Cancer metabolism in a snapshot: MRS(I)

The contribution of MRS(I) to the in vivo evaluation of cancer‐metabolism‐derived metrics, mostly since 2016, is reviewed here. Increased carbon consumption by tumour cells, which are highly glycolytic, is now being sampled by ¹³C magnetic resonance spectroscopic imaging (MRSI) following the injection of hyperpolarized [1‐¹³C] pyruvate (Pyr). Hot‐spots of, mostly, increased lactate dehydrogenase activity or flow between Pyr and lactate (Lac) have been seen with cancer progression in prostate (preclinical and in humans), brain and pancreas (both preclinical) tumours. Therapy response is usually signalled by decreased Lac/Pyr ¹³C‐labelled ratio with respect to untreated or non‐responding tumour. For therapeutic agents inducing tumour hypoxia, the ¹³C‐labelled Lac/bicarbonate ratio may be a better metric than the Lac/Pyr ratio. ³¹P MRSI may sample intracellular pH changes from brain tumours (acidification upon antiangiogenic treatment, basification at fast proliferation and relapse). The steady state tumour metabolome pattern is still in use for cancer evaluation. Metrics used for this range from quantification of single oncometabolites (such as 2‐hydroxyglutarate in mutant IDH1 glial brain tumours) to selected metabolite ratios (such as total choline to N‐acetylaspartate (plain ratio or CNI index)) or the whole ¹H MRSI(I) pattern through pattern recognition analysis. These approaches have been applied to address different questions such as tumour subtype definition, following/predicting the response to therapy or defining better resection or radiosurgery limits.     

1H and 13C HR-MAS spectroscopy of intact biopsy samples ex vivo and in vivo 1H MRS study of human high grade gliomas

High-resolution magic angle spinning (HR-MAS) one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy has been used to study intact glioblastoma (GBM) brain tumour tissue. The results were compared with in vitro chemical extract and in vivo spectra. The resolution of 1H one-dimensional, 1H TOCSY and 13C HSQC HR-MAS spectra is comparable to that obtained on perchloric extracts. 13C HSQC HR-MAS spectra have been particularly useful for the identification of 37 different metabolites in intact biopsy tumours, excluding water and DSS components. To our knowledge, this is the most detailed assignment of biochemical compounds obtained in intact human tissue, in particular in brain tumour tissue. Tissue degradation during the recording of the NMR experiment was avoided by keeping the sample at a temperature of 4 degrees C. Detailed metabolical compositions of 10 GBM (six primary, two secondary and two unclassified) were obtained. A good correlation between ex vivo and in vivo MRS has been found.     

Proton MRS of oral creatine supplementation in rats. Cerebral metabolite concentrations and ischemic challenge.

Proton magnetic resonance spectroscopy (MRS) was employed to determine the concentrations of N-acetylaspartate (NAA), total creatine (tCr), choline-containing compounds (Cho), myo-inositol (Ins), glucose (Glc), and lactate (Lac) in rat brain before and after 10 days of oral supplementation of 2.6 g Cr-monohydrate per kg body weight per day. Measurements were performed both in vitro (n = 16) and in vivo (n = 6). The neuroprotective potential of oral Cr was assessed by dynamically monitoring brain Glc and Lac in response to transient global ischemia (12 min). In comparison to controls the in vitro concentrations of Cr (13.1 +/- 9.3%) and Ins (12.7 +/- 14. 0%) were significantly increased in Cr-fed rats. Under in vivo conditions, the data revealed trends for elevated tCr (4.7%) and Ins (10.6%) which were enhanced in the concentration ratios of tCr:Cho (10.2%) and Ins:Cho (17.8%). Together with an increased Glc level (27.3%), the observation of a statistically significant decrease of brain Lac (-38.5 +/- 19.3%) in Cr-fed rats may reflect a shift of the energy metabolism from non-oxidative toward oxidative glycolysis. One hour after global ischemia most of the metabolic differences between Cr-fed rats and controls were retained. The increased Glc level (44.4 +/- 33.3%) reached statistical significance, but the accumulation of Lac and its time course during ischemia and early reperfusion showed no differences between Cr-fed rats and controls.     

Impact of intratumoral heterogeneity on the metabolic profiling of breast cancer tissue using high-resolution magic angle spinning magnetic resonance spectroscopy

High-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy (MRS) is a useful metabolic profiling technique for human tissue. However, the impact of intratumoral heterogeneity on the metabolite levels of breast cancers is not yet established. The purpose of this prospective study was to investigate whether the tumor cell fraction of core needle biopsy (CNB) specimens of breast cancers affect metabolic profiles assessed with HR-MAS MRS. From June 2015 to December 2016, 46 patients with 47 breast cancers were enrolled. HR-MAS MRS was used for the metabolic profiling of 285 CNB specimens from the 47 cancers. Multiple CNB samples (range 2–8) for the HR-MAS MRS experiment were obtained from surgical specimens under ultrasound guidance following surgical removal of the tumor. Tumor cell fraction was expressed as a percentage of the tumor cell volume relative to the total tumor volume contained in each CNB sample. Metabolite quantification levels were compared according to primary tumor characteristics using the t-test. Multivariate analyses were performed including primary tumor characteristics and tumor cell percentages as variables. Correlations between tumor cell percentage and metabolite levels in the CNB specimens were assessed according to the immunohistochemical status of the primary tumor. In univariate analysis, levels of choline-containing compounds, glutamate, glutamine, glycine, serine, and taurine were correlated with primary tumor characteristics. In multivariate analysis, most metabolite levels were not affected by tumor cell percentage. Tumor cell percentage showed poor correlation with metabolite levels in hormone receptor-positive cancer and triple-negative cancer, and poor to fair correlation with metabolite levels in HER2-positive cancer. This study showed that differences in the tumor cell fraction of CNB samples do not affect predictions on the primary cancer from which the samples are obtained.     

Investigation of metabolite changes in the transition from pre-invasive to invasive cervical cancer measured using (1)H and (31)P magic angle spinning MRS of intact tissue

The aim of this study was to determine the metabolic changes in the transition from pre-invasive to invasive cervical cancer using high-resolution magic angle spinning (HR-MAS) MRS. Biopsy specimens were obtained from women with histologically normal cervix (n = 5), cervical intraepithelial neoplasia (CIN; mild, n = 5; moderate/severe, n = 40), and invasive cancer (n = 23). (1)H HR-MAS MRS data were acquired using a Bruker Avance 11.74 T spectrometer (Carr-Purcell-Meiboom-Gill sequence; TR = 4.8 s; TE = 135 ms; 512 scans; 41 min acquisition). (31)P HR-MAS spectra were obtained from the normal subjects and cancer patients only (as acetic acid applied before tissue sampling in patients with CIN impaired spectral quality) using a (1)H-decoupled pulse-acquire sequence (TR = 2.82 s; 2048 scans; 96 min acquisition). Peak assignments were based on values reported in the literature. Peak areas were measured using the AMARES algorithm. Estimated metabolite concentrations were compared between patient diagnostic categories and tissue histology using independent samples t tests. Comparisons based on patient category at diagnosis showed significantly higher estimated concentrations of choline (P = 0.0001) and phosphocholine (P = 0.002) in tissue from patients with cancer than from patients with high-grade dyskaryosis, but no differences between non-cancer groups. Division by histology of the sample also showed increases in choline (P = 0.002) and phosphocholine (P = 0.002) in cancer compared with high-grade CIN tissue. Phosphoethanolamine was increased in cancer compared with normal tissue (P = 0.0001). Estimated concentrations of alanine (P = 0.01) and creatine (P = 0.008) were significantly reduced in normal tissue from cancer patients compared with normal tissue from non-cancer patients. The estimated concentration of choline was significantly increased in CIN tissue from cancer patients compared with CIN tissue from non-cancer patients (P = 0.0001). Estimated concentrations of choline-containing metabolites increased from pre-invasive to invasive cervical cancer. Concurrent metabolite depletion occurs in normal tissue adjacent to cancer tissue.     

Decreased lactate concentration and glycolytic enzyme expression reflect inhibition of mTOR signal transduction pathway in B‐cell lymphoma

The application of kinase inhibitors in cancer treatment is growing rapidly. However, methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on the tracking of changes in the tumor volume, a rather late and relatively insensitive marker of the therapeutic response. In contrast, MRS can detect changes in cell metabolism and has the potential to provide early and patient‐specific markers of drug activity. Using human B‐cell lymphoma models and MRS, we have demonstrated that the inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by the measurement of lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in lymphoma cell line cultures and also diminished steady‐state lactate levels in xenotransplants. The inhibition was time dependent and was first detectable 8 h after drug administration in cell cultures. In xenotransplants, 2 days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo, which were followed by tumor growth arrest and tumor volume regression. The rapamycin‐induced changes in lactate production were strongly correlated with the inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or ¹⁸F‐fluorodeoxyglucose positron emission tomography (FDG PET) detection of changes in glucose metabolism may represent effective noninvasive methods for the monitoring of mTOR targeting therapy in lymphomas and other malignancies. Furthermore, the measurement of glucose metabolic inhibition by MRS or FDG PET imaging may also prove to be effective in monitoring the efficacy of other kinase inhibitors given that the rapamycin‐sensitive mTOR lies downstream of many oncogenic signaling pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Ketone bodies effectively compete with glucose for neuronal acetyl‐CoA generation in rat hippocampal slices

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1‐¹³C]glucose and 1.0 mM [2‐¹³C]lactate + 0.1 mM pyruvate (combined [2‐¹³C]lactate + [2‐¹³C]pyruvate) to acetyl‐CoA production were measured both without and with 5.0 mM [U‐¹³C]3‐hydroxybutyrate in superfused rat hippocampal slices by ¹³C NMR non‐steady‐state isotopomer analysis of tissue glutamate and GABA. Without [U‐¹³C]3‐hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl‐CoA, respectively. With [U‐¹³C]3‐hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U‐¹³C]3‐hydroxybutyrate became the major acetyl‐CoA contributor (68 ± 3%) – about three‐times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U‐¹³C]3‐hydroxybutyrate contributed approximately the same acetyl‐CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1‐¹³C]glucose did not decrease significantly in the presence of 3‐hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3‐hydroxybutyrate. This indicates that, under these conditions, 3‐hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 John Wiley & Sons, Ltd.     

Hyperpolarized 13C‐lactate to 13C‐bicarbonate ratio as a biomarker for monitoring the acute response of anti‐vascular endothelial growth factor (anti‐VEGF) treatment

Hyperpolarized [1‐¹³C]pyruvate MRS provides a unique imaging opportunity to study the reaction kinetics and enzyme activities of in vivo metabolism because of its favorable imaging characteristics and critical position in the cellular metabolic pathway, where it can either be reduced to lactate (reflecting glycolysis) or converted to acetyl‐coenzyme A and bicarbonate (reflecting oxidative phosphorylation). Cancer tissue metabolism is altered in such a way as to result in a relative preponderance of glycolysis relative to oxidative phosphorylation (i.e. Warburg effect). Although there is a strong theoretical basis for presuming that readjustment of the metabolic balance towards normal could alter tumor growth, a robust noninvasive in vivo tool with which to measure the balance between these two metabolic processes has yet to be developed. Until recently, hyperpolarized ¹³C‐pyruvate imaging studies had focused solely on [1‐¹³C]lactate production because of its strong signal. However, without a concomitant measure of pyruvate entry into the mitochondria, the lactate signal provides no information on the balance between the glycolytic and oxidative metabolic pathways. Consistent measurement of ¹³C‐bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of ¹³C‐bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized ¹³C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of ¹³C‐lactate to ¹³C‐bicarbonate provides a more robust metric relative to ¹³C‐lactate for the assessment of the metabolic effects of anti‐angiogenic therapy. Our data suggest a potential application of this ratio as an early biomarker to assess therapeutic effectiveness. Furthermore, although further study is needed, the results suggest that anti‐angiogenic treatment results in a rapid normalization in the relative tissue utilization of glycolytic and oxidative phosphorylation by tumor tissue. Copyright © 2016 John Wiley & Sons, Ltd.     

In vivo 1H MRS of WSU‐DLCL2 human non‐Hodgkin's lymphoma xenografts: response to rituximab and rituximab plus CHOP

In order to identify early ¹H MRS metabolic markers of response to rituximab immunotherapy and to rituximab plus CHOP (cyclophosphamide, hydroxydoxorubicin, vincristine, and prednisone) combination therapy, we performed an in vivo MRS investigation of a non‐Hodgkin's lymphoma (NHL) xenograft model. Human WSU‐DLCL2 NHL cells were subcutaneously implanted into flanks of female severe combined immunodeficient mice. When tumor volumes reached ～600 mm³, rituximab was administered for three weekly cycles at a dose of 25 mg/kg per cycle with or without CHOP. Before and after treatment, tumor lactate (Lac) and total choline (tCho) were detected using the selective multiple quantum coherence sequence and the stimulated echo acquisition mode sequence, respectively. Rituximab produced a small tumor growth delay (～5 days), whereas treatment with rituximab plus CHOP (RCHOP) led to ～20% tumor regression after three cycles of therapy. After one cycle of rituximab, the tCho/H₂O ratio had decreased significantly (5%, P = 0.003), whereas the Lac/H₂O ratio had not changed (P = 0.58). Both Lac/H₂O and tCho/H₂O had decreased after one cycle of RCHOP treatment (26%, P = 0.001; 10%, P = 0.016, respectively). After two cycles of RCHOP, Ki67 assay of histological tumor specimens indicated ～40% decrease in proliferation (P < 0.001) in the RCHOP‐treated tumors; no change was detected after treatment with rituximab alone. This study suggests that decreases in tCho/H₂O are more sensitive indices of response to rituximab, whereas decreases in Lac/H₂O are more sensitive to response to CHOP combination therapy. Copyright © 2008 John Wiley & Sons, Ltd.     

Effects of endurance training on the gluconeogenic capacity of periportal and perivenous hepatocytes

This study was undertaken to assess the effect of 8 weeks of endurance training (treadmill, last 4 weeks: 60 min at 26 m/min, 8-10% grade) on the gluconeogenic capacity of periportal (PP-H) and perivenous (PV-H) hepatocytes of overnight fasted rats. Isolated PP-H and PV-H, obtained by selective destruction after liver perfusion with digitonin and collagenase, were incubated with saturating concentrations of a mixture of lactate and pyruvate (20:2 mM; Lac+Pyr) or alanine (20 mM; Ala) to determine the glucose production flux (J(glucose)) in the incubation medium. Endogenous J(glucose) as well as J(glucose) from substrates were significantly higher (P<.05) in PP-H than PV-H in the untrained state. Following training, a selective increase (P<.05) in J(glucose) from endogenous substrates and from Lac+Pyr was observed in PV-H only, resulting in the disappearance (P>.05) of the difference of J(glucose) between PP-H and PV-H. It is concluded that the increase in the gluconeogenic capacity of the liver following endurance training is first observed in PV-H.     

Biochemical characterization of muscle tissue of limb girdle muscular dystrophy: an 1H and 13C NMR study

The metabolic differences between the muscle biopsies of patients with limb girdle muscular dystrophy (LGMD) and normal controls were characterized using high-resolution 1H and 13C NMR spectroscopy. In all, 44 metabolites were unambiguously assigned in the perchloric acid extracts of skeletal muscle tissue, using 2D double quantum filtered (DQF COSY), total correlation (TOCSY), and 1H/13C heteronuclear multiple quantum coherence (HMQC) spectroscopy. The concentrations of glycolytic substrate, glucose (p=0.03), gluconeogenic amino acids, glutamine (p=0.02) and alanine (p=0.009) together with glycolytic product, lactate (p=0.04), were found to be significantly lowered in LGMD patients as compared with controls. The reduction in the concentration of glucose may be attributed to the decrease in the concentration of gluconeogenic amino acids in the degenerated muscle. Reduction in the rate of anaerobic glycolysis and lowered substrate concentration appear to be the possible reasons for the decrease in the concentration of lactate. A significant reduction in the concentration of choline in LGMD patients was also observed compared with controls. Lower concentration of choline may be the result of decreased rate of membrane turnover in LGMD patients. The data presented here provide an insight into the potentials of in-vitro NMR spectroscopy in the study of muscle metabolism.     

MR‐detectable metabolic consequences of mitogen‐activated protein kinase kinase (MEK) inhibition

Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS‐detectable metabolic consequences of mitogen‐activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF‐7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS‐detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF‐7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized ¹³C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3‐kinase pathway and AMP‐activated protein kinase. Our findings demonstrate the value of translatable non‐invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic profile of the hippocampus of Zucker Diabetic Fatty rats assessed by in vivo 1H magnetic resonance spectroscopy

Localized in vivo 1H magnetic resonance spectroscopy (MRS) was used to investigate metabolite levels in the brain of adult Zucker Diabetic Fatty (ZDF) rats, an animal model for type 2 diabetes mellitus. This study focussed on the hippocampus, assumed to be one of the main brain areas affected by this disease. Together with an almost 5-fold increase in blood glucose concentration measured by glucose oxidation, significant increases were found in the hippocampal concentrations of glucose (4.93 vs 1.66 mM p < 0.001), myo-inositol (6.52 vs 4.30 mM; p < 0.05), and total creatine (12.71 vs 10.50 mM; p < 0.05) in ZDF rats (n = 5) compared with littermates (n = 5). Although no obvious alterations were detected in the hippocampal levels of other metabolites, including NAA + NAAG and choline-containing compounds in the ZDF rats, the increase in Glc and Ins levels is in line with elevated brain tissue contents of these metabolites in patients with diabetes mellitus.     

Quantification of metabolites in breast cancer patients with different clinical prognosis using HR MAS MR spectroscopy

Absolute quantitative measures of breast cancer tissue metabolites can increase our understanding of biological processes. Electronic REference To access In vivo Concentrations (ERETIC) was applied to high resolution magic angle spinning MR spectroscopy (HR MAS MRS) to quantify metabolites in intact breast cancer samples. The ERETIC signal was calibrated using solutions of creatine and TSP. The largest relative errors of the ERETIC method were 8.4%, compared to 4.4% for the HR MAS MRS method using TSP as a standard. The same MR experimental procedure was applied to intact tissue samples from breast cancer patients with clinically defined good (n = 13) and poor (n = 16) prognosis. All samples were examined by histopathology for relative content of different tissue types and proliferation index (MIB‐1) after MR analysis. The resulting spectra were analyzed by quantification of tissue metabolites (β‐glucose, lactate, glycine, myo‐inositol, taurine, glycerophosphocholine, phosphocholine, choline and creatine), by peak area ratios and by principal component analysis. We found a trend toward lower concentrations of glycine in patients with good prognosis (1.1 µmol/g) compared to patients with poor prognosis (1.9 µmol/g, p = 0.067). Tissue metabolite concentrations (except for β‐glucose) were also found to correlate to the fraction of tumor, connective, fat or glandular tissue by Pearson correlation analysis. Tissue concentrations of β‐glucose correlated to proliferation index (MIB‐1) with a negative correlation factor (?0.45, p = 0.015), consistent with increased energy demand in proliferating tumor cells. By analyzing several metabolites simultaneously, either in ratios or by metabolic profiles analyzed by PCA, we found that tissue metabolites correlate to patients' prognoses and health status five years after surgery. This study shows that the diagnostic and prognostic potential in MR metabolite analysis of breast cancer tissue is greater when combining multiple metabolites (MR Metabolomics). Copyright © 2010 John Wiley & Sons, Ltd.     

Stimulus-induced brain lactate: effects of aging and prolonged wakefulness

Both aging and sleep deprivation disturb the functions of the frontal lobes. Deficits in brain energy metabolism have been reported in these conditions. Neurons use not only glucose but also lactate as their energy substrate. The physiological response to elevated neuronal activity is a transient increase in lactate concentrations in the stimulated area. We have previously shown that cognitive stimulation increases brain lactate. To study the effect of prolonged wakefulness on the lactate response we designed an experiment to assess brain lactate levels during a 40-h sleep deprivation period in young (19-24 years old; n = 13) and in aged (60-68 years old; n = 12) healthy female volunteers. Brain lactate levels were assessed with proton MR-spectroscopy ((1)H MRS) during the performance of a silent word generation task. The (1)H MRS voxel location was individually selected, using functional magnetic resonance imaging, to cover the activated area in the left frontal lobe. The degree of sleepiness was verified using vigilance tests and self-rating scales. In the young alert subjects, the silent word generation test induced a 40% increase in lactate, but during the prolonged wakefulness period this response disappeared. In the aged subjects, the lactate response could not be detected even in the alert state. We propose that the absence of the lactate response may be a sign of malfunctioning of normal brain energy metabolism. The behavioral effects of prolonged wakefulness and aging may arise from this dysfunction.     

Two-compartment model for determination of glycolytic rates of solid tumors by in vivo 13C NMR spectroscopy

Carbon-13 NMR spectroscopy of 13C enriched substrates is useful for non-invasively determining metabolic fluxes of cells and tissues. Our study demonstrates that for RIF-1 tumor cells, examined under monolayer culture with continuous perfusion and also grown as solid subcutaneously (sc) implanted tumors in vivo, the levels of intracellular glucose and intermediates of the glycolytic pathway are below the level of detection by NMR spectroscopy. For these tumors, glucose transport into the cell is the most probable rate limiting step of the glycolytic pathway. Under these limiting conditions a simple two-compartment model of glycolysis applies. This model yields two parameters: the average rate of glycolysis and the rate of lactate clearance through the vasculature. For the RIF-1 tumor these parameters were 0.022 +/- 0.01 and 0.034 +/- 0.006 min(-1), respectively.     

Online 1H‐MRS measurements of time‐varying lactate production in an animal model of glioma during administration of an anti‐tumoral drug

The aims of this study were to implement a magnetic resonance spectroscopy (MRS) protocol for the online profiling of subnanomolar quantities of metabolites sampled from the extracellular fluid using implanted microdialysis and to apply this protocol in glioma‐bearing rats for the quantification of lactate concentration and the measurement of time‐varying lactate concentration during drug administration. MRS acquisitions on the brain microdialysate were performed using a home‐built, proton‐tuned, microsolenoid with an active volume of 2 μL. The microcoil was placed at the outlet of the microdialysis probe inside a preclinical magnetic resonance imaging (MRI) scanner. C6‐bearing rats were implanted with microdialysis probes perfused with artificial cerebrospinal fluid solution and the lactate dehydrogenase (LDH) inhibitor oxamate. Microcoil magnetic resonance spectra were continuously updated using a single‐pulse sequence. Localized in vivo spectra and high‐resolution spectra on the dialysate were also acquired. The limit of detection and limit of quantification per unit time of the lactate methyl peak were determined as 0.37 nmol/√min and 1.23 nmol/√min, respectively. Signal‐to‐noise ratios (SNRs) of the lactate methyl peak above 120 were obtained from brain tumor microdialysate in an acquisition time of 4 min. On average, the lactate methyl peak amplitude measured in vivo using the nuclear magnetic resonance (NMR) microcoil was 193 ± 46% higher in tumor dialysate relative to healthy brain dialysate. A similar ratio was obtained from high‐resolution NMR spectra performed on the collected dialysate. Following oxamate addition in the perfusate, a monotonic decrease in the lactate peaks was observed in all animals with an average time constant of 4.6 min. In the absence of overlapping NMR peaks, robust profiling of extracellular lactate can be obtained online using a dedicated sensitive NMR microcoil. MRS measurements of the dynamic changes in lactate production induced by anti‐tumoral drugs can be assessed accurately with temporal resolutions on the order of minutes. The MRS protocol can be readily transferred to the clinical environment with the use of suitable clinical microdialysis probes.     

Noninvasive quantification of ascorbate and glutathione concentration in the elderly human brain

In this study, ascorbate (Asc) and glutathione (GSH) concentrations were quantified noninvasively using double-edited (1)H MRS at 4 T in the occipital cortex of healthy young [age (mean ± standard deviation) = 20.4 ± 1.4 years] and elderly (age = 76.6 ± 6.1 years) human subjects. Elderly subjects had a lower GSH concentration than younger subjects (p < 0.05). The Asc concentration was not significantly associated with age. Furthermore, the lactate (Lac) concentration was higher in elderly than young subjects. Lower GSH and higher Lac concentrations are indications of defective protection against oxidative damage and impaired mitochondrial respiration. The extent to which the observed concentration differences could be associated with physiological differences and methodological artifacts is discussed. In conclusion, GSH and Asc concentrations were compared noninvasively for the first time in young vs elderly subjects.     

A rapid inversion technique for the measurement of longitudinal relaxation times of brain metabolites: application to lactate in high‐grade gliomas at 3 T

The aim of this study was to develop a time‐efficient inversion technique to measure the T₁ relaxation time of the methyl group of lactate (Lac) in the presence of contaminating lipids and to measure T₁ at 3 T in a cohort of primary high‐grade gliomas. Three numerically optimized inversion times (TIs) were chosen to minimize the expected error in T₁ estimates for a given input total scan duration (set to be 30 min). A two‐cycle spectral editing scheme was used to suppress contaminating lipids. The T₁ values were then estimated from least‐squares fitting of signal measurements versus TI. Lac T₁ was estimated as 2000 ± 280 ms. After correcting for T₁ (and T₂ from literature values), the mean absolute Lac concentration was estimated as 4.3 ± 2.6 mm . The technique developed agrees with the results obtained by standard inversion recovery and can be used to provide rapid T₁ estimates of other spectral components as required. Lac T₁ exhibits similar variations to other major metabolites observable by MRS in high‐grade gliomas. The T₁ estimate provided here will be useful for future MRS studies wishing to report relaxation‐corrected estimates of Lac concentration as an objective tumor biomarker. Copyright © 2016 John Wiley & Sons, Ltd.     

31P and 13C NMR studies of isolated perfused hematopoietic cells from leukemic mice

The myeloproliferative leukemic virus (MPLV) induces within 2-3 weeks a massive infiltration of the adult mouse liver by hematopoietic leukemic cells. Since the metabolism of the infiltrated organ might be correlated with an interaction of two cell populations, it was decided to study the isolated hematopoietic cells separately. The metabolism of these cells embedded in an agarose gel and perfused with labeled substrates was investigated using 31P and 13C NMR. Using [1-13C]glucose as precursor, sequential 13C NMR spectra showed that the hematopoietic cells were able to store glucose as [1-13C]glycogen and to metabolize it through the glycolytic pathway to give [3-13C]lactate as sole end-product. The liver neoglucogenic substrates: [2-13C]pyruvate and [3-13C]alanine are not metabolized by these cells. This suggests that the tricarboxylic acid cycle was not efficient. To investigate further the glycolytic properties of the cells, 10 mM sodium azide was added to the medium containing [1-13C]glucose. When compared to the aerobic conditions, a 40% decrease of nucleotides (0.10 vs 0.17 mumole NTP/10(9) cells), a degradation of [1-13C]glycogen and an increase of ca 35% of the glycolytic rate were observed. The analysis of 13C NMR spectra of the perfusates at the end of the perfusion indicates a total conversion of [1-13C]glucose into [3-13C]lactate and [3-13C]pyruvate under anaerobic conditions. These results permit a better understanding of the metabolism of the perfused leukemic livers which are extensively infiltrated by these hematopoietic cells.