Cancer stem cell characteristics and their potential as therapeutic targets.

Cancer stem cells (CSCs) are a tumour subpopulation whose capacity for self-renewal, differentiation and proliferation generates unfavourable patient outcomes, including therapeutic resistance and metastasis. Much research has focused on the generation, biomarkers and therapeutic resistance of CSCs, as well as the development of CSC-targeted therapies. Reviews to date have either addressed general CSC characteristics or focused on CSCs from a well-studied cancer. Increasingly, specific treatment plans based on identification of molecular features and biomarkers of a patient's cancer, rather than classification according to tissue origin or bulk tumour properties, are leading to better patient outcomes. Here, we compare CSC characteristics, specifically their biomarkers and molecular features, and identify those that are common to a number of cancers. Identification of CSC markers that suggest therapeutic strategies has led to several successful in vitro and animal tests, recommending clinical trials of treatments with potentially enhanced therapeutic benefits, especially for recurring cancers.

pH and cell volume effects on H2O and phosphoryl resonance splitting in rapid-spinning NMR of red cells.

Two resonances are seen in the (1)H-NMR spectrum of water in erythrocyte suspensions spun at the magic angle, a broad signal from water inside the cells and a sharp signal from extracellular water. The splitting is a result of a true chemical shift difference between the two populations, as bulk magnetic susceptibility effects are negated at the magic angle. The pH dependence of this chemical shift difference in erythrocyte suspensions was investigated. Splittings of 16.7 +/- 0.1, 18.9 +/- 0.9, and 21.0 +/- 0.2 Hz were observed at pH 6.0, 7.0, and 8.5, respectively; however, this was accompanied by a change in the mean cell volume. To account for any contribution from the volume change, the osmolality of the pH 6.0 and 8.5 suspensions was adjusted to equalize the cell volume between samples at the three pHs. Under these conditions, the splitting was 18.3 +/- 0.1 and 18.6 +/- 0.1 Hz at pH 6.0 and 8.5, respectively. Thus the observed chemical shift difference between the two water resonances was independent of pH. Therefore the splitting of the water resonance was concluded to be directly proportional to the protein concentration within the cell. Measurements of the magnetic susceptibility difference between the two compartments were also carried out, yielding a value of 2.0 +/- 0.2 x 10(-7) (SI units) for erythrocytes in isotonic saline at pH 7.0.

A metabolomic approach to ionotropic glutamate receptor subtype function: a nuclear magnetic resonance in vitro investigation.

A range of behaviours are elucidated via ionotropic glutamate receptors (iGluR). In this work, we examined the acute activation of iGluRs by a range of receptor ligands and effectors to see whether distinguishable metabolic sequelae were elucidated by the activity. We used a guinea-pig brain cortical tissue slice model using targeted receptor ligands ((RS)-(tetrazol-5-yl)glycine (TZG), (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801, dizocilpine), cis-4-[phosphomethyl]-piperidine-2-carboxylic acid (CGS 19755), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, (2S, 3S, 4S)-2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetic acid (kainate) and D-serine (D-Ser), as well as compounds (quinolinic acid and kynurenic acid (KynA)) involved in some neuroinflammatory responses. The data were derived using 13C and 1H NMR spectroscopy, and analysed by metabolomic approaches and multivariate statistics. The metabolic effects of agonists at the three major classes of iGluR were easily separated from each other using this method. The classical N-methyl-D-aspartate receptor agonist TZG and the antagonist CGS 19755 produced excitatory and inhibitory metabolic responses, respectively, while the blocker MK-801 resulted in a significant decrease in net metabolism and produced the largest decrease in all metabolite pool sizes seen by any glutamatergic ligand we have studied. Quinolinic acid and KynA produced similar acute metabolic responses, which were unlike those to TZG or CGS 19755, but similar to that of D-Ser. D-Ser was highly stimulatory of net flux into the Krebs cycle. These data show that the metabolic response to iGluR perturbation in vitro is a sensitive discriminator of function.

Sensitizer-mediated photooxidation of histidine residues: evidence for the formation of reactive side-chain peroxides.

Exposure of proteins to visible light in the presence of a sensitizer results in the oxidation of Met, Trp, Tyr, Cys, and His side chains. These reactions are only partially understood, particularly with His. In this study, the oxidation of free His, His derivatives, and His-containing peptides has been examined using visible light and a range of sensitizers. It is shown that photooxidation gives rise to unstable peroxides, in a light-, illumination time-, and sensitizer-dependent manner. The yield of these materials is increased when reactions are carried out in solutions prepared with D2O, which prolongs the lifetime of 1O2, and decreased in the presence of the potent 1O2 scavenger azide, consistent with the involvement of this excited state. These peroxides have half-lives of hours, though the rate of decomposition is enhanced by elevated temperatures, reductants, and metal ions. Reducing metal ions catalyze the formation of radicals, which have been detected by EPR spin trapping. Structural analysis of His photo-products using NMR spectroscopy has provided evidence for the formation of oxygenated and cyclized compounds (e.g., 6a-hydroxy-2-oxo-octahydro-pyrollo[2,3-d]imidazole-5-carboxylic acid) and cross-linked materials. The latter materials may be partly responsible for the high yield of aggregated materials detected on photooxidation of His-containing proteins.

Brain metabolic markers reflect susceptibility status in cytokine gene knockout mice with murine cerebral malaria.

Treatment of cerebral malaria, a complication of the world's most significant parasitic disease, remains problematic due to lack of understanding of its pathogenesis. Metabolic changes, along with cytokine expression alterations and blood cell sequestration in the brain, have previously been reported during severe disease in human infection and mouse models leading to the "cytopathic hypoxia" and "sequestration" theories of pathogenesis. Here, to determine the robustness of the metabolic changes and their relationship to disease development, we investigated changes in cerebral metabolic markers in a mouse model of cerebral malaria (CM) in wildtype (C57BL/6) and cytokine knockout (TNF(-/-), IFNgamma(-/-) and LTalpha(-/-)) mice using multinuclear magnetic resonance spectroscopy. Mice susceptible to CM (wildtype, TNF(-/-)) showed decreased cerebral glucose use, decreased Krebs cycle metabolism and decreased high-energy phosphates. Conversely, mice resistant to CM (IFNgamma(-/-), LTalpha(-/-)) showed little sign of these effects, despite identical levels of parasitemia. Previously reported changes in lactate were shown to be strain dependent. Elevated glutamine and decreased phosphorylation potential emerged as robust metabolic markers of susceptibility, further implicating the trytophan/NAD(+) pathway in disease development. Thus these metabolic changes are firmly linked both to the immune system response to malaria and to the occurrence of pathogenic changes in experimental CM.

Apparatus for rapid adjustment of the degree of alignment of NMR samples in aqueous media: verification with residual quadrupolar splittings in (23)Na and (133)Cs spectra.

NMR spectra of (23)Na(+) and (133)Cs(+) in gelatine in a silicone rubber tube that was stretched to various extents showed remarkably reproducible resonance multiplicity. The relative intensities of the components of the split peaks had ratios, 3:4:3, and 7:12:15:16:15:12:7, respectively, that conformed with those predicted using a Mathematica program. The silicone-rubber tube was sealed at its lower end by a small rubber stopper and placed inside a thick-walled glass tube. Gelatine was injected in solution into the silicone tube and 'set' by cooling below 30 degrees C. A plastic thumb-screw held the silicone tube at various degrees of extension, up to approximately 2-fold. After constituting the gel in buffers containing NaCl and CsCl, both (23)Na and (133)Cs NMR spectroscopy revealed that after stretching the initial single Lorentzian line was split into a well-resolved triplet and a heptet, respectively. This was interpreted as being due to coupling between the electric quadrupoles of the nuclei and the average electric field gradient tensor of the collagen molecules of gelatine; these molecules became progressively more aligned in the direction of the main magnetic field, B(0), of the vertical bore magnet, as the gel was stretched. This apparatus provides a simple way of demonstrating fundamental physical characteristics of quadrupolar cations, some characteristics of gelatine under stretching, and a way to invoke static distortion of red blood cells. It should be useful with these and other cell types, for studies of metabolic and membrane transport characteristics that may change when the cells are distorted, and possibly for structural studies of macromolecules.

Investigation of methaemoglobin reduction by extracellular NADH in mammalian erythrocytes.

The effect of extracellular NADH on the rate of reduction of nitrite-induced methaemoglobin in erythrocytes from man, cattle, dog, horse, grey kangaroo, pig and sheep was investigated. Extracellular NADH was found to enhance the rate of methaemoglobin reduction in man, dog, pig and kangaroo erythrocytes, but had essentially no effect on the rate of methaemoglobin reduction in erythrocytes from cattle, horse and sheep. In erythrocytes of those animals affected by extracellular NADH the rate of reduction of metHb in the presence of NADH was the same or greater than that observed in the presence of nutrients such as glucose and inosine. The combination of nutrient and NADH produced a more profound increase in the rate of methaemoglobin reduction. The rate of methaemoglobin reduction in all cases was significantly less than that observed with methylene blue, the standard treatment of methaemoglobinaemia. Extracellular NADH was found to indirectly increase the intracellular NADH concentration through displacement of the pseudo-equilibrium of the intracellular LDH reaction and relied upon the presence of sufficient LDH activity released into the extracellular medium through haemolysis. The lack of response of cattle, horse and sheep RBCs to extracellular NADH was found to derive mainly from their low extracellular LDH activity, but also correlated with their lower NADH-methaemoglobin reductase activity compared to the other species.

Proton nuclear magnetic resonance-based metabonomics for rapid diagnosis of meningitis and ventriculitis.

BACKGROUND: Reduction of mortality associated with bacterial meningitis and postsurgical cerebral ventriculitis is dependent on early diagnosis and institution of appropriate therapy. Metabonomics rapidly defines metabolic profiles of biological fluids through the use of high-throughput analytical techniques combined with statistical pattern recognition tools. METHODS: Proton nuclear magnetic resonance (1H NMR)-based metabonomics was applied to (1) lumbar cerebrospinal fluid samples collected prospectively from a cohort of patients with bacterial, fungal, or viral meningitis and from control subjects without neurological disease and (2) ventricular cerebrospinal fluid samples from patients with ventriculitis associated with an external ventricular drain and from control subjects. 1H NMR spectra were analyzed by the unsupervised statistical method of principal components analysis. RESULTS: Metabonomic analysis clearly distinguished patients with bacterial or fungal meningitis (11 patients) from patients with viral meningitis (12) and control subjects (27) and clearly distinguished patients with postsurgical ventriculitis (5) from postsurgical control subjects (10). Metabolites of microbial and host origin that were responsible for class separation were determined. Metabonomic data also correlated with the onset and course of infection in a patient with 2 episodes of bacterial ventriculitis and with response to therapy in another patient with cryptococcal meningitis. CONCLUSIONS: Metabonomic analysis is rapid, requires minimal sample processing, and is not targeted to specific microbial pathogens, making the platform potentially suitable for use in the diagnostic laboratory. This pilot study indicates that metabonomic analysis of cerebrospinal fluid is feasible and a potentially more powerful diagnostic tool than conventional rapid laboratory indicators for distinguishing bacterial from viral meningitis and for monitoring therapy. This should have important implications for early management, reduced empirical use of antibiotics, and treatment duration.

Brain gene expression, metabolism, and bioenergetics: interrelationships in murine models of cerebral and noncerebral malaria.

Malaria infection can cause cerebral symptoms without parasite invasion of brain tissue. We examined the relationships between brain biochemistry, bioenergetics, and gene expression in murine models of cerebral (Plasmodium berghei ANKA) and noncerebral (P. berghei K173) malaria using multinuclear NMR spectroscopy, neuropharmacological approaches, and real-time RT-PCR. In cerebral malaria caused by P. berghei ANKA infection, we found biochemical changes consistent with increased glutamatergic activity and decreased flux through the Krebs cycle, followed by increased production of the hypoxia markers lactate and alanine. This was accompanied by compromised brain bioenergetics. There were few significant changes in expression of mRNA for metabolic enzymes or transporters or in the rate of transport of glutamate or glucose. However, in keeping with a role for endogenous cytokines in malaria cerebral pathology, there was significant up-regulation of mRNAs for TNF-alpha, interferon-gamma, and lymphotoxin. These changes are consistent with a state of cytopathic hypoxia. By contrast, in P. berghei K173 infection the brain showed increased metabolic rate, with no deleterious effect on bioenergetics. This was accompanied by mild up-regulation of expression of metabolic enzymes. These changes are consistent with benign hypermetabolism whose cause remains a subject of speculation.

For want of a nail. ramifications of a single gene deletion, dystrophin, in the brain of the mouse.

Lack of expression of a single gene, dystrophin, causes the severe, progressive muscle wasting and mental deficits characteristic of Duchenne muscular dystrophy. In this work, we investigated the impact of dystrophin deletion on expression of other genes in the brain cortex, hippocampus and cerebellum using the murine homologue, the mdx mouse, and RT-PCR. Expression of the brain glucose transporters GLUT1 and GLUT2 was found to be decreased, as were some subunits of the GABAA and nicotinic acetylcholine receptors. Genes involved in bioenergetic homeostasis, such as the mitochondrial creatine kinase and the gamma subunit of ATP synthase were also found to be abnormally expressed, while expression of the structural proteins beta-dystrobrevin and rapsyn was also significantly affected. We relate these findings to known functional deficits and discuss the possible mechanisms behind the altered gene expression.

Abnormalities in brain biochemistry associated with lack of dystrophin: studies of the mdx mouse.

Biochemical abnormalities have been reported in dystrophin-deficient muscle of boys with Duchenne (severe Xp21) muscular dystrophy or in the murine (mdx) model of the disease. These abnormalities include altered energy metabolism and responses to osmotic shock. In contrast, the situation in brain is less well understood and it is probable that dystrophin is playing a different role (or roles) in this organ. In this study we conclude that the elevation in choline-containing compounds reported in mdx brain is confined to cerebellum and hippocampus in older (> 6 months) mice. We report alterations in glucose metabolism in mdx brain under normal, awake conditions, and a reduced response of brain metabolism to the gamma-aminobutyric acid(A) receptor agonist muscimol. Using brain cortical slices we found no difference in the response of dystrophic tissue to hypoosmotic shock, but increased, substrate-dependent oxygen consumption rates at low oxygen partial pressures.

The effect of phosphate loading on erythrocyte 2,3-bisphosphoglycerate levels.

BACKGROUND: Phosphate supplementation has been used in an effort to enhance athletic performance by increasing erythrocyte 2,3-bisphosphoglycerate levels ([2,3-BPG]) and hence improve oxygen offloading from haemoglobin. Claimed effects of phosphate loading upon both exercise performance and erythrocyte [2,3-BPG] are inconsistent, and the basis of any change in [2,3-BPG] is unknown. METHODS: We analysed plasma inorganic phosphate concentration ([P(i)]) and erythrocyte [P(i)] and [2,3-BPG] in venous blood samples from 12 healthy subjects. We re-examined a subset of five of these subjects after 7 days of phosphate loading. RESULTS: There were significant positive correlations between plasma [P(i)] and erythrocyte [P(i)] (r(2)=0.51, p=0.009) and between erythrocyte [P(i)] and [2,3-BPG] (r(2)=0.68, p<0.001). Following phosphate loading, there was a 30% increase in plasma [P(i)] (1.02+/-0.22 to 1.29+/-0.15 mmol/l (mean+/-S.D.), p=0.03) and a 25% increase in erythrocyte [2,3-BPG] (6.77+/-1.12 to 9.11+/-1.87 mmol/l cells, p=0.03). There is no relation between [2,3-BPG] and plasma [P(i)]. CONCLUSIONS: Phosphate loading increases both plasma and erythrocyte phosphate pools and the rise in [2,3-BPG] is probably a consequence of the rise in cell [P(i)].

Singlet oxygen-mediated protein oxidation: evidence for the formation of reactive side chain peroxides on tyrosine residues.

Singlet oxygen (1O2) is generated by a number of enzymes as well as by UV or visible light in the presence of a sensitizer and has been proposed as a damaging agent in a number of pathologies including cataract, sunburn, and skin cancers. Proteins, and Cys, Met, Trp, Tyr and His side chains in particular, are major targets for 1O2 as a result of their abundance and high rate constants for reaction. In this study it is shown that long-lived peroxides are formed on free Tyr, Tyr residues in peptides and proteins, and model compounds on exposure to 1O2 generated by both photochemical and chemical methods. The yield of these species is significantly enhanced in D2O and decreased by azide. Nuclear magnetic resonance and mass spectroscopic analysis of reaction mixtures, or materials separated by high-performance liquid chromatography, are consistent with the initial formation of an (undetected) endoperoxide that undergoes rapid ring-opening to give a hydroperoxide situated at the C1 ring-position (i.e. para to the phenolic group). In the presence of a free alpha-amino group (e.g. with free Tyr), rapid ring-closure occurs to give an indolic hydroperoxide that decays into the corresponding alcohol, 3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acid. Hydroperoxides that lack a free alpha-amino group (e.g. those formed on 3-(4-hydroxyphenyl)propionic acid, N-Ac-Tyr and Tyr-containing peptides) are longer-lived, with half-lives of hours to days. These species undergo slow decay at low temperatures to give the corresponding alcohol. Their rate of decay is enhanced at 37 degrees C, or on exposure to UV light or metal ions, and gives rise to reactive radicals, via cleavage of the peroxide bond. These radicals have been characterized by electron paramagnetic resonance spin trapping. These studies demonstrate that long-lived Tyr-derived peroxides are formed on proteins exposed to 1O2 and that these may promote damage to other targets via further radical generation.

Metabolite profiling of the intraerythrocytic malaria parasite Plasmodium falciparum by (1)H NMR spectroscopy.

NMR spectroscopy was used to identify and quantify compounds in extracts prepared from mature trophozoite-stage Plasmodium falciparum parasites isolated by saponin-permeabilisation of the host erythrocyte. One-dimensional (1)H NMR spectroscopy and four two-dimensional NMR techniques were used to identify more than 50 metabolites. The intracellular concentrations of over 40 metabolites were estimated from the (1)H NMR spectra of extracts prepared by four extraction methods: perchloric acid, methanol/water, methanol/chloroform/water, and methanol alone. The metabolites quantified included: the majority of the biological alpha-amino acids; 4-aminobutyric acid; mono-, di- and tri-carboxylic acids; nucleotides; polyamines; myo-inositol; and phosphocholine and phosphoethanolamine. The parasites also contained a significant concentration (up to 12 mM) of the exogenous buffering agent, HEPES. Although the metabolite profiles obtained with each extraction method were broadly similar, perchloric acid was found to have significant advantages over the other extraction media.

Water chemical shift in 1H NMR of red cells: effects of pH when transmembrane magnetic susceptibility differences are low.

The (1)H magic angle spinning (MAS) NMR spectrum of water in erythrocyte suspensions shows peaks from each of the intracellular and extracellular water pools. The splitting is a true chemical shift and is brought about by the elimination of water exchange under MAS conditions due to physical separation of the two water populations. The size of the chemical shift difference is determined by the concentration of intracellular protein affecting the average extent of hydrogen bonding of water. We present here a model of the chemical shift behavior for water in erythrocytes under normal high-resolution NMR conditions based on results from MAS experiments on these cells exposed to different pH and osmotic conditions. The model accurately predicts the chemical shift of water for a static sample, and the results demonstrate that in high-resolution NMR experiments the chemical shift of water will appear to be invariant if differences in magnetic susceptibility across the cell membrane are minimal (<10% of the magnetic susceptibility of water). Thus, changes in the shape and chemical shift of the water resonance are not due to pH changes in the physiological range. The findings are fundamental to an interpretation of the mechanism of chemical shift effects on the water resonance that may occur in functional MRI.

Alanine metabolism, transport, and cycling in the brain.

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na(+)-dependent transporters with properties similar to system B(0) isoform B(0)AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.

Inhibitors of glutamate transport modulate distinct patterns in brain metabolism.

High affinity uptake of glutamate plays a major role in the termination of excitatory neurotransmission. Identification of the ramifications of transporter function is essential to understand the diseases in which defective excitatory amino acid transporters (EAAT) have been implicated. In this work we incubated Guinea pig cortical tissue slices with [3-(13)C]pyruvate and major currently available glutamate uptake inhibitors and studied the resultant metabolic sequelae by (13)C and (1)H NMR spectroscopy using a multivariate statistical approach. Perturbation of glutamate uptake produced significant effects on metabolic flux through the Krebs cycle, and on glutamate/glutamine cycling rates, with this effect accounting for 76% of the variation in the total data set. The effects of all inhibitors were separable from each other along three major principal components. The competitive inhibitor L-CCG III ((2S,1'S,2'R)-2-carboxycyclopropyl)glycine) differed most from the other inhibitors, showing negative weightings on both the first and second principal components, whereas the EAAT2-specific inhibitor dihydrokainate (DHK) showed metabolic patterns similar to that of anti-endo-3,4-methanopyrolidine dicarboxylate but separate from those of DL-threo-beta-benzyloxyaspartate (TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (L-tPDC). This indicates that different inhibition mechanisms or different colocalisation of the separate transporter subtypes with glutamate receptors can produce significantly different metabolic and functional outcomes for the brain.

Rapid exchange of fluoroethylamine via the Rhesus complex in human erythrocytes: 19F NMR magnetization transfer analysis showing competition by ammonia and ammonia analogues.

A remarkable recent discovery in red blood cell function is that the Rhesus antigen complex that for so long was considered to be simply a means of cell recognition is also the ammonia transporter. It catalyzes transmembrane exchange of ammonia on the subsecond time scale, and yet because of a lack of rapid-exchange methodology its kinetics had not been characterized. The flux of ammonia varies appreciably in diverse clinical states, and a convenient method for its characterization would be of basic and of clinical diagnostic value. Fluoroethylamine is water-soluble and when added to a suspension of human red blood cells (RBCs) displays the experimentally useful property of giving separate 19F NMR spectral peaks for the populations inside and outside the cells. By using two-site, one-dimensional magnetization exchange spectroscopy (1D-EXSY), the transmembrane exchange of fluoroethylamine was measured; it was found to occur on the subsecond time scale with an apparent first-order rate constant for efflux, under the equilibrium exchange conditions, of 3.4 s(-1). The method was used to characterize the concentration, temperature, and pH dependence of the exchange rate constant. We determined the extent of competitive inhibition exhibited by ammonia and two molecules that contain an amine group (ethylamine and methylamine). Inhibition of the exchange by incubating the suspension with anti-RhAG antibody, and no inhibition by anti-RhD antibody, suggested specificity of exchange via the RhAG protein of the Rh complex.

Antifungal effects on metabolite profiles of medically important yeast species measured by nuclear magnetic resonance spectroscopy.

Drug-induced inhibition of fungal growth is used in the diagnostic laboratory to predict therapeutic efficacy but is relatively slow, and determination of endpoints can be problematic. Nuclear magnetic resonance (NMR) spectroscopy identifies the metabolic complement of microorganisms while monitoring utilization of constituents of the incubation medium. This technique may provide a rapid and objective indicator of antifungal effects. We evaluated the effects of caspofungin, amphotericin B (AMB), and voriconazole on metabolic profiles of yeast species cultured in RPMI-2% glucose-morpholinepropanesulfonic acid buffer in microtiter plates in a proof-of-principle study. 1H NMR spectra were obtained using Bruker NMR spectrometers at 1H frequencies of 600 and 360 MHz. Metabolites were identified by two-dimensional correlation NMR spectra, and relative peak integrals were calculated from one-dimensional 1H NMR spectra. MICs were determined by a modification of the Clinical and Laboratory Standards Institute broth microdilution method M27-A. Utilization of glucose and branched-chain and aromatic amino acid substrates was accompanied by fungal production of acetate, acetaldehyde, ethanol, formate, fumarate, glycerol, lactate, pyruvate, and succinate. Clear-cut metabolic endpoints indicating a greater than 50% reduction in substrate utilization and fungal metabolite production which correlated with MICs were noted at 16 and 24 h for all three drugs. At 8 h, reductions of greater than 50% for selected metabolites were noted for caspofungin and AMB. Direct NMR-based observation of metabolic alterations in yeast cultures reveals changes in key metabolic pathways and should be evaluated formally as a rapid technique for determining susceptibility to antifungal drugs.

NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes.

Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a 13C-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-13C]glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using 1H spin-echo NMR, while 13C[1H-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.