An objective look at acid phosphatase determinations: a comparison of biochemical and immunological methods.

Measurements of serum and bone marrow acid phosphatase were made by 3 enzymatic methods, alpha-naphthyl phosphate, beta-glycerol phosphate, and thymolphthalein monophosphate, and ocmpared to a double antibody radioimmunoassay. Serum and bone marrow acid phosphatase levels were studied in 46 controls with histologically proven benign prostatic hyperplasia and in 135 patients with various stages of prostatic carcinoma. In the control group the upper limit for bone marrow acid phosphatase was found to be significantly higher than the corresponding serum limit with respect to the enzymatic assays studied. The radioimmunoassay was the only method suitable for the analysis of the prostatic acid phosphatase content of bone marrow. A larger number of elevations were noted in patients with extracapsular and metastatic disease when prostatic acid phosphatase measurement was carried out by radioimmunoassay as compared to enzymatic methods. However, only 8% of the patients with intracapsular disease had elevations of prostatic acid phosphatase as measured by radioimmunoassay. Additional standardisation of immunological methods and clinical trials is required before comparison can be made of results from various centres using immunological methods for the measurement of prostatic acid phosphatase and a true assessment made of the usefulness of this procedure.     

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.     

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.     

Immunoreactive prostatic acid phosphatase in prostatic cancer: diagnosis and followup of patients

We compared the measurements of serum acid phosphatase activity to those obtained by radioimmunoassay of prostatic acid phosphatase in the sera of 126 untreated prostatic cancer patients. The catalytic activity of prostatic acid phosphatase was elevated in 32 per cent of the patients and the serum concentration of prostatic acid phosphatase was elevated in 66 per cent. Of these 126 patients 16 had stage T0-2M0N0-x disease, and enzyme activity and prostatic acid phosphatase concentration were increased in 0 and 38 per cent, respectively, in this group. Of the 110 patients with proved extracapsular cancer the corresponding figures were 36 and 70 per cent, respectively. We followed 109 of these 126 patients for 1 or more years after orchiectomy. A salient finding was that return of elevated serum prostatic acid phosphatase concentration to the health-associated reference interval within 7 days following castration indicated no progression of the disease at 1 year irrespective of the initial staging. The same was not detected by the measurement of catalytic activity of serum acid phosphatase. Our findings substantiate data showing that the measurement of circulating prostatic acid phosphatase is achieved better by immunological techniques than by measurements of catalytic activity of the enzyme. A novel aspect is the usefulness of immunological prostatic acid phosphatase measurements in evaluation of the prognosis of patients with metastatic prostatic carcinoma following ablative endocrine treatment.     

Comparison of acid phosphatases in the rat prostatic complex and seminal vesicles

Acid phosphatase activity of different parts (ventral, lateral and posterior lobes and coagulating gland) of the rat prostatic complex and seminal vesicles were analyzed in homogenate and after fractionation with gel filtration and chromatofocusing. Significant differences between the various tissue homogenates were recorded in the hydrolysis of p-nitrophenyl phosphate, alpha-naphthyl phosphate and naphthol ASBI phosphate and the percentage of the tartrate-resistant activity varied from about 30 (in seminal vesicles) to 56 (in coagulating gland). Gel filtration resulted in the appearance of 2 (Sephadex G-200) to 3 (Sepharose 6B) activity peaks (GF-1 to GF-3). Studies with 6 substrates (p-nitrophenyl phosphate, alpha-naphthyl phosphate, beta-naphthyl phosphate, naphthol ASBI phosphate, phenolphthalein phosphate and thymolphthalein phosphate) showed some relative differences in the hydrolysis rates as well as indications for possible overlapping of multiple enzymes in these peaks. Chromatofocusing of the ventral prostate homogenate resulted in the appearance of 8 activities (CF-1 to CF-8) with different pI-values (8.2, 8.1, 7.9, 7.1, 6.4, 6.0, 5.5, and 5.0), substrate preferences, pH-optima (5.5, 4.2, 4.2, 6.0, 5.5, 5.0, 4.2, and 5.0), molecular weights and modifier characteristics. The other tissues contained a lower number of these activities: 4 (CF-1 to CF-3 and CF-6) in lateral and posterior lobes and coagulating gland and 5 (CF-1 to CF-4 and CF-6) in seminal vesicles. The correspondence of the enzyme peaks after gel filtration and chromatofocusing was analyzed by refractionation. This indicated that some of the enzymes (GF-1, CF-5, CF-8) may appear in polymeric/aggregate forms. The differences in the acid phosphatase pattern suggest characteristic functional features in the various parts of the rat prostatic complex.     

Current experience with radioimmunoassay techniques for prostatic acid phosphatase

Data are presented demonstrating that radioimmunoassay techniques for measurement of serum prostatic acid phosphatase are more sensitive than enzymatic methods in the detection of all stages of prostatic cancer. The possibility of using a solid phase RIA technique to screen for prostatic cancer is considered. Sixty-three hundred and twenty men over age 45 entering a clinical laboratory for any indication were evaluated using the RIA test for PAP. In this group 444 (7%) had elevated test values. Clinical recall and urologic review of the patients with elevated test results yielded 67 who were suspect for prostatic cancer, of whom 59 (88%) were confirmed by prostatic needle biopsy. These data suggest that the RIA for prostatic acid phosphatase as an isolated clinical procedure is not sufficiently specific to be used for screening due to the large number of false-positive results. However, the RIA-PAP test in combination with a follow-up urologic examination is quite specific and deserves further consideration as a screening method for prostatic malignancy.     

Correlation of clinical stage, serum prostatic acid phosphatase and preoperative Gleason grade with final pathological stage in 275 patients with clinically localized adenocarcinoma of the prostate

The usefulness of clinical stage, serum prostatic acid phosphatase and preoperative Gleason grade in predicting final pathological stage in patients with adenocarcinoma of the prostate remains controversial. To determine the predictive value of these 3 preoperative variables we reviewed 275 patients with clinically localized disease who were treated between April 1982 and February 1986. All patients were examined preoperatively and subsequently were operated upon by 1 urologist. Serum prostatic acid phosphatase was determined in all patients by the Roy method using thymolphthalein monophosphate as the substrate. The Gleason grade of each prostatic biopsy specimen was determined preoperatively by 1 pathologist, who also examined the final pathological specimen with respect to capsular penetration, and seminal vesicle and pelvic lymph node involvement. Using logistic regression analysis with the likelihood ratio chi-square test, clinical stage and Gleason grade had a direct correlation with capsular penetration (p less than 0.0001 and less than 0.0001, respectively), seminal vesicle involvement (p less than 0.0001 and less than 0.0001, respectively) and positive lymph nodes (p less than 0.0001 and less than 0.0002, respectively). Within the normal range of values (0.0 to 0.8 IU/l.) serum prostatic acid phosphatase correlated directly with capsular penetration (p less than 0.003) and seminal vesicle involvement (p less than 0.01) but not with lymph node involvement (p equals 0.08). Again with logistic regression analysis we determined that the best predictors of final pathological stage are not individual variables but models that use combinations of preoperative variables. The models generated are as follows: capsular penetration--serum prostatic acid phosphatase and Gleason grade (p less than 0.00001), seminal vesicle involvement--clinical stage and Gleason grade (p less than 0.00001), and lymph node involvement--clinical stage and Gleason grade (p less than 0.00001). With these models probability plots have been constructed so that the final pathological stage in patients with clinically localized prostatic cancer can be predicted preoperatively.     

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.     

SINGLE-STEP PURIFICATION OF PROSTATIC ACID PHOSPHATASE: IMMUNOAFFINITY CHROMATOGRAPHY WITH A MONOCLONAL ANTIBODY

Background:
Prostatic acid phosphatase (PAP) is an important protein which should be studied further as a tumor marker or as a biologically functional molecule. The purpose of the study was to establish a simple and reliable method to obtain highly pure PAP.
Methods:
Spleen cells from mice immunized with prostatic epithelial cells prepared from benign prostatic hyperplasia tissue were fused with myeloma cells X63Ag8–653. Hybrid cells of interest were selected using the indirect immunofluorescence method with unfixed frozen tissue sections. One clone of the hybrid cell lines was established which secreted the monoclonal antibody specifically reactive to prostatic acid phosphatase. Using this monoclonal antibody, we purified the antigen from human prostatic tissue by means of single-step immunoaffinity chromatography.
Results:
SDS-PAGE profiling under reducing conditions indicated that the protein recognized by this antibody consisted of several components of molecular weight 41,000–45,000. Partial amino acid sequence analysis of this protein indicated that these components involved a heterogeneously modified single polypeptide, and that this antigen is identical to human prostatic acid phosphatase. Conclusions: This single-step method saves the time needed to purify prostatic acid phosphatase and requires only half a day for the whole procedure. Moreover, the purity of the isolated protein was extremely high. This method seems to be useful not only for purifying prostatic acid phosphatase but also for purifying other proteins from the prostate gland and for analysis of antigenic macromolecules.     

Naphthol-ASBI phosphate as a preferred substrate for tartrate-resistant acid phosphatase isoform 5b.

Tartrate-resistant acid phosphatase (TRAP) isoform 5b is a potential serum marker for osteoclastic activity. Biochemical assays for serum TRAP activity with para-nitrophenylphosphate (pNPP) have low specificity for bone because of hydrolysis by unrelated nontype 5 TRAPs of blood cells and by related isoform 5a. Our purpose was to increase the specificity of TRAP assay for osteoclastic activity by using naphthol-ASBI phosphate (N-ASBI-P) as a substrate for serum type 5 TRAP activity and heparin as an inhibitor of isoform 5a. TRAP activity in individual and pooled sera of normal subjects and patients with endstage renal disease (ESRD) and rheumatologic diseases was quantitated using pNPP and N-ASBI-P as substrate at pH 5.5 and 6.1. For some experiments, heparin (23U/ml) was added as a specific inhibitor of isoform 5a activity. Isoforms 5a and 5b were separated from serum pools by cation exchange chromatography and identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). N-ASBI-P was selectively hydrolyzed by TRAP isoform 5b. TRAP assays with pNPP and N-ASBI-P correlated only in ESRD sera, which contained primarily isoform 5b. The two assays did not correlate in normal or rheumatic sera with significant amounts of 5a. Heparin inhibited isoform 5a activity approximately 50% but had little effect on isoform 5b activity. Biochemical assay of serum TRAP activity can be made specific for isoform 5b by using N-ASBI-P and heparin. This method can be adapted to simple microplate biochemical or immunochemical assays. This simplified method for assessment of osteoclastic TRAP 5b activity warrants a detailed investigation in diseases of bone metabolism.     

Cytochemistry and biochemistry of acid phosphatases VII: Immunohistochemistry of canine prostatic acid phosphatase

Acid phosphatase (E.C. 3.1.3.2.) has been isolated from canine prostatic gland homogenates by gel permeation chromatography (AcA34 or G150), by affinity chromatography (con A-Sepharose), or by using fluid phase liquid chromatography (FPLC) using Superose 12 and Mono P columns. Acid phosphatase-enriched fractions were submitted to analytical SDS-PAGE or to analytical isoelectric focusing. A protein with a molecular weight of 30 kD (on SDS gels) was used for immunization of rabbits. The antiserum produced was cross-reactive with prostatic acid phosphatase (canine and human) as shown by immunoblotting. When applied to paraffin or plastic sections of normal canine prostate, a positive immunoreaction was found exclusively in the secretory cells. In experimentally altered glands (castration and/or hormone treatment), a varying pattern of immunoreactive cells was found. In canine prostatic carcinomas, intensively reacting cell clusters were found along with nonreactive cells. The antiserum was also slightly cross-reactive with the respective human antigen, but the cross-reactivity of an antiserum prepared against human prostatic secretory acid phosphatase with canine prostatic acid phosphatase was far more pronounced.     

Radioimmunochemical measurement of bone marrow prostatic acid phosphatase.

Human acid phosphatases are ubiquitous phosphohydrolases that are present in most respiring tissues and cells. Specifically, human prostatic acid phosphatase is a unique enzyme within a vast family of acid phosphatases concerned with catabolic processes in cellular metabolism. The majority of serum and bone marrow acid phosphatases are of non-prostatic origin and are present chiefly in erythrocytes, leukocytes, platelets and other maturing cells in the bone marrow. The specific concentration of prostatic acid phosphatase in serum and bone marrow is normally relatively low compared to non-prostatic acid phosphatases. Many falsely positive assays for total serum acid phosphatases and bone marrow acid phosphatases have been reported, particularly after traumatic marrow biopsy procedures and mishandling of blood samples in the clinical laboratory and in hematologic disease states. The disruption and lysis of whole blood and marrow cells can liberate non-specific acid phosphatases into the serum. Since standard enzymatic assays do not discriminate accurately prostatic acid phosphatase from non-prostatic acid phosphatase present in the serum spurious results can be realized. A preliminary experience with a promising radioimmunoassay for the specific measurement of prostatic acid phosphatase in bone marrow and serum is presented.     

Radioimmunoassay for prostatic acid phosphatase in early prostatic carcinoma

A radioimmunoassay procedure has been used to measure prostatic acid phosphatase in the serum of 46 patients with intracapsular carcinoma of the prostate. The results obtained did not differ significantly from those obtained in a control group of similar size. It is concluded that the radioimmunoassay procedure for measurement of prostatic acid phosphatase has no advantage over enzyme activity measurements for the detection of early prostatic carcinoma.     

A solid phase radioimmunoassay for prostatic acid phosphatase

The sensitivity of a recently developed solid phase radioimmunoassay for human prostatic acid phosphatase was compared to that of an enzymatic method using p-nitrophenylphosphate as substrate. In 109 histologically verified untreated stages I to IV prostatic cancers and 200 men without such cancer the solid phase radioimmunoassay method demonstrated substantially greater sensitivity and specificity than the enzymatic technique. In the 109 prostatic malignancies the immunochemical method correctly classified 80 (73 per cent) versus 34 (31 per cent) for the p-nitrophenylphosphate enzymatic technique (p less than 10(-6). In 44 stages I and II cancers confined to the prostate the radioimmunoassay was abnormally elevated in 19 (43 per cent) with only 4 (9.1 per cent) enzymatic elevations (p less than 10(-3). In 65 stages III and IV extraprostatic cancers correct classifications were noted in 61 (94 per cent) of the radioimmunoassays and 30 (46 per cent) enzymatic tests (p less than 10(-6). The radioimmunoassay in 200 male controls yielded 11 (5.6 per cent) and the p-nitrophenylphosphate enzymatic test yielded 7 (3.5 per cent) falsely positive results. In 90 non-prostatic human cancer sera 85 (94.5 per cent) were correctly classified as negative by the radioimmunoassay for prostatic acid phosphatase versus 66 (73 per cent) as negative by the enzymatic method. These data are discussed in terms of the merits of a radioimmunochemical approach for the measurement of human serum prostatic acid phosphatase.     

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.     

An enzyme histochemical investigation on bone acid phosphatases: The use of fluoride to distinguish the isoenzymes

The histochemical distribution of acid phosphatase activity in chicken tibial metaphyses was investigated with the azo-dye method, using naphthol AS-BI phosphate as a substrate, and the lead-salt method, usingβ-glycerophosphate, p-nitrophenylphosphate or adenosine triphosphate as substrates. Tartrate-resistant activity was found in cartilage and bone matrices and in osteoclasts when naphthol AS-BI phosphate, p-nitrophenylphosphate or adenosine triphosphate were used. Fluoride-resistant activity was observed in the cytoplasm of osteoclasts with naphthol AS-BI phosphate or p-nitrophenylphosphate; this activity was also insensitive to tartrate. The tartrate-resistant acid adenosine triphosphatase activity, which is due to purple acid phosphatse (type V acid phosphatase isoenzyme), was significantly weaker in the cytoplasm of osteoclasts than the tartrate-resistant acid phosphatase (TRAP) activity with naphthol AS-BI phosphate or p-nitrophenylphosphate as substrates. Furthermore, the purple acid phosphatase activity was strongly inhibited by fluoride. Therefore, the TRAP activity detected with naphthol AS-BI phosphate or p-nitrophenylphosphate may be due to the combined activity of the purple acid phosphatase and another isoenzyme, which is termed fluoride-resistant acid phosphatase (FRAP).     

Prostatic trauma and release of acid phosphatase. Radioimmunochemical and enzymatic comparison

Radioimmunoassay for prostatic acid phosphatase and a conventional enzymatic method using alpha-naphthyl phosphate were employed to document the changes in serum levels of this enzyme following transurethral prostatectomy and prostatic massage. Thirty-four patients with histologically proved benign prostatic hyperplasia and 120 controls were studied. Consistent parallel elevations were noted after surgical trauma. A rapid clearance was observed with normal levels returning at twenty-four hours. Prostatic massage did not elicit a change by either method.     

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.     

针刺对小鼠实验性前列腺增生的防治作用

目的：探讨针刺治疗前列腺增生症的作用机理。方法：采用丙酸睾丸素皮下注射造成小鼠前列腺增生模型，通过针刺白环俞、会阴旁、委阳和三阴交穴，观察针刺对前列腺湿重、组织形态、血清睾丸酮、雌二醇、酸性磷酸酶含量的影响。结果：针刺对丙酸睾丸素引起的小鼠前列腺增生有显著的抑制作用，可使增生的前列腺重量明显减轻，腺上皮细胞增生明显减少，并能降低血清睾丸酮含量，抑制酸性磷酸酶活性，升高雌二醇含量。结论：针刺治疗前列腺增生症的作用机理与降低血清睾丸酮含量，升高雌二醇含量，抑制酸性磷酸酶的活性有关。