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 a-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.
Article
Serum metabolome analysis in hyperthyroid cats before and after radioactive iodine therapy
https://doi.org/10.21203/rs.3.rs-3221800/v1
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published 10 Jun, 2024
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hyperthyroidism
feline
metabolomics
metabolism
Hyperthyroidism is the most commonly diagnosed endocrinopathy in cats.1 Most cases are caused by benign adenomatous thyroid tissue. A small proportion of patients (approximately 2–3%) are due to thyroid carcinoma.2,3 The underlying etiology of hyperthyroidism in cats has yet to be identified; however, published studies suggest the cause of feline hyperthyroidism to be multifactorial, with dietary and environmental risk factors likely contributing.1, 4–6 Autonomous secretion of thyroxine and triiodothyronine (T4 and T3, respectively) are responsible for the disease's clinical and metabolic manifestations.
Clinical abnormalities associated with hyperthyroidism in cats include muscle catabolism, generalized weakness, polyphagia, polyuria and polydipsia, behavioral changes, hair coat changes, cardiomyopathy, hypertension, and vomiting/diarrhea. These sequelae are detrimental to hyperthyroid cats' health and quality of life. Hyperthyroid cats often have alterations in serum biochemical markers, the most common of which is an increased alanine transferase, reflecting hepatocellular injury or enzyme induction.7 It is posited that increased metabolic demand and oxidative stress drive increases in this enzyme.
Metabolomic profiles in human hyperthyroid patients (both naturally occurring and experimentally induced) demonstrate persistent metabolic alterations despite achieving euthyroid status in some cases.8 Persistent alterations in the metabolome after successful radioactive iodine I-131 treatment in cats would be attractive targets for developing novel therapeutic interventions.
We hypothesized that hyperthyroid cats would possess distinct metabolomic signatures and that some metabolomic changes would persist after (I-131) treatment. Additionally, an untargeted metabolomics approach has the potential to identify candidate biomarkers that may aid in diagnosing challenging hyperthyroid cases. Therefore, we leveraged an untargeted metabolomic approach (Fig. 1) to (i) define the metabolomic “fingerprint” of hyperthyroid cats, (ii) identify critical metabolic pathways affected in feline hyperthyroidism, and (iii) identify persistent alterations in metabolomic profiles in cats following I-131 treatment and achievement of euthyroid state.
Animals: Cats presenting for I-131 treatment (n = 96) between 9/14/2020 and 4/29/2022 were initially eligible for enrollment. Six cats did not undergo I-131 treatment for various reasons, leaving a total of 90 eligible cats. We initially enrolled 12 cats with informed client consent. Serum was collected before I-131 treatment (HYP-T0) and at one (HYP-T1) and three (HYP-T3) months post-treatment (Fig. 1). All cats treated cats in the study were euthyroid at both follow-up time points. Five cats were excluded from the study because a complete set of samples was not obtained due to loss to follow-up (n=4) or patient mortality (n =1). Seven hyperthyroid cats (n = 3 male, n = 4 female) completed the study time points and were included in the final analysis. The control group comprised 12 cats. Age, weight, and body condition scores were similar between hyperthyroid and control cats (Table 1).
Serum Metabolites: Hyperthyroid cats sampled before I-131 treatment (HYP-T0) had a distinct metabolic signature (Supplementary Table S1) with 277 metabolites significantly different than controls (70 increased, 207 decreased). Data reduction analysis (Fig. 2A) revealed separate clustering of hyperthyroid (HYP-T0) and CON cats with intermediate phenotypes after treatment (HYP-T1 & HYP-T3). Cluster analysis demonstrated a similar pattern with clades segregating hyperthyroid cats before I-131, after treatment, and control cats separately, except for one hyperthyroid cat after treatment that segregated with controls (Fig. 2B).
Metabolic Pathways: The altered metabolic signatures of hyperthyroid cats (HYP-T0) impacted several metabolic pathways (Supplementary Fig. S1). The pathway most impacted in hyperthyroid cats was taurine and hypotaurine metabolism (Fig. 3, Supplementary Table S2). After taurine and hypotaurine metabolism, amino acid metabolic and biosynthetic pathways were the most impacted pathways (Fig. 3, Supplementary Table S2). Other notable pathways included the TCA cycle, glycerophospholipid metabolism, arachidonic acid metabolism, nucleotide metabolism, steroid and steroid hormone (cortisol) biosynthesis, and B vitamin metabolism (Supplementary Table S2).
Metabolites defining feline hyperthyroidism: Several metabolites were significantly increased or decreased greater than two-fold in hyperthyroid (HYP-T0) cats compared to healthy controls (Fig. 4A), with a larger proportion of metabolites being decreased. When comparing cats in the hyperthyroid state (HYP-T0) to euthyroid control cats, cluster analysis segregated the groups into two distinct clades (Fig. 4B). Notably, ribose, cortisol, alpha-ketoglutarate, and mevalonate increased. While creatine phosphate, transurocanate, and several lipid metabolites were decreased. Many of these metabolites also correlated with thyroxine (Fig. 4C).
We conducted random forest analysis (RFA) to identify the metabolites that best-classified groups. When all groups were included in the model, thyroxine was the best distinguishing metabolite for the model. The accuracy of the mass spectrometry measured thyroxine was compared to total T4 by radioimmunoassay, and agreement by linear and non-linear regression was excellent (Fig. 5A). After thyroxine, the best model discriminators were 1-pentadecanoyl-GPC and mevalonate (Fig. 5 B&C).
We conducted RFA and univariate biomarker analysis (receiver-operator characteristics, ROC), comparing controls to HYP-T0. Three metabolites were shared with the RFA model, including post-treatment data, creatine phosphate, mevalonate, and ribose (Figure 6A). In this RFA model, mevalonolactone was the second-best metabolite. The out-of-bag error rate was 0.105, with class errors of 0.0833 and 0.143 for hyperthyroid and control cats, respectively. Complementing the RFA results, we identified five metabolites by ROC with area under the curves of 1.0, indicating excellent discrimination between healthy control and hyperthyroid (HYP-T0) cats. Two metabolites, mevalonate, and mevalonolactone, were increased (Figure 6B), while three metabolites, creatine phosphate, 1oleol-GPE (18:1), and transurocanate, were decreased in hyperthyroid cats (Fig. 6C).
Metabolites of additional clinical interest: Three metabolites were notable due to their clinical implications, persistent alteration after treatment, or both. Hyperthyroid cats (HYP-T0) had significantly higher cortisol and cortisone levels (Fig. 7 A&B). Changes in cortisol and cortisone combined with the impacts on lipid pathways prompted us to compare cholesterol concentrations from metabolomic and biochemistry analysis. Although cortisol was in the reference range for all hyperthyroid cats in the study, hyperthyroid cats had significantly lower (P < 0.02) cholesterol concentrations measured by biochemistry and metabolomic analysis (Supplementary Figure S2 and Supplementary Table S1). Median serotonin levels, a neurotransmitter that could contribute to some clinical signs of hyperthyroidism, were higher in hyperthyroid cats (Fig. 7C). Serum levels of two metabolites of nutritional relevance, carnitine, and alpha-tocopherol, were lower (T0 vs. CON), persisting after I-131 treatment (Fig. 7 D&E). Additionally, 66 (T1 vs. CON) and 64 (T3 vs. CON) metabolite differences persisted after treatment.
We aimed to define the metabolomic signature of hyperthyroid cats and identify persistent metabolic alterations after successful treatment. Hyperthyroidism results in a hypermetabolic state leading to altered lipid, protein, and carbohydrate metabolism with increased resting energy requirements.9–11 This hypermetabolic state is assumed to be reversed once the euthyroid state is achieved. Our study revealed a severe, global alteration in the metabolome of hyperthyroid cats. Although the metabolome trended towards that of the euthyroid control group profile post-treatment, metabolites in some lipid, protein, and carbohydrate metabolic pathways remained persistently altered despite achieving a euthyroid state. This mirrors findings in experimentally induced and naturally occurring human hyperthyroidism, where alterations in metabolomic signatures persist despite returning to the euthyroid state.12–15
The metabolomic signature of hyperthyroid cats in our study corroborated altered cholesterol and steroid hormonogenesis observed in hyperthyroid humans.16–18 Key components of the mevalonate pathway, the hydroxy fatty acids mevalonate and mevalonolactone, are essential for cholesterol biosynthesis. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase synthesizes these lipids and is the rate-limiting step in cholesterol biosynthesis.19 Although hypocholesterolemia is not a reported biochemical abnormality observed in hyperthyroid cats, in the authors’ experience, cholesterol levels in hyperthyroid cats tend to be near the low end of the reference interval. Additionally, in one study, serum cholesterol concentrations generally increased in treated hyperthyroid cats.20 Our data suggest cholesterol is lower in hyperthyroid cats, although typically within diagnostic laboratory reference ranges. In humans, hyperthyroidism augments cholesterol excretion and increases low-density lipoprotein turnover, decreasing total and low-density lipoproteins and cholesterol. In contrast, high-density lipoproteins are reduced or not affected.21 In people with hyperthyroidism, cholesterol normalizes once a euthyroid state is achieved, suggesting cholesterol metabolism normalizes.22 We did not observe this in our cohort, although cholesterol homeostasis may take longer than our observation period to normalize in cats after I-131 treatment. Similarly, higher mevalonate levels in hyperthyroid cats did not return to similar levels as euthyroid controls following I-131 therapy and achievement of euthyroid state.
Hypercortisolemia has previously been documented in hyperthyroid cats, which was corroborated by our results.23,24 However, to our knowledge, no studies have evaluated cortisol levels after treating hyperthyroidism in cats. Our data indicate persistent hypercortisolemia in hyperthyroid cats following I-131 therapy and euthyroid status. Ramspot et al. assessed the adrenal function of hyperthyroid cats via the adrenocorticotropic hormone stimulation test and by measuring urine cortisol to creatinine ratios (UCCR).23 Hyperthyroid cats had significantly higher post-stimulation cortisol concentrations; however, adrenal gland size and UCCRs did not differ significantly from controls. Those authors postulated that cortisol levels should eventually normalize after successful treatment. Alternatively, another study reported significantly higher UCCRs in hyperthyroid cats than in controls.24 It was presumed that this increase was associated with stimulation of the hypothalamic-pituitary-adrenal axis due to increased clearance of cortisol associated with a hyperthyroid state. When UCCRs were measured following I-131 therapy, ratios were significantly reduced compared to baseline in the hyperthyroid cats.24 However, our data suggest that despite achieving a euthyroid state, steroid biosynthetic pathways are persistently altered for at least three months, which is supported by increased adrenal size measured by ultrasound in treated hyperthyroid cats.25
Serum total T4 is the screening test of choice for diagnosing hyperthyroidism in cats,26–29 and this was corroborated by our finding that thyroxine measured by tandem mass spectrometry was the best model predictor by RFA. However, additional biomarkers for hyperthyroidism in cats may be valuable in some cases. Increased awareness of feline hyperthyroidism in cats has led to more routine total T4 monitoring, often paired with serum biochemistry profiles, during wellness evaluations for geriatric cats. As routine screening becomes increasingly common, cats are diagnosed earlier in the disease course with less severe clinical signs are often less severe than historically described.30,31 Early screening may partly explain why approximately 10% of hyperthyroid cats overall and up to 30% of cats with early or mild hyperthyroidism have a serum total T4 within the normal reference range.26,27,29 This is typically attributed to early-stage hyperthyroidism or concurrent non-thyroidal illness.26 Free T4, T3 suppression testing, and thyrotropin-releasing hormone stimulation tests have been suggested as adjuvant tests for the diagnosis of hyperthyroidism in these cases; however, tests are not always reliable in the face of non-thyroidal illness and can be challenging to perform in the clinical setting.29,32 Alternatively, repeating serum total T4 or free T4 measurements several weeks after equivocal testing or treating patients presumptively with methimazole and monitoring response to treatment (resolution of clinical signs) are common clinical approaches.32 As such, novel biomarkers capable of discriminating euthyroid from hyperthyroid cats with non-thyroidal illness or early hyperthyroidism would be helpful. Further evaluation of mevalonate/mevalonolactone, creatine phosphate, and other candidate biomarkers is required to determine their suitability for supporting a diagnosis of hyperthyroidism in cats with concurrent non-thyroidal illness. As these candidate biomarkers correlated with thyroxine in our cohort of hyperthyroid cats, it is possible that they would also not distinguish occult cases of hyperthyroidism.
Our results identified many metabolites that defined the feline hyperthyroid state. Changes in fatty acid biosynthesis and metabolism, including the acylcarnitines, that we discovered in hyperthyroid cats are similar to reported changes in hyperthyroid people.33 Although T4 was the best model discriminator (based on random forest analysis and corroborated by biomarker analysis), other discriminant metabolites were 1-pentadacenoyl-GPC, mevalonate, ribose, creatine phosphate, alpha-ketoglutarate, trans-urocanate, 2’-deoxycytidine, and pimelate. Other noteworthy metabolite alterations include serotonin, alpha-tocopherol, and carnitine. In humans, alpha-ketoglutarate, creatine (increased in the hyperthyroid state compared to controls), and alpha-tocopherol have been documented to be significantly altered in the hyperthyroid state vs. euthyroid controls.8,13 However, many metabolites identified in this study have not been previously observed or evaluated in humans.
We identified several significant metabolites in hyperthyroid cats that reflect altered cellular energy metabolism. Lower creatine phosphate (phosphocreatine) may reflect lower muscle masses often seen in hyperthyroid cats.34,35 As creatine phosphate serves as a rapidly mobilizable reserve of high-energy phosphates in skeletal muscle, myocardium, and the brain, reduced serum levels also reflect changes in energy metabolism at these sites.36,37 In this study, post-I-131 treatment creatine phosphate levels improved but were still significantly reduced compared to controls, further indicating persistent negative energy balance in previously hyperthyroid cats. Alpha-ketoglutarate was increased in hyperthyroid cats compared to control cats, which may be consistent with an overall increase in TCA cycle activity associated with a hypermetabolic state.38 Although these levels decreased post-I-131, they were still slightly elevated compared to control cats, indicating a persistent hypermetabolic state during our observation period.
Similarly, transurocanate was reduced in the hyperthyroid state and did not return to normal levels post-I-131 treatment, further suggesting persistent alterations in the TCA cycle. Ribose was elevated in hyperthyroid cats compared to controls but did not return to normal following I-131 treatment and euthyroid state. As a vital component of DNA, RNA, ATP, ADP, and AMP, this supports altered carbohydrate and energy metabolism in hyperthyroid cats, which is corroborated by differences in fructosamine levels between healthy and hyperthyroid cats.39
Although there was some overlap in serotonin levels in hyperthyroid and healthy cats, overall, concentrations were higher in hyperthyroid cats. While increased thyroid hormone concentrations can directly explain clinical signs of hyperthyroidism, other metabolic alterations, such as this potential increase in serotonin, may also contribute to clinical signs. Higher serotonin levels could partly explain some clinical manifestations in hyperthyroid cats, including behavioral changes, vomiting, and diarrhea. Increased serotonin and cortisol may also explain heterogeneity in clinical signs among individual hyperthyroid cats.
We identified two candidate nutritional targets for cats achieving euthyroidism post-I-131, vitamin E and carnitine. The most bioactive form of vitamin E is a-tocopherol, an essential antioxidant. Vitamin E reacts with the peroxyl radicals and protects membranes from excessive oxidative damage. Lower a-tocopherol levels have been documented in hyperthyroid people.40 Hyperthyroidism is associated with increased oxidative stress, and rodent models of hyperthyroidism have demonstrated vitamin E supplementation benefits.41,42 This has been corroborated by studies in hyperthyroid humans, where vitamin E supplementation was associated with improved time to achieve euthyroid status.43 Cats have a less favorable glutathione status than dogs, making them at higher risk for oxidant injury as a species.44 This further suggests that vitamin E supplementation is likely beneficial in hyperthyroid cats. However, in one study no differences were found in vitamin E concentrations between control cats, and hyperthyroid cats before treatment or 2 months after I-131.42 Therefore, additional work is necessary to determine if vitamin E supplementation is appropriate for hyperthyroid cats. An essential participant in b-oxidation is carnitine and other acylcarnitines. Carnitine facilitates long-chain fatty acid transport into the mitochondria for b-oxidation.45 Given its metabolic role, carnitine is concentrated in skeletal and cardiac muscle and other tissues that metabolize fatty acids as an energy source.46 The significantly lower carnitine levels post-I-131 could perpetuate altered energy metabolism, less efficient lipid metabolism, and impaired b-oxidation into the euthyroid state. Therefore, carnitine supplementation in euthyroid cats following I-131 therapy may be beneficial.
This study had a modest sample size obtained from a single clinical site. The sample size was targeted based on previous metabolomic studies conducted in cats and dogs and guidelines from the metabolomic lab that conducted the analysis. Further supporting the validity of our results is the robust metabolic phenotype occurring in hyperthyroidism. Future studies with multiple sites may be interesting in evaluating geographical differences in hyperthyroid cat metabolomes. This approach might also reveal candidate location-related risk factors. Our follow-up period was relatively short. Therefore, it is possible that a longer monitoring period would disclose the normalization of more metabolic perturbations. A longer longitudinal study would be needed to determine if supplementing any of the targeted metabolic disturbances described above is (i) beneficial and (ii) required long or short-term. Finally, the nature of untargeted metabolomic analysis only provides a semi-quantitative measurement of analytes. Therefore, follow-up studies quantitatively measuring metabolites of interest are needed to validate findings from untargeted metabolomic studies like ours.
Our study defined the metabolome of hyperthyroid cats, which demonstrates significant alterations in lipid, carbohydrate, and protein metabolism. Some of these metabolomic changes persisted after I-131 treatment despite a return to euthyroidism. These persistent changes were associated with oxidative stress, cholesterol and steroid hormonogenesis, energy metabolism, and lipid metabolism. These data suggest that recovery to normal serum T4 concentrations following I-131 therapy may belie persistent metabolic derangements in successfully treated hyperthyroid cats. Further studies are needed to determine if hyperthyroid cats could benefit from additional nutritional or medical treatments to address these lasting metabolomic derangements.
Animals
We conducted this case-control study from 9/14/2020 to 4/29/2022. Hyperthyroid case cats (HYP) were enrolled from client-owned patients presented to Cornell University Hospital for Animals (CUHA) Internal Medicine Service for I-131 treatment. Cats with mild kidney disease (International Renal Interest Society Stage 1 or 2), chronic enteropathy, or cardiac abnormalities attributable to hyperthyroidism were permitted. Patients demonstrating radiopharmaceutical uptake consistent with neoplasia were excluded from the study. Any prior treatment, such as methimazole, was discontinued for at least ten days before receiving I-131 therapy. The dosage of I-131 was selected based on each patient’s serum T4 level and thyroid scintigraphy results.
Privately owned control (CON) cats were recruited from staff and students at the Cornell University College of Veterinary Medicine, targeting a similar demographic population as we obtained for the HYP group. A normal serum T4 value was required for CON group inclusion. All other inclusion and exclusion criteria for the HYP group were also applied to the CON group.
For both case and control cats, a complete blood count, serum chemistry profile, total T4, and urinalysis were performed (by the New York State Veterinary Diagnostic Laboratory) as part of routine diagnostic testing. Additionally, these parameters were rechecked in case cats, and serum was collected for metabolomic analysis at 1 month and 3 months following I131 therapy (Fig. 1).
Sample Collection and Processing
Fasted blood samples were acquired by venipuncture of peripheral veins. The phlebotomist chose the site at their discretion. In most cases, blood was collected contemporaneously with diagnostic blood sampling. Whole blood was collected into 4 ml additive-free tubes (BD Vacutainer®). After clotting (~ 15 minutes), tubes were centrifuged at 676 x g for 10 minutes at room temperature to yield serum. Serum was collected with transfer pipettes and stored in 2 ml plastic tubes. All serum samples were stored at -80°C and shipped on dry ice for analysis.
Metabolomic Profiling
Metabolon (Metabolon, Inc, 617 Davis Drive, Suite 100, Morrisville, NC 27560) conducted metabolomic profiling on serum samples as previously described. Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000), followed by centrifugation to remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and recover chemically diverse metabolites. The resulting extract was divided into five fractions: two for analysis by two separate reverse phases (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.
All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution.
The informatics system consisted of four major components, the Laboratory Information Management System (LIMS), the data extraction and peak-identification software, data processing tools for QC and compound identification, and a collection of information interpretation and visualization tools for use by data analysts. These informatics components’ hardware and software foundations were the LAN backbone and a database server running Oracle 10.2.0.1 Enterprise Edition.
Metabolon maintains a library based on authenticated standards that contains the retention time/index (RI), mass-to-charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Furthermore, biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library +/- 10 ppm, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores are based on comparing the ions present in the observed spectrum to those in the library spectrum. While there may be similarities between these molecules based on one of these factors, all three data points can be used to distinguish and differentiate biochemicals.
The Human Metabolome Database (https://hmdb.ca/) was searched for reference information on selected metabolites.
Statistical analysis
A minimum of 7–10 cases was targeted based on Metabolon’s sample size guidelines. Descriptive statistics are reported as medians and ranges. Due to the sample size, we did not conduct normality testing and applied non-parametric statistics. We compared age, weight, body condition score, and total T4 by the Mann-Whitney test. Metabolon’s initial standard statistical analyses are performed in ArrayStudio/Jupyter Notebook on log-transformed data. For those analyses not standard in ArrayStudio/Jupyter Notebook, the programs R (http://cran.r-project.org/) or JMP are used. The Welch’s two-sample t-test compared control (CON) cats to hyperthyroid (HYP) cats at each time point (i.e., CON:HYP-T0, CON:HYP-T1, and CON:HYP-T3). The Matched Pair t-test compared hyperthyroid cats between time points (i.e., HYP-T0: HYP-T-1, HYP-T0:HYP-T3, and HYP-T1:HYP-T3).
Additional statistical analyses and data visualizations (including volcano plots, random forest analysis, and biomarker analysis) were performed with MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/home.xhtml). Normalized data were log-transformed, and Pareto data scaling was applied. Fold-change and T-tests further identified significantly increased or decreased metabolites. The sparse partial least squares - discriminant analysis (sPLS-DA) method was chosen for data reduction analysis.51 Pathway analysis was conducted using the human Kyoto Encyclopedia of Genes and Genomes (KEGG) and Small Molecule Pathway Database databases. A P value of < 0.05 established significance.
CON: control (euthyroid cat) group
HYP: hyperthyroid cat group
I-131: Radioactive iodine
MS: mass spectrometry
T3: triiodothyronine
T4: thyroxine (note: we refer to thyroxine/T4 measured by mass spectrometry as thyroxine to be consistent with laboratory reporting)
RFA: random forest analysis
UCCR: urine creatinine:cortisol ratio
Ethical approval and informed consent
This study was approved by the Cornell University Institutional Animal Care and Use Committee for the use of vertebrates in research (#2014-0052), experiments were conducted following all applicable guidelines, and regulations, and is reported consistent with ARRIVE guidelines. Informed consent was obtained from all owners before enrollment of any case or control cat.
Acknowledgments
We thank Seth A. Peng for his technical support. A Cornell Feline Health Center grant generously funded this study.
Author contributions
MAB, KWS, and JPL conceived the study. MAB, MLM, JMP, KWS, and JPL coordinated case recruitment and oversaw case management. MAB, CEF, and LLB organized sample collection, storage, and other logistics. JPL and YL compiled data. JPL conducted the statistical analyses. MAB, MEP, KWS, and JPL critically assessed the data and its clinical implications. All authors contributed to the writing and approved of the final manuscript.
Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Additional Information
The authors report no competing interests.
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Table 1.
No competing interests reported.