CLINICAL ENZYMOLOGY in veterinary medicine.pdf

CLINICAL ENZYMOLOGY
DR.MAGEJA,BVSc,UZ

Enzymes in medicine
Objectives
o List the clinically important enzymes and
isoenzymes.
o State which of the enzymes and isoenzymes are
found in which tissues
o Describe plasma enzyme changes in myocardial
infarction and liver disease
o Outline different ways of measuring plasma enzymes

Uses of enzymes in medicine
o Diagnostic – enzyme levels altered with disease
Heart attack:
E.g
 Lactate dehydrogenase
 Creatine kinase
 Serum glutamate-oxaloacetate transaminase (SGOT)
Pancreatitis:
 Amylase
 Lipase
o Analytical reagents – enzyme used to measure another substance
 Urea converted to NH3 via urease
 Blood urea nitrogen (BUN) measured
o Replacement therapy
 Administer genetically engineered β-glucocerebrosidase for Gaucher’s disease

Clinical enzymology
o Enzymes are catalysts that increase the rate or velocity of physiologic
reactions.
o Each and every reaction in our body takes place with the help of an
enzyme.
o In general, most enzymes are present in cells at much higher concentrations
than in plasma.
o Measurement of their levels in plasma indicates the integrity of their tissue
of origin whetherit is damaged leading to the release of intracellular
componentsinto the blood or it is intact thus functioningproperly.
o This forms the basis of clinical enzymology.
o Thus clinical enzymology refers to measurement of enzyme activity for the
diagnosis and treatment of diseases.

Clinical enzymology
o Enzymes present in plasma can be classified into 2 types,
they are
1. Functional Plasma enzymes and
2. Non-functional plasma enzymes
o Functional plasma enzymes:
 Present in plasma at higher concentration than tissues
 They function in plasma.
 Mostly synthesized by the liver
 Usually decreased in disease conditions
Eg. Clotting enzymes, lipoprotein lipase

Clinical enzymology
o Non-functional plasma enzymes:
 Present in plasma at lower concentration than tissues
 Do not have any function in plasma
 Mostly synthesized by liver, skeletal muscle, heart,
brain etc
 Usually increased in disease conditions
 Eg. Creatine kinase, Alanine transaminase etc
 Measurement of these enzymes in plasma can be
used to assess cell damage and proliferation i.e.
diagnosis of disease.

Measurement of serum enzymes
Diagnostic enzymology
 Enzymes are normally intracellular and LOW
concentration in blood
 Enzyme release (leakage)in the blood indicates cell damage
(cell –death, hypoxia, intracellular toxicity)
 Quantitative measure of cell/tissue damage
Organ specificity
 But not absolute specificity
 Most enzymes are present in most cells but differing in
amounts

Information from enzymes
measurements in serum
o Presence of disease
o Organs involved
o Aetiology /Nature of disease: differential
diagnosis
o Extent of disease - more damaged cells-more
leaked enzymes in blood
o Time course of disease

Measurement of enzyme activity
oEnzyme activity is expressed in
International unit (IU)
o It corresponds to the amount of enzyme that
catalyzes the conversion of one micromole
(mol) of substrate to product per minute

Isoenzymes
o Catalyse same reactions but are formed from structurally
different polypeptides.
o They perform the same catalytic function
o Different isoenzymes may arise from different tissues and their
specific detection may give clues to the site of pathology
o Various isoenzymes of an enzyme can differ in three major
ways:
- enzymatic properties
- physical properties (e.g heat stability)
- biochemical properties such as amino acid composition and
immunological reactivities

Isoenzymes
Isoenzymes (also known as isozymes) are enzymes that differ in amino
acid sequence but catalyze the same chemical reaction
o Believed to be originating from closely linked genes or from
multiple gene loci
o Evolution from a single form possibly due to long-term mutations
o They vary with respect to their kinetic parameters, electrophoretic
mobility, and localization
o They all have independent action
Eg. Lactate dehydrogenase have 5 isoenzymes (LDH1, LDH2,
LDH3,LDH4 & LDH5)
o They can be used to identify the specific affected tissues
o They can be differentiated from each other and can be clinically
quantified in the lab

Lactate dehydrogenase
o Lactate Dehydrogenase (EC 1.1.1.27; L-lactate: NAD+
oxidoreductase; LD) catalyses the reversible interconversion
of lactate and pyruvate.
o The enzyme is widely distributed in the body, with high
concentrations in cells of cardiac and skeletal muscle, liver,
kidney, brain and erythrocytes: measurement of plasma total
LD activity is therefore a non-specific marker of cell damage.
o LD has a molecular weight of 134 kDa and is composed of
four peptide chains of two types:
I. M
II. H

o Each under separate genetic control
o The subunit compositions of the five isoenzymes are listed
below in order of their decreasing anodal mobility in an
alkaline medium.
1. LD-1 (HHHH; H4) = migrates fastest towards the anode
2. LD-2 (HHHM; H3M)
3. LD-3 (HHMM; H2M2)
4. LD-4 (HMMM; HM3)
5. LD-5 (MMMM; M4)

• It is a tetrameric protein and made of two types of subunits
namely H = Heart, M = skeletal muscle
• It exists as 5 different isoenzymes with various combinations
of H and M subunits
• LDH is elevated in myocardial infarction, blood disorders

LDH
Clinical significance
Normal range of total LDH: 180-360 U/L= 3.1-6.1 μkat/L
 It is increased in plasma in Myocardial injury, acute leukemias,
 Generalized carcinomatosis and in acute hepatitis.
 Estimation of its isoenzymes in more useful in clinching
diagnosis between hepatic disease and Myocardial Injury.
Causes of Raised Plasma Total LD Activity
Artefactual: due to in vitro haemolysis or delayed separation of
plasma from whole blood.
Marked increase (more than 5 times the upper reference limit in
adults): Circulatory failure with ‘shock’ and hypoxia:

LDH
Myocardial infarction
 Some haematological disorders. In blood diseases such as
megaloblastic anaemia, acute leukaemias and lymphomas.
 very high levels (up to 20 times the upper reference limit in
adults) may be found.
Moderate increase. viral hepatitis: malignancy of any tissue:
skeletal muscle disease: pulmonary embolism: infectious
mononucleosis.

Isoenzymes of LDH
o LDH1 fraction predominatesin cells of cardiac muscle, erythrocytesand
kidneys.
o LDH5 is the most abundantform in the liver and in skeletal muscle.
Whereas in many conditionsthere is an increase in all fractions, the
finding of certain patternsis of diagnostic value.
o Predominant elevation of LDH1 and LDH5. (LDH1 greater than LDH5
occurs after myocardial infarction, in megaloblastic anaemia and after renal
infarction.
o Predominant elevation of LDH2 and LDH3 occurs in acute leukaemia:
LDH3 is the main isoenzyme elevated due to malignancy of many tissues.
o Elevation of LDH5 occurs after damage to the liver or skeletal muscles.

Creatine kinase
• Creatine phosphate acts as a
backup for rapid ATP
regeneration in active tissues
– Creatine phosphate is in energetic
equilibrium with ATP
– Creatine kinase (CK) catalyzes
the transfer of phosphate between
creatine and ATP/ADP
• Provides rapid regeneration of
ATP when ATP is low
• Creatine phosphate is
regenerated when ATP is
abundant
ADP ATP

Creatine kinase
o Phosphocreatine – serves
as an energy reserve
during muscle contraction
o Creatine kinase is a
dimer made of 2
monomers
o occurs in the tissues
 Skeletal muscle contains
M subunit, Brain contains
B subunits
 Three different isoenzymes
are formed

Creatine kinase isozymes
o Two different polypeptide chains (M and B) are differentially
expressed in tissues
o Combine at random to give three isozymes:
 CK-MM (primarily muscle)
 CK-MB (hybrid)
 CK-BB (primarily brain)
o The CK-MB has its highest concentration in heart muscle
o CK-MB >5% of total CPK strongly suggests myocardial
infarction

o Total CK activity is determined by a simple enzyme assay
(phosphocreatine + ADP  ATP)
o CK-MB mass is determined by a two-antibody “sandwich”
assay.
Determining CK-MB (mass) / CK (activity)
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
anti-CK-B coated tube
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Tagged anti-CK-M
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
POSITIVE
Y
Y Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Substrate

Creatine kinase
o Clinical significance
o Normal range for total CK: Male : 46-171U/L= 0.78-2.90 μkat/L
o Female: 34-145 U/L= 0.58-2.47μkat/L
o Serum CK activity is greatly elevated in all types of muscular dystrophy.
o In progressive muscular dystrophy(particularlyDuchennesex-linked
musculardystrophy),enzyme activity in serum is highest in infancy and
childhood(7-10 years of age) and may increase long before the disease is
clinicallyapparent.
o Serum CK activity characteristicallyfalls as patientsget olderand as the
mass functioningmuscle diminishes with the progression of the disease.
o About 50%- 80% of the asymptomatic female carriers of Duchenne
dystrophyshow threefold to six-fold increase of CK activity.

Creatine kinase
o Quite high values of CK are noted in viral myositis,
polymyositis and similar muscle disease.
o However in neurogenic muscle disease, such as:
 (a) Myasthenia gravis
 (b) Multiple sclerosis
 (c) Poliomyelitis
 (d) Parkinsonism
o Serum enzyme activity is normal

Isoenzymes of CK
o CK consists of two protein subunits, M (for muscle) and B (for brain),
which combine to form three isoenzymes. BB (CK-1), MB (CK-2) and
MM (CK-3).
o CK-MM is the predominant isoenzyme in skeletal and cardiac muscle and
is detectablein the plasma of normal subjects.
o CK-MB accountsfor about 35 per cent of the total CK activity in cardiac
muscle and less than five per cent in skeletal muscle: its plasma activity is
always high after myocardial infarction.
o It may be detectablein the plasma of patientswith a variety of other
disorders in whom the total CK activity is raised, but this accountsfor less
than six per cent of the total. CK-BB is present in high concentrationsin
the brain and in the smooth muscle of the gastrointestinal and genital tracts.

Alanine Transaminase (ALT) And Aspartate
Transaminase( AST)
 Alaninetransaminase(ALT) and Aspartatetransaminase(AST) enzymes
are most abundantlypresent in the liver and are elevated in blood as a result
of leakage from damaged cells
 Measurement of these transaminases is useful for the diagnosis of liverdiseases
 In viral hepatitis the enzyme levels are increased 20-50 times above the upper
limit of the normal range
 Alaninetransaminase(ALT) increase is specific for liver damage involving
hepatocellulardamage
 Aspartatetransaminase(AST) is also moderately increased in muscular
dystrophy and acute myocardial infarction

Transaminase( AST)
Markers of hepatocellulardamage
Aminotransferases/Transaminases
o The transaminases are enzymes involved in the transfer of an amino group
from a 2-amino- to a 2-oxoacid:they need the cofactor, pyridoxal
phosphate for optimal activity.
o They are widely distributedin the body.
o The 2-oxoglutarate/L-glutamatecoupleserves as one amino group acceptor
and donor pair in all amino-transfer reactions; the specificity of the
individual enzymes derives from the particularamino acid that serves as
the other donorof an amino group.
o Thus AST catalyzes the reaction:

Transaminase( AST)

Transaminase( AST)
o The reactions are reversible, but the equilibriumofAST and ALT reactions
favor formation of aspartateand alanine respectively.
o In the liver, the concentrationofALT per unit weight of the tissue is more
than AST.
o AST and ALT enzymes are more important in assessing and monitoring the
degree of liver cell inflammation and necrosis.
o Elevated plasma ALT are considered to be relatively specific for liver
disease.
o AST may be elevated in otherforms of tissue damage, such as myocardial
infarction, muscle necrosis and renal disorders.
o In liver disease, theALT level is increased markedly compared to AST.
o In acute viral hepatitis thereis a 100-1000times increase in both ALT and
AST but ALT level is increased more than that of AST

Transaminase( AST)
(a) Aspartate Transaminase(EC 2.6.1.1; L-aspartate:2-oxoglutarate
aminotransferase; AST)
Clinical Significance
o Normal values ofAST: Male: <35 U/L = <0.60 mkat/L
o Female: <31 U/L = <0.53 mkat/L
(b) AlanineTransaminase (EC 2.6.1.2;L-alanine:2-oxoglutarate
aminotransferase; ALT)
o Normal values ofALT: Male: <45 U/L = <0.77 mkat/L
o Female: <34 U/L = <0.58 mkat/L

Levels of enzymes in diseases
involving liver damage
In viral hepatitis
Rapid rise in
transaminases (AST &
ALT) in serum occurs
even before bilirubin rise
is seen

Levels of enzymes in myocardial infarction
CK
CK-MB
AST
LDH
HBDH
AST and CK rise in 6
hours following acute
myocardial infarction
Hydroxybutyrate
dehydrogenase
(HBDH) and LDH
are elevated much
later and remain high
for a longer period of
days

 Is a group of enzymes that have maximal activity at a high
pH 9.0-10.5
 Widely distributed throughout the body
 High levels are seen is liver, bone, placenta and intestine and
useful to assess hepatobiliary and bone diseases
 In hepatobiliary obstruction, hepatocytes lining the biliary
ducts induces the ALP synthesis.
 High levels of ALP is indicative of extrahepatic obstruction
rather than intrahepatic obstruction
 In bones, the enzyme is derived from osteoblasts. Hence
increased in bone diseases like rickets, osteomalacia,
neoplastic diseases with bone metastates and healing
fractures
Alkaline phosphatase (ALP)

o Alkaline phosphatase(EC 3.1.3.1; orthophosphoric-monoester
Phosphohydrolase[alkaline optimum]; ALP). Half-life= 10 days
o The alkaline phosphatasesare a group of enzymes that hydrolyse organic
phosphatesat high pH.
o They are present in most tissues but are in particularlyhigh concentration
in the osteoblastsof boneand the cells of the hepatobiliarytract, intestinal
wall, renal tubulesand placenta.
o The exact metabolicfunction of ALP is unknown but it is probably
important for calcification of bone
o In adultsplasma ALP is derived mainly from bone and liver in
approximatelyequal proportions:the proportion due to the bone fraction is
increased when there is increased osteoblasticactivity that may be
physiological

Causes of raised PlasmaALPactivity
o 1. Physiological: There is a gradual increase in the proportionof liver ALP
with age: in the elderly the plasma bone isoenzyme activity may increase
slightly.
 Bone ailment: rickets and osteomalacia
 Liver disease:
 Malignancy boneor liver involvement or direct tumour production.
o Possible Causes of Low Plasma ALP Activity
 Arrested bone growth
 Hypophosphatasia: an autosomal recessive disorder, associated with rickets
or osteomalacia.

Isoenzymes of Alkaline Phosphatase
o Bone disease with increased osteoblastic activity, or liver disease with
involvement of the biliary tracts, are the commonest causes of an increased
total alkaline phosphatase activity.
o The isoenzymes originating from cells of bone, liver, intestine and placenta
may be separated by electrophoresis, but interpretation may be difficult if the
total activity is only marginally raised.
Assays for ALP isoenzymes are needed when:
I. The source of an elevated ALP in serum is not obvious and should be
clarified.
II. The main clinical question is concerned with detecting the presence of liver
or bone involvement
III. In the case of metabolic bone disorders, to ascertain any modifications in the
activity of osteoblasts to monitor the disease activity and the effect of appropriate
therapies.

 The activity of the bone isoenzyme can be estimated by heat treating
a serum sample at 56oC.
 The bone ALP is heat liable and is destroyed or heat inactivated at
this temperature.
 Measurement of ALP before and after heat treatment gives a
measure of bone ALP
p-NPP + H2O p-NP (benzenoid form) + PO4
3-
p-NP (quinonoid form) + PO4
3-
Colorless
Yellow
Rearrangement
Color read at 405nm
Para nitro
phenylphosphate
ALP, Mg2+
pH 10.3

Acid phosphatase (ACP)
 Is a group of enzymes that have maximal activity at pH 5.0-6.0
 It is present in prostate gland, liver, spleen and RBC.
 The main source of ACP is prostate gland and so can be used as a
marker for prostate disease.
 Acid Phosphatase (EC 3.1.3.2; orthophosphoric acid-monoester
phosphohydrolase [acid optimum];ACP)
 Acid phosphatase is present in lysosomes, which are organelles
present in all cells with the possible exception of erythrocytes.
 Extra lysosomal ACPs are also present in many cells:

(a) prostate,
(b) bone (osteoclasts),
(c) spleen
(d) platelets
(e) erythrocytes.
o The lysosomal and prostatic enzymes are strongly inhibited by d-tartrate ions
(tartrate-labile ACP), whereas the erythrocyte and bone isoenzymes are not
(TRACP)
o Normal range of TR-ACP: 1.5-4.5 U/L= 0.03-0.08 ì kat/L
Elevated TR-ACP
(a) Paget disease
(b) Hyperparathyroidism with skeletal involvement
(c) Presence of malignant invasion of bones by cancers
o The only non-bone condition in which elevated activities of TR-ACP are found
in serum is Gaucher disease of the spleen, a lysosome storage disease.
.

The main indications for estimation are to help diagnose prostatic
carcinoma and to monitor its treatment
The estimation is gradually being replaced by the measurement of
plasma prostate specific antigen (PSA) a protein derived from
the prostate.
This test is more specific and sensitive for diagnosis and monitoring
treatment.
However, it may be raised in similar circumstances to those affecting
prostatic ACP and is more expensive to estimate.
ACP is more useful for monitoring the treatment of a known case of
disseminated prostatic carcinoma than for making the diagnosis.

Amylase
Is a digestive enzymes from the pancreas and salivary
glands which breaks down starch.
Elevated in acute pancreatitis.
It is used as a marker to detect acute pancreatitisAND appendicitis.
a-Amylase: (EC3.2.1.1;1,4-a-D-glucan glucanohydrolase;AML) belongs to
hydrolase class that catalyzes the hydrolysis of 1,4-a-glycosidic linkages in
polysaccharides.
They are low molecularweight proteins (54 to 62 kDa) that can pass the
glomeruli of the kidneys.
 It is the only plasma enzyme physiologically found in urine.
 The AMY activity present in normal serum and urine is of pancreatic(P -
AMY) and salivary gland (S-AMY)origin.
Normal values of amylase: 28-100U/L = 0.48-1.7ì kat/L

Amylase
Causes of Raised Plasma Amylase Activity
1. Marked increase (five to 10 times the upperreference limit):
 Acute pancreatitis
 Severe glomerular impairment
2. Moderateincrease (up to five times the upperreference limit):
 Perforated peptic ulcer
 Acute cholecystitis
 Intestinal obstruction
 Salivary gland disorders like mumps, salivary calculi

Lipase
Lipase: (EC 3.1.1.3; triacylglycerol acyl hydrolase; LPS) is a single
chain glycoprotein with molecular weight of 48 kDa.
Normal values: 40-200 U/L
o Plasma lipase levels are elevated in acute pancreatitis and carcinoma
of the pancreas.
o Serum amylase is increased in mumps, pancreatic disease or due to
some
o other cause, whereas lipase is increased only in pancreatitis.
o Therefore, the determination of both amylase and lipase together
helps in the diagnosis of acute pancreatitis.

Trypsin
Trypsin: (EC 3.4.21.4; no systemic name; TRY) is a serine
proteinase that hydrolyze the peptide bonds formed by the
carboxyl groups of lysine arginine with other amino acids.
Normal values of trypsin: 25 ± 5.3 μ g/L
Increased in pancreatic disease.
o But as there is no distinct role of trypsin estimation in
the routine management of patients with acute
pancreatitis, this test is therefore considered of limited
clinical value.

o Amino acid + Glutathione  -glutamyl amino acid +
cysteinylglycine
o It is involved in amino acid transport across the membranes.
o Found mainly in biliary ducts of the liver, kidney and pancreas.
o Enzyme activity is induced by a number of drugs and in particular
alcohol.
o -GT increased in liver diseases especially in obstructive jaundice.
o -GT levels are used as a marker of alcohol induced liver disease
and in liver cirrhosis.
( GT)
 Glutamyltransferase ( GT)

 Glutamyltransferase ( GT)
Gamma-glutamyl-transferase (EC 2.3.2.21;γ-glutamyl-peptide:amino acid
γ-Glutamyltransferase; GGT): catalyzes the transfer of the γ–glutamyl group
from peptides and compoundsthat contain it to an acceptor Gamma glutamyl
transferase occurs mainly in the cells of liver, kidneys, pancreas and prostate.
Plasma GGT activity is higher in men than in women.
Normal values for GGT Male: <55 U/L = <0.94 μkat/L
Female: <38 U/L = <0.65 μkat/L
Causes of raised plasma GGT activity
o Induction of enzyme synthesis, without cell damage, by drugs or alcohol.
o Hepatocellular damage, such as that due to infectious hepatitis:

Measurement of enzymes
o Enzymes are measured
End point assay
Kinetic assay
o Measurement of enzymes are affected by the presence of
inhibitors or activators.
o Hence most of the enzymes are measured by coupled assay.
o A coupled assay is one in which a second enzyme is used to act
on the product of the enzyme of primary interest.
o Second enzyme used NADH as a coenzyme. The rate can be
followed by measuring oxidation of NADH which can be done
conveniently at 340nm.

Measurement of Enzyme Activity
o In the assessment of enzyme levels in the clinical laboratory, the most
frequently used procedure consists of measuring the rate of the enzyme-
catalyzed chemical reaction.
o The initial rate of an enzymatic reaction is directly proportional to the amount
of enzyme present when the substrate concentrations are maintained at
saturating levels (i.e., zero-order kinetics with respect to substrate
concentration) and other factors (e.g., pH, temperature, and cofactors) are
maintained at optimal and constant levels
o Under these conditions, the rate of substrate removal or product formation in a
given time interval is directly related to enzyme activity.
o The reaction rate is determined by measuring product formation (or substrate
removal) between two fixed times or by continuously monitoring the reaction
with time.
o For accuracy, it is preferable to measure product formation rather than
substrate removal because the former involves measurement of a change in
concentration from an initial zero or low level to higher levels, which is
analytically more reliable than the reverse.

o Both methods, fixed-time and continuousmonitoring, are kinetic
procedures.
o The true rate of enzyme activity is obtainedonly when the reaction rate is
measured in the linear region (a period of constantreaction rate and of
maximum velocity).
o Inaccurateresults are obtainedif the rate is measured during nonlinearor
non maximum phases.
o In fixed-time procedures, in which a single given interval of time is chosen
to measure enzyme activity, it is essential to select reaction conditions, such
as concentration of substrate, pH, temperature, and cofactors, that will
provide linearity with maximum slopeduring the time period in question.
o False negative values for enzyme rates may arise from substratedepletion,
inhibition of the enzyme by product, increase in the reverse reaction due to
productaccumulation, denaturation of the enzyme, presence of certain
endogenousmetabolites, and drugs (or their metabolites).

o In the design of an enzyme assay, a key element is selection of a suitable
physical or chemical technique for following the appearance of a product or the
disappearance of a substrate.
o The rate of enzymatic reactions can be monitored continuously by photometric
procedures (such as light absorption, fluorescence, or optical rotation) if the
reaction is accompanied by a change in optical properties.
o For example, serum alkaline phosphatase, used in the diagnosis of
hepatobiliary and bone diseases, is measured by employing its ability to
catalyze the hydrolysis at alkaline pH of the colorless synthetic substrate p-
nitrophenylphosphate to the yellow-colored product p-nitrophenol.
o The rate of reaction can be measured from changes in absorption at 405 nm.
o Many enzymatic reactions can be followed by a change in absorbance at 340
nm
o if they are linked (either directly or indirectly) to a dehydrogenase reaction
requiring a nicotinamide adeninedinucleotide (NAD + or NADP +).

o The coenzyme absorbs ultraviolet light at 340 nm in the reduced state but
not in the oxidized state.
o Hence, changes in reaction rate in either direction can be followed by
measurement of the absorbanceat 340 nm.
o An example of one such enzyme assay is given below.
o Red cell glucose-6-phosphate dehydrogenase can be specifically assayed in
a red cell hemolysate.
o The enzyme catalyzes the reaction
D-Glucose 6-phosphate+ NADP + ⇌ D-glucono-6-1actone6-phosphate+
NADPH + H +
o Since NADPH absorbs light at 340 nm and NADP + does not , this reaction
can be followed by measurement of the change in absorbanceat this
wavelength with time.

o A similar assay (with lactateand NAD +) can be used for serum lactate
dehydrogenase.
o The difference in absorbanceis measured so readily that a number of
assays make use of it directly or indirectly.
o In the lattercase, the reaction to be measured is linked by one or more steps
to a reaction in which a nicotinamideadenine dinucleotideis oxidized or
reduced.
o Thus, in the measurement of aminotransferase, one of the productsof the
primary reaction serves as a substratefor a secondary reaction in which
NADH is required. For example, in the assay of aspartateaminotransferase,
L-Aspartate + α-ketoglutarate(also known as 2-oxoglutarate) ⇌ oxaloacetate+
L-glutamate (primary reaction)
o The oxaloacetateformed in the primary reaction is reduced to L-malate by
NADH in the presence of malate dehydrogenase:

L-matate + NAD+
Malate dehydrogenase
MDH
Principle involved in AST estimation
Aspartate
aminotransferase
(AST)
- Oxoglutarate + L-aspartate
L- glutamate + oxaloacetate
NADH + H+
+

FIGURE 11.1
o Absorption spectra of NAD +
(NADP+) and NADH (NADPH).
o At 340 nm, the reduced coenzymes
(NADH or NADPH) show
significant absorbance, whereas the
oxidized forms (NAD + or
NADP+) show negligible
absorbance.
o Thus, many enzymatic reactions can
be monitored at 340 nm if the
reaction is directly or indirectly
dependent upon a dehydrogenase
reaction involving a nicotinamide
adenine dinucleotide as a
coenzyme.

FIGURE 11.2
o Diagrammatic representation of the
measurement of the activity of an
enzyme, showing product formation
as a function of time.
o The true activity of the enzyme is
calculated from data obtained when
the reaction rate is linear with
maximum velocity.
o The reaction rate is directly
proportional to the amount of active
enzyme only when the substrate
concentrations are maintained at
saturating levels and when other
variables (e.g., pH, and
o cofactors) are held constant at
optimal conditions.

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