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Clinical Chemistry II - 4378 > Blakes Diagnositc enzymology > Flashcards

Blakes Diagnositc enzymology Flashcards

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1
Q

Alterations in Liver enzyme activities

A

1) Haptocellular damage: aminotransferase activity
2) Cholestasis: activities of alkaline phosphatase, 5’-nucleotidase, γ -glutamyltransferase

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2
Q

Cholestasis

A

is the suppression of normal bile flow

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3
Q

Amminotransferase has what Amino acids and coenzymes

A

2-oxo-acids
Pyrudoxal-5’-phosphate (P-5’-P)
Prydoxamine-5’-phosphate

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4
Q

Aspartate aminotransferase (AST)

Location

sources of error

what forms

A
  • Liver, Kidney, Striated Muscle, RBCs. Predominantly in the liver (& kidney)
    Source of error: Hemolysis lesser amount in Striated Muscle
    Cytoplasm & Mitochondrial forms
    increased Acute hepatocellular disorders and increased in myocardial muscle or other conditions
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5
Q

Alanine aminotransferase

Location

is in what part of the cell

A

-Predominantly in liver and kidneys
-less amount in Striated muscle
-exclusively in the cytoplasm

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6
Q

In acute liver disease
ALT in greater then

A

AST and remains elevated longer

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7
Q

ALT ref diseases

A

alcoholic hepatitis, hepatic cirrhosis, liver neoplastic

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8
Q

AST reaction

A

Aspartate + a-KG with enzymes AST and P-5-P —> Oxaloacetate + Glu

Oxaloacetate + NADH with enzyme MDG —–> H + Malate + NAD

Measures the decrease in OD of NADH at 340nm

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9
Q

ALT reaction

A

Alanin + α-KG with enzyme P-5-P and ALT —-> Pyruvate + Glu

Pyruvate + NADH + H+ with enzyme LDH —–> Lactate + NAD

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10
Q

Aminotransferase clinical signifance

Liver disease level

Thershold for diagnosing Acute liver Injury

Peak activity

A

LIVER disease : might 100 times the URL. (usually 10-40 fold increase)
Hepatic necrosis : prior to symptom, both ENZYMES activity increase
Threshold for Diagnosing Acute Liver Injury : 7 times URL
Peak activity of ALT & AST : 7 th and 12th day

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11
Q

Chronic hepatitis

A

ALT ( > 6 months) [Periodic measurement]

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12
Q

Toxic hepatitis V Infection hepatitis

A

Acetaminophen-induced hepatic injury:
The peak activity: 85 x URL (about 90% of case)
Infectious hepatitis: rarely seen this level of elevation

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13
Q

medication induced elevation of ALT and AST

A

NSAID, antibiotics, antiepileptic drug, statin, etc

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14
Q

Non-Alcohol Fatty Liver Disease: h ALT, AST

A

Metabolic syndrome
1) Higher body mass index, 2)increased waist circumstance, 3) ↑serum TG, 4) ↑ fasting insulin,
5) LDL/HDL cholesterol

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15
Q

Primary & metastatic malignancy of liver

A

AST>ALT; both enzymes 2-5 x h activity
Cf. Early stage: activity increases within normal range

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16
Q

AST/ALT ratio (AAR) greater than 1:

A

Presence of advanced fibrosis in patients with chronic liver diseases
(≈ 90% positive predictive value)

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17
Q

Alkaline Phosphatase (ALP)

Lipid transport in

A

lipid transport in the intestine and with the calcification process in bone

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18
Q

Genetic loci of ALP

A

Tissue nonspecific

Germ cell

Placental

Intestinal

Fetal Intestinal

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19
Q

ALP coenzymes

A

Co2+, Mg2+, Mn2+

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20
Q

ALP inhibitors

A

Phosphate, Borate, Oxalate, and cyanide ion

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21
Q

ALP hepatobiliary diasease

Serum enzyme activity could be increased to

Biliary tree obstruction–> blank–> blank

Extra hepatic obstruction blank vs intra hepatic activity

A

Serum enzyme activity could h up to 12 x
depend on the degree of obstruction

Biliary tree obstruction g hepatocytes g
synthesis of ALP

Extra hepatic obstruction 3 x elevation
of ALP activity vs. intra hepatic

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22
Q

ALP clinical sig

Primary liver what or widespread what

mild increase or normal in what

Blank therapy could also increase blank

A

-Primary liver cancer patients or widespread
secondary hepatic metastasis
-Infectious hepatitis : mild increase or
normal ALP activity.
-Drug therapy could increase ALP activity.
** narcotic pain medicines, NSAIDs, propranolol,
tranquilizers, tricyclic antidepressants

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23
Q

Hypophosphatasia ALT

examples diseases

A

-severe bone
disease and impaired bone growth
-Paget disease (osteitis deformans)
enlarged & deformed bones
-Vit D deficiency Osteomalacia, Ricket
2-4x URL
-Osteoporosis
-Bone cancer

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24
Q

Hypophosphatasia

ALP sig

Primary and secondary what

ALP is increased during what

A

Primary / secondary) hyperparathyroidism
Slight to moderate h
ALP activity is temporarily increased during healing from
the fracture

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25
Q

ALP reaction

A

-4-NPP or PNPP is the most popular chromogenic substrates for ALP
O.D. of p-nitrophenol at 405 nm alkaline pH (~10)
-Serum/plasma w/o hemolysis at room temp within 4 hrs after collection
-EDTA & citrate CANNOT be used
-Be preferentially measured in fasting sera

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26
Q

Gamma Glutamyltransferase trasfers what

Substrate

A

Transfer γ-glutamyl group from donor to an
acceptor

1) the γ-glutamyl
acceptor, 2) AA or peptide, 3) water.

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27
Q

GGT critical marker

Location

A

Hepatobiliary disease [critical marker]

Liver (hepatobiliary system), Kidney (PCT), Pancreas, & Intestine.
Cytoplasmic protein [Membrane Bound]

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28
Q

GGT intra and post what

5-15 URL increase

A

-: 5-30 x URL
Intra- & post-hepatic biliary obstruction

-Primary and metastatic liver neoplasm
-Alcoholic hepatitis
-Anticonvulsant drugs

5-15 URL increase
Acute and chronic pancreatitis
Pancreatic malignancies
(with biliary obstruction

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29
Q

GGT

Normal range

A
  • Adult: Male - 70 U/L / Female – 40 U/L
  • 2 fold higher in people of African ancestry
  • At birth: 6-7 times of adult reference interval
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30
Q

GGT reaction

A

γ-glutamyl-p-nitroanilide + glycylglycine with enzyme GGT—–> p-nitroaniline + γ-glutamyl-glycylglycine

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31
Q

GGT

P-nirtoaniline is measured at

A

p-nitroaniline (yellow) is measured spectophotometrically at 405 nm

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32
Q

γ-glutamyl-p-nitroanilide

A

less water soluble, It is difficult to reach the saturating concentration of substrate. derivatives of GGPNA in which various groups have been introduced into the benzene ring to
increase water solubility

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33
Q

GGT second reaction

A

γ-glutamyl-3-carboxy-4-nitroanilide + glycylglycine with enzyme GGT ——> 5-amino-2-nitrobenzoate + γ-glutamyl-glycylglycine

5-amino-2-nitrobenzoate is measured at 410 nm

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34
Q

GGT sample and storage

A

-GGT activity is stable for 1 month @ 4C/ 1 year @ -20 C
-Non hemolyzed serum (preferred)/ EDTA-plasma
-Heparin [Turbidity of sample]; Citrate, oxalate, Fluoride [↓ GGT acticvity 10-15%]

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35
Q

Amylase P type

Hydrolysis of what

Synthesized by what

Where is S-type amylase

A

Hydrolysis of 1,4-α-glucosidic linkages in polysaccharides
Ca++ metalloenzymes
Synthesized by acinar cells
pancreatic duct system
GI Track
Ovaries, Fallopian tubes, lungs, adipose tissue, salivary gland (S type)
Amylase (p type) & Lipase
Lack of specificity of total Amylase
direct measurement of P-AMY

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36
Q

Amylase requires what cofactor

A

Ca2+ Metalloenzymes- Ca2+ is required to maintain structure of amylase.

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37
Q

P type amylase

A

Pancrease

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38
Q

S type amylase

A

Salivary gland specific

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39
Q

Amylase can be activated by

A

Chloride and bromide

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40
Q

Amylase breaks down

A

Polysaccaride
1-4a-glucosidic

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41
Q

Amylase is the only

A

Enzyme present in the urine.

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42
Q

GGT is used in measuring what organ

A

Pancreas for acute and chronic pancreatitis

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43
Q

Amylase is delivered through the pancreatic duct to the

A

Intestines ( GI tract)

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44
Q

Total amylase activity is not good because

A

Other parts of the body can make amylase such as the salivary glands, ovaries, fallopian tubes, lungs, adipose tissue, and salivary glands

45
Q

Amylase in blood is mainly what two types

A

S and P types

46
Q

How can P-type amylase be quantified

A

By using a p-type amylase antibody

47
Q

ALP is increased in

A

Bone/liver/cancer conditions.

48
Q

ALT has a longer

A

Half life therefore the ALT/AST ratio is over one

49
Q

In chronic liver disease the ALT/AST ratio is

A

Flip and AST/ALT is over one.

50
Q

ALP and GGT will differentiate between what

A

Liver and bone disease

both GGT and ALP will be elevated in liver disease

only ALP is elevated in bone disease

51
Q

Enzyme use what units

A

U/L

dont use Mg/dl

52
Q

Essential coFactors for AST and ALT

A

P-5-P

53
Q

Pancreas specific enzymes

A

Amylase and Lipase
Stomach acid= painful

54
Q

Test material=

Test for amylase
P-amylase use

A

Selective S-amylase monoclonal antibody
= so All S amylase is trapped in sample and the remaining P-Amylase is quantified

55
Q

You can three forms of product of ESP reaction of amylase which are

A

2ED-GS + 2-4-NP-G2

2ED-G4 + 2-4 NP=G3

ED + 4-NP-G4

ED= Ethylidene

NP= nitrophenal

56
Q

Further hydrolysis of amylase happens by

NSP amylase reaction

A

Glycosidase= further breakdown of sugar and NP

57
Q

Test material

All anticoagulants inhibits

A

Amylase activity because of calcium binding

Exception is Heparin

58
Q

what anticoagulant can be used with amylase

What other specimen can be used

A

Heparin because it doesn’t inhibit amylase activity= Heparinized plasma

you can also use serum as a specimen

59
Q

Nitrophenols give off a huge

A

Color

60
Q

If you dont have a disease of the pancreases the

A

Amylase has low physiological level.

61
Q

There can be what amylase

A

Urine because the size of it is 54-62kD

62
Q

Levels of amylase are not related to

A

Severity of conditions.

63
Q

Total amylase activity goes up in

A

Pancreas and salivary gland in inflammation

64
Q

Acute pancreatitis=

A

Amylase activity can return to normal in 3-4 days

65
Q

Cholecystitis=

A

Bile duct obstruction

P-amy (4x increase URR)

66
Q

Ethylidene is used to

A

Stabilize the NP-G2 complex for measurement

67
Q

Nitrophenol is connected to sugar because phenol will give

A

Color once released from sugar

68
Q

Amylase is stable

A

Stable 4 days at room temp, 2 weeks at -4 °C, 1 year at -25 °C, 5 years at -80 °C

69
Q

Amylase is measured at what wavelength

A

405 nm

70
Q

Substrate used in the amylase reaction

A

4-nitrophenyl (4-NP)-glycoside [Substrate]

71
Q

2nd reaction in amylase reaction

A

2 4-NP-G2 + 2 4-NP-G3 + 10 H2O with enzyme Glycosidase —–> 4 4-NP + 10 G

72
Q

Lipase

cofactors

highest levels in

in what else

A

LPS
Cofactors: bile salts & colipase Pancreas, gastric and intestinal mucosa

highest levels in pancreas

in the gastric, and intestinal mucosa, also

73
Q

Lipase is a single

can it be detected in the urine

A

Single chain glycoprotein: 48 kD
100% filtered & 100% reabsorption
DOES NOT PHYSIOLOGICALLY DETECTED IN URINE

74
Q

Acute pancreatitis level of LPS

A

LPS in Serum: acute pancreatitis
[2-50 x URL]

75
Q

Serum LPS is 3X the upper in

A

Perforated gastric, or duodenal ulcer, GI obstruction, Mesenteric Vascular obstruction
serum LPS activity to greater than 3 x the URL (w/o renal failure)

patients with renal disease because of it is 100% reabsorbed so it will be detected alot in renal disease.

76
Q

More sensitive then

LPS remains elevated longer then

With reduce GFR then

A

More sensitive than increase in AMY activity
➡ LPS remains elevated longer than AMY does
➡ With reduced GFR, serum LPS activity is
increased

77
Q

Lipase analysis

A

-Use TG or non-TG substrate

Titrimetric, turbidimetric, spectrophotometry, fluorometric, immunologic techniques

Methylresorufin can be detected at 580 nm
๏ bluish purple chromophore

No sex and age related differences

LPS activity: stable for 1 week at room temp; 3 weeks in 4 °C; severl years at -20 °C

78
Q

Acid phosphatase (ACP)

All phosphatase with optimal activity is under

A

acidic pH (< pH 7.0)

79
Q

Acid phosphatase ( ACP)

A

acidic pH (< pH 7.0)

80
Q

ACP has what enzyme activity

A

Lysosomal and extralysosomal enzyme

81
Q

ACP greatest concentrations

A

1) prostate, 2) bone, 3)spleen, 4) platelet, 5) RBC

82
Q

Low ACP activity of serum

A

tartrate-resistant type (TR-ACP, TRAP or
TRAPase)

mAb for TR-ACP

83
Q

If you have prostate ACP elevated then it can be a marker for the

A

Prostate cancer

84
Q

Lysosomal ACP is

A

Prostate ACP

Low decreased activity level in blood

85
Q

Bone and RBC acid phosphatase

A

extra lysosomal enzymes

are not inhibited by tartrate

so called tartrate resistant Acid phosphatase

86
Q

Lysosomal tartrate will be inhibited by

A

Tartrate aka Prostate

87
Q

Acid phosphatase

add tartrate and measure

A

Activity= acid phosphatase activity goes down or no changes

Tartrate added to sample and activity is not changing it is Extralysosomal

Add tartrate and value goes down then lysosomal( Prostate)

88
Q

Extra lysosomal ACP=

A

Bone or RBC

89
Q

Bone ACP can be elevated in

A

Activities of this fraction (bone) are increased in growing children

conditions of increased osteolysis & bone remodeling

AKA: Paget, hyperparathyroidism, malignant invasion in bone

90
Q

Lysosomal acid phosphatase is a marker for

A

Prostate cancer

91
Q

ALP is measured the same way as

A

Measured the same way as ALP, but under ACIDIC conditions (pH = ~5)

Reference range: 0 – 3.5 ng/ml

92
Q

Cholinesterase (CHE)

Hydrolyzes esters of

Metabolizes/ clearance of

Deactivation of

A

choline, e.g acetylcholine

Metabolize/clearance of drugs

deactivation of octanoyl ghrelin

93
Q

Two main types of CHE

A

Actylcholinesterase

Pseudocholinesterase

94
Q

Acetylcholinesterase

A

Acetylcholinesterase (true cholinesterase, RBC Che, cholinesterase I) RBC, Lung, Spleen, Nerve endings, Gray matter of the brain.

95
Q

Pseudocholinesterase

A

Pseudocholinesterase (Acetylcholine acylhydrolase, Serum(/plasma) cholinesterase (CHE, SchE), cholinesterase II), current Butyylcholinesterase(BChe)
Liver, Pancreas, Heart, White matter of brain, Serum

96
Q

Cholinesterase (CHE)

Liver function tests

Used for monitoring

A

Decreased in CHE activity: impaired synthesis of the enzyme by liver

-used for monitoring of liver function after “liver transplantation”

97
Q

Cholinesterase is a indicator of

A

possible insecticide poisoning
Insecticides : CHE inhibitors (both cholinesterase)

CHE falls more rapidly

Decreased in n RBC enzyme is used as a measure of “chronic exposure”

98
Q

CHE

For the detection of patients with atypical forms of the enzyme

A

Muscle relaxants: Succinyldicholine, mivacurium

  • Hydrolyzed by CHE
99
Q

Cholinesterase (CHE) analysis

Substate

Chromogenic agents

what do you measure spectrophotometrically at what wavelength

Stable for

A

Subtrate: acylthiocholine ester (ATCl)

Chromogenic disulfide agents: DTNB (Elliman’s reagent)

Measure spectrophotometrically 5-thio-2-nitro-banzoic acid @ 410 nm

Serum: activity stable for several weeks @ 4 °C; several years @-20 °C

100
Q

Glucose-6-Phosphate Dehydrogenase

Enzyme is the first step in the

Leads to the production of

A

Enzyme is the first step in the pentose-phosphate shunt

Leads to the production of NADPH

101
Q

Glucose-6-Phosphate Dehydrogenase

very important enzyme in the RBC to

A

Functions to maintain NADPH levels.

NADPH (glutathione) helps protect cell/hemoglobin from oxidation. A deficiency in G6PDH results in an inadequate supply of NADPH and inability to protect cell

102
Q

Glucose-6-Phosphate Dehydrogenase

Hemolysis can lead to

A

Hemolysis can occur leading to hemolytic anemia

103
Q

Glucose-6-Phosphate Dehydrogenase analysis

A

Glucose-6-phosphate + NADP+ ——————>
6-phosphogluconate + NADPH

NADPH is measured spectrophotometrically (340 nm).

“Erythrocyte hemosylate” is assayed for enzyme deficiency.

104
Q

Elevated LDH

A

LD not specific to cardiac tissue – found in various other tissues as well.

Increase in serum level from ~12 - 24 h post infarc, peaks after ~ 48-72 h, gradually returns to normal by ~ 7 – 14 days

105
Q

Elevated Cardiac enzymes (CK)

A

CK-MB is heart specific
Rises about 4-8 h after infarct, peaks at 12-24 h, and returns to normal in 2-3
days

CKMB index = CKMB activity/Total CK activity x 100 CKMB: <6% total CK

106
Q

Troponins

A
  • Troponins are proteins involved in muscle contraction.
  • TnI, TnT, TnC: found in skeletal/cardiac muscle
  • Screen for cTnI and cTnT
  • Following AMI, levels begin to rise ~3 – 6 h, reach peak levels in 14-24 h, return to
    normal in 5 – 10 days
107
Q

Myoglobin

A

-For early detection (leaks 1-3 h of onset) of AMI.
-Peak is reached 5 – 12 hours.
-Myoglobin is a small molecule (kidney can freely filter) and thus returns to normal in 18 – 30 h after the AMI.
-Problem: NOT specific
Present in all muscle cells so non-specific

108
Q

Diagnosis of Acute myocardial infarction use what enzymes

A

LDH
Cardiac enzymes
Troponins
Myoglobin

Clinical Chemistry II - 4378 (28 decks)

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