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Michelle: Clinical Chem > Week 3: Enzymes > Flashcards

Week 3: Enzymes Flashcards

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1
Q

T/F: Enzymes alter both biochem rxn AND chem equilibrium point of rxn

A

FALSE

Alter rate of biochcem rxn but not chem equilibrium point

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2
Q

Why is the measurement of enzymes clinically relevant?

A

Enzyme concentration can be measured to detect cellular injury or altered production by specific organ tissue

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3
Q

What is the general enzyme rxn equation?

A

E + S -> E*S -> E + P

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4
Q

Define adduct

A

Enzyme-substrate complex

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5
Q

What is a catalyst?

A

Same as enzyme! Accelerates rate of reaction without being modified itself

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6
Q

Define cofactor and give examples

A

Definition: Non-protein, molecules essential for enzyme activity and must bind enzyme to alter configuration for optimum substrate binding

Examples: Mg2+, Cl-, Zn- (activators)

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7
Q

Define coenzyme and give examples

A

Definition: Organic molecules that loosely bind proteins and are required for enzyme function. Participate in rxn but are not substrates. Carry electrons, atoms, or functional groups

Example: NADH

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8
Q

Apoenzyme

A

protein portion of enzyme

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9
Q

Holoenzyme

A

enzyme + coenzyme

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10
Q

Zymogen

A

Inactive, secreted form of enzyme

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11
Q

Prosthetic group

A

Tightly bound non-protein molecules of organic OR non-organic (metal ion) origin

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12
Q

What type of bond is present in the adduct?

A

Noncovalent bonds

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13
Q

What are general enzyme reaction conditions?

A
  • Usually 37°C, no higher than 40°C
  • -5°C are inactivated and stored
  • Most pH ranges are 7-8
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14
Q

First-order kinetics

A

Constant [E] with increasing [S] and increasing reaction rate

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15
Q

Zero-order kinetics

A

All enzyme sites are saturated with substrate. Constant reaction rate that depends on [E]

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16
Q

Post-zone phenomenon

A

Antigen excess (substrate excess)

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17
Q

Prozone phenomenon

A

Antibody excess (enzyme excess)

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18
Q

Hook effect

A

Decrease in signal due to excess analyte concentration (outside of analytical range). See data increase and then “hook” down

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19
Q

Enzyme reaction conditions when measuring reaction rate

A
  • [S] > [E]
  • Change in [product] must solely depend on [E]
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20
Q

Km

A

[S] which is half of Vmax

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21
Q

Difference between non-competitive and uncompetitive inhibitors?

A

Non-competitive: Allosteric inhibitor binds enzyme and may allow substrate binding but can’t form product

Uncompetitive: Inhibitor binds enzyme-substrate complex and prevents product formation

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22
Q

As long as [S] > [E], enzyme velocity is proportional to ___

A

[E]

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23
Q

List clinically significant enzymes

A
  • Lactate dehydrogenase (LD)
  • Creatine kinase (CK)
  • Aspartate amino transferase (AST)
  • Alanine amino transferase (ALT)
  • Gamma glutamyl transferase (GGT)
  • Alkaline phosphatase (ALP)
  • Amylase (AMY)
  • Lipase (LIP)
  • Cholinesterase/pseudocholinesterase
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24
Q

Purpose of aminotransferase (aka transaminase?

A

To transfer amino groups for the formation of oxaloacetate and pyruvate

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25
Q

List the two aminotransferases and their end products

A

AST = oxaloacetate
ALT = pyruvate

26
Q

What can increase liver enzyme activity?

A

Inflamed liver tissue, infection

27
Q

Describe AST (aspartate aminotransferase)

A
  • In liver, heart, skeletal muscle, and kidney tissue
  • Cytoplasm + mitochondria
  • Short half-life increases clinician value
  • Normal: [AST] < [ALT]
28
Q

In which diseases is [AST] > [ALT]?

A
  • Alcoholic hepatitis
  • Hepatic cirrhosis
  • Hepatocellular carcinoma
29
Q

Why should you avoid hemolysis when measuring AST?

A

False increase in [AST]

30
Q

Describe ALT (alanine aminotransferase)

A
  • Primarily in liver + kidney tissue
  • Greater specificity for damaged liver than AST
  • Cytoplasm
31
Q

ALT is the best predictor of liver damage associated with which conditions?

A
  • Viral hepatitis
  • hepatic cirrhosis due to non-alcoholic, fatty liver disease
  • Hepatitis caused by drug abuse (e.g., fentanyl causes massive increase in AST and ALT)
32
Q

How do you measure AST and ALT (aminotransferase assay)?

A
  • Form oxaloacetate and oxidize NADH
  • Measure decrease in absorbance at 340 nm 3 times
33
Q

Describe alkaline phosphatase (ALP)

A
  • Catalyzes hydrolysis at alkaline pH
  • Membranes of small intestines, kidneys, liver, bone, and placenta
  • Clinical lab only analyzes liver ALP isoenzyme
  • Liver isoenzyme > bone isoenzyme > intestinal placenta isoenzyme
  • Magnesium activator
34
Q

List disease states associated with ALP

A
  • Obstructive hepatobiliary disease
  • Osteoblast-mediated bone disease
35
Q

Describe ALP assay

A
  • No NADH used
  • 4-NPP converts to 4-NP (yellow)
  • Measure increase in absorbance at 405 nm three times
36
Q

List interfering factors in ALP assay

A
  • Hemolysis causes false increase since intracellular ALP gets released
  • Chelating anticoags (e.g., citrate, oxalate, EDTA) should not be used bc can interfere with Zn and Mg cofactors
  • If liver disease not implicated (regular ALT + AST), then order ALP isoenzyme panel to find tissue of origin
37
Q

Describe acid phosphatase

A
  • optimal pH < 7 (acidic)
  • Prostate, bone, liver, spleen, kidney, RBCs, plts
  • Aids detection of prostatic carcinoma, especially metastatic
38
Q

Describe Gamma-glutamyl transferase (GGT)

A
  • Found in kidney, liver, pancreas, and intestines
  • Cell membrane and cytoplasm
  • Involved in glutathione metabolism + membrane transport of aa + peptides
39
Q

Disease states associated with GGT?

A
  • Mostly hepatobiliary disorders
  • Metastatic carcinoma (super high GGT)
  • Severe alcoholism + drug use
  • Normal during pregnancy + bone disorder pts
40
Q

Which enzyme should be measured to screen for alcohol use and why?

A

GGT because it takes longer to return to normal levels after drinking

41
Q

Which wavelength is used for the GGT assay?

A

Measure at 410 nm

42
Q

Describe acinar cells in pancreas

A
  • Create bicarb-heavy alkaline fluid to neutralize stomach acid in intestines
  • Get inflamed with super fatty foods
43
Q

Describe amylase

A
  • Functions to break down carbs into simple sugars
  • S-amylase secreted by salivary glands to start sugar digestion
  • P-amylase secreted by pancreatic acinar cells to further sugar breakdown
  • Good indicator of pancreatic injury but not best diagnostic test for pancreatitis
  • High concentration gradient between plasma and acinar cells
  • Amylase is small enough to be excreted through urine
44
Q

Disease states associated with high plasma amylase levels

A
  • Acute/chronic pancreatitis
  • Hereditary pancreatitis
  • Pancreatic carcinoma
45
Q

Describe amylase levels in populations that eat high carb vs high protein diets

A

High carb = higher amylase levels
High protein = lower amylase levels

46
Q

What is the time frame of detectable amylase activity in acute pancreatitis pts?

A
  • 5-8 hours after symptom onset
  • Activity peaks after 12-48 hrs
  • Return to normal 3-4 days post-symptom onset
47
Q

List interfering factors in amylase assay

A
  • Calcium-chelating anticoags (EDTA, citrate, oxalate) inhibit amylase
  • Only use heparinized plasma, tho most often assayed on serum
  • Add 24-hr urine amylase test to serum amylase test bc not specific to acute pancreatitis
48
Q

Describe lipase

A
  • Functions to hydrolyze triglycerides to glycerol
  • Only test needed to diagnose acute pancreatitis
  • Lipase level just above ref range considered to “reach threshold” for diagnosis
  • Usually low levels in normal ppl
49
Q

Disease states associated with abnormal lipase levels

A
  • Acute pancreatitis (super specific)
  • Chronic pancreatitis
  • Celiac disease
  • Crohn’s disease
  • Cystic fibrosis
  • Pancreatic carcinoma
50
Q

Lipase enzyme is almost exclusive to which organ?

A

Pancreas
[pancrea lipase] up to 5000x higher than in other tissues

51
Q

Lipase assay methods

A
  • Old = turbidemetry to measure fatty acid release
  • New = synthetic substrates in one coupled-enzyme, continuous monitoring method
52
Q

Describe creatine kinase (CK)

A
  • Uses ATP to convert creatine to creatine phosphate (energy source for muscles)
  • Fwd rxn occurs in mitochondria
  • Found primarily in cardiac + musculoskeletal tissue
  • In cytoplasm, CK also phosphorylates ADP to ATP for muscle contraction (rvs rxn)
53
Q

Where are the following CK isoenzymes found:
- CK-MB
- CK- MM
- CK-BB

A
  • CK-MB = cardiac tissue
  • CK-MM = muscle tissue
  • CK-BB = brain tissue
54
Q

Disease states associated with high CK levels

A
  • Muscular dystrophy
  • Acute rhabdomyolysis
  • Acute myocardial infarction (most common disease associated with high CK)
  • Severe fall
  • Trauma
  • Surgery
55
Q

Interfering factors with CK measurement?

A
  • CK is unstable, so quickly store after analysis
  • Hemolysis falsely increases [CK]
  • Coupled-enzyme test only measures total CK, so order specific immunoassay test or perform protein electrophoresis to ID isoenzyme
56
Q

Describe lactate dehydrogenase (LADH)

A
  • Functions to moderate interconversion between lactate and pyruvic acids
57
Q

Disease states associated with moderate increase of LADH?

A
  • Acute myocardial infarction (AMI)
  • Pulmonary embolism
  • Leukemia
  • Hemolytic anemia
  • Liver and renal diseases
58
Q

Disease states associated with severe increase of LADH?

A
  • Pernicious anemia
  • Megaloblastic anemia
  • Cancer
59
Q

LADH assay limitations?

A
  • Old assay
  • General disease marker, not specific
  • 5 isoenzymes exist
60
Q

Order of LADH isoenzyme concentrations in normal patients?

A

LD2 > LD1 > LD3 > LD4 > LD5

61
Q

Order of LADH isoenzyme concentration in AMI pt?

A

LD1 > LD2 > LD3 > LD4 > LD5

62
Q

Describe LADH assay

A

Labs measure lactic acid conversion to pyruvic acid and measure formation of NADH (increase in absorbance, NAD+ gets reduced)

Michelle: Clinical Chem (4 decks)

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