Note: Descriptions are shown in the official language in which they were submitted.
il2~833
The invention relates to the determination of lipase
activity in a biological or physiological fluid.
The usefulness of the determination of lipase
activity in physiological fluids, including serum and duodenal
fluid, for example, in early diagnosis of pancreatic disease
and monitoring the clinical course thereof has been generally
acknowledged in the past. However, technical difficulties as
well as pro~lems with specificit~ and sensitivity of existing
lipase methods have generally hindered the more widespread
use of this enzyme as a primary laboratory indicator of
pancreatic function.
The most widely accepted methods for measuring
lipase activity in biological fluids usually employ olive oil
as a substrate and rely on titration, with standardized sodium
hydroxide of the fatty acids liberated during a 24 hour
incubation period. Some major disadvantages of these types
of methods include long incubation times, resulting in non~
linearity and reduced specificity: large sample requirements:
~; the requirement for highly concentrated and stable olive oil
20 emulsions, which are difficult and cumbersome to prepare; and
the difficulties of reproducing the end point in the tit-
ration of the extremely weak long-chain fatty acids. Colori-
metry of the free fatty acids produced by lipolysis of an olive
oil substrate (in the form of their copper soaps, after
extraction into a lipid solvent) permits shorter incubation
times (10-30 minutes), but involves many tedious manipulations
which limits its usefulness in a routine laboratory. The pro-
cedures described above, including variations thereof employ-
ing sensitive fluorescent pH indicators, for example, in
addition to the described difficulties, lack the desired
sensitivity and precision in the normal range of lipase
activities.
In addition to the above described methods, turbidi-
metric methods of lipase analysi~ have been employed in the
past, but the substrate concentrations used are suboptimal to
permit the rate of substrate clearance to be measured in a
reasonably accurate photometric range and initial increases
in absorbance and nonlinear absorbance changes throughout
the entire reaction period have been reported for patients'
samples, indicating problems with some of these procedures.
In addition to the olive oil substrate, several synthetic
soluble chromogenic and fluorogenic substrates, mostly mono-
esters of long-chain fatty acids, have been proposed for use
in the measurement of lipase activity in serum. However,
it has been conclusively shown that pancreatic lipase does not
act on soluble esters but is active only when adsorbed at an
oil/water interface. Thus, the nonemulsified state of these
types of substrates indicates limited usefulness as analytical
tools for measuring pancreatic lipase activity. Lipase
activity has also been determined by radial enzyme diffusion,
based on measurement of the cross-sectional area of the
clearing of an olive oil emulsion suspended in buffered
~; agarose gel. The disadvantages of this method are that two
hour incubation times are required, accurately measuxing
small diameters of the clear zone is difficult, and it is
necessary to calibrate with seconda~y standards.
Thus, a method for the determination of lipase
activity in biological fluids which has reduced incubation
time, can employ relatively small amounts of sample fluids,
and which is highly specific and sensitive is desirable.
Further, a lipase activity measurement procedure which can
be performed simply, using readily available laboratory
equipment, would be advantageous~
The method of the present invention overcomes the
disadvantages of the lipase activity tests briefly described
above by employing a coupled enzymatic reaction sequence
to convert the primary product of lipolysis into a chemical
compound that may be measured with greater speed and ease
and with greater sensitivity, precision, and accuracy than
is possible in the analysis of free long~chain fatty acids
by either titrimetric or indirect photometric methods follow-
ing solvent extraction.
According to the invention there is provided a method
for determining the lipase activity of a biological fluid
comprising: contacting a sample of a biological fluid with an
emulsified substrate effective in the presence of lipase to
produce ~atty acid by lipolysis, contacting any fatty
acid produced with lipoxygenase in the presence of oxygen
to convert any fatty acid produced by-said lipolysis to
its acid hydroperoxide, said lipoxygenase being added in an
amount to effect the conversion at a rate sufficient to
ensure that lipolysis is the limiting reaction, and measuring
the production of the acid hydroperoxide as a measure
of the lipase activity of the biological fluid.
According to another aspect of the invention there
is provided a reagent for determining the lipase activity
of a biological fluid comprising: an emulsified substrate
effective in the presence of lipase to produce fatty
acid by lipolysis,and lipoxygenase in an amount effective,
in the presence of oxygen, to convert the acid produced
by lipolysis to its acid hydroperoxide at a rate sufficient
to ensure that lipolysis is the limiting reaction.
In a particular embodiment there is provided a
test system for determining the lipase activity of a biological
fluid comprising the reagent of the invention and means for
_ 3 _
measuring the production of linoleic acid hydroperoxide
as a measure of the lipase activity of the biological fluid.
Basically, in a particular embodiment the method
co~rises adding a sample of biological fluid to a ~ubstrate
emulsion to thereby induce lipolysi~. The substrate employed
i9 preferably trilinolein but can be an e~uivalent thereof
as further defined hereinbelow. Either at the same time or
shortly thereafter, an amount of lipoxygenase is added which
is effective to convert the fatty acid produced by lipolysis
to its acid hydroperoxide, the conversion being effected
at a rate substantially greater than that at which the
acid is produced. The hydroperoxide can then be measured
by, for example, colorimetry, uv-spectrophotometry, or kinetic
assay techniques.
A lipoxygenic endpoint assay requires only about 15
minutes and the kinetic version of the tests, which usually
employs an oxygen electrode, only a few munutes for completion
which makes the assay well suited for an emergency procedure.
Further, increased sen~itivity is achieved by the coupled
enzymatic reaction as compared to the standard lipase
methods that measure the weak long-chain fatty acids produced
during incubation of the sample with olive oil or triolein
emulsions by titration with alkali, thus permitting the use of
microsamples which makes the procedure ideally applicable to
pediatric applications.
The rapid enzymatic micromethod for specific deter-
mination of the diagno~tically important enzyme lipase (glycerol
ester hydrolase, E.C. No~ 3.1.1.3) of the present invention
employ~ a coupled enzymatic reaction sequence whereby the free
fatty acid, particularly linoleic acid produced by lioplysis
of an emulsified substrate, for example, a trilinolein
substrate or a substrate formulation employing one of the other
112~
triglycerides set forth in detail below, is converted to its
more easily identifiable acid hydroperoxide. The conversion is
accomplished by providing an excess amount of lipoxygenase (also
known as lipoxidase, linoleate:oxygen oxidoreductase E.C.1.99.2.1)
so that the reate of formation of the acid by lipolysis is the
rate limiting step of the coupled enzymatic reaction.
The basic coupled enzymatic reaction sequence can be
schematically depicted in the following manner, by reference to
the production of linoleic acid as the fatty acid:
tl) Trilinolein Li~a9e ) 1, 2 Dilinolein + Linoleic Acid
( 2 ? Linoleic Acid + 2 Li~oxyqenase ~ Linoleic Acid
Hydroperoxide
Because an excess amount of lipoxygenase is employed, the
rate o~ formation of linoleic acid, which i~ directly pro-
portional to the activity of lipase under the employed
conditions of substrate saturation, may be measured by the
rate of production of linoleic acid hydroperoxide, or the
rate of consumption of oxygen. Suitable reaction conditions
for the coupled en~ymatic reaction sequence are 30C at a
pH of 8.~. Under these conditions, the incubation time for
the coupled enzyme reaction is about 10 minutes and the
reaction can be stopped by the addition of alcohol. Thus,
when lipoxygenic endpoint assay techniques are employed, the
entire prccedure takes only about 15 minutes, and when kinetic
assay techniques are employed the actual measurement of lipase
activity takes place during the incubation period. In com-
parison, the standard titrimetric olive oil procedures
typically require from 3 to 24 hours of incubation to generate
adequate amounts of titratable acid and the continuous sampling
version of these types of methods requires a pH-stat instru-
ment which is generally unavailable to most clinical
laboratories. The coupled enzyme reaction sequence set forth
- 5 -
above is also superior ~o the presently available methods for
measuring lipase activity i~ that the soluble substrate~ used,
for example, trilinolein and its equivalents described herein-
below are not hydrolized by the various carboxylesterases and
therefore do not yield false positive results in cases where
these non-specific esterases are elevated in body fluids.
The ~pecific enzymatic measurement of linoleic
acid rather than of total acidity also eliminates the inter-
ference by proteolytic enzymes or any other non~specific H+
producing or consuming side-reactions. The coupled enzymatic
reaction also provides for a drastic increase in senqitivity
when compared to standard lipase methods that measure the
weak long-chain fatty acids produced during incubation of the
sample with olive oil emulsions by titration with alkali,
This increa~e in sensitivity permits the use of microsamples,
which makes the test well suited for pediatric patients.
A further advantage of the lipase method of the pre-
sent invention is that preparation of the substrate emulsion
can be performed rapidly and in a technically simple and non-
~0 critical manner since adequate oil/water interface i8 easilyobtained when microsamples are employed. This should be con-
tra~ted with the preparation of olive oil emulsions for the
existing standard titrimetric methods which preparation is a
technically involved cumbersome procedure re~uiring the
addition of an emulsifier such as gum acacia, in order to
obtain a stable emulsion of high oil/water interfacial area,
which is crucial for the success of the method.
While the naturally occuring glycerol ester tri-
linolein is the preferred substrate material, it will be
apparent to one ~killed in the art that other natural or
synthetic, simple or mixed triglycerides or triacylglycerols
- 6 -
1~Z0833
can be employed. Basically, the requirements of other useful
natural or synthetic, Qimple or mixed triglycerides are that
the component fatty acids, esterified to glycerol in 1,3-( or ~)
position meet the structural requirements for a good substrate
for the enzyme lipoxygenase, i.e. result in high rates of
formation of hydroperoxide in the presence of oxygen. Present
knowledge of the enzyme suggests that a methylene interrupted
cis-diene system with double bonds extending from carbon atom
No. 6 to 7 (w6) and from carbon atom No. 9 to 10 (w9) count-
ing from the methyl end of the fatty acid must be present
according to:
wl W3 ~w6- ~9 I CO
3 H2 CH(2)~H(2)-CH2 t CH=cH-cH2-cH=cH ~ cH2- -OH
Other double bonds (e.g. w3, w12, w15 ..... ) may or may not
be present.
The general structure for suitable triglycerides
may be depicted schematically as follows:
C- O- C - R
Il ~1 ..
~ R2- C -O -CH
1(2)
~ t3) 11
:112~33
W15 w6
1 ( 2)~ ( (2)~ (2)--CH2)2 (CH CH CH2)2
W3 wl
CH(2 ~CH(2)----~H2----C3
is 1, 3 or 5 for naturally occurring and 1, 2, 3, 4 or 5
for synthetic fatty acids.
R2 is Rl for the preferred simple triglyceride substrates.
Otherwise stated Rl may be a group of formula
(CH2)n-A2(CH=C~-CH2)2-~ CH3
in which A i~ -CH=C~-CH2- or -CH2-CH2-CH2~; and ~ i9 an
integer from 1 to 5.
R2 may be any saturated or (poly)unsaturated fatty
acid alkyl chain in ca~e of mixed triglyceride substrates.
Thi3 is so because the 2 ( or ~ ) esterlinkage is not
attacked by lipase during the initial reaction phase, and con-
sequently, there are no specific structural requirements or
the fatty acid bound in this po~ition.
Trilinolein, or an equivalent triglyceride of the
above specified structural formula, can be prepared in
emulsified form^~elatively easily for use as a substrate with
which lipase will conjugate according to the coupled enzymatic
re~action 3equence of the i~vention. For example, trilinolein,
which i~ available in 100 mg/ampules, can be dissolved in an
ethanol/acetone solvent and stored at 0-4C to form a substrate
stock solution which can be emulsified by blending with a
working buffer which provides the proper pH for the enzymatic
reactions. A suitable working buffer can be prepared from
sodium deoxycholate dissolved in tris(hydroxymethyl)amino-
methane (hereinafter sometimes referred to as Tris) with
sufficient hydrochloric acid to adjust the pH to the desired
112~
level. Alternatively, barbital buffer may be substituted for
Tris. Substrate emulsions can be prepared by mixing the worX-
ing buffer with the substrate stoc~ solution and blending, at
maximum speed, in a co~nercial blender for about 5 minutes.
Preferably the sub~trate emulsions are prepared freshly each
day.
Lipoxygenase (linoleate:oxygen oxidoreductase,
E.C. 1.99.2.1) necessary to perform the specific conversion
of the linoleic acid produced by lipolysis to linoleic acid
hydroperoxide, can be prepared, in stock solution form, by
admixing lyophilized-lipoxygenase of sufficient activity with
the appropriate volume of working buffer (described above with
relation to the sub~trate emulsion) to obtain the stock
solution having an activity of 1.40 X 106 S.U.*/ml (~ 168,000
I.U./ml at 25C). This lipoxygenase stock solution should be
stored at temperatures at from about 0 to about 4. A
lipoxygenase working solution having an activity of about
140,000 S.U./ml (~ 16,800 I.U./ml at 25C) can then be pre-
pared by diluting the lipoxygenase stock solution 1:10 with
more working buffer and should be prepared freshly each day.
The coupled enzyme reaction cequence of the present invention
i3 made possible by the high specificity of lipoxygenase for
free linoleic acid and the ~imilarity of the pH optima of both
enzym2s (pH 8.8 for lipase, pH 8.3 for lipoxygenase), The
lipoxygenase employed in the present invention can be isolated
* Definition of spectrophotometric unit (S.U.): One (1) S.U. will
cause an increase in absorbance at 234nm (~A ) of O.001 at 25C
when linoleic acid is the substrate in 3 ml vo~ume at pH 9.0
(1 cm light path). 1 S.U. is equivalent to the oxidation of
0.12 ~ mole of linoleic acid, i.e. 0.12 International Units
(I ~UoB ~ units or I.U.) at 25C.
_ g _
:~12~)~333
from soybean-flour extracts.
While any biological fluid suspected of containing
the enzyme lipase may be employed with the substrate emulsion,
and lipoxygenase working solution set forth above to perform
the coupled enzymatic reaction sequence of the present
invention, the biological fluids of greatest interest are
blood serum and duodenal fluid. Because of the extreme
sensitivity of the test procedure of the subject invention,
microsamples of blood serum and duodenal aspirate can be
employed. For example, 10 ~1 of serum sample (20 ~1 with
test blank) can be employed in most cases. A like amount
of diluted duodenal aspirate can be employed.
Thus, the above described reagents, that is substrate
emulsion, lipoxygenase working solution and biological sample
fluid are all the reagents necessary to form the coupled
enzymatic reaction sequence of the present invention. Of
course, other reagents will be useful depending upon the
analytical method employed to determine lipase activity.
However, for example, when kinetic assay techniques are
employed which measure directly the rate of oxygen consumption
by the reaction between linoleic acid and oxygen (see equation
2 above) no othar reagents need be employed.
The kinetic variables which influence the coupled
enzymatic reaction of the subject invention, including sub-
strate concentration, effects of sample matrix, effect of
blending times and speeds on the substrates, effect of bile
salts, effects of pH,effect of temperature, effect of
lipoxygenase activity and effect of concentration of oxygen
in the system were studied in order to better understand the
operating parameter~ of the system. The results of thase
studies will aid one skilled in the art in the practice of
the subject invention.
-- 10 _
llZ~ 3~
With regard to substrate concentration, it should be
noted that since the lipase acts on the oil/water interface
only, the interfacial area of the substrate/buffer emulsion
takes the place of the substrate concentration in the kinetic
equation for homogeneous systems. However, if the substrate
is insoluble in water, and the degree of emulsification is
identical for all substrate concentrations used, the inter-
facial area increases linearly with increasing substrate
concentration. Because of the technical difficulties of measur-
ing accurately the interfacial area of emulsions, the con-
ditions of emulsification were kept constant throughout the
study and the reaction rates were expressed as a function of
the trilinolein concentrations. Basically, with respect to
trilinolein, it was determined that at substrate concentrations
of less than about 1.0 X 10 4 mol/liter there is insufficient
3ubstrate for saturation of the enzyme and at substrate con-
centrations above approximately 4 X 10 4 mol/liter there is an
apparent decrease in the maximum velocity of the substrate-
lipase reaction, indicating some form of substrate inhibition.
The effect of the sample matrix was studied by com-
paring serial dilutions of pancreatitis serum with heat-
deactivated human serum and solutions of 10 grams of bovine
serum albumin in 1 liter of working buffer. The differences
in the sample matrix were not significant for lipase activities
up to 1,000 U/liter. Apparently, at the high substrate con-
centration chosen for the assay, the velocity of the reaction
is at a maximum whether the samples have been diluted or not
and is equal for identical lipase activities, independent
of the sample matrix.
The effect of blending times and speeds in pre-
paration of the substrate emulsion was also investigated.
Basically, it was found that substrates prepared simply by
-- 11 --
112~33
vortex-mixing trilinolein for 3 to 5 minutes with the sub-
strate buffer led to data indicating inadequate interface
area or instability of the emulsion, or both. It was deter-
mined that the best results were obtained with homogenized
substrates, prepared by blending the chilled working buffer
in a commercial blender at a maximum speed with trilinolein
for 3 to 5 minute~. Blending for more than S minutes does
not apparently further increase either the linear range or the
stability of the substrate and may result in undesirable heat--
ing of the ~ubstrate. Substrates prepared by the methodssuggested above are stable for three to four days, if kept
refrigerated.
The effect of various concentrations of bile salts,
and specifically deoxycholate on the overall reaction ràte
was studied. For both normal and pancreatitis serum samples
maximum activity was observed at 3.6 mmol of deoxycholate per
liter. Other bile salts (glycocholate, taurocholate) have a
similar effect.
The effect of pH on the rate of trilinolein hydrolysis
of both normal and pancreatitis serum was studied in the range
of 7,6 to 9,4 at 30C. A trilinolein substrate, prepared in
50 mmol/liter at 25C, was divided into several aliquots,
which were adjusted individually by dropwise addition of
12 mol/liter HCl to the desired pH values. The results of
these tests indicate that lipase of normal serum loses its
activity faster than that in diluted pancreatitis serum on
either side of the pH optimum which is about 8.8 at 30C for
both samples.
The effect of temperature on the rate of trilinolein
hydrolysis was studied in the range between 20and 40C. The
pH was maintained at exactly 8,8 at all reaction temperatures,
by computing the corresponding pH at room temperature from the
33
temperature coefficient of Tris buffer (0.025/C), and adjust-
ing the substrate pH values at 25C accordingly by dropwise
addition of 12 mol/liter HCl, or 10 mol/liter NaOH. The
study shows that between 37 and 40C an actual decrease in
activities is o~served, indicating progressive heat
denaturation of either lipase or lipoxygenase, or both.
These ~tudies indicate that 30C should be the recommended
reaction temperature for the assay of the present invention.
In order to determine the degree of saturation of
lipoxygenase, diluted pancreatitis serum was analyzed under
the conditions of the assay of the present invention, using
different lipoxygenase activities. At lipoxygenase activities
of 1000 S.U./liter of substrate (~ 120 I.U./liter at 25C), the
lipase-catalyzed hydroly~is of trilinolein apparently becomes
rate limiting. The actual lipoxygenase activity used in the
final assay is about 1400 S.U./liter (~ 168 I.U./liter) in
order to compen4ate for partial loss of enzyme activity by
potential serum inhibitor4 of this enzyme. Thus, lipoxygenase
activities of at least 1,000 S.U./liter of substrate (120
I.U./liter at 25C) are necessary in order that the conversion
of trilinolein to linoleic acid by lipase be the rate limiting
reaction.
Finally, the effect of the concentration (partial
pressure) of oxygen was studied. According to the second
equation of the coupled enzymatic reaction sequence of the
present invention, one mole of oxygen is consumed for each
mole of linoleic acid converted to its hydroperoxide. In
order to determine whether or not atmospheric conditions would
provide sufficient oxygen to insure the reaction rate would
remain unaffected, reactions using atmospheric conditions
were compared to those carried out under a continuous
- 13 -
llZ~33
stream of pure oxygen, using a preoxygenated buffer. No
difference in either the velocity or the linearity of the
reaction with respect to time and activity were noted for
the latter type reactions, thus indicating that the test
can be carried out with non-oxygenated buffer in open test
tubes in normal laboratory atmosphere.
EXAMPLE PROCEDURES
In order to more fully illustrate the present
invention, example procedures are set forth below wherein
colorimetric, spectrophotometric, and kinetic assay techniques
are employed in combination with the coupled enzymatic reaction
sequence set forth above to determine lipase activity. It is
not intended that the scope of the present invention be
limited to these methods of analysis, and they are set forth
as exemplary only,
The starting materials, and reagents prepared there-
from, are set forth below and are examplary of those which
can be employed. Modifications thereof will be apparent to
one skilled in the art:
Sodium deoxycholate, AR.
Acid-aLcohol solution, 2~62 g/liter. Dilute 6.0 ml
of 12 mol/liter hydrochloric acid (AR) to 1 liter with
absolute ethanol (AR).
Ferrous ammonium sulfate (AR), 0.13 mol/liter.
Dissolve 250 mg in 5,0 ml of hydrochloric acid, 30 g/liter.
Ammonium thiocyanate(AR)~ 2.63 mol/liter.
Dissolve 20 g in deionized water, dilute to 1 dl.
Lipoxygenase (linoleate:oxygen oxidoreductase,
E.C. 1,99.2.1.), activity, 165,000 S.U,/mg (~ 19,800 I.U./mg
at 25C.~
- 14 -
i8~3
Lipoxygenase stock solution, 1.40 X 106 S.U./ml.
Dissolve 85 mg in 10,0 ml of working buffer. Stora 0.1 ml
aliquots at 0-4C.
Lipoxygena~e working solution, 140,000 S.U./ml.
(~ 16,800 I.U./ml at 25 C). Dilute a 0,1 ml aliquot of
stock solution with 0.9 ml of working buffer. Prepare freshly
each day,
Trilinolein, 100 mg/ampule.
Trilinolein stock solution, 20 mg/ml. Dissolve the
contents of a 100 mg ampule in 5.0 ml of ethanol/acetone
(3/2 by vol.) Store at 0-4C.
Working buffer, Dissolve 1.5 g (3.6 mmol~ of
sodium deoxycholate in 1 liter of tris(hydroxymethyl)amino-
methane (Tris), 50 mmol/liter. Adjust the pH to 8.9 at 25~C
by dropwise addition of hydrochloric acid, 12 mol/liter.
Store at 2-6C.
Substrate emulsion, 0.34 mmol/liter. Transfer
50 ml of the working buffer to the blender vessel, add 0.75
ml of trilinolein stock solutionj blend at maximum speed
for 5 min. Prepare freshly each day.
Linoleic acid, 1 g/ampule.
Linoleic acid stock solution, 36 mm~ol/liter.
~ Dissolve the contents of a 1 g ampule in 1 dl o absolute
;~ ethanol. Store at 0-4C.
Linoleic acid working standard, 50 ~mol/liter.
Dilute 0.14 ml of stock solution to 1 dl with working buffer.
Solutions for linoleic acid standard curve. Dilute
280 ~1 of stock solution to 1 dl with working buffer, 100
~mol/liter. Serially dilute this standard with working
buffer to yield standards of 75, 50 and 25 ~mol/liter.
llZ~3~
Control ~era.
Bovine Serum Albumin, 1 g/dl in deionized water.
Colorimetric Endpoint Assay
The amount of linoleic acid hydroperoxide produced
by the coupled enzymatic reaction sequence of the invention
within a 10 minute incubation period can be measured photo-
metrically at 480 nm after dissolving the reaction mixture
in acid alcohol, using the oxidation of ferrous to ferric
ion by hydroperoxide and the detection of the ferric ion by
thiocyanate according to the following equations:
(3) R - OOH ~ 2 Fe2+ ~ 2 ~+ ~ R - OH + 2 Fe ~ H20
(4) Fe3+ + 3 SCN ) [Fe (SCN)3~ (red complex)
~ipase activity is computed in IUB units, with use of primary
linoleic acid standards that are submitted to the same reaction
sequence. The contribution of traces of free linoleic acid
and of lipid peroxides, present in commercial batches of
trilinolein, is subtracted in a substrate blank, the con-
tribution of endogenous a~d exogenous sample constituents is
compensated for in individual test blanks, and the contribution
of the color reagents i9 subtracted using reagent blanks.
The following procedure can be employed to perform
the colorimetric endpoint assay embodiment of the present
invention.
(1) Label a series of test tu~es, two for each
patient's serum (T and TB) plus one each for a standard (S),
"substrate blank" (SB), and 'reagent blank" (RB).
(2) To the tubes labeled T and SB, add 1 ml of
substrate emulsion. To the S tube add 1 ml of the 50 ~mol/
liter linoleic acid standard. To the tubes labeled TB and
RB, add 1 ml of working buffar.
(3) Place all the tubes in a 30C waterbath for 5
minutes.
- 16 -
1120~33
(4) In an exactly timed sequence (a 15 second
interval i~ recommended), add 10 ~1 of serum, add 10 ~1 of
lipoxygenase working solution, in that order, to the tubes
labeled T and TB, vortex-mixing for 5 seconds after each
addition, and replace it in the waterbath. To the tubes
labeled S and SB add 10 ~1 of lipoxygenase only, and vortex-
mix for 5 seconds replacing them in the waterbath.
(5) After exactly 10 minutes, and in the same timed
interval, remove each tube from the waterbath and add 6 ml
of acid/alcohol reagent from a dispenser and vortex-mix
briefly. It should be noted that because the absorbance of
the test blanks does not change measurably ~uring the 10
minute incubation period, it is not necessary to adhere to
a timed sequence for the test blanks, and the acid-alcohol
reagent may be added to the TB tubes at any time while the
T tubes are still incubating.
(6) Add 20 ~1 of ferrous ammonium sulfate reagent
to all tubes and vortex-mix briefly.
(7) Add 100 ~`1 of ammonium thiocyanate reagent to
all-tubes and vortex-mix briefly.
(8) Measure the absorbance of each sample at 480 nm
against the reagent blank (RB).
The enzymatic activity of the serum sample can be
calculated using the following formula:
~AT
Lipase activity (U/liter) = X 500 where
A~
~AT = AT ATB - ASB
he above formula was derived from the following ~nown factors:
sample dilution factor: 100 (10 ~1 ~ 1 ml)
incubation time: 10 minutes
- 17 _
llZ~)~333
linoleic acid standard: 50 ~mol/liter
50 ~ol/liter substrate is equivalent to 5 mmol/liter
sample
5 mmol/liter per 10 min is equivalent to S00 ~mol/
liter per min (500 U/liter)
Alternatively, the concentration of linoleic acid
corresponding to ~AT can be read from the linoleic acid
standard curve in ~mol/liter and multiplied by 10 to obtain
the lipase activity in U/liter.
W - Snectronhotometric End~oint Assay
The l1noleic acid hydroperoxlde concentration can
be measuréd directly in an ethanol diluted reaction mixture
via it~ W absorption~at 234 nm (E234 ~ 23,000 at 37C) using
a narrow-band W - Spectrophotometer. This procedure is
recommended especially for the assay of lipase activity in
duodenal aspirates.~
Duodenal~aspirates can be prepared for u~e as
amples in the method of the present invention by centrifuging
specimens obtained by incubation~at 0-8C at 4,000 g for
approximately 10 minutes to remove~suspended particles. The
duodenal aspirate~is then diluted 40-80 fold with a 1 g/liter
- solution of bovine~erum albumin~
Next, the procedure set forth~in steps (1) through
(5~3 above with respect t~o the colorimetric assay procedure,
are repeated sub~tituting the~diluted duodenal juice for
thé ~erum sample.
. ~ .
After the above steps and procedures have been per-
formed, the absorbance of each~sample is read in 1 cm quartz
cuvets at 234 nm against ~he reagent blank. Lipase activity
in the unknown sample can then be calculated using the same
equation set forth above with respect to the colorimetric assay
technique.
- 18 -
" .
1~20833
Alternatively, the concentration of linoleic acid
hydroperoxide (CLAH) corresponding to ~AT may be determined
directly (without use of ~tandards) once the molar extinction
coefficient of linoleic acid hydroperoxide at 234 nm (E234)
has been measured with the spectrophotometer employed. This
calculation can be performed using the following equations:
c r~moll
LAH literl
-- J E234*
Lipase activity (U/liter) = CLAH X 10**
* E234 (37 C) ~ 23,000
** Dilution factor of_sample = 100 = 10
Incubation time (min) 10
Kinetic Assay Using a P02 Electrode
Using this method of analysis the reaction is
monitored by the decrease in oxygentension (Po2) as linoleic
acid produced during lipolysis reacts with an equimolar amount
of dissolved oxygen to form linoleic acid hydroperoxide,
using a PO2 electrode. Lipase activity is computed with the
initial rate of decrease of P02 in the reaction mixture after
addition of sample lipoxygenase to the trili-nolein emulsion
~ubstrate. The apparatus necessary to perform this type
of analysis includes micro P02 electrodes suitable for blood
gas analysis with associated signal amplifier and read-out
device (strip-chart recorder, printer, etc.). A thermostat
controlled reactionchamber (30C), containing the sensing
element of the oxygenelectrode and a magnetic stirrer. The
assay is carried out in the following manner:
(1) 1 ml of the previously aerated substrate
emulsion is pipetted into the reaction chamber. The electrode
is allowed to stabilize. The recorder is set to maximum
reading.
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1120833
(2) 10 ~1 of sample (serum, duodenal aspirate,
control, etc.) and 10 ~1 of lipoxygenase working solution are
added with a micro-pipet. The magnetic stirrer is activated
for 5-10 seconds.
(3) The decrease in oxygen tension is recorded as
the reaction proceeds for approximately 3-6 minutes.
(4) The slope of the pO2~time curve is measured
as soon as it becomes linear.
t5) The system is zero-rated by repeating steps
1-4 as described, but omitting the sample addition of step 2.
(6) The rate obtained using the substrate blank
(SB) i~ subtracted from the rate obtained with the test
sa~ple (~) to obtain the net oxygen consumption rate
(~rT = rT ~ rSB)
(7) A suitable lipase reference standard is then
assayed the same way as the unknown sample and its net oxygen
consumption rate is calculated (~rref). The lipase activity
in the unknown te~t sample can be computed as follows:
~rT
Lipase activity (U/liter) = X Lipase activity of
r f reference standard
re (U/liter)
A}ternatively, the lipase activity in the unknown
sample may be calculated directly from the decrease in oxygen
tension per minute after calibration of the system with
primary linoleic acid standards (1 ~mol of linoleic acid will
consume 1 ~mol oxygen under the conditions of this assay).
1 (IUB) Unit result9 in the consumption of 1 ~mol of 2 per
minute under the conditions of the assay.
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