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Lab#2 BCH 471 Determination of Aspartate Aminotransferase(AST) in serum
Objectives -To evaluate the presence of tissue damage, By estimating the level of AST in serum.
Blood Enzymes: Plasma, serum or blood proteins, are proteins present in blood plasma which have several functions. Some blood proteins also act as enzymes. Enzymes are .? Clinical enzymology refers to measurement of enzyme activity in body fluids for the diagnosis and treatment of diseases. Most clinical enzyme measurements using serum or plasma, occasionally other fluids, such as urine and gut secretions are also investigated.
PlasmaEnzymes Functional Plasma Enzymes (plasma derived enzymes) Nonfunctional Plasma Enzymes (Cell derived enzymes) They perform no physiological function in the plasma and mostly arise from destruction of RBCs, Tissue damage caused by injury or disease can cause marked increase in their level, so measurement of these enzymes level can help in diagnosis and prognosis of diseases, depending on their tissue origin. Enzymes that are always present in the circulation and preform a physiological function in the blood.
PlasmaEnzymes 1- Functional Plasma Enzymes (plasma derived enzymes): Enzymes that are always present in the circulation and preform physiological function in the blood. 2- Nonfunctional Plasma Enzymes (Cell derived enzymes): they perform no physiological function in the plasma and mostly arise from destruction of RBCs, Tissue damage caused by injury or disease. - can cause marked increase in their level, so measurement of these enzymes level can help in diagnosis and prognosis of diseases, depending on their tissue origin.
Differences of Functional and Nonfunctional plasmaenzymes Functional plasma enzymes Non-functional plasmaenzymes Always present in the blood Absent from the blood (or present in very small amount). Different organs e.g. liver, heart, muscles, and brain Their substrate Site of synthesis synthesized and secreted by liver. Effect of diseases Decrease in liver diseases Different enzymes increase in different organ diseases - ALT - AST - LDH -Acid Phosphatase -Amylase Examples - Clotting factors - Lipoprotein - Lipase
Sources of Non-functional Plasma Enzyme Cell damage with the release of its content of enzymes into blood e.g. Myocardial infarction and viral hepatitis. Obstruction of normal pathways e.g. Obstruction of bile duct increases alkaline phosphatase. Increase of the enzyme synthesis e.g. In response to certain stimuli or damage, the body may increase the synthesis of specific enzymes. Increased permeability of cell membrane as in hypoxia
Medical Importance of Non Functional Plasma Enzymes Measurement of nonfunctional enzymes is important for: Diagnosis of diseases. Prognosis of the disease: following up of the treatment by measuring plasma enzymes before and after treatment.
Aspartate Aminotransferase(AST) AST, is an enzyme that catalyzes a type of reaction (transamination). Serum aspartate aminotransferase (AST) ,is a tissue enzyme that catalyzes the exchange of amino and keto groups between alpha-amino acids and alpha-keto acids. AST, is widely distributed in tissues, principally cardiac, hepatic, muscle and kidney. So, injury to these tissues results in the release of the AST enzyme to general circulation. For Men: up to 37 U/L For women: upt 31 IJ/L Normal range:
Elevated levels of aspartate aminotransferase (AST), in the blood can indicate potential liver damage or injury. However, it's important to note that AST is not specific to the liver, and increased levels can also be observed in conditions affecting other organs. 1.Liver Diseases: include hepatitis (viral, alcoholic, or autoimmune), liver cirrhosis, non-alcoholic fatty liver disease (NAFLD), and drug-induced liver injury. 2.Biliary Tract Obstruction. 3.Heart Diseases: AST is also found in cardiac muscle cells. Heart conditions that involve damage to cardiac tissue, such as a heart attack (myocardial infarction), congestive heart failure, or myocarditis, can cause increased AST levels. 4.Muscle Disorders: AST is present in skeletal muscle cells as well. Muscle damage or injury, including conditions like muscular dystrophy, rhabdomyolysis, or strenuous exercise, can lead to elevated AST levels. 5.Trauma or Injury: Severe trauma, such as extensive burns, physical trauma, or major surgery, can cause tissue damage and subsequently elevate AST levels. 6.Certain Medications: Some medications, such as statins (used for cholesterol management), acetaminophen (in high doses), or certain antibiotics, can cause liver cell injury and lead to increased AST levels.
Method Principle: The enzymatic reaction sequence employed in the assay of aspartate aminotransferase is as follows: AST 1. L-Aspartate + 2-Oxoglutarate -----------> Oxaloacetate + L-Glutamate. MDH 2. Oxaloacetate + NADH + H+ L-Malate + NAD+ + H2O. -----------> AST, catalyzes the transfer of an amino group between L-aspartate and 2-0xoglutarate. The oxaloacetate formed in the first reaction, is then reacted with NADH in the presence of malate dehydrogenase (MDH) to form NAD +. AST activity is determined by measuring the rate of oxidation of NADH at 340 nm. How?
https://proteopedia.org/wiki/index.php/Image:Ast-reaction_final_copy.JPGhttps://proteopedia.org/wiki/index.php/Image:Ast-reaction_final_copy.JPG
REAGENT COMPOSITION: 2-0xoglutarate. L-Aspartic Acid, NADH, MDH LDH, Buffer, pH 7.8 0.1 and nonreactive Preservatives and Fillers
Method: (Macro Method) Pipette into clean and dry test tubes (Blank and Test): Blank Test 3.0 ml 0 ml d.H2O Reconstituted reagent 0 ml 3.0 ml Pre-warm to 37 C, then add: Sample 0 ml 0.2 ml - Mix and incubate at 37 oc for 1 minute and read the absorbance at 340 nm against distilled water (A1). - Re-incubate at 37 oc and after exactly 5 min. read the absorbance (A2). Choose the following on the spectrophotometer: 2) Applications 2) Simple Kinetics wavelength (340 nm) Seconds Duration (300 sec =5 min) Print Data Table (off) Press start (2 times)
Results Time Absorbance 340nm 1 min A1 5 min A2 Calculations: (A1 A2) X 514 = AST ACTIVITY (U/L) For Men: up to 37 U/L For women: up to 31 U/L Normal range:
