Macro CK type 1 as a major component of serum
creatine kinase activity in pregnant sheep
I. Vojtic
*Biotechnology Research Centre, Maribor Veterinary Hospital, Sentiljska 109, SI-2000 Maribor, Slovenia
Accepted 30 June 1999
Abstract
Electrophoretic separation of creatine kinase (ATP: creatine phosphotransferase, E.C. 2.7.3.2) isoenzymes in agarose gel was made in the serum of 93 sheep of three Slovenian domestic breeds (Solcavska, Pramenka and Bovska breeds). 44 sheep were pregnant between 112 and 135 days at the time of the blood collection. The median value of serum total CK activity for all animals investigated was 82 U/l. After direct immunoinhibition of CK with anti-M-CK monoclonal antibodies the total CK activity remained the same (88 U/l, P0.354). There were signi®cant differences among breeds in CK activity for the Solcavska (101 U/l), Bovska (89 U/l) and Pramenka breeds (73 U/l), respectively (Kruskal±Wallis one way analysis of variance,P< 0.01), and between pregnant (105 U/l) and non-pregnant animals (76 U/l), irrespective of the breed (Mann± Whitney rank sum test,P< 0.05). According to electrophoresis, all non-pregnant sheep had exclusively free CK-BB serum bands activity. In all pregnant sheep coupled dimeric BB variant appeared as macro-CK type 1 in the range between 80% and 100% of total CK activity. The present study con®rms the existence of an elimination mechanism for CK from the plasma abundance free CK-BB enzyme.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Creatine kinase; Isoenzymes; Electrophoresis; Sheep
1. Introduction
Creatine kinase (CK; ATP: creatine phosphotrans-ferase, E.C. 2.7.3.2) is a widely distributed enzyme in the body of different mammal species. It has been recognized that CK plays a crucial metabolic role in cells with a high energy metabolism, such as cross-striated muscle or neural tissue. Also, it is the accepted opinion that CK catalyses the transfer of energy rich phosphate to ADP, to generate ATP (Wallimann and
Hemmer, 1994). Lefebvre et al. (1996) reviewed several factors that may in¯uence physiological var-iations in CK activity. CK is permanently released from muscles and other visceral organs into the blood plasma, but the effect of age, sex or diet have not been recorded. Beside this, under ®eld conditions, it would seem as if CK can be an excellent indicator of muscle damage. Studies on weaner wethers have shown that plasma activity of CK is signi®cantly correlated with muscle pathology during ovine nutritional myopathy (Smith et al., 1994), although parallel diagnostic combination of the different enzymes can overcome the lack of sensitivity of CK as an single diagnostic tool (Fry et al., 1994).
*Tel.:386-62-228-3708; fax:386-62-228-3701. E-mail address: [email protected] (I. Vojtic).
The total activity of CK in serum has been deter-mined as the sum of the enzymes activities originating from different organs such as muscle, kidney, spleen, liver or lung (Braun et al., 1992). The synthesis of enzyme subunits is controlled by two genes; one for the M and one for the B monomer subunit. Plasmatic pools consist of postsynthetic variants of three dimeric isoenzymes, noteworthy as MM (muscle), BB (brain) and a heterodimer composed of M- and B-subunits (MB; Stein et al., 1983). Using special techniques for subdifferentiation of human brain and muscle
isoenzymes, several isoforms can be isolated
(MM1±4 and MB1±2; Stein et al., 1983). Beside the
cytosolic enzymes, there are also two mitochondrial variants. They exhibit an octameric structure and
appear as muscle speci®c (Mia-CK) and ubiquitous
forms (brain, kidney, gut; Mib-CK). Both are
described as plasma macro-CK type 2 (Stein et al., 1983), whereas immunoglobulin bounded BB dimer is designated as macro-CK type 1 (Bohner and Stein, 1983).
The ®rst separation of CK isoenzymes, 35 years ago, was made on different rabbit tissues using elec-trophoresis on agar gel plates (Eppenberger, 1994). Later, numerous techniques have been evaluated in humans for isoenzymatic differentiation such as elec-trophoresis on cellulose gel strips, mass concentration by immunoassay or nephelometry, ion exchange chro-matography (Stein et al., 1983), radioelectrophoresis (Bohner and Stein, 1983), radioimmunoassay (Urdal, 1983), several immunological methods (WuÈrzburg, 1983) and solid phase separation (Gadsen et al., 1994). Although there have been many studies con-cerning total CK activity in ovine species (Pernthaner et al., 1993; Baumgartner and Pernthaner, 1994; Patra et al., 1996) until now, isoenzymatic patterns have not been well elucidated. Interestingly, most of the pub-lished methods used for enzyme separation have not been performed in sheep serum. Thus, this study was undertaken to investigate the presence and distribution of serum CK isoenzymes in the three Slovenian native breeds to better understanding the biology of CK. Using the combined method of electrophoresis with immunoinhibition, evidence will be presented to show that free BB dimer of CK is the only isoenzyme present in serum of non-pregnant adult sheep. On the other hand, in pregnant sheep macro CK type 1 comprises a major part of the total serum CK acitivity.
2. Material and methods
93 ewes aged between 2 and 6 years served as experimental animals. These ewes were native Slove-nian breeds from three ¯ocks: breeds from Solcava
(partially improved with Romanov breed, n27),
Bovska (n41) and Pramenka (n25). Several
months before (from November) and during the experiment (April) all animals were maintained in stalls on straw bedding and fed a basal diet consisting of hay (Bovska and Pramenka sheep) and grass silage with small amounts of hay (Solcava). Only the Pra-menka sheep were supplemented with a concentrate (18% of crude protein). A vitamin±mineral mix was freely available. The ewes were allowed to lamb and the gestational age at the time of blood collection was calculated according to the date of lambing and mean gestation period determined in the ¯ocks.
After collection, blood samples were centrifuged with a separation gel (Vacutainer, Becton Dicinson)
and the serum frozen at ÿ268C. All assays for CK
activity were performed within 14 days. The total CK activity was measured in a BM and Hitachi centrifugal analyzer (model 911) using the quantitative UV kinetic kits (Sys 2, No. 1273248) of Boehringer (Mannheim, Germany). The non-CK-M activity in the sample was measured with the same technique after incubating the aliquots of serum with anti-M monoclonal antibodies (Sys 1, No. 1127608 from Boehringer) with a binding capacity of up to 2000 U/l. This direct and speci®c immunoinhibition was performed according to Gerhardt et al. (1977). The quantitative analysis of CK isoenzymes was assessed by electrophoresis of 10ml serum on cellulose acetate, in tris-barbital sodium-barbital buffer (pH 8.8), based on procedure as described by Trainer and Gruenig (1968). Duration time of electrophoresis was 10 min at 300 V. The relative percentages of bands were evaluated by ¯uorescent development and automati-cally printed for each band on the scan.
performed (Dunn's post-hoc test) (Bechhofer et al., 1995). All computer algorithms used, were obtained from SigmaStat (Rev. 2.0, 1995, Jandel Scienti®c, San Rafael, CA).
3. Results
The median values of all CK activity measurements in the three groups of ewes grouped according to the breed, but irrespective of pregnancy status, are pre-sented in Table 1. The differences in the mean values among treatment groups, show statistically signi®cant
(P0.007) breed differences. The differences in
ranks (Q3.086), between the Bovska and Pramenka
breeds was the only signi®cant difference (P< 0.05), whereas no differences were detected between the other two breeds (Bovska vs. Solcavska and Solcavska vs. Pramenka breed, respectively). The median activ-ity of CK in all the ewes between 112 and 135 days of pregnancy was 105 U/l, and was signi®cantly different from that the non-pregnant ewes (76 U/l,P0.034),
although proportions of pregnant/non-pregnant ani-mals among breeds were not different (Solcavska 27/ 0, Bovska 13/28 and Pramenka ewes 4/21, respec-tively; chi-square43.906, d.f.2,P0.122). On the other hand, direct immunoinhibition with anti-M antibodies did not change the overall median CK activity in serum samples (82 U/l before vs. 88 U/l
after immnunoinhibition, respectivelyP 0.354).
The results of densitometry scanning of electro-phoresis sample plates show that non-pregnant ewes exhibit only the free BB dimer bands. No other bands were found in these animals. Conversely, all pregnant (112±135 days of pregnancy) animals have high rate of macro CK type 1 ranged between 80% and 100%, while the rest of CK bands was detected as a free BB dimer.
4. Discussion
As previously reported (Pernthaner et al., 1993) neither breed nor age differences were found among particularly mountain, milk and merino sheep. Dif-ferent level of CK observed may be attributed to various management and climate factors or malnutri-tion as reported by Pernthaner et al. (1993), Baum-gartner and Pernthaner (1994), Kumagai and White (1995), Patra et al. (1996) and Petrie et al. (1996).
The observation of median CK catalytical activity between pregnant and non-pregnant sheep (that were higher in pregnant) is consistent with widely accepted opinion of many authors in reviewed literature (see Wallimann and Hemmer, 1994). It shows that the CK activity in myometrium increases with gestation which leads to an increased potential for myo®laments contraction during parturition (Clark et al., 1997). In bovine, there was a doubling of CK activity in preg-nant myometrium cytosol fraction (Iyengar et al., 1980). Obviously, this phenomena must be hormonal related. The cause of elevated catalytical concentra-tion of CK has been reported by Reiss and Kaye (1981). It was identi®ed that estrogen induced protein in rat uterus was, in fact, CK BB isoenzyme. The CK activity was under stringent estradiol-17-b control at the level of messenger RNA transcription (Payne et al., 1993). Similar effects were detected using activation of rat's CK-B gen promoter by estradiol. The constructed estrogen receptors were able to activate gen promoter in a hormone-dependent manner, whereas no such effects were detected in the presence of progesterone, another hormone which had elevated concentrations during pregnancy (Crombie et al., 1994).
The most interesting and unexpected ®nding observed here was absence of any other isoenzymes than free BB dimer in serum of all non-pregnant sheep
Table 1
Descriptive statistics of kreatine kinase serum activity (U/l) in three group of ewes sorted by breeda
CK activity (U/l) Breed
Solcavska Bovska Pramenka
n 27 41 25
Min 35 51 50
Max 1116 568 175
Median 101 89 73
Skewness 2.038 2.908 3.078 Kurtosis 3.314 8.604 12.437 K±S distanceb 0.289 0.309 0.221
Pvalueb <0.001 <0.001 0.003
aKruskal-Wallis analysis of variance on ranks suggested
statistically significant among breed differences (P< 0.01).
investigated. Although there have been many studies concerning total CK activity, isoenzymatic pro®le in serum of healthy sheep has not been well elucidated. In contrast to our results, Beatty and Doxey (1983) reported four isoenzymes were present in serum of ®ve-month-old Cheviot lambs: two MM (subtypes
MM1 and MM2), MB1 and BB. The discrepancy
between these two studies cannot be easily explained. For the separation of isoenzymes electrophoresis was used. The main disadvantage of this method is low analytical sensitivity for MB and MM variants which frequently overlapped with other isoenzymes (Hen-derson et al., 1994).
To avoid this, two measurements of total CK were done: before and after inhibition with monoclonal anti-M antibodies. No signi®cant effect on total CK activity was detected after this treatment. Thus, there was no M activity in our samples and electrophoresis bands showed exactly a free BB isoenzyme without any overlapping as suggested by Gadsen et al. (1994). The results can be profoundly affected by abandoning a treatment with anti-M antibodies as seen in the study cited above (Beatty and Doxey, 1983). On the other hand, we would like to suggest another possibility, that can be attributed to the differences in age of experi-mental animals. As previously reported (Avallone et al., 1996), CK-MM was the only detectable isoenzyme in serum of buffalo calf during the ®rst week of life. Body growth and development of digestive system led to a change in the isoenzymatic pool. CK-BB appeared in the serum of 10-week old calves, while CK-MM diminished its activity to half. It is therefore possible, that the isoenzymatic pro®le of adult non-pregnant sheep re¯ect the contribution of particular organs and systems to the serum CK pool.
Contrarily, in all pregnant ewes the major compo-nent of total CK activity as macro-CK type 1 was identi®ed. The ®rst suggestion that CK-BB and IgG can form a macro-molecular complex was made by Stein and Bohner (1979). Binding between CK enzyme molecule and IgG is highly speci®c for cat-alytically active CK-BB (Bohner and Stein, 1983), which is quantitatively con®rmed in the present study. Macro-CK type 1 appears in older humans suffering from chronic diseases. Increased levels of CK were also present in serum through the pregnancy of ewes observed in this investigation. It seems reasonable to postulate, that the formation of a BB-IgG complex is
one of the mechanisms for enzyme elimination from the plasma abundance free CK-BB. This conclusion is supported by in vitro experiments using placental CK-BB determined by enzyme concentration, as well as enzyme activity (Bayer et al., 1983). During incuba-tion at 378C for 20 h the enzyme activity decreased, while enzyme concentration increased. This differ-ence con®rms the existdiffer-ence of an elimination mechan-ism for CK enzyme from the circulation and is in agreement with our hypothesis.
It can be concluded from this study that pregnant ewes have a substantially higher total CK activity then non-pregnant ewes, irrespective of the breed. The isoenzymatic pro®les showed a differential expression of CK isoenzyme BB in non-pregnant and pregnant animals. The latter had CK-BB coupled to the immu-noglobulins, which appeared on electrophoretic bands as macro-CK type 1. These results con®rm the exis-tence of an elimination mechanism for CK from the plasma abundance free CK-BB enzyme. Until now, such phenomenon in pregnant sheep has not been documented. This data can contribute to a better understanding of the biology of CK in ovine species.
Acknowledgements
This study was partialy supported by Krka, Phar-maceutical Industries, Slovenia. The author thanks Mrs. Mirjana Sauperl and Mr. Zlatko Jenko for expert technical assistance.
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