Enzymology

Definitions
CK
AST
ALT
ALP
GGT
Amylase
Lipase
LD
Cholinesterase

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Definitions
Terminology
Enzymes
Isoenzyme

Alleloenzyme
Hybrid isoenzymes
Isoforms
Inhibitors
Reversible inhibitors
Irreversible inhibitors

Product of different gene but able to catalyze the enzymes

characteristic reaction. Differ in structure.
Example: ALP(Liver) vs ALP(Placenta)
Isoenzymes that result from different allelic gene
Example: Cholinesterase(F) vs Cholinesterase(U)
Oligomeric enzymes with monomers derived from different
structural genes (e.g. LD-3, CK-MB)
Different forms of an enzyme, product of the same gene,
differing in post-translational modification
Activity fully restored when inhibitor removed from system
Combines covalently with enzymes so that physical
methods are unable to separate the two. (Occasionally,
chemical methods may work)
e.g. organophosphate, -1 antitrypsin

Inhibitor behaviors
Competitive

Binds to free enzyme on the substrate site

e.g. sulphonamides on dihydropteroate synthetase
Non-competitive
Binds to free enzyme on a site different from the substrate
site
e.g. fluoride on phosphopyruvate hydratase (enolase)
Uncompetitive
Binds to enzyme-substrate complex but not free enzyme
e.g. phenylalanine on intestinal and placental alkaline
phosphatase
Enzyme-related molecules
Activator
Simple molecules such as magnesium ion, or pyridoxal-5phosphate
Coenzyme
More complex molecules such as NADH and NADPH.
Measurement methods
Rate kinetic
Progress of reaction is monitored continuously
Fixed time
Amount of product produced measured after stopping of
measurement
reaction at a fixed time period. Requires longer reaction
time, more subject to interference, and subject to substrate
depletion.
Lag phase
In consecutive enzyme reaction, before the reactants reach
steady state, decreased by increased amount of indicator
enzymes
Units of activity
Katal (kat) (SI Unit)
1 mol/s
1 kat = 60 U/L
International units (U) 1 mol/min
1 U/L = 16.7 nkat

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Some other enzymes

Aldolase

Glycogen
phosphorylas
e

Chitotriosidas
e

Trypsin

Split Fructose-1,6-diphosphate into glyceraldehyde-3-phosphate

and dihydroxyacetone phosphate. The enzyme itself is a tetramer,
with three kinds of subunits, A, B, and C. A and B are found in most
tissue, whereas A and C are found in brain tissue. Aldolase is used
for diagnosis of muscular disease.
Assay reaction: 1. Aldolase, 2. Triosephosphate isomerase (converts
glyceraldehyde-3-phosphate into dihydroxyacetone phosphate),
3. Glycerol-3-phosphate dehydrogenase (consume NADH
monitored at 340 nm).
Plasma as specimen. Serum not suitable because of release of
platelet enzymes, hemolysed specimen not suitable because of red
cell enzymes
Breaks down glycogen into glucose-1-phosphate. First step of
glycogenolysis. Released in ischemic injury and tissue hypoxia.
Dimer of two identical subunits, three types of subunit: GP-MM, GPLL, GP-BB
MM: Muscle BB: Brain MM/BB: Myocardium, GP-BB predominant
LL: Liver, and all tissue except skeletal muscle, myocardium and
brain
Once used as an early marker for AMI, but not specific for heart
Assay: Immunoassay, isoenzymes, glucose-1-phosphate linked
enzyme reaction
Chitotriosidase is an enzyme that is elevated (>600 fold) in patients
with Gaucher disease, which decrease with treatment. It can be
used to monitor the treatment. However, in patients with a
hereditary 24-bp duplication, chitotriosidase cannot be used as a
biomarker for Gaucher disease. The absence of this enzyme per se
has no clinical significance.
Immunoreactive trypsin is used to screen for cystic fibrosis, and is
incorporated into screening programs.

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Creatine Kinase
Creatine phosphate is the storage form of ready-to-use energy in muscle and heart. After
ATP is consumed, creatine phosphate provides phosphate to transfer to ATP.
Reaction. Creatine kinase catalyzes the reaction between creatine and adenosine
triphosphate, yielding creatine phosphate and adenosine diphosphate (the forward
reaction). The forward reaction is alkaline-optimal (pH 9.0) whereas the reverse reaction is
neutral-optimal (pH 6.7).

Inhibitor and co-factors. The reaction requires a narrow range of magnesium ion, and is
inhibited by other divalent ions such as Ca, Zn, Cu, and Mn. The activity is also inhibited by
halides (F, Cl, Br, I), nitrate, acetate, citrate, malonate, and thyroxine. Creatine kinase
possess sulfhydryl group and is easily oxidized.
Isoforms and isoenzymes. Creatine kinase is a dimeric enzyme, consist of either B (for
brain) and M (for muscle) subunits; CK-1 is CK-BB, CK-2 is CK-MB, and CK-3 is CK-MM. There
is also a mitochondrial form.
Isoenzy
me
CK-1 (BB)

CK-2 (MB)

CK-3
(MM)

CK-Mt

Activity

Characteristics

100% of brain activity

92-96% in viscera
<1% in others
22% of cardiac muscle
activity
6% of urinary bladder activity
3% of slow-twitch fiber
activity
78% of cardiac muscle
activity
>95% of striated muscle
activity
Up to 15% of total CK activity
in heart

Most anodal
Least stable in neutral pH
Increased in muscular disease
because of fetal reversion; >5%
can be found in diseased muscle.
Also seen in uremia.
Slightly cathodal to application
point
Most stable in neutral pH
Mitochondrial; Most cathodal
Less stable than CK-MM at neutral
pH

Macro-CK. Two types of Macro CK exist; type 1 is Ig-CK complex (no pathological
significance); type 2 is oligomeric CK-Mt, found in severely ill patient and is associated with
a very poor prognosis.
Method of analysis. All available commercial assays utilize the reverse reaction, measuring
the ATP generated by hexokinase/glucose-6-phosphate dehydrogenase, generating NADPH
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which is detected by spectrophotometry at 340 nm. Consumption of ATP by adenylate

kinase is inhibited by AMP and Ap5A (Adenosine pentaphosphate), and the reaction mixture
is stabilized by EDTA (2 mmol/L; to bind calcium) with addition of magnesium (10 mmol/L,
as acetate). N-acetylcysteine is added to reactivate >99% CK. The IFCC reference method
measures creatine kinase at 37oC, with a lag phase of 2 minutes.
Moderate degree of haemolysis is tolerated (severe hemolysis interferes by release of red
cell enzymes such as AK, ATP, G6P) , and specimen is stable for 8 hours at room
temperature, 2 day at 4-8oC, and 1 month at -20oC.
Half-life. The half-life of creatine kinase (CK-MM) is 15-18 hours; for CK-MB, the half-life is
~13 hours.
Reference interval. CK is higher in male cf females, black cf whites, and newborn vs adults;
and for newborns, high levels in those delivered per vaginal cf caesarian section. Sky high
CK is seen in myositis, malignant hyperthermia, and rhabdomyolysis. High CK is seen in
other muscle disease, muscle injury, muscular dystrophy, and intramuscular injection.
Statin, fibrate, anti-retrovirals and angiotensin II RB causes drug-related rise in CK.
Consider hypothyroidism and Cushing disease (both causes of myopathy) in examination
cases with rise in CK.
Separation of isoenzymes.
Method
Ion exchange
chromatography

Electrophoresis
(Labour intensive)

Immunoinhibition

Immunoprecipitation

Immunoassay (Mass assay)

Note
DEAE-media (sephadex, cellulose, glycophase,
biogel) is used, with elution sequence of CK-3
(TRIS pH 8; 50 mM NaCl) CK-2 (TRIS pH 8;
100 mM NaCl) CK-1 (TRIS pH 7; 500 mM NaCl)
Problem of (1) dilution during elution, (2) carryover, (3) poor recovery.
Limit of quantitation ~ 1-5 U/L
Agarose electrophoresis with development by
concentrated mixture of CK reagent /
immunofixation. NADPH revealed by UV
fluorescence at 360 nm. Albumin may be
fluorescent in patients undergoing
haemodialysis and simulate the CK-1 band.
Limit of quantiation ~ 10 U/L
Based on inhibition of CK-M units; Assumes that
there are no CK-BB present; low
sensitivity/specificity
Three measurement can be taken (total, CK-M
precipitation[=CK-1], CK-B precipitation[=CK-3])
and CK-2 calculated from (total CK CK1 CK3)
Sandwich-type immunoassay. Commercially
available.
Non-catalytically active units also measured.
Various labels (ALP, Fluorescein)

Interpretation
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%CK-MB
CK-MB < 4%

CK-MB 4-25%

CK-MB > 25%

Interpretation
Skeletal muscle contain a small amount of CK-MB,
which can result in increased CK-MB activity. In trained
athletes, as well as in patients with myopathies, such
percentage may be higher.
With an elevated CK-MB activity (16 U/L), such CK-MB
activity is consistent with a diagnosis of myocardial
infarction.
In activity assay, such elevation reflects the presence of
significant amount of CK-BB or Macro-CK (both type 1
and 2). Further investigation is required.

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Aminotransferases
Alanine aminotransferase (ALT) catalyzes the reaction between alanine and 2-oxoglutarate,
to yield pyruvate and glutamate. Aspartate aminotransferase (AST) catalyzes the reaction
between aspartate and 2-oxoglutarate, to yield oxaloacetate and glutamate. In vivo, ALT
and AST generates ammonia which is utilized in the urea cycle.
Cofactors. Both enzyme requires a co-factor, pyridoxal-5-phosphate (and pyridoxamine-5phosphate), which is used as a carrier of amino group during the reaction. Pyridoxal-5phosphate, a derivative of vitamin B6, is deficient in patients with severe vitamin B
deficiency, such as alcoholics, and patients who are severely ill.
Isoforms and isoenzymes. Whereas ALT is exclusively cytoplasmic, there are cytoplasmic
and mitochondrial forms of AST. The two forms of AST are genetically distinct and are true
isoenzymes, with both having dimeric structures. Mitochondrial AST activity are reflective of
liver cell regeneration and necrosis, with most significant increase in alcoholic diseases.
Macro-AST. Macro-AST has been described, and relates to AST linked to immunoglobulins.
These are detected by PEG precipitation. It has no clinical significance other than leading to
unnecessary workups.
Method for analysis. Analysis of aminotransferases are by coupling with the respective
dehydrogenases [lactate dehydrogenase, malate dehydrogenease], oxidizing the oxo-acids
(pyruvate; oxaloacetate) to hydroxy-acids (lactate; malate), with consumption of NADH. The
reaction is measured by rate monitoring. The reaction requires preliminary incubation
period to endogenous oxo-acids are reduced before 2-oxoglutarate is added to start the
reaction. The IFCC reference procedure includes P-5-P in the reaction mixture to ensure
activation of all enzymes. (Wroblewski [ALT] / Karman [AST] method)
Addition of P-5-P would reduce on-board stability of reagents (from 30 days to 7 days) if
two-reagent is used. However, on platforms with separate reagent of P-5-P then there are
no such issues (Olympus AU series).

An alternative procedure is to conversion of pyruvate/oxaloacetate into its

dinitrophenylhydrazone, measured by colorimetric means; this procedure is less specific,
because of feedback inhibition by pyruvate/oxaloacetate, and cross-reaction with ketones
(though most do not absorb at the wavelength 505 nm selected).
A dry-film assay of AST utilize oxaloacetate decarboxylase to produce pyruvate, which is
then oxidized by pyruvate oxidase to acetyl-phosphate and hydrogen peroxide.
Half-life and stability. ALT is unstable in serum and plasma, whether stored at RT, 4 oC or
-25oC. AST is stable up to 48 hours in 4oC.
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Enzyme
Half-life
ALT
47 hours
AST
17 hours
m-AST (mitochondrial)
87 hours
Separation of isoenzymes. Mitochondrial AST can be separated from cytoplasmic AST by
electrophoresis, the cytoplasmic form being more anodal. 5-10% of AST activity in a normal
subject is from m-AST.
Interferences. Glutamate dehydrogenase catalyses the formation of 2-oxoglutate from
glutamate and ammonium ion, while generating NADH. This results in underestimation. This
interference is possible only if ammonia is present at high concentration, and is not present
in IFCC-tracable assays.
Reference intervals. Males have higher values of ALT than females. Healthy newborns has a
higher level of ALT compared with adults, but decrease to adult level by 3 months. Likewise,
AST activity in infants is twice the level in adults, and similar decrease to adult level by 6
months.
De Ritis ratio. AST:ALT ratio is increased in cirrhosis, alcoholic hepatitis, acute liver failure,
and hepatocellular carcinoma/metastatic carcinoma of liver [Biochemical picture: normal
bilirubin, grossly raised ALP/GGT, raised ALT/AST interpret a picture of partial biliary
obstruction]
Historically AST is also used as cardiac enzymes.
Derived values. De Ritis ratio (AST/ALT ratio). >2 alcoholic hepatitis, hepatocellular
carcinoma; 1-2 cirrhosis;
<1 viral hepatitis.

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Alkaline phosphatase
Alkaline phosphatase catalyses the hydrolysis of phosphate compounds with an alkalineoptimum pH.
Inhibitor and co-factors. Alkaline phosphatase requires an optimal Mg/Zn ratio, and is
activated by divalent ions Mg, Co and Mn. 2-amino-2-methylpropanol (AMP) is an activating
buffer, and is used in the candidate reference method for the CLSI/EPE (Expert Panel on
Enzymes).
Type of
buffer
Inert
Inhibiting
Activating

Note
Carbonate, Barbital
Glycine, Propylamine
2-amino-2-methyl-1-propanol, diethanolamine,
TRIS, ethylaminoethanol, N-methylglucamine

Isoforms and isoenzymes. Four genes exist (ALPL liver, bone, and kidney, ALPI intestinal,
ALPP placental, ALPPL2 placenta-like) for the enzyme, with the liver, bone and kidney
forms being isoforms, and the four gene products being isoenzymes.
Isoenzymes
ALPL

ALPI

ALPP

ALPPL2

Notes
Liver, bone and kidneys isoforms, with differences in
glycosylation.
Also known as tissue non-specific ALP
Intestinal isoenzyme is higher in those who are secretors of
blood group substances (group B and O); Increases after
meal;
Benign familial elevation due to intestinal isoenzyme;
Placental isoenzyme which is elevated up to 2-3 times normal
in third trimester of pregnancy; Regan isoenzyme (Tumor
marker)
Germ cell isoenzyme; Nagao isoenzyme (Tumor marker)

Method for analysis. Self-indicating substrates for the determination of ALP has been
developed (4-nitrophenylphosphate; PNPP), which, with ALP at alkaline pH, result in
cleavage into 4-nitrophenoxide and phosphate. 4-nitrophenoxide rearrange at alkaline pH
into the quinonoid form which is yellowish. AMP (2-amino-2-methylpropanol) provides the
necessary pH and also act as a phosphate group acceptor. The reaction is monitored at 403
nm. The provisional IFCC recommended method utilizes PNPP in AMP buffer, with Mg and Zn
added, and HEDTA used to control the amount of Zn and Mg ions.

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Half-life and stability. ALP is stable in whole blood for 4 days, and 7 days if separated, at
room temperature. Activity slowly increases during storage because of ?release of bound
enzymes. The half-life for ALP is 10 days.

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Hypophosphatasia. Mutation in ALPL (Also known as TnAP tissue non-specific alkaline

phosphatase) results in poor bone mineralization, and has pathology similar to rickets.
Bisphosphonates and vitamin D are contraindicated. Marker is plasma amino acid
Phosphoethanolamine.
Condition
Perinatal (lethal)
Perinatal (benign)
Infantile
Childhood
Adult
Odontohypophospha
tasia

Notes
Perinatal onset. Respiratory insufficiency, hypercalcaemia,
early death.
Perinatal onset. with skeletal manifestation resolve into
milder childhood/adult forms
Onset: 0-6 months. Rickets without elevated serum ALP
Low bone mineral density with unexplained fracture to rickets
Early loss of dentition, stress fracture/pseudofracture of lower
limb
Premature exfoliation of primary teeth or severe dental
caries.

Separation of isoenzymes.
Method
Electrophoretic mobility
(Anode LBPIII - Cathode)

Urea inhibition

Chemical Inhibition

Stability to heat

Lectin affinity and

Neuraminidase treatment
(Based on electrophoresis)

Note
L Liver; B, P Bone (diffuse) and placenta
(discrete)
III Three bands of intestinal forms in
decreasing intensity
High concentration of urea inhibits the activity
of alkaline phosphatase. Bone isoform is most
susceptible.
(Bone: 16%; Liver: 44%,
Intestinal/Placental: 69%)
Chemical
Inhibits
Phenylalanine
intestinal, placental and
Regan
Levamisole
bone, liver
Leucine
placental
Homoarginine
bone, liver
Heat Stability Index: 56oC for 10 minutes
(Bone: <0.25; Liver: >0.35)
Placental ALP: stable at 65oC for 30 minutes
Imprecision can arise from rate of heating,
cooling, and uncertainty from two
measurements.
Wheat germ lectin. Binds the Nacetylglucosamine; retarding bone isoform
more than liver isoform;
Neuraminidase treatment. Bone form more
readily attacked, mobility more reduced cf liver
isoform.
Intestinal forms are resistant because there are
NO terminal sialic acid in the form.
Short treatment: bone v liver
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Immunochemical
characteristics

Long treatment: intestinal

Ostase. Allows measurement by immunoassay
means.
More difficult to produce bone v liver because
they are product of the same gene, cross
reactivity:
5% cross-reactivity with intestinal ALP
15% cross-reactivity with liver ALP
<1% cross-reactivity with placental ALP
100 U/L = 37.5 ug/L

Gamma-glutamyltransferase
Gamma-glutamyltransferase catalyse the transfer of gamma-glutamyl groups from
peptides/compounds to an acceptor, typically glycine/glycylglycine. When the acceptor is
water, simple hydrolysis occurs. Despite the abundance of GGT in renal tubules (higher
than liver, pancreas and intestine), majority of plasma GGT is derived from liver. GGT is
both a microsomal and membrane-bound enzyme.
GGT participates, in vivo, in the transport of amino acids into cells, and in the proximal
tubules, it serve to conserve amino acids via transport.
Apart from the use as a diagnostic aid in alcoholic liver disease and as a ductal enzyme,
high GGT is predictive of treatment failure of hepatitis C towards interferon therapy. It is
also elevated by enzyme inducers.
Inhibitor and co-factors. The velocity of the reaction depends on the nature of the acceptor
substrate. Glycylglycine is a better acceptor substrate compared with glycine or tripeptide
glycylglycylglycine. GGT is inhibited by fluoride, oxalate, and citrate, but only to modest
extent (15% at 1 g/L).
Method for analysis. LGCN (-L-glutamyl-3-carboxy-4-nitroanilide) is used in the IFCCrecommended method. In the reaction, 5-amino-2-nitrobenzoate is formed and is monitored
at 410 nm. The analysis is performed in a TRIS-glycylglycine buffer which also serve as the
-glutamyl acceptor substrate.
Note, the conjugated double bond is what confer the color: however, this comes with a cost
of decreased solubility. Hence the benzene ring of LGCN is highly substituted with nitro
and carboxyl groups. Glycylglycine provides buffering and also act as a gamma-glutamyl
acceptor. A high concentration is used.
The volume fraction of gamma glutamyl transferase is high (1:11, usually for other
enzymes > 1:20), hence temperature control of serum is important.
Half-life and stability. -glutamyltransferase is relatively stable: 1 month at 4 oC and 1 year
at -20oC.
Reference interval. GGT activity is higher in man than women, and GGT is higher in black
than whites. Other causes of elevation of GGT includes cigrette smoking (10%), oral
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contraceptives (20%), and especially anticonvulsant therapy (phenytoin, phenobarbital)

(200% in adults, 400% in children). Full-term neonates exhibit GGT activity 6-7x the upper
normal limit of adults.

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Amylase
Amylase catalyses the hydrolysis of 1,4-glucosidic bonds, digesting amylose, amylopectin
and glycogen, into maltose and some glucose. The best diagnostic efficacy is to use
amylase with a cutoff of threefold the upper reference limit.
Inhibitor and co-factors. Amylase are metalloenzyme which requires calcium for optimal
function. It is activated by various anions such as chloride, bromide, cholate (a bile acid),
and monohydrogenphosphate. The optimal pH for amylase is 6.9-7.0.
Isoforms and isoenzymes. Five genes (1A-C for salivary; 2A-B for pancreatic) coding for two
types (S-AMY, P-AMY) exists. Pancreatic amylase is not glycosylated. Salivary amylase exist
in both glycosylated and non-glycosylated forms.
Isoenzymes
Pancreatic

Salivary

Notes
Elevated in pancreatic disease, biliary tract disease, and other
intraabdominal pathologies such as perforated peptic ulcer,
gastritis, etc.
Elevated in salivary gland lesion, alcoholism, diabetic
ketoacidosis, female genital tract lesion, as well as tumors
and drug-related hyperamylasemia.

Method for analysis. Historically, saccharogenic (measures the liberated sugar),

amyloclastic (measures the decrease in light scattering turbidimetry or nephelometry, or
adding iodine to the reaction mixture after an incubation period), and chromolytic starch
method (liberation of a dye coupled to polysaccharide) were used.

Dry-film procedures utilizes the chromolytic method in which the dyed saccharides in the
spreading layer diffuse into underlying reagent layer, where they are measured by
reflectance spectrophotometry.
The IFCC procedure utilizes a well-defined, ethylidene-protected (against alpha-glucosidase)
substrate 5-ethylidene-4-nitrophenyl-maltoheptaoside. This substrate is better hydrolyzed
by pancreatic than salivary amylase, with free nitrophenol detected at 405 nm.
Separation of isoenzymes.
Method
Electrophoresis
Ion-exchange
chromatography

Note
Salivary more anodal than pancreas
Has not achieved sufficient precision, reliability,
practicability and analytical speed to allow for
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Wheat germ inhibitor

Immunoprecipitation
Immunoinhibition

introduction to clinical use

Two antibody that target S-AMY; reagent cost
similar to total amylase.

Reference interval. IFCC recommended method ~31-107 U/L; Pancreatic amylase 15-53
U/L, not demonstrable in infants less than 6 months of age, rises slowly and reach adult
level by 5 years of age.

Macroamylase
Fraction of amylase excretion (also known as ACCR amylase creatinine clearance ratio) is
used to determine whether there are macroamylase.

ACCR=

U Amy U Cr

100
S Amy U Cr

Interpretation:

Normally 2-6% (some say 5-7%)

Increased in late presentation of pancreatitis
Decreased in macroamylasemia (typically <1%, one case in PMH 0.07%)

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Lipase
Lipase catalyses the hydrolysis of triacylglycerols. Only acyl groups in positions 1 and 3 can
be hydrolysed. Upon hydrolysis into 2-acylglycerol, such compound would spontaneously
isomerize into 1-acylglycerol, where the third acyl chain can be cleaved. Lipase is mainly
derived from pancreas in human, but salivary, gastric, pulmonary, and intestinal forms also
exist. Compared with amylase, lipase rise earlier, higher, peak later, and remain elevated
longer.

Rise
Peaks
Remain
elevated
Fold increase

Amylase
5-8 hours
12-48 hours
3-4 days

Lipase
4-8 hours
24 hours
7-14 days

4-6 fold

2-50 fold

Inhibitor and co-factors. Lipase requires the presence of bile salts and colipase, a protein
secreted also by the pancreas. Bile salt allows the reaction mixture to become an emulsion
because lipase only work at the aqueous-organic interface. Colipase attach to micelle of
bile salt, exposing compounds to lipase which hydrolyses the bonding.
Method for analysis.

Methodology
Titrimetric
Colorimetric
Immunological

Notes
Lipase digests triglycerides into fatty acid and monoglyceride,
using acid-base indicator or pH meter
Similar to titrimetric method, with pH indicator in the initial
reaction mixture
Latex agglutination. Antibody-coated latex beads aggregation
Competitive binding assay. Anti-lipase Fab linked to horseradish
peroxidase

Coupled enzyme
spectrophotome
tric reaction

Turbidimetric
method

Decrease in turbidity upon lipase action

Page | 16

Lactate dehydrogenase
Lactate dehydrogenase catalyses the reaction between L-lactate and pyruvate, while
utilizing NADH for its reducing power. The pH optimum for the forward reaction (L P) is
8.8-9.8, whereas for the backward reaction is 7.4-7.8; LD not only catalyses the reaction for
lactate, but also between other 2-oxoacids and 2-hydroxyacids.
Inhibitor and co-factors. LD is inhibited by reagents with thiol group reactivity (e.g. mercuric
ion) with effect reversed by N-acetylcysteine, cysteine, and glutathione; oxalate and borate
inhibit by competing on the lactate binding site. EDTA also inhibit LD activity, and such
inhibition may be related to Zn binding.
Isoforms and isoenzymes. Three types of unit, LD-M, LD-H, and LD-X, composes LD which is
a tetramer.
Isoform
LD1
LD2

Compositi
on
HHHH
HHHM

LD3

HHMM

LD4
LD5

HMMM
MMMM

LD-X

XXXX

Haemolysis, Myocardial infarction, ineffective

haemopoiesis, germ cell tumor, renal infarction,
muscular dystrophy
Reticuloendothelial system. Platelet, Lymphoid,
Spleen, Malignancies
Liver disease (Hepatitis, Cardiac liver injury),
Skeletal muscle injury, prostatic carcinoma,
Malignancies
Postpubertal human testes

Method for analysis. Kinetic spectrophotometry monitoring the forward reaction (LP).
Haemolysis is not acceptable because erythrocyte contain 4000x the amount of LD of
serum. Serum should be stored at room temperature (for 3 days) because LD is coldsensitive, with LD-4,5 activity being lost if specimen is stored at -20 oC.
The IFCC primary reference procedure, using forward reaction at 37 oC, has been optimized
for LD-1. Serum is preferred (Plasma can be contaminated by platelet, which is LDH-rich).
Utilizes lithium lactate, NAD and N-methyl-D-glucamine buffer.
Separation of isoenzymes. Lactate dehydrogenase isoenzymes can be separated by
electrophoresis, with reaction mixture layered over the separation medium and NADH
generated detected by fluorescence when excited by long wavelength ultraviolet light.
Method
Ion exchange
chromatography
Electrophoresis
Chemical inhibition

Immunoprecipitation
2-hydroxybutyrate DH

Note
-LD1 most anodal
1,6-hexandiol
Sodium perchlorate
all inhibit LD2LD5
Guanidinium thiocyanate
Goat antisera towards LD-5: binds LD2-LD5,
allows measurement of LD1
Lower in M than H
Page | 17

activity

Reference interval. LD levels are higher in children, with gradual decrease during childhood.
Lights Criteria: At least one of the following exudative
Protei Pleural fluid/Serum > 0.5
n
LDH
Pleural fluid/Serum > 0.6
LDH
> 2/3 of upper limit of normal (for serum)
Cholinesterase
Two major forms of cholinesterase exist: acetylcholinesterase (true cholinesterase, found in
red cells, lung, spleen, nerve endings, and gray matter), and pseudocholinesterase (found
in liver, pancreas, heart, serum, and white matter). Cholinesterase catalyses the reaction
breaking down choline esters, and the physiological role for the enzyme is to break down
acetylcholine (for true cholinesterase) and to break down octanoyl ghrelin (for
pseudocholinesterase), a hormone that stimulate feeding.
Pseudocholinesterase has slight specificity difference compared with acetylcholinesterase.
Pseudocholinesterase hydrolyses benzoylcholine but not -methylcholine, whereas the red
cell enzyme does the reverse.
Inhibitor and co-factors. Cholinesterase is inhibited by prostigmine, physostigmine, and
carbamates (competitive inhibitors) as well as organophosphates (non-competitive
inhibitors). Wild-type cholinesterase is inhibited by fluoride and dibucaine. Variants are
characterized by their resistance to these inhibitors.
Variants
U for Usual
A for Atypical
F for Fluorideresistant
S for Silent

Feature
100% activity, inhibited by dibucaine (84%) and fluoride
(80%)
22.5% when homozygous; dibucaine resistant (20%)
42.3% when homozygous; fluoride resistant (53.6%)
Little or no cholinesterase activity in homozygous state (SS
0.3%, SU 55%)

Method for analysis. Most contemporary assays utilizes acetylthiocholine as the substrate,
the thiocholine produced is detected by reaction with disulphide compounds such as 5,5dithio-bis(2-nitrobenzoate), also known as DTNB or Ellmans reagent. The resultant
compound 5-mercapto-2-nitrobenzoic acid is measured at 410 nm.
Alternatively, succinylthiocholine, and butyrylthiocholine can also be used. The reaction is
analogous.

Page | 18

Michaelis-Menten equation
The basic Michaelis-Menten equation is the following equation:
v=

V max [S]
K M +[ S]

Where v is the rate of reaction, V max is the maximum rate of reaction, K M is the
Michaelis constant (concentration of substrate where the reaction rate is half of
V max ), and [ S ] is the substrate concentration. It is trivial to prove that at a
concentration [ S ] =K M the rate of reaction is half V max .
v|[ S ]= K =
M

V max [S ] V max K M V max K M V max

=
=
=
K M +[S ] K M + K M
2 KM
2

This equation is derived from the law of mass action, which in a reaction
E+ S ES E+ P consists of 4 differential equations, the detail of which is not
relevant in the present discussion (but can be found in Wikipedia page for
Michaelis-Menten Kinetics. On the other hand, by taking the reciprocal of
Michaelis-Menten equation, the equation takes on a linear shape and can be used to
derive the parameters from experimentally derived results.
KM
[S]
1 K M +[ S ]

+
=
v V max [ S ]
V max [ S ] V max [ S ]
K
1
1
M
+
V max [S] V max

The result would be a linear plot in which the y intercept is 1/ V max , the X-intercept
being -1/ K M and the slope being K M / V max . The plot is called double
reciprocal plot as well as Line-Burkweaver plot.
There are three types of enzyme inhibitions: competitive, non-competitive, and
uncompetitive.
Competitive inhibition occurs when the inhibitor binds to the substrate site.
Theoretically, increasing substrate concentration to infinity would lead to same
maximum rate of reaction ( V max ). However, note that the concentration to achieve
half maximum ( K M ) would be increased.
Non-competitive inhibition occurs when the inhibitor binds to enzyme but not on the
substrate site. In such case, V max is decreased whereas K M is the same.

Page | 19

Uncompetitive inhibition occurs when the inhibitor binds to enzyme-substrate complex. In

this case, both V max and K M are decreased.

Competitive: Binds S
K M increased

Non-competitive: Binds E
V max decreased

Un-competitive: Binds ES
K M increased and V max
decreased

Units of catalysis
The conventional unit for catalytic activity is units (U). A unit of catalytic activity results in 1
mol of product generated per minute. On the other hand, the SI unit of catalytic activity is
katal, with a katal of catalytic activity generating 1 mol of product per second. The IFCC
endorsed both types of units in 2002.
Units (U)

1 micromole/min

Katal (kat)

1 mole/second

U/L
1 U/L =
kat/L
1 microkat/L

1 micromole/min/litre
16.7 nkat/L
1 mol/min/litre
60 U/L

Phase of reaction
Reactions can be divided into the lag phase, in which the intermediates do not reach steady
state yet, the linear phase, which is used for measurement, and the plateau phase, where
end product inhibition and saturation of detector occur.

IFCC Primary Reference Method

The IFCC primary reference methods are published in the journal CCLM (Clinical Chemistry
and Laboratory Medicine). They adhere to several principles:
1. Enzymes assayed are optimized at 37 degree Celsius
2. The measurand is well-defined. (e.g. for LD, optimized for LD-1 activity)
3. Activate as much enzyme as possible (e.g. use of acetylcysteine in CK)
Page | 20

4. Provide cofactor and coenzyme as appropriate (e.g. Ca++ in amylase, P5P in AST/ALT)
5. Kinetic method, at least 6 measurements
Problem with serum start
In general, modern enzymatic analysis is based on reagent start i.e. serum is added to the
buffer, then it is blanked. The reaction is started by adding one particular reagent (e.g. add
serum and reagent 1 to buffer, followed by blanking; then reagent 2 is added to start the
reaction). The following describes the problem with serum start:
1. Activation and lag phase starts at the same time. Activation phase refers to say, Nacetylcysteine reactivation of CK. At this time the reaction does not contribute to the
linearity and not all enzymes are active at the moment. Lag phase refers to the time
until intermediates saturates the enzyme.
Overall, this wastes the reagent and lead to high blank absorbance. High blank
absorbance decrease dynamic range and performance of the assay
2. Immunoinhibition is most effective (quickness and completeness) when there are no
substrate in solution, for immunoinhibition utilizes antibodies that target the active site.
3. Blank subtraction of serum cannot be done easily with serum start
4. Serum start cannot remove interfering substance in indicator reaction without the main
enzyme.

Page | 21

Appendix 1. Derivation of Michelis-Menten equation

This equation describes the action of
enzyme-substrate reaction to form product.
Rearranging,

E+S k -1 k 1 ES k 2 P

[ ES ] =

The rate of the reaction is defined as the

rate of synthesis of product

v=

d [P]
= k 2 [ES]
dt

The change in

is defined below.

d [ ES ]
= k 1 [E][S]k 1 [ES]k 2 [ES]
dt
However, during equilibrium, the amount of
enzyme-substrate complex remains
constant, and thus d[ES]/dt = 0

d [ ES ]
= k 1 [E][S]k 1 [ES]k 2 [ES]=0
dt

As

[ E ] =[E Total ][ES] ,

[ ES ] =

[ ETotal ][ ES]
Km

[S]

[ ETotal ] [S ]
K m +[S]

Define v max which is the velocity

maximum for the given enzyme
concentration:

v max = lim k 2

[ ETotal ] [S ]

[S ]

K m +[ S]

=k 2 [ ETotal ]

Therefore,

v=

Rearranging,

k 1 [E][S]=k 1 [ES]+k 2 [ES]

k
[ ES ] = 1 [E][S]
k 1 +k 2
1
[ ES ] = [E][S]
Km

K m +[S ]

v =k 2 [ES] ,

As

v =k 2
[ES]

[ ETotal ] [S]

v max [ S]
Km+ [ S ]

The physical meaning of

the following equation:

v
v max

When

Km

is given by

[S]
K m+[ S ]
K m=[S ] ,

v
v max

1
, thus
2

Km

is

the substrate concentration when the

velocity is half maximum.

Page | 22

Appendix 2. Cardiac enzymes

Enzymes and
Markers

First
detect
ed

Peaks

CK Total
CK-MB

6-12 h
3-6 h

12-24 h
12-24 h

Remai
n
elevat
ed
3d
--

MM3

1h

10 h

--

MM3/MM1

<6h

--

--

MB2
MB2/MB1
AST
LD Total
LD1
LD1/LD2
Troponin I

2h
2h
6-10 h
12-48 h
12-48 h
-4-6 h

-4-6 h
12-48
48-72
48-72
-12-24

--3-4 d
10 d
10 d
-10 d

Troponin T

4-6 h

12-24 h

10 d

Myoglobin
Myosin light
chains

1h
3-5 h

4-12 h
--

18 h
2w

h
h
h
h

Cutoffs

Notes

99
centile

Some say 4-8 hours

Non-specific, seen also in
skeletal muscle injury or
strenuous exercise
See above. More sensitive but
less specific than CK-MB

--> 1.0
99
centile
99
centile

Late presentation
Sensitivity 75-90%
First detectable <3 h if high
sensitivity
First detectable <3 h if high
sensitivity