Acid phosphatase test

Two methods commonly used to test for seminal stains are the acid phosphatase test and the Florence test. Both tests were developed on the basis of the reaction of an introduced compound with substances that are present in seminal fluid. Positive results for these tests are either the formation of a characteristic color or the formation of specific crystals. Since the substances tested are also present in other body fluids and in vegetable juices, the specificity of these tests has been questioned (lh). 

Specimen tubes should be centrifuged with stoppers in place. Closure reduces evaporation, which occurs rapidly in a warm centrifuge with the air currents set up by centrifugation. Stoppers also prevent aerosolization of infectious particles. Specimen tubes containing volatiles, such as ethanol, must be stoppered while they are spun. Centrifuging specimens with the stopper in place maintains anaerobic conditions, which are important in the measurement of carbon dioxide and ionized calcium. Removal of the stopper before centrifugation allows loss of carbon dioxide and an increase in blood pH. Control of pH is especially important for the enzymatic measurement of acid phosphatase, which is labile under alkaline conditions engendered by CO2 loss. Indeed, once the serum is separated for acid phosphatase tests, a tablet of disodium citrate should be added to stabilize the pH at about 6.2. 

Sample Collection and Enzyme Stability. Serum samples are collected with chemically clean, sterile glassware. Blood is allowed to clot at room temperature, the clot is gently separated from the test tube with an applicator stick, and the blood is centrifuged for 10 minutes at 1,000 g. If the red cells are known to contain the enzymes whose activity is being measured, as in the case of LD, even slightly hemolyzed serums must be discarded. When acid phosphatase is to be measured, the serum should be placed immediately in ice and processed as soon as possible, or it should be acidified by the addition of a small amount of sodium citrate. Anticoagulants such as EDTA, fluoride and oxalate inhibit some serum enzymes. However, heparin activates serum lipoprotein lipase. 

Red blood cells also contain sufficient acid phenylphospha-tase for mild hemolysis to cause false elevations. Therefore, inhibitors such as ethanol, formaldehyde, copper sulfate> and 1-tartrate have been used to inhibit selectively the enzyme of one or more tissues and enhance the specificity of the test (101). Ethanol is unsuitable because it inhibits the enzyme from erythrocytes and prostate simultaneously, and because it yields serum activities which correlate poorly with prostatic disease. Formaldehyde inhibits the erythrocytic enzyme and has been said to yield clinically satisfactory results. The copoper resistant acid phosphatase of serum is elevated by metastatic carcinoma of the breast, as well as by other metastatic cancers, and is also elevated by a wide variety of non-cancerous diseases. 

Oral administration of di(2-ethylhexyl) phthalate at levels of 0, 250, 500 or 1000 mg/kg bw to adult albino rats for 15 days resulted in a significant decrease in sperm count in the epididymus and increased activity of P-glucuronidase, y-GT and lactate dehydrogenase and a decrease in the activity of acid phosphatase in the testes. The authors interpreted these findings as indicating germ-cell depletion and deterioration of the germinal epithelium in the testes (Parmar et al, 1986). 

The incubation digest (7.0 ml) contained 1 ml of 0.022 M phenyl phosphate 2.5 ml of 0.1 M acetate buffer, pH 5.0 0.5 ml of test enzyme solution and 3.0 ml of solutions of acceptors giving a final concentration as shown in the third column. Incubation time, 30 min. Digests were inactivated by 3.0 ml of 10% trichloroacetic acid solution and were analyzed for phenol and inorganic phosphate. In the case of the standard acceptor, 1,4-butanediol, the expected transfer product, 1,4-butanediol phosphate, was isolated in a yield of 35% from a large-scale experiment. The hydrolysis of this phosphate ester by prostatic acid phosphatase liberated approximately equimolar amounts of 1,4-butanediol and inorganic phosphate. 

Scott (80) purified red cell acid phosphatase of homozygous types A and B by using ammonium sulfate and DEAE-cellulose chromatography. The relative activity of these isozyme preparations was the same when tested with a number of substrates. Type B enzyme showed small kinetic... 

Forensic biochemists perform blood typing and enzyme tests on body fluids in cases involving assault, and also in paternity cases. Even tiny samples of blood, saliva, or semen may be separated by electrophoresis and subjected to enzymatic analysis. In the case of rape, traces of semen found on clothing or on the person become important evidence the composition of semen varies from person to person. Some individuals excrete enzymes such as acid phosphatase and other proteins that are seldom found outside seminal fluid, and these chemical substances are characteristic of their semen samples. The presence of semen may be shown by the microscopic analysis for the presence of spermatozoa or by a positive test for prostate specific antigen. 

There are scattered reports that phenolic acids inhibit a variety of enzymes, and it is evident that these compounds can block the function of many enzymes if they are sufficiently concentrated at the site of enzymatic functions. Activities of amylase, maltase, invertase, acid phosphatase and protease were suppressed by ferulic acid in tests using maize seeds and seedlings.17,50 Exogenously applied gibberellic acid reversed the effect of ferulic acid on amylase and acid phosphate. 

Early reported applications of this technique were the preparation of a 24-member peptide library [83], of a 125-member tripeptide-substituted cinnamic acid library tested for inhibition of tyrosine phosphatase PTP1B [83], of a 64-member peptide-like library [83] and of libraries based on a natural product, epothilone, using also new polystyrene grafted solid supports [84], Other applications, ranging from l,5-benzodiazepin-2-one library synthesis [85] to chalcone library synthesis [86], were also recently reported. Commercialization of the basic components for this technique [87] (reaction supports and vessels, tags, software, sorters, reaction stations, and so on) will ensure its quick and effective use in combinatorial chemistry and also the implementation of new technical features and possibilities for more complex and demanding applications in future. 

Application and Principle This procedure is used to determine acid phosphatase activity in preparations derived from Aspergillus niger var. The test is based on the enzymatic hydrolysis of p-nitrophenyl phosphate, followed by the measurement of the released inorganic phosphate. 

Other enzymes are also useful indices of liver pathology. Serum alkaline phosphatase is often a useful indicator of liver and bone disease. The alkaline phosphatases are a diverse group of enzymes that catalyze reactions in which a phosphate is removed from a phosphate ester, especially at an alkaline pH. Physicians don t care about this. They do care that serum alkaline phosphatase levels often rise with bone breakdown (as in tumor infiltration) and in liver disease, especially where tliere is obstruction of the bile duct. Acid phosphatase is particularly rich in the prostate. A rise in its serum levels provides a test as to the presence of prostate carcinoma. This test has largely been replaced by assay for Prostate Specific Antigen (PSA), a serine protease that is elevated in prostatic carcinoma. 

Abul-Fadl and King (Al, A2, A3, A4) also investigated the effect of various ions and organic compounds on the acid phosphatase activity of these two tissues. Without describing the results in detail, some of the outstanding effects may be noted. A concentration of 0.5 X 10 M Cu inhibited erythrocytic phosphatase to the extent of 88-96%, but prostatic phosphatase only to the extent of 10-18%. Similarly, 0.5% formaldehyde inhibited completely the erythrocytic phosphatase, but had no effect on prostatic phosphatase. The reverse patterns were shown by 0.5 X 10 M Fe + (ferric) ion, which inhibited erythrocytic phosphatase slightly, about 5-9%, and inhibited the prostatic enzyme to the extent of 80%. Fluoride in 0.01 Af concentration also had comparatively little effect (8% inhibition) on erythrocytic phosphatase but exerted a marked inhibition, 96%, on prostatic phosphate. Of various organic radicals tested, only L-( + )-tartrate (0.01 A/) had a marked differential effect, with 94% inhibition of the prostatic phosphatase and none of the erythrocytic phosphatase. 

Two distinct peaks of acid phosphatase activity were detected in each phenotype, but the positions of these peaks differed. For example, in phenotype A, the peaks were approximately at tubes 150 and 190 in B, at about 130, 170, and 265 in BA, at 110 and 155. In these three, the first peaks showed minor enzyme activity. In CA, there was a major peak at about tube 130 and a smaller one at about tube 170. The shape of the curves varied according to the phenotype tested. In general, these results confirmed what gel electrophoresis originally showed, namely, that there are charge differences between the various isoenzymes. The electrophoretic patterns may also be influenced by the type of buffer used to make up the starch gel (K2). 

Of the several factors found to affect acid phosphatase in vitro, the one that could be tested safely in vivo was alteration of body temperature by application of the procedures of Ripstein et al. (R5). In two patients... 

Testing of the medium in which the fibroblasts had been growing for 3 days revealed significantly elevated levels of activity for the lysosomal enzymes that had been decreased within the cell. No mention was made of acid phosphatase activity. Wiesmann et al. (W5) considered several... 

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. 

Martin A Clynes M (1991) Acid phosphatase endpoint for in vitro toxicity tests. In Vitro Cell and Developmental Biology 21 A 183-184. 

Hemolysis is defined as the disruption of the red cell membrane and results in the release of hemoglobin. Serum shows visual evidence of hemolysis when the hemoglobin concentration exceeds 200 ing/L. Slight hemolysis has little effect on most test values. Severe hemolysis causes a slight dilutional effect on those constituents present at a lower concentration in the erythrocytes than in plasma. However, a notable effect may be observed on those constituents that are present at a higher concentration in erythrocytes other than in plasma. Thus plasma activities or concentrations of aldolase, total acid phosphatase, lactate dehydrogenase, isocitrate dehydro-... 

Although delays of a specimen in transit from a patient in a hospital to the laboratory are usually short, the time elapsing from the separation of serum and cells until analysis may be considerable. The specimen must be properly treated both during its transport to the laboratory and from the time the serum has been separated until it is analyzed. For some tests, specimens must be kept at 4 C from the time the blood is drawn mitil the specimens are analyzed, or until the serum or plasma is separated from the cells. Examples are specimens for ammonia and blood gas determinations, such as PCO2, PO2, and blood pH (see Chapter 27). Transfer of these specimens to the laboratory must be done by placing the specimen container in ice water. Specimens for acid phosphatase, lactate and pyruvate, and certain hormone tests (e.g., gastrin and renin activity) should be treated the same way. A notable decrease in pyruvate and increase in lactate concentration occurs within a few minutes at ambient temperature (see Chapter 25). 

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 S-3 Receiver operating characteristic curves of prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) assays for patients with benign prostatic hyperplasia and prostatic carcinoma. Because the PSA assay curve is above the PAP assay curve at ail points, the PSA assay is the better assay for the patients tested.

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