2.7.2. Biological Measures

Urine sampling commenced on the evening prior to the baseline and week 26 testing visits and comprised three separate samples—void at bedtime and the first and second voids of the following day (morning of the testing visit). If a participant needed to urinate during the night, then these voids were also collected in the same manner, as described below.

Urine was collected in a sterilized measuring cylinder. Void volume, time and date were recorded, before a 10 mL aliquot of urine was retained and refrigerated in a screw cap container, pre-labelled with the participant’s study details. The samples were taken to the laboratory at the baseline and week 26 testing visits, for further labelling and immediate storage at −80 °C for later analysis of the major melatonin metabolite 6-sulfatoxymelatonin (aMT6s), using radioimmunoassay [19].

Total excretion of aMT6s (ng) summed from all voids and the bedtime aMT6s (ng) values were calculated. Bedtime excretion of aMT6s specifically was also chosen to be analyzed independently from the total aMT6s, as a measure of melatonin production before sleeping, in an attempt to assess the effects of treatment on bedtime melatonin levels. This is because reduced evening melatonin production is associated with sleep disturbances [20], and urinary levels of aMT6s are seen to parallel those of melatonin in the blood, saliva, and urine [21].