CA1323554C - Simultaneous assay for cholesterol and triglyceride - Google Patents
Simultaneous assay for cholesterol and triglycerideInfo
- Publication number
- CA1323554C CA1323554C CA000578065A CA578065A CA1323554C CA 1323554 C CA1323554 C CA 1323554C CA 000578065 A CA000578065 A CA 000578065A CA 578065 A CA578065 A CA 578065A CA 1323554 C CA1323554 C CA 1323554C
- Authority
- CA
- Canada
- Prior art keywords
- cholesterol
- triglyceride
- determination
- substrates
- reagent system
- 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.)
- Expired - Fee Related
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 92
- 235000012000 cholesterol Nutrition 0.000 title claims abstract description 46
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 238000003556 assay Methods 0.000 title description 28
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 46
- 239000000758 substrate Substances 0.000 claims abstract description 46
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- 238000000034 method Methods 0.000 claims abstract description 30
- 108090001060 Lipase Proteins 0.000 claims abstract description 20
- 102000004882 Lipase Human genes 0.000 claims abstract description 20
- 239000004367 Lipase Substances 0.000 claims abstract description 19
- 238000002835 absorbance Methods 0.000 claims abstract description 19
- 235000019421 lipase Nutrition 0.000 claims abstract description 19
- 102000057621 Glycerol kinases Human genes 0.000 claims abstract description 16
- 108700016170 Glycerol kinases Proteins 0.000 claims abstract description 16
- 108010089254 Cholesterol oxidase Proteins 0.000 claims abstract description 15
- 108010029942 microperoxidase Proteins 0.000 claims abstract description 15
- 102000013009 Pyruvate Kinase Human genes 0.000 claims abstract description 13
- 108020005115 Pyruvate Kinase Proteins 0.000 claims abstract description 13
- 239000011541 reaction mixture Substances 0.000 claims abstract description 13
- 239000000376 reactant Substances 0.000 claims abstract description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000001301 oxygen Substances 0.000 claims abstract description 7
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 6
- 108010055297 Sterol Esterase Proteins 0.000 claims abstract description 5
- 102000000019 Sterol Esterase Human genes 0.000 claims abstract description 5
- 230000000694 effects Effects 0.000 claims abstract description 5
- 101710128527 DNA-directed RNA polymerase subunit alpha Proteins 0.000 claims abstract 6
- 101710112941 DNA-directed RNA polymerase subunit beta Proteins 0.000 claims abstract 6
- 101710126019 DNA-directed RNA polymerase subunit beta C-terminal section Proteins 0.000 claims abstract 6
- 101710122417 DNA-directed RNA polymerase subunit beta N-terminal section Proteins 0.000 claims abstract 6
- 101710185074 DNA-directed RNA polymerase subunit beta' Proteins 0.000 claims abstract 6
- 101710135457 DNA-directed RNA polymerase subunit beta'' Proteins 0.000 claims abstract 6
- 102100029880 Glycodelin Human genes 0.000 claims abstract 6
- 101710154444 Putative DNA-directed RNA polymerase subunit omega Proteins 0.000 claims abstract 6
- 101710116223 Tyrosine-protein phosphatase non-receptor type 22 Proteins 0.000 claims abstract 6
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims description 20
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 18
- 230000005670 electromagnetic radiation Effects 0.000 claims description 4
- 238000005259 measurement Methods 0.000 abstract description 10
- 239000000523 sample Substances 0.000 description 13
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 11
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 11
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 11
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 11
- 238000012544 monitoring process Methods 0.000 description 11
- 229950006238 nadide Drugs 0.000 description 8
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 8
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 7
- 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 7
- DTBNBXWJWCWCIK-UHFFFAOYSA-K phosphonatoenolpyruvate Chemical compound [O-]C(=O)C(=C)OP([O-])([O-])=O DTBNBXWJWCWCIK-UHFFFAOYSA-K 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 4
- 229940099352 cholate Drugs 0.000 description 4
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 239000001384 succinic acid Substances 0.000 description 4
- 150000003626 triacylglycerols Chemical class 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 102000003992 Peroxidases Human genes 0.000 description 3
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 3
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- GLDQAMYCGOIJDV-UHFFFAOYSA-N 2,3-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=CC(O)=C1O GLDQAMYCGOIJDV-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH 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](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 150000001840 cholesterol esters Chemical class 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229940071221 dihydroxybenzoate Drugs 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 239000001008 quinone-imine dye Substances 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
- 108010079522 solysime Proteins 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 125000005457 triglyceride group Chemical group 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
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
- C12Q1/26—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
- C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides
-
- 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
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- 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)
Abstract
ABSTRACT
A method for the simultaneous determination of cholesterol and triglyceride substrates with a single reagent system. The reagent system is reacted with the specimen such that each of the substrates react with their respective reactant simultaneously. The change in absorbance or fluorescence of the resulting reaction mixture is measured at a plurality of wavelengths which are characteristic for each of the substrates to be determined. The amount of cholesterol and triglyceride substrates can be determined by either an endpoint or reaction rate measurement. The reagent system comprises an enzyme having cholesterol esterase activity, a chromogenic oxygen acceptor, microperoxidase, and cholesterol oxidase for determination of cholesterol;
and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH and NAD(P)H or analogs thereof for determination of triglyceride.
A method for the simultaneous determination of cholesterol and triglyceride substrates with a single reagent system. The reagent system is reacted with the specimen such that each of the substrates react with their respective reactant simultaneously. The change in absorbance or fluorescence of the resulting reaction mixture is measured at a plurality of wavelengths which are characteristic for each of the substrates to be determined. The amount of cholesterol and triglyceride substrates can be determined by either an endpoint or reaction rate measurement. The reagent system comprises an enzyme having cholesterol esterase activity, a chromogenic oxygen acceptor, microperoxidase, and cholesterol oxidase for determination of cholesterol;
and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH and NAD(P)H or analogs thereof for determination of triglyceride.
Description
132'3~a~
SIMULTANEOUS ASSAY FOR
CHOLESTEROL AND TRIGLYCERIDES
BACKGROUND OF THE INVENTION
The present invention relates to the simultaneous measurement of a plurality of substrates with a single reagent by monitoring concurrent reactions which produce changes in the electromagnetic radiation absorbance characteristics of the sample. In one aspect, the invention relates to the simultaneous measurement of cholesterol and triglycerides in blood serum by monitoring two concurrent reactions at two or more different wavelengths.
In the field of diagnostics, various assays are designed to identify or quantify a substrate which may be present in a sample material. Unfortunately the assay is usually only specific to one type of substrate even though it may be desirable to diagnose more than one substrate for any given sample. This leads to multiple testing on the same sample which increases diagnosis cost and decreases efficiency. It is therefore desirable to develop diagnostic testing which can identify or quantify multiple substrates in an efficient manner.
For example, cholesterol and triglycerides are two of the more common tests performed in the clinical chemistry laboratory. Analysis of cholesterol is typically done using a cholesterol esterase, peroxidase (Trinder) method (Tietz, N.W., Textbook of Clinical Chemistry, 1986, p. 883). In the Trinder method cholesterol esters are hydrolyzed by cholesterol 1323~4 esterase to form free cholesterol and fatty acids. The free cholesterol is then oxidized by cholesterol oxidase which forms hydrogen peroxide. The hydrogen peroxide then reacts with peroxidase and a chromogenic oxygen acceptor to produce a color change in the 400-500 nm range.
Analysis of triglyceride is typically done using the lipase/glycerol kinase method (Tietz, N. W., Textbook of Clinical ChemistrY, 1986, p. 887).
Triglycerides are hydrolyzed by microbial lipase to produce glycerol and free fatty acids. Glycerol and ATP
in the presence of glycerol kinase form glycerol-3-phosphate + ADP. The ADP from this reaction together with phosphoenolpyr~lvate react with pyruvate to form ATP
and pyruvate. The pyruvate produced reacts with lactate dehydrogenase to produce lactate, with the concomitant oxidation of NADH producing a decrease in absorbance at 340 nm.
The assays mentioned above are performed with separate reagents in separate cuvettes. This costs the clinical chemistry lab time and money. By combining the two tests into one test the lab would be able to realize an increase in productivity and also a cost savings.
Combining the two tests is not a straightforward task. Conditions must be selected that allow precise measurement of both substrates. For example, combination of the traditional cholesterol oxidase and triglyceride lipase/glycerol kinase methods is eliminated by the fact that peroxidase in the cholesterol reaction would oxidize the NAD~ in the triglyceride reaction.
lt~23~4 One way of combining the two assays in a single reaction vessel is to do a sequential assay (U.S. Patent 4,425,427 to Luderer and EP Patent 133064 to Cam et al.). In a sequential assay, reagent for the first assay is added to the vessel and the reaction proceeds.
At some later time a concentration is determined for the first component. Then a second reagent, which either quenches the first reaction or is added after the first reaction is complete, is added to the vessel to trigger a reaction with the second component. At some later time the concentration of the second component is determined. These reactions can either be monitored at the same wavelength or at different wavelengths (either through the use of filter wheels or diode arrays).
U.S. Patent 3,925,162 describes the simultaneous measurement of enzyme activity in body fluids. In this approach the substrate for each of the enzymes to be identified are added to a reaction medium with other reagents and changes in the absorbance or fluorescence of the resulting reaction system are measured. The present invention utilizes an approach where a single reagent system is used to simultaneously identify or quantify substrate by monitoring the electromagnetic signal of the reaction mixture.
SUMMARY OF THE INVENTION
The present invention is directed toward a method for the simultaneous determination of cholesterol and triglyceride substrates with a single reagent system in a reaction mixture. The method comprises adding a reagent system containing a reactant for each of the substrates to be determined, each reactant being 1 ~235~
selected such that it is capable of giving a unique electromagnetic radiation absorbance for the particular substrate permits calculation of both substrate concentrations. The substrates are reacted with their respective reactant under conditions such that the reaction takes place simultaneously. The concentration of the substrates is determined by measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
In another aspect the present invention is a method for the simultaneous determination of cholesterol and triglyceride substrates with a single reagent system in a reaction mixture by adding a reagent system containing a chromophore for each of the substrates to be determined, each chromophore being selected such that it is capable of giving a unique absorbance band for the particular substrate and allows the determination of the other substrate. The substrates are reacted with their respective chromophore under conditions such that the reaction takes place simultaneously. The concentration of the substrates is determined by measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
The reagent system comprises an enzyme having cholesterol esterase activity, a chromogenic oxygen acceptor, microperoxidase, and cholesterol oxidase for determination of cholesterol; and lipase, adenosine ~123~
triphosphate (ATP), phosphoenolpyruvate (PEP), glycerol kinase, pyruvate kinase, lactate dehydrogenase (LDH) and NAD(P)H or analogs thereof for determination of triglyceride. The simultaneous assay can be performed with a reagent system comprising lipase, 4-aminoantipyrine, phenol, microperoxidase and cholesterol oxidase to allow for a reaction rate or endpoint determination of cholesterol; and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH, and NAD(P)H
in concentrations sufficient to allow an endpoint or reaction rate determination of triglyceride.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for simultaneously measuring a plurality of substrates in a biological fluid. The method utilizes a single reagent for measurement of each of the substrateg by monitoring several electromagnetic signals simultaneously.
The electromagnetic signals can be monitored simultaneously by a spectrophotometer, or spectrofluorometer. The measurement of changes in the reaction mixture can be carried out on any of the instruments by conventional procedures. The particular change in the system ,i.e., wavelength, is not critical, but it is preferable that the changes or differences in wavelength be as great as possible provided they can be monitored simultaneously.
In a simultaneous assay a reagent containing all the components for reaction with the substrates to be measured are added to the sample and the reactions 1323~
are monitored by the instrument. Typically, a simultaneous assay is done in a single cuvette with a single reagent, eliminating the need for a second reagent dispense or other optional steps generally associated with multiple substrate assays.
A key to the design of a simultaneous assay is the selection of reagents that will allow the reactions to proceed simultaneously, and permit accurate determination of both analytes in the clinically relevant range. Reactants are chosen for each of the substrates to be determined, such that each is capable of giving a unique electromagnetic radiation absorbance for the particular substrate. A reactant can be a chromophore or indicator dye where the reaction will be monitored by spectra wavelength. For example, by choosing appropriate chromophores an assay can be developed that will measure cholesterol and triglyceride simultaneously as described below.
After the proper reactant is chosen the sample is added to the reagent system which contains the appropriate reactants. The reagent and sample are mixed such that each of the substrates is contacted with their respective reactant under conditions such that the reaction takes place simultaneously. The addition and mixing of the sample and reagent is monitored by instrumentation appropriate for the reaction taking place such as measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
Preferably the monitoring of the reaction mixture is begun as soon as the reagent and sample are intermixed. This allows for monitoring of changes in either the reaction rate or endpoint reaction change for the particular electromagnetic signal being monitored.
The subject method allows for the simultaneous measurement of cholesterol and triglyceride in blood serum using a single reagent. The cholesterol and triglyceride reactions proceed at the same time, with measurement of the two different reactions monitored at two separate wavelengths by a spectrophotometer. The spectrophotometer employs a diode array detector having the capability of simultaneously monitoring many wavelengths.
Generally the reagent system comprises an enzyme having cholesterol esterase activity, a chromogenic oxygen acceptor, microperoxidase, and cholesterol oxidase for the determination of cholesterol; and lipase, adenosine triphosphate (ATP), phosphoenolpyruvate (PEP), glycerol kinase, pyruvate kinase, lactate dehydrogenase (LDH) and reduced nicotinamide-adenine dinucleotide, or reduced nicotinamide-adenine dinucleotide phosphate, jointly referred to as NAD(P)H, or analogs thereof for the determination of triglyceride.
The measurement of cholesterol is through the use of microperoxidase to combine with hydrogen peroxide, and a chromogenic oxygen acceptor such as a quinoneimine dye with a cosubstrate such as phenol or dihydroxybenzoate whose absorbance range is between 400-500 nm. Preferably the chromogenic oxygen acceptor is 4-aminoantipyrine (4-AAP) and phenol to produce the final dye. This allows coupling the cholesterol reagent with a triglyceride reagent uslng NAD(P)H or analogs thereof, since microperoxidase does not oxidize NAD(P)H. The triglyceride reaction monitors the oxidation of NAD(P)H at 340 nm.
To further describe the instant invention the following examples are provided.
Cholesterol/Triglyceride Simultaneous Assay The following procedure describes a method for performing a simultaneous assay for cholesterol and triglyceride by monitoring the endpoints of both the cholesterol and the triglyceride reactions. A reagent system was prepared by mixing the following (U/L is units per liter and mM is millimoles per liter):
Cholate,Na 3.OmM
4-Aminoantipyrine 0.8mM
Phenol 14~OmM
Lipase 250,000U/L
Cholesterol Oxidase 117U/L
Microperoxidase 12mg/L
NADH 0.4mM
Phosphoenolpyruvate (PEP)0.7mM
Adenosine triphosphate (ATP) 0.06mM
MgSO4 s.5mM
Tris buffer 100mM
Succinic Acid 26mM
Pyruvate Kinase 1667U/L
Glycerol Kinase 667U/L
Lactate Dehydrogenase (LDH)1000U/L
13~3~
Sample was added to the reagent at a ratio of 1:101 and the reaction was allowed to proceed. After 3 minutes the absorbance was read at 340 nm and at 500 nm. Concentrations were calculated by comparison with standard curves.
Cholesterol/Triglyceride Simultaneous Assay The following procedure describes a method for performing a simultaneous assay for cholesterol and triglyceride by monitoring the cholesterol rate of reaction and the triglyceride reaction endpoint. A
reagent system was prepared by mixing the following:
Cholate,Na 3.OmM
4-Aminoantipyrine (4-AAP)0.8mM
Phenol 14.OmM
Lipase 250,000U/L
Cholesterol Oxidase 10U/L
Microperoxidase 12mg/L
NADH 0.4mM
Phosphoenolpyruvate (PEP)a . 7mM
ATP 0.06mM
MgSO4 5.5mM
Tris buffer 100mM
Succinic Acid 26mM
Pyruvate Kinase 1667U/L
Glycerol Kinase 667U/L
Lactate Dehydrogenase (LDH)1000U/L
Sample was added to the reagent at a ratio of 1:101 and the reaction was allowed to proceed. The cholesterol rate of reaction was monitored at 500 nm by 13235~
taking a reading every 60 seconds for three minutes, starting at 60 seconds. After 3 minutes the absorbance is read at 340 nm. Concentrations are calculated by comparison with standard curves.
Cholesterol/Triglyceride Simultaneous Assay The following procedure describes a method for performing a simultaneous assay for cholesterol and triglyceride by monitoring the cholesterol reaction endpoint and the triglyceride reaction rate. A reagent system was prepared by mixing the following:
Cholate,Na 3.OmM
4-Aminoantipyrine 0.8mM
Phenol 14.OmM
Lipase 250,000U/L
Cholesterol Oxidase 117U/L
Microperoxidase 12mg/L
NADH 0.4mM
Phosphoenolpyruvate 0.7mM
ATP 0.06mM
MgSO4 . s.5mM
Tris buffer 100mM
Succinic Acid 26mM
Pyruvate Kinase 1667U/L
Glycerol Kinase 60U/L
Sample was added to the reagent at a ratio of 1:101 and the reaction was allowed to proceed. The triglyceride was followed at 340 nm by reading every 60 seconds for three minutes. After 3 minutes the absorbance is read at 500 nm. Concentrations were calculated by comparison with standard curves.
13,~3~
Cholesterol/Triglyceride Simultaneous Assay The following procedure describes a method for performing a simultaneous assay for cholesterol and triglyceride by monitoring the cholesterol reaction rate and the triglyceride reaction rate. A reagent system was prepared by mixing the following:
Cholate,Na 3.OmM
4-Aminoantipyrine 0.8mM
Phenol 14.OmM
Lipase 250,000U/L
Cholesterol Oxidase lOU/L
Microperoxidase 12mg/L
NADH 0.4mM
Phosphoenolpyruvate 0.7mM
ATP 0.06mM
MgS04 5.5mM
Tris buffer lOOmM
Succinic Acid 26mM
Pyruvate Kinase 1667U/L
Glycerol Kinase 60U/L
LDH lOOOU/L
Sample was added to the reagent at a ratio of 1:101 and the reaction was allowed to proceed. Every 60 seconds for three minutes the absorbance is read at 500 nm and at 340 nm. Concentrations were calculated by comparison with standard curves.
Cholesterol/Triglyceride Simultaneous Fluorescent Assay 13235~
The following procedure d.escribes a method for performing a simultaneous assay for cholesterol and triglyceride which employs fluorescence to determine the substrate concentration. In this method a spectrofluorometer is used to monitor the simultaneous reactions, The components of the assay are essentially the same as in Example 1. The cholesterol part of the assay is measured by following the fluorescence emission peak as the dye is formed. The triglyceride part of the assay is measured by following the fluorescence emission at 440 nm with excitation at 340 nm as NADH is oxidized to NAD.
SIMULTANEOUS ASSAY FOR
CHOLESTEROL AND TRIGLYCERIDES
BACKGROUND OF THE INVENTION
The present invention relates to the simultaneous measurement of a plurality of substrates with a single reagent by monitoring concurrent reactions which produce changes in the electromagnetic radiation absorbance characteristics of the sample. In one aspect, the invention relates to the simultaneous measurement of cholesterol and triglycerides in blood serum by monitoring two concurrent reactions at two or more different wavelengths.
In the field of diagnostics, various assays are designed to identify or quantify a substrate which may be present in a sample material. Unfortunately the assay is usually only specific to one type of substrate even though it may be desirable to diagnose more than one substrate for any given sample. This leads to multiple testing on the same sample which increases diagnosis cost and decreases efficiency. It is therefore desirable to develop diagnostic testing which can identify or quantify multiple substrates in an efficient manner.
For example, cholesterol and triglycerides are two of the more common tests performed in the clinical chemistry laboratory. Analysis of cholesterol is typically done using a cholesterol esterase, peroxidase (Trinder) method (Tietz, N.W., Textbook of Clinical Chemistry, 1986, p. 883). In the Trinder method cholesterol esters are hydrolyzed by cholesterol 1323~4 esterase to form free cholesterol and fatty acids. The free cholesterol is then oxidized by cholesterol oxidase which forms hydrogen peroxide. The hydrogen peroxide then reacts with peroxidase and a chromogenic oxygen acceptor to produce a color change in the 400-500 nm range.
Analysis of triglyceride is typically done using the lipase/glycerol kinase method (Tietz, N. W., Textbook of Clinical ChemistrY, 1986, p. 887).
Triglycerides are hydrolyzed by microbial lipase to produce glycerol and free fatty acids. Glycerol and ATP
in the presence of glycerol kinase form glycerol-3-phosphate + ADP. The ADP from this reaction together with phosphoenolpyr~lvate react with pyruvate to form ATP
and pyruvate. The pyruvate produced reacts with lactate dehydrogenase to produce lactate, with the concomitant oxidation of NADH producing a decrease in absorbance at 340 nm.
The assays mentioned above are performed with separate reagents in separate cuvettes. This costs the clinical chemistry lab time and money. By combining the two tests into one test the lab would be able to realize an increase in productivity and also a cost savings.
Combining the two tests is not a straightforward task. Conditions must be selected that allow precise measurement of both substrates. For example, combination of the traditional cholesterol oxidase and triglyceride lipase/glycerol kinase methods is eliminated by the fact that peroxidase in the cholesterol reaction would oxidize the NAD~ in the triglyceride reaction.
lt~23~4 One way of combining the two assays in a single reaction vessel is to do a sequential assay (U.S. Patent 4,425,427 to Luderer and EP Patent 133064 to Cam et al.). In a sequential assay, reagent for the first assay is added to the vessel and the reaction proceeds.
At some later time a concentration is determined for the first component. Then a second reagent, which either quenches the first reaction or is added after the first reaction is complete, is added to the vessel to trigger a reaction with the second component. At some later time the concentration of the second component is determined. These reactions can either be monitored at the same wavelength or at different wavelengths (either through the use of filter wheels or diode arrays).
U.S. Patent 3,925,162 describes the simultaneous measurement of enzyme activity in body fluids. In this approach the substrate for each of the enzymes to be identified are added to a reaction medium with other reagents and changes in the absorbance or fluorescence of the resulting reaction system are measured. The present invention utilizes an approach where a single reagent system is used to simultaneously identify or quantify substrate by monitoring the electromagnetic signal of the reaction mixture.
SUMMARY OF THE INVENTION
The present invention is directed toward a method for the simultaneous determination of cholesterol and triglyceride substrates with a single reagent system in a reaction mixture. The method comprises adding a reagent system containing a reactant for each of the substrates to be determined, each reactant being 1 ~235~
selected such that it is capable of giving a unique electromagnetic radiation absorbance for the particular substrate permits calculation of both substrate concentrations. The substrates are reacted with their respective reactant under conditions such that the reaction takes place simultaneously. The concentration of the substrates is determined by measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
In another aspect the present invention is a method for the simultaneous determination of cholesterol and triglyceride substrates with a single reagent system in a reaction mixture by adding a reagent system containing a chromophore for each of the substrates to be determined, each chromophore being selected such that it is capable of giving a unique absorbance band for the particular substrate and allows the determination of the other substrate. The substrates are reacted with their respective chromophore under conditions such that the reaction takes place simultaneously. The concentration of the substrates is determined by measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
The reagent system comprises an enzyme having cholesterol esterase activity, a chromogenic oxygen acceptor, microperoxidase, and cholesterol oxidase for determination of cholesterol; and lipase, adenosine ~123~
triphosphate (ATP), phosphoenolpyruvate (PEP), glycerol kinase, pyruvate kinase, lactate dehydrogenase (LDH) and NAD(P)H or analogs thereof for determination of triglyceride. The simultaneous assay can be performed with a reagent system comprising lipase, 4-aminoantipyrine, phenol, microperoxidase and cholesterol oxidase to allow for a reaction rate or endpoint determination of cholesterol; and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH, and NAD(P)H
in concentrations sufficient to allow an endpoint or reaction rate determination of triglyceride.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for simultaneously measuring a plurality of substrates in a biological fluid. The method utilizes a single reagent for measurement of each of the substrateg by monitoring several electromagnetic signals simultaneously.
The electromagnetic signals can be monitored simultaneously by a spectrophotometer, or spectrofluorometer. The measurement of changes in the reaction mixture can be carried out on any of the instruments by conventional procedures. The particular change in the system ,i.e., wavelength, is not critical, but it is preferable that the changes or differences in wavelength be as great as possible provided they can be monitored simultaneously.
In a simultaneous assay a reagent containing all the components for reaction with the substrates to be measured are added to the sample and the reactions 1323~
are monitored by the instrument. Typically, a simultaneous assay is done in a single cuvette with a single reagent, eliminating the need for a second reagent dispense or other optional steps generally associated with multiple substrate assays.
A key to the design of a simultaneous assay is the selection of reagents that will allow the reactions to proceed simultaneously, and permit accurate determination of both analytes in the clinically relevant range. Reactants are chosen for each of the substrates to be determined, such that each is capable of giving a unique electromagnetic radiation absorbance for the particular substrate. A reactant can be a chromophore or indicator dye where the reaction will be monitored by spectra wavelength. For example, by choosing appropriate chromophores an assay can be developed that will measure cholesterol and triglyceride simultaneously as described below.
After the proper reactant is chosen the sample is added to the reagent system which contains the appropriate reactants. The reagent and sample are mixed such that each of the substrates is contacted with their respective reactant under conditions such that the reaction takes place simultaneously. The addition and mixing of the sample and reagent is monitored by instrumentation appropriate for the reaction taking place such as measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
Preferably the monitoring of the reaction mixture is begun as soon as the reagent and sample are intermixed. This allows for monitoring of changes in either the reaction rate or endpoint reaction change for the particular electromagnetic signal being monitored.
The subject method allows for the simultaneous measurement of cholesterol and triglyceride in blood serum using a single reagent. The cholesterol and triglyceride reactions proceed at the same time, with measurement of the two different reactions monitored at two separate wavelengths by a spectrophotometer. The spectrophotometer employs a diode array detector having the capability of simultaneously monitoring many wavelengths.
Generally the reagent system comprises an enzyme having cholesterol esterase activity, a chromogenic oxygen acceptor, microperoxidase, and cholesterol oxidase for the determination of cholesterol; and lipase, adenosine triphosphate (ATP), phosphoenolpyruvate (PEP), glycerol kinase, pyruvate kinase, lactate dehydrogenase (LDH) and reduced nicotinamide-adenine dinucleotide, or reduced nicotinamide-adenine dinucleotide phosphate, jointly referred to as NAD(P)H, or analogs thereof for the determination of triglyceride.
The measurement of cholesterol is through the use of microperoxidase to combine with hydrogen peroxide, and a chromogenic oxygen acceptor such as a quinoneimine dye with a cosubstrate such as phenol or dihydroxybenzoate whose absorbance range is between 400-500 nm. Preferably the chromogenic oxygen acceptor is 4-aminoantipyrine (4-AAP) and phenol to produce the final dye. This allows coupling the cholesterol reagent with a triglyceride reagent uslng NAD(P)H or analogs thereof, since microperoxidase does not oxidize NAD(P)H. The triglyceride reaction monitors the oxidation of NAD(P)H at 340 nm.
To further describe the instant invention the following examples are provided.
Cholesterol/Triglyceride Simultaneous Assay The following procedure describes a method for performing a simultaneous assay for cholesterol and triglyceride by monitoring the endpoints of both the cholesterol and the triglyceride reactions. A reagent system was prepared by mixing the following (U/L is units per liter and mM is millimoles per liter):
Cholate,Na 3.OmM
4-Aminoantipyrine 0.8mM
Phenol 14~OmM
Lipase 250,000U/L
Cholesterol Oxidase 117U/L
Microperoxidase 12mg/L
NADH 0.4mM
Phosphoenolpyruvate (PEP)0.7mM
Adenosine triphosphate (ATP) 0.06mM
MgSO4 s.5mM
Tris buffer 100mM
Succinic Acid 26mM
Pyruvate Kinase 1667U/L
Glycerol Kinase 667U/L
Lactate Dehydrogenase (LDH)1000U/L
13~3~
Sample was added to the reagent at a ratio of 1:101 and the reaction was allowed to proceed. After 3 minutes the absorbance was read at 340 nm and at 500 nm. Concentrations were calculated by comparison with standard curves.
Cholesterol/Triglyceride Simultaneous Assay The following procedure describes a method for performing a simultaneous assay for cholesterol and triglyceride by monitoring the cholesterol rate of reaction and the triglyceride reaction endpoint. A
reagent system was prepared by mixing the following:
Cholate,Na 3.OmM
4-Aminoantipyrine (4-AAP)0.8mM
Phenol 14.OmM
Lipase 250,000U/L
Cholesterol Oxidase 10U/L
Microperoxidase 12mg/L
NADH 0.4mM
Phosphoenolpyruvate (PEP)a . 7mM
ATP 0.06mM
MgSO4 5.5mM
Tris buffer 100mM
Succinic Acid 26mM
Pyruvate Kinase 1667U/L
Glycerol Kinase 667U/L
Lactate Dehydrogenase (LDH)1000U/L
Sample was added to the reagent at a ratio of 1:101 and the reaction was allowed to proceed. The cholesterol rate of reaction was monitored at 500 nm by 13235~
taking a reading every 60 seconds for three minutes, starting at 60 seconds. After 3 minutes the absorbance is read at 340 nm. Concentrations are calculated by comparison with standard curves.
Cholesterol/Triglyceride Simultaneous Assay The following procedure describes a method for performing a simultaneous assay for cholesterol and triglyceride by monitoring the cholesterol reaction endpoint and the triglyceride reaction rate. A reagent system was prepared by mixing the following:
Cholate,Na 3.OmM
4-Aminoantipyrine 0.8mM
Phenol 14.OmM
Lipase 250,000U/L
Cholesterol Oxidase 117U/L
Microperoxidase 12mg/L
NADH 0.4mM
Phosphoenolpyruvate 0.7mM
ATP 0.06mM
MgSO4 . s.5mM
Tris buffer 100mM
Succinic Acid 26mM
Pyruvate Kinase 1667U/L
Glycerol Kinase 60U/L
Sample was added to the reagent at a ratio of 1:101 and the reaction was allowed to proceed. The triglyceride was followed at 340 nm by reading every 60 seconds for three minutes. After 3 minutes the absorbance is read at 500 nm. Concentrations were calculated by comparison with standard curves.
13,~3~
Cholesterol/Triglyceride Simultaneous Assay The following procedure describes a method for performing a simultaneous assay for cholesterol and triglyceride by monitoring the cholesterol reaction rate and the triglyceride reaction rate. A reagent system was prepared by mixing the following:
Cholate,Na 3.OmM
4-Aminoantipyrine 0.8mM
Phenol 14.OmM
Lipase 250,000U/L
Cholesterol Oxidase lOU/L
Microperoxidase 12mg/L
NADH 0.4mM
Phosphoenolpyruvate 0.7mM
ATP 0.06mM
MgS04 5.5mM
Tris buffer lOOmM
Succinic Acid 26mM
Pyruvate Kinase 1667U/L
Glycerol Kinase 60U/L
LDH lOOOU/L
Sample was added to the reagent at a ratio of 1:101 and the reaction was allowed to proceed. Every 60 seconds for three minutes the absorbance is read at 500 nm and at 340 nm. Concentrations were calculated by comparison with standard curves.
Cholesterol/Triglyceride Simultaneous Fluorescent Assay 13235~
The following procedure d.escribes a method for performing a simultaneous assay for cholesterol and triglyceride which employs fluorescence to determine the substrate concentration. In this method a spectrofluorometer is used to monitor the simultaneous reactions, The components of the assay are essentially the same as in Example 1. The cholesterol part of the assay is measured by following the fluorescence emission peak as the dye is formed. The triglyceride part of the assay is measured by following the fluorescence emission at 440 nm with excitation at 340 nm as NADH is oxidized to NAD.
Claims (8)
1. A method for the simultaneous determination of cholesterol and triglyceride substrates with a single reagent system in a reaction mixture, comprising:
adding a reagent system containing a reactant for each of the substrates to be determined, each reactant being selected such that it is capable of giving a unique electromagnetic radiation absorbance for the particular substrate and permits the determination of the other substrate;
simultaneously reacting each of the substrates with their respective reactant under conditions such that the reaction takes place simultaneously; and measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
adding a reagent system containing a reactant for each of the substrates to be determined, each reactant being selected such that it is capable of giving a unique electromagnetic radiation absorbance for the particular substrate and permits the determination of the other substrate;
simultaneously reacting each of the substrates with their respective reactant under conditions such that the reaction takes place simultaneously; and measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
2. A method for the simultaneous determination of cholesterol and triglyceride substrates with a single reagent system in a reaction mixture, comprising:
adding a reagent system containing a chromophore for each of the substrates to be determined, each chromophore being selected such that it is capable of giving a unique absorbance band for the particular substrate which permits determination of the other substrate;
simultaneously reacting each of the substrates with their respective chromophore under conditions such that the reaction takes place simultaneously; and measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
adding a reagent system containing a chromophore for each of the substrates to be determined, each chromophore being selected such that it is capable of giving a unique absorbance band for the particular substrate which permits determination of the other substrate;
simultaneously reacting each of the substrates with their respective chromophore under conditions such that the reaction takes place simultaneously; and measuring changes in absorbance or fluorescence of the resulting reaction mixture at a plurality of wavelengths which are characteristic for each of the substrates to be determined.
3. The method of Claim 2 wherein said reagent system comprises an enzyme having cholesterol esterase activity, a chromogenic oxygen acceptor, microperoxidase, and cholesterol oxidase for determination of cholesterol; and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH and NAD(P)H or analogs thereof for determination of triglyceride.
4. The method of Claim 3 wherein said reagent system comprises lipase, 4-AAP, phenol, microperoxidase, cholesterol oxidase in a concentration sufficient to allow an endpoint determination of cholesterol; and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH
and NAD(P)H in a concentration sufficient to allow an endpoint determination of triglyceride.
and NAD(P)H in a concentration sufficient to allow an endpoint determination of triglyceride.
5. The method of Claim 3 wherein said reagent system comprises lipase, 4-AAP, phenol, microperoxidase, cholesterol oxidase in a concentration sufficient to allow a rate determination of cholesterol; and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH and NAD(P)H in a concentration sufficient to allow an endpoint determination of triglyceride.
6. The method of Claim 3 wherein said reagent system comprises lipase, 4-AAP, phenol, microperoxidase, cholesterol oxidase in a concentration sufficient to allow an endpoint determination of cholesterol; and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH
and NAD(P)H in a concentration sufficient to allow a rate determination of triglyceride.
and NAD(P)H in a concentration sufficient to allow a rate determination of triglyceride.
7. The method of Claim 3 wherein said reagent system comprises lipase, 4-AAP, phenol, microperoxidase, cholesterol oxidase in a concentration sufficient to allow a rate determination of cholesterol; and lipase, ATP, PEP, glycerol kinase, pyruvate kinase, LDH and NAD(P)H in a concentration sufficient to allow a rate determination of triglyceride.
8. A reagent system for the simultaneous determination of cholesterol and triglyceride as described in Claim 3, 4, 5, 6 or 7.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US9989087A | 1987-09-22 | 1987-09-22 | |
| US099,890 | 1987-09-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1323554C true CA1323554C (en) | 1993-10-26 |
Family
ID=22277116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000578065A Expired - Fee Related CA1323554C (en) | 1987-09-22 | 1988-09-21 | Simultaneous assay for cholesterol and triglyceride |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0395654A4 (en) |
| JP (1) | JPH01108998A (en) |
| KR (1) | KR890701762A (en) |
| AU (2) | AU2486388A (en) |
| CA (1) | CA1323554C (en) |
| WO (1) | WO1989002925A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6380380B1 (en) | 1999-01-04 | 2002-04-30 | Specialty Assays, Inc. | Use of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucliotide phosphate (NADP) analogs to measure enzyme activities metabolites and substrates |
| WO2003025584A2 (en) | 2001-02-05 | 2003-03-27 | The Board Of Regents For Oklahoma State University | Direct serum lipids assays for evaluation of disease states |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2315501C3 (en) * | 1973-03-28 | 1980-02-21 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method for the determination of cholesterol |
| US4425427A (en) * | 1980-03-13 | 1984-01-10 | Vitafin N.V. | Simultaneous, kinetic, spectrophotometric analysis of blood serum for multiple components |
| US4309502A (en) * | 1980-06-30 | 1982-01-05 | Beckman Instruments, Inc. | Enzymatic assay for glycerol and triglycerides and a reagent for use therein |
| FR2547925B1 (en) * | 1983-06-23 | 1985-12-20 | Biotrol Sa Lab | PROCESS FOR THE SUCCESSIVE DETERMINATION OF VARIOUS COMPONENTS OF A BIOLOGICAL SAMPLING FROM A SAME TEST IN THE SAME REACTION ENCLOSURE AND REAGENTS USED |
-
1988
- 1988-09-20 EP EP19880908629 patent/EP0395654A4/en not_active Ceased
- 1988-09-20 KR KR1019890700883A patent/KR890701762A/en not_active Ceased
- 1988-09-20 WO PCT/US1988/003169 patent/WO1989002925A1/en not_active Application Discontinuation
- 1988-09-20 AU AU24863/88A patent/AU2486388A/en not_active Abandoned
- 1988-09-21 JP JP63237453A patent/JPH01108998A/en active Pending
- 1988-09-21 CA CA000578065A patent/CA1323554C/en not_active Expired - Fee Related
-
1992
- 1992-09-07 AU AU22808/92A patent/AU2280892A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO1989002925A1 (en) | 1989-04-06 |
| EP0395654A1 (en) | 1990-11-07 |
| EP0395654A4 (en) | 1991-01-09 |
| AU2280892A (en) | 1992-11-19 |
| AU2486388A (en) | 1989-04-18 |
| JPH01108998A (en) | 1989-04-26 |
| KR890701762A (en) | 1989-12-21 |
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