Metabolomics Studies on Feces and Serum from Type 2 Diabetes Mellitus
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摘要:
目的 研究2型糖尿患者与健康者血清和粪便的代谢组学差异，分析差异代谢物与2型糖尿病之间的相关性。 方法 选择2018年1月至2019年3月在昆明医科大学第二附属医院内分泌科住院的2型糖尿病患者53例，以及同期常规体检健康对照人群30例，采用超高效液相色谱-四级杆飞行时间质谱技术对两组研究对象的血清代谢物及30例糖尿病患者与对照组的粪便代谢物进行非靶向和靶向的代谢组学研究。应用Spearman相关性分析方法对血清和粪便中的差异代谢物与2型糖尿病相关指标的相关性进行分析。 结果 在2型糖尿病组与健康对照组血清样本中鉴定出15个差异代谢物，在粪便样本中鉴定出的差异代谢物则有6个。其中，糖尿患者血清中的谷氨酰胺、壬二酸、癸二酸及3-羟基癸二酸等二羧酸类羟基化衍生物明显低于健康对照组，差异有统计学意义（P < 0.01），而琥珀酰乙酰乙酸、缬氨酸、亮氨酸、葡萄糖、乳酸的含量则高于对照组，差异有统计学意义（P < 0.01），并且琥珀酰乙酰乙酸、乳酸与糖化血红蛋白、餐后2 h血糖及空腹血糖浓度具有较强的正相关性，而壬二酸、癸二酸及二羧酸类羟基化衍生物与血糖浓度则具有较强的负相关；在粪便代谢物中，去氧胆酸和鹅去氧胆酸等胆汁酸与受检者的血糖浓度也有着一定的正相关性，并且单变量分析结果显示，与健康对照组相比糖尿病患者血清中的去氧胆酸和鹅去氧胆酸均显著性升高，差异有统计学意义（P < 0.05）。 结论 2型糖尿患者的血清代谢组学与健康者具有明显的差异，这些差异代谢物与糖尿病的发生和发展具有较强的关联性。在粪便代谢组学中，糖尿患者的胆汁酸水平与血糖的浓度变化也密切相关。 -
关键词:
- 2型糖尿病 /
- 代谢组学 /
- 超高效液相色谱-四级杆飞行时间质谱技术
Abstract:Objective To study the metabolomic differences of serum and feces between patients with type 2 diabetes and healthy people, and analyze the correlation between different metabolites and type 2 diabetes. Methods From January 2018 to March 2019, 53 patients with newly diagnosed type 2 diabetes mellitus and 30 healthy controls were enrolled in the Department of Endocrinology, the Second Affiliated Hospital of Kunming Medical University. The serum metabolites of the two groups and the fecal metabolites of 30 diabetic patients and the control group were detected by ultra performance liquid chromatography quadrupole time of flight mass spectrometry （UPLC-QTOF/MS） To carry out non targeted and targeted metabolomics studies. Spearman correlation analysis method was used to analyze the correlation between the differential metabolites in serum and stool and related indicators of type 2 diabetes. Results Fifteen differential metabolites were identified in serum samples of type 2 diabetes group and healthy control group, and 6 differential metabolites were identified in stool samples. The levels of glutamine, azelaic acid, sebacic acid, 3-hydroxysebacic acid and other dicarboxylic acid hydroxylated derivatives in patients with diabetes were significantly lower than those in healthy controls （P < 0.01）, while the levels of succinylacetoacetate, valine, leucine, glucose and lactic acid in patients with diabetes were significantly higher than those in healthy controls （P < 0.01）. In the fecal metabolites, deoxycholic acid, chenodeoxycholic acid and other bile acids also had a certain positive correlation with the blood glucose concentration of the subjects, and univariate analysis results showed that compared with the healthy control group, the blood glucose concentration of the subjects was significantly higher in the two groups The serum levels of deoxycholic acid and chenodeoxycholic acid were significantly increased in patients with uropathy. Conclusions There are obvious differences in serum metabolomics between patients with type 2 diabetes mellitus and healthy people, and these metabolites are closely related to the occurrence and development of diabetes mellitus. In fecal metabonomics, the level of bile acid in diabetic patients is closely related to the change of blood glucose concentration. -
图 1 血清的非靶向代谢组学分析
a：ESI+模式下的血清代谢组PCA得分图；b：ESI-模式下的血清代谢组PCA得分图；c：ESI+模式下的血清代谢组OPLS-DA得分图；d：ESI-模式下的血清代谢组OPLS-DA得分图；e：ESI+模式下的血清代谢组S-plot：红色圆圈表示变化较为明显的代谢物（分子量）；f：ESI-模式下的血清代谢组S-plot：红色圆圈表示变化较为明显的代谢物（分子量）；3-HDDCA：3-Hydroxydodecanedioic acid；3-HTTCA：3-Hydroxytetradecanedioic acid。
Figure 1. The non-targeted metabolomics analysis of serum
图 2 粪便的非靶向代谢组学分析
a：ESI+模式下的粪便代谢组PCA得分图；b：ESI-模式下的粪便代谢组PCA得分图；c：ESI+模式下的粪便代谢组OPLS-DA得分图；d：ESI-模式下的粪便代谢组OPLS-DA得分图；e：ESI+模式下的粪便代谢组S-plot：红色圆圈表示变化较为明显的代谢物（分子量）；f：ESI-模式下的粪便代谢组S-plot：红色圆圈表示变化较为明显的代谢物（分子量）。
Figure 2. Non-target metabolomic analysis of fecal
图 3 差异代谢物的单变量靶向分析
a：血清差异代谢物的相对定量；b：粪便差异代谢物的相对定量；c：血清中的胆汁酸水平；与对照组比较，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 3. Univariate targeting analysis of different metabolites
图 4 差异代谢物与临床指标的相关性分析
Figure 4. Correlation analysis of different metabolites and clinical indicators
表 1 两组研究对象临床特征（
$\bar x\pm s$ ）Table 1. The clinical characteristics of the control and the T2DM groups （
$\bar x\pm s $ ）指标 对照组 2型糖尿病组 t P 年龄（岁） 51.77 ± 8.01 54.47 ± 11.40 −1.26 0.21 TC（mmol/L） 4.48 ± 0.71 4.78 ± 1.18 −1.459 0.15 TG（mmol/L） 1.31 ± 0.57 2.95 ± 1.98 −5.61 0.000** HDL-C（mmol/L） 1.52 ± 0.39 1.09 ± 0.24 5.501 0.000** LDL-C（mmol/L） 2.62 ± 0.51 3.05 ± 0.99 −2.63 0.01* FPG/mmol/L 5.12 ± 0.47 9.59 ± 3.64 −8.80 0.000** FINS（μIU/mL） 11.16 ± 3.29 13.39 ± 8.39 −1.71 0.09 FC-P（ng/mL） 2.75 ± 0.96 3.06 ± 1.18 −1.22 0.23 HbA1C（%） 5.19 ± 1.16 9.21 ± 2.18 −10.97 0.000** 尿素（mmol/L） 5.20 ± 1.72 5.29 ± 1.66 0.24 0.81 肌酐（mol/L） 67.50 ± 17.04 70.38 ± 17.30 −0.73 0.47 与对照组比较，*P > 0.05，**P > 0.01。 
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表 2 临床血清和粪便中差异代谢物的定性与定量信息表
Table 2. The qualitative and quantitative information of different metabolites in serum and fecal samples
保留
时间分子量 化合物 离子模式 偏相关系
数绝对值变量权
重值倍数
（糖尿病组/
对照组）质荷比
误差离子碎片 样本类型 0.87 202.047 Succinylacetoacetate ESI+ 0.75 8.74 1.66 4 84.96；116.06；
139.05；172.11serum 0.88 146.070 Glutamine* ESI+ −0.56 1.06 0.86 6 84.04；102.05；
130.05serum 1.00 117.079 Valine* ESI+ 0.52 2.92 1.18 0 55.05；72.08 serum 1.05 131.095 Leucine* ESI+ 0.57 4.57 1.24 3 30.03；44.05；
69.07；86.10serum 5.92 188.104 Azelaic acid* ESI+ −0.72 4.49 0.30 5 125.10；171.10 serum 6.63 246.147 3-Hydroxydodecanedioic acid ESI+ −0.83 5.89 0.50 1 171.10；229.15 serum 7.74 274.177 3-Hydroxytetradecanedioic acid ESI+ −0.86 9.84 0.42 4 171.10；239.16 serum 8.95 302.208 5-Hydroxyhexadecanedioic acid ESI+ −0.81 2.25 0.38 4 171.10；285.21 serum 0.84 180.063 Glucose* ESI− 0.64 1.38 1.72 2 45.00；59.01；
89.03serum 1.05 90.032 Lactic acid ESI− 0.71 8.62 1.48 3 43.02；71.01 serum 5.51 218.115 3-Hydroxydecanedioic acid ESI− −0.77 2.51 0.40 2 69.03；119.03；
158.24serum 5.91 188.104 Azelaic acid* ESI− −0.68 8.53 0.31 3 125.10；143.86；
169.08serum 6.60 202.120 Sebacic acid* ESI− −0.67 2.10 0.29 1 139.12；183.10 serum 6.64 246.147 3-Hydroxydodecanedioic acid ESI− −0.79 5.51 0.51 2 57.04；125.10；
187.10serum 7.25 216.136 Undecanedioic acid* ESI− −0.69 2.01 0.27 1 153.13；197.11 serum 8.95 302.209 5-Hydroxyhexadecanedioic acid ESI− −0.79 1.86 0.38 1 71.05；153.12；
197.12serum 8.66 372.267 Cervonoyl ethanolamide ESI+ 0.51 2.61 2.16 1 107.09；159.12；
173.13；337.25feces 9.35 356.272 Tetracosahexaenoic acid ESI+ 0.52 2.17 1.67 1 107.09；187.15；
247.17feces 9.39 392.293 7，12-Dihydroxycholanoic acid ESI− 0.57 3.85 1.47 1 345.28；373.26 feces 10.06 392.293 Chenodeoxycholic acid* ESI− 0.63 7.53 3.54 1 354.07；373.26 feces 8.65 408.290 Cholic acid* ESI− 0.57 10.72 3.04 6 95.05；251.20；
289.22；343.27feces 10.26 392.293 Deoxycholic acid* ESI− 0.71 8.40 1.61 1 327.27；345.28；
355.27；373.26feces *表示与标准品进行比对过的化合物。
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