CREATINE KINASE (new) LACTATE DEHYDROGENASE

CREATINE PHOSPHOKINASE (old)

SYSTEMATIC NAME ATP: Creatine N-Phosphotransferase L-Lactate: NAD Oxidoreductase
ENZYME CLASSIFICATION # 2.7.3.2 1.1.1.27
REACTION CATALYZED Reversible phosphorylation of Creatine by ATP Reversible Oxidation of L-lactate to pyruvate
with the mediation of NAD as hydrogen
acceptor or coenzyme
REACTION EQUATION
9.0 8.8-9.8
Creatine + Phosphocreatine L-lactate + Pyruvate +
ATP CK + ADP NAD+ LDH NADH + H+
6.8 7.2-7.8
*Reverse Reaction is 2-5x Faster
REACTION EXPLANATION Happens during muscle contraction where ATP is In the body, pyruvate is formed as end product of
hydrolyzed to ADP to produce chemical energy the EMBDEN-MEYERHOF pathway which converts
glucose to energy
CK is an anzyme generally associated with ATP
generation in contractile or transport systems It is the key branch point in this process and can be
further metabolized to a number of different
Phosphocreatine = regenerate ATP products

Every contraction cycle = Phosphocratine use + ATP When O2 level in the system is low, part of the
production = relatively constant levels of muscle pyruvate is converted to lactate (reduction)
ATP

Also involved in phosphorylation in the

Mitochondria
TISSUE DISTRIBUTION Widely Idstributed Widely Distributed

Highest in Skeletal Muscles High conc. Found in the Heart and Liver

Also found in: Significant amounts are found in the RBC

- Cardiac Muscle
- Brain tissues - Hemolysis produces an artefactual
- Intestinal tract increase in serum levels
- Kidney - 100-150x greater activity in RBC than
- Uterus serum
- Prostate
SAMPLE COLLECTION SERUM = specimen of choice SERUM = specimen of choice
*HEMOLYSIS MUST BE AVOIDED (False high)
PLASMA = must be aware of anticoagulants that are Freezing = may distort isoenzyme distribution
inhibitors (Oxalate, Fluoride, Citrate, EDTA) markedly (False low)

Exposure to light = False low CSF, Urine

Hemolysis = False high
Plasma inhibitor anticoagulants = false low
ACTIVATORS 1. SH- 1. SH-
- Must be in REDUCED form - Avoid delay in Processing to avoid
- Loses its activity when oxidized to S-S oxidation to S-S dimer
dimer (disulfide) *DELAY IN PROCESSING*
- To prevent oxidation:
 Incorporate CYSTEINE or its
derivtives
 NALC, Glutathione,
DIthiothreitol(Cleiand’s Rgt),
Monothioglycerol
- Helps maintain the 3D structure of the
enzyme
2. Mg ions
3. Mn and Ca (lesser extent)

INHIBITORS 1. Chloride and Sulfate 1. Merchuric chloride

2. Traces of Divalent ions like Zn, Cu, Hg, Ag 2. N-ethylmaleimide
3. Oxalate, Fluoride, Citrate, EDTA 3. Chloromercuribenzoate
4. L – thyroxine and iodoacetate - Prceipitants of SH-
- Inhibition can be made reversible by
CYSTEINE or GLUTATHIONE
4. Borate or Oxalate
- Compete with Lactate for its primary
binding site on the enzyme
5. Oxamate
- Compete with Pyruvate
6. Urea
- Selectively inhibits LD isoenzyme and may
be used as a basis for LD5 isoenzyme
studies
7. Sulfite
- Selectively inhibits LD1 and LD2
8. Iodate, EDTA

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MEASUREMENT OF TOTAL GENERAL PRINCIPLES CREATINE KINEASE LACTATE DEHYDROGENASE
ACTIVITY
*COUPLED ENZYME REACTION since neither substrates nor products of the main reaction have any * Most determination are based on the detection of NADH in the reaction
unique structural characteristics which would allow easy detection by spectrophotometric or *NADH = has a strong absorance at 340nm which permits direct
fluorometric means spectrophotometric measurement
*NADH can be detected by a coupled reaction with REDOX INDICATOR or
TETRAZOLIUM SALT
METHODS OF ESTIMATION CREATINE KINASE LACTATE DEHYDROGENASE
FORWARD REACTION UV ADP AUTHOR Tanzer and Gilvarg AUTHOR Amador et al
SPECTROPHOTOMETRY NADH Wacker, Ulmer, Valle
MODE OF ADP is measured by following the rate of DECREASE MODE OF NADH is measured by following the rate of
MEASUREMENT in Absorbance at 340nm MEASUREMENT INCREASE in Abs at 340nm
WAVELENGTH 340nm WAVELENGTH 340nm
REACTION 1. Creatine CK Phosphocreatine + Reaction LD
+ ATP -------> ADP L-lactate +NAD ------> Pyruvate + NADH +
H+
2. ADP PK Pyruvate + ATP
+ Phosohoenol pyruvate ------>

3. Pyruvate + LD Lactate + NAD+

NADH ----- >
PRIMIARY REACTION: Phosphorylation of Creatine by ATP
INTERMEDIATE REACTION: ADP Participates with PEP catalyzed by Pyruvate Kinase regenerating ATP
and producing pyruvate
INDICATOR REACTION: Reaction of Pyruvate with NADH to form lactate and NAD, catalyzed by LD

*NAD DOES NOT ABSORB UV LIGHT AT 340nm

*More rapid drop = higher enzyme activity
*Absorbance reading should be decreasing
*Drawback: initial 340m in the Abs system is rather high, so lower levels of activity are not measured
accurately since small changes in Abs are more difficult to detect
COLORIMETRY ADP AUTHOR Nuttal and Wedin NADH AUTHOR Babson and Phillips
Nechlass
Raabo and Scand
MODE OF Photometric measurement of MODE OF Photometric measurement of INT (2-p-
MEASUREMENT Pyruvate -2,4-dinitrophenylhydrazone (brown) in MEASUREMENT iodophenyl-3-p-nitrophenyl-5-phenyl
alkaline solution tetrazolium chloride) in its reduced form
(Formazan)
INT – redox indicator and is colorless unless
reduced by NADH to formazan

REACTION Pyruvate + Alkali Pyruvate-2,4- NADH AUTHOR Ellis

2,4 DNPHzine -----------> DNPHzone

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 CREATINE AUTHOR Hughes and Dreyfus et al MODE OF Photometric measurement of 2-6
MEASUREMENT DIchloroindophenol in its reduced form
DESCRIPTION Product of Acid Hydrolysis of Phosphocreatine PYRUVATE AUTHOR Cabaud and Roblewski
King
Zimmerman and Weinstein
MODE OF Colorimetric Measurement of red colored solution MODE OF Photometric measurement of hydrazone as
MEASUREMENT formed by reaction with diacetyl and α-naphthol in MEASUREMENT the product of 2,4-dinitrophenylhydrazine
alkaline solution made to reach with pyruvate
REACTION Alkali
Creatine + diacetyl + α-naphthol --------> red color
CREATINE AUTHOR Folin
MODE OF Measurement of CREATININE formed by Jaffe’s
MEASUREMENT reaction before and after heating of CREATINE with
ACID

Difference is taken as a measure of amount of

creatine present

PHOSPHATE AUTHOR Fiske and Subbarow; Gomori

DESCIPTION Product of Acid Hydrolysis of phosphocreatine
Any method for measurement used in serum
inorganic phosphate determination
MODE OF Photometric measurement of MOLYYBDENUM BLUE
MEASUREMENT formed by reduction of AMMONIUM
MOLYBDOPHOSPHATE (formed by inorganic
phosphate and ammonium molybdate) by p –
semidine
FLUOROMETRY CREATINE AUTHOR Sax and Moore; Armstrong et al NADH MODE OF NADH is measured by its fluorescence
MEASUREMENT
DESCRIPTION Product of Acid Hydrolysis of Phosphocreatine
MODE OF Assay of fluorescent compound (fluorophor)
MEASUREMENT produced by the reaction with triketohydrindene
hydrate (ninhydrin) in strong alkali
REACTION Alkali
Ninhydrin + creatine -------> fluorescence
(fluorophor)

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REVERSE REACTION UV ATP AUTHOR Oliver and Rosalki NAD AUTHOR Wrobleswki and Ladue
SPECTROPHOTOMETRY Kubowitz and Ott
Henry, mod.
MODE OF ATP is measured by following the rate of INCREASE in MODE OF NAD is measured by following the rate of
MEASUREMENT absorbance at 340nm MEASUREMENT DECREASE in absorbance at 340nm

*NAD does not absorb UV light at 340nm

REACTION CK REACTION LD
1. Creatine Phosphate ---------> Creatine + ATP Pyruvate + NADH + H+ -----> L-Lactate +
+ ADP NAD+
HK
2. ATP + Glucose -----------> ADP + Glucose-6-
phosphate

3. Glucose-6-phospate G6PD 6phospphogluconate

+ NADP ----------> +NADPH + H+
SAMPLE/CALIBRATOR 40 uL or 0.04mL SAMPLE/CALIBRATOR 20 uL / 0.02mL
BLANK 50uL or 0.05mL distilled water (WATER BLANK) BLANK 20 uL / 0.02mL
MONOREAGENT 1000uL or 1mL REAGENT 1: 1000uL / 1mL
INCUBATION TIME 5 minutes (Initial absorbance reading) INCUBATION TIME 1 minute
NO. OF SUCEEDING 1 min -> 2 min -> 3 min -> 4 min -> 5 min NO OF SUCCEEDING 1 min -> 2 min -> 3 min
ABS READINGS *Absorbance reading should be increasing ABS READINGS
TOTAL NO. OF 6 ( Initial + 5 succeeding 1 minute intervals) TOTAL NO. OF 4 (Initial + 3 succeeding 1 minute intervals)
READINGS READINGS
FORMULA WITHOUT CALIBRATOR: FORMULA WITHOUT CALIBRATOR:
CK-MB(U/L) = Abs/min x Factor LDH(U/L) = Abs/min x Factor
Abs/min = Sum of the differences of each interval / Abs/min = Sum of the differences of each
5 interval / 3
WITH CALIBRATOR:
CK-MB(U/L) WITH CALIBRATOR:
= Abs/min Sample x Conc of Calibrator LDH(U/L)
Abs/min Calibrator = Abs/min Sample x Conc of Calibrator
Abs/min Calibrator
FACTOR 8254 without CK-MB DS; 4127 with CK-MB DS FACTOR 8095
CONVERSION FACTOR 0.0167 CONVERSION 0.0167
TO ukat/L CK-MB(ukat/L) = CK-MB(U/L) x 0.01667 FACTOR TO ukat/L LDH(ukat/L) = LDH(U/L) x 0.01667
PERFORMANCE Developed to Determine CK-MB activities up to PERFORMANCE Automated system
CHARACTERISTICS 2000U/L CHARACTERISTICS - Developed to determine LDH
If value is exceeded, dilute with NaCl soln (9.0g/L) activities up to 1200 U/L

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 Manual Procedure
- Suitable for LDH activities which
correspond to a maximum of 0.15
average change of
absorbance/min at 340 nm and
0.08 at 365nm

If these values exceed, dilute sample with

1+10- NaCl solution (9.0g/L) and results
multiplied by 11

REFERENCE RANGES CK(MEN) = >190U/L (3.17ukat/L) REFERENCE RANGES FEMALE = <247 U/L (4.12ukat/L)
CK(WOMEN) = >167U/L (2.78ukat/L) MALE = <248 U/L (4.14ukat/L)
CK-MB = >24U/L (0.40ukat/L)
CK-MB activity is between 6 and 25% of total CK
activity
OTHERS AUTHOR Nielsen and Ludvigsen

Hess et al
ADENYLATE KINASE Found in high concentrations in RBCs

Enzyme that catalyzes the direct conversion of ADP to ATP and AMP

The above process distorts the measurement of CK because of the changes in concentration in ATP and ADP generated by AK

To minimize this problem, add to the system:

- Fluoride
- AMP
- Diadenosine-5-pentaphosphate
*AMP + Diadeneosine-5-pentaphosphate is better
* Presence of large amounts of AMP inhibit AK

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 CREATINE KINASE LACTATE DEHYDROGENASE
ISOENZYMES Exists as a dimer composed of 2 polypeptide chains Basically made up of 4 polypeptide chains comprised of 2
types of polypeptide chains designated as
M – Muscle
B – Brain M – Muscle
H – Heart
Form 3 isoenzymes:
Form 5 isoenzymes
CK – MM (CK3) = slowest
CK – MB (CK2) = intermediate ES AS % in
CK – BB (CK1) = fastest serum
HHHH LD1 LD5 α1 18-33
1. CK-MM (Muscle Type) HHHM LD2 LD4 α2 28-40
HHMM LD4 LD3 β 18-30
- 99% in Skeletal Muscles HMMM LD5 LD2 γ1 6-16
- 80% in Cardiac Muscles MMMM LD6 LD1 2-13
γ2
- Lung, Thyroid Liver, Spleen, Placenta
- MAJOR ISOENZYME (94-100%) in the serum and
NOMENCLATURE
of healthy people
- Major isoenzyme fraction found in striated
European System = based on how fast the isoenzymes moves
muscle and normal serum
towards the anode
2. CK-MB (Hybrid Type)
- LD1 = Fastest
- 15-20% of CK Activity in Cardiac Muscle
- LD5 = Slowest
- 5% of CK Activity in Skeletal Muscle
- Unpredictable to trace
American System = reverse of the European
3. CK-BB (Brain Type)
DISTRIBUTION
- 90% in Brain Tissues
- Increased in most tissues other than skeletal or
1. LD1 and Some LD2
cardiac muscles
- Heart Muscles
- Small quantities in the sera of healthy people
- RBCs
- Presence depends on method of detection
- Renal Cortex
(Mostly not detected)
2. LD3
- Lungs
MINOR IZOENZYMES
- Lymphocytes
- Lymph Nodes
1. Makro-CK
- Spleen
- Pancreas
- Largely composed of CK-BB + IgG (rarely IgA)
- Thyroid
- Usually migrates between the MM and MB
- Adrenals
fraction in electrophoresis
3. LD4 and LD5
- Complexes with Lipoprotein and CKMM
- Kidneys
2. CK-Mi / CK-Mito / CK-Mt
- Liver
- Skeletal Muscle
- Mitochondrial form
4. LD5
- Bound to the exterior surface of the inner
- Skeletal Muscles
mitochondrial membrane of the Muscle, Brain
- Liver
and Liver (most of the other isoenzymes are
found in the cytoplasm)
- Appears only in extensive tissue damage
(breakdown of mitochondrion and cell wall)
- Does not correlate with a specific disease
- Indicator of severe illness
- Does not react with antibodies to CK-B or CK-M
subunits
ASSAYS USED IN
STUDYING ISOENZYMES

ELECTROPHORESIS Method of Choice Cellulose Acetate or Agarose

Cellulose Acetate or Agarose
pH 8.6
Development of individual isoenzyme fraction is
developed by rolling a thin layer of assay solution over
the support mediumand allowing the system to incubate
for a set of time
OLIVER-ROSALKI MODIFICATION
- Fluorescent light = to detech NADPH
- Densitometer = to quantitate the degree of
fluorescence to determine the relative amount
of each isoenzyme
NITROBLUE TETRAZOLIUM AND SIMILAR COMPOUNDS
AND PMS (PHENAZINE METHOSULFATE)
- are added to react with NADPH
- Band of color produced
- Intensity of the color is proportional to the
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After separation the film is treated with substrate (pyruvate)
and other reagents to develop bands of activity
- After electrophoretic separation, the isoenzymes can
be detected either fluorometrically or
colorimetrically
WACKER METHOD
- Most widely used method
- Colorimetric
- Solution of BUFFER, LACTATE and NAD is gently rolled
onto the surface of the film
- Incubated at 30-370C for 30 MINTUES or more
- The NADH which forms as a result of enzyme reaction
is detected by:
 Treating the film with a solution of
NITROBLUE TETRAZOLIUM and PMS
(Phenazine monophosphate)
 Whaatever NADH is present will reduce
the nitro blue compound to form colored
band (blue purple) in each area of the
 amount of enzyme present in the fraction
- Not as sensitive as fluorometric method
- Underestimate MB and BB (because they are
undetectable to trace)
DRAWBACK: Heat sensitivity of isoenzymes (BB and MB)
at 600C which leads to the two fractions being
UNDERESTIMATED
Problem of special concern when quantitating CK-MB for
diagnosis of MI
IMMUNOINHIBITION Antibodies to the M subunit
Binds to the M subunit of the isoenzyme and inhibits it
If applied to a sample containing only CK-MM, it would
mean the loss of enzyme activity
Although a small residual activity is still detected in most
systems suggesting less than complete inhibition by the
Ab
*AK MUST BE COMPLETELY INHIBITED*
*MUST NOT USED HEMOLYZED SAMPLE*
DRAWBACK: Method assumes that B subunit activity =
CK activity (CK-MB activity may be attributed to CK-BB
and vice versa)
A small amount of MM fraction will still register activity
(because it is the major isoenzyme in the circulation
COLUMN Uses DEAE (Diethylamino ethyl)-SEPHADEX A-50 or
CHROMATOGRAPHY smiliar ANION exchange reasins as Stationary Phase
pH: 8.0 = CHONs have net (-) charge and bind to the
column which are weak attachments and can be
removed f a stronger anion is added
By changing the concentration of NaCl soln, one can
selectively elute each isoenzyme and determine activity
of each fraction itself
ADVANTAGES = NO AK INTERFERERNCE
- AK appears to be eluted with CK-MM fraction and
does not interfere with measurement of CK-
MB activity
- If CK-MM is to be estimated, an inhibitor for AK is
to be added so that MM is not
OVERESTIMATED
IMMUNOASSAY MASS IMMUNOASSAY
- MOST COMMONLY USED approach to measure
concentrations of the CK-MB CHON ( “mass”)
using CONAN MONOCLONAL ANTIBODY
- Uses SANDWICH TECHNIQUE
- Uses one antibody specifically recognizes only the
MB dimer
- Ensures that ONLY CK-MB IS ESTIMATED; neither
CK-MM nor CK-BB react with both antibodies
- MORE SENSITIVE; Limit of detection = 1 ug/L
-
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film where LD activity is found
 The absorbance of this product is
proportional to the amount of NADH
present
 Relative % of LD isoenzyme fractions are
determined and reported along with
total LD activity of the sample
 LD can use other substrates such as
α-hydrobutyrate (H subunits have
greater affinity for this than M subunit
and is thus used to measure LD1 activity)
 Allows ID and quantitation of each of the
5 fractions though time consuming
 Provides more useful information than
any other methods for separation and
quantitation of LD isoenzymes
Used in the detection of LD1
Ab to M Subunit of LD + Sample
- Any isoenzyme containing and M subunit binds Ab to
it rendering the CHON inactive
- Since only LD1 does not contain the M subunit, it will
be the only one showing activity when assayed for
total LD
By comparing total activity with and without antibody
present in the same, the relative % of LD1 can be determined
- Limited because only LD1 activity is assessed
- There are instances where increase in total LD is
attributed to the other isoenzymes so the method
cannot give information with regards to other
isoenzymes
Procedure is reasonably QUICK to perform and COST IS THE
SAME AS ELECTROPHORESIS
Some data suggests that LD isoenzyme changes associated
with MI measuring the relative amound of LD1 and LD2 by
electrophoresis (LD1 will be greater than LD2)
Isolate LD1 and LD2 along with CK-MB fraction
- CK-MB and LD1 = represent those isoenzymes which
are elevated in serum of patients with MI. If done,
useful clinical information can be obtained without
the need for electrophoretic fractionation of all
isoenzymes
• Sample is applied to an ion exchange collumn
• unwated fractions (CK-MM, LD3,4,5) are eluted with
TRIS BUFFER (low NaCl conc.)
• Desired CK and LD fractions are removed from the
column by increaing the amount of NaCl in the buffer
After removal, total activity for CK and LD in the
second fraction are determined
SHORTCOMINGS:
- Sample volume required is quite large
- Useful information is lost regarding damage to other
tissues
- Difficult to process a large number of samples
efficiently
 ADVANTAGES

- Sample stability
- Non-interference with hemolysis anticoagulants
- Adaptable to full automation
- Fast TAT
RIA (RADIOIMMUNOASSAY)

- Ab against B subunit
- Competitive ligand in the assay is frequently CK-
BB from and animal source is labeled with an
isotope (generally 125I)
- Patient’s serum containing CK and labeled CK-BB
are mixed with Ab to B subunit and separation
of bound and free materials are carried out
- Quantitation of B subunit allow estimation of CK-
MB present
- Basic principle is that Ag to be measured is
assayed indirectly through its competition with
labeled Ag for binding a limited amount of Ab
- Labeled reagent Ag + reagent Ab = Labeled Ag-Ab
complex
- Wash away free labeled Ag
- Add patient’s serum containing unlabeled Ag to
displace labeled Ag complexed to an Ab
- Unlabeled Ag can displace radiolabeled Ag from
immune complexes and the amount of labeled
Ag released to the system
- The greater the concentration the unlabeled Ag,
rhw moew labeled Ag will be free in the
solution (Direct proportionality)
INHIRENT DIFFICULTY

- If CK-BB is less present for some reason, either in

addition to (or instead of) CK-MB fraction,
there will be OVERESTIMATION of CK-MB
levels

ANSWER TO DIFFICULTY

- Sandwich assay employing antibodies to both B

and M subunits
- Ab to CK-B subunit is attached to a sold phase
when sample is added. All fractions containing
a B subunit bind to the Ab
- The MM fraction can then be washed away
- The Ab to the M subunit is added and attaches
only to the CK-MB resin
- The Ab is labeled with radioisotope or some tag
so that its degree of binding can be assessed
- The amount of M subunit Ab which attaches is an
estimate of the quantity of CK-MB present
- CK-BB does not interfere and there is no
overestimation of the quantity of MB present

SOURCES OF ERROR Grossly Hemolyzed

- RBC’s are rich in adenylate cyclase or adenylate

kinase which reacts with ADP to produce ATP
which will participate in the assay causing
FALSELY ELEVATED CK levels

Lactescense/Milky serum

- Result in variable absorbance readings in

sppectro
REFERENCE RANGES Adult Males = up to 160 U/L Normal LD depends on the assay technique employed
Adult Females = up to 130 U/L
Forward Reaction
Difference is related to muscle mass
Older patients have lower normal range due to - 35-90 U/L
decreased muscle activity and muscle mass
Reverse Reaction

- 95-200 U/L
- Infants have values up to 4x higher

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