PMID- 10759471
OWN - NLM
STAT- MEDLINE
DCOM- 20000421
LR  - 20220330
IS  - 0009-9147 (Print)
IS  - 0009-9147 (Linking)
VI  - 46
IP  - 4
DP  - 2000 Apr
TI  - Analysis of free prostate-specific antigen (PSA) after chemical release from the 
      complex with alpha(1)-antichymotrypsin (PSA-ACT).
PG  - 474-82
AB  - 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.
FAU - Peter, J
AU  - Peter J
AD  - Institut fur Organische Chemie und Biochemie, Technische Universitat Munchen, 
      Lichtenbergstrasse 4, 85748 Garching, Germany.
FAU - Unverzagt, C
AU  - Unverzagt C
FAU - Hoesel, W
AU  - Hoesel W
LA  - eng
PT  - Journal Article
PL  - England
TA  - Clin Chem
JT  - Clinical chemistry
JID - 9421549
RN  - 0 (Biomarkers, Tumor)
RN  - 0 (Indicators and Reagents)
RN  - 0 (alpha 1-Antichymotrypsin)
RN  - 5KV86114PT (Ethanolamine)
RN  - EC 3.4.21.77 (Prostate-Specific Antigen)
SB  - IM
MH  - Biomarkers, Tumor/*blood/chemistry/isolation & purification/standards
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Ethanolamine
MH  - Humans
MH  - Immunoassay
MH  - Indicators and Reagents
MH  - Male
MH  - Mass Spectrometry
MH  - Prostate-Specific Antigen/*blood/chemistry/isolation & purification/standards
MH  - Prostatic Neoplasms/blood
MH  - Reference Standards
MH  - alpha 1-Antichymotrypsin/*blood/chemistry/standards
EDAT- 2000/04/12 09:00
MHDA- 2000/04/29 09:00
CRDT- 2000/04/12 09:00
PHST- 2000/04/12 09:00 [pubmed]
PHST- 2000/04/29 09:00 [medline]
PHST- 2000/04/12 09:00 [entrez]
PST - ppublish
SO  - Clin Chem. 2000 Apr;46(4):474-82.