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Biochemistry 2 : practical

Biochemistry test : creatine kinase- NAC


Biochemistry method test : kinetic test UV. NACActivated

Principle
The kinetic determination of CK is bases upon IFCC and DGKC
recommendations. Creatine kinase catalyses the reversible transfer of a phosphate
group from phosphocreatine to ADP. This reaction is coupled to those catalysed
by hexokinase (HK) and glucose-9-phosphate dehydrogenase (G6P-DH):

Creatine-phosphate + ADP −−−−−−−−→creatine + ATP

ATP + D-glucose −−−−−−−−−−−−−−→glucose-6-P + ADP


Glucose-6-P + NADP−−−−−−−→gluconate-6-P + NADPH + H

Reagent composition

Reagent 1 Imidazole pH
Reagent 2 6,7..........................................125 mmol/L
D-
Glucose.....................................................
25 mmol/L
N-
acetylcysteine........................................25
mmol/L
Magnesium
acetate.....................................12,5mmolL
NADP.....................................................2,
52 mmol/L
EDTA........................................................
2,02 mmol/L
Hexokinase
(HK)............................................≥ 6800
U/L
ADP............................................................
15,2 mmol/L
AMP...........................................................
...25 mmol/L
Diadenosine-5-
P..........................................103 mmol/L
G-6-
PDH........................................................≥
8800 U/L
Creatine-
phosphate..................................250
mmol/L
*
Anti-CK-
M......................................................2000
U/L
Lyophilized human
serum..................................2 mL
Value (assay at 37°C) indicated on label

Storage and stability


All the components of the kit are stable at 2-8C up to the date of expiration as
specified, when stored tightly closed, protected from light and contaminations
prevented during their use. The reagent should be a clear solution. If turbidity or
precipitation has occurred or if blank absorbance at 340 nm >1,60, the reagent should
be discarded.

Calibration and quality control


Calibration by means of a factor , alternatively, you cna use the human calibrator
(HBC03) for calibration .Control sera arc recommended to monitor the performances
of assay procedures.use normal and pathological human (HBC01,HBC02)
sera.prepare and measure these controls the same as samples.if the control values are
found outside the defined range.check the instrument, reagents and calibrator for
problems.
Sample preparation
Blank sample standard
Standard 20ul
Sample 20ul
Working reagent(R1+R2) 1.0ml 1.0ml 1.0ul

Materials
Cyanstart analyser, vortex, test tube, mircopipette,beaker,distilled water,water bath,
liquid reagent.
Conclusion
Creatine kinase (CK) is a cellular enzyme with wide tissue distribution in
the body. Its physiological role is associated with adenosine triphosphate (ATP)
generation for contractile or transport systems. Elevated CK values are observed in
diseases of skeletal muscle and after myocardial infarction1,5,6. Clinical diagnosis
should not be made on a single test result; it should integrate clinical and other
laboratory data. .
References
1. cyanstart analyser insert or manual
2. Cyanpress reagent insect