Total Adenine Nucleotide Content Research Articles

Red Blood Cell Adenylate Energetics Is Related to Endothelial and Microvascular Function in Long COVID.

Adenine nucleotides play a critical role in maintaining essential functions of red blood cells (RBCs), including energy metabolism, redox status, shape fluctuations and RBC-dependent endothelial and microvascular functions. Recently, it has been shown that infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) might lead to morphological and metabolic alterations in erythrocytes in both mild and severe cases of coronavirus disease (COVID-19). However, little is known about the effects of COVID-19 on the nucleotide energetics of RBCs nor about the potential contribution of nucleotide metabolism to the long COVID syndrome. This study aimed to analyze the levels of adenine nucleotides in RBCs isolated from patients 12 weeks after mild SARS-CoV-2 infection who suffered from long COVID symptoms and to relate them with the endothelial and microvascular function parameters as well as the rate of peripheral tissue oxygen supply. Although the absolute quantities of adenine nucleotides in RBCs were rather slightly changed in long COVID individuals, many parameters related to the endothelial and microcirculatory function showed significant correlations with RBC adenosine triphosphate (ATP) and total adenine nucleotide (TAN) concentration. A particularly strong relationship was observed between ATP in RBCs and the serum ratio of arginine to asymmetric dimethylarginine-an indicator of endothelial function. Consistently, a positive correlation was also observed between the ATP/ADP ratio and diminished reactive hyperemic response in long COVID patients, assessed by the flow-mediated skin fluorescence (FMSF) technique, which reflected decreased vascular nitric oxide bioavailability. In addition, we have shown that patients after COVID-19 have significantly impaired ischemic response parameters (IR max and IR index), examined by FMSF, which revealed diminished residual bioavailability of oxygen in epidermal keratinocytes after brachial artery occlusion. These ischemic response parameters revealed a strong positive correlation with the RBC ATP/ADP ratio, confirming a key role of RBC bioenergetics in peripheral tissue oxygen supply. Taken together, the outcomes of this study indicate that dysregulation of metabolic processes in erythrocytes with the co-occurring endothelial and microvascular dysfunction is associated with diminished intracellular oxygen delivery, which may partly explain long COVID-specific symptoms such as physical impairment and fatigue.

Sequential Changes of Total Adenine Nucleotide and Adenylic-Acid Energy Charge in Muscles of Rats after Death.

Objective To determine the purine adenylate [adenosine triphosphate （ATP）, adenosine diphosphate （ADP）, adenosine monophosphate （AMP）] content in the muscles of both hind limbs of rats at different postmortem interval （PMI）, calculate the changes in the total adenine nucleotide （TAN） content and the adenylic-acid energy charge （AEC）, and explore their relationship with PMI. Methods Healthy rats were sacrificed by cervical dislocation and kept at 20 ℃. The muscles of their hind limbs were extracted at 0, 24, 48, 72, 96, 120, 144, and 168 h after death. Reversed-phase high performance liquid chromatography was used to determine the content of purine adenylates, the TAN and AEC of the muscles of the both hind limbs were calculated, and the related regression equations of their relationship with PMI were established. Results Within 168 h of death of rats, the trend of ATP change was different from ADP, and the content of AMP continuously increased. The TAN value gradually increased with the extension of PMI, and the AEC showed a downward trend within 168 h after death. Among them, the patterns of AEC changes with PMI were obvious, the correlation coefficient was high （ R2=0.903）, and the curve fitting relationship was good; the fitting relationship between ATP, ADP, AMP, TAN and PMI was poor （ R2=0.198-0.754）. Conclusion The postmortem change patterns of AEC provide new research ideas for PMI estimation in the forensic field.

Increased AMP deaminase activity decreases ATP content and slows protein degradation in cultured skeletal muscle

BackgroundProtein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. MethodsTo determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. ResultsOverexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. ConclusionsAltogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.

Synergistic effects of polydatin and vitamin C in inhibiting cardiotoxicity induced by doxorubicin in rats.

The purpose of this study was to assess the synergistic effect of polydatin and vitamin C on attenuating cardiotoxicity induced by doxorubicin (DOX) in rats. Polydatin could significantly increase the activity of superoxide dismutase (SOD) and the heart rate, attenuate myocardial pathological damage, decrease malondialdehyde (MDA) content, slightly increase arterial pressure and glutathione peroxidase (GSH-Px) activity, reduce intervals of QRS, QT, and ST, and lower free fatty acid (FFA) content. The combination of polydatin and vitamin C could significantly increase arterial pressure and heart rate, decrease QRS interval and slightly reduce ST and QT intervals, significantly attenuate myocardial pathological damage, increase the activities of GSH-Px,T-SOD, Na+ K+ -ATPase, and Ca2+ Mg2+ -ATPase, elevate phosphocreatine (PCr) and adenosine triphosphate (ATP) contents, slightly increase adenosine diphosphate (ADP) and total adenine nucleotide (TAN) contents and PCr/ATP, and significantly decrease the contents of MDA and FFA, when compared with those in the DOX group. Meanwhile, the improvement effects on FFA content, the activities of ATPase and SOD, and contents of ATP and TAN in combination group were more obvious than those in polydatin group, and the improvement effects on arterial pressure, heart rate, interval of QRS, GSH-Px activity, and MDA, ADP, and PCr contents in combination group were slightly obvious when compared with those in polydatin group. In addition, the mRNA expression levels of AMPK-α2 and PPAR-α were slightly improved in combination group. The results illustrate that the combination of polydatin and vitamin C has the ability to enhance the myocardial protective effects by its antioxidative effect and improve energy metabolism.

Dietary Supplementation with α-Ketoglutarate Activates mTOR Signaling and Enhances Energy Status in Skeletal Muscle of Lipopolysaccharide-Challenged Piglets

Background: Skeletal muscle undergoes rapid loss in response to inflammation. α-Ketoglutarate (AKG) has been reported to enhance muscle growth in piglets, but the underlying mechanisms are largely unknown.Objectives: This study tested the hypothesis that dietary AKG supplementation activates mechanistic target of rapamycin (mTOR) signaling and improves skeletal muscle energy metabolism in lipopolysaccharide (LPS)-challenged piglets.Methods: Forty-eight male piglets (Duroc × Landrace × Yorkshire) were weaned at 21 d of age to a corn- and soybean meal–based diet. After a 3-d period of adaptation, piglets with a mean weight of 7.21 kg were randomly assigned to control, LPS (intraperitoneal administration of 80 μg LPS/kg body weight on days 10, 12, 14, and 16), or LPS plus 1% dietary AKG (LPS+AKG) groups. On day 16, blood samples were collected from 8 piglets/group 3 h after LPS administration. On day 17, piglets were killed to obtain gastrocnemius muscle from 8 piglets/group for biochemical analysis.Results: Compared with the control group, LPS administration increased (P < 0.05) plasma concentrations of globulin (by 14%) and tumor necrosis factor α (by 59%) and the intramuscular ratio of AMP to ATP (by 93%) and abundance of phosphorylated acetyl–coenzyme A carboxylase (ACC) β protein (by 64%). Compared with the control group, LPS administration reduced (P < 0.05) weight gain (by 15%); plasma concentrations of glutamine (by 20%), glucose (by 23%), insulin, insulin-like growth factor I, and epidermal growth factor; intramuscular concentrations of glutamine (by 27%), ATP (by 12%), ADP (by 22%), and total adenine nucleotides; and intramuscular ratios of phosphorylated mTOR to total mTOR (by 38%) and of phosphorylated 70-kDa ribosomal protein S6 kinase (p70S6K) to total p70S6K (by 39%). These adverse effects of LPS were ameliorated (P < 0.05) by AKG supplementation.Conclusions: Dietary AKG supplementation activated mTOR signaling, inhibited ACC-β, and improved energy status in skeletal muscle of LPS-challenged piglets. These results provide a biochemical basis for the use of AKG to enhance piglet growth under inflammatory or practical postweaning conditions.

Energy response of swimming crab Portunus trituberculatus to thermal variation: Implication for crab transport method

Temperature variation and air exposure are two predominant abiotic stressors during the transport and storage of Portunus trituberculatus . To understand the effects of these factors on P. trituberculatus , we examined the hemolymph (hemocyanin, glucose and lactate level) and muscle indicators (glycogen content, metabolic enzymes activity and energy status) through laboratory experiments. Energy status was evaluated by measuring changes in total adenine nucleotide (TAN) concentration, ATP, ADP, AMP, ADP/ATP, AMPATP, adenylate energy charge value (AEC) and the apparent equilibrium constant for adenylate kinase ( K ′ ADEN ). It was found that thermal variation significantly influenced ( P < 0.05) the energy metabolism of crabs by increasing lactate level, ADP and AMP content, cytochrome c oxidase (CCO) and lactate dehydrogenase (LDH) activity and the ratio of ADP/ATP and AMP/ATP and by decreasing glucose and ATP content, AEC and K ′ ADEN value at high temperature. At lower temperature, all parameters except the hemocyanin and lactate level were similar between immersed and aerial exposed crabs ( P > 0.05), indicating that the maintenance of energy homeostasis was independent of environment media. However, at higher temperature, air exposure resulted in more perturbation of energy metabolism in the crabs compared to immersion, as a greater decrease in hemocyanin, ATP, AEC and K ′ ADEN level ( P < 0.05), and a greater increase in glucose, ADP, AMP, ADP/ATP and AMP/ATP level ( P < 0.05) were observed in aerial exposed crabs. In summary, high temperature especially during air exposure could lead to a severe stress in P. trituberculatus , and the perturbation of energy status as a result of mismatch between energy demand and supply is a possible inducer of crab death. • Energy produce capability is reduced in P. trituberculatus subject to thermal variation or aerial exposure. • Aerial exposure leads to more energy demand and consequently a more severe disturbance of energy status. • The transport of P. trituberculatus without water seems to be a better alternative at lower temperature.

Dietary supplementation of aspartate enhances intestinal integrity and energy status in weanling piglets after lipopolysaccharide challenge

The intestine has a high requirement for ATP to support its integrity, function and health, and thus, energy deficits in the intestinal mucosa may play a critical role in intestinal injury. Aspartate (Asp) is one of the major sources of ATP in mammalian enterocytes via mitochondrial oxidation. We hypothesized that dietary supplementation of Asp could attenuate lipopolysaccharide (LPS)-induced intestinal damage via modulation of intestinal energy status. Twenty-four weanling piglets were allotted to one of four treatments: (1) nonchallenged control, (2) LPS-challenged control, (3) LPS+0.5% Asp treatment, and (4) LPS+1.0% Asp treatment. On day 19, pigs were injected with saline or LPS. At 24 h postinjection, pigs were killed and intestinal samples were obtained. Asp attenuated LPS-induced intestinal damage indicated by greater villus height and villus height/crypt depth ratio as well as higher RNA/DNA and protein/DNA ratios. Asp improved intestinal function indicated by increased intestinal mucosal disaccharidase activities. Asp also improved intestinal energy status indicated by increased ATP, ADP and total adenine nucleotide contents, adenylate energy charge and decreased AMP/ATP ratio. In addition, Asp increased the activities of tricarboxylic acid cycle key enzymes including citrate synthase, isocitrate dehydrogenase and alpha-oxoglutarate dehydrogenase complex. Moreover, Asp down-regulated mRNA expression of intestinal AMP-activated protein kinase α1 (AMPKα1), AMPKα2, silent information regulator 1 (SIRT1) and peroxisome proliferator–activated receptor gamma coactivator-1α (PGC1α) and decreased intestinal AMPKα phosphorylation. These results indicate that Asp may alleviate LPS-induced intestinal damage and improve intestinal energy status.

The parameters of energetic status of the wild Atlantic salmon (Salmo salar L.) spermatozoa

The energetic status of fresh sperm of wild Atlantic salmon (Salmo salar L.) was presented in selected males (n=10) with motility rate ≥80%. Purine and pyridine nucleotides: adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, inosine monophosphate, nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; nucleosides: adenosine, guanosine and inosine; bases: hypoxanthine, xanthine and uric acid were determined using the high-performance liquid chromatography method. The average value of adenylate energy charge in the group was 0.77 ± 0.10, and the mean total adenine nucleotide content (TAN) was 65.1 ± 18.3 pmol × 10−6 spermatozoa. The mean concentrations of ATP, ADP and AMP were 43.5, 11.8 and 9.9 pmol × 10−6 spermatozoa respectively. The concentrations of the other energetic nucleotides studied were lower. In all males studied (n=23) with a motility rate from 0% to 95%, no statistically significant correlation between the per cent of motile sperm and ATP concentration was found (Rs=0.35), whereas the correlation between the per cent of motile sperm and ADP was statistically significant (Rs=0.60). A negative correlation was found between hypoxanthine and the per cent of motile spermatozoa (Rs=−0.44). Our results suggest that hypoxanthine is the final product of salmon spermatozoa of adenine nucleotide catabolism.

Ca2+‐CaM activation of AMP deaminase contributes to adenine nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes

Ca2+-calmodulin (Ca2+-CaM) activates erythrocyte adenosine monophosphate deaminase (AMPD) in conditions of disturbed calcium homeostasis, prompting us to investigate adenine nucleotide metabolic dysregulation in sickle cell disease (SCD). However, higher ATP concentrations in reticulocytes, compared to erythrocytes, confound a comparative evaluation of SCD and normal RBCs. Therefore, a combination of centrifugation and antiCD71-labelled magnetic bead selection was used to prepare reticulocyte-poor fractions (reticulocytes <4% of total RBCs) of SCD RBCs. ATP and total adenine nucleotide concentrations were 12% lower in sickle erythrocytes compared to normal erythrocytes and inosine monophosphate (IMP) concentrations were threefold elevated (all P < 0.05). Furthermore, preincubation with a diffusible CaM antagonist slowed IMP accumulation in sickle erythrocytes during an experimental period of energy imbalance, thus showing that Ca2+-CaM activates AMPD in SCD. Finally, adenine treatment (100 micromol/l) of ex vivo SCD RBCs significantly expanded ATP levels (16% higher) and reduced phosphatidylserine (PS)-exposure, specifically those cells with the highest levels of PS externalization (46% fewer events) (both P-values <0.05 compared to untreated samples). We conclude that Ca2+-CaM activation of AMPD contributes to increased turnover of the adenine nucleotide pool in sickle erythrocytes and that this metabolic dysregulation promotes PS exposure that may contribute to the pathogenesis of SCD.

AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside

AICA riboside (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in P(i) (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both alpha1 and alpha2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and P(i). ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and P(i). We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular P(i) depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution.

Effect of trimetazidine on the nucleotide profile in rat kidney with ischemia–reperfusion injury

Ischemia–reperfusion injury is often responsible for delayed graft function after transplantation. Trimetazidine (TMZ) is an antioxidant agent used to protect grafts from ischemia–reperfusion injury. The aim of the study was to examine the effect of TMZ on nucleotide profile in rat kidney with ischemia–reperfusion injury. The study was carried out on Wistar rats divided into two groups: animals treated with TMZ and control group receiving placebo. TMZ 10 mg/kg/day was administrated for 30 days. Concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (Ado), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), guanosine (Guo), inosine monophosphate (IMP), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), uric acid (UA), uridine (Urd), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) were determined in kidney tissues after ischemia–reperfusion using HPLC. The total adenine nucleotide concentration (TAN) and adenylate energy charge (AEC) were also determined. Moreover the kidneys were evaluated histologically. Tissue concentrations of ATP, ADP, AMP, TAN and AEC were significantly increased in kidneys from rats treated with TMZ in comparison with rats receiving placebo. Concentrations of products of nucleotide degradation: inosine (Ino), guanosine (Guo) and uridine (Urd), as well as oxypurines: Hyp and Xan, were significantly decreased in rats treated with trimetazidine. Moreover, significantly less pronounced acute tubular necrosis was observed in kidneys of rats treated with TMZ. These results suggest that trimetazidine protects against dephosphorylation of nucleotides and ischemic damage.

Adenine nucleotide regulation in pancreatic β-cells: modeling of ATP/ADP-Ca2+interactions

Glucose metabolism stimulates insulin secretion in pancreatic beta-cells. A consequence of metabolism is an increase in the ratio of ATP to ADP ([ATP]/[ADP]) that contributes to depolarization of the plasma membrane via inhibition of ATP-sensitive K+ (K(ATP)) channels. The subsequent activation of calcium channels and increased intracellular calcium leads to insulin exocytosis. Here we evaluate new data and review the literature on nucleotide pool regulation to determine the utility and predictive value of a new mathematical model of ion and metabolic flux regulation in beta-cells. The model relates glucose consumption, nucleotide pool concentration, respiration, Ca2+ flux, and K(ATP) channel activity. The results support the hypothesis that beta-cells maintain a relatively high [ATP]/[ADP] value even in low glucose and that dramatically decreased free ADP with only modestly increased ATP follows from glucose metabolism. We suggest that the mechanism in beta-cells that leads to this result can simply involve keeping the total adenine nucleotide concentration unchanged during a glucose elevation if a high [ATP]/[ADP] ratio exits even at low glucose levels. Furthermore, modeling shows that independent glucose-induced oscillations of intracellular calcium can lead to slow oscillations in nucleotide concentrations, further predicting an influence of calcium flux on other metabolic oscillations. The results demonstrate the utility of comprehensive mathematical modeling in understanding the ramifications of potential defects in beta-cell function in diabetes.

Comparison of the changes in adenine nucleotides of rat liver mitochondria induced by tamoxifen and 4-hydroxytamoxifen

The antiestrogen tamoxifen (TAM) inhibits the growth of different estrogen receptor (ER)-negative cells. Recently, multiple effects of TAM on mitochondrial bioenergetic functions have been pointed to explain its ER-independent cell death mechanisms. We have shown that TAM and its major active metabolite 4-hydroxytamoxifen (OHTAM) induce depolarization of the mitochondrial membrane potential (ΔΨ) and uncouple the mitochondrial respiration, depressing the oxidative phosphorylation efficiency. To clarify the biochemical mechanisms underlying the changes in the regulation of ATP synthesis and yield, in this work we evaluated the alterations of mitochondrial adenine nucleotides induced by both drugs and ascertained whether such changes could reflect a specific inhibition of either the adenine nucleotide translocase (ANT) or the phosphate carrier, as well as the activation of ATP hydrolysis due to ΔΨ depolarization. We found that both antiestrogens caused a concentration-dependent decrease in mitochondrial ATP levels. Mitochondrial ADP and AMP were concomitantly increased with a subsequent decrease in the ATP/ADP or ATP/AMP ratios. The total concentration of adenine nucleotides also changed. Additionally, both drugs decreased the ANT content of mitochondria, inhibited the phosphate carrier and induced ATP hydrolysis. However, the effects of TAM were more drastic than those induced by OHTAM. Therefore, the depletion of ATP might result from an activation of ATP catabolism, as well as from a decrease in the mitochondrial content of ANT and partial inhibition of the phosphate carrier. Our data may explain the ER-independent effects and cytotoxicity of both drugs and, in agreement with other previous studies, suggest that OHTAM is much less toxic to mitochondria than TAM.

The effects of four weeks of ribose supplementation on body composition and exercise performance in healthy, young, male recreational bodybuilders: a double-blind, placebo-controlled trial

Background: Ribose is a pentose sugar that is present in ribonucleic acids, riboflavin, nucleotides, and adenosine triphosphate. Whether exogenous ribose administration affects skeletal muscle concentrations of total adenine nucleotides is unknown. Whether supplementation with ribose positively affects body composition or exercise performance in recreational bodybuilders also is unknown. Objective: The purpose of this double-blind, placebo-controlled trial was to determine the effects of 4 weeks of ribose supplementation on body composition and exercise performance in healthy, young, male recreational bodybuilders. Methods: Healthy, male recreational bodybuilders aged 18 to 35 years were recruited and randomized to a ribose-supplemented group (10 g/d in powder formulation) or a placebo group (dextrose). Each subject participated in a heavy-resistance training program designed to increase skeletal muscle mass. Body composition (ie, body weight, body fat, lean body mass, fat mass, and bone mineral content) was assessed using dual-energy x-ray absorptiometry analysis. Muscular strength (as measured by a 1-repetition maximum-strength [1-RM] bench press) and total work performed (as measured by total repetitions for 10 sets of bench presses before muscular failure; 1-minute resting interval between sets) to muscular failure at a submaximal load (100% of pretest body weight) were ascertained. In addition, 24-hour dietary recalls were obtained before and after the study. Results: Twenty men (mean age ± SE, 23.9 ± 1.4 years) were enrolled; 19 subjects completed 24-hour dietary recalls and exercise performance testing; 12 subjects completed the study (24-hour dietary recalls, exercise performance, and body composition). No baseline differences were found between the 2 groups for any of the measured parameters. The ribose-supplemented group experienced a significant pretreatment-to-posttreatment increase in the total work performed, whereas the placebo group did not change significantly (24.5 ± 7.6 to 29.3 ± 7.5 repetitions; 19.6% ribose [ P = 0.028] vs 34.1 ± 8.6 to 38.2 ± 8.0 repetitions, 12.0% placebo). In addition, the ribose-supplemented group experienced a significant increase in 1-RM bench press strength, whereas the placebo group did not change significantly (114.1 ± 13.6 to 117.7 ± 14.0 kg, 3.2% ribose [ P = 0.008] vs 129.6 ± 14.2 to 131.8 ± 14.5 kg, 1.7% placebo). No pretreatment-to-posttreatment within-group or between-group differences were found for any of the measures of body composition or the 24-hour dietary data. Conclusion: The results of this study indicate that supplementation with ribose 10 g/d for 4 weeks resulted in significant increases in muscular strength and total work performed in recreational bodybuilders in this study, although no significant changes in body composition or 24-hour dietary data were found.

Effect of Adenosine Triphosphate–Sensitive Potassium Channel Openers on Lung Preservation

ATP-sensitive potassium channel (KATP) openers have been proven to be involved in ischemic preconditioning, which protects ischemic tissue. However, the effect of KATP openers on ischemia-reperfusion injury of the lungs remains unknown. We investigated whether a KATP opener, pinacidil, can attenuate ischemia-reperfusion injury using an ex vivo rat lung model. Heart-lung blocks were flushed and preserved with phosphate-buffered saline (control group) or with one of the solutions containing pinacidil (pinacidil group) or pinacidil + glibenclamide (a KATP blocker) (glibenclamide group). The control and glibenclamide groups showed significantly higher values with respect to shunt fraction, pulmonary arterial pressure, and peak inspiratory pressure than the pinacidil group. The concentrations of total adenine nucleotides and ATP in the lung after reperfusion became significantly lower in the control and glibenclamide groups than in the fresh group. Lipid peroxidation of the lungs increased significantly in the control and glibenclamide groups after reperfusion. State 3 mitochondrial respiration and State 3/4 ratios of mitochondrial respiration were significantly decreased in the lungs of the control and glibenclamide groups. These findings suggested that KATP openers would maintain the mitochondrial respiratory function during lung preservation, prevent lipid peroxidation after reperfusion, and attenuate ischemia-reperfusion injury.

Mitochondrial DNA deletion associated with the reduction of adenine nucleotides in human atrium and atrial fibrillation.

Structural changes in the number, size, and shape of mitochondria (mt) have been observed in the atrial muscles of patients with atrial fibrillation (AF) and of animals with rapid atrial pacing, however, it is not known whether the mitochondrial function is impaired in human atrium with AF. We determined adenine nucleotides concentrations and mtDNA deletions in 26 human right atria obtained at the time of cardiac surgery, using HPLC and PCR amplification, and studied the relationship between mtDNA deletions and clinical manifestations, the haemodynamic parameters of the patients and adenine nucleotide concentrations in their atrium. The age and the prevalence of AF were significantly higher in the patients with a mtDNA deletion of 7.4 kb than in those without a deletion; there were no significant differences regarding haemodynamic parameters between the two groups. The concentrations of ATP, ADP, AMP and total adenine nucleotides in the right atrium were significantly lower in the patients with mtDNA deletions than the patients without a deletion. In a gender- and diseased-matched population, the mtDNA deletion was still significantly associated with age and a decreased concentration of adenine nucleotides in the atrium. Using quantitative PCR analysis, the proportion of mtDNA deletion to normal mtDNA of the atrium, was estimated to be 0.3-2% in four cases. These results suggest that the deletion of mtDNA associated with ageing or AF can lead to a bioenergetic deficiency due to an impaired ATP synthesis in the human atrium; however, no conclusion can be made whether mtDNA deletion were the result or the cause of an impaired ATP synthesis, ageing, hemodynamic deterioration, or AF.

Storage pool defect in pooled buffy coat platelet concentrates within the shelf-life period.

Platelet concentrate (PC) transfusions are useful for maintaining haemostasis in a variety of clinical situations. The function of transfused platelets is of critical importance, and changes on storage of buffy coat-prepared PC may influence their haemostatic potential. Total platelet adenine nucleotide content and platelet aggregation responses were studied, serially, in pooled buffy coat-derived PCs (n = 7), stored under UK recommended blood bank conditions, over the stipulated shelf-life of 5 days. Mean platelet volume (MPV), platelet counts and platelet distribution width (PDW) were also quantified. Total platelet ADP content decreased from 4.45+/-0.78 to 3.71+/-0.69 nmol/108 platelets (P<0.01, day 1 versus day 5, mean +/- SEM) over the shelf-life period. This was associated with reduced aggregatory responses: responses (expressed as percentage of maximum height) to 5 and 10 microM ADP decreased from 10.8+/-2.8% to 1.0+/-1.0% (P<0.005, 5 microM, day 1 versus day 5) and from 18.0+/-5.4% to 4.7+/-2.2% (P<0.02, 10 microM, day 1 versus day 5) while the decreased responsiveness was more pronounced for 4 microg/ml of collagen: 49.0+/-13.3% to 7.2+/-7.1% (P<0.01, day 1 versus day 4) and 49.9 +/-13.3% to 2.1+/-1.9% (P<0.001, day 1 versus day 5). These data indicate an acquired storage pool defect that is maximal by day 4 or 5 and accompanied by decreased platelet function, characterized by significant decreases in platelet aggregation responses. Addition of freeze-thawed plasma (autologous day 1) to PCs on days 2, 3, 4 and 5 did not alter the responses to ADP and collagen.

MUSCLE IMP ACCUMULATION IN TRAINED AND UNTRAINED MEN DURING SUBMAXIMAL CYCLING TO FATIGUE

719 This study was undertaken to compare the accumulation of IMP within the contracting muscle of endurance trained and untrained men following prolonged submaximal exercise to fatigue. Seven endurance trained (28.3 ± 1.7 yrs; 67.7 ± 2.8 kg; VO2max = 65.8 ± 2.4 ml/kg/min, mean± SEM) and six untrained (21.8 ± 1.4 yrs; 79.7 ± 4.4 kg; VO2max = 46.2 ± 1.9 ml/kg/min) men cycled to fatigue at a work rate calculated to require 70% peak oxygen uptake. Time to exhaustion was 36% longer (P < 0.01) in trained compared with untrained men (148 ± 11 and 95 ± 8 min, respectively). Glycogen content was extremely low in both TR and UT (112 ± 38 and 76 ± 35 mmol/kg dw, respectively) at fatigue and no differences for this metabolite were observed between the groups at this point. Despite the low muscle glycogen content at fatigue in TR, inosine 5'-monophosphate (IMP), a marker of metabolic stress, was not different compared with rest. In contrast, significant (P < 0.01) accumulation of IMP was observed at fatigue in UT. Additionally, the exercise-induced change in total adenine nucleotide (TAN = ATP + ADP + AMP) content tended (P = 0.06) to be greater in UT compared with TR. These data demonstrate that fatigue was associated with a similar low level of intramuscular glycogen in both trained and untrained men. Furthermore, fatigue from prolonged exercise in untrained men resulted in a compromised ADP rephosphorylation rate, which may be related to fatigue; whereas in endurance trained men, fatigue is probably related to factors other than impaired ATP resynthesis.

Influence of ageing on functional recovery and guanine nucleotide levels of the heart following cold cardioplegic arrest.

The effect of age on metabolism and mechanical recovery of the heart after cardioplegic arrest is important, but remains a relatively unexplored subject. In this study, functional recovery and nucleotide levels were compared in the heart at different ages subjected to prolonged hypothermic cardioplegic arrest. Three different age groups of rats: 1 (A); 4 (B); and 16 months (C) were perfused in working mode and subjected to cardioplegic arrest (St. Thomas' No. 1) and ischemia for 4 h at 4 degrees C, followed by reperfusion for 35 min. Cardiac function (cardiac output and aortic pressure) was recorded before and after ischemia. Another series of hearts in all three age groups underwent 5 min of normoxic perfusion to obtain pre-ischemic baseline metabolite concentrations. Hearts were freeze-clamped at the end of each experiment and used for determination of nucleotide and creatine metabolites by HPLC. The post-ischemic recovery (% of the pre-ischemic value) of the cardiac power was 48.9 +/- 7.8% for group A, which was significantly higher than the functional recovery of group B (24.1 +/- 3.5%) or C (21.4 +/- 4.7%, P < 0.05, respectively). There was no difference in ATP or the total adenine nucleotide or creatine metabolite concentrations between the three age groups. In contrast, both GTP and the total guanine nucleotide concentration was highest in A (P < 0.05). Total guanylate pool was 1.52 +/- 0.10 1 micromol/g dry wt. in A, as compared to B (1.05 +/- 0.04) or C (1.12 +/- 0.04). NAD was significantly higher in B (4.1 +/- 0.1. P < 0.05), when compared to A (3.6 +/- 0.1) and C (3.8 +/- 0.1). Best post-ischemic functional recovery after cardioplegic arrest was observed in the 1-month-old hearts (A) and was associated with highest guanine nucleotide concentration; preservation of guanine nucleotide pool in the youngest hearts may be an important mechanism for improved cardioprotection due to the important role of GTP in signalling pathways.