Validation of a low-dose adrenocorticotropic hormone stimulation test in healthy neonatal foals

Allison J. Stewart Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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James C. Wright Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Ellen N. Behrend Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Linda G. Martin Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Robert J. Kemppainen Departments of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Katherine A. Busch Departments of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Abstract

Objective—To determine the lowest ACTH dose that would induce a significant increase in serum cortisol concentration and identify the time to peak cortisol concentration in healthy neonatal foals.

Design—Prospective randomized crossover study.

Animals—11 healthy neonatal foals.

Procedures—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) was administered IV. Serum cortisol concentrations were measured before and 10, 20, 30, 60, 90, 120, 180, and 240 minutes after administration of cosyntropin or saline solution; CBCs were performed before and 30, 60, 120, and 240 minutes after administration.

Results—Serum cortisol concentration was significantly increased, compared with baseline, by 10 minutes after cosyntropin administration at doses of 0.1, 0.25, and 0.5 μg/kg. Serum cortisol concentration peaked 20 minutes after administration of cosyntropin at doses of 0.02, 0.1, and 0.25 μg/kg, with peak concentrations 1.7, 2.0, and 1.9 times the baseline concentration, respectively. Serum cortisol concentration peaked 30 minutes after cosyntropin administration at a dose of 0.5 μg/kg, with peak concentration 2.2 times the baseline concentration. No significant differences were detected among peak serum cortisol concentrations obtained with cosyntropin administration at doses of 0.25 and 0.5 μg/kg. Cosyntropin administration significantly affected the lymphocyte count and the neutrophil-to-lymphocyte ratio.

Conclusions and Clinical Relevance—Results suggested that in healthy neonatal foals, the lowest dose of cosyntropin to result in significant adrenal gland stimulation was 0.25 μg/kg, with peak cortisol concentration 20 minutes after cosyntropin administration.

Abstract

Objective—To determine the lowest ACTH dose that would induce a significant increase in serum cortisol concentration and identify the time to peak cortisol concentration in healthy neonatal foals.

Design—Prospective randomized crossover study.

Animals—11 healthy neonatal foals.

Procedures—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) was administered IV. Serum cortisol concentrations were measured before and 10, 20, 30, 60, 90, 120, 180, and 240 minutes after administration of cosyntropin or saline solution; CBCs were performed before and 30, 60, 120, and 240 minutes after administration.

Results—Serum cortisol concentration was significantly increased, compared with baseline, by 10 minutes after cosyntropin administration at doses of 0.1, 0.25, and 0.5 μg/kg. Serum cortisol concentration peaked 20 minutes after administration of cosyntropin at doses of 0.02, 0.1, and 0.25 μg/kg, with peak concentrations 1.7, 2.0, and 1.9 times the baseline concentration, respectively. Serum cortisol concentration peaked 30 minutes after cosyntropin administration at a dose of 0.5 μg/kg, with peak concentration 2.2 times the baseline concentration. No significant differences were detected among peak serum cortisol concentrations obtained with cosyntropin administration at doses of 0.25 and 0.5 μg/kg. Cosyntropin administration significantly affected the lymphocyte count and the neutrophil-to-lymphocyte ratio.

Conclusions and Clinical Relevance—Results suggested that in healthy neonatal foals, the lowest dose of cosyntropin to result in significant adrenal gland stimulation was 0.25 μg/kg, with peak cortisol concentration 20 minutes after cosyntropin administration.

The low-dose ACTH stimulation test is an important tool in diagnosing mild adrenocortical insufficiency in several species and has been advocated for the diagnosis of the syndrome CIRCI,1–8 a condition associated with increased occurrence of shock, multiple organ dysfunction, and death.1,4 Critical illness–related corticosteroid insufficiency is characterized by dysfunction of any part of the HPA axis, including tissue corticosteroid resistance, and replaces the older term of relative adrenal insufficiency, which described transient adrenal gland dysfunction associated with severe illness.9 It has been estimated that 30% to 77% of critically ill humans have CIRCI,1,9–16 depending on the population of patients studied and the diagnostic criteria used. Recognition and treatment of CIRCI in human patients substantially improves the survival rate.1,15,17,18

Although the best method for diagnosing CIRCI is still a matter of controversy, the method 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,5,6 suggesting that CIRCI may exist in this population. 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.19 Recently, adrenal gland dysfunction has 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-dose and high-dose (10 μg/foal and 100 μg/foal) ACTH stimulation test.7,20

Traditionally, to diagnose absolute adrenal gland insufficiency in human patients, supraphysiologic ACTH doses (eg, 250 μg/patient or 2 to 5 μg/kg [0.9 to 2.3 μg/lb]) were used for the ACTH stimulation test.21,22 However, in critically ill humans, a normal response (ie, values within reference range) to a high dose of ACTH does not rule out CIRCI, which is believed, in part, to be caused by adrenal gland resistance to ACTH.4,23 Administration of 250 μg of ACTH to human patients induces circulating ACTH concentrations that are 100-fold stress-induced concentrations and can override adrenal gland resistance.24 Use of a low-dose ACTH stimulation test (1 μg of cosyntropin/patient or 0.007 to 0.02 μg/kg [0.003 to 0.009 μg/lb) induces a similar cortisol response as does a dose of 250 μg/patient.23 Low-dose ACTH testing is more sensitive than use of superphysiologic doses of ACTH for diagnosis of early and mild CIRCI.3,4,25

Serum cortisol concentrations are significantly increased after IV administration of cosyntropin at doses of 10, 100, and 250 μg/foal (approx 0.2, 2, and 5 μg/kg [0.091, 0.91, and 2.27 μg/lb]) to healthy 3- to 4-day-old foals but not after administration at a dose of 1 μg/foal (0.02 μg/kg).20 In adult horses, 0.1 μg of cosyntropin/kg elicits the same peak cortisol concentration as does 0.25 and 0.5 μg of cosyntropin/kg.26 Further investigation of serum cortisol concentrations in response to low-dose ACTH administration is indicated in foals, especially for doses of cosyntropin from 0.02 to 2 μg/kg.20 Furthermore, peak stimulated cortisol concentrations are reported to occur 30 minutes after administration of 1 and 10 μg of cosyntropin to foals,27 but cortisol concentrations at time points < 30 minutes after ACTH administration have not been investigated. Cortisol release from the adrenal gland occurs rapidly in response to ACTH stimulation, and peak responses to low ACTH doses may occur prior to 30 minutes after cosyntropin administration.26

Endogenous cortisol secretion or exogenous corticosteroid administration leads to an increase in neutrophil and monocyte counts and a decrease in lymphocyte and eosinophil counts. When ACTH stimulation tests are performed on critically ill patients, the possibility exists that ACTH administration may affect the results of a CBC and therefore interfere with clinical assessment. Cosyntropin administration to adult horses significantly affects WBC, neutrophil, and eosinophil counts and the neutrophil-to-lymphocyte ratio.26 The effect of low doses of ACTH on hematologic variables in foals and how long these effects persist in foals are unknown. In addition, whether assessment of changes in hematologic variables after ACTH administration could be used as an indirect assessment of adrenal gland production of cortisol has not been evaluated; assessment of CBC changes may be useful if measurement of cortisol concentrations is not immediately available.

The purpose of the study reported here was to determine the lowest ACTH dose that would consistently induce a significant increase in cortisol concentration in healthy neonatal foals, to identify the time to peak cortisol concentration after administration of low doses of ACTH, and to determine the effect of administration of low doses of ACTH on hematologic values. Our hypothesis was that an ACTH dose < 0.5 μg/kg can be used to stimulate the HPA axis in healthy neonatal foals.

Materials and Methods

Animals—All aspects of the study were approved by the Auburn University Institutional Laboratory Animal Care and Use Committee. Eleven apparently healthy university-owned foals (6 colts and 5 fillies) were studied. All foals were full term (> 330 days gestation) and born without assistance. Foals entered the study at 2 days of age and were 12 days of age by study completion. The foals weighed from 32 to 65 kg (70 to 143 lb; mean ± SD, 52 ± 11 kg [115 ± 24 lb]) at the commencement of the study and were weighed daily during the study. Foals gained 4.5 to 29 kg (10 to 64 lb; 13 ± 7 kg [29 ± 16 lb]) by the end of the study. Breeds represented included Quarter Horse (n = 4), Thoroughbred (3), Tennessee Walking Horse (2), and Warmblood (2). All foals 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 and immunoglobulin concentration (> 800 mg/dL). No foal had received any medication prior to the study.

The study was performed from March until August, and tests commenced at 7 AM. Several hours after birth, foals and their accompanying mares were moved from small paddocks to box stalls. At 1 day of age, the foals were sedated with diazepama (0.2 mg/kg, IV) and butorphanolb (0.06 mg/kg [0.027 mg/lb], IV) for placement of a central venous catheterc in 1 jugular vein. After recovery from the sedation, the foals remained with their mares and were allowed to nurse free-choice throughout the study.

ACTH stimulation tests—In a randomized crossover design, 5 ACTH stimulation tests were performed on each foal, with a 2-day washout 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)d was administered IV into the contralateral jugular vein via a transiently placed catheter.e Lyophilized cosyntropin (250 μg) 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 1 μg/mL and used immediately or stored at 4°C in the original plastic saline solution bag for use within the next week. Diluted cosyntropin solution remains fully stable (in concentrations as low as 0.5 μg/mL) for at least 4 months when refrigerated in a plastic container.28

Collection of blood samples—Blood samples were collected from the central venous catheter immediately before (0 minutes [baseline]) and 30, 60, 90, 120, 180, and 240 minutes after injection of cosyntropin or saline solution for measurement of cortisol concentration. Samples were also collected at 10 and 20 minutes in 6 of 11 foals. At each time point, 6 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 (approx 22°C) and were then centrifuged; serum was collected and frozen at −20°C until assayed.

Before injection of cosyntropin or saline solution, an additional 6 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 inhibitorf; final concentration, 500 kallikrein inactivator units/mL of blood), as described.29 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. Lastly, blood (6 mL) was collected into evacuated tubes containing EDTA before and 60, 120, and 240 minutes after injection of cosyntropin or saline solution for performance of a routine CBC. 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 heparing/mL.

Sample analysis—Plasma endogenous ACTH and serum total cortisol concentrations were measured in duplicate with a single foal's samples always run in the same assay. Plasma endogenous ACTH concentration was measured by use of a sandwich immunoradiometric assayh validated for use in horses.30 The sensitivity of the assay was 1.0 pg/mL, and the interassay and intra-assay coefficients of variation for values < 30 pg/mL were 9.2% and 4.5%, respectively.31 Serum cortisol concentrations were determined by use of a validated direct radioimmunoassay.30,i 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.30 Complete blood counts were performed with an automated analyzerj; differential cell counts were performed manually.

Statistical analysis—Results are summarized as mean ± SD values. For each time point after baseline for each foal, delta cortisol concentration was calculated as cortisol concentration measured at that time minus the baseline cortisol concentration. Differences in ACTH-stimulated cortisol concentrations, delta cortisol concentrations, and CBC results were analyzed via the mixed model for 2-D (time and dose) repeated-measures ANOVA and the Scheffe test for multiple comparisons. Analyses were performed with standard software.k 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. For all analyses, values of P < 0.05 were considered significant.

Results

Baseline ACTH and cortisol concentrations—Baseline endogenous serum cortisol and plasma ACTH concentrations in the 55 samples obtained prior to the 5 tests performed in the 11 foals ranged from 14 to 149 nmol/L (mean ± SD, 52.7 ± 27.3 nmol/L) and from 7 to 33 pg/mL (16.6 ± 6.3 pg/mL), respectively. Mean baseline serum cortisol and plasma ACTH concentrations did not differ among test days.

Cortisol response to administered doses of cosyntropin—A significant increase in cortisol concentration was not detected after administration of 0.02 μg of cosyntropin/kg; however, significance was approached when comparing baseline cortisol concentrations with cortisol concentrations at 20 (P = 0.061) and 30 minutes (P = 0.067; Figure 1). After the 0.1 and 0.25 μg/kg doses of cosyntropin, serum cortisol concentration was significantly (P = 0.02 to < 0.001) increased, compared with baseline concentrations, 10, 20, and 30 minutes after cosyntropin administration. For the 0.5 μg/kg dose, serum cortisol concentration was significantly (P = 0.002 to < 0.001) increased, compared with baseline concentrations, between 10 and 60 minutes.

Figure 1—
Figure 1—

Mean serum cortisol concentrations in 11 healthy neonatal foals administered 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. +Significantly (P ≤ 0.05) different from concentration measured at the same time point after administration of saline solution. *Significantly (P ≤ 0.05) different from baseline (time 0) concentration for that dose.

Citation: Journal of the American Veterinary Medical Association 243, 3; 10.2460/javma.243.3.399

Serum cortisol concentration peaked at 20 minutes after administration of doses of 0.02, 0.1, and 0.25 μg/kg, with peak concentrations 1.7, 2.0, and 1.9 times the baseline concentrations, respectively. Serum cortisol concentration peaked at 30 minutes after administration of a cosyntropin dose of 0.5 μg/kg, with peak concentration 2.2 times the baseline concentration. No significant differences were detected in the peak serum cortisol concentrations obtained in response to administration of cosyntropin at a dose of 0.25 μg/kg at 20 and 30 minutes and 0.5 μg/kg at 30 minutes. After administration of 0.25 and 0.5 μg/kg doses, no significant differences were detected among serum cortisol concentrations at 10, 20, 30, and 60 minutes.

Comparison of cortisol response between cosyntropin doses and saline solution—No significant differences were detected between serum cortisol concentrations after administration of saline solution at any time point or between cortisol concentrations achieved after any of the cosyntropin doses, compared with cortisol concentrations subsequent to saline solution administration at 10 and 20 minutes. There was a significant (P < 0.001) difference among cortisol concentrations after administration of saline solution, compared with cortisol concentrations after administration of 0.25 and 0.5 μg/kg doses of cosyntropin at 30 minutes and after administration of 0.5 μg/kg at 60 minutes (P < 0.001).

Comparison of cortisol response to administered doses of cosyntropin between foals and adult horses—The cortisol response to 4 identical doses of cosyntropin administered to the 11 foals from the present study and to eight 2- to 12-year-old adult mixed-breed horses in a previous study26 (mean ± SD, 7.4 ± 3.5 years) was compared (Figure 2). The ACTH-stimulated cortisol concentrations peaked earlier, were of lower magnitude, and were of a shorter duration in foals, compared with adult horses.

Figure 2—
Figure 2—

Mean serum cortisol concentrations in 11 healthy neonatal foals and 8 adult horses (mean ± SD age, 7. 4 ± 3.5 years; range, 2 to 12 years)26 administered saline solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 μg/kg) of cosyntropin.

Citation: Journal of the American Veterinary Medical Association 243, 3; 10.2460/javma.243.3.399

Delta cortisol response to administered doses of cosyntropin—No significant differences were detected among the delta cortisol concentrations after administration of saline solution (Figure 3). Compared with administration of saline solution, delta cortisol concentrations at 10, 20, and 30 minutes were significantly different after the administration of the 0.1, 0.25, and 0.5 μg/kg doses of cosyntropin and at 60 minutes after the administration of the 0.5 μg/kg dose. Delta cortisol concentration peaked 20 minutes after administration of the 0.1 μg/kg dose of cosyntropin, which was not different from the delta cortisol concentrations at 10 and 30 minutes. The peak delta cortisol concentration after administration of 0.25 μg of cosyntropin/kg occurred at 30 minutes but was not different from the delta cortisol concentrations at 10 and 20 minutes. Delta cortisol concentration peaked 30 minutes after administration of 0.5 μg of cosyntropin/kg and was not significantly different from the delta cortisol concentrations at 10, 20, and 60 minutes.

Figure 3—
Figure 3—

Mean delta cortisol concentrations in 11 healthy neonatal foals administered saline solution or 1 of 4 doses of cosyntropin. See Figure 1 for key.

Citation: Journal of the American Veterinary Medical Association 243, 3; 10.2460/javma.243.3.399

Effect of cosyntropin on hematologic variables—Significant effects on hematologic variables were found in response to cosyntropin administration but not in response to administration of saline solution. The neutrophil-to-lymphocyte ratio increased 2.4-fold, from 4.76 ± 1.71 at 0 minutes to 11.6 ± 11.6 at 120 minutes (P = 0.01), and the lymphocyte count decreased from 1,555 ± 498 cells/μL at 0 minutes to 1,122 ± 600 cells/μL at 120 minutes (P = 0.038) after administration of 0.25 μg of cosyntropin/kg. For all 4 doses of cosyntropin, no significant differences in neutrophil count, monocyte count, eosinophil count, Hct, or RBC count over time were detected.

Discussion

The results of this study provide information on the effects of administration of low cosyntropin doses on cortisol concentration and hematologic variables in healthy neonatal foals.8,20,27,32–34 Results suggested that the lowest cosyntropin dose that can be used to assess adrenal gland function in healthy neonatal foals is 0.25 μg/kg, with peak cortisol response obtained between 20 and 30 minutes after cosyntropin administration. No significant differences were detected among peak serum cortisol concentrations obtained in response to cosyntropin administration at doses of 0.25 and 0.5 μg/kg. A study27 in 3- to 4-day-old foals revealed an equivalent increase in cortisol concentrations 30 minutes after administration of 0.2, 2, and 5 μg/kg doses of cosyntropin; accordingly, a dose between 0.2 and 2 μg/kg was suggested for evaluation of adrenal gland function in neonatal foals. The present study investigated doses intermediate to these concentrations. In the study27 of 3- to 4-day-old foals, the peak response to the 0.2 μg/kg dose occurred at 30 minutes, returning to baseline concentrations by 90 minutes. In the present study, the peak response to a similar dose of 0.25 μg/kg occurred at 20 minutes, with equivalent responses obtained between 10 and 60 minutes.

Previous studies in adult horses26 and 3- to 4-day-old foals27 did not measure cortisol concentrations at times < 30 minutes after cosyntropin administration. The present study found that the adrenal gland response to cosyntropin was rapid, and responses equivalent to the peak response were obtained as early as 20 minutes after administration of cosyntropin at doses of 0.25 and 0.5 μg/kg in neonatal foals. This rapid response could shorten the time required to perform an ACTH stimulation test in healthy foals.

Results of the present study suggested that administration of cosyntropin at a dose of 0.02 μg/kg was insufficient to increase serum cortisol concentrations in healthy neonatal foals. Similarly, when eight 3- to 4-day-old foals received 1 μg of cosyntropin/foal (approx 0.02 μg/kg), no significant increase in cortisol concentrations after 30, 60, 90, or 120 minutes were detected.27 In the present study, the difference between cortisol concentrations at 20 and 30 minutes after administration of 0.02 μg of cosyntropin/kg and baseline concentrations approached significance (P = 0.06). A further study involving a larger number of foals may detect a difference at this low dose of cosyntropin. In a previous study26 of 8 healthy adult horses, cosyntropin administration at 0.02 μg/kg caused a significant increase in serum cortisol concentration 30 to 60 minutes after injection. However, we do not recommend the use of a 0.02 μg/kg dose for clinical testing of neonatal foals or adult horses because cortisol concentrations obtained are lower than those achieved with higher cosyntropin doses and the response can be variable in some foals.

When performing ACTH stimulation tests, clinicians are faced with a decision between use of a cosyntropin dose high enough to obtain a significant increase in cortisol concentration, compared with baseline concentration, versus use of 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 gland function.35 In dogs, the dose of cosyntropin used for ACTH stimulation testing has shifted from a traditional dose of 250 μg/dog to a dose of 5 μg/kg. The lowest ACTH dose that would cause maximum adrenal gland stimulation in healthy dogs was recently found to be 0.5 μg/kg, when doses of 1.0, 0.5, 0.1, 0.05, and 0.01 μg/kg (0.45, 0.23, 0.05, 0.023 and 0.005 μg/lb) were compared,35 but this dose has not yet been adopted for routine clinical testing in dogs. Even though the 0.5 μg/kg dose maximally stimulates the adrenal glands in healthy dogs, there is little research indicating that this still holds true in critically ill dogs or dogs with hypoadrenocorticism, which may explain the reluctance to use the 0.5 μg/kg dose in dogs. It was the recommendation of the authors of 2 canine studies35,36 to continue to investigate the use of a 0.5 μg/kg dose in dogs. Evidence that the lower dose is at least as accurate and appears to be more sensitive at diagnosing CIRCI in critically ill dogs is becoming availablel and is in agreement with studies3,4,25 in humans. Although lower doses of cosyntropin can be used to obtain peak cortisol concentrations equal to those obtained from higher cosyntropin doses, the duration of increased cortisol concentrations is reduced as the dose of cosyntropin is reduced. When low doses of cosyntropin are used, the postadministration sample can be collected earlier but timing of the collection needs to be more precise.35,36 The ability to use a low dose of ACTH for stimulation testing increases the affordability of the test. Multiple doses can be administered from the same 250-μg vial without affecting test results if the solution is stored properly. Reconstituted cosyntropin can be stored in plastic for as long as 4 months in a refrigerator or as long as 6 months at −20°C, with no adverse effects on its bioactivity.37

Neonatal foals are thought to lack a functionally mature and responsive HPA axis, with lower adrenal gland responses to endogenous and exogenous ACTH, compared with mature horses.8,38,39 During the first week of life, cortisol and ACTH concentrations are highest immediately after birth, followed by a gradual decrease.8,32 Endogenous serum cortisol concentrations in the 2- to 12-day-old foals in the present study were similar to those reported in 3- to 4-day-old foals.27 However, endogenous serum cortisol concentrations in foals are approximately one-third the values in adult horses (range, 76 to 264 nmol/L [mean ± SD, 172.4 ± 44.8 nmol/L]).26 Although no differences were detected in basal cortisol and ACTH concentrations among healthy foals that were 12 to 24 hours, 24 to 48 hours, and 5 to 7 days of age, foals that were 12 to 24 hours of age had higher delta cortisol concentrations after administration of 10 and 100 μg of cosyntropin than when the foals were < 1 hour, 36 to 48 hours (low dose only), and 5 to 7 days of age.8 Furthermore, plasma cortisol concentrations at 30 and 60 minutes after a 0.1 μg/kg dose of cosyntropin were significantly higher in 12-week-old foals, compared with foals that were 2 to 3 weeks of age.32 Because of the low numbers of foals tested at each age for each cosyntropin dose in the present study, it was not possible to determine whether there was any change in the delta cortisol response with age. In comparison to a similar study of adult horses,26 the ACTH-stimulated cortisol concentrations peaked earlier, were of lower magnitude, and were of a shorter duration in foals. It is essential that reference ranges for adult horses not be used to assess adrenal gland function in foals.

Plasma endogenous ACTH concentrations in healthy adult horses range from 6 to 30 pg/mL.26,40,41 The ACTH concentrations in the foals in the present study were similar (range, 7 to 33 pg/mL) to those of adult horses but slightly less than reported in other studies of healthy foals of similar age (mean, approx 31 pg/mL).8,32 Those studies used a chemiluminescent enzyme immunoassay, whereas the present study used an immunoradiometric assay, which may account for these slight differences.

Mean baseline serum cortisol and ACTH concentrations in the 2- to 12-day-old foals used in the present study did not differ throughout the study period, which ruled out day-to-day variations in environmental effects such as temperature, external stimulation, adrenal gland suppression from repeated ACTH administration, and age. The decrease in serum cortisol concentration 120 minutes after administration of the 0.1 and 0.25 μg/kg doses of cosyntropin in the present study was likely caused by negative feedback on the HPA axis attributable to the increases in cortisol concentrations induced by the exogenously administered ACTH.

In a similar study26 performed in adult horses, the neutrophil-to-lymphocyte ratio increased significantly at 120 to 240 minutes after administration of the 0.02, 0.1, and 0.5 μg/kg doses of cosyntropin, compared with baseline values. In the foals of the present study, the neutrophil-to-lymphocyte ratio doubled between 0 and 120 minutes only after administration of the 0.25 μg/kg cosyntropin dose. The neutrophil and total WBC counts increased significantly at 240 minutes after administration of both the 0.25 and 0.5 μg/kg doses in adult horses26 but not in the foals. Foals had significantly lower lymphocyte counts 120 minutes after administration of the 0.25 μg/kg dose, whereas lymphocyte counts did not change significantly in adult horses in response to any of the cosyntropin doses.26 Circulating WBCs appear to be sensitive to subtle changes in the HPA axis in healthy adult horses and foals, with reduction in lymphocyte counts occurring in foals and increases in neutrophil counts occurring in adult horses. Thus, it is advisable to collect blood for assessment of hematologic data prior to performing ACTH stimulation testing. Lack of consistency among individual foals precludes the use of CBC values to assess adrenal gland responsiveness after cosyntropin administration.

Results of the present study indicated that a dose of cosyntropin of 0.25 μg/kg with collection of blood samples for evaluation of serum cortisol concentration between 20 and 30 minutes after cosyntropin administration can be used to assess adrenal gland response in healthy neonatal foals. The best dose of cosyntropin for assessment of adrenal gland function in critically ill foals and the cutoff values that suggest an appropriate response are still debatable.20

ABBREVIATION

CIRCI

Critical illness–related corticosteroid insufficiency

HPA

Hypothalamic-pituitary-adrenal

a.

Diazepam, 5 mg/mL, Abbott Laboratories, North Chicago, Ill.

b.

Torbugesic, 10 mg/mL, Fort Dodge Animal Health, Iowa.

c.

14-gauge, 8-inch polyurethane guidewire system catheter, Mila International, Florence, Ky.

d.

Cortrosyn, Amphastar Pharmaceuticals Inc, Rancho Cucamonga, Calif.

e.

Angiocath 18-gauge, 3-inch PEP polymer, Becton-Dickinson Infusion Therapy Systems Inc, Sandy, Utah.

f.

Aprotinin lyophilized powder, USB Corp, Cleveland, Ohio.

g.

Heparin, 10,000 U/mL, Heparin sodium, Sagent Pharmaceuticals, Schaumburg, Ill.

h.

ACTH immunoradiometric assay, Nichols Institute, San Clemente, Calif.

i.

Coat-A-Count Cortisol direct radioimmunoassay, Diagnostic Products Corp, Los Angeles, Calif.

j.

Advia 120 Hematology System, Siemens, Tarrytown, NY.

k.

SAS software, version 9.1, SAS Institute Inc, Cary, NC.

l.

Martin LG, Behrend EN, Holowaychuk MK, et al. Comparison of low-dose and standard-dose ACTH stimulation tests in critically ill dogs by assessment of serum total and free cortisol concentrations (abstr). J Vet Intern Med 2010;24:685–686.

References

  • 1. Rivers EP, Gaspari M & Saad GA, et al. Adrenal insufficiency in high-risk surgical ICU patients. Chest 2001; 119: 889896.

  • 2. Marik PE, Zaloga GP. Adrenal insufficiency during septic shock. Crit Care Med 2003; 31: 141145.

  • 3. Beishuizen A, van Lijf JH & Lekkerkerker JFF, et al. The low dose (1 μg) ACTH stimulation test for assessment of the hypothalamo-pituitary-adrenal axis. Neth J Med 2000; 56: 9199.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Soliman AT, Taman KH & Rizk MM, et al. Circulating adrenocorticotropic hormone (ACTH) and cortisol concentrations 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: 209214.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Burkitt JM, Haskins SC & Nelson RW, et al. Relative adrenal insufficiency in dogs with sepsis. J Vet Intern Med 2007; 21: 226231.

  • 6. Martin LG, Groman RP & Fletcher DJ, et al. Pituitary-adrenal function in dogs with acute critical illness. J Am Vet Med Assoc 2008; 233: 8795.

  • 7. Wong DM, Vo DT & Alcott CJ, et al. Baseline plasma cortisol and ACTH concentrations and response to low-dose ACTH stimulation testing in ill foals. J Am Vet Med Assoc 2009; 234: 126132.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Hart KA, Heusner GL & Norton NA, et al. Hypothalamic-pituitary-adrenal axis assessment in healthy term neonatal foals utilizing a paired low dose/high dose ACTH stimulation test. J Vet Intern Med 2009; 23: 344351.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Marik PE, Pastores SM & Annane D, et al. Recommendations for the diagnosis and management of corticosteroid insufficiency in critically ill adult patients: consensus statements from an international task force by the American College of Critical Care Medicine. Crit Care Med 2008; 36: 19371949.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Marik PE. Critical illness related corticosteroid insufficiency. Chest 2009; 135: 181193.

  • 11. Sibbald WJ, Short A & Cohen MP, et al. Variations in adrenocortical responsiveness during severe bacterial infections. Ann Surg 1977; 186: 2933.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Moran JL, Chapman MJ & O'Fathartaigh MS, et al. Hypercortisolemia and adrenocortical responsiveness at onset of septic shock. Intensive Care Med 1994; 20: 489495.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Soni A, Pepper GM & Wyrwinski PM, et al. Adrenal insufficiency occurring during septic shock—incidence, outcome, and relationship to peripheral cytokine levels. Am J Med 1995; 98: 266271.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Beishuizen A, Thijs LG. Relative adrenal failure in intensive care: an identifiable problem requiring treatment? Best Pract Res Clin Endocrinol Metab 2001; 15: 513531.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Annane D, Sébille V & Charpentier C, et al. Effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock [Erratum published in JAMA 2008; 30:1652]. JAMA 2002; 288: 862871.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Annane D, Maxime V & Ibrahim F, et al. Diagnosis of adrenal insufficiency in severe sepsis and septic shock. Am J Respir Crit Care Med 2006; 174: 13191326.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Annane D. Glucocorticoids in the treatment of severe sepsis and septic shock. Curr Opin Crit Care 2005; 11: 449453.

  • 18. McKee JI, Finlay WEI. Cortisol replacement in severely stressed patients. Lancet 1983; 1: 484.

  • 19. Rossdale PD, Silver M & Ellis L, et al. Response of the adrenal cortex to tetracosactrin (ACTH1–24) in the premature and full term foal. J Reprod Fertil Suppl 1982; 32: 545553.

    • Search Google Scholar
    • Export Citation
  • 20. Hart KA, Slovis NM, Barton MH. Hypothalamic-pituitary-adrenal axis dysfunction in hospitalized neonatal foals. J Vet Intern Med 2009; 23: 901912.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Annane D, Sébille V & Troché G, et al. A 3-level prognostic classification in septic shock based on cortisol levels and cortisol response to corticotropin. JAMA 2000; 283: 10381045.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Marik PE, Zaloga GP. Adrenal insufficiency in the critically ill—a new look at an old problem. Chest 2002; 122: 17841796.

  • 23. Kozyra EF, Wax RS, Burry LD. Can 1 microg of cosyntropin be used to evaluate adrenal insufficiency in critically ill patients? Ann Pharmacother 2005; 39: 691698.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Oelkers W. Adrenal insufficiency. N Engl J Med 1996; 335: 12061212.

  • 25. Gonc EN, Kandemir N, Kinik ST. Significance of low-dose and standard-dose ACTH tests compared to overnight metyrapone test in the diagnosis of adrenal insufficiency in childhood. Horm Res 2003; 60: 191197.

    • Search Google Scholar
    • Export Citation
  • 26. Stewart AJ, Behrend EN & Wright JC, et al. Validation of a low-dose ACTH stimulation test in normal adult horses. J Am Vet Med Assoc 2011; 239: 834841.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Hart KA, Ferguson DC & Heusner GL, et al. Synthetic adrenocorticotropic hormone stimulation tests in healthy neonatal foals. J Vet Intern Med 2007; 21: 314321.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Dickstein G, Shechner C & Nicholson WE, et al. Adrenocorticotropin stimulation test: effects of basal cortisol level, time of day, and suggested new sensitive low dose test. J Clin Endocrinol Metab 1991; 72: 773778.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Kemppainen RJ, Clark TP, Peterson ME. Preservative effect of aprotinin on canine plasma immunoreactive adrenocorticotropin concentrations. Domest Anim Endocrinol 1994; 11: 355362.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Golland LC, Evans DL & Stone GM, et al. Plasma cortisol and beta-endorphin concentrations in trained and over-trained Standardbred racehorses. Pflugers Arch 1999; 439: 1117.

    • Search Google Scholar
    • Export Citation
  • 31. Marc M, Parvizi N & Ellendorff F, et al. Plasma cortisol and ACTH concentrations in the Warmblood horse in response to a standardized treadmill exercise test as physiological markers for evaluation of training status. J Anim Sci 2000; 78: 19361946.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Wong DM, Vo DT & Alcott CJ, et al. Adrenocorticotropic hormone stimulation tests in healthy foals from birth to 12 weeks of age. Can J Vet Res 2009; 73: 6572.

    • Search Google Scholar
    • Export Citation
  • 33. Hart KA, Barton MH & Ferguson DC, et al. Serum free cortisol fraction in healthy and septic neonatal foals. J Vet Intern Med 2011; 25: 345355.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Jellyman JK, Allen VL & Forhead AJ, et al. Hypothalamic-pituitaryadrenal axis function in pony foals after neonatal ACTH-induced glucocorticoid overexposure. Equine Vet J Suppl 2012;(41):3842.

    • Search Google Scholar
    • Export Citation
  • 35. Martin LG, Behrend EN & Mealey KL, et al. Effect of low doses of cosyntropin on serum cortisol concentrations in clinically normal dogs. Am J Vet Res 2007; 68: 555560.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Kerl ME, Peterson ME & Wallace MS, et al. Evaluation of a low-dose synthetic adrenocorticotropic hormone stimulation test in clinically normal dogs and dogs with naturally developing hyperadrenocorticism. J Am Vet Med Assoc 1999; 214: 14971501.

    • Search Google Scholar
    • Export Citation
  • 37. Frank LA, Oliver JW. Comparison of serum cortisol concentrations in clinically normal dogs after administration of freshly reconstituted and stored frozen cosyntropin. J Am Vet Med Assoc 1998; 212: 15691571.

    • Search Google Scholar
    • Export Citation
  • 38. Rossdale PD, Ousey JC & Silver M, et al. Studies on equine prematurity 6: guidelines for assessment of foal maturity. Equine Vet J 1984; 16: 300302.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39. Silver M, Ousey JC & Dudan FE, et al. Studies on equine prematurity 2: post natal adrenocortical activity in relation to plasma adrenocorticotrophic hormone and catecholamine levels in term and premature foals. Equine Vet J 1984; 16: 278286.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40. Funk RA, Stewart AJ & Wooldridge AA, et al. Seasonal changes in plasma alpha melanocyte stimulating hormone and adrenocorticotropic hormone in response to thyroid releasing hormone administration in normal aged horses. J Vet Intern Med 2011; 25: 579585.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41. Schreiber CM, Stewart AJ & Kwessi E, et al. Seasonal variation in results of diagnostic tests for pituitary pars intermedia dysfunction in older, clinically normal geldings. J Am Vet Med Assoc 2012; 241: 241248.

    • Crossref
    • Search Google Scholar
    • Export Citation
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