Isoforms of creatine kinase (creatine-N-phosphotransferase, CK, EC 2.7.3.2), BB and MB, were isolated from healthy human brain tissue and cardiac muscle, respectively, and were characterized by means of isoelectric focusing (IEF). CK-BB isoforms in Tris-HCl buffer were focused at pI 4.5 (a tissue form) and those in fresh sera from healthy adults were focused at pI 5.0, 5.1 and 5.2 (plasma forms). The IEF patterns of CK-BB isoforms were not altered following treatment with carboxypeptidase B and incubation in fresh serum at 37 degrees C; thus, it was found that there was no lysine at the C-terminal of the CK-B subunit and CK-BB isoforms were not results of the removal of lysine. Three CK-BB isoforms in fresh sera were identified to be oxidized, intermediate and reduced forms from the anodal side, respectively, by treatment with hydrogen peroxide and 2-mercaptoethanol. The oxidized form of CK-BB seemed to have higher affinity to IgG than the other two plasma forms of CK-BB. On the other hand, CK-MB isoforms in Tris-HCl buffer were focused at pI 5.4 (a tissue form) with a minor band at pI 5.2 and those in sera were focused at pI 5.0, 5.1, 5.2 (plasma forms) and 5.4. Four CK-MB isoforms were identified to be reduced, intermediate and oxidized forms without lysine from the anodal side, respectively, and the cathodal band was a tissue form with lysine.

Peroxiredoxins (Prxs) are versatile enzymes that demonstrate various cell functions as peroxidases, protein chaperones, functions of signal modulators and binding partners. It is well established that Prxs can interact with multiple proteins in cells, such as ASK1, Cdk5-p35, JNK, MIF, PDGF, TK R4 and others. In this study, we attempted to evaluate a possible association between ubiquitous Prx II and ATP/ADP buffering enzyme - brain-type creatine kinase (CK BB). Our co-immunoprecipitation (Co-IP) results from the A549 and HeLa cell lysates with overexpressed HA-Prx II and Flag-CK BB have demonstrated strong association between two proteins under non-stressed conditions. This protein interaction was enhanced by the heat treatment with further HA-Prx II precipitation to the immobilized Flag-CK BB depending on the temperature increase. Temperature induced oligomerization of Prx II may contribute to the formation of Prx II conglomerates, which in turn, can associate with CK BB and increase signal intensities on the blotted membranes. Thus, such association and oligomerization of Prx II could take part in recovery and protection of the CK BB enzyme activity from inactivation during heat-induced stress.

OBJECTIVE

In aortic arch surgeries, antegrade selective cerebral perfusion (SCP) combined with deep hypothermic circulatory arrest (DHCA) has been recently widely used in institutions as one of the most reliable methods for cerebral protection. However, some studies reported a 3.7-9.3% incidence of postoperative cerebral complications. To perform antegrade SCP more safely, we sought to examine the impact of pulsatile flow perfusion during DHCA on cerebral tissue metabolism, focusing on physiological effects of pulsatile flow perfusion.

METHODS

Sixteen pigs were divided into 2 groups. In each group, antegrade SCP combined with DHCA was conducted. During circulatory arrest, for SCP, a pulsatile flow (group P) and a nonpulsatile flow (group N) were used. We compared results between group P and group N. Jugular venous oxygen saturation (SjO(2)) and cerebral tissue oxygen partial pressure (PtO(2)) were measured at baseline, and continuously throughout the extracorporeal circulation. Hematocrit (Ht), and concentrations of S-100 protein and CK-BB in blood and the cerebrospinal fluid (CSF) were measured at baseline (before the beginning of extracorporeal circulation), following SCP, and after rewarming. Following rewarming, each brain under perfused fixation was removed, and histopathological examinations were conducted using Kluver-Barrera and Tunnel staining methods, electron micrograph.

RESULTS

SjO(2) was found to be within normal ranges until after SCP, but decreased with rewarming in both groups. In Group N, changes in SjO(2) were significant, with a decrease to < or =50%. In Group N, concentrations of S-100 protein and CK-BB in CSF after SCP and after rewarming were significantly higher than those in Group P. The time needed for rewarming to 36 degrees C in Group P was shorter than that in Group N.

CONCLUSIONS

Our results suggest that the pulsatile flow circulation method shows cerebral protection effects with increasing blood flow in small cerebral tissues. In addition, it is effective for improving the imbalance between oxygen supply and demand, especially in the process of rewarming from hypothermic conditions. This method seems to be useful as an adjunct in hypothermic circulatory arrest procedures.

BACKGROUND

To verify and evaluate the performance characteristics of a creatine kinase phosphokinase isoenzymes MB (CK-MB) assay kit, which produced by Xiamen Innodx Biotech Co. Ltd.

METHODS

Evaluation was carried out according to "Guidelines for principle of analysis performance evaluation of in vitro diagnostic reagent." The performance parameters included detection limit, linearity range, reportable range, recovery test, precision verification, interference test, cross-reactivity, matrix effect, and method comparison.

RESULTS

The detection limit was 0.1 ng/mL. The assay had clinical linearity over range of 0.1 ng/mL-500 ng/mL. Reportable range was from 0.1 ng/mL to 1000 ng/mL. The average percent of recovery was 99.66%. The coefficient of variation (CV) for within-run and between-run of low CK-MB sample was 5.55% and 6.16%, respectively. As for high-level sample, it was 7.88% and 7.80%. In medical decision level, the relative deviation (Bias) of all interference tests was lower than 15%. When the sample had mild-hemolysis; hemoglobin ≤15 g/L; triglyceride ≤17 mmol/L; bilirubin ≤427.5 μmol/L; rheumatoid factor ≤206U/mL, there was no significant interference to be found. Moreover, assay kit had no cross-reaction with CK-MM and CK-BB. At last, total diagnostic accuracy of kit was 93.24%, when compared with refer kit.

CONCLUSIONS

Overall the results of the verification study indicated the performance of kit is met the requirements of the clinical test.

The comparability of results for enzyme activity concentrations estimated with the methods recommended by the Scandinavian Committee on Enzymes(SCE) or the subcommittee on Enzymes of the European Committee for Clinical Laboratory Standards(ECCLS), and with the Kodak Ektachem methods, are discussed. As the Ektachem system in principle uses the IFCC methods at 37 degrees C as a reference, the results compare reasonably well for the alanine amino-transferase(ALAT), aspartate aminotransferase(ASAT), creatine kinase(CK) and gamma-glutamyltransferase(GT). The Ektachem reference method for lactate dehydrogenase(LD) is in principle the SCE method. The reasons for lower values with the SCE method are discussed. The interference on the Ektachem alkaline phosphatase (ALP) method from high concentrations of methotrexate(MTX) in serum, is shown. Results from our investigations on the interference by heparin in enzyme determinations on Ektachem are given. The problems with CK determinations in cancer patients regarding myocardial diseases are well known. When CK-BB is present in serum, the CK-B concentration is apparently lower using the Ektachem CK method than with use of the CK-MB slide, probably relating to the fact that chelator is omitted from the CK slide, while EGTA is supplied in the CK-MB slide. The user's dependence on the manufacturer for the choice of methods and the quality of the reagents, makes it important for laboratories and the Kodak Company to collaborate with the intention to continuously improve the Ektachem methodologies. The future possibilities of this technology seem to be nearly unlimited.

Serum creatine kinase (CK) and lactate dehydrogenase (LD) isoenzymes were determined electrophoretically, along with various other biochemical markers of malignancy, in 19 patients with metastatic carcinoma of the prostate. Mitochondrial CK appeared in 15 patients, the CK-BB isoenzyme in 6. As a result, CK activity not inhibited by anti-M-subunit antibodies, CK non-M, was above the reference value in altogether 17 patients. There was a cathodic shift among the LD isoenzymes, significantly more prominent with increasing total LD, and a positive correlation between elevations of CK non-M and LD-5, suggesting a relation to tumour burden for both. An LD 'flip' (LD-1 greater than LD-2) was present in 10/15 patients. The frequency of CK non-M elevations was similar to--but not quantitatively correlated with--elevations of prostatic acid phosphatase and alkaline phosphatase. Thus, changes in CK and LD patterns are frequent in patients with prostatic cancer and must be taken into consideration when acute cardiac symptoms are evaluated in such patients.

BACKGROUND

The point-of-care (POC) test Roche CARDIAC CK-MB is a new assay which has been developed for the existing Roche Cardiac reader system.

METHODS

We performed a multicentre evaluation at six sites to assess the analytical performance of the POC CK-MB assay and to compare it with a quantitative laboratory CK-MB assay.

RESULTS

Within-series coefficients of variation (CV) resulting from 34 ten-fold measurements with patient samples ranged from 4.3% to 16.4%. Using quality control material, the mean CV values for day-to-day imprecision were 6.5% for the low level control and 8.4% for the high level control. Based upon 847 pairs of values, the mean relative bias of three independently calibrated lots of the POC CK-MB assay ranged from -6% to -11% in method comparisons with the lab CK-MB assay. The mean relative lot-to-lot differences of POC CK-MB were between -2% and +1%. No interference was observed with lipaemic blood (triglyceride concentrations up to 8.1 mmol/L), icteric blood (bilirubin concentrations up to 513 micromol/L), haemolytic blood (haemoglobin concentrations up to 0.12 mmol/L), biotin (up to 30 mg/L) and rheumatoid factor (up to 119 IU/mL), or with 53 standard or cardiological drugs even in toxic concentrations. There was no influence on the results by varying haematocrit values in the range from 21% to 54%. A slight interference with human anti-mouse antibodies type 2 was found. No significant influence on the results with POC CK-MB was found by using sample volumes between 135 and 165 microL. High CK-MB concentrations above the measuring range of POC CK-MB (1-40 microg/L) did not lead to false low results due to potential high-dose hook effect. No significant effect of sample age on recovery occurred up to a sample age of 24 h. No cross-reactivity was found between the POC CK-MB assay and either CK-MM or CK-BB. A substudy with healthy individuals confirmed the reference limits of 3.8 microg/L for females and 6.7 microg/L for males.

CONCLUSIONS

The POC CK-MB assay showed a very good analytical performance with an excellent concordance with the calibration and reference laboratory method. It should be therefore suitable for its intended use in POC settings. Clin Chem Lab Med 2008;46:630-8.

Creatine kinase MB (CK-MB) was measured in serum by a fluorometric enzyme immunoassay on the Stratus analyzer and by an immunochemiluminometric assay using the Ciba Corning Magic Lite System. Both methods were standardized against purified CK-MB, with Stratus underestimating by 20 percent and Magic Lite overestimating by 28 percent. The assays proved sensitive and linear; however, at a CK-MB concentration of 7.0 micrograms per L, Stratus gave unacceptable inter-assay precision. No cross-reactivity was observed with CK-MM or CK-BB and elevated triglycerides, bilirubin, and hemoglobin did not interfere. Correlations with an immunoradiometric assay (Embria), using 522 samples, gave: Stratus = 0.999 (Embria) -3.3; r = 0.969, and Magic Lite = 1.225 (Embria) -3.03; r = 0.971. When using Magic Lite, results from 40 acute myocardial infarction (AMI) patients gave a mean CK-MB value of 93.8 micrograms per L (range: 9.2 to 428 micrograms per L) at the peak of enzyme release and a mean value of 69.6 micrograms per L (range: 6.7 to 319 micrograms per L) when using Stratus. Both methods proved to be highly sensitive and specific in the diagnosis of AMI; however, the need for standardization of CK-MB assays is stressed.

Seven (41%) out of 17 subarachnoid haemorrhage patients had MB isoenzyme of creatine kinase (CK) in the serum. The peak activity of MB was observed about 16 h after the onset of neurological symptoms. Of the patients with MB activity, five (71%) had pathological ECG changes suggesting acute cardiac injury. Of the patients examined, five died, and four of these had both CK-MB and CK-BB isoenzymes in the serum. In a preliminary study a test was made whether MM and BB were converted to MB in 48 h at 4 degrees C or 20 degrees C in a saline solution containing mercaptoethanol and bovine serum albumin. Such conversion was not observed. Human brain tissue extract obtained at autopsy was used in the study.

A CK isoenzyme migrating between CK-MB and CK-BB was detected in the serum of three patients with metastatic prostatic carcinoma. CK-BB was detected in the serum of all three patients and mitochondrial CK in two of the patients. Total CK activity was either normal or elevated, and the atypical CK isoenzyme, CK-BB and the mitochondrial CK isoenzyme were present in serum for up to 1.5 months. This atypical CK isoenzyme was not CK-MB, an albumin-ligand complex, or adenylate kinase, and was not bound to an immunoglobulin. This atypical CK isoenzyme did not contain immunologically normal CK-M subunits but had some CK-B subunits and could be a variant CK-BB or CK-MB isoenzyme. Its appearance in serum could be indicative of a serious illness.

We observed nonenzymic peaks when serum isoenzymes of lactate dehydrogenase (EC 1.1.1.27; LD) and creatine kinase (EC 2.7.3.2.; CK) were separated by "high-performance" liquid chromatography and detected by continuously monitoring the column effluent for enzyme activity. Such background peaks were particularly apparent in CK isoenzyme profiles obtained from human sera. We observed two nonenzymic peaks with fluorescence detection, one in the CK-MB region, the other in the CK-BB region. Serum albumin was a major component in the artifactual CK-MB peak, with lipoprotein as a minor component. We present evidence that the material responsible for the other peak fluoresced quite strongly and is mostly pre-albumin.

A marked intrapair discordance in placentas and in many body and organ measurements are risk factors influencing perinatal mortality and morbidity in twins. Asphyxia is the single most important perinatal cause of neurologic morbidity in newborn infants. The higher hypoxic risk for the second twin arises, however, from conclusions based on studies that did not consider the new diagnostic possibility of using blood measurements of the brain-type isoenzyme of creatine kinase (CK-BB) as a marker of perinatal asphyxia. CK-BB levels were measured in cord blood of 60 preterm infants (mean birth weight 1670 +/- 390 g, and mean gestational age 33 +/- 1.9 weeks) born of twin gestation in the last 3 years. The mean CK-BB values were 48 +/- 40 U/l versus 29 +/- 31 U/l (p less than 0.5). Skilful antepartum and perinatal care are the keys for optimal management of both babies, as demonstrated by similar CK-BB values obtained in their cord-blood specimens after birth.

Intermittent exposure to hypoxia (IHE) increases production of reactive oxygen and nitrogen species which, as signalling molecules, participate in tissue injury-repair-regeneration cascade. The process is also stimulated by arginine whose bioavailability is a limiting factor for NO synthesis. The effects of IHE in combination with arginine (Arg) intake on myogenesis and angiogenesis mediators were examined in a randomized and placebo-controlled trial. Blood samples were collected from 38 elite athletes on the 1st, 7th and 14th days during the training camp. The oral doses of arginine (2 × 6 g/day) and/or IHE using hypoxicator GO2Altitude (IHE and Arg/IHE) were applied. Serum NO and H₂O₂ concentrations increased significantly and were related to muscle damage (CK activity >900 IU/mL) in IHE and Arg/IHE compared to placebo. The changes in NO and H₂O₂ elevated the levels of circulating growth factors such as HGF, IHG-1, PDGF^BB, BDNF, VEGF and EPO. Modification of the lipid profile, especially reduced non-HDL, was an additional beneficial effect of hypoxic exposure with arginine intake. Intermittent hypoxic exposure combined with high-dose arginine intake was demonstrated to affect circulating mediators of injury-repair-regeneration. Therefore, a combination of IHE and arginine seems to be a potential therapeutic and non-pharmacological method to modulate the myogenesis and angiogenesis in elite athletes.

Keywords: athletes; growth factors; hydrogen peroxide; muscle damage; nitric oxide.

We present a measurement of the time-dependent CP-violating (CPV) asymmetries in B0->>K(0)(S)pi(0) decays based on 124x10(6) Upsilon(4S)->>BB decays collected with the BABAR detector at the PEP-II asymmetric-energy B factory at SLAC. In a sample containing 122+/-16 signal decays, we obtain the magnitudes of the direct CPV asymmetry CK(0)(S)(pi(0))=0.40(+0.27)(-0.28)+/-0.09 and of the CPV asymmetry in the interference between mixing and decay SK(0)(S)(pi(0))=0.48(+0.38)(-0.47)+/-0.06 where the first error is statistical and the second systematic.

The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB) specific monoclonal antibodies (mAb), and characterize the monoclonal antibody and further development of quantitative detection assay for CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then monoclonal antibodies were prepared according to conventional hybridoma technique and screened by indirect and capture ELISA method. To identify the epitopes and evaluate the classification, purchased creatine kinase isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes, with immunoblotting and synthetic CK-MM and CK-BB in different linear epitope. A double antibody sandwich ELISA was applied to screen the mAb pairs for CK-MB detection, and the quantitative detection assay for CK-MB was developed. We used 74 cases of clinical specimens for comparison of our assay with Roche's CK-MB assay. We successfully developed 22 strains of hybridoms against CK-MB, these mAbs can be divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross monoclonal antibody and CK-MB specific reaction with partial conformational monoclonal antibody, and CK-MB quantitative detection assay was developed by using partial conformational monoclonal antibody. The correlation coefficient factor r of our reagent and Roche's was 0.930 9. This study established a screening method for CK-MB partial conformational specific monoclonal antibody, and these monoclonal antibodies were analyzed and an established quantitative detection assay was developed. The new assay had a high concordance with Roche's.

We studied the behaviour of an atypical CK in one female patient. Using exclusion-diffusion chromatography of the serum, on Sephadex G 200, we observed the simultaneous elution of a CK-BB and IgA. We demonstrated that this eluate contained the components responsible for this atypical CK. The study of the behaviour of this enzyme complex reveals a resemblance with that of immune complexes.

We measured the BB isoenzyme of creatine kinase in the serum of 135 patients following cardiac surgery with cardiopulmonary bypass: 64 infants, 48 children and 23 adults, 56 of them have had, at least, one positive result. The duration of anaesthesia do not have any influence; we have found the same proportion of positive results in the two groups with anaesthesia less or more than 8 hours. Severity and complexity of the surgical procedure induce the CK-BB increase. In many cases, a high serum concentration of CK-BB is not related to clinical cerebral damage. Under this circumstances, it is possible that the rise in CK-BB concentration is not specific of central nervous system lesion.

A new two-site immunoenzymometric method using monoclonal antibodies was developed for measuring CK-BB mass concentrations in cerebrospinal fluid (CSF). Within- and between-assay coefficient of variation values for the method varied between 6 and 9%. Assay results are not affected by presence of sulfate and sialic acid groups on the enzyme. In comparison to catalytic activity measurements, a steady decline in the enzyme's specific activity was observed after acute head trauma. Repetitive measurements of CK-BB mass concentration in cerebrospinal fluid during the first 24 h after trauma enabled the estimation of brain lesion size. Clinical outcome of acute head trauma patients evaluated by Glasglow Outcome Scale, correlated well with cumulative CK-BB release after trauma. Also in neonates, CK-BB determinations in CSF correlated well with clinical findings.

Growth, protein synthesis and expression of creatine kinase (CK) by embryonic chick myogenic cells are inhibited by vitamin D and certain of its metabolites. 25-OH cholecalciferol was most active in concentrations of 10(-5) - 10(-6) M, with cholecalciferol and ergocalciferol less active in that order. Ergosterol had no activity of this sort. Inhibition of CK was most marked on the 4th and 5th day of culture and was due to suppression of the appearance of CK-NM and MB. CK-BB was not affected and CK-MB was more affected than CK-BB. Skin fibroblasts by comparison were slightly stimulated in growth at 10(-6) M and much less affected at 10(-5) M than the myogenic cells. It is suggested that vitamin D has a direct effect upon the muscle cell, to cause a selective diminution in the production of certain polypeptides.

We evaluated the analytical performance of the immunoprecipitation technique for quantification of creatine kinase (CK; EC 2.7.3.2) isoenzyme MB, focusing on specimens with increased blank activities. The samples we studied had blank values that were equal to or greater than either 15 U/L or the uncorrected CK-MB value. Of 134 specimens selected, 16 and 21 contained macro CK type 1 and CK-BB, respectively. All samples containing macro CK type 1 gave negative CK-MB values, even though four contained significant quantities of CK-MB. Correcting the blank value for the fluid displacement of the second antibody suspension eliminated all but three of the negative results. However, 45 specimens, negative for CK-MB by electrophoresis, gave CK-MB values ranging from 1 to 21 U/L. Furthermore, 11 of the specimens containing macro-CK type 1 were in this group. Thus, the presence of macro-CK type 1 definitely interferes with the immunoprecipitation technique. Our results indicate the need to re-evaluate the diagnostic accuracy of the immunoprecipitation technique, especially when the un-adjusted blank activities are increased.