Increased levels are found in

myocardial infarction, cerebrovascular diseases, muscular

dystrophy, pulmonary infarction and, electrical shocks.

Increased levels can also be caused by intramuscular

injections, strenuous exercise and recent surgery. Early

pregnancy may produce decreased levels.

Principle

Creatine kinase catalyzes the reaction between creatinine

phosphate and ADP to form creatine and ATP. The ATP

formed along with glucose is catalyzed by hexokinase

to form glucose 6 phosphate. The glucose 6 phosphate

reduces NADP to NADPH in the presence of glucose 6

phosphate dehydrogenase. The rate of reduction of NADP

to NADPH is measured as an increase in absorbance,

which is proportional to the CK activity in the sample.

 Creatine Kinase Creatine Phosphate Creatine + ATP

+ ADP

 Hexokinase Glucose + ATP Glucose 6 phosphate + ADP G-6-PDH

G - 6 - P + NADP Gluconate - 6 - P + NADPH + H

Normal Reference Values

Serum (male) : 24–195 U/L at 37°C

 (female) : 24–170 U/L at 37°C.

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 2 × 10 mL 2 × 25 mL

L1 : Enzyme reagent 2 × 8 mL 2 × 20 mL

L2 : Starter reagent 2 × 2 mL 2 × 5 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent: For sample start assays, a single reagent is

required. Pour the contents of 1 bottle of L2 (Starter Reagent)

into 1 bottle of L1 (Enzyme Reagent). This working reagent is

stable for at least 10 days when stored at 2 to 8°C. Alternatively

for flexibility as much of working reagent may be made as

and when desired by mixing together 4 parts of L1 (Enzyme

Reagent) and 1 part of L2 (Starter Reagent). Alternatively,

0.8 mL of L1 and 0.2 mL of L2 may also be used instead of

1 mL of the working reagent directly during the assay.

Sample Material

Serum. Free from hemolysis. CK is reported to be stable in

serum for 3 days at 2 to 8°C.

Procedure

Wavelength/filter : 340 nm

542 Concise Book of Medical Laboratory Technology: Methods and Interpretations Temperature : 37°C / 30°C / 25°C

Light path : 1 cm

Substrate Start Assay

Pipette into a clean dry test tube labeled as Test (T):

Addition

Sequence

(T)

25°C / 30°C

(T)

37°C

Enzyme reagent (L1) 0.8 mL 0.8 mL

Sample 0.05 mL 0.02 mL

Incubate at the assay temperature for 5 minutes and add

Starter reagent (L2) 0.2 mL 0.2 mL

Mix well and read the initial absorbance A0 and repeat

the absorbance reading after every 1, 2, and 3 minutes.

Calculate the mean absorbance change per minute (∆A/

min).

Sample Start Assay

Pipette into a clean dry test tube labeled as test (T):

Addition

Sequence

(T)

25°C / 30°C/

(T)

37°C

Working reagent 1.0 mL 1.0 mL

Incubate at the assay temperature for 1 minute and add

Sample 0.05 mL 0.02 mL

Mix well and read the initial absorbance A0 after

10 minutes and repeat the absorbance reading after every

1, 2, and 3 minutes. Calculate the mean absorbance change

per minute (∆A/min).

Calculations

Substrate/Sample start

CK Activity in U/L 25°C/30°C = ∆A / min × 3333

 37°C = ∆A / min × 8095

Temperature Conversion Factors

Assay

Temperature

Desired

25°C

Reporting

30°C

Temperature

37°C

25°C 1.00 1.56 2.44

30°C 0.64 1.00 1.56

37°C 0.41 0.63 1.00

Linearity

The procedure is linear up to 2000 U/L at 37°C. If the

absorbance change (∆A/min) exceeds 0.250, use only the

value of the first 2 minutes to calculate the result, or dilute

the sample 1+ 9 with normal saline (NaCl 0.9%) and repeat

the assay (Results × 10).

Note

Samples having a high activity show a very high initial

absorbance as most of the NADP is converted prior to the

start of measurement. If this is suspected then dilute the

sample and repeat the assay.

The working reagent or the combined reagent should

have an absorbance below 0.800 against distilled water

at 340 nm. Discard the reagent if the absorbance is above

0.800.

System Parameters

Reaction : UV Kinetic Interval : 60

Wavelength : 340 nm Sample volume : 0.02 mL

Zero setting : Distilled water Reagent volume : 1.00 mL

Incubation

temperature

: 37°C Standard :

Incubation

time

: - Factor : 8095

Delay time : 60 sec React. slope : Increasing

Read time : 180 sec Linearity : 2000 U/L

No. of read : 4 Units : U/L

CK MB (NAC Act) (Immunoinhibition/Mod. IFCC method)

(Courtesy: Tulip Group of Companies)

For the determination of CK-MB activity in serum (For in

vitro diagnostic use only).

Summary

CK is dimeric molecule composed of M and B subunits,

which are immunologically distinct. It exists as three main

isoenzymes CK - MM, CK - MB, and CK - BB. The CK -

MM is found in the muscle while CK - MB is found mainly

in the myocardial cells. The CK - BB is found mainly in

the brain and lungs, and enters the bloodstream only

on injury to these organs like cerebrovascular accident

or pulmonary infarction. Normally CK - MM is found in

the blood. CK - MB levels increase significantly 4–6 hours

following a myocardial infarction and peak at around 12

to 24 hours after the infarct. The levels return to normal,

in case of no further myocardial damage, after 24 to 48

hours. Hence, the increased levels of CK - MB along with

elevated levels of total CK is a good indicator of myocardial infarction. CK - MB levels usually do not rise in

chest pain caused by angina, pulmonary embolism or

congestive heart failure.

Enzymology 543

Principle

CK - M fractions of the CK - MM and the CK - MB in the

sample are completely inhibited by an anti CK - M antibody

present in the reagent. Then the activity of the CK - B fraction

is measured by the CK (NAC act) method, the CK - MB

activity is obtained by multiplying the CK - B activity by two.

Normal Reference Values

Serum up to 24 U/L at 37°C.

Indication of myocardial infarction is based on the

following factors:

Total CK (male) : < 195 U/L at 37°C

(female) : < 170 U/L at 37°C

CK - MB : < 24 U/L at 37°C

CK - MB to Total CK : < 6%

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 2 × 10 mL 2 × 25 mL

L1 : Enzyme reagent 2 × 8 mL 2 × 20 mL

L2 : Starter reagent 2 × 2 mL 2 × 5 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent: For sample start assays, a single reagent

is required. Pour the contents of 1 bottle of L2 (Starter

Reagent) into 1 bottle of L1 (Enzyme Reagent). This working

reagent is stable for at least 10 days when stored at 2–8°C.

Alternatively for flexibility as much of working reagent may

be made as and when desired by mixing together 4 parts

of L1 (Enzyme Reagent) and 1 part of L2 (Starter Reagent).

Alternatively, 0.8 mL of L1 and 0.2 mL of L2 may also be

used instead of 1 mL of the working reagent directly during

the assay.

Sample Material

Serum. Free from hemolysis.

Procedure

Wavelength/filter : 340 nm

Temperature : 37°C / 30°C / 25°C

Light path : 1 cm

Substrate Start Assay

Pipette into a clean dry test tube labeled as test (T):

Addition

Sequence

(T)

25°C / 30°C/37°C

Enzyme reagent (L1) 0.8 mL

Sample 0.05 mL

Incubate at the assay temperature for 1 minute and add

Starter reagent (L2) 0.2 mL

Mix well and read the initial absorbance A0 after

5 minutes and repeat the absorbance reading after every 1,

2, and 3 minutes. Calculate the mean absorbance change

per minute (∆A/min).

Substrate Start Assay

Pipette into a clean dry tube labeled as test (T).

Addition

Sequance

(T)

25°C/30°C/37°C

Working reagent 1.0 mL

Incubate at the assay temperature

for 1 minute and add

Sample 0.05 mL

Mix well and read the initial absorbance A0 after 10

minutes and repeat the absorbance reading after every 1, 2

and 3 minutes. Calculate the mean absorbance change per

minute (∆A/min).

Calculations

Substrate/Sample start

CK - B activity in U/L 25°C / = ∆A/min × 3333

30°C /37°C

CK-MB activity in U/L = ∆A/min × 6666

25°C / 30°C /37°C

Temperature Conversion Factors

Assay Desired Reporting Temperature

Temperature 25°C 30°C 37°C

25°C 1.00 1.56 2.44

30°C 0.64 1.00 1.56

37°C 0.41 0.63 1.00

Linearity

This procedure is linear up to 1000 U/mL. Inhibition of

CK - MM is up to 1500 U/L at 37°C. If the total CK activity

exceeds this limit dilute the sample 1 + 9 with normal saline

(NaCl 0.9%) before estimating CK - MB (Results × 10).

544 Concise Book of Medical Laboratory Technology: Methods and Interpretations Note

This method will also measure any CK - BB isoenzyme

present in the sample. The amount of CK - BB is usually

negligible in serum from normal individuals or in patients

with myocardial infarction.

A macro form of BB has been observed and this will be

measured as CK - B activity, if the CK - B activity exceeds

20% of the total CK activity the presence of macro BB

should be suspected.

The working reagent or the combined reagent should

have an absorbance below 0.800 against distilled water

at 340 nm. Discard the reagent if the absorbance is above

0.800.