IL33898A - Process and agent for the determination of the content of alkaline phosphatase in serum - Google Patents
Process and agent for the determination of the content of alkaline phosphatase in serumInfo
- Publication number
- IL33898A IL33898A IL33898A IL3389870A IL33898A IL 33898 A IL33898 A IL 33898A IL 33898 A IL33898 A IL 33898A IL 3389870 A IL3389870 A IL 3389870A IL 33898 A IL33898 A IL 33898A
- Authority
- IL
- Israel
- Prior art keywords
- determination
- serum
- content
- diethanolamine
- alkaline phosphatase
- Prior art date
Links
- 210000002966 serum Anatomy 0.000 title claims description 20
- 238000000034 method Methods 0.000 title claims description 19
- 102000002260 Alkaline Phosphatase Human genes 0.000 title claims description 9
- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims description 9
- 239000000758 substrate Substances 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 18
- VJLOFJZWUDZJBX-UHFFFAOYSA-N bis(2-hydroxyethyl)azanium;chloride Chemical compound [Cl-].OCC[NH2+]CCO VJLOFJZWUDZJBX-UHFFFAOYSA-N 0.000 claims description 9
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 4
- 238000006460 hydrolysis reaction Methods 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- FQPFAHBPWDRTLU-UHFFFAOYSA-N aminophylline Chemical compound NCCN.O=C1N(C)C(=O)N(C)C2=C1NC=N2.O=C1N(C)C(=O)N(C)C2=C1NC=N2 FQPFAHBPWDRTLU-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 description 20
- 108090000790 Enzymes Proteins 0.000 description 20
- 239000000243 solution Substances 0.000 description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 230000008033 biological extinction Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 description 4
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 description 4
- 235000021317 phosphate Nutrition 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 150000003014 phosphoric acid esters Chemical class 0.000 description 2
- 238000005375 photometry Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- AZUYLZMQTIKGSC-UHFFFAOYSA-N 1-[6-[4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methylindazol-5-yl)pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl]prop-2-en-1-one Chemical compound ClC=1C(=C2C=NNC2=CC=1C)C=1C(=NN(C=1C)C1CC2(CN(C2)C(C=C)=O)C1)C=1C=C2C=NN(C2=CC=1)C AZUYLZMQTIKGSC-UHFFFAOYSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- 208000020084 Bone disease Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000012928 buffer substance Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- CMPQUABWPXYYSH-UHFFFAOYSA-N phenyl phosphate Chemical compound OP(O)(=O)OC1=CC=CC=C1 CMPQUABWPXYYSH-UHFFFAOYSA-N 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
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
- C12Q2334/10—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases p-Nitrophenol derivatives
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
Process and agent for the determination of the content of alkaline phosphatase in serum T»03a nvv*%ypi?R ΠΤΧΠΒΟΙΒ η*7Τ3η nvzp1? »PSDKI T»*?rn The present invention is concerned with a process and agent for the determination of the content of alkaline phosphatase in serum.
The determination of the content of alkaline phosphatase in serum is of importance for the diagnosis and continuous control of bone diseases and tumorous diseases, as well as of gall and liver diseases.
Such an enzyme determination can be carried out by means of a large number of different processes, which differ from one another with regard to the substrate used, the concentration of the substrate, the nature of the detection of the reaction products, the amount of serum needed, the pH value, the period of incubation and the incubation temperature and the nature of the buffer employed.
In the case of one of the most important known processes, serum is incubated in a glycine-sodium hydroxide buffer at a pH of 10.5, together with £-nitro-phenyl phosphate as substrate, for 30 minutes at a temperature of 37°C. and the amount of j3-nitrophenol formed subsequently used for the calculation of the , enzyme content present. The £-nitrophenol formed can be determined photometrically. This discontinuous, so-called two-point method involves, in the same way as all other . previously known methods, various possibilities of error. Thus, for example, an enzyme inhibation occurring during the period of incubation, i.e. a non-linear course of the reaction, brought about, for example, by a displacement of the pH value, cannot be observed. This may well be one of the reasons why the Commission of Enzymes of that enzyme reactions should, whenever possible, be measured op ically-kinetically, i.e. for example, in the case of the determination of alkaline phosphates, the initial velocity of the hydrolysis of the phosphoric acid ester should be determined.
In the case of the use of j -nitrophenyl phosphate as substrate, the detection of the £-nitrophenol formed in the alkaline medium used by the action of the enzyme can be determined photometrically by direct measurement at kOO nm since the pK„ value of p-nitrophenol is 7.16.
Thus, the determination is possible by the op ical-kinetic method by mixing the buffered substrate solution with the serum to be tested, whereafter the extinction caused by the liberated p_-nitrophenol is continuously read off or is recorded with the help of a compensation recorder.
In the case of this process, the incubation is carried put in the known buffers of the conventional low molarity and thus with low buffer capacity.
A process is. also known for the determination of the content of alkaline phosphate in serum with the use of a buffered aqueous p_-nitrophenyl phosphate solution as substrate and with the help of a photometric measurement of the initial velocity of the hydrolysis of this phosphoric diethanolamine acid ester, 1 M/diethanolamine hydrochloride being used as the buffer substance* (a diethanolamine - HC1 buffer).
In the case of the use of diethanolamine hydrochloride buffer at a pH of 9.2+, the enzyme activity is, surprisingly, about 2.3 times higher than in all the previous known buffers. Due to this, it is possible to measure, with only a small amount of serum and in an extremely short Thus, this process permits the determination of the alkaline phosphate in an optical-kinetic way. An especially optimum increase of the enzyme activity is achieved, in the case of this process, when working at a pH of 9.-4. and with the use of a 1 molar buffer concentration.
An important advantage of the method consists in that it works quickly and simply and that, in particular, it gives a very good reproducibility. The other previously known methods are characterised by a very unsatisfactory reproducibility, which always leads to relatively uncertain measurement results.
We have now found, surprisingly, that the determination of the alkaline phosphate in serum proceeds espec- diethanolamine ially favourably when the/diethanolamine hydrochloride is used in a concentration of at least 1.5 and at most Z+.5 mole/litre, especially of about 2 mole/litre. Especially by the use of this relatively high buffer concentration, there is obtained a high buffer capacity which precludes the harmful action of carbon dioxide from the air and keeps the pH value constant during the whole period of the experiment.
Thus, the present invention provides a process for the determination of the content of alkaline phosphatase in' serum with the use of an aqueous solution of p-nitro- dietnano1amine phenyl phosphate as substrate buffered with/diethanolamine hydrochloride and with the help of a photometric measurement of the initial velocity of the hydrolysis of this phosphoric acid ester, which is characterised in that the diethanolamine /diethanolamine hydrochloride is used in a molar concentration of at least 1.5 and at most i+.5» especially of The present invention also provides an agent for carrying out this process, which comprises £-nitrophenyl diethanolamine phosphate as substrate buffered with diethanolamine/ hydrochloride in a molar concentration of at least 1.5 and at most 4.5, especially of about 2.
The following Examples are given for the purpose of illustrating the present invention: - Example . a) Preparation of the buffer. 210.28 g. diethanolamine (crystallisable) are dissolved in about 600 ml. double distilled water. Sub-sequently, about 160 ml. 2N hydrochloric acid are added, with stirring, using a glass stirrer. 500 mg. sodium azide are then added, together with 101.5 mg. magnesium chloride hexahydrate. The solution is made up with water to almost 1000 ml. Subsequently, the pH value is adjusted to precisely 9.8 by the dropwise addition of H hydrochloric acid and the solution then made up to precisely 1000 ml. with double distilled water. b) Preparation of the buffer substrate solution. 1000 A 1-00- mg. tablet which contains 131.5 mg. 100 disodium j>-nitrophenyl phosphate, the remainder being sodium chloride and possibly some moisture, is dissolved in 10 ml. of the above-described buffer solution. When this solution is stored at 1+°C> with the exclusion of light, it can be used for 2 days for carrying put the enzyme determination described hereinafter. c) Enzyme determination.
The buffer substrate and the serum to be tested are warmed to 25°C., the temperature being measured in the solution. 2 ml. of the buffered substrate solution are mixed with 0.02 ml. fresh serum and thoroughly mixed. After about 1 minute, the extinction is measured at 25°C. and, simultaneously, the time is ascertained with a stopwatch. The measurement is repeated every minute for 3 minutes.
The extinction measurement is carried out with, for example, a photometer which is equipped with a filter for the mercury line at i+05 nm. The layer thickness is 1 cm.
If the extinction differences Δ E/min at the beginning of the measurement are greater than 0.2 , then the determination is repeated with the serum diluted 1 : 5 with a physiological solution of sodium chloride and the result obtained multiplied by 5. 1 International Enzyme Unit (u) is the amount of enzyme which reacts with 1 μ, mole of substrate in one minute under standard conditions. 1 milli-unit (mil) = 1 /1 000 U.
The extinction difference per minute Δ E/+05 is introduced into the following calculation formula: enzyme concentration = _» Ei05 . 5kS0 mU/ml.
In this Example, 90 - 250 mU ( 25°C.)/ml. is to be regarded as the normal range.
Example 2. a) Preparation of the buffer.
The preparation of the buffer is carried out in a manner analogous to that described in Example , except that the magnesium chloride hexahydrate is used in an amount of 203.0 mg. b) Preparation of the buffered substrate solution.
A tablet which contains 39.45 mg. 00% disodium _-nitrophenyl phosphate is dissolved in 10 ml. of the buffer solution of Example 2a) in a manner analogous to that described in Example 1b). c) Enzyme determination.
The enzyme determination is carried out in a manner analogous to that described in Example 1c), except that 1 ml. of buffered substrate solution is added to 0.02 ml. serum.
The calculation of the enzyme content is carried out, in this Example, by means of the formula: enzyme concentration = ΔΕ/min . 2757 mU/ml.
When, in a variant of this Example, i+.O ml. buffered substrate solution are added to 0.02 ml. serum, .the following formula is used for the calculation of the enzyme content: enzyme concentration = ΔΣ/mln . 10865 mU/ml.
Example 3.
The preparation of the buffer is carried out in a manner analogous to that described in«Example 1a) but with the use of the following amounts of the individual components : 168.22 g. diethanolamine 128 ml. 2N hydrochloric acid 101.5 mg. magnesium chloride hexahydrate The remaining components are used in the same concentrations as in Example 1.
The preparation of the buffered substrate solution and the enzyme determination are also carried out in a manner analogous to that described in Example 1b) and c), respectively.
Example k* By way of variation of Example 1 , the buffer is . prepared from the following components: i+20.56 g. diethanolamine, without additional water 320 ml. 2N hydrochloric acid 101.5 mg. magnesium chloride hexahydrate.
The preparation of the substrate solution, the preparation of the buffered substrate mixture and the enzyme determination are carried out in a manner analogous to that described in Example 1.
Claims (6)
1. What we claim is :- 1. An agent for carrying out the determination of the content of alkaline phosphatase in serum, which comprises /diethano1amine an aqueous solution of £-nitrophenyl phosphate containing/ diethanolamine hydrochloride in a molar concentration of at least 1.5 and at most h.5.
2. An agent according to claim 1, wherein the molar diethano1amine concentration of the /diethanolamine hydrochloride is about 2.
3. An agent according to claim 1 for carrying out the determination of the content of alkaline phosphatase in serum, substantially as hereinbefore described and exemplified. k» Process for carrying out the determination of the content of alkaline phosphatase in serum, wherein, as substrate, there is used an aqueous solution of _-nitro- diethanolamine phenyl phosphate which contains, as a buffer/ diethanolamine hydrochloride in, a molar concentration of at least 1.5 and at most
4. » the initial velocity of the hydrolysis of the £-nitrophenyl phosphate being measured photometrically,
5. Process according to claim 2+, wherein the molar diethanolamine concentration of the/ diethanolamine hydrochloride is about 2.
6. Process according to claim i+ for carrying out the determination of the content of alkaline phosphatase in serum, substantially as hereinbefore described and exemplified.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19691910803 DE1910803A1 (en) | 1969-03-04 | 1969-03-04 | Method and means for determining the level of alkaline phosphatase in serum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| IL33898A0 IL33898A0 (en) | 1970-04-20 |
| IL33898A true IL33898A (en) | 1972-12-29 |
Family
ID=5726994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL33898A IL33898A (en) | 1969-03-04 | 1970-02-13 | Process and agent for the determination of the content of alkaline phosphatase in serum |
Country Status (9)
| Country | Link |
|---|---|
| BE (1) | BE746829A (en) |
| BR (1) | BR6915155D0 (en) |
| CH (1) | CH535433A (en) |
| DE (1) | DE1910803A1 (en) |
| FR (1) | FR2037607A5 (en) |
| GB (1) | GB1273506A (en) |
| IL (1) | IL33898A (en) |
| NL (1) | NL7003093A (en) |
| SE (1) | SE360929B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ZA756003B (en) * | 1975-09-22 | 1977-04-27 | Chembro Holdings Pty Ltd | Measurement of alkaline phophatase levels in body fluids |
| CN114624205B (en) * | 2022-02-28 | 2024-07-23 | 海南绿峰资源开发有限公司 | Alkaline phosphatase detection method |
-
1969
- 1969-03-04 DE DE19691910803 patent/DE1910803A1/en active Pending
- 1969-12-15 BR BR215155/69A patent/BR6915155D0/en unknown
-
1970
- 1970-01-19 CH CH67970A patent/CH535433A/en not_active IP Right Cessation
- 1970-02-12 GB GB6901/70A patent/GB1273506A/en not_active Expired
- 1970-02-13 IL IL33898A patent/IL33898A/en unknown
- 1970-03-03 SE SE02772/70A patent/SE360929B/xx unknown
- 1970-03-04 NL NL7003093A patent/NL7003093A/xx unknown
- 1970-03-04 FR FR7007740A patent/FR2037607A5/fr not_active Expired
- 1970-03-04 BE BE746829D patent/BE746829A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DE1910803A1 (en) | 1970-10-08 |
| CH535433A (en) | 1973-03-31 |
| GB1273506A (en) | 1972-05-10 |
| SE360929B (en) | 1973-10-08 |
| IL33898A0 (en) | 1970-04-20 |
| BR6915155D0 (en) | 1973-02-08 |
| BE746829A (en) | 1970-09-04 |
| FR2037607A5 (en) | 1970-12-31 |
| NL7003093A (en) | 1970-09-08 |
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