CA1113840A - Composition for the analysis of the alkaline phosphatase and method therefor - Google Patents
Composition for the analysis of the alkaline phosphatase and method thereforInfo
- Publication number
- CA1113840A CA1113840A CA315,458A CA315458A CA1113840A CA 1113840 A CA1113840 A CA 1113840A CA 315458 A CA315458 A CA 315458A CA 1113840 A CA1113840 A CA 1113840A
- Authority
- CA
- Canada
- Prior art keywords
- salt
- magnesium
- propanediol
- amino
- composition
- 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
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 40
- 102000002260 Alkaline Phosphatase Human genes 0.000 title claims abstract description 24
- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000004458 analytical method Methods 0.000 title claims abstract description 5
- 150000003839 salts Chemical class 0.000 claims abstract description 23
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 claims abstract description 16
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910021538 borax Inorganic materials 0.000 claims abstract description 8
- 159000000003 magnesium salts Chemical class 0.000 claims abstract description 8
- 235000010339 sodium tetraborate Nutrition 0.000 claims abstract description 8
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims abstract description 8
- PAFZNILMFXTMIY-UHFFFAOYSA-N cyclohexylamine Chemical class NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 claims description 10
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 7
- 239000011777 magnesium Substances 0.000 claims description 7
- 229910052749 magnesium Inorganic materials 0.000 claims description 7
- 229940091250 magnesium supplement Drugs 0.000 claims description 7
- IOAOAKDONABGPZ-UHFFFAOYSA-N 2-amino-2-ethylpropane-1,3-diol Chemical class CCC(N)(CO)CO IOAOAKDONABGPZ-UHFFFAOYSA-N 0.000 claims description 4
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 4
- UXFQFBNBSPQBJW-UHFFFAOYSA-N 2-amino-2-methylpropane-1,3-diol Chemical class OCC(N)(C)CO UXFQFBNBSPQBJW-UHFFFAOYSA-N 0.000 claims description 3
- 229960001983 magnesium aspartate Drugs 0.000 claims description 3
- RXMQCXCANMAVIO-CEOVSRFSSA-L magnesium;(2s)-2-amino-4-hydroxy-4-oxobutanoate Chemical compound [H+].[H+].[Mg+2].[O-]C(=O)[C@@H](N)CC([O-])=O.[O-]C(=O)[C@@H](N)CC([O-])=O RXMQCXCANMAVIO-CEOVSRFSSA-L 0.000 claims description 3
- ITZMJCSORYKOSI-AJNGGQMLSA-N APGPR Enterostatin Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N1[C@H](C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)CCC1 ITZMJCSORYKOSI-AJNGGQMLSA-N 0.000 claims description 2
- 102100024321 Alkaline phosphatase, placental type Human genes 0.000 claims description 2
- 108010031345 placental alkaline phosphatase Proteins 0.000 claims description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims 4
- 239000011654 magnesium acetate Substances 0.000 claims 2
- 235000011285 magnesium acetate Nutrition 0.000 claims 2
- 229940069446 magnesium acetate Drugs 0.000 claims 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims 2
- 229960002337 magnesium chloride Drugs 0.000 claims 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims 2
- 229960003390 magnesium sulfate Drugs 0.000 claims 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 11
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000005065 mining Methods 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 14
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 11
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 239000000370 acceptor Substances 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 230000003169 placental effect Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 230000002440 hepatic effect Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 230000001575 pathological effect Effects 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
- 239000002253 acid Substances 0.000 description 2
- -1 amine compounds Chemical class 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- SFRDXVJWXWOTEW-UHFFFAOYSA-N 2-(hydroxymethyl)propane-1,3-diol Chemical compound OCC(CO)CO SFRDXVJWXWOTEW-UHFFFAOYSA-N 0.000 description 1
- GDTSJMKGXGJFGQ-UHFFFAOYSA-N 3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound O1B([O-])OB2OB([O-])OB1O2 GDTSJMKGXGJFGQ-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000010191 Osteitis Deformans Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000027868 Paget disease Diseases 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-L aspartate group Chemical class N[C@@H](CC(=O)[O-])C(=O)[O-] CKLJMWTZIZZHCS-REOHCLBHSA-L 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000003841 chloride salts Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- AKJBMINTPVKBGZ-UHFFFAOYSA-L disodium;(2-nitrophenyl) phosphate Chemical compound [Na+].[Na+].[O-][N+](=O)C1=CC=CC=C1OP([O-])([O-])=O AKJBMINTPVKBGZ-UHFFFAOYSA-L 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000231 kidney cortex Anatomy 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000027202 mammary Paget disease Diseases 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- ULWHHBHJGPPBCO-UHFFFAOYSA-N propane-1,1-diol Chemical compound CCC(O)O ULWHHBHJGPPBCO-UHFFFAOYSA-N 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 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)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (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)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
COMPOSITION FOR THE ANALYSIS OF THE
ALKALINE PHOSPHATASE AND METHOD THEREFOR
ABSTRACT OF THE DISCLOSURE . -A novel composition is disclosed for deter-mining the activity of the alkaline phosphatase, which comprises a salt of p-nitrophenylphosphoric acid, a magnesium salt, sodium borate and 2-amino--2-hydroxymethyl-1,3-propanediol. The composition affords advantages over the known composition espe-cially as regards sensitivity.
ALKALINE PHOSPHATASE AND METHOD THEREFOR
ABSTRACT OF THE DISCLOSURE . -A novel composition is disclosed for deter-mining the activity of the alkaline phosphatase, which comprises a salt of p-nitrophenylphosphoric acid, a magnesium salt, sodium borate and 2-amino--2-hydroxymethyl-1,3-propanediol. The composition affords advantages over the known composition espe-cially as regards sensitivity.
Description
1384~
This invention relates to a novel composition which is adapted for the determination alkaline phosphatase, such composition being composed by a salt of para-nitrophenyl-phosphoric acid, a salt of magnesium, sodium borate and 2-amino-
This invention relates to a novel composition which is adapted for the determination alkaline phosphatase, such composition being composed by a salt of para-nitrophenyl-phosphoric acid, a salt of magnesium, sodium borate and 2-amino-
2-hydroxymethyl-1,3-propanediol, The invention also relates to a method for determining alkaline phosphatase, which method is based on the use of such a composition.
It is known that alkaline phosphatase is predominantly concentrated in the intertinal epithelium, in the portion of the bones which undergo growth, in the kidney cortex, in the mammary glands, in the liver, in the bile, in the blood and in the placenta as well. The action of the alkaline phosphatase is manifested in the catalysis of hydrolysis reactions of orthophosphates according to the reaction scheme:
R - O - PO(OH)z ~ H20 ~ R - OH + H3PO4 1) and its presence at high concentrations in the blood is evidence of a certain pathological condition of the hyman system:
an increase of the phosphatase activity is an indication, for example, of disease of hepatic, intestlnal or bone origin.
The first determination of the alkallne phosphatase in the human-blood-serum dates back to 1930 (Kay, H.D., J. Biol.
Chem., 89 235 (1930!, and since those early times, the methods of assay of the enzymic activity have undergone many changes, especlally due to the influence thereon of the medium in which the reaction is caused to take place.
A number of the methods which are adapted for the determination of the phosphatase activity are now based ( J. Biol., Chem., 172, 1, (1948) ) on the discovery, by Axelrod, of the fact that the phosphatase enzyme is also capable of catalytically spurring the transphosphorylation reaction between the orthophosphate indicated in the reaction pattern (1) reported ab~ve, and an alcohol, which acts as a , 84~
receptor.
An optical method is now being widely used for the determination of the activity of the alkaline phosphatase, such optical method providing for the use of p-nitrophenyl-phosphates as the first substrate for carrying out the trans-phosphorylation reaction mentioned above. The method provides for the admixing of the first substrate, to the second substrate (acceptor) and the enzyme, under the appropriate conditions, and the readout of the optical density at 400-415 nm, the readings being progressively taken during progress of the reaction. The increase of the optical density, as time goes by, is proportional to the quantity of the product which is being transformed so that an appropriate recordal of the data permits to deduce the activity of the enzyme.
Now, it is well known ( see, for example, the book "The Enzymes", by Academic Press, 1961, pages 55 and ff), that alkaline phosphatase has an optimum activity in the vicinity of a pH of 10, and that its activity is improved by the presence of mineral salts, especially magnesium salts. These facts have compelled all the preparations which are commercially used for the determination of the phosphatase enzyme, to use ingredients which are both rigorously defined and legally required.
Thus, the use has become widespread of derivatives of p-nitrophenylphosphoric acid in combination with organic magnesium salts and buffers as required to maintain the above indicated pH range. The reagents that were initially used, were the sodium salts of the acid and inorganic buffers.
It has been ascertained that the sodium-nitrophenyl-phosphate has a poor stability and thus, in more recent times,it has been preferred to resort to the use of salts of the acid in question with amine compounds.
~13~
.
The preparation of p-nitrophenylphosphates, including those of amine compounds, and their use in biological formula-tions, are well known (see, for example, JACS, 79, (1957), page 3741.
Attempts have also been made with a view to improv-ing the progress of the transphosphorylation reaction and, in the initial composition, the acceptor substances have been thus changed until arriving, lastly, at the use of buffers which simultaneously displayed the twofold action of keeping the pH
value constant while concurrently acting as acceptors in the transphosphorylation reaction.
The care which must be taken when preparing the several buffers must not be overlooked, and such a care has, of course, its bearing on the overall economy of the reagent and the analysis. However, all the mixtures as used hereto-fore still suffer from a number of drawbacks, the most prominent of which is the poor stability of the reagent in time. Even if they are stored under drastic conditions, the prior art composi-tions cannot be used with reliability as to the reproducibility of the result, more than two or three days from their prepara-tion, at the most.
In addition, many of the conventional reagents, such as those which use mannitol and like polyhydric alcohols as the acceptors in the transphosphorylation reaction, are capable of determining with a good sensitivity also the alkaline phos-phatase of placental origin.
In the majority of the cases, dosing the activity of the alkaline phosphatase is carried out in order to detect pathological conditions which can be attributed to the hepatic system (obstruction jaundice),the bone system (such as tumours, and Paget's disease), or the bowel system. Thus, in the case of pregnancy, the high physiological activity of the alkaline .. ,, :
13~34~
phosphatase in the blood may mask one or more of the patho-logical patterns mentioned a~ove.
The Applicants have now surprisingly ascertained that a novei composition permits that the determination of the alkaline phosphatase may be carried ou-t to give reproducible results without suffering from any of the shortcomings recalled hereinabove.
According to the present invention there is provided a composition for the analysis of alkaline phosphatase, said composition comprising a salt of p-nitrophenylphosphoric acid, a magnesium salt, sodium borate and 2-amino-2-hydroxymethyl-1,3-propanediol.
According to another aspect of the present invention there is provided a method for the determination of alkaline phosphatase comprising contacting the sample to be tested with a composition comprising a salt of p-nitrophenylphosphoric acid, a salt of magnesium, sodium borate and 2-amino-2-hydroxymethyl-1,3-propanediol and thereafter photometrically measuring the amount of alkaline phosphatase.
The present invention also provides a method for the determination of non-placental alkaline phosphatase which com-prises contacting the sample to be tested with a composition comprising a salt of p-nitrophenylphosphoric acid, a salt of magnesium, sodium borate and 2-amino-2-hydroxymethyl-1,3- propa-nediol and thereafter photometrically determining the amount of alkaline phosphatase.
The composition according to the present invention is stable and retains for many days its initial properties; it is only slightly sensitive to the presence of phosphatases of placental origin. Moreover, it does not require an accurate -preparation of a buffer, since the pH of the transphosphorilation reaction is provided by the ingredients of the composition them-selves.
.~ .
. . . r ~ ' "
' '.' ' ~. ' .
~1384~
The method suggested herein provides, obviously, the reaction sequence ()1'1 -: -¦¦ +ROH ~ + ROPO3 Mg++
I
again, in which ALP is the alkaline phosphatase (EC 3.13.1) and ROH is the propanediol mentioned above. Inasmuch as the reaction takes place at an alkaline pH value, the hydrolysis of the p-nitrophenylphosphate to p-nitrophenol is monitored by merely reading out the extinction at 405 nm.
As a matter of fact, under alkaline pH conditions, the p-nitrophenol is strongly coloured in yellow whereas the p-nitrophenylphosphate is colorless.
The method permits to meter the alkaline phosphatase activity with a single mixture of reagents: it can be used manually, or also by applying it to automatic discrete and -;
continuous-flow apparatuses. It can be used also for kinetic determinations and also for fixed time determinations by the addition o a blocking agent.
For performing the method many starting substrates can be used, even though, of course, it is preferred to avoid the use of the sodium salt of the p-nitrophenylphosphoric acid, on account of the tendency of the latter to become hydrolyzed spontaneously. Thus, there can be lndicated as Examples the salts of the 2-amino-2-methyl-],3-propanediol, of 2-amino-2-ethyl-1,3-propanediol, of 2-amino-2-hydroxymethyl-1,3-propanediol, of cyclohoxyla~ c, just to ciLc a Lcw oE tl~o~
The donor substrate, obviously, can be formulated as desired, since the affinity towards the enzyme is due to the p-nitrophenylphosphoric acid rather than to the salification agent.
:; '.
~3~4~
Likewise, the magnesium salt can be selected from among a wide variety of compounds such as acetates, aspartates, chlorides, sulfates.
Good results have been obtained, for example by employing .he salt of cyclohexylamine of p-nitrophenylphosphoric acid as the donor substrate and the acetate of magnesium as the activator of the enzymic reaction.
As regards the 2-amino-2-hydroxymethyl-1,3-propanediol, that is the aceptor, its concentration in the compositions con-templated herein must range from 0.7 molar to 2 molar.
- 6a -B
. . . :
. .
~$~3~34~ ~
The present Example relates to the preparation of 50 kilograms of reagent powder starting from the salts of the p-nitrophenylphosphoric acid (pNPP) with 2-amino--2-ethyl-1,3-propanediol (2A2E1, 3PD), with 2-amino-2--methyl-1,3-propanediol (2A2M1, 3PD~ and with cyclo-hexylamine (CEA). The acceptOr, that is~ 2-amino-2--hydroxymethyl-1,3-propanediol has been called herein TRIS for short. There have been dried about 1.5 kg of pNPP-CEA.H20 under vacuum and over P205 at a temperature not excee~ling 30C, for about 48 hours in the dark. The final moisture contents must be 1.5% as a maximum.
As rcgards the pNPP-2A2E1, 3PD~ or the pNPP-2A2M1, 3PD~ the drying step was carried out only if the moisture contents of the product was over 0.5%.
The grit size of the several ingredients (TRIS, pNPP, Mg acetate, Na borate) was made uniform by grinding and/or screening. ~ -There were mixecl subsequently 48.35 kilograms of ~
TRIS with 68.6 grams of Mg acetate.4H20 and with 683.5 - ~-grams of Na tetraborate 10H20 until homogeneization of the powders was achieved.
The powder was placed on stainless steel trays~
plates or other suitable supporting vessels and dried at 70C for about 14 hours.
The whole was cooled in an anhydrous environment and screened again to make the grit size of the mixture uniform. The final moisture contents had to be less than o.6%.
There have been obtained 48.76 kilograms of base powder. The base powder was combined with 1.37 kilo-grams of pNPP-CAE ( or with 1.49 kilograms of pNPP-2A2E1, 3PD~ or with 1 40 kilograms of pNPP-2A2M1, 3PD) and the entire lot was thoroughly blended j~
A statistical cbeck for ascertaining the homogeneity ~`:
7.
: -~13~
, of the mixture has been carried out by sampling random scantling~s (3-S grams of mixture plus 20 mls H20) and determining thereon the pH, the magnesium and the pNPP.
The final moisture contents was not over 0.8%.
S Distribution . -By using the different pNPP, there have been obtain-ed~ respectively, 50.1~ kilograms when using pNPP-CEA
50.25 kilograms when using pNPP-2A2E; ~3PD II
50.16 kilograms when using pNPP-2A2M1,3PD III
T~ the end of the practical use of the reagent~ the following considerations have been made. An individual ALP test (3 mls) corresponds to 483 milligrams of I or of III and to 484 milligrams of II.
Since, by dissolving the powder in a certain volume of H20, the volume is increased by about 12.5%~ it is possible to calculate the weight of powder to be distributed in the flask~ to which there must be added 20 mls of H20, 22.5 mls being obtained as the dissolution is completed.
483 by 22.5 : 3 = ~622 milligrams per flask of I or of III
484 by 22.5 : 3 = 3630 milligrams per flaslc of II
In the two-litre flasks of complete solution~ there must be distributed~ conversely :
322 grams of I or of III~ and 323 grams of II, respectively.
One of the reagents as prepared according to the previous Example has been employed for determining the alkaline phosphatase activity.
The reagent has been heated previously to the react-ion temperature and then a 3-ml scantlin~s has been admixed with 0.05 mls of the sample to be tested. The whole has been placed in a photometric readout apparatus at 405 mm at the preselected constant temperature.
8.
3B~?~
The variatiol~ of the optical density ( ~ O.D.min has becn measured. For ~ O.D.min higher than 0.750 (equivalent to about 2~500 mU/ml) the measurement was repeated on a sample diluted to 1:10. In this case the result ha~ to be multiplied by 10.
The ~ O.D.min which had been found was introduced in the following calculation, to obtain the mU/ml :
1000 Vt h O.D.min by ---~----~------ = mU/ml 18.6 .Lp.Vs wherein :
1000 is the factor to pass from units to thousandths of units.
Vt is the total reaction volume, i.e. 3.05 mls 18.6 is the millimola~extinction coefficient of pNP
L is the light path which is l cm V is the volume of the sample being tested~ which is 0.05 ml Now~ therefore :
mU/ml = /~ O.D.min multiplied by 3280 Comparison tests have been carried out between the composition hereof and a conventlonal composition for the . .
determination of the alkaline phosphatase~ the conventional composition being made up by the salt of pNPP with CEA
magnesium aspartate and a carbonate buffer. There have been used the same quantities as specified in the previous Example at a temperature of 37C.
The results are plotted in the graph of FIGURE 1 `
wherein the abscissae~.indicate the mU/ml which have been
It is known that alkaline phosphatase is predominantly concentrated in the intertinal epithelium, in the portion of the bones which undergo growth, in the kidney cortex, in the mammary glands, in the liver, in the bile, in the blood and in the placenta as well. The action of the alkaline phosphatase is manifested in the catalysis of hydrolysis reactions of orthophosphates according to the reaction scheme:
R - O - PO(OH)z ~ H20 ~ R - OH + H3PO4 1) and its presence at high concentrations in the blood is evidence of a certain pathological condition of the hyman system:
an increase of the phosphatase activity is an indication, for example, of disease of hepatic, intestlnal or bone origin.
The first determination of the alkallne phosphatase in the human-blood-serum dates back to 1930 (Kay, H.D., J. Biol.
Chem., 89 235 (1930!, and since those early times, the methods of assay of the enzymic activity have undergone many changes, especlally due to the influence thereon of the medium in which the reaction is caused to take place.
A number of the methods which are adapted for the determination of the phosphatase activity are now based ( J. Biol., Chem., 172, 1, (1948) ) on the discovery, by Axelrod, of the fact that the phosphatase enzyme is also capable of catalytically spurring the transphosphorylation reaction between the orthophosphate indicated in the reaction pattern (1) reported ab~ve, and an alcohol, which acts as a , 84~
receptor.
An optical method is now being widely used for the determination of the activity of the alkaline phosphatase, such optical method providing for the use of p-nitrophenyl-phosphates as the first substrate for carrying out the trans-phosphorylation reaction mentioned above. The method provides for the admixing of the first substrate, to the second substrate (acceptor) and the enzyme, under the appropriate conditions, and the readout of the optical density at 400-415 nm, the readings being progressively taken during progress of the reaction. The increase of the optical density, as time goes by, is proportional to the quantity of the product which is being transformed so that an appropriate recordal of the data permits to deduce the activity of the enzyme.
Now, it is well known ( see, for example, the book "The Enzymes", by Academic Press, 1961, pages 55 and ff), that alkaline phosphatase has an optimum activity in the vicinity of a pH of 10, and that its activity is improved by the presence of mineral salts, especially magnesium salts. These facts have compelled all the preparations which are commercially used for the determination of the phosphatase enzyme, to use ingredients which are both rigorously defined and legally required.
Thus, the use has become widespread of derivatives of p-nitrophenylphosphoric acid in combination with organic magnesium salts and buffers as required to maintain the above indicated pH range. The reagents that were initially used, were the sodium salts of the acid and inorganic buffers.
It has been ascertained that the sodium-nitrophenyl-phosphate has a poor stability and thus, in more recent times,it has been preferred to resort to the use of salts of the acid in question with amine compounds.
~13~
.
The preparation of p-nitrophenylphosphates, including those of amine compounds, and their use in biological formula-tions, are well known (see, for example, JACS, 79, (1957), page 3741.
Attempts have also been made with a view to improv-ing the progress of the transphosphorylation reaction and, in the initial composition, the acceptor substances have been thus changed until arriving, lastly, at the use of buffers which simultaneously displayed the twofold action of keeping the pH
value constant while concurrently acting as acceptors in the transphosphorylation reaction.
The care which must be taken when preparing the several buffers must not be overlooked, and such a care has, of course, its bearing on the overall economy of the reagent and the analysis. However, all the mixtures as used hereto-fore still suffer from a number of drawbacks, the most prominent of which is the poor stability of the reagent in time. Even if they are stored under drastic conditions, the prior art composi-tions cannot be used with reliability as to the reproducibility of the result, more than two or three days from their prepara-tion, at the most.
In addition, many of the conventional reagents, such as those which use mannitol and like polyhydric alcohols as the acceptors in the transphosphorylation reaction, are capable of determining with a good sensitivity also the alkaline phos-phatase of placental origin.
In the majority of the cases, dosing the activity of the alkaline phosphatase is carried out in order to detect pathological conditions which can be attributed to the hepatic system (obstruction jaundice),the bone system (such as tumours, and Paget's disease), or the bowel system. Thus, in the case of pregnancy, the high physiological activity of the alkaline .. ,, :
13~34~
phosphatase in the blood may mask one or more of the patho-logical patterns mentioned a~ove.
The Applicants have now surprisingly ascertained that a novei composition permits that the determination of the alkaline phosphatase may be carried ou-t to give reproducible results without suffering from any of the shortcomings recalled hereinabove.
According to the present invention there is provided a composition for the analysis of alkaline phosphatase, said composition comprising a salt of p-nitrophenylphosphoric acid, a magnesium salt, sodium borate and 2-amino-2-hydroxymethyl-1,3-propanediol.
According to another aspect of the present invention there is provided a method for the determination of alkaline phosphatase comprising contacting the sample to be tested with a composition comprising a salt of p-nitrophenylphosphoric acid, a salt of magnesium, sodium borate and 2-amino-2-hydroxymethyl-1,3-propanediol and thereafter photometrically measuring the amount of alkaline phosphatase.
The present invention also provides a method for the determination of non-placental alkaline phosphatase which com-prises contacting the sample to be tested with a composition comprising a salt of p-nitrophenylphosphoric acid, a salt of magnesium, sodium borate and 2-amino-2-hydroxymethyl-1,3- propa-nediol and thereafter photometrically determining the amount of alkaline phosphatase.
The composition according to the present invention is stable and retains for many days its initial properties; it is only slightly sensitive to the presence of phosphatases of placental origin. Moreover, it does not require an accurate -preparation of a buffer, since the pH of the transphosphorilation reaction is provided by the ingredients of the composition them-selves.
.~ .
. . . r ~ ' "
' '.' ' ~. ' .
~1384~
The method suggested herein provides, obviously, the reaction sequence ()1'1 -: -¦¦ +ROH ~ + ROPO3 Mg++
I
again, in which ALP is the alkaline phosphatase (EC 3.13.1) and ROH is the propanediol mentioned above. Inasmuch as the reaction takes place at an alkaline pH value, the hydrolysis of the p-nitrophenylphosphate to p-nitrophenol is monitored by merely reading out the extinction at 405 nm.
As a matter of fact, under alkaline pH conditions, the p-nitrophenol is strongly coloured in yellow whereas the p-nitrophenylphosphate is colorless.
The method permits to meter the alkaline phosphatase activity with a single mixture of reagents: it can be used manually, or also by applying it to automatic discrete and -;
continuous-flow apparatuses. It can be used also for kinetic determinations and also for fixed time determinations by the addition o a blocking agent.
For performing the method many starting substrates can be used, even though, of course, it is preferred to avoid the use of the sodium salt of the p-nitrophenylphosphoric acid, on account of the tendency of the latter to become hydrolyzed spontaneously. Thus, there can be lndicated as Examples the salts of the 2-amino-2-methyl-],3-propanediol, of 2-amino-2-ethyl-1,3-propanediol, of 2-amino-2-hydroxymethyl-1,3-propanediol, of cyclohoxyla~ c, just to ciLc a Lcw oE tl~o~
The donor substrate, obviously, can be formulated as desired, since the affinity towards the enzyme is due to the p-nitrophenylphosphoric acid rather than to the salification agent.
:; '.
~3~4~
Likewise, the magnesium salt can be selected from among a wide variety of compounds such as acetates, aspartates, chlorides, sulfates.
Good results have been obtained, for example by employing .he salt of cyclohexylamine of p-nitrophenylphosphoric acid as the donor substrate and the acetate of magnesium as the activator of the enzymic reaction.
As regards the 2-amino-2-hydroxymethyl-1,3-propanediol, that is the aceptor, its concentration in the compositions con-templated herein must range from 0.7 molar to 2 molar.
- 6a -B
. . . :
. .
~$~3~34~ ~
The present Example relates to the preparation of 50 kilograms of reagent powder starting from the salts of the p-nitrophenylphosphoric acid (pNPP) with 2-amino--2-ethyl-1,3-propanediol (2A2E1, 3PD), with 2-amino-2--methyl-1,3-propanediol (2A2M1, 3PD~ and with cyclo-hexylamine (CEA). The acceptOr, that is~ 2-amino-2--hydroxymethyl-1,3-propanediol has been called herein TRIS for short. There have been dried about 1.5 kg of pNPP-CEA.H20 under vacuum and over P205 at a temperature not excee~ling 30C, for about 48 hours in the dark. The final moisture contents must be 1.5% as a maximum.
As rcgards the pNPP-2A2E1, 3PD~ or the pNPP-2A2M1, 3PD~ the drying step was carried out only if the moisture contents of the product was over 0.5%.
The grit size of the several ingredients (TRIS, pNPP, Mg acetate, Na borate) was made uniform by grinding and/or screening. ~ -There were mixecl subsequently 48.35 kilograms of ~
TRIS with 68.6 grams of Mg acetate.4H20 and with 683.5 - ~-grams of Na tetraborate 10H20 until homogeneization of the powders was achieved.
The powder was placed on stainless steel trays~
plates or other suitable supporting vessels and dried at 70C for about 14 hours.
The whole was cooled in an anhydrous environment and screened again to make the grit size of the mixture uniform. The final moisture contents had to be less than o.6%.
There have been obtained 48.76 kilograms of base powder. The base powder was combined with 1.37 kilo-grams of pNPP-CAE ( or with 1.49 kilograms of pNPP-2A2E1, 3PD~ or with 1 40 kilograms of pNPP-2A2M1, 3PD) and the entire lot was thoroughly blended j~
A statistical cbeck for ascertaining the homogeneity ~`:
7.
: -~13~
, of the mixture has been carried out by sampling random scantling~s (3-S grams of mixture plus 20 mls H20) and determining thereon the pH, the magnesium and the pNPP.
The final moisture contents was not over 0.8%.
S Distribution . -By using the different pNPP, there have been obtain-ed~ respectively, 50.1~ kilograms when using pNPP-CEA
50.25 kilograms when using pNPP-2A2E; ~3PD II
50.16 kilograms when using pNPP-2A2M1,3PD III
T~ the end of the practical use of the reagent~ the following considerations have been made. An individual ALP test (3 mls) corresponds to 483 milligrams of I or of III and to 484 milligrams of II.
Since, by dissolving the powder in a certain volume of H20, the volume is increased by about 12.5%~ it is possible to calculate the weight of powder to be distributed in the flask~ to which there must be added 20 mls of H20, 22.5 mls being obtained as the dissolution is completed.
483 by 22.5 : 3 = ~622 milligrams per flask of I or of III
484 by 22.5 : 3 = 3630 milligrams per flaslc of II
In the two-litre flasks of complete solution~ there must be distributed~ conversely :
322 grams of I or of III~ and 323 grams of II, respectively.
One of the reagents as prepared according to the previous Example has been employed for determining the alkaline phosphatase activity.
The reagent has been heated previously to the react-ion temperature and then a 3-ml scantlin~s has been admixed with 0.05 mls of the sample to be tested. The whole has been placed in a photometric readout apparatus at 405 mm at the preselected constant temperature.
8.
3B~?~
The variatiol~ of the optical density ( ~ O.D.min has becn measured. For ~ O.D.min higher than 0.750 (equivalent to about 2~500 mU/ml) the measurement was repeated on a sample diluted to 1:10. In this case the result ha~ to be multiplied by 10.
The ~ O.D.min which had been found was introduced in the following calculation, to obtain the mU/ml :
1000 Vt h O.D.min by ---~----~------ = mU/ml 18.6 .Lp.Vs wherein :
1000 is the factor to pass from units to thousandths of units.
Vt is the total reaction volume, i.e. 3.05 mls 18.6 is the millimola~extinction coefficient of pNP
L is the light path which is l cm V is the volume of the sample being tested~ which is 0.05 ml Now~ therefore :
mU/ml = /~ O.D.min multiplied by 3280 Comparison tests have been carried out between the composition hereof and a conventlonal composition for the . .
determination of the alkaline phosphatase~ the conventional composition being made up by the salt of pNPP with CEA
magnesium aspartate and a carbonate buffer. There have been used the same quantities as specified in the previous Example at a temperature of 37C.
The results are plotted in the graph of FIGURE 1 `
wherein the abscissae~.indicate the mU/ml which have been
3 obtained when using the composition according to this invention~ whereas the ordinates indicate the mU/ml which have been obtained by using the conventional composition.
The straigrht line l is the ideal curve which would be obtained if both compositions should give the same -13~
results. The marking 0 (circled dots) are referred to samples of phosphatase of placental origin.~ The straight line 2 is referred to the actual linear regression curve of the conventional composition relative to the composition of the invention. Such a regression is evidence of the fact that the method disclosed herein is more sensitive than the conventional one and is such by more than 20% (see the angular deviation relative to the ideal curve).
Such an improved sensitivity is not experienced for the phosphatases of placental origin: in the case of this invention, thus the placental phosphatase is undetermined relative to the conventional methods. The uncircled dots, in fact, refer to samples which contain phosphatase of different origins, such as hepatic, bony, intestinal. The curve 2 reproduces the equation Y = 0.727x + 9.01, the curve 1 being obviously the reproduction with Y = x.
The straigrht line l is the ideal curve which would be obtained if both compositions should give the same -13~
results. The marking 0 (circled dots) are referred to samples of phosphatase of placental origin.~ The straight line 2 is referred to the actual linear regression curve of the conventional composition relative to the composition of the invention. Such a regression is evidence of the fact that the method disclosed herein is more sensitive than the conventional one and is such by more than 20% (see the angular deviation relative to the ideal curve).
Such an improved sensitivity is not experienced for the phosphatases of placental origin: in the case of this invention, thus the placental phosphatase is undetermined relative to the conventional methods. The uncircled dots, in fact, refer to samples which contain phosphatase of different origins, such as hepatic, bony, intestinal. The curve 2 reproduces the equation Y = 0.727x + 9.01, the curve 1 being obviously the reproduction with Y = x.
Claims (8)
1. A composition for the analysis of alkaline phosphatase, said conposition comprising a salt of p-nitro-phenylphosphoric acid, a magnesium salt, sodium borate and 2-amino-2-hydroxymethyl-1,3-propanediol.
2. A composition according to claim 1, wherein the 2-amino-2-hydroxymethyl-1,3-propanediol is present at a concentration variable from 0.7 molar to 2-molar.
3. A composition according to claim 1, wherein the salt of p-nitrophenylphosphoric acid is selected from the group consisting of the salts of 2-amino-2-methyl-1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, and cyclohexylamine.
4. A composition according to claim 2, wherein the salt of p-nitrophenylphosphoric acid is selected from the group consisting of the salts of 2-amino-2-methyl-1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, and cylcohexylamine.
5. A composition according to any one of claims 1, 2 and 3, wherein the magnesium salt is selected from the group consisting of magnesium acetate, magnesium aspartate, magnesium chloride and magnesium sulfate.
6. A composition according to claim 4, wherein the magnesium salt is selected from the group consisting of magnesium acetate, magnesium aspartate, magnesium chloride and magnesium sulfate.
7. A method for the determination of alkaline phospha-tase comprising contacting the sample to be tested with a compo-sition comprising a salt of p-nitrophenylphosphoric acid, a salt of magnesium, sodium borate and 2-amino-2-hydroxymethyl-1,3-propanediol and thereafter photometrically measuring the amount of alkaline phosphatase.
8. A method for the determination of non-placental alkaline phosphatase which comprises contacting the sample to be tested with a composition comprising a salt of p-nitrophenyl-phosphoric acid, a salt of magnesium, sodium borate and 2-amino-2-hydroxymethyl-1,3-propanediol and thereafter photometrically determining the amount of alkaline phosphatase.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT19458/78A IT1108964B (en) | 1978-01-20 | 1978-01-20 | COMPOSITION SUITABLE FOR THE ANALYSIS OF ALKALINE PHOSPHATASE AND METHOD USING THE SAME |
| IT19458A/78 | 1978-01-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1113840A true CA1113840A (en) | 1981-12-08 |
Family
ID=11158184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA315,458A Expired CA1113840A (en) | 1978-01-20 | 1978-10-31 | Composition for the analysis of the alkaline phosphatase and method therefor |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US4306020A (en) |
| JP (1) | JPS54104390A (en) |
| AR (1) | AR218954A1 (en) |
| BR (1) | BR7900801A (en) |
| CA (1) | CA1113840A (en) |
| DE (1) | DE2845156A1 (en) |
| ES (1) | ES477040A1 (en) |
| FR (1) | FR2415300A1 (en) |
| IT (1) | IT1108964B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4555484A (en) * | 1983-07-25 | 1985-11-26 | Eastman Kodak Company | Analytical element and method for alkaline phosphatase assay |
| JPS61110058A (en) * | 1984-11-02 | 1986-05-28 | Fuji Photo Film Co Ltd | Integrated type multilayer analysis element for measuring alkaline phosphatase activity |
| JP2668656B2 (en) * | 1994-09-05 | 1997-10-27 | コニカ株式会社 | Dry analytical element for alkaline phosphatase |
| AU2003298036A1 (en) * | 2002-12-12 | 2004-07-09 | Zoltan Laboratories | Placental alkaline phosphatase to control diabetes |
| US7048914B2 (en) * | 2002-12-12 | 2006-05-23 | Zoltan Laboratories | Placental alkaline phosphatase to control diabetes |
| GB0318110D0 (en) * | 2003-08-01 | 2003-09-03 | Isaeo Ltd | Methods and kits for detecting an enzyme capable of modifying a nucleic acid |
| WO2007075199A1 (en) * | 2005-12-28 | 2007-07-05 | Zoltan Laboratories Llc | Use of alkaline phosphatase to maintain healthy tissue mass in mammals |
| JP4871630B2 (en) * | 2006-04-12 | 2012-02-08 | 第一三共株式会社 | A method for inhibiting cell growth, comprising inhibiting phosphatase |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2359052A (en) * | 1940-07-01 | 1944-09-26 | Scharer Harry | Method for detecting enzyme activity |
| US3466306A (en) * | 1965-08-06 | 1969-09-09 | Warner Lambert Pharmaceutical | Process for the stabilization of organic esters of phosphoric acid |
| US3425912A (en) * | 1966-11-14 | 1969-02-04 | Smithkline Corp | Laboratory reagent for assay of alkaline phosphatase |
| US3595756A (en) * | 1968-11-26 | 1971-07-27 | Smith Kline French Lab | Laboratory reagent for assay of alkaline phosphatase |
| US4132598A (en) * | 1977-06-03 | 1979-01-02 | Modrovich Ivan Endre | Stabilized liquid phosphate containing diagnostic compositions and method of preparing same |
-
1978
- 1978-01-20 IT IT19458/78A patent/IT1108964B/en active
- 1978-10-17 DE DE19782845156 patent/DE2845156A1/en not_active Ceased
- 1978-10-23 JP JP12955678A patent/JPS54104390A/en active Granted
- 1978-10-31 CA CA315,458A patent/CA1113840A/en not_active Expired
- 1978-11-14 FR FR7832150A patent/FR2415300A1/en active Granted
-
1979
- 1979-01-12 ES ES477040A patent/ES477040A1/en not_active Expired
- 1979-01-17 AR AR275205A patent/AR218954A1/en active
- 1979-01-22 BR BR7900801A patent/BR7900801A/en unknown
-
1980
- 1980-09-25 US US06/190,794 patent/US4306020A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| ES477040A1 (en) | 1979-10-16 |
| BR7900801A (en) | 1979-09-04 |
| JPS54104390A (en) | 1979-08-16 |
| IT1108964B (en) | 1985-12-16 |
| JPS6130560B2 (en) | 1986-07-14 |
| AR218954A1 (en) | 1980-07-15 |
| DE2845156A1 (en) | 1979-07-26 |
| FR2415300B1 (en) | 1983-02-18 |
| FR2415300A1 (en) | 1979-08-17 |
| US4306020A (en) | 1981-12-15 |
| IT7819458A0 (en) | 1978-01-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nachlas et al. | The determination of lactic dehydrogenase with a tetrazolium salt | |
| Bowers Jr et al. | A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase | |
| Kramer et al. | Clinical enzymology | |
| Baginski et al. | Glucose-6-phosphatase | |
| Nostrandt et al. | A modified spectrophotometric method appropriate for measuring cholinesterase activity in tissue from carbaryl-treated animals | |
| US3968011A (en) | Test implement and test method for colorimetrically determining whether a female is fertile or pregnant | |
| EP0132830B1 (en) | Dry analytical element for alkaline phosphatase assay | |
| CA1113840A (en) | Composition for the analysis of the alkaline phosphatase and method therefor | |
| PT1456404E (en) | A method for determining the biological activity of defibrotide | |
| US3002893A (en) | Method for the determination of serum acid phosphatase and diagnostic preparation therefor | |
| Pette et al. | Kinetic microphotometric activity determination in enzyme containing gels and model studies with tissue sections | |
| US3493467A (en) | Reversible biochemical reaction employing a trapping agent | |
| US3876502A (en) | Reagent formulations for assaying urea nitrogen in biological specimens and methods of preparing and using same | |
| Zimmerman | A rapid assay for pectinesterase activity which can be used as a prescreen for pectinesterase inhibitors | |
| US3928137A (en) | Reagent formulation for uric acid assay | |
| Levin | Calorimetry, a time-honored technique with a potential in analytical work and cellular biology. | |
| US3278394A (en) | Method and composition for diagnosing glucose | |
| McGlothlin et al. | Thermochemical determination of glucose in serum, plasma, and whole blood without prior deproteinization | |
| US3546074A (en) | Reagent for assaying glutamate oxaloacetate transaminase | |
| US3530040A (en) | Test composition,device and method for detecting urea in aqueous fluids | |
| US3551296A (en) | Reagent and method for assaying malate dehydrogenase | |
| US3926735A (en) | Alkaline phosphatase assay | |
| US3527332A (en) | Method for assaying glutamate pyruvate transaminase | |
| JPS60501489A (en) | Method and composition for measuring γ-glutamyl transpeptidase | |
| Rinker et al. | IFCC methods for the measurement of catalytic concentration of enzymes Part 5. IFCC method for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, alkaline optimum, EC 3.1. 3.1) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |