DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404
Title:
Measurement of enzyme activity (Lactate dehydrogenase)

Objective:
 To determine the rate of enzyme reaction, by monitoring the rate of
absorbance changes
 To learn the principle of coupled enzyme reaction

Principle:
Two of the most usual or common reaction that occur in the living
organism is oxidation and reduction. One of the enzymes that represent
this reaction is lactate dehydrogenase that present in all human tissue.
Usually this enzyme can be found in high concentration in heart, liver and
skeletal muscle. Lactate dehydrogenase can do process oxidation and
reduction. So, the main focus for this experiment is to see and measure
the activity of lactate dehydrogenase in oxidation and reduction process.

In reduction process:
L − lactate + NAD + Lactatedeh
  → Pyruvate + NADH + H +
ydrogenase

In oxidation process:
Pyruvate + NADH + Lactatedeh
  → L − lactate + NAD +
ydrogenase

In measuring the activity of enzyme we must use international unit. One

international unit (1 U/I) is defined as the amount of enzyme that
catalyzes the transformation of one micromole of substrate per minute.

Reagent and sample: (Oxidation Process)

Reagent (NAD), sample A, sample B, control, water bath,
spectrophotometer

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Methods: (Oxidation Process)
Warm NAD reagent to 300C or 370C for 2-3 minutes

1000µl NAD reagent + 50 µl sample A/B/control

Mix and measure at wavelength 340nm with air as a

blank

Read initial absorbance after 0.5 min and start timer

simultaneously. Read again after 1, 2 and 3 minutes

Plot the graph and calculate lactate dehydrogenase

activity

Results:
Sample 0.5 min 1 min 2 min 3 min
Sample A 0.368 0.386 0.403 0.420
Sample B 0.445 0.461 0.474 0.490
Control 0.572 0.605 0.633 0.659

Calculation:
To calculate the lactate dehydrogenase, the following formula was used:
LD activity (U/I) = 3333 X ΔA340 nm/min
For control:
(0.63 − 0.57)
ΔA340 = = 0.04 nm/min
( 2 − 0.5) min
So LD activity for control: 3333 X 0.04 = 133.32
Control range: 157.99 ± 15.884 (2SD)

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For sample A:
(0.42 − 0.37)
ΔA340 = = 0.02 nm/min
(3 − 0.5) min
So LD activity for sample A: 3333 X 0.02 = 66.66

For sample B:
(0.49 − 0.45)
ΔA340 = = 0.02 nm/min
(2.75 − 0.75) min
So LD activity for control: 3333 X 0.02= 66.66

Reagent and sample: (Reduction Process)

Reagent (NADH), Color reagent (2, 4 – dinitrophenylhydrazine and
hydrochloric acid), sodium hydroxide (NaOH) 1.6% (0.4 N), sample A,
sample B, control, water bath, spectrophotometer, distilled water

Methods: (Reduction Process)

: To make a calibration curve

Tube 1: Tube 2: Tube 3: Tube 4: Tube 5: Tube 6:

0.9 ml 0.7 ml 0.5 ml 0.3 ml 0.2 ml 0.1 ml
NADH NADH NADH NADH NADH NADH

Place in water bath at 370C for 2 – 3 minutes

Tube 1: Tube 2: Tube 3: Tube 4: Tube 5: Tube 6:

0.1 ml 0.3 ml 0.5 ml 0.7 ml 0.8 ml 0.9 ml
distilled distilled distilled distilled distilled distilled
water + water + water + water + water + water +
1.0 ml 1.0 ml 1.0 ml 1.0 ml 1.0 ml 1.0 ml
color color color color color color
reagent reagent reagent reagent reagent reagent

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 Mix, and allow standing for 20 min at room
temperature

Add 10.0 ml NaOH 1.6%

Mix, and allow standing for 10 min at room

temperature

Measure absorbance at 520nm with distilled water

as a blank. Measure absorbance 3 times

Take the mean and draw graph absorbance vs

enzyme activity

Reference: enzyme activity (U/I) for lactate dehydrogenase

Sample 1 2 3 4 5 6
U/I 0 111.4 223.1 334.9 390.6 446.3

: For sample A, sample B and control

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 Add 0.9 ml NADH into test tube and place in water
bath at 370C for 2 – 3 minutes

Add 0.1 ml sample A/B/Control

Mix, and incubate in a water bath at 370C for 30

minutes

Add 1.0 ml of color reagent, mix and allow to stand

up at room temperature for 20 minutes

Add 10.0 ml NaOH 1.6%

Mix, and allow standing for 10 min at room

temperature

Measure absorbance at 520nm with distilled water

as a blank. Measure absorbance 3 times

Determine the enzyme activity using a reference

calibration curve

Results:

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Sample / Ctrl Absorbance
Reference st
1 reading nd
2 reading 3rd reading Average
1 0.226 0.231 0.269 0.242
2 0.206 0.197 0.197 0.200
3 0.154 0.146 0.170 0.157
4 0.134 0.149 0.102 0.128
5 0.093 0.077 0.083 0.084
6 0.044 0.075 0.047 0.055
Sample A 0.186 0.167 0.164 0.172
Sample B 0.151 0.154 0.132 0.146
Control 0.109 0.108 0.118 0.112

Calculation:
From the graph that was plotted when the control absorbance is 0.112, the
LD activity is 343.
Control range: 351.41 ± 15.44 (2SD)

From the graph that was plotted when the sample A absorbance is 0.172,
the LD activity is 152

From the graph that was plotted when the sample B absorbance is 0.146,
the LD activity is 250

Discussion:
Enzymes play an important role especially in disease detection. In
pathologic states involving tissue necrosis and neoplasia, leakage of enzyme
from even a small amount of damaged tissue can significantly raise levels in
various body fluids.
So, we can determine the serum enzyme level in indicating cellular
damage. In reduction process, pyruvate is converted to lactate, but in oxidation
process lactate are converted back to pyruvate.
Lactate dehydrogenase, also called lactic dehydrogenase, or LDH, is an
enzyme found in the cells of many body tissues, including the heart, liver,
kidneys, skeletal muscle, brain, red blood cells, and lungs. It is responsible for
converting muscle lactic acid into pyruvic acid, an essential step in producing
cellular energy.
Lactate dehydrogenase oxidizes the secondary alcohol in lactate to a
carbonyl in pyruvate. Lactate dehydrogenase removes 2 H atoms (not H+) from
the substrate, hence the enzyme name dehydrogenase. The 2 H atoms are
donated to a common biological oxidizing agent, NAD , +
(nicotinamide
adenine dinucleotide), which is reduced to NADH and H+.

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 NAD+ does not absorb NADH strongly absorbs
ultraviolet at 340 nm

The reaction progress can be monitored by measuring the ultraviolet

absorbance increase at 340 nm due to the formation of NADH. Absorbance is
measured in a spectrophotometer, which provides a light source with selectable
wavelength in the range 200-700 nm (ultraviolet and visible light).
Sample is contained in a square chamber or cuvette of exactly 1.00 cm
thickness. The light passing through the sample is then measured and recorded
by a detector. If light is absorbed by the sample, the measured intensity I passing
through the sample is less than the original intensity of the beam Io.
In biological systems, a coupled oxidation-reduction reaction is one in
which one substance is oxidized (loses electrons) while a second is reduced
(gains electrons). An example of one such reaction is the conversion of lactate to
pyruvate

1. Principles involve in this experiment:

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 L − lactate + NAD + Lactatedeh
  → Pyruvate + NADH + H +
ydrogenase

Formation of NADH results in an increase in absorbance at 340nm,

which is proportional to the rate of lactate dehydrogenase activity
in the sample

Pyruvate + NADH + Lactatedeh

  → L − lactate + NAD +
ydrogenase

The pyruvate that remains unchanged reacts with 2, 4 –

dinitrophenylhydrazine to give the corresponding phenylhydrazone,
which is determined colorimetry in alkaline medium

2. Discussion of the results:

From experiment 1 (oxidation of lactate to pyruvate), the control
value falls outside the range. So the graph that was draw are not
good enough to determine the LD activity for sample A and sample
B. These errors occur maybe due to some mistake during this
method was made. The example of mistake is the time when the
reading was taken are not accurate and the interference that occur
inside the spectrophotometer. From this experiment the LD activity
for control is 133.32 and the LD activity for sample A and sample B
is same (66.66). The reference for control value is 157.99 ± 15.884
(2SD)

From experiment 2 (reduction of pyruvate to lactate), the control

value falls inside the range. So the graph was accurate and can be
used to determine the LD activity for sample A and sample B. From
this experiment the LD activity for control is 343 and the LD
activity for sample A is 152 and sample B is 250. The reference
for control value is 351.41 ± 15.44(2SD)

3. Function of lactate dehydrogenase. The significance of measuring this

enzyme:
Lactate dehydrogenase found in almost all tissue of body. It plays
oxidation and reduction process in body system. In reduction
process, pyruvate is converted to lactate, but in oxidation process
lactate are converted back to pyruvate. By measuring this
enzyme activity we can determine the level or activity of lactate
dehydrogenase in human body. If any changes occur, the result
shows an abnormal activity of lactate dehydrogenase, so it can
indicate the present of disease in human body.

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 4. Principle involve, advantages and disadvantages in end point,
multipoint, and kinetic assay methods:
End point: in this method, after addition of the substrate, we set the
time, stop the reaction and finally stop the reaction by taking the
concentration of product that was formed. In order words, this
method only takes the final concentration of the product.
Advantages of this method are simple, fast and can be easily
automated. The advantages are this method can’t show the
substrate depletion and if enzyme activity is high, it can be under
estimation.

Multipoint: in this method, we read the reading (concentration of

product) at certain range of time until the final reading. So we can
get more result at different time. The advantages are this method
can show different types of reaction (for example linear graph,
show substrate depletion, and reaction with lag phase). The
disadvantages are this method need more time to finish (time
consuming)

Kinetic assay: this method also called continuous assay. It usually

use in determine the rate activity of enzyme to the substrate and
the principle same with multipoint assay. These advantages are this
method provide information about rate of enzyme reaction and
also provide more result at different time. The disadvantages are
this method also needs a long period of time to done (time
consuming)

Conclusion:
From experiment 1: the control value falls outside the range. So the
graph that was draw are not good enough to determine the LD
activity for sample A and sample B. LD activity for control is 133.32
and the LD activity for sample A and sample B is same (66.66). The
reference for control value is 157.99 ± 15.884 (2SD)

From experiment 2: the control value falls inside the range. So the
graph was accurate and can be used to determine the LD activity
for sample A and sample B. From this experiment the LD activity
for control is 343 and the LD activity for sample A is 152 and
sample B is 250. The reference for control value is 351.41 ±
15.44(2SD)

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