基于液相色谱-串联质谱法的轻症和重症急性胰腺炎差异的血清代谢组学研究

doi:10.12114/j.issn.1007-9572.2022.0451

摘要/Abstract

摘要： 背景轻症急性胰腺炎（MAP）有一定的概率转变为重症急性胰腺炎（SAP），SAP一旦发生，将对患者造成较大的危害，探讨MAP与SAP的转化机制对临床诊治急性胰腺炎（AP)具有一定意义。目的探讨MAP与SAP的差异代谢物，寻找异常代谢通路，探索区别MAP和SAP的潜在生物标志物，为SAP的早期诊断、治疗提供参考依据。方法收集2020年8月至2021年3月于湖南省人民医院住院的AP患者68例为研究对象，根据2012年修订版亚特兰大分类（RAC）标准分为MAP组40例、SAP组28例，利用液相色谱-串联质谱法（LC-MS），通过单变量分析〔t检验、差异倍数（FC）〕，多变量分析〔主成分分析（PCA）、偏最小二乘判别分析（PLS-DA）〕，最终以变量权重值（variable important in projection，VIP）>1、FC>1.5且P<0.05筛选出两组间的差异代谢物，分析两组间差异代谢物及代谢通路。结果经PCA、PLS-DA分析发现MAP与SAP代谢物轮廓具有明显差异。结合VIP>1、FC>1.5且P<0.05，在两组之间筛选出50种差异代谢物，5条代谢通路，其中牛磺酸和亚牛磺酸代谢、萜类骨架生物合成为影响最大的两条代谢通路；结合受试者工作特征（ROC）曲线进行分析发现，ROC曲线下面积（AUC）>0.9的差异代谢物有8种，分别为：2-苯基-1，3-丙二醇单氨基甲酸酯、苯海拉明N-葡萄糖醛酸、rac-5，6-环氧-视黄酰-β-D-葡萄糖醛酸、六氟异丙醇、NNAL-N-葡萄糖醛酸、赤藓醇四硝酸酯、3-羟基丁酸、四氢脱氧皮质酮，其中在SAP患者中表达升高的有：2-苯基-1，3-丙二醇单氨基甲酸酯、rac-5，6-环氧-视黄酰-β-D-葡萄糖醛酸、六氟异丙醇、赤藓醇四硝酸酯、3-羟基丁酸、四氢脱氧皮质酮，表达下调的有:苯海拉明N-葡萄糖醛酸、NNAL-N-葡萄糖醛酸。结论 MAP与SAP患者的血清代谢物存在明显差异，其中2-苯基-1，3-丙二醇单氨基甲酸酯、苯海拉明N-葡萄糖醛酸、rac-5，6-环氧-视黄酰-β-D-葡萄糖醛酸、六氟异丙醇、NNAL-N-葡萄糖醛酸、赤藓醇四硝酸酯、3-羟基丁酸、四氢脱氧皮质酮对MAP和SAP的鉴别诊断效能较高，可能为区别二者的潜在生物标志物。

关键词: 液相色谱-串联质谱法, 轻症急性胰腺炎, 重症急性胰腺炎, 代谢组学, 代谢通路

Abstract:

Background

Mild acute pancreatitis (MAP) has a certain probability of transforming into severe acute pancreatitis (SAP) . Once SAP occurs, it will cause greater harm to patients. It is meaningful for diagnosis and treatment of pancreatitis to explore the transformation mechanism between MAP and SAP.

Objective

This study aimed to explore the differential metabolites, abnormal metabolic pathways and potential biomarkers between MAP and SAP, thus providing reference for early diagnosis and treatment of SAP.

Methods

This study collected 68 AP patients who were hospitalized in Hunan Provincial People's Hospital from August 2020 to March 2021. These 68 patients were divided into MAP group (n=40) and SAP group (n=28) based on the 2012 Revised Atlanta Classification (RAC) criteria. This study used liquid chromatography-tandem mass spectrometry (LC-MS) , and screened out the differential metabolites between the two groups by using univariate analysis (T test, FC) , multivariate analysis (PCA, PLS-DA) , VIP>1, FC >1.5, and P<0.05, thus analyzing the differential metabolites and metabolic pathways between the two groups.

Results

PCA and PLS-DA analyses found that the metabolic profiles of MAP and SAP were significantly different. Combined with VIP>1, FC>1.5, and P<0.05, 50 differential metabolites and 5 metabolic pathways were screened between the two groups, taurine and hypotaurine metabolism, and terpenoid skeleton biosynthesis were the two largest metabolic pathways. Combination with receiver operator characteristic curve (ROC curve) , there were 8 differential metabolites with area under the curve (AUC) >0.9, including 2-phenyl -1, 3-propanediol monocarbamate, diphenhydramine N-glucuronic acid, rac-5, 6-epoxy-retinoyl-β-D-glucuronic acid, hexafluoroisopropanol, NNAL -N-glucuronic acid, erythritol tetranitrate, 3-hydroxybutyric acid, tetrahydrodeoxycorticosterone. Six of them elevated in patients with severe pancreatitis, including 2-phenyl-1, 3-propanediol monocarbamate, rac-5, 6-epoxy-retinoyl-β-D-glucuronic acid, hexafluoroisopropanol, erythritol tetranitrate, 3-hydroxybutyric acid, tetrahydrodeoxycorticosterone. Two of them decreased, including diphenhydramine N-glucuronic acid, NNAL-N-glucuronic acid.

Conclusion

There were significant differences in serum metabolites between MAP and SAP patients, 2-phenyl-1, 3-propanediol monocarbamate, diphenhydramine N-glucuronic acid, rac-5, 6-epoxy-retinoyl-β-D-glucuronic acid, hexafluoroisopropanol, NNAL-N-glucuronic acid, erythritol tetranitrate, 3-hydroxybutyric acid, and tetrahydrodeoxycorticosterone have the larger differential diagnosis efficacy of MAP and SAP. They may be the potential biomarkers between distinguishing MAP and SAP.

Key words: Liquid chromatography-tandem mass spectrometry, Mild acute pancreatitis, Severe acute pancreatitis, Metabolomics, Metabolic pathway

黄湘平,吴玲,谭超超. 基于液相色谱-串联质谱法的轻症和重症急性胰腺炎差异的血清代谢组学研究[J]. 中国全科医学, 2023, 26(09): 1118-1124. DOI: 10.12114/j.issn.1007-9572.2022.0451.
HUANG Xiangping,WU Ling,TAN Chaochao. Serum Metabolomic Study on the Difference between Mild and Severe Acute Pancreatitis Based on Liquid Chromatography-tandem Mass Spectrometry[J]. Chinese General Practice, 2023, 26(09): 1118-1124. DOI: 10.12114/j.issn.1007-9572.2022.0451.

图/表 6

图1 MAP组与SAP组患者血清样本差异代谢物的火山图注：Ａ为正离子模式，Ｂ为负离子模式；红色的点代表上调的代谢物，蓝色的点代表下调的代谢物，x轴对应log2 （fold change），y轴对应-log10 （p-value）

Figure 1 Volcano plot of differential metabolites in serum samples from patients in MAP group and SAP group

图2 MAP与SAP正负离子得分散点图注：A、B为PCA模式下MAP与SAP正负离子模式下的得分散点图，C、D为PLS-DA模式下MAP与SAP正负离子模式下的得分散点图

Figure 2 Scatter plots of MAP and SAP

图3 MAP组与SAP组患者血清代谢组学数据聚类分析热图注：Ａ为正离子模式，Ｂ为负离子模式

Figure 3 Heat map of cluster analysis of serum metabolomics data of patients in MAP group and SAP group

表1 筛选出的代谢通路及其相关信息

Table 1 Screened metabolic pathways and related information

相关代谢通路	-log（P）	通路影响因子	相关差异代谢物
牛磺酸和亚牛磺酸代谢	5.118	0.285	3-亚磺基丙氨酸
赖氨酸降解	3.886	0.151	赖氨酸
淀粉和蔗糖代谢	3.502	0.138	6-磷酸葡萄糖
甘氨酸、丝氨酸和苏氨酸代谢	3.381	0.051	甜菜碱
萜类骨架生物合成	2.256	0.183	焦磷酸法呢酯

表2 差异代谢物对MAP与SAP的鉴别诊断价值

Table 2 Differential diagnostic value of the differential metabolites screened for MAP and SAP

差异代谢物	AUC	灵敏度（%）	特异度（%）	阳性似然比	阴性似然比
2-苯基-1，3-丙二醇单氨基甲酸酯	0.982	96.4	95.0	19.28	0.04
苯海拉明N-葡萄糖醛酸	0.968	96.4	92.5	12.86	0.04
rac-5，6-环氧-视黄酰-β-D-葡萄糖醛酸	0.967	92.8	92.5	12.38	0.08
六氟异丙醇	0.945	92.8	90.0	9.29	0.08
NNAL-N-葡萄糖醛酸	0.942	96.4	87.5	7.71	0.04
赤藓醇四硝酸酯	0.938	92.8	87.5	7.42	0.08
3-羟基丁酸	0.912	92.9	87.5	7.43	0.08
四氢脱氧皮质酮	0.904	96.4	90.0	9.64	0.04

图4 筛选出的差异代谢物对MAP与SAP鉴别诊断价值的ROC曲线注：A、B、C、D、E、F、G、H分别为苯海拉明N-葡萄糖醛酸、赤藓醇四硝酸酯、六氟异丙醇、2-苯基-1，3-丙二醇单氨基甲酸酯、3-羟基丁酸、四氢脱氧皮质酮、NNAL-N-葡萄糖醛酸、rac-5，6-环氧-视黄酰-β-D-葡萄糖醛酸

Figure 4 ROC curves of the differential diagnostic value of the differential metabolites screened for MAP and SAP

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