Blakes Diagnositc enzymology Flashcards
Alterations in Liver enzyme activities
1) Haptocellular damage: aminotransferase activity
2) Cholestasis: activities of alkaline phosphatase, 5’-nucleotidase, γ -glutamyltransferase
Cholestasis
is the suppression of normal bile flow
Amminotransferase has what Amino acids and coenzymes
2-oxo-acids
Pyrudoxal-5’-phosphate (P-5’-P)
Prydoxamine-5’-phosphate
Aspartate aminotransferase (AST)
Location
sources of error
what forms
- 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
Alanine aminotransferase
Location
is in what part of the cell
-Predominantly in liver and kidneys
-less amount in Striated muscle
-exclusively in the cytoplasm
In acute liver disease
ALT in greater then
AST and remains elevated longer
ALT ref diseases
alcoholic hepatitis, hepatic cirrhosis, liver neoplastic
AST reaction
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
ALT reaction
Alanin + α-KG with enzyme P-5-P and ALT —-> Pyruvate + Glu
Pyruvate + NADH + H+ with enzyme LDH —–> Lactate + NAD
Aminotransferase clinical signifance
Liver disease level
Thershold for diagnosing Acute liver Injury
Peak activity
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
Chronic hepatitis
ALT ( > 6 months) [Periodic measurement]
Toxic hepatitis V Infection hepatitis
Acetaminophen-induced hepatic injury:
The peak activity: 85 x URL (about 90% of case)
Infectious hepatitis: rarely seen this level of elevation
medication induced elevation of ALT and AST
NSAID, antibiotics, antiepileptic drug, statin, etc
Non-Alcohol Fatty Liver Disease: h ALT, AST
Metabolic syndrome
1) Higher body mass index, 2)increased waist circumstance, 3) ↑serum TG, 4) ↑ fasting insulin,
5) LDL/HDL cholesterol
Primary & metastatic malignancy of liver
AST>ALT; both enzymes 2-5 x h activity
Cf. Early stage: activity increases within normal range
AST/ALT ratio (AAR) greater than 1:
Presence of advanced fibrosis in patients with chronic liver diseases
(≈ 90% positive predictive value)
Alkaline Phosphatase (ALP)
Lipid transport in
lipid transport in the intestine and with the calcification process in bone
Genetic loci of ALP
Tissue nonspecific
Germ cell
Placental
Intestinal
Fetal Intestinal
ALP coenzymes
Co2+, Mg2+, Mn2+
ALP inhibitors
Phosphate, Borate, Oxalate, and cyanide ion
ALP hepatobiliary diasease
Serum enzyme activity could be increased to
Biliary tree obstruction–> blank–> blank
Extra hepatic obstruction blank vs intra hepatic activity
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
ALP clinical sig
Primary liver what or widespread what
mild increase or normal in what
Blank therapy could also increase blank
-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
Hypophosphatasia ALT
examples diseases
-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
Hypophosphatasia
ALP sig
Primary and secondary what
ALP is increased during what
Primary / secondary) hyperparathyroidism
Slight to moderate h
ALP activity is temporarily increased during healing from
the fracture
ALP reaction
-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
Gamma Glutamyltransferase trasfers what
Substrate
Transfer γ-glutamyl group from donor to an
acceptor
1) the γ-glutamyl
acceptor, 2) AA or peptide, 3) water.
GGT critical marker
Location
Hepatobiliary disease [critical marker]
Liver (hepatobiliary system), Kidney (PCT), Pancreas, & Intestine.
Cytoplasmic protein [Membrane Bound]
GGT intra and post what
5-15 URL increase
-: 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
GGT
Normal range
- 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
GGT reaction
γ-glutamyl-p-nitroanilide + glycylglycine with enzyme GGT—–> p-nitroaniline + γ-glutamyl-glycylglycine
GGT
P-nirtoaniline is measured at
p-nitroaniline (yellow) is measured spectophotometrically at 405 nm
γ-glutamyl-p-nitroanilide
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
GGT second reaction
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
GGT sample and storage
-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%]
Amylase P type
Hydrolysis of what
Synthesized by what
Where is S-type amylase
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
Amylase requires what cofactor
Ca2+ Metalloenzymes- Ca2+ is required to maintain structure of amylase.
P type amylase
Pancrease
S type amylase
Salivary gland specific
Amylase can be activated by
Chloride and bromide
Amylase breaks down
Polysaccaride
1-4a-glucosidic
Amylase is the only
Enzyme present in the urine.
GGT is used in measuring what organ
Pancreas for acute and chronic pancreatitis
Amylase is delivered through the pancreatic duct to the
Intestines ( GI tract)
Total amylase activity is not good because
Other parts of the body can make amylase such as the salivary glands, ovaries, fallopian tubes, lungs, adipose tissue, and salivary glands
Amylase in blood is mainly what two types
S and P types
How can P-type amylase be quantified
By using a p-type amylase antibody
ALP is increased in
Bone/liver/cancer conditions.
ALT has a longer
Half life therefore the ALT/AST ratio is over one
In chronic liver disease the ALT/AST ratio is
Flip and AST/ALT is over one.
ALP and GGT will differentiate between what
Liver and bone disease
both GGT and ALP will be elevated in liver disease
only ALP is elevated in bone disease
Enzyme use what units
U/L
dont use Mg/dl
Essential coFactors for AST and ALT
P-5-P
Pancreas specific enzymes
Amylase and Lipase
Stomach acid= painful
Test material=
Test for amylase
P-amylase use
Selective S-amylase monoclonal antibody
= so All S amylase is trapped in sample and the remaining P-Amylase is quantified
You can three forms of product of ESP reaction of amylase which are
2ED-GS + 2-4-NP-G2
2ED-G4 + 2-4 NP=G3
ED + 4-NP-G4
ED= Ethylidene
NP= nitrophenal
Further hydrolysis of amylase happens by
NSP amylase reaction
Glycosidase= further breakdown of sugar and NP
Test material
All anticoagulants inhibits
Amylase activity because of calcium binding
Exception is Heparin
what anticoagulant can be used with amylase
What other specimen can be used
Heparin because it doesn’t inhibit amylase activity= Heparinized plasma
you can also use serum as a specimen
Nitrophenols give off a huge
Color
If you dont have a disease of the pancreases the
Amylase has low physiological level.
There can be what amylase
Urine because the size of it is 54-62kD
Levels of amylase are not related to
Severity of conditions.
Total amylase activity goes up in
Pancreas and salivary gland in inflammation
Acute pancreatitis=
Amylase activity can return to normal in 3-4 days
Cholecystitis=
Bile duct obstruction
P-amy (4x increase URR)
Ethylidene is used to
Stabilize the NP-G2 complex for measurement
Nitrophenol is connected to sugar because phenol will give
Color once released from sugar
Amylase is stable
Stable 4 days at room temp, 2 weeks at -4 °C, 1 year at -25 °C, 5 years at -80 °C
Amylase is measured at what wavelength
405 nm
Substrate used in the amylase reaction
4-nitrophenyl (4-NP)-glycoside [Substrate]
2nd reaction in amylase reaction
2 4-NP-G2 + 2 4-NP-G3 + 10 H2O with enzyme Glycosidase —–> 4 4-NP + 10 G
Lipase
cofactors
highest levels in
in what else
LPS
Cofactors: bile salts & colipase Pancreas, gastric and intestinal mucosa
highest levels in pancreas
in the gastric, and intestinal mucosa, also
Lipase is a single
can it be detected in the urine
Single chain glycoprotein: 48 kD
100% filtered & 100% reabsorption
DOES NOT PHYSIOLOGICALLY DETECTED IN URINE
Acute pancreatitis level of LPS
LPS in Serum: acute pancreatitis
[2-50 x URL]
Serum LPS is 3X the upper in
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.
More sensitive then
LPS remains elevated longer then
With reduce GFR then
More sensitive than increase in AMY activity
➡ LPS remains elevated longer than AMY does
➡ With reduced GFR, serum LPS activity is
increased
Lipase analysis
-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
Acid phosphatase (ACP)
All phosphatase with optimal activity is under
acidic pH (< pH 7.0)
Acid phosphatase ( ACP)
acidic pH (< pH 7.0)
ACP has what enzyme activity
Lysosomal and extralysosomal enzyme
ACP greatest concentrations
1) prostate, 2) bone, 3)spleen, 4) platelet, 5) RBC
Low ACP activity of serum
tartrate-resistant type (TR-ACP, TRAP or
TRAPase)
mAb for TR-ACP
If you have prostate ACP elevated then it can be a marker for the
Prostate cancer
Lysosomal ACP is
Prostate ACP
Low decreased activity level in blood
Bone and RBC acid phosphatase
extra lysosomal enzymes
are not inhibited by tartrate
so called tartrate resistant Acid phosphatase
Lysosomal tartrate will be inhibited by
Tartrate aka Prostate
Acid phosphatase
add tartrate and measure
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)
Extra lysosomal ACP=
Bone or RBC
Bone ACP can be elevated in
Activities of this fraction (bone) are increased in growing children
conditions of increased osteolysis & bone remodeling
AKA: Paget, hyperparathyroidism, malignant invasion in bone
Lysosomal acid phosphatase is a marker for
Prostate cancer
ALP is measured the same way as
Measured the same way as ALP, but under ACIDIC conditions (pH = ~5)
Reference range: 0 – 3.5 ng/ml
Cholinesterase (CHE)
Hydrolyzes esters of
Metabolizes/ clearance of
Deactivation of
choline, e.g acetylcholine
Metabolize/clearance of drugs
deactivation of octanoyl ghrelin
Two main types of CHE
Actylcholinesterase
Pseudocholinesterase
Acetylcholinesterase
Acetylcholinesterase (true cholinesterase, RBC Che, cholinesterase I) RBC, Lung, Spleen, Nerve endings, Gray matter of the brain.
Pseudocholinesterase
Pseudocholinesterase (Acetylcholine acylhydrolase, Serum(/plasma) cholinesterase (CHE, SchE), cholinesterase II), current Butyylcholinesterase(BChe)
Liver, Pancreas, Heart, White matter of brain, Serum
Cholinesterase (CHE)
Liver function tests
Used for monitoring
Decreased in CHE activity: impaired synthesis of the enzyme by liver
-used for monitoring of liver function after “liver transplantation”
Cholinesterase is a indicator of
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”
CHE
For the detection of patients with atypical forms of the enzyme
Muscle relaxants: Succinyldicholine, mivacurium
- Hydrolyzed by CHE
Cholinesterase (CHE) analysis
Substate
Chromogenic agents
what do you measure spectrophotometrically at what wavelength
Stable for
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
Glucose-6-Phosphate Dehydrogenase
Enzyme is the first step in the
Leads to the production of
Enzyme is the first step in the pentose-phosphate shunt
Leads to the production of NADPH
Glucose-6-Phosphate Dehydrogenase
very important enzyme in the RBC to
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
Glucose-6-Phosphate Dehydrogenase
Hemolysis can lead to
Hemolysis can occur leading to hemolytic anemia
Glucose-6-Phosphate Dehydrogenase analysis
Glucose-6-phosphate + NADP+ ——————>
6-phosphogluconate + NADPH
NADPH is measured spectrophotometrically (340 nm).
“Erythrocyte hemosylate” is assayed for enzyme deficiency.
Elevated LDH
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
Elevated Cardiac enzymes (CK)
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
Troponins
- 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
Myoglobin
-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
Diagnosis of Acute myocardial infarction use what enzymes
LDH
Cardiac enzymes
Troponins
Myoglobin
