Metabolic profiling in multiple sclerosis and other disorders by quantitative analysis of cerebrospinal fluid using nuclear magnetic resonance spectroscopy.

Cerebrospinal fluid (CSF) is being analyzed for the diagnosis of a variety of neurological diseases. Among the methods employed, metabolomics and proteomics are increasingly gaining popularity. At present, sensitivity and, in particular, specificity are limited in CSF metabolomics by nuclear magnetic resonance (NMR) spectroscopy. Nonetheless, progress is being made by studying more and more well-defined and homogeneous patient cohorts. This review starts off with a brief overview of classical CSF analysis in multiple sclerosis (MS), followed by a description of NMR spectroscopy in general metabolic CSF analysis. The subsequent sections focus on metabolomic profiling of CSF by NMR spectroscopy in MS and other neurological disorders. Currently existing results are reviewed and compared, and the potential and limits of this approach are discussed. In addition, several methodological questions are addressed, and the prospects for future developments are briefly outlined.

Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays.

KTCTL-26 and KTCTL-2 are renal cell carcinoma (RCC) lines with high and low expression of P-170 glycoprotein, respectively. Inherent differences between the two cell lines in terms of phosphate metabolites and growth characteristics in culture were examined for possible association with multidrug resistance (MDR). Differences in response to drug treatment were investigated for 40 h incubations with various doses of vinblastine (VBL) alone or as cotreatments with various concentrations of the calcium antagonist diltiazem (DIL) and/or interferon-alpha (IFN-alpha). Treatment effects were quantitated using the MTT survival assay and 31P magnetic resonance spectroscopy (MRS) to determine phosphate metabolite profiles in intact cells. KTCTL-2 and KTCTL-26 cells exhibited significant inherent differences in phosphocholine, glycerophosphocholine, glycerophosphoethanolamine, and phosphocreatine levels. KTCTL-26 cells were more sensitive than KTCTL-2 to 0.011 mircroM VBL alone (87% vs. 102% survival) or to 0.011 microM BL + 10 microM DIL (55% vs. 80% survival). The latter treatment resulted in a significant decrease in the ratio of phosphocholine to glycerophosphocholine in KTCTL-26 cells but no significant changes in phosphate metabolites in KTCTL-2 cells. Metabolomic 31P MRS detects different metabolite profiles for RCC cell lines with different MDR phenotypes and may be useful for noninvasive characterization of tumors in a clinical setting.

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

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

Non-growing Escherichia coli cells starved for glucose or phosphate use different mechanisms to survive oxidative stress.

Recent data suggest that superoxide dismutases are important in preventing lethal oxidative damage of proteins in Escherichia coli cells incubated under aerobic, carbon starvation conditions. Here, we show that the alkylhydroperoxide reductase AhpCF (AHP) is specifically required to protect cells incubated under aerobic, phosphate (Pi) starvation conditions. Additional loss of the HP-I (KatG) hydroperoxidase activity dramatically accelerated the death rate of AHP-deficient cells. Investigation of the composition of spent culture media indicates that DeltaahpCF katG cells leak nutrients, which suggests that membrane lipids are the principal target of peroxides produced in Pi-starved cells. In fact, the introduction of various mutations inactivating repair activities revealed no obvious role for protein or DNA lesions in the viability of ahp cells. Because the death of ahp cells was directly related to ongoing aerobic glucose metabolism, we wondered how glycolysis, which requires free Pi, could proceed. 31P nuclear magnetic resonance spectra showed that Pi-starved cells consumed Pi but were apparently able to liberate Pi from phosphorylated products, notably through the synthesis of UDP-glucose. Whereas expression of the ahpCF and katG genes is enhanced in an OxyR-dependent manner in response to H2O2 challenge, we found that the inactivation of oxyR and both oxyR and rpoS genes had little effect on the viability of Pi-starved cells. In stark contrast, the inactivation of both oxyR and rpoS genes dramatically decreased the viability of glucose-starved cells.

Metabolic effects of photodynamically induced apoptosis in an erythroleukemic cell line. A (31)P NMR spectroscopic study of Victoria-Blue-BO-sensitized TF-1 cells.

Victoria Blue BO (VB BO) is a new and promising photosensitizer currently being evaluated for photodynamic therapy (PDT). Its photochemical processes are mediated by oxygen radicals, but do not involve singlet oxygen. We used (31)P NMR spectroscopy of VB-BO sensitized TF-1 leukemic cells to gain further insight into the biochemical mechanisms underlying PDT-induced cell death. Sham-treatment experiments were performed to evaluate the effects of this photosensitizer in the absence of light irradiation. Significant metabolic differences were detected for TF-1 cells incubated with VB BO but not exposed to light, as compared with native cells (controls). These changes include reductions in phosphocreatine, UDP-hexose and phosphodiester levels (as percentage of total phosphate) and slightly reduced intracellular pH. Complete phosphocreatine depletion, significant acidification and concomitant inorganic-phosphate accumulation were observed for TF-1 cells irradiated after incubation with VB BO. Moreover, significant changes in phospholipid metabolites, i.e., accumulation of cytidine 5'-diphosphate choline and a decrease in phosphodiester levels, were observed for PDT-treated vs. sham-treated cells. Perturbations of phospholipid metabolism may be involved in programmed cell death, and the detection of a characteristic DNA ladder pattern by gel electrophoresis confirmed the existence of apoptosis in PDT-treated TF-1 cells.

Assignment and pH dependence of the 19F-NMR resonances from the fluorouracil anabolites involved in fluoropyrimidine chemotherapy.

Fluoropyrimidine chemotherapy relies on the intracellular anabolic conversion of 5-fluorouracil and the corresponding nucleosides to cytotoxic fluorinated nucleotides (F-Nuctd), such as 5-fluorouridine-5'-triphosphate (FUTP) or 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP), which can be detected by 19F-NMR spectroscopy. We have made 19F-NMR signal assignments at 11.7 T and 4 degrees C for model solutions containing 5-fluorouracil (FUra), 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorouridine-5'-monophosphate (FUMP), FdUMP, 5-fluorouridine-5'-diphosphate (FUDP), FUTP and 5-fluorouridine-5'-diphospho(1)-alpha-D-glucose (FUDPG), and we have studied the effects of pH over the range 4.5-7.8, of Mg2+ concentration and addition of EDTA. This information provides a basis for the analysis of 19F-NMR spectra obtained from cells, tissues or extracts following fluoropyrimidine treatment.

Experimental protocol for clinical analysis of cerebrospinal fluid by high resolution proton magnetic resonance spectroscopy.

High resolution magnetic resonance spectroscopy (MRS) of cerebrospinal fluid (CSF) is a non-destructive analytical method which allows rapid and simultaneous detection of molecules involved in intermediary and oxidative metabolic pathways. We developed a protocol suitable for routine MRS analysis of lyophilized CSF samples. This procedure guarantees sample integrity, from CSF collection to spectrum acquisition. MRS analysis of blood serum was included in our protocol as a complementary method to CSF analysis. This protocol can contribute to establish MRS of CSF as a new analytical tool to better understand the metabolic processes involved in neurological diseases.

Further assignment of resonances in 1H NMR spectra of cerebrospinal fluid (CSF).

A number of previously unidentified 1H NMR signals detected in CSF spectra of patients with various neurological and metabolic diseases are assigned to metabolites, drugs and drug excipients. Two-dimensional 1H NMR spectroscopy (COSY and J-resolved) is employed to resolve resonances which are hidden by superimposed peaks in one-dimensional spectra. Assignments obtained by making use of 2-D techniques, and of a 1-D 1H NMR data base created for ca. 150 authentic compounds, enable us to clarify the nature of complex signal patterns found in crowded spectral regions of CSF such as the aliphatic methyl region at ca. 1.0 ppm.

Perturbations of glucose metabolism associated with HIV infection in human intestinal epithelial cells: a multinuclear magnetic resonance spectroscopy study.

OBJECTIVE: To analyse the effect of HIV-1 infection on the glucose metabolism of human intestinal epithelial cells. METHODS: HT-29 cells were infected with HIV-1NDK and studied 3 weeks (acutely infected cells) or 9 months (chronically infected cells) post-infection. Perchloric acid extracts were analysed by high-resolution 1H, 31P and 13C nuclear magnetic resonance spectroscopy. Metabolite concentrations and specific 13C enrichments were quantified for chronically infected, acutely infected and control cells grown in Dulbecco's modified Eagle's medium containing natural-abundance or 1-13C-enriched glucose to determine significant differences between infected and non-infected cells. RESULTS: Chronically HIV-infected cells showed alterations in glycerol-3-phosphate (+40%), fructose-1,6-diphosphate (-66%), uridine diphosphate glucuronic acid (-33%), lactate (+75%) and [1-13C]glucose (+181%) levels, and in specific lactate 3-13C enrichment (+19%) when compared with controls. Acutely infected cells exhibited decreased fructose-1,6-diphosphate (-58%) and increased nicotinamide adenine dinucleotide (+33%) levels relative to controls. CONCLUSION: HIV-1 infection results in a disturbance of glycolytic and oxidative activities in human intestinal epithelial cells. This finding supports the concept that HIV-1 may directly impair some metabolic functions of the intestinal epithelium, and that it can be considered a potential aetiological agent for HIV-associated enteropathy.

Analysis of individual purine and pyrimidine nucleoside di- and triphosphates and other cellular metabolites in PCA extracts by using multinuclear high resolution NMR spectroscopy.

This work demonstrates that individual purine and pyrimidine NDP and NTP can be assigned in high resolution 31P NMR spectra from tissue extracts. To the best of our knowledge, it is shown for the first time that ATP, GTP, UTP, CTP, and the corresponding diphosphates can be quantitated in cell extracts without using HPLC or other biochemical methods. This work provides the basis for further optimization of nucleotide quantitation by 31P NMR spectroscopy, and for a full assessment of this method. Furthermore, a new technique was developed for 1H, 31P, and 13C NMR signal assignment and quantitation in cell extracts by using the same external reference capillary for all three nuclei. This allows for efficient, quantitative, multinuclear NMR spectroscopy without extract contamination by standard material.

A new method for the determination of specific 13C enrichment in phosphorylated [1-13C]glucose metabolites. 13C-coupled, 1H-decoupled 31P-NMR spectroscopy of tissue perchloric acid extracts.

A 31P-NMR method for the determination of 13C enrichment in phosphorylated [1-(13)C]glucose metabolites was developed by taking advantage of the 13C satellites detectable for 31P-NMR signals of metabolites such as UDP-hexoses, UDP-N-acetylhexosamines and other phosphorylated compounds generated during glycolysis and subsequent anabolism. HT-29 cells were incubated in culture medium containing 4.5 g/l [1-(13)C]glucose for 24 h prior to cell extraction, and high-resolution 31P-NMR spectra were acquired from perchloric acid extracts. Since glucose and its phosphorylated products are key metabolites for many different metabolic processes, this method may be very helpful for studying specific metabolic pathways involving phosphorylated glucose metabolites.

31P MRS of human tumor cells: effects of culture media and conditions on phospholipid metabolite concentrations.

31P magnetic resonance spectroscopy has been used to determine phosphate metabolite profiles in five human tumor cell lines in culture and as solid tumor xenografts in nude mice. Significant differences between cell lines, in particular in their phospholipid metabolite levels, were observed. The largest differences between metabolite profiles in vivo and in culture were observed for cell lines which exhibit low phosphoethanolamine levels in culture. One of these lines, the colon carcinoma CX-1, was studied in more detail in both incubated and perfused DMEM cultures with variation of the concentrations of glucose, choline and ethanolamine. Highly significant alterations of phospholipid metabolite concentrations and UDP-hexoses (primarily UDP-GlcNAc and UDP-GalNAc) were observed as a function of the precursor concentrations, culture time or perfusion time. A strong interaction between phospholipid metabolic pathways and UDP-hexose pathways could be demonstrated.

The influence of medium formulation on phosphomonoester and UDP-hexose levels in cultured human colon tumor cells as observed by 31P NMR spectroscopy.

High-resolution 31P NMR spectroscopy at 11.7 T was used to examine the influence of medium formulation (medium and serum type, and concentrations of glucose and inositol) on the cellular phosphate metabolism of CX-1 cells, a human colon cancer cell line derived from HT-29 cells. Striking differences in the 31P spectra of harvested CX-1 cells were observed. The largest variation was seen in the phosphocholine and UDP-hexose levels (up to seven-fold changes), with smaller differences in the levels of other phosphate metabolites. The major UDP-hexose species were found to be UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine (ca 2:1 ratio), which have been proposed in the literature to be markers of cell differentiation status. Medium-induced alterations in metabolite levels were much greater than the normal variations seen in CX-1 control samples grown under identical conditions. They even exceeded the characteristic differences observed between different human tumor cell lines grown under one set of culture conditions. The remarkable sensitivity of CX-1 cellular phosphate metabolism to the culture environment has implications for the comparison of in vitro vs in vivo spectra, and for the interpretation of effects due to growth and therapy.

A 1H-NMR method for determining temperature in cell culture perfusion systems.

This report describes a noninvasive 1H-NMR method for measuring absolute temperatures (+/- 0.2 degrees C) in biological samples and, in particular, in cell culture perfusion systems, utilizing the linear temperature dependence of the water chemical shift relative to the temperature-independent shift of one of the components of the biological medium, e.g., pyruvate, acetate or lactate. The effects of flow on temperature can be monitored and appropriate adjustment of the temperature controller can be made.

Selective suppression of lipid resonances by lipid-soluble nitroxides in NMR spectroscopy.

The ability of lipid-soluble nitroxides to suppress selectively the peaks of lipid resonances in 31P, 1H, and 13C NMR spectra was investigated in serum as part of studies aimed at using these contrast agents for magnetic resonance imaging and magnetic resonance spectroscopy in vivo. Nitroxides are especially interesting potential contrast agents because they can reversibly be converted in cells to diamagnetic hydroxylamines, with conversion rates that are dependent on the redox potential and the intracellular concentration of oxygen; the characterization of nitroxide-dependent changes in NMR spectra may therefore be a useful means to measure oxygen-dependent redox metabolism in vivo. The fatty acid analogs, doxyl stearates, suppressed the methyl resonance of choline and the methyl and methylene peaks of lipids in the 1H NMR spectra of serum samples. As a consequence, lactate peaks, which were not readily detected became clearly resolved and could be evaluated quantitatively. The 31P resonance of phosphatidylcholine in the 31P NMR spectrum was suppressed by 5-doxyl stearate and 4-(N,N-dimethyl-N-hexadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxy l,iodid e (Cat16). In the 13C NMR spectrum, the resonances of the methyl groups of choline and the lipids also were broadened significantly by addition of 5-doxyl stearate. Differential suppression of lipid resonances can be employed to facilitate quantitation of lactate.

Phosphorus magnetic resonance spectroscopy of patients with mitochondrial cytopathies demonstrates decreased levels of brain phosphocreatine.

The hypothesis that brain mitochondria are directly affected in several phenotypes associated with disordered oxidative phosphorylation was tested using phosphorus 31 (31P) magnetic resonance spectroscopy. Abnormal phosphorylation potentials in skeletal muscle have been demonstrated by 31P magnetic resonance spectroscopy in patients with mitochondrial cytopathies (heritable disorders of oxidative phosphorylation), but abnormalities of phosphorylation potentials in other organs have not been documented. Several lines of evidence suggest that these mutations may affect mitochondria in nonmuscle tissue. In this study we found that phosphocreatine-to-ATP ratios in brain were significantly reduced and that calculated brain ADP concentrations, phosphorylation potentials, and percentage of maximal rate of ATP synthesis were significantly altered in the 5 patients examined. This study indicates a primary abnormality of mitochondrial function in the brain, even in the absence of clinically evident cerebral dysfunction.

[Description of the magnetic resonance relaxation behavior of tissues using a suitable theoretical model]. / Beschreibung des MR-Relaxationsverhaltens von Gewebe mit Hilfe einer geeigneten Modellvorstellung.

An introduction into the physicochemical background of MR imaging is given by illustrating the MR relaxation processes that are relevant for body tissues. In particular the importance of molecular mobility (correlation time tau c) and resonance frequency omega o to NMR relaxation times T1 and T2 is explained.

[Phantom substances for quantitative evaluation of MRT images. III. Effect of various protein concentrations on MRT intensity values]. / Phantomsubstanzen zur quantitativen Auswertung von MR-T-Aufnahmen. III. Der Einfluss verschiedener Protonenkonzentrationen auf die MR-T-Intensitätswerte.

The use of heavy water (D2O) in paramagnetic agar phantoms can yield tissue equivalent values of the proton spin density p. In this manner all the MRI parameters that are typical of tissues can be realised in phantom substances. It is shown that the use of heavy water results in some enhancement of relaxation time, T2, that can be easily controlled via the agar concentration.

[Phantom substances in the quantitative evaluation of magnetic resonance T-images. II. Effect of relaxation times on magnetic resonance T-intensity values]. / Phantomsubstanzen zur quantitativen Auswertung von MR-T-Aufnahmen. II. Der Einfluss der Relaxationszeiten auf die MR-T-Intensitätswerte.

Reference material for quantitative MR Imaging (paramagnetic agar gel) is examined with regard to effects of small T1 and T2 variations on MRI signals. For some pulse sequences that are important in clinical practice, it can be shown that SE images are very sensitive to T2 variations, whereas IR images are influenced by both T1 and T2.