LAB4 - LIPASE and AMYLASE Flashcards
a turbidimetry assay used for lipase determination
tietz-fiereck turbidimetry assay
the kit for tietz-fiereck turbidimetry assay conists of
lipase buffer
olive oil substrate
lipase activity standard
the pH level of lipase buffer
pH 7
amount of lipase activity standard used
60 U/L
principle of tietz-fiereck turbidimetry assay
the lipolytic action of lipase in the serum hydrolyzes the olive oil emulsion to fatty acids, mono and di-glycerides and glycerol. The decrease in turbidity at 550 nm is proportional to the lipase activity of the serum
Give the values or the amount of reagent needed for cuvette 1 (STANDARD)
lipase buffer- 1200 ul
olive oil - 250 ul
standard - 25 ul
serum - no serum needed
Give the values or the amount of reagent needed for cuvette 2 (UNKNOWN/TEST SERA)
lipase buffer- 1200 ul
olive oil - 250 ul
standard - NO STANDARD
serum - 25 ul
after preparing the solution needed, how many minutes we do need to stand after shaking vigorously/
10 mns standing at room temp
wavelength needed to read lipase
550 nm
read blank tube needed for the method
distilled water
formula for lipase
abs of sample/ abs of standard
X 60 u/l
ref range of lipase
30 - 200 u/l by clsi, nccls
increased level of lipase are associated to what conditions
acute pancreatitis
carcinoma of pancreas
intestinal obstruction
perforated peptic ulcer
acute peritonitis
it is generally agreed that lipase values have about the same significance as ____ values in the diagnosis of acute pancreatitis
amylase
during pancreatic disturbances, the serum lipase activity may rise slowly that the serum amylase, but it may remain elevated for a longer time so that it may be useful in the diagnosis and follow up of acute pancreatitis
true or false?
true
what should we do if the chemistry analyzer minimum’s readability is 2.5 ml
multiply by 2 the volume of the standard/test/ and the control and reagent
the amylase reagent sit we used and studied is for the (quantitative, qualitative) kinetic determination of amylase activity
quantitative
the amylase reagent sit we used and studied is for the quantitative kinetic determination of amylase activity.
intended to use for (manual, automated, both) procedures in human serum and urine
both
amylase was first measured QUANTITATIVELY by an ___
iodometric method
amylase was first measured QUANTITATIVELY by an iodometric method by who?
Wohlegemuth in 1908
In 1938, he then introduced a procedure that STANDARDIZED the amounts of starch and iodin
somogyi
His work became the basis for the widely used amyloclastic and saccharogenic methods
Somogyi
Again, what is the method of somogyi that is widely used and became the basis for lipase determination?
amyloclastic 1956 and saccharogenic methods 1960
what is the disadvanatage of amyloclastic and saccharogenic methods of somogyi
long incubation time
endogenous glucose interference
unstable reaction colors resulting in poor reproducibility and reliability
several procedures have been suggested such as the ______, however they are still subjected to long incubation time,
endogenous glucose interference and interference in the formation of NADH
use of defined substrate maltoteraose
he introduce a substrate that will eliminate the interference from endogenous glucose and pyruvate
wallenfells et.al.
wallenfells et.al
he introduce a substrate that will eliminate the interference from endogenous glucose and pyruvate
what substrate is it?
p-Nitrophenylglycosides
the present procedure is based on the modification of __
wallenfells et.al
the present procedure we are doing right now is based on the modification of wallenfells et.al’s method.
What could be the modification?
p-Nitrophenyl-D-Maltoheptaoside with the terminal glucose blocked to reduce spontaneous degradation of substrate by glucosidase and glucoamylase
the modified method of wallenfells is performed in what mode
kinetic mode with very short time lag and offers much greater stability than previous amylase methodologies.
the determination of amylase activity in what sample is most commonly performed for the diagnosis of acute pancreatitis
serum and urine
in acute pancreatitis, amylase levels are elevated for longer periods of time in what preferred sample
urine than in serum
since urine amylase levels are elevated for longer periods of time in urine, determining the ratio of amylase and ____ is important in followng the course of the pancreatitis
creatinine clearances
give the first reaction of amylase determination
PNPG7 —– amylase ——- PNPG3+Maltotetraose
2nd reaction in amylase determination
PNPG3 —- glucoamylase — PNPG1 + glucose
3rd reaction of amylase determination
PNPG1— glucosidase —-p-Nitrophenol+glucose
amylase hydrolyze p-nitrophenyl-D-maltoheptaoside to form
p-nitrophenylmaltotriose
p-nitrophenylmaltotriose will be hydrolyze glucosidase to form
p-nitrophenylglucoside
p-nitrophenylglucoside will be hydrolyze again by glucosidase to form
p-nitrophenol + glucose
the end product, p-nitrophenol + glucose, will form a result color of
yellow
the rate of ____ in absorbance is measured in 405 nm and is proportional to the amylase activity in the sample
increase
nm use for amylase
405 nm
reagent compositions
p-nitrophenyl-D-maltoheptaoside
glucosidase
glucoamylase
sodium chloride
calcium chloride
buffer 50 ml
the reagents are intended to use only in ____
in vitro
we must store the dry reagent in what temp
2-8 * C refrigerated
reagent are stable until when
expiration label
reconstituted reagent is stable for at least how many days at room temp 15-30*C
10 days
reconstituted reagent is stable for at least how many days at refrigerated temp 2-8
30 days
the reagent should be discarded if
turbidity has occurred; turbidity may be a sign of contamination
moisture has penetrated the vial and caking has occurred
reagent does not dissolve completely upon reconstitution
the reconstituted reagent has an absorbance of 0.70 or greater when measured against water at 405 nm
if the reconstituted reagent has an absorbance of 0.90 upon measuring against water blank at 405 nm, what is the next course of action?
discard the reagent, the reconstituted reagent must have an absorbance of lower than 0.70
the ideal specimen for amylase
serum
can we use plasma in amylase?
yes, only heparinized plasma
why do other anticoagulant such as citrate and EDTA are not used for amylase determination
such anticoagulants bind with calcium which is the ion needed for amylase activity, so only heparin is allowed for plasma use
urine specimen must be collected in ____ period
24 hr period
urine specimen must be collected in 24 hr period and the pH must be adjusted to
pH 7 with either 0.1 N NaOH or 0.1 N HCl and keep refrigerated
do we need to add preservative to the urine sample?
no need for that
amylase in serum and urine is reported stable for how many days or weeks in room temp
18-25*C
1 week
amylase in serum and urine is reported stable for how many days or weeks in refrigerated temp and PROTECTED AGAINST EVAPORATION AND BACTERIAL CONTAMINATION
several months
macroamylasemia ___ activity of pancreatic amylase in serum
increases
a number of diseases can affect the determination of amylase
true or false
true the fire
who listed drugs and other substances that can interfere in amylase activity
young et.al
lipemia, hemolysis, will ____amylase values
increase
can insulin affect amylase determination?
yes
how many minutes we need to prewarm the reagent
3 minutes
how many minutes do we need to read the sample for amylase
read first at 15 seconds then read for every 30 sconds
the formula for amylase
abs/min X 1.025 X 1000
divide them by
8.5 X 0.025 X 1
shortcut for amylase
abs/min X 4824
how do you get the abs/min then?
minus minus plus then divide by 4
to convert to SI units (nkat/L) the amylase
multiply to 16.67
linearity for amylase
1500 IU/L
if the linearity exceed for amylase, what should we do
dilute with an equal volume of saline and reru the multiply the result by 2
ref range for amylase in serum
up yo 96 IU/L
ref range for amylase in URINE
18 to 330 IU/L
