Clinical Enzymology Flashcards
Enzyme activity in plasma/serum in normal healthy individuals (with steady cell turnover) represents what?
balance between its rate of liberation into the extracellular (E.C.) space and its rate of clearance/uptake from E.C. space (+ auto degradation of enzyme).
• General cases in which enzymes in plasma are elevated:
- Cell proliferation: There is an increase in cell turnover.
- Non-proliferative increase in the rate of cell turnover
- Cell damage
Enzymes are mainly influenced by:
pH (aminio acid sequence does not change with pH but protein’s shape may change)
Temperature (directly proportional with rate of rxn; very high > 37C =denaturation)
Characteristics of enzymes
• very efficient
• all are proteins (charge and conformation may be changed; (-) = slower)
specific to substrates (due to receptors on membranes)
• partly specific to tissues
• Assay by measure of rate of specific reaction catalyzed by
that enzyme
This model shows the concentration of substrate when the reaction velocity is equal to one half of the maximal velocity for the reaction
Michaelis menten model
*reaction slows down and plateaus after vmax/2
measure of how well a substrate complexes with a given enzyme
binding affinity
ATP’s role in elevated enzyme activities*
*??
maintenance of cell permeability (for retaining cytosolic enzyme within cells)
*In the absence of added ATP to medium containing cells, the rate of escape of intracellular enzyme is strictly related to the rate of depletion of intracellular ATP.
Factors which may affect elevated enzymes:
Anoxia
Hypoxia
Drugs/Poison
Lipid peroxidation
Factors which may affect elevated enzymes: How does anoxia affect elevated enzymes?
It may allow escape of intracellular enzyme from cells even in the absence of organ damage such as in sever cardiac/respiratory disease
Factors which may affect elevated enzymes: When does hypoxia happen?
during intense exercise or etc. (may allow transient escape of enzymes from muscle cells)
Factors which may affect elevated enzymes: examples of drugs which inhibit energy-yielding processes
chlorpromazine and promethazine (exert direct effect on cell membrane to accelerate enzyme release)
Poison (?) which causes increase in serum enzyme activities
bacterial toxins
Factors which may affect elevated enzymes: Role of electrolytes and osmotically active molecules
maintain cell integrity
This factor which affects elevated enzymes cases serum enzy elevations to be non-specific
drugs/poison
Factors which may affect elevated enzymes: Example of indices related to cell necrosis when patient is with drugs(??)
Mitochondrial enzymes (only liberated during cell necrosis e.g. Glu-DH, AST (exist in mitochondrial form, distinct from cytosolic form)
This affects release of membrane-associated enzymes
Lipid peroxidation
[Lipid Peroxidation: Hepatic (microsomal) enzyme induction] These are elevated in epileptic patients taking anti-convulsants
ALP, GGT, 5-nucleotdase
[Lipid Peroxidation: Hepatic (microsomal) enzyme induction] When do bile acids promote liberation of enzymes from damaged hepatic cell?
in biliary obstruction
[Lipid Peroxidation: Hepatic (microsomal) enzyme induction] The enzyme rate of clearance and elmination is measured through half lives. what are the half-lives of AST, ALT and CK
ALT - 6.3 days
AST - 2.0 days
CK - 1.4 days
Different mechanisms of enzyme clearance
• Amylase and lipase
- Excreted in urine (detection of urinary amylase is essential in acute pancreatitis)
• ALP excreted partly in the bile.
• Reticuloendothelial system clears some enzymes.
• Proteolytic degradation.
• Non-biologic decay (same as where serum specimens are not kept refrigerated).
• Conjugation of enzyme with Abs (enzyme loses its
biological potency) e.g. CK/LDH
Deals with the enzymatic reaction from the different parts or tissues of the body as it measures enzyme activity for the diagnosis and treatment of diseases (i.e.: calcium cytotoxic injury)
Diagnostic enzymology
unit for enzyme
IU: μmol of substrate transformed per minute under standard conditions.
• New unit: Katal: amount of activity that convert one mole of substrate/second (1 Katal = 6x107 IU)
- (amount of enzymes that catalyzes the conversion of one micromole of substrate to product per minute)
Why are there low concenctrations of enzymes in blood?
enzymes are normally intracellular (endoenzyme)
*detection only through tissue destruction (release from intracellular to extra)
examples of extracellular enzymes
amylase and lipase in digestive tract
T or F: enzyme levels are high when degree cell damage is high
true
This characteristic of enzymes is not absolute in spite of same gene content
Organ specificity
T or F: Cancer lowers enzyme activity; old people have more enzymes associated with bones
Cancer with higher ea; old people lesser amts in bones
What can be known from enzyme measurements in serum?
- Presence of disease
- Organs involved
- Etiology / nature of disease: differential diagnosis
- Extent of disease – more damaged cells – more leaked enzymes in blood
- Time course of disease
Two groups of enzyme assays according to their sampling method
Continuous assays (cont. reading) (Chemiluminiscence, Spectrophotometer, Fluorometric, Calorimetric) Discontinuous assays (Chromatography and Radiometric)
Assays used in measuring enzymes
end point
kinetic
one in which second enzyme is used to act on the product of the enzyme of primary interest.
*Second enzyme used NADH as coenzyme and rate is followed by measuring oxidation of NADH at 340 nm.
coupled assay
This enzyme is present throughout all body tissues but with high concentrations in: • Heart • Skeletal muscle • Liver • Kidney • Brain • RBCs
Lactate dehydrogenas
- non-specific index of cell damage
- used in conversion of pyruvate to lactate
What causes artefactual increase activity of LDH?
hemolysis or delayed separation of plasm due to LDH presence in RBCs
When is LDH elevated
myocardial infarctions (LD1), blood disorders
two types of subunits of LDH (a tetrameric protein)
Heart (H)- rich in LD1* and LD2 and Skeletal muscle (M)
*MI: LD1 > LD2
normal serum: LD2 > LD1
5 different isoenzymes of LDH
LD1(H4)- moves fastest to (+), LD2(H3M), LD3(H2M2), LD4(H1M3), LD5(M4)***(slowest)
- present in myocardium, RBC
- *present in kidney, skeletal muscle
- **present in skeletal muscle, liver (elevated in disorders re this)
High serum LDH is associated with what?
widespread metastases esp to liver
*LD4 and LD5 are predominant
What are liberated directly from circulating RBCs in hemolytic anemia?
LD1 and LD2
In myocardial infarctions, when does elevation commence?
elevation, >5x normal, commences 12-18 hrs after onset of symptoms and peaks on third day declining until reaching normality by 10th day
When is LDH assay more valuable in myocardial infarctions?
case presenting several days after suspected MI
Other diseases with elevation of LDH
- Hepatobiliary disease (increased serum LDH in acute liver necrosis: LD5- predominant)
- Blood disease (megaloblastic anemia and leukemia: >5x LD1 and LD2 due to red nucleated cells in BM; hemolytic anemias: LD1 and LD2 liberated from circulating RBCs)
- Cancer (LD4, LD5- predominant)
This serves as energy reserve during muscle contraction
Phosphocreatinine
*uses creatinine kinase (dimer) occuring in tissues
Skeletal muscle and brain contains these subunits of creatinine kinase
M subunit (slow mving)
B subunits (BB/CK-1 = moves rapidly towards (+))
*=3 diff isoenzymes
*2% of CK in normal serum and 20-40% found in heart: MB-type
*others: X, Y, Z
physiological increases of CK is due to:
- Males > Females
- Increase with age and body weight
- Neonates have high CK but fall rapidly during the first weeks
- Physical exercise
- Labor and parturition
CK present in acute myocardial infarction
CK-2 (composed of MB)
T or F: CK-3 (MM) is also present in myocardium
true
Creatinine Kinase is the most uesful test in establishing/refuting
myocardial infarction
*increase in serum CK after 6 hrs of MI onset; peak @ 36 hrs, declining after day 3
What can cause a delay in return to normal value or cause secondary spikes in creatinine kinase?
Re-infarction, thromboembolism, and arrhythmias
[Muscle diseases assoc with CK] The CK elevated mainly is CK-MM with other enzymes elevated also such as aldolase, LDH, aminotransferases
Duchenne Muscular Dystrophy
[Muscle diseases assoc with CK] more benign form of Duchenne MD
Becker MD
[Muscle diseases assoc with CK] An example of this would be neurogenic atrophy and malignant hyperpyrexia
Secondary disease of muscles
*other muscle diseases: polymyositis
What are the cerebral diseases associated with CK?
• Epileptic seizure due to muscular activity
• Tetanus
• Organic neurologic disease
- E.g. Cerebral infarction, meningitis and cephalitis (Ck - BB).
Other conditions where in CK is elevated
alcohol intoxication and hypothyroidism
These conditions cause moderate increase in serum level of CK
- Prolonged chest pain with transient ST-segment and T-waves changes (40% of cases).
- Prolonged chest pain with E.C.G. abnormalities (20% of cases).
- Classic angina pectoris (occasionally)
Why does MB-type have a short duration of increase after infarct within few hours?
rapid clearance from vascular space
*increase in the amount of CK-MB determines the severity of M.I. and hence prognosis
products of L-aspartate and L-alanine:
L-glutamate and oxaloacetate
alanine: L-glutamate and pyruvate
These enzymes are the most abundantly present in liver and is elevated in blood because of leakage from damaged cells
alanine transaminase and aspartate transaminase
*useful for diagnosis of liver diseases
Where is ALT almost exclusively found?
*increase specific for LIVER DAMAGE involving hepatocellular damage
cytosol and in high concentration in hepatocyte
Where is AST found?
*moderately increased in MUSCLE DYSTROPHY
cytosol and mitochondria (abundant in liver, hear and SM)
T or F: In viral hepatitis, enzyme levels are increased 20-60x above upper limit of normal range
False, 20-50x
*rapid rise in AST and ALT in serum occurs before bilirubin rise (10-100x) elevation at prodromal > 10 days before icterus
When is AST (elevated >10x) usually detected after onset of MI?
6-12 hours after; peaking at 20-48 hrs after onset of symptoms; returning to ref value by 5th day (delayed fall = heart failure)
What is detected in very severe MI?
mitochondrial AST in serum (prognostic index)
ALT is moderately elevated in uncomplicated MI. When does it rise >10x the normal value?
in event of cardiac failure, severe shock or other complciations
Where does elevated ALT in uncomp. MI come from?
released from damaged hepatocytes due to tissue anoxia
*little diagnostic value in MI but useful in detecting complication
Type of chronic hepatitis wherein continuing elevation of serum ALT and AST is seen
Chronic Active hepatitis
Type of chronic hepatitis wherein elevation is higher than moderate and AST: ALT >2
Chronic Aggressive Hepatitis
less favorable prognosis
Type of chronic hepatitis wherein elevation is moderate and AST: ALT < 1
Chronic Persistent Hepatitis
What does icterus indicate n portal cirrhosis?
slightly raised ALT/AST
- normal if no icterus
- elevation rarely more than 3x normal (AST>ALT)
- normal ALP
Elevation of ALT and AST in biliary cirrhosis
moderately pronounce (up to 4x normal) increase (ALT=AST)
range of AST/ALT in hepatobiliary obstruction
2-8x normal (depending on icterus)
ALT:AST in obstruction caused by a non-malignant disease
ALT>AST (usually)
*AST>ALT in jaundice caused by tumor
T or F: ALT is the dominant aminotransferase in hepatic metastases
false, AST
major disadvantage of aminotransferases
lack of specificity
These are group of enzymes that have maximal activity at a high pH 9.0-10.5
Alkaline phosphatase (ALP)
*Low substrate specificity catalyzes hydrolysis of phosphate esters at alkaline pH.
What does all ALP contain at active site?
Zn and Ser
High levels of ALP are seen in
*can also be increased in children
liver,* bone, placenta, and intestine and useful to assess hepatobiliary and bone diseases
*increased in mother’s placental insufficiency, liver and bone carcinoma (requested), and intestinal problems
cellular location of ALP
Brush borders of proximal convoluted tubule of kidney and intestinal mucosa.
(Other membranes: Sinusoidal and Canalicular surfaces of hepatocytes. Its location in membranes plays
a role in absorption and transport processes)
role of ALP in bones
mineralization since its activity changes with osteoblastic function
Isoenzymes of ALP
- Placental and small intestinal mucosa: ALP forms under separate genetic control.
- Bone, liver and kidney: ALP is a product of the same gene locus but subject to post translational modification within each tissue.
T or F: Placental ALP is unaffected at 65˚ C, whereas all other ALP isozymes lose their activity totally
true
*@56C for 10 mins: intestinal alp loses 20% of activity; liver 60%; bone 80%
Least stable at urea denaturation (2M) of all aLP
Bone ALP
*most= placental ALP
Physiological causes for raised serum ALP in infancy
due to predominance of bone ALP until about the age of puberty (2-2 ½ x adult normal)
Physiological causes for raised serum ALP in puberty
- At puberty: up to 5-6 x than in adult age.
- In adults most ALP activity is due to liver isoenzyme.
- Intestinal ALP appears only in serum if a person is of blood groups O or A, secretors of ABH red cell antigens and are positive for Lewis antigen
Physiological causes for raised serum ALP in pregnancy
- Pregnancy: placental ALP appears in serum of pregnant women only during second and third trimester
- Sharp reduction in PALP often indicates placental insufficiency and death of fetus
In hepatobiliary disease, what induces ALP synthesis?
hepatocytes lining the biliary ducts
*very high levels in biliary obstruction vs intrahepatic obstruction
T or F: moderate levels of ALP are seen in parenchymal liver disease
true
*impt in differential diagnosis of hepatic and obstructive jaundice
hepatobiliary disease where serum bilirubin and ALP rise and fall in parallel
extrahepatic obstruction
hepatobiliary disease where there is little change in ALP even when bilirubin concentration is rising dramaticall but if icterus fails to clear and patient goes into cholestatic phase, ALP rises substantially
Acute Hepatic Necrosis
ALP level in biliary cirrhosis
high; even when bilirubin is normal
Effect of alcoholic fatty liver to ALP
raised
This isoenzyme which resembles placental ALP has been detected in 1%-3% of patients of carcinoma/lymphatous infiltration of liver
Regan
*especially in bronchial carcinoma
These bone diseases can cause very high ALP level
Primary bone tumors (e.g. osteogenic sarcoma) and secondary osteoblastic bone tumors* (e.g. those originating from prostate)
*slight to moderate increase of ALP
bone disease which is associated with vitamin D deficiency and leads to high ALP
osteomalacia and rickets
bone disease wherein paralysis in thoracolumbar vertebra leads to fracture and increase in ALP (high)
Paget’s disease
*also seen in throacolumbar tuberculosis
Other bone diseases which lead to high ALP
- Primary hyperparathyroidism with bone involvement (often associated)
- Secondary hyperparathyoidism as in renal ostesodystrophy (Dramatic increase in ALP)
- Healing fractures
Why is a high level of ALP (bone) found after gastrectomy in intestinal disease?
impaired absorption of Ca2+, PO4-, and vit. D
This component is used in ALP activity measurement as it is directly proportional to it
p-nitrophenol (quinonoid form)
T or F: ALP has a low degree of temperature tolerance
False, high (only other enzyme)
What can be done to estimate the activity of bone isoenzyme?
heat treating a serum sample at 56C
- bone ALP = heat labile and destroyed or heat inactivated at 56C
- placental ALP: @65-67C
A group of enzymes that have maximal activity at pH 5.0-6.0
acid phosphatase
*activity increases rapidly at room temp unless pH is reduced below 6.0
Where is acid phosphatase present?
• It is present in the prostate gland (main source of ACP), liver, spleen, and RBC
responsibility of acid phosphatase
hydrolysis of a wide
range of phosphate ester bonds at acidic pH.
predominant form of isoenzymes of ACP (exists in all body cells)
lysosomal
soluble form in cytosol
replaced the ACP as marker for prostate disease
Prostatic specific antigen
[ACP]
prostate and RBC enzyme are sensitive to?
P: L-tartrate (along with other tissue forms; detectable in females and in males after prostactectomy)
R: formaldehyde (ACP release is inhibited)
ACP is only used in the diagnosis of prostatic cancer when?
disease has spread to adjacent bone i.e.: pelvis
When can high ACP level be seen?
when it spreads to soft tissue
*If tumor remains as nodule / not extended beyond prostate capsule: only ½ of patients may show elevated serum activity of ACP.
(why ACP use as diagnostic and screening test is limited in prostatic cancer.)
What rarely causes transient elevation of ACP?
Prostatic massage
Why is raised serum ACP activity expected in some liver storage diseases such as Gaucher’s disease and Niemann-Pick disease?
enzyme location predominantly in lysosome
Other causes of raised serum ACP activity
- acute retention of urine and passage of catheter
- thromboembolic and myeloproliferative disorders
[ACP]
What happens when the prostate’s capsule is destroyed?
ACP steeply increases along with exponential increase in PSA
[ACP]
What happens when the prostate’s capsule is still intact
ACP will still be contained, but an increase in ACP can already be expected
(Same goes for PSA)
• Digestive enzymes from the pancreas and salivary glands (For digestion of complex carbohydrates) which is used as a marker to detect acute pancreatitis and appendicitis
Amylase
when is amylase elevated
acute pancreatitis
- Not commonly used anymore for diagnosis of appendicitis
- Can help differentiate ovarian problems from appendicitis in female patients complaining of right lower quadrant pain
- Increased: appendicitis
- Normal: possibly due to ovarian torsion or other ovarian problems
This is involved in amino acid transport across the membranes found mainly in the biliary ducts of the liver, kidney and pancreas
𝛾-glutamyltransferase
*induced by drugs and alcohol (used as a very specific marker of alcohol induced liver disease and in liver cirrhosis)
highly toxic drug which induces 𝛾-GT
phenothiazines
seizure med which induces 𝛾-GT
valproic acid
When is 𝛾-GT increased?
liver diseases and obstructive jaundice
*Obstructive jaundice due to gall stones causes clogged biliary ducts due to accumulation of bile juice in gall bladder increasing degradation thus increasing 𝛾-GT
What usually causes Normal 𝛾-GT levels in suspected cirrhosis?
possibly due to
presence of damage to liver only, not actual cirrhosis
