Changes In Phosphate Metabolites Research Articles

Mitochondrial function assessed by 31P MRS and BOLD MRI in non-obese type 2 diabetic rats.

The study aims to characterize age‐associated changes in skeletal muscle bioenergetics by evaluating the response to ischemia‐reperfusion in the skeletal muscle of the Goto‐Kakizaki (GK) rats, a rat model of non‐obese type 2 diabetes (T2D). 31P magnetic resonance spectroscopy (MRS) and blood oxygen level‐dependent (BOLD) MRI was performed on the hindlimb of young (12 weeks) and adult (20 weeks) GK and Wistar (control) rats. 31P‐MRS and BOLD‐MRI data were acquired continuously during an ischemia and reperfusion protocol to quantify changes in phosphate metabolites and muscle oxygenation. The time constant of phosphocreatine recovery, an index of mitochondrial oxidative capacity, was not statistically different between GK rats (60.8 ± 13.9 sec in young group, 83.7 ± 13.0 sec in adult group) and their age‐matched controls (62.4 ± 11.6 sec in young group, 77.5 ± 7.1 sec in adult group). During ischemia, baseline‐normalized BOLD‐MRI signal was significantly lower in GK rats than in their age‐matched controls. These results suggest that insulin resistance leads to alterations in tissue metabolism without impaired mitochondrial oxidative capacity in GK rats.

Modeling of Oxygen Transport and Cellular Energetics Explains Observations on In Vivo Cardiac Energy Metabolism

Observations on the relationship between cardiac work rate and the levels of energy metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP), and phosphocreatine (CrP) have not been satisfactorily explained by theoretical models of cardiac energy metabolism. Specifically, the in vivo stability of ATP, ADP, and CrP levels in response to changes in work and respiratory rate has eluded explanation. Here a previously developed model of mitochondrial oxidative phosphorylation, which was developed based on data obtained from isolated cardiac mitochondria, is integrated with a spatially distributed model of oxygen transport in the myocardium to analyze data obtained from several laboratories over the past two decades. The model includes the components of the respiratory chain, the F0F1-ATPase, adenine nucleotide translocase, and the mitochondrial phosphate transporter at the mitochondrial level; adenylate kinase, creatine kinase, and ATP consumption in the cytoplasm; and oxygen transport between capillaries, interstitial fluid, and cardiomyocytes. The integrated model is able to reproduce experimental observations on ATP, ADP, CrP, and inorganic phosphate levels in canine hearts over a range of workload and during coronary hypoperfusion and predicts that cytoplasmic inorganic phosphate level is a key regulator of the rate of mitochondrial respiration at workloads for which the rate of cardiac oxygen consumption is less than or equal to approximately 12 μmol per minute per gram of tissue. At work rates corresponding to oxygen consumption higher than 12 μmol min−1 g−1, model predictions deviate from the experimental data, indicating that at high work rates, additional regulatory mechanisms that are not currently incorporated into the model may be important. Nevertheless, the integrated model explains metabolite levels observed at low to moderate workloads and the changes in metabolite levels and tissue oxygenation observed during graded hypoperfusion. These findings suggest that the observed stability of energy metabolites emerges as a property of a properly constructed model of cardiac substrate transport and mitochondrial metabolism. In addition, the validated model provides quantitative predictions of changes in phosphate metabolites during cardiac ischemia.

Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays

KTCTL-26 and KTCTL-2 are renal cell carcinoma (RCC) lines with high and low expression of P-170 glycoprotein, respectively. Inherent differences between the two cell lines in terms of phosphate metabolites and growth characteristics in culture were examined for possible association with multidrug resistance (MDR). Differences in response to drug treatment were investigated for 40 h incubations with various doses of vinblastine (VBL) alone or as cotreatments with various concentrations of the calcium antagonist diltiazem (DIL) and/or interferon-alpha (IFN-alpha). Treatment effects were quantitated using the MTT survival assay and 31P magnetic resonance spectroscopy (MRS) to determine phosphate metabolite profiles in intact cells. KTCTL-2 and KTCTL-26 cells exhibited significant inherent differences in phosphocholine, glycerophosphocholine, glycerophosphoethanolamine, and phosphocreatine levels. KTCTL-26 cells were more sensitive than KTCTL-2 to 0.011 mircroM VBL alone (87% vs. 102% survival) or to 0.011 microM BL + 10 microM DIL (55% vs. 80% survival). The latter treatment resulted in a significant decrease in the ratio of phosphocholine to glycerophosphocholine in KTCTL-26 cells but no significant changes in phosphate metabolites in KTCTL-2 cells. Metabolomic 31P MRS detects different metabolite profiles for RCC cell lines with different MDR phenotypes and may be useful for noninvasive characterization of tumors in a clinical setting.

Cyclical changes in high-energy phosphates during the cardiac cycle by pacing-Gated 31P nuclear magnetic resonance.

Whether cyclical changes in energy-related phosphate metabolites arise during a cardiac cycle in isolated rat hearts and are affected by differences in myosin isozyme composition was determined. Myocardial adenosine triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi), and intracellular pH in normal, hypothyroid and hyperthyroid rat hearts were measured using the pacing-gated 31P nuclear magnetic resonance technique. Maximal decrease in ATP and PCr, and maximal increase in Pi at the peak-systole in normal rat hearts were observed. In hypothyroid and hyperthyroid rats, similar cyclical changes in phosphate metabolites were observed during the cycle. However, the magnitude of fluctuations was smaller in hypothyroid rats and larger in hyperthyroid rats compared with that observed in normal rats. Cardiac myosin isozyme patterns were also different amongst the experimental groups. The results suggest that cyclical changes and the magnitude of fluctuations in energy-related phosphate metabolites during a cardiac cycle may depend on the cardiac workload and the intrinsic properties in the enzyme kinetics of myosin.

Phosphorus MR spectroscopy in the treatment of human extremity sarcomas

The application of 31P MR spectroscopy in the characterization and treatment of malignant human extremity tumors is reviewed and placed in the perspective of results obtained in murine sarcomas. Despite the now widespread acquisition of gradient localized spectral maps, the low spatial resolution that can be achieved at 1.5 or 2 T with 31P MRS, greatly limits its use in the study of tumor heterogeneity. The potential of 31P MRS is in the evaluation and monitoring of large inoperable extremity tumors. There are early spectral changes in human extremity sarcomas monitored after therapy, and recent studies have shown that the 31P MR spectra measured before treatment, and the changes in phosphate metabolites measured shortly thereafter, correlate with the clinical response after 2 or 3 months. Larger clinical studies are needed to confirm whether correlations of, for instance, pretreatment tumor pH with necrosis at resection and Pi decrease with tumor regression, can be used as a predictive test for clinical response. © 1998 John Wiley & Sons, Ltd.

Phosphorus MR spectroscopy in the treatment of human extremity sarcomas.

The application of 31P MR spectroscopy in the characterization and treatment of malignant human extremity tumors is reviewed and placed in the perspective of results obtained in murine sarcomas. Despite the now widespread acquisition of gradient localized spectral maps, the low spatial resolution that can be achieved at 1.5 or 2 T with 31P MRS, greatly limits its use in the study of tumor heterogeneity. The potential of 31P MRS is in the evaluation and monitoring of large inoperable extremity tumors. There are early spectral changes in human extremity sarcomas monitored after therapy, and recent studies have shown that the 31P MR spectra measured before treatment, and the changes in phosphate metabolites measured shortly thereafter, correlate with the clinical response after 2 or 3 months. Larger clinical studies are needed to confirm whether correlations of, for instance, pretreatment tumor pH with necrosis at resection and Pi decrease with tumor regression, can be used as a predictive test for clinical response.

Relation among regional O2 consumption, high-energy phosphates, and substrate uptake in porcine right ventricle.

Changes in phosphate metabolites may play a role in the regulation of myocardial oxidative phosphorylation in vivo. We tested the hypothesis that changes in phosphate metabolites with increased myocardial oxygen consumption (MVO2) depend on the mechanism by which MVO2 is increased. In 17 open-chest pigs, regional MVO2 of the right ventricular (RV) free wall was increased from control by isoproterenol infusion (Iso) and by pulmonary artery constriction (PAC). The phosphocreatine-to-ATP ratio (PCr/ATP), which is inversely related to free ADP concentration ([ADP]), was determined by 31P-nuclear magnetic resonance (NMR) spectroscopy. Regional MVO2 and lactate, glucose, and free fatty acid (FFA) uptake were determined in the myocardium directly beneath the NMR coil. Iso and PAC increased MVO2 nearly equally, to approximately twice control, but produced directionally opposite changes in PCr/ATP: a significant decrease with PAC (control 1.52 +/- 0.06, PAC 1.35 +/- 0.06, means +/- SE) but a significant increase with Iso (to 1.72 +/- 0.07). Thus increased [ADP] may have stimulated oxidative phosphorylation during PAC but could not have done so during Iso. With Iso, uptake of FFA was more than three times that with PAC, and the sum of the oxygen extraction ratios for lactate, glucose, and FFA was more than double that with PAC. Enhanced substrate uptake during Iso may have increased mitochondrial NADH, which in turn may have provided an alternative stimulus to the rate of oxidative phosphorylation. These results support multifactorial control of RV oxidative phosphorylation in vivo.

The time course of phosphate metabolites and intracellular pH using 31P NMR compared to recovery heat in rat soleus muscle.

1. The recovery time course of changes in phosphate metabolites and pH, after tetanic contractions of 6 and 9 s were studied using 31P NMR with 4 and 16 s resolution, in rat soleus muscles at 20 degrees C. Muscles were at a sarcomere length of 3.15 microns (active), being greater than optimum for force which was 2.88 microns (active). 2. The post-contraction recovery of chemical changes was compared with the heat production in parallel experiments. Initial and recovery heat production were measured in tetanically stimulated muscles. 3. During recovery from tetanic contractions the changes in phosphocreatine (PCr) matched the changes in inorganic phosphate (Pi). The change in intracellular pH (pHi) was biphasic. The pHi first became more alkaline after contraction and then decreased until it reached a point below the baseline value. There was then a final recovery. 4. The initial heat and recovery heat production were greater than that expected from the PCr hydrolysis estimated during the tetanus and the PCr resynthesis that follows. 5. These data support the presence of 'unexplained heat' during a contraction and its related recovery processes in rat soleus muscle.

Effects of repeated ischemia on cerebral blood flow and brain energy metabolism.

To determine if cerebral blood flow (CBF) and cerebral metabolic alterations during partial ischemia are affected by a prior interval of ischemia, 13 neonatal piglets were studied during two successive protocols (termed A and B), each consisting of an interval before, during and after partial ischemia induced by hypotension. Piglets were studied with either microspheres (n = 6) to measure CBF and calculate cerebral uptake of O2 and glucose or 31P nuclear magnetic resonance spectroscopy (n = 6) to measure intracellular pH (pHi) and cerebral phosphorylated metabolites. One piglet was used to determine time effects. Control values of all variables were similar during protocol A and B. In each protocol hypotension was associated with similar reductions in CBF and cerebral O2 uptake but cerebral glucose uptake differed (0.10 +/- 0.05 vs 0.05 +/- 0.02 mmol.min-1.100 g-1 during hypotension of A and B, respectively, p less than 0.05). During hypotension of protocol A and B similar changes in phosphorylated metabolites and pHi occurred and were characterized by a reduction in pHi, phosphocreatine and adenosine triphosphate and an increase in inorganic phosphate. Changes in phosphate metabolites and pHi were reversible within 25 min following hypotension in both protocols. Thus, changes in CBF, cerebral O2 uptake, pHi and cerebral energy metabolism are similar during repeated episodes of partial ischemia. However, differences in cerebral glucose uptake in protocol A and B raise the possibility that the balance between energy production and utilization is altered, or alternative substrates are metabolized or enzymatic activity in the glycolytic pathway is changed.

Functional pools of oxidative and glycolytic fibers in human muscle observed by 31P magnetic resonance spectroscopy during exercise.

Quantitative probing of heterogeneous regions in muscle is feasible with phosphorus-31 magnetic resonance spectroscopy because of the differentiation of metabolic patterns of glycolytic and oxidative fibers. A differential recruitment of oxidative and glycolytic fibers during exercise was demonstrated in 4 of 10 untrained young men by following changes in phosphate metabolites. Concentrations of inorganic phosphate (Pi), phosphocreatine, and ATP were estimated in the wrist flexor muscles of the forearm at rest, during two cycles of three grades of exercise, and in recovery. At high work levels (40% of maximum strength), two distinct Pi peaks were observed and identified with Pi pools at pH 6.9 and pH 5.9-6.4, respectively. These could be accounted for as follows. At the lowest level of work (using 20% of maximum strength), early recruitment primarily of oxidative (type I) and possibly some intermediate (type IIA) muscle fibers occurs with relatively little net lactate production and consequently little decrease in pH. At higher work loads, however, primarily glycolytic (type IIB) muscle fibers are recruited, which have relatively high net lactate production and therefore generate a second pool of Pi at low pH. ATP depletion (35-54%) and Pi losses accompanied the reduction in ability to perform during the first exercise cycle. When the cycle of graded exercise was repeated immediately, the total Pi remained high but gave rise to only one peak at pH 6.8-7.0. These observations indicated exhaustion of glycolytic type IIB fibers, removal of lactate by high local blood flow, and sustained contractions largely by oxidative type I and IIA fibers. A functional differentiation of fiber types could also be demonstrated during recovery if exercise was stopped while two pools of Pi were still apparent. In the first 3 min of recovery, the Pi peak at pH 6.8-6.9 disappeared almost entirely, whereas the Pi peak at pH 6.0 remained unaltered, reflecting the faster recovery of oxidative type I fibers. The potential of magnetic resonance spectroscopy to characterize oxidative and glycolytic fibers, predict capacity for aerobic performance, and signal the presence of muscle pathology is discussed.

Phosphagen and intracellular pH changes during contraction of creatine-depleted rat muscle.

To evaluate the functional role of phosphocreatine (PCr) and creatine in muscle metabolism, these compounds were depleted by feeding rats the creatine analogue, beta-guanidinopropionate (beta-GPA, 2% of diet). Changes in phosphate metabolites and intracellular pH were monitored in gastrocnemius muscle in situ by phosphorus nuclear magnetic resonance (31P-NMR) at 162 MHz using the surface coil technique. After 3 mo of feeding, 25 mumol/g of phosphorylated beta-GPA (beta-GPAP) had accumulated, and PCr, creatine, and ATP levels were reduced to 6, 17, and 56%, respectively, compared with muscles of control animals. In resting muscle, there was no measurable exchange of phosphate between beta-GPAP and ATP by the NMR saturation transfer method. During muscle stimulation at 1 and 5 Hz, the maximum net rate of beta-GPAP hydrolysis was 10% that of PCr in control muscles, so that after 150 s inorganic phosphate had increased to less than 50% of the level attained in control muscles. At both rates, peak twitch force declined toward a steady state more rapidly in beta-GPA-loaded muscles, but after 100 s force was either not different (1 Hz) or significantly greater (5 Hz) in the beta-GPA-fed animals. Intracellular pH initially decreased more rapidly during stimulation and recovered more rapidly afterward in the beta-GPA-loaded muscles compared with controls. This difference could be explained by the difference in expected proton consumption due to net PCr hydrolysis. However, despite buffering by PCr hydrolysis, pH ultimately decreased more in control muscle (6.1 vs. 6.3 for 5 Hz), indicating greater acid accumulation compared with beta-GPA-loaded muscles. In the superficial, predominantly fast-twitch glycolytic section of muscles clamp-frozen after 5-Hz stimulation for 150 s, lactate accumulation was twofold greater in controls. The results indicate that PCr is not essential for steady-state energy production but that the phosphate from PCr hydrolysis may be important for maximum activation of glycogenolysis and/or glycolysis.