CS240043B1 - Electron acceptor for lactate concentration determination by means of b2 cytochrome - Google Patents
Electron acceptor for lactate concentration determination by means of b2 cytochrome Download PDFInfo
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- CS240043B1 CS240043B1 CS846744A CS674484A CS240043B1 CS 240043 B1 CS240043 B1 CS 240043B1 CS 846744 A CS846744 A CS 846744A CS 674484 A CS674484 A CS 674484A CS 240043 B1 CS240043 B1 CS 240043B1
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- lactate
- cytochrome
- concentration
- electron acceptor
- lactate concentration
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 title claims abstract description 18
- 102000018832 Cytochromes Human genes 0.000 title claims 2
- 108010052832 Cytochromes Proteins 0.000 title claims 2
- 125000003831 tetrazolyl group Chemical group 0.000 claims abstract description 4
- 238000002835 absorbance Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 208000010125 myocardial infarction Diseases 0.000 abstract description 2
- 208000010444 Acidosis Diseases 0.000 abstract 1
- 108090000841 L-Lactate Dehydrogenase (Cytochrome) Proteins 0.000 abstract 1
- 230000007950 acidosis Effects 0.000 abstract 1
- 208000026545 acidosis disease Diseases 0.000 abstract 1
- 238000002845 discoloration Methods 0.000 abstract 1
- 238000011156 evaluation Methods 0.000 abstract 1
- 230000005484 gravity Effects 0.000 abstract 1
- 238000001429 visible spectrum Methods 0.000 abstract 1
- 102100025287 Cytochrome b Human genes 0.000 description 6
- 108010075028 Cytochromes b Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 3
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 3
- 229950006238 nadide Drugs 0.000 description 3
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 3
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 2
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108091006149 Electron carriers Proteins 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 150000001875 compounds Chemical class 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
- 229930195712 glutamate Natural products 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 208000006443 lactic acidosis Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 239000002824 redox indicator Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000005891 transamination reaction Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Použití tetrazoliové soli jako akceptoru elektronů při stanovení koncentrace laktátu pomocí cytochromu b2 má za následek významné zvýšení citlivosti reakce, dosažení pozitivní změny absorbance a v neposlední řadě i možnost měřit výsledné zabarvení ve viditelné oblasti spektra. Využití řešení je vhodné zejména pro laboratoře klinické biochemie a jiná pracoviště, kde se provádí stanovení koncentrace laktátu při hodnocení tíže laktátové acidózy u nemocných v těžkém stavu při určeni anaerobního práhu u pacientů po infarktu myokardu a u sportovcUse of a tetrazolium salt as an acceptor electrons in determining concentration of lactate by cytochrome b2 has resulting in a significant increase in response sensitivity, achieving a positive change in absorbance a Last but not least, the ability to measure the resulting visible spectrum discoloration. The use of the solution is particularly suitable for laboratories clinical biochemistry and other sites where the concentration determination is performed lactate in the evaluation of lactate gravity Acidosis in Patients in Severe State of the art determining the anaerobic threshold in patients after myocardial infarction and athletes
Description
Vynález se týká použití tetrazoliové soli jako akeeptoru elektronů při stanovení koncentrace laktátu pomocí cytochrómu b2·The invention relates to the use of tetrazolium salt as an electron aceeptor in the determination of lactate concentration by cytochrome b 2 .
Kýselina mléčný resp. laktát je důležitý metabolit, vznikající jako konečný produkt anaerobní glykolýzy. Stanovení koncentrace laktátu v plazmě má zásadní význam u nemocných v těžkém stavu, zejména v šoku, kdy pomáhá určit nejen diagnózu laktátové acidózy, ale uplatňuje se i jako ukazatel úspěšnosti léčby a prognózy onemocnění. U pacientů po infarktu myokardu se využívá stanovení koncentrace laktátu v krvi k určení velikosti zátěže, kterou pacient ještě dokáže zvládnout aerobní oxigenací, tj. lze určit vhodnou zátěž pro rehabilitační cvičení. U sportovců dovoluje stanovení koncentrace laktátu v krvi po fyzické zátěži posoudit stupeň trénovánosti.Lactic acid resp. Lactate is an important metabolite produced as the end product of anaerobic glycolysis. Determination of lactate concentration in plasma is essential in patients in severe condition, especially in shock, where it helps to determine not only the diagnosis of lactic acidosis, but it is also used as an indicator of treatment success and disease prognosis. In patients after myocardial infarction, blood lactate concentration is used to determine the amount of exercise that the patient is still able to cope with by aerobic oxigenation, ie to determine the appropriate load for rehabilitation exercise. In athletes, the determination of the lactate concentration in the blood after physical exercise allows to assess the degree of training.
Ke stanovení koncentrace laktátu v plazmě se obvykle užívá jedné ze dvou specifických enzymových metod. Prvá spočívá v oxidaci laktátu na pyruvát nikotinamidadenindinukleotidem za katalýzy živočišnou laktótdehydrogenasou. Vznik redukované formy nikotinamidadenindinukleotidu se projeví nárůstem absorbance v ultrafialové oblasti spektra. Protože reakční rovnováha je nepříznivá pro uvedený směr reakce, je třeba odčerpávat jeden z produktů, obvykle pyruvát. K tomu lze např. využít transaminační reakce s glutamátem za katalýzy alaninaminotransferasou. Tento princip je užit v práci Nolla (in: Bergmeyer, H. U., ed.: Methoden der enzymatischen Analyse, 2. vyd., díl 2, Akademie Verlag, Berlin 1970, s. 1433 eě 1437) a je na něm založeno i u nás užívané stanovení koncentrace laktátu pomocí soupravy Lactat vollenzymatisch firmy Boehringer Mannheim. Nutnost přítomnosti dvou enzymů, koenzymu a dalšího pomocného substrátu prodražuje stanovení. Další nevýhodou je nezbytnost užití fotometru s možností měřit v ultrafialové oblasti spektra; tento fotometr jeUsually one of two specific enzyme methods is used to determine the plasma lactate concentration. The first is the oxidation of lactate to pyruvate by nicotinamide adenine dinucleotide under catalysis by animal lactose dehydrogenase. The formation of a reduced form of nicotinamide adenine dinucleotide results in an increase in absorbance in the ultraviolet region of the spectrum. Since the reaction equilibrium is unfavorable for said reaction direction, one of the products, usually pyruvate, must be pumped off. For example, a transamination reaction with glutamate under alanine aminotransferase catalysis can be used. This principle is used in the work of Noll (in: Bergmeyer, HU, ed .: Methoden der Enzymatischen Analyze, 2nd Ed., Volume 2, Verlag Academy, Berlin 1970, pp. 1433 and 1437) and is also based on the determination of lactate concentration using the Lactat vollenzymatisch kit from Boehringer Mannheim. The need for the presence of two enzymes, coenzyme and another auxiliary substrate, makes the assay more expensive. Another disadvantage is the necessity of using a photometer with the possibility of measuring in the ultraviolet range of the spectrum; this photometer is
- 2 240 043 drahý a v menších laboratořích klinické biochemie nedostupný·- 2 240 043 expensive and unavailable in smaller clinical biochemistry laboratories ·
Druhá metoda využívá cytochrom b2, obvykle izolovaný z aerobních kvasinek, např. z pekařského droždí. Tento enzym působí také jako laktátdehydrogenasa a katalyzuje přeměnu laktátu na pyruvát. Enzym nepotřebuje žádný koenzym a jako umělý akceptor elektronů je využíván ferrikyanid draselný. Tuto metodu poprvé popsal Wieland (Biochem. Z. 329, 1958, č. 7, s. 568« 576) a užili ji později i jiní autoři, např. Durliat a spol. (Clin. Chem. 22, 1976, δ. 11, s. 1802 « 1805). Průběh reakce je sledován jako pokles absorbance v krátké oblasti viditelného světla, způsobený přeměnou žlutého ferrikyanidu na bezbarvý ferrokyanid. Nevýhodou stanovení je malá citlivost a obtížný způsob automatizace při záporné změně absorbance.The second method uses cytochrome b 2 , usually isolated from aerobic yeast, such as baker's yeast. This enzyme also acts as lactate dehydrogenase and catalyzes the conversion of lactate to pyruvate. The enzyme does not need any coenzyme, and potassium fericyanide is used as an artificial electron acceptor. This method was first described by Wieland (Biochem. Z. 329, 1958, No. 7, p. 568-576) and was later used by other authors such as Durliat et al. (Clin. Chem. 22, 1976, p. 11, p. 1802-1805). The course of the reaction is observed as a decrease in absorbance in the short region of visible light due to the conversion of yellow ferriyanide to colorless ferrocyanide. The disadvantage of the assay is its low sensitivity and difficult automation with negative absorbance changes.
Podstata akceptoru elektronů při stanovení koncentrace laktátu pomocí cytochromu b2 spočívá v použiti tetrazoliové soli. Tato sloučenina, obvykle užívaná jako redoxní indikátor k průkazu redukované formy nikotinamidadenindinukleotidu nebo sulfhydrylových skupin, je v tomto případě redukována laktétem za katalytického působení cytochromu b2 na barevný formazan; nárůst absorbance je měřen ve viditelné oblasti spektra.The principle of the electron acceptor in the determination of the lactate concentration by cytochrome b 2 is the use of the tetrazolium salt. This compound, commonly used as a redox indicator to detect a reduced form of nicotinamide adenine dinucleotide or sulfhydryl groups, is in this case reduced with lactate under the catalytic action of cytochrome b 2 to formazan; the increase in absorbance is measured in the visible region of the spectrum.
Výhodou tohoto stanovení je jednoduchost a vysoká citlivost, téměř desetkrát vyšší než při užití ferrikyanidu. Protože dochází k nárůstu absorbance, lze výsledky snadno automaticky vyhodnocovat. Vzniklé zabarvení je měřeno ve viditelné oblasti spektra, čímž je stanovení zpřístupněno i těm laboratořím, které nejsou vybaveny spektrofotometrem pro měření v ultrafialovém světle. Izolace cytochromu b2 je levné a pro jednoduchost postupu snadno realizovatelná.The advantage of this assay is its simplicity and high sensitivity, almost ten times higher than when using ferriic anhydride. As the absorbance increases, the results can be easily evaluated automatically. The resulting color is measured in the visible region of the spectrum, making the assay available to those laboratories that are not equipped with a spectrophotometer for ultraviolet light measurements. Isolation of cytochrome b 2 is cheap and easy to implement for simplicity of procedure.
Příklad 1Example 1
Při stanovení koncentrace laktátu pomocí cytochromu b2 ee jako akceptor elektronů užije 3,3’-dianisol-4,4’-bis[2-(4-nitrofenyl)-5-fenyltetrazoliumehlorid] v konečné koncentraci 0,4 mrnol/1 a jako přenašeč elektronů N-metylfenaziniummetylsulfát v konečné koncentraci 0,15 mmol/1. Vzniklé zabarvení se měří při vlnové délce 530 nm.When determining the lactate concentration using cytochrome b 2 ee, the electron acceptor used is 3,3'-dianisole-4,4'-bis [2- (4-nitrophenyl) -5-phenyltetrazolium chloride] at a final concentration of 0.4 mol / l and as electron carrier N-methylphenazinium methyl sulfate at a final concentration of 0.15 mmol / l. The resulting color is measured at a wavelength of 530 nm.
- 3 Příklad 2 240 «43- 3 Example 2 240 «43
Po elektroforéze v gradientovém polyakrylamidovém gelu následuje inkubace v roztoku obsahujícím laktát v koncentraci 50 mmol/1, 3-(4-, 5-dimetylthiazol-2-yl)-2,5-difenyltetrazoliumbromid v koncentraci 2,0 mmol/1 a N-metylfenaziniummetylsulfát v koncentraci 0,2 mmol/1. Postup slouží k charakterizaci laktátdehydrogenasy po její izolaci z kvasinek.Gradient polyacrylamide gel electrophoresis is followed by incubation in a solution containing lactate at a concentration of 50 mmol / l, 3- (4-, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide at a concentration of 2.0 mmol / l and N- methylphenazinium methyl sulfate at a concentration of 0.2 mmol / l. The procedure serves to characterize lactate dehydrogenase after its isolation from yeast.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS846744A CS240043B1 (en) | 1984-09-07 | 1984-09-07 | Electron acceptor for lactate concentration determination by means of b2 cytochrome |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS846744A CS240043B1 (en) | 1984-09-07 | 1984-09-07 | Electron acceptor for lactate concentration determination by means of b2 cytochrome |
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| Publication Number | Publication Date |
|---|---|
| CS674484A1 CS674484A1 (en) | 1985-06-13 |
| CS240043B1 true CS240043B1 (en) | 1986-02-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS846744A CS240043B1 (en) | 1984-09-07 | 1984-09-07 | Electron acceptor for lactate concentration determination by means of b2 cytochrome |
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1984
- 1984-09-07 CS CS846744A patent/CS240043B1/en unknown
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| Publication number | Publication date |
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| CS674484A1 (en) | 1985-06-13 |
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