On neglecting chemical exchange when correcting in vivo (31)P MRS data for partial saturation: commentary on: "Pitfalls in the measurement of metabolite concentrations using the one-pulse experiment in in Vivo NMR".

This article replies to Spencer et al. (J. Magn. Reson. 149, 251--257, 2001) concerning the degree to which chemical exchange affects partial saturation corrections using saturation factors. Considering the important case of in vivo (31)P NMR, we employ differential analysis to demonstrate a broad range of experimental conditions over which chemical exchange minimally affects saturation factors, and near-optimum signal-to-noise ratio is preserved. The analysis contradicts Spencer et al.'s broad claim that chemical exchange results in a strong dependence of saturation factors upon M(0)'s and T(1) and exchange parameters. For Spencer et al.'s example of a dynamic (31)P NMR experiment in which phosphocreatine varies 20-fold, we show that our strategy of measuring saturation factors at the start and end of the study reduces errors in saturation corrections to 2% for the high-energy phosphates.

On neglecting chemical exchange effects when correcting in vivo (31)P MRS data for partial saturation.

Signal acquisition in most MRS experiments requires a correction for partial saturation that is commonly based on a single exponential model for T(1) that ignores effects of chemical exchange. We evaluated the errors in (31)P MRS measurements introduced by this approximation in two-, three-, and four-site chemical exchange models under a range of flip-angles and pulse sequence repetition times (T(R)) that provide near-optimum signal-to-noise ratio (SNR). In two-site exchange, such as the creatine-kinase reaction involving phosphocreatine (PCr) and gamma-ATP in human skeletal and cardiac muscle, errors in saturation factors were determined for the progressive saturation method and the dual-angle method of measuring T(1). The analysis shows that these errors are negligible for the progressive saturation method if the observed T(1) is derived from a three-parameter fit of the data. When T(1) is measured with the dual-angle method, errors in saturation factors are less than 5% for all conceivable values of the chemical exchange rate and flip-angles that deliver useful SNR per unit time over the range T(1)/5 < or = T(R) < or = 2T(1). Errors are also less than 5% for three- and four-site exchange when T(R) > or = T(1)(*)/2, the so-called "intrinsic" T(1)'s of the metabolites. The effect of changing metabolite concentrations and chemical exchange rates on observed T(1)'s and saturation corrections was also examined with a three-site chemical exchange model involving ATP, PCr, and inorganic phosphate in skeletal muscle undergoing up to 95% PCr depletion. Although the observed T(1)'s were dependent on metabolite concentrations, errors in saturation corrections for T(R) = 2 s could be kept within 5% for all exchanging metabolites using a simple interpolation of two dual-angle T(1) measurements performed at the start and end of the experiment. Thus, the single-exponential model appears to be reasonably accurate for correcting (31)P MRS data for partial saturation in the presence of chemical exchange. Even in systems where metabolite concentrations change, accurate saturation corrections are possible without much loss in SNR.

Broadband proton decoupling for in vivo brain spectroscopy in humans.

A new decoupling sequence, PBAR, is described for broadband heteronuclear decoupling in vivo in humans at 1.5T. The sequence uses non-adiabatic, frequency- and amplitude-modulated inversion pulses designed to minimize decoupling sidebands at low applied gammaB(2) RF field levels and to cover only the narrow range of resonance offsets encountered in practice. The offset dependence of the decoupling efficiency of PBAR is demonstrated and compared to the conventional WALTZ-4 sequence. At the same average power levels, PBAR had slightly reduced bandwidth but significantly less intense decoupling sidebands. Applications of PBAR are shown in vivo in the human brain both for (31)P and natural abundance (13)C spectroscopy using volume decoupling coils. The PBAR sequence allows whole brain [(1)H]-[13]C decoupling to be performed at 1.5T with a standard head coil within FDA guidelines for RF power deposition. Magn Reson Med 45:226-232, 2001.

Mitral regurgitation: impaired systolic function, eccentric hypertrophy, and increased severity are linked to lower phosphocreatine/ATP ratios in humans.

BACKGROUND: A number of phosphorus (31P) magnetic resonance spectroscopy (MRS) studies link alterations of high-energy phosphate metabolism in valvular disease and cardiomyopathy to the clinical severity of heart failure. However, correlations between MRS and indexes of ventricular dysfunction are inconclusive to date. We examined whether changes in 31P MRS are associated with the impaired contractility, which predisposes to chronic congestive heart failure in patients with mitral regurgitation. METHODS AND RESULTS: Thirteen normal control subjects and 22 patients with echocardiographically characterized chronic mitral regurgitation were studied by 31P MRS. The apical phosphocreatine-to-ATP ratio (PCr/ATP) was lower in severe disease (P<.02) and those on therapy (n=13, 1.29+/-0.29, P<.01) in contrast to control subjects (n=13, 1.61+/-0.3). Compared to those with mild mitral regurgitation, patients with more severe incompetence had lower mean myocardial PCr/ATP ratios (mild, n=6, 1.73 [0.17], P<.05 and P<.01; moderate, n=5, 1.49 [0.18], P<.05; and severe, n=1, 1.29 [0.32], P<.01). PCr/ATP in those referred for mitral valve replacement was lower (n=8, 1.17+/-0.23) although not significantly decreased compared with the ratio among subjects on medical therapy alone (n=5, 1.48+/-0.29). PCr/ATP correlated with the end-systolic diameter (r2=.7, P<.001), end-diastolic diameter (r2=.32, P<.05), left ventricular wall thickness (r2=.38, P<.01), left atrial dimension (r2=.36, P<.05), and derived measurements such as the percent fractional shortening (2=.5, P<.01), and left ventricular mass/body surface area (r2=.5, P<.001) but not with wall stress. CONCLUSIONS: These results demonstrate that abnormalities of PCr/ATP in mitral regurgitation are related to disease severity as measured by dimensional indexes of left ventricular dilatation. They suggest that impaired high-energy phosphate metabolism is a marker of hypertrophy and heart failure.

Metabolic response of normal human myocardium to high-dose atropine-dobutamine stress studied by 31P-MRS.

BACKGROUND: 31P-MRS during cardiac stress may provide (patho)physiological insights into the high-energy phosphate metabolism of the myocardium. Accordingly, the purpose of the present study was to determine the metabolic response of normal human myocardium to severe atropine-dobutamine (A-D) stress. To corroborate the results from the present in vivo study, a 31P-MRS experiment was performed with a moving phantom to simulate respiratory motion. METHODS AND RESULTS: The phantom experiment showed no relation (P=.371) between the intensity ratio of two separate phosphate peaks and amplitude of phantom excursions. The phosphocreatine (PCr) and ATP signal strength and the PCr/ATP ratio were determined from the left ventricular wall in 20 healthy subjects (posttest likelihood for coronary artery disease was <2.5%) with 31P-MRS at rest and during high-dose A-D stress (rate-pressure product increased threefold). Stress-induced changes were -21% for PCr (P<.001) and -9% for ATP (P<.05). The average PCr/ATP value at rest was 1.42+/-0.18 and decreased by 14% to 1.22+/-0.20 during stress (P<.001). CONCLUSIONS: The phantom experiment shows that the in vivo decrease of myocardial PCr/ATP due to high-dose A-D stress we observed is not a motion artifact. Consequently, this indicates that myocardial high-energy phosphate metabolism of the normal human heart is altered at high workloads.

Optimum flip-angles for exciting NMR with uncertain T1 values.

T1 is often ill-determined. This means that an Ernst angle excitation often cannot be precisely defined for the simple pulse and acquire experiment. Here, published 31P T1 values of metabolites in human muscle, liver, heart, and brain are archived, some new data on heart and brain added, and overall confidence intervals determined. Strategies for setting the flip-angle based on the confidence intervals are examined, and an optimum flip-angle derived which minimizes the signal loss relative to what could have been realized if T1 were precisely known. With such optimized pulses, signal loss can be limited to < or = 14% for up to a 10-fold variation in T1, with TR < or = T1. The effect that an uncertainty in T1 by a factor of two has on the saturation corrected signal is limited to < or = 20% in the optimum flip-angle experiment. Adiabatic B1-independent rotation phase-cycled (BIRP) excitation pulses are ideal as optimum flip-angle pulses as they can be prescribed without calibration.

Phosphorus-31 magnetic resonance spectra reveal prolonged intracellular acidosis in the brain following subarachnoid hemorrhage.

Subarachnoid hemorrhage may be complicated by cerebral ischemia which, though reversible initially, can progress to an irreversible neurological deficit. 31P magnetic resonance spectroscopy, which can determine intracellular pH and thus detect areas of ischemia noninvasively, was applied to 10 patients on 30 occasions, at various times after subarachnoid hemorrhage. In 5 of them, there were focal areas of the brain in which the intracellular pH was reduced to < 6.8 compared with the normal range of 7.05 +/- 0.05. Consciousness was impaired in 4 of these patients. Repeat studies in these 4 patients showed that intracellular pH remained abnormally low for several days but eventually returned toward normal. The return of intracellular pH to normal paralleled an improvement in clinical condition in each case. In the fifth patient with lowered regions of intracellular pH, there had been an impaired level of consciousness and a transient focal deficit prior to the single study. In the other 5 patients there were no areas of reduced pHi even though in 3 of them there was intraventricular or cisternal blood shown on brain computerized tomography. In 2 of these 3 patients there were no abnormal neurological signs at the time of the magnetic resonance study. The third patient had a dense and persistent hemiparesis. The remaining two patients had no abnormal neurological signs at any stage. We suggest that the areas of acidosis may reflect ischemia which is potentially reversible. Since the technique is noninvasive, sequential 31P magnetic resonance spectroscopy of the brain offers a method of detecting cerebral ischemia and, more importantly, of assessing methods of treatment.

Global and depth resolved phosphorus magnetic resonance spectroscopy to predict outcome after birth asphyxia.

Twelve normal and 32 asphyxiated neonates were studied using global and depth resolved phosphorus magnetic resonance spectroscopy (31PMRS). Eight of the asphyxiated group died or survived with major neurodevelopmental abnormalities. A global phosphocreatinine/inorganic phosphate (PCr/Pi) ratio below the range of values from normal infants predicted adverse outcome after asphyxia with a positive predictive value of 64%, sensitivity 88%, and specificity 83%. Corresponding values for global inorganic orthophosphate/adenosine triphosphate (Pi/ATP) ratios were positive predictive value 88%, sensitivity 96%, and specificity 88%. Spatially localised MRS data, obtained using phase modulated rotating frame imaging, showed cerebral energy metabolism to be more abnormal in deep than superficial regions after birth asphyxia. However, in this population of full term infants none of the regional metabolite concentrations were superior to global data for prediction of outcome.

Detection of low phosphocreatine to ATP ratio in failing hypertrophied human myocardium by 31P magnetic resonance spectroscopy.

Phosphorus-31 magnetic resonance spectroscopy can be used to study intracellular biochemistry non-invasively by measuring the relative proportions of high energy phosphates. Study of deteriorating cardiac metabolism might be useful in the management of hypertrophy and heart failure. 31P magnetic resonance spectroscopy was carried out in fourteen patients with aortic valve disease (six with aortic stenosis, eight with aortic incompetence). Six patients were receiving treatment for symptoms of heart failure. The phosphocreatine (PCr) to ATP ratio in these patients (1.1 [SD 0.32]) was significantly lower than that in thirteen controls (1.5 [0.2], p less than 0.001) or in the eight patients who did not have symptoms of heart failure (1.56 [0.15], p less than 0.0035). These findings indicate that heart failure in aortic valve disease is associated with low PCr, which could be due to loss of intracellular creatine. The measurement could eventually have a role in helping to determine the optimum timing for aortic valve replacement.

Erythrocyte Na+/K+ ATPase activity measured with 23Na NMR.

A 23Na NMR assay for measurement of erythrocyte Na+/K+ ATPase activity is presented. Using the nonpermeant shift reagent dysprosium tripolyphosphate the signals of intra- and extracellular sodium are separated, enabling measurement of sodium fluxes nondestructively, without the need to physically separate the cells from their environment. By increasing membrane permeability with nystatin we have shown that the assay allows the detection of differences in membrane permeability. With low doses of nystatin the ouabain-sensitive sodium flux increased more than twofold. With high doses of nystatin the Na+/K+ pump could not prevent an almost total equilibration of intra- and extracellular sodium. All sodium that entered the cells remained NMR visible, proving that sodium influx can be measured quantitatively. 31P NMR spectra taken before and after the assay revealed a slight acidification of the cells and no significant change in ATP concentration. No evidence of Dy3+ entering the cell was observed.

Hexose monophosphate shunt activity in erythrocytes related to cell age.

Erythrocytes were separated by age using a combination of density centrifugation and counterflow centrifugation and tested for basal activity of the hexose monophosphate shunt (HMP-shunt) as well as the methylene blue-stimulated maximal capacity by measuring CO2 production. No significant differences were found in basal HMP-shunt activity, but the maximal methylene blue-stimulated activity of old erythrocytes reached only half of the activity of the total cell population. The maximal HMP-shunt activity showed a significant correlation with hexokinase activity, but not with glucose-6-phosphate dehydrogenase activity in all but the youngest cells. The sensitivity to oxidative stress was tested by measuring the kinetics of pyruvate kinase isolated from erythrocytes incubated in presence and absence of methylene blue. Pyruvate kinase kinetics were affected more in the old cell population than in the total cell population: the K0.5 for phosphoenol-pyruvate increased four times in the unseparated cells and eight times in old cells.

Intracellular free magnesium and phosphorylated metabolites in hexokinase- and pyruvate kinase-deficient red cells measured using 31P-NMR spectroscopy.

The erythrocyte metabolism of two patients with nonspherocytic hemolytic anemia caused by a hexokinase deficiency, and a pyruvate kinase deficiency, respectively, were studied with NMR. The complexing of ATP and 2,3-diphosphoglycerate (2,3-DPG) with Mg2+ and hemoglobin (Hb) was determined using 31P-NMR on oxygenated and deoxygenated cells to investigate the influences of these enzyme defects on intracellular magnesium distribution and on Hb oxygen dissociation. In the pyruvate kinase-deficient red blood cells, the 2,3-DPG concentration was almost twice the normal value and the ATP concentration was near the lower limit of the normal range. In the hexokinase-deficient red cell population, the predominance of young cells masked the deficiency. Therefore, reticulocyte control cells were included in this study. In the oxygenated pyruvate kinase-deficient cells, the fraction of ATP that is complexed to magnesium as well as the free Mg2+ concentration were normal, despite the abnormal concentration of 2,3-DPG. In the deoxygenated cells the free Mg2+ concentration was lower than in normal cells. The fraction of Hb complexed with 2,3-DPG was higher than normal in both oxygenated and deoxygenated pyruvate kinase-deficient cells, in accordance with the high p50 of the oxygen-hemoglobin dissociation curve. In hexokinase-deficient cells, two major abnormalities are found: when the cells were deoxygenated, the concentration of ATP and 2,3-DPG fell. This was not observed for any other sample and could, therefore, be a consequence of the hexokinase deficiency. Despite almost normal levels of magnesium-binding metabolites, the free Mg2+ concentration in oxygenated and deoxygenated cels is much lower than in normal cells. This could be a cell-age-related phenomenon, since lower free Mg2+ concentrations were also found in reticulocyte control cells.

Distribution of phosphorylated metabolites and magnesium in the red cells of a patient with hyperactive pyruvate kinase.

The intracellular distribution of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) was studied in the red cells of a patient with a "high-ATP syndrome" by using 31P nuclear magnetic resonance. In this patient, red cell ATP was increased 2.5-fold, whereas 2,3-DPG was decreased fourfold due to the presence of a hyperactive pyruvate kinase. In oxygenated red cells, these abnormal concentrations were reflected to the same extent in all complexes in which ATP and 2,3-DPG take part. The diminished amount of 2,3-DPG bound to hemoglobin was almost completely replaced by ATP-hemoglobin complexes. Therefore, free hemoglobin was only slightly increased. In deoxygenated cells, the relative distribution of ATP and 2,3-DPG complexes was significantly disturbed. The main difference was a shift in the ratio of magnesium ATP (MgATP) over the ATP-hemoglobin complex; 74% of total ATP was complexed to hemoglobin (45% in normal cells), whereas the concentration of MgATP was only slightly increased with respect to normal. The shortage in 2,3-DPG bound to hemoglobin could partially be replenished by an increase in hemoglobin (Mg) ATP complexes. Therefore, the amount of uncomplexed hemoglobin raised from 15% in normal cells to 38% in the patient's cells. As a result, the oxygen-dissociation curve was only moderately shifted to the left. It is concluded that the regulatory role of 2,3-DPG in oxygen transport is taken over in part by (Mg) ATP in this patient. In both aerobic and anaerobic cells, the increase in magnesium bound to ATP, either free or bound to hemoglobin, exceeds the decrease in 2,3-DPG Mg complex. In spite of this, the amount of intracellular free Mg++ was normal or slightly lowered. This suggests the presence of a compensatory mechanism by which the amount of total cellular magnesium could be increased.

Polyphosphoinositides undergo charge neutralization in the physiological pH range: a 31P-NMR study.

The charge state of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was determined as a function of pH by way of 31P-NMR spectroscopy. The pK values for the first protonation of the phosphomonoester residues in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were found to be 6.2 and 6.6, respectively, for the 4-phosphate moiety, and 7.7 for the 5-phosphate moiety.