Alice Watson: Determination of Multi-Steroid Profiles of Cats With Hyperaldosteronism or Other Diseases: A Retrospective Study

Primary hyperaldosteronism (PHA) involves excessive aldosterone secretion that is independent of normal physiological regulation by the renin-angiotensin system, with suppression of renin activity due to the resulting extracellular volume expansion [1, 2]. By contrast, secondary hyperaldosteronism arises when activation of the renin-angiotensin system results in high renin activity; this stimulates aldosterone secretion. PHA has been identified in cats as a result of unilateral disease (either benign or malignant adrenal neoplasia) [3] or bilateral disease (bilateral adrenal hyperplasia or tumors) [3, 4].

Immunoassays are commonly used to measure aldosterone concentrations in feline clinical samples. Immunoassays are considered less reliable than mass spectrometry because antibody cross-reactivity and matrix effects often lower specificity [5]. Antibody binding kinetics can limit immunoassay sensitivity [5], especially where other steroids are present in excess and can compete with antibody binding. Mass spectrometry-based methods are therefore often considered preferable due to higher specificity [5], and the possibility for simultaneous quantification of multiple steroid hormones [6]. Due to steroid hormones having similar structures and molecular weights resulting in similar mass to charge ratios (m/z), steroids require separation to facilitate differential detection. This can be achieved with liquid chromatography preceding tandem mass spectrometry, facilitating the quantification of multiple steroids in a single low-volume sample [6]. Targeted steroid metabolome analysis in humans has identified signature steroid profiles associated with disorders of steroid biosynthesis and metabolism, and provided insights into the pathophysiology of adrenal disease [7]. Steroid profiles have not been reported in the peer-reviewed literature in species with a single CYP11B enzyme for both aldosterone and cortisol synthesis, such as cats [8].

Renal disease impairs the quantification of aldosterone by immunoassay in human samples due to the accumulation of polar metabolites of aldosterone, which can cross-react with the antibodies used in immunoassays [9-11]. PHA usually arises in older cats, and as chronic kidney disease is a common condition within this population, we hypothesized that renal disease can falsely elevate the quantification of aldosterone by immunoassays. Furthermore, multiple steroid excesses, including progesterone [12], cortisol [13], estradiol [14], and corticosterone [15], occur in a subset of cats with hyperaldosteronism using immunoassays.

To date, no studies have directly compared immunoassay and mass spectrometry quantification of aldosterone (and other steroids) in cat blood samples. This study aimed to compare quantification of aldosterone by radioimmunoassay (RIA) and liquid chromatography tandem mass spectrometry (LC–MS/MS) and determine whether azotemic chronic kidney disease is associated with discrepant results for aldosterone quantification by RIA and LC–MS/MS. In addition, in an exploratory study, multi-steroid profiles of cats with PHA were compared with two groups of cats without overt PHA, subdivided into low-normal and high aldo groups based on aldosterone concentrations quantified by RIA.

2 Materials and Methods

2.1 Case Selection

This was a retrospective study using LC–MS/MS on residual serum or plasma samples from cats that [1] had documented overt PHA based on hyperaldosteronemia with otherwise unexplained hypokalemia and a unilateral adrenal mass (PHA group, n = 6); or [2] that had previously undergone serum or plasma aldosterone quantification by RIA for another study. These cats were selected from a longitudinal health monitoring program and had not had abdominal imaging. A large number of samples were available; samples for LCMS/MS were selected to cover a range of aldosterone and creatinine concentrations. These were sub-divided into those that had aldosterone concentrations above the RIA reference interval (high aldo group, n = 6) or within or below the reference interval (low aldo group, n = 15). Systemic hypertension was defined according to the local clinic protocol in place at the time of the case being managed, and was of sufficient severity to justify the prescribing of amlodipine. Cats were classified with azotemic chronic kidney disease (CKD) if the plasma creatinine was over the upper limit of the laboratory reference interval 177 μmol/L (2.0 mg/dL) with a concurrent urine specific gravity less than 1.035, or if creatinine was over 177 μmol/L on two occasions at least 2 weeks apart. Cats were classified as hyperthyroid if they were receiving medication for hyperthyroidism (thiamazole). Samples were only included in the analysis if the hyperthyroidism was clinically controlled and their total thyroxine was below the upper limit of the laboratory reference interval (55 nmol/L or 4.27 ng/dL) at the time of sampling.

Samples for the low and high aldo group cats were collected from cats seen in two first opinion practices in central London (People's Dispensary for Sick Animals, Bow and Beaumont Sainsbury Animal Hospital, Camden) between 2001 and 2021. All aldosterone measurements from these cats were performed in a single diagnostic reference laboratory (Michigan State University Veterinary Diagnostic Laboratory, USA), for which the upper limit of the reference interval was 388 pmol/L. Radioimmunoassay was performed on different aliquots of the same blood sample to those subsequently analyzed by LC–MS/MS. Biochemical data (creatinine, potassium) was obtained from a single reference laboratory (Idexx laboratories, Wetherby, UK) at the same time point.

PHA samples were obtained from cats seen in the above longitudinal study (n = 2) and cats seen in several different referral hospitals: Queen Mother Hospital for Animals (Royal Veterinary College, Hatfield, UK, n = 2), Cummings School of Veterinary Medicine (Tufts University, MA, USA, n = 1) and Pieper Veterinary (Pieper Veterinary, CT, USA, n = 1). Some cats were managed with surgical adrenalectomy, while others were managed medically. Biochemical data reported are from the closest available date to the collection of the sample used for LC–MS/MS but was not necessarily contemporaneous, and the biochemical results (including aldosterone quantification by RIA for three cats) were from local laboratories, so methodologies varied. PHA cases could receive potassium supplementation at the time of sampling. Methods of blood pressure measurement for cats in the PHA group from these referral hospitals were not standardized.

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