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BioMedChem M3 > 32 - Clinical Assays of Enzymes > Flashcards

32 - Clinical Assays of Enzymes Flashcards

1
Q

Immunoassays

A

Quick & Specific

But only one protein @ a time
no Parallel assays

Detected by:
Color Change / Fluorescence / Radiolabel

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2
Q

Electrophoresis & Chromatography

A

CHEAP but slower vs immunoasays

Parallel Assay
(more than 1 at a time)

Detection:
Stains / IV absorbance / Fluorescence

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3
Q

Electrophoretic Separations

and it is dependent on what?

A

SIZE

Larger Size of Particle –> larger drag force –> LESS IT MOVES

CHARGE = Z

Greater the E-Force on the particle –> MORE IT MOVES

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4
Q

Detecting Proteins with ELECTROPHORESIS

what is used?

A

To REDUCE diffusion & convection –> to IMPROVE resolution
proteins are often electrophoresed in a
GEL MEDIUM
Polyacrylamide / Agrose / Cellulose acetate

this is then treated with a dye or
STAIN
Bromcresol Green / Coomassie Blue

Intensity of the stain : amount of protein present

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5
Q
  • *Capillary Electrophoresis**
  • *CE**
A

AUTOMATED & NO GEL MEDIUM
uses a cool capillary tube that supresses the convection

Detected by:
UV Absorbance / MS

To detect:
DRUGS / Proteins

we can use micelles for un-charge analytes

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6
Q

Blood level of enzyme/protein is UNUSUALLY HIGH,
indicates what?

Diagnostic Enzymology

A

MAY indicate Pathology

different tissues have:
different levels of key ezymes / proteins
and might even have different forms (isozymes / isoforms)

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7
Q

If enzyme / protein levels are well BELOW the normal levels….

Diagnostic Enzymology

A

There MAY be some damage to the
SECRETING ORGAN

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8
Q

Assays help us do what for

Diagnostic Enzymology?

A

IDENTIFY the affected tissues

find the EXTENT of damage

and possibly the CAUSE of the damage

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9
Q

Examples of Enzymes and Proteins Assayed ​for

LIVER DAMAGE

A

Alkaline Phosphatase

AST / ALT

LDH

5’ nucleotidase / GGT / albumin / complement factors

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10
Q

Examples of Enzymes and Proteins Assayed ​for

HEART MUSCLE DAMAGE

A

MYOGLOBIN

TROPONIN I / T

isoforms of creatine kinase + LDH

AST / ALT

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11
Q

MULTPLE/Continous Enzyme Assay

How do we determine the AMOUNT OF ENZYME PRESENT?

A

Initially, Product increases LINEARLY w/ time
we are Following PRODUCT RELEASE at a rate of time

Δ[P] is proportional to the Amount of Enzyme

so we can COMPARE:
Δ[P]to the standard calibration curve
in order to get the amount of enzyme present

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12
Q

End-Point Assay
one of 2 main ACTIVITY ASSAY types

A

Start the rxn and incubate for a FIXED TIME @ constant T

then STOP reaction & measure amount of PRODUCT
simple but PRONE TO ERROR

can be automated and done quickly
also must be done in LINEAR REGION of the kinetic curve

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13
Q

Continuous / Multiple Point Assay

one of 2 main ACTIVITY ASSAY types

A

Start RX & follow it CONTINUOUSLY
with MANY time points

More accurate but MORE Complicated

can be automated and done quickly
also must be done in LINEAR REGION of the kinetic curve

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14
Q

Steps / Precautions AFTER drawing blood sample

before doing the Assay

A

Centrifugation
seperates Serum from RBC / leukocytes
serum may then be directly assayed or STORED

proteins denature / degrade upon storage
we can try to fight that by reducing temperature to help maintin stability & activity
BUT NOT ALWAYS

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15
Q

Enzyme Stability
<10% change in activity

A

ALT
can NOT be FROZEN

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16
Q

What does ONE Conventional UNIT ( U ) of enzyme activity

do?

A

CONVERT

1 MICROmole of substrate

to product in ONE MINUTE

17
Q
  • *Alkaline Phosphatase**
  • *ALP** or AP

Function & Catalytic Activity

A

HIGH ALP:
from Rapid Bone Growth @ puberty
from Placental Release @ 3rd trimester of pregnancy

group of enzymes that
Hydrolyze monoPhosphate esters @ ALKALINE pH

Highest activity in
LIVER / BONE / intestine / kidney / placenta
but is found in most tissues

18
Q

GGT
Gamma-Glutamyl Transferase

Function & Catalytic Activity

A

Induced by DRUGS & ALCOHOL

HIGH Serum GGT = Liver Disease
NORMAL in bone disease, ALP might be elevated

  • *Microsomal** enzyme that is major in:
  • *Glutathione Metabolism**
  • *Resorption** of AA’s in kidney

Activity is HIGHEST in KIDNEY

19
Q

5’-Ntase
5’-Nucleotidase

Function & Catalytic Activity

A

Serum levels Increased in LIVER DISEASE
but NOT in bone disease

Hydrolyzes Nucleoside 5’-phosphate Esters

microsomal enzyme

20
Q

Aminotransferases

ALT (Ala) / AST (Asp)

Function & Catalytic Activity

A

Converts:
AA’s –> corresponding a-Keto acids

ALT = highest in Liver

AST = roughly EQUAL levels in Heart / SkelMuscle / Liver

may be elevated in:
liver disease / MI / Renal infarction

21
Q

LDH
Lactate Dehydrogenase

Function & Catalytic Activity

A

Interconverts:
Lactate <–> Pyruvate

using NAD/NADH
cytoplasmic enzyme

HIGHEST levels found in kidney & heart
also found in other tissues

22
Q

Diagnosing a AMI (Acute Myocardial Infarction)

A

Presence of at least 2 of the following:

H/O Chest Pain

Evolutionary Changes in ECG
EKG / ECG = electrocardiogram

Elevation in serum of Cardiac Enzymes / Proteins
LDH / Creatine Kinase
Myoglobin / Troponin I & T

23
Q

Cardiac Markers

ENZYMES

and how do they differ?

A

LDH & Creatine Kinase isoforms

differ in:
location in cell
how quickly they are released after cell damage
how quickly they are cleared from serum

24
Q

Cardiac Markers

PROTEINS

A

Myoglobin** & **Troponins I & T

differ in:
location in cell
how quickly they are released after cell damage
how quickly they are cleared from serum​

25
Q

AMI Markers

A

1st to Peak = MYOGLOBIN
returns in 1 day

2nd = CK

  • *Troponin I** = 1st peak
  • *Troponin T** = 2nd peak

Last to Peak = LD

26
Q

LDH Isoforms

A
  • *Tetrameric enzyme** w/ 2 main subunit types:
  • *M & H**

with 5 principle isoforms that are MORE Tissue-specific:
Mixed Isoforms = LD2/3/4
found in a WIDE variety of tissues

Isoforms can be seperated with
ELECTROPHORESIS & QUANTIFIED

27
Q

LD-1 is found in highest concentrations where?

Lactate Dehydrogenase Isoform

A

H4
1 of 5 LDH isoforms, highest in

HEART muscle / KIDNEY / RBC

Think LD1 = #1 is heart muscle

28
Q

LD-5 is found highest concentrations where?

Lactate Dehydrogenase Isoform

A

M4
one of 5 LDH isoforms found highest in:

LIVER & SKELETAL MUSCLE

29
Q

What can cause a RISE in TOTAL Serum LDH?

A

STROKE / HEART ATTACK
LD-1 (H4)

blunt-force TRAUMA
LD-1 RBC

LIVER DAMAGE
LD-5 (M4)

heavy EXERCISE
LD-5 skeletal muscle

30
Q

Which indicates an ACUTE MI?

LDH Isoform Levels

A

B & C

LD1 = Heart / Kidney / RBC

HIGH LD1 indicates ACUTE MI

31
Q

Creatine Kinase
CK

Function & Catalytic Activity

A

Dimeric enzyme that catalyzes:

REVERSIBLE Phosphorylation of CREATINE
using ATP
Creatine Phosphate = resevoir for “High Potential” Phosphate
for re-phosporylation of ADP during EXERTION

Greatest activity in:
MUSCLE / Brain / Heart

32
Q

CK Isoenzymes

A

2 Subunit polypetides:

  • *B = Brain (comes FIRST)**
  • *M = Muscle**

Three main isoenzymes:
CK-1 = BB
CK-2 = MB
CK-3 = MM
CK-4 = mitochondrial form

Isoforms can be seperated by ELECTROPHORESIS

33
Q

Through electrophoresis if there is a SHIFT
in CK isoform concentrationfrom

CK-3 -> CK-2

WHAT DOES THAT INDICATE?

A

MYOCARDIAL INFARCTION

CK-3 = MM (Muscle concentration)
leaks down to:
CK-2 = MB (Mix of Muscle + Brain)

shows that there is a LEAK from MUSCLE -> BRAIN

34
Q

CK as a DISEASE MARKER

A

Duschenne’s Muscular Dystrophy
rise in CK-3 & 2 = skel muscle

HEART = MI / other heart conditions
leak from CK-3 –> CK-2 MB

CNS in cerebral ischemia / cerebrovascular disease
CK-3 RISES ALONE, comes likely from VASCULAR cells

TUMORS
neuronal damage –> raise in CK 1

35
Q
A

BioMedChem M3 (8 decks)

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