US3002893A

US3002893A - Method for the determination of serum acid phosphatase and diagnostic preparation therefor

Info

Publication number: US3002893A
Application number: US773571A
Authority: US
Inventors: Arthur L Babson
Original assignee: Warner Lambert Pharmaceutical Co
Current assignee: Warner Lambert Co LLC
Priority date: 1958-11-13
Filing date: 1958-11-13
Publication date: 1961-10-03
Anticipated expiration: 1978-10-03

Description

United States Patent METHOD FOR THE DETERMINATION OF SERUM ACID PHOSPHATASE AND DIAGNOSTIC PREP- ARATION THEREFOR Arthur L. Babson, Morris Plains, NJ., assignor to Warher-Lambert Pharmaceutical Company, Morris Plains, NJ., a corporation of Delaware No Drawing. Filed Nov. 13, 1958, Ser. No. 773,571

13 Claims. (Cl. 195103.5)

This invention relates to a new and improved process for the determination of serum acid phosphatase and relates more particularly to a stable composition for use in a simplified semi-quantitative determination of acid phosphatase in serum.

Acid phosphatase is an enzyme which has the ability to hydrolyze phosphate esters in an acid medium (pH 5). This enzyme is present in the prostate gland of humans and, in cases of cancer of the prostate, the enzyme is released with the result that the serum acid phosphatase level increases several fold above normal values. For this reason a reliable test for serum acid phosphatase activity levels is of great value as a diagnostic aid in detecting cancer of the prostate. A method for the determination of acid phosphatase in serum was developed by Shinowara et a1. and reported in the Journal of Biological Chemistry, vol. 142, page 921 (1942). This method employs a bufiered glycerophosphate solution and depends on the release of phosphate ion in the serum to be tested as a result of activity of the acid phosphatase enzyme. The released phosphate ion is determined bya colorimetric procedure which relies upon the conversion of the phosphate ion to a colored phosphomolbdate complex.

This method has several disadvantages which render it generally unacceptable as a rapid diagnostic test unless the test is carried out by trained personnel under carefully controlled conditions. The volume of the serum and of all test reagents must be measured accurately and at least two (2) ml. of serum is required. In addition, an incubation period of one hour is required. Also, the effectiveness of this test depends upon a measurement of the released phosphate ion which is formed by the activity of the acid phosphatase enzyme on the buttered glycerophosphate solution. However, serum normally contains an appreciable quantity of phosphate ion and the phosphate ion released from the glycero phosphate by the action of the enzyme will, in most cases, be only a small proportion of the phosphate ion normally occurring in the serum. For this reason, it is necessary to carry out a blank determination on the serum without the addition of buffered glycerophosphate in order to determine the phosphate ion normally present in the serum and the acid phosphatase enzyme activity is then determined by observing the dilference in color between the blank determination and the determination made with the addition of buttered glycerophosphate. Since 'any colorimetric determination is subject to certain inaccuracies, it is apparent that this method must be carried out with great care and precision to obtain any worthwhile and dependable results.

It is an object of this invention to provide a method of determining serum acid phosphatase which is capable of yielding reliable results without any necessity for highly skilled laboratory technicians.

It is a further object of the invention to provide a method for determining serum acid phosphatase where only a small volume of serum is required and where the need for precise and time-consuming measurements is eliminated.

It is a still further object of the invention to provide a substrate for use in the determination of serum acid phosphatase which is specific to acid phosphatase released by the prostate gland. Other objects and the advantages of the invention will appear hereinafter.

In accordance with the present invention, serum acid phosphatase is determined in a small volume of serum by a process in which the serum is incubated with a substrate comprising a solid preparation consisting of a metal salt of a phenolic phosphate ester and a buffer, and after incubation, an azonium salt of an aromatic amine containing from 1 to 2-NH groups per molecule is added and the color which then develops is compared with a standard calibrated color chart, the depth of color observed being a measure of the prostatic acid phosphatase activity in the serum being tested.

In accordance with a preferred embodiment of this invention, I employ a substrate comprising a buffer and a salt of ot-naphthyl phosphate. This substrate has been found to be specific to serum acid phosphatase released by the prostate gland and when test serum is incubated with this substrate and the azonium salt added, prostatic acid phosphatase is readily determined.

In the method of this invention, the acid phosphatase enzyme present in the serum hydrolyzes the substrate Which liberates phosphate ion and free phenolic compound, which then couples with the azonium salt to form an azo dye. Since the procedure does not depend on the colorimetric determination of liberated phosphate ion, it is not necessary to run a blank on each sample of serum tested to determine the phosphate ion normally present. Serum does not contain any free phenolic compound, so that any color developed during the test must be due solely to activity of acid phosphatase. The simplicity of the method of the invention eliminates the need for expensive equipment and highly trained technical personnel to perform the test. The simplicity and rapidity of the procedure renders the test valuable for the screening of serum acid phosphatase levels in large population groups and furnishes a convenient and inexpensive means for early diagnosis of cancer of the prostate.

The acid phosphatase enzyme which is present in the prostate gland is prostatic acid phosphatase which diifers from the acid phosphatase normally present in red blood cells, which is erythrocytic acid phosphatase. In cases where cancer of the prostate exists, prostatic acid phosphatase is released into the serum which will not normally contain any erythrocytic acid phosphatase. There is, however, always a possibility that as a result of hemolysis of the blood sample taken, some erythrocytic acid phosphatase may be released into the serum and the presence of this enzyme will give a false prostatic acid phosphatase reading. It would be desirable to have a test which is specific to prostatic acid phosphatase to eliminate the possibility of misleading results should the serum tested contain any erythrocytic acid phosphatase.

It has been found that the metal salts of u-naphthyl phosphate, for example, sodium a-naphthyl phospate, are specific in their property of being hydrolyzed far more rapidly by prostatic acid phosphatase than by erythrocytic acid phosphatase. It is for this reason that the salts of a-naphthyl phosphate are preferred ingredients in the substrate since any acid phosphatase activity determined is that of prostatic acid phosphatase. The use of this reagent eliminates the possibility that contamination of the sample with erythrocytic acid phosphatase will cause misleading results.

The preferred substrate also contains a buffer soluble in serum in an amount sufficient to maintain in the serum a pH of about 4.5 to 5.5 which is the optimum pH for acid phosphatase enzyme activity. In order to obtain reproducible results it is necessary that this pH be maintained in the serum throughout the entire test procedure. Citrate butters based on citric acid are particularly cffective, such as a mixture of citric acid and trisodium citrate, ,a mixture of disodium citrate and trisodium citrate, or disodium citrate alone. Salts of oxalic acid or malic acid can also be used as buffers. Salts of tartaric and phosphoric acids interfere with the acid phosphatase induced hydrolysis and should be avoided.

The quantities of the a-naphthyl phosphate salt and the buffer in the substrate can be varied within wide limits provided that there is an excess of the salt present. In addition there should be suflicient buffer so that the pH can be maintained in the critical range of about 4.5 to 5.5 throughout the incubation period. The presence in the substrate of about 0.4 to about 1 mg. of a-naphthyl phosphate salt and about to about 25 mg. buffer is a particularly effective range and will normally provide an excess of salt and effective buffering with the normal volumes of serum tested.

The substrate can be in the form of a liquid, with the v a-naphthyl phosphate salt and buffer in solution in a solvent such as water. It is preferred that the substrate be formulated as a solid mixture ofa-naphthyl phosphate salt and buffer which can be added to a small volume of serum in carrying out the test without the necessity for time-consuming measurements of liquid volumes. A substrate in the form of a tablet is a particularly convenient form. In preparing the tablet, the active ingredients comprising the a-naphthyl phosphate salt and the bufier are mixed with conventional tableting excipients including fillers such as lactose, sucrose and the like together with lubricants such as leucine, Carbowax-6000 and the like. Carbowax-6000 is a solid polyethylene glycol having a molecular weight of about 6000 and a melting point of about 60 C. produced by Carbide and Carbon Chemicals, South Charleston, West Virginia. Although salts of a-naphthyl phosphate are preferred, other phenolic phosphate ester salts can be used to form a solid substrate for use in the invention. Thus, the alkail and alkaline earth metal salts of a phosphoric acid ester of a hydroxy-substituted aromatic compound selected from the group consisting of benzene and naphthalene can be used. Suitable salts include the salts of sodium, potassium, lithium, barium calcium and magnesium, for example, but the alkali metal salts are preferred because of their more ready solubility in serum. The phosphoric acid esters of aromatic hydroxy compounds such as anaphthol, fl-naphthol, ortho-, metaand p-cresol, phenol, catechol, resorcinol, hydroquinone and the like have been found to be quite useful.

The formulation of a phenolic phosphate ester salt such as described above with a bulfer into a solid substrate is particularly effective in carrying out the test in accordance with this invention since no measurements of liquid volumes need be made. Where a salt of a phosphoric acid ester of a hydroxy substituted aromatic compound other than u-naphthol is used to form a solid substrate, care must be taken that no hemolysis of the blood sample to be tested takes place, since the phenolic phosphate ester salts other than a-naphthyl phosphate salts are hydrolyzed by erythrocytic acid phosphatase.

In carrying out the actual diagnostic test, a mixture of serum and substrate is incubated for a predetermined or standard time period causing the phosphate ester to be split by the activity of the acid phosphatase enzyme thus releasing the hydroxy substituted aromatic compound forming the ester. In the case of the preferred a-naphthyl phosphate salt, of course, a-naphthol is released. The incubation period employed will vary with the temperature. At a temperature of 65 F. the incubation period should be about 25 minutes; at 72 F. about minutes, at 86 F. about 15 minutes and at 98 F. about 12 minutes.

The incubation need not be carried out in complex temperature controlled baths, but can be carried out at normal room temperature with the time being adjusted depending on the temperature of the room.

At the conclusion of the incubation period, the colorforming ingredient which couples with the released anaphthol or other hydroxy substituted aromatic compound is added. Any of the well-known azonium salts useful in the manufacture of commercial dyes can be used, such as the azonium salts of aniline, o-dianisidine, 4-benzoylamino-2,5-dimethoxyaniline, 4-amino-2,5-diethoxybenzanilide, nitrobenzene-azo 2,5 dimethoxyaniline, 2-amino-4-chloroanisole, m-nitroaniline and the like. It has been found that tetrazotizcd o-dianisidine (Naphthanil Diazo Blue B) is particularly effective since with this material an unusually deep color is obtained with very small amounts of hydroxy aromatic compound. Normally about 0.2 to about 0.6 mg. of the azonium salt is added to the serum and substrate after completion of the incubation period. This component is preferably added in the form of a solid tablet prepared by mixing the azonium salt with a filler such as sucrose, lactose and the like and a lubricant followed by tableting in the conventional manner. For ease of handling, it is generally desirable that sutficient inert material be incorporated to produce a tablet weighing at least 15 mg.

After the addition of the azonium salt, the color is allowed to develop for a fixed period of time and then compared with a calibrated color standard. The period allowed for color development is not critical, but it should preferably be the same as that used in the preparation of the standards. Normally a time in the range of 2 to 4 minutes is preferred.

In the test procedure of the present invention, serum containing normal amounts of acid phosphatase will show a light yellow-brown color when the test is carried out using sodium a-naphthyl phosphate and tetrazotized odianisidine. Markedly elevated levels of serum acid phosphatase which indicate a suspicion of cancer of the prosstate show a color of deep reddish purple. These differences in color are easily determined by even unskilled people with the result that the method of the invention affords a convenient and effective diagnostic tool.

In order further to illustrate this invention the following examples are given:

Example I 0.77 gram sodium a-naphthyl phosphate, 5.32 grams citric acid monohydrate, 13.91 grams trisodium citrate dihydrate, 0.4 gram leucine and 0.6 gram Carbowax-6000 are well blended and formed into 21 milligram tablets, each containing 0.77 milligram sodium a-naphthyl phosphate and 19.23 milligrams citrate buffer.

Example II 0.67 gram sodium wnaphthyl phosphate, 10 grams disodium citrate, 8.3 grams trisodium citrate dihydrate, 6 grams lactose and 2 grams leucine are thoroughly blended and formed into 27 milligram tablets, each tablet containing 0.67 milligram sodium a-naphthyl phosphate and 18.33 milligrams citrate buffer.

Example III 0.4 gram tetrazotized o-dianisidine, 4.2 grams of sucrose and 0.25 gram Carbowax-6000 are blended and formed into 5 milligram tablets, each tablet containing 0.4 milligram tetrazotized o-dianisidine.

Example IV 0.4 gram tetrazotized o-dianisidine, 18.6 grams sucrose and 1 gram Carbowax-6000 were blended and formed into 20 milligram tablets, each tablet containing 0.4 milligram tetrazotized o-dianisidine.

The tablets obtained by the procedures described in Examples I and II are added before incubation to the serum whose acid phosphatase activity is to be determined. Tablets prepared in accordance with Examples III and IV are added to the incubated serum to develop the color, the depth of which is a measure of the acid phosphatase contained in the serum being tested.

Example V To approximately 0.2 millimeter of serum is added 1 tablet prepared in accordance with Example II. The tablet is broken up with a glass rod and the mixture is incubated at 72 F. for 20 minutes. To the incubated serum and substrate is then added a tablet prepared in accordance with Example IV, the tablet being broken up with a glass rod and stirred into the serum. After exactly 3 minutes, the developed color is matched against a standard color chart which has previously been prepared from serum containing known amounts of acid phosphatase enzyme and an accurate reading of the acid phosphatase activity of the test serum is thus conveniently obtained.

Any-departure from the foregoing description which conforms to the present invention is intended to be included within the scope of the claims.

I claim:

1. A substrate for use in the determination of prostatic acid phosphatase in serum which comprises a buffer adapted to maintain the serum pH within about 4.5 to about 5.5 and a salt of a-naphthyl phosphate.

2. A substrate according to claim 1, which comprises about to about 25 milligrams of said bufier and about 0.4 to about 1 milligram of said salt.

3. A substrate according to claim 2 wherein said bufier comprises salts of acids selectd from the group consisting of citric acid, oxalic acid and malic acid and said salt is an alkali metal salt.

4. A substrate according to claim 3 wherein said salt is a sodium salt.

5. A dry preparation for use in the determination of prostatic acid phosphatase in serum which comprises about 0.4 to about 1 milligram of a salt of u-naphthyl phosphate and about 10 to about 25 milligrams of a buffer adapted to maintain the pH of the serum within about 4.5 to about 5.5.

6. A dry preparation for use in the determination of serum acid phosphatase comprising about 10 to about 25 milligrams of a butler adapted to maintain the serum pH .vithin about 4.5 to about 5.5, said buffer comprising salts of acids selected from the group consisting of citric acid, oxalic acid and malic acid, and about 0.4 to about 1 milligram of a metal salt of a phenolic phosphate ester, said metal being selected from the group consisting of the alkali and alkaline earth metals and said ester being the phosphoric acid ester of a hydroxy-substituted aromatic compound selected from the group consisting of benzene and naphthalene.

7. A method for the determination of prostatic acid phosphatase in serum which comprises incubating a small volume of serum in a substrate comprising a buffer adapted to maintain the pH of said serum within about 4.5 to about 5.5 and a salt of a-naphthyl phosphate, adding to said incubated mixture an azonium salt of an aromatic amine and comparing the developed color to the color developed in serum in the presence of a known concentration of prostatic acid phosphatase.

8. A method for the determination of prostatic acid phosphatase in serum which comprises adding to a small volume of serum a substrate comprising about 10 to about 25 milligrams of a buffer adapted to maintain the pH of said serum within about 4.5 to about 5.5, said bufier comprising salts of acids selected from the group consisting of citric acid, oxalic acid and malic acid, and about 0.4 to about 1 milligram of an alkali metal salt of a-naphthyl phosphate, incubating the mixture of said serum and said substrate at a temperature of about 65 F. to 98 F. for about 25 to about 12 minutes to permit the prostatic acid phopshatase enzyme present to hydrolyze said a-naphthyl phosphate, adding to said incubated mixture an azonium salt of an aromatic amine containing 1 to 3 NI-I, groups per molecule and comparing the developed color to the color developed in serum in the presence of a known concentration'of prostatic acid phosphatase.

9. A method according to claim 8 wherein said bufier comprises salts of citric acid, said alkali metal salt of a-naphthyl phosphate is a sodium salt and said azonium salt is tetrazotized o-dianisidine.

10. A method for the determination of prostatic acid phosphatase in serum which comprises adding to a small volume of serum a first dry preparation comprising about 10 to about 25 milligrams of a citrate buffer adapted to maintain the pH of said serum within about 4.5 to about 5.5 and about 0.4 to about 1 milligram of an alkali metal salt of e-naphthyl phosphate, incubating the mixture of said serum and said substrate at a temperature of about 65 F. to about 98 F. for about 25 to about 12 minutes to permit the prostatic acid phosphatase enzyme present to hydrolyze said ot-naphthyl phosphate, adding to said incubated mixture a second dry preparation comprising about 0.2 to about 0.6 milligram of tetrazotized o-dianisidine and comparing the developed color to the color developed in serum in the presence of a known concentration of prostatic acid phosphatase.

11. A method for the determination of serum acid phosphatase which comprises adding to a small volume of serum a first dry preparation comprising a buffer adapted to maintain the pH of said scrum within about 4.5 to about 5.5 and a metal salt of a phenolic phosphate ester, said metal being selected from the group consisting of alkali and alkaline earth metals and said ester being a phosphoric acid ester of a hydroxy substituted aromatic compound selected from the group consisting of benzene and naphthalene, incubating the mixture to permit the acid phosphatase enzyme present to hydrolyze said phos phate ester, adding to said incubated mixture at second dry preparation comprising an azonium salt of an aromatic amine containing from 1 to 2 -NH, groups per molecule and comparing the developed color to the color developed in serum in the presence of a known concentration of acid phosphatase.

12. A method for the determination of serum acid phosphatase which comprises adding to a small volume of serum a first tablet comprising about 10 to about 25 milligrams of a bufier comprising salts of acids selected from the group consisting of citric acid, oxalic acid and malic acid adapted to maintain the pH of said serum within about 4.5 to about 5.5 and about 0.4 to about 1 milligram of an alkali metal salt of a phosphoric acid ester of a hydroxy substituted aromatic compound selected from the group consisting of benzene and naphthalene, incubating the mixture of said serum and said first tablet at a temperature between 65 F. and 98 F. for about 25 to about 12 minutes to permit the acid phosphatase enzyme present to hydrolyze said ester, adding to said incubated mixture a second tablet comprising about 0.2 to about 0.6 milligram of an azonium salt of an aromatic amine containing from 1 to 2 -NH; groups per molecule, and comparing the developed color with the color developed in serum in the presence of a known concentration of acid phosphatase.

13. A method according to claim 12 wherein said bufier is a citrate butter and said azonium salt is tetrazotized o-dianisidine.

Organic Chemistry," Fieser and Fieser, D. C. Heath & 00., 1944, Pp. 859868.

Claims

1. A SUBSTRATE FOR USE IN THE DETERMINATION OF PHOSTATIC ACID PHOSPHATASE IN SERUM WHICH COMPRISES A BUFFER ADAPTED TO MAINTAIN THE SERUM PH WITHIN ABOUT 4.5 TO ABOUT 5.5 AND A SALT OF A-NAPHTHYL PHOSPHATE.

7. A METHOD FOR THE DETERMINATION OF PORSTATIC ACID PHOSPHATASE IN SERUM WHICH COMPRISES INCUBATING A SMALL VOLUME OF SERUM IN A SUBSTRATE COMPRISING A BUFFER ADAPTED TO MAINTAIN THE PH OF SAID SERUM WITHIN ABOUT 4.5 TO ABOUT 5.5 AND A SALT OF A-NAPHTHYL PHOSPHATE, ADDING TO SAID INCUBATED MIXTURE AN AZONIUM SALT OF AN AROMATIC AMINE AND COMPARING THE DEVELOPED COLOR TO THE COLOR DEVELOPED IN SERUM IN THE PRESENCE OF A KNOWN CONCENTRATION OF PROSTATIC ACID PHOSPHATASE.