| Summary: | 博士 === 國立交通大學 === 應用化學系碩博士班 === 105 === Innovation in mass spectrometry (MS) in the last decade of the 20th century and in the beginning of the 21st century made it a versatile and accessible technique. MS is a very
powerful analytical technique because it can accurately identify and quantify various types of chemicals. The applications of MS in different research areas develop rapidly. MS has already entered clinical laboratories. In particular, hyphenating liquid and gas chromatography with MS is useful in medical diagnostics and biomarker discovery. Modern MS methods facilitated measurements of low-molecular-weight molecules present in various biological samples. This resulted in the development of a new subdivision of systems biology – metabolomics. In most standard protocols in clinical analysis, blood and
urine specimens are analyzed. In this thesis, I highlight analysis of unconventional biological specimens. In particular, I focus on sampling and analysis of skin excretions, which could be collected in totally non-invasive manner, and analyzed by MS without
extensive sample preparation. A brief introduction of the background of my research, including definition of metabolomics, basic principles of MS, application of MS for quantitative analysis of unconventional biological specimens and a summary of current status of skin excretions testing, are presented in Chapter 1 of this thesis.
Taking into account the usefulness of skin excretion analysis, we proposed a simple tool for fast and non-invasive collection of minute quantities of skin excretions that could directly be screened by MS (Chapter 2). A simple probe composed of biocompatible agarose hydrogel micropatches embedded within chemically inert probes was invented. The probe was combined on-line with an extraction-ionization technique – nanospray desorption electrospray ionization (nanoDESI). Tiny amounts of the collected skin specimens were found to be sufficient to perform chemical fingerprinting. Twenty four low-molecular-weight skin metabolites have been putatively identified based on MS/MS and high-resolution MS analysis (Chapter 2-4).
In one variant of the hydrogel micropatch probe, the micropatches were arranged in an array of spots (5 × 5) within a larger chip. So-called micropatch-arrayed pads (MAPAs) were used to acquire spatial distribution of chemicals on skin surface (Chapter 3). MS scanning of the probe was automated and a 3D-printed humidity chamber, preventing hydrogel from drying, was incorporated into the experimental system. MAPAs were applied to follow dispersion of topical drugs applied to human skin in vivo and to porcine skin ex vivo. Differences between drug dispersion in vivo and ex vivo were observed.
Furthermore, two skin metabolomic studies were conducted using hydrogel micropatch sampling and MS. In these studies, we investigated the skin metabolomes of
patients suffering from a common skin disease – psoriasis (Chapters 4 and 5). Obtaining information about the metabolomes of psoriatic skin may bring new insights into the complex pathophysiology of this disease. In the first metabolomic study, skin excretion specimens from 100 patients and 100 healthy individuals were collected (Chapter 4). A custom-developed algorithm automated processing of the large data sets obtained in the
course of this study. Further chemometric analysis revealed major differences between the metabolomes of psoriatic and healthy skin. Several polar metabolites correlated positively (choline and glutamic acid) or negatively (urocanic acid and citrulline) with the severity score values characterizing psoriatic plaques. Those metabolites are considered as biomarkers of the disease progression. In the second metabolomic study, we investigated dynamic changes of metabolic profiles of psoriatic skin and blood plasma of patients treated with the newest type of therapy – biologics (Chapter 5). Skin excretion and blood specimens were collected multiple times from 19 patients during the first 5-7 months of
therapy. As expected, not every patient reacted to the biologic therapy in the same way. Alterations to the metabolic skin profiles were observed in the course of the treatment.
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