Marquette Universitye-Publications@MarquetteBiomedical Sciences Faculty Research andPublications Biomedical Sciences, Department of

6-1-1961

Studies of the Rate of Disappearance of LabeledThyroxine from the Intravascular CompartmentEdward J. LennonMarquette University

Norman H. EngbringMarquette University

William W. EngstromMarquette University

Published version. Journal of Clinical Investigation, Vol. 40, No. 6 ( June 1961): 996-1005. DOI. ©1961 American Society for Clinical Investigation. Used with permission.

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STUDIES OF THE RATE OF DISAPPEARANCEOF LABELED THYROXINEFROMTHE INTRAVASCULARCOMPARTMENT*

By EDWARDJ. LENNON, NORMANH. ENGBRINGANDWILLIAM W. ENGSTROM

(From the Department of Medicine, Marquette University School of Medicine and MilwaukeeCounty Hospital, Milwaukee, Wis.)

(Submitted for publication June 23, 1960; accepted March 2, 1961)

The reversible combination of thyroxine withcertain of the plasma proteins clearly provides anefficient transport mechanism enabling microgramquantities of the hormone to be carried to tissuesites with minimal losses via glomerular filtrationand other nonselective excretory pathways. Thedemonstration of thyroxine-binding proteins inother extracellular fluids (1) and within cells (2)suggests a means by which thyroxine may be se-lectively distributed to tissues according to theirindividual needs.

The role, if any, of the plasma thyroxine-bindingproteins in various thyroid disorders is not clear.In vitro studies of the protein-thyroxine complexinvolve alterations of pH, Pco2, temperature andother aspects of the biochemical environment andmay not give an accurate index of in vivo rela-tionships. Furthermore, such studies neglect in-teractions of plasma thyroxine-binding proteinsand extravascular thyroxine-binding proteins.

It seemed reasonable to suppose that the rateof disappearance of I131-labeled L-thyroxine (T4*)from the intravascular compartment during anacute experiment might yield information aboutthe rate at which equilibrium between intra- andextravascular compartments was approached.The rate of disappearance should reflect the abilityof the serum proteins to bind thyroxine and re-tain it within the vascular compartment in the faceof all ambient forces favoring its removal. Con-ventional studies of labeled thyroxine survival(measured in days) are presumed to measure pri-marily the rate of thyroxine disposal after equili-bration between labeled and stable thyroxine hasbeen attained throughout the body. In preliminarystudies, attempts were made to measure the rate

* This investigation was supported in part by PublicHealth Service Grants A-1200 and 2A-5023, NationalInstitute of Arthritis and Metabolic Diseases, Bethesda,Md.

of disappearance of T4* from the circulation ofisolated extremities but technical problems pre-cluded accurate measurement. A whole-bodytechnic was therefore employed and found to besatisfactory.

The method consisted in determining the rateof disappearance of I131-L-thyroxine from the in-travascular compartment of human subjects dur-ing a 30-minute period after allowing 20 minutesfrom time of intravenous injection for thoroughintravascular mixing. The unsteady state pre-vailing shortly after the injection of T4* was de-liberately selected for the period of study becausethe rate of loss of T4* from the circulation is maxi-mal at this time and the apparent volume of dis-tribution is rapidly expanding. The rate of disap-pearance was calculated and was shown to bereproducible.

Experimental results indicate: a) a profoundinfluence of the liver on the acute disappearancerate of T4*, indicated by a markedly prolongedintravascular retention of thyroxine in subjectswith hepatic disease; b) rapid disappearance ofT4* in patients with diffuse toxic goiter (Graves'disease), but no significant change from normalin subjects fed large doses of desiccated thyroid,and slight prolongation of disappearance rate insubjects with toxic nodular goiter; and c) markedprolongation of disappearance rate in subjectswith spontaneous myxedema, but no similar pro-longation in subjects with Graves' disease who de-veloped myxedema after treatment of hyperthy-roidism.

MATERIAL AND METHODS

Each subject was evaluated by one or more of theauthors and a clinical impression of the state of thyroidalfunction was made without reference to laboratory stud-ies. Impressions were confirmed by serum protein-boundiodine (PBI) determinations and, in some instances, bymeasuring the thyroidal 24-hour uptake of P131. The

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THE ACUTE INTRAVASCULARDISAPPEARANCEOF THYROXINE

control group consisted of 15 euthyroid subjects rangingfrom 13 to 76 years of age. None had clinical evidence ofhepatic or renal disease, but some suffered from a varietyof other nonfebrile illnesses, e.g., generalized arterio-sclerosis, carcinoma of the lung, mild malnutrition, com-pensated heart disease, and so forth.

The study groups consisted of 9 euthyroid subjects withliver disease, 7 subjects with toxic nodular goiter, 9 withGraves' disease and thyrotoxicosis, 4 subjects with Graves'disease who had become euthyroid after treatment (oneof these receiving 3 grains of desiccated thyroid dailybecause of moderate exophthalmos), 5 subj ects withGraves' disease who were myxedematous as a result oftreatment and 7 subjects with spontaneous primary myx-edema. In addition, 10 subjects whose ranges of acutethyroxine disappearance rate approximated the rangesobserved among the various groups were subjected to asecond study 2 to 4 days after the initial measurementto estimate reproducibility of the method.

Subjects were brought to the radioisotope laboratoryin the early afternoon without special preparation. Ano. 18 needle was inserted and secured in an antecubitalvein and connected to one limb of a Radicoil (Abbott)resting in a shielded well-type scintillation counter.The other limb of the Radicoil was connected via athree-way stopcock to a 20 ml syringe and a solution of50 mg aqueous heparin in 500 ml of isotonic saline.Blood was withdrawn through the Radicoil to the 10ml mark of the syringe, counted for 1 minute, and re-injected into the subject. This step was repeated one ormore times, depending on background radioactivity. Attime zero, a 20 to 30 ,tc dose of T4* of high specific ac-tivity (Abbott) was injected into a vein in the oppositearm. The T4* was used within 7 days of delivery; itwas not rechromatographed before use since prior stud-ies indicated that free I13. had little influence on the func-tion being measured. The three-way stopcock was ad-justed to permit a slow drip of the heparin solution toprevent clotting in the indwelling needle. At 5-minuteintervals, blood was drawn into the Radicoil, countedfor 1 minute and reinjected. Care was taken to drawthe blood slowly (20 seconds) to the 10 ml mark of thesyringe to prevent variations in mixing and dilution.Counting at 5-minute intervals was continued for 50 to 60minutes after injection of the T4*. There was no sig-nificant adherence of thyroxine to the plastic Radicoil inthe presence of plasma proteins.

RESULTS

When the curve of thyroxine disappearance isderived on semilog paper by plotting the netcounts per minute (log scale) against time (linearscale), three distinct components of the curve areapparent. The first component. with most rap-idly declining concentration of To*, extends from0 to 10 or 20 minutes after injection and primarilyrepresents mixing and dilution of the T4* within

the vascular compartment. In normal subjectsthis brief, initial phase requires less than 10 min-utes; however, when circulation is slowed, as inmyexedma, it may extend to 20 minutes. Even inthis brief period of mixing, simultaneous egressof T4* from the circulation will occur, but it isnot possible to separate that decline in radioac-tivity due to mixing from that due to extravascu-lar movement. The moment when mixing is com-plete and when the decrements of T4* largelyrepresent egress from the circulation, cannot beascertained.

The second component is curvilinear and ex-tends for 1 or 2 days. It primarily represents mix-ing of T4* throughout the body with establishmentof an equilibrium between labeled and stable thy-roxine. The curvilinear regression clearly repre-sents the simiultaneous operation of a number ofprocesses: a) egress from the circulation-therate of fall in plasma radioactivity by this functionwill be greatest when intravascular concentrationof T4* is highest, i.e., the early part of the curve;b) re-entry of T4* into the circulation from theextravascular compartment-as extravascular con-centrations of T4* slowly increase, the contributionto plasma radioactivity increases; c) superimposedis the third component of the curve-the disposalof thyroxine from the body.

TIME (MINUTES)

FIG. 1. THREE ILLUSTRATIVE CURVESPLOTTED ON SEMI-LOG PAPER SHOWINGTHE REGRESSION OF NET COUNTSPERMINUTE ON TIME AFTER INTRAVENOUS INJECTION OF I"-L-THYROXINE. TTIE HALF-TIME VALUE IS DERIVED FRO-MCALCULATION OF THE SLOPE OF THAT SEGMENTOF THECURVEBETWEEN20 AND 50 MINUTES.

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EDWARDJ. LENNON, NORMANH. ENGBRINGAND WILLIAM W. ENGSTROM

TABLE I

Replicate determinations of acute thyroxine disappearance*

Acute thyroxinedisappearance rate

Half-time

First SecondSubject study study Difference Difference

min min min %A.K. 57.8 56.5 - 1.3 - 2.30R.B. 43.9 44.1 + 0.2 + 0.46B.P. 71.8 78.8 + 7.0 + 9.75E.K. 96.5 88.3 - 8.2 - 8.50F.H. 114.0 109.1 - 4.9 - 4.30C.B. 65.4 68.1 + 2.7 + 4.13H.S. 59.1 54.5 - 4.6 - 7.78F.T. 123.4 103.8 -19.6 -15.88L.J. 99.7 84.6 -15.1 -15.14L.B. 55.6 63.5 + 7.9 +14.21

Mean 78.7 75.1 - 3.6 - 2.5SDt : 8.9 i 9.9

* Second studies were performed 2 to 4 days after initialstudies.

t Standard deviation.

The third component is practically linear andcan be measured after equilibration is established(3). The normal half-time of thyroxine "sur-vival" or disposal is about 6 days. Since the

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half-time is 3 or more days even in hyperthyroidstates, the slope of this linear component, whenconsidered over a 30-minute period, is almost im-perceptible and could influence calculated resultsvery little.

Thus, while all of the above simultaneously oc-

curring processes can not be separated, to obtainsome measure or approximation of the net effectof forces favoring retention and removal of intra-vascularly injected (initially unbound) T4*, at-tention must be directed to the interval when T44concentrations are highest and decreasing mostrapidly. While the mixing phase is rapid in nor-

mals, it may require as long as 20 minutes in some

patients with myxedema. Therefore, so as to beable to compare different groups in a similar man-

ner and since the "rate" was obtained by calcula-tion rather than a visual fit, the regression from20 to 50 minutes was chosen for analysis (Figure1 and Table II). In this interval mixing shouldhave been completed, re-entry of T4* from ex-

travascular spaces should be small although per-

haps not insignificant in all circumstances, and

* EuthiroidX On thyroid 209r./dayX Toxic nodular goiter

Spontaneous myxedemr.A Graves' - hyperthyroido - euthtyroidAd Ad - hypothyroidA Liver disease

* X

x X X zK~~~~~~~~~

X

1* X

0 4 8 12 16 20 24 28

PBI

FIG. 2. THE RELATIONSHIP BETWEENPBI, REFLECTING LEVELS OF THYROIDAL

ACTIVITY, AND THE ACUTE THYROXINE DISAPPEARANCERATE.

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THE ACUTE INTRAVASCULARDISAPPEARANCEOF THYROXINE

TABLE II

Experimental results for control subjects and study groups

Thyroidal Acute thyroxine24 hr. I'M3 disappearance rate

Group Subject Age Sex Race PBI uptake Half-time

Healthy,euthyroid

M.P.E.R.A.D.A.Da.D.D.D.E.D.Z.I.J.L.C.M.S.P.W.W.S.R.Q.C.A.G.K.

yrs342944752813484247765336252942

FFMMMMFFMMMMMMM

N.Cau.Cau.Cau.Cau.Cau.Cau.N.,Cau.Cau.Cau.N.Cau.N.Cau.

Isg%5.23.83.95.83.37.55.84.53.55.94.45.85.46.76.8

21.9

min71.863.176.677.475.882.778.677.472.073.261.666.269.486.577.2

10.6

18.510.721.619.5

A.K.E.A.M.H.L.K.I.A.A.S.E.K.

M.R.S.B.M.G.S.T.M.S.D.A.E.H.K.M.M.P.

R.B. 27S.T. 54V.H. 28K.S. 48

S.N. 19M.R. 34B.W. 30D.A.* 32E.C. 56

50 F Cau.77 F Cau.70 F Cau.68 F Cau.76 F Cau.65 F Cau.49 F Cau.

21 F N.67 M Cau.39 F N.54 F Cau.26 F Cau.32 F Cau.54 M Cau.47 F Cau.30 F Cau.

M Cau.F Cau.F Cau.F Cau.

F Cau.F Cau.F Cau.F Cau.F Cau.

5.87.47.7

12.07.96.8

16.620.0

8.515.315.213.625.320.2

7.05.69.95.8

1.51.02.11.51.6

56.242.154.444.248.532.449.0

36.671.567.443.061.566.040.5

38.211.8

8.45.0

0.45.9

Mean 74.0SD -r 6.9

84.185.072.095.678.497.782.2

Mean 85.0SD 4 9.1

51.351.970.360.742.662.453.346.956.5

Mean 55.1SD -- 8.4

62.370.767.556.2

Mean 64.2SD 6.3

78.882.578.271.868.6

Mean 76.0SD 4 5.6

* Previously studied while thyrotoxic.

Toxicnodulargoiter

Graves'disease,toxic

Graves'disease,euthyroid

Graves'disease,myxedema

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EDWARI) J. LnNNON, NORMAN1H. ENGI3RING AND WILLIAM W. ERWSTROM

TABLE ii-( Continued)

Thyroidal Acute thyroxine24 hr I131 disappearance rate

Group Subject Age Sex Race PBI uptake Half-time

yrs lg% minSpontaneous E.M. 71 F Cau. 1.3 157.6primary G.E. 75 F Cau. 3.0 2.4 106.4myxedema H.B. 69 F Cau. 0.6 1.2 106.4

C.D. 68 F Cau. 0.5 3.2 107.5O.S. 59 M Cau. 1.0 86.5M.B. 55 F N. 0.4 1.4 97.1S.C. 85 F Cau. 0.2 4.3 111.5

Mean 110.4SD 4 22.4

Liver disease J.H.t 24 M Cau. 7.8 111.9L.T. 43 M Cau. 4.3 127.6R.L. 42 M Cau. 6.4 250.9O.T. 42 M Cau. 273.7A.W. 46 F N. 5.6 135.0F.T. 48 M Cau. 3.3 116.7M.W. 67 M Cau. 5.6 15.4 111.5J.E. 44 M Cau. 95.9M.B. 44 F N. 248.8

Mean 163.6SD 4 71.8

Normals W.S. 36 M N. 9.8 71.7fed R.Q. 25 M Cau. 7.0 59.0thyroid C.A. 29 M N. 10.0 62.4

G.K. 42 M Cau. 9.7 83.8

Mean 69.2SD :1 11.1

t Studied during acute phase of infectious hepatitis.

the effects of thyroxine disposal from the body asa whole should be negligible.1

Statistical analysis indicated the probabilitythat the portion of the curve for the relativelyshort 20 to 50 minute interval was linear in eachcase (p < 0.01). This linear portion of the re-gression curve was solved mathematically in eachinstance by the method of least squares and theslope was determined (log y = a + bx). Thetime required for the net counts per minute to de-cline to one-half the intercept value at time zero(time of injection of T4*) was obtained by ex-trapolation and is expressed as the "acute thy-

1 Our data, which does not contain simultaneously de-termined concentrations of extravascular T4*, does not per-mit the calculation of a precise rate constant for transferof T4 out of plasma, either in normals or in the differentdisorders studied. This would also require knowledgeof the rate constant for transfer of T4 from the ex-travascular compartment to plasma. Thus, our resultsare expressed merely as a "rate," rather than a rate con-stant, and the slopes can not be considered to be truezero-time slopes.

roxine disappearance half-time." The statisticalmethods used were those described by Snedecor(4).

Replicate studies. To determine reproducibilityin the same subject, ten subjects were restudiedafter 2 to 4 days without treatment. Subjects wereselected on the basis of the first study to includethe range of values noted within the study groups.Table I compares the results of first and secondstudies. The mean of the difference (first studyminus second study) was - 3.6 minutes. Groupmeans for first and second studies were not sta-tistically different (p > 0.50).

Euthyroid controls. The acute thyroxine disap-pearance rate for 15 euthyroid controls was 74.0minutes. Analysis of covariance indicated the like-lihood of a common population regression coeffi-cient for these subjects. Variations correlatedwith age, sex or race could not be detected in thisrelatively small group. Individual data for sub-jects in this and subsequent groups are given inTable II and in Figure 2.

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Liver disease. Marked prolongation of thyrox-ine disappearance rate was noted in the eight pa-

tients with Laennec's cirrhosis and in the one withacute infectious hepatitis. Details as to degree ofhepatic involvement are given in Table III. Themean half-time of disappearance was 163.6 min-utes. The slowest disappearance rates were ob-served in the subjects with ascites, indicating thatexpansion of the extracellular fluid volume neednot accelerate disappearance rate (as might be ex-

pected if this measured simple diffusion from theintravascular compartment). The wide range ofvalues observed undoubtedly reflects different de-grees of hepatic impairment, but could not be cor-

related with any of the recorded indices of liverfunction. Difference from the control group was

highly significant (p < 0.001).Hyperthyroidism. A sharp distinction between

patients with nodular toxic goiter and those withdiffuse toxic goiter (Graves' disease) was evident.The mean half-time of thyroxine disappearance forseven subjects with toxic nodular goiter (85.0minutes) was actually significantly longer thanthat observed in the controls (p < 0.01). Bycontrast, nine patients with diffuse toxic goiterhad a mean thyroxine disappearance half-time of55.1 minutes, very significantly shorter than thecontrol group (p < 0.001).

In an effort to clarify this difference fourhealthy, euthyroid subjects were given increasingamounts of desiccated thyroid over a 6-week pe-

riod, up to 20 grains daily. The thyroxine disap-pearance rate was determined at 2-week intervals.

The results are summarized in Table II. Asnoted by others (5-7), the subjects tolerated largedoses of thyroid well, although they manifestedweight loss, slight tremor, increased sweating andmild tachycardia. The highest PBI value pro-duced was 10.3 ,tg per 100 ml. Mean thyroxinedisappearance rate at the end of the experimentalperiod (after 20 grains of desiccated thyroid hadbeen administered daily for 1 week) was 69.2minutes. This was not significantly different fromthe euthyroid control group (p > 0.40) or fromthe results in the same subjects before treatmentwith desiccated thyroid. None of the subjects hadchanges in disappearance rate falling outside the95 per cent confidence limits for replicate studiesin untreated subjects.

Myxederna and treated hyperthyroidism. Meanhalf-time of acute thyroxine disappearance forseven subjects with spontaneous, primary myx-edema was 110.4 minutes, a highly significant dif-ference from the control group (p < 0.001).When five subjects with Graves' disease werestudied after they had become profoundly myx-edematous from treatment, their thyroxine disap-pearance rate (mean half-time of 76.0 minutes)was very significantly longer (p < 0.001) than inhyperthyroid subjects with Graves' disease (meanhalf-time of 55.1 minutes), but not significantlydifferent from that of euthyroid controls. Yet,interestingly, the disappearance half-time of 76.0minutes was significantly shorter than in thosewith spontaneous myxedema (mean 110.4 min-utes). Thus, although the development of myx-

TABLE III

Indices of hepatic function in subjects with liver disease

Acutethyroxine

BSP Pro- disap-reten- throm- pearancetion at bin rate45 min Serum time Choles- Half-

Subject Age Sex Race Ascites albumin Hgb. terol PBI time

yrs 7C g% sec g mg% 'Ug% minL.T. 43 M Cau. + 40.6 1.91 12.7 9.5 140 4.3 127.6A.W. 46 F N. 0 34.8 1.84 16.4 8.5 348 5.6 135.0J.E. 44 M Cau. 0 31.4 3.15 19.0 10.0 100 95.9J.H.* 24 M Cau. 0 t 3.90 13.4 14.0 215 7.8 111.9O.T. 42 M Cau. + 42.8 3.60 13.4 7.0 308 273.7M.W. 67 M Cau. 0 41.0 2.42 15.6 9.0 130 2.6 111.5F.T. 48 M Cau. 0 12.4 15.0 3.3 116.7R.L. 42 M Cau. + t 2.30 24.6 10.5 133 6.4 250.9M.B. 44 F Catu. + t 2.20 28.5 9.0 138 248.8

* Acute infectious hepatitis,t Icteric,

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EDWARDJ. LENNON, NORMANH. ENGBRINGAND WILLIAM W. ENGSTROM

edema slows the disappearance rate, a specific dif-ference in handling of labeled thyroxine seems toexist in Graves' disease even when functional thy-roid tissue is absent, or virtually so.

Euthyroid Graves' disease. Four subjects withGraves' disease were studied when they were eu-thyroid after treatment. One of these (V.H.) 'wasmaintained on 3 grains of desiccated thyroid dailybecause of moderately severe exophthalmos (theelevated PBI noted in this case was attributed toextraneous iodine ingestion). Subjects had beeneuthyroid for from 3 months to 2 years. Thegroup mean was 64.2 minutes and differed fromthe control group at marginal significance level(p < 0.05).

DISCUSSION

The early phases of disappearance of intrave-nously injected T4* from the circulation have notbeen studied in detail. Limited data from othersources (3, 8) and observations during the courseof the present studies implicate a complex seriesof physiological events, many of which proceedsimultaneously. These events include: a) mixingof T4* within the intravascular compartment withalmost immediate binding to the plasma proteins;b) distribution throughout the extrathyroidal thy-roxine pool with intravascular-extravascular com-petition for T4*; c) specific trapping of thyroxineby certain organs, particularly the liver; d) dis-posal of T4* from the body via the gastrointestinaltract, by excretion of small amounts in the urine(8) and by metabolic degradation with subse-quent appearance of free 1131 in the blood, the thy-roid and the urine. In the present studies, cover-ing only a 30-minute period, it is unlikely that deg-radation of T4* is significant since the total turn-over of endogenous thyroxine does not appear toexceed 30 per cent per day even in hyperthyroidstates (9). The present studies are believed toreflect primarily the events of b and c above,namely, the early distribution and specific trappingof thyroxine.

The half-time value determined from decreas-ing blood levels of radioactivity in each case re-flects the rate at which equilibrium between la-beled and stable thyroxine is being approachedwithin the body. This process of equilibration isoccurring at different rates between the intra-vascular and the many extravascular compart-

ments of the body. The method of measurementemployed in the present studies fails to distinguishbetween these various rates, but reflects them col-lectively. The technique intends only to estimatethe balance existing between forces regulating therate of removal of T4* from the circulation. Al-though the calculated value does not represent atrue rate constant, the results obtained were re-producible among groups of euthyroid individualsand among individuals with the various patho-logical states noted, and the results were statisti-cally different between certain groups.

Factors which influence the acute rate of dis-appearance of a tracer amount of T4* from theblood include: a) the relative size of intra- andextravascular thyroxine pools (e.g., a large in-travascular pool of thyroxine would slow the rateof disappearance); b) the relative thyroxine-binding affinity and saturation of intra- and ex-travascular proteins (e.g., increased intravascularthyroxine-binding capacity would be associatedwith a slower rate of removal of T4*); c) the in-tegrity of the vascular lining which prevents ex-travasation of protein-bound T4*; and d) theability of certain organs specifically to concentrateprotein-bound or free T4*.

The relationship of intravascular protein bindingof thyroxine to thyroidal economy in normal aswell as in pathological states has received consid-erable attention. In vitro measurements of thy-roxine-binding capacity permit comparison of thebinding affinity of plasma proteins in normal andabnormal states under the conditions imposed bythe technique of measurement. The effectivenesswith which plasma proteins retain thyroxine withinthe circulation of intact subjects, however, mustbe equally dependent upon the relative bindingaffinity of extravascular proteins as well as uponthe other factors mentioned above. The presentstudies suggest a reasonably good correlation be-tween the rate of removal of thyroxine from theblood and in vitro estimates of plasma-binding ca-pacity. In circumstances reported to exhibit in-creased thyroxine-binding capacity of the serumproteins, a delay in the acute disappearance ofthyroxine from the circulation was observed, i.e.,in seven patients with primary myxedema, in asingle subject with acute hepatitis, and in sub-jects given diethylstilbestrol (unpublished ob-servations).

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The findings in subjects with various types ofhyperthyroidism are of particular interest. Sub-jects with Graves' disease, at all levels of thyroidfunction, had more rapid thyroxine disappearancerates than other subjects with comparable levelsof metabolic activity. While it is clear, in com-paring hyperthyroid, euthyroid, and hypothyroidsubjects with Graves' disease, that hyperthyroid-ism has some influence on the function measured,the failure to find significant similar accelerationin subjects with toxic nodular goiter or in thyro-toxicosis medicamentosa suggests a more specificabnormality in Graves' disease. It may be arguedthat subjects with Graves' disease simply have alarger extravascular pool of thyroxine or thatthere was greater saturation of binding proteins(since PBI levels were somewhat higher in theGraves' group). However, the persistence of ac-celerated disappearance rate, in relation to otherswith comparable metabolic status, in Graves' sub-jects made hypothyroid, and probably also inthose made euthyroid, invalidates this argument.Also, it is likely that those individuals with toxicnodular goiter and thyrotoxicosis medicamentosahad ample time to expand their extravascular thy-roxine pools to a degree comparable to hyperthy-roid Graves' subjects.

The data support the concept of a qualitative,biological difference between Graves' disease andtoxic nodular goiter and suggest altered in vivoplasma or possibly even tissue protein binding ofthyroxine in Graves' disease. Indeed, the abnor-mality is apparent when these patients have be-come myxedematous after treatment. In a pre-liminary report, Richards, Dowling and Ingbar(10) noted that thyroxine-binding by prealbuminis usually decreased in untreated thyrotoxic pa-tients with Graves' disease, but a similar defect insubjects ill with other diseases was also observed.It may be that defective prealbumin binding ofplasma in Graves' disease plays a significant rolein the phenomena observed in the acute thyroxinedisappearance rate. Our data and those of othersdo not exclude the possible importance of extra-vascular forces. Since we have not observed theacute thyroxine disappearance rate to be accele-rated in illnesses other than Graves' disease, thedefective prealbumin binding associated with somenonspecific illnesses may in turn be balanced by acomparable decrease in extravascular thyroxine-

binding capacity. Certainly, patients with non-specific illnesses do not exhibit signs of hyper-thyroidism.

Our observations on acute thyroxine disap-pearance rate in Graves' disease are consonantwith the increased thyroxine turnover rates insubjects with treated Graves' disease (11) andwith the finding that heterologous serum labeledwith T4* disappears from the circulation in ac-cordance with the recipient's thyroid status ratherthan the donor's (12). Also, Hamolsky, Ellisonand Freedberg have shown that labeled thyroxineis more avidly incorporated by guinea pig dia-phragm in vitro from sera of patients with Graves'disease than from sera of subjects with toxicnodular goiter or sera from normals enriched invitro to hyperthyroid levels of PBI (13). Bycontrast, they found that labeled thyroxine mixedwith sera from subjects with Graves' disease disap-peared more rapidly from the circulation of re-cipient dogs than when mixed with sera fromnormals or subjects with toxic nodular goiter.This finding is not easily interpreted withoutknowledge of the relative binding affinity for thy-roxine of dog and human serum or of dog's ex-travascular proteins. Their findings may reflectpoorer thyroxine binding by Graves' plasma andrepresent distributional phenomena rather thanperipheral metabolic factors.

The finding of an actual, although slight, pro-longation of thyroxine disappearance rate in sub-jects with toxic nodular goiter may be due to ex-pansion of intravascular pool size out of propor-tion to increase in turnover. It is equally pos-sible that this prolongation is related to the in-creased age (although other older patients did notshare this finding) or to minor impairment of he-patic function (clinically not apparent).

The acute thyroxine disappearance rate wasconsistently and markedly prolonged in subjectswith cirrhosis of the liver; in most instances thedisappearance rates were even longer than in pa-tients with spontaneous myxedema. While in-creased binding of thyroxine by plasma globulinshas been described in hepatitis where the PBI isoften elevated (14), this seems unlikely in Laen-nec's cirrhosis. In the latter, the PBI is usuallynormal or even low, and increased red cell uptakeof triiodothyronine in vitro has been reported(15). The latter finding has been attributed to

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EDWARDJ. LENNON, NORMANH. ENGBRINGAND WILLIAM W. ENGSTROM

decreased binding affinity by the serum proteinsin this condition. If such is the case in most in-stances of cirrhosis, it would then seem that in-travascular factors (e.g., the ability of the plasmaproteins to bind thyroxine) are of much less im-portance than are extravascular phenomena ineffecting the acute thyroxine disappearance ratein liver disease. Thus, the liver may play a moreimportant role in the handling and metabolism ofthyroxine than has generally been appreciated.Preliminary observations during these studies andobservations by others (8) indicate that thyroxinerapidly accumulates in the liver. Since fecal ex-cretion of organic iodine is small (9, 16), thismust indicate that: a) thyroxine trapped by theliver is excreted in the bile and reabsorbed via theenterohepatic circulation; b) thyroxine is releasedto the circulation after hepatic trapping, free or inbound form; or c) the liver stores large amountsof thyroxine for its own metabolic needs. Quan-titative data are not available to decide which ofthe possibilities might predominate in affecting theacute handling of thyroxine.

The results of the present studies are very simi-lar to those recently reported by Peterson andPierce (17) regarding the disappearance of cor-tisol, cortisone and corticosterone from the in-travascular space. Alterations from normal inthe rates of disappearance of these steroids werenoted in cirrhosis, myxedema, thyrotoxicosis 2 andafter administration of estrogen to normal sub-jects. These qualitative changes were similar tothose we have reported for I131-thyroxine.

SUMMARYANDCONCLUSIONS

1. A method is described for the determinationof the rate at which equilibrium between intra-and extravascular thyroxine pools is approachedafter intravenous administration of labeled thy-roxine. This measurement, termed the "acutethyroxine disappearance rate" is accomplishedduring a 30-minute period beginning 20 minutesafter the intravenous injection of I131-labeledL-thyroxine.

2. Subjects with Graves' disease were found tohave shorter acute thyroxine disappearance rates

2 By personal communication, we are informed thatall of their patients with "thyrotoxicosis" suffered fromGraves' disease.

at all levels of thyroid function than other sub-jects with comparable thyroidal activity. Evenwhen functionally athyrotic, the patients withtreated Graves' disease handled thyroxine differ-ently from athyrotic subjects without Graves'disease. This observation suggests that plasma orextravascular protein binding of thyroxine in sub-jects with Graves' disease may be altered in vivoand that this alteration is not the direct result ofhypermetabolism or hyperthyroidism.

3. No significant shortening of thyroxine disap-pearance rate was noted in patients with toxicnodular goiter or in normals fed exogenous thy-roid. This finding indicates a qualitative differ-ence between the toxic nodular goiter and Graves'disease. These observations cannot be accountedfor by differences in the level of the PBI or deriva-tives of this measurement, since the thyroxinedisappearance rate in Graves' disease remainedrelatively rapid after treatment of hyperthyroidism.

4. Greatly prolonged thyroxine disappearancerates in subjects with liver disease suggest that theliver plays an important role in the handling ofthyroxine.

5. Replicate experiments indicate that the methodof study is reproducible and applicable to the studyof drug and hormone effects on the rate of egressof labeled hormone from the circulation.

ACKNOWLEDGMENT

It is a pleasure to acknowledge the technical assistanceof Miss Virginia Griffin and Mrs. Marian Wisniewskiand to thank Miss Griffin for reviewing the statisticalanalyses.

REFERENCES

1. Freinkel, N., Ingbar, S. H., and Dowling, J. T. Theinfluence of extracellular thyroxine-binding pro-tein upon the accumulation of thyroxine by tissueslices. J. clin. Invest. 1957, 36, 25.

2. Tata, J. R. A cellular thyroxine-binding proteinfraction. Biochim. biophys. Acta 1958, 28, 91.

3. Haddad, H. M. Rates of I'31-labeled thyroxine me-

tabolism in euthyroid children. J. clin. Invest.1960, 39, 1590.

4. Snedecor, G. W. Statistical Methods Applied toExperiments in Agriculture and Biology, 5th ed.Ames, Iowa State College Press, 1956.

5. Johnston, M. W., Squires, A. H., and Farquharson,R. F. The effect of prolonged administration ofthyroid. Ann. intern. Med. 1951, 35, 1008.

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6. Goldsmith, R., and Stanbury, J. B. The tolerance ofpatients with myxedema for thyroid. J. clin. En-docr. 1955, 15, 568.

7. Danowski, T. S., Huff, S. J., Tarail, R., Wirth, P.,Peters, J. H., Mateer, F. M., and Garver, K. Se-rum protein-bound iodine during ingestion ofdesiccated thyroid. J. clin. Endocr. 1952, 12, 1572.

8. Benua, R. S., Albert, A., and Keating, F. R., Jr.The metabolism of radiothyroxine in exophthalmicgoiter. J. clin. Endocr. 1952, 12, 1461.

9. Ingbar, S. H., and Freinkel, N. Simultaneous esti-mation of rates of thyroxine degradation and thy-roid hormone synthesis. J. clin. Invest. 1955, 34,808.

10. Richards, J. B., Dowling, J. T., and Ingbar, S. H.Alterations in the plasma transport of thyroxinein sick patients and their relation to the abnor-mality in Graves' disease (abstract). J. clin. In-vest. 1959, 38, 1035.

11. Ingbar, S. H., and Freinkel, N. Studies of thyroidfunction and the peripheral metabolism of I131-la-beled thyroxine in patients with treated Graves'disease. J. clin. Invest. 1958, 37, 1603.

12. Levy, R. P., Kelly, L. W., Jr., Cooper, G. W.,and Jefferies, W. McK. The volume of distri-bution and turnover of endogenously labelled hu-

man thyroid hormone from euthyroid and hyper-thyroid donors (abstract). J. clin. Invest. 1955,34, 948.

13. Hamolsky, M. W., Ellison, H. E., and Freedberg,A. S. The thyroid hormone-plasma protein com-

plex in man. I. Differences in different states ofthyroid function. J. clin. Invest. 1957, 36, 1486.

14. Vannotti, A., and Beraud, T. Functional relation-ships between the liver, the thyroxine-binding pro-tein of serum, and the thyroid. J. clin. Endocr.1959, 19, 466.

15. Hamolsky, M. W., Golodetz, A., and Freedberg, A. S.The plasma protein-thyroid hormone complex inman. III. Further studies on the use of the invZitro red blood cell uptake of I31-/-triiodothyronineas a diagnostic test of thyroid function. J. clin.Endocr. 1959, 19, 103.

16. Berson, S. A., and Yalow, R. S. Quantitative aspectsof iodine metabolism. The exchangeable organiciodine pool, and the rates of thyroidal secretion,peripheral degradation and fecal excretion of en-

dogenously synthesized organically bound iodine.J. clin. Invest. 1954, 33, 1533.

17. Peterson, R. E., and Pierce, C. E. The metabolismof corticosterone in man. J. clin. Invest. 1960, 39,741.

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