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.
Materials and Methods
EQUIPMENT: Paragon Electrophoresis System (Beckman) that includes: electrophoresiscell, power supply, sample applicator, incubator and incubation box, wetprocessor station.
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.
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%).
REFERENCE INTERVAL FOR ISOLIPASES: Reference values were establishedby calculating the mean values ± 2 DS, from a population of healthymale and female adults.
CLINICAL STUDIES: L1 and L2 percentages and L1/L2 ratio were calculatedand compared with the control group.
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.
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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/|
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