We describe a method to measure human leukocyte elastase (HLE) inhibitory capacity and compared it with porcine pancreatic elastase (PPE) inhibitory capacity and with a turbidimetric method using a specific antibody to alpha-1 antitrypsin (AAT), all performed on a Cobas Bio centrifugal analyser. This assay used methoxysuccinyl-dialanine-proline-valine-p-nitroanilide as substrate in the presence of 0.01% Brij 35, an HLE enzyme activator. Samples containing commonly used anti-coagulants and serum could be used in the assay, except for those containing heparin which strongly inhibited HLE.
This assay was used to determine the functional AAT concentrations in plasma from a number of normal volunteers and patient groups, and was compared to the immuno-turbidimetric AAT assay. No difference in the proportion of functional to immuno-turbidimetric AAT was noted between any of the groups studied except for the adult respiratory distress syndrome (ARDS), where this percentage was reduced (p < 0.05). An increase in both immuno-turbidimetric and functional AAT was seen for children (both p < 0.01), in emphysema (p < 0.05 and p < 0.01 respectively) and ARDS (both p < 0.05) when compared to adult non-smokers.
This assay was also used to determine the HLE inhibitory capacity of serum and bronchoalveolar lavage (BAL) fluid from normal volunteer smokers(n = 4) and non-smokers (n = 4), and in the serum and BAL fluid from patients with ARDS (n = 5). Serum AAT was 94% functional in non-smokers (91% with PPE functional assay) and 96% in smokers (97% with PPE assay). In BAL fluid, the presence of other HLE inhibitors as well as AAT was demonstrated by increased inhibition: 138% in nonsmokers (70% with PPE assay) and 153% in smokers (85% with PPE assay). In ARDS there was a two-fold increase in serum AAT (ARDS 4.50 -+ 2.43 g/L, normal volunteers 2.05 -+ 0.33) with more than a forty-fold increase in BAL AAT (ARDS 0.101 -+ 0.167 g/L, normal volunteers 0.0017 +-0.0013) as determined with the immuno-turbidimetric assay. The HLE and PPE inhibition assays both showed 76% inhibition in ARDS serum. However, inhibition of 118% (normal non-smokers and smokers were 138% and 153%) and 37% (normal non-smokers and smokers were 70% and 85%) respectively were seen in BAL fluid, reflecting the inactivation of AAT and other inhibitors in the HLE assay.
The HLE inhibition assay described is more rapid and sensitive for the estimation of AAT in serum or plasma than the comparable PPE inhibition and the immunoturbidimetric assays; it also has the advantage that in BAL all the functional inhibitory activity towards HLE is measured.