Oxidative stress mediated the inhibition of cerebral creatine kinase activity in silver catfish fed with aflatoxin B1-contaminated diet.

Aflatoxin B1 (AFB1) is an environmental toxicant and neurotoxic compound that induces the production of free radicals, causing oxidative stress. Creatine kinase (CK) is a central controller of energy metabolism in tissues with a large and fluctuating energy demand, and it is highly susceptible to inactivation by free radicals and oxidative damage. Thus, the aim of this study was to evaluate whether a diet for freshwater silver catfish (Rhamdia quelen) containing AFB1 inhibits cerebral CK activity, as well as the involvement of the oxidative stress on this inhibition. Brain CK activity was lower on days 14 and 21 post-feeding in animals that received AFB1-contaminated diet compared to the control group (basal diet), similarly to the brain sodium-potassium pump (Na+, K+-ATPase) activity. On the other hand, lipid peroxidation and protein carbonylation levels were higher on days 14 and 21 post-feeding in animals fed with AFB1-contaminated feed compared to the control group, while the antioxidant capacity against peroxyl radicals and thiol content was lower. Based on these evidences, the data demonstrated that diet containing AFB1 severely affects CK activity, an essential enzyme that plays an important role in brain energy homeostasis. Also, the impairment of energetic homeostasis linked with the use and generation of ATP via inhibition of CK activity elicited an inhibition of enzymes ATP-dependent, such as Na+, K+-ATPase. Moreover, the inhibition of brain CK activity appears to be mediated by the oxidation of lipids, proteins, and thiol group, as well as by a reduction in the antioxidant capacity.

Creatine kinase inhibition lowers systemic arterial blood pressure in spontaneously hypertensive rats: a randomized controlled trial.

OBJECTIVE: Creatine kinase is reported to be a main predictor of blood pressure (BP) in the general population, with a strong correlation between resistance artery creatine kinase expression and clinical BP in humans. The enzyme rapidly regenerates ATP near cytoplasmic ATPases involved in pressor responses, including resistance artery contractility and renal sodium retention. Therefore, we assessed whether creatine kinase inhibition reduces BP. METHODS: We implemented the 'Animal Research: Reporting of In Vivo Experiments' guideline. In a 4-week randomized controlled trial, male 16-week-old spontaneously hypertensive rats (N = 16) were randomly assigned to the specific competitive creatine kinase inhibitor beta-guanidinopropionic acid (3%)-supplemented chow vs. standard chow. BP measured by the tail-cuff method was the main outcome. Other outcomes included vasodilation in isolated arteries and renal renin expression. RESULTS: Creatine kinase inhibition reduced BP safely and reversibly. Mean baseline BP of, respectively, 191.5 (standard error 4.3) mmHg SBP and 143.1 (4.1) mmHg DBP was reduced by, respectively, 42.7 (5.5) mmHg SBP and 35.6 (5.0) mmHg DBP (P < 0.001) compared with controls, with evidence of enhanced vasodilation and a diuretic effect. CONCLUSION: To our knowledge, this is the first report on the BP-lowering effect of creatine kinase inhibition. Our data indicate that modulation of the creatine kinase system is a potential novel treatment target for hypertension.

The Effect of Creatine Kinase Inhibition on Contractile Properties of Human Resistance Arteries.

BACKGROUND: Creatine kinase (CK) is a main predictor of blood pressure, and this is thought to largely depend on high resistance artery contractility. We previously reported an association between vascular contractility and CK in normotensive pregnancy, but pregnancy is a strong CK inducer, and data on human hypertension are lacking. Therefore, we further explored CK-dependency of vascular contractility outside the context of pregnancy in normotensive and hypertensive women. METHODS AND RESULTS: Nineteen consecutive women, mean age 42 years (SE 1.3), mean systolic/diastolic blood pressure respectively 142.6 (SE 5.9)/85.6 (3.4) mm Hg (9 hypertensive), donated an omental fat sample during abdominal surgery. We compared vasodilation after the specific CK inhibitor 2,4-dinitro-1-fluorobenzene (DNFB; 10(-6) mol/l) to sodium nitroprusside (10(-6) mol/l) in isolated resistance arteries using a wire myograph. Additionally, we assessed predictors of vasoconstrictive force. DNFB reduced vascular contractility to 24.3% (SE 4.4), P < 0.001, compared to baseline. Sodium nitroprusside reduced contractility to 89.8% (SE 2.3). Maximum contractile force correlated with DNFB effect as a measure of CK (r = 0.8), and with vessel diameter (r = 0.7). The increase in contractile force was 16.5 mN [9.1-23.9] per unit DNFB effect in univariable and 10.35 mN [2.10-18.60] in multivariable regression analysis. CONCLUSION: This study extends on our previous findings in pregnant normotensive women of CK-dependent microvascular contractility, indicating that CK contributes significantly to resistance artery contractility across human normotension and primary hypertension outside the context of pregnancy. Further studies should explore the effect of CK inhibitors on clinical blood pressure.

Effect of Cd2+ on muscle type of creatine kinase: Inhibition kinetics integrating computational simulations.

Creatine kinase (EC 2.7.3.2, CK) plays an important role in cellular energy metabolism and homeostasis by catalyzing the transfer of phosphate between ATP and creatine phosphate. We investigated the effects of Cd2+ on muscle type of creatine kinase from Pelodiscus sinensis (PSCKM). Cd2+ conspicuously inactivated the activity of PSCKM (IC50=0.062 mM) in a first-order kinetic process and exhibited non-competitive inhibition with creatine and ATP. A conformational study showed that Cd2+ induced tertiary structure changes in PSCKM with exposure of hydrophobic surfaces. The addition of osmolytes, such as glycine and proline, partially reactivated the Cd2+-mediated inactive PSCKM. Additionally, molecular dynamics and docking simulations between PSCKM and Cd2+ were conducted to show that Cd2+ blocked the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd2+-induced inactivation of PSCKM. Our study demonstrates the effect of Cd2+ on PSCKM enzymatic function and unfolding, including the protective effects of osmolytes on PSCKM inactivation. This study provides important insights into the changes in the PSCKM metabolic enzyme of ectothermic animals in response to the environment.

Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness.

The acute effect of beta-guanidinopropionic acid versus creatine or placebo in healthy men (ABC Trial): study protocol for a randomized controlled trial.

BACKGROUND: Despite adequate treatment, up to 30% of treated antihypertensive patients with primary, uncomplicated hypertension remain uncontrolled. We proposed that high intracellular activity of the ATP regenerating enzyme creatine kinase (CK) increases pressor responses and hypertension risk. In line with this, we found that plasma CK activity after rest, a surrogate measure of tissue activity, is the main predictor of blood pressure levels and failure of antihypertensive therapy in the general population. In addition, the creatine analog and competitive oral creatine kinase inhibitor beta-guanidinopropionic acid effectively and safely reduced blood pressure in the spontaneously hypertensive rat. However, to our knowledge there are no human data on the safety of oral supplementation with this substance. Therefore, we will assess the tolerability of beta-guanidinopropionic acid in men, compared to creatine and placebo. METHODS/DESIGN: This is a randomized, active and placebo controlled, triple blind, double dummy, single center clinical intervention trial in 24 healthy male volunteers, 18 to 50 years old, recruited in the Netherlands. The intervention consists of one week of daily oral administration of beta-guanidinopropionic acid 100 mg, creatine 5 gram, or placebo. The primary outcome is the tolerability of beta-guanidinopropionic acid as a descriptive measure, in an intent-to-treat analysis. Other outcomes include the placebo-adjusted differences with baseline in biochemical and hemodynamic parameters, including plasma markers of muscle tissue damage, urine sodium excretion, resting sitting systolic and diastolic brachial blood pressure, supine systolic and diastolic central blood pressure, pulse wave velocity and augmentation index, heart rate, cardiac contractility, cardiac output, and total peripheral resistance. DISCUSSION: There is an unfulfilled need for new conservative options to treat resistant hypertension. This study will provide first-in-men data on creatine kinase inhibition as a potential new class of antihypertensive drugs. TRIAL REGISTRATION: The Netherlands National Trial Register Trialregister.nl (identifier NTR 4444) , registered 9 March 2014.

Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake.

Snake envenoming is an important public health problem around the world, particularly in tropics. Beyond deaths, morbidity induced by snake venoms, such as myotoxicity, is of pivotal consequence to population. Bothrops jararacussu is the main venomous snake in southeast region of Brazil, and particularly presents strong myotoxic effect. The only available therapy, antibothropic antivenom, poorly affects venom-induced myotoxicity. The aim of this study is to assess the ability of fucosylated chondroitin sulfate (fucCS), a glycosaminoglycan with anticoagulant and antithrombotic properties, and its derivatives to inhibit toxic activities of B. jararacussu crude venom and its isolated toxins, named bothropstoxins (BthTX-I and BthTX-II). The in vitro myotoxic activities induced by crude venom, by BthTX-I alone and by toxins together were abolished by fucCS. Carboxyl reduction (fucCS-CR) kept this ability whereas defucosilation (defucCS) abrogates myoprotection. We observed the same pattern in the response of these polysaccharides in antagonizing the increase in plasma creatine kinase (CK) levels, the reduction of skeletal muscle CK content and the rise of myeloperoxidase (MPO) activity induced by crude venom and isolated toxins. FucCS inhibited edematogenic activity and partially prevented the reduction of total leukocytes in blood when pre-incubated with crude venom. Furthermore, the venom procoagulant effect was completely antagonized by increasing concentrations of fucCS, although this polyanion could stop neither the tail bleeding nor the skin hemorrhage induced by Bothrops jararaca venom. The B. jararacussu phospholipase, hyaluronidase, proteolytic and collagenase activities were inhibited in vitro. The results suggest that fucCS could be able to interact with both toxins, and it is able to inhibit BthTX-II phospholipase activity. Light microscopy of extensor digitorum longus muscle (EDL) muscle showed myoprotection by fucCS, once necrotic areas, edema and inflammatory cells were all decreased as compared to venom injection alone. Altogether, data show that fucCS was able to inhibit myotoxicity and inflammation induced by B. jararacussu venom and its phospholipase toxins, BthTX-I and BthTX-II. Thus, fucosylated chondroitin sulfate is a new polyanion with potential to be used as an adjuvant in the treatment of snakebites in the future.

The adenosine salvage pathway as an alternative to mitochondrial production of ATP in maturing mammalian oocytes.

Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.

The minor structural difference between the antioxidants quercetin and 4'O-methylquercetin has a major impact on their selective thiol toxicity.

Antioxidants act as intermediates by picking up the high unselective reactivity of radicals and transferring it to other molecules. In this process the reactivity is reduced and becomes selective. This channeling of the reactivity can cause selective toxicity. The antioxidant quercetin is known to channel the reactivity towards thiol groups. The present study compares the thiol reactivity of quercetin with that of 4'O-methylquercetin (tamarixetin) towards creatine kinase (CK), a vital protein that contains a critical thiol moiety. Our results showed that oxidized quercetin and oxidized tamarixetin both adduct CK, which then loses its enzymatic function. Ascorbate, an important representative of the antioxidant network, is able to prevent adduction to and thus the inhibition of the enzyme by tamarixetin but not by quercetin. Apparently, tamarixetin is less thiol toxic than quercetin, because--rather than adduction to CK--tamarixetin quinone prefers to pass reactivity to the antioxidant network, i.e., to ascorbate. The findings exemplify that radical scavenging flavonoids pick up the reactivity of radicals and act as a pivot in directing the way the reactivity is channeled. A mere minor structural difference of only one methyl moiety between quercetin and tamarixetin appears to have a high impact on the selective, thiol toxicity.

Huperzine A ameliorates damage induced by acute myocardial infarction in rats through antioxidant, anti-apoptotic and anti-inflammatory mechanisms.

Huperzine A (HupA), an alkaloid used in traditional Chinese medicine and isolated from Huperzia serrata, has been shown to possess diverse biological activities. The present study was undertaken to evaluate the cardioprotective potential of HupA in myocardial ischemic damage using a rat model of acute myocardial infarction. HupA significantly diminished the infarct size and inhibited the activities of myocardial enzymes, including creatine kinase (CK), the MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin T (cTnT). A significantly reduced activity of malondialdehyde (MDA) and elevated activities of superoxide dismutase (SOD), of the non-enzymatic scavenger enzyme, glutathione (GSH), as well as of glutathione peroxidase (GSH-PX) were found in the HupA-treated groups. Furthermore, decreased protein levels of caspase-3 and Bax, and increased levels of Bcl-2 were observed in the infarcted hearts of the rats treated with various concentrations of HupA. In addition, treatment with HupA markedly inhibited the expression of the nuclear factor-κB (NF-κB) subunit p65, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). These findings suggest that the cardioprotective potential of HupA is associated with its antioxidant, anti-apoptotic and anti-inflammatory properties in acute myocardial infarction in rats.

Kinetic studies on the inhibition of creatine kinase activity by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one in the cerebral cortex of rats.

Tellurium has been used as an industrial component of many alloys and in the electronic industry. Organotellurium compounds can cause poisoning which leads to neurotoxic symptoms such as significant impairment of learning, spatial memory and are potentially neurotoxic to human beings. However, the molecular mechanisms of neurotoxicity of organotellurium compounds are not well understood. Considering that creatine kinase plays a key role in energy metabolism of tissues with intermittently high and fluctuating energy requirements, such as nervous tissue, the main objective of this study was to investigate the mechanisms by which 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one inhibit creatine kinase activity, a key enzyme of energy homeostasis, in the cerebral cortex of 30-day-old Wistar rats. For the kinetic studies, the Lineweaver-Burk plot was used to characterize the mechanisms of enzyme inhibition by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one. The results suggested that this compound inhibits creatine kinase activity by two different mechanisms: competition with ADP and oxidation of critical sulfhydryl groups for the functioning of the enzyme. The potential for inhibition of creatine kinase to occur in vivo may contribute to the neurotoxicity observed by this organochaocogen.

Chemical composition of three polysaccharides from Gynostemma pentaphyllum and their antioxidant activity in skeletal muscle of exercised mice.

PURPOSE: To investigate the effects of polysaccharide from Gynostemma pentaphyllum on antioxidant activity in skeletal muscle of mice exercised to exhaustion. METHODS: Three polysaccharide fractions were obtained from G. pentaphyllum polysaccharide (GPP) and termed GPP1-a, GPP2-b, and GPP3-a. Gas chromatography (GC) and infrared spectrum of the polysaccharides were determined. The fractions were orally administrated to mice once daily for 1 wk. The exercise time to exhaustion was assessed using a forced swim test of mice after a week. The glucose, creatine phosphokinase, and lactic dehydrogenase in serum; the activity of superoxide dismutase and glutathione peroxidase; and the levels of malondialdehyde (MDA) and glycogen in muscle were determined. RESULTS: The results of GC demonstrated that GPP1-a, GPP2-b, and GPP3-a were composed of different monosaccharides with distinct molar ratios. Infrared spectrum showed that the main typicals of GPP1-a and GPP2-b were ß-configuration and the main typical of GPP3-a was α-configuration. Among the 3 fractions of GPP, GPP1-a administration significantly prolonged exercise time to exhaustion of mice, increased glycogen level and some of antioxidant enzyme activities, and decreased MDA level in muscle. CONCLUSIONS: The mechanism by which GPP1-a prolonged exercise time to exhaustion in mice may be associated with scavenging reactive oxygen species excessively produced and further increasing glycogen levels in skeletal muscle.

Anti-proliferative effects of a novel isoflavone derivative in medullary thyroid carcinoma: an in vitro study.

Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line, might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor (ER) α mRNA expression in TT cells was more abundant than ERß, with a ratio of 48:1. Estradiol-17ß (E2) increased DNA synthesis in a dose dependent manner. [(3)H]-thymidine incorporation was also stimulated by the ERß agonist DPN and the ERα agonist PPT. cD-tBoc inhibited TT cell growth as assessed by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase specific activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise inhibited. The inhibitory effect of cD-tBoc on [(3)H]-thymidine incorporation could be blocked by the ERß antagonist PTHPP but not by the ERα antagonist MPP, suggesting that the antiproliferative effect of cD-tBoc on these cells is mediated through ERß. Furthermore, cD-tBoc potently increased apoptosis and cell necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell line TT suggest and that this property may be utilized to design effective anti-neoplastic agents.

Inhibition of vascular peroxidase alleviates cardiac dysfunction and apoptosis induced by ischemia-reperfusion.

Myeloperoxidase (MPO) is involved in myocardial ischemia-reperfusion (IR) injury and vascular peroxidase (VPO) is a newly identified isoform of MPO. This study was conducted to explore whether VPO is involved in IR-induced cardiac dysfunction and apoptosis. In a rat Langendorff model of myocardial IR, the cardiac function parameters (left ventricular pressure and the maximum derivatives of left ventricular pressure and coronary flow), creatine kinase (CK) activity, apoptosis, VPO1 activity were measured. In a cell (rat-heart-derived H9c2 cells) model of hypoxia-reoxygenation (HR), apoptosis, VPO activity, and VPO1 mRNA expression were examined. In isolated heart, IR caused a marked decrease in cardiac function and a significant increase in apoptosis, CK, and VPO activity. These effects were attenuated by pharmacologic inhibition of VPO. In vitro, pharmacologic inhibition of VPO activity or silencing of VPO1 expression significantly suppressed HR-induced cellular apoptosis. Our results suggest that increased VPO activity contributes to IR-induced cardiac dysfunction and inhibition of VPO activity may have the potential clinical value in protecting the myocardium against IR injury.

Creatine and creatine analogues in hypertension and cardiovascular disease.

BACKGROUND: The creatine kinase system, the central regulatory system of cellular energy metabolism, provides ATP in situ at ATP-ases involved in ion transport and muscle contraction. Furthermore, the enzyme system provides relative protection from tissue ischaemia and acidosis. The system could therefore be a target for pharmacologic intervention. OBJECTIVES: To systematically evaluate evidence regarding the effectiveness of interventions directly targeting the creatine kinase system as compared to placebo control in adult patients with essential hypertension or cardiovascular disease. SEARCH METHODS: Electronic databases searched: Medline (1950 - Feb 2011), Embase (up to Feb 2011), the Cochrane Controlled Trials Register (issue 3, Aug 2009), Latin-American/Caribbean databank Lilacs; references from textbooks and reviews; contact with experts and pharmaceutical companies; and searching the Internet. There was no language restriction. SELECTION CRITERIA: Randomized controlled trials comparing creatine, creatine phosphate, or cyclocreatine (any route, dose or duration of treatment) with placebo; in adult patients with essential hypertension, heart failure, or myocardial infarction. We did not include papers on the short-term use of creatine during cardiac surgery. DATA COLLECTION AND ANALYSIS: The outcomes assessed were death, total myocardial infarction (fatal or non-fatal), hospitalizations for congestive heart failure, change in ejection fraction, and changes in diastolic and systolic blood pressure in mm Hg or as percent change. MAIN RESULTS: Full reports or abstracts from 1164 papers were reviewed, yielding 11 trials considering treatment with creatine or creatine analogues in 1474 patients with heart failure, ischemic heart disease or myocardial infarction. No trial in patients with hypertension was identified. Eleven trials (1474 patients, 35 years or older) comparing add-on therapy of the creatine-based drug on standard treatment to placebo control in patients with heart failure (6 trials in 1226 / 1474 patients ), or acute myocardial infarction (4 trials in 220 / 1474 patients) or 1 in ischemic heart disease (28 / 1474 patients) were identified. The drugs used were either creatine, creatine phosphate (orally, intravenously, or intramuscular) or phosphocreatinine. In the trials considering heart failure all three different compounds were studied; creatine orally (Gordon 1995, Kuethe 2006), creatine phosphate via intravenous infusion (Ferraro 1996, Grazioli 1992), and phosphocreatinine orally (Carmenini 1994, Maggi 1990). In contrast, the acute myocardial infarction trials studied intravenous creatine phosphate only. In the ischemic heart disease trial (Pedone 1984) creatine phosphate was given twice daily through an intramuscular injection to outpatients and through an intravenous infusion to inpatients. The duration of the study intervention was shorter for the acute patients, from a two hour intravenous infusion of creatine phosphate in acute myocardial infarction (Ruda 1988, Samarenko 1987), to six months in patients with heart failure on oral phosphocreatinine therapy (Carmenini 1994). In the acute myocardial infarction patients the follow-up period varied from the acute treatment period (Ruda 1988) to 28 days after start of the symptoms (Samarenko 1987) or end of the hospitalization period (Zochowski 1994). In the other trials there was no follow-up after discontinuation of treatment, except for Gordon 1995 which followed the patients until four days after stopping the intervention.Only two out of four trials in patients with acute myocardial infarction reported mortality outcomes, with no significant effect of creatine or creatine analogues (RR 0.73, CI: 0.22 - 2.45). In addition, there was no significance on the progression of myocardial infarction or improvement on ejection fraction. The main effect of the interventions seems to be on improvement of dysrhythmia. AUTHORS' CONCLUSIONS: This review found inconclusive evidence to decide on the use of creatine analogues in clinical practice. In particular, it is not clear whether there is an effect on mortality, progression of myocardial infarction and ejection fraction, while there is some evidence that dysrhythmia and dyspnoea might improve. However, it is not clear which analogue, dose, route of administration, and duration of therapy is most effective. Moreover, given the small sample size of the discussed trials and the heterogeneity of the population included in these reports, larger clinical studies are needed to confirm these observations.

In vitro effect of lead, silver, tin, mercury, indium and bismuth on human sperm creatine kinase activity: a presumable mechanism for men infertility.

BACKGROUND: The aim of the present study was to investigate the in vitro effects of mercury (Hg+2), lead (Pb+2), silver (Ag+2), tin (Sn+2), bismuth (Bi+3) and indium (In+3) ions on sperm creatine kinase. METHODS: creatine kinase was isolated from human sperm homogenates after chromatography on a DEAE cellulose column. RESULTS: At 60 mug ml-1 metal concentration, 70% of the creatine kinase activity was inhibited by Hg+2, while at the same concentration, Pb+2, Ag+2, Sn+2, Bi+3 and In+3 caused 68%, 66.5%, 65.7%, 64.7% and 62.7% inhibition, respectively. All six metal ions displayed a competitive type of inhibition mechanism for the isolated creatine kinase as analyzed by Lineweaver-Burk plot. Ki values of Hg+2, Pb+2, Ag+2, Sn+2, Bi+3 and In+3 were calculated and 8.34 mM, 5 mM, 4.54 mM, 3.45 mM, 3.12 mM and 2.63 mM values were obtained, respectively. CONCLUSION: All the studied metal ions, at levels of 60 mug ml-1, may reduce normal sperm metabolism by inhibition of sperm creatine kinase, which probably is an important cause of infertility in men. However, further investigations, as in vitro and in vivo, are needed to elucidate the exact mechanism of heavy metals on male reproductive functioning at the molecular level.

ATP production rate via creatine kinase or ATP synthase in vivo: a novel superfast magnetization saturation transfer method.

RATIONALE: ³¹P magnetization saturation transfer (MST) experiment is the most widely used method to study ATP metabolism kinetics. However, its lengthy data acquisition time greatly limits the wide biomedical applications in vivo, especially for studies requiring high spatial and temporal resolutions. OBJECTIVE: We aimed to develop a novel superfast MST method that can accurately quantify ATP production rate constants (k(f)) through creatine kinase (CK) or ATP synthase (ATPase) with 2 spectra. METHODS AND RESULTS: The T1(nom) (T1 nominal) method uses a correction factor to compensate the partially relaxed MST experiments, thus allowing measurement of enzyme kinetics with an arbitrary repetition time and flip angle, which consequently reduces the data acquisition time of a transmurally differentiated CK k(f) measurement by 91% as compared with the conventional method with spatial localization. The novel T1(nom) method is validated theoretically with numeric simulation, and further verified with in vivo swine hearts, as well as CK and ATPase activities in rat brain at 9.4 Tesla. Importantly, the in vivo data from swine hearts demonstrate, for the first time, that within an observation window of 30 minutes, the inhibition of CK activity by iodoacetamide does not limit left ventricular chamber contractile function. CONCLUSIONS: A novel MST method for superfast examination of enzyme kinetics in vivo has been developed and verified theoretically and experimentally. In the in vivo normal heart, redundant multiple supporting systems of myocardial ATP production, transportation, and utilization exist, such that inhibition of one mechanism does not impair the normal left ventricular contractile performance.

Metabolism of motile zebrafish sperm.

As metabolism of motile fish sperm is not well understood, the current study examined the metabolism of saline-activated zebrafish (Danio rerio) sperm. Activation of sperm with inhibitors of oxidative phosphorylation (potassium cyanide, 2,4 dinitrophenol or carbonyl cyanide 3-cholorophenylhydrazone) negatively impacted sperm motility by 60-90 s postactivation. Incubation of quiescent sperm with 2,4 dinitrophenol prior to activation resulted in a 67% decrease in the percent motile sperm assessed 15s postactivation. Thus, production of ATP in quiescent sperm is important for motility upon activation and nascent ATP production via oxidative phosphorylation by motile sperm appears important at 60-90 s postactivation. Exposure of sperm to iodoacetamide, an inhibitor of creatine kinase, at activation was without effect. However, incubation of quiescent sperm with iodoacetamide prior to activation resulted in a 77% reduction in percent motile sperm and decreased velocity and wobble at 15s postactivation. These results suggest that creatine kinase and phosphocreatine shuttle are physiologically important at, or shortly after the initiation of motility. Finally, sperm were exposed to lactate, pyruvate, or acetate as well as to several monosaccharides upon activation. The results provided no evidence supporting any metabolic role of exogenous organics (potentially from the female via ovarian fluid) in sperm once motility has begun.

Carbamylated erythropoietin does not alleviate signs of dystrophy in mdx mice.

Erythropoietin promotes myoblast proliferation and inhibits fibrosis and thus it could impede the pathogenesis of muscle degenerative diseases. However, its stimulation of erythropoiesis limits its use as a therapeutic agent. An erythropoietin analog, carbamylated erythropoietin (C-EPO), retains these protective actions, yet it does not interact with the erythropoietin receptor. To determine whether treatment with C-EPO alleviates the signs of muscular dystrophy in an animal model of Duchenne muscular dystrophy, we treated mdx mice with intraperitoneal injections of 50 µg/kg and 100 µg/kg C-EPO for 4 and 12 weeks, and we monitored weight, serum creatine kinase levels, and changes in muscle histology. Moderate histological improvement was observed at 4 weeks, which did not translate into a significantly decreased level of serum creatine kinase. At the doses tested, C-EPO is not an effective therapeutic for the treatment of a mouse model of Duchenne muscular dystrophy.

Effects of creatine and ß-guanidinopropionic acid and alterations in creatine transporter and creatine kinases expression in acute seizure and chronic epilepsy models.

BACKGROUND: In order to confirm the roles of creatine (Cr) in epilepsy, we investigated the anti-convulsive effects of Cr, creatine transporter (CRT) and creatine kinases (CKs) against chemical-induced acute seizure activity and chronic epileptic seizure activity. RESULTS: Two hr after pilocarpine (PILO)-seizure induction, ubiquitous mitochondrial CK (uMtCK) immunoreactivity was unaltered as compared to control level. However, brain-type cytoplasm CK (BCK) immunoreactivity was decreased to 70% of control level. CRT immunoreactivity was decreased to 60% of control level. Following Cr or Tat-CK treatment, uMtCK or CRT immunoreactivity was unaffected, while BCK immunoreactivity in Cr treated group was increased to 3.6-fold of control levels. ß-Guanidinopropionic acid (GPA, a competitive CRT inhibitor) reduced BCK and CRT expression. In addition, Cr and tat-BCK treatment delayed the beginning of seizure activity after PILO injection. However, GPA treatment induced spontaneous seizure activity without PILO treatment. In chronic epilepsy rats, both uMtCK and CRT immunoreactivities were reduced in the hippocampus. In contrast, BCK immunoreactivity was similar to that observed in control animals. Cr-, GPA and tat-BCK treatment could not change EEG. CONCLUSION: Cr/CK circuit may play an important role in sustaining or exacerbating acute seizure activity, but not chronic epileptic discharge.