BIOCHEMISTRY AND ITS ROLE IN DISEASE DIAGNOSIS

1. BIOCHEMISTRY AND ITS ROLE IN DISEASE DIAGNOSIS Dr. A. K. Dwivedi

2. INTRODUCTION TOBIOCHEMISTRY

3. BIOCHEMISTRY deals with the chemical prosess taking place in all living organisms from smallest viruses to bacteria to largest living matter.

4. It is defined as , THE study of chemical composition of living matter and chemical changes that occur in it during life process.

5. HSTORY: First intrdused by CARL NEWBERG,a germen scientist in1903. Concept was given by:Karl william scheel

6. INCLUDES study of Carbohydrates Protiens,aminoacids,peptides Lipids Enzymes Vitamins etc.

7. INTRODUCTION TO ENZYMES

9. Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. Because of their role in maintaining life processes, the assay and pharmacological regulation of enzymes have become key elements in clinical diagnosis and therapeutics.

10. The macromolecular components of almost all enzymes are composed of protein, except for a class of RNA modifying catalysts known as ribozymes. Ribozymes are molecules of ribonucleic acid that catalyze reactions on the phosphodiester bond of other RNAs.

11. Enzymes are found in all tissues and fluids of the body. Intracellular enzymes catalyze the reactions of metabolic pathways.

12. Plasma membrane enzymes regulate catalysis within cells in response to extracellular signals,

13. and enzymes of the circulatory system are responsible for regulating the clotting of blood

14. Almost every significant life process is dependent on enzyme activity.

15. Enzyme Classifications

16. Currently enzymes are grouped into six functional classes by the International Union of Biochemists (I.U.B.).

20. Enzymes are also classified on the basis of their composition. Enzymes composed wholly of protein are known as simple enzymes in contrast to complex enzymes, which are composed of protein plus a relatively small organic molecule. Complex enzymes are also known as holoenzymes.

21. In this terminology the protein component is known as the apoenzyme, while the non-protein component is known as the coenzyme

22. Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. It should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes, which lack sufficient cofactors derived from vitamins to maintain homeostasis.

23. Enzymes that require a metal in their composition are known as metalloenzymes

26. Introduction - Enzyme Characteristics: • The basic mechanism by which enzymes catalyze chemical reactions begins with the binding of the substrate (or substrates) to the active site on the enzyme. The active site is the specific region of the enzyme which combines with the substrate. The binding of the substrate to the enzyme causes changes in the distribution of electrons in the chemical bonds of the substrate and ultimately causes the reactions that lead to the formation of products. The products are released from the enzyme surface to regenerate the enzyme for another reaction cycle.

27. The active site has a unique geometric shape that is complementary to the geometric shape of a substrate molecule, similar to the fit of puzzle pieces. This means that enzymes specifically react with only one or a very few similar compounds. • Lock and Key Theory: • The specific action of an enzyme with a single substrate can be explained using a Lock and Key analogy first postulated in 1894 by Emil Fischer. In this analogy, the lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme).

28. Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do not fit into the lock (enzyme). Only the correctly shaped key opens a particular lock. This is illustrated in graphic on the left. • QUES: Using a diagram and in your own words, describe the various lock and key theory of enzyme action in relation to a correct and incorrect substrate.

29. Induced Fit Theory: Not all experimental evidence can be adequately explained by using the so-called rigid enzyme model assumed by the lock and key theory. For this reason, a modification called the induced-fit

30. theory has been proposed. The induced-fit theory assumes that the substrate plays a role in determining the final shape of the enzyme and that the enzyme is partially flexible. This explains why certain compounds can bind to the enzyme but do not react because the enzyme has been distorted too much. Other molecules may be too small to induce the proper alignment and therefore cannot react. Only the proper substrate is capable of inducing the

31. Induced Fit Theory: Not all experimental evidence can be adequately explained by using the so-called rigid enzyme model assumed by the lock and key theory. For this reason, a modification called the induced-fit theory has been proposed. The induced-fit theory assumes that the substrate plays a role in determining the final shape of the enzyme and that the enzyme is partially flexible. This explains why certain compounds can bind to the enzyme but do not react because the enzyme has been distorted too much. Other molecules may be too small to induce the proper alignment and therefore cannot react. Only the proper substrate is capable of inducing the proper alignment of the active site. In the graphic on the left, the substrate is represented by the magenta molecule, the enzyme protein is represented by the green and cyan colors. The cyan colored protein is used to more sharply define the active site. The protein chains are flexible and fit around the substrate.

33. Enzymes in the Diagnosis of Pathology

34. The measurement of the serum levels of numerous enzymes has been shown to be of diagnostic significance. This is because the presence of these enzymes in the serum indicates that tissue or cellular damage has occurred resulting in the release of intracellular components into the blood. Hence, when a physician indicates that he/she is going to assay for liver enzymes, the purpose is to ascertain the potential for liver cell damage.

35. Commonly assayed enzymes are the amino transferases: alanine transaminase, ALT (sometimes still referred to as serum glutamate-pyruvate aminotransferase, SGPT) and aspartate aminotransferase, AST (also referred to as serum glutamate-oxaloacetate aminotransferase, SGOT); lactate dehydrogenase, LDH; creatine kinase, CK (also called creatine phosphokinase, CPK); gamma-glutamyl transpeptidase, GGT. Other enzymes are assayed under a variety of different clinical situations but they will not be covered here.

36. The typical liver enzymes measured are AST and ALT. ALT is particularly diagnostic of liver involvement as this enzyme is found predominantly in hepatocytes. When assaying for both ALT and AST the ratio of the level of these two enzymes can also be diagnostic. Normally in liver disease or damage that is not of viral origin the ratio of ALT/AST is less than 1. However, with viral hepatitis the ALT/AST ratio will be greater than 1. Measurement of AST is useful not only for liver involvement but also for heart disease or damage.

37. The level of AST elevation in the serum is directly proportional to the number of cells involved as well as on the time following injury that the AST assay was performed. Following injury, levels of AST rise within 8 hours and peak 24-36 hours later. Within 3-7 days the level of AST should return to pre-injury levels, provided a continuous insult is not present or further injury occurs. Although measurement of AST is not, in and of itself, diagnostic for myocardial infarction, taken together with LDH and CK measurements (see below) the level of AST is useful for timing of the infarct.

38. The measurement of LDH is especially diagnostic for myocardial infarction because this enzyme exist in 5 closely related, but slightly different forms (isozymes). The 5 types and their normal distribution and levels in non-disease/injury are listed below. LDH 1 - Found in heart and red-blood cells and is 17% - 27% of the normal serum total. LDH 2 - Found in heart and red-blood cells and is 27% - 37% of the normal serum total. LDH 3 - Found in a variety of organs and is 18% - 25% of the normal serum total. LDH 4 - Found in a variety of organs and is 3% - 8% of the normal serum total. LDH 5 - Found in liver and skeletal muscle and is 0% - 5% of the normal serum total.

39. Following a myocardial infarct the serum levels of LDH rise within 24-48 hours reaching a peak by 2-3 days and return to normal in 5-10 days. Especially diagnostic is a comparison of the LDH-1/LDH-2 ratio. Normally, this ration is less than 1. A reversal of this ration is referred to as a "flipped LDH.". Following an acute myocardial infart the flipped LDH ratio will appear in 12-24 hours and is definitely present by 48 hours in over 80% of patients. Also important is the fact that persons suffering chest pain due to angina only will not likely have altered LDH levels.

40. CPK is found primarily in heart and skeletal muscle as well as the brain. Therefore, measurement of serum CPK levels is a good diagnostic for injury to these tissues. The levels of CPK will rise within 6 hours of injury and peak by around 18 hours. If the injury is not persistent the level of CK returns to normal within 2-3 days.

41. PRESENTATION PRESENTATION Seminar Presentation On PROTEINS BY ANKUSH VANI

42. INTRODUCTION The proteins are complex molecules built mainly from a-amino acid linked together in chains. The linkage between the amino acids is called peptide bond; molecules built up from many (up to 100) amino acids are called polypeptides. Proteins consist of several polypeptide chains, cross-linkaged between specific amino acid units. Chains containing 2-10 amino acids are called peptides.

43. Amino Acids The principal amino acids obtained by breakdown of proteins are: Neutral amino acids - they contain one NH2 (basic) group and one COOH (acidic) group which mutually neutralize each other. Types: Amino acids with unsubstantiated C chains: glycine, almandine, valine, leucine, isolucine. (ii) Hydroxyl-substituted amino acids: serine, threonine. (iii) Sulphur containing amino acids: cytosine, cystine (oxidative product of cytosine), methionine. (iv) Aromatic amino acids, derived from almandine: phenylalanine, tyrosine,thyroxine, triiodothyro-nine

44. 2. Acidic amino acids - amino acids with acidic side chain: aspartic acid, asparagine, glutamic acid, glutamine. 3. Basic amino acids - amino acids with basic side chain: arginine, lysine, histidine. 4. Imino acids - contains imino group but no amino group: proline, hydroxyproline.

45. C. Digestion and absorption of proteins. Digestion of proteins in the stomach pepsin is the most important proteolytic enzyme of gastric juiceOptimum pH for the activite of pepsin is 2 to 3 and it is completely inactive at a pH above 5 . The hydrochloric acid in the gastric juice provides the ideal pH for the activity of pepsin . Pepsin acts on proteins and breaks them down into proteoses , peptones and large polypestides . So , the proteins reach the duodenum in these forms along with chyme.

46. Proteins Pepsin (Gastric Juice) Proteoses Peptones Large polypeptides Trypsin Chymotrypsin (Endopeptidases in Pancreatic juice) Dipeptides Tripeptides Polypeptides Carboxy peptidase Peptidases (Exopeptidase in (Succus pancreatic juice entericus) Amino acids

47. DIGESTION OF PROTEINS IN THE SMALL INTESTINE Most of the p[roteins are digested in the doudenum and jejunum by the proteolytic enzymes of the pancreatic juice and succus entericus. Pancreatic juice contains trypsin, chymotrypsin and carboxy peptidases. Trypsin and chymotrypsin are called endopeptidases as these two enzymes break the interior bonds of the protein molecules. Both the enzymes act on proteoses and peptonessplit then into dipeptide and tripeptide molecules are absorbed directly into the epithelial cells of the mucosa of the small intestine. Carboxypeptidase from pancreatic juice breaks the terminal bonds of the protein molecules. So, it is called exopeptidase. By the activity of carboxypeptidase, the dipeptides, tripeptides and the polypeptides are converted into amino acids. The last digestion of the proteins is by proteolytic enzymes present in the succus entericus. It contains dipeptidases, tripeptidases and aminopolypeptidases. These enzymes act on large polypeptides and some of the left over dipeptides and tripeptides and convert these proteins into the final stage of single amino acids, which can be easily absorbed. Pancreatic juice contains two more enzymes namely, collagenase and elastease. Collagenase acts on collagen and elastase acts on elastic fibers.

48. ABSORPTION OF PROTEINS The proteins are absorbed in the form of amino acids from small intestine. The levoamino acids are actively absorbed by means of sodium co-transport, whereas, the dextroamino acids are absorbed by means of facilitated diffusion. The absorption of amino acids is faster in duodenum and jejunum and slower in ileum.

49. D. Amino Acid Pool:- Most of the tissue proteins (structural as well as functional protein) are continuously undergoing disintegration to release amino acids. The amino acids derived from food (exogenous protein) and those derived from the tissues break down (endogenous protein) enter the circulation forming general ammo acid pool. It represents an availability of amino acid building units. From this common amino acid pool, amino acids are taken up by the cells, if a cell takes up as much amino acid as it loses, it is in a state of dynamic equilibrium; if the loss is greater, the cell degenerates; if the gain is greater, the cell grows. The proteins of the body are in a state of dynamic equilibrium i.e. a balance between simultaneous breakdown and synthesis. The endogenous protein turnover rate is about 80-100 gm/day being greatest in intestinal mucosa, followed by kidney, liver, brain and muscle in that order.

50. E. Essential Amino acids These are the amino acids needed for replacement and growth, but which cannot be synthesized by the body in amounts sufficient to fulfil its normal requirements. The rest of the amino acids are the non-essential amino acids and can be synthesized in the body. It has been found that the following amino acids are indispensable for human adults under normal conditions: valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, histidine and arginine .