31P and 1H NMR spectroscopic studies of liver extracts of carbon tetrachloride-treated rats.

NMR spectroscopy was used to examine hepatic metabolism in cirrhosis with a particular focus on markers of functional cellular hypoxia. (31)P and (1)H NMR spectra were obtained from liver extracts from control rats and from rats with carbon tetrachloride-induced cirrhosis. A decrease of 34% in total phosphorus content was observed in cirrhotic rats, parallelling a reduction of 40% in hepatocyte mass as determined by morphometric analysis. Hypoxia appeared to be present in cirrhotic rats, as evidenced by increased inorganic phosphate levels, decreased ATP levels, decreased ATP:ADP ratios (1.72 +/- 0.40 vs 2.48 +/- 0.50, p < 0.01), and increased inorganic phosphate:ATP ratios (2.77 +/- 0.48 vs 1.62 +/- 0.24, p < 0.00001). When expressed as a percentage of the total phosphorus content, higher levels of phosphoethanolamine and lower levels of NAD and glycerophosphoethanolamine were detected in cirrhotic rats. Cirrhotic rats also had increased phosphomonoester:phosphodiester ratios (5.73 +/- 2.88 vs 2.53 +/- 0.52, p < 0.01). These findings are indicative of extensive changes in cellular metabolism in the cirrhotic liver, with many findings attributable to the presence of intracellular hypoxia.     

31P NMR study of cisplatin- and doxorubicin-induced changes in tumour metabolism in rats with a cisplatin-sensitive or -resistant immunocytoma

The response of tumours to treatment with the cytostatic drugs cisplatin (CDDP) or doxorubicin (DXR) was followed in vivo by 31P NMR spectroscopy. A CDDP-sensitive parent line (IgM-I) and a CDDP-resistant subline (IgM/CDDP) of the IgM-immunocytoma grown s.c. on LOU/M WsL rats were used. Animals from both tumour groups (n = 33) were divided into 3 subgroups: CDDP-treated (1 mg/kg), DXR-treated (10 mg/kg) and control. In 3 out of the 4 treated subgroups where the tumours regressed to less than one half of the initial size, 31P NMR spectroscopy revealed alkaline shifts of 0.31-0.41 pH units at day 4, while the ratio of nucleoside triphosphate to Pi in the tumours, increased continuously to 250-435%. Following CDDP treatment, the 31P NMR spectra of the non-responding IgM/CDDP tumours showed a similar pH increase (0.37 units). The ratio of NTP/Pi showed a temporary decrease to 63 +/- 14% SEM at day 1, which was followed by a recovery to 130 +/- 12% at day 2 and 119 +/- 15% at day 4. The control tumours showed no change in pH and a gradual decrease in the ratio of NTP/Pi. In DXR-treated rats the concentrations of DXR in the immunocytoma tumour and its subline were similar, but in the CDDP-treated rats the IgM-I tumours contained significantly higher levels of platinum than the IgM/CDDP tumours, both measured at 3 and 4 days after administration. The continuous increase in NTP/Pi ratio observed in the responding tumours, is a phenomenon characteristic of tumour regression, while the early temporary decrease in tumour NTP/Pi ratio could be associated with resistance to CDDP. Whether the reported response-specific spectral change applies to other tumour types and other treatment regimens remains to be established.     

Changes in phosphate metabolism in thymoma cells suggest mechanisms for resistance to dexamethasone-induced apoptosis. A 31P NMR spectroscopic study of cell extracts

Treatment of the mouse thymoma-derived WEHI7.2 cell line with dexamethasone, a synthetic glucocorticoid, causes the cells to undergo apoptosis. Previous studies have shown that WEHI7.2 cell variants with an increased antioxidant defense exhibit increased resistance to dexamethasone-induced apoptosis, suggesting that oxidative stress may play a role in glucocorticoid-induced apoptosis. In this work we compared metabolic profiles of WEHI7.2 parental cells with those of WEHI7.2 variants with an increased antioxidant defense or overexpressing bcl-2, to determine whether bolstering the antioxidant defense results in altered metabolic parameters that could translate into increased resistance to dexamethasone-induced apoptosis. WEHI7.2 parental cells and cells overexpressing catalase, thioredoxin or bcl-2, or selected for resistance to 200 micro M H(2)O(2) were cultured in low-glucose DMEM medium supplemented with 10% calf serum, and extracted using chloroform-methanol-water (1:1:1). Metabolites contained in the aqueous and organic phases of the extracts were processed separately and subjected to high-resolution (31)P NMR spectroscopy. In most of the steroid-resistant variants, ATP levels and energetic status were decreased compared with the steroid-sensitive parental cell line, while the concentrations of hexose and triose phosphates were increased. Furthermore, the ratio of choline-containing phospholipids to ethanolamine-containing phospholipids was generally reduced in steroid-resistant cells. Phosphatidylethanolamine and its derivatives contain a higher amount of polyunsaturated fatty acids (PUFA) than the choline-containing analogs, and PUFA are readily oxidized by reactive oxygen species. Therefore, an increased initial amount of phosphatidylethanolamine may increase the 'buffering capacity' of this antioxidant and may thus contribute to the steroid resistance of WEHI7.2 variants.     

31P NMR spectroscopic and near infrared spectrophotometric studies of effects of anesthetics on in vivo RIF-1 tumors. Relationship to tumor radiosensitivity

Mice with subcutaneous RIF-1 tumors were anesthetized with sodium pentobarbital (PB) or ketamine plus acepromazine (KA) before acquisition of in vivo 31P nuclear magnetic resonance (NMR) spectra from tumors. The area of the inorganic phosphate resonance was significantly greater (relative to phosphomonoesters or the alpha-phosphate resonance of nucleoside triphosphate) in spectra obtained under PB anesthesia, suggesting that the hypoxic fraction of the tumor increased following PB anesthesia. In vivo near-infrared laser spectroscopy directly demonstrated that tumor oxyhemoglobin was reduced by more than 20% following PB but was not significantly affected by KA. Total hemoglobin (tumor blood volume) was reduced by 11% following PB anesthesia, but was not significantly affected by KA. Tumor growth delay induced by gamma-irradiation was shorter when tumors were irradiated under PB anesthesia than when irradiated under KA, showing that PB anesthesia had a radioprotective effect. These studies demonstrate that both the 31P NMR and near infrared methods can detect metabolic or physiological changes associated with an increase in tumor radioresistance (i.e., an increase in the radiobiological hypoxic fraction).     

Metabolic heterogeneity in RIF-1 tumours detected in vivo by 31P NMR spectroscopy

The spatial distribution of phosphate metabolites and pH within subcutaneously implanted RIF-1 tumours was determined by 1-dimensional phosphorus chemical shift imaging. 31P spectra from two to three 4 mm thick cross-sectional slices were obtained for each tumour. Quantitative morphometry was used to estimate the amount of necrosis within those slices. A spatially heterogeneous distribution of phosphate metabolites and pH was detected in most of the tumours. Levels of necrosis ranged from 0-24% for the tumours in this study. There was no significant correlation between the extent of necrosis over this range and levels of metabolites or pH, suggesting that factors besides necrosis can contribute to spectral heterogeneity.     

Quantification of phosphocholine and glycerophosphocholine with 31P edited 1H NMR spectroscopy

Choline and the related compounds phosphocholine (PC) and glycerophosphocholine (GPC) are considered to be important metabolites in oncology. Past studies have demonstrated correlations linking the relative ratios and concentrations of these metabolites with the development and progression of cancer. Currently, in vivo and tissue ex vivo magnetic resonance spectroscopy methods have mostly centered on measuring the total concentration of these metabolites and have difficulty in differentiating between them. Here, a new scheme that uses (31)P edited (1)H spectroscopy to quantify the concentrations of choline, PC and GPC in biological samples is reported and its applicability is demonstrated using samples of human brain tumor extracts. This method is particularly well-suited for analytical situations where the PC and GPC resonances are not sufficiently resolved and/or are obscured by other metabolites. Consequently, this scheme has the potential to be used for the analysis of choline compounds in ex vivo tissue samples.     

Broadband proton decoupling in human 31P NMR spectroscopy

The limited chemical shift dispersion of in vivo 31P NMR spectra obtained at the relatively low field strengths used for human applications is the cause of poor spectral resolution. This makes it difficult to obtain accurate quantitative information from overlapping resonances, and interesting resonances may be obscured. At 1.5 T unresolved 1H-31P couplings contribute significantly to the linewidth of in vivo 31P NMR resonances. Therefore, proton decoupling can improve spectral resolution substantially, resulting in better resolved resonances and more reliable quantitative information. In this work it is shown that well resolved resonances of glycerophosphocholine, glycerophosphoethanolamine and phosphoethanolamine are obtained in 1H decoupled 31P NMR spectra of human muscle, brain, and liver. In spectra of the human heart it has been possible to resolve the myocardial Pi signal from the signals of 2,3-diphosphoglycerate from blood. With surface coils it is difficult to achieve broadband decoupling over the entire sensitive region of the coil by using conventional decoupling sequences. This problem has been overcome by applying a train of frequency modulated inversion pulses to achieve proper decoupling despite B2 inhomogeneity. Broadband 1H decoupling of 31P NMR spectra was possible without exceeding specific absorption rate guidelines.     

Compatibility of superparamagnetic iron oxide nanoparticle labeling for 1H MRI cell tracking with 31P MRS for bioenergetic measurements

Labeling of cells with superparamagnetic iron oxide nanoparticles permits cell tracking by ¹H MRI while ³¹P MRS allows non‐invasive evaluation of cellular bioenergetics. We evaluated the compatibility of these two techniques by obtaining ³¹P NMR spectra of iron‐labeled and unlabeled immobilized C2C12 myoblast cells in vitro. Broadened but usable ³¹P spectra were obtained and peak area ratios of resonances corresponding to intracellular metabolites showed no significant differences between labeled and unlabeled cell populations. We conclude that ³¹P NMR spectra can be obtained from cells labeled with sufficient iron to permit visualization by ¹H imaging protocols and that these spectra have sufficient quality to be used to assess metabolic status. This result introduces the possibility of using localized ³¹P MRS to evaluate the viability of iron‐labeled therapeutic cells as well as surrounding host tissue in vivo. Published in 2010 by John Wiley & Sons, Ltd.     

Effect of photodynamic therapy on RIF-1 tumor metabolism and blood flow examined by 31P and 2H NMR spectroscopy

Photodynamic therapy utilizes the tumor localizing drug dihematoporphyrin ether and red laser light to produce both direct tumor cell destruction via damage to mitochondrial membranes, and also indirect cell kill via destruction of the tumor vasculature. As a first step towards examining the mechanistic relationship between metabolic and vascular effects of photodynamic therapy, murine RIF-1 tumors were treated with a subcurative treatment (500 J/cm2). Tumor metabolic status was monitored using in vivo 31P NMR before, during and after the treatment. The tumor blood flow immediately before and after treatment was measured by direct intratumor injection of D2O saline and observation of the tracer signal clearance from the tumor via 2H NMR. During the photodynamic therapy treatment, significant decreases were observed for the nucleoside triphosphate concentrations, tumor pH and tumor blood flow, while inorganic phosphate concentrations increased. Animals treated with laser light alone and those not given any treatment, demonstrated no significant changes in tumor metabolic status, tumor pH or tumor blood flow. Further studies are required to determine whether tumor blood flow or metabolic status is affected first.     

Esophageal cancer phospholipids correlated with histopathologic findings: a 31P NMR study.

We analyzed 36 esophageal tumor specimens for phospholipid content using phosphorus nuclear magnetic resonance spectroscopy (31P NMR) and correlated the individual phospholipid profiles with specific clinical and histopathologic features. Among the 18 phospholipids identified in the esophageal tumor specimens, the mean mole percentage concentration of dimethylphosphatidylethanolamine, lysoalkylacylphosphatidylcholine, lysophosphatidic acid, lysophosphatidylcholine (deacylated at the glycerol-1 carbon), and lysoethanolamine plasmalogen correlated with pathologic T stage, nuclear grade, or the presence of lymphatic invasion. 31P NMR produces well-dispersed phospholipid spectra and a precise determination of phospholipid relative mole percentages. These data provide a statistical correlation between histopathologic features and molecules known to play an important role in cellular activities and processes unique to malignant tissues.     

Assessment of magnesium concentrations by 31P NMR in vivo.

31P NMR spectra obtained in vivo reveal the presence of a few reasonably well defined chemical species, namely, ATP, orthophosphate (Pi), and, in brain, phosphocreatine. The chemical shifts of these resonances respond to changes in concentrations of ions such as H+ and Mg2+ in a manner that depends on both the chemical shifts intrinsic to individual complexes and the formation or binding constants for the several complexes. Values of the appropriate formation constants are well established in the literature. We have derived estimates of the chemical shifts intrinsic to the individual complexes by analyzing high resolution spectra of solutions whose composition brackets the domain of physiological relevance. This provides information sufficient to estimate intracellular concentrations of H+ and Mg2+ from chemical shifts seen with in vivo spectra. The primary finding is an estimate of 0.3 mM for the concentration of free magnesium in human brain. Differing values are obtained from other tissues.     

Quantification of in vivo 31P NMR brain spectra using LCModel

Quantification of ³¹P NMR spectra is commonly performed using line‐fitting techniques with prior knowledge. Currently available time‐ and frequency‐domain analysis software includes AMARES (in jMRUI) and CFIT respectively. Another popular frequency‐domain approach is LCModel, which has been successfully used to fit both ¹H and ¹³C in vivo NMR spectra. To the best of our knowledge LCModel has not been used to fit ³¹P spectra. This study demonstrates the feasibility of using LCModel to quantify in vivo ³¹P MR spectra, provided that adequate prior knowledge and LCModel control parameters are used. Both single‐voxel and MRSI data are presented, and similar results are obtained with LCModel and with AMARES. This provides a new method for automated, operator‐independent analysis of ³¹P NMR spectra. Copyright © 2015 John Wiley & Sons, Ltd.     

In vivo 31P NMR spectroscopy assessment of skeletal muscle bioenergetics after spinal cord contusion in rats

Purpose

Muscle paralysis after spinal cord injury leads to muscle atrophy, enhanced muscle fatigue, and increased energy demands for functional activities. Phosphorus magnetic resonance spectroscopy (³¹P-MRS) offers a unique non-invasive alternative of measuring energy metabolism in skeletal muscle and is especially suitable for longitudinal investigations. We determined the impact of spinal cord contusion on in vivo muscle bioenergetics of the rat hind limb muscle using ³¹P-MRS.

Methods

A moderate spinal cord contusion injury (cSCI) was induced at the T8–T10 thoracic spinal segments. ³¹P-MRS measurements were performed weekly in the rat hind limb muscles for 3 weeks. Spectra were acquired in a Bruker 11 T/470 MHz spectrometer using a 31P surface coil. The sciatic nerve was electrically stimulated by subcutaneous needle electrodes. Spectra were acquired at rest (5 min), during stimulation (6 min), and recovery (20 min). Phosphocreatine (PCr) depletion rates and the pseudo first-order rate constant for PCr recovery (k _PCr) were determined. The maximal rate of PCr resynthesis, the in vivo maximum oxidative capacity (V _max) and oxidative adenosine triphosphate (ATP) synthesis rate (Q _max) were subsequently calculated.

Results

One week after cSCI, there was a decline in the resting total creatine of the paralyzed muscle. There was a significant reduction (~24 %) in k _PCr measures of the paralyzed muscle, maximum in vivo mitochondrial capacity (V _max) and the maximum oxidative ATP synthesis rate (Q _max) at 1 week post-cSCI. During exercise, the PCr depletion rates in the paralyzed muscle one week after injury were rapid and to a greater extent than in a healthy muscle.

Conclusions

Using in vivo MRS assessments, we reveal an acute oxidative metabolic defect in the paralyzed hind limb muscle. These altered muscle bioenergetics might contribute to the host of motor dysfunctions seen after cSCI.     

Time-dependent effects of anesthetic agents on 31P NMR high-energy phosphates in KHT and RIF-1 fibrosarcomas

Previous studies have reported significant radiobiological and hemodynamic effects associated with sodium pentobarbital (PB) anesthetization. The present work contrasts the effects of PB with azaperone-ketamine (AZ) in RIF-1 and KHT tumors while animal body core temperature is maintained at 37 degrees C. The primary aims were to evaluate both agents in terms of: (i) duration of anesthetic; (ii) effect on absolute levels of 31P NMR phosphocreatine (PCr) + beta-nucleoside triphosphate (beta-NTP)/inorganic phosphate (Pi) ratios; and (iii) effect on temporal variability of PCr + beta-NTP/Pi ratios. In terms of overall duration, AZ was the clear preference. Although the maintenance of 37 degrees C core temperature significantly reduced overall durations for both anesthetics, AZ animals invariably remained immobile for a minimum of 80 min. For PB, durations were highly unpredictable. With AZ, mean PCr + beta-NTP/Pi ratios were constant over the entire 80 min period for both lines. With PB, PCr + beta-NTP/Pi ratios were lower in relation to AZ for KHT at select timepoints, but highly variable among RIF-1 tumours. Since ratios under PB varied substantially with time for RIF-1 lines, measurements taken with PB are clearly not representative of the control state. Furthermore, in light of the consistent and reproducible results obtained with AZ, this anesthetic is considered a marked improvement over PB for animal studies of this nature.     

Effect of anaerobic metabolic changes on the creatine kinase reaction in frog muscle studied by 31P saturation transfer NMR

The effect of anaerobic metabolic changes on the flux of the creatine kinase reaction in bullfrog skeletal muscle was evaluated by 31P saturation transfer NMR. During an anaerobic time course the reduction ratio (M+/M0) of the phosphocreatine signal and the spin-lattice relaxation time measured in the presence of saturating irradiation at the ATP gamma-phosphate resonance were decreased; however, the intrinsic spin-lattice relaxation time of phosphocreatine was constant (4 s). The pseudo-first-order rate constant for the forward reaction of creatine kinase (i.e., phosphocreatine to ATP) was increased during the time course, accompanied by a decrease in phosphocreatine concentration and an intracellular acidification. However, the flux was constant (4 mmol.kg-1 wet wt.s-1) over the anaerobic time course. The fluxes analysed in several locations along the fibers, which were simultaneously measured by means of the one-dimensional chemical shift imaging technique, were almost the same along the entire length of the fibers. These results indicate that the creatine kinase reaction is at equilibrium along the entire length of the frog muscle fibers.     

1H NMR spectroscopic method for diagnosis of malabsorption syndrome: a pilot study

Despite its well-documented limitations, colorimetry has been commonly used for the d-xylose test in the diagnosis of malabsorption syndrome (MAS). With a possibility of overcoming its limitations, the use of (1)H NMR spectroscopy for D-xylose test is explored herein. Urine samples from 35 adults with suspected MAS were obtained before and after oral ingestion of D-xylose. The diagnosis of MAS was based on fecal fat (72 h excretion using Van de Kamer's technique, normal < 7 g/24 h and/or Sudan III stain of spot stool specimen, normalor=1 g/5 g/5 h). In vitro experiments on the standard specimens of D-xylose were also performed independently using both methods. Colorimetry showed a lower value for the quantity of D-xylose excreted in urine than NMR [median 0.73 (0.17-1.89 g) vs 1.37 (0.17-3.23 g), respectively; p<0.0001, Wilcoxon's signed ranks test]. Colorimetry and NMR correctly diagnosed 11/12 and 10/12 (p=N.S.) patients with MAS and 14/23 and 20/23 (p<0.05) without MAS, respectively. Sensitivity and specificity of colorimetry and NMR were 91.6 and 60.7% vs 83.3 and 86.9%, respectively. In in vitro experiments, the values obtained for standard xylose using NMR showed a maximum error of 7%, whereas the colorimetric method showed 20%. The NMR method is simple and may be more accurate for the D-xylose absorption test. Colorimetry was found to be inferior as compared with NMR due to its low specificity.     

31P NMR study of the impact of dietary manipulation on tumor metabolism and response to methotrexate

The effects of nutritional manipulation and subsequent chemotherapeutic treatment upon growth and metabolism of a transplanted rat rhabdomyosarcoma were investigated by in vivo 31P NMR spectroscopy. Nutritional manipulation was accomplished by administration of a protein deprived diet containing no protein and 75.5% glucose. After 5 days the protein deprived rats (PD rats) were nutritionally replenished with a normal protein diet containing 27% protein and 47.3% glucose. Twenty-four hours after nutritional replenishment the PD rats and continuously well-fed controls (NP rats) received methotrexate (MTX, 30 mg/kg, i.p.). 31P NMR spectroscopy of the tumors 24 h after MTX administration showed a decreased ratio of nucleoside triphosphates to inorganic phosphate (referred to as 'ATP/Pi ratio') in PD rats in contrast to an unchanged ATP/Pi ratio in the NP controls. At the time of MTX administration the PD rats had a significantly lower tumor pH than the NP group (6.75 +/- 0.03 [SEM] vs 6.95 +/- 0.04; p less than 0.02). Tumor response in the PD group was significantly (p less than 0.01) enhanced compared to the NP group. These findings indicate that a period of dietary protein deprivation combined with a high glucose load and followed by nutritional replenishment impairs tumor metabolism. The altered metabolic status is expressed by acidification of the tumor and distinct changes in ATP/Pi ratio and appears to relate to an enhanced susceptibility to MTX chemotherapy.     

Chronic cardiotoxicity of adriamycin studied in a rat model by 31P NMR

Adriamycin induced cardiotoxicity is, among other factors, characterized by an impairment of mitochondrial function and altered energy metabolism. The possible merits of 31P NMR in timely detection of this cardiotoxicity were studied in perfused hearts of chronically treated rats after cumulative doses of 6, 8, 10, 12 and 13 mg adriamycin/kg body wt and compared to histological evaluation. After high cumulative doses the Phosphocreatine (PCr)/ATP ratio was significantly decreased in the hearts of treated animals, compared to the control animals (1.65 +/- 0.13 vs. 2.47 +/- 0.36 (p less than 0.001) at 12 mg/kg and 1.92 +/- 0.22 vs. 2.37 +/- 0.15 (p less than 0.01) at 13 mg/kg adriamycin, respectively). This decrease coincides with a sudden increase in the histological score from 2.0 at 10 mg/kg to 8.0 at 12 mg/kg adriamycin on a scale of 10.0. The Pi/PCr ratio, coronary flow and rate pressure product (RPP) of the isolated hearts of treated animals were not significantly different from controls. When heart rates were increased, parallel changes in PCr/ATP ratio, Pi/PCr ratio, RPP and coronary flow were observed in both control and treated groups, except for the 12 mg/kg adriamycin group in which pacing failed to increase RPP. In addition, in this group the Pi/PCr ratio at higher heart rates was significantly increased (p less than 0.001) compared to controls. At 13 mg/kg similar effects were observed but less pronounced. The decreased PCr/ATP ratio may indicate an increased ADP concentration and altered regulation of energy metabolism. Differences between control and treated groups in RPP and Pi/PCr ratio during pacing may also be related to cardiotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)