Glucagon Like ActivityのBioassayに関する研究
スポンサーリンク
概要
- 論文の詳細を見る
In the course of experimental studies on the carbohydrate metabolism of isolated tissue by the Warburg method, the author found a new method for quantitative estimation of glucagon like activity of ordinary insulin and human sera.<BR>The general experimental procedure used for this study was as follows : Rat liver slices were incubated in Warburgs apparatus. Incubation medium was 2ml. of modified Buchanan-Hastings solution with glucose to 1 gm. par dl. concentration. Glucose in the incubation medium was estimated by Hagedorn-Jensen's method and this was regarded as glucose output from the liver slices or glucose uptake from the medium. <I>Δ</I>-Glycogen contents of liver slices, prior and after incubation, were regarded as glycogenesis or glycolysis.<BR>When ordinary regular insulin (with glucagon) or NOVO-insulin (glucagon free) were added to the soaking medium, liver slices showed quite a diverse behaviour to <I>Δ</I>-glucose and <I>Δ</I>-glycogen in both experimental conditions. This difference is thought to be based on the co-existence of glucagon in ordinaly insulin.<BR>This view was verified through the experiment, in which it was proved, that glucagon free NOVO-insulin, acted under the addition of a known amount of glucagon (crystalline glucagon, Eli-Lilly & Co.) as ordinary regular insulin.<BR>In the course of critical studies on the amount of glucagon added to NOVO-insulin, we found a possibility of quantitative micro-bioassay for glucagon in the soaking medium.<BR>It was then clearly brought about the glycogenetic changes of liver slices were statistically unreliable, whereas the decrease of glucose uptake in the incubation medium made a linear regression with log doses of corresponding concentration added to the incubation medium. Here was found a possibility for micro-bioassay of a minute amount of glucagon.<BR>(A) Trials for assay of glucagon in ordinary insulin.<BR>Animals : Male albino rats of Wister strain, weighing 100 to 120gm. were used. The animals were made to fast for 24 hours prior to the experiments, then bled ; the liver was removed and slices were prepared to a size about 0.3mm thick and weighing 100mg. each.<BR>Method : Two doses of glucagon, 10<SUP>-2</SUP>μg. per ml. and 10<SUP>-4</SUP>/μg. per ml. were added to the incubation medium and a linear regression was drawn taking the decrease of glucose uptake caused by glucagon, as the standard.<BR>Samples of glucagon like substances, prepared from ordinary insulins following Sutherland's description (1949), were added to the incubation medium also in two dilution.<BR>From the comparison of two regressions, the following results were obtained.<BR>(B) Assay of glucagon like activities of sera.<BR>To exclude the action of unknown impurities of plasma extract, the glucagon like activity of serum had to be estimated under direct addition of serum itself to the soaking medium.<BR>Pretreatment of rats : To standardize the glycogen content of the rat liver slices to be used, the animals were fed a high carbohydrate diet for five days and then made to fast for 48 hours prior to the experiment. This procedure enabled us to get liver almost constant, i.e. 0.24±0.03mg. glycogen per 1gm. wettissue.<BR>Use of dehydroergotamine (D.H.K.) as a component of soaking medium, and influence of other hormones : To get rid of the disturbing effect of epinephrine and analogue compounds of the serum, which act glycogenolytically, 1μg. per ml. of D.H.K. (T214-B) was added to the soaking medium. It was also proved that several other hormones such as ACTH, growth hormone, TSH and cortisone, had no significant effect on the course of this assay method.<BR>Basic procedures for bioassay : Two kinds of control values were necessary for the calculation of glucagon like activity of the serum to be tested, the one for the maximal glucose uptake (C<SUB>1</SUB>) without glucagon activity,
- 日本内分泌学会の論文