LC/MS-Based Metabolomics Approach for Exploring Plasma Metabolome Modififications before and after 3,600 m Speed Race in Yili horse

doi:10.6048/j.issn.1001-4330.2023.02.030

Abstract

Abstract:

【Objective】 The aim of this study is to investigate the difference and change of plasma metabolites before and after 3,600 m speed race in Yili horses. 【Method】 According to the genetic pedigree provided by the equestrian club, 5(3♂2♀) unrelated, weight (384.40±34.18)kg, and age 2 speed raced-horses were selected.The horses were trained daily with equal intensity. On the 25 th day of the experimental stage, the 3,600 m speed race was performed 4 h after morning feeding. The horse completed the race at (11.75±0.87) m/s at average speed. The rate of heartbeat and the breathing were measured immediately before and after the race. At the same time, blood was collected, plasma samples were prepared, plasma mass spectrometry was collected, and metabolites were determined by non-target metabolomics method. 【Results】 Compared with the pre-competition heart rate (39.80±0.40) bpm and respiratory rate (14.00±1.80) bpm, the horse heart rate (80.80±7.70) bpm and respiratory rate (78.80±3.90) bpm were significantly increased (P<0.05). Compared with Yili horses plasma at pre-race, the citric acid cycle, metabolism of alanine, aspartic acid and glutamic acid, transformation between pentaccharide and glucuronic acid, metabolism of cystine and methionine, oxidative phosphorylation, fatty acid degradation and sheath lipid metabolism showed significant changes. On the basis of the LC/MS analysis of plasma metabolomics of Yili horse before and after the 3,600 m speed race, LC/MS metabolic profiles of plasma samples of Yili horse 3,600 m before and after the race were different. The differential metabolites involved in citric acid cycle, glycolysis system, fat metabolism, amino acid metabolism. 【Conclusion】 During the 3,600 m speed race, the Yili horse body presents anaerobic, aerobic and mixed oxygen metabolism characteristics show anaerobic, aerobic and mixed oxygen metabolism.

Key words: Yili horse; 3,600 m speed race; before and after speed race; non-targeted metabolomics; plasma metabolites

摘要：

【目的】 研究伊犁马3 600 m速度赛赛前赛后血浆中代谢物的差异变化。【方法】 根据马场提供的遗传系谱,挑选5匹(3♂+2♀)无亲缘关系、体重(384.40±34.18)kg、年龄为2岁的速度赛用伊犁马,每日进行等强度训练。第25 d晨饲后4 h进行3 600 m速度赛,试验马以(11.75±0.87)m/s的平均速度完成比赛。赛前安稳状态下和赛后即刻测定心率和呼吸频率,采集血液、制备血浆样品,采集血浆的质谱,采用非靶向性代谢组学方法测定代谢物。【结果】 与赛前心率(39.80±0.40)bpm、呼吸频率(14.00±1.80)bpm相比,3 600 m速度赛赛后即刻试验马心率(80.80±7.70)bpm、呼吸频率(78.80±3.90)bpm显著升高(P<0.05)。与赛前相比,试验马赛后即刻部分血浆代谢物产生显著变化,差异代谢物主要涉及柠檬酸循环、丙氨酸、天冬氨酸和谷氨酸代谢、五糖和葡萄糖醛酸间转化、半胱氨酸与蛋氨酸代谢、氧化磷酸化、脂肪酸降解、鞘脂代谢等生化代谢途径(P<0.05)。【结论】 伊犁马3 600 m速度赛前、赛后血浆样本的LC/MS代谢谱存在差异,差异代谢物涉及柠檬酸循环、糖酵解系统以及脂肪代谢、氨基酸代谢等过程,3 600 m速度赛伊犁马机体呈现出无氧、有氧以及混氧代谢的特点。

关键词: 伊犁马, 3 600 m速度赛, 运动前后, 非靶向代谢组学, 血浆代谢差异物

CLC Number:

S821

ZHANG Shiqi, LI Xiaobin, ZHANG Wenjie, HAN Ming, WANG Shichang, ZHENG Wenxiang, OUYANG Wen, QI Juzhong, YANG Kailun. LC/MS-Based Metabolomics Approach for Exploring Plasma Metabolome Modififications before and after 3,600 m Speed Race in Yili horse[J]. Xinjiang Agricultural Sciences, 2023, 60(2): 501-510.

张仕琦, 李晓斌, 张文杰, 韩明, 王世昌, 郑文祥, 欧阳文, 祁居中, 杨开伦. 基于LC/MS的伊犁马3 600 m速度赛赛前、赛后血浆代谢组学差异变化[J]. 新疆农业科学, 2023, 60(2): 501-510.

Figures/Tables 9

Table 1 Composition and nutrient levels of the diet (DM basis,%)

项目 Items		含量 Content	项目 Items		含量 Content
原料 Ingredients	黄玉米	15.30	营养水平 Nutrient levels²⁾	干物质 DM	91.74
	黄玉米	15.30		粗蛋白 CP	13.76
	小麦麸	6.00		粗蛋白 CP
	小麦麸	6.00	中性洗涤纤维 NDF	43.54
	大豆粕 (CP44%)	9.00	中性洗涤纤维 NDF	43.54
	大豆粕 (CP44%)	9.00	酸性洗涤纤维 ADF	27.72
	磷酸二氢钙 CaH₂PO₄	1.00	酸性洗涤纤维 ADF	27.72
	磷酸二氢钙 CaH₂PO₄	1.00	钙 Ca	0.62
	预混料 ¹⁾	0.33	钙 Ca	0.62
	预混料 ¹⁾	0.33	磷 P	0.43
	食盐	0.67	磷 P	0.43
	食盐	0.67	赖氨酸 Lys	0.32
	L-丙氨酸	1.03	赖氨酸 Lys	0.32
	L-丙氨酸	1.03	蛋氨酸+半胱氨酸 Met+Cys	0.11
	青干草	66.67	蛋氨酸+半胱氨酸 Met+Cys	0.11
	青干草	66.67	丙氨酸 Ala	0.94
	合计	100.00	丙氨酸 Ala	0.94

Table 2 Chromatographic gradient elution procedure

时间Time(min)	A(%)	B(%)
0	98	2
1	98	2
17	50	50
17.5	50	50
18	98	2
20	98	2

Table 3 Heart rate and respiratory rate of the Yili horse before and after 3 600 m speed race(bpm)

项目 Item	赛前 Pre-race	赛后即刻 Immediately at post-race	P值 P-value
心率 Heart rate	39.80±0.4	80.8±7.70^*	<0.001
呼吸频率 Respiratory rate	14.00±1.80	78.80±3.90^*	<0.001

Fig.1 Total ion chromatography of plasma metabolites in Yili horse Note: A is positive ion mode and B is negative ion mode

Fig.2 PCA score plot Note: The confidence ellipse is 95%.A is the positive ion mode and B is the negative ion mode,the same as below

Fig.3 PLS-DA score plot Note: R2 is model interpretation rate.Q2 is model prediction capability

Table 4 Plasma differential metabolites before and after 3 600 m Speed Race in Yili horse(n=5)

代谢物 Metabolites	分子式 Formula	保留时间 RT (min)	变量投影重要度 VIP	上调/ 下调 Up/ Down
葡萄糖Glucose	C₅H₁₁ClNaO₃PS	1.61	3.24	down
D-葡萄糖-6-磷酸 D-glucose-6-phosphate	C₆H₁₀N₃NaO₂	1.61	3.43	down
甘油Glycerin	C₃H₈O₃	1.41	5.27	up
柠檬酸Citric acid	C₆H₈O₇	1.34	1.05	up
琥珀酸 Butanedioic acid	C₄H₆O₄	2.47	6.52	up
精氨基琥珀酸 Argininosuccinic acid	C₁₀H₁₈N₄O₆	1.46	2.04	up
乳酸Lactic acid	C₃H₆O₃	2.03	2.64	up
丙酮酸Pyruvic acid	C₃H₄O₃	1.41	1.71	up
α-酮戊二酸 α-Ketoglutaric acid	C₅H₆O₅	1.31	1.16	up
尿酸Uric acid	C₅H₄N₄O₃	1.41	3.02	up
亮氨酸Leucine	C₈H₁₆NNaO₄	1.54	1.14	down
犬尿氨酸 Kynurenine	C₆H₉NO₄S	3.46	4.04	up
色氨酸 Tryptophan	C₁₁H₁₂N₂O₂	6.23	1.16	down
组氨酸 Histidine	C₆H₉N₃O₂	1.36	1.68	down
胱氨酸 Cystine;	C₆H₁₂N₂O₄S₂	1.69	1.19	up
甜菜碱 Betaine	C₅H₁₁NO₂	1.40	1.01	down
乙酰天冬氨酸 N-acetylaspartic acid	C₆H₉NO₅	2.46	1.91	up
苯丙氨酸 Phenylalanine	C₃H₇NNa₂P₂	2.90	1.48	up
谷氨酸Glutamate	C₇H₁₁NO₅	1.27	1.97	up
谷氨酰胺Glutamine	C₅H₁₀N₂O₃	1.52	1.60	down
脯氨酸Proline	C₅H₉NO₂	1.43	1.01	down
苏氨酸Threonine	C₄H₉NO₃	1.40	1.37	down
乙酰精氨酸 Acety-L-arginine	C₇H₁₅N₄NaO₄	1.59	1.41	down
混旋肉碱 DL-Carnitine	C₇H₁₅NO₃	1.58	1.39	down
乙酰左旋肉碱 Acety-L-carnitine	C₉H₁₇NO₄	1.41	1.73	up
辣椒素 Capsaicin	C₄₀H₅₆O₃	15.69	1.23	down
多巴胺 Dopamine	C₈H₁₁NO₂	3.99	1.08	down

Table 5 Changes of free amino acids in pasma before and after 3 600 m Speed Race in Yili horse(n=5, μmol/L)

项目 Item	赛前 Pre-race	赛后即刻 Immediately at post-race	P值 P-value
天门冬氨酸 Asp	2.60±0.45	8.79±0.91^**	<0.001
色氨酸 Trp	53.22±5.60	37.80±6.68^**	<0.001
赖氨酸 Lys	87.44±4.99	113.68±8.15^**	<0.001
组氨酸 His	81.74±4.93	62.14±9.00^**	0.002
天门冬酰胺 Asn	51.00±6.48	41.44±5.35^**	0.004
丙氨酸 Ala	333.80±40.94	426.00±43.3 $8^{^{*}}$	0.036
精氨酸 Arg	178.00±4.74	202.00±14.80^#	0.05
半胱氨酸 Cys	83.22±10.32	71.82±4.20^#	0.076
亮氨酸 Leu	125.98±5.80	150.80±23.32^#	0.085
异亮氨酸 Ile	73.34±6.06	80.20±4.09	0.108
谷氨酰胺 Gln	414.40±21.10	432.00±36.18	0.205
谷氨酸 Glu	15.00±1.53	55.12±7.54	0.205
苯丙氨酸 Phe	58.68±2.66	67.28±13.17	0.214
甘氨酸 Gly	688.20±61.37	734.40±88.17	0.313
丝氨酸 Ser	262.00±45.38	246.60±34.16	0.401
酪氨酸 Tyr	115.32±7.74	112.06±8.56	0.447
缬氨酸 Val	216.40±21.71	217.20±24.91	0.54
甲硫氨酸 Met	27.68±1.92	26.20±3.76	0.547
苏氨酸 Thr	103.08±6.88	101.32±10.61	0.778
脯氨酸 Pro	138.60±23.22	129.52±15.66	0.94
氨基酸总量 TAA	3 109.70±142.84	3 316.37±260.25	0.128
必需氨基酸 EAA	745.82±40.05	794.48±78.08	0.176
非必需氨基酸 NEAA	2 363.88±113.33	2 521.89±185.38	0.119
支链氨基酸 BCAA	415.72±28.38	448.20±46.78	0.139
芳香族氨基酸 AAA	227.22±11.05	217.14±17.71	0.117
必需氨基酸/非必需氨基酸	0.32±0.02	0.32±0.02	1.000
色氨酸/支链氨基酸	0.13±0.01	0.08±0.01	0.002

Table 5 Changes of free amino acids in pasma before and after 3 600 m Speed Race in Yili horse(n=5, μmol/L)

项目 Item	赛前 Pre-race	赛后即刻 Immediately at post-race	P值 P-value
天门冬氨酸 Asp	2.60±0.45	8.79±0.91^**	<0.001
色氨酸 Trp	53.22±5.60	37.80±6.68^**	<0.001
赖氨酸 Lys	87.44±4.99	113.68±8.15^**	<0.001
组氨酸 His	81.74±4.93	62.14±9.00^**	0.002
天门冬酰胺 Asn	51.00±6.48	41.44±5.35^**	0.004
丙氨酸 Ala	333.80±40.94	426.00±43.3 $8^{^{*}}$	0.036
精氨酸 Arg	178.00±4.74	202.00±14.80^#	0.05
半胱氨酸 Cys	83.22±10.32	71.82±4.20^#	0.076
亮氨酸 Leu	125.98±5.80	150.80±23.32^#	0.085
异亮氨酸 Ile	73.34±6.06	80.20±4.09	0.108
谷氨酰胺 Gln	414.40±21.10	432.00±36.18	0.205
谷氨酸 Glu	15.00±1.53	55.12±7.54	0.205
苯丙氨酸 Phe	58.68±2.66	67.28±13.17	0.214
甘氨酸 Gly	688.20±61.37	734.40±88.17	0.313
丝氨酸 Ser	262.00±45.38	246.60±34.16	0.401
酪氨酸 Tyr	115.32±7.74	112.06±8.56	0.447
缬氨酸 Val	216.40±21.71	217.20±24.91	0.54
甲硫氨酸 Met	27.68±1.92	26.20±3.76	0.547
苏氨酸 Thr	103.08±6.88	101.32±10.61	0.778
脯氨酸 Pro	138.60±23.22	129.52±15.66	0.94
氨基酸总量 TAA	3 109.70±142.84	3 316.37±260.25	0.128
必需氨基酸 EAA	745.82±40.05	794.48±78.08	0.176
非必需氨基酸 NEAA	2 363.88±113.33	2 521.89±185.38	0.119
支链氨基酸 BCAA	415.72±28.38	448.20±46.78	0.139
芳香族氨基酸 AAA	227.22±11.05	217.14±17.71	0.117
必需氨基酸/非必需氨基酸	0.32±0.02	0.32±0.02	1.000
色氨酸/支链氨基酸	0.13±0.01	0.08±0.01	0.002

Table 6 KEGG enrichment

途径 Pathway	P值 P-value	校正P值 Adjusted P-value
柠檬酸循环(TCA循环)Citrate cycle (TCA cycle)	0.01	0.46
丙氨酸、天冬氨酸和谷氨酸代谢Alanine, aspartate and glutamate metabolism	0.01	0.46
五糖和葡萄糖醛酸间转化Pentose and glucuronate interconversions	0.02	0.46
半胱氨酸与蛋氨酸代谢Cysteine and methionine metabolism	0.03	0.46
氧化磷酸化(作用)Oxidative phosphorylation	0.01	0.57
脂肪酸降解Fatty acid degradation	0.03	0.57
鞘脂代谢Sphingolipid metabolism	0.03	0.57

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