Yeast adenylic acid as a substrate for serum acid phosphatase determination
Reynolds, Orland Bruce
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In metastatic carcinoma of the prostate, high levels of acid phosphatase are found in the serum (19). The measurement of acid phosphatase in serum is a valuable aid in diagnosis of this disease and in following the response to treatment. The prostate produces large amounts of acid phosphatase, and cancerous prostate as well as distant metastases show this ability. When the prostate is normal, no significant amount of the enzyme is present in the serum. It is found in semen in high concentration, and spasmodically in urine. Large amounts of the enzyme usually leak into the blood, however, from carcinoma of the prostate and from metastases of this origin (55). About 20% of patients with prostatic carcinoma do not have abnormal levels even though the disease is far advanced (54). Since the acid phosphatase of serum is not all of prostatic origin, it is desirable to be able to distinguish the "prostatic" enzyme in dealing with prostatic disease. Serum contains variable amounts of non-prostatic acid phosphatases. This fraction consists of red cell acid phosphatase and others less well characterized. Acid phosphatases of different origin show different substrate specificities and respond differently to certain activators or inhibitors. A number of methods have been used to measure the fraction of the serum acid phosphatase which is of prostatic origin. The procedures adopted for this purpose are: 1. The substrate β-glycerophosphate is naturally selective toward prostatic acid phosphatase (54). 2. Inhibition by L-tartrate (14) using phenyl phosphate substrate. 3. Inhibition by alcohol and selective inactivation by heat (22). 4. Formaldehyde inactivation of the non-prostatic portion (33). 5. Comparison of hydrolysis rates between morpholine ethanol phosphate and β-glycerophosphate substrates (11). The phosphate group of yeast adenylic acid (3-adenylic) is auite labile to phosphatase action. It has been used as a substrate in a number of phosphatase studies. Fischman et al. (13) employed 3-adenylic acid for the assay of prostatic acid phosphatase in extracts of normal and abnormal prostatic tissue. They state: "This substrate, which is now commercially available, is particularly suitable for the determinations because of the rapid rate at which it is hydrolyzed by the enzyme. It is split by prostatic phosphatase about three times as rapidly as β-glycerophosphate. This difference might possibly be of value in blood analysis in order to distinguish prostatic phosphatase from acid phosphatases of other origin." In a recent review Rosenmund (46) notes that the use of 3-adenylic acid to distinguish prostatic phosphatase from other acid phosphatases in blood as suggested by Fischman et al. has not yet been confirmed. Three-Adenylic acid was employed by Bernhard and Rosenbloom (5) as a substrate for alkaline phosphatase in serum. They compared this substrate with adenosinetriphosphate and with β-glycerophosphate. They stated: "Yeast adenylic acid substrate gave the most consistent results, it is a stable compound, not subject to spontaneous hydrolysis in acid solutions." This investigation was undertaken to devise a practical procedure for acid phosphatase assay in serum using adenylic acid as substrate and to determine its suitability for routine clinical use in distinguishing the prostatic portion of the acid phosphatase of serum. Experimental A method for the assay of acid phosphatase in serum using 3-adenylic acid as substrate was developed. In most of the developmental procedures this substrate was compared with β-glycerophosphate. The procedure which was developed involves incubating serum with 3-adenylic acid at a pH of 5.75 in an acetate buffer. The concentration of 3-adenylic acid selected as giving near maximal hydrolysis was 200 mg. per 100 ml. of the buffered substrate mixture. Higher substrate concentrations interfered with color development. The incubation was allowed to proceed for one hour at 38°C; the hydrolysis was then arrested with trichloroacetic acid. The precipitated protein was separated by centrifugation. The inorganic phosphate hydrolyzed from the substrate was then determined colorimetrically by the procedure of Fiske and Subbarow (15). Slight modifications in the procedure were made to adapt it to the specific conditions of this test. The procedure of Fiske and Subbarow for inorganic P exhibits sensitivity to certain substances which may be present in the mixtures analyzed. Hence a study of the various components of these mixtures was made. When higher concentrations of either β-glycerophosphate or 3-adenylic acid were used in the buffered substrates, color development was strongly inhibited. A concentration of 3-adenylic acid of 250 mg. per 100 ml. allowed color development, but at 300 mg. per 100 ml. the development was slow. The rate of hydrolysis was determined for 3-adenylic acid at various pH's. Three-adenylic acid was made up in 0.2 M acetate buffers and the pH adjusted With acetic acid. A fairly broad and symmetrical maximum was obtained at about pH 5.75. This pH value was incorporated into the procedure. The effect of the concentration of these substrates on hydrolysis by prostatic acid phosphatase was investigated. Graphing the data by the method of Lineweaver and Burke (40) allows an estimate of the maximum rate to be made. The hydrolysis rate increases more sharply with β-glycerophosphate than with 3-adenylic as substrate concentrations are increased, so that 3-adenylic acid is only very little more sensitive to prostatic phosphatase at the substrate concentrations selected. The concentration of 3-adenylic which was selected (200 mg. per 100 ml.) gave about 89% of the maximum hydrolysis rate. Increasing the substrate concentration beyond this would not result in a great gain in sensitivity of the test and would interfere with subsequent color development. Three-adenylic acid seems to be well adapted for serum acid phosphatase assay. However, its adoption for this purpose would not seem warranted unless it were shown to possess distinct advantages over substrates in common usage. Its principle value as a substrate would be its selectivity for prostatic acid phosphatase relative to phosphatases of other origin found in serum. This selectivity makes it potentially useful in the detection of carcinoma of the prostate. This substrate was compared with β-glycerophosphate and with phenyl phosphate from the standpoint of its selectivity for prostatic phosphatase and for erythrocytic phosphatase. It was shown to be very similar to β-glycerophosphate in this respect - sensitive to prostatic phosphatase and insensitive to erythrocytic phosphatase. In addition, at the substrate concentrations selected 3-adenylic acid was hydrolyzed only about 6% more rapidly than β-glycerophosphate by prostatic phosphatase. Hence its advantage in lability is rather small. Any advantage in the use of 3-adenylic acid as a substrate to replace β-glycerophosphate in routine clinical determination of serum acid phosphatase would not seem to justify the trauma of "re-tooling the industry". Its selectivity for prostatic acid phosphatase is not greater, and it would be somewhat more expensive. This certainly does not imply that this substrate is not of value in investigational work with phosphatases.
Thesis (M.A.)--Boston University