Biochemical Signatures in Growth Hormone Deficiency: a Pilot Study Using Metabolomics Approaches by Direct Infusion-mass Spectrometry

Purpose: The discovery of biomarkers to detect growth hormone deficiency (GHD) and monitor growth hormone replacement therapy (GHRT) remains challenging. Among “omics” technologies used for screening biomarkers, metabolomics stands out as a powerful tool for large-scale identification and quantification of small molecules present in biological matrixes. Metabolomic profiles allow us to infer the phenotypic state; therefore, metabolomics is a great ally in investigating biomarkers and understanding biological processes. Methods: In this study, global metabolomics (N = 39; range scan 50-600 m/z) and lipidomics (N = 36; range scan 50-1500 m/z) approaches were performed by high-resolution direct infusion-mass spectrometry. Partial least-square discriminant analysis (PLS-DA) models were used for data-driven diagnosis of GHD and evaluation of GHRT (VIP score >1.5). Cross-validation, permutation test, and ROC curves confirmed the predictive accuracy of PLS-DA models. The features were annotated using accurate mass measurements matched against the metabolomics database. Results: Data analysis revealed changes in the class of proteinogenic/glycogenic acids, carnitines, n-acyl-amines, unsaturated fatty acids, and sulfur amino acids, and pathway analyzes revealed changes in glycerophospholipid metabolism. Regardless of GH therapy, GHD individuals remain with changes in lipids and amino acids compared to healthy control. Conclusion: GHRT influences the metabolism status of GHD patients in order to compensate for dysregulations caused by GHD. The data has corroborated the action of GHRT and indicated new potential biomarkers for treatment follow-up.

PSV-B-17 Additional dietary choline increases serum metabolites as determined by direct infusion mass spectrometry in post-gonadectomy kittens

This study aimed to investigate changes in serum metabolites using direct infusion mass spectrometry (Di-MS) after 12 weeks with or without additional choline in growing, post-gonadectomy kittens. The present study was approved by the University of Guelph Animal Care Committee (AUP#4118). Intact, male kittens (3-months old; n = 15) were fed a base-diet to growth requirements (3310mg choline/kg DM) over an 11-week acclimation. Kittens were gonadectomized (week 1) and assigned to base-diet (n = 7) or base-diet with 300mg/kgBW0.75 additional choline (n = 8) and fed to mimic ad libitum food intake (3 meals totaling thrice daily requirements) for 12-weeks. Fasted serum samples were obtained and analyzed for 130 metabolites via Di-MS (week 0 and 12). The proc GLIMMIX procedure (SAS) was used to analyze differences between groups as a repeated measure. Additional choline resulted in higher serum concentrations of one-carbon metabolites such as methionine, serine, betaine, and sarcosine (P < 0.05). As expected, serum concentrations of phosphatidylcholines increased with additional choline, as did serum concentrations of lysophosphatidylcholines and sphingomyelins, compared to no additional choline (P < 0.05). There was minimal impact on acyl carnitines with a decrease in medium length acyl carnitines (P < 0.05). Additionally, there was an increase in serum concentrations of spermine, trans hydroxyproline, methionine sulfoxide, proline, and indole acetic acid, and a decrease in serum trimethylamine N-Oxide compared to control (P < 0.05). Effects of growth stage or gonadectomy may have resulted in increased serum amino acid concentrations such as asparagine, glutamine, glutamic acid, histidine, lysine, methionine, threonine, and tyrosine in both groups (P < 0.05). These results may indicate benefits for providing additional choline to post-gonadectomy kittens on one-carbon metabolism and fatty acid oxidation which could promote healthy weight and body condition. However, more research is warranted to further understand choline’s role in energy balance and its potential in prevention and treatment of feline obesity.

Intrinsic Degradation of the Type-II Antitoxin ParD from Pseudomonas aeruginosa

Type-II Toxin Antitoxin (TA) systems are regulated by differential half-lives of the resulting non-secreted proteins, such that the neutralizing antitoxin undergoes continual degradation and replenishment to maintain neutralization of its cognate toxin. Antitoxin proteins are widely reported as labile, including upon purification and in vitro storage. During the course of studies on a ParDE TA system we noted a prevalent in vitro degradation of the ParD antitoxin. In efforts to combat this for practical use in assays, we characterized parameters impacting the degradation as well as the resulting products. These revealed a mechanism likely mediated by a serine or metal-dependent protease. Using Direct Infusion Mass Spectrometry, the cleavage products were identified as an essentially intact DNA binding region of the antitoxin and with the toxin binding domain completely removed. No other species were identified in the solution, such as a contaminant that may mediate such cleavage. Therefore, while our studies revealed viable strategies to mitigate the in vitro degradation they did not identify any protease, leaving open the possibility of a potential auto-catalytic proteolytic activity of the antitoxin proteins.