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Modulation of gut microbiome by green tea diet and its correlation with metabolic changes
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Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
rectal bleeding and diarrhea, and histological analysis results. Then, in order to explore the mechanism of celastrol’s effect on IBD, lipidomics was used to characterize the metabolic profiles of the control, DSS, positive drug/DSS-treated and celastrol/DSS-treated mice. PLS-DA scores plot showed clear clustering of the above four groups, either in serum or colon samples. Lipidomics analysis revealed the alteration of several lipid markers associated with IBD, including LPC and sphingomyelin, which were then restored after celastrol treatment. Accordingly, trends of some related fatty acids in the above groups were further confirmed using GCMS analysis. Messenger RNAs of genes in the related pathway were determined by qPCR analysis. It was revealed that expression of the key enzyme in the involved pathway was dramatically inhibited in IBD mice, which was then significantly recovered after celastrol treatment. These results for the first time revealed the mechanism of action of celastrol in alleviating colitis, and provided scientific basis for further development of celastrol in treating IBD.
levels and the 3'TS-UTR genetic polymorphism (p<0.05). From 5-FU pharmacokinetic (PK) analyses, the above each genotype did not show any effects on PK parameters. However, the combination of genetic polymorphisms at the MTHFR 222T/C, DPYD 1896T/C and 3'TS-UTR showed significant differences in AUC of 5-FU and 5-FU/5-FUD ratios (p<0.01). Therefore, this study provides molecular and clinical evidences for personalized medicine of 5-FU.
P122 MODULATION OF GUT MICROBIOME BY GREEN TEA DIET AND ITS CORRELATION WITH METABOLIC CHANGES
The host-microbe metabolic axis influences the development and maturation of human physiology from birth through adulthood. Two- to six-carbon long (C2-C6) isomeric short-chain fatty acids (SCFAs) are derived from gut microbial fermentation of dietary fiber. These critical endobiotics modulate energy metabolism pathways, regulate hormone secretion and influence immune cell function. Dysbiosis of intestinal flora results in differential production of these endobiotics and is linked to pathologies such as diabetes, obesity and allergic conditions. While much focus has been devoted to the more abundant SCFAs such as acetate, propionate, butyrate and isobutyrate, the levels and biological importance of other SCFAs are relatively unknown. To elucidate the significance of differential SCFA compositions in human systems biology, we aimed to develop a comprehensive LC/MS/MS methodology to quantitatively profile 15 SCFAs in human stool (Figure 1). SCFAs in infant (< 12 months) and adult stool were extracted using ice-cold 50% acetonitrile/water containing deuterated benzoic acid (50 mM) as an internal standard. Extracted SCFAs were then derivatized with 12C aniline and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) at 4 C for 120 min at a SCFA/aniline/EDC ratio of 1:10:5. The derivatization was subsequently quenched for 120 min at 4 C using 2mercaptoethanol and succinate. Calibration standards were constructed by spiking stool samples pooled from infants and adults with serial dilutions of 13C aniline-derivatized SCFA. Baseline separation was achieved for 12 of 15 SCFAs within a 15 min detection window, while 3,3-dimethylbutyrate, 2-methylvalerate and 3-methylvalerate were partially separated (Figure 1). Lower limits of detection and quantitation were < 10 and <30 fmol respectively, except for acetate (53.3 and 177.5 fmol), and pivalate (16.7 and 55.6 fmol). Derivatization with aniline allowed us to differentially append 12C and 13C isotopes to endogeneous SCFAs (analyte) and to exogeneous SCFAs (calibrants) respectively, permitting in-matrix quantitation of endogeneous SCFAs while eliminating matrix effects. Controls using 12C aniline-derivatized SCFAs which were then exposed to 13C aniline showed no formation of 13C aniline-derivatized products, confirming complete derivatization. Quenching of derivatization was a crucial step to prevent artefactual in system derivatization of SCFAs. This was critical as acetate, a common chromatographic modifier, was observed to cause a large background signal in unquenched samples. A pilot study revealed that acetate was the predominant SCFA in both infants (n¼3) and adults (n¼5). Although the absolute level of acetate was higher in adults (17.8 mmol/g) than infants (8.96 mmol/g), it was more highly represented in infants (92.7%) than adults (69.7%). For other SCFAs, absolute levels were 2- to 140-fold higher in adults than infants. Apart from the commonly measured acetate, propionate, butyrate and isobutyrate, we demonstrate that valerate, isovalerate, pivalate, 2methylbutyrate, caproate, 2-methylvalerate and 4-methylvalerate were additionally present in infant stool. In conclusion, we report the first attempt to profile all 15 SCFAs in stool matrices, using a novel derivatization strategy that minimizes background acetate signals and facilitates in-matrix quantitation of endobiotics. This methodology will enable a comprehensive determination of SCFA profiles to elucidate their roles in biology and health.
Eun Sung Jung 1, Hye Min Park 1, Seung Min Hyun 2, Jong-Cheol Shon 3, Kwang-Hyeon Liu 3, Jae Sung Hwang 2, Choong Hwan Lee 1. 1 Konkuk University, Seoul, South Korea; 2 Kyung Hee University, Yongin, South Korea; 3 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, South Korea Metabolomics approach was used to investigate the relationship between faecal microbial community and changes in endogenous metabolite of mice subjected to green tea diet (GTD). Following GTD, the diversity of faecal microbial community were increased and modulation of microbial community were observed, especially on Firmicutes. The Allobaculum, Lachnoclostridium, and Parvibacter was enriched in the faeces while Lactobacillus was inhibited. GTD affected microbial energy, carbohydrate, lipid, and bile acid metabolism through alterations of faecal metabolites. Abnormal bile acid metabolism was also detected in the liver, serum, and skin. Among them, methionine, malic acid, uracil, glyceric acid, glycerol, mannose, myo-inositol, xylitol, (2-(14,15-epoxyeicosatrienoyl) glycerol, cholic acid, and lysoPC (15:0) showed high positive correlation with bacteria which were enriched by GTD, while a number of amino acids, lipids, and fatty acids including isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, lanosterol, linoleic acid, monopalmitin, oleic acid, palmitic acid, palmitoleic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, and lysoPE (18:2) showed high negative correlations. Additionally, 42 catechin derivatives were detected in the faeces and liver. Epicatechin, methyl-epicatchin, 5-(3'4'dihyroxyphenyl)-g-valerolactone (M6), methyl-M6, and hydroxyphenylpropionic acid, known antioxidant and anti-inflammatory compounds, were detected in the liver. Our results suggested that GTD highly influenced to modulation of faecal microbiome and its related changes of endogenous metabolites and green tea catechin catabolites might highly relevant to positive effects on body. P123 RIGHT DRUGS AND RIGHT PATIENTS-I. PHARMACO-GENOMIC AND -GENETIC APPROACHES FOR EFFICIENT TREATMENT OF 5-FU Mihi Yang 1, Kyung Tae 2. 1 Sookmyung Women's University, Seoul, South Korea; 2 Hanyang University, Seoul, South Korea 5-fluorouracil (5-FU) has been used as a broad anticancer drug, however, its side effects have been monitored. Therefore, we performed pharmacogenomic and -genetic studies for right treatment of 5-FU for right patients. As results, we found genetic polymorphisms at metabolic enzymes of 5-FU, e.g. DPYD-85, -1627, and -1896 sites, TS-5'ER, and -3'UTR, and MTHFR-222, and -429 sites among Korean populations (normal, N¼105; head and neck patients, N¼28). There was a significant association between TS-mRNA
P124 TARGETED LC/MS-BASED QUANTITATIVE PROFILING OF 15 GUT MICROBIOME-DERIVED SHORT CHAIN FATTY ACIDS IN INFANT AND ADULT STOOL James Chun Yip Chan, Dorinda Yan Qin Kioh, Gaik Chin Yap, Eric Chun Yong Chan, Bee Wah Lee. National University of Singapore, Singapore, Singapore
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
rectal bleeding and diarrhea, and histological analysis results. Then, in order to explore the mechanism of celastrol’s effect on IBD, lipidomics was used to characterize the metabolic profiles of the control, DSS, positive drug/DSS-treated and celastrol/DSS-treated mice. PLS-DA scores plot showed clear clustering of the above four groups, either in serum or colon samples. Lipidomics analysis revealed the alteration of several lipid markers associated with IBD, including LPC and sphingomyelin, which were then restored after celastrol treatment. Accordingly, trends of some related fatty acids in the above groups were further confirmed using GCMS analysis. Messenger RNAs of genes in the related pathway were determined by qPCR analysis. It was revealed that expression of the key enzyme in the involved pathway was dramatically inhibited in IBD mice, which was then significantly recovered after celastrol treatment. These results for the first time revealed the mechanism of action of celastrol in alleviating colitis, and provided scientific basis for further development of celastrol in treating IBD.
levels and the 3'TS-UTR genetic polymorphism (p<0.05). From 5-FU pharmacokinetic (PK) analyses, the above each genotype did not show any effects on PK parameters. However, the combination of genetic polymorphisms at the MTHFR 222T/C, DPYD 1896T/C and 3'TS-UTR showed significant differences in AUC of 5-FU and 5-FU/5-FUD ratios (p<0.01). Therefore, this study provides molecular and clinical evidences for personalized medicine of 5-FU.
P122 MODULATION OF GUT MICROBIOME BY GREEN TEA DIET AND ITS CORRELATION WITH METABOLIC CHANGES
The host-microbe metabolic axis influences the development and maturation of human physiology from birth through adulthood. Two- to six-carbon long (C2-C6) isomeric short-chain fatty acids (SCFAs) are derived from gut microbial fermentation of dietary fiber. These critical endobiotics modulate energy metabolism pathways, regulate hormone secretion and influence immune cell function. Dysbiosis of intestinal flora results in differential production of these endobiotics and is linked to pathologies such as diabetes, obesity and allergic conditions. While much focus has been devoted to the more abundant SCFAs such as acetate, propionate, butyrate and isobutyrate, the levels and biological importance of other SCFAs are relatively unknown. To elucidate the significance of differential SCFA compositions in human systems biology, we aimed to develop a comprehensive LC/MS/MS methodology to quantitatively profile 15 SCFAs in human stool (Figure 1). SCFAs in infant (< 12 months) and adult stool were extracted using ice-cold 50% acetonitrile/water containing deuterated benzoic acid (50 mM) as an internal standard. Extracted SCFAs were then derivatized with 12C aniline and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) at 4 C for 120 min at a SCFA/aniline/EDC ratio of 1:10:5. The derivatization was subsequently quenched for 120 min at 4 C using 2mercaptoethanol and succinate. Calibration standards were constructed by spiking stool samples pooled from infants and adults with serial dilutions of 13C aniline-derivatized SCFA. Baseline separation was achieved for 12 of 15 SCFAs within a 15 min detection window, while 3,3-dimethylbutyrate, 2-methylvalerate and 3-methylvalerate were partially separated (Figure 1). Lower limits of detection and quantitation were < 10 and <30 fmol respectively, except for acetate (53.3 and 177.5 fmol), and pivalate (16.7 and 55.6 fmol). Derivatization with aniline allowed us to differentially append 12C and 13C isotopes to endogeneous SCFAs (analyte) and to exogeneous SCFAs (calibrants) respectively, permitting in-matrix quantitation of endogeneous SCFAs while eliminating matrix effects. Controls using 12C aniline-derivatized SCFAs which were then exposed to 13C aniline showed no formation of 13C aniline-derivatized products, confirming complete derivatization. Quenching of derivatization was a crucial step to prevent artefactual in system derivatization of SCFAs. This was critical as acetate, a common chromatographic modifier, was observed to cause a large background signal in unquenched samples. A pilot study revealed that acetate was the predominant SCFA in both infants (n¼3) and adults (n¼5). Although the absolute level of acetate was higher in adults (17.8 mmol/g) than infants (8.96 mmol/g), it was more highly represented in infants (92.7%) than adults (69.7%). For other SCFAs, absolute levels were 2- to 140-fold higher in adults than infants. Apart from the commonly measured acetate, propionate, butyrate and isobutyrate, we demonstrate that valerate, isovalerate, pivalate, 2methylbutyrate, caproate, 2-methylvalerate and 4-methylvalerate were additionally present in infant stool. In conclusion, we report the first attempt to profile all 15 SCFAs in stool matrices, using a novel derivatization strategy that minimizes background acetate signals and facilitates in-matrix quantitation of endobiotics. This methodology will enable a comprehensive determination of SCFA profiles to elucidate their roles in biology and health.
Eun Sung Jung 1, Hye Min Park 1, Seung Min Hyun 2, Jong-Cheol Shon 3, Kwang-Hyeon Liu 3, Jae Sung Hwang 2, Choong Hwan Lee 1. 1 Konkuk University, Seoul, South Korea; 2 Kyung Hee University, Yongin, South Korea; 3 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, South Korea Metabolomics approach was used to investigate the relationship between faecal microbial community and changes in endogenous metabolite of mice subjected to green tea diet (GTD). Following GTD, the diversity of faecal microbial community were increased and modulation of microbial community were observed, especially on Firmicutes. The Allobaculum, Lachnoclostridium, and Parvibacter was enriched in the faeces while Lactobacillus was inhibited. GTD affected microbial energy, carbohydrate, lipid, and bile acid metabolism through alterations of faecal metabolites. Abnormal bile acid metabolism was also detected in the liver, serum, and skin. Among them, methionine, malic acid, uracil, glyceric acid, glycerol, mannose, myo-inositol, xylitol, (2-(14,15-epoxyeicosatrienoyl) glycerol, cholic acid, and lysoPC (15:0) showed high positive correlation with bacteria which were enriched by GTD, while a number of amino acids, lipids, and fatty acids including isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, lanosterol, linoleic acid, monopalmitin, oleic acid, palmitic acid, palmitoleic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, and lysoPE (18:2) showed high negative correlations. Additionally, 42 catechin derivatives were detected in the faeces and liver. Epicatechin, methyl-epicatchin, 5-(3'4'dihyroxyphenyl)-g-valerolactone (M6), methyl-M6, and hydroxyphenylpropionic acid, known antioxidant and anti-inflammatory compounds, were detected in the liver. Our results suggested that GTD highly influenced to modulation of faecal microbiome and its related changes of endogenous metabolites and green tea catechin catabolites might highly relevant to positive effects on body. P123 RIGHT DRUGS AND RIGHT PATIENTS-I. PHARMACO-GENOMIC AND -GENETIC APPROACHES FOR EFFICIENT TREATMENT OF 5-FU Mihi Yang 1, Kyung Tae 2. 1 Sookmyung Women's University, Seoul, South Korea; 2 Hanyang University, Seoul, South Korea 5-fluorouracil (5-FU) has been used as a broad anticancer drug, however, its side effects have been monitored. Therefore, we performed pharmacogenomic and -genetic studies for right treatment of 5-FU for right patients. As results, we found genetic polymorphisms at metabolic enzymes of 5-FU, e.g. DPYD-85, -1627, and -1896 sites, TS-5'ER, and -3'UTR, and MTHFR-222, and -429 sites among Korean populations (normal, N¼105; head and neck patients, N¼28). There was a significant association between TS-mRNA
P124 TARGETED LC/MS-BASED QUANTITATIVE PROFILING OF 15 GUT MICROBIOME-DERIVED SHORT CHAIN FATTY ACIDS IN INFANT AND ADULT STOOL James Chun Yip Chan, Dorinda Yan Qin Kioh, Gaik Chin Yap, Eric Chun Yong Chan, Bee Wah Lee. National University of Singapore, Singapore, Singapore