The pancreatic lipase
is an organ-specific enzyme which is formed by the pancreas and is secreted
into the pancreatic duct. In the small intestine it hydrolyzes the exogenous
triglycerides. Pancreas lipase is activated by bile acid and colipase.
Increased activity
of pancreatic lipase in serum/plasma is indicative for a disease of the exocrine
pancreas. The analysis of the pancreatic lipase acitivity in serum/plasma
in the presence of upper abdominal symptons is helpful for differential
diagnosis. In acute pancreatitis and/or recurrent attacks in chronic pancreatitis
patients, the pancreatic lipase increases approx. 3 – 6 hours after
onset of the pain and is used for monitoring the course of the
disease.
Principle of the Test
The pancreatic
lipase test is based on the substrate
1-trinitrophenyl-amino-dodecanoyl-2-pyrendecanoyl-3-0-hexadecyl-sn-glycerol
(12-TA-10-P-H6), a triglyceride in which the pyrene fluorescence is
intramolecularly quenched by the trinitrophenyl group (Hermetter and colleagues
[1-2]). After reconstitution, the substrate is complexed with albumin in
a clear microemulsion. Colipase is also included.
Upon addition of serum
with pancreatic lipase activity, the fatty acid of the quencher is hydrolysed
and a fluorescing pyrene glyceride is formed. Specific conditions in the
test preparation and direct measurement of the fluorescence of the products
without secondary reactions allow precise measuring results and a very good
sensitivity and specificity even at low lipase acitivity in serum and plasma
samples. The kinetic fluorescence increase is measured at Ex 342 nm/Em 400
nm after a lag phase of 120 sec. The test does not measure lipoprotein lipase
and hepatic lipase in post heparin plasma and is, therefore, specific for
pancreatic lipase.
The standard provided is the unquenched fluorescent derivate of the substrate,
exhibiting endpoint fluorescence. The standard is required for calibration of
the fluorometer and needed for calculation of the molar fluorescence of the
pyrene group. Like the products of the lipase reaction, it is an unquenched
diglyceride, exihibiting end-point fluorescence.
Materials Required, But not
Included:
§
Fluorometer
(342 excitation [Ex] nm and 400 nm emission [Em] wavelength) with a thermostated
cuvette holder; the appropriate excitation and emission slit width (e.g.
10 nm / 10 nm) has to be set up for the instrument used.
§
Vials
for sample and sample dilution (1 ml, 5 ml)
§
Precision
pipettes (10 µl, 1000 µl)
§
Sterile
pipette
tips
§
UV-permeable
acrylic or quartz cuvettes (2 ml)
Contents
of the Test Kit
3 bottles of lipase Substrate C (lyoph.). Immediately before use, reconstitute with 16 ml Buffer C each. Mix well. Particles dissolve when warmed up to 37°C.
4
bottles of Buffer C, 30 ml each.
(pH 7.4; physiological salt concentration)
5 vials of Standard C (lyoph.). Immediately before use, reconstitute with 4 ml dist. water each to obtain the following concentrations:
Standard
C1:
80 pmol/ml
Standard
C2:
40 pmol/ml
Standard
C3:
10 pmol/ml
Standard
C4:
0
pmol/ml
Sample Material
Serum/plasma, cell
culture supernatant or cell extract, and purified lipase may be used. Appropriate
dilutions should be prepared in the buffer provided, e.g. predilute serum/plasma
1:10.
The Test
Requires Four Working Steps:
Step 1:
Adjustment of Sensitivity and Assay Range: Calibration of the
Fluorometer
The test requires a linear assay range. Serial dilutions of the provided standard are prepared. The linear range is obtained by adjustment of a suitable slit width and sensitivity at the fluorometer.
1.1
Transfer 2 ml of each standard into a cuvette and measure fluorescence
at Ex 342 nm and Em 400 nm at room temperature.
1.2 Adjust the sensitivity of the instrument first with the highest (which gives the highest fluorescence of all standards). Make sure that there is no signal overflow of the photomultiplier! Depending on the concentration, the other standards of the serial dilution will then show a lower fluorescence. The concentration of the unquenched standards S1 - S4 and their corresponding pyrene fluorescence (relative fluorescence units RFU) must result in a linear correlation.
Fig.
1: Example of a Calibration
Curve with Standards
|
Step
2: Quantification of the
Pyrene
Fluorescence
The „molar fluorescence“ of the pyrene group is needed for
the calculation of the concentration of the unquenched pyrene group which
accumulates over time in the kinetic lipase assay (see
below).
The molar fluorescence constant of the pyrene fluorescence of the
unquenched product is the slope of the straight line obtained with standards
S1-S4 and their fluorescence. It can be calculated using the ratio of the
differences of the standard concentrations and their corresponding fluorescence
values from the calibration straight line.
Example
of Calculation of Molar Fluorescence from Values in Fig.
1:
y
2
-
y1 |
= |
511.2
– 98.3 |
= |
5.15
RFUx pmol-1 x ml |
x
2
-
x1 |
80
-
0 |
Step
3: Kinetic Lipase
Assay
The lipase activity in the assay
corresponds to the appearance of the pyrene fluorescence of the unquenched
product over time. If the calibration range is not altered, the increase
of fluorescence in the lipase assay over time remains linear and no signal
overflow occurs.
3.1 Transfer 2 ml freshly reconstituted Substrate into a cuvette and warm up to 37°C.
3.2 Add 20 µl sample and mix well.
3.3 Start kinetic measuring after 2-3 min. Measure
the kinetics for 6-10 min. Very slow kinetics have to be measured over a
longer period of time.
Use fluorometer set-up as for calibration
(Ex 342 nm, Em 400 nm, slit width, amplification).
Fig. 2:
Example
 |
Step
4: Calculation of Lipase
Activity
The „molar fluorescence“ of the pyrene group calculated from the straight line of the standards above (Step 2) is used now and the pyrene fluorescence over time released from the substrate by the action of lipase (Step 3).
First, the unquenched pyrene fluorescence resulting from cleavage of the substrate by the lipase in the assay over time represents the slope of the straight line (x-axis: time; y-axis: pyrene fluorescence). It is calculated from the ratio of the differences between time points and the corresponding fluorescence values, respectively, of the values obtained under Step 3.
Example
of Calculation of
Fluorescence/Time
with Values of Fig. 2:
y
2-y1 |
= |
335.9 – 180.8
RFU
RFU |
= 29.2 RFU x min
-1 |
x
2-x1 |
9
-
3
min
min |
Second, the activity of the lipase
in the assay is now calculated as the ratio
D
fluorescence/time in the assay and the molar
fluorescence, calculated from the straight line of the
standards.
Example:
29.2 RFU x min
-1 |
= 5.66 pmol x ml-1 x
min-1 |
5.15 RFU x pmol
-1 x ml |
|
Limitations
With this test kit, up to now, only a limited number
of patient sera and sera of test persons has been analysed. As no clincial
evaluation studies are available, the test should be used for biomedical
research only and not for routine diagnostic workup.
References
(1)
Zandonella G, Haalck L, Spener F, Faber K, Paltauf F, Hermetter A
(1995) Eur J. Biochem 231:50-55
(2)
Duque M, Graupner M, Stütz H, Wicher J., Zechner R, Paltauf F,
Hermetter A (1996) JLR 37:868-876
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