LACTOSE

DEHYDROGENA
SE Group Members:
Marvin Ryan Duncan 105025624
Shatica Cambridge 105025612
Shaneek Natoya Dabrell 105025615
Junior Solomon Dare 105025619
Kevin Louis 105025628
INTRODUC
Lactate dehydrogenase (LDH) is
TION
an enzyme required during the
process of turning sugar into
energy for your cells. LDH is
present in many kinds of organs
and tissues throughout the body,
including the liver, heart,
pancreas, kidneys, skeletal
muscles, lymph tissue, and
blood cells.
 WHAT IS THE LACTOSE
DEHYDROGENASE TEST?
➤ This is the test conducted to measure the
lactose dehydrogenase level in blood, urine or
cerebrospinal fluid.
➤ It involves obtaining the sample from the
patient and then using an auto analyser to
test the level of LDH in the sample.
PRINCIPLE
➤Lactate dehydrogenase (LD or LDH) catalyzes the
oxidation of lactate by NAD+, to form pyruvate and NADH.
➤The catalytic concentration is determined from the rate of
increase of NADH, measured at 340 nm1,2,3.
➤Lactate + NAD+ Pyruvate + NADH
LDH
• Working Reagent:
➤ Pour the contents of the Reagent B into the Reagent A and mix
gently. Other volumes can be prepared in the proportion: 4 mL
Reagent A + 1 mL Reagent B.
➤ Stable for 3 days at 2-8ºC.

• Sample:
➤ Serum or plasma collected by standard procedures. They must be
separated from the clot as soon as possible.
➤ Ensure that the centrifugation is adequate to remove platelets in
plasma. Do not use hemolysed samples.
➤ Lactate dehydrogenase in serum or plasma is stable for 2 days at
room temperature and for 24 hours at 2-8ºC. Heparin is use as
anticoagulant.
MATERIALS
➤Reagent A: N-Methyl-D-glucamine 0.406 mol/L, lactate 62.5 mmol/L, pH 9.4
➤Reagent B: NAD+ 25 mmol/L.
➤ Sample – heparinized plasma (used in experiment) or serum.
➤Control sample
➤ 37oC water bath
➤Distilled water
➤Test tubes & racks
➤Micropipettes & tips

➤ Semi autoanalyser (spectrophotometer)

➤Vortex

➤Paraffin
 METHOD
1. Gather all the necessary materials and turn on the semi autoanalyser, allow it
to equilibrate and then set to 340 nm.

2 Obtain and label two new tubes Sa’ & ‘C’ for sample and control specimen
respectively.

3.Prepare the working reagent for the Sa tube (Sample) in a 4:1 (A:B) ratio. Add
200 μL of reagent B and 800 μL of reagent A into the test tube labelled "Sa."

4. Prepare the working reagent for the C tube (Control) in a 4:1 (A:B) ratio. Add
200 μL of reagent B and 800 μL of reagent A into the test tube labelled “C".

5. Cover both test tubes using paraffin strips.

6. While tube Sa is left in the rack, pre-warm tube C by incubating it in the 37oC
water bath for 5 minutes.

7. After the 5 minutes, pipette 25 μL of the control sample into tube C while tube C is still
inside the water bath.

8. Immediately after adding the control sample use the vortex to mix the content of Tube C for
5 - 10 seconds.

9. Immediately after mixing the contents of tube C thoroughly, bring it to the semi autoanalyser
for analysis.

10. Wait until the timer was elapsed and record the needed values from the display.

11. Pre-warm tube Sa incubating it in the 37℃ water bath for 5 minutes.

12. After the 5 minutes, pipette 25 μL of the patient's sample into tube Sa while tube Sa is
still inside the water bath.

13. Following steps 7 - 9, mix using the vortex then analyze using the semi auto analyzer for
Tube Sa. Record the results given by the analyzer.
 USING THE SEMI
AUTOANALYSER
➤The semi autoanalyser is pre setup with the setting needed to analyze LDH concentration such
as:

NADH absorbance wavelength 340 nm.

Initial Absorbance reading after first 30 seconds
Subsequent absorbance readings, each at 1 minute intervals for 3 minutes

➤Ensure that the analyzer suction tube is clean before analyzing your sample. If uncertain or the
suction tube is unclean wash the tube using distilled water

➤Washing: Half fill a test tube with distilled water. Place the analyzer suction tube into the test
tube. Ensure the suction tube is at the bottom of the test tube. Press the "WASH" button on the
analyzer interface. Wait until the suction tube uptakes the distilled water, washing is completed
automatically thereafter.
➤ Analyzing your sample: Place the analyzer suction tube into the test
tube containing the sample + working reagent mixture. Ensure the
suction tube is at the bottom of the test tube. Press down the suction
button. Wait until the suction tube uptakes the sample, the tube will
uptake twice.

➤ Thecontrol sample should be analyzed first. Wash the suction tube and
reset the analyser or the next analysis. Analyse the patient's sample.

➤ Thesemi autoanalyser will yield the recordings, as well as the

concentration of both control and sample thus the output should be
simply recorded and recalculated manually for certainty.
 CALCULATIONS
•The LDH concentration is calculated using the following general formula:
•U/L = Vt × 106 × ΔA/min
ε× l × VS
•The molar absorbance (ε) of NADH at 340 nm is 6300 and the light-path (l)
is 1 cm, the total reaction volume (Vt) is 1.025, the sample volume (Vs) is
0.025.
•The calculation of the catalytic concentration deduce the formula:
ΔA/min
x 6508 = U/L
x 108 = µkat/L ΔA/min
x 6508 = U/L
x 108 = µkΔA x 6508= U/L
ΔA/min
x 108 = µkat/L
REFERENCE
VALUES
Reaction Temperature Adults
U/L µKat/L

30ºC 83 - 156 1.38 - 2.59

37º C2 132 - 248 2.20 - 4.13

• Values at 30ºC are obtained from those at 37ºC by using a conversion

factor2. These ranges are given for orientation only; each laboratory should
establish its own reference ranges.
ELEVATED LEVELS
OF LACTOSE
DEHYDROGENASE
Blood flow deficiency. IS
Cerebrovascular
RELATED TO: accident, also
known as a stroke.
Blood flow deficiency.
Cerebrovascular accident, also
known as a stroke.
Certain cancers.
Heart attack.
Haemolytic anaemia.
Infectious mononucleosis.
Liver disease, such as hepatitis.
Muscle injury.
 DECREASED LEVELS
OF LACTOSE
DEHYDROGENASE
It is very rare for a person to have
lowIS RELATED
LDH TO: you may
levels. However,
have low LDH levels if you have
consumed a large amount of
ascorbic acid (vitamin C).
LIMITATIONS
Limitations of the experiment may include; prone to receiving false
results since there is a very slim window between mixing the
sample with the reagent and testing the concentration. The lab
technologist needs to have good lab skills and knowledge, and
malfunctioning of equipment’s can all contribute to an unsuccessful
experiment.