Phosphocreatinine Research Articles

Association between dietary phosphate intake and skeletal muscle energetics in adults without cardiovascular disease.

Highly bioavailable inorganic phosphate (Pi) is present in large quantities in the typical Western diet and represents a large fraction of total phosphate intake. Dietary Pi excess induces exercise intolerance and skeletal muscle mitochondrial dysfunction in normal mice. However, the relevance of this to humans remains unknown. The study was conducted on 13 individuals without a history of cardiopulmonary disease (46% female, 15% Black participants) enrolled in the pilot-phase of the Dallas Heart and Mind Study. Total dietary phosphate was estimated from 24-h dietary recall (ASA24). Muscle ATP synthesis was measured at rest, and phosphocreatinine (PCr) dynamics was measured during plantar flexion exercise using 7-T 31P magnetic resonance (MR) spectroscopy in the calf muscle. Correlation was assessed between dietary phosphate intake normalized to total caloric intake, resting ATP synthesis, and PCr depletion during exercise. Higher dietary phosphate intake was associated with lower resting ATP synthesis (r = -0.62, P = 0.03), and with higher levels of PCr depletion during plantar flexion exercise relative to the resting period (r = -0.72; P = 0.004). These associations remain significant after adjustment for age and estimated glomerular filtration rate (both P < 0.05). High dietary phosphate intake was also associated with lower serum Klotho levels, and Klotho levels are in turn associated with PCr depletion and higher ADP accumulation post exercise. Our study suggests that higher dietary phosphate is associated with reduced skeletal muscle mitochondrial function at rest and exercise in humans providing new insight into potential mechanisms linking the Western diet to impaired energy metabolism.NEW & NOTEWORTHY This is the first translational research study directly demonstrating the adverse effects of dietary phosphate on muscle energy metabolism in humans. Importantly, our data show that dietary phosphate is associated with impaired muscle ATP synthesis at rest and during exercise, independent of age and renal function. This is a new biologic paradigm with significant clinical dietary implications.

98-LB: Effects of SGLT2 Inhibition on Skeletal Muscle Bioenergetics and Myocellular Lipids in Patients with T2D and HFrEF

Background: The CV benefits of SGLT2i are well established. However, how skeletal muscle (SkM) bioenergetics respond to SGLT2i in T2D and HFrEF remains largely unknown. Objective: To explore the effects of empagliflozin on SkM bioenergetics (phosphorus metabolites &amp; myocellular lipid content) at rest and post-exercise (31P &amp; 1H MRS) and cardiac function (MRI). Methods: Subjects with T2D &amp; HFrEF (&amp;lt;40%) (n=6) were randomized 2:1 to empagliflozin 25mg (SGLT2i) or placebo for 12 wks (Age=59±2.6, BMI=33.3±1.1, A1c=7.1±0.3, EF 28±4%). A cardiac MRI, &amp; static/dynamic 31P &amp; 1H SkM by MRS, were performed before &amp; after treatment. Results: In the empagliflozin group, SkM 31P MRS at rest, showed decrease in phosphodiesterase [PDE]. 31P MRS post-exercise, showed significant improvement in Phosphocreatinine (PCr) recovery, expressed by the K value. 1H MRS showed a reduction of saturated intramyocellular lipids (IMCL-CH2:CH3 ratio). Conclusion: Empagliflozin in subjects with T2D and HFrEF had beneficial effects in SkM energy utilization and muscle recovery post-exercise as determined by a decrease in PDE and increase in K value. Additionally, empagliflozin decreased saturated IMCL, which could be expected to improve mitochondrial function and energy utilization. If similar findings were to occur in cardiac muscle, this could provide a novel mechanism for the beneficial CV effects of SGLT2i. Disclosure Y. Qin: None. N. D. Sanchez: None. A. Moody: None. F. M. Acosta: None. S. Neppala: None. M. Brown: None. H. Honka: None. L. A. Cruz Moreno: None. C. L. Triplitt: Speaker's Bureau; Novo Nordisk. G. D. Clarke: None. E. Cersosimo: None. R. A. DeFronzo: Speaker's Bureau; AstraZeneca. Advisory Panel; AstraZeneca, Bayer Inc., Boehringer-Ingelheim, Novo Nordisk. Research Support; AstraZeneca, Boehringer-Ingelheim, Merck &amp; Co., Inc. C. Solis-Herrera: None. Funding Doris Duke Charitable Foundation; Voelcker Foundation; National Institutes of Health

Abstract 10484: Cardiac Adaptation to Acute Haemodynamic Stress in Patients with Type 2 Diabetes

Background: Due to varying cardiac workloads, efficient demand-supply matching is crucial for maintaining normal cardiac function. Cardiac metabolism is profoundly affected by changes in cardiac workload. There is scarcity of non-invasive studies evaluating how the heart adapts to hemodynamic stress in type 2 diabetes (T2D) patients. Objectives: Using cardiovascular magnetic resonance (CMR) and 31 Phosphorus MR spectroscopy ( 31 P-MRS) we assessed changes in cardiac energetics, perfusion, strain, systolic and diastolic function in response to increases in cardiac workload with dobutamine stress in T2D patients and healthy volunteers (HV). Methods: 24 T2D patients and 18 matched HV were recruited. Participants with ischemic heart disease were excluded. Results: Table-1 shows clinical and CMR/ 31 P-MRS data. Compared to HV, T2D patients showed reductions in left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS) at rest, and a blunted increase in LVEF and GLS during stress. Diastolic function was impaired in T2D with lower mitral in-flow E/A ratio compared to HV at rest, which was further reduced during stress in both groups. Stress myocardial blood flow was lower in T2D patients.Compared to HV, rest phosphocreatinine (PCr)/ATP ratio was 17% lower in T2D patients, with a further 8% reduction during stress. However, there was a more pronounced reduction in the PCr/ATP with stress in HV (33% relative reduction), although stress PCr/ATP remained numerically higher in HV compared to T2D patients. Conclusions: T2D is associated with blunted ability of the myocardium to significantly increase contractile function from rest to stress. Moreover, T2D patients show exacerbated reductions in myocardial energetics and diastolic function during stress. Although reductions in myocardial energetics and diastolic function in response to acute stress were also detected in HV, these assessments remained higher in HV at stress compared to T2D patients.

Size-adjusted muscle power and muscle metabolism in patients with cystic fibrosis are equal to healthy controls \u2013 a case control study

BackgroundSkeletal muscle function dysfunction has been reported in patients with cystic fibrosis (CF). Studies so far showed inconclusive data whether reduced exercise capacity is related to intrinsic muscle dysfunction in CF.MethodsTwenty patients with CF and 23 age-matched controls completed an incremental cardiopulmonary cycling test. Further, a Wingate anaerobic test to assess muscle power was performed. In addition, all participants completed an incremental knee-extension test with 31P magnetic resonance spectroscopy to assess muscle metabolism (inorganic phosphate (Pi) and phosphocreatinine (PCr) as well as intracellular pH). In the MRI, muscle cross-sectional area of the M. quadriceps (qCSA) was also measured. A subgroup of 15 participants (5 CF, 10 control) additionally completed a continuous high-intensity, high-frequency knee-extension exercise task during 31P magnetic resonance spectroscopy to assess muscle metabolism.ResultsPatients with CF showed a reduced exercise capacity in the incremental cardiopulmonary cycling test (VO2peak: CF 77.8 ± 16.2%predicted (36.5 ± 7.4 ml/qCSA/min), control 100.6 ± 18.8%predicted (49.1 ± 11.4 ml/qCSA/min); p < 0.001), and deficits in anaerobic capacity reflected by the Wingate test (peak power: CF 537 ± 180 W, control 727 ± 186 W; mean power: CF 378 ± 127 W, control 486 ± 126 W; power drop CF 12 ± 5 W, control 8 ± 4 W. all: p < 0.001). In the knee-extension task, patients with CF achieved a significantly lower workload (p < 0.05). However, in a linear model analysing maximal work load of the incremental knee-extension task and results of the Wingate test, respectively, only muscle size and height, but not disease status (CF or not) contributed to explaining variance. In line with this finding, no differences were found in muscle metabolism reflected by intracellular pH and the ratio of Pi/PCr at submaximal stages and peak exercise measured through MRI spectroscopy.ConclusionsThe lower absolute muscle power in patients with CF compared to controls is exclusively explained by the reduced muscle size in this study. No evidence was found for an intrinsic skeletal muscle dysfunction due to primary alterations of muscle metabolism.

Hydrogen-Rich Saline Attenuates Lipopolysaccharide-Induced Heart Dysfunction by Restoring Fatty Acid Oxidation in Rats by Mitigating C-Jun N-Terminal Kinase Activation.

Sepsis is common in intensive care units (ICU) and is associated with high mortality. Cardiac dysfunction complicating sepsis is one of the most important causes of this mortality. This dysfunction is due to myocardial inflammation and reduced production of energy by the heart. A number of studies have shown that hydrogen-rich saline (HRS) has a beneficial effect on sepsis. Therefore, we tested whether HRS prevents cardiac dysfunction by increasing cardiac energy. Four groups of rats received intraperitoneal injections of one of the following solutions: normal saline (NS), HRS, lipopolysaccharide (LPS), and LPS plus HRS. Cardiac function was measured by echocardiography 8 h after the injections. Gene and protein expression related to fatty acid oxidation (FAO) were measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. The injection of LPS compromised heart function through decreased fractional shortening (FS) and increased left ventricular diameter (LVD). The addition of HRS increased FS, palmitate triphosphate, and the ratio of phosphocreatinine (PCr) to adenosine triphosphate (ATP) as well as decreasing LVD. The LPS challenge reduced the expression of genes related to FAO, including perioxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), perioxisome proliferator-activated receptor alpha (PPARα), Estrogen-related receptor alpha (ERRα), and their downstream targets, in mRNA and protein level, which were attenuated by HRS. However, HRS had little effect on glucose metabolism. Furthermore, HRS inhibited c-Jun N-terminal kinase (JNK) activation in the rat heart. Inhibition of JNK by HRS showed beneficial effects on LPS-challenged rats, at least in part, by restoring cardiac FAO.

Correlation of cardiovascular risk scores with myocardial high-energy phosphate metabolism

The aim of our current work was to prove the association of myocardial high-energy phosphate metabolism measured by MR spectroscopy with validated cardiovascular risk scores, such as the European Systematic COronary Risk Evaluation (SCORE) Project [1], the German PROspective CArdiovascular Munster (PROCAM) Study [2] and the US Framingham Heart and Offspring Studies [3]. We used the ratio of phosphocreatinine (PCr) to β-adenosine-triphosphate (β-ATP) as a surrogate for the energy metabolism of the myocardium [4,5]. PCr-β-ATP ratios are reduced in patients with coronary heart disease [4,5], hypertension [6], and our previous studies suggested cardiovascular risk factors such as hypercholesterolemia [7], diabetes [8] or age [9] to be involved in this process. Therefore we hypothesized a relationship between established primary-event risk scores [1–3] and myocardial high-energy phosphate metabolism. Fasting blood samples of ninety-nine healthy asymptomatic volunteers without history of coronary heart disease were obtained to evaluate cardiovascular risk factors. All of the volunteers underwent cycle ergometry and echocardiography to exclude a latent coronary insufficiency and/or a reduced left ventricular function. Tabulated cardiovascular risk factors and parameters are shown in Table 1. For each patient the cardiovascular risk was assessed by:

Mechanism of Post-Translational Modification by Tyrosine Phosphorylation of Apoptotic Proteins During Hypoxia in the Cerebral Cortex of Newborn Piglets

The present study aims to investigate the mechanism of phosphorylation of apoptotic proteins and tests the hypothesis that the hypoxia-induced increased tyrosine phosphorylation of apoptotic proteins Bcl-2 and Bcl-xl is Ca(2+)-influx-dependent. Piglets were divided in normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-pretreated with clonidine (Clo + Hx, n = 4) groups. Hypoxic animals were exposed to an FiO(2) of 0.06 for 1 h. Clonidine (12.5 microg/kg, IV) was administered to piglets 30 min prior to hypoxia. Hypoxia was confirmed by ATP and phosphocreatinine (PCr) levels. Cytosol was isolated and separated by 12% SDS-PAGE and probed with tyrosine phosphorylated (p) -Bax, Bad, Bcl-2 and Bcl-xl antibodies and bands were detected. The ATP levels (micromol/g brain) in the Nx, Hx, Clo + Hx were 4.3 +/- 1.0 (P < 0.05 vs. Hx, Clo-Hx), 0.9 +/- 0.8 and 1.5 +/- 0.3, respectively. The PCr levels in the Nx, Hx, Clo + Hx were 2.7 +/- 0.7 (P < 0.05 vs. Hx, Clo-Hx), 0.9 +/- 0.2 and 0.9 +/- 0.9, respectively. Ca(2+)-influx (pmoles/mg protein) was 4.96 +/- 0.94 in Nx, 11.11 +/- 2.38 in Hx, and 6.23 +/- 2.07 in Clo + Hx (P < 0.05 Nx vs. Hx and Hx vs. Clo + Hx). p-Bcl-2 density was 21.1 +/- 1.1 Nx, 58.9 +/- 9.6 Hx and 29.5 +/- 6.4 Clo + Hx (P < 0.05 vs. Hx). p-Bcl-xl density was 29.6 +/- 1.5 Nx, 50.6 +/- 7.4 Hx and 32.1 +/- 0.1 Clo + Hx (P < 0.05 vs. Hx). p-Bax density was 38.6 +/- 16.2 Nx, 46.1 +/- 5.5 Hx and 41.6 +/- 1.9 Clo + Hx groups (P = NS). p-Bad was 66.7 +/- 12.8 Nx, 71.2 +/- 6.8 Hx and 78.7 +/- 22.5 Clo + Hx groups (P = NS). Results showed that clonidine administration prior to hypoxia prevents the hypoxia-induced increased nuclear Ca(2+)-influx and increased phosphorylation of Bcl-2 and Bcl-xl while phosphorylation of Bad and Bax was not altered. We conclude that post-translational modification of anti-apoptotic proteins Bcl-2 and Bcl-xl during hypoxia is nuclear Ca(2+)-influx-dependent. We propose that blockade of nuclear Ca(2+)-influx that prevents phosphorylation of antiapoptotic proteins may become a neuroprotective strategy.

Foot Muscle Energy Reserves in Diabetic Patients Without and With Clinical Peripheral Neuropathy

OBJECTIVETo investigate changes in the foot muscle energy reserves in diabetic non-neuropathic and neuropathic patients.RESEARCH DESIGN AND METHODSWe measured the phosphocreatinine (PCr)/inorganic phosphate (Pi) ratio, total 31P concentration, and the lipid/water ratio in the muscles in the metatarsal head region using MRI spectroscopy in healthy control subjects and non-neuropathic and neuropathic diabetic patients.RESULTSThe PCr/Pi ratio was higher in the control subjects (3.23 ± 0.43) followed by the non-neuropathic group (2.61 ± 0.36), whereas it was lowest in the neuropathic group (0.60 ± 1.02) (P < 0.0001). There were no differences in total 31P concentration and lipid/water ratio between the control and non-neuropathic groups, but both measurements were different in the neuropathic group (P < 0.0001).CONCLUSIONSResting foot muscle energy reserves are affected before the development of peripheral diabetic neuropathy and are associated with the endothelial dysfunction and inflammation.

Effect of hypoxia on the expression of procaspase-9 and procaspase-3 in neuronal nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets

Previous studies have shown that cerebral hypoxia results in increased activity of caspase-9, a key initiator of programmed cell death, in the cytosolic fractions of the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia results in increased expression of procaspase-9 and procaspase-3 in neuronal nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets. To test this hypothesis, expression of procaspase-9 and procaspase-3 was determined in 10 newborn piglets divided into two groups: normoxic (Nx, n=5) and hypoxic (Hx, n=5). The hypoxic piglets were exposed to an FiO(2) of 0.06 for 1h. Tissue hypoxia was documented by ATP and phosphocreatinine (PCr) levels. Neuronal nuclear, mitochondrial and cytosolic fractions were isolated and the expression of procaspase-9 and procaspase-3 was determined by immunoblotting using specific anti-procaspase-9 and anti-procaspase-3 antibodies. ATP levels (micromol/g brain) were 4.34+/-0.36 in the Nx and 1.43+/-0.28 in the Hx (p<0.001 vs. Nx) groups. PCr levels (micromol/g brain) were 3.75+/-0.27 in the Nx and 0.69+/-0.26 in the Hx (p<0.001 vs. Nx) group. Cytosolic procaspase-9 density (ODxmm(2)) was 88.82+/-17.55 in the Nx and 215.54+/-22.77 in the Hx (p<0.001 vs. Nx). Mitochondrial procaspase-9 density (ODxmm(2)) was 104.67+/-12.75 in the Nx and 183.44+/-16.69 in the Hx (p<0.001 vs. Nx). Nuclear procaspase-9 density (ODxmm(2)) was 135.56+/-15.36 in the Nx and 190.66+/-29.35 in the Hx (p<0.001 vs. Nx). Cytosolic procaspase-3 density (ODxmm(2)) was 23.72+/-3.71 in the Nx and 92.44+/-8.46 in the Hx (p<0.001 vs. Nx). Mitochondrial procaspase-3 density (ODxmm(2)) was 22.12+/-2.97 in the Nx and 51.22+/-10.67 in the Hx (p<0.001 vs. Nx). Nuclear procaspase-3 density (ODxmm(2)) was 53.80+/-7.18 in the Nx and 84.67+/-5.63 in the Hx (p<0.001 vs. Nx). We conclude that procaspase-9 and procaspase-3 proteins increased in all cell compartments including cytosolic, mitochondrial and nuclear during hypoxia, indicating increased expression of procaspase-9 during hypoxia. We propose that following increased expression of procaspase-9 and procaspase-3, these molecules traffic among the various cell compartments and become available for their activation resulting in increased caspase-9 and caspase-3 activity.

Effect of neuronal nitric oxide synthase inhibition on caspase-9 activity during hypoxia in the cerebral cortex of newborn piglets

Previous studies have shown that cerebral hypoxia results in increased activity of caspase-9, a key initiator of programmed cell death. We have also shown increased nitric oxide (NO) free radical generation during hypoxia in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia-induced increase in caspase-9 activity in the cerebral cortex of newborn piglets is mediated by NO derived from neuronal nitric oxide synthase (nNOS). To test this hypothesis, cytosolic caspase-9 activity was determined in 15 newborn piglets divided into three groups: normoxic (Nx, n = 5), hypoxic (Hx, n = 5), and Hx pretreated with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, 1 mg/kg, i.p., 1 h prior to hypoxia (Hx + 7NI, n = 5). The hypoxic piglets were exposed to an FiO 2 of 0.06 for 1 h. Tissue hypoxia was documented by ATP and phosphocreatinine (PCr) levels. The cytosolic fraction was obtained from the cerebral cortical tissue following centrifugation at 100,000 × g for 1 h and caspase-9 activity was assayed using Ac-Leu-Glu-His-Asp-amino-4-methyl coumarin, a specific fluorogenic substrate for caspase-9. Caspase-9 activity was determined spectroflourometrically at 460 nm using 380 nm as excitation wavelength. ATP levels (μmol/g brain) were 4.35 ± 0.21 in the Nx 1.43 ± 0.28 in the Hx ( p < 0.05 versus Nx), and 1.73 ± 0.33 in the Hx + 7-NINA group ( p < 0.05 versus Nx, p = NS versus Hx). PCr levels (μmol/g brain) were 3.80 ± 0.26 in the Nx, 0.96 ± 0.20 in the Hx ( p < 0.05 versus Nx), and 1.09 ± 0.39 in the Hx + 7 NINA group ( p < 0.05 versus Nx, p = NS versus Hx). Cytosolic caspase-9 activity (nmol/mg protein/h), increased from 1.27 ± 0.15 in the Nx to 2.13 ± 0.14 in the Hx ( p < 0.05 versus Nx) compared to 1.10 ± 0.21 in the Hx + 7-NINA group ( p < 0.05 versus Hx, p = NS versus Nx). Caspase-3 activity (nmol/mg protein/h) also increased from 9.39 ± 0.73 in Nx to 18.94 ± 3.64 in Hx ( p < 0.05 versus Nx) compared to 8.04 ± 1.05 in the Hx + 7-NINA group ( p < 0.05 versus Hx, p = NS versus Nx). The data show that administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increase in caspase-9 activity that leads to increase in caspase-3 activity. Since nNOS inhibition blocked the increase in caspase-9 activity during hypoxia, we conclude that hypoxia-induced increase in caspase-9 activity is mediated by nNOS derived NO. We propose that the NO generated during hypoxia leads to activation of caspase-9 and results in initiation of caspase-cascade-dependent hypoxic neuronal death.

Phosphorous-31 Magnetic Resonance Spectroscopy of Cervical Cancer Using Transvaginal Surface Coil

We developed a new transvaginal coil tuned for phosphorous-31 and measured its magnetic resonance spectroscopy ((31)P MRS) of uterine cervical cancer. In a 50-year-old woman with uterine cervical cancer (International Federation of Gynecology and Obstetrics [FIGO] stage IIIb), (31)P MRS with the new coil clearly differentiated the low intensity of phosphocreatinine (PCr) and high intensity of phosphomonoester (PME) in tumor from those in muscle. Results suggests that this method will be useful for assessing uterine cervical cancer.

Metabolic dependency of ionophore A23187-induced contraction of ileal longitudinal smooth muscle.

1. Tonic contraction in response to K+ is well known to be highly dependent on aerobic metabolism in ileal muscle. The ionophore A23187 (10(-5) M) induced an initial contraction and successive rhythmic contraction in ileal muscle, but did not induce tonic contraction. This study, therefore, was performed to investigate the metabolic dependency during contraction induced by A23187. 2. Under hypoxic conditions, A23187 (10(-5) M) induced an initial contraction accompanied by an increase in lactate release. However, it induced only small rhythmic contractions and decreases in ATP, phosphocreatinine (PCr) and glycogen contents. 3. In glucose-free medium, A23187 (10(-5) M) induced an initial contraction and concomitant significant decrease in the ATP and glycogen contents. However, it did not induce successive rhythmic contractions. In 'glycogen-depleted muscle' stimulated repeatedly with 60 mM K+ in glucose-free medium, 60 mM K+ induced a phasic contraction without tonic contraction. A23187 induced no contraction under these conditions. 4. These results suggested that the initial contraction induced by A23187 was dependent on endogenous glycogen, while successive rhythmic contractions were dependent on aerobic metabolism after supply of external glucose to the ileal muscle.

REPLY TO "NONINVASIVE METABOLIC ASSESSMENT OF HUMAN DONOR LIVERS: METABOLITE ASSIGNMENT IN 31P MAGNETIC RESONANCE SPECTROSCOPY"

Changani et al. correctly state that it is conventional to reference the resonances in a 31P-nuclear magnetic resonance spectrum relative to the phosphocreatinine (PCr) resonance, which is-if present-set at 0 ppm. When, however, no PCr is present, another spectral peak has to be used for reference. As Changani et al. mention, one can use the α-ATP for reference, which is quite convenient when using experimental setups with high signal-to-noise ratios, for example, when working at field strengths of 11.7 Tesla. This is even more convenient when the investigator can use freshly harvested rat livers, tissue extracts, or cell cultures. Clinical liver transplantation, however, is different: Most of the livers that we studied came from long distances with accordingly long cold ischemia times. As is well known, the longer the cold ischemia time, the lower the total adenine nucleotide (TAN) levels. In most of the livers that we studied, there was hardly any α-ATP to be detected. In contrast, NADH was present in all livers and could be used for reference, because the NADH peak position is pH-independent and the peak is stable over longer periods of time. The use of NADH as a reference peak is adapted from previous experiments on rat livers by other authors (1,2). We have successfully used this method to study tissue pH changes in cold stored human livers during cold preservation (3). Changani et al. furthermore ask what could be the origin of the small but distinct peak attributed (by us) to PCr. Their statement that PCr levels drop very rapidly after the onset of ischemia is absolutely correct; this has been observed in (too?) many animal experiments. Their suggestion that PCr peaks may originate from muscle cells in the extrahepatic biliary tree and great vessels is interesting. However, if that were the case, we think all livers in our study should have shown the PCr peak, which was not observed. The spectrum that Changani et al. provide to convince us that a liver does not contain spectroscopically detectable PCr is recorded from a perchloric acid extract of normal human liver (presumably a liver biopsy). Our spectra were recorded from cold stored whole human livers after in situ preservation perfusion and several hours of cold ischemia. These events have probably had a large impact on liver adenine (and energy) metabolism, and this is probably the basic reason for the presence of a peak at the PCr site. Indeed, we did not prove (for example, by means of high-performance liquid chromatography or high-field strength nuclear magnetic resonance) whether this peak was actually PCr, since that was not the purpose of our study; thus, the true origin of the peak at the PCr site remains to be determined. Changani et al. furthermore remark that the sequential spectra show better resolution without indication of a second inorganic phosphate pool. As the figure to which they refer was taken from a previous publication(4) (as the figure legend mentions), different spectral processing parameters were employed which account for the optical difference between the respective spectra. Their remark that the concentration of inorganic phosphate in University of Wisconsin preservation solution is significantly higher than the intracellular phosphate level is true. For exactly that reason, we positioned the donor liver as close as possible against the precooled spectroscopy coil to prevent "drowning" of the hepatic inorganic phosphate signal in the University of Wisconsin inorganic phosphate signal. We agree with Changani et al.'s final conclusions that 31P magnetic resonance spectroscopy is the only rapid nonivasive means of acquiring biochemical information prior to transplantation. Based on its ability to monitor on-line energy metabolism in cold stored organs, this technique may help to develop better preservation solutions. On the basis of our results, however, we are not convinced that 31P magnetic resonance spectroscopy is yet a reliable clinical instrument to provide data that can convince a clinician to proceed to transplant the liver or to discard the liver because of an "abnormal spectrum." R.F.E. Wolf Department of Radiology; University Hospital; 9700 RB Groningen, The Netherlands

Metabolic changes in the rat brain after acute and chronic ethanol intoxication: A 31P NMR spectroscopy study

In this work, 31P phosphorus NMR (31P NMR) studies of the brain have been conducted in rats acutely and chronically intoxicated with ethanol. In both groups, changes in levels of high-energy phosphates were observed: increase of phosphocreatinine (PCr)/beta AaTP and PCr/inorganic phosphate (Pi) in acute and long-term ethanol exposure, and decrease of Pi/beta ATP after acute ethanol administration. These changes in high-energy phosphates, indicative of a reduction of adenosine triphosphate (ATP) and PCr consumption (PCr+ ADP+ H+ ATP+ Cr; ATP ADP+ Pi), suggest a reduction of cerebral metabolism both in acute and chronic ethanol exposure. In addition, in the group of rats chronically intoxicated with ethanol, there were variations in phosphodiester peak intensities (decrease of phosphomonoester (PME)/phosphodiester (PDE), increase of PDE/beta ATP), suggesting increased breakdown of membrane phospholipids. These changes could provide a metabolic explanation for the development of cerebral atrophy in chronic alcoholism.

Coupled in vivo activity of creatine phosphokinase and the membrane-bound (Na+,K+)-ATPase in the resting and stimulated electric organ of the electric fish Narcine brasiliensis

Physiological control of the plasma membrane sodium pump, (Na+,K+)-ATPase, is essential for proper function of eukaryotic cells. In the electric organ of the elasmobranch Narcine brasiliensis, the normal demands placed upon the pump during the process of generation of electrical currents call for large and rapid changes in activity of this enzyme, making this a good model for the study of its cellular regulation. 31P NMR spectroscopic techniques were used to study metabolic regulation of membrane pump function in resting and stimulated electric organ and in skeletal muscle of the live, intact N. brasiliensis. Because the ATP synthetic abilities of the electric organ by glycolysis or oxidative phosphorylation are extremely limited, depletion of phosphocreatinine (PCr) could be used to determine the activity of the (Na+,K+)-ATPase after the electric organ was stimulated to discharge, and to measure the net flux from PCr to ATP through the creatine phosphokinase (CPK) reaction in the electric organ. Saturation transfer, an NMR technique which measures exchange rates, was applied to determine the unidirectional flux in the forward direction through the same reaction in the electric organ and in skeletal muscle as a control. The pseudo first-order rate constant kf for the CPK reaction at 24 degrees C in resting electric organ was 0.000 +/- 0.002 s-1 (n = 10) and in skeletal muscle was 0.08 +/- 0.03 s-1 (n = 3). The results demonstrate that in resting electric organ, which is well supplied with CPK, there was no measurable flux through this reaction, although CPK when extracted is highly active. Measured and calculated levels of all substrates for the creatine kinase reaction in the electric organ are similar to those in unstimulated skeletal muscle, where the creatine phosphokinase reaction rates are high in vivo. In contrast to the resting electric organ, during stimulation of the electric organ the measured net rate constant was greater than 0.08 s-1. In addition, as shown by lack of PCr depletion, there was virtually no net turnover of ATP in the resting organ compared to the stimulated organ. The marked difference in the (Na+,K+)-ATPase activity in the resting and activated electric organ confirmed earlier results (Blum, H., Nioka, S., and Johnson, R. G., Jr. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1247-1251). Together, these results suggest that there is a novel method of coordinate regulation of cellular enzymes of great sensitivity and rapidity.

Tumor size dependent changes in a murine fibrosarcoma: use of in vivo 31P NMR for non-invasive evaluation of tumor metabolic status.

Tumor tissue contains viable hypoxic regions that are radioresistant and often chemoresistant and may therefore be responsible for some treatment failures. A subject of general interest has been the development of non-invasive means of monitoring tissue oxygen. Pulse Fourier transform 31P NMR spectroscopy can be used to estimate intracellular nucleotide triphosphates (NTP), phosphocreatinine (PCr), inorganic phosphate (Pi) and pH. We have obtained 31P NMR spectra as an indirect estimate of tissue oxygen and metabolic status in a C3H mouse fibrosarcoma FSaII. Sequential spectra were studied during tumor growth in a cohort of animals and peak area ratios for several metabolites were computed digitally by computer. During growth, tumors showed a progressive loss of PCr with increasing Pi, and most tumors greater than 250 mm3 in volume had little or no measurable PCr. The smallest tumors (38 mm3 average volume) had PCr/Pi ratios of 1.03 +/- .24, whereas tumors 250 mm3 or more had an average PCr/Pi ratio of 0.15 +/- .04. Similarly derived NTP/Pi ratios decreased with tumor size, but this change was not significant (p = .17). Radiobiologic hypoxic cell fractions were estimated using the radiation dose required to control tumor in 50% of animals (TCD50) or by the lung colony technique. Tumors less than 100 mm3 had a hypoxic cell fraction of 4% (TCD50) while tumors 250 mm3 had a 40% hypoxic cell fraction (lung colony assay). These hypoxic fraction determinations correlated well with the depletion of PCr and decline in NTP/Pi ratios seen at 250 mm3 tumor volumes. Tumor spectral changes with acute ischemia were studied after ligation of the tumor bearing limb and were similar to changes seen with tumor growth. PCr was lost within 7 minutes, with concurrent increase in Pi and loss of NTP. Complete loss of all high energy phosphates occurred by 40 minutes of occlusion. In vivo tumor 31P NMR spectroscopy can be used to estimate tissue metabolic status and may be useful in non-invasive prediction of hypoxic cell fraction, reoxygenation, and radiation treatment response.

31P-NMR studies on an animal model of human defective muscle glycolysis.

Sequential metabolic changes in leg muscles of a model of defective muscle glycolysis that was produced by intraaortic injection of sodium iodoacetate were observed by in vivo 31P-NMR spectroscopy with a surface coil. Spectra obtained from iodoacetate-treated muscles presenting exercise-induced cramp showed decreased levels of phosphocreatinine (PCr) and adenosine triphosphate (ATP) and an accumulation of sugar phosphate. The chemical shift of inorganic phosphate (Pi) resonance, which reflects the intracellular pH of the contracted muscles, showed a milder shift to the acidic condition than those of controls. In vivo 31P-NMR study showed dynamic information on the muscle energetics under defective glycolytic conditions.