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rev:   June 3, 2005


CONFLUOLIPä   Pancreatic Lipase Test

Continuous fluorometric enzyme test for quick determination of pancreas lipase in serum/
plasma and tissue

culture fluids/ cell extracts

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

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

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

The following monoclonal antibodies (Mab), antisera and polypeptides for lipid research are also available

Cat. No.             Product                           

61080:                Human LDL, Mab

61081:                Bovine Lipoprotein Lipase, Mab

61082:                Human HDL, Mab

61085/61086:     Human Apolipoprotein E, Mab

61088:                Human Apolipoprotein E2, Mab

61087:                Human LDL Receptor, Mab

61065:                Human LRP, 515 kDa, Mab

61066/61067:     Human LRP, 85 kDa, Mab

61068:                Human RAP, 39 kDa, Mab

61098:                Pleo-Lipoprotein Receptor, rabbit

61049/61549:     Human LDL Receptor, chicken

61099/61599:     Human LDL Receptor, rabbit

62220:                Cholesterylester Transfer Protein
                           (CETP), human recombinant (
inquire for availability)

62221:                RAP (Lipoprotein Receptor-associated
                           Protein), rat recombinant

NEW                  Inquire for human recombinant Apolipoproteins

http://www.researchd.com/miscabs/lipoabs.htm

 



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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