US2999793A - Diagnostic preparation and process for the determination of serum alkaline phosphatase - Google Patents
Diagnostic preparation and process for the determination of serum alkaline phosphatase Download PDFInfo
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- US2999793A US2999793A US671151A US67115157A US2999793A US 2999793 A US2999793 A US 2999793A US 671151 A US671151 A US 671151A US 67115157 A US67115157 A US 67115157A US 2999793 A US2999793 A US 2999793A
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- serum
- alkaline phosphatase
- determination
- sodium
- serum alkaline
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- 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
- C12Q2334/40—Triphenylmethane dye chromogens, e.g. fluorescein derivatives
Definitions
- This invention relates to a diagnostic preparation and relates more particularly to a stable composition for use in a simplified, semiquantitative determination of alkaline phosphatase in serum.
- the determination of serum alkaline phosphatase is of considerable value in the diagnosis or such conditions as bone cancer, liver damage, bile obstruction, and gallstone disorders.
- An object of this invention is the preparation of a stable, diagnostic composition in tablet form suitable for use in the determination of serum alkaline phosphatase.
- a further object of this invention is to provide a method for determining serum alkaline phosphatase where the use of solutions and the necessity for precise and timeconsurning measurements and reactions is eliminated.
- serum whose alkaline phosphatase content is to be determined is incubated with a solid preparation containing sodium phenolphthalein phosphate and a buffer and the reaction mixture thus formed is then made alkaline.
- the depth of color which develops and which is due to the free phenolphthalein formed by the action of the enzyme present is then compared with a standard color chart from which one is readily able to obtain a semiquantitative reading of the serum alkaline phosphatase concentration.
- the buflfer employed should be present in suflicient quantity to overcome the bufiering action of the serum and to maintain the serum pH in a range of about 9 to 11, the range in which the serum alkaline phosphatase enzyme is active.
- the incubation period employed varies with temperature. At a temperature of 67 F. the incubation period should be about thirty minutes, at 80 F. about twenty minutes and at 865 F. about sixteen minutes. The time and temperature relationship within this range is essentially a straight-line relationship. At 93 F. the incubation time should be thirteen minutes, and at 98.6 F. twelve minutes. The incubation is preferably carried out at temperatures within the range given which are the usual room temperatures.
- I preferably include an activator for the enzyme such as a magnesium salt, for example magnesium sulfate.
- the activator is employed to provide suflicient magnesium ions in the event that the magnesium concentration in the serum tested is abnormally low and insuflicient to activate the enzyme.
- the solid substrate tablet preparation of this invention preferably contains the active components in a ratio of about 1.5 parts by weight of sodium phenolphthalein phosphate to 0.1 part of magnesium sulfate and 98.4 parts of the buffer, e.g.: tris (hydroxymethyl)-aminomethane.
- the buffer e.g.: tris (hydroxymethyl)-aminomethane.
- This new and novel substrate tablet is convenient and stable. In use, no measurement of substrate volume is required since the tablet volume is essentially zero, the entire tablet being of the order of only about 20 mg. in weight. Since the test is run in whole serum in the presence of an excess of the substrate rather than in 5% buffered serum solution as in the prior testing method, the volume of serum used need not be measured accurately. Since the concentration of alkaline phosphatase in the reaction mixture determines the amount of color produced and the serum is essentially the whole volume of the reaction mixture, the concentration of alkaline phosphatase in the reaction mixture is essentially independent of the volume of serum used.
- a 20 mg. tablet may contain from about 0.2-0.6 mg. of sodium phenolphthalein phosphate, corresponding to about 1 to about 3 percent by weight of the tablet, up to about 0.02 mg. of magnesium sulfate that is, up to about 0.1 percent by weight of the tablet, with the remainder of the active components being the buffer.
- Example 1 To approximately 0.2 ml. of serum is added 10 to 30 mg. of a solid composition, in either powder or tablet form, containing approximately 1.5 parts sodium phenolphthalein phosphate, approximately 0.1 part magnesium sulfate, and approximately 98.4 parts tris (hydroxymethyD-aminomethane. The mixture is incubated for about twenty minutes at 80 F. and to the incubated mixture is then added one drop (about 0.04 ml.) of 0.5 N aqueous sodium hydroxide to make the mixture alkaline. The developed color is then matched against a standard color chart, as described. The said color chart is developed by matching the color densities of quantitatively determined serum alkaline phosphatase values against the color densities produced in the method of this invention. Thus, a direct reading of serum alkaline phosphatase concentration is had.
- a dry preparation for use in the determination of serum alkaline phosphatase which comprises about 1 to about 3 percent by weight of sodium phenolphth-alein phosphate, up to about 0.1 percent by weight of a magnesium salt and tris (hydroxymethyl)-aminomethane in an amount sufiicient to maintain the serum pH in a range of about 9 to about 11.
- a tablet for the determination of serum alkaline phosphatase consisting of about 0.3 mg. sodium phenolphthalein phosphate, about 0.02 mg. of magnesium sulfate, and about 19.68 mg. tris(hydroxymethyl)-aminomethane.
- a method for the determination of serum alkaline phosphatase which comprises adding to a sample volume of serum about 10 to about 30 milligrams of a dry preparation comprising about 1 to about 3 percent of the weight of the preparation of sodium phenolphthalein phosphate, up to about 0.1 percent of the Weight of the preparation of a magnesium salt and tris(hydroxymethyl)- aminomethane in an amount sufiicient to maintain the serum pH in a range of from about 9 to 11, incubating the mixture to permit the enzyme present to hydrolyze the phenolphthalein compound, making the incubated mixture alkaline, and comparing the developed color to the color developed in the presence of a known concentration of serum alkaline phosphatase.
- a method for the determination of serum alkaline phosphatase which comprises adding to approximately 0.2 ml. of serum a 10 to 30 mg. tablet containing about 1.5% by weight of sodium phenolphthalein phosphate, about 0.1% by Weight of magnesium sulfate, and about 98.4% by weight of tris (hydroxymethyl)-amino-methane, incubating the mixture at a temperature of from 67 to 98.6" F. from 30 to 12 minutes, adding from 0.02 to 0.10 cc. of 0.5 N aqueous alkali metal hydroxide, and comparing the developed color to the color developed in the presence of a known concentration of serum alkaline phosphatase.
- a method for the determination of serum alkaline phosphatase which comprises adding to approximately 0.2 ml. of serum a 20 mg. tablet containing about 0.3 mg. sodium phenolphthalein phosphate, about 0.02 mg. of magnesium sulfate, and about 19.68 mg. tris(hydroxymethyl)-amino-methane, incubating the mixture, making the mixture alkaline by adding about 0.4 cc. of a 0.5 N aqueous sodium hydroxide to the incubated mixture, and comparing the developed color to the color developed in the presence of a known concentration of serum alkaline phosphatase.
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
tnitetates This invention relates to a diagnostic preparation and relates more particularly to a stable composition for use in a simplified, semiquantitative determination of alkaline phosphatase in serum. The determination of serum alkaline phosphatase is of considerable value in the diagnosis or such conditions as bone cancer, liver damage, bile obstruction, and gallstone disorders.
An object of this invention is the preparation of a stable, diagnostic composition in tablet form suitable for use in the determination of serum alkaline phosphatase.
A further object of this invention is to provide a method for determining serum alkaline phosphatase where the use of solutions and the necessity for precise and timeconsurning measurements and reactions is eliminated. Other objects of this invention will appear from the following detailed description.
One of the present tests for determining serum alkaline phosphatase was developed by Huggins and Talalay (J. Biol. Chem. 159, 399 (1945)). In this assay, a buffered solution of sodium phenolphthalein phosphate containing 5% serum is incubated at 37 C. for one hour. The incubation is then halted and the reaction mixture is made alkaline with a solution of sodium pyrophosphate in glycine butfer. The amount of phenolphthalein released by the alkaline phosphatase present is measured in a colorimeter, and the alkaline phosphatase concentration in the serum is determined from a standard curve.
In accordance with the present invention and in accordance with the novel method which has now been developed, serum whose alkaline phosphatase content is to be determined is incubated with a solid preparation containing sodium phenolphthalein phosphate and a buffer and the reaction mixture thus formed is then made alkaline. The depth of color which develops and which is due to the free phenolphthalein formed by the action of the enzyme present is then compared with a standard color chart from which one is readily able to obtain a semiquantitative reading of the serum alkaline phosphatase concentration. The buflfer employed should be present in suflicient quantity to overcome the bufiering action of the serum and to maintain the serum pH in a range of about 9 to 11, the range in which the serum alkaline phosphatase enzyme is active.
The incubation period employed varies with temperature. At a temperature of 67 F. the incubation period should be about thirty minutes, at 80 F. about twenty minutes and at 865 F. about sixteen minutes. The time and temperature relationship within this range is essentially a straight-line relationship. At 93 F. the incubation time should be thirteen minutes, and at 98.6 F. twelve minutes. The incubation is preferably carried out at temperatures within the range given which are the usual room temperatures.
In addition to the sodium phenolphthalein phosphate and butter, I preferably include an activator for the enzyme such as a magnesium salt, for example magnesium sulfate. The activator is employed to provide suflicient magnesium ions in the event that the magnesium concentration in the serum tested is abnormally low and insuflicient to activate the enzyme.
While tris (hydroxymethyl)-aminomethane has proven to be a satisfactory buffer in my novel compositions, other bufiers of similar solubility and basicity may also be used *atet ice . 2 in order to maintain the pH. within the preferred range during the incubation period. Examples of other buffers which may also be employed are the sodium salt of 5,5-diethyl barbituric acid, sodium carbonate and guanidine carbonate.
The solid substrate tablet preparation of this invention preferably contains the active components in a ratio of about 1.5 parts by weight of sodium phenolphthalein phosphate to 0.1 part of magnesium sulfate and 98.4 parts of the buffer, e.g.: tris (hydroxymethyl)-aminomethane. Although inert binding materials may be used as an aid in forming these tablets, this is generally unnecessary.
This new and novel substrate tablet is convenient and stable. In use, no measurement of substrate volume is required since the tablet volume is essentially zero, the entire tablet being of the order of only about 20 mg. in weight. Since the test is run in whole serum in the presence of an excess of the substrate rather than in 5% buffered serum solution as in the prior testing method, the volume of serum used need not be measured accurately. Since the concentration of alkaline phosphatase in the reaction mixture determines the amount of color produced and the serum is essentially the whole volume of the reaction mixture, the concentration of alkaline phosphatase in the reaction mixture is essentially independent of the volume of serum used. Thus, a semiquantitative measure of serum alkaline phosphate is obtained, and no accurate measurements are necessary The present technique is simple and rapid requiring an incubation time of only twenty minutes at F. instead of the one hour needed in the older method. Further, only small volumes of serum are required in the test, the preferred volume range being 0.1 to 0.3 ml.
With such small serum samples, volumes of 0.5N aqueous sodium hydroxide of the order of 0.04 ml. are sufficient to stop the incubation and develop the full phenolphthalein color. The simplicity of the method of this invention obviates the need for expensive equipment and highly trained technical personnel to perform the test. The simplicity and rapidity of the present method makes it readily available for the screening of serum alkaline phosphatase levels in large population groups. In view of the known value of this diagnostic test, this convenient method of determining serum alkaline phosphatase levels could be of great value for the early diagnosis of cancer, liver disfunction, and gall bladder disfunction.
It is to be understood that the proportion of the active ingredients set forth above is the preferred one, but it may be subjected to some variations without affecting its use and value as mentioned herein. Thus, for example, a 20 mg. tablet may contain from about 0.2-0.6 mg. of sodium phenolphthalein phosphate, corresponding to about 1 to about 3 percent by weight of the tablet, up to about 0.02 mg. of magnesium sulfate that is, up to about 0.1 percent by weight of the tablet, with the remainder of the active components being the buffer.
It is to be understood that other salts of phenolphthalein phosphate, such as other alkali metal and alkaline earth metal salts may be used. Thus, in place of the sodium salt, the calcium and barium salts of phenolphthalein phosphate may be employed.
The following example will serve to illustrate my invention and sets forth the detailed method for the determination of serum alkaline phosphatase following my method.
Example To approximately 0.2 ml. of serum is added 10 to 30 mg. of a solid composition, in either powder or tablet form, containing approximately 1.5 parts sodium phenolphthalein phosphate, approximately 0.1 part magnesium sulfate, and approximately 98.4 parts tris (hydroxymethyD-aminomethane. The mixture is incubated for about twenty minutes at 80 F. and to the incubated mixture is then added one drop (about 0.04 ml.) of 0.5 N aqueous sodium hydroxide to make the mixture alkaline. The developed color is then matched against a standard color chart, as described. The said color chart is developed by matching the color densities of quantitatively determined serum alkaline phosphatase values against the color densities produced in the method of this invention. Thus, a direct reading of serum alkaline phosphatase concentration is had.
It is to be understood that the foregoing detailed de scription is given merely by way of illustration and that many variations may be made therein without departing from the spirit of my invention.
Having described my invention, what I desire to secure by Letters Patent is:
1. A dry preparation for use in the determination of serum alkaline phosphatase which comprises about 1 to about 3 percent by weight of sodium phenolphth-alein phosphate, up to about 0.1 percent by weight of a magnesium salt and tris (hydroxymethyl)-aminomethane in an amount sufiicient to maintain the serum pH in a range of about 9 to about 11.
2. A dry preparation according to claim 1 wherein said magnesium salt is magnesium sulfate.
3. A dry preparation according to claim 2 in the form of a tablet weighing about 10 to about 30 milligrams.
4. A tablet for the determination of serum alkaline phosphatase consisting of about 0.3 mg. sodium phenolphthalein phosphate, about 0.02 mg. of magnesium sulfate, and about 19.68 mg. tris(hydroxymethyl)-aminomethane.
5. A method for the determination of serum alkaline phosphatase, which comprises adding to a sample volume of serum about 10 to about 30 milligrams of a dry preparation comprising about 1 to about 3 percent of the weight of the preparation of sodium phenolphthalein phosphate, up to about 0.1 percent of the Weight of the preparation of a magnesium salt and tris(hydroxymethyl)- aminomethane in an amount sufiicient to maintain the serum pH in a range of from about 9 to 11, incubating the mixture to permit the enzyme present to hydrolyze the phenolphthalein compound, making the incubated mixture alkaline, and comparing the developed color to the color developed in the presence of a known concentration of serum alkaline phosphatase.
6. A method for the determination of serum alkaline phosphatase which comprises adding to approximately 0.2 ml. of serum a 10 to 30 mg. tablet containing about 1.5% by weight of sodium phenolphthalein phosphate, about 0.1% by Weight of magnesium sulfate, and about 98.4% by weight of tris (hydroxymethyl)-amino-methane, incubating the mixture at a temperature of from 67 to 98.6" F. from 30 to 12 minutes, adding from 0.02 to 0.10 cc. of 0.5 N aqueous alkali metal hydroxide, and comparing the developed color to the color developed in the presence of a known concentration of serum alkaline phosphatase.
7. A method for the determination of serum alkaline phosphatase which comprises adding to approximately 0.2 ml. of serum a 20 mg. tablet containing about 0.3 mg. sodium phenolphthalein phosphate, about 0.02 mg. of magnesium sulfate, and about 19.68 mg. tris(hydroxymethyl)-amino-methane, incubating the mixture, making the mixture alkaline by adding about 0.4 cc. of a 0.5 N aqueous sodium hydroxide to the incubated mixture, and comparing the developed color to the color developed in the presence of a known concentration of serum alkaline phosphatase.
References Cited in the file of this patent UNITED STATES PATENTS Scharer Sept. 26, 1944 OTHER REFERENCES
Claims (1)
1. A DRY PREPARATION FOR USE IN THE DETERMINATION OF SERUM ALKALINE PHOSPHATASE WHICH COMPRISES ABOUT 1 TO ABOUT 3 PERCENT BY WEIGHT OF SODIUM PHENOLPHTHALEIN PHOSPHATE, UP TO ABOUT 0.1 PERCENT BY WEIGHT OF A MAGNESIUM SALT AND TRIS(HYDROXYMETHYL)-AMINOMETHANE IN AN AMOUNT SUFFICIENT TO MAINTAIN THE SERUM PH IN A RANGE OF ABOUT 9 TO ABOUT 11.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US671151A US2999793A (en) | 1957-07-11 | 1957-07-11 | Diagnostic preparation and process for the determination of serum alkaline phosphatase |
| DE19581423469 DE1423469A1 (en) | 1957-07-11 | 1958-07-07 | Dry preparation and method for determining the alkaline phosphatase in serum |
| FR1199840D FR1199840A (en) | 1957-07-11 | 1958-07-10 | Preparation for diagnostics for the determination of alkaline phosphatase in serum |
| GB22153/58A GB863739A (en) | 1957-07-11 | 1958-07-10 | Diagnostic preparation for the determination of serum alkaline phosphatase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US671151A US2999793A (en) | 1957-07-11 | 1957-07-11 | Diagnostic preparation and process for the determination of serum alkaline phosphatase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| US2999793A true US2999793A (en) | 1961-09-12 |
Family
ID=24693320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US671151A Expired - Lifetime US2999793A (en) | 1957-07-11 | 1957-07-11 | Diagnostic preparation and process for the determination of serum alkaline phosphatase |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US2999793A (en) |
| DE (1) | DE1423469A1 (en) |
| FR (1) | FR1199840A (en) |
| GB (1) | GB863739A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3362886A (en) * | 1965-04-09 | 1968-01-09 | Miles Lab | Composition and method for the detection of galactose |
| US3466306A (en) * | 1965-08-06 | 1969-09-09 | Warner Lambert Pharmaceutical | Process for the stabilization of organic esters of phosphoric acid |
| US3539026A (en) * | 1969-04-07 | 1970-11-10 | Wayne N Sutliff | Fishing tool energizer |
| US3896008A (en) * | 1971-10-28 | 1975-07-22 | Owens Illinois Inc | Electrochemical potentiometric method for selectively determining alkaline phosphatase content in aqueous fluids |
| US3926735A (en) * | 1971-10-20 | 1975-12-16 | Mallinckrodt Inc | Alkaline phosphatase assay |
| US4045290A (en) * | 1975-02-28 | 1977-08-30 | Princeton Biomedix Incorporated | Diagnostic method and compounds for use therewith |
| US4076502A (en) * | 1975-02-14 | 1978-02-28 | Miles Laboratories, Inc. | Method, composition, and device for determining the specific gravity of a liquid |
| US4123384A (en) * | 1975-08-20 | 1978-10-31 | Boehringer Mannheim Gmbh | Control serum containing alkaline phosphatase of constant activity |
| WO2015035487A1 (en) * | 2013-09-11 | 2015-03-19 | Universidade Federal Do Rio De Janeiro - Ufrj | Method and kit for synthesizing phenolphthalein diphosphate tetrasodium |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1285217B (en) * | 1962-08-09 | 1968-12-12 | Inst Milchforschung Oranienbur | Reagent paper for the detection of alkaline phosphatase |
| US3791929A (en) * | 1968-09-08 | 1974-02-12 | Searle & Co | Phosphatase assay using lyophilized aryl phosphate monesters |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2359052A (en) * | 1940-07-01 | 1944-09-26 | Scharer Harry | Method for detecting enzyme activity |
-
1957
- 1957-07-11 US US671151A patent/US2999793A/en not_active Expired - Lifetime
-
1958
- 1958-07-07 DE DE19581423469 patent/DE1423469A1/en active Pending
- 1958-07-10 GB GB22153/58A patent/GB863739A/en not_active Expired
- 1958-07-10 FR FR1199840D patent/FR1199840A/en not_active Expired
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2359052A (en) * | 1940-07-01 | 1944-09-26 | Scharer Harry | Method for detecting enzyme activity |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3362886A (en) * | 1965-04-09 | 1968-01-09 | Miles Lab | Composition and method for the detection of galactose |
| US3466306A (en) * | 1965-08-06 | 1969-09-09 | Warner Lambert Pharmaceutical | Process for the stabilization of organic esters of phosphoric acid |
| US3539026A (en) * | 1969-04-07 | 1970-11-10 | Wayne N Sutliff | Fishing tool energizer |
| US3926735A (en) * | 1971-10-20 | 1975-12-16 | Mallinckrodt Inc | Alkaline phosphatase assay |
| US3896008A (en) * | 1971-10-28 | 1975-07-22 | Owens Illinois Inc | Electrochemical potentiometric method for selectively determining alkaline phosphatase content in aqueous fluids |
| US4076502A (en) * | 1975-02-14 | 1978-02-28 | Miles Laboratories, Inc. | Method, composition, and device for determining the specific gravity of a liquid |
| US4045290A (en) * | 1975-02-28 | 1977-08-30 | Princeton Biomedix Incorporated | Diagnostic method and compounds for use therewith |
| US4123384A (en) * | 1975-08-20 | 1978-10-31 | Boehringer Mannheim Gmbh | Control serum containing alkaline phosphatase of constant activity |
| WO2015035487A1 (en) * | 2013-09-11 | 2015-03-19 | Universidade Federal Do Rio De Janeiro - Ufrj | Method and kit for synthesizing phenolphthalein diphosphate tetrasodium |
Also Published As
| Publication number | Publication date |
|---|---|
| GB863739A (en) | 1961-03-22 |
| FR1199840A (en) | 1959-12-16 |
| DE1423469A1 (en) | 1968-10-10 |
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