Acid phosphatase method

An acid phosphatase method has been developed corresponding to that of the alkaline phosphatase already described. 

R4). It might have been expected that, with the presumably more specific method proposed by these investigators, the reverse situation would have held. There appear to have been no further clinical studies utilizing the copper-resistant acid phosphatase method. 

F6. Fishman, W. H., and Davidson, H. M., Determination of serum acid phosphatases. Methods Biochem. Anal. 4, 257-284 (1957). 

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). 

Pulse ultrasonic relaxation method, 32 18 Pump-and-probe techniques, 46 137 Purification, of actinide metals, see Actinide, metals, purification XjPj Purified protein, 36 94 Purple acid phosphatases, 40 371, 376, 43 362, 395-398, 44 243-245 biological function, 43 395 homology, 43 397... 

When acid DNase activity is assayed by the acid-solubility method the optimal DNA concentration is 0.4 mg/ml (9) and higher substrate concentrations appear to be inhibitory (16, 21, 34)- It has been shown, however, that this inhibition is because increasing substrate concentration decreases the efficiency of acid-soluble oligonucleotide release since the number of breaks per unit length of DNA is lower. If a direct method of estimating enzymic activity is used, such as the determination of phosphatase-sensitive phosphate, it can be shown that the inhibition by high substrate seen by the acid solubility method is only apparent (34)-... 

A spectrofluorometric method for the estimation of acid phosphatase has been devised. It uses a-naphthyl phosphate as substrate thus, it is somewhat more specific for prostatic acid phosphatase than most (37). 

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. 

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). 

Hakalahti, L., and Vihko, P. (1989). Purification of monoclonal antibodies raised against prostate-specific acid phosphatase for use in vivo in radioimaging of prostatic cancer. ]. Immunol. Methods 117, 131-136. 

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. 

Also, 5 -AMP has been incubated with MgS04 [14], the reaction terminated by trichloroacetic acid precipitation and inorganic phosphate determined in the supernatant by the method of Fiske and SubbaRow [163], Tartrate has been included in the assay medium in order to inhibit acid phosphatase [183]. Inorganic phosphate has been assayed by the method of King [184]. 

Methods of Determination of Acid Phosphatase Activity 2.1. Introduction... 

A considerable number of procedures have been utilized to assay the acid phosphatase activity of serum, blood cells, and tissues. These have involved different substrates or concentrations of substrates, different temperatures, buffers, or variations in other conditions. If the same acid phosphatase were being measured, then the results were naturally not comparable. But the possibility also exists that closely related but different acid phosphatases were present within the same tissue or in different tissues, and the rate of action of these acid phosphatases depended on the particular substrates, buffers that were employed, or other conditions of the reaction. It seems most appropriate then to preface our review and consideration of the literature by describing briefly the conditions characterizing the most frequently used procedures for the determination of acid phosphatase activity, particularly in the serum. Other methods, or modifications of those to be presented here, will be described in later sections of this review. 

In 1947, Hudson et al. (H15) developed a method for acid phosphatase which, like the procedure of Bessey et al. for alkaline phosphatase (B16), was based upon the use of p-nitrophenyl phosphate as substrate. The buffer substrate solution consisted of equal volumes of a 0.1 M sodium acetate-acetic acid buffer, pH 5.4, and 0.001 M magnesium chloride and of a 0.4% solution of approximately 50% pure disodium p-nitrophenyl phosphate in 0.001 N HCl. To 1 ml of this solution, 0.1 ml of the serum sample was added. The final concentrations in this reaction mixture were 0.045 M acetate buffer, pH 5.4 magnesium chloride. 0.00045 ilf substrate, 0.004 M. The reaction was allowed to run for 30 minutes at 38°C, and the reaction was stopped by the addition of sodium hydroxide. The liberated yellow p-nitrophenol was read at 400 nm and the amount was... 

CoMPAKisoN OP Acid Phosphatase Activities Determined by Different Methods... 

As the preceding considerations illustrate and as was noted at the beginning of this section (2.1), comparison of acid phosphatase activities obtained in different studies must take into account the method employed. Some workers have attempted to do this by using the terms 8-glycerophosphatase, phenylphosphatase, etc. to designate the substrate employed (B6, T6). However, such usage may imply that different acid phosphatases are responsible for these actions, and we shall therefore attempt to avoid this usage in the present review. 

Current Methods for Determination of Serum Acid Phosphatase Activity... 

The question may arise as to which is the preferred method. In the author s experience, and this will be documented more completely later, the use of the substrate, sodium (S-glycerophosphate, as in the Bodansky procedure (B18, 32), is more specific for elevations of serum acid phosphatase activity due to prostatic carcinoma. However, the use of other substrates, such as sodium phenyl phosphate in the Gutman method (GIO, G14), may elicit alterations of activity in the serum that reflect diseases in other tissues. 

As we have seen, practically all the methods on the determination of acid phosphatase activity in serum are calculated upon the amount of reaction product, such as inorganic phosphate, phenolphthalein, or p-nitrophenol, that would be produced under the conditions stated for the method by 100 ml of serum or, as in the method of Hudson et al. (H15), by 1 liter of serum. In the case of the acid phosphatase activity of tissues, some other basis for calculation is used, although the method may be the same as that used for serum. 

The purification of acid phosphatase from the human prostate was undertaken, and high degrees of purity were obtained, before any solid information was available concerning the intracellular distribution of this enzyme or its existence in multiple molecular forms or isoenzymes. Accordingly, in this review several methods of purification will be described first, and the other aspects will then be considered. 

The preceding description of the use of chromatographic methods in the purification of prostatic acid phosphatase (B24, 04) has already indicated that this enzyme exists in more than one molecular form, or isoenzyme. There is, in addition, immunological (S19) and starch gel electrophoretic evidence (L14, L15, S24, S31) of the existence of several forms. In order to ensure that no isoenzymes are lost during any purification, it is preferable to perform such studies on a homogenate of the whole tissue. It should be recognized that the isoenzymatic composition may not be characteristic of the prostatic cell per se, but may also represent components from blood cells, secretory ducts, connective tissue, and other sources. 

The presence of acid phosphatase in the human erythrocyte was recognized in 1934 (D4) and properties of this enzyme were studied for almost thirty years (A4, K6, Tl, T2, T4, T5) before its role in human genetics was revealed (H13). This role will be described in detail later. The properties of crude preparations of erythrocytic acid phosphatase have been previously noted in this review. At this point, we shall describe methods of purification, and the nature of the isoenzymes, particularly as they are related to the phenomenon of polymorphism. 

A much purer preparation of acid phosphatase from horse erythrocytes was obtained by Ito et al. (12) by adding the DEAE-chromatography procedure to the method of Tsuboi and Hudson (T2). Since this procedure may be applicable to human erythrocytes, it will be mentioned briefly. One liter of horse erythrocytes was washed and lysed by the addition of 4 liters of distilled water. One liter of calcium phosphate gel suspension was added to the hemolysate to remove most of the nonenzymatic protein, and the mixture was centrifuged. Five liters of the gel suspension were added to the supernatant, resulting in the adsorption of the enzyme. The enzyme was eluted with citrate-acetate buffer, pH 4.5, and solid ammonium sulfate was added to the eluate up to 60% saturation. The precipitate was collected, dissolved in 40 ml of water, dialyzed against water at 5°C for 10 hours, and again subjected to calcium phosphate gel adsorption, elution, and precipitation with solid ammonium sulfate to 60% saturation. 

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