US3823071A - Prostatic acid phosphatase determination - Google Patents
Prostatic acid phosphatase determination Download PDFInfo
- Publication number
- US3823071A US3823071A US00100345A US10034570A US3823071A US 3823071 A US3823071 A US 3823071A US 00100345 A US00100345 A US 00100345A US 10034570 A US10034570 A US 10034570A US 3823071 A US3823071 A US 3823071A
- Authority
- US
- United States
- Prior art keywords
- acid phosphatase
- prostatic acid
- thymolphthalein
- prostatic
- monophosphate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/40—Triphenylmethane dye chromogens, e.g. fluorescein derivatives
Definitions
- This invention relates to the detection and quantitative determination of enzymes in biological fluids. It relates more especially to the detection and quantitative determination of prostatic acid phosphatase in blood serum and in other biological fluids.
- prostatic acid phosphatase in biological fluids is useful in the clinical diagnosis of prostatic carcinoma, a known symptom of which is an increase in the content of prostatic acid phosphatase in blood, urine and other biological fluids.
- prostatic acid phoshatase there are several substrates which are known to be responsive to the catalytic activity of prostatic acid phoshatase, those most generally recognized in this regard being phenol-phosphate, phenolphthalein monophosphate; alphanaphthyl-phosphate, p-nitro-phenol-phosphate, and beta-glycerophosphate.
- acid phosphates other than prostatic acid phosphatase which may occur in biological fluids. Those which generally are present in significant amounts in blood are erythrocytes from blood cells and platelet acid phosphatase.
- any of the substrates hereinabove mentioned is employed in an attempted diagnosis for prostatic acid phosphatase difliculties are encountered because of activity induced by other acid phosphatases which, if present, result in a falsepositive reaction.
- those having the greatest specificity for prostatic acid phosphatase are beta-glycerophosphate and alpha-naphthylphosphate, but even these are deficient in specificity and While such deficiency to substantial extent may be lessened by resort to the technique of comparative results with the aid of known inhibitors such as tartrate or formalin, such techniques are relatively cumbersome.
- the test procedure using beta-glycerophosphate is a matter of considerable difficulty.
- the aforesaid objects have been obtained by the utilization of the sodium salt of thymolphthalein monophosphate as the substrate for prostatic acid phosphatase.
- the sodium salt is preferred, other salts of thymolphthalein monophosphate may be employed, namely the salts of other alkali metals than sodium, e.g., potassium and salts of the alkaline earth metals.
- reference to the salts of the alkaline earth metals includes magnesium salts. Salts of other alkaline earth metals also may be employed, i.e., calcium and barium.
- Magnesium thymolphthalein monophosphate has heretofore been proposed as a substrate for the detection and quantitative determination of alkaline phosphatase in blood serum. Elevated values of alkaline phosphatase oc our in increased osteoblastic activity, impairment of liver function, pregnancy and biliary obstruction. Lowered values are observed in hypophosphatasia and in malnutrition. Alkaline phosphatase determination is ordinarily carried out with the substrate, e.g., magnesium thymolphthalein monophosphate, dissolved in a solution buffered at a pH of 10.
- the substrate e.g., magnesium thymolphthalein monophosphate
- the sodium salt of thymolphthalein monophosphate possesses specificity as a substrate for prostatic acid phosphatase. Moreover, it may be employed in a determination procedure that is simple to perform. Quantitative readings merely require comparison of color in relation to similarly tested known standards which may, if desired, be indicated on a chart or curve.
- the other alkali metal and alkaline earth metal salts likewise may be availed of, the lesser solubility of the alkaline earth metal salts as compared with alkali metal salts being less of a limitation than with an alkaline phosphatase determination because of greater solubility of the alkaline earth metal salts at the acid pH which characterizes acid phosphatase determination as compared with their solubility at an alkaline pH.
- the technique involved in the practice of this invention is simple. A measured quantity of serum or other biological fluid is added to a solution of the substrate buffered at an acid pH. Incubation proceeds at a given temperature for a predetermined period of time. During incubation free thymolphthalein is released and at the end of the incubation period the solution is rendered alkaline, thereby stopping further reaction and fully developing the color of the thymolphthalein which will remain stable for at least two hours. The test is read against a blank and the absorbance obtained is compared with absorbances obtained with pure solutions of thymolphthalein in different amounts. The results are expressed in International Units (I.U.), i.e., as micromoles of substrate converted per minute per liter of biological fluid. It is of advantage in the practice of this invention not only that the technique is simple but also that it employs stable reagents and lends itself to standardization by a primary standard.
- I.U. International Units
- the method likewise is of advantage in that it follows zero kinetics over a wide range of enzyme concentrations and against incubation time. When tested with several.
- a most important advantage of the method of this invention is its good specificity for prostactic acid phosphatase, the method being less affected than other substrates by non-prostatic acid phosphatase isozymes that are responsible for false-positive reactions for prostatic acid phosphatase when other known substrates are employed.
- the activity or afiinity of erythrocytic acid phosphatase with respect to phenol-phosphate is approximately 88 times that of its activity with respect to sodium thymolphthalein monophosphate. This is of major diagnostic significance due to the fact that erythocytic acid phosphatase usually is contained in blood serum in addition to any prostatic acid phosphatase and its reactivity with phenol-phosphate is such as to produce a positive reaction that interferes with or obscures the detection of prostatic acid phosphatase.
- the sodium salt of thymolphthalein monophosphate is used as the substrate with serum containing prostatic acid phosphatase together with erythrocytic acid phosphatase
- the activity thereon of the erythrocytic acid phosphatase is so slight in relation to the activity thereon of prostatic acid phosphatase that a positive reaction is quantitatively indicative of the presence of the prostatic acid phosphatase in the serum sample to the extent that the indicated positive reaction is in excess of the normal values for the method that are obtained with serum obtained from males who are free of kidney and liver diseases as well as malignancies.
- the normal value for the method has been determined to be of the order of 0.04 and 0.37 I.U.
- Another acid phosphatase isozyme that commonly is encountered in blood serum in addition to any prostatic acid phosphatase that may be present is platelet acid phosphatase.
- the sodium salt of thymolphthalein monophosphate has distinct advantages in relation to other substrates as regards specificity in the detection of prostatic acid phosphatase.
- phenol-phosphate and phenolphthalein monophosphate are above five times more active toward platelet acid phosphatase than the sodium salt of thymolphthalein monophosphate.
- Alpha-naphthyl-phosphate has about the same activity.
- p-nitro-phenol-phosphate is about sixteen times more active and beta-glycerophosphate is about four times more active. Except for phenolphthalein monophosphate, neither tartrate nor formalin will eliminate these interferences.
- the sodium salt of thymolphthalein monophosphate may be employed in the detection of prostatic acid phosphatase in other body fluids such as urine, although the degree of specificity in relation to possible interferences is not as great as in the use of the method of this invention with blood sera.
- the method is carried out at an acid pH. While the pH is not critical, it is preferably between about 5.5 and about 6.4 and normally is at a pH of 6 at which maximum activity occurs. These pH values are measured at 25 C. Any compatible buffer may be employed, although that which normally is used is a conventional citrate or acetate buffer. Reactivity starts immediately after zero incubation time and is linear up to about 35 minutes. In normal procedure the incubation is for a period of 30 minutes at 37 C.
- the molarity of the new substrate is not critical in that activity in terms of International Units increases until an optimum occurs at about 2.2 mmol per liter, after which activity falls off gradually. The preferred range of molarity is of the order of 1.5 to 3 mmol per liter.
- the method of this invention may be illustrated by the following example of preferred practice.
- a 0.1 M citrate buffer solution citric acid plus sodium citrate
- suflicient sodium thymolphthalein monophosphate to provide a concentrate of 2.2 mmols per liter, the pH of the solution being 6 at 25 C.
- 1.0 ml. of the buffered substrate is incubated with 0.2 ml. of serum for 30 minutes at 37 C. If prostatic acid phosphatase is present the substrate is acted upon to release free thymolphthalein, the rate of release being greater for greater amounts of prostatic acid phosphatase present in the serum sample.
- the end of the incubation period 5 ml.
- the absorbance obtained is evaluated preferably by measurement with a spectrophotometer at 590 nm. in comparison with absorbances obtained with pure solutions of thymolphthalein which, for convenience, may have been recorded on a chart or curve. Alternatively, a colorimeter may be used. The test is read against a serum blank and any activity in excess of normal is, as aforesaid, expressed in International Units.
- an admixture of the substrate and buffer in the dry state may be prepackaged in a vial to which a given quantity of distilled water may be added to produce a solution having predetermined desired molarities for the substrate and for the buffer in a convenient amount for such number of tests as may be desired.
- the improvement which comprises incubating an aqueous solution of said biological fluid containing as the sole substrate an alkali metal or alkaline earth metal salt of thymolphthalein monophosphate, measuring the amount of substrate which is converted after incubation and comparing said amount with the normal amount of substrate which is converted when similarly measured employing a fluid from a male who is free of kidney or liver disease or malignancy, thereby determining any amount that is in excess of said normal amount.
- a method according to claim 2 wherein said salt of thymolphthalein monophosphate is the sodium salt.
- reaction mass is rendered alkaline thereby stopping the enzymatic reaction and developing the color of any released thymolphthalein and wherein the quantity of released thymolphthalein is meas- 10 ured colorimetrically.
Abstract
Description
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US00100345A US3823071A (en) | 1970-12-21 | 1970-12-21 | Prostatic acid phosphatase determination |
CA125,500A CA975268A (en) | 1970-12-21 | 1971-10-19 | Prostatic acid phosphatase determination |
GB4925571A GB1371209A (en) | 1970-12-21 | 1971-10-22 | Prostatic acid phosphatase determination |
IT53841/71A IT965018B (en) | 1970-12-21 | 1971-11-02 | SUBSTRATE FOR THE DETECTION AND QUANTITATIVE DETERMINATION OF EN ZIMI IN BIOLOGICAL FLUIDS AND REAGENT PACK CONTAINING THE SUBSTRATE AND A BUFFER |
ES397269A ES397269A1 (en) | 1970-12-21 | 1971-11-23 | Prostatic acid phosphatase determination |
FR7144735A FR2120776A5 (en) | 1970-12-21 | 1971-12-13 | |
JP46101521A JPS5137040B1 (en) | 1970-12-21 | 1971-12-16 | |
DE19712163315 DE2163315C3 (en) | 1970-12-21 | 1971-12-20 | Method for the detection and / or quantitative determination of acidic prostate phosphatase in biological fluids |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US00100345A US3823071A (en) | 1970-12-21 | 1970-12-21 | Prostatic acid phosphatase determination |
Publications (1)
Publication Number | Publication Date |
---|---|
US3823071A true US3823071A (en) | 1974-07-09 |
Family
ID=22279284
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US00100345A Expired - Lifetime US3823071A (en) | 1970-12-21 | 1970-12-21 | Prostatic acid phosphatase determination |
Country Status (7)
Country | Link |
---|---|
US (1) | US3823071A (en) |
JP (1) | JPS5137040B1 (en) |
CA (1) | CA975268A (en) |
ES (1) | ES397269A1 (en) |
FR (1) | FR2120776A5 (en) |
GB (1) | GB1371209A (en) |
IT (1) | IT965018B (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4045290A (en) * | 1975-02-28 | 1977-08-30 | Princeton Biomedix Incorporated | Diagnostic method and compounds for use therewith |
US4162194A (en) * | 1976-02-13 | 1979-07-24 | Beckman Instruments, Inc. | Kinetic assay for acid phosphotase and composition therefore |
US4206280A (en) * | 1975-12-24 | 1980-06-03 | Hoffmann-La Roche Inc. | Determination of acid phosphatase |
US4985414A (en) * | 1987-11-20 | 1991-01-15 | Boehringer Mannheim Gmbh | 1-naphtholphthalein monophosphates and diagnostic reagents containing them |
US5763490A (en) * | 1994-09-20 | 1998-06-09 | University Of South Carolina | Treating prostate cancer with tartrate ions |
US20060257879A1 (en) * | 2003-08-01 | 2006-11-16 | Stuart Wilson | Methods and kits for detecting an enzyme capable of modifying a nucleic acid |
WO2007149782A2 (en) * | 2006-06-23 | 2007-12-27 | University Of Medicine And Dentistry Of New Jersey | Selective inhibitors for transferases |
US20110105349A1 (en) * | 2008-04-15 | 2011-05-05 | Munodiagnostic Systems Limited | Use of a monophosphate ester of a phthalein compound as a substrate for tartrate-resistant acid phosphatase b5 (trap 5b) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH625833A5 (en) * | 1975-12-24 | 1981-10-15 | Hoffmann La Roche | Method for the determination of acid phosphatase |
US5981206A (en) * | 1992-05-20 | 1999-11-09 | Johnson & Johnson Clinical Diagnostic Systems, Inc. | Dry analytical element and method for the detection of prostatic acid phosphatase |
-
1970
- 1970-12-21 US US00100345A patent/US3823071A/en not_active Expired - Lifetime
-
1971
- 1971-10-19 CA CA125,500A patent/CA975268A/en not_active Expired
- 1971-10-22 GB GB4925571A patent/GB1371209A/en not_active Expired
- 1971-11-02 IT IT53841/71A patent/IT965018B/en active
- 1971-11-23 ES ES397269A patent/ES397269A1/en not_active Expired
- 1971-12-13 FR FR7144735A patent/FR2120776A5/fr not_active Expired
- 1971-12-16 JP JP46101521A patent/JPS5137040B1/ja active Pending
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4045290A (en) * | 1975-02-28 | 1977-08-30 | Princeton Biomedix Incorporated | Diagnostic method and compounds for use therewith |
US4206280A (en) * | 1975-12-24 | 1980-06-03 | Hoffmann-La Roche Inc. | Determination of acid phosphatase |
US4162194A (en) * | 1976-02-13 | 1979-07-24 | Beckman Instruments, Inc. | Kinetic assay for acid phosphotase and composition therefore |
US4985414A (en) * | 1987-11-20 | 1991-01-15 | Boehringer Mannheim Gmbh | 1-naphtholphthalein monophosphates and diagnostic reagents containing them |
US5763490A (en) * | 1994-09-20 | 1998-06-09 | University Of South Carolina | Treating prostate cancer with tartrate ions |
US20060257879A1 (en) * | 2003-08-01 | 2006-11-16 | Stuart Wilson | Methods and kits for detecting an enzyme capable of modifying a nucleic acid |
WO2007149782A2 (en) * | 2006-06-23 | 2007-12-27 | University Of Medicine And Dentistry Of New Jersey | Selective inhibitors for transferases |
WO2007149782A3 (en) * | 2006-06-23 | 2008-05-02 | Univ New Jersey Med | Selective inhibitors for transferases |
US20090306201A1 (en) * | 2006-06-23 | 2009-12-10 | University Of Medicine And Dentistry Of New Jersey | Selective inhibitors for transferases |
US20110105349A1 (en) * | 2008-04-15 | 2011-05-05 | Munodiagnostic Systems Limited | Use of a monophosphate ester of a phthalein compound as a substrate for tartrate-resistant acid phosphatase b5 (trap 5b) |
Also Published As
Publication number | Publication date |
---|---|
IT965018B (en) | 1974-01-31 |
ES397269A1 (en) | 1974-04-16 |
JPS5137040B1 (en) | 1976-10-13 |
DE2163315B2 (en) | 1976-09-16 |
CA975268A (en) | 1975-09-30 |
FR2120776A5 (en) | 1972-08-18 |
DE2163315A1 (en) | 1972-07-13 |
GB1371209A (en) | 1974-10-23 |
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Legal Events
Date | Code | Title | Description |
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AS | Assignment |
Owner name: FLOW GENERAL INC.; A CORP OF DE. Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:MILLIPORE CORPORATION;REEL/FRAME:004061/0349 Effective date: 19820302 |
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AS | Assignment |
Owner name: COOPERBIOMEDICAL, INC., 3145 PORTER DRIVE, PALO AL Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:FLOW GENERAL INC., A DE CORP;REEL/FRAME:004286/0086 Effective date: 19840307 |
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AS | Assignment |
Owner name: TECHNICON ISNTRUMENTS CORPORATION, 511 BENEDICT AV Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:COOPER DEVELOPMENT COMPANY;REEL/FRAME:004945/0566 Effective date: 19880711 Owner name: TECHNICON ISNTRUMENTS CORPORATION, NEW YORK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:COOPER DEVELOPMENT COMPANY;REEL/FRAME:004945/0566 Effective date: 19880711 |