DE3032377A1 - METHOD FOR DETERMINING TOTAL CHOLESTEROL - Google Patents
METHOD FOR DETERMINING TOTAL CHOLESTEROLInfo
- Publication number
- DE3032377A1 DE3032377A1 DE19803032377 DE3032377A DE3032377A1 DE 3032377 A1 DE3032377 A1 DE 3032377A1 DE 19803032377 DE19803032377 DE 19803032377 DE 3032377 A DE3032377 A DE 3032377A DE 3032377 A1 DE3032377 A1 DE 3032377A1
- Authority
- DE
- Germany
- Prior art keywords
- cholesterol
- bound
- dehydrogenase
- determination
- total cholesterol
- 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.)
- Withdrawn
Links
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 title claims abstract description 66
- 235000012000 cholesterol Nutrition 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims description 19
- 108010055297 Sterol Esterase Proteins 0.000 claims abstract description 7
- 102000000019 Sterol Esterase Human genes 0.000 claims abstract description 7
- 241000087134 Streptomyces hydrogenans Species 0.000 claims abstract description 3
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) 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](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims abstract 2
- 108010023417 cholesterol dehydrogenase Proteins 0.000 claims description 8
- 108010085346 steroid delta-isomerase Proteins 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 230000001419 dependent effect Effects 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 2
- -1 bound cholesterol Chemical compound 0.000 abstract 1
- 239000000758 substrate Substances 0.000 abstract 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate 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](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 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 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108010089254 Cholesterol oxidase Proteins 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- IZVFFXVYBHFIHY-UHFFFAOYSA-N (3alpha, 5alpha)-Cholest-7-en-3-ol, 9CI Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCCC(C)C)CCC33)C)C3=CCC21 IZVFFXVYBHFIHY-UHFFFAOYSA-N 0.000 description 1
- QYIXCDOBOSTCEI-QCYZZNICSA-N (5alpha)-cholestan-3beta-ol Chemical compound C([C@@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CCCC(C)C)[C@@]2(C)CC1 QYIXCDOBOSTCEI-QCYZZNICSA-N 0.000 description 1
- SEXXGJVBJDNZII-XKUXXBAGSA-N (6r)-6-[(9r,10s,13r,14r,17r)-10,13-dimethyl-2,3,4,5,6,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylheptan-1-ol Chemical compound C1CCC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)CCCC(CO)C)CC[C@H]33)C)C3=CCC21 SEXXGJVBJDNZII-XKUXXBAGSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 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 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- QYIXCDOBOSTCEI-UHFFFAOYSA-N alpha-cholestanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 QYIXCDOBOSTCEI-UHFFFAOYSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- GGCLNOIGPMGLDB-GYKMGIIDSA-N cholest-5-en-3-one Chemical compound C1C=C2CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 GGCLNOIGPMGLDB-GYKMGIIDSA-N 0.000 description 1
- NYOXRYYXRWJDKP-UHFFFAOYSA-N cholestenone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 NYOXRYYXRWJDKP-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000006877 oatmeal agar Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000005691 oxidative coupling reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229910052573 porcelain Inorganic materials 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108010009004 proteose-peptone Proteins 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000007127 saponification reaction Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- 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
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Verfahren zur Bestimmung von Gesamtcholesterin Method for the determination of total cholesterol
Die Erfindung betrifft ein Verfahren zur Bestimmung von Gesamtcholesterin einschließlich des gebundenen Cholesterins durch Freisetzung des gebundenen Cholesterins mit Cholesterinesterase und Umsetzung des freien Cholesterins mit einer NAD- oder NADP-abhängigen Cholesterindehydrogenase und Messung des reduzierten Cosubstrats.The invention relates to a method for determining total cholesterol including the bound cholesterol by releasing the bound cholesterol with cholesterol esterase and conversion of the free cholesterol with an NAD or NADP-dependent cholesterol dehydrogenase and measurement of the reduced cosubstrate.
Bei der üblichen Cholesterinbestimmung nach der Liebermann-Burchard-Reaktion bildet Cholesterin mit Essigsäureanhydrid und konzentrierter Schwefelsäure blau-grün gefärbte Verbindungen, deren Farbintensität gemessen wird. Wegen stark korrodierenden und viskosen Reagenzien eignet sich diese Reaktion nicht für die Automatisierung. Sie ist überdies nicht völlig spezifisch, sondern spricht auch auf andere Steroide, z.B. 3 % Dihydrocholesterol und 0,5-1,4 % A 7-Cholestenol an, die ebenfalls in Körperflüssigkeiten vorliegen können.With the usual cholesterol determination according to the Liebermann-Burchard reaction forms cholesterol with acetic anhydride and concentrated sulfuric acid blue-green colored compounds whose color intensity is measured. Because of highly corrosive and viscous reagents, this reaction is not suitable for automation. Moreover, it is not completely specific, but also speaks other Steroids, e.g. 3% dihydrocholesterol and 0.5-1.4% A 7-cholestenol, which also may be present in body fluids.
Verfahren zur Bestimmung von Gesamtcholesterin oder gebundenem Cholesterin durch Freisetzung des gebundenen Cholesterins mit Cholesterinesterase und Bestimmung des freien Cholesterins mit einem Cholesterin umsetzenden Enzym sind bekannt. Als Cholesterin umsetzendes Enzym wird Cholesterinoxidase genannt, welche in Anwesenheit von Luftsauerstoff Cholesterin unter Bildung von H 202 in Cholestenon überführt (DE-AS 22 24 132; DE-OS 22 65 121; DE-OS 22 65 122).Method for the determination of total cholesterol or bound cholesterol by releasing the bound cholesterol with cholesterol esterase and determining of free cholesterol with a cholesterol-converting enzyme are known. as Cholesterol converting enzyme is called cholesterol oxidase, which is in the presence by atmospheric oxygen, cholesterol is converted into cholestenone with the formation of H 202 (DE-AS 22 24 132; DE-OS 22 65 121; DE-OS 22 65 122).
Der besondere Vorteil dieses Verfahrens ist darin zu sehen, daß eine vollenzymatische Bestimmung des Cholesterins in biologischem Material ermöglicht wird, wo Cholesterin gewöhnlich sowohl in freier als auch in gebundener Form als Ester vorliegt. Durch diese vollenzymatische Bestimmung wird die früher übliche, mindestens zweistufige Bestimmung, bei der die Verseifung des Esters in einer gesonderten Stufe durchgeführt werden muß, wesentlich vereinfacht.The particular advantage of this method is to be seen in the fact that a enables fully enzymatic determination of cholesterol in biological material is where cholesterol is usually considered to be in both free and bound form Ester is present. Through this fully enzymatic determination, the previously common, at least two-stage determination in which the saponification of the ester takes place in a separate Stage must be carried out, much simplified.
Die Reaktionsprodukte aus der Oxidation des Cholesterins lassen sich nach verschiedenen Methoden bestimmen. Eine bekannte Methode zur Bestimmung von H202 besteht in der oxidativen Kupplung mit p-Amino-phenzazon und Phenol und in Gegenwart von Peroxidase. Bei dieser Reaktion wird ein Chromogn gebildet mit einem Absorptionsmaximum bei 500 nm, welches sich leicht mit einem Photometer quantitativ bestimmen läßt. Es ist bekannt, daß diese H 202-Bestimmung auch im Rahmen der Cholesterinbestimmung mit Cholesterinoxidase anzuwenden ist. Die Methode erlaubt zwar eine Bestimmung des Cholesterins mit einfachen Mitteln, in vielen Fällen werden jedoch starke Abweichungen festgestellt, die auf der Bildung einer Trübung beruhen. An diesem Verfahren sind mindestens zwei Enzyme beteiligt, woraus Fehlermöglichkeiten resultieren können.The reaction products from the oxidation of cholesterol can be determine according to various methods. A well-known method for determining H202 consists of the oxidative coupling with p-amino-phenzazon and Phenol and in the presence of peroxidase. During this reaction, a chromogen is formed with an absorption maximum at 500 nm, which can easily be seen with a photometer can be determined quantitatively. It is known that this H 202 determination also takes place in the context of the cholesterol determination with cholesterol oxidase is to be used. The method allows Although a determination of cholesterol by simple means can be done in many cases however, strong deviations were found, which are based on the formation of a haze. At least two enzymes are involved in this process, which leads to possible errors can result.
Bei analytischen und klinischen Bestimmungen wird sehr häfig von Methoden Gebrauch gemacht, die in gekoppelten Reaktionen schließlich zur Reduktion von NAD oder NADP unter Bildung von NADH bzw. NADPH führen, da sich letztere Umsetzung besonders einfach in üblichen und weit verbreiteten Photometern verfolgen läßt.Methods are used very frequently in analytical and clinical determinations Made use of that in coupled reactions eventually to reduce NAD or NADP with the formation of NADH or NADPH, since the latter implementation is particularly important can be easily tracked in common and widely used photometers.
Die bekannten vollenzymatischen Cholesterinbestimmungen lassen sich jedoch nur auf recht komplizierte und umständliche Weise mit den nachgeschalteten enzymatischen Reaktionen koppeln, daß schließlich NADH bzw. NADPH gemessen werden kann. Es ist bekannt, daß diese Schwierigkeit durch ein Verfahren beseitigt werden kann, bei dem das gebundene Cholesterin mit einer Cholesterinesterase freigesetzt und gleichzeitig oder anschließend das freigesetzte Cholesterin mit einer NAD- bzw. NADP-abhängigen Dehydrogenase aus einem anaeroben Mikroorganismus oder aus Warmblüterleber bestimmt wird (DE-OS 26 49 249). Der wesentliche Nachteil dieser Methode liegt jedoch darin, daß zur Gewinnung der Cholesterindehydrogenase schwierige Kulturbedingungen eingehalten werden müssen.The well-known fully enzymatic cholesterol determinations can be but only in a rather complicated and cumbersome way with the downstream Coupling enzymatic reactions that finally NADH or NADPH are measured can. It is known that this difficulty can be overcome by one method can, in which the bound cholesterol is released with a cholesterol esterase and at the same time or afterwards the released cholesterol with an NAD- or NADP-dependent dehydrogenase from an anaerobic microorganism or is determined from warm-blooded liver (DE-OS 26 49 249). The main disadvantage However, this method is that for the recovery of cholesterol dehydrogenase difficult culture conditions have to be observed.
Der vorliegenden Erfindung liegt nun die Aufgabe zugrunde, ein Verfahren anzugeben, mit dem die Nachteile bekannter Verfahren vermieden werden können.The present invention is now based on the object of a method indicate with which the disadvantages of known methods can be avoided.
Es hat sich gezeigt, daß sich diese Aufgabe in technisch fortschrittlicher Weise lösen laßtt wenn als Cholesterindehydrogenase ein Enzym aus einem aeroben Mikroorganismus verwendet wird. Vorzugsweise wird dieCh6lesterindehydrogenase aus Streptomyces hydrogenans- (ATCC 19631) gewonnen.It has been shown that this task is technically more advanced Wise, let it be solved if, as cholesterol dehydrogenase, an enzyme from an aerobic Microorganism is used. Preferably, the chlesterine dehydrogenase is selected from Streptomyces hydrogenans- (ATCC 19631) obtained.
Zur Freisetzung des gebundenen Cholesterins kann auch eine Cholesterinesterase aus dem gleichen Mikroorganismus verwendet werden.A cholesterol esterase can also be used to release the bound cholesterol from the same microorganism can be used.
Da aerobe Mikroorganismen eine Kultivierung in offenen Fermenten zulassen, werden die Bedingungen bei der Gewinnung der Cholesterindydrogenase wesentlich erleichtert.Since aerobic microorganisms allow cultivation in open ferments, the conditions in the production of cholesterol dehydrogenase are considerably facilitated.
Im folgenden wird die Herstellung der Cholesterindehydrogenase anhand eines lediglich einen Ausführungsweg darstellenden Beispiels näher erläutert.The production of cholesterol dehydrogenase is based on the following an example, which merely represents one embodiment, is explained in more detail.
Für die Kultivierung des Mikroorganismus wird Streptomyces hydrogenans (ATCC 19631) auf Haferflockenagar in Schrägagarröhrchen gehalten. Zusammensetzung des Nährbodens nach Heinz und Ring: 3 g Haferflocken, 2 g Agar, 250 mg NaCl, 90 ml Wasser und 10 ml Erdextrakt. Das Gemisch wird 30 min im siedenden Wasserbad erhitzt, über Gaze abgegossen, auf pH 7,2 eingestellt und 5 min bei 177 OC im Autoklaven sterilisiert. In Abständen von ca. 6 Wochen wird überimpft und jeweils nach 10 Tagen bei 30 OC im Brutschrank kann die ausgewachsene Stammkultur bei Zimmertemperatur auf bewahrt werden.Streptomyces hydrogenans is used to cultivate the microorganism (ATCC 19631) kept on oatmeal agar in agar slants. composition of the nutrient medium according to Heinz and Ring: 3 g oat flakes, 2 g agar, 250 mg NaCl, 90 ml of water and 10 ml of soil extract. The mixture is heated in a boiling water bath for 30 minutes, Poured over gauze, adjusted to pH 7.2 and 5 min at 177 ° C. in an autoclave sterilized. The vaccination takes place every 6 weeks and every 10 days at 30 OC in the incubator, the mature stock culture can be stored at room temperature be kept.
Für die Versuche werden Bakterien in Schüttelkulturen von 150 ml bzw. 30 ml Nährmedium in 1 1 bzw. 250 ml Erlenmeyerkolben kultiviert. Das Medium wird wie folgt hergestellt: 10 g Glucose, 1 g Hefeextrakt, 2,5 g Nach, 4 g Fleischextrakt, 4 g Caseinpepton und 1 1 Wasser werden erhitzt bis sich alle Bestandteile gelöst haben. Nach Filtration wird auf pH 7,5 eingestellt und bei 20 min 117 OC im Autoklaven erhitzt. Vom Schrägagar wird mit einer Platinöse angeimpft und bei 30 OC unter Schütteln mit ca. 100 Upm und 7 cm Amplitude eine Übernachtkultur gezüchtet. Diese Kultur kann entweder sofort verwendet werden, oder man impft mit je 20 ml Übernachtkultur eine neue 150-ml-Kultur an, die man nach 5 bis 6 Stunden Wachstum ernten kann. Die Zellen in der quasi-logarithmischen Phase werden in einer Porzellanfilternusche abgesaugt,und mit kalten Trispuffer (10 nM Tris, 1,5 mM MgCl2, 10 mM KCl, 1 mM Natriumazid, pll 7,4) gewaschen. In Fällen, in denen die Zellen schon stark sporulieren und die Filterporen verstopfen, werden sie dreimal in einer Christ-IIK-Zentrifuge bei 1500 x g jeweils 10 min zentrifugiert und gewaschen. Die Zellen werden dann in einem geeigneten Puffer resuspendiert und homogenisiert.For the experiments, bacteria in shaking cultures of 150 ml resp. 30 ml culture medium cultivated in 11 or 250 ml Erlenmeyer flasks. The medium becomes prepared as follows: 10 g glucose, 1 g yeast extract, 2.5 g Nach, 4 g meat extract, 4 g of casein peptone and 1 liter of water are heated until all of the components dissolve to have. After filtration, the pH is adjusted to 7.5 and 117 ° C. in the autoclave for 20 minutes heated. A platinum loop is inoculated from the agar slant and shaken at 30 ° C an overnight culture was grown at about 100 rpm and an amplitude of 7 cm. This culture can either be used immediately or inoculated with 20 ml overnight culture a new 150 ml culture that can be harvested after 5 to 6 hours of growth. the Cells in the quasi-logarithmic phase are placed in a porcelain filter shower sucked off, and with cold tris buffer (10 nM Tris, 1.5 mM MgCl2, 10 mM KCl, 1 mM sodium azide, pI 7.4). In cases where the cells already sporulate heavily and clog the filter pores, they will sporulate three times in a Christ IIK centrifuge centrifuged at 1500 x g for 10 min each time and washed. The cells will then resuspended in a suitable buffer and homogenized.
Zum Aufbrechen der Zellwände haben sich vor allem zwei Methoden bewährt: Ultraschallaufschluß und Aufschluß mit der X-Press. Bei der Ultraschallmethode werden die Zellen in 3 ml Tris-Pulver resuspendiert und im Branson-Sonifier ; 75 fir 90 sec. bei Stufe 8 und 4,5 A homogenisiert.Two methods in particular have proven effective for breaking the cell walls: Ultrasonic digestion and digestion with the X-Press. When using the ultrasound method the cells are resuspended in 3 ml of Tris powder and placed in the Branson sonifier; 75 fir 90 sec. Homogenized at level 8 and 4.5 A.
Wenn ein Detergens verwendet werden soll, setzt man es kurz vor Ende der Ultraschallbehandlung zu. Während der Beschallung wird die Probe mit Eis gekühlt. Zur Aufarbeitung der gesammelten Zellen in der X-Press werden diese in wenig entsprechendem Puffer je nach Extraktionsmethode und gegebenenfalls unter Zusatz von Detergenzien suspendiert und die tiefgekühlte, mindestens -25 OC kalte X-Press eingefüllt und noch eine halbe Stunde bei -30 OC gekühlt. Die gefrorenen Zellen werden dann mittels einer iiydraulischen Presse in 4 Durchläufen bei 1000 - 2000 N/mm2 homogenisiert.If a detergent is to be used, put it just before the end ultrasound treatment too. During the sonication, the sample is cooled with ice. In order to work up the collected cells in the X-Press, these are not very appropriate Buffer depending on the extraction method and, if necessary, with the addition of detergents suspended and the frozen, at least -25 OC cold X-Press filled and Chilled another half an hour at -30 OC. The frozen cells are then means homogenized in a hydraulic press in 4 passes at 1000 - 2000 N / mm2.
1)ie homogenisierten Zellen werden 20 min 0 - 4 OC in einer Sorvall Superspeed SR-2 mit 35.000 x g zentrifugiert.1) The homogenized cells are 20 min 0-4 OC in a Sorvall Superspeed SR-2 centrifuged at 35,000 x g.
Im Niederschlag gefinden sich sodann Zellwandtrümmer und Membranteile. Aus dem überstand können nun durch 3-stündige Zentrifugation bei 105.000 x g und 0 - 4 °C in einer Spinco L50 die Ribosomen sedimentiert werden.Cell wall debris and parts of the membrane are then found in the precipitate. The supernatant can now be centrifuged for 3 hours at 105,000 x g and 0 - 4 ° C the ribosomes are sedimented in a Spinco L50.
Die Gewinnung kann durch fraktionierte Ammonsulfatfällung in an sich bekannter Weise erfolgen. Eine weitere Möglichkeit, das Enzym zu höherer spezifischer Aktivität aufzureinigen, besteht in der Ionenaustauscher-Chromatographie. Das gewonnene Enzym kann in Lösuny mit dem freien Cholesterin unti NAD bzw. NADP als Cosubstrat in an sich bekannter Weise umgesetzt und NADH bzw. NADPH als Endprodukte photometrisch bestimmt werden.The recovery can be done by fractional ammonium sulfate precipitation in itself take place in a known manner. Another way of making the enzyme more specific Purifying activity consists in ion exchange chromatography. The won Enzyme can be used in solution with free cholesterol and NAD or NADP as a cosubstrate implemented in a manner known per se and NADH or NADPH as end products photometrically to be determined.
Claims (3)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19803032377 DE3032377A1 (en) | 1980-08-28 | 1980-08-28 | METHOD FOR DETERMINING TOTAL CHOLESTEROL |
| PCT/EP1981/000139 WO1982000833A1 (en) | 1980-08-28 | 1981-08-26 | Method for the determination of total cholesterol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19803032377 DE3032377A1 (en) | 1980-08-28 | 1980-08-28 | METHOD FOR DETERMINING TOTAL CHOLESTEROL |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DE3032377A1 true DE3032377A1 (en) | 1982-04-01 |
Family
ID=6110557
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE19803032377 Withdrawn DE3032377A1 (en) | 1980-08-28 | 1980-08-28 | METHOD FOR DETERMINING TOTAL CHOLESTEROL |
Country Status (2)
| Country | Link |
|---|---|
| DE (1) | DE3032377A1 (en) |
| WO (1) | WO1982000833A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3817747A1 (en) * | 1988-05-25 | 1989-11-30 | Johannes Dr Med Aufenanger | METHOD FOR DETERMINING THE RELATIVE VOLUME OF ALL CHOLESTERO-CONTAINING LIPOPROTEINS IN KOERPER FLUIDS |
| DE4438206A1 (en) * | 1994-10-26 | 1996-05-02 | Boehringer Mannheim Gmbh | Microbial cholesterol dehydrogenase, process for its preparation and use |
| US5856156A (en) * | 1994-10-26 | 1999-01-05 | Boehringer Mannheim Gmbh | Microbial cholesterol dehydrogenase, process for its production and use |
| DE69720230T2 (en) * | 1996-07-24 | 2003-11-13 | Helena Laboratories Corp., Beaumont | SEPARATING CHOLESTEROL AND FLUORESCENCE ANALYSIS |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2047147A5 (en) * | 1969-05-06 | 1971-03-12 | Kyowa Hakko Kogyo Kk | |
| DE2305232C3 (en) * | 1973-02-02 | 1978-06-08 | Boehringer Mannheim Gmbh, 6800 Mannheim | Process for the production of a cell-free and cholesterol-free enzyme preparation with cholesterol dehydrogenase activity and its use for the determination of cholesterol in serum |
| AT335626B (en) * | 1974-03-01 | 1977-03-25 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR THE DETERMINATION OF TOTAL CHOLESTEROL OR BONDED CHOLESTEROL |
| DE2649749A1 (en) * | 1976-10-29 | 1978-05-11 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR THE DETERMINATION OF TOTAL CHOLESTEROL OR BONDED CHOLESTEROL |
-
1980
- 1980-08-28 DE DE19803032377 patent/DE3032377A1/en not_active Withdrawn
-
1981
- 1981-08-26 WO PCT/EP1981/000139 patent/WO1982000833A1/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| WO1982000833A1 (en) | 1982-03-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DE2265121B2 (en) | Enzyme preparation for the determination of cholesterol and process for its production | |
| DE2316637A1 (en) | METHOD FOR DETERMINATION OF CHOLESTEROL | |
| DE2224133B2 (en) | Use of certain strains of microorganisms to obtain cholesterol oxidase, which oxidizes cholesterol with O low 2 to cholestenone + H low 2 O low 2 | |
| DE2649749C2 (en) | ||
| DE1768215C3 (en) | Process for the production of 1,4-androstadiene-3,17-dlon and 4-androstene-3,17-dione by microbiological degradation | |
| DE3127979C2 (en) | Use of a specific cholesterase to hydrolyze cholesterol fatty acid esters | |
| EP0120440B1 (en) | Nad(p)-independent glycerol dehydrogenase, process for its preparation and its use in the determination of glycerol and triglycerides | |
| DE69330671T2 (en) | METHOD FOR PRODUCING A SUBSTANCE FOR LOWERING THE CHOLESTERIN LEVEL | |
| DE2527068A1 (en) | PROCESS FOR THE PRODUCTION OF CHOLESTEROLESTERASE | |
| DE2842940C2 (en) | Process for the production of sarcosine oxidase | |
| DE3541242C2 (en) | ||
| DE3032377A1 (en) | METHOD FOR DETERMINING TOTAL CHOLESTEROL | |
| EP2333100B1 (en) | NAD(P)+-cofactor regeneration system und its use | |
| NO130897B (en) | ||
| DE3600563A1 (en) | HEAT-RESISTANT SARCOSINOXIDASE N AND METHOD FOR THE PRODUCTION THEREOF | |
| DE2929530C2 (en) | Glycerin oxidizing enzyme, process for its preparation and its use for the quantitative determination of glycerin | |
| DE3151616C2 (en) | Process for the production of a cholesterol oxidase | |
| DE3741198C2 (en) | ||
| DE3991428C2 (en) | Bile acid sulphate sulphatase produced by pseudomonas testosterone | |
| DE2917891C2 (en) | Process for the production of acyl-CoA synthetase | |
| DE2164018A1 (en) | Process for the biotechnological production of uricase | |
| DE3025424C2 (en) | Process for the production of galactose oxidase | |
| EP0139985B1 (en) | Process and reagent for the determination of transaminases | |
| DE2712004C2 (en) | Method for the determination of formate or compounds which can be converted into formate and a suitable reagent therefor | |
| DE3019450A1 (en) | METHOD FOR OBTAINING GLUCOSEDEHYDROGENASE AND MICROORGANISM SUITABLE FOR THIS |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 8130 | Withdrawal |