Determination of Acid Phosphatase Activity

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. 

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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 pH of serum, after it is separated from the clot, tends to go up as carbon dioxide is evolved and will eventually reach pH 8.5. This is an important consideration in the determination of acid phosphatase, which is rapidly destroyed at alkaline pH (W16a). Eighty-nine percent of serum acid phosphatase activity remains after 4 hours at pH 7.8 or 1 hour at pH 7.98 (B2). At pH 7.98 only 58% remains after 4 hours (B2). If serum is maintained over the clot, no rise in pH or inactivation of acid phosphatase occurs for as long as 5 hours at room temperature or 24 hours in the refrigerator (B17b). 

Subtract the blank absorbance from the sample absorbance (the difference should be between 0.100 and 1.000). Determine the acid phosphatase activity (HFU/mL) from the standard curve, and multiply by the dilution factor. For the activity of solid samples, use the following equation ... 

The report of these high incidences of elevations of acid phosphatase activity in miscellaneous disease and in nonprostatic neoplastic disease, determined by a method presumably more specific than the usual Gutman method (GIO, G14), is not in accord with earlier studies, such as those of Sullivan et al. (S30) shown in Table 10. The incidences of elevations were considerably lower than those reported by Reynolds et al. 

The level of acid phosphatase activity in the aboveground part indicated differences between cuitivars. Significant differences in activity were between cuitivars Zitnica and Novosadska rana 2 in all Pi treatments except Po. An increase in acid phosphatase activity was determined in all oases with the decrease of Pi concentration in thenutrient solution in the aboveground part as well as in the root (Fig. 3). 

FIGURE 1.4 Manifold of a multicommutated flow injection system (MCFA) developed for simultaneous determination of acid phosphatase and alkaline phosphatase activity consisting of substrate/buffer delivery module (a), sample delivery cell module (b), reaction module (c), and detection module (d). V—valves, P—pumps, HC—holding coils, PEDD—paired emitter detector diode, NPP— p-nitrophenyl phosphate, DEA— diethanolamine. (Adapted from Tymecki, L., K. Strzelak, and R. Koncki. 2013. Anal. Chim. Acta 797 57-63.)... 

Recently the amino acid sequence of vanadium chloroperoxidase was determined to have similar stretches with three families of acid phosphatases, which were previously considered unrelated [72], This sequence raises questions about the phosphatase activity of apo-V-ClPO and whether the acid phosphatases can coordinate vanadate and carry out peroxidative halogenation chemistry. In fact, apo-V-C1PO does have phosphatase activity, catalyzing the hydrolysis of/i-nitrophe-nol phosphate (p-NPP). In addition, /i-NPP displaces vanadate from V-CIPO. At this point, the haloperoxidase activity of the acid phosphatases containing coordinated vanadium(V) has not been reported. 

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. 

In 1950 Seligman and his co-workers (S13) suggested the use of sodium j8-naphthyl phosphate as a substrate for the determination of acid or alkaline phosphatase activity. For the former, 1 ml of 1 20 diluted serum was added to 5 ml of 0.4 mM sodium 8-naphthyl phosphate in 0.1 M acetate buffer of pH 4.8, and the reaction was allowed to proceed for 2 hours at 37.5°C. The addition of 4 drops of IM sodium carbonate solution served to retard the reaction as well as to raise the pH to the optimal level for coupling with 1 ml of a solution of tetrazotized ortho-dianisidine. After 3 minutes, the protein was precipitated with trichloroacetic acid, the dye extracted with ethyl acetate, and the color density determined in the region of 540 nm. The unit of phosphatase activity was defined as that amount of enzyme which liberates the color equivalent of 10 ml of j8-naphthol per hour at 37.5° in 1 hour. The serum acid phosphatase in a group of normal adults ranged from 0.7 to 1.6 units and averaged 1.0 unit per 100 ml of serum. 

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

The acid phosphatase appeared to be associated in the saclike structure with other hydrolytic enzymes, such as 3-glucuronidase and cathepsin, which also acted optimally at acid pH levels. Further studies were undertaken to isolate this structure (A13, G2). By means of a differential centrifugation procedure which will be described in detail later, de Duve and his associates (D9, DIO) determined the intracellular distribution of total and free acid phosphatase activity and of other enzymes as well. The mean values, expressed as percent of total acid phosphatase activity, were nuclear, 3.6 mitochondrial, 24.1 light mitochondrial, 40.7 microsomal, 20.1 final supernatant, 13.3. 

Attempts to determine the intracellular distribution of acid phosphatase in the prostate must take into account the presence of this enzyme in the extracellular secretion. Employing centrifugal methods, Siebert et al. (S20) found that of the total acid phosphatase present in bull prostate homogenate, 0.7% was in the nuclear fraction, 41% in the mitochondrial fraction which presumably included the lysosomal component, and 84% in the microsomal and supernatant components. The finding that the sum of these activities exceeded that in the homogenate was considered to represent removal of inhibitors during separation of the fractions. 

As long ago as 1935, Kutscher and Wolbergs (K12) observed that semen and the prostate are among the richest sources of acid phosphatase in the human body. In a more recent survey (Bll) the acid phosphatase activities of seminal plasma in various species, determined as milligrams of nitrophenol liberated by 100 ml seminal plasma from 0.006 M p-nitrophenyl phosphate, in 60 minutes at 37°C and pH 4.9 were human, 274,000 cock, 15,000 turkey, 4000 bull, 570 rabbit 85. Human seminal plasma is made up by the secretory fluids produced in the epididymides, vasa deferentia, ampullae, seminal vesicles, the prostate and the bulbourethral (Cowper s) and urethral (Littre s) glands (M4). The semen contains many particulate bodies. Best known, of course, are the spermatozoa, which are formed in the seminiferous... 

If it is assumed that all the acid phosphatase activity observed in the various types is determined by the three genes, P , P , and P then the question arises whether the quantitative effects of these genes are additive in a simple way. It may be seen from Table 5 that half the mean activity, 122.4, in type A plus half the mean activity, 188.3, in type B is equal to 61.2 + 94.2 or 155.4. This value is in good agreement with 153.9, the mean value actually observed for type BA. Other equations... 

The next most common method for determination of serum acid phosphatase activity was based on the use of sodium )3-glycerophosphate as substrate. This method and its modifications have been described in Section 2.3. Values obtained by these methods in normal males are shown in Table 7 a liberal summary of these values indicates a mean value of about 0.4 0.2 Bodansky units. 

As was previously noted (Section 6.2), the total and prostatic serum acid phosphatase levels were determined by the method of Fishman and Lerner (FI) in a series of 365 males attending a cancer-prevention clinic (D6). The values for the total acid phosphatase activities in the 315 patients of this group who had no prostatic enlargement have already been described (Table 6). In groups of the size under consideration, values within 2.5 standard deviations of the mean can be considered as normal ... 

Determinations of the prostatic serum acid phosphatase activity in... 

Whatever may be the reason for the low incidence of elevations of total serum acid phosphatase activity in Fishman and his associates (F2) series of proven cases of carcinoma, the higher incidence of elevations of prostatic acid phosphatase activity indicates that in the patients of this series the determination of the latter was a more sensitive indicator of the presence of prostatic carcinoma. Moreover, when serum prostatic acid phosphatase activities were determined during the course of a patient s illness, they paralleled the exacerbation or remission of the disease. Whitmore et al. (W4) have considered the relationship of clinical status to the total and prostatic acid phosphatase activities in 20 patients with proven carcinoma of the prostate. 

One female and one male patient had hyperparathyroidism with elevated serum alkaline phosphatase activities and extensive bone changes characteristic of generalized osteitis fibrosa cystica. In both instances, the serum acid phosphatase activity of the serum fell to normal values after removal of the parathyroid adenoma despite transitorily increased serum alkaline phosphatase activity. The fifth patient was a female with osteopetrosis involving the major part of the skeleton. The serum acid phosphatase was 8.7 K.A. units, the highest in the control series— yet the serum alkaline phosphatase was within normal limits. It would appear, therefore, that some patients with skeletal disease may have a slight but definitely elevated serum acid phosphatase activity, at least as determined by the Gutman method (GIO, G14), which cannot be explained by concurrent prostatic carcinoma or by a spillover of alkaline phosphatase activity to a pH of 5.0. 

Using the Bodansky (B18, 52) procedure with 8-glycerophosphate as substrate, Woodard (W8) was unable to obtain such elevations. She determined the serum acid phosphatase activities in 83 females and 342 males, or a total of 425 patients with miscellaneous diseases. Of these, 61 had various types of infectious or metabolic disorders, including 11 cases of inflammatory disease of bone and 12 cases of hepatic cirrhosis. The remainder had some type of neoplastic disease and about one-third had metastases to bone from cancer of various primary sites. There were 15 cases of osteogenic sarcoma and 32 cases of osteitis deformans. All these cases, whether their serum alkaline phosphatase activities were elevated or not, had serum acid phosphatase values that were essentially within the normal range, 0.06-0.89 Bodansky unit for females and 0.11-0.88 unit for males. In contrast to the Gutman method (GIO, G14), there-... 

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