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Keywords:

  • horse;
  • adrenocorticotropic hormone;
  • circadian rhythm;
  • Cushing's disease;
  • pituitary pars intermedia dysfunction;
  • ultradian rhythm

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

Reasons for performing study

There is little published information on whether measurement of plasma ACTH concentration at a single timepoint is a repeatable indicator of pituitary pars intermedia dysfunction (PPID).

Objectives

To determine whether ultradian or circadian fluctuations in ACTH production influence plasma ACTH concentration in normal horses and horses with PPID.

Study design

Prospective observational study.

Methods

Plasma ACTH concentration in 8 non-PPID horses and 8 horses with PPID was measured at 08.00, 11.00, 14.00 and 17.00 h on 5 nonconsecutive days within a 3 week period. In addition, at 08.30 h on one day, 6 samples were collected from each horse at precisely 5 min intervals over a period of 25 min. Descriptive and graphical analysis was performed and a linear mixed effects model was fitted to assess the effect of time of day on ACTH concentration in non-PPID and PPID horses.

Results

Evidence of ultradian fluctuation in ACTH production was not identified in either non-PPID or PPID horses. Evidence for circadian fluctuation was identified in non-PPID horses; plasma ACTH concentrations were highest at 08.00 h and decreased through the day. There was no evidence of circadian fluctuation in PPID horses. In non-PPID horses, the magnitude of circadian changes in ACTH concentration was smaller than variations in concentration that occurred at random. Intrahorse variability of ACTH concentration was greater in PPID horses than in non-PPID horses.

Conclusions

Ultradian and circadian fluctuations in ACTH concentration are unlikely to influence clinical decision making; however, variations of potential clinical relevance do occur in individual horses, for reasons that remain to be determined, and increase in magnitude with progression of PPID. Results of the current study indicate that when an ACTH concentration between 19 and 40 pg/ml is measured, further testing should be considered to increase the accuracy of PPID diagnosis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

Pituitary pars intermedia dysfunction (PPID) is a neurodegenerative condition of horses associated with ageing [1]. In one survey, 21% of horses aged over 15 years had endocrine changes consistent with PPID [2]. Measurement of plasma adrenocorticotropic hormone (ACTH) concentration has been demonstrated to be an accurate means by which to diagnose PPID [3-5] and is also used to assess responses to treatment in horses with the condition [6, 7]. Assessment of a single blood sample offers practical and financial advantages over the use of dynamic tests such as the dexamethasone suppression test, thyrotropin releasing hormone (TRH) stimulation test or domperidone stimulation test. In non-PPID horses, ACTH is released from the pars distalis and is subject to an inhibitory feedback loop that regulates its concentration in plasma. In horses with PPID, loss of dopaminergic inhibition of melanotrophs in the pars intermedia results in increased and unregulated production of ACTH [1].

In order for ACTH concentration to be of value in the diagnosis and monitoring of horses with PPID, expected intra-animal variations in both health and disease need to be established. The reliability of a single measurement of ACTH concentration has been questioned when only a single measurement is made [8, 9] and variation in resting ACTH concentration has been reported to occur in both non-PPID horses and horses with PPID when paired blood samples have been collected 5 min apart [10-13].

Variation in ACTH concentration may be due, at least in part, to its pulsatile release into pituitary venous blood [14-16]. Fluctuations of over 50% of the mean ACTH concentration were identified in jugular venous blood in one study [17]. Circadian fluctuations in ACTH production and secretion may also influence the repeatability of ACTH concentration when samples are collected at different times of day. Circadian rhythms for cortisol and ACTH production have been investigated in non-PPID horses [18]; however, only limited investigation of circadian effects on ACTH concentration in horses with PPID has been performed [19].

The primary objective of this study was to determine whether a single measurement of ACTH concentration was a repeatable means of assessing pituitary ACTH production. Specifically, the existence and magnitude of ultradian and circadian fluctuations in ACTH concentration were investigated in non-PPID horses and horses with PPID.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

The institution's animal care and ethics committee approved the study. Eight non-PPID horses from the institution's research herd and 8 client-owned horses with PPID were included in the study. Client-owned horses were loaned to the institution with informed consent. Horses were classified as PPID if they had a resting ACTH concentration >40 pg/ml when originally tested for PPID at the clients' premises. The diagnosis of PPID was confirmed by performing a TRH stimulation test at least 24 h after horses were admitted to the institution; horses were considered positive if ACTH concentration measured >100 pg/ml 10 min after 1 mg TRH was administered i.v [11]. The interval between screening and commencing the study ranged from 4 to 8 weeks. Four of the horses in the PPID group exhibited hypertrichosis, the remaining 4 did not exhibit clinical signs of the disease. Non-PPID horses had no clinical signs of PPID and had resting ACTH concentrations <25 pg/ml and ACTH concentrations of <100 pg/ml 10 min after i.v. injection of 1 mg TRH. The group of non-PPID horses comprised 8 geldings (5 Thoroughbred and 3 Standardbred) aged 5–19 years (median 10 years). The PPID group comprised 4 ponies of mixed breeding, 2 Shetland ponies, one Thoroughbred and one Standardbred. The ages of the PPID group ranged between 15 and 34 years (median 23 years).

Horses were acclimatised to housing in dirt yards at longitude 35° S, 7, 0 and latitude 147°22, 0, E for a minimum of 2 days prior to sampling. All samples were collected via an indwelling i.v. catheter placed in one jugular vein at least one day prior to sampling. Horses remained in the yards throughout the study period and were not exercised. Blood samples were collected from the non-PPID group in September (spring) and from the PPID group in December (summer). Blood was collected into chilled Vacutainer tubes containing ethylenediaminetetra-acetic acid, placed on ice immediately, centrifuged within 1 h, and the harvested plasma frozen at -80°C prior to analysis within 2 months of sample collection. Analysis was performed using an automated chemiluminescent immunoassay (Immulite)a validated for use in horses [5]. Intra-assay variation for the laboratory was determined from repeat analysis of 23 samples spanning a range of ACTH concentrations from 0 to 250 pg/ml.

Part 1. Investigation of ultradian effects

For each group, samples were collected simultaneously from each horse at precisely 5 min intervals over a 25 min period commencing at 08.30 h.

Part 2. Investigation of circadian effects

Horses were sampled at 08.00, 11.00, 14.00 and 17.00 h on 5 predetermined and nonconsecutive days within a 3 week period. Horses were sampled sequentially and a random order for sampling was assigned on each occasion.

Data analysis

Data were collated in Excelb and statistical analyses performed using GraphPad Prism 5.0 dc and IBM SPSS Statistics v 20d. Descriptive and graphical analysis was performed initially and linear mixed effects models were fitted to assess the effect of time of day on ACTH concentration in non-PPID horses, PPID horses and both groups combined. For these models, ACTH concentration was specified as the outcome, time of day that sampling occurred (08.00, 11.00, 14.00 and 17.00 h) as a fixed effect and day that sampling was undertaken (Days 1–5) and horse as random effects.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

The median coefficient of variation for the assay in the test laboratory was 6%, consistent with results obtained when the analyser was validated for use in horses [5].

Part 1. Investigation of ultradian effects

Results of repeated analyses performed over a 25 min period in 8 non-PPID and 8 PPID horses are presented in Figure 1. Following graphical analysis, it appears that intrahorse variability was greater for PPID horses than for non-PPID horses. In one non-PPID horse a peak in ACTH concentration was identified. In the remaining non-PPID horses and the horses with PPID there was no evidence of a cyclical pattern of ACTH concentration.

figure

Figure 1. Plasma ACTH concentrations in 8 non-PPID horses (blue lines) and 8 horses with PPID (red lines) sampled 6 times over a 25 min period. Dashed lines indicate that results have been reduced by a factor of 5 to enable presentation on the same scale.

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Part 2. Investigation of circadian effects

The linear mixed effects models identified a significant association between time of day and ACTH concentration (P = 0.001) in non-PPID horses. A significant reduction in ACTH concentration was found between samples taken at 08.00 and 11.00 h (P = 0.001), 14.00 h (P<0.001) and 17.00 h (P<0.001) and between 11.00 and 17.00 h (P = 0.009) but not between 11.00 and 14.00 h (P = 0.5) or 1400 and 1700 h (P = 0.05). No significant effects of time of day were identified for PPID horses (P = 0.6), or when both groups were combined (P = 0.8).

Results for individual horses are displayed in Figure 2 as medians for each timepoint across the 5 days of sampling and reflect the statistically significant decrease in ACTH concentration identified for non-PPID horses and the absence of such an effect in PPID horses. In Figure 3, group means for all 5 days are displayed and there is a progressive decrease in mean ACTH concentrations through the day in non-PPID horses. In PPID horses the mean ACTH concentration increases initially before decreasing. However, the interquartile ranges are wide, particularly in PPID horses, indicating that when looking at individual results random effects are likely to be exerting a greater influence than circadian effects.

figure

Figure 2. Adrenocorticotropic hormone concentrations in 8 non-PPID horses (blue lines) and 8 horses with PPID (red lines). Each line connects medians of results for each horse from 5 nonconsecutive days. Dashed lines indicate that results have been reduced by a factor of 10 to enable presentation on the same scale.

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figure

Figure 3. Mean plasma ACTH concentrations for 8 non-PPID horses (blue bars) and 8 horses with PPID (red bars) sampled on 5 nonconsecutive days. Error bars show standard error of means.

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Results for both Parts 1 and 2 and from an additional sample from each horse obtained for another investigation (5 min before each ultradian investigation) are displayed graphically (Fig 4) to show the range of 27 results for each horse within the 3 week study period irrespective of time of collection. Variation was greater for horses with PPID and increased as median ACTH concentration increased. Whereas most results for non-PPID horses were below typical cut-offs used to diagnose PPID, 2 non-PPID horses had an occasional ACTH concentration that would typically be considered positive for PPID. Furthermore, in 4 of the 8 PPID horses (those that did not exhibit clinical signs) the majority of results were below typical cut-off concentrations [5] for PPID. However, median results in these 4 PPID horses were higher than in the non-PPID horses. None of the non-PPID horses had ACTH concentrations >40 pg/ml and none of the PPID horses had ACTH concentrations <19 pg/ml.

figure

Figure 4. Scatter plot showing plasma ACTH concentrations measured on 27 occasions over a 3 week period in 8 non-PPID horses (blue dots) and 6 horses with PPID (red dots). Results for 2 horses with PPID have been reduced by a factor of 10 to enable presentation on the same scale (orange dots). Medians and interquartile ranges are also displayed. The dashed lines indicate the maximum values for non-PPID horses and minimum values for PPID horses. Two datapoints for Horse 13 and 2 datapoints for Horse 16 lie above the upper axis limit.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

In the current study no evidence was identified for the existence of ultradian fluctuations of ACTH concentration in venous blood either in non-PPID horses or horses with PPID. Statistically higher ACTH concentrations were identified in morning samples than in afternoon samples in non-PPID horses, suggesting that there may be a circadian influence on ACTH concentration in non-PPID horses. Differences between ACTH concentrations at different times of day were not statistically significant in horses with PPID. Large variations in ACTH concentration of sufficient magnitude to influence clinical decision-making were identified in individual animals, although the explanation for these changes was not determined.

In non-PPID horses, release of ACTH into pituitary venous blood occurs in a pulsatile manner [14-16]; however, concentrations detected in jugular venous blood have been estimated to be approximately 2% of those of pituitary effluent, leading to doubt as to whether such pulses would be detected in peripheral blood [3]. In the current study, failure to identify defined peaks and troughs consistent with ultradian cycles of release was contrary to the findings of Cudd et al. [17], who identified 11–17 min ultradian cycles in 6 horses and 27–40 min cycles in the other 2 horses examined in that study. The 25 min sample period in the current study was selected to encompass the length an ultradian cycle identified to occur in 6/8 horses of the study by Cudd et al. [17] but may not have been long enough to detect longer cycles. This may explain the discrepancy between the findings of the current study and the study by Cudd et al. [17].

Circadian fluctuations of ACTH concentration in horses have been the subject of 2 previous investigations in non-PPID horses [18, 19] and one investigation of horses with PPID [19]. Lee et al. [19] did not identify a clear circadian pattern of release in non-PPID or PPID horses; however, the investigation was limited by a restriction on the number of samples that could be collected in one day, resulting in sample collection at 27 h intervals across a 10 day period. Cordero et al. [18] identified a circadian rhythm of low magnitude in non-PPID horses in September but not in May; in September, ACTH concentration peaked at 11:48 pm. The aim of the current study was to investigate fluctuations that might be relevant to clinicians, and samples were not collected out of normal working hours. It is possible that ACTH concentrations in the non-PPID horses in the current investigation may have peaked overnight when samples were not collected. Although the present study identified significant intra-day variation in non-PPID horses, which is in contrast to the findings of Lee et al. [19] and Cordero et al. [18], the difference is likely to be of little clinical importance as small fluctuations within the reference range in non-PPID horses are of little relevance in the diagnosis or exclusion of PPID. Furthermore, the variation that may occur as a result of circadian effects was small compared to the variation that occurred due to other (as yet undetermined) factors. In horses with PPID, diurnal effects were not apparent, which may indicate a loss of circadian fluctuation in ACTH production in horses with PPID, as is thought to occur for cortisol [21, 20]. Although it may still be preferable, the results of the current study do not indicate a need to standardise the timing of sample collection for the diagnosis or monitoring of PPID.

The causes of the random fluctuations in ACTH concentrations identified in both PPID and non-PPID horses in this study are unclear. As animal housing, exercise, feeding, blood collection and sample handling were rigorously controlled throughout the study period and the maximum variation introduced by the assay was comparatively very small, the variation in maximum and minimum recorded ACTH concentrations is assumed to be the result of fluctuations in pituitary ACTH production. The effects of stress on ACTH concentrations in horses have received limited investigation [22]; however, measures were taken in the current study to limit, and hopefully eliminate, stress as a potentially confounding effect. The horses were acclimatised to their environment, handlers and routine kept constant, and samples collected via indwelling jugular catheters. Fluctuations in ACTH concentration were larger in the PPID group, with the interquartile ranges for some horses being very wide. This suggests that as pituitary dysfunction progresses and resting ACTH concentration increases, ACTH may become a less reliable means of monitoring the disease. However, one of the horses with high resting ACTH concentrations had very consistent results, and it is possible that differences between individuals may relate to the heterogeneous nature of histological changes that occur in horses with PPID. Further studies of patterns of ACTH concentrations with greater numbers of horses subsequently examined histologically, are required.

Although caution may be required when using ACTH concentrations to assess relative improvements or deteriorations in pituitary dysfunction in response to treatment, the results of this study support previous assertions that a single measurement of ACTH concentration is a reliable means of distinguishing non-PPID horses from horses with PPID. None of the non-PPID horses had ACTH concentrations >40 pg/ml and none of the PPID horses had ACTH concentrations <19 pg/ml. As PPID is a progressive condition which encompasses a range of histological changes [23], it is likely that there will always be a transitional period when horses with early pituitary dysfunction are difficult to distinguish from horses with normal pituitary function. Figure 4 highlights the crossover where horses that tested negative for PPID using a TRH stimulation test and were thus classified as non-PPID exhibited ACTH concentrations which overlapped with horses that tested positive for PPID using a TRH stimulation test but did not exhibit any clinical signs. Therefore, based on the results of the current study, further testing would be recommended for horses that have resting ACTH concentrations between 19 and 40 pg/ml.

In future, rather than focusing on a classification of normal or abnormal pituitary pars intermedia function, it may be more appropriate to focus on the ACTH concentration at which clinical signs (particularly laminitis, which is associated with the highest rates of morbidity and mortality) are at increased risk of developing, or on the ACTH concentration at which instigation of treatment may be beneficial either in reducing the severity of clinical signs or in altering the rate of progression of the disease. However, PPID encompasses a spectrum of histological changes [23] involving different clonal populations of cells within the pars intermedia [25, 26, 24] that have variable hormone producing and processing activity. Given this variation, it is likely that any system of classification based upon one product from melanotrophs will have inherent limitations.

A limitation of the current study was the failure to sample the non-PPID and PPID horses simultaneously. However, sampling was performed in spring and midsummer when pituitary production of ACTH remains consistent [28, 27]. Whether the findings of the current study are applicable to autumn months when pituitary activity is increased is not known. A further limitation was the failure to match the non-PPID and PPID groups for age and breed, as it is unknown how these factors may influence the repeatability of ACTH concentration or response to TRH stimulation. Although horses were tested for PPID prior to inclusion using resting ACTH concentration and a TRH stimulation test performed on different occasions, examination post mortem was not conducted and it is impossible to eliminate the possibility that horses may have been assigned incorrectly to the PPID or non-PPID group.

In conclusion, results of the current study indicate that ultradian and circadian fluctuations are likely to have little effect on the clinical interpretation of ACTH concentrations in horses. Clinically relevant fluctuations in ACTH concentration occur in individual horses, for reasons that remain to be determined, and increase in magnitude with progression of pituitary dysfunction. These fluctuations should be considered when making serial assessments of ACTH concentration in individual horses, especially horses with higher resting ACTH concentrations. Our results support previous assertions that ACTH is a reliable means of differentiating non-PPID and PPID horses but indicate that there is a transitional zone, around currently recommended cut-offs for diagnosing the disease, at which further testing should be considered.

Authors' declaration of interests

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

None of the authors have personal or commercial affiliations that might prejudice the results.

Ethical animal research

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

The study was approved by The Animal Care and Ethics Committee of Charles Sturt University. Client-owned horses were included with informed consent.

Source of funding

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

The study was funded by an internal grant from the School of Animal and Veterinary Sciences, Charles Sturt University.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

The authors are grateful to staff and students at Charles Sturt University for their assistance. Particular thanks to Ms Sarah Gough, Mrs Jacqui Price and Ms Kristie Hann.

Authorship

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References

All of the authors contributed to study design, data collection or analysis, interpretation and preparation of the manuscript.

Manufacturers' addresses
  1. aSiemens, Bayswater, Victoria, Australia.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Ethical animal research
  9. Source of funding
  10. Acknowledgements
  11. Authorship
  12. References
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