Application of in vitro bioaccessibility and bioavailability methods for calcium, carotenoids, folate, iron, magnesium, polyphenols, zinc, and vitamins B(6), B(12), D, and E.

A review of in vitro bioaccessibility and bioavailability methods for polyphenols and selected nutrients is presented. The review focuses on in vitro solubility, dialyzability, the dynamic gastrointestinal model (TIM)™, and Caco-2 cell models, the latter primarily for uptake and transport, and a discussion of how these methods have been applied to generate data for a range of nutrients, carotenoids, and polyphenols. Recommendations are given regarding which methods are most justified for answering bioaccessibility or bioavailability related questions for specific nutrients. The need for more validation studies in which in vivo results are compared to in vitro results is also discussed.

Production of stable-isotope-labeled bovine heme and its use to measure heme-iron absorption in children.

BACKGROUND: The use of stable isotopes has provided valuable insights into iron absorption in humans, but the data have been limited to nonheme iron. OBJECTIVE: Our objectives were to produce heme iron enriched in (58)Fe and to use it to study the absorption of heme iron and the effect of iron and zinc intakes on heme-iron absorption in children. DESIGN: Labeled bovine heme was produced in a bovine model. Forty-eight children were randomly assigned to consume identical meals containing 1 of 3 doses of labeled heme iron (2, 4, or 8 mg as hemoglobin) and 1 of 2 doses of inorganic zinc (1 or 9 mg); successful measurements of iron absorption, zinc absorption, or both were made in 40 of these subjects. We hypothesized that fractional heme-iron absorption would decrease as heme-iron intake increased and that higher zinc intakes would decrease heme-iron absorption. RESULTS: (58)Fe heme was produced with an enrichment (mass/mass) of 9.5%. Fractional iron absorption in children was significantly affected by the intake of heme iron (P = 0.0013) and of zinc (P = 0.0375), but, contrary to expectations, heme-iron absorption was higher at higher zinc intakes. Absolute heme-iron absorption was higher in the group with higher zinc intakes, but only for those with the lowest heme-iron intake (2 mg; P = 0.0147). Although fractional zinc absorption decreased as zinc intake increased (P = 0.031), absolute zinc absorption continued to increase across the intake range studied (P = 0.018). CONCLUSIONS: Heme iron intrinsically labeled with (58)Fe can be produced at sufficient enrichments for use in human studies. In children, heme iron and zinc absorption decrease as the dose of each mineral increases. Heme iron did not inhibit zinc absorption. At lower heme intakes, zinc intakes may increase heme-iron absorption.

Effect of beef and soy proteins on the absorption of non-heme iron and inorganic zinc in children.

BACKGROUND: Iron and zinc deficiency remain substantial problems in small children in both developed and developing nations. Optimizing mineral absorption is an important strategy in minimizing this problem. OBJECTIVES: To assess the effects of beef and soy proteins on the bioavailability of non-heme iron and zinc in children. METHODS: We measured iron (n = 26) and zinc (n = 36) absorption in 4-8 y old children from meals differing only in protein source (beef or a low-phytate soy protein concentrate). Iron and zinc absorption were measured using multi-tracer stable isotope techniques. Iron absorption was calculated from the red blood cell iron incorporation measured after 14 days and zinc absorption from the ratio of the oral and intravenous excretion of the zinc tracers 48 hours after dosing. RESULTS: Iron absorption from the beef meal was significantly greater (geometric mean, 7.6%) than from the soy meal (3.5%, p = 0.0015). Zinc absorption from the beef meal was greater (mean +/- SD, 13.7 +/- 6.0%) than from the soy meal (10.1 +/- 4.1%, p = 0.047). CONCLUSION: These findings indicate that beef protein increases both non-heme iron and zinc absorption compared to soy protein. The effect of protein source on non-heme iron and inorganic zinc absorption should be one of the factors taken into account when designing diets for children. The inhibitory effect of the soy based meal on iron and zinc absorption could be overcome by fortifying the soy protein with these minerals during the production process.

The effect of calcium salts, ascorbic acid and peptic pH on calcium, zinc and iron bioavailabilities from fortified human milk using an in vitro digestion/Caco-2 cell model.

The calcium, zinc, and iron bioavailabilities of human milk with commercial and noncommercial human milk fortifiers (HMFs) were evaluated under a variety of conditions: peptic digestion at pH 2 and pH 4, supplementation of ascorbic acid, and addition of three calcium salts. The noncommercial HMFs consisted of casein phosphopeptides (CPPs), alpha-lactalbumin, colostrum, and hydrolyzed whey protein concentrate (WPC). They were mixed with human milk (HM) and calcium, zinc, and iron were added. Ascorbic acid (AA) was added in certain studies. The commercial HMFs were Nestlé FM-85, Similac HMF (SHMF), and Enfamil HMF (EHMF). All HMFs were compared to S-26/SMA HMF. Results showed that the peptic pH (2 vs. 4) had no effect on mineral bioavailability. Addition of different calcium salts had no effect on calcium cell uptake and cell ferritin levels (an indicator of iron uptake), however, the addition of calcium glycerophosphate/gluconate increased zinc uptake by Caco-2 cells. Addition of AA significantly increased ferritin levels, with no effect on calcium or zinc uptake. Among the commercial HMFs, FM-85 was significantly lower in zinc uptake than S-26/SMA, and HM+EHMF was significantly higher than HM+S-26/SMA. Cell ferritin levels were significantly higher for HM+S-26/SMA than for all other commercial fortifiers. None of the commercial HMFs were different from HM+S-26/SMA in calcium uptake.

A low-molecular-weight factor in human milk whey promotes iron uptake by Caco-2 cells.

The iron bioavailability of human milk (HM) is substantially greater than that of cow's milk (CM), but the factor responsible for this high bioavailability is unknown. This study evaluated the effects of various HM and CM fractions on iron bioavailability. Milk was separated into fat, casein and whey fractions by ultracentrifugation. Whey was further fractionated by ultrafiltration with a 10-kDa membrane to produce a 10-kDa retentate (10kR) and a 10-kDa filtrate (10kF). Samples were prepared by mixing various combinations of the fractions, bringing the samples to prefractionation weight with minimum essential medium (MEM), and adding iron (10 micro mol/L) as ferrous sulfate. Samples were divided into two aliquots: one was subjected to in vitro digestion, the other was not. Bioavailability was assessed by applying the samples to Caco-2 cell monolayers and incubating for 24 h. Ferritin formation in the cells was used as an index of iron uptake. Removing the fat from undigested HM samples doubled the ferritin formation, but removing the whey or casein had no effect. Results with digested HM samples were similar, except that removing the whey decreased ferritin formation by 48%. Removing the fat from digested CM samples had no effect, but removing the casein doubled the ferritin formation. Removing the 10kF from HM reduced ferritin formation by 60%, but removing the 10kR had no effect. These data suggest that a low-molecular-weight factor (<10 kDa) in human milk enhances iron absorption.

Simultaneous determination of (45)calcium and (65)zinc uptake by caco-2 cells.

A simple method for simultaneously determining cell-associated Ca and Zn in Caco-2 cells is described. Calcium and zinc uptake was measured via radioisotopes (45)Ca and (65)Zn. Preliminary studies revealed that (65)Zn, a positron (beta(+)) and gamma emitter, contributed to (45)Ca counts in a liquid scintillation counter (LSC). However, (45)Ca, being a true beta emitter, did not contribute to the counts in a gamma counter (gammaC). To differentiate the counts of (45)Ca from those of (65)Zn, first a (65)Zn-labeled cell suspension was read in a gammaC and an LSC, thus obtaining the relationship between the radioactive counts obtained from the gammaC and LSC. This information defined the linear relationship between gammaC (65)Zn counts per minute (CPM) and LSC (65)Zn CPM. Because the (45)Ca and (65)Zn counts obtained in the LSC are additive, giving total LSC CPM, the value of LSC (45)Ca CPM was obtained by subtracting LSC (65)Zn CPM from total LSC CPM for the dual-labeled cell sample, obtaining then LSC (45)Ca CPM. To determine the absolute activity or disintegrations per minute (DPM) of each isotope in the dual-labeled sample, the linear relationship between DPM and CPM was determined for each isotope. The method is simple and straightforward for the determination of (45)Ca counts from a sample also containing (65)Zn, using gamma and liquid scintillation counters.