Functional and metabolic evaluation of the hypertrophied heart using MRI and31P-MRS

4. Conclusions Diastolic LV function and myocardial HEP metabolism are impaired only when LVH is caused by permanent pressure or volume overload, and not by a temporary increase in cardiac workload during part of the day as in elite athletes. Therefore, training-induced and pressure/volume-overload-induced LVH seem to represent different phenotypes of LVH, possibly related to genetic reprogramming which only occurs during permanent cardiac overload [17]. Moreover, there is an association between impaired LV diastolic function and altered myocardial HEP metabolism in patients with hypertension and in patients with aortic valve disease. Finally we did not find a correlation between myocardial HEP metabolism and LV mass in any of the groups studied. The latter indicates that LVH should be regarded as an epiphenomenon to cardiac overload, and not as a primary factor causing abnormal HEP metabolism.     

Mechanisms of31P relaxation in phosphorus metabolites

Measurements have been made of the longitudinal relaxation timeT ₁ of³¹P for the individual resonances of the metabolites AMP, ADP, ATP, P_ir and PCr (phosphocreatine) in H₂O and D₂O solutions from 5 to 60°C at various concentrations and at frequencies of 40 MHz (2.3 T) and 120 MHz (7 T). The contributions of dipolar, chemical shift anisotropy, and spin-rotation mechanisms have been separated, and activation parameters of the underlying molecular reorientations have been determined.     

Combined 1H and 31P MR spectroscopic imaging: impaired energy metabolism in severe carotid stenosis and changes upon treatment

OBJECT: To evaluate if combined (1)H and (31)P MR spectroscopic imaging (MRSI) before and after treatment of severe internal carotid artery (ICA) stenosis detects significant changes in energy metabolism in the basal ganglia of both hemispheres. MATERIALS AND METHODS: A group of 14 patients with high-grade ICA stenosis and 11 healthy control subjects were examined with 2D (1)H MRSI and 3D (31)P MRSI at 3 T before and after treatment of severe ICA stenosis. Spectroscopic data were processed with LCModel and jMRUI software. Changes of the phosphorylated metabolites, pH, N-acetyl-acetate, creatine and choline-containing compounds prior/post intervention were analyzed and patients' data were compared with that of control subjects. RESULTS: Untreated patients had significantly higher Adenosindiphosphate (ADP) in basal ganglia ipsi- and contralateral to the side of ACI stenosis compared to controls. After treatment, ADP of both hemispheres significantly decreased by approximately 20% compared to the pre-treatment values. Further, significant decreases of phosphorylated metabolites prior/post intervention were found for patients compared to controls. CONCLUSION: This spectroscopic study reveals that unilateral high-grade ICA stenosis has an effect on cerebral high-energy metabolism of both hemispheres, which is at least partially reversible after treatment. Therefore the restoration of blood flow in high-grade ICA stenosis recovers the impaired energy balance of the brain.     

The magnitude of signal errors introduced by ISIS in quantitative31P MRS

It is well known that the quality of a quantitative³¹P MRS measurement relies largely on the performance of the volume selection method, and that image selected in vivo spectroscopy (ISIS) suffers from contaminating signal caused mostly by Tl smearing. However, these signal errors and their magnitude are seldom addressed in clinical studies. The aim of this study was therefore to investigate the magnitude of signal errors in³¹P MRS when using ISIS. The results from the measurements with a homogeneous head phantom are as follows: at low TR/T1 ratios the contamination increases rapidly, especially for small (< 27 cm³) VOI sizes; at TR/T1 = 1, the signal from a 27 cm³ VOI was 20% too high, and from an 8 cm³ VOI 150% too high. The signal obtained from different VOI positions varied between 80 and 127%. The signal varied linearly with the³¹P concentration in the object. However, a too high signal was obtained when the concentration was lower in the region of interest (inner container) than in the rest of the phantom. The agreement between the simulations and measurements shows that the results of this study are generally applicable to the measurement geometry and the ISIS experiment order rather than being specific for the MR system studied. The errors obtained both experimentally and in computer simulations are too large to be ignored in clinical studies using the ISIS pulse sequence.     

Reduced purine catabolite production in the postischemic rat heart: a31P NMR assessment of cytosolic metabolites

Ischemia can cause release of adenosine and purine catabolites from the heart, through the breakdown of ATP. If repeated periods of ischemia are induced, the efflux of purines is markedly reduced, although it is not clear if this is beneficial for the long-term survival of the heart. We have investigated changes in high-energy phosphates and purine release in the isolated perfused rat heart using³¹P NMR spectroscopy and high-performance liquid chromatography. Hearts were subjected to one of the following protocols: Group A—1 min of total global ischemia (TGI) after 40 min, 60 min, and 85 min of perfusion (a total of 3 × 1 min ischemia); Group B—1 min of TGI after 40 min of perfusion, 10 min of TGI after 50 min of perfusion, and a final 1 min of TGI after 85 min of perfusion. The profile of high-energy phosphate metabolites, P_i accumulation and purine release was similar for each 1-min period of TGI in Group A, whereas phosphocreatine content was increased and ATP content reduced by an extended period of TGI in Group B, leading to a less severe acidosis and purine efflux in the final 1 min of TGI at 85 min of perfusion. In conclusion, the reduced purine release observed in Group B may be related to the preischemic ATP pool size and accessibility and the increased myocardial energy reserve in the form of phosphocreatine.     

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 lowexpression 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–α (IFN-α). 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 0.011μM VBL alone (87% vs. 102% survival) or to 0.011μM BL + 10μM 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.This revised version was published online in August 2005 with a corrected sequence of authors.     

Experimental31p nmr study of the influence of ionic strength on the apparent dissociation constant of mgatp

The classical method for³¹P NMR determination of intracellular free magnesium concentration ([Mg _free ²⁺ ]) requires an accurate knowledge of the apparent dissociation constant (K _D ) of MgATP. There is a large difference between the previously determined values ofK _D . Although the value of 50 µM, determined by a³¹P NMR method, is now largely accepted, a value of 86 µM has more recently been measured with a fitting method derived from the original one, and with a different ionic strength. The purpose of our study was to assess if the cause of the difference between these two previously reportedK _D values was due to the measuring method or to the ionic strength value used.Working at pH=7.2,T=37°C, and [KCl]=0.25 M, we performedK _D measurements with the original³¹P NMR method and with the fitting method. The results (67±13 µM and 61±20 µM, respectively) were not significantly different. Then, with the first method, we measured K_D at [KCl]=0.12 M and found a value of 19±5 µM. We conclude that the main cause of difference between theK _D values measured by³¹P NMR reside in the disparity of ionic strength values used for their measurement. OurK _D measurements at [KCl]=0.25 and 0.12 M demonstrate the importance of the ionic strength value used for imitating the intracellular medium on the absolute value of ([Mg _free ²⁺ ]) measured by³¹P NMR spectroscopy.Address for correspondence: Université Catholique de Louvain, Unité CPMC, Bâtiment Lavoisier, Place Louis Pasteur n°1, B-1348 Louvain-la-Neuve, Belgium. Additional reprints of this chapter may be obtained from the Reprints Department, Chapman & Hall, One Penn Plaza, New York, NY 10119.     

Glycolytic atp production estimated from31p magnetic resonance spectroscopy measurements during ischemic exercisein vivo

In an oxygen-depleted muscle, glycolytically produced ATP is inversely related to the ([ATP] + creatine phosphate [PCr]) decrease because ATP, PCr, and glycolysis are virtually the only energy sources under these conditions. In particular, the onset of glycolysis or any appreciable increase in the rate of glycolytic ATP production will lead to a slower rate of ([ATP] + [PCr]) breakdown at a given energy consumption. To quantify this relationship, endurance athletes performed isometric foot plantar flexion (20% of a test force [TF],n=10; 50% TF,n=5) during local arterial occlusion. Parameters of energy metabolism were measured with³¹P magnetic resonance spectroscopy (³¹P-MRS). During exercise, [PCr] decreased to 80±10 (20% TF) and 11±4% (50% TF) of its resting concentration, and pH dropped from 7.04 0.01 to 6.98±0.10 (20% TF) and from 7.03±0.02 to 6.70 0.10 (50% TF). In both experiments, two phases of ([ATP] + [PCr]) decrease were observed: an initial faster decrease was followed by a slower decline. The latter phase started at about the time when the pH began to drop. The difference between a line extrapolated from the slope of the initial phase and the measured ([ATP] + [PCr]) decrease was used as an estimate for glycolytically produced ATP. This estimate and pH were significantly correlated withr=–0.97 (20% TF) andr=–0.99 (50% TF). These results indicate that glycolytically produced ATP can be estimated from the ([ATP] + [PCr]) decrease during exercise.     

Physiological constraints on changes in pH and phosphorus metabolite concentrations in ischemically exercising muscle: implications for metabolic control and for the interpretation of31P-magnetic resonance spectroscopic studies

Relationships between pH and the concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), and lactate during ischemic exercise depend on passive buffering, proton consumption as a consequence of net PCr breakdown, the control of glycogenolysis, (particularly in relation to the concentration of Pi, a substrate of glycogen phosphorylase that is produced by net PCr breakdown), and the creatine kinase equilibrium. The author analyzes the implications of these relationships for the interpretation of³¹P-magnetic resonance spectroscopic data and for the control of glycogenolysis. For realistic adenosine diphosphate (ADP) concentrations, given the constraints of the creatine kinase equilibrium, the pH must be near-linear with lactate, with an apparent buffer capacity (i.e., the ratio of lactate accumulation to pH change) that is nearly twice the true buffer capacity (i.e., the ratio of net proton loading to pH change). The implications for glycogenolytic control depend on adenosine triphosphate (ATP) turnover, but an upper limit of activation of glycogen phosphorylase (i.e., the amount of thea form) that would permit no increase in ADP concentration can be calculated. Phosphorylase activation during ischemic exercise seems approximately proportional to the power output, consistent with calcium stimulation of phosphorylaseb kinase. In simulations, ADP concentration is highly sensitive to this proportionality, as (unlike in purely oxidative exercise) ADP concentration is not known to participate in any closed feedback loops in ischemic exercise.     

Signal enhancement through heteronuclear polarisation transfer in in-vivo <Superscript>31</Superscript>P MR spectroscopy of the human brain

Significant ³¹P NMR signal enhancement through heteronuclear polarisation transfer was obtained in model solutions and in vivo on a 1.5-T whole-body MR scanner equipped with two RF channels. The much higher population differences involved in proton Zeeman energy levels can be transferred to the ³¹P levels with the refocused INEPT (insensitive nucleus enhancement by polarisation transfer) double-resonance experiment by means of a series of simultaneously applied broadband RF pulses. INEPT achieves a polarisation transfer from ¹H to ³¹P spin states by directly reordering the populations in spin systems with heteronuclear scalar coupling. Thus, only the ³¹P NMR signal of metabolites with scalar ¹H–³¹P coupling is amplified, while the other metabolite signals in the spectra are suppressed. Compared to Ernst-angle excitation, a repetition-time-dependent signal enhancement of η=(29±3)% for methylene diphosphonic acid (MDPA) and η=(56±1)% for phosphorylethanolamine (PE) was obtained on model solutions through optimisation of the temporal parameters of the pulse experiment. The results are in good agreement with numerical calculations of the theoretical model for the studied spin systems. With optimised echo times, in-vivo ³¹P signal enhancement of the same order was obtained in studies of the human brain.     

31P MR spectroscopy and in vitro markers of oxidative capacity in type 2 diabetes patients

Background: Skeletal muscle mitochondrial function in type 2 diabetes (T2D) is currently being studied intensively. In vivo ³¹P magnetic resonance spectroscopy (³¹P MRS) is a noninvasive tool used to measure mitochondrial respiratory function (MIFU) in skeletal muscle tissue. However, microvascular co-morbidity in long-standing T2D can interfere with the ³¹P MRS methodology. Aim: To compare ³¹P MRS-derived parameters describing in vivo MIFU with an in vitro assessment of muscle respiratory capacity and muscle fiber-type composition in T2D patients. Methods: ³¹P MRS was applied in long-standing, insulin-treated T2D patients. ³¹P MRS markers of MIFU were measured in the M. vastus lateralis. Muscle biopsy samples were collected from the same muscle and analyzed for succinate dehydrogenase activity (SDH) and fiber-type distribution. Results: Several ³¹P MRS parameters of MIFU showed moderate to good correlations with the percentage of type I fibers and type I fiber-specific SDH activity (Pearson’s R between 0.70 and 0.75). In vivo and in vitro parameters of local mitochondrial respiration also correlated well with whole-body fitness levels (VO _2peak) in these patients (Pearson’s R between 0.62 and 0.90). Conclusion: Good correlations exist between in vivo and in vitro measurements of MIFU in long-standing insulin-treated T2D subjects, which are qualitatively and quantitatively consistent with previous results measured in healthy subjects. This justifies the use of ³¹P MRS to measure MIFU in relation to T2D.     

31P-NMR spectroscopy of human blood and serum: first results from volunteers and patients with congestive heart failure,diabetes mellitus and hyperlipidaemia

³¹P-containing metabolites in human blood, serum and erythrocytes were measured or calculated. Phosphodiesters were found in serum, but not in erythrocytes. 2,3-diphosphoglycerate and 2,3-diphosphoglycerate/ATP ratios were increased in patients with congestive heart failure (2,3-diphosphoglycerate by 13% in mild to moderate, 31% in severe congestive heart failure, 2,3-diphosphoglycerate/ATP ratio by 9% in mild to moderate, 38% in severe congestive heart failure); phosphodiesters were increased in diabetes mellitus (by 26%) and even more so in hyperlipidaemia (by 57%). Changes of blood³¹P compounds with disease states may have diagnostic potential and should be recognized for correction of organ spectra.     

Use of spin-traps during warm ischemia-reperfusion in rat liver: comparative effect on energetic metabolism studied using31P nuclear magnetic resonance

Detection of free radicals by electron spin resonance (ESR) proves the involvement of reactive oxygen species (ROS) in reperfused organ injuries. Spin-traps are known to ameliorate hemodynamic parameters in an isolated postischemic heart. The effects of 5 mmol/L DMPO (5,5-dimethyl-1-pyrroline-N-oxide) or DEPMPO (5-(diethylphosphoryl)-5-methyl-1-pyrrolineN-oxide) on intracellular pH (pH_in) and ATP level were evaluated by³¹P nuclear magnetic resonance on isolated rat liver submitted to 1 hour of warm ischemia and reperfusion. At the end of the reperfusion period, during which pH_in recovered to its initial value (7.16±0.03) in all groups, the ATP recovery level (expressed in percentage of initial value) was similar in controls and DEPMPO (60%±5%,n=6 and 54%±4%,n=6, respectively), but only 37%±1% in DMPO-treated livers (n=6) (p<0.05 versus controls andp<0.05 versus DEPMPO). Oxidative phosphorylation was not affected by an addition of nitrones on isolated mitochondria extracted from livers not submitted to ischemia-reperfusion. In contrast, mitochondria extracted at the end of the ischemia-reperfusion showed an impairment in the phosphorylation parameters, particularly in the presence of DMPO. Mass spectrum of ischemic liver perchloric acid extracts evidenced probable catabolites in treated groups. The differences in the effect of the two nitrones on energetic metabolism may be explained by the production of deleterious catabolites by DMPO as compared to DEPMPO. Even though a specific radical scavenging effect could be operative in the liver, our results indicate that catabolic effects were predominant. The absence of deleterious effects of DEPMPO in contrast to DMPO on the liver energetic metabolism was evidenced, allowing the use of DEPMPO for ESR detection.     

Multinuclear MR-spectroscopy on ion-homeostasis and energetics during ischemia and reperfusion

4. Conclusion Voltage-gated Na⁺-channels constitute a major port of Na⁺ entry in the initial minutes following the onset of ischemia. The persistent component of the Na⁺ current does not play a significant role in the guinea pig heart even thereafter. Due to intracellular acidification. NHE is activate dand forms the dominant influx pathway in the next 30 min of ischemia, but is partially inactivated in the later course of ischemia. Alternative, not yet characterised ports of entry gain importance in this phase. Blocking either voltage-gated Na⁺-channels or NHE improved post-ischemic contractile function. Thus reducing Na⁺-overload in ischemia (and reperfusion) is a promising therapeutical aporoach for cardio-protection.     

Sodium ion distribution in the vitreous body

We have studied the nuclear magnetic resonance (NMR) relaxation behavior, and thus the dynamic properties, of the sodium ion in the vitreous body at different temperatures. The²³Na NMR spectrum exhibits a resonance, the intensity of which accounts for an ion visibility of 100%. The²³Na longitudinal and transverse relaxation times, at all temperatures but the highest, present two components, suggesting that the sodium ions are present in two states of different mobility, whose populations are in slow exchange on the NMR time scale. The correlation times and quadrupole coupling constants for the two sodium pools have been derived. The faster relaxation of a fraction of the vitreal sodium has tentatively been ascribed to the influence of the macromolecular framework of the vitreous body. The reported information may be of use for the understanding of the diagnostic applications of²³Na magnetic resonance imaging of the ocular structures.     

Temporal analysis of valence & electrostatics in ion-motive sodium pump

The present work establishes a unique framework for the simulation study of ion-motive pumps in general and the Na⁺/K⁺-ATPase, or Na⁺ pump, in particular. We shall discuss the implications of electrostatic analysis, valence calculations, and protein cavity data, each carried over data extracted from molecular dynamics (MD) simulations, on the structure-function relationship of Na⁺/K⁺-ATPase. This diverse set of tools will be used to investigate atomic-level characteristics that remain undetermined such as ion binding and accessibility.     

Large increases in1H metaboliteT 2's after cerebral hypoxia-ischemia correlate with ATP depletion

In vivo proton (¹H) magnetic resonance spectroscopy (MRS) can measure cerebral metabolite concentrations and nuclear relaxation times. Function of the sodium (Na⁺)/potassium (K⁺) pump in cell membranes depends on adequate adenosine triphosphate (ATP) levels: intracellular Na⁺ is normally extruded in exchange for extracellular K⁺. Low ATP will cause pump dysfunction and loss of K⁺ accompanied by influx of Na⁺and water. Raised intracellular water may increase molecular mobility and this might be detectable as increased apparent transverse relaxation times (T ₂'s).¹H-MRS of the brains of newborn piglets during acute hypoxia-ischemia revealed enigmatic increases in the peak area of creatine + phosphocreatine (Cr) relative to those of choline-containing compounds (Cho) andN-acetylaspartate (NAA). Interleaved¹H and phosphorus (³¹P) MRS showed that theT ₂'s of both Cr and lactate (Lac) increased during acute hypoxia-ischemia and these changes correlated with reductions in nucleotide triphosphate (NTP; largely ATP). Within 50 h of metabolic recovery from the primary insult, as delayed energy failure developed, theT ₂'s of Cho, Cr, NAA, and Lac increased greatly. TheseT ₂ changes also correlated with NTP depletion. These observations demonstrate important relationships betweenT ₂'s and function of the ATP-dependent Na⁺/K⁺ pump.     

In vivo1H-MR spectroscopy of the human heart

4. Conclusions Combined respiratory and cardiac triggering improves the localization accuracy and spectral quality in cardiac¹H-MRS dramatically leading to substantially increased spectral reproducibility. The best practical realization of double triggering turned out to be the use of the ECG amplitude when making use of the fact that it is modulated by respiration. In spite of the spectral quality achieved in most subjects, we still fail to record satisfactory spectra in a minority of subjects. The reasons for this are not understood at present but must be some particulars of either a given subject or the experimental setup. The cardiac¹H-MR spectra contain quantifiable contributions from creatine, TMA, lipids, and probably taurine. It is possible that the spectral contributions of creatine are subject to dipolar coupling similar to the observations for skeletal muscle.     

Exploration of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase ion permeation pathways <Emphasis Type="Italic">via</Emphasis> molecular dynamic simulation and electrostatic analysis

Biologically-inspired nanodevices can serve as “natural” alternatives to conventional semiconductor devices in many applications from information storage to mechanical rotors. In this work we consider an ATP-powered transmembrane protein, the Na⁺,K⁺-ATPase, which has appealing functionality but still lacks an “atomistic” picture capable of elucidating its operation. The vast collection of experimental literature on the Na⁺,K⁺-ATPase gives a unique advantage to this protein in developing and validating computational tools. We have performed extensive molecular dynamic simulations of the Na⁺,K⁺-ATPase in an accurate biological environment, followed by time-averaged electrostatic analysis, to investigate the ion-binding loci and access/egress pathways that cations may take through the protein as they are transported across the membrane.