CREATINE KINASE REAGENT (Colorimetric Method)

For the quantitative determination of Creatine Kinase in serum.

 

INTRODUCTION

Creatine Kinase (CK) plays an important role in the energy-storing mechanism of tissue by catalyzing the reversible reaction between creatine and ATP to form creatine phosphate and ADP. CK is distributed in various organs; the highest activities (in decreasing order) are skeletal muscle, heart and brain.¹ Thus determination of CK is an aid in diagnosing muscular dystrophy and other diseases of the skeletal muscles, myocardial infarction, hypothyroidism, renal diseases and/or dysfunction.²

 

The early procedure for determining CK was based on the rate of ATP formation.³ A modified method was described by Nielson by adding a sulfhydryl compound and AMP to assure maximum CK activity and inhibit adenylate kinase activity.⁴   Optimized conditions for measuring CK were published by Szasz in 1976 as well as by the Scandinavian committee on enzyme.^5,6  The above procedure was modified again in 1979 to include EDTA.⁷ The UV kinetic method is a modification of the above revision.  A tetrazolium salt coupled with diaphorase is incorporated in the colorimetric determination in the CK activity.⁸

 

PRINCIPLE

                                                   CK

Creatine Phosphate + ADP   ----------->  Creatine + ATP

 

                                       HK

ATP + D-Glucose⁺   ---------> Glucose-6-Phosphate + ADP

                                 

                                  6-PDH

Glucose-6-Phosphate + NAD ------------->  6-Phosphogluconate

       +  NADH  +  H⁺

                        

NADH + INT -----------------> NAD + INTH (red formazan) Diaphorase                                     (500 nm)

 

CK catalyzes the conversion of creatine phosphate and ADP to creatine and ATP. The ATP and glucose are converted to ADP and glucose-6-phosphate by hexokinase (HK). Glucose-6-phosphate de-hydrogenase (G-6-PDH) oxidizes at the D-glucose-6-phosphate and reduces the nicotinamide adenine dinucleotide (NAD). The NADH reacts with 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) catalyzed by diaphorase and forms a formazan (INTH). The INTH is measured colorimetrically and is proportional to the concentration of CK present.

 

REAGENT COMPOSITION

1.     CK REAGENT:  Concentration based upon reconstitution:  30 mmol/L creatine phosphate, 20 mmol/L D-glucose, 20 mmol/L N-acetyl-L-cysteine, 2 mmol/L EDTA, 2 mmol/L adenosine diphosphate, 2 mmol/L adenosine monophosphate. 2 mmol/L NAD, £ 3000 U/L hexokinase, £ 3000 U/L glucose-6-phosphate dehydrogenase from L, mesenteroides, £ 1000 U/L diaphorase; buffer 100 mmol/L also contains filler and stabilizers.

2.     CK COLOR REAGENT: 0.2% w/v INT with preservative. Keep tightly capped and protect from contamination.

3.     CK CALIBRATOR: A lyophilized serum which has been tested and found to be non-reactive for hepatitis  B  surface antigen (HBsAg) by a method of third generation sensitivity using reagents licensed by the Bureau of Biologics.  No known test method can offer complete assurance that products from human blood will not transmit hepatitis; therefore, blood derived products should be handled as potentially hazardous and with appropriate care.

 

The CK value for each lot is determined via NADH standardization, i.e. based upon the relationship 1 IU/L CK generates 10 µmoles NADH/L per 10 minute reaction time; measurement of NADH solution made at 340 nm in a 1-cm light path and utilizing the molar absorptivity of NADH (6.22 x 10³). The CK Calibrator may be aliquoted after reconstitution and frozen.  Do Not Repeatedly Freeze and Thaw.

 

WARNINGS AND PRECAUTIONS

1.     For in vitro diagnostic use.

2.     Exercise the normal precautions required for the handling of all laboratory reagents.  Pipetting by mouth is not recommended for any laboratory reagent.

 

REAGENT PREPARATION

Reconstitute with volume of distilled water specified on each vial,

swirl to dissolve.

 

STORAGE AND STABILITY

The reagent should be stored at 2-8°C prior to reconstitution. The reagent may be used until the expiration date indicated on the package label.  After reconstitution, the reagent is stable for twenty four (24) hours at room temperature or seven (7) days at refrigerator temperature.

 

REAGENT DETERIORATION

1.     Physical Appearance, If reagent appears damp and clumped, deterioration may have occurred and the product should be discarded.

2.     Blank Absorbance, If the reconstituted CK REAGENT without added sample has an absorbance greater than 0.70 at 340 nm versus reagent grade water, the reagent is considered to be unsatisfactory for use and should be discarded.

3.     Control Assays, Failure to obtain accurate results in the assay of control materials may indicate reagent deterioration.

4.     We cannot guarantee the stability of reagents which have been:  

        a.  transferred from their original containers

        b. improperly stored prior to or during use

        c. contaminated during use

 

SPECIMEN COLLECTION

Collect whole blood by non-traumatic venipuncture and allow to clot. Centrifuge and remove serum immediately. Serum is reportedly stable for four (4) hours at room temperature, 8 - 12 hours at 4°C, and 2 - 3 days when frozen.⁹

 

 

INTERFERING SUBSTANCES

Certain drugs and medications may affect the activity of CK, see Young et al. ¹⁰

 

MATERIALS REQUIRED BUT NOT PROVIDED

Sample and reagent pipettes, test vials or cuvettes, timer, test tube rack, 37°C heating bath, control serum, 0.1 N Hydrochloric Acid, spectrophotometer.

 

PROCEDURE (MANUAL)

Reconstitute CK reagent according to instructions

1.     Transfer 0.5 ml of CK Reagent to test tubes labeled: UNKNOWN(S), CONTROL(S), CALIBRATOR, and REAGENT BLANK.

2.     Prewarm tubes at 37°C for 3 - 5 minutes.

3.     At timed intervals add 0.010 ml (10 ul) of sample to its respective tube, mix and return to 37°C heating bath for exactly 10 minutes.

4.     Following the same timed intervals, add 0.10 ml (100 ul) of CK COLOR REAGENT to all tubes, mix and incubate for five (5) minutes at 37°C.

5.     Stop the reaction by adding 2.5 ml of O. 1 N Hydrochloric Acid to all tubes and mix.

6.     Zero the spectrophotometer at 500 nm using the REAGENT   

        BLANK. Read and record the absorbance for each vial.

        (Wavelength range:500-520).

* USE TC - MUTI PURPOSE CALIBRATOR TO REPLACE STANDARD.

 

PROCEDURE NOTES

1.       In the case of very icteric or lipemic serum. a serum blank
          should be measured. This may be performed as follows:

          a.    Add 0.010 ml (10 ul) of the sample to 3.1 ml of deionized
                water.
          b.   Zero the spectrophotometer at 500 + 5 nm with deionized
                water.
          c.    Read and record absorbance of "serum blank".
          d.   Subtract this "serum blank" absorbance from the sample

                absorbance measured in step #6 above. Use this corrected
                absorbance value to calculate CK activity.

2.       Traumatic muscle injury (i.e intramuscular injections) as well 

          as vigorous physical exercise, labor, and delivery of pregnancy
          will elevate the CK value.
3.       CK is a light sensitive enzyme and excessive light exposure
          reportedly will cause CK values to decrease in the serum sample.

 

PROCEDURE LIMITATIONS

1.       Some inhibitors of CK activity¹¹

               a.    Excessive Mg⁺⁺  Cl^-  SO₄ ^--

          b.   Most heavy metals, i.e. Zn⁺⁺, Cu⁺⁺, Mn⁺⁺
               c.    Iodoacetate and other sulfhydryl binding agents
          d.   Excess ADP, citrate, fluoride, L-thyroxine
          e.    Excess uric acid

2.       This procedure measures total CK activity irrespective of its
          tissue or organ of origin.

3.       Lower than expected CK values have been reported in samples
          having high alkaline phosphatase activity.

 

CALCULATIONS
Use the absorbance readings of the  CALIBRATOR and
UNKNOWNS(S) to calculate CK values as follows:

(where A = absorbance)

 

A(UNKNOWN)     x       _{CK value of CALIBRATOR}

A(CALIBRATOR)                               (IU/L)

 

                                                         = CK in uknown (IU/L)

EXAMPLE OF CALCULATION

Assume that the CALIBRATOR had a CK value of 200 IU/L and that it gave an absorbance of 0.15 while the UNKNOWN gave an absorbance of 0.21. The CK value of the UNKNOWN may then be calculated as follows:

 

0.21  x  200 IU/L =  280 IU/L

0.15

 

QUALITY CONTROL

Use control sera with known normal and abnormal values to monitor the integrity of the reaction.  Values should be those acceptable for this method and temperature.

 

EXPECTED VALUES¹²

25 - 192 IU/L (37°C)

 

It is strongly recommended that each laboratory establish its own normal range.

 

PERFORMANCE

Lineanty: 1000 IU/L

Sensitivity:  Based on an instrument resolution of A = 0.001, this procedure has a sensitivity of 1.5 IU/L.

Comparison:  Studies done between this procedure and a UV procedure yield a correlation coefficient of 0.99 with a regression equation of Y = 0.87x + 12.99.

 

Precision:
                                    Within Run
Mean (mg/dl)                         S.D.                             C.V.%
 154                                          8.6                             5.6
 405                                        22.5                             5.5

 

                                    Run to Run
Mean (mg/dl)                         S.D.                             C.V.(%)
144                                           8.6                             5.9
413                                         19.1                             4.6

 

REFERENCES

1.         Faulker, W.R. and Meites, S.:  Selected Method of Clinical
Chemistry. vol 9, p. 185 (1982).

2.         Rosalki, S.B.: J. Lab. Clin. Med. 69:696 (1967).

3.         Oliver, I.T.: Biochem.. 1. 61:116 (1955).

4.         Nielson, L., Ludvigsen, B.:  J. Lab. Clin. Med. 62:159
(1963).

5.         Szasz, G., et al.: Clin. Chem.22:650 (1976).

6.         The Committee on Enzymes of the Scandinavian Society for
Clinical Chemistry and Clinical Physiology.  Scand. J. Clin. Lab. Invest. 36:711 (1976).

7.         The Committee on Enzymes of the Scandinavian Society for
Clinical Chemistry and Clinical Physiology.   Scand. J. Clin. Lab. Invest. 36:711 (1979).

8.         Avigad, G., and Levin, N.:  European 1. Biochem  1:102
(1969).

9.         Kaehmar, J.F. and Moss, D.W.: Fundamentals of Clinical Chemistry.  Tietz N.W. ed. Saunders, W.B. Co., Philadelphia, 686 (1976).

10.       Young, D.S., et al.: Clin Chem 21:10 (1975).

11.       Moren, L.G.: Clin. Chem. 23:1569 (1977).

12.       Tietz, N.W.:  Fundamentals  of  Clinical  Chemistry.
Philadelphia, W.B. Sanders, 1210 (1976).

 

Date revised: 04/97