Suboptimal dietary zinc intake is increasingly recognised as an important public health issue. Although the lack of generally accepted biomarkers of zinc status has impeded estimation of the global prevalence of zinc deficiency, based on information regarding the amount of zinc present in national food supplies, it has been estimated that the risk of low dietary intake of absorbable zinc and consequent zinc deficiency affects between one-third and one-half of the world’s population [1] and rates of deficiency may approach 73% in some regions [2]. Although severe zinc deficiency is uncommon in European populations, marginal deficiency is likely to be much more prevalent [3], with associations to immune system dysfunction and restricted physical development [4]. Children are particularly vulnerable to suboptimal zinc status during periods of rapid growth that create increased zinc needs that may not be met [5,6]. It is estimated that over 450,000 deaths per year (4.4% of all mortalities) among children between six months and five years of age are attributable to zinc deficiency [7]. Zinc deficiency also has an impact of child morbidity, impairing growth and contributing to childhood stunting [8,9].

Physiological requirements for zinc peak at the time of the pubertal growth spurt, the onset of which varies according to gender. In girls the onset of the growth spurt (OGS) occurs at 10.1 years and peak height velocity (PHV) occurs at 12.0 years. In boys OGS and PHV occur at 11.8 and 14.2 years, respectively [10]. Even after the growth spurt has ceased, adolescents may require additional zinc to replete tissue zinc pools depleted during puberty [11]. Marginal zinc status during the pubertal growth spurt has been associated with slower skeletal growth, maturation, and reduced bone mineralisation [12,13,14]. As nearly a third of total skeletal mineral is accumulated in the 3–4 year period immediately after the onset of puberty [15] suboptimal zinc intake may have long-term consequences on bone health.

Although a sensitive and specific biomarker has yet to be identified, a recent systematic review concluded that plasma (or serum) zinc concentration was responsive to both zinc supplementation and depletion and is the most widely used biomarker for zinc [16]. However, meta-analytic methods have not yet been used to model zinc status as a function of zinc intake levels. Understanding the relationship between dietary intake and micronutrient status is essential for deriving dietary recommendations.

The recommendations for zinc intake during childhood vary widely across Europe and comparisons are difficult due to differences in categorisation. For example between 4 and 6 age categories are used to describe micronutrient recommendations in childhood with different age cut-off points being used by different European countries [17,18]. Recommendations for zinc intake differ between males and females at the age of 15 years in most countries, but also differ at the age of 10 years in some countries. Zinc intake values range from 2.9 to 10.0 mg/day in children aged 5 years, 5.7 to 15.5 mg/day (boys) and 4.6 to 15.0 mg/day (girls) in children aged 10–15 years [17]. The EURRECA project attempts to consolidate the basis for the definition of micronutrient requirements across Europe, taking into account relationships among intake, status and health outcomes, in order to harmonise these recommendations [19].

This paper presents a systematic review of the data from all available randomised controlled trials (RCTs) meeting EURRECA’s quality standard [20], which investigated zinc intake and biomarkers of zinc status, and combines these studies in meta-analyses to model zinc concentrations in serum or plasma as a function of zinc intake.

Twenty-four estimates of zinc intake and serum/plasma zinc status in 18 RCTs with children were eligible for meta-analysis. All studies were RCTs published between 1975 and 2009 which reported zinc intake and plasma/serum zinc concentrations. The 24 estimates included 1722 participants in total with sample sizes ranging from 10 to 122. The median duration of the trials was 24 weeks (range 6–52 weeks). In 11 studies zinc was supplemented alone at doses ranging from 3 to 70 mg/day and in 7 studies participants also received other micronutrients. Zinc was provided with iron supplements [23] or iron fortified milk [36], as part of a fortified meal [24,35], and with other micronutrients [27,31,37]. The zinc dose ranged from 10 to 20 mg/day when combined with other vitamin/minerals and 2.57 to 9 mg/day when provided in fortified meals. Most studies (n = 7) provided the zinc supplements in the form of zinc sulphate, but others used zinc citrate [32], zinc gluconate [33], zinc carbonate [23], zinc methionine [29,34], amino acid chelate [27], and elemental zinc in a syrup [28,40]. Studies were conducted in Latin America (n = 9), North America (n = 4), Asia (n = 3), Africa (n = 1) and Europe (n = 1). Habitual zinc intakes ranged from 4.6 to 7.1 mg/day (where data was provided) and ages of children ranged from 2 to 17 years. Most studies included, but did not differentiate between, males and females, but one study provided separate male and female data [24], one included only females [32], and one only males [39].

The majority of studies (n = 13) reported that zinc supplementation significantly increased zinc plasma/serum status or significantly reduced the decline in zinc serum values compared to placebo. Of these, two studies also reported increased plasma/serum zinc concentrations in the placebo group. Five studies failed to find a significant relationship between zinc supplementation and zinc status [23,25,26,30,35], four of which provided zinc supplements or fortified meals with a zinc concentration of 10 mg/day or less.

Our meta-analysis of available studies suggested that zinc supplementation was associated with increased serum/plasma zinc concentrations. Combining the 18 RCTs in one meta-analysis yielded an overall pooled β-coefficient of 0.12 (95% CI 0.04, 0.20; p < 0.005; I² 97.6%) (Figure 2). Since we applied a base-e logarithmic transformation on the zinc intake and serum/plasma zinc concentration before calculation of the study-specific ’s, the overall represents the difference in the log_e transformed predicted value of serum/plasma zinc status for each one-unit difference in the log_e transformed value in zinc intake. Therefore, an overall of 0.12 means that for every doubling in zinc intake, the difference in zinc serum or plasma concentration is 2 (2^0.12 = 1.09), which is 9%. This means that a person with a zinc intake of 14 mg/day has a zinc serum/plasma concentration that is 9% higher than a person who has a zinc intake of 7 mg/day.

As the physiological requirements for zinc peak at the time of the pubertal growth spurt, which generally occurs in girls between 10 and 15 years and in boys between 12 and 15 years, a separate subgroup analysis compared zinc intake and status according to age. As the onset of puberty was rarely assessed in papers, arbitrary age groups of <10 year, and ≥10 year were used. One study for which mean serum/plasma zinc values were given for children whose ages spanned both age groups were excluded from this analysis [23]. A meta-analysis of 18 studies performed in children under 10 years yielded an overall β of 0.13 (95% CI 0.04, 0.22; I² 69.7%), compared to a meta-analysis of 3 studies performed in children over 10 years which yielded a β of 0.08 (95% CI 0.02, 0.15; I² 97.9%), although care should be taken with interpreting this finding as the latter analysis is based on limited available data.

There is statistical evidence for substantial between-study heterogeneity in the overall meta-analysis (I² 97.6%, p < 0.0001). To evaluate potential sources of heterogeneity, the variables duration, age, dose of zinc, and zinc status of the placebo groups (as a proxy for baseline zinc status) were added simultaneously to a meta-regression model as continuous variables. The analysis revealed that only zinc status of the placebo group was a statistically significant determinant of the overall β. The model explained 26.5% of between-study variance but the residual variation due to heterogeneity remained high (91.7%). It is important to note that serum/plasma zinc levels of the placebo groups were used as a proxy of baseline serum/plasma zinc as the mean baseline serum/plasma zinc levels were infrequently reported in the papers, however it does suggest that the β vary according to zinc status.

Table 3 summarises the internal validity of the included studies, assessed as described in the methods section. The risk of bias was high in all but three where the risk was low/moderate [25,37,38]. Papers were given a high risk of bias rating due to insufficient information provided on sequence generation and/or allocation, drop-outs and funding bodies.

This study is unique in providing an estimate of the dose-response relationship of zinc intake and serum/plasma zinc concentrations in children aged 1–17 years. Similar to findings published in an earlier systematic review [44], this meta-analysis of 24 estimates in 18 RCTs found a significant effect of zinc intake and serum/plasma zinc concentrations in children. In addition we have provided an estimate of the dose-response relationship between zinc intake and serum/plasma concentrations. An overall of 0.12 means that for every doubling in zinc intake, the difference in zinc serum or plasma concentration is 9%. In other words, a child with a zinc intake of 14 mg/day has a zinc serum/plasma concentration that is 9% higher than a person who has a zinc intake of 7 mg/day. It is important to note however that, due to homeostatic regulatory mechanisms, the amount of dietary zinc absorbed decreases as intake increases, and plasma zinc concentration is homeostatically controlled within a narrow physiological range, therefore this dose response relationship can only be applied to the range of intakes used to derive this relationship. The studies included in this meta-analysis were different in a number of aspects, such as using various designs, follow-up times, zinc doses, and populations. Therefore, it is no surprise that, when combining these studies in a meta-analysis, a large heterogeneity is observed between the studies (I² = 97.6% p = 0.0001). This between-study heterogeneity may be caused by methodological factors, such as biological factors, e.g., differences in study population characteristics (age, socio-economic status), differences in doses of provided zinc (amount, one or more doses per day, study duration). We have considered the dose of zinc provided, study duration, age, and supplement type and these factors did not significantly explain the between-study heterogeneity. An individual participant data meta-analysis may have provided a more conclusive explanation of the between-study heterogeneity in this meta-analysis. However, this type of analysis would involve the input of raw individual participant data provided by the original study investigators for re-analysis and combination in a pooled analysis and as such would be a major undertaking in terms of time, costs, and collaboration. Moreover, an inability to include individual participant data from all relevant studies could introduce selection bias. The meta-analytic approach used in this paper is not an attempt to accurately describe the biological relation between actual zinc intake and zinc concentrations in blood under strict experimental conditions and on an individual level, but rather to simulate a dose-response relationship between zinc intake and status that is useful for surveillance studies with a public health point of view and, as such, deliberately incorporates the differences between dietary assessment methods, laboratory assessment methods and participant characteristics to ensure a broad external validity. Thus, the heterogeneity reflects the lack of standardisation of methods and the true heterogeneity between study populations and necessarily enters as uncertainty into the application of such data for public health purposes [45].

The relationship observed between serum/plasma zinc concentration and zinc intake may have been weakened by the limitation of this particular biomarker for zinc status. It is well established that plasma zinc concentration can fall in response to factors unrelated to zinc status or dietary zinc intake, such as infection, inflammation, exercise, stress or trauma. Conversely, tissue catabolism during starvation can release zinc into the circulation, causing a transient increase in circulating zinc levels. Postprandial plasma zinc concentrations have been reported to fall up to 19% [46]. Twelve studies used fasting blood samples in their analyses (usually overnight). Other factors related to the adequacy of serum/plasma sampling, such as storage and separation of samples, was often inadequately reported. Whilst all studies included in the analysis were undertaken in individuals without chronic disease or severe protein-energy malnutrition, other factors such as stress, infection and inflammation may also have gone unreported. For example, only three studies screened for parasitic infection [28,30,36]. Clearly such confounders have a strong influence on the interpretation of plasma zinc concentrations. However, as more sensitive indices of zinc status have yet to be identified, plasma serum zinc remains by far the most commonly used biomarker of zinc status [16].

It is important to note that the majority of studies in our meta-analysis (n = 13 of 18) were conducted in countries where participants are likely to have dietary patterns with low-moderate zinc bioavailability with higher fibre and phytate content which may have weakened the overall β. Although suboptimal zinc status may be caused by inadequate dietary intake of zinc in some cases, inhibitors of zinc absorption are likely the most common causative factor [47], and recent evidence in adults suggests that the inhibitory effect of dietary factors such as phytate on zinc absorption is likely to be much greater than previously recognised [48], although whether this is the case for children is less certain [49]. Indeed, a proxy measure for initial nutritional status of participants (zinc concentration in placebo groups) was found to be a significant effect modifier of β. However as very few studies reported baseline zinc or gave details of the concentration of indigestible zinc binding ligands in participants’ diets we were unable to investigate this important effect further.

Zinc was given in combination with other micronutrients including iron in several studies. As iron and zinc are known to compete for absorption [50] it is possible that iron supplements may impair child zinc status [47]. It is possible therefore that additional supplementation of iron may have reduced the effect of zinc supplementation on zinc plasma levels. A review on the interaction between zinc and iron in supplementation trials reported that, in the 4 RCTs reviewed, addition of iron to zinc supplementation did not affect plasma zinc status in children [51]. As three of the included trials were in infants aged 4–6 months and as the iron-regulatory mechanisms of infants may differ before and after 9 months of age [52], further studies in older age groups are needed to understand more fully the interaction effects of micronutrient supplementation.

To conduct our meta-analysis some assumptions related to the availability of the required data or related to statistical issues had to be made. The meta-analysis required transformations of the intake and biomarker data to a common scale, as the studies included in our meta-analyses had different ways of reporting the relation between zinc and biomarkers of zinc status in blood. We standardised the different ways of reporting by transformation of both the intake and biomarker data to double log_e-scale, which allowed us to derive a standardised estimate from each study of the regression coefficient and its standard error as a basis for comparing these heterogeneously reported results. We also assumed a linear relationship on the double log_e-scale. This rigorous but flexible transformation allowed us to pool β’s and report these as a dose-response relationship between zinc intake and serum/plasma zinc concentrations. As compared to a conventional meta-analysis of mean differences between high and low exposed subjects, a linear relationship on the double log_e-scale with a slope lower than 1 allows us to model biomarker levels as a non-linear but monotonic concave function of dose, which is considered a more likely shape in biology. The meta-analyses were conducted within the context of the EURRECA project as a means to provide additional evidence for underpinning reference values for zinc intake of populations [53]. Whether the dose-response relationship, as provided in this paper, could be used as either qualitative or quantitative evidence to substantiate the daily zinc intake dose necessary to achieve normal or optimal levels of biomarkers for zinc status, remains a matter of discussion regarding the cut-off levels for biomarkers of zinc and the predictive value of serum/plasma zinc concentration for relevant functional health outcomes such as growth, immune function, cognitive function and psychomotor development.

Due to the wide heterogeneity that exists in the published literature on the relation between zinc intake and zinc status, such data cannot be combined in a conventional meta-analysis. Our paper not only provides a useful summary of this data in a systematic review, but also demonstrates a new meta-analytic approach to summarise all this data while appreciating the heterogeneity of it. The mathematical basis of this novel approach has recently been published [41] and is beyond the scope of the paper under review here, but in summary, we modelled a dose-response relation as a monotonic concave function between zinc intake and biomarkers. This is an innovative way to use all the data available, albeit heterogeneous data, to model the dose-response relationship; information which is essential when appraising micronutrient recommendations. When more research on zinc intake and status becomes available, this meta-analytical approach can be improved to strengthen the evidence on which we base our zinc recommendations.

In conclusion, based on 24 estimates among 1722 participants, the results indicate that a doubling of zinc increases plasma/serum levels by 9%. Although it is recognised that serum/plasma zinc is a somewhat flawed biomarker of zinc status, in the absence of suitable alternatives it remains by far the most commonly used method and as such this review provides a valuable source of information for those seeking to assess zinc nutriture. As serum/plasma zinc levels are considered intermediates in the causal path to health benefits and because the heterogeneity between study results reflects real uncertainty in the evidence base, these issues must be taken into account when our dose-response data are used as complementary evidence for underpinning zinc reference values.