Serum biochemical parameters TOTAL Protein assay Biochemistry Clinical practice CLS 432 Dr. Samah Kotb Lecturer of Biochemistry 2015

1. Total and conjugated bilirubin 2. ALT 3. AST 4. Total protein 5. GGT 6. ALP 7. 5′-nucleotidase 8. Albumin 9. prothrombin time Liver Function tests

Total protein was estimated according to the method of (Burtis A and Koller A., 1999), Colorimetric method. Principle

Proteins give an intensive violet –blue complex with copper salts in an alkaline medium. Iodide is included as an antioxidant. Principle

The intensity of the color formed is proportional to the total protein concentration in the sample.

Total protein Standard : Bovine serum albumin standard (7g/dL) (70 g/L). Biuret reagent: R1 Sodium Potassium tartrate (21 mmol/L) Sodium hydroxide (0.75 mol/L) Potassium iodide (6 mmol/L) Cupric Sulfate (6 mmol/L) Reagents

1- 1 ml of Biuret reagent was added into both unknown test and control tubes, then 0.02 ml of serum were added to unknown. Procedure

ml of Bovine albumin primary standard and 1 ml of Biuret reagent were added into standard tube, 1 ml of Biuret reagent was added into blank tube.

3- The contents were mixed well, incubated at 37°C for 5min, standard and blank tubes.

4- The absorbance read at 540 nm. 5- Blank was used for zero adjustment of standard, and the test.

A sample Concentration of protein ( g/dL) = x 7.0 Where: A standard A: absorbance of recorded sample 7.0: Standard concentration Calculation:

Adults: g/dL (66-87g/L). Prematures: g/dL (36-60 g/L). Newborns : g/dL (53-89 g/L) Pregnancy: Concentration lowers from 69 to 61 g/L Reference values