Introduction to basic Chromatography.pptx

Introduction to basic Chromatography

1. 7/25/2019 sumit prajapati 1 Research Methodology: Separation Techniques

2. CONTENTS Introduction to chromatography History Principles Importance Chromatographic terms Classification of chromatography Adsorption chromatography Partition chromatography Gas-liquid phase chromatrography Solid-liquid phase chromatrography Liquid-gas phase chromatrography Liquid-liquid phase chromatrography Important properties of liquid phase Conclusion 2

3. Chromatography Chromatography (from Greek chroma "color and graphein "to write") is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases . 3

4. History Chromatography, literally "color writing", was first employed by Russian scientist Mikhail Tsvet in 1900. He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. Since these components have different colors (green, orange, and yellow , respectively ) they gave the technique its name. 4

5. Principles Chromatography usually consists of mobile phase and stationary phase. The mobile phase refers to the mixture of substances to be separated dissolved in a liquid or a gas. The stationary phase is a porous solid matrix through which the sample contained in the mobile phase percolates. The interaction between the mobile phase and the stationary phase results in the separation of the compound from the mixture. 5

6. Applications of chromatography The chromatographic technique is used for the separation of amino acids , proteins & carbohydrates. It is also used for the analysis of drugs , hormones,vitamins . Helpful for the qualitative & quantitative analysis of complex mixtures. The technique is also useful for the determination of molecular weight of proteins. 6

7. The chromatographic method of separation, in general, involves following steps Adsorption or retention of substances on the stationary phase Separation of the adsorption of substances by the mobile phase Recovery of the separated substances by a continuous flow of the mobile phase; the method being called elution Qualitative and Quantitative analysis of the eluted substances 7

8. Chromatographic terms The analyte is the substance to be separated during chromatography. A chromatogram is the visual output of the chromatograph. The eluate is the mobile phase leaving the column. The eluent is the solvent that carries the analyte The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation. 8

9. Classification of chromatography 1. Based on mechanism of separation adsorption chromatography Partition chromatography 2. Based on phases Solid phase chromatography Solid-liquid chromatography Solid-gas chromatography Liquid phase chromatography Liquid-liquid chromatography Liquid –gas chromatography 3. Based on shape of chromatographic bed Planner chromatography Paper chromatography Thin layer chromatography Column chromatography Packed column chromatography Open tubular column chromatography 9

10. Flow chart diagram of chromatography 10

11. Adsorption chromatograohy It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase The equilibriation between the mobile and stationary phase accounts for the separation of different solutes. Adsorption chromatography is process of separation of components in a mixture introduced into chromatography system based on the relative difference in adsorption of components to stationary phase present in chromatography column 11 sumit prajapati

12. Partition chromatography This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase Solute equilibrates between the mobile phase and the stationary liquid. Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase or on differences between the solubility of the components in the mobile and stationary phases 12 sumit prajapati

13. Gas-Solid chromatography(G.S.C.) Gas chromatography employs an inert gas as the mobile phase Separation depends on the relative partial pressures of the sample components above the stationary phase. Gas-solid chromatography is relatively rare, but it is used to separate atmospheric gases Common solids are charcoal, a synthetic zeolite called "molecular sieve", or a combination of the two . The mobile phase is a gas, often nitrogen, but sometimes helium, hydrogen or occasionally another gas. It is called the "carrier gas". 13

14. Solid-Liquid chromatography Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid . Liquid chromatography can be carried out either in a column or a plane In liquid-solid chromatography the porous adsorbent is polar and separation is based on the properties of classes of compounds—e.g., amines (alkaline) from alcohols (neutral) and esters (neutral) from acids The preferred mobile phase is a nonpolar or slightly polar... Popular adsorbents are Silica and Alumina . 14

15. Liquid-Gas Chromatography Dimethyl Polysiloxane (350 o C) Hydrocarbons, Polynuclear aromatics Poly(phenyl methyl) siloxane (250 o C) Steroids, Pesticides, Glycols Stationary phase used in (LGC) The mobile phase is an unreactive gas, such as nitrogen (the carrier gas ) The stationary phase comprises of a small amount of liquid held on a finely-divided inert solid support. Gas-liquid chromatography is very sensitive and can be used to detect small quantities of substances it is often used in forensic tests 15

16. Liquid-Liquid Chromatography Liquid-liquid chromatography is a chromatography separation technique in which the mobile phase is a liquid ( usually a solvent or a simple binary solvent mixture ) and the stationary phase is also a liquid ( which must be immiscible and insoluble in the liquid mobile phase). The first liquid-liquid system was reported by A. J. P. Martin who used water supported on silica gel as the stationary phase and n-heptane as the mobile phase The system is inherently unstable, as the stationary phase will always have some solubility in mobile phase 16

17. Planner chromatography Planar chromatography is a separation technique in which the stationary phase is present on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography ) or a layer of solid particles spread on a support such as a glass plate (Thin layer chromatography ). Different compound s in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (R f ) of each chemical can be used to aid in the identification of an unknown substance. 17

18. Column Chromatography Column chromatography is a separation technique in which the stationary bed is within a tube . The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube ( packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube ( open tubular column ). Differences in rates of movement through the medium are calculated to different retention times of the sample 18

19. Important properties of liquid stationary phase Liquid phase should have low volatility and high stability at elevated temperatures Liquid phase should not permeate too deeply into the fine pores of the support structure as slow diffusion in and out of pores affects column efficiency Small particles of support give higher efficiency as HETP is proportional to particle diameter but particle size reduction increases back pressure Support should be deactivated before use as undesirable surface impurities can cause decomposition of the sample or stationary liquid 19

20. Conclusion In overall ranking Chromatography techniques , it can be judge SFC falls somewhere between HPLC or GC. In field of pharmaceutical chemistry and bioanalytical application gained its applications 20

21. Gel Filtration Gel permeation chromatography Size exclusion chromatography Separation of molecules on the basis of size (and shape)

22. Theory Column matrix Porous beads Large molecules are “excluded” from the pores and travel through the column fastest Small molecules are “included” – can diffuse into the pores and elute later

23. Theory

24. Elution Profile Ve Ve Ve Ve = Elution volume (volume of solvent between injection and elution). Dictated by proportion of porous matrix available to molecules (Kd).

25. Column Parameters Vs= volume of solvent held in the pores. This is normally approximated to Vt-Vo = volume of beads Vo = void volume Vt = total volume Vo = Elution volume of a large “totally excluded” molecule such as blue dextran Vt = Physical volume of column

26. Calculation of Ve For a molecule that can partially enter the pores: Ve = Vo + Kd (Vs) or Ve = Vo + Kav (Vt-Vo) Kav = proportion of pores available to the molecule. Totally “exclude” Kav = 0 and Ve = Vo Totally “included” Kav = 1 and Ve = Vt

27. Behaviour of Molecule on any Column Kav = Ve – Vo Vt - Vo

28. Resolution Resolution proportional to square root of column length. Also affected by rate at which column is run

29. Design of Column Column size Analytical or preparative Solvent Inert matrix most solvents OK Matrix Most important consideration Many different types Material Pore size

30. Matrix Types Material Sephacryl dextran Sephadex dextran Sepherose agarose Superdex mixture Sephacryl Protein (kD) Dextrans (kD) S-100 1-100 NS S-200 5-250 1-80 S-300 10-1500 2-400 S-400 20-8000 10-2000 S-500 NS 40-20,000

31. Running the column Sample size / Fraction size 0.5 – 5% of total bed volume (Vt). Concentration limited by viscosity Running time Determined by “trial and error” Slow rates allow efficient partitioning into pores and thus increase resolution Slow rates increase diffusion of sample on column thus increasing peak width and reducing resolution. Protein about 5mL cm -2 . h -1

32. Types of Column Systems Liquid Chromatography High Performance Liquid Chromatography (HPLC)

33. Determination of Molecular Weight Calibrate column with known standards Plot Kav against lg Mol Wt

34. Other Types of Column Chromatography Ion-Exchange Chromatography Separation on basis of charge DEAE- sephadex Hydrophobic Interaction Chromatography Separation on basis of hydrophobicity Phenyl-sepherose Affinity Chromatography Affinity of enzyme for substrate or other ligand

35. ION EXCHANGE CHROMATOGRAPHY

36. Principle…… Different affinity of the different components to stationary phase causes the separation

37. Ion Exchange Chromatography Ion exchange chromatography -- is a separation based on charge Used for almost any kind of charged molecules --- large proteins, small nucleotides and amino acids Ion-exchange chromatography preserves analyte molecules on the column based on ionic interactions Mobile phage – buffer, pH and salt concentration---opposite charged solute ions attracted to the stationary phage by electrostatic force Stationary phage– resin is used to covalently attach anions or cations onto it

38. Principle………. Ion Exchange Chromatography relies on charge-charge interactions between the proteins

39. Types of IEC…. anion exchangers cation exchangers

40. Cation exchange chromatography ---positively charged molecules are attracted to a negatively charged solid support. Commonly used cation exchange resins are S-resin, sulfate derivatives; and CM resins, carboxylate derived ions      

41. Anion exchange chromatography ---negatively charged molecules is attracted to a positively charged solid support. Commonly used anion exchange resins are Q-resin, a Quaternary amine; and DEAE resin, DiEthylAminoEthane

42. Buffers Used In IEC Buffer system 1 : Buffer A = 20 mM Tris, pH=8. Buffer B = 20 mM Tris, 1 M NaCl, pH=8.0 Buffer system 2: (Common CEC buffer system): Buffer A = 30 mM sodium acetate, pH=4.5. Buffer B = 30 mM sodium acetate, 1 M NaCl, pH=4 Buffer system 3: (AEC for proteins which are very insoluble or have a very high pI) Buffer A = 30 mM Ethanolamine, 8M urea, pH=10.0 Buffer B = 30 mM Ethanolamine, 8M urea, 1 M NaCl, pH=10.0

43. Chromatography Methods Column washed with buffer A to equilibrate Buffer B is used to equilibrate again Equilibrate the column with buffer A Sample loading Flow through collection Elute protein

44. Advantages   It is a non-denaturing technique. It can be used at all stages and scales of purification An IEX separation can be controlled by changing pH, salt concentration and/or the ion exchange media It can serve as a concentrating step. A large volume of dilute sample can be applied to a media, and the adsorbed protein subsequently eluted in a smaller volume It offers high selectivity; it can resolve molecules with small differences in charge.

45. Disadvantages costly equipment and more expensive chemicals turbidity should be below 10ppm Conclusion Ion exchange chromatography is more efficient than other chromatography. It could be widely used for commercial purposes.

46. High Performance Liquid Chromatography

47. Introduction HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.

48. HPLC system

49. FOUR TYPES OF LIQUID CHROMATOGRAPHY Partition chromatography Adsorption, or liquid-solid Ion exchange chromatography Size exclusion, or gel, chromatography

50. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM Solvent Solvent Delivery System (Pump) Injector Sample Column Detectors (Diode Array) Waste Collector Recorder (Data Collection)

51. Picture of HPLC instrument

52. Uses of HPLC This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation. In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.

53. HPLC Chromatograph injectors The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column. When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered to the vial.

54. HPLC columns The column is one of the most important components of the HPLC chromatograph because the separation of the sample components is achieved when those components pass through the column. The High performance liquid chromatography apparatus is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm. Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, is inert to most compounds and has a high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible.

55. Picture of an HPLC column

56. WHAT AFFECTS SYSTEM Column Parameters Column Material Deactivation Stationary Phase Coating Material Instrument Parameters Temperature Flow Signal Sample Sensitivity Detector

57. WHAT AFFECTS SYSTEM Sample Parameters Concentration Matrix Solvent Effect Sample Effect

58. Several column types (can be classified as ) Normal phase Reverse phase Size exclusion Ion exchange

59. Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

60. Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile -water.

61. Size exclusion In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size . Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.

62. Ion exchange In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid , usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.

63. Selectivity Factor K’ values tell us where bands elute relative to the void volume. These values are unaffected by such variables as flow rate and column dimensions. The value tell us where two peaks elute relative to each other. This is referred to as the selectivity factor or separation factor (now and then as the chemistry factor).

64. Types of Liquid Column Chromatography (LCC) LLC (Liquid Liquid) LSC (Liquid Solid - adsorption) SEC ( Size Exclusion) GLC GSC SFC (Supercritical Fluid)

65. Types of Detectors Absorbance (UV with Filters, UV with Monochromators) IR Absorbance Fluorescence Refractive-Index Evaporative Light Scattering Detector (ELSD) Electrochemical Mass-Spectrometric Photo-Diode Array

66. EVALUATION PARAMETERS EFFICIENCY RESOLUTION INERTNESS RETENTION INDEX COLUMN BLEED CAPACITY FACTOR

67. Electrophoresis Electrophoresis is the migration of charged molecules,particles or ion in a liquid medium under the influence of an electric field Various types – defined by support used Paper – amino acids, small peptides Polyacrylamide – Proteins, small DNA/RNA (<500bp) Agarose – DNA/RNA Good preparative and analytical method

68. From large to small and simple

69. Principle Proteins move in the electric field. Their relative speed depends on the charge, size, and shape of the protein

70. instrumentation and reagents: (1) Two buffer boxes contain the buffer used in the process. (2) Each buffer box contains an electrode made of either platinum or carbon, the polarity of which is determined by the mode of connection to the power supply. (3)The electrophoresis support on which separation takes place may contact the buffer directly, or by means of wicks (4)The entire apparatus is covered to minimize evaporation and protect the system (5) The power supply to provide electrical power. Technique of electrophoresis

71. General operations performed in conventional electrophoresis include: (1) separation (2) staining (3) detection (4) Quantification General Procedure

72. SAMPLE APPLICATION : The sample may be applied as a spot (about 0.5 cm in diameter) or as a uniform streak. ELECTROPHORETIC RUN : The current is switched on after the sample has been applied to the paper and the paper has been equilibrated with the buffer. .The types of buffer used depends upon the type of separation. Once removed, the paper is dried in vacuum oven . DETECTION AND QUANTITATIVE ASSAY: To identify unknown components in the resolved mixture the electrophoretogram may be compared with another electrophoretogram on which standard components have been electrophoresced under identical conditions. Physical properties like fluorescence, ultraviolet absorption or radioactivity are exploited for detection.

73. Electro-osmosis:

74. A simplified schematic drawing of a protein pattern separated by cellulose acetate paper electrophoresis

75. What is a gel? Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules. Types of Gel: Agarose gel. Polyacrylamide gel. GEL ELECTROPHORESIS

76. Gel Electrophoresis Gel electrophoresis uses a cross-linked polymers ( agarose ) that contain various pores. Pores allow molecular sieving, where molecules e.g. DNA, can be separated based upon there mobility through the gel.

77. A highly purified uncharged polysaccharide derived from agar. Used to separate macromolecules such as nucleic acids, large proteins and protein complexes. It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40 °C. It is fragile because of the formation of weak hydrogen bonds and hydrophobic bonds. AGAROSE GEL

78. Used to separate most proteins and small oligonucleotides because of the presence of small pores . POLYACRYLAMIDE GEL

79. Detection Dye e.g. ethidium bromide Audioradiography 32 P, Blotting (see later) Uses Analytical- Can determine size of DNA fragment, Preparative – Can identify a specific fragment based on size DNA Gel Electrophoresis

80. 2D-gel ( coomassie stained )

81. Example of silver stained gel Silver staining is usually 10-100 times more sensitive than Coomassie Blue staining, but it is more complicated. Faint but still visible bands on this gel contain less than 0.5 ng of protein!

82. Electrophoretic method that separates proteins according to the iso-electric points Is ideal for seperation of amphoteric substances Seperation is achieved by applying a potential difference across a gel that contain a pH gradient Isoelectric focusing requires solid support such as agarose gel and polyacrylamide gel ISOELECTRIC FOCUSING

83. IEF Separates proteins by their isoelectric points ( pI ) Each protein has own pI = pH at which the protein has equal amount of positive and negative charges (the net charge is zero)

84. IEF example Zavialov A. IEF 4-6.5 pH gradient

85. IEF Mixtures of ampholytes , small amphoteric molecules with high buffering capacity near their pI , are used to generate the pH gradient. Positively and negatively charged proteins move to – and +, respectively, until they reach pI . PI of proteins can be theoretically predicted. Therefore, IEF can also be used for protein identification.

86. A TYPICAL ISOELECTRIC FOCUSING GEL

87. Using specific probes that are labelled specific sequences of DNA can be identified. There are three main hybridization techniques which vary in the sample blotted and the probes used; Northern Blot -Transfer of an RNA sample separated and identified using DNA or RNA probes. Southern Blot -Transfer of an DNA sample separated and identified using DNA or RNA probes. Western Blot - Transfer of an Protein sample separated and identified typically using an antibody. Blotting Techniques

88. Blotting – Transfer of DNA, RNA or Proteins, typically from a electrophoresis gel to a membrane e.g. nitrocellulose. This membrane can then be subject to further techniques such as hybridization. Hybridization – Process where two complementary single strands of nucleic acid (DNA or RNA) form a double helix. Blotting Techniques

89. Western Blotting (WB) WB is a protein detection technique that combines the separation power of SDS PAGE together with high recognition specificity of antibodies An antibody against the target protein could be purified from serum of animals (mice, rabbits, goats) immunized with this protein Alternatively, if protein contains a commonly used tag or epitope, an antibody against the tag/epitope could be purchase from a commercial source (e.g. anti-6 His antibody)

90. WB: 4 steps Separation of proteins using SDS PAGE 2. Transfer of the proteins onto e.g. a nitrocellulose membrane (blotting) 3. Immune reactions 4. Visualization

91. WB, Step 2: Blotting

92. WB, Steps 3-4: Detection

93. This technique combines the technique IEF (first dimension), which separates proteins in a mixture according to charge (PI), with the size separation technique of SDS-PAGE second dimension). The combination of these two technique to give two-dimension(2-D)PAGE provides a highly sophisticated analytical method for analysing protein mixtures. TWO-DIMENSIONAL ELECTROPHORESIS

94. Using this method one can routinely resolve between 1000 and 3000 proteins from a cell or tissue extract and in some cases workers have reported the separation of between 5000 and 10000 proteins. The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used stains are silver and coomasie staining.

95. Capillary electrophoresis

96. In CE, the classic techniques of electrophoresis are carried out in a small-bore, fused silica capillary tube, the outer diameter of such tubes typically varies from 180 to 375 micrometer, the inner diameter from 20 to 180 micrometer, and the total length from 20 cm up to several meters. This capillary tube serves as a capillary electrophoretic chamber that is connected to a detector at its terminal end and, via buffer reservoirs, to a high-voltage power supply The main advantage of CE comes from efficient heat dissipation compared with traditional electrophoresis. Improved heat dissipation permits the application of voltages in the range of 20 to 30 kV, which enhances separation efficiency and reduces separation time in some cases to less than 1 minute Capillary electrophoresis

97. Microchip electrophoresis

98. 1. Discontinuities in sample application : may be due to dirty applicators, which are best cleaned by agitating in water followed by gently pressing the applicators against absorbent paper. Caution must be used, and it is inadvisable to clean wires or combs by manual wiping. 2. Unequal migration of samples across the width of the gel may be due to dirty electrodes causing uneven application of the electrical field or to uneven wetting of the gel. 3. Distorted protein zones may be due to bent applicators. incorporation of an air bubble during sample application. over application of sample. excessive drying of the electrophoretic support before or during electrophoresis. The following problems may be encountered when peforming gel electrophoresis .

99. 4. Irregularities (other than broken zones) in sample application probably are due to excessively wet agarose gels. Parts of the applied samples may look washed out. Unusual bands are usually artifacts that may be easily recognized. Atypical bands in an isoenzyme pattern may be the result of binding by an immunoglobulin. An irregular , but sharp protein zone at the starting point that lacks the regular, somewhat diffuse appearance of proteins may actually be denatured protein resulting from a deteriorated serum.

100. Isoenzymes 110

101. Isozymes (also known as isoenzymes ) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction . These enzymes usually display different kinetic parameters (e.g. different K m values), or different regulatory properties. Catalyze the same reactions but are formed from structurally different polypeptides. They perform the same catalytic function. Various isoenzymes of an enzyme can differ in three major ways: Enzymatic properties Physical properties ( e.g. heat stability) Biochemical properties such as amino acid composition and immunological reactivities . 111

102. Lactate dehydrogenase (LDH) Pyruvate → Lactate (anaerobic glycolysis) LDH is elevated in myocardial infarction, blood disorders It is a tetrameric protein and made of two types of subunits namely H = Heart, M = skeletal muscle It exists as 5 different isoenzymes with various combinations of H and M subunits 112

103. Isoenzyme name Composition Present in Elevated in LDH 1 ( H 4 ) HHHH Myocardium, RBC myocardial infarction LDH 2 (H 3 M 1 ) HHHM Myocardium, RBC LDH 3 (H 2 M 2 ) HHMM Kidney, Skeletal muscle LDH 4 (H 1 M 3 ) HMMM Kidney, Skeletal muscle LDH 5 (M 4 ) MMMM Skeletal muscle, Liver Skeletal muscle and liver diseases 113

104. Creatine Kinase (CK) Creatine + ATP → phosphocreatine + ADP (Phosphocreatine – serves as energy reserve during muscle contraction) Creatine kinase is a dimer made of two monomers occurs in the tissues. Skeletal muscle contains M subunit, Brain contains B subunits. Three different isoenzymes are formed. 114

105. Isoenzyme name Composition Present in Elevated in CK-1 BB Brain CNS diseases CK-2 MB Myocardium/ Heart Acute myocardial infarction CK-3 MM Skeletal muscle, Myocardium 115

106. Allosteric modulation Allosteric sites are sites on the enzyme that bind to molecules in the cellular environment. The sites form weak, noncovalent bonds with these molecules, causing a change in the conformation of the enzyme. This change in conformation translates to the active site, which then affects the reaction rate of the enzyme. Allosteric interactions can both inhibit and activate enzymes and are a common way that enzymes are controlled in the body. 116

107. 117

108. Clinical Enzymology 118

109. Serum enzymes increases may be due to:- Cell death - this results in a small short-lived increase (e.g., following myocardial infarction). Increased cell membrane permeability in living cells (due to hypoxia, inflammation, drugs/poisons, cellular swelling) gives rise to a large protracted increase in serum enzymes as there is ongoing enzyme synthesis (e.g., Duchenne muscular dystrophy, acute viral hepatitis). Increased synthesis in a specific cell type (e.g., gamma glutamyl transferase in liver cells is induced by alcohol or anticonvulsant drugs, alkaline phophatase in liver cells is induced by obstruction, lactate dehydrogenase is induced in neoplastic tissues). 119

110. CAUSES OF CELL DAMAGE OR DEATH 120

111. CREATINE KINASE (CK) 121

112. Important in tissues where significant metabolic energy is stored as creatine phosphate. Distribution : skeletal muscle, heart, brain. Isoenzyme composition : CK is a dimer consisting of sub-units M or B coded by two distinct genes. Thus 3 possible isoenzymes - M M ; M B ; B B Skeletal muscle - all MM; Heart - 80% MM, 20% MB; Brain - all BB. Normal pattern in serum - predominantly MM present, with MB < 6% of total CK. 122

113. Myocardial infarction Early increase of total CK, specifically the MB isoenzyme . Total CK levels start to rise at about 6 hours , peak at 18 to 30 hours, and return to normal by 3 days. If total CK raised, measurement of CK-MB is indicated. CK-MB levels may be raised by 4 hours, are almost certain to be raised by 12 hours, and may return to normal by 24 hours after MI. Used as a diagnostic test before 24 hours, and as a prognostic indicator (amount of increase reflects extent of cardiac damage ). 123

114. Skeletal muscle damage e.g., trauma, surgery (especially in cardiac surgery), over-exercise, convulsions, ischaemia , inflammation (myositis), malignant hyperthermia, congenital muscular dystrophy. increase of total CK, but MB isoenzyme not increased. In neurogenic muscle disease, e.g., poliomyelitis and Parkinsonism, CK levels are normal. In Duchenne Muscular Dystrophy, CK elevation precedes onset of symptoms by years, and falls as disease progresses. In chronic muscle disease there is reversion to the foetal isoenzyme pattern (MB appears in skeletal muscle and serum). Hypothyroidism is associated with high total CK levels. 124

115. LACTATE DEHYDROGENASE (LDH, LD ) 125

116. Important in the disposal of glycolytically generated NADH, particularly when mitochondrial disposal is impaired by hypoxia. Distribution : ubiquitous, including heart, skeletal muscle, liver and RBCs - specificity is improved by isoenzyme analysis. LD is a tetramer consisting of sub-units H or M coded by two different genes. 126

117. Anoxic tissues (liver, muscle) express M subunit, thus LD5 predominates. Erythrocyte precursors and heart express the H subunit, hence LD1 predominates in these tissues. Normal pattern in serum - predominantly LD2, with slightly less LD1, and even less LD3, LD4 and LD5. 127

118. Myocardial infarction - late and long-lasting increase in total LD. Total LD levels start to rise at about 8 to 12 hours, peak at 24 to 48 hours and return to normal by 10 days. The predominant isoenzyme is LD1, which is present in greater concentration than the normal LD2 (flipped pattern ). Liver damage , including viral or toxic hepatitis (ethanol, paracetamol overdose, carbon tetrachloride), cardiac failure (liver congestion) - increase in total LD, exclusively due to LD5. 128

119. Hematological disorders - often isolated elevation in total LD. Due to breakdown of circulating red cells or red cell precursors in bone marrow. Intra-vascular (or in vitro) haemolysis , e.g., due to an auto-immune disorder, prosthetic heart valve, inherited enzyme deficiency (G6PD, PK) - both LD1 and LD2 increased. Associated features include elevated serum unconjugated bilirubin, increased urine and stool urobilinogen , and decreased haptoglobin . Megaloblastic anaemia due to folate or vitamin B 12 deficiency : failure of cell division leads to cell lysis and enzyme release from the bone marrow - predominant increase in LD1 (as in myocardial infarction). Extremely high levels can be achieved. 129

120. Malignant tumours may manifest an isolated increase in serum LD due to enhanced synthesis of glycolytic enzymes by a wide variety of neoplasms (even measured in aqueous humour to diagnose retinoblastoma). Typically centripetal isoenzyme pattern (LD2, LD3 and LD4) due to expression of both subunits (H and M). An exception is seen in germ cell tumours which show an increase in LD1. 130

121. TRANSAMINASES 131

122. ASPARTATE TRANSAMINASE (AST) (aspartate + α- ketoglutarate -----> oxaloacetate + glutamate ). ALANINE TRANSAMINASE (ALT) (alanine + α- ketoglutarate -----> pyruvate + glutamate ). Wide distribution in tissues, including liver, skeletal muscle, heart, kidney and RBCs . Cofactor vitamin B6 (pyridoxine) - carries amino acid intermediates. 132

123. Acute hepatitis - high, sustained elevations of both ALT and AST (up to 100 x normal). ALT higher especially in mild injury; precedes onset of jaundice (allows early diagnosis. Disproportionate AST elevation indicates liver cell necrosis (involving mitochondria) or ethanol induced damage (acetaldehyde formed during ethanol metabolism depletes cytosolic pyridoxine, hence ALT activity selectively lost). Myocardial infarction - AST elevation of 5 - 10 x normal. AST levels start to rise at about 6 to 8 hours, peak at 18 to 24 hours and return to normal by 4 to 5 days. ALT hardly increases at all. AST always greater than ALT (de Ritis quotient AST/ALT > 5. In liver disease this ratio is usually < 1). 133

124. ALKALINE PHOSPHATASE (ALP) 134

125. Located in membranes, specifically the brush borders of the PCT of the kidney, the small intestinal mucosa, both the sinusoidal and canalicular surfaces of the hepatocyte, in osteoblasts in bone and in the placenta. Important in the formation of new bone by osteoblasts. Normal level in serum is determined by age and sex, reflecting periods of active bone growth, i.e., very high in young children. Normal pattern in children is preponderance of bone isoenzyme , while in adults liver isoenzyme predominates. 135

126. Bone disease Reflects increased osteoblastic activity i.e., bone synthesis. High levels of total ALP, due to increased bone isoenzyme , seen in: active bone growth (young children, at puberty, healing fractures ). Primary bone tumours ( osteogenic sarcoma). secondary tumours evoking a sclerotic response e.g., prostatic and breast metastases. Rickets (children) and osteomalacia (adults). long-standing primary or secondary hyperparathyroidism (e.g., chronic renal disease where calcium is resorbed from bone, leading to renal osteodystrophy ) 136

127. Liver disease Classic marker of cholestasis due to extra-hepatic (gallstones ) or intra-hepatic (drugs, inflammation) obstruction. Synthesis of liver isoenzyme induced by biliary obstruction - differentiates obstruction from hepatocellular damage. Elevated liver ALP without jaundice suggests : Intermittent or incomplete obstruction (gallstone). Intra-hepatic space-occupying mass ( tumour ). 137

128. Placental isoenzyme is found in the serum in late pregnancy and remains elevated a week or two after delivery. 138

129. GAMMA GLUTAMYL TRANSFERASE (GGT) 139

130. γ- glutamyl -N-donor + acceptor --> γ- glutamyl -N-acceptor + donor. Distribution : It present in all cells except muscle. Located in cell membranes and endoplasmic reticulum of hepatocytes . Role : Synthesis of reduced glutathione (GSH) required for drug detoxification e.g., paracetamol . Normal level in serum derived from liver. 140

131. Hepatic synthesis of GGT is induced by biliary obstruction. Serum levels correlate with liver ALP and 5’ nucleotidase . Very sensitive and specific marker of liver disease. Hepatic synthesis of GGT is also induced by drugs (especially barbiturates, antidepressants and anticonvulsants) and alcohol. Serum GGT is increased not just in patients with alcoholic liver disease, but also in people who are heavy drinkers. 141

132. ACID PHOSPHATASE (ACP) 142

133. Distribution Prostate , lysosomes of all cells, red blood cells (avoid hemolysis ). Number and genetics of isoenzymes not known, but at least prostatic and red cell isoenzymes exist. Identify prostatic isoenzyme by L-tartrate inhibitable activity. ACP is temperature and pH labile. So, specimens for enzyme assay should be submitted on ice. 143

134. Prostatic CA . High activity of ACP, especially the prostatic isoenzyme , indicates disseminated disease (usually spreads to bone). CA-in-situ and benign hypertrophy of the pr normal levels of ACP. Recently, ACP assays have been largely replaced by measurement of prostate-specific antigen. 2. Gaucher’s disease, bone destruction by infection and neoplasia . Lysosomal isoenzyme - tartrate insensitive, normal RIA result. ostate usually have 144

135. AMYLASE 145

136. Distribution Amylase secreted by the pancreas and salivary glands; also present in Fallopian tubes and small intestine. Two common isoenzymes occur, a pancreatic (P) and salivary (S) type, which can be differentiated using a wheat germ lectin which selectively inhibits the S isoenzyme . 146

137. Acute pancreatitis an increase in total amylase, in the appropriate clinical setting, is suggestive of acute pancreatitis. In cases of an acute abdomen, determination of isoenzyme type is seldom helpful - P isoenzyme is increased not only in acute pancreatitis, but also in perforated peptic ulcer and intestinal obstruction/ infarction Amylase only remains elevated in the serum for 3 to 4 days, but remains elevated in the urine for longer (6 days). Levels of both amylase and lipase are normal in chronic pancreatitis. 147

138. 7/25/2019 148 sumit prajapati Thank You All