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Quantification of the Disaccharide Trehalose from Biological Samples: A Comparison of Analytical Methods.

ABSTRACT: Trehalose is a disaccharide that is biosynthesized by many different organisms subjected to extreme conditions, such as dehydration, heat, oxidative stress, and freezing. This disaccharide allows organisms to better survive these environmental stresses; however, the mechanisms by which trehalose exerts its protective effects are not well understood. Methods to accurately measure trehalose from different organisms will help us gain better understanding of these protective mechanisms. In this study, three experimental approaches for the quantification of trehalose from biological samples were compared: an enzymatic trehalose assay (Trehalose Assay Kit; Megazyme International), a high-performance liquid chromatography coupled with refractive index detection-based assay, and a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay. Limits of detection and quantification for each assay were compared, as were the dynamic ranges for all three assays. The percent recoveries for known amounts of trehalose spiked into bacterial and mammalian cellular lysates were also determined for each of the assays. Finally, endogenous trehalose produced by Escherichia coli cells was detected and quantified using these assays. Results from this study indicate that an LC-MS/MS-based assay is the most direct and sensitive method for the quantification of low concentrations of trehalose from biological samples; however, the enzymatic assay is suitable for the rapid quantification of higher concentrations of trehalose when an LC-MS/MS is unavailable.

SUBMITTER: Hayner GA

PROVIDER: S-EPMC6044988 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Multiple analytical methods are used for quantification of total polyphenols and total flavanols in fruit juices and beverages. Four methods were evaluated in this study: Folin-Ciocalteu (F-C), Lowenthal permanganate (L-P), 4-dimethylaminocinnamaldehyde (DMAC), and the bovine serum albumin (BSA) precipitation method. Method validation parameters, including working range, limit of detection, limit of quantitation, precision (repeatability), accuracy, and specificity, were assessed and compared. The F-C method was not specific to polyphenols, and the L-P method had the widest working range but lacked accuracy. The DMAC method was the most specific to flavanols, and the BSA method was not suitable for quantification of smaller flavanols, such as catechin and epicatechin. Quantitative performance was evaluated using commercial fruit juice samples (n = 14), apple juice samples of different cultivars (n = 22), and commercial ciders (n = 17). In general, the L-P titration method and DMAC method resulted in higher quantitative values than the F-C method and BSA precipitation method, respectively. However, ratios of results obtained by the L-P and F-C method ranged from 1 to 28, and ratios of results obtained by the DMAC and BSA precipitation method ranged from <1 to 280. This tremendous variation is likely due to variation in polyphenol composition and sample matrix. This information provides perspective for comparison of results obtained through these different methods, and a basis for choosing the most appropriate analytical method for quantification of polyphenols to address a specific research question when working with commercial fruit juice, apple juice from different apple cultivars, and commercial ciders. PRACTICAL APPLICATION: This study compared results obtained when four common polyphenol quantification methods were applied to a diverse selection of fruit juices and beverages with distinct polyphenol composition and sample matrix. The matrix and polyphenol composition of the samples significantly influenced the results. Our findings can help manufacturers of fruit-based products choose the most appropriate analytical method for polyphenol quantification as part of a quality assurance program or to convey information on dietary polyphenol content to consumers. An assessment of analytical method validation parameters is provided for each of the four methods, which will help users of these methods to understand their limitations.

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Quantification of the Disaccharide Trehalose from Biological Samples: A Comparison of Analytical Methods.

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