Carlos Velasco-Reynold

Miguel Navarro-Alarcon

López -Gª De

L A Serrana

Vidal Perez-Valero

María C Lopez-

Dr Miguel Navarro-Alarcón

Determination of daily dietary intake of chromium by duplicate 1 diet sampling: in vitro availability study 2 3

Keywords: Duplicate diets, GFAAS, Health significance, Metals -bioavailability Additives/Contaminants: Metals, Metals -nutritional, Trace elements (nutritional), Trace elements (toxic) Chromium, ETAAS, daily dietary intake, duplicate diet method, in vitro 20 availability

Determination of daily dietary intake of chromium by duplicate diet sampling: in vitro availability study

Miguel Navarro

Spain) corresponding to 36 consecutive days. The total and dialyzable Cr levels were 4 measured by a validated electro thermal atomic absorption spectrometry (ETAAS) method. A 5 mean Cr fraction of 26 ± 12 µg meal -1 was found. The Cr supply from meals was directly and 6 significantly (p < 0.001) correlated with their macronutrient (carbohydrates, fiber and 7 protein) content. Cereals and by-products, legumes, dry fruit, meat, potatoes, dairy products 8 and seafood are the primary sources of Cr. The mean Cr fraction dialyzed through the 9 dialysis tubing was 1.2 ± 1.1 µg meal -1 (4.6 ± 3.8% as mean Cr dialysability). The Cr intakes 10 found for breakfasts were significantly lower (p < 0.001). A correlation between the 11 logarithmic data of total and dialyzable fraction of Cr in meals (p= 0.020) was found. The 12 dialysis ratio enhancement and therefore bioavailability increased with the total Cr amount. [START_REF] Nielsen | Copper In: Modern 14 nutrition in health and disease[END_REF] The dialysed element content present in meals was significantly correlated with fibre, protein, 14 Fe, Na, I, F, sodium, ascorbic acid and vitamin A levels (p < 0.05). At Fe contents in meals 15 higher than ≅7.5 mg meal -1 the net absorption of Cr decreased significantly. The mean Cr 16 daily dietary intake (DDI) was 77 ± 17 µg day -1 which indicates no adverse effects in relation [START_REF] Rowbotham | Chromium in the environment: an evaluation of 18 exposure of the UK general population and possible adverse health effects[END_REF] to Cr nutrition (deficiency or toxicity) should occur in individuals from the area. 18 Chromium is an essential element that forms part of the glucose tolerance factor and helps to 4 maintain normal glucose metabolism (Sola-Larrañaga and Navarro-Blasco 2006). [START_REF] Stoecker | Cromo[END_REF] Specifically Cr has been identified as a factor that enhances insulin activity (Goldhaber 6 2003). The National Institute of Health's programme reported that this element could act as 7 an adjuvant in the therapy for the type 2 diabetes and impaired glucose tolerance (Vincent 8 2004). In humans submitted to total parenteral nutrition not supplemented with Cr, the 9 addition of this element as CrCl 3 led to a decrease in the insulin needs and ameliorated the 10 glucose tolerance [START_REF] Jeejeebhoy | Chromium deficiency, glucose intolerance and 24 neuropathy reversed by chromium supplementation in a patient receiving long term total 25 parenteral nutrition[END_REF]. It seems that this element can have a biochemical 11 function that would enhance the capacity of the insulin receptor to interact with its hormone. 12 It has been observed from in vitro studies that the RNA synthesis increases by the Cr binding 13 to DNA. This finding suggests that this element could exert a regulation of a molecule 14 synthesis that would reinforce the insulin action and therefore as such would influence 15 carbohydrate, lipid and protein metabolism [START_REF] Anderson | Minerals[END_REF]Navarro-Alarcon et al. 2005). 16 Nevertheless, other researchers have indicated that individuals with type 2 diabetes did not 17 obtain any benefit from yeast-based Cr supplements of 100 µg Cr day -1 [START_REF] Trow | Lack of effect of dietary chromium supplementation on glucose tolerance, plasma 9 insulin and lipoprotein levels in patients with type 2 diabetes[END_REF]. 18 [START_REF] Schachter | Oral chromium picolinate and control of glycemia 24 in insulin treated diabetic dogs[END_REF] also suggested that at a dosage range of 20-60 µg kg -1 day -1 the Cr 19 picolinate caused no beneficial or harmful effects in insulin-treated diabetic dogs. Berner et 20 al. (2004) also reported how one particular form of Cr 3+ , the Cr picolinate (Chromax) was 21 determined to be safe for use as a nutrient supplement in foods consumed by human beings. 22

Other studies have indicated the positive effect exerted by this mineral on the immune system 23 [START_REF] Moonsie | Supplemental chromium on performance, serum constituents, 5 and immune status of etressed feeder calves[END_REF]. Other researchers have found that the intake of Cr supplements 24 by malnourished children produced a significant enhancement of the growth rate (Gurson and 25 Saner 1973). Similarly, others reported that organic Cr tended to improve growth 26 performance of pigs (as Cr picolinate; [START_REF] Cho | Effects of chromium picolinate, 29 L-carnitine and thyroxine on the performance, nutrient digestibility and nitrogen balance in 30 pigs weaned at 21 days of age[END_REF] or broiler chicks (as Cr picolinate or 27 Cr yeast; Suksombat and Kanchanatawee 2005). Recently, the Cr has been used as an agent 28 for increasing lean body mass and decreasing percent body fat [START_REF] Kobla | Chromium, exercise, and body composition[END_REF]. 29

Lately, due to the hypothesized anabolic capacity of Cr, an enahncement of the consumption 30 of Cr supplements by sportsmen in order to rise or to keep their muscle mass (Crawford et al. 31 1999), has been observed. A beneficial effect of the Cr on lipid profiles, with a diminishment 32 of the total cholesterol and low-density-lipoproteins (LDL) and triglyceride levels and a 33 The toxic effects produced by the Cr were directly related with industrial exposure 3 and associated with the different Cr +6 species carried by the air (Von [START_REF] Burg | Chromium and hexavalent chromium[END_REF]. 4

The main health effects related to Cr 6+ are allergenic dermatitis, lesions in the skin and in the 5 nose wall as well as a higher lung cancer incidence (Von [START_REF] Burg | Chromium and hexavalent chromium[END_REF][START_REF] Losi | Environmental biochemistry of chromium. 32 Review of Environmental Contamination and Toxicology 136:91-121. 33 iron availability from meals[END_REF], 6 Rowbotham et al. 2000) associated with some occupational exposure scenarios. Contrarily, 7 the Cr 3+ is an essential nutrient with a broad safety range and low toxicity (Rowbotham et al. 8 2000). Like the absorption of the Cr 3+ is scarce, their oral intakes have to be very high in 9 order to rise damage levels. 10

Cr absorption depends of the elemental species and the content in food components of 11 meals [START_REF] Stoecker | Cromo[END_REF]. The fraction of the Cr 3+ form usually absorbed in the bowel from 12 dietary intakes mainly by passive paracellular absorption is very low and usually ranges 13 between 0.5-2% (García et al. 2001a). Nevertheless, the absorption of Cr 6+ species seems to 14 be until 6 times higher. Although the intestinal absorption mechanism for Cr has still not been [START_REF] Offenbacher | Promotion of chromium absorption by ascorbic acid[END_REF] clearly identified, it seems that in addition to the passive route previously noted, active 16 mechanisms also exist (Navarro Alarcon et al. 2005). 17

The absorption (15-20%) and bioavailability of Cr is affected by dietary factors like 18 chemical form, meal composition, lumen intestinal content, interaction with other nutrients as 19 well as endogenous physiological factors (status of the intestinal mucosa, nutritional status of 20 individuals, homeostatic mechanisms, carrier proteins, etc.; Garcia et al., 2001a; Anderson 21 2004;Navarro Alarcon et al. 2005). On the other hand, the Cr present in meals interacts with 22 other nutrients that influence its net absorption. Specifically, the amino acids and starch 23 [START_REF] Seaborn | Effect of starch: sucrose, fructose, and glucose on chromium 26 absorption and tissue concentrations in obese and lean mice[END_REF]Stoecker 1989, Anderson 2004), and the ascorbic acid [START_REF] Offenbacher | Promotion of chromium absorption by ascorbic acid[END_REF]) 24 facilitate the net Cr transport and absorption by forming soluble complexes. The 25 bioavailability of this is also element enhanced by Fe deficiency [START_REF] Anderson | Minerals[END_REF]). This 26 finding could be due to problems of competence existing between both minerals by joint at 27 the same intestine absorption sites (Navarro Alarcon et al. 2005). 28 In the present study, the assessment of the Cr DDI by adult inhabitants from south eastern 29 Spain by one retrospective method, was performed. A duplicate portion sampling method of 30 108 duplicate meals (36 breakfasts, 36 lunches and 36 dinners) corresponding to 36 31 consecutive days from the restaurant of the Hospital of Motril (SE Spain), and analysis by an 32 electro thermal atomic absorption spectrometry (ETAAS) method, was carried out. Due to the 33 analytical techniques are required for its measurement, as well as new standard reference 2 materials for accurate determinations [START_REF] Anderson | Minerals[END_REF]). Consequently, a previous 3 development and optimization of a specific time-temperature program for the Cr 4 measurements by ETAAS was performed. The interaction of other nutrients present in 5 analyzed meals with Cr and their influence on the element absorption was also studied. In 6 order to determine the available Cr fraction an in vitro method that simulates the gastro 7 intestinal conditions with the employment of dialysis tubing, was used (Garcia e tal.2001a). 8

The determination of levels of several nutrients considered present in meals was carried out 9 by the employment of food composition tables compiled in the programme Dietsource 2.0 10 (Moreira [START_REF] Ingles | Valoración y soporte 8 nutricional del paciente hospitalizado y ambulatorio[END_REF]. The study of the interaction of the levels of these nutrients on the total 11 and dialyzable Cr contents in analyzed meals was also performed. Additionally, to assess the 12 magnitude of inadequate intakes of Cr (deficiency or excess and toxicity) we employed the 13 dietary adequate intakes (AIs) cut off points proposed by the USA Institute of Medicine 14 (IOM, 2002) (35 and 25 µg day -1 for male and female healthy adults older than 19 y). 15 modifier. Nitric acid (65% v v -1 ) and perchloric acid (70% v v -1 ) were employed to mineralize 1 the diet samples. 2 Pepsin (Sigma P7000, porcine pancreas, Sigma-Aldrich Chemie Gmbh, Steinheim, 3 Germany), pancreatin porcine pancreas (Sigma p1500, Sigma Chemical co., St Louis, MO) 4 and bile salts (Sigma B8756) were used to simulate gastric and intestinal digestion. A pepsin 5 solution was prepared by dissolving 16 g pepsin in 100 ml of 0.1 M HCl. Pancreatin-bile 6 extract mixture was prepared by dissolving 4 g pancreatin and 25 g bile extract in 1 l of 0.1 M 7 NaHCO 3 . The solutions of 0.5 M NaOH and 6 M HCl were employed to adjust the pH. 8

Dialysis tubing Spectra with molecular mass cut-off (MMCO) of 12-14 kDa (Visking size 3-9 20/32), Medicell International, London, UK) were used. The dialysis tubing was freed of 10 trace element impurities by boiling in a 2% w v -1 NaHCO 3 0.1 % w v -1 sodium dodecyl 11 sulphate and 0.01 M ethylenediaminetetraacetic acid disodium salt (EDTA) for 30 min, 12 followed by thoroughly washing with double distilled deionized water, and preserved in 20% 13 ethanol solution. 14 All solutions were prepared from analytical reagent grade reagents (Merck, Germany, 15 Suprapur). The water employed for preparing the standards for calibration and dilutions was 16 deionised double distilled water with a specific resistivity of 18 MΩ cm obtained by filtering 17 distilled water through a Milli-Q purifier (Millipore, Waters, Mildford, MA) immediately 18 before use. [START_REF]Food and Nutrition Board. Dietary references intakes for 20 vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine[END_REF] 20 ETAAS 21 Duplicate portion samples of 108 different meals, corresponding to 36 breakfasts, 36 lunches 22 and 36 dinners, were taken over a 36-days consecutive period from May to June 2002 from 23 the restaurant of the Hospital of Motril. The dietician was instructed to collect duplicates of 24 the three meals consumed during each 24-hr period in 2-2.5 l previously cleaned polyethylene 25 containers. As a second instruction all food and beverage items corresponding to each menu 26 were recorded and weighed on balances available in the hospital. The containers were then 27 stored at -18ºC. Meal samples were transported to the laboratory of the Department of 28 Nutrition and Food Science of the University of Granada. The analysed meals (n= 108) were 29 sliced and mixed in a blender (Stomacher 400), of which various parts were Teflon coated, 30 noting the menu weight. Aliquots of about 100 g of the mixed food were dried at temperature 31 ranged from 60-80ºC. After drying, liquid content was calculated and dried samples were 32 homogenized in previously cleaned Teflon containers. About of 400 mg of the pulverized 33 meal sample was placed in a flask and subjected to an acid mineralization in a multiple place 1 digestion block by adding 5 ml of HNO 3 cc and heating at 60º C for 30 min. Another 1 ml of 2 4:1 HNO 3 -HClO 4 (4:1) was added and heating continued at 120ºC for 90 min. The digest was 3 then cooled and the resulting solution diluted to 25 ml with double distilled water. The 4 analytical solution obtained was suitable for the Cr ETAAS determination. 5

Duplicate portion sampling of meals and Cr analysis by

The Cr determination was directly performed in acidified samples adequately diluted 6 with 0.2% HNO 3 solution. The element was directly determined by the linear calibration 7 method. A sample volume of 10 µl was manually injected with a micropipette through a 8 graphite tube with L'Vov platform. The chemical modifier (10 µl) [0.5% Mg(NO 3 ) 2 (equivalent 9 to 40 µg Mg(NO 3 ) 2 ] was also injected. The appropriate conditions for Cr measurements were 10 optimised by us on the basis of time-temperature studies using certified standards (CBR CRM 11 278R mussel tissue and CBR CRM 185R bovine liver). Peak area was used as the analytical 12 signal. Furnace conditions for Cr determination by ETAAS at 357.9 nm are summarised in table 13 I. The Cr hollow cathode lamp used at 18 mA and a spectral slit width of 0.7 nm was selected. 14

The samples were analysed in triplicate. 15 16 17

Duplicate portion sampling of meals and estimation of nutrient intakes by food 18 composition tables 19

The estimation of contents of several nutrients and energy in analysed meals was done by the 20 employment of food composition tables compiled in the programme (Dietsource version 2.0, 21 Novartis, Barcelona). It has allowed us to calculate their daily dietary intakes as well as that of 22 energy. These values have also been used to study the correlations existing among total and 23 dialyzable Cr levels and contents of other nutrients and energy in the analysed meals. 24

Unfortunately, these food composition tables do not include levels for Cr. Consequently, the 25 comparison of Cr DDI determined in the present method vs. that estimated using the food 26 composition tables compiled in the referred programme was not possible. 27 28

Estimation of the absorbable fraction by the in vitro method 29

The simulated gastrointestinal digestion procedure and the in vitro absorption estimation was 30 doubly distilled water and the pH was adjusted to 2.0 with 6 M HCl. After 15 min, we 1 determined again the pH adjusting again to 2 if it was necessary. Titritable acidity corresponded 2 to the number of equivalents of 0.5 NaOH required to titrate the amount of combined pepsin 3 digest pancreatin-bile extract mixture to pH= 7.5. For its determination, a 20 g aliquot of the 4 pepsin digest which 5.0 ml of the pancreatin-bile extract mixture were added employing 0.5 M 5 NaOH in the titration. 6 7

Statistics 8

For interpreting data the statistical package SPSS 12.0 for Windows program was employed. 9

Results are expressed as arithmetic mean and standard deviation. They were analysed in order 10 to evaluate the existence of statistical differences according to the meal (breakfast, lunch or 11 dinner) considered for the estimation of Cr intakes. The normal distribution of variables and 12 the homogeneity of variances were checked by the Kolmogorov-Smirnov and Bartlett's tests, 13 respectively. The comparisons were done using the Student's t-test when the variable fulfilled 14 parametric conditions and the Kruskall-Wallis test when conditions were non-parametric. [START_REF] Offenbacher | Promotion of chromium absorption by ascorbic acid[END_REF] Additionally, correlations by the Pearson or Spearman's tests (for parametric and non-16 parametric conditions, respectively), linear regression models and correlation of logarithmic 17 data were also used, to check the association between determined total and dialyzable Cr 18 levels from meals and among them and total levels present of other nutrients and energy 19 intake. [START_REF] Santos | Assessment of dialy intake of trace 21 elements due to consumption of foodstuffs by adult inhabitants of Rio de Janeiro city[END_REF]

22

Results and discussion 23 24

Analytical quality assurance 25

The performance characteristics of the proposed method were determined following the 26 instructions indicated elsewhere (Delgado-Andrade et al. 2003). The detection limit and 27 characteristic mass were 187 ng g -1 and 3 pg, respectively. Mean recovery for spiked samples 28 was 100%. The mean Cr concentrations determined in the BCR CRM 278R and 185R (0.78 ± 29 0.06 and 0.80 ± 0.20 µg g -1 , respectively) were 0.80 ± 0.08 and 0.83 ± 0.26 µg g -1 , respectively. 30

The relative standard deviation (n= 7) ranged from 5 to 12% (mean value= 7%) for the three 31 different samples for which between-day variability was determined. 32 In the present study an ETAAS method has been developed for measuring total and dialysed 1 Cr levels in 108 meals. The results obtained for the standard reference materials show an 2 appropriate accuracy and precision of the program with the time-temperatures employed. The 3 detection limit and characteristic mass were suitable for the Cr concentrations found. 4

Additionally, the values obtained for the interday repeatability and percentage of recovery of 5 previously added samples with Cr show the adequacy of this technique for determining this 6 element in meals in order to check its total dietary intake as well as its dialyzable fraction. 7

Consequently, it could be used to assess the magnitude of inadequate intakes of Cr 8 (deficiency or excess and toxicity). 9 10

Total Cr and other nutrient intakes and energy levels from duplicate meals of the hospital 11 restaurant 12

The mean Cr intake determined by this method for the three groups of principal meals eaten 13 every day (breakfast, lunch and dinner) are given in table II. The Cr intakes found for 14 breakfasts were significantly lower than those determined for lunch and dinner (p < 0.001). 15 Lunch (43%) is the meal that contributes mainly to the 24-hr Cr DDI followed by dinner 16 (38%) and finally breakfast (19%). By using of the Pearson or Spearman's tests significant 17 correlations among Cr intake levels and supply of several nutrients and energy by the meals 18 analysed (p < 0.05, table III) were found. The corresponding correlation coefficients are also [START_REF]Food and Nutrition Board. Dietary references intakes for 20 vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine[END_REF] collected in this table . 20 There was a positive and linear correlation between total Cr intake levels, and energy, 21 carbohydrates, fibre and protein and several amino acid supplied by the meals (table III). 22 Garcia et al. (2001a) also reported a statistically significant correlation between the presence 23 of Cr and the protein and carbohydrate content of the diet and energy intake. Our findings 24 establish that Cr supply by meals is directly related with macronutrient contents in them. [START_REF] Seaborn | Effect of starch: sucrose, fructose, and glucose on chromium 26 absorption and tissue concentrations in obese and lean mice[END_REF] Consequently, food like cereals and by-products, legumes, dry fruits, meats, potatoes, dairy 26 products and seafood would be primary sources of Cr in the meals as other researchers had 27 previously noted [START_REF] Haro | Chromium content in foods and dietary 17 intake estimation in the Northwest of Mexico[END_REF], Garcia et al. 2001b[START_REF] Santos | Assessment of dialy intake of trace 21 elements due to consumption of foodstuffs by adult inhabitants of Rio de Janeiro city[END_REF][START_REF] Anderson | Minerals[END_REF], 28 Leblanc et al. 2005, Navarro Alarcon et al. 2005). [START_REF] Bratakos | Chromium content of selected Greek foods[END_REF] found that in 29 addition to meat and cereals the importance of vegetables as contributors to Cr dietary intake 30 from Greek foods. Consequently, the food with high carbohydrate and/or protein content 31 would also be a better source for Cr. [START_REF] Van Cauwenbergh | Daily dietary chromium 12 intake in Belgium, using duplicate portion sampling[END_REF] also suggested that Cr 32 intake may increase parallel to energy intake. Garcia et al. (2001b) As it can be checked in table II the mean Cr DDI for the 24-hr meals was 77 ± 17 µg day -1 3 corresponding to the range 46-132 µg Cr day -1 for the 36 consecutive days studied. All daily 4 menus of the restaurant of the hospital considered, supplied element levels higher to the daily 5 adequate intakes (AIs) established by the US IOM (IOM, 2002; 35 and 25 µg Cr day -1 for 19-6 50 y old men and women, respectively). Even when comparing with AIs indicated for the 7 physiological periods of pregnancy and lactation (AIs: 30 and 45 µg Cr day -1 for 31-50 y old 8 women, respectively) the Cr supplied by all daily menus studied were higher than them. For 9 the Cr no upper levels (ULs) defined as the maximum level of daily Cr intake that is likely to 10 pose risk of adverse effects, have been noted by the IOM. Nevertheless, the US National 11 Research Council (NRC) established a daily intake recommended for Cr 3+ (main form of Cr 12 presents in consumed food in meals with low toxic effects) between ≤50-200 µg day -1 . In our 13 study the 24-hr daily menus that supplied the highest Cr DDI contributed with a 66% to the 14 upper level noted by the NRC. Consequently, no adverse effects in relation to Cr nutrition 15 (deficiency or toxicity) are present in the individuals that usually intake the meals of the 16 restaurant of the Motril Hospital (SE Spain), namely staff, users of ambulatory services and 17 patients' family. 18

The Cr DDIs previously reported for healthy adults by other researchers in different countries 19 ranged from 23 µg day -1 in individuals from Rio de Janeiro (Brazil) [START_REF] Santos | Assessment of dialy intake of trace 21 elements due to consumption of foodstuffs by adult inhabitants of Rio de Janeiro city[END_REF]) to 20 283 µg day -1 in inhabitants from Ibaraki (Japan) [START_REF] Shiraishi | Dietary intakes of eighteen elements and 40 K in eighteen food categories 29 by Japanese subjects[END_REF]. We can observe a high 21 variability among the range reported. The mean Cr DDI determined in our study (77 ± 17 µg 22 day -1 , table II) is similar to that found by [START_REF] Leblanc | Dietary 29 exposure estimates of 18 elements from the 1st French total diet study[END_REF] in French individuals aged ≥15 23 y (77 µg day -1 ). Taking into consideration the three ranges of daily Cr intakes pointed out by 24 Garcia et al. (2001b) (high: >104 µg day -1 ; moderate: 42-104 µg day -1 ; and low: <42 µg day - 25 1 ), almost all the daily menus evaluated by us correspond to moderate element intake levels. 26 27

Dialyzable Cr levels and Cr dialysabilities from duplicate meals of the hospital restaurant 28

The interpretation of Cr intake is complicated and may indicate higher or lower level of 29 deficiency, "adequacy" and toxicity than intake alone. In this sense, the determination of the 30 element fraction that would be absorbed and transformed into a biologically active form in 31 the organism would be more informative that the measurement of the total mineral content 32 and supply from the daily diet alone. Consequently, in the present study we analyzed the 1 dialyzable Cr fraction from duplicate meals by an in vitro method that simulates the gastro 2 intestinal conditions because as a previous step to be absorbed each mineral has to be present 3 in a soluble form able to cross the intestinal mucosa. The mean Cr amount dialysed in the in 4 vitro study performed was 1.2 ± 1.1 µg meal -1 (table IV) from a total mean content of 26 µg 5

Cr meal -1 . The corresponding mean percentage of Cr dialysed from the total element content 6 of the meals (Cr dialysability) is 4.63% (table IV). 7

In the regression analysis a correlation between the logarithmic data of total and the 8 dialyzable fraction of Cr in the hospital meals (p= 0.020) was found (table III). Additionally, 9 dialyzable Cr levels were linearly correlated with the Cr dialysability of the meals considered 10 (p= 0.000, r= 0.978). The mean dialysed Cr determined was 1.2 ± 1.1 µg meal -1 (table IV) 11 which corresponds to a Cr dialysability of 4.6 ± 3.8% in meals. Contrarily, Nielsen (1994) 12 collected data on different human studies indicating that when the Cr DDI was 10 µg day -1 13 the net absorption was 2%, which diminished with the rise of the Cr content. Mahan and 14 Arlin (1992) observed that when the Cr DDIs were higher than 240 µg day -1 the amount 15 absorbed was constant while the excretion increased proportionally with the element intake. 16 [START_REF] Anderson | Minerals[END_REF] noted that with intakes ≥40 µg day -1 , Cr absorption reaches and remains at a 17 plateau. The differences observed between these human studies and the in vitro study 18 performed by us are related with the endogenous physiological factors of individuals like the 19 status of their bowel mucosa, nutritional status, homeostatic mechanisms, carrier proteins, 20 etc. (Navarro Alarcon et al. 2005). In the present study we only determine the soluble Cr 21 fraction that forms complexes able to dialyze through the dialysis tubing and therefore to be 22 absorbed. 23 The statement that the bioavailability of a single element may depend on interactions with 24 other components, and both competitive and cooperative interdependence, has been reported 25 (Garcia et al. 2001a). In the present study significant correlations among the dialyzable Cr 26 fraction and the levels of other nutrients like protein, Fe, Na, I, F, ascorbic acid, fiber and 27 vitamin A (p < 0.05) were found. Specifically, it has been noted by others that the ascorbic 28 acid stimulates the Cr bioavailability (Seaborn and Stoecker, 1990;Offenbacher, 1994) 29 similarly to the result found by us. This finding could be related with the capacity of the 30 ascorbic acid to facilitate the formation of soluble complexes with Cr 3+ and consequently to 31 stimulate its crossing through the dialysis tubing. 32 In relation to Fe, [START_REF] Anderson | Minerals[END_REF] reported that the deficiency in this element stimulates the 1 net absorption and bioavailability of Cr suggesting that this element shares some similarities 2 with the Fe absorption pathway. In our study a quadratic correlation (p= 0.031) between the 3 Fe and dialyzed Cr levels in meals, has been observed. This result reported that from 0 to 4 ≅7,5 mg Fe meal -1 the dialyzable Cr fraction enhances significantly with the Fe content of 5 meals. Nevertheless, at Fe contents higher than ≅7,5 mg meal -1 the net absorption decreased 6 significantly as was also reported by [START_REF] Anderson | Minerals[END_REF]. 7

We have also found that dialyzed Cr levels were correlated with fiber content of meals 8 by a logarithmic model (p= 0.011). Therefore, the possibility that fiber could facilitate the net 9 absorption of soluble complexes with Cr 3+ through the dialysis tubing should be considered. 10 Anderson (2004) reported that oxalate increases Cr absorption. 11

Despite the results reported in the present study, future research leading to better 12 knowledge of Cr bioavailability from diet using in vivo studies performed in human beings is 13 needed. Although the in vitro studies of bioavailability can be useful for evaluating the 14 percentage of Cr that could be transformed into absorbable forms in the digestive tract and 15 the influence exerted by other nutrients of the diet, they do no exactly reproduce the 16 physiological conditions and are insufficient to enable a comprehensive evaluation of it. 17

Consequently, they should be combined with in vivo studies in order to reach a better 18 understanding of the bioavailability process in the human organism.

  the high-density-lipoproteins (HDL), was also noticed (Navarrro-Alarcon 1 et al. 2005). 2

  Cr in food, body tissues and body fluids, careful and appropriate 1

20 A

 20 Perkin-Elmer 1100B double-beam atomic absorption spectrophotometer equipped with a 21 deuterium background corrector (Perkin-Elmer, Norwalk CT, USA) and a Cr hollow cathode 22 lamp were used together an HGA-700 furnace with pyrolytically-coated graphite tubes and a 23 L'Vov platform. The samples were injected manually with a Pipetman micropipeptte. Argon 24 of 99.999% purity at a flow rate of 300 ml min -1 was used as the internal gas. A Stomacher 25 blender, model 400, of which different parts were Teflon-coated, and Teflon containers were 26 used for sample homogenization. A thermostatic multiplace digestion block (Selecta S.A., 27 Barcelona, Spain) and a Radiometer model 26 pHmeter (Radiometer, Copenhagen N.V., 28 Denmark) were used for in vitro assays. 29Reagents 30Commercially available standard solution of Cr (1,000 µg ml -1 ; Tritisol, Merck, Darmstadt, 31Germany) was used to prepare calibration graphs. Magnesium nitrate was used as chemical 32

  done according to a modification of the method ofGarcia et al. (2001a), which was based in 31 other methods previously developed(Miller et al. 1981;[START_REF] Shen | Estimation of selenium bioavailability from 31 human, cow's goat and sheep milk by an "in vitro" method[END_REF]. To simulate the 32 gastric digestion, a portion of 5 g of homogenized sample was added to 40 ml of deionised 33

  Cr presence and the protein and carbohydrate content in the diet and 1 the energy intake as we found in the present study. 2

19

 19

  Furnace conditions for Cr determination in duplicate meal samples by ETAAS technique

Table II .

 II Mean intake and DDI of total Cr determined by the technique of duplicate portion sampling 1 of meals (breakfast, lunch and dinner) and analysis by ETAAS 2

			Food Additives and Contaminants
	Meal		N	Total Cr (µg)	% of Cr DDI
				Range	Mean ± SD
	Breakfast		36	8.2-26	15 ± 7.0 a	19
	Lunch		36	20-75	33 ± 14	43
	Dinner		36	17-45	29 ± 8.4	38
	24-hr meals (DDI)	36	46-132	77 ± 17	100
	a p < 0.001.	F o r
			P
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			R
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				v i e
				w
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					y
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Table III .

 III Significant correlation coefficients and significance levels among the total and dialyzable 1 Cr levels of the analyzed hospital meals (n= 108) and other nutrients estimated from the food 2 composition tables compiled in the statistics programme Dietsource 2.0 (Novartis)

	3
	Cr form	Nutrient		r	Nutrient	R	Nutrient	R
	Total Cr	Energy		0.736 a	Vitamin E	0.375 c	Protein	0.725 a
		Carbohydrates	0.732 a	Folate	0.411 b	Trp	0.453 b
		Fiber Fe		0.717 a 0.657 a	Vitamin B 12 Monounsaturated acids	0.462 a 0.370 c	Thr Ile	0.451 b 0.473 a
		P		0.527 a	C16:1	0.301 c	Leu	0.448 b
		K		0.502 a	C18.1	0.364 c	Lys	0.453 b
		F		0.357 c	Polyunsaturated acids	0.342 c	Met	0.447 b
	Ascorbic acid Niacin Pyridoxine Vitamin A Dialyzable Cr Na I F o r	0.373 c 0.519 a 0.553 a 0.314 c 0.437 b 0.340 c	C20:5 C22:6 Total cholesterol Ascorbic acid Fiber	0.369 c 0.426 b 0.362 c 0.389 b 0.375 c	Phe Val Ethanol Vitamin A Total Cr	0.462 a 0.462 a 0.351 c 0.414 b 0.345 c
	a p ≤ 0.001.	F	0.300 c P	Protein	0.423 c	Fe	0.390 c
			e
				e r
					R
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Table IV .

 IV Mean levels of dialyzable Cr and Cr dialysabilities determined by the technique of duplicate 1 portion sampling of meals (breakfast, lunch and dinner) and analysis by ETAAS 2

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		Meal	n	Dialyzable Cr (µg)	Percentage of Cr dialysed (Cr
						dialysability)
				Range	Mean ± SD	Range	Mean ± SD
		Breakfast	36	0.001-1.1	0.535 ± 0.405 a	0.006-8.0	3.6 ± 2.6
		Lunch	36	0.003-3.2	1.3 ± 0.956	0.005-12	4.0 ± 3.3
		Dinner	36	0.001-4.2	1.8 ± 1.3	0.006-16	6.3 ± 4.9
		Mean value	36	0.001-4.2	1.2 ± 1.1	0.006-16	4.6 ± 3.8
	3	a p < 0.001.
	4
	5
	6

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Food Additives and Contaminants

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nutrition, & diet therapy, 8 th edn. Philadelphia: Saunders.

Miller D, Shcricker B, Rasmussen R, Campen D. 1981. An in vitro method for estimation of

b p < 0.01.


Torben ; Leth

Udo Jensen

Sisse Fagt

Rikke Andersen

non-alcoholic beverages in Denmark 2005

Keywords: Chromatography -HPLC, Exposure assessment, Health significance, Total diet Additives/Contaminants: Sweeteners Estimated intake, intense sweeteners, Denmark 2005, cyclamate, acesulfame-K, aspartame, saccharin, children

Estimated intake of intense sweeteners from non-alcoholic beverages in Denmark 2005

Introduction

In 2007 the National Food Institute published an investigation from 1999 about intense sweeteners in non-alcoholic beverages showing an estimated intake of cyclamate in 1-3 years old over the ADI value for more than 10% of the children [START_REF] Leth | Estimated intake of intense sweeteners from non-alcoholic beverages in Denmark[END_REF]. Since then the EU on the advice of the European Food Safety Authority (EFSA) and on the behest of several member states has reduced the maximum permitted level (MPL) for cyclamate from 400 mgl -1 to 250 mgl -1 in soft drinks (EU 2004).

The ADIs of sweeteners are: 15 mg kg -1 bw day -1 for acesulfame-K, 40 mg kg -1 bw day -1 for aspartame, 7 mg kg -1 bw day -1 for cyclamate and 5 mg kg -1 bw day for saccharin. Other intense sweeteners like sucralose or neohesperidinedihydrochalcone are not found on the Danish market in 2005. The Danish Dietary Survey [START_REF] Andersen | The Danes dietary habits 1995. Main results[END_REF] provides extensive information on the intake of foodstuffs in a representative part of the population aged 1 to 80 years, and it is so far the only Danish source for information of the food intake of small children. With this survey an estimation of the intake of specific compounds is possible if the level of the compound in all potential foodstuffs is known. The Danish monitoring programme for food additives samples foodstuffs for chemical analysis. The primary objective is to check that the additives are used legally and within the maximum permitted levels, and the results are continuously sent to EU to fulfil our legal duties according to EU directives, but the analyses are also utilized to survey the intake of food additives. Non-alcoholic beverages, which are the major source of intake for intense sweeteners [START_REF] Bergsten | Intake of artificial sweeteners. Acesulfame-K, aspartame, cyclamate and saccharin[END_REF]) and confirmed by the Danish monitoring programme, were monitored in 1999 and again in 2005. In a recent review of intake estimations of intense sweeteners (Renwick 2006) it appears that very few intake estimations based on large representative parts of the population have been carried out and none at all after the reduction in EU of the MPL for cyclamate from 400 to 250 mgl -1 . Thus it was the purpose of this investigation to determine the intake of intense sweeteners with nonalcoholic beverages in different age groups of the Danish population based on the monitoring results from 2005 and to compare the results with the similar investigation based on the results from 1999 [START_REF] Leth | Estimated intake of intense sweeteners from non-alcoholic beverages in Denmark[END_REF].

Materials and methods

Monitoring programme

In 2005, the project "Monitoring of additives: Intense sweeteners, colouring agents and preservatives in non-alcoholic beverages" was carried out as part of the Danish Veterinary and Food Administrations programme for monitoring of additives [START_REF] Jensen | Monitoring and control of food additives: Intense sweeteners, colouring agents and preservatives in non-alcoholic beverages[END_REF]. A total of 177 samples were taken by the local authorities all over Denmark from producers, importers and wholesalers from June 2005 to July 2006, and the samples were stored in a refrigerator at 4ºC until analysis. All the samples were analysed, but only 76 samples had a declared content of intense sweeteners, so-called "light" soft drinks, with 56 samples in the category of light soft drinks with carbon dioxide and 20 samples in the category of light soft drinks without carbon dioxide. The 101 sugar sweetened soft drinks with and without carbon dioxide and without a declared content of intense sweeteners were also analysed. Apart from the intense sweeteners the samples were also analysed for the preservatives sorbic and benzoic acid, coffein and synthetic coulorants, if there presence were suspected. However these results will not be presented here. A total of 142 samples were of Danish origin and 35 were imported.

Chemical analysis

The chemical analysis for acesulfame-K, aspartame, cyclamate and saccharin was carried as described earlier [START_REF] Leth | Estimated intake of intense sweeteners from non-alcoholic beverages in Denmark[END_REF]. In short the analytical samples were diluted, if necessary, with deionised water to bring the concentrations of intense sweeteners within the calibration range of the method. Acesulfame-K, aspartame and saccharin were separated by HPLC on a C-18 column and measured at 220 nm, while cyclamate was derivatized with sodium hypochlorite to N,Ndichlorocyclohexylamine, separated by HPLC on a C-18 column and measured at 314 nm. The laboratory was accredited to perform these methods according to ISO 17025 (ISO 2005). Also the quality control was carried out as described earlier [START_REF] Leth | Estimated intake of intense sweeteners from non-alcoholic beverages in Denmark[END_REF]) with a duplicate and a recovery experiment in each series of analysis. The results showed that the methods had been under control at all times with no values exceeding the control limits for double determinations or for recoveries.

Dietary survey

The survey was conducted in 1995 on a random sample of Danes aged from 1 to 80 years drawn from the Central Populations Register and stratified by sex and age [START_REF] Andersen | The Danes dietary habits 1995. Main results[END_REF] as described earlier [START_REF] Leth | Estimated intake of intense sweeteners from non-alcoholic beverages in Denmark[END_REF]. Dietary data was based on recording on 7 consecutive days. The questionnaire included 20 beverages (e.g. different types of milk, beer, wine soft drinks and fruit juices). Sweetened beverages included two types of beverages with carbon dioxide: soft drinks "light" and soft drinks sugar sweetened; and two types of beverages without carbon dioxide: soft drinks "light" and soft drinks sugar sweetened including fruit juices. This survey, although more than 10 years old, is the only Danish survey with data for the most critical group of 1-3 years old children and must therefore be used again for the intake estimation.

Estimated intake calculation

The 177 samples of beverages analysed were aggregated into four different types of beverages for which intake data was present: with carbon dioxide, light (average intake 24 mlday -1 ) and sugar sweetened (average intake 108 mlday -1 ) and without carbon dioxide, light (average intake 71 mlday - 1 ) and sugar sweetened (average intake 136 mlday -1 ). For each person the registered average daily intake of the four categories was multiplied with the content of intense sweetener, and a calculation of basic statistic parameters of the intake distribution was carried out in the form of mean, standard error of mean (not shown in the paper) and percentiles including the median with the persons divided in age groups, males, females and the total population. For the content of intense sweetener was used two scenarios: One with the analytical determined mean value for all the light samples with content of the specific sweetener called "high intake" to simulate situations for brand-loyal consumers and one with the analytical determined mean value for all the light samples in the category called "average intake", which will be lower than "high intake", since many of the light samples were not sweetened with all the four artificial sweeteners. The variability in the levels of sweeteners inside the four categories of soft drinks has not been used in the estimated intake calculation.

Results

Concentration of intense sweeteners in non-alcoholic beverages

Insert table 1 here

Table I gives the average and range of sweeteners in the light categories with and without carbon dioxide and the number of samples in each category, both average of all the samples in the category for the average intake estimation, and the average of only the samples with content of the specific sweetener for high intake estimation.

It could be assumed that the reduction in the use of cyclamate will lead to increased use of the other intense sweeteners. The development from 1999 to 2005 is also illustrated in table I for light soft drinks without and with carbon dioxide. No illegal use of the four examined sweeteners was found, that is sweeteners used in food for which they are not allowed, and the contents for cyclamate as well as for the other sweeteners exceeded in no cases the maximum permitted levels shown in table I. Only three faulty declarations for sweeteners were found (4% of the samples), two samples with an intense sweetener declared but not present and one sample with an intense sweetener not declared but present, compared to 19 faults in 1999 (23% of the samples).

No sugar sweetened soft drinks contained intense sweeteners, which means, that the two sugar sweetened categories, soft drinks with and without carbon dioxide, do not contribute to the intake of intense sweeteners in contrast to the 1999 investigation, where a few sugar sweetened samples also contained aspartame and acesulfame-K.

Pattern of intense sweetener use in non-alcoholic beverages

Insert figure 1 and2 here.

The sum of all the intense sweeteners in the light products is shown in Figures 1 and2 for soft drinks without carbon dioxide and with carbon dioxide, respectively. When used at all, cyclamate is most often added in amounts over 150 mgl -1 , however the content does not exceed the MPL of 250 mgl -1 in any sample, and average values are about 100 mgl -1 lower than in 1999. For the other sweeteners the content is usually much lower than the maximum permitted levels. To replace the cyclamate the producers have used both acesulfame-K and aspartame in soft drinks without carbon dioxide. However, in soft drinks with carbon dioxide only the content of aspartame increases,while acesulfame-K decreases, but the levels of both aspartame and acesulfame-K are still much higher in soft drinks with carbon dioxide than in soft drinks without carbon dioxide (see table I).

In soft drinks without carbon dioxide cyclamate is still like in 1999 the dominating sweetener. In soft drinks with carbon dioxide, a mixture of aspartame and acesulfame-K is more often used to sweeten the products. Cyclamate is not used by the large soft drinks companies like Coca-cola, Pepsi-cola and Faxe.

Estimated intake of intense sweeteners from non-alcoholic beverages

Two intake calculations were carried out. One using an average only of the samples with a content of the specific sweetener called high intake (Figure 3) in light soft drinks with (n=29) and without (n=12) carbon dioxide, and another with an average of all samples in light soft drinks with (n=56) and without (n=20) carbon dioxide called average intake (Figure 4). Estimated intake in mgkg - 1 bwday -1 was calculated from an average for each age group of the actual body weights registered during the food survey.

Insert Fig. 3 and 4 about here.

Both high intake and average intake of all four sweeteners are shown in

Discussion

To take into account the possibility of people being faithful towards their usual brands [START_REF] Leclercq | Stochastic modelling of human exposure to food chemicals and nutrients within the Montecarlo project. An exploration of the influence of brand loyalty and market share on intake estimates of intense sweeteners from sugar-free soft drinks[END_REF] we have found it relevant to carry out a high intake calculation by averaging only the samples with the analytical determined content of intense sweeteners. Even higher estimated intakes could be found under the assumption that a person only drinks a beverage with the highest found content of intense sweetener, but that would be a different brand for each of the four sweeteners and therefore not a very likely scenario. However, it could also be argued that the average for all samples in each of the four categories of beverages is a more reasonable figure to put into the intake calculation, so that extremes do not dominate the intake calculation. Therefore this calculation has also been carried out.

The dietary survey used for the intake estimation is from 1995, as this is the only Danish data available for the small children of 1-3 years old, which was shown to be the most critical group in the 1999 investigation. A new dietary survey for children is under way in the National Food Institute but with no data yet. It can not be excluded that the pattern of soft drink intake has changed over the last 10 years, for instance with higher intake of soft drinks with intense sweeteners in view of the often discussed increasing obesity in the society. However, unpublished data from our new Dietary Survey 2000-2004 show in the group 4-6 years old a small increase in the average intake of light soft drinks with carbon dioxide from 12 to 18 mlday -1 but a much larger decrease for light soft drinks without carbon dioxide from 115 to 75 mlday -1 suggesting an overall decrease in the intake of intense sweeteners. A similar development could be assumed for the group 1-3 years old as a result of a health-wave now leading to an overall increase in the intake of non-sweetened juice. The results for cyclamate are presented in Figure 3 and4 and for all the sweeteners in Table II. The difference between the estimated high and average intake is not so great, since especially in soft drinks without carbon dioxide, which is responsible for 75% of the intake of artificially sweetened soft drinks, a mixture of the sweeteners is often used. In soft drinks with carbon dioxide a mixture of only aspartame and acesulfame-K is more often used. In 2005 no sugar sweetened soft drinks contained intense sweeteners, which has caused the high intake of especially aspartame to be decreased significantly compared to 1999, where just one sugar sweetened sample with aspartame got to represent the whole category in the intake calculations [START_REF] Leth | Estimated intake of intense sweeteners from non-alcoholic beverages in Denmark[END_REF]). The 99 th percentile of the estimated intakes of acesulfame-K, aspartame and saccharin are as in 1999 far below the ADI values and give no cause for alarm.

After the reduction of the MPL for soft drinks from 400 to 250 mgl -1 the high estimated intake of cyclamate is almost halved compared with 1999. Only in the group of 1-3 year olds is the 99 th percentile of the intake just over the ADI value of 7 mgkg -1 bwday -1 . For all the groups the 90 th percentile is half or less of the ADI value (see figure 3). For the average calculation of the estimated intake is also the 99 th percentile well below the ADI value in all the groups (see figure 4). The child with the highest cyclamate intake gets 9.5 mgkg -1 bwday -1 in contrast to 18 mg in 1999. For cyclamate the estimated intake is still closer to the ADI value and in a few cases even over the ADI value, than for any of the other intense sweeteners used in soft drinks.

As in 1999 boys 7-10 year old have a significantly higher intake of cyclamate and the other intense sweeteners than girls in the same age group. With the same dietary survey used for the intake calculation it is not so surprising that the same conclusion as in 1999 is reached, although the intake in 2005 has decreased for both boys and girls.

The other significant source of intense sweeteners is tabletop sweeteners. High consumers of cyclamate based tabletop sweeteners ate 230 mg/day corresponding to 35% of the ADI [START_REF] Bergsten | Intake of artificial sweeteners. Acesulfame-K, aspartame, cyclamate and saccharin[END_REF]). However, tabletop sweeteners are typically not used by healthy children, and will therefore not increase the cyclamate intake in children to any large extent.

Thus, it has been demonstrated with this first publication of intake data for cyclamate after the reduction of the MPL value from 400 to 250 mgl -1 that the estimated intake of cyclamate for small children is now decreased to a level well below the ADI value. However, the Codex still recommends 400 mgl -1 and an ADI of 11 mgkg -1 bwday -1 (Codex 2006), so the cyclamate intake outside EU probably remains high.

Conclusions

In an investigation from 2005 on 177 samples of non-alcoholic beverages, 76 of these light soft drinks with content of intense sweeteners, it was demonstrated that the estimated intake of cyclamate after reduction of the maximum permitted level in EU from 400 to 250 mgl -1 in soft drinks was decreased to a level well below the ADI value also in small children with only the 99 th percentile for 1-3 year olds a little over the ADI value of 7 mgkg -1 bwday -1 . The estimated intake of acesulfame-K, aspartame and saccharin was far below their respective ADI values. I. Intense sweeteners in light non-alcoholic beverages: Average concentration levels, (average from 1999 99 ), (range) and (number of samples) in each category Table II. Average and high intake estimation of intense sweeteners in the whole population and in children from 1 to 10 years (mgkg -1 bwday -1 ), 50 th , 90 th and 99 th percentiles. Data for cyclamate corresponds to data in figure 3 and4.

  R. 2006. The intake of intense sweeteners -an update review. Food Additives and Contaminants 23:327-338.

Figure 1 .

 1 Figure 1. Intense sweeteners in light soft drinks without carbon dioxide Figure 2. Intense sweeteners in light soft drinks with carbon dioxide Figure 3. High estimated intake of cyclamate from light non-alcoholic beverages -males and females Figure 4. Average estimated intake of cyclamate from light non-alcoholic beverages -males and females

F

  Table II for all persons, four of the age groups of the intake distribution and for boys and girls in one age group based on the values from table I. Only the intake for boys and girls, and for the whole group of 1-80 year olds males and females for comparison, is shown in the table, as it is only for young people with low bodyweight that the intake appears high compared to ADI values.

	F o r
	P
	e
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	R
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	v i e
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	Insert tableII here.

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	Sex	Age	Percen-	Acesulfame-K,	Aspartame,	Cyclamate,	Saccharin,
		(years)	tile	average and high	average and high	average and high	average and high
	Soft drinks		Acesulfame-K (mgkg -1 bwday -1 )	Aspartame (mgkg -1 bwday -1 )	Cyclamate (mgkg -1 bwday -1 )	Saccharin (mgkg -1 bwday -1 )
	All	1-80	50		(mgl -1 ) 0.02			(mgl -1 ) 0.03	(mgl -1 ) 0.04	(mgl -1 ) 0.01
	With carbon dioxide, light		48 (49 99 ) 0.04		197 (152 99 ) 0.04	60 (152 99 ) 0.06	12 (15 99 ) 0.01
	All samples	90		0.35			0.52	0.78	0.10
	n = 56				0.60			0.64	0.93	0.17
			99		1.35			1.77	2.65	0.40
	With carbon dioxide, light		93 (129 99 ) 2.34		212 (159 99 ) 2.11	151 (245 99 ) 3.58	24 (29 99 ) 0.64
	Samples with sweetener		(17 -166)		(31 -560)	(21 -240)	(13 -60)
	F o r No carbon dioxide, light All samples n = 20 All 1-3 50 F 90 o r 99	(n = 29) 65 (42 99 ) 0.35 0.60 1.40 2.42 2.79		(n = 52) 64 (36 99 ) 0.40 0.50 1.61 2.08 3.26	(n = 22) 132 (152 99 ) 0.60 0.91 2.42 3.68 4.88	(n = 27) 19 (26 99 ) 0.11 0.16 0.41 0.65 0.85
	No carbon dioxide, light Samples with sweetener Maximum permitted level MPL All 4-6 50 90 99	P e e r 108 (62 99 ) (35 -260) (n = 12) 350 4.83 P 0.24 e 0.42 1.14 e 1.96 r 2.30	85 (56 99 ) (11 -310) (n = 15) 600 4.28 0.29 0.40 1.43 1.76 2.80	165 (333 99 ) (87 -225) (n = 16) 250 7.37 0.44 0.64 2.14 2.99 4.19*	31 (37 99 ) (10 -54) (n = 12) 80 1.30 0.07 0.11 0.35 0.53 0.67
	Boys Girls	7-10 7-10	50 90 99 50		3.98 0.25 0.43 1.12 1.93 2.27 3.94 0.20	R e v i e w 3.81 R 0.29 e 0.40 1.22 v 1.62 i 2.63 e 3.37 w 0.26	6.09# 0.53 0.66 2.45* 2.95* 4.95* 6.00* 0.45	1.09 0.07 0.11 0.34 0.52 0.69 1.05 0.06
			90 99		0.35 0.83 1.45 1.35 2.34			0.34 1.02 1.30 1.73 2.21	O n l 0.53 1.92* 2.22* O 2.98* n l 3.59*	0.10 0.26 0.40 0.40 0.64
	ADI				15			40	y y 7	2.5
	*The t-test shows a significant difference between boys and girls at the 99.9% probability level with
	highest intake for boys.
	#The t-test shows a significant difference between boys and girls at the 95% probability level with
	highest intake for girls.

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