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Lab 13: Determination of Creatinine concentration in serum • Creatinine is a west product formed in musulecreatine metabolism. Creatine is synthesized in the liver (from three amino acids—arginine, methionine, and glycine), • passes in to the circulation and is taken up almost entirely by skeletal muscle for conversion to creatine phosphate, which serves as a storage from of the energy in skeletal muscles.
Determination of Creatinine concentration in serum • About 2% of the total createne is converted daily to cratinine so that the amount of creatinine produced is related to the total muscle mass remains approximately the same in plasma and urine form day to day unless the muscle mass changes
Creatinine coefficient • The number of milligrams of creatinine excreted in the urine within a 24-hr period per kilogram of body weight is the creatinine coefficient of that individual. The creatinine coefficient of the normal male is 20 to 26; that of females is 14 to 22 • So urinary cratinine is used as a very rough check of the accuracy of 24 hr urine collection
Increased serum creatinine: • Renal Diseases • False high values may obtained in diabetic mellitus • Excessive creatinine has also been observed in hyperthyroidism during pregnancy • Low serum creatinine is not significant.
Principle of the method • Creatinine in alkaline medium react with picric acid to form a red or yellow of creatininepicrate, the intensity of which is measured at 520 nm. At a low picric acid concentration used in this method a precipitation of protein does not take place. As a result of rapid reaction between creatinene and picric acid later secondary reaction do not cause interference
Procedure: Waite for 2 min then read absorbance at 520 nm. Plot a graph with Creatinine concentration against the absorbance
CreatineKinase (CK) • Phosphocreatine is a high-energy compound that can be used by cells to drive energy-requiring reactions. This compound is formed from creatine and ATP by the action of creatinekinase (CK).
This enzyme is also often called creatinephosphokinase (CPK). The reaction performed by this enzyme is readily reversible: ADP and phosphocreatine can be converted to creatine and ATP. Surprisingly, increased levels of this enzyme in the blood have been found to be associated with a number of serious yet seemingly unrelated conditions including heart attacks, alcoholism, muscular dystrophy, stroke, epilepsy, and other neurological and endocrine disorders. In general, elevated serum CK levels are associated with conditions leading to tissue damage and cell death (necrosis).
Creatinekinase is a dimeric enzyme composed of two subunits. The monomer subunit M and the monomer subunit B. these subunits combine to form 3 distinct CK isoenzymes: CK-BB (CK-1), CK-MB (CK-2) and CK-MM (CK-3). • CK-MM is the main form in skeletal muscle. CK-BB is found in brain and smooth muscle. CK-MB is found in high level in myocardium (SK-MB activity represents between 10 and 20% total CK activity) and in lesser amount in skeletal muscle (CK-MB activity represents lesser than 2% of total CK activity).
In absence of disease, most CK activity in serum is due to CK-MM. acute myocardial infarction will result in increased CK-MB isoform circulating in serum. This one increases between 4 and 6 hours following the beginning of the attack, then peaks between 12 and 24 hours and returns to normal within 48 hours.
Procedure • Pipette into 1 cm path length thermostatedcuvette 1ml of reagent. • bring to 37⁰C, then add 50µl of sample • Mix. Read initial absorbance after 5min at 340nm and then record absorbance every minute during 5 minutes. • Calculate absorbance change per minute (Δabs/min).
Lactate dehydrogenase (LDH) Lactate dehydrogenase (LDH):- is an enzyme that helps the process of turning sugar into energy for your cells to use. LDH is present in many kinds of organs and tissues throughout the body, including the liver, heart, pancreas, kidneys, skeletal muscles, brain, and blood cells.
LDH and damages • When illness or injury damages your cells, LDH may be released into the bloodstream, causing the level of LDH in your blood to rise. High levels of LDH in the blood point to acute or chronic cell damage, but additional tests are necessary to discover its cause. Abnormally low LDH levels occur only rarely and usually aren’t harmful.
Types of LDH • There are five different forms of LDH, and they are distinguished by slight differences in their structure. Each form of the LDH enzyme is called an isoenzyme • LDH-1: heart • LDH-2: red blood cells • LDH-3: lungs • LDH-4: kidneys, placenta, and pancreas • LDH-5: liver and skeletal muscle
Principle • This method is based on the reduction of pyruvate to lactate in the presence of NADH by the action of lactate dehydrogenase. • The pyruvate that remains unchanged reacts with 2.4-dinitrophenylhydrazine to give the corresponding phenylhydrazone, which is determined calorimetrically in an alkaline medium.