Enzymology

MLSCLIN23 LABORATORY
Methods of Enzyme Activity Determination

Spectrophotometric Assays
• most common
• the substrate does not absorb light at a particular
wavelength at which the product absorbs strongly
- example: Lactate dehydrogenase
Methods of Enzyme Activity Determination

Ultraviolet
• By Manometry
o Measures the evolution of a gas or disappearance of a gas
as the reaction proceeds
o Can be made into a radioactive technique
• By Fluorometry
o oxidized nucleotides do not fluoresce while reduced ones
do
o Reaction can be traced by measuring conversion of NAD
or NADP (cofactors for many enzyme reaction) to NADH or
NADPH (fluoresce)
Enzyme Activity Units

• Enzymes are reported in International units (IU/liter)

• IU = amount of substrate utilized, or product produced, in

terms of micromoles/minute/liter of blood or other body
fluid, under controlled conditions

• Standard way of expressing enzyme activity

Enzyme Activity Units

All enzyme measurements are one of two kinds:

• Fixed time – measurement of a product or substance

produced over a given period of time

• Kinetic (continuous) – measurement of a substrate

product per minute (or hour) produced constantly over a
period of time
Considerations and Storage:

 Avoid hemolysis
 Specimen of choice: serum
 Some anticoagulants may inhibit enzymatic activities

Storage:
 6°C - for 24 hours time storage
 24°C - for storage of less than 24 hours
 -20°C - for longer storage (months-years)
 Samples for CK determination is stored at -70°C
Factors That Influence Enzymatic Reactions

 Substrate Concentration
 Enzyme Concentration
 pH
 Temperature
 Cofactors
 Inhibitors
Clinically Significant
Enzymes
 Creatine kinase
(EC 2.7.3.2.)
• Generally associated with ATP
regeneration

• Major tissue sources: skeletal muscles

and cardiac muscles, and brain tissues

• Very sensitive indicator of AMI with

highest elevations seen in Duchenne’s
muscular dystrophy
CK
 Tanzer Gilvarg Assay (forward/direct method)

Creatine + ATP CK
----------> Creatine phosphate + ADP

PK
ADP + Phosphoenolpyruvate <-----------> Pyruvate + ATP

LD
Pyruvate + NADH + H <----------> Lactate + NAD+
+

pH 9.0 @ 340 nm

CK
 Oliver Rosalki Assay (reverse/indirect method)

CK
Creatine phosphate + ADP -----------> Creatine + ATP

HK
ATP + glucose <------------> ADP + glucose-6-phosphate

G-6-PD
Glucose-6-phophate + NADPH -------------->6–phosphogluconate + NADPH
+

pH 6.8 @ 340 nm

CK
Electrophoretic Migration

Cathode Anode
(-) (+)

CK Mi MM Macro MB BB

ORIGIN

CK
 Sources of Error

• Hemolysis greater than 320 mg/L hgb

• Exposure to light

• elevated baseline – people who are physically well trained

CK
 Reference Range
Total CK:
 Male, 46–171 U/L (37°C)
 Female, 34–145 U/L (37°C)
 CK-MB: <5% total CK

CK
 Lactate Dehydrogenase
(EC 1.1.1.27.)

• Catalyzes the interconversion of lactic and pyruvic acids

• Tissue source: heart, rbcs, kidneys (LD1 and LD2), lungs,

pancreas, spleen (LD3), skeletal muscles, liver, intestine (LD4
and LD5)

• Highest levels are seen in pernicious anemia and hemolytic

disorders
LD
 Lactate Dehydrogenase
(EC 1.1.1.27.)

LD
Wacker Method (forward/direct reaction)

Lactate + NAD LD
------------ > Pyruvate + NADH
pH 8.3 – 8.9

Wroblewski La Due Method (reverse/indirect reaction)

LD > Lactate + NAD

Pyruvate + NADH ------------
pH 7.1 – 7.4
LD
Electrophoretic Migration

Cathode Anode
(-) (+)

LDH6 LDH5 LDH4 LDH3 LDH2 LDH1

ORIGIN

LD
 Sources of Error

• Hemolysis
• LDH4 and LDH5 is labile at 4˚C
• should not be frozen
• Within 3 days storage: Total LDH decreases
• May be stored for 24 hours at room temperature

LD
 Reference Range
125–220 U/L (37˚C)

LD
 Aspartate aminotransferase (EC
2.6.1.1.)
• Involved in the transfer of an amino group between
aspartate and α-keto acids
• Previously known as SGOT
• Major tissue source: cardiac tissue, liver, and
skeletal muscle
• Other source: kidney, pancreas, RBC
• Highest elevation of AST can be found in Acute Viral
AST
Hepatitis
 Aspartate aminotransferase (EC
2.6.1.1.)

AST during AMI:

• Rises: 6-8 hours
• Peaks: 24 hours
• Returns to normal: 5 days

AST
Methods
Methods

• Based on the principle of Karmen method which

incorporates a coupled enzymatic reaction using malate
dehydrogenase (MD) as the indicator reaction and
monitors the change in absorbance at 340 nm
continuously as NADH is oxidized to NAD+.

• The optimal pH is 7.3 to 7.8.

AST
 Karmen Method
Karmen Method

AST
Aspartate + α-ketoglutarate oxaloacetate + glutamate

MD
Oxaloacetate + NADH + H malate + NAD

AST
 Sources
SOURCES of Error
OF ERROR
• Hemolysis

• AST activity is stable in serum for 3 to 4 days at refrigerated

temperatures.

AST
 Reference Range
REFERENCE VALUE

5–35 U/L (37°C)

AST
 Alanine aminotransferase
(EC 2.6.1.2.)
• Catalyzes the transfer of an amino group from alanine to α-
ketoglutarate with the formation of glutamate and pyruvate
• Old term: SGPT
• Has enzymatic activity similar to AST
• Evaluation of hepatic disorders
• Major tissue source: liver
• Other sources: kidney, pancreas, rbc, heart, skeletal muscles,
lungs
ALT
 Methods

• Coupled enzymatic reaction using LDH as the indicator

enzyme, which catalyzes the reduction of pyruvate to lactate
with the simultaneous oxidation of NADH.

• The change in absorbance at 340 nm measured continuously

is directly proportional to ALT activity.

ALT
 Coupled Enzymatic
Coupled Enzymatic Reactions Reaction

ALT
Alanine + α-ketoglutarate pyruvate + glutamate

LD
Pyruvate + NADH + H lactate + NAD

Optimal pH: 7.3 to 7.8

ALT
 AST/SGOT ALT/SGPT
Major organ affected Heart Liver

Substrate Aspartic alpha ketoglutaric acid Alanine alpha ketoglutaric acid

End products Glutamic acid + oxaloacetic acid Glutamic acid + pyruvic acid

Color developer 2,4 DNPH 2,4 DNPH

Color intensifier 0.4N NaOH 0.4N NaOH

Methods Reitman and Frankel Reitman and Frankel

ALT
 Sources of Error

• ALT is stable for 3 to 4 days at 4C.

• It is relatively unaffected by hemolysis.

ALT
 Reference Range
REFERENCE VALUE

7 – 45 U/L (37°C)

ALT
Alkaline Phosphatase
( EC 3.1.3.1.)

• “Alkaline Orthophosphoric Monoester

Phosphohydrolase”
• Activity occurs in pH 9.0 – 10.0
• In healthy sera, ALP levels are derived from liver and
bone (osteoblast)
• Mg2+ - is a necessary cofactor in ALP activity
• Major tissue sources:
 Liver, bone, Placenta, intestinal
ALP
Alkaline Phosphatase
( EC 3.1.3.1.)

Isoenzymes:
Placental isoenzyme
Intestinal isoenzyme
Liver isoenzyme
Bone isoenzyme

ALP
Alkaline Phosphatase
( EC 3.1.3.1.)

| Electrophoresis
- most useful single technique for ALP
isoenzyme analysis

ALP
Electrophoretic Mobility of ALP:

Liver | Bone | Placental | Intestinal

(Fastest to slowest)

ALP
Assays to separate isoenzymes

Heat Stability
• ALP activity is measured before and after heating the serum
at 56oC for 10 minutes.

 Residual activity is less than 20% the total activity before

heating – bone phosphatase (heat labile)

 Residual activity is greater than 20% - liver phosphatase

(heat stable)
ALP
ALP Heat Stability:

Placental | Intestinal | Liver | Bone

(Stable to labile)

Most heat stable:

Regan isoenzyme
(carcinoplacental ALP)
ALP
ALP isoenzymes in cancer patients:

Regan isoenzyme – Lung cancer

Nagao isoenzyme – Adenocarcinoma

Kasahara isoenzyme - Hepatoma

ALP
 Selective Chemical Inhibition

 Phenylalanine inhibits intestinal and placental

ALP and Regan ALP, Nagao ALP.
 3M urea inhibits bone ALP.
 Levamisole reagent inhibits liver and bone ALP.
 L- leucine inhibits Nagao ALP.

ALP
 | ALP Methods|

ALP
 Bowers and McComb Method
ALP
p-nitrophenylphosphate < -------- > p-nitrophenol + phosphate ion

 P – nitrophenolphosphate (colorless) is hydrolysed to

p – nitrophenol (yellow) and the increase in absorbance at
405 nm and at a pH of 10.15, which is directly proportional to
ALP activity, is measured

Reference Method for ALP!

ALP
Clinical Significance
• Often used in evaluation of hepatobiliary (obstructive
conditions) and bone disorders (osteoblast involvement)
• Highest elevation of ALP is seen in Paget’s Disease
• Physiologic elevation of ALP can be seen in pregnant and
growing children

ALP
 Paget’s
Disease

ALP
 Sources of Error
• Hemolysis
• ALP increases at 3% to 10% on standing at 25 oC or 4oC for several
hours.
• Diet may induce elevations in ALP activity of blood group B and O
individuals who are secretors.
• Values may be 25% higher following ingestion of a high – fat meal.
• ALP is inhibited by phosphorus.
The addition of 2–amino–2– methyl–1 –propanol (AMP) buffer
binds phosphorus under Bowers and McComb method.

ALP
 Reference Range
ALP, total (37°C)
Males-Females 4 – 15 y 54 – 369 U/L

Males 20 – 50 y 53 – 128 U/L

> 60 y 56 – 119 U/L

Females 20 – 50 y 42 – 98 U/L
> 60 y 53 – 141 U/L
ALP
 Acid Phosphatase
(EC 3.1.3.2.)
• A hydrolase that catalyzes the same type of reactions with ALP

• Can be found in high concentrations in prostate gland secretions,

bone, liver, spleen, kidneys, RBCs and platelets

• Optimal pH is at 5.0

• Useful in forensic clinical chemistry in the investigation of rape cases –

vaginal washings are examined for seminal fluid – ACP activity which
ACP can persist up to 4 days.
 Diagnostic Significance
• ACP measurement has been used as an aid in the
detection of prostatic carcinoma.

• Prostate–Specific Antigen (PSA) is a more useful

screening and diagnostic tool.

ACP
 ACP Methods

ACP
 Roy and Hillman Method

Substrate: thymolphthalein monophosphate

(specific substrate for PROSTATIC ACP)
- substrate of choice for quantitative endpoint reaction
• End product: Free thymolphthalein

ACP
 Babson, Reed and Phillips Method

- Substrate: alpha – naphthyl phosphate (preferred for

continuous monitoring methods)
- Endpoint: Alpha naphthol

ACP
 Selective Chemical Inhibition

 L - tartrate ions inhibit prostatic ACP.

 Cupric ions and formaldehyde inhibit red cell/

erythrocytic ACP.

ACP
 Source of Error

• Serum ACP decreases within 1 – 2 hours if left at room

temperature.

• If not assayed asap, serum should be frozen or acidified to

a pH lower than 6.5. With acidification, ACP is stable for 2
days at room temperature.

• Hemolysis
ACP
 Reference Range
REFERENCE VALUE

Prostatic ACP, 0–3.5 ng/mL

Tartrate-resistant ACP:
• adults: 1.5 - 4.5 U/L (37°C)
• children: 3.5 - 9.0 U/L (37°C)

ACP
 GGT
( EC 2.3.2.2)

• involved in the transfer of the γ-glutamyl residue from γ-glutamyl peptides

to amino acids, H2O, and other small peptides

• found primarily in tissue of the kidney, brain, prostate, pancreas,

and liver

• Increased in hepatobiliary disorders

GGT
 GGT
( EC 2.3.2.2)

• Measurement: 405 -420 nm

• Szasz Method

-Glutamyl ------ GGT ----- p-nitroaniline

p-nitroanilide

measured by the addition of 10% NaOH

GGT
 Reference Range
REFERENCE VALUE
Male 6 - 55 U/L (37°C)
Female 5 - 38 U/L (37°C)

GGT
 Amylase
(EC 3.2.1.1.)

• Alpha-1-4 Glucan-4-Glucohydrolase
• Digestion of starches
• Requires calcium and chloride ions for its activation.
• Normally filtered by renal glomerulus and also appears in the
urine
• Earliest pancreatic marker!
• Major tissue source: acinar cells of the pancreas and the
salivary glands
AMY
 Amylase
(EC 3.2.1.1.)

• Acute pancreatitis (AP)

• Rise: 5 – 8 hours after onset of attack

• Peak: 24 hours
• Normalize : 3 – 5 days

AMS
 Methods Description
1. Amyloclastic • Measures amylase activity by following the decreases in substrate
concentration (degradation of starch)

2. Saccharogenic • The classic reference method expressed in somogyi units

• Measures the amount of reducing sugars produced by the
hydrolysis of starch by the usual glucose method

3. Chromogenic • Measures amylase activity by the increase in color intensity of the

soluble dye-substrate solution produced in the reaction

4. Continuous • Coupling of several enzyme systems to monitor amylase activity

monitoring (Coupled-
enzyme)

AMS
 Continuous Monitoring

Maltopentose AMS
Maltrotriose + Maltose

Maltotriose + maltose
glucosidase
5-glucose

5-glucose + 5 ATP
hexokinase
5-glucose-6-phosphate + ADP

G-6-PD
5-glucose-6-phosphate + NAD 5,6-phosphogluconolactone + 5 NADH

Optimal
AMS pH 6.9; 340 nm
 Source of Error

• Heparin may inhibit the activity of AMS (using some, but not all methods)
• TAG may inhibit serum AMS activity
• Samples with high activity of AMS should be diluted with NaCl to prevent
inactivation
• Many endogenous inhibitor of AMS, such as wheat germ are present in
serum
• The administration of morphine and other opiates for pain relief before
blood sampling will lead to falsely elevated serum AMS level

AMS
 Reference Range
REFERENCE VALUE
Serum: 28–100 U/L (37°C)
Urine: 1–15 U/h

AMS
 Lipase
(EC 3.1.1.3.)

• Hydrolyzes the ester linkages of fats to produce alcohols and fatty

acids
• Most specific pancreatic marker
• Other name: triacylglycerol acylhydrolase
• In acute pancreatitis (AP)
• levels rise 4 – 8 hours after onset of attack
• peak at 24 hours
• remains elevated for 7 days
LPS • normalize in 8 - 14 days
 Lipase
(EC 3.1.1.3.)

1. Cherry Crandal Method (Reference Method)

Principle:hydrolysis of olive oil after incubation for 24 hours at 37OC and

titration of fatty acids using NaOH

LPS
TAG (Olive oil) + 2H2O <------- > 2-monoglyceride + 2 Fatty Acids

LPS
 Lipase
(EC 3.1.1.3.)

2.Tietz and Fiereck Method

3. Peroxidase Coupling Method
– most commonly used method
- does not use 50% olive oil

LPS
 Reference Range
REFERENCE VALUE
< 38 U/L (37°C)

LPS
 Glucose-6-Phosphate Dehydrogenase
(EC 1.1.1.49.)

• Catalyzes the oxidation of glucose-6-phosphate to 6-

phosphogluconate or the corresponding lactone

• Found in adrenal cortex, spleen, thymus, lymph nodes, lactating

mammary gland, and RBCs

G6PD
 Glucose-6-Phosphate Dehydrogenase
(EC 1.1.1.49.)

Glucose-6-phosphate + NADPH+ --G-6-PD--

6-phosphogluconate + NADPH + H+

G6PD
 Reference Range

7.9–16.3 U/g Hgb

G6PD
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