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CC LAB EXPE 8 Flashcards

1
Q

Amylase was first measured quantitatively by an _____ introduced by ______

A

Iodometric method; by Wohlegemuth

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2
Q

Who introduced a procedure in 1938 that standardized the amounts of starch and iodine?

A

Somogyi

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3
Q

His work became the basis for the widely used amyloclastic and saccharogenic methods introduced in 1956 and 1960

A

Somogyi

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4
Q

What are the disadvantages of the methods used by wohlegemuth and Somogyi?

A

Long incubation times, endogenous glucose interference, and unstable reaction colors resulting in poor reproducibility and reliability

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5
Q

Wallenfels et Al introduced____ as defined substrates for amylase determination in a procedure that eliminated interference from endogenous glucose and pyruvate

A

P-nitrophenylglycosides

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6
Q

The present procedure is based on modifications of Wallenfels, using a substrate ________ with the terminal glucose blocked to reduce spontaneous degradation of the substrate by ____ and _____

A

P-nitrophenyl- D-maltoheptaoside (PNPG7); glucosidase and glucoamylase

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7
Q

The determination of amylase activity in serum and and urine is most commonly performed for the diagnosis of _____

A

Acute pancreatitis

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8
Q

True or False: in acute pancreatitis, amylase levels are elevated for longer periods of time in serum than in urine

A

False: in acute pancreatitis, amylase levels are elevated for longer periods of time in urine than in serum

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9
Q

_____ hydrolyzes PNPG7 to PNPG3 and _____

A

Amylase; Maltotetraose

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10
Q

____ hydroxides PNPG3 to PNPG1 and glucose

A

Glucoamylase

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11
Q

___ hydrolyzed to glucosidase to glucose and p-nitrophenol, which produces a _____ color

A

PNPG1; Yellow color

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12
Q

The rate of increase in absorbance of p-nitrophenol is measured at _____ and is ____ to the amylase activity in the sample

A

405nm; directly proportional

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13
Q

PNPG3 +____

A

Matlotetraose

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14
Q

PNPG1 +____

A

Glucose

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15
Q

P-nitrophenol + ____

A

Glucose

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16
Q

Assay for amylase involves the determination of both ______ and the _____

A

Salivary amylase and pancreatic amylase

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17
Q

This method measure the breakdown of starch substrate

A

Amyloclastic method

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18
Q

What are under amyloclastic methods

A

Viscosimetric
Iodometric
Turbidimetric
Nephelometric

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19
Q

An instrument used to measure the change in flow rate of starch

A

Viscosimeter

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20
Q

This procedure involves the hydrolysis of starch that results in the disruption of its molecular structure with reduction of viscosity

A

Viscosimetric

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21
Q

This method are very inaccurate, highly dependent on the nature of starch substrate and unsuitable for the measurement of low amylase activities

A

Viscosimetric

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22
Q

This procedure measure the decrease in absorbance of a turbid starch substrate due to reduction in starch granule size as a result of amylase activity.

A

Turbidimetric

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23
Q

These methods are simple and rapid, but poor in precision at near normal activity of amylase and inadequate substrate stability.

A

Turbidimetric

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24
Q

True or False: the Turbidimetric change is linearly related to amylase activity, except perhaps in the first two minutes of the reaction

A

The Turbidimetric change is NOT linearly related to amylase activity, except perhaps in the first two minutes of the reaction

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25
What procedure involves; The endpoint is indicated by the absence of the blue-starch iodine color
Iodometric
26
Only___ gives a blue color with iodine, ____ forms a weak red color
Amylose; Amylopectin
27
The characteristic blue color is due to the helical configuration which amylose forms with iodine; each turn of helix contains ___ glucose units and surrounds __ molecule of iodine
6 glucose units and 1molecule of iodine
28
The blue color is obtained only when the overall chain length of amylose is greater than ____
45 glucose units
29
What are the disadvantages of Iodometric Methods
-only the hydrolysis of the amylose portion of the starch is being measured yet amylase has greater activity in hydrolyzing amylopectin - starches of different origin vary in their proportions of amylose and amylopectin - amylase does not act under the substrate saturation conditions - interference from proteins, particularly albumin, and from lipids
30
___ based on reduction of light-scattering of starch substrate by amylase activity
Nephelometric assays
31
What are the advantages of Nephelometric Approach
Simplicity, Speed, Precision, and Zero Order kinetics
32
It is claimed to be suitable for emergency purposes, results being available in less than 3 minutes with direct read-out, bench working time and manipulation are minimal, sample blanking is NOT required
Nephelometric
33
In this method, a starch bound to a dye is hydrolyzed by amylase with the release of dye-substrate fragments which are water soluble
Chromogenic Methods
34
This method is based on the hydrolysis of starch by amylase to produce carbohydrates that have reducing properties
Saccharogenic Method
35
To convert Somogyi units to IU, multiply values by _____
1.85
36
This involves the measurement of amylase activity by HLPC with fluorescence detection, using a fluorogenic substrate
Fluorogenic methods
37
HPLC product analysis revealed a treated production of substituted oligosaccharide by ____ than by ____, and this observation was used as a the basis of a differential assay for these isoamylases in ____
Pancreatic; salivary amylase ; Human serum
38
This method involves the usage of fluorescein-labeled amylose
Fluorescence Depolarization Method
39
Fluorescence Depolarization Method: the degree of polarization of light emitted from this fluorophore when excited by a polarized beam of light is ____ by amylase ____
Decreased; hydrolysis
40
True or False: (Fluorescence Depolarization Method) : The smaller molecules formed are less polarized because they rotate faster than the intact substrate
True
41
This method is the basis of the Abbott TDX Amylase assay and this method provides a simple and sensitive direct assay but requires special instrumentation
Fluorescence Depolarization Method
42
This method is for measuring amylase activity using radio labeled starch substrate
Radiometric Method
43
Amylase Hydrolysis of the radio-labeled substrate releases ____ which is ___ proportional to amylase activity
Free radioisotope; proportional
44
This method involves immunoassay using polyclonal antibodies produced against salivary or pancreatic isoamylase as antigen
Immunologic Method
45
Maltopentose ——> maltotriose + maltose
Amylase
46
Maltotriose + maltose ——> glucose
Alpha glucosidase
47
Glucose + ATP ——-> glucose-6-phosphate + ADP
Hexokinase
48
Glucose-6-phosphate + NAD ——-> 6-phosphogluconolactone + NADH
G6PD
49
The IFCC has optimized this method at 37C, recommending it as a reference method for amylase determination
5 ethylidene- 4- nitrophenyl glycosides as subtrates
50
This involves the use of 2-chloro-p-nitrophenol as an indicator
Use of 2-chloro-p-nitrophenyl- alpha-D-maltotrioside as substrate
51
Disadvantages of 2-chloro-p-nitrophenyl-D-alpha-matlotrioside as a subtracted
Slow reaction rate Variation in molar absorptivity of chloro-p-nitrophenol associated with changes in pH Temperature Protein content Presence of potassium thiocyanate = allosteric changes to amylase and precluding the use of antibodies for p-amylase determination
52
True or false: In amylase determination, heparinized plasma can also be used
True
53
Anticoagulants such as ______, ______, and _____ should NOT be used as these bind to calcium (an ion needed for amylase activity) , this May lower amylase activity (EDTA by 10%)
Citrate EDTA Oxalate
54
Urine specimens should be collected for a ____, adjusted to a pH of ___ with either 0.1N NaOH or 0.1 HCl and kept refrigerated until assayed
24 hour period, ph 7
55
Amylase in serum and urine is reported stable for ____ at ____ and for several months when stored at _____ degrees Celsius and protected against evaporation and bacterial contamination.
1 week at RT; 2-8C
56
Stability may decrease in urine samples with ph less than ____
5
57
What causes the increase in pancreatic amylase in serum?
Macroamylasemia Lipemia Bilirubin concentration greater than 20 mg/dL (falsely increase) Insulin and some bacteria Administration of morphine and opiates (falsely increase)
58
Contamination of sample with ___ and ____ should be avoided as these contain alpha-amylase
Sweat and saliva
59
What is the linearity of the method?
1,500 IU/L
60
What are the isoenzymes of amylase?
Pancreatic and Salivary fractions
61
What methods can be used to differentiate the isoenzymes of amylase?
Electrophoresis Ion exchange chromatography Isoelectric focusing Selective inhibition of S-AMY by a wheat germ inhibitor Immuno precipitation by a monoclononal antibody Immune inhibition
62
Only methods based on selective isoenzyme inhibition by monoloclonal antibodies have shown sufficient——-
Precision Reliability Practicability Analytical speed to allow the introduction of P-AMY determination into a clinical practice
63
WHAT IS ACCR?
Amylase-creatinine clearance ratio
64
It was established as an index for screening of acute pancreatitis
ACCR (Amylase-Creatinine Clearance Ratio)
65
ACCR is increased in cases of _______________
Diabetes keto acidosis Renal insufficiency Severe burns
66
Reference range of ACCR?
1-5%
67
Reagent composition of AMYLASE DETERMINATION
P-nitrophenyl-D-Maltoheptaoside Glucosidase-yeast Glucoamylase- rhizopus spp. Sodium chloride Calcium chloride Buffer PH
68
P-nitrophenyl- D- Maltoheptaoside (____)
0.9 mM
69
Glucosidase- yeast (_____)
25, 000 IU/L
70
Glucoamylase- rhizopus spp (___)
10,000 IU/L
71
Sodium chloride [____]
50mM
72
Calcium chloride (_____)
5 mM
73
Buffer (___)
50 mM
74
Ph [______]
6.9
75
TV=
1.025
76
MMA=
8.5
77
SV=
0.025 mL
78
Multiplication factor of AMYLASE
4824
79
Linearity of amylase
1,500 IU/L
80
Expected values in serum
Up to 96 IU/L
81
Expected values in urine
18-330 IU/L

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