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Prostate-specific antigen in ascitic fluid.

Although previously thought to be produced almost exclusively by the epithelial cells of the prostate [1], prostate-specific antigen (PSA) is produced and secreted by several extraprostatic sources [2, 3]. Considering the high degree of homology of PSA with the human glandular kallikrein (hKGK1) [4, 5] and the activation of the kallikrein-kinin system in peritoneal effusions [6, 7], we undertook the study of PSA distribution and expression in ascitic fluids. Between May 1996 and January 1997 we collected ascitic fluids from 44 consecutive women of ages 29-61 years (mean 49 [+ or -] 7) undergoing ultrasound examination and a diagnostic paracentesis. After collection (~10 mL), ascitic fluids were centrifuged at 20 120g for 20 min at +4 [degrees]C and the supernatants stored at -30 [degrees]C until processed. Blood samples were also taken, and after clotting were centrifuged at 360g for 5 min at +4 [degrees]C and stored at -30 [degrees]C until assay. In 24 patients (ages 28-81 years), the ascites was associated with malignancies (ovarian, pancreatic, breast, gastrointestinal, and lung). In the other 20 patients (18-75 years), ascites arose from chronic liver diseases, bacterial peritonitis, congestive heart failure, thrombosis, and other nonmalignant diseases. Albumin, serum-ascites albumin concentration gradients, total protein, lactate dehydrogenase, and cholesterol concentrations were also measured in the fluids (data not shown) [8]. PSA was measured by two methods [9-11]: a solid-phase two-site IRMA (PSA-RIACT[TM]) from CIS Bio International (Gif-sur-Yvette, France) and a microparticle capture enzyme immunoassay (MEIA) (IMx[R]) from Abbott Labs, Abbott Park, IL. Patients with malignant ascites had not yet received cytotoxic drugs and (or) chemotherapeutic agents before sample collection. Results are expressed as means [+ or -] SE. Statistical analyses were performed through the StatView v.4.1 package (Abacus Concepts, Berkeley, CA) on Macintosh Power PC (Apple Computer, Cupertino, CA).

The work was carried out in accordance with the Helsinki Declaration of 1975, as revised in 1983.

Among the 44 patients examined, 41% of ascitic fluids contained detectable amounts of PSA, tested with both assay methods (mean [+ or -] SE 0.278 [+ or -] 0.045 [micro]g/L, range 0.06-0.78 [micro]g/L, n = 18). Matrix effects of ascitic fluid constituents in the PSA assays were excluded by performing dilutions of samples having high PSA content. A good linearity (r = 0.973) between PSA content and dilution was obtained with the IMx method. The PSA-RIACT (y) and IMx-PSA (x) agreed (n = 15, y = 0.019 + 0.863x, means [+ or -] SE of y = 0.251 [+ or -] 0.068 and of x = 0.268 [+ or -] 0.077, [r.sup.2] = 0.958, P <0.0001). In agreement with others [12, 13], we found a plasma PSA content [less than or equal to]0.05 [micro]g/L in ~91% (40 of 44) of the women examined. The PSA mean concentration in our series of ascitic fluid samples did not show a significant difference between malignant-related and nonmalignant ascitic fluids (0.262 [+ or -] 0.074 [micro]g/L, 0.297 [+ or -]0.047 [micro]g/L, respectively; t = -0.317271, P = 0.761). The total PSA content in ascitic fluids was significantly greater than in plasma (0.278 [+ or -] 0.045 [micro]g/L and 0.032 [+ or -] 0.011 [micro]g/L, respectively; n = 18, P <0.0001). PSA was not statistically significantly correlated with patient's age.

A major 33-kDa immunoreactive band (due to the free form of this serine protease) was seen on Western blots with an anti-human PSA monoclonal antibody (Dako, Milan, Italy). The 100-kDa immunoreactive protein due to the [[alpha].sub.1]-antichymotrypsin-bound form was not detected, nor were other bands (Fig. 1).

Several sources could be suggested for PSA expression in ascitic fluids: (a) plasma ultrafiltration and accumulation, at an increased rate in the peritoneal space through a vascular hyperpermeability of the inflamed peritoneal tissue. Previous data have shown the liberation, accumulation, and activation of the kallikrein-kininogens-kinin system [6, 7] and the role of a vascular permeability factor in the pathogenesis of ascitic fluid accumulation [14]. (b) Local secretion mainly due to the enhanced protease synthesis by the neoplastic ascitic cells. Several reports have documented the activity of proteolytic enzymes in peritoneal fluid [6, 15, 16] as well as in an experimental animal model [17]. (c) Enhanced PSA expression modulated throughout the steroid receptors. Previous studies have revealed the presence of steroid hormones and their receptors in peritoneal fluid and mesothelium [18, 19].

[FIGURE 1 OMITTED]

The present report is the first evidence of PSA in ascitic fluids at measurable concentrations with commercial methods, even though it does not contribute to the discrimination of malignant-related and nonmalignant ascites. The detectable amounts of PSA in peritoneal effusions give further evidence of the distinctiveness of this widespread serine protease, even though the biological effects and the mechanism causing its increase still remain unexplained. Several hypotheses have been previously suggested for the new functions of PSA in nonprostatic sources [2, 3, 20]: The presence in ascitic fluids of several mitogens and growth factors could be related to the enhanced expression of PSA in peritoneal effusions [21-24].

We are currently investigating the potential role of PSA in nonprostatic tissues and in other biological fluids as a possible sensitive molecular marker implicated in hormone responsiveness and (or) in the inflammatory/neoplastic processes, which could, in part, be responsible for ascitic fluid proteolytic activities [15, 16].

References

[1.] Oesterling JE. Prostate-specific antigen: a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate [Review]. J Urol 1991;145:907-23.

[2.] Diamandis EP, Yu H. New biological functions of prostate-specific antigen? [Editorial]. J Clin Endocrinol Metab 1995;80:1515-7.

[3.] Diamandis EP. New diagnostic applications and physiological functions of prostate specific antigen [Review]. Scand J Clin Lab Invest 1995;55 (Suppl 221):105-12.

[4.] Schedlich LJ, Bennetts BH, Morris BJ. Primary structure of a human glandular kallikrein gene. DNA 1987;6:429-37.

[5.] Riegman PHJ, Klaassen P, Van der Korput JAGM, Romijin JC, Trapman J. Molecular cloning and characterization of novel prostate antigen cDNA's. Biochem Biophys Res Commun 1988;155:181-8.

[6.] Waldner H, Vollmar B, Conzen P, Gotz A, Lehnert P, Fink E, et al. Enzyme liberation and activation of the kallikrein-kinin system in experimental pancreatitis. Studies of portal vein blood, pancreatic lymph and peritoneal effusion. Langenbecks Arch Chir 1993;378:154-9.

[7.] Karlsrud TS, Buo L, Aasen AO, Johansen HT. Characterization of kininogens in human malignant ascites. Thromb Res 1991;63:641-50.

[8.] Castaldo G, Oriani G, Cimino L, Topa M, Mostarda I, Castellano L, et al. Total discrimination of peritoneal malignant ascites from cirrhosis- and hepatocarcinoma-associated ascites by assays of ascitic cholesterol and lactate dehydrogenase. Clin Chem 1994;40:478-83.

[9.] Mannello F, Bocchiotti GD, Bianchi G, Marcheggiani F, Gazzanelli G. Quantification of prostate-specific antigen immunoreactivity in breast cyst fluids. Breast Cancer Res Treat 1996;38:247-52.

[10.] Mannello F, Bianchi G, Gazzanelli G. Immunoreactivity of prostate-specific antigen in plasma and saliva of healthy women [Tech Brief]. Clin Chem 1996;42:1110-1.

[11.] Mannello F, Miragoli G, Bianchi G, Gazzanelli G. Immunoreactive prostate-specific antigen in pleural effusions [Tech Brief]. Clin Chem 1997;43: 847-8.

[12.] Armbruster DA. Prostate-specific antigen: biochemistry, analytical methods, and clinical application [Review]. Clin Chem 1993;39:181-95.

[13.] Diamandis EP, Yu H, Melegos DN. Ultrasensitive prostate-specific antigen assays and their clinical application [Opinion]. Clin Chem 1996;42: 853-7.

[14.] Nagy JA, Masse EM, Herzberg KT, Meyers MS, Yeo KT, Yeo TK, et al. Pathogenesis of ascites tumor growth: vascular permeability factor, vascular hyperpermeability and ascites fluid accumulation. Cancer Res 1995;55: 360-8.

[15.] Buo L, Karlsrud TS, Dyrhaug G, Bell H, Engstrom L, Johansen HT, Aasen AO. The fibrinolytic system in human ascites. Scand J Gastroenterol 1995;30: 1101-7.

[16.] Abe H, Campeas JD, Rodgers KE, Ellefson DD, Girgis W, diZerega GS. Peritoneal lavage fluid protease levels after in vivo administration of tolmetin in hyaluronic acid. J Surg Res 1993;55:451-6.

[17.] Isidoro C, Demoz M, De Stefanis D, Baccino FM, Bonelli G. High levels of proteolytic enzymes in the ascitic fluid and plasma of rats bearing the Yoshida AH-130 hepatoma. Invasion Metastasis 1995;15:116-24.

[18.] Prentice A, Randall BJ, Weddell A, McGill A, Henry L, Horne CH, Thomas EJ. Ovarian steroid receptor expression in endometriosis and in two potential parent epithelia: endometrium and peritoneal mesothelium. Hum Reprod 1992;7:1318-25.

[19.] Kim-Bjorklund T, Landgren BM, Hamberger L. Peritoneal fluid volume and levels of steroid hormones and gonadotrophins in peritoneal fluid of normal and norethisterone-treated women. Hum Reprod 1991;6:1233-7.

[20.] Graves HCB. Nonprostatic sources of prostate-specific antigen: a steroid-dependent phenomenon? [Editorial]. Clin Chem 1995;41:7-9.

[21.] Koutsilieris M, Allaire-Michaud L, Fortier M, Lemay A. Mitogen(s) for endometrial-like cells can be detected in human peritoneal fluid. Fertil Steril 1991;56:888-93.

[22.] Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR. Transforming growth factor-beta activity is increased in peritoneal fluid from women with endometriosis. Obstet Gynecol 1994;83:287-92.

[23.] Munker R, Darsow M, Stotzer O, Kremer JP, Mezger J. Expression of trasforming growth factor beta-3 (TGF[[beta].sub.3]) on reactive and malignant cells in ascites and pleural effusion. Med Klin 1994;89:248-51.

[24.] Giudice LC, Dsupin BA, Gargosky SE, Rosenfeld RG, Irwin JC. The insulin-like growth factor system in human peritoneal fluid: its effects on endometrial stromal cells and its potential relevance to endometriosis. J Clin Endocrinol Metab 1994;79:1284-93.

Ferdinando Mannello, (1) * Giovanni Miragoli, (2) Giuseppe Bianchi, (3) and Giancarlo Gazzanelli (1)

((1) Ist. Istol. & Anal. Lab., Facolta Sci. MFN Universita, Via E. Zeppi, 61029 Urbino, Italia; (2) Div. Med. and (3) Lab. Anal. Ospedale Civile, Urbino, Italia; *author for correspondence: fax 139-722-322370, e-mail mannello@bio.uniurb.it)
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Title Annotation:Technical Briefs
Author:Mannello, Ferdinando; Miragoli, Giovanni; Bianchi, Giuseppe; Gazzanelli, Giancarlo
Publication:Clinical Chemistry
Date:Aug 1, 1997
Words:1610
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