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rev: 5/15/2008
CONFLUOLIPä
Continuous fluorometric enzyme test for
quick determination of pancreas lipase in serum/plasma
culture fluids/ cell extracts
Cat. No.: RDI-PROPR2002, new cat#: 55R-PROPR2002
Pancreatic Lipase Test
$412.50/kit
Storage: 2 - 8° C
For In Vitro Research Use only-Not for use
in diagnostics procedures
Introduction
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
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.
bottles of Buffer
C, 30 ml each.
(pH 7.4; physiological salt concentration)
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
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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 |
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
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