THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 20, Issue of October 25, PP. 4686-4693, 1966

Printed in U.S.A.

Conversion of Iodate to Iodide in Viva and in Vitro*

(Received for publication, March 8, 1966)

ALVIN T~UROG,$ EARL M. HOWELLS,~ AND HAROLD I. NACHIMSON~

From the Department of Pharmacology, The University of Texas Southwestern Medical School, Dallus, Texas 75235

SUMMARY

Iodate is almost immediately reduced to iodide following its intravenous administration to rats or rabbits. Even when it is given in relatively large doses, reduction is com- plete within 2 to 3 min. Following oral administration also, radioiodate is so rapidly transformed to radioiodide that 2-hour thyroid 13rI uptakes in rats that received radioiodate by stomach tube were not significantly different from those of rats that received radioiodide by the same route. These tidings support the view that iodate can substitute for iodide in all instances in which supplementary iodine is required for human or animal nutrition.

Whole blood, resuspended washed red cells, and various tissue extracts reduce iodate to iodide very rapidly in vitro. However, plasma is much less effective than whole blood in reducing iodate.

Iodate is rapidly reduced by glutathione at pH ‘7.4, but not by a number of other biological reducing agents. The stoichiometry of the reaction between GSH and iodate in- dicates that the reaction proceeds as follows.

6 GSH -I- IOa- --3 3 G-S-S-G + I- + 3 Hz0

Because red cells and most tissues contain a fairly high concentration of GSH, it might appear that GSH is the active agent in blood and tissues responsible for reducing iodate. However, the iodate-reducing capacity of red cells and of tissue extracts far exceeds their potential GSH content, and it is suggested that -SH groups in hemoglobin and in other proteins may also be oxidized by iodate.

Results of incubations carried out at 0” suggest that iodate reduction in blood is not enzymatic, at least for iodate levels below 1 x low3 M.

Previous reports on the metabolism of iodate have dealt pri- marily with its toxicology, or with its ability to replace iodide in animal nutrition (see “Discussion”). Very little information has been available on the biochemical aspects of iodate metabo- lism. Leblond and Sue (1) prepared iodate labeled with the

* This work was supported by Grant AM-03612 from the United States Public Health Service.

$ Career Research Awardee, United States Public Health Serv- ice.

0 Medical student summer fellow.

short lived isotope rz*I (25-min half-life), and showed that it was rapidly transformed into iodide following intravenous injection into rats. Following injection of 1250 pg of KIOS, they observed that 25% of the blood lz81 was in the form of iodate after 25 min, and almost 0% after 40 min. Anghileri recently reported (2) that ?I-iodate is largely transformed to 1311-iodide upon pro- longed incubation (6 hours) with rat blood or tissue homogenates.

At the time we began our studies, the brief report of Leblond and Sue (1) comprised the major evidence for the widespread view that iodate is rapidly reduced to iodide in the animal body, and it seemed worthwhile to obtain additional data on this conversion and to attempt to elucidate the mechanism of the iodate to iodide transformation. Since reduction of iodate in the chemical laboratory is always performed in a strongly acid medium, this suggested that iodate reduction in the body might occur enzymatically.

Some of the results reported here have appeared previously in preliminary form (3).

ME!t’HODS

Preparation of 1311-Iodate-A standard analytical procedure for the determination of stable iodide involves oxidation of iodide to iodate with elemental bromine (4). This procedure was adapted for the preparation of radioiodate as follows. One milliliter of solution containing 10 pg of iodide, 25 ,ul of 1 y0 sodium acetate-IO% acetic acid, and the desired amount of 131I- was mixed with 10 ~1 of Brz in a 3-ml conical centrifuge tube. After a few minutes, the colored supernatant was withdrawn from the excess BrZ with a capillary pipette and transferred to a small flask. Two small Carborundum boiling chips were added and the contents of the flask were carefully brought to boiling in the hood over a micro gas burner. The excess Brz was completely eliminated after 1 to 2 min of boiling, and the reaction mixture was diluted to 10 ml with 0.067 M phosphate buffer, pH 7.8. The final pH of the resulting radioiodate solution was approxi- mately 7. Carrier potassium iodate was added to provide the concentrations indicated in the various experiments.

The identity of the radioiodate was established by ascending paper chromatography (Whatman No. 3MM) in two different solvents: l-butanol-95$??o ethanol-2 N NHAOH, 5:1:2, and 95% ethanol-pyridine-water-concentrated NH,OH, 60:20:16:4 (5). The RF for iodate in the former solvent was approximately 0.080; in the latter solvent, approximately 0.15. In both solvents the major peak of 1311, determined by radioautography, coincided exactly with stable iodate carrier, visualized by spraying the paper strip with a ceric sulfate-arsenite reagent (6). The purity

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of the radioiodate was determined by butanol-ethanol-NHdOH TABLE I chromatography, since, in this solvent, there was minimal streak- Thyroid uptake of la1I after administration of radioiodate ing of the iodate peak. As shown in Fig. 1 for a tvnical radio- or radioiodide to rats iodate preparation, approximately 98’% of the IQ1 oh the chro- matogram was present as iodate, 1% as iodide, and 1 y. as an unidentified component that appeared just below the main peak of radioiodate.

Chromatographie and Counting Procedure-For routine analy- sis, iodate and iodide were separated by chromatography on Whatman No. 3MM paper in butanol-ethanol-Z .N NHIOH (5: 1:Z). In experiments with whole blood 10 11 were applied over a line 3.5 cm long, or 25 al over a line 8 cm long. In all other experiments 25 ~1 of the sample were applied over a 3.5-cm line.

After chromatography, the filter paper strips were placed in contact with x-ray film for 24 to 48 hours. The areas correspond- ing to radioiodate and to radioiodide were located by radioautog- raphy, excised, and counted in a well-type scintillation counter. Generally, radioactivity remaining at the origin of the chromato- gram was also determined, although iodate plus iodide comprised 98% or more of the total la11 on the chromatogram.

FIG. 1. Radioautograms of paper chromatograms. Left, typi- cal starting radioiodate preparation. Right, urine sample col- lected over a a-hour period following intravenous injection of 0.08 pmole of radioiodate into rat. The numbers indicate the per- centage of the total Ia11 in the corresponding section of the chroma- togram. Chromatography solvent, 1-butanol-ethanol-2 N NHdOH (5:1:2). 0, origin; SF, solvent front.

Oral administration Form of

admiiis- tered 18’1 Dose ‘tif- Uptake in thyroid

--- pm& hrs %

Iodide 0.002 4 6.05 zk 0.68~ (‘3) (‘3)

Iodate Iodate 0.002 0.002 4 4 9.1 9.1 i i 2.2 2.2 (6) (6)

Iodide Iodide 0.2 0.2 2 2 1.8 1.8 f f 0.19 0.19 (4) (4)

Iodate Iodate 0.2 0.2 2 2 2.5 2.5 i i 0.23 0.23 (4) (4)

0 Mean i S.E. Numbers in parentheses refer to number of animals.

-

_ Intraperitoneal administration

Dose It;;- U$‘t$~;

-- ptmlc hrs %

0.002 4 12.1 i 0.33 (6)

0.002 4 11.5 f 0.37 (f-3

0.8 4 1.6 i 0.25 (4)

0.8 4 1.4 f 0.12 (4)

Incubation Procedure-In those experiments in which radio- iodate was incubated with blood (heparinized to prevent clot- ting), tissue extracts, or various chemical agents, the samples were preheated in an aluminum block to 37” before the addition of radioiodate. Samples removed at intervals after addition of radioiodate were applied to filter paper strips, and the strips were then placed immediately in the chromatography jars (7). In this way little time was allowed for the reaction to continue after application of the sample to the paper strip. It required approximately 5 min for the arrival of solvent at the origin of the chromatogram, but it was assumed that the reaction was stopped immediately by the highly ammoniacal atmosphere in the chromatography jar.

Animals--Adult Sprague-Dawley rats were maintained on a modified McCollum diet (8). In previous experiments this diet was found to contain approximately 0.2 pg of iodine per g. Rabbits were maintained on Purina rabbit chow. In experi- ments in which it was desired to obtain blood samples at very early intervals after radioiodate injection, the animal was anesthetized with pentobarbital, and a carmula was placed in the jugular vein (rats), or femoral artery (rabbits). Intravenous injections were made via the tail vein in rats, and via the margi- nal ear vein in rabbits.

Miscellaneous-Reduced glutathione in blood and in red cells was measured by the nitroprusside method of Gruenert and Phillips, as modified by Beutler et al. (9). Additional details of the various procedures used are provided with the results of individual experiments. Although results of only single experi- ments are presented, all experiments were performed at least twice (except as noted).

RESULTS

Thyroid Uptake of la11 after Administration of Radtiioduts or Radioiodide-As shown in Table I, iodate iodine was just as readily available to the thyroids of rats as was iodide, following oral or intraperitoneal injection. This was true both for in- jections of 0.25 pg (0.002 pmole) or 25 pg (0.2 pmole) of iodine.

The distribution of the 1311 among the various iodinated amino acids of the thyroid was the same for both radioiodate- and radio- iodide-injected rats (Table II).

Absorption of Orally Administered Iodate and Iocla&---& would be anticipated from the results in Table I, iodate iodine

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4688 Metabolism of Iodate Vol. 241, No. 20

TABLE II Is11 distribution in thyroid S hours following oral administration

of radioiodate or radioiodide to rats The values in the table represent results obtained on the pooled

thyroids of four rats. The uptake figures are calculated per individual rat.

~__ ~- % mole % % % %

Iodide.. 2.3 0.003 4.6 47.6 34.1 8.3 Iodate... 2.9 0.003 5.0 47.6 33.0 8.7 Iodide.. 1.5 0.25 5.2 49.7 31.3 8.3 Iodate... 2.5 0.25 4.8 45.8 32.6 8.2

was just as readily absorbed from the gastrointef

-

rt

. . I- “,oS

-- % %

4.3 1.0 5.0 0.6 4.1 1.0 7.3 0.9

inal tract as was iodide. This was demonstrated by measuring residual 1311 in various portions of the gastrointestinal tract of rats that had received radioiodate or radioiodide by stomach tube. No significant differences between the two groups could be observed 2 to 3 hours after administration of the radioactive compounds. This was true both for normal rats and for rats that had been made hypothyroid by the feeding of 0.05% propyl thiouracil in the diet for 30 days. Presumably, under the conditions used here radioiodate was very rapidly converted to radioiodide in the gastrointestinal tract, since it has been reported that iodate as such is not absorbed by the isolated small intestine (10). Pre- liminary experiments of our own with everted sacs of rat small intestine also confirmed the inability of iodate to move across the gut wall.

Distribution and Excretion of l31I after Administration of Radio- iodute or Radioiodide-Only single experiments were performed in which 1311 distribution in tissues or urinary and fecal excretion of Is11 was measured. In the distribution experiment, three rats were injected intravenously with 1.25 mpmoles of either 1311- iodate or 1311-iodide, and the la11 concentration in the following tissues was measured after 2 hours: blood, liver, kidney, brain, heart, skeletal muscle, small intestine, stomach, testes, sub- maxillary, skin and hair, and thyroid. No essential differences were observed between the radioiodate- and the radioiodide- injected groups.

In the excretion experiment, rats that received intravenously 4.0 mpmoles of either radioiodate or radioiodide (four animals per group) were placed in,metabolism cages for separate collec- tion of urine and feces. I311 measurements, made after a 48-hour collection period, showed no significant differences between the two groups.

In several short term experiments, urine was chromatographed after oral or intravenous administration of radioiodate. Results of a typical experiment are shown in Fig. 1. In no case was radioiodate detected in urine after its administration to rats.

Uptake of Radioiodate or Radioiodide by Thyroid Slices Thyroid slice experiments were performed to determine whether iodate can be concentrated by thyroid tissue. When thyroid slices were incubated in Krebs-Ringer-bicarbonate medium con- taining either radioiodate or radioiodide, the initial rate of up- take of 1311 was greater in the case of radioiodide (Table III). However, after 90 min of incubation there was very little differ- ence between the samples with respect to either total uptake or l311 distribution. In the thyroid slices exposed to radioiodate, no evidence could be obtained that iodate as such was concen- trated by the tissue. The results suggest that iodate-i311 was converted to iodide-1311 before it was concentrated. Similar findings were reported by Wolff and Maurey (11).

Rate of Conversion of Iodate to Iodide in VivtiRadioiodate was very rapidly converted to radioiodide following its intravenous administration to rats or rabbits. This is illustrated in Fig. 2. Within 3 min following the administration of 0.75 pmole of radio- iodate (95 fig of iodine) to rats, or 8 hmoles of radioiodate (1000 pg of iodine) to a rabbit, radioiodate could no longer bs detected in the blood, and all the administered i311 was in the form of iodide.

Rate of Conversion of Iodate to Iodide by Blood in V&+-The rapid rate of reduction of iodate in rats and rabbits observed in Fig. 2 suggested that the transformation of iodate to iodide occurred directly in blood. When radioiodate (4 x 1O-5 M) was incubated in vitro at 37” with whole blood (Fig. 3A), all the radioiodate was converted to iodide within 3 mm. Plasma was not nearly as effective as whole blood, reducing only about 24yo of the radioiodate in 15 min.

Even when the concentration of radioiodate was raised to 8.3 x 10m4 M (105 pg of iodine per ml), complete reduction to iodide occurred within 2 min in rat whole blood (Fig. 3B). Washed,

TABLE III Distribution of Is11 in hog thyroid slices incubated with radioiodide or radioiodate

Approximately 200 mg of slices were incubated in 3 ml of Krebs-Ringer-bicarbonate buffer containing 0.66 pg of iodine either as 1311-iodate or ‘alI-iodide. Each value in the table is the average of closely agreeing duplicates.

Form of added ‘8’1 Incubation time

min

Iodide 10 30 90

Iodate 10 30 90

1

-

- I

“‘I uptake Origin IOr

%$‘s”s”,,8

18.1 37.3 71.9

% %

I-

%

0.4 0 99.6 1.4 0 98.7

8.5 0 62.5 37.5 23.5 0.3 29.9 69.8 68.7 0.5 3.3 96.2

Ial1 in medium

- I

Origin IOC

% %

2.1 0 14.1 0 58.1 0

1.3 0.1 6.2 0

54.4 0

I-

%

97.9 85.9 41.9

98.6 43.8 45.6

- I la’1 in slice digesta

d-monoiodo- tyrosine

3, S-Diiodo- tyrosine

% %

6.8 4.4 29.6 19.9

4.4 3.0 27.4 18.0

o Homogenate of slices in NaCl-Tris buffer, pH 8.5, digested with pancreatin for 16 hours.

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resuspended red cells were about equally as effective as whole blood in reducing iodate. Blood that had been heated to 65” for 10 min still retained its high capacity for reducing iodate, suggesting that an enzymatic reaction was not involved. When the concentration of radioiodate was raised to 3.3 X 10-s M (420

pg of iodine per ml), the rate of reduction by whole blood was slowed, and approximately 15yo of the 103 remained unchanged after 15 min of incubation.

Reduction of Ioclate by Various Reducing Agents at pH ?‘.S-As shown in Fig. 4, the sulfhydryl-containing compounds, cysteine, t,hioglycolate, and reduced glutathione, readily reduced iodate

100 -

a0 -

P‘Zr cant 60 -

of Total 1’3’ ..” Blood

Present 40 - aslodtdc

20 -

1

A Normal Rat 0.75,~motes Iodate

0 Normal Rabb,t 80 pmoles Iodate . Hypothyrord Rat 0 75,~ moles Iodata

0 0 2 4 6 a 10 12 14

Mtnutos after Injection of Radiotodate

FIG. 2. Time curve showing rapid rate of conversion of intra- venously injected radioiodate to radioiodide in rat and rabbit.

100

a0

60 Per cent

Iodtdo-I’“’

40

20

0

A

0 Rat Wholo Blood a Rabb,t Whole Blood . Rat Plasma . Phosphate Buffer, pH 73

60 Per Cent Iodtdo-I”’

40

1

n Rat Whole Blood + 8 3x&M Iodate 0 Rat Whole Blood + 33xlO”M Iodate 0 Rat Rod Coils + 8 3x10-4M Iodate V Rat Wholo Btood

Provwsly Heated to Go for 10 Mm + 4.2xlO“M Iodete

I3 I I 1 I I I I

0 2 4 6 a 10 12 14 Mtnutes of Incubation at 37%

FIG. 3. Time curves showing conversion of radioiodate to radio- iodide under various conditions of incubation in vitro. In A, the concentration of iodate was 4 X 10m5 M. In B, the concentra- tion of iodate is indicated.

60 Per cent

Iodide-I”’

40

. Thtqlycolate 5 x10-+ M Bx@M 0 GSH 5 x10-6 r-l 9x10-5r-l A Moth<marola 5xwt-l 9XlPt-7 V Asccrb,c Asrd 5 x10-+ M 9x 10-s M 0 SLJlf,te 5x10-+ M 9x10-5 M I Th<ouroa 5x10-3 r-l 9x1O-5 M

-I 0 2 4 6 a 10 12 14 16

Mmutos of Incubatton at 37OC

FIG. 4. Time curves showing conversion of radioiodate to radio- iodide upon incubation with various reducing agents at pH 7.3. All incubations were performed in 0.1 M phosphate buffer.

TABLE IV Reduction of iodate by various preparations

All incubations were made at 37” without shaking and with air as the gas phase. Red cells were washed twice with a volume of cold 0.15 M NaCl approximately equal to the original volume of plasma, and the washed cells were suspended in 0.15 M NaCl to provide a 50’% suspension. Phosphate buffer (0.1 M, pH 7.3 to 7.4) was used for preparing the tissue homogenates and as the incubation medium for the experiments with ergothioneine, DPNH, and TPNH. Tissues were perfused with cold 0.15 M

NaCl before homogenization, and supernatants from 30% ho- mogenates that had been centrifuged in the cold at 1500 rpm for 10 min were used for incubation.

Human whole blood. Human wrtshed red blood cells Human plasma. Rat liver extract, pH 7.1. Rat kidney extract, pH 7.1. Rat brain extract, pH 7.4. Ergothioneine, 1O-3 M, pH 7.4. DPNH, 1OW M, pH 7.4.. TPNH, 1O-3 M, pH 7.4.

Iodate concentration

- M

3.9 x 10-4 3.9 x 10-4 3.9 x 10-4 7.5 x 10-4 7.5 x 10-4 7.6 X lo-+

1 x 10-4 1 x 10-d 1 X lo-’

Reduction -

1 min _

%

98.4

89.4

25.1 96.9 83.9 99.8

-

15 min 6

%

99.8

94.4

25.6 99.9 98.4 99.9

0.4 0.0 0.4

Q min

%

0.1 0.0 0.5

at pH 7.3. However, the reducing compounds 1-methyl-2- mercaptoimidazole, ascorbic acid, sulfite, and thiourea did not react with iodate at this pH.

The effect of several additional compounds and of various tissue extracts is shown in Table IV. The biological reducing agents, DPNH and TPNH, did not reduce iodate at pH 7.4. On the other hand, extracts prepared from 0.15% NaCl-perfused rat liver, kidney, or brain readily reduced iodate at pH 7.1 to 7.4, as did human whole blood and resuspended, washed red cells.

Effect of pH on Reaction between Iodate and I-Methyl-d-Mercap- toimidaxole-The reaction between 1-methyl-2-mercaptoimida- zole and iodate was pH-dependent, as shown in Fig. 5. Above pH 5 no reaction occurred, but at pH 2.3 iodate was almost completely reduced to iodide in 30 min at room temperature.

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In other experiments it was shown that ergothioneine, which was ineffective at pH 7.3, readily reduced iodate at pH 3.7.

It is not apparent why -SH compounds such as GSH, cysteine, and thioglycolate readily reduce iodate at pH 7, whereas ergo- thioneine and 1-methyl-2-mercaptoimidazole do not. This is probably another instance of the lack of true mercaptan be- havior on the part of the thiolimidazoles (12). The oxidation- reduction potential for the couple, I- + 3 Hz0 -+ 103 + 6 H+ + 6 e-, at pH 7 is $0.665 volt (as calculated from the standard oxidation-reduction potential). Although this is considerably lower than the potential in strong acid solution, it is, neverthe- less, a relatively high value compared with the oxidation-reduc- tion potential for ergothioneine, which is reported to be -0.06 volt at pH 7 ((13) for RSH + + R-S-S-R + H+ + e-). The corresponding potential for I-methyl-2-mercaptoimidazole could not be found in the literature, but it would not be expected to differ greatly from that for ergothioneine, since both are thiolimidazole derivatives. Both ergothioneine and l-methyl-Z mercaptoimidazole, therefore, are probably thermodynamically capable of reducing iodate at pH 7, but apparently the reactions lack a suitable mechanism. The fact that both compounds readily reduce iodate in acid solution suggests that the undis- sociated form of the sulfhydryl is the reactive one. However,

100

80

60 Per cant

Iodide -113’

40

2c

09X10-3M MMI+l.lxlO-‘MIO;

A i.l~iO-~ M IO; Alom

(30 Minutes Incubation at 23-C)

2 3 4 5 6 7 8

PH

FIG. 5. Effect of pH on reduction of radioiodate by 1-methyl-2- mercaptoimidazole. Acetate buffer, 0.1 N, was used for pH values in the range of 3.45 to 5.2, and 0.067 M phosphate buffer for pH values above 6. The most acid sample was incubated in approxi- mately 0.01 N HCl.

60 tica Line for o uSH +IO; + 3 G-S-S-G +?

0 Starting Conc.of GSH - 10~‘Pl

Obsewod Mold Rati 0

I I I I I I I I

2 3 4 5 6 7 6 9 10

Mold Ratio GSH/IO;

FIG. 6. Stoichiometry of reaction between iodate and GSH. See the text for details.

TABLE V Effect of iodate on GSH level of red cells

One milliliter of 50yo red cell suspension (see the legend to Table IV) was incubated with 0.5 pmole of 103 for 5 to 10 min at 37”.

GSH founda

Source of cells

Experiment 1 Rat............................

Experiment 2 0.88 0.00

Rat............................ 0.98 0.16 Human......................... 1.04 0.13 Human......................... 0.98 0.16

@ The nitroprusside procedure used for determining GSH may be affected by other nonprotein sulfhydryl compounds, although it was reported by Beutler et al. (9) that estimation of red cell GSH levels by the nitroprusside method gave results quite com- parable to those obtained with a more specific method.

this alone would not explain the results in Fig. 5, if, as seems likely, the pK for the dissociation of the SH group in l-methyl- 2-mercaptoimidazole is assumed to be close to that for GSH (pk = 8.7 (13)). Further experiments are necessary to explain the differences in the reactivity of the various -SH compounds toward iodate.

Reaction between Iodate and GSH-Since GSH readily reduced iodate at normal blood pH, and since this peptide is widely distributed in animal tissues, including red blood cells, it seemed logical to postulate that GSH may be the active agent that re- duces iodate injected into animals. The stoichiometry of the reaction between GSH and iodate at pH 7.3 was studied, as shown in Fig. 6. The starting concentration of GSH was fixed at 1 x 10-S M, and radioiodate was added to provide molal ratios of GSH to iodate varying from 2 to 10. Iodate was com- pletely reduced when the molal ratio of glutathione to iodate exceeded 6. When the ratio fell below 6, the percentage of initial iodate that remained was very close to that calculated for the following over-all reaction.

6 GSH + IOs- + 3 G-S-S-G + I- + 3 Hz0

The data in Fig. 6, therefore, may be taken as evidence that iodate oxidizes reduced glutathione to the disulfide form at pH 7.3, and not to the more highly oxidized sulfinic or sulfonic acid forms. However, it remains possible that, in the red cell, iodate oxidized GSH beyond the disulfide stage, so that the stoichiome- try in Fig. 6 may not be strictly applicable.

Effect of Iodate on GSH Level of Red Cells--Incubation of red cell suspensions with iodate quickly reduced the GSH level, as shown in Table V. These results indicate that the GSH in red cells is readily available for reaction with iodate. It should be recognized, however, that the concentration of iodate used here (5 X 10m4 M) was relatively high. It is unlikely that iodate administered to animals only in amounts necessary to provide the daily requirement of iodine would have any detectable effect on the reduced glutathione level of red blood cells.

Capacity of Red Cell Suspensions for Reducing loo&e---Al- though it first appeared that GSH might be the active agent in red cells responsible for reduction of injected iodate, it was soon

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discovered that the reaction was more complicated. The capac- ity of red cells for reducing iodate was observed to exceed many fold that which could be attributed to the GSH present. This is shown by the results in Table VI. When 4 pmoles of iodate (50 pg of iodine) were incubated for 25 min with 1 ml of rat whole blood, or resuspended washed red cells, more than 85y0 of the iodate (3.5 pmoles) was reduced to iodide. Assuming that the GSH concentration was 0.31 mg per ml (I pmole per ml, a maximal value obtained in other experiments), this could have accounted for the reduction of only 0.17 pmole of iodate, based on the stoichiometry shown in Fig. 6.

The possibility was considered that GSH might be continuously produced by the glutathione reductase-TPNH system known to be present in red blood cells (14), and that this might account for the high capacity of red cells to reduce iodate. However, this seemed unlikely since washed red cells, incubated without added glucose, showed nearly the same capacity for reducing iodate as did an equivalent amount of whole blood. Neverthe- less, it was considered of interest to test blood from a patient with a red cell deficiency of glucose 6-phosphate dehydrogenase. Such a sample was kindly provided by Dr. Eugene Frenkel, and it showed a completely normal capacity for reducing iodate when compared with blood from normal human subjects. It appears

TABLE VI

Capacity of whole blood and washed red cells for reducing added iodate

One milliliter of whole blood or of washed, resuspended red cells (see the legend to Table IV) was incubated at 37” with radio- iodate.

Iodate reduced in whole blood Iodate reduced in washed red cells Iodate added

lmin / lmin 1 25min lmin j 7min j 25min

pV?WleS /moles

0.5 0.49 0.50 0.50 0.48 0.50 0.50 1.0 0.95 1.0 1.0 0.91 0.98 1.0 2.0 1.75 1.86 2.00 1.43 1.69 1.97 3.0 2.25 2.55 2.86 1.86 2.20 2.82 4.0 2.58 2.88 3.57 2.03 2.56 3.50

TABLE VII

E$ect of NEM on reduction of iodate by GSH and by

Concentration of NEM

M

0.0 0.005 0.01 0.02

red cell suspension

I Red cell@ Reduction of

iodate by GSHa Redu&& of GSHC level

% % w/ml

99.6 98.9 0.24 3.3 93.8 0.04 2.2 67.0 1.9 38.0

o GSH, 3 X lo+ M, was incubated for 5 min with NEM at pH 7. Then 5 X 1OP M radioiodate was added, and incubation was continued for an additional 10 min.

b One milliliter of red cell suspension was incubated with NEM for 10 min. Then 5 X lo+ M radioiodate was added, and incuba- tion was continued for an additional 10 min.

c See the footnote to Table V.

‘L~x!O-~M lodote

Per cent

13’1- Iodide

Minufes of Incubation

FIG. 7. Time curve showing rate of reduction of iodate by rat blood at 0” and at 37”.

likely, therefore, that red cells contain some component in addi- tion to GSH that is capable of reducing added iodate. Further evidence for this was obtained in experiments with NEM.’ Addition of NEM to red cells has been reported to reduce the GSH level of red cells to very low values (15). This was also observed in the present study. However, as shown in Table VII, a concentration of NEM which greatly reduced the GSH level had only little effect on the reduction of added iodate. This lends further support to the view that a component other than GSH in red cells is capable of reducing iodate.

Extracts of various tissues from which red cells had been re- moved by prior perfusion were very active in reducing iodate (Table IV). In the case of these tissues, also, the capacity of the extracts for reducing iodate greatly exceeded their estimated GSH content (16). Apparently, compounds capable of reduc- ing iodate at pH 7 are widespread in tissues other than red cells.

To obtain further evidence on the question of enzyme in- volvement in the reduction of iodate by blood, incubations were performed at 0”. As shown in Fig. 7A, iodate reduction was practically complete after 1 min of incubation at O”, when the initial concentration of iodate was 4.5 X 10m4 M. Although this result alone does not necessarily exclude an enzymatic mechanism at 37”, when examined in relation to the results shown in Fig. 7B, it does provide evidence against an enzyme reaction. In Fig. 7B, the initial concentration of iodate was

raised to 5 X 10W3 M, and the rate curves at 0” and 37” indicate that two components, with markedly differing temperature coeficients, were involved in the reaction. The fast component was independent of temperature, and it seems unlikely, there- fore, that it represents an enzyme reaction. This component accounted for the reduction of 1.75 pmoles of iodate per ml of blood, an amount greatly in excess of the entire iodate reduction in Fig. 7A (0.45 I.tmole of iodate per ml). It seems reasonable to suggest, therefore, that even at 37” the reduction of 4.5 x lo-’ M iodate in blood proceeds nonenzymatically. The slow component in Fig. 7B has the appearance of an enzyme reaction,

1 The abbreviations used are: NEM, N-ethylmaleimide; Ta, 3’,3,5-triiodothyronine; Tp, thyroxine.

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but it could also represent nonenzymatic oxidation of -SH Although the present investigation does not provide a defini- groups in protein (see below). It was observed in a separate tive answer to the question of the site and mechanism of reduc- experiment that the reaction between GSH (6.4 x 10-3 M) tion of iodate, the data do suggest certain possibilities. It has and 103 (1.0 X 10e3 M), at pH 7.3, was complete within 1 min been shown here that iodate, even in relatively high concen- at 0”. However, the rapid component in Fig. 7B must represent tration, is very rapidly reduced to iodide in vitro upon incuba- oxidation of some component in addition to GSH, since there is tion with blood or with tissue extracts. Although the active insufficient GSH in 1 ml of blood to account for the reduction of reducing agent in blood has not been determined, it has been 1.75 pmoles of iodate. shown that GSH, which occurs abundantly in red blood cells

DISCUSSION and in other tissues, is capable of reducing iodate at pH 7.4. However, since the capacity of red cells to reduce iodate far ex-

The use of iodate instead of iodide for the iodization of salt ceeds their potential GSH content, and since treatment with has been recommended for certain conditions in which iodide NEM (which greatly reduces the GSH level of red cells) has loss from the salt may occur, as, for example, in open air salt only a minor effect on iodate reduction, there must be some blocks used in the feeding of livestock (17), or in moist, crude component of red cells, in addition to GSH, capable of reducing salt exposed to heat., sunlight, or acidic conditions (see Kelly iodate. In this connection the studies of Hird and Yates (26) (18) for review). are of particular interest, since these workers showed that iodate

The availability of iodate-1311 to the thyroid was studied in reacts readily with -SH groups in certain proteins, and that human subjects by Murray and Pochin (19), and in sheep by this reaction is probably the basis for the use of iodate as a dough Wright and Andrews (20). Although in both studies it was conditioner in the baking industry. It seems possible, therefore, observed that iodate-1311 is readily available to the thyroid, the that in red cells the cysteine residues in hemoglobin (6 per mole results suggested that, in some instances, iodate iodine was not in human hemoglobin A (27)) contribute to the reduction of quite as readily available to the thyroid as was iodide. However, iodate. Experiments with purified bovine hemoglobin indicated the number of subjects was quite small in both investigations, that this protein is, indeed, capable of reducing iodate, but the and the significance of any differences between iodide and iodate iodate-reducing capacity was not nearly great enough to account could not be established. In the present study with rats, there for the high activity of red cells. Purified hemoglobin was, was no indication that iodate-13rI was any less readily available nevertheless, much more effective than bovine serum albumin to the thyroid than was iodide-‘311. Moreover, our results in reducing iodate. Proteins containing -SH groups might also -indicate that iodate is so rapidly converted to iodide following be involved, together with GSH, in the rapid rate of reduction its injection that administration of iodate is practically tanta- of iodate by liver, kidney, and brain extracts (Table IV). mount to iodide administration. The recent note of Anghileri (2) indicates that rat tissues such

Scrimshaw et al. (21) administered iodate, iodide, or placebo as intestine, lung, stomach, and muscle are also capable of re- to school children in endemic goiter areas of Central America, ducing appreciable amounts of iodate in vitro, although only and they concluded that iodate given by mouth was as effective long incubation intervals (6 and 24 hours) were employed in his as iodide in the treatment or prophylaxis of endemic goiter. studies. There is an unexplained discrepancy between Anghi- They suggested that iodate could be used for iodizing salt in leri’s findings and ours with regard to rat blood. We observed areas of the world in which the purification and drying of salt 80% reduction of 5 x 10-S M iodate incubated with rat blood is economically or culturally impractical. for 60 min (Fig. 7B), whereas he observed only 37% reduction

Toxicity studies have shown that there are no detectable with 2 X 10-a M iodate after 6 hours of incubation. He also adverse effects of iodate, even when administered in relatively observed a considerable decrease in the iodate-reducing capacity large doses. Murray (22) reported results of experiments in of tissue homogenates and of blood after heating at 90” for 30 which rabbits were given 1 mg of NaI03 per kg by mouth bi- min, and suggested that a thermolabile system is responsible weekly for 4 or 8 months. Offspring from these rabbits, born for iodate reduction. A decreased iodate-reducing capacity of and suckled during iodate administration, were also given 1 mg tissue homogenates and blood, heated for as long as 30 min, of NaI03 per kg biweekly for 53, 7, or 14 months. No overt could be due simply to air oxidation of thiol groups, and the effects of iodate feeding were observed, and no abnormal histo- results reported by Anghileri, therefore, do not, in themselves, logical changes were detected in liver, kidney, thyroid, or retina provide evidence for an enzyme reaction. at autopsy. Murray concluded that there were no contraindica- A recent note by London, Vought, and Brown (28) indicates tions to the use of 103 for the iodization of salt intended for human that the use of iodate in the conditioning of dough is fairly wide- use. However, extremely large doses of K101 or NaI03 ( >lOO spread, at least in the area of Washington, D. C. They re- mg per kg) have been shown to be toxic in mice (23, 24). ported that ingestion of bread prepared with iodate-conditioned

Brumbaugh, Mehring, and Titus (25) compared the effects dough could easily lead to iodine intakes as high as 1 mg per of iodide and iodate fed to young chickens for 10 weeks at levels day, a level which could affect the interpretation of thyroid ranging up to 15 mg of iodine per kg of diet. They observed no 13rI uptakes in human subjects. Presumably the iodine in such differences in growth, efficiency of food utilization, or mortality bread is already in the form of iodide, but it would be predicted rate. They also investigated the possibility of increased met- from the present study that any unreduced iodate remaining in hemoglobin formation in those animals receiving iodate, since the bread would have the same effect as an equivalent amount of it is known that chlorates may induce methemoglobinemia. iodide. However, even at the highest dose level of iodate, no increase in the blood methemoglobin level was observed, and the blood REFERENCES

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Alvin Taurog, Earl M. Howells and Harold I. Nachimsonin Vitro and in VivoConversion of Iodate to Iodide

1966, 241:4686-4693.J. Biol. Chem. 

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