Subjects and Methods

9.2.1 Ethical issues

This study was approved by the LRECs detailed in section 7.2.1. This was an

invasive study involving the collection o f both capillary and venous blood samples and the administration o f stable isotopes. The Ethics Advisory Committee o f the Royal College o f Paediatrics and Child Health (2000) considers that blood sampling

from children is a low, but not minimal, risk procedure. The blood collection

procedures were explained in detail to the parents, emphasising that if they felt their child became unacceptably upset they could halt the procedure, fron stable isotopes present no known risk to children as they occur in naturally in food and have been used successfully in term and low birthweight infants (Fairweather-Tait et al 1995a, Zlotkin et al 1995). All parents gave written consent for their children to take part in the study and parents were informed o f blood test results and copies o f test results were sent to the children’s GPs.

9.2.2 Subjects and recruitment

9.2.2.1 Sample size

The study described here employed a factorial design such that subjects were randomised to one o f four treatment groups. The following equation was used to calculate a total sample size. Cole (1997) reports that;

where n is the total number o f subjects in two groups and f is the fraction o f a

standard deviation o f the variable to be detected. The standard deviation o f

percentage iron bioavailability measured in young children is known to be 2.2% (Tom

Fox, personal communication % unpublished data). Assuming that it would be

desirable to detect a one standard deviation difference in iron bioavailability between two groups, then f= l.

Therefore, n = 1 6 /f^

n = 16/1 = 16

That is, 16 children in two groups ie 8 in each o f the four groups and 32 in total will

allow the detection o f one standard deviation in iron bioavailability between the groups at 80% power and 5% significance.

Once field-work had started, it became clear that there were problems with recruitment and with administration o f the isotope. The outcomes o f the recruitment o f the first six children are presented and reviewed in Chapters 9 and 10 as a pilot study. This review provided an opportunity to revise the study aims and protocol. The revised study is reported in Chapters 11 and 12.

9.2.2.2 Recruitment

Recruitment was via three General Practice surgeries in Hackney, London. Families with children aged 1.0-1.25 years and registered with the practices were sent an invitation pack. This included an invitation letter, a covering letter from the GP partners at their practice, a parent information sheet, a reply form and a reply paid envelope (Appendix 6). Reminder letters were sent after three weeks. Home visits

were made to the families who volunteered to take part. A capillary blood sample o f up to 0.5ml was collected from each child for a full blood count (Bayer H3) and

haemoglobin electrophoresis (Biorad Variant). The author collected all capillary

blood samples. Six children, four girls and two boys, met all the inclusion criteria and entered the pilot study.

Table 9.1 Study inclusion and exclusion criteria

Inclusion criteria Exclusion criteria

Aged 1.0-1.25 years old <1.0 years or >1.25 years old

Apparently healthy Unwell, history o f chronic illness

Not taking iron medication Taking iron medication

Haemoglobin concentration o f 9.5- 11.5g/dl

Haemoglobin concentration <9.5g/dl or >11.5g/dl

Normal birthweight (>2500g) Low birthweight (<2500g)

Consuming a mixed diet including some meat

Following a vegetarian diet

Note

1. Recruitment rates were low and the upper end o f the age inclusion criterion, was in practice, slightly extended. The oldest children were 1.3 years old.

9.2.3 Study design

The effects o f meat and cow ’s milk on the bioavailability o f non-haem iron were estimated by measuring the percentage incorporation o f ferrous sulphate doses enriched in the isotopes ^^Fe and ^^Fe and administered with the different food combinations, into erythrocytes. Subjects were randomly allocated to one o f four groups, and, depending on group assignment, received a different combination o f foods and drinks. Each child received a test meal with the ^^Fe on days 1 and 2 o f the study period and a reference dose o f ^^Fe on day 3 taken with an apple and orange juice drink (Cow & Gate Nutricia Ltd). Dividing the isotope doses over two days was carried out to reduce variability relating to changes in the efficiency o f iron absorption within individuals. The reference dose was used to normalise the results, that is, to allow for the large inter-individual variation in iron absorption, and was given under

conditions that favour iron absorption. The degree o f iron absorption from the

reference dose is designated 40% and absorption from the isotopic dose given with the test meals is expressed in relation to it (Magnusson et al 1981). Randomisation was stratified so that equal numbers should be entered into each group. The study employed a factorial design such that each o f the four groups received a different combination o f foods under test, as indicated below.

Group 1 Group 2