The Development of Circadian Rhythms in Human Infants

Alternative method of urine collection - pads
The Newcastle urine collector Uricol pads (Redlands, Newcastle UK) were used as a
third alternative method by mothers to collect the night time/day time urine samples.
Firstly the infant‟s pubic and perianal area was cleaned and clear of powder, oils and
creams. The Uricol pad was accurately positioned over the expected void area cotton
side down. The adhesive tape on the back of the pad was removed and the sticky
backing attached to the baby nappy. The nappy was then secured. At 10 minute
intervals the pad was checked for wetness. As soon as this was confirmed the pad
was removed and laid on a flat surface, wet side up. Using the 5ml syringe provided,
the tip was placed on the pad at an angle of 45 degrees and 5 ml of urine extracted.
The urine was then decanted into a sterile universal container and stored.
Parents placed collected urine in universal containers which were placed in household
refrigeration facilities. These were then transported the following day to university
where they were kept at -20 degrees till processing.
2.4 Estimation of creatinine
Creatinine was estimated using the RANDOX Creatinine Colorimetric method, using
commercially available reagents (piric acid and sodium hydroxide). The creatinine
reacts with the piric acid to form a coloured complex. The amount of complex
formed is proportional to the concentration of creatinine (Stockgrand Ltd, Surrey
UK). Equal volumes of piric acid and sodium hydroxide are added to make the
standard solution. The urine samples are diluted to 1:29 concentration with water.
The standard piric acid/sodium hydroxide solution was added to aliquots of the
diluted urine samples and quality control high/low creatinine solutions. The samples
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