DK144643B - METHOD AND REAGENT FOR DETERMINING TRIGLYCERIDES - Google Patents
METHOD AND REAGENT FOR DETERMINING TRIGLYCERIDES Download PDFInfo
- Publication number
- DK144643B DK144643B DK245273AA DK245273A DK144643B DK 144643 B DK144643 B DK 144643B DK 245273A A DK245273A A DK 245273AA DK 245273 A DK245273 A DK 245273A DK 144643 B DK144643 B DK 144643B
- Authority
- DK
- Denmark
- Prior art keywords
- triglycerides
- lipase
- serum
- glycerol
- reagent
- Prior art date
Links
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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- 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
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- 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
- C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides
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)
Description
(19) DANMARK ( W) , \Ra/(19) DENMARK (W), \ Ra /
W (12) FREMLÆGGELSESSKRIFT <n> 14^643 BW (12) PUBLICATION <N> 14 ^ 643 B
DIREKTORATET FOR PATENT- OG VAREMÆRKEVÆSENETDIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM
(21) Ansøgning nr. 2452/73 (51) |ntC|3 ¢12 fll 1/44 (22) Indleveringsdag raaJ 1973 (24) Løbedag ^ · maJ 1 973 (41) Aim. tilgængelig 20. dec. 1973 (44) Fremlagt 26. apr. 1982 (86) International ansøgning nr. “ (86) International indleveringsdag -(85) Videreførelsesdag “ (62) Stamansøgning nr. ~(21) Application No. 2452/73 (51) | ntC | 3 ¢ 12 fll 1/44 (22) Date of filing raaJ 1973 (24) Running day ^ · maJ 1 973 (41) Aim. available Dec. 20; 1973 (44) Posted Apr 26 1982 (86) International Application No. "(86) International Filing Day - (85) Continuation Day" (62) Master Application No. ~
(30) Prioritet 19· jun. 1972, 2229849, DE(30) Priority 19 · Jun. 1972, 2229849, DE
(71) Ansøger BOEHRINGER MANNHEIM GMBH., 68 Mannheim-Waldhof, DE.(71) Applicant BOEHRINGER MANNHEIM GMBH., 68 Mannheim-Waldhof, DE.
(72) Opfinder August Wilhelm Wahlefeld, DE: Hans Moellering, DE: Wolf= gang Gruber, DE: Erich Bernt, DE: Peter Roeschlau, DE.(72) Inventor August Wilhelm Wahlefeld, DE: Hans Moellering, DE: Wolf = gang Gruber, DE: Erich Bernt, DE: Peter Roeschlau, DE.
(74) Fuldmægtig Firmaet Chas. Hude.(74) Associate Company Chas. Hude.
(54) Fremgangsmåde og reagens til be= stemmelse af triglycerider.(54) Method and reagent for the determination of triglycerides.
Opfindelsen angår en fremgangsmåde og et reagens til bestemmelse af triglycerider ved forsæbning af glyceriderne og bestemmelse af den derved frigjorte glycerol.The invention relates to a method and reagent for determining triglycerides by saponifying the glycerides and determining the glycerol released thereby.
Bestemmelsen af triglyceriderne får en stadig større betydning inden for såvel levnedsmiddelanalysen som inden for den medicinske diagno->Q stik, navnlig ved diagnose af hyperlipæmier inden for den kliniske Ύ) kemi.The determination of triglycerides is becoming increasingly important in both the food analysis and in the medical diagnosis, especially in the diagnosis of hyperlipaemia in clinical chemistry.
tf Ώ * 3tf Ώ * 3
Ifølge en kendt fremgangsmåde sker bestemmelsen på den måde, at man først forsæber triglyceriderne med alkoholisk alkalimetalhydroxid oq derpå 2 144643 bestemmer den dannede glycerol. Bestemmelsen sker hensigtsmæssigt ved hjælp af enzymatiske metoder. Herved bliver glycerolen phosphoryleret med adenosintriphosphat (ATP) i nærværelse af glycerolkinase (GK) til dannelse af glycerin-l-phosphat og adenosindiphosphat (ADP). Dannet ADP overføres atter ved hjælp af phosphoenolpyruvat (PEP) i nærværelse af pyruvatkinase til pyruvat og ATP. Herved dannet pyruvat hydreres med nikotinamidadenindinucleotid i hydreret form (NADH) i nærværelse af lactatdehydrogenase (LDH) til lactatet, hvorved NADH oxideres til nikotinamidadenindinucleotidet (NAD). Den ved reaktionen forbrugte NADH-mængde er ækvivalent med glycerolmængden. NADH kan let bestemmes kvantitativt på grund af dets absorption ved 366, 340 eller 334 nm.According to a known method, the determination is made by first saponifying the triglycerides with alcoholic alkali metal hydroxide and then determining the glycerol formed. The determination is conveniently done by enzymatic methods. Thereby, the glycerol is phosphorylated with adenosine triphosphate (ATP) in the presence of glycerol kinase (GK) to form glycerin-1-phosphate and adenosine diphosphate (ADP). Formed ADP is again transferred by phosphoenol pyruvate (PEP) in the presence of pyruvate kinase to pyruvate and ATP. The pyruvate thus formed is hydrogenated with the nicotinamide adenine dinucleotide (NADH) in the presence of lactate dehydrogenase (LDH) to the lactate, thereby oxidizing the NADH to the nicotinamide adenine dinucleotide (NAD). The amount of NADH consumed by the reaction is equivalent to the amount of glycerol. NADH can be readily determined quantitatively due to its absorption at 366, 340 or 334 nm.
En væsentlig ulempe ved denne kendte fremgangsmåde består i forsæbningen med ethanolisk elkalimetalhydroxid. Dette forsæbningstrin gør den i øvrigt specifikke, nøjagtige og let gennemførlige metode omstændelig og tidsrøvende, da alene forsæbningen varer 20-30 minutter ved en temperatur på ca. 70°C. Derefter skal der neutraliseres og centrifugeres, før der kan begyndes med selve glycerolbestemmelsen.A major disadvantage of this known process is the saponification with ethanolic alkali metal hydroxide. This saponification step makes the otherwise specific, accurate and easily practicable method cumbersome and time consuming, since the saponification alone lasts 20-30 minutes at a temperature of approx. 70 ° C. Then, neutralize and spin before the glycerol assay can begin.
Denne ulempe afhjælpes ifølge en kendt fremgangsmåde ved hjælp af den enzymatiske forsæbning af triglyceriderne, hvorved der anvendes en lipase fra Rhizopus arrhizus. Ved denne fremgangsmåde var det overraskende, at der kunne findes frem til en lipase, som i vandig puffer kan spalte triglyceriderne fuldstændigt til fedtsyre og glycerol på 5-10 minutter ved 35-37°C. Ved en anden kendt fremgangsmåde anvendes en lipase. sammen med en protease til forsæbningen, og med en lignende virkning. Andre lipaser, navnlig den kendte pankreaslipase, viste sig uegnet.This disadvantage is remedied according to a known method by the enzymatic saponification of the triglycerides, using a lipase from Rhizopus arrhizus. By this method, it was surprising that a lipase could be found which in aqueous buffer can completely cleave the triglycerides to fatty acid and glycerol in 5-10 minutes at 35-37 ° C. In another known method, a lipase is used. together with a protease for saponification, and with a similar effect. Other lipases, particularly the known pancreatic lipase, proved unsuitable.
En ulempe ved den enzymatiske spaltning består imidlertid i, at forsæbningen kræver en væsentlig mængde af det meget kostbare enzym. For at opnå en brugbar reaktionstid er ca. 1 mg af enzymet for hver 0,1 ml serumprøve nødvendig. Endelig dannede de frigjorte fedtsyrer uopløse-lige sæber med eventuelt tilstedeværende calcium- og magnesiumioner, hvilket atter førte til uklarheder og dermed til forfalskning af måleresultaterne, hvis der ikke centrifugeres.However, one disadvantage of the enzymatic cleavage is that the saponification requires a substantial amount of the very expensive enzyme. To achieve a useful reaction time, approx. 1 mg of the enzyme for each 0.1 ml serum sample is needed. Finally, the released fatty acids formed insoluble soaps with any calcium and magnesium ions present, which in turn led to fuzziness and thus to falsification of the measurement results if not centrifuged.
Opfindelsen tager derfor sigte på at afhjælpe disse ulemper og angive en fremgangsmåde til bestemmelse af triglycerider i en vandig væske ved hjælp af enzymatisk forsæbning, ved hvilken den nødvendige lipase-mængde samt den nødvendige besteirmelsestid formindskes væsentligt, og nødvendigheden af en fraskillelse af de eventuelt udfældede sæber endvidere fjernes.The invention therefore aims to alleviate these drawbacks and to provide a method for determining triglycerides in an aqueous liquid by enzymatic saponification, which substantially reduces the amount of lipase and the time required to precipitate and the need to separate the precipitates, if any. soaps are further removed.
U4643 3U4643 3
Dette opnås ifølge opfindelsen ved hjælp af en fremgangsmåde til bestemmelse af triglycerider ved enzymatisk forsæbning ved hjælp af lipase og måling af den frigjorte glycerol, som er ejendommelig ved, at forsæbningen foretages i nærværelse af såvel lipase som carboxyl= ester ase og et alkalimetal- eller jordalkalimetalalkylsulfat med 10-15 carbon-atomer i alkylgruppen. Fortrinsvis arbejdes der herved i nærværelse af serumalbumin.This is achieved according to the invention by a method for determining triglycerides by enzymatic saponification by lipase and measuring the released glycerol, which is characterized by the saponification being carried out in the presence of both lipase and carboxyl ester ase and an alkali metal or alkaline earth metal alkyl sulfate having 10-15 carbon atoms in the alkyl group. Preferably, this is done in the presence of serum albumin.
Den til forsæbningen ifølge opfindelsen benyttede reagenskombination muliggør overraskende en meget stor nedsættelse af den nødvendige li-pasemængde og tilmed fra ca. 1 mg pr. 0,1 ml serumprøve til 20 pg pr.0,1 ml serumprøve uden samtidig forøgelse af reaktionstemperaturen eller reaktionstiden.The reagent combination used for saponification according to the invention surprisingly allows a very large reduction in the required amount of lipid, and even from approx. 1 mg per 0.1 ml serum sample to 20 µg per 0.1 ml serum sample without simultaneously increasing the reaction temperature or reaction time.
Som lipase foretrækkes en lipase fra Rhizopus arrhizus. Som carboxyl= esterase EC 3.1.1.1, carboxylesterhydrolase, anvendes fortrinsvis et pattedyr-leverpræparat, navnlig en svineleveresterase. Der kan dog også anvendes andre carboxylesteraser. En mængde på ca. 100 pg este= rase pr. 0,1 ml serumprøve har vist sig at være fuldt tilstrækkelig.As lipase, a lipase from Rhizopus arrhizus is preferred. As carboxyl = esterase EC 3.1.1.1, carboxylester hydrolase, a mammalian liver preparation, in particular a pig liver esterase, is preferably used. However, other carboxylesterases may also be used. An amount of approx. 100 pg este = race per day. 0.1 ml of serum sample has been found to be fully adequate.
Blandt alkylsulfaterne foretrækkes alkalirretalsaltene, da de ikke giver anledning til dannelse af uopløselige sæber med de frigjorte fedtsyrer. I den foretrukne udførelsesform for fremgangsmåden, hvor der anvendes serumalbumin, kan der imidlertid også anvendes jordal kalimet al -alkylsulfater. Dodecylsulfat foretrækkes, da det fremskynder forsæbningen af triglyceriderne kraftigst under disse betingelser. Alkyl= sulfatet er virksomt i en mængde ned til 0,001 vægt%/vol. i prøvematerialet. Mængder over 0,1% fører til en forstyrrende skumdannelse og en let hæmning og er derfor i almindelighed uhensigtsmæssige.Among the alkyl sulfates, the alkali urethral salts are preferred as they do not give rise to the formation of insoluble soaps with the liberated fatty acids. However, in the preferred embodiment of the method using serum albumin, terrestrial potassium al-alkyl sulfates may also be used. Dodecyl sulfate is preferred as it greatly accelerates the saponification of the triglycerides under these conditions. The alkyl = sulfate is effective in an amount down to 0.001% w / v. in the sample material. Amounts above 0.1% lead to a disturbing foaming and a slight inhibition and are therefore generally inappropriate.
Fremgangsmåden ifølge opfindelsen gennemføres hensigtsmæssigt ved pH-værdier mellem 6 og 9, fortrinsvis mellem 7 og 8,5. Dette har den fordel, at der ved påvisning af den dannede glycerol med den bekendte reaktion under anvendelse af ATP, GK, PEP, PK, NADH og LDH ikke behøver at foregå en pH-værdiændring, men at forsæbning og måling af den dannede glycerol kan gennemføres i et enkelt reaktionsmateriale.The process according to the invention is conveniently carried out at pH values between 6 and 9, preferably between 7 and 8.5. This has the advantage that when detecting the glycerol formed with the known reaction using ATP, GK, PEP, PK, NADH and LDH, there is no need for a pH change, but that saponification and measurement of the glycerol formed can is carried out in a single reaction material.
I den foretrukne udførelsesform, hvor der tilsættes serumalbumin, fortrinsvis kvægserumalbumin, undgås en uklarhed som følge af udfældede sæber pålideligt, så at sådanne sæber ikke skal fraskilles, f.eks. ved centrifugering. Serumalbumin tilsættes herved hensigtsmæssigt i en koncentration mellem 0,1 og 2,0 mg/ml prøvemateriale.In the preferred embodiment, where serum albumin, preferably bovine serum albumin, is added, obscurity due to precipitated soaps is reliably avoided so that such soaps should not be separated, e.g. by centrifugation. Consequently, serum albumin is conveniently added at a concentration between 0.1 and 2.0 mg / ml of test material.
U4643 4U4643 4
Kobolt- og/eller magnesiumioner har en vis aktiverende virkning på lipasen eller esterasen og kan derfor ligeledes tilsættes til yderligere reaktionsfremskyndelse.Cobalt and / or magnesium ions have some activating effect on the lipase or esterase and can therefore also be added for further reaction speed.
Som puffere er alle inden for ovennævnte pH-område virksomme puffere egnede med undtagelse af phosphatpuffere. Eksempler på egnede puffere er triethanolaminpuffer,trispuffer, imidazolpuffer, veronalpuffer, glycinpuffer samt, mindre foretrukket, amediolpuffer, boratpuffer og collidinpuffer.As buffers, all buffers active within the above pH range are suitable except for phosphate buffers. Examples of suitable buffers are triethanolamine buffer, tris buffer, imidazole buffer, veronal buffer, glycine buffer, and, less preferably, amediol buffer, borate buffer and collidine buffer.
Reagenset til gennemførelse af fremgangsmåden ifølge opfindelsen indeholder enzy= matisk lipase son forskningsmiddel, puffer og et system til påvisning af glycerol og er ejendommeligt ved et indhold af såvel lipasen som carboxylesterase og et al= kalimetal- eller jordalkalimetalalkylsulfat med 10-15 carbonatomer i alkylgruppen.The reagent for carrying out the process of the invention contains enzymatic lipase containing research agent, buffer and glycerol detection system and is characterized by a content of both the lipase and carboxylesterase and an alkali metal or alkaline earth metal alkyl sulfate having 10-15 carbon atoms in the alkyl group.
Som system til påvisning af glycerol kan der i reagenset ifølge opfindelsen principielt anvendes ethvert sådant system. Et påvisningssystem, som består af NADH, ATP, PEP, LDH, PK,GK samt magnesiumioner og puffer, foretrækkes. Sidstnævnte påvisningssystem er velegnet i reagenset ifølge opfindelsen og har tillige den fordel, at bestanddelene ikke forstyrrer hinanden, og at alle enzymer er aktive ved samme pH-værdi.As a system for detecting glycerol, any reagent according to the invention can in principle be used for any such system. A detection system consisting of NADH, ATP, PEP, LDH, PK, GK as well as magnesium ions and buffer is preferred. The latter detection system is well-suited in the reagent of the invention and also has the advantage that the components do not interfere with each other and that all enzymes are active at the same pH.
Inden for rammerne af den foretrukne reagenskombination består et særlig egnet reagens af 0,1-10,0 mg lipase fra Rhizopus arrhizus 0,5-20,0 mg carboxylesterase 0,01-0,2 mg alkylsulfatIn the context of the preferred reagent combination, a particularly suitable reagent consists of 0.1-10.0 mg of lipase from Rhizopus arrhizus 0.5-20.0 mg of carboxylesterase 0.01-0.2 mg of alkyl sulfate
1- 20 mM NADH 10-100 mM ATP1- 20 mM NADH 10-100 mM ATP
2- 20 mM PEP 0,5-5 mg LDH 0,2-5 mg PK 0,05-10 mg GK 0,1-2,0 mg serumalbumin 3- 30 mM magnesiumioner eventuelt 0,5-1,0 mM koboltioner og 0,03-0,3 M puffer, pH = 6-9.2- 20 mM PEP 0.5-5 mg LDH 0.2-5 mg PK 0.05-10 mg GK 0.1-2.0 mg serum albumin 3- 30 mM magnesium ions optionally 0.5-1.0 mM cobalt ions and 0.03-0.3 M buffer, pH = 6-9.
De ovenfor angivne mængder refererer til 1 ml pufferopløsning med den angivne koncentration. Som puffer foretrækkes 0,1 M triethanolaminpuf- 5 144643 fer med en pH-værdi fra 7 til 8,5 i det ovennævnte reagens.The above amounts refer to 1 ml of buffer solution at the concentration indicated. As buffer, 0.1 M triethanolamine buffers having a pH of 7 to 8.5 in the above reagent are preferred.
Som allerede nævnt muliggør fremgangsmåden og reagenset ifølge opfindelsen en meget stor billiggørelse af triglyceridbestemmelsen. I forhold til den kendte fremgangsmåde, som anvender forsæbning med ethanolisk kalilud, forkortes varigheden af bestemmelsen fra mere end 1 time til mindre end 1/2 time. Samtidig bliver talrige pipetteringstrin, opvarmning af forsæbningsmaterialet, neutralisering og centrifugering af bundfaldet overflødig.As already mentioned, the method and reagent according to the invention enable a very large lowering of the triglyceride determination. Compared to the known method which uses saponification with ethanolic potash, the duration of the determination is shortened from more than 1 hour to less than 1/2 hour. At the same time, numerous pipetting steps, heating of the saponification material, neutralization and centrifugation of the precipitate become superfluous.
Sammenlignet med den kendte teknik, der anvender enzymatisk forsæbning, opnås en meget betydelig formindskelse af den nødvendige enzymmængde.Compared to the prior art using enzymatic saponification, a very significant reduction in the amount of enzyme required is achieved.
De følgende eksempler belyser opfindelsen nærmere.The following examples further illustrate the invention.
Eksempel 1Example 1
Man fremstillede et lagringsbestandigt reagens bestående af fem komponenter, som blandes før brugen og derpå er holdbare i blandet tilstand i 1-2 dage.A storage-resistant reagent consisting of five components was prepared which were mixed before use and then durable in the mixed state for 1-2 days.
Komponenter Sammensætning 1 0,1 M triethanolaminpuffer pH 7,6 indeholdende 3 mM MgSO^, 1,5 mg kvægserumalbumin/ml og 0,1 mg natrium= dodecylsulfat/ml.Components Composition 1 0.1 M triethanolamine buffer pH 7.6 containing 3 mM MgSO 4, 1.5 mg bovine serum albumin / ml and 0.1 mg sodium = dodecyl sulfate / ml.
2 Opløsning af 6 mM NADH, 33 mM ATP og 11 mM PEP i destilleret vand.2 Solution of 6 mM NADH, 33 mM ATP and 11 mM PEP in distilled water.
3 Krystalsuspension af 2 mg LDH/ml og 1 mg PK/ml (fås i handelen).3 Crystal suspension of 2 mg LDH / ml and 1 mg PK / ml (commercially available).
4 Opløsning af 0,2 mg lipase fra Rhizopus arrhizus/ml og 4,0 mg carboxylesterase/ml.4 Resolution of 0.2 mg of lipase from Rhizopus arrhizus / ml and 4.0 mg of carboxylesterase / ml.
5 Krystalsuspension af 2 mg GK/ml.5 Crystal suspension of 2 mg GK / ml.
2,9 ml komponent 1, 0,1 ml komponent 2 og 0,02 ml komponent 3 blandes og indstilles på 25°C. Derpå iblandes 0,1 ml serum, som indeholder de triglycerider, der skal bestemmes, og der inkuberes i 5 minutter ved den angivne temperatur. Derpå iblandes 0,1 ml komponent 4, og blandingen holdes i 15-20 minutter ved den angivne temperatur, og ekstinktionen ved 366 nm eller 340 nm aflæses derpå i et fotometer. Den aflæste værdi betegnes med E^. Prøven tilsættes derpå 0,02 ml komponent 5, ogMix 2.9 ml of component 1, 0.1 ml of component 2 and 0.02 ml of component 3 and adjust to 25 ° C. Then 0.1 ml of serum containing the triglycerides to be determined is incubated and incubated for 5 minutes at the specified temperature. Then 0.1 ml of component 4 is added and the mixture is kept for 15-20 minutes at the specified temperature and the extinction at 366 nm or 340 nm is then read in a photometer. The value read is denoted by E ^. The sample is then added 0.02 ml of component 5, and
Claims (2)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2229849A DE2229849C2 (en) | 1972-06-19 | 1972-06-19 | Method and reagent for the determination of triglycerides |
| DE2229849 | 1972-06-19 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| DK144643B true DK144643B (en) | 1982-04-26 |
| DK144643C DK144643C (en) | 1982-09-27 |
Family
ID=5848134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK245273A DK144643C (en) | 1972-06-19 | 1973-05-04 | PROCEDURE AND REAGENT FOR DETERMINING TRIGLYCERIDES |
Country Status (17)
| Country | Link |
|---|---|
| US (1) | US3862009A (en) |
| JP (1) | JPS4964495A (en) |
| AR (1) | AR195328A1 (en) |
| AT (1) | AT324282B (en) |
| BE (1) | BE801106A (en) |
| CA (1) | CA994658A (en) |
| CH (1) | CH573117A5 (en) |
| CS (1) | CS166842B2 (en) |
| DD (1) | DD104367A5 (en) |
| DK (1) | DK144643C (en) |
| FR (1) | FR2190277A5 (en) |
| GB (1) | GB1395126A (en) |
| HU (1) | HU167097B (en) |
| IT (1) | IT990535B (en) |
| NL (1) | NL165845C (en) |
| SE (1) | SE413326B (en) |
| SU (1) | SU639487A3 (en) |
Families Citing this family (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AT339505B (en) * | 1974-03-14 | 1977-10-25 | Boehringer Mannheim Gmbh | ENZYMATIC ANALYSIS PROCEDURE |
| JPS50142090A (en) * | 1974-04-30 | 1975-11-15 | ||
| US4014744A (en) * | 1975-01-30 | 1977-03-29 | Miles Laboratories Inc. | Processes for measuring tri-, di- and monoglycerides |
| AT354406B (en) * | 1975-08-12 | 1979-01-10 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR DETERMINATION OF TRIGLYCERIDES |
| US4012287A (en) * | 1975-11-18 | 1977-03-15 | Dr. Bruno Lange Gmbh | Method and reagent for the quantitative analysis of triglycerides |
| US4045297A (en) * | 1975-12-15 | 1977-08-30 | Monsanto Company | Triglycerides determination method |
| DE2558536B2 (en) * | 1975-12-24 | 1979-07-12 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method of kinetic substrate determination and reagent for its implementation |
| US4056442A (en) * | 1976-06-01 | 1977-11-01 | The Dow Chemical Company | Lipase composition for glycerol ester determination |
| US4179334A (en) * | 1976-08-19 | 1979-12-18 | Eastman Kodak Company | Hydrolysis of protein-bound triglycerides |
| US4275151A (en) | 1977-02-03 | 1981-06-23 | Eastman Kodak Company | Hydrolysis of protein-bound cholesterol esters |
| US4275152A (en) | 1977-02-03 | 1981-06-23 | Eastman Kodak Company | Hydrolysis of protein-bound cholesterol esters |
| US4241178A (en) * | 1978-01-06 | 1980-12-23 | Eastman Kodak Company | Process and composition for the quantification of glycerol ATP and triglycerides |
| DE2831580C2 (en) * | 1978-07-18 | 1980-09-18 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method and reagent for the determination of glycerin |
| US4264589A (en) * | 1978-12-20 | 1981-04-28 | Felts James M | Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system |
| US4178285A (en) * | 1978-12-20 | 1979-12-11 | Felts James M | Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system |
| US4394445A (en) * | 1979-02-22 | 1983-07-19 | Nix Paul T | Enzymatic glyceride hydrolysis |
| FR2449726A1 (en) * | 1979-02-22 | 1980-09-19 | Millipore Corp | Fast reacting lipase compsn. for hydrolysis of glycerol ester(s) - contg. Rhizopus arrhizus lipase and Pseudomonas Fluoresens lipase |
| US4259440A (en) * | 1979-05-21 | 1981-03-31 | Miles Laboratories, Inc. | Hydrolysis and assay of triglycerides |
| JPS55156598A (en) * | 1979-05-25 | 1980-12-05 | Mitsubishi Chem Ind Ltd | Enzymatic determination of monobasic fatty acid |
| JPS561895A (en) * | 1979-06-20 | 1981-01-10 | Mitsubishi Chem Ind Ltd | Enzymic determination of monofunctional fatty acid |
| DE2950381A1 (en) * | 1979-12-14 | 1981-06-19 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD AND REAGENT FOR DETERMINING TRIGLYCERIDES |
| US4322496A (en) * | 1980-04-17 | 1982-03-30 | Eastman Kodak Company | Inhibition of lactate oxidase |
| US4309502A (en) * | 1980-06-30 | 1982-01-05 | Beckman Instruments, Inc. | Enzymatic assay for glycerol and triglycerides and a reagent for use therein |
| EP0045031A1 (en) * | 1980-07-22 | 1982-02-03 | Baker Instruments Corporation | Glycerol detection reagent and its use |
| DE3413118A1 (en) * | 1984-04-06 | 1985-10-24 | Miles Laboratories, Inc., Elkhart, Ind. | ANALYSIS METHOD AND MEANS FOR DETECTING ESTEROLYTIC AND / OR PROTEOLYTIC ENZYMS |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2000127C3 (en) * | 1970-01-02 | 1974-12-12 | Boehringer Mannheim Gmbh | Method for the quantitative cleavage and for the quantitative detection of tri-, di- and monoglycerides |
| US3703591A (en) * | 1970-12-16 | 1972-11-21 | Calbiochem | Triglyceride hydrolysis and assay |
-
1973
- 1973-03-12 AT AT216473A patent/AT324282B/en active
- 1973-04-10 IT IT22837/73A patent/IT990535B/en active
- 1973-04-11 HU HUBO1423A patent/HU167097B/hu unknown
- 1973-04-16 AR AR247572A patent/AR195328A1/en active
- 1973-04-17 NL NL7305350.A patent/NL165845C/en not_active IP Right Cessation
- 1973-04-25 DD DD170421A patent/DD104367A5/xx unknown
- 1973-05-04 DK DK245273A patent/DK144643C/en not_active IP Right Cessation
- 1973-05-08 CS CS3274*BA patent/CS166842B2/cs unknown
- 1973-05-30 US US365355A patent/US3862009A/en not_active Expired - Lifetime
- 1973-06-11 GB GB2769573A patent/GB1395126A/en not_active Expired
- 1973-06-13 CA CA174,004A patent/CA994658A/en not_active Expired
- 1973-06-14 CH CH864573A patent/CH573117A5/xx not_active IP Right Cessation
- 1973-06-14 SE SE7308385A patent/SE413326B/en unknown
- 1973-06-14 FR FR7321759A patent/FR2190277A5/fr not_active Expired
- 1973-06-19 BE BE132423A patent/BE801106A/en not_active IP Right Cessation
- 1973-06-19 JP JP48069109A patent/JPS4964495A/ja active Pending
- 1973-12-13 SU SU731975398A patent/SU639487A3/en active
Also Published As
| Publication number | Publication date |
|---|---|
| AR195328A1 (en) | 1973-09-28 |
| FR2190277A5 (en) | 1974-01-25 |
| IT990535B (en) | 1975-07-10 |
| AT324282B (en) | 1975-08-25 |
| CS166842B2 (en) | 1976-03-29 |
| NL7305350A (en) | 1973-12-21 |
| NL165845C (en) | 1981-05-15 |
| US3862009A (en) | 1975-01-21 |
| CA994658A (en) | 1976-08-10 |
| NL165845B (en) | 1980-12-15 |
| JPS4964495A (en) | 1974-06-21 |
| DD104367A5 (en) | 1974-03-05 |
| SE413326B (en) | 1980-05-19 |
| CH573117A5 (en) | 1976-02-27 |
| HU167097B (en) | 1975-08-28 |
| DK144643C (en) | 1982-09-27 |
| SU639487A3 (en) | 1978-12-25 |
| BE801106A (en) | 1973-12-19 |
| GB1395126A (en) | 1975-05-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DK144643B (en) | METHOD AND REAGENT FOR DETERMINING TRIGLYCERIDES | |
| Wahlefeld | Triglycerides determination after enzymatic hydrolysis | |
| US4259440A (en) | Hydrolysis and assay of triglycerides | |
| Sinensky | Temperature control of phospholipid biosynthesis in Escherichia coli | |
| CS212729B2 (en) | Method of determining glycerol ester and fatty acid contents in aqueous media,and an enzymatic agent for carrying out the method | |
| US3759793A (en) | Process for the quantitative determination of tri di and monoglycerides | |
| US4543326A (en) | Stabilization of oxidase | |
| US3898130A (en) | Rapid enzymatic hydrolysis of triglycerides | |
| CA1066210A (en) | Synergistic effect of microbial lipases for the hydrolysis of glycerol esters | |
| DK151396B (en) | PROCEDURE AND REAGENT FOR DETERMINING CHOLESTERIN | |
| Meijer | Histochemical method for the demonstration of myosin adenosine triphosphatase in muscle tissues | |
| US4343897A (en) | Reagent for the determination of lipase and process for preparing same | |
| US4394445A (en) | Enzymatic glyceride hydrolysis | |
| US4338395A (en) | Method for the analysis of triglycerides | |
| US4223090A (en) | Reagents for the enzymatic determination of triglycerides | |
| CS219875B2 (en) | Method of fixing the triglycerine | |
| US4309502A (en) | Enzymatic assay for glycerol and triglycerides and a reagent for use therein | |
| JPS5948098A (en) | Measurement of lipase | |
| US4476223A (en) | Process for the stabilization of aqueous solutions of cholesterol esterase from pseudomonas | |
| JPH0474000B2 (en) | ||
| Knoche et al. | Some characteristics of a lipase preparation from the uredospores of Puccinia graminis tritici | |
| SU505382A3 (en) | The method of determination of triglycerides in substances | |
| EP0045032B1 (en) | A triglyceride analysis composition and a method for triglyceride determination | |
| Volkin | [82] Phosphomonoesterase of bone | |
| O'Brien et al. | Carboxytransphosphorylase: VIII. Ligand-mediated interaction of subunits as a possible control mechanism in Propionibacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUP | Patent expired |