Estimation of alkaline phosphate

Estimation of alkaline phosphate

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  Presentation on Estimation ofAlkaline Phosphatase in Serum Presented by: Dr. Monika Jangid, Assistant Professor, Dept. of Chemistry , Pacific University, Udaipur
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  Description Why to bedone Normal and Abnormal values Symptoms How to be done Applied method Application
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   Alkaline phosphatase(ALP, ALKP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides proteins and alkaloids.  The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment.  It is sometimes used synonymously as basic phosphatase.
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   Medical testingof Alkaline Phosphatases is concerned with a group of enzymes found primarily the liver (isoenzyme ALP-1) and bone (isoenzyme ALP-2).  Small amounts are also produced by cells lining the intestines (isoenzyme ALP-3), the placenta, and the kidney (in the proximal convoluted tubules).  Since the mucosal cells that line the bile system of the liver are the source of alkaline phosphatase, the free flow of bile through the liver and down into the biliary tract and gallbladder are responsible for maintaining the proper level of this enzyme in the blood.  When the liver, bile ducts or gallbladder system are not functioning properly or are blocked, this enzyme is not excreted through the bile and alkaline phosphatase is released into the blood stream.
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   The optimalrange for alkaline phosphatase depends on age. Children will have a much higher alkaline phosphatase than a full grown adult because his/her osteoblasts are laying down bone very rapidly. The normal range for serum ALP level is 20 to 140 IU/L, but this can vary from laboratory to laboratory. The normal range runs higher in children and decreases with age. Abnormal Level- Increased serum Alkaline Phosphatase causes Congestion or obstruction of the biliary tract Paget's Disease, Hyperthyroidism, Rickets-Vitamin D deficiency.  Decreased serum alkaline phosphatase causes Zinc deficiency, Hypothyroidism, Vitamin C deficiency/Scurvy, Folic acid deficiency. Excess Vitamin D intake, Low phosphorus levels(hypophosphatasia).
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   Signs andSymptoms of liver involvement may include:  Weakness, fatigue  Loss of appetite  Nausea, vomiting  Abdominal swelling and/or pain  Jaundice  Dark urine, light colored stool  Itching  Some examples of the signs and symptoms suggesting a bone disorder include:  Bone and/or joint pain  Increased frequency of fractures  Deformed bones
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   Alkaline phosphataseis measured by combining the person's serum with specific substances with which alkaline phosphatase is known to react. The end product of this reaction is measured and from that measurement, the amount of alkaline phosphatase in the person's serum is determined.  It is done in the pathology lab by kit method.  One of the method is kind and king’s method to estimate this in the lab.
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  Principle  ALP atan alkaline pH hydrolyses disodium Phenylphosphate to form phenol. The Phenol formed reacts with 4-Aminoantipyrine in the presence of Potassium Ferricyanide, as an oxidising agent, to form a red coloured complex. The intensity of the colour formed is directly proportional to the activity of ALP present in the sample.  Reaction
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  Reagents: • Reagent 1:Buffered substrate, pH 10 , Reagent 2: Chromogen reagent, Reagent 3: Phenol substrate • We have taken two serum samples for this. One is of 21 years and another is of 34 years. Reagents Blank Standard Control Test Working buffer substrate 0.5 0.5 0.5 0.5 Distilled water 1.5 1.5 1.5 1.5 Mix well and incubate at 37 0C for 3 mins. Pipetting chart To be continue ...
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  Serum - -- 0.05 phenol substrate - - 0.05 - After that: Mix well and incubate at 370 C for 15 mins. Chromogen reagent 1.0 1.0 1.0 1.0 Serum - - 0.05 - Mix well and measure the Optimum Density against purified water at 520 nm. To be continue ...
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  Absorbance at 520nm O.D. of Blank - ……………………….. O.D. of STD - ………………………… O.D. of Test - ……………………........ Calculation: ALP activity (IU/L)= Result: Interpretation: O.D. of Test- O.D. of Blank O.D. of STD- O.D. of Blank X concentrate STD To be continue ...
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   Alkaline phosphatasehas become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA from ligating there by keeping DNA molecules linear until the next step of the process for which they are being prepared.  Removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA.  Undifferentiated pluripotent stem cells have elevated levels of alkaline phosphatase on their cell membrane, therefore alkaline phosphatase staining is used to detect these cells and to test pluripotency (i.e. embryonic stem cells or embryonal carcinoma cells) .