alkaline phosphatase estimation
TRANSCRIPT
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FUNDAMENTALS OF DIAGNOSTIC ENZYMOLOGY
Dr. Gangadhar ChatterjeeMBBS;MD
Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India
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FUNDAMENTALS OF ENZYME ESTIMATION
You are NOT measuring the enzyme molecule or its concentration like sugar or urea……….
Because- Enzymes are biocatalysts……and catalysts in reaction remains in VERY MINUTE quantity……….even less than picogram per liter………making it almost impossible to measure directly.
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FUNDAMENTALS OF ENZYME ESTIMATION
- enzymes, being very delicate molecule and extreme minute presence in body fluids, it is very difficult to extract it in pure form to prepare STANDARDS. ( without which it’s NOT possible to estimate)
………………………..Hence ESTIMATION OF ENZYME is a WRONG term…..as we are not doing that.
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then …………what's the possible way out???????
We can measure the SUBSTRATE or PRODUCT of an enzyme appreciably.
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So what actually we can do………..
• We can look for the enzyme ACTIVITY in terms of decreasing SUBSTRATE or an increasing PRODUCT of certain enzyme mediated reaction.
…………….So actually we are estimating the ENZYMATIC ACTIVITY.
*******right term is Estimation of ABC enzyme ACTIVITY in XYZ sample*******
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Control tube
Used to nullify effect of the INDIGENOUS (S/P) molecule in the final colour in the TEST tube………………………………………………………………………………………………………………………………………………………………………………….as the colour produced by the indigenously present molecule is NOT evolved due to enzyme action……………………………………………………………………………………………………………………….NOTE some modification in the equation for this CONTROL TUBE.
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Some facts about ALP- Alkaline phosphatase ( EC 3.1.3.1; orthophosphoric
monoester phosphohydrolase) catalyzes the alkaline hydrolysis of organic phosphates.
- Zn2+ is a constituent metal ion of this enzyme…………………Metalozyme
- Mn2+ , Co2+ , Mg2+ are activator of this enzyme.- Phosphate, Borate, Oxalate and Cyanide are inhibitor
of this enzyme.
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PRINCIPLE OF THE METHOD
Alkaline phosphatase (ALP) catalyzes the hydrolysis of p-nitrophenyl phosphate at pH 10.4, liberating p-nitrophenol and phosphate, according to the following reaction:
ALPp-Nitrophenylphosphate + H20 p-Nitrophenol + Phosphate
The rate of p-Nitrophenol formation, measured photometrically, is proportional to the catalytic concentration of alkaline phosphatase present in the sample
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Isoenzymes of ALP
Hepatic, bone, intestinal and placental are major subtypes of ALP isoenzymes.
On electrophoresis, Liver form is fastest moving. Bone and intestinal forms give a diffuse large band, little slow moving than liver form.