C1 - Section 3. ENZYMES OF CLINICAL IMPORTANCE Flashcards
(135 cards)
1
Q
Catalyze interconversions of the amino acids & alpha-ketoacids by transfer of amino groups
A
AMINOTRANSFERASES
2
Q
as obligate coenzyme
A
Pyridoxal phosphate
3
Q
will be bound to the apoenzyme and serves as a true prothetic group
A
Pyrodoxal-5’-phosphate
4
Q
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 –
A
oxaloacetate and pyruvate
5
Q
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
A
oxoglutarate
6
Q
AMINOTRANSFERASES Function:
A
Amino acid metabolism
7
Q
Ketoacids formed are ultimately oxidized by the
A
TCA Cycle
8
Q
Formerly SGOT (Serum glutamic-oxalocacetic transaminase)
A
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
9
Q
involved in the transfer of an amino group between aspartate and a-keto acids
A
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
10
Q
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
Reaction catalyzed:
A
11
Q
widely distributed in human tissue
A
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
12
Q
Highest concentration: cardiac tissue, liver & skeletal muscle
A
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
13
Q
Smaller amounts: kidney, pancreas & RBCs
A
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
14
Q
Isoenzymes of AST in the cytoplasm & the mitochondria
A
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
15
Q
Both mitochondria and cytoplasmic forms of AST are found in cells.
A
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
16
Q
About 5-10% of the AST activity in serum from healthy individuals is of mitochondrial origin
A
ASPARTATE AMINOTRANSFERASE (AST); E.C. 2.6.1.1
17
Q
Evaluation of hepatocellular disorders & skeletal muscle involvement
A
AST
18
Q
Liver disease-most important cause of elevated transaminase activity in serum
A
AST
19
Q
In most liver disease, ALT is higher than AST (Except:?)
A
Alcoholic hepatitis, Hepatic Cirrhosis and liver neoplasia
20
Q
Mild degree of liver tissue injury: cytoplasmic isoenzyme is predominant
A
AST
21
Q
Severe tissue damage: release of mitochondrial isoenzyme
A
AST
22
Q
shows marked increase in patients with extensive liver cell degeneration and damage
A
mitochondrial AST activity in serum
23
Q
Formerly SGPT (Serum glutamic pyruvic transaminase)
A
ALANINE AMINOTRANSFERASE (ALT); E.C. 2.6.1.2
24
Q
specifically, catalyze the transfer of an amino group from alanine to a-ketoglutarate with the formation of glutamate and pyruvate
A
ALANINE AMINOTRANSFERASE (ALT); E.C. 2.6.1.2
25
ALANINE AMINOTRANSFERASE (ALT); E.C. 2.6.1.2
Reaction catalyzed:
26
Distributed in many tissues
ALT
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High concentrations in the liver (more liver-specific enzyme of the transferases)
ALT
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low levels in the heart and skeletal muscle
ALT
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Isoenzyme: exclusively cytoplasmic form
ALT
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RBC contains 5-8X as much ALT activity as does the serum
ALT
31
Evaluation of hepatic disorders (hepatocellular)
ALT
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Progressive inflammatory liver conditions: higher ALT elevations than AST
ALT
33
Higher elevations are found in hepatocellular disorders than extrahepatic ot intrahepatic obstructive disorders
ALT
34
diagnostic marker of alcoholic liver disease
De Ritis Ratio (AST:ALT ratio)
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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)
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(also in viral hepatitis)
o Normally <1.0
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: associated with cirrhosis
o If >1.0 but <2.0
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: associated with alcoholic hepatitis or hepatocellular carcinoma
o If >2.0
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: AST>ALT initially; w/in 24-48 hrs., ALT>AST
o Acute hepatocellular injury
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Higher AST activity in
hepatocytes
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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
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Transaminase reactions coupled to specific dehydrogenase reactions
Continuous Monitoring Method
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multiple measurement of absorbance change during the reaction is observed
Continuous Monitoring Method
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The oxo-acids formed in the reaction are measured indirectly by enzymatic reduction to the corresponding hydroxyl acids
Continuous Monitoring Method
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The accompanying change in NADH concentration is being monitored spectrophotometrically
Continuous Monitoring Method
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coupled enzymatic reaction w/ MDH (indicator reaction)
Assay reaction for AST Activity
Karmen Method
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Monitors change in absorbance at 340 nm (NADH to NAD)
Assay reaction for AST Activity
Karmen Method
48
Optimal pH: 7.3 – 7.8
Assay reaction for AST Activity
Karmen Method
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Hemolysis: increase serum
AST
50
Pyruvate formed is converted to lactate by LDH
Assay reaction for ALT Activity
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LDH as the indicator enzyme
Assay reaction for ALT Activity
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NADH formed is oxidized to NAD
Assay reaction for ALT Activity
53
The accompanying change in NADH concentration is being monitored spectrophotometrically
Assay reaction for ALT Activity
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the disappearance of NADH is followed by measuring he decrease in absorbance
Assay reaction for ALT Activity
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Change in absorbance is proportional to the micromoles of NADH oxidized that reflects the number of substrate transformed
Assay reaction for ALT Activity
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activity in RBC is 15x higher than in serum
AST
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activity is stable in serum for 3 – 4 days at ref °T
AST
58
AST Reference Range:
5 – 30 U/L (37°C)
59
Relatively unaffected by hemolysis
ALT
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activity in RBCs is 7x higher than in normal serum)
ALT
61
Stable for 3 – 4 days at 4°C
ALT
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ALT Reference Range:
6 – 37 U/L (37°C)
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Coupling w/ 2,4- DNPH (Reitman-Frankel)
Colorimetric Method
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Still feasible
Colorimetric Method
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Phenylhydrazones of oxaloacetate & pyruvate are more chromogenic
Colorimetric Method
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Simple; limited but acceptable accuracy
Colorimetric Method
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Derivative formed will give a strong blue color measured as 505 nm
Colorimetric Method
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An enzyme w/ MW of approximately 82,000
Creatine Kinase (E.C. 2.7.3.2)
69
Catalyzes reversible phosphorylation of Creatine by ATP
Creatine Kinase (E.C. 2.7.3.2)
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it is generally associated w/ ATP regeneration in transport system
Creatine Kinase (E.C. 2.7.3.2)
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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)
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CK Reaction catalyzed:
73
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)
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Mg-forms complexes with ATP and ADP with narrow concentration because excess will be inhibitory
Creatine Kinase (E.C. 2.7.3.2)
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Greatest in striated muscle, brain tissue & heart tissue
CK
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Smaller quantities: bladder, placenta, GIT, Thyroid, uterus, kidney, lung, prostate, spleen & pancreas
CK
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liver and erythrocyte is devoid of the activity of CK
CK
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Dimer w/ 2 sub-units: B (brain) & M (muscle)
CK
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Each sub-unit w/ a MW of ~ 40,000
CK
80
CK Three major isoenzymes:
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Found in the brain, prostate, gut, lung, bladder, uterus, placenta & thyroid
CK – BB (brain type) CK1
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present in varying degrees in heart muscle (25 – 46% of CK activity) and minor degree in skeletal muscle
CK – MB (hybrid type) CK2
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predominates in skeletal & cardiac muscle
CK – MM (muscle type) CK3
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All three CK isoenzymes are found in the
cell cytosol
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Unusual CK isoenzymes
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migrates cathodic of CK-MM
CK-Mt : 4th isoenzyme
87
Located b/w the inner & outer membranes of the mitochondria
CK-Mt : 4th isoenzyme
88
Differs immunologically & in electrophoretic mobility
CK-Mt : 4th isoenzyme
89
Constitutes up to 15% CK activity in the heart
CK-Mt : 4th isoenzyme
90
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
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CK1 associated with IgG or CK3 w/ IgA
Macro CK Type 1
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Oligomeric CK-Mt
Macro CK Type 2
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All types of muscular dystrophy, esp. Duchenne type sex-linked progressive muscular dystrophy)
Disease of the Skeletal Muscle
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which may show up to 50x the ULN
Disease of the Skeletal Muscle
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Normal Serum CK activity is seen in
Neurogenic muscle disease
96
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
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Injury on both heart and skeletal will mean elevation of CKMM
Disease of the Skeletal Muscle
98
CK level: sensitive indicator of AMI (Total CK & CK-2)
Disease of the Heart
99
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
100
Elevation of Total CK: Cardiac trauma ff. heart surgery (including transplantation)
Disease of the Heart
101
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
102
The time course of CK is unique in AMI
Disease of the Heart
103
isoenzyme will be elevated in conditions such as cerebral ischemia and cerebrovascular ischemia where blood flow to the brain is insufficient
CKBB
104
Elevations are also noted during head injury and acute cerebrovascular disease
Disease of the CNS
105
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
106
60% of hypothyroid subjects
Disease of the Thyroid
107
Major isoenzyme is CK-3
Disease of the Thyroid
108
Hypothyroidism results in (?) elevations because of the involvement
CK-MM
109
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
110
is rarely seen in the serum bec. Of its molecular size
CKBB
111
Extensive damage to the brain may lead to the leakage of it in the serum
CK activity in Malignancy
112
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
113
CKBB can be a useful tumor marker
CK activity in Malignancy
114
Makes use of coupled enzymatic reaction, either the forward or reverse reaction
CK
115
is set at a pH of 9.0 and the reduction of NADH to NAD is monitored spectrophotometrically
The forward reaction
116
is proportional to the activity of CK
The change in absorbance
117
– an enzyme released from erythrocytes in hemolyzed samples and appearing as a band cathodal to CK-MM.
Adenylate kinase (AK) Effect
118
may interfere with chemical or immunoinhibition methods, causing a falsely elevated CK or CK-MB value
Adenylate kinase (AK) Effect
119
reacts w/ ADP to produce ATP
120
causes falsely elevated CK activity
121
Hemolysis – RBCs are devoid of (?) but are rich in AK, thus hemolysis should be avoided
CK
122
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
123
Separation Techniques
124
– reference method and the most useful method
Electrophoresis
125
bands are visualized by incubating the support with a concentrated CK assay using the reverse reaction
Electrophoresis
126
NADPH formed is observed with the bluish-white fluorescence after excitation by ultra violet light
Electrophoresis
127
Allows visualization of AK
Electrophoresis
128
Potential for being more sensitive & precise than electrophoresis
129
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
130
Measure the conc. of Enzyme protein rather than Enzyme activity
Immunoassays
131
detects enzymatically inactive CK-2
Immunoassays
132
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
133
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
134
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
135
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:
