Pituitary pars intermedia dysfunction and EMS are the most commonly reported endocrine disorders of adult horses.1–4 Although both conditions have well-described clinical signs, diagnostic testing is recommended to fully assess the patient and determine disease severity. Clinical signs of advanced PPID include hypertrichosis, skeletal muscle loss, redistribution of body fat, polyuria, polydipsia, and laminitis, and this disorder is commonly detected in older horses (> 10 years of age).1,2,5,6 The signs of EMS include insulin dysregulation, regional or generalized adiposity, and an increased predisposition for laminitis, and this syndrome is typically first recognized in somewhat younger horses.3,4,7,8 Although a definitive etiopathogenesis has yet to be established for EMS,9 genetic and environmental factors likely contribute to development of the disorder.3,4 It can be difficult to differentiate between the 2 conditions because some horses and ponies affected by EMS can subsequently develop PPID.
The TRH stimulation test is a dynamic test for PPID that makes use of the presence of TRH receptors on melanotrophs within the pituitary pars intermedia.10–13 It has been described as a sensitive diagnostic test for PPID, with the ability to attain a diagnosis when resting endogenous ACTH concentrations are still within the reference range.10–13 The OST is a dynamic test for insulin dysregulation, which is a key component of EMS and the factor most closely linked with the development of laminitis.3,4,14–16 This test was first described in 2010, and it is used in the field to detect glycemic and insulinemic responses to an orally administered bolus of sugar.14 A previous study14 found that horses and ponies affected by EMS had higher circulating glucose and insulin concentrations 60 and 90 minutes after sugar administration than did healthy horses. These 2 tests are currently performed on separate days and under different conditions. Whereas there are no specific instructions for the timing of TRH stimulation testing in regard to feeding, the OST is performed after food is withheld overnight.10,14–17
The ability to test a given horse for both EMS and PPID on the same day could lower associated costs for the owner, and this in turn could potentially increase the frequency of testing for insulin dysregulation. Given that current recommendations for the OST include withholding of feed prior to the test, any combined testing for the 2 endocrine disorders would require that this step be included. Therefore, the purpose of the study reported here was to determine the effects of withholding feed on ACTH concentrations in horses during the TRH stimulation test, and the effects of combined testing on ACTH concentrations in the TRH stimulation test and on glucose and insulin concentrations in the OST. We hypothesized that withholding feed or allowing access to hay would not significantly affect plasma ACTH concentrations in the TRH stimulation test results and that measured values for the OST and the TRH stimulation test performed in combination would not differ significantly from those obtained when each test was performed alone.
Materials and Methods
Horses
Thirty horses (a convenience sample; 22 geldings and 8 mares) of the teaching herd at Johnson and Wales University Equine Center were included in the study, which was performed over 6 weeks from March 9 until April 13, 2014. The time period was chosen to avoid the August to October time frame, for which appropriate reference ranges for the TRH stimulation test have not been established.12 The horses had a mean ± SD age of 14.8 ± 3.6 years (range, 6 to 21 years) and mean ± SD body weight of 513 ± 45 kg (range, 430 to 565 kg). Body weight was estimated by use of a weight tape because no platform scale was available on the premises. Body condition scoring was performed by 1 investigator (MMR). All horses were estimated to have body condition scores of 5 to 7 on a 9-point scale (where 1 = poor and 9 = extremely fat). Breeds included Oldenberg (n = 5), Hanoverian (4), Thoroughbred (4), Warmblood (4), Holsteiner (3), Dutch Warmblood (3), Quarter Horse or Quarter Horse cross (2), and Westphalin, Swedish Warmblood, Paint, Irish Sport Horse, and Canadian Sport Horse (1 each). Three horses in the study (a 14-year-old mare and 2 geldings [each 20 years old]) were determined to have PPID by previous TRH stimulation testing. These horses received pergolide (1 to 2 mg/horse/d, PO; prescribed by the veterinarian for the facility) throughout the study. The remaining 27 horses were deemed healthy on the basis of medical history and physical examination findings. The study protocol was reviewed and approved by the Cummings School of Veterinary Medicine Clinical Studies Review Committee, and written informed consent was obtained for inclusion of the horses in the study by a representative of Johnson and Wales University.
Study design
All horses underwent the same series of tests: a TRH stimulation test alone under fed (day 1) and nonfed (day 14) conditions, an OST alone under nonfed conditions (day 28), and then combined testing (an OST and a TRH stimulation test under nonfed conditions; day 35). A physical examination was conducted on each test day before experiments commenced, and rectal temperature, heart rate, and respiratory rate were recorded. Testing started by 8:30 am on each test day (all times were Eastern Standard Time), with testing completed by 10:30 am when the TRH stimulation test or OST was performed alone (study days 1, 14, and 28) or by 11:00 am on the day of combined testing (day 35). Horses were monitored, and adverse effects associated with study treatments were recorded by veterinarians as tests were performed; when testing was complete, students and staff of the equine center observed the horses for the remainder of the day. When testing required horses to be in a nonfed state, a previously described feed withholding protocol for the OST3 was followed, in which 1 flake (approx 2 kg) of hay was fed at 10:00 pm, and presumably consumed within 1 to 2 hours, with no additional feed provided until testing was complete (12 to 13 hours later). Horses were permitted free access to hay, but not grain, when the TRH stimulation test was performed under fed conditions. Water was available ad libitum.
TRH stimulation test
Thyrotropin-releasing hormone solution was prepared as a single batch. Ninety milligrams of synthetic TRHa was reconstituted with a sterile technique under a biohazard hood. Sterile water was used to create a solution containing 1 mg of TRH/mL. One-milliliter aliquots were filtered with a 0.22-μm syringe filter and stored in sterile 3-mL syringes at −20°C until the time of use.
Each horse underwent TRH stimulation testing under fed (access to hay ad libitum; day 1) and nonfed (feed withheld overnight; day 14) conditions. Briefly, jugular venipuncture was performed to obtain a 10-mL blood sample immediately prior to TRH treatment (a baseline sample), and the sample was transferred to chilled tubes containing EDTA as an anticoagulant. One milligram (1 mL) of TRH was then administered into a jugular vein, and horses were observed for adverse effects associated with TRH administration during the following 10 minutes. A 10-mL poststimulation blood sample was obtained by jugular venipuncture 10 minutes after TRH administration. Blood samples were placed in a cooler with ice and centrifuged ≤ 6 hours after collection. Plasma was collected after low-speed centrifugation (1,000 × g for 15 minutes) at 4°C and stored at −20°C. Plasma samples were sent to a diagnostic laboratoryb ≤ 3 weeks after collection, and plasma ACTH concentrations were measured by chemiluminescent immunoassay.c The immunoassay used to measure ACTH concentrations was previously validated for use with equine samples,18 and the testing laboratory also conducted its own validation and quality control procedures. Results of the TRH stimulation test were considered positive if the baseline ACTH concentration was > 35 pg/mL11 or the poststimulation ACTH concentration was > 110 pg/mL, as described in Equine Endocrinology Group recommendations.d
OST
Each horse underwent an OST on day 28 under nonfed conditions as previously described.3,14 Immediately prior to corn syrup administration, jugular venipuncture was performed to obtain a 10-mL blood sample (the baseline sample for the OST), and blood was transferred to tubes containing EDTA as an anticoagulant. Horses received 0.15 mL of corn syrupe/kg of body weight (assumed to be equivalent to 0.15 g of glucose/kg of body weight), administered orally by use of 60-mL catheter-tip syringes, and were monitored for signs of adverse effects. Additional 10-mL blood samples were obtained by jugular venipuncture 60 and 90 minutes after syrup administration. Plasma was collected as described for TRH testing, and samples were stored at −20°C for ≤ 3 weeks until they were shipped to the diagnostic testing laboratory.b Plasma insulin and glucose concentrations were measured in samples collected at all 3 time points. Insulin concentrations were measured with a porcine insulin radioimmunoassay.f The radioimmunoassay for measurement of insulin was previously validated for use with equine samples,19 and the laboratory conducted its own validation and quality control procedures for the specific kit used. Glucose concentrations were measured at the same laboratory by means of the glucohexokinase method through the use of an automated chemistry analyzer.g Insulin concentrations > 20 μU/mL at baseline for the OST or ≥ 45 μU/mL 60 or 90 minutes after syrup administration were interpreted as abnormal. The glucose response was considered excessive if the plasma glucose concentration was > 125 mg/dL at baseline or at 60 or 90 minutes after syrup administration.3,14
Combined testing
On day 35, horses underwent combined endocrine testing under nonfed conditions with monitoring as described for the individual tests. The TRH stimulation test and OST protocols were performed as previously described, but the TRH stimulation test was performed during the longer OST protocol. The baseline (pretreatment) blood sample was collected for the OST, and corn syrup was administered as described; 60 minutes later, 2 blood samples were collected. One was used as the 60-minute OST sample, and the other was used for baseline measurement of ACTH for the TRH stimulation test. The TRH was administered IV immediately after this sample was collected, and the post-TRH stimulation sample was collected 10 minutes later (70 minutes after the start of the OST). The final blood sample for the OST was then collected 90 minutes after corn syrup administration. Blood samples collected at each of these 4 time points were transferred into EDTA-containing tubes, and plasma was collected, stored, and analyzed as described for the individual tests.
Statistical analysis
Visual assessment of data distributions and Shapiro-Wilk testing revealed that only glucose concentration data for the OST were normally distributed. Wilcoxon matched-pairs, signed rank tests (for plasma ACTH and insulin concentrations), or paired Student t tests (for plasma glucose concentrations) were selected for comparisons according to the distribution of the data. Baseline and poststimulation plasma ACTH concentrations were compared between horses undergoing TRH stimulation testing alone in fed and nonfed conditions, between fed horses undergoing TRH stimulation testing alone and nonfed horses for which the test was combined with the OST, and between nonfed horses undergoing TRH stimulation testing alone and nonfed horses for which the test was combined with the OST. Plasma glucose and insulin concentrations at baseline and 60 and 90 minutes after corn syrup administration were compared between horses undergoing the OST alone and those undergoing combined OST and TRH stimulation testing. Agreement between pairs of test methods (TRH stimulation testing alone under fed vs nonfed conditions, TRH stimulation testing performed as the sole test [fed or nonfed conditions] vs combined with the OST, and the OST performed as the sole test vs combined with TRH stimulation testing) was assessed by Bland-Altman analysis. In this analysis, the difference in results for 2 test methods is plotted against the mean result for the 2 methods, which allows for numerical assessment of the discrepancy between methods (bias) and variability in bias across the range of measured values. Correlations between pairs of methods were assessed by calculation of the Spearman correlation coefficient. All calculations were performed with commercial statistical software,h and values of P < 0.05 were considered significant.
Results
None of the 3 horses with PPID had clinical signs compatible with uncontrolled PPID; however, the presence of hypertrichosis was difficult to assess because horses' coats were clipped at the time of the study. All 30 horses remained apparently healthy throughout the study on the basis of medical records maintained by the farm, as well as rectal temperature, heart rate, and respiratory rate data, which were recorded on each study day and were within expected ranges at all time points for all horses. No clinically important complications were observed during testing. Minor procedural problems included breakage of an oral syringe tip when the OST was performed (as the sole test) for 1 horse and poor compliance during syrup administration for 4 horses (2 on the day that OST was performed alone and 2 on the day that the OST and TRH stimulation test were combined). The broken syringe tip was recovered in the stall shortly after the incident, with no ill effects to the horse, and horses that resisted oral corn syrup administration only lost a small amount (estimated to be < 5 mL [ie, < 6% to 8% of the total dose]) of syrup. No adverse events were observed during the OST at any time points.
Several adverse effects were associated with TRH administration; all of these signs were first detected ≤ 3 minutes after TRH administration and resolved without intervention ≤ 5 minutes after TRH administration. For the 90 TRH stimulation tests performed in the 30 study horses, the observed adverse effects included chewing (n = 39 tests), coughing (20), licking (18), flehmen response (9), yawning (7), sneezing or nose rubbing (6), pawing (5), head shaking (3), and stretching of the neck (2). On days 1 and 14 (with TRH stimulation performed as the sole test under fed and nonfed conditions, respectively), 21 of 30 horses had adverse effects. On day 35 (when the TRH stimulation test was performed during the OST), 19 of 30 horses had adverse effects. Ten horses had adverse effects 1 of 3 times that the TRH stimulation test was performed, 6 horses had adverse effects 2 of 3 times, and 13 horses had adverse effects all 3 times. Only 1 horse did not have any adverse effects observed.
Effects of feeding conditions and combined testing on TRH stimulation test results
Neither baseline nor poststimulation plasma ACTH concentrations differed significantly between the fed and nonfed conditions when TRH stimulation was performed alone (Figure 1). At baseline (immediately prior to TRH injection), median ACTH concentrations were 19.7 pg/mL (range, 6.1 to 94.7 pg/mL) and 20.6 pg/mL (range, 4.5 to 88.7 pg/mL) in fed and nonfed horses, respectively (P = 0.968). The median poststimulation concentrations (10 minutes after TRH injection) were 93.4 pg/mL (range, 27.5 to 285.0 pg/mL) and 101.4 pg/mL (range, 29.4 to 347.0 pg/mL) for fed and nonfed horses, respectively (P = 0.598). Plasma ACTH concentrations increased by a lower magnitude when the TRH stimulation test was performed under fed (vs nonfed) conditions in 12 of 30 (40%) horses and increased by a higher magnitude in the other 18 (60%) horses.
Scatterplots depicting baseline (A) and poststimulation (B) plasma ACTH concentrations in 30 horses that underwent TRH stimulation tests (TRHSTs) and OSTs under various conditions. The TRH stimulation test was performed alone in fed horses (with access to hay ad libitum) on day 1 and in nonfed horses (with feed withheld overnight) on day 14; on day 35, the test was performed in combination with an OST. For the TRH stimulation test, baseline plasma ACTH concentration was measured in a blood sample collected immediately prior to administration of TRH (1 mg/horse, IV), and the poststimulation ACTH concentration was measured in a sample collected 10 minutes later. The OST (performed under nonfed conditions) required collection of a blood sample for measurement of baseline plasma glucose and insulin concentrations, immediately followed by oral administration of corn syrup (0.15 mL/kg; assumed equivalent to 0.15 g of glucose/kg), with posttreatment blood samples collected 60 and 90 minutes later. On the day of combined testing, the OST was initiated as described; 60 minutes later, 2 blood samples were collected (1 used as the 60-minute posttreatment sample for the OST, and the other used for baseline measurement of ACTH). The TRH stimulation test was performed as described, and a final blood sample was collected 90 minutes after sugar administration to complete the OST. The horizontal lines depict median values.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Scatterplots depicting baseline (A) and poststimulation (B) plasma ACTH concentrations in 30 horses that underwent TRH stimulation tests (TRHSTs) and OSTs under various conditions. The TRH stimulation test was performed alone in fed horses (with access to hay ad libitum) on day 1 and in nonfed horses (with feed withheld overnight) on day 14; on day 35, the test was performed in combination with an OST. For the TRH stimulation test, baseline plasma ACTH concentration was measured in a blood sample collected immediately prior to administration of TRH (1 mg/horse, IV), and the poststimulation ACTH concentration was measured in a sample collected 10 minutes later. The OST (performed under nonfed conditions) required collection of a blood sample for measurement of baseline plasma glucose and insulin concentrations, immediately followed by oral administration of corn syrup (0.15 mL/kg; assumed equivalent to 0.15 g of glucose/kg), with posttreatment blood samples collected 60 and 90 minutes later. On the day of combined testing, the OST was initiated as described; 60 minutes later, 2 blood samples were collected (1 used as the 60-minute posttreatment sample for the OST, and the other used for baseline measurement of ACTH). The TRH stimulation test was performed as described, and a final blood sample was collected 90 minutes after sugar administration to complete the OST. The horizontal lines depict median values.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Scatterplots depicting baseline (A) and poststimulation (B) plasma ACTH concentrations in 30 horses that underwent TRH stimulation tests (TRHSTs) and OSTs under various conditions. The TRH stimulation test was performed alone in fed horses (with access to hay ad libitum) on day 1 and in nonfed horses (with feed withheld overnight) on day 14; on day 35, the test was performed in combination with an OST. For the TRH stimulation test, baseline plasma ACTH concentration was measured in a blood sample collected immediately prior to administration of TRH (1 mg/horse, IV), and the poststimulation ACTH concentration was measured in a sample collected 10 minutes later. The OST (performed under nonfed conditions) required collection of a blood sample for measurement of baseline plasma glucose and insulin concentrations, immediately followed by oral administration of corn syrup (0.15 mL/kg; assumed equivalent to 0.15 g of glucose/kg), with posttreatment blood samples collected 60 and 90 minutes later. On the day of combined testing, the OST was initiated as described; 60 minutes later, 2 blood samples were collected (1 used as the 60-minute posttreatment sample for the OST, and the other used for baseline measurement of ACTH). The TRH stimulation test was performed as described, and a final blood sample was collected 90 minutes after sugar administration to complete the OST. The horizontal lines depict median values.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Baseline plasma ACTH concentration also did not differ significantly (P = 0.153) for horses undergoing the TRH stimulation test alone under fed conditions (median, 19.7 pg/mL; range, 6.1 to 94.7 pg/mL), compared with that for the same horses when the test was combined with the OST on day 35 (median, 17.7 pg/mL; range, 2.0 to 99.7 pg/mL; Figure 1). However, the median poststimulation plasma ACTH concentration was significantly (P = 0.002) lower when the OST and TRH stimulation test were combined (70.75 pg/mL; range, 34.6 to 265.0 pg/mL), compared with that measured when TRH stimulation was performed alone under fed conditions (93.4 pg/mL; range, 27.5 to 285.0 pg/mL). Plasma ACTH concentrations increased by a lower magnitude during combined testing, compared with the increase measured when the TRH stimulation test was performed alone under fed conditions, in 23 of 30 (77%) horses and increased by a higher magnitude in the other 7 (23%) horses.
Baseline plasma ACTH concentration did not differ significantly (P = 0.167) when TRH stimulation tests were performed alone under nonfed conditions (median, 20.6 pg/mL; range, 4.5 to 88.7 pg/mL), compared with that measured when the test was completed in combination with the OST (median, 17.7 pg/mL; range, 2.0 to 99.7 pg/mL; Figure 1). In contrast, the median poststimulation ACTH concentration was significantly (P = 0.004) lower when the OST and TRH stimulation test were combined (70.75 pg/mL; range, 34.6 to 265.0 pg/mL) than when the TRH stimulation test was performed alone under nonfed conditions (101.4 pg/mL; range, 29.4 to 347.0 pg/mL). Plasma ACTH concentrations increased by a lower magnitude during combined testing, compared with that recorded for TRH stimulation testing under nonfed conditions alone, in 21 of 30 (70%) horses and increased by a higher magnitude in the other 9 (30%) horses.
Sixteen horses had negative results for all of the TRH stimulation tests performed, and 14 horses had a positive TRH stimulation test result on the basis of an abnormal baseline or post-TRH stimulation ACTH concentration (or both). Two horses had normal baseline ACTH concentrations on days 1 (fed conditions) and 35 (combined testing) and abnormal baseline ACTH concentrations on day 14 (nonfed conditions; 35.6 and 49.7 pg/mL), with normal post-TRH stimulation ACTH values. One other horse had an abnormal baseline ACTH concentration; this horse had a prior diagnosis of PPID and had abnormal baseline and poststimulation ACTH concentrations for all 3 TRH stimulation tests. Twenty-seven horses had normal baseline ACTH concentrations throughout the study.
Twelve horses had poststimulation ACTH concentrations above the diagnostic cutoff for ≥ 1 TRH stimulation test, including the horse with PPID that had abnormal baseline values. The other 11 horses had normal baseline ACTH concentrations but ≥ 1 abnormal post-TRH stimulation ACTH concentration. Seven of these horses had abnormal post-TRH stimulation ACTH concentrations for all 3 TRH stimulation tests, including the 2 other horses with a prior diagnosis of PPID. Three horses had abnormal post-TRH stimulation ACTH concentrations on days 1 (fed conditions) and 14 (nonfed conditions) but not day 35 (combined test conditions), and 1 horse only had an abnormal result on day 14. This horse had a poststimulation ACTH concentration that was just above the diagnostic cutoff value on that day (with results of 93 vs 112 pg/mL under fed and nonfed conditions, respectively).
Effects of combined testing on OST results
Median baseline plasma insulin concentration (obtained immediately prior to oral sugar [corn syrup] administration) did not differ significantly (P = 0.316) when the OST was performed alone on day 28 (6 μU/mL; range, 3 to 17 μU/mL), compared with that for the same horses when TRH stimulation testing was performed in combination with the OST on day 35 (7 μU/mL; range, 4 to 12 μU/mL). Similarly, median insulin concentrations in horses 60 (22 μU/mL [range, 12 to 43 μU/mL] vs 23 μU/mL [range, 9 to 55 μU/mL]) and 90 (18 μU/mL [range, 9 to 36 μU/mL] vs 17 μU/mL [range, 10 to 45 μU/mL]) minutes after oral sugar administration did not differ significantly (P = 0.10 and P = 1.0, respectively) between the solitary OST and that performed in combination with the TRH stimulation test (Figure 2).
Scatterplots depicting plasma insulin concentrations for the same horses as in Figure 1 60 (A) and 90 (B) minutes after oral sugar administration during OSTs performed alone on day 28 and in combination with the TRH stimulation test on day 35. See Figure 1 for key.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Scatterplots depicting plasma insulin concentrations for the same horses as in Figure 1 60 (A) and 90 (B) minutes after oral sugar administration during OSTs performed alone on day 28 and in combination with the TRH stimulation test on day 35. See Figure 1 for key.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Scatterplots depicting plasma insulin concentrations for the same horses as in Figure 1 60 (A) and 90 (B) minutes after oral sugar administration during OSTs performed alone on day 28 and in combination with the TRH stimulation test on day 35. See Figure 1 for key.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Baseline plasma glucose concentration in horses did not differ significantly (P = 0.07) when the OST was performed alone (mean ± SD, 82.1 ± 4.1 mg/dL), compared with that measured during combined testing (83.6 ± 3.9 mg/dL). Glucose concentration 60 minutes after oral sugar administration was significantly (P = 0.012) lower when the OST was performed alone (mean ± SD, 99.9 ± 8.7 mg/dL), compared with the concentration for the same time point during combined testing (104.9 ± 10.5 mg/dL; Figure 3). However, glucose concentrations did not differ significantly (P = 0.06) between the 2 methods 90 minutes after the oral sugar treatment (mean ± SD, 95.3 ± 8.1 mg/dL vs 98.5 ± 11.9 mg/dL for the OST alone and in the combined test, respectively).
Scatterplots depicting plasma glucose concentrations for the same horses as in Figure 1 60 (A) and 90 (B) minutes after oral sugar administration during OSTs performed alone on day 28 and in combination with the TRH stimulation test on day 35. The horizontal lines depict mean values; error bars represent SD. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Scatterplots depicting plasma glucose concentrations for the same horses as in Figure 1 60 (A) and 90 (B) minutes after oral sugar administration during OSTs performed alone on day 28 and in combination with the TRH stimulation test on day 35. The horizontal lines depict mean values; error bars represent SD. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Scatterplots depicting plasma glucose concentrations for the same horses as in Figure 1 60 (A) and 90 (B) minutes after oral sugar administration during OSTs performed alone on day 28 and in combination with the TRH stimulation test on day 35. The horizontal lines depict mean values; error bars represent SD. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Two horses had abnormal plasma insulin concentrations (above the cutoff value) during the OST when the test was performed in combination with the TRH stimulation test, but had normal results for this variable when the OST was performed alone. This discrepancy was found for one horse 60 minutes after oral sugar administration (48 vs 43 μU/mL for combined and solitary tests, respectively) and for the other horse 90 minutes after the treatment (45 vs 35 μU/mL, respectively). A third horse had normal plasma glucose concentrations at the 60- and 90-minute time points (119 and 108 mg/dL, respectively) when the OST was performed alone, but had excessive glucose concentrations at both time points when the OST was performed together with the TRH stimulation test (129 and 132 mg/dL, respectively). All remaining horses had OST results that were consistent for both time points.
Agreement and correlation analysis
Significant positive correlations were identified among poststimulation plasma ACTH concentrations (10-minute postinjection values) measured under different testing conditions, with Spearman correlation coefficients ranging from 0.86 to 0.92 (Table 1). Bland-Altman analysis revealed that the mean bias for poststimulation ACTH concentrations under fed versus nonfed conditions was small (−3.57 pg/mL) when TRH stimulation tests were performed alone. However, mean bias was substantial when the results for TRH stimulation testing alone under nonfed or fed conditions were compared with those measured in combined tests (25.54 pg/mL and 21.97 pg/mL, respectively), and the 95% CIs were wide (Figure 4).
Bland-Altman plot of the difference between poststimulation plasma ACTH concentrations during TRH stimulation tests performed alone in 30 adult horses under nonfed (A) and fed (B) conditions on day 14 and in combination with OSTs on day 35. The difference in ACTH concentrations was calculated as follows: poststimulation ACTH concentration for specified feeding condition – poststimulation ACTH concentration during combined testing. The mean of ACTH concentrations was calculated as (poststimulation ACTH concentration for specified feeding condition + poststimulation ACTH concentration during combined testing)/2.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Bland-Altman plot of the difference between poststimulation plasma ACTH concentrations during TRH stimulation tests performed alone in 30 adult horses under nonfed (A) and fed (B) conditions on day 14 and in combination with OSTs on day 35. The difference in ACTH concentrations was calculated as follows: poststimulation ACTH concentration for specified feeding condition – poststimulation ACTH concentration during combined testing. The mean of ACTH concentrations was calculated as (poststimulation ACTH concentration for specified feeding condition + poststimulation ACTH concentration during combined testing)/2.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Bland-Altman plot of the difference between poststimulation plasma ACTH concentrations during TRH stimulation tests performed alone in 30 adult horses under nonfed (A) and fed (B) conditions on day 14 and in combination with OSTs on day 35. The difference in ACTH concentrations was calculated as follows: poststimulation ACTH concentration for specified feeding condition – poststimulation ACTH concentration during combined testing. The mean of ACTH concentrations was calculated as (poststimulation ACTH concentration for specified feeding condition + poststimulation ACTH concentration during combined testing)/2.
Citation: American Journal of Veterinary Research 77, 7; 10.2460/ajvr.77.7.738
Results of Spearman correlation testing and Bland-Altman analysis for agreement between variables of interest in a study to assess the effects of withholding feed on TRH stimulation test results and the effects of combined testing on TRH stimulation test and OST results in 30 adult horses.
| Spearman correlation | Bland-Altman analysis* | |||||
|---|---|---|---|---|---|---|
| Measurement | Experiment | r | P value | Bias | SD | 95% CI |
| ACTH concentration | ||||||
| Baseline | TRHST (fed vs nonfed) | 0.52 | 0.003 | −0.77 | 7.82 | −16.10 to |
| TRHST (fed) vs combined testing | 0.66 | < 0.001 | 1.32 | 4.87 | −8.22 to | |
| TRHST (nonfed) vs combined testing | 0.48 | 0.007 | 2.09 | 8.55 | −14.67 to | |
| 10 min after injection | TRHST (fed vs nonfed) | 0.92 | < 0.001 | −3.57 | 30.98 | −64.29 to |
| TRHST (fed) vs combined testing | 0.86 | < 0.001 | 21.97 | 36.66 | −49.89 to | |
| TRHST (nonfed) vs combined testing | 0.90 | < 0.001 | 25.54 | 43.80 | −60.30 to | |
| Insulin concentration | ||||||
| Baseline | OST vs combined testing | 0.79 | < 0.001 | −0.20 | 2.11 | −4.33 to |
| 60 min after treatment | OST vs combined testing | 0.76 | < 0.001 | −1.97 | 5.99 | −13.70 to |
| 90 min after treatment | OST vs combined testing | 0.66 | < 0.001 | −0.20 | 6.49 | −12.92 to |
| Glucose concentration | ||||||
| Baseline | OST vs combined testing | 0.45 | 0.012 | −1.47 | 4.22 | −9.73 to |
| 60 min after treatment | OST vs combined testing | 0.49 | 0.006 | −4.93 | 9.81 | −24.16 to |
| 90 min after treatment | OST vs combined testing | 0.67 | < 0.001 | −3.20 | 8.90 | −20.65 to |
Each horse received each treatment in sequential order (TRH stimulation test alone under fed [access to hay; day 1] and nonfed [feed withheld overnight before testing; day 14] conditions, OST alone [day 28], or combined OST and TRH stimulation test [day 35]). For TRH stimulation testing, baseline and poststimulation plasma ACTH concentrations were measured in blood samples collected immediately prior to IV injection of TRH (1 mg/horse) and 10 minutes after the injection, respectively. For the OST, baseline plasma concentrations of insulin and glucose were measured in blood samples collected immediately prior to oral corn syrup administration (0.15 mL/kg), with posttreatment concentrations measured in samples collected 60 and 90 minutes after the treatment. On the day of combined testing, the TRH stimulation test was initiated immediately after 60-minute posttreatment sample collection for the OST.
Data for Bland-Altman analysis are shown as pg/mL, μU/mL, and mg/dL for ACTH, insulin, and glucose concentrations, respectively.
TRHST = TRH stimulation test.
Plasma glucose and insulin concentrations 60 and 90 minutes after oral sugar delivery during combined testing were positively correlated with those detected when the OST was performed alone (Table 1). Spearman correlation coefficients for insulin concentration were 0.76 and 0.66 at 60 and 90 minutes after treatment, respectively, and those for glucose concentration were 0.49 and 0.67, respectively. The degree of bias between results for the 2 testing conditions was considered small for both variables at both time points.
Discussion
In the present study, feeding conditions (fed [access to hay ad libitum] vs nonfed [feed withheld overnight]) did not significantly affect median plasma ACTH concentrations measured during TRH stimulation tests in horses. Although the ACTH concentration at baseline (immediately prior to TRH administration) was not affected by a combination with the OST, the poststimulation ACTH concentration (10 minutes after TRH injection) during combined testing was significantly lower than that measured in the same horses when the TRH stimulation test was performed alone under fed or nonfed conditions.
The finding that withholding feed versus allowing access to hay did not significantly affect median ACTH concentrations during TRH stimulation testing in the present study was in contrast to findings from another recent study17 involving horses. Diaz de Castro et al17 reported that healthy horses undergoing TRH stimulation testing for PPID under fed conditions had significantly higher ACTH concentrations, compared with results for the same horses when feed was withheld.17 Potential explanations for this discrepancy include differences in housing and feeding conditions or the number of horses studied (617 vs 30). It was noted that for 18 of 30 (60%) horses in the present study, ACTH concentrations increased by a higher magnitude in response to TRH under fed conditions than under nonfed conditions. An important consideration is that within-horse variability for ACTH concentrations 10 minutes after TRH administration can be substantial, and some horses in the present study had discrepancies of up to 82 pg/mL between fed and nonfed conditions. These differences might or might not alter the interpretation of the test with respect to PPID diagnosis, and it is important to recognize this potential for variability when applying cutoff values.
Food consumption has been associated with higher plasma ACTH concentrations in rats and humans, compared with those measured after food withholding or fasting,20–22 with proposed mechanisms including α-1 adrenoceptor,20 cholinergic agent,22 and vasoactive intestinal peptide21,23 activity. Investigators of another study24 assessed stress responses to withholding of feed by evaluating plasma ACTH concentrations (without TRH stimulation) in 6 healthy Thoroughbreds with and without feed withheld, but the results revealed no difference in ACTH concentrations between the 2 conditions. Studies25–27 have also been performed in recent years to explore relationships between plasma ACTH concentrations and anorexia in mice used to study human disease; results of some studies25,26 revealed relationships between anorexia and increased hypothalamic concentrations of proopiomelanocortin and its derivatives α-melanocyte–stimulating hormone and ACTH, and food withholding exacerbated this effect.27 It is conceivable that concentrations of these hormones increase after feeding in horses as a part of a hypothalamic satiety mechanism, and these relationships require further study.
Potential ramifications of the effects of combination with the OST on TRH stimulation test results in our study were highlighted by the finding that 2 horses would have been deemed to have PPID on the basis of results for TRH stimulation tests performed alone (under both fed and nonfed conditions) but had negative results for the same test when combined with the OST. In contrast to this result in horses, effects of oral glucose administration on plasma ACTH and cortisol concentrations after fasting have been studied in healthy men,28 and pulsatile ACTH secretion was found to increase after glucose administration, with a concomitant increase in cortisol concentration. The amount of abdominal visceral fat (estimated by CT) was positively associated with the magnitude of the glucose-induced increase in ACTH secretion and cortisol secretory-burst mass.28 It is thought that this exaggerated increase in ACTH occurs as a result of gut-derived insulinotropic peptides being released, which amplifies secretion of cortisol and corticotrophin-releasing hormone in addition to insulin.23,29,30 Visceral fat mass was not measured in the study reported here, but should be examined in the future.
Transient adverse effects of TRH administration were observed in most horses in the present study, but were considered mild, with no intervention required. Twenty-nine of 30 horses had adverse effects observed in ≥ 1 of the 3 tests, and 13 of 30 had adverse effects with every TRH administration. Although investigators of previous studies11,17 have reported adverse effects of exogenous TRH in horses, to our knowledge, this was the first study to assess the range, frequency, and consistency of adverse effects occurring in a population of horses with tests performed on 3 days. Results of 1 study11 indicated that generalized muscle fasciculations were the most common adverse effects, with licking, yawning, flehmen response, and coughing noted with lower frequency. In contrast, the most common adverse effect in horses of the present study was chewing (observed in 39/90 [43%] TRH stimulation tests), and muscle fasciculations were not observed in any tests. Other commonly observed signs included coughing and licking, consistent with results of another recent study.17 In other species, reported adverse effects of TRH administration vary, with people having nausea, vomiting, light headedness, urgency of micturition, and facial flushing.31 No adverse effects were reported when TRH was administered in a study32 of healthy Beagles; however, lip-licking and chewing have been anecdotally reported as a common signs of nausea in dogs receiving TRH, and the same relationship might exist in horses. No clinically important complications were noted during the OST when performed alone or in combination with the TRH stimulation test. Although compliance issues led to the loss of a small amount of syrup during the OST for 4 horses, this was deemed unimportant because it represented only a small percentage of the total volume administered. Because this compliance issue was noted for 2 horses on the day that the OST was performed alone and 2 horses on the day of combined OST and TRH stimulation testing, it was unlikely to have contributed to any differences in results between the 2 methods.
Three horses in the present study were previously determined to have PPID on the basis of TRH stimulation test results. Results of our tests confirmed these findings, with all 3 having poststimulation plasma ACTH concentrations greater than the diagnostic cutoff of 110 pg/mLd and 1 of the 3 having a high baseline ACTH concentration (> 35 pg/mL11) as well. Five other horses had abnormal poststimulation ACTH concentrations on all 3 tests, and 3 horses had abnormal poststimulation ACTH concentrations on days 1 and 14. Owing to a lack of clinical signs compatible with PPID and baseline ACTH concentrations below the diagnostic cutoff for PPID, we considered it likely that these 8 horses were in the early stages of PPID, exemplifying a situation in which the TRH stimulation test is clinically useful. The horse that tested positive only under nonfed conditions had a poststimulation ACTH concentration that was just above the diagnostic cutoff value on that day, and the difference between test results for this horse (93 vs 112 pg/mL under fed and nonfed conditions, respectively) was well within the reported within-laboratory range of SDs for plasma ACTH concentrations in duplicate samples from individual horses in a recent repeatability study.33 Within-horse variability could also explain why 2 horses with a baseline ACTH concentration above the diagnostic cutoff on a single occasion had post-TRH stimulation ACTH concentrations that were considered normal. It is also conceivable that variation in sample handling or assay variability33 contributed to the discrepancy in results for these 2 horses, considering that both had baseline and poststimulation ACTH concentrations below the respective diagnostic cutoffs under fed conditions and during combined testing with the OST. Technical problems (eg, lack of TRH solution stability or error during IV administration) could also contribute to inconsistent test results.
An influence of combined testing was also indicated by some of the OST results, with a significantly lower mean plasma glucose concentration detected 60 minutes after oral sugar administration when the OST was performed alone, compared with that for the same time point during combined testing (mean ± SD, 99.9 ± 8.7 mg/dL vs 104.9 ± 10.5 mg/dL, respectively). It is not clear why glucose concentrations differed between the 2 test protocols at this time point because during combined testing, the 60-minute OST blood sample was collected just before injection of the hormone for the TRH stimulation test, and glucose concentrations did not differ between the solitary OST and combined tests 90 minutes after sugar administration (30 minutes after TRH injection). To our knowledge, there are no published studies of OST repeatability in horses, but considerable variation in repeatability has been reported for other dynamic tests of insulin sensitivity,34–36 as well as effects of stress and concurrent disease on glucose concentrations,37 with coefficients of variation in plasma glucose levels ranging from 8% to 32% in 1 study.34 In the present study, although the difference in mean glucose concentrations between the test methods at this time point was statistically significant, when results for individual horses were assessed, only 1 of 30 would have been recharacterized from having a normal test result for the OST alone to having an abnormal result for the combined test on the basis of this variable. This horse had glucose concentrations that were considered normal at all time points when the OST was performed alone, but had abnormally high concentrations at the 60- and 90-minute time points on the day of combined testing. Two other horses had insulin concentrations considered abnormal when the OST was performed in combination with the TRH stimulation test (one at 60 minutes and the other at 90 minutes), yet had normal results at all time points when the OST was performed alone. All of these values were just above the cutoff indicating an abnormal OST result,14 and differences were attributed to within-horse variability, rather than clinically relevant differences between tests.
Mean bias represents the systematic discrepancy between 2 test methods and shows the difference of the mean of the 2 tests from zero. Mean bias for plasma ACTH concentrations measured 10 minutes after TRH administration was high (indicative of a large amount of variability), and the 95% CIs were wide when results for combined testing were compared with those for TRH stimulation tests performed alone. It was also noted that bias varied considerably among horses, as reflected by the high SD and wide CIs. Clinically, this raises concerns about the consistency of results when using the TRH stimulation test to diagnose PPID, despite the positive reports published to date.10–12,17 However, bias is more evident with higher ACTH concentrations and is less likely to impact ACTH concentrations that lie close to the cutoff value, which would influence the negative or positive outcome of the test. This suggests that variability is less important if ACTH concentrations are lower, as expected in healthy or mildly affected horses. Baseline ACTH concentrations had a smaller amount of bias between test methods, compared with that for post-TRH stimulation values. There was good agreement between tests for glucose and insulin concentrations measured during the OST performed alone versus in combination.
A crossover design was not used in this study, and this represents a limitation. Application of a Latin square design would have allowed effects of time to be assessed, given that the study was conducted over 35 days, and ACTH responses to TRH on day 1 might have differed from those detected on day 35 regardless of whether the tests were performed alone or in combination with the OST. Although a crossover design would have been ideal, the housing arrangement for the horses did not allow for feeding half of the study horses and withholding feed from the others without causing stress. Although feed withholding alone has not been shown to result in altered circulating ACTH concentrations in horses,24 short-term and chronic food withholding have been shown to increase ACTH concentrations in mice.25 An effect of food availability on hypothalamic hormones has also been identified in 2 species of bats.38 Selective withholding of food would be expected to induce stress, and other stressors such as transport, competition, training, illness, and surgical procedures have all been reported to cause changes in plasma ACTH and cortisol concentrations in horses as well as other species.39–42 Ideally, a placebo would also have been included in the present study design to control for any stress associated with additional handling, treatment administration, or blood sample collection.
Timing was also a potential source of variability in the present study because the time of day for tests performed during combined testing did not exactly match those for OST or TRH simulation tests performed alone, although previous investigations on the effects of time of day on circulating ACTH concentrations in horses found no significant circadian effect on basal17,43,44 or poststimulation17 ACTH concentrations. Although a difference of approximately 60 minutes (for the TRH stimulation test) or less (for the OST) was unlikely to have impacted the study results, the possibility of such an effect must still be considered. Season was accounted for in this study, with the period from March to May chosen to avoid the autumnal rise in circulating ACTH concentrations.11,12,15,44–47 Reference values have not been established for TRH stimulation testing of horses during this time of year, and recent studies have reported seasonal discrepancies in poststimulation ACTH concentrations in this species.11,12,17 Finally, most (27/30) horses included in this study were considered clinically normal, and it would be interesting to include a larger number of horses with PPID or EMS in future studies to better assess the consistency of TRH stimulation test results in affected animals. It should also be considered that horses were monitored for immediate adverse effects of TRH administration during the tests by veterinarians, and the students and staff of the facility observed the horses for the rest of the day. The observed effects were transient in the study performed here, and no horses required intervention, but an extended monitoring period should be considered in future studies because of the potential for additional adverse effects of TRH administration to develop after a few hours.
On the basis of our study results, we conclude that the TRH stimulation test can be performed in fed (access to hay) or nonfed (feed withheld overnight) conditions. Although mean plasma glucose concentration 60 minutes after oral sugar administration was significantly lower when the OST was performed alone than during combined testing, this difference could not be attributed to TRH administration, and no conclusions could be drawn in regard to the effect of combined testing on the OST results. However, because a combination of the TRH stimulation test with the OST resulted in significantly lower poststimulation plasma ACTH concentrations and a substantial degree of bias was detected for this variable between the combined test and TRH stimulation tests performed alone, combined testing as evaluated in this study is not recommended.
Acknowledgments
Supported by Boehringer Ingelheim Vetmedica Inc.
Dr. Frank consults for Boehringer Ingelheim on research study design. The authors declare that there were no other conflicts of interest.
The authors thank Beth Beukema, Kelly O'Neil, and John Richards for technical assistance, and Dr. Bruce Barton for assistance with statistical analysis.
ABBREVIATIONS
| CI | Confidence interval |
| EMS | Equine metabolic syndrome |
| OST | Oral sugar test |
| PPID | Pituitary pars intermedia dysfunction |
| TRH | Thyrotropin-releasing hormone |
Footnotes
Sigma-Aldrich Co, St Louis, Mo.
Cornell University Animal Health Diagnostic Center, Ithaca, NY.
Immulite ACTH chemiluminescent assay, Siemens Medical Solutions Diagnostics, Los Angeles, Calif.
Equine Endocrinology Group website. Available at: sites.tufts.edu/equineendogroup. Accessed Jan 15, 2014.
Karo syrup, ACH Food Co Inc, Summit, Ill.
EMD Millipore, Billerica, Mass.
Roche Diagnostics, Indianapolis, Ind.
GraphPad Prism, version 6.0, Graphpad Software, La Jolla, Calif.
References
1. McFarlane D. Equine pituitary pars intermedia dysfunction. Vet Clin North Am Equine Pract 2011; 27: 93–113.
2. Schott HC II. Pituitary pars intermedia dysfunction: equine Cushing's disease. Vet Clin North Am Equine Pract 2002; 18: 237–270.
3. Frank N. Equine metabolic syndrome. Vet Clin North Am Equine Pract 2011; 27: 73–92.
4. Frank N, Tadros EM. Insulin dysregulation. Equine Vet J 2014; 46: 103–112.
5. McGowan TW, Pinchbeck GP, McGowan CM. Prevalence, risk factors and clinical signs predictive for equine pituitary pars intermedia dysfunction in aged horses. Equine Vet J 2013; 45: 74–79.
6. Donaldson MT, Jorgensen AJ, Beech J. Evaluation of suspected pituitary pars intermedia dysfunction in horses with laminitis. J Am Vet Med Assoc 2004; 224: 1123–1127.
7. de Graaf-Roelfsema E. Glucose homeostasis and the enteroinsular axis in the horse: a possible role in equine metabolic syndrome. Vet J 2014; 199: 11–18.
8. Asplin KE, Sillence MN, Pollitt CC, et al. Induction of laminitis by prolonged hyperinsulinaemia in clinically normal ponies. Vet J 2007; 174: 530–535.
9. Sojka-Kritchevsky JE, Johnson PJ. Current status and future directions: equine pituitary pars intermedia dysfunction and equine metabolic syndrome. Equine Vet J 2014; 46: 99–102.
10. Beech J, Boston R, Lindborg S. Comparison of cortisol and ACTH responses after administration of thyrotropin releasing hormone in normal horses and those with pituitary pars intermedia dysfunction. J Vet Intern Med 2011; 25: 1431–1438.
11. Beech J, Boston R, Lindborg S, et al. Adrenocorticotropin concentration following administration of thyrotropin-releasing hormone in healthy horses and those with pituitary pars intermedia dysfunction and pituitary gland hyperplasia. J Am Vet Med Assoc 2007; 231: 417–426.
12. Funk RA, Stewart AJ, Wooldridge AA, et al. Seasonal changes in plasma adrenocorticotropic hormone and alpha-melanocyte-stimulating hormone in response to thyrotropin-releasing hormone in normal, aged horses. J Vet Intern Med 2011; 25: 579–585.
13. Beech J, McFarlane D, Lindborg S, et al. α-Melanocyte–stimulating hormone and adrenocorticotropin concentrations in response to thyrotropin-releasing hormone and comparison with adrenocorticotropin concentration after domperidone administration in healthy horses and horses with pituitary pars intermedia dysfunction. J Am Vet Med Assoc 2011; 238: 1305–1315.
14. Schuver A, Frank N, Chameroy KA, et al. Assessment of insulin and glucose dynamics by using an oral sugar test in horses. J Equine Vet Sci 2014; 34: 465–470.
15. Frank N, Elliott SB, Chameroy KA, et al. Association of season and pasture grazing with blood hormone and metabolite concentrations in horses with presumed pituitary pars intermedia dysfunction. J Vet Intern Med 2010; 24: 1167–1175.
16. Geor R, Frank N. Metabolic syndrome—from human organ disease to laminar failure in equids. Vet Immunol Immunopathol 2009; 129: 151–154.
17. Diez de Castro E, Lopez I, Cortes B, et al. Influence of feeding status, time of the day, and season on baseline adrenocorticotropic hormone and the response to thyrotropin releasing hormone-stimulation test in healthy horses. Domest Anim Endocrinol 2014; 48: 77–83.
18. Perkins GA, Lamb S, Erb HN, et al. Plasma adrenocorticotropin (ACTH) concentrations and clinical response in horses treated for equine Cushing's disease with cyproheptadine or pergolide. Equine Vet J 2002;34: 679–685.
19. Freestone JF, Wolfsheimer KJ, Kamerling SG, et al. Exercise induced hormonal and metabolic changes in Thoroughbred horses: effects of conditioning and acepromazine. Equine Vet J 1991; 23: 219–223.
20. Al-Damluji S, Iveson T, Thomas JM, et al. Food-induced cortisol secretion is mediated by central alpha-1 adrenoceptor modulation of pituitary ACTH secretion. Clin Endocrinol (Oxf) 1987; 26: 629–636.
21. Alexander LD, Evans K, Sander LD. A possible involvement of VIP in feeding-induced secretion of ACTH and corticosterone in the rat. Physiol Behav 1995; 58: 409–413.
22. Dodt C, Hansen K, Uthgenannt D, et al. Cholinergic potentiation of the meal-related rise in ACTH and cortisol concentrations in men. Exp Clin Endocrinol 1994; 102: 460–466.
23. Nussdorfer GG, Bahcelioglu M, Neri G, et al. Secretin, glucagon, gastric inhibitory polypeptide, parathyroid hormone, and related peptides in the regulation of the hypothalamus-pituitary-adrenal axis. Peptides 2000; 21: 309–324.
24. Ohmura H, Boscan PL, Solano AM, et al. Changes in heart rate, heart rate variability, and atrioventricular block during withholding of food in Thoroughbreds. Am J Vet Res 2012; 73: 508–514.
25. Mercer AJ, Stuart RC, Attard CA, et al. Temporal changes in nutritional state affect hypothalamic POMC peptide levels independently of leptin in adult male mice. Am J Physiol Endocrinol Metab 2014;306:E904–E915.
26. Schulz C, Paulus K, Lobmann R, et al. Endogenous ACTH, not only alpha-melanocyte-stimulating hormone, reduces food intake mediated by hypothalamic mechanisms. Am J Physiol Endocrinol Metab 2010;298:E237–E244.
27. Al-Barazanji KA, Miller JE, Rice SQ, et al. C-terminal fragments of ACTH stimulate feeding in fasted rats. Horm Metab Res 2001; 33: 480–485.
28. Iranmanesh A, Lawson D, Dunn B, et al. Glucose ingestion selectively amplifies ACTH and cortisol secretory-burst mass and enhances their joint synchrony in healthy men. J Clin Endocrinol Metab 2011; 96: 2882–2888.
29. Larsen PJ, Tang-Christensen M, Jessop DS. Central administration of glucagon-like peptide-1 activates hypothalamic neuroendocrine neurons in the rat. Endocrinology 1997; 138: 4445–4455.
30. Malendowicz LK, Nussdorfer GG, Nowak KW, et al. Exendin-4, a GLP-1 receptor agonist, stimulates pituitary-adrenocortical axis in the rat: investigations into the mechanism(s) underlying Ex4 effect. Int J Mol Med 2003; 12: 237–241.
31. Crowther CA, Alfirevic Z, Han S, et al. Thyrotropin-releasing hormone added to corticosteroids for women at risk of preterm birth for preventing neonatal respiratory disease. Cochrane Database Syst Rev 2013;11:CD000019.
32. Yagi K, Ohashi E, Tanabe S, et al. Serum thyrotropin response to TRH administration in six healthy Beagle dogs. Vet Rec 2000; 146: 706–707.
33. Gehlen H, Bradaric Z. Study on the reproducibility of ACTH concentrations in plasma of horses with and without pituitary pars intermedia dysfunction (PPID) [in German]. Berl Munch Tierarztl Wochenschr 2013; 126: 350–356
34. Bröjer J, Lindáse S, Hedenskog J, et al. Repeatability of the combined glucose-insulin tolerance test and the effect of a stressor before testing in horses of 2 breeds. J Vet Intern Med 2013; 27: 1543–1550.
35. Pratt SE, Geor RJ, McCutcheon LJ. Repeatability of 2 methods for assessment of insulin sensitivity and glucose dynamics in horses. J Vet Intern Med 2005; 19: 883–888.
36. Frank N, Elliott SB, Boston RC. Effects of long-term oral administration of levothyroxine sodium on glucose dynamics in healthy adult horses. Am J Vet Res 2008; 69: 76–81.
37. Eiler H, Frank N, Andrews FM, et al. Physiologic assessment of blood glucose homeostasis via combined intravenous glucose and insulin testing in horses. Am J Vet Res 2005; 66: 1598–1604.
38. Lewanzik D, Kelm DH, Greiner S, et al. Ecological correlates of cortisol levels in two bat species with contrasting feeding habits. Gen Comp Endocrinol 2012; 177: 104–112.
39. Ayala I, Martos NF, Silvan G, et al. Cortisol, adrenocorticotropic hormone, serotonin, adrenaline and noradrenaline serum concentrations in relation to disease and stress in the horse. Res Vet Sci 2012; 93: 103–107.
40. Fazio E, Medica P, Aronica V, et al. Circulating beta-endorphin, adrenocorticotrophic hormone and cortisol levels of stallions before and after short road transport: stress effect of different distances. Acta Vet Scand 2008; 50: 6.
41. Cayado P, Munoz-Escassi B, Dominguez C, et al. Hormone response to training and competition in athletic horses. Equine Vet J Suppl 2006;(36):274–278.
42. Goldstein DS, Kopin IJ. Adrenomedullary, adrenocortical, and sympathoneural responses to stressors: a meta-analysis. Endocr Regul 2008; 42: 111–119.
43. Cordero M, Brorsen BW, McFarlane D. Circadian and circannual rhythms of cortisol, ACTH, and alpha-melanocyte-stimulating hormone in healthy horses. Domest Anim Endocrinol 2012; 43: 317–324.
44. Lee ZY, Zylstra R, Haritou SJ. The use of adrenocorticotrophic hormone as a potential biomarker of pituitary pars intermedia dysfunction in horses. Vet J 2010; 185: 58–61.
45. Beech J, Boston RC, McFarlane D, et al. Evaluation of plasma ACTH, alpha-melanocyte-stimulating hormone, and insulin concentrations during various photoperiods in clinically normal horses and ponies and those with pituitary pars intermedia dysfunction. J Am Vet Med Assoc 2009; 235: 715–722.
46. Copas VE, Durham AE. Circannual variation in plasma adrenocorticotropic hormone concentrations in the UK in normal horses and ponies, and those with pituitary pars intermedia dysfunction. Equine Vet J 2012; 44: 440–443.
47. Donaldson MT, McDonnell SM, Schanbacher BJ, et al. Variation in plasma adrenocorticotropic hormone concentration and dexamethasone suppression test results with season, age, and sex in healthy ponies and horses. J Vet Intern Med 2005; 19: 217–222.
