Dr Atef Masad PhD Biomedicine

1. Clinical Chemistry Dr Atef Masad PhD Biomedicine Dr Atef Masad

2. Chapter 2 Amino Acids and Proteins Dr Atef Masad

3. Amino Acids and Proteins • The word proteins is derived from the Greek word proteis, meaning "first rank“ • Proteins and amino acids have unique structures that allow them to participate in some specific types of chemical reactions. Dr Atef Masad

4. Serum contains a large variety of total proteins, averaging 7.0 g/dL in the adult • There are two main types of proteins, albumin and globulins. • They are grouped into five classes as determined by their electrophoretic separation: • albumin, alpha1 globulins, alpha2 globulins, beta globulins, and gamma globulins. • Serum albumin, at around 4 g/dL, makes up roughly half of the total serum proteins. Dr Atef Masad

5. A simple way to assess the balance between the patient’s albumin and globulins in serum is to calculate the albumin:globulin, or A/G, ratio. • Globulins (G) are calculated as albumin (A) subtracted from total serum proteins. Albumin is then divided by globulin. Dr Atef Masad

6. 1- Albumin • represents the highest concentration in serum "60%". • synthesized in the liver. • t½=15-19 days • F: 1-Maintaining fluid balance and contributes to intravascular colloid osmotic pressure • 2- Carrier: • binds various substances in blood e.gbilirubin, salicylic acid, fatty acids, Ca++, Mg++, cortisol and others. • 3-Anti-oxidant activity 4-Buffer Dr Atef Masad

7. Clinical significance • Hyperalbuminemia • rare and of no clinical significance • It is increased in dehydration. • Hypoalbuminemia • Increased loss (nephrotic syndrome) loss in urine due to the damage of glomeruli, • Decreased production (nutritional deficit, liver failure) Dr Atef Masad

8. Protein glycation • Protein glycation has important implications for protein activity, unfolding, and degradation, oxidative stress, as well as for cell functioning. • Glycated Albumin can be used as a sensitive indicator of short term hyperglycemic control than HBA1c • It is replaced in the body approximately every 20-25 days. Dr Atef Masad

9. As with other proteins in the body, it is subject to glycation by excess sugar. • A comparison with total albumin provides a simple, stable index of glycation over a three weeks to one month test period. Dr Atef Masad

10. MECHANISM OF ALBUMIN GLYCATION • The glycation process is a condensation reaction between carbohydrate and free amino acid at the amino terminus of proteins or at the epsilon amino groups of lysine residues of proteins. • The reaction is initiated with attachment of the aldehyde function of acyclic glucose to a protein amino group via nucleophilic addition, forming an aldimine, also known as a Schiff base. Dr Atef Masad

11. This intermediate product subsequently undergoes an Amadori rearrangement to form a 1-amino-1-deoxyfructose derivative in a stable ketoamine linkage, which in turn can cyclize to a ring structure. • This bimolecular condensation of free saccharide with protein constitutes a mechanism by which proteins are subject to post-ribosomal modification without the influence of enzymatic activities. Dr Atef Masad

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13. Non-enzymatic glycation of albumin occurs at multiple sites: glucose can attach at • Lysine199, Lysine281, Lysine439, and Lysine525, other minor lysine and arginine • residues, and also at N-terminal residues of polypeptides. Lysine525 accounts for 33% of all glycation Dr Atef Masad

14. Complex multi-step reactions follow that cause formation of early and advanced glycation end-products (AGEs). • Recent clinical studies showed a significant correlation between increased serum Glycated albumin level and the presence and severity of coronary artery disease in patients with type 2 diabetes mellitus and suggest the use of glycated albumin as a screening test for CAD Dr Atef Masad

15. 2- Prealbumin "Transthyretin". • Thyroxine-binding protein • binds with retinol and form a complex that transports retinol "vitamin A". • Sensitive marker of nutritional status, since its t½ is only 2 days • It is decreased in hepatic damage, acute phase inflammatory response, and tissue necrosis. • It is increased in patients receiving steroids, in alcoholism, and in chronic renal failure. Dr Atef Masad

16. 3- Globulins • 1, 2,  and  fractions. • (a) 1-antitrypsin • Protease inhibitor that binds to, and inactivates, trypsin • It neutralizes trypsin-like enzymes "e.g elastase" that can cause hydrolytic damage to structural protein • A deficiency of 1-antitrypsin is associated with Pulmonary emphysema(expansion of lung air spaces), liver cirrhosis "the protein is synthesized but not released from hepatocytes". Dr Atef Masad

17. B- 1-Fetoprotein "AFP“ • It is synthesized initially by the fetal yolk sac and then by parenchymal cells of the liver. • It is believed that AFP protects the fetus from immunolytic attack by its mother. • Serum AFP is used as tumor marker. • High concentrations are found in hepatocellular carcinoma and certain gonadal tumors in adults. Dr Atef Masad

18. (c)2-Haptoglobin • Binds free Hb by its  chain. • It is increased in inflammatory conditions e.g Rheumatic diseases, burns and nephrotic syndrome. • Hemolytic diseases can deplete Hp levels Dr Atef Masad

19. (d) 2-ceruloplasmin • Copper-containing 2-glycoprotein • Copper transport protein "6-8Cu++ ions • Participates in plasma redox reactions • Cp measurement is usually to screen for Wilson’s disease • More than 90% of serum copper is found in ceruloplasmin. • Deficiency is associated with deposition of Cu++ in skin, liver, brain, cornea. • This may cause liver cirrhosis & neurologic damage. Dr Atef Masad

20. (e) - 2-Macroglobulin • And abbreviated as α2M and A2M • Largest non-immunoglobulin in plasma • Synthesized mainly in liver, but also locally by macrophages, fibroblasts, and adrenocortical cells. • Protease inhibitor • It functions as an inhibitor of coagulation by inhibiting thrombin. Dr Atef Masad

21. May act as a carrier protein because it also binds to numerous growth factors and cytokines, such as platelet-derived growth factor, basic fibroblast growth factor, insulin, and IL-1β. • Increased in nephrotic syndrome & diabetes • Rises 10-fold or more in the nephrotic syndrome when other lower molecular weight proteins are lost in the urine. Dr Atef Masad

22. (f) Transferrin "siderophilin“ • Glycoprotein, synthesized by the liver. • The major component of the -globulin. • Its function is the transport of iron and prevention of iron loss by the kidney. • Two molecules of Fe+3 can bind to each molecule of transferrin. • Transferrin transports iron to its storage sites, where it is incorporated into another protein apoferritin to form ferritin. Dr Atef Masad

23. Deficiency of transferrin may result in accumulation of iron & it may precipitate in tissues as hemosiderin. • iron bound to transferrin • increased in hereditary disorder of iron metabolism. Dr Atef Masad

24. Hemochromatosis • Iron overload , excess iron is deposited in the tissues, "liver, & pancreas" • this disorder is associated with bronze skin, cirrhosis, diabetes mellitus, and low plasma transferrin levels. • Transferrin is increased in iron deficiency anemia, as well as pregnancy and estrogen therapy • Decreased in inflammation, malignancy, or liver disease Dr Atef Masad

25. (g) Hemopexin • Synthesized by paranchymal liver cells. • It removes circulating heme which is formed during the breakdown of Hb, myoglobin, or catalase, the complex is removed by liver. Dr Atef Masad

26. (h) 2-Microglobulin • 2M is synthesized in all nucleated cells, and is associated with the heavy chain of the class I major histocompatibility complex. • It is the light chain component of human leukocyte antigen (MHC ) on the surface of most nucleated cells. • Urinary BMG levels are a sensitive measure of renal tubular function • Increased in renal failure Dr Atef Masad

27. 2M is mainly catabolized by renal glomerular filtration followed by reabsorption in the proximal tubules and subsequent degradation. • Upon activation of the immune system both B and T lymphocytes actively release 2M into circulation. Dr Atef Masad

28. In several disorders the 2M level has been found to be higher than expected when considering the glomerular filtration rate. • These disorders are AIDS, rheumatoid arthritis, many lymphoproliferative and myeloproliferative diseases as well as several non-hematological malignancies • The 2M /cystatin C ratio has also been shown to be a promising marker of lymphoproliferation in patients with normal or mildly impaired renal function Dr Atef Masad

29. (i) -Complement proteins and Immunogobulins • Compliment proteins are decreased in genetic deficiencies, and increased in inflammation • The complement system consists of a number of small proteins found in the blood, generally synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). • When stimulated by one of several triggers such as cytokineses. • Region • Includes immunoglobulins (IgG, IgA, IgM) and C-reactive protein Dr Atef Masad

30. (j) Fibrinogen • Glycoprotein, synthesized by liver. • It forms fibrin clot when activated by thrombin. • Increased during the acute phase of inflammatory process. • Recent research has shown that fibrin plays a key role in the inflammatory response and development of rheumatoid arthritis, cardiovascular disease (>3.43 g/L). Dr Atef Masad

31. It may be elevated in any form of inflammation, as it is an acute-phase protein; for example, it is especially apparent in human gingival tissue during the initial phase of periodontal disease. Dr Atef Masad

32. (k) C-Reactive Protein "CRP" • Synthesized in the liver. • CRP was so named because it was shown to bind with the C-Polysaccaride on the CW of Streptococcus pneumonia • It enhances phagocytosis of bacteria. • It is elevated in acute rheumatic fever, bacterial infections, myocardial infarction, rheumatoid arthritis, carcinomatosis, gout & viral infection Dr Atef Masad

33. CRP is a general marker for inflammation and infection, so it can be used as a very rough proxy for heart disease risk. • Since many things can cause elevated CRP, this is not a very specific prognostic indicator. • A level above 2.4 mg/L has been associated with a doubled risk of a coronary heart disease compared to levels below 1 mg/L • Whether elevated CRP has any predictive value of acute coronary events in the general population of all age ranges remains unclear. Dr Atef Masad

34. Cystatin C • Small protein, 120 aa, is a cysteine proteinase inhibitor • Produced and destroyed at a constant rate • Freely filtered by the glomeruls and completely reabsorbed and catabolised by the proximal tubular cells • Proposed as a sensitive endogenous serum marker for the GFR • When the GFR decreased indicates decreased function rate Dr Atef Masad

35. Recently, it has been studied for its role in predicting new-onset or deteriorating cardiovascular disease. • It also seems to play a role in brain disorders involving amyloid (a specific type of protein deposition), such as Alzheimer's disease. Dr Atef Masad

36. If kidney function and glomerular filtration rate decline, the blood levels of cystatin C rise. • Serum levels of cystatin C are a more precise test of kidney function (as represented by the glomerular filtration rate, GFR) than serum creatinine levels • It has been suggested that cystatin C might predict the risk of developing chronic kidney disease, thereby signaling a state of 'preclinical' kidney dysfunction Dr Atef Masad

37. Cystatin C levels have been reported to be altered in patients with cancer, thyroid dysfunctionand glucocorticoid therapy in somebut not allsituations. • Levels are influenced by cigarette smoking and levels of C-reactive protein. • The role of cystatin C to monitor GFR during pregnancy remains controversial. Like creatinine, the elimination of cystatin C via routes other than the kidney increase with worsening GFR. Dr Atef Masad

38. Amyloid • Insoluble protein aggregates formed due to alteration in protein structure known as b pleated sheets • Proteins abnormally deposited in organs and/or tissues • Amyloidosis can be inherited or due to chronic diseases, such as malignancies, Rheumatic disorders which cause overabundant or abnormal protein production. Dr Atef Masad

39. Considered as a common link between many unrelated diseases. Associated with considerable damage to tissues. Could be due to generation of free radicals and reactive oxygen species (ROS) by aggregating proteins. Applied and Natural Sciences International Conference - Alaqsa Univerity

40. For example amyloid deposits derived from a peptide called ‘amylin’ are accumulates specifically in the islets of Langerhans of the pancreas in the vast majority of cases of T2DM. • Could be due to protein misfolding Applied and Natural Sciences International Conference - Alaqsa Univerity

41. The ‘protein misfolding diseases’ or ‘proteopathies’ include several different neurodegenerative diseases, such as prion disease, Alzheimer, Parkinson, RA and more than 40 other diseases , • where various different proteins and peptides accumulate in the brain and central nervous system. Applied and Natural Sciences International Conference - Alaqsa Univerity

42. MYOGLOBIN • Heme protein found in striated and cardiac muscles. • It is released upon the damage of striated muscles, in acute myocardial infarction "AMI", the increase is seen within 1-3 h of onset and reaches peak in 5-12h. • For diagnosis of AMI, serum myoglobin should be measured serially. • CK-MB peak (6-18h). Dr Atef Masad

43. TROPONIN • Troponin is a complex of 3 proteins that bind to the thin filaments of striated muscles "cardiac and skeletal" but not smooth muscles. • Troponin complex consists of: • Troponin T "TnT". • Troponin 1 "Tn1". • Troponin C "TnC". • All function to regulate muscle contraction. Dr Atef Masad

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45. Cardiac troponin T levels in serum begin to rise within 3-4 h following the onset of myocardial damage, peak in 10-24 h and remain elevated for 10 days – 14 days following AMI. • Its relative increase is greater than CK-MB (6-18h) or myoglobin. Dr Atef Masad

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48. Clinical significance of total proteins • Hypoproteinemia • Nephritic syndrome- Excessive loss - alb kidney leakage • Decreased intake in diet or due to malabsorption. • Liver disease. • Inherited immunodeficiency. • Burns, bleeding - fluid loss with rapid water replacement and slower protein replacement • Accelerated catabolism • Salt retention – leads to water retention and protein dilution Dr Atef Masad

49. Hyperproteinemia • Dehydration – increases the total protein 10-20% due to decreased body water – from decreased intake or excessive water loss. • Multiple Myeloma - Excessive production of  globulin (myeloma proteins) 1/2/2020 Dr Atef Masad 49

50. Measuring proteins By reactivity Biuret reaction, Lowry method By chemical properties Absorption at =260 nm (Phe) or 280 nm (Tyr, Trp) By activity Enzymes By immunogenicity Dr Atef Masad