Challenges of using MR spectroscopy to detect neural progenitor cells in vivo.

A recent report of detection of neural progenitor cells (NPCs) in living human brain by using in vivo proton MR spectroscopy ((1)H-MR spectroscopy) has sparked great excitement in the field of biomedicine because of its potential influence and utility in clinical neuroscience research. On the other hand, the method used and the findings described in the report also caused heated debate and controversy. In this article, we will briefly detail the reasons for the debate and controversy from the point of view of the in vivo (1)H-MR spectroscopy methodology and will propose some technical strategies in both data acquisition and data processing to improve the feasibility of detecting NPCs in future studies by using in vivo (1)H-MR spectroscopy.

Time-dependent effects of imatinib in human leukaemia cells: a kinetic NMR-profiling study.

The goal of this study was to evaluate the time course of metabolic changes in leukaemia cells treated with the Bcr-Abl tyrosine kinase inhibitor imatinib. Human Bcr-Abl(+) K562 cells were incubated with imatinib in a dose-escalating manner (starting at 0.1 microM with a weekly increase of 0.1 microM imatinib) for up to 5 weeks. Nuclear magnetic resonance spectroscopy and liquid-chromatography mass spectrometry were performed to assess a global metabolic profile, including glucose metabolism, energy state, lipid metabolism and drug uptake, after incubation with imatinib. Initially, imatinib treatment completely inhibited the activity of Bcr-Abl tyrosine kinase, followed by the inhibition of cell glycolytic activity and glucose uptake. This was accompanied by the increased mitochondrial activity and energy production. With escalating imatinib doses, the process of cell death rapidly progressed. Phosphocreatine and NAD(+) concentrations began to decrease, and mitochondrial activity, as well as the glycolysis rate, was further reduced. Subsequently, the synthesis of lipids as necessary membrane precursors for apoptotic bodies was accelerated. The concentrations of the Kennedy pathway intermediates, phosphocholine and phosphatidylcholine, were reduced. After 4 weeks of exposure to imatinib, the secondary necrosis associated with decrease in the mitochondrial and glycolytic activity occurred and was followed by a shutdown of energy production and cell death. In conclusion, monitoring of metabolic changes in cells exposed to novel signal transduction modulators supplements molecular findings and provides further mechanistic insights into longitudinal changes of the mitochondrial and glycolytic pathways of oncogenesis.

1H-NMR metabolic profiling of human neonatal urine.

OBJECT: The measurement of different urine components and their changes over time may provide comprehensive and early information about perinatal metabolic processes and physiological changes. We hypothesized that (1) H-NMR-spectroscopy generating a complex spectral profile without pre-selection of urinary metabolites could identify metabolites determining the neonatal physiological status and discriminating between different metabolic states. MATERIALS AND METHODS: We studied spot urine of three groups of neonates (healthy term-born, term-born with non-specific bacterial infections, and preterm neonates) for the first 6 days of life using (1) H-NMR-spectroscopy. In the group of healthy neonates metabolites changing were identified and their excretion patterns compared between groups. RESULTS: Six metabolites indicating physiological changes were identified: N-methylnicotinamide (NAD (+)-pathway), formate, hippurate, betaine (kidney development), taurine (neuronal development), and bile acids (hepatic clearance). While the dynamic changes over the first 6 days were the same for all metabolites in both groups of term-born neonates, the excretion of N-methylnicotinamide and taurine was significantly higher in preterm neonates compared to healthy term neonates and neonates with bacterial infections from the third day after birth (P < 0.05). CONCLUSION: Urine analysis using (1) H-NMR-spectroscopy could identify markers for perinatal metabolic changes. Further studies have to clarify if the proposed physiological interpretation will correlate with long-term physiological development.

Effects of environmental hypercapnia on animal physiology: a 13C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus.

Global climate change is associated with a progressive rise in ocean CO(2) concentrations (hypercapnia) and, consequently, a drop in seawater pH. However, a comprehensive picture of the physiological mechanisms affected by chronic CO(2) stress in marine biota is still lacking. Here we present an analysis of protein biosynthesis rates in isolated muscle of the marine invertebrate Sipunculus nudus, a sediment dwelling worm living at various water depths. We followed the incorporation of (13)C-labelled phenylalanine into muscular protein via high-resolution NMR spectroscopy. Protein synthesis decreased by about 60% at a medium pH of 6.70 and a consequently lowered intracellular pH (pHi). The decrease in protein synthesis rates is much stronger than the concomitant suppression of protein degradation (60% versus 10-15%) possibly posing a threat to the cellular homeostasis of structural as well as functional proteins. Considering the progressive rise in ocean CO(2) concentrations, permanent disturbances of cellular protein turnover might seriously affect growth and reproductive performance in many marine organisms with as yet unexplored impacts on species density and composition in marine ecosystems.

Effect of TEGDMA on the intracellular glutathione concentration of human gingival fibroblasts.

Previous studies revealed that primarily small and relatively hydrophilic comonomers, such as TEGDMA, leach out of resin-based restorative materials into aqueous media. Subsequently, these compounds may cause detrimental reactions with intracellular metabolic systems. The present experiments attempted to elucidate the interactions of TEGDMA with the important intracellular reducing agent glutathione (GSH). The influence of various concentrations of TEGDMA (0.5-7.5 mM) on viability and intracellular GSH concentration of primary human gingival fibroblasts was determined by means of a fluorescence assay (monobromobimane) performed in microtiter plates. Cells were treated with TEDGMA between 2 and 24 h. The incubation of fibroblasts with TEGDMA even at subtoxic concentrations quickly decreased the intracellular glutathione level to 30-50% of controls within the first 2-6 hours. However, no simultaneous adverse effect on cell viability was found. Longer incubation periods up to 24 h caused a regulatory reincrease at TEGDMA concentrations

Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain.

Clinical studies have shown enhancement of cyclosporine toxicity when co-administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the effects of sirolimus with those of the structurally related RAD. Two-week-old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg(-1) d(-1)), III. sirolimus (3 mg kg(-1) d(-1)), IV. RAD (3 mg kg(-1) d(-1)), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78+/-6% of control; GABA: 67+/-12%; NAD(+): 76+/-3%; P<0.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81+/-3% of control, RAD: 69+/-2%; P<0.02). Sirolimus enhanced cyclosporine-induced inhibition of mitochondrial glucose metabolism (glutamate: 60+/-4%; GABA: 59+/-8%; NAD(+): 45+/-5%; P<0.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast, RAD antagonized the effects of cyclosporine (glutamate, GABA, and NAD(+), not significantly different from controls). The results can partially be explained by pharmacokinetic interactions: co-administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co-administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their effects on brain high-energy metabolism and tissue distribution in the rat.

13C isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes.

After incubation of glial cells with both (13)C-labeled and unlabeled glucose and alanine, (13)C isotopomer analysis indicates two cytosolic pyruvate compartments in astrocytes. One pyruvate pool is in an exchange equilibrium with exogenous alanine and preferentially synthesizes releasable lactate. The second pyruvate pool, which is of glycolytic origin, is more closely related to mitochondrial pyruvate, which is oxidized via tri carbonic acid (TCA) cycle activity. In order to provide 2-oxoglutarate as a substrate for cytosolic alanine aminotransferase, glycolytic activity is increased in the presence of exogenous alanine. Furthermore, in the presence of alanine, glutamate is accumulated in astrocytes without subsequent glutamine synthesis. We suggest that the conversion of alanine to releasable lactate proceeds at the expense of flux of glycolytic pyruvate through lactate dehydrogenase, which is used for ammonia fixation by alanine synthesis in the cytosol and for mitochondrial TCA cycle activity. In addition, an intracellular trafficking occurs between cytosol and mitochondria, by which these two cytosolic pyruvate pools are partly connected. Thus, exogenous alanine modifies astrocytic glucose metabolism for the synthesis of releasable lactate disconnected from glycolysis. The data are discussed in terms of astrocytic energy metabolism and the metabolic trafficking via a putative alanine-lactate shuttle between astrocytes and neurons.

Metabolic effects of dental resin components in vitro detected by NMR spectroscopy.

Earlier studies have shown that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) and the photostabilizer 2-hydroxy-4-methoxybenzophenone (HMBP) are cytotoxic and inhibit cell growth. It was the aim of this study to elucidate the underlying metabolic effects of TEGDMA and HMBP on immortal contact-inhibited Swiss albino mouse embryo cells (3T3 fibroblasts) by nuclear magnetic resonance (NMR) spectroscopy. Cell extracts and culture media were analyzed by NMR spectroscopy for metabolic changes after incubation for 24 hours with ED20-concentrations of TEGDMA and HMBP. TEGDMA could be detected in all fractions (cytosol, lipid fractions, and culture media) of 3T3 cells, while HMBP was found only in the lipid fraction accumulated at a maximum rate (51 nmol/mg DNA) compared with TEGDMA (27 nmol/mg DNA). TEGDMA increased the concentration of phosphomonoesters to 180+/-36% and decreased the phosphodiesters to 65+/-5% of controls (control = 100%). Thus, the turnover of phospholipids was enhanced, whereas content and composition of phospholipids of membranes did not alter markedly. Additionally, TEGDMA changed the metabolic state of cells, indicated by slight decreases of nucleoside triphosphates and an increase in the ratio of nucleoside diphosphates to nucleoside triphosphates, while HMBP had no effect. The most remarkable effect of TEGDMA was a nearly complete decline of the intracellular glutathione levels. Analysis of our data shows that NMR spectroscopy of cell-material interactions may reveal metabolic effects of organic test substances which are not detectable by standard in vitro assays. The comonomer TEGDMA affected the metabolism of the cells on different levels, while HMBP accumulated in the lipid fraction and induced significantly fewer effects on cell metabolism.

Magnetization transfer MRS.

This review deals with magnetization transfer (MT) effects observed in in vivo NMR spectroscopy. The basic experimental methods of MT experiments, the underlying kinetic mechanisms as well as the evaluation of measured data by fits to two- or three-pool models are described. Experimental results of both (31)P and (1)H in vivo MRS are reviewed showing the potential of MT experiments to characterize kinetic equilibrium reactions. This includes reactions where all involved components are MR visible, as well as situations where one indirectly measures pools of bound spins which cannot directly be observed in vivo. In particular, MT effects are described which have been observed in in vivo (1)H NMR spectra measured on the animal or human brain or on skeletal muscle. Possible mechanisms for the strong MT effects observed for the signals of creatine/phosphocreatine, lactate, alcohol and other metabolites are discussed. It is also emphasized that MT effects caused by water suppression techniques may lead to systematic errors in the quantification of in vivo (1)H NMR spectra.

Changes in apparent diffusion coefficients of metabolites in rat brain after middle cerebral artery occlusion measured by proton magnetic resonance spectroscopy.

Diffusion-weighted proton MR spectroscopy and imaging have been applied to a rat brain model of unilateral middle cerebral artery occlusion between 1 and 4 hr post occlusion. Similar apparent diffusion coefficients (ADC) of most metabolites were observed within each hemisphere. In the ischemic ipsilateral hemisphere, the ADCs were (0.083--0.116). 10(-3) mm(2)/sec for lactate (Lac), alanine (Ala), gamma-amino butyric acid (GABA), N-acetyl aspartate (NAA), glutamine (Gln), glutamate (Glu), total creatine (tCr), choline-containing compounds (Cho), and myo-inositol (Ins), in the contralateral hemisphere (0.138--0.158). 10(-3) mm(2)/sec for NAA, Glu, tCr, Cho, and Ins. Higher ADCs was determined for taurine (Tau) in the ipsilateral (0.144. 10(-3) mm(2)/sec) and contralateral (0.198. 10(-3) mm(2)/sec) hemisphere. In the ischemic hemisphere, a relative ADC decrease to 65--75% was observed for NAA, Glu, tCr, Cho, Ins and Tau, which was similar to the decrease of the water ADC (to 67%). The results suggest a common cause of the observed ADC changes and provide a broader experimental basis to evaluate theories of water and metabolite diffusion. Magn Reson Med 45:383-389, 2001.

NMR spectroscopic study on the metabolic fate of [3-(13)C]alanine in astrocytes, neurons, and cocultures: implications for glia-neuron interactions in neurotransmitter metabolism.

Nuclear magnetic resonance (NMR) spectroscopy and biochemical assays were used to study the fate of [3-(13)C]alanine in astrocytes, neurons, and cocultures. (1)H- and (13)C-NMR analysis of the media demonstrated a high and comparable uptake of [3-(13)C]alanine by the cells. Thereafter, alanine is transaminated predominantly to [3-(13)C]pyruvate, from which the (13)C-label undergoes different metabolic pathways in astrocytes and neurons: Lactate is almost exclusively synthesized in astrocytes, while in neurons and cocultures labeled neurotransmitter amino acids are formed, i.e., glutamate and gamma-aminobutyric acid (GABA). A considerable contribution of the anaplerotic pathway is observed in cocultures, as concluded from the ratio (C-2-C-3)/C-4 of labeled glutamine. Analysis of the multiplet pattern of glutamate isotopomers indicates carbon scrambling through the TCA cycle and the use of alanine also as energy substrate in neurons. In cocultures, astrocyte-deduced lactate and unlabeled exogenous carbon substrates contribute to glutamate synthesis and dilute the [2-(13)C]acetyl-CoA pool by 30%. The coupling of neuronal activity with shuttling of tricarboxylic acid (TCA) cycle-derived metabolites between astrocytes and neurons is concluded from the use of [4-(13)C]-monolabeled glutamate leaving the first TCA cycle turn already for glutamine and GABA synthesis, as well as from the labeling pattern of extracellular glutamine. Further evidence of a metabolic interaction between astrocytes and neurons is obtained, as alanine serves as a carbon and nitrogen carrier through the synthesis and regulated release of lactate from astrocytes for use by neurons. Complementary to the glutamine-glutamate cycle in the brain, a lactate-alanine shuttle between astrocytes and neurons would account for the nitrogen exchange of the glutamatergic neurotransmitter cycle in mammalian brain.

Effects of ammonia exposition on glioma cells: changes in cell volume and organic osmolytes studied by diffusion-weighted and high-resolution NMR spectroscopy.

NH(4)Cl (10 mM) caused a sustained increase in the cell volume in immobilized, perfused F98 glioma cells to approx. 125% of control after 3 h, as measured by diffusion-weighted (1)H NMR spectroscopy. Concomitantly, the glutamine (Gln) concentration increased by 130%, accompanied by a marked decrease in cytosolic osmolytes, i.e. myo-inositol and taurine, determined from (1)H NMR spectra of PCA extracts. Inhibition of Gln synthetase partially prevented the increase in water content. While losses of organic osmolytes are also observed under hypotonic conditions, the rapid cell swelling is followed by the regulatory cell volume decrease (RVD), and is accompanied by decreased cytosolic Gln. We suggest that the rise in intracellular osmolarity, which is attributed to NH(4)Cl metabolism to Gln, but also to alanine (Ala), is not compensated by the release of other osmolytes, and causes cell swelling without RVD.

A new method for fast proton spectroscopic imaging: spectroscopic GRASE.

A new fast spectroscopic imaging method is presented which allows both a very short minimum total measurement time and effective homonuclear decoupling. After each excitation, all data points from N(GE) k(x)-k(y)-slices at different k(omega)-values are acquired by using a gradient and spin echo (GRASE) imaging sequence. The delay between consecutive gradient echoes, which are measured with uniform phase encoding between consecutive refocusing alpha-pulses, is the inverse of the spectral width (SW). A refocusing 180 degrees pulse, which is applied within a constant delay between excitation and the GRASE sequence, is shifted in a series of measurements by an increment N(GE)/(2 * SW) to cover the whole k(omega)-k(x)-k(y)-space. Spectroscopic GRASE was implemented on a 4.7 T imaging system and tested on phantoms and normal rat brain in vivo. Measurements were performed with a nominal voxel size of 1.5 x 1.5 x 3 mm(3) and a spatial 64 x 64 matrix. The total measurement time was 2 or 4 min using a repetition time of 1.9 sec, 96 chemical shift encoding steps, SW = 800 Hz, N(GE) = 3, and 2 or 4 accumulations.

Fast echo planar based correlation-peak imaging: demonstration on the rat brain in vivo.

Two-dimensional correlation spectroscopy (2D-COSY) was combined with a fast echo planar based spectroscopic imaging technique in a new sequence. It can be optimized according to the coupling patterns of particular metabolites by using a constant time (CT) variant of COSY with chemical shift selective excitation and refocusing. Experiments were performed with an evolution time of 110 ms which was determined by simulating the CT-COSY experiment at several evolution times for the spin systems of myo-inositol (Ins) and taurine (Tau). The sequence has a minimum total measurement time of 17 min and was tested on a spherical phantom filled with a solution of Ins. The in vivo application of this method on the healthy rat brain demonstrates its improved spectral resolution as cross-peak signals from both Ins and Tau can be separated clearly. Magn Reson Med 44:23-28, 2000.

The novel immunosuppressant SDZ-RAD protects rat brain slices from cyclosporine-induced reduction of high-energy phosphates.

1. SDZ-RAD, 40-O-(2-hydroxyethyl)-rapamycin, is a novel macrolide immunosuppressant. Because of its synergistic interaction, SDZ-RAD is under clinical investigation as immunosuppressant in combination with cyclosporine after organ transplantation. Neurotoxicity is a critical side-effect of cyclosporine. 2. We studied the effect of SDZ-RAD and its combination with cyclosporine on high-energy phosphates, phosphocreatine (PCr) and nucleoside triphosphates (NTP), in brain slices using 31P-magnetic resonance spectroscopy (MRS). 3. Cyclosporine significantly reduced high-energy phosphates after 2 h in a dose-dependent manner (100 micrograms l-1: 93 +/- 3% of control (NTP), 91 +/- 3% (PCr); 500 micrograms l-1: 84 +/- 2% (NTP), 73 +/- 2 (PCr); 5000 micrograms l-1: 68 +/- 3% (NTP), 55 +/- 5% (PCr); n = 6; P < 0.02). 4. In contrast, after perfusion for 2 h, SDZ-RAD (500 micrograms l-1 and 5000 micrograms l-1) significantly increased high-energy phosphate concentrations in the brain slices (P < 0.02). Even at the lowest concentration, SDZ-RAD protected brain energy metabolism against cyclosporine toxicity: 100 micrograms l-1 SDZ-RAD + 5000 micrograms l-1 cyclosporine: 86 +/- 3% (NTP), 83 +/- 7% (PCr), n = 3, P < 0.03 compared to cyclosporine alone. 5. As evaluated using an algorithm based on Loewe isobolograms, the effects of SDZ-RAD/cyclosporine combinations on brain energy reduction were antagonistic. Both drugs were found in mitochondria using h.p.l.c-MS analysis. 6. We conclude that cyclosporine inhibits mitochondrial high-energy phosphate metabolism, which can be antagonized by SDZ-RAD.

A fast variant of (1)H spectroscopic U-FLARE imaging using adjusted chemical shift phase encoding.

So far, fast spectroscopic imaging (SI) using the U-FLARE sequence has provided metabolic maps indirectly via Fourier transformation (FT) along the chemical shift (CS) dimension and subsequent peak integration. However, a large number of CS encoding steps N(omega) is needed to cover the spectral bandwidth and to achieve sufficient spectral resolution for peak integration even if the number of resonance lines is small compared to N(omega) and even if only metabolic images are of interest and not the spectra in each voxel. Other reconstruction algorithms require extensive prior knowledge, starting values, and/or model functions. An adjusted CS phase encoding scheme (APE) can be used to overcome these drawbacks. It incorporates prior knowledge only about the resonance frequencies present in the sample. Thus, N(omega) can be reduced by a factor of 4 for many (1)H in vivo studies while no spectra have to be reconstructed, and no additional user interaction, prior knowledge, starting values, or model function are required. Phantom measurements and in vivo experiments on rat brain have been performed at 4.7 T to test the feasibility of the method for proton SI.

Lipid oxidation in blood plasma of patients with neurological disorders.

Nuclear magnetic resonance (NMR) spectra of blood plasma lipids from lyophilized plasma samples from patients with neurological disorders stored for several weeks in an evacuated exsiccator show characteristic differences compared to freshly lyophilized plasma samples. The main differences concern the unsaturated fatty acids, e.g., the extent of unsaturation and their structural composition. The total amount of double bond signals of unsaturated fatty acids are noticeably reduced in intensity and new signals arise from conjugated double bonds. These signals can be assigned to keto-octadecadienoic acid (KODE) or hydroxy-octadecadienoic acid (HODE). The proton and carbon NMR chemical shifts and their structural assignment to the main molecular components are given. Whereas the KODE and HODE signals occur only as storage artifacts in the spectra, we have found small amounts of 9,11-octadecadienoic acid also in fresh blood plasma of controls. Its concentration is about 60 microM. In two-dimensional H,H total correlation spectroscopy spectra also a very low amount (6-7 microM) of 13-HODE can be detected.

Functional MRI of the human motor cortex using single-shot, multiple gradient-echo spiral imaging.

In this study, we combined the advantages of a fast multi-slice spiral imaging approach with a multiple gradient-echo sampling scheme at high magnetic field strength to improve quantification of BOLD and inflow effects and to estimate T2* relaxation times in functional brain imaging. Eight echoes are collected with echo time (TE) ranging from 5 to 180 ms. Acquisition time per slice and echo time is 25 ms for a nominal resolution of 4 x 4 x 4 mm3. Evaluation of parameter images during rest and stimulation yields no significant activation on the inflow sensitive spin-density images (rho or I0-maps) whereas clear activation patterns in primary human motor cortex (M1) and supplementary motor area (SMA) are detected on BOLD sensitive T2*-maps. The calculation of relaxation times and rates of the activated areas over all subjects yields an average T2* +/- standard deviation (SD) of 46.1+/-4.5 ms (R2* of 21.8+/-2.2 s(-1)) and an average increase (deltaT2* +/- SD) of 0.93+/-0.47 ms (deltaR2* of -0.4+/-0.14 s(-1)). Our findings demonstrate the usefulness of a multiple gradient echo data acquisition approach in separating various vascular contributions to brain activation in fMRI.

Oxidative stress-induced metabolic alterations in rat brain astrocytes studied by multinuclear NMR spectroscopy.

Oxidative stress in cultured astrocytes exerted by 30-min treatment with 50-200 microM H(2)O(2) caused time- and concentration-dependent effects on cellular metabolism. These changes were accompanied by alterations in cellular morphology. Using (31)P nuclear magnetic resonance (NMR) spectroscopy, the data demonstrate that the energy status of the cells was greatly affected directly after the stress, as indicated by the loss of high energy phosphates, i.e., phosphocreatine (PCr) and nucleoside triphosphates (NTP). Oxidative stress also involves a dysregulation of the osmotic control in astrocytes, which is accompanied by a dramatic loss of myo-inositol, taurine, and hypotaurine, as monitored by (1)H and (13)C NMR spectroscopy. While the energy state of the cells was essentially restored during a 7-hr recovery period, the changes in osmolyte concentrations lasted longer and went on throughout the recovery period. Even after 24-hr recovery, organic osmolyte concentrations were still below the control levels. (13)C NMR spectra of astrocyte cell extracts also demonstrated an enhanced glucose metabolism via the pentose phosphate pathway (PPP) and a reduced glycolysis. Additionally, the appearance of (13)C glutamate points to a distortion of glutamine synthetase (GS), leading to the accumulation of glutamate. Glycolysis as well as GS activity were back to control levels after 7 hr recovery. Thus, in contrast to the energy metabolism, osmoregulatory processes and complex glucose metabolism was impaired not only directly after oxidative stress, but occurred with a later onset during a 2-hr recovery period, and cells only slowly recovered during the next 24 hr.