FE A. BARTOLOME, MD, DPASMAP
Dept. of Pathology & Laboratory Diagnosis
Our Lady of Fatima University
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
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
Creatine kinase (CK)
• primary tissue sources:
1. Brain, smooth muscle, prostate, thyroid, gut,
lung CK-BB
2. Cardiac muscle – MB (20-30%) & MM (70-
80%)
3. Skeletal muscle – MB (1-2%) & MM (98-99%)
4. Plasma – predom. MM with > CK >> AST and LD
• released during ischemia, injury or inflammation
• also increased in:
1. Chronic myopathies
2. Chronic renal failure
3. Acute respiratory exertion – respiratory muscles
with more CK than other muscles
Diagnostic Applications
CK-BB
• Brain trauma or brain surgery
1. Injury to smooth muscles (e.g. intestinal
ischemia)
2. Patients with malignancies, esp. prostate
cancer, small cell lung CA, intestinal
malignancies synthesize B subunit
3. Transient increase after cardiac arrest
reflect cerebral ischemia
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
Diagnostic Applications
Total Serum CK
Normal: 24 – 170 U/L (women) 24 – 195 U/L (men)
• Marked elevation (> 5x normal)
1. After trauma from electrocution, crush injury,
convulsion, tetany, surgical incision or IM injection
2. Athletic individuals – inc. muscle mass & inc.
release during strenuous activity
3. Muscular dystrophies
4. Chronic inflammation of muscle (dermatomyositis
or polymyositis)
Diagnostic Applications
Total Serum CK
• Mild or moderate elevation (2 – 4x normal)
1. Hyper- or hypothermia
2. Hypothyroidism
3. After normal vaginal delivery – BB isoenzyme
from myometrial contractions
4. Reye’s syndrome
Atypical
Isoenzymes
Forms that migrate electrophoretically in positions different
from standard ones
1. Adenylate kinase
Catalyze formation of ATP & AMP from ADP
Released from erythrocytes
2. Macro CK type 1 – complex of CK (BB) with antibody (IgG)
mistaken for CK-MB; no known clinical significance
3. Macro CK type 2 – oligomeric variant of CK; mitochondrial
If (+) in serum – poor prognostic sign
(+) in patients with malignancies & moribund patients
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)
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 > LD1 > LD3 > LD4 > LD5
• Highest in newborns and infants; values do not change
with age in adults
• No gender difference
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 > ALT cardiac or skeletal
muscle injury
• AST & ALT inc. > LD transiently in liver disease
(toxic or ischemic liver injury)
Myocardial damage –
• Myocardium normally with LD1 > LD2 similar to
rbc
• Acute myocardial infarction:
Inversion or flipped LD1/LD2 ratio to a value >
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
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)
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
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
Myoglobin
• In normal individuals,
Levels related to muscle mass and activity
Males > females
Africans > Europeans
Increase with increasing age due to
decreasing GFR
Troponins
• Bind tropomyosin and govern excitation-contraction
coupling
• Three subunits
1. Troponin C (TnC) – calcium-binding subunit
2. Troponin I (TnI) – bind to actin inhibitory
3. 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
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
Troponins
• Cardiac troponins released in two phases:
1. Initial damage (acute MI) – leave myocardial cells
enter circulation the same time that CK-MB
does peak at 4-8 hrs
2. 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
Troponins
• General advantages:
1. cTnT and cTnI are released only following
cardiac damage.
2. 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
3. cTnT and cTnI are very sensitive.
Troponins
• General disadvantages:
1. Elevation can occur as a result of causes other
than MI myocarditis, severe cardiac failure,
cardiac trauma, pulmonary embolus with cardiac
damage
2. Failure to show a rise in cTnT or cTnI does not
exclude the diagnosis of ischemic heart disease.
3. Both may be elevated in patients with chronic
renal failure with sustained levels of elevation.
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
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)
Acid phosphatase (ACP)
• Prostatic ACP distinguished from others using
thymolphthalein monophosphate highly specific for
prostatic ACP
• Major applications:
1. Evaluation of prostatic CA (metastatic & local growth)
Not elevated in CA confined within prostate, BPH,
prostatitis or ischemia of prostate
2. Medicolegal evaluation of rape – vagina with little or
no ACP
• Measured by radioimmunoassay acidify serum with
citric acid to stabilize ACP activity
Alkaline phosphatase (ALP)
• Widely distributed along surface membranes of
metabolically active cells
• Encoded for by four different genes expressed in:
1. Placenta
2. Intestines
3. Germ cell and lung
4. Tissues including bone, liver, kidney & granulocyte
• Very high activity in bone, liver, intestine, kidney, wbc
and placenta
Alkaline phosphatase (ALP)
• Methods for distinguishing ALP isoenzymes:
1. 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
2. Chemical inhibition
Urea – block placental ALP
Phenylalanine – block liver & bone ALP
3. Electrophoresis - definitive
DIAGNOSTIC APPLICATIONS
Liver ALP
Derived from epithelial cells of biliary tract excreted by
bile into intestine
• Used for establishing diagnosis in jaundice
• Pronounced increase (> 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
Bone ALP
Elevation part of osteoblastic growth
• Pronounced increase:
Paget’s disease
Osteogenic sarcoma
Hyperparathyroidism
• Moderate increase: metastatic tumors in bone;
metastatic bone disease (rickets, osteomalacia)
• Slight increase: healing fractures; normal growth
patterns in children
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
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
Aldolase
• Glycolytic enzyme split fructose-1,6-diphosphate into two
triose phosphate molecules in glucose metabolism
• Distributed in all tissues
• Elevated in serum following:
1. Skeletal muscle disease or injury – reflect severity of
dermatomyositis
2. Metastatic CA to liver 5. Hemolytic anemia
3. Granulocytic leukemia 6. Tissue infarction
4. Megaloblastic anemia
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
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
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:
1. Shock or circulatory collapse from any cause
2. Acute pancreatitis
3. Cardiac arrhythmias or ischemic insult that do not
progress to infarction
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
Diagnostic
Applications
• increased activity:
1. In urine – renal tubular damage
2. Hepatocellular & hepatobiliary diseases
correlates better with obstruction &
cholestasis than with pure hepatocellular
damage “obstructive” enzyme
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
Diagnostic
Applications
• GGT & drugs
Barbiturates, phenytoin & other drugs
(acetaminophen) inc. microsomal activity of
GGT
Potentially useful in drug treatment protocols
Amylase (Diastase)
• digestive enzyme
• Acts extracellularly to cleave starch into smaller
groups & finally to monosaccharides
• Major sources: salivary glands
exocrine pancreas
Pancreatic amylase
• secretion stimulated by pancreozymin
(cholecystokinin)
• enter duodenum at ampulla of Vater via
sphincter of Oddi
• Low levels found in:
1. Fallopian tubes 3. Small intestine
2. Adipose tissue 4. Skeletal muscle
• readily cleared in urine
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
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
DIAGNOSTIC
APPLICATIONS
Malabsorption & liver disease
• (+) circulating complexes of amylase with a
high MW compound such as Ig’s
macroamylasemia prevent renal clearance
• no diagnostic significance
DIAGNOSTIC
APPLICATIONS
Tumors
1. serous ovarian tumors
• epithelium similar to FT produce cyst
fluid with amylase appear in serum &
urine
2. Lung CA
• ectopic production of amylase
Conditions Affecting
Serum Amylase
Pronounced Elevation (> 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
Angiotensin Converting
Enzyme (ACE)
• Action: convert angiotensin I to angiotensin II
• Site: lungs
• Main tissue source: macrophages & epithelioid cells
• Diagnostic use:
1. Active sarcoidosis – primary use
2. Other granulomatous diseases (e.g. TB)
3. Disorders of macrophage function (e.g. Gaucher’s
disease & leprosy)
4. Normal adults & persons LD2)
Troponins I and T
Skeletal muscle Creatine kinase (MM)
Lactate DH (LD5)
Aldolase
ENZYME-ORGAN
ASSOCIATIONS
Brain Creatine kinase (BB)
Bone Alkaline phophatase (heat labile)
Prostate Acid phosphatase
Pancreas Amylase
Lipase