Incidence of antibodies blocking thyrotropin effect in vitro in patients with euthyroid or hypothyroid autoimmune thyroiditis

Autoantibodies blocking the TSH-dependent production of cAMP in thyroid cells (TSH-BAb) have been described in atrophic thyroiditis (AT; idiopathic myxedema) and in neonates with transient hypothyroidism, but their incidence in autoimmune thyroiditis in relation to thyroid status remains to be completely established. To this purpose TSH-BAb were evaluated in a group of 140 consecutive patients with autoimmune thyroiditis, which included 26 cases of AT and 114 subjects with goitrous Hashimoto's thyroiditis (HT); among the goitrous group 27 were euthyroid (HT-E), 32 had subclinical hypothyroidism (HT-SH), and 55 had clinical hypothyroidism (HT-H). TSH-BAb were measured in immunoglobulin G prepared by DEAE-Sephadex A-50 by determining their ability to inhibit TSH-dependent cAMP production in a differentiated strain of cultured rat thyroid cells (FRTL-5). Using this sensitive and reproducible method, TSH-BAb were detected in 12 of 26 (46%) patients with AT, in 1 of 27 (3.7%) subjects with HT-E, in 3 of 32 (9.4%) with HT-SH, and in 20 of 55 (36%) with HT-H. The prevalence of TSH-BAb was higher in AT vs. HT-H (P less than 0.001), HT-SH (P less than 0.001), or HT-E (P less than 0.001), and in HT-H vs. HT-SH (P less than 0.001) or HT-E (P less than 0.001). Mean TSH-BAb levels in AT were higher than those in HT-H (P less than 0.005) and HT-SH (P less than 0.025); the difference was not significant between HT-H and HT-SH. An inverse correlation was found between TSH-BAb levels and estimated goiter weight (P less than 0.005). The results of the present study indicate that 1) in autoimmune thyroiditis TSH-BAb are detectable almost exclusively in hypothyroid patients, their prevalence being higher in overt hypothyroidism than in subclinical thyroid failure; 2) the prevalence of TSH-BAb and their mean levels are higher in hypothyroid patients with AT than in those with HT; and 3) therefore, the presence of circulating TSH-BAb appears to be related to the development of hypothyroidism and thyroid atrophy.     

Peripheral metabolism of homologous thyrotropin in euthyroid and hypothyroid rats: acute effects of thyrotropin-releasing hormone, triiodothyronine, and thyroxine.

The peripheral metabolism and metabolic clearance rate (MCR) of homologous TSH was studied in euthyroid and hypothyroid rats. Incubation of freshly labeled [125I]iodo-TSH with rat serum revealed a labeled nonimmunoreactive protein in the void volume of a Sephadex G-100 column which could not be detected by conventional chromatographic purification. Removal of this contaminant from the tracer reduced the nonspecific binding in the absence of serum and increased the binding of tracer in the absence of added exogenous TSH. Injection of [125I]iodo-TSH into rats was followed within 15 min by the appearance of at least three labeled protein components. Gel filtration showed that these peaks were trichloroacetic acid (TCA)-precipitable proteins of larger molecular weight than TSH, but not all were precipitable by antibody to rat TSH. The disappearance rate of TCA-precipitable 125I (t1/2 = 28 min) was significantly longer than the disappearance rate of immunoprecipitable 125I (t1/2 = 22 min). The disappearance rate of immunoprecipitable [125I]iodo-TSH was identical to that of injected purified rat TSH and of the TRH-induced TSH increment in euthyroid rats. The disappearance rate os suppressible TSH (after 100 microgram T3) in hypothyroid animals was only slightly longer than the rate of disappearance of immunoprecipitable [125I]iodo-TSH (40 vs. 36 min) in the same rats. The calculated MCR of TSH was slightly lower (P less than 0.05) in hypothyroid rats (18.3 +/- 3.0 ml/h/100 g BW, mean +/- SD) than it was in euthyroid rats (22.6 +/- 2.1). The pituitary TSH concentration in hypothyroid rats was 29 mU/mg wet wt, similar to that of euthyroid animals. These results indicate that the turnover rate of pituitary TSH in hypothyroid rats with serum TSH concentrations of 1400-3000 microunit/ml is 7-14 times/day. Therefore, the significant increase we observed in pituitary TSH concentration 1 h after T4 (1.5 microgram/100 g BW) or T3 (0.15 microgram/100 g BW) administration indicates that the 35% decrease in plasma TSH at this interval is due to inhibition of TSH release, not to inhibition of TSH synthesis.     

Binding of thyrotropin to lentil lectin is unchanged by thyrotropin-releasing hormone administration in three patients with thyrotropin-producing pituitary adenomas.

Glycoproteins have increased affinity for lentil lectin when fucose residues are bound to N-acetylglucosamine in the "core region" of their asparagine-linked oligosaccharides. In three patients with thyrotropin (TSH)-producing pituitary tumors, the proportion of serum TSH isoforms that bound to lentil (70.8% +/- 15%) was higher than that seen for TSH from normal persons (32.5 +/- 8%). Unlike normal subjects, the concentration of TSH circulating in the tumor patients after acute administration of TSH-releasing hormone (TRH) did not rise, and the TSH did not exhibit increased binding to lentil compared to basal TSH. The TSH binding to lentil in one tumor patient decreased after metoclopramide, but TSH binding to lentil generally remained unchanged after metoclopramide or L-dopa administration. We conclude that human thyrotropic tumor tissue, unlike normal thyrotrophs, generally fails to release more highly fucosylated isoforms of TSH after pharmacologic stimulation, perhaps because the tumor tissue is less readily modulated by endocrine stimuli, or because the TSH is already relatively highly fucosylated.     

Alpha 1-adrenoreceptors and alpha 1-adrenoreceptor-mediated thyrotropin release in cultures of euthyroid and hypothyroid rat anterior pituitary cells

TSH responses to adrenergic agonists have been measured in 3-day monolayer cultures of euthyroid and hypothyroid male rat anterior pituitary (AP) cells. Responses were qualitatively similar in that (-)epinephrine and (-)norepinephrine had the same ED50 in each culture (ED50 = approximately 6 and 16 nM, respectively) and demonstrated the same alpha 1-adrenergic specificity. Hypothyroid cultures secreted approximately twice as much TSH per cell as euthyroid cultures over the 2-h experimental period. (-)Epinephrine produced a 95 +/- 8% (mean +/- SE) release of TSH relative to basal secretion in euthyroid cultures and only 62 +/- 7% release in the hypothyroid cultures (P less than 0.01). The comparable figures for (-)norepinephrine were 62 +/- 7% and 38 +/- 5%, respectively (P less than 0.05). In absolute terms, adrenergic agonists released the same amount of TSH from euthyroid and hypothyroid cultures. In contrast, TRH (and the Ca+2 channel ionophore A23187) released twice as much TSH from the hypothyroid cells as in the euthyroid cultures. Epinephrine-induced TSH release was significantly impaired (P less than 0.001) when either euthyroid or hypothyroid cells were cultured without thyroid hormones. In contrast, TRH-induced TSH release was enhanced (P less than 0.001) in the euthyroid cultures. [3H]Dihydroergocryptine [( 3H]DHE) was used to quantify alpha 1-adrenoreceptors on the same cell preparations as those used to derive the functional data (see above). Prazosin (1 microM) was used to define nonspecific binding of [3H]DHE. Specific binding to euthyroid cells had a Kd of 5.8 +/- 4 nM and a maximum binding capacity of 2.2 +/- 0.4 fmol/10(5) cells (n = 5). In parallel cultures of hypothyroid cells, the Kd (6.2 +/- 5 nM) was not significantly different, whereas the maximum binding capacity (1.4 +/- 0.3 fmol/10(5) cells) was significantly reduced (P less than 0.05). Adrenergic compounds showed a rank order of potency of prazosin greater than (-)epinephrine greater than or equal to (-)norepinephrine greater than or equal to yohimbine greater than clonidine against the binding of 5 nM [3H]DHE to euthyroid and hypothyroid cells. The amount of [3H]DHE binding per cell that each adrenergic compound was able to displace at saturating concentrations was less in hypothyroid cells than in euthyroid cells. There was no change in the ED50 values of these compounds in the same experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic differences shown by HLA typing among Japanese patients with euthyroid Graves'' ophthalmopathy, Graves'' disease and Hashimoto''s thyroiditis: genetic characteristics of euthyroid Graves'' ophthalmopathy

Euthyroid Graves' ophthalmopathy (EO) is an ophthalmic disorder without persistent hyperthyroidism. To elucidate genetic differences among EO, Graves' disease (Gr) and Hashimoto's thyroiditis (H), we analysed HLA-A, B, C, DR, DQ, D and DP types in 23 Japanese EO patients, 88 Gr patients, 46 H patients and 186 control subjects utilizing assays of lymphocyte cytotoxicity and restriction fragment length polymorphism (RFLP). When compared with the control subjects, EO patients showed significant associations with HLA B40 (w61), DR9, DQw3, and Dw15 (P less than 0.01) and with HLA B12 and Cw1 (P less than 0.05). When allowance was made for the number of antigens tested, only DQw3 was significant. Significant differences were found between EO and Gr (DPw2), and between EO and H (Cw1) even after correction of P values. Comparisons between EO and related subgroups of Gr confirmed the heterogeneity of EO again. It is concluded from these results that EO is associated with different HLA types from Gr and H.     

Increased arterial and venous plasma noradrenaline levels in patients with primary hypothyroidism during hypothyroid as compared to euthyroid state.

The use of venous plasma noradrenaline levels as a marker of general sympathetic tone has been questioned as changes in local sympathetic activity may influence the venous levels. To compare arterial and venous plasma noradrenaline levels in patients with primary hypothyroidism, arterial and venous blood were sampled during strictly standardized conditions during hypothyroid and euthyroid states. The patients were hospitalized for 5 days at a metabolic ward on a standardized sodium and potassium intake. On the fourth day catheters were positioned in the axillary artery and vein. Blood samples were drawn simultaneously for noradrenaline and adrenaline determinations during resting conditions. The arterial and venous plasma noradrenaline levels did not differ significantly, neither during hypothyroidism nor during euthyroidism. The arteriovenous difference in plasma adrenaline was similar during hypothyroidism compared to euthyroidism, indicating similar peripheral extraction rate of catecholamines during hypothyroidism as compared to euthyroidism. During hypothyroidism venous and arterial noradrenaline were significantly higher as compared to euthyroidism. In conclusion, there is no difference between arterial and venous noradrenaline levels either in the hypothyroid or the euthyroid state, and the peripheral extraction rate of plasma noradrenaline seems to be similar in hypothyroidism and euthyroidism. The local contribution of noradrenaline from the arm, reflecting local sympathetic nervous activity, is limited during resting conditions. In hypothyroid patients plasma noradrenaline levels are increased as compared to the euthyroid state, indicating increased general sympathetic activity in hypothyroidism.     

The effect of prednisone on serum thyrotropin, thyroxine and triiodothyronine concentrations in hypothyroid patients.

The effect of exogenous prednisone on serum thyrotropin (TSH), thyroxine (T4), and triiodothyronine (T3) concentrations was investigated in four patients with non-functioning thyroid glands receiving T4 replacement therapy. Orally administered prednisone, in a dose of 20 mg each day for nine days, resulted in a significant decrease in mean serum TSH levels (p less than 0.01) without significant changes in levels of serum T4, T3, and thyroxine binding globulin (TBG). These findings suggest an inhibitory action of relatively low pharmacologic doses of prednisone on TSH release without changes in circulating thyroid hormone concentrations or inhibition of the peripheral conversion of T4 to T3.     

Free thyroxine, free triiodothyronine, and thyrotropin concentrations in hypothyroid and thyroid carcinoma patients receiving thyroxine therapy

Free thyroxine (FT4) and free triiodothyronine (FT3) concentrations in serum were measured by direct equilibrium dialysis methods in patients receiving thyroxine replacement or suppression therapy. Four of 50 hypothyroid patients euthyroid on replacement therapy (mean thyroxine dose 120 micrograms/day) had supranormal FT4 concentrations, whereas the FT3 concentrations were normal in all. Forty-one of 56 operated thyroid carcinoma patients on suppressive therapy (mean thyroxine dose 214 micrograms/day) had raised FT4 concentrations, whereas the FT3 concentrations was elevated in only one patient. There was a large difference in mean FT4 values for hypothyroid and thyroid carcinoma patients (17.2 vs 29.5 pmol/l), whereas the difference in mean FT3 values was small (5.0 vs 6.1 pmol/l), suggesting a decreased peripheral conversion of T4 to T3 with increasing concentrations of FT4. Serum TSH concentrations, as determined by an immunoradiometric assay, varied from less than 0.02 to 11.9 mU/l in treated hypothyroid patients; 21 patients (42%) had values outside the reference limits. As a single test, serum TSH is therefore not very useful for the assessment of adequate thyroxine dosage in patients with primary hypothyroidism. In thyroid carcinoma patients, the TSH concentrations were less than 0.18 mU/l; 45 patients had values less than 0.02 mU/l indicating sufficient suppression of TSH secretion in the majority of cases. On the basis of these results we recommend the combination of FT3 and TSH tests for monitoring thyroxine replacement and suppression therapy. FT4 appears less useful than FT3 for this purpose even if special reference values values were adopted for each patient group.     

Differences in the metabolic clearance of pituitary and serum thyrotropin (TSH) derived from euthyroid and hypothyroid rats: effects of chemical deglycosylation of pituitary TSH

We compared the MCR, volume of distribution, and rapid phase (rt1/2) and slow phase half-lives of purified pituitary rat (r) TSH, TSH from crude pituitary extracts of normal and hypothyroid rats, TSH from hypothyroid rat sera, and TSH secreted from hypothyroid rat pituitaries incubated in vitro. For 3 h after iv bolus injection into euthyroid rats, 125I-labeled rTSH was determined by acid precipitation in serum and various organs, and unlabeled TSH was measured by RIA. The MCR of TSH from normal pituitary extracts (0.53 +/- 0.02 ml/min) was similar to that of unlabeled purified rTSH (0.52 +/- 0.03), while those from hypothyroid pituitary extracts (0.32 +/- 0.03) and hypothyroid sera (0.33 +/- 0.01) were decreased. The reduced MCR of TSH from hypothyroid pituitaries was due to a decreased distribution volume (8.4 +/- 0.6 ml) compared to that from normal pituitaries (11.4 +/- 0.7) and hypothyroid sera (10.9 +/- 0.8). The decreased MCR of circulating TSH from hypothyroid sera reflected an increase in its rt1/2 (12.6 +/- 0.5 min) vs. that from both normal (5.1 +/- 0.5) and hypothyroid (5.7 +/- 0.4) pituitaries. The rt1/2 of secreted TSH from incubated hypothyroid rat pituitaries (8.5 +/- 0.9) was intermediate between those of circulating and pituitary forms of hypothyroid rTSH. The clearances of intact bovine TSH (bTSH) and deglycosylated bTSH (dg-bTSH) were compared. The dg-bTSH MCR was found to be increased (0.71 +/- 0.02 ml/min) compared to that of bTSH (0.59 +/- 0.02), primarily due to a decreased rt1/2 for dg-bTSH (3.8 +/- 0.1 min) vs. bTSH (4.7 +/- 0.2). Uptake of purified [125I]rTSH was highest in the kidney at all times, varying from 43% of the injected dose at 5 min to 54% at 180 min. We conclude that in the euthyroid rat, 1) the metabolic clearance of TSH differs between pituitary and serum forms and appears to depend on specific molecular features that vary with the physiological state of the animal from which the TSH is derived; 2) since chemical deglycosylation increased the clearance of TSH, we speculate that the chemical basis for changes in TSH clearance may be related to alterations in its carbohydrate structure; and 3) for normal pituitary TSH, the kidney is the major organ of clearance.     

Changes in the sialylation and sulfation of secreted thyrotropin in congenital hypothyroidism.

We have examined the oligosaccharide structure of secreted thyrotropin (TSH) in perinatal and mature rats with congenital primary hypothyroidism. Rat pituitaries from euthyroid control animals and those rendered hypothyroid by methimazole treatment were incubated with [3H]glucosamine in vitro. Secreted TSH was purified, and oligosaccharides were enzymatically released and characterized by anion-exchange HPLC. In perinatal hypothyroid animals compared with control animals, oligosaccharides from TSH alpha and beta subunits contained more species with three or more negative charges. Moreover, perinatal hypothyroid animals demonstrated a dramatic increase in the ratio of sialylated to sulfated species within oligosaccharides of the same negative charge (2.9- to 7.4-fold increase for TSH-alpha; 15.1- to 25.5-fold increase for TSH-beta). In mature hypothyroid 9-week-old animals compared with control animals, changes were less pronounced, suggesting that endocrine regulation of oligosaccharide structure is dependent upon the maturational state of the animal. These changes were specific for TSH because glycosylation of free alpha subunit (synthesized by the thyrotroph and gonadotroph) and of total glycoproteins was minimally altered by hypothyroidism. Together, these data provide direct evidence and characterization of specific changes in the structure of a secreted pituitary glycoprotein hormone occurring as a result of in vivo endocrine alterations during early development. Moreover, they provide a potential structural basis to explain the delayed clearance of both TSH and the gonadotropins with end-organ deficiency, which may have important implications for the in vivo biological activities of these hormones. Specifically, such posttranslational changes may be an important adaptive response to prevent the consequences of endocrine deficiency during early development.     

Effects of thyroid status on thyroid autoimmunity expression in euthyroid and hypothyroid patients with Hashimoto's thyroiditis

OBJECTIVE In patients with hypothyroid goitrous Hashimoto's thyroiditis, the recovery from hypothyroidism seems to be due to a spontaneous decrease of antibodies (Ab) to the TSH-receptor (R). In contrast, in patients with Graves' disease made euthyroid by antithyroid drug therapy, the suppression of TSH secretion by thyroid hormone during antithyroid drug treatment decreases the production of Ab to TSH-R. We investigated in patients with initially euthyroid or hypothyroid goitrous Hashimoto's thyroiditis the relationships between thyroid status and the serum TSH-R, peroxidase (TPO) and thyroglobulin (Tg) Ab concentrations in untreated or l -thyroxine (T4) treated patients. PATIENTS A prospective study of 174 consecutive patients, referred with goitrous Hashimoto's disease in an initially euthyroid (group I, n= 78) or hypothyroid (group II, n= 96) state. The patients with positive (±=7%) TSH-RAb (group I, n = 18; group II, n = 22) were reinvestigated 12 months after the initiation of L-T4 therapy. After which, (1) L-T4 was continued and an evaluation performed 2 months later (i.e. 14 months after l -T4 initiation) in 9 patients of group I and in 11 patients of group II or (2) l -T4 was withdrawn and an evaluation performed 2 months later in 9 patients of group I and in 11 patients of group II. MEASUREMENTS Measurements of basal plasma TSH, free T4 (FT4) and total T3 and serum TSH-R, TPO and TgAb. RESULTS The prevalence of positive TSH-RAb levels did not differ between group I (23.1%) and group II (22.9%). However, the mean TSH-RAb level in group I (9.4 ± 0.4%) was lower (P<0.01) than in group II (11.6 ± 0.5%). In the patients with positive TSH-R Ab, (1) the prevalences of positive TSH-RAb decreased (P<0.001) under l -T4 therapy (group I = 22.2%, group II = 21.2%) and increased again (P<0.01) 2 months after l -T4 cessation (group I = 77.7%, group II = 63.6%) to reach lower levels (group I, P<0.05; group II, P<0.01) than those obtained prior to l -T4 treatment. Statistical analysis of TSH levels through the course of the study confirmed these results. (2) In contrast to the variations of the mean TgAb values, the variations of the mean TPOAb levels in each group were in good agreement with those of TSH-RAb through the course of the study. (3) There were significant correlations between some parameters of thyroid status and both TSH-RAb (TSH, r= 0.43, P< 0.001; FT4, r=-0.35, P<001) and TPOAb (TSH, r= 0.42, P<0.001; FT4, r= -0.31, P<0.01) levels. In contrast, no correlations were found between thyroid status and TgAb values. CONCLUSIONS This study demonstrates that thyroid status can modulate thyroid autoimmunity expression, such as TSH-RAb and TPOAb, in patients with euthyroid or hypothyroid goitrous Hashimoto's thyroiditis. Similar results have been reported in patients with Graves' disease made euthyroid by the administration of thyroid hormone during antithyroid drug treatment.     

Microheterogeneity of thyroid-stimulating hormone from the pituitaries of euthyroid, hypothyroid and hyperthyroid rats.

The microheterogeneity of pituitary thyroid-stimulating hormone (TSH) is dependent on variations in the hormone's carbohydrate moieties. In this study, changes in the pattern of heterogeneity have been assessed by chromatofocusing, which separates the isospecies on the basis of their isoelectric points (pI). Rats (n = 6 per group) were either untreated or rendered hypo- or hyperthyroid by including in the drinking water either propylthiouracil (0.05% for 8 weeks) or thyroxine (T4; 4 mg/l for 6 weeks) before they were killed at 16 weeks. On autopsy, serum TSH and total T4 were (means +/- S.E.M.): 2 +/- 0.3 micrograms TSH/l and 64 +/- 5 nmol T4/l (control); < 1 microgram TSH/l and 133 +/- 6 nmol T4/l (hyperthyroid); 58 +/- 6 micrograms TSH/l and 32 +/- 6 nmol T4/l (hypothyroid). The pituitaries were individually homogenized and the TSH isoforms separated by chromatofocusing over a pH range of 7-4. Fractions were assayed for TSH by radioimmunoassay. TSH from the control group was distributed into seven major peaks with pI values of (means +/- S.E.M., n = 6) 6.9 +/- 0.1, 6.6 +/- 0.1, 6.2 +/- 0.1, 5.8 +/- 0.1, 5.5 +/- 0.1, 5.2 +/- 0.1 and 4.8 +/- 0.1; 7 +/- 3% of the TSH had a pI of < 4.0. Six peaks of TSH were conserved in the hypothyroid group (with pI values of 6.8 +/- 0.1, 6.5 +/- 0.1, 6.2 +/- 0.1, 5.8 +/- 0.1, 5.4 +/- 0.1 and 5.2 +/- 0.1), and 11 +/- 4% of the hormone had a pI of < 4.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of recombinant human thyrotropin receptors versus porcine thyrotropin receptors in the thyrotropin binding inhibition assay for thyrotropin receptor autoantibodies.

Thyrotropin receptor autoantibodies (TRAb) are most commonly measured in a thyrotropin-binding inhibition (TBI) assay using solubilized porcine thyrotropin receptors (pTSHR). Recently, we reported modifications in recombinant human thyrotropin receptor (hTSHR) production and extraction that made substitution of this antigen for the pTSHR practical. We now report the first comparison of the behavior in a TBI assay of the recombinant, solubilized hTSHR with the pTSHR in a large series of clinically characterized patients with autoimmune thyroid disease. We studied 227 patients with Graves' disease (32 untreated patients, 156 patients receiving antithyroid medications, 24 patients in remission, 9 patients with recurrence of disease, and 6 thyroidectomized patients), as well as 32 patients with Hashimoto's thyroiditis and 28 normal individuals. In patients with untreated Graves' disease, 29 of 32 (90.6%) were TBI positive with either antigen, although two sera gave discrepant data in the two assay. Of the patients receiving antithyroid drugs, 94 of 156 (60.3%) were positive with the pTSHR and 106 of 156 (67.9%) were positive with the hTSHR TBI assay (p < 0.05%). In all other respects, however, there was no difference between the two TBI assays. Neither assay performed well in providing clinical guidance in the remission or relapse of disease. Of the 24 Graves' patients in remission, 75.0% and 79.2% were TBI negative with the hTSHR and pTSHR assays, respectively. The TBI assay at the time of relapse was even less informative; 6 of 9 (66.7%) being TBI negative in the pTSHR assay and 3/9 (33.3%) being negative in the hTSHR assay. In TBI assays with both species of TSHR, 3 of 32 hypothyroid patients with Hashimoto's thyroiditis were TBI positive. In summary, production of the recombinant hTSHR is now a practical reality and this antigen can clearly substitute at least as well for the pTSHR in the imperfect, although most commonly used, TBI assay. It is, therefore, likely that the hTSHR will supplant the pTSHR in this important assay. However, the use of the hTSHR rather than pTSHR does not appear to provide a major advantage, at least in terms of TBI assay sensitivity, specificity and predictive value.     

Osmotic control of the release of prolactin and thyrotropin in euthyroid subjects and patients with pituitary tumors.

The effects of acute changes in serum osmolality on basal serum PRL and TSH levels and on responses of prolactin (PRL) and thyrotropin (TSH) to the thyrotropin-releasing hormone (TRH) analogue, N3im-methyl-TRH, were studied in ten euthyroid subjects and in three patients with PRL-secreting pituitary tumors. An oral water load of 20 ml/kg had no effect on basal serum PRL or TSH levels but did result in an increased PRL response to methyl-TRH in the ten euthyroid patients. Intravenous infusion of 5% sodium chloride in the ten euthyroid subjects significantly depressed basal serum PRL levels but had no effect on the PRL response to methyl-TRH. Infusion of hypertonic saline significantly decreased the TSH response to methyl-TRH. In the three patients with pituitary tumors, oral water loading and hypertonic saline infusion had no significant effect on the basal serum PRL and TSH or the PRL and TSH responses to methyl-TRH. The patients with pituitary tumors had a higher basal serum osmolality and a proportionately higher serum concentration of arginine vasopressin than the euthyroid patients. These data suggest that changes in osmolality in euthyroid patients may have a direct effect on the anterior pituitary's PRL and TSH response to a releasing factor.