Acid phosphatase assay

E675 Saini, M., Chambers, D. and Arter, T. (1990). Acid phosphatase assay for Ek-tachem analyzers. Clin. Chem. 36, 1198, Abstr. 1150. 

Colorimetric assays that can be miniaturized into 96-well plates and measured using an ELISA reader allow many samples to be analysed rapidly, and also reduce medium and plastics costs (Cook Mitchell, 1989). The acid phosphatase assay, terminated by NaOH addition (AP NaOH assay), has been found to be particularly useful for both serum batch testing and toxicity assessment. The usefulness and limitations of the following colorimetric assays are described. 

Wlien comparing two procedures, confusion is avoided by using the ROC curves instead of accepting statements such as Test A is more sensitive, but Test B is more specific. For example, the usefulness of the prostatic acid phosphatase assay has been compared for years with that of the PSA assay for diagnostic and foUow-up purposes. Various claims have been made regarding the relative sensitivity and specificity of the two assays. 

Figure 15-3 compares the performance of a thymol-phthalein acid phosphatase assay with that of the PSA assay for discrimination between BPH and prostatic carcinoma stages A through D. Although each has been claimed to be... 

Very recently, a sandwich assay for prostatic acid phosphatase antigen was carried out using two cascaded enzyme reactions to provide amplification of the immunochemical event. In one format, an optical readout was used whereby a forma-zan dye was generated by reaction of a dye precursor and NADH generated from the second enzyme cycle. In the electrochemical format, the NADH generated in the second enzyme cycle was used to reduce Fe(CN) to FeCCN) " which was then detected amperometrically. While the use of Fe(CN) in ECIA has appeared in the... 

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

In the application of alkaline-phosphatase-sensitive, triggerable 1,2-dioxetanes, the nucleic-acid hybridization assay is nowadays quite popular . Such techniques include viral load assays for hepatitis B and C and for human immunodeficiency viruses (HBV,... 

Enzyme-sensitive supramolecular polymers also hold promise in analytical applications such as the screening of enzyme inhibitors. A simple visual assay based on the hydrogelation of small molecules has been developed for screening the activities of inhibitors of enzymes like acid phosphatase. A number of inhibitors for... 

Although casein is a substrate for milk acid phosphatase, the major caseins, in the order as(asl + as2) > (3 > k, also act as competitive inhibitors of the enzyme when assayed on p-nitrophenylphosphate, probably due to binding of the enzyme to the casein phosphate groups (the effectiveness of the caseins as inhibitors is related to their phosphate content). 

Two major difficulties must be considered in any assay for acid phosphatase. The enzyme is subject to surface inactivation (23, 24). Accordingly, reproducible initial hydrolytic rates are not always obtained, and the kinetic behavior should be checked in any new assay developed. Discrepancies between the amount of inorganic phosphate produced and phenol liberated from phenolic phosphates may be substantial if extensive phosphotransferase activity occurs because of phosphoryl acceptor action on the part of hydroxylic buffers or other constituents of the incubation mixture (25, 26). Fluorogenic assays have been developed with very high sensitivity (27). Reference will be made to particular assays in the discussion of the specific enzymes. 

Fig. 3. Surface inactivation rate of prostatic acid phosphatase by shaking and protection by added surface-active agent. Shaking mixtures (20 ml) contained purified enzyme (056 /ug of protein/ml) in 0.05 M acetate buffer at pH 5.5. The solutions were shaken in 50 ml volumetric flasks using a mechanical shaker (Burrell, model CC). Temperatures were maintained by immersion of the flasks in an appropriately set water bath. After specified intervals of shaking, duplicate 0.1 ml ahquots were removed into tubes containing Triton X-100. All tubes were assayed simultaneously, following the shaking procedure, with 0.05 M phenyl phosphate as substrate. Curve 1 Enzyme + Triton X-100 at 0°C and 29°C. Curve 2 Enzyme alone at 0°C. Curve 3 Enzyme alone at 29°C. From Tsuboi and Hudson (88).

Fig. 12. Diagram of elution pattern of red cell acid phosphatase and various markers on Biogel P 60. The position of the various protein markers was determined both by optical density determination and by starch gel electrophoresis of the individual fractions (83). The experiment was carried out using a polyacrylamide gel (Biogel P 60, 50-150 mesh exclusion limit >60,000 Bio-Rad Laboratories, California) in 0.05 M tris buffer, pH 8.0, containing 0.08% (v/v) Tween 80 and 0.1% (v/v) 2-mercaptoethanol to stabilize the enzyme. Column 60 X 4 cm. Flow rate 20 ml/hr, 4 ml fractions. (A) OD at 280 nm, ( ) OD at 540 nm, ( ) LDH assay with p-nitrophenyl phosphate for AcP. From Hopkinson and Harris (85).

MacDonald (99) showed that mouse liver acid phosphatase required active sulfhydryl groups for activity and that malonate buffer, pH 5.9, was useful for the assay of this enzyme because it stabilized the enzyme during the period of the assay. 

Besides his fundamental research in the carbohydrate field, the functions of Courtois as the head of a hospital laboratory for many years led him to publish a number of papers dealing with clinical chemistry, among which may be cited determination of ethyl alcohol, proteins, acidic phosphatases, and trehalase in blood determination of the basic groups of proteins by phytic acid study of the phytosoluble glycoproteins in biological fluids and identification and determination of scyllitol in urine. Under the aegis of the International Pharmaceutical Federation, he participated in the standardization of the methods proposed for the assay of such enzymes as cellulases and hemicellulases. 

Calculation One acid phosphatase unit (HFU) is the amount of enzyme that liberates, under the conditions of the assay, inorganic phosphate from p-nitrophenyl phosphate at the rate of 1 nmol/min. 

The HPLC method has been used to assay a number of activities usually found associated with lysosomal vesicles. All these assays utilize the fluorometric compound 4-methylumbelliferone (4-MU). When carbohydrates, lipids, phosphates, or sulfates are conjugated with 4-MU, these compounds can be used as substrates for glycosidase, lipases, acid phosphatases, and arylsulfatases. The activity is determined by the release of 4-MU. 

Alkaline and acid phosphatase are organ-specific enzymes that are assayed in the diagnosis of many diseases. These activities are phosphomonoesterases that dephosphorylate a number of compounds, including nucleoside monophosphates, to their respective nucleosides and free phosphates. However, such dephosphorylations have traditionally been assayed with 4-nitrophenyl... 

Stor e characteristics of the native enzyme are generally good, with activities maintained over years. Alkaline phosphatase conjugates are usually prepared via amino or carboxylic acid side chains and purified by gel filtration chromatography. Conjugates are very stable, but the enzyme is costly due to the limited supply of calf intestine. Alkaline (and also neutral and acid) phosphatase enzymes in biological samples are a potential problem with the use of this enzyme label. Careful washing of solid phases may be required to ensure no interference in assays. 

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