Radioimmunoassay for 6-Sulphatoxymelatonin in Urine Using an Iodinated Tracer

Abstract

SUMMARY. A radioimmunoassay (RIA) for urinary 6-sulphatoxymelatonin (aMT6s) using an 1251_aMT6s tracer is described. Iodinated aMT6s used as the label was synthesised by direct iodination of aMT6s using 1.3,4.6 tetrachloro 3.6 diphenylglycouril as the oxidant. The assay shows low cross reactivity with related compounds. Serial dilutions of 1:25() and I: 125 diluted urine gave parallel displacement curves.Comparison of the new RIA using 12.~I-aMT6s with the RIA using 3H-aMT6s label gave good correlation. as did comparison with a gas chromatography mass spectroscopy (GCMS) for total free and conjugated 6-hydroxymelatonin.It is reported that 6-sulphatoxymelatonin is the major urinary metabolite of the pineal hormone melatonin in humans. I. 2 It is synthesised in the liver as a conjugate of 6-hydroxymelatonin the primary metabolite of melatonin." It has been shown to be a good index of pineal melatonin secretion and hence pineal function.v" Since urine is convenient to collect, it provides a simple non-invasive method for long-term study of the pineal, and opens up the possibility of studying melatonin in many clinical situations.We have previously reported a direct RIA for aMT6s in plasma and urine using a tritiated tracer." Whilst this assay is sensitive specific and reproducible, it is expensive in time and reagents.This report describes an assay using an iodinated tracer which is as specific and more sensitive than the previously reported assay. It uses a much simpler method of label production and avoids costly and cumbersome scintillation counting. Materials and methods MATERIALS

Ocular Measures of Sleepiness Are Increased in Night Shift Workers Undergoing a Simulated Night Shift Near the Peak Time of the 6-Sulfatoxymelatonin Rhythm

“…After each collection, participants recorded the volume of the sample and the time of urine collection. A 5-mL urine aliquot was frozen (−20°C) and subsequently analyzed by aMT6s radioimmunoassay 19 at the Adelaide Research Assay Facility, University of Adelaide using reagents purchased from Stockgrand, Ltd., University of Surrey (Guildford, UK). The aMT6s intra-assay coefficient of variation (CV) was 7.2% and the inter-assay CVs were 23%, 7%, and 11% at 3.6 ng/mL, 15.6 ng/mL, and 29.6 ng/mL, respectively.…”

Sleep and Circadian Rhythms in Hospitalized Patients with Decompensated Cirrhosis: Effect of Light Therapy

“…The Horne-Östberg (HÖ) questionnaire determines a subject's diurnal preference (definitely morning [score 70-86], moderately morning [59-69], intermediate [42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58], moderately evening [31][32][33][34][35][36][37][38][39][40][41] and definitely evening [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]) based on 19 self-administered questions [24].…”

“…Sequential urine samples were collected from patients over the first two nights after inpatient admission, and the day in between (4-hour intervals during the day and 8-hour intervals overnight, for a total of 36 hours) [31]; 3 ml aliquots were collected from each sample and kept at -20°C until assayed for 6-sulpahtoxymelatonin (aMT6s), in duplicate, using a commercial radioimmunoassay [32]. Urine collections were repeated every 7 days, depending on the length of hospitalization.…”

On the Origin and the Consequences of Circadian Abnormalities in Patients With Cirrhosis

“…Aliquots of 3 ml were extracted from each individual collection and kept at -20 0 C until assayed for aMT6s, using a radioimmunoassay (RIA) (18). The intra-assay coefficients of variation (CV) ranged from 10-11%; the inter-assay CV ranged from 6-12%; the limit of detection was 0.3 ng/mL.…”