Isozymes of acid phosphatase in normal and cancerous human prostatic tissue.

The supernatants of the homogenates from normal and cancerous human prostatic tissue run on polyacrylamide gel electrophoresis have 2 major electrophoretic bands when stained for prostatic acid phosphatase. The ratios of the electrophoretically distinguishable isoenzymes differ in normal and cancerous tissues. Similar distinctions between isoenzymes in normal and cancerous prostates are observed following column chromatographic separation or isoelectric focusing. The faster electrophoretic band can be separated by diethylaminoethyl cellulose column chromatography or by isoelectric focusing into at least five fractions with different electrophoretic mobilities. We could not find any differences in normal and cancerous tissues among these subfractions of the faster-moving electrophoretic band. Analysis by gel electrophoresis does not show association between these fractions after chromatographic or isoelectric separation of the prostatic acid phosphatase fractions. Quantitative, but no qualitative, differences in prostatic acid phosphatase isozymes occur in normal versus cancerous prostates.

A solid-phase radioimmunoassay for human prostatic acid phosphatase.

A solid-phase technique for radioimmunoassay of human prostatic acid phosphatase (EC 3.1.3.2) is described. Human prostatic acid phosphatase was purified from prostatic fluid. Monospecific antisera to the purified acid phosphatase were produced in rabbits. Disposable polypropylene tubes were coated with antiserum and used for radioimmunoassay with 125I-acid phosphatase. The nonspecific binding was minimized by saturating the binding sites of the tubes with bovine serum albumin. The working range of the technique was 1 to 30 ng of antigen. The solid-phase radioimmunoassay is rapid, sensitive, and efficient. In preliminary clinical trials it was shown that (a) patients with advanced prostatic cancer had elevated prostatic acid phosphatase levels by both enzymatic assay and radioimmunoassay assays, and (b) patients with other cancers were in the normal range for prostatic acid phosphatase.

A radioimmunoassay for prostatic acid phosphatase.

A solid phase radioimmunoassay for human prostatic acid phosphatase has demonstrated substantially greater biochemical sensitivity than a standard enzymatic method for which p-nitrophenylphosphate was used as substrate. Preliminary data indicate that the radioimmunochemical approach can precisely classify 43% stage I-II and 94% stage III-IV prostate cancers. In contrast, the standard enzymatic methods correctly classified only 9% stage I-II and 46% stage III-IV cancers. It is clinically apparent that a radioimmunochemical approach for the measurement of human prostatic phosphatase may have distinct potential in the clinical diagnosis of prostate cancer.

Measurement of prostatic acid phosphatase in various cell lines.

In the last few years, we have developed a radioimmunoassay for the measurement of human prostatic acid phosphatase. This method, which requires samples of the patient's serum, has been proved to be more accurate than the conventional enzymatic assay for the detection of early stages of carcinoma of the prostate. We used the enzymatic assay and radioimmunoassay for the quantitation of prostatic acid phosphatase in cultured prostatic cell lines. We were unable to show any difference in the concentration of prostatic acid phosphatase between prostatic and any other established cell lines.

Counterimmunoelectrophoresis in determination of prostatic acid phosphatase in human serum.

We evaluated counterimmunoelectrophoresis for use in measuring prostatic acid phosphatase in detection of prostatic cancer. After staining for acid phosphatase, we could detect as little as 0.3 ng of purified enzyme standard complexed with antibody by this technique. However, when serum samples were used as antigen, the method was less sensitive (1.5-2.0 ng) because some of the serum proteins migrate with the phosphatase and decrease the intensity of the stain for acid phosphatase. For this reason we could not detect the phosphatase in serum samples of normal persons; only patients with moderately (or greater) increased activity in their serum showed positive results. In contrast, by radioimmunoassay as little as 1.0 ng of the phosphatase can be detected in serum.

Comparison of human prostatic acid phosphatase by measurement of enzymatic activity and by radioimmunoassay.

We compared results of measurement of prostatic acid phosphatase activity in serum and various tissues by enzymatic assay and radioimmunoassay. By enzymatic assay, activity in serum is lost rapidly, even at room temperature. In contrast, there was no change in antigenic activity during 48 h by radioimmunoassay. The radioimmunoassay was more specific in 12 tissues and in serum than were several enzymatic assays that make use of inhibitors of the enzyme. The enzymatic assay resulted in 26.6% (24/90) false positives from non-prostatic cancer patients. In contrast, with radioimmunoassay there were only 5.5% (5/90) false positives. We conclude that immunological detection of prostatic acid phosphatase is the more reliable technique.

Fasting plasma cyclic AMP levels in an adult diabetic and non-diabetic group.

Fasting plasma cyclic adenosine monophosphate (cAMP) was measured in 50 mature-onset diabetic patients and in 111 non-diabetic patients. Methods used to determine plasma cAMP are described. The addition of sepharose agar beads to the bovine adrenocortical binding protein has considerably improved the sensitivity and simplified the radioligand-receptor assay of cAMP. No statistical differences in plasma cAMP were noted in relation to sex in either group, to the presence of diabetes mellitus or to age or weight in the non-diabetic patients. Plasma cGMP levels are now being studied to determine if these may prove better indicators of insulin activity than plasma cAMP.

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.

Detection of prostatic cancer by solid-phase radioimmunoassay of serum prostatic acid phosphatase.

We compared our radioimmunoassay with the standard enzyme assay for prostatic acid phosphatase in the diagnosis of prostatic cancer. Serum samples from 50 controls, 113 patients with prostatic cancer, 36 with benign prostatic hyperplasia, 83 with other cancers, 20 with gastrointestinal disorders and 28 with total prostatectomies were randomized and studied by radioimmunoassay and enzyme assay. When the upper limit was set at 8.0 ng per milliliter (mean + 4 S.D.) the radioimmunoassay diagnosed prostatic cancer in 33, 79, 71 and 92 per cent of the patients with Stage I, II, III and IV disease. In contrast, the enzyme assay detected elevations of enzyme in the serum of 12, 15, 29, and 60 per cent respectively. No false-positive results were detected by either assay in normal controls but the radioimmunoassay test was positive in two patients with benign prostatic hyperplasia, in one patient after total prostatectomy, in nine with other cancers and in one of the group with gastrointestinal disorders. In contrast to the enzyme assay, the radioimmunoassay distinguished over half the cases of intracapsular prostatic cancer.

Enhancing effect of surfactant and protein on hydrolysis of thymolphthalein monophosphate by purified prostatic acid phosphatase.

Purified prostatic acid phosphatase catalyzes the hydrolysis of thymolphthalein monophosphate 10-fold faster if an optimal concentration of Brij 35 (a wetting agent) or protein (bovine serum albumin or human serum proteins) is present. Results of gel filtration, dialysis, and sucrose density-gradient centrifugation analysis suggest that the substrate must combine with detergent or protein before the enzyme can catalyze its hydrolysis.