CS269205B1 - Stabilized solution of chromogene for urea's enzymatic determination by means of berthelot reaction - Google Patents
Stabilized solution of chromogene for urea's enzymatic determination by means of berthelot reaction Download PDFInfo
- Publication number
- CS269205B1 CS269205B1 CS881177A CS117788A CS269205B1 CS 269205 B1 CS269205 B1 CS 269205B1 CS 881177 A CS881177 A CS 881177A CS 117788 A CS117788 A CS 117788A CS 269205 B1 CS269205 B1 CS 269205B1
- Authority
- CS
- Czechoslovakia
- Prior art keywords
- solution
- chromogen
- mmol
- urea
- concentration
- Prior art date
Links
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 239000004202 carbamide Substances 0.000 title claims abstract description 23
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 22
- 230000002255 enzymatic effect Effects 0.000 title claims description 4
- 239000000243 solution Substances 0.000 claims abstract description 42
- 108010046334 Urease Proteins 0.000 claims abstract description 22
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000872 buffer Substances 0.000 claims abstract description 6
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 6
- 238000002835 absorbance Methods 0.000 claims abstract description 5
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 5
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims abstract description 4
- 239000008139 complexing agent Substances 0.000 claims abstract description 4
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- 230000002195 synergetic effect Effects 0.000 claims abstract 2
- MGSRCZKZVOBKFT-UHFFFAOYSA-N thymol Chemical compound CC(C)C1=CC=C(C)C=C1O MGSRCZKZVOBKFT-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 claims description 6
- 230000000873 masking effect Effects 0.000 claims description 4
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 claims description 3
- -1 alkali metal citrate Chemical class 0.000 claims description 3
- 235000010323 ascorbic acid Nutrition 0.000 claims description 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 239000011668 ascorbic acid Substances 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 claims description 2
- 229910052783 alkali metal Inorganic materials 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims 2
- NGSJNQJPYFYNJV-UHFFFAOYSA-N 5-methyl-2-propylphenol Chemical compound CCCC1=CC=C(C)C=C1O NGSJNQJPYFYNJV-UHFFFAOYSA-N 0.000 claims 1
- 239000002250 absorbent Substances 0.000 claims 1
- 230000002745 absorbent Effects 0.000 claims 1
- 150000001540 azides Chemical class 0.000 claims 1
- 239000007979 citrate buffer Substances 0.000 claims 1
- XEYBHCRIKKKOSS-UHFFFAOYSA-N disodium;azanylidyneoxidanium;iron(2+);pentacyanide Chemical compound [Na+].[Na+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].[O+]#N XEYBHCRIKKKOSS-UHFFFAOYSA-N 0.000 claims 1
- 239000008055 phosphate buffer solution Substances 0.000 claims 1
- 239000003755 preservative agent Substances 0.000 claims 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims 1
- 235000010265 sodium sulphite Nutrition 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 3
- PXNNOCWWPXGOFZ-UHFFFAOYSA-N 5-ethyl-2-propan-2-ylphenol Chemical compound CCC1=CC=C(C(C)C)C(O)=C1 PXNNOCWWPXGOFZ-UHFFFAOYSA-N 0.000 abstract 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract 1
- 239000002253 acid Substances 0.000 abstract 1
- 229910052799 carbon Inorganic materials 0.000 abstract 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 21
- 235000013877 carbamide Nutrition 0.000 description 17
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 16
- 229910021529 ammonia Inorganic materials 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 5
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical group [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229960004025 sodium salicylate Drugs 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- 239000003054 catalyst Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000008094 contradictory effect Effects 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 238000001952 enzyme assay Methods 0.000 description 3
- RSAZYXZUJROYKR-UHFFFAOYSA-N indophenol Chemical compound C1=CC(O)=CC=C1N=C1C=CC(=O)C=C1 RSAZYXZUJROYKR-UHFFFAOYSA-N 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- BVQVLAIMHVDZEL-UHFFFAOYSA-N 1-phenyl-1,2-propanedione Chemical group CC(=O)C(=O)C1=CC=CC=C1 BVQVLAIMHVDZEL-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001860 citric acid derivatives Chemical class 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000005691 oxidative coupling reaction Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- JXYWFNAQESKDNC-BTJKTKAUSA-N (z)-4-hydroxy-4-oxobut-2-enoate;2-[(4-methoxyphenyl)methyl-pyridin-2-ylamino]ethyl-dimethylazanium Chemical compound OC(=O)\C=C/C(O)=O.C1=CC(OC)=CC=C1CN(CCN(C)C)C1=CC=CC=N1 JXYWFNAQESKDNC-BTJKTKAUSA-N 0.000 description 1
- 208000037157 Azotemia Diseases 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 206010018366 Glomerulonephritis acute Diseases 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- NULAJYZBOLVQPQ-UHFFFAOYSA-N N-(1-naphthyl)ethylenediamine Chemical compound C1=CC=C2C(NCCN)=CC=CC2=C1 NULAJYZBOLVQPQ-UHFFFAOYSA-N 0.000 description 1
- 206010061876 Obstruction Diseases 0.000 description 1
- 241000161214 Pelates Species 0.000 description 1
- 206010037596 Pyelonephritis Diseases 0.000 description 1
- 101100410826 Salmo salar pym1a gene Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 231100000851 acute glomerulonephritis Toxicity 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000318 alkali metal phosphate Inorganic materials 0.000 description 1
- 125000003282 alkyl amino group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- FSEUPUDHEBLWJY-HWKANZROSA-N diacetylmonoxime Chemical compound CC(=O)C(\C)=N\O FSEUPUDHEBLWJY-HWKANZROSA-N 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 150000004673 fluoride salts Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000000687 hydroquinonyl group Chemical class C1(O)=C(C=C(O)C=C1)* 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 150000003891 oxalate salts Chemical class 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- 229940031826 phenolate Drugs 0.000 description 1
- 229960000969 phenyl salicylate Drugs 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 150000003672 ureas Chemical class 0.000 description 1
- 208000009852 uremia Diseases 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Stabilizovaný roztok chromogenu sestává z vodného roztoku 3-»ethyl-6-isopropylfenolu o koncentraci 1 až 5 mmol/1 obsahujícího komplexotvorné látky schopné maskovat těžké kovy o koncentraci 0,05 až 5 mmol/1, jako jeou například kyselina ethylendiamintetraoctová nebo azid sodný, stopové množství redukující látky o koncentraci 10 až 500 pmol/l a alifatický alkohol o celkovém počtu 1 až 3 atomy uhlíku o koncentraci 0,1 až 2 mol/1. Stabilizovaný roztok chromogenu může být připraven spolu β pufrem o pH 5 až 9, který je kompatibilní jak β enzymem ureázou, tak i s Berthelotovou reakcí. Vlivem nového typu chromogenu a specifického složení roztoku chromogenu je dosa ženo synergického účinku projevujícího se vysokou stálostí roztoku chromogenu i kompletního roztoku. Je potlačena absorbance kontrolního roztoku a roztok chromogenu uvedeného složení neihinbuje enzym ureózu,jejíž obsah v kompletním roztoku může být několikanásobně snížen. Roztok je velmi stabilní a zjednoduší se tak analytické stanovení močoviny o jeden stupeň při současném zvýšení přesnosti a správnosti a snížení ceny vlivem úspory enzymu ureáza.The stabilized chromogen solution consists from an aqueous solution of 3-ethyl-6-isopropylphenol at a concentration of 1 to 5 mmol / l containing complexing agents capable of mask heavy metals at a concentration of 0.05 up to 5 mmol / l, such as acid ethylenediaminetetraacetic or sodium azide, Trace amount of reducing substance at concentration 10 to 500 pmol / l and aliphatic alcohol of 1 to 3 atoms 0.1 to 2 mol / l carbon. The stabilized chromogen solution may be prepared with β buffer at pH 5 to 9, which is compatible with β enzyme urease, as well as Berthelot reaction. Due to the new type of chromogen and specific the composition of the chromogen solution is dosa synergistic effect manifested with high stability of the chromogen solution i complete solution. Absorbance is suppressed control solution and chromogen solution of said composition does not inhibit the enzyme urea, the content of which in complete solution can be reduced several times. The solution is very stable and simplified thus the analytical determination of urea by one step while increasing accuracy and accuracy and price reduction due to savings enzyme urease.
Description
CS 269205 Bl 1CS 269205 B1
Vynález se týká stabilizovaného roztoku chromogenu pro enzymová stanovení močovi-ny Berthelotovou reakcí.BACKGROUND OF THE INVENTION The present invention relates to a stabilized solution of a chromogen for the enzymatic determination of urea by Berthelot reaction.
Stanovení močoviny je Široce využíváno v krmivářství, biochemii, v humánní a ve-terinární diagnostice. Její stanovení v biologickém materiálu, zejména v séru, patřímezi základní analýzy, nebot močovina Je spolu s kreatininem ukazatelem funkce ledvin.Její hladina se zvySuJe u akutní glomerulonefritis, pyelonefritis, obstrukci, uremii,ale i při chorobách jater, při gastrointestinálních poruchách a při nedostatečné čin-nosti srdce.Urea determination is widely used in feed, biochemistry, human and veterinary diagnostics. Its determination in biological material, especially in serum, belongs to the basic analysis, because urea together with creatinine is an indicator of kidney function. Its level is increased In acute glomerulonephritis, pyelonephritis, obstruction, uremia, but also in liver diseases, gastrointestinal disorders and inadequate heart.
Pro stanovení močoviny byla postupnS v biochemii využita celá řada metod. Z fo-tometrických metod, které jsou stále nejrozčířenějčí, je to reakce močoviny a někte-rými alfa-diketony a jejich oximy, jako je diacetyl, acetylbenzoyl a diacetylmonoxim.Močovina s nimi reaguje za zvýSené teploty 80 až 100 °C v silně kyselém prostředí astanovení lze jen obtížně automatizovat. Dalčí skupinu tvoří metody vhodné pro kine-tický způsob stanovení, například chemické stanovení s o-ftaldialdehydem a N-(l-naf-tyDethylendiaminem nebo enzymové stanovení s ureázou, glutamátdehydrogenázou a redu-kovaným nikotinamidadenindinukleotidem v ultrafialové oblasti spektra.A variety of methods have been used to determine urea in biochemistry. Of the photometric methods, which are still the most prominent, urea and some alpha-diketones and their oximes, such as diacetyl, acetylbenzoyl and diacetylmonoxime, react with ureas at elevated temperatures of 80 to 100 ° C under strongly acidic conditions. it is difficult to automate the determination. Another group is a method suitable for a cine-based assay, for example, a chemical assay with o-phthaldehyde and N- (1-naphthylethylenediamine or an enzyme assay with urease, glutamate dehydrogenase, and reduced nicotinamide adenine dinucleotide in the ultraviolet region of the spectrum).
Hlavní metodou stanovení močoviny zůstává stále v biochemii a klinické chemiijejí enzymové stanovení s ureázou, která katalyzuje rozklad močoviny na amoniak aoxid uhličitý. Fotometricky se obvykle stanovuje amoniak po Jeho převedení na indofe-nol postupem podle Berthelota. Toto stanovení, které Je vysoce specifické a citlivé,má ale několik nevýhod, nebot představuje spojení dvou samostatných a současně znač-ně protichůdných reakcí. Protichůdnost požadavků reakčních podmínek pro enzymovýrozklad močoviny a na stanovení amoniaku podle Berthelota je hlavním zdrojem potíží.Protože enzym ureáza obsahuje thiolové skupiny, je konverze močoviny na amoniak kata-lyzovaná ureázou inhibována těžkými kovy. Ureázu inaktivují i látky s oxidačními účin-ky, která oxidují Její thiolové skupiny. Při rozkladu močoviny ureázou lze stálostenzymu prodloužit přítomností látek maskujících těžká kovy, jako jsou komplexotvornélátky, například kyselina ethylendiamintetraoctová, kyselina citrónová a látky a thio-lovými skupinami, například glut athlon (W.Schneider a kol.: Ger. Offen, 2 612 726,1977, Chem. Abstr. Θ7, 196 469, 1977). Pychlost enzymová hydrolýzy se zvyěuje za pří-tomnosti některých aminokyselin, které ale ruěí vlastní stanovení amoniaku oxidačníkopulací podle Berthelota. Mezi látky ručící Berthelotovu reakci patří zejména aminy,thioly, redukující látky, komplexující činidla a obecně látky s nukleofilním účin-kem na chinony, s nimiž tvoří přednostně monoalkyl až bis(alkylamino)hydrochinony, abrání tak vzniku indofenolu (T. T. NGo a kol.: Anal. Chem. 54, 46, 1932). Podle Pymaa Milhama (Anal. Chem. 48, 1413, 1976) snižují výtěžek Berthelotovy reakce o 20 až90 % fluoridy, citráty, oxaláty a kyselina ethylendiamintetraoctová. Podmínkou Bert-helotovy reakce je, aby byl fenol přítomen v roztoku ve formě fenolátového aniontu.Jeho aktuální koncentrace v roztoku je závislá nejen na pH, ale i na disociační kons-tantě užitého fenolu. Citlivost reakce i rychlost vzniku indofenolu bude záviset natypu fenolu a druhu subatituentů na fenolovém jádře. Stanovení amoniaku podle Bert-helota s fenolem je pro analýzu séra přílič citlivé, se vzorkem o objemu 0,02 ml jevýsledný objem roztoku přílič vysoký a dosahuje až 10 ml. Enzymové stanovení močovinys ureázou v kombinaci s Berthelotovou reakcí se provádí jako několikastupňová aznačně komplikovaná analýza. Postupuje se větčinou tak, že se vzorek nejprve inkubu-je s roztokem ureázy, pak se přidá roztok obsahující fenol a nltroprussid, kterýslouží Jako katalyzátor, nakonec se přidá oxidační roztok obsahující nejčastěji alka-lický chlornan nebo i jiné látky Jako zdroje aktivního chloru. Vzniklé modré zbarve-ní se měří fotometricky. Spojit enzymovou směs s fenolem a katalyzátorem do jednohoinkubačního roztoku je obtížné, nebot fenol inhibuje ureázu. CS 269205 BlThe main method of urea determination is still in biochemistry and clinical chemistry is the enzyme determination with urease, which catalyzes the decomposition of urea to ammonia and carbon dioxide. Typically, ammonia is determined photometrically after its conversion to indophenol according to the Berthelot method. However, this determination, which is highly specific and sensitive, has several drawbacks, since it is a combination of two separate and at the same time contradictory reactions. The contradictory requirements of the reaction conditions for urea enzyme decomposition and for the determination of ammonia by Berthelot is a major source of difficulty. Because the urease enzyme contains thiol groups, urease-converted urea catalyzed ammonia is inhibited by heavy metals. Urease also inactivates substances with oxidizing effects that oxidize its thiol groups. In urea decomposition, urease can be prolonged by the presence of heavy metal masking agents such as complex-forming agents such as ethylenediaminetetraacetic acid, citric acid, and substances and thiol groups such as glut athlon (W Schneider et al .: Ger. Offen, 2,612,726, 1977, Chem Abstr., 7, 196, 469 (1977). The enzymatic hydrolysis rate increases with the presence of some amino acids which, however, interfere with the determination of ammonia by the oxidation-coupled Berthelot. In particular, the Berthelot-leaching agents include amines, thiols, reducing agents, complexing agents, and generally quinone-nucleophilic agents with which they preferably form monoalkyl to bis (alkylamino) hydroquinones, thus preventing the formation of indophenol (TT NGo et al .: Anal., Chem., 54, 46, 1932). According to Pyma Milham (Anal. Chem. 48, 1413, 1976), the Berthelot reaction yield reduced by 20 to 90% fluorides, citrates, oxalates and ethylenediaminetetraacetic acid. The condition of the Bert helot reaction is that the phenol is present in the solution in the form of a phenolate anion. Its actual concentration in the solution depends not only on the pH but also on the dissociation constant of the phenol used. The sensitivity of the reaction and the rate of indophenol formation will depend on the phenol type and the type of sub-substituents on the phenolic core. The determination of ammonia by Bert-Helot with phenol is sensitive to serum analysis, with a sample volume of 0.02 ml, the resulting solution volume is high and reaches up to 10 ml. The urea urease enzyme assay in combination with the Berthelot reaction is performed as a multi-stage, elaborately complicated analysis. Most of the time, the sample is incubated with the urease solution, then a solution containing phenol and nltroprusside is added to serve as a catalyst, and an oxidizing solution containing mostly alkali hypochlorite or other substances is added as active chlorine sources. The resulting blue color is measured photometrically. Combining the enzyme mixture with phenol and the catalyst in a single incubation solution is difficult because phenol inhibits urease. CS 269205 Bl
Spojení lnkubačního roztoku s oxidačním činidlem vylučuje charakter jejich kom-ponent. Proto byly hledány dalSÍ deriváty fenolu, která by byly s ureázou alespoňčástečné kompatibilní. Jedním z nich je salicylan sodný (Tabacco a kol.: Clin. Chem.25, 336, 1979), který lze sice přidat přímo do lnkubačního roztoku, ale za cenu pod-statného zvýšení koncentrace ureázy v roztoku z obvyklých 15 ač 20 U/ml až na 50 U/ml,nebot i salicylan sodný snižuje katalytickou aktivitu 1 stabilitu ureázy. Tím sicelze stanovení močoviny o jeden etupeň zjednodušit, současně se ale značně zvýší jehocena. Další nevýhodou, která provází zvySování obsahu ureázy v inkubačním roztoku,je růst absorbance slepého pokusu. Ureáza obsahuje vidy stopy amonných solí, kterázvyšují absorbanci slepého pokusu úměrné se zvyšováním navážky enzymu.Combining the incubation solution with the oxidizing agent eliminates the nature of their components. Therefore, other phenol derivatives were sought which would be at least partially compatible with urease. One of these is sodium salicylate (Tabacco et al., Clin. Chem. 25: 336, 1979), which can be added directly to the incubation solution but at the expense of a substantial increase in urease concentration in the solution of the usual 15 to 20 U /. ml up to 50 U / ml because sodium salicylate reduces the urease catalytic activity 1. Thereby, the determination of urea can be simplified by one stage, but at the same time its cost is greatly increased. Another disadvantage associated with the increase in urease content of the incubation solution is the increase in absorbance of the blank. Urease contains traces of traces of ammonium salts, which increase the absorbance of the blank by proportional to the increase in enzyme load.
Uvedené nevýhody, spočívající v rozdílných a protikladných požadavcích na slože-ní činidel pro enzymovou hydrolýzu močoviny ureázou a pro stanovení amoniaku iB.erthe-lotovou reakcí, spolu s komplikovaným několikastupňovým postupem analýzy, jsou důvo-dem k hledání jiných, výhodnějších řešení, která by uvedené nevýhody odstranila neboalespoň potlačila. Jedné se zejména o nalezení vhodnějšího .typu fenolu jako chromoge-nu pro oxidační kopulaci při Berthelotově reakci, který by nelnhiboval ureázu, a je-hož lndofenol by měl nižší absorpční molární koeficient, aby nebylo nutné pracovats velkými výslednými objemy při fotometrickém měření. K odstranění uvedených nedostatků směřuje vodný stabilizovaný roztok chromogenupro enzymové stanovení močoviny Berthelotovou reakcí podle vynálezu, jehož podstataspočívá v tom, že se skládá z vodného roztoku 3-methyl-6-lsopropylfenolu o koncentra-ci 1 až 5 mmol/1, komplexotvorné látky schopné maskovat těžké kovy, například kyseli-ny ethylendiamintetraoctové nebo azidu sodného o koncentraci 0,05 až 5 mmol/1, stopo-vého množství redukující látky, s výhodou kyseliny askorbové, siřičitanu nebo kyseli-ny thiobarbiturové o koncentraci 10 až 500 ^umol/1 a alifatického alkoholu o koncen-traci 0,1 až 2 mol/1 o celkovém počtu 1 až 3 atomy uhlíku a pufr o pH 5 až 9 kompa-tibilní s enzymem ureázou i s Berthelotovou reakcí, s výhodou barbituran, fosforeč-nan nebo citronan alkalického kovu.These disadvantages, due to the differing and contradictory requirements for urea enzyme hydrolysis reagents for urease and for ammonia determination by the iBerthe-Lot reaction, together with a complicated multi-step analysis procedure, are the reason for finding other, more advantageous solutions that would these disadvantages have been eliminated or at least suppressed. In particular, it is desirable to find a more suitable phenol type as a chromogen for oxidative coupling in a Berthelot reaction that does not inhibit urease, and indenophenol would have a lower absorption molar coefficient in order to avoid large volumes of photometric measurements. To overcome these drawbacks, the aqueous stabilized solution of chromogen is directed to the urea enzyme determination of the invention by a Berthelot reaction consisting of an aqueous solution of 3-methyl-6-isopropylphenol having a concentration of 1 to 5 mmol / l, a complexing agent capable of masking heavy metals such as ethylenediaminetetraacetic acid or sodium azide at a concentration of 0.05 to 5 mmol / l, a trace amount of a reducing agent, preferably ascorbic acid, sulfite or thiobarbituric acid at a concentration of 10 to 500 µmol / l; an aliphatic alcohol having a concentration of 0.1 to 2 mol / l of a total of 1 to 3 carbon atoms and a buffer of pH 5 to 9 compatible with the enzyme urease and Berthelot reaction, preferably barbituran, phosphate or alkali metal citrate .
Použití 3-Bethyl-6-isopropylfenolu jako chromogenu pro enzymové stanovení močo-viny ve spojení s Berthelotovou reakcí má proti užívaným chromogenúm, jimiž jsou fe-nol a salicylan sodný, řadu výhod. Fenol inhibuje enzym a roztok chromogenu nelzespojovat s enzymem. Salicylan sodný, který má disoclační konstantu pK& asi 13,4 musíbýt k dosažení optimální analytické koncentrace v reakční směsi, kde musí být příto-men v disociované, reaktivní formě, přidáván do reakční směsi v množství 62 mmol/1,zatímco 3-methyl-6-lsopropylfenol postačuje v koncentraci o více než řád nižší 1 až5 mmol/1. Množství ureázy lze s chromogenem podle tohoto vynálezu snížit až čtyřnásob-ně, tím podstatně snížit absorbanci kontrolního roztoku a celou analýzu zpřesnit. Látky maskující těžké kovy spolu se stopovým množstvím redukující látky chráníroztok chromogenu před jeho spontánní oxidací, způsobující postupné vybarvování roz-toku chromogenu, která je nežádoucí vlastností provázející oxidaci prakticky všechfenolů. Koncentrace obou látek, které Interferují při Berthelotově reakci, je volenaspecificky tak, aby tyto látky nerušily oxidační kopulaci amoniaku se 3-methyl-6--isopropylfenolem a s alkalickým chlornanem.The use of 3-methyl-6-isopropylphenol as a chromogen for the enzymatic determination of urea in conjunction with the Berthelot reaction has a number of advantages over the chromogens used, which are phenol and sodium salicylate. Phenol inhibits the enzyme and the chromogen solution does not bond with the enzyme. Sodium salicylate having a dissociation constant of pK & about 13.4 must be added to the reaction mixture in an amount of 62 mmol / l to achieve the optimum analytical concentration in the reaction mixture, where it has to be added in a dissociated, reactive form, while 3-methyl-6-isopropylphenol is sufficient in a concentration of more than orders of magnitude less than 1-5 mmol / L. The amount of urease can be reduced by up to four times with the chromogen of the present invention, thereby substantially reducing the absorbance of the control solution and refining the entire analysis. Heavy metal masking agents together with a trace amount of reducing agent protect the chromogen solution from its spontaneous oxidation, causing gradual staining of the chromogen, which is an undesirable property accompanying the oxidation of virtually all phenols. The concentration of both substances that interfere with the Berthelot reaction is freely specific so that these substances do not interfere with the oxidative coupling of ammonia with 3-methyl-6-isopropylphenol and with alkaline hypochlorite.
Alifatický alkohol usnadňuje přípravu roztoku chromogenu a spolu a ním součas-ně potlačují bakteriální kontaminaci. Obě' látky tedy roztok současně konzervují.The aliphatic alcohol facilitates the preparation of the chromogen solution and simultaneously and simultaneously suppresses bacterial contamination. Thus, both substances simultaneously preserve the solution.
Pro enzymové stanovení močoviny lze ve spojení a Berthelotovou reakcí užít ome-zený okruh pufrů, nebot řada z nich, například všechny aminoalkoholy, při kopulaciinterferují. Požadavku kompatibility pufru s oběma reakčními systémy splňují napří-klad barbituran a fosforečnany nebo citronany alkalických kovů, které mají současněpříznivý vliv nejen na stálost roztoku chromogenu, ale i na stálost kompletního roz-toku obsahujícího současně i enzym ureázu a katalyzátor kopulace. StabilizovanýFor the urea enzyme assay, a limited range of buffers can be used in conjunction with the Berthelot reaction since many of them, for example, all aminoalcohols, interfere with the coupling. For example, barbituran and alkali metal phosphates or citrates, which have a beneficial effect not only on the stability of the chromogen solution, but also on the stability of the complete solution containing both the urease enzyme and the coupling catalyst, meet the requirement of buffer compatibility with both reaction systems. Stabilized
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS881177A CS269205B1 (en) | 1988-02-24 | 1988-02-24 | Stabilized solution of chromogene for urea's enzymatic determination by means of berthelot reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS881177A CS269205B1 (en) | 1988-02-24 | 1988-02-24 | Stabilized solution of chromogene for urea's enzymatic determination by means of berthelot reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CS117788A1 CS117788A1 (en) | 1989-09-12 |
| CS269205B1 true CS269205B1 (en) | 1990-04-11 |
Family
ID=5345501
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS881177A CS269205B1 (en) | 1988-02-24 | 1988-02-24 | Stabilized solution of chromogene for urea's enzymatic determination by means of berthelot reaction |
Country Status (1)
| Country | Link |
|---|---|
| CS (1) | CS269205B1 (en) |
-
1988
- 1988-02-24 CS CS881177A patent/CS269205B1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CS117788A1 (en) | 1989-09-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gochman et al. | Automated determination of uric acid, with use of a uricase—Peroxidase system | |
| US3886045A (en) | Process for the enzymatic determination of glucose with a glucose-oxidase/peroxidase enzyme system | |
| Kageyama | A direct colorimetric determination of uric acid in serum and urine with uricase-catalase system | |
| Fossati et al. | Use of 3, 5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. | |
| Trivedi et al. | New enzymatic method for serum uric acid at 500 nm. | |
| US4350762A (en) | Aminopyrine improved Trinder's reagent and dosing process for hydrogen peroxide from enzymatic oxidation of metabolic substrata with the same | |
| US4385114A (en) | Oxidation indicators comprising 3,3',5,5'-tetraalkylbenzidine compounds | |
| JPS61146196A (en) | Method and reagent for improving photometry of h202 | |
| US3558435A (en) | Diagnostic agents for use in the determination of hydroperoxides and of peroxidate-active substances and methods for manufacturing and using the same | |
| Morgenstern et al. | Automated determination of NAD-coupled enzymes. Determination of lactic dehydrogenase | |
| JPS6134799B2 (en) | ||
| FI77896B (en) | KOMPOSITION FOER BESTAEMNING AV URINSYRA ELLER KOLESTEROL I ETT VAETSKEPROV. | |
| US5055388A (en) | Process and reagent composition for determination of fructosamine in body fluids | |
| Watts | Determination of uric acid in blood and in urine | |
| EP0396584B1 (en) | Assay of salicylates or reduced pyridine nucleotides | |
| US5106753A (en) | Method for determining manganese level in blood using a porphyrin composition | |
| Scheibe et al. | Uric acid | |
| CS269205B1 (en) | Stabilized solution of chromogene for urea's enzymatic determination by means of berthelot reaction | |
| JP2838866B2 (en) | Method for suppressing the activity of reducing substances in oxidative colorimetric analysis | |
| US4409328A (en) | Method and reagent for the determination of glycerol | |
| US5888828A (en) | Kit for measuring urea nitrogen | |
| JPH0635966B2 (en) | Analytical reagent useful for detecting thiol compound and detection method using the same | |
| US4230798A (en) | Unitized uric acid test composition and device | |
| JPH0675515B2 (en) | Method for quantifying biological substances using ammonia as reaction product | |
| CZ281122B6 (en) | Stabilized enzyme agent with urease for urea determination by employing berthelot reaction |