Alkaline phosphate

Moderator
dr.sahr Iqbal ..

1. Alkaline Phosphate Dr.Ghulam Murtaza Resident Chemical Pathology DIMC

2. Behind the name : ALP functions best under alkaline pH environments and has the physiological role of dephosphorylating compounds A phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol Half live 3-7 days • Physiologic increase occurs at growing age

3. Distribution in the body : enzyme present in the blood, and its subtypes are present in the liver, intestine, bones, and placenta Liver and bone ALP are predominantly more in the serum

4. Alkaline phosphatase isoenzymes in Humans? All isoenzyme & isoforms are glycoprotein Bone ,liver , kidney forms share a common primary structure coded for same genetic locus ,but differ in carbohydrates content .

5. Liver ALP Bone ALP Renal ALP Intestinal ALP Placental ALP moderately heat-stable Blood group B or O   heat stable 50%  inactivated by heat  heat inactivates 90% of the bone isoenzymes increase ALP after 2 hours of the meal. Secreted by epithelial cells of the biliary tract Secreted by renal tubular cell ∼25% of the normal sera. placental in origin. (16th to 20th week of gestation) Excreted in urine A fasting  ALP level is indicated

6. Activator of enzymes Inhibitor of enzymes

7. Clinical significance Any obstruction in biliary tree induce the synthesis of ALP by hepatocytes, newly formed ( ecto enzymes ) is released from cell membrane by the action of bile salts & enter the circulation to increase the enzymatic activity in serum, ( > 4 URL ).

8. Sample collection Complexing anticoagulant such as citrate, oxalate ,& EDTA must be avoided because they bind cation such as Mg² & Zn² which act as co factor for ALP activity measurement . freshly collected sample should be done with in 4 hours . Preferred:  Serum gel tube Red-top tubes should be centrifuged and aliquoted within 2 hours of collection

9. Methods ; Alkaline phosphatase method is based on a procedure published by Bowers & McCombs & more recently received by Rej Endorsed by the international federation of clinical chemistry This assay respond to all ALP isoenzymes in human serum required temperature 37 C .

10. Principle of procedure : Alkaline phosphatase catalyzes the transphosphorylation of p-nitro phenyl phosphate (p-NPP) to p-nitrophenol in the presence of transphosphorylating buffer , 2 amino-2-methyl-1 –propanol (AMP) . The reaction is enhanced through the use of Mg² to Zn² ions The change in absorbance at 410nm/ 478 (bi chromatic ) due to formation of p-ANP is directly proportional to alkaline phosphatase concentration HEDTA hydro oxyethylene diamine triacetic acid act as metallic ion buffer maintain concentration of both ions Mg² to Zn²

11. Isomerization of alkaline phosphate differ in the number of Sialic acid residues (charged group ) attached to enzymes – Post transcription modification Position of alkaline phosphatase in electrophoresis Location Alpha 2 Liver Gamma Intestine Pre beta Bone ( heat labile ) Pre beta Placenta

12. Comparison of isoenzymes of alkaline phosphatase S.No Isoenzymes of ALP Occurrence % of total ALP in serum Clinical signific 1 ALPHA ONE LIVER 10 OBSTRUCTIVE JAUNDICE , HEPATOMA 2 ALPHA 2 ( HEAT LABILE ) – DENATURED AT 65 C ( 30 MINTS ) LIVER 20 HEPATITIS 3 ALPHA 2 ( HEAT STABLE ) PLACENTA 10 LUNG /LIVER/GIT CARCINOMA 4 PRE BETA (HEAT LABILE ) BONE 50 BONE CARCINOMA 5 GAMMA INTESTINAL 10 ULCERATIVE COLITIS

13. ANODE – THE LIVER ALP TYPICALLY MOVES TOWARDS ANODE BONE ALP HAS SLIGHLY REDUCED ANODAL MOVEMENT SO GIVES DIFFUSE ZONE PLACENTAL ISOENZYME COMMONLY APPEARS AS DISCRETE BAND INTESTINAL ALP MIGRATES MORE SLOWLY THAN BONE PRETREATMENT APPROACH IS BIENG USED TO DIFFERENTIATE BETWEEN BONE & LIVER ALP

14. Why Blood levels of alkaline phosphatase increase two to four times during pregnancy ? Additional alkaline phosphatase produced by the placenta