Cytochemistry and biochemistry of acid phosphatases. VI: Immunoelectron microscopic studies on human prostatic and leukocytic acid phosphatases

Using different antisera against secretory and lysosomal prostatic acid phosphatases, the localization of the respective antigens was studied in the human prostate at the ultrastructural level. Secretory acid phosphatase was confined exclusively to the secretory vacuoles of the glandular cells. Discharge of the secretory material occurs in a merocrine type of secretion. The identical antigen could be localized in the primary and secondary granules of neutrophil and eosinophil granulocytes separated from human peripheral blood. The antiserum used was also cross-reactive with the canine prostate, where a very distinct immunoreaction was observed with the secretory granules of the glandular cells. The antibodies directed against lysosomal acid phosphatases prepared from prostatic homogenates consistently gave a positive immunoreaction with dense bodies, lipofuscin, and secretory granules. The respective antigens were present also in neutrophil and eosinophil granulocytes. These findings do not identify the existence of a prostate-specific acid phosphatase, which does not exist. The secretory form of the isoenzymes, however, is clearly distinct from the lysosomal form, both of which are present in granulocytes. Therefore the origin of acid phosphatases elevated in peripheral blood in cases of metastatic prostatic cancer could be either the carcinomatous cells or leukocytes destroyed during the process of metastasis.     

Immunohistochemistry of prostatic acid phosphatase

The human prostatic acid phosphatase is a specific marker for the prostatic epithelial cells. By using an immunoperoxidase staining method for this enzyme, it is possible both to identify the prostatic epithelial cells and to recognize the prostatic origin of metastatic lesions of prostate cancer. Of the tissues containing prostatic epithelial cells from 120 patients, positive staining reaction was detected in 114 (95%), and negative in 6. In nonprostatic tissues from 242 patients, weak but positive staining reaction was detected in 8 (3.3%), including tissues from one renal cell carcinoma and 7 breast carcinomas. Of 27 patients in whom tumor tissues were tested at a time when tumor origin was unknown, the staining reaction was positive in 14 patients later found to have prostate cancer. It was negative in 6 patients with nonprostatic carcinoma and 7 patients with carcinoma of unknown primary. Although this immunohistochemical technique for prostatic acid phosphatase appears promising in diagnosing metastatic prostate cancer, its clinical significance and limitations remain unclear, and there are considerable technical problems yet to be solved. These problems are best approached by joint collaborative efforts of the various investigators interested in prostate cancer.     

Phosphotyrosine phosphatase activity of human and canine acid phosphatases of prostatic origin

Human and canine prostatic specimens containing high levels of acid phosphatase (AP) activity were tested, at acid pH, for their ability to hydrolyze the major phosphoaminoacids present in phosphorylated proteins, phosphoserine (p-ser), phosphothreonine (p-thr), and phosphotyrosine (p-tyr). The cleavage of a synthetic substrate, para-nitrophenyl-phosphate (p-npp), was also measured as an indicator of AP activity; its inhibition by sodium-L-tartrate (T) was used as a criterion to identify prostatic acid phosphatase (PAP). It was found that: 1) the Km of p-tyr and p-npp were 2.0 mM and 0.41 mM, respectively, with similar Vmax values (0.078 and 0.087 mumoles of phosphate (Pi) liberated per minute per milligram of protein); 2) the ID50 were 0.25 mM and 0.50 mM with sodium orthovanadate (VO4) and T, respectively, using p-npp as substrate-with p-tyr as substrate, the values obtained were 0.016 mM and 0.11 mM, respectively; 3) activity toward p-ser and p-thr was minimal; 4) native PAP from dog seminal plasma, with a molecular weight of 90-100 kD, as determined by gel filtration on HPLC, hydrolyzed p-tyr preferentially, and this phosphatase (Pase) activity was also strongly inhibited by both T and VO4; and 5) the AP present in human and canine prostatic tissue and cells, as well as in their secretions, also preferentially hydrolyzed phosphotyrosine, and it was inhibited by T and VO4. It is proposed that these p-tyr Pases may be involved in the local regulation of prostatic growth.     

Evidence against a zinc binding peptide in pilocarpine-stimulated canine prostatic secretions

Pilocarpine-stimulated canine prostatic secretions were subjected to gel filtration chromatography over Sephadex G-100 and Bio Gel P-4. Free zinc and the zinc in pilocarpine-stimulated canine prostatic secretions were observed to elute in the same fractions from the Sephadex G-100 column. In addition, Sephadex G-100 chromatography could not resolve free zinc from the small peptide bacitracin. However, a column packed with Bio Gel P-4 could resolve free zinc and bacitracin. When pilocarpine-stimulated canine prostatic secretions were chromatographed over the Bio Gel P-4 column, the major portion of the zinc eluted in the same fractions as did free zinc. No zinc was observed in the fractions where the small peptide bacitracin was found to elute. These results indicate that contrary to a previous report, zinc in pilocarpine-stimulated canine prostatic secretions was not bound to an eight amino acid peptide, but, rather, behaved chromatographically like free zinc.     

A clinical assay for prostatic acid phosphatase using choline phosphate as a substrate: Comparison with thymolphthalein phosphate

We describe an assay method using choline O-phosphate as a substrate for the measurement of serum prostatic acid phosphatase as an aid in the diagnosis of prostatic cancer. Choline phosphate is hydrolyzed by homogeneous prostatic acid phosphatase, and it is also hydrolyzed by an acid phosphatase present in the serum of prostatic carcinoma patients. In contrast, serum samples from apparently healthy persons do not exhibit any significant choline O-phosphate phosphatase activity. There is a correlation of 98% (n = 46) between choline O-phosphate phosphatase activity and typical measurement for prostatic acid phosphatase activity carried out using thymolphthalein monophosphate as the substrate. The new method appears to be as accurate as colorimetric methods based on thymolphthalein phosphate as a substrate. Although not as sensitive as immunologically based methods, the present technique for measuring prostatic acid phosphatase activity using choline phosphate as a substrate is economical and relatively simple.     

Radioimmunoassay of bone marrow prostatic acid phosphatase

The clinical value of prostatic acid phosphatase (PAP) measurements in the bone marrow aspirate of patients with prostatic adenocarinoma has been unclear. Using a radioimmunoassay (RIA) to measure PAP, we have evaluated this potential indicator of occult metastases in 127 controls and in 300 patients with prostatic adenocarinoma. Elevations of the tumor marker were found in 9%, 10%, 19%, and 82% of patients with stages B, C, D₁, and D₂ adenocarcinoma respectively. Clinical follow-up ranging from 7 to 43 months (average 23 months) was available for 97 patients without any initial indication of metastasis by bone scan. In this group 11 patients had elevated levels of bone marrow acid phosphatase (BMAP) by RIA and four developed radiological evidence of bone metastais 21 – 25 months following initial staging. However, only three of the 86 patients with normal BMAP levels have developed bone metastasis. Our results indicate that measurement of bone marrow PAP by immunological methods has prognostic significance. Dilution of the bone marrow aspirate by peripheral blood, however, may limit the application of this technique.     

Isocitric and citric acid in human prostatic and seminal fluid: Implications for prostatic metabolism and secretion

Human prostatic secretion is remarkably rich in citric acid but the mechanisms to account for this accumulation are not well understood. One factor may be the extent of citrate oxidation to isocitrate, catalyzed by aconitase. The citrate-to-isocitrate ratio will help characterize the relative significance of this reaction in prostatic production and secretion of citrate. Isocitric acid and citric acid were measured in samples of seminal fluid and expressed prostatic secretion (EPS). A constant ratio between citrate and isocitrate of about 33:1 was found (r = 0.93, P < 0.0001) despite the wide variation in concentrations. Citrate ranged from 1 to 180 mM in EPS and from 13 to 50 mM in seminal fluid while isocitrate varied between 0 to 4.8 mM in EPS and from 0.4 to 1.5 mM in seminal fluid. Isocitrate is present in EPS and semen at much higher levels than found in most other animal or plant tissues or fluids and may be actively secreted by the same mechanism as citrate. The high citrate to isocitrate ratio of about 33:1, compared to the expected value of about 10:1, supports suggestions that citrate to isocitrate oxidation by aconitase is a rate limiting step in prostatic citrate metabolism. A low aconitase activity will therefore play a significant role in enabling accumulation of high citrate levels in prostatic epithelia and acini. © 1994 Wiley-Liss, Inc.     

Fundamental biochemical and immunological aspects of prostatic acid phosphatase

Prostatic acid phosphatase (PAP) was purified from human malignant prostate tissue by means of ammonium sulfate fractionation followed by sequential chromatographies of ion exchange, affinity column, and gel filtration. PAP has a molecular weight of 100,000 and consists of two subunits of 50,000. Owing, in part, to sialic acid contents in the molecule, PAP has multiple isoelectric points (pIs) at 4.2-5.5. In 0.2 M citrate, PAP has the highest affinity (Km 9.2 × 10^?5 M) in hydrolyzing α-naphthyl phosphate among the phosphomonoesters. Tartrate and heat at 37?C for 2 hours almost completely inhibit PAP enzymic activity. By immunoprecipitate technique, anti-PAP heteroantiserum exhibited a distinct immunologic characteristics. Further, PAP possessed different antibody-binding site from enzyme hydrolytic site.     

Altered distribution of acid phosphatase in neoplastic prostatic cells

Summary Because of the well-known problem of variable cell differentiation encountered in the electron-microscopic evaluation of prostatic cancer, histochemical ultrastructural studies have been performed to assess whether an altered intracellular distribution of acid phosphatase is a more reliable index of malignant change. The results indicate that acid phosphatase activity not restricted to lysosomes is common in malignant cells, and that it may be an intermediate stage in the release of the enzyme into the serum.     

Production of specific antibody to purified prostatic acid phosphatase

Summary Prostatic acid phosphatase may well be a prime antigenic protein in prostatic tissue and fluid. Extraction of the enzyme in highly purified form from prostatic fluid and benign hypertrophic prostatic tissue provides a unique antigen capable of inducing a prompt and specific antibody response in the goat and rabbit as manifested by immunodiffusion, immunoelectrophoresis, and immuno-fluoresence techniques. In prostatic cancer patients with elevated serum acid phosphatase levels it is possible to detect humoral circulating PAP antigen by standard immunoelectrophoretic methods and to confirm the existence of the enzyme by radioautography, L-tartrate inhibition, and the Gomori or Burstone staining procedures. Preliminary indirect prostatic immunofluorescence studies consistently demonstrated characteristic fluorescent foci in the paranuclear areas of benign prostatic epithelial cells, the presumed area of synthesis of prostatic acid phosphatase. Consideration has been given to the possibility of the development of a radioimmunoassay for prostatic acid phosphatase utilizing a heterologous antiserum to the enzyme extracted from human prostatic fluid.     

Solid-phase immunofluorescent and immunoadsorbent assays of serum prostatic acid phosphatase

Human prostatic acid phosphatase was purified to homogeneity from malignant prostatic tissue by Tween 80 extraction and 40-75% ammonium sulfate precipitation, followed by Con A-Sepharose, DEAE-cellulose, and gel filtration chromatography. A specific antiserum was raised by immunizing female goats or rabbits with the purified enzyme. This antiserum did not cross-react with the acid phosphatase of other human tissues. Two immunochemical methods, a solid-phase fluorescent immunoassay and a solid-phase immunoadsorbent assay, were developed. The IgG antibody fraction from antiprostatic acid phosphatase was conjugated to CNBr-activated Sepharose 4B, which was then used in the two immunoassays to separate serum prostatic acid phosphatase from other acid phosphatases or serum proteins. The enzyme activity was subsequently measured by incubating the solid-phase bound prostatic acid phosphatase with α-naphthyl phosphate and quantitating the fluorescent product with a spectrophotofluorometer (immunofluorometric assay) or quantitating the α-naphthol-FRBS colored complex with a spectrophotometer (immunoadsorbent assay). The sensitivity of this immunofluorometric assay was 60 pg/ml, more sensitive than other immunoassays. The results obtained from clinical evaluation indicate that serum prostatic acid phosphatase in prostate cancer can be detected in significant percentage with early stages of prostatic cancer. The sensitivity of the immunoadsorbent assay was 0.22 IU/l of enzyme activity or 0.88 ng of prostatic acid phosphatase protein per ml serum. Initial clinical evaluation demonstrated that 19 of 25 patients with early stages of prostate cancer and 12 of 14 patients with metastatic prostate cancer exhibited an elevated serum PAP level (over all 79%), as compared with only six and eight patients respectively (overall 35%), by a conventional chemical method.     

Comparison of prostate secretory protein with prostate specific antigen and prostatic acid phosphatase as a serum biomarker for diagnosis and monitoring patients with prostate carcinoma

Serum prostate secretory protein (PSP) levels were measured in 49 patients with benign prostatic hyperplasia (BPH), 144 patients with various stages of prostatic carcinoma (CaP), and 82 CaP patients who were followed serially. PSP values were compared with serum levels of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). In the BPH group, PSP was elevated (> 10 ng/ml) in 41% of patients, whereas PSA (> 4 ng/ml) and PAP (> 3.3 ng/ml) were elevated in 39% and 23% of the cases, respectively. PSP levels were elevated in 48% of the CaP pretreatment specimens, compared to 79% for PSA and 40% for PAP. PSP levels in cancer patients who had intracapsular disease were about two to three times higher than those observed for PAP. PSP was found to be the only marker elevated in eight (6%) pretreatment CaP patient serum specimens, while PAP was never found to be elevated when PSA was normal. PSP serum concentrations correlated with the clinical course of the disease in 79% of patients, compared with 90% for PSA and 66% for PAP. In certain patients, monitored over time, disease correlation was reflected in serum values with only a single biomarker, i.e., 1% with PAP, 8% with PSP, and 10% with PSA. This study has shown that PSP is a less sensitive serum biomarker than PSA, but more sensitive than PAP for detection and monitoring the early stages of prostate cancer. This suggests that PSP as a biomarker may be a useful adjunct for the management of a subpopulation of low-stage and -grade CaP. © 1993 Wilcy-Liss, Inc.     

Two-dimensional characterization of prostatic acid phosphatase, prostatic specific antigen and prostate binding protein in expressed prostatic fluid

Specimens of pooled prostatic fluid, collected by rectal massage from men under 50 years of age with no apparent prostatic disorders, were subjected to two-dimensional gel electrophoresis to study the composition of its proteins. In a preliminary study, a total of 57 major protein groups were detected. In the present study, we attempted to identify, in the two-dimensional gels, those that are related to prostate-associated proteins, i.e., prostatic acid phosphatase (PAP), prostatic specific antigen (PSA), and prostate binding protein (PBP). Individual proteins were recognized by the procedure of Western Blot using specific antisera with peroxidase-antiperoxidase as the staining reagent. Each protein spot in the two-dimensional gel was expressed, along the abscissa, by its isoelectric point (pI) and, along the ordinate, by the molecular weight (MW). PAP consisted of a train of more than ten protein spots that occupied an area in the gel from pI 7.0, MW 45,000 to pI 6.0, MW 50,000. Four protein spots with a MW of 34,000 and a pI range of 8.2-8.8 were identified as PSA. PBP was observed as having three protein spots that were located at pI 5.6-6.6 with a single MW of 15,000. For PAP and PSA, additional protein spots with lower MWs also stained positively with the specific antisera, suggestive of the presence of degradative products of these proteins. Following the removal of the serum-related proteins by an extensive absorption with anti-human serum antibody by affinity chromatography, the prostatic fluid contained 27 major groups of non-serum proteins. These non-serum proteins in the prostatic fluid included PAP, PSA, PBP, and their related smaller molecular species. These results indicate that the prostatic fluid contains PAP, PSA, PBP and that their presence and the patterns of their distribution in the two-dimensional gels should be considered as the characteristic property of the prostatic secretions.     

Cytochemistry and biochemistry of acid phosphatases V: Electrophoretic studies on the heterogeneity of acid phosphatases from human prostate, seminal fluid, and leukocytes

Comparison of zymograms of acid phosphatases (orthophosphoric monoester phosphohydrolase, acid optimum, E.C.3.1.3.2) from human prostate, leukocytes, seminal vesicles, and seminal fluid, separated by analytical isoelectric focusing (IEF), resulted in the identification of three individual groups, particularly of the prostate. These groups contain three molecular forms at different molecular weights and activities in varying quantities and combinations. The molecular weights, estimated by SDS gel electrophoresis, were 1) 86,000, 2) 76,000, and 3) 46,000/50,000 (in a doublet) daltons. None of these isoenzymes were restricted to the prostate, but they were present in very high concentrations in the prostate. Compared to the prostate, the seminal vesicles and isolated leukocytes had very closely related zymograms of acid phosphatases. No specific inhibitor has been found that would selectively inhibit one particular isoenzyme without affecting the others. Protein titration and staining activity of IEF gels at different pH values showed that the isoenzymes from all three groups have high hydrolytic activity of orthophosphoric monoesters beyond the acidic pH range of pH 4-5. Incubation of acidic isoforms of acid phosphatases with neuraminidase did not result in the formation of a homogeneous stem molecule. Further analysis of the secretory moiety using the western blotting method showed a binding of peroxidase-conjugated concanavalin A (Con A), indicating that these isoenzymes are glycoproteins. Moreover, isoenzymes extracted from prostate, seminal fluid, and human leukocytes are immunologically identical.     

Latent and clinically manifest prostatic carcinoma

In a histological study of 17 prostates excised in toto for stages B and C primary carcinoma, we found one focus of well differentiated carcinoma in each of six prostates, which, in addition to these foci, had large tumors of invasive prostatic carcinoma. In four of the six prostates, the foci were located near but separate from the main tumor, and in the remaining two they were invaded by the main tumor. These findings strongly suggest that prostatic carcinoma is multifocal in origin and that focal well differentiated carcinomas are different in biological behavior from invasive poorly differentiated carcinomas.     

Counterimmunoelectrophoretic studies of serum prostatic acid phosphatase

A counterimmunoelectrophoretic (CIEP) assay for the specific determination of prostatic acid phosphatase (PAP) is described. PAP was obtained from benign human prostatic tissue and a specific antiserum to this enzyme was produced in rabbits and goats. The lowest detectable activity of PAP was at 0.3 IU/l or 4 ng./0.1 ml. This CIEP method was compared to a standard biochemical method (Roy) on a wide spectrum of prostatic and nonprostatic disease. Nonprostatic malignancies and other disorders associated with hyperacidphosphatasemia by the biochemical method were found to be nonreactive for PAP by CIEP. Patients under treatment with various stages of prostatic carcinoma showed comparable elevations by both methods (35%). In untreated patients, the CIEP was statistically most sensitive in stage A (39% by CIEP and 14% by chemical).     

Immunohistochemical prostatic acid phosphatase level as a prognostic factor of prostatic carcinoma

To determine whether prostatic acid phosphatase (PAP) immunoreactivity in prostatic adenocarcinoma is a reliable prognostic factor, the PAP immunohistochemical distribution has been examined in 78 prostatic carcinoma cases. The intensity of PAP immunostaining was graded from 0 to 2, and the scores of the primary and the secondary staining patterns were added to assess the extent of the PAP expression in needle biopsy specimens. As a result, a higher cancer-specific survival rate was observed in patients showing a greater PAP immunostaining (P less than 0.01). Further, a multivariate analysis was made of possible prognostic factors (age, stage, Gleason score, serum PAP, PAP-immunostaining score, and the initial treatment) to estimate the extent of their impact on cancer-specific survival. Results have confirmed that the difference in PAP immunoreactivity is an excellent, independent prognostic factor for prostatic carcinoma.     

Secretion of bromide by the canine prostate

Four dogs with surgically produced prostatic fistulas were given single oral doses of 1.13 gm of sodium bromide daily for five consecutive days. Two days later the mean (+/- SE) serum level of bromide was 28.0 +/- 4.0 meq/L and the serum chloride level had decreased from a pretreatment value of 112.5 +/- 1.0 meq/L to 86.5 +/- 3.7 meq/L; in the basal prostatic secretion, the mean prostatic fluid to serum (PF/S) ratios for bromide and chloride were 0.56 +/- 0.15 and 0.53 +/- 0.11, respectively, and were not different (P greater than 0.05, paired t-test); at higher rates of secretion provoked by intravenous pilocarpine the corresponding PF/S ratios of 1.48 +/- 0.04 and 1.32 +/- 0.01 were significantly different (P less than 0.05, paired t-test). It is concluded that the processes involved in forming the basal and pilocarpine-induced prostatic secretions must differ and that the ability of the chloride-transporting system to transport bromide is slightly greater than that for chloride. Because it may impair sperm motility, bromide secreted in prostatic fluid potentially could adversely affect reproduction.     

Antibiotic therapy--rationale and evidence for optimal drug concentrations in prostatic and seminal fluid and in prostatic tissue

The theoretical background of drug penetration into the prostate is outlined, emphasizing the phenomenon of ion-trapping and the role of nonionic diffusion of weak acids, bases and amphoteric drugs across biological membranes with a pH gradient. Determination of drug concentrations in human prostatic secretion are problematic because of possible urinary contamination. Studies have been carried out mainly in healthy volunteers. The results have to be interpreted with caution, if not care was taken to rule out or at least identify urinary contamination. Analysing the concentrations of various fluoroquinolones in prostatic and seminal fluid as well as in prostatic tissue, it becomes obvious that the fluoroquinolones differ not only in plasma concentrations but also in their penetration ability to these sites. In spite of intensive investigations, our knowledge is still limited concerning the mechanisms that govern the transport of antibiotic drugs into and their activity in the various prostatic compartments and how the findings can be applied clinically. Nevertheless, overall the concentrations at the site of infection of most of the fluoroquinolones should be sufficient for the treatment of chronic bacterial prostatitis and vesiculitis caused by susceptible pathogens.