Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF T~IE INVENTION
1. Field of the Invention: The presen-t invention
relates to a method for determining the lipase activity
of a sample by reacting the sample with a triglyceride
emulsion, and to a method of preparing the triglyceride
reagent used to form the emulsion.
2. Discussion of the Prior Art: Lipase is an enzyme
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produced by the pancreas. Lipase activity in blood or blood
serum is increased by pancreatitis and other diseases of the
pancreas. A measure of lipase activity in blood or blood
serum is, therefore, a useful diagnostic tool to physicians.
A traditional method of measurinq lipase activity
is the turbidimetri.c method described ~y Shihabi and Bishop
in Clinical Chemistry, 17, pp. 1150-1153 (1971), in which
the decrease in absorbance oE an olive oil/water emulsion
is related to lipase activity. Olive oil is a mixture of
substances called collectively triglyceride. When mixed in
aqueous solution, triglyceride forms an emulsion. Light
incident on this emulsion is scattered by the oil droplets
so that very little light is transmitted through the emul~
sion. Lipase hydrolyzes triglyceride into fragments which
are soluble in the reaction mixture. As the triglyceride
is hydrolyzed, light scattering by the emulsion is decreased
and transmission of the light through the medium is increased.
It is currently believed that the lipase attacks
the triglyceride at the liquid-oil interface so that the
emulsified state of the triglyceride is important to the
reaction. Important as it is, current techniques of forming
the triglyceride emulsion are inadequate. Triglyceride
emulsions formed in a homogenizer, such as a blender,or by
a solvent/aqueous mixture, exhibit limited stability ~four
to eight weeks). In addition, they are difficult to make
and in general lack reproducibility. Both deficiencies
limit the usefulness of the assay in automated analyzers.
SUMMARY OF T~IE INVENTION
The present invention provides a method for deter-
mining the lipase activity of a sample by reacting the sample
with an emulsion of a triglyceride in an aqueous reaction
solution, and measuring the rate at which the triglyceride
is hydrolyzed to soluble fragments. The emulsion is formed
by providing as the triglyceride reagent, a solid matrix of
an excipient uniformly impregnated with the triglyceride,
the excipient being soluble in the aqueous reaction solution,
and dissolving the solid matrix to release the triglyceride,
as an emulsion, into thé aqueous reaction solution.
In the preferred embodiment, the excipient com-
prises polyethylene glycol and a sugar or sugar alcohol
such as mannitol, inositol, sorbitol, maltose, dextrose,
lactose, dextran and mixtures thereof; and it is impreg-
nated with triolein, one of the major triglycerides.
The invention also provides a process for pre-
paring a reagent for use in conjunction with an aqueous
reaction solution to determine the lipase activity of a
sample, comprising the steps of:
(a) ~orming a liquid solution of at least one trigly-
ceride (e.g., triolein) with a solvent for that triglyceride;
(b) mixing the liquid solution with a predetermined
quantity of an excipient which is soluble in the aqueous
reaction solution, the volume of solvent used to form the
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liquid solution being chosen to substantially saturate the
excipient; and
(c) evaporating the solvent to leave a solid matrix
of the excipient impregnated with the triglyceride, the
amount of triglyceride used to form the liquid solution
being chosen to give the desired concentration of trigly-
ceride per weight of solid matrix.
DESCRIPTION OF THE PREFERRF,D EMBODIMEN~
The Eirst step in the process of preparing the
triglyceride reagent for use in determining the lipase
activity of a sample, particularly a sample of blood or
blood serum, is to dissolve the triglyceride in a suitable
solvent. The triglyceride is a substrate for the lipase.
In the past, purified olive oil or triolein, one of the
major triglycerides, have been used. For convenience, the
discussion which follows will be limited to a discussion
of triolein, but lipase will react with any triglyceride,
so any -triglyceride or combination of triglycerides, can
be used in the present invention.
The solvent used to dissolve the triglyceride
will, of course, depend upon the triglyceride chosen.
Numerous solvents for triglyceride are known to those
skilled in the art. The solvent used to form the trigly-
ceride reagent of the present invention should satisfy
four criteria. It must dissolve the particular trigly-
ceride used. It must not dissolve the particular excipient
chosen. It must evaporate under conditions which will not
harm the triglyceride or the excipient. Finally, it must
leave no residue or, at least, only an inert residue,
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i.e., one that is not reactive with or harmEul to the
other reagent used.
~ olvents such as chloroform and ethanol will
dissolve triolein. It has been found that chloroform or
ethanol or mixtures of chloroform, preferably mixtures con-
taining up to about 50~ ethanol,will produce a uniform
solution of triolein.
The volume of solvent usecl to dissolve the tri-
glyceride is chosen so that when the liquid triglyceride
solution is applied to the excipient, the liquid will just
saturate the solid material. If too little solvent is
used, a non-uniform distribution of the solvent, and hence
the triglyceride, throughout the solid will occur. I~hen
the powder is divided, this will produce reagent portions
with variable concentrations of triglyceride. If too much
solvent is used, not all of the solution, and in particular,
the triglyceride which it contains, will impregnate the
solid. If the solid is drained before the drying step,
this will lead to a loss of some of the triglyceride; if
not, that portion of the triglyceride contained in the
solution which does not impregnate the solid will coat
the solid in a non-uniform manner. Depending upon the
precision required, each of these conditions can be toler-
ated, but for best results, the amount of solvent used
should be chosen so that the triglyceride solution sub-
stantially saturates the solid material.
The amount of triglyceride used in the solution
is a matter of choice. The amount used should be chosen
to yield the desired concentration of triglyceride per
given volume of the solid matrix formed when the solvent
is evaporated.
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By definition, an excipient is an inert substance
that forms a vehicle for a particular reagent, in this case,
the triglyceride. The excipient chosen to ~orm the solid
matrix of the present invention should satisfy three criteria.
It must not dissolve in the solvent chosen to dissolve the
triglyceride. It must dissolve in the medium in which the
lipase assay takes place. Such dissolu-tion is required
to release ~he triglyceride into the reaction solution to
form the emulsion. The reaction solution is normally an
aqueous solution which is maintained at a pH of between
about 8.5 and about 9.5. Finally, the excipient should be
inert and nondetectable in the assay. There are many
materials known to those skilled in the art which qualify
for use as excipients in the present invention. Common
among them are sugars and sugar alcohols, such as mannitol,
inositol~ sorbitol, maltose, dextrose, lactose, dextran
and mixtures thereoE.
A more uniform distribution of the triglyceride
in the solid matrix will be formed if, in addition to the
2d substances referred to above, polyethylene glycol is
used. Carbowax~ 6000 is one suitable substance. This
substance prevents agglomeration of the triglyceride
droplets in the solid matrix and aids in producing a
uniform coating of the triglyceride solution on the sur-
face of the solid matrix; The polyethylene glycol, how-
ever, is not absolutely necessary.
Once the triglyceride and the solvent for the
triglyceride have been chosen and mixed to form the desired
solution, the triglyceride solution and the excipient are
blended together. This can be done in a number of ways
known to those skilled in the art. In particular, a ~lobart~
mechanical mixer can be used. ~ixing continues until the
excipient is just saturated. The saturated excipient is
then dried to evaporate the solve~nt used to dissolve the
triglyceride, and a solid matrix uniformly impregnated with
the triglyceride is formed. Drying can be accomplished in
any number of ways well known to those skilled in the art.
In particular, air drying in a fume hood can be used. The
impregnated solid matrix can be used as a powder, or it
1~ can be tableted using conventional tableting techniques.
As a particular example of the formation oE the
triglyceride impregnated matrix, 94 parts of mannitol and
5 parts of Carbowax~ 6000 are blended together to form an
excipient. One part of triolein is dissolved in a 50~
chloroform, 50~ ethanol mixture, the amount of solvent
being chosen such that when blended with the excipient,
a just saturated solid blend is formed. The solvent mix-
ture containing the triolein is then added to the mannitol-
Carbowax~ 6000 blend in a Hobart~ mechanical mixer and
blended for 30 minutes. The saturated excipient so formed
is then air dried for 60 minutes, or until the solvent has
completely evaporated, in a fume hood. A solid matrix
uniformly impregnated with triolein is formed. This
solid matrix is in powder form. The impregnated blend is
then tableted to yield approximately 85 mg. tablets contain-
ing about 0.87 mg. of triolein for each tablet.
An assay to determine lipase activity in human
serum is then performed by a kinetic turbidimetric approach
in which the rate of "clearing" of the triolein emulsion
is monitored at 340 nm. This rate is proportional to the
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lipase activity of the sample. Calcium chloride and bile
salts are used as activators for the lipase. The system
is also buffered using tris(hydroxylmethyl) aminomethane
("Tris") buffer which serves as a pH control (pH of 8.6
to 9.0) for the aqueous reaction solution. The reaction
proceeds as follows:
Triolein Emulsion ~ ~ Free Fatty ~cids
pH 8.8 (25C) Mixed Glycerides
(Turbid) (Decreasing turbidity)
As a particular example of a lipase assay, a
test kit containing the following reagents is prepared:
Tris Buffer, pH 8.8 -~ 0.2; 50 ~Mol
Deoxycholate 35.5 ~Mol
Calcium chloride 0.7 ~Mol
Triolein 0.98 ~Mol (in the tableted form
described above)
These reagents are added to 4.8 ml. of water, and 0.20 ml.
of sample containing an unknown amount of lipase is then
added to the solution. With a small amount of mixing, the
excipient in the tablet dissolves and a triolein emulsion
is formed in the aqueous reaction solution. ~s the lipase
enzyme contained in the sample reacts with the triolein
emulsion, free fatty acids and mixed glycerides are formed
and the mixture becomes increasing optically transparent.
The rate of "clearing" of the triolein emulsion as moni-
tored at 340 nm. is found to be proportional to the lipase
activity.
The use of a tablet described above yields highly
reproducible emulsions and imparts long-term stability to
the substrate (at least 12 months).
The above description is intended to teach those
skilled in the art how to make and use the triglyceride
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reagent of the pre.sent invention. It is not intendcd to
limit the scope of the invention as set forth in the
appended claims.
