Differential effects of insulin-like growth factor 1 on the hormonal product and proliferation of glycoprotein-secreting human pituitary adenomas.

The effects of human recombinant insulin-like growth factor 1 (IGF-1) on the secretion, viability, and proliferation of dispersed human anterior pituitary adenomas secreting FSH, LH, and alpha-subunit (alpha-su) were examined in vitro over 4 h and 4 days. The acute effect of IGF-1 on secretion over 4 h was examined in four tumors secreting FSH, LH, and alpha-su. IGF-1 (100 nmol/L) reduced LH compared to control (100%) in one tumor (61%, P < 0.01), and three tumors remained unaffected. FSH and alpha-su secretion were insufficient to measure over 4 h. Nine tumors were studied over 4 days; relative to control, IGF-1 (100 nmol/L) increased FSH secretion in all seven tumors secreting FSH (28-266%, P < 0.05) and increased alpha-su secretion in all four tumors studied (36%, 63%, 91%, and 121%, P < 0.05). IGF-1 reduced LH secretion in four/nine tumors (13%, 23%, 32%, and 50%, P < 0.05). Dose response curves (1-100 nmol/L IGF-1) were performed on three tumors cosecreting FSH and LH. Stimulation of FSH was achieved with either 1 or 10 nmol/L IGF-1, a single tumor in which alpha-su was measured showed maximal stimulation at 10 nmol/L IGF-1, and one of three tumors showed LH inhibition with 100 nmol/L IGF-1. In situ viability of attached cells was assessed with fluorescein and propidium iodide in seven tumors. After 4 days' exposure to 100 nmol/L IGF-1, in situ viability was increased in five tumors (range 12-19%, 15 +/- 1.3% SEM, P < 0.05). The effects of IGF-1 on the adenoma cell proliferative S-phase fraction was determined in six tumors after 4 days of treatment using double immunostaining with bromodeoxyuridine incorporation for 1 h. In four/six adenomas that stained positive for bromodeoxyuridine in the controls (1-5.6%), the S-phase fraction was increased by 100 nmol/L IGF-1 [(range 2.1-10.6%, increase 90-220%) (P < 0.05)]. These results show that IGF-1 has differential effects on gonadotropins from human pituitary adenomas, stimulating intact FSH and alpha-su, inhibiting or being without effect on intact LH in vitro, and increasing both viability and number of tumorous glycoprotein-secreting cells entering into the S-phase of proliferation.

Comparison of thyroid stimulating hormone and triiodothyronine response to thyrotrophin releasing hormone in the assessment of thyroid status.

The response to an intravenous dose of 200 microng of thyrotrophin releasing hormone (TRH) has been studied by estimating, by radioimmunoassay, baseline levels followed by further estimations of thyroid stimulating hormone (TSH) 20 minutes after the injection and triiodothyronine (T3) three hours after the injection in 112 patients referred for routine thyroid assessment. Comparison of diagnostic accuracy of the response to TRH gave similar results with both procedures but slightly better overall accuracy for the response measured by TSH assay. However, estimation of baseline T3 is a valuable test for hyperthyroidism, in contrast to baseline TSH, and combined with the estimation of T3 three hours after TRH injection provides an accurate additional test in borderline cases.

Assessment of an enhanced chemiluminescent immunometric assay for TSH in 1127 patients.

An enhanced immunochemiluminometric assay for serum TSH ('Amerlite', Amersham, Bucks, UK) was studied in 1127 patients in routine clinical practice to assess its value as a first-line test of thyroid status. Good correlation with clinical thyroid status was found in the untreated euthyroid patients, in the untreated hyperthyroid and hypothyroid patients, in pregnancy and in the sick euthyroid. However, a large proportion of clinically euthyroid patients with nodular goitre, as well as those treated by thyroidectomy, radioiodine or antithyroid drugs and those on replacement l-thyroxine showed TSH values outside the reference range. Therefore, additional tests are likely to be needed frequently in these categories.

Thyroid stimulating hormone measurement using a third generation immunometric assay.

After an initial evaluation of the standard procedure for performance of a third generation TSH (thyroid stimulating hormone) assay (Amerlite TSH-30) modifications were made to standardize the timing of measurement of light emission following signal reagent addition. By adopting this optimized procedure, a significant improvement in assay sensitivity was achieved when compared to a second generation TSH assay (DAKO). Using the optimized assay the sensitivity was 0.003 mU/L (20 replicates of zero) or 0.009 mU/L [22% CV (coefficient of variation) from the precision profile]. Recovery of added TSH and parallelism of the assay were good. A significant negative bias was detected for the Amerlite TSH-30 assay when compared to the DAKO assay (log y = 0.92 log x-0.33, n = 210). Excellent discrimination was achieved between euthyroid, hypothyroid and thyrotoxic subjects. A high percentage of thyrotoxic patients had undetectable TSH and the spread of values between thyrotoxic and euthyroid was greater with the third generation assay. In patients receiving thyroxine therapy a higher percentage had detectable TSH values. The optimized Amerlite TSH 30 assay offers improved assay performance when compared to a second generation assay.

Assessment of optimal L-thyroxine replacement dose by the TRH test.

Two hundred patients taking varying L-thyroxine replacement doses were studied using a normal TRH test as the index of optimal replacement dose. The mean optimal dose was 141 microgram/day. Normal serum T3 and FT3I were found in most patients, whatever the TRH response, and they are probably too unspecific. Serum T4 and FT4I were elevated in many patients with a normal TRH response. A higher range for FT4I of 102-166, although only 66.5% accurate, gave the best index of optimal L-thyroxine replacement of the single in-vitro tests.

Factors predicting hypothyroidism in long-term follow-up after 131I therapy.

187 patients, euthyroid for more than a year after radioiodine treatment for hyperthyroidism, were studied for 10 years; 81 (43%) became hypothyroid. The incidence of hypothyroidism was lower in patients initially presenting with large thyroids (28%) or with nodular thyroids (22%) and in those without thyroid autoantibodies (31%). During follow-up, an elevated serum TSH was present in all 81 patients when they became hypothyroid (sensitivity and negative predictive value 100%), and was present for at least a year in 98% of these. However, an elevated serum TSH was also present in 48% of 106 patients remaining euthyroid (positive predictive value 61%). FT4I was low in 94% of patients who became hypothyroid and normal in 80% of patients who remained euthyroid (positive predictive value 78%, negative predictive value 93%). Serum TSH and FT4I were the best biochemical predictors. FT3I and serum cholesterol were less satisfactory. A palpable thyroid becoming impalpable, though readily assessed, was limited in usefulness. Clinical appraisal remains important and a progressive, though perhaps less rapid, later increase in the incidence of hypothyroidism appears likely.

The effect of carbimazole following radioiodine therapy on radiation dose to the thyroid.

Radioiodine in the thyroid gland after a therapy dose of 131I was measured serially in 7 patients without Carbimazole, and in 11 patients starting Carbimazole 60 mg daily fourteen days after the therapy dose. Effective half-life for radioiodine in the gland initially 5.53 plus or minus 1.08 days fell to 4.26 plus or minus 1.12 days (p less than 0.01) during Carbimazole, and returned to 5.83 plus or minus 1.21 days (NS) after stopping the drug. The radiation dose to the thyroid from a given therapy dose of 131I followed by Carbimazole was calculated to be 97% of that without Carbimazole when he drug was started after 14 days, and 90% and 75% when the drug was started after 7 days and 1 day respectively.

The use of discriminatory values for thyroid uptake and free thyroxine index.

The distributions of 4 hour 132I neck uptake and 20 minute 99mTcO4 thyroid uptake in euthyroid patients were found to conform closely to a log normal distribution, from which a statistical normal range may be obtained. More accurate discriminatory values for determining thyroid status may be established by plotting intersecting distribution curves for hypo-, hyper- and euthyroid groups. Subdivision by age and by the presence or absence of a palpable thyroid reveals considerable variations from which a series of more accurate discriminatory values may be established for these subgroups. For Free Thyroxine Index (T4RT3 index) more accurate discriminatory values were also obtained by similar methods; while no significant variations related to age or palpable thyroid were observed, subdivision of the patients into two groups referred as suspected hypothyroidism and suspected hyperthyroidism yielded a further small improvement in accuracy.

Pitfalls in the use of thyrotropin concentration as a first-line thyroid-function test.

Measurement of thyrotropin concentration alone as a first-line thyroid-function test fails to indicate hypopituitarism in a number of patients. Using a combination of thyrotropin and thyroxine assays, we analysed 56,000 tests for a population of 471,000 over 12 months. 15 patients with clinically unsuspected hypopituitarism were detected, indicating that the occurrence of hypopituitarism might be underestimated.

Leucine aminopeptidase-like activity in Aplysia hemolymph rapidly degrades biologically active alpha-bag cell peptide fragments.

We have investigated the role that proteolytic enzymes in Aplysia hemolymph play in the inactivation of the neurotransmitter alpha-bag cell peptide (alpha-BCP(1-9), Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu). alpha-BCP fragments containing Pro in positions 1 or 2, or Tyr in position 1, were degraded relatively slowly (half-life, t1/2 = 10-64 min), whereas fragments lacking these residues were degraded relatively rapidly (t1/2 = 0.5-2.7 min). Of 12 peptidase inhibitors tested, only bestatin, amastatin, and phenanthroline significantly inhibited alpha-BCP(3-9) degradation. alpha-BCP(3-9) yielded only four observable cleavage products (in order of decreasing abundance at early time points): alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), and alpha-BCP(7-9). Degradation of alpha-BCP(3-9), alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), or alpha-BCP(7-9) was strongly inhibited by bestatin, moderately inhibited by amastatin, and not inhibited by arphramenine B. The rates of degradation of eight alpha-BCP fragments and three other peptides in plasma were well correlated with their rates of degradation in mammalian leucine aminopeptidase (LAP, EC 3.4.11.1). Collectively our data support the following ideas. 1) In hemolymph one or more LAP-like enzymes rapidly and sequentially cleave alpha-BCP(3-9) or other small peptides lacking Pro at positions 1 or 2 or Tyr at position 1. 2) LAP-like peptidases in hemolymph may act in concert with previously described ganglionic peptidases to degrade neurally released alpha-BCP(1-9) and alpha-BCP(1-8) into inactive fragments.

Aplysia peptide neurotransmitters beta-bag cell peptide, Phe-Met-Arg-Phe-amide, and small cardioexcitatory peptide B are rapidly degraded by a leucine aminopeptidase-like activity in hemolymph.

We have been investigating the role of proteolytic enzymes in the inactivation of peptide neurotransmitters in the marine snail Aplysia. Previous studies (Squire, C. R., Talebian, M., Menon, J. G., Dekruyff, S. D., Lee, T. D., Shively, J. E., and Rothman, B. S. (1991) J. Biol. Chem. 266, 22355-22363) showed that neuroactive fragments of the neurotransmitter alpha-bag cell peptide (alpha-BCP) were rapidly degraded (t1/2 = 0.5-2.7 min) in plasma, hemolymph that had been cleared by centrifugation. Degradation was caused by one or more enzymes resembling mammalian leucine amino-peptidase (LAP, EC 3.4.11.1). In this report we show that three other Aplysia peptide neurotransmitters, beta-BCP(1-5) (Arg-Leu-Arg-Phe-His), FMRFa (Phe-Met-Arg-Phe-amide), and SCPB(1-9) (Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met-amide) are rapidly degraded (t1/2 = 0.3-2.4 min) in plasma by apparently the same LAP-like enzyme(s). Our findings strongly suggest that the LAP-like enzyme(s), by means of its broad substrate specificity and access to the extracellular spaces of the nervous system in vivo, plays a significant role in the inactivation of many Aplysia peptide neurotransmitters, and they raise the possibility that proteolytic enzymes in the extracellular fluid contribute significantly to the inactivation of peptide neurotransmitters in other animal species.