Application of ABTS Radical Cation for Selective On-Line Detection of Radical Scavengers in HPLC Eluates

The radical cation 2,2 ' -azinobis-(3 -ethylbenzothiazoline-6-sulfonate), (ABTS(.+)) was utilized in an on-line HPLC method for the detection of radical scavengers in complex matrixes. The HPLC-separated analytes react postcolumn with the preformed ABTS(.+), and the induced bleaching is detected as a negative peak by an absorbance detector at 734 nm, An optimized instrumental and experimental setup is presented. The method is suitable for both isocratic and gradient HPLC runs using mobile phases containing 100% organic solvent or,its solution in water, weak acids, or buffers (pH 3-7.4), The method is sensitive, selective, relatively simple, applicable to compounds of different chemical natures; uses common instruments and inexpensive reagents; and has a time-saving, non-laborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The minimum detectable concentration varied from 0.02 to 0.13 mug/mL, depending on the compound tested, The method can be applied to perform kinetic studies, which is illustrated by determination of Trolox equivalent antioxidant capacities (TEAC) of several known antioxidants in now injection mode.</p

The ALP-Enigma Protein ALP-1 Functions in Actin Filament Organization to Promote Muscle Structural Integrity in Caenorhabditis elegans

Mutations that affect the Z-disk–associated ALP-Enigma proteins have been linked to human muscular and cardiac diseases. Despite their clear physiological significance for human health, the mechanism of action of ALP-Enigma proteins is largely unknown. In Caenorhabditis elegans, the ALP-Enigma protein family is encoded by a single gene, alp-1; thus C. elegans provides an excellent model to study ALP-Enigma function. Here we present a molecular and genetic analysis of ALP-Enigma function in C. elegans. We show that ALP-1 and α-actinin colocalize at dense bodies where actin filaments are anchored and that the proper localization of ALP-1 at dense bodies is dependent on α-actinin. Our analysis of alp-1 mutants demonstrates that ALP-1 functions to maintain actin filament organization and participates in muscle stabilization during contraction. Reducing α-actinin activity enhances the actin filament phenotype of the alp-1 mutants, suggesting that ALP-1 and α-actinin function in the same cellular process. Like α-actinin, alp-1 also interacts genetically with a connectin/titin family member, ketn-1, to provide mechanical stability for supporting body wall muscle contraction. Taken together, our data demonstrate that ALP-1 and α-actinin function together to stabilize actin filaments and promote muscle structural integrity

Metabolomics meets functional assays: coupling LC-MS and microfluidic cell-based receptor-ligand analyses

IntroductionMetabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds.ObjectiveWe aimed to combine LC–MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics.MethodsWe developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Capsicum varieties. Two were selected with contrasting degrees of spiciness for characterization by HPLC coupled to high mass resolution MS. Subsequently, the biosensor was then used to link individual pepper compounds with TRPV1 activity.ResultsAmong the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Capsicum fruits as well as a commercial product. Sensitivity of the biosensor to this commercial product was similar to the sensory threshold of a human sensory panel.ConclusionOur results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC–MS