Assessment of Thyrotropin-Releasing Hormoneand Thyrotropin Reserve in Man

PETERA. SINGERand JOHNT. NICOLOFFFrom the Department of Medicine, University of Southern California School ofMedicine and the Los Angeles County-USC Medical Center,Los Angeles, California 90033

A B S T R A C T Endogenous thyrotropin-releasing hor-mone (TRH) reserve and pituitary thyrotropin (TSH)reserve were assessed in four normal subjects, threepatients post-cryohypophysectomy, one patient with ahypothalamic lesion secondary to trauma, and four pa-tients with Sheehan's syndrome. TSH reserve was de-termined by the immunoassayable TSH response to 500Ag TRH given i.v. (TRH stimulation test). TRH re-serve was assessed by the rebound response in thyroidaliodine release (TIR) following withdrawal of phar-macologic doses of prednisolone (glucocorticoid with-drawal test). When compared with normals, the post-cryohypophysectomy patients demonstrated parallel im-pairment of TRH stimulation and glucocorticoid with-drawal testing. The patient with the hypothalamic lesionand the four patients with Sheehan's syndrome all hadnormal TRH stimulation tests, indicating adequate TSHreserve capacity, yet had abnormal glucocorticoid with-drawal tests, indicative of impairment in endogenousTRH reserve or neurohumoral transport. Three of thepatients (hypothalamic injury and two Sheehan's) withimpaired TRHreserve were euthyroid.

The following conclusions were reached: (a) A com-bination of the TRH stimulation test and glucocorticoidwithdrawal test may allow for differentiation betweenpituitary and suprahypophyseal disorders. (b) Certaincases of Sheehan's syndrome appear to have impairedendogenous TRH reserve or transport. (c) Euthyroid-ism can be maintained in spite of diminished TRHreserve.

INTRODUCTION

The availability of synthetic thyrotropin-releasing hor-mone (TRH)1 for clinical investigation has led to the

Received for publication 13 November 1972 and in revisedform 8 January 1973.

' Abbreviations used in this paper: KGS, ketogenic ster-

report of numerous studies concerning the assessmentof pituitary thyrotropin (TSH) reserve capacity innormal man. TRH administration has been employed inthe evaluation of TSH reserve in patients with Graves'disease (1, 2), primary hypothyroidism (1, 3, 4), mono-

trophic TSH deficiency (5), and panhypopituitarism (3,4, 6). Much interest has also been recently directed atthe separation of secondary hypothyroidism into pitui-tary and hypothalamic causes. The patient with hypo-thalamic hypothyroidism is presently defined as havingsubnormal circulating thyroid hormone levels associatedwith normal responses of the thyroid and pituitaryglands to exogenous TSH and TRH stimulation re-

spectively (7, 8). Presumably these patients have a

deficiency in endogenous hypothalamic TRH secretion.This is a conclusion which is reached by indirectionhowever, for as yet, no direct measurement of endogenousTRH secretory capacity is possible. Thus, a diagnosisof hypothalamic hypothyroidism is now established byexcluding thyroidal and pituitary etiologies.

Recently we have proposed a qualitative method forassessing endogenous TRH and TSH reserve in man(9). This method is based on the observation that glu-cocorticoid administration in pharmacologic doses willacutely inhibit endogenous TRH secretion without di-rectly affecting other facets of pituitary or thyroidfunction. Following the withdrawal of glucocorticoidadministration, there is an apparent surge in TRH se-cretion leading to a measurable augmentation in TSH re-lease and secondarily in thyroidal iodine release (TIR).The purpose of the present investigation is to report theusefulness of the surge in TIR following glucocorticoidwithdrawal in conjunction with the assessment of pitui-tary TSH reserve capacity by exogenous TRH adminis-

oid; RAIU, radioactive iodine uptake; T4, thyroxine; TIR,thyroidal iodine release; TRH, thyrotropin releasing hor-mone; TSH, thyrotropin.

The Journal of Clinical Investigation Volume 52 May 1973 -1099-1107 1099

TABLE IEndocrine Function Tests in Patient Groups

Plasma growth Thyroidalhormone Urinary 17KGS excretion 24 hr RAIU

Urinary Base Maxi- Base After After Serum T4t Free T4§ Before AfterPatients Age Sex gonadotropins line mum* line methopyrapone ACTH TSH TSH

mU/24 hr ng/ml mg/24 h pg/100 ml ng/100 ml %Normal Subjects

C. C. 42 F >100 2.3 11.3 8.3 42.9 - 6.8 2.0 -

T. C. 16 F 16-50 <2.0 6.6 8.1 38.2 5.1 1.5 -

B. P. 33 F 16-50 <2.0 9.0 7.5 55.1 6.7 2.0 -

H. F. 30 F 16-50 1.1 7.1 6.9 49.4 5.8 1.6 - -Post-cryohypophysectomy patients

J. Z. 37 M 6-16 2.1 3.8 7.8 20.4 - 2.2 0.8 5F. M. 35 M 6 1.5 1.2 3.9 4.5 - 5.2 2.0 11C. S. 32 F 6 1.5 2.8 4.1 16.4 - 1.4 0.7 4

Hypothalamic injury patientL. L. 23 M 16-50 3.6 52.0 6.1 15.8 - 5.2 1.4 22

Sheehan's syndrome patientsJ. B. 25 F 16-50 <2.0 <2.0 4.2 5.9 24.1 5.4 1.4 17 -D.C. 44 F <6 0.2 1.9 1.1 1.6 8.1 3.0 1.2 21P. T. 23 F <6 2.0 <2.0 4.3 6.6 22.3 2.0 0.9 8 19V. N. 29 F 16-50 <2.0 <2.0 2.3 2.8 26.7 2.0 0.7 17 31

* Following insulin-induced hypoglycemia.$ Expressed as T4 iodine (normal values: 3.0-7.0 .ug/100 ml) (performed by Bio-Science Laboratories, Van Nuys, Calif.).§ Normal values: 1.0-2.3 ng/100 ml (performed by Bio-Science Laboratories).

tration as a more definitive means for distinguishing be-tween pituitary and hypothalamic derangements in thy-roid hormone regulation.

METHODS

Study groups12 subjects were included in this study (see Table I):

Four normal subjects had been referred to our Center forevaluation of possible pituitary lesions but were found tobe free of disease. Of the normals, C. C. was referred forevaluation of headaches and oligomenorrhea; T. C. wasreferred for amenorrhea, which during 6 mo of observation,spontaneously cleared without specific therapy; H. F. wasevaluated for postpartum oligomenorrhea which subse-quently cleared; and B. P. was referred to suspected visualfield impairment which by more extensive examination wasfound not to be present. Of the other study patients, threehad undergone cryohypophysectomy for acromegaly, onepatient had sustained a traumatic hypothalamic injury mani-fested later by a disordered thirst mechanism with hyper-natremia, and four patients had Sheehan's syndrome. Diag-noses in individual cases were made clinically and were con-firmed by appropriate laboratory testing. All studies wereperformed on the Clinical Research Center. of the LosAngeles County-USC Medical Center. Patients who hadbeen taking thyroid medication were withdrawn from thy-roid hormone for at least 4 wk prior to testing.

Pituitary testingGrowth hormone reserve was determined by the rise in

plasma growth hormone in response to insulin-induced hypo-

glycemia (10). Growth hormone determinations were per-formed by a modification of the radioimmunoassay methodof Greenwood and Hunter (11). ACTH reserve was as-sessed by the rise in the 24 h urinary ketogenic steroid(KGS) excretion following the administration of metho-pyrapone given orally on a schedule of 750 mg every 4 hfor a 2 day period. Urinary KGS excretion was determinedby the method of Rutherford and Nelson (12). When themethopyrapone test was abnormal, the adequacy of adrenalfunction was verified by measuring urinary 17KGS excre-tion in response to the intravenous administration of syn-thetic ACTH. Pituitary gonadotropins were assessed bymeasurement of urinary bioassayable gonadotropins (13).

Thyroid testing proceduresSerum TSH values were determined by the double anti-

body radioimmunoassay technique described by Odell, Wil-ber, and Utiger (14). Serum thyroxine was measured bythe competitive protein binding method of Murphy andPattee (15) and free thyroxine estimates by the method ofSterling and Brenner (16) (performed by Bio-Science Lab-oratories, Van Nuys, Calif). 24-h thyroidal radioactiveiodine uptake measurements (RAIU) before and followingTSH stimulation were performed in selected cases.

Thyroidal iodine release (TIR) methodA dual iodine isotopic release method was used to mea-

sure changes in TIR. This method has been described indetail in previous reports (17, 18). To summarize briefly,each subject was administered carrier-free 'fI orally inorder to label the thyroid gland endogenously. The dose of'I administered was calculated on the basis of RAIU

1100 P. A. Singer and J. T. Nicoloff

l1

8

THYROIDALIODINE

RELEASE

125I, '1311

6

4

2

RELEASESLOPE

PHASE

6 8 10 12 14 16DAYS

FIGURE 1 A thyroidal iodine release slope in a normal euthyroid subject is depicted. Thesolid line is drawn through the base-line control slope. The notation "days" on the abscissarepresents time following ['HI]- and [mI]T4 administration. Each numeral coincides withmidnight.

measurements so that 10-15 AiCi would be delivered to thethyroid gland. 50 AGCi of ['Ithyroxine (T4) (specific ac-tivity was greater than 40 ,Ci/ig at time of administration)was then given intravenously to serve as a reference iso-tope. The site of measurement of the thyroid release prod-ucts was the urine. Urinary ratios of `2I/P'lI were usedto detect changes in TIR. Since T4 is deiodinated at a rela-tively constant rate (19), alterations in renal clearance donot change 'lJI/'~I ratio values, and therefore any changesin ratio values would reflect alterations in TIR. Fig. 1depicts a normal TIR release slope following equilibrationof the isotopes. This predictable logarithmic linear slopewe have termed the release slope phase of TIR. All studiesin this investigation were performed during this phase.Superimposed on this linear slope are daily variations inTIR, which are a reflection of the normal circadian varia-tions in serum TSH (9).

Study protocolGlucocorticoid withdrawal testing. After a base-line TIR

slope had been established, each subject was administered20 mg of prednisolone orally every 8 h for 3 days. Gluco-corticoids were then withdrawn and the response of TIRmeasured for. an additional 7-8 days until the presuppressionbase-line slope had been reestablished.

TRH stimulation test. Following completion of the glu-cocorticoid withdrawal test, each subject was administered500 lAg of synthetic TRH in 5 ml of sterile saline over a15-30 s time interval. 10 ml of blood was obtained by anindwelling 19 gauge scalp vein needle at -30 min, 0 min,+ 15 min, + 20 min, + 30 min, + 45 min, + 60 min, and+ 120 min following TRH administration. The blood wascentrifuged shortly after collection and the sera separatedand frozen until TSH determinations were performed.

RESULTS

Table I summarizes the endocrine function tests in boththe normal subjects and the patient groups. Fig. 2 il-lustrates the effects of glucocorticoid administrationand withdrawal on TIR in the four normal subjects.Following administration of prednisolone, there is nor-

mally a rapid suppression of TIR, which is reflected bya decrease in '1I/"I ratio values. Within 12-24 h follow-ing withdrawal of prednisolone, there is a marked surgeand overshoot in TIR with resumption of the originalrelease slope within 4-8 days. The linear interrupted lineconnects the pre-glucocorticoid suppression and post-glucocorticoid withdrawal base-line TIR slopes. A simi-lar pattern of suppression and prompt rebound and over-shoot following withdrawal of glucocorticoids has beenpresent in all of the more than 20 normal subjectswhom we have studied in this manner (9). Note thatin subject H. F. there is suppression of TIR be-low the base line 4 days following glucocorticoidwithdrawal, lasting until 8 days post-steroid withdrawalbefore the control release slope is reestablished. A simi-lar oscillation pattern below baseline following with-drawal of glucocorticoid has been noted in several of theother normal subjects we have studied.

Site and nature of glucocorticoid suppression. Pres-ent evidence suggests that glucocorticoids suppress hy-pothalamic release of TRH. The studies of Wilber and

TRH and TSH Reserve in Man 1101

PREDNfI SOI.0tNF20 rnq

80

X ~~~~~~~~~~~~~~~~~~~~~~~C

1 RH

20-

40 104

SUBJECTS

0

T S HCC

20-~~~~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~0

THYROIDALIODINE

RELEASE

125 131

8 20-

10-

20-

2

-30 0 30 60

MIN'JTES POSTINJECI8 1 0 12 1 4 16 8 20 22

DAYS

FIGURE 2 The effects of glucocorticoid administration and withdrawal on TIR in normaleuthyroid subjects is shown. The broken line is drawn through the base-line control TIRslopes. Also shown are the TSH responses to ixv. administered synthetic TRH. The notation"days" on the abscissa represents time following [11] - and ['SI T4 administration. Eachnumeral coincides with midnight. Prednisolone administered every 8 h.

1102 P. A. Singer and J. T. Nicoloff

TION

PREDN ISO LONE2Omg i. |d

4 4

TRH 100 pg v. PULSE DOSES

4 4 4

4 16 18 20 22 24 26DAYS

FIGURE 3 The TIR response to repetitive doses of i.v. TRH are shown in a-normal euthyroidsubject before and during administration of pharmacological doses of glucocorticoids (de-picted by the shaded area). The notation "days" on the abscissa represents time following["I]- and [131I]T4 administration. Each numeral coincides with midnight. Prednisolone ad-ministered every 8 h.

Utiger (20) in the rat have shown that normal TSHresponsiveness to exogenous TRH was maintained dur-ing a period of glucocorticoid administration, thus elimi-nating the anterior pituitary gland as the site of gluco-corticoid suppression. Wehave confirmed these findingsin man, as shown in Fig. 3, which illustrates that repeti-tive doses of synthetic TRH before and during a periodof glucocorticoid administration produced similar re-

sponses in TIR. Thus, acutely, glucocorticoid administra-tion appears not to significantly alter pituitary respon-siveness to exogenous and presumably endogenous TRHstimulation. However, with chronic glucocorticoid ad-ministration, some reduction in pituitary TSH respon-

siveness to TRHadministration has been reported (21).It seems then that the rebound in TIR following glu-cocorticoid withdrawal is mediated via an increase inendogenous TRH release. Thus, the sudden withdrawalof glucocorticoids, or what we have termed the "gluco-corticoid withdrawal test" would appear to offer an

indirect assessment of endogenous TRH reserve ca-

pacity. Because of the indirect nature of the methodsemployed, it becomes apparent that the glucocorticoidwithdrawal test can serve as a measure of TRH reserve

only when an intact pituitary-thyroid axis is present.Also depicted in Fig. 2 are the TSH responses to intra-venously administered synthetic TRH in the normalcontrol subjects. The TSH values are commensurate

with those obtained in normal subjects by other workersemploying similar doses of TRH. The dose of 500 Ag ofTRHwas used in the present study since previous workhas shown that this is the maximal stimulating doseof pituitary TSH reserve capacity (3, 22).

Glucocorticoid withdrawal and TRH stimulation testsin patients with pituitary lesions. In order to test thehypothesis that diminished endogenous TSH reserveshould be reflected by both abnormal glucocorticoid with-drawal and TRH stimulation tests, three patients whohad undergone cryohypophysectomy for acromegaly were

studied. The degree and severity of pituitary insufficiencyin this group varied (see Table I). Fig. 4 demonstratesthe loss of overshoot in TIR and a rather slow return tothe base-line release slope following glucocorticoid with-drawal in all three of these subjects. While some over-

shoot is observed in patient J. Z., the degree is smalland delayed compared with the normal subjects depictedin Fig. 2. Also noted are the markedly impaired TSHresponses to synthetic TRH administration. It is ap-parent, however, that in spite of the pituitary unrespon-siveness to exogenous TRH, there is some suppressionof TIR following glucocorticoid administration. Thiswould imply that some TSH reserve remains, and thatthis residual TSH secretion can be further diminishedby the suppressive effect of glucocorticoids on hypo-thalamic TRH. Also noteworthy is that one of the sub-

TRH and TSH Reserve in Man 110.3

THYROI DALIODINE

RELEASE

125I/ 131I

SUJ B J ECTS

PREDNISOLONE

20 mgqi- , ....... .... TSH

PU/ml

lOr

1or

Ic.

10~R~-

C S.

8 '0 12 14 6 18DAYS

FIGURE 4 TIR rebound following glucocorticoid withdrawal, as well as TSH response toiv. TRH, is noted to be impaired in patients with primary pituitary insufficiency. The notation"days" on the abscissa represents time following [HI]- and [mI]T4 administration. Eachnumeral coincides with midnight. Prednisolone administered every 8 h.

jects, F. M., is euthyroid. Thus it would also appear thata euthyroid state can be maintained in the presence ofmarkedly diminished TSH reserve. The results of thisstudy then would indicate that patients with primarypituitary insufficiency exhibit the expected parallel im-pairment in glucocorticoid withdrawal and TRH stim-ulation tests, and that a normal glucocorticoid reboundrequires intact pituitary TSH secretion.

Study of a patient with hypothalamic lesion. Fig.5 depicts a study performed on a 23-yr old malewho had sustained a traumatic hypothalamic injury5 yr earlier. This patient was clinically and chemicallyeuthyroid. Noted is the abnormal glucocorticoid with-drawal test and the normal TSH response to TRH ad-ministration. While there is some TIR above the baseline following glucocorticoid withdrawal, clearly the

PREDN I SOLONE20man a

TSH

uU/ml

14 6 le

500tgTRH

20 -

10 .

o . .-30 0 30 60 120

MINUTES POSTINJECTION

DAYS

FIGURE 5 The abnormal TIR response to glucocorticoid withdrawal is contrasted to thenormal response to i.v. TRH in this patient with known hypothalamic lesion. The notation

"days" on the abscissa represents time following ['MI]- and [1KI]T4 administration. Eachnumeral coincides with midnight. Prednisolone administered every 8 h.

1104 P. A. Singer and J. T. Nicoloff

10I

6

THYROIDALIODINE

RELEASE125 3 I

2~

500sgTRH

-30 0 30 60 120MINUTES POSTINJECTION

10

6

THYROI DALIODINE

RELEASE12 IN1

2

0

a lo

I

PREDNISOLONE20mg C

TSH

pU/ml

500ug

8 U D 14 0 0 -30 0 30 60 120DAYS

MINUTES POSTINJECTION

FIGURE 6 Glucocorticoid withdrawal and TRH stimulation responses in four patients withSheehan's syndrome are depicted. Noted is that the TIR response to glucocorticoid with-drawal is abnormal, while TSH responsiveness to TRH is intact. The notation "days" onthe abscissa represents time following ['1]- and [I]T4 administration. Each numeral coin-cides with midnight. Prednisolone administered every 8 h.

response is blunted when compared to the normal sub-jects. This patient then displays a pattern consistentwith diminished endogenous TRH reserve capacity,while retaining normal pituitary TSH reserve. This isat variance with the parallel impairment of tests notedin the patients with pituitary lesions.

Glucocorticoid withdrawal and TRH tests in patientswith Sheehan's syndrome. Fig. 6 depicts the gluco-

corticoid withdrawal tests and TSH responses to TRHadministration in four patients with Sheehan's syndrome.In every case the glucocorticoid withdrawal test is ab-normal, as indicated by the lack of overshoot in TIR andslow return to the base-line release slope. Although allof the patients in this group had abnormal glucocorticoidwithdrawal responses, they all responded normally toadministration of exogenous TRH, indicating adequate

TRH and TSH Reserve in Man 1105

pituitary TSH reserve capacity. Thus, these patientsdisplay patterns similar to that of the patient with thehypothalamic lesion, depicted in Fig. 5, by exhibitingimpaired endogenous TRH reserve, while retaining nor-mal pituitary TSH reserve. Additional evidence for di-minished endogenous TRH reserve or transport is thefact that two of the patients in this group were hypo-thyroid, yet responded normally to TRH stimulation.However, it is also apparent that the euthyroid statecan be maintained in the presence of impaired endoge-nous TRH reserve, since two of the four patients in thisstudy group were euthyroid.

DISCUSSION

The results of this study indicate that the combined useof the glucocorticoid withdrawal test for endogenousTRHreserve and the TRHstimulation test for pituitaryTSH reserve allows for differentiation between cer-tain pituitary and suprahypophyseal lesions. Patientswith pure pituitary lesions exhibit parallel impairmentof both glucocorticoid withdrawal and TRH stimulationtests, in contrast to patients with apparent suprahypo-physeal lesions who only demonstrate impaired gluco-corticoid withdrawal testing, while maintaining normalTSH reserve capacity. The numerous recent studies inman attest to the validity of the use of synthetic TRH toassess endogenous TSH reserve. Employing the gluco-corticoid withdrawal maneuver also seems to be a validmethod for assessing endogenous TRH reserve capacity,as the animal work of Wilber and Utiger (20) and ourpresent study (Fig. 3) provide in vivo evidence for theglucocorticoid suppressive effect on endogenous TRHrelease. Also, recent work by Reichlin (21) providesevidence that TRH synthetase activity is altered by glu-cocorticoid administration. Thus the magnitude and re-bound of TIR noted following glucocorticoid withdrawalwould seem to directly reflect augmented release of en-dogenous TRH.

It would appear that the four patients with Sheehan'ssyndrome have diminished endogenous TRH reserve ortransport rather than primary pituitary lesions affectingthe thyrotroph population. This does not imply that allpatients with this disorder have suprapituitary lesions.Fleischer and coworkers (4) for example, have shownthat administration of 500 ,g of synthetic TRH to sev-eral patients with Sheehan's syndrome produced no de-tectable increase in serum TSH levels. Indeed, pituitarynecrosis often characterizes the pathologic findings inthis disorder (23). On the other hand, it has been shownby Sheehan and Stanfield (24) that the lesion in thisdisorder may occasionally involve only the vascular sup-ply of the pituitary stalk with secondary pituitary in-sufficiency. Sheehan (23) also has shown that the pitui-tary gland is capable of some regeneration and prolifera-

tion of the parenchyma following vascular necrosis. Thefact that pregnancy had occurred in two of our patients,and also has been reported by others (25, 26), adds sul)-port to the contention that not a simple destructive lesionof the pituitary had occurred in this group, as in the post-cryohypophysectomy patients. It would follow then thatother pituitary trophic hormone deficiencies in Sheehan'ssyndrome may involve impaired hypophysiotrophic hor-mone reserve or transport capacity.

Despite the apparent discrimination provided by themethods employed in this study, certain inherent limi-tations are evident. The interpretation of the glucocorti-coid withdrawal test is at present qualitative in nature.No firm conclusions regarding the amount of suppressionby glucocorticoids and the degree of rebound of TIRfollowing glucocorticoid withdrawal can be made bysimple inspection of the graphic data. However, weare currently assessing various measurable parametersin order to quantitate the degree of suppression and re-bound. These data will be included in a subsequent report.Presently though, it seems evident that the differences inrebound responses in normal subjects and those in pa-tients with pituitary and suprahypophyseal disorderscan be appreciated on a qualitative basis. Another metho-dological limitation is that several weeks of frequent dailyurine collections are required as a part of this studyprocedure. This tends to be somewhat cumbersome andtedious, therefore limiting its potential clinical usefulness.

Possibly the most interesting finding in this studywas that euthyroidism could be maintained in spite ofimpaired endogenous TRH reserve. This was apparentin the patient with the hypothalamic lesion and in twoof the four patients with Sheehan's syndrome. Perhapsthe wide distribution of TRH in the anterior and basalportions of the hypothalamus, and the compensating na-ture of the negative feedback action of thyroid hormoneon pituitary release of TSH allows for maintenance of aeuthyroid state in the presence of this apparent dimin-ished TRH reserve. Recent studies performed in othereuthyroid patients with suspected or demonstrated hypo-thalamic lesions have revealed similar findings (27, 28).Thus a future major application of the TRH stimulationand glucocorticoid withdrawal tests may be in the detec-tion of patients with abnormal endogenous TRH re-serve, in whomadequate TSHreserve and normal periph-eral thyroid hormone economy are maintained.

ACKNOWLEDGMENTSThe authors express their gratitude to Dr. Michael S. An-derson, Abbott Laboratories (North Chicago, Ill.) forthe generous supply of synthetic TRH. The authors alsowish to thank Misses Josie Jaramillo and Ursula Finck fortheir excellent technical assistance, and Mrs. Anne Santofor her help in preparing the manuscript.

1106 P. A, Singer and J. T. Nicoloff

This investigation was supported in part by ResearchGrant AM-11727, General Clinical Research Center GrantRR-43, and Research Fellowship AM-53093 from the Na-tional Institutes of Health, Public Health Service.

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TRH and TSH Reserve in Alan 1107