A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients'' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.     

An integrated chip for rapid, sensitive, and multiplexed detection of cardiac biomarkers from fingerprick blood

Cardiovascular diseases are the major cause of death among adults worldwide. Electrocardiogram (ECG) is a first test when a patient suffering from chest pain sees a doctor, however, it is lack of the required sensitivity. Standard assays to detect cardiac biomarkers, like enzyme-linked immunosorbent assay (ELISA) are sensitive, but suffer from important sample and reagent consumption in large-scale studies. Moreover they are performed in central laboratories of clinics and hospitals and take a long time, which is highly incompatible with the quick decisions needed to save a heart attack patient. Herein, we describe an integrated chip allowing rapid, sensitive, and simultaneous analysis of three cardiac biomarkers in fingerprick blood. The integrated chip is composed of a filtration chip for plasma separation from blood and a silicon nanowire (SiNW) array sensor chip for protein detection. These two chips are fabricated separately and bonded to form a single unit after alignment. The integrated chip is capable of reducing the dead volume of the sample by eliminating the tubing between the two chips. After the plasma is filtrated by the filtration chip, the SiNW sensor, spotted with three different antibodies, enabled us to detect three cardiac biomarkers, troponin T (cTnT), creatine kinase MM (CK-MM) and creatine kinase MB (CK-MB), simultaneously. The integrated chip is able to attain a low detection limit of 1 pg/ml for the three cardiac biomarkers from 2 μl blood in 45 min.     

Clinical and analytical performance of automated immunochemiluminometric assays for determination of cardiac markers in serum

Three new immunochemiluminometric assays for quantitation of cardiac markers, i.e. creatine kinase isoenzyme MB (CK-MB), myoglobin and cardiac troponin I (cTnI), were evaluated with the Sanofi Access analyser. The complete profile requires 20 min to perform, the method being suitable in true stat situations. In patients with early myocardial infarction (median time of sample collection: 210 min from onset, range 30–450; n = 44), the diagnostic sensitivity of Access cTnI was 66%, compared with 80% for myoglobin, and 43% for CK-MB. For comparison, cTnI, with an automated immunofluorimetric assay was also measured (sensitivity, 45%; p < 0.05 vs. Access cTnI). Our data confirmed myoglobin as the first biochemical marker to appear elevated after infarction. However, cTnI may be a more sensitive marker for early detection of cardiac damage than initially thought, when determined by an ultrasensitive method such as an immunochemiluminometric assay. © 1998 John Wiley & Sons, Ltd.     

Myofibrillar creatine kinase and cardiac contraction

This article is a review on the organization and function of myofibrillar creatine kinase in striated muscle. The first part describes myofibrillar creatine kinase as an integral structural part of the complex organization of myofibrils in striated muscle. The second part considers the intrinsic biochemical and mechanical properties of myofibrils and the functional coupling between myofibrillar CK and myosin ATPase. Skinned fiber studies have been developed to evidence this functional coupling and the consequences for cardiac contraction. The data show that creatine kinase in myofibrils is effective enough to sustain normal tension and relaxation, normal Ca sensitivity and kinetic characteristics. Moreover, the results suggest that myofibrillar creatine kinase is essential in maintaining adequate ATP/ADP ratio in the vicinity of myosin ATPase active site to prevent dysfunctioning of this enzyme. Implications for the physiology and physiopathology of cardiac muscle are discussed.     

最大等速离心运动诱发肌肉损伤程度与肌酸激酶水平的关系

为探讨人体进行最大等速离心运动(ECC)诱发血液肌酸激酶(CK)水平变化、血清肌酸激酶水平与肌肉损伤(EIMD)的关系,本研究筛选出150名"缺乏运动"的健康大学生为受试者,进行血样采集,进行前测包括血清肌酸激酶(CK)、最大等长肌力(MVC)、肘关节活动角度(ROM)、上臂围(CIR)、肌肉感受(VAS)。受试者进行5组×12次最大等速离心运动,运动后恢复期,将全部受试者血清肌酸激酶值进行排序:血清肌酸激酶值最高和最低20%样本,高肌酸激酶水平组(HCK组)和低肌酸激酶水平组(low LCK组),利用SPSS18.0统计学软件,以方差分析和多元回归分析进行统计分析。本研究发现全部受试者、高肌酸激酶水平组、低肌酸激酶水平组在最大等速离心运动后各评估指标均显著高于比前测结果,p<0.05。全部受试者、高肌酸激酶水平组受试者在最大等速离心运动后各指标变化皆明显大于低肌酸激酶水平组受试者,p<0.05。受试者血清肌酸激酶峰值与最大等长肌力、肘关节活动角度、上臂围、肌肉感受最大变化值有相关,p<0.05。本研究认为肌肉损伤程度与肌酸激酶水平具有显著相关,尤其高血清肌酸激酶水平者肌酸激酶水平较大程度反映肌肉损伤程度趋势。本研究表明,肘关节活动角度、上臂围具有预测肌酸激酶峰值的效果。     

Baseline serum levels of cardiac biomarkers in patients with stable coronary artery disease

Stable coronary artery disease (CAD) can cause repetitive reversible myocardial ischaemia, and it seems to be possible that reversibly injured myocardium releases small amounts of soluble cytoplasmic proteins. Hence, the aim was to evaluate the effect of stable CAD on baseline serum levels of cardiac biomarkers. We studied 68 consecutive outpatients referred for gated myocardial perfusion imaging. Before a treadmill exercise test, blood samples for measurement of creatine kinase (CK), CK-myocardial band (CK-MB) mass, myoglobin, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were collected. Normal perfusion patterns were detected in 29 (43%) patients (group 1) and perfusion defects were detected in 39 (57%) patients (group 2). Baseline serum levels of biomarkers except CK were significantly higher in group 2 (p=0.001). Stable CAD increases baseline levels of CK-MB mass, myoglobin, AST and LDH in the serum and this increase is related to the extent and severity of the perfusion defect and to some extent the ejection fraction of the left ventricle.     

S-thiolation of cytoplasmic cardiac creatine kinase in heart cells treated with diamide

Two methods for quantitation of protein S-thiolation, by isoelectric focusing or by enzyme activity, were used for studying S-thiolation of cytoplasmic cardiac creatine kinase. With these methods, creatine kinase was identified as a major S-thiolated protein in both bovine and rat heart. In rat heart cell cultures, creatine kinase became 10% S-thiolated during a 10 min incubation with 0.2 mM diamide. This enzyme became S-thiolated more slowly than other heart cell proteins and it also dethiolated more slowly. Two sequential additions of diamide at a 25 min interval caused twice as much S-thiolation after the second addition as compared to the first. This increased sensitivity to the second diamide treatment may have resulted from glutathione loss during the first addition which produced a higher GSSG-to-GSH ratio after the second treatment. The GSSG-to-GSH ratio was highest prior to the maximum S-thiolation of creatine kinase, but, in general, the time course of glutathione was similar to the S-thiolation of creatine kinase. This study demonstrates that cytoplasmic creatine kinase is S-thiolated and, therefore, inhibited during a diamide-induced oxidative stress in heart cells. Implications for regulation of cardiac metabolism during oxidative stress are discussed.     

Baseline serum levels of cardiac biomarkers in patients with stable coronary artery disease

Stable coronary artery disease (CAD) can cause repetitive reversible myocardial ischaemia, and it seems to be possible that reversibly injured myocardium releases small amounts of soluble cytoplasmic proteins. Hence, the aim was to evaluate the effect of stable CAD on baseline serum levels of cardiac biomarkers. We studied 68 consecutive outpatients referred for gated myocardial perfusion imaging. Before a treadmill exercise test, blood samples for measurement of creatine kinase (CK), CK-myocardial band (CK-MB) mass, myoglobin, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were collected. Normal perfusion patterns were detected in 29 (43%) patients (group 1) and perfusion defects were detected in 39 (57%) patients (group 2). Baseline serum levels of biomarkers except CK were significantly higher in group 2 (p=0.001). Stable CAD increases baseline levels of CK-MB mass, myoglobin, AST and LDH in the serum and this increase is related to the extent and severity of the perfusion defect and to some extent the ejection fraction of the left ventricle.     

Diaphragm and cardiac mitochondrial creatine kinases are impaired in sepsis.

Previous studies indicate that ATP formation by the electron transport chain is impaired in sepsis. However, it is not known whether sepsis affects the mitochondrial ATP transport system. We hypothesized that sepsis inactivates the mitochondrial creatine kinase (MtCK)-high energy phosphate transport system. To examine this issue, we assessed the effects of endotoxin administration on mitochondrial membrane-bound creatine kinase, an important trans-mitochondrial ATP transport system. Diaphragms and hearts were isolated from control (n = 12) and endotoxin-treated (8 mg.kg(-1).day(-1); n = 13) rats after pentobarbital anesthesia. We isolated mitochondria using techniques that allow evaluation of the functional coupling of mitochondrial creatine kinase MtCK activity to oxidative phosphorylation. MtCK functional activity was established by 1) determining ATP/creatine-stimulated oxygen consumption and 2) assessing total creatine kinase activity in mitochondria using an enzyme-linked assay. We examined MtCK protein content using Western blots. Endotoxin markedly reduced diaphragm and cardiac MtCK activity, as determined both by ATP/creatine-stimulated oxygen consumption and by the enzyme-linked assay (e.g., ATP/creatine-stimulated mitochondrial respiration was 173.8 +/- 7.3, 60.5 +/- 9.3, 210.7 +/- 18.9, was 67.9 +/- 7.3 natoms O.min(-1).mg(-1) in diaphragm control, diaphragm septic, cardiac control, and cardiac septic samples, respectively; P < 0.001 for each tissue comparison). Endotoxin also reduced diaphragm and cardiac MtCK protein levels (e.g., protein levels declined by 39.5% in diaphragm mitochondria and by 44.2% in cardiac mitochondria; P < 0.001 and P = 0.009, respectively, comparing sepsis to control conditions). Our data indicate that endotoxin markedly impairs the MtCK-ATP transporter system; this phenomenon may have significant effects on diaphragm and cardiac function.     

应用高敏感性肌钙蛋白T检测冠心病的临床意义初探

目的：探讨应用高敏感性肌钙蛋白T检测冠心病的临床意义。方法：2010年8月到2013年1月选择来我院治疗的126例冠心冠心病患者者作为观察组,同期选择在我院体检的健康人126例作为对照组,对血清高敏感性肌钙蛋白T、普通肌钙蛋白T与肌酸肌酶同工酶进行检测。结果：两组在4小时与12小时的高敏感性肌钙蛋白T、普通肌钙蛋白T与肌酸肌酶同工酶检测结果组间对比有明显差异（P〈0．05）。而在2小时,只有高敏感性肌钙蛋白T在组间对比有明显差异（P〈0．05）。同时观察组高敏感性肌钙蛋白的阴性检出率明显高于普通肌钙蛋白T与肌酸肌酶同工酶（P〈0．05）。而阳性检出率也同样高于前两者,但是无统计学差异（P〉0．05）。结论：高敏感性肌钙蛋白T检测技术的出现为冠心病的检测与心肌损伤的防治带来了有效的方法,在临床检测中可以作为心肌损伤的一个重要的标志。     

Phosphorylcreatine shuttle enzymes during perinatal heart development

Mammalian heart development, from the time of weaning until adulthood, is characterized by progressive and significant enhancement in functional performance. Aerobic metabolism and contractile protein ATPase activity increase in parallel with augmented cardiac function. The present studies examined the potential contribution of phosphorylcreatine shuttle enzymes to the developmentally linked alterations in heart performance. Mitochondrial ATPase specific activity was not altered between weanling and adult heart; however, creatine kinase activity was enhanced approximately threefold. Myofibrillar ATPase activity doubled over the developmental time course, while creatine kinase activity increased to an even greater extent. Enhanced myofibrillar ATPase activity was not due to alterations in either calcium sensitivity or ATPase activity measured in purified myosin. Both the mitochondrial and myofibrillar creatine kinase enzyme activities are enhanced during normal heart growth; however, relatively greater enhancement of the myofibrillar component occurs. Thus, enzymatic reactions comprising the phosphorylcreatine shuttle system are dramatically increased during normal heart development. This mechanism deserves consideration as a potentially powerful contributor to enhanced cardiac function during the perinatal period.     

Ultrastructure studies on the binding of creatine kinase and the 165,000 molecular weight component to the M-band of muscle

Binding of creatine kinase and the 165,000 molecular weight component to the M-band of rabbit skeletal muscle and bovine cardiac muscle, following their removal by low ionic strength extraction, has been accomplished. Examination of the myofibrils in the electron microscope shows that creatine kinase and the 165,000 M_r component will bind to the M-band independently of each other. In the skeletal system, rabbit skeletal creatine kinase and the 165,000 M_r protein were used, while in the bovine cardiac case, bovine cardiac creatine kinase and rabbit skeletal 165,000 M_r protein were employed. The latter conditions gave results that suggest the presence of a bovine analogue of M_r = 165,000 in the M-band of bovine cardiac myofibrils. The fact that bovine creatine kinase will bind to the M-band firmly establishes its presence, for the first time, as an integral part of the M-band structure of mammalian cardiac muscle.     

Compartmentalized energy transfer in cardiomyocytes: use of mathematical modeling for analysis of in vivo regulation of respiration.

The mathematical model of the compartmentalized energy transfer system in cardiac myocytes presented includes mitochondrial synthesis of ATP by ATP synthase, phosphocreatine production in the coupled mitochondrial creatine kinase reaction, the myofibrillar and cytoplasmic creatine kinase reactions, ATP utilization by actomyosin ATPase during the contraction cycle, and diffusional exchange of metabolites between different compartments. The model was used to calculate the changes in metabolite profiles during the cardiac cycle, metabolite and energy fluxes in different cellular compartments at high workload (corresponding to the rate of oxygen consumption of 46 mu atoms of O.(g wet mass)-1.min-1) under varying conditions of restricted ADP diffusion across mitochondrial outer membrane and creatine kinase isoenzyme "switchoff." In the complete system, restricted diffusion of ADP across the outer mitochondrial membrane stabilizes phosphocreatine production in cardiac mitochondria and increases the role of the phosphocreatine shuttle in energy transport and respiration regulation. Selective inhibition of myoplasmic or mitochondrial creatine kinase (modeling the experiments with transgenic animals) results in "takeover" of their function by another, active creatine kinase isoenzyme. This mathematical modeling also shows that assumption of the creatine kinase equilibrium in the cell may only be a very rough approximation to the reality at increased workload. The mathematical model developed can be used as a basis for further quantitative analyses of energy fluxes in the cell and their regulation, particularly by adding modules for adenylate kinase, the glycolytic system, and other reactions of energy metabolism of the cell.     

Peroxynitrite-induced inhibition and nitration of cardiac myofibrillar creatine kinase

Although cardiac peroxynitrite formation and attendant protein nitration is an established event in both acute and chronic settings of cardiac failure, the putative intracellular targets involved remain incompletely defined. We have recently shown that the myofibrillar isoform of creatine kinase (a critical energetic controller of cardiomyocyte contractility) may be a particularly sensitive target of peroxynitrite-induced nitration and inactivation in vivo. However, the kinetic and mechanistic aspects of this interaction remain undefined. Here we tested the hypothesis that myofibrillar creatine kinase is sensitive to inhibition by peroxynitrite, and investigated the mechanistic role for tyrosine nitration in this process. Peroxynitrite potently and irreversibly inhibited myofibrillar creatine kinase capacity (Vmax), at concentrations as low as 100 nM, while substrate affinity (Km) was unaffected. Concentration-dependent nitration of myofibrillar creatine kinase was observed. The extent of nitration was linearly related to peroxynitrite concentration and highly correlated to the extent of myofibrillar creatine kinase inhibition. This inhibition was not reversible by treatment with free cysteine (250 microM), but pre-incubation with substrate (phosphocreatine and/or ATP) provided significant protection of MM-CK from both nitration and inhibition. These results suggest that myofibrillar creatine kinase is a highly sensitive target of peroxynitrite-mediated inhibition, and that nitration may mediate this inhibition.     

Identification of potential serum biomarkers for Wilms tumor after excluding confounding effects of common systemic inflammatory factors

Wilms tumor is the most common pediatric tumor of the kidney. Previous studies have identified several serum biomarkers for Wilms tumor; however, they lack sufficient specificity and may not adequately distinguish Wilms tumor from confounding conditions. To date, no specific protein biomarker has been confirmed for this pediatric tumor. To identify novel serum biomarkers for Wilms tumor, we used proteomic technologies to perform protein profiling of serum samples from pre-surgery and post-surgery patients with Wilms tumor and healthy controls. Some common systemic inflammatory factors were included to control for systemic inflammation. By comparing protein peaks among the three groups of sera, we identified two peaks (11,526 and 4,756 Da) showing significant differential expression not only between pre-surgery and control sera but also between pre-surgery and post-surgery sera. These two peaks were identified as serum amyloid A1 (SAA1) and apolipoprotein C-III (APO C-III). Western blot analysis confirmed that both proteins were expressed at higher levels in pre-surgery sera than in post-surgery and control sera. Using the method of leave-1-out for cross detection, we demonstrate that detection of these two candidate biomarkers had high sensitivity and specificity in discriminating pre-surgery sera from post-surgery and normal control sera. Taken together, these findings suggest that SAA1 and APO C-III are two potential biomarkers for Wilms tumor.     

Phosphocreatine production coupled to the glycolytic reactions in the cytosol of cardiac cells

Phosphocreatine production catalyzed by a cytosolic fraction from cardiac muscle containing all glycolytic enzymes and creatine kinase in a soluble form has been studied in the presence of creatine, adenine nucleotides and different glycolytic intermediates as substrates. Glycolytic depletion of glucose, fructose 1,6-bis(phosphate) and phosphoenolpyruvate to lactate was coupled to efficient phosphocreatine production. The molar ratio of phosphocreatine to lactate produced was close to 2.0 when fructose 1,6-bis(phosphate) was used as substrate and 1.0 with phosphoenolpyruvate. In these processes the creatine kinase reaction was not the rate-limiting step: the mass action ratio of the creatine kinase reaction was very close to its equilibrium value and the maximal rate of the forward creatine kinase reaction exceeded that of glycolytic flux by about 6-fold when fructose 1,6-bis(phosphate) was used as a substrate. Therefore, the creatine kinase raction was continuously in the state of quasiequilibrium and the efficient synthesis of phosphocreatine observed is a result of constant removal of ADP by the glycolytic system at an almost unchanged level of ATP ([ATP] ? [ADP]), this leading to a continuous shift of the creatine kinase equilibrium position.When phosphocreatine was added initially at concentrations of 5–15 mM the rate of the coupled creatine kinase and glycolytic reactions was very significantly inhibited due to a sharp decrease in the steady-state concentration of ADP. Therefore, under conditions of effective phosphocreatine production in heart mitochondria, which maintain a high phosphocreatine: creatine ratio in the myoplasm in vivo, the glycolytic flux may be suppressed due to limited availability of ADP restricted by the creatine kinase system. The possible physiological role of the control of the glycolytic flux by the creatine kinase system is discussed.     

心肌型脂肪酸结合蛋白胶体金检测试纸条的制备和初步应用

目的应用胶体金免疫层析法制备检测全血或血清样本中心肌型脂肪酸结合蛋白（H-FABP）的检测试纸条,用于急性心肌梗塞（AMI）的早期辅助诊断。方法采用柠檬酸三钠还原法制备胶体金,标记鼠抗心肌型脂肪酸结合蛋白单克隆抗体,喷于玻璃纤维膜上制成胶体金结合物垫,将另一株鼠抗心肌型脂肪酸结合蛋白单克隆抗体和抗鼠二抗分别包被检测线和质控线,组装成试纸条进行灵敏性、特异性、精密性、稳定性及临床样品检测。结果该试纸条的检测灵敏度为10ng／mL,15min内可判定结果;与肌钙蛋白I、C反应蛋白、肌酸激酶、人心肌肌红蛋白无交叉反应。检测240份临床标本,与临床诊断结果进行配对分析,阳性符合率95．83％、阴性符合率100％、总符合率97．92％。结论制备的H-FABP检测试纸条有良好的灵敏性、特异性,可用于早期AMI的辅助诊断。     

Characterization of a protein kinase from human seminal plasma.

Localization of creatine kinase in isolated cardiac cells nuclei has been studied. Carefully purified cardiac nuclei preparations contain creatine kinase electrophoretically similar to the mitochondrial creatine kinase isoenzyme. Histochemical electron microscopic investigations have shown that nuclear creatine kinase is localized inside nuclei closely to chromatin.     

On a chip

The future of clinical and POC BioMEMS is very bright. With an increasing emphasis on the personalization of medicine and the rising costs of health care, early detection and diagnostics at the POC will be even more important. Early detection implies early intervention, resulting in the saving of lives and reducing overall spending. The potential impact of these technologies on the early diagnosis and management of both communicable and noncommunicable diseases is very high. Many grand challenges applications are possible, e.g., routine tests such as complete blood cell count on a chip that an individual can perform at home; detection of cardiac markers from blood after a perceived heart attack; detection of cancer markers such as exosomes, CTCs from blood, or protein biomarkers in serum; and detection of infectious agents such as virus and bacteria for public health. These applications are expected to result in new diagnostic assays for home, doctor's office, clinical laboratories, and various POC settings.     

ATP produced by myocardial sarcolemmal-bound creatine kinase is not preferentially used by the Na+ pump

It has been proposed (Grosse et al. (1980) Biochim. Biophys. Acta 603, 142–156) that membrane-bound creatine kinase and the ATP-dependent Na⁺ pump form a functional complex in cardiac sarcolemmal vesicles. In this model ADP produced at Na⁺ pump sites would be rephosphorylated by the creatine kinase for preferential delivery back to the Na⁺ pump. We have reexamined this hypothesis and find that under some conditions active Na⁺ transport can be stimulated by ATP produced by sarcolemmal creatine kinase. However, the characteristics of this stimulation are no different than stimulation produced by an added soluble ATP-regenerating system (phosphoenolpyruvate/pyruvate kinase). Thus, we are unable to detect coupling between the Na⁺ pump and sarcolemmal creatine kinase.