血清Sulfation Factorに関する研究

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The determination of SF in human serum may be used as a measure of growth hormone activity. In this paper, the author established an improved method of the in vitro bioassay of SF activity in human serum, based on S35 uptake of cartilage obtained from hypophysectomized puppies. Material and method Animals : Hypophysectomized puppies were used. The hypophysectomy was performed at the age of 2-3 months. The puppies were killed 10-14 days after hypophysectomy. Costal cartilage segments were taken from the puppies ribs and carefully trimmed free of soft tissue and bone. The weight of each segment was kept constant about 15 mg. Incubation : Two cartilage segments were placed into individual flasks (height : 8 cm, diameter of base : 2 cm). To each flask was added a medium containing 4 mg of glucose, 120 i.u. of penicillin, 120 μg of streptomycin, 60 μc of carrier-free S35-sulfate and Krebs' phosphosaline buffer, making up a total volume of 2.0 ml. Pooled serum obtained from 5 healthy male adults was used as control serum. To measure the activity of SF in a given serum, simultaneous determination of SF of control serum was carried out : 0.1 ml and 0.2 ml, or 0.2 ml and 0.4 ml of control serum and the given serum were added to the test flask before fiilling the flask with phosphosaline buffer. Then two cartilage segments were placed into each flask and all flasks were incubated in a metabolic shaker at +37°C for 24 hours. Removal of inorganic sulfates : After incubation, the cartilage segments were washed sufficiently by water in order to remove the inorganic S35-sulfate and placed overnight in saturated Na2SO4 solution at +37°C and then placed in water at +37°C for three hours. Drying and weighing procedures : Each cartilage segments were then dried thoroughly under an infrared lamp and weighed on an electrobalance to the nearest 0.1 mg. The weight of most segments was between 3 and 5 mg. Counting : Each segment was placed in separate test tubes containing 0.5 ml 6N-HCl. The cartilage was hydrolyzed in a water bath at 100°C for three hours. Each hydrolysate was transfered with 0.5 ml of water to individual counting cups. The water and hydrolysate were evaporated under an infrared lamp and the cups were counted for radioactivity with a GM tube. The S35 uptake has been expressed as counts per minute per mg of dried cartilage. Correction for self-absorption was omitted. Statistical evaluation of human SF activity : In all cases a 4-point-parellel line assay was employed with a two-serum volume (0.1 and 0.2 ml or 0.2 and 0.4 ml) of the standard serum and the unknown serum. Experiments carried out to make up standard conditions of the assay system. (1) The doses of S35 sulfate added to the medium were fixed at 60 μc/Flask from the results of examinations of the relation between the doses of S35 sulfate added in the medium and the S35 uptake of cartilage. (2) The incubation time was fixed at 24 hours from the result of investigations of the relation between the rate of S35 uptake by cartilage and the incubation time. (3) The comparison of the S35 uptake rate of cartilage in different conditions, in which one or two pieces of cartilage were added in the medium, respectively, were examined. There were no differences when one or two pieces of cartilage were added to the medium. So, two pieces of cartilage were added to the medium to simplify the assay method. (4) The S35 uptake by costal cartilage segment taken from various parts of ribs was examined. Costal cartilages of the 1st, 2nd and 3rd rib were excluded from the test material, since they sometimes gave unstable S35 (5) The effect of 4 in vitro additions of normal human serum (0.1, 0.2, 0.4 and 0.8 ml) on the S35 uptake by cartilage was examined.
日本内分泌学会の論文

著者

藤沢義朗
神戸医科大学辻内科

血清Sulfation Factorに関する研究

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