Sensitive Method for Quantitative Estimation of Epinephrine in Blood

Abstract

Meltzer and Auer 1 observed that epinephrine causes dilatation of the pupil much more readily after the corresponding superior cervical ganglion has been excised, in rabbits. By means of the “paradoxical” pupil reaction, Joseph and Meltzer 2 demonstrated liberation of epinephrine from the adrenals on stimulation of the splanchnic nerves. This was confirmed by Elliott, 3 who found that dilatation of the pupil and retraction of the nictitating membrane, resulting from electrical excitation of the splanchnic nerve, do not occur in the absence of the adrenals.

Following these qualitative observations, Stewart and Rogoff 4 employed the eye reactions (pupil dilatation, retraction of nictitating membrane and widening of palpebral aperture), sensitized by corresponding superior cervical ganglionectomy, for quantitative studies on the epinephrine output of the adrenals. A method of “auto-assay” was devised based upon reactions of the sensitized eye when adrenal vein blood was collected in a “cava pocket” for a given time and then released into the circulation. The intensity of the epinephrine reactions was measured by intravenous injections of appropriate amounts of epinephrine.

In our experience with various biological test objects used for detecting epinephrine, it was observed that sensitivity of the reagent usually increases in the course of an experiment. An increase of 5 to 25 or more times may occur after the test object has been used for an hour or two. This is quite common with segments of rabbit's intestine and uterus. In earlier work we observed increased sensitivity to epinephrine with the so-called “denervated” or sensitized eye reactions but not of such magnitude as was seen with intestine segments.

The present report will show that it is possible to increase sensitivity of the denervated eye to epinephrine to such a degree that the reactions can be utilized for detection of epinephrine in small amounts in blood-serum or in saline solutions. Quantitative estimation of the epinephrine concentration in the liquid can be made by comparing the reactions with those obtained by injection of epinephrine solutions of different concentrations. By this method it was found possible to detect, with certainty, epinephrine in serum or in physiological saline solution in dilutions of 1 : 100,000,000 to 1:500,000,000. Sometimes the sensitivity is not so great and occasionally an animal may yield reactions with even higher dilutions.

Procedure. The method depends upon further sensitizing the eye to epinephrine after it has already been sensitized by superior cervical ganglionectomy. This is accomplished by repeated injections of epinephrine in amounts sufficient to elicit small but definite reactions. Cats were used 5 or 6 days to 2 weeks after unilateral excision of the superior cervical ganglion. Sensitive reactions have been obtained up to about a month after ganglionectomy. The animal is anesthetized (urethane, 0.75 to 1.0 gm. per kg. body weight by stomach tube). A portion of the femoral or external jugular vein is exposed and a loose ligature is adjusted to facilitate injection into the vein. The carotid artery on the side of the sensitized eye is similarly prepared. A syringe with a very small hypodermic needle is used for the injections.

Epinephrine in saline solution is injected intravenously at intervals of about 5 minutes. When it is observed that the reacting structures are becoming more sensitive the injections are made directly into the carotid artery on the side of the sensitized eye. The injections are repeated, as in the case of the intravenous injections, until sensitivity of the reacting structures is markedly increased. When this occurs the animal can be used for assay of an unknown which contains an epinephrine concentration within the range of sensitivity of the test object.

Quantities ranging from 0.1 cc. to 1.0 cc. of the epinephrine solutions are used, usually beginning with 0.25 cc, 0.5 cc. and 1.0 cc.