An increase in circulating glucocorticoid concentrations is an essential component of the stress response, which is necessary for homeostasis during critical illness. Inflammatory mediators such as tumor necrosis factor-α, interleukin-1, and interleukin-6 are released in response to illness and can suppress secretion of corticotropin-releasing hormone and ACTH, cause ACTH resistance, and alter cortisol secretion and metabolism.1–4 It has been estimated that 30% to 77% of high-risk critically ill humans (ie, patients with conditions such as severe sepsis, septic shock, trauma, head injury, burns, liver failure, or pancreatitis and patients that have undergone cardiac surgery) have CIRCI,2,5–12 depending on the population of patients studied and the diagnostic criteria. The transient adrenal gland dysfunction associated with severe illness has previously been known as relative adrenal gland insufficiency but is now identified as CIRCI. Critical illness-related corticosteroid insufficiency is characterized by dysfunction of the entire HPA axis, including tissue corticosteroid resistance, and by an excessive and protracted inflammatory response. This condition is associated with increased morbidity and mortality rates,7,10,13,14 and recognition and treatment of CIRCI in human patients dramatically improves the survival rate.10,11,15,16
Critical illness–related corticosteroid insufficiency may also exist in animals. Although the best method for diagnosing CIRCI is controversial, the variable most commonly used in humans is identification of a low delta cortisol concentration, where delta cortisol concentration is the difference between baseline and ACTH-stimulated cortisol concentrations. Low delta cortisol concentrations have been identified in critically ill dogs, suggesting that CIRCI may exist in this population.17,18 Transient CIRCI has been reported in a septic neonatal foal, a cat, and a dog that were all successfully treated with glucocorticoids.19–21 A high-dose (125 μg/foal) ACTH stimulation test has been used to diagnose CIRCI in premature foals and was advocated as a method of assessing maturity.22 Another parameter used to identify CIRCI in human patients is a high ACTH-to-cortisol concentration ratio, and in 2 populations of foals with sepsis, nonsurvivors had significantly higher ratios than did surviving foals, suggesting that the former group possibly had CIRCI.23,24 Lastly, adrenal dysfunction has recently been identified in neonatal foals with sepsis by means of a low-dose (0.1 μg/kg [0.045 μg/lb]) ACTH stimulation test and a paired low- (10 μg/foal) and high-dose (100 μg/foal) ACTH stimulation test.25,26
Critical illness–related corticosteroid insufficiency likely occurs in critically ill adult horses. Unfortunately, little is known about the effects of severe illness on function of the HPA axis in adult horses. In a small study27 of horses examined because of colic at a referral institution, a high serum cortisol concentration at the time of patient admission was significantly associated with an increased risk of death. The high baseline cortisol concentration in these horses may have been associated with a low delta cortisol concentration, but ACTH stimulation tests were not performed. In addition, adrenal function throughout the period of hospitalization has not been assessed in adult horses. In hospitalized horses that subsequently underwent necropsy, extensive adrenal gland hemorrhage, venous thrombosis, and adrenocortical necrosis are common findings,28 which could provide a structural as well as a functional basis for development of CIRCI.
Traditionally, to diagnose absolute adrenal insufficiency in human patients, supraphysiologic ACTH doses have been administered (eg, 250 μg/human patient or 2 to 5 μg/kg [0.9 to 2.3 μg/lb]).29,30 However, in critically ill humans, a normal response to high doses of ACTH does not rule out CIRCI, which is believed to be caused in part by adrenal resistance to ACTH.14,31 Administration of 250 μg of ACTH to human patients creates circulating ACTH concentrations 100 times concentrations secreted endogenously in response to stress and can override adrenal resistance.32 In comparison, a low-dose (1 to 2 μg/human patient) ACTH stimulation test appears to be more sensitive at diagnosing early and mild CIRCI and, accordingly, is the preferred test in human patients.14,33,34
Doses of ACTH that have been administered to adult horses range from 2.5 to 2,000 μg/horse or approximately 0.0025 to 4 μg/kg (0.0011 to 1.8 μg/lb) IV or IM,35–37 with the lowest published ACTH dose used for stimulation tests being 250 μg/horse or approximately 0.5 μg/kg (0.23 μg/lb) IV.38,39 Although the maximal adrenal response in horses has not been determined, an ACTH dose of 0.5 μg/kg IV results in a higher cortisol concentration than that achieved with maximal treadmill exercise.36 Similarly, the lowest dose of exogenous ACTH that will maximally stimulate the adrenal glands in horses has not been identified.
When ACTH stimulation tests are performed on critically ill patients, some concern exists that administration of ACTH may affect the results of a CBC and therefore subsequently interfere with clinical assessment. Endogenous cortisol secretion or exogenous steroid administration leads to an increase in neutrophil and monocyte counts and a decrease in lymphocyte and eosinophil counts. It is unknown whether low doses of ACTH would affect hematologic parameters and, if so, how long these effects would persist. In addition, if immediate assessment of cortisol concentrations were not available, it is unknown whether assessment of changes in hematologic parameters after ACTH administration could be used as an indirect assessment of adrenal production of cortisol.
Our hypothesis was that lower ACTH doses than the currently recommended 0.5 μg/kg dose can be used to maximally stimulate the HPA axis in healthy adult horses. Thus, the purposes of the study reported here were to determine the lowest ACTH dose that would consistently induce a significant increase in cortisol concentration in healthy adult horses, determine the lowest ACTH dose required to induce a maximum increase in cortisol concentration, identify the time to peak cortisol concentration after administration of low doses of ACTH, and determine the effect of administration of low doses of ACTH on hematologic values.
Materials and Methods
Animals—All aspects of the study were approved by the Auburn University Institutional Laboratory Animal Care and Use Committee. Eight healthy university-owned horses (4 mares and 4 geldings) were studied. Horses ranged in age from 2 to 12 years (mean ± SD, 7.4 ± 3.5 years) and in weight from 405 to 598 kg (891 to 1,316 lb; mean ± SD, 493 ± 67 kg [1,085 ± 147 lb]). Breeds represented included Quarter Horse (n = 3), Thoroughbred (2), Arabian (2), and Tennessee Walking Horse (1). All horses were deemed to be healthy on the basis of history, physical examination findings, and results of a CBC and serum biochemical profile and measurement of fibrinogen concentration. All horses were free from any signs of pituitary pars intermedia dysfunction. No horse had received any medication in the preceding 2 months.
The study was performed in late January and early February, and tests commenced at 7 am. The day prior to a test, horses were moved from small paddocks to box stalls. They continued to eat the same coastal Bermuda grass hay throughout the study. Hay and water were offered free choice.
ACTH stimulation tests—In a randomized crossover design, 5 ACTH stimulation tests were performed on each horse, with a 3-day period between tests. For each test, saline (0.9% NaCl) solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 μg/kg [0.009, 0.045, 0.114, and 0.227 μg/lb]) of cosyntropin (synthetic ACTH)a was administered IV into a jugular vein. Lyophilized cosyntropin was diluted with 1.0 mL of sterile saline solution in accordance with the manufacturer's instructions. Cosyntropin was then further diluted with sterile saline solution to a final concentration of either 10 μg/mL (for administration of the 0.25 and 0.5 μg/kg doses) or 1 μg/mL (for administration of the 0.02 and 0.1 μg/kg doses). Unused reconstituted cosyntropin solution (1 μg/mL) was refrigerated in the original plastic saline solution bag for use within the next week. Diluted cosyntropin solution has been shown to remain fully stable (in concentrations as low as 0.5 μg/mL) for at least 4 months when refrigerated in plastic containers.40
Collection of blood samples—An IV catheterb was aseptically placed in 1 jugular vein for collection of blood samples. Blood samples were collected before (0 minutes [baseline]) and 30, 60, 90, 120, 180, and 240 minutes after injection of cosyntropin or saline solution. At each time point, 10 mL of blood was obtained and placed in a standard serum clot tube. Blood samples were allowed to clot for 30 minutes at room temperature and were then centrifuged; serum was collected and frozen at −20°C until analyzed. Before injection of cosyntropin or saline solution, an additional 10 mL of blood was obtained for measurement of endogenous ACTH concentration; the sample was placed in a cold evacuated tube containing EDTA and was mixed with 0.5 mL of aprotinin (a protease inhibitorc; final concentration, 500 kallikrein inactivator units/mL of blood), as described.41 Samples were transported on ice and centrifuged within 20 minutes after collection. The plasma was removed, placed in a plastic tube, and frozen at −20°C until assayed. Blood (10 mL) was also collected into evacuated tubes containing EDTA before and 60, 120, and 240 minutes after injection of cosyntropin or saline solution for routine CBCs. Prior to the collection of each blood sample, the catheter was flushed with 5 mL of saline solution and then 5 mL of blood was withdrawn and discarded. After sample collection, the catheter was flushed with 5 mL of saline solution containing 10 U of heparin/mL.
Sample analysis—Plasma endogenous ACTH and serum total cortisol concentrations were measured in duplicate. Plasma endogenous ACTH concentration was measured by use of a sandwich immunoradiometric assayd previously validated for use in horses.36,38 The sensitivity of the assay was 1.0 pg/mL, and the interassay and intra-assay coefficients of variation were, for values < 30 pg/mL, 9.2% and 4.5%, respectively.38 Serum cortisol concentrations were determined by use of a previously validated direct radioimmunoassay.36,e The sensitivity of the assay was 14.0 nmol/L. The interassay and intra-assay coefficients of variation ranged from 7.1% to 7.7% and from 6.1% to 8.1%, respectively.36 Complete blood counts were performed with an automated analyzerf; differential cell counts were performed by hand.
Statistical analysis—Results were summarized as mean ± SD. For each time point in each horse, delta cortisol concentration was calculated as cortisol concentration measured at that time minus the baseline cortisol concentration. Differences in endogenous cortisol and ACTH concentrations, ACTH-stimulated cortisol concentrations, delta cortisol concentrations, and CBC results over time were analyzed by means of repeated-measures ANOVA followed by the least significant difference test. Analyses were performed with standard software.g The dose effect at each time point was determined by comparing the ACTH-stimulated cortisol concentration with the cortisol concentration measured after administration of saline solution. For each cosyntropin dose, cortisol concentration measured at each time point was compared with the baseline concentration. Baseline endogenous serum cortisol and plasma ACTH concentrations were also compared among days that samples were collected. Differences in maximum cortisol concentration and time of maximum cortisol concentration between doses were determined by use of the Friedman repeated-measures test, with post hoc comparisons done by use of the Student-Newman-Keuls method.h For all analyses, values of P < 0.05 were considered significant.
Results
Baseline endogenous serum cortisol and plasma ACTH concentrations in the 40 samples obtained prior to the 5 tests performed in the 8 horses ranged from 76 to 264 nmol/L (mean ± SD, 172.4 ± 44.8 nmol/L) and from 6 to 37 pg/mL (19.2 ± 5.6 pg/mL), respectively. Mean baseline serum cortisol and plasma ACTH concentrations did not differ among test days. No significant increases in serum cortisol concentration were detected after administration of saline solution; however, there was a significant decrease in serum cortisol concentration 240 minutes after administration of saline solution, compared with the baseline concentration (Figure 1).
Mean serum cortisol concentration in 8 healthy adult horses before (0 minutes) and after administration of saline (0.9% NaCl) solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 μg/kg [0.009, 0.045, 0.114, and 0.227 μg/lb]) of cosyntropin in a crossover study. *Significantly (P ≤ 0.05) different from baseline (time 0) concentration for that dose. †Significantly (P ≤ 0.05) different from concentration measured at the same time point after administration of saline solution.
Citation: Journal of the American Veterinary Medical Association 239, 6; 10.2460/javma.239.6.834
Mean serum cortisol concentration in 8 healthy adult horses before (0 minutes) and after administration of saline (0.9% NaCl) solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 μg/kg [0.009, 0.045, 0.114, and 0.227 μg/lb]) of cosyntropin in a crossover study. *Significantly (P ≤ 0.05) different from baseline (time 0) concentration for that dose. †Significantly (P ≤ 0.05) different from concentration measured at the same time point after administration of saline solution.
Citation: Journal of the American Veterinary Medical Association 239, 6; 10.2460/javma.239.6.834
Mean serum cortisol concentration in 8 healthy adult horses before (0 minutes) and after administration of saline (0.9% NaCl) solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 μg/kg [0.009, 0.045, 0.114, and 0.227 μg/lb]) of cosyntropin in a crossover study. *Significantly (P ≤ 0.05) different from baseline (time 0) concentration for that dose. †Significantly (P ≤ 0.05) different from concentration measured at the same time point after administration of saline solution.
Citation: Journal of the American Veterinary Medical Association 239, 6; 10.2460/javma.239.6.834
For all 4 doses of cosyntropin, serum cortisol concentration was significantly increased, compared with baseline concentrations, 30 and 60 minutes after cosyntropin administration, and concentration 30 minutes after cosyntropin administration was significantly increased, compared with concentration 30 minutes after administration of saline solution. Serum cortisol concentration peaked 30 minutes after administration of cosyntropin at a dose of 0.02 μg/kg, with peak concentration 1.5 times the baseline concentration, and was significantly increased, compared with the baseline concentration, 30 and 60 minutes after cosyntropin administration (Table 1). Serum cortisol concentration peaked 30 minutes after administration of cosyntropin at a dose of 0.1 μg/kg, with peak concentration 1.9 times the baseline concentration, and was significantly increased, compared with the baseline concentration, 30, 60, and 90 minutes after cosyntropin administration. Serum cortisol concentration peaked 90 minutes after administration of cosyntropin at a dose of 0.25 μg/kg, with peak concentration 2.0 times the baseline concentration, and was significantly increased, compared with the baseline concentration, 30, 60, 90, and 120 minutes after cosyntropin administration. Serum cortisol concentration peaked 90 minutes after administration of cosyntropin at a dose of 0.5 μg/kg, with peak concentration 2.3 times the baseline concentration, and was significantly increased, compared with the baseline concentration, 30, 60, 90, 120, and 240 minutes after cosyntropin administration.
Serum cortisol concentrations (nmol/L) in 8 healthy adult horses before (0 minutes) and after administration of saline (0.9% NaCl) solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 μg/kg [0.009, 0.045, 0.114, and 0.227 μg/lb]) of cosyntropin in a crossover study.
| Time (min) | |||||||
|---|---|---|---|---|---|---|---|
| Cosyntropin dose (μg/kg) | 0 | 30 | 60 | 90 | 120 | 180 | 240 |
| 0 (saline solution) | 182 ± 54 | 169 ± 67 | 174 ± 81 | 198 ± 77 | 164 ± 32 | 176 ± 43 | 119 ± 14* |
| (109–264) | (78–284) | (59–311) | (77–313) | (122–209) | (112–235) | (103–143) | |
| 0.02 | 182 ± 52* | 277 ± 59a | 227 ± 46 | 176 ± 46* | 169 ± 74* | 120 ± 49* | 108 ± 51* |
| (120–241) | (165–290) | (165–290) | (140–272) | (99–311) | (76–206) | (42–185) | |
| 0.1 | 167 ± 35* | 323 ± 54b | 322 ± 53 | 246 ± 55 | 219 ± 61* | 158 ± 31* | 135 ± 70* |
| (104–210) | (251–394) | (249–414) | (193–337) | (159–314) | (128–203) | (66–283) | |
| 0.25 | 175 ± 39* | 337 ± 80 | 341 ± 86 | 343 ± 83b | 295 ± 77 | 212 ± 65* | 170 ± 50* |
| (120–229) | (211–489) | (236–468) | (248–486) | (189–425) | (134–314) | (107–265) | |
| 0.5 | 156 ± 48* | 304 ± 55 | 340 ± 80 | 363 ± 74b | 352 ± 95 | 285 ± 78 | 201 ± 54* |
| (76–236) | (240–402) | (214–468) | (243–466) | (240–555) | (196–386) | (151–318) | |
Data are reported as mean ± SD (range).
Significantly (P ≤ 0.05) different from peak concentration for that dose.
Superscript letters indicate peak concentration for each dose; peak concentrations with different superscript letters were significantly (P ≤ 0.05) different.
After administration of cosyntropin at a dose of 0.02 μg/kg, no significant difference was detected between serum cortisol concentrations at 30 and 60 minutes. After administration of cosyntropin at a dose of 0.1 μg/kg, no significant differences were detected among serum cortisol concentrations at 30, 60, and 90 minutes. After administration of cosyntropin at a dose of 0.25 μg/kg, no significant differences were detected among serum cortisol concentrations at 30, 60, 90, and 120 minutes. After administration of cosyntropin at a dose of 0.5 μg/kg, no significant differences were detected among serum cortisol concentrations at 30, 60, 90, 120, and 180 minutes. No significant differences were detected among maximum serum cortisol concentrations obtained in response to administration of cosyntropin at doses of 0.1, 0.25, and 0.5 μg/kg. Therefore, a maximum adrenal response was obtained between 30 and 90 minutes after administration of cosyntropin at a dose of 0.1 μg/kg, between 30 and 120 minutes after administration of cosyntropin at a dose of 0.25 μg/kg, and between 30 and 180 minutes after administration of cosyntropin at a dose of 0.5 μg/kg.
No significant differences were detected among delta cortisol concentrations 30 minutes after administration of cosyntropin at a dose of 0.02 μg/kg and delta cortisol concentrations 30 and 60 minutes after administration of cosyntropin at a dose of 0.1 μg/kg (Table 2). Delta cortisol concentration peaked 90 minutes after administration of cosyntropin at a dose of 0.25 or 0.5 μg/kg, and for both doses, peak delta cortisol concentration was significantly higher than peak delta cortisol concentration after administration of cosyntropin at the 2 lower doses.
Delta cortisol concentration (nmol/L; calculated as cortisol concentration measured at each time point minus the baseline [time 0] cortisol concentration) in 8 healthy adult horses given saline solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 μg/kg) of cosyntropin in a crossover study.
| Time (min) | ||||||
|---|---|---|---|---|---|---|
| Cosyntropin dose (μg/kg) | 30 | 60 | 90 | 120 | 180 | 240 |
| 0 (saline solution) | −13 ± 75 | −7.5 ± 77 | 17 ± 63 | −17 ± 41 | −27 ± 62 | −62 ± 46 |
| (−109 to 125) | (−81 to 152) | (−56 to 98) | (−80 to 49) | (−133 to 49) | (−123 to −6) | |
| 0.02 | 95 ± 28a | 41 ± 16 | −6.0 ± 49 | −9.3 ± 54 | −55.8 ± 51* | −78 ± 34* |
| (42 to −133) | (21 to 70) | (−95 to 54) | (−85 to 70) | (−136 to 28) | (−129 to −25) | |
| 0.1 | 156 ± 40a | 156 ± 45a | 79 ± 46 | 58 ± 54 | 4.8 ± 38* | −31.5 ± 65* |
| (97 to 215) | (87 to 207) | (24 to 133) | (−8 to 117) | (−33 to 62) | (−83 to 114) | |
| 0.25 | 162 ± 60 | 166 ± 64 | 167 ± 61b | 120 ± 63 | 40 ± 42* | −5.4 ± 43* |
| (91 to 267) | (81 to 264) | (81 to 264) | (11 to 203) | (−25 to 92) | (−74 to 43) | |
| 0.5 | 148 ± 35 | 184 ± 50 | 207 ± 49b | 196 ± 53 | 115.5 ± 41 | 44 ± 28* |
| (115 to 209) | (89 to 242) | (118 to 254) | (152 to 319) | (71 to 175) | (7.0 to 82) | |
Significantly different from peak delta cortisol concentration for that dose.
See Table 1 for remainder of key.
Significant effects on hematologic variables were found in response to cosyntropin administration but not in response to administration of saline solution (Table 3). Significant increases in WBC count, neutrophil count, and the neutrophil-to-lymphocyte ratio and significant decreases in eosinophil count were detected. The magnitude and timing of the changes were dependent on the dose of cosyntropin. The most clinically important change was in the neutrophil-to-lymphocyte ratio; there was a doubling of the ratio, compared with the baseline ratio, 240 minutes after administration of cosyntropin at a dose of 0.25 or 0.5 μg/kg, although a significant difference was detected only after administration of the higher dose. For all 4 doses of cosyntropin, there were no significant differences in lymphocyte count, monocyte count, Hct, or RBC count over time.
Results of hematologic testing for 8 healthy adult horses before (0 minutes) and after administration of saline solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 μg/kg) of cosyntropin in a crossover study.
| Time (min) | |||||
|---|---|---|---|---|---|
| Variable | Cosyntropin dose (μg/kg) | 0 | 60 | 120 | 240 |
| Total WBC count (cells/μL) | 0 (saline solution) | 6,852 ± 1,271 | 6,161 ± 1,336 | 6,136 ± 1,314 | 6,536 ± 1,847 |
| (4,300–8,260) | (3,810–8,060) | (4,040–7,880) | (3,650–9,260) | ||
| 0.02 | 6,903 ± 1,433 | 5,779 ± 1,070 | 6,575 ± 1,271 | 7,189 ± 1,615 | |
| (4,350–9,660) | (3,220–6,740) | (4,240–8,220) | (4,760–9,740) | ||
| 0.1 | 6,740 ± 1,137 | 5,980 ± 1,296 | 6,327 ± 696 | 7,208 ± 1,542 | |
| (4,700–7,950) | (3,730–7,640) | (4,560–7,290) | (5,110–9,300) | ||
| 0.25 | 6,963 ± 1,420 | 6,373 ± 713 | 7,066 ± 1,623 | 8,567 ± 1,559 | |
| (4,400–8,950)a | (4,810–6,910)a | (5,660–10,590)a | (7,000–11,550)b | ||
| 0.5 | 6,561 ± 1,273 | 5,595 ± 1,106 | 6,412 ± 1,342 | 8,079 ± 1,141 | |
| (4,320–8,080)a | (4,020–6,880)a | (4,150–7,910)a | (5,720–9,320)b | ||
| Neutrophil count (cells/μL) | 0 (saline solution) | 3,935 ± 1,108 | 3,373 ± 770 | 3,749 ± 1,178 | 3,843 ± 1,279 |
| (2,322–6,112) | (2,438–4,627) | (2,060–5,201) | (1,862–6,112) | ||
| 0.02 | 3,770 ± 978 | 3,322 ± 773 | 4,244 ± 1,236 | 4,592 ± 1,332 | |
| (2,262–5,313) | (1,996–4,326) | (2,451–6,165) | (2,881–6,623) | ||
| 0.1 | 3,803 ± 806 | 3,337 ± 679 | 4,080 ± 657 | 4,913 ± 1,143 | |
| (2,444–5,009)a | (2,126–4,228)a | (2,964–4,977)a,b | (3,475–5,972)b | ||
| 0.25 | 4,245 ± 857 | 3,560 ± 652 | 4,649 ± 1,880 | 6,312 ± 1,754 | |
| (2,816–5,566)a | (2,939–4,906)a | (3,193–8,790)a,b | (4,831–9,933)b | ||
| 0.5 | 3,550 ± 622 | 2,973 ± 786 | 4,091 ± 1,012 | 5,654 ± 731 | |
| (2,722–4,755)a,b | (1,918–3,881)a | (2,532–5,234)b | (4,519–6,645)c | ||
| Neutrophil-to-lymphocyte ratio | 0 (saline solution) | 1.7 ± 0.90 | 1.9 ± 0.96 | 1.9 ± 0.96 | 1.6 ± 0.54 |
| (0.75–3.4) | (1.1–3.9) | (0.8–3.9) | (0.96–1.4) | ||
| 0.02 | 1.4 ± 0.38 | 1.6 ± 0.54 | 2.2 ± 0.85 | 2.1 ± 0.67 | |
| (0.83–2.1)a | (0.79–2.6)a,b | (0.85–3.4)b | (0.78–3.0)a,b | ||
| 0.1 | 1.6 ± 0.64 | 1.4 ± 0.14 | 2.0 ± 0.53 | 2.5 ± 1.0 | |
| (0.90–2.6)a | (1.3–1.6)a | (1.3–2.8)a,b | (1.4–3.9)b | ||
| 0.25 | 1.9 ± 0.73 | 1.6 ± 0.63 | 2.27 ± 1.5 | 4.1 ± 3.1 | |
| (1.2–3.0) | (0.9–2.7) | (1.1–4.9) | (1.4–11) | ||
| 0.5 | 1.4 ± 0.4 | 1.4 ± 0.59 | 2.1 ± 0.65 | 2.99 ± 1.0 | |
| (0.92–2.1)a | (0.73–2.4)a,c | (1.1–3.0)c | (1.7–4.4)d | ||
| Eosinophil count (cells/μL) | 0 (saline solution) | 117 ± 124 | 72 ± 66 | 95 ± 160 | 125 ± 254 |
| (0–298) | (0–161) | (0–465) | (0–747) | ||
| 0.02 | 119 ± 116 | 87 ± 76 | 71 ± 64 | 145 ± 140 | |
| (0–386) | (0–202) | (0–153) | (0–425) | ||
| 0.1 | 165 ± 133 | 67 ± 60 | 8.4 ± 22 | 67 ± 106 | |
| (0–398)a | (0–146)b | (0–59)b | (0–241)a,b | ||
| 0.25 | 143 ± 181 | 133 ± 115 | 114 ± 212 | 25 ± 67 | |
| (0–537) | (0–333) | (0–631) | (0–176) | ||
| 0.5 | 150 ± 179 | 110 ± 164 | 112 ± 96 | 67 ± 118 | |
| (0–465) | (0–493) | (0–316) | (0–321) | ||
Data are reported as mean ± SD (range).
Within each row, values with different superscript letters were significantly (P ≤ 0.05) different.
Discussion
Results of the present study suggested that administration of cosyntropin at a dose as low as 0.02 μg/kg was sufficient to cause a significant increase in serum cortisol concentration 30 minutes after injection in healthy adult horses. No significant differences were detected among maximum serum cortisol concentrations obtained in response to administration of cosyntropin at doses of 0.1, 0.25, and 0.5 μg/kg, although the time to maximum cortisol concentration was delayed at the 2 higher doses. Maximum adrenal response was obtained between 30 and 90 minutes after administration of cosyntropin at a dose of 0.1 μg/kg, between 30 and 120 minutes after administration of cosyntropin at a dose of 0.25 μg/kg, and between 30 and 180 minutes after administration of cosyntropin at a dose of 0.5 μg/kg. Further studies involving administration of cosyntropin at these doses to critically ill horses with possible CIRCI are needed to determine whether a low-dose ACTH stimulation test can be used to identify CIRCI in critically ill horses.
Previous reports35,38,39,42–45 of ACTH tests performed in adult horses described the use of much higher doses than used in the present study. To our knowledge, there have been no previous studies comparing various doses of ACTH to determine the lowest dose that would consistently cause a significant increase in serum cortisol concentration. In these previous studies, the lowest dose of ACTH administered to adult horses for use in an ACTH stimulation test was 0.5 μg/kg IV, and this dose resulted in cortisol concentrations that were higher than those obtained with maximum exertion during treadmill exercise.38,39 For this reason, we selected 0.5 μg/kg as the highest dose in our study.
Recently, serum cortisol concentrations were found to be significantly increased after IV administration of cosyntropin at doses of 0.2, 2, and 5 μg/kg (0.09, 0.9, and 2.3 μg/lb) to healthy 3- to 4-day-old foals but not after administration at a dose of 0.02 μg/kg.46 No significant difference was detected in the area under the cortisol concentration-versus-time curve between the 2 highest doses, and the authors suggested using a dose between 0.2 and 2 μg/kg to evaluate adrenal function in neonatal foals. Mean ± SD baseline serum cortisol concentrations in the neonatal foals in that study were 70.0 ± 24.8 nmol/L (range, 19.3 to 126.9 nmol/L), and concentrations peaked 30 minutes after administration of cosyntropin at a dose of 0.2 μg/kg, with mean ± SD peak concentration of 135 ± 24.8 nmol/L. When cosyntropin was administered at doses of 2 and 5 μg/kg, mean ± SD peak cortisol concentrations were 204.2 ± 63.4 nmol/L and 220.7 ± 52.4 nmol/L, respectively. Baseline concentrations of cortisol were approximately 40% of baseline cortisol concentrations for the adult horses in the present study, although different analytic techniques were used. Further investigation of serum cortisol concentrations in response to low-dose ACTH administration is indicated in foals, especially for doses of cosyntropin between 0.02 and 2 μg/kg, to determine the lowest dose that will result in maximum adrenal gland stimulation in foals.
When performing ACTH stimulation tests, the clinician is faced with a decision between using a cosyntropin dose high enough to obtain a significant increase in cortisol concentration, compared with baseline concentration, versus using a dose that will result in a maximum increase. In dogs, for example, lower ACTH doses are more sensitive in detecting subtle changes in adrenal function.47 For ACTH stimulation testing in dogs, there has been a shift from using a traditional dose of 250 μg/dog to using a dose of 5 μg/kg. The lowest dose of ACTH that would cause maximum adrenal stimulation in dogs was recently found to be 0.5 μg/kg.47 Because the cost of cosyntropin has recently increased substantially, the ability to use a low dose of ACTH for stimulation testing increases the affordability of the test in adult horses. Cortisol concentrations 30 minutes after administration of each cosyntropin dose in the present study were significantly greater than baseline cortisol concentrations and concentration obtained 30 minutes after administration of saline solution. However, although the 0.02 μg/kg dose of cosyntropin resulted in a significant increase in cortisol concentrations in all 8 horses, it did not cause maximum adrenal stimulation, in that peak concentration after administration of cosyntropin at this dose was lower than peak concentrations after administration at the other 3 doses. Because no significant differences were detected in peak cortisol concentrations after administration of the 0.1, 0.25, and 0.5 μg/kg doses of cosyntropin, we conclude that a dose of cosyntropin of 0.1 μg/kg with collection of blood samples for evaluation of serum cortisol concentration between 30 and 60 minutes after cosyntropin administration can be used to assess maximum adrenal response in healthy horses. Cortisol concentrations between 0 and 30 minutes after cosyntropin administration were not assessed and require further investigation to determine whether the maximum cortisol response occurs earlier than 30 minutes at the 0.1 μg/kg dose of cosyntropin. Further investigation of various doses of cosyntropin in critically ill adult horses need to be performed to determine the most appropriate dose to be used for assessing adrenal response in this population.
Use of lower doses of cosyntropin will reduce the cost of ACTH stimulation testing in horses. Multiple doses can be administered from the same 250-μg vial without affecting test results if the vial is stored properly. Reconstituted cosyntropin can be stored in plastic for as long as 4 months in the refrigerator or as long as 6 months at −20°C with no adverse effects on its bioactivity.48
Mean baseline serum cortisol and ACTH concentrations in the horses used in the present study did not differ throughout the study period, ruling out day-today variations in environmental effects such as temperature, external stimulation, and adrenal gland suppression from repeated ACTH administration. Plasma ACTH concentrations in healthy adult horses reportedly range from 6.5 to 30 pg/mL (mean, 18.7 pg/mL),49 and similar results were obtained in the present study. Seasonal variation in plasma ACTH concentrations has been reported, with markedly higher ACTH concentrations in September, compared with concentrations in January or May.50 The present study was performed in January and February.
The decrease in serum cortisol concentration 240 minutes after administration of saline solution in the present study was likely due to a reduction in endogenous stress levels. By that time, the horses were accustomed to the procedure, and the environment was quieter at the time of blood sample collection.
In a small previous study35 in which a cosyntropin dose of approximately 2 μg/kg was administered IV to 3 mature Thoroughbred or American Saddlebred mares, no significant differences were observed in total leukocyte or eosinophil counts at 30, 60, 120, and 240 minutes, compared with values for 4 control mares that were only subjected to blood sampling. In the present study, statistically significant increases, compared with baseline values, were found in the neutrophil-to-lymphocyte ratio 120 minutes after administration of the 0.02 μg/kg dose of cosyntropin, 240 minutes after administration of the 0.1 μg/kg dose, and 120 and 240 minutes after administration of the 0.5 μg/kg dose. There were also increases in the neutrophil and total WBC counts 240 minutes after administration of both the 0.25 and 0.5 μg/kg doses. Therefore, circulating WBCs may be sensitive to subtle changes in the HPA axis in healthy horses. Differences were possibly not detected in the previous study35 because of a smaller sample size. Thus, it is advisable to collect blood for assessment of hematologic data prior to performing ACTH stimulation testing. There was a doubling of the neutrophil-to-lymphocyte ratio between 0 and 240 minutes after administration of both the 0.25 and 0.5 μg/kg doses of cosyntropin. Subjectively, less variability was observed with the 0.5 μg/kg dose, with an increase in the ratio by a factor of 1.5 and 2.4 between 0 and 240 minutes. Lack of consistency between individual horses would preclude the use of other CBC parameters to assess adrenal responsiveness after cosyntropin administration.
ABBREVIATIONS
| CIRCI | Critical illness-related corticosteroid insufficiency |
| HPA | Hypothalamic-pituitary-adrenal gland |
Cortrosyn, Amphastar Pharmaceuticals Inc, Rancho Cucamonga, Calif.
Angiocath 14-gauge 5.25-inch PEP polymer, Becton-Dickinson Infusion Therapy Systems Inc, Sandy, Utah.
Aprotinin lyophilized powder, USB Corp, Cleveland, Ohio.
ACTH immunoradiometric assay, Nichols Institute, San Clemente, Calif.
Coat-A-Count Cortisol direct radioimmunoassay, Diagnostic Products Corp, Los Angeles, Calif.
Advia 120 Hematology System, Siemens, Tarrytown, NY.
SAS software, version 9.1, SAS Institute Inc, Cary, NC.
SigmaStat for Windows, version 1.0, Jandel Scientific, SPSS Inc, Chicago, Ill.
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