Iron Dialyzability in a Multiple Nutrient Formulation and Effect of the Addition of Separate Nutrients

Introduction: Different nutrients added to nutritional formulations can facilitate, reduce or b lock the iron absorption. Objective: The aims of this study was to assess the modulation of iron bioavailabily by other minerals and vitamins in an multip le nutrient formulation and in an aqueous iron solution. Material and Methods: We analyzed the iron dialyzability in the multiple nutrients formulation developed to simulate an enteral diet. Furthermore, the iron dialyzability was determined in aqueous solutions containing 25 mg of iron, which were added separately usual amounts of soluble fiber, salt mixture, vitamin mixture, calcium and v itamin C. The d ialysed iron was measured by atomic absorption spectrometry. Results: In the multip le nutrients formulation, we documented low bioavailab ility of iron (0.80 ± 0.01%). Compared with the aqueous iron solution (70.0 ± 6.0%), the addit ion of 135 mg of vitamin C increased the iron d ialyzability (90.0 ± 3.0%). There was reduction of iron dialysis after addit ion of soluble fiber (1.00 ± 0.01%), the v itamin mixture (25.00 ± 0.12%), salt mixture (2.00 ± 0.06%) and calcium (0.80 ± 0.02%). Conclusion: The low iron bioavailability in the multip le nutrients formulation can be attributed to the protein source and supply of fiber and calcium, thereby affecting the absorption of iron.


Introduction
Iron absorption is affected by body iron stores, by the chemical structure of the salt, and by dietary factors (1) which have positive or negative effects on iron bioavailability (2,3). The solubility, dialyses, and entry/transport of iron by Caco-2 cells are in vitro methods used to assess the addition of nutrients on bioavailability of iron. In vitro dialyzability analysis the iron dialyzes through a semi-permeab le memb rane after simulat ion of gastric and duodenal digestion (4) represents a methodological option for the assessment of iron bioavailability after the addition of different nutrients. This methodology may provide useful information on iron b ioavailab ility in foods and dietary products (5), like oral supplement and enteral diet.
Patients unable to consume sufficient quantities of food to satisfy their nutrient requirements need oral supplement or enteral nutrition (6). The possible interactions between the components of the formu la may alter the bioavailab ility of specific nutrients and interfere with the clin ical course of sick individuals. The aims of this study was to assess the modu lation of iron bioavailab ily by other minerals and vitamins in an mult iple nutrient formu lation and in an iron

Preparati on of The Experi mental Formula and of the Aqueous Solutions
We prepared a mult iple nutrient formu lation that would reproduce the nutrient composition of products used for oral supplementation or poly meric enteral d iet (Tab le 1). A ll components (protein soy isolate, maltodext rin, canola and corn oils, soy lecithin, part ially hydroly zed guar gum, and a mixtu re of minerals and vitamins) used to prepare the formu lation were purchased. In parallel, we p repared an aqueous ferrous sulphate solution containing 25 mg elemental iron to which the fo llo wing nutrients were later added: partially hydroly zed guar gum (25 g); salt mixture (3 g); vitamin mixtu re (10 g); calciu m (800 mg); and vitamin C (135 mg ). A total volu me of 250 mL and an iron concentration of 25 mg were kept constant regardless of the nutritional co mposition of the enteral formu la or aqueous iron solution.

Digestion and Dialyzability of the Samples
The in vitro bioavailability of iron in the samples was determined by the method of Miller and others (7) and modified by Luten and others (8). For the simulat ion of the digestive process, a 250mL samp le of the mu ltip le nutrient and Effect of the Addition of Separate Nutrients formulat ion was ho mogenized and 6 N HCl was added until a pH value of 2 was reached. Five 20g aliquots were separated and pepsin was added at the proportion of 0.125 g/g protein. The solution was incubated at 37 °C in a water bath with shaking for 2 h. Finally, t itration with 0.5 N KOH was performed up to p H 7. A sodiu m b icarbonate solution was prepared and added to the dialysis tube until pH 5 was reached after 30 min under constant shaking. The pancreatin-bile solution was then prepared at the proportion of 25 mg pancreatin/g protein in the sample and of 0.4 g pancreatin/2.5 g bile extract. The pancreatin-bile solution (4 mL) was added to 3 beakers containing 20 g of the digest and the mixture was incubated in a water bath with shaking for 2 h.
The process was finalized by removing the dialysis tubes fro m the solutions and the content of the beakers was transferred to a 25mL volu metric round-botton flask and reconstituted to its final volu me with deionized water. For the samples of aqueous solutions containing 25.0 mg iron, only pH control was performed by acidification and neutralization with the reagents used in the method, without the addition of digestive enzy mes.
For the evaluation o f iron d ialy zability, 20 g of the d igest or of the aqueous solutions was placed in a beaker together with the d ialysis tube previously hydrated in deionized water for 10 min and filled with 25 mL o f an NaHCO 3 solution. The flasks were covered and kept in a water bath at 37 °C with shaking fo r 30 min. Four mL of the bile-pancreatin suspension was added to each flask and incubation was continued for 2 additional hours. At the end of the incubation period the dialy zed content was transferred to volumetric balloons and deionized water was added to complete the volume to 25 mL, followed by storage in a freezer at -20 º C until the time for reading.

Determination of Total and Di alyzed iron
For the determination of total iron in the aqueous solutions and in the various formu lations tested, 2 g samples were obtained and digested with nitric acid (HNO 3 ) and hydrogen peroxide (H 2 O 2 ) at a 5:1 proportion at 100 ºC in a block digestor (Pyrotec ® ). The material was diluted with deionized water in a 50 mL round-botton flask. The analyses were performed using a Sh imadzu® ato mic absorption spectrophotometer model AA 6200 (Shimadzu Corporation, Tokio, Japan) with an air/acetylene o xidant under the following conditions: hollow cathode lamp, 248.3 wavelength for iron and a 0.2 n m slit. The solutions for the standard iron curve were prepared with Tritisol ferric chloride (Merck  9972) at concentrations of 0.5, 2.0, and 4.0 µgFe/ mL. A ll determinations were carried out in triplicate and data are reported as means + SD.
Iron dialy zability was estimated as the proportion of dialyzed iron in relat ion to iron concentration at the beginning of the in vitro d igestion process after a period of equilibriu m through the dialysis membrane. Table 2 shows that percent dialyzed iron was very low in the mu ltip le nutrient formu lation (0.80 ± 0.01%), indicating a possible impairment of iron b ioavailability in products used in enteral formu la, like this mult iple nutrient formu lation. In aqueous solutions, iron dialy zability was facilitated by the addition of ascorbic acid (90.00 ± 3.00%), and was reduced by the addition of guar gum (1.00 ± 0.01%), and calciu m carbonate (0.80 ± 0.02%).

Discussion
In the present study, iron dialyzability in a mu ltip le nutrient formu lation was very lo w (0.80 ± 0.01%). This study's results confirm the inhib itory effect of calciu m and of soluble fiber on iron bioavailability assessed by the method of in vitro dialy zability.
Ascorbic acid has long been known to enhance absorption of iron fro m test meals (9). Despite the presence of vitamin C, the iron dialyzab ility was lo w fro m mu ltip le nutrient supplements, indicating that other factors were harming the bioavailability of iron. Calciu m carbonate was used in the present study, that possibly determined the format ion of insoluble co mp lexes with iron, exp laining the inhibitory effect of the addition of calciu m on the iron dialyzab ility (10,11). A negative effect on iron absorption has been documented with calciu m phosphate (12) and calciu m carbonate (13) supplementation, although this effect did not occur in a radio isotope study on humans (14) or in experiments of iron dialy zability (15).   In vitro methods are relat ively simp le, rapid and inexpensive and can simulating the digestion gastric and duodenal, followed by dialysis. The proportion of the element diffused through the semi permeable membrane during the process, is the dialysability element after an equilibration period, being used as an estimate of nutrient bioavailability (7)(8).
In a collaborative study to compare the methods using in vitro and in vivo to assess the absorption of non-heme iron, found a statistically significant correlation indicating that the results obtained using the method in vitro can be extrapolated to humans (8).
In vitro studies have shown that iron absorption from breakfast cereals was reduced after the addition of fibers (16) and also in foods such wheat, rice, corn and soy (17). In contrast to these findings, rats fed with partially hydrolyzed guar gum presented greater intestinal iron absorption than rats receiving a diet without the addition of fiber (18). In a study on healthy humans, the daily addition of 40 g soluble fiber to a mixed diet did not alter the apparent absorption of iron (17). The discordance in the literature regarding the effect of fibers on iron bioavailability may be attributed to differences in the potential of soluble or insoluble fibers to form precip itate that limit iron absorption. There is also the hypothesis that the consumption of soluble fiber will increase the bacterial flora, resulting in a stimu lus of iron absorption by the colonic mucosa (18,19) and explaining the difference between in vitro and in vivo studies. Thus, when the content of soluble fiber is modified in formu lations, it is possible that in vitro methods will not be feasible for the assessment of iron bioavailability.
This multip le nutrit ional formu lation was studied because is similar to enteral nutrition who co mposited by multip le nutrients and offered to people in the same t ime.
In various commercial enteral diet products, percent dialyzed iron ranged fro m 2.34 to 9.67 % according to the presence of fiber and to the calciu m-iron proportion (20). The study of optimization of d ialy zed iron in a mu ltip le nutrient solution showed that the ideal formu lation for a ma ximu m o f 7 % d ialy zability required 10 mg/ L iron, 10 g/L fiber, 320 mg/ L calciu m and no addition of medium-chain triacylg lycerides (21).
In the present study, the highest concentration of nutrients of the solution can justify poor results of both the iron dialy zabilidade formu la with mult iple nutrients and in aqueous solution of ferrous sulfate. The enteral formu la used in this study contains soy protein extract, wh ich is known to decrease iron absorption (22)(23)(24). The inhibitory effect of soybean products is thought to be due to the protein component (7) than phytic acid (25).
Cook et al. (14) assessed the effect of calciu m salts commonly used as supplements on iron absorption when administered during the interval between meals and observed that calcium carbonate at the dose of 600 mg did not inhibit the absorption of ferrous sulfate (18 mg), at an iron/calciu m proportion of 1:33). When the same assays were repeated using citrate and phosphate salts as a source of calciu m at the same concentrations, iron absorption was reduced to 44 % and 62 %, respectively, showing that the type of salts used can also affect the bioavailability of minerals. Reddy & Cook (26) observed that different iron/calciu m proportions (above 1:40) and the types of salt sources of the minerals interfere with the bioavailability of iron.
Yoon et al. (27) d iscussed the possibility of fiber acting on the human gastrointestinal tract by causing changes in the utilization of nutrients and showed that greater amounts of fiber (> 20 g/day) can affect the b ioavailability of minerals. The supplements studied here contained 25 g fiber that may have represented a factor capable of reducing iron absorption.
Nu merous interactions exist between the different trace elements affecting absorption via the gastrointestinal tract. Factors affecting bioavailability of trace elements include the actual chemical form of the nutrient (eg, organic fo rm of iron is better absorbed than the ionic form), antagonistic ligands (eg, zinc absorption is decreased by phytate and fiber; iron absorption is decreased by fiber), facilitatory ligands (eg, zinc absorption is aided by citric acid or iron absorption is increate by amino acids or fermented products ), and competitive interactions (eg, iron depresses the absorption of copper, and zinc; zinc depresses copper absorption and vice versa) (28).
Studies evaluating the effect of different protein, fiber and calciu m sources on iron dialy zability in mu ltip le nutrition formulation are needed in order to provide an adequate nutrition supply without a reduction in iron bioavailability. The iron bioavailability in a mu ltip le nutrient solution proved to be low, a fact that may harm the health of individuals who use enteral nutrition.