Analysis of plasma lipids by NMR spectroscopy: application to modifications induced by malignant tumors.

Lipid extracts of plasma were studied by 31P and 1H NMR spectroscopy at 9.4 T. Signals recorded on lipid mixtures were assigned to different lipid classes using a data base built with two-dimensional 1H COSY spectra of seven standard lipids. Signals unique to glycerophospholipids, sphingolipids, and triacylglycerols were identified. 31P and 1H NMR spectroscopy was used to study qualitative and quantitative modifications induced in plasma by malignant tumors. The results show a significant increase in triglyceride/phospholipid ratio and a concomitant decrease of total phospholipids in patients with cancer. In order to check for the possible presence of particular lipids such as glycolipids in these patients, 1H COSY spectra were recorded on the intact plasma and on extracts of plasma lipids in patients with cancer and in healthy subjects. Only in one case of ovarian cancer, a cross-peak at 1.35 and 4.15 ppm, corresponding to fucose residue in glycolipids, was detected.

Graphic-aided study of metabolic modifications of plasma in cancer using proton magnetic resonance spectroscopy.

Proton high-resolution MRS of human plasma allows the rapid detection, on the same spectrum, of many compounds originating from different metabolic pathways. In this paper, we illustrate the modifications of the plasma metabolic profiles recorded by proton NMR spectroscopy in different classes of cancers. These modifications can be easily monitored with graphic aids such as 'star plots' which define for each type of cancer a particular pattern describing the most altered metabolic pathways. By using 'star plots' three types of metabolic patterns have been distinguished: (i) the 'inflammatory' pattern characterized by an increase of glycosylated moieties of glycoproteins; (ii) a 'lipid modified' pattern, characterized by various modifications occurring mainly in the lipid moieties detected by MRS; and (iii) a pattern which is often observed in sarcomas and mainly characterized by an alteration in the N-acetyl glucosamine/N-acetyl neuraminic acid ratio. This study demonstrates the ability of proton MRS of plasma to rapidly detect the occurrence of metabolic modifications brought about by cancer evolution or therapy.

High resolution NMR spectroscopy of physiological fluids: from metabolism to physiology.

High resolution NMR spectroscopy of physiological fluids provides quantitative, qualitative and dynamic information on the metabolic status of the interstitial and plasma compartments under a variety of pathophysiological conditions. The simultaneous detection and quantitation by NMR spectroscopy of numerous compounds of the intermediary metabolism offers a new insight in the understanding of the milieu intérieur. NMR spectroscopy of physiological fluids offers a unique way to define and monitor the global metabolic homeostasis in humans. The development of this analytical approach is still limited by the scarcity of pluridisciplinary teams able to fully exploit the wealth of information present on the NMR spectrum of a fluid. While application in pharmacology and toxicology is already established, the main areas of current development are cancer, hereditary metabolic disorders, organ transplantation and neurological diseases.

Two-dimensional 1H NMR spectroscopy of normal and pathological human plasma: complete water suppression and further assignment of resonances.

Two-dimensional J-resolved and correlated 1H NMR spectra with complete water suppression have been obtained to further characterize a metabolic pattern for normal and pathological human plasma samples. 1H COSY spectra have been recorded on plasma from 12 patients with cancer in order to check for the possible presence of fucose. Our results show that there is no evidence for the presence of fucosylated lipids in the plasma of these patients.

Energetic metabolism of glucose, mannose and galactose in glucose-starved rat insulinoma cells anchored on microcarrier beads. A phosphorus-31 NMR study.

Insulin-secreting cells (RINm5F) have successfully been grown on a large scale on poly-L-lysine coated-polystyrene microcarriers, providing a high cell number in a restricted volume under conditions that respect the metabolic integrity of these anchorage-dependent cells. The energetic metabolism of the perfused cells has been followed non-invasively by phosphorus-31 nuclear magnetic resonance spectroscopy. Glucose starvation induced a rapid decrease in nucleoside triphosphates (mainly ATP) pools, correlated with an increase in Pi level. The initial ATP level was rapidly recovered when the cells were refed with glucose or with mannose, but not with galactose, even after 2 h of perfusion. These differential effects of hexoses on energetic metabolism might be related to their various insulin-release actions on tumor islet cells.

Quantitation of metabolites in human blood serum by proton magnetic resonance spectroscopy. A comparative study of the use of formate and TSP as concentration standards.

Formate has been evaluated as an alternative standard to quantitate human serum metabolites in 1H NMR spin-echo spectra. The comparison between added formate and 3-(trimethylsilyl) 3,3,3,3-tetradeutero-propionic acid (TSP) shows that, unlike TSP, formate does not interact with serum macromolecules. Transverse and longitudinal proton relaxation times have been measured on several serum metabolites, in the presence of ammonium chloride. With the exception of glucose, values of metabolite concentrations derived from Hahn spin-echo spectra recorded on serum containing 15.4 mM exogenous formate as a standard, are in excellent agreement with the results of biochemical and chromatographic assays, after correction for differential relaxation effects. This approach can be readily used for quantitation of metabolites from blood serum (and eventually other physiological fluids) in normal and in pathological situations not involving disorders of endogenous formate metabolism.

[Study of biological fluids by nuclear magnetic resonance spectroscopy]. / Etude des liquides biologiques par spectroscopie de résonance magnétique nucléaire.

The use of nuclear magnetic resonance (NMR) spectroscopy in the study of biofluids is rapidly developing and might soon constitute a new major medical application of this technique which benefits from technological and methodological progress such as higher magnetic fields, new probe design, solvent suppression sequences and advanced data processing routines. In this overview, the clinical and pharmacological impact of this new approach is examined, with emphasis on the NMR spectroscopy of plasma, cerebrospinal fluid and urine. Applications to pharmacokinetics and toxicology are illustrated. Interestingly, a number of biochemical components of fluids which are not usually assayed by conventional biochemical methods are readily detected by NMR spectroscopy which is clearly a new competitive entrant among the techniques used in clinical biology. Its ease-of-use, cost effectiveness and high informational content might turn it into a major diagnostic tool in the years to come.

Variations of plasma sialic acid and N-acetylglucosamine levels in cancer, inflammatory diseases and bone marrow transplantation: a proton NMR spectroscopy study.

Proton NMR spectroscopy allows the detection in plasma of resonances arising from N-acetyl-glucosamine (NAG) and N-acetyl-neuraminic acid (NANA) which have been shown to be borne by acute phase glycoproteins. These resonances can be identified using 2 different protocols of spectrum acquisition detecting different physical states in the global pool of glycoproteins, ie mobile and less mobile moieties of glycosylated chains. In this study we demonstrate that NMR spectroscopy allows a precise monitoring of the variations of glycosylated residues in cancers, inflammatory processes and bone marrow transplantation. The most important findings are that: i), the distribution of glycosylated residues varies with the origin of the cancerous tissue; ii), the level of these residues is a function of tumor development; iii), the concentrations in NAG and NANA are well correlated with the standard biological parameters of acute phase and leucocyte activation. Proton NMR spectroscopy of glycosylated residues in plasma may offer a new means of monitoring sialic acid in cancer and other pathological conditions.

[High resolution NMR spectroscopy of CSF: methodological issues and perspective clinical applications]. / La spectroscopie de RMN à haute résolution du LCR: problèmes méthodologiques et perspectives d'applications cliniques.

High resolution proton nuclear magnetic resonance (NMR) spectroscopy is a new analytical technique which allows to readily identify and quantitate a variety of key metabolites in cerebrospinal fluid (CSF) in relation to normal and pathological brain activity. Proton NMR spectroscopy can be performed on native CSF, with or without addition of exchange reagent (NH4Cl). The analysis of native CSF provides qualitative information (identification) of metabolites or xenobiotics present in the fluid. Alternately, CSF can be lyophilized and dissolved in deuterated water. This concentration offers 2 advantages: additional compounds are detected and a precise quantification of all CSF metabolites can be obtained. Both protocols require a very small volume of CFS (1-2 ml). The high informational content available on the NMR spectra of CSF, the ease-of-use of NMR spectroscopy and its cost effectiveness concur to predict that this analytical approach will keep developing to completement the array of existing tests which are already routinely performed on CSF.

Phosphorus-31 nuclear magnetic resonance study of the C6 glioma cell line cultured on microcarrier beads.

The energetic metabolism of perfused C6 glioma cells anchored and cultured on polystyrene microcarrier beads has been studied by phosphorus-31 nuclear magnetic spectroscopy (NMR). The observation of intracellular phosphorylated compounds demonstrates the metabolic long-lasting viability of the perfused cells. The effect of glucose deprivation on energetic metabolism and intracellular pH illustrates the existence of an active aerobic glycolysis. The non-invasive study of anchored C6 cells by NMR provides a direct means to investigate the metabolism of glioma cells.

[Modification of the relative plasma concentrations of methyl and methylene groups in cancer: a study using proton NMR spectroscopy]. / Modification des concentrations relatives des groupements méthyle et méthylène plasmatiques dans la pathologie cancéreuse: une étude par spectroscopie RMN du proton.

Fossel et al. have recently proposed the proton NMR examination of plasmatic lipoproteins--and more precisely the determination of an index obtained from the averaged linewidth of the CH2 and CH3 resonances--as a possible tool for detection of cancer. Many evaluations conducted on an international basis have demonstrated that initial expectations were not met and that the test lacked sensitivity, specificity, and predictive value to be accepted as a screening and diagnostic tool. In our evaluation we have collected plasma from healthy subjects, from patients with various kinds of cancer at different stages of evolution and therapy, and from patients suffering from a variety of pathologies, including benign tumors. In accordance with Chmurny et al., we observed that the linewidth index (LWI) is precise and reproducible when care is taken in the handling and storage of samples and in the fasting of subjects. After finding no predictive value to the test, we have reanalyzed the spectra and studied the variations of the ratio defined by the methylene signal area over the methyl signal area. This ratio is significantly increased in cancer. Furthermore, it offers a better separation of statistical populations permitting a more precise discrimination between cancer, other pathologies and controls. We have also found that malignant tumors arising from mesenchyma (sarcoma, leukemia, lymphoma) induce less important variations in the CH2/CH3 ratio than adenocarcinoma or glioma, when such differences cannot be documented using the LWI. These observations are particularly interesting since they might bring new information on the metabolic modifications of the LWI and the CH2/CH3 ratio might reflect the embryologic origin of the tumors and raise the issue of the heterogeneity of cancer disease.