TOTAL PROTEIN IN PLASMA The level of total serum proteins found in healthy young and middle-aged adults is 6,0 {0 8.2 8/100 mi of serum. In plasma, fibrinogen inerexsce this value by an additional 0.2 fo Ot g/100 mi of plasma. Total plasma Proteins tend to be increased in dehydration due to haemo-concentration and’ in various diseases such as lymphogranuloma venereum, granuloma inguinale, leprosy and multiple myeloma. The latter disease, which represents a tumor of the bone marrow, is characterized by the petal ane er called Bence-Jones proteins in the plasma and urine aa increases the teal plasma protein content in excess of 10 g%. Infections cauce marked increases in the gamma-globulin fraction due to antibody formation while is importance unless certain additional measurements are performed. A minimum analysis Should include the following estimation (average values ana normal ranges in g%) MEASUREMENT AVERAGE RANGE Total plasma/serum protein 7A 6.0 - 8,28 Albumin 43 3,8- 4,7 Total globulin 29 23-35 Albumin-globulin (A/G) ratio Ls 11-18 “Serum2 TOTAL PROTEIN (KINGSLEY) The diluted plasma or serum is treated with a special Biuret reagent. The colour developed is compared with that of a standard protein solution trected similarly, By Suitable fractionation of serum before treatment with Biuret. albumin may be determined separately. PROCEDURE Transfer 0.5 ml unhaemolyzed serum to a centrifuge tube and add 23% sodium sulfate wea cam mark. Mix by inversion only and immediately pipette out 2 ml of ae uniform fluid to a test tube. This is Tube 1 which will be used for tour protein Gitimation. To the remainder of the centrifuge tube add 3 ml of ether, cud shake vigorously. Centrifuge for 5 minutes at 3500 r.p.m. After centrifuging remove 2 ml of the clear fluid beneath the globulin precipitate and transfer to a test cuhe, This is Tube 2 which will be used for albumin estimation. For photometric measurement, place 2 mil of standard protein solution in a third tube and 2'ml of distilled water te a fourth tube as a blank. To each of the four tubes add 4 mi of biuret reagent, followed by 2,5 ml of ether. Shake vigorously and centrifuge for 5 minutes. By means of a pipette, transfer the Total protein (g%) = QD. of tube 1 x Cone. of standard O.D. of tube 3 Albumin (g%) = Q.D, of Tube 2 x Cone. of standard O.D. of Tube 3 Globulin (g%) = Total protein - Albumin Calculate the A/G ratio.3. 5. Sodium sulfate (23%) Biuret reagent Dissolve 230 g anhydrous sulfate (AR) in 600- 700 mi of distilled water by heating and stirring. ‘Transfer immediately to 1 volumetric flask and dilute to the mark. ‘Stopper and maintain temperature at 37°C for experimental work, Prepare a saturated solution of NaOH (carbonate-free) by shaking up 110 ¢ NaQH (AR) with 100 ml of distilled water. Stopper and allow to stand for 3 days to allow carbonate to settle out leaving a carbonate. free solution. Measure 92 mi of this solution into @ graduated cylinder (500 mi) and add distilled water to the 300 ml mark. Add 100 ml of 1% crystalline Cu-sulfate and mix. Transfer to clean bottle and stopper. The reagent is stable for months despite the formation of a slight sediment. Dilute 5 mi of a stock serum solution to 100 ml in a 100 ml volumetric flask with a 15% NaCl solution, is .. Refrigerate but do not use this solution after one month of storage.