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rev: June 3, 2005
Continuous fluorometric enzyme test for quick determination of pancreas lipase in serum/plasma and tissue
culture fluids/ cell
Cat. No.: RDI-PROPR2002 Pancreatic Lipase Test
$375.00/1kit $344.00/kit 5 or more
Size of the Kit: 24 Determinations
Storage: 2 - 8� C
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.
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
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 �l, 1000 �l)
� Sterile pipette tips
� UV-permeable acrylic or quartz cuvettes (2 ml)
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.
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 C2: 40 pmol/ml
Standard C3: 10 pmol/ml
Standard C4: 0 pmol/ml
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.
1.2 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
2: Quantification of the
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 �l 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.
of Calculation of
with Values of Fig. 2:
335.9 � 180.8 RFU RFU
= 29.2 RFU x min -1
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.
29.2 RFU x min -1
= 5.66 pmol x ml-1 x min-1
5.15 RFU x pmol -1 x ml
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.
(1) Zandonella G, Haalck L, Spener F, Faber K, Paltauf F, Hermetter A (1995) Eur J. Biochem 231:50-55
Duque M, Graupner M, St�tz H, Wicher J., Zechner R, Paltauf F,
Hermetter A (1996) JLR 37:868-876
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