Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 46, No. 6 Printed in U.S.A.
MERRITT RUDOLPH, TOSHIRO SAKURADA, SHIH-LIEH FANG, APOSTOLOS G. VAGENAKIS, LEWIS E. BRAVERMAN, AND SIDNEY H. INGBAR The Thorndike Laboratory of Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts, and the Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts labeled hormone measured by these techniques will be in error, because pre-T.t will be included. Thus, the MCR of labeled T3 and rT.-t measured by Sephadex chromatography will always be higher than values obtained by the other two separative techniques and the magnitude of change will be similar to the ratio of preT:J to precursor T 3 or rTa. The Sephadex chromatographic technique is laborious, but appears to be the method of choice where greatest accuracy of measurement is required. As the generation of pre-T.i from labeled T4 is sufficiently slow, the present chromatographic technique is not necessary in studying peripheral T,( turnover. (J Clin Endocrinol Metab 46: 923, 1978)
ABSTRACT. Chromatography of serum on columns of Sephadex G-25 superfine after the iv administration of 12r 'I-labeled T4 consistently yielded labeled iodide, iodoprotein, T3, and a labeled peak that eluted from the column before the T3, termed "pre-T3." Much larger quantities of pre-T3 were generated after the iv administration of l25I-labeled T3 and rT3. The ratio of [12r'I]pre T3:[12SI]T3 and [l25I]pre T3:[12BI]iT3 plateaued at approximately 10 h and 2 h, respectively, after the iv administration of the labeled hormone, and averaged approximately 15% in euthyroid subjects. As pre-T3 behaves like its precursors in the TCA precipitation-ethanol extraction or anion exchange chromatographic procedures used to separate labeled iodide and iodoprotein from administered labeled T3 and rT3) concentrations of
A
S PART of studies of the rate of periph- rial that eluted from the column in a peak just L eral conversion of T4 to T3 in man, it before the peak of T3 (pre-T.3).1 The present was necessary to develop a method of treating report describes the analytic method by which large volumes of serum in order to separate this material can be both separated from other from one another the administered radioio- iodine-containing compounds in serum and dine-labeled T4 and the major radioiodinated measured, the probable sources from which it products of T4 metabolism known to appear is formed, and its quantitative significance as in serum (iodide, iodoprotein, and T3). A suit- a source of error in studies of the peripheral able method, involving chromatography of se- metabolism of various iodothyronines. A porrum in columns of Sephadex G-25 superfine, tion of this work has been described in abwas adapted from methods published earlier stract form (2). (1). When this technique was applied to the study of sera from patients given [125I]T4, they Methods and Results were found to contain, in addition to the three labeled products expected, I25I-labeled mate- Sephadex chromatography of serum The basic method employed was that described by Green (1), modified for use in the Received August 3, 1977. present studies. Address requests for reprints to: Dr. Lewis E. BraverThe following solutions were employed: man, Division of Endocrinology and Metabolism, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01605. * This work was supported in part by Grants RR-01032, AM-18416, and AM-18919 from the NIH, Bethesda, Md.
1
In the abstract in which these studies were described (2), what is here termed "pre-T.i" was designated "labeled unknown products" or LUP.
923
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Appearance of Labeled Metabolites in the Serum of Man after the Administration of Labeled Thyroxine, Triiodothyronine (T3), and Reverse Triiodothyronine (rT3)*
924
RUDOLPH ET AL.
JCK & M V0U6
1978 No 6
500
Evaluation of Sephadex chromatography of serum To assess the extent to which T4 might be deiodinated during the column chromatographic procedure, 5 ml normal human serum was enriched with 10 /*Ci I25I-labeled T42 and was subjected to chromatography on the Sephadex column, as described above. An aliquot of the fraction that contained the highest concentration of [125I]T4 was added to another specimen of serum and was rechromatographed. The resulting elution pattern, shown in Fig. 1, revealed only a peak of [I25I]T4, with 2 [>25i]'j\| and rT;J were obtained from Abbott Laboratories and ['"IjT.j was purchased from Amersham-Searle.
50 100 FRACTION NUMBER
150
FIG. 1. Rechromatography of l2f'I-labeled T4 on a column of Sephadex G-25 superfine. Serum enriched with [l2r>I]T.i was chromatographed and an aliquot of an eluate from the [I25I]T4 zone was added to a fresh sample of serum and rechromatographed. Raw counts (background not subtracted) are shown. In this system, iodoprotein is eluted in fractions 3-5, iodide in fractions 30-33, and Ty in the fractions demarcated by brackets (see also Fig. 2). rT.) is eluted with the leading portion of the T^ zone (data not shown). The study reveals the absence of artifactual generation of detectable quantities of other labeled compounds during the chromatographic procedure.
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equilibrium solution, 0.1 M NaOH-1.0 M NaCl, no radioactivity being seen in other zones, containing 3.8 g/liter Na2S2O5 (pH 12.5); dil- including those corresponding to iodide, T3, uent solution, 0.10 M phosphate buffer, pH 6.5, and pre-T.3. containing 1.0 M NaCl; eluent I, 0.10 M NaOHRecovery of radioactivity from the Sepha0,01 M NaCl, containing 3.8 g/liter Na2S2O5 dex column during chromatography of serum (pH 10.5); eluent II, 0.15 M NaOH-0.01 M NaCl was also assessed. Total 125I concentration was solution, containing 3.8 g/liter Na2S2O5 (pH measured in samples of serum obtained from 12.5). patients given 125I-labeled T3 or rT 3 from 6 h Sephadex G-25 superfine was allowed to to 3 days earlier. Samples were then subjected swell in equilibration solution for at least 24 h to chromatography on Sephadex columns and before use and was then used to pack a 50.0 the total recovery of 125I was determined. In X 2.5-cm glass column to a height of 35 cm. 45 studies, recovery of radioactivity averaged Equilibration solution was passed through the 97.6 ± 3.0% (mean ± SD). The virtually comcolumn by gravity overnight, after which the plete recovery of total radioactivity suggests column was ready for use. The feeder tube that recovery of each of the individual radioiwas then run through a peristaltic pump and odinated compounds contained in the samples the flow rate was adjusted to 0.9-1.0 ml/min. was also nearly complete. To assess the reproducibility of chromatoAn aliquot (5-10 ml) of whole serum was mixed with an equal volume of diluent solu- graphic separations provided by the Sephadex tion to which 9 mg/ml 8-anilino-l-naphtha- column, as it might be used for studies of lene sulfonic acid (ANS) had been added. The peripheral T3 or rT 3 metabolism, duplicate latter agent served to dissociate labeled io- samples of serum from patients given [125I]T3 dothyronines from the plasma proteins and or [125I]rT3 were subjected to chromatographic permitted essentially complete adsorption of analysis. In two paired observations, 125I in the iodothyronines onto the column. The dilute T 3 zone differed between duplicates by only sample was passed onto the column through 0.6 and 3.8%, while in five duplicate observathe feeder tubing, which was then shifted to a flask containing eluent I, and the collection of 1000 5-ml fractions was begun. After 250 ml eluent I had passed into the column, the feeder tubing was shifted to a flask containing eluent II and the chromatography was continued until a total of 160 fractions had been collected.
LABELED METABOLITES OF T4, T3, AND rT3
925
ible quantities of [125I]pre-T3 has not been determined.
Generation ofpre-Tz from T4
Rechromatography ofpre-Tz
The foregoing method was applied to separate [I25I]T3 from its precursor [125I]T4 in sera obtained from patients given a single iv injection of [125I]T4 (100 jLiCi, 3 /ig) 2-6 days earlier. Such sera were consistently found to contain 125 I-labeled iodoprotein and iodide, 125I-labeled T 3 as judged from coelution with marker [131I]T3, and the residue of administered [125I]T4. In addition, there always was observed a small peak of 125I eluting just before [I25I]T3, sometimes well separated from T 3 and other times forming a shoulder on the leading segment of the [125I]T3 peak (Fig. 2). For present purposes, we have designated the materials in this peak as pre-T3. Despite its relatively low radioactivity and often incomplete separation from [125I]T3, a 125I-labeled pre-T3 peak, separable from [125I]T3 as judged from the peak of marker [131I]T3, has been seen in more than 100 samples of serum from more than 25 patients given [125I]T4 from 2-6 days earlier. The shortest interval between the administration of [125I]T4 and the appearance of discern-
When samples of pre-T3 were pooled and rechromatographed, the radioactivity once again eluted in the pre-T3 zone. In the case of pre-T3 generated from [125I]T3, a very small amount of [125I]T3 was also seen, but no [125I]rT3 was seen on rechromatography of pre-T3 generated from rT3.
5000 r
50 100 FRACTION NUMBER l2r>
150
FIG. 2. Generation of Uabeled pre-T;) (and T:i) from [I25I]T4 in a euthyroid patient. Sephadex G-25 chromatography of serum from a patient given 100 /xCi of [l2r'I]T4 4 days earlier. , elution zones of pre-T.j and of T;), as revealed by marker [1:ilI]T.i. Raw counts (background not subtracted) as shown.
Sources ofpre-Ta To determine whether pre-T3 is generated directly and exclusively from T4 or is derived, in turn, from other products of T4 metabolism, euthyroid volunteers were given a single iv injection of 100 juCi [I25I]T3 (1.5 /ig) or [125I]rT3 (0.2 jug) and serial samples of serum were obtained thereafter for column chromatographic analysis. After the administration of [125I]T3, a peak of I25I, eluting just before T3, appeared in serum obtained as soon as 1 h after [125I]T3 administration (Fig. 3). The peak in the pre-T3 zone could be clearly separated from that of both the administered [125I]T3 and the coincident peak of [131I]T3 added as a marker. In sera drawn at progressively later intervals, the magnitude of the pre-T3 peak relative to that of the [125I]T3 peak increased progressively until, in the example shown, at approximately 10 h an apparently constant 125 I-labeled pre-T3:T3 ratio was achieved. Similar findings were made after the acute iv administration of 125I-labeled rT 3 (Fig. 4). 125 I in the pre-T3 zone was evident in sera obtained at 30 min after [125I]rT3 administration. The [125I]pre-T3:[125I]rT3 ratio in serum increased rapidly with time, reaching an apparent plateau at 1-2 h. Because rT 3 elutes in the same general zone as T4 does in this system, labeled pre-T3 and rT 3 were very widely separated. Quantitative aspects ofpre-Tz formation Multiple samples of serum were obtained from patients given a single iv injection of 100 [125I]T3 in whom the metabolic clearance
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tions with [125I]rT3, percentage differences between duplicates ranged between 1.0-4.3%.
RUDOLPH ET AL.
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JCK&M • 1978 Vol46 • No 6
4000 '/2-HOUR 3000
(8%)
1000 I25J
(cpm) 4000
3000
10 Hours (19%)
12 Hours (16%)
2000
1000
OL
FIG. 3. Generation of 125I-labeled pre-T:, from [I25I]T3 in a euthyroid patient. Only a portion is shown of the elution patterns yielded by Sephadex G-25 chromatography of sequential samples of serum obtained at the indicated times after administration of 100 /xCi [l2r'I]T.) to a euthyroid patient. Values in parentheses indicate the ratio between radioactivity in the pre-T.) and T.( zones, expressed as a percent. • • ••, elution pattern of [l:ilI]T.i added to serum as a marker. Some I3II eluting in the preT;) zone is occasionally seen in preparations of [I;)II]T3.
FIG. 4. Generation of 125I-labeled pre-T:i from [l2r>I]rT.i in a euthyroid patient. Only a portion is shown of the elution patterns yielded by Sephadex G-25 chromatography of sequential samples of serum obtained at the indicated times after administration of 100 /iCi [l25I]rTa to a euthyroid patient. Values in parentheses indicate the ratio between radioactivity in the pre-T:J and rT:J zones, expressed as a percent. Pre-T3 is here designated as an unknown (UNK).
before and during a total fast of at least 6-day duration, ratios increased in all; the extent of increase ranging from 1.4- to 2.3-fold. Similar observations were made in the case of six euthyroid subjects given [125I]rT3. [125I]Pre-T3:[125I]rT3 ratios achieved plateau values no later than 4 h after administration rate of T 3 was to be determined by noncomof [125I]rT3 and were maintained for the repartmental analysis. For each patient, at least maining period of observation. For each of the three samples obtained from 24-72 h after six subjects, the equilibrium value of the ratio [125I]T3 administration were subjected to chro- was calculated as the average of the values matographic analysis on Sephadex columns. obtained in three to four samples of serum From the elution patterns, and with the aid of collected between 4 and 12 h after administramarker [131I]T3, 125I in the pre-T3 zone was tion of [125I]rT . In the group of six subjects, 3 compared to that in the T 3 zone by calculating equilibrium ratios averaged 14.9 ± 2.7%. Al125 125 the ratio of [ I]pre-T3:[ I]T3, expressed as though this mean was similar to that obtained a percent. Numerical values for the ratio in the 15 patients studied with [125I]T , paired 3 showed no significant trend with time, in- values of the ratio in 4 subjects given labeled dicating that secular equilibrium between T 3 and labeled rT 3 were higher in the case of [125I]pre-T3 and its precursor [125I]T3 had been rT 3 (mean for T3, 6.2%; mean for rT3, 14.5%). achieved. Values of the ratios were averaged, therefore, to obtain a mean value for each patient at equilibrium. In 15 euthyroid sub- Behavior of pre-Ts in other separative sysjects, the ratios averaged 17.8 ± 7.4% (mean tems ± SD). Ratios were similar in six hyperthyroid As compounds comprising pre-T3 appeared patients (24.2 ± 7.3) and in two patients with to constitute a significant fraction of the total hypothyroidism (17.8 and 25.8%). Fasting ap- radioiodine in the serum of patients given peared to increase the equilibrium value of radioiodine-labeled T 3 or rT3, and to contain the pre-T3:T3 ratio. In four patients studied a moderately large number of counts relative
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2000
LABELED METABOLITES OF T4) T3, A N D rT3
Effect of pre-Tz on the measured kinetics of T3 and rT3 metabolism Because the foregoing studies had shown that the radioiodinated compounds in pre-T3 are largely or entirely included in the iodothyronine-containing fractions yielded by techniques commonly employed to separate labeled iodide and iodoprotein from labeled T3 and rT 3 in studies of their peripheral metabolism, comparative studies were carried out in six patients who received [125I]T4 and two who received [l25I]rT3. Samples of serum were obtained at 7.5, 15.0, and 30.0 min and 1, 2, 3, 4, 5, and 8 h in patients who received [125]rT3, and at 12-h intervals between 24 and 72 h as well, in patients who received [125I]T3. All samples of serum were divided into two aliquots, one of which was treated by either TCA precipitation-ethanol extraction or anion exchange chromatography, while the other
was chromatographed on Sephadex columns and the radioactivity of the administered compound was specifically determined. Plasma disappearance curves generated by the two methods were then compared. As would be expected, at all time points later than 1 h, radioactivity in Sephadex isolates was clearly less than that in the TCAprecipitable ethanol-extractable fraction or in the iodothyronine eluates of anion exchange chromatography. Because of the establishment of virtually constant values of the [I25I]pre-T3:[125I]precursor ratio, disappearance curves generated by the Sephadex technique and those generated by the other procedures were virtually parallel after 1-2 h in the case of rT 3 and after 10-12 h in the case of T3. As would be expected from the lower concentrations of I25I in Sephadex isolates than the iodothyronine fractions yielded by the other techniques, calculated values of the MCR of T 3 and rT 3 were consistently higher when based on the Sephadex isolation procedure than when based on data generated by the other two methods. Moreover, the proportionate extent by which the MCR yielded by the Sephadex method exceeded that yielded by the other methods was approximately the same as the equilibrium value of the [125I]preT3:125I-precursor ratio. Discussion It had previously been thought that iodide and iodoprotein were the sole radioiodine-labeled products that appeared in the serum of man in significant quantities after administration of radioiodine-labeled T 3 or rT3. The present studies have demonstrated, however, that another class of radioiodinated products also appears in serum soon after the administration of labeled T 3 or rT3. These products, whose identification and isolation are described in a companion report (5) have here been termed pre-T3 because they elute just before T 3 in the Sephadex chromatographic system in which they were initially demonstrated. 125I-labeled pre-T3 appears in serum within 1 h after administration of [125I]T3 or [125I]rT3 and increases in proportion to the
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to the counts contributed by its radioactive precursor, studies were performed to ascertain the extent to which the precursor would be separated from pre-T3 by procedures commonly employed to separate the precursor from its other labeled products, iodide and iodoprotein. Sera from patients given either [125I]T3 or 125 [ I]rT3 were chromatographed on columns of Sephadex G-25, and fractions eluting in the pre-T.3 zone were pooled. Pooled eluates were concentrated by adsorption onto small columns of Sephadex LH-20 and elution into small volumes of methanol-ammonia. Eluates were dried, the residues were dissolved in normal human serum, and aliquots were subjected to TCA precipitation-ethanol extraction (3) or anion exchange chromatography (4), using methods applied in previous studies of the kinetics of peripheral T.3 or rT,3 turnover. Between 85-87% of 125I in pre-T3 was recovered in the TCA-precipitable, ethanol-extractable fraction, a value similar to that observed for T3 itself. During anion exchange chromatography of pre-T3, significant quantities of radioiodine were eluted only in those fractions in which iodothyronines, including T3 and rT3, are recovered.
927
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JCK & M V0U6
in measurements of the MCR of T 3 and rT 3 would be on the order of about 15%. Although the Sephadex chromatographic method we have described is time-consuming and laborious, particularly when applied to the many samples of serum whose analysis is required for measurements of the MCR of T 3 or rT3, it would appear to be the method of choice where greatest accuracy of measurement is required. This would be true in studies aiming to assess the rate of conversion of T4 to T 3 and rT3, the relative production rates of T 3 and rT3, and the relative contributions of peripheral T4 deiodination and direct thyroid secretion to total T 3 and rT 3 production. As with the rate of generation of iodide and iodoprotein, generation of pre-T3 after administration of labeled T4 is sufficiently slow so that refined techniques for the treatment of serum in studies of peripheral T4 turnover are not required. References 1. GREEN, W. L., Separation of iodocompounds in serum by chromatography on sephadex columns, J Chromatogr 72: 83, 1972. 2. RUDOLPH, M., T. SAKURADA, A. VAGENAKIS, S. FANC, L.
BHAVEBMAN, and S. INGBAR, Demonstration of sequential
monodeiodination as the major pathway of iodothyronine metabolism, Clin Res 24: 429A, 1976. 3. SURKS, M., and J. H. OPPENHEIMER, Formation of iodopro-
tein during the peripheral metabolism of 3,5,3'-triiodothyronine in the euthyroid man and rat, J Clin Invest 48: 685,1969. 4. NICOLOFF, J. T., J. C. Low, J. H. DUSKAULT, and D. A.
FISHER, Simultaneous measurements of thyroxine and triiodothyronine peripheral turnover kinetics in man, J Clin Invest 51: 473, 1971. 5. SAKURADA, T., M. RUDOLPH, S. L. FANC:, A. G. VAGENAKIS,
L. E. BRAVERMAN, and S. H. INGBAR, Evidence that triiodothyronine and reverse triiodothyronine are sequentially deiodinated in man, J Clin Endocrinol Metab 46: 916, 1978.
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concentration of residual 125I-precursor until a constant ratio is achieved between the two which persists until neither compound is any longer demonstrable. In almost all previous studies of the kinetics of peripheral T 3 or rT 3 metabolism, either TCA precipitation-ethanol extraction or anion exchange chromatographic procedures have been employed to exclude labeled iodide and iodoprotein from the measurement of the administrated labeled compound. As pre-T3 behaves like its precursors in both techniques, the measured concentration of labeled precursor will be in error by virtue of the inclusion of labeled pre-T3. By direct comparative studies, we have shown that this is indeed the case, and that plasma disappearance curves which specifically reflect the concentration of [I25I]T3 or [125I]rTa are at almost all time points lower than those generated by either of the other two techniques. As a consequence, values for the MCR of T 3 or rT 3 based on measurements in Sephadex isolates are always higher than values of the MCR derived from data generated by the other two techniques. The percentage of difference by which the MCR values yielded by the present technique exceeds those yielded by previous techniques will be slightly less than the equilibrium value of the [125I]pre-T3:[125I]precursor ratio, and we have shown this to be the case. The slightly lesser value of the percentage of difference in MCR values is due to the time required for equilibrium values of the [125I]pre-T3: ^^-precursor ratio to be achieved. Our studies indicate that the average magnitude of the error
1978 No 6
Vol. 46, No. 6 Printed in U.S.A.
MERRITT RUDOLPH, TOSHIRO SAKURADA, SHIH-LIEH FANG, APOSTOLOS G. VAGENAKIS, LEWIS E. BRAVERMAN, AND SIDNEY H. INGBAR The Thorndike Laboratory of Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts, and the Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts labeled hormone measured by these techniques will be in error, because pre-T.t will be included. Thus, the MCR of labeled T3 and rT.-t measured by Sephadex chromatography will always be higher than values obtained by the other two separative techniques and the magnitude of change will be similar to the ratio of preT:J to precursor T 3 or rTa. The Sephadex chromatographic technique is laborious, but appears to be the method of choice where greatest accuracy of measurement is required. As the generation of pre-T.i from labeled T4 is sufficiently slow, the present chromatographic technique is not necessary in studying peripheral T,( turnover. (J Clin Endocrinol Metab 46: 923, 1978)
ABSTRACT. Chromatography of serum on columns of Sephadex G-25 superfine after the iv administration of 12r 'I-labeled T4 consistently yielded labeled iodide, iodoprotein, T3, and a labeled peak that eluted from the column before the T3, termed "pre-T3." Much larger quantities of pre-T3 were generated after the iv administration of l25I-labeled T3 and rT3. The ratio of [12r'I]pre T3:[12SI]T3 and [l25I]pre T3:[12BI]iT3 plateaued at approximately 10 h and 2 h, respectively, after the iv administration of the labeled hormone, and averaged approximately 15% in euthyroid subjects. As pre-T3 behaves like its precursors in the TCA precipitation-ethanol extraction or anion exchange chromatographic procedures used to separate labeled iodide and iodoprotein from administered labeled T3 and rT3) concentrations of
A
S PART of studies of the rate of periph- rial that eluted from the column in a peak just L eral conversion of T4 to T3 in man, it before the peak of T3 (pre-T.3).1 The present was necessary to develop a method of treating report describes the analytic method by which large volumes of serum in order to separate this material can be both separated from other from one another the administered radioio- iodine-containing compounds in serum and dine-labeled T4 and the major radioiodinated measured, the probable sources from which it products of T4 metabolism known to appear is formed, and its quantitative significance as in serum (iodide, iodoprotein, and T3). A suit- a source of error in studies of the peripheral able method, involving chromatography of se- metabolism of various iodothyronines. A porrum in columns of Sephadex G-25 superfine, tion of this work has been described in abwas adapted from methods published earlier stract form (2). (1). When this technique was applied to the study of sera from patients given [125I]T4, they Methods and Results were found to contain, in addition to the three labeled products expected, I25I-labeled mate- Sephadex chromatography of serum The basic method employed was that described by Green (1), modified for use in the Received August 3, 1977. present studies. Address requests for reprints to: Dr. Lewis E. BraverThe following solutions were employed: man, Division of Endocrinology and Metabolism, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01605. * This work was supported in part by Grants RR-01032, AM-18416, and AM-18919 from the NIH, Bethesda, Md.
1
In the abstract in which these studies were described (2), what is here termed "pre-T.i" was designated "labeled unknown products" or LUP.
923
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Appearance of Labeled Metabolites in the Serum of Man after the Administration of Labeled Thyroxine, Triiodothyronine (T3), and Reverse Triiodothyronine (rT3)*
924
RUDOLPH ET AL.
JCK & M V0U6
1978 No 6
500
Evaluation of Sephadex chromatography of serum To assess the extent to which T4 might be deiodinated during the column chromatographic procedure, 5 ml normal human serum was enriched with 10 /*Ci I25I-labeled T42 and was subjected to chromatography on the Sephadex column, as described above. An aliquot of the fraction that contained the highest concentration of [125I]T4 was added to another specimen of serum and was rechromatographed. The resulting elution pattern, shown in Fig. 1, revealed only a peak of [I25I]T4, with 2 [>25i]'j\| and rT;J were obtained from Abbott Laboratories and ['"IjT.j was purchased from Amersham-Searle.
50 100 FRACTION NUMBER
150
FIG. 1. Rechromatography of l2f'I-labeled T4 on a column of Sephadex G-25 superfine. Serum enriched with [l2r>I]T.i was chromatographed and an aliquot of an eluate from the [I25I]T4 zone was added to a fresh sample of serum and rechromatographed. Raw counts (background not subtracted) are shown. In this system, iodoprotein is eluted in fractions 3-5, iodide in fractions 30-33, and Ty in the fractions demarcated by brackets (see also Fig. 2). rT.) is eluted with the leading portion of the T^ zone (data not shown). The study reveals the absence of artifactual generation of detectable quantities of other labeled compounds during the chromatographic procedure.
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equilibrium solution, 0.1 M NaOH-1.0 M NaCl, no radioactivity being seen in other zones, containing 3.8 g/liter Na2S2O5 (pH 12.5); dil- including those corresponding to iodide, T3, uent solution, 0.10 M phosphate buffer, pH 6.5, and pre-T.3. containing 1.0 M NaCl; eluent I, 0.10 M NaOHRecovery of radioactivity from the Sepha0,01 M NaCl, containing 3.8 g/liter Na2S2O5 dex column during chromatography of serum (pH 10.5); eluent II, 0.15 M NaOH-0.01 M NaCl was also assessed. Total 125I concentration was solution, containing 3.8 g/liter Na2S2O5 (pH measured in samples of serum obtained from 12.5). patients given 125I-labeled T3 or rT 3 from 6 h Sephadex G-25 superfine was allowed to to 3 days earlier. Samples were then subjected swell in equilibration solution for at least 24 h to chromatography on Sephadex columns and before use and was then used to pack a 50.0 the total recovery of 125I was determined. In X 2.5-cm glass column to a height of 35 cm. 45 studies, recovery of radioactivity averaged Equilibration solution was passed through the 97.6 ± 3.0% (mean ± SD). The virtually comcolumn by gravity overnight, after which the plete recovery of total radioactivity suggests column was ready for use. The feeder tube that recovery of each of the individual radioiwas then run through a peristaltic pump and odinated compounds contained in the samples the flow rate was adjusted to 0.9-1.0 ml/min. was also nearly complete. To assess the reproducibility of chromatoAn aliquot (5-10 ml) of whole serum was mixed with an equal volume of diluent solu- graphic separations provided by the Sephadex tion to which 9 mg/ml 8-anilino-l-naphtha- column, as it might be used for studies of lene sulfonic acid (ANS) had been added. The peripheral T3 or rT 3 metabolism, duplicate latter agent served to dissociate labeled io- samples of serum from patients given [125I]T3 dothyronines from the plasma proteins and or [125I]rT3 were subjected to chromatographic permitted essentially complete adsorption of analysis. In two paired observations, 125I in the iodothyronines onto the column. The dilute T 3 zone differed between duplicates by only sample was passed onto the column through 0.6 and 3.8%, while in five duplicate observathe feeder tubing, which was then shifted to a flask containing eluent I, and the collection of 1000 5-ml fractions was begun. After 250 ml eluent I had passed into the column, the feeder tubing was shifted to a flask containing eluent II and the chromatography was continued until a total of 160 fractions had been collected.
LABELED METABOLITES OF T4, T3, AND rT3
925
ible quantities of [125I]pre-T3 has not been determined.
Generation ofpre-Tz from T4
Rechromatography ofpre-Tz
The foregoing method was applied to separate [I25I]T3 from its precursor [125I]T4 in sera obtained from patients given a single iv injection of [125I]T4 (100 jLiCi, 3 /ig) 2-6 days earlier. Such sera were consistently found to contain 125 I-labeled iodoprotein and iodide, 125I-labeled T 3 as judged from coelution with marker [131I]T3, and the residue of administered [125I]T4. In addition, there always was observed a small peak of 125I eluting just before [I25I]T3, sometimes well separated from T 3 and other times forming a shoulder on the leading segment of the [125I]T3 peak (Fig. 2). For present purposes, we have designated the materials in this peak as pre-T3. Despite its relatively low radioactivity and often incomplete separation from [125I]T3, a 125I-labeled pre-T3 peak, separable from [125I]T3 as judged from the peak of marker [131I]T3, has been seen in more than 100 samples of serum from more than 25 patients given [125I]T4 from 2-6 days earlier. The shortest interval between the administration of [125I]T4 and the appearance of discern-
When samples of pre-T3 were pooled and rechromatographed, the radioactivity once again eluted in the pre-T3 zone. In the case of pre-T3 generated from [125I]T3, a very small amount of [125I]T3 was also seen, but no [125I]rT3 was seen on rechromatography of pre-T3 generated from rT3.
5000 r
50 100 FRACTION NUMBER l2r>
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FIG. 2. Generation of Uabeled pre-T;) (and T:i) from [I25I]T4 in a euthyroid patient. Sephadex G-25 chromatography of serum from a patient given 100 /xCi of [l2r'I]T4 4 days earlier. , elution zones of pre-T.j and of T;), as revealed by marker [1:ilI]T.i. Raw counts (background not subtracted) as shown.
Sources ofpre-Ta To determine whether pre-T3 is generated directly and exclusively from T4 or is derived, in turn, from other products of T4 metabolism, euthyroid volunteers were given a single iv injection of 100 juCi [I25I]T3 (1.5 /ig) or [125I]rT3 (0.2 jug) and serial samples of serum were obtained thereafter for column chromatographic analysis. After the administration of [125I]T3, a peak of I25I, eluting just before T3, appeared in serum obtained as soon as 1 h after [125I]T3 administration (Fig. 3). The peak in the pre-T3 zone could be clearly separated from that of both the administered [125I]T3 and the coincident peak of [131I]T3 added as a marker. In sera drawn at progressively later intervals, the magnitude of the pre-T3 peak relative to that of the [125I]T3 peak increased progressively until, in the example shown, at approximately 10 h an apparently constant 125 I-labeled pre-T3:T3 ratio was achieved. Similar findings were made after the acute iv administration of 125I-labeled rT 3 (Fig. 4). 125 I in the pre-T3 zone was evident in sera obtained at 30 min after [125I]rT3 administration. The [125I]pre-T3:[125I]rT3 ratio in serum increased rapidly with time, reaching an apparent plateau at 1-2 h. Because rT 3 elutes in the same general zone as T4 does in this system, labeled pre-T3 and rT 3 were very widely separated. Quantitative aspects ofpre-Tz formation Multiple samples of serum were obtained from patients given a single iv injection of 100 [125I]T3 in whom the metabolic clearance
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tions with [125I]rT3, percentage differences between duplicates ranged between 1.0-4.3%.
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(8%)
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12 Hours (16%)
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OL
FIG. 3. Generation of 125I-labeled pre-T:, from [I25I]T3 in a euthyroid patient. Only a portion is shown of the elution patterns yielded by Sephadex G-25 chromatography of sequential samples of serum obtained at the indicated times after administration of 100 /xCi [l2r'I]T.) to a euthyroid patient. Values in parentheses indicate the ratio between radioactivity in the pre-T.) and T.( zones, expressed as a percent. • • ••, elution pattern of [l:ilI]T.i added to serum as a marker. Some I3II eluting in the preT;) zone is occasionally seen in preparations of [I;)II]T3.
FIG. 4. Generation of 125I-labeled pre-T:i from [l2r>I]rT.i in a euthyroid patient. Only a portion is shown of the elution patterns yielded by Sephadex G-25 chromatography of sequential samples of serum obtained at the indicated times after administration of 100 /iCi [l25I]rTa to a euthyroid patient. Values in parentheses indicate the ratio between radioactivity in the pre-T:J and rT:J zones, expressed as a percent. Pre-T3 is here designated as an unknown (UNK).
before and during a total fast of at least 6-day duration, ratios increased in all; the extent of increase ranging from 1.4- to 2.3-fold. Similar observations were made in the case of six euthyroid subjects given [125I]rT3. [125I]Pre-T3:[125I]rT3 ratios achieved plateau values no later than 4 h after administration rate of T 3 was to be determined by noncomof [125I]rT3 and were maintained for the repartmental analysis. For each patient, at least maining period of observation. For each of the three samples obtained from 24-72 h after six subjects, the equilibrium value of the ratio [125I]T3 administration were subjected to chro- was calculated as the average of the values matographic analysis on Sephadex columns. obtained in three to four samples of serum From the elution patterns, and with the aid of collected between 4 and 12 h after administramarker [131I]T3, 125I in the pre-T3 zone was tion of [125I]rT . In the group of six subjects, 3 compared to that in the T 3 zone by calculating equilibrium ratios averaged 14.9 ± 2.7%. Al125 125 the ratio of [ I]pre-T3:[ I]T3, expressed as though this mean was similar to that obtained a percent. Numerical values for the ratio in the 15 patients studied with [125I]T , paired 3 showed no significant trend with time, in- values of the ratio in 4 subjects given labeled dicating that secular equilibrium between T 3 and labeled rT 3 were higher in the case of [125I]pre-T3 and its precursor [125I]T3 had been rT 3 (mean for T3, 6.2%; mean for rT3, 14.5%). achieved. Values of the ratios were averaged, therefore, to obtain a mean value for each patient at equilibrium. In 15 euthyroid sub- Behavior of pre-Ts in other separative sysjects, the ratios averaged 17.8 ± 7.4% (mean tems ± SD). Ratios were similar in six hyperthyroid As compounds comprising pre-T3 appeared patients (24.2 ± 7.3) and in two patients with to constitute a significant fraction of the total hypothyroidism (17.8 and 25.8%). Fasting ap- radioiodine in the serum of patients given peared to increase the equilibrium value of radioiodine-labeled T 3 or rT3, and to contain the pre-T3:T3 ratio. In four patients studied a moderately large number of counts relative
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2000
LABELED METABOLITES OF T4) T3, A N D rT3
Effect of pre-Tz on the measured kinetics of T3 and rT3 metabolism Because the foregoing studies had shown that the radioiodinated compounds in pre-T3 are largely or entirely included in the iodothyronine-containing fractions yielded by techniques commonly employed to separate labeled iodide and iodoprotein from labeled T3 and rT 3 in studies of their peripheral metabolism, comparative studies were carried out in six patients who received [125I]T4 and two who received [l25I]rT3. Samples of serum were obtained at 7.5, 15.0, and 30.0 min and 1, 2, 3, 4, 5, and 8 h in patients who received [125]rT3, and at 12-h intervals between 24 and 72 h as well, in patients who received [125I]T3. All samples of serum were divided into two aliquots, one of which was treated by either TCA precipitation-ethanol extraction or anion exchange chromatography, while the other
was chromatographed on Sephadex columns and the radioactivity of the administered compound was specifically determined. Plasma disappearance curves generated by the two methods were then compared. As would be expected, at all time points later than 1 h, radioactivity in Sephadex isolates was clearly less than that in the TCAprecipitable ethanol-extractable fraction or in the iodothyronine eluates of anion exchange chromatography. Because of the establishment of virtually constant values of the [I25I]pre-T3:[125I]precursor ratio, disappearance curves generated by the Sephadex technique and those generated by the other procedures were virtually parallel after 1-2 h in the case of rT 3 and after 10-12 h in the case of T3. As would be expected from the lower concentrations of I25I in Sephadex isolates than the iodothyronine fractions yielded by the other techniques, calculated values of the MCR of T 3 and rT 3 were consistently higher when based on the Sephadex isolation procedure than when based on data generated by the other two methods. Moreover, the proportionate extent by which the MCR yielded by the Sephadex method exceeded that yielded by the other methods was approximately the same as the equilibrium value of the [125I]preT3:125I-precursor ratio. Discussion It had previously been thought that iodide and iodoprotein were the sole radioiodine-labeled products that appeared in the serum of man in significant quantities after administration of radioiodine-labeled T 3 or rT3. The present studies have demonstrated, however, that another class of radioiodinated products also appears in serum soon after the administration of labeled T 3 or rT3. These products, whose identification and isolation are described in a companion report (5) have here been termed pre-T3 because they elute just before T 3 in the Sephadex chromatographic system in which they were initially demonstrated. 125I-labeled pre-T3 appears in serum within 1 h after administration of [125I]T3 or [125I]rT3 and increases in proportion to the
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to the counts contributed by its radioactive precursor, studies were performed to ascertain the extent to which the precursor would be separated from pre-T3 by procedures commonly employed to separate the precursor from its other labeled products, iodide and iodoprotein. Sera from patients given either [125I]T3 or 125 [ I]rT3 were chromatographed on columns of Sephadex G-25, and fractions eluting in the pre-T.3 zone were pooled. Pooled eluates were concentrated by adsorption onto small columns of Sephadex LH-20 and elution into small volumes of methanol-ammonia. Eluates were dried, the residues were dissolved in normal human serum, and aliquots were subjected to TCA precipitation-ethanol extraction (3) or anion exchange chromatography (4), using methods applied in previous studies of the kinetics of peripheral T.3 or rT,3 turnover. Between 85-87% of 125I in pre-T3 was recovered in the TCA-precipitable, ethanol-extractable fraction, a value similar to that observed for T3 itself. During anion exchange chromatography of pre-T3, significant quantities of radioiodine were eluted only in those fractions in which iodothyronines, including T3 and rT3, are recovered.
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in measurements of the MCR of T 3 and rT 3 would be on the order of about 15%. Although the Sephadex chromatographic method we have described is time-consuming and laborious, particularly when applied to the many samples of serum whose analysis is required for measurements of the MCR of T 3 or rT3, it would appear to be the method of choice where greatest accuracy of measurement is required. This would be true in studies aiming to assess the rate of conversion of T4 to T 3 and rT3, the relative production rates of T 3 and rT3, and the relative contributions of peripheral T4 deiodination and direct thyroid secretion to total T 3 and rT 3 production. As with the rate of generation of iodide and iodoprotein, generation of pre-T3 after administration of labeled T4 is sufficiently slow so that refined techniques for the treatment of serum in studies of peripheral T4 turnover are not required. References 1. GREEN, W. L., Separation of iodocompounds in serum by chromatography on sephadex columns, J Chromatogr 72: 83, 1972. 2. RUDOLPH, M., T. SAKURADA, A. VAGENAKIS, S. FANC, L.
BHAVEBMAN, and S. INGBAR, Demonstration of sequential
monodeiodination as the major pathway of iodothyronine metabolism, Clin Res 24: 429A, 1976. 3. SURKS, M., and J. H. OPPENHEIMER, Formation of iodopro-
tein during the peripheral metabolism of 3,5,3'-triiodothyronine in the euthyroid man and rat, J Clin Invest 48: 685,1969. 4. NICOLOFF, J. T., J. C. Low, J. H. DUSKAULT, and D. A.
FISHER, Simultaneous measurements of thyroxine and triiodothyronine peripheral turnover kinetics in man, J Clin Invest 51: 473, 1971. 5. SAKURADA, T., M. RUDOLPH, S. L. FANC:, A. G. VAGENAKIS,
L. E. BRAVERMAN, and S. H. INGBAR, Evidence that triiodothyronine and reverse triiodothyronine are sequentially deiodinated in man, J Clin Endocrinol Metab 46: 916, 1978.
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concentration of residual 125I-precursor until a constant ratio is achieved between the two which persists until neither compound is any longer demonstrable. In almost all previous studies of the kinetics of peripheral T 3 or rT 3 metabolism, either TCA precipitation-ethanol extraction or anion exchange chromatographic procedures have been employed to exclude labeled iodide and iodoprotein from the measurement of the administrated labeled compound. As pre-T3 behaves like its precursors in both techniques, the measured concentration of labeled precursor will be in error by virtue of the inclusion of labeled pre-T3. By direct comparative studies, we have shown that this is indeed the case, and that plasma disappearance curves which specifically reflect the concentration of [I25I]T3 or [125I]rTa are at almost all time points lower than those generated by either of the other two techniques. As a consequence, values for the MCR of T 3 or rT 3 based on measurements in Sephadex isolates are always higher than values of the MCR derived from data generated by the other two techniques. The percentage of difference by which the MCR values yielded by the present technique exceeds those yielded by previous techniques will be slightly less than the equilibrium value of the [125I]pre-T3:[125I]precursor ratio, and we have shown this to be the case. The slightly lesser value of the percentage of difference in MCR values is due to the time required for equilibrium values of the [125I]pre-T3: ^^-precursor ratio to be achieved. Our studies indicate that the average magnitude of the error
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