Estimation Total Protein & Albumin .pptx

Total Protein & Albumin

1. Estimation of Total protein & Albumin By: ABDULKAREEM MOHAMMED

2. Introduction Proteins are polymers of amino acids produced by living cells in all forms of life The key roles which plasma proteins play in bodily function, together with the relative ease of assaying them, makes their determination a valuable diagnostic tool as well as a way to monitor clinical progress. In very general terms, variations in plasma protein concentrations can be due to any of three changes: in the rate of protein synthesis, the rate of removal, and in the volume of distribution.

3. Proteins: Common properties In spite of functional differences between the various serum proteins, they have certain common biophysical and biochemical properties. These include: a basic composition of carbon, hydrogen, nitragen and oxygen . backbone of covalent peptide bonds which join the amino acid units together; and absorption maxima in the ultraviolet region.

4. Proteins: Common properties Based on these properties, laboratory methods have been developed to determine the concentration of proteins in serum often with the assumption that: each of the several hundred individual proteins present in serum reacts similarly in chemical reactions

5. Serum Total Protein Protein in the plasma is made up of albumin and globulins. "The globulin in turn is made up of a1, a2, B, and y globulins. These fractions can be quantitated using protein electrophoresis, but the total protein test is a faster and cheaper test that estimates the total of all fractions together. The traditional method for measuring total protein uses the biuret reagent, but other chemical methods are also available.

6. Methods of Total Protein Analysis Kjeldahl: quantitative, protein nitrogen determination Ultraviolet absorption: quantitative, absorption at 210 nm Refractometry: quantitative, RI Biuret: quantitative, increased absorption at 540 nm Dye-binding Total nitrogen

7. Estimation of Total protein Protein : are polymers of amino acid . Amino Acids : are organic compound , which contain both an amino acid (NH2) and carboxyl group (COOH). The building blocks of all protein molecules are amino acids . Chemical classification of protein : Simple protein : Contain only amino acid ( Albumin – Globulin . Conjugated protein: consist of two parts, one is protein and the another one is not protein. e.g Phosphoprotein (casein). Derived protein: result from hydrolysis of simple or conjugated protein (peptone) .

8. Biochemical Importance and functions of protein: 1- Structural component 2- Antibody. 3- Enzyme . 4- Hormones . 5- Transport. 6- Blood coagulation .

9. Estimation of Total protein Purpose of the test : It is a part of your routine health checkup. Total protein test measures the total amount of protein in your blood and specifically looks for the amount of albumin and globulin . Principle : Biuret Method Peptide bonds of proteins react with tartrate-complexed cupric ions in alkaline solutions to form a colored product. In a positive test, a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. The intensity of the color, and hence the absorption at 546(520-570) nm, is directly proportional to the protein concentration.

10. Estimation of Total protein Reagent : Working reagent (WR): Contains the reaction compounds, It is ready to use. Standard (STD): Contains 5 g/dl . Sample: Serum or plasma free of hemolysis. Procedures: Collects the blood sample and prepare serum (without anticoagulant) or plasma (with anticoagulant). Bring reagents and samples to room temperature .

11. Procedures: Take 3 clean and dray test tube and label them as following ( Blank – Standard – Test ) Method Blank tube STD tube Test tube Reagent (ml) 1.0 1.0 1.0 STD (µ) 20 Serum (µ) 20

12. Procedures: Mix well and incubate for 5 min at 37Cº or 10 min at Room temperature (15- 25ºC) . Switch on the spectrophotometer and adjust the wave length (WL) to 546 nm. Zero the spectrophotometer with blank . Read the absorbance (Abs) of standard &sample.

13. Reference R ange : Reference range for total proteins is 66.6 to 81.4 g/L • Results for males are approximately 1 g/L higher than results for females; this difference is probably not of clinical significance. • In newborns, the mean serum protein concentration is 57 g/L, increasing to 60 g/L by 6 months and to adult levels by about 3 years of age.

14. Interpretation : Hypoproteinemia : Malnutrition and/or malabsorption Excessive loss as in renal disease, GI leakage, excessive bleeding, severe burns Excessive catabolism Liver disease Hyperproteinemia : Dehydration Monoclonal increase Polyclonal increase

15. Estimation of Albumin Albumin is a carbohydrate-free protein representing 55 – 65% of the total plasma proteins. It is synthesized in the liver and is the main protein of human blood plasma and consist of polymers of amino acid that are linked to each other by peptide bonds & source of endogenous amino acids. Function of albumin : Vascular: maintenance of plasma osmotic pressure Transport of : (Steroid hormone – free fatty acid –bilirubin – Ca …) Metabolic : Acid base balance . Albumin also binds toxic heavy metal ions and many drugs, which is why a decrease in albumin in the blood can have important pharmacokinetic consequences

16. Estimation of Albumin Hyperalbuminemia is of little diagnostic significance except in dehydration. Hypoalbuminemia is very common in many diseases and stems from various factors: impaired synthesis, either primary as a result of liver disease or secondary due to diminished protein intake; increased catabolism because of tissue damage (severe burns) or inflammation; malabsorption of amino acids (Crohn’s disease); proteinuria due to nephrotic syndrome; protein loss by way of feces (neoplastic disease). In severe hypoalbuminemia plasma albumin levels are below 2.5 g/dl. The low plasma oncotic pressure allows water to move out of the blood capillaries into the tissues (edema).Albumin measurements also allow monitoring of the patient’s response to nutritional support and are a useful test of liver function .

17. Methods of A l b u m i n Analysis Precipitation : quantitative Salt fractionation , Solvent fractionation , Acid fractionation Principle of analysis : Changes of net charge of protein result in precipitation . Tryptophan content : quantitative Principle of analysis : • Glyoxylic acid + tryptophan in globulin Purple chromogen ( Amax , 540 nm ) . Total protein -globulin = albumin . Electrophoresis : quantitative Principle of analysis : Albumin is separated from other proteins in electrical field ; percent staining of albumin fraction multiplied by total protein value .

18. Methods of A l b u m i n Analysis Immunochemical : Radial immunodiffusion , Turbidimetry , , Nephelometry , & Radioimmunoassay , Enzyme immunoassay Dye binding : quantitative Methyl orange ; BCG ( bromcresol green ) ; BCP ( bromcresol purple ) Dye binding : semi - quantitative Bromphenol blue in test strip changes color from yellow to blue in presence of albumin most commonly used test for urine protein

19. Estimation of Albumin Purpose of the test : To screen for and help diagnose a liver disorder or kidney disease; sometimes to evaluate nutritional status . Principle : Albumin in the presence of bromocresol green at a slightly acid pH, produces a colour change of the indicator from yellow-green to - green-blue. The intensity of the color formed is proportionalto the albumin concentration in the samples . Albumin + Bromocresol . Colored complex pH > 7

20. Procedures: Reagent : Working reagent (WR): Contains the reaction compounds, It is ready to use. Standard (STD): Contains 5 g/dl . Sample: Serum or plasma free of hemolysis. Procedures: Collects the blood sample and prepare serum (without anticoagulant) or plasma (with anticoagulant). Bring reagents and samples to room temperature .

21. Procedures: Take 3 clean and dray test tube and label them as following ( Blank – Standard – Test ) Method Blank tube STD tube Test tube Reagent (ml) 1.0 1.0 1.0 STD (µ) 5 Serum (µ) 5

22. Procedures: Mix well and incubate for 5 min at 37Cº or 10 min at Room temperature (15- 25ºC) . Switch on the spectrophotometer and adjust the wave length (WL) to 630 nm. Zero the spectrophotometer with blank . Read the absorbance (Abs) of standard &sample. Normal range : Adults: ( 3.5 - 5.0 g/dl)

23. Interpretation : Hypoalbuminaemia : is a medical sign in which the level of albumin in the blood is abnormally low : Malnutrition Renal disease (nephrotic syndrome ) Liver disease (hepatitis , cirrhosis ) Sever burn Infection Heart failure Hypoalbuminemia my cause generalized edema swelling via decrease osmotic pressure .

24. Interpretation : Hyperalbuminemia : is only importance in case of dehydration Interfering Factors : Albumin is decreased in : Pregnancy ( last trimester , owing to increased plasma volume ) Oral birth control ( estrogens ) and other drugs Prolonged bed rest IV fluids , rapid hydration , overhydration