Research on mammalian antidiuretic hormone (ADH) has been seriously limited by the lack of techniques for detection of physiological levels of ADH in blood and plasma.The bioassay of Jeffers, Livezey, and Austin (1), using the ethanol-anesthetized rat for ADH in mammalian plasma, was significantly refined by Dicker (2), who was able to detect as little as 2 uU ADH per ml of plasma. More recently, Heller and Stulc (3), using rats with exteriorized urinary bladders, could detect as little as 0.65 ,uU per ml of plasma. With this technique Heller and Stulc (4) found mean levels of 1.67 ,uU per ml of ADH in the plasma of "normally" hydrated men.The present study was undertaken to evaluate Heller's procedure and, if possible, to utilize it to study the release, turnover, and action of ADH. MethodsA. Assay method for ADH 1) Exteriorization of the urinary bladder. Female rats of the Sprague-Dawley strain, weighing 100 to 120 g, were anesthetized with ether. After shaving the skin about 1 cm above the symphysis pubis, the linea alba was incised and the peritoneum was opened. The top of the bladder was drawn out with fine forceps. The upper half of the bladder was cut off, and the edges were sutured with thin catgut (50) to the edges of the skin * Submitted for publication May 11, 1963 incision. Eight sutures were utilized, the first two being placed at the superior and inferior margins in the mid-line of the bladder. The mid-line can be identified by a fine dorsal or posterior mesentery. Care was taken not to twist the bladder during suturing. Distortion of the trigone could lead to ureteral obstruction. Four to five days after surgery the rats could be used for the assay. The same rats could be used twice at an interval of 4 to 6 days. After this period there frequently developed ascending pyelitis and pyelonephritis.2) Assay. The rats were hydrated as well as anesthetized by giving 12% ethanol in water by stomach tube. The rats were given two doses, each equal to 3% body weight, at 30-minute intervals; a third dose, given after 20 minutes, was about 2% of the body weight. The amount of ethanol given with the third dose varied according to the state of the individual animal. Some rats were completely anesthetized by the first two doses, and in this case only tap water was administered as the third dose; others were quite awake and therefore needed an additional dose of 12%o ethanol. Infrequently, in-between doses were given. Care was taken not to give larger amounts of ethanol than necessary to anesthetize the rats as this could bring on shock and even death.When anesthesia was sufficiently deep, at the longest, 10 to 15 minutes after the third dose, a little vaseline was applied around the exteriorized bladder, and the animal was laid prone on a table-like board. A small funnel that fitted through a hole in the board was put in contact with the skin around the exteriorized bladder so that the urine flowed directly through this funnel into graduated tubes that could be read to 0.05 ml. These tubes were made from 5.0-ml ...

The exact mechanism (s) responsible for the impaired water diuresis of primary and secondary adrenal cortical insufficiency is still unknown. Previous studies from this (1-3) and other laboratories (4-6) have suggested that this defect is due in part to the loss of a permissive effect of cortisol-like steroids on the diluting segments of the nephron, i.e., a normal amount of glucocorticoid is essential to allow maximal impermeability to water to develop in these segments in the absence of circulating antidiuretic hormone (ADH). This concept implies that the release, metabolism, and action of ADH are normal in the adrenal-insufficient patient (1-3). Others (7-12), however, imply that adrenal glucocorticoids directly affect the release and metabolism of ADH. This implies that the impaired water diuresis of adrenal insufficiency is due to the altered ADH physiology or its unopposed activity. These conflicting points of view have in general developed from experiments in which the release, metabolism, and action of ADH were studied indirectly rather than by direct measurement of the hormone in biologic fluids. Detection of physiological levels of circulating ADH can provide direct evidence that may settle this conflict.In the previous paper (13) the authors have described a sensitive and accurate bioassay for ADH. With this technique it has been possible to study the release, metabolism (turnover), and action of ADH in untreated patients and animals with adrenal insufficiency. The results conclusively demonstrate that the impaired water diuresis of adrenal insufficiency and its correction by glucocorticoids are not due to an alteration of release, metabolism, or action of ADH. MethodsThe studies were carried out on patients, dogs, and rats with adrenal insufficiency. The patients, three with secondary and one with primary adrenal insufficiency, took no glucocorticoid replacement therapy for 3 to 5 days before the study. In two males, J.B., age 68, and G.R., age 47, the adrenal insufficiency resulted from chromophobe adenomas of the pituitary, whereas in one female, P.S., age 42, it was secondary to total hypophysectomy for metastatic carcinoma of the breast. One male, A.B., age 43, had Addison's disease, probably due to primary atrophy of the adrenal cortex. In all patients the diagnosis had been unequivocally confirmed by blood and urine measurements of Porter-Silber chromogen before and after administration of ACTH.A comparable experiment was run on each patient.Water and liquids were withheld for 11 to 12 hours before the experiment. One hour after a light, dry breakfast, an indwelling venous needle was inserted after previous local procaine infiltration and left in place throughout the experiment. After a prehydration sample of blood was obtained, three of the patients drank 1,500 ml of tap water over 10 to 15 minutes. This level of positive balance was maintained throughout the experiment by additional oral ingestion of tap water. In patient P.S. hydration was attained by the rapid iv infusion of 0.45%o sodium c...

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To evaluate the rate of production and turnover of antidiuretic hormone (ADH), it is essential to know its volume of distribution. Our previous paper (1), and those in the literature (2-4), have suggested that this volume approximates the plasma volume, and most authors have explained this as the result of binding of ADH to plasma proteins. In the accompanying study (1) on the physiology of ADH in man, we were unable to demonstrate the binding of the hormone to plasma proteins using in vitro ultrafiltration and dialysis techniques. Despite this, the volume of distribution of arginine-vasopressin, calculated from the injected dose and the extrapolated zero time concentration in the plasma, was only 2.5 % of the body weight, a volume significantly less than plasma volume. We concluded, however, that under the circumstances of these experiments the volume calculated from the extrapolation technique was erroneously low. In the same study we found that the turnover rate of both endogenous and exogenous ADH (arginine-vasopressin) in humans was related to the state of hydration. Three days of dehydration significantly accelerated while a comparable period of sustained over hydration slowed the observed normal turnover rate.The present study was undertaken in an effort to determine the true volume of distribution in animals, using a constant infusion technique, and to determine whether the fractional turnover rate * Submitted for publication December' 18, 1963; ac- was truly influenced by the state of hydration per se or by the different levels of circulating ADH resulting from the various states of hydration. MethodsThe fractional turnover rate and volume of distribution of endogenous and exogenous ADH Dog experiments. 1) The disappearance curve of endogenous ADH and injected arginine-vasopressin in the water-loaded animal. Four trained female dogs were deprived of fluids for 12 hours. At the start of the experiment a sample of blood was drawn from an indwelling needle in the foreleg vein, and the animals were hydrated by stomach tube with 800 to 1,000 ml of tap water (5% of body weight). This state of hydration was maintained by a continuous iv infusion of 2.5% glucose in 0.45% sodium chloride at a rate equivalent to urine output. At suitable intervals blood samples were obtained for ADH determination. Timed urine specimens were collected by an indwelling catheter. The osmolality of the urine samples was measured by freezing point depression, using a Fiske osmometer.1 ADH was assayed by a modification of the bioassay of Heller and Stulc (1, 5). After maximal water diuresis was attained, two of the dogs were injected intravenously with 2.5 mU of synthetic arginine-vasopression,2 and its disappearance from the circulation was followed. From these data the fractional turnover rates and apparent volume of distribution were calculated.

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