Analysis of free prostate-specific antigen (PSA) after chemical release from the complex with alpha(1)-antichymotrypsin (PSA-ACT).
BACKGROUND
Prostate-specific antigen (PSA), a marker for prostate cancer (CaP), forms a covalent complex with alpha(1)-antichymotrypsin (ACT) in human blood. Structural analysis of the PSA-ACT complex is difficult, and complexation may be a reason for biased immunological assays when compared with the analysis of free PSA. We developed a method to cleave the PSA-ACT complex chemically. The liberated PSA was thus available for analysis as free PSA (F-PSA).


METHODS
PSA was released from the PSA-ACT complex by cleaving the interprotein ester bond with ethanolamine under alkaline conditions. The release was followed by reversed-phase HPLC and an immunoassay for F-PSA. Released PSA obtained from human blood was further immunopurified and analyzed by matrix-assisted laser desorption-induced time of flight (MALDI-TOF) mass spectrometry.


RESULTS
In vitro-prepared PSA-ACT complex was completely cleaved by treatment with nucleophilic compounds such as ethanolamine at pH 9-10. The released PSA was stable under these conditions and could be measured by reversed-phase HPLC as well as the ENZYMUN immunoassay for F-PSA. When plasma from a CaP patient [containing 190 microg/L F-PSA and 1890 microg/L total PSA (T-PSA)] was treated under similar conditions, a concentration of approximately 1600 microg/L F-PSA was measured at the end of the incubation, indicating that the PSA-ACT complex was completely cleaved. Two benign prostatic hyperplasia and CaP sera panels (12 and 13 sera, respectively) containing 4-45 microg/L T-PSA were similarly treated. The concentrations of F-PSA measured after incubation were, on average, 85% of the T-PSA values of the untreated sera. Finally, the PSA released from the complex of the CaP plasma was isolated by immunosorption, analyzed by MALDI-TOF mass spectrometry, and compared to PSA obtained from semen. The intact PSA as well as the peptides observed after digestion with endoproteinase Lys C did not reveal any structural difference between the PSA from these two sources.


CONCLUSIONS
PSA complexed to ACT in plasma of a CaP patient seems to be structurally very similar to the PSA reference material from semen. The release of PSA from the PSA-ACT complex allows F-PSA and T-PSA to be measured by the same immunological assay, thus eliminating any possible bias between two different assays.
Peter
J
J
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany.
Unverzagt
C
C
Hoesel
W
W
D014408:Biomarkers, Tumor / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification / Q000592:standards; D004591:Electrophoresis, Polyacrylamide Gel; D019856:Ethanolamine; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D008297:Male; D013058:Mass Spectrometry; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification / Q000592:standards; D011471:Prostatic Neoplasms / Q000097:blood; D012015:Reference Standards; D000514:alpha 1-Antichymotrypsin / Q000097:blood* / Q000737:chemistry / Q000592:standards
D014408:Biomarkers, Tumor; D007202:Indicators and Reagents; D000514:alpha 1-Antichymotrypsin; D019856:Ethanolamine; D017430:Prostate-Specific Antigen