Biochemistry Lab College of Pharmacy Determination of Total Serum Protein (T.S.Protien) Method:- the Biuret method Aim: to estimation total serum protein in the given sample INTRODUCTION:- -protein are the most abundant compound in serum ( the rest of blood when you remove all the cell after closing) Amino acids are the building blocks of all the proteins in turn proteins are the building blocks of all cells and body tissues. -they are the basic componets of enzyme many hormones,antibodies and clotting agents -protein acts as transport substances for hormones, vitamins minerals, lipids and other compounds. -in addition, protien help to balnce the osmotic pressure of the blood and tissue osmotic pressure is part of what keeps water inside a particular compartment of your body protein play a major role in maintaining the delicate acid-alkaline balance of your blood. -,serum proteins serve as a reserve source of energy for your tissues and muscle when you are not ingesting an adequate amount. -The major measured serum proteins are divided into tow groups albumin and globulin there are four major types of globulin each with specific properties and actions. -A typical blood panel will provide four different measurements the total protein, albumin, globulin,and albumin-globulin ratio Clinical significanc: 1-Protein important for control of extracellular fluid distribution and transport (hormones vitamins lipids bilirubin Ca and drugs) protein transport poorly water soluble substance in plasma and inflammatory response and control of infection 2-The proteins are macromolecular organic compounds, widely distributed in the.organism. They act like structural and transport elements 3-The determination of total proteins is useful in the detection of High protein levels caused by hemoconcentration like in the dehydrations or increase in.the concentration of specific proteins Low protein level caused by hemodilution by an impared synthesis or loss (as by - hemorrhage) or excessive protein catabolism 4-This test is often done to diagnose nutritional problems, kidney disease or liver disease Msc pure Biochemistry- AHMED KAREEM THAMER
Total protein may be elevated due to: Total protein may be decreased due to: -Chronic infection ( including tuberculosis) -Liver disease Adrenal cortical hypofunction -Hormone -imbalances that favor breakdowen -Hypersessitivity states of tissue Sarcoidosis -loss through the -urine in severe kidney disease -Hemolysis -Cryoglobulinemia -low albumin and globulins -pergnancy (dilution pf protein due to extra held Vascular system) -Alcoholism -Malnutrition and malabsorption -Leukemia -Respiratory distress The Albumin/Globulin (A/G) ratio: 1-Elevated in: -hypothyroidism -High prptein/high carbohydrate diet whith poor nitrogen retention -low globulin 2-decreased in: -Liver dysfunction -Multiple myeloma
-Cirrhosis -Autoimmune disease -Nephrotic syndrome PRINCIPLE: Proteins give violet-blue complex with copper salts in an alkaline medium The intensity of the color formed is proportional to the total protein concentration in the sample (Biuret reaction). Biuret reagent reacts with an alkaline solution of CuSO 4 to form a violet chelate compound. Specimen: serum or heparin zed plasma class ware and Equipment test tubes, micro pipette with 1ml and 20 µl, cuvette (glass, quartz) Reagents: R (1) Alkaline: potassium sodium tartrate 12mmol /L Reagent : sodium hydroxide 0.6 mmol/l, Potassium iodide 30 mmol/l R(2) Colouring: Cooper sulfate (Novice) 0.6mol/L R(3) Standard : Bovine albumine 5g/ dl, 50g/ L Working Reagent(W.R) Mix 3ml of Reagent R 2 in I container R1, Mix 5ml of reagent R 2 in I container R 1, Working reagent is stable 6months at 12-8C.
Procedure:. Wavelength 546nm Temperature.. 37C Cuvette 1cm light path Read against blank reagent Calculation: (A) sample x 7 (standard con.) =gm/dl of total protein in sample (A) std. Normal value: Total serum proteins 6-8 gm/dl Albumin - 3.7-5.3 gm/dl Globulin - 1.8-3.6 gm/dl Fibrinogen - 200-400 gm/dl The Albumin-globulin ratio varies from about 2,5:1 about 1.2:1(1.5:1) RESULT: 1- A? Con? 2-The result compared with normal values: discuss the result 3- Drawing A vs con
The report questions: 1-What is the difference between serum and blood plasma? Conversion of fibrinogen to fibrin 2- the serum globulin level elevated and decreased in 3-what happens -High levels of albumin -Low levels of albumin 4-what is the importance of blank and standard in quantitative analysis?