THE NATURE OF PLASMA IODINE AS REVEALED

BY FILTER PAPER PARTITION

CHROMATOGRAPHY*

BY ALVIN TAUROG, I. L. CHAIKOFF, AND W. TONG

(From the Division of Physiology, University of California Medical School, Berkeley)

(Received for publication, December 5, 1949)

Despite the fact that crystalline thyroxine completely replaces the

function of the thyroid gland in thyroidectomized animals, it is not uni-
versally accepted that thyroxine itself is the actual form of the circulating
thyroid hormone. The reasons for this have been pointed out in a pre-

Downloaded from http://www.jbc.org/ by guest on March 7, 2019

vious report from this laboratory (1). In that report, experimental evi-
dence was also presented favoring the view that the greatest part of the
so called “protein-bound iodine” of plasma is actually thyroxine loosely
attached to protein.
The sensitivity of the technique of filter paper partition chromatography,
as described in the preceding paper, suggested a new approach to a study
on the nature of plasma iodine. The application of this technique to the
fractionation of plasma iodine is described here.

EXPERIMENTAL

Rats which had been raised on a fairly low iodine diet (0.3 y per gm.)
were injected with 150 /IC. of carrier-free P. 24 hours later, blood was
removed by heart puncture and pooled, and the plasma separated. It
has been shown previously in this laboratory that plasma 1131at that in-
terval is largely in the form of protein-bound iodine (2). When a portion
of the pooled plasma was treated with 10 per cent trichloroacetic acid,
85 to 90 per cent of the total radioactivity was precipitated with the pro-
teins. It may therefore be concluded that only about 10 to 15 per cent
of the radioiodine in this plasma was in the form of inorganic iodide.
Butyl alcohol extracts were prepared from 4 cc. portions of the radio-
active plasma. Each 4 cc. portion was extracted with 10 cc. of butyl
alcohol in 40 cc., narrow necked, round bottom centrifuge tubes. The
layers were separated by centrifugation, and the clear butanol layer was
drawn off. The residue was extracted a second time with 10 cc. of butanol,
and the clear butanol layer obtained by centrifugation was combined with
the first extract. The final butanol extract contained about 70 per cent
of the total radioactive iodine.
*This work was supported by grants from the United States Public Health
Service.
99
100 NATURE OF PLASMA IODINE

The butanol solutions were delivered to filter paper strips exactly as

described in the previous paper (3). However, since the volumes of
butanol were larger (usually 22 cc.) and the amounts of radioactivity
contained therein were considerably less than in the case of the thyroid
extracts, it was necessary to transfer more of the solution to the filter
paper. A total of 500 ~1. was delivered in 25 ~1. portions. The chromato-
grams were prepared with the collidine-water-NH3 solvent as previously
described. Chromatograms were also prepared from the same butanol ex-
tract to which had been added an appropriate amount of crystalline
thyroxine in 0.1 cc. of weakly alkaline solution. This provided 30 to 40 y
of thyroxine in the butanol extract delivered to the paper. These chro-
matograms were sprayed with the diazotized sulfanilic acid reagent to

Downloaded from http://www.jbc.org/ by guest on March 7, 2019

locate the added thyroxine.
Radioautographs were prepared by exposing the dried chromatograms
to no screen x-ray film for 5 days.

Results
The radioautographs of the chromatograms are shown in Figs. 1 and 2.
Fig. 1 is that of the untreated butanol extract, Fig. 2 that of the same
butanol extract to which thyroxine had been added. The intense band
seen in Fig. d (Rp = 0.47’) coincided exactly, both in shape and in position,
with the added thyroxine as determined by the color test. The fact that this
band was somewhat different in shape from that in Fig. 1 may be attributed
to the tremendous difference in the quantity of thyroxine present in the
two cases. In the case of the butanol extract containing added thyroxine,
the quantity of P2’ delivered to the paper was approximately 30 y. In
the case of the untreated butanol, the amount of iodine delivered to the
paper was only about 0.004 y, an amount below the limits of sensitivity
of the micromethods now available for measurement of iodine. This is
an excellent demonstration of the sensitivity of the chromatographic
technique.
In one experiment the filter paper chromatograms were divided into
sections as shown in Fig. 1. The radioiodine in each section was then de-
termined by digesting the filter paper with chromic acid, distilling the
iodine, and counting it with a sensitive mica window counting tube. 2 y
of iodide carrier were added before the digestion to improve the recovery
of radioiodine in the distillate.
The recoveries of P31 in the various sections of the chromatogram are
shown in Table I for three different conditions: (1) the untreated butanol
extract of plasma, (2) the butanol containing added thyroxine, and (3)
the butanol extract containing added diiodotyrosine. In all cases, the
largest fraction of the added radioiodine was recovered in the thyroxine
 A. TAUROG, I. L. CHAIKOFF, AND W. TONG 101

section. When thyroxine carrier was added, 75 per cent of the radioiodine
appeared in the thyroxine section, but in the untreated butanol extract,
or when diiodotyrosine carrier was added, only 55 per cent was recovered
in this section. At the same time, only 16 per cent of the radioiodine was
found in the inorganic section when thyroxine carrier was added, whereas
24 per cent appeared in this section in the case of the untreated butanol

Downloaded from http://www.jbc.org/ by guest on March 7, 2019

-- -
FIG. 1. Radioautogram of ascending filter paper chromatogram prepared from a
butanol extract of the plasma of rats injected for 24 hours with P. Exposure time
5 days.

extract. The latter figure is considerably higher than that found in the
trichloroacetic acid-soluble fraction of this plasma (13 per cent).
These findings may be explajned on the assumption that considerable
breakdown of thyroxine (or whatever the major organic iodine component
of the plasma is) occurs during the chromatography, and that this break-
down is proportionately less when larger amounts of thyroxine are present.
102 NATURE OF PLASMA IODINE

The possibility of exchange between radioactive inorganic iodide and

added thyroxine must also be considered. That this may not be appre-

Downloaded from http://www.jbc.org/ by guest on March 7, 2019

FIG. 2. Left, ascending filter paper chromatogram of same butanol extract as

that used in Fig. 1, but containing added thyroxine. The position taken by the
added thyroxine was determined by spraying with diazotized sulfanilic acid. Right,
the radioautogram of this filter paper. Note the correspondence in position and
shape between the intense spot in the radioautogram and the line drawing of the
thyroxine in the chromatogram.

ciable, however, is suggested by the absence of any exchange between

inorganic iodide and added diiodotyrosine (Table I).
In several experiments, the butSan extracts were reextracted with 4 ~\j
NaOH-5 per cent Na2C03 before being applied to the filter paper. This
reagent removes inorganic iodide and diiodotyrosine, but not thyroxine,
 A. TAUROG, I. L. CHAIKOFF, AND W. TONG 103

from a butanol solution. It was expected, in this case, that the autograph
would show only a single band, that corresponding to thyroxine. How-
ever, the autograph prepared from the chromatogram of this reextracted
butanol showed not one, but three, bands. In addition to that of thyrox-
ine, the bands corresponding to inorganic iodine and diiodotyrosine were
very prominent. Apparently a good deal of breakdown of organic iodine
occurs when the highly alkaline butanol extract is applied to the paper,
or during its chromatography with collidine-NHS.

TABLE I
Filter Paper Partition Chromatography of Butanol Extract of Plasma Obtained from
P-Injected Rats

Downloaded from http://www.jbc.org/ by guest on March 7, 2019

Recovery of added radioactive iodine

Untreated 28.4 -y 10.1 y diio-

thyroxm dotyrosine
section(of~~il~togram Component extract iodine added iodine added
represented
Counts zit Counts gt Counts E;t
P-
set of per
sec. of P- of
tots1 total sec. total
-__ - __ __- __
1 Origin 2.4 1.5 2.0 1.2 2.4 1.4
2 Diiodotyrosine 6.4 3.9 4.4 2.6 6.8 4.0
3 4.0 2.4 5.2 3.1 5.2 3.0
4 Thyroxine 90.0 55 125.6 75 91.6 54
5 Inorganic 39.2 24 27.2 16 48.0 28
6 21.2 13 9.6 5.7 0 0
----___--
Total, Sections l-6.. 163 174 154
‘I added to paper. . . 164 167 171
_.--. ___----

Comment
A previous report (1) points out that the very minuteness of the con-
centration of iodine in plasma made its chemical identification difficult.
By employing the radioactive isotope of iodine, 1131,together with a re-
fined method for determining small quantities of iodine, we were, however,
able to provide evidence that circulating hormonal iodine is actually
thyroxine. In the present investigation, the nature of plasma iodine was
further studied by means of filter paper partition chromatography. The
sensitivity of this new procedure permitted us to identify the iodine equiv-
alent of 0.1 cc. of plasma, or approximately 0.004 y of iodine.
A butanol extract of the plasma of rats injected with 1131shows only
two major Irsl- containing components when examined by filter paper
partition chromatography with collidine-NH3. One of these corresponds
exactly to the position taken up by added thyroxine, and the other, to the
104 NATURE OF PLASMA IODINE

position taken up by added inorganic Ps1. In some experiments, a faint

band corresponding to diiodotyrosine also appeared, as did a faint darken-
ing at the solvent front.
These results support the view, set forth in a previous communication
from this laboratory (l), that the circulating thyroid hormone consists
of thyroxine loosely attached to plasma protein. However, it is not
certain that the procedure used here could be used to distinguish thyroxine
from one of its peptides. The possibility, therefore, that thyroxine cir-
culates in the organism as a small peptide has not been ruled out. A
further study, with synthetic small peptides of thyroxine, should help
resolve this problem.

Downloaded from http://www.jbc.org/ by guest on March 7, 2019

SUMMARY

1. Plasma obtained from rats that had been injected 24 hours earlier
with 1’31 was extracted with butanol, and the nature of the organic P31-
containing components of the extract was examined by filter paper chro-
matography.
2. The position taken by the major radioactive component, as de-
termined by radioautographs of the filter paper, corresponded exactly
to that of thyroxine carrier as determined by color test. The organic
iodine of plasma, therefore, appears to be identical with thyroxine.
Addendum-After this manuscript had been submitted there came to our atten-
tion a report by Laidlaw (4), who also employed filter paper partition chromatog-
raphy to study the nature of the circulating thyroid hormone. His conclusions are
essentially in agreement with those presented here.

BIBLIOGRAPHY

1. Taurog, A., and Chaikoff, I. L., J. Biol. Chem., 176, 639 (1948).
2. Chaikoff, I. L., Taurog, A., and Reinhardt, W. O., Endocrinology, 40,47 (1947).
3. Taurog, A., Tong, W., and Chaikoff, I. L., J. Biol. Chem., 164, 83 (1950).
4. Laidlaw, J. C., Nature, 164, 927 (1949).
THE NATURE OF PLASMA IODINE AS
REVEALED BY FILTER PAPER
PARTITION CHROMATOGRAPHY
Alvin Taurog, I. L. Chaikoff and W. Tong
J. Biol. Chem. 1950, 184:99-104.

Access the most updated version of this article at

http://www.jbc.org/content/184/1/99.citation

Downloaded from http://www.jbc.org/ by guest on March 7, 2019

Alerts:
• When this article is cited
• When a correction for this article is posted

Click here to choose from all of JBC's e-mail

alerts

This article cites 0 references, 0 of which can be

accessed free at
http://www.jbc.org/content/184/1/99.citation.full.ht
ml#ref-list-1