CLINICAL CHEMISTRY Flashcards
Functions of Enzymes
1)Hydration of Carbon Dioxide (respiration)
2)Nerve induction
3)Muscle contraction
4)Nutrient degradation (digestion)
5)Growth and Reproduction
6)Energy storage and use
Aspartate Aminotransferase (AST)
A.K.A
Serum Glutamic Oxaloacetic Transaminase (SGOT)
enzyme not specific to the liver, widely distributed, highest activities in cardiac, liver, and skeletal muscles
AST
most abundant isoenzyme of AST
Cytoplasmic
Increased AST isoenzyme that indicates cell necrosis
Mitochondrial
AST reaction:
Aspartate + a-ketoglutarate ⇌
Oxaloacetate + Glutamate
After MI, AST begins to rise in
6-8 hours
AST level peaks at ____ after MI
24 hours
AST returns to normal state ___ after MI
5 days
AST is increased in
Hepatocellular and Skeletal muscle disease
Chronic disease of the liver with progressive damage
Cofactor tightly bound to an enzyme
Prosthetic group
Oxidized form of NAD measures:
decreased absorbance
Reduced form of NADH measures
Increase absorbance
Assay for AST that uses malate dehydrogenase and monitors absorbance at 340 nm
Karmen method
Reference range for AST
5-30 u/L
Hemolyzed samples in AST results
Falsely increase
Karmen Method:
Oxaloacetate + NADH+H ⇌ (MD) ⇌
end product
malate + NAD+ (oxidized form)
Serum glutamic-pyruvic transaminase (SGPT)
AKA
Alanine Aminotransferase (ALT)
Liver specific enzymes
ALT
Increased in acute inflammatory conditions of the liver
Used to monitor the course of hepatitis treatment and the effects of drug therapy
Screening post transfusion hepatitis
Screen blood donors
Markedly increased in jaundice
Used as test for occupational toxic exposure
ALT
ALT reaction:
Alanine + a-ketoglutarate ⇌
pyruvate + glutamate
shows the result of the AST and ALT measurements
De Ritis Ratio
De Ritis Ratio:
Ratio > 1
AST> ALT
Non-viral origin
De Ritis Ratio:
Ratio < 1
AST < ALT
Viral origin
Assay for ALT uses
Lactate dehydrogenase
Assay for ALT end product
Alanine + a-ketoglutarate ⇌ pyruvate + glutamate
Pyruvate + NADH + H ⇌
Lactate + NAD+
Methods to Detect AST and ALT
Reitman and Frankel
Major organ affected in AST/SGOT
Heart
Major organ affected in SGPT/ALT
Liver
Substrate for SGOT/AST
Aspartic alphaketoglutaric acid
Substrate for SGPT/ALT
Alanine Alphaketoglutaric acid
End products for SGOT/AST
Glutamic acid + Oxaloacetic acid
End products for SGPT/ALT
Pyruvic acid + glutamic acid
Color developer for SGPT and SGOT
2,4 DNPH
Color intensifier for ALT/AST
0.4 N NaOh
Used for the diagnosis of hepatobiliary disorders and chronic alcoholism, obstructive jaundice
GGT
GGT is located in the canaliculi of the hepatic cells specifically in
epithelial lining of the biliary ductules
Sensitive marker for ethanol intoxication, occult alcoholism
GGT
Glutathione + amino acid ⇌
End product
glutamyl-peptide + L-cysteinylglycine
Szaz assay for GGT:
absorbs ____ and is measured at 405-430 nm
p-nitroaniline
End product of Szas Assay:
y-glutamyl-p-nitroanilide + glycerine → GGTP →
y-glutamyl-glycylglycine + p-nitroaniline
Alkaline Phosphatase
AKA
Alkaline orthophosphoric monoester phosphohydrolase
Liberates inorganic phosphate from an organic phosphate ester with production of alcohol
Alkaline phosphatase
Reference range of ALP in adult
30-90 u/L (adult)
Reference range of ALP in 0-3 months
70-220 u/L
Reference range of ALP in 3-10 years
50-260 u/L
Reference range of ALP in 10 years to puberty age
60-295 u/L
Source of ALP
Liver, Bone (osteoblast), placenta, intestine, and renal tissues
In vivo activity of ALP requires — activator (example of cofactor that enhances enzyme activity by altering the spatial configuration of the active site of the enzyme for substrate finding)
Mg2+
Useful for hepatobiliary disease and bone marrow disorders
ALP
ALP is significantly increased in
Paget’s disease or Osteitis deformans
Predominant isoenzyme of ALP
Bone and Liver
Assay for ALP based on molar absorptivity of p-nitrophenol
pH of 10.2
Bowers and McComb
End product of Bowers and McComb ALP
p-Nitrophenyl-phosphate ⇌ ALP ⇌
p-nitrophenol + phosphate ion
Color result for Bowers and McComb
yellow (405 nm)
Substrate for Bodansky, Shinowara, Jones, Reinheart - ALP
B-glycerophosphate
Substrate for Bessy, Lowry and Brock
p-nitrophenyl phosphate
Bowers and McComb substrate
p-nitrophenyl phosphate
King and Armstrong substrate
Phenyl phosphate
End product for bodansky, shinowara, jones, reinhart
ACP
Inorganic PO4 + glycerol
End product for bessy, lowry, and brock
p-nitrophenol (yellow)
King and Armstrong end product
phenol
Electrophoresis for ALP
Most anodal to Least Anodal
Liver > Bone > Placental > Intestinal
Fastest isoenzyme of ALP in electrophoresis
Liver
Heat labile fraction of ALP
Bone
Bone ALP is increased in
bone disease
healing of bone fractures
physiologic bone growth
Most heat stable fraction in ALP
Placental
Physiologic increase of ALP
Increased in 16th or 28th week of pregnancy
Pathologic increase in placental ALP
Malignancy or cancer (carcinoplacental)
Placental ALP withstand heating at
65 degC for 30 mins
Slowest moving fraction in blood group B or O
Intestinal ALP
Increased in fatty meal consumption and GIT disorder (Isoenzyme)
Intestinal ALP
Placental and Intestinal ALP are inhibited by
phenylalanine
Inhibits Bone ALP
3M Urea
Inhibits bone and liver isoenzyme
Levamisole
Total ALP elevations by liver or Bone ALP is differentiated by heating of serum at
56 deg C for 10 minutes
Most stable to Most Labile in heat stability of ALP
Placental > Intestinal > Liver> Bone
ALP residual activity is ↓ to >20%
Liver ALP
ALP residual activity is ↓ to <20%
BONE ALP
Lung, Breast, and Gynecological cancers, bone ALP co-migrator, most heat stable ALP
Specific ALP that is most heat stable
REGAN ALP
Adenocarcinoma of the Pancreas and Bile duct, Pleural cancer
Nagao ALP
Inhibited by Phenylalanine reagent only
REGAN
Inhibited by both Phenylalanine and L-leucine. (Alp isoenzyme)
Nagao
Acid Phosphatase AKA
Acid Orthophosphoric Monoester phosphohydrolase
Phosphomonoester + H2O ⇌ ACP ⇌
End product
Alcohol + Phosphate ion
Catalyze the hydrolysis of various phosphomonoester at an acid pH
ACP
Liberate inorganic phosphate from an organic phosphate ester with production of alcohol
ACP
ACP is present in
Prostate, RBCs, Platelets, Bone (osteoclasts)
The activity of ACP in the seminal fluid is only demostrated for up to
4 days
inhibits specific prostatic ACP
L-tartrate ions
inhibits red cell ACP
Formaldehyde and Cupric ions
Reference Range: Prostatic ACP
0-3.5 ng/mL
Assay for ACP quantitative substrate
thymolpthalein monophosphate
Assay for ACP continuous substrate
a-napthyl phosphate
Assay for ACP gutman and gutman substrate
phenyl po4
Assay for ACP shinowara substrate
pnpp
Assay for ACP babson, read, and phillips substrate
alpha napthyl po4
Assay for ACP roy and hillman substrate
thymolphthalein mono PO4
End product of gutman and gutman
inorganic phosphate
End product of shinowara
p-nitrophenol
End product of babson, read, and phillips
ACP
alpha napthol
End product of roy and hillman
free thymolphthalein
Type of transferase enzyme that catalyze transfer of phosphate group between substrate
Creatine Kinase
Creatine kinase is widely distributed and has highest activities in
skeletal muscle
heart (main source)
brain
Creatine kinase end product:
creatine + atp ⇌ CK ⇌
creatine phosphate + ADP
Creatine originates in the liver from
arginine
glycine
methionine
Reference range of CK for Male
15-160 U/L
Reference range of CK for female
15 to 130 U/L
Forward reaction for CK
Tanzer-Gilvarg
Primary enzyme in Tanzer Gilvarg
CK
Tanzer Gilvarg reaction end product
Lactate + NAD
Tanzer Gilvarg reaction:
Creatine + ATP ⇌CK⇌ Creatine phosphate + ADP
ADP + phosphoenolpyruvate ⇌ PK⇌ pyruvate + ATP
Pyruvate + NADH + H ⇌ LD⇌
Lactate + NAD
Reverse Reaction for CK
Oliver Rosalki
Increase absorbance at 340 nm for CK determination
Oliver Rosalki
Decrease in absorbance at 340 nm for CK determination
Tanzer Gilvarg
Secondary enzyme for Oliver Rosalki
HK and G6PD
Secondary enzyme for Tanzer Gilvarg
PK and LD
Hemolysis effect on CK
falsely increase
Enzyme that has the same activity with CK
Adenelyte kinase
inactivated by light (protect the tube from direct light exposure)
Creatine Kinase
Physical activity and IM injections cause in CK
increase
Bed ridden patients: CK activity is
decreased
CK:
Slowest mobility toward the anode
Major isoenzyme in striated muscle and normal serum
CK-3/CK-MM/ Muscle type
CK
Hybrid type (linked with acute myocardial infarction)
2nd fastest to migrate toward the anode
Significant quantities are found in heart tissues
CK-2/CK-MB
CK
Migrate fastest towards the anode
Highest concentration in CNS, GI tract and uterus (pregnancy)
A large molecule that is not allowed to pass the blood brain barrier
CK-1/CK-BB/Brain type
Healthy human serum CK-1 level
less than 1%
After MI, CK-MB (>6%) levels begin to rise within
4-8 hours
CK-MB peaks at _____ after MI
12-24 hours
CK-MB returns to normal levels at
48-72 hours
First enzyme to elevate during acute MI
CK-MB
CK-MM reference value
94-98%
CK-MB reference level
2-6%
CK that complex with antibodies IgG/IgA
CK-BB
CK complex with lipoproteins (Macro-CK)
CK-MM
CK
Located midway between CK-MM and CK-MB
Macro-CK
Increase level of indicates cell necrosis
Migrates cathodal to CK-MM
Mitochondrial CK-Mi
Oxidoreductase enzyme that catalyzes interconversion of lactic and pyruvic acids
Lactate dehydrogenase
Widely distributed, highest level activities in heart, hepatic, skeletal muscle and RBC
Lactate dehydrogenase
Storage of the sample could affect the activity of LD
When the serum/plasma is frozen this could
decrease the activity of LD
The activity of LD is maintained when the serum and plasma is stored at room temperature up to
2 days
Assay for Lactate Dehydrogenase: forward reaction
Wacker Method
LD assay:
Increase in absorbance is monitored at 340 nm
Optimal pH is ____
8.3-8.9
Commonly used method for LD measurement
Wacker Method
Wacker Method end product:
Lactate + NAD ⇌ LD ⇌
Pyruvate + NADH + H
Reverse reaction for Lactate dehydrogenase
Wrobleuski La Due
LD:
Decrease in absorbance is monitored at 340 nm
Three times faster but more susceptible to substrate exhaustion
Wrobleuski La Due
Optimal pH for Wrobleuski La Due
7.1 to 7.4
end product for wrobleuski la due
Pyruvate + NADH ⇌
Lactate + NAD
Has greater affinity of H subunits
Can represent LDH-1 (the only isoenzyme that has 4H; HHHH)
a-hydroxybutyrate dehydrogenase
a-hydroxybutyrate dehydrogenase end product:
a-ketobutyrate + NADH + H ⇌ a-HBD⇌
a-hydroxybutyrate
after acute MI, LD begins to rise within
10 - 24 hours
LD peaks at
48-72 hours
LD remains elevated for
10 days
Reference range of A-hydroxybutyrate dehydrogenase
100-225 u/L
Markers for acute MI
CK-MB, Troponin I, CRP
Last enzyme to persist in the serum of MI patient
can be used to monitor patients response to therapy
LDH
LDH found in HEART AND RBC
LDF-1 (HHHH) and 2 (HHHM)
LDH 1 is increased in
MI, Hemolytic anemia
LDH 2 (HHHM) is increased in
RI, Megaloblastic anemia
LDH 3 (HHMM) tissue source
Spleen, Pancreas, Lung
LDH 4 (HMMM) tissue source
Liver
LDH 5 (MMMM) tissue source
Skeletal muscle
HHMM is increased in
pulmonary embolism
HMMM is increased in
Hepatic injury
MMMM is increased in
skeletal muscle injury
LD concentration in healthy human serum
2, 1, 3, 4, 5
HHHM, HHHH, HHMM, HMMM, MMMM
Acute MI and intravascular hemolysis concentration of LD
1,2,3,4,5 (FLIPPED PATTERN)
LDH 6 is demonstrated in
Obstructive jaundice
Atherosclerotic failure
Hepatotoxicity
↑ LDH-1 and ↑ CKMB results
possible AMI
↑ LDH 1 and normal CKMB results
Hemolytic conditions
CK MB appears —- after MI within
4-8 hours
AST appears —- after MI
6-8 hours
LDH appears within
10-24 hours
CKMB peaks at
12-24 hours
AST peaks at
24 hours
LDH peaks at
48-72 hours
CKMB stays elevated
3 days
AST stays elevated for
5 days
LDH stays elevated for
10 days
Amylase and Lipase are released from the
acinar cells of the pancreas
First enzyme to elevate in acute pancreatitis
Amylase
Amylase breakdown starch and glycogen via
a,1-6 branching linkages
Major tissue source of amylase
Pancreas, Salivary gland
Minor tissue source of amylase:
adipose tissues, fallopian tubes, small intestine, skeletal muscles
Amylase is increased in
acute pancreatitis, renal failure, and parotitis
Amylase is ____ in cases of renal failure
increased
amylase is increased after onset of acute pancreatitis in
2-12 hours
Amylase peaks at
24 hours
Amylase persists for
3-5 days
Salivary Amylase
(fast moving) (more anodal)
ptyalin
Pancreatic Amylase
amylopsin (slow moving)
Assay for Amylase:
Measures the disappearance of starch substrate
Amyloclastic
Amyloclastic:
Starch-Iodine Complex (Dark-blue) →
decrease in color intensity
Amylase assay:
Measures the appearance of the product
Saccharogenic
Saccharogenic assay:
Starch →
reducing sugars
Assay for amylase:
directly proportional amylase activity with the reducing sugar
saccharogenic
Amylase assay:
Activity of amylase is inversely proportional with the absorbance
Amyloclastic method
Amylase methodologies:
measures the increasing color from production of product-chromogenic dye fragment
Chromogenic
Insoluble starch dye →
Chromogenic end point:
soluble starch-dye fragment
Coupling of several enzymes to monitor amylase activity
Continuous monitoring
Endpoint of continuous monitoring for amylase:
5,6,-phosphogluconolactone + 5 NADH
Secondary enzyme for amylase continuous monitoring
A-glucosidase, hexokinase, G6PD
not excreted but is reabsorbed in plasma; amylase with antibodies
macroamylase
Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids
Lipase
Specific to pancreatitis; early and specific marker for acute pancreatitis
Lipase
Hydrolysis of dietary triglycerides in the intestine to 2-monoglyceride and fatty acids
Lipase
Lipase increases after onset of acute pancreatitis in
6 hours
Lipase peaks at
24 hours
Lipase persists for
7 days
Assay for Lipase:
Substrate for Cherry Crandall and Tietz
50% olive oil (triolein)
Cherry Crandall and Tietz titrating agent
0.4N NaOH
Indicator for Cherry Crandall
Phenolpthalein
Indicator for Tietz
Thymolpthalein + Veronal
End point for cherry crandall and tietz
fatty acid (oleic acid)
End color cherry crandall
pink
end color for tietz
blue
Estimation of liberated fatty acids
Turbidimetric methods
Measurement of the amount of light blocked by particles in soluble agents in the sample
Turbidimetric
Reagent for turbidimetric methods
Triglyceride (hydrophobic; not polar, insoluble)
Turbidimetric methods
Triacylglycerol + 2H2O ⇌
End product:
2-monoglyceride + 2 fatty acids
A PHOSPHORIC MONOESTER HYDROLASE
Predominantly secreted in the liver
Marker for hepatobiliary disease
5’ NUCLEOTIDASE (5’N)
Reference value for 5’ Nucleotidase (5’N)
0-1.6 UNITS
INDEX OF PARENCHYMAL FUNCTION
USED TO MONITOR THE EFFECT OF MUSCLE
RELAXANTS (SUCCINYLCHOLINE) AFTER SURGERY
Secreted in the liver
Used as a marker for insecticide/pesticide poisoning (organophosphate)
CHOLINESTERASE/PSEUDOGHOLINESTERASE
CHOLINESTERASE/PSEUDOGHOLINESTERASE REFERENCE VALUE:
0.5-1.3 UNITS (PLASMA)
a.k.a PEPTIDYLDIPEPTIDASE A OR KININASE II
ANGIOTENSIN-CONVERTING ENZYME (ACE)
Increased in angiotensin-converting enzyme (ACE)
Sarcoidosis, Acute and Chronic Bronchitis, and Leprosy
Main source of angiotensin-converting enzyme
Macrophage And epithelioid cells (specifically in the lungs)
important for vasoconstriction of the renal arterioles to increase blood pressure
Stimulates adrenal cortex to release aldosterone for reabsorption of sodium
Angiotensin II
Used as a possible indicator of neuronal dysfunction
Angiotenstin Converting Enzyme
Copper-Carrying protein which acts as an enzyme
Ceruloplasmin
Ceruloplasmin is a marker for
Wilson’s disease
Other enzyme marker for hepatobiliary diseases
Ornithine Carbamoyl Transferase (OCT)
Ornithine Carbamoyl Transferase (OCT) reference values
8-20 mU/mL
It functions to maintain NADPH in the reduced form in the erythrocytes
G6PD
responsible for maintaining and stabilizing the membrane integrity of erythrocytes
Reduced NAD(PH)
G6PD is found in
adrenal cortex
spleen
RBC
Lymph nodes
G6PD Deficiency can lead to —- drug-induced hemolytic anemia
primaquine (antimalarial drug)
Increased levels of G6PD
Myocardial infarction, megaloblastic anemia
Specimen for G6PD
Red cell Hemolysate, Serum
Reference values for G6PD
10 — 15 U/g Hgb or 1200 - 2000 mU/mL packed RBC
