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In vitro 1H MRS of human bile has shown potential in the diagnosis of various hepatopancreatobiliary (HPB) diseases. Previously, in vivo 1H MRS of human bile in gallbladder using a 1.5 T scanner demonstrated the possibility of quantification of choline-containing phospholipids (chol-PLs). However, other lipid components such as bile acids play an important role in the pathophysiology of the HPB system. We have employed a higher magnetic field strength (3 T), and a custom-built receive array coil, to improve the quality of in vivo 1H MRS of human bile in the gallbladder. We obtained significant improvement in the quality of 1D spectra (17 healthy volunteers) using a respiratory-gated PRESS sequence with well distinguished signals for total bile acids (TBAs) plus cholesterol resonating at 0.66 ppm, taurine-conjugated bile acids (TCBAs) at 3.08 ppm, chol-PLs at 3.22 ppm, glycineconjugated bile acids (GCBAs) at 3.74 ppm, and the amide proton (-NH) arising from GCBAs and TCBAs in the region 7.76-8.05 ppm. The peak areas of these signals were measured by deconvolution, and subsequently the molar concentrations of metabolites were estimated with good accuracy, except for that of TBAs plus cholesterol. The concentration of TBAs plus cholesterol was overestimated in some cases, which could be due to lipid contamination. In addition, we report the first 2D L-COSY spectra of human gallbladder bile in vivo (obtained in 15 healthy volunteers). 2D L-COSY spectra will be helpful in differentiating various biliary chol-PLs in pathological conditions of the HPB system.

Colorectal cancer is one of the most common cancers in the western world. Its early detection has been found to improve the prognosis of the patient, providing a wide window of opportunity for successful therapeutic interventions. However, current diagnostic techniques all have some limitations; there is a need to develop a better technique for routine screening purposes. We present a new methodology based on magnetic resonance spectroscopy of fecal extracts for the non-invasive detection of colorectal cancer. Five hundred twenty-three human subjects (412 with no colonic neoplasia and 111 with colorectal cancer, who were scheduled for colonoscopy or surgery) were recruited to donate a single sample of stool. One-dimensional 1H magnetic resonance spectroscopy (MRS) experiments were performed on the supernatant of aqueous dispersions of the stool samples. Using a statistical classification strategy, several multivariate classifiers were developed. Applying the preprocessing, feature selection and classifier development stages of the Statistical Classification Strategy led to ∼87% average balanced sensitivity and specificity for both training and monitoring sets, improving to ∼92% when only crisp results, i.e. class assignment probabilities ≥75%, are considered. These results indicate that 1H magnetic resonance spectroscopy of human fecal extracts, combined with appropriate data analysis methodology, has the potential to detect colorectal neoplasia accurately and reliably, and could be a useful addition to the current screening tools. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

It has been generally found that solid tumours in vivo are more susceptible to destruction by heat than normal tissues. Hyperthermia has, thus, been employed in the treatment of cancer either applied alone or in combination with other modalities such as chemotherapy and radiotherapy. However, the critical mechanism(s) by which heat sensitizes and kills cells in the solid tumour remains poorly defined. Magnetic resonance spectroscopic monitoring of tumour metabolism during application of hyperthermia may provide important insight into the response to hyperthermic challenge. The implementation of dual antenna-coil methodology that provides for NMR spectroscopic monitoring (31P at 121 MHz) concomitant with applied 4 MHz RF hyperthermia in murine tumours is described herein, in some detail. This technology, which does not require advanced (and expensive) magnetic resonance imaging systems, should be readily adaptable by other laboratories with an interest in murine tumour models. [ABSTRACT FROM AUTHOR]

Described herein are the initial findings from an 'in-magnet' 31 P NMR compatible hyperthermia system capable of concurrently heating and monitoring the metabolic response of murine tumours; the murine radiation induced fibrosarcoma (RIF-1) was employed for these studies. At thermal doses sufficient to raise tumour temperature to 41.5 and 43°C for a period of 30 min, a marked and rapid decrease in nucleoside triphosphate concentration and in pH was observed during the heating period, while inorganic phosphate concentration increased significantly but more gradually. These 31 P NMR determined metabolic indices remained depressed/elevated throughout a 1.5 h post-hyperthermia monitoring period. Importantly, these metabolic indices correlated significantly with specific growth delay. This suggests a possible role for NMR spectroscopy in early assessment, and perhaps control, of therapeutic response to hyperthermia. [ABSTRACT FROM AUTHOR]

Apoptosis and necrosis need to be differentiated in order to distinguish drug-induced cell death from spontaneous cell death due to hypoxia. The ability to differentiate between these two modes of cell death, especially at an early stage in the process, could have a significant impact on accessing the outcome of anticancer drug therapy in the clinic. Nuclear magnetic resonance spectroscopy was used to distinguish apoptosis from necrosis in human cervical carcinoma (HeLa) cells. Apoptosis was induced by treatment with the topoisomerase II inhibitor etoposide, whereas necrosis was induced by the use of ethacrynic acid or cytochalasin B. We found that the intensity of the methylene resonance increases significantly as early as 6 h after the onset of apoptosis, but that no such changes occur during necrosis. The spectral intensity ratio of the methylene to methyl resonances also shows a high correlation with the percentage of apoptotic cells in the sample (r²=0.965, P < 0.003). [ABSTRACT FROM AUTHOR]

Abstract Background The aim of this study was to analyze the potential of proton magnetic resonance spectroscopy (1H MRS) in diagnosing early inflammatory bowel disease (IBD). Methods Thirty male Sprague Dawley rats were fed 2% carrageenan in their diet for either 1 or 2 weeks. 1H MRS was performed ex-vivo on colonic mucosal samples (n = 123) and the spectra were analyzed by a multivariate method of analysis. The results of the multivariate analysis were correlated with histological analysis performed using H & E stain for the presence of inflammation in the samples from each group. Results Multivariate analysis classified the samples in their respective groups with an accuracy of 82%. Our region selection algorithm identified four regions in the spectra as being discriminatory. The metabolites assigned to these regions include creatine, phosphatidylcholine, the -CH2HC= group in fatty acyl chain, and the glycerol backbone of lipids. The differences in concentration of these metabolites in each group offer insight into the biochemical changes occurring during IBD and confer diagnostic potential to 1H MRS as a tool to study colonic inflammation in conjunction with biopsy. Conclusion 1H MRS is a sensitive tool to detect early colonic inflammation in an animal model of IBD.

Among human genetic diseases, Fanconi Anemia (FA) tops all with its largest number of health complications in nearly all human organ systems, suggesting the significant roles played by FA genes in the maintenance of human health. With the accumulated research on FA, the encoded protein products by FA genes have been building up to the biggest cell defense signaling network, composed of not only 22+ FA proteins but also ATM, ATR, and many other non-FA proteins. The FA D2 group protein (FANCD2) and its paralog form the focal point of FA signaling to converge the effects of its upstream players in response to a variety of cellular insults and simultaneously with downstream players to protect humans from contracting diseases, including aging and cancer. In this review, we update and discuss how the FA signaling crucially eases cellular stresses through understanding its focal point.

Serum metabolites of healthy controls and esophageal cancer (EC) patients have previously been compared to predict cancer-specific profiles. However, the association between metabolic alterations in serum samples and esophageal tissues in EC patients remains unclear. Here, we analyzed 50 pairs of EC tissues and distant noncancerous tissues, together with patient-matched serum samples, using 1 H NMR spectroscopy and pattern recognition algorithms. EC patients could be differentiated from the controls based on the metabolic profiles at tissue and serum levels. Some overlapping discriminatory metabolites, including valine, alanine, glucose, acetate, citrate, succinate and glutamate, were identified in both matrices. These results suggested deregulation of metabolic pathways, and potentially revealed the links between EC and several metabolic pathways, such as the tricarboxylic acid cycle, glutaminolysis, short-chain fatty acid metabolism, lipometabolism and pyruvate metabolism. Perturbation of the pyruvate metabolism was most strongly associated with EC progression. Consequently, an optimal serum metabolite biomarker panel comprising acetate and pyruvate was developed, as these two metabolites are involved in pyruvate metabolism, and changes in their serum levels were significantly correlated with alterations in the levels of some other esophageal tissue metabolites. In comparison with individual biomarkers, this panel exhibited better diagnostic efficiency for EC, with an AUC of 0.948 in the test set, and a good predictive ability of 82.5% in the validation set. Analysis of key genes related to pyruvate metabolism in EC patients revealed patterns corresponding to the changes in serum pyruvate and acetate levels. These correlation analyses demonstrate that there were distinct metabolic characteristics and pathway aberrations in the esophageal tumor tissue and in the serum. Changes in the serum metabolic signatures could reflect the alterations in the esophageal tumor profile, thereby emphasizing the importance of distinct serum metabolic profiles as potential noninvasive biomarkers for EC., (© 2021 John Wiley & Sons, Ltd.)

Previous studies have compared fecal metabolites from healthy and colorectal cancer (CRC) patients to predict the pro-CRC signatures. However, the systemic mechanistic link between feces and colonic tissues of CRC patients is still limited. The current study was a paralleled investigation of colonic tumor tissues and their normal adjacent tissues alongside patient-matched feces by using 1 H nuclear magnetic resonance spectroscopy combined with pattern recognition to investigate how fecal metabolic phenotypes are linked to the changes in colorectal tumor profiles. A set of overlapping discriminatory metabolites across feces and tumor tissues of CRC were identified, including elevated levels of lactate, glutamate, alanine, succinate and reduced amounts of butyrate. These changes could indicate the networks for metabolic pathway perturbations in CRC potentially involved in the disruptions of glucose and glycolytic metabolism, TCA cycle, glutaminolysis, and short chain fatty acids metabolism. Furthermore, changes in fecal acetate were positively correlated with alterations of glucose and myo-inositol in colorectal tumor tissues, implying enhanced energy production for rapid cell proliferation. Compared to other fecal metabolites, acetate demonstrated the highest diagnostic performance for diagnosing CRC, with an AUC of 0.843 in the training set, and a good predictive ability in the validation set. Overall, these associations provide evidence of distinct metabolic signatures and metabolic pathway disturbances between the colonic tissues and feces within the same individual, and changes of fecal metabolic signature could reflect the CRC tissue microenvironment, highlighting the significance of the distinct fecal metabolic profiles as potential novel and noninvasive relevant indicators for CRC detection., (© 2019 UICC.)

Primary sclerosing cholangitis (PSC) has been considered to be either an "autoimmune disease" or a "bile acid-induced injury." In vitro MRS studies on PSC patients have limitations due to the contamination of bile with contrast agent (commonly administered during endoscopic retrograde cholangiopancreatography) and/or the use of patient cohorts with other diseases as controls. The objective of this study was to quantify biliary metabolites using in vivo 1 H MRS and gain insight into the pathogenesis of PSC. Biliary metabolites in 10 PSC patients and 14 healthy controls were quantified in vivo using 1 H MRS on a 3 T MR scanner. The concentrations of total bile acids plus cholesterol, glycine-conjugated bile acids, taurine-conjugated bile acids, and choline-containing phospholipids (chol-PLs) were compared between the two groups. There were statistically significant decreases in the levels of the above mentioned biliary metabolites in the PSC patients compared with controls. The reduction in bile acid secretion in bile of PSC patients indicates accumulation of bile acids in hepatocytes. Moreover, reduction in the levels of chol-PLs in bile may increase the toxic effects of bile acids. Our findings suggest that the bile duct injury in PSC patients is most likely due to "bile acid-induced injury.", (© 2019 John Wiley & Sons, Ltd.)

The field of metabolomics has been growing tremendously over the recent years and, consistent with that growth, a number of investigators have been looking at the potential of NMR-based urinary metabolomics for several applications. While such applications have shown promising results, there still remains an enormous amount of work to be done before this approach becomes accepted and widely used in clinical diagnostics and other biomedical applications. To achieve such goals, optimization of parameters and standardization of protocols are of paramount importance. In view of this, in this chapter, we present some recommended methods and procedures that can help researchers in the field. Furthermore, we have highlighted some of the challenges encountered in such applications and suggested some possible ways to overcome those challenges.

Metabolomics is a rapidly growing field with potential applications in various disciplines. In particular, metabolomics has received special attention in the discovery of biomarkers and diagnostics. This is largely due to the fact that metabolomics provides critical information related to the downstream products of many cellular and metabolic processes which could provide a snapshot of the health/disease status of a particular tissue or organ. Many of these cellular products eventually find their way to urine; hence, analysis of urine via metabolomics has the potential to yield useful diagnostic and prognostic information. Although there are a number of analytical platforms that can be used for this purpose, this review article will focus on nuclear magnetic resonance-based metabolomics. Furthermore, although there have been many studies addressing different diseases and metabolic disorders, the focus of this review article will be in the following specific applications: urinary tract infection, kidney transplant rejection, diabetes, some types of cancer, and inborn errors of metabolism. A number of methodological considerations that need to be taken into account for the development of a clinically useful optimal test are discussed briefly., Competing Interests: DECLARATION OF CONFLICTING INTERESTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objectives: Lung cancer is one of the most lethal cancers. Currently, there are no biomarkers for early detection, monitoring treatment response, and detecting recurrent lung cancer. We undertook this study to determine if 1 H magnetic resonance spectroscopy (MRS) of sputum and exhaled breath condensate (EBC), as a noninvasive tool, can identify metabolic biomarkers of lung cancer., Materials and Methods: Sputum and EBC samples were collected from 20 patients, comprising patients with pathologically confirmed non-small cell lung cancer ( n = 10) and patients with benign respiratory conditions ( n = 10). Both sputum and EBC samples were collected from 18 patients; 2 patients provided EBC samples only. 1 H MR spectra were obtained on a Bruker Avance 400 MHz nuclear magnetic resonance (NMR) spectrometer. Sputum samples were further confirmed cytologically to distinguish between true sputum and saliva., Results: In the EBC samples, median concentrations of propionate, ethanol, acetate, and acetone were higher in lung cancer patients compared to the patients with benign conditions. Median concentration of methanol was lower in lung cancer patients (0.028 mM) than in patients with benign conditions (0.067 mM; P = 0.028). In the combined sputum and saliva and the cytologically confirmed sputum samples, median concentrations of N -acetyl sugars, glycoprotein, propionate, lysine, acetate, and formate were lower in the lung cancer patients than in patients with benign conditions. Glucose was found to be consistently absent in the combined sputum and saliva samples (88%) as well as in the cytologically confirmed sputum samples (86%) of lung cancer patients., Conclusion: Absence of glucose in sputum and lower concentrations of methanol in EBC of lung cancer patients discerned by 1 H MRS may serve as metabolic biomarkers of lung cancer for early detection, monitoring treatment response, and detecting recurrence., Competing Interests: Authors disclose no potential conflicts of interest.

Metabolomics is a relatively new technique that is gaining importance very rapidly. MRS-based metabolomics, in particular, is becoming a useful tool in the study of body fluids, tissue biopsies and whole organisms. Advances in analytical techniques and data analysis methods have opened a new opportunity for such technology to contribute in the field of diagnostics. In the MRS approach to the diagnosis of disease, it is important that the analysis utilizes all the essential information in the spectra, is robust, and is non-subjective. Although some of the data analytic methods widely used in chemical and biological sciences are sketched, a more extensive discussion is given of a 5-stage Statistical Classification Strategy. This proposes powerful feature selection methods, based on, for example, genetic algorithms and novel projection techniques. The applications of MRS-based metabolomics in breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, hepatobiliary cancers, gastric cancer, and brain cancer have been reviewed. While the majority of these applications relate to body fluids and tissue biopsies, some in vivo applications have also been included. It should be emphasized that the number of subjects studied must be sufficiently large to ensure a robust diagnostic classification. Before MRS-based metabolomics can become a widely used clinical tool, however, certain challenges need to be overcome. These include manufacturing user-friendly commercial instruments with all the essential features, and educating physicians and medical technologists in the acquisition, analysis, and interpretation of metabolomics data.

Bile acids, phospholipids, and cholesterol are the major lipid components in human bile. The composition of bile is altered in various cholestatic diseases, and determining such alterations will be of great clinical importance in understanding the pathophysiology of these diseases. A robust method for the simultaneous quantification of major biliary lipids--glycine-conjugated bile acids (GCBAs), taurine-conjugated bile acids (TCBAs), total bile acids (TBAs) and choline-containing phospholipids (choline-PLs) has been devised using (1)H NMR spectroscopy. Bile samples were obtained from patients with various hepatopancreatobiliary diseases (n=10) during an endoscopic retrograde cholangiopancreatography (ERCP) examination. Peak areas of metabolite-signals of interest were obtained simultaneously by deconvoluting the experimental spectrum, making the present method robust. GCBAs and TCBAs have been quantified using the peak areas of their characteristic methylene (CH(2)) signals resonating at 3.73 and 3.07 ppm, whereas TBA and choline-PLs were quantified using their methyl (CH(3)) and trimethylammonium (-N(+)(CH(3))(3)) signals resonating at 0.65 and 3.22 ppm respectively. The present method was compared with an NMR-based literature method (which involves dissolving bile in DMSO), and a good correlation was observed between the two methods with regression coefficients - 0.97, 0.99, 0.98 and 0.93 for GCBAs, TCBAs, TBAs, and choline-PLs respectively. This method has the potential to be extended to in vivo applications for the simultaneous quantification of various biliary lipids non-invasively., (Crown Copyright (c) 2010. Published by Elsevier B.V. All rights reserved.)

People with inflammatory bowel disease (IBD) are at risk for developing colorectal cancer, and this risk increases at a rate of 1% per year after 8-10 years of having the disease. Saturated and omega-6 polyunsaturated fatty acids (PUFAs) have been implicated in its causation. Conversely, omega-3 PUFAs may have the potential to confer therapeutic benefit. Since proton magnetic resonance spectroscopy ((1)H MRS) combined with pattern recognition methods could be a valuable adjunct to histology, the objective of this study was to analyze the potential of (1)H MRS in assessing the effect of dietary fatty acids on colonic inflammation. Forty male Sprague-Dawley rats were administered one of the following dietary regimens for 2 weeks: low-fat corn oil (omega-6), high-fat corn oil (omega-6), high-fat flaxseed oil (omega-3) or high-fat beef tallow (saturated fatty acids). Half of the animals were fed 2% carrageenan to induce colonic inflammation similar to IBD. (1)H MRS and histology were performed on ex vivo colonic samples, and the (1)H MR spectra were analyzed using a statistical classification strategy (SCS). The histological and/or MRS studies revealed that different dietary fatty acids modulate colonic inflammation differently, with high-fat corn oil being the most inflammatory and high-fat flaxseed oil the least inflammatory. (1)H MRS is capable of identifying the biochemical changes in the colonic tissue as a result of inflammation, and when combined with SCS, this technique accurately differentiated the inflamed colonic mucosa based on the severity of the inflammation. This indicates that MRS could serve as a valuable adjunct to histology in accurately assessing colonic inflammation. Our data also suggest that both the type and the amount of fatty acids in the diet are critical in modulating IBD.

NMR-based metabolomics is becoming a useful tool in the study of body fluids and has a strong potential to contribute to disease diagnosis. While applications on urine and serum have been the focus to date, there are a number of other body fluids that are readily available and could potentially be used for metabolomics-based disease diagnosis. One such body fluid is stool or fecal extract. Given its contact with and transient stay in the colon and rectum, stool carries a lot of useful information regarding the health/disease status of both the colon and the rectum. This could be particularly useful for the non-invasive diagnosis of colorectal cancer and inflammatory bowel disease--the two bowel diseases that are very common and pose significant public health problems. Different methodological considerations including the collection of sample, the storage of sample, the preparation of sample, NMR acquisition parameters, experimental conditions and data analysis methods are discussed. Results obtained in the detection of colorectal cancer and in the differentiation of the two major forms of inflammatory bowel disease (i.e. ulcerative colitis and Crohn's disease) are presented. This is concluded with a brief discussion on the future of MR metabolomics of fecal extracts.

Objective: There are no specific biomarkers available for the definitive diagnosis of pancreatic cancer. Analysis of D-glucuronic acid (GlcUA) in bile could be valuable in this regard., Materials and Methods: Bile samples obtained from patients with pancreatic cancer (n = 4), chronic pancreatitis (n = 3) and control patients with biliary obstruction (n = 10) were analyzed by (1)H NMR spectroscopy. GlcUA was quantified from the peak area of the alpha-(1)CH signal (at 5.24 ppm) obtained by deconvolution., Results: GlcUA was detected in human bile by one-dimensional (1)H NMR and two-dimensional (1)H-(1)H COSY and TOCSY experiments. Quantification of GlcUA was achieved by measuring the peak area of the alpha-(1)CH signal using CPMG experiment, and the quantities of GlcUA were calibrated to account for the attenuation due to T (2) relaxation. GlcUA was observed at elevated levels in bile samples obtained from pancreatic cancer patients, whereas it was either absent or found in negligible amounts in control and chronic pancreatitis patients. The reason for the presence of elevated levels of GlcUA could be the hydrolysis of biliary bilirubin diglucuronide by beta-glucuronidase, released excessively from pancreatic tissue during the course of malignancy., Conclusion: Analysis of D-glucuronic acid in bile could be valuable in the detection of pancreatic cancer, and detecting GlcUA by in vivo (1)H MRS has the potential to help in the non-invasive diagnosis of pancreatic cancer. Given that only four cancer patients have been studied so far, the new biomarker is regarded as a preliminary finding, but one that warrants further investigation.

Hypoxia may contribute to the pathogenesis of synovitis in rheumatoid arthritis (RA). Magnetic resonance spectroscopy (MRS) is a technique that uses radiofrequency waves to generate a signal which allows a qualitative and quantitative assessment of the biochemical composition of tissue. MRS was used to evaluate RA synovial tissue for evidence of hypoxia and anaerobic metabolism. Synovial tissue samples obtained from eighteen RA patients and four osteoarthritis control patients undergoing total knee replacement were analyzed using proton MRS, processed for histopathology and scored for inflammation and vascularity. Spectra from severely and mildly inflamed tissue differed in peak intensity at regions 1.3 ppm (representing lactic acid and lipid), 3.0 ppm (representing creatine), 3.2 ppm (representing choline containing metabolites), and 3.8 ppm (representing carbohydrates, possibly glucose). With increasing inflammation, the intensities of the peak resonance at 1.3 ppm increased and that at 3.8 ppm decreased. The intensities of the 3.8 and 3.0 ppm peaks were reduced in highly vascular tissue. Specific MR spectral features reflect the anaerobic metabolism that is evident with progressively increasing degrees of RA synovial inflammation and vascularity. These features correlate partially with synovial histopathology.

Metabolic profiling of biological specimens is emerging as a promising approach for discovering specific biomarkers in the diagnosis of a number of diseases. Amongst many analytical techniques, nuclear magnetic resonance spectroscopy and mass spectrometry are the most information-rich tools that enable high-throughput and global analysis of hundreds of metabolites in a single step. Although only one of the two techniques is utilized in a majority of metabolomics applications, there is a growing interest in combining the data from the two methods to effectively unravel the mammoth complexity of biological samples. In this article, current developments in nuclear magnetic resonance, mass spectrometry and multivariate statistical analysis methods are described. While some general applications that utilize the combination of the two analytical methods are presented briefly, the emphasis is laid on the recent applications of nuclear magnetic resonance and mass spectrometry methods in the studies of hepatopancreatobiliary and gastrointestinal malignancies.

The utility of (1)H MR spectroscopy in detecting chronic cholestasis has been investigated. The amide proton region of the (1)H MR spectrum of human bile plays a major role in differentiating cholestatic (Ch) patterns from the normal ones. Bile obtained from normal bile ducts contains both taurine and glycine conjugates of bile acids--cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA). Absence of a glycine-conjugated bile acid glycochenodeoxycholic acid (GCDCA) has been observed in bile samples obtained from primary sclerosing cholangitis (PSC) patients. A total of 32 patients with various hepatobiliary diseases were included in the study. Twenty-one patients had PSC and 11 had normal cholangiograms. One PSC patient was excluded from the study because of a bad spectrum. Seventeen out of the 20 PSC patients showed an absence of GCDCA in their (1)H MR spectrum of bile. Six of the 11 reference patients with normal cholangiogram also showed spectra similar to those of PSC, indicating the possibility of cholestasis. DQF-COSY and TOCSY experiments performed on bile samples from PSC patients also revealed absence of phosphatidylcholine (PC) in some of the bile samples, suggesting possible damage to the cholangiocytes by the toxic bile. These observations suggest that analysis of human bile by (1)H MRS could be of value in the diagnosis of chronic Ch liver disorders., (2008 John Wiley & Sons, Ltd.)

N-linked oligosaccharides obtained from total serum of mice with implanted head and neck tumors were analyzed and compared with those from control samples of healthy mice. Methods used include a combination of a derivatization procedure with phenylhydrazine (PHN) and analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Oligosaccharides were enzymatically released from total serum with PNGaseF and purified by high-performance liquid chromatography (HPLC) on a reversed-phase column. Mass spectra contained ion peaks of labeled oligosaccharides and MS/MS experiments provided useful data for the structural elucidation of these compounds. More than 40 N-glycans with compositions characteristic of high-mannose, hybrid, complex, neutral, and sialylated structures were identified in the serum of tumoral mice. Significant differences between samples were observed with respect to the abundances of high mannose and hybrid glycans. These oligosaccharides showed higher relative intensities in the spectra obtained from the cancer sera. Complex sialylated oligosaccharides had similar abundances in both types of sera, with the exception of fucosylated biantennary disialylated oligosaccharide, which was mostly detected with lower abundance in control samples. In the MALDI spectra, several minor species corresponded to uncommon carbohydrates. These structures have been investigated in detail by MS/MS. Among these novel glycoforms, a few sialylated oligosaccharides without a free reducing end were identified. Also, glycans with an extra 60 u were observed and likely feature the presence of a 2-acetamido-2-deoxyoctose residue attached on antennae of 3- or 6-linked mannose.

The significance of glycoproteins in living systems instigates the ceaseless expansion of new techniques and procedures for the analysis of biological samples. Many of these applications are focused on improving the detection limit of analyzed material. In a previous study, we described a procedure for the detection of oligosaccharides cleaved from tryptic glycopeptides. Treatment of deglycosylated fractions with phenylhydrazine gave rise to peaks consistent with labeled glycans, and both types of compounds--deglycosylated peptides and oligosaccharides--were recorded from one spot and observed in one matrix-assisted laser desorption/ionization (MALDI) mass spectrum for the first time. Here, we added an additional step to this simple procedure of deglycosylating glycopeptides directly from the target spot of the first analyzed glycosylated peptides. For the purpose of this new study, a mixture of 2-aza-2-thiothymine and phenylhydrazine hydrochloride showed to be an excellent matrix for glycopeptides, oligosaccharides, deglycosylated peptides and moreover it allowed PNGaseF to be active enough to cleave oligosaccharides from peptides. The efficiency of this procedure is demonstrated on a series of intact glycoproteins and on the analysis of tryptic peptides obtained from IgG and total mouse serum. This one-step on-target deglycosylation method with subsequent derivatization on the same spot makes MALDI-MS analyses of glycopeptides fast, simple and accessible for biological samples, where classical procedures cannot produce useful results.

Tumor hypoxia and its downstream effects have remained of considerable interest for decades due to its negative impact on response to various cancer therapies and promotion of metastasis. Diagnosing hypoxia non-invasively can provide a significant advancement in cancer treatment and is the dire necessity for implementing specific targeted therapies now emerging to treat different aspects of cancer. A variety of techniques are being proposed to do so. However, none of them has yet been established in the clinical arena. This review summarizes the methods currently available to assess tumor hypoxia in vivo and their respective advantages and shortcomings. It also points out the impedances that need to be overcome to establish any particular method in the clinic, along with a broad overview of requirements for further advancement in this sphere of cancer research.

Background and Purpose: Poor treatment outcome remains high in patients with squamous cell carcinoma (SCC) of the head and neck region. Treatment of head and neck cancer could be improved and perhaps standardized if reliable markers for tumor progression and poor prognosis could be developed. MR spectroscopy has been used previously to differentiate between malignant and adjacent normal specimen in these cancers. This study explores the capability of MR spectroscopy in providing an indication of the aggressiveness of a tumor and its response to treatment., Methods: Thirty-six SCC patients with treatment failure, and 22 other patients who were treated concurrently at the same center but with no indication of failure for a period of 3 years, were selected for the study. Tumor specimens were kept frozen at -70 degrees C, and later subjected to 1H-MR spectroscopy at 25 degrees C. The resonance areas for 6 spectral regions were determined, and their ratio calculated. The mean values of the ratios were then compared between the 2 groups by using the Student t test., Results: The choline-to-creatine (3.2/3.0 parts per million [ppm]) and the 1.3/0.9 ppm spectral intensity ratios (signal due to lipid or lactic acid) were the 2 most notable ones to be significantly elevated in the group with poor response. Using these ratios, a sensitivity of 83% and a specificity of 82% were obtained in predicting which head and neck cancer patients would fail treatment., Conclusions: These preliminary results suggest that MR spectroscopy has the potential to contribute to an accurate and early prediction of tumor behavior and response to treatment in squamous cell carcinoma of the head and neck region.

Background: Definitive diagnosis of head and neck cancer is generally made by histopathologic evaluation. Management and prognosis largely depend on accurate and timely diagnosis. We have explored the use of (1)H magnetic resonance spectroscopy in search of a better or complementary diagnostic technique., Methods: Tumor and adjacent normal tissue specimens (n = 135) from untreated head and neck cancer patients (n = 40) were obtained and subjected to spectroscopic evaluation followed by histopathologic analysis. Data were partitioned into training and test sets and subjected to multivariate analysis., Results: The resonances from taurine, choline, glutamic acid, lactic acid, and lipid were found to have diagnostic potential by our optimal region selection algorithm. Multivariate analysis of the spectral data differentiated between normal and malignant tissues, with an overall accuracy of 92.6% (training set, 97.3%; test set, 87.3%), an overall sensitivity of 93% (test set, 90%), and an overall specificity of 92% (test set, 82.6%)., Conclusions: (1)H magnetic resonance spectroscopy combined with multivariate methods of analysis can distinguish between normal and malignant squamous cell tissue, and this may lead to the development of an objective and noninvasive diagnostic procedure., (Copyright 2002 Wiley Periodicals, Inc.)

Purpose: Histological grading is currently one of the best predictors of tumor behavior and outcome in soft tissue sarcoma. However, occasionally there is significant disagreement even among expert pathologists. An alternative method that gives more reliable and non-subjective diagnostic information is needed. The potential use of proton magnetic resonance spectroscopy in combination with an appropriate statistical classification strategy was tested here in differentiating normal mesenchymal tissue from soft tissue sarcoma., Methods: Fifty-four normal and soft tissue sarcoma specimens of various histological types were obtained from 15 patients. One-dimensional proton magnetic resonance spectra were acquired at 360 MHz. Spectral data were analyzed by using both the conventional peak area ratios and a specific statistical classification strategy., Results: The statistical classification strategy gave much better results than the conventional analysis. The overall classification accuracy (based on the histopathology of the MRS specimens) in differentiating normal mesenchymal from soft tissue sarcoma was 93%, with a sensitivity of 100% and specificity of 88%.The results in the test set were 83, 92 and 76%, respectively. Our optimal region selection algorithm identified six spectral regions with discriminating potential, including those assigned to choline, creatine, glutamine, glutamic acid and lipid., Conclusion: Proton magnetic resonance spectroscopy combined with a statistical classification strategy gave good results in differentiating normal mesenchymal tissue from soft tissue sarcoma specimens ex vivo. Such an approach may also differentiate benign tumors from malignant ones and this will be explored in future studies.