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Clinic of Nuclear Medicine (J.R., D.B., C.R.), University of Würzburg, D-97080 Würzburg, Germany; and Merck KGaA (T.G.), D-64271 Darmstadt, Germany
Address all correspondence and requests for reprints to: Dr. Johann Rendl, Clinic of Nuclear Medicine, University of Wuerzburg, Josef-Schneider-Straße 2, D-97080 Wuerzburg, Germany. E-mail: rendl{at}nuklearmedizin.uni-wuerzburg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we present a rapid urinary iodide test that is very easy to perform and does not require any technical equipment or apparatus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Included were: disposable columns, 65 x 10.5 mm, packed
with purified activated charcoal (Merck patent number WO 96/27794) for
removing interfering substances; column support; three test cups
(25 x 50 mm); and color scale (Fig. 1
pictogram).
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Included were: buffer solution [citrate-hydrochloric acid (pH 4.0), Merck catalog no. 1.09435]; peracetic acid/H2O2 (dropping bottle), 1.2% in 30% H2O2; and 3,3',5,5'-TMB (dropping bottle), 2.5 mmol/L analytical grade in ethanol.
Apparatus
Apparatus included a spectrophotometer (Shimadzu Corp., Kyoto, Japan); and HPLC equipment (all components from Waters Chromatography Div., Millipore, Milford, MA (Ref.12).
Procedures
Collection of urine samples. For a period of 4 weeks, we collected 370 random urine samples from 370 consecutive patients who were referred to our clinic for thyroid examination. For dilution experiments, an additional 51 random urine specimens were assembled from patients within 1 week.
Performance of the rapid urinary iodide test. As discussed below, each urine sample must be tested within 2 h after collection. Further, the test cannot be performed on frozen specimens.
Dilution of the urine sample Using a pipet, 1 mL of the urine sample was placed into test cup 1, and 4 mL of the buffer solution was added.
Column preparation Columns were initially filled with a weak alcoholic solution. This solution was removed by pouring it out. Then the column was mounted to test cup 2 using the column support. First, the column was equilibrated with 3 mL of the buffer solution. After the column run dry, 2 mL of the diluted urine sample in test cup 1 was applied to the column. The column was allowed to run dry again. Then, the solution in test cup 2 was discarded.
Separation procedure (removal of interfering substances) The column was mounted to test cup 3 again using the column support. Using a pipet, 2 mL of the diluted urine sample in test cup 1 was applied to the column, and the eluate in test cup 3 was collected. The column was discarded, and the column support was removed. It is necessary to carry out the following step within 10 min after collection of the eluate to avoid deterioration of the sensitivity of the color reaction.
Iodide determination
Using a pipet, 150 µL (or 6 drops) TMB was applied to the
solution in test cup 3. The catalytic reaction was started by adding 25
µL (one drop) of peracetic acid/H2O2, mixing
by gently shaking the cup, waiting approximately 30–60 sec, and then
comparing the color of the solution with the color ranges of the
pictogram (Fig. 1
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Spectral characteristics/colorimetry. After addition of
peracetic acid/H2O2 to the solution in test cup
3, containing the eluate and TMB (see above), optical spectra were
recorded after precisely 90 sec. In the visible region, two peaks
appeared at 370 and 655 nm (Fig. 2).
Because of correspondence with the visual appearance of the solution,
the absorbance was measured at 655 nm, in a 1-cm path cell, against a
water blank.
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Photometric detection. The catalytic effect of iodide in the
redox reaction between the colorless 3,3',5,5'-TMB and the peracetic
acid/H2O2, to yield colored products, is the
basis of the photometric method used by the rapid urinary iodide test
for determination of iodide in urine. The first colored product is a
blue charge-transfer complex (Fig. 3) of
the parent diamine and the diamine oxidation product (14). This species
exists in rapid equilibrium with the TMB-radical cation (Fig. 3).
Incubations with high iodide concentrations turn blue, pass through a
green stage, and finally become yellow. The green solution (see
pictogram, Fig. 1) is simply a mixture of the initial blue product and
final yellow component (14).
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), were
diluted 1:1 with buffer solution (500 µL buffer and 500 µL urine).
The test procedure was carried out on these diluted urines, and the
results were compared with those obtained from the undiluted ones. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distribution of urinary iodide in 370 consecutive
patients is given in Fig. 4. The median
of the distribution is 91 µg/L (0.72 µmol/L). The iodide
concentrations varied between 5 and 6600 µg/L (0.04–51.97 µmol/L).
If patients on medical treatment for goiter or with a history of
exposure to large amounts of exogenous iodine caused by radiological
procedures were excluded (n = 76), the median of the resulting
distribution would be 71 µg/L (0.56 µmol/L). This result is in
agreement with corresponding data from other regions in Germany
(15).
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The mean (± SD) of the final column eluate, as determined by weighing 39 eluates, was 2.007 (0.044) mL. The volumes, therefore, are uniform and can be extrapolated back to the starting urine sample, because 2 mL of the diluted sample were applied to the column (see previous Separation procedure).
Interobserver reproducibility was excellent and was tested by having four observers read the color of the reaction of the first 50 samples and by having two observers for the remainder of the urine samples. Because of the clear appearance of the color, 30–60 sec after starting the reaction, no differences were found between the different observers.
Possible disturbing factors and influences
To assess the stability of columns and reagents under conditions of storage, 50 columns and two sets of reagents were stored for 10 months at room temperature (varying from 18 C in wintertime to approximately 30 C in summertime). No difference was observed, with respect to the intensity of the color reaction between tests performed with these columns and reagents and tests carried out with new kits stored at 7 C in the refrigerator.
To investigate whether environmental temperature, especially heat, can affect the results of the chemical reaction, five urine samples with iodide concentrations of 72, 95, 139, 160, and 250 µg/L (0.57, 0.75, 1.09, 1.26, and 1.97 µmol/L) were tested at room temperature (24 C) and at 43 C incubation temperature after heating urines, columns, and reagents up to this temperature for 2 h in an incubator. The tests done in the incubator and at room temperature gave the same correct results.
Possible effects of interfering substances were assessed by adding known amounts of potassium thiocyanate, L-ascorbic acid, sodium sulfide, and sodium chloride to three urine samples with 59, 169, and 287 µg iodide/L (0.46, 1.33, and 2.26 µmol/L) to a final concentration of 300 µmol/L for thiocyanate, 20 mmol/L for ascorbic acid, 50 µmol/L for sulfide, and 100 mmol/L for chloride. Correct results were obtained, and no differences were observed when testing these urines, native and spiked with the compounds above.
The test carried out on aqueous solutions of potassium iodate did not show any color reaction; the method, therefore, does not detect iodine in any other biologically relevant form than iodide.
Comparison between spectrophotometry and HPLC
For comparison, the iodide content of the 370 urine samples was
determined by both spectrophotometry and HPLC. The results, in terms of
absorbance vs. HPLC data, are given in Fig. 5. There is an almost-linear relationship
up to 500 µg/L (3.94 µmol/L) iodide. At higher iodide
concentrations, the absorbance shows a nonlinear increase, reaching a
plateau at about 1500 µg/L (11.81 µmol/L). The r value is 0.94,
calculated by nonlinear regression analysis.
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The data obtained, using the rapid urinary iodide test
for determining the iodide content of the same 370 urine samples, are
plotted in Fig. 6, with HPLC as a
reference method. Corresponding to the color ranges of the pictogram
(see Fig. 1), four panels (A-D) are shown, each representing the
classification of urines, with respect to the color of the chemical
reaction observed in the rapid test. As can easily be seen (Fig. 6, A,
C, and D), there is good agreement between the rapid test and the HPLC
data for urines with high (Fig. 6, C and D) and low (Fig. 6A) iodide
concentrations. The deviations from the HPLC values (shaded
areas in Fig. 6) are relatively small, amounting to approximately
8% per color range. Larger discrepancies with the HPLC data are seen
only in the lower range of color scales 2+3 (Fig. 6B), where 17%
false-positive results are found. In these cases, the rapid test shows
a color reaction despite the relatively low urinary iodide
concentrations between 50 and 100 µg/L (0.39–0.79 µmol/L). To
solve the problem of ambiguous results in color ranges 2 or 3, dilution
experiments were carried out on an additional 51 urines [median
urinary iodide, 228 µg/L (1.80 µmol/L); range: 72–478 µg/L
(0.57–3.76 µmol/L)]. In comparison with the iodide concentrations
determined by HPLC, the results allow the following classification: if
the diluted urine gives no color reaction, the iodide concentration of
the undiluted urine is usually less than 200 µg/L (<1.57 µmol/L),
but at most, equal to 250 µg/L (1.97 µmol/L); in the case of a
color reaction despite dilution, the iodide content of the undiluted
urine is usually more than 200 µg/L (>1.57 µmol/L) but not lower
than 180 µg/L (1.42 µmol/L).
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. A
very good agreement among HPLC, spectrophotometry, and the rapid
urinary iodide test is obtained. The same is true for the calculated
median values.
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Data collected between 1993 and 1994 in a prospective
nationwide survey in Germany show that it is possible to determine with
sufficient precision the median of a given distribution of urinary
iodine concentrations solely from the percent of samples with urinary
iodine less than 100 µg/L (0.79 µmol/L). A total number of 6815
subjects, belonging to various age groups and spread over 34 regions
throughout in Germany, could be enrolled in this study. Random samples
of urine were collected from 6381 clinically euthyroid persons without
history of thyroid disease, and the samples were measured for iodine
concentration using a modification of the colorimetric method of
Sandell and Kolthoff. Part of these data was published in 1996 by
Hampel (16). With kind permission by the author (R. Hampel, University
of Rostock, FRG) and by the sponsor of the study (Merck KGaA), we could
use the original data of the whole study to establish a relationship
between the median values of 37 distributions of urinary iodine
concentrations encompassing 7849 subjects (3 data sets from Wuerzburg
collected in 1986, 1990, and 1997, with a total number of 1468 persons,
were included) and the corresponding percentages of samples below 100
µg/L (0.79 µmol/L). The correlation shown in Fig. 7 is excellent (r = -0.99) and
statistically highly significant (P <
10-6).
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), with an SE of
estimate (SEE) of only 2.6 µg/L (0.02 µmol/L). To evaluate the precision of the test in determining the percentage of samples, we constructed (by random number assignment) 50 subgroups, each containing 50 samples from our original data set (n = 370) and calculated the corresponding percentages of samples below 100 µg/L (0.79 µmol/L), as classified by the rapid test. The mean value of the percentages of samples below 100 µg/L (0.79 µmol/L) was 51.96%, with a coefficient of variation (CV) of 11.7%.
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Discussion |
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Bourdoux (17) has shown that information
about the iodine status of a population can easily be obtained from a
set of 50–100 casual urine samples. Because of the important scatter
of urinary iodide concentrations yielding skewed distributions,
Bourdoux (18) proposed a classification (Table 2) of iodine status, based on his
experience with thousands of samples from several countries throughout
the world, using an arbitrary categorization of urinary iodide
concentrations: 
20, 50, and 100 µg/L (0.16, 0.39, 0.79
µmol/L). These three ranges are related to 3 levels of iodine
deficiency (mild, moderate, and severe) suggested by international
organizations (2, 7, 19, 20), who have adopted a simplified
classification based only on single median values (2, 7). Table 2
suggests that these median values may be correlated with the percentage
of samples below 100 µg/L (0.79 µmol/L). We could indeed derive
from 37 distributions a corresponding relationship showing a high
correlation (r = -0.99). Therefore, it is possible to extrapolate
the median of a given distribution from the percentage of samples below
100 µg/L (0.79 µmol/L) by using Fig. 7. As can easily be seen, the
correlation shown in Fig. 7 is in very close agreement with Bourdoux’s
classification (Table 2). If, for example, 50–80% of all samples are
below 100 µg/L (0.79 µmol/L), the corresponding median is between
50 and 100 µg/L (0.39–0.79 µmol/L), and the iodine intake is
mildly deficient; if more than 80% of all samples are below 100
µg/L, the median lies below 50 µg/L (0.39 µmol/L), and the iodine
intake is to be classified as moderately deficient. Evaluating the
precision of the rapid test at the cut-off level of 100 µg/L (0.79
µmol/L), yields a CV of 11.7%. For comparison, the mean CV of method
A in Ref. 6 is 11.0%.
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. The data show a very good agreement among HPLC,
spectrophotometry, and rapid test results, with respect to the
classification of all samples by number and percentage of samples in
each category. In more than 50% of all samples, iodide
concentrations 100 µg/L (0.79 µmol/L) were found by HPLC,
spectrophotometry, and the rapid test. This is equivalent to a mildly
deficient iodine intake, according to Table 2, and corresponds to the
median of 91 µg/L (0.72 µmol/L) of the whole distribution. The
median values calculated by using the regression equation of Fig. 7
also are given in Table 1; they are in good agreement with the exact
value. Furthermore, because of the correct classification of samples
also in the ranges between 100–300 µg/L (0.79–2.36 µmol/L) and
more than 300 µg/L (>2.36 µmol/L), this test allows the rapid
identification of the percentage of subjects who, in case of
supplementation, move from one category to another. Compared with other simple methods (6, 21, 22), the rapid test is extremely easy to perform and does not require any instrumentation or apparatus. Only columns with purified activated charcoal are needed. One technician can analyze, in parallel, 8–10 samples; the total analysis time for one sample is 15 min. About 60–80 samples can be tested this way within 2 h, at a cost of 0.5–1.0 $(US) per sample (depending on the number of kits ordered), labor not included. This rapid test allows on-site monitoring of iodine deficiency under conditions of heat and storage usually found in developing countries, and it makes shipping of samples unnecessary. For that reason, the test is well suited to epidemiological investigations, especially in these countries.
Suitability of the rapid urinary iodide test for estimating the iodine intake of a single individual
Fig. 6 shows that an unambiguous interpretation of a
single test result is possible if the color of the chemical reaction
can be related to ranges 4 or 5, meaning that this sample is suspected
to be iodine contaminated, or if no color reaction occurs (
color
range 1). In the latter case, iodine contamination can be excluded
because the iodide concentration is below 100 µg/L (0.79 µmol/L) in
more than 90% of the samples, and it does not exceed 200 µg/L (1.57
µmol/L) (Fig. 6A). An equivocal result is obtained (Fig. 6B) if the
color of the reaction corresponds to ranges 2 or 3 100–300
µg/L (0.79–2.36 µmol/L), where 17% of the urine samples of this
category gave a color reaction despite the fact that the iodide
concentrations were below 100 µg/L (0.79 µmol/L), as measured by
HPLC. Interfering substances and matrix effects are very likely to
cause this ambiguity. One of the interfering compounds is possibly
ammonium, which originates from the decomposition of urea in
long-standing urines. During freezing and thawing, the same process may
be responsible for the formation of interfering products. By adding
ammonium to urine samples, for example, the color reaction can be
completely suppressed. Such compounds are only partially removed by the
activated carbon. So, if the concentration of interfering substances in
a urine sample is relatively high, color reaction may not occur, even
when the iodide concentration is above 100 µg/L (0.79 µmol/L). If,
on the contrary, the amount of interfering substances is relatively
low, a color reaction may be observed despite iodide concentrations
below 100 µg/L (0.79 µmol/L), because the color reaction itself is
sensitive also to small iodide concentrations (as observed when giving
the reagents to pure iodide calibrators diluted only with buffer). In
the special matrix of urines, however, the cut-off level is 100 µg/L
(0.79 µmol/L). The problem of ambiguous results in color ranges 2 or
3 can be solved by repeating the test with the same urine diluted 1:1
with buffer. The test result obtained from the diluted urine allows a
final statement, with respect to the iodide content of the undiluted
sample. A second determination from a dilution, however, is required
only if the iodine intake of a single individual is to be estimated,
especially if iodine contamination should be excluded. In field
studies, on the other hand, it is not necessary to dilute any sample,
because the test yields the correct percentages of samples in each
range (Table 1).
In conclusion, comparison with the HPLC method clearly demonstrates that this rapid test for determination of urinary iodide is well suited to the assessment of iodine deficiency and to the monitoring of iodine supplementation in epidemiological surveys. With respect to the determination of iodide in a single urine sample, the test allows the detection or exclusion of iodine contamination (excess iodide), which is important in iodide-induced hyperthyroidism (23).
Received March 13, 1997.
Revised August 27, 1997.
Revised November 7, 1997.
Accepted November 20, 1997.
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