Unit 8 Enzymology

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  1. Define: Coenzyme (cofactor)
    Required by some enzymes.

    • Some are Hydrogen acceptors or donors (NAD+,
    • NADH, NADP+, NADPH, etc.)

    Some are Vitamins (B-6, B-12, etc.)
  2. Define: Activator (Enzyme)
    Required by some enzymes.

    • Usually metal cations (Mg2+, Mn2+, Ca2+,
    • etc) that form ionic bonds with the enzyme and change it’s shape.
  3. Define: Isoenzyme
    Two or more forms of an enzyme that catalyze the same reaction. Composed of more than one protein subunit.
  4. Explain the four (4) mechanisms by which cellular enzymes appear in a patient's blood:
    Stress-induced cell leakage (stress from disease)

    Cellular necrosis – cells die, rupture, and release their cellular contents.

    Induction of synthesis – a normal % of enzymes leaks out, so if cells are synthesizing more enzymes, blood levels will increase.

    Proliferation of cells – more cells to leak the normal mounts resulting again in increased blood levels of enzymes.
  5. Define: an International Unit of enzyme activity.
    Amount of enzyme activity that will catalyze the conversion of one micromole of substrate into product per minute under standardized conditions.
  6. Explain what is meant by Zero Order and First Order enzyme kinetics, with respect to the substrate concentration of the reaction mixture.
    Zero Order – enzyme activity independent of changes in substrate concentration.

    First Order – enzyme activity dependent on changes in substrate concentration.
  7. Creatine Kinase (CK or CPK)
    Has 2 subunits so there are 3 isoenzymes: CK-MM, CK-MB, and CK-BB.

    • Reaction catalyzed in the laboratory:
    • Creatine–Phos + ADP ------CK-----> Creatine + Phos + ATP

    ATP + Glucose -------hexokinase----> ADP + Glucose-6-PO4

    Glucose-6-PO4 + NADP+ -------------> NADPH + 6-Phosphogluconate

    • Tissue(s) of enzyme abundance:
    • Heart Muscle
    • MM (CK-3) 80%
    • MB (CK-2) 20%

    • Skeletal Muscle
    • MM (CK-3) 96%
    • MB (CK-2) 4%

    Brain Tissue: predominantyly the BB isoenzyme

    Clinical significance: Useful marker for muscle disease and myocardial infarct (MI or AMI). 

    Calculate CK-MB Relatvie Index Ratio (it is the same for kinetic and mass quantitation):

    •     CK-MB UL or ug/mL                         
    •     ----------------------        
    •          Total CK UL            

    • ≥6% (or 7%) = AMI (heart).
    • ≤4% = different muscle tissue

    • Special Specimen handling precautions:
    • Serum or Heparanized Plasma. All other anti-coagulants inhibit the enzyme.

    Hemolysis will inhibit the enzyme.

    Freeze if not assaying immediately.

    Sulfhydryl groups of active site or easily oxidized by heat or bright light (light labile).
  8. Lactic Dehydrogenase (LH or LDH)
    Removes an H from its preferred substrate.  There are 5 isoenzymes: LLLL, LLLH, LLHH, LHHH, and HHHH – LD 5, 4, 3, 2, 1.

    • Reaction catalyzed in the laboratory:
    • Wacker Reaction (most common)
    • Lacic Acid + NAD+ -----------> Pyruvate + NADH

    • Wroblewski Reaction (Vitros-Preferred)
    • Pyruvate + NADH --------------------> Lactic Acid + NAD+

    • Tissue(s) of enzyme abundance:
    • All cells of all tissues have this enzyme.

    LD isoenzyme electrophoresis can isolate isoenzymes.

    Typically: LD-2 > LD-1 > LD-3 > LD-5 > LD-4

    • LD-1 and LD-2:
    • RBCs and WBCs (and bone marrow).
    • Heart (LD-1 > LD-2 – LD Flip)

    LD-3:  Lung Tissue. Seen in Pulmonary Embolisms with Pulmonary Infarcts.

    • LD-4 and LD-5:
    • Smooth and Skeletal Muscle (Trauma & MD)
    • Liver (Hepatic Disease)
    • Kidney (Renal Necrosis and Renal Infarcts)

    • Elevations >3x normal range
    • RBCs and WBCs
    • Heart
    • Skeletal Muscle and Smooth Muscle.

    Liver or other tissues (pancreas, bone, kidney, etc) usually won’t double normal range.

    • Special Specimen handling precautions:
    • Serum or heparinized plasma may be used on most instruments.

    Hemolyzed RBCs will release tons of LD. Hemolysis is completely unacceptable.

    Should not be frozen.  Will give slightly lower values.
  9. Alpha-Hydroxybutyric Dehydrogenase (a-HBD or HBDH)
    LD-1 and LD-2 with a different substrate that LD-3,4, and 5 won’t use.  

    • Reaction catalyzed in the laboratory:
    • α-Hydroxybutyric Acid + NAD+ ------LD-1/2-----> α-ketobutyric acid + NADH

    • Tissue(s) of enzyme abundance:
    • Heart,
    • RBCs and WBCs
    • Bone marrow

    • Clinical significance:
    • “Quick and dirty” alternate to LD isoenzyme studies to rule-in/out MI as cause of LD elevation.  Mostly replaced by other tests like CK-MB and Troponin-I that is more specific to the heart.

    LD High and α-HBD elevated --> consistent with AMI.  The elevated LD is from LD-1 and 2.

    • LD High and α-HBD normal --> rules out AMI. 
    • The elevated LD is not from LD-1 or 2.
  10. Aspartate Transaminase (AST) (aka SGOT or GOT)
    Primarily a cytoplasmic enzyme in almost all cells.  Marginal serum elevations can come from any tissue.  Previously known as Serum Glutamic-Oxaloacetic Trasaminase (SGOT).

    • Reaction catalyzed in the laboratory: 
    • Aspartic Acid + α-ketoglutarate <--------AST-----------> Oxaloacetic Acid + Glutamic Acid

    • Tissue(s) of enzyme abundance: 
    • High content:
    • Heart
    • Skeletal Muscle and Smooth Muscle
    • Liver.
    • *Elevates by twice normal values.

    • Lesser but still significant:
    • RBCs and WBCs (RBCs > WBCs)
    • Special Specimen handling precautions: 
    • Serum or heparinized plasma.  No known anticoagulant inhibits AST.

    No hemolyzed specimens. RBCs have 10x serum levels.
  11. Alanine Transaminase (ALT) (aka: SGPT or GPT)
    Primarily a mitochondrial enzyme, formerly known as serum glutamate-pyruvate transaminase.

    • Reaction catalyzed in the laboratory: 
    • Alanine + α-ketoglutaric acid <--------ALT----------> Pyruvic Acid + Glutamic Acid

    • Tissue(s) of enzyme abundance: 
    • Liver (the most specific for liver disease)

    • Slight elevations --> from any tissue
    • 10% above normal range --> Liver (most likely). 
    • > 20% is coming from the liver.

     AST/ALT Ratio.

    • Special Specimen handling precautions:
    • No hemolyzed specimens (RBCs have 5x normal serum level.)
  12. Gamma Glutamyl Transferase (g-GT) (also GGT or g-GTP)
    The most sensitive liver enzyme there is.

    • Reaction catalyzed in the laboratory: 
    • g-L-glutamyl-p-Nitrolanilide (colorless) + Glycylglycine <--------g-GT--------> p-Nitroaniline (yellow) + g-L Glutamylglycylglycine

    Tissues that have it: Liver, Pancreas (elevates greatly), Kidney (doesn’t elevate reliably).

    Clinical significance: Used in Detoxification Centers to assess abstenance from drinking. 

    In hepatitis, it is the first enzyme to elevate; but it never elevates very high and does not correlate with the severity of disease.  ALT levels do, so it is the better marker in hepatic disease.

    • Causes of elevation:
    • Hepatobiliary Obstruction (gallbladder disease.  Elevates higher than in hepatitis)
    • Hepatitis
    • Acute Pancreatitis

    Does not elevate from bone disease or pregnancy.

    • Special Specimen handling precautions: 
    • No anticoagulants inhibit it.
    • None in RBCs, so hemolysis will not affect results.
  13. Alkaline Phosphatase (ALP) (aka Alk. Phos.)
    Removes phosphate groups.

    • Reaction catalyzed in the laboratory:
    • Bessie-Lowry-Brock Method
    • At pH 10.3 with Mg++.
    • p-Nitrophenyl Phosphate (colorless) -------ALP--------> p-Nitrophenoxide (yellow) + PO4

    • Tissue(s) of enzyme abundance:
    • Liver
    • Bone (osteoblastic or osteoclastic disease – except multiple myeloma)
    • Placenta (pregnancy or Regan isoenzyme—cancer marker))
    • Intestine
    • Kidney

    Clinical significance: Elevates higher in Hepatobiliary Obstuction than it does in Hepatic disease.  Most common sources of elevation are either liver or bone, and a g-GT can determine which is the cause.

    • Isoenzyme Electrophoresis
    •  (+)
    • Liver         <-- These two don’t separate well
    • Bone         <--  enough to tell them apart.
    • Placenta (Regan Isoenzyme)
    • Renal
    • Intestine                <--  POA 
    • (-)

    • Heat to 56°C for 10 minutes:
    • > 80% loss of original activity --> bone isoenzyme (the most heat labile)
    • 50 – 75% loss of activity --> liver isoenzyme (slightly more stable)
    • < 50% loss of activity --> intestinal isoenzyme (most heat stable)

    • Special Specimen handling precautions: 
    • Fresh serum or heparinized plasma.  All other anticoagulants inhibit the enzyme (Mg++ activator).  ALP activity increases with specimen age.

    Unhemolyzed.  RBCs have 6x more ALP than normal serum.

    Refrigerate. Otherwise activity increases with age of sample, causing increasing trends for controls and samples.
  14. Acid Phosphatase (ACP) (aka Acid Phos.)
    Removes phosphate groups.

    Reaction catalyzed in the laboratory: Uses same Bessie-Lowry-Brock Method but buffered at pH 5.0.  colorless --> yellow

    • Tissue(s) of enzyme abundance:
    • Osteoclasts
    • Platelets
    • Spleen
    • RBCs
    • Liver
    • Prostate Gland is the major source.

    • Current uses limited to:
    • Medical-Legal Rape Investigations
    • Vaginal secretions have no ACP.  Seminal fluid has lots. Vaginal washing is aliquoted into two parts, one with Tartrate the other without.

    • Diagnosis of Metastatic Prostatic Carcinoma
    • Benign Prostatic Enlargement – Normal
    • Prostate Cancer, no metastasis – Normal to 10% elevation
    • Metastatic Prostatic Carcinoma – Signigicantly elevated (above 10%).

    • Monthly ACP assays are done to assess success of therapy.
    • ACP does not rise over 5 years --> Cured
    • ACP does rise --> Carcinoma has metastasized and has recurred.

    • Special Specimen handling precautions: 
    • Fresh serum only.  All anticoagulants inhibit ACP.

    Unhemolyzed specimen. ACP will elevate if hemolyzed.

    Transport on ice, centrifuge, remove serum and acidify serum by adding Sodium Acetate or Disodium Citrate to drop pH to 4.5. 50% activity lost per hour if not processed in this manner.  (It is very heat and pH sensitive.)
  15. Prostate Specific Antigen (PSA)
    Better assay than ACP for detecting and monitoring prostate cancer.

    Clinical significance: Rises above normal 4.0 ng/mL range before any sings or symptoms of Prostate Cancer arise.  It is 98% sensitive to disease.

    Common sources of error: Elevations may be from benign prostatic hypertrophy (4 – 6 ng/mL), so a biopsy is the next step. 

    Newer and more sensitive assays for Total and Loosely-Bound PSA are increasing specificity for cancer.
  16. Amylase (AMS)
    • Pancreatic enzyme
    • Breaks starch into smaller polysaccharides and glucose

    • Reaction catalyzed in the laboratory:
    • Saccrogenic – measures amount of glucose formed.
    • Amyloclastic – rate of reduction of starch
    • Chromolytic – BEST METHOD.  Dye-labeled starch as substrate with little color, but glucose-dye products are blue chromophore.

    • Tissue(s) of enzyme abundance: 
    • Pancreatic Amylase (P-AMS) – a biggie for ruling in or out Acute Pancreatitis.
    • Salivary Amylase (S-AMS) – never a biggie.

    Clinical significance: These specimens usually come from the emergency room where they are trying to rule out several conditions that have nearly the same symptoms (fever, WBC left-shift, acute abdominal pain, shocky, sometimes tetany due to low calcium):

    Acute Pancreatitis, Acute Cholestasis (gall bladder disease), and Acute Perforated Ulcer.

    • Common sources of error: 
    • Serum amylase can also elevate due to amylase complexes with immunoglobulins (Macroamylasemia).

    • Special Specimen handling precautions:
    • Either serum or urine.

    If filtration is hindered, blood amylase will elevate and urine will be low.  Calculate ACCR.

    It’s half life in the blood is 12 hours, so blood levels will be back to normal within 2 days.
  17. Lipase (LPS)
    Pancreatic enzyme

    Reaction catalyzed in the laboratory: Most methods are lousy, which is why amylase methods are more popular, but Lipase stays in the blood longer.  The best method is similar to Trinder TG assay which measures Glycerol.

    • Clinical significance:
    • Significant elevation is always due to pancreatitis.

    Stays elevated 5 days after a pancreatic attack (longer than amylase).

    • Special Specimen handling precautions:
    • Unhemolyzed serum. 
    • All anticoagulants inhibit it as does RBC hemolysis.
  18. What are some other less important enzymes?
    Cholinesterase – Produced by liver and thought to function in serum. Breaks down major surgery muscle relaxant so that patient wakes up.  In insecticide poisoning this is inhibited.

    5’ Nucleotidase – Exact same correlation as g-GT only g-GT is cheaper. Elevates higher in Obstructive deasease than in Hepatitis.

    Isocitrate Dehydrogenase (ICD) – A Kreb’s Citric Acid Cycle enzyme. Correlation is identical to AST.  Western region assays AST and some eastern labs use ICD.

    Aldolase (ALD) – An Embden-Meyerhoff pathway enzyme.  Found in all tissues, particularly muscle.  Was used to assess muscle damage and AMI before good CK assays.  Correlation is very similar to LD.
  19. Explain the clinical rationale of performing Amylase/Creatinine clearance ratios.
    Important in telling the difference between a pancreatic increase in amylase and an increase due to renal dysfunction or Macroamylasemia.

    Collect 2-hour urine specimen and serum specimen.  Assay both for amylase and creatinine.

    ACCR =   Urine AMS/ Serum AMS   X   Serum Creatinine/ Urine Creatinine X 100%

    • Acute Pancreatisis: ≥ 5%
    • Macroamylasemia and Renal Disease: < 2%

    Although checking levels of g-GT could tell you whether it’s due to pancreatitis as well and is easier and cheaper.
  20. Explain the clinical utility of AST/ALT (DeRitis) ratios.  Additionally, given an AST/ALT ratio, correctly interpret the significance of the ratio.
    Assesses severity of hepatic disease.  In hepatic stress we expect the cytoplasmic enzymes (AST) to leak out faster than the mitochondrial enzymes (ALT).

    AST > ALT = Hepatic Stress (top would be bigger than bottom, therefore: >1)

    ALT ≥ AST = Hepatic Necrosis (top would be smaller than bottom, therefore: ≤1)
  21. Enzyme panel and pathophysiology for:  

    Myocardial Infarction
    CK elevates within 2 – 4 hours (as long as you get a base line) and peaks at 24 hours.  Returns to normal by 2 -3 days.  CK-MB follows same pattern.

    AST is second to elevate. Elevates within 4 – 8 hours. Peaks at 36 hours and returns to normal after five days.

    LD is last to elevate (LD-1 and LD-2 flip).  12-18 hours. Peaks by 3rd day. Remains elevated for 12 -15 days.  More than 3x normal range. α-HBD follows the same pattern.
  22. Enzyme panel and pathophysiology:  

    Skeletal muscle damage
    • Enzyme panel:
    • Elevated total CK with a CK-MB/Total CK ratio < 6%

    Elevated LD-4 and LD-5 more than 3x the normal range.

    Pathophysiology: muscular dystrophy, muscle trauma
  23. Enzyme panel and pathophysiology for:  

    Hepatocellular disease
    • Enzyme panel:
    • AST and ALT both elevate.  AST/ALT ratio:
    • > 1 Hepatic stress.  
    • ≤ 1 Hepatic Necrosis (ALT from mitochondria is elevating)

    Elevated g-GT (2 – 5x increase), although it elevates higher in hepatobiliary obstruction than in Hepatitis.

    Elevated Alkaline Phosphatase (ALP), although it elevates higher in hepatobiliary obstruction than in Hepatitis.

    Elevated Acid Phosphatase (ACP), I think.

    Elevated LD-4 and LD-5 but won’t usually double the normal range.
  24. Enzyme panel and pathophysiology for:  

    Hepatobiliary obstruction
    • Enzyme panel:
    • Elevated ALP (Alkaline Phosphatase), which elevates higher in hepatobiliary obstruction than in Hepatitis.

    Elevated g-GT (5 – 30x increase) (as well as 5’-NT), which elevates higher in hepatobiliary obstruction than in Hepatitis and tells us that ALP elevation is due to liver rather than bone disease.

    Elevated AST and ALT but not as elevated as in Hepatocellular disease.
  25. Enzyme panel and pathophysiology for:  

    Pancreatic disease (Acute Pancreatitis)
    • Enzyme panel: 
    • Elevated Pancreatic Amylase (a pancreatic enzyme) – a biggie for ruling in Acute Pancreatitis.

    Elevated Lipase (pancreatic enzyme) –more specific to pancreatitis but less sensitive to detection.

    Elevated g-GT.

    I think there is elevated LD-4 and LD-5 but it won’t double the normal range.

    • Pathophysiology:
    • High fat intake and severe alcohol abuse triggers a gigantic production of pancreatic enzymes (proteases, lipase, amylase), so many that the proteases autodigest the pancreas.

    • Gallstone gets stuck in common bile duct below the pancreatic duct.  Normal production of
    • pancreatic enzymes plus bile regurgitate up the pancreatic duct and autodigest the pancreas.
  26. Enzyme panel and pathophysiology for:  

    Prostatic disease (cancer)
    • Enzyme panel:
    • Elevated ACP (Acid Phosphatase), significantly elevated in metastatic prostatic carcinoma
    • *Also used to evaluate effectiveness of treatment.  5 years without elevation = cured.

    • Elevated PSA above 4.0 (Prostate Specific Antigen) (could also be benign prostatic
    • enlargement)
  27. Enzyme panel and pathophysiology for:  

    Bone disease
    • Enzyme panel:
    • Elevated Alkaline Phosphatase (ALP) (osteoblastic or osteoclastic disease except multiple myeloma.)

    Differentiate liver disease from bone disease through g-GT and 5’NT.  Does not increase in bone disease.

    Elevated LD-1 and LD-2 (at least for bone marrow)

    Elevated Acid Phosphatase (ACP) (osteoclasts are rich in Acid Phosphatase).
  28. Enzyme panel and pathophysiology for:  

    Hemolytic disease
    • Enzyme panel:
    • Elevated CK.

    Elevated LD-1 and LD-2 (more LD-2 than LD-1) more than 3x the normal range.

    Elevated ALP (slightly?).

    Pathophysiology: leukemias maybe, anemias.
  29. Utility of this cardiac marker to rule-in/rule-out a Myocardial Infarct:

    CK-MM Isoforms
    NH3+ of lysines on ends of enzyme are positively charged in the blood and get whacked off (by a deaminase).

    • MM3 – initial form produced
    • MM2 – one NH3+ chopped off
    • MM1 – both NH3+ chopped off

    • Ratio of MM3 to MM1 is >1 following acute heart or muscle damage.  Hours after release there
    • is essentially no MM3 so ratio is very small.

    Very sensitive indicator of acute muscle damage.  Not specific for heart, so non-MI causes would still have to be ruled out.

    Same principle for CK-MB isoform, except it only has one NH3 to chop off so there are only 2 forms.
  30. Utility of this cardiac marker to rule-in/rule-out a Myocardial Infarct:

    CK-MB (Mass Quantitation) and Relative CK-MB Index Ratios
    Heart muscle has a higher percentage of CK-MB than skeletal and smooth muscle, so it is a useful marker for muscle disease and myocardial infarct (MI or AMI).  Calculate CK-MB Relatvie Index Ratio.

    At least 6% (or 7% in some labs) means it is AMI (heart).

    4% or less indicates it is a different muscle tissue.
  31. Utility of this cardiac marker to rule-in/rule-out a Myocardial Infarct:

    Myoglobin is in all muscle but mainly used as a myocardial muscle marker, unfortunately there are many other things that can elevate it (low glomerular filtration or any other muscle damage).  Mostly replaced by the better TN-I assays.
  32. Utility of this cardiac marker to rule-in/rule-out a Myocardial Infarct:

    Troponin is part of the contractile apparatus of muscles and heart.

    Troponin-T – both skeletal muscle and heart have TN-T.

    Troponin-I (TN-I)  Most significant marker currently in use.  It is the very best cardiac marker to date.  Only heart has TN-I and it doen’t elevate from muscle trauma, exercise, or a renal condition.

    • 0.4 – 0.9 is questionable and requires further evaluation of patient
    • 1.0 – 2.0 is suspicious of AMI
    • > 2.0 ng/mL is chemically diagnostic of AMI
  33. Utility of this cardiac marker to rule-in/rule-out a Myocardial Infarct:

    B-Type Naturetic Peptide (BNP)
    Marker for Congestive Heart Failure

    Produced and secreted by ventricles of the heart and opposes the Renin-Angiotensin-Aldosterone System, reversing hypertension.  It causes sodium and water loss in urine and vasodilation.  It elevates in blood when the heart is not able to pump enough oxygenated blood to the body.

    >100 ng/mL congestive heart failure is ruled in.

    Method: BioSite Diagnostic’s BNP test.  Uses fluorescent tag read by special fluorimeter.
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Unit 8 Enzymology
2014-04-04 03:16:30
Exam4 Enzymology Clinical Chem unit8

Enzymology for Exam 4
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