The use of metabolomics for the discovery of new biomarkers of effect

1. The use of metabolomics for the discovery of newbiomarkers of effect 主讲人：宋彩霞

2. Contents： • 1. Basic concept(基本概念)： • 2. Introduction(引言)： • 3. Materials and methods(材料及方法)： • 4. Results and Discussion (结果讨论)： • 5. Summary(总结)：

3. 1. Basic concept： • Metabonomics和Metabolomics： 在本文中Metabonomics，是特指用NMR技术进行分析，而Metabolomics，是特指新的、灵敏的LC-MS和GC-MS技术进行分析。

4. PCA(主成分分析)：经典PCA线性降维的基本思想是对原变量空间进行旋转变换(方差最大化)以形成新的变量矩阵和误差矩阵;即:先计算原变量(n维)相关系数的协方差矩阵再按矩阵特征根由大到小顺序确定出原变量线性组合后的新变量主成分,目标是尽量用较少(一般2或3维)的独立主成分综合体现原多维变量中蕴含的绝大部分(习惯上>85%)整体信息;在代谢组学分析中,常通过得分图(score plot)以获得对样品分类的信息;通过载荷图(loading plot)以发现可作为生物标记物的变量。

5. PLS-DA(偏最小二乘判别分析 )：是一种稳健的判别分析统计方法,特别适合于解释变量数多且存在着多重共线性,样本观测数少,且干扰噪声大的情况。该法可以进行变量筛选，有效地克服变量间的多重相关性，具有较好的可解释性。

6. 2. Introduction： • 2.1 Advantages of metabolomics： ① Analyses of last step, integration; ② Known function, lower number; ③ Detectable in body fluids;

7. 2. Introduction： 2.2 The aim of this study： ①论证小鼠的血液代谢图谱(metabolomic profiling)能够可靠、一致地分辨循环代谢物水平; ②识别并且确定在用已知作用模式的药物处理后,循环代谢物特征性变化的标志; ③试图从代谢途径方面解释这些变化;

8. 3. Materials and methods： • 3.1 Animals and maintenance conditions • 3.2 Treatment of animals with compounds PB(Phenobarbital,本巴比妥):Diet PTU(propylthiouracil,丙基硫尿嘧啶):gavage FLU(flutamide,氟他胺或氟硝基酰胺):gavage HPPDInhibitors:BAS 670 H ,BAS 660 H: Diet • 3.3 Metabolite profiling • 3.4 Statistics

9. 3.3 Metabolite profiling： • MS-based 的代谢图谱分析的血浆样品首先经过喷射产生极性或非极性碎片： GC-MS:非极性碎片在酸性条件下水解形成脂肪酸甲酯,在分析之前所有碎片要进行甲硫烷基化； LC-MS:两类碎片要在合适的溶液中进行复原(reconstituted)； HPLC:样品在反相分离柱(reversed phase separation columns)上进行梯度洗脱。 • MS检测技术:MRM(multiple reaction monitoring) profiling－－Full screen(285,80,135;400.)

10. 3.4 Statistics • 各实验组实验数据都是通过与各自对照进组行对照分析的； • Normalized treatment group的意义是用于进一步数据分析和处理； • PCA 和 PLS-DA 是通过 Simca-P+ Version 11 (Umetrics AB, Umea,Sweden) 进行的.

11. 4. Results and Disscussion • 4.1 Studies with untreated control animals • 4.2 Study with PB(Dose) • 4.3 Studies with compounds with different toxicological modes of action(PB,PTU,FLU) • 4.4 Studies with HPPD inhibitors

12. 4.1 Studies with untreated control animals Fig. 1. PCA of 670 plasma profiles of male and female control animals from 11 experiments performed within 1 year (distribution of profiles in space of first two principle components).

13. 4.2 Study with PB： Fig. 2. PLS-DA of plasma profiles of male and female rats dosed with PB at dose of levels of 10, 100 and 1000 ppm for 7 days.

14. 4.3 Studies with compounds with different toxicological modes of action Fig. 3. PLS-DA of plasma profiles from male rats dosed with PB, PTU and FLU representing the three different ToxMoA (PB: liver enzyme inducer, PTU: thyroid hormone formation blocker, and FLU: anti-androgenic agent (plasma of each animal measured in triplicate).

15. 4.4 Studies with HPPD inhibitors Table 1 Blood metabolites up/down regulated by HPPD inhibitors BAS 670 H and BAS 660 H in females (changes expressed in fold of control values)

16. Table 2 Blood metabolites up/down regulated by HPPD inhibitors BAS 670 H and BAS 660 H in males (changes expressed in fold of control values)

17. 5-oxoproline 4-hydroxyphenylpyruvate glutamate Tyrosine BAS 660 H, BAS 670 H, … … glutamine x HPPD 4.4 Studies with HPPD inhibitors HPPD inhibitors

18. 5. Summary • 实验小结： 1. Studies with untreated control animals 2. Study with PB(Dose) 3. Studies with compounds with different toxicological modes of action(PB,PTU,FLU) 4. Studies with HPPD inhibitors • 应用及展望： 1. biomarkers,monitors, development of new active ingredients, 2. REACH framework(REACH 28 day toxicity studies)

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