1. Clinical diagnostic biochemistry - 4 Dr. Maha Al- Sedik

2. What are Proteins?  Proteins are made of amino acids (CHON)  They contain carbon, hydrogen, oxygen (like carbohydrates and fats).  In addition, also contain nitrogen  Each amino acid has: Acid group (-COOH) Amine group (-NH 2 ) Side chain (unique)

3. Amino Acid

5. Amino acids Essential a.a Non essential a.a

6. Amino Acids Proteins are made up of 20 AAs o 9 are essential ( can not be formed by the body ) o 11 are nonessential : Can be made in the body from other amino acids

9. Proteins are a link of Amino acid with peptide bonds:  Dipeptides: have two amino acids bonded together.  Tripeptides: have three amino acids bonded together.  Polypeptides: have more than two amino acids bonded together.

10. Amino acid sequence: determines shape and function and what protein is made. INSULIN

11. Protein Denaturing  Unfolding of protein shape by heat, acids, bases or salts that changes its ability to function.  After a certain point, denaturation cannot be reversed.  Stomach acid untangles proteins to aid in digestion.

12. Denaturation

13. Protein Digestion and Absorption Mouth  Crushed and moisten  No digestion of protein in the mouth Stomach  protein is denatured  Enzymes start to break the peptide bonds  HCl activates pepsinogen to pepsin  Pepsin cleaves proteins into smaller polypeptides.

14. small intestine:  In the small intestine, polypeptides broken down into tri-, dipeptides and amino acids by pancreatic protease (trypsin).  Cells in the small intestine absorb amino acids to be used by the body.

15. Protein and the kidney:  Amino acids in blood are filtered through the glomerular membranes, but normally are reabsorbed in the renal tubules by transport systems.  When the transport mechanisms become saturated or are defective, amino acids spill into urine, resulting in a condition known as aminoaciduria. Aminoaciduria

16. Two types of aminoaciduria have been identified : 1. Overflow aminoaciduria: occurs when the plasma concentration of one or more amino acids exceeds the renal threshold (capacity for reabsorption). 2. Renal aminoaciduria: occurs when plasma concentrations are normal, but the renal tubular reabsorption system has a congenital or acquired defect. Aminoaciduria

17. Causes of aminoaciduria: Primary aminoaciduria ( overflow ): Due to:  Inherited enzyme defect e.g. defect in the pathway of amino acid metabolism e.g. ( cysteinuria ).  DNA abnormality. Secondary amino aciduria ( Renal ): Due to:  generalized renal tubular dysfunction.

18. Analysis of amino acids Many procedures are available to measure amino acids in biological samples. To diagnose pathological disorders, the following three groups of tests for amino acid analysis are important: 1)Screening tests, including thin-layer chromatography (TLC) and photometric screening test for urine proteins. 2)Quantitative tests to monitor treatment or confirm an initial diagnosis. 3)Specific tests that identify an unknown amino acid or metabolite.

19. Analysis of aminoacids Screening tests Quantitative tests Specific tests

20. 1- TLC: TLC analysis of amino acids is conducted in three stages: (1)preparation of the sample, (2) chromatographic separation, (3) identification of the separated amino acids. 2- Photometric Screening Tests for Urine Screening tests

21. Amino acids are measured quantitatively in body fluids with a variety of techniques, including: 1.Electrophoresis. 2.Gas chromatography (GC). 3.High-performance liquid chromatography (HPLC). 4.Ion-exchange chromatography. Quantitative tests

22. In addition to the general analytical techniques discussed previously, a variety of simple tests exist that are specific for individual amino acids. These tests are used in the diagnosis of specific disorders. Tests for Specific Amino Acids

23. Plasma proteins

24. Functions of plasma proteins: (1) Enzymes are proteins that catalyze biochemical reactions essential to metabolism. (2) Hormones regulate body functions. (3) Immunity: Antibodies and complement system protect against infection. (4) Osmotic pressure: Plasma proteins maintain the osmotic pressure of plasma. (5) They transport hormones, vitamins, metals, and drugs. (6) Hemostasis: coagulation system.

25. Causes of increased plasma protein levels: 1.Dehydration: decreased volume of distribution due to relative water deficiency. 2.Hemoconcentration: due to stasis of blood during venipuncture and prolonged application of tourniquet. 3.Paraproteinaemia: increase in abnormal protein synthesis. 4.Hypergammaglobulinemia: increase in the antibody concentration.

26. Causes of decreased plasma protein levels: 1.Overhydration: increased volume of distribution ( IV fluid administration ). 2.Artifactual: blood samples from drip arm. 3.Decrease in protein synthesis: sever liver disease or immunodiffeciency. 4.Excessive protein loss: in urine e.g. nephrotic syndrom or from skin e.g. sever burns.

27. o Normal total plasma protein concentration: 6 – 8.5 gm / dl. o Electrophoresis separates serum Proteins into 5 distinct zones or bands at barbital buffer at pH 8.6 :  Prealbumin.  Albumin.  Alpha globulin ( Alpha 1 and Alpha 2 ).  Beta globulin ( Beta 1 and Beta 2 ).  Gamma globulin: IgG, IgA, IgM, IgD, IgE and C-reactive protein.

28. The width of each band is dependent upon the number of proteins that are present in that fraction.

29. Q – WHAT is the difference between serum proteins and plasma proteins in electrophoresis?

30. Serum is a clear yellowish fluid that remains from blood plasma after clotting factors (fibrinogen, prothrombin ect.) that have been used in the formation of a clot. Plasma is a clear yellowish fluid that still contains all of the clotting factors and have not been solidified into clot.

31. If plasma is used in electrophoresis, there will be an extra band ( fibrinogen ) between beta and gama bands. Fibrinogen