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Human Nutrition and Metabolism
Research Communication

Dephytinization of a Complementary Food Based on Wheat and Soy Increases Zinc, but Not Copper, Apparent Absorption in Adults¹

Laboratory for Human Nutrition, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology Zurich, 8803 Rüschlikon, Switzerland

²To whom correspondence should be addressed. E-mail: lena.davidsson{at}ilw.agrl.ethz.ch.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Complementary foods based on cereals may contain high amounts of phytic acid, which binds strongly to minerals and trace elements. The objective of the study was to evaluate the effect of dephytinization of a cereal-based complementary food on zinc and copper apparent absorption in adults. A dephytinized complementary food (<0.03 mg phytic acid/g) and one containing the native phytic acid concentration (4 mg/g) were labeled extrinsically with stable isotopes (⁷⁰Zn and ⁶⁵Cu). Apparent zinc and copper absorption was based on fecal excretion of nonabsorbed labels in 9 adults, using a crossover design. Stable isotopes were quantified by thermal ionization MS. Apparent fractional zinc absorption was significantly higher (P = 0.005; Student’s paired t test) from the dephytinized complementary food (34.6 ± 8.0%; mean ± SD) than from the complementary food with native phytic acid concentration (22.8 ± 8.8%). Apparent fractional copper absorption did not differ (P = 0.167; 19.7 ± 5.1% dephytinized vs. 23.7 ± 8.1% native phytic acid). These results clearly demonstrate the beneficial effect of dephytinization of a complementary food on fractional absorption of zinc but not of copper in adults. The long-term nutritional benefits of dephytinization of complementary foods should be evaluated in young children.

KEY WORDS: • zinc • copper • phytic acid • stable isotopes • complementary food

Complementary foods based on cereals are often one of the first semisolid foods introduced into the diet of infants. To improve protein quality, cereals are commonly combined with milk or legumes. However, both cereals and legumes contain relatively high amounts of phytic acid, a compound that binds strongly to nutritionally essential minerals and trace elements and that can impair their bioavailability. The negative influence of phytic acid on iron absorption was clearly demonstrated in both adults (1–3) and in infants (4). Phytic acid can also inhibit zinc absorption, but the evidence is less conclusive than for iron (5–8). The information available concerning the influence of phytic acid on copper absorption is very limited, and data based on animal and human studies report contradictory results (9–12).

Adequate absorption of minerals and trace elements is of special importance during periods of rapid growth and development. Strategies to improve the bioavailability of minerals and trace elements from foods consumed by infants and young children, in particular from complementary foods based on cereals and legumes with high concentration of phytic acid, are urgently needed. Recently, a novel approach to produce dephytinized complementary foods was developed, based on the utilization of the phytic acid–degrading enzyme phytase naturally occurring in whole-grain cereals (13,14). The aim of the present study was to evaluate the effect of dephytinization of a complementary food based on wheat and soy on zinc and copper apparent absorption in healthy adults. Zinc and copper apparent absorption was measured by a stable isotope technique, based on fecal monitoring.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
All chemical and reagents were of analytical grade quality. For trace element analysis, all tubes and containers were acid-washed. Water was purified by reverse osmosis (Nanopure Cartridge System, Skan AG) for laboratory analysis, preparation of test meals, and drinking water.

    Study subjects. Apparently healthy Caucasian premenopausal women (n = 8) and adult men (n = 2) were recruited in Zurich, Switzerland. Exclusion criteria included self-reported pregnancy, lactation, gastrointestinal disorders, and metabolic or chronic diseases. No medication (except for oral contraceptives) or vitamin, mineral, or trace element supplements were allowed 2 wk before the start of the study and during the entire study. The aims and procedures of the study were explained orally and in writing, and written informed consent was obtained from each subject. The study protocol was approved by the Ethical Committee of the Swiss Federal Institute of Technology Zurich.

    Production and analysis of complementary foods. Complementary foods based on wheat and soy were produced at the Nestlé Product Technology Centre, Orbe, Switzerland following good manufacturing practice. The complementary foods were based on 70% wheat flour (low extraction rate), 20% soy flour (from dehulled soybeans), and 10% whole-grain wheat flour. One batch was produced to contain the native phytic acid concentration of the ingredients, whereas the other batch was dephytinized, using a newly developed methodology (13,14). The microbiological safety of the complementary foods was ensured before release from the factory. The phytic acid concentration was determined in triplicate by an HPLC technique (15,16) with modifications (17) and expressed as the sum of pentainositol phosphate and hexainositol phosphate. After mineralization in a nitric acid-hydrogen peroxide mixture using microwave digestion (MLS 1200 MEGA, MLS GmbH), zinc and calcium concentrations were determined by flame atomic absorption spectrometry with a standard addition technique, and copper and iron concentration by the atomic absorption spectrometry graphite furnace technique with external calibration (SpectrAA-400 and GTA-96 graphite furnace atomizer, Varian, Techtron Pty.). The CV based on triplicate analysis was <7% for mineral and trace element determinations. Protein concentration was based on analysis of nitrogen using a standard Kjeldahl technique and applying the conversion factor 5.7.

    Stable isotope labels. Elemental zinc highly enriched in ⁷⁰Zn (73.7%) and elemental copper highly enriched in ⁶⁵Cu (99.2%) were purchased from Chemgas. ⁷⁰Zn was dissolved in concentrated HCl and diluted with water to a final concentration of 0.8 mg/g in 0.1 mol/L HCl. ⁶⁵Cu was dissolved in concentrated nitric acid and transformed into its chloride form by fuming with concentrated HCl. The solution was diluted with water to a final concentration of 1 mg/g in 0.1 mol/L HCl. The isotopic composition of the isotopic labels was determined by thermal ionization MS (MAT 262, Finnigan MAT). The zinc and copper concentrations of the isotopic labels were determined by reversed isotope dilution MS. Elemental concentrations of zinc were determined against a commercial standard (Titrisol, Merck Darmstadt). For the characterization of the copper isotopic label, a standard was prepared gravimetrically from an isotopic reference material in elemental form (SRM 976, National Institute of Standards and Technology).

    Test meals. The test meals were prepared by mixing 40 g dry complementary food (native phytic acid or dephytinized) with 5 g sugar and 300 g hot water. The test meals were labeled extrinsically with stable isotope solutions (0.4 mg ⁷⁰Zn and 0.5 mg ⁶⁵Cu) and were administered immediately after preparation. Cold water (200 g) was served as a drink with all test meals.

    Study protocol. A crossover design was used. The first test meal was randomly allocated, either complementary food with the native phytic acid concentration or dephytinized complementary food. The two study periods were separated by a time lapse of 2–4 wk. Women started each study period immediately after a menstrual cycle. On d 1, body weight and height were measured and a venous blood sample was drawn into an acid-washed glass tube after an overnight fast. Fecal markers (100 mg brilliant blue in a gelatin capsule, Apoteksbolaget) and 1.5 mg dysprosium [dysprosium(III) chloride hexahydrate, Sigma-Aldrich] dissolved in 150 g water] were administered on d 1 to avoid any potential interaction of dysprosium and phytic acid. On d 2, after the subjects fasted overnight, labeled test meals were administered in the morning and again 4 h later. No food or drink was allowed between intake of the two identical test meals and 4 h after intake of the second test meal. A standardized dinner and drinking water were provided on d 2. At all other times, the subjects consumed their normal self-selected diet. The complete collection of fecal material started immediately after intake of the fecal markers on d 1 (brilliant blue and dysprosium) and continued until the excretion of the second fecal marker (brilliant blue), administered 7 d later was completed.

    Blood analysis. Serum was separated from blood cells collected on d 1 within 3 h and analyzed for zinc and copper concentration using atomic absorption spectrometry (SpectrAA-400 and GTA-96 graphite furnace atomizer). Accuracy of the determinations was verified by analyzing a reference material, Seronorm trace element serum (Nycomed Pharma SA) in parallel. The CV based on triplicate determinations of zinc was <10% and <5% for copper.

    Analysis of feces. Fecal samples were freeze-dried, homogenized, and dried to a constant weight. Dried fecal samples (including the first fecal sample dyed by brilliant blue and including all samples before the appearance of the second dose of brilliant blue) were milled (0.75-mm mesh, centrifugal mill, Retsch GmbH), pooled, and dried to constant weight. Pooled fecal samples were mineralized using a nitric acid-hydrogen peroxide mixture by microwave digestion (MLS 1200 MEGA, MLS GmbH) after addition of an aqueous ⁶⁷Zn isotopic label. ⁶⁷Zn isotopic label was added to determine the amount of natural zinc in the sample based on isotope dilution principles including natural zinc introduced during sample preparation. Each sample was mineralized in duplicate. Together with each series of samples, 2 blanks containing known amounts of ⁶⁵Cu isotopic label were analyzed to monitor copper contamination. The solutions were centrifuged at 1000 x g for 10 min to separate insoluble silicates; the supernatant was divided for separation of copper and zinc from the matrix and subsequent thermal ionization MS analysis and for determination of copper and dysprosium concentration.

Copper in mineralized fecal samples was analyzed using flame atomic absorption spectrometry with a standard addition technique. The difference relative to the mean value of duplicate analysis was <5%. Dysprosium in mineralized fecal samples was determined by inductively coupled plasma MS (Perkin-Elmer Elan 6000, Perkin-Elmer Europe) using external calibration and rhodium as an internal standard. The difference relative to the mean value of duplicate analysis was <7%. Results were expressed as dysprosium recovery, i.e., dysprosium determined in the fecal pool as a proportion of the administered dose. Separation of copper and zinc from the mineralized matrix was performed by anion exchange chromatography similar to previously described techniques (18,19).

    Analysis of isotopic composition of feces. Isotopic ratios of zinc were determined by thermal ionization MS based on the generation of Zn⁺ ions in a rhenium double-filament ion source similar to the technique described by Turnlund and Keyes (19). Copper isotopic analysis was performed with a measurement technique using Cu(CN)₂^– ions. Samples (5–10 µg copper) were loaded as CuCl₂ (in aqueous solution) together with 40 µg zinc as ZnCl₂ and 100 µg NaCN on top of the evaporation filament. The solution was dried electrothermally at 0.8 Å while the ionization filament remained unloaded. Measurements were performed at ionization filament temperature of 930°C and evaporation filament temperature of 350°C, at ion intensities of 1–2 x10^–11 A for the main signal (personal communication, Walzcyk, T., Laboratory for Human Nutrition, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology, Zurich, Switzerland).

    Calculations of apparent fractional zinc and copper absorption. Apparent absorption was calculated on the basis of fecal excretion during 6 d of ⁷⁰Zn and ⁶⁵Cu isotopic labels, following previously described principles (20,21). The amount of zinc isotopic label (⁷⁰Zn) in the fecal pools was calculated on the basis of the measured isotopic ratios, ⁶⁷Zn/⁶⁴Zn and ⁷⁰Zn/⁶⁴Zn. The absolute amounts of nonabsorbed copper isotopic labels were derived from the total copper concentration in the fecal sample as determined by atomic absorption spectrometry. Calculations were performed following isotope dilution principles, considering that the isotopic labels were not monoisotopic (22). Results were expressed as apparent fractional absorption, i.e., the amount of the absorbed isotopic label relative to the total amount of orally administered isotopic label.

    Statistical methods. Results are presented as arithmetic means and SD. Paired Student’s t test was used to evaluate statistical significance of differences in zinc and copper apparent absorption between test meals. Differences with P-values < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Subject characteristics. The mean age of the subjects was 26 y (range: 22–34 y) and the mean BMI was 20.2 kg/m² (range: 18.1–23.3 kg/m²). Serum zinc concentrations (range: 9.7–17.8 µmol/L, first study period; range: 9.8–19.4 µmol/L, second study period) were in the normal range compared with data from the Department of Clinical Nutrition at University Göteborg, Sweden (23). Serum copper concentrations (range: 11.8–27.3 µmol/L, first study period; range: 13.9–26.2 µmol/L, second study period) were in the normal range according to Sauberlich (24). Serum zinc and copper concentrations did not change significantly between the two study periods.

    Fecal collections. Low dysprosium recovery (60%) in one fecal pool indicated that the fecal collection was incomplete. After exclusion of this subject (both study periods), the mean dysprosium recovery was 106% (range: 95–114%; 18 fecal pools), similar to those reported by Schuette et al. (25).

    Complementary food analysis. The phytic acid concentration of the complementary foods (native phytic acid vs. dephytinized) was 4 ± 0.2 mg/g vs. <0.03 mg/g. The native concentrations (µg/g) of minerals and trace elements were zinc: 20.4 vs. 20.3, copper: 4.2 vs. 4.3, iron: 21.4 vs. 19.9 and calcium: 171 vs. 741. Protein concentration was 183 vs. 186 mg/g. After the addition of the stable isotopes ⁷⁰Zn and ⁶⁵Cu, the molar ratio phytic acid:zinc was 11:1 and the molar ratio phytic acid:copper was 19:1 in the complementary food with the native phytic acid concentration.

    Apparent zinc and copper absorption. Apparent fractional zinc absorption was significantly (P = 0.005) lower from the complementary food with native phytic acid concentration (22.8 ± 8.8%) compared with the dephytinized complementary food (34.6 ± 8.0%). No difference (P = 0.167) was found between the apparent copper absorption from the complementary food with native phytic acid concentration (23.7 ± 8.1%) and the dephytinized complementary food (19.7 ± 5.1%) (Fig. 1).

View larger version (26K): [in this window] [in a new window]   FIGURE 1 Individual data on apparent fractional zinc and copper absorption from the complementary food with native phytic acid concentration and the dephytinized complementary food (n = 9; zinc P = 0.005; copper P = 0.167). The lines connect the data for each subject.

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Contrary to the positive effect on zinc absorption, dephytinization did not increase copper apparent absorption in the present study, nor in the recent study evaluating dephytinized soy formula in healthy infants (30). The lack of effect on copper absorption confirmed the results reported in a previous study in young men (9). Similarly, results of previous animal experiments by Lönnerdal et al. (12) indicated no difference in copper absorption from regular and dephytinized soy formula in rats and monkeys.

The noninhibitory effect of phytic acid on copper absorption in vivo is somewhat unexpected because in vitro phytic acid binds more strongly to copper than to zinc (31). However, the complexes are soluble over a wide pH range and are presumably not strong enough to prevent the transfer of the soluble copper to the transport system in the mucosal cells. Nolan et al. (32) investigated the solubility of phytic acid-copper complexes at different pH and at different molar ratios. At a 1:1 (or above) molar ratio of phytic acid:copper, the formation of soluble phytic acid-copper complexes was reported at pH 3.5. Champagne and Fisher (33) demonstrated that at pH 7 and a high molar ratio of phytic acid:copper (10:1), phytic acid-copper complexes stayed in solution. Also, when solubility of the phytic acid-copper complex was tested in cereal products at pH 7, there was little precipitation (34).

The native phytic acid concentration of the complementary food (4 mg/g) evaluated in this study can be expected to be representative of many commercial infant cereals, and even higher levels could be expected in home-prepared complementary foods based on less refined cereals. The dephytinized complementary food contained no detectable phytic acid; thus in the present study, the effect of complete dephytinization was evaluated under realistic conditions. It is important to note that the sometimes contradictory data presented on the effect of phytic acid did not compare foods before and after complete dephytinization (7).

In the present study, we used a stable isotope technique to evaluate the effect of dephytinization on zinc and copper absorption. One of the methodological limitations with this technique is that the molar ratio of phytic acid to the total amount of zinc and copper is modified by the addition of the isotopic labels. This potential problem can be minimized by a careful study design; for example, in this study, we administered the total dose of stable isotope labels in two identical test meals consumed on the same day. The molar ratio phytic acid:zinc of 11:1 in the complementary food with native phytic acid concentration is in the range that has been described to be inhibiting based on animal studies (35). Due to the addition of the stable isotope label, the molar ratio phytic acid:copper of 19:1 was lower than in previous studies, which had similarly reported no effects of phytic acid on copper absorption in adults (9,11).

Furthermore, the methodology used in this study is based on fecal monitoring of nonabsorbed stable isotope labels; the duration and completeness of the fecal collections are therefore of major importance. The completeness of the fecal collection was monitored by analysis of a nonabsorbable fecal marker, dysprosium. The usefulness of this approach was clearly demonstrated because 1 subject had to be excluded due to incomplete fecal collection. Furthermore, this methodology allows the determination of apparent, but not true zinc and copper absorption because no correction was made for reexcreted, absorbed isotopic label during the fecal collection period. Previous studies demonstrated that the difference between apparent and true zinc and copper absorption can be expected to be on the same order of magnitude. Turnlund et al. (21) reported a mean of 7.7% of an intravenous dose of ⁶⁵Cu excreted in feces during a 6-d period after administration, and Fairweather-Tait et al. (6) showed that true zinc absorption was 7% higher than apparent zinc absorption in men, using a double-label stable isotope technique. Although the difference between apparent and true absorption of trace elements is important in some contexts, it is of limited relevance in studies designed to make direct comparisons between test meals.

In conclusion, the results from the present study clearly demonstrate the beneficial effect of dephytinization of a cereal-based complementary food on fractional absorption of zinc in adults. No effect was observed on copper apparent absorption, and these results thus add to the body of evidence that phytic acid is not an inhibitor of copper absorption in humans. The usefulness of the newly developed technique to dephytinize cereal-based complementary foods by using phytase naturally occurring in whole-grain cereals should be explored in large-scale production, and the long-term nutritional benefits of dephytinization of foods consumed during early life should be evaluated in infants and young children.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the excellent cooperation of all volunteers in this study. Special thanks to Josef Burri, Johan de Reu, and the staff at the pilot plant at Nestlé Product Development Centre, Orbe, Switzerland for producing the complementary foods and Peter Kastenmayer, Nestlé Research Centre, Lausanne, Switzerland for performing the dysprosium analysis.

	FOOTNOTES

 
¹ Supported by Nestec Ltd., Vevey, Switzerland.

Manuscript received 14 December 2003. Initial review completed 13 January 2004. Revision accepted 16 February 2004.

	LITERATURE CITED

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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