This study was performed at the University of Georgia's equine breeding facility, which is affiliated with both the College of Veterinary Medicine and the Department of Animal and Dairy Science, University of Georgia, Athens, GA.
Hypothalamic-Pituitary-Adrenal Axis Assessment in Healthy Term Neonatal Foals Utilizing a Paired Low Dose/High Dose ACTH Stimulation Test
Version of Record online: 3 FEB 2009
Copyright © 2009 by the American College of Veterinary Internal Medicine
Journal of Veterinary Internal Medicine
Volume 23, Issue 2, pages 344–351, March/April 2009
How to Cite
Hart, K.A., Heusner, G.L., Norton, N.A. and Barton, M.H. (2009), Hypothalamic-Pituitary-Adrenal Axis Assessment in Healthy Term Neonatal Foals Utilizing a Paired Low Dose/High Dose ACTH Stimulation Test. Journal of Veterinary Internal Medicine, 23: 344–351. doi: 10.1111/j.1939-1676.2008.00271.x
- Issue online: 3 MAR 2009
- Version of Record online: 3 FEB 2009
- Submitted August 28, 2008; Revised November 1, 2008; Accepted December 11, 2008.
- HPA axis;
Background: Hypothalamic-pituitary-adrenal (HPA) axis function is dynamic in the neonatal foal. The paired low dose/high dose cosyntropin (ACTH) stimulation test allows comprehensive HPA axis assessment, but has not been evaluated in neonatal foals.
Hypothesis: Foal age will significantly affect cortisol responses to a paired 10 and 100 μg dose cosyntropin stimulation test in healthy neonatal foals.
Animals: Twenty healthy neonatal foals.
Methods: HPA axis function was assessed in 12 foals at birth and at 12–24, 36–48 hours, and 5–7 days of age. At each age, basal cortisol and ACTH concentrations were measured and cortisol responses to 10 and 100 μg cosyntropin were assessed with a paired ACTH stimulation test protocol. Eight additional 36–48-hour-old foals received saline instead of 10 μg cosyntropin in the same-paired ACTH stimulation test design.
Results: At birth, foals had significantly higher basal cortisol and ACTH concentrations and higher basal ACTH : cortisol ratios compared with foals in all other age groups. A significant cortisol response to both the 10 and 100 μg doses of cosyntropin was observed in all foals. The magnitude of the cortisol response to both doses of cosyntropin was significantly different across age groups, with the most marked responses in younger foals. There was no effect of the paired ACTH stimulation test design itself on cortisol responses.
Conclusions and Clinical Importance: A paired 10 and 100 μg cosyntropin stimulation test can be used to evaluate HPA axis function in neonatal foals. Consideration of foal age is important in interpretation of HPA axis assessment.
The hypothalamic-pituitary-adrenal axis (HPA axis) tightly regulates systemic cortisol concentrations in both health and disease, and thus plays an integral role in the maintenance of cellular, organ, and whole body homeostasis. Hypothalamic integration of input from the peripheral and central nervous systems induced by environmental and endogenous physiologic stressors culminates in modulation of the release of corticotropin-releasing hormone (CRH) into the hypothalamic-pituitary portal circulation. CRH acts on the adjacent anterior pituitary gland to induce the release of ACTH into the systemic circulation, which ultimately stimulates the adrenal cortices to synthesize and release cortisol, the primary mammalian stress hormone. Increased systemic cortisol concentrations exert a negative feedback effect on both the pituitary gland and hypothalamus, resulting in subsequent downregulation of both CRH and ACTH secretion and maintaining systemic cortisol concentrations at a level appropriate for the existing degree of physiologic stress.
Because cortisol is not stored in either the adrenal cortices or peripheral tissues, any disruption in HPA axis activation or cortisol synthesis can rapidly result in systemic cortisol insufficiency. In illness, this systemic cortisol insufficiency is often referred to as relative adrenal insufficiency (RAI) or critical illness-related corticosteroid insufficiency (CIRCI), and is characterized by transient serum cortisol concentrations inappropriately low for the existing degree of illness and abnormal responses on HPA axis function testing.1–6 The clinical impact of RAI/CIRCI in humans is exemplified by recent reports indicating that critically ill patients with concurrent HPA axis dysfunction often have significantly increased disease severity and mortality as compared with critically ill patients with intact HPA axes.1–3 Recent reports in both dogs7,8 and foalsa9,10 suggest that these syndromes may be of similar importance in veterinary critical care.
Thus, a means for thorough evaluation of HPA axis function in the neonatal foal is needed for further investigation of HPA dysfunction in foals in a clinical setting. Comprehensive HPA axis function assessment can include both measurement of basal ACTH and cortisol concentrations, to provide information regarding endogenous HPA axis activation, and determination of cortisol responses to both physiologic and supraphysiologic doses of ACTH to assess adrenocortical sensitivity to ACTH and maximal corticosteroid synthetic capacity. Because of the dynamic changes observed in HPA axis activity in foals during the 1st week of life,11,12 serial assessment of these parameters in healthy neonatal foals during this period is necessary to determine appropriate age-matched criteria for HPA axis function testing in foals. Previous studies on ACTH stimulation testing in fetal and neonatal foals utilized 125–250 μg of ACTH,11–13 resulting in supraphysiologic blood concentrations of ACTH that reach almost 300 times those that can be achieved physiologically.14 Although a supraphysiologic dose of exogenous ACTH is appropriate when testing for absolute adrenal gland insufficiency, lower physiologically relevant doses of ACTH (ie, 1–10 μg) may be more appropriate for investigation of relative, transient dysfunction of the adrenal gland (RAI/CIRCI).14–17 The timing of serial ACTH stimulation tests also is of clinical importance in the neonatal foal, because HPA activation and response to ACTH change rapidly in the 1st 6–48 hours after birth.11 Thus, a paired low dose/high dose ACTH stimulation test design that assesses cortisol response to both physiologic and supraphysiologic doses of ACTH over a short time period (ie, 2–3 hours) may be the most efficient method for comprehensive HPA axis evaluation in neonatal foals.
The cortisol response to a range of cosyntropin (synthetic ACTH, α 1–24 corticotropin) doses (1–250 μg) recently has been described in healthy 3–4-day-old foals.18 Three-to-four-day-old foals exhibit a significant dose-dependent increase in cortisol after administration of both low (10 μg) and high (100 and 250 μg) doses of cosyntropin, but do not exhibit a significant cortisol response to the 1 μg cosyntropin dose used for low dose ACTH stimulation testing in humans.14,16,19 Cortisol responses to the 10 and 100 μg cosyntropin doses have not been evaluated in younger or older foals, and a paired low dose/high dose ACTH stimulation test has not been evaluated in foals of any age.
The purpose of this study thus was to compare cortisol responses to a paired low dose/high dose ACTH stimulation test (Fig 1) in healthy full term foals at 4 time points during the 1st week of life. We tested the following 3 hypotheses: (1) administration of a low dose of cosyntropin (10 μg) 90 minutes before a high dose (100 μg) of cosyntropin in a paired low dose/high dose ACTH stimulation test protocol will have no effect on the cortisol response to the high dose of cosyntropin; (2) a low dose (10 μg) of cosyntropin will be equally effective as a high dose (100 μg) of cosyntropin in eliciting a cortisol response in healthy neonatal foals within this paired ACTH stimulation test design; and (3) foal age will significantly impact the cortisol response to both the low and the high doses of cosyntropin.
Materials and Methods
The study population (“paired ACTH stim group”) consisted of 12 neonatal Quarter Horse foals (7 males, 5 females). These foals were evaluated at 4 time points during the 1st week of life: within 1 hour of birth (n = 11), between 12 and 24 hours of age (n = 10), between 36 and 48 hours of age (n = 10), and between 5 and 7 days of age (n = 11). Mean foal body weight was 49.9 ± 8.6 kg (range, 38.7–61.4 kg). A separate group of 8 foals (5 Quarter Horses, 2 Tennessee Walking Horse crosses, and 1 pony) served as a control group to determine if any effects specific to the paired ACTH stimulation test protocol used in the study population were present. The control group consisted of 4 males and 4 females, and mean foal body weight was 46.0 ± 14.2 kg (range, 20.5–65.9 kg). Foals in this group were only available for evaluation at 1 time point, at 36–48 hours of age.
Only full term foals (>330 days gestation) born without assistance were included in the study. Foals were determined to be healthy before inclusion in the analysis and during the study based on a lack of abnormalities identified on serial physical examinations. Adequate passive transfer of immunoglobulin was confirmed by a serum immunoglobulin concentration ≥800 mg/dL at 12–24 hours of age in all foals.b Hematologic analysis and serum biochemical profiles were not routinely performed in foals in either group. Each mare and foal pair in both groups was stabled with daily paddock turnout during the study period, and animals were cared for according to the principles and guidelines in an Animal Use Protocol approved by the University of Georgia's Department of Animal Resources.
In the paired ACTH stim group, a repeated measures design was used to assess HPA axis function at 4 time points during the 1st week of life: within 1 hour of birth, between 12 and 24 hours of age, between 36 and 48 hours of age, and between 5 and 7 days of age. Time of birth was determined by use of a Foalertc system placed on each mare before her anticipated foaling date, and defined as the time at which the Foalert alarm and automatic dialer was activated. At each age, blood was collected for measurement of basal serum total cortisol and endogenous plasma ACTH concentrations, and a paired low (10 μg) and high (100 μg) dose cosyntropinc (synthetic ACTH, α 1–24 corticotropin) stimulation test was performed (Fig 1).
Foalings were not attended and Foalert™ systems were not utilized for the control group. The dams of these foals were observed every 2–8 hours from the time at which foaling appeared imminent until parturition occurred, and foaling time was estimated based on these observations to determine foal age. HPA axis assessment was performed once in this group of foals, at 36–48 hours of age. At this time, blood was collected for measurement of baseline serum total cortisol and endogenous plasma ACTH concentrations, and a “sham” paired low/high dose cosyntropin stimulation test was performed. After collection of basal blood samples, these foals received an equivalent volume of sterile saline (0.9% sodium chloride) instead of the 10 μg dose of cosyntropin, and then received the 100 μg dose of cosyntropin 90 minutes later.
In the paired ACTH stim group, the dosage of cosyntropin administered to each foal on the basis of weight was 0.21 ± 0.03 μg/kg (range, 0.16–0.26 μg/kg) for the 10 μg dose and 2.1 ± 0.03 μg/kg (range, 1.6–2.6 μg/kg) for the 100 μg dose. In the control group, the dosage of cosyntropin based on foal weight for the 100 μg dose was 2.5 ± 1.1 μg/kg (range, 1.5–4.9 μg/kg).
Preparation of Cosyntropin
Cosyntropind was supplied as a lyophilized powder in glass vials. Each vial contained 250 μg of cosyntropin, which was reconstituted with 1 mL sterile saline according to the manufacturer's directions. The resulting solution then was diluted 10-fold with sterile saline to produce a 25 μg/mL solution of cosyntropin. The 10 and 100 μg doses consisted of 0.4 and 4 mL of this solution, respectively. The 10 μg doses were additionally diluted with sterile saline to an equivalent total volume of 4 mL. Reconstituted and diluted cosyntropin solutions were stored frozen at −80 °C in sterile glass vials until immediately before use (not longer than 4 months). Reconstituted cosyntropin solutions are stable when frozen in glass vials at −80 °C for up to 6 months.20
All blood samples were collected by jugular venipuncture under brief restraint by experienced foal handlers. Foals were restrained in sternal recumbency for collection of the basal (time 0) samples at the 1st sampling age (within 1 hour of birth) if the foal had not yet stood on its own at this time. All other samples were collected from standing foals. Before administration of 10 μg of cosyntropin, blood was collected for measurement of basal serum total cortisol and plasma endogenous ACTH concentrations. Blood was placed into plastic tubes containing 3.6 mg potassium EDTA for measurement of plasma endogenous ACTH concentration. Blood for cortisol concentration measurement was placed into glass tubes without additives and allowed to clot at room temperature for 30–60 minutes.
After collection of basal samples and before withdrawal of the venipuncture needle, 10 μg cosyntropin (paired ACTH stim group) or the equivalent volume of sterile saline (control group) was administered IV as a rapid bolus. Blood was then collected 30 minutes later for assessment of the peak cortisol response to the 10 μg cosyntropin dose. Ninety minutes after administration of 10 μg cosyntropin, blood again was collected for measurement of cortisol concentration, and before removal of the venipuncture needle, 100 μg of cosyntropin was administered IV as a rapid bolus (both groups). Blood was collected 30 and 90 minutes later for assessment of the cortisol response to the 100 μg cosyntropin dose.
Blood samples were stored at 4 °C until centrifugation and separation of the serum or plasma within 3 hours of collection. Serum and plasma samples were stored frozen at −80 °C until assays were performed (within 30 days). Plasma ACTH and serum total concentrations are stable frozen at −80 °C for 30 and 90 days, respectively.e
ACTH and Cortisol Assay
Plasma endogenous ACTH concentrations (henceforth referred to as ACTH concentrations) and serum total cortisol concentrations (henceforth referred to as cortisol concentrations) were determined on an automated analyzer using chemiluminescent enzyme immunoassaysf validated for use in horses.21–23 For the ACTH assay, the interassay and intraassay coefficients of variation were 7–9% and 9%, respectively, and the limit of detection was 9 pg/mL.21 The interassay and intra-assay coefficients of variation were <20%23 and the limit of detection was 0.05 μg/dL for the cortisol assay.22
For the paired ACTH stim group, basal ACTH and cortisol concentrations, basal ACTH : cortisol ratio, and cortisol responses to both the 10 μg and the 100 μg dose of cosyntropin were determined for each individual foal at each of the 4 ages. Low-dose peak cortisol concentration (LDpeak) was defined as the cortisol concentration achieved 30 minutes after administration of the 10 μg cosyntropin dose. High-dose peak cortisol concentration (HDpeak) similarly was defined as the highest cortisol concentration reached after administration of the 100 μg cosyntropin dose (either 30 or 90 minutes after cosyntropin administration, whichever was higher).
The relative increase in serum cortisol concentrations from basal results also was determined for each cosyntropin dose in each individual foal at each of the 4 ages. The low-dose delta serum cortisol concentration (LDdelta) was defined as the peak cortisol concentration reached after administration of 10 μg cosyntropin minus the basal (time 0) cortisol concentration. The high-dose delta cortisol concentrations (HDdelta) was defined similarly as the peak cortisol concentration reached after administration of 100 μg cosyntropin minus the basal cortisol concentration. The low-dose fold change in cortisol concentration (LD fold change) and high-dose fold change in cortisol concentration (HD fold change) were calculated by dividing the LDpeak and HDpeak cortisol concentrations, respectively, by the basal cortisol concentration.
For the control group, basal ACTH and cortisol concentrations and ACTH : cortisol ratios, HDpeak cortisol concentrations, HDdelta cortisol concentrations, and HD fold change were similarly defined and determined for each individual foal at the 1 sampling age. LDpeak and LDdelta cortisol concentrations and LD fold change were not determined for this group because these foals did not receive the low dose of cosyntropin.
Basal ACTH and cortisol concentrations, basal ACTH : cortisol ratios, LDpeak and HDpeak cortisol concentrations, LDdelta and HDdelta cortisol concentrations, and LD fold change and HD fold change in cortisol concentrations were compared among all foal age groups using the mixed procedure for repeated measures.g Multiple comparisons were conducted with Tukey's test. Spearman's rank correlation testh was used to evaluate for an effect of foal weight and foal sex on cortisol response to cosyntropin (LDdelta and HDdelta cortisol concentrations and LD and HD fold change in cortisol concentrations) for each of the 4 foal age groups. Data also were compared between the control group and the 36–48 hour old foals in the paired ACTH stim group using analysis of variance for repeated measures and Student's t test.g,h Statistical significance was set at P < .05 for all analyses. Data are expressed as mean ±standard deviation.
Effect of Foal Age on Basal ACTH and Cortisol Concentrations
An overall effect of foal age on basal ACTH and cortisol concentrations and ACTH : cortisol ratios was present (Table 1; P = .005, P < .001, and P = .006, respectively). At birth, foals had significantly higher basal ACTH (P = .002 to .003) and cortisol (P < .001) concentrations than at any other time point, but no significant differences in either basal ACTH or basal cortisol concentrations were found between any other age group comparisons. An overall effect of age also was present for comparisons of basal ACTH : cortisol ratios (P = .006), but significantly higher ACTH : cortisol ratios only were present in foals at birth as compared with 12–24 hours of age (P= .004).
|Birth (n = 11)||12–24 Hours (n = 10)||36–48 Hours (n = 10)||5–7 Days (n = 11)|
|Basal cortisol (μg/dL)||10.2 ± 2.3a (7.6–13.5)||3.6 ± 1.6b (2.1–6.7)||2.6 ± 1.0b (1.3–4.4)||2.0 ± 0.8b (1.0–2.7)|
|Basal ACTH (pg/ml)||285.5 ± 284.8a (19.4–968)||19.6 ± 5.3b (13.8–30.5)||32.7 ± 26.4b (16.6–35.7)||33.8 ± 23.1b (10.3–110)|
|ACTH:cortisol ratio||27.4 ± 23.8a (2.6–63.4)||6.2 ± 2.0b (2.6–8.7)||13.9 ± 7.4a,b (5.8–23.9)||15.9 ± 5.2a,b (9.8–30.6)|
|LDpeak cortisol (μg/dL)||13.8 ± 3.9a (8.9–21.7)||9.1 ± 2.1b (5.2–12.3)||6.0 ± 1.6c (3.8–8.1)||3.3 ± 0.8d (2.3–4.4)|
|HDpeak cortisol (μg/dL)||16.6 ± 5.1a (12.1–29)||12.9 ± 3.8a (6.8–17.5)||9.2 ± 2.9b (5.5–14.2)||5.5 ± 1.1c (3.5–6.1)|
|LDdelta cortisol (μg/dL)||3.6 ± 2.0a (0.4–8.2)||5.5 ± 2.1b (2.8–9.4)||3.5 ± 1.7a (1.6–6.8)||1.3 ± 0.6c (0.6–2.6)|
|HDdelta cortisol (μg/dL)||6.4 ± 4.1a (0.3–15.5)||9.4 ± 3.5b (4.6–15)||6.8 ± 3.4a,b (3.2–12.7)||3.5 ± 1.3c (1.7–5.9)|
|LD fold change in cortisol||1.4 ± 0.2a (1.0–1.6)||2.9 ± 1.0b (1.4–4.2)||2.8 ± 1.6b (1.6–6.2)||1.8 ± 0.5a,b (1.2–2.7)|
|HD fold change in cortisol||1.6 ± 0.4a (1–2.1)||4.0 ± 1.7b (1.6–6.2)||4.5 ± 2.8b (2–9.5)||3.2 ± 1.5a,b (1.5–5.7)|
Cortisol Responses to Cosyntropin in the Paired ACTH Stim and Control Groups
There were no adverse effects noted after administration of either dose of cosyntropin in any foal in either group. Basal ACTH and cortisol concentrations and cosyntropin dosages based on weight were not significantly different between the control group and age-matched foals (36–48 hours old) in the paired ACTH stim group (Fig 2; P= .995, .742, and .218, respectively). In the control group, cortisol concentrations at 30 and 90 minutes after administration of the “sham” low dose were not significantly different from basal cortisol concentration (P= .997 to .999). Thirty-minute cortisol concentrations however were significantly higher in the 36–48-hour-old paired ACTH stim group than 30-minute cortisol concentrations in the control group (P < .001). There were no significant differences in the response to the high dose of cosyntropin between the 2 groups (HDpeak: P= .705; HDdelta: P= .549; HDfold change: P= .900).
Effect of Cosyntropin Dose on Cortisol Responses within Age Groups
Data from the paired ACTH stim test group are shown in Table 1 and Figure 3. Foals in all age groups showed a significant cortisol response to both the 10 and 100 μg doses of cosyntropin (P < .001). Cortisol concentrations were significantly increased from basal (time 0) concentrations at 30 minutes after administration of 10 μg cosyntropin at all ages (P < .001 to .002). Ninety minutes after administration of 10 μg cosyntropin, cortisol concentrations returned to concentrations statistically indistinguishable from basal concentrations in all foal age groups (P = .46 to 1.0). A significant increase in cortisol concentration from basal values also was observed in all age groups at both 30 and 90 minutes after administration of 100 μg cosyntropin (P < .001). In addition, in all 4 age groups, the HDpeak cortisol concentration was significantly higher than the LDpeak cortisol concentration (P < .001 to .008). A similar pattern was observed for the delta cortisols, with all age groups showing significantly higher HDdelta than LDdelta cortisol concentrations (P < .001 to .006). There was no effect of foal weight on delta cortisol concentration or fold change in cortisol concentration after administration of either dose of cosyntropin (P = .287 to .924).
Effect of Foal Age on Cortisol Response to Cosyntropin
An overall effect of foal age also was present for cortisol response to cosyntropin (Table 1). LDpeak and HD peak cortisol concentrations were significantly higher in foals at birth than other age groups (P < .001 to .013), and decreased significantly over the 1st week of life. Similarly, area under the cortisol concentration curve was greatest in foals at birth, and decreased significantly with increasing age (P < .001 for all age comparisons). In addition, the HDpeak cortisol concentration was reached more rapidly in foals at birth, with 6/11 foals reaching the HDpeak at 30 minutes after administration of 100 μg cosyntropin. All foals in the 3 older age groups did not attain their HDpeak cortisol concentration until the 90-minute sample, with the exception of 2 foals at 12–24 hours of age and 1 foal at 36–48 hours of age.
LDdelta cortisol was significantly higher at 12–24 hours than at any other age (P < .001 to .038) and significantly different among all foal age groups except between foals at birth and 36–48 hours of age (P= .999). The HDdelta cortisol showed a similar pattern, but the difference among age groups only reached statistical significance for comparisons between 12 and 24 hours versus 5–7 days (P < .001) and 36–48 hours versus 5–7 days (P = .038). LD fold change and HD fold change in cortisol concentration were significantly lower in foals at birth than at 12–24 and 36–48 hours (P < .001 to .005). No significant differences in LD fold change or HD fold change between other foal age groups were found.
Effect of Foal Sex on Cortisol Response to Cosyntropin
There was no effect of foal sex on LDdelta or HDdelta cortisol concentration or LD fold change or HD fold change in cortisol concentration in any age group (P= .370 to .861), except for foals in the oldest age group (5–7 days). In this group, males had a significantly higher LDdelta cortisol (P= .025), LD fold change (P= .001), and HD fold change (P= .017) than did females. HDDelta cortisol also was higher in males in this age group, but the difference did not reach statistical significance (P= .063).
The results of this study indicate that a paired low dose (10 μg)/high dose (100 μg) cosyntropin stimulation test may be utilized for evaluation of HPA axis function in the neonatal foal. The lack of significant difference in cortisol response to the 100 μg cosyntropin dose between foals in the control group, who received sterile saline instead of the initial 10 μg cosyntropin dose, and age-matched foals in the paired ACTH stim group, who received 10 μg cosyntropin 90 minutes before the 100 μg dose, suggests that this paired ACTH stimulation test protocol does not produce a significant priming or suppressive effect on the cortisol response to the 100 μg dose and provides support for our 1st hypothesis. In addition, the lack of significant increase in cortisol concentrations from baseline concentrations in the foals in the low-dose saline group during the 1st 3 sampling points (0, 30, and 90 minutes) suggest that even with brief periodic restraint and direct venipuncture, the test procedure itself did not induce endogenous activation of the foal's HPA axis.
The results of the present study are consistent with previous reports illustrating the dynamic nature of HPA axis function in the neonatal foal.11–13,24–26 At birth, basal ACTH and cortisol concentrations were significantly higher than resting concentrations previously reported both in older foals and in adult horses,27–29 and were similar to cortisol concentrations recently reported in adult horses with colic (13.6 ± 7.6 μg/dL).i This provides further evidence that the healthy full-term foal is capable of mounting a cortisol response to periparturient stressors.12,30 However, by 12 hours of age, basal ACTH and cortisol concentrations decreased significantly in foals in this study as compared with concentrations at birth, reaching concentrations well below reported means in resting adult horses (approximately 5.4–6.4 μg/dL).28,29 Resting HPA axis function was consistent among 12–24, 36–48 hours, and 5–7 days of age in the foals in this study, and remained at a level below that described in adult horses. This finding is consistent with previous reports in older foals, aged 3 days to 13 weeks.18,27 Thus, whereas the increased cortisol and ACTH concentrations in foals at birth indicate that the neonatal foal's HPA axis is capable of mounting a response to clinically relevant physiologic stress (eg, the stress of parturition), endogenous HPA axis tone appears to be maintained at a lower level in foals during the 1st week of life than in adult horses.
Despite the variations in basal HPA axis tone between foals at birth and the older age groups in this study, foals at all ages produced a significant cortisol response to both the 10 and 100 μg doses of cosyntropin, as evidenced by significant increases in cortisol concentrations from basal concentrations after administration of both doses. These findings support previous reports indicating that the healthy full-term foal is capable of producing a cortisol response to a supraphysiologic amount (1–2 μg/kg, equivalent to the 100 μg dose used in this study) of exogenous ACTH shortly before, at and after the time of parturition.11–13,18 In addition, despite high basal HPA axis activation at birth and the subsequent decreased HPA axis tone during the 1st week of life, healthy full-term foals also are capable of producing a cortisol response to a much lower, more “physiologically relevant,” 10 μg (0.2 μg/kg) dose of exogenous ACTH during this period. Thus, these finding provide support for our 2nd hypothesis, and suggest that this paired ACTH stimulation test protocol, in conjunction with measurement of basal endogenous ACTH and cortisol concentrations, may be utilized to comprehensively evaluate HPA axis function in neonatal foals in a clinical setting. A paired ACTH stimulation test protocol utilizing standard doses of 10 and 100 μg cosyntropin appears to be appropriate for most foals, as evidenced by the lack of a significant effect of foal weight on the cortisol response to either dose of cosyntropin in this study. However, because the majority of the foals evaluated were a similar size and weight, adjustment of the cosyntropin dose on a microgram per kilogram basis occasionally may be necessary in very small or very large foals. The apparent effect of foal sex on cortisol responses seen in the oldest foals in this study may represent the development of sex-related differences in HPA axis function as foals age. However, further investigation of this potential effect in a larger number of older foals is necessary to determine if foal sex significantly impacts responses to HPA axis function testing in a clinical setting.
The results of the present study also provide support for our 3rd and final hypothesis. Whereas differences in endogenous HPA axis function (as evidenced by baseline cortisol and ACTH concentrations and ACTH : cortisol ratios) and response to exogenous ACTH (peak and delta cortisol concentrations) did not reach statistical significance between all foal age groups for all results, a significant effect of foal age on the cortisol response to ACTH during the neonatal period was observed. Although the pattern of cortisol response to both the low and high doses of cosyntropin was similar across age groups in this study, the magnitude of basal HPA axis activation and peak and delta cortisol responses to exogenous ACTH was highest in the 24 hours after parturition, and steadily decreased over the 1st week. A trend toward lower basal cortisol concentrations and significantly lower delta cortisols was apparent as foals aged to 5–7 days. These findings, and the decrease in the magnitude of both LDpeak and HDpeak cortisol concentrations observed over the 1st week of age, may represent some degree of limited corticosteroid synthetic capacity in the foal. Induction of the steroidogenic enzyme 17-α-hydroxylase necessary for corticosteroid synthesis occurs just before parturition in the foal.31,32 If this enzymatic machinery is not fully mature during the 1st week of life, the newborn foal may be unable to maintain cortisol synthesis at adult levels much beyond the time of parturition.
This trend toward a decreasing response to cosyntropin over the 1st week of life, however, could be artifactual, because of some degree of exhaustion of corticosteroid synthetic capacity caused by the serial ACTH stimulation tests utilized in this study design. This seems unlikely, however, because serial ACTH stimulation tests were performed closer together (12–24 hours apart) in the initial 3 age groups, versus at least 72 hours apart between foals aged 36–48 hours and 5–7 days. Thus, the oldest foals had the longest time to recover corticosteroid synthetic capacity between serial stimulation tests. Alternatively, serial administration of cosyntropin in this study may have induced tolerance to cosyntropin, perhaps mediated by ACTH receptor downregulation or production of anti-cosyntropin antibodies, and contributed to the decreased cortisol response to cosyntropin seen with increasing foal age. Evaluation of ACTH stimulation tests at a single time point in individual foals of different ages would have eliminated any effects of serial testing, but would have required a much larger group of foals and greatly increased interhorse variability in this analysis.
These findings also provide evidence to support previous work indicating that adrenocortical sensitivity to both endogenous and exogenous ACTH may be generally decreased in some foals,9,10 suggesting the observed differences across the 1st week of life in this study may indeed represent true differences in the neonatal foal. Despite lower basal cortisol concentrations than adult horses, basal ACTH concentrations in the foals in all age groups were notably higher than endogenous ACTH concentrations previously reported in healthy adult horses during the spring (foaling season; median plasma ACTH in adult horses in January and May = 16.1–17.1 pg/mL)33 except for foals at 12–24 hours of age. This finding may represent decreased adrenocortical sensitivity to endogenous ACTH in neonatal foals, with higher ACTH concentrations required to produce a comparable cortisol response. Cortisol responses to both doses of exogenous ACTH in the foals in this study (Table 1) also were lower than responses reported in human infants (basal cortisol = 14.8 ± 1.9 μg/dL; LDpeak = 17.4 μg/dL; HDpeak = 24.5 μg/dL) receiving comparable doses of ACTH.17,34 In addition, except for foals at 12–24 hours of age, the delta cortisol responses to even a supraphysiologic (100 μg, 2 μg/kg) dose of cosyntropin observed in foals in this study were substantially lower than the delta cortisol values reported in both adult horses (approximately 9 μg/dL) and human infants (approximately 69 μg/dL) in response to an equivalent dose of cosyntropin.17,35 Thus, both basal HPA axis activation and the cortisol response to both low and high doses of exogenous ACTH appear to be blunted in neonatal foals in comparison with mature horses and human infants. However, considering the large degree of individual variation in endogenous ACTH concentrations and delta cortisol concentrations in the foals in this study, investigation of these parameters in a larger group of foals would be needed to allow more definitive conclusions.
If some degree of HPA axis immaturity does indeed persist into the postnatal period, the neonatal foal's ability to cope with the substantial physiologic stresses induced by illness may be limited, potentially increasing the risk for development of HPA axis dysfunction (RAI/CIRCI) in critically ill foals. However, the differences in HPA axis function in neonatal foals found in this and other studies11–13 alternatively may represent beneficial periparturient adaptations unique to the foal or the neonatal period. Comprehensive HPA axis assessment thus should be considered in critically ill neonatal foals to determine if clinically important associations between severe illness, HPA axis dysfunction and decreased survival occur in foals, as in other species.3,6,7,9,15
In summary, these findings suggest that evaluation of basal ACTH and cortisol concentrations, basal ACTH : cortisol ratios, and cortisol responses to a paired low dose (10 μg)/high dose (100 μg) cosyntropin stimulation test can provide comprehensive assessment of the HPA axis in the foal. However, due to the unique species and age-related differences in HPA axis function in the neonatal foal, appropriate interpretation of both basal hormone concentrations and ACTH stimulation testing responses requires careful consideration of foal age. These findings also provide evidence for persistent HPA axis immaturity during the 1st week of life in the foal, which may substantially impact the foal's ability to cope with the stress of illness during the neonatal period.
aHart KA, Slovis NM, Barton MH. 2008. Hypothalamic-pituitary-adrenal axis dysfunction in critically ill neonatal foals. 26th Annual Forum of the American College of Veterinary Internal Medicine, San Antonia, TX, June 2008 (research abstract)
bSNAP Foal IgG Test, IDEXX Laboratories Inc, Westbrook, ME
cFoal Alert Inc, Atlanta, GA
dCortrosyn, Amphastar Pharmaceuticals Inc, Rancho Cucamonga, CA
eImmulite cortisol assay package insert, Diagnostics Product Corporation, Los Angeles, CA
fImmulite, Diagnostics Product Corporation
gSAS Statistical Software (Version 9.1), SAS Institute Inc, Cary, NC
hGraphPad Prism (Version 4), GraphPad Software Inc, San Diego, CA
iSherlock CE, Mair TS. 2008. Serum cortisol concentrations in horses with colic. 9th International Equine Colic Research Symposium, Liverpool, UK, June 2008 (research abstract)
The authors acknowledge Loralei Jones and Paul Stamper for assistance with and care of the animals used in this study; Jon Gabbard, Michelle Coleman, Erin Master, Dee Whelchel, Jenn Winnick, Chrissy Abreu, and Carly Whittal for assistance in sample collection; Shay Bush for technical assistance; and Dr Deborah Keys for statistical analyses. This study was funded by the American College of Veterinary Internal Medicine Foundation.
- 17Circulating adrenocorticotropic hormone (ACTH) and cortisol concentration in normal, appropriate-for-gestational-age newborns versus those with sepsis and respiratory distress: Cortisol response to low-dose and standard-dose ACTH tests. Metabolism 2004;53:209–214., , , et al.
- 32Endocrinology of the late gestational mare in health and disease. 26th Annual American College of Veterinary Internal Medicine Forum, San Antonio, TX, 2008.