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The relation between concentration of thymolphthalein monophosphate substrate and catalytic activity was investigated for the determination of prostatic acid phosphatase. This study, an extension of previously reported work (Clin. Chem. 27: 1372, 1981), shows that lot-to-lot variation in purity of thymolphthalein monophosphate preparations is reflected in substrate-velocity curves. Plateau regions in these curves at 1.5-2.5 g/L result from the combined effects of (a) substrate concentrations that are an order of magnitude below Km and (b) a further decrease in available substrate caused by formation of substrate aggregates in the presence of serum. To simplify the identification of superior lots of thymolphthalein monophosphate, we give a mixed-substrate protocol for testing different lots.

A retrospective review of 102 consecutive patients with surgically staged, clinically localized prostatic carcinoma was performed to determine the relationship between pre-treatment enzymatic acid phosphatase values and histopathological extent of the tumor. Of 96 patients with normal pretreatment acid phosphatase titers (thymolphthalein monophosphate substrate) 77 (80 per cent) had values in the lower and 19 (20 per cent) had values in the upper half of the normal range. Of the latter 19 patients 16 (84 per cent) had histological evidence of extraprostatic tumor extension. Similarly, 5 of 6 patients (83 per cent) with elevated pre-treatment acid phosphatase titers had extraprostatic extension and 1 had a persistent postoperative acid phosphatase elevation that normalized with megestrol acetate therapy. Thus, 22 of 25 patients (88 per cent) with acid phosphatase values in or above the upper half of the normal range had either histological or clinical evidence of extracapsular tumor extension. By contrast, 41 of the 77 patients (53 per cent) with acid phosphatase titers in the lower half of the normal range had extracapsular extension. The predictive value for extraprostatic tumor extension of an acid phosphatase level in the upper half of the normal range was 84 per cent. Furthermore, in the 96 patients with normal acid phosphatase titers the incidence of extraprostatic tumor extension was significantly greater (p less than 0.01, chi-square) in those with values in the upper rather than the lower half of the normal range. Acid phosphatase titers in the upper half of the normal range were proportionately more common among patients with high grade and high clinical stage tumors. However, among patients with low grade and low stage tumors an acid phosphatase value in the upper half of the normal range was an independent variable that correlated with the presence of extracapsular tumor extension. These results confirm previously reported adverse prognostic implications of enzymatic acid phosphatase titers in or above the upper half of the normal range.

We measured serum prostatic acid phosphatase in ostensibly normal controls and a selected patient population, using both a modified radioimmunoassay and an enzymic method with thymolphthalein monophosphate as substrate. The upper limit of normal for the radioimmunoassay was 2.2 micrograms/L; its sensitivity and specificity for prostatic cancer were 71 and 95%, respectively, vs 51 and 99% for the enzymic method. For both methods the correlation between clinical staging and values for acid phosphatase was poor. Our data suggest that adjunctive use of the radioimmunoassay may help further discriminate those patients requiring needle biopsy.

The use of sodium thymolphthalein monophosphate in measuring alkaline phosphatase activity of serum is described. Thymolphthalein liberated in the reaction is conveniently measured by raising the pH of the medium, which produces a color and also stops the hydrolysis. The effects of pH, kind and molarity of buffer, temperature, and enzyme and magnesium con-centrations were studied. Results by this procedure correlate well with those obtained by a standard procedure in which liberated p-nitrophenol is de-termined. Advantages of the present method include excellent linearity, high precision, short incubation, and reagent stability.

Methodological modifications have been made in our "SMA 12/60 AutoAnalyzer" system, to improve the reproducibility and accuracy of six commonly performed procedures. These include glucose with the glucose oxidase method, albumin with the bromcresol green method, and alkaline phosphatase with the sodium thymolphthalein monophosphate method. We have also modified the composition of the reagents used in the determination of protein, inorganic phosphate, and aspartate aminotransferase. A general approach that is useful for adapting a new method to a multichannel AutoAnalyzer system is discussed.

The use of sodium thymolphthalein monophosphate for presumptive seminal acid phosphatase testing is discussed. Recent evidence reveals that the dye (o-dianisidine) used in conjunction with α-naphthyl phosphate for seminal acid phosphatase testing is a carcinogenic hazard. Numerous tests for seminal acid phosphatase were conducted with swabs from sexual assault kits to compare both substrates. Results of tests using sodium thymolphthalein or α-naphthyl phosphate correlated exactly. Sodium thymolphthalein is an effective alternative for preliminary seminal fluid analysis because of its high degree of selectivity and stability, and because it eliminates the potential health hazard.

β-Glycerophosphate and sodium thymolphthalein monophosphate were compared clinically and methodologically as substrates for alkaline phosphatase assay. The relative simplicity of the thymolphthalein procedure, its adaptability to semi-automated and micro methods, and its apparent clinical reliability indicate that it is a better substrate for serum alkaline phosphatase assay than is β-glycerophosphate.

An automated method is described for determining alkaline phosphatase activity in serum, with use of sodium thymolphthalein monophosphate as the substrate. The system makes use of standard AutoAnalyzer components, has a simple flow diagram, and does not involve dialysis. The method has good precision, and the results correlate well with those from the manual method of which it is an adaptation

Summary: A critical evaluation of the determination of acid phosphatase of prostatic origin with the Du Pont ACA is reported. The affinity of the enzyme towards certain substrates, and the specificity of sodium thymolphthalein monophosphate as a substrate for prostatic acid phosphatase were investigated. The validity of this method, which is supposed to be specific for prostatic phosphatase is, however, limited; clinical data showed that related human acid phosphatases interfere and decrease the specificity of the determination.

Use of radioimmunoassay (RIA) for determinations of prostatic acid phosphatase has recently received considerable attention because of reported higher sensitivity and specificity than previous enzymatic assays. We have compared the sensitivity and specificity of a commercially available RIA to a highly specific enzymatic assay (thymolphthalein monophosphate) using 37 patients with prostatic cancer and 34 patients with surgically proved benign prostatic hyperplasia. Seventeen of the cancer patients and all 34 of the BPH patients were studied prospectively. We further evaluated specificity by performing the RIA on 25 specimens of bone marrow from patients with nonprostatic disease. Our results indicate the radioimmunoassay is not, at this time, an adequate screening tool, and we question its accuracy in staging patients anymore reliably than by enzymatic assay.