Sample Collection and Assessment
First spot urine samples were taken in the morning from the case and
control groups. Each of the samples was divided into 4.5 mL tubes and
then kept in storage at -20 ° C. Urine samples from the patients and the
matched control group were processed in the same way and tested at the
same time for the same study. All of the samples were simultaneously
taken from the freezer and transferred to the laboratory on dry ice. The
laboratory staff was blinded to the all samples’ case control status.
Two standard samples were included in each test to check the analytical
error. Morning urine measurements revealed good sensitivity and
specificity in determining individual differences in nocturnal plasma
melatonin levels19.
A solid-phase type of enzyme immunoassay was used to measure urinary
melatonin sulfate was measured (Melatonin Sulphate ELISA Kit, GenWay
Biotech Inc. San Diego, USA). Test sensitivity was 1.0 ng/dL. Intra- and
inter-assay coefficients of variation had the upper limits ranged
between 5.0-12.5% and 5.1– 15.1%, respectively, at 5.7–205 ng/dL and
12.5–230 ng/dL. automatic photometer (ELx808 ™ Absorbance Microplate
Reader) was used to measure optical density at 450 nm.
A chemiluminescent immunoassay was used to measure urine cortisol
performed on an ADVIA Centaur XP Immunoassay System analyzer (Siemens
Healthcare Diagnostics Ltd. Frimley, Camberley, UK). Test sensitivity
was obtained as 0.1 μg/dL. The upper limits of inter-assay and
intra-assay coefficients of variation were 1.85–5.50% and,
2.91–3.79% respectively, at 3.90–36.95 ng/dL. The modified Jaffe
reaction method was used to measure the urine creatinine levels. Urinary
creatinine levels corrected the urine melatonin-sulfate and cortisol
levels 19.