Clinical Chemistry

Clinical Chemistry

1. Metabolic Levels

   • Glucose
     • Glucose + ATP
       • Glucose 6-P + ADP
         • GLucose 6-P + NADP
           • Glucono-lactone-6P + NAPDH
             • Look at NADP/NADPH ratio
               • Determines how well rxn is going forward
           • Glucose 6-P dehydrogenase
       • Hexokinase

2. Tissue Damage

   • Serum Enzymes
     • Diagnostic indicators
     • AST, ALT
       • Viral hepatitis
       • Liver disease
     • Amylase
       • Acute pancreatitis
     • Ceruloplasmin
       • Wilson's disease
     • Creatine phosphokinase
       • Creatine-P + ADP --> Creatine + ATP Use ATP to phosphorylate glucose with hexokinse --> glucono-lactone-6-P + NADPH
       • Use this with the glucose --> glucose 6-P --> glucono-lactone-6-P to measure CPK activity
       • Can be confused with adenylate kinase activity because you can also get ATP from it. 2 ADP --> ATP + AMP
       • Control -- measure ADP --> ATP with no creatine This will give level of ATP produced by adenylate kinase. Subtract from total level of ATP produced, can determine the amount that is produced by creatine kinase
       • Muscle disorders, MI
         • Isozymes
           • MM MB - MI possible BB
           • Muscle (M)
           • Brain (B)
           • MB is more rapid diagnosis
             • Myocardial Infarcation
             • MB can also be present in well trained athletes
               • Even with no MI
     • Gamma-glutamyl transpeptidase
     • Lactate dehydrogenase
       • LDH activity can be visualized by coupling the reaction to a stain The compounds created in the reaction will eventually reduce NBT which is a blue color. The more blue, the more the reaction has occurred.
       • MI
         • Used to measure Rxn from lactate --> pyruvate
           • Isozymes LDH
             • Form Tetramers
               • HHHH, Myocardium, RBC HHHM, Myocardium, RBC HHMM, Brain, Kidney HMMM, -- MMMM, Liver, Skeletal Muscle
               • Increase in fraction of HHHH and HHHM
                 • MI!
             • Muscle (M)
             • Heart (H)
     • Lipase
     • Phosphatase, acid
       • Metastatic carcinoma of prostate
     • Phosphatase, alkaline
       • Bone disorders, obstructive liver disease
     • Limitations
       • Specificity
         • How specific are enzymes to this particular disease?
           • Look at isozymes for specificity
             • Isozymes are closely related enzymes that catalyze the same reaction but have different amino acid composition (different genes)
             • Isozymes usually have a different charge and you can use electrophoresis to separate them
             • Isozymes also have different kinetic properties, which can make them more useful in specific tissues where they are used. Tissue specific.
       • Speed
         • The faster the better, but not always get the fastest results
           • Measure LDH in MI patients
             • Can take >24 hours
               • Want to triage quickly
       • No ID of cause
         • Could be due to numerous reasons
           • Just know there is damage, not why
       • Tissue may not be in contact with circulation
         • Blood flow occluded

3. Measuring Enzyme Levels

   • Enzyme Assays
     • Ideal
       • Linear relationship
         • Amount and activity
     • Radioimmunassay (RIA)
       • Antibody I on polymer support
         • Protein binds antibody I
           • Add radioactive antibody II
             • Binds protein, shows where it is
     • Enzyme Linked Immunoabsorbant Assay (Elisa)
       • Same as RIA
         • Antibody II has enzyme
           • Enzyme will convert substrate to fluorescent
   • Best Enzyme for MI Detection
     • Must be specific to heart muscle
       • Troponin I
         • Has extension in cardiac muscle
           • Bigger than skeletal muscle Troponin I
       • Troponin T
         • Heart sequence specificity
       • These 2 are preferred indicators of MI
         • Handheld measuring
         • Fast results