大鼠血浆瘦素结合活性的年龄相关性变化:简单酸-乙醇沉淀法与柱层析法的比较。

Arshag D. Mooradian, R. Hurd, J. Chehade, K. Pun, M. Haas
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引用次数: 12

摘要

建立了一种新的测定游离瘦素的方法,并对色谱技术进行了验证。该试验采用酸乙醇萃取(AEE)分离结合/游离瘦素片段。试验间变异系数为3.9%。与1微克未标记的大鼠瘦素孵育证实了瘦素结合的特异性,该瘦素有效地与放射性标记的瘦素竞争,而人类生长激素和白细胞介素-6在与放射性标记的瘦素结合竞争中无效。与大鼠血浆竞争结合实验的Scatchard分析表明,与0.3-0.6 x 109 M-1的结合亲和力呈线性关系。这种新的测定方法被用来确定年龄相关的对瘦素作用的不敏感是否继发于血清瘦素结合改变。研究了不同年龄组大鼠体脂、血清总瘦素和游离瘦素浓度的变化。24月龄大鼠血清游离瘦素浓度(ng/ml平均+/- SEM)显著升高(5.56 +/- 0。21)与18月龄大鼠(4.76 +/- 0.17)相比(P < 0.01),尽管两个年龄组的体重和脂肪相似。12月龄大鼠血浆游离瘦素浓度(3.86 +/- 0.28)和6月龄大鼠血浆游离瘦素浓度(2.05 +/- 0.06)相对于3月龄大鼠(1.37 +/- 0.06)的升高(P < 0.001)与衰老大鼠体脂增加不成比例。由此可见,衰老与大鼠对瘦素的相对不敏感有关。这种变化不能归因于血浆结合增加或瘦素游离部分减少。
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Age-related changes in plasma leptin binding activity in rats: A comparison of a simple acid-ethanol precipitation technique with column chromatography.
A novel assay for measuring the free leptin fraction was developed and validated against a chromatographic technique. The assay used acid-ethanol extraction (AEE) for separation of bound/free leptin moieties. The interassay coefficient of variation was 3.9%. The specificity for leptin binding was confirmed by incubation with 1 microg of unlabeled rat leptin that effectively competed with radiolabeled leptin whereas human growth hormone and interleukin-6 were ineffective in competing with radiolabeled leptin binding. Scatchard analysis of competitive binding experiments with rat plasma demonstrated a linear relationship with a binding affinity of 0.3-0.6 x 109 M-1. This novel assay was used to determine if age-related insensitivity to leptin action is secondary to altered serum leptin binding. Rats at various age groups were studied for changes in body adiposity and serum total and free leptin concentrations. Serum free leptin concentrations (ng/ml mean +/- SEM) were significantly increased in 24-month-old rats (5.56 +/- 0. 21) compared with 18-month-old rats (4.76 +/- 0.17) (P < 0.01) despite similar body weight and adiposity of the two age groups. The increase in plasma free leptin concentrations in 12-month-old rats (3.86 +/- 0.28) and 6-month-old rats (2.05 +/- 0.06) relative to 3-month-old rats (1.37 +/- 0.06) (P < 0.001) was out of proportion to the increase in body adiposity in aging rats. It is concluded that aging in rats is associated with relative insensitivity to leptin. This change cannot be attributed to increased plasma binding or to a reduction in the leptin free fraction.
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