Abnormalities of endogenous glucagon and insulin in unstable diabetes.

The responses of glucagon, growth hormone, and insulin secretion to the oral administration of glucose and to the intravenous infusion of saline, arginine, and insulin were measured in seven patients who had stable diabetes, eight who had unstable diabetes, and seven healthy volunteers. Hyperglycemia suppressed secretion of glucagon in normal subjects but not in diabetics. The oral glucose and arginine infusion tests demonstrated partial preservation of insulin-secretory ability in stable diabetics and its virxual absence in unstable diabetics. Glucagon responses to arginine infusion were similar in all three groups. In response to hypoglycemia induced by insulin infusion, the concentrations of plasma glucagon increased in normal subjects and, to a lesser extent, in stable diabetics but increased in only two of the unstable diabetics. The impairment in glucagon response during hypoglycemia in diabetics correlated positively with the degree of diabetic instability and insulin deficiency during glucose and arginine testing. The severity of the insulin deficiency also correlated with the degree of diabetic instability. These findings support the hypothesis that inherent abnormalities of insulin and glucagon secretion may account for many of the clinical characteristics of unstable and stable diabetic patients.

Glucagon-blood catecholamine test: use in isolated and familial pheochromocytoma.

In 149 cases, blood pressure response to glucagon test did not exceed 20/10 mm Hg more than the response in the cold pressor test control and was considered negative. Plasma catecholamine level increases may be seen in 95% of patients without pheochromocytomas. Among six patients with pheochromocytomas, urinary metanephrine levels were of diagnostic importance in two with isolated pheochromocytoma and in one with the multiple endocrine neoplasia of type 2 (MEN-type 2). Urinary metanephrine determinations yielded false-negative results in three patients with MEN-type 2, while vanilmandelic acid level was normal in one and nephrotomograms were positive in two of these three. These results suggest that the early diagnosis of pheochromocytoma in patients with MEN-type 2 may be difficult and may require multiple biochemical and roentgenographic investigations.

The effects of transforming growth factor-beta on growth and differentiation of the continuous rat thyroid follicular cell line, FRTL-5.

Transforming growth factor-beta (TGF beta) has been shown to influence the growth and differentiation of many widely varied cell types in vitro, including some that are endocrinologically active. We have investigated the previously unknown effects of this unique growth factor in the differentiated rat thyroid follicular cell line FRTL-5. The cells demonstrated specific, high affinity binding of TGF beta, and as with other epithelial cells, the growth of these thyroid follicular cells was potently inhibited by addition of TGF beta to the culture medium. TGF beta caused a significant reduction in TSH-sensitive adenylate cyclase activity in the cells. The addition of (Bu)2cAMP along with the growth factor to cultures partially reversed the characteristic morphological changes seen with TGF beta, but did not reverse the growth inhibition. To further investigate the possible mechanisms of the effects of TGF beta on the cells, we measured the influence of the growth factor on [125I]TSH binding. TGF beta did not compete for specific TSH-binding sites; however, exposure of the cells to TGF beta for 12 or more h resulted in a dose-dependent down-regulation of TSH receptors that was fully reversible. While cellular proliferation was potently inhibited by TGF beta, differentiated function, as manifest by iodine-trapping ability, was stimulated by the growth factor. This stimulation of iodine uptake was independent of, and additive to, the stimulatory effects of TSH. Finally, FRTL-5 cells in serum-free medium and in response to TSH were shown to secrete TGF beta-like activity that competed for [125I]TGF beta in a RRA. These studies suggest that TGF beta may represent an autocrine mechanism of controlling the growth response to TSH in thyroid follicular cells, while allowing the continuance of differentiated function.

The effects of synthetic alpha-subunit peptides on thyrotropin interaction with its receptor.

Synthetic peptides of the alpha-subunit of human glycoprotein hormones have been shown previously to inhibit binding of [125I]iodo-hCG to ovarian membranes, thus indicating the importance of the alpha-subunit in the structure-function relationships of the gonadotropic hormone. These same synthetic alpha-subunit peptides, the sequences of which are common to all human glycoprotein hormones, were found to inhibit the binding of [125I]iodo-TSH to human thyroid membrane preparations and FRTL-5 rat thyroid cells. The active portions of the subunit were represented in synthetic peptides alpha 21-35, alpha 31-45, alpha 26-46, and alpha 81-92, indicating that 2 separate sites within the alpha-subunit have binding activity for TSH. Peptides alpha 26-46 and alpha 31-45 were also found to potently inhibit the stimulation of adenylate cyclase activity by bovine TSH in TSH bioassay using FRTL-5 cells. Seven other synthetic peptides, including the remainder of the 92-amino acid sequence of the alpha-subunit, demonstrated little or no ability to inhibit binding of the tracer or inhibit the bioactivity of intact TSH. The findings were very similar to those of previous studies involving hCG binding, except that the two active sites appeared to be somewhat shifted towards the COOH-terminal end of the subunit. These studies support the concept of the importance of the alpha-subunit in receptor binding of all glycoprotein hormones and demonstrate the utility of the overlapping synthetic peptide strategy in investigations of protein structure-function relationships.

Distribution and metabolism of L- and D-triiodothyronine (T3) in the rat: preferential accumulation of L-T3 by hepatic and cardiac nuclei as a probable explanation of the differential biological potency of T3 enantiomers.

Previous studies have reported markedly lower biological potency of D-T3 than would be predicted from its relative affinity for the specific nuclear L-T3 receptor as measured in vitro. Studies were undertaken to define the basis of this discrepancy. In vitro studies of the relative binding affinities of [125I]L-T3 and [125I]D-T3 using either whole nuclei or partially purified solubilized receptor confirmed several previous reports which had indicated only small relative differences. The mean L/D binding ratio of all in vitro studies was 1.43. In contrast, we determined from measurement of final alpha-glycerophosphate dehydrogenase activity that the potency ratio (L/D) after seven daily injections of graded doses of L- and D-T3 was 6.7 for liver, with 95% confidence limits of 4.1 to 10.8, and 15.9 for heart, with 95% confidence limits of 5.1 to 50.2. Difference in biological potency could not be attributed to differences in the metabolism of L- and D-T3. Thus, tracer [125I]L- and [125I]D-T3 studies showed that the mean residence time, t, of L-T3 was not different from that of D-T3 [10.6 +/- (SE) 3.1 h vs. 11.4 +/- 3.8 h]. The MCR for L-T3 was 19.1 +/- 4.4 ml/h . 100 g BW and for D-T3, 26.1 +/- 5.6 ml/h . 100 g BW. The volume of distribution of L-T3 (181 ml/100 g BW) was 68% that of D-T3 (263 ml/100 g BW), in part owing to the 27% greater plasma binding (bp) of L-T3. Moreover, the calculated free hormone clearance (MCR X bp) of D-T3 was only 8% greater than for L-T3. Throughout the 24 h after injection of the [125I]L- or D-T3, concentrations of the D-enantiomer in liver were almost twice those of [125I]L-T3, whereas in heart [125I]D-T3 concentrations were half or less those of [125I]L-T3. Nevertheless, in both liver and heart, the integrated nuclear occupancy of L-T3 was 5 to 6 times that of D-T3, approaching the ratio in biological potency observed in the tissues. Thus, selective nuclear binding of L-T3 vs. D-T3 in vivo appears to be a major determinant in the relative biological effects of these enantiomers.

Thyrotropin receptors in normal and pathological human thyroid tissues.

The properties of TSH receptors in normal and pathological human thyroid tissues were studied. Highly purified bovine TSH after lactoperoxidase iodination retained full biological activity, as assessed by radioreceptor assay. Binding of bovine [125I]TSH to 1000 x g pellets of human thyroid homogenate was specific with respect to hormone and tissue. Total binding amounted to 50-60% of total radioactivity using 10 mg (wet weight) normal thyroid tissue. Nonspecific binding was only 6% of total radioactivity. Normal thyroid tissue contained two orders of binding sites, which were shown to be independent of each other by Hill plot analysis. The high affinity sites [equilibrium dissociation constant (Kd), 0.015-0.16 x 10-9 M] were present in concentrations of 1.05-9.30 pmol/mg protein and concentrations of low affinity sites (Kd, 1.2-2.4 x 10-9 M) were 35.9-213 pmol/mg. In all pathological thyroid tissue studied, two orders of binding sites were found with dissociation constants similar to those of normal tissues, but the number of binding sites was markedly reduced. Both orders of binding sites in solitary "cold" adenomas and only the low affinity sites in thyroid tissue from patients with Graves' disease were significantly reduced in number (P less than 0.01). There was a questionable decrease in high affinity sites in the Graves' tissue (P = 0.05). We have found the definite presence but a decreased number of binding sites in both orders of receptors in papillary and follicular carcinomas. There were few or no binding sites in medullary carcinoma.

Analysis of the estrogen receptor from human uterus and breast tumor tissue by isoelectric focusing.

Cytosolic estrogen receptors from human breast carcinoma (approximately 50 patients) and uterine myometrium tissue (approximately 20 patients) were analyzed by isoelectric focusing on agarose gels, by their ability to bind to isolated nuclei, and by ultracentrifugation, under diverse conditions. Through the use of diisopropylfluorophosphate (DFP) and sodium molybdate (Mo), two in vitro processes were examined by these techniques: receptor activation and receptor degradation. These agents are capable of inhibiting these processes selectively so that the resulting stabilized forms of the estrogen receptor can be characterized in more detail. The predominant molecular forms, as determined by the parameters stated above, vary dramatically depending on the presence of DFP or Mo. At least six distinct species of the estrogen receptor were resolved by isoelectric focusing. Of these, four appear to be products of proteolysis. These had isoelectric points of 6.9, 6.8, 6.4, and 6.3, with a combined sedimentation coefficient of approximately 3.2S. The nonactivated form of the receptor, prepared in the presence of both DFP and Mo, focused at pH 4.8 and had a sedimentation coefficient of approximately 9.5S. Finally, the activated form detected in the presence of DFP sedimented at 8.0S and had an apparent pI of 5.4.

Comparative effectiveness of dextrothyroxine and levothyroxine in correcting hypothyroidism and lowering blood lipid levels in hypothyroid patients.

Data reported here establish that treatment regimens of 4 mg dextrothyroxine and 0.15 mg levothyroxine in hypothyroid subjects produce similar degrees of lowering of serum TSH, cholesterol, triglycerides, and phospholipid levels and equal stimulation of metabolic rate. The Murphy-Pattee total T4 determination applied to blood samples drawn 24 h after the last dose of dextrothyroxine can be used to assess adequacy of treatment. Correction of hypothyroidism requires high serum levels of dextrothyroxine than of levothyroxine. Serum T3 levels increase in patients treated with dextrothyroxine. In the treatment of hypothyroidism, the cholesterol-lowering and metabolic rate-stimulating effects of dextrothyroxine do not appear to be dissociated. Further studies are needed to determine whether such an effect can be demonstrated in euthyroid hypercholesterolemic subjects with doses established herein as equivalent in terms of the stimulating effect on metabolic rate.

Urinary free cortisol and cortisone determined by high performance liquid chromatography in the diagnosis of Cushing's syndrome.

To determine the efficacy of cortisol and its metabolite, cortisone, measured simultaneously by high performance liquid chromatography (HPLC) in the diagnosis of Cushing's syndrome, we retrospectively reviewed the histories of 29 surgically proven Cushing's syndrome patients (20 Cushing's disease, 5 ectopic ACTH syndrome, and 4 adrenal Cushing's syndrome) and 6 patients with exogenous Cushing's syndrome. These 35 patients had urinary free cortisol determined by both HPLC and competitive binding methods. The efficacy of the HPLC assay using cortisol alone was equivalent to that of the competitive binding assay; 22 of 29 (76%) patients had increased cortisol. Cortisone also aided in the diagnosis; 25 of 29 (86%) had increased cortisone. Twenty-seven of the 29 (93%) patients had either both cortisone and cortisol (n = 19) or at least 1 of the 2 (n = 8) increased. All 6 patients with exogenous Cushing's syndrome had suppressed urinary free cortisol, cortisone, and the presence of prednisone and prednisolone. In the competitive binding assay, all exogenous Cushing's patients had falsely increased cortisol results. In conclusion, urinary free cortisol plus cortisone determined simultaneously by HPLC added a new dimension to the diagnosis of Cushing's syndrome. It should be considered when exogenous Cushing's syndrome is suspected or when only one urinary cortisol test is allowed to be ordered.

Effect of metoclopramide on the secretion and metabolism of aldosterone in man.

We performed this study to determine whether metoclopramide increases the concentration of plasma aldosterone in normal man by increasing the secretion rate of aldosterone or by decreasing aldosterone metabolic clearance. On the first day that metoclopramide was administered orally to seven normal subjects, the secretion rate of aldosterone increased significantly (P less than 0.05) from the rate during the preceding placebo period. By the fourth day of treatment, the secretion rate had returned to control values and remained there during an ensuing placebo period. The excretion rate of aldosterone followed a similar pattern. The increase in aldosterone secretion was accompanied by a transient but significant decrease in urinary sodium excretion. Metoclopramide administered iv had no effect on the metabolic clearance of aldosterone. Metoclopramide stimulated aldosterone-producing adenomas and nodular hyperplastic adrenal tissue resected from patients with primary aldosteronism to produce aldosterone in vitro. We conclude that metoclopramide increases the concentration of aldosterone in plasma by stimulating the secretion of aldosterone rather than by decreasing aldosterone metabolic clearance, and that metoclopramide probably stimulates aldosterone secretion by acting directly on adrenal tissue.

The effects of synthetic alpha-subunit peptides on thyroid-stimulating immunoglobulin activity.

Synthetic peptides, representing specific portions of the alpha-subunit of the human glycoprotein hormones, can inhibit both the binding of labeled TSH to thyroid membranes and adenylate cyclase stimulation by TSH in vitro. The same synthetic peptides (alpha 26-46 and alpha 31-45) significantly (P less than 0.05) inhibited the adenylate cyclase-stimulating activity of thyroid-stimulating immunoglobulins (TSI) from 10 patients with hyperthyroid Graves' disease. Peptide alpha 26-46 was the most potent, resulting in 79.1 +/- 8.8% (+/- SE) inhibition at 133 micrograms/mL, while peptide alpha 31-45 inhibited TSI activity by 36.3 +/- 5.2%. Peptides alpha 61-75 and alpha 81-92, that had only minimal ability to inhibit TSH-mediated cAMP generation, did not significantly inhibit TSI activity. The inhibitory action of alpha 26-46 was dose dependent, and a significant negative correlation was found between the maximum TSI activity of the serum sample and the inhibition achieved by the synthetic peptide, suggesting that differences in TSI affinity and/or titer may account for the variable inhibitory activity of the peptides. These results suggest that TSI interact with the TSH receptor at the site that recognizes the portion of the TSH alpha-subunit represented by the synthetic peptide alpha 26-46 and, thus, support the concept that the TSH-binding site of the TSH receptor is the site of antigen binding between TSI and the thyroid cell.

The effect of metoclopramide and dopamine on plasma aldosterone concentration in normal man and rhesus monkeys (Macaca mulatta): a new model to study dopamine control of aldosterone secretion.

Metoclopramide, a dopamine receptor antagonist, increases plasma aldosterone concentration in man, suggesting that dopamine regulates the secretion of aldosterone. In the current study, we administered metoclopramide to rhesus monkeys and normal subjects and compared the time-course and dose-response characteristics of plasma aldosterone. We also examined the effect of dopamine on the plasma aldosterone response to metoclopramide in both species. Six male rhesus monkeys and several normal subjects (five women and two men) were studied on diets providing an estimated daily sodium intake of 70 mg/kg. In both species the peak increase in plasma aldosterone occurred 15 min after metoclopramide was injected. The peak plasma aldosterone value was 3-fold higher than control values. There were no significant changes in PRA, cortisol or potassium, whereas plasma PRL increased 7-fold in the monkeys and 11-fold in the normal subjects. After 0.04 mg/kg metoclopramide, there was no change in plasma aldosterone concentration in the monkeys, whereas aldosterone increased significantly (delta = 3.7 +/- 0.68 ng/dl) in the human subjects. The half-maximal dose of metoclopramide was also higher in the monkeys than in the normal subjects. A dopamine infusion at 4.0 to 8.0 micrograms/kg . min partially suppressed the plasma aldosterone response to metoclopramide in both the human subjects and the monkeys. This study demonstrates that metoclopramide produces dose-related increases in plasma aldosterone concentration in the nonhuman primate that are similar to those in normal man and that the increases can be inhibited by dopamine. We conclude that aldosterone secretion may be under dopamine control and that the rhesus monkey should be an excellent model in which to study further the regulation of aldosterone by dopamine.

L-thyroxine contamination of pharmaceutical D-thyroxine: probable cause of therapeutic effect.

Studies have shown that pharmaceutic preparations of the stereo isomers of thyroxine differ with respect to thyromimetic potency and lipid level-lowering effects. We applied a stereospecific assay for dextrothyroxine (DT4) and levothyroxine (LT4) to determine whether the biologic effects observed after the administration of DT4 (Choloxin; Flint Laboratories) resulted from inherent biologic activity of DT4, conversion of DT4 to LT4 in vivo, or LT4 contamination of Choloxin tablets. Choloxin was administered in a dose of 8 mg/day for 5 mo to nine athyreotic subjects who were then treated with pharmaceutic LT4 (Synthroid), 0.2 mg/day for an additional 5 mo. Analysis showed that LT4 contamination of Choloxin tablets ranged from 0.50% to 2.30%. This degree of contamination resulted in physiologically significant doses of LT4 in the 8 mg/day doses of Choloxin. During the treatment with two different lots of Choloxin, serum LT4 accounted for 33% to 53% of the measurable serum total thyroxine. The degree of LT4 contamination in Choloxin tablets was sufficient to account for the observed serum LT4 levels and casts doubt on the conclusions derived from previous studies in which Choloxin was used as the source of "DT4."

Serum nicotine and cotinine levels during nicotine-patch therapy.

We related serum nicotine and cotinine levels while subjects were smoking their usual numbers of cigarettes to levels while wearing a nicotine patch under carefully controlled, smoke-free conditions in a clinical research center. Twenty-four volunteers who needed intensive treatment for severe nicotine dependence were admitted to the clinical research center and were treated with a 22 mg transdermal nicotine patch each day and an intensive smoking-cessation program. Serum nicotine and cotinine levels, withdrawal symptoms, and hours and quality of sleep were noted. The steady-state serum nicotine and cotinine levels produced with the nicotine patch were lower than those observed when the subjects were smoking. Mean nicotine and cotinine levels were inversely related to mean withdrawal scores for the first 6 days. A fixed dose of transdermal nicotine will not be effective for all smokers. Individualization of therapy should be based on objective biologic measures such as serum cotinine and subjective assessment of withdrawal relief.

Toward a biochemical classification of depressive disorders, VII: urinary free cortisol and urinary MHPG in depressions.

The authors measured urinary free cortisol and urinary 3-methoxy-4-hydroxyphenylglycol (MHPG) levels in 24 severely depressed patients and 22 normal control subjects. Urinary free cortisol levels were significantly higher in the depressed patients than in the control subjects, but mean urinary MHPG levels were not significantly different in these two groups. A very high positive correlation between urinary MHPG levels and urinary free cortisol levels was found in the depressed patients but not in the control subjects.

The dexamethasone suppression test in normal control subjects: comparison of two assays and effect of age.

he authors used competitive protein binding assay and radioimmunoassay to measure cortisol levels in 38 normal control subjects three times before and three times after administration of 1 mg of dexamethasone. They found significant interassay differences at 11:00 p.m. before dexamethasone and at all three postdexamethasone times. Analysis of variance revealed significant overall positive relationships between age and cortisol levels measured by both techniques. Age correlated significantly with postdexamethasone cortisol levels measured by radioimmunoassay but not when measured by competitive protein binding assay. Clinicians should obtain data from their laboratories as to appropriate cutoffs for cortisol suppression on the specific assay used.

Aging and aldosterone.

We measured urinary and plasma aldosterone in normal subjects, aged 20 to 59 years, during a period of unrestricted sodium intake and after sodium depletion, using furosemide or a 20 meq sodium diet. Before and after sodium depletion, the mean and the upper limit of the range of urinary aldosterone excretion were considerably lower in subjects over 50 years compared with subjects under 30 years. Aging had no effect on plasma aldosterone concentration when the subjects were on an unrestricted sodium diet and blood was sampled while they were recumbent. In contrast, when the subjects were upright, both before and after sodium depletion, the mean and the upper limit of the range of plasma aldosterone concentration were lower in the subjects over 50 years compared with those under 30 years. The metabolic clearance of aldosterone was the same in the younger and the older subjects. Of eight patients over 40 years of age with aldosterone-producing adenoma, four had normal aldosterone excretion rates when the normal range was not age adjusted, but with age adjustment, all of the patients had clearly elevated excretion rates. Similarly, six of seven patients over 40 years of age had normal upright plasma aldosterone concentrations if the normal range of plasma aldosterone concentration was not age adjusted. We conclude that aldosterone secretion declines with advancing age. The effect of age on aldosterone secretion is an important consideration when evaluating older hypertensive patients for primary aldosteronism.

Diagnostic evaluation of pheochromocytoma.

Progress in the diagnostic evaluation of pheochromocytoma has taken place in biochemical studies and localization techniques. Measurement of fractionated catecholamines and their metabolites has been subjected to sensitivity and specificity assessment. Magnetic resonance imaging and isotopic imaging have led to much better localization of extra-adrenal tumors. Flow cytometry of the DNA of the tumor cells very likely indicates the malignant nature of the tumor.

Further study on the two-column plasma catecholamine assay.

The two-column procedure for plasma catecholamine determination was adopted as a routine diagnostic test. It was found that the recovery of catecholamine added to properly preserved plasma was 65%. The most efficient recovery occurred when the water content of alumina used was above 23%. Both interassay and intra-assay precision were +/- 10% (coefficient of variation). The catecholamine was stable, in properly preserved plasma, for at least 18 days at -20 degrees C.

Clinical use of the dexamethasone suppression test for psychiatric disorders. Need for sensitive method for measuring corticosteroids and cautious interpretation of results.

The corticosteroid levels in 256 plasma samples from 112 patients with the clinical diagnosis of depression were assayed in two ways: by the routine method (sensitivity of 5 to 40 micrograms/dl) and by a newer, more sensitive method (sensitivity of 1 to 10 micrograms/dl). Of the 152 "true-negative" samples measured by the more sensitive method, 81 (53%) were reported as positive ("false-positive") by the routine method. The routine method does not yield accurate results on borderline levels of corticosteroids (5 to 7 micrograms/dl) for the dexamethasone suppression test for psychiatric disorders.