The level of free thyroxine (T4) in the blood isan important correlate of an individual's thyroidstatus. Evidence for this concept has been recentlyreviewed (1 ) . The association constants for thebinding of the hormone to its specificcarrier proteinsin plasma are such that approximately 1 molecule ofT4 lfl 2,000 is in the free state (1 ) . In recent yearsseveral methods have been developed to estimatethe free-thyroxine fraction (F T4 F), i.e., the percent of total serum T4 that exists in the free form.All these methods involve some procedure to separate “free―and “bound―fractions of labeled T4added to the serum. In some of these techniquesequilibrium dialysis is used (2—4); in others, ultrafiltration (5), charcoal particles (6) or columns ofDextran gel (Sephadex) (7) are used.

In an attempt to improve the existing methods ofestimating F T4 F we have developed a simple, inexpensive technique suitable for routine use in adiagnostic laboratory. The principle of our methoddepends on the competition for labeled T4 betweenthyroxine-binding plasma proteins and Sephadex.

MATERIALS AND METHODS

Buffer. Potassium phosphate buffer, hereafter referred to as buffer, is made from 0.075 M K2HPO4(405 ml) and 0.075 M KH2PO4 (95 ml) dilutedto 1,000 ml with water. The final pH should be7.40 ±.02.

“Workingsolution―of labeled thyroxine. ‘311-L-thyroxine is obtained every 2 weeks from a commercial supplier with a specific activity from 30 to40 mCi/mg. A working solution was prepared asfollows: approximately 0.5 ml of 1311-T4 solutionis added to 2.5 ml of 2% serum albumin in phosphate buffer. In place of albumin, 1% serum (0.025ml serum to 2.5 ml buffer) can be used here. Theprotein is necessary to prevent losses of T4 by absorption to surfaces. The exact volume of 1311-T4solution used in preparing this working solution iscalculated from the concentration of carrier T4 inthe original shipment, so as to yield a concentration

of approximately 1@ T4/ml. The working solutionis kept at 4°Cin the dark and should be made freshevery week. The ‘251-T4can also be used in thismethod.

Sephadex G-25, coarse grade, is obtained from acommercial source (Pharmacia Fine Chemicals, Piscathaway, N.J.). Exactly 0.20-gm portions areplaced into dry, disposable plastic tubes, 16 X 125mm. A precalibrated plastic spoon which delivers0.20 gm Sephadex is recommended for this purpose.See Ref 17 for a description of the spoon which wehave found satisfactory. This type of spoon delivers0.2-gm Sephadex with 1% reproducibility.

Control serum. A pool of sera obtained from theClinical Laboratory was used for quality control.One-milliliter aliquots of this pool were kept frozenuntil used.

PROCEDURE

To 1 ml of the serum to be tested add 20@ ofthe working solution containing labeled T4. This stepresults in the addition of 0.02 @gT4/ml serum. Two0. 1-mi aliquots of the labeled T4-serum mixture arediluted 32-fold by adding 3. 1-ml phosphate buffer.An automatic diluting device is recommended atthis step. The diluted sample is added quantitativelyto 0.20 gm Sephadex, premeasured in plastic tubes.The tube is agitated on a Vortex ‘mixerfor at least10 sec. The exact duration of mixing is not critical,but should be the same for each sample. The tubeis set aside to allow the Sephadex to settle (about ½mm). Approximately ¾of the supernatant is removed by aspiration and saved (“initial supernatant―) . The sedimented Sephadex particles arewashed with 3 ml of buffer, agitated for 5—10secand allowed to settle. About ¾of the supernatantis removed by aspiration and discarded. The washing

Received Sept. 10, 1968; revision accepted March 28,1969.

For reprints contact: Ralph R. Cavalieri, Veterans Administration Hospital, 4150 Clement St., San Francisco, Calif.94121.

565Volume 10, Number 9

A SIMPLIFIED METHOD FOR ESTIMATING

FREE-THYROXINE FRACTION IN SERUM

RalphR. Cavalieri,JamesN. Castleand Gilbert 1. Searle

Veterans Administration Hospital, San Francisco, California

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CAVALIER!, CASTLE AND SEARLE

procedure is repeated four times (total of fivewashes). Following the final wash, the particles adhering to the side of the tube are washed to thebottom by addition of 1 ml buffer. Both the washedSephadex and exactly 1 ml of the “initialsupernatant―are counted in a well counter.

In a typical run using normal serum, the countingrate in the Sephadex is approximately 3% of thecounting rate in 1 ml of the initial supernatant. Ina typical case, the Sephadex counting rate was 292cpm (net), and 1 ml of the supernate yielded 8,488cpm (net). Duplicates should agree within ±5%of the mean. To maintain uniformity, the results areexpressed as a percent of the value obtained in thecontrol serum. Thus

Patient cpm Sephadex/cpm 1 ml SupernatantControl: cpm Sephadex/cpm 1 ml Supernatant

X 100=FT4F.The normalized free T4 fraction (F T4 F) in percentof the control value is used to calculate the free T4concentration index: F T4 F (% of control) X T4iodine (,@g/100 ml) = Free T4 Conc. Index wherethe T4 iodine is determined by chemical analysisfollowing separation on an ion-exchange column

(8). The serum PBI can also be used at this pointas an approximation of total T4 iodine. No significance should be attached to the units in which freeT4 concentration is expressed (hence an “index―).

RESULTS

The effects of several variables were tested. Increasing the quantity of Sephadex from 0.07 to 0.20gm resulted in a linear increase in the proportionof labeled T4 taken up (Fig. 1A). Quantities ofSephadex greater than 0.2 gm gave proportionatelyhigher uptake values. Although the absolute quantityof Sephadex added is important, it is more essentialto add exactly the same amount to each tube becausethe final results for F T4 F are expressed in percentof the control value.

The effect of varying the number of washes isshown in Fig. lB. The first two or three washes removes most of the protein-bound radioactivity. Increasing the number of washes beyond five results inonly a slight decrease in the percent of label associated with Sephadex. For routine use, five washesproved to be adequate.

The duration of exposure to Sephadex was foundnot to be critical. As shown in Fig. 2A, only a

6.0varying the amount ofsephadex

varying the number ofwashes

w

@ 4.0a.

I—zwUa:wa. 2.0

1.0

.1 .2 I 3 5 7 9A GRAMS SEPHADEX B NUMBER OF WASHES

FIG. 1. A givesproportionof labeledT@(ml) takenup by tidsandall otherflguresdatapointsindicatemeanandbracketsSephadex as function of quantity of Sephadex added. Normal show ±1 s.d. of five determinations. S gives @uptake―of ml.T4 byserum W05 diluted 1:32. Sephadex was washed 6ve times as de- Sephadex (0.20 gm) as function of number of washes followingscribed in Methods. All other variables were kept constant. In 10-sec exposure. Normal serum (1:32) was used.

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ESTIMATING FREE-THYROXINE FRACTION IN SERUM

effect of duration of exposureto sephadex @effect of temperature

2.0

Ui

a.

I—zwl.O0

a:Ui

0.5

2.0

1.0

0.5

I 234 5lB° 25°370ATIME(minutes) B TEMP (C.)

FIG. 2. A gives“uptake―of ml@T4by S.phadex(0.20gm)as function of duration of exposure before washing. Tubes contaming mixture of Sephadex, labeled T4 and dilute serum wereagitated continuously for period indicated. Normal serum (@:32),

negligible increase in Sephadex uptake occurredwhen the duration of exposure before the initial washwas increased from 1 to 5 mm. In other experimentsit was determined that mixing for 10 sec yielded thesame value as mixing for 1 min. It is apparent,therefore, that equilibration of distribution of labeled T4 between protein and Sephadex is attainedrapidly and is virtually complete within 10 sec.

Temperature affects the uptake of T4 by Sephadex(Fig. 2B). In the temperature range between 18°and 37°C, uptake increased linearly by 0.05% foreach degree rise in ambient temperature. Thereforewide fluctuations in temperature during the test areto be avoided. The inclusion of a reference controlserum with each run helps to avoid errors due today-to-day variations in room temperature.

The reproducibility of the method was evaluatedby periodic testing of aliquots of a pool of normalsera stored frozen. In 17 determinations over aperiod of 5 weeks using three lots of 1311-T4,uptakeby Sephadex as a percent of the counting rate in1 ml of supernate ranged from 2.82 to 3.62%.(Mean = 3.28 ± 0.24 s.d.)

The effect of radioiodide which is usually presentas a contaminant in 1811-T4or ‘25I-T4was tested.In three separate experiments, in which radioiodide

five washes of Sephadex. B shows effect of temperature on Sepha.dcx “uptake―of ‘@l.T4using normal serum (1:32) with 10-sec cxposure and five washes. Uptake increased linearly by 0.05% foreach degree rise in ambient temperature.

was added to diluted serum and the procedure wascarried through five washes, the radioiodide uptakeby Sephadex averaged only one-fifth of the uptakeof labeled T4. Since in all lots of labeled T4 testedwithin 2 weeks after arrival, the content of radioiodide never exceeded 10% of total radioactivity(usually 3—8%), then the error from the source willbe less than 2%.

Organic radioiodinated impurities which have beendetected in commercial preparations of 131I-T4 canbe removed by dialysis in the presence of serum(5,9) . We tested three lots of 131I-T4by determiningthe Sephadex uptake from normal (pooled) serumbefore and after dialysis of 131I-T4as recommendedby Schussler and Plager (5) . The results were notsignificantly affected by this dialysis. Therefore, preliminary purification of ‘31I-T4appears unnecessaryprovided the preparation is used within 2 weeksafter it is received.

The sensitivity of the method was tested by adding unlabeled T4 to normal serum. As the concentration of total (labeled plus unlabeled) T4 increases,more T4-binding sites on serum proteins are occupied and an increasing proportion of T4 is free andavailable for uptake by Sephadex. The results, givenin Fig. 3, show that Sephadex uptake does in fact

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FT4FTotalT@FreeT4(%ofiodineconc.Clinical

Status control)(@g/100 ml)index

80—120 3.0—7.0 3.0—7.0

CAVALIER!, CASTLE AND SEARLE

more, the range within each group was narrower bythe present method, indicating improved precision.

Free T4 fraction and free T4 concentration index:Table 1 lists the results of determining the F T4 Fby the Sephadex method, total serum T4 iodine by acolumn chromatographic method and the free T4concentration index calculated from these values inhyperthyroid and hypothyroid patients and in 5everal clinically euthyroid individuals selected for abnormalities in serum T4-binding proteins. In thehyperthyroid group (diagnosis made by clinicalexamination and by ‘@‘Ithyroid uptake) the F T4 Franged from 121 to 159 % of control, the total T4iodine values were above 9 @g/100 ml and the calculated free T4 concentration index ranged from13.3 to 19.5. In hypothyroid patients (clinical diagnosis) the F T4 F was below 75% in five of the sixpatients, total T4 iodine was 3.0 or lower in all andcalculated free T4 concentration index was 1.8 orless. The euthyroid group showed extreme variationsin F T4 F and total T4 iodine ranging from low tovery high values. In all of these patients the free T4conc. index ranged from 3.7 to 6.8, within the norma! range and consistent with the clinically euthyroidstate of these individuals.

Q)0'C0U

CQ)U

0.

0.10 020

pgm 14 added per ml. serum

FIG. 3. Effectof addedunlabeledL-T4onSephadexuptakeof“@l-T4at various dilutions of serum (normal pool). L-T4was addedto serum before dilution with buffer. Ordinate gives value ob.tamed as percent of control value obtained with @l.T4but noadditional unlabeled T4.

serum free thyroxine fraction by two methods

200

@ 60a:I-

wo0 U 20<LLI0WI(n@ 80

Ua:Wa. 40

TABLE 1. FREET4 FRACTION (F T4 F), TOTALT4 IODINE AND FREET4 CONCENTRATION

INDEX IN PATIENTS WITH THYROID DISEASESAND IN EUTHYROID PATIENTS WITH VARIOUS

ABNORMALITIES IN T4-BINDING PROTEINS40 80@ 20 60 200 240PERCENT OF CONTROL

DIALYSIS

Normal rangeHyperthyroid

AA 145 9.2 13.3RT 121 16.1 19.5RC 126 12.2 15.4JR 121 12.6 15.2BH 142 10.0 14.2BC 159 11.8 18.8

HypothyroidRG 75 2.0 1.5CD 60 3.0 1.8BMcG 64 1.0 0.6NB 71 2.2 1.5HS 93 1.0 0.9BF 70 1.5 1.0

EuthyroidBB Oral contraceptive

(high TBG') 58 10.2 5.9DRViral hepatitis

(highTBG) 55 7.2 4.0VC Idiopathic(low TBG) 169 2.2 33MZCirrhosis(lowTBG) 203 1.9 3.9

* @180= Serumthyroxine-bindingglobulin(capacity).

568 JOURNAL OF NUCLEAR MEDICINE

x

xx

@ __________________ SeuthyroidI ° @J * thyrotoxic6 °0 0 hypothyroid

FIG. 4. Comparisonof resultsobtainedusingtwomethodsofestimating serum free thyroxine fraction (F T@F). On vertical axisis shown value by present Sephadex method. In both methodsresults are expressed as percent value obtained in control pooledserum.On horizontal axis is value obtained by equilibrium dialysismethod of Sterling and Brenner (4). Both tests were performedon serum obtained at same time from each patient. Boxes encloseranges in each group of patients.

increase with addition of unlabeled T4 to serum.The highest sensitivity to loading occurred when theserum was diluted 1 :32 with buffer. It was decidedtherefore to use this dilution routinely.

Comparison with equilibrium dialysis method: Todetermine the efficacy of this method over a widerange of values, sera from patients with hypothyroidism, thyrotoxicosis and from euthryroid individuals were tested using both the present method andthe equilibrium dialysis technique of Sterling andBrenner (4) . The results are shown in Fig. 4. Ingeneral, the values obtained by the two methodscorrelated well (correl. coeff. = +0.72). Further

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ESTIMATING FREE-THYROXINE FRACTION IN SERUM

in differentiating hyper- and hypothyroid from euthyroid individuals and especially in determining thyroidstatus in patients with alterations in binding proteins.The free T4 conc. index is calculated from the F T4 Fand the level of total T4. We routinely estimate thelatter from the chemical determination of T4 iodine(T4 by column) or the PBI. Alternatively, the technique of displacement analysis developed by Murphyand Pattee (13,14) can be used to measure total T4.

In the case of many other tests of thyroid hormonebinding, results must be interpreted with caution inpatients taking drugs known to affect T4-binding.

Diphenylhydantoin (Dilantin—Parke Davis and Co.,Detroit, Mich.) and salicylates, the latter in dosesgreater than 3 gm/day, competitively inhibit thebinding of T4 to plasma proteins (15) . As shownpreviously by Chin and Schussler (16) using anultrafiltration method, F T4 F tends to be elevatedby the addition of diphenylhydantoin when undilutedserum is tested but remains relatively unchanged ina system containing dilute serum. Using our presentSephadexmethod (serum diluted 1:32) we have alsofound that F T4 F is insensitive to addition ofdiphenyihydantoin at therapeutic levels of the drug.We have not yet tested the effects of salicylates inour system, but on theoretical grounds the resultsshould be relatively unaffected by the presence ofthis drug.

The test is useful in evaluation of patients takingthyroid hormones as replacement therapy, dependingon the preparation of hormone. In patients takingdesiccated thyroid, the F T4 F, total T4 and free T4concentration index are usually within the normalrange if the patient is euthyroid. In the case of individuals on adequate L-thyroxine replacement therapy, all three parameters tend to be high-normal. Inpatients taking L-triiodothyronine, however, all threevalues are below normal and these tests are thereforenot diagnostically useful.

Further clinical experience is needed before all ofthe advantages and limitations of this test are known.

SUMMARY

A method has been developed for estimating relative changes in free T4 fraction (F T4 F) in serum.The principle involves the partition of added labeledT4 between binding sites on specific proteins andsites on Dextran gel (Sephadex) . By using diluteserum and rapidly sedimenting particles of Sephadex,the method avoids the necessity for prolonged incubation and centrifugation. The Sephadex “uptake―of labeled T4 from patient's serum is expressed as apercent of the uptake from a control serum. Theresults correlate well with those obtained by equilibrium dialysis over a wide range of thyroid states.

DISCUSSION

Each of the various methods already available fordetermination of the F T4 F in serum has disadvantages limiting its usefulness as a routine diagnostic test. The techniques involving equilibrium dialysis(2—4), ultraffitration (5) or charcoal adsorption(6) require prolonged incubation or repeated centrifugation. The present method was specificallydesigned to avoid such drawbacks without sacrificingsensitivity.

In principle the method involves the competitivebinding of labeled hormone between specific sites onserum proteins and Dextran gel. A technique basedOn the same principle using columns of Sephadex

has already been described (7) but has not receivedwidespread application. In the present method thedisadvantages of the column technique have beenavoided. Our results indicate that within the rangetested the “uptake―of labeled T4 by Sephadex partides @5proportional to the F T4 F and that distribution equilibrium of the hormone between proteinand Sephadex is achievedwithin a few seconds. Thustwo important requirements, sensitivity and speed,have been met.

It must be emphasized that the present methodprovides a relative, not an absolute, estimate ofF T4 F. The “uptake―of labeled T4 by Sephadexdepends on several factors, including the amount ofSephadex added, the number of washes, the ambienttemperatures, etc. We do not consider it a drawback of the method that it does not give an absolutevalue for F T4 F because, as has been pointed outby Oppenheimer (10), it is doubtful that any available in vitro method yields a truly reliable andabsolute estimate of F T4 F in the circulation. Theclinical usefulness of the test depends on its abilityto reflect relative changes in F T4 F under variousphysiologic or pathologic conditions. The resultsshow that our method meets this requirement.

The question arises, what advantage if any doesthe determination of F T4 F offer over the oldertests involving T3 uptake by red cells or resin?From a priori considerations one would expectF T3 F (or T3-uptake) to be proportional to F T4 F.However, since TBPA binds T4 but not T3 (11),the T3 uptake only indirectly reflects overall binding of T4 by plasma proteins. Now with a simple,rapid technique for F T4 F, it is no longer necessary to depend on the use of T3 tests to estimateF T4 F for routine diagnostic purposes.

The importance of the free-T4 concentration as acorrelate of thyroid status has already been mentioned. Our experience and that of others (3,4,12)provide ample evidence that the free T4 conc. indexis superior to either F T4 F or total T4 values alone

569Volume 10, Number 9

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CAVALIERI, CASTLE AND SEARLE

The normalized F T4 F, obtained by this method, isused together with the total T4 value to calculate anindex of free T4 concentration. The latter is considered to be the parameter which best reflects thelevel of thyroid function.

ACKNOWLEDGMENTS

The authors are grateful to Paul R. Jensen and MissPatricia Lazarus for their cooperation in obtaining samplesof serum and the results of chemical thyroxine analyses.The expert technical assistance of Mrs. Suzanne Embertonis greatly appreciated.

REFERENCES

1. ROBBINS, J. *Nn RALL, J. E.: The iodine-containinghormones.In Hormones in Blood, 2nd ed., vol. 1, Gray, C.H. and Bacharach, A. L., eds., Academic Press, New York,1967, p. 383.

2. OPPENHEIMER, J. H. AND SURKS, M. I. : Determination of free thyroxine in human serum : A theoretical andexperimental analysis. I. Clin. Endocrinol. 24:785, 1964.

3. INGBAR, S. H., BRAVERMAN, L. E., DAwai@it, N. A.AND LEE, G. Y. : A new method for measuring the free

thyroid hormone in human serum and an analysis of thefactors that influence its concentration. I. Clin. Invest.44:1,679, 1965.

4. SmauNG, K. AND BRENNER, M. A. : Free thyroxinein human serum; Simplified measurement with the aid ofmagnesium precipitation. I. Clin. Invest. 45 :153, 1966.

5. SCHUSSLER, G. C. AND PLAGER, J. E. : Effect of preliminary purification of @9-thyroxine on the determinationof free thyroxine in serum. 1. Clin. Endocrinol. 27:242,1967.

6. KUMAGAI, L. F., JUBIZ, W. AND JESSOP, L. D.: A sim

ple and rapid method for determining free thyroxine bycharcoal absorption. Clin. Research 15:124, 1967.

7. LEE, N. D., HENRY, R. J. AND GOLUB, 0. J.: Dc

termination of free thyroxine content in serum. I. Clin.Endocrinol. 24:486, 1964.

8. PELLEGI, V. I., LEE, N. D., GOLUB, 0. 1. AND HnNRY,R. J. : Determination of iodine compounds in serum. I.Serum thyroxine in the presence of some iodine contaminants. I. Clin. Endocrinol. 21 : 1,272, 1961.

9. VOLPERT, E. M., MARTINEZ,M. AND OPPENHEIMER,J. H. : Radioiodinated impurities in commercial preparations of 19-thyroxine and their effect on measurement offree thyroxine in human serum by equilibrium dialysis.I. Clin. Endocrinol. 27:421, 1967.

10. OPPENHEIMER,I. H. : Role of plasma proteins in thebinding, distribution and metabolism of the thyroid hormones. New Engi. I. Med. 278:1,153, 1968.

11. INOBAR, S. H. AND FREINKEL, N. : Regulation of theperipheral metabolism of the thyroid hormones. RecentProgr. Hormone Res. 16:353, 1960.

12. ARANGO, 0., MAYBERRY, W. E., HOCKERT, T. I. ANDELVEBACK, L. R. : Total and free human serum thyroxine

in normal and abnormal thyroid states. Mayo Clin. Proc.43:503,1968.

13. MURPHY, B. E. P. AND PATrEE, C. J. : Determinationof thyroxine utilizing the property of protein-binding. I.Clin. Endocrinol. 24 :187, 1964.

14. MURPHY, B. E. P. : The determination of thyroxineby competitive protein-binding analysis employing ananion-exchange resin and radiothyroxine. I. Lab. Clin. Med.66:161, 1965.

15. WOLFF, J., STANDAERT,M. E. AND RAIL, J. E. : Thyroxine displacement from serum proteins and depressionof serum protein-bound iodine by certain drugs. I. Clin.invest. 40:1,373, 1961.

16. CHIN, W. ANDSCHUSSLER,G. C.: Decreasedserumfree thyroxine concentration in patients treated with diphenyihydantoin. I. Clin. Endocrinol. 28:181, 1968.

17. Muiu@w, B. E. P. : Some studies of the proteinbinding of steroids and their application to the routine microand ultra micro measurement of various steroids in bodyfluids by competitive protein-binding radioassay. I. Clin.Endocrinol. 27:973, 1967.

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1969;10:565-570.J Nucl Med.   Ralph R. Cavalieri, James N. Castle and Gilbert L. Searle  A Simplified Method for Estimating Free-Thyroxine Fraction in Serum

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