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Technical Briefs |
1-Antichymotrypsin-Prostate-specific Antigen in Patients with Prostate Carcinoma but not in Those with Benign Prostate Hyperplasia
1
Urology and
2
Laboratory Medicine, University Hospital Charité, Humboldt University Berlin, Berlin, Germany;
a address correspondence to this author at: Department of Urology, University Hospital Charité, Humboldt University, Schumannstrasse 20/21, D-10098 Berlin, Germany
Aproximately 70–90% of the total serum prostate-specific
antigen (t-PSA) in serum is complexed to
1-antichymotrypsin (ACT), whereas 10–30%
of t-PSA is not bound to serum proteins and is called free PSA (f-PSA).
The determination of f-PSA and the calculation of the ratio of free to
total PSA has proven to be a promising tool for differentiating between
prostate cancer (PCa) and benign prostate hyperplasia (BPH), because
the ratio is lower in PCa than in BPH (1)(2). Although the
determination of ACT-PSA would have the analytical advantage of
measuring the major and not the minimal portion and the clinical
advantage of measuring the portion of serum PSA apparently directly
related to PCa (3)(4)(5), overrecovery problems related to
interferences with the ACT-cathepsin G complex for ACT-PSA assays are
obstructive to accurate ACT-PSA measurement (6). Thus, it is
generally assumed that the sum of f-PSA plus ACT-PSA yields t-PSA,
whereas the small amounts of PSA being bound to other proteins such as
1-antitrypsin and protein C may be neglected.
However, there is no information concerning whether the occurrence of
minor forms of PSA is different between patients with PCa and those
with BPH. We have now studied this problem by using a reliable ACT-PSA
assay.
t-PSA, f-PSA, and ACT-PSA were measured on an ES immunoanalyzer (Boehringer Mannheim GmbH). The principle of the tests is based on the one-step (for t-PSA; cat. no. 1555332) and/or two-step (for f-PSA, cat. no. 1776444; for ACT-PSA, a research assay) sandwich technique. For the determination of t-PSA, samples (calibrators, patient samples, and control materials) are incubated together with biotinylated mouse monoclonal anti-t-PSA antibody (mAb M10) solution and horseradish peroxidase-labeled mouse anti-PSA conjugate in streptavidin-coated plastic tubes. For the determination of f-PSA and ACT-PSA, the newly developed assay, samples are incubated in the first step with specific monoclonal biotinylated antibodies, forming a PSA-antibody sandwich complex that is bound by the biotinylated antibodies to the streptavidin-coated tubes. ACT-PSA is similarly captured as t-PSA with anti-PSA mAb M10. After the tubes are washed, a horseradish peroxidase-labeled mouse anti-PSA conjugate for the determination of f-PSA or anti-ACT mAb 53 (CanAg) for the determination of ACT-PSA is added and bound to the previously formed complex in a second incubation cycle. The substrate diammonium 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonate) is then added, and the absorbance is measured after 30 min. Calibration of the ACT-PSA assay was performed with ACT-PSA (Scripps Laboratory; diluted in 100 mmol/L phosphate buffer, pH 6.8, containing 60 g/L bovine serum albumin) determined with the t-PSA assay.
This study was approved by the Ethical Standard Committee of the hospital and included 69 patients with BPH (median age, 65 years; range, 50–88 years) and 96 patients with PCa (median age, 65 years; range, 50–87 years) diagnosed histologically before treatment with total PSA concentrations up to 20 µg/L. The diagnosis of BPH was established clinically (by digital rectal examination and/or transrectal ultrasonography, n = 34) or histologically (either with tissue obtained by transurethral resection of the prostate or by biopsy, n = 35). Because there were no differences between these two BPH groups, in the results described in the following, they were considered as one group.
Blood samples were collected before any diagnostic procedures. After
the blood was allowed to clot for 1 h at room temperature, the
samples were centrifuged at 1600g for 15 min at 4 °C. The
sera were generally frozen at -80 °C within 2 h and then
analyzed during 12 weeks. Statistical calculations (t-test
of paired data, Wilcoxon test, Mann–Whitney U-test, and
2 test) were performed using the statistical
package SPSS 7.5 for Windows (SPSS Software). Regression analysis for
methodical evaluation was performed using the EVAPAK for Windows
software, Ver. 3.01 (7). P <0.05 was considered
statistically significant.
The assay characteristics for t-PSA and f-PSA corresponded to data obtained previously with those tests (8)(9). According to the manufacturer, the t-PSA assay is an equimolar test, and recent data from the IFCC standardization study confirmed its comparability with an equimolar assay (Hybritech) when the calibrators in the test or the Stanford 90:10 PSA calibrator (10) are used. The data for the new ACT-PSA assay were as follows: lower limit of detection (21 replicate measurements of the zero calibrator, calculated on the basis of the mean value + 2 SD) of 0.068 µg/L; intraassay imprecision (CV; n = 21) of 1.51–3.48% in five pooled human sera with ACT-PSA values between 1.08 and 22.0 µg/L; interassay CV (n = 8–17) of 2.08–6.32% in four pooled sera and one control material with ACT-PSA values between 0.32 and 32.2 µg/L. Measurement of 53 female sera (41 healthy females and 12 with pronounced inflammation indicated by highly increased C-reactive protein between 144 and 284 mg/L; reference value, <5 mg/L) showed a mean value of <0.01 µg/L of ACT-PSA, with all concentrations being lower than twice the lower detection limit. Addition of ACT-PSA to female sera (PSA <0.02 µg/L) yielded the following relationship: yACT-PSA=1.010xt-PSA - 0.068; n = 63; r = 0.998 by the regression analysis of Passing and Bablok (7). The mean recovery (± SD) of ACT-PSA based on the t-PSA measurement was 98.2% ± 6.9%.
PCa patients, although having higher median t-PSA concentrations than
BPH patients (6.19 vs 3.49 µg/L; P <0.0001),
were characterized by similar f-PSA concentrations (0.79 vs 0.73
µg/L; P = 0.223) but lower f-PSA% values compared
with BPH patients (12.4% vs 24.8%; P <0.0001).
However, in contrast to that typical behavior of f-PSA% in PCa and BPH
patients, the ratio ACT-PSA to t-PSA did not differ between PCa and BPH
(78.9% vs 77.2%; P = 0.591). Consequently, the f-PSA
and ACT-PSA values of each patient in both groups were summed and
related to the corresponding measured t-PSA. When related to the t-PSA
value, the sum of f-PSA and ACT-PSA amounted to 102% ± 10% in BPH
patients and 91% ± 13% in PCa patients and differed significantly
(P <0.0001). Regression analysis according to Passing and
Bablok (7) showed that the slope of the regression line
between the sum of f-PSA plus ACT-PSA and t-PSA differed from one in
PCa patients (P <0.05), but not in BPH patients (Fig. 1
). The difference plots (Fig. 1
) underlined this effect
and showed that it was independent of the concentration of t-PSA. The
2 test confirmed that significantly
(P <0.0001) more PCa patients (78 of 96) than BPH patients
(30 of 69) were below the zero line. These data demonstrate that there
is a gap between t-PSA and the sum of f-PSA plus ACT-PSA in patients
with prostatic carcinoma but not in those with BPH. Thus, sera of PCa
patients are characterized by the occurrence of additional PSA forms
that are detected by the t-PSA assay but by neither the f-PSA nor by
the ACT-PSA assay.
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It may be possible that PSA in the sera of PCa patients is increasingly
complexed with other proteins. In vitro and in vivo experiments showed
complexation of PSA with inhibitors such as
1-protease inhibitor and protein C
(4)(11)(12). A changed glycation of the PSA forms occurs
in the presence of prostate carcinoma and could be the reason for the
change of the complex formation of PSA with various proteinase
inhibitors (13). Thus, these minor PSA complexes produce
such concentrations in PCa patients that the above mentioned gap
appears. Other possible explanations for these discrepancies between
BPH and PCa patients include the calibration of the assays, different
recognition of multiple forms of f-PSA or t-PSA in the two groups of
patients, and the lack of equimolarity of the tests. However, we
believe these rather technical artifacts are ruled out as far as
possible by the analytical data described above.
To our knowledge of the literature, this phenomenon was not clearly pointed out until now (3)(4)(5)(14). One reason might be that, because of the analytical problems of overestimation mentioned above, the measurement of ACT-PSA concentrations could not be performed reliably in the past (6). When an improved assay for ACT-PSA was used, t-PSA values were frequently greater than the sum of f-PSA plus ACT-PSA, similar to our study (6). However, no additional conclusions were given by these authors.
In conclusion, we believe that our results contradict the high
expectations concerning the determination of ACT-PSA or the ratio of
ACT-PSA to t-PSA to improve the differentiation between PCa and BPH
(15). The f-PSA/t-PSA ratio, and not the ACT-PSA/t-PSA
ratio, allowed the best discrimination between BPH and PCa patients.
These data correspond to the findings described previously by
Björk et al. (14). Other studies also showed that
ACT-PSA concentration alone did not improve the specificity for PCa
diagnostics over t-PSA (16). Therefore, a recently described
PSA immunoassay that detects all complexed PSA forms, such as ACT-PSA,
as well as the minor forms except PSA complexed to
2-macroglobulin may eliminate the problem of
minor PSA forms as found in our study (17).
Acknowledgments
This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (Ju 365/2-1) and the Fund of the German Chemical Industry (No. 400770 to K.J.). We thank Roche Diagnostics Boehringer Mannheim and CanAg Diagnostics (Gothenburg, Sweden) for providing us with the PSA test kits and detailed technical data of the tests. We thank Sabine Becker, Silke Klotzek, and Katrin Krüger for valuable technical assistance. The study includes parts of the doctoral thesis of A.K.
Footnotes
fax 4930 28021402, e-mail klaus.jung{at}charite.de
References
1-antichymotrypsin. Clin Chem 1991;37:1618-1625.
1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.
1-antichymotrypsin as an indicator of prostate cancer. J Urol 1993;150:100-105.
[Web of Science][Medline]
[Order article via Infotrieve]
1-protease inhibitor in vitro. Prostate 1997;33:87-96.
[Web of Science][Medline]
[Order article via Infotrieve]
1-anti-chymotrypsin. Prostate 1995;27:166-175.
[Web of Science][Medline]
[Order article via Infotrieve]
1-antichymotrypsin complex for the screening and management of prostate cancer. J Clin Lab Anal 1998;12:32-40.
[Web of Science][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
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  K. Jung, U. Elgeti, M. Lein, B. Brux, P. Sinha, B. Rudolph, S. Hauptmann, D. Schnorr, and S. A. Loening Ratio of Free or Complexed Prostate-specific Antigen (PSA) to Total PSA: Which Ratio Improves Differentiation between Benign Prostatic Hyperplasia and Prostate Cancer? Clin. Chem., January 1, 2000; 46(1): 55 - 62. [Abstract] [Full Text] [PDF]
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B. L. Croal, I. Mitchell, A. Dickie, P. A. Duff, N. P. Cohen, I. S. Ross, K. Jung, B. Brux, P. Sinha, M. Lein, et al. Complexed Prostate-specific Antigen and the ""Prostate-specific Antigen Gap"" • The authors of the Technical Brief cited above reply: Clin. Chem., November 1, 1999; 45(11): 2040 - 2041. [Full Text] [PDF]
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