Enseñanza aprendizaje de la ortografía

It was not possible to assess the results in the manner intended or to draw any conclusions from the pilot study. This was because three subjects did not complete the protocol and there were analytical failures o f the remaining three subjects’ blood samples. A review o f the study design revealed a number o f issues that needed resolving before the main study commenced. The major issues were recruitment, analytical method failures and the difficulties o f getting young children to eat and drink the test meals, drinks and isotope doses.

10.2.1 Recruitment

There was a poor response to the invitation to take part in the study. This was despite the invitation letter being accompanied by a letter o f introduction from the children’s GPs and reminder letters. A further issue was that recruitment had been excessively time consuming. It took six months to recruit six children to the project. It is probable that some parents were unwilling to submit their children to venous blood sampling. The protocol was revised so that this was replaced by capillary sampling.

This is a simple procedure, more likely to be acceptable to parents and children. A disadvantage is that blood samples can be diluted by tissue fluid released by the

incision through the skin so affecting the MS ratio measurements. This can be

minimised by good sampling technique. This method yields small samples o f

approximately 0.5ml, a further disadvantage. At this stage, the prospect o f increased recruitment outweighed the disadvantages. Parent information sheets were rewritten to reflect this change. Alternative methods o f recruitment were considered. The researcher attended four different child health clinics per week, speaking to parents who had children o f the appropriate age. An eight-week trial o f this method o f recruitment proved unsuccessful as no children were recruited. It was decided that invitation packs would be posted to families with children o f the appropriate age who were registered with the Camden & Islington Community Health Services Trust. This represented a much larger pool o f potential subjects than registered with individual GP practices.

The response rates were further eroded by failure to meet the inclusion criteria o f

haemoglobin concentration <11.5g/dl. Ideally subjects should be less than iron

replete as the influence o f dietary components on non-haem bioavailability is reduced in those with good iron status. The cut-off o f <11.5g/dl was an arbitrary choice, a possible way o f improving recruitment was relaxing this criterion. The range was therefore broadened to 9.5-11.9g/dl.

10.2.2 Analytical method failures

The poor precision for the isotopic ratios o f in all three samples, suggest that the

quantities o f ^^Fe in the blood samples were below the limits o f detection for the TIMS. This could be explained by a number o f reasons. The children could have genuinely absorbed very small percentages o f the isotope labels. One o f the children, subject 6, only consumed h alf o f the total 2mg dose. This may explain why the quantity o f ^^Fe dose in the blood in her sample, was not measurable by this method. It was also noted that the other subject, despite consuming a more than adequate amount o f iron (8.5mg/day), had a low haemoglobin concentration (9.6g/dl). There

was also a family history o f IDA. It seemed possible that this child absorbed

particularly small amounts o f iron which may explain the low isotopic enrichment in her blood sample. It was thought imprudent to increase the doses as they were

already large in comparison with the amount o f natural iron found in the test meals. Another explanation for the poor precision is that the blood samples were perhaps insufficiently mixed before the sub-samples were taken so that they were not representative o f the total samples. Alternatively the procedure to isolate and purify iron from blood samples could have been sub-optimal leading to low recoveries. Kastenmayer (1996) notes that errors in the sample preparation are usually greater than the error contributed by the TIMS.

10.2.3 Young children’s eating habits

A further problem was trying to ensure the children consumed the labelled foods and

drinks. Every attempt was made to encourage the children to eat but on some

occasions they refused. A particular problem was giving the reference dose o f iron with the juice drink. Children o f this age do not have sufficiently developed oro- motor skills to maintain a tight mouth seal and keep fluid in their mouth. Attempts were made to collect possible losses for later quantification. However in one case isotope and juice soaked into clothing and in two other cases was spat out as a fine spray. It was impossible to collect this type o f loss. Obvious practical measures such having the child wear a plastic bib with a lip proved fruitless as most o f the children refused to wear the bib. Attempting to use a transfer pipette failed. The two children who managed to consume the reference dose drank from a cup with a spout and a bottle respectively. The problem was not so much with the transfer o f juice and isotope into the mouth but more about whether the child could keep the fluid in their mouth and then swallow it. In retrospect, a medicine syringe probably would have been the best option for administrating the reference dose. This delivers the liquid to the back o f the mouth, which should force the swallow action, this perhaps would have prevented the losses. An additional issue here may have been the palatability o f the food. Limiting vitamin C rich ingredients in the recipes resulted in probably less sweet dishes which the children may have found unappealing. As the protocol was revised (see below) the lamb mince recipe was altered to include dried apricots that had the twin advantages o f increasing the palatability and non-haem iron content o f the dish and but also the possible disadvantages o f increasing the vitamin C and

calcium contents. Both nutrients may influence non-haem iron bioavailability

although in practice, the cooking and storage procedures probably reduce the vitamin C content to minimal levels.

10.2.4 Conclusions and protocol revisions

It was clear the protocol had to be revised in view o f the above comments. The research question was revised as a consequence o f the publication by Englemann et al (1998) that the addition o f 25g meat (beef) to a vegetable based meal significantly improved non-haem iron bioavailability in infants. Eight infants aged 7-8 months old were given vegetable puree test meals labelled with iron stable isotopes with and without b eef in a cross over design. The meals were isocaloric and contained similar amounts o f enhancers (vitamin C) and inhibitors (calcium, polyphenols and phytic acid) o f iron absorption. Geometric mean iron absorption was greater from the test meal with meat than without, 15% compared to 9.9%, a statistically significant difference (p=0.002). The authors concluded that adding meat to vegetable weaning foods is a strategy for increasing the bioavailability o f non-haem iron in infancy. Other issues taken into consideration when revising the protocol were the time available to complete the research, the feeding problems, the remaining quantity o f ^^Fe and ^^Fe isotopes available and the low volunteer rates. Although it would have been useful to attempt to replicate Englemann and colleagues (1998) findings, it was decided that the available resources should be used to clarify the effects o f cow ’s milk. The research question was therefore reformulated to examine the effects o f a drink o f cow ’s milk on non-haem iron bioavailability in a meat-based meal in young children.

In the new protocol each child acted as his/her own control instead o f using a reference dose o f isotope to normalise differences in inter-individual iron absorption. According to the revised protocol children would have a test meal mixed with one isotope and milk on Day 1 and the same test meal mixed with the other isotope and water as a substitute for milk on Day 2 o f the study period or vice versa. This resolved three particular problems. First, it was no longer necessary to administer one o f the isotopic doses to the children in a drink. Second, the study period was reduced by one day. It was hoped reducing the time commitment would improve recruitment. Third, although randomisation to the four groups specified in the original protocol was stratified to ensure even numbers in groups, in practice attrition after randomisation was so great it was likely that group numbers would have been uneven. As the children would act as their own controls this would no longer be a problem. The full revised study design is described in Chapter 11.

10.3 Chapter Summary

The original aims o f Study 2 were to examine the effects o f meat and cow ’s milk, independently and in combination on the non-haem iron bioavailability in young children. However, the study design was not feasible as originally planned. This was largely because o f practical issues such as lack o f recruitment, difficulties in administering the isotopic doses and in obtaining and preparing venous blood samples for TIMS. Extensive changes were made to the study protocol as a result. The revised aim was to examine the effects o f cow ’s milk on non-haem iron bioavailability in children.

Chapter 11

Study 2: Non-haem Iron Bioavailability in Young Children