Kanabrocki E L, Kanabrocki J A, Sothern R B, Futscher B, Lampo S, Cournoyer C, Rubnitz M E, Zieher S J, Greco J, Bushnell D L
Nuclear Medicine Service, Veteran's Administration Hospital, Hines, IL 60141.
Chronobiol Int. 1990;7(5-6):433-43. doi: 10.3109/07420529009059155.
Urine samples were collected at 3-hr intervals over a single 24-hr period from each of seven clinically healthy men who ranged in age from 21-25 years. Urines at each collection time were subsequently pooled using 20% of each volume and serially dialyzed against ammonium-barbituric acid buffer (pH 7.35 +/- 0.02), using a cellulose membrane permeable to compounds of less than 12,000-14,000 molecular weight (mw). When the dialyzed portions were then analyzed for total proteins, the sum of proteins in eight pools amounted to 74 mg. A 1 ml aliquot of each pool, representing approximately 50 micrograms of proteins, was concentrated and reconstituted. Approximately 20 micrograms of reconstituted proteins were then subjected to polyacrylamide gel electrophoresis. The stained gel was then scanned by laser densitometry and planimetry. Each aliquot revealed eight segments as identified by Coomassie and silver staining. Their molecular weights, estimated by extrapolation from concurrently run protein standards, and their total protein amounts were: 116,000 mw (9.44 mg), 91,000 mw (3.3 mg), 68,000 mw (11.58 mg), 53,000 mw (2.58 mg), 43,000 mw (9.12 mg), 32,000 mw (7.13 mg), 24,000 mw (4.52 mg) and 20,000 mw (5.27 mg). A statistically significant rhythm (P = 0.022 from ANOVA and 0.011 from Single Cosinor) was found for the excretion of total proteins, with an acrophase in the afternoon (1537) for these diurnally-active subjects.
在一个24小时的时间段内，每隔3小时从7名年龄在21至25岁之间的临床健康男性中收集尿液样本。随后，将每个收集时间点的尿液按各体积的20%混合，并使用分子量小于12,000 - 14,000的化合物可透过的纤维素膜，对混合尿液进行连续透析，透析液为巴比妥酸铵缓冲液（pH 7.35 ± 0.02）。当对透析后的部分进行总蛋白分析时，八个样本池中的蛋白质总量为74毫克。从每个样本池中取出1毫升等分试样，约代表50微克蛋白质，进行浓缩和重构。然后对约20微克重构后的蛋白质进行聚丙烯酰胺凝胶电泳。之后，用激光密度计和平面测量法对染色后的凝胶进行扫描。通过考马斯亮蓝染色和银染色鉴定，每个等分试样显示出八个条带。根据同时运行的蛋白质标准品外推估计，它们的分子量和总蛋白量分别为：116,000道尔顿（9.44毫克）、91,000道尔顿（3.3毫克）、68,000道尔顿（11.58毫克）、53,000道尔顿（2.58毫克）、43,000道尔顿（9.12毫克）、32,000道尔顿（7.13毫克）、24,000道尔顿（4.52毫克）和20,000道尔顿（5.27毫克）。对于这些昼间活动的受试者，发现总蛋白排泄存在统计学显著节律（方差分析P = 0.022，单余弦法P = 0.011），高峰相位在下午（1537）。
