Unraveling Compositional Study, Chemometric Analysis, and Cell-Based Antioxidant Potential of Selective High Nutraceutical Value Amaranth Cultivars Using a GC-MS and NMR-Based Metabolomics Approach.

Amaranthus (family Amaranthaceae) is a potentially nutritious pseudocereal also known as a functional food owing to its high nutritional quality grains especially rich in essential amino acids. Emerging study, however, unambiguously indicates that apart from essential nutrients like protein, other phytochemicals present in amaranth seeds provide excellent health benefits. Squalene is one such phytonutrient found in Amaranthus seeds, which is also its largest vegetal source. In this research work, GC-MS and NMR spectroscopy-based metabolomics have been utilized for the compositional analysis of Amaranthus seeds coupled with a multivariate data set. Investigation of nonpolar and polar seed extracts of six different cultivars of amaranth identified 47 primary and secondary metabolites. One-way ANOVA showed significant quantitative metabolic variations in different cultivars of amaranth. Multivariate principal component analysis of both the GC-MS and NMR analyzed data broadly classified in two groups showed significant variations in the polar (lysine, arginine, GABA, and myoinositol) and nonpolar (squalene, tryptophan, and alkylated phenols, which are potential nutraceutical agents) metabolites. The squalene content estimated using HPLC varied significantly (1.61 to 4.72 mg g-1 seed dry weight) among six different cultivars. Positive correlations were found among the cellular antioxidant activity and squalene content. Cultivar AM-3 having the maximum squalene content showed the highest antioxidant activity evaluated on the cellular level over human embryonic kidney cells, clearly revealing potent intercellular reactive oxygen species scavenging capacity and strong membrane lipid peroxidation inhibition potential. Oxidative stress markers such as MDA, SOD, GSH, and CAT levels in cells further corroborated the research work. The study also indicated high concentrations of lysine (80.49 mg g-1 dry seeds) in AM-2, squalene (0.47% by weight) in AM-3, and 2,4-di-tert-butyl phenol (18.64% peak area) and myoinositol (79.07 mg g-1 dry seeds) in AM-5. This novel comparative metabolomic study successfully profiles the nutrient composition of amaranth cultivars and provides the opportunity for the development of nutraceuticals and natural antioxidants from this functional food.

1H NMR Using Metabolic Study in Body Fluids for Diagnosis of Cryptococcal Meningitis in Adults.

Background: Cryptococcal meningitis is considered to affect HIV patients and those with impaired immune systems. Early identification and treatment are the keys to decreasing morbidity and mortality related to CM. Using 1H NMR spectroscopy, a prospective case-control study will assess the metabolic profile of adults' serum, urine, and CSF. Methodology: The present multicentric study was conducted at Lucknow. The study included 150 participants, out of which there were 31 cryptococcal meningitis cases, 34 positive meningitis controls, and the rest, 85, were disease controls. Result: The discriminant function analysis (DFA) of the three biofluids was used to find significant metabolites between the cases and the control group collectively. A group categorization between control group and the cases in serum, urine, and CSF samples was also made possible by the NMR spectral bin-based orthogonal signal correction and principal component analysis score plots of important metabolites produced from DFA. The cases group had a higher proportion of patients with higher CSF protein levels than the positive control group (BM and TM). Acetone was found among urine samples in both control samples, i.e., positive and negative. Conclusion: This is the first study to explore biomarkers in serum, urine, and CSF in addition to radiological features and clinical symptoms. Hence, a quick, non-invasive prognosis and diagnosis of cryptococcal meningitis in adults can be made using clinical and microbiological investigation, as well as metabolomic analysis of urine samples. This study shows that urine can be used as a biofluid to differentiate between Cryptococcus meningitis in adults. However, when compared to the negative control, our sample size was significantly smaller, necessitating further confirmation on a larger sample size.

Malignancy prediction among tissues from Oral SCC patients including neck invasions: a 1H HRMAS NMR based metabolomic study.

INTRODUCTION: Oral cancer is a sixth commonly occurring cancer globally. The use of tobacco and alcohol consumption are being considered as the major risk factors for oral cancer. The metabolic profiling of tissue specimens for developing carcinogenic perturbations will allow better prognosis. OBJECTIVES: To profile and generate precise 1H HRMAS NMR spectral and quantitative statistical models of oral squamous cell carcinoma (OSCC) in tissue specimens including tumor, bed, margin and facial muscles. To apply the model in blinded prediction of malignancy among oral and neck tissues in an unknown set of patients suffering from OSCC along with neck invasion. METHODS: Statistical models of 1H HRMAS NMR spectral data on 180 tissues comprising tumor, margin and bed from 43 OSCC patients were performed. The combined metabolites, lipids spectral intensity and concentration-based malignancy prediction models were proposed. Further, 64 tissue specimens from twelve patients, including neck invasions, were tested for malignancy in a blinded manner. RESULTS: Forty-eight metabolites including lipids have been quantified in tumor and adjacent tissues. All metabolites other than lipids were found to be upregulated in malignant tissues except for ambiguous glucose. All of three prediction models have successfully identified malignancy status among blinded set of 64 tissues from 12 OSCC patients with an accuracy of above 90%. CONCLUSION: The efficiency of the models in malignancy prediction based on tumor induced metabolic perturbations supported by histopathological validation may revolutionize the OSCC assessment. Further, the results may enable machine learning to trace tumor induced altered metabolic pathways for better pattern recognition. Thus, it complements the newly developed REIMS-MS iKnife real time precession during surgery.

Post-periodontal surgery propounds early repair salivary biomarkers by 1H NMR based metabolomics.

INTRODUCTION: Oral microflora is a well-orchestrated and acts as a sequential defense mechanism for any infection related to oral disease. Chronic periodontitis is a disease of a microbial challenge to symbiosis and homeostasis. Periodontal surgery is the most promising cure with repair process during periodontal regeneration. It has an encouraging outcome in terms of early recovery biomarkers. OBJECTIVE: Saliva of periodontal surgery subjects with the chronic periodontitis have been evaluated by 1H NMR spectroscopy in search of possible early metabolic differences that could be obtained in order to see the eradication of disease which favours the symbiotic condition. METHOD: The study employed 1H NMR spectroscopy on 176 human saliva samples in search of distinctive differences and their spectral data were further subjected to multivariate and quantitative analysis. RESULT: The 1H NMR study of periodontal surgery samples shows clear demarcation and profound metabolic differences when compared with the diseased condition. Several metabolites such as lactate, ethanol, succinate, and glutamate were found to be of higher significance in periodontal surgery in contrast to chronic periodontitis subjects. The PLS-DA model of the studied group resulted in R2 of 0.83 and Q2 of 0.70. CONCLUSION: Significant metabolites could be considered as early repair markers for chronic periodontitis disease as they are being restored to achieve symbiosis. The study, therefore, concluded the early recovery process of the diseased subjects with the restoration of possible metabolomic profile similar to the healthy controls.

NMR Spectroscopy for Metabolomics Research.

Over the past two decades, nuclear magnetic resonance (NMR) has emerged as one of the three principal analytical techniques used in metabolomics (the other two being gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled with single-stage mass spectrometry (LC-MS)). The relative ease of sample preparation, the ability to quantify metabolite levels, the high level of experimental reproducibility, and the inherently nondestructive nature of NMR spectroscopy have made it the preferred platform for long-term or large-scale clinical metabolomic studies. These advantages, however, are often outweighed by the fact that most other analytical techniques, including both LC-MS and GC-MS, are inherently more sensitive than NMR, with lower limits of detection typically being 10 to 100 times better. This review is intended to introduce readers to the field of NMR-based metabolomics and to highlight both the advantages and disadvantages of NMR spectroscopy for metabolomic studies. It will also explore some of the unique strengths of NMR-based metabolomics, particularly with regard to isotope selection/detection, mixture deconvolution via 2D spectroscopy, automation, and the ability to noninvasively analyze native tissue specimens. Finally, this review will highlight a number of emerging NMR techniques and technologies that are being used to strengthen its utility and overcome its inherent limitations in metabolomic applications.

Comprehensive metabolite profiling in distinct chemotypes of Commiphora wightii.

Commiphora wightii (Arn.) Bhandari, known as guggul, produces a medicinally important gum resin which is used extensively by Ayurvedic physicians to treat various ailments. However, most of the studies on C. wightii have been limited to its gum resin. Comprehensive metabolic profiling of leaves, stem and gum resin samples was undertaken to analyse aqueous and non-aqueous metabolites from three distinct chemotypes (NBRI-101, NBRI-102 and NBRI-103) shortlisted from different agro-climatic zones. GC-MS, HPLC and NMR spectroscopy were used for comprehensive metabolomics. Multivariate analysis showed characteristic variation in quinic and citric acids, myo-inositol and glycine (aqueous metabolites) and 2,6-di-tert-butyl-phenol, trans-farnesol and guggulsterones (non-aqueous metabolites) amongst the three chemotypes. Quinic acid, citric acid and myo-ionositol were detected in substantial quantities from leaves and stem samples which provide opportunities for novel nutraceutical and pharmaceutical formulations. Quinic acid, from the leaves, was identified as a marker metabolite for early selection of high guggulsterones-yielding cultivars.

A case study of primary malignancy of buccal mucosa using proton HR-MAS NMR spectroscopy on tissue specimens.

The diagnosis and confirmation of oral SCC (squamous cell carcinoma) is still dependent on histopathology report in spite of development of radiological investigations. It is, thus important to understand the underlying molecular mechanisms and how the alterations in metabolic pathways effect the tumor development and progression. The simultaneous and comprehensive information about the presence and absence of small molecule metabolites and their relative concentrations has been provided by 1H HR-MAS NMR spectroscopy on tissue specimens. In this paper a unique case study was presented in order to correlate histological and NMR spectroscopic findings. The patient's initially lesion was found to be non-malignant in nature based on histological findings but its periodic localized recurrence even after laser ablation prompted us to perform HR-MAS based analysis and its role in identifying the metabolic alterations in known pathways occurring during its progressions. Thus it was confirmed after analysis that HR-MAS NMR can also be used as an analytical tool which is reliable in order to distinguish between malignant and non-malignant tissues, in combination with histopathology.

Recommended strategies for spectral processing and post-processing of 1D 1H-NMR data of biofluids with a particular focus on urine.

1H NMR spectra from urine can yield information-rich data sets that offer important insights into many biological and biochemical phenomena. However, the quality and utility of these insights can be profoundly affected by how the NMR spectra are processed and interpreted. For instance, if the NMR spectra are incorrectly referenced or inconsistently aligned, the identification of many compounds will be incorrect. If the NMR spectra are mis-phased or if the baseline correction is flawed, the estimated concentrations of many compounds will be systematically biased. Furthermore, because NMR permits the measurement of concentrations spanning up to five orders of magnitude, several problems can arise with data analysis. For instance, signals originating from the most abundant metabolites may prove to be the least biologically relevant while signals arising from the least abundant metabolites may prove to be the most important but hardest to accurately and precisely measure. As a result, a number of data processing techniques such as scaling, transformation and normalization are often required to address these issues. Therefore, proper processing of NMR data is a critical step to correctly extract useful information in any NMR-based metabolomic study. In this review we highlight the significance, advantages and disadvantages of different NMR spectral processing steps that are common to most NMR-based metabolomic studies of urine. These include: chemical shift referencing, phase and baseline correction, spectral alignment, spectral binning, scaling and normalization. We also provide a set of recommendations for best practices regarding spectral and data processing for NMR-based metabolomic studies of biofluids, with a particular focus on urine.

Serum metabolomics study in a group of Parkinson's disease patients from northern India.

BACKGROUND: Parkinson's disease (PD) is the result of progressive degeneration of the nigrostriatal dopaminergic pathway and depletion of neurotransmitter dopamine in the striatum. METHODS: We included 17 patients with PD along with 7 patients of progressive supranuclear palsy (PSP), 6 patients of multiple system atrophy (MSA) and 22 age and sex-matched healthy controls. We analyzed metabolite profiles in the serum of these patients and controls using 1H NMR spectroscopy. RESULTS: Isoleucine, valine, alanine, glutamine and histidine in PD, PSP and MSA were significantly (P < 0.001) higher than controls, whereas, glutamate and glucose were significantly increased in PD (P < 0.001), PSP and MSA (P < 0.05) vs. CONTROL: Citrate was increased in PD, PSP and MSA (P < 0.05) vs. CONTROL: While, acetone, lactate and formate were higher at P < 0.001, threonine is increased at P < 0.05. The 3D scattered score plot of OPLS-DA model revealed clear differentiation among the groups, R2 = 0.92 and Q2 = 0.78. CONCLUSION: Significant differences in various metabolite levels were found between control and disease groups. Common amino acids that are significantly higher in all groups include branched chain amino acids, which could increase neuronal excitability.

Proton NMR based serum metabolic profile correlates with the neurological recovery in treated acute spinal cord injury (ASCI) subjects: A pilot study.

BACKGROUND: Acute Spinal Cord Injury (ASCI) is still having substantial morbidity and mortality despite of advanced therapeutics. Major obstacles are paucity of monitoring tools or biomarkers for severity determination, recovery and prognostication. A prospective case control pilot study with serum 1H NMR spectroscopic metabolic profiling was carried out to evaluate metabolites perturbations and its relationship with recovery and to see role of stem cells in facilitating neurological recovery. METHODOLOGY: Twenty subjects with ASCI were classified on the basis of therapeutic modality into surgical fixation alone (Group-1, n = 10), stem cell adjuvant (Group-2, n = 10) and healthy controls (Group-0, n = 10). Serum samples were collected at admission (baseline) and after six months (follow-up). NMR data of serum sample were quantified and subjected to Wilcoxon and ANOVA tests. Further validation was performed using supervised OSC-PCA and OPLS-DA by incorporating substantial control samples. RESULT: Twenty-eight metabolites were identified; well resolved resonances of fifteen metabolites were quantified wherein seven were statistically significant. Predominantly amino acids and ketone bodies played vital role in the differentiation of groups. CONCLUSIONS: Serum NMR spectroscopy reveals certain metabolites perturbations having clear correlation with pattern of recovery in treated ASCI subject. Stem cells treatment group had comparatively effective recovery.

Alteration in lipid composition differentiates breast cancer tissues: a 1H HRMAS NMR metabolomic study.

INTRODUCTION: Breast cancer is the most frequent diagnosed cancer among women with a mortality rate of 15% of all cancer related deaths in women. Breast cancer is heterogeneous in nature and produces plethora of metabolites allowing its early detection using molecular diagnostic techniques like magnetic resonance spectroscopy. OBJECTIVES: To evaluate the variation in metabolic profile of breast cancer focusing on lipids as triglycerides (TG) and free fatty acids (FFA) that may alter in malignant breast tissues and lymph nodes from adjacent benign breast tissues by HRMAS 1H NMR spectroscopy. METHODS: The 1H NMR spectra recorded on 173 tissue specimens comprising of breast tumor tissues, adjacent tissues, few lymph nodes and overlying skin tissues obtained from 67 patients suffering from breast cancer. Multivariate statistical analysis was employed to identify metabolites acting as major confounders for differentiation of malignancy. RESULT: Reduction in lipid content were observed in malignant breast tissues along with a higher fraction of FFA. Four small molecule metabolites e.g., choline containing compounds (Chocc), taurine, glycine, and glutamate were also identified as major confounders. The test set for prediction provided sensitivity and specificity of more than 90% excluding the lymph nodes and skin tissues. CONCLUSION: Fatty acids composition in breast cancer using in vivo magnetic resonance spectroscopy (MRS) is gaining its importance in clinical settings (Coum et al. in Magn Reson Mater Phys Biol Med 29:1-4, 2016). The present study may help in future for precise evaluation of lipid classification including small molecules as a source of early diagnosis of invasive ductal carcinoma by employing in vivo magnetic resonance spectroscopic methods.

Mn(II) based T1 and T2 potential MRI contrast agent appended with tryptamine: Recognition moiety for Aß-plaques.

We report the synthesis and characterization of manganese(II) complexes having pentadentate ligands L1 (2,6-bis(1-(2-phenyl-2-(pyridin-2-yl)hydrazono)ethyl)pyridine), L2 (methyl 2,6-bis((E)-1-(2-phenyl-2-(pyridin-2yl)hydrazono)ethyl)isonicotinate), L3 (N-(2-(1H-indol-3-yl)ethyl)-2,6-bis((E)-1-(2-phenyl-2-(pyridin2yl)hydrazono)ethyl)isonicotiamide) and their application as dual contrast agents for simultaneous T1 and T2 weighted magnetic resonance imaging. Single crystal analysis of all the complexes [MnIIL1, MnIIL2 and MnIIL3] confirm the formation of novel seven-coordinate manganese complexes with an inner sphere water and perchlorate ion. The Magnetic Resonance Imaging (MRI) contrast agent [MnL2] was further modified by incorporating tryptamine as a binding moiety specific to Amyloid Beta-fibrils (Aß-fibrils) in Alzhiemer's disease (AD) and it's in vitro evaluation for specific binding with Aß-fibrils indicated as a bio-marker of AD.

Proton NMR metabolic profiling of CSF reveals distinct differentiation of meningitis from negative controls.

BACKGROUND: Cerebrospinal fluid (CSF) is an essential bio-fluid of the central nervous system (CNS), playing a vital role in the protection of CNS and performing neuronal function regulation. The chemical composition of CSF varies during onset of meningitis, neurodegenerative disorders (positive controls) and in traumatic cases (negative controls). METHODS: The study design was broadly categorized into meningitis cases, negative controls and positive controls. Further differentiation among the three groups was carried out using Principal Component Analysis (PCA) followed by supervised Partial Least Square Discriminant Analysis (PLS-DA). RESULTS: The statistical analysis of meningitis vs. negative controls using PLS-DA model resulted in R2 of 0.97 and Q2 of 0.85. There was elevation in the levels of ketone bodies, total free amino acids, glutamine, creatine, citrate and choline containing compounds (choline and GPC) in meningitis cases. Similarly, meningitis vs. positive controls resulted in R2 of 0.80 and Q2 of 0.60 and showed elevation in the levels of total free amino acids, glutamine, creatine/creatinine and citrate in the meningitis group. Four cases of HIV were identified by PLS-DA model as well as by clinical investigations. CONCLUSION: On the basis of metabolic profile it was found that negative control CSF samples are more appropriate for differentiation of meningitis than positive control CSF samples.

Etiological study of lymphadenopathy in HIV-infected patients in a tertiary care hospital.

INTRODUCTION: Human immunodeficiency virus (HIV) infection has become a global pandemic. Persistent generalized lymphadenopathy (PGL) is very common manifestation of HIV infection. Moreover, different opportunistic infections such as tuberculosis (TB) and malignancies may present with lymphadenopathy. Mycobacterium avium complex (MAC) infection is most common with cluster of differentiation (CD)4+ count ≤50 cells/µL. Fine-needle aspiration cytology (FNAC) offers a simple and effective modality for obtaining a representative sample of the material from lymph nodes, permitting cytological evaluation and other investigations. AIMS AND OBJECTIVES: The aim of this study is to find out the different etiologies of lymphadenopathy in HIV-infected patients and to establish a possible correlation with CD4+ count. MATERIALS AND METHODS: A total of 100 HIV-infected patients having significant (>1 cm) extrainguinal lymphadenopathy were studied in 1 year at the Department of Pathology by FNAC and the stains used were Leishman-Giemsa, Ziehl-Neelsen (ZN), Papanicoloau, and Gram stains. For tubercular culture, Löwenstein-Jensen (LJ) medium was used. CD4+count was done by flow cytometer. RESULT: The present study revealed four types of cytomorphological variants in lymphadenopathy cases by FNAC, which include: Reactive hyperplasia and caseation necrosis; caseation necrosis and ill-formed granuloma; well-formed granuloma without any necrosis; and non-Hodgkin lymphoma (NHL). The highest acid-fast bacilli (AFB) positivity was among the patients showing caseation necrosis. Tubercular culture in LJ media turned out as a more sensitive method for diagnosis than routine ZN staining. The 2 cases that showed well-formed epithelioid granuloma without any necrosis turned out to be histoplasmosis and cryptococcosis, respectively. In this study, we found 2 cases of NHL. The study also revealed that caseation necrosis and AFB positivity along with opportunistic infections increases with decreased CD4+ count.

The application of absolute quantitative (1)H NMR spectroscopy in drug discovery and development.

INTRODUCTION: The identification of a drug candidate and its structural determination is the most important step in the process of the drug discovery and for this, nuclear magnetic resonance (NMR) is one of the most selective analytical techniques. AREA COVERED: The present review illustrates the various perspectives of absolute quantitative (1)H NMR spectroscopy in drug discovery and development. It deals with the fundamentals of quantitative NMR (qNMR), the physiochemical properties affecting qNMR, and the latest referencing techniques used for quantification. The precise application of qNMR during various stages of drug discovery and development, namely natural product research, drug quantitation in dosage forms, drug metabolism studies, impurity profiling and solubility measurements is elaborated. To achieve this, the authors explore the literature of NMR in drug discovery and development between 1963 and 2015. It also takes into account several other reviews on the subject. EXPERT OPINION: qNMR experiments are used for drug discovery and development processes as it is a non-destructive, versatile and robust technique with high intra and interpersonal variability. However, there are several limitations also. qNMR of complex biological samples is incorporated with peak overlap and a low limit of quantification and this can be overcome by using hyphenated chromatographic techniques in addition to NMR.

Serum procalcitonin levels in combination with (1)H NMR spectroscopy: A rapid indicator for differentiation of urosepsis.

BACKGROUND: Urosepsis, a severe form of sepsis requires immediate medical attention for prognosis. It is clinically diagnosed by estimating serum procalcitonin (PCT) levels along with time taking urine and blood cultures. We explored NMR based profiling, deriving metabolites that could potentially aid diagnosis. METHODS: The proton NMR of serum and urine samples of healthy control subjects (n=32) and urosepsis cases (n=35) based on PCT levels, were analyzed. Four clinically identified non-urosepsis cases with high PCT levels were also differentiated through principal component analysis (PCA) of the serum samples. RESULTS: Quantification of serum and urine through Discriminant Function Analysis (DFA) afforded 93.7% and 91.7% correct classification respectively, along with identification of malonate and urea as potential biomarkers for the disease in both urine and serum samples. The partial least square discriminant analysis (PLS-DA) showed an R(2) value of 0.97 in both biofluids with Q(2)=0.87 and 0.85 for serum and urine respectively. The training set of serum samples provided precise prediction of the test set in a small cohort through random re-sampling method, while in urine samples, the predictions were inconclusive. CONCLUSIONS: Our pilot study reveals that (1)H NMR of serum metabolic profiling in combination with PCT levels may provide a rapid method for differentiation of urosepsis.

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.

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

The 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.

Metabolic profiling of Commiphora wightii (guggul) reveals a potential source for pharmaceuticals and nutraceuticals.

Guggul gum resin from Commiphora wightii (syn. Commiphoramukul) has been used for centuries in Ayurveda to treat a variety of ailments. The NMR and GC-MS based non-targeted metabolite profiling identified 118 chemically diverse metabolites including amino acids, fatty acids, organic acids, phenolic acids, pregnane-derivatives, steroids, sterols, sugars, sugar alcohol, terpenoids, and tocopherol from aqueous and non-aqueous extracts of leaves, stem, roots, latex and fruits of C. wightii. Out of 118, 51 structurally diverse aqueous metabolites were characterized by NMR spectroscopy. For the first time quinic acid and myo-inositol were identified as the major metabolites in C. wightii. Very high concentration of quinic acid was found in fruits (553.5 ± 39.38 mg g(-1) dry wt.) and leaves (212.9 ± 10.37 mg g(-1) dry wt.). Similarly, high concentration of myo-inositol (168.8 ± 13.84 mg g(-1) dry wt.) was observed from fruits. The other metabolites of cosmeceutical, medicinal, nutraceutical and industrial significance such as α-tocopherol, n-methylpyrrolidone (NMP), trans-farnesol, prostaglandin F2, protocatechuic, gallic and cinnamic acids were identified from non-aqueous extracts using GC-MS. These important metabolites have thus far not been reported from this plant. Isolation of a fungal endophyte, (Nigrospora sps.) from this plant is the first report. The fungal endophyte produced a substantial quantity of bostrycin and deoxybostrycin known for their antitumor properties. Very high concentrations of quinic acid and myo-inositol in leaves and fruits; a substantial quantity of α-tocopherol and NMP in leaves, trans-farnesol in fruits, bostrycin and deoxybostrycin from its endophyte makes the taxa distinct, since these metabolites with medicinal properties find immense applications as dietary supplements and nutraceuticals.

Duodenal morphometry and small bowel permeability in children with portal hypertension.

OBJECTIVE: We prospectively studied children with portal hypertension (PHT) for portal hypertensive duodenopathy (PHTD) and small bowel intestinal permeability (SIP) with the objectives of defining histopathological parameters for PHTD and to find out whether any association existed among structural changes, SIP, and nutritional status. METHOD: SIP was assessed by using lactulose and mannitol sugar probes in 31 children with PHT (cirrhosis n = 15 and extrahepatic portal venous obstruction n = 16) and 15 healthy children as controls. Morphometric assessment from duodenal biopsies was done in children with PHT. SIP and morphometric parameters were correlated with nutritional status and dietary intake. RESULTS: Among children with PHT, 48% had PHTD defined as presence of villous atrophy (villous to crypt ratio < 2.5:1), dilated capillaries (capillary diameter > 16.8 µm, capillary area > 151 µm, capillary perimeter > 56 µm), and thickened muscularis mucosae (>22.2 µm). Lactulose excretion alone was increased in children with PHT as compared with healthy children (median %: 0.03, 0.02, and 0.01 for cirrhosis, extrahepatic portal venous obstruction, and controls, respectively [P < 0.01]) signifying increased paracellular permeability in PHT. Children with PHT had significantly lower z scores for height, weight, and triceps skin-fold thickness (<-2SD), whereas no differences were found in dietary intake between patients and controls. Increased SIP, nutritional compromise, and PHTD in our patients had no correlation. CONCLUSIONS: PHT is often associated with duodenopathy. SIP does occur as a result of increased paracellular permeability. Factors of increased SIP, undernutrition, and PHTD do not have correlation in childhood PHT.