1. 285 PHL Lab # 2 Plasma Proteins

2. Proteins: Classification Proteins: Classification 1- Simple proteins e.g. albumin and globulin 2- Conjugated proteins Protein + prosthetic gp. e.g. lipoprotein 3- Derived proteins Derived from simple proteins (denaturation) e.g. peptones

3. Plasma Proteins Plasma contains both simple and conjugated proteins Examples Albumin, globulin, lipoproteins e.t.c. Albumin Albumin Globulin Globulin - Low mol. wt. - Soluble in ½ sat. soln. of ammonium sulfate - High mol. wt. - Insoluble in ½ sat. soln. of ammonium sulfate

4. Determination of plasma proteins Steps: 1- Estimate total protein 2- Fractionation (e.g. salt fractionation) to ppt. globulin and measure albumin 3- Globulin = total protein – albumin

5. Determination of plasma proteins BIURET METHOD BIURET METHODPrinciple Cupric ions complex with the groups involved in the peptide bond. In an alkaline medium and in the presence of at least 2 peptide bonds, a violet- colored chelate is formed Methods: 1- Manual 2- Kit

6. Determination of plasma proteins Procedure (manual) Procedure (manual) 1- Total protein stand. 2- Albumin stand. Pipette 6 ml of Sulfate –sulfite Soln. Add 0.4 ml stand. serum Invert to mix. Remove at once 2 ml into t.t labeled total Protein stand. Add 3 ml ether to the rest of the mix. Put a stopper Invert 20 times to mix Don’t shake vigorously Cap the tube Centrifuge for 5 mins The globulin layer will be formed Tilt the tube & insert Pasteur pipette into the clear soln. below globulin layer (don’t disturb globulin layer) Pipette the clear soln. into clean t.t. Take 2 ml of this soln. into anther t.t. Label it Albumin stand.

7. Determination of plasma proteins Procedure Procedure 3- Total protein sample 4- Total albumin sample 5- Blank In test tube labeled Blank add 2 ml of sulfate-sulfite soln. Repeat all above steps using serum sample instead of standard sample

8. Determination of plasma proteins Procedure Procedure T Ptn StAlb. StBlankAlb. ST Ptn S -Add 5 ml of Biuret reagent to each tube -Place in water bath at 37°C for 10 ‘ - Read the absorbance at λ 540 nm

9. Calculations Serum total protein (gm/dl)= abs. total protein sample x conc. of std (7 g/dl) abs. total protein std Serum albumin conc. (gm/dl)= abs. albumin sample x conc. of std (3.5 g/dl) abs. albumin std Serum globulin conc. (gm/dl)= Total protein – albumin

10. Determination of plasma proteins Procedure (kit) Standard StandardSample Serum sample -- --0.2ml Standard 0.2 ml -- -- Working reagent 1 ml -Mix and place in water bath at 37°C for 15 min. -Read the absorbance at λ 540 nm

11. Determination of plasma proteins Calculation: Conc. Of total proteins (g/dl)= A sample x conc. Of st.(7) A stand. A stand.

12. Normal values Total protein: 4.2 - 5.6 g/dl Albumin: 2.3 - 3.5 g/dl Globulin: 1.9 – 2.1 g/dl

13. Clinical significance Hyperproteinemia HyperproteinemiaDefinition An increase in total plasma proteins CausesDehydrationShock

14. Clinical significance Hypoproteinemia HypoproteinemiaDefinition A decrease in total plasma proteins below the reference causes Renal diseases BleedingMalabsorptionMalnutritionBurns High fever Liver diseases