Serum metabolome analysis in hyperthyroid cats before and after radioactive iodine therapy

Abstract

Hyperthyroidism is the most common feline endocrinopathy. In hyperthyroid humans, untargeted metabolomic analysis identified persistent metabolic derangements despite achieving a euthyroid state. Therefore, we sought to define the metabolome of hyperthyroid cats and identify ongoing metabolic changes after treatment. We prospectively compared privately-owned hyperthyroid cats (n = 7) admitted for radioactive iodine (I-131) treatment and euthyroid privately-owned control (CON) cats (n = 12). Serum samples were collected before (T0), 1-month (T1), and three months after (T3) I-131 therapy for untargeted metabolomic analysis by MS/MS. Hyperthyroid cats (T0) had a distinct metabolic signature with 277 significantly different metabolites than controls (70 increased, 207 decreased). After treatment, 66 (T1 vs. CON) and 64 (T3 vs. CON) metabolite differences persisted. Clustering and data reduction analysis revealed separate clustering of hyperthyroid (T0) and CON cats with intermediate phenotypes after treatment (T1 & T3). Mevalonate/mevalonolactone and creatine phosphate were candidate biomarkers with excellent discrimination between hyperthyroid and healthy cats. We found several metabolic derangements (e.g., decreased carnitine and α-tocopherol) do not entirely resolve after achieving a euthyroid state after treating hyperthyroid cats with I-131. Further investigation is warranted to determine diagnostic and therapeutic implications for candidate biomarkers and persistent metabolic abnormalities.

Fig 1. Methodology overview.

Serum was collected from hyperthyroid cats (n = 7) before (T = 0) and after (at T = 1 mo and T = 3 mo) I131 treatment. A single sample was collected from control cats (n = 12). Samples were stored for subsequent untargeted metabolic profiling and analysis. Created with Biorender.com.

Fig 2. Cluster analysis of feline metabolome data.

(A) Sparse Partial Least Squares—Discriminant Analysis (sPLS-DA) plot. Dots indicate individual cats, and shading highlights clustering. (B) Heatmap of 25 metabolites with the smallest p values, segregated into clades based on patterns of relative magnitude. Colors indicating groups are provided in legends. CON = healthy control cats, HYP-T0 = hyperthyroid time 0, HYP-T1 = hyperthyroid cats 1-month post-I131 treatment, and HYP-T3 = hyperthyroid cats 3 months post-I131 treatment.

Fig 3. Pathway analysis.

Topology maps of affected Kyoto Encyclopedia of Genes and Genomes (A) and Small Molecule Pathway Database (B) pathways impacted in serum metabolomes of hyperthyroid cats (n = 7) versus healthy control cats (n = 12). Highly significant and impacted pathways are annotated. Each circle represents a pathway, circle sizes correspond to pathway impact, and the color gradient corresponds to pathway significance.

Fig 4. Comparison of healthy controls to hyperthyroid state.

(A) Volcano plots of significant metabolites in hyperthyroid cats (n = 7) versus healthy control cats (n = 12). Metabolites significantly (P < 0.05) increased (red) or decreased (blue) greater than 2-fold are depicted by single dots. (B) Heatmap of 25 metabolites with the smallest P values, segregated into clades based on patterns of relative magnitude. Colors indicating groups are provided in legends. CON = healthy control cats, HYP-T0 = hyperthyroid time 0. (C) Correlation plot of metabolites best correlated to thyroxine.

Fig 5. Model analysis in hyperthyroid cats.

(A) Comparison of total T4 values (measured by radioimmunoassay) vs. thyroxine values obtained through metabolomic profiling. (B) Random Forest Analysis variation importance plots for serum metabolites are depicted. (C) Individual plots of the top nine metabolites identified by random forest analysis. CON = healthy control cats, HYP-T0 = hyperthyroid time 0, HYP-T1 = hyperthyroid cats 1-month post-I131 treatment, and HYP-T3 = hyperthyroid cats 3 months post-I131 treatment.

Fig 6. Features distinguishing cats in a hyperthyroid state from healthy cats.

(A) Random forest analysis was conducted on metabolomic results from hyperthyroid cats before treatment (HYP-T0) and healthy controls. Univariate biomarker analysis conducted by receiver-operator characteristics identified robust novel candidate biomarkers. Mevalonate and mevalonolactone were higher (A), and creatine phosphate, 1-oleoyl-GPE (18:1), and trans-urocanate (B) were lower in hyperthyroid cats.

Fig 7. Selected metabolites of clinical interest.

Violin plots of (A) cortisol, (B) cortisone, (C) serotonin, (D) carnitine, and (E) alpha-tocopherol. Dots indicate individual cat values. CON = healthy control cats, HYP:T0 = hyperthyroid time 0, HYP:T1 = hyperthyroid cats 1-month post-I131 treatment, and HYP:T3 = hyperthyroid cats 3 months post-I131 treatment.