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Clinical Chemistry > Enzyme > Flashcards

Enzyme Flashcards

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1
Q

What are Enzymes?

A 
Proteins, Biological catalysts
Created in the body
Supply energy/chemical changes in the body
Muscle contraction
Nerve conduction
Respiration, reproduction
Digestion or nutrient degradation, growth
Maintaining body temp
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2
Q

Apoenzyme

A

heat-sensitive protein portion. Requires a coenzyme

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3
Q

Coenzyme

A

organic co-factors that resemble vitamins (NAD🡪NADH)

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4
Q

Haloenzyme:

A

apoenzyme + cofactor

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5
Q

Cofactor

A

nonprotein molecule necessary for enzyme activity

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6
Q

Metalloenzymes

A

inorganic cofactor (Cl-, Zn2+, Cu 2+, Ca2+Mg2+)

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7
Q

Zymogen

A

Inactive Form

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8
Q

Absolute specificity

A

catalyze 1 specific substrate or reaction

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9
Q

Group specificity

A

catalyze substrates with similar structural groups

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10
Q

Bond Specificity

A

catalyzing reaction based upon a certain type of bond

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11
Q

Stereospecificity

A

Stereoisomer specificity - Catalyze reactions with only certain optical isomers

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12
Q

Oxidoreductases

A

catalyze an oxidation–reduction reaction between two substrates (LD)

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13
Q

Transferases:

A

catalyze transfer of a group other than hydrogen from one substrate to another (AST)

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14
Q

Hydrolases

A

catalyze hydrolysis of various bonds (Amylase)

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15
Q

Lyases

A

catalyze removal of groups from substrates without hydrolysis; product contains double bonds

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16
Q

Isomerases

A

catalyze interconversion of geometric, optical, or positional isomers (phosphohexose isomerase) α glucose🡪β glucose

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17
Q

Ligases

A

catalyze joining of two substrate molecules, coupled with breaking of pyrophosphate bond in ATP

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18
Q

Energy of Activation (EA)

A

energy required to raise 1 mole of substrate to form the activated complex (IU/L)
Enzymes catalyze reactions by lowering EA level.

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19
Q

Enzyme-Substrate Complex

A

provides free energy required for the reaction.
Reaction is allowed to proceed without additional energy
↓ energy barrier = ↑ product created

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20
Q

Vmax

A

substrate concentration high enough that all enzyme is bound to substrate and all active sites are engaged

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21
Q

Michaelis-Menten constant (Km)

A

substrate concentration in moles/Liter when the speed of enzymatic reaction = ½ Vmax
Represents relationship between reaction speed & substrate concentration
Km is a constant and remains the same for a given enzyme-substrate pair under given conditions

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22
Q

First-order kinetics

A

The velocity is directly proportional to the substrate concentration

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23
Q

Zero-order kinetics

A

The reaction rate is independent of substrate concentration

Clinical lab- most common measurements

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24
Q

Unit of Measurement

A 
International Unit (IU) = 1 µmol of product per minute under standard conditions
Expressed as U/L (units/liter)
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25
Q

Substrate Concentration

A

Increases rate of reaction

Zero-order rxn: excess substrate required so that no more than 20% is converted to product in a normal reaction

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26
Q

Enzyme concentration

A

Higher the enzyme level, the faster the reaction will proceed
More substrate converted to product

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27
Q

pH

A

Optimal body pH from 7.0 – 8.0

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28
Q

Temperature

A

↑ temp will ↑ rate of reaction
↓ temp reversibly inactivates enzymes
25oC, 30oC, 37oC
Temperature > 40 - 50oC result in enzyme denaturation

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29
Q

Cofactors

A

Metallic (Ca2+, Fe2+, Mg2+, Mn2+, K+

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30
Q

Inhibitors

A

Competitive: physically bind to enzyme active site
Noncompetitive: reversibly binds to a site other than enzyme active site
Uncompetitive: binds to ES complex → [substrate]

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31
Q

Isoenzymes

A

Multiple forms of an enzyme that can catalyze the enzyme’s characteristic reaction
LD 1: found in rbc; LD5- found in liver
Used to determine origin of disease
Differentiated by: electrophoresis, resistance to heat or chemicals

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32
Q

Measurement of Enzyme Mass

A

Immunoassay methodologies are used to quantify enzymes

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33
Q

Enzymes as Reagents

A

Used to measure many non-enzymatic constituents in serum
Used as reagents to quantify analytes that are substrates for corresponding enzyme
Used as reagents in competitive and noncompetitive immunologists (HIV, therapeutic drugs, cancer antigens)
Horseradish perioxidase, ALP, G6PD

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34
Q

Creatine Kinase (CK)

A

cytoplasmic and mitochondrial enzyme that catalyzes the reversible phosphorylation of creatine by adenosine triphosphate (ATP)
Equilibrium of the CK reaction is dependent on pH
very important in muscle tissue.
Allows high-energy phosphate to be stored in a more stable form than ATP
Dimer with 2 subunits
B (brain) and M (muscle)
Tissue source: (1) muscle (2) brain (3) heart
CK highest in infancy and childhood; decreases as we age
Inverse relationship with thyroid function
Hypothyroidism = ↑CK

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35
Q

CK-2

A

rises 4-6 hrs post AMI, peaks at 24 hrs and returns to normal within 2-3 days

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36
Q

CK-3

A

rises with muscular dystrophy (Duchenne type

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37
Q

CK-1

A

tumor marker for prostate and lung cancer
Childbirth & hypothermia
Hypothyroidism

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38
Q

Specimen Collection CK

A

Serum is the specimen of choice
Store in dark place; CK is light sensitive
Not affected by hemolysis but adenylate kinase (AK) released by rbcs reacts with ADP- ATP (Oliver-Rosalki) causing increase CK
CK activity is unstable and rapidly lost during storage
4 hrs at room temperature
48 hrs at 4oC
1 month at -20oC

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39
Q

CK Reference Range

A 
Males: 52–236 U/L
Females :38–176 U/L
Affected by:
Age, Physical activity 
Race, Bed rest (even overnight can decrease CK)
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40
Q

Testing Methods: Oliver-Rosalki

A

Creatinine phosphate + ADP →┴(𝐶𝐾, 𝑀𝑔2+@ 𝑝ℎ=6.7)Creatine + ATP

ATP + Glucose →┴𝐻𝑒𝑥𝑜𝑘𝑖𝑛𝑎𝑠𝑒 Glucose-6-phosphate + ADP
Inhibited by:
Ca2+ : fix by adding EDTA or add extra 𝑀𝑔2+
Preferred lab method
Measured at 340 nm
Optimal pH = 6.8
Other Methods
Electrophoresis
Ion-exchange chromatography
RIA, EIA
Immunoinhibition
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41
Q

Lactate Dehydrogenase (LD)

A

Tissue source
Highest concentration in heart, liver, skeletal muscle
Also found in kidney, erythrocytes
LD is a nonspecific marker
Important enzyme in the Embden-Meyerhoff
Tissue concentration 500 times higher than serum levels

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42
Q

Diagnostic significance LD

A

pernicious anemia, hemolytic disorders, viral hepatitis, cirrhosis, acute myocardial infarction, pulmonary infarct, skeletal muscle disorders, leukemia
In healthy people the major fractions appear as follows:
LDH-2 ⇾ LDH-1 ⇾ LDH-3 ⇾ LDH-4 ⇾ LDH-5

43
Q

Heart Attack (AMI)

A

LDH Flip: when LD-1 > LD-2 = heart attack

Rises 12-24 hrs, peaks 48-72 hrs and returns to normal within 7-14 days after AMI

44
Q

Two subunits LD

A

H (heart) and M (muscle) form five isoenzymes.

45
Q

LD-1 (HHHH)

A

Heart, RBC and kidney

46
Q

LD-2 (HHHM)

A

Heart, RBC and kidney

47
Q

LD-3 (HHMM)

A

Spleen, lungs, and many tissues

48
Q

LD-4 (HMMM)

A

Liver and skeletal muscle

49
Q

LD-5 (MMMM)

A

Liver and skeletal muscle

50
Q

Specimen Collection LD

A

Serum, no hemolysis
Stored 72 hrs at room temp= no loss of activity
LD-5 decreased at freezing -20oC

51
Q

Reference range: LD

A

L🡪P: 100-224 U/L at 37°C

P🡪L: 80 - 300 U/L at 37°C

52
Q

Source of error: LD

A

any degree of hemolysis
Enzyme unstable in serum
Store samples at 25oC rather than 4oC and measure within 24 hrs of collection

53
Q

LD analysis

A 
Most current methods measure the interconversion of NAD+ to NADH at 340 nm. 
Wacker procedure
Lactate to pyruvate reaction
Most often used
pH 8.8-9.8 = forward reaction
pH 7.4 – 7.8 = reverse reaction
54
Q

Troponin

A 
Cardiac-specific troponin I (cTnI)
Cardiac-specific troponin T (cTnT)
Specific for cardiac tissue
High diagnostic specificity and sensitivity
Released early after AMI
Remain elevated for long period of time
Very low or undetectable in serum of normal patients
Very few interfering substances
55
Q

Troponin False (+)

A

Heterophile antibodies

Rheumatoid factor

56
Q

Troponin False (-)

A

Bilirubin, hemoglobin
Interfering factor
Circulating cTnI autoantibodies

57
Q

Natriuretic Peptides (NP)

A

Regulate fluid volume, blood pressure and electrolyte balance
Cleared and made by the kidneys

58
Q

Type NP

A

Atria natriuretic peptide (ANP): cardiac atria
Brain natriuretic peptide (BNP): cardiac ventricles
ANP & BNP released in response to atrial. Ventricular stretching from volume overload, renal and liver disorders
Biomarker for congestive heart failure
↑BNP = severe heart failu

59
Q

Cytokines

A

Protein secreted by cells to attract and direct target cells

60
Q

interleukin-6 (IL-6)

A

Produced by monocytes & lymphocytes
Acute phase reactant
Elevated in patients with AMI

61
Q

Leptin

A

Hormone
Regulates body weight, expressed by adipocytes
Independent risk factor associated with CVD
Low levels associated with unstable coronary artery disease (CAD)

62
Q

Pregnancy-Associated Plasma Protein A (PAPP-A)

A

Typically measured to detect Down’s Syndrome

Detects unstable acute coronary syndrome (ACS) without increased concentration of cTn

63
Q

Placental Growth Factor (PlGF)

A

Attracts monocytes
Regulates vascular endothelial growth factor (VEGF)
Biomarker for plaque instability, myocardial ischemia, ACS

64
Q

Aspartate Aminotransferase (AST)

A

Serum glutamic oxaloacetic transaminase (SGOT or GOT)

65
Q

Tissue source: AST

A

cardiac tissue, liver, skeletal muscle

66
Q

Diagnostic significance: AST

A

hepatocellular disorders (viral hepatitis, cirrhosis), skeletal muscle disorders (muscular dystrophies, inflammatory conditions), pulmonary embolism

67
Q

Source of error: AST

A

Hemolysis
stable at room temp for 48 hrs
3–4 days at refrigerated temperatures

68
Q

Specimen Collection: AST

A

serum

69
Q

Assay: Karmen method AST

A 
2 reactions
pH = 7.3 – 7.8
340 nm
Malate dehydrogenase 
is the indicator reaction measuring the decrease in absorbance at 340 nm as NADH is oxidized to NAD+.
70
Q

Reference range: AST

A

5–35 U/L (37°C)

71
Q

Alanine Aminotransferase (ALT)

A

Glutamic pyruvic transaminase (SGPT or GPT)
More specific than AST for liver disorders
found in Liver tissue

72
Q

De Ritis ratio (AST/ALT)

A

Aids in diagnosing liver disease
Normal ratio: 0.7 – 1.4
Alcoholic liver disease/cirrhosis: AST>ALT and is greater than 1
Ratio >2 alcoholic hepatitis or alcoholism
Viral hepatitis, obstructive liver disease and acute inflammatory disease ALT>AST and is less than 1 (ratio < 1

73
Q

Diagnostic significance ALT

A

hepatic disorders

74
Q

Reference range ALT

A

7 - 45 U/L (37°C)

75
Q

Specimen Collection ALT

A

Serum; measure within 24 hrs; decrease activity at 4o and -20°C
ALT stable at -70°C
avoid hemolysis: ALT 5-8 times higher in rbcs than serum

76
Q

Assay: ALT

A

Wroblewski and LaDue Method
pH = 7.3 – 7.8
340 nm

77
Q

Alkaline Phosphatase (ALP)

A

found in all tissues of the body esp. at or near cell membranes
Highest concentration in hepatocytes

78
Q

Diagnostic significance: ALP

A

hepatobiliary (biliary tract obstruction)
bone (Paget’s disease, osteomalacia, rickets, hyperparathyroidism, osteogenic sarcoma) disorders
Separation of isoenzymes led to discovery of abnormal enzyme carcinoplacental(Regan)

79
Q

Reference range: ALP

A

42-128 U/L (30°C)

(M/F 20-50 y/o)

80
Q

Specimen Collection: ALP

A 
Serum or heparin samples
Stored at room temp; measure in 4 hrs
Levels drop slowly at 4oC (2%/day)
Hemolysis acceptable
Cannot use any other anticoagulant due to the binding of Mg2+ and Zn2+ to Ca2+
81
Q

Source of error: ALP

A

Hemolysis
assays should be run as soon as possible after collection
Enzyme activity ↑ upon standing at RT or 4o
high-fat meal
Group O or B secretors = ↑ ALP

82
Q

Assay: Bowers & MaComb (ALP)

A

p-nitrophenyl-phosphate (colorless) is hydrolyzed to p-Nitrophenol (yellow)
405 nm

83
Q

γ-Glutamyltransferase (GGT)

A

Present in serum and all cells except muscle

kidney, brain, prostate, pancreas, liver

84
Q

Diagnostic significance: GGT

A

hepatobiliary disorders (biliary tract obstruction), hepatic parenchyma, alcoholism,
acute pancreatitis, diabetes mellitus,
myocardial infarction
NOT elevated in bone disease
Used to diagnose drug & alcohol abuse
Used to measure growth during pregnancy and in children

85
Q

Assay for enzyme activity: GGT

A

γ-glutamyl-p-nitroanilide is most widely accepted substrate used in GGT analysis
Absorbance @ 405-420 nm

86
Q

Specimen collection:

A

Serum

87
Q

Source of error: GGT

A

stable for 1 week at 4°C

hemolysis not a concern

88
Q

Reference range: GGT

A

male: 6–55 U/L (37°C)
female: 5–38 U/L (37°C)

89
Q

Amylase (AMS)

A

acinar cells of pancreas & salivary glands

90
Q

Diagnostic significance: AMS

A

Nonspecific finding
acute pancreatitis
disorders causing salivary gland lesions (mumps, parotitis)
intraabdominal diseases

91
Q

Assay for enzyme activity: AMS

A 
Requires Ca2+ and Cl- for activation
Immunoassay
Interference: opiates, morphine
Serum / urine stable
RT for 1 week or 4oC for 2 months
92
Q

AMY Methodologies

A

Maltotetraose reaction
Used in many instruments
Most common
AMY → Maltose phosphorylase → β-Phosphoglucose mutase → Glucose-6-phosphate

93
Q

Reference Range: AMS

A

Serum: 40-140 U/L
Urine: 24-400 U/L

94
Q

Lipase (LPS)

A

primarily in pancreas

also in stomach & small intestine

95
Q

Diagnostic significance: LPS

A

acute pancreatitis
other intraabdominal diseases (penetrating duodenal ulcers, perforated peptic ulcers, intestinal obstruction, acute cholecystitis)

96
Q

Assay for enzyme activity: LPS

A

turbidimetric (simple and rapid)
Colorimetric (perioxidase or glycerol kinase)
Enzymatic LPS reactions have largely replaced titrimetric and turbidimetric methodologies

97
Q

Source of error: LPS

A

stable in serum for 1 week at room temperature & 3 weeks at 4°C; hemolysis

98
Q

Reference range: LPS

A

<45 U/L

LPS 🡪 Glycerol kinase 🡪 L-α-glycerophosphate kinase 🡪 Peroxidase

99
Q

Acid Phosphatase (ACP)

A

prostate, bone, liver, spleen, kidney, erythrocytes, platelets

100
Q

Diagnostic significance: ACP

A

prostatic carcinoma, hyperplasia of prostrate, prostatic surgery,
osteoclasts, Paget’s disease,
breast cancer with bone metastases, Gaucher’s disease

101
Q

Assay for enzyme activity: ACP

A

same techniques as in alkaline phosphatase, except performed in an acid pH

102
Q

Reference range: ACP

A

prostatic ACP: 0–3.5 ng/mL

103
Q

Source of error: ACP

A

Serum should be separated from red cells as soon as blood has clotted; no hemolysis
serum should be used immediately, frozen
acidified to pH = 6.5 stable for 2 days at room temp

Clinical Chemistry (8 decks)

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