McConway M G, Johnson D, Kelly A, Griffin D, Smith J, Wallace A M
Department of Clinical Biochemistry, Royal Infirmary, Glasgow, UK.
Ann Clin Biochem. 2000 Sep;37 ( Pt 5):717-23. doi: 10.1258/0004563001899771.
We describe a radioimmunoassay (RIA) for total leptin and a gel filtration procedure for the separation of free and bound leptin in human serum. The RIA, based on a locally prepared antibody, has a minimum detection limit of 0.9 ng/mL, a working range (CV < 10%) of 2.5-50 ng/mL, inter-assay precision of 10.2, 7.2 and 8.9%CV at 7.9, 15.4 and 30.0 ng/mL, respectively, 94% recovery of exogenous leptin (range 81.1-120.6%), exhibited parallelism and demonstrated no significant cross-reactivity or interferences. A difference plot of results from this method and those from a commercially available kit (Linco Research) demonstrated satisfactory agreement up to concentrations of 50 ng/mL total leptin, with no significant bias. A gender-dependent correlation was obtained between body mass index (BMI) and total leptin (r = 0.91, P<0.001, n = 75 for men; r = 0.79, P<0.001, n = 72 for women), with women having higher leptin concentrations than men for any given BMI. Gel filtration studies (inter-assay precision: 4.7%CV, n = 18) demonstrated that a variable fraction (between 10% and 40%) of total leptin in serum was bound with high affinity (Keq = 1.0-1.45 x 10(9) L/mol) to a non-albumin, non-lipid macromolecule. Binding affinities were found to be similar irrespective of gender or fat mass. A significant positive correlation between free or bound leptin concentrations and BMI was obtained for both men and women (r = 0.87-0.94); free and bound leptin concentrations were also significantly higher in women (P<0.01) than in men for any given BMI, and higher in obese (P<0.01) than in lean individuals. We conclude that leptin 'resistance' associated with obesity cannot be accounted for by reduced free leptin concentrations in serum and that the methods described are suitable for the investigation of total, free and bound leptin for both clinical and research purposes.
我们描述了一种用于检测人血清中总瘦素的放射免疫分析法（RIA）以及一种用于分离游离和结合态瘦素的凝胶过滤方法。基于本地制备抗体的放射免疫分析法，其最低检测限为0.9 ng/mL，工作范围（变异系数<10%）为2.5 - 50 ng/mL，批间精密度在7.9、15.4和30.0 ng/mL时分别为10.2%、7.2%和8.9%变异系数，外源性瘦素回收率为94%（范围81.1 - 120.6%），呈现平行性，且无显著交叉反应或干扰。该方法与市售试剂盒（Linco Research）结果的差异图显示，在总瘦素浓度达50 ng/mL时一致性良好，无显著偏差。体重指数（BMI）与总瘦素之间存在性别依赖性相关性（男性r = 0.91，P<0.001，n = 75；女性r = 0.79，P<0.001，n = 72），对于任何给定的BMI，女性的瘦素浓度高于男性。凝胶过滤研究（批间精密度：4.7%变异系数，n = 18）表明，血清中总瘦素的可变部分（10%至40%）以高亲和力（平衡常数Keq = 1.0 - 1.45 x 10(9) L/mol）与一种非白蛋白、非脂质大分子结合。发现结合亲和力与性别或脂肪量无关。男性和女性的游离或结合态瘦素浓度与BMI均呈显著正相关（r = 0.87 - 0.94）；对于任何给定的BMI，女性的游离和结合态瘦素浓度也显著高于男性（P<0.01），肥胖者高于瘦者（P<0.01）。我们得出结论，与肥胖相关的瘦素“抵抗”不能用血清中游离瘦素浓度降低来解释，且所描述的方法适用于临床和研究目的的总瘦素、游离瘦素和结合态瘦素的研究。
