Metabolic Clearance and Secretion Rates of Subunitsof Human Thyrotropin

IONE A. KOURIDES, RICHARDN. RE, BRUCED. WEINTRAUB, E. CHESTERRIDGWAY,and FARAHEMALOOF

From the Thyroid and Endocrine Units, Department of Medicine, Massachusetts General Hospitaland Harvard Medical School, Boston, Massachusetts 02114, and the Clinical Endocrinology Branch,National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health,Bethesda, Maryland 20014

A B S T RA C T Metabolic clearance rates (MCR) of thealpha and beta subunits of human thyrotropin (hTSH-aand hTSH-,8) were determined by a constant infusionto equilibrium method. In 15 normal individuals(six men, six premenopausal women, and three post-menopausal women), the mean MCR of hTSH-a(68 ml/min per m2) was significantly faster than that ofhTSH-,B (48 ml/min per M2) was significantly fasterthan that of hTSH-,B (48 ml/min per m2); both weretwo to three times more rapid than the previouslydetermined MCRof hTSH. In patients with primaryhypothyroidism, MCRwere significantly slower witha mean value of 55 ml/min per m2 for hTSH-aand 37 ml/min per m2 for hTSH-,B. However, MCRof subunits were not significantly faster than normalin hyperthyroid patients.

Serum concentrations of alpha subunits and hTSH-,8were measured by radioimmiunoassay, and secretionrates of alpha and hTSH-p8 from the pituitary werecalculated using hTSH-a and hTSH-,8 MCR, respec-tively. In the normal individuals, alpha secretionrates averaged 91 ,.ug/day per 112, greater than thosepreviously determined for hTSH and human follicle-stimulating hormone. Alpha secretion rates weresignificantly elevated in the normal postmenopausalwomen (211 ,uglday per m2) and in the premenopausalhypothyroid women (202 ,ug/day per m2); they werealso elevated in the postmenopausal hypothyroidwomen (277 ,ugg/day per m2) Alpha secretion rateswere significantly decreased in the premenopausalhyperthyroid women (66 gg/day per m2). Usually,the secretion rates of hTSH-,3 could not be calculated

This study was presented in part at the 57th AnnualMeeting of the Endocrine Society, 18-20 June 1975, NewYork.

Received for publication 22 April 1976 and in revisedform 24 November 1976.

in normal individuals, and the rates in hyperthyroidpatients could never be calculated because seruinhTSH-,B was not detected. Six normals had detectablehTSH-,8 secretion rates (17 ,.ug/day per M2); hTSH-,fsecretion rates were significantly increased in patientswith primary hypothyroidism (28 ,ug/day per m2).Although we had previously demonstrated a 50-foldincrease in hTSH secretion rates in primary hypothy-roidism, there was only a 2-fold increase in alphaand hTSH-,8 secretion rates. Thus, increased subunitsynthesis appears to be utilized predominantly forproduction of complete hTSH.

INTRODUCTION

The development of sensitive and specific radio-immunoassays (1-4) for the immunologically indis-tinguishable alpha subunits (a)' of the human glyco-protein hormones, thyrotropin (hTSH), follicle-stimni-lating hormone (hFSH), luteinizing hormone (hLH),and chorionic gonadotropin (hCG), and for the betasubunit of hTSH (hTSH-,8), has permitted measure-ment of subunits in biological fluids. Wehave previ-ously reported that both the alpha subtunit and hTSH-p8are present in the serum of patients with primaryhypothyroidism and are secreted from the pituitary

I Abbreviations used in this paper: alpha or a, immunologi-cally common alpha subunit of the glycoprotein hormones;fT4, free thyroxine; hCG, human chorionic gonadotropin;hCG-a, alpha subunit of hCG; hFSH, human follicle-stimu-lating hormone; hLH, human luteinizing hormone; hTSH,human thyrotropin; hTSH-a, alpha subunit of hTSH; hTSH-,3,beta subunit of hTSH; Kav, partition coefficient; LHRH,luteinizing hormone-releasing hormone; MCR, metabolicclearance rates; r, correlation coefficient; TRH, thyrotropin-releasing hormone; T3, total triiodothyronine by radioim-munoassay; T4; total thyroxine by competitive protein bindingassay; Vo, void volume; Vt, total bed volume.

The Journal of Clinical Investigation Volume 59 March 1977*508-516508

after the administration of thyrotropin-releasing hor-mone (TRH) (2, 5). In addition, normal postmeno-pausal womenhave higher serum alpha concentrationsthan premenopausal women or men (2, 6); further-more, serum alpha concentrations in normal in-dividuals demonstrate greater increases after luteiniz-ing hormone-releasing hormone (LHRH) than afterTRH (7-10).

In previous studies, we have determined themetabolic clearance rates (MCR) and production ratesof hTSH in various disorders of thyroid function andfound that changes in the serum concentration of hTSHare mainly due to altered pituitary hTSH secretionrather than to changes in hTSH MCR(11). In thepresent study, we have determined the MCR ofhTSH-a and hTSH-,3 in normal individuals and inpatients with thyroid disorders. Wehave also measuredendogenous alpha and hTSH-,B serum concentrationsby radioimmunoassay and calculated the pituitarysecretion rate of stubunits (12). Thus, we have beenable to compare changes in pituitary secretion rateof hTSH to that of hTSH subtunits in variouis statesof thyroid dysfuniction.

METHODS

Determination of MCR of subunits. hTSH-a was agift of Dr. C. H. Li, University of Califomia, San Francisco,Calif.; hTSH-,8 was obtained from Calbiochem, San Diego,Calif.; and hCG-a was a gift of Dr. 0. Bahl, State Uni-versity of New York at Buffalo, Buffalo, N.Y. Immuno-logical and physicochemical testing showed that these sub-units had properties indistinguishable from those pre-viously prepared by our laboratory (1). Each subunit waslabeled with either 1251 or 131I to a specific activity of 50-80,uCi/,g by a modification (13) of the chloramine-T method(14) utilizing one to three additions of 180 ng of chlora-mine-T without sodium metabisulfite. Labeled subunit wasseparated from aggregated products of iodination and un-reacted iodine by gel chromatography on a 1.5 x 90-cmcolumn of Sephadex G-100 (Pharmacia Fine Chemicals Inc.,Piscataway, N. J.). Further preparation of labeled subunitfor infusion was identical to that previously described forhTSH (11). 70-75 ml of infusate containing a mixture oflabeled subunits, 0.15 M sodium chloride, and 1.0% humanserum albumin was injected, 20-25 ml being given initiallyas a bolus to achieve equilibrium more rapidly. After 15min, the remainder was infused at a constant rate (0.15-0.17 ml/min) for 4-5 h. Apparent equilibrium was definedas that time after which subsequent measurements of im-munoprecipitable labeled subunit levels showed no variationthat exceeded +±10% of the mean immunoprecipitablelabeled subunit concentration during the preceding 1-hperiod (11, 15). After infusion to equilibrium, labeled sub-unit was separated from serum or infusate by a doubleantibody technique with either excess rabbit anti-hTSH-aor anti-hTSH-,B at a final concentration of 1:1,000. At thetime of infuision, 70-90% of each tracer was immunoprecip-itable. Each subject received hTSH-a labeled with 1251or 1311; hTSH-,p labeled with 125J or 1311 was separatelyinfused 4-7 days before or after the infusion of hTSH-a.Both isotopes were used to label each subunit in separateexperiments, and no significant differences between isotopes

BOLUSI CONSTANT

INFUSIONi200 r-

1000

TOTAL 125 I

flodP

800 I-

600 H

400 H

200H

0

I MMUNOPRECIPITABLE125 I-hTSH - d

1 2 3 4 5

HOURSAFTER INFUSIONFIGURE 1 Total 125I and immunoprecipitable 1251-hTSH-aduring infusion of 1251-hTSH-a to a representative normalindividual. Apparent equilibrium of immunoprecipitablecounts per minute per milliliter was achieved by 3 h, whereastotal 25I cpm/ml continued to increase with time. In datanot shown, at all time points precipitability of labeled sub-unit with phosphotungstic acid was greater than with anti-serum to alpha subunit. Therefore, the increase in total1251 represented both deiodination of labeled subunit andformation of nonimmunoreactive species.

were noted. The total radioactivity administered to a singlesubject was 50 ,uCi or less. The measurement of serumimmunoprecipitable counts at equilibrium and the calculationof MICR were performed in the same fashion as previouslydescribed for hTSH (11).

In several subjects, a blood sample was obtained 30min after beginning constant infusion of 1251-hTSH-aor '311-hTSH-a and separate infusion of '251-hTSH-,8 or 1311_hTSH-,3; a 2-ml aliquot of this serum was co-chromatographedwith free 125I (to determine the total bed volume of thecolumn, Vt) on a 1.5 x 86-cm column of Sephadex G-100.The void volume of the column, Vo, was determined with1251-bovine thyroglobulin in separate experiments. 2-ml frac-tions were collected and counted. All peaks of radioactivitywere tested for immunoprecipitability with either excess anti-hTSH-a or anti-hTSH-13.

Radioimmunoassays and other assays. All radioim-munoassays were performed using a double antibody tech-nique. The immunoassays of hTSH (16), hFSH (17), hLH(17, 18), and total triiodothyronine (T3) (19) were performedby our previously published methods. The methodologyand specificity of the subunit immunoassays have alsopreviously been reported (1, 2, 5). In the current studies,the sensitivity of the hTSH-13 immunoassay was increasedto 0.02 ng/ml in the assay tube or 0.2 ng/ml in serumby a 48-h delay in addition of tracer to other assay re-agents. Measurement of total thyroxine (T4) was by competi-tive protein displacement assay and free thyroxine (fT4)by equilibrium dialysis (16).

Subjects and experimental protocol. 15 normal in-

Metabolic Clearance and Secretion Rates of hTSH Subunits 509

2000 [-

0 20 22 24

VoI t

\ VII I~~~~~~~~~~~~~~~~~~~~

I# I0

.Ar Oak _

26 28 30 32 34 36 38 40 42 44 46 48 50 52 73

FRACTION NUMBER

FIGURE 2 Fractionation by gel chromatography on a Sephadex G-100 column of a 2-mlaliquot of serum obtained 30 min after beginning constant infusion of 1311-hTSH-a. Monomeric'311-hTSH-a had an elution volume (Kav, 0.36) similar to that of standard unlabeled alphasubunit (Kav, 0.35). Identical technique was used for fractionation of 1251-hTSH-a, 125I1hTSH-,3, and '311-hTSH-,3, which had been separately infused into other individuals. Theelution volumes of labeled hTSH-13 (Kav, 0.44) were identical to that of standard un-

labeled hTSH-,f (1). Small amounts of larger molecular weight aggregates (peak for 131I-hTSH-a atfraction 33) were nonimmunoreactive (see text) and, therefore, were not used in calculationof MCR.

100

90

80

70

. C

\~

60

50

40

30

20

10

MEAN± SE

FIGURE 3 MCR(milliliters per minute per square meters)of hTSH-a and hTSH-,3 in 15 normal subujects (6 men

[0], 6 premenopausal women [0], and 3 postmenopausalwomen [A]. MCR(mean+SE) for each subunit in the 15normal subjects is shown at the bottom of the figure.

dividuals, 6 men (ages 18-60 yr; mean T4, 6.5 ,ug/dl;fT4, 1.3 ng/dl; and T3 113 ng/dI), 6 premenopausal women

with normal menstrual cycles (ages 21-30 yr; mean T4,6.7 ,tg/dl; fT4, 1.3 ng/dl; and T3, 91 ng/dl), and 3 post-menopausal women (ages 50-58 yr; mean T4 6.7 ,ugldl;fT4, 1.1 ng/dl; and T3, 76 ng/dl) were studied. In addition,eight women with primary hypothyroidism, four premeno-pausal and four postmenopausal, participated in the study;each had elevated serum hTSH (18-164 ,uU/ml), low T4(0.5-3.5 ,ug/dl), and fT4 (0.1-0.6 ng/dl), and, all except one,low T3 (10-60 ng/dl). Two patients with central (pituitaryor hypothalamic) hypothyroidism and inappropriately lowgonadotropins, one of whomhad a pituitary tumor, were alsostudied. Finally, five women (three premenopausal and twopostmenopausal) with hyperthyroidism, four with a diffusegoiter and one with an autonomous nodule, were includedin the study. Each had undetectable serum hTSH (<0.5,uU/ml), elevated T3 (290-540 ngldl), and, all except one,

elevated T4 (13.5-19 ,ugldl) and fT4(3.0-5.4 ng/dl). No subjectwas taking any medication, and all gave informed consentto participate in this study.

All subjects received an i. v. 200-,ug boluis of TRH (AbbottLaboratories, North Chicago, Ill.) and 200 ,ug of LHRH(Ayerst Laboratories, New York) with a 4-7-day intervalbetween tests; serum samples were collected at varioustimes over a 180-min period for measurement of alpha,hTSH-A3, hTSH, hLH, hFSH, T4, fT4, and T3. MCRofhTSH-a and hTSH-i3 were separately determined with a 4-7-day interval between tests (see above); all euthyroid patientsreceived 270 mg of potassium iodide immediately beforeinfusion of labeled subunits. No change in T4, fT4, T3, or

hTSH was noted between the beginning and end of thedetermination of each MCR. All 15 normal subjects alsohad MCRof hCG-a determined simultaneously with hTSH-a;one subunit was labeled with 1251 or 1311, and the other was

labeled with the opposite isotope. In several of the studysubjects, serum samples were also collected for measurementof alpha at 30-min intervals for 41/2 h. In six other normalsubjects, two men, two premenopausal women, and two post-menopausal women, serum alpha was measured at 20-minintervals for 1 day. (These samples were kindly supplied

510 1. Kourides, R. Re, B. Weintraub, E. Ridgway, and F. Maloof

8000 r

6000 F

4000Cl)

I'l

hTSH-d hTSH-0

0

0

-S o0~~~

0~~~

0

0

68±3 47+3

0---O '3' I- h TSH- ct

by Dr. Robert Boyar, University of Texas SouthwestemMedical School, Dallas, Tex.).

Calculation of endogenous secretion rates. The secretionrate of alpha subunits (,ug/day per m2) was calculated as theproduct of the endogenous serum concentration of alpha(ng/ml) and the MCRof hTSH-a (ml/min per m2) times 1,440min/day. Secretion rate of hTSH-,B was similarly calculatedutilizing serum concentration of hTSH-/3 and MCRof hTSH-f8.

RESULTS

MCRof hTSH-a and hTSH-f3. Validity of the deter-mined MCRof these subunits depended on iodinatedsubunit concentrations being at equilibrium in serum.Apparent equilibrium was indeed achieved between2'/2 and 3 h after the beginning of infusion in allsubjects since there was no variation of immuno-precipitable counts after that time that exceeded+10% of the mean counts in at least three serumsamples drawn during the preceding 1-h period(Fig. 1). Fractionation by gel chromatography on aSephadex G-100 column of serum obtained 30 min

after the start of constant infusion in several patientsrevealed that small quantities of larger molecularweight aggregates of iodinated subunits were present.However, these aggregates were not immunoprecipit-able (< 1%), and, thus, only the immunoreactive mono-meric forms were included in the calculation of MCR(Fig. 2). In addition, monomeric hTSH-a, labeled witheither 125I or 131I, had a partition coefficient (Kav)of 0.36, similar to that of standard unlabeled alphasubunits; likewise, monomeric hTSH-,3 labeled eitherwith 1251 or 1311 had Kav identical to each other andto standard unlabeled hTSH-,8 (Kav, 0.44).

There were no significant differences in MCRofhTSH-a and hTSH-,B between the six normal premeno-pausal womenand six normal men when the clearancerates were expressed in terms of body surface area (Fig.3 and Table I), as we have previously reported forMCRof hTSH (11). These 12 normal subjects hada mean MCRof hTSH-a of 70+4 ml/min per m2,which was significantly faster than the mean hTSH-,3

TABLE IEndocrine Data

Peak alphaSecretion MCR Secretion

No. of MCR rate, post- post- hTSH- rate, Peak hTSH-8Patient grotup patients hTSH hLH hFSH Alpha hTSH-a Alpha TRH LHRH hTSH-,B 3 hTSH-,3 post-TRH

AU/ml inlUImi ng/ml ml/minim 2 pg/day/M 2 ng/ml ng/ml ml/min/m2 pg/day/M 2 ng/ml

Normal (0.5-3.2) (6-26)1 (5-25)1 (0.5-2.0)" (<0.5)Men 6 1.8±0.4* 10±1 7±2 0.9±0.2 75±6 90±8 1.2±0.1 3.7±0.7 <0.2 .54±5 < 19 <0.2Premenopatisal 6 1.7±0.5 24±10 9±2 1.0±0.1 66±5 92±7 1.6±0.1 3.3±0.6 0.25±0.03** 45±5 17±4** 0.3±0.04**

womenPostmenopauisal 3 3.3±2.3 57±16 92±7 2.5±0.3 58±3 211±28 2.6±0.4 8.2±1.9 0.3±01t 41±1 18±0.5tt 0.3±0.1

women

Primary hypo-thyroid

Premenopatusal 4 89±31 19±6 23±11 2.5±0.6 60±8 202±26 5.9±1.3 4.8±1.3 0.6±0.1 40±4 33±8 2.6±1.0women

Postmenopatisal 4 97±30 78±17 112±11 3.9±1.6 49±7 277±138¶ 5.9±2.3 8.6±4.0 0.5±0.1 34±3 23±5 1.6±0.2women

Central hypo-thyroid

Womanwith 1 1.0 1.2 2.5 <0.5 43 <31 <0.5 <0.5 <0.2 - - <0.2normal sellatutrcica

Womanwith 1 5.2 23§ 335 0.8§ 61 71 2.0 - <0.2 - - 0.3enlargedsella turcica

HyperthyroidPremenopausal 3 <0.5 12±5 12±5 0.7±0.1 71±4 66±10 0.7±0.2 2.2±0.4 <0.2 61±6 <19 <0.2

womenPostmenopausal 2 <0.5 71±10 92±25 2.5±0.6 65±8 237±81 2.3±0.4 9.5±5.0 <0.2 58±8 <19 <0.2

women

* Values are presented as mean±SE.In postmenopausal women, normal hLH values were 40-95 mIU/ml and hFSH values, 35-210 mIU/ml.This woman was postmenopausal.

"In postmenopausal women, normal alpha values were 1-7 ng/ml.I Alpha secretion rates were 181, 550, and 98 pAg/day/M2; serum alpha concentrations were 3.4, 8.3, and 1.1 ng/ml, respectively.** Two normal premenopausal women had undetectable hTSH-(3 concentrations (<0.2 ng/ml) basally and post-TRH.tt One normal postmenopausal woman had an undetectable hTSH-,B concentration (<0.2 ng/ml) basally.

Metabolic Clearance and Secretion Rates of hTSH Subunits 511

100

90

80

70

60

Z 50

F 40

30

20

10

FIGURE 4 MCR(milliliters per minute per square meters)of hTSH-a and hTSH-,8 in hypothyroid and hyperthyroidpatients. The shaded areas represent MCRof the normalindividuals studied (mean±2SE). Mean MCRof both subunitswere significantly slower in the hypothyroid patients than inthe normals. MCRof subunits were not faster in the hyper-thyroid patients than in normals.

MCRof 49+4 ml/min per m2 (paired t test, P < 0.005).In addition, the mean MCR of hCG-a of 61+3ml/min per m2 was significantly slower (P < 0.001)than the simultaneously determined hTSH-a MCR.MCRof both hTSH-a and hCG-a were determinedin the normal individuals since the structures ofthe alpha subunits of hTSH, hFSH, and hLH are

thought to be virtually identical, whereas hCG-ahas been suggested to have a higher carbohydratecontent. The three normal postmenopausal women

studied had MCRof subunits similar to those of theother normal subjects.

MCRof subunits were also determined in patientswith disorders of thyroid function (Fig. 4 and TableI). MCRin nine hypothyroid patients (seven primaryand two central) were significantly slower at 55+5ml/min per m2 for hTSH-a (unpaired t test, P < 0.05)than in normals. Likewise, MCRof hTSH-13 in eightpatients with primary hypothyroidism were signif-icantly slower at 37+2 ml/min per m2 (P < 0.05)than in normals. However, in the five hyperthyroidpatients studied, MCRof hTSH-a (69+4 ml/min per m2)and hTSH-,8 (59+4 ml/min per m2) were not signif-icantly different from those of the normal individuals.Similarly to normals, hTSH-a MCRwere more rapid

than those of hTSH-,8 in the hypothyroid and thehyperthyroid patients.

Correlation of MCRof subunits of hTSH withthyroid hormone concentrations and creatinineclearance. The correlation between serum thyroidhormone concentrations and MCRof both subunitsof hTSH in the 15 normal, 10 hypothyroid, and 5hyperthyroid subjects studied were investigated. T4was related to MCRof hTSH-a with a correlationcoefficient (r) = 0.41 (P < 0.05), and to MCR ofhTSH-,3 with r = 0.53 (P < 0.005) (Fig. 5). T3 was re-

lated to MCRof hTSH-a with r = 0.32 (P > 0.05), andto MCRof hTSH-,8 with r = 0.54 (P < 0.005). Like-wise, creatinine clearance of all the subjects was

related to the MCRof hTSH-a with r = 0.46 (P < 0.02),and MCRof hTSH-,8 with r = 0.28 (P > 0.10) (Fig. 6).All of the subjects studied had normal serum creatinineconcentrations, and none had known renal disease.

Secretion rates of subunits of hTSH. Fluctuationsin serum alpha concentrations were small in six nor-

mal individuals (two men, two premenopausal women,

and two postmenopausal women), who had serum

alpha measured every 20 min throughout a day (Fig. 7).In addition, several individuals in this study had serum

alpha measured every 30 min for 41/2 h with similarresults. These findings permitted calculation of dailysecretion rates of free alpha subunits. The alpha secre-

tion rates (mean±SE) in the six normal men and sixnormal premenopausal women studied were similarand averaged 91±5 ,ug/day per m2 (Table I). The threenormal postmenopausal womenhad an increased alphasecretion rate of 211±28 ag/day per M2. The fourpremenopausal women with primary hypothyroidismhad an alpha secretion rate of 202±26 ,ug/day per M2;the three postmenopausal hypothyroid women, 277±138 ,ug/day per M2. The postmenopausal hypo-thyroid women had wide variations in their alphasecretion rates because of marked differences in theirserum alpha concentrations rather than in their MCRof hTSH-a. However, the alpha secretion rate was

significantly increased in the patients with primaryhypothyroidism as compraed to the normals whetherthe postmenopausal individuals were included (P< 0.02) or excluded (P < 0.001). The three premeno-

pausal hyperthyroid women had an alpha secretionrate of 66±10 u.g/day per M2; the two postmeno-pausal hyperthyroid women, 237±81 ,ug/day per M2.The alpha secretion rate was significantly decreasedonly when the three premenopausal hyperthyroidwomen were compared to the six normal men and sixnormal postmenopausal women(P < 0.05). In addition,the two patients with central hypothyroidism had de-creased alpha secretion rates of <31 and 71 ,ug/dayper m2(Table I). Alpha secretion rates in all the subjectsstudied were significantly related to the peak serum

alpha after TRH (r = 0.84, P < 0.001) and after LHRH(r = 0.82, P < 0.001) (Table I).

512 I. Kourides, R. Re, B. Weintraub, E. Ridgway, and F. Maloof

hTSH-a hTSH-,

HYPOTHY. HYPERTHY. HYPOTHY. HYPERTHY.

...... ...:. .:.:-:::.-: ,:.:.: :.:

.......;!

~.............

_0*:-:-...... *---:- -

* ~~~~~~~~::::::: ::::::::::::::::::::::

_ 00

0..................0..0..................

100

90

80

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zt

70

60

50

40

30-O_ P<0.001

20

10 _

.I0 2 4 6 8 10 12 14 16 18 20

T4 (,g9/d/)

FiGuRE 5 Correlation of serum T4 with MCR(milliliters per minute per square meters)of hTSH-a (0) and of hTSH-13 (0). The regression line for the correlation between T4and complete hTSH MCRis reproduced from our previously published data (11).

Endogenous serum hTSH-,B was <0.2 ng/ml in all thehyperthyroid patients and in 9 of the 15 normal in-dividuals studied, and was 0.3+0.02 ng/ml in the sixnormals with detectable concentrations; hTSH-,3 waselevated in the patients with primary hypothyroidismat 0.6+0.1 ng/ml (Table I). Significant increases inserum hTSH-,8 occurred after TRHonly in the patientswith primary hypothyroidism in spite of the greatersensitivity of this hTSH-,B immunoassay. In the sixnormal womenwith detectable serum hTSH-,p, the cal-culated secretion rate of hTSH-,B was 17+2 ug/dayper M2; in the other normals and the hyperthyroidpatients, hTSH-,3 secretion rate was < 19 ug/day per M2.The secretion rate of hTSH-,8 in the eight patientswith primary hypothyroidism was significantly in-creased at 28+5 ,ug/day per m2 when compared tothat of normals (P < 0.005).2

DISCUSSION

The method of constant infusion to equilibrium fordetermination of MCRrequires that a steady state oflabeled subunit in serum has been achieved (Fig.

2 For tests of significance, the normal individuals with un-detectable serum hTSH-/3 were assigned a serum concentra-tion equivalent to the detection limit of the assay. Thisserum concentration (0.2 ng/ml) was multiplied by the respec-tive MCRof hTSH.,8 to yield a maximum hTSH-,8 secre-tion rate.

1) and that labeled and unlabeled subunits have similarclearance rates. It has previously been shown thatlabeled and unlabeled hTSH and hLH had similarMCRin humans (20, 21), and that labeled and un-labeled hTSH had similar MCRin dogs (22). Dueto insufficient quantities of subunits available, studieswith unlabeled subunits have not yet been performed.However, the method used for iodination of subunitsin this study has previously been shown to preservethe biological activity of complete TSH (13). More-over, labeled subunits had similar elution volumes tounlabeled preparations by gel chromatography, andonly the monomeric labeled subunits were immuno-reactive and thus included in the calculation of MCR(Fig. 2).

We have found MCRof hTSH-a and hTSH-,8 innormal individuals to be two to three times as rapidas that of hTSH (Fig. 3), which was 25 ml/min per m2(11), and as much as 5-30 times as rapid as that ofthe gonadotropins (23-25). The clearance of subunitsof hLH has recently been reported by a single injec-tion method to be twice as rapid as that of hLH(21). Although hCG-a and hTSH-a were simultane-ously infused for determination of MCRin the normalindividuals, hTSH-a had a more rapid MCRthanhCG-a. The reason for the slower MCRof hCG-amay be either a greater carbohydrate and sialic acidcontent than the other alpha subunits (26) or arti-factual differences in the particular two preparations

Metabolic Clearance and Secretion Rates of hTSH Subunits 513

200

180

160

140

120

100

0hTSH- a (0)r - 0.46P< 0.02

00.

0 0

00

0

80- , H hTSH-(0)0 r9-0. r0.28

60~~~ 0600. 9 P>0*100 0 o 0

40 h~\TSH20

~~~~00r a0.612-0 J*# ~~~~P<0O.001

0 20 40 60 80 100 120 140 160 180

CREArTIN/NE CLEARANCE(mi/m/n )FIGURE 6 Correlation of creatinine clearance with MCR(milliliters per minute) of hTSH-a(a) and of hTSH-,8 (0). MCRis here expressed as milliliters per minute since creatinineclearance is dependent on body surface area. The regression line for the correlationbetween creatinine clearance and hTSH MCRis reproduced from our previously publisheddata (11).

3

2 p 41.85 ± 0.42( mean ± SD)

1~~~~~~~~~~~~08±0.17.4(mean ± SD)

8 16 24 32 40 48 56 64 72SAMPLES AT 20 min INTERVALS

43.25 ± 0.32

Q.4 34 mean SD)

2

1' 0.85±0.46(mean ±SD)

i2 3 4 5HOURS

FIGURE 7 Serum alpha concentrations in a representative normal postmenopausal woman(A) and a premenopausal woman (0) every 20 min for 1 day (upper panel). Serum alphaconcentrations in representative study subjects, a postmenopausal hypothyroid woman (A)and a normal premenopausal woman (0) every 30 min for 4Y2 h (lower panel). Alphafluctuations were only slightly greater than could be accounted for by the 8% coefficientof variation in the alpha immunoassay.

514 1. Kourides, R. Re, B. Weintraub, E. Ridgway, and F. Maloof

of alpha subunits arising during their dissociationand purification from complete glycoprotein hormones.In addition, hTSH-a had a faster MCRthan hTSH-,8in all the individuals studied (Figs. 3, 4) even thoughboth subunits have approximately the same molecularweight and hTSH-a has a greater carbohydrate content(27). It has been shown previously that in certaincases proteins with more carbohydrate, specificallysialic acid, are protected from degradation in the liverand have slower clearances (28). However, our datahave shown that other factors must also be importantin determining the clearance of glycoproteins.

Since MCRof subunits were influenced by bodysurface area (Table I and Fig. 3), all MCRin thisreport have been expressed as milliliters per minuteper square meters. MCRof subunits of hTSH weresignificantly correlated with the serum T4 levels of thesubjects studied (Fig. 5), but not always with the serumT3 levels. MCRof subunits in hypothyroid patients(Fig. 4) were significantly slower than those of normalindividuals; furthermore, study of a larger group ofhyperthyroid patients might have yielded significantlyfaster MCRof subunits than in normal individuals.Because MCRwas also correlated with creatinineclearance (Fig. 6), excretion of subunits in urine wasinvestigated but could not be adequately determinedbecause of renal deiodination of the trace quantitiesof labeled subunits.

The lack of significant diurnal variation in serumalpha concentrations (Fig. 7), in spite of fluctuationsthroughout the day in serum gonadotropins (29, 30),allowed the calculation of daily secretion rates of alphasubunits. Since immunologically indistinguishablealpha subunits are secreted from both hTSH- andgonadotropin-secreting pituitary cells (2, 7), secretionrates of total alpha subunits from the pituitary werecalculated (Table I). Secretion rate of subunits hasbeen used instead of production rate since subunitsproduced in the pituitary can also be utilized to makecomplete glycoprotein hormones.

The patients with primary hypothyroidism had ele-vated daily alpha secretion rates in spite of lowerMCRof hTSH-a, whereas the normal postmenopausalwomenhad elevated alpha secretion rates with normalhTSH-a MCR. The premenopausal hyperthyroidwomen had significantly decreased alpha secretionrates in spite of MCR of hTSH-a not differentfrom normals. The two patients with central hypo-thyroidism, who also had inappropriately low gonado-tropins, had even smaller alpha secretion rates thanthe hyperthyroid patients, presumably because ofdecreased alpha secretion from both gonadotrophs andthyrotrophs (Table I). Therefore, as we had previouslyreported for hTSH (11), changes in serum alphawere due more to altered pituitary alpha secretionrather than to changes in MCR. Secretion rates ofhTSH-,1 were also significantly elevated in patients

with primary hypothyroidism. Wehave previously re-ported that there was a 50-fold increase in hTSHsecretion rates in primary hypothyroidism (11); how-ever, there was only a two-fold increase in alphasecretion rates in primary hypothyroidism (202 ,g/dayper m2) as compared to normal (91 ,tg/day per in2), aswell as in hTSH-,B secretion rates (28 vs. 17 ,ug/dayper m2). Thus, it appears that most of the increasedsynthesis of subunits of hTSH, particularly of theunique beta subunit, is utilized for production of hTSH.Nevertheless, in primary hypothyroidism acute secre-tion of subunits of hTSH after TRH (Table I) wasmore comparable in percent increments to that of comll-plete hTSH (2, 5), suggesting different "pools" of hTSHand subunits involved in acute vs. chronic secretion.

These studies have implications for the mechanismof biosynthesis of the glycoprotein hormones. Thealpha secretion rates were much greater than those ofhTSH-,3 in all patients. Furthermiore, the mean alphasecretion rate of 91 ,ug/day per m2 in normal individualswas also greater than those of both hTSH (11)and hFSH (24) and comparable to that of hLH (23) whenexpressed in molar terms. The high secretion ratesof free subunits have not previously been appreciatedin measurements of serum concentrations because ofthe much more rapid MCRof subunits comparedto complete glycoprotein hormones. Much higher alphathan hTSH-,8 secretion rates were consistent with theprevious finding of a marked predominance in normalpituitary glands of alpha subunits relative to bothhTSH-,B (2) and hLH-,8 (31, 32). Furthermore, certainpituitary tumors have been shown to secrete largequantities of alpha subunit without hTSH-,8 (7). Inaddition, there have been previous reports of isolatedproduction of either alpha or beta subunit (18, 33), aswell as differential response of alpha and beta sub-units of hTSH in response to thyroid hormone (2).These data, as well as our current findings, are con-sistent with the concept that alpha and beta subunitsof the glycoprotein hormones are independentlysynthesized.

ACKNOWLEDGMENTS

The authors are grateful to Mary Ann L. Martorana fortechnical assistance, to Dr. R. Boyar for serum samplesprovided for measurement of diurnal alpha fluctuations, toDr. C. H. Li for hTSH-a, and to Dr. 0. Bahl for hCG-a. Weappreciate various reagents provided by the hormone distribu-tion program of the National Pituitary Agency. We wouldalso like to thank Dr. B. Mordicai of Abbott Laboratoriesfor TRH and Dr. J. Guy Rochefort of Ayerst Laboratoriesfor LHRH.

This investigation was supported by U. S. Public HealthService grants CA-18280 and AM-16791.

REFERENCES

1. Kourides, I. A., B. D. Weintraub, M. A. Levko, and F.Maloof. 1974. Alpha and beta subuinits of human thyrot-

Metabolic Clearance and Secretion Rates of hTSH Subunits 515

ropin: Purification and development of specific radio-immunoassays. Endocrinology. 94: 1411-1421.

2. Kourides, I. A., B. D. Weintraub, E. C. Ridgway, and F.Maloof. 1975. Pituitary secretion of free alpha and betasubunit of human thyrotropin in patients with thyroiddisorders.J. Clin. Endocrinol. Metab. 40: 872-885.

3. Vaitukaitis, J. L., G. T. Ross, L. E. Reichert, Jr., andD. N. Ward. 1972. Immunologic basis for within andbetween species cross-reactivity of luteinizing hormone.Endocrinology. 91: 1337-1342.

4. Binoux, M., J. G. Pierce, and W. D. Odell. 1974. Radio-immunological characterization of human thyrotropin andits subunits: Applications for the measurement of humanTSH.J. Clin. Endocrinol. Metab. 38: 674-682.

5. Kourides, I. A., B. D. Weintraub, E. C. Ridgway, andF. Maloof. 1973. Increase in the beta subunit of humanTSH in hypothyroid serum after thyrotropin releasinghormone.J. Clin. Endocrinol. Metab. 37: 836-840.

6. Hagen, C., and A. S. McNeilly. 1975. The specificityand application of a radioimmunoassay for the a-subunitof luteinizing hormone in man. Acta Endocrinol. 78:664-674.

7. Kourides, I. A., B. D. Weintraub, S. W. Rosen, E. C. Ridg-way, B. Kliman, and F. Maloof. 1976. Secretion of alphasubunit of glycoprotein hormones by pituitary adenomas.

J. Clin. Endocrinol. Metab. 43: 97-106.8. Hagen, C., and A. S. McNeilly. 1975. Changes in circulat-

ing levels of LH, FSH, LH/3- and a-subunit after gonado-tropin-releasing hormone, and of TSH, LHJ3- and a-subunit after thyrotropin-releasing hormone. J. Clin.Endocrinol. Metab. 41: 466-470.

9. Edmonds, M., M. Molitch, J. G. Pierce, and W. D. Odell.1975. Secretion of alpha subunits of luteinizing hormone(LH) by the anterior pituitary.J. Clin. Endocrinol. Metab.41: 551-555.

10. Edmonds, M., M. Molitch, J. G. Pierce, and W. D. Odell.1975. Secretion of alpha and beta subunits of TSH bythe anterior pituitary. Clin. Endocrinol. 4: 525-530.

11. Ridgway, E. C., B. D. Weintraub, and F. Maloof.1974. Metabolic clearance and production rates of humanthyrotropin. J. Clin. Invest. 53: 895-903.

12. Kourides, I. A., R. N. Re, B. D. Weintraub, E. C. Ridg-way, and F. Maloof. 1975. Metabolic clearance (MCR)and production rates (PR) of subunits of human thyrot-ropin (hTS H), Endocrinology. 96: 155. (Abstr.)

13. Goldfine, I. D., S. M. Amir, A. W. Petersen, and S. H. Ing-bar. 1974. Preparation of biologically active '25I-TSH.Endocrinology. 95: 1228-1233.

14. Greenwood, F. C., W. M. Hunter, and J. S. Glover.1963. The preparation of 131I-labelled growth hormoneof high specific radioactivity. Biochem. J. 89: 114-123.

15. Tait, J. F. 1963. Review: The use of isotopic steroidsfor the measurement of production rates in vivo. J. Clin.Endocrinol. Metab. 23: 1285-1297.

16. Ridgway, E. C., B. D. Weintraub, J. L. Cevallos, M. C.Rack, and F. Maloof. 1973. Suppression of pituitaryTSH secretion in the patient with a hyperfunctioningthyroid nodule.J. Clin. Invest. 52: 2783-2792.

17. Rosen, S. W., and B. D. Weintraub. 1971. Monotropicincrease of serum FSH correlated with low sperm countin young men with idiopathic oligospermia and aspermia.

J. Clin. Endocrinol. Metab. 32: 410-416.18. Weintraub, B. D., and S. W. Rosen. 1973. Ectopic

production of the isolated beta subunit of humanchorionic gonadotropin. J. Clin. Invest. 52: 3135-3142.

19. Gautvik, K. M., A. H. Tashjian, Jr., I. A. Kourides,B. D. Weintraub, C. T. Graeber, F. Maloof, K. Suzuki,and J. E. Zuckerman. 1974. Thyrotropin-releasing hor-mone is not the sole physiologic mediator of prolactinrelease during suckling. N. Engl. J. Med. 290: 1162- 1165.

20. Odell, W. D., R. D. Utiger, J. F. Wilber, and P. G.Condliffe. 1967. Estimation of the secretion rate of thyrot-ropin in man.J. Clin. Invest. 46: 953-959.

21. Pepperell, R. J., D. M. de Kretser, and H. G. Burger.1975. Studies on the metabolic clearance rate and produc-tion rate of human luteinizing hormone and on the initialhalf-time of its subunits in man. J. Clin. Invest. 56: 118-126.

22. Ridgway, E. C., F. R. Singer, B. D. Weintraub, L. Lorenz,and F. Maloof. 1974. Metabolism of human thyrotropinin the dog. Endocrinology. 95: 1181-1185.

23. Kohler, P. O., G. T. Ross, and W. D. Odell. 1968.Metabolic clearance and production rates of humanluteinizing hormone in pre- and postmenopausal women.

J. Clin. Invest. 47: 38-47.24. Coble, Y. D., Jr., P. 0. Kohler, C. M. Cargille, and G. T.

Ross. 1969. Production rates and metabolic clearancerates of human follicle-stimulating hormone in premeno-pausal and postmenopausal women. J. Clin. Invest. 48:359-363.

25. Wide, L., E. Johannisson, K. -G. Tillinger, and E. Dicz-falusy. 1968. Metabolic clearance of human chorionicgonadotropin administered to nonpregnant women. ActaEndocrinol. 59: 579-594.

26. Bellisario, R., R. B. Carlsen, and 0. P. Bahl. 1973.Human chorionic gonadotropin. Linear amino acid se-quence of the a-subunit. J. Biol. Chem. 248: 6796-6809.

27. Cornell, J. S., and J. G. Pierce. 1973. The subunits ofhuman pituitary thyroid stimulating hormone. Isolation,properties, and composition. J. Biol. Chem. 248: 4327-4333.

28. Ashwell, G. 1973. The role of sialic acid in the im-munological and biological properties of glycoproteinhormones. In Proceedings of the 4th InternationalCongress of Endocrinology. R. 0. Scow, editor. ExcerptaMedica, Amsterdam. 601-604.

29. Boyar, R., M. Perlow, L. Hellman, S. Kapen, and E. Weitz-man. 1972. Twenty-four hour pattern of luteinizing hor-mone secretion in normal men with sleep stage record-ing.J. Clin. Endocrinol. Metab. 35: 73-81.

30. Naftolin, F., H. L. Judd, and S. S. C. Yen. 1973. Pulsa-tile patterns of gonadotropins and testosterone in man:The effects of clomiphene with and without testosterone.

J. Clin. Endocrinol. Metab. 36: 285-288.31. Prentice, L. G., and R. J. Ryan. 1975. LH and its sub-

units in human pituitary, serum and urine. J. Clin.Endocrinol. Metab. 40: 303-312.

32. Hagen, C., and A. S. McNeilly. 1975. Identification ofhuman luteinizing hormone, follicle-stimulating hor-mone, luteinizing hormone S-subunit and gonadotrophina-subunit in foetal and adult pituitary glands. J. Endo-crinol. 67: 49-57.

33. Rosen, S. W., and B. D. Weintraub. 1974. Ectopic produc-tion of the isolated alpha subunit of the glycoproteinhormones. A quantitative marker in certain cases ofcancer. N. Engl. J. Med. 290: 1441-1447.

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