Iron bioavailability from commercially available iron supplements
- 897 Downloads
Iron deficiency anaemia (IDA) is a global public health problem. Treatment with the standard of care ferrous iron salts may be poorly tolerated, leading to non-compliance and ineffective correction of IDA. Employing supplements with higher bioavailability might permit lower doses of iron to be used with fewer side effects, thus improving treatment efficacy. Here, we compared the iron bioavailability of ferrous sulphate tablets with alternative commercial iron products, including three liquid-based supplements.
Iron bioavailability was measured using Caco-2 cells with ferritin formation as a surrogate marker for iron uptake. Statistical analysis was performed using one-way ANOVA followed by either Dunnett’s or Tukey’s multiple comparisons tests.
Spatone Apple® (a naturally iron-rich mineral water with added ascorbate) and Iron Vital F® (a synthetic liquid iron supplement) had the highest iron bioavailability. There was no statistical difference between iron uptake from ferrous sulphate tablets, Spatone® (naturally iron-rich mineral water alone) and Pregnacare Original® (a multimineral/multivitamin tablet).
In our in vitro model, naturally iron-rich mineral waters and synthetic liquid iron formulations have equivalent or better bioavailability compared with ferrous iron sulphate tablets. If these results are confirmed in vivo, this would mean that at-risk groups of IDA could be offered a greater choice of more bioavailable and potentially better tolerated iron preparations.
KeywordsIron supplements Anaemia Pregnancy Bariatric surgery Micronutrient deficiency Caco-2 cells
Iron deficiency is a global health problem; 2 billion people suffer from anaemia, and the World Health Organization (WHO) estimates that iron deficiency accounts for 50 % of these cases . Although the prevalence of iron deficiency anaemia (IDA) is highest in developing countries, inadequate iron status is also a significant public health problem in developed countries in high-risk groups including the elderly, post-bariatric surgery patients, toddlers, and pregnant women and women of child-bearing age [2, 3].
Three ferrous iron salts, sulphate, fumarate and gluconate, are the standard recommended supplements for IDA [4, 5]. Clinical trials with pregnant women document that these iron supplements do improve IDA ; however, non-compliance secondary to supplement-associated gastro-intestinal (GI) problems, such as constipation, black stools, nausea, reflux and vomiting, means that real-life efficacy may be low [7, 8, 9]. Pregnancy itself induces gastrointestinal symptoms, related to the mechanical effects of the growing foetus, changes in water homoeostasis, and hormonal effects on GI motility—all of which contribute to increased incidence of reflux, constipation and bloating [10, 11]. Higher doses of iron increase GI symptoms in pregnant women , and studies document that lower doses are associated with fewer GI symptoms [6, 13], thus it is possible that the use of iron preparations with higher bioavailability, enabling lower absolute doses of iron to be ingested, may cause less GI upset.
IDA is associated with increased health risks to both mother and foetus (reviewed in ), and it is therefore vital that effective supplement forms are available to pregnant women. A human volunteer study demonstrated that iron absorption from a naturally iron-rich mineral water (Spatone Iron-Plus) was higher compared with ferrous sulphate tablets (FeSO4) . Furthermore, a subsequent study looking specifically at pregnant women with IDA found that the mean absorption from 25 mg of Spatone-derived Fe was 28 %—comparable or greater to that reported from a higher dose of FeSO4 .
The physical form of iron administered in supplements could have a bearing on bioavailability. A recent in vitro study found that dissolution rates varied significantly amongst different iron tablet formulations; FeSO4 conventional-release tablets dissolved most quickly at 48–64 min, and this was associated with the highest iron uptake in the in vitro Caco-2 cell model used in this study . We hypothesized therefore that liquid iron formulations, whether naturally occurring or synthetic, might provide alternative supplement forms that have higher bioavailability compared with iron delivered in tablet form.
In this study, we have employed the Caco-2 cell in vitro digestion model to assess iron bioavailability, using cell ferritin as a surrogate marker for iron absorption, from five commercially available iron formulations including three liquid iron supplements . This model is used worldwide to measure iron bioavailability from different substances and has been validated by human studies . We compared two naturally mineral-rich waters, Spatone® and Spatone Apple® (Spatone with added apple flavour and ascorbate), Iron Vital F® (a synthetic liquid iron supplement with added ascorbate and other micronutrients), immediate release FeSO4 tablets, and Vitabiotics Pregnacare® (a multivitamin and mineral tablet marketed to pregnant women).
Materials and methods
Chemicals, hormones and enzymes were purchased from Sigma-Aldrich, UK, unless otherwise noted. Cell culture media, flasks, tissue culture plates and culture reagents were obtained from Thermo Fisher Scientific, and acids used in ICP analysis, in digestions and for glassware cleaning were purchased from VWR, UK. Glassware and plasticware used in experiments were soaked in 10 % trace metal grade 68 % nitric acid for 24 h prior to use and then rinsed with 18 mΩ pure water.
Commercial iron preparations were purchased from Boots Pharmacy, UK. These consisted of: Spatone® and Spatone Apple® (A Nelson and Co Ltd, UK); Iron Vital F® (Anton Hübner GmbH and Co, Germany); Pregnacare Original® (Vitabiotics Ltd, UK) and Almus Ferrous Sulphate tablets (Almus Pharmaceuticals, UK). Table 1 provides reported iron concentration for the different formulations.
Caco-2 cells (TC7 clone), gifted to the Sharp laboratory by Monique Rousset and Edith Brot-Laroche , were used from cell passages 46–49. The TC7 clone has been validated for use in studies on iron metabolism  and has been used in our previous published work on iron bioavailability . Cells were grown in T75 tissue culture flasks and subcultured every 7 days. For experiments, cells were grown in six-well tissue cultures plates seeded at a density of 1 × 10 4 cells/cm2 and used on days 13–15 post-seeding in the method developed by the Glahn laboratory [18, 19]. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 41965) supplemented with 10 % v/v foetal bovine serum (LCG Standards, 30-2020), 1 % penicillin–streptomycin, 4 mmol/L l-glutamine, 1 % non-essential amino acids and Plasmocin 5 mg/mL (Source Bioscience).
Twenty-four hours prior to the initiation of in vitro digestion experiments, medium was changed to supplemented MEM without foetal bovine serum but with 10 mmol/L PIPES [piperazine-N, N′-bis- (2-ethanesulfonic acid)], 1 % antibiotic/antimycotic solution, 11 mmol/L hydrocortisone, 0.87 mmol/L insulin, 0.02 mmol/L sodium selenite (Na2SeO3), 0.05 mmol/L triiodothyronine and 20 mg/L epidermal growth factor, as developed by Glahn et al. [18, 19], in order to ensure adequate cell growth but with low basal media iron levels.
Iron supplements and in vitro digestions
Iron-containing tablets were sealed inside a disposable plastic sleeve and crushed with a mortar and pestle; the particles were then solubilized in 250 mL 0.2 mol/L hydrochloric acid (HCl) . Liquid formulations were used directly from the sachets (Spatone®and Spatone Apple®) or from the bottle (Iron Vital F®). The supplements all had different iron concentrations; therefore, the volumes of prepared solutions, or liquid supplements, were adjusted (based on the manufacturers reported iron concentrations) to achieve a final digest iron concentration of 50 μmol/L.
Fresh tablet solutions were prepared, and new sachets were used for every experiment. In addition, all experiments had a set of controls consisting of: a digest with no added iron to ensure no iron contamination of our system; a reference digest of 50 μmol/L Fe added as Fe solubilized in 1 % HCl (High-Purity Standards, 100026-2); and a positive control digest of 50 μmol/L Fe and 500 μmol/L ascorbate.
Iron levels of digests were quantitatively analysed by inductively coupled plasma-optical emission spectrometry (ICP-OES). All iron-containing digests, and undiluted samples of Spatone® and Spatone Apple®, were subjected to microwave digestion using an accelerated reaction system (CEM MARS 5H with XP-1500 vessels). 0.5 mL of the solutions (in triplicate) was added to 5.0 mL concentrated 68 % trace analysis grade nitric acid. Samples were processed for 20 min at 400 psi pressure and 1200 W power. After digestion, samples were reconstituted with 50 mL 18 mΩ water and aliquots used to measure iron levels on a Perkin–Elmer Optima 4300 DV ICP-OES.
Digests were prepared as previously described . Briefly, iron solutions were added to 10 mL of 140 mmol/L NaCl and 5 mmol/L KCl followed by the sequential addition of pepsin (1 h in the shaking incubator, pH 2 digests) and then bile and pancreatin digestive enzymes (1 h in the shaking incubator, pH 7 digests) to mimic the digestive process. 1.5 mL of the above digests was then placed in a chamber suspended over a layer of Caco-2 cells grown on the bottom of the tissue culture wells of a six-well plate. The upper chamber was created using a 15,000 molecular weight cut-off dialysis membrane (Tubing Spectra/Por 7 dialysis membrane, Fisher Scientific) fitted over a Transwell insert and held in place with a silicon ring (Web Seal). Plates were placed on a platform-fitted multi-function 3D rotator (Fisher Scientific PSM3D) set at six oscillations per minute in a 37 °C incubator with a 5 % CO2/95 % air atmosphere at constant humidity for 60 min. Inserts were then removed, and an additional 1 mL of supplemented MEM was added to the cells, which were returned to the incubator for a further 22 h and then harvested for ferritin. All supplements were tested on three separate occasions, with n = 6 for each treatment.
Direct application of iron supplements onto Caco-2 cells to assess dose and treatment duration responses
Caco-2 cells were seeded and maintained as described for in vitro digestion experiments. On the day of the experiment, fresh solutions of Spatone®, Spatone Apple® and ferric ammonium citrate (FAC) were mixed with unsupplemented MEM (herein referred to as MEM) and placed directly onto the cell monolayers at iron concentrations of 10, 30 and 100 μmol/L. In order to assess ferritin formation as a function of treatment duration/time, one set of cells was treated continuously for 24 h, while another set was treated for 4 h (begun 20 h after medium was changed to MEM). At the end of both time periods, cells were harvested for ferritin. Experiments were repeated on three separate occasions with n = 6 for each treatment.
Cell harvest and ferritin analysis
Cells were harvested as previously described . Briefly, rinsed cell monolayers were detached with the proprietary cell lysis buffer CelLytic™ (Sigma-Aldrich) with added protease inhibitor. Cells were shaken for 15 min on ice on an orbital shaker at 300 rpm and then spun at 6,000×g for 6 min in a 5804R Eppendorf centrifuge. The supernatant was aspirated and stored in a −80 °C freezer. For analysis, samples were thawed on ice and ferritin was measured with SpectroFerritin MT Enzyme Linked Immunoassay (ELISA; RAMCO, USA). Final ferritin levels were adjusted for cell protein also measured from the supernatants using Pierce Protein BCA assay (Fisher Scientific, 23227).
Statistical analysis of the data was performed using GraphPad Prism (version 6.0c GraphPad Software, San Diego, CA, USA). Digest experiments were analysed using the statistical methods of Motulsky . Data are presented as mean ± SEM and were analysed by one-way ANOVA followed by Tukey’s multiple comparisons test (all-pairwise across experimental groups); where the comparison was made to a single control, Dunnett’s post hoc test was used. Except as noted, differences between means were considered significant at p ≤ 0.05.
Manufacturers’ information including reported iron content for tested iron preparations
Reported iron content
0.25 mg FeSO4/mL
A Nelson and Co Ltd (UK)
0.20 mg FeSO4/mL
A Nelson and Co Ltd (UK)
Iron Vital F®
1 mg iron-II-gluconate/mL
Anton Hübner GmbH and Co (Germany)
51.4 mg ferrous fumarate equivalent to 17 mg ferrous iron/tablet
Vitabiotics Ltd (UK)
Almus ferrous sulphate tablets
200 mg FeSO4 equivalent to 65 mg ferrous iron/tablet
Almus Pharmaceuticals (UK)
Iron digest levels and Caco-2 cell ferritin after treatment with iron supplements—unadjusted for iron digest levels
Digest iron concentrations (mg/L)
ng ferritin/mg protein (unadjusted)
2.80 ± 0.10 (0 %)
11.82 ± 0.67 (0 %)
Iron Vital F® (IVF)
2.90 ± 0.37 (4 %)
25.76 ± 2.07a (118 %)
Pregnacare Original® (PG)
2.83 ± 0.68 (1 %)
10.35 ± 0.82 (−12 %)
3.40 ± 0.62 (18 %)
12.24 ± 0.91 (4 %)
4.00 ± 0.75* (30 %)
83.94 ± 4.46b (610 %)
Current strategies for the treatment of IDA are based on oral ferrous iron supplements; however, these may be poorly tolerated by patients due to a range of GI side effects [6, 7, 8, 9]. This in turn may lead to poor compliance with therapy, which has consequences for the efficacy of treatment. Iron preparations with higher bioavailability, thus enabling lower absolute doses of iron to be ingested, may cause less GI upset and be better tolerated by patients. This notion is supported by a study undertaken in pregnant women who had been non-compliant with taking FeSO4 tablets and were switched to Spatone iron-rich water; 57 % of subjects were compliant with the new iron supplement compared with 67 % in the controls given placebo (plain water), and dyspepsia scores did not differ between the two groups .
We have compared the bioavailability of a range of commercially available iron supplements using a well-characterized in vitro digestion model [18, 19]. Spatone and ferrous sulphate tablets had equivalent bioavailability in the in vitro model, which is consistent with previous absorption studies in human volunteers . The iron in Spatone® is FeSO4 (i.e. the same as in ferrous sulphate tablets); this suggests that being in liquid form per se does not increase iron bioavailability. Spatone Apple® demonstrated the highest iron bioavailability in the in vitro model, followed by the synthetic liquid iron formulation Iron Vital F®; this result remained highly significant even after adjustment for the increased iron in Spatone Apple® digests. Both Spatone Apple® and Iron Vital F® contain added ascorbate, a known enhancer of iron uptake. While Spatone Apple® has 80 mg ascorbate/sachet (E Hunt, Product and Consumer Information Officer for Nelson and Co., personal communication, 2014) giving an iron:ascorbate molar ratio of 1:16, the iron:ascorbate ratio in Iron Vital F is 1:6 according to the supplement label. The enhancing effects of ascorbate on iron bioavailability are dose dependent ; therefore, the higher ascorbate:iron ratio in Spatone Apple® may explain the difference in iron uptake between the two supplements. Furthermore, Iron Vital F®, according to the manufacturer’s ingredients list, also contains plant extracts, pectin and thickening agents. These dietary factors are sources of polyphenols and phytates, both of which inhibit iron absorption and are only partly counteracted by ascorbate . Interestingly, Vitabiotics Pregnacare® had the same bioavailability as FeSO4, despite having added ascorbate (70 mg/tablet). The iron:ascorbate molar ratio, however, is only 1:4, which may not be optimal for improving iron uptake. In addition, the presence of other minerals contained in Pregnacare®, such as zinc, may partially inhibit iron uptake .
Pregnant women are at high risk of IDA ; however, iron supplement recommendations during pregnancy differ between countries. The WHO and the USA recommend that all pregnant women receive prophylactic prenatal iron [29, 30, 31]. Currently, the UK does not suggest routine prenatal iron supplementation ; historically, pregnant women in the UK were advised to take iron but this was found either to be ineffective  or to have no demonstrated benefit on maternal or foetal outcomes [34, 35]. Recent research may challenge the current UK guidelines ; two large systematic reviews with meta-analyses found that daily prenatal iron reduced maternal anaemia, IDA and risk of foetal low birth weight [37, 38]. However, one of the reviews noted that dose and regimen recommendations for routine iron supplementation need refining and updating , and within this context, our results suggest that further research with the tested formulations used in our study is warranted.
Another group that might benefit from both Spatone Apple® and Iron Vital F® are post-bariatric surgery patients who are at high risk of IDA [39, 40, 41] and in whom treatment with standard iron tablets is often ineffective [42, 43]. In one study, post-operative gastric bypass patients given 100 mg FeSO4 tablets as an oral challenge absorbed inadequate amounts of iron as measured by change in serum iron concentration ; altered gut physiology after bariatric surgery may not allow for iron absorption from FeSO4 tablets. Furthermore, several studies have also documented low vitamin C levels in patients after bypass procedures [45, 46]; therefore, liquid iron preparations with added ascorbate—such as Spatone Apple® and Iron Vital F® (which also has other micronutrients)—may be helpful in this population.
The data presented here for Spatone Apple® suggest that this, or similar products, are highly bioavailable as they gave rise to a rapid increase in cell ferritin in our in vitro model. Furthermore, 10 µmol/L iron given as Spatone Apple® gave a comparable ferritin response to 100 µmol/L FAC and a higher ferritin level than 100 µmol/L FeSO4 (from Spatone®). An important caveat is that we have only performed in vitro studies to date. The bioavailability of Spatone Apple® has not been assessed in human volunteers, and our data suggest that these studies are warranted. The Caco-2 in vitro digestion model provides a validated method for screening iron bioavailability of a range of compounds and test meals. Furthermore, it has been shown to predict the direction, but not necessarily the magnitude, of iron bioavailability in humans . If our in vitro results are confirmed in vivo, it would suggest that lower doses of a more bioavailable iron form could be effective in combating iron deficiency. This would be particularly advantageous for groups who are both at increased risk of IDA and less tolerant to high doses of iron, such as pregnant women.
In conclusion, the results of this in vitro study demonstrate that naturally iron-rich mineral waters, or synthetic liquid iron formulations, are equivalent to the standard of care FeSO4 recommended for IDA, and those with added ascorbate have increased bioavailability. If these results are confirmed in randomized control studies in human volunteers at risk of IDA, such subjects could then be offered a greater choice of more bioavailable and potentially better tolerated iron preparations.
This work was supported by the Faculty of Engineering and Science at the University of Greenwich, and the Diabetes and Nutritional Sciences Division at King’s College London. We thank David Scott Ganis for assistance with statistical and numerical analysis.
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
- 7.Hyder SM, Persson LA, Chowdhury AM, Ekstrom EC (2002) Do side-effects reduce compliance to iron supplementation? A study of daily- and weekly-dose regimens in pregnancy. J Health Popul Nutr 20(2):175–179Google Scholar
- 12.Beard JL (2000) Effectiveness and strategies of iron supplementation during pregnancy. Am J Clin Nutr 71(5 Suppl):1288S–1294SGoogle Scholar
- 13.Ekstrom EC, Kavishe FP, Habicht JP, Frongillo EA Jr, Rasmussen KM, Hemed L (1996) Adherence to iron supplementation during pregnancy in Tanzania: determinants and hematologic consequences. Am J Clin Nutr 64(3):368–374Google Scholar
- 18.Glahn RP, Lee OA, Yeung A, Goldman MI, Miller DD (1998) Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion/Caco-2 cell culture model. J Nutr 128(9):1555–1561Google Scholar
- 19.Yun S, Habicht JP, Miller DD, Glahn RP (2004) An in vitro digestion/Caco-2 cell culture system accurately predicts the effects of ascorbic acid and polyphenolic compounds on iron bioavailability in humans. J Nutr 134(10):2717–2721Google Scholar
- 24.Motulsky H (2010) Intuitive biostatistics, 2nd edn. Oxford University Press, OxfordGoogle Scholar
- 28.Scholl TO (2005) Iron status during pregnancy: setting the stage for mother and infant. Am J Clin Nutr 81(5):1218S–1222SGoogle Scholar
- 29.CDC (1998) Recommendations to prevent and control iron deficiency in the United States. MMWR Recomm Rep, vol 47. Centers for Disease Control and PreventionGoogle Scholar
- 30.WHO (World Health Oreganization) (2012) Guideline: daily iron and folic acid supplementation in pregnant women. Geneva, World Health OrganizationGoogle Scholar
- 31.WHO (World Health Organization) (2012) Guideline: intermittent iron and folic acid supplementation in non-anaemic pregnant women. Geneva, World Health OrganizationGoogle Scholar
- 32.NICE (2008) Antenatal care: routine care for the healthy pregnant woman. National Institute for Clinical Excellence, LondonGoogle Scholar
- 43.Gasteyger C, Suter M, Gaillard RC, Giusti V (2008) Nutritional deficiencies after Roux-en-Y gastric bypass for morbid obesity often cannot be prevented by standard multivitamin supplementation. Am J Clin Nutr 87(5):1128–1133Google Scholar
- 45.Clements RH, Katasani VG, Palepu R, Leeth RR, Leath TD, Roy BP, Vickers SM (2006) Incidence of vitamin deficiency after laparoscopic Roux-en-Y gastric bypass in a university hospital setting. Am Surg 72(12):1196–1202Google Scholar
- 47.Fairweather-Tait S, Lynch S, Hotz C, Hurrell R, Abrahamse L, Beebe S, Bering S, Bukhave K, Glahn R, Hambidge M, Hunt J, Lonnerdal B, Miller D, Mohktar N, Nestel P, Reddy M, Sandber AS, Sharp P, Teucher B, Trinidad TP (2005) The usefulness of in vitro models to predict the bioavailability of iron and zinc: a consensus statement from the HarvestPlus expert consultation. Int J Vitam Nutr Res 75(6):371–374CrossRefGoogle Scholar