Iodine-129 Tracer Method for Investigating Human Iodine Metabolism

Overview

Current iodine requirements defined for pregnancy and lactation are rough factorial estimates extrapolated from older studies in adults that used radioactive iodine tracers. To ensure optimal thyroid function in these vulnerable groups, a tracer method that could be safely used to accurately define iodine requirements would be valuable. Iodine-129 (129I), a long-lived semi-stable isotope with no health risks, could be used as a tracer, but the analytic challenges are formidable. However, we have developed an ICP-MS method to measure pp-billion (10-9) to pp-trillion (10-12) quantities of 129I in biological samples. In this project we will perform a study in which iodine-replete adult subjects will consume an oral dose of 129I. We will quantify 129I kinetic patterns in plasma, urine and stools after the oral dose, and use these data to derive tracer absorption, retention and excretion rates. This trial will allow us to define optimized procedures for the routine application of this method to assess iodine metabolism in humans. The use of 129I may prove to be a breakthrough technique to safely assess iodine metabolism and requirements in pregnant/lactating women in order to ensure healthy thyroid function in these age groups.

Experimental design. The experimental design of this pilot-trial foresees the administration of a physiological oral dose of 12 µg of 129I (the tracer) in healthy adults coupled with quantitative measurement of the tracer concentration in plasma, urine, and stool samples collected prior and following tracer administration. A single dose of 12 µg 129I corresponds to a level of activity of 78.5 Bq and an exposure in adults of about 0.16 mSv for the thyroid as the target organ. Baseline plasma sample will be collected just before the administration of the tracer (-5 min) and follow-up samples will be collected 15, 30, 45, 60, and 90 min, as well as 2, 4, 8, 24, 48, 72, and 96 hours following tracer administration. Complete urine will be collected over a period of 8 days: baseline samples 1 day before the tracer dose (Day 3) and follow-up samples 7 days thereafter (until Day 10). Similarly, complete stool collection will be done on the day before the tracer dose (Day 3) as well as on the 3 following days (until Day 6). At Day 20 and 40 (follow-ups), blood sampling and spot urine collection will be repeated to verify washed out period. Baseline samples will be used for baseline-correction of post-administration samples. In order to minimize variability in the response for the purpose of this pilot-trial, the participants will consume a standardized diet during the first 6 days of the trial and will keep a food diary during the last 4 days of the trial. After suitable preparation, plasma, urine and stool samples will be analyzed for tracer quantification (determination of 129I mass on 127I mass; 127I being the naturally occurring iodine). From this data, we will construct pharmacokinetic curves of tracer appearance and clearance in blood, urine, and stool and we will derive rates and timing of human iodine absorption, retention and excretion. In addition, iodine speciation will be performed using iodine speciation based on a chromatography method coupled with ICP-MS for direct mass quantification of iodine species as plasma inorganic iodine (PII) and protein-bound iodine (PBI). This will provide an additional level of information on separate pharmacokinetics of metabolized and not metabolized 129I. Because uptake by the thyroid will be a determinant of the fraction of the tracer that is retained, the characterization of the participants' thyroid function is important to control for potential variation of calculated retention in our subjects, and thyroglobulin and thyroid hormones in whole blood will be assessed. Methods of analysis. The tracer concentration in plasma, urine and stool will be measured by a high resolution multi collector ICP-MS with isotope dilution analysis (IDA) using a tellurium internal standard following sample digestion according to a method recently developed in our laboratory \[Dold et al, Thyroid 2015\]. For the additional speciation of 129I in plasma, analysis will be performed by using ion chromatography ICP-MS analysis (IC-ICP-MS) at the Federal Food Safety and Veterinary Office, Swizterland. Urinary iodine concentration will be measured by means of the Sandell-Kolthoff reaction \[Pino et al, Clin Chem 1996, 42:239-243\] in our laboratory. Thyroglobulin will be assessed using an ELISA dried blood spot method recently developed in our laboratory \[Stinca et al, Thyroid 2015, 25:1297-1305\]. Thyroid hormones will be measured by the University Hospital in Zurich, Switzerland, using a DELFIA (dissociation-enhanced lanthanide fluorescent immunoassay) method. Iodine content in the standardized diet will be will be measured in our laboratory by ICP-MS analysis and data from the food diaries will be evaluated by using the nutrition software EBIS-Pro (University of Hohenheim). Data analysis and evaluation. The data generated by this pilot-trial will be used to construct pharmacokinetic curves of tracer appearance and clearance in the collected samples (total plasma, plasma PII-fraction, plasma PBI-fraction, urine, stools). Curves will be constructed by fitting a LOESS multiple regression model, a non-parametric locally weighted scatter plot smoothing of tracer concentration in samples as a function of time, and provide uncertainty range to visually represent the tracer's pharmacokinetic patterns and variability of response. For the evaluation of tracer kinetic patterns we will generate classic pharmacokinetic modelling parameters, including maximum tracer concentration (cmax), time at maximum tracer concentration (Tmax), elimination rate constant (ke), absorption rate constant (ka), and area under the curve (AUC), by using the simulation analysis and modelling software SAAM II (University of Washington). Total tracer retention will be calculated as total tracer intake - total tracer excreted (AUCurine + AUCstool). Total tracer absorption will be calculated as total tracer intake - total tracer appearance as plasma inorganic iodine (AUCPII). The optimization of procedures for routine application of this method will mainly include the definition of minimal requirements of specimen's specific sampling duration (number of days) after the administration of the tracer dose. This will be assessed by using ROC curves. Expected output and significance/ expected impact. This study will be the first to use 129I as a biologic tracer in humans. Once developed, the availability of this novel method would provide a unique tool to address several key issues with regards to human iodine metabolism and thyroid function. These include the precise definition of the iodine requirement for important life stages, investigation of potential up-regulation of iodine absorption during iodine deficiency, thyroid uptake and turnover of iodine in varying iodine status, assessment of placental and breast milk iodine transfer, as well as the assessment of iodine bioavailability from different foods. Thus, this project could have significant long term impact in the field of iodine and the thyroid.