基于~1H NMR技术的奶牛Ⅰ型和Ⅱ型酮病血浆代谢谱分析

~1H NMR-Based Plasma Metabolic Profiling of Dairy Cows with Type Ⅰ and Type Ⅱ Ketosis

【目的】运用代谢组学中1H NMR技术方法筛选出Ⅰ型酮病、Ⅱ型酮病与健康对照组之间血浆差异性代谢物。【方法】选取产后7—28 d,平均胎次为2—3胎的实验奶牛50头。根据血糖(Glc)、β-羟丁酸(BHBA)和游离脂肪酸(NEFA)的含量与临床发病特点分为Ⅰ型酮病、Ⅱ型酮病与健康对照组,其中Ⅰ型酮病20头,Ⅱ型酮病为20头,健康对照组为10头。当患病牛血中BHBA>1.20 mmol?L-1,Glc<2.50 mmol?L-1,NEFA>0.50 mmol·L-1时,被认为患I型酮病;当患病牛血浆中BHBA>1.20 mmol?L-1,Glc>2.80 mmol?L-1,NEFA>0.50 mmol?L-1时,被认为患II型酮病;当奶牛血中BHBA<1.00 mmol?L-1,Glc>3.75 mmol?L-1,NEFA<0.40 mmol?L-1时,被认为健康对照组。运用代谢组学中1H NMR技术对实验奶牛的血浆代谢物分析,获得相应的代谢图谱,并结合多元统计分析中的主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)的模式判别,从而寻找潜在的生物标记物。【结果】通过1H NMR分析,Ⅰ型酮病、Ⅱ型酮病与健康对照的代谢图谱差异明显,3组代谢产物各自聚集,分散区域显著。Ⅱ型酮病与健康对照比较,获得7种血浆差异代谢物,主要为丙氨酸、赖氨酸、β-羟丁酸、丙酮、乳酸等,其中血浆中β-羟丁酸、丙酮、乳酸浓度升高;丙氨酸、赖氨酸、酪氨酸、肌酸浓度呈现下降。Ⅰ型酮病与健康对照组比较,获得19种血浆差异代谢物,主要为酪氨酸、苯丙氨酸、肌酸、β-羟丁酸、丙酮等,其中β-羟丁酸、丙酮浓度升高;酪氨酸、苯丙氨酸、赖氨酸、组氨酸、丙氨酸、肌酸、肌醇、β-葡萄糖、谷氨酰胺、谷氨酸、柠檬酸、α-葡萄糖、甲酸、甘氨酸、O-乙酰葡萄糖胺、磷酸胆碱浓度呈现下降。Ⅰ型酮病与Ⅱ型酮病比较,获得24种血浆差异代谢物,主要为柠檬酸、组氨酸、β-葡萄糖、异亮氨酸、极低密度脂蛋白/低密度脂蛋白等,其中β-羟丁酸、低密度脂蛋白和极低密度脂蛋白、异亮氨酸、缬氨酸、丙酮、亮氨酸、乙酸浓度升高;柠檬酸、酪氨酸、组氨酸、肌醇、谷氨酰胺、β-葡萄糖、苯丙氨酸、谷氨酸、α-葡萄糖、赖氨酸、甲酸、甘氨酸、磷酸胆碱、丙氨酸、O-乙酰葡萄糖胺浓度呈现下降。【结论】1H NMR技术与多元统计分析的有效结合能够有效的筛选出Ⅰ型酮病、Ⅱ型酮病与健康对照组之间血浆差异性代谢物,为进一步探究奶牛Ⅰ型酮病、Ⅱ型酮病的发病机理和诊断与防治提供了新的方向。

【Objective】To identify the differential metabolites in plasma among the cows of type I ketosis, type II ketosis and normal controls, the plasma metabolic profiles from all experimental cows were analyzed using 1H nuclear magnetic resonance technology（1H NMR）.【Method】A total of 50 cows with 2 to 3 parities were selected at 7-28 days postpartum and divided into type I ketosis（K1, 20 cows）, type II ketosis（K2, 20 cows） and normal control（C, 10 cows） according to glucose（Glc）, 3-hydroxybutyrate acid（BHBA）, non-esterified fatty acid（NEFA） level and clinical signs of the disease. Type I ketosis was defined as having plasma BHBA1.20 mmol·L-1, plasma Glc2.50 mmol·L-1, and plasma NEFA0.50 mmol·L-1. Type II ketosis was defined as having plasma BHBA1.20 mmol·L-1, Glc2.80 mmol·L-1, and plasma NEFA0.50 mmol·L-1. Healthy controls were those with plasma BHBA1.00 mmol·L-1, plasma Glc3.75 mmol·L-1, and plasma NEFA0.40 mmol·L-1. The plasma metabolic profiles from all experimental cows were analyzed using 1H NMR. The data were processed by principal component analysis（PCA）, and orthogonal partial least-squares discriminant analysis（OPLS-DA） was performed to identify the differential metabolites in plasma among the three groups.【Result】The results showed that OPLS-DA model was better to distinguish the three plasma samples. Compared with healthy controls, 7 differential metabolites were obtained from type II ketosis and healthy controls, such as alanine, lysine, 3-hydroxybutyrate, acetone, lactate, and so on, type II ketosis had higher plasma levels of BHBA, acetone, and lactate and lower levels of alanine, lysine, tyrosine, and creatine. Nineteen differential metabolites were obtained from type I ketosis and healthy controls, for example tyrosine, phenylalanine, creatine, 3-hydroxybutyrate, acetoneet, and so on. Type I had higher levels of BHBA and acetone and lower levels of tyrosine, phenylalanine, lysine, histidine, alanine, creatine, myo-inositol, β-Glc, glutamine, glutamate, citrate, α-Glc, formate, glycine, O-acetyl glycoprotein（OAG）, and phosphocholine. Twenty-four differential metabolites were obtained from type I ketosis and type II ketosis, for instance citrate, histidine, β-glcose, isoleucine, VLDL/LDL, and so on. Compared with type II ketosis, type I ketosis had higher levels of BHBA, low density lipoprotein（LDL）, very low density lipoprotein（VLDL）, isoleucine, valine, acetone, leucine, and acetate and lower levels of citrate, tyrosine, histidine, creatine, glutamine, β-Glc, phenylalanine, glutamate, α-Glc, lysine, formate, glycine, phosphocholine, and OAG. 【Conclusion】The combination of 1H NMR and multivariate statistical analysis techniques could effectively distinguish different metabolites among type I, type II ketosis and the healthy control group, It is an important basis for further studying the clinical pathology, early detection, diagnosis and detailed pathogenesis of type I and type II ketosis in dairy cows in the future, which lays a foundation for preventing type I and type II ketosis in dairy cows effectively.