WO1991015597A1 - Procedure for determination of clinically significant metabolites - Google Patents

Procedure for determination of clinically significant metabolites Download PDF

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Publication number
WO1991015597A1
WO1991015597A1 PCT/FI1991/000094 FI9100094W WO9115597A1 WO 1991015597 A1 WO1991015597 A1 WO 1991015597A1 FI 9100094 W FI9100094 W FI 9100094W WO 9115597 A1 WO9115597 A1 WO 9115597A1
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WO
WIPO (PCT)
Prior art keywords
peroxide
hppa
transformed
procedure according
cholesterol
Prior art date
Application number
PCT/FI1991/000094
Other languages
French (fr)
Inventor
Osmo Suovaniemi
Matti HÄRKONEN
Original Assignee
Elomit Oy
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 Elomit Oy filed Critical Elomit Oy
Publication of WO1991015597A1 publication Critical patent/WO1991015597A1/en

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    • 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
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • the present invention concerns a procedure-for determining clinically significant metabolites, such as cholesterol, HDL cholesterol, triglycerides, creatinine and uric acid, in body fluids.
  • the object of the present invention is to dis ⁇ close a novel procedure for determining, in a simple way, rapidly and economically, significant metabolites, such as cholesterol, HDL cholesterol, triglycerides, creatinine and uric acid. It is a particular object of the invention, to disclose a novel procedure for simul ⁇ taneous determination of total cholesterol and HDL cho ⁇ lesterol, e.g. in plasma or serum.
  • the invention is based on the fundamental idea that the respective metabolite is made to produce per ⁇ oxide.
  • the peroxide which has been formed is determin ⁇ ed, based on the dimer constituted by 3-(4-hydroxy- phenyl)propionic acid (HPPA) with peroxide (dihydroxy- diphenyl-dipropionic acid) .
  • HPPA 3-(4-hydroxy- phenyl)propionic acid
  • peroxide dihydroxy- diphenyl-dipropionic acid
  • cholesterolester for instance, may be hydrolyzed to cholesterol and fatty acid: cholesterolester + H 2 0 —> cholesterol + fatty acids
  • the cholesterol is oxidized e.g. enzymatically with the aid of cholesteroloxidase:
  • the peroxide and HPPA are constituted to form the powerfully fluorescent HPPA dimer, e.g. with the aid of peroxidase.
  • the strongly fluorescent HPPA dimer (HPPA-HPPA) can be quantitatively determined e.g. at excitation wavelength 320 nm and emission wavelength 405 nm.
  • the glycerol is transformed into glycerol-3- phosphate, e.g. enzymatically with the aid of glycero- kinase:
  • the glycerol-3-phosphate is oxidized to di- hydroxyacetonephosphate and peroxide, e.g. enzymatical ⁇ ly with the aid of glycerol-3-P-oxidase:
  • creatinine For determination of creatinine, it is hydro- lyzed to creatine, e.g. enzymatically with the aid of creatinase:
  • the creatine is further hydrolyzed to sarco- sine, e.g. enzymatically with the aid of creatinase:
  • the sarcosine is oxidized to glycine, form- aldehyde and peroxide, e.g. enzymatically with the aid of sarcosinoxidase:
  • uric acid For determining uric acid, it is oxidized to allantoin, carbon dioxide and peroxide, e.g. enzymati ⁇ cally with the aid of uricase:
  • the peroxide is determined as HPPA dimer in the manner described.
  • the procedure is applicable in principle in determination of all clinically significant metabo ⁇ lites, hormones and proteins which can be made to pro ⁇ Jerusalem peroxide (H 2 0 2 ) , e.g. with the aid of various purified enzymes.
  • the metabolite may be reacted with peroxidase-labelled antibodies which pro ⁇ Jerusalem peroxide quantitatively.
  • the peroxide determina ⁇ tion is ultimately made as described, as HPPA dimers.
  • the procedure of the invention can be imple ⁇ mented most rapidly, e.g. within 6 to 7 min. after drawing a blood sample. As a whole, the procedure is readily implementable and applicable in automated sample handling and analysis.
  • the proce ⁇ dure is appropriate for determining total cholesterol and HDL cholesterol.
  • a blood sample was taken into a heparinized tube, plasma and cells were separated in the centri ⁇ fuge, 30 sec, 8000 g.
  • dextran sulphate-Mg ++ precipitation was carried out, alternatively phosphotungstic acid-Mg ++ precipitation, the precipitate was separated in the centrifuge, 2 min., 8000 g.
  • Cholesterolesterase 1.0 U/ml Cholesteroloxidase 1.0 U/l
  • Serum or plasma can be used for sample.
  • the sample can be stored one week at +4°C, and for instance several months at -20°C.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
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  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
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  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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Abstract

A procedure for determination of clinically significant metabolites, such as cholesterol, HDL cholesterol, triglycerides, creatinine and uric acid, in body fluids, the compound being made to produce peroxide, and the peroxide thus produced being determined with the aid of the dimer of peroxidase and 3-(4-hydroxyphenyl)propionic acid (HPPA), whereby a powerfully fluorescent HPPA dimer is produced, which is fluorometrically assayed.

Description

PROCEDURE FOR DETERMINATION OF CLINICALLY SIGNIFICANT METABOLITES
The present invention concerns a procedure-for determining clinically significant metabolites, such as cholesterol, HDL cholesterol, triglycerides, creatinine and uric acid, in body fluids.
Recently, various methods for determining total cholesterol, based on dry chemistry, have become known in the field. Regarding the state of art refer¬ ence is made to the References.
Procedures of prior art are not appropriate for simultaneous determination of total cholesterol and HDL cholesterol. The object of the present invention is to dis¬ close a novel procedure for determining, in a simple way, rapidly and economically, significant metabolites, such as cholesterol, HDL cholesterol, triglycerides, creatinine and uric acid. It is a particular object of the invention, to disclose a novel procedure for simul¬ taneous determination of total cholesterol and HDL cho¬ lesterol, e.g. in plasma or serum.
Regarding the features characteristic of the invention, reference is made to the claims. The invention is based on the fundamental idea that the respective metabolite is made to produce per¬ oxide. The peroxide which has been formed is determin¬ ed, based on the dimer constituted by 3-(4-hydroxy- phenyl)propionic acid (HPPA) with peroxide (dihydroxy- diphenyl-dipropionic acid) . This dimer is powerfully fluorescent and can be quantitatively determined by fluorometry.
When the procedure of the invention is used specifically for determination of cholesterol or of a cholesterol derivative, cholesterolester, for instance, may be hydrolyzed to cholesterol and fatty acid: cholesterolester + H20 —> cholesterol + fatty acids
The cholesterol is oxidized e.g. enzymatically with the aid of cholesteroloxidase:
cholesterol + 02 —> cholest-4-en-3-one + H202
The peroxide and HPPA are constituted to form the powerfully fluorescent HPPA dimer, e.g. with the aid of peroxidase.
H 2O2 + 2HPPA —> HPPA-HPPA + 2H20
The strongly fluorescent HPPA dimer (HPPA-HPPA) can be quantitatively determined e.g. at excitation wavelength 320 nm and emission wavelength 405 nm.
Other clinically significant metabolites can be determined in like manner, in principle. For determination of triglycerides, these are hydrolyzed to glycerol and fatty acids, e.g. enzymati¬ cally with the aid of lipase:
triglyceride + 3H 0 —> glycerol + fatty acids
The glycerol is transformed into glycerol-3- phosphate, e.g. enzymatically with the aid of glycero- kinase:
glycerol + ATP —> glycerol-3-phosphate + ADP
The glycerol-3-phosphate is oxidized to di- hydroxyacetonephosphate and peroxide, e.g. enzymatical¬ ly with the aid of glycerol-3-P-oxidase:
glycerol-3-phosphate + 0 2 —> dihydroxyacetone phosphate + H202 Finally, the peroxide is quantitatively deter¬ mined in like manner as before, as HPPA dimer.
For determination of creatinine, it is hydro- lyzed to creatine, e.g. enzymatically with the aid of creatinase:
creatinine + H20 —> creatine
The creatine is further hydrolyzed to sarco- sine, e.g. enzymatically with the aid of creatinase:
creatine + H20 —> sarcosine + urea
The sarcosine is oxidized to glycine, form- aldehyde and peroxide, e.g. enzymatically with the aid of sarcosinoxidase:
sarcosine + H20 + 02 —> glycine + HCHO + H202
Finally the peroxide is determined as described above, as HPPA dimer.
For determining uric acid, it is oxidized to allantoin, carbon dioxide and peroxide, e.g. enzymati¬ cally with the aid of uricase:
uric acid + 2H. *0 + 0,* —> allantoin + CO.2 + H202
Finally the peroxide is determined as HPPA dimer in the manner described. The procedure is applicable in principle in determination of all clinically significant metabo¬ lites, hormones and proteins which can be made to pro¬ duce peroxide (H202) , e.g. with the aid of various purified enzymes. Alternatively, the metabolite may be reacted with peroxidase-labelled antibodies which pro¬ duce peroxide quantitatively. The peroxide determina¬ tion is ultimately made as described, as HPPA dimers. The procedure of the invention can be imple¬ mented most rapidly, e.g. within 6 to 7 min. after drawing a blood sample. As a whole, the procedure is readily implementable and applicable in automated sample handling and analysis. Furthermore, the proce¬ dure is appropriate for determining total cholesterol and HDL cholesterol.
The invention is described in detail in the following, with the aid of an embodiment example.
Example: Total cholesterol and HDL cholesterol
A blood sample was taken into a heparinized tube, plasma and cells were separated in the centri¬ fuge, 30 sec, 8000 g. For determining HDL cholesterol, dextran sulphate-Mg++ precipitation was carried out, alternatively phosphotungstic acid-Mg++ precipitation, the precipitate was separated in the centrifuge, 2 min., 8000 g.
For total cholesterol determination, 5 μl of plasma diluted 1:10 in water and for HDL cholesterol determination, plasma precipitated in the foregoing and diluted 1:5 in water, were both added to 500 μl reac¬ tion solution containing 100 mmol/1 Tris-HCl buffer (pH 8.1), 50 mmol/1 MgCl2, 10 mmol/1 Na cholate, 0.5 mmol/1 HPPA, 0.3% Genapol X-080, 0.02% BSA (bovine serum albumin), 0.25 U/ml peroxidase, 1.0 U/ml cholesterol- esterase and 1.0 U/ml cholesteroloxidase. The reaction was finished in less than 3 min. The reaction is linear up to 20 mmol/1 cholesterol plasma concentration. The determinations were carried out using a Transcon 102 FN fluoronephelometer (Elomit Oy, Finland) . The intra- determination and interdetermination standard devia¬ tions at 5 mmol/1 total cholesterol level amounted to 1.0 and 1.6%, respectively on 1.2 mmol/1 HDL cholester- ol level 1.7 and 3.3%. Haemoglobin and bilirubin did not interfere with the measurement at concentrations of 4 g/1 and 250 μmol/1. Comparison with results obtained by the CHOD-PAP method (Boehringer) of prior art yield¬ ed for correlation the equations:
y = 1.05 x 0.37 (r=0.88, N=56) for cholesterol, and y = 1.08 x 0.004 (r=0.99 , N=20) for HDL cholesterol.
The reagent used in the studies was prepared with said chemicals, dissolving them in water, where¬ upon the solution was freeze-dried. Addition of water before determination produced the following end concen¬ tration:
Tris-HCl buffer, pH 8.1 100 mmol/1
BSA 0.02% Genapol X-080 0.3%
HPPA 0.5 mmol/1
MgCl2 50 mmol/1
Sodium cholate 10 mmol/1
Cholesterolesterase 1.0 U/ml Cholesteroloxidase 1.0 U/l
Peroxidase 0.25 U/l
Serum or plasma can be used for sample. The sample can be stored one week at +4°C, and for instance several months at -20°C.
In the measurements fluorometric filters were used, absorption maximum 320 nm (290 to 340 nm) , emis¬ sion maximum 405 nm (390 to 440 nm) . The incubation temperature was room temperature, or e.g. 37°C. The results were calculated by the formula
F/ sampl .e
Cholesterol (mm.ol/1 ) - x calibrator cone .
^calibra or (mmθl/1) The embodiment example is only meant to illustrate the invention, and the invention may be varied within the scope of the claims following below.

Claims

1. A procedure for determination of clinical¬ ly significant metabolites, such as cholesterol, HDL cholesterol, triglycerides, creatinine and uric acid, in body fluids, c h a r a c t e r i z e d in that the compound is made to produce peroxide or reacted with a peroxidase-labelled antibody and made to produce peroxide, and the peroxide thus produced is determined with the aid of the HPPA dimer of peroxidase and 3-(4- hydroxyphenyl)propionic acid, which is fluorometrically assayed.
2. Procedure for determining a cholesterol derivative according to claim 1, c h a r a c t e r - i z e d in that the cholesterol derivative is oxidized with cholesteroloxidase to produce peroxide, the pero¬ xide is transformed into the corresponding HPPA dimer and fluorometrically assayed.
3. Procedure according to claim 1 or 2, c h a r a c t e r i z e d in that the reagent is free¬ ze-dried and after water addition admixed to the plas¬ ma/serum sample.
4. Procedure according to any one of claims 1-3, c h a r a c t e r i z e d in that the reaction of the HPPA compound is monitored and the maximum rate or endpoint recorded at excitation wavelength 320 nm and emission wavelength 405 nm.
5. Procedure according to any one of claims 1-4, c h a r a c t e r i z e d in that the deter- mination is carried out either as a one-point or a two- point measurement.
6. Two-point measuring procedure according to claim 5, c h a r a c t e r i z e d in that reagent without cholesteroloxidase and sample are mixed and the initial fluorescence is determined, and that hereafter an enzyme specific to cholesterol (cholesteroloxidase) is added, and the endpoint or maximum rate of fluores- cence increment is determined.
7. Procedure according to any one of claims 1-6, c h a r a c t e r i z e d in that blood plasma, or serum, and cells are separated in a centrifuge.
8. Procedure according to any one of claims
1-7, c h a r a c t e r i z e d in that for HDL cho¬ lesterol determination from plasma/serum the chylomi- crones, VLDL and LDL are separated by dextransulphate- Mg++ or phosphotungstic acid-Mg++ precipitation and cent- rifuging (8000 g, 2 min.) and thereafter the HDL cho¬ lesterol is determined from the plasma sample by the HPPA dimer method.
9. Procedure according to claim 1 or 2 for determining a triglyceride derivative, c h a r a c - t e r i z e d in that the triglyceride derivative is transformed into glycerol, the glycerol is transformed into glycerol-3-phosphate and the glycerol-3-phosphate is transformed into dihydroxyacetone phosphate and peroxide, and the peroxide is assayed by the HPPA dimer method.
10. Procedure according to claim 1 or 2 for determining a creatinine derivative, c h a r a c t e¬ r i z e d in that the creatinine is transformed into creatine, the creatine is transformed into sarcosine, the sarcosine is transformed into glycerol and peroxi¬ de, and the peroxide is assayed by the HPPA dimer met¬ hod.
11. Procedure according to claim 1 or 2 for determining uric acid, c h a r a c t e r i z e d in that the uric acid is transformed into allantoin and peroxide and the peroxide is assayed by the HPPA dimer method.
PCT/FI1991/000094 1990-04-04 1991-04-02 Procedure for determination of clinically significant metabolites WO1991015597A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI901703 1990-04-04
FI901703A FI901703A (en) 1990-04-04 1990-04-04 FOERFARANDE FOER BESTAEMMANDE AV KLINISKT RELEVANTA METABOLITER.

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006061646A1 (en) * 2004-12-11 2006-06-15 The Science And Technology Facilities Council Assay for generation of a lipid profile using fluorescence measurement

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0218127A1 (en) * 1985-09-18 1987-04-15 Roche Diagnostics GmbH Method and reagent for the specific determination of HDL-Cholesterol in serum

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0218127A1 (en) * 1985-09-18 1987-04-15 Roche Diagnostics GmbH Method and reagent for the specific determination of HDL-Cholesterol in serum

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMCIAL ABSTRACTS, Volume 94, No. 21, 25 May 1981, (Columbus, Ohio, US), see page 354, Abstract 170650v; & JP,A,56 014 157, (OKURA, OYSUKE), 1981. *
CHEMICAL ABSTACTS, Volume 102, No. 23, 10 June 1985, (Columbus, Ohio, US), HAMADA, CHIZUKO et al.: "Enzymic Microassay of Glucose, Uric Acid and Cholesterol using 3-(P-Hydroxyphenyl), Propionic Acid as Fluorogenic Substrate for Horseradish Peroxidase", see page 306, Abstract 200512e; & RINSHO KAGAKU, 1984, 13(5), *
CHEMICAL ABSTRACTS, Volume 104, No. 16, 21 April 1986, (Columbus, Ohio, US), HAYASHI, YOHJI et al.: "Flow-Injection Analysis for Hydrogen Peroxide by using Immobilized Horseradish Peroxidase and its Fluorogenic Substrate 3-(P-Hydroxyphenyl) Propionic Acid", see page 757, Abstract 141252m; & ANAL. SCI., 1985, 1(1), 65-68. *
CHEMICAL ABSTRACTS, Volume 109, No. 9, 29 August 1988, (Columbus, Ohio, US), GU, YANSONG et al.: "A Flurogenic Substrate for Horseradish Peroxidase and its use in Elisa", see page 361, Abstract 69853s; & ZHONGHUA YIXUE JIANYAN ZAZHI, 1987, 10(6), 343-345. *
CHEMICAL ABSTRACTS, Volume 94, No. 3, 19 January 1981, (Columbus, Ohio, US), ZAITSU, KIYOSHI et al.: "New Fluorogenic Substrates for Horseradish Peroxidase: Rapid and Sensitive Assays for Hydrogen Peroxide and the Peroxidase", see page 125, Abstract 12014m; & ANAL. BIOCHEM., 1980, 109(1), 109-113. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006061646A1 (en) * 2004-12-11 2006-06-15 The Science And Technology Facilities Council Assay for generation of a lipid profile using fluorescence measurement

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FI901703A0 (en) 1990-04-04

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