Adachi K, Yasuda K, Fuwa Y, Goshima E, Yamakita N, Miura K
Third Department of Internal Medicine, Gifu University School of Medicine, Japan.
Nihon Naibunpi Gakkai Zasshi. 1990 Feb 20;66(2):113-26. doi: 10.1507/endocrine1927.66.2_113.
The authors aimed at developing a simplified method for the measurement of plasma free (unbound) steroids with ultrafiltration using Grace MPS device, and its clinical application. In this method, the movement of free steroids from plasma into ultrafiltrate was monitored with [14C]glucose added to the plasma. Plasma free steroids (cortisol, testosterone, estradiol and prednisolone) were measured as follows: Plasma was incubated with [14C]glucose (1.2 X 10(4) dpm/5 microliters) for 30 min at 37 degrees C. A 0.5 ml of aliquot was transferred to MPS device containing a single YMT membrane, and centrifuged at 1100 x g, at 37 degrees C, for 30 min in a 45 degree fixed angle head with the special temperature controller. After centrifugation, [14C] in 30 microliters of plasma and ultrafiltrate were counted. For the calculation of plasma free level, steroid concentration of ultrafiltrate measured directly by radioimmunoassay (RIA) was multiplied by the ratio of [14C]glucose (dpm) in plasma to [14C]glucose (dpm) in ultrafiltrate. For RIA of cortisol, testosterone and estradiol, commercially available kits were used, and for prednisolone, anti-prednisolone antibody developed in our laboratory was utilized. Since plasma free cortisol level showed a parallel increase with rise in temperature, strict temperature control during ultracentrifugation was required. On the other hand, the duration of centrifugation and the sample volume applied to MPS did not show any significant effect on the estimated values. Intraassay and interassay variations were 4.4% and 6.1% in free cortisol, 6.3% and 8.7% in free testosterone, 8.5% and 9.2% in free estradiol, and 8.8% and 9.9% in free prednisolone, respectively. The correlations between the plasma free steroid levels obtained by ultrafiltration (y) and equilibrium dialysis (x) were as follows, respectively: free cortisol; y = 1.16x + 0.017 (r = 0.95, n = 10), free testosterone; y = 1.17x-0.027 (r = 0.92, n = 10), free estradiol; y = 1.33x + 8.55 (r = 0.98, n = 12), free prednisolone; y = 1.03x + 1.00 (r = 0.98, n = 7). The mean plasma free cortisol levels and percent free fractions (%FF) were 1.16 +/- 0.40 (+/- SD) micrograms/dl and 10.9 +/- 3.0% in 10 control patients, 4.4 +/- 1.6 micrograms/dl and 15.6 +/- 3.3% in 8 patients with Cushing's syndrome, and 1.45 +/- 0.48 micrograms/dl and 5.9 +/- 2.4% in 11 normal pregnant women (10-40 weeks of pregnancy), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
作者旨在开发一种使用Grace MPS装置通过超滤测量血浆游离（未结合）类固醇的简化方法及其临床应用。在该方法中，向血浆中添加[¹⁴C]葡萄糖来监测游离类固醇从血浆进入超滤液的移动情况。血浆游离类固醇（皮质醇、睾酮、雌二醇和泼尼松龙）的测量方法如下：将血浆与[¹⁴C]葡萄糖（1.2×10⁴ dpm/5微升）在37℃孵育30分钟。取0.5毫升等分试样转移至装有单个YMT膜的MPS装置中，并在37℃下以1100×g在45度固定角度转头中离心30分钟，配备特殊温度控制器。离心后，对30微升血浆和超滤液中的[¹⁴C]进行计数。为计算血浆游离水平，通过放射免疫分析（RIA）直接测量的超滤液中类固醇浓度乘以血浆中[¹⁴C]葡萄糖（dpm）与超滤液中[¹⁴C]葡萄糖（dpm）的比值。对于皮质醇、睾酮和雌二醇的RIA，使用市售试剂盒，对于泼尼松龙，使用我们实验室开发的抗泼尼松龙抗体。由于血浆游离皮质醇水平随温度升高呈平行升高，因此超速离心期间需要严格控制温度。另一方面，离心时间和应用于MPS的样品体积对估计值没有任何显著影响。游离皮质醇的批内和批间变异分别为4.4%和6.1%，游离睾酮为6.3%和8.7%，游离雌二醇为8.5%和9.2%，游离泼尼松龙为8.8%和9.9%。通过超滤获得的血浆游离类固醇水平（y）与平衡透析（x）之间的相关性分别如下：游离皮质醇；y = 1.16x + 0.017（r = 0.95，n = 10），游离睾酮；y = 1.17x - 0.027（r = 0.92，n = 10），游离雌二醇；y = 1.33x + 8.55（r = 0.98，n = 12），游离泼尼松龙；y = 1.03x + 1.00（r = 0.98，n = 7）。10名对照患者的平均血浆游离皮质醇水平和游离分数百分比（%FF）分别为1.16±0.40（±标准差）微克/分升和10.9±3.0%，8名库欣综合征患者为4.4±1.6微克/分升和15.6±3.3%，11名正常孕妇（妊娠10 - 40周）为1.45±0.48微克/分升和5.9±2.4%。（摘要截短于400字）
