Classification of tumour 1H NMR spectra by pattern recognition.

1H spectra of tumours or normal tissues, which include signals from all hydrogen-containing metabolites, are too complex for the human eye to interpret. We have studied 58 1H spectra from perchloric acid extracts of three normal tissues (liver, kidney and spleen) and five rat tumours (GH3 pituitary, fibrosarcoma, Morris Hepatomas 7777 and 9618a and Walker carcinosarcoma). Instead of editing them or quantifying individual metabolites, we have used statistical pattern recognition techniques to classify them into groups. This automatic, objective method differentiated spectra from normal and malignant rat tissue biopsies, and from different types of cancer. It seems likely that this technique can be applied to human tissues and thus used for cancer diagnosis.     

An assessment of the effects of sample ischaemia and spinning time on the metabolic profile of brain tumour biopsy specimens as determined by high-resolution magic angle spinning (1)H NMR

High-resolution magic angle spinning (HRMAS) (1)H NMR of biopsy tissue provides a biochemical profile that has potential diagnostic and prognostic value, and can aid interpretation of the lower-resolution (1)H-NMR spectra obtained in vivo. However, the biochemical profile obtained may be affected by experimental factors such as a period of ischaemia before snap-freezing of the biopsy tissue for subsequent analysis and the mechanical stress of the spinning procedure of HRMAS itself. We have used normal rat brain cortex as a 'gold standard', either funnel-frozen or deliberately allowed to become ischaemic for set periods of time before snap-freezing, to quantitatively investigate these two effects. In addition, we have compared biochemical changes that occur in normal rat brain during HRMAS (spun continuously at 5 kHz for 4 h at 4 degrees C as could be required for a two-dimensional acquisition) with those that occur in biopsy samples from low-grade and high-grade adult human astrocytomas, during the same HRMAS procedure. Significant changes due to delayed initial sample freezing were noted in metabolites associated with glycolysis (alanine, glucose and lactate), as expected. However, for the funnel-frozen rat tissue at 4 degrees C, there were even more significant changes, which appear to be the result of extended spinning at 5 kHz. In particular, the 18% total creatine increase observed is unlikely to be de novo synthesis of creatine. More likely, the asymptotic exponential increase in creatine suggests an exponential release of an NMR-invisible bound creatine store as a result of tissue damage from mechanical stress of sample spinning. Overall, it appears that tissue ischaemia during biopsy excision and delays in snap-freezing may have less significant effects on metabolite profile than the prolonged spinning times required for two-dimensional HRMAS, and this must be accounted for when results are being interpreted.     

Lipid biomarkers of glioma cell growth arrest and cell death detected by 1H magic angle spinning MRS

Biomarkers of early response to treatment have the potential to improve cancer therapy by allowing treatment to be tailored to the individual. Alterations in lipids detected by in vivo MRS have been suggested as noninvasive biomarkers of cell stress and early indicators of cell death. An improved understanding of the relationship between MRS lipids and cell stress in vitro would aid in the translation of this technique into clinical use. Rat BT4C glioma cells were treated with 50 µ m cis‐dichlorodiammineplatinum II (cisplatin), a commonly used chemotherapeutic agent, and harvested at several time points up to 72 h. High‐resolution magic angle spinning ¹H MRS of cells was then performed on a 600‐MHz NMR spectrometer. The metabolites were quantified using a time domain fitting method, TARQUIN. Increases were detected in saturated and polyunsaturated fatty acid resonances early during the exposure to cisplatin. The fatty acid CH₂/CH₃ ratio was unaltered by treatment after allowing for contributions of macromolecules. Polyunsaturated fatty acids increased on treatment, with the group –CH = CH–CH₂–CH = CH– accounting for all the unsaturated fatty acid signals. Transmission electron microscopy, in addition to Nile red and 4',6‐diamino‐2‐phenylindole co‐staining, revealed that the lipid increase was associated with cytoplasmic neutral lipid droplets. Small numbers of apoptotic and necrotic cells were detected by trypan blue, annexin V–fluorescein isothiocyanate‐labelled flow cytometry and DNA laddering after up to 48 h of cisplatin exposure. Propidium iodide flow cytometry revealed that cells accumulated in the G1 stage of the cell growth cycle. In conclusion, an increase in the size of the lipid droplets is detected in morphologically viable cells during cisplatin exposure. ¹H MRS can detect lipid alterations during cell cycle arrest and progression of cell death, and has the potential to provide a noninvasive biomarker of treatment efficacy in vivo. Copyright © 2012 John Wiley & Sons, Ltd.     

Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high‐resolution magic angle spinning 1H NMR spectroscopy

High‐resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi‐core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9‐ and 1.3‐ppm signals) and metabolite quantification using HR MAS ¹H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16–0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one‐way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.     

Characterization of metabolites in infiltrating gliomas using ex vivo 1H high‐resolution magic angle spinning spectroscopy

Gliomas are routinely graded according to histopathological criteria established by the World Health Organization. Although this classification can be used to understand some of the variance in the clinical outcome of patients, there is still substantial heterogeneity within and between lesions of the same grade. This study evaluated image‐guided tissue samples acquired from a large cohort of patients presenting with either new or recurrent gliomas of grades II–IV using ex vivo proton high‐resolution magic angle spinning spectroscopy. The quantification of metabolite levels revealed several discrete profiles associated with primary glioma subtypes, as well as secondary subtypes that had undergone transformation to a higher grade at the time of recurrence. Statistical modeling further demonstrated that these metabolomic profiles could be differentially classified with respect to pathological grading and inter‐grade conversions. Importantly, the myo‐inositol to total choline index allowed for a separation of recurrent low‐grade gliomas on different pathological trajectories, the heightened ratio of phosphocholine to glycerophosphocholine uniformly characterized several forms of glioblastoma multiforme, and the onco‐metabolite D‐2‐hydroxyglutarate was shown to help distinguish secondary from primary grade IV glioma, as well as grade II and III from grade IV glioma. These data provide evidence that metabolite levels are of interest in the assessment of both intra‐grade and intra‐lesional malignancy. Such information could be used to enhance the diagnostic specificity of in vivo spectroscopy and to aid in the selection of the most appropriate therapy for individual patients. © 2014 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.     

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

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

An investigation of human brain tumour lipids by high-resolution magic angle spinning 1H MRS and histological analysis

NMR-visible lipid signals detected in vivo by 1H MRS are associated with tumour aggression and believed to arise from cytoplasmic lipid droplets. High-resolution magic angle spinning (HRMAS) 1H MRS and Nile Red staining were performed on human brain tumour biopsy specimens to investigate how NMR-visible lipid signals relate to viable cells and levels of necrosis across different grades of glioma. Presaturation spectra were acquired from 24 adult human astrocytoma biopsy samples of grades II (8), III (2) and IV (14) using HRMAS 1H MRS and quantified using LCModel to determine lipid concentrations. Each biopsy sample was then refrozen, cryostat sectioned, and stained with Nile Red, to determine the number of lipid droplets and droplet size distribution, and with Haematoxylin and Eosin, to determine cell density and percentage necrosis. A strong correlation (R=0.92, P<0.0001) was found between the number of Nile Red-stained droplets and the approximately 1.3 ppm lipid proton concentration by 1H MRS. Droplet sizes ranged from 1 to 10 microm in diameter, and the size distribution was constant independent of tumour grade. In the non-necrotic biopsy samples, the number of lipid droplets correlated with cell density, whereas in the necrotic samples, there were greater numbers of droplets that showed a positive correlation with percentage necrosis. The correlation between 1H MRS lipid signals and number of Nile Red-stained droplets, and the presence of lipid droplets in the non-necrotic biopsy specimens provide good evidence that the in vivo NMR-visible lipid signals are cytoplasmic in origin and that formation of lipid droplets precedes necrosis.     

Metabolomic studies of human lung carcinoma cell lines using in vitro (1)H NMR of whole cells and cellular extracts

We report principal component analysis (PCA) of (1)H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and (1)H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumour origin with different chemotherapy drug resistance patterns. For whole cells, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene mobile lipid (1)H resonance intensities, which correlate with our knowledge of the level of resistance displayed by the different cells. Similarly, in the (1)H NMR spectra of the aqueous and lipophilic extracts, significant quantitative differences in the metabolite distributions were apparent, which are consistent with the PCA results. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Quantification of amounts and 13C content of metabolites in brain tissue using high‐ resolution magic angle spinning 13C NMR spectroscopy

Metabolic pathway mapping using ¹³C NMR spectroscopy has been used extensively to study interactions between neurons and glia in the brain. Established extraction procedures of brain tissue are time consuming and may result in degradation of labile substances. We examined the potential of mapping ¹³C‐enriched compounds in intact brain tissue using high‐resolution magic angle spinning (HR‐MAS) NMR spectroscopy. Sprague–Dawley rats received an intraperitoneal injection of [1,6‐¹³C]glucose, and 15 min later the animals were subjected to microwave fixation of the brain. Quantification of concentration and ¹³C labelling of metabolites in intact rat thalamus were carried out based on exogenous ethylene glycol concentrations measured from ¹H NMR spectra using an ERETIC (Electronic REference To access In vivo Concentrations) signal. The results from intact tissue were compared with those from perchloric acid‐extracted brain tissue. Amounts of ¹³C labelling at different positions (C2, C3 and C4) in glutamate, glutamine, γ‐aminobutyric acid and aspartate measured in either intact tissue or perchloric acid extracts were not significantly different. Proton NMR spectra were used for quantification of six different amino acids plus lactate, inositol, N‐acetylaspartate, creatine and phosphocreatine. Again, results were very similar when comparing the methods. To our knowledge, this is the first time quantitative ¹³C NMR spectroscopy measurements have been carried out on intact brain tissue ex vivo using the HR‐MAS technique. The results show that HR‐MAS ¹³C NMR spectroscopy in combination with ¹H NMR spectroscopy and the ERETIC method is useful for metabolic studies of intact brain tissue ex vivo. Copyright © 2008 John Wiley & Sons, Ltd.     

Polymer conformation and NMR chemical shifts, 5 1H and 13C NMR spectra of poly(methyl methacrylate)

Tacticity-dependent splittings observed in ¹H and ¹³C NMR spectra of poly(methyl methacrylate) are elucidated by a theoretical procedure which consists of conformational analysis of the polymer chain and shielding calculation. The conformation is analyzed by the statistical mechanical method combined with the force field energy calculation on the dyad sequence models. As significant shielding factors, diamagnetic, anisotropic, and electrostatic terms are taken into account for the chemical shifts of ¹H NMR, while paramagnetic, diamagnetic, and anistropic terms are considered for ¹³C NMR. The calculation is used to assing the α-methyl proton, methoxyl proton, methylene proton, quaternary carbon, methylene carbon, α-methyl carbon, carbonyl carbon, and methoxyl carbon peaks split due to dyad and triad cotacticities. The results are generally consistent with the observed spectra. Especially, the calculated splitting widths are in quantitative agreement with the observed splittings of the methoxyl and α-methyl protons resonances as well as of the carbonyl and α-methyl carbons resonances. Effects of temperature and stereoregularity on the spectra are also discussed.     

A metabonomic approach to early prognostic evaluation of experimental sepsis by 1H NMR and pattern recognition

This study proposes an NMR‐based metabonomic approach to early prognostic evaluation of sepsis. Forty septic rats receiving cecal ligation and puncture (CLP) were divided into the surviving group and nonsurviving group on day 6, while 20 sham‐operated rats served as the control group. Serum samples were collected from septic and sham‐operated rats at 12 h after surgery and analyzed using ¹H NMR spectroscopy. Orthogonal partial least squares (OPLS) were applied and showed clustering according to predefined groups, indicating that NMR‐based metabolic profiling could reveal pathologic characteristics in the serum of sham‐operated, surviving, and nonsurviving septic rats. In addition, six characteristic metabolites including lactate, alanine, acetate, acetoacetate, hydroxybutyrate, and formate, which are mainly involved in energy metabolism, changed markedly in septic rats, especially in the nonsurvivors. Using these metabolites, a predictive model for prognostic evaluation of sepsis was constructed using a radial basis function neural network (RBFNN) with a prediction accuracy of about 87% by test samples. The results indicated that the NMR‐based metabonomic approach is a potential technique for the early prognostic evaluation of sepsis. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation of relative metabolic changes in the organs and plasma of rats exposed to X‐ray radiation using HR‐MAS 1H NMR and solution 1H NMR

Excess exposure to ionizing radiation generates reactive oxygen species and increases the cellular inflammatory response by modifying various metabolic pathways. However, an investigation of metabolic perturbations and organ‐specific responses based on the amount of radiation during the acute phase has not been conducted. In this study, high‐resolution magic‐angle‐spinning (HR‐MAS) NMR and solution NMR‐based metabolic profiling were used to investigate dose‐dependent metabolic changes in multiple organs and tissues – including the jejunum, spleen, liver, and plasma – of rats exposed to X‐ray radiation. The organs, tissues, and blood samples were obtained 24, 48, and 72 h after exposure to low‐dose (2 Gy) and high‐dose (6 Gy) X‐ray radiation and subjected to metabolite profiling and multivariate analyses. The results showed the time course of the metabolic responses, and many significant changes were detected in the high‐dose compared with the low‐dose group. Metabolites with antioxidant properties showed acute responses in the jejunum and spleen after radiation exposure. The levels of metabolites related to lipid and protein metabolism were decreased in the jejunum. In addition, amino acid levels increased consistently at all post‐irradiation time points as a consequence of activated protein breakdown. Consistent with these changes, plasma levels of tricarboxylic acid cycle intermediate metabolites decreased. The liver did not appear to undergo remarkable metabolic changes after radiation exposure. These results may provide insight into the major metabolic perturbations and mechanisms of the biological systems in response to pathophysiological damage caused by X‐ray radiation. Copyright © 2016 John Wiley & Sons, Ltd.     

1H and 13C NMR spectra of poly(propyl α-bromoacrylate) and poly(methyl α-bromoacrylate)

The ¹³C NMR spectra measured at 25 MHz of the methyl and propyl esters of isotactic and syndiotactic poly(α-bromoacrylate) were sufficiently resolved to be analysed for pentad tacticity sequences. The pentad tacticity of the syndiotactic polymers prepared with free radical initiators at ?40°C agreed with those calculated for Bernoullian sequence distributions based on P_r values of 0,83–0,87. The tacticities of the isotactic polymers prepared with heterogeneous catalysts were determined on the basis of these assignments. Good internal consistency was obtained between the calculated and observed pentad proportions from the quaternary and carbonyl carbon peaks in the spectra of these polymers. The order of chemical shifts for the meso and racemic dyads and tetrads in these polymers were opposite to those found in the equivalent methacrylate polymers, but as with the latter, the ¹³C-T₁ values were longer in the isotactic than in the syndiotactic polymers.     

Molecular characterization of human healthy and neoplastic cerebral and renal tissues by in vitro (1)H NMR spectroscopy (review)

The clinical impact of (1)H NMR spectroscopy in the study of human organs, brain and kidney in particular, is well demonstrated. The in vitro (1)H NMR technique is a powerful tool for monitoring changes in intracellular metabolites of human normal and neoplastic cerebral and renal tissues. Healthy and tumoral tissues of different histologic types have been fully characterized from a biochemical standpoint. Molecular characterization is performed on both the aqueous and lipid extracts of surgically removed tissue biopsies yielding a full picture of tissue biochemistry. These analyses have disclosed markers of healthy brain and kidney and of their respective neoplastic lesions. Moreover, some biochemical features can differentiate neoplasms within the same histological type. In particular, lipidic components, like cholesteryl esters (namely oleate), detected in highest grade tumors, warrant further investigation. A better understanding of the biochemistry of diseased human tissues could open the way to new diagnostic and treatment strategies.     

The 13C NMR spectra of poly(1-pentenylene) and poly(1,3-cyclopentylenevinylene)

¹³C NMR spectra were obtained for polymers made by ring-opening polymerization of cyclopentene and bicyclo[2.2.1]hept-2-ene respectively. The fraction of cis double bonds could be determined with much greater precision from ¹³C NMR spectra than from IR spectra and varied from 0,66 to 0,31 for the samples of poly(1-pentenylene), ( 2 ), and from 1,0 to 0,14 for the samples of poly(1,3-cyclopentylenevinylene), ( 4 ). This is the first time an all-cis polymer of 4 has been reported. The spectra of 2 showed a cis (upfield) and trans (downfield) peak for each of ?CH and α-CH₂, but only one peak for β-CH₂. The spectra of 4 showed multiple fine structure, the main splittings corresponding to a cis (upfield) and trans (downfield) peak for α-CH, and a reverse line order for the other three carbons; subsidiary splittings were observed for all but the olefinic carbons, interpreted in terms of sensitivity of the chemical shifts to the cis/trans structure at the next nearest double bond. A complete interpretation of the line orders in 4 is given in terms of steric compression effects. The possibility that ring tacticity accounts for some of the fine structure cannot be entirely discounted. The stereochemistry of 4 is discussed in relation to the four possible modes of addition of monomer to a carbene chain carrier during polymerization.     

In vitro (1)H NMR studies of RD human cell infection with echovirus 11

The effects of echovirus 11 infection on RD human cell line (derived from rhabdomyosarcoma) were studied using (1)H NMR spectroscopy and optical microscopy. Both uninfected and infected cells consumed glucose and produced lactate, acetate and formate as extracellular metabolites. In infected whole cells, phosphocholine and uridine-sugar were observed in addition to the metabolites observed in uninfected cells. Water-soluble intracellular metabolites of infected cells showed glutamine, phosphocholine and glycine which were not observed in uninfected cells. Cellular metabolites except lipid components gradually decreased and disappeared during 24-48 h of viral infection. The quantity of lipid components in infected cells was comparable with that in uninfected cells, indicating that echovirus 11 does not utilize cell lipid molecules. Unlike optical microscopy, (1)H NMR spectroscopy identified early stages of infection through metabolic changes. These results may have potential implications in probing virus-cell interactions using NMR-based metabolomics.     

Galactose metabolism in normal human lymphoblasts studied by (1)H, (13)C and (31)P NMR spectroscopy of extracts

The development of tools to follow and quantitate the fate of galactose in mammalian cells is crucial to the study and understanding of the inherited disorders of galactose metabolism. In this study we incubated normal human lymphoblasts with 1- or 2-(13)C galactose for 2.5 or 5 h and prepared TCA extracts of the cells. The various galactose metabolites were identified and quantified using a combination of proton, carbon and phosphorus NMR spectra. Galactose-1-phosphate (gal-1P), uridine diphosphogalactose, uridine diphosphoglucose and galactitol were present in the extracts. Average levels for gal-1P were around 10 nmol/mg protein and for uridine diphosphoglucose, uridine diphosphogalactose and galactitol in the range of 0.5-2 nmol/mg protein. Galactonate was never found in any conditions. Percentage labeling could be estimated for gal-1P and for the ribose carbons of AMP. The labeling agrees with a conversion of galactose to glucose through the Leloir pathway.     

Overexpression of KIT (CD117) in chromophobe renal cell carcinoma and renal oncocytoma

KIT expression has not been studied substantially in renal tumors. We analyzed the immunohistochemical expression for KIT in 256 conventional renal cell carcinomas (RCCs), 29 chromophobe RCCs, 25 papillary RCCs, 6 collecting duct RCCs, 6 unclassified RCCs, 7 renal oncocytomas, 20 urothelial carcinomas, 7 nephroblastomas, and 23 angiomyolipomas. We found that 24 chromophobe RCCs (83%) and 5 renal oncocytomas (71%) revealed membranous immunoreactivity for KIT while none of the RCCs of other types expressed KIT immunohistochemically. Sporadic cases of urothelial carcinoma and nephroblastoma were focally positive for KIT. All angiomyolipomas were negative. Genomic DNA extracted from the chromophobe RCCs and renal oncocytomas was submitted for polymerase chain reaction and direct sequencing of the juxtamembrane (exons 9 and 11) and tyrosine kinase (exons 13 and 17) domains. No mutation was found. Our results demonstrate that KIT could be a useful immunophenotypic marker for chromophobe RCC and renal oncocytoma; therefore, it has value for the precise classification of renal cortical epithelial tumors. However, the therapeutic relevance of KIT overexpression in these tumors is uncertain owing to the lack of mutations that would lead to constitutive activation of the protein.