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Articles |
1
Pharmaceutical Research Laboratory, Hitachi Chemical Co., Ltd., 13-1, Higashi-cho 4-chome, Hitachi-shi, Ibaraki-ken 317-8555, Japan.
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International Council for Control of Iodine Deficiency
Disorders, New Delhi 110029, India.
3
International Council for Control of Iodine Deficiency
Disorders, Tokyo 112-0001, Japan.
a Author for correspondence. Fax 81-294-24-3602; e-mail tos-oohashi{at}hitachi-chem.co.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Using a specially designed sealing cassette to prevent loss of vapor and cross-contamination among wells, ammonium persulfate digestion was performed in a microplate in an oven at 110 °C for 60 min. After the digestion mixture was transferred to a transparent microplate and the Sandell–Kolthoff reaction was performed at 25 °C for 30 min, urinary iodine was measured by a microplate reader at 405 nm.
Results: The mean recovery of iodine added to urine was 98%
(range, 89–109%). The theoretical detection limit, defined as 2 SD
from the zero calibrator, was 0.11 µmol/L (14 µg/L iodine). The
mean intra- and interassay CVs for samples with iodine concentrations
of 0.30–3.15 µmol/L were 
10%. The new method agreed well with the
conventional chloric acid digestion method (n = 70;
r = 0.991; y =
0.944x + 0.04; Sy|x = 0.10) and
with the inductively coupled plasma mass spectrometry method (n =
61; r = 0.979; y =
0.962x + 0.03; Sy|x = 0.20). The
agreement was confirmed by difference plots. The distributions of
iodine concentrations for samples from endemic areas of iodine
deficiency diseases showed similar patterns among the above three
methods.
Conclusions: Our new method, incorporating the whole process into a microplate format, is readily applicable and allows rapid monitoring of urinary iodine.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the other hand, an alternative method that uses ammonium persulfate digestion has been reported recently as a nonhazardous, nonexplosive, and easy-to-use method (4). The persulfate digestion makes possible a comparatively nonhazardous (no chlorine gas) measurement. However, this method is still not completely suitable for testing because it is time-consuming and produces a nonnegligible amount of toxic waste.
One of our objectives in this study was to seek an easy-to-use method. We applied a microplate format to all processes so that we could minimize the amount of toxic wastes as well as simplify and speed up the procedure. We tried using a closed system during the digestion process to keep the reaction mixtures in all wells volumetrically equal and to avoid contamination throughout the process; in other words, to prevent the leakage of vapor and to achieve an accurate measurement of urinary iodine.
In this study, we evaluated the analytical performance of the ammonium persulfate digestion on microplate (APDM) method.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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The colorimetric measurements were performed in a microplate reader (IMMUNO-MINI; Nalge Nunc International).
chemicals
Potassium iodate for calibrators and analytical grade
arsenic trioxide, potassium chlorate, perchloric acid (700
g/L), ammonium persulfate, tetraammonium cerium (IV) sulfate
dihydrate, sodium chloride, and sulfuric acid were obtained from Wako
Pure Chemical Industries. Glass-distilled deionized water was used for
preparation of reagent solution and dilution procedures.
solutions
Chloric acid solution (3.3 mol/L).
Potassium chlorate (500 g)
was dissolved in 1000 mL of water in a 2000-mL Erlenmeyer flask with
heating for 60 min in a boiling water bath, after which 375 mL of
perchloric acid was added slowly with constant stirring. The solution
was then stored at -25 °C in a freezer overnight. The resulting
suspension was filtered with a glass filter (5–10 µm mesh). The
filtrate was stored in a refrigerator (4 °C) until use.
Ammonium persulfate solution (1.31 mol/L).
Ammonium persulfate
(30 g) was dissolved in water to a final volume of 100 mL. This
solution was prepared fresh just before use.
Arsenious acid solution (0.05 mol/L).
Arsenic trioxide (5 g)
was dissolved in 100 mL of 0.875 mol/L sodium hydroxide solution.
Concentrated sulfuric acid (16 mL) was then added slowly to the
solution in an ice bath. After cooling, 12.5 g of sodium chloride
was added to the solution, and the mixture was diluted to 500 mL with
cold water and filtered.
Ceric ammonium sulfate solution (0.019 mol/L).
Tetraammonium
cerium (IV) sulfate dihydrate (6 g) was dissolved in 1.75 mol/L
sulfuric acid and adjusted to a final volume of 500 mL with the same
acid solution.
Iodine calibrators.
In a 100-mL volumetric flask, 168.6 mg of
potassium iodate was dissolved in water to make a 7.88 mmol/L stock
solution (1000 mg/L iodine). The stock solution was diluted 100- and
10 000-fold, and working solutions of 0.039–4.73 µmol/L (5–600
µg/L iodine) were prepared.
urine samples
Urine samples were collected from the following: (a)
individuals (children and adults) staying in the suburbs of
Ulaanbaatar, Mongolia; (b) patients (children and adults)
attending clinics in Baluchistan, Pakistan; (c) nurses and
other technical staff working in the All India Institute of Medical
Sciences, New Delhi, India; and (d) the general
population in Khopasi, Nepal (suburbs of Kathmandu).
experimental procedure
APDM method.
Calibrators and urine samples (50 µL each) were
pipetted into the wells of a polypropylene (PP) plate, followed by the
addition of 100 µL of ammonium persulfate solution (final
concentration, 0.87 mol/L). The PP plate was set in a cassette. The
cassette was tightly closed and was kept for 60 min in an oven adjusted
to 110 °C. After digestion, the bottom of the cassette was cooled to
room temperature with tap water to avoid condensation of vapor on the
top of wells and to stop the digestion. The cassette was opened, and
50-µL aliquots of the resulting digests were transferred to the
corresponding wells of a polystyrene 96-well microtiter plate
(MicroWell; Nalge Nunc International). Arsenious acid solution (100
µL) was added to the wells and mixed; 50 µL of ceric ammonium
sulfate solution was then added quickly (within 1 min), using a
multichannel pipette (Finnpipette Varichannel; Labsystems). The
reaction mixture was allowed to sit for 30 min at 25 °C, and the
absorbance was measured at 405 nm with a microplate reader.
Inductively coupled plasma mass spectrometry (ICP/MS) method.
An SPQ 8000A1 ICP/MS analyzer (Seiko Instruments) was used. The
procedure was carried out according to the method of Yoshinaga and
Morita (5). The plasma gas was argon at flow of 16 L/min,
the auxiliary gas was argon at a flow of 0.4 L/min, and the nebulizer
gas was argon at a flow of 0.4 L/min. The sample flow rate was 1
mL/min. The output frequency was 1 kW, 12.2 MHz, and the detector was
set at 2 kV.
Conventional chloric acid digestion method in a test tube.
Calibrators and urine samples (250 µL) were added to 16 x 160
mm test tubes, and 750 µL of chloric acid solution was added. The
tubes were covered with plastic caps, placed into the wells (75 mm in
depth) of an aluminum block, and left for 60 min at 110 °C in a fume
hood. Because the test tubes were much longer than the depth of the
heating block wells, the vapor refluxed within the test tubes.
After the test tubes were cooled, 3.5 mL of arsenious acid solution was
added into each tube and mixed. Three hundred fifty microliters of
ceric ammonium sulfate solution was added and mixed by vortex-type
mixer; a stopwatch was used to keep a constant interval. Exactly 20 min
after the addition of the ceric ammonium sulfate solution, the
absorbance at 405 nm was measured with a spectrophotometer.
evaluation of sealing cassette
Airtightness.
The airtightness of the sealing cassette was
evaluated by comparing the weight of water in each well of the PP plate
before and after heating with the cassette in an oven, using the
following method: Water (150 µL) was pipetted into one well of the PP
plate on a balance, and the increased weight of the PP plate was
determined. The above step of pipetting and weighing was repeated
sequentially for remaining all wells. The PP plate was then loaded into
the sealing cassette and heated for 60 min in a 110 °C oven. After
cooling, the plate was weighed. The water in one well was completely
removed by sucking with a pipette and drying with a cotton swab, and
the plate was reweighed. This step was repeated for all other wells.
Cross-contamination.
Cross-contamination between wells was
evaluated by comparing two 96-well plates: (a) a control
plate in which urine was placed into every second well (48 wells); and
(b) a test plate, in which the same urine was placed into
every second well (48 wells) and KI urine was placed into the remaining
wells (48 wells). KI urine was prepared by mixing KI solution (0.1 mL
of a 1000 µmol/L solution) and the urine (0.9 mL). After digestion
with ammonium persulfate, the concentration of iodine was measured
separately.
optimization of apdm method of digestion
Optimization is performed using eight Mongolian urine samples
(iodine concentration, 0.30–1.35 µmol/L). Optimum digestion was
defined as having sufficient recoveries of iodine added to various
urine samples (final added iodate concentration, 0.79 µmol/L)
according to the method described below.
assay evaluation
Calibration curve and calculation.
A calibration curve was
prepared for each plate by plotting the logarithmic conversion of the
means of absorbance at 405 nm (n = 2) on the y axis vs
the iodine concentrations [0.20, 0.39, 0.79, 1.57, 2.36, 3.15 µmol/L
(25, 50, 100, 200, 300 and 400 µg/L iodine)] on the x
axis. The urinary iodine concentration was determined using linear
regression. Water was used for the zero calibrator.
Detection limit.
A pooled urine sample at a low iodine
concentration was serially diluted with water. The detection limit of
urinary iodine, defined as 2 SD from the zero calibrator (replicates of
10), was characterized from five analyses.
Precision.
Pooled urine samples with low, medium, and high
concentrations of iodine were used to determine the intra- and
interassay CVs. In an intraassay experiment, each urine sample was
assayed in eight replicates on the same plate. Using the same samples,
an interassay experiment was performed on 30 different days.
Recovery.
The recovery of iodine was estimated by assaying in
triplicate 12 different urine samples supplemented with potassium
iodate solution, and comparing the results with those of water-added
urine samples. The iodate-added urine was prepared by adding a given
volume (1/10 volume of the urine sample) of potassium iodate solution
(3.94 µmol/L) to the urine samples. For the water-added urine, water
was added instead of potassium iodate solution. The percentage of
recovery was calculated by the following equation:
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Effect of interfering substances.
The effects of three
interfering substances (potassium thiocyanate, L-ascorbic
acid, and ferrous ammonium sulfate) on the assay were assessed in
triplicate using the following series: (a) iodate solution
plus interfering substance, without digestion; (b) iodate
solution plus interfering substance, with digestion; and (c)
urine plus interfering substance, with digestion. The interfering
compounds were added to urine or iodate solution to final
concentrations of 0.1–64 mmol/L. The iodine concentrations of urine
sample and iodate solution without interfering substances were measured
as controls.
Digestion of iodocompounds.
Four organic iodocompounds
(p-iodobenzoic acid, m-iodophenol, 2-iodophenol,
and 3-iodotyrosine) were examined with the digestion process. The
recovery of iodine from each iodocompound was estimated by comparing
the measured iodine concentration with the calculated concentration.
Linearity.
Pooled urine samples containing low, medium, and
high concentrations of iodine were serially diluted with water. The
testing was carried out in triplicate.
Comparison with other methods.
Pearson and Spearman
correlations were applied to the results. Comparison of the APDM method
and the ICP/MS method was performed using a total of 61 urine samples
from Mongolia and Pakistan with iodine concentrations of 0.079–3.15
µmol/L. Comparison of the APDM method with the conventional chloric
acid digestion method was performed using 70 urine samples from Nepal.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cross-contamination.
The mean urinary iodine concentration of
the control plate was 1.17 ± 0.03 µmol/L (range, 1.13–1.28
µmol/L), and that of test plate was 1.21 ± 0.05 µmol/L
(range, 1.11–1.34 µmol/L). The recovery of iodine added to urine as
potassium iodide (100 µmol/L) was 96.8% ± 6.7% (range,
87.8–118.6%).
optimization of apdm method of digestion
The recovery of iodine added to eight urine samples was compared
with various combinations of three variables; i.e., final concentration
of ammonium persulfate, oven temperature, and digestion time. Based on
analysis of the data, the combination of 0.87 mol/L (200 g/L )
ammonium persulfate (final concentration), an oven temperature of
110 °C, and a 60 min-incubation was found to be optimum and gave the
highest recovery with little scatter (Fig. 2
). When digestion was performed under submaximal conditions, the
recovery was 0–100%, depending on the urine samples.
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assay evaluation
Calibration curve.
The absorbance on a log scale was linear
for iodine concentrations between 0 and 3.15 µmol/L. The correlation
coefficient for the linearity was >0.998, using six calibrators. An
example of a calibration curve is shown in Fig. 3
.
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Detection limit.
The theoretical detection limit of urinary
iodine, defined as 2 SD from the zero calibrator, was 0.11 µmol/L
(range, 0.07–1.4 µmol/L), based on five analyses.
Precision.
At medium and high concentrations of urinary
iodine, the intraassay CVs were 1.7–2.0%, and the interassay CVs were
4.4–4.5% (Table 1
). At a concentration of 0.30 µmol/L (Table 1
, low 2), the
interassay CV was 
10%, whereas at 0.15 µmol/L (Table 1
, low 1),
the CV was 20%. The working detection limit (tentatively defined as
the lowest concentration measured with an interassay CV <10%
) was estimated as 0.3 µmol/L.
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Recovery.
The recoveries of iodine added to urine samples at
different concentrations were 89–109% (Table 2
).
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Effect of interfering substances.
In the assay without
digestion, the addition of potassium thiocyanate (
0.1 mmol/L),
ascorbic acid (4 mmol/L), or ferrous ammonium sulfate (16 mmol/L)
significantly increased the estimated concentration of iodine in the
iodate solution. On the other hand, in the assay with digestion, the
addition of up to 16 mmol/L (final concentrations) of these interfering
compounds did not affect the results for the urine sample or the iodate
solution (Table 3
).
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Digestion of iodocompounds.
The recovery of iodine from four
organic iodinated compounds (p-iodobenzoic acid,
m-iodophenol, 2-iodophenol, and 3-iodotyrosine) was assayed
with digestion. The mean recovery (as inorganic iodine) for these
compounds was 101% of the expected value (Table 4
).
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Linearity.
The recovery was linear for the medium- and
high-concentration samples with urinary iodine >0.20 µmol/L. The
recovery of urinary iodine was 92–106% for concentrations >0.20
µmol/L (Table 5
).
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Comparison with other methods.
The APDM method was compared
with two other methods by the regression analysis. The correlation
between the APDM and the ICP/MS was good for measurements of urine
samples with iodine concentrations <3.15 µmol/L (n = 61;
r = 0.979; y = 0.962x+0.03;
Sy|x = 0.20). In a subset of the urine samples
with iodine concentrations <0.79 µmol/L, which were classified as
iodine deficient, the correlation between the two methods also was
linear (n = 27; r = 0.978; y =
1.124x; Sy|x = 0.05). Comparison with
the conventional chloric acid digestion method showed a higher
correlation than that with ICP/MS, with a small standard error of the
estimate (n = 70; r = 0.991; y =
0.944x+0.04; Sy|x = 0.10). Using the
difference plot recommended by Bland and Altman (6),
we compared APDM with the other methods: the conventional chloric acid
digestion method and ICP/MS, respectively. On the abscissa, we plotted
mean values of the two methods compared instead of a reference method
because, to date, none of the three methods has been accepted as the
reference method (7)(8)(9). Comparison between APDM and the
conventional method gave a mean difference (d) of 0.01
µmol/L, a SD for the differences of 0.11 µmol/L, and a distribution
of d + 1.96 SD = 0.23 µmol/L to d -
1.96 SD = -0.21 µmol/L for urinary iodine concentrations of
0.16–3.15 µmol/L (Fig. 4
A). For the samples with iodine concentrations <0.79 µmol/L
as described above in the regression analysis, we obtained a much
narrower distribution (SD = 0.05 µmol/L) of differences between
APDM and the conventional method. The difference plot for APDM vs
ICP/MS is shown in Fig. 4B
. The result suggested that the difference
plot analysis between the two methods is consistent with the regression
analysis, showing a wider distribution than that of APDM vs the
conventional method.
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evaluation of the method as a public health application
We evaluated APDM as a public health application by comparing it
and the conventional and ICP/MS methods, using the two sets of urinary
samples described above. The proportion of samples below specific
cutoff points and medians are shown in Table 6
. The World Health Organization and ICCIDD proposed the use of
these cutoffs for interpreting urinary iodine results as follows:
"severe deficiency", urinary iodine concentration <0.16 µmol/L;
"moderate deficiency", urinary iodine concentration, 0.16–0.39
µmol/L; "mild deficiency", urinary iodine concentration,
0.39–0.79 µmol/L; and "normal", urinary iodine concentration
>0.79 µmol/L (1)(10).
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The three methods gave similar distribution patterns. Particularly, in the case of APDM vs the conventional method, we obtained good agreement for 70 samples between the two methods. The median values obtained with the conventional and APDM methods were 0.67 and 0.68 µmol/L, respectively; the median values obtained with the ICP/MS and APDM methods were 0.44 and 0.47 µmol/L, respectively. The Spearman rank correlation coefficients for both comparisons were 0.990 (APDM vs the conventional method) and 0.957 (APDM vs the ICP/MS), respectively (P <0.0001). These results indicated acceptable interpretative agreement between two methods in each comparison.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Using this cassette (commercially
available at approximately $1500), we observed no substantial loss of
sample volume and no cross-contamination between wells. In our study, the recovery of iodine added to urine showed very large variation when digestion was insufficient. We tried raising the temperature and/or prolonging the digestion time, and found the optimum conditions: 60-min digestion in 110 °C standard oven in our system. Although it is natural that the recovery is less with insufficient digestion, a long digestion time also decreased recovery. We surmise that some substances produced from urine during digestion may have been responsible for the decrease in recovery. This may depend mainly on the characteristics of urinary samples. Further study will be required to make this clear. Regarding interference, ascorbic acid, potassium thiocyanate, and ferrous ammonium sulfate in concentrations up to 16 mmol/L did not affect the Sandell–Kolthoff reaction after digestion in our study, which is consistent with a previous report by Pino et al. (4).
The cutoff points proposed by WHO/UNICEF/ICCIDD for classifying iodine deficiency are based on median urinary iodine concentrations. As an indicator of iodine deficiency "elimination", they proposed that the median iodine concentration should be 0.79 µmol/L, i.e., 50% of samples should be above 0.79 µmol/L, and not more than 20% of samples should be below 0.39 µmol/L.
The intra- and interassay CVs for samples with iodine concentrations of
0.30–3.15 µmol/L were 10% for the APDM method. The APDM method
has sufficient precision to assess the indicator of iodine deficiency
"elimination" (10). On the other hand, because the
interassay CV was 20% for samples at 0.16 µmol/L, in the case of
monitoring of urinary iodine for severe iodine deficiency it is
necessary to take into consideration the measurement error.
The APDM method was compared with the conventional chloric digestion method and the ICP/MS method, which is said to be the most sensitive method for urinary iodine detection (5)(7)(8)(11)(12). There were good correlations between the APDM method and other two methods. In the difference plot, trends or shifts between these methods were not observed.
In addition, we compared the distributions and medians of urinary
iodine concentrations measured by three methods (Table 6
). The Spearman
rank correlation coefficients for the APDM method vs the conventional
and ICP/MS methods were 0.99 and 0.95, respectively. The three methods
gave similar distributions. In particular, in comparison of the APDM
method and the conventional chloric digestion method, good agreement
was obtained for 70 samples between the two methods.
The only equipment required for the APDM method is an automated
microplate reader, which is widely used in assays for
thyroid-stimulating hormone (one of the biochemical indicators of IDD).
Because the Sandell–Kolthoff reaction is a kinetic reaction, ideally
the interval between addition of the cerium solution to a well and
reading by the microplate reader would be the same for all
wells. However, if an automated reader (reading time, 20–50 s)
and a multichannel pipette (pipetting time, <60 s) are used, the time
lag between pipetting and reading is at most 40 s, which is only
2% error for a 30-min reaction. The number of samples that could be
assayed in a day is 300–500 if three sealing cassettes are used.
Recently, one of the co-authors (M.G. Karmarker) has successfully
applied this technique in Nepal to analyze 7740 urine samples in the
IDD survey with a proper internal quality assessment. The required
testing period, which had been estimated as >3–4 months for the
conventional method, was only 20 days by our method.
In conclusion, our new method has a good detection limit
(0.11 µmol/L) and precision with a dynamic range of 0.30–3.15
µmol/L (CV 10%), and also reduces assay time to 2 h for 80
samples per microplate. The microplate digestion method is almost
identical to the conventional method in classifying iodine deficiency
or sufficiency. The APDM method is advantageous from the viewpoint of
safety, ease of operation, and stability of ammonium persulfate. The
APDM method enables easy, portable, rapid, and nonhazardous urinary
iodine detection. Although further interlaboratory comparisons are
necessary, we believe that the performance of the APDM method may be
useful for the purpose of urinary iodine monitoring.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  S. Fallouch, P.-J. Lejeune, J. Barbaria, P. Carayon, and B. Mallet Urinary Iodine Analysis: An Alternative Method for Digestion of Urine Samples Clin. Chem., April 1, 2004; 50(4): 780 - 782. [Full Text] [PDF]
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 M. B Zimmermann, S. Y Hess, L. Molinari, B. de Benoist, F. Delange, L. E Braverman, K. Fujieda, Y. Ito, P. L Jooste, K. Moosa, et al. New reference values for thyroid volume by ultrasound in iodine-sufficient schoolchildren: a World Health Organization/Nutrition for Health and Development Iodine Deficiency Study Group Report Am. J. Clinical Nutrition, February 1, 2004; 79(2): 231 - 237. [Abstract] [Full Text] [PDF]
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D. Gnat, A. D. Dunn, S. Chaker, F. Delange, F. Vertongen, and J. T. Dunn Fast Colorimetric Method for Measuring Urinary Iodine Clin. Chem., January 1, 2003; 49(1): 186 - 188. [Full Text] [PDF]
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 C. Yamada, D. Oyunchimeg, P. Enkhtuya, A. Erdenbat, A. Buttumur, and T. Umenai Current Status of Iodine Deficiency in Mongolia in 1998-1999 Asia Pac J Public Health, January 1, 2000; 12(2): 79 - 84. [Abstract] [PDF]
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