CA1120833A - Analytical determination of lipase - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A test for lipase activity is provided wherein trilinolein, or an equivalent as defined herein, is employed in an emulsified form as a substrate. Reduced incubation times and improved accuracy are obtained by providing an excess of lipoxygenase to convert the linoleic acid product of lipolysis to linoleic acid hydroperoxide which is measur-able with great accuracy at low concentrations by spectro-photometric or kinetic assay techniques, for example.

Description

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 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 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 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 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 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.~

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.

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.

" .

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.

(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.

Claims (40)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. 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 fatty acid by lipolysis, contacting any fatty acid produced with lip-oxygenase 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.

2. A method according to claim 1, wherein said substrate is a triglyceride substrate.

3. A method for the specific determination of lipase activity in a biological fluid comprising:
a) adding a sample of said biological fluid to an emulsified substrate, said substrate having the structural formula:

wherein R1 is ? is an integer of 1 to 5, and R2 is a member selected from the group consisting of R1 and saturated or (poly)unsaturated fatty acid alkyl chains;
b) simultaneously, or immediately thereafter, adding lipoxygenase, in the presence of oxygen, said lipoxy-genase being added in an amount effective to convert the fatty acid produced by lipolysis to its acid hydro-peroxide at a rate sufficient to ensure that lipolysis is the limiting reaction; and c) measuring the production of said acid hydroperoxide.

4. A method according to claim 3, wherein the substrate is trilinolein.

5, A method according to claim 3, carried out at a pH of about 8.8 and at a temperature of about 30°C.

6. A method according to claim 3, 4 or 5, wherein the activity of said lipoxygenase is at least about 1,000 S.U./
liter of substrate.

7. A method according to claim 3, 4 or 5, wherein the activity of said lipoxygenase is about 1,400 S.U./liter of substrate.

8. A method according to claim 4, wherein the con-centration of said substrate is between about 1.0 X 10-4 mol/
liter and 4 X 10-4 mol/liter.

9, A method according to claim 3, wherein step c) comprises measuring spectrophotometrically the amount of acid hydroperoxide produced in a fixed incubation period.

10. A method according to claim 9, wherein said fixed incubation period is effected by stopping the lipolytic reaction in step b) by adding an acid/alcohol mixture.

11. A method according to claim 9 or 10, wherein said fixed incubation period is from about 5 to about 15 minutes.

12. A method according to claim 9, wherein the spectro-photometric measuring comprises colorimetric measuring.

13. A method according to claim 12, wherein the colori-metric measuring employs the capability of the acid hydroperoxide to oxidize ferrous ion to ferric ion.

14. A method according to claim 9, wherein the spectro-photometric measuring comprises UV spectrophotometric measuring.

15. A method according to claim 3, wherein said measuring in step c) comprises kinetically measuring the rate of oxygen consumption in step b).

16. A method according to claim 3, wherein said substrate is based on naturally occurring fatty acids and ? is 1, 3 or 5.

17. A method according to claim 3, wherein said substrate is based on a synthetic fatty acid and ? is 1, 2, 3, 4 or 5.

18. An analytical procedure for the determination of lipase activity comprising adding an unknown sample fluid to a substrate reagent comprising an emulsion of trilinolein, adding lipoxygenase, in the presence of oxygen, said lipoxy-genase being added in an amount sufficient to insure that lipolysis is the rate limiting reaction and thereafter calculating lipase activity on the basis of production of acid hydroperoxide.

19. A method according to claim 18, carried out at a pH of about 8.8 and at a temperature of about 30°C.

20. A method according to claim 19, wherein the activity of said lipoxygenase is at least about 1,000 S.U./liter of substrate.

21. A method according to claim 18, wherein said trilinolein is present in a concentration of between about 1.0 X 10-4 mol/liter and about 4 X 10-4 mol/liter.

22. A method according to claim 18, wherein the pro-duction of acid hydroperoxide is determined by spectrophotometry.

23. A method according to claim 18, wherein the pro-duction of acid hydroperoxide is measured kinetically by monitoring the accompanying decrease in oxygen tension.

24. In a test for lipase activity wherein the triglyceride substrate in emulsified form is contacted with a test sample and lipase activity is measured as a function of the product formed by lipolysis, the improvement comprising:
employing a substrate having the general formula:

wherein R1 is ? is 1, (2), 3, (4), 5, and R2 is a member selected from the group consisting of R1 and saturated or (poly)unsaturated fatty acid alkyl chains; and further employing a sufficient amount of lipoxygenase, in the presence of oxygen, to ensure that the conversion of sub-strate to fatty acid by lipolysis is the rate limiting reaction, and thereafter determining lipase activity as a function of the acid hydroperoxide produced.

25. A test according to claim 24, wherein said substrate is trilinolein.

26. A test according to claim 24, wherein said test sample, substrate and lipoxygenase are allowed to react at about 30°C. and at a pH of about 8.8.

27. A test according to claim 24, wherein the activity of said lipoxygenase is at least about 1,000 S.U./liter.

28. 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 fatty acid produced by lipolysis to its acid hydro-peroxide at a rate sufficient to ensure that lipolysis is the limiting reaction.

29. A reagent according to claim 28, wherein said substrate is trilinolein.

30. A reagent according to claim 29, wherein said lipoxygenase has an activity of about 16,800 I.U./ml at 25°C,

31. A test system 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, 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; and means for measuring the production of acid hydroperoxide as a measure of the lipase activity of the biological fluid.

32. A test system according to claim 31, wherein said substrate is trilinolein.

33. A test system according to claim 31 or 32, wherein said means comprises spectrophotometric measuring means.

34. A test system according to claim 31 or 32, wherein said means comprises kinetic assay means for kinetically measuring the rate of oxygen consumption.

35. A method for the specific determination of lipase activity in a biological fluid constituting a hetero-geneous system comprising:
(a) adding a sample of a biological fluid sample to an emulsified substrate containing at least 1.0 x 10-4 mole per liter of a water-insoluble triglyceride having the structural formula:

R1 = ? is 1, (2), 3, (4), 5, and R2 = a member selected from the group consisting of R1 and saturated or (poly)unsaturated fatty acid alkyl chains and wherein said substrate also contains a bile salt;
(b) simultaneously, or immediately thereafter, adding an amount of lipoxygenase high enough to convert the acid produced by lipolysis, in the presence of oxygen, to its acid hydroperoxide at a rate sufficient to ensure that lipolysis is the limiting reaction but low enough to ensure that the rate of reaction with said substrate itself remains low, and (c) measuring the production of said acid hydro-peroxide.

36. A method according to claim 1,2 or 3, wherein said fatty acid produced by lipolysis comprises linoleic acid.

37. A method according to claim 18, 19 or 20, wherein said acid hydroperoxide comprises linoleic acid hydro-peroxide.

38. A test according to claim 24, 25 or 26, wherein said fatty acid comprises linoleic acid.

39. A reagent according to claim 28, 29 or 30, wherein said substrate is effective in the presence of lipase to produce linoleic acid by lipolysis.

40. A test system according to claim 31 or 32, wherein said substrate is effective in the presence of lipase to produce linoleic acid by lipolysis.