Abstract
The following procedure has been used in obtaining extracts of the growth hormone of the anterior hypophysis for producing experimental gigantism in rats and provoking the growth of hypophysectomized puppies in the hands of Dr. Frederick L. Reichert.
2,000 gm. of beef anterior hypophysis. The anterior lobes of beef hypophyses had been stored for a period of between 18 and 24 months with solid CO2 in an insulated container in a room held at −5.0° F., after having been ground in a Wiley laboratory mill.
12,000 cc. distilled water.
3,000 cc. 0.2/N Ba (OH)2
Centrifugation: The alkaline mass was mixed by an electric household stirrer (“kitchen mechanic”); then centrifuged in a Sharpies laboratory centrifuge. Large nozzles were used in the first centrifuging in order to remove the bulk of the insoluble material; the extract was then centrifuged at a rate of about 10 cc. per minute. The centrifuge was cooled with salt and ice at all times and the extract was kept surrounded with ice.
Neutralization: 0.2/N H2SO4 was added slowly with constant stirring by the same stirrer. Phenol red was used as indicator and the end point chosen was about pH 8.0, about 500 cc. being required. The Ba not already precipitated by H2SO4 was removed by a slight excess of Na2SO4.
Centrifugation: Sharples centrifuging was then repeated, finishing as before at a rate of 10 cc. per minute.
Filtration: A standard 14 cm. Seitz asbestos filter was used. A pressure of about 10 1hs. per square inch was applied by the use of a tire pump. Time required for filtration of I liters was about 45 minutes; towards the end, the rate of flow through the filter had decreased to half the original rate. Precautions were taken to maintain sterility of the solution after filtration. The extract was stored in 25 cc. lots in rubber-capped bottles and kept frozen until needed for injection.
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