Clinica Chimica Acta, 70 (1976) 285-288 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 7881

CREATINE

WILLIAM Division Scotland

(Received

KINASE ACTIVITY

A. WARREN

IN SERUM AND URIC ACID SOLUTION

* and VERGINE

MADELIAN

of Laboralories and Research, New York Avenue, Albany, NY 12201 (U.S.A.)

January

State Departmeni

of Health,

I20

New

11,1976)

Skeletal muscle creatine kinases from man and rabbit, activated to measured specific activities by thiol agents, were inactivated in human serum and in uric acid solution, and the extent of their subsequent reactivation by dithiothreitol was determined. Both enzymes were completely reactivated from uric acid solution but were only 60-70% reactivated from serum. No inactivation occurred within 14 days if serum samples contained dithiothreitol (0.01 M) and were stored at -17°C.

Introduction Determination of the amount of creatine kinase (CK) (ATP:creatine Nphosphotransferase, EC 2.7.3.2) in serum is important for clinical diagnosis of heart and skeletal muscle diseases. A major technical problem in assaying CK is inactivation of the enzyme in serum. We have recently demonstrated that uric acid is one serum component which participates in this inactivation [l] and that CK inactivated in uric acid solution can be reactivated by adding thiol compounds to the assay system, a method that has long been used to partially restore CK activity in serum [ 2-121. The availability of CK of known activity has now permitted us to determine the extent to which CK can be reactivated in serum and uric acid solution and to devise methods to maintain full enzyme activity during storage. Methods Human skeletal muscle CK was prepared as previously described [ 131. Rabbit skeletal muscle CK was purchased from Sigma Chemical Co. Each enzyme, -.__

286

dissolved in 0.05 M Tris acetate buffer (1 mg/ml, pH 7.51, was activated to maximum catalytic activity by adding 2-mercaptoethanol (0.02 M). The residual 2-mercaptoethanol was removed by dialysis against Tris acetate buffer for 16 h at 4°C. When assayed immediately after dialysis, the human enzyme was usually only about 80% activated, i.e. the rate when assayed in the absence of a thiol compound was 80% of that obtained in its presence. The activity of the dialyzed rabbit enzyme was the same when assayed in the presence or absence of a thiol compound (100% activated). To study the reactivation of CK by dithiothreitol (DTT), activated human and rabbit skeletal muscle CK were added either to serum or to a solution, pH 7.4, containing potassium phosphate (0.1 M), sodium chloride (0.05 M), and uric acid (0.005 M). The serum or solution was incubated at 25”C, and aliquots were assayed at intervals. When the CK activity had decreased to the levels indicated in Table I (“After inactivation”), DTT (0.01 M) was added, and CK activity was measured 1 h later. CK activity was measured by a coupled-enzyme spectrophotometric procedure [II] with and without DTT in the assay mixture (see Fig. 1). Reaction rates were measured at 340 nm with a Gilford spectrophotometer. One unit (U) was defined as that amount of CK which catalyzes transphosphorylation between creatine phosphate and ADP at a rate of 1 @mole per min per ml. The data are presented as U/ml of solution in the assay cuvette. Protein concentrations of CK were determined by absorbance at 280 nm, using c:2m = 10.1 for the human [13] and 8.8 for the rabbit [14] enzyme. Results After CK was inactivated at 25°C in serum and in uric acid solution, the extent of reactivation was tested by subsequent addition of DTT (Table I). Roth human and rabbit skeletal muscle enzymes were completely reactivated by DTT in uric acid solution but could be only 60-70% reactivated in serum. #CK activity was fully retained for at least 14 days in serum containing DTT (0.01 M) stored at -17°C and for 1 day in serum containing DTT (0.01 M) stored at 4°C (Fig. 1A). A minimum DTT concentration of 0.003 M (0.006 M TABLE

I

R~ACTI~?ATI~N

OF

CREATINE

KfNASE

AFTER

INACTIVATION

IN SERUM

AND

IN

URIC

SOLUTION _______~______^_

---.

___~~

Enzymr

Inactivation

Creatinr

s*urce

solution

~._

kinase

activity

(U/ml)

Before

After

After

inactivation

inactivation

reactivation

0.0350

0.0032

0.0350

lirir

acid

SeIX1Tl Rabbit

Uric

acid

Serum -__---~~

.-..

Percentage

100

0.0450

0.0035

0.0463

103

0.0340

0.007

0.0370

reactivation ____~

--_--

_.___-._ Human

DTT

0.0226

66

0.0068

0.0232

63

0.0690

0.0042

0.0720

104

0.0525

0.0089

0.0525

100

0.0675

0.0113

0.0420

62

o.osfi5

0.0232

0.0645

67

1

ACID

ASSAYED

WITH

1 ASSAYED

DTT

WITHOUT

DTT

.06-Ei is ;

.04-

3 F: (;: 5

.02 -

0

4

8

12

0

4

8

12

DAYS Fig. 1. Effect of storage on creatine kinase activity in serum. Crystalline rabbit CK, dissolved in Tris acetate buffer (0.05 M, PH 7.5) and activated with P-mercaptorthanol, was added to human serum. Aliquots were stored as shown (boxes) with or without DTT (0.01 kf), Samples were assayed periodically with or without DTT (0.01 M) in the assay mixture. The frozen samples were in small aliquots: one of each was thawed and assayed on the same day.

SH) was required for full pres~~ation of CK activity in serum stored at -17°C. Samples preserved in this way did not require addition of thiol compounds in the assay mixture for full activity (Fig. lB, r!). Discussion We have previously established that uric acid is an inhibitor of CK [I]. The present study indicates that other unidentified serum components also contribute to the modification of CK and that serum-inactivated CK cannot be fully reactivated by treatment with thiol compounds. Thus the current clinical method for CK reactivation by inclusion of a thiol compound in the assay mixture is an uncertain procedure and may fail to provide 100% recovery of activity in samples in which appreciable enzyme inactivation has occurred. In contrast to an earlier report [6], these results show that the only certain way to preserve CK activity in serum is to add the thiol compound to the specimen immediately. The sample can then be kept at 4°C for 1 day or at -17OC for at least 14 days before analysis. The effect of this procedure upon the CK isoenzyme distribution in serum has not been determined. N-Acetyl-L-cysteine, DTT, and dithioerythritol are effective thiol agents for preventing serum inactivation of CK, whereas L-cysteine, Z-mercaptoethanol, and glutathione fail to provide complete protection. In addition to preserving CK activity, immediate introduction of the thiol agent int.o serum eliminates

288

the requirement for a thiol compound in the assay mixture and reduces extended and unpredictable lag period of the assay to about 1 min [ 121.

the

Acknowledgments The authors are grateful to Mrs. Elizabeth Austin for technical assistance and to the Eastern New York State and Mid-Hudson Heart Associations for grants in support of this work. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Warren, W.A. (1975) Clin. Biochem. 8. 247.--253 Oliver, I.T. (1955) Biochem. J. 61, 116-122 Hughes, B.P. (1962) Clin. Chim. Acta 7, 597403 V., Watanabe, T., Ebashi, F. and Ebashi, S. (1964) Okinaka, S., Sugita, H., Momoi, H.. Toyokura, Lab. Clin. Med. 64. 299-305 Kar. N.C. and Pearson, C.M. (1965) Proc. Sot. Exp. Biol. Mrd. 118. 662-+64 Nuttall, F. and Wedin. D. (1966) J. Lab. Clin. Med. 68.324-332 Rosalki, S.B. (1967) J. Lab. Clin. Med. 69, 696.-705 Hess, J.W., MacDonald, R.P., Natho, G.J.W. and Murdock, K.J. (1967) 13, 994-1005 Fleisher, G.A. (1967) Clin. Chem. 13. 233-241 Wright, R.K. and Alexander, Jr., R.L. (1970) Clin. Chem. 16, 294-299 Warren. W.A. (1972) Clin. Chem. 18.473-475 Miyada, D.S., Dinovo, E.C. and Nakamura, R.M. (1975) Chim. Clin. Acta 58,97-99 Warren. W.A. (1973) Prep. Biochem. 3.199-208 Kuby, S.A., Mahowald. T.A. and Noltmann, E.A. (1962) Biochemistry 1, 748-762

J.

Creatine kinase activity in serum and uric acid solution.

Clinica Chimica Acta, 70 (1976) 285-288 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands CCA 7881 CREATINE WILLI...
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