Suruchi Singh

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

ABSTRACT 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 1H 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 1H 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.

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 &mgr;m, capillary area > 151 &mgr;m2, capillary perimeter > 56 &mgr;m), and thickened muscularis mucosae (>22.2 &mgr;m). 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.

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

BACKGROUND Parkinsons 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.

Elevated CA 125 in Disseminated Tuberculosis: A Case Report

### Learning Point for Clinicians Tuberculosis should be kept as a possibility in patients with multisystem disease, especially in developing countries like India. A simple test like CT guided FNAC can help detect infection with M. tuberculosis and hence a major surgery can be avoided CA-125 is an epithelial marker derived from coelomic epithelium. It is elevated in 90% of advanced ovarian cancers and has a sensitivity of 57–80% and specificity of 100%. Therefore, CA-125 is an important investigation in evaluating a pelvic mass. However, any process that disrupts the epithelial lining of the peritoneum has the potential to raise its level. Therefore, it can be elevated in many non-neoplastic conditions such as hepatitis, pancreatitis, menstruation etc.1 We illustrate a patient who presented with elevated CA-125 levels but was ultimately diagnosed as …

Comprehensive metabolite profiling in distinct chemotypes of Commiphora wightii

Abstract 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.

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.

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.