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
]. 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 loge
-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 loge
-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 loge
-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.