Physiologic Studies of Antidiuretic Hormone by Its Direct Measurement in Human Plasma *

Abstract

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 ...

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