C1 - Section 3. ENZYMES OF CLINICAL IMPORTANCE Flashcards
Catalyze interconversions of the amino acids & alpha-ketoacids by transfer of amino groups
AMINOTRANSFERASES
as obligate coenzyme
Pyridoxal phosphate
will be bound to the apoenzyme and serves as a true prothetic group
Pyrodoxal-5’-phosphate
Pyrodoxal-5’-phosphate
will accept the amino group from the first substrates (aspartate/alanine) to form pyridoxamine-5-phosphate and the first product of the reaction –
oxaloacetate and pyruvate
the coenzyme in amino form will then transfer the amino group to the acceptor/second substrate (?)-to form the second products of the reaction-p5p is regenerated
oxoglutarate
AMINOTRANSFERASES Function:
Amino acid metabolism
Ketoacids formed are ultimately oxidized by the
TCA Cycle
Formerly SGOT (Serum glutamic-oxalocacetic transaminase)
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
involved in the transfer of an amino group between aspartate and a-keto acids
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
Reaction catalyzed:
widely distributed in human tissue
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
Highest concentration: cardiac tissue, liver & skeletal muscle
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
Smaller amounts: kidney, pancreas & RBCs
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
Isoenzymes of AST in the cytoplasm & the mitochondria
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
Both mitochondria and cytoplasmic forms of AST are found in cells.
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
About 5-10% of the AST activity in serum from healthy individuals is of mitochondrial origin
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
Evaluation of hepatocellular disorders & skeletal muscle involvement
AST
Liver disease-most important cause of elevated transaminase activity in serum
AST
In most liver disease, ALT is higher than AST (Except:?)
Alcoholic hepatitis, Hepatic Cirrhosis and liver neoplasia
Mild degree of liver tissue injury: cytoplasmic isoenzyme is predominant
AST
Severe tissue damage: release of mitochondrial isoenzyme
AST
shows marked increase in patients with extensive liver cell degeneration and damage
mitochondrial AST activity in serum
Formerly SGPT (Serum glutamic pyruvic transaminase)
ALANINE AMINOTRANSFERASE (ALT); E.C. 2.6.1.2
specifically, catalyze the transfer of an amino group from alanine to a-ketoglutarate with the formation of glutamate and pyruvate
ALANINE AMINOTRANSFERASE (ALT); E.C. 2.6.1.2
ALANINE AMINOTRANSFERASE (ALT); E.C. 2.6.1.2
Reaction catalyzed:
Distributed in many tissues
ALT
High concentrations in the liver (more liver-specific enzyme of the transferases)
ALT
low levels in the heart and skeletal muscle
ALT
Isoenzyme: exclusively cytoplasmic form
ALT
RBC contains 5-8X as much ALT activity as does the serum
ALT
Evaluation of hepatic disorders (hepatocellular)
ALT
Progressive inflammatory liver conditions: higher ALT elevations than AST
ALT
Higher elevations are found in hepatocellular disorders than extrahepatic ot intrahepatic obstructive disorders
ALT
diagnostic marker of alcoholic liver disease
De Ritis Ratio (AST:ALT ratio)
implication: most causes of liver cell injury, associated w/ greater ALT than AST, however, there are cases where in AST to ALT ratio is 2:1 or greater
De Ritis Ratio (AST:ALT ratio)
(also in viral hepatitis)
o Normally <1.0
: associated with cirrhosis
o If >1.0 but <2.0
: associated with alcoholic hepatitis or hepatocellular carcinoma
o If >2.0
: AST>ALT initially; w/in 24-48 hrs., ALT>AST
o Acute hepatocellular injury
Higher AST activity in
hepatocytes
In (?) of the liver, ALT elevations are frequently higher than those of AST and tend to remain elevated longer as a result of the longer half-life of ALT in serum (16 and 24 hours, respectfully).
acute inflammatory conditions
Transaminase reactions coupled to specific dehydrogenase reactions
Continuous Monitoring Method
multiple measurement of absorbance change during the reaction is observed
Continuous Monitoring Method
The oxo-acids formed in the reaction are measured indirectly by enzymatic reduction to the corresponding hydroxyl acids
Continuous Monitoring Method
The accompanying change in NADH concentration is being monitored spectrophotometrically
Continuous Monitoring Method
coupled enzymatic reaction w/ MDH (indicator reaction)
Assay reaction for AST Activity
Karmen Method
Monitors change in absorbance at 340 nm (NADH to NAD)
Assay reaction for AST Activity
Karmen Method
Optimal pH: 7.3 – 7.8
Assay reaction for AST Activity
Karmen Method
Hemolysis: increase serum
AST
Pyruvate formed is converted to lactate by LDH
Assay reaction for ALT Activity
LDH as the indicator enzyme
Assay reaction for ALT Activity
NADH formed is oxidized to NAD
Assay reaction for ALT Activity
The accompanying change in NADH concentration is being monitored spectrophotometrically
Assay reaction for ALT Activity
the disappearance of NADH is followed by measuring he decrease in absorbance
Assay reaction for ALT Activity
Change in absorbance is proportional to the micromoles of NADH oxidized that reflects the number of substrate transformed
Assay reaction for ALT Activity
activity in RBC is 15x higher than in serum
AST
activity is stable in serum for 3 – 4 days at ref °T
AST
AST Reference Range:
5 – 30 U/L (37°C)
Relatively unaffected by hemolysis
ALT
activity in RBCs is 7x higher than in normal serum)
ALT
Stable for 3 – 4 days at 4°C
ALT
ALT Reference Range:
6 – 37 U/L (37°C)
Coupling w/ 2,4- DNPH (Reitman-Frankel)
Colorimetric Method
Still feasible
Colorimetric Method
Phenylhydrazones of oxaloacetate & pyruvate are more chromogenic
Colorimetric Method
Simple; limited but acceptable accuracy
Colorimetric Method
Derivative formed will give a strong blue color measured as 505 nm
Colorimetric Method
An enzyme w/ MW of approximately 82,000
Creatine Kinase (E.C. 2.7.3.2)
Catalyzes reversible phosphorylation of Creatine by ATP
Creatine Kinase (E.C. 2.7.3.2)
it is generally associated w/ ATP regeneration in transport system
Creatine Kinase (E.C. 2.7.3.2)
Its dominant physiologic function is in muscle cells where it is involved in storage of high-energy creatine phosphate (phosphocreatine - major phosphorylated compound in muscle)
Creatine Kinase (E.C. 2.7.3.2)
CK Reaction catalyzed:
When muscle contracts, ATP is consumed (to form ADP) & CK catalyzes the rephosphorylation of ADP to form ATP, using Phosphocreatine
Creatine Kinase (E.C. 2.7.3.2)
Mg-forms complexes with ATP and ADP with narrow concentration because excess will be inhibitory
Creatine Kinase (E.C. 2.7.3.2)
Greatest in striated muscle, brain tissue & heart tissue
CK
Smaller quantities: bladder, placenta, GIT, Thyroid, uterus, kidney, lung, prostate, spleen & pancreas
CK
liver and erythrocyte is devoid of the activity of CK
CK
Dimer w/ 2 sub-units: B (brain) & M (muscle)
CK
Each sub-unit w/ a MW of ~ 40,000
CK
CK Three major isoenzymes:
Found in the brain, prostate, gut, lung, bladder, uterus, placenta & thyroid
CK – BB (brain type) CK1
present in varying degrees in heart muscle (25 – 46% of CK activity) and minor degree in skeletal muscle
CK – MB (hybrid type) CK2
predominates in skeletal & cardiac muscle
CK – MM (muscle type) CK3
All three CK isoenzymes are found in the
cell cytosol
Unusual CK isoenzymes
migrates cathodic of CK-MM
CK-Mt : 4th isoenzyme
Located b/w the inner & outer membranes of the mitochondria
CK-Mt : 4th isoenzyme
Differs immunologically & in electrophoretic mobility
CK-Mt : 4th isoenzyme
Constitutes up to 15% CK activity in the heart
CK-Mt : 4th isoenzyme
Its presence does not correlate with any specific disease however it correlates with severe illness and cases of malignant tumor and cardiac abnormality
CK-Mt : 4th isoenzyme
CK1 associated with IgG or CK3 w/ IgA
Macro CK Type 1
Oligomeric CK-Mt
Macro CK Type 2
All types of muscular dystrophy, esp. Duchenne type sex-linked progressive muscular dystrophy)
Disease of the Skeletal Muscle
which may show up to 50x the ULN
Disease of the Skeletal Muscle
Normal Serum CK activity is seen in
Neurogenic muscle disease
CKMM major CK in serum of healthy people where Skeletal muscle contains almost exclusively of CKMM and heart muscle activity is attributed to CKMM
Disease of the Skeletal Muscle
Injury on both heart and skeletal will mean elevation of CKMM
Disease of the Skeletal Muscle
CK level: sensitive indicator of AMI (Total CK & CK-2)
Disease of the Heart
CK-MB levels begin to rise w/in 4 – 8 hrs. Peak at 12 – 24 hrs. & return to normal levels w/in 48 – 72 hrs.
Disease of the Heart
Elevation of Total CK: Cardiac trauma ff. heart surgery (including transplantation)
Disease of the Heart
CKMB activity has been observed in other cardiac conditions, not entirely specific for AMI although specificity can increase if tested in conjunction with LDH
Disease of the Heart
The time course of CK is unique in AMI
Disease of the Heart
isoenzyme will be elevated in conditions such as cerebral ischemia and cerebrovascular ischemia where blood flow to the brain is insufficient
CKBB
Elevations are also noted during head injury and acute cerebrovascular disease
Disease of the CNS
It is also observe that the activity may increase in conditions that affects children such as Reye’s syndrome where the liver and the brain is swollen as a side effect of viral infections
CK BB
60% of hypothyroid subjects
Disease of the Thyroid
Major isoenzyme is CK-3
Disease of the Thyroid
Hypothyroidism results in (?) elevations because of the involvement
CK-MM
of muscle tissue (increased membrane permeability), the effect of thyroid hormone on enzyme activity, and, possibly, the slower clearance of CK as a result of slower metabolism.
CK MM
is rarely seen in the serum bec. Of its molecular size
CKBB
Extensive damage to the brain may lead to the leakage of it in the serum
CK activity in Malignancy
is associated also with patients with carcinoma of various organs such as adenocarcinoma, lung tumors, tumors of the prostate, kidney breasts and ovary the
CKBB
CKBB can be a useful tumor marker
CK activity in Malignancy
Makes use of coupled enzymatic reaction, either the forward or reverse reaction
CK
is set at a pH of 9.0 and the reduction of NADH to NAD is monitored spectrophotometrically
The forward reaction
is proportional to the activity of CK
The change in absorbance
– an enzyme released from erythrocytes in hemolyzed samples and appearing as a band cathodal to CK-MM.
Adenylate kinase (AK) Effect
may interfere with chemical or immunoinhibition methods, causing a falsely elevated CK or CK-MB value
Adenylate kinase (AK) Effect
reacts w/ ADP to produce ATP
causes falsely elevated CK activity
Hemolysis – RBCs are devoid of (?) but are rich in AK, thus hemolysis should be avoided
CK
Reference range
TOTAL CK:
Male :
Female :
CK – MB :
TOTAL CK:
Male : 15 – 160 U/L (37°C)
Female : 15 – 130 U/L (37°C)
CK – MB : < 6% Total CK
Separation Techniques
– reference method and the most useful method
Electrophoresis
bands are visualized by incubating the support with a concentrated CK assay using the reverse reaction
Electrophoresis
NADPH formed is observed with the bluish-white fluorescence after excitation by ultra violet light
Electrophoresis
Allows visualization of AK
Electrophoresis
Potential for being more sensitive & precise than electrophoresis
On unsatisfactory column:
may merge into CK-MB
may be eluted w/ CK-MB
may elute w/ CK-MB
CK-MM
CK-BB
Macro-CK
Measure the conc. of Enzyme protein rather than Enzyme activity
Immunoassays
detects enzymatically inactive CK-2
Immunoassays
an anti-CK-M subunit antiserum is used to inhibit both M subunits of CK-MM and the single M subunit og CK-MB and allows determination of the enzyme activity of the B subunit of CK-MB and the B subunit of CK-BB
Immunoinhibition
The residual activity after inhibition is multiplied by 2 to account for MB activity (50% inhibited). The major disadvantage of this method is that it detects BB activity, which, although not normally detectable, will cause falsely elevated MB results when BB is present.
Immunoinhibition
In addition, the atypical forms of CK-Mi and macro-CK are not inhibited by anti-M antibodies and also may cause erroneous results for MB activity.
Immunoinhibition
catalyzes the interconversion of lactic acid and pyruvic acids NAD - a hydrogen transfer enzyme that uses the coenzyme NAD as a hydrogen acceptor Reaction catalyzed:
