The metabolic profiling of cheetahs (Acinonyx jubatus): a systems biology approach to understanding the chronic diseases they suffer in captivity
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In captivity, cheetahs (Acinonyx jubatus) are known to frequently suffer from several chronic diseases including lymphoplasmacytic gastritis, glomerulosclerosis, renal amyloidosis, veno-occlusive disease of the liver, adrenal hyperplasia and several ill-defined neurological disorders that are rare in free-ranging animals. Stress, lack of exercise, low genetic variability and the provision of unnatural diets in captive facilities have been proposed as potential causal factors, but to date convincing pathophysiological explanations for these diseases have been lacking or unsatisfactory. Using a systems biology approach, we used untargeted metabolomic analysis of serum and urine from captive and free-ranging cheetahs, generating new physiological data for this species in the hope of developing a better understanding of their metabolism. Prior to the actual quantification of serum or urine metabolites, we created a new, more objective, method of approximating body condition in cheetahs by means of a body mass index value. As expected, the morphometric data and body mass indices obtained in the study population showed significant differences between males and females, but importantly, all the animals fell within a healthy body mass index range. The impact of body condition on various serum and urine metabolites could thus be objectively assessed. We also evaluated the use of either urine creatinine concentrations or urine specific gravity values for the correction of spot urine samples obtained from the cheetahs. Creatinine excretion was found to be highly variable in the study animals - influenced by individual, largely age-related differences in creatinine production rather than accurately reflecting changes in glomerular filtration rate. Relying on urine creatinine concentrations to correct or "standardize" urine metabolite concentrations would therefore result in overestimation of metabolite concentrations in younger and older cheetahs. The variability in urine specific gravity was considerably lower and shown to provide a better indication of urine dilution. Urine specific gravity was therefore used to correct urine metabolite concentrations in this study. Using gas chromatography-mass spectrometry, 339 different organic acids were annotated and quantified in the urine of 56 captive and two free-ranging cheetahs. Phenolic compounds, thought to be produced by the anaerobic fermentation of aromatic amino acids in the distal colon, as well as their corresponding glycine conjugates, were present in high concentrations in the urine of the captive cheetahs. It is suggested that the required detoxification of these phenolic compounds through glycine conjugation could result in the chronic depletion of both glycine and sequestration of Coenzyme A, with associated negative metabolic consequences. We suggest that the high urine levels of these phenolic compounds may be caused by an excess in dietary protein as most captive cheetahs are fed a diet rich in muscle meat and low in fat and other so-called animal fibre. Concentrations of these phenolic compounds correlated negatively with the end-stage metabolites of dopamine and catecholamines, providing a potential mechanism for significant neuroendocrine dysregulation. Potential mechanisms by which dopamine depletion may play a central role in the pathophysiology of both gastric and renal disease in cheetahs are discussed. Using gas chromatography-mass spectrometry as well as liquid chromatography-tandem mass spectrometry we established serum and urine amino acid profiles in captive cheetahs. Although the serum concentrations of most of the amino acids in cheetahs were comparable to those in published data for domestic cats, the serum arginine and ornithine concentrations were substantially higher. Finally, the serum fatty acid and acylcarnitine profiles of 35 captive and 43 free-ranging cheetahs were evaluated through the use of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. The profiles obtained from the free-ranging animals provide a unique, healthy control group for comparison. Significant differences were noted for most of the fatty acid and acylcarnitine concentrations between these two populations, indicating dramatic differences in the dietary fat intake, composition and/or metabolism of these nutrients. Most of the serum polyunsaturated fatty acid and mono-unsaturated fatty acid concentrations were significantly lower in the free-ranging cheetahs, compared to the captive animals, suggesting that the fatty acids in the wild cheetah diet are largely saturated. Fatty acids not only provide a valuable source of energy, but also perform other vital functions in the body, including hormone production, cellular signalling and the provision of structural components of biological membranes. Altered serum fatty acids could thus have a dramatic impact on health and, since their concentrations are largely influenced by diet, the values obtained from free-ranging cheetahs potentially provide valuable healthy target values for their captive counterparts. Through this unique approach, we have established new baseline data for a large range of serum and urine metabolites in cheetahs. The results raise many questions and provide valuable new insights and hypotheses into the potential mechanisms of metabolic disorders in captive cheetahs, creating a platform for future research in this species.