Abstract
The determination of chromogranine released in exocytosis as well as of the catecholamines released from the adrenal medulla should greatly contribute not only to unraveling the mechanism of exocytosis but also to clinically diagnosing pheochromocytoma. The determination of chromogranine, however, has not been amply studied as yet.
In the first place, the catecholamine-storing vesicle-rich fraction was collected from the pig adrenal medulla by the density gradienter. In order to lyse these cell particles, they were first frozen with cold methanol, then thawed, and centrifuged. The supernatant was developed by DEAE chromatography to collect only the peak fraction, from which chromogranine was isolated. Rabbits were then injected with this chromogranine to obtain the anti-chromogranine antiserum. The successful immunization of the rabbits was demonstrated by immunodiffusion on the Ouchterlony plate.
The anti-pig chromogranine antiserum was demonstrated to be common in antigenicities with the human pheochromocytoma extract; and a serological method of chromogranine assay was then developed. For the preparation of the sensitized sheep red blood cells, the sheep red blood cells were first made to bind to the pig chromogranine by means of treating them with a formaldehyde solution and tannic acid. For the assay of the pheochromocytoma extract for chromogranine 25μl of the extract were added to two-fold dilution series of 25μl of the anti-chromogranine antiserum, then 25μl of the sensitized red blood cells was added. The dilution number of the antiserum that completely inhibited hemagglutination reaction, that is, inhibition titer, was defined as the level of chromogranine. This method demonstrated the occurrence of large amounts of catecholamines in the resected tissue extract and also of chromogranine at the 1: 8 inhibition titer.
