Lab # 9 224 PHL.  Phosphatases are enzymes which catalyze the splitting of phosphoric acid from mono-phosphate esters.  They are hydrolases.  Organic.

Slides:



Advertisements
Similar presentations
ALT/sGPT activity xiaoli.
Advertisements

Faculty of nursing CHEM 203 Biochemistry UNIT VIII Diagnostic Enzymes Dr.Ola Fouad Talkhan.
Determination of plasma enzymes using the clinical analyzer
Factors that affect cellobiase
TRACE METALS - FROM DEFICIENCY TO TOXICITY Quest – July 22, 2004 Yeala Shaked, Yan Xu and Francois Morel, Geosciences Dept, Ecology and Evolutionary Biology.
AST, ALT & ALP Lab. 5.
1 EXPERIMENT THREE ASSAY OF ACTIVITY OF ALKALINE PHOSPHATASE
Dr. Samah Kotb Nasr Eldeen.  1. Total and conjugated bilirubin  2. ALT  3. AST  4. Total protein  5. GGT  6. ALP  7. 5′-nucleotidase  8. Albumin.
Assay the Activity of Creatine Kinase (CK)-total in Serum Dept.of Biochemistry.
Estimation of serum bilirubin (total and direct)
Enzymes Lab Section 2.4 Enzymes Protein catalysts Have complex 3-D structures Pockets act as active sites –catalyze specific chemical reactions E + S.
1 Assay the Activity of Alkaline Phosphatase (ALP) in Serum (Disodium phenyl phosphate Method)
Enzymes Part 2 M. Zaharna Clin. Chem
DESCRIPTION  Alkaline phosphatase (ALP) (EC ) catalyzes the hydrolysis of phosphate esters in an alkaline environment, resulting in the formation.
CLINICAL ENZYMOLOGY.
Liver function tests Lecture 3.
Quantitative Determenation of Alanine aminotransferase (ALT) CLS 431.
Clinical significance & Methods
BCH302 [Practical] 1. There are three main methods of estimation the reducing sugar content in solution : 1. Reduction of cupric to cuprous salts. 2.
Determination of Alkaline Phosphatase activity
Experiment 8: Assay The Activity of ALT in Serum.
Serum biochemical parameters (ALT) (AST) assay Biochemistry Clinical practice CLS 432 Dr. Samah Kotb Lecturer of Biochemistry 2015.
Methods of Enzyme Assay
BIOCHEMISTRY 285 PHL Introduction Blood Glucose. Blood Blood is vascular tissue that circulates in the closed system of blood vessels Functions: TransportationTransportation.
Enzyme Clinical Application
Measuring the Enzyme Function of Alkaline Phosphatase What is the effect of substrate concentration on alkaline phosphatase activity?
224 PHL Lab#5. Non-protein nitrogen (NPN) NPN includes the nitrogen from all nitrogenous substances other than proteins. The NPN could be measured as.
Cellular Biochemistry and Metabolism (CLS 333 ) Dr. Samah Kotb Nasr Eldeen Serum biochemical parameters (ALT) (AST) assay.
Differential Diagnosis of Alkaline Phosphatase B 陳建佑.
CLINICAL ENZYMOLOGY. Measurements of the activity of enzymes in plasma are of value in the diagnosis and management of a wide variety of diseases.
Enzymes AST, ALT & ALP Lab. 6.
Manual Extraction of DNA from The Blood. - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket.- Materials.
Biochemistry Clinical practice Lecturer of Biochemistry
Manual Extraction of DNA from The Blood. Materials - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket.-
Phosphatases Lab # 7.
Lab2 Lactate Dehydrogenase Quantitative determination of lactate dehydrogenase LDH Daheeya AlEnazi.
The effect of temperature on the rate of an enzyme catalyzed reaction. The effect of temperature on the rate of an enzyme catalyzed reaction. Amani Alghamdi.
Lab 3 ALT and AST Daheeya AlEnazi.
285 PHL Lab # 2 Plasma Proteins. Proteins: Classification Proteins: Classification 1- Simple proteins e.g. albumin and globulin 2- Conjugated proteins.
Lab 5 Acid Phosphatase(ACP) Quantitaive determination of acid phosphatase (Total, Prostatic and Non-prostatic) in serum by a- naphthylphosphate colorimetric.
Lab # 2 Liver Function Tests (LFTs) ALT&AST T.A. Bahiya M. Osrah.
Khadija Balubaid KAU-Faculty of Science- Biochemistry department Clinical biochemistry lab (BIOC 416) 2013 Liver Function profile (LFT) Enzymes.
Determination of plasma enzymes
Clinical usage of enzymes
Lab (2): Liver Function profile (LFT)
THE ISOENZYME PROFILE OF LACTATE DEHYDROGENASE Assaying the serum levels of lactate dehydrogenase (LDH) activity combined with the results of other clinically.
 Assaying the serum levels of lactate dehydrogenase (LDH) activity combined with the results of other  Clinically important enzyme assays (GOT, SGOT,
Determination Of Albumin In Serum By Bromocresol Green Method
Methods of Enzyme Assay
The effect of temperature on the rate of an enzyme catalyzed reaction
Lab (3): Liver Function profile (LFT)
(USING TURBIDIMETRIC METHOD)
Lab 4 Alkaline Phosphatase(alp)
ALKALINE PHOSPHATASE This is a widely distributed enzyme which releases inorganic phosphate from many organic phosphomonoesters and also pyrophosphates.
Lab (2): Liver Function profile (LFT)
Measuring the rate of the reaction
322 BCH Method of Enzyme Assay.
Practical Analysis Using Spectrophotometer
Estimation of Serum Bilirubin (Total & Direct)
LAB 8 LIPASE Daheeya ALEnazi
In this experiment, we will continue to study acid phosphatase kinetics.
Lab2 Lactate Dehydrogenase
QUANTITATIVE DETERMENATION OF GAMMA-GLUTAMYL TRANSFERASE ( ϒ-GT)
Determination of plasma enzymes
Measuring the rate of the reaction
Part 1 Liver enzyme(ALT-AST-ALP-GGT)
Quantitative determination of Acid Phosphatase (ACP)
Determination of plasma enzymes
Bilirubin.
Presentation transcript:

Lab # PHL

 Phosphatases are enzymes which catalyze the splitting of phosphoric acid from mono-phosphate esters.  They are hydrolases.  Organic phosphate esters + water alcohol + phosphate ion Two types are commonly estimated in the serum :  Alkaline phosphatase with maximum activity at pH10.  Acid phosphatase with maximum activity at pH5.

Occurrence: in most tissues of the body, mainly in: ▪ Osteoblasts in bone. ▪ Bile canaliculi in liver. ▪ Small intestinal epithelium. ▪ Proximal tubules of kidney. ▪ Breasts during lactation.  In all these sites ALP seems to be involved in transport of phosphates across cell membranes.

 ALP is activated by Mg +2, Mn +2,Co +2.  ALP is inactivated by Zn +2,Cu +2,Hg +2,EDTA.

P-nitrophenylphosphate + H2O ALP Phosphate + p-nitrophenol.

Pipette in to clean dry test tube 1 ml from working reagent Put the test tube in the 37⁰C water bath “beaker” Add 0.2 ml from the sample Mix and put in the cuvette, and wait for 30 second Record the absorbance at λ= 405 nm every 1 minute interval for 3 minutes The absorbance of the sample: A ⁰, A 1, A 2, and A 3.

Calculation: The ALP activity (U/L) = (∆ A / min.) X 2720 = (A ⁰ - A 1 )+ (A 1 - A 2 )+ (A 2 - A 3 )/3 X 2720 Normal value: 98 – 279 U/L

Increase in ALP occurs mainly in: 1) Bone diseases like Paget’s disease (highest level), rickets, hyperparathyroidism, bone cancer. 2) Liver diseases like obstructive jaundice, biliary cirrhosis, carcinoma liver abscess. 3) Drugs producing cholestasis like androgens, sulfonamides. or hepatotoxic drugs like aspirin, gentamycin, cyclophosphamide, and halothane. Decrease in ALP occurs in: anemia, scurvy, and cretinism.

Occurrence: The highest concentration of ACP is found in prostate (prostatic ACP), also in RBCs, leucocytes and platelets (non prostatic ACP).  ACP has a maximum activity at pH5.6

A variety of substrates have been used for determination of serum ACP activity. These include:  Nitrophenylphosphate-attacked by phosphatases of non-prostatic origin.  Β-Glycerophosphate, α naphthylphosphate, phenolphthalein monophosphate are all non specific substrates for both. Prostatic Acid Phosphatase is obtained by subtracting the results of the Non-Prostatic Acid Phosphatase assay from the results of the Total Acid Phosphatase assay on the same sample.

Principle:(ACP) 1-naphthyl phosphate + H2O ACP Phosphate + 1- naphthol 1-naphthol + 4-chloro-2-methylphenyldiazonium salt Azo dye.

a)Determination of Serum Total Acid phosphatase Activity. b)Determination of Serum Non- Prostatic Acid phosphatase Activity

Pipette in to clean dry test tube 1 ml from working reagent (R2A) Put the test tube in the 37⁰C water bath “beaker” Add 0.1 ml from the sample, Mix and incubate for 5 minute at water bath Put in the cuvette Record the absorbance at λ= 405 nm every 1 minute interval for 3 minutes The absorbance of the sample: A ⁰, A 1, A 2, and A 3.

Pipette in to clean dry test tube 1 ml from working reagent (R2B) Put the test tube in the 37⁰C water bath “beaker” Add 0.1 ml from the sample, Mix and incubate for 5 minute at water bath Put in the cuvette Record the absorbance at λ= 405 nm every 1 minute interval for 3 minutes The absorbance of the sample: A ⁰, A 1, A 2, and A 3.

Calculation:  Total ACP activity (U/L) = (∆ A / min.) X 743 = (A ⁰ - A 1 )+ (A 1 - A 2 )+ (A 2 - A 3 )/3 X 743  Non-prostatic ACP activity (U/L) = (∆ A / min.) X 743 = (A ⁰ - A 1 )+ (A 1 - A 2 )+ (A 2 - A 3 )/3 X 743  Prostatic ACP activity (U/L) = Total ACP activity - Non-prostatic ACP activity.

Normal value: Total ACP Up to 4.7 U/L. Prostatic ACP Up to 1.6 U/L.

Prostatic ACP is used mostly to detect prostatic carcinoma when it may reach very high level. In benign hypertrophy of prostate ACP is normal.

POP QUIZ