WO1982000833A1 - Procede de determination du cholesterol total - Google Patents

Procede de determination du cholesterol total Download PDF

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Publication number
WO1982000833A1
WO1982000833A1 PCT/EP1981/000139 EP8100139W WO8200833A1 WO 1982000833 A1 WO1982000833 A1 WO 1982000833A1 EP 8100139 W EP8100139 W EP 8100139W WO 8200833 A1 WO8200833 A1 WO 8200833A1
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WO
WIPO (PCT)
Prior art keywords
cholesterol
bound
determination
dehydrogenase
nad
Prior art date
Application number
PCT/EP1981/000139
Other languages
German (de)
English (en)
Inventor
Inst Battelle
Original Assignee
Betz J
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Betz J filed Critical Betz J
Publication of WO1982000833A1 publication Critical patent/WO1982000833A1/fr

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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

Definitions

  • the invention relates to a method for determining total cholesterol including the bound cholesterol by releasing the bound cholesterol with cholesterol esterase and reacting the free cholesterol with a NAD- or NADP-dependent cholesterol dehydrogenase and measuring the reduced cosubstrate.
  • cholesterol esterase To determine total cholesterol, the bound cholesterol is generally released with cholesterol esterase and the free cholesterol is reacted with an enzyme specific for cholesterol.
  • the cholesterol-converting enzyme is called cholesterol oxidase which, in the presence of atmospheric oxygen, converts cholesterol into the formation of H 2 O 2 in cholestenone.
  • the particular advantage of this method is that it enables a fully enzymatic determination of cholesterol in biological material, where cholesterol is usually present as an ester both in free and in bound form.
  • This fully enzymatic determination considerably simplifies the previously customary, at least two-stage method, in which the saponification of the ester has to be carried out in a separate stage.
  • the reaction products from the oxidation of cholesterol can be determined using various methods.
  • a known method for determining H 2 O 2 is based on the oxidative coupling with p-amino-phenazone and phenol, in Presence of peroxidase.
  • a chromogen is formed with an absorption maximum at 500 nm, which can easily be determined quantitatively with a photometer.
  • This method can be used to determine cholesterol with cholesterol oxidase.
  • Cholesterol is determined using simple means. In many cases, however, large deviations are found, which are due to the formation of turbidity. Furthermore, at least two enzymes are involved in this reaction, which can result in errors.
  • Nocardia erythropolis It is known that an enzyme preparation with cholesterol dehydrogenase activity can be obtained from Nocardia erythropolis (DE-OS 23 05 232).
  • Nocardia erythropolis is an aerobic microorganism, but has no NAD or NADP dependency. For this reason, the detection of the reaction products formed is difficult and prone to failure.
  • the present invention is based on the object of specifying a method with which the disadvantages of known methods can be avoided.
  • Streptomyces hydrogenans as an aerobic microorganism allows cultivation in open ferment, the conditions for the recovery of the cholesterol dehydrogenase are considerably facilitated.
  • Streptomyces hydrogenans (ATCC 19631) is kept on oatmeal agar in inclined agar tubes.
  • Composition of the nutrient medium according to Heinz and Ring 3 g oatmeal, 2 g agar, 250 mg NaCl, 90 ml water and 10 ml soil extract.
  • the mixture is heated in a boiling water bath for 30 min, poured off over gauze, adjusted to pH 7.2 and sterilized in an autoclave at 177 ° C. for 5 min.
  • Vaccinations are carried out at intervals of approx. 6 weeks and after 10 days at 30 ° C in the incubator, the fully grown stock culture can be stored at room temperature.
  • bacteria are cultivated in shaking cultures of 150 ml or 30 ml nutrient medium in 1 l or 250 ml Erlenmeyer flasks.
  • the medium is prepared as follows: 10 g glucose, 1 g yeast extract, 2.5 g NaCl, 4 g meat extract, 4 g casein peptone and 1 l water are heated until all components have dissolved. After filtration, the pH is adjusted to 7.5 and the mixture is heated at 117 ° C. in an autoclave at 20 min. The slanted agar is inoculated with a platinum loop and an overnight culture is grown at 30 ° C. with shaking at approx. 100 rpm and 7 cm amplitude.
  • This culture can either be used immediately, or you inoculate a new 150 ml culture with 20 ml overnight culture, which you can harvest after 5 to 6 hours of growth.
  • the cells in the quasi-logarithmic phase are aspirated in a porcelain filter and washed with cold Tris buffer (10 nM Tris, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM sodium azide, pH 7.4).
  • Tris buffer 10 nM Tris, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM sodium azide, pH 7.4
  • the cells are already very sporulating and the filter pores are clogged, they are centrifuged and washed three times in a Christ IIK centrifuge at 1500 xg for 10 min each.
  • the cells are then resuspended in a suitable buffer and homogenized.
  • the homogenized cells are centrifuged for 20 min at 0-4 ° C in a Sorvall Superspeed SR-2 at 35,000 x g. Cell wall debris and membrane parts are then in the precipitation. The supernatant can now be sedimented by centrifugation at 105,000 x g and 0 - 4 oC in a Spinco L50 for 3 hours.
  • the extraction can be carried out by fractional ammonium sulfate precipitation in a manner known per se.
  • Another possibility to purify the enzyme to a higher specific activity is the ion exchange chromatography.
  • the enzyme obtained can be reacted in solution with the free cholesterol and NAD or NADP as cosubstrate in a manner known per se and NADH or NADPH as the end product can be determined photometrically.

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  • 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

Pour la determination du cholesterol total, y compris le cholesterol lie, on libere le cholesterol lie au moyen de cholesterol-esterase et on met en presence le cholesterol libre avec une cholesterol-hydrogenase obtenue a partir de streptomyces hydrogenans, dependante de NAD ou NADP, puis on mesure le co-substrat reduit.
PCT/EP1981/000139 1980-08-28 1981-08-26 Procede de determination du cholesterol total WO1982000833A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19803032377 DE3032377A1 (de) 1980-08-28 1980-08-28 Verfahren zur bestimmung von gesamtcholesterin
DE3032377800828 1980-08-28

Publications (1)

Publication Number Publication Date
WO1982000833A1 true WO1982000833A1 (fr) 1982-03-18

Family

ID=6110557

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1981/000139 WO1982000833A1 (fr) 1980-08-28 1981-08-26 Procede de determination du cholesterol total

Country Status (2)

Country Link
DE (1) DE3032377A1 (fr)
WO (1) WO1982000833A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0344580A1 (fr) * 1988-05-25 1989-12-06 IMMUNO Aktiengesellschaft Méthode pour déterminer les quantités relatives de toutes les lipoprotéines contenant du cholestérol dans les liquides corporels
EP0709456A2 (fr) * 1994-10-26 1996-05-01 Roche Diagnostics GmbH Cholestérol-déhydrogénase microbienne, procédé de préparation et utilisation
WO1998003675A1 (fr) * 1996-07-24 1998-01-29 Helena Laboratories Corporation Separation de cholesterol et analyse de celui-ci par fluorescence
US5856156A (en) * 1994-10-26 1999-01-05 Boehringer Mannheim Gmbh Microbial cholesterol dehydrogenase, process for its production and use

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2047147A5 (fr) * 1969-05-06 1971-03-12 Kyowa Hakko Kogyo Kk
DE2305232A1 (de) * 1973-02-02 1974-08-22 Thomas Patterson Pro Whitehead Zell- und cholesterinfreies enzympraeparat mit cholesterindehydrogenaseaktivitaet, verfahren zu seiner herstellung und seine verwendung
DE2506712A1 (de) * 1974-03-01 1975-09-04 Boehringer Mannheim Gmbh Verfahren zur bestimmung von cholesterin
FR2369564A2 (fr) * 1976-10-29 1978-05-26 Boehringer Mannheim Gmbh Procede et reactif pour la determination de la teneur en cholesterol total ou en cholesterol combine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2047147A5 (fr) * 1969-05-06 1971-03-12 Kyowa Hakko Kogyo Kk
DE2305232A1 (de) * 1973-02-02 1974-08-22 Thomas Patterson Pro Whitehead Zell- und cholesterinfreies enzympraeparat mit cholesterindehydrogenaseaktivitaet, verfahren zu seiner herstellung und seine verwendung
DE2506712A1 (de) * 1974-03-01 1975-09-04 Boehringer Mannheim Gmbh Verfahren zur bestimmung von cholesterin
FR2369564A2 (fr) * 1976-10-29 1978-05-26 Boehringer Mannheim Gmbh Procede et reactif pour la determination de la teneur en cholesterol total ou en cholesterol combine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, vol. 79, No. 19, 12 November 1973, (Columbus Ohio, US) H.H. Flegg:"Determination of serum cholesterol by an enzymic method", page 139, abstract 112935w; & Ann. Clin. Biochem. 1973, 10 (Pr3). 79-84 *
Chemical Abstracts, vol. 91, No 19, 5 November 1979, (Columbus Ohio,US) H. Duchmann u.a.: "Purification and characterization of a 17-béta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans" page 250, abstract 153358b; & Z. Naturforsch. C: Biosci. 1979, 34 C(7-8), 533-40 *
Chemical Abstracts, vol.83, No. 7, 18 August 1975 (Columbus Ohio, US) B. Kohler u.a.: "delta5-3 bétahydroxysteroid dehydrogenase from streptomyces hydrogenans" page 200, abstract 55098 g; & Naturwissenschaften 1975, 62 (6), 299 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0344580A1 (fr) * 1988-05-25 1989-12-06 IMMUNO Aktiengesellschaft Méthode pour déterminer les quantités relatives de toutes les lipoprotéines contenant du cholestérol dans les liquides corporels
US5385828A (en) * 1988-05-25 1995-01-31 "Immuno" Aktiengesellschaft Fur Chemisch-Medizinische Produkte Method for determining the relative amounts of all cholesterol-containing lipoproteins in body fluids
EP0709456A2 (fr) * 1994-10-26 1996-05-01 Roche Diagnostics GmbH Cholestérol-déhydrogénase microbienne, procédé de préparation et utilisation
US5856156A (en) * 1994-10-26 1999-01-05 Boehringer Mannheim Gmbh Microbial cholesterol dehydrogenase, process for its production and use
EP0709456A3 (fr) * 1994-10-26 1999-08-11 Roche Diagnostics GmbH Cholestérol-déhydrogénase microbienne, procédé de préparation et utilisation
WO1998003675A1 (fr) * 1996-07-24 1998-01-29 Helena Laboratories Corporation Separation de cholesterol et analyse de celui-ci par fluorescence

Also Published As

Publication number Publication date
DE3032377A1 (de) 1982-04-01

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