液相色谱质谱法对47例非透析慢性肾脏病患者色氨酸-犬尿氨酸通路的分析

doi:10.3877/cma.j.issn.2095-3216.2020.06.002

目的

利用液相色谱-质谱(LC-MS)代谢组学方法探讨慢性肾脏病(chronic kidney disease，CKD)1-4期非透析患者色氨酸(tryptophan, Trp)-犬尿氨酸(kynurenine, Kyn)通路的改变及意义。

方法

2018年3月至2018年6月苏州大学附属第一医院肾内科CKD1～4期非透析患者47例，同期30例健康志愿者作为对照组。应用LC-MS法对受试者血浆进行代谢物分析。应用Human Metabolome Database (HMDB)，MassBank，LipidMaps等数据库匹配不同CKD分期Kyn通路差异代谢物，绘制受试者曲线(receiver operating characteristic, ROC)评估差异代谢物敏感性与特异性。采用Metaboanalyst软件进行代谢通路拓扑分析。以Kyn/Trp比值(KT值)表示Kyn通路限速酶吲哚胺-2，3-双加氧酶(indoleamine-2，3-dioxygenase, IDO)活性。

结果

Kyn在CKD2期即有明显升高(P<0.05)，犬尿喹啉酸(kynurenine acid, KA)在CKD3期明显升高(P<0.01)，二者随CKD分期增加呈上升趋势(P<0.01)。KT值在CKD2期即有升高(P<0.05)，且随CKD分期增加呈上升趋势(P<0.01)。Trp代谢在CKD早期即出现紊乱。

结论

Trp-Kyn通路在CKD早期即增强，Kyn、KA、IDO可能作为CKD早期诊断及进展的生物标志物。

关键词: 慢性肾脏病, 色氨酸-犬尿氨酸通路, 代谢组学, 吲哚胺-2, 3-双加氧酶

Objective

To explore the changes and significance of the tryptophan (Trp)- kynurenine (Kyn) pathway in non-dialysis patients with chronic kidney disease (CKD) stage 1-4 by liquid chromatography-mass spectrometry (LC-MS) metabolomics method.

Methods

From March 2018 to June 2018, 47 non-dialysis patients with CKD stage 1-4 from the Department of Nephrology, First Hospital Affiliated to Suzhou University were recruited, together with 30 healthy volunteers as the control group. LC-MS was used to analyze the blood samples of the subjects. Databases as Human Metabolome Database (HMDB), MassBank, and LipidMaps, etc, were used to match different metabolites of the kynurenine pathway of different CKD stages, and draw the receiver operating characteristic (ROC) curve to evaluate the sensitivity and specificity of the different metabolites. The Metaboanalyst software was used for metabolic pathway topology analysis. The ratio of Kyn to Trp (KT value) was used to express the activity of the indoleamine-2, 3-dioxygenase (IDO), a rate-limiting enzyme in the kynurenine pathway.

Results

In comparison with those of the control, Kyn increased in CKD stage 2 (P<0.05) while kynurenic acid (KA) increased in CKD stage 3 (P<0.01). Both Kyn and KA increased with the progressive loss of renal function (P<0.01). KT value increased in CKD stage 2 (P<0.05), and also increased with the progressive loss of renal function (P<0.01). Disorder of Trp metabolism was observed in the early stage of CKD.

Conclusion

The Trp-Kyn pathway was enhanced in the early stage of CKD. Kyn, KA, and IDO might be used as biomarkers of both CKD early diagnosis and CKD progression.

Key words: Chronic kidney disease, Tryptophan-kynurenine pathway, Metabonomics, Indoleamine-2, 3-dioxygenase

表1 47例非透析CKD患者及30例对照一般临床资料

项目		对照组(n＝30)	CKD1期(n＝13)	CKD2期(n＝11)	CKD3期(n＝11)	CKD4期(n＝12)	P值
基本情况
	年龄(岁)	48±11	47±16	51±21	56±17	58±14	0.232
	性别(男/女)	10/20	5/8	7/4	7/4	7/5	0.223
BMI^a(kg/m²)		1.37±0.07	1.38±0.06	1.36±0.58	1.40±0.04	1.38±0.05	0.755
实验室指标
	肌酐^a(μmol/L)	1.80±0.08	1.74±0.10	1.97±0.10	2.14±0.09	2.37±0.07	<0.001
	白蛋白(g/L)	48(46，49)	36(29，41)	40(37，43)	43(39，44)	41(38，43)	<0.001
	尿素氮^a(mmol/L)	0.67±0.10	0.66±0.14	0.79±0.12	0.95±0.12	1.09±0.16	<0.001
	总胆固醇(mmol/L)	4.5(3.9，5.2)	5.2(4.1，5.6)	4.7(4.5，5.5)	4.5(4.2，5.1)	4.5(3.8，5.2)	0.419
	甘油三酯^a(mmol/L)	0.12±0.23	0.20±0.29	0.16±0.17	0.20±0.11	0.24±0.19	0.493
	血糖(mmol/L)	4.9(4.6，5.2)	4.6(4.5，4.8)	4.6(4.1，4.9)	4.7(4.1，4.8)	4.6(4.3，5.0)	0.086
	血红蛋白(g/L)	140.0±11.4	128.5±17.7	126.6±11.4	121.9±12.8	111.3±17.9	<0.001
	尿酸(μmol/L)	344.5±91.0	379.8±122.7	430.3±59.8	431.2±129.7	420.4±138.4	0.060
临床用药情况
	ACEI类/ARB类	0/30	1/13	2/11	6/11	5/12	-
	前列腺素E1	0/30	0/13	0/11	5/11	6/12	-
	调脂药	0/30	1/13	1/11	6/11	5/12	-
	抗凝药	0/30	1/13	1/11	5/11	6/12	-
	α-酮酸	0/30	1/11	1/11	6/11	6/12	-
	中成类药	0/30	1/13	2/11	6/11	6/12	-

表1 47例非透析CKD患者及30例对照一般临床资料

项目		对照组(n＝30)	CKD1期(n＝13)	CKD2期(n＝11)	CKD3期(n＝11)	CKD4期(n＝12)	P值
基本情况
	年龄(岁)	48±11	47±16	51±21	56±17	58±14	0.232
	性别(男/女)	10/20	5/8	7/4	7/4	7/5	0.223
BMI^a(kg/m²)		1.37±0.07	1.38±0.06	1.36±0.58	1.40±0.04	1.38±0.05	0.755
实验室指标
	肌酐^a(μmol/L)	1.80±0.08	1.74±0.10	1.97±0.10	2.14±0.09	2.37±0.07	<0.001
	白蛋白(g/L)	48(46，49)	36(29，41)	40(37，43)	43(39，44)	41(38，43)	<0.001
	尿素氮^a(mmol/L)	0.67±0.10	0.66±0.14	0.79±0.12	0.95±0.12	1.09±0.16	<0.001
	总胆固醇(mmol/L)	4.5(3.9，5.2)	5.2(4.1，5.6)	4.7(4.5，5.5)	4.5(4.2，5.1)	4.5(3.8，5.2)	0.419
	甘油三酯^a(mmol/L)	0.12±0.23	0.20±0.29	0.16±0.17	0.20±0.11	0.24±0.19	0.493
	血糖(mmol/L)	4.9(4.6，5.2)	4.6(4.5，4.8)	4.6(4.1，4.9)	4.7(4.1，4.8)	4.6(4.3，5.0)	0.086
	血红蛋白(g/L)	140.0±11.4	128.5±17.7	126.6±11.4	121.9±12.8	111.3±17.9	<0.001
	尿酸(μmol/L)	344.5±91.0	379.8±122.7	430.3±59.8	431.2±129.7	420.4±138.4	0.060
临床用药情况
	ACEI类/ARB类	0/30	1/13	2/11	6/11	5/12	-
	前列腺素E1	0/30	0/13	0/11	5/11	6/12	-
	调脂药	0/30	1/13	1/11	6/11	5/12	-
	抗凝药	0/30	1/13	1/11	5/11	6/12	-
	α-酮酸	0/30	1/11	1/11	6/11	6/12	-
	中成类药	0/30	1/13	2/11	6/11	6/12	-

图1 47例非透析CKD患者与30例对照血浆基峰色谱图

图2 47例非透析CKD患者与30例对照血浆样本OPLS-DA图

图3 色氨酸-犬尿氨酸代谢通路

表2 CKD1期患者和对照组色氨酸-犬尿氨酸通路血浆差异代谢物鉴定

	差异代谢物	AUC值	灵敏度	特异度	Youden指数	Ci	Ci2
1	犬尿氨酸	0.524	0.615	0.533	0.148	0.338	0.718
2	犬尿喹啉酸	0.596	0.538	0.700	0.238	0.474	0.756

表2 CKD1期患者和对照组色氨酸-犬尿氨酸通路血浆差异代谢物鉴定

	差异代谢物	AUC值	灵敏度	特异度	Youden指数	Ci	Ci2
1	犬尿氨酸	0.524	0.615	0.533	0.148	0.338	0.718
2	犬尿喹啉酸	0.596	0.538	0.700	0.238	0.474	0.756

图4 色氨酸-犬尿氨酸通路关键代谢物变化趋势

图5 色氨酸代谢通路影响因子

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Analysis of tryptophan-kynurenine pathway in 47 non-dialysis patients with chronic kidney disease by liquid chromatography-mass spectrometry

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Analysis of tryptophan-kynurenine pathway in 47 non-dialysis patients with chronic kidney disease by liquid chromatography-mass spectrometry