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The effects of hypothyroidism and adrenalectomy (ADX) on PRL receptors as well as the subcellular distribution of PRL-binding sites in adult male rat target tissues were investigated. In ventral prostate, kidney, and adrenal, PRL-binding activity was enriched in the 15,000 X g pellet (mitochondrial/Golgi fraction). The zona fasiculata/reticularis plus medulla of the adrenal had higher PRL-binding activity than the zona glomerulosa (plus capsule). Propylthiouracil-induced hypothyroidism significantly decreased PRL binding to kidney (65%) and testis (50%), and 16-h replacement with T4 restored PRL binding to control levels. T4 was, however, without effect on renal PRL binding in hypothyroid castrated rats, indicating that androgen may be required for this response. The increase in PRL binding to ventral prostate and adrenal due to hypothyroidism was not reversed within 16 h of T4 injection, whereas T3 injection decreased PRL binding to ventral prostate within 12 h. ADX increased PRL binding to rat testis (38%) and kidney (18%), and 5-day replacement with dexamethasone decreased PRL binding to levels lower than those in control rats. ADX had no effect, and dexamethasone treatment decreased (16-20%) PRL binding to ventral prostate and hCG binding to rat testis. The modulation of PRL receptors by hypothyroidism and ADX suggests that thyroid and adrenal hormones play a role in the regulation of PRL receptors.

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The Dunning R3327 rat prostatic adenocarcinoma contains specific binding sites for iodo-oPRL (ovine prolactin) in the membrane fraction (100,000 X g pellet). These are normally present at a low level, but immunization with oPRL produces a dramatic increase in tumor binding in either sex (females bear an implant of testosterone propionate pellet). Under these experimental conditions, the ventral prostate of rats bearing the tumors have no detectable PRL receptors. 5 alpha-Reductase activity, on the other hand, is lower in the tumors than in the normal prostates. Immunization also decreased somewhat the rate of growth of the tumors.

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Three experiments were performed to determine whether human prolactin (hPr) affects prostatic uptake and metabolism of testosterone (T). 1) Patients with prostatic cancer were infused twice with radio-labelled androgens, the first time with basal hPR, the second time with oral thyrotrophin-releasing hormone (TRH)-elevated hPr. In 5/7, significant increases in metabolic clearance of dihydrotestosterone (DHT) and in conversion of T to DHT accompanied increased hPR. 2) The incorporation of labelled T into minced benign prostatic hypertrophy (BPH) tissue from subjects with high (40 ng/ml) hPR was measured and was found to be more than twice the uptake into tissue from those with low hPR (6.5 +/- 1.9 ng/ml). 3) Uptake and metabolism in vivo of a bolus of 3H-T by BPH and carcinomatous prostates was measured and was far greater in subjects whose hPR was elevated by chlorpromazine than in untreated controls. It is concluded that prolactin increases prostatic uptake and metabolism of T. It is suggested that the best management of prostatic cancer should include depletion of prolactin as well as androgen.

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The current method labeling small molecules for radioimmunoassay by coupling iodohistamine to haptens through a mixed anhydride reaction is unacceptable to clinical laboratories. Therefore, we propose the use of a simple two-step procedure: treatment of 125I-2-iodohistamine with the activated ester of a small molecule followed by thin layer chromatography to remove unlabeled ligand. Only one radioactive substance, 125I-2-iodohistamine, need be stocked, and the availability of labeled ligands is limited only by the number of nonradioactive activated esters. This principle is illustrated by the use of testosterone. N-Hydroxysuccinimidyl esters of testosterone hemisuccinate and of testosterone-3-carboxymethyloxime were coupled to 2-iodohistamine, 125I-2-iodohistamine or to 125I-2,5-diiodohistamine. Optimum conditions required reaction of 20-50 fold molar excess of ester in 75 microL of tetrahydrofuran with iodohistamine in 75 microL of buffer at pH 8.5 for 30 min at 4 degrees. The reaction mixture was applied directly to a pre-absorbent TLC plate coated with silica gel and run in the system, benzene:ethanol:acetic acid, 75:24:1 (v:v:v). The desired radioligand was eluted in 85% yield.

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Recently radioactively labeled iodohistamines have been claimed to have superior shelf-life to the iodophenols more commonly used in radioimmunoassay of small molecules. This claim is based largely on theoretical considerations; no systematic study has appeared. We found that iodination of histamine on a macroscale proceeds rapidly at pH 7-8.4 to yield principally 2-iodohistamine. With a large excess of iodine, substantial diiodination can be achieved. In 0.5 N sodium hydroxide solution, triiodination produces 1,2,5-triiodohistamine; the N-I bond, however, is somewhat labile. 125I-2,5-Diiodohistamine is also somewhat unstable, having a first order decomposition rate of 1.6 X 10(-3) day-1 (t1/2, 182 days), while 125I-2-iodohistamine shows a barely perceptible change in 60 days (7.5 X 10(-5) day-1). The assignment of the first iodine introduced to C-2 is based on a comparison of the NMR spectra of monoiodohistamine and histamine. Iodination with carrier-free iodine-125 using the Hunter-Greenwood procedure (chloramine-T) produces a 76% yield of mono- and a 19% yield of diiodo product which are easily isolable by a single TLC using silica gel in the solvent system, ethanol:ethyl ether:water, 5:5:2.

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PIP: The effects of hypophysectomy and prolactin replacement therapy on prostatic response to androgen in orchiectomized rats were studied. Castration 23 days prior to treatment with testosterone propionate (TP), followed by hypophysectomy 13 days before TP treatment, and then treatment with 1 mg TP every other day for 16 days caused a greater decrease in body weight, prostatic weight, and the level of citric acid in the prostate than did TP treatment, castration, and sham hypophysectomy. This suggests the existence of a pituitary factor in the maintenance of prostatic integrity. Prolactin replacement therapy in hypophysectomized, castrated,TP-treated rats significantly (p less than .005) increased the prostatic weight and both the content and concentration of citric acid. The results confirm previously reported observations that the prostatic response to androgens is markedly reduced by hypophysectomy in castrated rats, and that prolactin acts synergistically wtih testosterone in promoting prostatic growth and the concentration of citric acid in the prostate. The direct effects of both hypophysectomy and prolactin replacement therapy on the ventral and dorsolateral lobes of the prostate were also demonstrated.^ieng

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A four step synthesis of 6-(0-carboxymethyl) oximinoethynylestradiol is reported. This compound, 6-(0-carboxymethyl) oximinomestranol, the 3-(0-carboxymethyl) oximes of norethindrone and norgestrel and the 3-hemisuccinate of ethynylestradiol were synthesized and conjugated with bovine serum albumin. Rabbits were immunized at 3 dose levels of haptene (20, 66 and 200 nmoles) and eight weeks later with a booster containing 66 nmoles of haptene. The antibody titer and association constant of responding rabbits was nearly independent of dose although most antibody production occurred after the booster injection. Antibodies to mestranol crossreacted more than 100 percent with ethynylestradiol and to a small extent with norethindrone and norgestrel.

PIP: The methods of synthesis of the 6-(0-carboxymethyl) oxime derivative of 6-oxoethynylestradiol-17beta, the 3-succinyl derivative of ethiny estradiol-17beta and the 3-carboxymethyl oxime derivative of norethindrone and norgestrel as well as the synthesis of the corresponding bovine serum albumin (BSA) conjugates required for the specific antisera for mestranol, ethinyl estradiol, norethindrone, and norgestrel in rabbits are described. The synthesis of 6-(0-carboxymethyl) oxime derivative involves a 4-step synthesis and a chromatographic purification which is an improvement over past methods. Conjugation to BSA was by the carbodiimide method. Response of immunization was measured in terms of titer, association constant, and cross-reactivity as a function of time after immunization with 3 dose levels of each antigen. Titer response was independent of dose for the original injection. Ethiny estradiol-3-conjugates had very low titers.

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R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), a synthetic progestin, was tested for its binding to purified human transcortin, albumin and human serum. Scatchard analysis showed that R5020 bound relatively weakly to transcortin (Kass = 8 X 10(6)M-1); in 20% human serum or albumin greater than 90% was bound with low Kass and an indeterminate number of binding sites.

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In most rapid radioimmunoassay methods, absorbents are used that disturb the equilibrium of the ligand-antibody interaction; thus the separation process must be rigidly timed. We show here that polyethylene glycol (mol wt. 6000) abolishes this constant. We measured gamma-globulin precipitation by polyethylene glycol (150g/liter) in a thyroxine radioimmunoassay system involving use of a sheep anti-thyroxine antiserum, quantitatively and qualitatively, with either normal human serum or completely precipitated at pH 6-9 at 4 and 25 degrees C, but incompletely if salt concentration was high (1 mol/liter). Specificity of gamma-globulin precipitation increased with increasing pH and temperature. All gamma-globulin-bound thyroxine was precipitated, and also some not bound to gamma-globulin. This was taken into account by including "blank" tubes (no antibody, but with normal sheep serum) in all assays. Because this reaction is predominantly entropic and temperature independent, the entire procedure can be done at 37 degrees C and room temperature without rigid timing. The assay requires 10 mul of serum, its reproducibility is 4-8% (CV) in the euthyroid and hyperthyroid range, and its accuracy is close to 100%.

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A study of plasma concentration (P1T, ng/ml), metabolic clearance rate (MCRT, L/day) and blood production rate (PBT, mg/day) was done on seven XYY subjects of various ages and four pair-matched control XY subjects by a radioinfusion technique of 1,2-3H-testosterone. Although MCRT showed no significant difference between the groups, P1T and PBT were significantly lower (P less than 0.05) in XYY subjects. Therefore, increased aggressive behavior of the XYY subjects can not be attributed to increased levels or production rates of testosterone.

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A pharmacodynamic model was developed to facilitate computer analysis of the circadian-episodic influx of cortisol into plasma from the adrenal gland. The model consists of a catenary system of a biorhythmic control, the adrenal gland, and a body compartment containing circulating cortisol. Computer nonlinear regression analysis was carried out on data consisting of plasma cortisol concentrations measured at 30-min intervals over 1 day in a group of normal children. Circadian rhythmic synthesis of cortisol by the adrenal gland was described by a sinusoidal function which provides the level (284 plus or minus 50 mugg/h/m2) and amplitude (245 plus or minus 50 mug/h/m2) of the synthesis rate and the period (24 hours) and acrophase (8.14 plus or minus 1.95 h) of the cycle. Cosinor analysis of the data confirmed a highly significant circadian rhythm in the daily synthesis rate of cortisol. Episodic secretion, described by an empirical switch function, is assumed to result from accumulation of small amounts of cortisol precursors in the adrenal gland and intermittent stimulation of cortisol release by ACTH. This was found to take place over 46.6 plus or minus 2.4% of a 24-h day. Circulating cortisol is contained in a single body compartment with an apparent volume of distribution (5.3 plus or minus 0.55 liters/m2) from which elimination occurs by first-order metabolic clearance. The biological half-life averaged 0.96 plus or minus 0.18 h. Upon least-square optimization of selected kinetic parameters, the circadian-episodic model increases the accountable variation (r2 x 100) to 89% in comparison with the 35% obtained by use of only a periodic function.

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