CLINICAL AND PHYSIOLOGICAL OBSERVATIONSIN A PATIENTWITH AN IDIOPATHIC DECREASEIN THE THYROXINE-

BINDING GLOBULIN OF PLASMA*

By SIDNEY H. INGBARt(From the Thorndike Memorial Laboratory, Second and Fourth (Harvard) Medical Services,

Boston City Hospital and the Department of Medicine, Harvard Medical School,Boston, Mass.)

(Submitted for publication June 13, 1961; accepted August 4, 1961)

Since its discovery in 1952 (1), the thyroxine-binding globulin of plasma (TBG) has been thesubject of extensive investigation designed toelucidate its physicochemical characteristics, al-terations in abnormal states, and physiologicalfunction. Recent reviews have summarized avail-able knowledge in this area (2, 3).

The present report describes a male patientwhose plasma appears to be virtually devoid ofthyroxine binding by TBG. This unusual ab-normality, fortuitously discovered, afforded aunique opportunity to assess further the role ofTBG in regulating the peripheral metabolism ofthe thyroid hormone. A number of physiologicalstudies was therefore performed in this patient,and the results are reported herein. While thesestudies were in progress, clinical features of amale patient with a similar thyroxine-binding ab-normality were reported by Tanaka and Starr(4). It is likely that the physiological disturb-ances currently described were also present in theearlier case and that both patients are affected bythe same distinct, though rare, disorder in plasmaprotein metabolism.

CASE HISTORY

History. F.B., a 58 year old unmarried white male,was admitted to the Harvard Medical Service of the'Boston City Hospital on September 14, 1958. His chiefcomplaint at that time was increasingly severe exertionaldyspnea, orthopnea, and episodes of paroxysmal nocturnaldyspnea of 2 years' duration. He had taken digitalis ir-

* This investigation was supported in part by ResearchGrant A-267 from the National Institute of Arthritis andMetabolic Diseases, and in part by the Medical Researchand Development Board, Office of The Surgeon Gen-eral, Department of the Army, under Contract DA-49-007-MD-412. Read by title at the Annual Meeting ofthe American Society for Clinical Investigation, May 2,1960, Atlantic City, N. J.

t Investigator, Howard Hughes Medical Institute.

regularly during that period, but had had none during theyear prior to entry. The patient had been known to havea heart murmur since childhood. Symptoms of acuterheumatic fever were denied. The patient's only othercomplaint was weakness of the arms and legs of 2 year'sduration. This was especially prominent in the distalmusculature and was of moderate, but not incapacitating,severity.

Details of early development were not readily recalled,but pubertal maturation apparently began at about age 14.At age 16 a bilateral inguinal herniorrhaphy was per-formed. Subsequently, if not immediately thereafter,normal masculine psychosexual development ceased.Libido was minimal throughout the patient's life, al-though rare erections and nocturnal emissions occurred.Pubic and axillary hair remained scant, and balding didnot take place. The patient shaved infrequently.

There were no other symptoms referable to the endo-crine organs, including the thyroid, adrenals and pituitarygland, and no symptoms of an intracranial lesion. Re-view of systems was otherwise negative.

The patient's parents had died when he was a youngboy; he had no siblings. He knew of no other close rela-tives who were alive. The patient was raised by mem-bers of a religious order and was living and serving asjanitor in their institution at the time he entered thehospital.

Physical examination. T, 98.8°; P, 100; R, 20; BP,130/70. Salient features of the physical examinationwere confined to the cardiovascular, endocrine and neu-romuscular systems. Examination of the heart revealedcardiomegaly and a prominent precordial heave. Cardiacrhythm was regular. The pulmonic second sound wasincreased, split, and louder than the aortic second sound.The first sound in the mitral area was booming; therewas no opening snap. A grade 4, harsh systolic murmur,radiating widely, was audible over the entire precordium,but was heard best in the fourth left intercostal space inthe parasternal area.

Bilateral herniorrhaphy scars and reducible herniaewere present. Small, firm testes were felt in the scrotalsac. The phallus was normal in size, the prostate smalland firm. Pubic hair was scant, the escutcheon female.Only a few fine strands of axillary hair were present.The skin was fine and smooth, typical of that associatedwith hypogonadism. Skeletal proportions were not eu-

2053

SIDNEY H. INGBAR

nuchoid. There was a slight temporal calvietes, but noother balding.

There were no peripheral stigmata of hyper- or hypo-thyroidism. There were no pigmentary abnormalities.The eyes were highly myopic, but there were no cata-racts. Visual fields were normal to gross confrontation.

There was moderate weakness of the distal muscu-lature of the arms and legs with atrophy of the interos-seous muscles. No myotonia was evident. Deep tendonreflexes and sensory examination were normal.

Laboratory. The urine and blood were within normallimits. Lumbar puncture was normal. Serum chemis-tries included: urea nitrogen, 15 mg per 100 ml; glucose(fasting), 100 mg per 100 ml; calcium, 10.2 mg per 100ml; phosphorus, 3.1 mg per 100 ml; cholesterol, 210 mgper 100 ml; sodium, 138 mEq per L; potassium, 4.1 mEqper L; chloride, 104 mEq per L; C02, 25.6 mEq per L.Serum iron concentration was 110 Ag per 100 ml andiron-binding capacity 320 Ag per 100 ml, both withinnormal limits.

Skull films were normal. A bone survey revealed noabnormalities; the epiphyses of the iliac crests wereclosed. Visual fields were within normal limits. Twenty-four hour urinary excretion of 17-ketosteroids was 9.9mg (5), of 17-hydroxycorticoids, 4.3 mg (6).1 Urinarygonadotropins were positive at 50 and equivocal at 100mouse units per 24 hours (7).2

Twenty-four hour thyroidal uptake of I131 was 28 percent. BMR's ranged between - 5 and + 4 per cent.Serum protein-bound iodine (PBI) was 2.0 ug per 100ml (8).

Course. The patient's cardiac complaints respondedwell to digitalization and a low salt diet. Mercurial di-uretics were not employed. Cardiac catheterization stud-ies were interpreted as consistent with a persistent leftvena cava and atrial septal defect, probably of thesecundum type.

In view of the patient's obvious hypogonadism, a thor-ough endocrine evaluation was undertaken. The as-sociated subnormal PBI, repeatedly confirmed, stronglyraised the possibility of pituitary hypofunction, but thisdiagnosis seemed clearly excluded by the neurologicaland X-ray examinations and by other endocrinologicalfindings, especially the increased urinary excretion ofgonadotropins. The dissociation between the PBI andother laboratory, as well as clinical, indices of thyroidfunction raised the possibility of abnormal binding ofthyroxine in the patient's plasma. This hypothesis wasconfirmed by repeated electrophoretic studies of the pa-tient's serum, all of which revealed a virtual absence ofTBG.3

1 Performed at the Boston Medical Laboratory, Bos-ton, Mass. Normal values: 17-ketosteroids, 10 to 24 mgper 24 hours; 17-hydroxycorticoids, 1 to 10 mg per 24hours.

2 Performed at the Gynecological-EndocrinologicalLaboratory, Peter Bent Brigham Hospital, Boston, Mass.

3 In the present report, for purposes of brevity, achange in the thyroxine-binding activity of TBG will be

Studies of the peripheral turnover of IP3`-labeled thy-roxine were then performed. Several days after thesewere complete, the left inguinal hernia became incarcer-ated and emergency repair was performed. At this timethe testis was seen to be grossly atrophic and a testicularbiopsy was taken. In view of the muscular weakness, abiopsy of the anterior tibial muscle and overlying skinwas also made. The latter revealed no abnormality.Histologically, the testis was fibrotic, with atrophy andfibrous replacement of tubular elements and decrease ininterstitial cells, consistent with chronic restriction ofblood supply. Genetic sex was masculine, as assessed inboth testicular and cutaneous biopsies.

When the patient's convalescence was complete, oraladministration of diethylstilbestrol, 30 mg daily, was be-gun. This was continued for 7 weeks. During the lat-ter portion of this period, the patient developed increas-ing dyspnea, pretibial edema, marked areolar pigmenta-tion, and pronounced tender gynecomastia. Urinary ex-cretion of gonadotropin declined to less than 5 mouse Uper 24 hours. Electrophoretic studies of hormonal bind-ing in the patient's serum were performed at weekly in-tervals during the administration of diethylstilbestrol.During the last 10 days of this regimen, studies of theperipheral metabolism of labeled thyroxine were repeated.

Two months later, after clinical evidence of estrogeniceffect had disappeared, a course of adrenocortical sup-pressive therapy was begun. The patient was given 10.0mg prednisolone 4 by mouth, each day for a period of 6weeks. Under this treatment urinary 17-ketosteroids de-clined to 1.1 mg daily. Bloods were drawn weekly forelectrophoretic analysis of thyroxine binding.

After gradual withdrawal of corticoid therapy, a 6-weekcourse of sodium-L-thyroxine, 0.2 mg by mouth daily,was instituted. The patient experienced no symptomsfrom this medication; physical examination, PBI, andelectrophoretic findings with regard to thyroxine bindingremained unaltered. Twenty-four hour thyroidal uptake ofI" was suppressed from its control value of 28 per cent to6 per cent, however.

After withdrawal of exogenous thyroid hormone, thepatient remained well and has continued thus, save forone episode of congestive heart failure that followed dis-continuance of digitalis, which quickly responded to re-digitalization. Bloods have subsequently been obtainedat intervals for electrophoretic analysis; these have con-tinued to show a virtual absence of TBG.

MATERIALS AND METHODS

Thyroxine-binding capacities of individual serum pro-teins were determined by zonal electrophoresis in filterpaper of serum enriched with variable quantities of P'-labeled and stable L-thyroxine.

referred to as a change in TBG; no implications con-cerning the absolute concentration of the protein areintended.

4 11,,17a,21-trihydroxy-4-pregnadiene-3,20-dione.

2054

IDIOPATHIC DECREASEOF THYROXINE-BIN'DING GLOBULIN

IJ3"-labeled hormones (L-thyroxine, T,; 3,5,3'-L-triiodo-thyronine, T3; 3,5,3'-triiodothyroacetic acid, triac; 3,5,3'5'-tetraiodothyroacetic acid, tetrac) obtained from a com-mercial source,5 were 90 to 95 per cent pure, as assessedchromatographically, and were diluted with 1.0 per centhuman serum albumin to a final concentration of 160 Acper ml immediately upon arrival. This procedure reducedboth the degradation of labeled compounds and their ad-sorption to glassware. Nevertheless, labeled materialswere not employed for studies after 1 week of storage inthis form.

The procedure employed in preparing samples of se-rum for the electrophoretic estimation of thyroxine bind-ing by TBG and thyroxine-binding prealbumin (TBPA)was as follows:

To 6.2 ml of serum, 75 Al of I31-labeled T4 in humanserum albumin was added, yielding a final concentrationof approximately 2 Ac per ml (Solution I). A 3.0-mlaliquot of Solution I was pipetted into a separate tube,and to this was added 100 1.l of solution of 1.0 per centhuman serum albumin containing 14.0 Ag of stable T4.This yielded a solution containing 451 ,ug of added stableT4 per 100 ml (Solution G). Aliquots of Solution I,Solution G, and mixtures of the two were employed toprepare samples containing 87, 112.5, 130, 340, 395, and 451Ag of added stable T4 per 100 ml. The three lower con-centrations were employed to calculate the binding ca-pacity of TBG and the three higher concentrations, thebinding capacity of TBPA.6 Two additional samples ofserum containing IP1`-labeled but no stable T4 were pre-pared. The first, Solution A, is a mixture of equal partsof Solution I with fresh serum (1 ,uc I" per ml) and thesecond (Solution H) a 1: 10 dilution of Solution A withfresh serum (0.1 ,c I"~ per ml). In general, the specificactivity of the `131-labeled T4 is such that these two sam-ples contain approximately 4 Ag and 0.4 Ag of added stableT4 per 100 ml, respectively. Electrophoresis was per-formed in sheets of Whatman no. 3 filter paper, 22.5 X 12inches, large enough to accommodate the 8 separate speci-mens. After being moistened in buffer medium andblotted, filter paper sheets were placed upon a carefullyground, siliconized glass plate,7 15 inches long, 13 incheswide, and 1 inch thick. Aliquots of serum (20 Al) werethen applied in thin lines, equidistant from the ends of thesheet, to areas carefully predetermined from a template.Usually, duplicate aliquots of serum were applied to asecond filter-paper sheet carefully placed directly uponthe first sheet. A minute quantity of bromphenol blue(BPB) was applied to each serum band to serve as amarker for the electrophoretic migration of albumin.Sheets were then covered by a second glass plate, similar

5 Abbott Laboratories, Oak Ridge, Tenn.6 Evidence that thyroxine-binding sites of TBG and

TBPA in normal serum are saturated at the concentra-tions indicated has been presented briefly in a previousreport (2) and will be presented in detail in a latercommunication.

7Available from the Pittsburgh Plate Glass Co., Bos-ton, Mass.

in dimension to the first. When, as is often the case, itis desired to analyze an additional 8 samples upon thesame machine, a thin glass plate (13 X 15 X 1/8 inch) isplaced upon the first pair of duplicate sheets. A secondpair of sheets containing the additional samples canthen be placed upon the thin plate and the entire "sand-wich" covered by the second thick glass plate. To mini-mize evaporation, the hiatus between the top and bottomglass plates at the front and rear of the apparatus wassealed with cellophane tape. At the lateral margins ofthe plates, filter paper sheets largely filled this hiatus andwere allowed to hang into buffer baths. Gravitational flowof buffer across the filter paper was eliminated by meansof a horizontal leveling device in the buffer reservoirs.

Buffer systems employed in the present studies wereeither 0.05 M veronal or a (hydroxymethyl) amino-methane (Tris) -maleate buffer, both at pH 8.6.8 Throughplatinum electrodes immersed in the buffer reservoirs, apotential of 110 was applied across the filter paper fora period sufficient to produce approximately a 4-inch mi-gration of the serum albumin. This usually requiredabout 18 hours at normal room temperature. No at-tempts were made to cool the apparatus, as heating isonly slight under these conditions.

When migration of proteins was sufficient, filter papersheets were dried. One sheet was set aside for radio-autography and subsequent staining with BPB, makingpossible the localization of major protein components.The second sheet was cut into 8 strips and, except inthe case of Sample H, radioactive scanning of individualelectrophoretograms was performed by means of a con-tinuous scanner and dual-channel recorder. Areas un-der the individual peaks were determined with an elec-tronic integrator whose output is recorded synchronouslywith the scan itself. The distribution of labeled T, wascalculated from the recorded integral as the proportionof added hormone bound by each of the demonstratedbinding proteins. The total quantity of labeled T4 boundby each protein per unit volume of serum was calculatedas the product of the proportion bound and the estimatedtotal concentration of T4 in the specimen. The latter es-timate is derived from the total concentration of addedT, (I"3-labeled plus stable) plus an estimate of endoge-nous T4, assuming the endogenous PBI to be comprisedentirely of this hormone. Sample H was divided intosegments 0.5 cm wide; these were counted individually ina well-type scintillation counter.

In view of the decreased thyroxine-binding capacity ofTBG in the present patient's serum, samples were alsoassayed which had been enriched with both labeled andstable T4 in a number of concentrations below the usuallowest enrichment with 87 ,tg of stable T4 per 100 ml.

The concentration of the maj or electrophoreticallydistinguishable groups of plasma proteins was assessedby densitometric scanning of electrophoretic strips stainedwith BPB.

8 Preparation of the Tris-maleate buffer has been de-scribed in an earlier communication (2). The final solu-tion is 0.073 M with respect to both Tris and maleate.

2055'

SIDNEY H. INGBAR

Thyroxine turnover studies. Analyses of the peripheralturnover of I"'l-labeled T4 in vivo were performed accord-ing to methods described in detail elsewhere (9). Serialsamples of serum and complete 24-hour urinary collectionswere obtained daily for 8 to 10 days after the intravenousadministration of a tracer dose of IP'-labeled T4. Thesewere analyzed for their concentration of I". Aliquotsof urine were also subjected to a butanol extraction pro-cedure, similar to that described for serum (10), and theproportion of total urinary I" present as butanol-ex-tractable I" was determined. The several functions cal-culated and their derivations are described in the Ap-pendix.

Red blood cell uptakes of I`31-labeled hormones. Bloodwas drawn from a single normal donor (Type 0) intoa heparinized syringe. After centrifugation, plasma wasremoved and the erythrocytes were washed 4 times in 3volumes of normal saline. After the final wash, suffi-cient normal saline was added to bring the red bloodcell suspension to a Standard hematocrit. A sufficientvolume of the suspension was pipetted into a series of 13X 100 mmcounting tubes to provide 0.5 ml of packed redblood cells in each. Samples of fresh plasma (oxa-late anticoagulant) from the patient and from a group ofnormal control subjects were enriched with 0.02 uc perml of I"'-labeled compounds; 0.5-ml aliquots of plasmawere pipetted into tubes containing the packed red bloodcells. Tubes were stoppered and agitated at 370 C for 1hour. The radioactivity in each tube was then assesseddirectly in a well-type scintillation counter. Tubes werecentrifuged, the plasma removed by suction and the redblood cells were then washed 5 times with 3 volumes ofphysiological saline solution at room temperature. Af-ter the last wash, 0.5 ml of saline was added to the packedred blood cells to bring the final volume to 1 ml. Thecells were resuspended, and the radioactivity in the tubewas again measured.

Red blood cell uptakes of labeled hormones were cal-culated as the ratio of final to initial counting rates, ex-pressed as a per cent. All analyses were performed induplicate. Electrophoretic analyses of thyroxine-protein-binding interactions in the sera studied were performedin all instances.

RESULTS

Electrophoretic analyses (Figures 1, 2; TableI). Repeated electrophoretic analyses of the pa-tient's serum failed to reveal more than a trace ofT4 in the inter-a-globulin (TBG) area, duringelectrophoresis of serum in either the Tris-maleateor the veronal buffer systems.9 Radioactivity inthe TBG zone was most frequently found whenelectrophoretograms of serum enriched with only

9 Values described for thyroxine-binding by TBG andTBPA represent the means of those found during elec-trophoretic analysis of at least 3 specimens obtained dur-ing each treatment period.

0.4 ,ug of T4 per 100 ml were cut into strips andcounted in a well counter. Even at this concen-tration, however, no more than an approximate 5per cent of total radioactivity was localized in theTBG zone. Often, both before and during ad-ministration of diethylstilbestrol, no TBG couldbe demonstrated. In sera enriched with approxi-mately 4.0 ug of T4 per 100 ml, a small upwardconvexity in the radioactive scan was occasionallyseen in the TBGzone. This was evident on radio-autographs as a faint zone of darkening. Directwell counting of such strips indicated that no morethan 2 to 3 per cent of added radioactivity was lo-calized with TBG. In sera enriched with higherconcentrations of T4, no band in the TBG zonecould be discovered. It thus appears likely thatthis patient's serum was either not completely ornot always devoid of TBG. The binding capacityof TBG, when demonstrable, could not be assessedwith any accuracy but clearly seemed no morethan a small fraction of 1 Mg of T4 per 100 ml ofserum.

Thyroxine-binding by TBPAwas evident in allspecimens subjected to electrophoresis in the Tris-maleate buffer system. The thyroxine-binding ca-pacity of TBPAwas usually near the normal meanof approximately 120 Mug of T4 per 100 ml of serum

BASAL' POST-OPERATIVE ESTROGENPfT TBM ALO TOG JBPA ALB TBG TBPA AL8 TBG

ADDED(,&g. / OOmL.) i

4

158

348

FIG. 1. THE EFFECT OF INGUINAL HERNIORRHAPHYAND OF DIETHYLSTILBESTROL ON THE BINDING OF THY-ROXINE IN THE SERUMOF A PATIENT WITH DECREASEDTHYROXINE-BINDING GLOBULIN (TBG). Postoperativeserum obtained 2 days after surgery. Serum obtainedduring therapy (obtained after 5 weeks of treatmentwith diethylstilbestrol, 30 mg daily, p.o.). Curves shownare scans of I"l-labeled thyroxine added in the indicatedconcentrations to sera subjected to electrophoresis inTris-maleate buffer, pH 8.6. The small peak seen in theTBG zone during estrogenic therapy was not evident inother samples obtained during the same treatment period.

2056

IDIOPATHIC DECREASEOF THYROXINE-BINDING GLOBULIN

(2), but varied with the patient's clinical state, de-clining to subnormal values during exacerbationsof congestive heart failure or in the -period fol-lowing emergency inguinal herniorrhaphy. Azone of radioactivity corresponding in electropho-retic migration to that of serum albumin was theonly other area in which added I'31-labeled T4 was

localized.As previously reported (11, 12), J131-labeled

triac and tetrac were bound primarily to TBPAand secondarily to albumin during electrophoresisin either Tris-maleate or veronal buffers. Atstandard concentrations of added labeled com-

pounds, the percentile distribution of the deriva-tives between these two proteins in the patient'sserum, obtained during the control state, was notappreciably different from normal.

During the administration of estrogen, predni-solone, or sodium-L-thyroxine, appreciable or con-

sistent differences in thyroxine binding by TBG,as compared with those found during the controlstate, could not be discerned; the small radioactivepeak seen in Figure 2 was not found in other sam-

CONTROL8.0 -

t % '30days

6.0 TDS c 13.2Liters

C = 3.05 LitersPPBI - 2.0,g. %

40= 61.0,%/Dry

THYROXINE(% dose/Liter)

2.0 _

1.0 ,\

ples obtained during the same treatment period.The thyroxine-binding capacity of TBPA was

slightly reduced during administration of diethyl-stilbestrol, but was unaffected by treatment withprednisolone and L-thyroxine.

Red blood cell uptakes of labeled hormones(Table I). In vitro uptakes of labeled T4 and T3from the patient's plasma by red blood cells were

greatly increased in the basal state and were notappreciably altered during the several treatmentperiods. The red blood cell uptake of labeled triacwas normal during both the basal state and theadministration of diethylstilbestrol, but under-went a pronounced increase during the immediatepostoperative period.

Thyroxine metabolism in vivo (Table I). Theperipheral metabolism of 1131-labeled T4, meas-

ured while the patient was in the basal state, was

grossly abnormal (9, 13). Fractional rate ofturnover of administered hormone was rapid, 23.1per cent per day. The T4 distribution space was

moderately increased when related to total bodyweight (0.22 L per kg), and hormonal clearance

ESTROGEN

SERUMELECTROPHORESIS

at .P 2 ALB a P 7, ;ALB

FIG. 2. THE EFFECT OF DIETHYLSTILBESTROL ON THE PROTEIN BINDING AND

PERIPHERAL TURNOVEROF THYROXINE IN A PATIENT WITH DECREASEDTBG.Thyroxine turnover studies during estrogenic therapy begun on Day 41 oftreatment with 30 mg of diethylstilbestrol daily, p.o. Distribution of I131-labeled thyroxine (0.4 /Ag per 100 ml) in sera obtained during each thyroxineturnover study and subjected to electrophoresis in Tris-maleate buffer, pH8.6. The small peak of radioactivity seen in the TBG zone during estro-genic therapy was not evident in other samples obtained during the same

treatment period.

tX = 3.5 daysTDS 15.7 Liters

C- 3.11UlersPBI 2 2.IAq.%

D = 65.3,6gJDay

.

2057

SIDNEY H. INGBAR

TABLE I

Values for several aspects of thyroid hormone economy in normal subjects and in apatient with idiopathic decrease in thyroxine-binding globulin *

Patient F.B.

Diethyl- Predni- Postop-Function Normal valuest Control stilbestrol solone erative L-thyroxine

Thyroid I131 uptake (% dose) 20-50 28 26 6Basal metabolic rate -15-+15 +3 +6 -1 +4Serum cholesterol (mg %) 150-270 210 204 198Protein-bound iodine (pg %) 4.0-8.0 2.0 2.1 2.1 1.8 2.3

Thyroxine turnover studies (9)Thyroxine space (TDS; L) 9.4 ± 2.0 13.2 15.7Turnover rate (k; %/day) 10.6 4± 0.9 23.1 19.9Clearance rate (C; Llday) 1.0 i 0.3 3.05 3.11Organic iodine pool (ETT; pug I) 508 d 146 264 329Daily hormone disposal (D; pug I/day) 53.6 i 16.5 61.0 65.3Fecal fraction (Fm..; %dose) 26.9 1 9.2 25.7 27.2Urinary fraction (Umax; %dose) 73.1 ± 9.3 74.3 72.8Fecal clearance (L/day) 0.27 0.11 0.78 0.85Degradative clearance (Llday) 0.73 i 0.21 2.27 2.26Fecal excretion (fig I/day) 14.6 4- 6.8 15.6 17.8Degradation rate (pug I/day) 39.0 i 12.7 45.3 47.5

Hormonal binding studies (2, 36)TBGcapacity (iug thyroxine/100 ml) 18-25 :14 4 41 4+TBPAcapacity (pug thyroxine/100 ml) 90-160 136 85 120 18 121RBCthyroxine uptake (%) 0.6-0.9 1.5 1.5 1.7RBCtriiodothyronine uptake (%) 5.0-8.5 13.2 12.8 14.4RBCtriiodothyroacetic acid uptake (%) 1.2-2.1 1.5 1.9 2.9

* For a description of functions and abbreviations shown under thyroxine turnover studies, see Appendix. Fordetails regarding the several treatment periods, see text.

t Values presented are normal range or mean :i standard deviation. Normal values are those presented in previousstudies or calculated from data contained therein, and were obtained by methods similar to those employed in PatientF.B. Numbers in parentheses provide references to these sources.

±i Indicates traces of TBGinconstantly seen, and, when demonstrable, present in concentrations too low to permitcalculation of thyroxine-binding capacity.

rate was greatly augmented (3.05 L per day).Because of the subnormal PBJ, however, totalhormonal disposal, 61.0 ,ug of iodine per day, waswithin normal limits. The extrathyroidal organiciodine pool was greatly reduced.

Radioiodine appeared in the urine more rapidlythan normal after administration of labeled thy-roxine. Butanol extractions of urine on the third,fourth, and fifth daily collections revealed that 3.3,4.6, and 4.0 per cent, respectively, of urinary I131was soluble in butanol and relatively insoluble inaqueous alkali; these values were similar to thosefound in normal individuals (14). Calculated val-ues for the ultimate urinary and fecal excretion ofI131 were 74.3 and 25.7 per cent of the adminis-tered dose, respectively. Both fecal clearance(0.78 L per day) and degradative clearance of T4(2.27 L per day) were greatly increased, but thedaily fecal excretion rate and daily degradationrate were within normal limits.

Neither the peripheral metabolism of T4 northe concentration of hormonal iodine in the plasmawas significantly altered by the prolonged adminis-tration of large doses of diethylstilbestrol. Neitherprednisolone nor thyroid-suppressing doses of so-dium-L-thyroxine increased the PBJ.

DISCUSSION

There is general agreement that TBG serves asa rate-regulating factor in the peripheral metabo-lism of T4. TBG, like other proteins with whichT4 is associated, is thought to limit the concentra-tion of free or unbound hormone, thereby retard-ing its cellular penetration and decreasing theproportion of extrathyroidal hormone that is af-fixed to the cell. If, then, a constant proportion ofthe hormone within cellular confines is metabo-lized per unit time, the fractional rate of disposalof the entire peripheral pool of hormone will varyinversely with the binding activity of TBG (2, 3,

2058

IDIOPATHIC DECREASEOF THYROXINE-BINDING GLOBULIN

14). This hypothesis is largely based on inferen-tial evidence, consisting of a series of correlationsbetween changes in extracellular thyroxine-TBGinteractions and changes in the metabolism of thehormone, either in vitro or in vivo. Thus, the up-take of T4 by cellular systems in vitro varies in-versely with the concentration of TBGand directlywith the concentration of hormone in the suspend-ing medium (15). In vivo correlations havemainly required the use of pharmacological agentsto alter the thyroxine-binding capacity of TBG,associated effects on the peripheral metabolism oflabeled T4 being then determined. Diethylstil-bestrol and natural estrogens, for example, in-crease the binding capacity of TBG '(13, 16) anddecrease the fractional rate of turnover of the hor-mone (13, 17), while methyltestosterone producesconverse effects (18). These in vivo observa-tions, while consistent with the major hypothesis,have not been entirely conclusive because of thepossibility that the agents employed might directlyaffect cellular mechanisms for the disposal of hor-mone. This seemed especially possible in thecase of diethylstilbestrol, in view of reports thatthis compound inhibits the deiodination of T4 bothby intact cellular preparations and by a partiallypurified thyroxine-deiodinase (19, 20).

More compelling evidence in favor of the postu-lated function of TBG has been afforded by therecently described patient in whose plasma therewas found an apparently idiopathic increase inTBG (21). This abnormality was accompaniedby changes in the peripheral metabolism of T4similar to those found in association with the in-creased TBG induced by estrogens (13). Nogross evidence of endogenous excess of estrogenswas evident in the male patient described. How-ever, values for urinary estrogens were ratherhigh and those for urinary gonadotropin ratherlow. These findings, together with the familialnature of the disorder, raised the possibility thatsome inherited disturbance in the secretion or me-tabolism of estrogens may have produced the in-crease in TBGwhich this patient manifested.

In the present patient, the apparently coinci-dental association of testicular hypofunction withvirtual absence of TBG in the plasma afforded anunusual opportunity to complement existing dataconcerning the function of TBG. In view of thetheoretical objections to the in vivo studies de-

scribed above, it appeared desirable to obtain con-clusive evidence that the abnormality in TBGdis-played by this patient was not due to disturbancesin known hormonal or metabolic factors whichmight, in themselves, influence the metabolism ofT4.

This patient's distinct hypogonadism excludedexcessive secretion of normal androgens as thecause of decreased TBG. It appeared possible,however, that the patient's abnormal testes weresecreting an unusual, nonandrogenic steroid ca-pable of decreasing TBG. Apparently the ca-pacity of certain steroids to decrease TBG is not afunction of their androgenic potency, since nor-ethandrolone,10 a steroid with relatively moreanabolic than androgenic activity (22), has provedcapable of inducing pronounced reductions inTBG within a period as short as 1 week (23).During this time fractional peripheral turnover ofT4 was accelerated, but androgenic effects werenot evident, even in hypogonadal patients (23).The large doses of diethylstilbestrol administeredto the present patient produced gynecomastia andedema, suppressed gonadotropin production, andshould have been adequate to suppress any linger-ing function that his testes may have retained.Nevertheless, diethylstilbestrol failed to restorethe binding capacity of TBG to normal, and, in-deed, any increase in TBG that may have beeninduced by this agent was too small to be recog-nized consistently during electrophoretic analyses.

Finally, it seems unlikely that an androgen-likesteroid of adrenocortical origin was responsiblefor the decrease in TBG, since prolonged adreno-cortical suppression failed to influence the ab-normality in hormonal binding. The patient withdecreased TBGreported by Tanaka and Starr hada history of hepatitis and displayed abnormalcephalin flocculation and thymol turbidity tests.In the present patient there was no clinical orlaboratory evidence of hepatic or renal disease.Therefore, currently recognized causes of de-creased TBGappear to have been excluded in thepresent case.

The abnormalities in the peripheral metabolismof T4 which accompanied the decrease in TBG inthis patient were consonant with the postulatedfunction of TBG. The greatly increased red

10 17a-ethyl-17-hydroxynorandrostenone.

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SIDNEY H. INGBAR

blood cell uptakes from the patient's plasma of T4and T3, hormones bound primarily by TBG (14),provided evidence that an abnormally high. pro-portion of these hormones was unbound or "free."As would therefore be expected, the T4 distributionspace was moderately increased in vivo and thefractional turnover of hormone was greatly ac-celerated. Accordingly, total hormonal clearancerate was markedly increased. However, the PBIwas abnormally low, with the result that totalhormonal disposal was well within the normalrange.

Studies of urinary I$'s and estimates of fecalexcretion made possible an assessment of the gen-eral metabolic pathways by which the increasedfractional rate of turnover was effected. Al-though fecal clearance of T4 was increased several-fold, a finding in accord with earlier evidence thatprotein binding of T4 limits its gastrointestinalexcretion (24, 25), this increase accounted foronly a small fraction of the increase in total T4clearance which the patient displayed. In thepresence of an increased proportion of unboundhormone in the plasma, an increased renal clear-ance of T4 would also be expected (14). Nor-mally, however, this route of disposal contributesso little to total hormonal clearance that an enor-mous increase in this function would have beenrequired to contribute significantly to the ab-normality in hormonal turnover (26, 27). Meas-urements of the butanol-extractable I'll in the pa-tient's urine confirmed the relative unimportanceof urinary excretion of T4 as a factor in the uri-nary excretion of I18l and in total hormonal dis-posal. Thus, increased hormonal turnover wasdue, in greatest part, to increased disposal of T4by those metabolic pathways which ultimatelylead to hormonal deiodination.

It seemed important to exclude the possibilitythat this patient's subnormal PBI was not due tothe abnormality in hormonal binding, but ratherreflected the secretion of some thyroid hormone,like T3, which, in amounts sufficient to maintaina normal metabolic state, does not contributegreatly to the PBI (28). If this were the case,then the metabolism of labeled T4 would not haveaccurately reflected the metabolism of endogenoushormone. However, if T3 were indeed the majorhormone in the patient's plasma, then doses ofL-thyroxine adequate to suppress endogenous thy-

roid function should have restored the PBI tonormal. Since they did not, it seems likelyin the present patient, as is normally the case(29), that T4 constituted the major secretoryproduct of the thyroid gland and the major con-stituent of the PBI. Calculated values for hor-monal disposal were therefore validated.

The present association of normal thyroid func-tion and metabolic status with normal values fordaily T4 disposal and deiodination, despite a sub-normal PBI, supplements previous evidence, re-viewed elsewhere (30), that both the secretion ofthyrotropin and the metabolic state of the patientare more closely related to the quantity of hor-mone available to the tissues than to the total con-centration at which they are delivered. The con-centration of unbound hormone in the plasma,could it be readily determined, would likely con-stitute a better index of metabolic state than doesthe total PBI.

The failure of diethylstilbestrol to alter the ki-netics of T4 metabolism in this patient indicatesthat, in man, in contradistinction to certain cellu-lar preparations in vitro, this agent apparentlydoes not directly affect the cellular degradation ofT4. It therefore follows that the changes in T4metabolism that accompany the administration ofestrogen to patients with normal TBGare indeeddue to the resulting increases in TBG. Further-more, the present findings support the belief thatthe changes in T4 metabolism induced by andro-genic hormones are not due to a direct cellulareffect of the androgens, but rather to the associ-ated decrease in TBG. Thus, the data lend con-siderable weight, both directly and indirectly, tothe postulated function of TI3G, described above.They do not, however, conclusively prove the hy-pothesis. Alternative interpretations of the func-tion of TBG in the light of the present and previ-ous observations concerning the metabolism of T4may also be envisioned (2)-.' Furthermore, onecannot logically exclude the possibilty that the ab-normalities in T4 metabolism which this patientdisplayed arose, not from the decreased TBG, butfrom unrelated abnormalities in the cellular me-tabolism of'tlie hormone.

In the absence qf significant hormonal bindingby TBG, an unusually large share of the transportof T4 must dvolv'e' upon other proteins,-notablyTBPA (31). 'However, a consideration'of the

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IDIOPATHIC DECREASEOF THYROXINE-BINDING GLOBULIN

possible physiological role of TBPA in the pres-ent patient, or indeed, in general, is beyond thescope of this report. While, in a number of cir-cumstances, correlated changes in thyroxine bind-ing by TBPA and in T4 metabolism indicate thatthis protein contributes significantly to hormonalbinding in vivo (2, 12), other evidence suggeststhat it may not (32, 33). Nevertheless, hormonalbinding by TBPAseems adequate to explain severalphenomena observed in the present studies. Thus,the normal values for thyroxine binding by TBPAfound in the patient's serum when he was in a basalstate correlated well with normal values for the redblood cell uptake of triac, since this derivative isprimarily bound to TBPA, and not to TBG (11,12). Similarly, the increased red blood cell up-take of triac obtained when the patient was in thepost-operative period is probably explained by thereduced hormonal binding by TBPA which oc-curred at that time, a change similar to that foundpreviously in sera from a large number of pa-tients with other nonthyroidal illnesses (34).Subsequent studies in a large number of patientshave indicated that the in vitro uptake of de-aminated derivatives of T4 and T3 by an adsorp-tive particulate system varies inversely with, andis a convenient means of estimating, the thyroxine-binding capacity of TBPA (35).

As was the case in the patient reported byTanaka and Starr (4), the defect in TBGin thepresent patient was associated with no gross ab-normalities in the concentration of the major elec-trophoretically distinguishable groups of plasmaproteins. Of the many transport functions of theplasma proteins only the transport of iron wasassessed, and this proved to be normal. Thus, itis uncertain whether the abnormality describedhere is an isolated defect. In neither patient withthis disorder has it been possible to ascertainwhether the decrease in TBG was congenital oracquired, or whether it represented an inheritedabnormality. The male patient with absent TBGdescribed by Tanaka and Starr had no evidence ofcardiac disease (4). Consequently, it is uncertainwhether the congenital cardiac lesion in the pres-ent patient with decreased TBG represents a re-lated abnormality or merely a fortuitous associ-ation.

Although the defect in the present patient hasbeen referred to as a decrease in TBG, the concen-

tration of the protein itself was not measured.Conceivably, decreased hormonal binding couldhave resulted from the presence of a highly ef-fective inhibitor of thyroxine binding or from aminor structural abnormality in the protein thatimpaired the attachment of- T4. More likely,however, the abnormality described in this paperrepresents, as in other dysproteinemias (36), adecrease in the concentration of the protein itself.Whether, if this be true, the virtual absence ofTBGfrom this patient's plasma is due to excessivedestruction or defective protein synthesis, as isthe case in agammaglobulinemia and analbumine-mia (36), can not be determined at present. Reso-lution of these and other intriguing questions con-cerning the function and metabolism of TBGawaitample supplies of the purified protein.

SUMMARY

1. Clinical features have been described of aeuthyroid male patient with postoperative primaryhypogonadism and a congenital atrial septal de-fect, whose plasma was virtually devoid of thyrox-ine binding by the thyroxine-binding globulin(TBG).

2. Prolonged administration of large doses ofsynthetic estrogen or adrenal corticosteroid failedto alter significantly the binding activity of TBG,suggesting that the abnormality in this proteinwas not produced by an androgen of testicular oradrenocortical origin.

3. This seemingly idiopathic decrease in TBGwas associated with pronounced abnormalities inthe peripheral metabolism of thyroxine. Both thevolume of distribution and fractional rate of turn-over of IJ31-labeled thyroxine were increased; hor-monal clearance rate was markedly augmented.However, the serum protein-bound iodine was sub-normal, with the result that daily disposal of hor-mone by both excretory and degradative routeswas within normal limits.

4. In contrast to its action in normal individuals,in whom it induces an increased binding activityof TBG, estrogenic therapy had no significant ef-fect upon the concentration or peripheral turn-over of thyroxine in this patient.

5. The several findings in this patient are viewedas consistent with the hypothesis that TBG regu-lates the peripheral metabolism of thyroxine by

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SIDNEY H. INGBAR

limiting its cellular penetration and hence itsperipheral metabolism and action.

APPENDIX

In this manuscript and Table I the following termshave been employed. These were described in detail inan earlier communication (9).

TDS, thyroxine distribution space; the virtual volumeof body fluids through which exchangeable thyroxinewould be distributed were it present throughout at thesame concentration at which it exists in the plasma.

PBI, concentration of hormonal iodine in the plasma,and, by definition, the geometrical mean concentration ofhormonal iodine in the TDS; assumed to represent solelyiodine in thyroxine.

ETT, extrathyroidal thyroxine or the organic iodinepool; the total quantity of thyroxine within the TDS, interms of its content of iodine.

k, fractional rate of turnover of thyroxine; the pro-portion of ETT removed from the TDS per unit time.

T1, thyroxine half-time; the time required for one-halfof the ETT to be removed from the TDS.

C, thyroxine clearance rate; the volume of plasma con-taining an amount of thyroxine equal to that removedfrom the TDS per unit time. Defined as "volume turn-over, V" in an earlier communication (9).

D, hormonal disposal rate; the quantity of hormone re-moved from the TDS per unit time, in terms of its con-tent of iodine.

Fmax, the proportion of D accounted for by fecal ex-cretion of hormone; Umax, the proportion of D accountedfor by urinary excretion of organic iodine and iodide re-leased during hormonal deiodination; fecal clearance, thevolume of plasma that contains a quantity of hormoneequal to that lost in the feces per unit time; degradativeclearance, the volume of plasma containing a quantity ofhormone equal to that removed by nonexcretory path-ways per unit time. As calculated, degradative clearanceincludes the renal clearance of thyroxine, but the lattercomprises a negligible proportion of the total; fecal ex-cretion, the quantity of hormone excreted in the feces perunit time; degradation rate, the quantity of hormoneremoved by nonexcretory pathways per unit time.

ETT=PBIXTDS. C=TDSxk. D=CXPBI=ETT X k. T = 0.693/k. Fecal clearance = C X Fmax.Degradative clearance = C - fecal clearance = C X Una.X.Fecal excretion = fecal clearance X PBI = DX Fmax. Deg-radation rate = degradative clearance X PBI = D-fecalexcretion.

ACKNOWLEDGMENTS

The author is indebted to Miss Patricia Eveleth andMiss Rebecca Feretos for valuable technical assistance.

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