Transmural metabolite distribution in regional myocardial ischemia as studied with 31P NMR

Phosphorus-31 nuclear magnetic resonance (31P NMR) has been applied to study the canine heart prior to and during regional myocardial ischemia induced by partial flow reduction in the left anterior descending coronary artery (LAD). NMR data were acquired in a transmural fashion by restricting the signal to a column perpendicular to the heart wall using B0 gradients and obtaining spectroscopic spatial resolution along the third dimension using the B1 gradient and adiabatic excitation. With this approach, transmural spectra were accumulated in five separate voxels spanning the wall of the left ventricle from the epicardium to the endocardium. In the normal canine myocardium the levels of high-energy phosphates CP and ATP were relatively constant throughout the left ventricular wall, with only minor evidence of free inorganic phosphate in any of the transmural voxels. However, during sustained partial occlusion of the LAD, significant regional differences between the epi- and the endocardium were noted. The data demonstrate the importance of studying cardiac bioenergetics with transmural differentiation.     

In vivo evaluation of intracellular pH and high-energy phosphate metabolites during regional myocardial ischemia in cats using 31P nuclear magnetic resonance

Phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMR) was used to assess the temporal changes of high-energy phosphate metabolites in the region of acute myocardial ischemia of open-chest cats. Eight anesthetized cats were studied following ligation of the left anterior descending coronary artery. Creatine phosphate showed a 79 +/- 16% (mean +/- SD) reduction by 4 min after the onset of ischemia. Prominent qualitative reductions of the spectral peak of creatine phosphate occurred by 40 s after ischemia. Adenosine triphosphate measured under the beta spectral peak (beta-ATP) decreased 37 +/- 9% by 20-25 min after ligation of the left anterior descending coronary artery. These reductions developed more slowly and were of smaller magnitude than those of creatine phosphate. Intracellular pH decreased from 7.39 +/- 0.07 to 7.13 +/- 0.09 units by 40 s after ischemia. By 30 min, pH decreased to 6.07 +/- 0.40 units. The study shows, therefore, the temporal changes of high-energy phosphate metabolites during ischemia in localized regions of the myocardium of open-chest animals.     

31P MRS of myocardial inorganic phosphate using radiofrequency gradient echoes.

Determination of the chemical shift and integral of the myocardial intracellular inorganic phosphate (Pi) resonance by 31P magnetic resonance spectroscopy (MRS) is often precluded due to a large overlapping signal from 2,3-diphosphoglycerate (2,3-DPG) from chamber and myocardial blood. This report demonstrates the use of radiofrequency (RF) magnetic field gradient echoes (RFGE) to eliminate signals from 2,3-DPG in flowing blood, while retaining signals from intracellular myocardial Pi, ATP, and phosphocreatine (PCr). The ECG-triggered 31P spectra were acquired from the myocardium of open chest pigs using a Philips Gyroscan 2-T magnetic resonance spectrometer. A 2.5-cm-diameter surface coil attached to the myocardium was used to provide the RF gradient as well as for excitation and detection of signals. Optimal performance of the RFGE pulse sequence was obtained when the RF gradient pulses were centered at peak diastole or peak systole. Under these conditions, 2,3-DPG signals were completely suppressed, and sensitivity was usually sufficient to allow detection of a well-resolved Pi signal. Myocardial pH determined from RFGE experiments was 7.16 +/- 0.10, and the ratio of the integrals of the Pi and ATP resonances (Pi/ATP) was 0.24. The mean signal-to-noise ratio (S/N) for PCr in control spectra acquired in 4 min was 19/1, while the mean S/N for PCr in RFGE-edited spectra acquired in 15 min was 11/1, demonstrating that the present implementation of the RFGE method results in significant loss in sensitivity. These experiments demonstrate that RFGE-editing allows accurate determination of the chemical shift and integral of the Pi resonance in blood-perfused myocardium in situ.     

NMR determination of myocardial pH in vivo: separation of tissue inorganic phosphate from blood 2,3-DPG.

Phosphorus NMR can measure myocardial tissue pH from the chemical shift of inorganic phosphate (Pi) in isolated buffer-perfused hearts, but in vivo the Pi peak originating from the myocardium is obscured by the resonance of 2,3-diphosphoglycerate (DPG) in the blood, making pH difficult to determine. Taking advantage of the fact that most of the interfering DPG is within the cardiac chambers and is rapidly flowing out of the sensitive volume of our coil, we developed a pulse sequence which would separate myocardial Pi signal from interfering DPG. We tested this method on a flow phantom and in living rat heart, using exogenous glycerol phosphate as a blood-pool marker. The results indicated that signal from moving and nonmoving substances could be separated, and derived values for myocardial pH and PCr/Pi ratio were consistent with previous estimates. This method should be useful for studying myocardial acid-base physiology with NMR.     

Altered phosphate metabolism in myocardial infarction: P-31 MR spectroscopy

The high-energy myocardial phosphate metabolism of four patients with acute anterior myocardial infarction after coronary angioplasty and drug therapy was evaluated with cardiac-gated phosphorus magnetic resonance (MR) depth-resolved surface coil spectroscopy (DRESS) 5-9 days after the onset of symptoms. Significant reductions (about threefold) in the phosphocreatine (PCr) to inorganic phosphate (Pi) ratio and elevations in the Pi to adenosine triphosphate (ATP) ratio were observed in endocardially or transmurally derived MR spectra when compared with values from epicardially displaced spectra and values from seven healthy volunteers (P less than .05). High-energy phosphate metabolites and Pi ratios did not vary significantly during the cardiac cycle in healthy volunteers. However, contamination of Pi resonances by phosphomonoester components, including blood 2,3-diphosphoglycerate, precluded accurate spectral quantification of Pi and pH. The results indicate that localized P-31 MR spectroscopy may be used to directly assess cellular energy reserve in clinical myocardial infarction and to evaluate metabolic response to interventions.     

Tumor 31P NMR pH measurements in vivo: A comparison of inorganic phosphate and intracellular 2-deoxyglucose-6-phosphate as pHnmr indicators in murine radiation-induced fibrosarcoma-1

Uncertainty regarding the intracellular/extracellular distribution of inorganic phosphate (P_i) in tumors has raised concerns that pH calculated from the tumor P_i chemical shift may not accurately represent the intracellular pH (pH_in). This issue was addressed in subcutaneously transplanted murine radiation induced fibrosarcoma-1 by directly comparing pH measured via P_i with pH measured via the in situ generated intracellular xenometabolite 2-deoxyglucose-6-phosphate (2DG6P). In 131 comparative measurements employing eight tumor-bearing mice under both control and hyperglycemic conditions (the latter to extend the range of tumor pH examined), the pH as derived from either 2DG6P or P_i showed only a small, but statistically significant, difference (0.07 ± 0.11 SD; P = 0.0001). Scatter in the comparative analysis over the pH range examined (ca. 5.5-7.5) was not uniform. Above pH 6.6, 2DG6P indicated a pH lower than that of P_i by 0.088 ± 0.105 SD (n = 107, P = 0.0001); below pH 6.6, 2DG6P indicated a pH essentially identical to and not statistically different from that of P_i (mean difference 0.003 ± 0.128 SD (n = 24, P = 0.92)). Evidence is presented in support of this differential arising from a systematic measurement error due to peak overlap between 2DG6P and endogenous phosphomonoester species. These results support the use of P_i as a tumor ³¹P NMR pH_in indicator, at least in RIF-1 tumors under control and hyperglycemic conditions.     

Human in vivo phosphate metabolite imaging with 31P NMR

Phosphorus (31P) spectroscopic images showing the distribution of high-energy phosphate metabolites in the human brain have been obtained at 1.5 T in scan times of 8.5 to 34 min at 27 and 64 cm3 spatial resolution using pulsed phase-encoding gradient magnetic fields and three-dimensional Fourier transform (3DFT) techniques. Data were acquired as free induction decays with a quadrature volume NMR detection coil of a truncated geometry designed to optimize the signal-to-noise ratio on the coil axis on the assumption that the sample noise represents the dominant noise source, and self-shielded magnetic field gradient coils to minimize eddy-current effects. The images permit comparison of metabolic data acquired simultaneously from different locations in the brain, as well as metabolite quantification by inclusion of a vial containing a standard of known 31P concentration in the image array. Values for the NMR visible adenosine triphosphate in three individuals were about 3 mM of tissue. The ratio of NMR detectable phosphocreatine to ATP in brain was 1.15 +/- 0.17 SD in these experiments. Potential sources of random and systematic error in these and other 31P measurements are identified.     

31P and 1H NMR spectroscopy to study the effects of gallopamil on brain ischemia

Studies were performed on 16 cats to evaluate the potential protective effects of Gallopamil on brain ischemia. Brain energy state was determined by 31P NMR and lactate concentration was determined by 1H NMR. Double-tuned surface coils (tuned to 35.8 and 88.4, respectively) were placed on the head after skin and muscle were removed from the calvarium. A 2.1-T, 25-cm-bore Oxford magnet interfaced to a Phosphoenergetics 250-80 spectrometer was used. The cats were bled to 50 mm Hg for 10 min with subsequent application of bilateral carotid occlusion for 10 min to produce ischemia. In all animals, brain energy state as measured by Pi/PCr and lactate concentrations were determined over 5-min intervals (before, during, and after the onset of ischemia). While Gallopamil did not prevent decreases in brain energy state or attenuate the rise in lactate concentration seen during ischemia, brain from animals treated with Gallopamil had a more rapid return of pHi to baseline during the recovery period. In Gallopamil-treated cats, higher levels of lactate were necessary to cause a similar decrease in pHi when compared to controls. The rate of lactate recovery to baseline levels was similar in both groups (control = -0.38 +/- 0.14 mM/min; Gallopamil = -0.44 +/- 0.32 mM/min). In conclusion, Gallopamil appears to lessen the acidosis caused by cerebral ischemia. In addition, we have demonstrated that multinuclear NMR spectroscopy is a powerful tool to study the effects of drugs on cerebral metabolism.     

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.     

The effect of cardiopulmonary bypass on brain and heart metabolism: a 31P NMR study

The development of a large animal preparation using 31P nuclear magnetic resonance (NMR) spectroscopy for the study of cerebral and myocardial metabolism during cardiopulmonary bypass (CPB) is reported. The effect of normothermic CPB on myocardial and cerebral metabolism was evaluated. Adolescent sheep were used which have low levels of 2,3-diphosphoglycerate, a compound which can interfere with the calculation of intracellular pH and inorganic phosphate content. CPB was performed using standard procedures modified for the presence of a high magnetic field and limited access to the animal. High quality 31P NMR data were obtained from the brains and hearts of these animals before and during normothermic CPB. These results demonstrate that the initiation of normothermic CPB does not change high energy phosphate levels or intracellular pH. In particular, the decreased myocardial oxygen demand associated with CPB is not associated with improvement in the levels of adenosine triphosphate or phosphocreatine. The measurements of energy metabolism and intracellular pH of the brain and heart during CPB were possible within the constraints of the NMR experiment without compromising the CPB procedure. Combining NMR and CPB techniques permits future studies of cerebral and myocardial metabolism, especially those relating to ischemia.     

Scalar couplings as pH probes in compartmentalized biological systems: 31P NMR of phosphite

The use of scalar couplings in nuclear magnetic resonance (NMR) spectra was investigated as a possible tool for the measurement of pH in different compartments of biological systems. The proposed method is attractive because no internal reference is required, unlike more widely used chemical shift titrations. The phosphite anion is shown to be ideal for the measurement of pH in the physiological range. In isotonic solution, the divalent anion PHO has a one‐bond ¹J_PH of 568.1 Hz, increasing to 620.7 Hz for the monovalent anion PH(OH)O, with a measured pK_a of 6.19. The technique was applied to the measurement of pH in a suspension of human erythrocytes. The ³¹P spectra of these species give well‐resolved doublets for the intra‐ and extracellular spaces, and can be used to measure the pH difference across the cell membrane with errors on the order of about 0.01 pH units in the range of pH 5–7. Variations in erythrocyte pH due to metabolic changes are clearly observed. Magn Reson Med 50:693–696, 2003. © 2003 Wiley‐Liss, Inc.     

缺血及再灌注大鼠心肌细胞内pH的~（31）P核磁共振论著测定

建立了用３１Ｐ－核磁共振（３１Ｐ－ＮＭＲ）非损伤性测定大鼠心肌细胞内ｐＨ（ｐＨｉ）的方法，并用该方法研究了Ｌａｎｇｅｎｄｏｒｆ灌流大鼠心脏在常温及低温缺血以及再灌注过程中ｐＨｉ的动态变化过程。结果发现，正常灌流情况下Ｌａｎｇｅｎｄｏｒｆ大鼠心脏的ｐＨｉ为７．０７±０．０５（ｎ＝１６），缺血后ｐＨｉ逐渐下降。常温缺血３０ｍｉｎ后大鼠心肌的ｐＨｉ降至５．４０±０．１６（ｎ＝６）；低温缺血３０和９０ｍｉｎ后ｐＨｉ分别降至６．６９±０．０５和５．８８±０．１４（ｎ＝６）。两种缺血条件下心脏经再灌注后ｐＨｉ均迅速恢复至缺血前的水平。     

The role of Na+/K+ ATPase activity during low flow ischemia in preventing myocardial injury: A 31P, 23Na and 87Rb NMR spectroscopic study

An increase in intracellular Na⁺ during ischaemia has been associated with myocardial injury. In this study, we determined whether inhibition of Na⁺/K⁺ ATPase activity contributes to this increase and whether Na⁺/K⁺ ATPase activity can be maintained by provision of glucose to perfused rat hearts during low flow, 0.5 ml/min, ischemia. We used ³¹P NMR spectroscopy to determine changes in myocardial energetics and intracellular and extracellular volumes. ²³Na NMR spectroscopy, with DyTTHA^3- present as a shift reagent, was used to measure changes in intracellular Na⁺ and ⁸⁷Rb NMR spectroscopy was used to estimate Na⁺/K⁺ ATPase activity from Rb⁺ influx rates, Rb⁺ being an NMR-sensitive congener of K⁺. In hearts provided with 11 mM glucose throughout ischemia, glycolysis continued and ATP was twofold higher than in hearts without glucose. In the glucose-hearts, Rb⁺ influx rate was threefold higher, intracellular Na⁺ was fivefold lower at the end of ischemia and functional recovery during reperfusion was twofold higher. We propose that continuation of glycolysis throughout low flow ischemia allowed maintenance of sufficient Na⁺/K⁺ ATPase activity to prevent the increase in intracellular Na⁺ that would otherwise have led to myocardial injury.     

Simultaneous monitoring of coronary blood flow and 31P NMR detected myocardial metabolites

We report a method of dynamically measuring coronary blood flow in lambs, while simultaneously monitoring cardiac phosphate metabolism with 31P NMR at 81 MHz. This method uses an ultrasonic transit time probe in conjunction with a 4.7-T CSI spectrometer with a 33-cm magnet bore.     

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.     

Human focal cerebral ischemia: evaluation of brain pH and energy metabolism with P-31 NMR spectroscopy.

The authors investigated early human focal ischemia with phosphorus-31 nuclear magnetic resonance spectroscopy at 1.89 T to characterize the temporal evolution and relationship of brain pH and phosphate energy metabolism. Data from 65 symptomatic patients were prospectively studied; none of the patients had had ischemic stroke in the internal carotid artery territory before. Twenty-eight neurologically normal individuals served as control subjects. Serial ischemic brain pH levels indicated a progression from early acidosis to subacute alkalosis. When acidosis was present there was a significant elevation in the relative signal intensity of inorganic phosphate (Pi) and significant reductions in signal intensities of alpha-adenosine triphosphate (ATP) and gamma-ATP compared with those of control subjects. Ischemic brain pH values directly correlated with the relative signal intensity of phosphocreatine (PCr) and the PCr index and inversely correlated with the signal intensity of Pi. There was a general lack of correlation between either ischemic brain pH or phosphate energy metabolism and the initial clinical stroke severity. The data suggest a link between high-energy phosphate metabolism and brain pH, especially during the period of ischemic brain acidosis, and the authors propose that effective acute stroke therapy should be instituted during this period.     

Phospholipid profile of the human brain: 31P NMR spectroscopic study

Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, and ethanolamine plasmalogen represent the six most abundant phospholipids of brain cell membrane. The ratio of the phospholipid contents (phospholipid profile) of the brain is remarkably consistent under various metabolic conditions and alteration of the phospholipid profile is believed to reflect changes in the membrane system. We describe here a simple but sensitive method to analyze the phospholipid profile of the human brain utilizing the acidified chloroform-methanol lipid extraction method of Folch et al. and 31P nuclear magnetic resonance (NMR) spectroscopy. Unique regional phospholipid profiles were consistently obtained. Although the large chemical-shift anisotropy of the 31P confined to rigid structures such as the cell membrane precludes direct observation of phospholipid profiles in living tissue, a reflection of the membrane phospholipid profiles can nevertheless be obtained by studying "NMR visible" water soluble intermediate metabolites of membrane phospholipids in vivo.     

Epicardial and endocardial localized 31P magnetic resonance spectroscopy: evidence for metabolic heterogeneity during regional ischemia

Previous studies have noted that myocardial blood flow and high energy phosphates are heterogeneous across the myocardial wall during ischemia. In order to determine whether differences in metabolites between the subendocardium and subepicardium could be detected using 31P magnetic resonance spectroscopy, the Fourier series window (FSW) experiment was implemented on a porcine model of graded regional ischemia. FSW experiments using a planar phantom showed a 46% improvement in localization to the subendocardium compared to a one-pulse experiment. Animal studies of graded ischemia demonstrated a gradient in the phosphocreatine to inorganic phosphate ratio in the myocardium that paralleled the gradient in blood flow. These studies demonstrate the ability of spatially localized 31P magnetic resonance spectroscopy to detect regional changes in myocardial high energy phosphates localized to the subepicardium and subendocardium.     

Effect of 6-aminonicotinamide on the pentose phosphate pathway: 31P NMR and tumor growth delay studies

6-aminonicotinamide (6AN) has been shown to enhance radio-sensitivity in vitro, although previous in vivo studies failed to show an effect. ³¹P NMR spectra were obtained by using a one-dimensional chemical shift imaging technique on a first generation transplant of the CD8FI spontaneous mammary carcinoma tumor model. Spectra were obtained both before and 10 h after treatment with 6AN (20 mg/kg). Changes in pH, nucleoside triphosphate/inorganic phosphate, and phosphocreatine/inorganic phosphate measured at 10 h post-6AN were not significant. A new peak was detected 10 h post-6AN, which was assigned to 6-phosphogluconate (6PG), indicating inhibition of the pentose phosphate pathway (PPP). Based on the spectral data demonstrating inhibition of the PPP at 10 h post-6AN, tumor-bearing mice were irradiated (15 Gy × 3 fractions) on Days 1, 10 or 11, and 21 10 h after administration of 6-aminonicotinamide (20 mg/kg). Tumor-bearing mice receiving 6AN alone (20 mg/kg × 3), radiation alone (15 Gy × 3), or saline were also studied. Tumor growth delay studies indicated that 6AN alone induced a small but significant tumor growth delay (4.3 ± 0.8 days). Radiation alone induced a tumor growth delay of 34.5 ± 2.7 days. Treatment with 6AN followed by radiation induced a tumor growth delay of 57.0 ± 3.8 days. This was significantly greater than the TGD values for treatment with 6AN alone or radiation (P < 0.01). No complete regressions were noted after treatment with 6AN or radiation alone. Concomitant therapy with 6AN plus radiation yielded 6/28 complete regressions (21%), which was significantly greater than radiation (P < 0.05) or 6AN alone (P < 0.01) on this mammary carcinoma.     

The water resonance as an alternative pH reference: relevance to in vivo 31P NMR localized spectroscopy studies.

pH measurements require a suitable pH reference within the 31P NMR spectrum with respect to which the chemical shift of Pi, and hence pH, may be calculated. In muscle spectra PCr is prominent and provides a reference frequency. However, recent localized tumor studies have reported the absence of PCr, for example, in breast tumors. The use of the alpha-, beta-, and gamma-ATP peaks as suitable references has been suggested, but the position of the beta- and gamma-ATP peaks is dependent upon the intracellular Mg2+ concentration. The alpha-ATP is not affected by ionic concentrations; however, it contains UDPG and NAD+, the presence of which can lead to peak-shape distortion. This paper considers the use of the H2O resonance from the proton spectrum used for shimming as a suitable pH reference, provided this is also localized to the same region of interest, using a sequence giving rise to eddy current effects comparable to those of the 31P NMR sequence. Localized in vivo measurements in the muscle and brain of volunteers indicate good agreement between the proton and phosphorus chemical shifts, allowing the PCr position to be predicted to within 0.01 ppm in all cases.