CREATINE KINASE & LACTATE DEHYDROGENASE Flashcards
(94 cards)
1
Q
CREATINE KINASE (CK)
EC
A
EC 2.7.3.2
2
Q
CK
Also known as:
A
Creatine Phosphokinase
ATP:Creatine N-phosphotransferase
3
Q
CK function
A
ATP regeneration in contractile or transport system
4
Q
CK
In muscle cells, it is involved in the storage of_______
Each contraction cycle uses _____to produce ATP
A
high-energy creatine phosphate
creatine phosphate
5
Q
TISSUE SOURCE
CREATINE KINASE (CK)
• Highest activities in:
A
Skeletal muscle
Heart muscle
Brain tissue
6
Q
TISSUE SOURCE
CREATINE KINASE (CK)
Present in much smaller quantities in:
BKPPP GLLUTS
A
Bladder
Placenta
Gastrointestinal tract
Thyroid
Uterus
Kidney
Lung
Prostate
Spleen
Liver
Pancreas
7
Q
CREATINE KINASE (CK)
• More specific indicator of disorders than total CK levels
A
ISOENZYMES
8
Q
• CK is a dimer; 2 subunits:
“B” for____ and “M” for____
A
Brain
Muscle
9
Q
CK exists as three isoenzymes:
A
CK-BB (brain type)
CK-MB (hybrid type)
CK-MM (muscle type)
10
Q
СК-BB (СК1)
A
Brain
gastrointestinal tract
uterus during pregnancy
11
Q
not usually measurable in serum due to blood-brain barrier
A
СК-BB (СК1)
12
Q
CK-MB (CK2)
A
Heart muscle
13
Q
elevated levels indicate myocardial damage
A
CK-MB (CK2)
14
Q
CK-MM (CK3)
A
Striated muscle and normal
serum
primary isoenzyme in skeletal muscle
15
Q
The major isoenzyme in healthy individuals
Significant in myocardial tissue and a good indicator of myocardial damage
A
CK-MM (CK3)
16
Q
ISOENZYMES
CREATINE KINASE (CK)
Atypical CK-isoenzymes
A
Macro-CK
CK-Mi
17
Q
Complexes of CK-BB with immunoglobulins, often IgG;
age and sex-related
A
Macro-CK
18
Q
Found in muscle, brain, liver;
indicates severe illness
A
CK-Mi
19
Q
Which travels fastest to the ANODE
A
BB
20
Q
Arrange from most to least fastest
A
BB
MB
Macro
MM
21
Q
DIAGNOSTIC SIGNIFICANCE
CREATINE KINASE (CK)
• Elevated in disorders of…
A
cardiac and skeletal muscle
22
Q
Increased CK
• Common conditions:
A
Myocardial infarction (MI)
rhabdomyolysis
muscular dystrophy
23
Q
CK
• Source of greatest elevation:
A
Duchenne muscular dystrophy
24
Q
METHODS OF CK ISOENZYME MEASUREMENT
CREATINE KINASE (CK)
A
1.) ELECTROPHORESIS
2.) ION-EXCHANGE CHROMATOGRAPHY
3.) IMMUNOINHIBITION
4.) IMMUNOASSAYS
25
CK
• Reference method
ELECTROPHORESIS
26
• Advantages:
• Can separate atypical bands, including strongly fluorescent bands near the CK-BB form.
ELECTROPHORESIS
27
Detects unsatisfactory separations and allows visualization of adenylate kinase (AK), which can interfere with chemical or immunoinhibition methods.
ELECTROPHORESIS
28
2.)ION-EXCHANGE CHROMATOGRAPHY
Procedure:
This method uses_____ to separate____
ion-exchange columns
CK isoenzymes.
29
Advantages:
Potentially more sensitive and precise than electrophoresis.
ION EXCHANGE CHROMATOGRAPHY
30
IEC
Disadvantages:
On an unsatisfactory column, _____may merge into_____, and _____may elute with_____.
Additionally,_____ can also elute with____, causing inaccuracies.
CK-MM -> CK-MB
CK-BB -> CK-MB
macro-CK -> CK-MB
31
Antibodies target M and B subunits to measure CK-MB activity.
IMMUNOINHIBITION
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Anti-M antibodies inhibit M activity but not B activity.
IMMUNOINHIBITION
33
IMMUNOINHIBITION
CK activity is measured_____
Remaining activity post-inhibition indicates B subunit activity.
Residual activity is doubled to account for MB (50% inhibited).
BB activity detection can falsely elevate MB results.
before and after M inhibition
34
IMMUNOINHIBITION
_________ are not inhibited, causing potential errors.
Atypical forms (CK-Mi, macro-CK)
35
This leads to the possibility of permitting detection of infarction earlier than other methods.
IMMUNOASSAYS
36
Measure the concentration of enzyme protein rather than enzymatic activity and can, therefore, detect enzymatically inactive CK-MB.
IMMUNOASSAYS
37
Detect CK-MB reliably with minimal cross-reactivity.
IMMUNOASSAYS
38
ASSAY FOR ENZYME ACTIVITY
CREATINE KINASE (CK)
For analysis, CK activity is coupled with other enzyme systems and measured by______ at ______
absorbance change at 340 nm.
39
Coupled with the PK-LD-NAD+ System
pH 9.0
FORWARD/DIRECT
Tanzer-Gilvarg
40
Most common method
2-6x faster
Less interference
REVERSE/INDIRECT
Oliver-Rosalki
41
Coupled with the HK-G6PD-NADP
System
pH 6.7 or 6.8
The starting chemicals are more expensive
REVERSE/INDIRECT
Oliver-Rosalki
42
STABILITY
CREATINE KINASE (CK)
• CK activity in serum is unstable due to oxidation of______
sulfhydryl groups.
43
CK
Inactivation can be partially reversed by the addition of sulfhydryl compounds to the assay reagent.
N-acetylcysteine
Mercaptoethanol
Thioglycerol
Dithiothreitol
44
STORAGE
CREATINE KINASE (CK)
• CK activity in_____ is relatively unstable and is rapidly lost during storage.
serum
45
CK
Average stabilities are:
at RT
at 4°C
at -20°C.
<8 hours
48 hours
1 month
46
SOURCE OF ERROR
CREATINE KINASE (CK)
• Hemolysis of serum samples can cause….
elevated CK activity
47
• Erythrocytes lack CK but contain _______which produces ATP.
Increased ATP can cause falsely_____ CK levels in assays.
Significant hemolysis (______) releases enough AK to overwhelm inhibitors.
adenylate kinase (AK)
elevated
above
320 mg/L hemoglobin
48
SOURCE OF ERROR
CREATINE KINASE (CK)
• _______ and ______affect CK levels:
• Physically trained individuals may have higher____
Prolonged bed rest may lead to_____
Muscular activity and muscle mass
baseline levels
decreased CK activity.
49
REFERENCE RANGE
CREATINE KINASE (CK)
Total CK:
• Males:
• Females:
• CK-MB:
46 to 171 U/L (37°C) (0.8 to 2.9 ukat/L)
34 to 145 U/L (37°C) (0.6 to 2.4 ukat/L)
<5% total CK
50
LACTATE DEHYDROGENASE (LDH)
EC
E.C. 1.1.1.27
L-Lactate NAD+ Oxidoreductase
51
LDH function
• Catalyzes the interconversion of lactic and pyruvic acids
• Hydrogen-transfer enzyme that uses the coenzyme NAD+
52
LDH Reference rangee
Reference range: 125 - 220 U/L
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DIAGNOSTIC SIGNIFICANCE
LACTATE DEHYDROGENASE (LDH)
• Pernicious Anemia & Hemolytic Disorders
• Liver Disorders
• Acute Myocardial Infarction (AMI)
• Skeletal Muscle Disorders & Leukemias
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• Highest levels of total LD due to erythrocyte destruction.
Pernicious Anemia & Hemolytic Disorders
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LDH
• Liver Disorders
• Viral____ and ____:_______
hepatitis and cirrhosis
Slight elevation (2-3x ULN).
56
LDH
Acute Myocardial Infarction (AMI)
______.
LD levels rise______
peak in______
may remain elevated for up to____
Slight elevation (2-3x ULN)
12-24 hours post-infarction
48-72 hours
10 days
57
Skeletal Muscle Disorders & Leukemias
Marked_____, especially in_____
elevations
acute lymphoblastic leukemia
58
LACTATE DEHYDROGENASE (LDH)
Structure
has 4 subunits
either H (heart) or M (muscle) in five arrangements
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LDH-1 (HHHH)
Heart - Myocardial infarction
Red blood cells - Hemolytic anemia
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Disorder
Myocardial infarction
Hemolytic anemia
LDH-1 (HHHH)
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LDH-2 (HHHM)
Heart - Megaloblastic anemia
Red blood cells - Acute renal infarct
Hemolyzed specimen
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Megaloblastic anemia
Acute renal infarct
Hemolyzed specimen
LDH-2 (HHHM)
63
LDH-3 (HHMM)
Lung - Pulmonary embolism
Lymphocytes - Extensive
Spleen - Pulmonary pneumonia
Pancreas - Lymphocytosis
Acute pancreatitis
Carcinoma
64
Pulmonary embolism
Extensive
Pulmonary pneumonia
Lymphocytosis
Acute pancreatitis
Carcinoma
LDH-3 (HHMM)
65
LDH-4 (HMMM)
Liver - Hepatic injury or inflammation
66
Hepatic injury or inflammation
LDH-4 (HMMM)
67
LDH
Liver
LDH-4 (HMMM)
68
LDH-5 (MMMM)
Skeletal muscle - Skeletal muscle injury
69
LDH
Skeletal muscle injury
LDH-5 (MMMM)
70
Relative Percentage of LD Isoenzymes in Various Tissues
Serum
Highest - LD2 (35)
Lowest - LD5 (5)
71
Relative Percentage of LD Isoenzymes in Various Tissues
Heart
H - LD1(45)
L - LD5(0)
72
Relative Percentage of LD Isoenzymes in Various Tissues
RBC
H - LD1(40)
L - LD5(0)
73
Relative Percentage of LD Isoenzymes in Various Tissues
Renal cortex
H - LD1(35)
L - LD5(0)
74
Relative Percentage of LD Isoenzymes in Various Tissues
Lung
H - LD3(40)
L - LD5(5)
75
Relative Percentage of LD Isoenzymes in Various Tissues
Skeletal muscle
H - LD5(60)
L - LD1/2(0)
76
Relative Percentage of LD Isoenzymes in Various Tissues
Liver
H - LD5(70)
L - LD1(0)
77
ISOENZYMES
LACTATE DEHYDROGENASE (LDH)
• LD Flipped Pattern
• Normal LD pattern:______
•_______ indicates AMI.
• May also occur in hemolyzed serum samples.
LD-2 > LD-1
LD-1 > LD-2
78
LDH
Normal serum
LDH2 > LDH1
79
LDH
Acute hepatitis
> LDH5
80
MEASUREMENT OF ISOENZYMES
LACTATE DEHYDROGENASE (LDH)
• Electrophoresis
• mmunoinhibition
• Chemical inhibition
• Differences in substrate affinity
81
MEASUREMENT OF ISOENZYMES
LACTATE DEHYDROGENASE (LDH)
• ELECTROPHORESIS
• _____migrates fastest,____ migrates slowest.
LD-1
LD-5
82
MEASUREMENT OF ISOENZYMES
LACTATE DEHYDROGENASE (LDH)
Measures atypical bands
(LDH complexed with immunoglobulin)
• ELECTROPHORESIS
83
Migrates between LD-3 and LD-4
After separation -> fluorometry or
colorimetry
Not associated with clinical abnormalities
LD-IgA/IgG
84
LD-IgA/IgG
Migrates between_______
After separation ->_______
Not associated with clinical abnormalities
LD-3 and LD-4
fluorometry or
colorimetry
85
LDH
Differences in substrate affinity
Measurement of________
hydroxybutyrate dehydrogenase activity
86
Differences in substrate affinity
Measurement of hydroxybutyrate dehydrogenase activity
Substrate:
___________
(with increased affinity to H subunit)
a-HBD represents LD-1 activity but not specific
a-hydroxybutyrate
87
ASSAY FOR ENZYME ACTIVITY
LACTATE DEHYDROGENASE (LDH)
FORWARD/DIRECT - Wacker
REVERSE/INDIRECT - Wrobleuski-Ladue
88
FORWARD/DIRECT
• Wacker
• Optimal pH:
8.3 - 8.9
89
LDH
• Most common method because it is not affected by product inhibition
FORWARD/DIRECT
• Wacker
90
REVERSE/INDIRECT
• Wrobleuski-Ladue
Optimal pH:
7.1 - 7.4
91
LDH
• This is ~3x faster but more susceptible to substrate exhaustion and loss of linearity
REVERSE/INDIRECT
• Wrobleuski-Ladue
92
LDH errors
Erythrocyte Contamination
LD concentration in erythrocytes:
________higher than in serum
Any degree of_____ renders a sample unacceptable for analysis.
100-150 times
hemolysis
93
LDH error
Stability of LD Activity
LD activity is unstable in______ regardless of storage temperature.
Ideal storage:_____, analyze within _____hours.
serum
25°C
48 hours
94
LDH Errors
Most labile isoenzyme.
Loses activity quicker at_____ compared to_____.
For LD isoenzyme analysis: Store serum samples at____, analyze within____ hours of collection.
LD-5 Isoenzyme
4°C; 25°C
25°C; 24 hours
