Measurement of both native and inactivated forms of alpha1 proteinase inhibitor in human inflammatory extracellular fluids.
BACKGROUND
Inactivation of the elastase inhibitor, alpha1 proteinase inhibitor (alpha1PI), may be of pathogenic significance in inflammatory diseases like periodontal disease. Two key mechanisms of inactivation appear to be (a) the formation of an alpha1PI-elastase complex and (b) proteolytic cleavage by elastase or other enzymes such as metalloproteinases of host origin or enzymes of bacterial origin. Based on the different heat stabilities of the intact, complexed and proteolytically cleaved forms of alpha1PI, an enzyme-linked immunosorbent assay (ELISA) that allowed the simultaneous measurement of native and inactive forms of alpha1PI was developed.


METHODS
The ELISA method described employs a commercially available antibody and represents a rapid, reproducible and sensitive method for studying alpha1PI inactivation in human inflammatory diseases. The assay was applied to normal human plasma and to human extracellular fluids obtained from patients with inflammatory diseases such as adult periodontitis and rheumatoid arthritis. Samples from patients with osteoarthritis, a "non-inflammatory" joint disease, were also studied.


RESULTS
The findings expressed as the mean percentage (+/-SD) of the total alpha1PI that was inactivated were as follows: gingival crevicular fluid from adult periodontitis patients: 73.5+/-16.6% (n=12); normal human plasma: 8.4+/-4.9% (n=13); knee-joint synovial fluid (SF) from rheumatoid arthritis patients: 12.5+/-4.5% (n=15); plasma from rheumatoid arthritis patients: 8.0+/-1.8% (n=15); knee-joint SF from osteoarthritis patients: 8.6+/-8.2% (n=14); plasma from osteoarthritis patients: 5.7+/-4.8% (n=14). The results obtained by ELISA were in good agreement with those obtained by the semi-quantitative method of SDS-PAGE and Western blotting.


CONCLUSIONS
We have shown that the differential heat stability of alpha1PI may be utilised as the basis for a rapid, sensitive and reproducible ELISA assay of alpha1PI inactivation. In gingival crevicular fluid from periodontal disease patients, alpha1PI is mainly inactivated and the extent of this inactivation is much higher than in inflammatory fluids from other chronic diseases such as rheumatoid arthritis. This assay could be useful in monitoring the progression of periodontal disease.
Petropoulou
P
P
Inflammation Research Group, Barts and The London, Queen Mary's School of Medicine and Dentistry, University of London, UK.
Zhang
Z
Z
Curtis
M A
MA
Johnson
N W
NW
Hughes
F J
FJ
Winyard
P G
PG
D000328:Adult; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000378:metabolism; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D045604:Extracellular Fluid / Q000737:chemistry*; D005883:Gingival Crevicular Fluid / Q000737:chemistry; D006801:Humans; D007719:Knee Joint / Q000378:metabolism; D010003:Osteoarthritis / Q000097:blood / Q000378:metabolism; D010518:Periodontitis / Q000097:blood / Q000378:metabolism; D015203:Reproducibility of Results; D015842:Serine Proteinase Inhibitors / Q000032:analysis* / Q000097:blood; D013582:Synovial Fluid / Q000737:chemistry; D000515:alpha 1-Antitrypsin / Q000032:analysis*
D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin