Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign; Urbana, Illinois, United States of America.
Fuqua School of Business, Duke University, Durham, North Carolina, United States of America.
PLoS One. 2024 May 30;19(5):e0303500. doi: 10.1371/journal.pone.0303500. eCollection 2024.
Untargeted metabolomics investigations have characterized metabolic disturbances associated with various diseases in domestic cats. However, the pre-analytic stability of serum metabolites in the species is unknown. Our objective was to compare serum metabolomes from healthy cats stored at -20°C for up to 12 months to samples stored at -80°C. Serum samples from 8 adult, healthy cats were stored at -20°C for 6 months, -20°C for 12 months, or -80°C for 12 months. Untargeted liquid chromatography-mass spectrometry was used to generate serum metabolite profiles containing relative abundances of 733 serum metabolites that were compared among storage conditions. Unsupervised analysis with principal component analysis and hierarchical clustering of Euclidian distances revealed separation of samples from individual cats regardless of storage condition. Linear mixed-effects models identified 75 metabolites that differed significantly among storage conditions. Intraclass correlation analysis (ICC) classified most serum metabolites as having excellent (ICC ≥ 0.9; 33%) or moderate (ICC 0.75-0.89; 33%) stability, whereas 13% had poor stability (ICC < 0.5). Biochemicals that varied significantly among storage conditions and classified with poor stability included glutathione metabolites, amino acids, gamma-glutamyl amino acids, and polyunsaturated fatty acids. The benzoate; glycine, serine and threonine; tryptophan; chemical (xenobiotics); acetylated peptide, and primary bile acid sub pathways were enriched among highly stable metabolites, whereas the monohydroxy fatty acid, polyunsaturated fatty, and monoacylglycerol sub-pathways were enriched among unstable metabolites. Our findings suggest that serum metabolome profiles are representative of the cat of origin, regardless of storage condition. However, changes in specific serum metabolites, especially glutathione, gamma-glutamyl amino acid, and fatty acid metabolites were consistent with increased sample oxidation during storage at -20°C compared with -80°C. By investigating the pre-analytic stability of serum metabolites, this investigation provides valuable insights that could aid other investigators in planning and interpreting studies of serum metabolomes in cats.
非靶向代谢组学研究已经描述了与各种疾病相关的猫代谢紊乱。然而，该物种血清代谢物的预分析稳定性尚不清楚。我们的目的是比较在-20°C 下储存长达 12 个月的健康猫的血清代谢组与在-80°C 下储存的样本。从 8 只成年健康猫采集的血清样本分别在-20°C 下储存 6 个月、12 个月或-80°C 下储存 12 个月。使用非靶向液相色谱-质谱法生成包含 733 种血清代谢物相对丰度的血清代谢物图谱，然后在储存条件之间进行比较。使用主成分分析和欧几里得距离的层次聚类进行无监督分析，结果显示无论储存条件如何，均能分离来自个体猫的样本。线性混合效应模型确定了 75 种代谢物在储存条件之间存在显著差异。组内相关分析（ICC）将大多数血清代谢物分类为具有极好的稳定性（ICC≥0.9；33%）或中等稳定性（ICC 0.75-0.89；33%），而 13%的代谢物稳定性差（ICC<0.5）。在储存条件之间差异显著且稳定性差的生化物质包括谷胱甘肽代谢物、氨基酸、γ-谷氨酰基氨基酸和多不饱和脂肪酸。苯甲酸；甘氨酸、丝氨酸和苏氨酸；色氨酸；化学（异源生物）；乙酰化肽和初级胆汁酸亚途径在高度稳定的代谢物中富集，而单羟基脂肪酸、多不饱和脂肪酸和单酰基甘油亚途径在不稳定的代谢物中富集。我们的研究结果表明，无论储存条件如何，血清代谢组谱都代表了原始猫的特征。然而，与-80°C 相比，在-20°C 下储存时，特定血清代谢物（特别是谷胱甘肽、γ-谷氨酰基氨基酸和脂肪酸代谢物）的变化表明样品氧化增加。通过研究血清代谢物的预分析稳定性，本研究提供了有价值的见解，可帮助其他研究人员规划和解释猫血清代谢组学研究。
