Amylas Flashcards
Measure the decrease in absorbance of a tubid starch
Turbidimetric
Simple and rapid
Turbidimetric
Disadvantage:
Poor precision in near normal activity of amylase
Inadequate subrate stability
Turbidimetric
Turbidimetric change is not linear to amylase activity, except the first _______ of the reaction
Two minutes
Amylase activity is measured by determining the time required for the amylase to completely hydrolyze all-iodine bound starch
Iodometric
Dark blue colored complex
Iodometric
Indication of the endpoint of iodometric method
Absence of blue starch-iodine color
Gives a blue color with iodine
Amylose
Forms a weak red color
Amylopectin
This is due to the helical configuration formed by amylose with iodine
Formation of blue color
In iodometric method, the blue color is obtained only when the overall chain length of amylose is greater than _________
45 glucose units
Acute pancreatitis
Rise: ?
Peak: ?
Return to normal: ?
R: 5-8h
P: 24h
Rtn: 3-5d
Based on the reduction of light scattering of substrate by amylose activity
Nephelometric
Nephelometric method has the advantage of (?)
Simplicity
Speed
Precision
Zero-order kinetics
Suitable for emergency purposes, results available less than three minutes
Nephelometric
Bench-working time and manipulation is minimal. Does not require blanking
Nephelometric
Nephelometric method standardization requires arbitrary standards such as (?)
Formazine
Major tissue sources of amylase
Pancreas and salivary glands
Minor tissue source of amylase
Small intestine
fallopian tubes
skeletal muscle
Amylase isoenzyme present in normal urine
P-type isoenzyme
S-type isoenzyme is present in (?) of normal serum
2/3
Separation basis of amylase determination
Physical characteristics/difference
Chromatography
Electrophoresis
Iso-electric focusing
Immune inhibition
Immuno precipitation
Selective inhibition
Hydrolysis of starch to produce carbohydrates with reducing properties
Saccharogenic
In saccharogenic method, the activity of amylase if expressed by
Somogyi unit
What is Somogyi unit
Expression on the number of milligrams of glucose released in 30mins at 37°C
Somogyi unit to IU
multiply by 1.85
A starch bound to dye is hydrolyzed by amylase
Chromogenic BASTA PAG DYE
Increasing color is proportional to anylase activity
CHROMOGENIC
Disappearance of starch substrate
Amyloclastic method
Measurement of amylase by high performance liquid chromatography (HPLC)
Fluorogenic method
Involves the use of fluorescein labelled amylose
Fluorescence depolarization methods
Basis of abbott TDX amylase assay
Fluorescence Depolarization Methods
Provides simple and sensitive direct assay but requires special instrumentation
Fluorescence Depolarization Method
PNPG7
p-nitrophenyl-D-maltoheptaoside
PNPG3
p-nitrophenylmaltotiose
PNPG1
p-nitrophenylglucoside
Uses radio labelled starch substrat for amylase activity determination
Radiometric method
Uses polyclonal antibodies to produce against salivary or pancreatic isoamylase as antigen
Immunologic method
Can be used for total amylase measurement
Immunologic methods
(Optimized at 37°C)
Recommended reference method for amylase determination by IFCC
5 ethylidene-4-nitrophenyl glucosides as substrate
In the use if 2–chloro-P-nitrophenyl-alpha-D-maltotriose, the presence of (?) causes allosteric changes to amylase
Potassium thiocyanate
Specimen of choice in maylase determination
Unhemolyzed serum and heparinized plasma
These should nit be used as these will bind to calcium
Oxalate
Citrate
Ethylenediamine tetra-acetic acid (EDTA)
Stability of amylase in serum and in urine
One week at room temp
Several months at 2-8°C
(Urine) Decrease stability with pH less than 5
Increases the activity of pancreatic amylase in serum
Macroamylasemia
This type of sample will increase the amylase values
Lipemic sample (lipemia)
Bilirubin concentration (?) falsely increases amylase activity
> 20mg/dL
This hormone will increase amylase activity
Insulin
Administration of (?) before sampling will lead to falsely increased amylase activity
Morphine and opiates
Contamination of sample with (1) should be avoided as these contain (2)
1 sweat and saliva
2 alpha-amylase
Linearity of amylase
1500 IU/L
(exceeded linearity: dilute with equal vol of saline and rerun, result x 2)
Two major isoenzyme of amylase
Pancreatic (P) and Salivary (S) fractions
Differentiation of major isoenzyme includes selective inhibition of the S-AMY by what inhibitor
Wheat germ inhibitor
ACCR
Amylase-creatinine clearance ratio
Established as an index for screening acute pancreatitis
Amylase-creatinine clearance ratio
No longer employed becuase of its insensitivity and less specificity for Px with acute pancreatitis
Amylase-creatinine clearance ratio
How many absorbance is needed for amylase determination?
What is the trend of absorbance?
5 absorbance
Increasing trend
Long-unbranched polypeptide
alpha 1,4- glycosidic branch
Amylose
Branched polypeptide
Alpha 1,6-glycosidic branch
Amylopectin
Amylase determination methods
1 amyloclastic method
a viscosimetric
b turbidimetric
c iodometric
d nephelometric
2 saccharogenic method
3 chromogenic method
4 fluorogenic method
5 fluorescence depolarization method
6 radiometric method
7 immunologic method
8 other enzymatic method/ continous monitoring assays
a use of alpha-glucosidase-hexokinase-G6PD system
b use of 5 ethylidene-4-nitrophenyl glycosides as substrate
c 2-chloro-p-nitrophenyl-alpha-D maltrotriose
Amylase is detemined by the formula:
mean abs difference/ min x (?)
4824
Normal values of amylase
Serum: up to 96 IU/L
Urine 18 - 330 IU/L
Sensitivity of amylase
5 IU/L
Activators of Amylase
Calcium and chloride ions