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.

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

 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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