Alkaline Phosphatase

Question 1

ALP Systematic name

Answer 1

E.C. 3.1.3.1

Question 2

Hydrolyzes a large variety of naturally occuring synthetic substrates at an alkaline pH

Answer 2

Alkaline Phosphate (ALP)

Question 3

Exact functions of ALP is well-known (T/F)

Answer 3

False.

Question 4

What is ALP somewhat important for?

Answer 4

Lipid transfer in the intestines, and bone formation

Question 5

Optimum Alkaline Phosphate pH

Answer 5

10.3

Question 6

Major ALP isoenzymes

Answer 6

Bone, Liver, Intestines, Placental Isoenzymes

Question 7

ALP will only take place in the presence of co-factors:

Answer 7

Disulfide ions: Co2+, Mg, Mn, Zinc

Question 8

Constituent metal ion that MUST combine w/ ALP for a RXN to take place

Answer 8

Zinc

Question 9

ALP is incomplete without it

Answer 9

Zinc

Question 10

ALT inhibitors

Answer 10

1. Phosphates 2. Borate 3. Oxalate 4. Cyanide Ions

Question 11

These are anticoagulants with borate & oxalate that may never be used when measuring ALP

Answer 11

Borate, Oxalate

Question 12

This measures the rate of p-NPP rxn

Answer 12

Bowers & McComb

Question 13

p-NPP is measured at:

Answer 13

405nm at 10.3pH

Question 14

This will falsely lowers activity; they will falsely bind to co-factors/cations

Answer 14

Chelators

Question 15

ALP Samples should be tested as soon as possible, but incase of delay how long should it be before the test should proceed

Answer 15

Within 4Hrs after collection

Question 16

The reason for enzyme activity increase slightly in storage

Answer 16

This is due to loss of inhibition (especially when refrigerated)

Question 17

How much enzyme activity is lost per day

Answer 17

2%

Question 18

ALP is relatively stable at what temp

Answer 18

4C for up to a week

Question 19

What do you do with frozen samples before testing

Answer 19

Thaw and leave at RT for 18-24hrs before testing

Question 20

Separation of Isoenzymes

Answer 20

1. Inhibition 2. Heat Fractionation (measurement before and after heating) 3. Electrophoresis 4. Use of enzymes or lectins

Question 21

Substrates that cause inhibition in the separation of isoenzymes

Answer 21

Phenylalanine, Levamisole

Question 22

Phenylalanine inhibits

Answer 22

Intestinal, Placental Isoenzymes

Question 23

Levamisole inhibits

Answer 23

Bone and Live Isoenzymes

Question 24

This measures based on heat stability (separation)

Answer 24

Heat fractionation

Question 25

How is heat fractionation measured

Answer 25

Measure isoenzyme, apply heat, measure once more; Exposes serum @ 56C for 10mins, measure base concentration of ACP and then apply heat, measure again and if value is high then isoenzyme causing increase in concentration is heat stable (VV)

Question 26

Most heat stable to most heat labile isoenzymes

Answer 26

Placental, Intestinal, Liver, Bone Isoenzyme

Question 27

Electrophoresis gel being used:

Answer 27

Agarose gel, polyacrylamide gel, and cellulose acetate

Question 28

This maintains the pH at 10.3

Answer 28

Buffer

Question 29

Electrophoresis measurement:

Answer 29

Gel is taken and incubated into a buffer, and overime you will see bands form in the support media

Question 30

Fastest to slowest isoenzyme in electrophoresis

Answer 30

Liver, Bone, Placental, Intestinal

Question 31

Electrophoresis visualization, liver and bone bands are stuck together, and placental band overlaps the bone isoenzyme. How do you mitigate this:

Answer 31

Expose sample to neuramidase rgt (pre-treatment for 15mins for 37C) before electrophoresis

Question 32

What does neuramidase do

Answer 32

Slows down other isoenzymes except the liver isoenzyme causing separation of bands

Question 33

ALP working rgt measurement

Answer 33

1mL or 1000uL

Question 34

Serum measurement

Answer 34

20uL

Question 35

ALP standard value

Answer 35

2757U/L

Question 36

Conversion factor to Ukat

Answer 36

0.0167

Question 37

Expected values

Answer 37

Males: 35-104 U/L (0.58-1.74 ukat/L) Females: 40-129 U/L (0.67 - 2.15 ukat/L)

Question 38

This reaction catalyzes the hydrolysis of p-nitrophenylphosphate to release p-nitrophenol under alkaline conditions read at 405nm

Answer 38

Alkaline Phosphatase (ALP)

Question 39

Present method of ALP is made according to who?

Answer 39

IFCC

Question 40

Reagent Composition

Answer 40

2-amino-2-methyl-1-propanol buffer (10.4), Magnesium acetate, Zinc sulfate, HEDTA, p-nitrophenylphosphate

Question 41

Specimen stability for ALP

Answer 41

Serum kept at RT usually show a slight increase in activity; varies from 1% over 6hr period, to 3-6% over 1-4 day period; Sera stored in Ref Temp increases slowly; Frozen sera, activity decreases slowly but slowly recovers after thawing the serum