Jill Beech1, Raquel M. Walton2, and Melissa Blauvelt3 1 School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, USA 2 IDEXX Laboratories, Inc., Langhorne, PA, USA 3 IDEXX Laboratories, Inc., Worthington, OH, USA ACTH, adrenocorticotropic hormone; alpha‐MSH, alpha‐melanocyte stimulating hormone; DST, dexamethasone suppression test; fT4D, free thyroxine dialysis method; fT4, free thyroxine; fT3, free triiodothyronine; L–T4, levothyroxine sodium; ODST, overnight dexamethasone suppression test; PPID, pituitary pars intermedia dysfunction; PI, pars intermedia; PTU, propylthiouracil; rT3, reverse T3; T3, triiodothyronine; T4, thyroxine; TT3, total triiodothyronine; TT4, total thyroxine; TRH, thyrotropin‐releasing hormone; TSH, thyroid‐stimulating hormone (thyrotropin). Equine endocrinology research over the past few decades has contributed greatly to our current understanding of mechanisms, triggers and disease manifestations, and it will likely remain a very active area of ongoing investigation. The intentions of this chapter are to characterize selected equine endocrinopathies and to provide a brief overview of these diseases and the diagnostics that are currently available. The hypothalamus functions as the interface between the nervous and endocrine systems. The pituitary gland interacts closely with the hypothalamus and orchestrates the signaling needed for initiating and terminating many endocrine functions. Pituitary pars intermedia disease was previously known as equine Cushing’s disease. The disease is now referred to as PPID to more accurately reflect the pathophysiology of the disease which, unlike Cushing’s disease, is not typically characterized by hypercortisolemia. An abnormal haircoat is considered the most common and specific clinical presentation of PPID (Figure 11.1) [1]. Hypertrichosis or other changes in the haircoat (e.g., retention of guard hairs; failure to shed; lightening of color), laminitis, and muscle wasting are characteristic clinical manifestations of PPID. A recent study of 301 laminitic horses identified PPID in 38% [2]. Other clinical findings can include obesity, polyuria/polydipsia (PU/PD), abnormal hidrosis, and lethargy [3]. Pituitary pars intermedia dysfunction occurs in horses, ponies, and donkeys. In horses, it is usually a disease seen in patients over 15 years of age, but has been documented in horses as young as 8 years. It is believed to occur in approximately 3% of the entire equine population and in approximately 20–30% in horses over the age of 20 years [3, 4]. No predisposing factors are known except for age. The general pathogenetic mechanism of PPID is rooted in the loss of periventricular dopaminergic neurons located mostly in the hypothalamus, which serve as inhibitory regulators for the pars intermedia [5]. With the loss of tonic inhibition, there is hyperplasia and hypertrophy of the pars intermedia with either a single macroadenoma or multiple microadenomas with concomitant increases in the production of pars intermedia hormones such as ACTH, alpha‐melanocyte stimulating hormone (α‐MSH), corticotropin‐like intermediate lobe peptide (CLIP), and proopiomelanocortin (POMC) peptides, amongst others [1]. Unlike ACTH release by corticotropes of the pars distalis, ACTH release by melanotropes in the pars intermedia does not stimulate adrenal hyperplasia or cortisol production and this seems to be the reason that hypercortisolemia is not a common finding in these patients [6]. Figure 11.1 (Left panel) PPID horse showing poor coat shedding. Source: Photo courtesy of Dina Duplantis DVM. (Right panel) Abnormal coat in a horse with PPID. Source: Photo courtesy of Cathy Lombardi DVM. Over the years, a number of endocrine tests have been introduced as diagnostic tests for PPID. Although horses with PPID may have hyperglycemia, hypertriglyceridemia, and other changes in serum biochemical analysis, and measuring nonhormonal blood components may help in managing individual patients with PPID, the data are not specific or sensitive for diagnosis. A leading group of equine endocrinology experts (the Equine Endocrinology Group; https://sites.tufts.edu/equineendogroup) has generated recommendations regarding the diagnosis and treatment of PPID. This chapter presents the individual hormone tests that most commonly have been used to diagnose PPID, with emphasis on those currently accepted as the most accurate by the Equine Endocrinology Group. It is important to remember that most of the tests have been evaluated in small populations, and it is possible that they may not represent large diverse equine populations in different geographic regions. Plasma cortisol concentration is usually measured by radioimmunoassay (RIA) (Diagnostic Products Corp., Los Angeles, CA) or chemiluminescent immunoassay (Immulite, Siemens, Los Angeles, CA). Concentrations are affected by stress, including transport, exercise, stage of pregnancy, drugs, and disease states such as colic [7–10]. Cortisol concentrations in donkeys are reported to be similar to those of horses [11]. Normal horses may have diurnal variation with higher concentrations in the morning than afternoon but this variation can be eliminated if a horse is moved to a novel environment [12]. In a study of 50 healthy horses, 64% had less than a 30% difference between the a.m. and p.m. cortisol concentrations [6]. A lack of rhythmicity has also been reported in horses with various health problems. Basal cortisol concentrations and diurnal variation do not reliably distinguish between normal horses and those with PPID; horses with PPID may have basal cortisol concentrations that are lower than those in normal horses [13–15]. These tests are not appropriate for diagnosis of PPID. Salivary cortisol concentrations measure free, not total, cortisol concentrations. Free cortisol is believed to be metabolically active while protein‐bound cortisol is not. Saliva contains only free cortisol and has been examined to determine if free levels in equine patients correlated with serum levels in response to ACTH. Although the study population was very small (five horses), and did not include patients with endocrine disease, free salivary and total serum cortisol ratios appeared to correlate [16]. A recent study showed measurement of free cortisol increased in horses with PPID, equine metabolic syndrome (EMS), obesity, and hyperinsulinemia [17], but there are insufficient studies to determine whether a high free cortisol is useful in identifying specific endocrine disorders. At present, their measurement is not recommended as a diagnostic test for PPID. The urinary cortisol concentration and the cortisol: creatinine ratio have been reported to be higher in horses with PPID compared to normal horses [18, 19]. Reports comparing seven healthy normal horses and seven with PPID indicated a 100% sensitivity for the urinary corticoid:creatinine ratio in horses, with ratios greater than 16 × 10−6 seen only in horses with PPID; however, there were also some false‐negative test results [19]. Another report comparing clinically normal horses with horses with PPID and with dysautonomia found no difference in the ratios between the groups, but reported that a diagnostic cut‐off in the cortisol:creatinine ratio of ≥6.9 × 10−6 gave positive results in 3/12 healthy horses, 12/13 horses with PPID, and 7/8 horses with dysautonomia. An increase in the cut‐off value to >12.5 × 10−6 (the highest ratio in the normal animals) resulted in positive classification in 8/13 horses with PPID and 6/8 horses with dysautonomia [18]. The low specificity and sensitivity limit the value of the test in diagnosing PPID. The test has not become widely used, especially with the availability of plasma ACTH assays, which are more sensitive and specific. Administering ACTH elicits cortisol release from the adrenal cortex, the magnitude of which depends on the number of adrenal cortical cells and their activity [20, 21]. Responses in horses with PPID have been variable. Although PPID horses as a group have been reported to have a greater response than a group of normal horses, the test does not determine whether an individual horse is affected and the test is not appropriate for diagnosing PPID. For many years, the overnight dexamethasone suppression test (ODST) was considered the gold standard for diagnosing PPID. Administration of dexamethasone in healthy horses suppresses ACTH secretion, resulting in decreased cortisol concentrations. However, both false‐negative and false‐positive tests have been reported and repeated testing can yield varying responses in the same horse. Positive ODST results have been reported in clinically normal horses between May and October, and abnormal cortisol responses have been reported in normal and in previously laminitic ponies in all seasons except spring [13, 22–24]. Individual horses with PPID can have inconsistent test responses. For these reasons and because of the current availability of an ACTH assay, this test is not advocated as a primary test for diagnosis of PPID. The potential link between the dexamethasone suppression test (DST) and laminitis is of concern to some. Although the test has been widely used without apparent problems, the risk has not been adequately studied. The TRH stimulation test was originally used based on the premise that TRH stimulates the release of ACTH; cortisol was initially measured because of a lack of any commercially available ACTH assay [14]. The TRH stimulation test is based on the observation that horses with PPID have a 30–50% increase in serum cortisol concentration following administration of TRH, whereas normal horses do not respond [14]. The initial report of a significant increase in cortisol from baseline following TRH administration was attributed by other investigators to the lower baseline level of cortisol in the PPID horses, and a diminution in response was later reported in PPID horses with high baseline cortisol concentrations [25, 26]. The validity of this test was questioned and it is not considered appropriate for PPID diagnosis. The DST/TRH administration test was reported to have a high specificity and sensitivity in differentiating between normal horses and those with PPID [15, 25]. In this test, blood is obtained before dexamethasone administration (40 μg/kg IV), a blood sample is obtained three hours later, and 1 mg of TRH is administered intravenously; blood samples are then obtained at 15, 30, 45, 60, 90 minutes, and 21 hours after TRH administration. The test is considered positive if (i) plasma cortisol concentration is ≥1 μg/dL (10 ng/mL) at 24 hours, or (ii) plasma cortisol concentration is increased ≥66% above the three‐hour baseline 30 minutes after TRH. A comparison of 17 PPID and 25 clinically normal horses with no pituitary pathology reported a sensitivity of 88%, specificity of 76%, positive predictive value of 71%, and negative predictive value of 90%. Two out of 17 PPID horses had a negative test and 6/25 normal horses had a positive test [15]. Currently, this test is not recommended for PPID testing. With the availability of commercial laboratories measuring ACTH, measuring basal plasma ACTH concentrations has become widely used to test for PPID. Normal reference interval depends on the laboratory and methodology. Both RIAs and chemiluminescent immunoassays (CIAs) are used for measurement of ACTH concentration in horses, but one study showed poor agreement and only moderate correlation between a commercial RIA and CIA [27]. Thus, ACTH measurements using different methodologies should not be considered interchangeable. The chemiluminescent immunoassay (Immulite, Siemens, Los Angeles, CA) is commonly used by commercial laboratories in the United States and current cut‐offs recommended for diagnosis of PPID are based on ACTH values obtained using this assay (Figure 11.2). Veterinarians should determine the basis for the reference interval of the laboratory they expect to use. In the US, generally the same reference interval (9–35 pg/mL) is used for both horses and ponies. One study reported ACTH concentrations were more likely to exceed the reference interval in ponies than in horses in late summer/autumn, which is in contrast to a study in England on ponies where ACTH concentrations were generally within the reference interval in summer but above reference range in winter [28, 29]. Normal mammoth donkeys (n = 45) tested in May/June were reported to have ACTH concentrations that ranged from 36 to 115 pg/mL, which was higher than the laboratory’s normal horse range (11.9–25.5 pg/mL) [11]. Larger numbers of donkeys in other geographic locations should be tested to further clarify normal ranges. Variable seasonal increases in basal ACTH concentrations in both ponies and horses have been reported from various geographic locations [23, 24, 30–34]. Concentrations can exceed the reference interval from mid‐August to mid‐October in normal horses and ponies, particularly in the latter. In one study on clinically normal equids, ACTH concentrations sometimes exceeded 200 pg/mL, and in another study several ponies had ACTH concentrations greater than 65 pg/mL between August and mid‐October [23, 29]. A study in the UK on circannual variation in ACTH concentrations in normal equids (n = 156) and those clinically suspected to have PPID (n = 941) suggested a normal upper reference value of 47 pg/mL between August and October and 29 pg/mL from November to July [30]. Another UK study with a smaller group of animals suggested an appropriate cut‐off value in March and June of 40–50 pg/mL with a value almost doubled in September and December [32]. As the same CIA was used in these studies, albeit in different laboratories, different populations probably explain the differing results amongst studies. As ACTH levels vary with season, season‐dependent reference intervals are determined. Better sensitivity and specificity are reported during nonfall months (80% sensitive and 83% specific) and are recommended for testing, but reference intervals for autumn months are established [35]. Current cut‐offs recommended by the Equine Endocrinology Group are different for fall and nonfall months (see Fig ure 11.2). Although basal concentrations of ACTH can be useful for diagnosing PPID, false‐negative tests can also occur [13, 29]. In one study in the UK, 556 of 1497 plasma samples submitted from untreated horses suspected to have PPID were within the reference interval [30]. Also, some horses with PPID may have basal ACTH concentrations that vary quite widely within a very short time period and there appears to be greater variability when concentrations are high [13, 29]. When measuring basal ACTH concentrations, collection of paired sampling did not improve sensitivity or specificity of the test [36]. If a horse suspected to have PPID has normal basal plasma ACTH concentrations, measuring the ACTH concentration change in response to TRH administration is advised (see Fig ure 11.2). Figure 11.2 Schema for PPID diagnosis using the TRH stimulation test measuring plasma ACTH. The TRH stimulation test measuring plasma ACTH following intravenous administration of TRH is currently recommended for diagnosis of PPID in horses with early clinical signs or equivocal resting ACTH results (see Fig ure 11.2). Horses can eat hay prior to testing, but no grain within 12 hours of the test. It is recommended that horses less than 250 kg receive 0.5 mg and horses greater than 250 kg should receive 1.0 mg intravenously (https://sites.tufts.edu/equineendogroup/files/2019/12/2019‐PPID_EEGbooklet.pdf). Blood should be collected into plastic or silicon‐coated glass EDTA tubes and refrigerated prior to plasma separation. It is currently recommended to spin blood and collect plasma within eight hours of collection, although a recent study demonstrated ACTH concentrations remained stable when collected in EDTA and refrigerated prior to plasma separation for up to 36 hours [37]; plasma may also be frozen. A preinjection blood ACTH sample should be collected and then a second sample for ACTH measurement collected 10 minutes after TRH administration. The magnitude of the increase in plasma ACTH at 10 minutes after stimulation is an indication of the hormone content stored in both the pars intermedia melanotropes and pars distalis corticotropes [38]. Measuring TRH‐stimulated ACTH concentrations has been shown to be useful in the identification of PPID and pituitary hyperplasia, including in some subclinically affected horses [13, 39]. However, because of the large variability in TRH response among horses in the fall, seasonal reference intervals may not be useful in the diagnosis of PPID. To date, there are insufficient data to permit establishment of accurate cut‐offs for the diagnosis of PPID in the fall and TRH stimulation testing is recommended in nonfall months. Domperidone is a dopamine (D2) receptor antagonist and its administration should release ACTH and α‐MSH secreting cells (melanotrophs) from dopaminergic inhibition, resulting in increased concentrations of these hormones in the blood. An advantage of using domperidone is that its administration PO is approved for treating mares with agalactia and it is more readily available than TRH. Disadvantages are variable blood levels, potentially due to altered gastrointestinal absorption, delayed gastric emptying, and variable food within the stomach. Both age and pituitary pathology have been reported to correlate with domperidone response [40]. Horses with PPID were reported to have greater increases in ACTH concentration compared to normal horses four and eight hours following administration of doses ranging from 1.25 to 5 mg/kg domperidone PO [40, 41]. An increase ≥2‐fold basal concentrations was considered a positive response and it was suggested that the basal and the four hours postdomperidone administration samples were best for differentiating between PPID and clinically normal horses [41]. In another study, a ≥2‐fold increase in ACTH concentration was seen in 3/15 clinically normal and 4/12 PPID horses at two hours and in 4/16 clinically normal and 3/12 PPID horses at four hours, leading the authors to conclude that the test did not appear as accurate as measuring ACTH or α‐MSH response to TRH administration [39]. If one looked at ACTH concentrations >36 pg/mL as a cut‐off for diagnosing PPID, administering domperidone did not increase sensitivity or specificity for making the diagnosis above using baseline ACTH concentrations [39]. Further evaluation of the test using larger numbers of horses is needed to determine its clinical value, but currently the test is not recommended (https://sites.tufts.edu/equineendogroup/files/2019/12/2019‐PPID_EEGbooklet.pdf). α‐MSH is considered a more specific marker of pars intermedia secretion than is ACTH as it is secreted by the melanotropes of the pars intermedia, whereas ACTH is secreted by both pars intermedia melanotropes and pars distalis corticotropes [38]. The assay is not commercially available to date; however, as this may change, information on its use as a marker for PPID follows. Studies cited in this chapter reporting its use have quantified the hormone using a RIA (American Laboratory Products Co., Windham, NH). A positive correlation has been found between α‐MSH and obesity/body mass index in healthy horses greater than 10 years of age, although there was huge individual variation [42]. Concentrations also rise significantly in autumn [33, 43]. As the pars intermedia (PI) area, PI:total pituitary ratio, and total pituitary area increase in the fall, the observed increase in α‐MSH is not unexpected [44]. A greater seasonal effect was reported for α‐MSH than for ACTH [6, 24, 29, 31]. Although greater increases were reported in ponies than in horses in one study, reports are inconsistent and geographic location and body condition scores could have affected results [29, 44]. It is apparent that the range of α‐MSH concentration that is considered normal may vary geographically and potentially with the populations being studied. Stabling does not appear to influence seasonal changes [29, 33]. α‐MSH concentration does not appear to be affected by circadian rhythm [43]. Reports comparing seasonal increases in α‐MSH with ACTH have varied, and both geographic location and breed appear influential [29, 33]. Whether α‐MSH appears superior to ACTH for diagnosing PPID appears unresolved at this time as reports have varied regarding comparative sensitivity and specificity [29, 33, 35, 39]. Increases in both ACTH and α‐MSH concentration after TRH administration have been documented to be higher in horses with PPID compared to normal horses and can identify horses with PPID that have normal basal hormone concentrations [13, 29, 38, 39]. The protocol is to obtain two baseline plasma samples (5–10 minutes apart), administer 1 mg TRH intravenously, and then obtain a sample 30 minutes later. Although additional samples can be obtained (e.g., at 15 and 45 minutes), sampling at 30 minutes appears to be sensitive and specific for differentiating between clinically normal horses and those with PPID using a cut‐off of <36 pg/mL for normal ACTH concentration and ≤50 pmol/L for α‐MSH concentration [29, 39]. Although concentrations of both hormones increase after administration of TRH, in normal horses they have usually returned to baseline by 30 minutes after TRH administration. In one study on 53 clinically normal horses and 25 horses with PPID, at baseline 10/60 tests from normal horses and 26/37 tests from PPID horses had ACTH concentrations >36 pg/mL; at 30 minutes post TRH administration, these respective numbers were 16/60 and 36/38. Numbers of clinically normal horses with ACTH >36 pg/mL were decreased when the group was limited to horses without pituitary histological changes; ACTH >36 pg/mL was seen in only 1/23 baseline and 2/23 30‐minute samples. For α‐MSH, a concentration >50 pmol/L was seen in 1/30 baseline samples and 9/30 30‐minute samples in clinically normal horses and in 12/18 baseline samples and 18/18 30‐minute samples in PPID horses. In the clinically normal horses without pituitary changes, no baseline samples and only 1/15 30‐minute samples had α‐MSH >50 pmol/L [39]. It is unknown whether the TRH stimulation test will identify horses with subclinical PPID that potentially might benefit from early dopaminergic treatment prior to onset of overt signs of PPID. The advantages of measuring α‐MSH or ACTH response to TRH compared to measuring the cortisol response to the combined DST/TRH test are that the hormones of interest are being measured, fewer samples over a shorter period of time during one patient visit are needed, and dexamethasone administration is avoided. Results from one study evaluating cortisol response to the combined DST/TRH test and another evaluating ACTH response to the TRH test showed 15/17 PPID horses and 6/25 normal horses (with no PI hyperplasia or adenomas) had a positive response to the former test and 36/38 tests in PPID horses and 1/23 tests in clinically and pathologically normal horses had a positive response to the latter test [15, 39]. However, as different populations were included in each of these studies, further studies would be needed to adequately compare the two tests. Chemical‐grade TRH has been used in published reports of the test, although it is not approved for this use. Acquisition, storage, and preparation of TRH may limit its use among veterinarians. Most studies have not reported side‐effects, but minor transient to generalized moderate muscle trembling for several minutes after administration of TRH has been seen in a few horses. This response was inconsistent when the test was repeated in the same horses, and the cause of the trembling remains speculative. Transient licking, yawning, and sometimes flehmen have been seen in a few horses [29, 39]. In summary, currently the best screening test for PPID is measurement of basal ACTH concentration in horses with moderate to severe clinical signs of PPID, and TRH stimulation testing in horses with equivocal signs or an equivocal ACTH concentration but appropriate clinical signs (see Fig ure 11.2). Adiposity associated with insulin resistance has been reported in 15–30% of horses with PPID and hyperinsulinemia has been reported in some horses with PPID [6, 33]. One study reported that although insulin concentrations differed between normal horses and those with PPID, hyperinsulinemia was rare [45]. Normal ponies were reported to have more variable concentrations than horses and more individual clinically normal ponies had insulin concentrations above the reference interval (24/48 tests in six normal ponies versus 0/48 tests in 14 normal horses); however, this could be associated with the greater body condition score in the ponies [29]. When evaluating donkeys, it is important to know that they are reported to have lower insulin concentrations than horses. In one study on 45 healthy donkeys, all but five had insulin concentrations below the normal horse reference limit [11]. Nonobese donkeys were reported to have lower insulin concentrations than nonobese horses. Concentrations were significantly higher in obese donkeys and those with a history of laminitis or current laminitis compared to nonobese donkeys. Veterinarians evaluating donkeys should ascertain whether the laboratory they use has reference intervals for donkeys. There has been growing interest in better defining hyperinsulinemia and equine metabolic syndrome (see Chapter 9) and investigating the relationship with PPID. It may be useful to monitor insulin concentrations in horses as high concentrations have been predictive for the development of laminitis and low insulin concentrations have been associated with improved survival [46, 47]. Factors such as diet can explain hyperinsulinemia and insulin resistance (see Chapter 9), and this combined with highly variable concentrations throughout the day complicates interpretation of values from single samples. Despite very few documented cases of thyroid dysfunction in horses and the unreliability of single measurements of thyroid hormones, basal serum concentrations of total triiodothyronine (TT3) and total thyroxine (TT4) are frequently evaluated by veterinarians. This section provides information on tests that have been used to evaluate thyroid function and factors that can influence results. There appear to be few studies documenting the prevalence of primary thyroid dysfunction in horses. Naturally occurring hypothyroidism is reported in foals and there are several case reports of mature/aged horses with hyperthyroidism [48–51]. Low thyroid hormone concentrations have been measured in horses with various conditions in the absence of evidence of hypothyroidism. Although hypothyroidism has been implicated as a contributing cause of laminitis, it should be noted that no evidence has been found to support measurement of thyroid hormones as a predictor of pasture‐associated laminitis in horses, and horses with experimentally induced hypothyroidism have not developed laminitis [52–56]. Horses that have been thyroidectomized have shown various clinical signs, most frequently haircoat abnormalities and decreased tolerance to cold. In younger animals, stature is affected. Mares can continue to show normal estrous cycles, conceive and deliver normal foals, and although stallions have decreased libido, they are fertile [55, 57, 58]. Propylthiouracil (PTU
11
Endocrine Evaluation
Abbreviations
11.1 Pituitary Pars Intermedia Dysfunction (PPID)
11.1.1 General Overview
11.2 Testing for PPID
11.2.1 Cortisol Concentrations
11.2.1.1 Baseline Cortisol Concentration
11.2.1.2 Salivary Cortisol Concentrations
11.2.1.3 Urinary Corticoids: Creatinine Ratio and Cortisol
11.2.1.4 ACTH Stimulation of Endogenous Cortisol Concentration
11.2.1.5 Dexamethasone Suppression of Cortisol Concentrations
11.2.1.6 Thyrotropin‐Releasing Hormone (TRH) Stimulation with Measurement of Cortisol
11.2.1.7 Combined Dexamethasone Suppression and TRH Stimulation Test
11.2.2 Adrenocorticotropic Hormone (ACTH) Concentrations
11.2.2.1 Basal Plasma ACTH Concentration
11.2.2.2 TRH Stimulation with Measurement of ACTH
11.2.2.3 Domperidone Stimulation of ACTH
11.2.3 α‐MSH Concentrations
11.2.4 ACTH and α‐MSH Concentration Responses Following TRH Administration
11.2.5 Insulin Concentrations
11.3 Testing Thyroid Function in Horses
11.3.1 Thyroid Dysfunction
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