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Poster Contents






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
Board

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Measurement of Pancreatic Lipase Isoforms by Agarose Gel Electrophoretic Fractionation


Contact Person: Rosa Sánchez (rsanchez@hsc.sas.cica.es)


Introduction

We describe and evaluate a new method for determining isolipases inserum.Two forms of lipase, L1 and L2, were identified by this method. Adifferent surface charge is the basis by which both isolipases are separatedby electrophoresis in a buffered agarose gel. After electrophoresis, thebands in the gel are detected by the following specific colorimetric chemicalreaction sequence.

A violet complex is formed at the site of each fraction; this patternmay be visually interpreted or quantitated by scanning with a densitometerat 550 nm. All studied samples showed two bands of lipase activity. Themost anodal fraction to the application point is L1 and the most cathodalfraction is L2, numbered in conformance to the convention of the InternationalUnion of Biochemistry.


Fig. 1: A:isolipasesin normal serum (L1/L2=0,18). B:isolipases in acute pancreatitis (L1/L2=0,52).

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Materials and Methods

EQUIPMENT: Paragon Electrophoresis System (Beckman) that includes: electrophoresiscell, power supply, sample applicator, incubator and incubation box, wetprocessor station.

REAGENTS:
Lipase gels: 1.0% agarose gels (CK/Paragon), 1.3% Tris AspartateBuffer, 0.1% SodiumAzide, and non-reactive ingredients necessary for optimumperformance.
Lipase buffer: MOPSO ( 3-(N-morpholino)-2-hydroxypropane sulfonicacid ) 22 mmol/L, and MOPSO sodium salt 28 mmol/L (CK/Paragon). It is necessaryto adjust pH at 7,8 with NaOH 1,5 M. Lipase substrate: Colour-substrate: Lipase-PS reactive (Sigma Diagnostics), is intended for the quantitative,kinetic determination of pancreatic lipase activity in serum: substrate(Lipase-PS Substrate reagent), reconstituted with Lipase-PS substrate diluent,and activator (Lipase-PS activator reagent). One substrate vial was dissolvedwith 6 ml of diluent ; reconstituted substrate stored in a closed containerat 2-4ºC is stable for 8 days. Just prior to use, mix 0.9 ml of reconstitutedsubstrate and 0.5 ml of activator reagent. SAMPLES: serum samples werecollected in the manner normally used for any laboratory test. Samplesshowing evidence of hemolysis were avoided. Specimens not analyzed on thesame day were stored at –20ºC. To test the clinical usefulness ofisolipases, 46 hyperamylasemic patients were studied, 30 of them with pancreatitisand 16 of nonpancreatic cause establishing reference values from a populationof 44 apparently healthy adults. Lipase total activity was measured at37ºC by Hitachi 704 analyzer (Boehringer Mannheim). Samples with totalmeasurable lipase activity in excess of 1000 UI/L were diluted with lipasebuffer just prior to electrophoresis.

ELECTROPHORETIC PROCEDURE: we used a template (identical to the recommendedfor SPE/Paragon/Beckman), samples (1-3 microliters) were applied acrosseach template slot and electrophoresed for 20 min. at a constant 100 volts.For visualitation of the bands a saturate blotter (100 ´50 mm) with a specific substrate were placed onto gel surface and incubatedfor 25 min. and 45ºC in a humid chamber. After incubation, evaluategel visually or scan with a suitable densitometer at 540 nm. Prolongedexposure to light will cause a rapid decrease of colour, so gels shouldbe evaluated as soon as possible, and stored in a clean dark area at –4ºC.

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Results

LINEARITY AND SENSITIVITY: we prepared a specimen mixture which wasdiluted in saline solution (0,9%) in order to have six samples with respectivetotal lipase activities of 1153, 576, 288, 144, 72 and 36 U/L. These studiesyielded a linear range of 72 IU/L to 1018 IU/L. Lower detection limit wasfixed at 9 IU/L and was obtained studing L1 percentages from lower totallipase activity samples.

Fig. 4:Linearity studyof the electrophoretic method for pancreatic lipase fraction.

PRECISION: the CV within-assay were calculated using 10 replicate analysesof 3 samples with different antivities and between-assay by making 6 consecutiveassays on the 3 controls (CV<10%).
 
   Within-run (n=10)a           Between-run (n=6)b
 
 
Lipase % of total
 
Lipase % of total
 
Mean
SD
CV.%
 
Mean
SD
CV.%
Total acty,1153 U/L       
L1
30.6
4.9
15.8
 
35.8
4.2
11.7
L2
69.4
4.9
7.1
 
64.2
4.2
6.5
Total acty, 476 U/l       
L1
37.7
3.0
7.9
 
38.2
2.1
5.4
L2
62.3
3.0
4.8
 
61.8
2.1
3.3
Total acty, 92 U/L       
L1
15.2
2.2
14.6
 
1.,6
3.0
16.6
L2
84.8
2.2
2.6
 
84.4
3.0
2.5
a- 10 is the maximun number ofsamples suitable of be appliying on the same agarose gel. 

b- 6 is the maximun number of gels that can be processedwith the same substrate vial.

 Fig. 5:Precision of the isolipases fractionationby Electrophoresis.

REFERENCE INTERVAL FOR ISOLIPASES: Reference values were establishedby calculating the mean values ± 2 DS, from a population of healthymale and female adults.
L1( 0 to 16%) and L2 (100 to 84%) of total lipase activity(77 ± 43 U/L). We calculated L1/L2 ratio, with a reference valueof L1/L2 = or <0,25.

 CLINICAL STUDIES: L1 and L2 percentages and L1/L2 ratio were calculatedand compared with the control group.
 
 
Isolipases
 
 
 n
%L1
%L2
L1/L2 ratio
     
Healthy adults
44
6.8 ± 5.2
93.2 ± 5.2
0.10 ± 0.07
Acute pancreatitis
30
38.5 ± 9.8*
61.5 ± 9.8*
0.69 ± 0.38*
 * p<0.001

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Discussion and Conclusion

 The method presented is rapid, sensitive and sufficiently preciseby separation of isolipases. The specificity is very good, no interferenceby bilirrubin, albumin and others lipolytic substances present in serum.This method has the great advantage of being adaptable in most clinicallaboratories for routine use, because it requires conventional electrophoresisequipment commercially reagents.
It was found a significant increase of L1 and L1/L2 ratio in pancreaticpatients (p<0,001).

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References

  • Lott JA, (1986) Assays of serum lipase:Analytical and clinical considerations.Clin Chem 32:1920-32.
  • Tetrault GA, (1991) Lipase activity in serum measured with Ektachem isoften increased in nonpancreatic disorders. Clin Chem,37:447-51.
  • Pantegnini M,(1992) Electrophoresis fractionation of pancreatic lipase.Clin Chem, 38:1712-6.
  • Lott JA, (1991) Lipase isoforms and amylase isoenzymes: Assays and applicationin the diagnosis of acute pancreatitis. Clin Chem,37:361-8.
  • Lin XZ, (1989) Serum amylase, isoamylase and lipase in the acute abdomen.J Clin Gastroenterol, 11:47-52.
  • IVB Nomenclature Committe. (1984). Enzyme nomenclature. New York:AcademicPress, Inc.,

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Sánchez, MR; Oliver, C; (1998). Measurement of Pancreatic Lipase Isoforms by Agarose Gel Electrophoretic Fractionation. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/
© 1998 Author(s) Hold Copyright