Developing and Standardizing a Protocol for Quantitative Proton Nuclear Magnetic Resonance ( ¹ H NMR) Spectroscopy of Saliva

Metabolic profiling by ¹H NMR spectroscopy is an underutilized technology in salivary research, although preliminary studies have identified promising results in multiple fields (diagnostics, nutrition, sports physiology). Translation of preliminary findings into validated, clinically approved knowledge is hindered by variability in protocol for the collection, storage, preparation, and analysis of saliva. This study aims to evaluate the effects of differing sample pretreatments on the ¹H NMR metabolic profile of saliva. Protocol considerations are highly varied in the current literature base, including centrifugation, freeze–thaw cycles, and different NMR quantification methods. Our findings suggest that the ¹H NMR metabolite profile of saliva is resilient to any change resulting from freezing, including freezing of saliva prior to centrifuging. However, centrifugation was necessary to remove an unidentified broad peak between 1.24 and 1.3 ppm, the intensity of which correlated strongly with saliva cellular content. This peak obscured the methyl peak from lactate and significantly affected quantification. Metabolite quantification was similar for saliva centrifuged between 750<i>g</i> to 15 000<i>g</i>. Quantification of salivary metabolites was similar whether quantified using internal phosphate-buffered sodium trimethylsilyl-[2,2,3,3-²H₄]-propionate (TSP) or external TSP in a coaxial NMR tube placed inside the NMR tube containing the saliva sample. Our results suggest that the existing literature on salivary ¹H NMR will not have been adversely affected by variations of the common protocol; however, use of TSP as an internal standard without a buffered medium appears to affect metabolite quantification, notably for acetate and methanol. We include protocol recommendations to facilitate future NMR-based studies of saliva

Determining bacterial and host contributions to the human salivary metabolome

BACKGROUND: Salivary metabolomics is rapidly advancing. AIM AND METHODS: To determine the extent to which salivary metabolites reflects host or microbial metabolic activity whole-mouth saliva (WMS), parotid saliva (PS) and plasma collected contemporaneously from healthy volunteers were analysed by (1)H-NMR spectroscopy. Spectra underwent principal component analysis and k-means cluster analysis and metabolite quantification. WMS samples were cultured on both sucrose and peptide-enriched media. Correlation between metabolite concentration and bacterial load was assessed. RESULTS: WMS contained abundant short-chain fatty acids (SCFAs), which were minimal in PS and plasma. WMS spectral exhibited greater inter-individual variation than those of PS or plasma (6.7 and 3.6 fold, respectively), likely reflecting diversity of microbial metabolomes. WMS bacterial load correlated strongly with SCFA levels. Additional WMS metabolites including amines, amino acids and organic acids were positively correlated with bacterial load. Lactate, urea and citrate appeared to enter WMS via PS and the circulation. Urea correlated inversely with WMS bacterial load. CONCLUSIONS: Oral microbiota contribute significantly to the WMS metabolome. Several WMS metabolites (lactate, urea and citrate) are derived from the host circulation. WMS may be particularly useful to aid diagnosis of conditions reflective of dysbiosis. WMS could also complement other gastrointestinal fluids in future metabolomic studies

The BRAF Inhibitor Vemurafenib Activates Mitochondrial Metabolism and Inhibits Hyperpolarized Pyruvate–Lactate Exchange in BRAF-Mutant Human Melanoma Cells

Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long-term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity, and potential as noninvasive imaging response biomarkers. H-1 NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAF(WT) (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine, and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. C-13 NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of hexokinase 2, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase, and monocarboxylate transporters (MCT) 1 and 4 in BRAF-mutant but not BRAF(WT) cells and, interestingly, decreased BRAF-mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized C-13-pyruvatelactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as noninvasive imaging of response. (C) 2016 AACR

Dissociation between iron accumulation and ferritin upregulation in the aged substantia nigra:Attenuation by dietary restriction

Despite regulation, brain iron increases with aging and may enhance aging processes including neuroinflammation. Increases in magnetic resonance imaging transverse relaxation rates, R2 and R2*, in the brain have been observed during aging. We show R2 and R2* correlate well with iron content via direct correlation to semi-quantitative synchrotron-based X-ray fluorescence iron mapping, with age-associated R2 and R2* increases reflecting iron accumulation. Iron accumulation was concomitant with increased ferritin immunoreactivity in basal ganglia regions except in the substantia nigra (SN). The unexpected dissociation of iron accumulation from ferritin-upregulation in the SN suggests iron dyshomeostasis in the SN. Occurring alongside microgliosis and astrogliosis, iron dyshomeotasis may contribute to the particular vulnerability of the SN. Dietary restriction (DR) has long been touted to ameliorate brain aging and we show DR attenuated age-related in vivo R2 increases in the SN over ages 7 – 19 months, concomitant with normal iron-induction of ferritin expression and decreased microgliosis. Iron is known to induce microgliosis and conversely, microgliosis can induce iron accumulation, which of these may be the initial pathological aging event warrants further investigation. We suggest iron chelation therapies and anti-inflammatory treatments may be putative ‘anti-brain aging’ therapies and combining these strategies may be synergistic

Phosphorus-nitrogen compounds. Part 47. Conformations and phosphorus–phosphorus spin–spin coupling constants in phosphazenylcyclophosphazenes

4JPP Spin–spin coupling constants have been measured in a series of phosphazenylcyclophosphazenes. They can be correlated with the conformations adopted by the phosphazenyl substituents in the solid state, which are also believed to be the predominant ones in solution. The conformations are depicted by suitable orthographic projections based on published X-ray crystallographic data