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The kinetics of diiodotyrosine metabolism in normal human subjects
ARCHIVES
OF
BJOCHEMISTRY
The Kinetics
AND
BIOPHYSICS
77,
403-416 (1958)
of Diiodotyrosine Metabolism Human Subjects
in Normal
W. R. Ruegamer and R. B. Chodos From
the Radioisotope Service, Veterans Administration Hospital and Departments of Biochemistry and Medicine, State University of New York College of Medicine, Syracuse, New York
Received February
the
10, 1958
Mono- and diiodotyrosine (MIT and DIT) are found in peptide linkage in the thyroid gland and are presumed to be precursors of thyroid hormone (1). Although the free amino acid forms are not detectable in the circulating plasma under normal circumstances, Roche et al. (2) have suggested that these substances might be released from thyroglobulin during proteolysis. Probably a large percentage of the free acids so released are catabolized in part before leaving the gland (2, 3), and any MIT or DIT which escapesinto the circulation must be destroyed so rapidly that the circulating concentration never becomes detectable. Several studies (3-9) have been made of the breakdown products resulting from the feeding and injection of exogenous DIT into experimental animals and from the in vitro incubation of DIT with tissue slices and homogenates. Foster et al. (8) and later Hartmann (9) fed large doses of DIT to animals and showed that at least a portion of the ingested DIT is converted to the lactic acid derivative (3,5-diiodo-4hydroxyphenyllactic acid). Albert et al. (4, 5) and Tong et al. (3) also studied DIT catabolism in human subjects and rats, and concluded that deiodination is unquestionably one of the first and most important steps in DIT degradation, although alteration of the side chain may produce detectable amounts of the lactic acid and pyruvic acid analogs in plasma and urine. Roche et al. (2) have emphasized the importance of the thyroid in catabolizing endogenousDIT, but Tong et al. (3) found that thyroidectomy in rats had little if any effect on the ability of the rat to deiodinate exogenous DIT. They subsequently concluded that 403
404
RUEGAMER
AND CHODOS
the thyroid is much lesseffective than the liver and kidney in catabolizing DIT. The in vitro experiments of Tong et al. (3) and those from this laboratory (10, 11) also support this contention since the greatest concentration of DIT deiodinase activity was observed in liver and kidney tissue. We were able to show further that DIT is deiodinated just as rapidly and completely in the nephrectomized dog as in the intact animal, and it might be postulated that the liver is the most important deiodination site for DIT. To test this hypothesis, we have proposed to compare the deiodination of DIT in human subjects suffering from liver disease with that observed in normal persons. Since such a study must necessarily involve comparisons of kinetic data, we have first of all developed a working model for DIT deiodination in the normal subject. The evidence for and the details of this model constitute the subject matter of this report. METHODS One-hundred-microcurie doses of Ir*r-labeled L-DIT* were administered intravenously to hospitalized patients under treatment for minor illnesses. There was no clinical or laboratory evidence for thyroid, liver, or kidney disease in any of these subjects. Heparinized blood samples and 3-min. saliva specimens (obtained under the stimulation of gum chewing) were collected from these individuals 15 min. after the injection of the PVabeled DIT, and then at frequent intervals thereafter. Complete individual urine specimens were obtained during the first 24-hr. period, and 24-hr. pooled specimens were collected during the remaining days of the experiment. Aliquots from all collections were counted on the same day in a Nancy Wood scintillation well counter to an accuracy of f2%. Protein-free plasma filtrates and their corresponding protein precipitates, prepared by precipitation with 20% trichloroacetic acid, were also counted in the well counter. Determinations were not continued beyond 72 hr. because the counting rates of collected samples became too low to be determined accurately. In order to determine the relative percentages of I ‘*r-labeled substances present in the protein-free plasma filtrate, saliva, and urine samples, aliquots of these materials were subjected to paper chromatographic analysis. Approximately ten samples of 16-560~1. were applied as a single spot and dried on 23 in. X 23 in. single sheets of Whatman #2 filter paper. The volumes applied depended entirely upon the radioactivities of the samples. The sheet was rolled into a cylinder and placed in a chromatography jar for ascending development in either 60% phenol-water, 33:2:22 butanol-acetic acid-water, or 106:35 collidine-water solvents. After approximately 16 hr. development, the sheets were dried and the individual chro1 The L-DIT was randomly labeled in the 3,5 positions, with a single label on any given molecule. This material was supplied by Abbott Laboratories with an average specific activity of 340 microcuries/mg.
DIIODOTYROSINE
METABOLISM
405
matograms were sectioned into lo-mm. horizontal strips which were then assayed for radioactivity in a scintillation well counter. The iodinated substances were identified from their RI values, and the relative amour1t.s present, could be determined from the counting rates observed for each spot. Identification was made possible by comparison of the R, values for these spots with those observed for standard solutions of P-labeled DIT and iodide which were chromatographed on the same sheet of paper in corresponding nonradioactive saliva, urine, and plasma filtrate carriers. In addition to the above determinations, the disappearance of P activity from the thigh, liver, and thyroid was followed by monitoring these areas with a scintillation counter recessed 5 cm. within a x-in. lead shield. A lead filter ss in. in thickness was used to eliminate secondary radiation. Thigh and liver measurements were made with the probe positioned at a fixed distance of 5 cm. from the body surface. Thyroid-uptake measurements on the other hand were made by two methods. In the first method, measurements were performed at a fixed distance of 30 cm. from the neck surface, with and without a l-in. lead brick interposed between the detector and the thyroid area. The counting rate obtained with tho brick in place was then used to correct the thyroid counting rate for body background. The second measurement was made according to a method previously described (la), in which the thigh disappearance curve was used to correct, fol body background. Both procedures gave essentially the same results.
~ZESULTS
Plasma-disappearance curves of total 1131activity were obtained by plotting plasma concentrations of radioactivity against time (Fig. 1). Theoretical zero-time values were obtained by dividing the total injected counts per minute by the plasma volume. The latter was calculated for each individual from hematocrit values and total blood volume data obtained by a previously described P32tagged red-cell method (13). Since DIT and iodide do not bind appreciably to plasma proteins, protein-free plasma-disappearance curves were essentially superimposable on the whole plasma curves. On the other hand, saliva 1131activity disappearance curves showed a somewhat different form. The concentration of 1131activity reached a maximum within a few hours and then disappeared at an exponential rate comparable to that observed for plasma activity (Fig. 1). Thigh- and liver-disappearance curves obtained from monitoring data were of the samegeneral shapeas the plasma curves although a simple exponential rate of disappearance was never observed (Fig. 2). Cumulative urinary excretion curves of Pa1activity were plotted as shown in Fig. 3, and excretion rate curves (Fig. 4) were prepared from these by plotting the amounts of radioactivity excreted per 4-hr. interval as chords against time. Smooth curves were drawn through these chords,
406
RUEGAMER
AND
CHODOS
PLASMA
0.11
I IO
20
40
50
60
H O”u”R S
Fro. 1. Saliva and plasma disappearance of II31 activity following of 100 microcuries of P-labeled diiodotyrosine. I
ZOOO(
0 100: c” so= 8
the injection
THIGH -*
60. 40 -
20 /
I
OO
FIQ.
5
IO
15 HOURS
20
25
3
2. Liver and thigh disappearance of 1131activity the injection of I lal-labeled diiodotyrosine.
following
FIG. 3. Cumulative urinary excretion of 1131activity administration of labeled diiodotyrosine.
following
the
60 40
WOB0 _ *OA-
O,'o
FIQ. 4. Urinary
IO
20
tiO",",S
40
50
60
Pa1 excretion rate curve derived from the cumulative urinary excretion curve of Fig. 3. 407
408
RUEGAMER
Third-Component
Half-Time
AND
CHODOS
TABLE Values Obtained
Thuroid
and
Urine
I Ilal
from Normal Rate Curves TI/Z values,
Human Plasma,
Saliva,
hr.
Patient Whole
JL RM GG AC ss WL AS cw Average0 (2Arithmetic
plasma
Saliva
8 11 8 8 10 9
8 12 8 8 9 12
10 11
10 10
9.4 f
1.6
9.6 f
means with corresponding
Thyroid
Urine
7 11 10 8 11 12 10 10 1.7
standard
9.8 f
8 10 10 7 9 9 9 10 1.6
9.0 i
1.0
deviations.
and final-component slopesappeared to be equivalent to those observed for plasma and saliva disappearance of radioactive iodine (Table I). Thyroid-accumulation rates could be calculated in a similar manner from cumulative thyroid 1131uptake curves as shown in Fig. 5. In this instance, gross thyroid 1131uptake measurements were corrected for background by subtracting a simulated throat-disappearance curve (curve C) from the grossthyroid-uptake curve (curve A). The simulated throat curve was prepared by drawing a curve having the sameshape as the thigh-disappearance curve (curve B) through the zero-time value of the gross thyroid-uptake curve (curve A). This manipulation is believed to be valid since throat and thigh tissue disappearance curves were found to parallel one another in thyroidectomized dogs receiving iodide-1131(12). Thyroid accumulation rate curves (curve E) were then prepared by plotting the counts/mm. accumulated by the thyroid per hour as chords against time. As in the case of urinary excretion rate curves, smooth curves were drawn through these chords, and finalcomponent slopes appeared to approximate those observed for plasma and saliva disappearance of iodine (Fig. 5, Table I). Paper chromatograms of saliva and urine specimens revealed that essentially all of the saliva and urine radioactivity could be attributed to iodide-1’31. The percentages of I 131-labelediodide and DIT remaining in all protein-free plasma filtrates were also determined from paper
DIIODOTYROSINE
METABOLISM
409
FIG. 5. Thyroid accumulation of 1131 activity following the administration of I13r-labeled diiodotyrosine. Curve A: gross thyroid uptake of 1131 activity. Curve B: thigh disappearance of 1131 activity. Curve C: simulated throat background disappearance. Curve D: net thyroid accumulation of P activity. Curve B: thyroid accumulation rate curve.
chromatograms (Fig. 6), and these were used to construct appearance and iodide-appearance curves (Fig. 7).
DIT-dis-
DISCUSSION
The plasma total IL3’ activity disappearance curve for normal suhjects appears to have at least three major components (Fig. 7). The first is very rapid and cannot be followed accurately beyond 15 min. The second requires approximately 4 hr. for the attainment of a steady state, and is then followed by a third and slower component with a half life of approximately 9 hr. (Fig. 1). It is postulated that the first two components probably represent a combination of DJT deiodination, DIT redistribution into body fluids, and iodide-P3* appearance. The third component undoubtedly represents iodide-1131 disappearance from the plasma, since plasma activity after 10 hr. is predominantly iodideI131. Consequently, third-component half-time values obtained from
410
RUEGAMER
AND CHODOS
70 w o 60 5 0
ul =O cl : 40 5 c P 30 ITi -2 20 8
FIG.
FIG.
6. Relative
per cent of plasma II31 activity
as
iodide ion.
7. Per cent of injected 1131-labeled DIT appearing plasma as DIT and iodide,
in
DIIODOTYROSINE
411
METABOLISM
plasma 113’ activity disappearance curves are not measurements of the diiodotyrosine disappearance rate, but rather of the disappearance of iodide-P. Such half-time values should be comparable to similar values calculated from plasma P activity disappearance curves obtained in whole body iodide-1131 clearance studies. A correlation has indeed been observed between the average half-time value of 9.4 hr. obtained for 8 normal subjects receiving P-labeled DI’I‘ (Table I) and an average value of 8.2 hr. for 12 normal persons receiving P-labeled iodide (12). The plasma total P activity disappearance curve was resolved into its DIT-disappearance and iodide-appearance components by determining the amounts of labeled DIT and iodide present on paper chromatograms of protein-free plasma filtrates (Fig. 7). The DIT-disappearance curve obtained by plotting the plasma DIT concentration against time also appears to have three components. The first is complete in approximately 15 min., whereas the second requires approximately 4 hr. for the attainment of a steady state. These two components probably represent a combination of DIT mixing with circulating plasma, redistribution in body fluids, and deiodination. After DIT has become distributed and a steady state exists between the DIT plasma and body fluid compart’ments, DIT disappearance appears to follow an exponential rate which may be attributed almost entirely to deiodination. Although this interpretation of the plasma total radioactivity and 1131-labeled DITdisappearance curves may be oversimplified, it provides an opportunity for deriving several mathematical expressions which appear to fit the experimental curves reasonably well. Such equations can then be used to calculate the plasma activity of DIT at any time and to express the rate of DIT deiodination. Accordingly, plasma DIT disappearance may be described by the following expression, provided that both the iodide ion and DIT are able to diffuse freely hack and fort,h between the plasma and body fluid compartments. -dP dt
__
= K,,s,P
- Kjl.pF1
+ K,.j,P
- K&‘o
where Lf, , Kflrp, KP,f2 and Kfq,p, respectively,
+ KJ
(1)
represent rate constants for the disappearance of DIT from plasma (P) into body fluid compartment (Fl), from this compartment into plasma, from plasma into other body fluids (Fz) and from these body fluids back into the plasma. KL represents the rate constant for the deiodination of DIT presumably by the liver. P, F1 , and Fz represent the concentrations of
412
RUEGAMER
AND
CHODOS
DIT in plasma, body fluid compartment (F1), and the remaining body fluid compartment (Fz), respectively. The term F1 is used to designate that tissue space immediately available to plasma DIT diffusion, whereas Fz is the space which is less available and requires approximately 4 hr. for the attainment of a steady state with plasma DIT. This differentiation of plasma compartments is arbitrary and is based on the differences in diffusion rates as evidenced by the first two slopes of the plasma DIT-disappearance curve. Obviously neither anatomical nor physiological boundaries can be assigned to these compartments at the present time. Although the constant, KL , in the above equation is used to denote the liver deiodination rate, there may be other important deiodination sites, and K, may actually represent the sum of the metabolic constants for all tissues involved. As indicated earlier, the first and most rapid component of the plasma DIT-disappearance curve appears to have reached a steady state within 15 min. At this time, mixing should be complete and the disappearance of DIT from the plasma (P) into the first and more readily available body fluid compartment (FJ should equal the return of DIT from this compartment into the plasma compartment. Or Kp,f, P = KflpFl , and the first two terms of Eq. (1) drop out for 2 = 15 min. The disappearance of DIT from the second body fluid compartment can now be expressed as - clF, = &,.,Fz dt
- Kmd’
and the solution of Eqs. (1) and (2) becomes P, = Cle-m’f + C2e-mzt
(3)
where Pt is the concentration of diiodotyrosine in the plasma at any time (t), Cl is the y intercept of the extrapolated slope of the third and last component (ml) and C2 is the y intercept of the second component having a slope of m2 . The latter curve can be obtained by extrapolating the third component slope back to the ordinate, and subtracting values on the extrapolated portion of this curve from the observed plasma DIT values. When theoretical plasma DIT concentrations were calculated for numerous values of t, usingEq. (3) and the slopes and intercepts obtained
DIIODOTYROSINE
413
METABOLISM
from any given experimental curve, it was found that these values defined a theoretical curve which could be superimposed over the experimental curve. The theoretical curve encompassed only the last two components of the experimental curve, however, because it was assumed in the derivation of Eq. (3) that a steady state had already been reached between the plasma DIT compartment and the body fluid compartment (F1). It is possible to describe the first extremely rapid component of the curve by adding the term C3Crnzt to Eq. (3). In this expression, CT5is the y intercept of the first component having an extrapolated slope (m3). This slope was obtained in a manner similar to that previously described for the second component. Since the theoretical and experimental curves coincide, it would appear that the assumptions made with respect to the attainment of equilibria between the various fluid compartments are justified and that a membrane permeable to DIT and iodide is operative between these compartments. It can be seen from Fig. 7 that the concentration of plasma iodide1131never exceeds 5-10 % of the administered dose. As iodide-1131 is produced by DIT deiodination, it equilibrates with body fluids and is eventually excreted into the urine, or taken up by the thyroid gland. Therefore, the amount of deiodination that has taken place cannot be deduced from the plasma concentration of iodide-1131, but it can be calculated in another way. As stated previously, the amount of iodide formed with respect to time should be equal to the amount of DIT deiodinated and should be a function of a deiodination constant, (KL) and the plasma concentration of DIT (P). Or:
d-L=KP dt
(4)
L
Since the plasma concentration of DIT for t 5 15 min. is given by Eq. (3), t,he deiodination of DIT can be expressed as dL x = KI,(Cle-mlt Solut,ion of this equation L
where
=
L represents
K&l -+-ml
+ C&m*f)
(5)
yields the following: KLCZ m2
the amount
K &le-m’t
- K LCBe-mZt
ml
of DIT
m2
which
(6)
has been deiodinated
414
RUEGAMER
AND
CHODOS
by the liver or other tissues at time 1. A value for KL can be deduced from the experimental data since it can be shown that ml = k - (15 -
1%;
m2 = k + C/i? + ~>KL 2
2
(7)
and consequently K,
=
m2 - ml /ii
Therefore, using these equations and the constants obtained from the experimental curves, it is possible to calculate the plasma concentration of DIT at any time using Eq. (3), the rate with which DIT is deiodinated using Eq. (8), and the amount of DIT deiodinated or the amount of iodide formed at any time using Eq. (6). Individual DIT half-time values and KL values are summarized in Table II. Since the saliva P1 activity disappearance curve parallels the plasma total 1131activity curve after 5 or 10 hr., (Fig. l), it might be postulated that the amount of 1131activity appearing in the saliva is a function of the plasma concentration of iodide-1131 and some clearance constant for the salivary glands. This supposition gains support from the fact that only iodide-1131 can be found in the saliva in detectable amounts and that the average half-time value of 9.6 hr. for saliva P activity disappearance agrees well with that of 9.4 hr. for plasma disappearance (Table I). If the salivary glands deiodinate DIT as has been suggested by Fawcett et al. (14), then a significant portion of the iodide secreted into the saliva might have originated from this deiodination reaction. However, other work has failed to support the concept that salivary glands are important deiodination sites, and it is believed that the iodide secretory mechanism is separate from the deiodination reaction (10, 15). Furthermore, the saliva to whole plasma 1131activity ratios used by Thode et al. (16, 17) are of questionable physiological significance TABLE
Arithmetic
Means Disappearance
ml
TX/P hr.
3.1
f
II
and Standard Deviations of the Plasma Diiodotyrosine Constants Obtained from Eight Normal Human Subjects
hr.3
1.3
a T1,2 is the
0.26 final
component
f
0.09 half-time
2.24 value.
ml
KL
hr.-’
hr.-l
f
0.87
0.88
f
0.35
DIIODOTYROSINE
415
METABOLISM
because they represent a comparison of saliva iodide-1131 concentration with plasma protein-bound iodine-P31 concentration. Such ratios may still be of practical importance in evaluating thyroid function, but saliva to plasma iodide-I131 ratios or salivary gland iodide secretion rate constants should be employed in physiological studies of possible thyroidsalivary gland interdependencies (18). Half-time values calculated from the thyroid accumulation and urinary excretion rate curves averaged 9.8 and 9.0 hr., respectively, as compared to an average value of 9.4 hr. for the plasma disappearance of total II31 activity (Table I). Furthermore, these values compare favorably with t’hose obtained from a similar kinetic study made with 1131-labeled sodium iodide (10). Therefore, the amount of activity either accumulated by the thyroid or excreted by the kidneys is probably a function of the plasma concentration of iodide-1131 and the respective metabolic rate constants for the thyroid and kidney. Thigh and liver 1131activity disappearance curves, on the other hand, are only approximately exponential in form (Fig. 2). This is probably due to the fact that the disappearance of radioactive iodine from the body tissues is more complicated than that found for plasma since the disappearance of iodide and diiodotyrosine activity from both the plasma and extravascular fluid compartments must be considered. ACKNOWLEDGMENT We lvish
to acknowledge
the technical
assistance
of Mr.
Raymond
Wells,
Jr.
SUMMARY
The kinetics of 1131-labeled DIT deiodination has been studied in normal human subjects, and a kinetic model has been formulated for use in future comparisons of DIT deiodination in subjects with various metabolic disorders. By employing the equations offered in this model, the plasma concentration of DIT at any time and the rate of DIT deiodination can be calculated. The model also predicts that the amount of iodide-1131 secreted by the salivary glands, excreted by the kidneys, and accumulated by the thyroid gland is a function of the plasma concentration of iodide-1131 and the respective tissue rate constant. REFERENCES 1. TAUROG, 2. ROCHE,
(1952).
A., TONG, J.,
MICHEL,
W., R.,
AND CHAIKOFF, AND LISSITZKY,
I. L., J. Biol. S., Biochim.
Chem. 184, 83 (1959). et Biophys. Acla 9, 161
416 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
RUEGAMER
AND
CHODOS
TONG, W., TAUROG, A., AND CHAIKOFF, I. L., J. Biol. Chem. 207, 59 (1964). ALBERT, A., AND KEATINO, R. F. JR., J. Clin. Endocrinol. 11, 996 (1951). JOHNSON, H., AND ALBERT, A., Endocrinology 48, 669 (1951). STANBURY, J. B., KASSENAAR, A. A. H., AND MEIJER, J. W. A., J. Clin. Endocrinol. 16, 735 (1956). STANBURY, J. B., MEIJER, J. W. A., AND KASSENAAR, A. A. H., J. Clin. Endocrinol. and Metabolism 16, 848 (1956). FOSTER, G. L., AND GUTMAN, A. B., J. Biol. Chem. 84, 289 (1930). HARTMANN, N., 2. physiol. Chem. 286,l (1959). RUEGAMER, W. R., Proc. Sot. Exptl. Biol. Med. 90, 146 (1955). RUEGAMER, W. R., AND CHODOS, R. B., Federation Proc. 16,343 (1956). RUEGAMER, W. R., Arch. Biochem. Biophys. 47, 119 (1953). Ross, J. F., CHODOS, R. B., BABER, W. H., AND FREIS, E. D., Trans. Assoc. Am. Physicians 66, 75 (1952). FAWCETT, D. M., AND KIRKWOOD, S., Science 120, 547 (1954). TONG, W., POTTER, G. D., AND CHAIKOFF, I. L., Endocrinology 67, 637 (1955). THODE, H. G., JAIMET, C. H., AND KIRKWOOD, S., New Engl. J. Med. 261, 129 (1954). JAIMET, C. H., AND THODE, H. G., Peaceful Uses of Atomic Energy 10, 272 (1955). FREINKEL, N., AND INGBAR, S. H., New Engl. J. Med. 262, 125 (1955).
OF
BJOCHEMISTRY
The Kinetics
AND
BIOPHYSICS
77,
403-416 (1958)
of Diiodotyrosine Metabolism Human Subjects
in Normal
W. R. Ruegamer and R. B. Chodos From
the Radioisotope Service, Veterans Administration Hospital and Departments of Biochemistry and Medicine, State University of New York College of Medicine, Syracuse, New York
Received February
the
10, 1958
Mono- and diiodotyrosine (MIT and DIT) are found in peptide linkage in the thyroid gland and are presumed to be precursors of thyroid hormone (1). Although the free amino acid forms are not detectable in the circulating plasma under normal circumstances, Roche et al. (2) have suggested that these substances might be released from thyroglobulin during proteolysis. Probably a large percentage of the free acids so released are catabolized in part before leaving the gland (2, 3), and any MIT or DIT which escapesinto the circulation must be destroyed so rapidly that the circulating concentration never becomes detectable. Several studies (3-9) have been made of the breakdown products resulting from the feeding and injection of exogenous DIT into experimental animals and from the in vitro incubation of DIT with tissue slices and homogenates. Foster et al. (8) and later Hartmann (9) fed large doses of DIT to animals and showed that at least a portion of the ingested DIT is converted to the lactic acid derivative (3,5-diiodo-4hydroxyphenyllactic acid). Albert et al. (4, 5) and Tong et al. (3) also studied DIT catabolism in human subjects and rats, and concluded that deiodination is unquestionably one of the first and most important steps in DIT degradation, although alteration of the side chain may produce detectable amounts of the lactic acid and pyruvic acid analogs in plasma and urine. Roche et al. (2) have emphasized the importance of the thyroid in catabolizing endogenousDIT, but Tong et al. (3) found that thyroidectomy in rats had little if any effect on the ability of the rat to deiodinate exogenous DIT. They subsequently concluded that 403
404
RUEGAMER
AND CHODOS
the thyroid is much lesseffective than the liver and kidney in catabolizing DIT. The in vitro experiments of Tong et al. (3) and those from this laboratory (10, 11) also support this contention since the greatest concentration of DIT deiodinase activity was observed in liver and kidney tissue. We were able to show further that DIT is deiodinated just as rapidly and completely in the nephrectomized dog as in the intact animal, and it might be postulated that the liver is the most important deiodination site for DIT. To test this hypothesis, we have proposed to compare the deiodination of DIT in human subjects suffering from liver disease with that observed in normal persons. Since such a study must necessarily involve comparisons of kinetic data, we have first of all developed a working model for DIT deiodination in the normal subject. The evidence for and the details of this model constitute the subject matter of this report. METHODS One-hundred-microcurie doses of Ir*r-labeled L-DIT* were administered intravenously to hospitalized patients under treatment for minor illnesses. There was no clinical or laboratory evidence for thyroid, liver, or kidney disease in any of these subjects. Heparinized blood samples and 3-min. saliva specimens (obtained under the stimulation of gum chewing) were collected from these individuals 15 min. after the injection of the PVabeled DIT, and then at frequent intervals thereafter. Complete individual urine specimens were obtained during the first 24-hr. period, and 24-hr. pooled specimens were collected during the remaining days of the experiment. Aliquots from all collections were counted on the same day in a Nancy Wood scintillation well counter to an accuracy of f2%. Protein-free plasma filtrates and their corresponding protein precipitates, prepared by precipitation with 20% trichloroacetic acid, were also counted in the well counter. Determinations were not continued beyond 72 hr. because the counting rates of collected samples became too low to be determined accurately. In order to determine the relative percentages of I ‘*r-labeled substances present in the protein-free plasma filtrate, saliva, and urine samples, aliquots of these materials were subjected to paper chromatographic analysis. Approximately ten samples of 16-560~1. were applied as a single spot and dried on 23 in. X 23 in. single sheets of Whatman #2 filter paper. The volumes applied depended entirely upon the radioactivities of the samples. The sheet was rolled into a cylinder and placed in a chromatography jar for ascending development in either 60% phenol-water, 33:2:22 butanol-acetic acid-water, or 106:35 collidine-water solvents. After approximately 16 hr. development, the sheets were dried and the individual chro1 The L-DIT was randomly labeled in the 3,5 positions, with a single label on any given molecule. This material was supplied by Abbott Laboratories with an average specific activity of 340 microcuries/mg.
DIIODOTYROSINE
METABOLISM
405
matograms were sectioned into lo-mm. horizontal strips which were then assayed for radioactivity in a scintillation well counter. The iodinated substances were identified from their RI values, and the relative amour1t.s present, could be determined from the counting rates observed for each spot. Identification was made possible by comparison of the R, values for these spots with those observed for standard solutions of P-labeled DIT and iodide which were chromatographed on the same sheet of paper in corresponding nonradioactive saliva, urine, and plasma filtrate carriers. In addition to the above determinations, the disappearance of P activity from the thigh, liver, and thyroid was followed by monitoring these areas with a scintillation counter recessed 5 cm. within a x-in. lead shield. A lead filter ss in. in thickness was used to eliminate secondary radiation. Thigh and liver measurements were made with the probe positioned at a fixed distance of 5 cm. from the body surface. Thyroid-uptake measurements on the other hand were made by two methods. In the first method, measurements were performed at a fixed distance of 30 cm. from the neck surface, with and without a l-in. lead brick interposed between the detector and the thyroid area. The counting rate obtained with tho brick in place was then used to correct the thyroid counting rate for body background. The second measurement was made according to a method previously described (la), in which the thigh disappearance curve was used to correct, fol body background. Both procedures gave essentially the same results.
~ZESULTS
Plasma-disappearance curves of total 1131activity were obtained by plotting plasma concentrations of radioactivity against time (Fig. 1). Theoretical zero-time values were obtained by dividing the total injected counts per minute by the plasma volume. The latter was calculated for each individual from hematocrit values and total blood volume data obtained by a previously described P32tagged red-cell method (13). Since DIT and iodide do not bind appreciably to plasma proteins, protein-free plasma-disappearance curves were essentially superimposable on the whole plasma curves. On the other hand, saliva 1131activity disappearance curves showed a somewhat different form. The concentration of 1131activity reached a maximum within a few hours and then disappeared at an exponential rate comparable to that observed for plasma activity (Fig. 1). Thigh- and liver-disappearance curves obtained from monitoring data were of the samegeneral shapeas the plasma curves although a simple exponential rate of disappearance was never observed (Fig. 2). Cumulative urinary excretion curves of Pa1activity were plotted as shown in Fig. 3, and excretion rate curves (Fig. 4) were prepared from these by plotting the amounts of radioactivity excreted per 4-hr. interval as chords against time. Smooth curves were drawn through these chords,
406
RUEGAMER
AND
CHODOS
PLASMA
0.11
I IO
20
40
50
60
H O”u”R S
Fro. 1. Saliva and plasma disappearance of II31 activity following of 100 microcuries of P-labeled diiodotyrosine. I
ZOOO(
0 100: c” so= 8
the injection
THIGH -*
60. 40 -
20 /
I
OO
FIQ.
5
IO
15 HOURS
20
25
3
2. Liver and thigh disappearance of 1131activity the injection of I lal-labeled diiodotyrosine.
following
FIG. 3. Cumulative urinary excretion of 1131activity administration of labeled diiodotyrosine.
following
the
60 40
WOB0 _ *OA-
O,'o
FIQ. 4. Urinary
IO
20
tiO",",S
40
50
60
Pa1 excretion rate curve derived from the cumulative urinary excretion curve of Fig. 3. 407
408
RUEGAMER
Third-Component
Half-Time
AND
CHODOS
TABLE Values Obtained
Thuroid
and
Urine
I Ilal
from Normal Rate Curves TI/Z values,
Human Plasma,
Saliva,
hr.
Patient Whole
JL RM GG AC ss WL AS cw Average0 (2Arithmetic
plasma
Saliva
8 11 8 8 10 9
8 12 8 8 9 12
10 11
10 10
9.4 f
1.6
9.6 f
means with corresponding
Thyroid
Urine
7 11 10 8 11 12 10 10 1.7
standard
9.8 f
8 10 10 7 9 9 9 10 1.6
9.0 i
1.0
deviations.
and final-component slopesappeared to be equivalent to those observed for plasma and saliva disappearance of radioactive iodine (Table I). Thyroid-accumulation rates could be calculated in a similar manner from cumulative thyroid 1131uptake curves as shown in Fig. 5. In this instance, gross thyroid 1131uptake measurements were corrected for background by subtracting a simulated throat-disappearance curve (curve C) from the grossthyroid-uptake curve (curve A). The simulated throat curve was prepared by drawing a curve having the sameshape as the thigh-disappearance curve (curve B) through the zero-time value of the gross thyroid-uptake curve (curve A). This manipulation is believed to be valid since throat and thigh tissue disappearance curves were found to parallel one another in thyroidectomized dogs receiving iodide-1131(12). Thyroid accumulation rate curves (curve E) were then prepared by plotting the counts/mm. accumulated by the thyroid per hour as chords against time. As in the case of urinary excretion rate curves, smooth curves were drawn through these chords, and finalcomponent slopes appeared to approximate those observed for plasma and saliva disappearance of iodine (Fig. 5, Table I). Paper chromatograms of saliva and urine specimens revealed that essentially all of the saliva and urine radioactivity could be attributed to iodide-1’31. The percentages of I 131-labelediodide and DIT remaining in all protein-free plasma filtrates were also determined from paper
DIIODOTYROSINE
METABOLISM
409
FIG. 5. Thyroid accumulation of 1131 activity following the administration of I13r-labeled diiodotyrosine. Curve A: gross thyroid uptake of 1131 activity. Curve B: thigh disappearance of 1131 activity. Curve C: simulated throat background disappearance. Curve D: net thyroid accumulation of P activity. Curve B: thyroid accumulation rate curve.
chromatograms (Fig. 6), and these were used to construct appearance and iodide-appearance curves (Fig. 7).
DIT-dis-
DISCUSSION
The plasma total IL3’ activity disappearance curve for normal suhjects appears to have at least three major components (Fig. 7). The first is very rapid and cannot be followed accurately beyond 15 min. The second requires approximately 4 hr. for the attainment of a steady state, and is then followed by a third and slower component with a half life of approximately 9 hr. (Fig. 1). It is postulated that the first two components probably represent a combination of DJT deiodination, DIT redistribution into body fluids, and iodide-P3* appearance. The third component undoubtedly represents iodide-1131 disappearance from the plasma, since plasma activity after 10 hr. is predominantly iodideI131. Consequently, third-component half-time values obtained from
410
RUEGAMER
AND CHODOS
70 w o 60 5 0
ul =O cl : 40 5 c P 30 ITi -2 20 8
FIG.
FIG.
6. Relative
per cent of plasma II31 activity
as
iodide ion.
7. Per cent of injected 1131-labeled DIT appearing plasma as DIT and iodide,
in
DIIODOTYROSINE
411
METABOLISM
plasma 113’ activity disappearance curves are not measurements of the diiodotyrosine disappearance rate, but rather of the disappearance of iodide-P. Such half-time values should be comparable to similar values calculated from plasma P activity disappearance curves obtained in whole body iodide-1131 clearance studies. A correlation has indeed been observed between the average half-time value of 9.4 hr. obtained for 8 normal subjects receiving P-labeled DI’I‘ (Table I) and an average value of 8.2 hr. for 12 normal persons receiving P-labeled iodide (12). The plasma total P activity disappearance curve was resolved into its DIT-disappearance and iodide-appearance components by determining the amounts of labeled DIT and iodide present on paper chromatograms of protein-free plasma filtrates (Fig. 7). The DIT-disappearance curve obtained by plotting the plasma DIT concentration against time also appears to have three components. The first is complete in approximately 15 min., whereas the second requires approximately 4 hr. for the attainment of a steady state. These two components probably represent a combination of DIT mixing with circulating plasma, redistribution in body fluids, and deiodination. After DIT has become distributed and a steady state exists between the DIT plasma and body fluid compart’ments, DIT disappearance appears to follow an exponential rate which may be attributed almost entirely to deiodination. Although this interpretation of the plasma total radioactivity and 1131-labeled DITdisappearance curves may be oversimplified, it provides an opportunity for deriving several mathematical expressions which appear to fit the experimental curves reasonably well. Such equations can then be used to calculate the plasma activity of DIT at any time and to express the rate of DIT deiodination. Accordingly, plasma DIT disappearance may be described by the following expression, provided that both the iodide ion and DIT are able to diffuse freely hack and fort,h between the plasma and body fluid compartments. -dP dt
__
= K,,s,P
- Kjl.pF1
+ K,.j,P
- K&‘o
where Lf, , Kflrp, KP,f2 and Kfq,p, respectively,
+ KJ
(1)
represent rate constants for the disappearance of DIT from plasma (P) into body fluid compartment (Fl), from this compartment into plasma, from plasma into other body fluids (Fz) and from these body fluids back into the plasma. KL represents the rate constant for the deiodination of DIT presumably by the liver. P, F1 , and Fz represent the concentrations of
412
RUEGAMER
AND
CHODOS
DIT in plasma, body fluid compartment (F1), and the remaining body fluid compartment (Fz), respectively. The term F1 is used to designate that tissue space immediately available to plasma DIT diffusion, whereas Fz is the space which is less available and requires approximately 4 hr. for the attainment of a steady state with plasma DIT. This differentiation of plasma compartments is arbitrary and is based on the differences in diffusion rates as evidenced by the first two slopes of the plasma DIT-disappearance curve. Obviously neither anatomical nor physiological boundaries can be assigned to these compartments at the present time. Although the constant, KL , in the above equation is used to denote the liver deiodination rate, there may be other important deiodination sites, and K, may actually represent the sum of the metabolic constants for all tissues involved. As indicated earlier, the first and most rapid component of the plasma DIT-disappearance curve appears to have reached a steady state within 15 min. At this time, mixing should be complete and the disappearance of DIT from the plasma (P) into the first and more readily available body fluid compartment (FJ should equal the return of DIT from this compartment into the plasma compartment. Or Kp,f, P = KflpFl , and the first two terms of Eq. (1) drop out for 2 = 15 min. The disappearance of DIT from the second body fluid compartment can now be expressed as - clF, = &,.,Fz dt
- Kmd’
and the solution of Eqs. (1) and (2) becomes P, = Cle-m’f + C2e-mzt
(3)
where Pt is the concentration of diiodotyrosine in the plasma at any time (t), Cl is the y intercept of the extrapolated slope of the third and last component (ml) and C2 is the y intercept of the second component having a slope of m2 . The latter curve can be obtained by extrapolating the third component slope back to the ordinate, and subtracting values on the extrapolated portion of this curve from the observed plasma DIT values. When theoretical plasma DIT concentrations were calculated for numerous values of t, usingEq. (3) and the slopes and intercepts obtained
DIIODOTYROSINE
413
METABOLISM
from any given experimental curve, it was found that these values defined a theoretical curve which could be superimposed over the experimental curve. The theoretical curve encompassed only the last two components of the experimental curve, however, because it was assumed in the derivation of Eq. (3) that a steady state had already been reached between the plasma DIT compartment and the body fluid compartment (F1). It is possible to describe the first extremely rapid component of the curve by adding the term C3Crnzt to Eq. (3). In this expression, CT5is the y intercept of the first component having an extrapolated slope (m3). This slope was obtained in a manner similar to that previously described for the second component. Since the theoretical and experimental curves coincide, it would appear that the assumptions made with respect to the attainment of equilibria between the various fluid compartments are justified and that a membrane permeable to DIT and iodide is operative between these compartments. It can be seen from Fig. 7 that the concentration of plasma iodide1131never exceeds 5-10 % of the administered dose. As iodide-1131 is produced by DIT deiodination, it equilibrates with body fluids and is eventually excreted into the urine, or taken up by the thyroid gland. Therefore, the amount of deiodination that has taken place cannot be deduced from the plasma concentration of iodide-1131, but it can be calculated in another way. As stated previously, the amount of iodide formed with respect to time should be equal to the amount of DIT deiodinated and should be a function of a deiodination constant, (KL) and the plasma concentration of DIT (P). Or:
d-L=KP dt
(4)
L
Since the plasma concentration of DIT for t 5 15 min. is given by Eq. (3), t,he deiodination of DIT can be expressed as dL x = KI,(Cle-mlt Solut,ion of this equation L
where
=
L represents
K&l -+-ml
+ C&m*f)
(5)
yields the following: KLCZ m2
the amount
K &le-m’t
- K LCBe-mZt
ml
of DIT
m2
which
(6)
has been deiodinated
414
RUEGAMER
AND
CHODOS
by the liver or other tissues at time 1. A value for KL can be deduced from the experimental data since it can be shown that ml = k - (15 -
1%;
m2 = k + C/i? + ~>KL 2
2
(7)
and consequently K,
=
m2 - ml /ii
Therefore, using these equations and the constants obtained from the experimental curves, it is possible to calculate the plasma concentration of DIT at any time using Eq. (3), the rate with which DIT is deiodinated using Eq. (8), and the amount of DIT deiodinated or the amount of iodide formed at any time using Eq. (6). Individual DIT half-time values and KL values are summarized in Table II. Since the saliva P1 activity disappearance curve parallels the plasma total 1131activity curve after 5 or 10 hr., (Fig. l), it might be postulated that the amount of 1131activity appearing in the saliva is a function of the plasma concentration of iodide-1131 and some clearance constant for the salivary glands. This supposition gains support from the fact that only iodide-1131 can be found in the saliva in detectable amounts and that the average half-time value of 9.6 hr. for saliva P activity disappearance agrees well with that of 9.4 hr. for plasma disappearance (Table I). If the salivary glands deiodinate DIT as has been suggested by Fawcett et al. (14), then a significant portion of the iodide secreted into the saliva might have originated from this deiodination reaction. However, other work has failed to support the concept that salivary glands are important deiodination sites, and it is believed that the iodide secretory mechanism is separate from the deiodination reaction (10, 15). Furthermore, the saliva to whole plasma 1131activity ratios used by Thode et al. (16, 17) are of questionable physiological significance TABLE
Arithmetic
Means Disappearance
ml
TX/P hr.
3.1
f
II
and Standard Deviations of the Plasma Diiodotyrosine Constants Obtained from Eight Normal Human Subjects
hr.3
1.3
a T1,2 is the
0.26 final
component
f
0.09 half-time
2.24 value.
ml
KL
hr.-’
hr.-l
f
0.87
0.88
f
0.35
DIIODOTYROSINE
415
METABOLISM
because they represent a comparison of saliva iodide-1131 concentration with plasma protein-bound iodine-P31 concentration. Such ratios may still be of practical importance in evaluating thyroid function, but saliva to plasma iodide-I131 ratios or salivary gland iodide secretion rate constants should be employed in physiological studies of possible thyroidsalivary gland interdependencies (18). Half-time values calculated from the thyroid accumulation and urinary excretion rate curves averaged 9.8 and 9.0 hr., respectively, as compared to an average value of 9.4 hr. for the plasma disappearance of total II31 activity (Table I). Furthermore, these values compare favorably with t’hose obtained from a similar kinetic study made with 1131-labeled sodium iodide (10). Therefore, the amount of activity either accumulated by the thyroid or excreted by the kidneys is probably a function of the plasma concentration of iodide-1131 and the respective metabolic rate constants for the thyroid and kidney. Thigh and liver 1131activity disappearance curves, on the other hand, are only approximately exponential in form (Fig. 2). This is probably due to the fact that the disappearance of radioactive iodine from the body tissues is more complicated than that found for plasma since the disappearance of iodide and diiodotyrosine activity from both the plasma and extravascular fluid compartments must be considered. ACKNOWLEDGMENT We lvish
to acknowledge
the technical
assistance
of Mr.
Raymond
Wells,
Jr.
SUMMARY
The kinetics of 1131-labeled DIT deiodination has been studied in normal human subjects, and a kinetic model has been formulated for use in future comparisons of DIT deiodination in subjects with various metabolic disorders. By employing the equations offered in this model, the plasma concentration of DIT at any time and the rate of DIT deiodination can be calculated. The model also predicts that the amount of iodide-1131 secreted by the salivary glands, excreted by the kidneys, and accumulated by the thyroid gland is a function of the plasma concentration of iodide-1131 and the respective tissue rate constant. REFERENCES 1. TAUROG, 2. ROCHE,
(1952).
A., TONG, J.,
MICHEL,
W., R.,
AND CHAIKOFF, AND LISSITZKY,
I. L., J. Biol. S., Biochim.
Chem. 184, 83 (1959). et Biophys. Acla 9, 161
416 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
RUEGAMER
AND
CHODOS
TONG, W., TAUROG, A., AND CHAIKOFF, I. L., J. Biol. Chem. 207, 59 (1964). ALBERT, A., AND KEATINO, R. F. JR., J. Clin. Endocrinol. 11, 996 (1951). JOHNSON, H., AND ALBERT, A., Endocrinology 48, 669 (1951). STANBURY, J. B., KASSENAAR, A. A. H., AND MEIJER, J. W. A., J. Clin. Endocrinol. 16, 735 (1956). STANBURY, J. B., MEIJER, J. W. A., AND KASSENAAR, A. A. H., J. Clin. Endocrinol. and Metabolism 16, 848 (1956). FOSTER, G. L., AND GUTMAN, A. B., J. Biol. Chem. 84, 289 (1930). HARTMANN, N., 2. physiol. Chem. 286,l (1959). RUEGAMER, W. R., Proc. Sot. Exptl. Biol. Med. 90, 146 (1955). RUEGAMER, W. R., AND CHODOS, R. B., Federation Proc. 16,343 (1956). RUEGAMER, W. R., Arch. Biochem. Biophys. 47, 119 (1953). Ross, J. F., CHODOS, R. B., BABER, W. H., AND FREIS, E. D., Trans. Assoc. Am. Physicians 66, 75 (1952). FAWCETT, D. M., AND KIRKWOOD, S., Science 120, 547 (1954). TONG, W., POTTER, G. D., AND CHAIKOFF, I. L., Endocrinology 67, 637 (1955). THODE, H. G., JAIMET, C. H., AND KIRKWOOD, S., New Engl. J. Med. 261, 129 (1954). JAIMET, C. H., AND THODE, H. G., Peaceful Uses of Atomic Energy 10, 272 (1955). FREINKEL, N., AND INGBAR, S. H., New Engl. J. Med. 262, 125 (1955).