Afford S C, Burnett D, Campbell E J, Cury J D, Stockley R A
Lung Immunobiochemical Research Laboratory, General Hospital, Birmingham, United Kingdom.
Biol Chem Hoppe Seyler. 1988 Sep;369(9):1065-74. doi: 10.1515/bchm3.1988.369.2.1065.
The form and function of alpha 1 proteinase inhibitor in lung lavage fluid from healthy smoking and non smoking individuals has been accurately assessed using critically appraised techniques. The present study demonstrated that it is possible to accurately assess alpha 1 PI function in unconcentrated lavage fluid but that sample collection, storage and subsequent processing may all affect the results. Absolute levels of alpha 1 PI were elevated in subjects who smoke and a substantial quantity of inactive protein was found in both smokers and non smokers. The proportion of inactive alpha 1 PI was similar for both groups, which by inference implies that normal smoking subjects do not have decreased protection by this inhibitor at the bronchoalveolar level. Physicochemical analysis of the alpha 1 PI in these normal subjects showed that it was different from alpha 1 PI previously reported from patients with established disease and this may have important implications regarding the pathogenesis of their condition. Western immunoblotting of bronchoalveolar lavage fluid (BALF) showed that all of the alpha 1 PI was present in the native molecular mass form (54,000 Da). Pre-incubation of samples with methionine sulphoxide peptide reductase restored alpha 1 PI function only by approximately 10% suggesting the presence of little reversibly oxidised alpha 1 PI in either group. Anion exchange HPLC of BALF revealed the presence of two alpha 1 PI species, one of which co-eluted with native, oxidised or proteolyzed forms and the other which was more cationic and did not inhibit porcine pancreatic elastase. Finally, thirteen out of sixteen BALF samples inhibited more neutrophil elastase than could be accounted for by the amounts of functional alpha 1 PI present, suggesting that the presence of other inhibitors is a feature of normal lavage fluids.
采用严格评估的技术，已对健康吸烟者和非吸烟者肺灌洗液中α1蛋白酶抑制剂的形式和功能进行了准确评估。本研究表明，有可能在未浓缩的灌洗液中准确评估α1抗胰蛋白酶（α1PI）的功能，但样本采集、储存及后续处理均可能影响结果。吸烟者的α1PI绝对水平升高，且在吸烟者和非吸烟者中均发现大量无活性蛋白。两组中无活性α1PI的比例相似，由此推断，正常吸烟受试者在支气管肺泡水平上并不会因该抑制剂而降低保护作用。对这些正常受试者的α1PI进行物理化学分析表明，其与先前报道的已确诊疾病患者的α1PI不同，这可能对其病情的发病机制具有重要意义。支气管肺泡灌洗液（BALF）的western免疫印迹显示，所有α1PI均以天然分子量形式（54,000 Da）存在。样品与甲硫氨酸亚砜肽还原酶预孵育仅使α1PI功能恢复约10%，这表明两组中可逆氧化的α1PI含量均很少。BALF的阴离子交换高效液相色谱显示存在两种α1PI，其中一种与天然、氧化或蛋白水解形式共洗脱，另一种带更多正电荷且不抑制猪胰弹性蛋白酶。最后，16份BALF样本中有13份对中性粒细胞弹性蛋白酶的抑制作用超过了其中功能性α1PI含量所能解释的范围，这表明存在其他抑制剂是正常灌洗液的一个特征。
