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Effect of varicocelectomy on sperm creatine kinase, HspA2 chaperone protein (creatine kinase-M type), LDH, LDH-X, and lipid peroxidation product levels in infertile men with varicocele.

OBJECTIVES: To determine the total sperm creatine kinase, HspA2 chaperone protein (creatine kinase-M isoform), lactate dehydrogenase (LDH), LDH-X activities, and lipid peroxidation product (malondialdehyde [MDA]) levels in infertile men with varicocele and to examine the possible effect of varicocelectomy on these parameters. METHODS: Eighty-one men were enrolled in this study. The study population consisted of a control group (n = 25) and a varicocele group (n = 56). Of the 56 patients with varicocele, 26 underwent microsurgical subinguinal varicocelectomy at our institution between July 2002 and July 2003. The standard semen parameters (sperm concentration and motility and Kruger morphology) and total sperm creatine kinase, HspA2, LDH, and LDH-X activities, and MDA levels were assessed in the control and varicocele groups. The differences were compared between the two groups. The same parameters were repeated at 6 months postoperatively in the varicocelectomy group and the preoperative and postoperative results were compared. RESULTS: The sperm concentration was significantly lower in the varicocele group than in the control group (P = 0.01). The mean sperm HspA2 activities were significantly lower and the LDH activities and MDA levels were significantly greater in the varicocele group than in the control group (P = 0.005 and P = 0.001, P < 0.001, respectively). No statistically significant difference was found in the semen parameters in the varicocelectomy group preoperatively and postoperatively. Sperm HspA2 activities increased significantly after varicocelectomy compared with preoperatively (P < 0.001). CONCLUSIONS: Our data suggest that sperm HspA2 activities are lower and LDH activities and MDA levels are greater in infertile men with varicocele. These data also suggest that varicocelectomy increases HspA2 activities in these patients.

Adult↗

Troponin T, creatine kinase MB mass, and creatine kinase MB isoform ratio in the detection of myocardial damage during non-surgical coronary revascularization.

The presence of myocardial injury during non-surgical coronary revascularization has been evaluated by means of highly specific and sensitive biochemical markers. Troponin T, creatine kinase-MB isoenzyme mass concentration, and creatine kinase MB2/MB1 isoform ratio have been determined in 80 patients who underwent coronary revascularization with percutaneous transluminal coronary angioplasty (PTCA). Forty-five patients underwent balloon angioplasty, 15 rotational atherectomy, 10 directional atherectomy, and 10 elective coronary stenting. Serum concentration of the evaluated markers did not increase significantly after 57 uncomplicated revascularization procedures, including 15 rotablation procedures, nor after 8 PTCAs complicated by localized coronary type B and C dissections. Significant elevation of all markers above the upper limits of the reference interval (P < 0.05) was detected after occlusion of small side branches (< 0.5 mm diameter) in 5 patients. Creatine kinase MB2/MB1 isoform ratio was the earliest marker to increase. After recanalization of occluded vessels in 8/10 patients with 6-60 days old myocardial infarction only troponin T concentrations increased from a baseline of 0.28 microgram/l to a median peak of 0.80 microgram/l. This increase was statistically not significant (P = 0.12). In conclusion, myocardial damage was not detected following uncomplicated non-surgical revascularization obtained with different techniques. Markers of myocardial injury provide high sensitivity after small side branch occlusion.

Aged↗

Preincubation with creatine enhances levels of creatine phosphate and prevents anoxic damage in rat hippocampal slices.

We have investigated the relationship between energy metabolism, NMDA-receptor antagonism, and anoxic damage in vitro. Anoxic damage was assessed by measuring protein synthesis, defined as the incorporation of [14C]lysine into perchloric acid-insoluble tissue extracts. The concentrations of energy metabolites were measured by ion-exchange HPLC. Anoxia caused an inhibition of protein synthesis, a reduction in phosphocreatine and adenosine triphosphate, and extensive neuronal damage. The reduction of protein synthesis depended on the duration of anoxia and the time allowed for recovery. Preincubation with the creatine dose-dependently (0.03-3 mmol/L) increased baseline levels of phosphocreatine, reduced the anoxia-induced decline in phosphocreatine and adenosine triphosphate, prevented the impairment of protein synthesis, and reduced neuronal death. Incubation with (R,S)-3-guanidinobutyric acid, a synthetic analogue of creatine that cannot be phosphorylated, did not prevent the anoxia-induced impairment of protein synthesis and did not enhance the levels of phosphocreatine and adenosine triphosphate. Incubation with a combination of both creatine and the noncompetitive NMDA antagonist MK-801 provided complete protection. These results indicate that energy status is a major factor controlling anoxic damage in the rat hippocampal slice.

Animals↗

High content of creatine kinase in chicken retina: compartmentalized localization of creatine kinase isoenzymes in photoreceptor cells.

Two isoforms of creatine kinase (CK; ATP:creatine N-phosphotransferase, E.C. 2.7.3.2), brain type (BB-CK) and mitochondrial type (MiMi-CK), but not the muscle types (MM- or hybrid MB-CK), were identified by cellulose polyacetate electrophoresis and immunoblots in retina from adult chickens. Indirect immunofluorescence labeling of cryosections of retinas revealed high concentrations of BB-CK in both rod and cone photoreceptor cells. Most of the fluorescence staining with anti-B-CK antibodies was found within the myoid and the ellipsoid portions of inner segments and the peripheral region of the outer segments. Significant staining with anti-B-CK antibodies was also found in horizontal cells and in the optical nerve fibers, with additional stratified staining in the inner plexiform layer. MiMi-CK was solely demonstrated in the ellipsoid portion of the photoreceptor cells. The presence of high concentrations of compartmentalized CK isoenzymes within photoreceptor cells (approximately equal to 30 enzyme units/mg) as well as the relatively high concentration of total creatine in these cells (approximately equal to 10-15 mM) indicates an important physiological function for CK and phosphocreatine in the energy transduction of vision.

Animals↗

Creatine supplementation affects muscle creatine during energy restriction.

INTRODUCTION: Anaerobic performance and body protein may decrease with energy restriction practiced by some athletes for weight loss. METHODS: This investigation examined the effects of creatine (Cr) supplementation during energy restriction on muscle Cr, exercise performance (10 sprints of 6 s, with 30-s rest), nitrogen balance, and body composition in male resistance trainers. Creatine supplemented (CrS, 20 g x d(-1) of Cr) and those given a placebo (P1) consumed a formula diet of 75.3 kJ (18 kcal) x kg(-1) x d(-1) (54.7% C, 21.3% P, 24% F) for 4 d. A control group was unsupplemented and continued their normal diet. There were no changes in body composition or performance of the control group. RESULTS: CrS and P1 demonstrated similar decreases in body weight and percent body fat. The percent change in fat-free mass was more for P1 (2.4+/-0.3% reduction) than CrS (1.4+/-0.4%), but urinary nitrogen losses were similar. Significant increases in muscle total Cr and CrP of 15-16% were demonstrated by CrS over the energy restriction period, whereas P1 had no changes in muscle Cr. Total work done during the sprints expressed per body weight tended to be 3.8% higher in CrS and 0.5% less in P1 after the energy restriction (P = 0.058). CONCLUSION: It was concluded that Cr supplementation increased muscle Cr during short-term energy restriction but did not affect body fat or protein loss. The change in muscle creatine was reflected in a tendency for higher total sprint work for the Cr group.

Adipose Tissue↗

Carbohydrate ingestion augments skeletal muscle creatine accumulation during creatine supplementation in humans.

This study investigated the effect of carbohydrate (CHO) ingestion on skeletal muscle creatine (Cr) accumulation during Cr supplementation in humans. Muscle biopsy, urine, and plasma samples were obtained from 24 males before and after ingesting 5 g Cr in solution (group A) or 5 g Cr followed, 30 min later, by 93 g simple CHO in solution (group B) four times each day for 5 days. Supplementation resulted in an increase in muscle phosphocreatine (PCr), Cr, and total creatine (TCr; sum of PCr and Cr) concentration in groups A and B, but the increase in TCr in group B was 60% greater than in group A (P < 0.01). There was also a corresponding decrease in urinary Cr excretion in group B (P < 0.001). Creatine supplementation had no effect on serum insulin concentration, but Cr and CHO ingestion dramatically elevated insulin concentration (P < 0.001). These findings demonstrate that CHO ingestion substantially augments muscle Cr accumulation during Cr feeding in humans, which appears to be insulin mediated.

Adenosine Diphosphate↗

Increase of total creatine in human brain after oral supplementation of creatine-monohydrate.

The effect of oral creatine supplementation on brain metabolite concentrations was investigated in gray matter, white matter, cerebellum, and thalamus of healthy young volunteers by means of quantitative localized proton magnetic resonance spectroscopy in vivo (2.0 T, stimulated echo acquisition mode sequence; repetition time = 6,000 ms, echo time = 20 ms, middle interval = 10 ms, automated spectral evaluation). Oral consumption of 4 x 5 g creatine-monohydrate/day for 4 wk yielded a statistically significant increase (8.7% corresponding to 0.6 mM, P < 0.001) of the mean concentration of total creatine (tCr) when averaged across brain regions and subjects (n = 6). The data revealed considerable intersubject variability (3.5-13.3%), with the smallest increases observed for the two male volunteers with the largest body weights. A regional analysis resulted in significant increases of tCr in gray matter (4.7%), white matter (11.5%), and cerebellum (5.4%) and was most pronounced in thalamus (14.6% corresponding to 1.0 mM). Other findings were significant decreases of N-acetyl-containing compounds in cerebellum and thalamus as well as of choline-containing compounds in thalamus. All cerebral metabolic alterations caused by oral Cr were reversible, as evidenced by control measurements at least 3 mo after the diet. This work demonstrates that excess consumption of Cr yields regionally dependent increases of the tCr concentration in human brain over periods of several weeks.

Administration, Oral↗

Detection of cardiac-specific creatine kinase isoenzyme in sera with normal or slightly increased total creatine kinase activity.

We describe a spectrophotometric kinetic assay for detecting creatine kinase MB isoenzyme activity in the 1 to 10 U/liter range. The MB isoenzyme was isolated [Clin. Chem. 20, 36 (1974)] and assayed (Rosalki method) with an Abbott ABA-100. Good reproducibility was demonstrated for MB isoenzyme activities near 1 U/liter (CV = 2.6%). Sera with normal or slightly increased total creatine kinase activity were evaluated. Sera of 14 patients with acute myocardial infarction contained, per liter, 84 to 236 U of total creatine kinase activity and 4.6 to 28.0 U of isoenzyme MB activity; corresponding ranges for sera from healthy lab technicians and patients with noncardiac disease were 36 to 277 and 0 to 2.6 U. MB isoenzyme activity for infarction patients rose and fell sharply within three days after the infarction. Atypical time-course patterns, MB isoenzyme activity remaining abnormally great for five days, were observed in serum from patients with prolonged atrial fibrillation and congestive heart failure or cardiomyopathy; the BB isoenzyme (1 to 5 U/liter) was also detected in sera of such patients but was absent in sera from infarcation patients. Quantification of column-isolated MB by the assay described is rapid, easy, specific, and extremely sensitive for measuring MB in the 1 to 10 U/liter range.

Chromatography, DEAE-Cellulose↗

[Changes in creatine kinase activity and protein spectrum of the developing muscle culture treated with creatine phosphate].

A study was made of the influence of creatine phosphate (CP) on creatine kinase activity and the total protein status of the primary chicken myoblast culture. It has been established that the two-hour exposure of the developing culture in the presence of 2 and 4 mM CP is accompanied by the lowering of the content of water extractable protein and a considerable increase of creatine kinase activity. Meanwhile the total activity of the enzyme rises in the non-differentiated and falls in the differentiated one. Electrophoretic analysis has shown CP to produce not only quantitative but also deep qualitative changes in the myoblast protein spectrum. It is assumed that the changes revealed are nonspecific.

Animals↗

Creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) activity in serum before and after intravenous salbutamol administration of patients with bronchial asthma.

The effect of salbutamol, a selective beta 2-adrenergic agonist, on creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) activity in serum of 20 asthmatic patients was investigated. Venous blood was obtained 30 min, 2 h and 4 h after 0.5 mg salbutamol intravenous injection of 0.5 mg salbutamol. Total creatine kinase activity was assayed on a Technico RA-1000 analyzer with the IFCC recommended method. CK-MB activity was determined using a centrifugal analyzer (Cobas Fera, Roche, Switzerland). Reagent kits were provided by Boehringer Mannheim (FRG). We observed no increase of CK-activity after salbutamol, and found a statistically significant increase of CK-MB activity. Serum CK-MB activity before treatment was 13 +/- 10/IU/l after 30 minutes drug, 31.8 +/- 19 IU/l after 2 h 27.2 +/- 17.79 and after 4 h 22.6 +/- 12 IU/l. We conclude that salbutamol exerts a cardiotoxic effect.

Adult↗

Creatine kinase determination: a European evaluation of the creatine kinase determination in serum, plasma and whole blood with the Reflotron system.

We evaluated a new dry-reagent carrier system for the determination of creatine kinase (EC 2.7.3.2) activity, Reflotron CK, with special attention to analytical performance with whole blood. We found a good within series imprecision. The median coefficient of variation was 3.1% for Reflotron CK (blood, serum and plasma) and 0.9% for the automatic analysers (serum and plasma only). The between-days imprecision with Reflotron CK (median CV: less than or equal to 3%) was similar to that for the comparison method on different analysers. Fresh samples of human blood, plasma and serum were examined by Reflotron CK and by a N-acetylcysteine activated creatine kinase method in six different clinical laboratories and in the Evaluation Department of Boehringer Mannheim GmbH. The correlation between these methods was excellent (r greater than or equal to 0.99), the median systematic deviation (bias) for all samples being smaller than -5%. Haematocrits between 0.25 and 0.50, haemolysis up to 6 g/l haemoglobin, and icteric samples with bilirubin concentrations up to 0.2 g/l showed no interference. No drug in therapeutic concentration was found to affect the Reflotron CK results; ascorbic acid, calcium dobesilate and sulphamethoxazole lowered the values only when present in high concentrations. Reflotron CK may be considered as a suitable alternative for decentralized testing sites, especially in situations where creatine kinase results are needed quickly.

Autoanalysis↗

A nonlinear tissue-binding model for creatine: estimation of creatine turnover time and creatinine production rate.

Intravenous bolus injections of 14C-labeled creatine were made in rabbits. Plasma and urine concentrations were measured. Plasma specific activity and urinary cumulative radioactivity data were examined by several means including the use of NONLIN. Utilization of urine and blood data suggested that a nonlinear model was more appropriate. The selected model has two nonlinear binding parameters and an elimination rate constant. The creatine turnover time was calculated to be about 22 minutes. The model allows the predication of creatine production rate that is in general agreement with the literature values.

Animals↗

The fall in creatine levels and creatine kinase isozyme changes in the failing heart are reversible: complex post-transcriptional regulation of the components of the CK system.

Decreases in total creatine kinase (CK) activity and creatine [Cr] combine to limit the capacity of the failing heart to rapidly re-synthesize ATP (energy reserve). If the loss in energy reserve could be reversed, cardiac contractile reserve may be improved. Here we test whether these changes are reversible during recovery from heart failure. Left ventricular (LV) contractile function was measured in chronically instrumented conscious dogs with heart failure (CHF) induced by cardiac pacing for 3-4 weeks, and after recovery from heart failure (Recovery) (unpaced) for 5-6 weeks. LV contractile function and contractile reserve were depressed in CHF but returned to control in Recovery. CK capacity fell by 55% in CHF due to decreases in [Cr] (-39%) and CK activity (-25%), but was fully restored in Recovery. CK-B isozyme activity, protein (Western) and mRNA levels (real time PCR), respectively, were higher by 2-, 5.4- and 11-fold in CHF and higher by 3-, 2- and 2-fold in Recovery. CK-MM activity was decreased (-30%) in CHF but returned to normal levels during Recovery; CK-M protein was 30% lower in both CHF and Recovery even though there were no changes in mRNA levels. A similar pattern was found for mitochondrial CK (sMtCK). Deceases in CK activity and [Cr] in CHF are reversible. Decreases in CK-MM and sMtCK activities, but not the increases in CK-BB and CK-MB, also reversed. Neither the changes in protein nor mRNA levels for CK-B and CK-M correlated to their activities, suggesting that CK is under complex post-transcriptional regulation.

Animals↗

Creatine metabolism and the consequences of creatine depletion in muscle.

Currently, considerable research activities are focussing on biochemical, physiological and pathological aspects of the creatine kinase (CK)-phosphorylcreatine (PCr)-creatine (Cr) system (for reviews see [1,2]), but only little effort is directed towards a thorough investigation of Cr metabolism as a whole. However, a detailed knowledge of Cr metabolism is essential for a deeper understanding of bioenergetics in general and, for example, of the effects of muscular dystrophies, atrophies, CK deficiencies (e.g. in transgenic animals) or Cr analogues on the energy metabolism of the tissues involved. Therefore, the present article provides a short overview on the reactions and enzymes involved in Cr biosynthesis and degradation, on the organization and regulation of Cr metabolism within the body, as well as on the metabolic consequences of 3-guanidinopropionate (GPA) feeding which is known to induce a Cr deficiency in muscle. In addition, the phenotype of muscles depleted of Cr and PCr by GPA feeding is put into context with recent investigations on the muscle phenotype of 'gene knockout' mice deficient in the cytosolic muscle-type M-CK.

Animals↗

Oligosaccharide and creatine supplementation on glucose and urea nitrogen in blood and serum creatine kinase in basketball athletes.

The effects of oligosaccharide and creatine (Cr) supplementation on glucose, lactic acid and urea nitrogen levels in blood and activity of serum creatine kinase (CK) were explored. Twenty CUBA male athletes were divided into 4 groups: group A (supplementation of Cr alone), group B (supplementation of oligosaccharide), group C (supplementation of oligosaccharide and Cr) and group D (placebo control group). By using orthogonal L4 table (2(3)), the experiment was performed. There were factors including oligosaccharide (carbohydrate, CHO), Cr and their correlation. Each factor had two levels: supplementation and no-supplementation. The results showed that the supplementation of CHO or Cr alone, combined supplementation of CHO and Cr could significantly reduce the glucose, urea nitrogen levels in blood and serum CK activity after competition in the athletes. Moreover, the effects of combined supplementation of CHO and Cr were more satisfactory. It was concluded that supplementation of CHO and Cr could promote the recovery of physical performance and athletic abilities after athletics in basketball athletes.

Athletes↗

Creatine kinase kinetics, ATP turnover, and cardiac performance in hearts depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed a diet containing 1% of the creatine substrate analogue beta-guanidinopropionic acid for 6-10 weeks. 31P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 mumol/g dry wt in guanidinopropionic acid-fed animals but no change in [Pi], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine----ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 mumol/g dry wt per s in control hearts and decreased 4-fold to 2.5-2.8 mumol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2-7.8 mumol/g dry per s. These rates are 1.5-3-times greater than the rate of the phosphocreatine----ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.

Adenosine Triphosphate↗

Creatine kinase equilibration follows solution thermodynamics in skeletal muscle. 31P NMR studies using creatine analogs.

The hypothesis tested was whether creatine kinase (CK) equilibrates with its substrates and products in the cytosol as if in solution. We used the creatine analogs cyclocreatine (cCr) or beta-guanidopropionate (beta GPA) to test if mass action ratios (gamma) for CK in muscle could be predicted from combined equilibrium constants (Kcomb) measured in solutions mimicking the intracellular environment. Mice were fed cCr or beta GPA and their muscles assayed for substrates and products of the CK reaction by 31P NMR spectroscopy and high performance liquid chromatography. After three weeks of feeding, gamma was indistinguishable from Kcomb in cCr-treated muscles demonstrating both PCr/Cr and phospho-analog/analog must have equilibrated with a constant and uniform cellular ATP/ADP ratio. In beta GPA-treated muscles, gamma was smaller than Kcomb due to a higher content of muscle beta GPA. Feeding beta GPA for 9-12 weeks resulted in a closer agreement between Kcomb and gamma, suggesting ATP/ADP ratios are not uniform within the muscle perhaps due to transient metabolic stress in some cells. From this analysis it follows that calculation of free ADP from the CK equilibrium for a heterogeneous population of cells with respect to total Cr and ATP content is correct only if chemical potentials of these cells are uniform.

Adenosine Diphosphate↗

Does muscle creatine phosphokinase have access to the total pool of phosphocreatine plus creatine?

Two fundamental assumptions underlie currently accepted dogma on creatine phosphokinase (CPK) function in phosphagen-containing cells: 1) CPK always operates near equilibrium and 2) CPK has access to, and reacts with, the entire pool of phosphocreatine (PCr) and creatine (Cr). We tested the latter assumption in fish fast-twitch or white muscle (WM) by introducing [14C]Cr into the WM pool in vivo. To avoid complications arising from working with muscles formed from a mixture of fast and slow fibers, it was advantageous to work with fish WM because it is uniformly fast twitch and is anatomically separated from other fiber types. According to current theory, at steady state after [14C]Cr administration, the specific activities of PCr and Cr should be the same under essentially all conditions. In contrast, we found that, in various metabolic states between rest and recovery from exercise, the specific activity of PCr greatly exceeds that of Cr. The data imply that a significant fraction of Cr is not free to rapidly exchange with exogenously added [14C]Cr. Releasing of this unlabeled or "cold" Cr on acid extraction accounts for lowered specific activities. This unexpected and provocative result is not consistent with traditional models of phosphagen function.

Adenosine Diphosphate↗