3 Enzymology Part 2

Clinical Chemistry 2 | Lecture

CREATINE KINASE OTHER DIAGNOSTIC SIGNIFICANCE
o Associated with ATP generation in contractile o AMI (Acute Myocardial Infarction)
system (muscular system) o Muscular Dystrophy-Duchenne Type – highest
o FUNCTION: in the muscle cells it stores Creatine concentration of CK-MB is seen; as high as 50-100x
phosphate that is important in ATP production the upper limit of CK
o Non-specific o CVA (stroke) & other brain conditions
Creatine phosphate + ADP ← → Creatine +ATP o Hypothyroidism, malignant hyperpyrexia, Reye’s
MAJOR TISSUE SOURCES: syndrome, Vibrio Vulnificus
1. Skeletal muscle ATYPICAL FORM OF CK (ABNORMAL ENZYME)
2. Heart muscle MACRO CK
3. Brain tissue o Found midway between MM AND MB
ISOENZYMES: dimer with two sub-units o Two theories:
CK-1 (CK-BB) → < 1% - brain type (most anodal) ▪ CK-BB is bound to IgG
CK-2 (CK-MB) → < 6% - hybrid type ▪ CK-MM is bound to lipoproteins
CK-3 (CK-MM) → 94-100% - muscle type (least anodal o No clinical significance except that it is related to age
but most cathodal) and sex; seen in female of >50 years old
ISOENZYMES MITOCHONDRIAL CK (CK-MI)
CK-MM o Found before the MM
o Major fraction in serum o Bound to the exterior surface of mitochondrial
o Elevated also in hypothyroidism, muscle activity, IM membrane of the Muscle, brain, & liver.
injection o indicator of severe illness, malignant tumors, and
▪ Hypothyroidism – increase membrane cardiac abnormalities
permeability, decrease CK clearance due to slow METHODS USED FOR THE MEASUREMENT OF
metabolism ISOENZYMES OF CK
▪ Muscle activity – due to vigorous exercise and o ELECTROPHORESIS – considered as reference
intramuscular injection method
o Found in skeletal muscles and cardiac muscles o ION EXCHANGE CHROMATOGRAPHY
CK-BB ▪ More sensitive and pricey
o Seldomly found in the plasma or circulation ▪ Problem with the bad column: CK-MM merge
▪ Short half-life:1-5 hours with CK-MB
▪ Have a high molecular size that is why it cannot ▪ CK-BB eluted with CK-MB
past through the blood brain barrier o ANTIBODIES – used specifically for CK-MB
o Confined in the brain due to the blood brain barrier determination and diagnosis of AMI
o increase only when there is a brain injury ▪ ANTI-M ANTIBODY inhibits all the activity of M
CK-MB subunit.
o Most important among the isoenzymes of CK ▪ To get the entire activity of the CK-MB, multiply
o 20% of cardiac tissue contains CK-MB, and very little the activity of CK-B into 2
to other tissues o IMMUNOASSAY
o Myocardium is the only tissue from which CK-MB ▪ Detects MB reliably with minimal reactivity
enters the serum, meaning it is quite specific to the ▪ Detects enzyme protein rather than activity
heart muscle METHODS FOR THE DETRMINATION OF CK
o Only myocardium can increase the level of CK-MB in TONSER-GILBERG ASSAY (9.0 AT 340NM)
the blood. Creatinine + ATP ←CK→ Creatine phosphate + ADP
o Indicator of myocardial damage (AMI or heart attack) (forward or direct reaction)
o RISE = 4-8hrs ADP + Phosphoenolpyruvate ←phosphokinase→
o PEAK = 12-24hrs pyruvate + ATP
o NORMALIZE = 48-72hrs Pyruvate + NADH + H ←lactate dehydrogenase→
▪ OTHER CARDIAC MARKERS: LDH, AST, troponin lactate + NAD
(not an enzyme but much specific and sensitive)
 OLIVER-ROSALKI ASSAY (6.8 AT 340NM) 3. SUBSTRATE AFFINITY
Creatinine phosphate + ADP ←CK→ Creatinine + ATP DIAGNOSTIC SIGNIFICANCE
(reverse or indirect method) o ELEVATED LEVEL: Renal, hepatic, cardiac, skeletal,
ATP + glucose ←hexokinase→ ADP + G6P hematologic, & neoplastic disorder
G6P + NADPH ←G6PD→ 6-phosphogluconate + NADPH o HIGHEST LEVEL: pernicious and hemolytic disorder
o When measuring CK, avoid hemolysis of RBC will o Serves as a cardiac marker
release adenylate kinase because it will give false AMI
elevated values. o RISE: 12-24 hours
o Storage o PEAK: 48-72 hours
▪ 4C: up to 7 days o NORMALIZE: after 10 days
▪ -20C: up to 1 month METHODS
o Reference value 1. WACKER METHOD (8.3-8.9 AT 340 NM)
▪ Male: 15-160 U/L ▪ Forward or direct reaction
▪ Female: 15-130 U/L 2. WROBLEUSKI AND LA DUE (7.1-7.4 AT 340NM)
▪ CK-MB: <6% ▪ Reverse or indirect reaction
LACTATE DEHYDROGENASE (LDH) ▪ 3 times faster, small sample is needed
o Catalyzes the interconversion of lactic acid and ▪ Susceptible to substrate exhaustion & loss of
pyruvic acid linearity
o Can convert lactic acid to pyruvic acid and vice-versa o Avoid hemolysis because RBC contains 100-150x the
Lactate + NAD ←LDH→ Pyruvate + NADH concentration of the LDH inside the red cells
o COENZYME: NAD o LDH is a cold labile enzyme (stored at room
ISOENZYMES temperature)
Tetrametric molecules containing 4 subunits of two o LD5 – most labile
possible forms; nonspecific REFERENCE VALUE: 100-225 U/L
I. LD1 – HHHH 14-26% - RBC and heart ASPARTATE AMINOTRANSFERASE (AST)
II. LD2 – HHHM 29 – 39% - RBC and heart o also called the serum glutamic oxaloacetic
III. LD3 – HHMM 20-26% - lungs, transaminase (SGOT)
lymphocyte, spleen, and o involved in the transfer of amino group between
pancreas aspartate and alpha keto acids.
IV. LD4 – HMMM 8-16% - liver o COENZYME: pyridoxal phosphate
V. LD5 – MMMM 6-16% - skeletal muscle Aspartate + a-ketoglutarate ←AST→ oxaloacetate +
o HM – heart and muscle glutamate
o LD1 and LD2 – indicators of AMI and intravascular ISOENZYMES
hemolysis o Cytoplasmic Isoenzyme
o LD3 – indicator of pulmonary disorders o Mitochondrial Isoenzyme
o LD4 and LD5 – indicator of intrahepatic disorders and No clinical significance; measured as a whole
muscular dystrophies TISSUE SOURCES
o Flipped pattern – the concentration of LD1 > LD2 1. Cardiac tissue
(AMI/hemolyzed sample) 2. Liver
o LD 6 - Alcohol Dehydrogenase (abnormal enzyme) 3. Skeletal muscle
▪ 6TH Band to the electrophoresis AMI
▪ Clinically significant; presence signifies great o RISE: after 6-8 hours after attack
prognosis and impending death o PEAK: after 24 hours
▪ Arteriosclerotic cardiovascular failure; can lead o NORMALIZE: after 5 days
to liver damage Highest concentration of AST is seen in acute
MEASUREMENT OF ISOENZYMES hepatobiliary disorder while in viral hepatitis the
1. ELECTROPHORESIS – widely used method; patten of concentration of AST goes as high 100x the normal value
migration is the same as their arrangement of AST and 4x in liver cirrhosis
2. IMMUNOINHIBITION OR CHEMICAL INHIBITION
 METHOD ▪ Bone and Intestine – co-migrators; to separate
Karmen Method – for the determination of AST add muramidase
Aspartate + a-ketoglutarate ←AST→ oxaloacetate + 2. HEAT STABILITY TEST – serum is subjected to 56C for
glutamate 10-15 minutes
Oxaloacetate + NADH + H ←Malate dehydrogenase→ ▪ Most heat stable – placental ALP
malate + NAD ▪ Heat labile – bone ALP
o Avoid hemolysis because it can increase the value of 3. CHEMICAL INHIBITION TEST
AST o Phenylalanine – inhibit activity of placental and
o Stored at ref temp – will last up to 3-4 days intestinal ALP
REFERENCE RANGE: 5-30 U/L o 3 molar urea – inhibit activity f placental ALP
ALANINE AMINOTRANSFERASE o Levamisole – inhibit activity of the bone ALP
o Also known as serum glutamic pyruvic transaminase CARCINOPLACENTAL ALP: isoform of the placental ALP
or SGPT o REGAN ALP - Lungs, breast, ovaries,
o Involved with the transfer of an amino group from gynecological problem
alanine to a- ketoglutarate with the formation of o NAGAO ALP - adenocarcinoma of pancreas, bile
glutamate and pyruvate duct & pleural cancer
o More specific METHOD: bowers & mccomb method (continuous
o Has a higher concentration; elevated longer because monitoring technique)
of its half life of 16-24 hours during liver disorders p-nitrophenyl phosphate ←ALP→ p-nitrophenol
Alanine + a-ketoglutarate ←→ Pyruvate + glutamate and phosphate ion
TISSUE SOURCE: Liver REFERENCE VALUE: 30-90U/L
Coupled Enzymatic Reaction (7.3 – 7.8 AT 340 nm) INCREASED ALP
Alanine + a-ketoglutarate ←ALT→ pyruvate + glutamate o Osteitis deformans – bone ALP
Pyruvate + NADH +H ←LD→ lactate + NAD o Obstructive jaundice – liver ALP
o Stored at 4C and ay last up 3-4 days o Osteomalacia – bone ALP
REFERENCE VALUE: 6-37 U/L o Rickets – bone ALP
ALKALINE PHOSPHATASE (ALP) o Bone CA – bone ALP
o Catalyze the hydrolysis of various o Sprue – intestinal ALP
phosphomonoester at an alkaline pH o Hepatitis and cirrhosis – liver ALP
o It functions to liberate inorganic phosphate from an o Hyperparathyroidism
organic phosphate ester with the concomitant ACID PHOSPHATASE
production of an alcohol. Catalyzes the same reaction made by ALP except that it
INHIBITOR: phosphorus is active at acidic pH (5.0)
TISSUE SOURCES: intestine, placenta, liver, bone, small MAJOR SOURCE: prostate gland
amount in kidney MINOR SOURCES: RBC, platelets, bone
ISOENZYMES: intestinal ALP, placental ALP, liver ALP, USES: forensic chemistry, detection of cancer
bone ALP METHOD: Shinowara Method – has the same reaction
BONE ISOENZYMES → elevated in children specially in for the method of ALP
periods of growth; as well in adults older than 50 years Special consideration on enzyme activity
old o Prostatic ACP – inhibited by L-tatrate
PLACENTAL ISOENZYMES → 16-20 weeks of gestation o Red cell ACP – inhibited by cupric anion and
SIGNIFICANCE: formaldehyde
o Obstructive jaundice – highest concentration of ALP o Prostatic CA
o Paget’s disease/ osteitis deformans – destruction of For more specific diagnosis of prostatic cancer ACP is
the bone combined with tumor marker called prostatic specific
SEPARATED BY: antigen (PSA)
1. ELECTROPHORESIS REFERENCE VALUE: 2.5 -11.7 U/L (total ACP)
▪ Liver ALP – most anodal but least cathodal 0-3.5 ng/mL (prostatic ACP)
▪ Intestinal ALP – least anodal but most cathodal
 AMYLASE (AMS/AMY) o Pancreas specific
o it catalyzes the breakdown of starch and glycogen; MAJOR TISSUE SOURCE:
helps in the digestion of carbohydrates SIGNIFICANCE:
o smallest enzyme o Acute pancreatitis
o If urine is tested with amylase, it will yield a positive ▪ RISE: after 6 hours
result ▪ PEAK: within 24 hours
ISOENZYMES: ▪ NORMAL: within 8-14 days
o S-TYPE (ptyalin) – salivary glands o Chronic pancreatitis – result in the degradation of
o P-TYPE (amylopsin) – pancreas acinar cells leading to the decrease concentration of
MAJOR TISSUE SOURCES: salivary glands and pancreas lipase
OTHER TISSUE SOURCES: adipose tissue, fallopian tube, METHOD
small intestine, skeletal muscle o SUBSTRATE: OLIVE OIL, TRIOLEIN
SIGNIFICANCE: o addition of COLIPASE – makes the method become
o Acute pancreatitis more sensitive and specific for determination of
▪ RISE: 2-4 hours acute pancreatitis
▪ PEAK: 24 hours o Cherry Crandal method - hydrolysis of olive oil after
▪ NORMALIZE: within 3-5 days incubation for 24 hours at 37 *C & titration of fatty
o Parotitis acids using NaOH
o Renal failure - increase of amylase to blood because TAG + H2O ←LPS→ monoglyceride and fatty acids
of failure of it to be excreted REFERENCE VALUE: 0-1.0 U/mL
Macro amylase → abnormal form of amylase; bound to ALDOLASE
immunoglobulins splits fructose-1,6-diphosphate into two triose
REFERENCE VALUE: 60-180 SU/L phosphate molecules
95-290 U/L ISOENZYMES
METHOD Aldolase A → found in the skeletal muscle
INHIBITOR: wheat germ lectin (salivary amylase) & TAG Aldolase B → found in the WBC, liver, kidney
(serum amylase) Aldolase C → found in the brain
SUBSTRATE: starch INCREASE LEVEL: skeletal muscle disease, leukemia, HA,
1. SACCHAROGENIC hepatic carcinomas
o Measures the amount of reducing sugar 5’ NUCLEOTIDASE
produced by the hydrolysis of starch by the usual Marker for hepatobiliary disease
glucose methods. REFERENCE VALUE: 0-1.6 units
o Classic reference method expressed in SU. GAMMA GLUTAMYL TRANSFERASE
2. AMYLOCLASTIC – destruction It catalyzes the transfer of glutamyl groups between
o Measures AMS activity by following the peptides or amino acid through linkage at a gamma
decreases in substrate concentration carboxyl group.
(degradation of the starch). SOURCES: liver, kidney, prostate & pancreas
3. CHROMOGENIC – color Sensitive indicator of alcoholism (Occult alcoholism) and
o Measures AMS activity by the increase in color acute alcoholic hepatitis
intensity of the soluble dye-substrate solution ELEVATED AMONG INDIVIDUAL: warfarin,
produced in the reaction. phenobarbital, & phenytoin therapy
4. COUPLED-ENZYME METHOD: Rosalki & Tarrow Method
o Measures AMS activity by the continuous SUBSTRATE: gamma- glutamyl-p-nitroanilide
monitoring technique REFERENCE VALUE: 5-30 U/L → Female; 6-45U/L → Male
▪ alpha glucosidase, hexokinase and G6PD CHOLINESTERASE/PSEUDOCHOLINESTERASE
LIPASE (LPS) o Produced from the liver parenchyma
o An enzyme that hydrolyzes the ester linkages of fats o Marker for insecticide or pesticide poisoning
to produce alcohol and fatty acid. specifically organophosphate; concentration
o Produced by the acinar cell of the pancreas decreases due to these poisoning
METHOD: Ellman techniques or potentiometry
REFERENCE VALUE: 0.5-1.3 pH unit
ANGIOTENSIN CONVERTING ENZYME
Also known as the peptidyl dipeptidase A or kininase II
INDICATOR OF NEURONAL DYSFUNCTION → specifically
Alzheimer’s disease
SOURCES: macrophage and epithelioid cells
CERULOPLASMIN
Copper carrying protein & an enzyme
Hepatolenticular disease (Wilson’s’ disease) - decrease
value
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
Deficiency of this enzyme can lead to drug-induced
hemolytic anemia (antimalarial drug, primaquine)
REFERENCE VALUE: 10-15U/g Hb
ORNITHINE CARBAMOYL TRANSFERASE
Marker for Hepatobiliary disease
8-20 mU/mL