Analysis Of Plasma Metabolomics Of Type Ⅰ And Type Ⅱ Ketones In Dairy Cows Based On ~ 1H NMR Technique

Ketosis is an important energy metabolic disorder that commonly occur inhigh-producingdairy cows during transition period, is biochemically characteristic of low blood glucose, highketone body and high free fatty acid. Neg-ative energy balance (NEB) and excessive fatmobilization are the pathological foundation for ketosis. It is usually classified by clinical andsubclinical ketosis based on the level of blood ketone body and clinical symptoms, which therehave been a lot of reports on their etiology. However, ketosis in dairy cows may be divided intotype I and typeⅡ ketosis based on the level of blood ketone, glucose, and free fatty acids like theclassification of diabetes. At present, study on metabolomics of typeⅡ ketosis is rather little indairy cows. Therefore, objective of research is to understand the overall changes of the plasmametabolism between type I and type Ⅱo f ketosis andto discovery the difference characteristicmetabolites using nuclear magnetic resonance (NMR) technology and multivariate statisticalanalysis. At the same time, it will provide a new scientific basis for enriching etiology andpreventing ketosis in dairy cows.50cows with two or three parities were selected at7to28days postpartum based on theglucose (Glc) and3-hydroxybutyrate acid (BHBA) and non-esterified fatty acid(NEFA) andinsulin (Ins) levels.20dairy cows with type I ketosis (K1) were chosen if they had no otherdiseases and plasma BHBA>1.20mmol/L, plasma Glc <2.50mmol/L, plasma NEFA>0.50mmol/L.20dairy cows with type II ketosis (K2) were defined if they had no other diseases andplasma BHBA>1.20mmol/L, Glc>2.80mmol/L, plasma NEFA>0.50mmol/L.10healthycontrols (C) were chosen if they had any diseases and plasma BHBA <1.00mmol/L, plasma Glc>3.75mmol/L, plasma NEFA <0.40mmol/L. First, plasma samples from three groups of dairycows were detected by nuclear magnetic resonance (NMR) technology. Second,1H NMR spectrawas analyzed by multivariate statistical analysis including principal component analysis(PCA),Partial Least Squares-discriminant Analysis(PLS-DA), and Orthogonal PartialLeast-Squares Discriminant Analysis(OPLS-DA). Then, all the biological differencesmetabolites were analyzed using the biological information technology. Finally, the differencemetabolites of type I and type II ketosis were verified by Enzyme Linked Immunosorbent Assaysmethod (ELISA). The results shown that there were26different metabolites among type I, type II ketosis andhealthy control. Compared to healthy controls,2metabolites like3-Hydroxybutyrate and acetonein plasma increased markedly, and15metabolites decreased significantly in type I ketosis, suchas alanine, glutamate, glutamine, glycine, histidine, lysine, citrate, creatine, creatine, lipid,myo-Inositol, O-acetyl glycoprotein signals, Phosphocholine, α-Glucose, β-Glcose (P<0.05).Meanwhile,3metabolites in plasma rose significantly, like3-Hydroxybutyrate, acetone andlactate, and15metabolites reduced markedly in type II ketosis，alanine，lysine，tyrosine andcreatine (P<0.05). Compared to type II ketosis,8metabolites in plasma increased significantly,like acetate,3-Hydroxybutyrate, acetone, isoleucine, leucine, valine, LDL and VLDL, and14metabolites deceased markedly in type I ketosis, such as alanine, citrate, formate, glutamate,glutamine, glycine, histidine, lipid, lysine, myo-Inositol, O-acetylglycoproteinsignals,Phosphocholine, α-Glucose, β-Glcose (P<0.05). The different metabolites analyzed throughKyoto Encyclopedia of Genes and Genomes(KEGG) not only involved in carbohydratemetabolism, fat metabolism, amino acid metabolism, and energy metabolism, and also in theKetone synthesis.11selected metabolites, like pyruvic acid, α-ketoglutaric acid, citrate,phosphatidylcholine, oxaloaceticacid, glycine, tyrosine, isoleucine, leucine, alanine and lactate,were verified by ELISA method, which had similar results as1H NMR technology.Conclusion obtained that plasma different metabolites for type I and type II ketosis may bescreened and verified using1H NMR technology, and the panoramic metabolic disorders for typeI and type II ketosis have been validated in carbohydrates metabolism, fat metabolism, andamino acids metabolism. It will lay the foundation to further explore the mechanism and preventstrategies for type II ketosis in the future.