FAILURE OF LEUCINE, ARGININE, AND EPINEPHRINE TO ALTER PLASMA INSULIN LEVELS IN VITRO
FAILURE OF LEUCINE, ARGININE, AND EPINEPHRINE TO ALTER PLASMA INSULIN LEVELS IN VITRO
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亮氨酸、精氨酸和肾上腺素无法在体外改变血浆胰岛素水平
DOI：
10.1055/s-0028-1096764
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发表时间：
1971
影响因子：
2.2
通讯作者：
J. J. Wyk
中科院分区：
文献类型：
--
作者：
S. Voina;L. Underwood;J. J. Wyk
最近有报道(Szabo和Mahler 1970)在体外向血浆中加入L亮氨酸、i-精氨酸或肾上腺素会改变可免疫测定的胰岛素水平。加入亮氨酸和精氨酸后，血浆胰岛素水平明显升高，加入肾上腺素后胰岛素水平明显降低。这些实验是使用放射免疫测定法进行的，其中TALE被用来分离游离胰岛素和抗体结合胰岛素(Rosselin，Assan，Yalow和Berson，1966)。我们使用双抗体沉淀法进行了类似的实验(Morgan和Lazarow，1963)，在体外添加这些物质后，没有观察到对血浆胰岛素水平的影响。材料和方法：3只健康受试者在口服葡萄糖负荷30分钟后采集血样。所有的血浆样本都被冷冻，直到化验，所有的研究都在同一个化验中进行.Poreine Ins*用于碘标记(1131)，并作为标准制剂。L-亮氨酸(Mann)、i-精氨酸(Mann)和甲苯丁胺(Orinase Upjohn Co.)在水中溶解并稀释，使反应混合物中的浓度为50/ug/ml。肾上腺素的反应浓度为0.125微克/毫升。据报道，在这个浓度下，亮氨酸、精氨酸和肾上腺素会影响滑石粉放射免疫测定(Szabo和Mahler 1970年)。放射免疫分析混合液包括0.02/UU-131-胰岛素、豚鼠抗胰岛素血清(1：85,000)、2.0~40个/UU/ml标准胰岛素或1：10或1：20稀释度的受试者血浆。反应混合量为500/ul，加入0.02M EDTA和2.5%牛血清白蛋白的0.05M藜芦醛缓冲液PR8.2。4℃孵育4d后，加入50Lul兔抗豚鼠血清，孵育16h。加入50/ul正常豚鼠血清后，离心分离上清液。结果：与未添加亮氨酸、精氨酸、甲苯丁胺或肾上腺素的标准曲线相比，添加亮氨酸、精氨酸、甲苯丁胺或肾上腺素的双重标准曲线对胰岛素-抗胰岛素反应无影响(图1)。来自3个正常亚组的血浆样本在不加和加有亮氨酸、精氨酸、肾上腺素和甲苯丁胺的情况下进行了三倍体检测。从不加添加剂的标准曲线读出的平均血浆胰岛素值分别为67(受试者1)、69(受试者2)和400uu/ml(受试者3)。增加了
It has reeently been reported (Szabo and Mahler 1970) that the in vitro addition to plasma of l-leueine, I-arginine, or epinephrine alters the level of immunoassayable insulin. An apparent increase in the plasma insulin levels was observed with the addition of leueine and arginine and a deerease was seen after the addition of epinephrine. These experiments were done using a radioimmunoassay in which tale was used to separate free insulin from antibodybound insulin (Rosselin, Assan, Yalow and Berson 1966). We have performed similar experiments using a double antibody precipitation radioimmunoassay (Morgan and Lazarow 1963) and have observed no effect on plasma insulin levels after the in vitro addition of these substances. Materials and Methods: Blood sampIes were obtained from 3 healthy subjeets 30 minutes after the administration of an oral glucose load. All plasma samples were kept frozen until assay amall studies were done in the same assay. Poreine insulin* was used for iodination (1131 ) and as the standard preparation. L-Ieucine (Mann), I-arginine (Mann), and tolbutamide (Orinase Upjohn Co.) were dissolved in water and diluted so that a coneentration of 50/ug/ml was present in the reaetion mixture. The reaetion mixture eoneentration of epinephrine was 0.125/ug/ml. At these concentrations, leucine, arginine, and epinephrine have been reported to affect the talc radioimmunoassay (Szabo and Mahler 1970). The radioimmunoassay mixture contained 0.02 /uU I 131-insulin, guinea pig anti-insulin serum (final dil., 1:85,000), 2.0 to 40/uU/ml standard insulin, or a 1:10 or 1:20 dilution of test plasma. A 500 /ul reaction mixture volume was aehieved with 0.05 M veranal buffer pR 8.2 with 0.02 M EDTA and 2.5% bovine serum albumin. After 4 days incubation at 4 0 C, 50Lul rabbit anti-guinea pig serum was added and incubated for 16 hours. After addition of 50/ul normal guinea pig serum the precipitate and supernatant were separated by centrifugation and deeantation. Results: Duplieate standard curves with the addition of leucine, arginine, tolbutamide, or epinephrine had no effect on the insulin-anti-insulin reaction when eompared with the eurve with no additions (Fig. 1). Plasma samples from the 3 normal subjeets were assayed in triplieate without and with the addition of leueine, arginine, epinephrine, and tolbutamide. The mean plasma insulin values read from the standard curve with no additive were 67 (Subject 1), 69 (Subjeet 2), and 400 uU/ml (Subject 3). The addition of the