CC1 Flashcards
Enumerate Prostate Enzymes
ACP
G-6-PDH
Enumerate Miscellaneous Enzymes
5’ nucleotidase
Cholinesterase/pseudocholinesterase
Angiotensin Converting Enzyme (ACE)
Ceruloplasmin
Ornithine Carbamoyl Transferase (OCT)
G-6-PD
a phosphoric monoester hydrolase
5’ N
5’ N source
predominantly secreted by the liver
Marker for hepatobiliary disease and infiltrative lesions of the liver
5’ N
5’ N is a marker for these conditions
Hepatobiliary disease
Infiltrative lesions of the liver
reference range for 5’N
0-1.6 units
index of parenchymal function of the liver
Cholinesterase/
Pseudocholinesterase
Cholinesterase/Pseudocholinesterase source
Liver
Monitor effects of muscle relaxants (succinylcholine) after surgery
Cholinesterase/
Pseudocholinesterase
Marker for insecticide/pesticide poisoning (organophosphate – poisonous agent)
Cholinesterase/
Pseudocholinesterase
reference range for cholinesterase/pseudocholinesterase
0.5-1.3 units (plasma)
a.k.a Peptidyldipeptidase A or Kininase II
Angiotensin Converting Enzyme (ACE)
aka Angiotensin Converting Enzyme (ACE)
Peptidyldipeptidase A or Kininase II
Converts angiotensin I to angiotensin II within the lungs (RAAS)
ACE
Primary enzyme of RAAS
Angiotensin Converting Enzyme (ACE)
Promotes vasoconstriction of the renal arterioles to increase blood pressure and stimulates the adrenal cortex to release aldosterone so that aldosterone will promote Na reabsorption
Angiotensin II
Events that activates RAAS
Blood pressure/volume is decrease
Low plasma sodium level
source of ACE
Macrophage and epithelioid cells of the lungs
Possible indicator of neuronal dysfunction
(Alzheimer’s/neurodegenerative disease)
ACE
ACE is increased in these conditions
Sarcoidosis
Acute and chronic bronchitis
Leprosy
Copper-carrying protein which acts as an enzyme
Ceruloplasmin
Marker for Wilson’s disease (hepatolenticular disease)
Ceruloplasmin
Clin. significance of Ornithine Carbamoyl Transferase (OCT)
Hepatobiliary diseases
Reference range for Ornithine Carbamoyl Transferase (OCT)
8-20 mU/mL
Maintain NADPH in the reduced form in the RBCs
G-6-PD
Specimen for G6PD
Red cell hemolysate
Serum
Responsible for maintaining and stabilizing
the membrane integrity of RBCs
reduced NADH (maintained by G6PD)
Protects RBCs from toxic agents that can
induce hemolytic reactions
Reduced NADH (maintained by G6PD)
Sources of G6PD
• Adrenal cortex
• Spleen
• RBC
• Lymph nodes
Enzyme used as a newborn screening marker
G6PD
G6PD is INCREASED in these conditions
MI
Megaloblastic anemia
G6PD is DECREASED in these conditions
Drug-induced hemolytic anemia (intake of primaquine, antimalarial drugs)
Reference range for G6PD
10-15 U/g Hgb
1200-2000 mU/mL pRBC
Biologic intracellular proteins that catalyze biochemical reactions
Enzymes
Affects the reaction of the organic matter
Enzymes
NOT consumed/changed in composition (only substrate → product)
Enzymes
Increased enzymes in serum is due to the following conditions
Cell injury/degradation
Increased membrane permeability
Organ damage (severely increased enzyme in serum/plasma)
What happen when there is an increased membrane permeability?
INCREASED ENZYMES in serum, allowing these proteins to move out easily from the cells
Enzymes are found in _____________
all body tissue (intracellular)
Concentration in serum is very low as it is very abundant in cytoplasm
Enzymes
Indication when there is a SEVERELY INCREASED enzyme in serum/plasma
Organ damage
FUNCTION OF ENZYMES
- Hydration of Carbon Dioxide (respiration)
- Nerve Induction (fast nerve impulse transmission)
- Muscle Contraction (locomotion)
- Nutrient Degradation (digestion)
- Growth and Reproduction (work with hormones)
- Energy Storage and Use
Enzymes with similar catalytic activity, but differ in their physical, biochemical, and immunologic properties
Isoenzymes
What are the difference between enzymes and its isoenzymes?
physical, biochemical, and immunologic properties
What is the similarity between enzymes and its isoenzymes?
catalytic activity
Type of cofactor that serve as a second substrate for enzyme
COENZYME
coenzyme tightly bound to an enzyme
Prosthetic group
T/F
If there is a coenzyme in the reaction, enzyme involved is oxidoreductase – ends with “dehydrogenase” (promotes redox reaction)
T
Part of the reagent since the primary enzyme is the target in the serum
Secondary coupling/indicator enzyme
Requires a coenzyme: pyridoxal phosphate/Vit B6
Aminotransferases
Coenzyme required by aminotransferases
pyridoxal phosphate/Vit B6
A cofactor that enhances enzyme activity by altering the spatial configuration of the active site of enzyme or
enhanced substrate finding
Activator
Types of cofactor
Coenzyme
Activator
These can be grouped as metallic or nonmetallic
Activators
Metallic activators
Mg, Iron, Zinc, Ca
Nonmetallic activators
Cl, Br
when used as coenzyme, it is converted into two forms when acted upon by oxidoreductase
NAD
Oxidized form acted upon by Oxidoreductase
NAD
Reduced form acted upon by Oxidoreductase
NADH
Measured absorbance if the product is OXIDIZED form
NAD
Measured: DECREASE in absorbance
Measured absorbance if the product is REDUCED form
NADH
Measured: INCREASE (HIGH) in absorbance
Enumerate liver enzymes
AST/SGOT
ALT/SGPT
GGT
ALP/Alkaline Orthophosphoric Monoester Phosphohydrolase
ACP/Acid Orthophosphoric Monoester Phosphohydrolase
5’ N
Enumerate cardiac enzymes
CK
LDH
AST
Enumerate non-enzymatic cardiac markers
Myoglobin
Troponins
Enumerate pancreatic enzymes
AMS
LPS
Type of enzyme:
Aspartate Aminotransferase (AST)/
Serum glutamic-oxaloacetic
transaminase (SGOT)
TRANSFERASE
Type of enzyme:
Alanine Aminotransferase (ALT)/
Serum glutamic-pyruvic
transaminase (SGPT)
TRANSFERASE
Type of enzyme:
Gamma-Glutamyltransferase
(GGT)
TRANSFERASE
Type of enzyme:
Acid Phosphatase (ACP)/
Acid Orthophosphoric Monoester
Phosphohydrolase
HYDROLASE
Type of enzyme:
Alkaline Phosphatase (ALP)/
Alkaline Orthophosphoric
Monoester Phosphohydrolase
HYDROLASE
Type of enzyme:
CK/CKP
TRANSFERASE
Type of enzyme:
LDH
OXIDOREDUCTASE
Type of enzyme:
5’ nucleotidase (5’N)
phosphoric monoester HYDROLASE
Type of enzyme:
Amylase (AMS)
HYDROLASE
Type of enzyme:
Lipase (LPS)
HYDROLASE
Transfer of an amino group between aspartate and α-keto glutaric acid
Aspartate Aminotransferase (AST)/
Serum glutamic-oxaloacetic transaminase (SGOT)
Involved in the synthesis and degradation of
AA (protein catabolism, deamination)
Aspartate Aminotransferase (AST)/
Serum glutamic-oxaloacetic
transaminase (SGOT)
Major organ affected of AST/SGOT
HEART
Substrate for AST/SGOT
Aspartic acid (aspartate)
a-ketoglutaric acid (a-ketoglutarate)
End products of AST/SGOT
Glutamic acid (glutamate)
Oxaloacetic acid (oxaloacetate)
Color developer for AST/SGOT
2,4 DNPH
(2,4-Dinitrophenylhydrazine)
Color intensifier for AST/SGOT
0.4 N NaOH
Colorimetric method
for aminotransferase
Reitman and Frankel
Major source of AST/SGOT
Heart
T/F
AST/SGOT is widely distributed
T
Tissue sources of AST/SGOT with increased activities
- Cardiac tissue
- Liver
- Skeletal muscle
AST/SGOT is increased in these conditions
- AMI
- Hepatocellular disorder: chronic liver disorder with progressive damage
- Skeletal muscle disorder: muscular dystrophy (Duchenne)
- Trichinosis
Enzyme increased in
chronic liver disorder with progressive damage
AST/SGOT
(a hepatocellular disorder)
Enzyme increased in muscular dystrophy (Duchenne)
AST/SGOT
Enzyme increased in Trichinosis
AST/SGOT
Isoenzymes of AST/SGOT
Cytoplasmic AST
Mitochondrial AST
most abundant AST isoenzyme in normal serum
Cytoplasmic AST
AST isoenzyme present in mitochondrial membrane
Mitochondrial AST
AST isoenzyme that is increased in cell necrosis
Mitochondrial AST
Assay for AST/SGOT
KARMEN METHOD
2°/indicator enzyme used in Karmen method for AST
Malate Dehydrogenase
Coenzyme used in Karmen method for AST
Pyridoxal phosphate/Vit B6
Monitored in Karmen method for AST and the wavelength used
decrease in absorbance at 340 nm
(measures oxidized NAD)
Variables in Karmen method? What will be the effect?
Hemolysis (False ↑) – very sensitive
Reference range for AST/SGOT using Karmen method
5 – 30 U/L
T/F
Product formed by the 1° enzyme (AST) will become the substrate for the 2° enzyme (MD)
T
In Karmen method, what is the substrate used by MD which is also the product formed by the primary enzyme AST?
oxaloacetate
In Karmen method, what are the products formed acted upon by MD?
malate + NAD
Transfer of an amino group between alanine
and α-ketoglutarate
Alanine Aminotransferase (ALT)/
Serum glutamic-pyruvic transaminase (SGPT)
More liver specific than AST
Alanine Aminotransferase (ALT)/
Serum glutamic-pyruvic
transaminase (SGPT)
Major organ affected by ALT/SGPT
LIVER
Substrates used by ALT/SGPT
Alanine
a-ketoglutaric acid (a-ketoglutarate)
End products of ALT/SGPT
Glutamic acid (glutamate)
Pyruvic acid (pyruvate)
Color developer for ALT/SGPT
2,4 DNPH
Color intensifier for ALT/SGPT
0.4 N NaOH
Major source of ALT/SGPT
LIVER
Minor sources of ALT/SGPT
- Kidneys
- Pancreas
- RBC
- Heart
- Skeletal muscle
- Lungs
ALT/SGPT is increased in these conditions
- Hepatocellular disorders
(liver specific enzyme) - Acute liver inflammation
Enzyme used to monitor hepatitis treatment
and drug therapy effects
ALT/SGPT
Enzyme used to screen post transfusion hepatitis
ALT/SGPT
Screens blood donors (not routine; only with jaundice)
ALT/SGPT
Sensitive test for
occupational toxic exposure
ALT/SGPT
Assay for ALT/SGPT
LACTATE DEHYDROGENASE (LD)
2°/indicator enzyme for ALT/SGPT assay
LACTATE DEHYDROGENASE (LD)
Coenzyme used in LD for ALT
Pyridoxal phosphate/Vit B6
Monitored in LD for ALT? What is the wavelength?
decrease in absorbance at 340 nm
(measures oxidized NAD)
Reference range for ALT using LD assay
6 – 37 U/L
In LD assay, what is the substrate used by LD which is also the product formed by the primary enzyme ALT?
pyruvate
In LD assay for ALT, what are the products formed by LD?
lactate + NAD
aka De Ritis Ratio
AST/ALT Ratio
SGOT/SGPT Ratio
Used to differentiate the cause of hepatic disorder
De Ritis Ratio (AST/ALT Ratio)
De Ritis Ratio (AST/ALT Ratio):
> 1
nonviral origin
De Ritis Ratio (AST/ALT Ratio):
<1
viral origin
what is the cause of hepatic disorder when the AST is higher than ALT?
non-viral
>1 (high AST: low ALT)
what is the cause of hepatic disorder when the ALT is higher than AST?
viral
<1 (low AST: high ALT)
T/F
In De Ritis Ratio, a ratio of exactly 1 is possible.
FALSE!
Ratio of 1 is not possible because AST has MANY tissue sources
Catalyze transfer of γ-glutamyl residue from γ-glutamyl peptides to amino acids, H20, etc.
Gamma-Glutamyltransferase
(GGT)
Common donor (biologic system) in GGT
glutathione
Substrates used in GGT
Glutathione + AA
Products formed when GGT is used as an enzyme
glutamyl-peptide +
L-cysteinylglycine
Source of GGT
Canaliculi of hepatic cells, specifically in the
epithelial lining of biliary ductulus
GGT is used to diagnose these conditions
Hepatobiliary disorders (obstructive jaundice)
Chronic alcoholism (ethanol intoxication)
Marker for occult alcoholism
GGT
Most sensitive marker for acute alcoholic hepatitis
GGT
Assay for GGT
SZAZ ASSAY
measures the absorbance of p-nitroaniline at 405-420 nm
SZAZ ASSAY for GGT
Wavelength used in Szaz assay to measure p-nitroaniline
405-420 nm (visible light region)
T/F
Wavelength requirement may be a clue to the detected product
T
400-700 nm – visible light region
Nonvisible regions:
<400 – UV region
>700 – IR region
substrates used in Szaz assay using GGT
y-glutamyl-p-nitroanilide +
glycylglycine
products formed in Szaz assay using GGT
y-glutamyl-glycylglycine +
p-Nitroaniline
Catalyze hydrolysis of phosphomonoesters
at an acid pH (5.0)
Acid Phosphatase (ACP)/
Acid Orthophosphoric Monoester
Phosphohydrolase
Liberate inorganic PO4 from an organic PO4
ester with alcohol production at an acid pH
Acid Phosphatase (ACP)/
Acid Orthophosphoric Monoester
Phosphohydrolase
pH requirement in ACP
5.0
NOT prostate specific
Acid Phosphatase (ACP)/
Acid Orthophosphoric Monoester
Phosphohydrolase
Incorporated in the prostatic fluid secreted by
the prostate gland (normally present in
seminal fluid)
Acid Phosphatase (ACP)/
Acid Orthophosphoric Monoester
Phosphohydrolase
Substrates used in ACP
phosphomonoester + H2O
Products formed by ACP
alc + phosphate ion
Sources of ACP
- Prostate (male)
- RBCs
- Platelets
- Bone (osteoclast – for bone resorption)
Detects metastatic prostatic cancer
ACP
Significant in forensic rape investigation
ACP
Sample used in forensic rape investigation using ACP
vaginal washings from rape victim
Detectability of ACP in forensic rape investigation
4 days or less
(Options if >4 days: vaginal laceration)
Added to differentiate
prostatic form (specific) from nonspecific
form like RBC ACP
Inhibitor
Inhibitors used for ACP
L-tartrate ions
Formaldehyde,
Cupric ions
L-tartrate ions inhibits these ACP isoenzymes
prostatic ACP
lysosomal ACP
Formaldehyde,
Cupric ions inhibits this ACP isoenzyme
RBC ACP
Reference range for Prostatic ACP
0-3.5 ng/ml
In Shinowara method, what is the substrate used and the products formed?
substrate: p-nitrophenyl-phosphate (PNPP) - COLORLESS
products: p-nitrophenol -YELLOW
phosphate ion
general methods for ACP
Quantitative end point
Continuous monitoring
Substrate used is Thymolphthalein monophosphate (most commonly used; sensitive and specific)
Quantitative end point
Substrate used is a-naphthyl phosphate for ACP
Continuous monitoring
Catalyze hydrolysis of phosphomonoesters at an Alk pH (9-10) (cleaves monoester bonds present in substrates)
Alkaline Phosphatase (ALP)/
Alkaline Orthophosphoric Monoester Phosphohydrolase
Liberate inorganic PO4 from an organic PO4
ester with alcohol production at an Alk pH
Alkaline Phosphatase (ALP)/
Alkaline Orthophosphoric
Monoester Phosphohydrolase
pH required in ALP
9-10
Hydrolase enzyme that requires an activator
ALP
Activator used in ALP
Mg2+
T/F
ALP is liver specific
F
Predominant ALP isoenzyme in normal serum
Liver ALP
Bone ALP
Sources of ALP
- Liver
- Bone (osteoblast)
- Placenta
- Intestine
- Renal tissues (not measured)
Enzyme significantly ↑ in Paget’s disease/Osteitis deformans
ALP
ALP is significantly increased in this condition
Paget’s disease/
Osteitis deformans
Important for evaluation of hepatobiliary (obstructive types) and bone disorders
ALP
ALP Isoenzymes
- Liver ALP
- Bone ALP
- Placental ALP
- Intestinal ALP
Assay for ALP
Bowers and McComb
Principle: molar absorptivity of p-Nitrophenol, Absorbance is measured at 405 nm (colorimetric measurement of yellow-colored p-Nitrophenol), pH 10.2
Bowers and McComb for ALP
optimal pH used in Bowers and McComb
10.2
In Bowers and McComb, absorbance is measured at this wavelength
405 nm
colorimetric measurement of yellow-colored p-Nitrophenol as product of ALP
Bowers and McComb
ALP Reference range in ADULTS
30 – 90 U/L
ALP Reference range in 0-3 months
70 – 220 U/L
ALP Reference range in 3-10 years
50 – 260 U/L
ALP Reference range in 10 yr - puberty
60 – 295 U/L
highest due to active bone development; involves osteoblast resulting to release of ALP in serum)
Fastest ALP isoenzyme
Liver ALP
2 fractions of Liver ALP
Major liver band
Fast liver (a1) band
responsible for predominant liver ALP level in normal serum
Major liver band
responsible for fast-migrating liver ALP in electrophoresis
Fast liver (a1) band
Liver ALP is increased PATHOLOGICALLY in
Liver diseases
Most anodal (fastest) ALP isoenzyme
Liver ALP
3rd most heat stable ALP isoenzyme
Liver ALP
ALP residual activity after heating:
decreased to >20%
Liver ALP
ALP isoenzyme inhibited by Levamisole
Liver ALP
Bone ALP
Heat labile ALP isoenzyme (If the temp is ↑, activity is markedly ↓)
Bone ALP
Bone ALP is increased PHYSIOLOGICALLY in
Bone growth
Bone ALP is increased PATHOLOGICALLY in
- Bone disease
- Healing of bone fractures
2nd most anodal ALP isoenzyme
Bone ALP
Least heat stable ALP isoenzyme
Bone ALP
ALP residual activity after heating:
decreased to <20%
Bone ALP
ALP isoenzyme inhibited by 3M urea
Bone ALP
Most heat stable ALP isoenzyme (withstand heating at 65ºC for 30 minutes)
Placental ALP
Placental ALP can withstand this temperature for how many minutes
65ºC for 30 minutes
Placental ALP is increased PHYSIOLOGICALLY in
Pregnancy (16th & 20th week of gestation)
Placental ALP is increased PATHOLOGICALLY in
Malignancy/cancer
(carcinoplacental ALP)
3rd most anodal ALP isoenzyme
Placental ALP
Inhibited by Phenylalanine
Placental ALP
Intestinal ALP
Regan ALP
Nagao ALP
Slowest moving ALP fraction
Intestinal ALP
Intestinal ALP is increased PHYSIOLOGICALLY in
- Blood group B and O
- Fatty meal consumption
Intestinal ALP is increased PATHOLOGICALLY in
GIT disorders
Least anodal ALP isoenzyme
Intestinal ALP
2nd most heat stable ALP isoenzyme
Intestinal ALP
Total ALP elevations by Liver or Bone ALP are
differentiated by heating of serum at 56°C for 10 mins.
(focuses on liver and bone due to predominance)
HEAT STABILITY
Adding of chemical reagent to the
sample to inhibit the activity of certain
isoenzyme
CHEMICAL INHIBITION
CARCINOPLACENTAL ALP
REGAN ALP
NAGAO ALP
Most heat stable ALP
REGAN ALP
Bone ALP co-migrator
REGAN ALP
(2nd most anodal)
Regan ALP is associated with these conditions
Lung, breast, gynecological cancers
Nagao ALP is associated with these conditions
Adenocarcinoma of the pancreas and bile duct, pleural cancer
Carcinoplacental ALP inhibited by Phenylalanine only
REGAN ALP
Carcinoplacental ALP inhibited by both Phenylalanine and L-leucine
NAGAO ALP
CARDIAC ENZYMES (MI PROFILE)
CK/CPK
LDH
AST
Involved in the storage of high-energy
creatine phosphate in muscle cells
Creatine Kinase (CK)/
Creatine Phosphokinase
(CPK)
transferase enzyme that catalyzes
the transfer of PO4 group betw. substrates
Kinase
(CPK – inappropriate term)
high energy
reservoir in muscle cells; utilized by muscle
cells to form waste product - creatinine
Creatine phosphate
originates in the liver from the amino acid arginine, glycine and methionine
Creatine
amino acids where creatine originates
methionine
arginine
glycine
formed from muscle metabolism; excreted in the nephrons of the kidney at a constant rate
Creatinine
T/F
From the liver, creatine is transferred to the
muscles. In the muscles, creatine is converted
to creatine phosphate through the action of
the enzyme CK
T
substrates used in CK
creatine + ATP
products formed in CK
creatine phosphate + ADP
T/F
CK is widely distributed
T
CK has inc. activities in these tissues
- skeletal muscle
- heart
- brain
First cardiac enzyme to elevate after AMI
CK2 (CK-MB)
(>6% of total CK)
Percentage of CK-MM
94-98%
Percentage of CK-MB
2-6%
Percentage of CK-BB
<1%
Reference range of Total CK in MALE
15-160 U/L
(higher than female due to inc. activity & muscle mass)
Reference range of Total CK in FEMALE
15-130 U/L
Reference range of CK-MB
<6% of total CK
Dimeric enzyme
CK
M - muscle, B - brain
CK isoenzymes
CK 1 (CK-BB)
CK 2 (CK-MB)
CK 3 (CK-MM)
Macro-CK
CK-Mi
CK isoenzyme that migrate fastest toward anode (most anodal)
CK1
CK-BB
Brain type
CK isoenzyme: 2nd fastest to migrate toward anode
CK2
CK-MB
Hybrid type
CK isoenzyme that migrate slowest toward anode (least anodal)
CK3
CK-MM
Muscle type
CK1 (CK-BB) - Brain type has inc. concentration in these sites
CNS
GI tract
Uterus (pregnancy)
Large CK molecule – cannot pass BBB
CK1
CK-BB
Brain type
CK isoenzyme ↑ in heart tissue
(cardiac muscle specific)
CK2
CK-MB
Hybrid type
↑ CK-MB denotes?
AMI
Major isoenzyme in striated muscles and normal serum
CK3
CK-MM
Muscle type
Macro-CK composition
CK-BB + antibodies (IgG/IgA)
CK-MM + lipoproteins
Migrate midway betw. CK-MM and CK-MB
Macro-CK
Migrates cathodal to CK-MM
Mitochondrial CK (CK-Mi)
CK isoenzyme located in mitochondrial membrane; used to diagnose cell necrosis/severe damage
Mitochondrial CK (CK-Mi)
Assay for CK
TANZER-GILVARG (FORWARD)
OLIVER-ROSALKI (REVERSE)
Measured in TANZER-GILVARG (FORWARD)
↓ in absorbance at 340 nm
Optimum pH in TANZER-GILVARG (FORWARD
9.0
Measured in OLIVER-ROSALKI (REVERSE)
↑ in absorbance at 340 nm
Optimum pH in OLIVER-ROSALKI (REVERSE)
6.8
SOURCES OF ERROR for CK measurement
Hemolysis (false inc.)
Physical activity and IM injections (false inc.)
Photosensitive (false dec.)
Immobilized/bedridden (false dec.)
Abundant enzyme inside the RBC that mimics the activity of CK causing hemolysis as a source of error
Adenylate kinase
Catalyzes interconversion of lactic and pyruvic acids
Lactate Dehydrogenase (LDH)
T/F
LDH is tissue specific
False
*Widely distributed
This may affect LDH activity
Storage
↓: frozen/low temp.
Maintained: RT for 2 days
Substrate for LD
Lactate + NAD
Products of LD
Pyruvate + NADH + H+
Inc. activities of LD are found in:
- Heart
- Liver
- Skeletal muscle
- RBC
Late cardiac marker (not practical for AMI dx)
LDH
SOLE PURPOSE: Monitor px response to therapy for cardiac diseases (i.e. AMI)
LDH
LDH reference range
100-225 U/L
Tetramer enzyme
LDH
(4 subunits/monomers of two active sub-unit forms [H & M]) H - heart, M - muscle
Tissue source of LDH 1
Heart,
RBC
Tissue source of LDH 2
Heart,
RBC
Tissue source of LDH 3
Lung,
Spleen,
Pancreas
Tissue source of LDH 4
Liver
Tissue source of LDH 5
Skeletal muscle,
Liver
Disorder associated with LDH 1
AMI,
Hemolytic anemia
Disorder associated with LDH 2
Renal Infarction,
Megaloblastic anemia
Disorder associated with LDH 3
Pulmonary embolism
Disorder associated with LDH 4
Hepatic injury
Disorder associated with LDH 5
Muscle dystrophy
Hepatic disorders
percentage of LDH 1 in total LDH
20-30%
percentage of LDH 2 in total LDH
30-40%
percentage of LDH 3 in total LDH
20-25%
percentage of LDH 4 in total LDH
7-15%
percentage of LDH 5 in total LDH
5-15%
LDH isoenzyme NOT normally seen in healthy people
LDH 6 / Alcohol dehydrogenase
LDH 6 / Alcohol dehydrogenase is present in these conditions
Drug hepatotoxicity
Obstructive jaundice
Atherosclerotic failure
has high affinity to α-hydroxybutyrate
LDH 1
LDH Normal Electrophoretic migration pattern
LDH 1>2>3>4>5
LDH Relative conc. in normal serum
LDH 2>1>3>4>5
LDH pattern in AMI/IV hemolysis
LDH 1>2>3>4>5
(FLIPPED PATTERN)
Used to differentiate AMI from IV hemolysis with flipped pattern
LDH 1
CK-MB
Differentiate AMI from IV hemolysis
AMI: high LDH1, high CK-MB
IV hemolysis: high LDH1, normal CK-MB
Assays for LDH
WACKER METHOD (FORWARD)
WROBLEUSKI LA DUE (REVERSE)
α-hydroxybutyrate dehydrogenase (α-HBD)
Assay: Lactate —> Pyruvate
WACKER METHOD (FORWARD)
Assay: Pyruvate —> Lactate
WROBLEUSKI LA DUE (REVERSE)
Measured in WACKER METHOD (FORWARD)
↑ in absorbance at 340 nm
Optimal pH in WACKER METHOD (FORWARD)
8.3 – 8.9
Commonly used LDH measurement due to:
o Production of positive rate
o Not affected by product inhibition
WACKER METHOD (FORWARD)
Measured in WROBLEUSKI LA DUE (REVERSE)
↓ in absorbance at 340 nm
Optimal pH in WROBLEUSKI LA DUE (REVERSE)
7.1 – 7.4
3x faster but more susceptible to substrate exhaustion
WROBLEUSKI LA DUE (REVERSE) for LDH
Has greater affinity of H subunit
α-hydroxybutyrate dehydrogenase (α-HBD)
α-hydroxybutyrate dehydrogenase (α-HBD) represent this LDH isoenzyme
LDH 1
(only isoenzyme with complete 4 subunits)
CK-MB (>6%) in AMI
Appearance (rise):
Peak:
Normalize:
Appearance (rise): 4-8 hours (EARLIEST)
Peak: 12-24 hours
Normalize: 3 days
AST in AMI
Appearance (rise):
Peak:
Normalize:
Appearance (rise): 6-8 hours
Peak: 24 hours
Normalize: 5 days
LDH in AMI
Appearance (rise):
Peak:
Normalize:
Appearance (rise): 10-24 hours (late to rise)
Peak: 48-72 hours
Normalize: 10 days (longest to persist)
Normalize after 10 days
Secreted by the acinar cells of the pancreas, useful in the digestion process
PANCREATIC ENZYMES
Catalyzes breakdown of starch and glycogen via α, 1-6 branching linkages
Amylase (AMS)
First enzyme to elevate in acute pancreatitis but nonspecific
Amylase (AMS)
Smallest enzyme in terms of molecular weight
Amylase (AMS)
Reaction eq. of AMS
CHO –(AMS)–> Maltodextrins
Major source of AMS
- Pancreas (acinar cells)
- Salivary gland
Minor source of AMS
- Fallopian tube
- Adipose tissues
- Small intestine
- Skeletal muscle
AMS is inc. in these conditions
- Acute pancreatitis
- Parotitis
- Renal failure
- Macroamylasemia
Earliest nonspecific marker of acute pancreatitis
Amylase (AMS)
Inflammation of carotid gland (viral infections), AMS hypersecretion in serum
Parotitis
T/F
AMS is normally filtered in glomerulus due to its low mol. weight; normally present in urine. During renal failure (altered kidney filtration) → AMS cannot pass through → reabsorbed → returned back to serum.
T
AMS + Ab/Ig; not filtered by glomerulus; reabsorbed
Macroamylasemia
aka Salivary Amylase
Ptyalin
fast moving; more anodal AMS isoenzyme (lower conc. in serum)
Ptyalin (salivary amylase)
slow moving AMS isoenzyme (highest conc. in serum)
Amylopsin (Pancreatic amylase)
aka Pancreatic amylase
amylopsin
AMYLASE METHODOLOGIES
AMYLOCLASTIC
SACCHAROGENIC
CHROMOGENIC
CONTINUOUS MONITORING
Measures disappearance of starch substrate
AMYLOCLASTIC
Indicator used in AMYLOCLASTIC method for AMS
Iodine (only react with polysaccharide)
Color of Starch-Iodine complex
dark-blue
Color of Glycogen-Iodine complex
mahogany-brown (a substitute for starch)
T/F
Decrease in color intensity is due to the conversion by AMS of polysaccharide substrates to simpler form (Thus, absorbance measured is also decreased)
T
(amyloclastic method)
T/F
In AMYLOCLASTIC method for amylase,
AMS activity = Absorbance
F
AMS activity ∝ Absorbance
Measured in this method is the product appearance (liberated reducing sugar)
SACCHAROGENIC
Starch → reducing sugars
T/F
In SACCHAROGENIC method for amylase,
AMS = Reducing sugar formed
T
Measures the increasing color from production of product acted by AMYLASE
CHROMOGENIC
(uses chromogenic dye fragment)
T/F
In CHROMOGENIC method for amylase,
AMS activity = soluble starch-dye fragment formed
T
Coupling of several enzyme to monitor AMS activity
CONTINUOUS
MONITORING
Measured in CONTINUOUS MONITORING for amylase
↑ in absorbance at 340 nm
Several enzymes used in cont. monitoring for ams
AMS
a-glucosidase
HK
G6PD
Target of LPS
ester bonds (present in lipids/fats)
Hydrolyzes ester linkages of fats to produce alcohols and fatty acids
Lipase (LPS)
Hydrolysis of dietary TAG in the intestine to 2-monoglyceride and fatty acids (enhances fat absorption)
Lipase (LPS)
Larger molecule of pancreatic enzyme
Lipase
Pancreatic enzyme that remains longer in circulation
Lipase
Reaction eq. of LPS
Triacylglycerol + 2H2O —LPS—> 2-monoglyceride + 2 FA
Only source of LPS
Pancreas (acinar cells)
Early and specific marker of acute pancreatitis
Lipase
(but not as fast as AMS)
Assays for lipase
CHERRY CRANDALL
TIETZ
Turbidimetric methods
Substrate in Cherry Crandall
50% olive oil (triolein – a purer form of fat reagent)
Substrate in Tietz
50% olive oil (triolein – a purer form of fat reagent)
Titrating agent in Cherry Crandall
0.4N NaOH (fatty acid titration is done)
Titrating agent in Tietz
0.4N NaOH (fatty acid titration is done)
Indicator and end color in Cherry Crandall
Phenolphthalein
Pink
Indicator and end color in Tietz
Thymolphthalein + Veronal
Blue
End point of both Cherry Crandall and Tietz
Fatty Acid (Oleic Acid)
Estimation of liberated fatty acids by measuring the amount of light blocked by the insoluble particles in the sample
Turbidimetric methods for Lipase
Reagent used in turbidimetric methods for LPS
TAG (hydrophobic, nonpolar, insoluble – turbid soln)
T/F
When LPS act
on TAG, it will be converted to a more polar, soluble – clear soln)
**Measured absorbance is DECREASED (inversely proportional)
T
AMYLASE in acute pancreatitis
Rise:
Peak:
Persists:
Rise: 2-12 hours (earliest)
Peak: After 24 hours
Persists: 3-5 days
LIPASE in acute pancreatitis
Rise:
Peak:
Persists:
Rise: 6 hours
Peak: After 24 hours
Persists: 7 days (longest to persist)
