During the past several decades, a variety of bioassay procedures for prolactin have been developed. Crop-sac of pigeons, mammary gland and corpus luteum of rats and mice have been employed for the determination of prolactin. Recently many improved methods for prolactin bioassay were reported, but the relationship among them was not yet precisely demonstrated. In order to clarify sensitivity and stability, several bioassay procedures were investigated by using rabbits, rats, mice and pigeons.
In vivo mammary gland assay method, such as systemic and intraductal administration into pseudopregnant rabbits revealed to be insensitive, variable and laborious. The minimal effective dose in systemic prolactin injections is 1 IU of prolactin. But there are no different findings of mammary gland between 1 IU and 8 IU. On the other hand 0.5 IU of prolactin shows milk formation in the mammary gland after intraductal administration, but there are no difference between 0.5 IU and 3.0 IU.
Organ culture of mammary glands of female rats were also used for the prolactin determination. Alveolar formation of incubated mammary gland is shown as a positive sign, but this method is insensitive and time-consuming.
Prolactin has been known to have a luteotropic action in some animals. Corpus luteum cell nuclei of hypophysectomized mice were counted after PMS, HCG and prolactin had been treated for 10 days. Prolactin stimulates growth of corpus luteum cell nuclei and therefore number of corpus luteum cell nuclei decreases per unit area.
Prolongation of diestrus of mice was observed following prolactin injection. Small amount of prolactin such as 0.01 IU shows prolongation of diestrus. Decidual reaction of mice's uteri is not as sensitive as diestrus method.
β-glucuronidase activity of rat testis is also used for prolactin bioassay but is not sensitive.
Recently, the pigeon assay method has been widely used for measuring prolactin because of its simplicity and specificity. Many pigeon crop-sac assay procedures are available for prolactin determination either by systemic or local injection. The crop-sac response to systemic injection can be quantified but its sensitivity is not sufficient to detect 0.01 IU of prolactin.
The intradermal prolactin injections therefore were precisely investigated. Injection schedule of two times a day for 2 days is most sensitive in local administration. Several assay procedures of intradermal injection; such as response diameter, area, dry weight of stimulated area and dry weight of mucosal epithelium, were compared with respectively. But dry weight of mucosal epithelium is found to be a most sensitive and stable method, and minimal effective dose of prolactin is 5-10 mU of prolactin.
From the results mentioned above, it may be concluded that Nicoll's method for dry weight of pigeon crop-sac mucosal epithelium is the best bioassay procedure for prolactin determination.

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Adrenocorticotropic hormone is a relatively poor antigen, and difficulties in obtaining antisera which are sufficiently sensitive for radioimmunoassay of plasma ACTH have been reported by several workers.
In the present study, guinea pigs were immunized by subcutaneous injections at biweekly intervals with 20 U of commercial porcine ACTH emulsified in complete Freund's adjuvant or acetylated wax D (AD) which contained less competing antigens than bacillary bodies of Mycobacteria. Antisera obtained by 8 injections were not sensitive for the measurement of plasma ACTH, though animals injected with acetylated wax D induced slightly increased production of the antibody.
Guinea pigs in another immunization schedule were given 30 U of commercial porcine ACTH mixed with metyrapone and complete Freund's adjuvant by monthly subcutaneous injections. The antiserum used for assay was obtained from one guinea pig after 14 months.
Highly purified porcine ACTH supplied by Mann Laboratories was iodinated by the chloramine T method and purified with QUSO G32. Specific activities ranged from 300 to 400 mc/mg. The reference standard used was highly purified human ACTH supplied by Dr. Lee.
The assay was performed by a modified method of Berson & Yalow. ¹²⁵I-porcine ACTH of 1 μμg, antiserum at a final dilution of 1 : 50,000 and unknown plasma of 0.2 ml were added to a final incubation volume of 1.0 ml. The mixtures were incubated at 4 °C for 7 days. Talc was used for separation of antibody bound and free labeled hormone.
A minimum of 16 μμg/ml of ACTH was detected in the assay. Therefore, 80 μμg ACTH/ml of whole plasma was measurable in plasmas assayed at 1 : 5 dilutions. Plasma concentrations in normal subjects ranged from less than 80 to 160 μμg/ml at 9 a.m.. Increased concentrations were observed in patients with Addison's disease and Cushing's syndrome associated with bilateral adrenal hyperplasia. Plasma ACTH levels were undetectable in normal subjects pretreated with dexamethasone and patients with Cushing's syndrome associated with adrenal adenoma. The administration of metyrapone in patients with Cushing's syndrome produced an increase in plasma ACTH concentration in those with bilateral adrenal hyperplasia, whereas no significant change in those with adrenal adenoma.

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In order to study the pituitary reserve of various pituitary hormones in patients with throid disorders, plasma growth hormone (HGH), adrenocorticotropic hormone (ACTH), luteinizing hormone (LH) and plasma 11-hydroxycorticosteroids (11-OHCS) in response to insulin-induced hypoglycemia and HGH to arginine infusion were examined. Twenty patients with hyperthyroidism (including 6 patients with periodic paralysis) and 8 patients with primary hypothyroidism were subjected to the studies. In some of the cases, studies were repeated after treatments when their condition became euthyroid. Insulin tolerance test was performed by injecting 0.1 u/kg of regular insulin and arginine infusion test by infusing 0.5 g/kg of 1-arginine for 30 min. Plasma pituitary hormones were measured by coated charcoal radioimmunoassay and plasma 11-OHCS by fluorometric technique with De Moor's method.
1. Responses of plasma HGH to both insulin-induced hypoglycemia and arginine infusion were deficient in both hyperthyroid and hypothyroid groups, especially in the hypothyroid group. The average peak value of plasma HGH to insulin-induced hypoglycemia was 14.0 mμg/ml after insulin injection in the hyperthyroid group and 2.8 mμg/ml in the hypothyroid group compared with 27.3 mμg/ml in normal subjects. Blood sugar levels fell comparably to normal subjects in both groups. The average peak value of plasma HGH to arginine infusion was 6.0 mμg/ml in the hyperthyroid group and 1.3 mμg/ml in the hypothyroid group compared with 30.2 mμg/ml in normal subjects.
2. In order to assess the role of adrenergic mechanism in HGH secretion in hyperthyroidism, 6 patients with hyperthyroidism were also subjected to insulin tolerance test after administration of propranolol, a beta blocker, either 30 mg/day orally for 2 weeks or by a single intravenous injection of 6 mg. But the responses of plasma HGH were not significantly different from those values which were obtained before administration.
3. The responses of plasma ACTH to insulin-induced hypoglycemia were diminished in both groups. The average peak value of ACTH to insulin was 142 μμg/ml in the hyperthyroid group and 91 μμg/ml in the hypothyroid group compared with 181 μμg/ml in normal subjects.
4. The responses of plasma LH to insulin were also impoverished in both groups. The average peak value of plasma LH after insulin was 39 μg/dl in the hyperthyroid group and 26 μg/dl in the hypothyroid group compared with 61 μg/dl in normal subjects.
5. The increments of plasma 11-OHCS in response to insulin-induced hypoglycemia were 9 μg/dl in the hyperthyroid group and 10μg /dl in the hypothyroid group compared with 15 μg/dl in normal subjects.
6. The disappearance rate of ¹³¹I-HGH from the plasma was measured in 2 patients with hyperthyroidism and a patient with hypothyroidism. Following a single intravenous injection of ¹³¹I-HGH using 0.1 μg (200 mc/mg), the disappearance curve in plasma plotted semilogarithmically against time indicated that the system was composed at least of 2 exponentials, the average half-life for the 2nd component was 68 min. in 2 patients with hyperthyroidism and 101 min. in a case of hypothyroidism against 59 min. in 2 normal subjects.
7. After treatment, the responses of plasma HGH, ACTH, LH and 11-OHCS almost returned to normal, though the recovery was better and sooner in patients with hyperthyroidism. For example, the average peak value of plasma HGH to insulin-induced hypoglycemia rose up to 34.2 mμg/ml in 6 patients treated for hyperthyroidism and 13.3 mμg/ml in 3 patients treated for hypothyroidism.
8. From the above results, it was concluded that the pituitary reserve of HGH, ACTH and LH was decreased in patients with thyroid disorders. As causative factors for diminished secretion of HGH which showed the most marked diminution among pituitary hormones examined,

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