Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Objective: We describe and evaluate a new method for determining isolipases in serum. Bases: two forms of lipase, L1 and L2, were identified by this method. A different surface charge is the basis by which the two isolipases are separated by electrophoresis in a buffered agarose gel. After electrophoresis the bands were detected with a specific colorimetric chemical reaction and quantified by densitometry. Samples and method: EQUIPMENT: Beckman´s Paragon Electrophoresis System. REAGENTS: 1% agarose gels (CK/Paragon). Buffer MOPSO and sodium salt MOPSO (CK/Paragon) was adjusted to pH 7,8 with NaOH 1,5M. Color-substrate, Lipase-PS (Sigma Diagnostics) reagent for the quantitative, kinetic determination of pancreatic lipase. SAMPLES: serum samples were obtained in the manner normally used for any laboratory test. Specimens not analyzed on the same day were stored at -20ºC. To test the clinical usefulness of isolipases, 46 hyperamylasemic patients were studied, 30 of them with pancreatitis and 16 of nonpancreatic cause establishing reference values on 44 healthy adult populations. ELECTROPHORETIC PROCEDURE: We used a template (for SPE/Paragon), samples (1-3 microliters) were applied across each template slot and electrophoresed for 25 min. at a constant 150 V. For visualitation of the bands a saturate blotter (100 x 50 mm) with a specific substrate were placed onto gel surface and incubated for 30 min. and 45ºC in a humid chamber. The relative quantity of the bands was set at 540 nm in an Appraise densitometer. Evaluation of the Method. Linearity and detection limit:were evaluated usin a high level control serum. These studies yielded a linear range of 72 to 1018 IU/L; sensitivity (9 IU/L). Precission: the CV within-assay were calculated using 10 replicate analyses of 3 samples with different activities and between-assay by making 6 consecutive assays on the 3 controls (CV < 10%). Reference values: L1 (0-20) and L2 (100-80), % of total lipase activity and the ratio L1/L2 = or < 0,25. Conclusions: The method presented is rapid, sensitive and precise by separation of isolipases and adaptable for routine use, because it requires conventional electrophoresis equipment and commercially available reagents. Clinical studies: L1 and L2 percentages and L1/L2 ratio were calculated and compared with the control group. It was found a significant increase of L1 and L1/L2 ratio in pancreatic patients (p < 0,001).

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Poster Number PAsánchez0680
Keywords: lipase, isolipase, electrophoresis, agarose gel