Bull. Equine Res. Inst. No. 17, 21-31 (1980)

Analysis of Creatine Kinase Isoenzyme in Racehorse Serum

and Tissues

Yoshikazu FujIT,* Syozi IKEDA*•˜ and Hiromasa WATANABE*

For the application to the clinical diagnosis of various equine diseases, creatine kinase (CK) isoenzyme analysis was conducted on racehorse serum and tissues by electrophoresis on cellulose acetate which was applied from that in human serum. This electrophoresis was evaluated in regard to simplicity, accuracy and sensitivity, as compared with column chromatography. The electrophoretic pattern of CK isoenzyme in normal horse serum possessed 5 bands: BB, MB, sub-band, MM1 and MM2. The mean values of percentage concentrations of these bands were 18.9%, 2.2%, 1.9%, 44.3% and 32.7%, respectively, in the sera of 150 clinically healthy racehorses. By electrophoresis of CK isoenzyme, MM1, MM2, MB and BB were obtained from almost all the equine tissues. Tissues with high CK activity possessed specificity to a respective organ, such as BB in cerebrum and spinal cord, MB in smooth muscle, and MM1 in skeletal and cardiac muscles. Further, this electro phoresis enabled the combined isoenzyme analysis of CK and LDH. Information from these analyses may make it easy to recognize the degrees and changes of clinical conditions, and may contribute to the accurate diagnosis of racehorse diseases by considering the organ specificity.

(hybrid type) 2) In the case of human Introduction serum, there are numerous reports3-13) Creatine kinase (ATP: creatine phos on CK isoenzymes, particularly on in photransferase, EC 2.7.3.2; CK) has creasing MB isoenzyme in myocardial been known as an important enzyme on infarction.3-8) The analysis of these iso muscular energy metabolism, and as being enzymes, in addition to that of total CK, involved mostly in brain and skeletal, has been performed as a routine clinical cardiac and smooth muscles.1) The iso examination. In the case of horse serum, enzyme of this enzyme has been proved the increases of total CK activities in to be composed of 3 forms of MM various diseases have been described, for (muscular type), BB (brain type) and MB example, in myodegeneration,14) tying up syndrome,15,16)slight muscle disorders Received for publication May 15, 1980. *:Equine Research Institute, Japan Racing Association. 27-7, after training,17,18) colic19) and pneu Tsurumaki 5 chome, Setagaya-ku, Tokyo 154 JAPAN.•˜ monia.19) On CK isoenzyme, however, : Present address. Ritto Training Center, Japan Racing As sociation. there are only a few reports by Anderson20) The outline of this paper was read before the 88th Meeting of the Japanese Society of veterinary Science in Hokkaido, 1979. and Ohishi et al.19)

-21- Y. Fujii et al.

In this paper, to separate 3 CK iso Enzyme activity analysis. Serum and enzymes from horse serum more simply tissue CK activities were assayed with a and accurately, cellulose acetate elec CPK monotest kit (Boehringer Mann trophoresis and column chromatography heim GmbH, West Germany) by the were compared. The normal values of method of Rosalki.21)Optical density was serum and tissue CK isoenzymes were determined by means of rate-assay with determined by this electrophoresis to ap a Spectronic 400 spectrophotometer ply them to the precise diagnosis of various (Baush & Lomb, U.S.A.) at 340nm. diseases in the horse. Serum CK activities were recorded in U/l of serum, and specific actitvies of Materials and Methods tissue extracts in U/g wet tissue.

Serum and tissue preparation. The Electrophoretic analysis of CK isoenzyme.

Thoroughbred racehorses used in this Electrophoretic separation of CK iso study were 150 clinically healthy males enzyme on cellulose acetate membrane

and females ranging from 2 to 6 years of (Titan ‡V-Iso Flur, Helena Lab., U.S.A.) age. They had been stabled at the Miho was performed in Tris-barbital buffer

Training Center and the Utsunomiya (pH 8.6) under the following conditions Rearing Farm, Japan Racing Associa which had been proved to be optimal for

tion, and received routine training. Serum horse serum. Usually, about 0.3 to 1.0

samples were separated from jugular of sample was simply applied withƒÊl venous blood of these horses, and stored a sample applicator and electrophoresis

at -20•Ž. The analyses of total CK performed at 300V for 25mm at 4•Ž. activity and CK isoenzyme were done After electrophoresis, the cellulose acetate

within five days. Several clinically ab strips were layered with a fluorescent

normal samples with high total CK CPK reagent (Helena Lab.). They were

activities were obtained from cases of dried quickly after incubation at 37•Ž

muscle, heart and digestive diseases. for 30min, and viewed under ultraviolet

Tissue samples were obtained from 3 light. Electrophoretic patterns were scan

clinically healthy horses within 2 h after ned on a CCU-fluorescent densitometer

death and stored at -20•Ž until they were (Helena Lab.). The values for each CK assayed. Those analyzed were skeletal and isoenzyme were recorded from integrated

cardiac muscles, cerebrum, spinal cord, scanning as percentage of total area under jejunum, cecum, stomach, spleen, the curve.

pancreas, kidney, lung and liver. Extracts In addition to the analyses of CK, the were prepared from them by homog total activities and isoenzyme patterns of

enizing 0.5g of tissue diluted to 10ml serum LDH were determined as indi

with cold 8.5% NaCI solution for 2 min cators of tissue damage in the clinically by using a polytron PCU-2-110 homog abnormal cases. Serum LDH activity was enizer (Kinematica GmbH, Switzer determined with the LDH monotest kit land). The supernatants after centrifuga (Boehringer Mannheim GmbH). LDH tion (12000•~g, 3min) were used for isoenzyme analysis was performed by the electrophoretic analyses. same electrophoretic procedure for CK,

- 22- Analysis of CK Isoenzyme except that the strips were stained with tion of high MM sample and of high BB a colorimetric LDH reagent (Helena sample. After separating these samples Lab.). into 3 isoenzyme fractions by column Column chromatography. Ion-exchange chromatography, each fraction was con column chromatography was performed centrated tenfold on Minicon (Amicon largely by the method of Mercer and Corp., U.S.A.). Then the concentrate was Varat.3,4) Minicolumns of DEAE run by this electrophoretic method.

Sephadex A-50 (Pharmacia Fine Chem.,

Sweden) 7•~60mm in size were prepared Results with 50mmol Tris buffer (pH 8.0). Electrophoresis.Fig. 1-a shows the elec Initially, 2ml of serum sample was ap trophoretic pattern of CK isoenzymeon plied to the top of the column, and 2ml cellulose acetate of normal racehorse of effluent collected as the first one of MM serum. Usually, this pattern consistsof 5 fractions. Then, stepwise elution was bands of CK isoenzyme, BB, MB, sub carried out, with a buffer containing band, MM, and MM2, as listed from the 50-300mmol NaC1, as shown in Fig. 3, anode. MB and sub-band, however, are at a flow rate of 0.5ml/min. The eluate too small in quantity of activity to be was a collection of 2ml of each of the alwaysfound. fractions. CK activity of an aliquot of Another pattern (Fig. 1-b) is obtained each isoenzyme fraction was assayed by from electrophoresis by using a blank the above-mentioned rate assay. reagent without creative phosphate. A The problem of contamination which fluorescentband in this pattern was con may occur in each CK isoenzyme frac firmed to be an albumin complex band tion5csl was investigated by the above by simultaneouselectrophoresis of serum described electrophoretic analysis with protein, and to be not due to any CK each concentrated chromatographic frac specificreaction.

Fig. 1. Cellulose acetate electropherograms (left) and fluorescent densitometric scans (right) of horse serum (a) and serum blank (b)

- 23- Y. Fujii et al. The accuracy and reproducibility of this electrophoresis are demonstrated in Table 1. The data of this table were obtained by extracting the homogenate of horse cecum and with normal serum containing slightly higher BB activity. The variation coefficient (CV) of MB in normal horse serum was high, because MB activity in Fig. 2. Relative mobilities of various CK isoenzymes normal serum is usually low. However, in relation to the serum proteins the CV of other isoenzyme was low and less than 10%. These results indicate the reliability and accuracy of this assay. Table 1. Within-run reproducibility on electro Column chromatography. Fig. 3 shows phoresis CK isoenzyme patterns determined with

10normal sera from racehorses by column

chromatography. The isoenzymes were

fractionated into 3 parts, as shown in

Fig. 3. The mean values and standard

deviations of percentage concentrations

of MM, MB and BB, were 73.3•}6.2,

3.2•}3.6 and 23.5•}5.2, respectively.

Remarks: *Control sample was prepared from homog

enate of horse cecum.

In Fig. 2, the relative mobility of CK isoenzymes revealed by cellulose acetate electrophoresis is illustrated, as compared with the mobility of serum protein bands. The mobilities of CK isoenzymes are as follows.

BB: adjacent to albumin band on

the anode side Fig. 3. CK isoenzyme patterns of normal racehorse MB: ƒ¿2-globulin serum by column chromatography sub-band: between ƒÀ1 and ƒÀ2 Remarks. 1. 50mmol Tris HCI buffer containing globulin 50mmol NaCI (pH 8.0). MMl: between ƒÀ2-globulin and II. The same buffer containing 100mmol origin NaCI (pH 8.0). MM2: ƒÁ-globulin III. The same buffer containing 200mmol A good separation of horse serum CK NaCI (pH 8.0). was obtained by this method even when IV. The same buffer containing 300mmol the total activity was as low as 5 10U/1. NaCI (pH 7.0).

- 24- Analysis of CK Isoenzyme a case of muscle disorder, which contained high MM activity. Other patterns shown in Fig. 4 (right side) are the electro phoretic patterns of the concentrates of MM, MB and BB fractions. Contamina tion with MM isoenzyme was observed in the MB fraction. Fig. 5 shows the elution pattern and electrophoretic patterns of Fig. 4. Elution pattern (left) of serum CK isoenzyme a sample with high BB activity. Contami in a case of muscle disorder by column nation with BB isoenzyme was found in chromatography and electrophoretic patterns the the MB fraction. (right) of these effluents GK isoenzyme patterns of normal sera and tissues of horse. Mean values and standard deviations of CK isoenzyme in normal horse sera are shown in Table 2. They were obtained from sera of 150 clinically healthy racehorses with total activity ranging from 10 to 40U/1. No significant sex or age difference was noted at P<0.05. Table 3 summarizes the total CK Fig. 5. Elution pattern (left) of serum CK isoenzyme activities and the relative proportions of with high BB activity by column chromato the isoenzyme activities in 12 tissues from graphy and electrophoretic patterns (right) each of the 3 racehorses. Fig. 6 presents of these effluents CK electropherograms from extracts of these horse tissues. As shown in Table 3, Figs. 4 and 5 show the results of total activities and percentage of each investigation on the contamination in band were different among these 12 column chromatography with high CK tissues, but CK isoenzymes of MM, MB activity samples. Fig. 4 (left side) shows and BB were observed in almost all the

Table 2. Total CK activities and percentages of CK isoenzymes in normal racehorse serum

Remarks: *SD=Standard deviation

- 25- Y. Fulii et al.

Table 3. Distribution of CK isoenzyme in horse tissue extracts

Fig. 6. Electrophoretic patterns of different horse

tissue extracts on cellulose acetate Remarks: *Enzyme activity is expressed as units per membrane gram of wet tissue.

activity were also found to possess this tissues. Within tissues with activities specificity, such as BB in pancreas and greater than 100U/g wet weight, the kidney, MB in spleen, and MM1 in lung greatest mean activity was found in and liver. As shown in Fig. 6, MM, skeletal muscle, followed by cardiac bands were not clear and had the tailing muscle, jejunum, cerebrum, cecum and in electropherograms of liver, kidney and stomach. Others showed a low CK ac pancreas. tivity. Tissues with high percentage of Abnormal CK isoenzymepatterns and ap MM, isoenzyme activity were skeletal plication to clinical diagnosis. In many muscle (75.2%) and cardiac muscle cases with total CK activities greater than (70.5%). Next, tissues with high MB 100U/1, MM1 activities increased to over isoenzyme activity were those originated 70%. Also in cases with slightly higher from smooth muscle, including cecum total activities, considerably large a (34.1%), stomach (36.9%) and jejunum mounts of MM1 activities were seen. In (32.8%). Further, those with high BB a few cases, BB activities increased to over isoenzyme activity were cerebrum 70%. (75.8%) and spinal cord (89.4%). Thus, Abnormal CK isoenzyme patterns with these tissues were found to possess an high MM activity were seen in clinical organ specificity. Tissues with low CK cases of various diseases, including

- 26- Analysis of CK Isoenzyme

Muscle disease (tying-up syndrome)

Heart disease (myocardial damage)

Digestive disease (serious colic)

Fig. 7. Diagrams of serum CK and LDH isoenzymes in various diseases myodegeneration, tying-up syndrome, of serum samples with normal total CK muscular stiffness, myalgia, obstruction activity, 2 separated MMl bands were of the esophagus and the first stage of found. colic. Abnormal patterns with increasing The diagnostic values of CK and LDH MB activity were observed in cases of zymograms considered together appeared myodegeneration, serious colic, chronic to be much greater than that of either diarrhea and colitis X. As other types of zymogram alone. Fig. 7 shows 3 diagrams abnormal pattern, in a few zymograms obtained from the combined analyses of

- 27- Y. Fujir et al. sera with various diseases. In the case of human serum. So the contamination with the tying-up syndrome, MM1 of CK, BB isoenzyme in MB isoenzyme should be LDH5 and LDH4 activities, based on the taken into consideration. To improve these disorder of skeletal muscle, increased problems just described, the following significantly. On the other hand, in the means have beenn required; concentrating case of myocardial damage with the oc enzyme reagent9) and of uent,5'6) and currence of a temporary cardiac arrest diluting the serum sample of high CK under general anesthesia, MM1, LDH1 activity.3) and LDH2 activities, based on cardiac On cellulose acetate electrophoresis, muscle, increased and MB activity in the sample volume is only 1 pl and several creased only slightly. In the case of samples can be applied simply and serious colic, an increase in MB activity uniformly by a sample applicator. The

based on smooth muscle was found. procedure is simpler in this technique than in column chromatography, but Discussion can analyze many samples simultaneously Various analytical methods of CK in the former within a short period. Also isoenzyme have been reported. Recently, by the electrophoresis, a good separation an electrophoretic method with a fluo of CK iso enzyme and high accuracy and rescent reagent has attracted attention for reproducibility are obtained, and the its high sensitivity. In this study, electro actual activity estimated is very likely phoresis was compared with column to reflect the true value of CK isoenzyme. chromatography which had previously Thus, this technique is supposed to be been performed in the authors' Institute. useful for the routine analysis of CK On column chromatography, the pro isoenzyme. cedure is said to be relatively simple, but Kiyose and Kyo10) obtained good elec requires a long period of time to be trophoretic patterns in human sera at performed. Therefore, this method may 350V for 9 min on the same cellulose be inadequate to analyze many samples. acetate membrane. In the present study, The effluents from the column are diluted the electrophoretic conditions, at 300V about 10 times, and these actual CK for 25min, were considered optimal for activities are very low. So in this method, horse serum to separate BB band from it may be possible to increase determina the albumin complex band in order to tion errors and to have low reliability. obtain the best electrophoretic pattern. Griffiths and Handschuh5) and Fiolet et Under these conditions, MM band was al.6) suggested that the false positivity of further separated into MM1 and MM2, MB isoenzyme might arise from the and sub-band was found between MB and contamination in high CK activity sample. MM1 bands. The present study could also confirm these Although Leroux et al.11) reported the contaminations by electrophoresis with presence of sub-band in abnormal human concentrated effluents from some serum serum, the sub-band observed in normal samples. In horse serum, more cases with horse serum is guessed to be a band sep high BB activity have been found than in arated from MM12,13) or MB band.12,3)

- 28- Analysis of CK Isoenzyme

It is also guessed to be a band origi described just above. From these findings, nated from some tissue with low CK in the application of this CK isoenzyme activity, because it has the same mobility analysis to the clinical diagnosis, the as the position of tailing between MM1 species difference should be considered. and MB observed in the isoenzyme pat In some serum samples from horses terns of liver, kidney and pancreas. with slight muscle disorders, and even In the present study, MM1 band was from clinically healthy horses with slightly further separated into 2 bands in a few higher total activity than a normal level, cases. The appearance of this abnormal the elevation of MM activity was electrophoretic pattern may be based observed. Thus, the activity of MM on 3 small bands separable from MM isoenzyme is supposed to serve as an band.12,13) Besides, it may also be a product indicator for the diagnosis of some muscle of complex formation from serum im diseases. MM activity is also assumed to munoglobulin22) or serum R-lipoprotein.23) be a good parameter of rest, proper The mobility of CK isoenzyme in therapy and therapeutic effect. An in normal horse serum on electrophoresis was crease in MB activity in human serum similar to that of isoenzyme in human has been used for the diagnosis of myo serum.24) The mean values of BB activities cardial infarction,3-8) but that in horse were reported to be less than 3% of total serum, as mentioned above, is supposed activity in normal human sera and to be useful for the diagnosis of such 18.5% in normal horse sera. Concerning diseases as originated from a smooth BB isoenzyme, there are a few reports on muscle disorder. In the CK isoenzyme some human diseases, such as enceph pattern of equine heart disease, an in alitis, 25) brain surgery, 25) infarction of crease in MM1 and a slight increase in the colon26) and carcinoma.27) In these re MB were observed. So, it seemed impos ports, however, discussion has scarcely sible to make a differential diagnosis been made on a normal value. The reason between heart disease and muscle disease why the BB activities are higher in horse with a similar pattern. By analyzing LDH serum than in human serum is not yet isoenzyme run simultaneously, an increase clear. Then, the problem mentioned above in LDH1 and LDH2 was observed in should be settled in future. heart disease, and an increase in LDH5 Tissue CK isoenzyme patterns were and LDH4 in muscle disease. Therefore, recognized to be different among animal the combined analyses of CK and LDH species, such as horse, human,28,29) isoenzymes seemed to enable the dif baboon,30) and dog.10) The CK isoenzyme ferential diagnosis of these diseases. Thus, patterns of skeletal muscle in horses informations from them in this study, to resembled those in man and other gether with the results of LDH isoenzyme animals. The patterns of cardiac and shown in some previous reports, 31, 32) smooth muscles possessed lower BB ac may make it possible to recognize the tivity and higher MM activity in horses degrees and changes of clinical conditions, than in man and other animals. Other and may contribute to the accurate tissues possessed similar patterns to those diagnosis of various equine diseases.

-29- Y. Fujir et al.

Acknowledgments kinase Z in human sera using a DEAE-cellulose mini-column method. Clin. Chim. Acta 80, 253 The authors wish to thank Professor I. Tomoda 264. of the Faculty of Agriculture, University of Tokyo, 12) Wevers, R. A., M. Delsing, J. A. G. Klein for helpful review on the manuscript. And, grate Gebbink and J. B. J. Soons. 1978. Post-synthetic ful appreciation to the staff' of the Veterinary changes in creatine kinase isoenzymes. Clan. Chim. Clinic, Miho Training Center, Japan Racing As Acta 86, 323-327. sociation, for their supply of useful clinical data and 13) Wevers, R. A., H. P. Olthuis, J. C. C. van samples for this work. Thanks are also given to Mrs. Nail, M. G. M. van Wilgenburg and J. B. J. M. Ijima, pharmacist, and Miss Y. Ishii, of the Soons. 1977. A study on the dimeric structure of authors' Institute, for the performance of important creatine kinase. Clin. Chim. Acta 75, 377-385. analyses of enzyme and isoenzyme activities. 14) Wilson, T. M., H. A. Morrison, N. C. Palmer, G. G. Finley and A. A. van Dreumel. 1976. Myodegeneration and suspected selenium vitamin E deficiency in horses. J. Amer. Vet. Med. Ass. Literature Cited 169, 213-217. 15) Yamaoka, S., S. Ikeda, H. Watanabe, Y. 1) Kachmar, J. F. 1970. Enzyme: Creatine Nagasawa, I. Takizawa and M. Hasegawa. 1978. phosphokinase, p. 464-470, In N. W. Tietz [ed.], Clinical and enzymological findings of tying-up Fundamentals of clinical chemistry. W. B. Saunders syndrome in thoroughbred racehorses in Japan. Co., Philadelphia. Exp. Rep. Equine Hlth Lab. No. 15, 62-78. 2) Shoji, S. 1980. CPK and CPK isoenzyme. 16) Lindholm, A., H. E. Johansson and P. Kjmrsgaard. 1974. Acute rhabdomyolysis (tying [transl, from Japanese]. JVihonRinsho (Jap. J. Gun. Med.) 38, 1320-1331 [inJapanese]. up) in standardbred horses. Acta Vet. Scand. 15, 3) Mercer, D. W. 1974. Separation of tissue and 325-339. serum creatine kinase isoenzymes by ion-exchange 17) Murakami, M. and S. Takagi. 1974. Effects of column chromatography. Gun. Chem. 20, 36-40. continuous long distance running exercise on 4) Mercer, D. W. and M. A. Varat. 1975. Detec plasma enzyme level in horses. Exp. Rep. Equine tion of cardiac-specific creatine kinase isoenzyme Hlth Lab. No. 11, 106-119. in sera with normal or slightly increased total 18) Anderson, M. G. 1975. The influence of exercise creatine kinase activity. Gun. Chem. 21, 1088-1092. on serum enzyme levels in the horse. Equine Vet. 5) Griffiith, J. and G. Handschuh. 1977. Creatine J. 7, 160-165. kinase isoenzyme MB in myocardial infarction; 19) Ohishi, H., S. Tamura, Y. Shimizu and Y. Methods compared. Clin. Chem. 23, 567-570. Ikemoto. 1978. Clinico-biochemical studies of 6) Fiolet, J. W. T., A. F. Willebrands, K. I. Lie creatine phosphokinase isoenzyme in horses. Res. and H. F. Ter Welle. 1977. Determination of Bull. Obihiro Univ. Ser. 11, 57-71 [in Japanese creatine kinase isoenzyme MB (CK-MB): Com with English summary]. parison of methods and clinical evaluation. Clin. 20) Anderson, M. G. 1976. The effect of exercise on Glum. Acta 80, 23-35. the lactic dehydrogenase and creatine kinase 7) Wolf, P. L., T. Kearns, J. Neuhoff and J. Lau isoenzyme composition of horse serum. Res. Vet. ridson. 1974. Identification of CPK isoenzyme Sci. 20, 191-196. MB in myocardial infarction. Lab. Med. 5, 48-50. 21) Rosalki, S. B. 1967. An improved procedure for 8) Ljungdahl, L., S. Hofvendahl, W. Gerhardt serum creatine phosphokinase determination. J. and J. Borjesson. 1977. Evaluation of S-CK B Lab. Clin. Med. 69, 696-701. subunit activity in the diagnosis of acute myocardial 22) Urdal, P. and S. Landaas. 1979. Macro creatine infarction. Clin. Chins. Acta 78, 43-53. kinase BB in serum, and some data on its 9) Takada, A., O. Sugita and M. Ogata. 1977. prevalence. Clan. Chem.25, 461-465. Fundamental studies on CPK isoenzyme by 23) Velletri, K., W. C. Grifpiths and I. Diamond. column chromatography. [transl, from Japanese]. 1975. Abnormal electrophoretic mobility of a Jap. J. Clan. Pathol. 25 (Suppl.), 381 [in Japanese, creatine kinase MM isoenzyme. Clin. Chem. 21 summary of oral presentaion]. 1837-1838. 10) Kiyose, H. and S. Kyo. 1977. Studies on analysis 24) Kawahara, T. and K. Ohtsuki. 1978. Studies and clinical application of CPK-MB isoenzyme. on CPK isoenzyme. [transl. from Japanese]. Eisei [transl. from Japanese]. Jap. J. Clin. Pathol. 25 Kensa (Jap. J. Med. Technol.) 27, 706-709 [in (Suppl.), 360 [in Japanese, summary of oral Japanese]. presentation]. 25) Nealon, D. A. and A. R. Henderson. 1975. 11) Leroux, M., H. K. Jacobs, S. W. Rabkin and Measurement of brain-specific creatine kinase P. R. Desjardins. 1977. Measurement of creatine isoenzyme activity in serum. Clin. Chem. 21, 1663

- 30- Analysis of CK Isoenzyme

1666. human tissues and sera by an immunological 26) Doran, G. R. 1979. Appearance of creatine kinase method. Clin. Chim. Acta 58, 223-232. BB isoenzyme in the serum of a patient suffering 30) Yasmineh, W. G., R. B. Pyle, N. Q. Hanson from infarction of the colon. Gun. Chim. Acta 92, and B. K. Hultman. 1976. Creatine kinase iso 415-419. enzymes in baboon tissues and organs. Gun. Chem. 27) van Lente, F. and R. Galen. 1978. Electro 22, 63-66. phoretic identification of the brain isoenzyme of 31) Ikeda, S., S. Yamaoka, H. Watanabe and T. creatine kinase following treatment with anti Kameya. 1978. Analysis of serum LDH isoenzyme BB antisera. Gun. Chim. Acta 87, 211-217. using a Cellogel membrane in Thoroughbred 28) Akanuma, S. and K. Kamishima. 1978. Studies racehorse. Exp. Rep. Equine Hlth Lab. No. 15, 1-7 on analysis and clinical application of CPK [in Japanese with English summary]. isoenzyme. [transl. from Japanese]. Eisei Kensa 32) Littlejohn, A. and D. J. Blackmore. 1978. (Jap. J. Med. Technol.) 27, 695-698 [in Japanese]. Blood and tissue content of the isoenzymes of 29) Jokers-Wretou, E. and G. Pfleiderer. 1975. lactate dehydrogenase in the thoroughbred. Res. Quantitation of creatine kinase isoenzymes in Vet. Sci. 25, 118-119.

競 走 馬 のCKア イ ソエ ンザ イ ム分 析

藤井良和*・ 池 田正二*§・渡辺博正*

競 走 馬 の疾 病 診 断 へ の応 用 を 目的 と して, CKア イ 馬150例 の正 常 分 画 比 は, BB…18.9%, MB…2.2%,

ソエ ンザ イ ム分 析 につ い て,セ ル ロー ス.ア セテ ー ト 亜 分 画 …1.9%, MM1…44.3%, MM2…32.7%で あ り,

膜 に よ る電 気 泳 動 法 とDEAE-Sephadexに よ る カ ラ ム 性 差,年 齢 差 は ほ とん ど認 め られ な か った.組 織CK

ク ロ マ トグ ラ フ ィ ー の操 作 性,感 度,精 度 等 を比 較 し アイ ソ エ ンザ イ ム にお い て,ほ とん ど の組 織 は,各 々

検 討 した.さ らに こ の泳 動 法 に よ って,競 走 馬 の正 常 総 活 性 ・分 画 比 は異 な る が, MM, MB, BBの3分 画

分 画 比 及 び診 断 の指 標 とな る臓 器 分 布 を検 討 し,臨 床 を有 す る こ とが 判 明 した.ま た 骨 格 筋 と心 筋 はMM1

例 にお け るCK, LDHア イ ソ エ ン ザイ ム の両 分 析 所 見 分 画,脳 と脊 髄 はBB分 画,平 滑 筋 はMB分 画 が 高 活

の 組 合 せ に よ る診 断 価 値 につ い て も検 討 した. 性 で あ り,組 織 特 異 性 の あ る こ とが確 認 され た. CK

電 気 泳 動 法 は カ ラ ム ク ロ マ トグ ラ フ ィー に比 べ,操 及 びLDHア イ ソ エ ンザ イ ム分 析 所 見 の組 合 せ,す な

作 が 簡 便 で,短 時 間 に多 検 体 を測 定 で き,分 離 能 及 び わ ち筋 障 害 はMM1分 画, LDH5・LDH4分 画 の 増

精 度 も良 好 で あ っ た.さ らにLDHア イ ソエ ンザ イ ム 加,心 筋 障 害 はMM1分 画 とLDH1・LDH2分 画 の 増

と同 時 泳 動 で き る利 点 が あ り,ル ー チ ンの 臨 床 検 査 法 加,及 び 消 化 器 障 害 はMB分 画 の 増 加 等 の 所 見 が 得

と して の 有 用 性 が 認 め られ た.馬 の 正 常血 清 に お け る られ る こ とか ら,各 々 の 障 害 の 病 勢 の 把 握 及 び鑑 別診

CKア イ ソ エ ンザイ ム の泳 動 パ タ ー ンに は, BB, MB, 断 等 に役 立 つ こ とが 判 明 した. MM1, MM2及 び 亜 分画 の5分 画 が 認 め られ た.競 走

*日 本 中央競馬会 競走馬総合研 究所 〒154 東京都世田谷区弦巻5 丁 目27番7号 § 同本中央競馬会 栗東 トレー ニング ・セ ンター 本 研究の要旨は第88回 日本獣 医学 会(北 海道, 1979年)に おいて発表 された.

- 31-