Lisbeth Grinder-Pedersen, Klaus Bukhave, Mikael Jensen, Liselotte Højgaard, Marianne Hansen, Calcium from milk or calcium-fortified meals doesn’t inhibit nonheme-iron absorption from a complete food regimen consumed over a 4-d interval, The American Journal of Medical Diet, Quantity 80, Concern 2, August 2004, Pages 404–409, https://doi.org/10.1093/ajcn/80.2.404
ABSTRACT
INTRODUCTION
Enough calcium consumption is important for acquiring optimum peak bone mass in youth and for minimizing bone loss later in life (1). Nonetheless, calcium consumption might have a damaging impact on each nonheme- and heme-iron absorption, as proven by a number of single-meal research (2–5). To counteract this putative impact, calcium dietary supplements, dairy merchandise wealthy in calcium, or calcium-fortified meals may very well be taken both with meals which are usually low in iron content material or between meals. Nonetheless, this will make it troublesome for weak teams to attain the advisable every day calcium consumption (6).
As a result of quite a few dietary elements affect iron absorption (7), the impact of calcium is probably not as pronounced when calcium is served as half of an entire food regimen (8). Subsequently, measurements of iron absorption from the entire food regimen over an extended time period are warranted (9). Earlier multiple-meal research on the impact of calcium on nonheme-iron absorption have additionally proven inconsistent and conflicting outcomes, partly due to variations in experimental design. When nonheme-iron absorption was measured for a 5-d interval, a 30% increased absorption was discovered for dairy calcium served at breakfast and dinner than for dairy calcium distributed extra equally all through the day (10). In distinction, a 70% discount in nonheme-iron absorption was noticed in a 1-d examine by which topics consumed supplemental calcium carbonate with the three essential meals (11). These outcomes battle with these obtained in a examine by Reddy and Prepare dinner (12), by which no distinction in nonheme-iron absorption between a low- and a high-calcium food regimen was discovered. The inhibitory impact of calcium on iron absorption has been reported to be dose associated, with no impact at <40 mg Ca/meal and no further inhibitory effect at >300 mg Ca/meal (4). Along with variations in calcium sources (ie, endogenous or supplemental) and within the iron standing of the topics investigated, this vary for inhibition might, not less than partly, clarify the aforementioned discrepancies. Subsequently, the intention of the current examine was to match the impact on nonheme-iron absorption of serving completely different sources of calcium with the three essential meals with that of serving a primary food regimen (BD) with a low calcium content material (ie, 60–88 mg/meal) in younger girls with low iron shops who consumed 4 strictly managed, 4-d complete diets.
SUBJECTS AND METHODS
Topics
Fourteen girls aged 24.2 ± 3.0 y (x̄ ± SD) with a imply weight of 65 ± 10 kg and a imply physique mass index (in kg/m2) of twenty-two.3 ± 2.6 participated within the examine. One lady dropped out of the examine earlier than the start of the final interval, ie, earlier than her BD interval. All the ladies had been nonsmokers, and none of them had been pregnant or lactating or took any vitamin or mineral dietary supplements for ≥2 mo earlier than or in the course of the examine. Ten topics used oral contraceptives all through the examine, however not one of the girls routinely took every other drugs. Blood donation was not allowed for ≥2 mo earlier than or in the course of the examine. Twenty-nine topics had been screened 3 wk earlier than the start of the examine, and 14 topics had been chosen for the examine on the idea of comparatively low iron shops, ie, a hemoglobin focus ≥ 110 g/L and a serum ferritin focus ≤40 μg/L. The members had been recruited from universities within the close by space and obtained oral and written details about the examine earlier than they gave their written consent. The examine was authorised by the Analysis Moral Committee of Copenhagen and Frederiksberg [(KF) 01-238/98] and the Nationwide Institute of Radiation Hygiene, Denmark.
Experimental design
The 4 diets within the examine consisted of the BD, which was low in calcium, and the BD supplemented with 1 of the three following calcium sources: a glass of milk served at every essential meal, calcium lactate (Merck 102102; Merck KGaA, Darmstadt, Germany), or a milk mineral isolate (MM) containing calcium (Arla Meals A/S, Aarhus, Denmark). The two latter calcium sources had been added to chose meals of the BD: rye bread, white bread, chocolate cake, and orange juice. The content material of calcium was evenly distributed between the meals (Desk 1). The examine was double blinded with regard to the management food regimen and the two diets fortified with calcium lactate or MM. The examine had a randomized crossover design with 4 durations, every of which lasted 18 d and featured strict management of dietary consumption on days 1–4, when the topics obtained the dietary intervention. The meals had been extrinsically labeled with the radioisotope 59Fe, and the absorption of the isotope was measured by whole-body retention on day 17 of every interval. Fasting blood samples had been collected within the morning on day 18 of every interval. The themes had been instructed to abstain from heavy bodily train for 36 h, to not devour alcohol for twenty-four h, and to quick for 12 h earlier than blood sampling and earlier than the primary meal in every intervention interval. They had been allowed to drink 0.5 L water offered by the Division of Human Diet, The Royal Veterinary and Agricultural College, Frederiksberg, Denmark, inside the 12-h fasting interval.
Formulation of take a look at meals and serving process
The intervention diets included 4 menus consumed on days 1, 2, 3, and 4, respectively. The menus and the meals portions used within the 4 intervention diets had been similar. The composition of the menus is proven in Desk 2. The calculated every day intakes of fats, carbohydrate, and protein had been 92 g (35% of vitality), 297 g (51% of vitality), and 84 g (14% of vitality), respectively, at an vitality consumption of 10 MJ/d [calculated with the use of DANKOST 2000, version 1.20 (Danish Catering Center, Herlev, Denmark), a computer program that is based on the Danish Veterinary and Food Administration food-composition database (13)]. Particular person parts of the meals had been weighed in line with the estimated vitality requirement of every topic (14).
In producing the fortified rye bread, white bread, and chocolate cake, the calcium supply was combined with rye flour (for rye bread) or white-wheat flour (for white bread and chocolate cake) in a meals processor for 90 min at low pace. After all of the substances had been combined, the dough for the rye bread was allowed to ferment for 72 min at 34°C and was then baked for 70 min at 220°C. The dough for the white bread was allowed to rise for 90 min at room temperature and once more for 60 min instantly earlier than baking for 45 min at 180°C. The chocolate cake was baked instantly after mixing of the substances for 45 min at 165°C. Within the 3 nonmilk durations, a small roll was ingested with every essential meal to compensate for the elevated vitality consumption within the milk interval (0.79 MJ/d at a complete vitality consumption of 10 MJ/d). The rolls had been ready from the identical recipe and batch of flour as used for the white-wheat bread. No meals or drinks apart from these offered by the examine employees had been allowed, and every topic maintained a relentless consumption throughout all 4 intervention durations. All the primary meals (ie, breakfast, lunch, and dinner) had been eaten within the eating room on the Division of Human Diet, whereas the night fruit was offered by the Division however eaten at residence. Lunch was served 4 h after breakfast, and dinner was served 5 h after lunch. The dinner meal was thawed in a single day in a fridge (4°C) and heated in a microwave oven simply earlier than serving.
Isotopes and labeling procedures
The take a look at meals, together with bread, had been thawed in a single day earlier than serving. The meals had been extrinsically labeled ≥16 h earlier than serving by pipetting the radioisotope answer (59FeCl3 in 0.1 mol HCl/L; Amersham Biosciences UK Restricted, Buckinghamshire, United Kingdom) onto the bread served at every meal in quantities that gave precisely the identical particular exercise of 59Fe (0.80 kBq 59Fe/mg Fe) in all meals. The overall dose in every interval was 42 kBq 59Fe.
Milk mineral isolate
The MM used was produced from skim milk by acid precipitation of casein adopted by ultrafiltration of the acidified whey, centrifugation (8500 × g for 30 s at 65°C), and spray drying. To make sure uniform composition of the MM, the isolate was provided in batches of 5 kg taken from a pool. Every pool of MM was composed from 5 equal samples taken out of the manufacturing line.
Complete-body counting and calculation of nonheme-iron absorption
Complete-body retention of 59Fe was measured in a lead-lined metal chamber with 4 NE110 plastic scintillator blocks (Nuclear Enterprises Restricted, Edinburgh) related to traditional nuclear digital modules and a multichannel analyzer system. The counting effectivity and vitality window settings had been established via measurements of water-filled phantoms whose outlines and weights are roughly equal to these of people. The phantoms had been full of recognized concentrations of the isotope. Within the precise setup and with the vitality window used, the general counting effectivity for 59Fe evenly distributed in a 77-kg phantom was ≈20%. To attenuate contamination by atmospheric background exercise, all topics had a bathe, washed their hair, and had been wearing hospital clothes earlier than every measurement. The counting time was 10 min, and the outcomes had been corrected each for chamber background radiation and for the topics’ personal background radioisotope exercise measured 1 wk earlier than the start of the primary intervention interval. All radioactivity measurements had been corrected for the bodily decay of the isotope. Outcomes are expressed as fractional absorption (share of ingested quantity).
Dietary analyses
Duplicate parts of the 4 menus had been freeze-dried, homogenized, and analyzed in duplicate for whole iron, phytic acid, and calcium. The vitality content material was calculated with using a food-composition laptop program (Danish Tables of Meals Composition, DANKOST 2000, model 1.20; Danish Catering Middle). Whole iron and calcium had been decided by atomic absorption spectrophotometry (Spectra-AA 200; Varian, Mulgrave, Australia) after wet-ashing in an MES 1000 Solvent Extraction system (CEM Corp, Matthews, NC) with 65% (by wt) suprapure nitric acid (Merck KgaA). The Normal Reference Materials 1548a (typical food regimen; Nationwide Institute of Requirements and Know-how, Gaithersburg, MD) was used because the reference for iron [35.30 ± 3.77 μg/g (x̄ ± SD)] and calcium (1.97 ± 0.11 mg/g), and the analyzed values had been 36.83 ± 4.04 μg/g (n = 6) and 1.90 ± 0.09 mg/g (n = 9), respectively. Phytic acid (inositol phosphates) evaluation was carried out by utilizing high-performance ion chromatography as described beforehand (15), and outcomes are given because the sum of inositol tri-, tetra-, penta-, and hexaphosphates.
Iron-status measurements
The blood samples taken on day 18 of every interval had been analyzed for hemoglobin and serum ferritin. After the topics rested for 10 min in a supine place within the morning, blood samples had been drawn with 20 G needles and minimal stasis from the cubital vein into evacuated EDTA-coated tubes and plain tubes (BD Vacutainer Techniques; Becton Dickinson, Franklin Lakes, NJ). Hemoglobin was decided on a Sysmex analyzer (SysmexKX-21; Sysmex Company, Kobe, Japan). Serum ferritin evaluation was carried out by utilizing a 2-site fluoroimmunometric assay with a Delfia fluorometer 1235 (PerkinElmer Life Sciences–Wallac Oy, Turku, Finland) and an AutoDELFIA Ferritin Equipment (B069-101; PerkinElmer Life Sciences–Wallac Oy).
Expression of iron absorption
Statistical analyses
The pattern dimension was calculated from our earlier information on iron absorption from single meals containing comparable quantities of meat and phytic acid (16). To acquire an influence of 90%, 12 topics are wanted to detect a 1.5% change in iron absorption at a significance stage of 0.05 (17). Serum ferritin and absorption information had been transformed to logarithms earlier than statistical analyses, and the outcomes had been reconverted to antilogarithms. The logarithmic information had been usually distributed, with variance homogeneity examined by plots and histograms of residuals. Shapiro-Wilk’s take a look at for regular distribution was carried out. Nonheme-iron absorption information are offered as means with 95% CIs. The impact of intervention kind on nonheme-iron absorption and interactions between interval and intervention kind had been analyzed by parametric two-way repeated-measures evaluation of variance, adopted by pairwise a number of comparisons with Tukey’s actually vital distinction methodology. One-way evaluation of variance was used to check for variations between absorption ratios and the impact of consumption of phytic acid on nonheme-iron absorption. The distinction between the imply absorption ratios and unity was examined in one-sample t checks. The info from the one that dropped out of the examine earlier than the final interval are included in all of the statistical analyses, aside from the analyses of the absorption ratios. All statistical analyses had been carried out with using SPSS 11.0 for WINDOWS (SPSS Inc, Chicago).
RESULTS
All topics had comparatively low iron shops however had been most likely not anemic as indicated by their serum ferritin concentrations (geometric x̄: 15 μg/L; vary: 6–44 μg/L) and hemoglobin concentrations (geometric x̄: 123 g/L; vary: 115–137 g/L). The themes’ imply (± SD) vitality consumption was 10.7 ± 0.8 MJ/d. The imply calcium consumption within the primary dietary interval was 224 mg/d, and the consumption was distributed as 60–88 mg/meal. The imply calcium intakes within the milk, calcium lactate, and MM dietary durations had been 826, 802, and 801 mg/d, respectively, and the distribution of consumption was within the vary of 253–291 mg/meal (Desk 3). The distinction within the calcium consumption between the BD and the opposite 3 diets was vital (P < 0.001). The imply iron consumption within the 4 dietary durations was 13.2 mg/d (Desk 3). The imply phytic acid intakes, expressed because the sum of the intakes of inositol tri-, tetra-, penta-, and hexaphosphates, within the primary, milk, calcium lactate, and MM dietary durations had been 481, 480, 592, and 628 mg/d, respectively, and the distribution of consumption was within the vary of 110–253 mg/meal. Nonheme-iron absorption from the food regimen within the primary dietary interval was 7.4% (95% CI: 5.3%, 10.5%) (Desk 4). Nonheme-iron absorption from the food regimen within the milk, calcium lactate, and MM dietary durations was 5.2% (95% CI: 3.5%, 7.9%), 6.7% (95% CI: 5.0%, 8.9%), and 5.1% (95% CI: 3.2%, 7.9%), respectively (P = 0.34). Absorption ratios calculated relative to the essential dietary interval had been 0.83 (95% CI: 0.45, 1.20), 0.91 (95% CI: 0.71, 1.11), and 0.80 (95% CI: 0.53, 1.08) for the milk, calcium lactate, and MM dietary durations, respectively (P = 0.62). The absorption ratios weren't considerably completely different from unity (P = 0.34, P = 0.36, and P = 0.15 for the milk, calcium lactate, and MM dietary durations, respectively). Nonheme-iron absorption didn't considerably correlate with serum ferritin concentrations (P = 0.39).
DISCUSSION – “calcium and iron absorption”
Within the current examine, total nonheme-iron absorption was decided throughout 4 4-d durations consisting of a BD with low calcium (224 mg/d) and three experimental diets with further calcium (575–600 mg/d) from completely different sources that resulted in a every day consumption of 800 mg/d, which complied with the advisable intakes for women and men within the Nordic Nutrient Suggestions (18). No results of a glass of milk or of an equal quantity of calcium from fortified meals on nonheme-iron absorption had been noticed.
A dose-related inhibition of iron absorption by a low quantity of calcium (40–300 mg) was reported in a single-meal examine, however no additional inhibition was noticed with calcium quantities >300 mg/meal (4). This dose-dependency of inhibition is additional supported by a examine by which no vital impact on iron absorption was noticed by the addition of 172 mg Ca within the type of milk to a meal already containing 320 mg Ca (19). Thus, the absence of inhibitory results on nonheme-iron absorption by calcium in some research could also be on account of a excessive background quantity of calcium. Nonetheless, this was not the case within the current examine, by which the quantity of calcium within the BD was <90 mg/meal. The calcium-supplemented diets contributed 200 mg further Ca to every meal. In accordance with Hallberg et al (4), a 50% discount in nonheme-iron absorption from the meals within the current examine would have been anticipated, if the meals had been served as single meals. Phytic acid has been considered some of the potent inhibitors of nonheme-iron absorption, and even small quantities of phytate can have a marked impact on nonheme-iron absorption. In 2 single-meal research, 18–36 mg phytic acid from bread lowered nonheme-iron absorption by ≈50%, and 88 mg phytic acid lowered absorption by 64% (20, 21). Analyses of phytic acid confirmed a considerably increased content material within the fortified diets (calcium lactate and MM diets) than within the BD and the milk food regimen (Desk 3). Bread was the merchandise chargeable for the upper quantities of phytic acid (information not proven), which signifies that addition of calcium earlier than leavening of the dough reduces the degradation of phytic acid throughout fermentation, in accordance with an earlier examine by which the addition of 40 mg Ca to dough containing 80 g white-wheat flour almost halved phytic acid degradation (4). However, the upper quantity of phytic acid within the 2 fortified diets didn't have any impact on nonheme-iron absorption (P = 0.13). Moreover, calculations of the inhibitory impact of phytic acid, as proposed by Hallberg et al (22), point out a 65–70% discount in iron absorption with the quantity of phytic acid within the meals within the BD and the BD plus milk and a 67–72% discount in iron absorption within the meals within the fortified diets. Thus, solely a minor impact of the upper quantity of phytic acid within the fortified diets is anticipated. As well as, it's doable that the relatively excessive content material of phytic acid within the meals within the BD (110–194 mg/meal) might have been chargeable for a robust inhibitory impact that might have masked a putative impact of calcium.
Along with ascorbic acid, one of many generally accepted enhancers of iron absorption is meat, and even small quantities of meat can have a big impact. In a single-meal isotope examine, nonheme-iron absorption elevated by 44% with the addition of fifty g pork to a meal containing 220 mg phytic acid (16). In one other single-meal examine, the addition of fifty g beef to a bread meal with a phytic acid content material of 88 mg had no impact on nonheme-iron absorption (21), whereas absorption elevated by 86% with the addition of fifty g beef when the phytic acid content material was raised to 882 mg. This implies that the impact of meat is extra pronounced within the presence of excessive quantities of phytic acid. Fish has additionally been proven to extend nonheme-iron absorption (a 12% enhance with 40 g fish) (23). Within the current examine, meat within the type of beef, pork, turkey, and tuna fish was served on the 3 essential meals in quantities starting from 20 g at breakfast to 40–75 g at lunch and 100 g at dinner, ie, in quantities consultant of a Western food regimen. Thus, it's doubtless that these quantities might have elevated nonheme-iron absorption from the calcium-supplemented diets. It has been proven that single-meal research might overestimate the impact of enhancers and inhibitors on iron absorption from the food regimen. Comparability of single meals and complete diets carried out by serving the identical meal as soon as or 3 occasions/d confirmed 80% increased iron absorption from the one meal (24). Prepare dinner et al (8) in contrast the impact of complete diets designed to maximally inhibit or improve nonheme-iron absorption with the impact of consultant single meals and located a higher inhibitory and enhancing impact from single meals than from complete diets. The explanations for the dearth of impact of calcium on nonheme-iron absorption when measured from a complete food regimen relatively than from a single meal are unknown. The size of the interval of fasting could also be one clarification as a result of it has been proven to have an effect on intestinal iron absorption (25). One other chance is that in whole-diet experiments, residual meals within the gut might reasonable the impact of inhibitors and enhancers current in single components of the food regimen. The long-term impact of calcium supplementation on iron standing has been assessed in only some research. In premenopausal girls, no impact on serum ferritin was noticed after the consumption of 1000 mg CaCO3/d for 3–12 mo (26–28). These observations are in settlement with the outcomes of cross-sectional research displaying no impact of dietary calcium on iron standing (29, 30) however are in disagreement with the outcomes of different cross-sectional research displaying decrease serum ferritin concentrations in teams with a excessive consumption of calcium from dairy merchandise than in teams with a decrease consumption (31, 32). Nonetheless, the impact of dietary calcium on iron standing is small, ie, solely a 1.6% lower in serum ferritin focus for each 100-mg/d enhance in calcium consumption (31). A possible method for growing calcium consumption in individuals who don't devour dairy merchandise is calcium fortification of meals merchandise. Nonetheless, little is understood in regards to the impact of such meals on nonheme-iron absorption. Within the current examine, 2 varieties of calcium had been used for fortification: calcium lactate and an MM with a excessive content material of calcium. Neither of those fortified diets had any damaging impact on nonheme-iron absorption. In conclusion, within the current examine, portions of calcium that corresponded to the advisable quantities within the Nordic Nutrient Suggestions and had been served with the three essential meals as milk or fortified meals for a 4-d interval didn't lower total nonheme-iron absorption. Nonetheless, variations in strategies and age teams in a lot of these research warrant additional investigations on the impact of calcium fortification on nonheme-iron absorption. We thank Hanne Lysdal Petersen, Division of Human Diet, The Royal Veterinary and Agricultural College, Frederiksberg, Denmark, and Susanne Svalling, Division of Medical Physiology and Nuclear Drugs, The Nationwide College Hospital, Copenhagen, for offering glorious technical help. The define of the examine was deliberate by MH. LG-P did a lot of the detailed planning, carried out the absorption research, did statistical therapy of the info, ready the primary draft of the manuscript, and interpreted the leads to collaboration with MH and KB. MJ estimated the whole-body retention of isotopes, calculated the radioactive doses, and described the strategy of whole-body measurements. LH was the clinician chargeable for the radioisotope research. All contributors had been concerned in preparation of the ultimate draft of the manuscript. Not one of the authors had any conflicts of curiosity in relation to the examine.REFERENCES
FOOTNOTES