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Articles |
1-Antichymotrypsin (PSA-ACT)
1
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany.
2
Roche Diagnostics GmbH, Nonnenwaldstrasse 2, 82372
Penzberg, Germany.
a Author for correspondence. Fax 49-8856-603341; e-mail wolfgang.hoesel{at}roche.com
Background: Prostate-specific antigen (PSA), a marker for prostate
cancer (CaP), forms a covalent complex with
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
µg/L F-PSA and 1890 µg/L total PSA (T-PSA)] was treated
under similar conditions, a concentration of 
1600 µg/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 µg/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.
The following articles in journals at HighWire Press have cited this article:
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  M. Tajiri, C. Ohyama, and Y. Wada Oligosaccharide Profiles of the Prostate Specific Antigen in Free and Complexed Forms from the Prostate Cancer Patient Serum and in Seminal Plasma: a Glycopeptide Approach Glycobiology, January 1, 2008; 18(1): 2 - 8. [Abstract] [Full Text] [PDF]
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G. Tabares, C. M. Radcliffe, S. Barrabes, M. Ramirez, R. N. Aleixandre, W. Hoesel, R. A. Dwek, P. M. Rudd, R. Peracaula, and R. de Llorens Different glycan structures in prostate-specific antigen from prostate cancer sera in relation to seminal plasma PSA Glycobiology, February 1, 2006; 16(2): 132 - 145. [Abstract] [Full Text] [PDF]
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 S. D. Mikolajczyk, K. M. Marker, L. S. Millar, A. Kumar, M. S. Saedi, J. K. Payne, C. L. Evans, C. L. Gasior, H. J. Linton, P. Carpenter, et al. A Truncated Precursor Form of Prostate-specific Antigen Is a More Specific Serum Marker of Prostate Cancer Cancer Res., September 1, 2001; 61(18): 6958 - 6963. [Abstract] [Full Text] [PDF]
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J. Peter, C. Unverzagt, T. N. Krogh, O. Vorm, and W. Hoesel Identification of Precursor Forms of Free Prostate-specific Antigen in Serum of Prostate Cancer Patients by Immunosorption and Mass Spectrometry Cancer Res., February 1, 2001; 61(3): 957 - 962. [Abstract] [Full Text]
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 W. Hoesel, J. Peter, H. Lenz, and C. Unverzagt Purification of Prostate-specific Antigen from Serum by Indirect Immunosorption and Elution with a Hapten Clin. Chem., September 1, 2000; 46(9): 1490 - 1491. [Full Text] [PDF]
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