Prostate isoenzymes

Acetoacetic acid,p-aminosalicylic acid, gentisic acid, pansporin, and bilirubin react with diazo dye Fast Red TR Salt. The addition of sodium tartrate to a final concentration of 0.05 mol/1 (pH S.S.) inhibits the prostatic isoenzyme of acid phosphatase and permits the measurement of the result due primarily to these interferents. AcP in samples suspected to contain these interferents may be analyzed before and after treatment with sodium tartrate to get the AcP and Blank results, respectively. 

Most of the substrates that have been used in measuring alkaline phosphatase activity have also been used to measure acid phosphatase, e.g. p-nitrophenylphosphate, phenylphosphate and a-naphthyl phosphate. In most cases of acid phosphatase estimation, it is the level of the prostatic phosphatase which needs to be known, and so specific inhibitors are included in the reaction mixtures. The prostatic isoenzyme is strongly inhibited by tartrate and so in many methods it is tartrate-labile acid phosphatase which is measured. On the pther hand, the red cell enzyme which contributes significantly to the total serum activity is inhibited by formaldehyde and cupric ions. Many laboratories therefore measure formaldehyde-stable acid phosphatase as an indication of prostatic acid phosphatase. 

A term which is synonymous with prostatic acid phosphatase. The total acid phosphatase activity in serum is derived from a number of different sites, e.g. the red blood cell, bone and prostate. The prostatic isoenzyme, unlike the red cell enzyme, is stable in the presence of formaldehyde. This property is used to measure the prostatic isoenzyme. 

Pharmacology Dutasteride inhibits the conversion of testosterone to 5 -dihydrotestosterone (DHT), the androgen primarily responsible for the initial development and subsequent enlargement of the prostate gland. Testosterone is converted to DHT by the enzyme 5 -reductase, which exists as 2 isoforms, type 1 and type 2. The type 2 isoenzyme is primarily active in the reproductive tissues while the type 1 isoenzyme is also responsible for testosterone conversion in the skin and liver. 

Cornford, P, Evans J, Dodson A, Parsons K, Woolfenden A, Neoptolemos J, Foster CS (1999) Protein kinase C isoenzyme patterns characteristically modulated in early prostate cancer. Am J. Pathol 154 137-144... 

Males with deficiency of the 5a-reductase isoenzyme do not develop acne, male pattern baldness, or enlarged prostates.274 The last fact was some of the impetus for development of the steroid 5a-reductase inhibitor finasteride, which is widely used to treat benign prostate enlargement 274/277/278 It is an enzyme-activated inhibitor in which the NADH reduces the C= C bond in the A ring, which is not in the same position as in the substrate. The resulting anion cannot become protonated but instead adds to the NAD+ as shown in Eq. 22-15. 

Because the intermediary metabolism of various organs is virtually the same, organ-specific enzymes are very rare. One example usually cited is the acid phosphatase of the prostate. However, the enzyme complement of the various organs may differ with respect to relative activities of the enzymes, the time dependence of their appearance in plasma, and the pattern of their isoenzymes (see below). Table 5.2 presents a list of enzymes commonly used for organ- and disease-specific diagnoses. 

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. 

There is no information concerning the isoenzymatic composition of the purified prostatic phosphatases that were used in the preceding kinetic studies. Nor do there appear to be any kinetic studies on the individual isoenzymes. The possibility exists that substantial differences in kinetic characteristics, such as the value for Ki, for l-(4-)-tartrate. 

G6. Goldberg, A. F., Takakura, K., and Rosenthal, R. L., Electrophoretic separation of serum acid phosphatase isoenzymes in Gaucher s disease, prostatic carcinoma and multiple myeloma. Nature London) 211, 41-43 (1966). 

Lactate dehydrogenase (human isoenzyme) Prostatic acid phosphatase (hmnan prostate) Alanine aminotransferase (pig heart) r a-Amylase (human pancreas)... 

Continuous-monitoring methods for assay of TR-ACP activity are based on the principle introduced by Hillmann in which a-naphthoi released from its phosphate ester forms a colored product with the stabilized diazonium salt of 2-amino-5-chlorotoluene-1,5-naphthalene disulfonate (Fast Red TR). The introduction of alcohols, such as 1,5-pen-tanediol, accelerates the reaction and increases sensitivity by acting as phosphate acceptors in transfer reactions. The addition of sodium tartrate inhibits the sensitive isoenzymes (i.e., prostatic and lysosomal ACPs) if they are present in the sample. 

With few exceptions, an increase in the activity or mass of an enzyme or isoenzyme is not specific or sensitive enough to be used for identifying the type of cancer or the specific organ involvement. An exception is PSA. PSA has mild protease activity and amino acid sequence homology with serine protease of the kallikrein family.It is expressed by normal, benign, hyperplastic, and cancerous prostate glands and minimally by other tissue. Until the application of PSA as a marker for prostate cancer, tumor enzymes had lost most of their popularity for use as cancer markers. Enzymes were used historically as tumor markers before the discovery of oncofetal antigens and the advent of monoclonal antibodies. The abnormalities of enzymes as a marker for cancer are either the expression of the fetal form of the enzyme (isozyme) or the ectopic production of enzymes. 

Creatine kinase (CK) catalyzes the phosphorylation of creatine by adenosine triphosphate. CK is a dimer consisting of two subunits, M (muscle) and B (brain). There are three isoenzymes, CKl (BB), CK2 (MB), and CK3 (MM). CKl is present in the brain, prostate gland, gastrointestinal tract, lung, bladder, uterus, and placenta. Cardiac muscle has the highest concentration of CK2 (= 20%). CK3 is present in skeletal and cardiac muscles. 

Elevated levels of CKl have been demonstrated in prostate cancer and small cell carcinoma of the lung. Although it is also elevated in other malignancies, such as those of the breast, colon, ovary, and stomach, the clinical usefulness of CKl as a tumor marker requires further investigation. CK isoenzymes have been included in a prostate cancer panel, ProstAsure. ... 

The ProstAsure index, developed by Zhang, Stamey, and Chan, was designed to increase the sensitivity and specificity of cancer detection, while maintaining a reasonable false positive detection rate. The index is derived firom several values, including age, total PSA, creatine kinase isoenzymes, and prostatic acid phosphatase, which are input... 

Immunological methods for enzymes, more specifically isoenzymes, such as lactate dehydrogenase-1 (167, 168), mitochondrial aspartate aminotransferase (169), prostatic acid phosphatase (170, 171,172), and creatine kinase-MB (173, 174, 175), have been in use in the clinical laboratory for 10 years. However, the use of the immunological rather than catalytic properties of enzymes has not provided the opportunities for standardization that was anticipated a number of years ago (176, 177, 178). It is only within the last year that a working group on CK-MB mass assay was formed under the auspices of the Standards Committee of the American Association for Clinical Chemistry (AACC). The objective of this working group is to prepare a reference material to calibrate methods that are based on the principle of CK-MB mass measurement. 

Similar to finasteride, dutasteride is a competitive and mechanism-based inhibitor not only of type 2 but also of type 1 5a-reductase isoenzymes, with which stable enzyme-NADP adduct complexes are formed, inhibiting the conversion of testosterone to DHT (106). The suppression of both type 1 and type 2 isoforms results in greater and more consistent reduction of plasma DHT than that observed for finasteride (107,108,109). The more effective dual inhibition of type 1 and type 2 5a-reductase isoforms lowers circulating DHT to a greater extent than with finasteride and shows advantages in treating BPH and other disease states (e.g., prostate cancer) that are DHT-dependent. 

A minor isoenzyme of human prostatic acid phosphatase (pi 5.5) has been separated from the major isoenzyme (pi 4.9). Immunological similarities between the two enzymes were demonstrated. 

Determination of the acid tartrate-sensitive P. in serum is important in the diagnosis of prostate cancer, which is accompanied by a marked increase in the serum level of this enzyme. Measurement of alkaline P in serum is used in the diagnosis of bone disease, especially bone tumors, and diseases of the liver (hepatitis) and the gall bladder (e.g. obstructive jaundice), all of which result in a several fold increase in serum P. The isoenzymes of both acid and alkaline P. differ in their neuraminic acid contents. The most widely used substrate for P. assay is p-nitrophenyl phosphate the released p-nitrophenol can be determined directly by photometry (k , 405 nm). 

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