The study was designed to investigate whether regular consumption of micronutrient fortified yoghurt (delivering 30% RDA of zinc, iron, iodine and vitamin A) over a period of 1 year could improve the micronutrient status and physical growth of school children. To our knowledge, this is one of the first reports of a study that used yoghurt as a vehicle for delivery of micronutrients and evaluated the impact among school children in a double-blind RCT. Although consumption of fortified yoghurt did not result in improvement in iron status indicators, it did show a significant improvement in Hb, and an impact of iodine status indicated by significantly lower decline urinary iodine levels when compared to the NFY group. One year of fortification resulted in an improvement in linear growth with a statistically significant change in height velocity and HAZ. A systematic review , evaluated the effect of the multi-micronutrient (MMN) fortification of foods compared to unfortified foods on the micronutrient status of school children and measured a statistically significant improvement in micronutrient status in the intervention group compared to the control group, taking baseline values into account. Some studies have reported positive effects on growth but the overall effects on these outcomes are equivocal . The response to fortification in the present study showed an improvement in Hb concentrations, even though no iron deficiency was present at baseline, and although RBP and iodine concentrations fell in both groups, they were significantly higher in the FY groups. A decline in the intervention group may be suggesting 30% RDA of selected micronutrients in the fortified yoghurt may not have been adequate to reverse decline in vitamin A and iodine concentrations in this population of school children.
In the present study, iron deficiency was not observed i.e. ferritin, TfR and estimated body iron stores were in normal range. Consistent with our finding, Winichagoon et al.  did not find an effect on iron status indicators after adding 5 g of elemental iron via multi-micronutrient (MMN) powder to the school lunches of Thai children. It has been demonstrated that the maximal inhibitory effect of calcium on iron absorption is reached at a level of ~300 mg of calcium . The possibility of the calcium content having been responsible for lack of effect on iron status markers is very unlikely as the calcium content of yoghurt was less than 100 mg. However cannot be totally ruled out given the study design and fairly adequate iron status of the population and therefore a possibility of reduced absorption and potential for minor impact.
In this study, the prevalence of elevated plasma CRP and AGP was low; indicating the presence of ongoing or recent acute inflammation in the study population was low, however that may not rule out some chronic infections but then their presence would have caused anemia rather than contrary. Prevalence of hemoglobinopathy, another possible cause for high ferritin concentrations in the presence of anemia, needs consideration. Anemia was present in a little more than half of the children in both the groups at baseline with a mean Hb concentration of 114.7 g/L. But levels of Hb in this population and distribution of Hb do not favor hemoglobinopathy as a cause of high ferritin. In fact in population with much higher levels of anemia in Delhi contribution of hemoglobinopathy to levels of ferritin has not been demonstrated (Unpublished-data). There was a significant difference in Hb levels of 1.5 g/L between FY and NFY groups after one year of fortification. A similar effect on Hb status was recently reported among Kenyan subjects with HIV infection in whom MMN supplementation resulted in increased Hb concentrations only among those without signs of inflammation . Although there are studies which found significant effects of multiple micronutrient fortification on concentrations of Hb, TfR, ferritin and on body iron stores , in the present study, the difference in Hb between the FY and NFY groups, despite the lack of difference in the iron status markers suggest better utilization of iron, which may have been caused by bio-available zinc and vitamin A. Vitamin A supplementation has been shown to increase erythropoietin (EPO) . Retinoic acid, a vitamin A metabolite, has been reported to regulate the EPO gene, which, in turn enhances EPO production in vitro in an animal model . Providing vitamin A in the fortified yoghurt may have increased the mobilization of iron from storage [29, 30], resulting in an improvement in Hb concentration, although the mechanism of this phenomenon is still to be verified.
At baseline, some of the children were mild to moderately deficient in iodine. The population where the study was conducted were found to consume plain salt more than the iodized salt, although, the national salt iodization program of 1989 in Bangladesh is ongoing on. In our study, iodine and RBP decreased in both the groups between base- and end-line, but the decrease was significantly less in FY group, suggesting compliance to and success of intervention in delivering nutrients. Similarly, all five studies included in the systematic review also found a beneficial effect of MMN fortification on urinary iodine excretion levels .
Zinc status was less than the optimum at the baseline. Zinc status improved but the differences in mean zinc levels were not statistically significant and concentrations were still below the accepted cutoff for the age group at end-line (9.94 μmol/L). A review of six studies investigated the effect of MMNs (including zinc) in fortified food compared to unfortified food on zinc status, only two studies found a significant beneficial effect . While other studies, did not find any effect on biochemical zinc status or deficiency [30–32]. Although dairy products have shown to increase zinc absorption , the sub-optimum effect of the zinc in the fortified yoghurt on zinc status could be due to the level of zinc added being too low, to a possible negative interaction between iron and zinc  or because plasma zinc may not be a good marker of zinc status. Indeed, recent studies have shown that iron inhibits zinc absorption  and vice versa. However, iron has little effect on zinc absorption when zinc-iron ratios are 1:1, which is similar to the ratio in our study . Therefore, the interaction of iron and zinc from the fortified yoghurt should be further investigated and the dosage of zinc should be re-evaluated. Further, the beneficial effects on observed height gain, which are commonly attributable to zinc intake rather than iron [37, 38] would argue for an improvement in zinc status which plasma zinc was not able to reflect.
One year of fortification resulted in an improvement in linear growth with a statistically significant change in height velocity and height for age Z- scores, but not on weight and BMIZ which was likely attributed to the synergetic effects of the micronutrients including the presence of zinc . A meta-analysis on the effects of micronutrients on growth in children up to 18 years of age found that interventions with iron or vitamin A alone did not have an effect on either height or weight gain, whereas MMN interventions, 80% of which included vitamin A, iron, and zinc, significantly improved linear growth and also had a small (not statistically significant) positive effect on weight gain . These findings would suggest that providing specific micronutrients results in a better height gain, and reversal of growth faltering over and above of macronutrients . A systematic review that tested the impact of multiple micronutrients provided via fortification on the micronutrient status, growth, health, and cognitive development of school children showed mixed results i.e. no effect to a positive effect on growth. Two of seven studies found a significant beneficial effect on height gain, with mean differences of about 0.6 to 1.0 cm in height increments between the groups after 6 or 14 months of intervention . Finally, improvement in Hb levels, and decline in the excretion of urinary iodine levels and improvement in growth points towards a global improvement in child’s health - a functional endpoint of multiple metabolic processes. This global improvement could be due to a combination of effects of individual constituents of the intervention and/or synergistic effects among the components.
Limitation of the study
The results of the study need to be interpreted with consideration to the initial micronutrient status of the population. The children in this study did not have severe anemia or iron deficiency at baseline. Given the design of the study we cannot attribute effects to a specific component but only describe the collective impact of the specific micronutrients tested as the experimental intervention in this study.