Natalia Sinitskaya

Sylviane Gourmelen

Carole Schuster-Klein

Béatrice Guardiola-Lemaitre

Paul Pevet

Etienne Challet

Increasing fat-to-carbohydrate ratio in a high-fat diet prevents the development of obesity but not prediabetic state in rats

Keywords: Obesity, insulin resistance, metabolic syndrome, leptin, adiponectin BMI, body mass index, CHO, carbohydrate, HCD, high-carbohydrate diet, HFD, high-fat diet HDL-C, high-density lipoprotein-cholesterol, LDL-

Metabolic disorders induced by high-fat feeding in rodents evoke some, if not all the features of the human metabolic syndrome. The occurrence and severity of metabolic disorders, however, varies according to rodent species, and even strain, as well as the diet. Therefore we investigated obesogenic and diabetogenic long-term effects of 3 high-fat diets differing by their fat/carbohydrate (CHO) ratios. Sprague-Dawley rats were fed for 10 weeks with either a control, high-carbohydrate, low-fat diet (HCD; 12/64/24 ratio of fat/CHO/protein, 3.7 kcal/g), a high-fat, medium-carbohydrate diet (HFD1; 53/30/17 ratio of fat/CHO/protein, 4.7 kcal/g), a very high-fat, low-carbohydrate diet (HFD2; 67/9/24 ratio of fat/CHO/protein, 5.2 kcal/g), or a very high-fat, carbohydrate-free diet (HFD3; 75/0/25 ratio of fat/CHO/protein, 5.9 kcal/g).

Compared to the control diet (HCD), rats fed with high fat combined with more (HFD1) or less (HFD2) of CHO exhibited higher body mass index (+13 and +10% BMI, respectively; P<0.05) and abdominal fat (+70% in both HFD1 and HFD2; P<0.05), higher plasma leptin (+130 and +135%, respectively; P<0.05), lower plasma adiponectin levels (-23 and -30%, respectively; P<0.05), and impaired glucose tolerance. Only the HFD1 group showed insulin resistance. By contrast, a very high-fat diet devoid of CHO (HFD3) led to impaired glucose tolerance, insulin resistance and hypoadiponectinemia (-50%, P<0.05) while BMI, adiposity and plasma leptin did not differ from respective values in animals fed with the control diet.

We conclude that increasing fat-to-carbohydrate ratio to the uppermost (i.e., carbohydratefree) in a high-fat diet prevents the development of obesity, but not the prediabetic state (i.e., altered glucose tolerance and insulin sensitivity).

INTRODUCTION

Spontaneous mutations and gene targeting in rodents have triggered remarkable progress for identifying key genes involved in the regulation of adiposity. In humans, however, monogenic causes of obesity are rare. Rather, the risk of developing obesity appears largely polygenic in combination with a large spectrum of environmental obesogenic influences.

Food and beverages rich in energy, fat and/or sugar are now commonly found in modern societies. Besides genetic predisposition [START_REF] Krosnick | The diabetes and obesity epidemic among the Pima Indians[END_REF][START_REF] Williams | Individual estimates of European genetic admixture associated with lower body-mass index, plasma glucose, and prevalence of type 2 diabetes in Pima Indians[END_REF], physical inactivity [START_REF] Keller | From obesity to diabetes[END_REF][START_REF] Mohan | Association of physical inactivity with components of metabolic syndrome and coronary artery disease -the Chennai Urban Population Study (CUPS no. 15)[END_REF] and perinatal environment [START_REF] Plagemann | Perinatal programming and functional teratogenesis: impact on body weight regulation and obesity[END_REF][START_REF] Stocker | Prevention of diet-induced obesity and impaired glucose tolerance in rats following administration of leptin to their mothers[END_REF], such diets have long been recognized as major causes to the obesogenic environment in humans [START_REF] Astrup | The role of dietary fat in obesity[END_REF][START_REF] Malik | Intake of sugar-sweetened beverages and weight gain: a systematic review[END_REF]. The interactions of these diets with the physiological systems that regulate energy metabolism and body composition are thus an area of considerable importance. Major complications of obesity include type 2 diabetes, insulin resistance and increased risk of cardiovascular disease [START_REF] Keller | From obesity to diabetes[END_REF][START_REF] Trost | Impaired fibrinolysis and risk for cardiovascular disease in the metabolic syndrome and type 2 diabetes[END_REF]. The simultaneous occurrence of obesity, and particularly of visceral origin, dyslipidemia, insulin resistance and hypertension is classically referred to as the so-called "metabolic syndrome" [START_REF] Trost | Impaired fibrinolysis and risk for cardiovascular disease in the metabolic syndrome and type 2 diabetes[END_REF][START_REF] Robinson | Metabolic syndrome, a cardiovascular disease risk factor: role of adipocytokines and impact of diet and physical activity[END_REF][START_REF] Spinler | Challenges associated with metabolic syndrome[END_REF].

A number of different types of high-fat and/or high-energy diets have already been used to induce obesity and/or mimic the human metabolic syndrome in rodents [START_REF] Archer | Normal distribution of body weight gain in male Sprague-Dawley rats fed a high-energy diet[END_REF][START_REF] Axen | Very low-carbohydrate versus isocaloric highcarbohydrate diet in dietary obese rats[END_REF][START_REF] Bartol-Munier | Combined effects of high-fat feeding and circadian desynchronization[END_REF][START_REF] Buettner | Defining high-fat-diet rat models: metabolic and molecular effects of different fat types[END_REF][START_REF] Jang | Role of dietary fat type in the development of adiposity from dietary obesity-susceptible Sprague-Dawley rats[END_REF][START_REF] Woods | A controlled high-fat diet induces an obese syndrome in rats[END_REF]. Most studies have used only one high-fat formula compared with the standard chow diet. To take into account the relative amounts of macronutrients, we aimed at testing specifically the possible importance of the fat/carbohydrate ratio in a high-fat diet.

Therefore, the current study was undertaken to characterize the occurrence of various aspects of the metabolic syndrome (namely, prevalence of obesity, dyslipidemia, glucose tolerance and insulin resistance) in rats placed on a high-carbohydrate, low-energy diet (chow) compared to three high-fat, high-energy diets differing in their respective amounts of saturated fat and carbohydrate.

MATERIALS AND METHODS

Ethics

All experiments were performed in accordance with the rules of the European Committee Council Directive of November 24, 1986 (86/609/EEC) and the French Department of Agriculture (license n°67-88). Animals 48 male Sprague-Dawley rats (Charles River Laboratories, L'arbresle, France) weighing around 120 g upon their arrival, were housed individually, kept at 21 ± 1°C under a 12:12 h light-dark cycle (lights on at 07:00 AM) and had ad libitum access to food (standard low-fat diet, 105, SAFE, 89290 Augy, France) and tap water for 2 weeks.

Experimental design

Rats were then divided into 4 groups: the first group of 12 control rats was fed a standard high-carbohydrate, low-fat pelleted diet (HCD; 3.7 kcal/g; 229H, SAFE; Table 1). The second group of 12 rats received a high-fat, medium-carbohydrate pelleted diet (HFD1; 4.7 kcal/g, SAFE; Table 1). The third group of 12 rats were fed with a very high-fat, low-carbohydrate diet (HFD2; 5.2 kcal/g; 231H, SAFE; Table 1). The fourth group of 12 rats received a very high-fat, carbohydrate-free diet (HFD3; 5.9 kcal/g, SAFE; Table 1). Diets were vacuumpacked and stored in a fresh (8°C) room before use. Animals were given fresh food once a week. Body mass was measured every week. Due to considerable wasting (especially for HFD2 and 3), we were not able to quantify with precision daily food intake according to the diet. One ill animal from the HCD group had to be taken out of the experiment, leading to n=11 control animals.

After 8 weeks of experimental diet, rats were fasted overnight and tested for glucose tolerance 2 h after light onset. After 10 weeks of experimental diet, rats were fasted overnight and tested for insulin sensitivity 2 h after light onset. In the morning of the following day, all rats were deeply anesthesized with isoflurane and their body length and mass were determined before decapitation. Thereafter, blood was immediately collected and centrifugated. Fat tissues were dissected and weighed. The sum of the mass of epididymal fat pads and retroperitoneal fat expressed as percentage of body mass was used as an index of adiposity. To test oral glucose tolerance, blood glucose was assessed in overnight fasted rats just before, 30 min, 1 and 2 h following an oral administration of glucose (2 g/kg, Sigma, St Louis, MO, USA) or water. Blood samples were collected via tail veins and blood glucose was immediately determined (Glucotrend premium kit, Roche Diagnostics, Meylan, France). To optimize timing of blood sampling and avoid any time-of-day effect, 24 rats were sampled, half of them receiving an either glucose or water. For each condition, 3 rats per diet were tested.

Methods of analysis

To assess insulin-induced hypoglycaemia, overnight fasted rats received an s.c. injection of 1 IU/ kg human insulin (Umuline NPH, Lilly France, Suresnes, France) 2 h after light onset.

Blood glucose collected as above was assessed just before, 30 min, 1 and 2 h after insulin treatment. During the sampling period, rats had no access to food. Again, to optimize timing of blood sampling and avoid any time-of-day effect, 24 rats (n=6 per diet) were tested with insulin only, because no major effect of manipulation and/or water injection was detected in the glucose tolerance test. Plasma insulin was determined by an ELISA kit for rats (EZRMI-13K, Linco Research, Inc., St Charles, MO, USA) with intra-and inter-assay coefficients of variation (%CV) of 1.9 0.6 and 7.6 0.8 %, respectively. The limit of sensitivity of insulin assay was 0.2 ng/ml. Plasma leptin was determined by an ELISA kit for rats (EZRL-83K, Linco Research) with 2.2 0.2 intra-and 3.4 0.3 inter-assay %CV. The limit of sensitivity of leptin assay was 0.04 ng/ml. Plasma adiponectin was determined by an ELISA kit for rats (EZRADP-62K, Linco Research) with 1.3 0.2 intra-and 7.0 0.4 inter-assay %CV. The limit of sensitivity of adiponectin assay was 0.155 ng/ml. Homeostatic model assessment (HOMA) used to assess B-cell function and insulin resistance (IR) was calculated as follows:

HOMA-IR=(Fasting plasma glucose Fasting plasma insulin)/22.5 as described by Matthews et al. [START_REF] Matthews | Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man[END_REF].

Statistical analysis

To test the effects of diet (HCD, HFD1, HFD2 or HFD3) and time (weeks), data were processed with analyses of variance (ANOVA) with repeated measures or not, depending on the parameter considered. If significant main effects or significant interactions were detected (P<0.05), post-hoc comparisons were performed with Tukey HSD test. Values are means SEM. For assessment of correlations, data were fitted to the following equation (SigmaPlot software, Jandel Scientific, Chicago, USA): y = y0+a•x where a was the slope and y0 the intercept.

RESULTS

Body mass and adiposity

Both HFD1 and HFD2 led to increased body mass compared to HCD control animals (+11 2 and +13 4 %, respectively; P<0.05) 10 weeks after the start of the experiment. Note that such a difference was significant after 6 weeks of high-fat diets up to the end of the experiment. In contrast, HFD3 without carbohydrate did not significantly modify gain in body mass over the experiment compared to rats fed with HCD (+3 2 %; NS; Fig. 1A). This lack of body mass difference cannot be attributed to a slower growth in the HFD3 group (Fig. 1B). Accordingly, the body mass index was larger in rats fed with HFD1 and HFD2, but not with HFD3, compared to control animals fed with HCD (Fig. 1C).

Interestingly, adiposity index (epididymal and retroperitoneal white fat as percentage of body mass) was larger in animals with diets containing both high-fat and a certain amount of carbohydrate (i.e., HFD1 and HFD2) compared to rats fed with either low-fat, high carbohydrate diet (HCD) or high-fat, carbohydrate-free diet (HFD3; Fig. 2).

Glucose tolerance

Fasting blood glucose, determined in the morning (i.e., 2 h after the onset of light), did not differ significantly between the feeding conditions (P>0.05; Fig. 3A). Whatever the diet considered, handling and oral administration of water did not modify significantly basal blood glucose during the 2-h period of blood sampling (P>0.05; Fig. 3B). In control rats, an oral bolus of glucose led to a significant increase in blood glucose, which returned to basal levels 2 h later (Fig. 3A). Blood glucose was increased by almost two-fold in rats with either HFD2 or HFD3 (Fig. 3A), while only a trend for increase was visible for the HFD1 group (Fig. 3A). In addition, 2 h after glucose administration, in contrast to the control group, blood glucose in the three groups of animals fed with high-fat diet (HFD1-3) was still larger than the basal values (P<0.05; Fig. 3A), indicating a reduction in glucose tolerance.

Insulin resistance

In response to an acute administration of insulin after an overnight fast, control rats fed with HCD displayed the expected lowering in blood glucose that levelled back to normal values 2 h after the treatment (Fig. 3C). Albeit reduced, insulin-induced hypoglycaemia was not significantly impaired in rats fed with HFD2 compared to control rats, whatever the time point considered (P>0.05; Fig. 3C). Unlike these limited alterations in the HFD2 group, blood glucose in HFD1 and HFD3 rats did not change at all during the testing period, leading to higher glucose levels 30 min and 1 h after insulin treatment in comparison with those in control rats (P<0.05; Fig. 3C), suggesting a reduced whole-body sensitivity to insulin.

Hormonal changes

Morning levels of plasma insulin in overnight fasted rats tended to be higher in the three HFD1-3 groups compared to the control HCD, but these differences did not reach significance (Fig. 4A). In keeping with adiposity data (Fig. 2), plasma leptin in HFD1 and HFD2 rats was significantly higher than that in HCD rats (P<0.05), while intermediate (but not significant) values of plasma leptin were found in HFD3 rats (Fig. 4B). Finally, plasma adiponectin was significantly reduced in the three groups of animals fed with high-fat diet (HFD1-3) compared than the control group (P<0.05). Surprinsingly, the lowest level of plasma adiponectin was detected in rats fed with HFD3 (Fig. 4C).

Homeostatic model assessment and insulin resistance (HOMA-IR) and correlations with adipokins HOMA-IR tended to higher in HFD1 (8.6 1.8 a.u.), HFD2 (9.1 2.5 a.u.) and HFD3 groups (8.9 2.5 a.u.) compared to control group (HCD: 3.5 0.9 a.u.), but these differences did not reach significance (P>0.05). Plasma adiponectin correlated neither with fat mass (Fig. 5A), nor with HOMA-IR (Fig. 5B). By contrast, plasma leptin was significantly correlated with both fat mass (Fig. 5C) and HOMA-IR (Fig. 5D) Circulating lipids Concerning total cholesterol, there was an apparent increase in all rats fed with high-fat diet, but the differences with control values were only significant in rats with HFD2 and HFD3 Clinical Science Immediate Publication. Published on 4 Jul 2007 as manuscript CS20070182 (P<0.05; Fig. 6A). Furthermore, LDL-cholesterol was clearly altered in HFD3--fed rats, but not with the HFD1 diet (Fig. 6B). Although not significant, LDL-cholesterol level tended to be increased in HFD2-fed rats. Plasma triglycerides were not markedly modified by the diet, except a higher level in rats fed with HFD1 compared to that in the HFD3 group, Fig. 6C). Moreover, free fatty acids were increased by high-fat diet in HFD2 rats (P<0.05), but this higher level did not reach the threshold of statistical significance in HFD1 and HFD3 animals (Fig. 6D).

Clinical Science Immediate Publication. Published on 4 Jul 2007 as manuscript CS20070182

DISCUSSION

The present study demonstrates that depending on respective amounts of saturated fat and carbohydrate, high-fat/high-energy diets lead to differential metabolic abnormalities. The two high-fat diets combined with moderate-to low-carbohydrate content (HFD1 and HFD2) were both obesogenic (i.e., causing high adiposity and plasma leptin as well as low plasma adiponectin) and diabetogenic (i.e., inducing insulin resistance), while the high-fat, carbohydrate-free diet did not produce abdominal obesity but led to insulin resistance and, by increasing LDL-C, also led to higher risk of cardiovascular disease.

High-fat diets have been widely used in rodents to provide experimental models of the human metabolic syndrome that simultaneously associate obesity, dyslipidemia, diabetes-related alterations, and eventually atherosclerotic cardiovascular disease. The present study confirms the usefulness of diets enriched in saturated fat to produce metabolic abnormalities mimicking the metabolic syndrome. A large number of experiments studying the metabolic consequences of high-fat diets have been already performed in various strains of rats such as Sprague-Dawley (e.g., [START_REF] Archer | Normal distribution of body weight gain in male Sprague-Dawley rats fed a high-energy diet[END_REF][START_REF] Axen | Very low-carbohydrate versus isocaloric highcarbohydrate diet in dietary obese rats[END_REF][START_REF] Jang | Role of dietary fat type in the development of adiposity from dietary obesity-susceptible Sprague-Dawley rats[END_REF]], this study), Wistar (e.g., [START_REF] Buettner | Defining high-fat-diet rat models: metabolic and molecular effects of different fat types[END_REF]) or Long-Evans (e.g., [START_REF] Bartol-Munier | Combined effects of high-fat feeding and circadian desynchronization[END_REF][START_REF] Woods | A controlled high-fat diet induces an obese syndrome in rats[END_REF]). The increase in body mass we obtained with both HFD1 (i.e., +11%) and HFD2 (i.e., +13%) diets is roughly consistent with previous experiments considering all the experimental animals (i.e., with no post-hoc selection according to body mass gain) and having the same duration as ours (e.g., +10% after 10 wks, [START_REF] Woods | A controlled high-fat diet induces an obese syndrome in rats[END_REF]; +10 % after 12 wks, [START_REF] Buettner | Defining high-fat-diet rat models: metabolic and molecular effects of different fat types[END_REF]). The accretion of body fat in HFD1 and HFD2 rats was even clearer when considering BMI and adiposity values.

Moreover, we aimed at investigating the possible importance of the saturated fat/carbohydrate ratio in a high-fat diet. This is why we compared the effects of three diets enriched in saturated fat differing mainly by their fat/carbohydrate ratio, while their protein content remained largely unchanged (range 17-25% in metabolizable energy). The most pronounced manifestations of abdominal obesity and insulin intolerance were observed after feeding with HFD1, that is, a diet containing high-fat, medium-carbohydrate proportions (in energy). Rats with this diet gained more body mass, had larger body mass index, were fattier with high leptinemia and low adiponectinemia, and displayed physiological signs not only of impaired glucose tolerance, but also of whole-body insulin resistance, compared to animals fed a standard, high-carbohydrate diet (HCD). HFD2, the second diet which the metabolic consequences were evaluated, contained a very high-fat (>50%), low-carbohydrate diet.

HFD2 led roughly to metabolic abnormalities comparable with HFD1, except that the glycemic responses to insulin treatment were less altered, being close to values in the control Clinical Science Immediate Publication. Published on 4 Jul 2007 as manuscript CS20070182 group (HCD). Dyslipidemia has been difficult to obtain in rats, even after modifications of the fat/carbohydrate ratio. Here only HFD2 and HFD3 rats showed altered profiles of circulating lipids. The increase in plasma free fatty acids was only significant with this HFD2 diet.

Nevertheless, except for dyslipidemia, the present results suggest that HFD1 is a better experimental model of the human metabolic syndrome than the two other high-fat diets tested here.

Diets with different ratios of fat/carbohydrate ratios have been already studied (e.g., in mice, [START_REF] Takahashi | Effect of the fat/carbohydrate ratio in the diet on obesity and oral glucose tolerance in C57BL/6J mice[END_REF]; in rats, [START_REF] Boozer | Dietary fat and adiposity: a dose-response relationship in adult male rats fed isocalorically[END_REF][START_REF] Lee | Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites[END_REF][START_REF] Morens | Low-carbohydrate diets affect energy balance and fuel homeostasis differentially in lean and obese rats[END_REF][START_REF] Pichon | A high-protein, high-fat, carbohydrate-free diet reduces energy intake, hepatic lipogenesis, and adiposity in rats[END_REF]). The overall conclusion of these studies is as follows: the more the caloric content derives from dietary saturated fat, the fattier and the less glucose-tolerant become the animals. To our knowledge, however, the previous studies did not investigate directly a carbohydrate-free, high-fat diet compared to high-fat diet with variable amounts of carbohydrate, while keeping protein content comparable between the diets to avoid any deficit in body growth.

A question that arises from the present study in which diets were not isoenergetic is whether the increase in body fat is due to the larger caloric content of the high-fat diets or to the larger percentage of energy as fat. Overall, the degree of obesity was not correlated with the caloric diet because the highest body mass index and adiposity were found in rats fed with the lowest energy content (i.e., HFD1 with 4.7 kcal/g) among the three high-fat diets studied.

Conversely, the highest caloric diet (i.e., HFD3 with 5.9 kcal/g) did not lead to a significant increase of body mass index or adiposity in comparison with control animals. Therefore, these opposite responses clearly indicate that the different caloric contents in the diets proposed here are not the main factor causing fat accretion. Alternatively, even if we could not measure caloric intake in the present experiment, changes in palatability and/or texture may have altered food intake to some extent.

In addition to the fat content in the diet, its composition in fatty acids has been shown to trigger differentially obesogenic factors. In particular, a higher proportion of saturated fatty acids is closely associated with resulting increased adiposity and insulin resistance [START_REF] Storlien | Diet composition and insulin action in animal models[END_REF]. By contrast, dietary unsaturated fats, particularly n-3 fatty acids from fish, have been demonstrated to limit fat accretion and hyperleptinemia [START_REF] Storlien | Diet composition and insulin action in animal models[END_REF][START_REF] Wang | Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression[END_REF] and to increase the thermogenic activity of brown adipose tissue [START_REF] Oudart | Brown fat thermogenesis in rats fed high-fat diets enriched with n-3 polyunsaturated fatty acids[END_REF]. In addition, unsaturated fatty acids, again especially n-3 [START_REF] Storlien | Fish oil prevents insulin resistance induced by high-fat feeding in rats[END_REF][START_REF] Samaha | Effect of very high-fat diets on body weight, lipoproteins, and glycemic status in the obese[END_REF] but also n-6 polyunsaturated fatty acids provided in a semisynthetic diet [START_REF] Lee | Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites[END_REF], have beneficial consequences on plasma cholesterol and insulin resistance. Therefore, a potential bias in the effects of the HFD3 diet is that it contains a larger proportion of corn oil (21%) than the other (including control) groups (6-12%). Because the rats fed with the HFD3 regimen were insulin-resistant, displayed impaired glucose tolerance, had the highest plasma total cholesterol and cholesterol with LDL, these effects are actually opposite to those expected from known benefits of (poly-)unsaturated fat consumption. First, it should be reminded that that the larger proportion of mono-and poly-unsaturated fats in the HFD3 comes from vegetal (i.e., corn), and not fish source. Second, corn oil contains mainly n-6 fatty acids (linoleic acid) and this oil is not recognized as a main source for n-3 fatty acids. Taken together, the most parcimonious hypothesis to explain the lack of obesogenic effects and the clear diabetogenic properties of HFD3 is that these differential changes are due to the largest proportion of energy derived from fat (both saturated and unsaturated) and/or to the absence of carbohydrate in this diet. Because the adiposity in HFD3-fed rats did not match that expected from the two other high-fat regimens with variable content in carbohydrate (HFD1 and HFD2), we rather favour the second hypothesis (i.e., carbohydrate-free diet) to be the main cause of the observed metabolic changes in HFD3-fed rats. In accordance with this interpretation, a recent study showed that a carbohydrate-free, high-fat diet also leads to a reduction in the body mass gain and development of body fat [START_REF] Pichon | A high-protein, high-fat, carbohydrate-free diet reduces energy intake, hepatic lipogenesis, and adiposity in rats[END_REF], even if in this study the lipid/protein ratio was close to 0.8 compared to 3 in the present work. Rather than a lack of dietary carbohydrate-induced thermogenesis, enzymatic activities measured by Pichon and colleagues in the white adipose tissue and the liver suggest that such a reduced gain of body fat in carbohydrate-free diets can be attributed to reduced hepatic lipogenesis [START_REF] Pichon | A high-protein, high-fat, carbohydrate-free diet reduces energy intake, hepatic lipogenesis, and adiposity in rats[END_REF].

When rats were challenged with exogenous insulin, there was a whole-body insulin resistance, indicative of compromised -cell function, in both HFD1-and HFD3-fed rats, while only a trend for alteration was noted in HFD2-fed rats. Probably as a consequence of insulin resistance, all the high-fat diets tended to produce a hyperinsulinemia that might have been significant if the experiment was continued further.

The increase in body fat in HFD1-and HFD2-fed rats was associated with higher levels of plasma leptin, a hormone secreted by the adipocytes and thought to signal metabolic status from the adipocytes to peripheral tissues and the brain. Accordingly, in the HFD3 group that did not display increased adiposity, leptinemia was not significantly different from animals fed with the control diet. As expected, there was a strong correlation between plasma leptin and abdominal fat mass irrespective of the diet.

Adiponectin is another adipokine secreted by the adipocytes. Its role and regulation differ largely from leptin, because the development of the metabolic syndrome and diabetes is associated with a downregulation of adiponectinemia. Furthermore, adiponectin is now known to have potent insulin-sensitizing effects [START_REF] Kadowaki | Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome[END_REF]. It is noteworthy that low levels of plasma adiponectin was observed not only in HFD1 and HFD2 animals (i.e., those with increased body fat), but also in HFD3 rats (i.e., those with an adiposity close to normal values). This observation raised doubts of whether low plasma adiponectin can be used as a reliable marker of the metabolic syndrome [START_REF] Santaniemi | Low plasma adiponectin concentration is an indicator of the metabolic syndrome[END_REF]. In humans it has been proposed hypoadiponectinemia is more closely related to insulin resistance than adiposity per se [START_REF] Abbasi | Discrimination between obesity and insulin resistance in the relationship with adiponectin[END_REF][START_REF] Weyer | Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia[END_REF]. The relationship was not clearly confirmed by our data in rats.

In conclusion, we propose that the carbohydrate/fat ratio of the diet may be important in the etiology of the metabolic syndrome linked to high fat diets. Still, we do not mean that certain high-carbohydrate, low-fat diets may not also predispose and/or generate a metabolic syndrome, but the present study was specifically focused on the carbohydrate/fat ratio during high-fat diet. The two diets combining high-fat and significant amount of carbohydrate led to a pre-diabetic state associated with an increase in both adiposity and leptinemia. Of interest, the high-fat, carbohydrate-free diet had only diabetogenic properties, but no obvious obesogenic features. Further experiments will be needed to understand the mechanisms underlying such a limited fat accretion (reduced hepatic lipogenesis ?).

Even if it is clearly premature to consider the present data in a clinical perspective, defining appropriate diets on a long-term scale for preventing and/or treating the metabolic syndrome is still a crucial and complex issue. New dietary strategies using low-carbohydrate diets are emerging [START_REF] Gardner | Comparison of the Atkins, Zone, Ornish, and LEARN diets for change in weight and related risk factors among overweight premenopausal women[END_REF][START_REF] Dansinger | Low-carbohydrate or low-fat diets for the metabolic syndrome ?[END_REF].

  Commercial enzymatic assays were used to determine plasma free fatty acids with RxL Dade Boehring analyzer (Diamond Diagnostics, Holliston, MA, USA) and plasma triglycerides, total cholesterol and high high-density lipoprotein-cholesterol (HDL-C) using Advia 1650 analyzer (Bayer Diagnostics, Tarrytown, NY). Low-density lipoprotein-cholesterol (LDL-C) was calculated as follows: LDL-C = Total Cholesterol -(HDL-C) -(Triglycerides/2.2).

Figure 1 :

 1 FIGURES LEGENDS

Figure 2 :

 2 Figure 2: Adiposity, as determined by the ratio of abdominal (i.e., epididymal and retroperitoneal) white fat, in rats fed either with low-fat, high carbohydrate diet (HCD, white bar), high-fat, medium-carbohydrate diet (HFD1, light grey bar), very high-fat, lowcarbohydrate diet (HFD2, dark grey bar) or very high-fat, carbohydrate-free diet (HFD3, black bar). * P<0.05 compared to the control HCD group.

Figure 3A :

 3A Figure3A: Time-course of glucose tolerance following an oral administration of glucose (2 g/kg) in rats fed either with low-fat, high carbohydrate diet (HCD, white circles), high-fat, medium-carbohydrate diet (HFD1, light grey squares), very high-fat, low-carbohydrate diet (HFD2, dark grey triangles) or very high-fat, carbohydrate-free diet (HFD3, black circles). a, P<0.05 for all groups compared to T0 min; b, P<0.05 for all HF groups compared to T0; n.s., non-significant (HCD group at T120 min compared to T0 min).3B: Time-course of changes in plasma glucose after an oral administration of water in rats fed either with low-fat, high carbohydrate diet (HCD, white circles), high-fat, mediumcarbohydrate diet (HFD1, light grey squares), very high-fat, low-carbohydrate diet (HFD2, dark grey triangles) or very high-fat, carbohydrate-free diet (HFD3, black circles). No significant effect was detected.3C: Time-course of changes in plasma glucose after insulin (1 IU/kg) injection in rats fed either with low-fat, high carbohydrate diet (HCD, white circles), high-fat, mediumcarbohydrate diet (HFD1, light grey squares), very high-fat, low-carbohydrate diet (HFD2, dark grey triangles) or very high-fat, carbohydrate-free diet (HFD3, black circles). * P<0.05 in HFD1 and HFD3 groups compared to the control HCD group at the same time-point.

Figure 4 :

 4 Figure 4: Plasma insulin (A), leptin (B) and adiponectin (C) in rats fed either with low-fat, high carbohydrate diet (HCD, white bar), high-fat, medium-carbohydrate diet (HFD1, light

Figure 5 :

 5 Figure 5: Plasma adiponectin expressed according to fat mass (panel A) and homeostatic model assessment and insulin resistance (HOMA-IR; panel B). Plasma leptin plotted to fat mass (panel C) and HOMA-IR (panel D). Only plasma leptin was positively correlated with fta mass and HOMA-IR. Rats were fed either with low-fat, high carbohydrate diet (HCD, white circles), high-fat, medium-carbohydrate diet (HFD1, light grey squares), very high-fat, low-carbohydrate diet (HFD2, dark grey triangles) or very high-fat, carbohydrate-free diet (HFD3, black circles). a.u., arbitrary unit.

Figure 6 :

 6 Figure 6: Plasma total cholesterol (A), Low-density lipoprotein (LDL)-cholesterol (B), triglycerides (C) and free fatty acids (D) in rats fed either with low-fat, high carbohydrate diet (HCD, white bar), high-fat, medium-carbohydrate diet (HFD1, light grey bar), very high-fat, low-carbohydrate diet (HFD2, dark grey bar) or very high-fat, carbohydrate-free diet (HFD3, black bar). * P<0.05 compared to the control HCD group. # P<0.05 between HFD1 and HFD3.

  Fig.1

Fig. 3

 3 Fig.3

Fig. 6 Clinical

 6 Fig.4

Clinical Science Immediate Publication. Published on 4 Jul 2007 as manuscript CS20070182

Clinical Science Immediate Publication. Published on 4 Jul 2007 as manuscript CS20070182

TABLE LEGENDS Table 1 .

 LEGENDS1 Diets composition

	HCD: low-fat, high carbohydrate diet
	HFD1: high-fat, medium-carbohydrate diet
	HFD2: very high-fat, low-carbohydrate diet
	HFD3: very high-fat, carbohydrate-free diet
	All diets contain cellulose (5-10% in mass), minerals (3-11% in mass) and vitamins (1 % in
	mass). CHO, carbohydrate.

Clinical

Science Immediate Publication. Published on 4 Jul 2007 as manuscript CS20070182 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 1 .

 1 Composition of the diets

		HCD	HFD1	HFD2	HFD3
	Energy content (kcal/g)	3.7	4.7	5.2	5.9
	Protein (%)	24	17	24	25
	CHO (%)	64	30	9	0
	Fat (%)	12	53	67	75
		(corn oil 12)	(corn oil 6)	(corn oil 12)	(corn oil 21)
			(lard 47)	(lard 55)	(lard 54)

Clinical Science Immediate Publication. Published on 4 Jul 2007 as manuscript CS20070182 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society


MD° Salvatore Paterna

Gaspare Parrinello

Sergio Fasullo Md°°

Filippo M Md°°°

MD°°°°° Pietro Sarullo

Pasquale Md°°° Di

MD Pietro Di Pasquale

NORMAL SODIUM DIET VERSUS LOW SODIUM DIET IN COMPENSATED CONGESTIVE HEART FAILURE: IS SODIUM AN OLD ENEMY OR A NEW FRIEND ? Running Title: Normal sodium diet vs low sodium diet in CHF

Keywords: Furosemide, normosodic diet, hyposodic diet, congestive heart failure

The study evaluated the effects of a normal (120 mmol Na) versus a low sodium diet (80 mmol Na) on readmissions for HF during six months of follow-up in compensated congestive heart failure (CHF) patients. 232 (88 F/144 M) compensated CHF pts (from IV to II Class NYHA) aged 55 to 83 years, with EF <35% and serum creatinine <2 mg/dl were randomized in 2 groups: group 1: 118 pts (45 F/73 M) received a normal sodium diet plus oral furosemide (250-500 mg bid); Group 2: 114 pts (43 F/71 M): received a low sodium diet plus oral furosemide (250-500 mg bid),and the treatment received after discharge (30 days) and for 180days after, associated with a drink intake of 1000 ml daily. Signs of CHF, BW,BP,HR, laboratory parameters, ECG, echocardiogram, BNP;Aldosterone values and plasma renin activity (PRA) were examined at baseline, and 180 days after. Normal sodium group showed a significant reduction (p<0.05) in readmissions. BNP values were lower in normal sodium group (685+255 pg/ml vs 425+125 pg/ml; p<0.0001) than low sodium group. The aldosterone and PRA showed significant increase (p<0.0001) in low sodium group during follow up, while normal sodium showed a little significant reduction (p=0.039) in aldosterone levels and not significant difference in PRA levels. After 6 months of follow up, aldosterone and PRA were (p<0.0001) significantly higher in low sodium group. Normoal sodium group showed a lower incidence of rehospitalization during the follow up and a significant reduction in BNP, aldosterone plasma levels and PRA.

Our date show that a normal sodium diet improves outcome and sodium depletion has detrimental renal and neurohormonal effects with worse clinical outcome in compensated HF patients. Further studies are required to determine if this is due to high dose diuretic or low-sodium diet.

INTRODUCTION

The management of patients with advanced CHF classically consists in sodium restriction, moderate physical activity and a tailored treatment that include digitalis, diuretics and ACE-inhibitors [START_REF] Cowie | The epidemiology of heart failure[END_REF]. Diuretics, particularly loop diuretics, have long been accepted as first-line treatment of patients with severe CHF and important fluid retention [START_REF]American Heart Association Task Force on Practice Guidelines: Guidelines for the evaluation and management of heart fallure[END_REF][START_REF] Smith | Management of heart failure[END_REF]. However, a lack of response to diuretic therapy is a common event particularly in elderly patients with advanced disease. Previously we reported the safety and tolerability of the combination of high dose furosemide associated with small-volume hypertonic saline solution (HSS) i.v. infusion [START_REF] Paterna | Tolerability and efficacy of high dose furosemide and small-volume hypertonic saline solution in refractory congestive heart failure[END_REF][START_REF] Arnold | Canadian cardiovascular society consensus conference:recommendations on heart failure update 2007:Prevention,management during intercurrent illness or acute decompensation,and use of biomarkers[END_REF][START_REF] Paterna | Small-Volume hypertonic saline solution and high-dosage furo-semide in the treatment of refractory congestive heart failure.A pilot study[END_REF], in patients with refractory CHF, and this combination in comparison with an i.v. high dose infusion of furosemide as bolus, determined a significant reduction of hospitalization time. In addition, we observed that patients receiving a normal sodium diet maintained the achieved NYHA functional class at discharge, a reduction of hospital readmissions for worsening CHF [START_REF] Paterna | Effects of high-dose furosemide and small-volume hypertonic saline solution infusion in comparison with a high dose of furosemide as a bolus,in refractory congestive heart failure[END_REF][START_REF] Paterna | High-dose torasemide is equivalent to high-dose furosemide with hypertonic saline in the treatment of refractory congestive heart failure[END_REF], and a significant mortality rate reduction (55% vs 13% survival after 48 months) [START_REF] Licata | Effects of high-dose furosemide and small-volume hypertonic saline solution in comparison with a high dose of furosemide as bolus in refractory congestive heart failure:long term effects[END_REF][START_REF] Drazner | Hypertonic saline: A novel therapy for advanced heart failure?[END_REF]. Successively, systematic data on BNP plasma levels changes and bioelectrical variables occurring in HF patients before and after treatment showed that patients receiving HSS reached their dry weight more rapidly than those not receiving HSS solution [START_REF] Paterna | Changes in BNP Levels and bioelectrical impedance measurements after treatment with High dose furosemide and hypertonic saline solution versus high dose of furosemide alone, in refractory congestive heart failure. A double blind study[END_REF][START_REF] Stevenson | Torrent or Torment From the Tubules? Challenge of the Cardiorenal Connections[END_REF]. The study was evaluating the effects of a normal sodium diet (120 mmol Na) versus low sodiu diet (80 mmol), with highdose furosemide and severe fluid restriction (1.000 ml/day), in patients compensated after recently decompensated CHF, on readmissions for worsening HF (primary endpoint). In addition, were also evaluated the combination of readmissions and mortality (secondary endpoint), and BNP, ALD plasma levels and plasma renin activity (PRA) during six months of follow up.

MATERIALS AND METHODS

Patient population: From January 2000 to May 2005, 1244 patients were consecutively admitted to hospital with worsening HF.

Eligibility criteria

We included into the study only compensated patients who previously were hospitalized (previous 30 days) for recently decompensated CHF with the following characteristics: patients had to have, according to the definition of refractory CHF [START_REF] Cody | Management of Refractory Congestive Heart Failure[END_REF] and according to Framingham criteria and New York Association Functional classification for congestive heart failure [START_REF] Rodeheffer | Congestive Heart Failure: Diagnosis, Evaluation, and Surgical Therapy[END_REF], uncompensated CHF (dyspnoea, weakness, lower limbs oedema or anasarca), NYHA IV functional class that was unresponsive to treatment with oral high doses of furosemide up to 250-500 mg/day and/or combinations of diuretics (thiazide, loop diuretic and spironolactone), ACE-inhibitors (captopril 75-150 mg/day), digitalis, beta-blockers and nitrates and to be under this therapy at least 2 weeks before hospitalization. The patients were judged unresponsive when they showed, during the treatment as above reported, a reduction of urine volume and constant increase of body weight (BW) and impairment of clinical signs of CHF as above reported, in spite the increase of furosemide and the combination of other diuretics (including thiazide). Additionally, they had to have a left ventricular ejection fraction (EF) <35%, serum creatinine <2 mg/dl, BUN <60 mg/dl, a reduced urinary volume (<500 ml/24h) and a low natriuresis (<60 mEq/24h) despite receiving the established treatments. reduced drink fluid intake (1000 ml/day) during hospitalization..When compensated state was achieved (II NYHA Class) patients received oral furosemide (250-500 mg/bid), normal sodium diet (120 mmol Na), and a drink fluid intake of 1000 ml daily according to our previous reports. [START_REF] Paterna | Tolerability and efficacy of high dose furosemide and small-volume hypertonic saline solution in refractory congestive heart failure[END_REF][START_REF] Arnold | Canadian cardiovascular society consensus conference:recommendations on heart failure update 2007:Prevention,management during intercurrent illness or acute decompensation,and use of biomarkers[END_REF][START_REF] Paterna | Small-Volume hypertonic saline solution and high-dosage furo-semide in the treatment of refractory congestive heart failure.A pilot study[END_REF][START_REF] Paterna | Effects of high-dose furosemide and small-volume hypertonic saline solution infusion in comparison with a high dose of furosemide as a bolus,in refractory congestive heart failure[END_REF][START_REF] Paterna | High-dose torasemide is equivalent to high-dose furosemide with hypertonic saline in the treatment of refractory congestive heart failure[END_REF][START_REF] Licata | Effects of high-dose furosemide and small-volume hypertonic saline solution in comparison with a high dose of furosemide as bolus in refractory congestive heart failure:long term effects[END_REF][START_REF] Drazner | Hypertonic saline: A novel therapy for advanced heart failure?[END_REF][START_REF] Paterna | Changes in BNP Levels and bioelectrical impedance measurements after treatment with High dose furosemide and hypertonic saline solution versus high dose of furosemide alone, in refractory congestive heart failure. A double blind study[END_REF] and the treatment was continued after discharge. Patients were considered clinically compensated when they reached a change in NYHA functional class to at least II and the accomplishment of ideal BW, calculated by the Lorenz formula. and bioelectrical impedance measurements [START_REF] Paterna | Changes in BNP Levels and bioelectrical impedance measurements after treatment with High dose furosemide and hypertonic saline solution versus high dose of furosemide alone, in refractory congestive heart failure. A double blind study[END_REF] Diuretic doses ranged from 250 to 500 mg/bid because these doses allowed maintenance of body weight and water balance. The diuretic doses were titrated according to the doses used during hospitalization. In addition, after discharge patients received tailored therapy (ACEinhibitors, digitalis, antialdosterone, beta-blockers and nitrates) too. Only patients in II NYHA class 30 days after discharge were included into the study and randomized.

Exclusion criteria: Patients with concomitant other important co-morbidity, cerebral vascular disease, dementia, cancer, uncompensated diabetes, inability to give informed consent, severe and hepatic disease, and inability to follow the assigned treatment. Patients requiring pace-maker implantation and alcoholic habit, who did not follow the protocol of treatment, programmed clinical visits, drink fluid intake of 1.000 ml day, and reduction or discontinuation of prescribed treatments. In addition, patients with side effects for ACEinhibitors (cough), even if these patients were given angiotensin-2 blockers, to obtain as more homogeneous as possible treatments were excluded too.

Protocol of the study and follow up (figure 1):

Pre-randomization evaluation: After discharge (first 30 days) the patients were controlled with clinical and laboratory evaluations, as outpatients every week for the first 30 days and those who just 30 days after the discharge meet the eligibility criteria were included into the study. During 30-days period after discharge, the treatments (where necessary) were corrected according to clinical status and laboratory measurements (reduction and/or increase of diuretics, ACE-inhibitors, digitalis, antialdosterone, beta-blockers and nitrates), in this way, during the follow up it was not necessary to correct the assigned treatments. In fact, all the enrolled patients were stable and receiving the best treatments which were able to maintain the compensated state obtained after discharge and before randomization During the follow-up period, other treatments were not added to those administered. Corrections of therapy were carried out only in readmitted patients Randomization:. Only patients judged compensated in II NYHA Class, by an independent external team (at least two) of blinded physicians about study protocol, were included into the study and randomized in two groups: a group received 120 mmol Na and continued oral furosemide (250-500 mg bid) and a group received 80 mmol Na and continued oral furosemide (250-500 mg bid), associated with the above reported therapy.

Both groups received a drink fluid intake of 1000 ml daily. Randomization was carried out using a preliminary computer algorithm and the assignment of all patients was decided at the baseline (just 30 days after discharge) before performing clinical and laboratory measurements Written informed consent was obtained from each patient before starting the study The protocol was aapproved by local ethical committee.

Baseline characteristics of the study subjects are shown in tables 1 and 2.

Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193

After randomization evaluation: After randomization (just after the first 30 days from discharge) and after the baseline clinical and laboratory assessments, the patients were evaluated every week for the first 1 month, every two weeks for the subsequent two months and every month for the subsequent periods. A complete physical examination, with a careful check of CHF signs and symptoms, including measurement of BW (in the morning before breakfast), supine and standing arterial blood pressure (BP) (mean of 3 measurements) and heart rate (HR) was performed at every follow up assessment. At baseline (before receiving treatments) fasting blood samples were drawn to determine serum Na, K, Cl, bicarbonate, albumin, uric acid, creatinine, urea and glycaemia. Aldosterone, plasma renin activity (PRA) and BNP plasma levels were also controlled.

Chest-x ray, ECG and echocardiogram (to obtain EF according to the modified Simpson' rule, which uses 2 cross section views (4 and 2 chamber apical views) were also evaluated. All these parameters, and BNP plasma levels, were also obtained at at 90 and 180 days of follow up, while plasma aldosterone and PRA were measured by radioimmunoassay in core laboratories at baseline and after 6 months of follow-up in both groups. In addition, all the patients with diabetes were receiving insulin therapy, oral anti-diabetes drugs were discouraged also after discharge and follow up. All patients after randomization received multiple written standard diets with a Na aumount of 80 mmol and prepared preliminarily by our dietitians. The 120 mmol group received the same diets with the addion of 40 Na mmol/day. In this way,all patients received the same aumount in satured fat,fruit,vegetable and etc, In addition, patients were also contacted every week during follow up by physicians and dietitians for a telephone interview to control the adherence to reduced fluid intake as well as the prescribed diet. Other informations were also obtained from a systematic contact with family doctors, who were informed about the study end points. Any of the patients experiencing worsening HF according to family doctors were invited to attend hospital for evaluation. Two blinded physicians performed the evaluations to verify the clinical status and if worsening HF were present.

Statistical analysis

We performed a multiple regression sample size calculating basing on a β value of 0.10 (90% power) and α value of 0.05. Therefore the sample size obtained was 67 for group and this number was assumed as minimum one for this study patients. We estimate a 20% of reduction in readmissions for worsening HF, on the basis of the data from our previous reports [START_REF] Licata | Effects of high-dose furosemide and small-volume hypertonic saline solution in comparison with a high dose of furosemide as bolus in refractory congestive heart failure:long term effects[END_REF]. Analysis was by intention to treat and according to absolute risk reduction; we calculated the number needed to treat (NNT) to prevent one event. We calculated event distributions with the Kaplan-Meier method and compared them by log-rank analysis. Data were analysed using the two-tailed t-test to identify differences between groups and analysis of variance (ANOVA) for repeated measures with Bonferroni post hoc test correction for intra-group data. Nominal data was analysed by the Chi-square test and p < 0.05 was assumed as statistically significant. All calculations were done with SPSS statistical software and the results are expressed as mean ± SD.

RESULTS

Of the 1244 patients admitted in hospital for worsening HF, 76 did not show reduced urinary volume (<500 ml/24h) and a low natriuresis (<60 mEq/24h), 92 were with severe co-morbidity (see exclusion criteria), 25 with side effects for ACE-inhibitors, 58 were requiring pace-makers, 18 were with alcoholic habit, 136 showed creatinine > 2.0 and/or BUN > 60 mg, (on hospital admission) and 451 were classified as NYHA Class III on hospital admission. In addition, 79 patients refused consent to participate to study either after discharge and 30 days after discharge, 54 patients during the 30 days period after discharge did not follow the prescribed limited drink fluid intake (1000 ml), and were also excluded from the study, but they accepted to be controlled as those included into the study. Twenty three patients classified as NYHA class III after hospital discharge were also excluded from the study. We obtained 232 (female/male: 88/144) compensated HF patients (NYHA class II) of different etiologies (110 coronary artery disease (CAD), 76 hypertensive heart disease (HHD), 46 dilatative cardiomyopathy (DCM), aged 53 to 85 years, who met the entry criteria and agreed to be enrolled and were randomized in two groups: Group 1 (118 pts) receiving normal sodium diet (120 mmol Na) and Group 2 (114 pts) receiving low sodium diet (80 mmol Na). (Table 1). The 120 mmol Na group maintained during the follow up daily diuresis as at baseline, whereas 80 mmol Na group showed a significant reduction in daily diuresis compared with baseline and with 120 mmol Na group. The same result was observed for natriuresis as well as serum Na. Serum K decreased significantly but the value remained in the normal range in both groups (Table 2). In addition, the renal function parameters controlled after 3 and 6 months in 120 mmol groups did not show significant differences from baseline values in contrast with increased creatinine and BUN values in 80 mmol group (Table 2). The 120 mmol group showed no significant variation in BW weight. In both groups systolic and diastolic values of BP were decreased without important clinical manifestations (Table 2). A significant ( p< 0.001 ) intergroup difference was observed in BNP plasma levels at the end of the study. These differences were significantly favorable in normal sodium group but with lower values at 90 (525+215 pg/ml vs 415+115 pg/ml; p<0.001) and 180 days (685+255 pg/ml vs 425+125 pg/ml; p< 0.0001) in comparison with low sodium group (Figure 2). In the 80 mmol group, aldosterone (normal value 7-150 pg/ml) (baseline, 188 ± 171 pg/mL) increased at 6 months (296+224 pg/ml, p<0.0001), while in the 120 mmol group, showed a little but significant (p=0.002) reduction (baseline, 192 ± 160 pg/ml versus 152+102 pg/ml) at 6 months. The PRA (normal value 0.2-0.8 ng/ml/h ) showed not significant difference ( p = 0.6) between baseline ( 3.91 + 3.71) and at 6 months ( 3.65 + 3.28) of follow up in 120 mmol group, while showed a significant (p=0.039) increase (3.98+3.68 vs 5.74 + 4.12) in 80 mmol group. The baseline values were similar in both groups, while after 6 months of follow up the values in ALD and PRA levels were significantly higher (p<0.0001) in 80 mmol than 120 mmol group (Figure 3).

Follow up:

The 120 mmol group showed a significant reduction (p<0.05) in readmission rate (primary endpoint) in comparison with 80 mmol group (figure 4,table 3), and a significant reduction (p<0.001) in combined mortality and readmissions (secondary endpoint) (figure 5, table 3). During the 180 days follow-up period 9 patients from 120 mmol group were readmitted for worsening HF (III-IV NYHA Class) and six patients died (1 sudden death and 5 irreversible HF). The 80 mmol group showed 30 patients readmitted to the hospital for clinical signs of HF and they presented at entry higher functional class than at discharge (III/IV NYHA Class), Fifteen patients died during the 180 days study period in 80 mmol group, (4 sudden death and 9 irreversible HF and 2 other causes, (neoplastic disease and stroke)) (Table 3). Figure 4 early (first 30 days) beneficial effects of a normal sodium intake that was more consistent during the subsequent months. In 80 mmol group 26.32 % of patients had an adverse outcome in comparison with only 7.63% in 120 mmol group. The Absolute Risk Reduction was 18.69%.(CI 9.29-28.08) and numbers of patients needed to treat to reduce a readmission ranges from 3.6 to 10.8 (NNT=6). No patients were receiving beta-blockers at time of the admission in hospital. Beta-blockers were also started when patients were compensated during hospitalization and after discharge, but only 18 patients (from both groups) tolerated beta-blockers (carvedilol), while the other patients showed signs of HF worsening and beta blockers were discontinued All patients continued ACE-inhibitors as previously reported (captopril 75-150 mg/day). No patient of both groups during follow up was excluded from the study for renal deterioration (creatinine > 2.0 mg) but the readmitted patients.

Echocardiographic data during follow up showed a significant slight improvement of EF in 120 mmol patients.

All the enrolled patients showed a good compliance with the prescribed treatments, only 21 patients (12 from 120 mmol group and 9 from 80 mmol group) during the first month (during summer) increased drink fluid intake (1.500 ml) and this violation of the protocol was corrected at subsequent control (7 days after). In addition, tables 4-5-6 show the clinical characteristics, laboratory parameters and results of CHF patients who refused consent but continued the prescribed treatments, and who did not follow the prescribed fluid intake (1000 ml day) after discharge, but they continued oral furosemide (250-500 mg bid) and normal sodium intake (120 mmol). These patients were not analyzed because they were not randomized and because the study was not aimed to study the effects of free fluid intake, but they were also controlled subsequently. The patients assuming a drink fluid intake > 1.000 ml (1.500-2000 ml/day) showed a high incidence of readmissions for HF worsening (30%) (16 patients) in the first 6 months. We also observed that the refused consent group showed similar results to those randomized to normal sodium diet (120 mmol), while the free fluid intake group (1.500-2000 ml/day) showed a increase in readmissions similar to patients with low sodium diet.

DISCUSSION

To our knowledge, this is the first investigation to assess the outcome response to different dose of Na intake in medically treated patients with compensated HF. In the present study we randomized low sodium (80 mmol Na) vs normal sodium (120 mmol Na) diet in compensated HF patients receiving the same treatment during hospitalization and 30 days after discharge. In this way, this criterion allowed us to evaluate the effects of normal sodium versus low sodium diet in the follow up period in patients matched for clinical characteristics and treatments. We choose 120 mmol of Na because epidemiological studies showed that in Europe and Italy it was estimated an assumption of a mean of 3.5 g (150 mmol) Na/day [START_REF] Leclercq | Total and domestic consumption of salt and their determinants in three regions of Italy[END_REF][START_REF]Nutrient and energy intakes for the European Community[END_REF] Our data suggests that a normal sodium sodium diet with a limited drink fluid intake (1 litre) associated with loop diuretic is able to reduce both hospital readmission for worsening of HF (primary endpoint) and neurohormonal activation (BNP,PRA and ALD plasma levels) after six months of follow up. The beneficial effects obtained with this association suggest that low sodium diet and free water intake usually recommended in clinical practice [START_REF] Yan | Narrative review: Pharmacoterpy for chronic heart failure:evidence from recent clinical trials[END_REF][START_REF] Hunt | ACC/AHA Guidelines for the evaluation And management of chronic heart failure in th adult[END_REF][START_REF] Nohria | Medical management of advanced heart failure[END_REF] could not be the best treatment for these patients and merits further investigations. In addition, more recently, the accepted link between Na intake and cardiovascular events was not confirmed. In fact, it was evidenced that high Na intake does not induce total body water storage but induces a relative fluid shift from the interstitial into the cardiovascular space [START_REF] Heer | High dietary sodium chloride consumption may not induce body fluid retention in humans[END_REF]. Conversely, revised NHANES II study showed an inverse association of Na with cardiovascular mortality [START_REF] Cohen | Sodium intake and mortality in the NHANES II follow up study[END_REF] while other papers refuted this link [START_REF] He | Salt, blood pressure and cardiovascular disease[END_REF]. Unfortunately, the two studies included different populations, a general population in the NHANES II study and a selected population of prehypertensive patients in the TOHP study [START_REF] Cook | Long term effects of dietary sodium reduction on cardiovascular disease outcomes: observational follow-up of the trials of hypertension prevention (TOHP)[END_REF], while compensated heart failure patients were included in the present paper. These data does not allow conclusive considerations about the management of Na in cardiovascular diseases. Our results indicate that renal hemodynamic and neurohormonal responses in Na intake were tolerated without any excessive Na and water retention and were associated with lower hospital readmissions for worsening HF. In the arm receiving 120 mmol of Na, only 9 patients were readmitted during the follow-up period in comparison with 30 patients readmissions in the arm receiving 80 mmol of Na. These findings oppose the results of previous studies, in which untreated and conventionally treated patients with HF have been shown to accumulate Na and water in excess during dietary Na loading [START_REF] Braunwald | A method for the detection and quantification of impaired sodium excretion. Results of an oral sodium tolerance test in normal subjects and in patients with heart disease[END_REF][START_REF] Cody | Sodium and water balance in chronic congestive heart failure[END_REF][START_REF] Hollander | The relationship of cardiovascular and renal hemodynamic function to sodium excretion in patients with severe heart disease but without edema[END_REF][START_REF] Proger | The effects of the ingestion of excessive amounts of sodium chloride and water on patients with heart disease[END_REF][START_REF] Volpe | Abnormalities of sodium handling and of cardiovascular adaptations during high salt diet in patients with mild heart failure[END_REF]. In view of these clinical data, we hypothesized that normal Na intake maintains a suitable arterial blood volume that per se inhibits the neurohormonal activation observed during low sodium diet, and improve both renal and hormonal alterations. In fact, the maintained plasma volume expansion determines an increased diuresis and natriuresis associated with reduced levels of AVP, plasma renin activity or ang II, and ALD [START_REF] Magga | Brain Natriuretic peptide in plasma, atria, and ventricles of vaspressin-and phenylephrine-infused conscious rats[END_REF]30]. These data could explain that despite the small difference (27 mmol Na) in urinary sodium between the two groups, probably the observed large difference in PRA could be due to the additive or synergistic effects of reduction in plasma volume and Na plasma levels in 80 mmol Na group with a subsequent RAAS stimulation. On the contrary, the additive or synergistic effects of plasma volume expansion and the normal Na plasma levels observed in 120 mmol Na group determine an inhibition of RAAS. system [START_REF] Neri Serneri | Evidence for the existence of a functional cardiac renin angiotensin system in humans[END_REF]. In addition,the difference in natriuresis confirmed that all patients followed the assigned diets. Maintenance of normal blood volume depends upon a balance between Na intake and renal excretion. The reduced tissue perfusion, operating in CHF patients, stimulate the proximal tubule avidly reabsorbition of Na and water, limiting the amount of dilute urine that can be generated and excreted. Thus, given the well-documented influence of the renal blood supply on Na handling and the reversal of the antinatriuresis when renal perfusion is increased in these patients, it seems likely that the renal vascular response participates in the Na retention in patients with advanced HF [START_REF] Neri Serneri | Evidence for the existence of a functional cardiac renin angiotensin system in humans[END_REF].

Recently, it was showed that altered Na intake elicited the same absolute and relative hemodynamic responses in HF patients as healthy controls. In addition, a suppression of vasoconstrictor hormones was determined in HF compensated patients receiving high Na intake [START_REF] Pickkers | Direct vascular effects of furosemide in humans[END_REF][START_REF] Gabrielsen | Neuroendocrine and renal effects of intravascular volume expansion in compensated heart failure[END_REF][START_REF] Damgaard | Hemodynamic and neuroendocrine responses to changes in sodium intake in compensated heart failure[END_REF]. It is possible that regular Na administration is able to restore fluid balance and to inhibit vasoconstrictor systems. These results suggest that Na combined with appropriate diuretic therapy and fluid intake might be beneficial to maintain a good intravascular volume and thereby to improve cardiac and renal blood flow. On the contrary, during Na restriction, Na and water retaining hormonal systems are activated and cardiac forward flow is impaired [START_REF] Gabrielsen | Neuroendocrine and renal effects of intravascular volume expansion in compensated heart failure[END_REF][START_REF] Damgaard | Hemodynamic and neuroendocrine responses to changes in sodium intake in compensated heart failure[END_REF][START_REF] Schnermann | Juxtaglomerular cell complex in the regulation of renal salt excretion[END_REF]. In addition it has been demonstrated that intravascular volume depletion induced by low Na intake can compromise renal function, especially if associated with ACE-inhibitors or diuretics [START_REF] Packer | Interaction of prostaglandins and angiotensin II in the modulation of renal function in congestive heart failure[END_REF]37] intake. Worsening indices of renal function limit symptomatic and neurohormonal therapy, lead to longer hospital stay, and predict higher rate of early readmissions and death. [START_REF] Weinfeld | Aggravated renal dysfunction during intensive therapy for advanced chronic heart failure[END_REF][START_REF] Gottlieb | The prognostic importance of different definitions of worsening renal function in congestive heart failure[END_REF][START_REF] Geisberg | Addressing the challenges of cardiorenal syndrome[END_REF] The marked increase and/or normalization in serum Na levels, observed in patients receiving normal Na intake, is consistent with vasopressin and RAAS inhibition [START_REF] Shankar | Loop diuretics: from the Na-K-2Cl transporter to clinical use[END_REF]. Problably, the association of high diuretic dose did not allow excessive increases in plasma volume, and the normal Na intake maintained normal Na plasma levels avoiding the effects of hyponatremia and stimulation of RAAS system. The effects on hyponatriemia is very important, because its has long been associated with worse outcome [START_REF] Lee | Prognostic importance of serum sodium concentration and its modification by converting-enzyme inhibition in patients with severe chronic heart failure[END_REF][START_REF] Gheorghiade | Lower Serum Sodium is associated with increased short-term mortality in hospitalized patients with worsening heart failure. Resulta from the Outcomes of a prospective trial of intravenous milrinone for exacerbations of Chronic Heart Failure (OPTIME-CHF) Study[END_REF]. It is possible that the intensity of diuretic therapy created an opportunity for protective benefit from higher Na intake that would not be realized with lower diuretic doses. In addition, it was shown that furosemide at therapeutic concentrations exerts a direct venodilator effect [START_REF] Braunwald | A method for the detection and quantification of impaired sodium excretion. Results of an oral sodium tolerance test in normal subjects and in patients with heart disease[END_REF][START_REF] Dikshit | Renal and eztrarenal hemodynamic effects of furosemide in congestive heart failure after acute myocardial infarction[END_REF]. The normosodic diet and oral dose of furosemide enough to maintain a stable body weight may explain the significant reduction in BNP, ALD and PRA plasma in comparison with hyposodic group [45]. The little variation in ALD and PRA plasma levels suggest that a normosodic diet, fluid restriction and oral dose of furosemide enough to maintain a stable body weight did not allow a further stimulation of the RAAS system which maintained similar values to those observed at baseline. However, it is possible that the therapeutic effects of this treatment are not only mediated by the direct effects on renal hemodynamics, but also by neurohormones modulation [START_REF] Damgaard | Hemodynamic and neuroendocrine responses to changes in sodium intake in compensated heart failure[END_REF]46,47]. It is possible that the Na intake (120 mmol) allowed a continue esponse to diuretics also by a reduction of intrarenal PGE2 inactivation, and an improvement of medullary microcirculation with reduction of hyper-osmolality and a vasopressin inhibition PGE2-induced. [48]. These last mechanisms could also justify the maintenance of the reduction in BNP levels observed after 90 and 180 days. The data obtained from patients who refused consent and continued the treatments (even if not randomized) were similar to those randomized to 120 mmol Na diet while those who assumed a higher fluid intake (1.500-2000 ml/day) and 120 mmol Na diet showed similar results to those randomized to 80 mmol of Na (perhaps for dilutional mechanism). These results confirm that the combination of reduced fluid intake (1000 ml/day) and a normal sodium diet (120 mmol) is able to explain the reduction both hospital readmissions and in PRA, ALD, and BNP levels. Before planning the present study, we evaluated in two groups (15 patients for group) of compensated (II Class NYHA) patients (similar to those included into the study) the effects of lower doses of furosemide (100-250 mg/day) after discharge associated with normal sodium diet (120 mmol) or with low sodium diet (80 mmol) and a reduced fluid intake (1 litre). We observed that the group receiving normal sodium diet showed a rapid worsening of HF within the first two weeks from discharge (10 patients were readmitted for HF worsening), and the group with low sodium diet showed a dramatic rapid worsening of HF within the first week from discharge (11 patients were readmitted for HF worsening) (unshown data). These data suggested us that higher doses of loop diuretics needed to maintain body weight and water balance and determined the diuretic dose of the present study. Further studies comparing a normal sodium diet plus free fluid intake versus a normal sodium diet plus reduced fluid intake are necessaries. In conclusion, the present investigation suggests a surprising efficacy of a new strategy to improve chronic diuretic response by Na intake increase and limited drink fluid intake (1000 ml daily). Our study shows that the use of high doses of loop diuretics were not detrimental also in the long term, as previously reported [START_REF] Licata | Effects of high-dose furosemide and small-volume hypertonic saline solution in comparison with a high dose of furosemide as bolus in refractory congestive heart failure:long term effects[END_REF]. The difference between our Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society studies and those reported in literature, probably are due to association of the normal sodium diet and higher diuretic dose. In fact, the studies reporting detrimental effects of long-term treatment with loop diuretics were performed in CHF patients receiving low sodium diet and different diuretic doses [START_REF] Damgaard | Hemodynamic and neuroendocrine responses to changes in sodium intake in compensated heart failure[END_REF]49,50]. In addition, all the readmitted patients were treated with HSS solution and high dose of loop diuretics which determined rapid compensation (<6 days), especially the patients from low sodium group. To date, this counterintuitive approach underlines the need for better understanding of factors that regulate Na and water handling in chronic CHF. The observation that a normosodic diet improves outcome in compensated CHF patients.and that high dose of loop diuretics if associated with normosodic diet do not determine detrimental effects in renal function.

suggest that Na depletion has detrimental renal and neurohormonal effects with worse clinical outcome in patients with compensated CHF. Further studies are required to determine if this is due to the high dose diuretic or low sodium diet.

Limits of the study: the more important limit was the brief follow-up, but the interim analysis (by ethical committee) after six months of follow up showed a significant reduction in readmissions rate and recommended the discontinuation of the study. These data were sufficient to suggest that a normosodic diet and a reduced drink fluid intake associated with a tailored therapy with furosemide may improve outcome in compensated HF patients. Another important limit was the unblinded design, but we attempted to perform a double blinded study but it was not possible because the patients receiving the prescribed diets perceived the difference in salt amount. In addition, to reduce bias, the randomization was performed by an external team of physicians blinded to the study, and other different blinded physicians performed evaluation in the programmed controls and evaluated the readmitted HF patients during follow up. The laboratory and neurohormonal controls were also performed by blinded physicians. The adherence to diet, fluid intake and doses of furosemide were also controlled by two blinded physicians. All these physicians communicated the results of controlled data to responsible of research after the conclusion of the study. Another limit was the low rate of beta-blocker therapy but the enrolled patients were compensated CHF (from advanced IV to II class NYHA): unresponsive, before hospitalization, to best conventional treatment (high doses of furosemide, combination of diuretics, betablockers, ace-inhibitors, etc), and in previous hospitalizations beta-blocker treatment was discontinued for repeated episodes of pulmonary oedema. This low rate of beta-blocker treatment did not influence the outcome of patients. It is also possible that the intensive follow up may have affected the outcome, but this intensive follow up was mandatory because of novelty of the study.

Clinical

  None of the patients had to take non-steroid anti-inflammatory drugs (NSAID). All patients received high dose furosemide (250-1000 mg bid), hypertonic saline solution, a normal sodium diet (120 mmol), and a Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical

  Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  -5 show the Kaplan-Meier cumulative event curves for primary end point (hospital readmission) and combined readmissions and Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society mortality (secondary endpoint) in the two groups during 180 days follow-up. Curves analysis show an evident

Clinical

  Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  . Thus not only neuroendocrine activation but also a compromised cardiorenal function could result from intravascular volume depletion induced by low Na Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 : 22 Figure 2 :

 1222 Figure 1: Flow chart of the study.

Figure 3 :

 3 Figure 3: Changes in PRA (plasma renin activity and aldosterone plasma levels during the

Figure 4 :

 4 Figure 4: Kaplan-Meier cumulative event curves for primary end point (hospital redmissions)

Figure 5 :

 5 Figure 5: Kaplan-Meier cumulative event curves for combined end points (hospital

Table 1 : Clinical Characteristics and ethiology of CHF. ________________________________________________________________________ 80 mmol Na 120 mmol Na

 1

	Patient No.	232	114	118
	Age (years)		73.3+9	72.1+7
	Sex	F/M	88/144	43/71	45/73
	Diabetes	55	25	30
	Captopril (75-150 mg day)	232	114	118
	Carvedilol (6.25-25 mg bid)	18	10	8
	digitalis (0.125-0.25 day)	47	23	24
	Furosemide (250 mg bid)	142	73	69
	Furosemide (500 mg bid)	90	46	44
	Spironolattone(25 mg day) 202	99	103
	CHF Ethiology
	CAD		110	53	57
	HHD		76	37	39
	DCM		46	24	22
	AF		51	26	25
	_______________________________________________________________________
	CAD=Coronary Artery Disease;HHD= Hypertensive Heart Disease; DCM= Dilatative
	Cardiomyopathy; A.F=Atrial fibrillation.

Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical SocietyTable 2 : Clinical and laboratory parameters at entry and after 90 and 180 days. Data are expressed as mean +SD.

 2

	___________________________________________________________________________________________________
			80 mmol Group		120 mmol group
		_________________________________	________________________________
		Entry	90 days 180 days P<	entry	90 days	180 days	P<
	__________________________________________________________________________________________
	Patients No	114	106	99		118	114	112
	SBP (mmHg)	126+15	116.+11	107+13	0.001	125+15	114+11	111+11	0.01
	DBP (mmhg)	82.+13	76.+11	77+9	0.001	83+14	78+12	75+11	0.001
	HR (beats/min)	85+14	83+11	*82+12	ns	84+11	76+9	*72+8	0.0001
	Ejection Fraction	29.3+5	30.1+6	30.2+6	ns	29.5+4	32.2+6	32.5+5	0.001
	Body Weight (Kg)	76.9+15	79.1+13 *80.2+13	0.001	75.2+14	74.8+15	*75.3+10	ns
	Diuresis (ml/24h)	2560+425	1580+555 *1525+565 0.0001	2495+635	2250+565 *2150+480 0.048
	Serum Na (mEq/L)	138.3±5	132.3+4	*132.1+5	0.001	138.7±7	140.4+3	*139.5+6	0.001
	Serum K (mEq/L)	4.6+0.8	3.7+0.4	3.6+0.8	0.0001	4.7+0.5	3.9+0.4	3.7+05	0.0001
	Urinary Na (mEq/24h)	102+11	77+9	* 76+7	0.01	105+14	*106+12	*103+11	ns
	BUN (mg/dl)	56.5+3.6	101±12	*105+5.8	0.0001	58.5+7	67± 9	*68.4+7.2 0.001
	Serum Creatinine (mg/dl) 1.55+0.05	1.89+0.3 *2.1+0.5	0.001	1.56+0.2	1.48+0.3	*1.45+0.4 0.01
	Uric Acid (mg/dl)	6.7 ± 2.1	9.8+3.5 10.2+2.3	0.0001	6.6±2.5	9.8+3.5	9.9+4.3	0.0001
	__________________________________________________________________________________
	(*) = p < 0.001, differences between the two groups : 80 mmol versus 120 mmol after 180 days .

Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 3 : Results of the study (180 days): Readmissions and Mortality. ___________________________________________________________________________________

 3

		80 mmol Na	120 mmol Na	ARR (95% CI)	P<
	Patient No.	114	118
	Sex F/M	43/71	45/73
	Readmissions	30 (26.32%)	9 (7.63%)	18.69%	0.05
	Mortality	15 (13.15%)	6 (5.08%)	8.07%	ns
	sudden death	4	1
	Irreversible HF	9	5
	other causes	2	-
	Readmissions and Mortality 45 (39.47%)	15 (12.71%)	26.76%	0.001

____________________________________________________________________________________ Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 4 : Clinical Characteristics and ethiology of CHF patients receiving normal sodium diet (120 mmol Na), who refused consent and patients excluded for protocol violation (fluid intake > 1000 ml/die).

 4

	________________________________________________________________________
			Refused Consent	Fluid intake violation	p<
	Patient No.	79	54
	Age (years)	75.4+13	71.2+11	ns
	Sex	F/M	33/46	23/31	ns
	Diabetes	21	14	ns
	Captopril (75-150 mg day)	79	54	ns
	Carvedilol (6.25-25 mg bid)	7	3	ns
	digitalis (0.125-0.25 day)	29	14	ns
	Furosemide (250 mg bid)	68	49	ns
	Furosemide (500 mg bid)	11	5	ns
	Spironolactone (25 mg day)	64	51	ns
	CHF Ethiology
	CAD		50	35	ns
	HHD		22	13	ns
	DCM		7	6	ns
	AF		37	21	ns
	________________________________________________________________________________
	CAD=Coronary Artery Disease;

HHD= Hypertensive Heart Disease; DCM= Dilatative Cardiomyopathy; A.F=Atrial fibrillation. Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 5 : Results (180 days) in readmissions and mortality of CHF patients receiving 120 mmol Na and who refused consent and patients excluded for protocol violation (fluid intake > litre).

 5

	_____________________________________________________________________________
	Refused consent	Fluid intake violation	P<
	Patient No.	79	54
	Readmissions	7	16	0.019
	Mortality	5	10	0.098
	Sudden death	1	3	ns
	Irreversible HF	2	6	ns
	Other causes	2	1	ns

____________________________________________________________________________ Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society 20

Table 6 : Clinical and laboratory parameters at entry and after 180 of CHF patients receiving 120 mmol Na. who refused consent and patients excluded for protocol violation (fluid intake > 1 L). Data are expressed as mean +SD.

 6

	________________________________________________________________________________
		Refused Consent		Fluid intake violation
		____________________		_____________________
		Entry	180 days	p<	entry	180 days	p<
	___________________________________________________________________
	Patients No	79	74		54	44
	SBP (mmHg)	128+17	119.+14	0.0001	131+11	124+16	0.012
	DBP (mmhg)	85.+15	76.+11	0.0001	83+14	78+12	0.064
	HR (beats/min)	88+14	83+11	0.016	84+11	91+9	0.0001
	Ejection Fraction	27.2+7	29.5+4	0.015	29.5+4	28.2+4	ns
	Body Weight (Kg)	76.9+15	77.1+12	ns	74.9+11	78.7+9	0.068
	Diuresis (ml/24h)	2560+425	2585+675	ns	2465+435	2125+665 0.003
	Serum Na (mEq/L)	138.3±5	141.3+5	0.0001	138.7±7	132.3+4	0.0001
	Serum K (mEq/L)	4.8+0.7	3.7+0.3	0.0001	4.6+0.7	3.5+0.4	0.0001
	Urinary Na (mEq/24h)	107+13	102+17	ns	104+14	101+15	ns
	BUN (mg/dl)	55.7+3.7	61.4±12	0.0001	58.5+7	67± 9	0.0001
	Serum Creatinine (mg/dl)	1.52+0.06	1.49+0.07	ns	1.66+0.5	1.71+0.7	ns

_________________________________________________________________________________________________

Clinical

Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society 21

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Stevenson, L.W. Design of therapy for advanced heart failure (2005). Eur. J. Heart Fail. 7, 323-331 Clinical Science Immediate Publication. Published on 9 Aug 2007 as manuscript CS20070193 © 2007 The Authors Journal compilation © 2007 Biochemical Society

No conflict of interest. No fund for this work was received. Approved by ethic committee