LIPASE KITS
CONFLUOLIP ™ ( Continuous Fluorometric Lipase Test)
-For Research use Only $200.00/1kit $175.00/kit 2 or more any combination
-Fluorometric tests for the analysis of triglyceride hydrolysis capacity in postheparin plasma (PHP) and tissue culture fluids/ cell extracts
Cat. No.: RDI-PROPR2003 Total Lipase Test
Cat. No.: RDI-PROPR2004 Hepatic Lipase Select Test
cat. No. RDI-PROPR2002 Pancreatic Lipase Kit
Size of the Kit: 24 Determinations
Storage: 2 - 8° C
sample protocol for total and hepatic lipase kits (see actual insert with each kit)
Introduction
Two lipases are involved in the catabolism of triglyceride-rich lipoproteins: lipoprotein lipase (LPL; EC 3.1.1.34) located on glucosaminoglycan chains, anchored to the luminal surface of the capillary epithelium in adipose tissue, heart and skeletal muscle; hepatic lipase (HL; EC3.1.1.3) which is almost exclusive to the endothelial cells of the liver. Both lipases are triglyceride lipases [1].
LPL is a multifunctional protein with a central role in homeostasis and a rate-limiting enzyme for the metabolism of triglyceride-rich chylomicrons and VLDL. Triglycerides of chylomicrons and very low density lipoprotein (VLDL) are the preferred substrates of LPL. An inherited deficiency of LPL causes defective chylomicron metabolism [1]. Patients with autosomal recessive LPL defects show the symptoms of chylomicronemia and type I hyperlipidemia [2]. Heterozygote LPL deficiency has been identified as a defect of some cases of familial combined hyperlipidemia, one of the most common causes of genetic hyperlipidemia [3]. Many metabolic disorders such as diabetes, obesity, insulin resistence, hyperinsulinemia, hypothyroidism, gestational hyperlipidemia, and renal disease as well as alcohol ingestion are associated with elevated plasma triglycerides. The lipid abnormalities in these diseases can be explained as modulated or changed by LPL activity [4,5].
HL hydrolyses the triglycerides of intermediate density lipoprotein (IDL) and high density lipoprotein-2 (HDL2) [6-9]. It was further shown that the hepatic removal of chylomicron remnants is primarily mediated by mechanisms involving HL (and apolipoprotein E). After the chylomicron remnant particles have bound to the hepatocyte surface, endocytosis is predominantly mediated by the hepatic LDL receptor and at a slower rate by the LDL receptor-related protein (LRP) which is regulated by the receptor-associated protein (RAP) [10]. LPL and HL have been shown to bind directly to LRP [11]. HL and LPL should, therefore, be an important determinant of lipoprotein receptor pathways [12]. Retention of lipoproteins by the extracellular matrix may be another process modulated by the lipases [13].
As the affinity of lipases for heparin is higher than the heparin-sulfate-like anchor, injection of an intravenous heparin bolus displaces both enzymes into postheparin plasma (PHP), where their activity can be quantified.
The Total Lipase Test [Cat. No.: RDI-PROPR2003] can also be used to determine the activity of bacterial lipases (data available for Pseudomonas lipase).
Principle of the Test
This kit's lipase substrate is 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 [14-16]. Upon addition of active lipase the quencher is hydrolysed and the pyrene becomes fluorescing. The kinetic increase in fluorescence intensity at 37°C is proportional to lipase activity. Fluorescence intensity is measured at 342 nm excitation and 400 nm emission wavelength. Distinct physical conditions (pH, NaCl and Triton X100 concentration) allow the selective determination of Hepatic Lipase [cat#RDI-PROPR2004]. The substrate is not hydrolized by esterases and phospholipase A2.
The standard provided is the unquenched fluorescent derivate of the substrate, exhibiting endpoint fluorescence. The standard is used for the calibration curve, to determine the linear measurement range of the fluorometer and to calculate the substrate turnover in the samples.
Materials Required, But not Included:
Fluorometer (342 excitation nm and 400 nm emission wavelength) with a thermostated cuvette holder; the appropriate excision and emission slit width (e.g. 10 nm / 10 nm) has to be evaluated for the instrument used.
Vials for sample and sample dilution (1 ml, 5 ml)
Precision pipettes (10 µl, 1000 µl)
Sterile pipette tips
Acrylic cuvettes (2 ml)
Contents of the Test Kit
3 vials of lipase Substrate (lyoph.), for 8 determinations each. Immediately before use reconstitute one vial with 16 ml buffer.
Cat#RDI-PRIPR2003: Substrate A;
cat#RDI-PROPR2004: Substrate B
4 bottles of Buffer, 30 ml each; PR2003: Buffer A (pH 8.2; physiological salt concentration)
cat#PROPR2004: Buffer B (pH 8.8; high salt)
3 vials of Standard (lyoph.). Immediately before use reconstitute one vial with 4 ml buffer to obtain a final concentration of 20 pmol/ml. The Standards for PR2003 and PR2004 are identical.
Sample Material
Postheparin plasma (PHP), heparin cell culture supernatant or cell extract, and purified lipase may be used. Appropriate dilutions should be prepared in the buffer provided, e.g. predilute postheparin plasma 1:10.
The Test Requires Four Working Steps:
1) Adjustment of sensitivity and Assay Range:Calibration of the fluoremeter:
The test requires a linear range.Serial dilutions of the provided standard are prepared. The linar range is obtained by adjustment of a suitable slit width and sensitivity at the fluoremeter.
Reconstitute 1 vial of standard with 4 ml buffer, mix well, and prepare standard dilutions in consecutive steps:
S1: reconstituted standard 20 pmol/ml
S2: 1 ml S1 + 3 ml buffer 5 pmol/ml
S3: 1 ml S2 + 3 ml buffer 1.25 pmol/ml
S4: 1 ml S3 + 3 ml buffer 0.30 pmol/ml
Transfer 2 ml of each standard into a cuvette and measure fluorescence at Ex 342 nm and Em 400 nm at room temperature.
Adjustrthe sensitivity of the instrument first with S1 (which give sthe highest fluorescence of all standards) .The other standards of the serial dilution will then show a lower fluorescence than S1. The concentration of the unuenched standards S1-S4 and their corresponding pyrene fluorescence must result in a linera correlation over the whole range (0.3 to 20 pmol/ml)
Fig. 1: Example of a Standard Curve
Step 2:Quantification of the Pyrene Fluorescence:
The molar fluorescence of the pyrene group is needed for the calculation of the unquenched pyrene group which accumulates over time in the kinetical lipase assay .
The molar fluorescence constant of the pyrene fluorecence of the unquenched product is the slope of the staright 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 calibartion straight line.
y2-y1 = 128.6-5.6 (RFC) = 6.2 RFU X pmol/L X ml
X2-X1 20-0.3 (pmol/ml)
This applies only to the fluorometer and fluorometer configuration used! DO NOT USE TO CALCULATE RESULTS.
Step 3: Kinetical Lipase Activity:
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.
Test Procedure For Sample Measurement
Transfer 2 ml freshly reconstituted substrate into a cuvette and warm up to 37°C.
Add 20 µl sample (diluted sample resp.) to the substrate and mix well.
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 configuration as for calibration (Ex 342 nm, Em 400 nm, slit width, amplification).
Fig. 2: Example of a Kinetic Measurement
Calculation of Lipse Activity:
The molar fluorescence of the pyrene group claculated 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-xis:pyrene fluorescence). It is calculated from the ratio of the differences between time points and the corresponding fluorescence values, repectively, of the values obtained under Step 3.
Example of Calculation of Fluorescence/Time Values of Fig 2
y2-y1 = 86.8-44.8 (RFU) = 6.0 RFU X {min -1)
X2-X1 10-3 (min)
Second the activity of the lipase in the assay is now calculated as the ratio delta fluorescence/time in the assay and the molar fluorescence, calculated from the straight line of the standards
6 RFU of sample per min = 0.97 pmol ml-1 min-1
RFU of 1 pmol/ml standard
For the example shown in fig. 2:
6.0 RFU / min = 0.97 [pmol ml-1 min-1]
6.2 RFU / pmol ml-1
This calculation does not yet correct for a possible dilution factor of the probe. If ou work with postheparin plasma or cell culture supernatants, it may be helpful to calculate the lipase activity of a test sample perr ml PHP and cell culture supernatant, respectively.
The results are given as pmol min-1 per ml assay volume, which is 2 ml. Multiply result by 2.
If different assay volumes or sample dilutions are used, multiply result accordingly.
Limitations:
Optimal conditions for HL activity are found at high ionic strength and pH 8.8, independent from activators or stimulators. LPL activity is almost completely inhibited under these conditions.
At pH 8.2 and physiological salt concentration are optimal for total lipase activity measurements. However, under these conditions both enzymes (LPL and HL) are not additive.
As a consequence, most PHP show a lower total lipase activity than the measured HL activity. The LPL activity can not be calculated from the results of Hepatic Lipase and Total Lipase activity.
Additionally, the test result may be influenced by competitive inhibition of serum lipids present in the sample. For interpretation of the results it could become necessary to analyse the lipid status of post heparin plamsa and , particularly the triglyceride values.
References
1) Olivecrona T, Bengtsson-Olivecrona G (1990) Lipoprotein Lipase and Hepatic Lipase. Curr Opin Lipidol 1:222-30
2) Brunzell JD (1989) in: The Metabolic Basis of Inherited Disease (Ed: Scriver CR, Beaudet AL, Sly WS and Valle D) 6th edition, pp 1165-1180, MacGraw-Hill Book Co., NY
3) Babirak SP, Iverius PH, Fukimoto WY, Brunzell JD (1989) Arteriosclerosis 9:326-334
4) ckel RH (1989) N. Engl. J. Med. 320:1060-1068
5) Taskinen MR (1987) in: Lipoprotein Lipase (Borensztaijn J, ed) pp 201-228, Evener Publ., Chicago
6) Patsch JR, Prasad S, Gotto AM, Patsch W (1987) J Clin Invest 80:341-347
7) Zambon A, Austin MA, Brown GB, Hokanson JE, Brunzell JD (1993) Arterioscler Thromb 13:147-153
8) Despres JP, Ferland M, Moorjani S, Nadeau A, Tremblay A, Lupien PJ et al (1989) Arteriosclerosis 9:485-492
9) Knusi T, Ehnholm C, Viikari iJ, Harkonen R, Vartiainen E, Puska P, Taskinen MR (1989) J Lipid Res 30:1117-1126
10) Herz J, Qin S-Q, Oesterle A, DeSilva HV, Shafi S, Havel RJ (1995) Proc. Natl. Acad. Sci. USA 92:4611-4615
11) Chappell DA, Fry GL, Waknitz MA, Iverius PH, Williams SE, Strickland DK (1992) J. Biol. Chem. 267:25764-25767
12) Beisiegel K, Weber W, Bengtsson-Olivecrona G (1991) Proc. Natl. Acad. Sci. 88:8342-8346
13) Eisenberg S, Sehayek E, Olivecrona T, Vlodovsky I (1992) J Clin Invest 90:2013-2021
14) Zandonella G, Haalck L, Spener F, Faber K, Paltauf F, Hermetter A (1995) Eur J. Biochem 231:50-55
15) Duque M, Graupner M, Stütz H, Wicher J., Zechner R, Paltauf F, Hermetter A (1996) JLR 37:868-876
16) PCT/EP95/01919 (patent pending)
For Research Only