Title Clinical Chemistry Review Booklet Part 2 SY2024-2025.docx.pdf ✅

Clinical Enzymology Enzyme Classification and Nomenclature: ∙ The first digit represents the class of enzymes. The second and third digits represent subclass of enzymes. The final number represents the serial number specific to each enzyme. 1. Oxidoreductases. Catalyze an oxidation-reduction reaction between two substrates 2. Transferases. Catalyze the transfer of a group other than hydrogen from one substrate to another 3. Hydrolases. Catalyze hydrolysis of various bonds 4. Lyases. Catalyze removal of groups from substrates without hydrolysis 5. Isomerases. Catalyze the interconversion of geometric, optical or positional isomers 6. Ligases. Catalyze the joining of two substrate molecules, coupled with breaking of the pyrophosphate bond in ATP or a similar compound Factors that Influence Enzyme Reaction: 1. Enzyme and substrate concentration Michaelis - Menten Theory V = Vmax [S] Km + [S] Where V stands for velocity, Vmax for maximum velocity, S for substrate and Km stands for Michaelis-Menten constant of enzyme for specific substrate ∙ Addition of substrate concentration will lead to increase in the rate of reaction E > S = ↑ S ↑ rate of reaction (FIRST-ORDER KINETICS) ∙ The reaction rate depends on enzyme concentration, enzyme becomes the rate-limiting factor S > E (ZERO-ORDER KINETICS) 2. pH ∙ Optimum range for most enzymes is at 7.0 – 8.0 ∙ Sudden changes may cause denaturation or alter the charge of amino acid residue on the active site; however, it can be prevented by means of buffer solutions 3. Temperature ∙ Enzyme are active at 5C, 30C, or 37C ∙ 37C is the optimum temperature for enzymatic activity ∙ The rate of denaturation is significant at 40 – 50C ∙ 60 – 65C may cause inactivation of enzymes ∙ Temperature coefficient (Q10) – for every 10C increase in temperature, enzyme reaction doubles
∙ In lab determinations, temp should not deviate from + 0.1C from the required temp to maintain accuracy of results o Creatine kinase – 37C o Amylase – 40C 4. Storage ∙ Repeated freezing and thawing denatures enzymes ∙ -20C – preservation for longer period of time ∙ 2 – 8C – ideal storage temp for substrate and coenzymes ∙ RT – ideal storage for LDH4 and LDH5; should not be stored less than 0C without loss of enzyme activity 5. Cofactors – nonprotein substances that may be necessary for enzyme reaction to take place ∙ Inorganic cofactors/activators – calcium, magnesium, manganese and chloride ∙ Organic cofactors/coenzymes a. NAD and flavine – required by oxidoreductases and dehydrogenases b. Pyridoxal phosphate – required by transaminases ∙ Metalloenzymes – inorganic ion attached to a molecule (catalase and cobalt) ∙ Hydrolases require no cofactors Coenzyme Reaction Type Deficiency Thiamine pyrophosphate Aldehyde transfer Beri-beri Folic acid coenzymes One-C transfer Megaloblastic anemia Cobamide (B12) Alkylation Pernicious anemia Nicotinamide Coenzyme Redox Pellagra 6. Inhibitors a. Competitive inhibition ∙ With a substrate concentration higher than the inhibitor concentration, the inhibition is reversible ∙ Physically binds to the active site of an enzyme b. Non-competitive inhibition ∙ Binds an enzyme at a place other than the active site ∙ Increasing substrate concentration does not reverse the inhibition c. Uncompetitive inhibition ∙ Inhibitor binds to enzyme-substrate complex ∙ Increasing the substrate concentration results in more ES complices thereby increasing inhibition 7. Hemolysis – falsely elevates most enzyme 8. Lactescence – falsely decreases enzyme concentration
Measurement of Enzyme Activity 1. Fixed-time / Endpoint Method ∙ Determines concentration of substrate or product at specific time after addition of sample ∙ Reaction proceeds for a designated time ∙ Weak acid is added to stop reaction 2. Continuous Monitoring / Kinetic Assay ∙ Multiple measurements at specific time intervals by using continuously recording spectrophotometer Tissue Specificity of Enzymes High Specificity ACP ALT Amylase Prostate, erythrocytes Liver Pancreas, salivary gland Moderate Specificity AST CK Liver, heart, skeletal muscles Bone, heart, skeletal muscles Low Specificity ALP LD Liver, bone, kidney All tissues ALKALINE PHOSPHATASE Major tissue source: Liver, bone, intestine, placenta Other tissue source: kidney, pancreas Clinical Significance: 1. ↑ 3 – 10x ULN in HEPATOBILIARY DISEASE (biliary cirrhosis, bile stone, cholelithiasis) 2. ↑ Paget’s disease (highest elevation), osteomalacia, osteogenic sarcoma, hyperthyroidism, rickets 3. Between 16 – 20 weeks of pregnancy 4. B and O groups increases intestinal ALP after consumption of fatty meal PRONOUNCED ELEVATION MODERATE ELEVATION SLIGHT ELEVATION (5x or more X ULN) Biliary cirrhosis Osteitis deformans (Pagets) Bile duct obstruction Hyperparathyroidism Osteogenic sarcoma (3-5X ULN) Granulomatous or infiltrative disease of liver Infectious mononucleosis Metastatic bone carcinoma Metabolic bone disease (rickets, osteomalacia) Up to 3X ULN Viral hepatitis Cirrhosis Pregnancy Normal growth pattern in children Healing fractures Methods: 1. Electrophoresis – L (most anodal) BPI 2. Heat Stability 56C for 10 minutes P (same as carcinoplacental isoenzymes) ILB 65C for 30 minutes (Placental ALP resists denaturation at this temp) 3. Chemical Inhibition ∙ All are inhibited by phenylalanine
∙ Liver and bone – inhibited by Levamisole reagent ∙ Bone – inhibited by 3M urea 4. Requires a pH of 9 - 10 Carcino Placental Isoenzymes 1. Regan – found in lung, breast and gynecological cancers 2. Nagao – found in adenocarcinoma of pancreas, and bile duct, and pleural cancer, inhibited by leucine **Same structure and heat stability with PLACENTAL isoenzyme but same mobility with BONE isoenzyme Measurement of Total ALP Activity Method Substrate Comments Shinowara-Jones-Reinhart Beta-glycerophosphate Long incubation time; ↑ blank values Bodansky Bessy, Lowry, and Brock P-nitrophenylphosphate Endpt or kinetic, rapid King-Armstrong Phenylphosphate Endpt, requires protein removal Bowers-McComb PNPP Uses phosphate-accepting buffer; reference method Huggins and Talalay Phenolphthalein diphosphate Phenolphthalein red Moss Alpha-naphthol phosphate Alpha-naphthol Klein, Bobson and Read Buffered phenolphthalein phosphate Free phenolphthalein ACID PHOSPHATASE Richest source: Prostate Other tissue source: RBCs, liver, bone, kidneys, spleen and platelets Clinical Significance: 1. Tumor marker for PROSTATIC CARCINOMA. ACP is tissue specific but not cancer specific because it also shows elevated amounts in BENIGN HYPERPLASIA OF THE PROSTATE 2. RAPE CASES – can persist up to 4 days in the vagina 3. Paget’s disease 4. Gaucher’s disease 5. ITP Methods: 1. Requires a pH of 5.0 2. Inhibited by fluoride, oxalate, heparin and prolonged storage at RT and tartrate