Acid phosphatase: New developments

ACID PHOSPHATASE: NEW DEVELOPMENTS Nicholas A. Romas, M.D., * Noel R. Rose, M.D., Ph.D., t and Myron Tannenbaum, M.D., Ph.D. oJ;.

Abstract Acid phosphatase was the first "tumor marker" to be measured in the blood, and over 40 years have passed since an elevation of the serum acid phosphatase level was observed in patients with prostatic carcinoma. However, significant elevations in the level of this enzyme have been observed in other diseases, as well as elevations of other tissue phosphatases, Many improvements in the colorimetric technique have been introduced, but none has been used successfully to detect the tissue origin of this ubiquitous enzyme. The finding that prostatic acid phosphatase is antigenically distinct from acid phosphatase of other tissues opened a new horizon in the measurement of acid phosphatase in prostatic cancer. On the basis of this immunochemical specificity, several immunoassays have been employed for determining the prostatic acid phosphatase level.

The acid phosphatases (orthophosphoric monoester phospho hydrolase) are a group of enzymes capable of hydrolyzing esters of orthophosphoric acid in an acid medium. Enzymatic hydrolysis results in the splitting of the O-P bond with the release of phosphoric acid. In man, acid phosphatases were first demonstrated in erythrocytes in 1924, in urine in 1925, and in spleen and liver in

1934.1-3 Dmochowski et a1. 4 and Kutsher et al. 5 , 6 found that the human ejaculate and the prostate were rich in add phosphatase, the properties of which were similar to those of urinary acid phosphatase. These findings were confirmed by Gutman and associates, and from 1936 to 1942 this group proceeded to study acid phosphatase extensively and established its clinical significance in human diseases.

*Associate Professor of Clinical Urology, Columbia University College of Physicians and Surgeons. Associate Attending Urologist, Columbia-Presbyterian Medical Center, New York, New York. tProfessor and Chairman, Department of Immunology and Microbiology, Wayne State University, Detroit, Michigan. :f:Associate Professor of Pathology, Columbia University College of Physicians and Surgeons. Associate Attending in Pathology, Columbia-Presbyterian Medical Center, New York, New York.

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These findings were summarized by Gutman" in 1968. Acid phosphatase was found to be abundant in the human prostate and the caudal lobe of the prostate gland of the rhesus monkey. 8 Prostatic acid phosphatase activity is negligible before adolescence in human subjects and in the rhesus monkey." It is subject to hormonal influence and can be induced in the immature rhesus monkey by the administration of testosterone but not by estradiol. It was found that the serum acid phosphatase level was markedly increased in patients with prostatic carcinoma, especially in those with bone metastases.P: 11 Gutman-" postulated that the increased acid phosphatase activity resulted from enzyme leakage into the circulation when the carcinomatous prostatic tissue metastasized and invaded lymph nodes or blood channels. These investigators also recognized that erythrocytes are a rich source of acid phosphatase and showed that the enzyme from the red blood cell is resistant to sodium fluoride and thereby different from that in the prostate.P In 1941 Huggins and Hodges" published their classic article on acid phosphatase and prostatic carcinoma. Their results confirmed the work of Gutman and associates and extended their findings by concluding that the growth of normal as well as malignant prostatic epithelia is under hormonal control. Antiandrogen therapy either in the form of estrogens or by removal of testosterone (by castration) decreased the activity of prostatic carcinoma and reduced serum acid phosphatase activity. These findings form the cornerstone of modern antiandrogen therapy for prostatic carcinoma. The clinical ambiguities in measuring the serum acid phosphatase level soon became apparent. Although the acid phosphatase concentration is highest in the cell's and secretions of the prostate glands, this enzyme is also present in erythrocytes, leukocytes, platelets, liver, spleen, kidney, and other tissues." Various non prostatic diseases were found to cause hyperacid phosphatasemia, and it was also noted that the properties of acid phosphatase in the serum of normal subjects differ from those of that in the prostate." Therefore the heterogeneous and ubiquitous nature of this enzyme has

complicated the interpretation of serum levels and has given rise to numerous biochemical methods in an attempt to achieve specificity. Current biochemical tests are incapable of detecting the tissue origin of the enzyme and are merely based on the capacity of acid phosphatase to hydrolyze phosphate esters. AbdulFadl and King'? and Fishman and associatesv- 19 utilized various inhibitors and indicated that the tartrate inhibitable fraction was most likely of prostatic origin. This view has not been universally accepted. 2o , 21 Other attempts to differentiate the different fractions of acid phosphatase in normal subjects and in patients with various diseases have met with variable degrees of success. Recent evidence suggests that prostatic acid phosphatase is antigenically distinct from acid phosphatase of other tissue origin." On the basis of this immunochemical specificity several immunologic methods have been employed for detecting prostatic acid phosphatase.w?" DISTRIBUTION OF ACID PHOSPHATASE IN HUMAN TISSUES Comori'" and Burstone'" introduced histochemical techniques to study acid phosphatases in human tissues. As a result of this work it is now possible to study the distribution of acid phosphatase activity by light microscopy and in ultrathin sections by transmission electron microscopy. The acid phosphatases are widely distributed in all human body fluids and tissues. Histochemical studies indicate that the tissue enzyme activity is located mainly in the glandular epithelium and that much of the acid phosphatase is of lysosomal origin. In human neoplasms enzyme activity is greatest in the prostate, with variable activity in the breast, stomach, and colon and low activity in the thyroid, kidney, and ovary.!" ISOENZYMES IN PROSTATIC NEOPLASIA The use of isoenzymes in the diagnosis of prostatic neoplasia is relatively new in the clinical laboratory. Moncure!' re-

ACID PHOSPHATASE-RoMAs cently summarized the use of isoenzymes in the diagnosis of prostatic carcinoma and indicated how advances in basic research have expanded the clinical application of this technique in urology. Isoenzymes are enzymatically active proteins, which catalyze similar reactions but differ in certain physicochemical properties. The biochemical reaction in question should be the physiologic one that occurs in vivo. However, the physiologic function of acid phosphatase is unknown, and, as with many other enzymes, it is detected only through the use of artificial substrates reacting under laboratory conditions. Separation of isoenzymes is most commonly accomplished by electrophoresis, but other physicochemical separation techniques based on molecular weight or antigenicity may also be utilized. The molecular charge (and therefore electrophoretic mobility) on an enzyme at a given pH may vary as a result of either amino acid substitution or variation in charged side groups (such as sialic acid) that are not an integral part of the polypeptide chain. Alterations of this type may occur in a molecule without changing the enzyme's physiologic function or its antigenicity. Methods for separation of the acid phosphatase isoenzymes by inhibitors or by substrate selectivity have not been able to be prostate specific. L( +)tartrate, the most widely utilized inhibitor of prostatic acid phosphatase, has also been found to inhibit other acid phosphatase isoenzyrnes.P Electrophoretic studies combined with the evaluation of preferred substrates suggest that none of the currently employed substrates is truly prostate specific."

ELECTROPHORETIC SEPARATATION OF ACID PHOSPHATASE Electrophoretic separation of acid phosphatase isoenzymes has been performed with serum and numerous tissue extracts. The laboratory techniques employed have varied considerably, and therefore no standard diagnostic method or isoenzyme pattern has gained widespread acceptance. Moncure'" has sum-

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marized the methodology and results obtained in 26 articles from 1959 to 1974 on acid phosphatase isoenzymes. He claims that the techniques of electrophoresis and enzyme staining are an important source of variation in results. Moncure suggests the following tentative conclusions: 1. At least three and probably four enzyme populations exist, which may have little physiologic relationship with each other. 2. Each major acid phosphatase population may be further subdivided into isoenzymes that may share common physiologic functions. 3. The major group of acid phosphatases includes intracellular red blood cell acid phosphatases, intracellular acid phosphatases associated with lysosomes, and acid phosphatases that are found as secretory products with extracellular function.

No attempt will be made to discuss all tissue isoenzymes, but emphasis will be placed on human plasma, serum, and the prostatic epithelium. Acid phosphatase isoenzymes are especially prominent in the reticuloendothelial system, and besides interfering in the measurement of prostatic acid phosphatase they may become valuable diagnostic aids in hematology. The numeric designations 0, 1, 2, 3B, 4, and 5 based on electromobility using polyacrylamide gel electrophoresis employed by Li et al.33 and by Yam et al.3 4 will be utilized for discussing this group of isoenzymes. Isoenzyme 0 moves very slowly and stays in the space gel layer, whereas isoenzyme 5 moves the fastest. Normal plasma contains tartrate resistant isoenzyme 5 but no demonstrable isoenzyme from red blood cells or prostate. Normal serum contains isoenzymes 5 and 3 (tartrate inhibited), which are released from the platelets during blood clotting. Osteoclasts are rich in acid phosphatase, which is tartrate resistant and migrates in position 5. Osteoclastic acid phosphatase is probably responsible for serum enzyme elevation in bone disorders. Therefore, it appears that the platelets (zone 3) and osteoclasts (zone 5) may be the primary sources of normal serum acid phosphatase. When prostatic extracts are studied by acrylamide gel electrophoresis, pro-

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static acid phosphatase shows activity in zone 2 with weak activity in zone 4. Both zones are tartrate sensitive. Granulocyte and other tissues have tartrate inhibitable forms of acid phosphatase. Prostatic acid phosphatase appears to be primarily a secretory product, although lysosomal forms of this enzyme may also occur in prostatic cells. It reveals extreme heterogeneity when starch gel electrophoresis is utilized. Sur et al. 35 have demonstrated up to 13 bands of activity. They also noted that only a single fast band of activity was observed when seminal plasma was studied. These findings suggest that the heterogeneity of the prostatic tissue enzyme may represent variation in side groups on a single molecular form of the enzyme. The cells may contain numerous incomplete forms of the final secretory product. This view is supported by Smith and Whitby'" and Ostrowski et al.,37 who demonstrated that apparently multiple molecular forms of acid phosphatase can be reduced to a single protein enzyme by neuraminidase digestion. Recently Chu et al.38 confirmed and expanded on this work. After a series of purifications the acid phosphatase isoenzymes from prostatic tissue were separated into two chromatographic fractions. Treatment with neuraminidase removed the sialic acid content of the molecule, changed the isoelectric focusing patterns, and abolished the chromatographic heterogeneity. In prostatic neoplasia the possibility of finding an "incomplete" variant of acid phosphatase has been entertained, but such variants have not been recognized. Although zymogram studies by electrophoresis are preferable to the biochemical assay methods in identifying the enzyme changes, polyacrylamide gel electrophoresis is a time consuming procedure. Li et aJ.33 concluded that this was not a practical clinical approach.

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In 1960 the existence of prostate specific antigens in dogs was suggested by the in vivo studies of Flocks et a1. 39 Shulman et al. 40 in 1964 demonstrated antibodies that reacted with human prostatic

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acid phosphatase. Subsequent studies by Moncure 23.41-44 and others 45,46 attested to the apparent antigenic specificity of prostatic acid phosphatase. The practical application of an immunologic assay was first demonstrated by Milisauskas and Rose.:" They employed electrophoresis of an antigen into an antiserum containing gel, thereby inducing precipitating zones in the shape of a cone. The antigenantibody complexes were stained by histochemical means. The height of the "rockets" made visible by staining were proportional to the prostatic acid phosphatase concentration. Since only acid phosphatase stained, antigen-antibody reactions did not interfere with this method. Because a pure enzyme is essential for the development of a highly specific antiserum for immunologic assays, there has been investigative work in several laboratories on the purification and characterization of human prostatic acid phosphatase, Purification procedures for prostatic acid phosphatase have been described by Ostrowski and Rybarska.v' Pais et al.,48 Lam et al.,49 and van Etten and Saini. 50 Lam et al.49 demonstrated the presence of three acid phosphatase isoenzymes in water extract from the prostate. After precipitation with ammonium sulfate (55 to 75 per cent saturated) and after double DEAE-cellulose chromatography, they obtained one isoenzyme. Vihko et al.51 purified the main isoenzyme of human prostatic acid phosphatase by affinity chromatography on L( + )-tartrate linked to agarose and by isoelectric focusing. The enzyme was a single protein when examined by polyacrylamide gel electrophoresis, either as a native protein or in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme, as measured by gel filtration, was 109,000 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 54,000, indicating that the enzyme had been isolated in the dimer form. Choe and associates" performed extensive studies on the purification and characterization of human prostatic acid phosphatase and also described a new purification procedure for it. The procedure included the use of carboxymethyl-

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Sephadex and concanavalin A affinity column chromatography. The purified enzyme could be further purified on the Sephadex G-200 or by polyacrylamide gel electrophoresis. The enzyme, thus purified, showed a single band on an SDSpolyacrylamide gel and was free of any extraneous proteins as judged by immunochemical criteria. The molecular weight of the enzyme was estimated to be 98,000 by gel filtration on a Sephadex G-200. When antiprostatic acid phosphatase sera were raised in rabbits and mice using purified prostatic acid phosphatase as the antigen, monospecific antisera were produced. Human acid phosphatase from liver, lung, pancreas, and human cell cultures did not cross react. Charge heterogeneity of prostatic acid phosphatase has been demonstrated by polyacrylamide gel electrophoresis, ion exchange chromatography, and isoelectric focusing. 32 , 53 It has been demonstrated that removal of sialic acid from prostatic acid phosphatase isoenzymes reduced the electrophoretic heterogeneity without causing any reduction of the enzyme activity. Choe et a1. 52 have shown that the neuraminidase digestion of prostatic acid phosphatase isoenzymes did not alter their antigenic specificity. Since the antigenic specificity of isoenzymes of prostatic acid phosphatase is retained after the removal of sialates, these residues are clearly not involved in immunologic specificity. So far, antiprostatic acid phosphatase and anti-isoenzyme antibodies are recognized antigenic determinants on prostatic acid phosphatase isoenzyrnes and subunit proteins; therefore, isoenzymes of prostatic acid phosphatase may have similar tertiary structures. However, these investigators noted that they used antibodies elicited against prostatic acid phosphatase that were purified from pooled human prostate glands or ejaculates, which cannot exclude the possible occurrence of substitution mutation of prostatic acid phosphatase among individual glands.

STABILITY OF ACID PHOSPHATASE ACTIVITY The three most important factors in the stability of serum acid phosphatase

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are pH, the temperature at storage, and the substrate acidity of the acid phosphatase being measured. 15 Of these three, pH is the most critical and temperature the least important.s" When serum is separated from the clot and kept at room temperature (25 C.), enzyme activity decreases considerably within one to two hours owing to the increase in pH as a result of the loss of carbon dioxide. This increase in pH, which may go from 7.6 to 8.5 in several hours, may be slowed by storage of the material in tightly stoppered containers. Serum enzyme activity is well preserved at room temperature when the serum is not separated from the clot or when the serum is buffered to pH 6.2 to 6.6 with disodium hydrogen citrate." Because the serum acid phosphatase level is influenced by factors such as inactivation of the enzyme and the unpredictable contribution of platelet acid phosphatase during blood clotting, it is probable that the ideal specimen for determining acid phosphatase activity is fresh plasma buffered with citrate to a pH of 6.2 to 6.6. 32 The foregoing applies to biochemical testing for acid phosphatase, but there is evidence from our laboratory that the addition of citric acid to serum when utilizing immunologic methods such as counterimmunoelectrophoresis will cause erroneous results.P" 0

CLINICAL CONSIDERATIONS Effect of Prostatic Massage An elevation of the serum acid phosphatase level has been recorded following massage of the prostate gland. Hock and Tessier'" recorded an elevation of serum acid phosphatase levels in 17 of 20 patients one hour after prostatic massage. Daniel and Van Zyl58 reported elevation of these levels for up to 24 hours in three of 24 patients with benign prostatic hypertrophy following routine clinical palpation of the prostate. Other investigators have found no significant change in the serum acid phosphatase level following digital prostatic examination.59, 60 There is evidence to suggest that in the presence of prostatic carcinoma, massage of the gland more readily produces a rise in the leve1.61, 62 A recent study by Khan

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et al. 63 investigated the effect on the serum acid phosphatase level in 100 elderly male patients after routine digital examination and 35 control subjects who did not undergo rectal examination. They utilized two different chemical methods and detected a fall rather than a rise in the serum level. The utilization of the newer immunologic assays for measuring prostatic acid phosphatase may help clarify this particular clinical problem. Until this information is forthcoming, blood specimens for prostatic acid phosphatase determinations should be obtained before a prostate examination or 24 to 48 hours after the examination.

Effect of Prostatic Infarction and Operative Manipulation Prostatic infarction may be accompanied by an elevated prostatic acid phosphatase level. 64 We have observed elevated levels by biochemical and immunologic methods in several patients who were shown to have prostatic infarction by pathologic examination. 56 Transurethral resection of the prostate is also accompanied by increased acid phosphatasemia. There is a correlation between the degree of rise and the amount of prostatic tissue resected. If more than 30 gm. is resected, nearly all patients manifest a rise in prostatic acid phosphatase activity that persists beyond 24 hours.f" Urinary retention and catheterization have also been noted to elevate the level. 66 This has been documented by our laboratory utilizing counterimmunoelectrophoresis to measure the level."

Nonprostatic Disorders and Hyperacid Phosphatasemia Batsakis et al." have listed the nonprostatic causes of elevated serum or plasma acid phosphatase levels as follows: primary disease of bone (Paget's disease, osteogenesis imperfecta, osteogenic sarcoma, osteopetrosis, and osteoporosis), secondary disorders of bone (hyperparathyroidism, multiple myeloma, and

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metastatic carcinoma), hepatobiliary disease (viral hepatitis, chlorpromazine hepatitis, extrahepatic obstruction, and cirrhosis), diseases of the kidney (chronic glomerulonephritis and gouty nephropathy), diseases of the reticuloendothelial system with hepatic or osseous manifestations or both (Gaucher's disease, Niemann-Pick disease, eosinophilic granuloma, reticulum cell sarcoma, and Hodgkin's disease), and carcinomas with hepatic or osseous metastases or both (breast, stomach, colon, kidney, and adrenal cortex). Bodansky'" has presented statistics relating to a large number of patients with nonprostatic disorders and hyperacid phosphatasemia. He reports the following: 19 per cent in patients with skeletal metastases, 2 per cent in patients with liver metastases, 6 per cent in patients with neoplasms without either bone or liver involvement, and 10 per cent in patients with primary bone tumors. In the category of non-neoplastic disease of bone, elevations were present in 21 per cent of 96 patients with Paget's disease, in 33 per cent of nine patients with hyperparathyroidism, and in 4 per cent of patients with miscellaneous diseases of bone. Romas et al.27 employing counterimmunoelectrophoresis to measure the prostatic acid phosphatase level studied a group of patients with nonprostatic disorders with hyperacid phosphatasemia and were able to eliminate these false elevations.

RADIOIMMUNOASSAY FOR DETERMINATION OF PROSTATIC ACID PHOSPHATASE Several laboratories have developed radioimmunoassays for the detection of prostatic acid phosphatase. Foti et aJ.25 have reported a clinical study with the use of solid phase radioimmunoassay. The method has been described in detail in a previous publication." In brief, human prostatic acid phosphatase was purified from the prostatic fluid of healthy adult males, and antiserum was produced by intramuscular inoculation of female New Zealand rabbits. This study presents

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randomized comparative data on the merits of a solid phase radioimmunoassay versus a standard enzymatic spectrophotometric method in the measurement of the serum prostatic acid phosphatase level. 68 In this study 113 patients with prostatic carcinoma (stages I to IV), along with 167 without prostatic cancer but with other disorders, and 50 normal control subjects were evaluated. The level of prostatic acid phosphatase did not exceed 6.6 ng. per 0.1 mi. in normal controls, but with 6.6 ng. as a cutoff, 15 per cent of the patients with benign prostatic hypertrophy were classified as "elevated." With the upper limit of normal at 8 ng. per 0.1 ml. (mean ±4 SD), radioimmunoassay identified prostatic carcinoma in 33, 71, 79, and 92 per cent of stage I, II, III, and IV patients, respectively. The enzyme assay detected serum elevations in 12, 15, 29, and 60 per cent, respectively. No false positive results were obtained by either assay in normal control subjects, but the radioimmunoassay was positive in two patients with benign prostatic hypertrophy (5 per cent), one who had undergone total prostatectomy (4 per cent), nine with other cancers (11 per cent), and 11 with gastrointestinal disorders (5 per cent). In contrast to the standard enzyme assay, their data suggest that the use of radioimmunoassay for prostatic acid phosphatase has the potential for detecting well over half the cases of intracapsular prostatic carcinoma. The use of solid phase radioimmunoassay for serum prostatic acid phosphatase appears to have distinct advantages over the standard enzyme assay. These authors also conclude that there appears to be no advantage in using the enzyme assay with the radioimmunoassay for measuring the serum prostatic acid phosphatase in order to increase the fre quency of correct diagnosis. Crayhack'" questioned the staging designation in that 12 per cent of stage I and 15 per cent of stage II tumors were associated with elevated acid phosphatase levels by the enzymatic method. False positive rates of 5 per cent with gastrointestinal disorders and 11 per cent in patients with other malignant tumors were not reassuring.

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Carroll"? has provided a statistical review of these data. If only the hospital patients are considered, the results comparing the two methods are as follows: sensitivity (percentage positivity of the test in disease), 69 per cent (79/113) for radioimmunoassay and 28 per cent (32/ 113) for enzyme assay; specificity (percentage negativity of the test in health), 92 per cent (160/167) for radioimmunoassay and 96 per cent (160/167) for enzyme assay; and confidence, 86 per cent (79/92) for radioimmunoassay and 82 per cent (32/39) for enzyme assay. It is immediately apparent that the radioimmunoassay is more sensitive than the enzyme assay. It is not more specific, however, and at this prevalence for the index of disease (40 per cent or 113/~ 80). the confidence in or predictive value of a high reading is not substantiallv better for radioimmunoassay . Carroll 'concludes th at the ad vantage is its sensitivity: it detects more cases. Cho~ and associates -v " have reported a double antibody radioimmunoassay for prostatic acid phosphatase. The source of antigen was human hypertrophic prostate glands and human ejaculate ; antisera were raised in rabbits. The upper limit of the serum prostatic acid phosphatase levels was set at 2.4 ng. per 0.1 ml. in 162 controls. The serum prostatic acid phosphatase levels in patients with nonprostatic malignant tumors fell in the normal range, whereas levels higher than 4.0 ng. per 0.1 ml. were found in patients with prostatic carcinoma . The number of cases studied (36) was small, and there was no attempt to indicate the specific stage of the disease. Attempts to utilize radioimmunoassay by the National Prostatic Cancer Project and participating institutions were not successful. Murphy'" reported that the major reasons for failure of radioimmunoassay in the national studies were technical. The most common problems were nonspecific binding and difficulties in establishing standard curves. Radioimmunoassay was found to be a tedious procedure, which required three to four days for completion, but excellent results could be expected by institutions having experience with this procedure that have developed the necessary specific antisera.

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COUNTERIMMUNOELECTROPHORESIS

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Several investigators have utilized counterimmunoelectrophoresis for the detection of prostatic acid phosphatase. McDonald et aI. 73 purified prostatic acid phosphatase from normal human prostate. A specific antiserum to the purified enzyme was produced in rabbits. The rabbit antiserum was shown to be specific for prostatic acid phosphatase and showed no reaction with acid phosphatase preparations from normal liver, lung, and pancreas or tissue cultures from lung, breast, and uterine cancers. A counterimmunoelectrophoresis method was developed utilizing the specific antisera and a chemical staining technique. The essence of this technique is the movement of antigen in an electric field to form a precipitin line. At pH 7.6 purified prostatic acid phosphatase moves toward the anode, whereas purified IgG migrates toward the cathode. Therefore, by placing prostatic acid phosphatase in the well at the cathodic side, the enzyme and its antibody should meet and precipitate between the cells during electrophoresis. Detection of the enzymeantibody precipitin line could be achieved by the application of enzyme activity staining with naphthol AS-MIX phosphoric acid and fast garnet GEe in acetate buffer at pH 5.1. The sensitivity of the counterimmunoelectrophoresis assay varied somewhat depending upon the antiserum used. The lowest detectable activity of prostatic acid phosphatase was about 2.5 ng. per 0.1 mI. of prostatic acid phosphatase protein. The original report indicated no positive reactions in 11 patients with benign prostatic hypertrophy or in 162 healthy volunteers. Positive results were obtained in 21 of 24 serum samples from patients with prostatic cancer as well as in 10 of 13 bone marrow samples. Murphy et al." reported recently on the use of counterimmunoelectrophoresis in a national field triaJ.26 For stage A and B prostatic carcinoma the biochemical methods used by the different hospitals revealed an elevated acid phosphatase level in 15 and 10 per cent, respectively, whereas the counterimmunoelectrophoresis method showed a positive test in

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39 per cent with stage A disease and 38 per cent in stage B. In stage C disease 20 per cent of the patients had an elevated enzyme level with the biochemical methods, whereas the immunoassay showed a positive reaction in 59 per cent. In stage D disease 51 per cent of the patients had an elevated acid phosphatase level by standard assays in contrast to a 69 per cent correlation by counterimmunoelectrophoresis testing. In stages A. B. and C only two patients in each group had an elevated acid phosphatase level with biochemical methods and a negative resuIt with counterimmunoelectrophoresis. There is no doubt that this study indicates a much higher sensitivity with counterimmunoelectrophoresis than with conventional biochemical testing for acid phosphatase. The limitations of this study are that the authors did not indicate which patients had been treated and also did not indicate which biochemical tests were utilized. We are all aware of the dramatic changes in the prostatic acid phosphatase level with conventional antiandrogen therapy. Romas et alY developed a counterimmunoelectrophoresis method for prostatic acid phosphatase and utilized it on bone marrow fluid as well as sera in patients with prostatic carcinoma and other disorders. They obtained a crude extract of prostatic acid phosphatase from benign hypertrophic prostate tissue and formed an antiserum in rabbits and, recently, in goats. This antiserum was absorbed to remove unwanted antibodies to other human proteins. A modification of the counterimmunoelectrophoresis method of Lee 74 was used and the precipitin band was stained by the naphthol phosphate and fast TR salt method (l\'FR).75 The lowest detectable activity of prostatic acid phosphatase was at 0.3 I. U. per L. (Roy method) or 4 ng. per 0.1 ml. The data presented indicated the clinical usefulness of having a specific laboratory test for prostatic acid phosphatase. In the evaluation of a patient for prostatism there is often a slight elevation of the serum acid phosphatase level and no other evidence of prostatic malignant disease. The counterimmunoelectrophoresis determination for prostatic acid phosphatase was negative in all but one case (1159), whereas the determination by a

ACID PHOSPHATASE-RoMAs well accepted spectrophotometric method yielded falsely elevated results in 20 per cent (12/59).76 A spectrum of diseases was studied that commonly produces false elevations of the serum acid phosphatase level by most chemical methods. There were no false positive results by counterimrnunoelectrophoresis in the sera of patients with nonprostatic disorders causing hyperacid phosphatasemia. Two weakly false positive results were found in the bone marrow determination for prostatic acid phosphatase. One patient had Paget's disease and the second, non-Hodgkin lymphoma. Other investigators have also reported false positive findings with immunologic methods. Choe et al." observed a case of pancreatic islet cell carcinoma in which metastasis occurred to the liver, producing an antigen that immunologically cross reacted to prostatic acid phosphatase. Drucker et al. 78 reported a false positive determination in bone marrow in a patient with chronic granulocytic leukemia. Preliminary results in studying early stages of prostatic carcinoma revealed 0 to 13 per cent elevations in stages A and B by the Roy method and 29 and 20 per cent elevations by counterimmunoelectrophoresis, Choe et al.24 and McDonald et al." have also reported the use of the counterimmunoelectrophoresis method for the determination of human prostatic acid phosphatase levels. They utilized purified prostatic acid phosphatase from human prostate and pooled human ejaculate. Localization of the immune precipitin band in agarose gel was visualized according to the procedure of Burstone.P They did not report an extensive clinical survey but made some excellent correlations between the values obtained by counterimmunoelectrophoresis assay and radioimmunoassay. The radioimmunoassay measures prostatic acid phosphatase concentrations as low as 0.1 ng. per 0.1 ml. sample, whereas the detection limit of counterimmunoelectrophoresis assay is 2.5 ng. per 0.1 ml. The normal serum prostatic acid phosphatase level for normal males is 1.6 ± 0.8 ng. per 0.1 ml, by radioimmunoassay. The normal serum prostatic acid phosphatase level is not de-

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tectable by counterimmunoelectrophoresis assay; however, prostatic acid phosphatase concentrations higher than 4 ng. per 0.1 mL are readily detected. According to their radioimmunoassay survey of serum prostatic acid phosphatase levels for prostatic carcinoma (beyond stage II), concentrations higher than 4 ng. per 0.1 ml. imply an elevation of the serum prostatic acid phosphatase level. There are very few false positive results with either immunoassay method.

IMMUNOENZYME ASSAY An improved and simplified immunoassay was recently described by Choe et aU 9 Termed "immunoenzyme assay," the method dispenses with the need for a radioactive label. The serum samples are mixed with specific antiserum and the resulting complexes are precipitated by ammonium sulfate. The amount of precipitated prostatic acid phosphatase is then estimated directly by its enzymatic activity. With this assay as little as 1 ng. per 0.1 mI. of prostatic acid phosphatase can be detected.

BONE MARROW ACID PHOSPHATASE The search [or improved and earlier staging in prostatic carcinoma has led several investigators to identify tumor cells in bone marrow and recently to determine the acid phosphatase level. Gutman and associates'" in 1936 showed an increase in the acid phosphatase concentration at the site of metastasis; they believed that tumor cells produce acid phosphatase similar to that of normal prostatic tissue. In 1949 Alyea and Rundles'" studied bone marrow aspirates in 32 cases of carcinoma of the prostate and found metastases in 18 cases. The sternum was the site of metastasis in 15 cases, the ilium in two, and the vertebral body in one. In 1952 Clifton and associates 82 demonstrated in random biopsy specimens that carcinoma cells from the prostate are found in equal frequency in the sternum and

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ilium . In 1963 Flocks 83 noted approximately 10 per cent positive bone marrow cytologic findings in prostatic malignant tumor. In 1969 Chua and associates'" studied 70 patients with documented cancer of the prostate by means of random bone marrow biopsy of the posterior iliac spine or selective biopsy of suspicious areas in the skeletal survey. Of these, 32 patients had radiographic findings diagnostic of bone metastasis. Nineteen of 32 patients underwent selective marrow biopsy, and bone marrow metastasis was confirmed in 18. The 13 random biopsy specimens in these 32 patients showed carcinoma cells in the bone marrow in eight patients. In three of 27 patients the positive random or verte br al bone marrow biopsy specimen was the only evidence of extension of disease. In reviewing 556 bone marrow aspirations performed on 449 patients within a 10 year period, Nelson and associates'" found an overall 7.6 per cent rate of positive cytologic studies . The authors concluded that the low yield in routine cytology made this technique one of questionable value in evaluating patients with carcinoma of the prostate. Chua and associates" were the first to indicate the potential value of measuring acid phosphatase in bone marrow. In 12 patients with benign prostatic hypertrophy the levels were normal in serum and bone marrow fluid. Eight of 12 patients with clinically localized carcinoma of the prostate (stages I and II) also had normal serum and bone marrow acid phosphatase levels. Four patients in the group had elevated bone marrow acid phosphatase levels with normal serum acid phosphatase levels and skeletal surveys negative for metastasis. Three of these four patients had negative random biopsy studies, but one patient had positive biopsy findings . In patients with extensive disease there was a high correlation between serum and bone marrow acid phosphatase levels. The method utilized in this study was a modification of the Bodansky technique with sodium {3glycerophosphate as a substrate. Woodard'" believed that the acid phosphatase elaborated by bone was relatively inactive to this substrate. Pontes.j" Kabler.f" Veenerna," and

Bruce? ' and their associates presented data supporting the value of this procedure as an early indicator of metastatic prostate carcinoma. But reservations about the value of this procedure have been expressed by Cittes." Khan;" and Boehme." who found a large number of falsely positive results in patients with nonprostatic disease. Boehme et al,94 showed that the levels of bone marrow acid phosphatase varied predictably with the aspiration technique used. In brief, it was difficult to accept the value of a single chemical determination in the absen ce of histologic proof of metastatic disease. Also assays for acid phosphatase determination were not specific for prostatic acid phosphatase. With the advent of immunologic methods for specific determination of prostatic acid pho sphatase, a renewed interest in bone marrow acid phosphatase has developed. Pontes et al. 95 analyzed 50 bone marrow samples collected at random from their hematology service. The samples were assayed fo r acid phosphatase content by a standard colorimetric method (Roy) and by two immunochemical assays : radioimmunoassay and counterimmunoelectrophoresis. They found a high percentage (61 per cent) of falsely positive results in patients with various hematologic diseases without evidence of prostatic carcinoma by the colorimetric evaluation. In all these patients except one there were negative immunochemical assays. Our laboratory studied the bone ma rrow acid phosphatase with the Roy and counterimmunoelectrophoresis methods in over 100 cases of benign prostatic hypertrophy and in various stages of prostatic carcinoma. We also found a high percentage (74 pe r cent) of falsely positi ve results by the colorimetric method. Moncure et al.96 employed radial immunodiffusion to compare prostatic acid phosphatase levels in serum and bone maITOW. In their experience, levels of prostatic acid phosphatase in a patient's marrow higher th an concurrent serum levels indicate stage IV disease and usually correlate with the demonstration of tumor cells in the marrow. In only one female patient with chronic myelogenous leukemia was there a questionable positive marrow reaction.

ACID PHOSPHATASE-RoMAS

A very recent report by Drucker et a1.9 7 emphasized the importance of immunologic testing (radial immunodiffusion) for staging prostatic carcinoma. When the bone surveys were negative, the immunodiffusion was positive in 45 per cent of the patients studied. In the same group of patients the serum acid phosphatase level was elevated in 18 per cent and bone marrow aspirates were positive in 16 per cent. In patients with negative isotope scans 16 per cent were positive by the radial immunodiffusion method for detecting prostatic acid phosphatase. Thus immunodiffusion may be a more accurate means of identifying metastatic disease than bone surveyor isotope scan. Belville et a1.98 utilized a double antibody radioimmunoassay to measure prostatic acid phosphatase in bone marrow aspirates. An upper limit of the prostatic acid phosphatase level in bone marrow in patients with benign prostatic hypertrophy was chosen as 12.0 ng. per ml. Eighty per cent of the patients with proven stage Dz disease had levels elevated above 12.0 ng. per ml. In stages C and D 1 the values were 18 and 25 per cent, respectively. In stages A and B only one value exceeded this upper limit. The experience of these investigators with the use of enzymatic method showed a poor correlation with the presence or absence of prostatic adenocarcinoma. A limited clinical study by Cooper et a1.99 of the use of radioimmunoassay for bone marrow acid phosphatase indicated that the technique was technically superior to the p-nitrophenyl phosphate enzymatic method in bone marrow and serum samples. Sadlowski'?" studied 47 patients with early stage prostatic carcinoma with pelvic lymphadenectomy and bone marrow acid phosphatase determinations. Thirteen (28 per cent) had tumor in the pelvic lymph nodes, and in no instance was the bone marrow acid phos phatase level elevated more than the normal value for serum by the use of phenylphosphate as a substrate. The recent development of immunologic methods for detecting prostatic acid phosphatase has generated a new enthusiasm for this oldest of tumor

ET AL.

markers. Romas and Tannenbaum 101 and Fink and Galen 10z have recently written critiques on these methods. It is hoped that further studies from several laboratories will better define the most reprod ucible method as well as the limitations of this new testing methodology.

REFERENCES* 7. Gutman, A. B.: The development of the add phosphatase test for prostatic carcinoma. Bull. N. Y. Acad. Med., 44:63, 1968. 10. Gutman, A. B., and Gutman, E. B.: An "acid" phosphatase occurring in the serum of patients with metastasizing carcinoma of the prostate gland. J. Clin. Irwest., 17:473, 1938. 11. Robinson, J. N., Gutman, E. B., and Gutrnan, A. B.: Clinical significance of increased serum "acid" phosphatase in patients with bone metastases secondary to prostatic carcinoma.]. Urol.,42:602, 1939. 12. Gutman, A. B.: Add phosphatase in patients with carcinoma of the prostate gland: present status. .l.A.M.A., 120: 1112, 1942. 14. Huggins, C., and Hodges. C. V.: Studies on prostatic cancer. 1. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res., 1 :293, 1941. 15. Sodernan, T. M. and Batsakis, J. GO' Acid phosphatase. In Tannenbaum. M. (Editor): Urologic Pathology: The Prostate. Philadelphia. Lea & Febiger, 1977, p. 129. 16. Batsakis, J. G., et al.: Diagnostic Enzymology. Chicago, American Society of Clinical Pathologists, 1970. 17. Abul-Fadl, M. A. M., and King, E. J.: Properties of the acid phosphatases of erythrocytes and of the human prostate gland. Biochem. J.,45:51, 1949. 22. Shuman, S., et al.: The detection of prostatic acid phosphatase by antibody reactions in gel diffusion. Immunology, 93:474, 1964. 23. Moncure, C. W., et al.: Immunological and histochemical evaluation of marrow aspirates in patients with prostatic carcinoma. ]. Ural., 108:609, 1972. 24. Choe, B. K., et al.: Immunochernical studies of prostatic acid phosphatase., Cancer Treat. Rep., 61 :2, 1977. 25. Foti, A. G., et al.: Detection of prostatic cancer by solid-phase radioimmunoassay of serum prostatic acid phosphatase. N. EngL J. Med.,

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*A complete list of references can be obtained from the authors.

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26. Chu, T. M.. et al.: Immunochemical detection of serum prostatic acid phosphatase. Invest. Ural., 15:319, 1978. ~7. Romas, ~. A.. et aI.: Ccunterimmunoelectrophoresis for detection of human prostatic acid phosphatase. Urology, 12:79-83, 1978. 3!. Moncure, C. W.: Isoenzymes in prostatic carcinoma. In Tannenbaum, M. (Editor): Urologic Pathology: The Prostate. Philadelphia, Lea & Febiger, 1977, p. 141. 32. Yam, L. T.: Clinical significance of the human acid phosphatase. Amer. J. Med., 56:605, 1974. 33. Li, C. Y., et al.: Acid phosphatases in human plasma, J, Lab. Clin. Med., 82:446, 1973, 37. Ostrowski, W., et aI.: 'The role of neuraminic acid in the heterogeneity of acid phosphomonoesterase from human prostate gland. Biochim. Biophys. Acta, 221:~97, 1970. 38. Chu, 'T, M., et aI.: Tumor antigen and acid phosphatase isoenzyme in prostatic cancer. Cancer Chemother, Rep., 59:97, 1975. 41. Moncure, C. W., et al.: Prostatic acid phosphatase antisera. Invest. Urol., 5:331,1968. 42. Moncure, C. W., and Prout, G. R., Jr.,: Antigenicity of human prostatic acid phosphatase. Cancer, 25:463, 1970. 45. Milisauskas, V., and Rose, N. R.: Irnmunochemical quantitation of prostatic phosphatase. Clin. Chern., 18: 1529, 1972. 47. Ostrowski, W. and Rybarska, J., Studies on human prostatic acid phosphomonoesterase. Further purification and molecular weight of the enzyme. Biochim. Biophys, Acta, 105:196,1965. 49. Lam, K. W., et al.: Biochemical properties of human prostatic acid phosphatase. CHn. Chem., 19:483,1973. 5!. Vihko, P., Kontturi, M., and Korhonen, L. K.: Purification of human prostatic acid phosphatase by affinity chromatography and isoelectric focusing. Part 1. Clin. Chern" 24 :466, 1978. 52. Choe, B. K., et aI.: Purification of human prostatic acid phosphatase preparative. Biochemistry, 8:73, 1978. 63. Khan, A. N., et aI.: The effect of routine digital examination of the prostate on serum acid phosphatase. Br. J. UroI., 10:349,1978. 64. Silber, 1., et al.: The incidence of elevated acid phosphatase in prostatic infarction. J. Uro!., 103:765, 1970. 67. Fori, A. G., et al.: A solid-phase radioimmunoassay for human prostatic acid phosphatase. Cancer Res., 35:2446, 1975. 70. Carroll, B. J.: Radioimmunoassay of prostatic acid phosphatase in carcinoma of the prostate (correspondence). New Engl. J. Med., 298:912,1978. 71. Choe, B. K., et al.: Human prostatic acid phosphatase. II. A double-antibody radioimmunoassay. Arch. Andro!., 1:227,1978. 72. Murphy, G. P., et al.: A national field trial of two new, direct and specific methods of acid phosphatase determination. Paper read at annual meeting, American Urological Association, Washington, D.C., May 21-25, 1978. 73. McDonald, 1., et a!': Human prostatic add

74.

77. 78. 79. 80.

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101. 102.

phosphatases. UI. Counterimmunoelectrophoresis for rapid identification. Arch. Andra!., 1:235, 1978. Lee, L. T., et al.: Quantitative precipitin analysis of influenza virus host antigen and of sulfated mucopolysaccharides of chicken embryonic allantoic fluid. J. Immunol., 102:1144,1969. Choe, B. K., et al.: Expression of human prostatic acid phosphatase in pancreatic islet cell carcinoma. Invest. Urol., 15:812, 1978. Drucker, J. R., et al.: Immunologic staging of prostatic carcinoma: three years of experience.J. Urol., 119:94,1978. Choe, B. K., et al.: Immunoenzyme assay for human prostatic acid phosphatase. Clin. Chern. (In press.) Gutman, E. B., Sproul, E. E., and Gutman, A. B.: Significance of increased phosphatase activity of bone at the site of osteoblastic metastasis secondary to carcinoma of the prostate gland. Amer. J. Cancer, 28:485, 1936. Chua, D. T., et al.: Acid phosphatase levels in bone marrow: value in detecting early bone metastasis from carcinoma of the prostate. J. UroL, 103 :462, 1970. Pontes, J. E., et al.: Bone marrow acid phosphatase in staging prostatic carcinoma. J. U 1'01., 114:422,1975. Veenerna, R. j., et al.: Bone marrow acid phosphatase: prognostic value in patients undergoing radical prostatectomy. J. Urol., 117:81,1977. Bruce, A. W., et al.: Carcinoma of the prostate: a critical look at staging. J. Urol., 117:319, 1977. Pontes, j. E., et al.: Bone marrow acid phosphatase in staging of prostatic cancer: How reliable is it? j. Urol., 119:772, 1978. Moncure, C. W., et al.: Immunological and histochemical evaluation of marrow aspirates in patients with prostatic carcinoma. J. UroL, 108:609, 1972. Drucker, J. R., et al.: Immunologic staging of prostatic carcinoma: three years of experience. J. Urol., 119 :94, 1978. Belville, W. D., et al.: Bone marrow acid phosphatase by radioimmunoassay. Cancer, 41:2286,1978. Cooper, J. F., et al.: Radioimmunochernical measurement of bone marrow prostatic acid phosphatase. J. Urol., 119 :392, 1978. Sadlowski, R. W.: Early stage prostatic cancer investigated by pelvic lymph node biopsy and bone marrow acid phosphatase. J. Urol., 119:89, 1978. Romas, N. A., and Tannenbaum, M.: Immunologic detection of prostatic acid phosphatase: critique 1. Hum. Pathol., 9:620, 1978. Fink, D. J., and Galen, R. S.: Immunologic detection of prostatic acid phosphatase: critique II. Hum. Pathol., 9:621,1978.

Department of Urology Columbia University College of Physicians and Surgeons 630 West 168th Street New York, New York !0032 (Dr. Tannenbaum)