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Problems of Interfering Substances Identified

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Problems of Interfering Substances Identified [Reprinted from CLINICAL CHEMISTRY, 36, 865 (1990).] ' Copyright 1990 by the American Association of Clinical Chemistry and reprinted by permission of the copyright owner. Evaluation of Automated Urinary lodine Methods: Problems of Interfering Substances Identified Warwick May,' Diana Wu,' Creswell Eastman,' Pierre Bourdoux,^ and Glen Maberly' We evaluated automated methods for measurement of uri­ biochemical déterminations have advanced rapidly. nary iodine (Ul) over a range expected in iodine­replete and Our aim w£is to evaluate the performance of the auto­ iodine­deficient populations. Results obtained with Tectini­ mated Ul System available since 1973, which is based on con AutoAnalyzer II Systems, based on either dialysis or acid dialysis of urine (method A). Results from samples assayed digestion, were compared witfi those obtained by a manual at Westmead Hospital, Australia, were correlated with alkaline ashing techinique. Results of automated dialysis results fi^m the Saint Pierre University Hospital, Bel­ were consistently higher than those obtained by the other gium, which were obtained with an even earlier automated methods. The apparently higher concentrations of Ul we Ul détermination, based on acid digestion (method B). measured were due to interfering substances crossing the Results from thèse automated methods were also compared with results obtained by a more technically demanding dialysis membrane and participating in the catalytic reaction. manual technique (method G, performed at Westmead Thiocyanate (SCN) was one endogenous substance contrib­ Hospital), based on the oSicial method of the Association of uting to the increased measurement of Ul. For urinary SCN OflRcial Analytical Chemists (AOAC) (6), in which alkaline concentrations of 5 to 15 mg/L, the amount of overestimation ashing is used to destroy organic matter in the sample in the Ul measurement attributable to SCN ranged from 21.8 before the final colorimetric measurement of iodine. to 61 Atg/L. However, SCN may account for only 40-50% of the apparent increase in Ul. In samples with lower Ul (<50 Materials and Methods /ng/L), interfering substances produced a 100% error in Apparatus results. We conclude that the automated dialysis System should not be used to assess iodine­deficient populations. Automated measurement of Ul was performed with a This leaves a major dilemma for researchers wanting to Technicon AutoAnalyzer II system (Technicon Instrument assess the iodine status of populations, because the auto­ Corp., Tarrytown, NY) with either a dialysis module (meth­ od A) or acid digestion unit (method B). Ashing of urine mated digestion method is no longer commercially available. samples (method C) was carried out in a spécial 102C muffle fumace (Scientific Equipment Manufacturers, Addltlonal Keyphrases: dialysis and acid digestion techniques Magill, South Australia). To measure absorbance, we used compared • analytical error • thiocyanate • nutritional status a Spectronic 20 spectrophotometer (Milton Roy, Rochester, NY). Endémie goiter, resulting from nutritional iodine defi­ ciency, is one of the oldest recognized disorders aflfecting Reagents whole populations. Recently, Hetzel (1) proposed the term Glass­distilled de­ionized water, used to prépare ail solu­ "iodine­deficiency disorders" to reflect the broader spec­ tions and for dilutions, was passed through an ion­ex­ trum of this condition, the eflFects of which include sponta­ change colimm containing anion­exchange resin AG501­ neous abortion, increased infant mortality, and cretinism. X8(D) (BioRad, Richmond, CA) to remove any traces of In fact, the stunting of intellectual functioning in entire iodine. AU other reagents were of analytical grade and iodine­deficient communities has been demonstrated (2). were found to have negligible iodine content. The International Council for the Control of lodine Defi­ ciency Disorders has estimated that 800 million people Urine Samples Worldwide, living mostly in Asia, Africa, and South Amer­ Casual (untimed) urine samples from 51 patients and ica, are at risk of iodine-deficiency disorders (3). staff (Westmead Hospital) and from 11­year­old schoolchil­ Accordingly, many countries have made or implemented dren (Tasmania, Australia) were selected to cover the plans to set up centers to assess and monitor iodine defi­ range of Ul concentrations found in the Australian popu­ ciency. This usually includes automated laboratories for lation. After collection, samples were acidified and refing­ measuring urinary iodine (Ul). Although Ul excrétion has erated until assayed. been considered the définitive criterion for grading the severity of iodine deficiency in communities (4), over the Procédures past two décades this test has become rare in clinical Alkaline ashing. Urine samples (3 mL) were treated with chemistry departments in the developed world. Indeed, 1 mL of 1 mol/L potassium hydroxide and ashed at 600 °C since modification of the earlier automated acid digestion according to the method of Belling (7). We measured the System in 1973 (5), there has been no real progress in iodine in the ashed solution by assessing the catalji;ic eflfect automated Ul meeisurement, whereas methods for other of iodine on the Sandell­Kolthofi" reaction, as described by Potter et al. (8). Because the potassium hydroxide used for ashing the samples depresses the catalytic eflfect of iodine ' Australian Centre for the Control of lodine Disorders, West- on the Sandell­Kolthoflf reaction, the calibration curve mead Hospital, Sydney, Australia. ^ Department of Nuclear Medicine, Saint Pierre University obtained with nonashed aqueous standards is always Hospital, Free University of Brussels, Brussels, Belginm. steejjer than the curve produced by adding iodine to ashed Received J^UEu-y 3, 1990; accepted March 21,1990. solutions. To compensate for this, we derived a correction CLINICAL CHEMISTRY, Vol. 36, No. 6,1990 865 fadbr by adding known amounts of iodine (5 or 10 ng per doux et al. (13). tube), in triplicate, to a séries of urine and blank samples Data analysis. Statistical analysis—linear régression, (n = 17). In several assays (n = 9), we calculated the slope Pearson's corrélation coefficient (r)—was performed with of the line obtained by plotting the amoimt of added iodine StatView 512+™ (Brainpower Inc., Calabassa, CA) on an against the change in absorbance at 340 nm (a), and Apple Macintosh computer. compared this with the slope of a line similarly produced with use of nonashed aqueous standards (6). We deter- Results mined the correction factor (expressed as b:a) to be 1.31 ± Recovery and précision. The mean (± SD) analytical 0.08 (mean ± SD); i.e., values obtained for urine samples by recovery we obtained in method C, after the addition of interpolation from a standard curve prepared with use of radioisotopic and nonlabeled iodine, was 100.3 (2.9)% and aqueous standards must be multiplied by 1.31 to obtain the 97.8 (7.2)%, respectively. Thèse results are in close agree- correct resuit. ment with the analytical recoveries published by others Analytical recovery of iodine after alkaline ashing was who used this method (7,14). Similarly, method A gave a assessed in two ways. First, we prepared urine samples mean analytical recovery of 99% (SD 3.7%), which accords (n = 8) as usual, but added 15 IJL of [^^^I]NaI to give about favorably with the mean recovery of 97% obtained by Gary 70 000 coimts/min. We counted the radioactivity before and et al. (5). lodine-supplemented urine assayed by method B after the ashing procédure and expressed the final count as also showed an analytical recovery of 96-97% (authors' a percentage of the initial counts. We also assessed the unpublished data). analytical recovery of nonlabeled iodine from urine after The between-assay précision (CV) for the assay of a the ashing and assay stages as follows: 2,4, or 6 ng of iodine control urine with a mean UI concentration of 11.3 /^/L, in was added, in triplicate, to 3-niL aliquots of urine (n = 4). a total of 30 alkaline ashing assays, was 10.6%. In method After ashing, we determined the final concentration of A, the between-assay CV for the three urine controls with iodine in each tube as described above. Recovery was mean UI concentrations of 24, 45, and 94 /xg/L was 3.8%, calculated as the amount of added iodine measured, ex• 2.5%, and 2.4%, respectively (n = 35 assays). pressed as a percentage of the amount of iodine added. Discrepancy between methods. Figure 1 compares the UI Reproducibility was evtduated with use of a control urine results for the samples in each method. UI is plotted on a spécimen stored frozen in 3-mL aliquots. Aliquots of this log scale, with results of the alkaline ashing method used to control were then thawed, ashed, and assayed in duplicate rank samples in decreasing order. This Figure demon- with each run. strates an increase in results obtained in automated dial• Automated analysis of urinary iodine. The Technicon ysis, especially in samples with UI <200 jug/L. Régression continuous-flow system used for determining UI by method analysis further highlights the bias between the ashing B is a modification of the procédure used for measuring method (x) and automated dialysis (y), yielding a corréla• protein-bound iodine in sérum, in which an automated tion coefficient (r) of 0.94, but a régression équation oiy = digester hélix is used to digest the samples with concen- 0.91x + 62.9. On the other hand, the corrélation between trated minerai acids at 320 °C (9). UI measurements by ashing (x) and automated digestion (y) was very close for method A were performed with the automated system the range studied (r = 0.99, y = 0.93x + 7.9). described by Gary et al. (5), in which the digester unit, just Interfering substances. To try to explain the apparent described, is replaced by the simpler and safer dialysis overestimation in UI in method A, we investigated the module. Results were calculated by a personal computer possible eflFect of thiocyanate, an ion that might cross the programmed to correct for baseline drift and nonlinearity dialysis membrane and interfère with the Sandell-Kolthoff of the standard curve.
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