.8
	Body weight
	(g)
		16 mo	1.995 b ± 0.149	2.007 b ± 0.056	7.890 a ± 0.474 7.337 a ± 0.320
		21 mo 1.357 b * ± 0.085 1.331 b * ± 0.162 7.749 a ± 0.445 7.192 a ± 0.900
		9 mo	1.127 ± 0.072	1.221 ± 0.050	1.184 ± 0.066	1.272 ± 0.060
	sodium
	(mmol/24 h)	16 mo	1.124 ± 0.086	1.255 ± 0.048	1.130 ± 0.071	1.163 ± 0.083
		21 mo	1.122 ± 0.080	1.180 ± 0.097	1.151 ± 0.086	1.093 ± 0.104
	magnesium	9 mo	44.03 ± 7.00	57.37 ± 6.58	47.74 ± 8.64	49.38 ± 6.58	Age: P=0.001
	(µmol/24 h)	16 mo	68.72 † ± 6.58	78.89 † ± 2.06	62.55 † ± 5.35	70.37 † ± 7.00
		21 mo	66.25 † ± 7.82	78.24 † ± 8.64	60.49 † ± 4.53	71.19 † ± 4.53
	Values are expressed as means±SEM, n=10 per group.
	*: significantly different from 9 and 16 mo of age.
	† : significantly different from 9 mo of age.

a

Table 3 .

 3 Calcium intake, fecal excretion, urinary excretion and retention in rats fed a normal protein diet, a high protein diet, a normal protein potassium citrate supplemented diet and a high protein potassium citrate supplemented diet for 9, 16 and 21 months. ± 2.50 15.50 a ± 3.25 11.25 b ± 2.25 11.00 b ± 1.25 21 mo 12.25 b ± 1.25 17.00 a ± 2.75 10.75 b ± 2.50 12.50 b ± 2.50 -d : significance of difference between NP, HP, NPKcit and HPKcit groups at a given age (Means without a common letter differ, P≤0.05). Figure 1. Effect of protein intake and potassium citrate supplementation on urinary citrate (A), phosphate (B) and sulphate (C) excretion (mmol/24 h) at 9, 16 and 21 mo of age. Values are expressed as means±SEM, n=10 per group. : significantly different from 9 and 16 mo of age a-d: significance of difference between NP, HP, NPKcit and HPKcit groups at a given age (Means without a common letter differ, P≤0.05). Figure 2. Effect of protein intake and potassium citrate supplementation on total femoral BMD (A), femoral biomechanical resistance (B), plasma osteocalcin (C) and the urinary DPD excretion rate (D) at 9, 16 and 21 mo of age. Values are expressed as means±SEM, n=10 per group. : significantly different from 9 and 16 mo of age †: significantly different from 9 mo of age

			NP	HP	NPKcit	HPKcit	Statistical significance, P values :
	Ca intake	9 mo	1402 ± 24	1407 ± 21	1409 ± 17	1396 ± 16
	(µmol/24 h)	16 mo	1412 ± 27	1417 ± 16	1401 ± 24	1404 ± 25	Age: P<0.0001
		21 mo	1293* ± 9	1306* ± 16	1291* ± 10	1299* ± 18
	Fecal calcium	9 mo	1158 b ± 38	1267 a ± 41	1206 b ± 37	1173 b ± 26
	excretion	16 mo	1339 b † ± 24	1444 a † ± 51	1326 b † ± 36	1329 b † ± 53	Age: P<0.0001 Protein x Kcit: P=0.027
	(µmol/24 h)	21 mo	1370 b † ± 31	1503 a † ± 56	1350 b † ± 48	1365 b † ± 51
	Urinary Ca	9 mo	8.00 b ± 1.50	15.25 a ± 2.25	8.50 b ± 1.25	9.75 b ± 1.75
	excretion (µmol/24 h) 9.25 b Ca retention 16 mo 9 mo 236 ± 52 (µmol/24 h) 16 mo 63 ± 41	105 ± 49 -32.5 ± 56	199 ± 52 64 ± 53	214 ± 31 64 ± 80	Protein: P=0.003 Kcit: P=0.041 Protein x Kcit: P=0.0009 Age x Protein x Kcit: P=0.044 Age: P<0.0001
		21 mo	-89 a † ± 51	-214 b † ± 42	-70 a † ± 55	-78 a † ± 79
	Values are expressed as means±SEM, n=10 per group.

*: significantly different from 9 and 16 mo of age. † : significantly different from 9 mo of age. a**


Mehmet-Ali Akinci

Relative clauses in the written texts of French and Turkish bilingual and monolingual children and teenagers

 as a unified predicate describing a single situation (an activity, event, or state). The results show that bilingual primary school students use more relative clauses in French written texts than their monolingual counterparts contrary to bilingual high school students which use fewer relative clauses. Moreover, Turkish-French bilinguals and Turkish monolinguals present similar use of relative clauses by invalidating the intuitive idea that bilinguals would present inferior linguistic ability in either language.

Theoretical Focus

One of the important aspects of constructing texts is to be cohesive and coherent from the beginning to the end. If a text is well formed, that's because it establishes a local relationships between propositions and a well-organized information about characters or events into a globally defined unit [START_REF] Bamberg | What holds a narrative together? The linguistic encoding of episodic boundaries[END_REF]. It is crucial for the text producer to mention clearly characters, events, and other concepts in order to allow the reader to understand the story through cohesive ties. Therefore, the competence to specify referents in the discourse permits to create a coherent oral or written discourse [START_REF] Lautamatti | Coherence in Spoken Discourse[END_REF][START_REF] Enkvist | Coherence, pseudo-coherence, and non-coherence[END_REF].

In the last few decades there has been an increase in the number of studies on the use of particular grammatical structures in discourse. Especially those that contribute organization of sequentially related events in both temporally linearized level and in thematic level has received a considerable amount of focus. In this body of research, many scholars have been concerned with the functional analysis of relative clauses in contributing connectivity between events by establishing a hierarchical relationship between two clauses. For example, Aksu-Koç & Erguvanlı-Taylan (1994) look at the functions of relative clauses in oral narratives and find correlations between the morphosyntactic and semantic properties of relative clauses, their information status and their functions in narrative discourse. Another original work is [START_REF] Dasinger | The development of relative clause functions in narrative[END_REF] crosslinguistic study which considers both formal and functional complexity of relative clauses.

According to [START_REF] Dasinger | The development of relative clause functions in narrative[END_REF] increasing competence in the expression of narrative organization influences the use of relative clauses varying with referential context across ages.

Since relative clauses as one of the structural options for introducing and maintaining referents across clauses create referential cohesion in clause combining, the developmental use of relative clauses both in form and in function indicates increasing levels of discourse competence and textual organizations underscoring the cognitive and pragmatic proficiency at different ages [START_REF] Dasinger | The development of relative clause functions in narrative[END_REF][START_REF] Jisa | Relative clauses in French children's narrative texts[END_REF].

Although the studies mentioned above provide a thorough comparative analysis of relative clause in the texts produced by both children and adults in different languages, all of the subjects recruited in these studies are monolinguals. Our aim, in this study, is to include a young bilingual population in order to understand the relation between development of linguistic forms and textual coherence by means of comparison between bilingual and monolingual children across ages. In this manner, we also hope to shed light on their ways to produce coherent texts as being bilinguals. In this study, we recruited French-Turkish bilinguals in addition to French and Turkish monolinguals and asked them to write one narrative text and one expository text successively. Our bilingual population write first in their home language which is Turkish and then in French which is considered as their dominant productive language by the age of ten (Akinci, 1999).

Another line of developmental research on child language has focused on the relative clause formation as being one of the most complex structures acquired by children with difficulty. Some of these studies have particularly directed their attention to the relation between the relativized head nouns in relative clause and their grammatical function in respective clause. For example, according to parallel function hypothesis [START_REF] Sheldon | The parallel function in the acquisition of relative clauses in English[END_REF][START_REF] Clancy | Processing strategies in the acquisition of relative clauses: Universal principles and language-specific realizations[END_REF]MacWhinney & Pleh, 1988), relative clause sentences in which the head noun plays the same role in the relative clause and the main clause will be easier to understand for young children than sentences in which the roles differ. In our study, we will also investigate if there is any difference for the use of parallel function in relative clause sentences in Turkish written texts by bilinguals and monolinguals. To the best of our knowledge, this analysis has not been done in Turkish written texts.

Our particular concern is to depict a developmental path of the acquisition of relative clauses as a mean of subordination in comparison to monolinguals versus bilinguals. The relative clauses used in Turkish-French bilinguals' texts compared to those of Turkish monolinguals and French ones across age groups are categorized in terms of the morphosyntactic properties of relative clauses to understand whether the children's production of relativized constituent is influenced by their grammatical function in the clause it immediately belongs to. In this analysis we attempt to figure out whether, and to what extent it plays a role in bilingual population. Before going into details of our analysis presented in this study, a small review of relative constructions in French and in Turkish is needed.

Relative constructions in French

There are two types of relativizers in French. Simple relativizers (qui, que, quoi, dont, où) and compound relativizers (lequel, duquel…). These vary according to genre and number.

Noun subject relativizer: qui

Qui is the more frequent and simplest relativizer as its use does not imply change in constituents' order. Similar to English relative constructions, French subject relativizer "qui" preserves the canonical subject-verb-(object) word order in relative clause. Qui has no genre neither number its form remains the same whether it may relativises a plural or a singular subject.

Therefore, its use is quiet simple for children, who use them in oral texts very frequently.

(1) j'ai frappe une fille qui s'appelle Léa (Pri Mono WN) (I beat a girl who is called Léa) Notice that the verb of the relative clause agrees with the antecedent (in this case 'une fille') when it acts as the subject.

Noun object relativizer: que Que is less frequently used than qui. This can be explained because que implies constituents' order change. Indeed the object relativizer que is removed in front of the verb's subject.

(2)

Un élève que je ne connais pas m'a poussé dans les buissons (Sec. Bi WN)

(A pupil (that) I don't know pushed me in the bush)

According to Arrivé, Gadet & Galmisch, "the more forms change the canonical order of sentences' words, the less they are used in oral practices and, also in written texts of children and less educated adults" (1986: 605). Relative clauses are early acquired structures in French but their functional diversity increases with development [START_REF] Jisa | Relative clauses in French children's narrative texts[END_REF].

Relative constructions in Turkish

Turkish relative clauses are pronominal, that is the relative clause is positioned before the relativized noun. There are two basic relative clause types in Turkish; namely subject relative, and non-subject relative differed in the suffixes attached to the participial form of the verb preceding the head noun. Where the target of relativization is a subject or part of a subject, -An suffix gets attached to the verb. The target of the relativization is phonologically unexpressed and subject-verb order is reversed.

(3) a. Adam koşuyor. man run-PROG&3SG "The man is running."

b. koş-an adam run-SUBJECT RELATIVIZER man "the man who is running"

Where the target of relativization is an object, -DIK suffix is attached to the verb. Similar to subject relative, the target is phonologically silent. The participial form of the verb of the relative clause is followed by agreement for the subject of the modifier clause. As in other embeddings, that subject receives Genitive Case. Turkish has also headless relative clauses where the head noun of the relative clause is phonologically unexpressed. And the morphology of the head noun which is missing in the relative construction gets attached to the participial form of the relative clause.

(5) a. Koşan adamı gördüm. run-SUBJECT RELATIVIZER man-ACC see-PAST-1SG "I saw the man who is running." b.

Koşanı gördüm. run-SUBJECT RELATIVIZER-ACC see-PAST-1SG "I saw the man who is running."

Method

The conceptual and methodological basis for this study is derived from an international research project on the development of text production abilities as a critical indicator of literacy across and beyond school ages [START_REF] Berman | Cross-Linguistic Perspectives on the Development of Text-Production Abilities in Speech and Writing. Part 1 & Part 2[END_REF]. Similarly, in our study, 60 participants in three age groups (20 students from primary school, 20 from junior school and 20 from high school for each population) were asked to produce two types of text (personal narration and expository) in two modalities (spoken and written), amounting to two narratives and two expository discussions from each speaker. The texts were elicited from participants in two successive sessions. In session I, they were asked to tell and write a story about an incident of interpersonal conflict that they had experienced personally. In the following session, expository discussions were gathered, where participants were asked to give a talk as if they are in front of their class, or write an essay or composition, discussing the issue of interpersonal conflict. All subjects produced both the narrative and expository texts in two modalities of speech and writing yielding a total of 4 texts per subject. Mode of presentation was balanced across the tasks, so that half of the subjects performed the spoken task first, and then produced a written text, while the other half started with the written text and continued with the spoken task. The sessions and the texts elicited in these sessions are depicted in Table 1. As mentioned before, at the end of the data collection phase, each subject ended up with 4 texts. These are oral narrative (ON), written narrative (WN), oral expository (OE) and written expository (WE) texts. All of the subjects produced narratives first and then came their expository texts. Only the mode of presentation was balanced across the four texts, that is half of the subjects yielded their spoken tasks first, after that they began writing what they told to the researcher. This spoken-first group formed Group A. At the same time, the other half of the subjects who are in Group B performed their tasks in written mode first.

In this paper, only the written texts produced by the subjects in both types of texts will be discussed. Therefore, in the remaining part of this paper only the written texts will be discussed and all the analysis will be based on written texts of monolingual and bilingual children.

Research population

The research groups of bilingual and monolingual children were organized according to their school-grade level. The texts were elicited from one group of primary school children, one group of secondary school, and one group of high school students. Each group consists of at least 20 subjects. The following Tables 2 and3 give the information about number and age of the bilingual informants in each group. The bilingual informants for this study were selected from the Turkish immigrant community living in Rouen and Grenoble. In order to control for the gender factor, we included equal numbers of males and females. They are sons and daughters of the first generation immigrants in France all of whom were born there. They start to acquire French, which will become their dominant language, essentially at nursery school entering at around the age of 3 (Akinci, 2001).

To complete the study which aims to compare and contrast the developing written texts production of Turkish-French bilingual children, we collected cross-sectional data in Turkey in from Turkish monolinguals in a little town of Turkey and in France from French monolinguals. The monolingual subjects are presented in Table 4 and in Table 5. The French monolinguals are raised and educated in French with low educational and socio-economic family backgrounds. They attend the same schools and live in the same neighborhoods as the immigrant children above mentioned.

Data from Turkish monolinguals were collected in a little town of Turkey that matched the place of origin of the parents of the bilingual informants. Two schools in a district of Denizli were cooperated to this study. These monolingual groups can be in that in terms of socioeconomic status, compared to our bilingual group. (1992), there has been a slight shift in the occupational monolinguals' fathers belong much more often to the liberal professions, qualified technician, trade or office workers than the others. As for the Turkish monolinguals' fathers, they often belong to the farmer, craftsmen and storekeepers, teachers and retired.

As far mothers occupation is concerned, we observed that mothers of bilinguals and Turkish monolinguals are much more often housewives than French monolinguals. This situation concerns indeed respectively 72% and 73% of bilingual and Turkish monolinguals' mothers. In contrast, the mothers of French monolinguals are overrepresented in almost all occupations. They more often work as manager than Turkish counterparts do. They also work as office manager or as cleaning ladies.

Data elicitation and collection

All subjects in France and in Turkey were given similar motivational instructions. At the very beginning, all the informants were told that they were recruited for an international project about the literacy skills of Turkish bilingual children living in Europe and Turkish monolingual children in Turkey. Three researchers were present during the data collection phase to help out the subjects. First, the subjects saw a short video film with no words before task elicitation. The film is three-minute long and it shows different kinds of problems depicted in a school setting. These scenes of interpersonal conflicts between people are categorized by [START_REF] Berman | Cross-Linguistic Perspectives on the Development of Text-Production Abilities in Speech and Writing. Part 1 & Part 2[END_REF] in three groups: moral conflicts (cheating in an exam, and finding money somebody has dropped); social conflict (to exclude somebody in a class); and physical conflict (fighting in a school, and spilling water on somebody). The events in the film do not have any resolution or concluding part.

The data collection phase began as dividing the subjects into two groups: Group A and Group B. The students in Group A were asked one by one to tell a story about a problem that they had experienced personally. As in Berman and Verhoeven's study (2002), they were clearly instructed not to describe the scenes in the video but to tell an event they experienced personally, and their elicitations were recorded to a minidisc. After they completed their elicitation, they were sent to another room to write down the same event that they told us. Once they completed their writings, they were asked to discuss the problems between people as they gave a talk in class. They were instructed not to tell a story but discuss the issue and state their ideas while they were recorded in a minidisc. In the same time with Group A, students in Group B did the same processes. The only difference between Group A and Group B was that students in Group B started with writing their structure from blue-collar (89.9% in 1982, 80% in 1989) to white-collar jobs and self-employment (both, 6.6% in 1982, 18.5% in 1989), the majority of the working Turkish population can still be identified as blue-collar.

personal experience narratives. They first wrote down and then told what they have written down.

Again, the sessions of Group B began with the personal narratives and continued with expository texts.

The sessions were carried out on the same day, or with one day interval. At the end of each session, the subject has produced two texts, one is written and one is spoken. The narratives are on the same event which happened to them, and the expository texts discuss the same issue that is conflict between people. At the end of the data collection phase, each subject has 4 texts produced under the same instructions. For our analysis, we summed up all relative clauses in narrative and expository texts in Group A and Group B.

Coding procedures

In this study, the main unit of analysis is "the clause" for the linguistic analysis which is defined by [START_REF] Berman | Relating events in narrative: a crosslinguistic developmental study[END_REF] as a unified predicate describing a single situation (an activity, event, or state). We first identified the relative clauses produced by our monolingual and bilingual subjects across all age groups.

After the relative clauses occurred in the database were identified, all relative clauses were classified according to the grammatical function of its head noun in the relative clause and in the clause it immediately belongs to. Two clause types which form our analytical framework are:

1. the relativizer is always subject in the RC, the head noun of RC can be subject or nonsubject in the broader clause:

(6) J'avais une copine qui habitait Paris (Sec BIL NW) I had a girlfriend who lived in Paris Paris'te yaşayan bir arkadaşım vardı (7) mon copain qui était malin n'a pas voulu se battre (Sec BIL NW) My boyfriend who was intelligent did not wanted to fight Akıllı olan erkek arkadaşım kavga etmek istemedi 2. the relativizer is always non-subject in the RC, the head noun of RC can be subject or nonsubject in the broader clause:

(8) je venais d'arriver à un collège que je ne connaissais pas (High BIL NW) I had just arrived to a high school that I did not know Tanımadığım bir okula yeni gelmiştim (9) L'histoire que j'ai racontée s'est déroulée au lycée (High BIL NW)

The story that I told was held at high school Anlattığım hikaye lisede geçti

Results

In this section, it will be presented the number of total clauses as well as the number and percentages of relative clause across every school age. Moreover, we also separated the use of subject relativizers and object relativizers within relative clauses by means of percentages. For every written language, we carried out a X 2 test of independence between bilinguals and monolinguals calculated by the number of clause occurrences. We will mention this analysis only when it reveals to be significant with a 5% error margin. We will particularly look into comparisons between close age groups like primary versus secondary or secondary versus high school students across linguistic category (bilingual, monolingual). Firstly, before going into further details about relative clause use, we have conducted an analysis about total clause distribution. The distribution of total clauses produced by bilinguals and monolinguals in French written texts was significantly different X 2 (df= 2, N= 3282) =12.701, p< .002, across three school groups. Overall, Turkish-French bilinguals produce more clauses than

French written texts

French monolinguals (1791 versus 1491 clauses). The main difference came from monolingual clause production which is less (360) in secondary school than their bilingual counterparts production (522) when we test them with high school bilinguals and monolinguals X 2 (df= 1, N= 2571) =12.511, p<.001.

The X 2 analysis revealed that relative clause production from bilinguals and monolinguals differ across three school ages X 2 (df=2, N=199) = 16.849, p<.001. The difference between monolingual primary (7) and secondary (25) relative clause production versus bilingual primary (20) and secondary (25) relative clause production is significative X 2 (df=1, N=77) = 4.184, p<.05.

Indeed, bilingual primary school students use more relative clauses than their monolingual counterparts. While this finding looks surprising, it might be linked to the fact that bilingual pupils felt confident with a bilingual researcher with whom they communicated.

However, another significant difference concerns high school subjects: bilinguals have fewer relative clauses (40) than monolinguals (82) when compared to secondary school students in French written texts X 2 (df=1, N= 172)= 4.47, p<.05.

It's also worth mentioning that, overall use of relative clause is slightly fewer (4,7%) in bilinguals than monolinguals (7,6%) although this difference does not seem to be statistically significant. Moreover, the use of subject relativizers seems to be more dominant in every category (bilingual or monolingual versus school ages) in French written texts. The X 2 analysis did not revealed any significant results neither in total clause comparison nor relative clause comparison between bilinguals and monolinguals across school ages. Since relative clauses as one of the structural options for introducing and maintaining referents across clauses create referential cohesion in clause combining, both monolingual and bilingual children show a similar developmental course in terms of relative clause constructions.

Turkish written texts

When we look at the Turkish texts of French-Turkish bilinguals, we observe the tendency to use the relative clause, similar to that of Turkish and French monolinguals. Such a tendency gradually develops after primary school. Quantitative data show that bilinguals and monolinguals use nearly same percentage of relative clauses as their monolingual counterparts in Turkish.

Parallel function analysis in Turkish written texts

According to [START_REF] Sheldon | The parallel function in the acquisition of relative clauses in English[END_REF], parallel function hypothesis claims that children will be following a strategy of interpreting the grammatical function of the relative pronoun as being the same as its antecedent. "It follows from this hypothesis that those sentences in which the shared nominals have the same grammatical function in their respective clauses like The man saw the boy who the girl hit will be easier to process than sentences in which the co-referential NPs have different grammatical functions, like The man saw the boy who hit the girl (pg:275)". Since 1974, others confirmed this idea by highlighting that is a crosslinguistic effet (for a review about relative clauses in French, see [START_REF] Jisa | Relative clauses in French children's narrative texts[END_REF][START_REF] Jisa | Relative clauses in French children's narrative texts[END_REF][START_REF] Amy | Stratégies de traitement des phrases relatives: quelques considérations d'ordre génétique[END_REF][START_REF] Amy | Stratégies de traitement des phrases relatives: quelques considérations d'ordre génétique[END_REF].

In order to see if this hypothesis applies in Turkish written texts, we also calculated the number of relative clauses with the same grammatical function than their nominals and with different grammatical functions. Moreover, we also wanted to see if there is a difference in the use of parallel functional relatives among Turkish-French bilinguals and Turkish monolinguals. In this analysis, we coded as a parallel relativiser when:

• the head noun of RC have the same grammatical functions in the relative clause and in the The X 2 analysis did not revealed any significant results in parallel effect distribution among French-Turkish bilinguals and Turkish monolinguals. This means that, indeed, there is a higher use of parallel functional relativiser in any case (153 versus 101 among monolinguals and 64 versus 47 among bilinguals). It seems that in every linguistic category, children prefer to use parallel functional relativizes without any difference. However, when we look at the Turkish texts of French-Turkish bilinguals, we observe the tendency to use parallel grammatical functions in the relative clause and in the broader clause, similar to that of Turkish monolinguals. However, such a tendency gradually develops after primary school. It seems that for further analyses, we should in our coding take into account the function of the head noun in the broader clause, the diversification of relativizers and the difference between expositive and narrative texts.

Conclusion and discussion

In order to make any discourse easily understandable, we have to present any temporal, spatial and characteristic information in a coherent and integrated way. Relative clauses are, probably, the most solicited grammatical structure to achieve this aim. In language development, this capacity is one of the latest structures acquired by children probably because of its need of more complex cognitive resources [START_REF] Evans | Cognitive Linguistics: An Introduction[END_REF]. This is why, in order to study the complexity of text production between French monolinguals and Turkish-French bilinguals, we chose to investigate the use of relative clauses. Moreover, to the best of our knowledge, this is the first time an investigation of relative clauses in French monolinguals' text production has been compared to Turkish-French bilinguals living in France across school ages.

According to our results, first of all, our study shows that Turkish-French bilingual young people stemming from Turkish immigration have very similar use of relative clauses compared to those of French and Turkish monolinguals in Turkish produced texts. For instance, X 2 analysis did not revealed any significant results neither in total clause comparison nor relative clause comparison between bilinguals and monolinguals. While an absence of significance does not mean that those two populations have the same developmental course, this finding clearly shows that both monolingual and bilingual children show a similar developmental course in terms of relative clause constructions.

On the other hand, the X 2 analysis did revealed a significant difference between monolingual primary and secondary relative clause production versus bilingual primary and secondary relative clause production calculated all together. This difference is best explained by higher production of relative clauses in Turkish-French primary school bilinguals' texts than their monolingual counterparts (20 to 7 production). Nevertheless, another significant finding comes again from high school bilinguals: they have fewer relative clauses than monolinguals (40 to 82) in French written texts. This is probably due to the fact that overall use of relative clause is slightly fewer in bilinguals than monolinguals.

Another novelty of our work is that for the first time we investigated parallel function analysis in Turkish written texts by comparing monolinguals and bilinguals. The parallel function hypothesis [START_REF] Sheldon | The parallel function in the acquisition of relative clauses in English[END_REF] was also observed in French written texts [START_REF] Jisa | Relative clauses in French children's narrative texts[END_REF][START_REF] Amy | Stratégies de traitement des phrases relatives: quelques considérations d'ordre génétique[END_REF] but only within monolingual children. As expected, we find that there is higher production of parallel relativisers than non-parallel relativisers in any case which means that there is no difference between Turkish monolinguals and Turkish-French bilinguals for their preference to use parallel relativisers. This gives us an additional idea of the similarity between Turkish monolinguals' and Turkish-French bilinguals' text production's complexity level.

In conclusion, our work seems to indicate that if there is a difference between Turkish monolinguals' and Turkish-French bilinguals' text complexity, this is more likely to manifest itself in French written texts. Moreover, this difference tends to show itself only among high school pupils. Further studies comparing monolinguals and bilinguals should investigate this matter by counting not only relative clauses but also by counting lexical richness index which is another determiner of language complexity.

  get-OBJECT RELATIVIZER-POSS money "the money that I got" There are other relativizer suffixes in Turkish as well such as -(y)EcEK, -mIş or aorist suffixes, but they are used relatively rare. They can function as subject or non-subject relatives.

  broader clause it immediately belongs to (these are the subject of both the broader clause & the relative clause (SS) and the non-subject of both the broader clause & the relative clause (OO)). and we coded as a non parallel relativiser when:• the head noun of RC have different grammatical functions in the relative clause and in the broader clause it immediately belongs to (these are the head noun is both the non-subject of the broader clause & the subject of RC (OS) and the head noun is both the subject of the broader clause & the non-subject of RC (SO)).

Table 1 :

 1 Order of presentation across the tasks and groups

		Session I	Session II	Number of subject
	ORDER A	Narrative spoken /	Expository spoken /	5 girls and 5 boys
		Narrative written	Expository written
	ORDER B	Narrative written /	Expository written /	5 girls and 5 boys
		Narrative spoken	Expository spoken

Table 2 :

 2 Age, number, mean age, range of the ages of Turkish-French bilingual informants in Turkish written texts

	School type	Primary		Secondary		High
	Nb. Of subject	20		20		20
	Sex	M	F	M	F	M	F
	Number	12	8	10	10	8	12
	Mean age	11;00		13;00		16;08
	Range ages	09;07-11;11		12;07-13;09		15;00-18;09
	Table 3: Age, number, mean age, range of the ages of Turkish-French bilingual informants in French written texts
	School type	Primary		Secondary		High
	Nb. Of subject	22		22		22
	Sex	M	F	M	F	M	F
	Number	13	9	9	13	10	12
	Mean age	11;00		13;00		16;01
	Range ages	10;00 -11;11	12;07 -13;11	14;10 -18;06

Table 4 :

 4 Age, number, mean age, range of the Turkish monolingual informants

	School type	Primary		Secondary		High
	Nb. Of subject	20		20		20
	Sex	M	F	M	F	M	F
	Number	9	11	9	11	10	10
	Mean age	11;00		12;09		15;06
	Range ages	10;04-11;07		12;06-13;04		13;10-16;11

Table 5 :

 5 Age, number, mean age, range of the French monolingual informants

	School type	Primary		Secondary		High
	Nb. Of subject	22		22		22
	Sex	M	F	M	F	M	F
	Number	10	12	11	11	12	10
	Mean age	11;01		13;04		16;03
	Range ages	09;09 -11;09	12;03 -14;06	15;09 -17;06
	It seems almost stereotypic, but all the fathers of bilingual informants except one person, are
	factory employees or unskilled workers and the free-lance masons. These two occupations
	constitute more than half of the fathers' activity (56/106). They are widely under-represented in
	contrast to very qualified jobs: technician, trade or office workers) 1 . On the other hand, French

1 According to

Echardour & Maurin (1993)

, 43.7% of Turks are working in production, 28.5% in construction and 23.5% in the services. Although, following the study of Brabant

Table 6 .

 6 Distribution of relative clauses for Turkish-French bilinguals in French written texts

		Primary school	Secondary school	High school	Total
	Total clauses	393	522	876	1791
	Total RC	20	25	40	85
	% RC of total clauses	5%	4,7%	4,5%	4,7%
	% subject relativizers	72%	60%	52%
	% object relativizers	12%	40%	18%

Table 7 .

 7 Distribution of relative clauses for French monolinguals in written texts

		Primary school	Secondary school	High school	Total
	Total clauses	318	360	813	1491
	Total RC	7	25	82	114
	% RC of total clauses	2,2%	7%	10%	7,6%
	% subject relativizers	57%	73%	56,5%
	% object relativizers	28,5%	11,5%	22,5%

Table 8 :

 8 Distribution of relative clauses for Turkish-French bilinguals in Turkish written texts

	Grade	Primary school	Secondary school	High school	Total
	Total clauses	292	385	658	1335
	Total RC	20	34	61	115
	% RC of total clauses	6,8%	8,8%	9,2%	8,6%
	% subject relativizers	50%	51%	67%
	% non-subject relativizers	17%	29%	23%
	Table 9 distribution of relative clauses for Turkish monolinguals in written texts
	Grade	Primary school	Secondary school	High school	Total
	Total clauses	609	851	1418	2878
	Total RC	47	68	139	254
	% RC of total clauses	7,7%	8%	9,8%	8,8%
	% subject relativizers	52%	54%	45%
	% non-subject relativizers	33%	36%	42%

Table 10 .

 10 Distributions of SS+OO and SO+OS types of relative clauses across Turkish monolinguals

	Type	Monolinguals	SS+OO	SO+OS
	Primary		34	13
	Secondary		44	30
	High		75	58
		Total	153	101

Table 11 .

 11 Distributions of SS+ OO and SO+OS types of relative clauses in Turkish written texts of Turkish-French

	bilinguals
	Type	Bilinguals	SS+OO	SO+OS
	Primary		6	10
	Secondary		15	13
	High		43	24
		Total	64	47