Raja Roy

Standardizing the experimental conditions for using urine in NMR-based metabolomic studies with a particular focus on diagnostic studies: a review

AbstractThe metabolic composition of human biofluids can provide important diagnostic and prognostic information. Among the biofluids most commonly analyzed in metabolomic studies, urine appears to be particularly useful. It is abundant, readily available, easily stored and can be collected by simple, noninvasive techniques. Moreover, given its chemical complexity, urine is particularly rich in potential disease biomarkers. This makes it an ideal biofluid for detecting or monitoring disease processes. Among the metabolomic tools available for urine analysis, NMR spectroscopy has proven to be particularly well-suited, because the technique is highly reproducible and requires minimal sample handling. As it permits the identification and quantification of a wide range of compounds, independent of their chemical properties, NMR spectroscopy has been frequently used to detect or discover disease fingerprints and biomarkers in urine. Although protocols for NMR data acquisition and processing have been standardized, no consensus on protocols for urine sample selection, collection, storage and preparation in NMR-based metabolomic studies have been developed. This lack of consensus may be leading to spurious biomarkers being reported and may account for a general lack of reproducibility between laboratories. Here, we review a large number of published studies on NMR-based urine metabolic profiling with the aim of identifying key variables that may affect the results of metabolomics studies. From this survey, we identify a number of issues that require either standardization or careful accounting in experimental design and provide some recommendations for urine collection, sample preparation and data acquisition.n

Lipid profiling of developing jatropha curcas L. seeds using 1H NMR spectroscopy

Seed development in Jatropha curcas L. was studied with respect to phenology, oil content, lipid profile and concentration of sterols. Seeds were collected at various stages of development starting from one week after fertilization and in an interval of five days thereafter till maturity. These were classified as stage I to stage VII. Moisture content of the seeds ranged from 8.8 to 90.3%; the lowest in mature seeds in stage VII and highest in stage I. The seed area increased as the seed grew from stage I to stage VI (0.2-10.2mm(2) per seed), however, the seed area shrunk at stage VII. Increase in seed area corresponded to increase in fresh weight of the seeds. (1)H NMR spectroscopy of hexane extracts made at different stages of seed development revealed the presence of free fatty acids (FFA), methyl esters of fatty acids (FAME) and triglycerol esters (TAG), along with small quantity of sterols. The young seeds synthesized predominantly polar lipids. Lipid synthesis was noticed nearly three weeks after fertilization. From the fourth week the seeds actively synthesized TAG. Stage III is a turning point in seed development since at this stage, the concentration of sterols decreased to negligible, there was very little FAME formation, accumulation of TAG increased substantially, and there was a sudden decrease in FFA concentration. The findings can be helpful in understanding the biosynthesis and in efforts to improve biosynthesis of TAG and reduce FFA content in the mature seeds.

Recommendations and Standardization of Biomarker Quantification Using NMR-Based Metabolomics with Particular Focus on Urinary Analysis

NMR-based metabolomics has shown considerable promise in disease diagnosis and biomarker discovery because it allows one to nondestructively identify and quantify large numbers of novel metabolite biomarkers in both biofluids and tissues. Precise metabolite quantification is a prerequisite to move any chemical biomarker or biomarker panel from the lab to the clinic. Among the biofluids commonly used for disease diagnosis and prognosis, urine has several advantages. It is abundant, sterile, and easily obtained, needs little sample preparation, and does not require invasive medical procedures for collection. Furthermore, urine captures and concentrates many “unwanted” or “undesirable” compounds throughout the body, providing a rich source of potentially useful disease biomarkers; however, incredible variation in urine chemical concentrations makes analysis of urine and identification of useful urinary biomarkers by NMR challenging. We discuss a number of the most significant issues regarding NMR-based urinary metabolomics with specific emphasis on metabolite quantification for disease biomarker applications and propose data collection and instrumental recommendations regarding NMR pulse sequences, acceptable acquisition parameter ranges, relaxation effects on quantitation, proper handling of instrumental differences, sample preparation, and biomarker assessment.

Polycyclic aromatic hydrocarbons and their quinones modulate the metabolic profile and induce DNA damage in human alveolar and bronchiolar cells.

The release of particulate pollutants into the air through burning of coal, crude oil, diesel, coal tar, etc. raises concerns of potential health hazards to the exposed human population. Polycyclic aromatic hydrocarbons (PAHs) are major toxic constituents of particulate matter (PM), which upon ingestion get metabolized to even more toxic metabolites such as quinones. The PAHs levels were assessed in both respirable particulate matter (RSPM, <10μM size) and suspended particulate matter (SPM, >10μM size) of urban ambient air (UAA) and that of major contributors viz. diesel exhaust particles (DEPs) and coal tar combustions emissions (CTCE). Seven US Environmental Protection Agency (USEPA) prioritized PAHs in RSPM and 10 in SPM were detected in UAA. Ten and 15 prioritized PAHs, respectively, were also detected in diesel exhaust particles (DEP) and coal tar combustion emission (CTCE) evidencing their release in the air. These PM associated PAHs for UAA, DEP and CTCE showed significant increase (p<0.05) in mutagenicity and mammalian genotoxicity in the order CTCE>DEP>UAA. Human lung alveolar (A549) and bronchiolar (BEAS-2B) cells when treated with PAH-metabolites viz. 1,4-benzoquinone (1,4-BQ), hydroquinone (HQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ) and 9,10-phenanthroquinone (9,10-PQ) showed metabolic modulation in these cell lines with significant depletion of principal cellular metabolites viz. NADP, uracil, asparagines, glutamine, and histidine and accumulation of di-methyl amine and beta-hydroxybutyrate, identified using (1)H NMR spectroscopy. These results suggest that PAH-quinones induce genotoxic effects by modulating the metabolic machinery inside the cells by a combined effect of oxidative stress and energy depletion. Our data for metabolic profiling of human lung cells could also help in understanding the mechanism of toxicity of other xenobiotics.

Application of HR-MAS NMR spectroscopy for studying chemotype variations of Withania somnifera (L.) Dunal.

Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plants with a number of pharmaceutical and nutraceutical applications. Metabolic profiling has been performed by HR‐MAS NMR spectroscopy on fresh leaf and root tissue specimens from four chemotypes of W. somnifera. The HR‐MAS NMR spectroscopy of lyophilized defatted leaf tissue specimens clearly distinguishes resonances of medicinally important secondary metabolites (withaferin A and withanone) and its distinctive quantitative variability among the chemotypes. A total of 41 metabolites were identified from both the leaf and root tissues of the chemotypes. The presence of methanol in leaf and root tissues of W. somnifera was detected by HR‐MAS NMR spectroscopy. Multivariate principal component analysis (PCA) on HR‐MAS 1H NMR spectra of leaves revealed clear variations in primary metabolites among the chemotypes. The results of the present study demonstrated an efficient method, which can be utilized for metabolite profiling of primary and secondary metabolites in medicinally important plants. Copyright

Metabolic and histopathological alterations of Jatropha mosaic begomovirus-infected Jatropha curcas L. by HR-MAS NMR spectroscopy and magnetic resonance imaging

Alterations in the anatomical structures, sap translocation and metabolic profiles in Jatropha curcas L. (Euphorbiaceae), infected with Jatropha mosaic virus (JMV) have been investigated using MRI and HR-MAS NMR spectroscopy. The contrast of MRI images distinguishes abnormalities in anatomical structures of infected and healthy stem. The HR-MAS NMR spectroscopic analysis indicated that viral infection significantly affected the plant metabolism. Higher accumulation of TCA cycle intermediates, such as citrate and malate, in JMV-infected plants suggested a higher rate of respiration. The respiration rate was more than twofold as compared to healthy ones. The viral stress also significantly increases the concentrations of alanine, arginine, glutamine, valine, GABA and choline as compared to healthy ones. Microscopic examination revealed severe hyperplasia caused by JMV with a considerable reduction in the size of stem cells. Lower concentration of glucose and sucrose in viral-infected stem tissues indicates decreased translocation of photosynthates from leaves to stem due to hyperplasia caused by JMV. The MR images distinguished stele, cortical and pith regions of JMV-infected and healthy stems. Contrast of T1- and T2-weighted images showed significant differences in the spatial distribution of water, lipids and macromolecules in virus-infected and healthy stem tissues. The results demonstrated the value of MRI and HR-MAS NMR spectroscopy in studying viral infection and metabolic shift in plants. The present methodology may help in better understanding the metabolic alterations during biotic stress in other plant species of agricultural and commercial importance.

Aluminum‐mediated metabolic changes in rat serum and urine: A proton nuclear magnetic resonance study

The toxic effects of Al3+ have been studied in 90‐days AlCl3 orally treated male albino rats (n = 7) using 1H NMR spectroscopy‐based metabolic profile of rat serum and urine, serum enzyme tests, behavioral impairment, and histopathology of kidney and liver. Metabolic profile of 90‐days Al3+‐treated rat sera showed significantly elevated levels of alanine, glutamine, β‐hydroxy‐butyrate, and acetoacetate and significantly decreased level of acetone when compared with that of control rats. However, metabolic profile of 90‐days Al3+‐treated rat urine showed significantly decreased levels of citrate, creatinine, allantoin, trans‐aconitate, and succinate and significantly increased level of acetate when compared to control rats. The overall perturbations observed in the metabolic profile of serum and urine demonstrate the impairment in the tricarboxylic acid cycle, liver and kidney metabolism, which was further reinstated by clinical chemistry and histopathological observations. Moreover, “in vivo” behavioral impairment has also been observed as the indication of aluminum neurotoxicity.

Abnormal Small Intestinal Permeability in Patients with Idiopathic Malabsorption in Tropics (Tropical Sprue) Does Not Change Even After Successful Treatment

BackgroundThough tropical sprue (TS) is common in tropics, studies on small intestinal permeability (SIP) in TS are scant.MethodSIP was evaluated using 1H nuclear magnetic resonance (NMR) spectroscopy of urinary lactulose and mannitol in 24 patients with TS (22 before and 15 after treatment with tetracycline and folate) and in 31 healthy subjects (HS). Effect of treatment of TS on SIP and its relationship with outcome were studied.ResultSubjects were comparable in terms of age and gender. Before treatment, urinary lactulose (0.24xa0mmol, 0–1.09xa0mmol versus 0.09xa0mmol, 0–0.68xa0mmol, Pxa0=xa00.02) and lactulose-to-mannitol (L/M) ratio (0.11, 0–0.41 versus 0.042, 0–0.26, Pxa0=xa00.001) were higher in TS than in HS, though mannitol was comparable (2.7xa0mmol, 0.61–10.5xa0mmol versus 3.8xa0mmol, 1.3–16.4xa0mmol, Pxa0=xa00.08). Patients improved after treatment [stool frequency (9, 4–20/day versus 1, 1–2/day, Pxa0<xa00.0001), weight (44.4, 32–69xa0kg, versus 56, 39–84xa0kg, Pxa0<xa00.0001), fecal fat (10.1, 6–26xa0g/24xa0h versus 4.4, 3.0–6.7xa0g/24xa0h, Pxa0<xa00.0001), d-xylose (0.57, 0.28–1.2xa0g/5xa0g/5xa0h versus 1.1, 0.2–2.1xa0g/5xa0g/5xa0h, Pxa0<xa00.0001), and small intestinal bacterial overgrowth (SIBO) resolved in 10/24 (41.7%) versus 1/15 (6.6%), Pxa0=xa00.02]. Though urinary lactulose (0.17, 0–4.3xa0mmol versus 0.09, 0–0.68xa0mmol, Pxa0=xa00.11) and mannitol (2.17, 0.8–36.7xa0mmol versus 3.84, 1.3–16.4xa0mmol, Pxa0=xa00.06) were comparable, L/M ratio was higher in TS than in HS (0.09, 0–0.22 versus 0.042, 0–0.26, Pxa0=xa00.002). L/M ratio was more often abnormal (cutoff 0.078) in TS than in HS [14/22 (63.6%) versus 3/31 (9.7%); Pxa0=xa00.0001], which persisted even after treatment [9/15 (60%) as compared with HS; Pxa0=xa00.0006]. Persistently abnormal SIP was associated with less weight gain and frequent stools following treatment.ConclusionSIP is often abnormal in TS and remains unchanged even after successful treatment that was associated with less weight gain and more frequent stool.

Proton HR-MAS MR spectroscopy of oral squamous cell carcinoma tissues: an ex vivo study to identify malignancy induced metabolic fingerprints

Oral squamous cell carcinoma (SCC) represents more than 90% of all head and neck cancers as reported by Hermans (Cancer Imaging, 5(Spec No A), S52–S57, 2005), which draws attention of investigative research for novel predictive metabolic biomarkers to understand the malignancy induced biochemical perturbations occurring at molecular level. In the present work, proton HR-MAS NMR spectroscopic studies have been performed on resected human oral SCC tumor tissues, its neighboring margins and bed tissues (nxa0=xa0159), obtained from 36 patients (nxa0=xa027 training set; nxa0=xa09 unknown test set), for the identification of metabolic fingerprints. The proton NMR spectra were then subjected to chemometric unsupervised PCA and supervised OSC-filtered PCA and PLS-DA multivariate analysis. Application of PLS-DA on orthogonally signal corrected training data-set (nxa0=xa0120 tissue specimens; 27 patients) allowed >95% correct classification of malignant tissues from benign samples with >98% specificity and sensitivity. The OSC-PLS-DA model thus constructed was used to predict the class membership of unknown tissue specimens (nxa0=xa039) obtained from nine patients. These tissue samples were correctly predicted in its respective histological categories with 97.4% diagnostic accuracy. The regression coefficients obtained from OSC-filtered PLS-DA model indicated that malignant tissues had higher levels of glutamate, choline, phosphocholine, lactate, acetate, taurine, glycine, leucine, lysine, isoleucine and alanine, and lower levels of creatine and PUFA, representing altered metabolic processes (lipidogenesis, protein synthesis, and volume regulation) during tumor progression. Thus proton HR-MAS MR spectroscopy could efficiently identify the metabolic perturbations of malignant tumor from non-malignant bed and margins tissue specimens, which may be helpful in understanding the extent of tumor penetration in neighboring tissues.

Real time HR-MAS NMR: application in reaction optimization, mechanism elucidation and kinetic analysis for heterogeneous reagent catalyzed small molecule chemistry.

A novel application of in situ 1H high‐resolution magic angle spinning (HR–MAS) NMR technique for real‐time monitoring of H2SO4‐silica promoted formation of 2, 2‐disubstituted quinozolin‐4(3H)‐ones is reported. The detailed NMR spectroscopic data led to elucidation of the mechanism, reaction optimization, kinetics and quantitative analysis of the product accurately and efficiently. The translation of the optimized parameters obtained by 1H HR–MAS NMR in the wet laboratory provided similar results. It is proposed that 1H HR‐MAS has a potential utility for optimization of various organic transformations in solid supported catalyzed reactions. Copyright