Note: Descriptions are shown in the official language in which they were submitted.
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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
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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.
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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
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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
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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
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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.
EXAMPLE 1
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
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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.
EXAMPLE 2
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
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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.
EXAMPLE 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
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
LDH 1000U/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.
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EXAMPLE 4
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.
EXAMPLE 7
Cholesterol/Triglyceride Simultaneous Fluorescent Assay
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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.
