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Articles

Calcium Intake Is Weakly but Consistently Negatively Associated with Iron Status in Girls and Women in Six European Countries¹

L.P.L. van de Vijver^*,2, A.F.M. Kardinaal^*, J. Charzewska, M. Rotily^**, P. Charles, M. Maggiolini, S. Ando, K. Väänänen, B. Wajszczyk, J. Heikkinen^#, A. Deloraine^§ and G. Schaafsma^*,3

^* Division of Human and Animal Nutrition, TNO Nutrition and Food Research Institute, 3700 AJ Zeist, The Netherlands, Department of Epidemiology, National Institute for Food and Nutrition, 02-903 Warsaw, Poland, ^** ORS PACA INSERM and CIC APHM-INSERM, 13006 Marseille, France, Osteoporosis Clinic, Aarhus Amtssygehus, 8000 Aarhus, Denmark, Centro Sanitario, University of Calabria, 87030 Arcavacata di Rende, Italy, Department of Anatomy, University of Oulu, 90220 Oulu, Finland, ^# Deaconess Institute of Oulu, 90100 Oulu, Finland, and ^§ CAREPS, 38043 Grenoble, France

²To whom correspondence should be addressed.

	ABSTRACT

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Several studies indicate that intake of calcium can inhibit iron absorption especially when taken simultaneously. In the CALEUR study, a cross-sectional study among girls (mean 13.5 y) and young women (mean 22.0 y) in six European countries, the association between calcium intake and iron status was studied. In 1,080 girls and 524 women, detailed information on calcium intake was collected by means of a 3-d food record, and serum ferritin, serum iron, serum transferrin and transferrin saturation were measured as indicators of iron status. The mean levels of serum iron, ferritin and transferrin were 15.8 ± 6.1 mmol/L, 34.5 ± 19.1 µg/L and 3.47 ± 0.47 g/L, respectively, in girls and 16.9 ± 7.5 mmol/L, 40.2 ± 30.5 and µg/L, 3.59 ± 0.60 g/L, respectively, in women. A consistent inverse association between calcium intake and serum ferritin was found, after adjusting the linear regression model for iron intake, age, menarche, protein, tea and vitamin C intake and country, irrespective of whether calcium was ingested simultaneously with iron. The adjusted overall regression coefficients for girls and women were -0.57 ± 0.20 and -1.36 ± 0.46 per 100 mg/d increase in calcium intake, respectively. Only in girls, transferrin saturation as a measure for short-term iron status was inversely associated with calcium intake (adjusted overall coefficient -0.18 ± 0.08). However, analysis per country separately showed no consistency. We conclude that dietary calcium intake is weakly inversely associated with blood iron status, irrespective of whether calcium was ingested simultaneously with iron.

KEY WORDS: • dietary calcium • iron status • ferritin • transferrin saturation • cross-sectional • girls • women

	INTRODUCTION

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Iron deficiency is the most prevalent nutritional deficiency in the world, highest in developing countries. Also in developed countries some populations show a high incidence of iron deficiency (Turnlund et al. 1990 ). Differences in bioavailability of dietary iron may be one of the factors explaining regional differences (Galan et al. 1991 ). Less than 10% of daily iron intake has to be absorbed to compensate for daily losses (up to 20% for menstruating and pregnant women). Therefore, factors determining absorption may be more relevant to iron status than the absolute intake of iron (Passmore and Eastwood 1986 ). Also, the form in which iron is present in the diet is important. Heme iron is absorbed more efficiently than nonheme iron and is thought to be less affected by other dietary constituents (Fairbanks 1994 ).

Studies of Hallberg et al. (1991 , Hallberg et al. 1992a , Hallberg et al. 1992b ) showed that calcium inhibits iron absorption when taken during the same meal. This inhibitory effect was observed for both heme and nonheme iron. The inhibitory effect of calcium seems to be limited to the meals in which both calcium and iron are consumed (Cook et al. 1991 , Dawson et al. 1986 , Deehr et al. 1990 , Galan et al. 1991 , Gleerup et al. 1993 , Hallberg et al. 1992a ). Further, a dose-dependent relationship was found in which calcium intake in excess of 300 mg/d did not lead to further inhibition of iron absorption (Hallberg et al. 1991 ).

The risk of iron deficiency is elevated in rapidly growing persons such as teenagers and in women during their reproductive years. Thus far, studies have focused mainly on the influence of supplementary calcium intake on iron absorption (Hallberg et al. 1991 , Hallberg et al. 1992a , Hallberg et al. 1992b ). To study the influence of nonsupplement calcium intake on iron in a group of persons most at risk of iron deficiency, we investigated the association between calcium intake and iron status in the CALEUR study, a large cross-sectional study among girls and women in six European countries. (Kardinaal et al. 1999 )

	MATERIALS AND METHODS

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The CALEUR study was conducted in Denmark, Finland, France, Italy, The Netherlands and Poland, initially to study the association between calcium intake and peak bone mass (Kardinaal et al. 1999 ). In each of the participating countries, girls aged 11–15 y and young adult women aged 20–23 y with high and low calcium intake were selected from random population samples of about 750 girls and 375 women. All subjects were of Caucasian origin. In Finland (Oulu), Denmark (Aarhus), Poland (Warsaw) and The Netherlands (Zeist), random samples from the local population registries were obtained and subjects invited to participate; response rates varied from 25.4 to 51.5% for girls and from 28.6 to 62% for women. In France, girls and women were recruited via general practitioners and gynecologists in two geographical areas, Rhone-Alps and Marseille; in Italy, girls were recruited from all eight secondary schools in the town of Rende, and women from the University of Calabria (response rates were 100%). Of those selected, the participation rate varied between 64 and 84% for girls and between 52 and 91% for women: data were collected for 1,116 girls and 526 women. The study was approved by local medical-ethical committees, and all participants (and their parents, if required) gave their informed consent. Subjects were excluded when indicating a chronic disease in general, diseases related to bone or calcium metabolism in particular, use of corticosteroids, participating in sports more than 7 h/wk, current or prior pregnancy, menstruation at irregular intervals (for the women only), vegetarianism or macrobiotism or a prescribed diet other than an energy-restricted diet.

Dietary intake.

To estimate calcium intake in a comparable way in the six countries, a 3-d food record method was used. The subjects were asked to record everything they consumed during a consecutive Wednesday, Thursday and Friday, the week before their visit to the institute. Time of day, food, quantity and recipes of composite dishes were recorded. The parent responsible for meal preparation was asked to assist in completing the food records. At the visit to the institute, the food records were checked by a dietitian for completeness; household measures were verified by comparison with standard measures. Daily consumption of food products in grams were converted to nutrient intake using local food composition tables. Mean intakes of calcium, iron and energy were calculated as the average over 3 d.

Height and weight were measured with the subjects wearing light clothing and no shoes. Subjects completed a self-administered questionnaire on menstrual function and use of oral contraceptives, smoking habits, alcohol use, time spent outdoors, height, weight and education of parents and physical activity. Physical activity was determined for the previous month and covered activities at school, at work and in leisure time (sports and household activities). For the girls, the questionnaire comprised 58 items, and for the women 88. The questionnaire was checked in an interview setting.

A 10-mL blood sample was drawn from nonfasting subjects in a 10-mL tube with clot activator and cooled to 4°C. Serum was prepared within 2 h by centrifugation at 3000 x g for 10 min. Serum was stored at -20°C. Serum ferritin was measured as the main marker for iron status. Serum ferritin levels reflect stored iron. Serum iron and the main iron-binding protein, transferrin, reflect the iron in transit and were used to calculate the transferrin saturation {(transferrin saturation (%) = iron (g/L)/[transferrin (g/L) x 1.41)]} (Wick 1996 ). Transferrin saturation can be used as a short-term marker of iron concentration (Wick 1996 ). Serum iron, transferrin and ferritin were measured with the Hitachi 911 (Boehringer Mannheim, Mannheim, Germany). Ferritin and transferrin were analyzed immunoturbidimetrically with a ferritin test kit (No. 1661400; Boehringer Mannheim) and a transferrin test kit (No. 1360752; Boehringer Mannheim). Serum iron was analyzed after separation of Fe³⁺ from transferrin. After reduction, Fe²⁺ forms colored complexes with FerroZine(TM) (Hach Chemical Co., Ames, IA). The coefficient of variation for the analysis of serum ferritin was 8.1 and 3.9% at mean values of 30 and 60 µg/L in the quality control samples, respectively. The variation coefficient for analysis of transferrin was 2.5% at a mean level of 3.3 g/L and for analysis of serum iron the variation coefficient was 1.8% at a mean value of 18 µmol/L in the quality control sample.

Blood samples were available for 1,083 girls and 525 women. In a selection of samples with extremely high or low ferritin levels (ferritin <3.0 or >10.0 µg/L), duplicate measurement was performed. When the second measurement was comparable to the first measurement, the first value was used. Samples of four subjects (three girls and one woman) were excluded from statistical analyses because of instability of the sample, leaving data for 1,080 girls and 524 women for analysis.

Statistical analyses.

Mean and SD of serum iron, serum ferritin, serum transferrin and transferrin saturation and of dietary intake levels were calculated. Pearson correlations between potential confounding factors and calcium intake and parameters for iron status were calculated. As potential confounders, age, height, weight, menses, smoking, tea and coffee consumption, alcohol consumption, energy intake, protein intake and vitamin C intake were considered. Variables associated with calcium intake and serum ferritin levels or transferrin saturation were included as a covariable in the statistical models with serum ferritin or transferrin saturation as independent variables. Categorical variables were put into the model as dummy variables. Though the covariables were not significantly associated with all the parameters of iron status, we choose to use a fixed set of covariables in the statistical models. With analysis of covariance, mean levels of the serum iron parameters were calculated per quartile of calcium intake, adjusted for the covariables. Linear regression analysis was used to calculate the effect of calcium intake on serum levels overall and per country, unadjusted and adjusted for covariables. The influence of the simultaneous intake of iron and calcium during a meal was measured by dividing calcium intake into calcium ingested simultaneously with iron and the remaining calcium. Simultaneous intake of iron and calcium was defined as a moment of the day when at least 20% of the total daily iron and at least 20% of the total daily calcium was consumed. For statistical analysis the BMDP statistical package was used (BMDP version 7.0; BMDP Statistical Software Inc., Los Angeles, CA). P values <0.05 were considered significant.

	RESULTS

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The study sample included girls with a mean age of 13.5 ± 1.5 y (37.8% were premenarcheal) and women with a mean age of 22.0 ± 1.1 y. Use of oral contraceptives was reported by 1.0% of the girls and 48.1% of the women. Smoking was reported by 7.0% of the girls and 29.4% of the women.

The ranges for (serum iron, serum ferritin, serum transferrin and the transferrin saturation) the different countries were comparable (Table 1 ).For the pooled data, serum iron ranged from 2.9 to 46.8 mmol/L for girls and from 2.1 to 45.1 mmol/L for women. Ranges for serum ferritin and serum transferrin were 2.3–161.1 µg/L and 2.0–5.3 g/L, respectively, for girls and 1.1–191.6 µg/L and 2.1–5.5 g/L, respectively, for women. Transferrin saturation ranged from 3.4 to 60.3% for girls and from 3.0 to 63.3% for women, with the lowest levels in Italy and the highest in Denmark. The lowest levels of serum iron and serum ferritin were seen in Finland, both for girls and women. Low-serum iron and ferritin levels were also seen in Italian women. Latent iron deficiency (ferritin <12 µg/L) was found in 4.3% of the girls and 7.4% of the women (Fig. 1 ).

View larger version (37K): [in this window] [in a new window]   Figure 1. FIG. 1 . Percentage of women and girls in each country with latent iron deficiency (ferritin < 12 µg/L).

In Table 3 the mean levels of serum iron, ferritin, transferrin and transferrin saturation per quartile of calcium intake are presented. The means were adjusted for a fixed set of covariables, age, country, tea, protein, vitamin C and iron consumption and for menses (in girls only). Adjusted means differed significantly between quartiles of calcium intake for transferrin levels in girls and serum iron and transferrin saturation in women.

The dose dependence of the association between calcium intake and serum ferritin levels was checked over strata of calcium intake levels. A division was made between intake levels of <300, 300–600, 600–900, 900–1200, and >1200 mg/d. No indication of a threshold effect was seen. None of the linear regression coefficients in the strata showed a significant association between calcium intake and serum ferritin levels. In all strata an inverse association was observed except for a positive linear regression coefficient in women with a calcium intake lower than 300 mg. No trend could be detected.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this cross-sectional study, performed in six European countries, a weak but consistent inverse association was seen between calcium intake and serum ferritin status, irrespective of whether calcium was ingested simultaneously with iron. Further, no dose-response relationship between calcium intake and serum ferritin was detected.

The study was conducted in six countries throughout Europe, which contributed to a wide variety in intakes of both calcium and iron. In each country, two groups of subjects were recruited, a group of girls and a group of young women. To ensure comparability of the data between groups and among countries, all procedures were similar. Because the study populations are not necessarily representative for the countries, prudence is called for when using these data as a measure of intake for a specific country. Also, blood was collected according to the same protocol, and all blood samples were analyzed centrally in one laboratory.

Dietary assessment took place on three consecutive days. From the Dutch National Food Consumption Survey, it was calculated that the day-to-day variation in calcium intake in The Netherlands was small (Van Erp-Baart 1996 ). A 3-d dietary record could thus give a representative estimate of usual calcium intake. Day-to-day variation in iron is expected to be more pronounced and consequently the 3-d record is not likely to accurately reflect habitual individual intake. On the other hand, measurement of serum iron parameters variable is also exposed to biological and analytical variances. The consequence of these factors is that whenever an association really exists, the association is either not found, or it seems to be weaker than in reality.

In this study dietary information was collected at different periods over the year for the separate countries. Therefore, comparison of intakes between countries can give a biased view. Blood collection took place in the same period as the dietary assessment was made. Because blood levels reflect intake, seasonality plays only a minor role in the association between calcium intake levels and iron status.

Several studies show an inhibitory effect of calcium intake on iron absorption (Deehr et al. 1990 , Cook et al. 1991 , Galan et al. 1991 , Hallberg et al. 1992a ). In these intervention studies, calcium was provided via a supplement or added to the meal (Hallberg et al. 1991 , Hallberg et al. 1992a , Hallberg et al. 1992b ). Some studies provide extra yogurt or milk during the meal (Gleerup et al. 1993 , Gleerup et al. 1995 ). An effect of calcium on iron absorption was reported to be most pronounced when calcium was provided during the same meal in which iron was consumed. Gleerup et al. (1993) showed that calcium given 2 or 4 h before a meal had no inhibitory effect on iron absorption. Some studies, however, did not find an inhibitory effect of milk on iron absorption (Turnlund et al. 1990 , Tidehag et al. 1995 ), and in a study among lactating Gambian women, no effect of calcium supplementation on serum ferritin level was observed (Yan et al. 1996 ).

A far as we know, our study is the first to look at normal dietary intake levels instead of supplementation with calcium. Contrary to most studies which evaluate iron absorption, our interest was the influence of calcium intake on iron status. This study, therefore, may yield a better assessment of the long-term effect of calcium on iron absorption. Our study did not reveal an effect of time of calcium consumption on iron status. It may well be that the effect of simultaneous consumption of calcium and iron is readily reflected in iron absorption, whereas the iron status of blood is a more long-term variable on which this effect is less clear. In our study we did not distinguish between heme and nonheme iron. However, because the inhibitory effect of calcium was observed in both forms of iron (Gleerup et al. 1995 , Hallberg et al. 1991 ), we assume that the distinction is of less importance. On the other hand, it is therefore not possible to assess iron availability from food as proposed by Tseng et al. (1997) .

The mechanism of inhibition of iron absorption by calcium is not yet clear. A complex formation with iron and phytate was suggested but, on the other hand, a complex formation may enhance rather than inhibit calcium absorption by forming a calcium phytate complex. As both heme and nonheme iron absorption is inhibited, Hallberg and colleagues (1992a) argued that the mechanism must involve inhibition of iron extrusion from the enterocyte. Recent studies suggest that calcium competes for iron-binding sites on the intestinal iron-binding protein mobilferrin.

It can be argued whether the finding that higher calcium intake is associated with reduced serum ferritin levels is of biological relevance. The normal absorption of iron from the diet is estimated to be 10% (about 1 mg/d). To maintain the iron balance, several mechanisms are involved. In case of an iron deficiency, the uptake of iron may be increased by up to 20–30% (Wick et al. 1996 ). Several studies showed that iron absorption is strongly and inversely associated with serum ferritin level (Hultén et al. 1995 ). Therefore, reducing calcium intake does not necessarily lead to increased serum ferritin levels. Because inadequate calcium intake may lead to other serious health problems such as osteoporosis (Heaney 1993 ), it seems inappropriate to advise strongly against the consumption of calcium to prevent iron deficiency.

From our results we conclude that dietary calcium intake is weakly, inversely associated with the iron status of blood in girls and young women, irrespective of whether calcium was ingested simultaneously with iron.

	ACKNOWLEDGMENTS

 
We thank all those who have contributed to data collection, questionnaire development, data management, biochemical analysis and technical and administrative support.

	FOOTNOTES

 
¹ Supported by the European Commission DG XII (grant BMH1-CT94-1523), the Dairy Foundation for Nutrition and Health, CERIN, Laboratoire Innothera, the Dutch Ministery of Public Health, Oda og Hans Svenningsens Fond, Mejeriforeningen, Danish Dairy Board, the Polish State Committee of Scientific Research (grant 4P05D02910), Tetra Laval Service GmbH-Warsaw Branch, Dairy Farmers of Canada, the Swedish Dairy Board and the Centre de Recherche et d'Information Nutritionelles, France.

³ Current address: DMV international, 5460 BA Veghel, The Netherlands.

Manuscript received June 10, 1998. Initial review completed September 24, 1998. Revision accepted January 20, 1999.

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