Discussion

The results of the current study demonstrate that inadequate intake of Zn was present in a healthy population cohort. There was no strong relationship between dietary Zn intake and Fe biochemical parameters in this healthy cohort. However, there was a statistically significant difference in Zn intake among groups of women with dissimilar Hb concentration. Zn and Fe dietary intakes were strongly correlated. Grains and meat were identified as major sources of dietary Fe intake and meat and dairy products as main foods consumed contributing to Zn intake. Generally, no differences were seen for Fe and Zn intakes and status among various SES groups, except for Fe intake between those on low income and affluent. Plasma Zn and Fe concentrations were within the reference ranges despite the insufficient dietary intakes of these minerals. However, a prolonged inadequate intake of Zn and Fe may contribute to the development/manifestation of Zn and/or Fe deficiencies, so regular monitoring of mineral intake and status in this population cohort is necessary.

The mean Fe and Zn intakes of our study population were similar to the values for these elements reported for the adult population in other developed countries: Hungary, Croatia, Eastern and Central European countries, Australia and Canada⁽ Reference Novaković, Cavelaars and Geelen ^{29 ,} Reference Mandic Puljek, Mandic and Perl ^{43 –} Reference Gurinović, Kadvan and Vukotić ^{45 )}.

A recent review of available micronutrient intake and status data in Europe⁽ Reference Novaković, Cavelaars and Geelen ^{29 )} showed that Fe and Zn intake data for a number of countries are very limited. More current measurements of adult Fe and Zn intake and status data are rare⁽ Reference Rodler, Bíro and Greiner ^{46 )}. In this sense, results presented in the current study can be considered a valuable resource.

The intakes of Fe and Zn were different between males and females, and there was a strong correlation between Zn and Fe intakes, which is in agreement with previous reports⁽ Reference Brants, LoÈwik and Westenbrink ^{47 –} Reference Pennington and Schoen ^{49 )}. There was also a positive correlation between mineral intakes and energy and this relationship has been highlighted by others⁽ Reference Sanchez, Lopez-Jurado and Planells ^{28 ,} Reference Galan, Hercberg and Soustre ^{50 )}. The EAR cut-point method was used to assess the adequacy of Fe and Zn intakes. The EAR cut-point method is identified as an appropriate dietary reference intake to use when assessing the adequacy of group intakes and has been adopted for assessing nutrient inadequacy⁽ Reference Murphy ^{51 )}. In addition, we also compared the intake data with other available dietary recommendations^{( 52 , 53 )} and showed that the risk of inadequate intakes was very different. The fact that the method employed can affect the estimation of nutrient intake inadequacy^{( 53 ,} Reference Lauzon, Volatier and Martin ^{54 )} has been confirmed again. The Nordic and FAO/WHO recommendations overestimate the true prevalence of inadequate intake compared with the EAR cut-point method. Still, it must be noted that while intakes below the recommendation are not synonymous with deficiency, they certainly indicate increased risk of deficiency⁽ Reference Lauzon, Volatier and Martin ^{54 )}.

The estimation of and adjustment for bias (such as under- or over-reporting of food intake) is a relatively unexplored field. An evaluation of the effect of misreporting on energy and nutrient intake assessment indicated that low energy reporters had lower mean intakes for several micronutrients (from 25 to 36 %) compared with non-under-reporters⁽ Reference Poslusna, Ruprich and de Vries ^{55 )}. Likely under-reporting of food consumption was estimated to occur in 34 % (260/754) of the present study participants. Of our study population, 28 % had low Fe intake and 52 % had Zn intake below current dietary recommendations^{( 33 )}. Taking under-reporting into account, it is estimated that 5 % of the study population had inadequate Fe and 15–25 % had unsatisfactory Zn intake. Sixty-five per cent of our study population were women within the age range of 20–50 years. Women in this age group are known as the most vulnerable group for the development of Fe and Zn deficiencies because they are more likely to have excessive menstrual bleeding, infrequently consume sources of bioavailable Zn and Fe, rarely consume rich sources of enhancers and often consume rich sources of inhibitors of Zn and Fe absorption⁽ Reference Sandstead ^{56 )}. We found that women with dietary Fe intake below recommendations (EAR of 8·1 mg/d) were at greater risk of inadequate dietary Zn intake: 38 % of women in the present study had inadequate dietary Fe intake and 76 % of these with low dietary Fe intake show concurrent insufficient intake of Zn.

In many developed countries, micronutrient deficiencies are not related to the quantity of food consumed, but rather to the quality of the diet⁽ Reference Kaganov, Margherita and Mazur ^{57 )}. Inadequate dietary intakes of Zn and Fe that failed to meet the high physiological demands of adolescent girls in Australia and New Zealand were shown to be related to food choices⁽ Reference Gibson, Heath and Ferguson ^{3 )}. Relevant to these findings is the observation in fifty-two non-pregnant premenopausal women from Seattle, Washington, USA, whose diets provided similar amounts of Fe, where the consumption of red meat five times per week was more efficacious for body Fe stores than the consumption of lacto-ovo vegetarian foods, or the flesh of chicken and fish⁽ Reference Worthington-Roberts, Breskin and Monsen ^{58 )}. In further support of this are the findings of the National Food Survey of the British population⁽ Reference Heath and Fairweather-Tait ^{59 )} showing that Fe intake and red meat consumption have declined during the past three decades, leading to low Fe stores in females aged 11–64 years.

Being overweight is a worldwide problem that affects developed and developing countries similarly^{( 37 , 40 )}. Approximately 30 % of our study population was overweight and 10 % were obese. Other European countries have shown a similar percentage of overweight and obesity^{( 37 )}. However, even with excess dietary intake of energy and macronutrients, it is still uncertain if people are taking the recommended intakes of micronutrients. For instance, a number of national epidemiological surveys conducted in several developed countries have reported the co-occurrence of obesity with inadequate intakes of certain vitamins and minerals, particularly Ca, Fe, Zn, thiamin, riboflavin, vitamins B₆ and D, and folate⁽ Reference Elmadfa ^{15 ,} Reference Brussaard, den Berg and Hermus ^{60 ,} Reference Harvey, Berti and Casgrain ^{61 )}.

In the present study we found higher intake of Fe in the obese population than in non-obese individuals. A possible explanation for the higher intake of Fe in obese people is that their energy intake was higher because of the greater consumption of fatty (energy-dense) foods (i.e. meats) and refined grain products (white bread is a staple food). The correlations of energy intake, total grain products and meat with Fe intake support this explanation. However, not only inappropriate intakes, but also impaired bioavailability and utilization of micronutrients may be involved in the inadequate micronutrient status in obesity. The low-level inflammation that accompanies obesity is likely to decrease Fe absorption⁽ Reference Cepeda-Lopez, Aeberli and Zimmermann ^{62 )}. This needs to be taken into account when evaluating the adequacy of Fe intake in a population.

Zn and Fe nutrition are often associated. Red meat and grain products are the most important common dietary sources of Fe and Zn. Our findings confirm observations that food sources of Zn are also important food sources of Fe, as well as support the theory that usual low intake of either mineral increases the likelihood of low intake of the other⁽ Reference Gibson, Heath and Ferguson ^{3 ,} Reference Lee, Folsom and Jacobs ^{17 ,} Reference Cole, Grant and Swaby-Ellis ^{18 )}. Furthermore, other factors influence daily intakes of Zn and Fe: their concentration in food, the amount of food consumed and the consumption of dietary components that delay Fe and Zn absorption⁽ Reference Lonnerdal ^{23 )}. Zn and Fe are most bioavailable from many of the same foods and their absorption is repressed by many of the same dietary substances⁽ Reference Sandstead ^{56 )}. Although we were not able to estimate the exact phytate:Zn molar ratio of the diets, by employing the method of Jati et al.⁽ Reference Jati, Widmer and Purwestri ^{42 )} we showed that there was a high bioavailability of both nutrients in the diets of the people we studied. The food composition database needs to be updated because, as in many other databases, our data did not contain information on the content of phytate.

Food preparation and processing methods can be used to reduce the phytate content of foods based on cereals and legumes. These methods are based on the enzymatic hydrolysis of phytic acid to lower inositol phosphates that are induced by germination and fermentation⁽ Reference Gibson, Heath and Prosser ^{26 )}. Soaking, followed by empting out of the soaking water, can also be used to reduce the phytate content of cereal and legume flours by passive diffusion of the water-soluble sodium, potassium and magnesium phytates⁽ Reference Lestienne, Icard-Verniére and Mouquet ^{63 )}. Household-level information was obtained for people where cereals and legumes contributed a large proportion to total Fe and Zn intakes. Almost all of our participants (97 %) employed preparation techniques that are known to reduce phytate content of foods and increase Zn bioavailability (i.e. soaking of beans, fermentation of bread dough).

Overall, the plasma concentrations of Fe and Zn in the present study were within the expected normal ranges for the adult population and similar to reported levels of these elements in healthy individuals elsewhere⁽ Reference Koram, Addae and Ocran ^{64 ,} Reference Fakayode, Owen and Pollard ^{65 )}. Similarly, the mean values of Fe-related biochemical parameters were within the expected ranges for a healthy population of subjects⁽ Reference Koram, Addae and Ocran ^{64 ,} Reference Calenti, Sanchez and Lorenzo-Lopez ^{66 ,} Reference Dosoo, Kayan and Adu-Gyasi ^{67 )}. Mean plasma levels of both elements were significantly different between males and females. Obesity, age, education and marital status did not contribute to the differences in plasma levels of the studied minerals. These findings are in agreement with the results of Sian et al. ⁽ Reference Sian, Xiang and Miller ^{68 )}, Bailey et al. ⁽ Reference Bailey, Maisey and Southon ^{69 )} and Sanchez et al. ⁽ Reference Sanchez, Lopez-Jurado and Planells ^{28 )}.

Our observation of a strong association between usual Fe and Zn intakes was not accompanied by a similar relationship between Fe and Zn status. The lack of association may be due to several factors.

First, the inter-individual variation in biological availability of dietary Fe and Zn is determined by the degree of intestinal absorption that depends on the composition of meals; the absorption from two different meals with similar Fe/Zn content may vary considerably due to the interaction between dietary Fe/Zn and absorption enhancers and inhibitors⁽ Reference Skikne and Baynes ^{70 ,} Reference Heitmann, Milman and Hansen ^{71 )}. It could also be that good homeostatic regulation of Zn contributes to these dissimilarities⁽ Reference Lowe, Dykes and Skinner ^{44 ,} Reference Johnson, Hunt and Milne ^{72 )}. Second, the calculation of reported dietary intake is performed using food composition tables (calculated and chemically measured content of the diet may be different). Finally, the reported intake of dietary Zn and Fe may deviate to some degree from the true intake due to diet reporting error. In the present study we attempted to correct for some of these discrepancies by taking under-reporting into account; however, we need to acknowledge that a certain level of error may still be present.

The correlation of Zn intake and Zn status with Fe biochemical parameters in healthy populations has not been investigated widely. Only a few studies have been initiated and provide inconsistent results⁽ Reference Sanchez, Lopez-Jurado and Planells ^{28 ,} Reference Yokoi, Sandstead and Egger ^{73 ,} Reference Lim, Booth and Szymlek-Gay ^{74 )}. Various studies in Fe- and Zn-deficient populations have shown a positive link between Zn intake and Fe status indicators⁽ Reference Gibson, Hess and Hotz ^{6 ,} Reference Shamim, Kabir and Merrill ^{27 )}. In our healthy population cohort we did not find any significant correlation, most likely due to having a small number of people with inadequate Fe status. Still, it was evident that Hb concentration was statistically different between women with appropriate and insufficient Zn intakes. Further work may be needed to examine the role of dietary Zn on Fe status indicators more fully. Measurements of ferritin, transferrin and total Fe-binding capacity may provide more detailed analysis of this relationship as low Hb, which was used in the present study, is not the best indicator of Fe status and is mainly apparent during the late stages of Fe deficiency. In addition, as a number of studies over the years have demonstrated the positive link between Zn intake and Fe status and the role of Zn in the Fe absorption process⁽ Reference Knez, Graham and Welch ^{21 ,} Reference Shamim, Kabir and Merrill ^{27 ,} Reference Yokoi, Sandstead and Egger ^{73 ,} Reference Kelkitli, Ozturk and Aslan ^{75 )}, further work in this area should aim to elucidate the specific point in Fe deficiency progression when inadequate Zn intake is beginning to have its role.

As shown by many, SES, conventionally measured by education, occupation or inco.me, can contribute to inequalities in health⁽ Reference Darmon and Drewnowski ^{76 ,} Reference Aggarwal, Monsivais and Cook ^{77 )}. Those with limited incomes or lower educational levels are more likely to have poor-quality diets⁽ Reference Giskes, Avendano and Brug ^{78 )}. Energy-dense and nutrient-poor diets are cheap and usually consumed by those with limited budgets⁽ Reference Aggarwal, Monsivais and Cook ^{77 )}.

A limited number of studies have shown inconsistent results on the variation in Fe and Zn intakes between SES groups. Higher (2–5 %) Zn levels were found in low-SES groups⁽ Reference Sanchez, Lopez-Jurado and Planells ^{28 ,} Reference Arnaud, Touvier and Galan ^{79 )}, while others found no differences among the groups⁽ Reference Friel, Kelleher and Nolan ^{80 )}. Similarly, higher values of plasma Fe have been reported in high-SES groups⁽ Reference Van Rossum, Van de Mheen and Witteman ^{81 ,} Reference Hulshof, Brussaard and Kruizinga ^{82 )}, while again there are studies that showed no differences in Fe intake or status among various SES groups⁽ Reference Roos, Prattala and Lahelma ^{83 )}.

In the current study we looked at the association of marital status, educational level and occupation with Fe and Zn intakes and status, and found no differences in intake or status between SES groups. The only difference was seen in the intake of Fe between the affluent and those on poor income, which is most likely due to higher consumption of grain products and pulses (staple foods) by people in the low-income group.

The findings of the current study should be interpreted in view of its strengths and limitations. The strength of our study is that we investigated a homogeneous population with similar demographic and genetic background. In this way, we were able to look at the influence of nutrition on Fe and Zn intakes and status and their relationship in the healthy adult population. Another strong point of our work is the collection of dietary data. We administered three 24h recall questionnaires, on three non-consecutive days, which is the gold standard method for assessing the adequacy of Fe and Zn intakes⁽ Reference Hotz ^{84 )}.

We identify a few limitations of the present study. Plasma (serum) Zn is a good indicator of Zn deficiency; however, plasma Zn levels do not reflect marginal status and may not reflect dietary intakes⁽ Reference Wood ^{85 )}. Plasma Zn is insensitive to Zn intakes within this range⁽ Reference Heitmann, Milman and Hansen ^{71 ,} Reference Wada, Turnlund and King ^{86 )}, and the Institute of Medicine ruled out plasma Zn as a useful status indicator for evaluating human Zn requirements for Canada and the USA^{( 33 )}. It is also accepted that plasma Zn concentration is a valid indicator of whole-body Zn status in the absence of confounding factors, such as infection or stress⁽ Reference Lowe, Fekete and Decsi ^{87 )}. Nevertheless, with all its limitations, it is the best biomarker currently available. Studies have pointed out that the ratio of blood fatty acids (linoleic acid:dihomo-γ-linolenic acid) may be a more appropriate biomarker of Zn status that is able to differentiate between various Zn deficiency/adequacy states⁽ Reference Reed, Qin and Ran-Ressler ^{88 ,} Reference Knez, Stangoulis and Zec ^{89 )}; however, additional research is needed to fully determine the efficacy of this biomarker. Another weakness of our study was the inability to calculate the phytate:Zn molar ratios due to the lack of available data on phytate content of foods in our database. However, we believe that this was overcome by estimating the bioavailability of Fe and Zn using the method of Jati et al.⁽ Reference Jati, Widmer and Purwestri ^{42 )}.

In countries where fresh vegetables, fruits and proteins are freely available and well embodied in the average diet, as is the case in many European countries, nutritional deficiencies of Fe and Zn are expected to be rare. However, easy access to food does not ensure healthy food choices required to achieve adequate nutrition⁽ Reference Kaganov, Margherita and Mazur ^{57 )}. Similarly, lifestyle changes occurring over the last few decades in many developed countries are characterized by increased consumption of low-cost but energy-dense foods and by reduced physical activity levels. These lifestyle changes are the major factors implicated in the aetiology of the current obesity epidemic⁽ Reference James ^{90 )}. It is well known that an unbalanced diet can contribute to micronutrient malnutrition⁽ Reference Suskind ^{91 )}. Dietary decisions made by individuals may lead to Fe and Zn deficiencies. Finally, with the present global trend of people in developed countries to decrease meat intake and increase intake of grains, there is a tendency that more individuals may end up having inadequate intakes of these important minerals.

In conclusion, considering that more than 20 % of our study population had inadequate dietary intake of Zn and bearing in mind the health consequences of prolonged inappropriate Zn intake on both Zn and Fe nutritional status, more regular follow-ups are needed to monitor the intake and status of these nutrients and to make sure that potential deficiencies do get recognized and addressed in a timely manner.