31P NMR and triple quantum filtered 23Na NMR studies of the effects of inhibition of Na+/H+ exchange on intracellular sodium and pH in working and ischemic hearts

The triple quantum filtered ²³Na NMR method is applied here to measure the effects of EIPA, a specific inhibitor of the Na⁺/H⁺antiporter, on relative intracellular sodium concentrations in isolated working hearts at baseline, during ischemia, and at subsequent reperfusion. In analogy to the spectrophotometric isosbestic point, an approach is developed that defines a value of τ at which the effect of the relaxation times on the TQF signal intensities is minimized, and the signals are proportional to the sodium concentration for both ischemic and working hearts. EIPA at 1.5 μ significantly inhibited (P < 0.01) the influx of intracellular Na⁺ during 20 min of ischemia at 36.2°C in this rat heart model. In parallel³¹P NMR studies, EIPA had no effect on either the development of acidosis during ischemia or on the recovery of pH, during reperfusion despite its profound effect on intracellular Na⁺ influx. Thus, under our conditions the Na⁺/H⁺ antiporter did not play a critical role in the maintenance of intracellular pH. EIPA treatment resulted in improved recovery (P < 0.005) of mechanical function after 20 min of ischemia. [ATP] was higher in treated hearts during ischemia and reperfusion.     

Assessment of glucosylifosfamide mustard biodistribution in rats with prostate adenocarcinomas by means of in vivo 31P NMR and in vitro uptake experiments

A combined in vitro/in vivo study was performed to evaluate the possible application of phosphorus (³¹P) NMR spectros-copy for therapy monitoring and to investigate glucosylifos-famide mustard (Glc-IPM) transport and biodistribution by radiotracer techniques. Dynamic in vivo ³¹P NMR measurements were performed in rats with prostate adenocarcinoma after iv injection of 1 mmol/kg body weight (bw) of ifosfamide (IFO) (n = 4) and 1 mmol/kg bw (n = 4) or 2.15 mmol/kg bw (n = 9) of Glc-IPM. In a biodistribution study with ¹⁴C-labeled Glc-IPM and a final dose of 0.8 mmol Glc-IPM/kg bw, the animals were killed 5, 30, 60, and 120 min after drug administration, an ethanol extraction was performed from several tissues, and the dose per g tissue was calculated. The same tumor cell line was used in saturation and competition experiments to further elucidate the transport mechanism. The ³¹P NMR signals of IFO and Glc-IPM showed no overlap with the endogenous phosphorus peaks. A rapid washout with a half-life between 25.9 ± 5.6 min for the lower dose and 34.3 ± 4.2 min for the higher dose of Glc-IPM was observed in the tumor. No statistically significant change of the pH value was observed during the examination period. The β-nucleoside 5′-triphosphate (NTP)/inorganic phosphate (Pi) signal intensity ratio showed a tendency to decrease but without statistical significance. A rapid elimination was demonstrated by both the noninvasive NMR technique and the biodistribution study. No saturation was found in vitro for the Glc-IPM uptake, even at the concentration of 5 m/W. Furthermore, the Glc-IPM uptake was not inhibited by the presence of 2-deoxyglucose and vice versa. The data show that the pharmacokinetics of Glc-IPM in the tumor can be followed in vivo by ³¹P NMR. The results presented are evidence for diffusion as the transport mechanism for Glc-IPM in this tumor model. However, the better visualization of Glc-IPM as compared to ifosfamide may be due to metabolic trapping of a negatively charged metabolite after deglycosylation.     

Simultaneous 31P NMR spectroscopy and EMG in exercising and recovering human skeletal muscle: Technical aspects

The bioenergetics of human skeletal muscle can be studied by ³¹P NMR spectroscopy (³¹P-MRS) and by surface electromyography (SEMG). Simultaneous ³¹P-MRS and SEMG permit accurate and noninvasive studies of the correlation between metabolic and electrical changes in exercising and recovering human skeletal muscle, a relationship that is still poorly understood. This study describes the optimization of skeletal muscle ³¹P-MRS in a whole-body magnet, involving surface coil design, utilization of adiabatic radio frequency pulses and advanced time-domain fitting, to the technical design of SEMG. A nonmagnetic ergometer was used for ankle dorsi-flexions that activated only the anterior tibia1 muscle as verified by post exercise imaging. The coil design and the adiabatic sechltanh pulse improved sensitivity by 45% and 56% respectively, compared with standard techniques. Simultaneous electromyographic recordings did not deteriorate the NMR spectra. The VARPRO time domain fitting routine was very suitable for estimating ³¹P muscle spectra. With these methods it was possible to accurately estimate parameters describing metabolic and electrical changes during rest, exercise and the entire recovery period with a 20-s time resolution on a standard 1.5 T whole-body NMR scanner.     

pH control in rat skeletal muscle during exercise,recovery from exercise,and acute respiratory acidosis

We used ³¹P magnetic resonance spectroscopy to compare the response of rat skeletal muscle to three kinds of proton load. During exercise (tetanic sciatic nerve stimulation), protons from lactic acid were buffered passively and consumed by net hydrolysis of phosphocreatine (PCr). During recovery from exercise, the pH-dependent efflux of protons produced by PCr resynthesis could be partially inhibited by amiloride or 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS), implicating both sodiudproton and bicarbonatelchloride exchange, but was not inhibited by simultaneous respiratory acidosis. In early recovery, up to 30% of proton efflux was mediated by lactatelproton cotransport. During acute respiratory acidosis at rest, the eventual change in muscle pH was consistent with passive buffering and was unaffected by amiloride or DIDS, implying no significant contribution of proton fluxes.     

Phosphocreatine resynthesis during recovery in different muscles of the exercising leg by 31P‐MRS

To investigate the high‐energy phosphate metabolism by ³¹P‐nuclear magnetic resonance spectroscopy during off‐transition of exercise in different muscle groups, such as calf muscles and biceps femoris muscles, seven male long‐distance runners (LDR) and nine untrained males (UT) performed both submaximal constant and incremental exercises. The relative exercise intensity was set at 60% of the maximal work rate (60%Wmax) during both knee flexion and plantar flexion submaximal constant load exercises. The relative areas under the inorganic phosphate (P_i) and phosphocreatine (PCr) peaks were determined. During the 5‐min recovery following the 60%Wmax, the time constant for the PCr off‐kinetics was significantly faster in the plantar flexion (LDR: 17.3 ± 3.6 s, UT: 26.7 ± 6.7 s) than in the knee flexion (LDR: 29.7 ± 4.7 s, UT: 42.7 ± 2.8 s, P < 0.05). In addition, a significantly faster PCr off‐kinetics was observed in LDR than in UT for both exercises. The ratio of P_i to PCr (P_i/PCr) during exercise was significantly lower during the plantar flexion than during the knee flexion (P < 0.01). These findings indicated that the calf muscles had relatively higher potential for oxidative capacity than that of biceps femoris muscles with an association of training status.     

Identification of different lipid phases and calcium phosphate deposits in human carotid artery plaques by MAS NMR spectroscopy

Accumulation of lipids in atherosclerotic plaques causes progressive narrowing of the arterial lumen, often followed by thrombosis and ischemia. Currently several different methods, most requiring disruption of the plaque, are used to study the physical properties of lipids accumulated in plaques, and lipid composition is typically determined by chemical analysis of completely disrupted plaques. In this study, ¹³C magic angle spinning NMR spectroscopy (MAS NMR) was used to determine in situ the lipid composition and molecular organization of all lipid phases in human carotid artery plaques (ex vivo). Protocols were developed to observe signals from one lipid phase without interference from other phases. In addition, ³¹P MAS NMR detected calcification in plaques by the signals from inorganic phosphate complexed to calcium. Together, ¹³C and ³¹P MAS NMR comprise a powerful nondisruptive approach for determining the quantity and phase state of components in arterial plaques.     

Glucose metabolism in RIF-1 tumors after reduction in blood flow: An in Vivo 13C and 31P NMR study

Low pH appears to enhance the effectiveness of therapeutic hyperthermia. ¹³C and ³¹P NMR spectroscopy have been employed to examine the possibility that elevating glucose in a solid tumor while simultaneously reducing tumor blood flow would induce a more profound acidosis than either treatment alone. When blood flow in RIF-1 tumors was acutely reduced by administration of hydralazine and additional glucose was delivered locally by intratumoral injection, tumor acidosis (as determined by ³¹P NMR spectroscopy) during the period of reduced blood flow was not enhanced, relative to administration of hydralarine alone. Tumor NTP/P₁ ratios decreased significantly within 20 min of hydralazine administration, whether or not glucose was injected, although NTP/P₁ ratios were slightly higher in tumors that received extra glucose. Tumor lactate concentrations were not significantly different in glucose-supplemented tumors, despite glucose concentratlons that were 4 to 5 times higher. When the added glucose was labeled with ¹³C, no correlation was detected between the pH in an individual tumor and the intensity of the 3-[¹³C]-lactate resonance in the same tumor.     

Impact of salbutamol on muscle metabolism assessed by 31P NMR spectroscopy

The potential ergogenic effects of oral salbutamol intake were demonstrated for decades but the underlying mechanisms remain to elucidate. We hypothesized that improved exercise performance after acute oral salbutamol administration is associated with changes in muscle metabolism. Twelve healthy, nonasthmatic, moderately trained, male subjects were recruited to compare in a double‐blind crossover randomized study, an oral dose of salbutamol (4 mg) and a placebo. After treatment administration, subjects performed repetitive plantar flexions to exhaustion in a 3T magnet. Continuous ³¹P nuclear magnetic resonance spectroscopy assessment of the calf muscles was performed at rest, during exercise, and during recovery. No significant difference between treatments was detected in metabolite concentration at rest (P > 0.05). Creatine phosphate and inorganic phosphate changes during and immediately after exercise were similar between treatments (P > 0.05). Intramuscular pH (pHi) was significantly higher at rest, at submaximal exercise but not at exhaustion with salbutamol (pHi at 50% of exercise duration, 6.8 ± 0.1/6.9 ± 0.1 for placebo and salbutamol, respectively, P < 0.05). The maximal power (28 ± 7 W/23 ± 7 W; P = 0.001) and total work (1702 ± 442 J/1381 ± 432 J; P = 0.003) performed during plantar flexions were significantly increased with salbutamol. Salbutamol induced significant improvement in calf muscle endurance with similar metabolic responses during exercise, except slight differences in pHi. Other mechanisms than changes in muscle metabolism may be responsible for the ergogenic effect of salbutamol administration.     

In Vivo 31P NMR measurement of glucose-6-phosphate in the rat muscle after exercise

Comparison of ³¹P NMR spectra of the rat gastrocnemius, obtained in vivo and from PCA extracts, after electrically induced contractions, demonstrates that glucose-6-phosphate (G6P) is the major metabolite in the low-field part of the PME spectral region. In vivo ³¹P NMR can thus be used to measure the muscle G6P concentration after exercise.     

Simultaneous 3D NMR spectroscopy of fluorine and phosphorus in human liver during 5-fluorouracil chemotherapy

Simultaneous multivoxel ³¹P and ¹⁹F 3D localized NMR spec-troscopy is demonstrated on a phantom and in the liver of patients undergoing bolus-infusion 5-fluorouracil chemotherapy. The ¹⁹F and ³¹P spectra were localized with 8 × 8 × 8 3D chemical-shift imaging, with both nuclei sharing the same field of view and voxel size (27 and 64 ml in phantom and liver, respectively) using a 1.5-Tesla clinical imager with two RF channels and a dual-tuned surface coil. The repetition time (TR = 0.26 s) and Ernst nutation angles (θ_E = 32° for ¹⁹F, 28° for ³¹P) were chosen to optimize the signal-to-noise ratio (SNR) per-untt time for the 0.5- to 2-s T₁ range of the ¹⁹F and ³¹P metabolites of interest The overall examination time, including tuning, imaging, shimming and dual-nuclear spectroscopy, was under 90 min. Simultaneous acquisition of ³¹P and ¹⁹F spectra will permit the study of the influence of hepatic and/or tumor metabolism on the uptake and catabolism of fluoropyrimidine drugs with no extra measurement time.     

The effects of isoflurane and halothane on blood flow and 31P NMR spectra in murine RIF-1 tumors

The principal aim of these studies was to evaluate the utility of isoflurane and halothane for NMR investigations of tumor physiology. In vivo ³¹P and ²H NMR were used to examine RIF-1 tumors before, during, and (for ³¹P) after anesthesia. In tumors, halothane decreases blood flow, [PCR]:[NTP], and pH indicated by the P₁ chemical shift (pH_nmr), while it increases [P₁:[NTP]; effects consistent with well-established cardiovascular effects of halothane. Isoflurane does not affect tumor blood flow or [PCr]:[NTP], but increases tumor [P₁:[NTP] and decreases tumor pH_nmr. In vivo ³¹P NMR measurements of normal mouse liver (upper abdomen) indicate that isoflurane has a similar effect in the liver. Although the mechanism for these effects is unknown, observation of a split P₁ peak during isoflurane anesthesia suggests that a pool of P₁ in a lower pH environment may become evident under isoflurane anesthesia. Regardless of the cause for increased [P₁:[NTP] and decreased pH_nmr the utility of isoflurane anesthesia for ³¹P NMR studies of energy metabolism is limited.     

Intracellular and extracellular spaces and the direct quantification of molar intracellular concentrations of phosphorus metabolites in the isolated rat heart using 31P NMR spectroscopy and phosphonate markers

To quantify metabolite and cation concentrations using NMR spectroscopy, the volumes of intracellular and extracellular spaces must be known. We describe a simple ³¹P NMR spectroscopic method that employs dimethyl methylphosphonate (DMMP) as a marker of total water space and phenylphosphoriate (PPA) as a marker of extracellular space to determine intracellular and extracellular space volumes in the isolated, perfused rat heart. In vivo and in vitro radiolabel studies were used to verify this method. The difference between the total and extracellular water spaces, determined as milliliters/heart, gave the intracellular volume and allowed direct calculation of myocardial creatine phosphate, ATP, and inorganic phosphate concentrations, which were 13.4 mM, 10.1 mM, and 3.4 mM, respectively, for the glucose-perfused rat heart. The extracellular volume decreased by 84% in hearts subjected to 28 min total, global ischemia and increased by 15% during reperfusion. The method described allows the determination of intracellular energy metabolite concentrations in perfused rat heart directly from a single, fully relaxed ³¹P NMR spectrum.     

A 31P NMR study of preconditioned isolated perfused rat heart exposed to intermittent ischemia

Exposure to a short ischemic period (ischemic preconditioning, IP) will protect the heart from damage following a subsequent longer ischemic episode. The aim of the study was to test whether IP is cardioprotective in the setting of repeated ischemia-reperfusion cycles. Thus, Langendorff-perfused hearts, exposed to IP, were subjected to three consecutive ischemia-reperfusion (10/15 min) cycles. Myocardial energetics, manifested by ³¹P NMR spectroscopy, was correlated with hemodynamics. ATP recovery was significantly higher for the IP group compared with control (P < 0.02) during reperfusions. However, there was no significant difference in ATP recovery during the three ischemic intervals. The supernormal recovery of phosphocreatine recorded during reperfusion was lower for the IP group (～120%) compared with control (～135%, P < 0.065). Better recovery of the left ventriculardeveloped pressure was noted during reperfusions for the IP group and became significant only during the last reperfusion (86% versus 68%, P < 0.025). In conclusion, the above results support prolonged IP cardioprotection.     

Resolution enhanced NMR spectroscopy in biological systems via magnetic susceptibility matched sample immersion chambers

A technique is described which reduced the magnetic susceptibility induced line broadening in NMR spectra obtained from three biological systems at 4.7 Tesla. Proton spectra from a sealed suspension of HL60 leukemic myeioblasts yielded minimum linewidths of 1.3 Hz at 200 MHz (0.0065 ppm) after 10 min of automated shimming. ³¹P spectra from an in vivo murine MCa mammary carcinoma yielded a well-resolved phospharylcholine resonance without proton decoupling and with the resistive shim coil currents set to zero. ³¹P spectra from a perfused suspension of RIF-1 fibrosarcoma cells exhibited a γ-nucleoside triphosphate resonance which was resolved into purine and pyrimidine components.     

Metabolic recovery in professional road cyclists: a 31P-MRS study

PURPOSE: Aerobic training of professional road cyclists is linked to tremendous aerobic capacities that have never been clearly related to what occur in skeletal muscles submitted to a specific exercise. The aim of the present study was to examine specifically metabolic recovery after an incremental cycling exercise performed until exhaustion in professional road cyclists as compared with moderately trained subjects and so using 31P- MRS. METHODS: Subjects performed a progressive cycling exercise on a cycloergometer until exhaustion, then they were positioned back in the magnet (delay lower than 45 s) for recovery scanning. 31P spectra of thigh muscles were time averaged in 2-s blocks at rest and for 15 min throughout the recovery period. RESULTS: For a significantly more intense exercise (477 +/- 28 vs 334 +/- 24 W in controls; P < 0.001), professional road cyclists displayed similar end-of-exercise extrapolated pH values (6.43 +/- 0.16 vs. 6.34 +/- 0.05 in controls) and a significantly higher PCr concentration (20.1 +/- 0.8 vs. 13.3 +/- 0.5 mM in controls, P < 0.001). The pH recovery kinetics provided the evidence of metabolic adaptations related to a specific training in professional cyclists with a significantly faster rate (P < 0.01) of pH return toward basal values (32.8 +/- 18.9 vs 10.8 +/- 6.7 mM x min(-1)). On the contrary, no significant difference was measured for the PCr recovery kinetics. At rest, PDE concentration was significantly higher in professional cyclists (2.50 +/- 0.80 vs 1.76 +/- 0.42 mM), likely indicating a difference regarding fiber-type composition. DISCUSSION: The present data demonstrated for the first time that the tremendous aerobic capacity in professional cyclists is linked to faster pH recovery kinetics after a specific cycling exercise.     

pH mapping in living tissues: An application of in vivo31P NMR chemical shift imaging

Chemical shifts were extracted from in vivo³-dimensional³¹P NMR CIS data and pH images were constructed. The images could spatilly resolve tissue pH ranging from 5.8 to 7.2 (with uncertainty of 0.11-0.17 pH unit)in an ischemia-reperfusion model of diabetic rat calf muscles.     

Determination of saturation factors in 31P NMR spectra of the developing human brain

In order to assess the influence of longitudinal relaxation on previously reported variations in ³¹P NMR signals during brain development, we used an accelerated two-point technique to determine T¹ at 2.35 Tesla in 8 min. Comparison between 10 normal neonates (age range 37–46 weeks postconception) and 10 healthy infants (age range 80–157 weeks postconception) indicated that T¹ does not vary substantially during the first year of life, except in the phosphomonoester (PME) region of the spectra. T¹ of total PME decreases with age which we could explain by its variable multicomponent nature: The signal from (unresolved) components at the downfield shoulder of PME (attributed mostly to phosphorylethanolamine at 6.72 ppm) with a T¹ of at least 6.4 s decreases with age relative to contributions from other phosphomonoester compounds resonating predominantly at the upfield side of the peak (approximately 6.3 ppm), with T¹ below 2.9 s. Because the T¹ heterogeneity of PME may depend on its relative composition, quantitative ³¹P NMR spectroscopy may require an assessment of the influence of longitudinal relaxation on the signal amplitudes in each measurement.     

Effects of real and sham whole‐body mechanical vibration on spinal excitability at rest and during muscle contraction

We examined the effects of whole‐body mechanical vibration (WBV) on indices of motoneuronal excitability at rest and during muscle contraction in healthy humans. Real and sham WBV at 30 Hz had no effect on reflexes measured during muscle contraction. Real WBV at 30 and 50 Hz depressed the H‐reflex ～45%. These depressions diminished across the five inter‐bout rest intervals. The depression converted to 27% and 7% facilitation over the 15‐min long recovery period following real WBV at 30 and 50 Hz, respectively. The depression, measured during the inter‐bout rest, correlated r = 0.48 (P = 0.007) with the subsequent facilitation, measured during the follow‐up. The depression produced by sham vs real WBV was significant but less (23%), recovered faster, and the facilitation was absent in the 15‐min long follow‐up period. WBV produced time‐varying depression followed by facilitation of the H‐reflex at rest. A lack of change in volitional wave suggests that WBV did not affect the efferent neural drive.     

A perifusion loop-gap resonator NMR probe for aerobic cell suspensions

NMR studies of aerobic cell metabolism require that spectral data of high sensitivity and resolution be acquired from samples maintained under suitable conditions. The loop-gap resonator ³¹P NMR probe described here was designed for investigations of highly aerobic-dependent renal proximal tubules in an environment similar to those of conventional incubation flasks. The inherently higher sensitivity of the loop-gap resonator design and associated circuitry made it possible to obtain adequate spectra in short periods of time with small amounts of sample. Cellular physiological properties of perifused aerobic renal proximal tubule cell suspensions in the loop-gap resonator probe were found to be similar to equivalent tissue samples incubated in conventional flasks. Furthermore, the loop-gap perifusion probe was found to provide useful ³¹P NMR spectra of the kidney tubule system after acquisition times as short as 3 min.     

Simultaneous 3D NMR spectroscopy of proton-decoupled fluorine and phosphorus in human liver during 5-fluorouracil chemotherapy

Simultaneous acquisition of ¹H-decoupled ³¹P and ¹⁹F 3D CSI is demonstrated in the liver of a patient undergoing 5-fluorouracil chemotherapy. Both ³¹P and ¹⁹F shared the same voxel size (64 or 27 ml), bi-level ¹H-decoupling and 0.35 s TR. The measurements were done in a 1.5 Tesla clinical imager with three radio-frequency (RF) channels and a triple-tuned surface-coil. The overall MRI and MRS examination time was under 90 min. Simultaneous acquisition of ³¹P and ¹⁹F permits localized study of the influence of hepatic metabolism on the uptake and catabolism of fluoropyrimidine drugs without extra measurement time or higher SAR.