[TRANS] LABORATORY UNIT 02: AMS, LIP, CK and LDH (LABORATORY)

ENZYMES
Table 1. Two Types of Isoenzymes
Review of Concepts:
S – Type P – Type
• Enzymes are proteins and it catalyzes different
biochemical reaction in the body Derived from Salivary tissue,
Derived from Pancreatic tissue
• Enzymes require substrates to yield a product(s) fallopian tube and lung
• Requires specific: S1, S2, S3 P1, P2, P3
o Environmental condition(s) to be active a.k.a. ptyalin
▪ E.g., pH - a pH above or below could denature
the proteins rendering it to be inactive • For electrophoresis:
o Cofactors to be active o S-type: Migrates more quickly than P-type.
o P-type: Migrates slower than S-type.
▪ E.g., Ca2+ in alpha-amylase
• For activity:
o Enzymes can be activated or inhibited
o S-type: Terminated in the stomach because of the
gastric pH.
Alpha-Amylase
Tissue Sources
Properties
• Two major sources of the alpha-amylase:
• IUB Nomenclature:
o Acinar cells of the pancreas
o E.C. 3.2.1.1; 1,4-α-D-glucan glucanohydrolase o Salivary glands
(AMY)
• Other sources: skeletal muscle, small intestine and
• Classification: Hydrolase fallopian tubes
• Molecular weight:
Diagnostic Significance
o 50,000 to 55,000 Da
▪ Amylase is normally occurring in the plasma • Serum and urine AMY measurements is in the
because of its small size. diagnosis of Acute pancreatitis.
▪ Since it is small, it can pass through the o Alpha-AMY - not specific for acute pancreatitis
glomeruli of the kidneys. because it can also be present in other tissues.
o NOTE: Amylase is the only plasma-enzyme found in ▪ If the other tissues are injured, there will also be
the urine. an elevation in the alpha-amylase.
• Activity: • Elevates: 5 to 8 hours after the onset of an attack
o Catalyse the breakdown of starch and glycogen • Peak: 24 hours
• Return to normal levels within 3 to 5 days.
• Substrates: • Values generally range from 250 to 1,000 Somogyi
o Amylose, amylopectin, and glycogen units per dL (2.55×ULN).

▪ Substrates are bigger molecules.

Table 2. Assay for Enzyme Activity
• Products:
o Glucose, Maltose and Dextrins Characteristics Amylase Methodologies
▪ Products are smaller molecules. Measures the disappearance of starch
Amyloclastic
• Optimum pH: 6.9 – 7.0 substrate.
• Cofactors: Calcium and Chloride Saccharogenic Measures the appearance of the product.
o For them to be active. Measures the increasing color from
Chromogenic production of product coupled with a
• Activators: Bromide, nitrate, cholate, or monohydrogen chromogenic dye.
phosphate
Continuous Coupling of several enzyme systems to
• Isoenzyme: S-type and P-type monitoring monitor amylase activity.

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 1
AQUINO. CAGAS
 LABORATORY UNIT 02: AMS, LIP, CK AND LDH

Specimen Collection, Storage, and Sources of Error • Reaction Principle

• Specimen 5 𝐶𝑁𝑃𝐺3 →
α−𝐴𝑚𝑦𝑙𝑎𝑠𝑒
3 CNP + 2 CNPG2 + 3 G3 + 2 G
o Ideal: Serum, Heparinized plasma, urine
• Storage: Table 3. Contents of the Reagent
o Room temperature for 1 week or at 4°C for 2
months Reagent Solution Measurement
• Sources of Error:
MES buffer 36 mmol/l
o Elevated plasma triglycerides
CNPG3 1.6 mmol/l
▪ suppress or inhibit serum amylase activity
Calcium acetate 3.6 mmol/l
o Morphine and other opiates
Sodium chloride 37 mmol/l
▪ Numb the sensation of pain and it will lead to
falsely elevated amylase levels when Potassium thiocyanate 253 mmol/l
administered
Sodium azide 0.095%

Reference Range
• Content of Reagent
• Serum: 28 to 100 U/L (37°C) (0.5 to 1.7 µkat/L)
o NOTE: µkat/micro-Katal – unit of an enzyme’s
catalytic activity
• Urine: 1 to 15 U/h
Package Insert
• Reagent Preparation and Stability
• Specimen
• Assay Parameters
• Method
o The α-amylase liquicolor colorimetric test comprises
a new substrate, 2-chloro-4-nitrophenyl-
maltotrioside (CNPG3).
o The substrate reacts directly with α-amylase and
does not require the presence of ancillary enzymes.
The release of 2-chloro-4-nitrophenol (CNP) from
the substrate and the resulting absorbance increase
per minute is directly related to the α-amylase
activity in the sample.
o The method is calibrated for the new IFCC method
to obtain the same Reference value.
o The different substances stabilize the enzymes.
o Specific pH range – for an enzyme to be active
▪ MES buffer –stabilizes the solution and pH (6.0),
an ideal pH for α-amylase
o Activators
▪ Calcium
▪ Chloride
o Reagent is stable unopened up to the stated expiry
date when stored at 2-8°C.
▪ If the reagent is stable at room temperature or
lower temperature.
▪ For enzymes or other proteins, if it is beyond or
below the required temperature, the enzymes
may be destroyed or inactivated.
o Reagent is ready for use. After opening, it is stable
for 12 weeks when stored light protected at 2-8°C
and for 4 weeks at 15-25°C.
o Contamination of the reagent solution must be
avoided.
o Ideal: Serum, heparinized plasma, and urine
▪ Found in Urine since α-amylase is small in size
o No loss of activity within 5 days at 4-25°C.
o Wavelength: Hg 405 nm, (400-410 nm)
o Optical path: 1cm
o Temperature: 25°C, 37°C
o Measurement: against H2O (increasing absorbance)
o Warm the reagent solution and the cuvettes to the
desired temperature. Temperature must be kept
constant (±0.5°C) for the duration of the test.

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 2
AQUINO. CAGAS
 LABORATORY UNIT 02: AMS, LIP, CK AND LDH

Table 4. Procedure Table 6. Reference Values

Pipette into cuvettes 25°C 37°C Values

Sample 20 µl 10 µl Temperature 25OC 37OC IFCC

Serum, plasma 120 U/I 220 U/I 28-100 U/I

Reagent 1000 µl 1000 µl
Spontaneously
600 U/I 1000 U/I ≤ 460 U/I
voided urine
• Procedure 24-hr urine ** 450 U/24h 900 U/24h ≤ 410 U/I
o (1) Mix well, incubate for 1 minute at the desired
temperature. ** Calculated Values
o (2) Read the absorbance and at the same time start
the stopwatch. • Quality Control
o (3) Read the absorbance again after 1, 2, and 3 o All control sera with α-amylase values determined
minutes. by the α-AMYLASE liquicolor method may be used
o Take note of the sample volume to be used for each o We recommended to use our HumaTrol quality
incubation temperatures (25°C/37°C) and the control sera, based on animal serum, or our
amount of reagent to be added. SERODOS made of human serum
o E.g., 25°C
• Automation
▪ Sample: 20 µl
▪ Reagent: 1000 µl o Proposals to apply the reagents on analysers are
available on request. Each laboratory has to validate
• Take note of the calculations and factors to be used: the application in its own responsibility.
o For amylase, CK and LDH • Notes
o Incubation: 25°C or 37°C
o Saliva and sweat contain α-amylase. To avoid
possible contamination do not pipette by mouth and
Calculation for Alpha-Amylase avoid contact of the reagent and pipette tips with the
skin.
• From the readings determine the mean absorbance o RGT may turn yellowish during shelf life. This does
change per minute (∆𝐴/𝑚𝑖𝑛) and employ this for the not affect the test performance as long as the
calculation of the α-amylase activity in the sample. Use reagent blank is < 0.200 A. Otherwise RGT should
the following factors: no longer be used.
o RGT contains sodium azide (0.095%). Do not
Table 5. Calculation for Alpha-Amylase swallow. Avoid contact with skin and mucous
membranes.
Factors Mean Absorbance Change per minute Lipase

U/I (25°C) = ∆𝐴/ min 𝑥 9864 Properties

U/I (37°C) = ∆𝐴/ min 𝑥 24820 • IUB Nomenclature:
IFCC: U/I (37°C) = ∆𝐴/ min 𝑥 10183 o E.C. 3.1.1.3 : Triacylglycerol Acylhydrolase
• Classification: Hydrolase
• Activity:
• Conversion factors:
o Hydrolyzes the ester linkages of fats to produce
o 1 U/I = 16.67 x 10-3 µkat/I
alcohols and fatty acids
o 1 µkat/I = 60 U/I
Tissue Source
Performance Characteristics
• Pancreas
• Stomach and small intestine
• Linearity
o If the absorbance change per minute exceeds Diagnostic Significance
∆𝐴/ min = 0.300, dilute 0.1 ml sample with 0.5 ml
NaCl solution (0.9%) and repeat the assay using this • Diagnosis of acute pancreatitis
dilution. Multiply the result by 6. • Acute pancreatitis: increases 4 to 8 hours after an
o Typical performance date can be found in attack
Verification Report • Peak: 24 hours
• Decrease within 8 to 14 day
• Persist for approximately 8 days in acute pancreatitis
o The best diagnostic test to use is lipase

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 3
AQUINO. CAGAS
 LABORATORY UNIT 02: AMS, LIP, CK AND LDH

o Lipase arises early and persist for up to 8 days as • Optimum pH:

compared to α-amylase which only persist 2-3 days
o 9.0 (Forward reaction)
after the acute attack.
o 6.7 (Backward reaction)
Assays For Enzyme Activity
Tissue Sources
• Classic Cherry-Crandall Method
• Skeletal muscles
o Reference method • Heart muscle
o Substrate: Olive Oil • Brain tissues
o Endpoint: Measurement of the liberated fatty acids • Other sources: Bladder, placenta, GIT, thyroid, uterus,
by titration after a 24-hour incubation kidney, lung, prostate, spleen, liver, and pancreas
▪ Modifications of the Cherry-Crandall Method are
available but lacks stable and uniform substrate. NOTE: If a tissue needs ATP, it contains creatine kinase.
▪ Triolein (substrate) – pure form of triglyceride; Almost all tissues in the body have creatine kinase
used today to address the problem with modified
methods. Table 7. Creatine Kinase

• Turbidimetric Method
Isoenzymes Tissue Source Electrophoresis
o Fats in the solution create a cloudy emulsion
o As the fats are hydrolyzed by LPS, the particles CK-MM Skeletal muscle CK-3
disperse
CK-MB Heart muscle CK-2
▪ It is a turbidimetric method due to the
involvement of particles.
CK-BB Brain tissues CK-1
o The rate of clearing can be measured as an
estimation of LPS activity
o Relatively simpler and more rapid than the reference
method
• Colorimetric Method
o Based on coupled reactions with enzymes such as
peroxidase or glycerol kinase

Specimen Collection, Storage, and Sources of Error
• Specimen: Serum (ideal specimen)
• Storage: in activity at room temperature for 1 week or
for 3 weeks at 4°C
• Sources of error: Hemolysis should be avoided
because hemoglobin inhibits the activity of serum LPS
o Hemolysis causes falsely low serum LPS values.
• On electrophoretic separation, CK-BB will migrate
fastest towards the anode and is therefore called CK1
o Followed by CK-MB (CK-2)
o Finally, CK-MM (CK-3)
▪ Exhibit the slowest mobility
NOTE: The MM-4 from the skeletal muscles is the major
isoenzyme in the sera of healthy people.

Reference Range Diagnostic Significance

• LPS (serum): <38 U/L (37°C) or (<0.6 µkat/L) • Used to determine if the person/patient have
Myocardial Infarction
o U/L – units per liter
o µkat/L – microkatal per liter o Rise: within 4-8 hours after the onset
o Peak: 12-24 hours
Creatine Kinase o Return to normal: within 48-72 hours

Properties • Elevated levels of the CK-MB, ≥6% of the total CK is

considered a good indicator of myocardial damage,
• IUB Nomenclature: particularly acute myocardial infarction
o E.C. 2.7.3.2 o There is obstruction in the flow of oxygenated blood
supply in the cardiac muscles itself
• ATP: Creatine N-phosphotransferase o If prolonged, cardiac cells are damaged, resulting to
• Classification: Transferase the leaking of CK enzymes inside of the cells
• Molecular weight:
• Troponins
o MW: 82,000 Da
o Non-enzyme proteins
• Activity: o More specific and elevated in the absence of CK-MB
elevations
o Catalyzes the rephosphorylation of ADP to ATP
using creatine phosphate as the phosphorylation
reservoir

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 4
AQUINO. CAGAS
 LABORATORY UNIT 02: AMS, LIP, CK AND LDH

Assays for Enzyme Activity Reference Range

• Tanzer-Gilvarg • Total CK:

o Forward reaction o Males: 46 to 171 U/L (37°C) (0.8 to 2.9ukat/L)
o Reaction Principle:
▪ a bit higher due to muscle mass
▪ Starts with Creatine and ends with lactate
o Females: 34 to 145 U/L (37°C) (0.6 to 2.4ukat/L)
• CK-MB: <5% total CK

Package Insert

o Enzymes involved:
▪ Creatine kinase (CK)
▪ Phosphoenolpyruvate kinase
▪ Lactate dehydrogenase
• Oliver-Rosalki
o Reverse Reaction
o Most commonly performed method
o Proceeds 2-6 times faster than the forward reaction
▪ There is less interference from the side reaction
o Reaction Principle:
▪ Starts with Creatine phosphate and ends with 6-
phosphogluconate

o Enzymes involved:
▪ Creatinine kinase
▪ Glucose-6-phosphate dehydrogenase

Specimen Collection, Storage, and Sources of Error

• Method
• Specimen:
o HumaZym CK-MB is based on an enzymatic CK
o Serum, Heparinised Plasma, EDTA Plasma
determination accompanied by an immunoinhibition
• Storage: method. An antibody is incorporated into the reagent
which will bind specifically to the M-subunit,
o <8hrs at room temperature inhibiting the enzymatic activity of that subunit.
▪ 48hrs at 4°C, and ▪ Thus only the remaining activity of the B-subunit
▪ 1 month -20 C is measured.
• Sources of error: o Due to negligible concentrations of CK-BB in the
o Moderate and severely hemolysed specimens are circulation, the remaining activity, multiplied by the
not accepted for analysis or a request for creatine factor 2, represents the activity of the CK-MB
kinase is made isoenzyme.

▪ RBCs do not contain CK; they are rich in

adenylate kinase • Reaction Principle
▪ Adenylate kinase reacts with ADP to produce 𝐶𝐾
ATP → ATP participates in the assay → causing 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐴𝐷𝑃 ↔ 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒 + 𝐴𝑇𝑃
a falsely elevated CK level. 𝐻𝐾
𝐴𝑇𝑃 + 𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 ↔ 𝐴𝐷𝑃 + 𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 − 6 − 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 (𝐺6𝑃)
𝐺6𝑃−𝐷𝐻
𝐺6𝑃 + 𝑁𝐴𝐷𝑃 ↔ 6 − 𝑝ℎ𝑜𝑠𝑝ℎ𝑜 − 𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑛𝑜 − 𝑙𝑎𝑐𝑡𝑜𝑛𝑒 + 𝑁𝐴𝐷𝑃𝐻 + 𝐻 +

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 5
AQUINO. CAGAS
 LABORATORY UNIT 02: AMS, LIP, CK AND LDH

Table 8. Contents, Reagent Composition in the Test • Procedure

Reagents and its Contents
[BUF] 1 x 60 ml buffer solution
Imidazole buffer (pH 6.7)
Glucose
Mg-acetate
o Prior to determining CK-MB activity it is
Measurement recommended to measure total CK activity using the
CK-NAC activated method (CK-NAC liquiUV, REF
12015, HumaZym M-Test, REF 12005) in order to
ensure correct diagnostic interpretation of the test
0.10 mol/l results.
o Warm reagents and cuvettes to the desired
20 mmol/l
temperature. Temperature must be kept constant
10 mmol/l (±0.5°C) for the duration of the test.

EDTA 2.00 mmol/l

Table 9. Macro and Micro
[ENZ] 20 x 3 ml enzyme/antibody reagent (lyoph.)

ADP 2.00 mmol/l Macro Micro

Pipette directly into the

AMP 5.00 mmol/l Pipette into cuvettes
reconstituted working reagent
Diadenosine pentaphosphate 10 mmol/l Sample/[CAL] 40 µl
Sample/[CAL] 100 µl
Working Reagent 1000 µl
NADP 2.00 mmol/l (1) Mix and incubate at the
(1) Mix and transfer the solution
desired temperature for 10
to a cuvette.
HK > 2.50 U/ml minutes.
(2) Incubate at the desired
G6P-DH > 1.50 U/ml temperature for 10 minutes. (2) Read the absorbance A1.
Read the absorbance A1.
N-Acetylcysteine 20 mmol/l (3) Exactly after 5 minutes read (3) Exactly after 5 minutes read
the absorbance A2. the absorbance A2.
Creatine phosphate polyclonal
30 mmol/l
Antibody to CK-M subunit
Additional material but not supplied with the kit
Calculation
[REF] 13611
• Calculate the absorbance change per minute (ΔA/min)
[LowHigh] 4 x 2 ml CK-MB Control Human Serum (lyophilised) according to:
[REF] 13612 o ΔA/min = A2-A1: 5.

[CAL] 2 x 1 ml CK-MB Calibrator Human Serum (lyophilised) • The CK-MB activity is calculated using the following
factors:

• Reagent Preparation and Stability Table 10. CK-MB Activity (U/I)

o (1) Reconstitute one vial ENZ with exactly 3ml BUF.
o (2) Swirl gently and incubate for 5 min. at room Semi-micro Macro
temperature prior to use.
o The reagents are stable up to the stated expiry date Hg 334 nm 8414 x ΔA/min 10032 x ΔA/min
when stored at 2…8°C
o The reconstituted working reagent is stable for 5 340 nm 8254 x ΔA/min 9842 x ΔA/min
days at 2…8°C
Hg 365 nm 14858 x ΔA/min 17716 x ΔA/min
• Specimen
o Serum • Conversion factor of traditional units (U/I) in SI-units
o Heparinised plasma or EDTA plasma (kat/l)
o Loss of activity within 1 day at 2…8°C: <10%
o 1 U/l = 16.67x10-3 µkat/l
• Assay o 1 µkat/l = 60 U/l
o Wavelength: Hg 334nm, 340 nm, Hg 365 nm Performance Characteristics
o Optical path: 1 cm
o Temperature: 25°C, 30°C or 37°C • Linearity
o Measurement: against air (increasing absorbance)
o The antibody inhibits the activity of M-subunits up to
2000 U/I of CK MM
• Reference Range, Myocardial Infarction (MI)
o The likelihood of myocardial damage is high if the
following 3 criteria are met:

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 6
AQUINO. CAGAS
 LABORATORY UNIT 02: AMS, LIP, CK AND LDH

Table 11. Reference Range (MI) Table 12. Lactate Dehydrogenase (LDH) Isoenzymes –
Tissue Localization and Sources of Elevation
25℃3 30℃4 37℃
Isoenzyme Tissue Disorder
Total CK
Heart Myocardial infarction
Men > 80 U/I > 130 U/I > 195 U/I LDH-1 (HHHH)
Red blood cells Hemolytic anemia
Women > 70 U/I > 110 U/I > 170 U/I
Megalobalastic anemia
Heart
LDH-2 (HHHM) Acute renal infarct
CK-MB > 10 U/I > 16 U/I > 25 U/I Red blood cells
Hemolyzed specimen
CK-MB activity ranging between 6% and 25% of the total CK Pulmonary embolism
activity Lung Extensive Pulmonary
Lymphocytes pneumonia
LDH-3 (HHMM)
Spleen Lymphocytosis
• Quality control Pancreas Acute pancreatitis
Carcinoma
o All control sera with CK-MB values determined by Hepatic injury or
this method can be employed LDH-4 (HMMM) Liver
inflammation
• Automation LDH-5 (MMMM) Skeletal muscle Skeletal muscle Injury
o This kit is designed for manual use. For use on
automates we recommend [REF] 12118. Proposals
to apply this reagents on analysers are available on
request. Each laboratory has to validate the Table 13. Lactate Dehydrogenase (LDH) Isoenzymes
application in its own responsibility. as a Percentage of Total LDH
• Note
o [BUF] contains sodium azide (< 0.095%) as Isoenzyme %
stabiliser. Avoid contact with the skin and mucous
membranes. LDH-1 14-26

Lactate Dehydrogenase LDH-2 29-39

Properties LDH-3 20-26

LDH-4 8-16
• IUB Nomenclature:
o E.C. 1.1.1.27 LDH-5 6-16

• ATP: L-Lactate: NAD+ Oxidoreductase

• Classification: Oxidoreductase • LDH-2 - The major isoenzyme infraction in the sera of
• Activity: healthy individuals
o Catalyzes the interconversion of lactic & pyruvic Diagnostic Significance
acids
• Myocardial Infarction
• Cofactor: Zinc
• Optimum pH: o Similar to CKMB, LDH is also elevated in MI
because of its wide distribution to the body.
o Forward reaction: 8.3-8.9
o Backward reaction: 7.1-7.4 ▪ However, the heart tissue and red blood cells
contains higher concentration of LDH. Thus, it is
not specific for MI and not preferred marker for
Tissue Sources diagnosing acute myocardial infraction.
• Widely distributed in the body: o Rise: within 12-24 hours after the onset
o Peak: 48-72 hours
o Heart o Remain elevated: 10 days
o Liver
o Skeletal muscle ▪ LD 1 > LD 2
o Kidney  LD 1 is the dominant isoenzyme
o Erythrocytes
o Lung  LD flipped pattern
o Smooth muscle  Suggestive AMI
o Brain
• Lactate dehydrogenase is elevated in a variety of
disorders due to its widespread activity in numerous
body tissue, with the highest levels noted in pernicious
anemia and hemolytic disorders

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 7
AQUINO. CAGAS
 LABORATORY UNIT 02: AMS, LIP, CK AND LDH

Assays for Enzyme Activity • Method

o “Modified method” based on the recommendations
of the SCE (Scandinavian Committee on Enzymes).
• Reaction Principle
• Forward reaction 𝐿𝐷𝐻
o Substrate: lactate 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 + ↔ 𝐿𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷 +
o Also known as _______
o Produces NADH Table 14. Contents, Reagent Composition in the Test
o Not affected by the product inhibition
o LD-1
Reagents and its Contents Measurement
• Reverse reaction
o Substrate: pyruvate [REF] 12214 12014 12024
o Also known as ________
o LD 5 [BUF] 16 x 4 ml 10 x 8 ml 8 x 40 ml
o Rate of the reverse reaction is approx. 3 times faster [SUB] 1 x 16 ml 2 x 10 ml 8 x 10 ml
but more susceptible to substrate exhaustion and
loss of linearity [BUF] Buffer / Substrate

▪ Allowing smaller samples and volumes and TRIS Buffer (pH 7.35) 62.5 mmol/l
shorter reaction times
Pyruvate 1.5 mmol/l
o Optimal pH
Sodium azide 0.095%
▪ Forward: 8.3 – 8.9
▪ Backward: 7.1 – 7.4 [SUB] Substrate

Specimen Collection, Storage, and Sources of Error NADH 0.75 mmol/l

Sodium azide 0.095%

• Specimen:
o Serum, Heparinised or EDTA plasma • Reagent Preparation
• Storage: o Procedure 1 with reagent start
o 25℃ analysed within 48hrs ▪ The reagents are ready for use
▪ The reagents are stable, even after opening, up
• Source of error: Hemolysis to the stated expiry date when stored at 2-8oC.
o Erythrocytes contains an LD concentration ▪ BUF must be kept light protected.
approximately 100 to 150 times found in the serum. ▪ Contamination of reagents must be avoided
o If there is any red coloration after centrifugation of o Procedure 2 with sample start
the serum or plasma, it is unacceptable for analysis
because it causes falsely elevation of LDH. ▪ REF 12024: Pour the entire contents of one
bottle SUB into one bottle BUF, mix thoroughly.
Reference range ▪ REF 12214: Pipette 1 mL from bottle SUB into
one bottle BUF, mix thoroughly.
• LD: 125 to 220 U/L (37℃) ▪ REF 12014: Pipette 2 mL from bottle SUB into
one bottle BUF, mix thoroughly.
Package Insert o The working reagent is stable for 3 weeks at 2…8oC
and 3 days at 15…25oC.
o The working reagent must be light protected.
• Specimen
o Serum, heparinized or EDTA plasma
o Avoid Hemolysis
o Loss of activity within 3 days 8% at + 4℃, 2% at
15…25℃
• Assay
o Wavelength: Hg 334 nm, 340 nm, Hg 365 nm
o Optical path: 1 cm
o Temperature: 25℃, 30℃ or 37℃
o Measurement: against air (decreasing absorbance)
o Warm the reagents and the cuvettes to the desired
temperature.
o Temperature must be kept at constant (±0.5℃) for
the duration of test.

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 8
AQUINO. CAGAS
 LABORATORY UNIT 02: AMS, LIP, CK AND LDH

Table 15. Procedure 1 Performance Characteristics

Pipette into • Linearity

25℃, 30℃ 37℃
cuvettes
o If the absorbance change per minute (∆𝐴/min )
Sample 20 µl 10 µl exceeds 0.150 at Hg 334 nm, 340 nm or 0.070 at Hg
[BUF] 1000 µl 1000 µl 365 nm.
Mix, incubate for 1-5 min. at 25℃, 30℃, or 37℃ o Dilute 0.1 mL of the sample with 0.9 mL
physiological saline (0.9%) and repeat the assay
[SUB] 250 µl using this dilution.
o Multiply the result by 10.
Mix, read the absorbance after 1 minute and at the same time start
the stop watch. Read the absorbance again exactly after 1, 2 and 3
minutes. Table 19. Reference values

Table 16. Procedure 2 Temperature 25 30 37 IFCC4

Pipette into Adults [U/I] 120-240 160-320 225-450

25℃, 30℃ 37℃
cuvettes
Men [U/I] < 243
Sample 20 µl 10 µl
Working reagent 1000 µl 1000 µl
Mix, read the absorbance after 1 minute and at the same time start Women [U/I] < 244
the stop watch. Read the absorbance again exactly after 1, 2 and 3 Children [U/I]
minutes. (up to 12 Up to 500
months)
• Semi-micro method; for macro methods double the
volumes.
• Quality Control
Calculation
o All control sera with LDH values determined by this
• Using the absorbance readings calculate the mean method can be employed.
absorbance change per minute (ΔA/min) o We recommended to use our animal serum based
• Calculate the LDH activity in the sample by multiplying HumaTrol or our human serum based SERODOS
ΔA/min using the following factors: quality control sera.
• Automation
Table 17. Calculation for Procedure 1
o Proposals to apply the reagents on analysers are
available on request.
Hg 334 nm 340 nm Hg 365 nm o Each laboratory has to validate the application in its
own responsibility.
U/I (25℃, 30℃) =
10275 10080 18675 • Note
ΔA/min. x
U/I (37℃ ) = o [BUF] and [SUB] contain sodium azide (0.095%). Do
20390 20000 37060
ΔA/min. x
not swallow. Avoid contact with skin and nucous
membranes.
Table 18. Calculation for Procedure 2
SUMMARY
• A single enzyme is not diagnostic and not specific for a
Hg 334 nm 340 nm Hg 365 nm
disease
U/I (25℃, 30℃) =
• To avoid falsely increased/falsely decreased values we
8250 8095 15000 must take note of the different sources of errors for
ΔA/min. x
U/I (37℃ ) = different enzymes
ΔA/min. x
16345 16030 29705 • Observe the proper storage of specimen if we can’t
analyse them right away

• Conversion factor of the traditional units (U/I) in SI-units

(kat/I):
o 1 U/I = 16.67 x 10-3 ukat/I
o 1 µkat/I = 60 U/I
• Factor to convert results to the new IFCC
recommended method:
o U/I (LDH SCE) x 0.4796 = U/I (LDH IFCC)

ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 9
AQUINO. CAGAS