Assay the Activity of Creatine

Kinase (CK)-total in Serum

Dept.of Biochemistry

Creatine

• Creatine is a nitrogenous organic acid that occurs naturally in vertebrates – produced from amino acids primarily in the ki

dney and liver. – transported in the blood for use by muscles. – helps to supply energy to muscle.– Approximately 95% of creatine is located in sk

eletal muscle. – The rest is located in the brain or heart.

Glycine

Creatine and phosphocreatine

Creatine by way of conversion to and from phosphocreatine is present and functions in all vertebrates as energy buffer system.

Keeps the ATP/ADP ratio high at subcellular places where ATP is needed.

Creatine supplements are sometimes used by athletes, bodybuilders, and others who wish to gain muscle mass.

CreatinineCreatinine is a break-down product of creatine phosphate in muscle, and is usually produced at a fairly constant rate by the body (depending on muscle mass).

Creatinine is chiefly filtered out of the blood by the kidneys. The level of creatinine excretion (clearance rate) is a measure of renal function.

Creatine kinase (CK)

• also known as creatine phosphokinase (CPK) or phospho-creatine kinase, is an enzyme expressed by various tissues and cell types.

• CK catalyzes the transfer of phosphate between creatine and ATP/ADP, the reaction is reversible – phosphocreatine is regenerated whe

n ATP is abundant– Provides rapid regeneration of ATP w

hen ATP is low

CK

CK isoenzymes• Creatine Kinase activity is found gre

atest in skeletal muscles, brain and heart tissue.

• It is a dimer containing B (Brain) and M(Muscle) subunits.

• Three isoenzymes: – CK1 (CK-BB): in the brain– CK2 (CK-MB): mostly in the cardiac musc

les– CK3(CK-MM): in both the skeletal and th

e cardiac muscles.• CK-MB is more specific for cardiac m

uscle, so it is a more sensitive marker of myocardial injury than total CK activity.

Muscle-Type CKMuscle-Type CK

(Dimer)(Dimer)

Clinical significance

• Elevation of CK is an indication of damage to muscle. CK value is increased in myocardial infarction and muscle injury such as muscular dystrophy, acute rhabdomyolysis due to strenuous exercise, myocarditis, alcoholic myopathy and so on.

• Following a myocardial infarction, CK rises measurably within a 4-6 hour period. Maximal values are observed within 24 hours, after which time, the activity returns to normal.

Assay the Activity of CK-Total in Serum• Principle

– Creatine kinase (CK) transfers phosphate from ATP to creatine, forming creatine phosphate and ADP.

– Pyruvate kinase(PK) then transfers phosphate from phosphoenolpyruvate (PEP) to ADP, yielding pyruvate and ATP.

– The pyruvate which combines with 2,4 – dinitrophenylhydrazine(2,4-DNPH) to give a brown colored complex (pyruvate-2,4 – dinitrophenylhydrazone)in alkaline medium.

– The change of absorbance of the colored complex is proportional to the activity of CK.

alkaline medium

PEP + ADP pyruvate + ATPPK

Creatine + ATP Creatine phosphate + ADPCK

pyruvate + 2,4-DNPH pyruvate-2,4 – dinitrophenylhydrazone(brown colored

complex)

– The pyruvate which combines with 2,4 – dinitrophenylhydrazine(2,4-DNPH) to give a brown colored complex (pyruvate-2,4 – dinitrophenylhydrazone)in alkaline medium.

Specimens & Materials• Specimen: serum • Working reagent:

– Creatine,ATP,PEP,PK • Color reagent:

– 2,4-DNPH– Function: stop the reaction, reveal color

• 0.4N NaOH• CS=200 U/L (Standard: pyruvate)• Water bath• Test tubes• Pipettes• Spectrophotometer

MethodB S T

Working reagent (l) 250 250 250

dH2O (l) 50 - -

standard (l) - 50 -

serum (l) - - 50

Mix, incubate for 15 mins at 37CColor reagent (l) 250 250 250

Mix, incubate for 15 mins at 37CNaOH (ml) 2.0 2.0 2.0

Mix, 2-3 mins at room temperature

•Mix the contents of each tube, measure the absorbance of test(T) and standard(S) setting zero with blank(B), λ=510nm.

Calculation

• CK activity (U/L)=AT /AS x CS

• Cs=200 U/L

• Expected normal value: 27-184 U/L

Spectrophotometry

1． Power switch2．Wavelength select

ion3．“Mode”4. “100%T／ 0A”5．“ 0％ T” 6． Cuvette holder(sample compartment)7． Pole

sample compartment

Pole

1 、 distinguish transparence and opaque 2 、 control solution at 2/3 volume 3 、 sop up water with paper 4 、 cleanout ， upend it

Cuvette

1. Switch on, allow 20 min for warm up before use.2. Adjust wave length of maximal absorption.3. Prepare test, blank and standard sample. sop up liq

uid with paper, Place them in the cuvette holder.(Notice: put the blank in position 1, Make sure the cuvette is ali

gned with the light source.)4. Mode “A” , press“100%T ／ 0A”, Set A=0 or T=10

0. 5. Pull the pole once time. 6. Change mode to “T”, press“0%T”, Set T =0 7. Change mode to “A”. 8. pull the pole second time, record A1; third time, rec

ord A2; forth time ,record A3.

Operating steps of spectrophotometry

Next experiment

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