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© 2006 American Society for Nutrition J. Nutr. 136:2970-2974, December 2006


Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Chili, but Not Turmeric, Inhibits Iron Absorption in Young Women from an Iron-Fortified Composite Meal1

Siriporn Tuntipopipat2, Kunchit Judprasong2, Christophe Zeder3, Emorn Wasantwisut2, Pattanee Winichagoon2, Somsri Charoenkiatkul2, Richard Hurrell3 and Thomas Walczyk3,*

2 Institute of Nutrition, Mahidol University, Nakhon Pathom, Thailand and 3 Laboratory of Human Nutrition, Institute of Food Science and Nutrition, CH-8092 Zurich, Switzerland

* To whom correspondence should be addressed. E-mail: thomas.walczyk{at}ilw.agrl.ethz.ch.


    ABSTRACT
 TOP
 ABSTRACT
 Introduction
 Material and Methods
 Results
 Discussion
 LITERATURE CITED
 
Chili and turmeric are common spices in indigenous diets in tropical regions. Being rich in phenolic compounds, they would be expected to bind iron (Fe)3 in the intestine and inhibit Fe absorption in humans. Three experiments were conducted in healthy young women (n = 10/study) to assess the effect of chili and turmeric on Fe absorption from a rice-based meal containing vegetables and iron fortified fish sauce in vivo. Iron absorption was determined by erythrocyte incorporation of stable isotope labels (57Fe/58Fe) using a randomized crossover design. Addition of freeze-dried chili (4.2 g dry powder, 25 mg polyphenols as gallic acid equivalents) reduced Fe absorption from the meal by 38% (6.0% with chili vs. 9.7% without chili, P = 0.0017). Turmeric (0.5 g dry powder, 50 mg polyphenols as gallic acid equivalents) did not inhibit iron absorption (P = 0.91). A possible effect of chili on gastric acid secretion was indirectly assessed by comparing Fe absorption from acid soluble [57Fe]-ferric pyrophosphate relative to water soluble [58Fe]-ferrous sulfate from the same meal in the presence and absence of chili. Chili did not enhance gastric acid secretion. Relative Fe bioavailability of ferric pyrophosphate was 5.4% in presence of chili and 6.4% in absence of chili (P = 0.47). Despite the much higher amount of phenolics in the turmeric meal, it did not affect iron absorption. We conclude that both phenol quality and quantity determine the inhibitory effect of phenolic compounds on iron absorption.



    Introduction
 TOP
 ABSTRACT
 Introduction
 Material and Methods
 Results
 Discussion
 LITERATURE CITED
 
Iron deficiency is the most common micronutrient deficiency in the world (1). Iron deficiency, and consequently iron deficiency anemia (IDA),4 develops when iron intake does not meet iron requirements over the long term. Children and women of childbearing age, with higher demands for iron due to growth and menstrual blood loss, are most vulnerable. Low iron status is more frequently observed in populations living on plant-based diets that are low in muscle tissue (2). Although meat can provide highly bioavailable heme iron (3) and is known to enhance dietary nonheme iron absorption (47), plant-based diets are commonly high in phytic acid and polyphenolic compounds as potent inhibitors of iron absorption. Primary sources of polyphenols in the diet are vegetables and beverages, including wine. Herb tea, black tea, or coffee strongly inhibit iron absorption in a dose-dependent manner from a simple meal of bread (8). Similar observations have been made in humans for phenolic-rich extracts from rosemary (9). In Thailand, the local vegetable Yod kratin (lead tree leaves, Leucaena glauca), reduced iron absorption by 90% in a composite meal of rice, fish, and vegetables (10).

Herbs and spices are used extensively as condiments in countries across the tropical belt, including Thailand (1113), and may affect iron absorption due to high phenolic content (14). In the present study, we evaluated the effect of chili and turmeric, two of the most common spices in Asia, on iron absorption. Chili in its various forms (genus Capsicum) contains high amounts of phenolic compounds, including capsaicin (15,16). On the other hand, peppers (including chili) and capsaicin were found to increase gastric acid secretion in the rat model (1719). Gastric acid is essential for releasing iron from the food matrix and for solubilization in the stomach (20). This refers in particular to iron compounds that are poorly soluble in water and need to be dissolved in the gut to make the iron accessible. Turmeric (Curcuma longa), the other spice under investigation, is used extensively in the Indian subcontinent and parts of South East Asia (1113). Turmeric is well known for its antioxidative and anti-inflammatory properties (21,22). Curcumin, a low molecular weight polyphenolic diketone, is the most active constituent of turmeric and forms complex solubilized iron in aqueous solution (2325).

The aim of the present study was to 1) evaluate the effect of chili pepper and turmeric on human iron absorption and 2) identify in vivo a possible effect of chili on gastric function by comparing iron absorption from ferric pyrophosphate as an acid-soluble iron compound to ferrous sulfate as a water-soluble iron compound in the presence and absence of chili. Absorption studies were conducted in Thailand by stable isotope techniques and based on erythrocyte incorporation of iron stable isotopic labels from an iron-fortified, rice-based meal.


    Material and Methods
 TOP
 ABSTRACT
 Introduction
 Material and Methods
 Results
 Discussion
 LITERATURE CITED
 
    Subjects. Thirty apparently healthy, adult women (~18–35 y of age with a maximum body wt of 60 kg) were recruited at Mahidol University, Thailand. Exclusion criteria included pregnancy, lactation, gastrointestinal disorders, or metabolic diseases. The absence of thalassemia was verified by hemoglobin typing using a commercial HPLC-based auto-analyzer (Variant, Bio-Rad). The volunteers were asked to refrain from vitamin or mineral supplements and medication, except oral contraceptives, starting 2 wk before the study until the last blood sample was drawn. The study protocol was approved by the Ethical Committees of Mahidol University, Thailand, and ETH Zurich, Switzerland. Written informed consent was obtained from all volunteers.

    Study design. Thirty women were randomly allocated to the 3 separate studies. All volunteers consumed 2 test meals on consecutive days: reference meal (R) and reference meal + chili (R+C) in study 1; and R and reference meal + tumeric (R+T) in study 2. Meal R consisted of rice and a vegetable soup that was seasoned with iron-fortified fish sauce. Isotopically labeled iron was added as ferrous sulfate to the fish sauce in the meals and administered in a randomized crossover design (R/R+C or R+C/R).

Iron absorption from isotopically labeled [57Fe]-pyrophosphate was measured from meal R in absence (meal P) or presence of chili (meal P+C). Iron absorption from meals P and P+C was compared directly in each subject to iron absorption from water-soluble [58Fe]-FeSO4 (meal R). Test meals were administered pair wise (R/P and R/P+C) on 2 consecutive days using a randomized crossover design with a 2 wk interval between the 2 feeding regimens.

All test meals were consumed in the morning between 0700 and 0900 after an overnight fast under standardized conditions and under close supervision of the investigators. Before serving the first meal, a venous blood sample was drawn to determine iron status parameters (hemoglobin and plasma ferritin concentration) and body weight and height were measured. In addition, C-reactive protein (CRP) concentration was measured to determine the presence of infection. No intake of food or fluids was allowed for 3 h after test meal intake. A second venous blood sample was drawn 14 d after intake of the second test meal (d 16). In study 3, a third blood sample was taken 14 d after administration of the last test meal (d 31). Recovery of isotopic labels in the drawn blood samples were used to calculate fractional iron absorption.

    Test meals. All test meals were based on a basic Thai-style dish used in an earlier study (26). The basic meal consisted of steamed white rice (50 g dry weight) served with a soup prepared from local vegetables (50 g white cabbage, 50 g Chinese cabbage, 30 g Thai mushrooms and 20 g steamed carrots). Vegetables and rice were purchased in bulk and individual portions were prepared using standardized procedures (26). For consumption, 120 mL of water and 12 mL commercial fish sauce were added to the defrosted vegetables and the soup was warmed in a microwave oven using a standardized heating program.

Each meal contained ~4 mg of isotopically labeled fortification iron, either as [57Fe/58Fe]-ferrous sulfate or [57Fe]-ferric pyrophosphate (see Table 1). In studies 1 and 2, isotopically labeled ferrous sulfate was added to the fish sauce before adding to the soup. This corresponds to the use of ferrous sulfate fortified fish sauce at a fortification level of ~350 mg Fe/L (26). In study 3, [58Fe]-ferrous sulfate was added likewise to the fish sauce (meal R), whereas meals P and P+C were prepared by adding [57Fe]-ferric pyrophosphate to the steamed rice and by seasoning the vegetable soup with unfortified fish sauce.


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TABLE 1 Administered test meals and added amounts of isotopically labeled iron and spices1

 
For preparation of test meals R+C and P+C, 4.2 g of ground lyophilized Thai chili (Capsicum annuum) was added to the soup shortly before consumption (14.2 g fresh wt, 25.2 ± 0.2 mg polyphenols as gallic acid equivalents). Test meal R+T was prepared by steaming 0.50 g ground lyophilized turmeric per serving (2.8 g fresh wt containing 50.13 ± 0.06 mg total polyphenols as gallic acid equivalents) with the rice. Ferric pyrophosphate was weighed in individual portions in plastic containers and added to the meal. The exact amount added to the meal was determined by reweighing the plastic containers. High purity water (18 mol/L{Omega}, 250 g) was served as a drink with all test meals. Quantitative intake of isotopic labels was ensured by rinsing the bowls 3 times with 10 mL deionized water and by letting the subject consume the washings.

    Preparation of isotopically labeled iron. Isotopically labeled [57Fe]-ferrous sulfate and [58Fe]-ferrous sulfate were prepared at ETH Zurich by dissolution of isotopically enriched elemental iron ([57Fe]-metal: 95.9% enriched; [58Fe]-metal: 93.2% enriched; both Chemgas) in diluted sulphuric acid. [57Fe]-Ferric pyrophosphate was prepared in food grade quality from 57Fe enriched elemental iron ([57Fe]-metal: 95.9% enriched, Chemgas) by Lohmann Chemicals (Emmerthal). The compound was prepared using a scaled-down procedure used for the production of the commercial product. Eighteen hours before the test meal was administered, labeled iron solutions ([57Fe]-ferrous sulfate and [58Fe]-ferrous sulfate) were added gravimetrically to the fish sauce in bulk along with citric acid (3 g/L) and potassium iodide (4.2 mg/L) to comply with the formulation of the fortified product previously described (26).

    Iron absorption measurements. Isotopically labeled compounds and collected blood samples were analyzed for isotopic composition at ETH Zurich by multicollector negative thermal ionization mass spectrometry as described earlier (27,28). Based on the shift of the Fe isotope ratios in the blood samples and the amount of Fe circulating in the body, amounts of 57Fe and 58Fe isotopic labels presented in the blood 14 d after test meal administration were calculated based on the principles of isotope dilution and considering that the used Fe isotopic labels were not mono-isotopic (28). Circulating Fe was calculated based on blood volume and hemoglobin concentration (29). Blood volume calculations were based on height and weight according to Brown et al. (30). For calculations of fractional iron absorption, 80% incorporation of the absorbed Fe into red blood cells was assumed (31).

    Blood analysis. Venous blood samples (5 mL) were collected in EDTA-treated tubes and analyzed for complete blood counts (CBC) using an ADVIA 120 Hematology System (Bayer). Plasma ferritin (normal range: 15–300 µg/L) and CRP (<5 g/L in absence of infection) were analyzed by chemiluminescent immunometric assay (IMMULITE/Ferritin and IMMULITE/High Sensitive CRP, DPC, CA) using low- and high-level quality control materials supplied by the company.

    Food analysis. Food samples were analyzed for iron and calcium by flame atomic absorption (SpectrAA 400, Varian) after mineralization by wet digestion using a HNO3/perchloric acid mixture. The phytic acid content was determined by anion-exchange chromatography (32). Ascorbic acid was analyzed by HPLC according to the methods described by Sapers (33) and Parviainen and Nyssonen (34) using a reversed phase column and photometric detection. Dehydroascorbic acid was reduced to ascorbic acid prior to analysis by addition of dithiothritol (Fluka). Total polyphenol content as gallic acid equivalents was determined by the Folin-Ciocalteu method (35).

    Statistics. All calculations and statistical analyses were performed using commercially available spreadsheet programs and in-house calculation programs developed by ETH Zurich. Iron absorption was logarithmically transformed before statistical analysis. Student paired t test was used to evaluate data within each study. Student unpaired t test was used to compare relative bioavailability (RBV) of ferric pyrophosphate to ferrous sulfate between studies 3.1 and 3.2. Results are presented as means (± 1 SD) unless otherwise noted. Differences in iron absorption between test meals were considered significant at P < 0.05. Studies were powered to resolve a 30% difference in iron absorption between test meals using each volunteer as her own control (studies 1–3) and to resolve a 50% difference in RBV in study 3 (95% CI).


    Results
 TOP
 ABSTRACT
 Introduction
 Material and Methods
 Results
 Discussion
 LITERATURE CITED
 
    Subjects. None of the volunteers were anemic. Their hemoglobin concentration was 134 g/L (range: 124–148 g/L). Plasma ferritin concentrations were in the normal range for most subjects (geometric mean: 53 µg /L, range: 13–262). Two subjects in study 2 and 1 subject in study 3 were marginally iron deficient (plasma ferritin concentration <15.0 µg/L). One subject showed a marginally elevated CRP concentration of 5.6 g/L, indicative of a mild infection (mean concentration: 0.9 g/L, range: 0.1–5.6 for all subjects).

    Test meals. Total iron content of the meal was 4.70–4.84 mg (4.00 mg from fortified compound, 0.56 mg from vegetables, 0.14 mg from rice, and 0.14 mg from chili) (Table 2). Calcium contents were low (29.4–32.7 mg/meal). Phytic acid was higher in test meals with added chili (76.2 mg, meals R+C and P+C) than meals R and R+T (58.9 mg). Vitamin C was found to be negligible in all meals, which can be attributed to the warming of meals before serving and the use of lyophilized chili and turmeric.


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TABLE 2 Content of iron, calcium, phytic acid and ascorbic acid of test meal ingredients

 
    Iron absorption. Results are presented as geometric means (–1 SD, +1 SD) for iron absorption (studies 1–3) and as RBV relative to ferrous sulfate for ferric pyrophosphate (study 3) (see Table 3). In study 1, iron absorption from ferrous sulfate fortified fish sauce with and without addition of chili was 6.0 (2.2, 16.5)% and 9.7 (3.9, 24.6)%, respectively. This translates into an inhibition of iron absorption by 38% from the added chili (P = 0.0017). In study 2, turmeric had no influence on iron absorption. Mean iron absorption was 8.9 (3.2, 24.4)% in the presence of tumeric and 8.7 (3.8, 19.8)% in the absence of turmeric (P = 0.91). In study 3.1, iron absorption from ferric pyrophosphate was much lower than that from ferrous sulfate (geometric mean 0.7 vs. 10.4%, P < 0.0001). In study 3.2, the addition of chili pepper did not improve iron absorption from ferric pyrophosphate. Thus, chili did not significantly effect iron absorption from ferric pyrophosphate relative to ferrous sulfate (RBV with chili was 5.4% vs. without chili 6.4%, P = 0.47).


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TABLE 3 Effect of chili (studies 1 and 3) and turmeric (study 2) ingestion on iron absorption in women after a basic test meal1

 
Based on the observed intra- and inter-individual differences in iron absorption, the power of the study (95% CI) would have allowed detection of a minimal relative difference in iron absorption between test meals of 32% in study 1, 49% in study 2, 26% in study 3.1, and 49% in study 3.2, respectively. The detection limit for a relative difference in RBV in study 3 was 50%.


    Discussion
 TOP
 ABSTRACT
 Introduction
 Material and Methods
 Results
 Discussion
 LITERATURE CITED
 
In this study we have demonstrated that freeze-dried, ground chili pepper (14.2 g fresh wt; 25 mg polyphenols as gallic acid equivalents) reduced iron absorption from a basic rice and vegetable meal by 38% (P = 0.0017). A cross-sectional pilot dietary survey in Northeast Thailand (36) showed that chili intake varies considerably in adult Thais depending on their preference for spicy foods. The intake of chili per dish at each meal was assessed by weighing individual ingredients of popular northeast dishes and ranged from 1.0 to 17.2 g chili (fresh wt), which corresponds to 0.25–4.0 g dry wt. Thus, the amount of chili in our test meals was on the high end but still within the range of habitual chili intake. The polyphenol content of the fresh chili, however, was at 620 mg/100 g, which was relatively low compared with the range of 750–1300 mg/100 g that we found in 8 samples obtained from different local markets in Bangkok and Northeast Thailand during the dry and wet season.

By adding a relatively large amount of chili to the meal, phytic acid content was increased from 57 to 76 mg in the meal. However, at a molar ratio of 1.3:1 of phytic acid to iron in the chili meal, it appeared unlikely that phytic acid had a significant effect on iron absorption (37). This allowed us to conclude that iron binding to phenolic compounds is the primary mechanism by which chili pepper inhibits nonheme iron absorption in humans. The known enhancing effects of chili and capsaicin on gastric acid secretion are apparently not of relevance to iron nutrition. Chili had no significant effect on iron absorption from acid-soluble ferric pyrophosphate relative to ferrous sulfate (RBV with chili 5.4% vs. without chili 6.4%, P = 0.47) in our study. Higher output of gastric juice and a lowering of gastric pH from chili would have resulted in an increased dissolution of ferric pyrophosphate and, therefore, an increase in RBV.

Although chili inhibited iron absorption, turmeric did not, despite the greater content of total phenols in the meal containing added turmeric. Total phenols amounted to 25 mg and 50 mg for added chili and turmeric, respectively. In an earlier study, intake of 20–50 mg total polyphenols from black tea or herb tea containing polymers of flavonoid-gallic acid esters and monomeric flavonoids reduced iron absorption from an iron-fortified bread roll by 50–70% (8). This compares well with our findings for chili, but not for turmeric, in our more complex meal. These apparently inconsistent observations reflect the principal difficulties in assessing the effect of plant polyphenols on iron absorption. Phenolic metabolism in plants is complex and yields a wide array of compounds ranging from monomeric tannins and flavonoids and their condensation and glycosylation products to the complex lignans of the plant cell wall (38). Accordingly, polyphenol composition and structures vary substantially between plant species and even within the plant. In turmeric, curcumin is the major bioactive compound and contains 2 phenolic groups and 1 diketone group whereas capsaicin is the most prominent phenolic compound in chili but contains only 1 phenolic hydroxyl group (39) (see Fig. 1). At the same time, both spices contain the flavonoid quercetin (Fig. 1) but at much different concentrations, i.e., 92.5 mg/kg dry wt for turmeric compared with 829 mg/kg dry wt for chili (15,16).


Figure 1
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Figure 1  Chemical structures of curcumin, capsaicin, and quercetin as major phenolic compounds in turmeric and chili pepper.

 
The difficulty in making comparisons across studies or between different plant foods or polyphenol-containing drinks lies in the structural diversity of the phenolic compounds. Although phenolic compounds can bind iron, in principle, they may differ considerably in the mechanisms of iron binding as well as in their potency to make iron unavailable for absorption at intestinal pH. As an example, capsaicin can bind iron only through its single phenolic hydroxyl group whereas curcumin can bind iron both through interaction with its 2 aromatic hydroxyl groups as well as by chelation through its diketone functionality (23,24,40). The stability of the formed complexes depend not only on pH and redox potential of the digested food but also on interaction with other iron-binding compounds such as phytic acid and ascorbic acid in the gut, which are difficult to predict for a complex meal such as the one used in our study. These differences are not reflected in the total phenol contents as determined by the Folin-Ciocalteau method. The Folin-Ciocalteau reagent oxidizes phenolates and reduces the heteropoly acids to a blue molybdenum-tungsten complex (35). The method delivers, therefore, only an estimate for the total number of aromatic hydroxyl groups and no information about the stability of the iron complex at gastric pH. Other available methods for polyphenol analysis are only sensitive to certain phenolic structural units, such as condensed polyphenols (proanthocyanidins) for the vanillin assay (41). HPLC analysis allows quantification of individual phenolic compounds in a sample (42) but, likewise, delivers no information about their potency to inhibit iron uptake in the gut. In conclusion, dietary contents of phenolic compounds per se, despite the applied analytical method, are of limited value in predicting their inhibitory effect on iron absorption in vivo. Although phenolic content may serve as a qualitative predictor of iron bioavailability, it is difficult or even impossible to predict its quantitative effect on iron absorption by the human body, which became apparent in our study.

The findings from our study are important insofar as chili is a common spice across the tropical belts. However, our findings do not necessarily indicate that chili intake is a potential risk factor for iron nutrition. The basic test meal in our study was fairly neutral regarding enhancers and inhibitors of iron absorption. In a meal that is already high in phytic acid and polyphenols, the additional polyphenol intake from chili may have a much less pronounced effect on iron absorption. The effect of polyphenols on iron absorption was found to reach a plateau at ~100–200 mg polyphenols per serving (8,42). In addition, the inhibitory effect of chili would be expected to be lower with the use of fresh chili instead of dried chili powder as used in our study. Lyophilization of chili would be expected to decrease its content in ascorbic acid as a known enhancer of iron absorption. For both chili and turmeric, the presence of a dose-response effect must also be considered when setting findings into a dietary context. Although amounts of chili were relatively high in our meals, as typically found in Southeast Asia, the Thai meal under investigation contained relatively low amounts of turmeric. At higher amounts, such as those used in the Indian subcontinent, turmeric may have an inhibitory effect on iron absorption, but this remains to be investigated.


    ACKNOWLEDGMENTS
 
We thank Dr. Ratchanee Kongkachuichai and Dr. Visith Chavasith from the Institute of Nutrition at Mahidol University for technical assistance; Ms. Pudsadee Siriprapa, Ms. Atitada Boonpraderm, Ms. Napaporn Riabroy, Mr. Somjai Klinkuan, Mr. Sueppong Gowachirapant, and Ms. Laksana Chaimongkol for test meal preparation.


    FOOTNOTES
 
1 Supported by the International Atomic Energy Agency (IAEA) Vienna under the Doctoral Coordinated Research Project on isotopic and complementary tools for the study of micronutrient status and interactions in developing country populations. Back

4 Abbreviations used: CRP, C-reactive protein; P: reference meal with iron added as [57Fe]-ferric pyrophosphate; P+C, reference meal with added chili and iron added as [57Fe]-ferric pyrophosphate; RBV, relative bioavailability; R, reference meal with iron added as [57Fe/58Fe]-FeSO4; R+C, reference meal with added chili and iron added as [57Fe/58Fe]-FeSO4; R+T, reference meal with added turmeric and iron added as [57Fe/58Fe]-FeSO4. Back

Manuscript received 31 May 2006. Initial review completed 7 July 2006. Revision accepted 14 September 2006.


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 Material and Methods
 Results
 Discussion
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