ENZYMES OF CLINICAL IMPORTANCE: AMINOTRANSFERASES diagnosis because of

their wide distribution

AMINOTRANSFERASES
 Are also called transaminases. ELEVATIONS
 They are cytoplasmic enzymes that play an important role in Hepatocellular disorders
exchanging the carbon skeleton between amino acids and alpha Congestive heart failure
ketoacids. Hepatocellular disorders
 There are several transaminase isoenzymes but only AST and Hepatocellular disorders Highest
ALT are measured in the clinical laboratory. Viral hepatitis May reach 100 times ULN
 Enzymatic activity: reversible Moderate levels: approximately 4
o Cofactor: Pyridoxal phosphate (REQUIRED) Cirrhosis
times ULN
 Reaction optimum pH: 7.3-7.8 Though the heart may contain ALT, it is NOT elevated in acute
 ASPARTATE AMINOTRANSFERASE (AST/ Glutamate myocardial infarction
oxaloacetate transaminase/GOT/ E.C. 2.6.1.1) Reference values: 5-35 U/L at 37⁰C
 ALANINE AMINOTRANSFERASE (ALT/Glutamyl pyruvate
transaminase (GPT)/ E.C. 2.6.1.2) ALANINE AMINOTRANSFERASE
 Tissue distribution  ALT
o AST and ALT are both widely distributed in human tissues.  E.C. 2.6.1.2
o AST: highly concentrated in cardiac and skeletal
muscles, and liver
 Little amount: seen in kidneys, pancreas, and RBC
o ALT: liver (highest) – can be considered a liver-specific enzyme
of the transferases family
*Hepatocytes contain 3-4x more AST than ALT

ASPARTATE AMINOTRANSFERASE
 AST
 E.C. 2.6.1.1

 ALT transfers an amino group from alanine to alpha-

ketoglutarate with the formation of glutamate and pyruvate

EVALUATION OF LIVER DISORDERS

Higher elevations are seen than in
Hepatocellular disorders extrahepatic or intrahepatic
obstructive disorders
ALT elevations are higher than
AST and ten to remain elevated
longer as a result of longer half-
 Transamination reaction: important in intermediary Acute inflammatory conditions
life of ALT in serum
metabolism because of its function in the synthesis and *AST: 16 hours
degradation of amino acids ALT: 24 hours
o The ketoacids formed are oxidized by the tricarboxylic acid
Also for non-alcoholic asymptomatic patients: for screening of
cycle to provide a source of energy
prospective blood donors; more sensitive and specific screening
 AST has 2 isoenzymes: cytoplasmic and mitochondrial ASTS test for posttransfusion hepatitis or occupational toxic exposure
o Cytoplasmic isoenzyme: more predominant in serum compared to AST
o Intracellular concentration is 7,000 times higher in serum
Though the heart may contain ALT, it is NOT elevated in acute
than the extracellular concentration
myocardial infarction
o Cellular necrosis: Mitochondrial form can be elevated
Reference values: 7-45 U/L at 37⁰C
Diagnostic Significance
DIAGNOSTIC SIGNIFICANCE  Assay General Conditions
Hepatocellular Skeletal muscle Acute myocardial o Haemolysis: AST = affected; ALT = not affected
disorders involvement infarction o ALT and AST are stable in serum for 3-4 days at
Muscular dystrophies Rises within 6-8 refrigerator temperature
and inflammatory hours; peaks at 24 o Specimen: serum or plasma – no anticoagulant inhibits
conditions: 4-8 times hours; returns to its reaction
ULN normal within 5 days o Turbidity (icteric samples): create difficulties with
*AST levels are NOT blanking
useful in AMI o Alcohol: lowers AST values
 o Muscle trauma (intramuscular injections, exercise, *Molecular weight: Approximately 82,000 – associated with ATP
surgery): increase AST levels generation in contractile or transport systems
Methods for Analysis  Catalyses the reversible phosphorylation of creatine using ATP as
 Assays for both enzymes utilise the same types of reactions a cofactor
for the detection of products and are based on three major
approaches
 All the reactions involve the measurement of ketoacid
formation
REACTION METHOD REMARKS
Reaction with
Disadvantage:
dinitrophenyl Reitman-Frankel
Lack of specificity
hydrazine (DNPH)
Disadvantage:
*Creatine kinase predominant function: occurs in muscle cells –
Though the
involved in the storage of high energy creatine phosphate
procedure may be
*Every contraction cycle of muscle = creatine phosphate use with
simple and
Coupling with the production of ATP → relatively constant ATP levels
Babson et al straightforward,
diazonium salt  Tissue distribution
problems may also
still occur because o High concentration
of some  Skeletal muscle
interferences  Heart muscle
 Brain
Coupled enzyme
Karmen or Walker Preferred method o Smaller amount
kinetics
 Bladder
*The reaction proceeds at pH 7.3-7.8
 Placenta
*The ketoacid formed by the aminotransferase reacts in a system
 Thyroid
using NADH
 Intestinal tract
*The coenzyme is oxidised to NAD and the decrease of
 Kidney
absorbance is measured at 340 nm
 Uterus
*ALT: Pyruvate formed in the reaction is converted to lactate by
 Spleen
lactate dehydrogenase
 Pancreas
*AST: Oxaloacetate produced is reduced to malate by the enzyme
 Prostate
malate dehydrogenase
 Creatine Kinase Isoenzymes
*Pyridoxal phosphate: necessary as a coenzyme to carry the
o Exists as a dimer: composed of 2 polypeptide chains
amino group from one acid to the other
o 2 possible structures for the protein subunit
 M chain
ASPARTATE ALANINE
 B chain
AMINOTRANSFERASE AMINOTRASNFERASE
o The two subunits combine to form 3 different coenzymes
(AST) (ALT)
 CK-BB/CK1: Brain type
Major
 CK-MB/CK2: Hybrid type
organ Heart Liver
 CK-MM/CK3: Muscle type
affected
 CK-BB or CK1
Aspartic alpha Alanine alpha
Substrate o Brain type
ketoglutarate ketoglutarate
o Fastest moving isoenzyme during electrophoresis
End Glutamic acid + Glutamic acid + pyruvic o Half-life: 1-5 hours
products oxaloacetic acid acid o Rarely in serum because of its greater molecular size and
Colour inability to pass the blood-brain barrier
2,4 DNPH 2,4 DNPH
developer ELEVATED LEVELS OF CK-BB/CK1
Colour In serum
0.4 NaOH 0.4 NaOH
intensifier
Indicative of diseases/injury
Methods Reitman and Frankel Reitman and Frankel Intestinal tract
of the following organs
 ALT-AST Ratio Prostate
o De Ritis Ratio Kidney
o High: alcoholic or toxic liver diseases
Bladder
o Low (< 10): Acute or chronic viral hepatitis (High ALT)
Breast
Brain
CREATINE KINASE
 CK
Also seen in CNS Shock
 Old name: Creatine phosphokinase (CPK)
Anoxic Encephalopathy
 ATP: Creatine-N-phosphotransferase
Cerebrovascular accident
 EC 2.7.3.2
Seizure
Placental/uterine trauma
 Carcinoma *CK-MB result must read with LDH in acute myocardial infarction
Reye’s Syndrome diagnosis
Carbon monoxide poisoning  Atypical Creatine kinase
Malignant hyperthermia o Macro-CK: Migration occurs midway between CK-MM and
Acute renal failure CK-MB
Chronic renal failure  Complexed with immunoglobulins or lipoproteins
 Occurrence appears to be age-related and frequent
 CK-MB/CK2 among females older than 50 years old.
o Hybrid type *IgG and IgA: immunoglobulins associated with Macro-CK in
o Tissue distribution most instances
 Skeletal muscle *Macro-CK Incidence in Serum: 0.8-1.6%
 Cardiac muscle o Mitochondrial CK(CK-Mi): bound to the exterior surface
 Tongue of the inner mitochondrial membranes of muscle, brain,
 Oesophagus & liver.
 Diaphragm  Detected in malignant tumours and cardiac
ELEVATED LEVELS OF CK-MB/CK2 abnormalities
Myocardial infarction *CK-Mi: Not present in normal serum and myocardial infarction
Subarachnoid injury *In some analyses, atypical forms can be measured as CK-MB
resulting in falsely elevated values
Ischemia
 Optimum reaction pH
Angina
o Forward reaction: 9.0
Inflammatory heart disease
o Backward reaction: 6.8
Cardiac surgery
 Assay General Considerations
Duchenne-type Muscular Dystrophy
o Best specimen: Serum
Polymyositis  Specimen = light sensitive
Malignant hyperthermia o Avoid haemolysis: Elevates CK levels
Reye’s Syndrome *RBCs do NOT have CK but are rich in AK: AK reacts with ADP
Rocky Mountain Spotted Fever to produce ATP which participates in the reaction → Falsely
Carbon monoxide poisoning elevated serum CK
 CK-MM/CK3 o Specimen cannot be stored for long periods: Enzyme
o Muscle type activity = diminished
o Tissue distribution *Activity regeneration: Addition of sulfhydryl groups –
 Greater concentration: Skeletal and cardiac muscles cysteine, dithiothreitol, or mercaptoethanol
o Major isoenzyme in serum of healthy people  CK Relative Index (CKI)
o Slowest migrating isoenzyme in the electrophoretic field o Expression of the percentage of the total CK that is
o Has three fractions: CK-MM1, CK-MM2, CK-MM3 attributed to CK-MB
ELEVATED LEVELS OF CK-MM/CK3 o To know the possible release of CK-MB from non-cardiac
Myocardial infarction tissues when total CK is very high
Skeletal muscle disorder
Muscular dystrophy
Polymyositis CKI = CK-MB µg/L or IU/L x 100
Malignant hyperthermia Total CK IU/L
Physical activity
Intramuscular injection
 Diagnostic Significance
o Creatine kinase levels are frequently elevated in the
following
 Myocardial infarction
 Rhabdomyolysis
 Muscular dystrophy
CK-MB LEVELS FOLLOWING MYOCARDIAL INFARCTION
4-8 hours Begin to rise
12-24 hours Peak
48-72 hours Return to normal
*Creatine kinase is elevated in Duchenne-type Muscular dystrophy
(50-100 X ULN)
*Total CK: an early diagnostic tool in the identification of Vibrio
vulnifincus infections
Methods for Analysis
TYPE OF ASSAY METHOD PRINCIPLE REMARKS
Decrease in absorbance due to
Enzymatic Assay Tanzer-Gilvarg Forward/direct method
NADH consumption at 340 nm
Reverse/indirect method; Most
Increase in the absorbance of commonly performed in the clinical
Oliver-Rosalki-Hess NADPH produced by G6PD laboratory; Reaction proceeds 2-6
reaction times faster than the forward
reaction

CK reacts with ninhydrin to

Colorimetric Method Sax and Moore
produce fluorophore
Creatine is made to react with
Hughes Method diacetyl and alpha naphthol to
produce a pink end product

Separate atypical forms and

Electrophoresis Reference method
visualise the presence of AK
Ion exchange chromatography

Immunoassay

Column chromatography

LACTATE DEHYDROGENASE *LDH-5: most labile isoenzyme

 LD  Methods of LD Isoenzyme Analysis
 L-Lactate:NAD oxidoreductase PHYSICAL CHEMICAL
 E.C. 1.1.1.27 Electrophoretic Substrate-product relationship
 Tissue distribution Selective absorption of
o Greater concentration diethylaminoethyl cellulose Coenzyme affinity
 Myocardium (DEAE)
 Liver Differential chemical inhibition
 Kidneys Solvent precipitiation of LD activity urea, sulphate,
 Skeletal muscles choloroform
o Significant amount: RBC Heat denaturation at 56ºC for
*LD is widely distributed 30 minutes
 Total LD can increase in many conditions
 Isoenzyme fractions give specific clinical significance  Reference Range
 LDH Isoenzymes FORWARD (L-P) 100-225 U/L (8.3-8.9)
o There are 5 isoenzymes BACKWARD (P-L) 80-280 U/L (7.1-7.4)
o Each is a tetramer (composed of 4 protein subunits) AT 35ºC: 125-220 U/L (BISHOP)
LDH ISOENZYMES
H (Heart) Polypeptide More affinity to lactate than
pyruvate
M (Muscle) Polypeptide More affinity to pyruvate than
lactate
*Molecular weight: 32,000 daltons
 Assay General Considerations
o Heparin, EDTA, and Oxalates: interfere with LD
activity
o Haemolysis: should be avoided
*RBC contains 100-150 times LD
o If not examined: preserve at 25ºC analysis is done
within 24 hours
*Sample should be kept at 25ºC and analysed within 48
hours
*LDH activity is unstable in serum regardless of the temperature at
which it is stored
Summary of LDH Isoenzymes
ISOENZYME % IN TOTAL LD ANODAL MOBILITY TISSUE SOURCE DISORDER COMMENTS
Myocardial infarction,
LD1/LD-I (HHHH/H4) 14-26 Greatest
haemolytic anaemia
Megaloblastic
Heart, RBCs Most heat stable
LD2/LD-II anaemia, acute renal
29-39
(HHHM/H3M) infarction, haemolysed
specimen
Pulmonary embolism,
extensive pulmonary
LD3/LD-III Lungs, lymphocytes, pneumonia,
20-26
(HHMM/H2M2) spleen, pancreas lymphocytosis, acute
pancreatitis,
carcinoma
LD4/LD-IV Hepatic injury or
8-16 Liver
(HMMM/HM3) inflammation
LD5/LD-V
6-16 Least Skeletal muscle Skeletal muscle injury
(MMMM/M4)
*LDH has been shown to complex with IgA IgG
>Migrates between LDH3 and LDH4 – this macromolecular complex is NOT associated with any specific clinical abnormality
*LDH is NOT specific to cardiac tissue and is NOT a preferred marker for acute myocardial infarction diagnosis
>Acute myocardial infarction and pulmonary infarction: show slight elevations of approximately THE SAME degree (2-3 times ULN)
*Skeletal muscle disorders and some leukaemia: contribute to increased LDH levels
>Marked elevations: observed in most patients with acute lymphoclastic leukaemia in particular
Extra LD Isoenzyme
EXTRA LD ISOENZYME
LD-6 Alcohol dehydrogenase
LD-6 migrates cathodic to LD-5
Present in arteriosclerotic cardiovascular failure
May reflect liver injury secondary to severe circulatory insufficiency
*Presence of LD-6: Grave prognosis and impending death

CONDITION FINDINGS
Normal serum LD2 > LD1 > LD3 > LD4 > LD5
Acute myocardial infarction LD1 > LD2 > LD3 > LD4 > LD5 (Flipped LD)
Acute renal infarction LD1 > LD2 > LD3 > LD4 > LD5 (Flipped LD)
Haemolysis LD1 > LD2 > LD3 > LD4 > LD5 (Flipped LD)
Normal CSF LD1 > LD2 LD3 > LD4 > LD5
Hydrocephalus and seizure LD2 > LD1 > LD3 > LD4 > LD5
Bacterial meningitis LD5 > LD4 > LD3 > LD2 > LD1

Methods of Total LD Analysis

WROBLEWSKI-CABAUD WROBLEWSKI-LADUE WACKER
Colorimetric method Final reaction: decrease in absorbance Final reaction: increase in absorbance
Measurement: 440-525 nm Spectrophotometric measurement: 340 nm Spectrophotometric measurement: 340 nm
Reference range: 200-450 Worblewski
Reference range: 80-280 U/L at 37 ºC Reference range: 100-225 U/L at 37 ºC
units/mol or 85-185 U/L at 30ºC

Clinical Application
Normally 2 times ULN in 1-3 day old infants
PATHOLOGIC CONDITIONS
MEGALOBLASTIC MYOCARDIAL PULMONARY METASTATIC LIVER ACUTE
ANAEMIA INFARCTION INFARCTION CANCER PANCREATITIS
Increase 12-24 hours after
High LD1 and LD2 High LD3 High LD4 and LD5 High LD3
pain onset
Aldolase and Aldolase and
Vitamin B12/folate Elevation is seen in 24 phosphohexose phosphohexose
Peaks at 48-72 hours
deficiency hours isomerase: increased isomerase: increased
(higher) (even higher)
 Aldolase and
AST: within normal range 2
phosphohexose Remains elevated for 10
days after pain onset =
isomerase: within days
pulmonary infarction
reference range
Flipped LD pattern occurs in
12-24 hours
Aldolase and
phosphohexose
isomerase: increased
*LD-5: has the greatest clinical significance in hepatic disorder detection, particularly intrahepatic disorders
>Skeletal muscles disorders: will reveal elevated LDH-5 levels
OTHER CLINICAL APPLICATIONS OF HIGH LD
Skeletal muscle involvement
Kidney damage
Bacterial meningitis