For the quantitative determination of Pancreatic Lipase in serum.
Serum
in lipase is recognized as an important indicator for the diagnosis, and
therapeutic monitoring, of pancreatic diseases. There have been many methods
developed for the assay of lipase activity: alkaline titration of fatty acid
liberated from triglyceride gum arabic emulsion, measurement of the decrease in
turbidity of a triglyceride (olive oil) emulsion, and a colorimetric method
using a synthetic substrate containing thiol ester of a short chain acid.
However, each of these methods has specific deficiencies, including difficulty
of the procedure, lack of specificity, lack of precision near the normal level,
poor reproducibility, susceptibility to interferences, and poor adaptability to
automated instrumentation.
This
new colorimetric Lipase reagent uses a clear substrate solution of 1,2-diglyceride,
a natural lipase substrate derived from egg lecithin. It is a highly specific
method for pancreatic lipase, using co-lipase and deoxycholate as activators.
The colorimetric measurement of the rate formation of the quinone dye from TOOS
provides a highly sensitive reaction with excellent reproducibility and
stability. Finally, because of the simple two-part reagent system, the
procedure is adaptable to many automated chemistry analyzers.
Pancreatic Lipase
1,2-diglyceride
+ H2O -------------------- >
2-monoglyceride + fatty acid
MGLP
2-monoglyceride + H2O -------- > glycerol + fatty acid
GK
glycerol + ATP -------- > glycerol-3-phosphatase + ADP
GPO
glycerol-3-phosphatase + O2 ------- >
dihydroxyacetone phosphate + H2O2
POD
2H2O2
+ 4-aminoantipyrine +TOOS -------- >
quinone dye +
4 H2O2
Serum
pancreatic lipase acts on a natural type of substrate, 1,2-diglyceride to
liberate 2-monoglyceride. The 2-monoglyceride is hydrolyzed by monoglyceride
lipase (MGLP) to produce glycerol and fatty acid.
Glycerol
Kinase (GK) then acts on the glycerol to produce glycerol-3-phosphatase which
is converted to dihydroxyacetone phosphate and hydrogen peroxide in a reaction
catalyzed by glycerol-3-phosphate oxidase (GPO). The hydrogen peroxide then
reacts with 4-aminoantipyrine and
N-Ethyl-N-(2_hydroxy-3-sulfopropyl)-m-toluidine sodium salt (TOOS) in a
reaction catalyzed by peroxidase (POD) to yield a quinone dye. The rate of
increase in absorbance at 550nm is directly proportional to the Lipase activity
of the sample.
Serum
Lipase activity is found to be elevated in acute pancreatitis or obstruction of
the pancreatic duct. The half –life of pancreatic lipase is longer than that of
pancreatic amylase making it a very reliable marker of pancreatic disease.
Since this colorimetric Lipase is specific for pancreatic Lipase, it is ideally
suited to run in conjunction with a total amylase reagent, which measures both
pancreatic and salivary type amylase.
1.
Lipase Substrate– 1,2-diglyceride 63mg%, MGLP 87u/100ml, GK 133u/100ml,
GPO 4,000u/100ml, TOOS 67mg%, ATP 40mg%, Peroxidase 133u/100ml, Co-lipase 4,000u/100ml, Buffer.
2.
Lipase Substrate Buffer – Buffer, Cholic Acid 217mg%, pH = 6.8 ±
0.1.
3.
Lipase Activator – Deoxycholate 1414mg%, 4-aminoantipyrine 120mg%,
Buffer pH = 8.7 ± 0.1.
1.
Reagent is for in vitro diagnostic use only.
2.
Avoid ingestion.
Reconstitute the Lipase Substrate vial with the
amount of Lipase Substrate Buffer indicated on the vial label. Swirl to
dissolve.
The Lipase Activator solution is ready to use.
Reconstitute the Lipase Standard with 3 ml of
distilled water.
Store reagent at 2-8°C
Unopened reagents are stable until the expiration
date on the label when stored at 2-8°C.
Upon reconstitution, the Lipase Substrate solution
is stable for four days at 18-25°C, and 21 days at 2-8°C.
Upon reconstitution, the Lipase Standard solution is
stable for 30 days at 2-8°C.
Do not use if reagents show turbidity or other
evidence of contamination or deterioration.
1.
Fasting, non-hemolyzed serum is the preferred specimen.
2.
Separate the serum from the clot immediately after collection and
measure the lipase activity promptly. If the assay is not performed
immediately, the serum must be refrigerated or frozen until use. Never repeat
freeze and thaw as the lipase can be inactivated
3.
Lipase is reported stable in serum for at least three days at 2-8°C.
1.
Free glycerol will not interfere with the assay as long as
concentrations are 100mg/dl or below. If the serum contains free glycerol above
100mg/dl, dilute with saline to make the level of free glycerol below 100mg/dl
before the assay.
2.
Microbial lipase and cholesterol esterase can affect the assay.
3.
For a listing of substrates which may affect serum lipase levels, see
Young et al.
1.
Lipase substrate and buffer reagents.
2.
Lipase activator solution.
3.
Lipase standard.
1.
Accurate pipettes.
2.
Test tubes.
3.
Timer.
4.
Heating block or water bath (37°C)
5.
Spectrophotometer with a temperature controlled cuvette.
6.
Controls
6.0 Test Procedure
6.1 Procedure (Automated)
See appropriate instrument application instructions.
6.2 Procedure (Manual)
1.
Label test tubes “Blank”, “Standard”, “Control”, “Patient”, etc.
2.
Pipette 300ml of reconstituted Lipase
Substrate reagent to all tubes.
3.
Pipette 5ml of distilled water to the
blank tube and 5ml of the appropriate sample
to the tubes labeled “Standard”, “Control”, etc.
4.
Mix each tube well and incubate for 3-5 minutes at 37°C.
5.
After the pre-incubation, add 100ml of Lipase activator to the
blank tube. Mix well and incubate for 3 minutes at 37°C.
Then measure the rate of increase in absorbance per minute at 550nm
(540-560nm).
6.
Repeat step 5 for all tubes.
7.
See “Calculations” to obtain results.
The above volumes may be multiplied by an
appropriate factor if a larger total volume of reaction mixture is necessary
for reading.
Use the lipase standard provided in the kit.
The integrity of the reaction should be monitored by
use of normal and abnormal control sera with known lipase activity.
Samples with Lipase activity exceeding 600 U/L should
be diluted with an appropriate amount of saline, re-assayed, and the final
result multiplied by the appropriate dilution factor.
DA Sample – DA Blank x
Conc of = Lipase
DA Standard – DA
Blank Std. (U/L) activity (U/L)
0-62 U/L
It is strongly recommended that each laboratory
establish its own normal range.
1.
Linearity: 600U/L
2.
Comparison: A study performed comparing the Lipase (Colorimetric)
methods to a turbidimetric Lipase procedure yielded a correlation coefficient
of 0.956 with a regression equation of y = 0.48x + 9.1.
3.
Precision: The lipase activity of three samples was measured ten times
each with the following results:
46.7 1.70 3.64
254.0 1.70 1.47
516.5 4.65 0.90
1.
Imamura, S. and Misaki, H., Selected Topics in Clinical Enzymology,
2:73, 1984.
2.
Imamura S. et al, Collection of summaries of lectures in the 126th
general meeting of Kinki branch, analytical section, Japan Society of Clinical
Chemistry, p 11-31, 1986.
3.
Hayashi, C., et al, Clinical Examination, Instrument and Reagent, 2:25,
1986.
4.
Kitaura, S., et al, Collection of summaries of lectures in the 6th
general meeting of The Japanese Biochemical Society, 848, 1988.
5.
Imamura, S., et al, clin., chem., Abstract Issue in the 41st
National Meeting, 1120, 1989.
6.
Young, D.S., et al, Clin. Chem. 21:1D, 1975.