BACKGROUND

Adverse outcomes after cardiopulmonary bypass (CPB) are often related to systemic inflammation triggered by complement and leukocyte activation. To determine how inhibition of the alternative complement pathway affects systemic inflammation and tissue injury, we studied a novel monoclonal antibody (Mab), anti-human factor D murine Mab 166-32, in baboons.

METHODS

Fourteen baboons (mean weight, 15 kg) underwent hypothermic CPB. The treatment group (n = 7) received a single injection of anti-factor D Mab 166-32 (5 mg/kg), and the control group (n = 7) was given saline solution. After initiation of CPB, all animals were subjected to 20 minutes of core cooling (rectal temperature, 27 degrees C), followed by 60 minutes of aortic cross-clamping, 25 minutes of rewarming, and 30 minutes of normothermic CPB. Blood samples were collected before CPB, during CPB, and 1, 2, 3, 6, and 18 hours after CPB. To measure neutrophil and monocyte activation, we performed flow cytometry for CD11b expression, ELISA for complement activation (Bb, C3a, C4d, and sC5b-9) and interleukin-6 (IL-6) production, and tissue injury studies for creatine kinase MB isoenzymes (CK-MB), creatine kinase (CK), and lactic dehydrogenase (LDH) levels.

RESULTS

Anti-factor D Mab almost completely inhibited plasma Bb, C3a, and sC5b-9 production during CPB (P < .001). CD11b expression on neutrophils (129 +/- 5% vs. 210 +/- 42%; P = .0006) and on monocytes (139 +/- 14% vs. 245 +/- 43%; P = .0002) was also lower in the treatment group during CPB. The treated animals had a significantly smaller increase in plasma IL-6 concentrations than did the control animals (71 +/- 27 pg/mL vs. 104 +/- 54 pg/mL; P = .0002). CK-MB levels were also lower in the treatment group 6 hours after the end of CPB (204 +/- 30 vs. 335 +/- 59 IU/L; P = .003) and 18 hours after the end of CPB (P < .05). Creatine kinase levels (6 and 18 hours after the end of CPB) and LDH levels (3 and 6 hours after the end of CPB) showed patterns similar to those of CK-MB (P < .05).

CONCLUSIONS

The alternative complement pathway plays a major role in systemic inflammation during CPB. Inhibition of complement activation via the alternative pathway by anti-factor D Mab 166-32 significantly reduces leukocyte activation and tissue injury in our baboon model.

Creatine kinase isoenzyme BB (CK-BB) is found in the serum of patients with various types of cancer. Using both radioimmunoassay and agarose electrophoresis, we found abnormal amounts of this isoenzyme in the serum of 15 of 17 patients with untreated prostatic carcinoma. Among patients with other types of adenocarcinomas and metastatic disease, serum CK-BB was increased in most of those who were unresponsive to therapy. In benign or malignant prostate tissue and in malignant pleural effusions, cytoplasmic CK-BB as determined by immunoperoxidase staining was found in epithelial cells. Prostatic fluid had high concentrations of CK-BB, as did malignant, but not benign, pleural fluid supernates. We conclude that glandular epithelial cells contain CK-BB, which is released in benign and malignant states and may appear in higher concentrations in the circulation in malignant states. These conclusions are consistent with predictions we have made from a model experimental system concerning characteristics of possible tumor markers. The observations indicate a role for CK-BB as a tumor marker, particularly for adenocarcinoma of the prostate.

OBJECTIVE

To examine the value of sperm creatine phosphokinase M-isoform (CK-MM) measurements toward predicting fertilizing potential of men.

METHODS

In 84 in vitro fertilization (IVF) couples without knowing the semen parameters, reproductive history or the outcome of the IVF cycles, we determined the sperm CK-MM ratios (the proportion of sperm CK-MM versus CK-MM+CK-BB). Husbands with less than 10% or greater than or equal to 10% CK-MM ratios were classified as "low likelihood for fertilization" (CKMM-Infertile, n = 22) or "high likelihood for fertilization" (CKMM-Fertile, n = 62), respectively.

RESULTS

Both the CKMM-Infertile and CKMM-Fertile groups (CK-MM ratios: 4.9% +/- 0.6% versus 31.1% +/- 1.8%) were in the normospermic range (31.5 +/- 6.9 versus 78.4 +/- 5.9 x 10(6) sperm/mL and 45.6% +/- 5.0% versus 54.0% +/- 2.0% motility). The fertilization rates (6.2 versus 4.9 oocytes inseminated) were 14.2% versus 53.4%, and 72.7% versus 25.8% of the couples failed to achieve any oocyte fertilization. All 14 pregnancies (16.7% rate) occurred in the CKMM-Fertile group. The pregnancy rate in the 62 CKMM-Fertile couples was 22.6%, and considering only the 46 CKMM-Fertile women in whom oocyte fertilization occurred, it was 30.4%. Among the 22 CKMM-Infertile men, 9 were normospermic and 9 of the 62 CKMM-Fertile men were oligospermic. Within the CKMM-Fertile group, 12 and 2 of the 14 pregnancies occurred by the 53 normospermic and 9 oligospermic men (22.6% versus 22.2% rate).

CONCLUSIONS

Sperm CK-MM ratios, a measure of normal sperm development, predict fertilizing potential independently from sperm concentrations. Sperm CK-MM ratios also detect unexplained male infertility (infertile men with normospermic semen), a diagnosis that until now could not be substantiated.

We have studied the expression of creatine kinase (CK) and the accumulation of creatine phosphate during the differentiation of human and mouse peripheral blood monocytes. Mouse monocytes cultured for 24 h do not contain detectable levels of CK and creatine phosphate. However, resident tissue macrophages and inflammatory elicited macrophages obtained from the peritoneal cavities of mice have 70 and 300 mU per mg protein of CK activity and contain 3 and 6 mol of creatine phosphate per mol of ATP, respectively. The major isozyme of CK in these cells has been identified as the brain form. These findings suggest that the differentiation of monocytes into macrophages is associated with the expression of CK and the accumulation of creatine phosphate. We have found a similar pattern in human monocytes. Human blood monocytes, maintained in culture for 24 or 48 h, do not contain detectable levels of CK or creatine phosphate. Monocyte-derived macrophages (monocytes maintained in tissue cultures for 1 to 2 wk) have up to 100 mU per mg protein of CK activity and contain 0.5 mol of creatine phosphate per mol of ATP. Human macrophages express multiple isozymes of CK including the brain (BB) and possibly the mitochondrial forms of this enzyme. Thus, the expression of CK and the accumulation of creatine phosphate in human monocytes is induced by their in vitro cultivation. The induction of CK during in vitro cultivation occurs independently of the concentration of creatine in the medium. However, the size of the creatine phosphate pool varies with respect to extracellular creatine concentration. Creatine phosphate and CK are not detectable in freshly isolated human lymphocytes, polymorphonuclear leukocytes or erythrocytes, but are found in freshly isolated human platelets.

Permanent human small cell lung cancer (SCLC) cell lines established in our laboratory were investigated for their expression of the enzymatic neuroendocrine markers L-DOPA decarboxylase (DDC), neuron-specific enolase (NSE), and creatine kinase (CK), including its BB isoenzyme (CK-BB), the classical tumor markers carcinoembryonic antigen (CEA), the alpha and beta subunits of human chorionic gonadotropin (alpha-HCG, beta-HCG), and alpha-fetoprotein (alpha-FP), and their chromosomal characteristics. DDC activities were detectable in 5/6 SCLC cell lines and absent in non-SCLC. NSE levels ranged from 160 to 1422 ng/mg soluble protein and were less than 290 ng/mg soluble protein in non-SCLC. Activities of CK and levels of CK-BB clearly distinguished SCLC from non-SCLC with CK activities greater than 1000 munits/mg soluble protein and CK-BB levels greater than 3000 ng/mg soluble protein in SCLC and less than 300 munits/mg soluble protein and less than 2000 ng/mg soluble protein in non-SCLC. CEA was detectable in 5/6 SCLC cell lines but absent in non-SCLC, and its level seemed to correlate with those of DDC, NSE, and CK. One cell line, SCLC-16H, lost some of its neuroendocrine properties and CEA after 1 year of in vitro cultivation. Generally, marker levels were low in fast growing cell lines and high in slow growing cell lines. HCG alpha and beta subunit and alpha-FP were not detectable in SCLC cell lines. All SCLC cell lines examined had near diploid DNA indices and modal chromosome numbers. Double minute chromosomes and homogeneously staining regions were found in 2/5 and 4/5 SCLC cell lines respectively. With respect to chromosomal aberrations, we found a deletion of the short arm of at least one chromosome 3 in all SCLC cell lines (5/5). These data show that SCLC expresses neuroendocrine markers and CEA; CK is the most sensitive marker, and DDC and CEA are the most specific markers for SCLC in vitro; individual marker levels correlate with each other and the in vitro malignancy of SCLC; and SCLC cell lines have relatively uniform chromosomal characteristics. Our results suggest that patients whose tumors have high levels of DDC, NSE, CK-BB, and CEA have a better prognosis than those with low marker levels. This hypothesis could be proved by comparing pairs of patients that are matched for all known prognostic parameters, in particular tumor spread, for their serum and tumor marker levels with respect to the patients' outcome and prognosis.

To change the levels of expression and isoenzyme distribution of creatine kinase (CK) in muscle, transgenic technology was used to express the B subunit of CK in mouse muscle. Normally, mammalian skeletal muscle contains the MM dimer of CK. The BB dimer and MB heterodimer of CK can be found in brain and heart, respectively. Heterologous genes consisting of skeletal and cardiac muscle-specific actin promoters fused to the genomic coding region of the B form of CK were used to create transgenic mice. Lines were established from the three highest expressing founders. Analysis of skeletal muscle extracts revealed that all three lines had an increase in total CK activity measured under maximal velocity conditions. The highest expressing line, 7001, had a CK activity 150% that of control muscle. Nuclear magnetic resonance saturation transfer was used to measure the in vivo rate of the CK reaction. In 7001 hindlimb muscles, the CK catalyzed reaction was 200% that of control muscle. The elevation in CK activity in transgenic muscle was accompanied by significant changes in the composition of the cytosolic isoenzyme ratio of CK. In control, 100% of CK was MM, whereas 7001 had 60 +/- 18% MM, 32 +/- 10% MB, and 8 +/- 2% BB. There were no changes in ATP, phosphocreatine, Pi, or creatine levels in transgenic muscle compared with control. Immunofluorescence of myofibrils isolated from control and transgenic muscle revealed specific association of CK to the M line. Small amounts of MB CK were detected on myofibrils from transgenic mice. Transgenic mice expressing the B subunit of CK in muscle represent a first step toward altering CK isoforms so as to elucidate the specific roles of these isoforms in energy metabolism.

The creatine kinase (CK) isoenzyme composition was determined in serial gastrocnemius muscle biopsies obtained from 12 male marathon runners. The mean muscle CK-MB composition significantly increased after chronic exercise (training) from 5.3% (pretraining) to 7.7% (premarathon) as well as after acute exercise (postmarathon) to 10.5% of the total CK activity (P less than 0.05). However, no significant differences in total CK activities were detected. Additionally, mitochondrial CK and CK-BB isoenzymes were present in muscle homogenates. A significant correlation was observed in the increase in mean serum total CK (3,322 U/l) and CK-MB (174 U/l) activities 24 h after the race (r = 0.98, P less than 0.05). These results show that gastrocnemius muscle adapts to long-distance training and racing with increased CK-MB activities and imply that skeletal muscle is the major source of elevated serum CK-MB activities in marathon runners.

Changes in creatine kinase (CK) activity and CK isoenzyme profiles in plasma after exercise were studied in rats in order to establish the source of the exercise-induced rise in CK activity. Male and female rats ran on a treadmill for 2 h and blood samples, taken before and after exercise, were assayed for total CK, CK isoenzymes and aminoaspartate transaminase (AST) activity. These enzymes were also assayed in homogenates of liver and several muscles. We found that the isoenzyme composition of liver, plasma and muscle did not differ between the sexes. However, the exercise-induced CK and AST responses did differ: CK and AST increased after exercise in males (101% and 15% resp.), but much less in females (47% and 1%). Although the isoenzyme profiles in rest did not differ, significant differences were observed after running: in males CK-MM increased with 678%, but females only showed a 114% increase. In contrast, CK-BB showed a small increase that was about the same for both sexes (males 41%, females 35%). We conclude that both males and females show a small and similar increase in CK-BB activity after exercise, and that a large release of CK-MM from skeletal muscle, observed only in males, accounts for sex-linked differences reported earlier.

We have studied the effects of human leukocyte interferon produced in bacteria and diterpene phorbol ester tumor promoters on differentiation of normal human myoblast cultures derived from mature skeletal muscle. Interferon (100-5,000 units/ml) induced an acceleration of myotube formation and creatine kinase (CK; EC 2.7.3.2) isoenzyme transition from CK-BB to CK-MM. Heat-inactivated or trypsin-treated interferon did not affect the differentiation process. In contrast, the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), but not its inactive structural analogues phorbol and 4 alpha-phorbol 12,13-didecanoate, caused a dose-dependent (0.01-100 ng/ml) inhibition of myotube formation and CK isoenzyme transition. Neither interferon nor TPA had a significant effect on myoblast proliferation prior to fusion, and the cloning efficiencies were similar as well. Opposing effects of interferon and TPA were also demonstrated by simultaneous application of these agents to the cultures. These studies suggest that some of the antitumor effects of interferon may relate to its capacity to modulate cellular differentiation.

The enzyme activities of creatine kinase (CK), its isoenzyme MB (CK-MB) and of lactate dehydrogenase isoenzyme 1 (LD-1) have been used for years in diagnosing patients with chest pain in order to differentiate patients with acute myocardial infarction (AMI) from non-AMI patients. These methods are easy to perform as automated analyses, but they are not specific for cardiac muscle damage. During the early 90's the situation changed. First creatine kinase MB mass (CK-MB mass) replaced the measurement of CK-MB activity. Subsequently cardiac-specific proteins troponin T (cTnT) and troponin I (cTnI) appeared on the scene, displacing LD-1 analysis. However, troponin concentrations in blood increase only from four to six hours after onset of chest pain. Therefore a rapid marker such as myoglobin, fatty acid binding protein or glycogen phosphorylase BB could be used in early diagnosis of AMI. On the other hand, CK-MB isoforms alone may also be useful in rapid diagnosis of cardiac muscle damage. Myoglobin, CK-MB mass, cTnT and cTnI are nowadays widely used in diagnosing patients with acute chest pain. Myoglobin is not cardiac-specific and therefore requires supplementation with some other analyses such as troponins to support the myoglobin value. Troponins are very highly cardiac-specific. Only the sera of some patients with severe renal failure, which requires hemodialysis, have elevated cTnT and/or cTnI without there being any evidence of cardiac damage. On the other hand, the latest studies have shown that elevated troponin levels in sera of hemodialysis patients point to an increased risk of future cardiac events in a similar manner to the elevated troponin values in sera of patients with unstable angina pectoris. In addition, the bedside tests for cTnT and cTnI alone or together with myoglobin and CK-MB mass can be used instead of quantitative analyses in the diagnosis of patients with chest pain. These rapid tests are easy to perform and they do not require expensive instrumentation. For routine clinical laboratory practice we suggest that in diagnosis of patients with chest pain, myoglobin and CK-MB mass measurements should be performed whenever they are requested (24 h/day) and cTnT or cTnI on admission to the hospital and then 4-6 and 12 hours later.

BACKGROUND

Although there is comprehensive information on traditional biomarkers of muscle and cardiac damage following exercise, less is known on the kinetics of innovative markers, including ischemia modified albumin (IMA), glycogen phosphorylase isoenzyme BB (GPBB), carbonic anhydrase III (CAIII) and heart-type fatty acid-binding protein (H-FABP) in athletes performing a sub-maximal exercise.

METHODS

A total of 10 healthy trained Caucasian males performed a 21-km run. Blood samples were collected before the run, immediately after (post), 3, 6 and 24 h thereafter. Cardiac troponin I (cTnI), myoglobin, creatine kinase isoenzyme MB (CK-MB), GPBB, CAIII and H-FABP were assayed using a new diagnostic system based on protein biochip array technology. IMA was measured by a commercial colorimetric assay on a Roche Modular system P.

RESULTS

Significant variations by one-way analysis of variance were observed for CK-MB (p=0.013), myoglobin (p<0.001), GPBB (p=0.029), H-FABP (p<0.001), CAIII (p=0.006), but not for cTnI (p=1.00) and IMA (p=0.881). In particular, values of all the biomarkers tested, but cTnI and IMA, increased significantly immediately after the run. GPBB and H-FABP values returned to baseline 6 and 3 h thereafter, those of CAIII, CK-MB and myoglobin remained significantly elevated from the pre-run value up to 24 h after the run. The major variation over pre-run values was recorded for myoglobin (nearly 4-fold increment), whereas CAIII, CK-MB, GPBB and H-FABP increased by 2.9-, 1.8-, 1.4- and 1.2-fold, respectively.

CONCLUSIONS

We conclude that a sub-maximal aerobic exercise influences the concentration of several markers of muscle damage. Except for IMA, not one of the emerging biomarkers tested can be safely used to rule out myocardial damage as well as cardiospecific troponins in patients who had undergone recent physical activity.

Creatine kinase isoenzyme activities in extracts of plasma, skeletal muscle, heart and brain tissue of domestic fowls were separated by anion exchange chromatography and tissue specific distributions of the isoenzyme designated MM-CK, BB-CKBB-CKCK) was the predominant form in plasma (99 per cent) and its activity increased in response to an episode of acute heat stress.

New clinical requirements for triaging chest pain patients challenge the abilities of the current cardiac markers. Serial measurements of myoglobin, creatine kinase (CK) isoenzyme MB (CKMB) mass, or CK isoforms in emergency rooms help to rapidly rule out acute myocardial infarction (AMI). However, within the first 3 to 4 h from chest pain onset, their sensitivities are too low to contribute significantly to AMI diagnosis during this period. CKMB and lactate dehydrogenase (LDH) isoenzyme 1 are not heart-specific, which hampers reliable diagnosis in patients with concomitant skeletal muscle damage. By contrast, the regulatory proteins troponin I and troponin T are expressed in three different isoforms: one for slow-twitch skeletal muscle fibers, one for fast-twitch skeletal muscle fibers, and one for cardiac muscle (cTnI, cTnT); cardiac-specific cTnI and cTnT assays are already available for routine use. cTnT and cTnI are the most promising markers for risk stratification in patients with unstable angina pectoris. Recent reports on increased cTnT in patients with renal failure or myopathy without evidence of myocardial injury and undetectable cTnI suggest that cTnT could be reexpressed similar to CKMB and LDH-1 in chronically damaged human skeletal muscle. Therefore, cTnI is probably the most heart-specific marker. Among the recently proposed new markers for early AMI diagnosis: glycogen phosphorylase isoenzyme BB (GPBB), fatty acid binding protein, phosphoglyceric acid mutase isoenzyme MB, enolase isoenzyme alpha beta, S100a0, and annexin V, GPBB is the most promising because it increases as early as 1 to 4 h from chest pain onset and its early release appears to be essentially dependent on ischemic myocardial injury.

Creatine kinase (CK) is responsible for the creatine/creatine phosphate level which that is known to alter in the brain of patients with schizophrenia. A comparative estimation of CK enzymatic activity and immunoreactivity of CK BB was carried out in readily soluble extracts from frontal cortex, anterior and posterior cingulate cortex, hippocampus and cerebellum from brains of individuals with schizophrenia versus normal controls. CK activity was determined using a commercial diagnostic kit. CK BB immunoreactivity was evaluated by ECL -immunoblotting using monoclonal antibody. A drastic drop of CK activity and CK BB immunoreactivity was observed in all the examined brain areas in schizophrenia patients compared to controls (p<0.01), with the maximum drop in the cerebellum. The reduction was independent of age, postmortem interval or chlorpromazine equivalent. The decreased level of CK BB in schizophrenia was confirmed by purification of CK BB from brains of patients with schizophrenia and control brains: the yield of the purified enzyme was significantly lower in schizophrenia, wherein molecular masses of CK B-subunits were equal. Possible causes and consequences of the decrease in CK BB level observed in brain of patients with schizophrenia are discussed.

Acute brain damage-cerebrovascular or cardiovascular, traumatic or infectious-released brain-type isoenzyme of creatine kinase (BB-CK) into the circulation within a few hours in 16 of 62 patients (26%). Occurrence of BB-CK was transient in the serum. BB-CK activity was found in the peripheral blood in 13 of 23 patients with diffuse brain damage compared to three of 39 patients with a local cerebrovascular accident (P less than .0005). The mortality of patients having BB-CK in their serum was 63% compared to 39% of those without BB-CK activity in their serum (P less than .05).

During a period of 2 months the activity of creatine kinase BB (CK-BB) was measured in the cerebrospinal fluid (CSF) from 93 consecutive patients admitted as emergencies to the Neurosurgical Department. Fourteen of the 15 patients with verified brain contusion showed an increased activity of CK-BB in the CSF whereas all patients with various other acute neurological disorders, such as epilepsy and acute headache, had a normal CK-BB activity. Two of 5 patients with subarachnoidal haemorrhage and 13 of 58 patients classified as concussion also showed an increased CK-BB activity. Spinal fluid pressure, number of red cells and activity of CK (total) were less useful than CK-BB in diagnosing acute brain damage. Even diagnostic ventricular puncture with a Fisher cannula, producing a tiny (diameter = 2.8 mm) brain lesion, gave rise to an increased CK-BB activity. CSF sampled repeatedly from 10 other patients with brain contusion showed CK-BB activities that suggest the optimum period for sampling to be between one and 15 hours after head injury. The results obtained suggest that CK-BB is a reliable indicator of brain damage following head injury.

New treatment approaches in the fight against SCLC are clearly on the horizon and some are already in clinical trials. With this in mind, several comments concerning future directions in staging this disease can be made: 1. Staging is important and complete staging is needed in order to continue to build meaningful information. 2. Limited/Extensive disease categories are in use and remain important; yet this system is not completely adequate. There are subsets within each group that do better: minimal disease versus bulky disease in limited stage, extraabdominal v intraabdominal in extensive disease, and single organ versus multiple organ involvement. Therefore, a new staging system is needed. The TNM system is designed primarily to define surgical resectability and will thus not adequately address the issues for SCLC unless the N and M categories are markedly enlarged. A staging symposium was recently held in Europe to begin to address potential approaches to staging and an American staging conference is planned. 3. Biomarkers: In the broad range of possible markers, most are not sufficiently sensitive or specific to supplant clinical exam and routine testing. But newer tests such as NSE, CK-BB and tumor surface antigen expression and recognition may impact on staging in the near future. 4. Finally, as the biology of SCLC is further understood, much of the derived understanding will likely change the staging and prognostic factors.

The cytosolic creatine kinases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have an important role in energy metabolism and display characteristic tissue- and stage-specific patterns of expression in mammals. To study the functional role of the distribution of the CK isoenzymes we have focussed on the modulation of expression of the genes encoding the individual B and M subunits, starting at the muscle creatine kinase (CKM) gene which is transcriptionally inactive during early embryogenesis. Using repeated rounds of gene targeting in mouse embryonic stem (ES) cells, two types of mutant cell lines were obtained. First, we generated a cell line in which insertion of a neomycin resistance (neor) gene had disrupted one of the CKM alleles. Subsequently, from this cell line, following introduction of an insertion type vector designed for replacement of the muscle specific CKM-enhancer by the constitutively acting polyoma virus enhancer PyF441, several independent doubly targeted clones were isolated which all had insertions in the previously neo-disrupted CKM allele. In some of these ES clones, the targeted enhancer replacement resulted in gene correction and functional activation of the silent CKM gene. Dimerisation between the ectopically expressed CKM subunits and CKB subunits which are normally present at high levels in ES cells, led to the formation of the BM isoform of CK in these clones.

Signal transduction pathways involved in the hypertrophic effect of neuropeptide Y (NPY) were investigated in adult cardiomyocytes. Reduction of transforming growth factor-beta activity in serum-supplemented media abolished the induction of hypertrophic responsiveness to NPY. In responsive cells, NPY (100 nM) increased protein synthesis, determined as incorporation of [14C]phenylalanine, by 35 +/- 15% (P < 0.05, n = 16 cultures). In these cells, NPY activated pertussis toxin (PTx)-sensitive G proteins and phosphatidylinositol (PI) 3-kinase. PTx and inhibition of PI 3-kinase abolished the hypertrophic effect of NPY. NPY also activated protein kinase C (PKC) and mitogen-activated protein (MAP) kinase. Inhibition of these two kinases attenuated the induction of creatine kinase (CK)-BB but not the growth response to NPY. In conclusion, NPY stimulates protein synthesis in adult cardiomyocytes via activation of PTx-sensitive G proteins and PI 3-kinase and it induces the fetal-type CK-BB via activation of PKC and MAP kinase.

Mechanism of amyloid beta-peptide (A beta) toxicity in cultured neurons involves the development of oxidative stress in the affected cells. A significant increase in protein carbonyl formation was detected in cultured hippocampal neurons soon after the addition of preaggregated A beta(1-40), indicating oxidative damage of proteins. We report that neurons, subjected to A beta(1-40), respond to A beta oxidative impact by activation of antioxidant defense mechanisms and alternative ATP-regenerating pathway. The study demonstrates an increase of Mn SOD gene expression and the restoration of Cu, Zn SOD gene expression to a normal level after temporary suppression. Partial loss of creatine kinase (CK) BB activity, which is the key enzyme for functioning of the creatine/phosphocreatine shuttle, was compensated in neurons surviving the A beta oxidative attack by increased production of the enzyme. As soon as the oxidative attack triggered by the addition of preaggregated A beta (1-40) to rat hippocampal cell cultures has been extinguished, CK BB expression and SOD isoenzyme-specific mRNA levels in surviving neurons return to normal. We propose that the maintenance of a constant level of CK function by increased CK BB production together with the induction of antioxidant enzyme gene expression in A beta-treated hippocampal neurons accounts for at least part of their adaptation to A beta toxicity.

Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.

Brain-type creatine kinase (CK) isoenzyme (CK-BB) was detected in the serum in 13 out of 26 patients with acute brain injury (50%). The peak of CK-BB activity ranged from 5 to 188 U/liter, constituting, on average, 10.5% of the total CK activity. The highest activities were seen in patients with gunshot wounds. High CK-BB activity was associated with poor prognosis, but minimal CK-BB elevations did not have prognostic significance. Heart-type creatine kinase isoenzyme (CK-MB) was detected in the serum in 17 out of 26 patients (65%). The peak activity ranged from 5 to 115 U/liter, constituting, on average, 6.6% of total CK activity. Electrocardiogram taken from 20 patients revealed transient T-wave inversions in the precordial leads in four patients; three of them also showed serum CK-MB activity. Subendocardial hemorrhage was detected at autopsy in three of the five CK-MB-positive patients, but in none of the four CK-MB-negative cases. Present findings suggest that acute brain injury may be secondarily cause myocardial damage.

OBJECTIVE

Monitoring of cardiotoxicity of conventional and high-dose chemotherapy (HD-CT) with multiple biomarkers of cardiac injury - glycogen phosphorylase BB (GPBB), heart-type fatty acid binding protein (H-FABP), cardiac troponins (cTnT, cTnI), creatine kinase MB (CK-MB mass), myoglobin.

METHODS

A total of 47 adult acute leukemia patients were studied - 24 patients treated with conventional CT containing anthracyclines (ANT) and 23 patients treated with HD-CT (myeloablative preparative regimen) followed by hematopoietic cell transplantation (HCT). Cardiac biomarkers were assessed prior to treatment (before CT/HD-CT), after first CT with ANT, after last CT with ANT in the first group, after HD-CT and after HCT in the second group. Values above the reference range were considered elevated.

RESULTS

Before CT/HD-CT, all biomarkers of cardiac injury were below the cut-offs in all patients. GPBB increased above the cut-off (7.30 microg/L) in 4 (16.7%) patients after first CT and in 5 (20.8%) patients after last CT with ANT. GPBB increased above the cut-off in 5 (21.7%) patients after HD-CT and remained elevated in 5 (21.7%) patients after HCT. CTnI became elevated (above 0.40 microg/L) in 2 (8.3%) patients after first and last CT with ANT. Both patients with cTnI positivity had elevated GPBB. Other tested biomarkers remained below the cut-offs during the study.

CONCLUSIONS

Our results suggest that GPBB could become a sensitive biomarker for detection of acute cardiotoxicity associated with conventional CT containing ANT and HD-CT followed by HCT. The predictive value for development of cardiomyopathy in the future is not known and should be evaluated during a prospective follow-up. Based on our data, a larger prospective and multicenter study would be most desirable to define the potential role of new circulating biomarkers in the assessment of cardiotoxicity in oncology.

The proportion of creatine kinase (CK; EC 2.7.3.2) isoenzyme MB activity was increased in skeletal muscle biopsies obtained from five long-distance runners, both 2 h before (mean 7.7%, SD 2.4%) and 30 min after (mean 7.2%, SD 1.2%) a marathon race, as compared with that in biopsies from five nonrunners (controls less than or equal to 1.0%). Further, mitochondrial CK and CK-BB isoenzymes were present in homogenates of the runners' skeletal muscle samples but not in those of the nonrunners. However, there were no substantial differences in the mean total CK activities per gram (wet wt.) of muscle tissue among premarathon samples, postmarathon samples, and nonrunners' samples (3148, 3365, and 3049 U/g, respectively). We conclude that the metabolically active gastrocnemius muscle of long-distance runners is qualitatively similar to the heart muscle in its CK isoenzyme composition.