Acid phosphatase in the lysosomal and microsomal fractions of human prostatic epithelium.

Polyacrylamide gel electrophoresis of acid phosphatase from prostatic tissue reveals one more band than seminal plasma. It was attempted to ascertain which subcellular fraction was responsible for that intracellularly localized enzyme. Prostatic epithelium from patients with prostatic hyperplasia was homogenized, and a lysosomal and microsomal fraction were prepared by differential centrifugation. These two fractions were further centrifuged on an isopycnic Percoll gradient. The intracellularly localized form of acid phosphatase was associated with the lysosomal as well as with the microsomal fraction. In a fused rocket electrophoresis experiment these acid phosphatases cross-reacted with antiserum from seminal plasma. After neuraminidase treatment of the acid phosphatase of lysosomal and microsomal origin, only one activity band was found in polyacrylamide gels. It is concluded that only one acid phosphatase protein exists in prostatic epithelium; differences in electrophoretic mobility are caused mainly by different amounts of sialic acid residues, coupled to the same protein backbone.

Evaluation of an enzyme-linked immunosorbent assay for prostatic acid phosphatase.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure prostatic acid phosphatase in human sera. The value of this method was tested on four groups: patients with non-prostatic disease, with prostatic hypertrophy, and with untreated and treated prostatic carcinoma. The results of ELISA were also compared with those of enzyme immunoassay (EIA) and the conventional, enzymatic method. The specificity of ELISA as calculated from the hypertrophy group, was 76% against 68% for EIA and 71% for the conventional method. The sensitivity of ELISA, calculated from the untreated carcinoma group, was 57% against 60% for EIA and 70% for the conventional method. ELISA did not prove better than EIA or the conventional method in quantifying prostatic acid phosphatase in patients' sera.

Development and clinical evaluation of two immunoassays for prostatic acid phosphatase in serum.

Polystyrene tubes were coated with IgG, isolated from antiserum against human prostatic acid phosphatase, and incubated with a known amount of purified antigen as a standard, and sera from patients. The amount of bound prostatic acid phosphatase was established by using its enzyme activity. This was performed either spectrophotometrically with p-nitrophenylphosphate (enzyme immunoassay) or fluorometrically with alpha-naphthylphosphate as a substrate (immunofluorescence assay). Results of both methods were compared with those of the conventional method and were evaluated in four groups of patients with: non-prostatic disease, hypertrophy, treated and untreated prostatic carcinoma. The first group was used to establish the upper limit of normal. In the hypertrophy group, the specificity of the immunological methods when compared with the conventional method, improved from 79% to 91%. Sensitivity, calculated from the untreated prostatic carcinoma group, was 74% for the enzyme immunoassay (EIA) and 71% for the immunofluorescence assay (IFA). The probability of having either carcinoma or hypertrophy, given observed EIA or IFA values, was calculated by the statistical method of the logistic regression.

The immunohistochemical detection of prostatic acid phosphatase: its possibilities and limitations in tumour histochemistry.

The immunological specificity of the Amsterdam rabbit antiserum against human prostatic acid phosphatase was studied on paraffin sections of 200 prostatic carcinomas and 330 control tissues using an indirect peroxidase technique. Peripheral blood leucocyte smears were also investigated with a fluorescent technique. In a limited number of cases, the mixed aggregation immunocytochemical method was also applied as post-primary incubation procedure. The diaminobenzidine (DAB) final reaction product of the peroxidase technique, carried out under standard conditions, was quantified in some cases using the Leyden Television Analysis System (LEYTAS) with a built-in standard. A positive reaction was obtained in 96.5% of the prostatic carcinomas. Only 2.1% of the non-prostatic tumour cases (23 types) showed a positive reaction, namely six out of 10 insulomas and one out of 10 carcinoid tumours. The beta-cells of the normal islet of Langerhans and the leucocytes in the smears showed a positive reaction. The sensitivity of the peroxidase method, judged subjectively, is not only dependent on the circumstances of fixation, embedding and incubation but also on the degree of tumour differentiation. None of the three prostatic carcinomas studied reached the level of DAB staining intensity shown by the hyperplastic prostatic epithelium.

Enzyme histochemical studies on the conducting system of the human heart.

In this communication, the results of applying various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases in the human heart are presented. The Purkinje fibres of the atrioventricular conducting system of the human heart differ from the myocardium proper in containing a slightly higher activity of most of the glycolytic and gluconeogenetic enzymes investigated. The relatively higher activity of 6-phosphofructokinase, the key enzyme in anaerobic carbohydrate metabolism, is especially noteworthy. On the other hand, the activities of some of the enzymes that play a part in the aerobic energy metabolism is slightly less than those in the myocardium fibres. As for the activity of the NADPH regenerating enzymes, the activity of 6-phosphogluconate dehydrogenase and malate dehydrogenase (oxaloacetate-decarboxylating) is somewhat higher, and the activity of glucose-6-phosphate dehydrogenase similar, in the Purkinje fibres compared to that in the myocardial fibres. The activity of myosin ATPase is similar for both types of fibre. Likewise, the fibres of the conducting system and of the myocardium show a similar activity of acid phosphatase, beta-glucuronidase, non-specific naphthylesterase and peroxidase. The neurogenic function of the conducting system of the human heart was demonstrated by the high activity of acetylcholinesterase in the Purkinje fibres and in the atrioventricular node. All these histochemical findings in Purkinje fibres are similar at widely differing levels of the conducting system.

Prostate-specific acid phosphatase: purification and specific antibody production in rabbits.

Demonstration of prostate-specific acid phosphatase by immunologic methods in tissue sections or in plasma necessitates a monospecific antiserum. This is produced by immunizing rabbits with pure prostate-specific acid phosphatase antigen, prepared from seminal fluid. The ejaculate is centrifuged, dialyzed against a citrate buffer, pH 4.8, and centrifuged again. The supernatant is brought onto a Sephacryl S-200 Superfine column and eluted with the same buffer. Prostate-specific acid phosphatase-positive fractions are concentrated, brought onto and stepwise eluted from a Sulphopropyl Sephadex column with citrate buffers at pH 4.8, 5.2, and 5.5. Fractions containing prostate-specific acid phosphatase are concentrated again and purified over a Blue Sepharose CL-6 B column. After elution, a new concentration step yields a protein concentration of 0.5-1.0 mg . ml-1. This sample in polyacrylamide gel electrophoresis shows a single protein band and is used to immunize female rabbits. The purity of the antiserum is then tested by immunoelectrophoresis and an Ouchterlony technic. Besides anti-prostate-specific acid phosphatase, two other antibodies against serum proteins are present, one of which is anti-albumin. Absorption of these impurities results in a monospecific antiserum against prostate-specific acid phosphatase.

Comparative investigation of the mixed aggregation immunocytochemical technique and the indirect peroxidase technique for the detection of prostate specific acid phosphatase in paraffin or paraplast sections.

The results obtained with the indirect peroxidase technique for the identification of prostate specific acid phosphatase in formalin fixed, paraffin or paraplast embedded autopsy material are compared with the results obtained with the mixed aggregation immuno-cytochemical technique. When using a monospecific antiserum the former technique is prefered. However, when a monospecific antiserum is not available, one has to balance the advantages of the mixed aggregation immuno-cytochemical technique against the disadvantages of having to prepare a monospecific antiserum, necessary for the indirect peroxidase technique. Both methods appeared positive in 20 prostatic carcinomas and in 36 metastases of prostatic carcinomas. In the epithelium of the seminal vesicles and in osteoclasts no acid phosphatase could be detected with the antiserum. A comparison of both techniques, as well as different types of preincubation to diminish nonspecific background staining are discussed.

Demonstration of the prostatic origin of metastases: an immunohistochemical method for formalin-fixed embedded tissue.

An indirect immunohistochemical technique is described for identification of the prostatic origin of metastases in formalin fixed, paraffin or paraplast embedded material. A rabbit antiserum against the prostate specific acid phosphatase isoenzyme was developed. The method is applicable with or without previous decalcification. In 30 cases of prostatic carcinoma there was only one negative result, and in 20 cases of metastases from prostatic carcinoma positive results were obtained in every instance. All carcinomas (primary focus or metastasis) of non prostatic origin (55) stained negatively with the developed antiserum. The application and possible limitations of the method are discussed.

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. VI. D-glucose 6-phosphate isomerase and phosphoglucomutase.

Improved histochemical multi-step techniques for the demonstration of glucose 6-phosphate isomerase and phosphoglucomutase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the glucosephosphate isomerase converts the substrate D-fructo-furanose 6-phosphoric acid to D-gluco-pyranose 6-phosphoric acid, and the phosphoglucomutase converts the substrate alpha-D-glucose 1-phosphate to the same reagent, which in turn is oxidized, by exogenous and endogenous glucose 6-phosphate dehydrogenase to D-glucono-delta-lactone 6-phosphoric acid. Concomittantly the electrons are transferred via NADP+, phenazine methosulphate and menadione to nitro-BT. Sodiumazide and amytal are incorporated to block electron transfer to the cytochromes.

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.