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MAKERERE

UNIVERSITY

COLLEGE OF VETERINARY MEDICINE, ANIMAL RESOURCES AND BIOSECURITY. SCHOOL OF BIOSECURITY, BIOTECHNOLOGY AND LABORATORY SCIENCE. BACHELORS OF BIOMEDICAL LABORATORY TECHNOLOGY. COURSE NAME: CLINICAL CHEMISTRY COURSE CODE: BBLT1208 RE: A PRACTICAL REPORT OF MEASUREMENT OF PLASMA TOTAL PROTEIN DONE ON 18TH NOVEMBER 2012.

A PRACTICAL CARRIED OUT ON PLASMA TO MEASURE TOTAL PROTEIN

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ABSTRACT Proteins are compounds of high molecular weight consisting of amino acid chains linked by peptide bonds. Plasma is the liquid part of blood. It is a pale yellow fluid that consists of about 91% water and 9% other substances like proteins, electrolytes, nutrients, gases and waste products. Most suspended substances in plasma are proteins. Plasma proteins can be classified based on charge and size as globulin, albumin and fibrinogen. Increased total plasma protein levels result from acute dehydration, shock, inflammation, multiple myeloma, Hodgkins lymphoma, leukemia, macroglobulinemia, tuberculosis, kidney and liver diseases and autoimmune disease. Low total plasma protein levels could be due to malnutrition, severe burns, kidney disease, gastrointestinal tract mal-absorption syndrome, uncontrolled diabetes mellitus, hyperthyroidism and heart failure. Two methods were used during the practical that is; The Refractometer method where a refractometer was used. Biuret method using biurets reagent and a Sherwood calorimeter 254 and wave length of 580nm. Using the refractometer method a total plasma protein concentration was 55g/l which was slightly lower than the lower limit of the reference value. In the biuret method, the values were 70g/l and 62.5g/l in tube 1 and 2 respectively.

INTRODUCTION
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The practical was done to determine the total plasma protein concentration in the sample. Determination of the total plasma protein concentration was to evaluate cases of abnormalities in the body portrayed by marked increase or decrease in the different components of the plasma proteins. The values would help check liver and kidney functions, to find out dietary components of proteins, determination of edema or ascites, to determine chances of developing infections and the existence of rare blood diseases like multiple myeloma. Plasma protein alterations are not usually specific for a particular disease condition. However certain alterations in total concentration or variation in the components may be significant both for diagnosis and prognosis. (Seeley, Stephens and Tate 2008). Plasma proteins can be classified into albumin, globulin and fibrinogen. Albumin makes up 58% of the plasma protein and is important in regulation of water movement between the tissue and blood, used to transport hydrophobic molecules like thyroid hormones. Globulins account for 38% and are divided into alpha, beta and gamma used to transport substances like iron and they are part of immunity against micro-organisms. Fibrinogen is important in the formation of blood clots hence providing protection to the body against pathogen infection. Any abnormality in plasma protein concentration indicates that some pathological, physiological or other induced factors are responsible.

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BACKGROUND A protein is a compound of high molecular weight consisting of amino acid chains linked by polypeptide bonds. There are two major types of plasma proteins; albumin and globulins. Globulin is further divided in alpha, beta and gamma globulins according to their different molecular weights. ALBUMIN This is the smallest plasma protein with a molecular weight of 69000 and constitutes 40-60% of total plasma proteins in a normal individual. It has greater effects on osmotic pressure of blood compared to the other plasma proteins. It is the primary source of reserve amino acids for tissue proteins, combine d with a variety of substances to prevent rapid excretion of drugs and assist in transportation of hydrophobic substances (Rod R. Seeley, Trent D. Stephens and Philip Tate, 2008). GLOBULINS These plasma proteins are insoluble in water but dissolve in dilute acids, bases, and salt solutions. They are divided into alpha, beta and gamma globulins. The alpha and beta globulins are carriers of various lipids, lipid hormones, and vitamins which are bound to the proteins to form lipoproteins. Alpha globulins also contain a glycoprotein and heptaglobulin components which transport copper and hemoglobin respectively. Beta globulin has transferrin component which is a carrier of iron. Gamma globulins are associated with antibodies in the body for example in the opsonisation of target cells for phagocytosis (Embert H. Cole 1980). The normal total plasma protein range is 60-85g/l. However variation in the concentration of one the components will result into varied concentrations of the total plasma concentration. Alteration in plasma protein concentration is usually not specific for a particular disease but is significant in the diagnosis, prognosis, to indicate pathological and physiological conditions in the body. It can also be used to determine the water balance of the body, detect kidney and liver diseases. Lowered plasma albumin concentration occurs in starvation, malnutrition, chronic gastrointestinal disease due to interfered digestion and absorption. It can be seen in cases of increased protein demand if dietary intake is
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insufficient, in chronic hepatic disease and in excessive breakdown as occurs in fever, uncontrolled diabetes mellitus, trauma and in ascites where the ascitic fluid contains a lot of albumin. In disease condition, it may also decrease due to inhibition of synthesis in the damaged organs. Hypoalbuminemia may also result from excessive loss in the nephrons due to acute nephrosis and nephritis. Increased levels are rarely seen except in cases of acute dehydration and shock. (Embert H. Cole, 1980). The different globulin concentrations occur in different conditions as mentioned; Increased alpha globulin may be seen in inflammatory reactions against bacterial and viral infections. This may be due to increased secretion of histamin. Increased gamma concentration is normally due to bacterial infection, parasitism, in liver disease like diffuse fibrosis and hepatitis, in certain types of neoplasms especially lymphosarcoma, plasmocytoma, and in multiple myeloma which is characterized immunoglobulin A (Embert H. Cole, 1980). Total plasma protein levels are measured using the refractometer method which measures in grams per 100 mls and the biuret method which uses reaction of the peptide bonds in the protein molecules with copper(II) ions reducing it to copper(I) forming a purple solution and the optical density read calorimetrically (Monica Cheesbrough, 2005).

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MATERIALS AND METHODS During the practical, two methods were used to determine the total plasma concentration in the sample, the Biuret and the refractometer methods. BIURET METHOD This was used to determine total plasma concentration. PRINCIPLE: In this method, copper (II) ions react with the peptide bonds in the proteins in a basic solution to form a violet- purple color, its self being reduced to copper (I) ions. The intensity of the color produced is read calorimetrically at a particular wavelength and is proportional to the number of peptide bonds reacting and therefore the amount of the proteins present in the reaction system. MATERIALS: Materials used during the test included the following; The plasma sample to be analyzed Biuret standard albumin (20mg/ml) Biuret reagent (alkaline copper sulfate) Distilled water Colorimeter (Sherwood calorimeter 254) Pipettes, test tubes, pipette fillers, test tube Vortex mixer PROCEDURE: Five test tubes were labeled B, S1, S2, 1 and 2 using clear labels to avoid wrong results. 1ml of distilled water was added in tube B. 0.1 ml of 20mg/ml Biuret standard albumin and 0.9 mls of distilled water added to tube S1. 0.3 mls of standard albumin and 0.7 mls of distilled water was added to tube S2. 0.05 mls of the test plasma, 0.95mls of distilled water pipetted into test tube1.
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0.1 ml of test sample and 0.9 mls of distilled water pipetted into tube2. To all the five tubes, 0.4 mls of biuret reagent was added and each mixture vortexed for complete mixing. The tubes were then allowed to stand at room temperature for thirty minutes to allow the reaction take place. The absorbance of the solution in each tube was then measured using a colorimeter at a wavelength of 580nm. A blank was used to zero the equipment before each measurement was read to reduce errors in the results. REFRACTOMETER METHOD Here, a refractometer was used to directly measure the total plasma protein concentration in g/100mls. PRINCIPLE Light enters the instrument in a beam parallel to the prism and, its refracted by the solution and is projected against the eye piece. The eyepiece is marked with scales in g/100mls. The refracted ray of light is viewed from the eye piece and the field of view appears as separated by a demarcation into a light and blue zone. The scale is directly read at the demarcation. MATERIALS Refractometer Plasma sample Pipettes Tissue paper PROCEDURE The slanted surface of the refractometer was cleaned to avoid interference with the real reading. A small drop of the sample was pipetted and put on the surface, the cover put back and the sample viewed through the eyepiece. The values were read from the scale for plasma protein concentration and recorded in g/100mls RESULTS
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TABLE OF RESULTS SHOWING TUBE ABSORBANCE AND PROTEIN (ALBUMIN) IN mg. Tube Absorbance Mg protein(as albumin S1 0.06 2.00 S2 0.24 6.00 1 0.14 3.50 2 0.25 6.25

TABLE OF RESULTS FOR THE PROTEIN CALIBRATION CURVE. Tubes 1 B Standard Albumin Serum Distilled water Biuret Total Volume Absorbanc e Mg Albumen 0 0 1.0 4.0 5.0 0 0 2 S1 0.2 0 0.8 4.0 5.0 0.08 2 3 S2 0.4 0 0.6 4.0 5.0 0.16 4 4 S3 0.8 0 0.2 4.0 5.0 0.24 8 5 S4 1.0 0 0 4.0 5.0 0.32 10

CALCULATIONS OF TOTAL PROTEIN IN PLASMA

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TUBE 1 Total protein (mg) = absorbance tube1 6 Absorbance S2 = 0.25 6 0.24 = 6.52mg/o.1ml or 0.00625g/0.1ml 0.1ml of solution contain 0.00625g of protein 1000ml 0f solution contain 0.00625 1000 0.1 = 62.5g/l TUBE 2 Total protein (mg) = absorbance tube 2 6 Absorbance S2 = 0.14 6 0.24 =3.5 mg/0.1 or 0.0035g/l 0.1ml of solution contain 0.0035g of protein 1000ml of solution contain 0.0035 1000 0.1 = 7g/l

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DISCUSSION Basing on the above results in relation to the background, the total plasma protein in both tube 1 and 2 were lying within the reference values, therefore the test sample was normal. However the refractometer method gave total plasma protein values of 55g/l which was slightly below the lower limit of the reference value. This could have been due to optical errors during viewing, dirty surface of the instrument and eyepiece.

CONCLUSION According to the test results, the sample was normal hence the patient did not have any disorder related to plasma protein abnormalities.

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REFERENCES 1. Embert H Coles. Veterinary clinical pathology 1980 Third Edition Wb Saunders Company. Phildelphia, London, Toronto. Pages 193-199. 2. Rod R.Seeley, Trent Stephens and Philip Tate. Anatomy and physiology2008 Eighth edition McGraw-Hill companies publications. New York. Pages 652-653. 3. Monica Cheesbrough. District laboratory practice in tropical countries 2005 Second Edition Cambridge University Press. Cambridge. Page 355-358.

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