Plasma Adrenocorticotropin Concentration in Healthy Horses and in Horses With Clinical Signs of Hyperadrenocorticism

Plasma Adrenocorticotropin Concentration in Healthy Horses and in Horses With Clinical Signs of... denomas of the pars intermedia of the pituitary gland produce a recognized syndrome in older horses known as equine Cushing’s disease (ECD),’.’ characterized by hypercorticism secondary to adrenocorticotropin (ACTH) hypersecretion from a pituitary tumor.4.’ There are some fundamental differences between this syndrome in horses and people, including anatomic location of the pituitary tumor and proopiomelanocortin (POMC) post-translational processing.’ Clinical signs of the disease are mainly related to secondary adrenal stimulation from an excess production of adrenocorticotropin (ACTH) from the pars intermedia.”7 Wilson et a1 showed that equine ACTH may be quantified accurately with radioimmunoassay (RIA) methods using antibodies against ACTH (rabbit antiporcine ACTH antibod- i 5 plasma concentration of POMC peptides and, in e High particdar, ACTH have been reported in horses with ECD4.5 and are well documented in humans with ectopic or pituitary Cushing’s syndrome.‘ Nevertheless, as of today, the importance of plasma ACTH measurement in the diagnosis of ECD has not been critically evaluated. Variability in sample handling conditions prior to ACTH quantification has been reported to influence results both in humans’ and dogs.’ ACTH is adsorbed onto glass surfaces and is degraded by proteolytic enzymes in whole blood, as well as in plasma.’,’0 Therefore, it is recommended to collect blood in cold plastic tubes (1” to 4°C) containing disodium EDTA as an anticoagulant, to separate the plasma immediately, and to store it frozen in plastic tubes for less than 1 month. These recommendations increase the difficulty of using ACTH concentrations as a diagnostic test for the equine practitioner. At the present time, the diagnosis of ECD is made on the basis of the typical clinical signs of hirsutism, polyuriapolydipsia, muscle wasting, hyperglycemia, and chronic laminitis.’-’ In addition, hypophyseal-adrenocortical axis function may be evaluated using various tests.’.’ However, these tests are not specific for the diagnosis of pituitary dysfunction’ and they often require serial sampling, which is more difficult to apply in the field. In addition, because the use of dexamethasone has a slight risk of inducing lami- nitis,” people are often reluctant to perform dexamethasonesuppression tests, especially in horses with a history of chronic laminitis. The study had 2 main objectives: first, to determine whether plasma ACTH concentrations differed between healthy equids and horses or ponies with clinically diagnosed pituitary adenomas, as well as between normal horses and ponies. Second, to determine whether ACTH quantification in plasma was affected by blood sample handling techniques. A prerequisite to conducting the study was to validate the commercially available human ACTH RIA kit (RSL ‘*’I hACTH; ICN Biomedicals, Inc, Diagnostic Division, Costa Mesa, CA) for equine ACTH measurement. Materials and Methods ACTH RIA Validation Using Equine Plasma Assay validation was performed on pooled equine plasma. Multiple aliquots of samples containing low, medium, and high concentrations of ACTH were frozen until assayed a single time to avoid effects of thawing and refreezing. Intra-assay precision was determined by assaying replicates on 3 equine plasma pools measured in one assay. Interassay precision was determined by 10 repeated measurements of equine plasma pools with medium and high ACTH concentrations on 3 days. An equine plasma pool containing high ACTH concentration was assayed undiluted, and at 3:4, 1:2, and 1 :4 ’ From the T u f s University School of Veierinaty Medicine, North Crafton, MA. Accepted Februavy 23, 1995. Supported by a Grantfrom the Mussachusetts Horse Racing Fund. Data from this study were presented at the Twelfth ACVIM Forum in Sun Francisco, 1994. The authors thank Ms. Julie Reid ,for her technical help, Dr. William Rand and Dr. Andy Hoffman f o r the statistical analysis, and Ms. Elaine Berglund f o r typing this manuscript, Reprint requests: Laurent Couetil, DVM, Purdue University, Department of Veterinary Clinical Sciences, 1248 Lynn Hall, West Lafayette, IN 47907-1248. Copyright 0 1996 by the American College of Veterinary Internal Medicine 0891-6640/96/1001-0001$3.00/0 Journal of Veterinary lnternal Medicine, Vol 10, No 1 (January-February),1996: pp 1-6 COUETIL, PARADIS, AND KNOLL Table 1. ACTH Concentration and Cortisol Increase Factor in Control Equids and Equids With ECD of Different Age Categories ~~~~~~~~~~~~~~ Control Groups Age Categories (yr) age 5 SD ACTH pg/mL* ACTH stimt 11-15 Mean age 2 SD ACTH pg/mL ACTH stim 16-20 Mean age 2 SD ACTH pmlmL ACTH stim >20 Mean age 2 SD ACTH pg/mL (range) ACTH stim 1 1 0 Mean 5.0 19.5 4.0 13.2 20.5 2.3 18.7 12.7 3.0 25.0 21.9 ECD Groups Horses 6) = 6) 2 0.3 (n = 2) 5 1.2 (n = 5) I (n = 5) 4.0 i_ 0.3 (n = 2) I (n = 4) 1.6 2 4.3 (n = 4) t 0.7 (n = 4) t 1.6 (n = 3) 2 6 (n = 3) ? 2.2 Ponies Horses = = Ponies (n t 7.4 ( n (n (n 7) 7) (n = 1) (n = 1) 13 (n = 1) 23.0 (n = 1) 5.3 In = 1) 18 (n = 1) 70.0 (n = 1) 22.8 ? 3.4 ( n = 9) 241.7 ? 326.2 In = 9) 123.0-1,018) 3.8 2 1.8 (n = 3) 11.5 2 0.5 (n 8.9 t 2.1 (n = = 2) 2) 3.3 -t 1.5 (n = 2) 11.0 i- 1 (n = 2) 135.0 t 58.9 (n = 2) 5.4 (n = 1) 17 In = 1) 71.1 In = 1) 4.8 In = 1) 27.1 t 2.1 (n = 7) 202.3 (n = 7) 243.2 I (40.8-669.9) 3.6 -t 0.3 (n = 2) Abbreviations: n, Number of animals in each category; ACTH, adenocorticotropin; ECD, equine Cushing’s disease. * Mean Values f SD. t ACTH stim, cortisol increase factor during ACTH stimulation test. dilutions. Results were evaluated for dilutional parallelism comparing observed (measured) values with expected values. Expected values were calculated multiplying the undiluted pool ACTH concentration by the dilution factor. The correlation coefficient between observed and expected ACTH concentrations was calculated. I 3 Clinical Study All horses and ponies included in the study presented to Tufts University School of Veterinary Medicine over a 1-year period. Eighteen horses and 9 ponies were used to establish reference ranges for plasma ACTH concentrations (control group). Four age categories were represented ( < I 0 years, 11 to 15 years, 16 to 20 years, and 220 years) (Table 1). These animals had no recent history of corticosteroid or exogenous ACTH administration and were determined to be clinically normal through a physical examination, CBC, and serum biochemistry profile. A second group, or ECD group, included I 1 horses and 11 ponies with either clinical signs of Cushing’s disease or postmortem confirmation of a pituitary tumor. The clinical diagnosis of pituitary adenoma was based on signs of hair coat abnormalities (hirsutism, delayed shedding, incomplete shedding) and at least one of the following signs: chronic laminitis, polyuria and polydipsia, muscle wasting, and hyperglycemia. Nine horses and 9 ponies admitted in the ECD group based on pathognomonic clinical signs were not available for necropsy. One horse and 2 ponies with typical clinical signs of ECD had histological evidence of a pituitary adenoma. One horse with normal hair coat and no clinical signs consistent with pituitary dysfunction had histological evidence of a pars intermedia adenoma and was, therefore, included in the ECD group. Each animal was evaluated using the same protocol as the control group consisting of physical examination, CBC, and serum biochemistry profile. During blood sample collection, horses and ponies were kept quiet in a stall with water and hay available. Sample collection for CBC, biochemistry profile, ACTH concentrations, and cortisol pre-ACTH gel injection were performed simultaneously between 9 AM and noon. ACTH stimulation tests were performed in a limited number of patients because of client compliance. Serum for cortisol quantification was harvested within 30 minutes of blood collection in tubes with no additive (Monoject; Sherwood Medical, St Louis, MO) (red top) and kept refrigerated until assayed, no longer than 3 days. Blood samples for ACTH quantification were collected in EDTA glass tubes (Monoject; Sherwood Medical, St Louis, MO) (lavender top), kept chilled in iced water (temperature approximately lac), and then centrifuged at 1,500 X g at room temperature for 10 minutes, within 15 minutes of blood sample collection. The plasma was then transferred into plastic (polypropylene) tubes and stored frozen at -20°C until assayed within a week of sample collection. Plasma ACTH concentrations in frozen samples stored 1 week or more in glass tubes are significantly different from those stored in plastic tubes.’ However, this phenomenon is not significant within 90 minutes of sample collection.’ ACTH concentration was determined using a commercially available kit (RSL r2sI hACTH) for human ACTH. The kit utilized ACTH antiserum generated in rabbits with purified porcine ACTH conjugate. ACTH standard consisted of human synthetic ACTH,.,,. Similar RIAs have been used by others to quantify directly equine plasma ACTH concentration^.^^'^ ACTH measurements for each plasma sample were done in duplicate. Statistical analysis was performed using ACTH concentrations and cortisol increase factors in control and cushingoid groups as dependent variables. Independent variables were ponies versus horses. Normality of data was tested using Kolmogorov-Smirnov goodness of fit test. Dependent variables were normally distributed in each group. Equality of variance was tested using Levene’s test and showed equal variance between control and cushingoid animals. However, Levene’s test for normal versus cushingoid ponies showed unequal variance. Dependent variables (ACTH concentration) were then log transformed, resulting in equal variances across groups. Then, plasma ACTH measurements in normal ponies and horses, and in cushingoid animals were compared with each other using two-way analysis of variance (ANOVA) on the logarithm of ACTH concentration.13 Sensitivity and specificityr3of plasma ACTH concentrations in horses with a clinical diagnosis of ECD were calculated. Breakpoint values for ACTH concentration (ie, ACTH values separating ECD group from healthy animals) were chosen arbitrarily as halfway between logarithm of mean ACTH concentration for ECD and control groups. Therefore, breakpoints for ponies and horses were 26.96 and 50 pg/mL, respectively. The protocol used for ACTH stimulation test was described by Dybdal* and included collection of a blood sample to quantify basal EQUINE HYPERADRENOCORTICISM Table 2. Intra-Assay Precision for Equine Plasma Assayed 10 Times With a Single ACTH Radioimmunoassay Sample (No. of Replicates) High (10) Medium (10) Low (5) Table 3. Interassay Precision for 2 Equine Plasma Pools Assayed 10 Times, Each on 3 Different Days Day of Assay Day1 Day6 Day7 Sample Pool 1 Pool 2 Pool 1 ACTH Concentration (pg/mL) Mean i SD 117.9 2 8.40 56.9 f 5.0 22.9 t 1.3 Coefficient of Variation (%) Mean ACTH (pg/mL) i S D 126.47 5 9.8 61.31 2 2.72 130.83f 19.31 54.82 2 4.93 117.91 -t 9.38 56.94 f 4.99 Coefficient of Variation x 100 Pool 2 Pool 1 Abbreviation: ACTH, adenocorticotropin. Pool 2 Abbreviation: ACTH, adenocorticotropin. cortisol concentrations and then injecting ACTH gel (H.P. Acthar Gel [with gelatin]; Rh6ne-Poulenc Rorer, Collegeville, PA, 40 U1 mL) (1 U k g body weight) IM. A second blood sample was collected 4 hours after ACTH administration for quantification of cortisol concentration. Cortisol was quantified using a commercial solidphase RIA kit (Coat-A-Count; Diagnostic Products Corp, Los Angeles, CA) previously validated for equine serum.” To compare the magnitude of adrenal response to ACTH stimulation among horses and ponies, a cortisol increase factor was introduced. Cortisol increase factor during ACTH stimulation test was defined as the ratio of cortisol concentration 4 hours after ACTH gel administration divided by the basal cortisol concentration. Differences in cortisol increase factor between ECD and control horses were analyzed using two-sample r-test.” Effects of Sample Handling on ACTH QuantiJication A separate experiment was designed to evaluate the effects of sample holding temperature after blood collection and time interval to plasma separation on ACTH concentration. Blood samples were collected by jugular venipuncture from 5 clinically normal horses. Forty milliliters were obtained from each horse and 5 mL of this blood was placed into each of 8 Monoject lavender top blood collection tubes containing sodium EDTA. Four of these tubes were kept chilled in iced water (1”C), and the remaining 4 tubes at room temperature (19°C). Blood samples of each set of 4 tubes collected at room temperature or in iced water were centrifuged successively at 15, 30. 60, and 180 minutes after blood collection. Each tube was centrifuged at 1,500 X g for 10 minutes at room temperature. The plasma was aspirated carefully, placed into a plastic (polypropylene) tube, and stored at -20°C. Plasma was assayed for ACTH within 1 month of sample collection, as recommended by Hegstad et al.9 All 8 samples from each horse were assayed simultaneously in duplicate or triplicate. Results were analyzed using 3-way ANOVA, with horse, time, and temperature as factors. pooled for ponies and horses within ECD and control groups (Table 1). The mean plasma ACTH concentration for horses and ponies in the control groups, 18.68 ? 6.79 pg/mL (mean 5 SD) and 8.35 2 2.92 pg/mL, respectively, were significantly different ( P = .009) (Table 5). The mean plasma ACTH concentrations for horses (n = 1 1 ) and ponies (n = 11) with ECD were 199.18 -+ 182.82 pg/mL (mean ? SD) and 206.21 ? 319.56 pg/mL, respectively, and were not significantly different using two-way ANOVA on the logarithm of ACTH concentration ( P > .05). Cushingoid horses and ponies had significantly higher plasma ACTH concentrations when compared with controls using a two-way ANOVA on the log of ACTH concentration ( P < .OOl). Sensitivity of plasma ACTH concentration as an indicator of ECD was 90.9% in horses and 8 1.8% in ponies. Specificity of the test for both groups was 100%. The 4 ponies and 4 horses from ECD groups tested had resting cortisol concentrations within the normal range (1.9 to 9.2 pg/dL). Cortisol increase factors after ACTH administration did not differ significantly between ECD and control horses (P = ,067). Among the other clinicopathologic parameters measured, hyperglycemia was the most consistent finding in ECD groups, with a glucose concentration above 110 mg/dL in 45% of the animals (Table 6). Hyperglycemia was also observed in 22% and 33% of control horses and ponies, respectively. Data analysis from the study of sample handling effect on plasma ACTH measurement showed that there was no significant differences between the 2 temperatures and for the 4 different times. Results Validation of the RIA for ACTH revealed intra-assay coefficients of variation of 7.1%, 8.8%, and 5.8% for samples containing high, medium, and low concentrations of ACTH, respectively (Table 2). The interassay coefficient of variation ranged from 4.4% to 14.7%, with an average of 8.7%, which was considered acceptable (Table 3). Serial dilution of plasma assayed for ACTH concentrations showed a good parallelism between observed (measured) and expected (calculated) ACTH values (Table 4), with a correlation coefficient of .9987. There was no significant difference in ACTH concentrations among age groups ( P > .05); therefore, data were Table 4. Serial Dilutions of Equine Plasma for Evaluation of Parallelism Using a Human Radioimmunoassay Kit for ACTH Quantification Plasma Dilution Undiluted 3 in 4 (25%) 2 in 4 (50%) 1 in 4 (75%) ACTH Observed 10) pg/mL (No. of Replicates) 117.91 (10) 98.0 (9) 56.9 (10) 22.9 (5) ACTH Expected (E)pglmL OiE x 100% Abbreviation: ACTH, adenocorticotropin. COUETIL, PARADIS, AND KNOLL Table 5. Summary of Results of Endocrine Tests Control Horses Mean ACTH t SD, pg/mL (range) Mean cortisol t- SD pg/dL Mean cortisol increase factor 18.68 2 6.79 (6.50-30.85) 5.02 i 2.07 3.05 2 1.07 Control Ponies 8.35 t- 2.92 (4.90- 13.6) 3.84 5 1.31 ECD Horses 199.18 ? 182.82 (40.8-669.93) 5.78 ? 4.3 4.74 ? 3.37 ECD Ponies 206.21 ? 319.56 (23.0-1,018.0) 3.84 t- 1.31 3.54 2 1.50 Abbreviations: ACTH, adenocorticotropin; ECD, equine Cushing's disease. Discussion In the horse, ACTH is secreted from the pituitary gland in a pulsatile manner consisting of at least 2 to 4 pulses per hour.'6 However, ACTH concentration in jugular vein plasma is 50 times lower than that in pituitary venous effluent; therefore, most of the pituitary spikes are not detectable in jugular blood.'6 Because of that dilution effect, ACTH in jugular blood follows a diurnal rhythm with minor hourly fluctuations. Therefore, a single ACTH measurement is a good reflection of pituitary secretory activity. The range for plasma ACTH found in this study is consistent with previous report^.^.^ The significant difference in mean plasma ACTH concentration between the control Table 6. Signalment, Clinical Signs, and Clinicopathologic Findings in Normal Horses and Ponies and in Those With ECD ECD Horses (n = 11) 21.5 (8-31) 6 5 Identification Mean age (yr) (range) Gender Stallionlgelding Female Clinical Signs Haircoat changes Weight loss Lethargy Laminitis PUIPD Hyperhidrosis Tachypnea Sinusitis Skin infection Other infections Bulging supra-orbital fat Clinical Pathology Hypercortisolemia (>9.2 fig/dL) Hyperglycemia ( > I 1 0 mg/dL) Leukocytosis ( > I 4,3OO/fiL) Leukopenia (<5,4OO/p,L) Neutrophilia (>8,6OO/fiL) Lymphopenia. (<1,5OO/p,L) ECD Ponies (n = 11) 21.4 (13-28) Control Control Horses Ponies In = 18) ( n = 9) 13.7 (2-27) 9 9 6.7 (4-12) Abbreviation: ECD, equine Cushing's disease. horses (18.68 ? 6.79 pg/mL) and the control ponies (8.35 ? 2.92 pg/mL) was unexpected, and to our knowledge has not been thus far reported. Although a cause for this difference was not elucidated in this project, other studies have established that ponies may be metabolically different from horses in their lipid and glucose metabolism.'7~'8 This is perhaps because of endocrine differences and not due to intrinsic differences in lipid or glucose metabolism. The results of this study indicate that plasma ACTH concentrations are valuable predictors of ECD in horses and ponies; an ACTH concentration higher than 27 pg/mL for ponies and 50 pg/mL. for horses strongly supports a clinical diagnosis of ECD. These results correlate well with previous reports involving small numbers of cushingoid The sensitivity and specificity of the test were excellent for both horses and ponies. Plasma ACTH concentration in horses and ponies with ECD varied widely. Significant variations in tumor size and ACTH content may account for the disparity, although it is still not clear if there is a correlation between tumor ACTH concentration and plasma ACTH concentration in horses with ECD.'.I9 High plasma ACTH concentrations were detected in horses with more severe clinical signs. However, this was not true for horses with mildly increased ACTH concentration. For example, postmortem examination of one affected horse revealed a pituitary adenoma, adrenocortical hyperplasia, and a plasma ACTH concentration of 146.9 pg/mL; however, none of the typical clinical signs were present. Again, it is conceivable that sustained exposure to high concentrations of POMC-derived peptides, in particular ACTH, is necessary for clinical signs to develop. Although most of the clinical signs in ECD are attributed to hypercortisolemia (ie, PUPD, muscle wasting, chronic laminitis, chronic infection, poor wound healing), the pathogenesis of hirsutism is still unknown.' Even though high plasma ACTH concentration appears to be a specific indicator of ECD, other factors that might affect ACTH concentration are stress and ectopic sources of ACTH-like peptides. The latter possibility has not been reported in the horse but is known to occur in humans?.'' Isolation stress does not seem to significantly affect ACTH secretion in horses.'" Higher stress level, as acute exercise, result in a significant increase in ACTH secretion; however, ACTH concentration returns to pre-exercise values within 30 minutes after horses finish the stress test." Furthermore, the marked increase in plasma ACTH increase observed during anesthesia in horses returns to normal within 2 hours of recovery.22Stress of illness might result in increased ACTH concentration.6 Nevertheless, samples collected from 4 sick EQUINE HYPERADRENOCORTICISM perplasia. Hillyer et alZ3reported normal ACTH stimulation responses in 2 of 6 horses with pituitary dependent hyperadrenocorticism. ACTH We could speculate that if a significant portion of the Animal concentration immunoreactive ACTH (IR-ACTH) originating from the piDescription Clinical Signs/Diagnosis PdrnL tuitary adenoma was biologically inactive one might expect 27 yo Arab Weight loss for 2 rnohntestinal 13.7 to find a high plasma ACTH concentration despite an abGelding lyrnphosarcorna sence of adrenal hyperplasia. Orth and Nicholson7 showed 18 yo Appaloosa Chronic nasal discharge/tooth 29.4 that a significant fraction of the IR-ACTH originating from Gelding root abscess 5 yo STB Dysphagia/EPM 16.1 pituitary adenomas in horses is bioactive ACTH; however, Gelding they only investigated 5 horses with pituitary adenomas, and 21 yo THB Anorexia/gastric adenornatous 11.3 all had bilateral adrenal hyperplasia. The structures of equine Gelding hyperplasia ACTH and cwmelanocyte-stimulating hormone are similar to Abbreviations: ACTH, adenocorticotropin; EPM, equine protozoal those of other species, including human being^.^,^' Therefore, myeloencephalitis; STB, standardbred; THB, thoroughbred; yo, year the kit used in this study might measure ACTH as well old. as POMC and related peptides. Wilson et a15 and the kit manufacturer, ICN Biomedicals, Inc, report low cross-reactivity percentage (0.1 %) in RIAs between cwMSH and ACTH. hospitalized horses that did not have any of the typical cliniIn addition, horses with pituitary adenoma secrete large cal signs of ECD revealed normal plasma ACTH concentraamounts of other peptides, in particular corticotropin-like tion (L. Couetil, unpublished data; Table 7). This anecdotal intermediate lobe peptide (CLIP), which might result in observation would require further investigation to better asfalsely increased IR-ACTH concentration. These peptides sess the specificity of high plasma ACTH concentration vispresumably have no corticotrophic activity; therefore, they a-vis ECD diagnosis. would account for those cases with high IR-ACTH and lack In this study, resting cortisol concentration appeared to be of adrenal hyperplasia. The effects of CLIP are unknown, a poor indicator of ECD. This is a surprising finding because but perhaps account for some of the clinical signs of ECD. an increase in plasma ACTH concentration is normally assoAnother tentative explanation for the absence of adrenal hyciated with a rise in cortisol concentration.I6 Nevertheless. perplasia observed in some cushingoid horses is that this it is consistent with previous report^.'.'^.'^ phenomenon requires ACTH hypersecretion over a sustained Horses with pituitary adenoma typically have less suppresperiod of time in order to result in adrenal hyperplasia. sion of cortisol secretion after dexamethasone administration Hyperglycemia has been reported as a fairly consistent than normal horses. Therefore, the dexamethasone suppresfinding in horses with ECD'32Y our results did not seem but sion test has been recommended for the diagnosis of pituto differ between the control and ECD groups. Stress of However, the itary-dependent hyperadrenocorti~ism.~.~~ blood collection and environment might have accounted for dexamethasone suppression was not used in this study bethis phenomenon, although during the experiment these cause, when informed on the hypothetical risk of inducing stress factors were reduced to a minimum, and the ponies laminitis or worsening a pre-existing laminitis," all clients and horses did not appear visibly stressed. Typical leukodeclined. gram change associated with hypercortisolemia is an inHorses with ECD typically have an exaggerated cortisol crease in neutrophiVlymphocyte ratio.26 However, in this response to exogenous ACTH administration, consistent study, neutrophilia and lymphopenia were equally common with adrenal hyperpla~ia.'.~.'~ However, our results support in healthy and cushingoid animals, as reported in a recent those of previous studies showing that the ACTH stimulation test is neither a sensitive nor a specific indicator of ECD.23-25 study.30 Plasma ACTH appeared stable in whole blood at 19°C up If we apply the concept of cortisol factor increase to previous to 3 hours; therefore, an equine practitioner could collect reports in the literat~re,~.~." find that a cortisol factor we blood for plasma ACTH determinations under field condiincrease of more than 3 is suggestive of Cushing's disease, tions. Special considerations regarding collection and shipand a factor between 2 and 3 reflects normal pituitary-adrenal ment of the sample include blood collection in an EDTA function. However, in this study, 4 normal horses showed tube (2 to 5 mL) and plasma separation within 3 hours. cortisol increase factors ranging from 3.7 to 4.7. The reason Separated plasma should be frozen at -20°C in a plastic for this exaggerated response to ACTH stimulation is un(polypropylene) tube and can be kept for up to 1 month clear. Two of these horses were race horses in training, in before shipment on dry ice to the appropriate laboratory. which the adrenal hyperplasia may be secondary to the stress Many endocrine function tests used in the diagnosis of of exercise. However, recent evidence suggests that training does not affect a horse's response to exogenous ACTH.19,26 ECD are time-consuming, difficult to interpret, and not readily available to practitioners. Plasma ACTH concentraConversely, one cushingoid pony had a cortisol increase tion appears to be a better clinicopathologic indicator of factor of only 1.3 in spite of a high plasma ACTH concentraECD than the ACTH stimulation test, cortisol concentration, tion (60.2 pg/mL). This finding is consistent with a study by and routine hematologic and biochemical analysis. Besides Heinrichs et alZ7who reported that only 4 of 19 horses with being a safe and easy test for diagnosis of ECD, in the future histological evidence of pituitary adenoma had adrenal hy- Table 7. Plasma ACTH Concentration of Horses Hospitalized for Various Illnesses COUETIL, PARADIS, AND KNOLL it may be useful for monitoring the progress of different treatment modalities. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Veterinary Internal Medicine Wiley

Plasma Adrenocorticotropin Concentration in Healthy Horses and in Horses With Clinical Signs of Hyperadrenocorticism

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Abstract

denomas of the pars intermedia of the pituitary gland produce a recognized syndrome in older horses known as equine Cushing’s disease (ECD),’.’ characterized by hypercorticism secondary to adrenocorticotropin (ACTH) hypersecretion from a pituitary tumor.4.’ There are some fundamental differences between this syndrome in horses and people, including anatomic location of the pituitary tumor and proopiomelanocortin (POMC) post-translational processing.’ Clinical signs of the disease are mainly related to secondary adrenal stimulation from an excess production of adrenocorticotropin (ACTH) from the pars intermedia.”7 Wilson et a1 showed that equine ACTH may be quantified accurately with radioimmunoassay (RIA) methods using antibodies against ACTH (rabbit antiporcine ACTH antibod- i 5 plasma concentration of POMC peptides and, in e High particdar, ACTH have been reported in horses with ECD4.5 and are well documented in humans with ectopic or pituitary Cushing’s syndrome.‘ Nevertheless, as of today, the importance of plasma ACTH measurement in the diagnosis of ECD has not been critically evaluated. Variability in sample handling conditions prior to ACTH quantification has been reported to influence results both in humans’ and dogs.’ ACTH is adsorbed onto glass surfaces and is degraded by proteolytic enzymes in whole blood, as well as in plasma.’,’0 Therefore, it is recommended to collect blood in cold plastic tubes (1” to 4°C) containing disodium EDTA as an anticoagulant, to separate the plasma immediately, and to store it frozen in plastic tubes for less than 1 month. These recommendations increase the difficulty of using ACTH concentrations as a diagnostic test for the equine practitioner. At the present time, the diagnosis of ECD is made on the basis of the typical clinical signs of hirsutism, polyuriapolydipsia, muscle wasting, hyperglycemia, and chronic laminitis.’-’ In addition, hypophyseal-adrenocortical axis function may be evaluated using various tests.’.’ However, these tests are not specific for the diagnosis of pituitary dysfunction’ and they often require serial sampling, which is more difficult to apply in the field. In addition, because the use of dexamethasone has a slight risk of inducing lami- nitis,” people are often reluctant to perform dexamethasonesuppression tests, especially in horses with a history of chronic laminitis. The study had 2 main objectives: first, to determine whether plasma ACTH concentrations differed between healthy equids and horses or ponies with clinically diagnosed pituitary adenomas, as well as between normal horses and ponies. Second, to determine whether ACTH quantification in plasma was affected by blood sample handling techniques. A prerequisite to conducting the study was to validate the commercially available human ACTH RIA kit (RSL ‘*’I hACTH; ICN Biomedicals, Inc, Diagnostic Division, Costa Mesa, CA) for equine ACTH measurement. Materials and Methods ACTH RIA Validation Using Equine Plasma Assay validation was performed on pooled equine plasma. Multiple aliquots of samples containing low, medium, and high concentrations of ACTH were frozen until assayed a single time to avoid effects of thawing and refreezing. Intra-assay precision was determined by assaying replicates on 3 equine plasma pools measured in one assay. Interassay precision was determined by 10 repeated measurements of equine plasma pools with medium and high ACTH concentrations on 3 days. An equine plasma pool containing high ACTH concentration was assayed undiluted, and at 3:4, 1:2, and 1 :4 ’ From the T u f s University School of Veierinaty Medicine, North Crafton, MA. Accepted Februavy 23, 1995. Supported by a Grantfrom the Mussachusetts Horse Racing Fund. Data from this study were presented at the Twelfth ACVIM Forum in Sun Francisco, 1994. The authors thank Ms. Julie Reid ,for her technical help, Dr. William Rand and Dr. Andy Hoffman f o r the statistical analysis, and Ms. Elaine Berglund f o r typing this manuscript, Reprint requests: Laurent Couetil, DVM, Purdue University, Department of Veterinary Clinical Sciences, 1248 Lynn Hall, West Lafayette, IN 47907-1248. Copyright 0 1996 by the American College of Veterinary Internal Medicine 0891-6640/96/1001-0001$3.00/0 Journal of Veterinary lnternal Medicine, Vol 10, No 1 (January-February),1996: pp 1-6 COUETIL, PARADIS, AND KNOLL Table 1. ACTH Concentration and Cortisol Increase Factor in Control Equids and Equids With ECD of Different Age Categories ~~~~~~~~~~~~~~ Control Groups Age Categories (yr) age 5 SD ACTH pg/mL* ACTH stimt 11-15 Mean age 2 SD ACTH pg/mL ACTH stim 16-20 Mean age 2 SD ACTH pmlmL ACTH stim >20 Mean age 2 SD ACTH pg/mL (range) ACTH stim 1 1 0 Mean 5.0 19.5 4.0 13.2 20.5 2.3 18.7 12.7 3.0 25.0 21.9 ECD Groups Horses 6) = 6) 2 0.3 (n = 2) 5 1.2 (n = 5) I (n = 5) 4.0 i_ 0.3 (n = 2) I (n = 4) 1.6 2 4.3 (n = 4) t 0.7 (n = 4) t 1.6 (n = 3) 2 6 (n = 3) ? 2.2 Ponies Horses = = Ponies (n t 7.4 ( n (n (n 7) 7) (n = 1) (n = 1) 13 (n = 1) 23.0 (n = 1) 5.3 In = 1) 18 (n = 1) 70.0 (n = 1) 22.8 ? 3.4 ( n = 9) 241.7 ? 326.2 In = 9) 123.0-1,018) 3.8 2 1.8 (n = 3) 11.5 2 0.5 (n 8.9 t 2.1 (n = = 2) 2) 3.3 -t 1.5 (n = 2) 11.0 i- 1 (n = 2) 135.0 t 58.9 (n = 2) 5.4 (n = 1) 17 In = 1) 71.1 In = 1) 4.8 In = 1) 27.1 t 2.1 (n = 7) 202.3 (n = 7) 243.2 I (40.8-669.9) 3.6 -t 0.3 (n = 2) Abbreviations: n, Number of animals in each category; ACTH, adenocorticotropin; ECD, equine Cushing’s disease. * Mean Values f SD. t ACTH stim, cortisol increase factor during ACTH stimulation test. dilutions. Results were evaluated for dilutional parallelism comparing observed (measured) values with expected values. Expected values were calculated multiplying the undiluted pool ACTH concentration by the dilution factor. The correlation coefficient between observed and expected ACTH concentrations was calculated. I 3 Clinical Study All horses and ponies included in the study presented to Tufts University School of Veterinary Medicine over a 1-year period. Eighteen horses and 9 ponies were used to establish reference ranges for plasma ACTH concentrations (control group). Four age categories were represented ( < I 0 years, 11 to 15 years, 16 to 20 years, and 220 years) (Table 1). These animals had no recent history of corticosteroid or exogenous ACTH administration and were determined to be clinically normal through a physical examination, CBC, and serum biochemistry profile. A second group, or ECD group, included I 1 horses and 11 ponies with either clinical signs of Cushing’s disease or postmortem confirmation of a pituitary tumor. The clinical diagnosis of pituitary adenoma was based on signs of hair coat abnormalities (hirsutism, delayed shedding, incomplete shedding) and at least one of the following signs: chronic laminitis, polyuria and polydipsia, muscle wasting, and hyperglycemia. Nine horses and 9 ponies admitted in the ECD group based on pathognomonic clinical signs were not available for necropsy. One horse and 2 ponies with typical clinical signs of ECD had histological evidence of a pituitary adenoma. One horse with normal hair coat and no clinical signs consistent with pituitary dysfunction had histological evidence of a pars intermedia adenoma and was, therefore, included in the ECD group. Each animal was evaluated using the same protocol as the control group consisting of physical examination, CBC, and serum biochemistry profile. During blood sample collection, horses and ponies were kept quiet in a stall with water and hay available. Sample collection for CBC, biochemistry profile, ACTH concentrations, and cortisol pre-ACTH gel injection were performed simultaneously between 9 AM and noon. ACTH stimulation tests were performed in a limited number of patients because of client compliance. Serum for cortisol quantification was harvested within 30 minutes of blood collection in tubes with no additive (Monoject; Sherwood Medical, St Louis, MO) (red top) and kept refrigerated until assayed, no longer than 3 days. Blood samples for ACTH quantification were collected in EDTA glass tubes (Monoject; Sherwood Medical, St Louis, MO) (lavender top), kept chilled in iced water (temperature approximately lac), and then centrifuged at 1,500 X g at room temperature for 10 minutes, within 15 minutes of blood sample collection. The plasma was then transferred into plastic (polypropylene) tubes and stored frozen at -20°C until assayed within a week of sample collection. Plasma ACTH concentrations in frozen samples stored 1 week or more in glass tubes are significantly different from those stored in plastic tubes.’ However, this phenomenon is not significant within 90 minutes of sample collection.’ ACTH concentration was determined using a commercially available kit (RSL r2sI hACTH) for human ACTH. The kit utilized ACTH antiserum generated in rabbits with purified porcine ACTH conjugate. ACTH standard consisted of human synthetic ACTH,.,,. Similar RIAs have been used by others to quantify directly equine plasma ACTH concentration^.^^'^ ACTH measurements for each plasma sample were done in duplicate. Statistical analysis was performed using ACTH concentrations and cortisol increase factors in control and cushingoid groups as dependent variables. Independent variables were ponies versus horses. Normality of data was tested using Kolmogorov-Smirnov goodness of fit test. Dependent variables were normally distributed in each group. Equality of variance was tested using Levene’s test and showed equal variance between control and cushingoid animals. However, Levene’s test for normal versus cushingoid ponies showed unequal variance. Dependent variables (ACTH concentration) were then log transformed, resulting in equal variances across groups. Then, plasma ACTH measurements in normal ponies and horses, and in cushingoid animals were compared with each other using two-way analysis of variance (ANOVA) on the logarithm of ACTH concentration.13 Sensitivity and specificityr3of plasma ACTH concentrations in horses with a clinical diagnosis of ECD were calculated. Breakpoint values for ACTH concentration (ie, ACTH values separating ECD group from healthy animals) were chosen arbitrarily as halfway between logarithm of mean ACTH concentration for ECD and control groups. Therefore, breakpoints for ponies and horses were 26.96 and 50 pg/mL, respectively. The protocol used for ACTH stimulation test was described by Dybdal* and included collection of a blood sample to quantify basal EQUINE HYPERADRENOCORTICISM Table 2. Intra-Assay Precision for Equine Plasma Assayed 10 Times With a Single ACTH Radioimmunoassay Sample (No. of Replicates) High (10) Medium (10) Low (5) Table 3. Interassay Precision for 2 Equine Plasma Pools Assayed 10 Times, Each on 3 Different Days Day of Assay Day1 Day6 Day7 Sample Pool 1 Pool 2 Pool 1 ACTH Concentration (pg/mL) Mean i SD 117.9 2 8.40 56.9 f 5.0 22.9 t 1.3 Coefficient of Variation (%) Mean ACTH (pg/mL) i S D 126.47 5 9.8 61.31 2 2.72 130.83f 19.31 54.82 2 4.93 117.91 -t 9.38 56.94 f 4.99 Coefficient of Variation x 100 Pool 2 Pool 1 Abbreviation: ACTH, adenocorticotropin. Pool 2 Abbreviation: ACTH, adenocorticotropin. cortisol concentrations and then injecting ACTH gel (H.P. Acthar Gel [with gelatin]; Rh6ne-Poulenc Rorer, Collegeville, PA, 40 U1 mL) (1 U k g body weight) IM. A second blood sample was collected 4 hours after ACTH administration for quantification of cortisol concentration. Cortisol was quantified using a commercial solidphase RIA kit (Coat-A-Count; Diagnostic Products Corp, Los Angeles, CA) previously validated for equine serum.” To compare the magnitude of adrenal response to ACTH stimulation among horses and ponies, a cortisol increase factor was introduced. Cortisol increase factor during ACTH stimulation test was defined as the ratio of cortisol concentration 4 hours after ACTH gel administration divided by the basal cortisol concentration. Differences in cortisol increase factor between ECD and control horses were analyzed using two-sample r-test.” Effects of Sample Handling on ACTH QuantiJication A separate experiment was designed to evaluate the effects of sample holding temperature after blood collection and time interval to plasma separation on ACTH concentration. Blood samples were collected by jugular venipuncture from 5 clinically normal horses. Forty milliliters were obtained from each horse and 5 mL of this blood was placed into each of 8 Monoject lavender top blood collection tubes containing sodium EDTA. Four of these tubes were kept chilled in iced water (1”C), and the remaining 4 tubes at room temperature (19°C). Blood samples of each set of 4 tubes collected at room temperature or in iced water were centrifuged successively at 15, 30. 60, and 180 minutes after blood collection. Each tube was centrifuged at 1,500 X g for 10 minutes at room temperature. The plasma was aspirated carefully, placed into a plastic (polypropylene) tube, and stored at -20°C. Plasma was assayed for ACTH within 1 month of sample collection, as recommended by Hegstad et al.9 All 8 samples from each horse were assayed simultaneously in duplicate or triplicate. Results were analyzed using 3-way ANOVA, with horse, time, and temperature as factors. pooled for ponies and horses within ECD and control groups (Table 1). The mean plasma ACTH concentration for horses and ponies in the control groups, 18.68 ? 6.79 pg/mL (mean 5 SD) and 8.35 2 2.92 pg/mL, respectively, were significantly different ( P = .009) (Table 5). The mean plasma ACTH concentrations for horses (n = 1 1 ) and ponies (n = 11) with ECD were 199.18 -+ 182.82 pg/mL (mean ? SD) and 206.21 ? 319.56 pg/mL, respectively, and were not significantly different using two-way ANOVA on the logarithm of ACTH concentration ( P > .05). Cushingoid horses and ponies had significantly higher plasma ACTH concentrations when compared with controls using a two-way ANOVA on the log of ACTH concentration ( P < .OOl). Sensitivity of plasma ACTH concentration as an indicator of ECD was 90.9% in horses and 8 1.8% in ponies. Specificity of the test for both groups was 100%. The 4 ponies and 4 horses from ECD groups tested had resting cortisol concentrations within the normal range (1.9 to 9.2 pg/dL). Cortisol increase factors after ACTH administration did not differ significantly between ECD and control horses (P = ,067). Among the other clinicopathologic parameters measured, hyperglycemia was the most consistent finding in ECD groups, with a glucose concentration above 110 mg/dL in 45% of the animals (Table 6). Hyperglycemia was also observed in 22% and 33% of control horses and ponies, respectively. Data analysis from the study of sample handling effect on plasma ACTH measurement showed that there was no significant differences between the 2 temperatures and for the 4 different times. Results Validation of the RIA for ACTH revealed intra-assay coefficients of variation of 7.1%, 8.8%, and 5.8% for samples containing high, medium, and low concentrations of ACTH, respectively (Table 2). The interassay coefficient of variation ranged from 4.4% to 14.7%, with an average of 8.7%, which was considered acceptable (Table 3). Serial dilution of plasma assayed for ACTH concentrations showed a good parallelism between observed (measured) and expected (calculated) ACTH values (Table 4), with a correlation coefficient of .9987. There was no significant difference in ACTH concentrations among age groups ( P > .05); therefore, data were Table 4. Serial Dilutions of Equine Plasma for Evaluation of Parallelism Using a Human Radioimmunoassay Kit for ACTH Quantification Plasma Dilution Undiluted 3 in 4 (25%) 2 in 4 (50%) 1 in 4 (75%) ACTH Observed 10) pg/mL (No. of Replicates) 117.91 (10) 98.0 (9) 56.9 (10) 22.9 (5) ACTH Expected (E)pglmL OiE x 100% Abbreviation: ACTH, adenocorticotropin. COUETIL, PARADIS, AND KNOLL Table 5. Summary of Results of Endocrine Tests Control Horses Mean ACTH t SD, pg/mL (range) Mean cortisol t- SD pg/dL Mean cortisol increase factor 18.68 2 6.79 (6.50-30.85) 5.02 i 2.07 3.05 2 1.07 Control Ponies 8.35 t- 2.92 (4.90- 13.6) 3.84 5 1.31 ECD Horses 199.18 ? 182.82 (40.8-669.93) 5.78 ? 4.3 4.74 ? 3.37 ECD Ponies 206.21 ? 319.56 (23.0-1,018.0) 3.84 t- 1.31 3.54 2 1.50 Abbreviations: ACTH, adenocorticotropin; ECD, equine Cushing's disease. Discussion In the horse, ACTH is secreted from the pituitary gland in a pulsatile manner consisting of at least 2 to 4 pulses per hour.'6 However, ACTH concentration in jugular vein plasma is 50 times lower than that in pituitary venous effluent; therefore, most of the pituitary spikes are not detectable in jugular blood.'6 Because of that dilution effect, ACTH in jugular blood follows a diurnal rhythm with minor hourly fluctuations. Therefore, a single ACTH measurement is a good reflection of pituitary secretory activity. The range for plasma ACTH found in this study is consistent with previous report^.^.^ The significant difference in mean plasma ACTH concentration between the control Table 6. Signalment, Clinical Signs, and Clinicopathologic Findings in Normal Horses and Ponies and in Those With ECD ECD Horses (n = 11) 21.5 (8-31) 6 5 Identification Mean age (yr) (range) Gender Stallionlgelding Female Clinical Signs Haircoat changes Weight loss Lethargy Laminitis PUIPD Hyperhidrosis Tachypnea Sinusitis Skin infection Other infections Bulging supra-orbital fat Clinical Pathology Hypercortisolemia (>9.2 fig/dL) Hyperglycemia ( > I 1 0 mg/dL) Leukocytosis ( > I 4,3OO/fiL) Leukopenia (<5,4OO/p,L) Neutrophilia (>8,6OO/fiL) Lymphopenia. (<1,5OO/p,L) ECD Ponies (n = 11) 21.4 (13-28) Control Control Horses Ponies In = 18) ( n = 9) 13.7 (2-27) 9 9 6.7 (4-12) Abbreviation: ECD, equine Cushing's disease. horses (18.68 ? 6.79 pg/mL) and the control ponies (8.35 ? 2.92 pg/mL) was unexpected, and to our knowledge has not been thus far reported. Although a cause for this difference was not elucidated in this project, other studies have established that ponies may be metabolically different from horses in their lipid and glucose metabolism.'7~'8 This is perhaps because of endocrine differences and not due to intrinsic differences in lipid or glucose metabolism. The results of this study indicate that plasma ACTH concentrations are valuable predictors of ECD in horses and ponies; an ACTH concentration higher than 27 pg/mL for ponies and 50 pg/mL. for horses strongly supports a clinical diagnosis of ECD. These results correlate well with previous reports involving small numbers of cushingoid The sensitivity and specificity of the test were excellent for both horses and ponies. Plasma ACTH concentration in horses and ponies with ECD varied widely. Significant variations in tumor size and ACTH content may account for the disparity, although it is still not clear if there is a correlation between tumor ACTH concentration and plasma ACTH concentration in horses with ECD.'.I9 High plasma ACTH concentrations were detected in horses with more severe clinical signs. However, this was not true for horses with mildly increased ACTH concentration. For example, postmortem examination of one affected horse revealed a pituitary adenoma, adrenocortical hyperplasia, and a plasma ACTH concentration of 146.9 pg/mL; however, none of the typical clinical signs were present. Again, it is conceivable that sustained exposure to high concentrations of POMC-derived peptides, in particular ACTH, is necessary for clinical signs to develop. Although most of the clinical signs in ECD are attributed to hypercortisolemia (ie, PUPD, muscle wasting, chronic laminitis, chronic infection, poor wound healing), the pathogenesis of hirsutism is still unknown.' Even though high plasma ACTH concentration appears to be a specific indicator of ECD, other factors that might affect ACTH concentration are stress and ectopic sources of ACTH-like peptides. The latter possibility has not been reported in the horse but is known to occur in humans?.'' Isolation stress does not seem to significantly affect ACTH secretion in horses.'" Higher stress level, as acute exercise, result in a significant increase in ACTH secretion; however, ACTH concentration returns to pre-exercise values within 30 minutes after horses finish the stress test." Furthermore, the marked increase in plasma ACTH increase observed during anesthesia in horses returns to normal within 2 hours of recovery.22Stress of illness might result in increased ACTH concentration.6 Nevertheless, samples collected from 4 sick EQUINE HYPERADRENOCORTICISM perplasia. Hillyer et alZ3reported normal ACTH stimulation responses in 2 of 6 horses with pituitary dependent hyperadrenocorticism. ACTH We could speculate that if a significant portion of the Animal concentration immunoreactive ACTH (IR-ACTH) originating from the piDescription Clinical Signs/Diagnosis PdrnL tuitary adenoma was biologically inactive one might expect 27 yo Arab Weight loss for 2 rnohntestinal 13.7 to find a high plasma ACTH concentration despite an abGelding lyrnphosarcorna sence of adrenal hyperplasia. Orth and Nicholson7 showed 18 yo Appaloosa Chronic nasal discharge/tooth 29.4 that a significant fraction of the IR-ACTH originating from Gelding root abscess 5 yo STB Dysphagia/EPM 16.1 pituitary adenomas in horses is bioactive ACTH; however, Gelding they only investigated 5 horses with pituitary adenomas, and 21 yo THB Anorexia/gastric adenornatous 11.3 all had bilateral adrenal hyperplasia. The structures of equine Gelding hyperplasia ACTH and cwmelanocyte-stimulating hormone are similar to Abbreviations: ACTH, adenocorticotropin; EPM, equine protozoal those of other species, including human being^.^,^' Therefore, myeloencephalitis; STB, standardbred; THB, thoroughbred; yo, year the kit used in this study might measure ACTH as well old. as POMC and related peptides. Wilson et a15 and the kit manufacturer, ICN Biomedicals, Inc, report low cross-reactivity percentage (0.1 %) in RIAs between cwMSH and ACTH. hospitalized horses that did not have any of the typical cliniIn addition, horses with pituitary adenoma secrete large cal signs of ECD revealed normal plasma ACTH concentraamounts of other peptides, in particular corticotropin-like tion (L. Couetil, unpublished data; Table 7). This anecdotal intermediate lobe peptide (CLIP), which might result in observation would require further investigation to better asfalsely increased IR-ACTH concentration. These peptides sess the specificity of high plasma ACTH concentration vispresumably have no corticotrophic activity; therefore, they a-vis ECD diagnosis. would account for those cases with high IR-ACTH and lack In this study, resting cortisol concentration appeared to be of adrenal hyperplasia. The effects of CLIP are unknown, a poor indicator of ECD. This is a surprising finding because but perhaps account for some of the clinical signs of ECD. an increase in plasma ACTH concentration is normally assoAnother tentative explanation for the absence of adrenal hyciated with a rise in cortisol concentration.I6 Nevertheless. perplasia observed in some cushingoid horses is that this it is consistent with previous report^.'.'^.'^ phenomenon requires ACTH hypersecretion over a sustained Horses with pituitary adenoma typically have less suppresperiod of time in order to result in adrenal hyperplasia. sion of cortisol secretion after dexamethasone administration Hyperglycemia has been reported as a fairly consistent than normal horses. Therefore, the dexamethasone suppresfinding in horses with ECD'32Y our results did not seem but sion test has been recommended for the diagnosis of pituto differ between the control and ECD groups. Stress of However, the itary-dependent hyperadrenocorti~ism.~.~~ blood collection and environment might have accounted for dexamethasone suppression was not used in this study bethis phenomenon, although during the experiment these cause, when informed on the hypothetical risk of inducing stress factors were reduced to a minimum, and the ponies laminitis or worsening a pre-existing laminitis," all clients and horses did not appear visibly stressed. Typical leukodeclined. gram change associated with hypercortisolemia is an inHorses with ECD typically have an exaggerated cortisol crease in neutrophiVlymphocyte ratio.26 However, in this response to exogenous ACTH administration, consistent study, neutrophilia and lymphopenia were equally common with adrenal hyperpla~ia.'.~.'~ However, our results support in healthy and cushingoid animals, as reported in a recent those of previous studies showing that the ACTH stimulation test is neither a sensitive nor a specific indicator of ECD.23-25 study.30 Plasma ACTH appeared stable in whole blood at 19°C up If we apply the concept of cortisol factor increase to previous to 3 hours; therefore, an equine practitioner could collect reports in the literat~re,~.~." find that a cortisol factor we blood for plasma ACTH determinations under field condiincrease of more than 3 is suggestive of Cushing's disease, tions. Special considerations regarding collection and shipand a factor between 2 and 3 reflects normal pituitary-adrenal ment of the sample include blood collection in an EDTA function. However, in this study, 4 normal horses showed tube (2 to 5 mL) and plasma separation within 3 hours. cortisol increase factors ranging from 3.7 to 4.7. The reason Separated plasma should be frozen at -20°C in a plastic for this exaggerated response to ACTH stimulation is un(polypropylene) tube and can be kept for up to 1 month clear. Two of these horses were race horses in training, in before shipment on dry ice to the appropriate laboratory. which the adrenal hyperplasia may be secondary to the stress Many endocrine function tests used in the diagnosis of of exercise. However, recent evidence suggests that training does not affect a horse's response to exogenous ACTH.19,26 ECD are time-consuming, difficult to interpret, and not readily available to practitioners. Plasma ACTH concentraConversely, one cushingoid pony had a cortisol increase tion appears to be a better clinicopathologic indicator of factor of only 1.3 in spite of a high plasma ACTH concentraECD than the ACTH stimulation test, cortisol concentration, tion (60.2 pg/mL). This finding is consistent with a study by and routine hematologic and biochemical analysis. Besides Heinrichs et alZ7who reported that only 4 of 19 horses with being a safe and easy test for diagnosis of ECD, in the future histological evidence of pituitary adenoma had adrenal hy- Table 7. Plasma ACTH Concentration of Horses Hospitalized for Various Illnesses COUETIL, PARADIS, AND KNOLL it may be useful for monitoring the progress of different treatment modalities.

Journal

Journal of Veterinary Internal MedicineWiley

Published: Jan 1, 1996

References

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