Clinical Enzymology

1
Clinical Enzymology
FE A. BARTOLOME, MD, DPASMAP Dept. of Pathology
Laboratory Diagnosis Our Lady of Fatima University
2
ENZYMES

• protein molecules
• catalyze chemical reactions without themselves
  being altered chemically
• contained primarily within cells
• essential enzymes present in virtually all
  organs but with slightly different forms in
  different locations ? isoenzymes
• classified according to biochemical functions
• unit of enzyme activity
• 1 IU transform 1 ?mol of substrate/minute
• 1 SI (katal) transform 1 mol substrate/second

3
Indicators of Cardiac Injury
Creatine kinase (CK)

• creatine phosphokinase (CPK)
• catalyze transfer of a PO4 group between
  creatine PO4 ADP to form creatine ATP
• requires magnesium as cofactor
• dimeric with pair of two different monomers ? M
  B
• 3 isozymes CK1 (BB), CK2 (MB), CK3 (MM)
• M and B subunits antigenically distinct proteins
  encoded by different genes

4
Creatine kinase (CK)

• primary tissue sources
• Brain, smooth muscle, prostate, thyroid, gut,
  lung ? CK-BB
• Cardiac muscle MB (20-30) MM (70-80)
• Skeletal muscle MB (1-2) MM (98-99)
• Plasma predom. MM with lt 6 MB
• relatively small molecular size ? allows leakage
  out of ischemic muscle or brain cells

5
Creatine kinase (CK)

• reference ranges in serum affected by
• Amount of lean muscle mass
• Thin, sedentary 30 50 U/L
• Muscular, exercising regularly 500 1000 U/L
• Age in neonates, CK-MB 5-10 of total CK
• Gender
• Race Africans 30 higher than Europeans
• Muscle activity direct relationship between
  intensity of exercise and CK level

6
Creatine kinase (CK)

• persons exercising periodically at usual
  intensity levels with lower CK than those who do
  not exercise at all
• decrease with severe inactivity
• short-term strenuous exercise ? 10-100 fold
  increase
• marathon runners ? up to 2000 U/L as resting value

7
Diagnostic Applications
CM-MM

• released from damaged muscles CK, AST, LD,
  myoglobin
• Myoglobin gtgt CK gtgt AST and LD
• released during ischemia, injury or inflammation
• also increased in
• Chronic myopathies
• Chronic renal failure
• Acute respiratory exertion respiratory muscles
  with more CK than other muscles

8
Diagnostic Applications
CK-BB

• Brain trauma or brain surgery
• Injury to smooth muscles (e.g. intestinal
  ischemia)
• Patients with malignancies, esp. prostate cancer,
  small cell lung CA, intestinal malignancies ?
  synthesize B subunit
• Transient increase after cardiac arrest ? reflect
  cerebral ischemia

9
Diagnostic Applications
CK-MB

• primary clinical use detection of acute MI
• Following MI
• Total CK 98 sensitive but 68-85 specific
  peak value 18-30 hrs duration 2-5 days level
  5-10x normal
• CK-MB rise proportional to extent of
  infarction appears in serum within 6 hrs after
  AMI peak value 12-24 hrs duration 1.5-3 days ?
  persistence indicates extension or infarction or
  re-infarction

10
Diagnostic Applications
Total Serum CK

• Normal 24 170 U/L (women) 24 195 U/L (men)
• Marked elevation (gt 5x normal)
• After trauma from electrocution, crush injury,
  convulsion, tetany, surgical incision or IM
  injection
• Athletic individuals inc. muscle mass inc.
  release during strenuous activity
• Muscular dystrophies
• Chronic inflammation of muscle (dermatomyositis
  or polymyositis)

11
Diagnostic Applications
Total Serum CK

• Mild or moderate elevation (2 4x normal)
• Hyper- or hypothermia
• Hypothyroidism
• After normal vaginal delivery BB isoenzyme from
  myometrial contractions
• Reyes syndrome

12
Atypical Isoenzymes

• Forms that migrate electrophoretically in
  positions different from standard ones
• Adenylate kinase
• Catalyze formation of ATP AMP from ADP
• Released from erythrocytes
• Macro CK type 1 complex of CK (BB) with
  antibody (IgG) ? mistaken for CK-MB no known
  clinical significance
• Macro CK type 2 oligomeric variant of CK
  mitochondrial
• If () in serum poor prognostic sign
• () in patients with malignancies moribund
  patients

13
Lactate dehydrogenase (LD)

• Zinc-containing part of glycolytic pathway
• Catalyze conversion of lactate to pyruvate using
  NAD as cofactor
• CH3 CH3
• HCOH NAD C O NADH H
• COOH COOH
• Tissue source present virtually in all tissues ?
  cytoplasm of all cells and tissues in the body
• Tetramers with 4 subunits of 2 possible forms H
  (heart) and M (muscle)

14
Lactate dehydrogenase (LD)

• Five isoenzymes
• LD1 LD2 high in heart muscle, erythrocytes,
  kidney
• LD4 LD5 high in skeletal muscle in liver
• Normal pattern in serum
• LD2 gt LD1 gt LD3 gt LD4 gt LD5
• Highest in newborns and infants values do not
  change with age in adults
• No gender difference

15
DIAGNOSTIC APPLICATIONS

• Total LD activity increase in any disease state
  where there is cell damage or destruction ?
  non-specific ? correlate with AST, ALT and CK
• Markedly inc. LD with normal or minimally inc.
  AST, ALT CK ? damage to biochemically simple
  cells (e.g. rbc, wbc), kidney, lung, LN or tumors
• Inc. LD CK inc. AST gt ALT ? cardiac or
  skeletal muscle injury
• AST ALT inc. gt LD ? transiently in liver
  disease (toxic or ischemic liver injury)

16
Myocardial damage

• Myocardium normally with LD1 gt LD2 ? similar to
  rbc
• Acute myocardial infarction
• Inversion or flipped LD1/LD2 ratio to a value gt
  1.0 in serum ? stay flipped for several days
• Levels inc. after 12-24 hrs, peak (2-10x normal)
  at 48-72 hrs, return to normal after 8-10 days
• used to confirm diagnosis of MI when CK isoenzyme
  analysis equivocal or after total CK CK-MB
  release has returned to normal

17
Other Applications

• Total LD used to estimate tumor mass including
  metastases
• LD1 or LD2 inc. in germ cell tumors (seminoma
  dysgerminoma) serve as tumor marker
• Flip LD1/LD2 ratio
• Extreme exercise
• Acute myocardial infarction
• Hemolytic anemia
• Megaloblastic anemia
• Renal cortical disease (renal infarct, renal cell
  CA)

18
Other Applications

• Inc. LD2, 3 and 4 malignancy large tumor
  burden
• Inc. LD3 4, dec. LD1 2 WBC tumors
  (leukemia, lymphoma, MM), pulmonary disease
• Inc. LD4 5 skeletal muscle injury, ischemic
  or toxic hepatic injury
• Isomorphic pattern inc. total LD, normal
  isoenzymes with tombstone pattern (relative
  amounts of isoenzymes the same) ? diffuse tissue
  damage accompanied by shock or hypoxemia

19
Myoglobin

• Small functions in storage and transfer of O2
  from Hgb in the circulation to intracellular
  respiratory enzymes of contractile cells
• With greater affinity for oxygen than Hgb
• Only one molecular form
• One of the first to diffuse out of ischemic
  muscle cells, even before CK
• Cleared from circulation by kidneys
• Measurement in serum with high sensitivity for
  muscle injury, including acute MI ? measure by
  immunoassay

20
Myoglobin

• In normal individuals,
• Levels related to muscle mass and activity
• Males gt females
• Africans gt Europeans
• Increase with increasing age due to decreasing GFR

21
Troponins

• Bind tropomyosin and govern excitation-contraction
  coupling
• Three subunits
• Troponin C (TnC) calcium-binding subunit
• Troponin I (TnI) bind to actin ? inhibitory
• Troponin T (TnT) bind to tropomyosin
• TnI and TnT with unique forms expressed in
  myocardial cells but not in other muscle types ?
  presence of cTnI or cTnT in serum highly specific
  for myocardial injury

22
Troponins

• cTnT
• 84 sensitivity for MI 8 hrs after onset of
  symptoms
• 81 specificity for MI 22 specificity for
  unstable angina
• cTnI
• 90 sensitivity for MI 8 hrs after onset of
  symptoms
• 95 specificity for MI 36 specificity for
  unstable angina

23
Troponins

• Cardiac troponins released in two phases
• Initial damage (acute MI) leave myocardial
  cells ? enter circulation the same time that
  CK-MB does ? peak at 4-8 hrs
• Sustained release from intracellular contractile
  apparatus occurs up to days after acute event
• First appear in circulation ff. myocardial injury
  slightly later than when myoglobin enters the
  blood ? rises after 3-6 hrs ? peaks at 20 hrs

24
Troponins

• General advantages
• cTnT and cTnI are released only following cardiac
  damage.
• Unlike CK CK-MB, cTnT and cTnI are present ,
  and remain elevated, for a long time ? cTnI
  detectable up to 5 days cTnT for 7-10 days
  following MI
• cTnT and cTnI are very sensitive.

25
Troponins

• General disadvantages
• Elevation can occur as a result of causes other
  than MI ? myocarditis, severe cardiac failure,
  cardiac trauma, pulmonary embolus with cardiac
  damage
• Failure to show a rise in cTnT or cTnI does not
  exclude the diagnosis of ischemic heart disease.
• Both may be elevated in patients with chronic
  renal failure with sustained levels of elevation.

26
Troponins

• Measured in serum by immunoassay
• Ideal time to check is between 6 and 9 hours from
  onset of symptoms
• If onset of symptoms indistinct take sample on
  admission, 6 9 hrs after and at 12 24 hrs
  after admission

27
(No Transcript)
28
Other enzymes useful in clinical diagnosis
Acid phosphatase (ACP)

• Optimal activity pH 5.0
• Tissue source
• Common to many tissues, esp. prostate
• Small amounts in rbc, platelets (during clot
  formation), liver and spleen
• Human milk and seminal fluid (very concentrated)

29
Acid phosphatase (ACP)

• Prostatic ACP distinguished from others using
  thymolphthalein monophosphate ? highly specific
  for prostatic ACP
• Major applications
• Evaluation of prostatic CA (metastatic local
  growth)
• Not elevated in CA confined within prostate, BPH,
  prostatitis or ischemia of prostate
• Medicolegal evaluation of rape vagina with
  little or no ACP
• Measured by radioimmunoassay ? acidify serum with
  citric acid to stabilize ACP activity

30
Alkaline phosphatase (ALP)

• Widely distributed along surface membranes of
  metabolically active cells
• Encoded for by four different genes expressed in
• Placenta
• Intestines
• Germ cell and lung
• Tissues including bone, liver, kidney
  granulocyte
• Very high activity in bone, liver, intestine,
  kidney, wbc and placenta

31
Alkaline phosphatase (ALP)

• Methods for distinguishing ALP isoenzymes
• Heat fractionation easiest most common heat
  serum sample at 56oC x 15 min. then compare with
  unheated sample
• Bone ALP extremely labile ? retain 10-20 of
  original activity
• Liver placental ALP heat stable ? liver ALP
  30-50 retained, placental ALP with all retained
• Chemical inhibition
• Urea block placental ALP
• Phenylalanine block liver bone ALP
• Electrophoresis - definitive

32
DIAGNOSTIC APPLICATIONS
Liver ALP

• Derived from epithelial cells of biliary tract ?
  excreted by bile into intestine
• Used for establishing diagnosis in jaundice
• Pronounced increase (gt 5x)
• Intra- or extrahepatic bile duct obstruction
• Biliary cirrhosis
• Moderate increase (3-5x normal) granulomatous
  or infiltrative liver disease
• Slight increase (up to 3x normal) viral
  hepatitis, cirrhosis

33
Bone ALP

• Elevation part of osteoblastic growth
• Pronounced increase
• Pagets disease
• Osteogenic sarcoma
• Hyperparathyroidism
• Moderate increase metastatic tumors in bone
  metastatic bone disease (rickets, osteomalacia)
• Slight increase healing fractures normal growth
  patterns in children

34

• Placental ALP
• With oncofetal form ? turned on and expressed by
  tumor cells in adults ? called Regan isoenzyme
• Slight increase in pregnancy
• Intestinal ALP
• Inc. in inflammatory bowel disease (ulcerative
  colitis regional enteritis)
• Secreted into the circulation after a meal in
  persons with blood type O and B ? inc. total
  ALP in non-fasting specimens

35

• Renal ALP
• Normally excreted into urine from renal tubular
  cells
• Granulocyte ALP
• Used as marker of granulocyte maturity in
  leukocytosis
• Lymphocytes infected with HIV ? release specific
  ALP fraction (band-10) ? surrogate marker for HIV
  infection in children

36
Aldolase

• Glycolytic enzyme ? split fructose-1,6-diphosphate
  into two triose phosphate molecules in glucose
  metabolism
• Distributed in all tissues
• Elevated in serum following
• Skeletal muscle disease or injury reflect
  severity of dermatomyositis
• Metastatic CA to liver 5. Hemolytic anemia
• Granulocytic leukemia 6. Tissue infarction
• Megaloblastic anemia

37
Aminotransferase (Transaminase)

• Catalyze reversible transfer of an amino group
  between an amino acid and an alpha-keto acid
• R R R R
• HCNH2 C O C O HCNH2
• COOH COOH COOH COOH
• Requires pyridoxal phosphate (vitamin B6) as
  cofactor

38
Alanine aminotransferase (ALT)

• glutamate-pyruvate transaminase (GPT)
• Rich amounts in hepatocytes ? with high
  specificity for liver damage
• Moderate amount kidney, heart, skeletal muscle
• Small amount pancreas, spleen, lung, red blood
  cells

39
Diagnostic Applications

• Inc. AST ALT excellent indicators of liver
  damage
• ALT increased in serum ff. acute MI 6 hrs after
  onset, peaks at 24-48 hrs, returns to normal in
  3-4 days
• AST inc. in conditions that can be confused with
  acute MI or that may complicate or co-exist with
  acute MI
• Shock or circulatory collapse from any cause
• Acute pancreatitis
• Cardiac arrhythmias or ischemic insult that do
  not progress to infarction

40
Gamma glutamyltransferase (GGT)

• gamma glutamyltranspeptidase
• Catalyze transfer of glutamyl groups between
  peptides or amino acids through linkage at a
  ?-COOH group ? important in transfer or movement
  of amino acids across membranes
• Large amounts in
• Pancreas and renal tubular epithelium
• Hepatobiliary cells

41
Diagnostic Applications

• increased activity
• In urine renal tubular damage
• Hepatocellular hepatobiliary diseases ?
  correlates better with obstruction cholestasis
  than with pure hepatocellular damage ?
  obstructive enzyme

42
Diagnostic Applications

• GGT alcohol
• Alcohol induces microsomal activity ? inc. GGT
  synthesis ? indicator of alcohol use
• GGT levels return to normal after 3-6 wks of
  abstention from alcohol ? test for compliance in
  alcohol-reduction programs

43
Diagnostic Applications

• GGT drugs
• Barbiturates, phenytoin other drugs
  (acetaminophen) inc. microsomal activity of GGT
• Potentially useful in drug treatment protocols

44
Amylase (Diastase)

• digestive enzyme
• Acts extracellularly to cleave starch into
  smaller groups finally to monosaccharides
• Major sources salivary glands
• exocrine pancreas

45
Pancreatic amylase

• secretion stimulated by pancreozymin
  (cholecystokinin)
• enter duodenum at ampulla of Vater via sphincter
  of Oddi
• Low levels found in
• Fallopian tubes 3. Small intestine
• Adipose tissue 4. Skeletal muscle
• readily cleared in urine

46
DIAGNOSTIC APPLICATIONS

• Acute Pancreatitis
• Levels rise within 6-24 hours ?remain high for a
  few days ? return to normal in 2-7 days
• Serum amylase normal but with suspicion of
  pancreatitis ? measure 24-hour urine amylase or
  serum lipase

47
DIAGNOSTIC APPLICATIONS

• Morphine administration
• Constrict pancreatic duct sphincter ? dec.
  intestinal excretion inc. absorption in the
  circulation
• Renal failure
• Failure to clear normally released amylase from
  the circulation ? no diagnostic significance

48
DIAGNOSTIC APPLICATIONS

• Malabsorption liver disease
• () circulating complexes of amylase with a high
  MW compound such as Igs ? macroamylasemia ?
  prevent renal clearance
• no diagnostic significance

49
DIAGNOSTIC APPLICATIONS

• Tumors
• serous ovarian tumors
• epithelium similar to FT ? produce cyst fluid
  with amylase ? appear in serum urine
• Lung CA
• ectopic production of amylase

50
Conditions Affecting Serum Amylase
Pronounced Elevation (gt 5x normal) Acute pancreatitis Pancreatic pseudocyst Morphine administration
Moderate Elevation (3-5x normal) Pancreatic CA (head of pancreas) Mumps Salivary gland inflammation Perforated peptic ulcer Ionizing radiation
51
Angiotensin Converting Enzyme (ACE)

• Action convert angiotensin I to angiotensin II
• Site lungs
• Main tissue source macrophages epithelioid
  cells
• Diagnostic use
• Active sarcoidosis primary use
• Other granulomatous diseases (e.g. TB)
• Disorders of macrophage function (e.g. Gauchers
  disease leprosy)
• Normal adults persons lt 20 y/o high levels

52
Cholinesterase

• Pseudocholinesterase (CHS)
• Serum
• Synthesized in hepatocytes
• Able to act on a wider variety of choline esters
• Active at both high and low substrate
  concentration
• Inhibited by organophosphorous compounds (e.g.
  insecticides)

53
Cholinesterase

• True Acetylcholinesterase (AcCHS)
• Erythrocytes and nerve endings
• Breaks down Ach into acetate and choline at
  post-synaptic sites
• Active at low substrate concentration inhibited
  by high Ach concentration
• Inhibited by organophosphorous compounds

54
DIAGNOSTIC APPLICATION

• Serum CHS
• Decreased in organophosphate poisoning ? falls
  early after exposure rises soon after exposure
  ceases ? used to document acute toxicity
• Decreased in hepatocellular disease
• Completely inactivates succinylcholine (muscle
  relaxant) in the circulation over a short
  interval (several seconds) ? () reversal of
  paralysis
• Normal activity inhibited by dibucaine

55
DIAGNOSTIC APPLICATION

• Erythrocyte AcCHS
• Inhibited less rapidly by organophosphate
  insecticides ? remains depressed even after serum
  CHS returns to normal ? used to document prior
  exposure
• Does not act efficiently on succinylcholine

56
Lipase

• Alimentary lipase
• Cleave dietary TGs into free fatty acid
  glycerol
• Secreted by exocrine pancreas into the duodenum
• Found almost exclusively in pancreas ? highly
  specific

57
Lipase

• Alimentary lipase
• Not cleared into the urine ? remain elevated
  after amylase has returned to normal
• Highest levels in acute pancreatitis
• Moderate increase pancreatic CA
• Inc. after administration of morphine or
  cholinergic drugs ? () constriction of sphincter
  of Oddi

58
Lipase

• Blood lipase
• Cleaves fatty acids from lipoproteins and clears
  chylomicrons from the circulation
• Bound to vascular endothelium membrane
• Released into plasma by administration of heparin
  ? occurs within minutes of IV heparin dose ?
  post-heparin lipolytic activity (PHLA)

59
Lysozyme (Muramidase)

• Low MW hydrolytic enzyme
• Catalyze breakdown of bacterial cell walls
• Sources tears, saliva, sputum, granulocytes,
  monocytes
• Readily cleared into urine
• Quantitated by ability to lyse the bacterium
  Micrococcus lysodeikticus ? convert turbid
  suspension into a clear one

60
DIAGNOSTIC APPLICATIONS

• Makedly elevated in serum urine acute
  monocytic acute myelomonocytic leukemias
• Low levels lymphoctic leukemia chronic
  granulocytic anemia
• Serial levels used in detecting relapse in acute
  leukemias

61
ENZYME-ORGAN ASSOCIATIONS
ORGAN ENZYME
Liver Aminotransferases (AST, ALT) Lactate DH (LD5) Gamma-glutamyltransferase Alkaline phosphatase
Heart Creatine kinase (MB) Lactate DH (LD1 gt LD2) Troponins I and T
Skeletal muscle Creatine kinase (MM) Lactate DH (LD5) Aldolase
62
ENZYME-ORGAN ASSOCIATIONS
Brain Creatine kinase (BB)
Bone Alkaline phophatase (heat labile)
Prostate Acid phosphatase
Pancreas Amylase Lipase
63
Thank You!