Increased nutrient demand and decreased intake during early lactation may results in a NEB status 24,25. Because of this, fatty acids and amino acids are mobilized from adipose and muscle tissues, respectively, predisposing animals to the risk of metabolic disease such as ketosis. Subclinical ketosis is less studied in dairy ewes but not less important because it can significantly affect farm economy 1,4. Further studies about the metabolomics profile of animals would be of critical importance to improve the understanding about ketosis on small ruminants and to develop biomarkers for an early diagnosis 26,27. Because of this, the aim of this study is the metabolomic analysis of serum in hyperketonemic ewes by ¹H-NMR.
The presence of hyperketonemia, hypoglycemia and uremia are consistent with an early subclinical ketosis status 28. The group K showed a higher concentration of BHB and urea, and a lower concentration of glucose. The urea serum concentration was slightly over physiological range in sheep (2.86–7.14 mmol/L), whereas glucose concentration was within it (2.78–4.44 mmol/L), although reduced compared with group H 29. The rPCA analysis is a multivariate statistical method used as an explanatory clustering technique to identify differences between groups of metabolome 30. Our rPCA revealed a clear difference between the serum metabolome of groups. This might suggest that the metabolomic profile of ewes was firmly associated with different BHB concentration, even though group K was only hyperketonemic. From this point of view, hyperketonemic ewes may be considered separately from properly healthy animals.
The mobilization of adipose tissue is related to triacylglycerols break down into its components, NEFA and glycerol, with a release in blood stream 31,32. In our study, the similarity both in NEFA and glycerol concentrations, may indicate that adipose tissue was not mobilized in group K. However, it is possible that an initial increase in NEFA and glycerol may be used by mammary gland to synthetize milk fat: an increase in fat percentage in milk is a common feature during ketosis 33. The NEFA can be partially oxidized to ketone bodies such as acetoacetate, BHB and acetone in hepatic tissue 34. BHB and acetone were identified by metabolomic approach in our study and they showed an increase in group K. Ketone bodies are responsible for alterations of inflammatory response 18. They may also derive from ketogenic amino acids (lysine, leucine and isoleucine) 22 which did not changed in this study suggesting non-use to produce ketone bodies.
Leucine, isoleucine, and valine are branched-chain amino acids (BCAAs) used for protein synthesis in muscle. Low concentration of BCAAs are positively related to alanine concentration, which is a glucogenic amino acid highly concentrate in muscle 23,35,36. Both alanine and valine showed a reduction in group K. The 3-methylhistidine come from muscle protein breakdown and it is considered as a biomarker of protein mobilization 4. This metabolite showed an increment in group K. The lower concentrations of valine and alanine, and the higher concentration of 3-methylhistidine suggest an amino acids mobilization in group K. Tyrosine is also related to muscle metabolism. Indeed, its low concentration is an indicator of reduced muscle growth 37. In this study, tyrosine was decreased in group K, in agreement with previously hypothesized. Creatine and its breakdown product, creatinine, are related to total muscle mass 19,38. Creatine concentration is related to subclinical ketosis in dairy cows and weight loss in different ewe breeds 37,39. The absence of changes in their concentrations may suggest that the total muscle mass was not still affected by protein break down.
There was an increase in methanol and ethanol concentrations in hyperketonemic ewes as well as in dairy cows13. Methanol is a potentially toxic compound which target is the retina in eyes and may explain the clinical sign of impaired vision during ketosis 14. Methanol can be derived from methane, a gas produced during ruminal fermentation by microbial cells 40. However, dimethylsulfone and formate showed similar concentrations between groups indicating that methane production was not affected 41. Increase in methanol may also be related to an increase in ethanol concentration that inhibits the methanol’s utilization by microorganisms 42. Ethanol derives from anaerobic fermentation by yeasts. This alcohol is an agonist of GABA receptors, so it has a depressive effect. The major product of ethanol in hepatic tissue is acetate to provide energy. Acetate is a volatile fatty acid (VFA) produced by ruminal fermentation that increases during ketosis as reported in the present study. Acetate is an important energy substrate when bound to coenzyme A to produce acetyl-CoA and enter in tricarboxylic acid cycle (TCA). Moreover, it may be used in brain metabolism, specifically in glial cells, potentially causing mitochondrial permeability and excitotoxic neuronal death 12,23,43. Acetyl-CoA can also be derived from 2,3-butanediol 44, a ruminal and intestinal microbial product that was increased in group K. Another VFA is propionate 12,45 which was not identified in our study, although the 3-hydroxyisobutyrate may result from propionate and it increased in group K. The above changes may suggest an increment and alteration in ruminal fermentations in hyperketonemic ewes with potential relationships with the pathogenesis and symptoms of ketosis.
Myo-inositol is a stereoisomeric form of inositol and represents an insulin mimetic metabolite because it promotes adipose tissue lipid storage and limits lipolysis rate 46. In our study, myo-inositol was similar between groups suggesting that the lipolysis process was not limited. Choline supports the transport of fatty acids, increases their oxidation and reduces the risk of hepatic lipidosis 22. Choline can be converted in TMAO, a marker of oxidative stress because it reduces glycolysis and enhances β-oxidation of fatty acids 13. Another metabolite related to β-oxidation is allantoin, a product of uric acid. Uric acid is related to triglycerides metabolism and its increase may limit enzymatic activity for their catabolism 39. The analogous concentration of choline, TMAO, and allantoin may suggest that the β-oxidation of fatty acids was not influenced in our hyperketonemic ewes. Although subclinical ketosis is associated with an increment and alteration in lipid metabolism, group K did not show these characteristics. As previously mentioned, a possible increment of NEFA may be hidden by mammary gland, leading to a lack of fatty acids oxidation’s alterations.
TCA begins with the combination between acetyl-CoA and oxaloacetate. Acetyl-CoA may derive from fatty acid catabolism or pyruvate oxidation. Pyruvate may derive from amino acids (glycine, serine and alanine) 47 among them only the alanine reduction in group K was significant. Glycine and serine are biosynthetically linked and they represent important regulators of glutathione synthesis to manage the oxidative stress 48. However, they did not change suggesting the absence of an oxidative stress state, in accordance with the lack of influence of fatty acids oxidation. Alanine represents one of the major resources for gluconeogenesis, therefore it affects carbohydrate metabolism. Its lower concentration is related to ketosis and fatty liver in dairy cows 49,50. Pyruvate did not change in group K as well as glucose identified by metabolomic analysis. Indeed, pyruvate can be used for gluconeogenesis to produce glucose 51. These findings confirm that glucose concentration depends on pyruvate and a reduction of pyruvate due to lack of its precursors (glycine, serine and alanine) can significantly affect glycemia and the development of ketosis. Asparagine is one of oxaloacetate precursors 47 and showed a reduction in our hyperketonemic ewes in agreement with other studies 52. This metabolite is involved in cell functions of nerve and brain tissue, and it is a nontoxic carrier of residual ammonia. In this study, oxaloacetate was not identified. However, the reduction of its precursor may indicate an oxaloacetate reduction and an alteration of TCA.
The following intermediates of TCA are citrate and isocitrate, which are maintained in equilibrium in the cell 53. Isocitrate is subsequently converted to α-ketoglutarate that may derive from glutamate. Histidine, proline, glutamine and arginine are all metabolites related to glutamate production 54,55. In accordance with the study of Zhang et al. (2017a), glutamate is an immune-regulator amino acid because it is involved in the activation and proliferation of immune system cells; genetic expression and production of cytokines and antibodies; and cellular oxide-reduction. Histidine presents antioxidant and anti-inflammatory qualities for scavenging reactive oxygen species (ROS) generated by cells during acute inflammation 56 and for suppressing pro-inflammatory cytokine expression 57. In this study, glutamate and histidine described a reduction in hyperketonemic ewes suggesting a potential oxidative stress state and immune suppression if the metabolic state progresses to ketosis. Glutamate may act as a neuroactive ligand for glutamate receptor 1, with a consequent excitatory effect. The reduction of glutamate may play an important role in nervous depression if ketosis develops. Glutamine can be converted in glutamate and after pyrroline-5-carboxylate, which links TCA and urea cycle. Arginine is converted into ornithine and urea in the final phase of the urea cycle 55. The reduction of glutamine, glutamate, and arginine may suggest an alteration of this cycle, with a possible lack of urea synthesis in hyperketonemic ewes.
Succinate is the subsequent intermediate of TCA whose precursors are threonine and methionine 32. Threonine is related to collagen production, regulation of the immune system, and secondary ketosis due to fatty liver syndrome 58,59. Methionine is involved in protein synthesis, antioxidant production and methyl group donation. It can also be synthetized from choline oxidation. The choline production from methionine is relatively high in ruminants due to its degradation in the rumen 60. In this study, threonine and methionine described a reduction in group K. These findings suggest a potential alteration of the inflammatory response, immune system functions and management of oxidative stress status other than their use to produce succinate, which was increased in group K, to obtain oxaloacetate for gluconeogenesis at the end of the cycle. However, fumarate is the next intermediate of TCA and not showed difference between groups. This metabolite may be synthetized by phenylalanine and tyrosine 47. As previously mentioned, tyrosine showed a reduction in group K as reported in other studies 61. These results may indicate that there was a disturbance of succinate dehydrogenase function and fumarate was maintained by its precursor. Succinate dehydrogenase is the only enzyme involved in TCA and in the electron transport chain (ETC) 62. An alteration in succinate dehydrogenase might suggest an influence on ETC in hyperketonemic ewes as well as in hyperketonemic dairy cows 13.
The main function of aminoacyl-tRNA biosynthesis is to catalyze the aminoacylation of transfer RNAs (tRNAs) involved in protein synthesis, angiogenesis and immune regulation 63. During Alanine, Aspartate, and Glutamate metabolism there is the biosynthesis of some amino acids (alanine, aspartate, asparagine, glutamate, and glutamine) and intermediates of TCA (oxaloacetate, citrate, succinate, fumarate). Therefore, this metabolic pathway is related with lipid, carbohydrates, and amino acid metabolisms. Furthermore, in human patient this pathway can be involved in the pathogenesis of metabolic syndrome 64. The D-Glutamine and D-Glutamate metabolism was linked to Alanine, Aspartate, and Glutamate metabolism and concerns the glutamine/ glutamate cycling. The influence of Glycolysis/Gluconeogenesis further emphasizes how much energy production and glucose synthesis are required in hyperketonemic ewes. Glyoxylate and Dicarboxylate metabolism is used to produce carbohydrates from fatty acids in bacteria, protozoa, protists, and fungi. The influence of this metabolic pathway could result from an alteration of animal’s microbiomes. Phenylalanine, Tyrosine, and Tryptophan biosynthesis might be important because it was related to ubiquinone synthesis, which was involved in oxidative phosphorylation during TCA.