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GC-TOF-MS and CE-MS based metabolic profiling of plasma and liver tissues from mice fed high-fat diet with cyanidin 3-O-β-D-glucoside

Title
GC-TOF-MS and CE-MS based metabolic profiling of plasma and liver tissues from mice fed high-fat diet with cyanidin 3-O-β-D-glucoside
Authors
소민영
Issue Date
2014
Department/Major
대학원 식품공학과
Publisher
이화여자대학교 대학원
Degree
Master
Advisors
김영석
Abstract
Metabolite profiling of plasma and liver tissues in mice induced obesity was employed to demonstrate the effect of cyanidin 3-O-β-D-glucoside on obesity. The plasma and liver metabolites of mice rendered obese by intaking of high-fat diet (HFD) were analyzed using gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) and capillary electrophoresis mass spectrometry (CE-MS). As multivariate statistical analysis, principle component analysis (PCA) and partial least squares regression (PLSR) were conducted to visualize and group the obtained complex data sets. The mice studied were divided into three groups depending on diets such as high-fat diet group (HFD), fenofibrate induced group (FF), and C3G induced group (C3G). A total of 29 plasma metabolites were found using GC-TOF-MS analysis, including 4 carbohydrates, 7 fatty acids, 6 organic acids, and 12 amino acids, whereas CE-MS analysis revealed 82 metabolites. The group fed C3G had lower levels of glucose, butyric acid, palmitic acid, linoleic acid, oleic acid, glycine, and branched chain amino acid (BCAA) compared to HFD group. In liver tissues, a total of 27 metabolites were found using GC-TOF-MS, including 3 carbohydrates, 8 fatty acids, 4 organic acids, and 12 amino acids. On the other hand, CE-MS detected 92 metabolites. The levels of arabitol, mannose, and glutathione (GSSG) in liver tissues were lower in the C3G group than those in the HFD group. Especially, the level of BCAA, which could contributed to the development of obesity-associated insulin resistance, was increased in liver tissues and decreased in plasma of C3G group. In principal component analsis (PCA) derived score plot, the C3G group was clearly differentiated from other groups from 8 weeks clearly. The major metabolites in plasma related to the differentiation were acetic acid, arachidonic acid, glucose, palmitic acid, glutamine, methionine, fumaric acid, arabitol, aspartic acid, succinic acid, phopsphoenolpyruvic acid, arginine, alanine, AMP, 3-phosphoglyceric acid, glycine, glutamine, glutathione, glycolic acid, asparagine, malic acid, cystathione, 2-hydroxyglutaric acid, homoserine, γ-aminobutyric acid, N,N-dimethylglycine, pyruvic acid, betaine, creatine, ADP, citric acid, carnitine, and ornithine. One the other hands, sorbitol, palmitoleic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid, mannose, 2,3-diphosphoglyceric acid, glutathione, arginine, AMP, pyruvic acid, argininosuccinic acid, putrescine, adenine, UDP-glucose, creatine, 3-phosphoglyceric acid, citrulline, NADP+, phosphoenolpyrvic acid, cystathionine, 2-phosphoglyceric acid, 2-hydroxyglutaric acid, ATP, aspartic acid, adenylosuccinic acid, malic acid, glutamic acid, β-alanine, guanosine, and acetyl COA were the major metabolites, responsible for the differentiation in liver tissues group separation. Regarding partial least squares analysis (PLSR) analysis in C3G group, several biochemical parameters for evaluating obesity and diabetes, such as TG and glucose, were found to be associated with HFD group, whereas HDL and HDL/LDL ratio were related to C3G group.;본 연구에서는 cyanidin 3-O-β-D-glucoside (C3G)를 투여한 쥐 생체시료 (혈액, 간조직)의 대사체 프로파일링의 차이를 분석하였다. 동물실험은 고지방식이를 통하여 비만이 유도된 쥐에 C3G를 추가적으로 투여하고 8주간 실험을 진행하였다. C3G 그룹 이외에도 고지방식이만을 투여한 그룹, fenofibrate를 투여한 그룹으로 총 3종류의 그룹이 대조군 및 양성대조군으로 비교되었다. 동물의 생체시료 내 대사물질은 gas chromatography-time-of-flight-mass spectrometry (GC-TOF-MS)와 capillary electrophoresis-mass spectrometry (CE-MS)를 통하여 분석하였으며, 다변량 통계기법을 이용하여 GC-TOF-MS 분석에서는 당류 4 종, 지방산류 7 종, 유기산류 6 종 및 아미노산류 12 종 등 총 29 가지의 혈액 내 대사체들이 동정되었다. 이에 비하여 CE-MS 분석을 통해서는 총 82가지의 혈액 내 대사체들이 동정되었다. C3G를 투여한 그룹은 고지방식이 그룹에 비하여 glucose, butyric acid, palmitic acid, linoleic acid, oleic acid, glycine 및 branched chain amino acids (BCAA)의 함량이 유의적으로 낮게 나타났다. 간조직에서는 GC-TOF-MS를 통하여 당류 3 종, 지방산류 8 종, 유기산 4 종 및 12종류의 아미노산 등으로 총 27가지의 대사체들이 동정되었다. 한편 CE-MS를 이용해서는 92가지의 물질이 동정되었다. C3G 투여 그룹의 간조직 내에서는 arabitol, mannose, and glutathione (GSSG) 함량이 고지방식이에 비해 낮게 측정되었으나, 반면 지방대사의 중간물질인 betaine과 carnitine은 유의적으로 더 높게 측정되었다. 주성분 분석에 따르면 C3G 그룹은 고지방식이 그룹과 확연히 구분되는 경향을 나타냈다. 부분최소평방회귀분석으로 CE-MS를 통하여 분석한 대사체 결과와 다양한 생화학 지수를 비교 적용해 본 결과 triglycerols (TG)와 glucose가 valine, threonine, serine, cystathione 등의 물질과 함께 고지방식이 그룹과 연관이 있는 것으로 나타났으며, C3G 투여 그룹은 high-density lipoprotein (HDL)과 HDL/low density lipoprotein (LDL)과 함께 fructose 1,6-diphosphate, 2-hydroxyglutaric acid 등의 물질이 서로 관련이 높은 것으로 나타났다. 결과적으로 C3G의 투여는 혈액 내 glucose 수준을 낮추고 지방대사 중간물질들의 간조직 내 함량을 높임으로서, 체내 TG와 glucose 수치 증진에 기여할 수 있는 물질로의 이용 가능성을 확인하였다.
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