RESUMEN
Adequate micronutrient intake and status are global public health goals. Vitamin and mineral deficiencies are widespread and known to impair health and survival across the life stages. However, knowledge of molecular effects, metabolic pathways, biological responses to variation in micronutrient nutriture, and abilities to assess populations for micronutrient deficiencies and their pathology remain lacking. Rapidly evolving methodological capabilities in genomics, epigenomics, proteomics, and metabolomics offer unparalleled opportunities for the nutrition research community to link micronutrient exposure to cellular health; discover new, arguably essential micronutrients of microbial origin; and integrate methods of molecular biology, epidemiology, and intervention trials to develop novel approaches to assess and prevent micronutrient deficiencies in populations. In this review article, we offer new terminology to specify nutritional application of multiomic approaches and encourage collaboration across the basic to public health sciences to advance micronutrient deficiency prevention.
Asunto(s)
Biomarcadores , Micronutrientes , Salud Pública , Humanos , Micronutrientes/deficiencia , Metabolómica/métodos , Proteómica/métodos , Genómica , Estado Nutricional , Epigenómica/métodos , MultiómicaRESUMEN
Hippuric acid is an abundant metabolite in human urine. Urinary hippuric acid levels change with toxic exposure to aromatic compounds, consumption of fruits and vegetables, cancers, chronic kidney disease, schizophrenia and Crohn's disease. While urinary hippuric acid can be detected and quantified via mass spectrometry or nuclear magnetic resonance spectroscopy, a colorimetric assay would be preferable for a low-cost, point-of care clinical assay. Two colorimetric methods, that use p-dimethylaminobenzaldehyde (DMAB) or benzenesulfonyl chloride (PhSO2Cl), respectively, have been previously developed to detect hippuric acid but these assays have many limitations. We replaced PhSO2Cl with p-toluenesulfonyl chloride (p-TsCl), to create a simpler, faster and more accurate method that works with human urine. This modified colorimetric assay detects from 60 µM to 1000 µM hippuric acid in urine in 2 min. We also corrected for the effects of interfering compounds present in urine such that the assay works across many urine backgrounds. We validated this improved assay on multiple hippurate-spiked urine samples, observing an excellent correlation (R2 > 0.94) between observed and known hippurate concentrations. These data suggest that this colorimetric assay is accurate and should greatly facilitate the measurement of hippuric acid in urine to detect a variety of human conditions.
Asunto(s)
Líquidos Corporales , Colorimetría , Humanos , Bioensayo , HipuratosRESUMEN
The presence of ascorbate in human urine has been shown to be a useful dietary, fruit or vitamin C intake biomarker. More recently it has been discovered that ascorbate levels in urine can be used to facilitate the detection of precancerous colorectal polyps. While there are a number elaborate HPLC, MS or multi-step enzymatic "kit" methods to detect and quantify urinary ascorbate, these are time consuming and expensive. There are also a number of low-cost paper-based ascorbate detection dipsticks. However, the limits of detection and quantification accuracy for these dipsticks are not adequate for applications with human urine. To address these limitations, we have developed a fast, sensitive, single-step colorimetric assay that can be used to quantify ascorbate in urine and other biological fluids. The assay uses the tetrazolium salt, methylthiazolyldiphenyl-tetrazolium bromide (MTT), with the electron carrier phenazine methosulfate (PMS), in a chelated acidic phosphate-buffer to produce a vivid purple color in the presence of ascorbate. Confirmation of the performance of the assay and of its standard curve in human urine was also done using independent LC-MS/MS and NMR analyses. The lower limit of detection of the ascorbate dipstick assay described here was found to be 3.2⯵M. The paper dipsticks are stable over a wide range of temperatures and can be stored for up to 150-days.
Asunto(s)
Ácido Ascórbico/orina , Colorimetría/métodos , Voluntarios Sanos , Humanos , Metosulfato de Metilfenazonio/química , Sales de Tetrazolio/químicaRESUMEN
Alzheimer's disease (AD) is a progressive neurological disease marked by the accumulation and deposition of misfolded amyloid beta or Abeta (Aß) peptide. Two species of Aß peptides are found in amyloid plaques, Aß1-40 and Aß1-42, with the latter being the more amyloidogenic of the two. Understanding how and why Aß peptides misfold, oligomerize and form amyloid plaques requires a detailed understanding of their structure and dynamics. The poor solubility and strong aggregation tendencies of Aß1-42 has made the isolation and characterization of its different structural isoforms (monomer, dimer, oligomer, amyloid) exceedingly difficult. Furthermore, while synthetic Aß1-42 peptides (Aß42syn) are readily available, the cost of isotopically labeled peptide is substantial, making their characterization by NMR spectroscopy cost prohibitive. Here we describe the design, cloning, high-level production, isotopic labeling and biophysical characterization of a modified (solubility-tagged) Aß1-42 variant that exhibits excellent water solubility and shares similar aggregation properties as wildtype Aß1-42. Specifically, we attached six lysines (6K) to the C-terminus of native Aß1-42 to create a more soluble, monomeric form of Aß1-42 called Aß42C6K. A gene for the Aß42C6K was designed, synthesized and cloned into Escherichia coli (E. coli) and the peptide was expressed at milligram levels. The Aß42C6K peptide was characterized using circular dichroism (CD), NMR, electron microscopy and thioflavin T fluorescence. Its ability to form stable monomers, oligomers and fibrils under different conditions was assessed. Our results indicate that Aß42C6K stays monomeric at high concentrations (unlike Aß1-42) and can be induced to oligomerize and form fibrils like Aß1-42. Our novel construct could be used to explore the structure and dynamics of Aß1-42 as well as the interaction of ligands with Aß1-42 via NMR.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Agregado de Proteínas , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Humanos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Multimerización de Proteína , SolubilidadRESUMEN
Cannabis is widely used for medicinal and recreational purposes. As a result, there is increased interest in its chemical components and their physiological effects. However, current information on cannabis chemistry is often outdated or scattered across many books and journals. To address this issue, we used modern metabolomics techniques and modern bioinformatics techniques to compile a comprehensive list of >6000 chemical constituents in commercial cannabis. The metabolomics methods included a combination of high- and low-resolution liquid chromatography-mass spectrometry (MS), gas chromatography-MS, and inductively coupled plasma-MS. The bioinformatics methods included computer-aided text mining and computational genome-scale metabolic inference. This information, along with detailed compound descriptions, physicochemical data, known physiological effects, protein targets, and referential compound spectra, has been made available through a publicly accessible database called the Cannabis Compound Database (https://cannabisdatabase.ca). Such a centralized, open-access resource should prove to be quite useful for the cannabis community.