MEDT 360 Enzymes of Clinical Significance PDF

Brief review of Enzymology, Enzymes of clinical significance MEDT 360 Chemistry Topic 5 Paul Johnson, Ph.D., DABCC, MLS(ASCP)  History of Enzymes  1835: Alpha-amylase was identified to break down starch more efficiently than chemical methods (sulfuric aci...

Brief review of Enzymology, Enzymes of clinical significance MEDT 360 Chemistry Topic 5 Paul Johnson, Ph.D., DABCC, MLS(ASCP)  History of Enzymes  1835: Alpha-amylase was identified to break down starch more efficiently than chemical methods (sulfuric acid)  “enzyme” derives from Greek (en = in + zyme = yeast) since initial research studies were on investigation of sugar fermentation in yeast 2 Basic Principles  Enzymes are proteins  They have the usual protein structures: primary, secondary, tertiary, (quaternary)  Have antigenic properties when injected into another species  Used for Immunoassay testing of:  isoenzymes  isoforms  Enzymes as catalysts  Involved in biological metabolic processes  Used in many clinical lab reagents  Spectrophotometry-based testing (many)  Labeled-enzyme Immunoassays (some formats) 3  Enzyme classification Enzymes are broadly categorized into 1 of 6 main groups, shown in the table: 1. “Redox” reactions: hydrogen transfer, addition or removal 2. Transfers a functional group other than Hydrogen 3. Uses water (H2O) to break a chemical bond 4. Removal of group w/o hydrolysis (H2O) 5. Catalyze interconversion of isomers 6. Join two substrates together coupled with breaking of high “energy” bond (e.g., ATPADP) 4  Definitions Enzymes may be further described when they exist as isoeyzmes or isoforms: “Isoenzyme” versus “Isoform” 5  Isoenzymes are defined at the genetic level Confers different tissue/organ expression of an enzyme, while still catalyzing the same reaction (same substrate to product formation) 6  Isoenzymes  Different genetic form(s) of an enzyme that catalyze the same reaction  Can result in slight differences in primary amino acid sequence (protein translation)  However, active tertiary structures have similar substrate affinity (and catalytic function)  Isoenzyme examples: tissue/organ differences  Creatine Kinase (CK) isoenzymes: (M) muscle and (B) brain  Lactate dehydrogenase (LD) isoenzymes: (H) heart and M (muscle)  Alkaline phosphate (ALP) isoenzymes; more extensive tissue expression (e.g., bone, kidney, liver, placenta) 7  Isoforms: NON-genetic modification of enzymes They are described as “post-translational” modifications, meaning there is a slight change/functional group added to the enzyme after protein synthesis 8  Basic Review of Enzyme Kinetics 9  E-S complex  The enzyme binds with the substrate to form an enzyme-substrate (ES) complex  Product (P) forms, then enzyme (E) dissociates from the complex  Enzyme is not depleted and can catalyze additional reactions 10  Reaction Rates and Activation Energy  Enzymes greatly speed up metabolic reactions  Life could not proceed without enzyme involvement  To understand how they do this, we’ll examine the concept of activation energy 11  Enzymes lower the energy barrier needed for a reaction to take place. Activation energy is defined as amount of energy required to energize 1 mole of substrate to the activated E-S complex 12  Michealis-Menten Kinetics V-max: when the substrate concentration is high enough that all enzyme molecules are bound to the substrate and all active sites are engaged Michealis-Menten Constant (Km): equals substrate molar concentration [S] when the reaction velocity is at ½ V-max As substrate concentration increases, reaction velocity increases…up to a saturation point That point is called “maximum velocity”, or V-max ½ V-max = Km (provides estimate of [S] while reaction is still at linear relationship) 13  “First order” and “Zero order” Kinetics First order kinetics: occurs when reaction velocity is directly proportional to substrate concentration [S] Zero order kinetics: occurs at enzyme saturation point; all sites on enzyme are occupied so that no addition ES complex can form 14  Factors Influencing Enzyme Activity Michaelis-Menten kinetics are based on defining the following parameters for an enzyme: 1. Substrate concentration 2. Temperature 3. Buffer conditions  pH  Salt concentration and other constituents 4. Cofactors (e.g., B-vitamins, minerals)  Such conditions will be standardized in clinical test assays to ensure consistent results. 15  Enzyme Inhibitors Inhibitors of enzymes are also studied because their effects can be serious Clinically, enzyme inhibitors may cause metabolic disorders in a person that can be harmful or even deadly  They are usually drugs or toxic metals  Example: the element lead (Pb) inhibits several heme synthesis enzymes needed for heme production, which is used by heme-containing enzymes (e.g., catalase, cytochrome P450) and hemoglobin for oxygen transport. 16  Enzyme Inhibitors  While a detailed discussion of enzyme inhibitors are beyond the scope of this course, there are a few key points that are useful to know: 1. Because of non-linear “zero order kinetics” in the M-M graph, when approaching V-max, it may be difficult to determine the effects of an enzyme inhibitor on the reaction rate  Sometimes enzyme inhibition can be reversed by adding more substrate to a reaction to speed it up; other times this will have no effect when it’s irreversible 2. To make this easier to visualize and describe, we rearrange the M-M equation by taking the reciprocal (1/x) and then graph those data  This is called the “Lineweaver-Burk plot” 17  Lineweaver-Burk transformation plot 1 Vo = Vmax[S] Michael- Menten [S] + Km equation Y= mꞏ X+b This plot shows enzyme kinetics without an inhibitor present. 18 Clinical applications  Disease correlation/tissue location  Since enzymes play critical roles in metabolism, and other processes, they are found across several different organs and cells within the body:  Muscle  Liver  Pancreas  Heart  Bone  Erythrocytes (and Lymphocytes)  In general, release of cellular enzymes into blood (serum) indicates a pathological process such as cellular destruction. 20  Summary table of clinically-important enzymes This table highlights the enzymes of interest, its primary tissue source(s), and most-commonly associated clinical interpretation 21  Enzyme coenzymes/cofactors  Enzymes require these to reach V-max (maximal reaction velocity) in reaction kinetics  Play a principal role in metabolism  Additions of coenzymes and cofactors to test reagents ensures Vmax is achieved and therefore we are not reporting out lower enzyme values on patients  Termed a “negative bias”  Enzyme test result with and without the coenzyme demonstrates this effect  The addition of these to test reagent is most essential with patients who have deficient intakes of one or more of these (see next slide) 22  Examples of coenzymes/cofactors Coenzyme: an organic molecule (contains carbon) Cofactor: same as a coenzyme, but is present as an inorganic mineral or metal These are essential nutrients to prevent a wide range of diseases Through the evolutionary process to “conserve energy”, humans have lost the ability to synthesize most of them Many B vitamins are coenzymes (NAD+/NADH, as example) Henry's Clinical Diagnosis and Management by Laboratory Methods, 22nd ed. (2011) 23  Reporting units for clinically-measured enzymes  International Unit (IU) describes an enzyme’s catalytic rate  Also expressed in liter (L) of blood and commonly written as U/L or IU/L  (This is the unit of measure used primarily by U.S. labs; it is not the SI unit for enzyme activity)  Definition: the international unit (IU) is the amount of enzyme that catalyzes 1 umol* of substrate to product per minute under standardized conditions of: temperature, pH, substrate, and coenzymes/cofactors * umol = micromole 24  Enzymes of Clinical and Diagnostic Use 25  Creatine kinase (CK)  General marker of muscle cell damage Clinical significance 1. Increased blood levels seen in muscular disorders (rhabdomyolysis, muscular dystrophy) 2. Severe trauma 3. Acute myocardial infarction (heart attack), although this test is no longer recommended as a primary marker to diagnose AMI  Newer markers have replaced it (cardiac troponin) 26 Creatine Kinase (CK) reactions Note the influence of pH driving the reaction in the “forward direction” or “reverse direction” Inorganic magnesium (Mg2+) as cofactor 27  Creatine Kinase (CK) – physiological role Forward reaction helps to create storage form of “energy” in muscle during times of excess ATP  Reverse reaction used by muscle, cardiac tissue to regenerate ATP rapidly in times of demand 28  CK Test Methodologies  CK reverse reaction proceeds two to six times more rapidly than the forward reaction; a reason this reaction design is more common in lab reagents  CK → Hexokinase → G-6-Phosphate Dehydrogenase at pH 6.7 1 HK 2 ATP + glucose  Glucose-6-P + ADP measured G6-PD 3 Glucose-6-P + NADP  6-Phosphogluconate + NADPH + H+ 29  Outline of measuring CK in serum Contains substrates: Add patient’s serum Contains other substrates: Creatine phosphate, Contains CK for the (2) Glucose, hexokinase ADP, Mg+2 first reaction (3) NADP, G6-PD ATP + Creatine NADPH 340 nm ABS proportional to CK activity concentration (U/L) 30  Typical Reference Ranges for Total CK activity  Males: 46-300 U/L  Females: 15-180 U/L  Affected by:  Age  Race  Muscle mass  Physical activity  CK enzyme has 3 isoenzymes (BB, MB, MM)  B = brain; M=muscle  The combined catalytic activity of all isoenzymes = total activity 31  CK isoenzyme distribution Of clinical interest, CK-MB is the only isoenzyme still in clinical use It is a marker of acute myocardial infarction (heart attack), although this isoenzyme test has been largely replaced by cardiac troponin 32 protein measurements  Concept of CK-MB immunoassay “Detection” Ab used to measure CK-MB of patient Anti-M Anti-B UH Ref. Range Male < 6.70 ng/mL Female < 3.80 ng/mL 33 Hepatic Enzymes  Liver Enzymes  Part of a clinical panel called: “Liver function tests” (LFTs)  Increased levels of these markers, in blood, occur from liver cell destruction or blockage of bile flow Clinical significance (liver diseases)  Hepatitis (liver inflammation); Cirrhosis (cell death); Liver cancer; Fatty liver disease  Bile tract or bile duct obstruction 35 Liver function tests  Enzymes measured as indicators of liver damage due to hepatocyte destruction or biliary tract disease  AST Transaminase enzymes  ALT  GGT  ALP 36  Transaminase enzyme reactions  Transaminase enzymes catalyze the interconversion of amino acids and α-ketoacids by the transfer of amino groups  Transamination is important for several metabolic reactions:  Providing substrate used in urea cycle (nitrogen metabolism)  Products of amino acid metabolism to cross between cytosol and mitochondria of cells  Gluconeogenesis to allow for amino acids to cross mitochondrial membrane  Vitamin B-6 required for full activity  AST, ALT are clinically important transaminases 37  Aminotransferases (transaminase)  AST  cytoplasmic and mitochondrial forms  ALT  cytoplasmic form only 38  Cellular distribution of liver enzymes Clarke W. Contemporary Practice in Clinical Chemistry Cytoplasm: AST, ALT, LDH Mitochondria: AST Membrane: ALK (ALP), GGT 39  Aspartate Aminotransferase (AST) 40 41  AST Methodologies – Step 1 (α‐ketoglutarate) (Patient serum) Needs vitamin B6 (P-5-P) as a coenzyme 42  AST Methodologies – Step 2  Malate dehydrogenase is the indicator reaction measuring the decrease in absorbance at 340 nm (UV λ) as NADH is oxidized to NAD+. Typical RR for AST < 35 U/L (IU/L), but can vary by lab 43  Outline of serum enzyme measurement procedures (AST) Contains substrates: Add patient’s serum Contains other substrates: Aspartate, α-ketoglutarate, Contains AST enzyme (2) Malate dehydrogenase Vitamin B6 (P-5-P) for the first reaction (3) NADH Oxaloacetate + glutamate NADH NAD+ ABS (indirectly) proportional 340 nm to AST enzyme activity 44 concentration  Alanine Aminotransferase (ALT) 45  Alanine Aminotransferase (ALT)  coupled reaction to LD to measure ALT (α-ketoglutarate) LD Uses vitamin B6 as a cofactor Measure decrease in NADH ABS at 340 nm Typical Ref. Range < 60 U/L males, < 42 U/L females) 46  ALT Clinical Significance  ALT’s diagnostic significance is confined mainly to hepatocellular disorders and it is more specific than AST.  Because ALT is entirely cytoplasmic, its levels also tend to be greater than AST in liver disease 47 Pincus MR et al. Clinical Enzymology. In: Henry's Clinical Diagnosis and Management by Laboratory Methods, 22nd ed. (2011)  GGT helps synthesize glutathione  Gamma glutamyltransferase (GGT) is believed to regulate and maintain extracellular reduced glutathione (GSH)  Glutathione is the body’s major antioxidant  It is a tri-peptide (3 a.a.) made from: Glycine-Cysteine (N-acetylated)-Glutamine  GGT enzyme often uses glutathione as the γ-glutamyl donor 48  GGT Clinical Significance  The primary role of GGT in the clinical chemistry lab is the detection and differential diagnosis of:  hepatobiliary disease with cholestasis (loss of bile flow) or  biliary obstruction associated with the highest elevation 49  GGT Clinical Significance  One other useful role of GGT is to differentiate liver disease from bone disease  GGT is not elevated in bone disease; therefore, if a patient has an elevated ALP and a normal GGT, the ALP would most likely be of bone origin Disorder ALP GGT Bone Elevated Normal Liver Elevated Elevated 50  GGT test method Transfer of γ-Glutamyl residue to glycylglycine (Gly-Gly) releases p-nitroanilide which has a strong absorbance at 405-420 nm 51  Alkaline phosphatase (ALP)  Biochemistry  Areas of concentration in body  Functions  Multiple isoenymes  Major clinical significance  Hepatobiliary disease (bile ducts of liver)  Bone disease 52  Alkaline Phosphatase (ALP) Best understood functions:  Bile acid production (liver)  Osteoblast synthesis and growth (bone)  Facilitates lipid transport (intestine) 53 Multiple Forms of ALP Major contributions of ALP in healthy human serum are from liver and bone 54  Alkaline Phosphatase (ALP)  Clinical significance (liver): thought to aid in bile production  ALP frees inorganic phosphate from an organic phosphate monoester, resulting in the production of an alcohol at an optimal pH 10 55 R=phosphate group  Alkaline Phosphatase (ALP)  ALP is also needed for normal bone growth  Elevated levels are seen during adolescence  It is important to differentiate cause of elevated ALP (especially in adults) due to liver disease rather than a bone disorder 56  ALP (relative differences) Ref. ranges will depend greatly on age and gender Plays a large role in bone growth during adolescence (good bone marker in early ages) 57  ALP Methodologies  4-nitrophenol-phosphate (4-NPP) is the substrate, and the yellow product,  4-nitrophenoxide is measured at 405 nm (visible range), at pH 10.2 58 Pancreatic Enzymes  Pancreatic digestive functions 60  Digestive and Pancreatic Enzymes Amylase  hydrolase enzyme that catalyzes the hydrolysis of complex carbohydrates including starch, amylopectin, glycogen, and their partially hydrolyzed products.  It is secreted by salivary glands to start digestive process  As food travels to intestinal region, pancreatic amylase gets secreted (this is the isoenzyme of major clinical interest)  Major clinical significance: Pancreatitis 61  Lipase  Lipase hydrolyzes glycerol esters of long-chain fatty acids (triglycerides) to produce monoglyceride and free fatty acids.  Produced by a variety of cell types 62  Pancreatic lipase clinical significance  Lipase is produced by the pancreatic acinar cells (meaning, the functional cells)  Serum Lipase activity is found to be elevated in acute pancreatitis or obstruction of the pancreatic duct  The half-life of pancreatic lipase is longer than that of pancreatic amylase  Expected values (ref. range) = < 38 U/L 63 Lipase has longer detection window than Amylase post-acute pancreatitis 64  Lipase Clinical Significance  Lipase is less affected by other conditions that can cause elevated Amylase  This makes lipase test more specific (true negatives) for acute pancreatitis  Although lipase is considered a more diagnostically specific test for acute pancreatitis, though both tests often ordered together 65 Hematology and Oncology RBCs, WBCs Lactate dehydrogenase (LD) glucose  Function  Role in anaerobic metabolism of glucose (glycolysis)  Especially important for Red Blood Cells (RBC)  Has five isoenzymes, although “Total LD” activity is the major measurement of use now  Isoenzyme/tissue differences are now rare to measure clinically 67  Lactate Dehydrogenase (LD) isoenzymes Specific elevations could provide clues as to the organ or cells involved. Isoenzyme separation test is rarely performed; instead measure “total LD activity” 68  Lactate Dehydrogenase (LD)  LD enzyme transfers a hydrogen to catalyze the oxidation of L-lactate to pyruvate with NAD+ as a hydrogen acceptor.  Enzyme functions in both directions 69  Total LD activity measurement  Reaction (lactate  pyruvate) is most often used for clinical test assay  Measure NAD+/NADH changes at 340 nm (UV range) 70  Lactate dehydrogenase (LD) LD measurement is most relevant in hematology and oncology Clinically significant elevations observed in red blood cell and white blood cell disorders  Hemolytic anemias  Megaloblastic anemias  Folate and/or B12 deficiency  Pernicious anemia  B12 absorption problem  Leukemias  CLL, for example Platelets and RBCs are rich sources of LD  Serum preferred sample  Reject hemolyzed samples; false increases in LD 71  Acid Phosphatase (ACP) ACP is in the family of hydrolase enzymes  Uses water to break bonds Clinical significance, increased values seen in: 1. Prostate cancer 2. Several bone disorders (from excess bone breakdown) 3. Gaucher's disease (fatty deposits in organs, including bone)  Isoform analysis to make better diagnosis 72  Additional enzymes of clinical importance  Clinically important but activity is not measured as routinely as others described in this presentation:  G-6-PD (deficiency)  CYP450 drug metabolizing enzymes  Aid in determining the enzymatic rate of an individual’s drug metabolism/elimination (slow, “normal”, or fast) 73  END 74