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.