Plasma steroid profiles before and after ACTH stimulation test in healthy horses

Journal Pre-proof Plasma steroid profiles before and after ACTH stimulation test in healthy horses A. Kirchmeier, A.E. van Herwaarden, J.H. van der Kolk, J. Sauer F, V. Gerber

PII:

S0739-7240(19)30098-0

DOI:

https://doi.org/10.1016/j.domaniend.2019.106419

Reference:

DAE 106419

To appear in:

Domestic Animal Endocrinology

Received Date: 3 July 2019 Revised Date:

31 October 2019

Accepted Date: 17 November 2019

Please cite this article as: Kirchmeier A, van Herwaarden AE, van der Kolk JH, Sauer F J, Gerber V, Plasma steroid profiles before and after ACTH stimulation test in healthy horses, Domestic Animal Endocrinology (2019), doi: https://doi.org/10.1016/j.domaniend.2019.106419. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

1

Plasma steroid profiles before and after ACTH stimulation test in healthy horses

2 3

a, *

b

a

a

a

Kirchmeier A. , van Herwaarden A. E. , van der Kolk J. H. , Sauer F. J. , Gerber V.

4 5

a

6

Faculty, University of Bern, and Agroscope, Länggassstrasse 124, 3012 Bern, Switzerland

7

b

Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse

Radboud University Medical Center, Department of Laboratory Medicine, Nijmegen, the Netherlands

8 9 10

* Corresponding author: Swiss Institute of Equine Medicine ISME, Vetsuisse Faculty, University of Bern, Länggassstrasse 124, 3012 Bern, Switzerland, [email protected]

11 12

Abstract

13

This study describes steroid profiles in equine plasma before and after ACTH stimulation. In human

14

medicine, other steroid metabolites have been shown to have a more pronounced reaction to an

15

ACTH stimulation test than cortisol. This study aimed to determine if the same was true for the horse.

16

A total of 11 clinically healthy horses were selected for this study. EDTA plasma samples were taken

17

before and 60 minutes after stimulation with 1 µg/kg BW of synthetic ACTH administered

18

intravenously. The samples were analyzed for cortisol, 11-deoxycortisol, 21-deoxycortisol, cortisone,

19

corticosterone, 11-deoxycorticosterone, androstenedione, 17-OH-progesterone, progesterone, and

20

testosterone with a liquid chromatography tandem mass spectrometry (LC-MS/MS). Cortisol, 11-

21

deoxycortisol, cortisone, corticosterone, and 11-deoxycorticosterone showed a significant increase

22

after ACTH stimulation. In conclusion, the LC-MS/MS represents a viable method to measure

23

glucocorticoids and related precursors or metabolites in equine plasma samples. In addition, we were

24

able to show a more pronounced increase of 11-deoxycorticosterone, 11-deoxycortisol, and

25

corticosterone compared to cortisol. These three metabolites could potentially serve as more sensitive

26

biomarkers for stress in horses than cortisol.

27 28

Keywords: equine steroid profiles, ACTH stimulation test, liquid chromatography tandem mass

29

spectrometry (LC-MS/MS), equine stress marker

30

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1. Introduction

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The intravenous ACTH stimulation test is a common tool to induce physiological stress and assess

33

adrenal function and related diseases in horses [1-5]. Cortisol is the response parameter that is

34

usually evaluated. However, there are many other steroids that may be stimulated by ACTH in addition

35

to cortisol. Cholesterol is the precursor of steroid biosynthesis and is converted into different

36

progestogens that subsequently form the three main pathways of steroid biosynthesis: the

37

glucocorticoid, the mineralocorticoid and the androgen pathway (Figure 1). Glucocorticoids are

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stimulated primarily by ACTH and are produced in the zona fasciculata of the adrenal cortex,

39

mineralocorticoids are stimulated by angiotensin II and potassium and are synthesized in the zona

40

glomerulosa and the adrenal androgens are subject to reproductive activity and are produced in the

41

zona reticularis [6-11].

42

In human medicine, different steroid metabolites including 11-deoxycortisol, 11-deoxycorticosterone,

43

androstenedione and 17-OH-progesterone are used as a diagnostic tool for adrenal disorders and

44

endocrinological oncology [1, 2, 4, 12]. Steroid profiles are assessed by a liquid chromatography

45

tandem mass spectrometry (LC-MS/MS) assay. A recent study in healthy human subjects showed that

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11-deoxycorticosterone, 11-deoxycortisol and corticosterone displayed a more pronounced increase

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after an ACTH stimulation test compared to cortisol. Corticosterone had a 15-fold increase compared

48

to cortisol which had a 1.4-fold increase [13]. The development and application of LC-MS/MS in

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equine urine and serum samples has previously been described [14, 15]. Concerning equine serum

50

analysis, a total of 17 baseline steroid hormones were tested in two mares, a stallion and a gelding.

51

The main steroids found were corticosterone and cortisol followed by 17-OH-pregnenolone,

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dihydrotestosterone, and pregnenolone [14].

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To the authors' knowledge, there is no study investigating steroid profiles in equine plasma samples

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before and after ACTH stimulation. The objectives of this study were to describe the application of the

55

LC-MS/MS in equine plasma and to investigate the stimulation of the different steroids 60 minutes

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after ACTH administration.

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2. Materials and Methods

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2.1. Population, Sample collection and storage

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All experimental procedures were approved by the cantonal committee of animal protection and

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welfare issues of Berne (Approval number: 27608/BE26/16).

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Samples from this study were taken from a bank of frozen plasma samples collected from 70 horses

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between 2016 and 2018 as part of a larger series of studies investigating the relationship between

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equine glandular gastric disease and the cortisol response to ACTH. For this purpose, horses

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underwent an ACTH challenge test and a gastroscopy. On the day before, a venous catheter was

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placed in the jugular vein to administer 1 µg/kg BW synthetic ACTH (Synacthen tetracosactidum 0.25

67

mg/ml equivalent to 25 IU/ml; Novartis, Vilvoorde, Belgium) and collect the blood samples. For the

68

test, saliva and blood samples were taken before (T0) and 60 minutes (T60) after ACTH application

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and were directly centrifuged at room temperature for 10 minutes at 185 X g. After separation of the

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cellular components of the blood, EDTA plasma samples were frozen at -20 °C in Eppendorf tubes.

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From that study population, clinically healthy, client owned horses were selected, if they matched the

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following criteria: a salivary cortisol value that increased less than 4 ng/ml after ACTH stimulation and

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no concurrent glandular gastric disease. A total of 11 horses met these inclusion criteria with a mean

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age of 9 ± 2 (6-14) yr. There were three mares and eight geldings consisting of Franches Montagnes

75

(n = 4), Swiss Warmblood (n = 3), Hanoverian (n = 1), Quarter Horse (n = 1), Trotter (n = 1) and Pura

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Raza Espagñola breeds (n = 1).

77

The samples were analyzed before stimulation to detect baseline values (T0) and 60 minutes after

78

stimulation (T60) to measure the difference between the baseline values and the values after ACTH

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stimulation.

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2.2 Sample analysis

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Plasma cortisol, 11-deoxycortisol, 21-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone,

83

androstenedione, 17-OH-progesterone, progesterone and testosterone were measured by liquid

84

chromatography tandem mass spectrometry (LC-MS/MS) after protein precipitation and solid phase

85

extraction as described previously [16, 17].

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2.3 Statistical analysis

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The statistical analysis was performed with NCSS 12 Statistical Software 2018 (NCSS, LLC. Kaysville,

89

Utah, USA). The data were tested for normality with normality plots and the Shapiro Wilk test and

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descriptive statistics were performed. Data were expressed as mean ± standard deviation unless

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otherwise stated. The increase after ACTH stimulation was calculated as follows: (T60-T0)/T0. Paired

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t-tests were used to compare the values before and after the ACTH stimulation test and the

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proportional increases of cortisol and the other steroids after stimulation. The Wilcoxon (Rank Sum)

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Test was used if the data were not normally distributed. The level of significance for all tests was set at

95

p < 0.05. If the values were below the detection range of the assay, they were excluded from the

96

statistics. Graphics were created with Microsoft Office Excel 2007.

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3. Results

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The results of the steroid metabolites are shown in Table 1. There was a significant increase after the

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ACTH

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deoxycorticosterone (Figure 2). At T0, seven values of 11-deoxycortisol were below the detection

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range (< 0.17 nmol/l) as well as five values of 11-deoxycorticosterone (< 0.01 nmol/l). Nevertheless, a

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significant increase after stimulation could be demonstrated in both steroids based on the available T0

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values. For the most part, values for androstenedione, 17-OH-progesterone, progesterone, and

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testosterone were below the detection range of the LC-MS/MS (see Table 1). Therefore, we cannot

106

conclusively evaluate if these steroids increase after ACTH stimulation and the corresponding p-

107

values have to be interpreted with caution.

108

All values were below the detection limit for 21-deoxycortisol (<1.0 nmol/l).

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The most pronounced increase showed 11-deoxycorticosterone with a 7.31-fold increase, followed by

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11-deoxycortisol with a 5.28-fold increase and corticosterone with a 4.73-fold increase 60 minutes

111

after ACTH stimulation. In comparison, cortisol showed a 1.96-fold increase. Table 2 shows the

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proportional increase of the steroids, which increased significantly and the significance of the increase

113

compared to the cortisol increase.

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Table 3 shows the comparison between the calculated mean values of the study of Genangeli et al.

115

[14] and our baseline value (T0) results.

116

stimulation

test

of

cortisol,

11-deoxycortisol,

cortisone,

corticosterone

and

11-

117

4. Discussion

118

In this study, we described steroid profiles before and after an ACTH stimulation test in healthy horses.

119

We were able to show a significant increase of some steroids after ACTH stimulation. The most

120

pronounced increase was found for 11-deoxycorticosterone (7.31-fold), followed by 11-deoxycortisol

121

(5.28-fold) and corticosterone (4.73-fold).

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Several studies in human medicine sampled their subjects 30 and 60 minutes after ACTH stimulation,

123

respectively [1, 2, 13]. We decided to analyze the samples 60 minutes after stimulation, based on the

124

findings of Sauer et al (18), who found this time point to have the highest combined diagnostic

125

sensitivity and specificity of increase in cortisol. In our study, cortisol, 11-deoxycortisol, cortisone,

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corticosterone, and 11-deoxycorticosterone showed a significant difference 60 minutes after the ACTH

127

stimulation test compared to baseline values (T0) (Table 1).

128

Baseline steroid profiles have already been performed successfully in serum samples in several

129

mammals and birds [e.g. 6, 14, 19, 20]. To the authors' knowledge, there is no study investigating

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steroid profiles before and after ACTH simulation in serum or plasma samples of domestic animals.

131

There is a similar study in human medicine [12], which demonstrated that 11-deoxycortisol, 11-

132

deoxycorticosterone and corticosterone showed a more pronounced response after ACTH stimulation

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compared to cortisol. Another study in humans [4] also demonstrated that 11-deoxycorticosterone and

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corticosterone increased more than cortisol. In our study, 11-deoxycorticosterone, 11-deoxycortisol,

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and corticosterone showed a more pronounced increase 60 minutes after ACTH stimulation compared

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to cortisol. Therefore, we expect that the three steroids have a more sensitive response to ACTH

137

stimulation in both humans and horses than the currently used cortisol value.

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Based on our results and the similarity to studies in human medicine, we conclude that 11-

139

deoxycorticosterone, 11-deoxycortisol, and corticosterone may be considered as new biomarkers to

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assess stress in horses [4, 13].

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Regarding androstenedione, 17-OH-progesterone, and progesterone, we could not assess if there

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was a significant increase, due to too many of the baseline values remaining undetectably low. An

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increase following ACTH stimulation seems possible, leading to the conclusion that all steroids

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synthesized in the adrenal cortex might be stimulated by ACTH. Future studies with a larger sample

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size investigating more steroids might help to clarify this hypothesis. Therefore, further investigation is

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needed to completely understand the stimulating effect of ACTH on the steroid biosynthesis in the

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adrenal cortex.

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Table 3 shows our results converted to ng/ml so that they are comparable to a previous steroid profile

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study in equine serum samples [14]. We had a mixed population of mares and geldings and therefore

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compared our results to the mean values of the two mares, the gelding and the stallion in the other

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study. The results of both studies were in the same range except for corticosterone, 17-OH-

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progesterone, and progesterone. The progestogens depend on the sex and cycle stage which can

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explain the discrepancy between the results. Our corticosterone values were markedly lower than in

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the study of Genangeli et al. [14]. Interestingly, the baseline value of corticosterone of Przewalski's

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wild horses in another study [19] is in the same range as our corticosterone value. Future studies

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should investigate if a methodological difference could explain the discrepancies of the corticosterone

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values in these studies.

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Based on previous findings, we only analyzed the samples taken 60 minutes after stimulation [1, 13,

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18]. Therefore, we might have missed peak values for some steroid metabolites occurring prior to or

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after this time point. It would be interesting to evaluate other time points after ACTH stimulation to

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receive a better impression about the dynamics of the different steroids.

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This study generated a baseline steroid profile for healthy horses before and 60 minutes after an

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ACTH stimulation test. It would be interesting for future studies to compare horses with induced

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stimulation of the hypothalamic-pituitary-adrenal axis and horses with pituitary pars intermedia

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dysfunction (PPID). This would lead to a better understanding of the pathophysiology of these

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diseases.

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Conclusion

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Our study showed a significant difference in several investigated steroids 60 minutes after an ACTH

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stimulation test. 11-deoxycorticosterone, 11-deoxycortisol, and corticosterone showed an increased

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response to ACTH stimulation compared to cortisol. Therefore, these three steroids are potential new

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biomarkers for stress in horses. Future studies should verify if these steroids are reliable stress

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markers in different stress situations of horses.

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Acknowledgements

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The authors would like to thank Dr. Shannon Axiak Flammer for her help with the manuscript.

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Funding: This work was supported by the Federal Food Safety and Veterinary Office (ARAMIS-Nr.

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2.17.02) as well as the ISMEquine Research.

180 181 182

Declarations of interest: None.

183

References

184

[1]

profiles in determining the cause of adrenal insufficiency. Steroids. 2007;72(1):71-84.

185 186

Holst JP, Soldin SJ, Tractenberg RE, Guo T, Kundra P, Verbalis JG, et al. Use of steroid

[2]

Kao PC, Machacek DA, Magera MJ, Lacey JM, Rinaldo P. Diagnosis of adrenal cortical

187

dysfunction by liquid chromatography-tandem mass spectrometry. Ann Clin Lab Sci.

188

2001;31(2):199-204.

189

[3]

Peeters M, Sulon J, Beckers JF, Ledoux D, Vandenheede M. Comparison between blood

190

serum and salivary cortisol concentrations in horses using an adrenocorticotropic hormone

191

challenge. Equine Vet J. 2011;43(4):487-93.

192

[4]

Peitzsch M, Dekkers T, Haase M, Sweep FC, Quack I, Antoch G, et al. An LC-MS/MS method

193

for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism.

194

J Steroid Biochem Mol Biol. 2015;145:75-84.

195

[5]

Scheidegger MD, Gerber V, Ramseyer A, Schupbach-Regula G, Bruckmaier RM, van der Kolk

196

JH. Repeatability of the ACTH stimulation test as reflected by salivary cortisol response in

197

healthy horses. Domest Anim Endocrinol. 2016;57:43-7.

198

[6]

Martins-Junior HA, Simas RC, Brolio MP, Ferreira CR, Perecin F, Nogueira Gde P, et al.

199

Profiles of Steroid Hormones in Canine X-Linked Muscular Dystrophy via Stable Isotope

200

Dilution LC-MS/MS. PLoS One. 2015;10(5):e0126585.

201

[7]

adrenal cortex. Mol Cell Endocrinol. 2017;441:146-55.

202 203

[8]

Rainey WE. Adrenal zonation: clues from 11beta-hydroxylase and aldosterone synthase. Mol Cell Endocrinol. 1999;151(1-2):151-60.

204 205

Pignatti E, Leng S, Carlone DL, Breault DT. Regulation of zonation and homeostasis in the

[9]

Sanderson JT. The steroid hormone biosynthesis pathway as a target for endocrine-disrupting chemicals. Toxicol Sci. 2006;94(1):3-21.

206 207

[10]

Selye H. Stress in Health and Disease. Boston: Butterworths; 1976.

208

[11]

Vinson GP. Functional Zonation of the Adult Mammalian Adrenal Cortex. Front Neurosci. 2016;10:238.

209 210

[12]

Arlt W, Biehl M, Taylor AE, Hahner S, Libe R, Hughes BA, et al. Urine steroid metabolomics

211

as a biomarker tool for detecting malignancy in adrenal tumors. J Clin Endocrinol Metab.

212

2011;96(12):3775-84.

213

[13]

Lindner JM, Suhr AC, Grimm SH, Mohnle P, Vogeser M, Briegel J. The dynamics of a serum

214

steroid profile after stimulation with intravenous ACTH. J Pharm Biomed Anal. 2018;151:159-

215

63.

216

[14]

Genangeli M, Caprioli G, Cortese M, Laus F, Matteucci M, Petrelli R, et al. Development and

217

application of a UHPLC-MS/MS method for the simultaneous determination of 17 steroidal

218

hormones in equine serum. J Mass Spectrom. 2017;52(1):22-9.

219

[15]

Genangeli M, Caprioli G, Cortese M, Laus F, Petrelli R, Ricciutelli M, et al. Simultaneous

220

quantitation of 9 anabolic and natural steroidal hormones in equine urine by UHPLC-MS/MS

221

triple quadrupole. J Chromatogr B Analyt Technol Biomed Life Sci. 2019;1117:36-40.

222

[16]

Aguirre-Gamboa R, Joosten I, Urbano PCM, van der Molen RG, van Rijssen E, van

223

Cranenbroek B, et al. Differential Effects of Environmental and Genetic Factors on T and B

224

Cell Immune Traits. Cell Rep. 2016;17(9):2474-87.

225

[17]

Engels M, Pijnenburg-Kleizen KJ, Utari A, Faradz SMH, Oude-Alink S, Van Herwaarden AE,

226

et al. Glucocorticoid activity of adrenal steroid precursors in untreated patients with congenital

227

adrenal hyperplasia Accepted for publication in the J. Clin. Endocrinol. Metab.

228

[18]

Sauer FJ, Bruckmaier RM, Ramseyer A, Vidondo B, Scheidegger MD, Gerber V. Diagnostic

229

accuracy of post-ACTH challenge salivary cortisol concentrations for identifying horses with

230

equine glandular gastric disease. J Anim Sci. 2018;96(6):2154-61.

231

[19]

Koren L, Ng ES, Soma KK, Wynne-Edwards KE. Sample preparation and liquid

232

chromatography-tandem mass spectrometry for multiple steroids in mammalian and avian

233

circulation. PLoS One. 2012;7(2):e32496.

234

[20]

Regal P, Vazquez BI, Franco CM, Cepeda A, Fente C. Quantitative LC-MS/MS method for the

235

sensitive and simultaneous determination of natural hormones in bovine serum. J Chromatogr

236

B Analyt Technol Biomed Life Sci. 2009;877(24):2457-64.

237 238 239

[21]

Mostaghel EA. Beyond T and DHT - novel steroid derivatives capable of wild type androgen receptor activation. Int J Biol Sci. 2014;10(6):602-13.

240

Tables

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Table 1: Results of the steroids presented as mean ± standard deviation before (minute 0 = T0) and

242

60 minutes after stimulation (T60) with 1 µg/kg BW of adrenocorticotropic hormone (ACTH). The

243

values of the steroids were compared, before and after stimulation using the paired t-test or Wilcoxon

244

(Rank Sum) Test.

245 246

Steroids

T0

n

T60

n

P-value

Cortisol [nmol/l]

138 ± 54

11

363 ± 112

11

<0.001

11-Deoxycortisol [nmol/l]

0.67 ± 0.14

4

2.94 ± 1.18

11

0.004

Cortisone [nmol/l]

6.10 ± 1.66

11

19.51 ± 7.75

11

<0.001

Corticosterone [nmol/l]

4.69 ± 2.87

11

19.28 ± 5.74

11

<0.001

11-Deoxycorticosterone [nmol/l]

0.04 ± 0.03

6

0.19 ± 0.15

11

0.018

Androstenedione [nmol/l]

0.06 ± 0.01

2

0.24 ± 0.10

11

0.335

17-OH-Progesterone [nmol/l]

0.59 ± 0.83

3

0.52 ± 0.47

11

0.250

Progesterone [nmol/l]

16.00

1

4.67 ± 9.47

5

NA

Testosterone [nmol/l]

NA

0

0.03 ± 0.00

3

NA

n: number of horses; NA: all measurements below the detection limit or not available

247

Table 2: The mean increase from before (minute 0) to 60 minutes after ACTH stimulation of five

248

steroids. P-values indicate if the increases of the corresponding steroids are significantly higher than

249

the increase of cortisol.

250 251

Steroid metabolite

Increase [x-fold]

P-value

Cortisol (Reference)

1.96

11-Deoxycortisol

5.28

0.01

Cortisone

2.36

0.08

Corticosterone

4.73

0.01

11-Deoxycorticosterone 7.31

0.03

252

Table 3: Comparison of the mean values of this study's baseline (minute 0) results to the results of

253

Genangeli et al. (2017) [14]. Steroid metabolite

254

Present study

Genangeli et al.

[nmol/l]

[ng/ml]

[ng/ml]

Cortisol

138.27

50.12

42.93

11-Deoxycortisol

0.67

0.23

0.19

Cortisone

6.10

2.20

NA

Corticosterone

4.69

1.62

45.51

11-Deoxycorticosterone 0.04

0.01

0.03

Androstenedione

0.06

0.02

0.22

17-OH-Progesterone

0.59

0.19

13.46

Progesterone

16.00

5.03

0.93

NA: not available

255

List of figures

256

Figure 1: Pathways of the steroid biosynthesis

257

Figure adapted from Mostaghel (2014) [21]. The figure shows the pathways of the steroid biosynthesis

258

in the adrenal gland (Zona glomerulosa, Zona fasciculata, and Zona reticularis) and the gonads.

259

Steroids written in bold showed a significant stimulation 60 minutes after ACTH administration in the

260

current study. Steroids written in grey were not analysed.

261 262

Figure 2: Increase after ACTH stimulation of six steroids

263

The graphs show the increase of the steroids from baseline values before (minute 0 = T0) to 60

264

minutes after stimulation (T60) with 1µg/kg ACTH intravenously: cortisol (A), 11-deoxycortisol (B),

265

cortisone (C), corticosterone (D), 11-deoxycorticosterone (E), and androstenedione (F). Lines indicate

266

the pre- and post-stimulation values of one horse, asteriks indicate a significant increase after

267

stimulation. Levels of significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

268

269 270 271

Figure 1

A

B

C

D

E

F

272 Figure 2

273

Plasma steroid profiles before and after ACTH stimulation test in healthy horses

a, *

b

a

a

a

Kirchmeier A. , van Herwaarden A. E. , van der Kolk J. H. , Sauer F. J. , Gerber V.

a

Swiss Institute of Equine Medicine ISME, Vetsuisse Faculty, University of Bern, Länggassstrasse

124, 3012 Bern, Switzerland b

Radboud University Medical Center, Department of Laboratory Medicine, Nijmegen, the Netherlands

Highlights •

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a viable method to measure steroids in equine plasma.

•

ACTH stimulates different pathways of the steroid biosynthesis in horses.

•

11-deoxycorticosterone, 11-deoxycortisol and corticosterone might be potential new stress markers in horses.