<?xml version="1.0" encoding="UTF-8" standalone="no"?>
<!DOCTYPE article PUBLIC "-//NLM//DTD Journal Publishing DTD v2.3 20070202//EN" "journalpublishing.dtd">
<article xml:lang="EN" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article">
<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Vet. Sci.</journal-id>
<journal-title>Frontiers in Veterinary Science</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Vet. Sci.</abbrev-journal-title>
<issn pub-type="epub">2297-1769</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fvets.2022.1014792</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Veterinary Science</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Serum steroid profiling of hepatocellular carcinoma associated with hyperadrenocorticism in dogs: A preliminary study</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name><surname>Oo</surname> <given-names>Thandar</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/1891434/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Sasaki</surname> <given-names>Noboru</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/665371/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Ikenaka</surname> <given-names>Yoshinori</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/1687951/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Ichise</surname> <given-names>Takahiro</given-names></name>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
</contrib>
<contrib contrib-type="author">
<name><surname>Nagata</surname> <given-names>Noriyuki</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
</contrib>
<contrib contrib-type="author">
<name><surname>Yokoyama</surname> <given-names>Nozomu</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/1951246/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Sasaoka</surname> <given-names>Kazuyoshi</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/919734/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Morishita</surname> <given-names>Keitaro</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
</contrib>
<contrib contrib-type="author">
<name><surname>Nakamura</surname> <given-names>Kensuke</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/919230/overview"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name><surname>Takiguchi</surname> <given-names>Mitsuyoshi</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="corresp" rid="c001"><sup>&#x0002A;</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/257080/overview"/>
</contrib>
</contrib-group>
<aff id="aff1"><sup>1</sup><institution>Laboratory of Veterinary Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University</institution>, <addr-line>Sapporo</addr-line>, <country>Japan</country></aff>
<aff id="aff2"><sup>2</sup><institution>Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Hokkaido University</institution>, <addr-line>Sapporo</addr-line>, <country>Japan</country></aff>
<aff id="aff3"><sup>3</sup><institution>Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University</institution>, <addr-line>Sapporo</addr-line>, <country>Japan</country></aff>
<author-notes>
<fn fn-type="edited-by"><p>Edited by: F. Capela e Silva, University of Evora, Portugal</p></fn>
<fn fn-type="edited-by"><p>Reviewed by: Ana Faustino, University of Evora, Portugal; Sandra Branco, University of Evora, Portugal</p></fn>
<corresp id="c001">&#x0002A;Correspondence: Mitsuyoshi Takiguchi <email>mtaki&#x00040;vetmed.hokudai.ac.jp</email></corresp>
<fn fn-type="other" id="fn001"><p>This article was submitted to Comparative and Clinical Medicine, a section of the journal Frontiers in Veterinary Science</p></fn></author-notes>
<pub-date pub-type="epub">
<day>28</day>
<month>09</month>
<year>2022</year>
</pub-date>
<pub-date pub-type="collection">
<year>2022</year>
</pub-date>
<volume>9</volume>
<elocation-id>1014792</elocation-id>
<history>
<date date-type="received">
<day>08</day>
<month>08</month>
<year>2022</year>
</date>
<date date-type="accepted">
<day>05</day>
<month>09</month>
<year>2022</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x000A9; 2022 Oo, Sasaki, Ikenaka, Ichise, Nagata, Yokoyama, Sasaoka, Morishita, Nakamura and Takiguchi.</copyright-statement>
<copyright-year>2022</copyright-year>
<copyright-holder>Oo, Sasaki, Ikenaka, Ichise, Nagata, Yokoyama, Sasaoka, Morishita, Nakamura and Takiguchi</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/"><p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p></license> </permissions>
<abstract>
<sec>
<title>Background</title>
<p>Hepatocellular carcinoma (HCC) is one of the most common primary liver tumors in humans and dogs. Excessive adrenocortical hormone exposure may cause steroid hepatopathy, which may develop into HCC. In our previous study, hyperadrenocorticism (HAC) was a highly concurrent disease in dogs with HCC. Therefore, this study hypothesized that adrenal steroid alterations might be involved in hepatocarcinogenesis and aimed to specify the relationship between HAC and HCC in dogs.</p></sec>
<sec>
<title>Materials and methods</title>
<p>This study included 46 dogs brought to the Hokkaido University Veterinary Teaching Hospital between March 2019 and December 2020. Owners gave their signed consent for blood collection on their first visit. A total of 19 steroids (14 steroids and 5 metabolites) in the baseline serum of 15 dogs with HCC, 15 dogs with HAC, and 10 dogs with both diseases were quantitatively measured using the developed liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method.</p></sec>
<sec>
<title>Results</title>
<p>In each group, 11 steroids were detected higher than 50%. The detection rate of steroid hormones did not significantly differ between the groups (<italic>p</italic> &#x0003E; 0.05). Principle component analysis (PCA) showed that the steroid profiles of the three groups were comparable. Median steroid hormone concentrations were not significantly different between the study diseases (<italic>p</italic> &#x0003E; 0.05).</p></sec>
<sec>
<title>Conclusion</title>
<p>The developed LC/MS/MS was useful for measuring steroid hormones. Although it was clear that HAC was concurrent in dogs with HCC, none of the serum steroids was suggested to be involved in HCC.</p></sec></abstract>
<kwd-group>
<kwd>adrenocortical hormones</kwd>
<kwd>hepatocarcinogenesis</kwd>
<kwd>baseline serum</kwd>
<kwd>LC/MS/MS</kwd>
<kwd>dogs</kwd>
</kwd-group>
<counts>
<fig-count count="4"/>
<table-count count="7"/>
<equation-count count="0"/>
<ref-count count="42"/>
<page-count count="12"/>
<word-count count="6033"/>
</counts>
</article-meta>
</front>
<body>
<sec sec-type="intro" id="s1">
<title>Introduction</title>
<p>The hepatocellular tumor is a common hepatobiliary disease in dogs. Hepatocellular carcinoma (HCC) accounts for 50&#x02013;70% of all hepatic tumors in dogs, also being the sixth most common cancer globally in humans (<xref ref-type="bibr" rid="B1">1</xref>&#x02013;<xref ref-type="bibr" rid="B4">4</xref>). Major etiopathologies of HCC in humans are viral infections and metabolic diseases (<xref ref-type="bibr" rid="B5">5</xref>). HCC frequently manifests in dogs aged over 10 years, and there are anecdotal reports of over-presentation in male dogs. However, gender predisposition, specific risk factors, and the precise mechanisms of hepatocarcinogenesis are not well-characterized in dogs (<xref ref-type="bibr" rid="B6">6</xref>, <xref ref-type="bibr" rid="B7">7</xref>). Retrospective studies revealed that Scottish Terriers with vacuolar hepatopathy, certain breeds of dogs, such as Miniature Schnauzers, Shih Tzus, Welsh Corgis, and Beagles, have high risks of developing HCC, and concurrent hyperadrenocorticism (HAC) is reportedly associated with the development of HCC (<xref ref-type="bibr" rid="B8">8</xref>&#x02013;<xref ref-type="bibr" rid="B11">11</xref>). In Scottish Terriers, increased corticosteroid isoforms of alkaline phosphatase and hepatocellular vacuolation on histology are very common (<xref ref-type="bibr" rid="B12">12</xref>&#x02013;<xref ref-type="bibr" rid="B14">14</xref>). HCC was detected in 34% of Scottish Terriers with vacuolar hepatopathy (<xref ref-type="bibr" rid="B8">8</xref>). A recent study suggested that a single nucleotide polymorphism in HSD17B2 causes an increase in adrenal sex steroids (progesterone and androstenedione) in Scottish Terriers, which results in increased alkaline phosphatase in those dogs (<xref ref-type="bibr" rid="B15">15</xref>, <xref ref-type="bibr" rid="B16">16</xref>). Leela-Arporn et al. investigated the concurrent diseases of 44 massive HCC cases and found that 10 dogs with HCC had hypercortisolemia with HAC suggestive clinical signs (<xref ref-type="bibr" rid="B9">9</xref>). Thus, it is not surprising that excess adrenal steroids affect the liver and play a role in HCC development.</p>
<p>In humans, metabolic syndrome is a potential risk factor for non-alcoholic fatty liver disease (NAFLD) that may progress to HCC. However, the mechanism of hepatic lipid accumulation and developing HCC remains ambiguous. The clinicopathologic abnormalities of HAC, such as steroid hepatopathy caused by chronic over-production of adrenocortical hormone in dogs, somewhat resemble NAFLD-induced HCC (<xref ref-type="bibr" rid="B17">17</xref>, <xref ref-type="bibr" rid="B18">18</xref>). An association between HAC and HCC in dogs may contribute to understand HCC development from NAFLD, a liver phenotype of the metabolic syndrome in humans. Cortisol is the main and potent glucocorticoid in dogs with HAC. Meanwhile, other adrenal steroids, such as 17&#x003B1;-OH progesterone, corticosterone, or 11-deoxycortisol, have been considered to cause HAC (<xref ref-type="bibr" rid="B19">19</xref>&#x02013;<xref ref-type="bibr" rid="B21">21</xref>). Therefore, it may be helpful to measure multiple adrenal steroids, so-called steroid profile, to understand the link between HAC and HCC in dogs.</p>
<p>In veterinary medicine, steroid hormones have been traditionally measured using immunoassay because of their convenience and high sensitivity (<xref ref-type="bibr" rid="B22">22</xref>&#x02013;<xref ref-type="bibr" rid="B24">24</xref>). However, the major disadvantage of immunoassay is that it is less specific due to the cross-reactivity among structurally similar steroids (<xref ref-type="bibr" rid="B25">25</xref>). Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) has become more popular in clinical laboratories since LC/MS/MS can measure multiple steroid hormones at a single runtime with high specificity and sensitivity (<xref ref-type="bibr" rid="B26">26</xref>, <xref ref-type="bibr" rid="B27">27</xref>). Although various LC/MS/MS methods have been continuously published for analyzing multiple steroid hormones in biofluids, some are particularly sensitive for a few steroids; others are feasible for detecting dozens but have poor sensitivity (<xref ref-type="bibr" rid="B28">28</xref>). The chemical derivative reagent is an additional sample pretreatment method that reacts to the functional groups of target analytes (amine, hydroxyl, or carbonyl groups) to enhance better ionization (<xref ref-type="bibr" rid="B29">29</xref>). In our previous LC/MS/MS method, it was feasible to measure the serum levels of nine steroid hormones; however, nine steroids may not be enough for the steroid profile (<xref ref-type="bibr" rid="B19">19</xref>). Thus, we combined the conventional non-derivatization and keto-derivatization methods for measuring multiple steroid hormones and investigated the relationship between HAC and HCC in dogs using serum steroid profiling.</p>
</sec>
<sec sec-type="materials and methods" id="s2">
<title>Materials and methods</title>
<sec>
<title>Study population</title>
<p>From September 2019 to December 2021, a retrospective study was conducted at the Hokkaido University Veterinary Teaching Hospital (HUVTH). A total of 46 dogs, 31 massive HCC and 15 HAC cases, were included in this study. Among the 31 massive HCC cases, 10 dogs had concurrent HAC, while 21 were HAC-free. Of the 15 dogs with HAC, abdominal ultrasonography revealed no hepatic lesion. The other endocrine disorders were 2 dogs with hypothyroidism and 1 dog with hyperparathyroidism. Myxomatous mitral valve disease (MMVD) in 5 dogs, biliary sludge in 3 dogs, chronic kidney disease (CKD) in 2 dogs, bladder stones in 2 dogs, atopic dermatitis in 2 dogs, and one each had pyoderma, mast cell tumor (MCT), hypercalcemia, and breast cancer were presented as other medical complications. Informed consent was obtained from all owners of the dogs involved in this study.</p>
<p>Liver tumors were collected when HCC-suspected dogs were undergoing surgery at HUVTH. Formalin-fixed liver tissues were sent to a private laboratory and HCC was histologically confirmed by a board-certificated pathologist. The pathological diagnosis of HCC was defined according to the guidelines of the World Small Animal Veterinary Association (WSAVA) Liver Standardization Group (<xref ref-type="bibr" rid="B30">30</xref>). Diagnostic criteria of HAC were the presence of one or more HAC-suggestive clinical signs, a positive result of the adrenocorticotropic hormone (ACTH) stimulation test, and clinical response to the trilostane treatment following 3 months (<xref ref-type="bibr" rid="B31">31</xref>). Six HCC dogs without HAC received corticosteroid treatment for more than 2 weeks. Those dogs were excluded from the further analysis, and serum profiling was performed on 15 dogs with HAC, 15 dogs with HCC, and 10 dogs with both diseases.</p>
<p>Blood of 2 ml volume was collected at their first visit to our hospital and serum was obtained by centrifuging at 3,000 rpm for 10 min. Serum was stored at &#x02212;80&#x000B0;C until analysis of steroid profile. The breed, age, body weight, gender distribution, and neutered status of all dogs are described in <xref ref-type="table" rid="T1">Table 1</xref>.</p>
<table-wrap position="float" id="T1">
<label>Table 1</label>
<caption><p>Demographic information of dogs in hepatocellular carcinoma (HCC), hyperadrenocorticism (HAC), and HCC with HAC.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th/>
<th valign="top" align="left"><bold>HCC</bold></th>
<th valign="top" align="left"><bold>HAC</bold></th>
<th valign="top" align="left"><bold>HCC with HAC</bold></th>
</tr>
<tr>
<th/>
<th valign="top" align="left"><bold><italic>N =</italic> 15</bold></th>
<th valign="top" align="left"><bold><italic>N =</italic> 15</bold></th>
<th valign="top" align="left"><bold><italic>N =</italic> 10</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">Breeds (<italic>n</italic>)</td>
<td valign="top" align="left">Miniature Dachshund (2)</td>
<td valign="top" align="left">Miniature Dachshund (2)</td>
<td valign="top" align="left">Miniature Dachshund (1)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Shiba (1)</td>
<td valign="top" align="left">Shiba (2)</td>
<td valign="top" align="left">Shiba (1)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Miniature Schnauzer (1)</td>
<td valign="top" align="left">Miniature Schnauzer (1)</td>
<td valign="top" align="left">Miniature Schnauzer (1)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Chihuahua (1)</td>
<td valign="top" align="left">Chihuahua (4)</td>
<td valign="top" align="left">Chihuahua (1)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Beagle (1)</td>
<td valign="top" align="left">West Highland White Terrier (2)</td>
<td valign="top" align="left">Shih Tzu (1)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Shih Tzu (1)</td>
<td valign="top" align="left">Miniature Pinscher (1)</td>
<td valign="top" align="left">Boston Terrier (1)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Siberia Huskey (1)</td>
<td valign="top" align="left">American Cocker Spaniel (1)</td>
<td valign="top" align="left">Beagle (1)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Japanese Spitx (1)</td>
<td valign="top" align="left">Yorkshire Terrier (1)</td>
<td valign="top" align="left">Mix breed (3)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Samyoed (1)</td>
<td valign="top" align="left">Mix breed (1)</td>
<td/>
</tr>
<tr>
<td/>
<td valign="top" align="left">Bichon Frise (1)</td>
<td/>
<td/>
</tr>
<tr>
<td/>
<td valign="top" align="left">Mix breed (4)</td>
<td/>
<td/>
</tr>
<tr>
<td valign="top" align="left">Gender (<italic>n</italic>)</td>
<td valign="top" align="left">Male (1)</td>
<td valign="top" align="left">Male (1)</td>
<td valign="top" align="left">Male (0)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Female (2)</td>
<td valign="top" align="left">Female (1)</td>
<td valign="top" align="left">Female (2)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Neutered male (6)</td>
<td valign="top" align="left">Neutered male (6)</td>
<td valign="top" align="left">Neutered male (4)</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Spay female (6)</td>
<td valign="top" align="left">Spay female (7)</td>
<td valign="top" align="left">Spay female (4)</td>
</tr>
<tr>
<td valign="top" align="left">Median age (range) (years)</td>
<td valign="top" align="left">10.7 (7.2&#x02013;14.8)</td>
<td valign="top" align="left">12.5 (5.5&#x02013;14.0)</td>
<td valign="top" align="left">10.8 (7.7&#x02013;14.3)</td>
</tr>
<tr>
<td valign="top" align="left">Median body weight (range) (kg)</td>
<td valign="top" align="left">7.0 (2.2&#x02013;27.0)</td>
<td valign="top" align="left">8.7 (2.7&#x02013;17.7)</td>
<td valign="top" align="left">9.8 (3.9&#x02013;21.6)</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec>
<title>Chemicals and reagents</title>
<p>A total of 19 steroids (<xref ref-type="fig" rid="F1">Figure 1</xref>) in the serum were quantified using LC/MS/MS. The standard (STD) and stable isotope-labeled internal standard (IS) of 19 steroids were purchased from Toronto Research Chemicals (Toronto, Canada), Cerilliant (Round Rock, TX, USA), or Sigma-Aldrich (St. Louis, MO, USA). Double-distilled water (DDW) and liquid chromatography grade-analytical reagents (methanol and acetonitrile) were bought from Kanto Chemical Co., Ltd. (Tokyo, Japan). Formic acid (abt. 99%) was obtained from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). The phospholipid removal MonoSpin<sup>&#x000AE;</sup> column was purchased from GL Sciences Co., Ltd. (Tokyo, Japan). Amplifex<sup>TM</sup> Keto Reagent Kits were acquired from AB SCIEX Pte. Ltd. (Framingham, MA, USA). Working solutions for STD and IS mixtures were dissolved in methanol at 10 ng/ml and 100 ng/ml concentrations, respectively.</p>
<fig id="F1" position="float">
<label>Figure 1</label>
<caption><p>Schematic pathway of steroids and metabolites. CYP17, 17&#x003B1;-hydroxylase, CYP21, 21-hydroxylase, CYP 11 B1, 11&#x003B2; hydroxylase, 3&#x003B1; HSD, 3&#x003B1;-hydroxysteroid dehydrogenase, and 3&#x003B2; HSD, 3&#x003B2;-hydroxysteroid dehydrogenase. Italic: Metabolites.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-1014792-g0001.tif"/>
</fig>
</sec>
<sec>
<title>Sample preparation</title>
<p>In total, 19 steroids (14 adrenal steroids and 5 metabolites) in the baseline serum of 40 dogs were quantitatively measured using two sample preparation methods. The preparation procedure is described below.</p>
<sec>
<title>Non-derivatization method</title>
<p>First, 25 &#x003BC;l of serum was thoroughly mixed with 10 &#x003BC;l IS mixture working solution and 90 &#x003BC;l of 1% formic acid containing acetonitrile. The concentration of each steroid in the IS mixture was 10 ng/ml. The mixture was centrifuged at 10,000 &#x000D7; g for 10 min at 25&#x000B0;C. Afterward, 100 &#x003BC;l of the supernatant was applied to the MonoSpin column and centrifuged at 3,000 &#x000D7; g for 2 min to remove serum phospholipids. The eluate was evaporated at 60&#x000B0;C using a centrifugal vaporizer CVE-2000D (EYELA, Bohemia, NY, USA). Then, 100 &#x003BC;l of 50% methanol-double distilled water (DDW) containing 0.1% formic acid was added to reconstitute the dried tube.</p>
</sec>
<sec>
<title>Keto-derivatization method</title>
<p>First, 25 &#x003BC;l of serum was mixed with 10 &#x003BC;l of the IS mixture working solution and 90 &#x003BC;l of 1% formic acid in acetonitrile. The mixture was vortexed and centrifuged at 10,000 &#x000D7; g for 10 min. Afterward, 100 &#x003BC;l of the organic layer was loaded into the MonoSpin column and the phospholipids were removed by centrifuging at 3,000 &#x000D7; g for 2 min. The eluate was dried at 60&#x000B0;C using the speed vacuum evaporation. A keto-derivatization reagent was prepared according to the instructions of the manufacturer. The dried eluate was reconstituted with 50 &#x003BC;l of the keto-derivatization reagent and incubated for 60 min on a vortex mixer. The derivatization reaction was stopped by combining with 50 &#x003BC;l DDW. The final 100 &#x003BC;l solution was transferred to the LC/MS/MS system.</p>
</sec>
</sec>
<sec>
<title>LC/MS/MS analytical conditions</title>
<p>Liquid chromatography coupled with tandem mass spectrometry was performed using an Agilent 6495B Triple Quadrupole LC/MS (Agilent Technologies, Santa Clara, CA, USA) coupled with a high-performance liquid chromatography system (1260 Infinity II, Agilent Technologies). The 50 &#x003BC;l solution was injected into the LC/MS/MS system. Chromatographic separation was carried out on the Shim-pack Biphenyl Column (&#x003D5;2.6 &#x003BC;m, 100 &#x000D7; 3 mm, Shimadzu, Kyoto, Japan) for the non-derivatization method and Poroshell 120/EC-C18 Column (&#x003D5;2.7 &#x003BC;m, 100 &#x000D7; 3 mm, Agilent Technologies (L-293) Santa Clara, USA) for the keto-derivatization method, respectively. The mass spectrometry was run in the positive electrospray ionization (ESI) multiple reaction monitoring (MRM) mode. The data acquisition and processing were operated using the Agilent Mass Hunter Workstation software (Agilent Technologies).</p>
</sec>
<sec>
<title>Validation of the method</title>
<p>Serum samples required for method validation were collected from 6 healthy, intact female Beagle dogs owned by the animal facility of the Graduate School of Veterinary Medicine, Hokkaido University. The method performance was determined by calculating the instrumental detection limit (IDL) and method detection limit (MDL) of the steroid hormones.</p>
</sec>
<sec>
<title>Calibration curves</title>
<p>The calibration curve was created using 9 concentrations of the STD mixture (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 ng/ml) dissolved with 1 ng/ml of the IS mixed in methanol. Peak areas of steroid hormones were manually identified with the corresponding IS peak area in each sample. The analyte peak area was less than the peak area of the lowest calibration point (0.001 ng/ml) and the concentration less than the MDL value was set as the zero concentration. The concentration of steroid hormone was calculated using the following equation.</p>
<p>Cs = Css &#x000D7; (As/Ass-b)/a</p>
<p>Cs: amount (ng) of target steroids</p>
<p>Css: amount (ng) of IS</p>
<p>As: Peak area of target steroids</p>
<p>Ass: Peak area of IS</p>
<p>a: slope of the calibration curve</p>
<p>b: intercept of the calibration curve</p>
</sec>
<sec>
<title>Statistical analysis</title>
<p>Statistical analyses were performed using commercial software (JMP Pro<sup>&#x000AE;</sup>16, version 16.0.0, SAS Institute Inc, Cary, NC, USA). A comparison of age and body weight was carried out using the Kruskal-Wallis test. Gender distribution and detection rate of steroid hormones among the groups were analyzed using the chi-square test. Principle component analysis (PCA) was performed to see the steroid pattern difference between the disease groups. The Steel-Dwass test was used for a non-parametric multiple comparison of median steroid concentration among HCC, HAC, and HCC with HAC dogs. <italic>p</italic>-value (&#x0003C; 0.05) was considered statistically significant for all comparisons.</p>
</sec>
</sec>
<sec sec-type="results" id="s3">
<title>Results</title>
<p>Breeds were not over-presented in 15 dogs with HCC, 15 dogs with HAC, and 10 HCC with HAC dogs (<xref ref-type="table" rid="T1">Table 1</xref>). Age, body weight, and gender distribution were not significantly different between the groups (<italic>p</italic> &#x0003E; 0.05).</p>
<p>A gradient program and column settings are described in <xref ref-type="table" rid="T2">Tables 2A</xref>,<xref ref-type="table" rid="T3">B</xref> for the non-derivatization and keto-derivatization methods, respectively. The mass-to-charge transition of 19 steroids and ISs in positive MRM mode are presented in <xref ref-type="table" rid="T4">Tables 3A</xref>,<xref ref-type="table" rid="T5">B</xref> for the non-derivatization and keto-derivatization methods, respectively. The assay performance, i.e., IDL and MDL values of each steroid, is described in <xref ref-type="table" rid="T6">Table 4</xref>.</p>
<table-wrap position="float" id="T2">
<label>Table 2A</label>
<caption><p>Gradient program and column temperature in the non-derivatization method.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th valign="top" align="left"><bold>Pump system</bold></th>
<th valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Mobile phase A</bold></th>
<th valign="top" align="center" colspan="2" style="border-bottom: thin solid #000000;"><bold>0.1% formic acid in DDW</bold></th>
</tr>
<tr>
<th/>
<th valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Mobile phase B</bold></th>
<th valign="top" align="center" colspan="2" style="border-bottom: thin solid #000000;"><bold>0.1% formic acid in methanol</bold></th>
</tr>
<tr>
<th valign="top" align="center" colspan="2" style="border-bottom: thin solid #000000;">Temperature</th>
<th valign="top" align="center" colspan="2" style="border-bottom: thin solid #000000;">50&#x000B0;C</th>
</tr> <tr>
<th valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Gradient condition pump system</bold></th>
<th valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Mobile phase A (%)</bold></th>
<th valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Mobile phase B (%)</bold></th>
<th valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Flow rate (ml/min)</bold></th>
</tr> 
</thead>
<tbody>
<tr>
<td valign="top" align="left">0 min</td>
<td valign="top" align="center">60.00</td>
<td valign="top" align="center">40.00</td>
<td valign="top" align="center">0.4</td>
</tr>
<tr>
<td valign="top" align="left">0.50 min</td>
<td valign="top" align="center">60.00</td>
<td valign="top" align="center">40.00</td>
<td valign="top" align="center">0.4</td>
</tr>
<tr>
<td valign="top" align="left">0.51 min</td>
<td valign="top" align="center">60.00</td>
<td valign="top" align="center">40.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">3.00 min</td>
<td valign="top" align="center">60.00</td>
<td valign="top" align="center">40.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">19.00 min</td>
<td valign="top" align="center">15.00</td>
<td valign="top" align="center">85.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">19.50 min</td>
<td valign="top" align="center">0.00</td>
<td valign="top" align="center">100.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">21.00 min</td>
<td valign="top" align="center">0.00</td>
<td valign="top" align="center">100.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">21.01 min</td>
<td valign="top" align="center">60.00</td>
<td valign="top" align="center">40.00</td>
<td valign="top" align="center">0.4</td>
</tr>
<tr>
<td valign="top" align="left" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Ion source</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Sheath gas temp (</bold><bold>&#x000B0;</bold><bold>C)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Sheath gas flow rate (L/min)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Drying gas temperature (</bold><bold>&#x000B0;</bold><bold>C)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Drying gas flow rate (L/min)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Nebulizer (psi)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Capillary (V)</bold></td>
</tr> <tr>
<td valign="top" align="left">Electrospray Ionization</td>
<td valign="top" align="center">350.00</td>
<td valign="top" align="center">12.00</td>
<td valign="top" align="center">290</td>
<td valign="top" align="center">19</td>
<td valign="top" align="center">40</td>
<td valign="top" align="center">3,500</td>
</tr>
</tbody> 
</table>
</table-wrap>
<table-wrap position="float" id="T3">
<label>Table 2B</label>
<caption><p>Gradient program and column temperature in the keto-derivatization method.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th valign="top" align="left"><bold>Pump system</bold></th>
<th valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Mobile phase A</bold></th>
<th valign="top" align="center" colspan="2" style="border-bottom: thin solid #000000;"><bold>0.1% formic acid in DDW</bold></th>
</tr>
<tr>
<th/>
<th valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Mobile phase B</bold></th>
<th valign="top" align="center" colspan="2" style="border-bottom: thin solid #000000;"><bold>0.1% formic acid in methanol</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left" colspan="2" style="border-bottom: thin solid #000000;">Temperature</td>
<td valign="top" align="center" colspan="2" style="border-bottom: thin solid #000000;">50&#x000B0;C</td>
</tr> <tr>
<td valign="top" align="left" style="border-bottom: thin solid #000000;"><bold>Gradient condition pump system</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Mobile phase A (%)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Mobile phase B (%)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000;"><bold>Flow rate (mL/min)</bold></td>
</tr> <tr>
<td valign="top" align="left">0 min</td>
<td valign="top" align="center">65.00</td>
<td valign="top" align="center">35.00</td>
<td valign="top" align="center">0.4</td>
</tr>
<tr>
<td valign="top" align="left">1.50 min</td>
<td valign="top" align="center">65.00</td>
<td valign="top" align="center">35.00</td>
<td valign="top" align="center">0.4</td>
</tr>
<tr>
<td valign="top" align="left">1.51 min</td>
<td valign="top" align="center">65.00</td>
<td valign="top" align="center">35.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">17.00 min</td>
<td valign="top" align="center">40.00</td>
<td valign="top" align="center">60.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">17.01 min</td>
<td valign="top" align="center">0.00</td>
<td valign="top" align="center">100.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">18.50 min</td>
<td valign="top" align="center">0.00</td>
<td valign="top" align="center">100.00</td>
<td valign="top" align="center">0.8</td>
</tr>
<tr>
<td valign="top" align="left">18.51 min</td>
<td valign="top" align="center">65.00</td>
<td valign="top" align="center">35.00</td>
<td valign="top" align="center">0.4</td>
</tr>
<tr>
<td valign="top" align="left" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Ion source</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Sheath gas temp (</bold><bold>&#x000B0;</bold><bold>C)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Sheath gas flow rate (L/min)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Drying gas temperature (</bold><bold>&#x000B0;</bold><bold>C)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Drying gas flow rate (L/min)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Nebulizer (psi)</bold></td>
<td valign="top" align="center" style="border-bottom: thin solid #000000; border-top: thin solid #000000;"><bold>Capillary (V)</bold></td>
</tr> <tr>
<td valign="top" align="left">Electrospray Ionization</td>
<td valign="top" align="center">350.00</td>
<td valign="top" align="center">12.00</td>
<td valign="top" align="center">290</td>
<td valign="top" align="center">17</td>
<td valign="top" align="center">220</td>
<td valign="top" align="center">3,500</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap position="float" id="T4">
<label>Table 3A</label>
<caption><p>Multiple reaction monitoring conditions of steroids and their internal standards in the non-derivatization method.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th valign="top" align="left"><bold>Native STD/Stable isotope labeled IS</bold></th>
<th valign="top" align="center"><bold>Precursor ion (<italic>m/z</italic>)</bold></th>
<th valign="top" align="center"><bold>Quantifier</bold><break/> <bold>(<italic>m/z</italic>)</bold></th>
<th valign="top" align="center"><bold>Qualifier</bold><break/> <bold>(<italic>m/z</italic>)</bold></th>
<th valign="top" align="center"><bold>Collision energy (V) quantifier</bold></th>
<th valign="top" align="center"><bold>Collision energy (V) qualifier</bold></th>
<th valign="top" align="center"><bold>Polarity</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td/>
<td/>
<td/>
<td/>
<td/>
<td/>
<td/>
</tr>
<tr>
<td valign="top" align="left">Progesterone</td>
<td valign="top" align="center">315.2</td>
<td valign="top" align="center">97.0</td>
<td valign="top" align="center">108.9</td>
<td valign="top" align="center">24</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Progesterone-d9</td>
<td valign="top" align="center">325.0</td>
<td valign="top" align="center">100.1</td>
<td valign="top" align="center">113.2</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">32</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">17&#x003B1; -OH progesterone</td>
<td valign="top" align="center">331.2</td>
<td valign="top" align="center">109.1</td>
<td valign="top" align="center">97.2</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">44</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">17&#x003B1; -OH progesterone-13C3</td>
<td valign="top" align="center">334.0</td>
<td valign="top" align="center">100.2</td>
<td valign="top" align="center">81.2</td>
<td valign="top" align="center">32</td>
<td valign="top" align="center">68</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Cortisol</td>
<td valign="top" align="center">363.2</td>
<td valign="top" align="center">121.1</td>
<td valign="top" align="center">91.0</td>
<td valign="top" align="center">32</td>
<td valign="top" align="center">68</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Cortisol-d4</td>
<td valign="top" align="center">367.4</td>
<td valign="top" align="center">121.0</td>
<td valign="top" align="center">97.1</td>
<td valign="top" align="center">24</td>
<td valign="top" align="center">52</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Cortisone</td>
<td valign="top" align="center">361.2</td>
<td valign="top" align="center">163.0</td>
<td valign="top" align="center">91.1</td>
<td valign="top" align="center">25</td>
<td valign="top" align="center">70</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Cortisone-d8</td>
<td valign="top" align="center">369.2</td>
<td valign="top" align="center">168.2</td>
<td valign="top" align="center">93.4</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">72</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Corticosterone</td>
<td valign="top" align="center">347.2</td>
<td valign="top" align="center">121.2</td>
<td valign="top" align="center">91.0</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">68</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Corticosterone-d4</td>
<td valign="top" align="center">351.4</td>
<td valign="top" align="center">121.0</td>
<td valign="top" align="center">97.1</td>
<td valign="top" align="center">44</td>
<td valign="top" align="center">40</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">11-deoxycorticosterone</td>
<td valign="top" align="center">331.5</td>
<td valign="top" align="center">109.2</td>
<td valign="top" align="center">97.0</td>
<td valign="top" align="center">32</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">11-deoxycorticosterone-13C3</td>
<td valign="top" align="center">334.1</td>
<td valign="top" align="center">100.0</td>
<td valign="top" align="center">112.1</td>
<td valign="top" align="center">24</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">11-deoxycortisol</td>
<td valign="top" align="center">347.2</td>
<td valign="top" align="center">97.1</td>
<td valign="top" align="center">109.0</td>
<td valign="top" align="center">32</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">11-deoxycortisol-d5</td>
<td valign="top" align="center">352.0</td>
<td valign="top" align="center">100.3</td>
<td valign="top" align="center">113.1</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">21-deoxycortisol</td>
<td valign="top" align="center">347.5</td>
<td valign="top" align="center">91.1</td>
<td valign="top" align="center">311.6</td>
<td valign="top" align="center">72</td>
<td valign="top" align="center">16</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">21-deoxycortisol-d8</td>
<td valign="top" align="center">355.3</td>
<td valign="top" align="center">319.3</td>
<td valign="top" align="center">125.2</td>
<td valign="top" align="center">16</td>
<td valign="top" align="center">24</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Aldosterone</td>
<td valign="top" align="center">361.2</td>
<td valign="top" align="center">343.3</td>
<td valign="top" align="center">91.1</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">80</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Aldosterone-d4</td>
<td valign="top" align="center">365.3</td>
<td valign="top" align="center">347.2</td>
<td valign="top" align="center">97.0</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">36</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Androstenedione</td>
<td valign="top" align="center">287.2</td>
<td valign="top" align="center">97.1</td>
<td valign="top" align="center">109.1</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">24</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Androstenedione&#x02212;13C3</td>
<td valign="top" align="center">290.0</td>
<td valign="top" align="center">100.0</td>
<td valign="top" align="center">112.0</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Testosterone</td>
<td valign="top" align="center">289.2</td>
<td valign="top" align="center">97.1</td>
<td valign="top" align="center">109.0</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Testosterone-13C3</td>
<td valign="top" align="center">292.0</td>
<td valign="top" align="center">100.1</td>
<td valign="top" align="center">111.9</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap position="float" id="T5">
<label>Table 3B</label>
<caption><p>Multiple reaction monitoring conditions of steroids and their internal standards in the keto-derivatization method.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th valign="top" align="left"><bold>Native STD/Stable isotope labeled IS</bold></th>
<th valign="top" align="center"><bold>Precursor ion (<italic>m/z</italic>)</bold></th>
<th valign="top" align="center"><bold>Quantifier</bold><break/> <bold>(<italic>m/z</italic>)</bold></th>
<th valign="top" align="center"><bold>Qualifier</bold><break/> <bold>(<italic>m/z</italic>)</bold></th>
<th valign="top" align="center"><bold>Collision energy (V) quantifier</bold></th>
<th valign="top" align="center"><bold>Collision energy (V) qualifier</bold></th>
<th valign="top" align="center"><bold>Polarity</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td/>
<td/>
<td/>
<td/>
<td/>
<td/>
<td/>
</tr>
<tr>
<td valign="top" align="left">Pregnenolone</td>
<td valign="top" align="center">431.4</td>
<td valign="top" align="center">372.2</td>
<td valign="top" align="center">126.1</td>
<td valign="top" align="center">24</td>
<td valign="top" align="center">44</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Pregnenolone-d4</td>
<td valign="top" align="center">435.5</td>
<td valign="top" align="center">376.3</td>
<td valign="top" align="center">130.1</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">48</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">17&#x003B1; -OH pregnenolone</td>
<td valign="top" align="center">447.3</td>
<td valign="top" align="center">370.2</td>
<td valign="top" align="center">388.2</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">20</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">17&#x003B1; -OH pregnenolone-13C3d2</td>
<td valign="top" align="center">451.4</td>
<td valign="top" align="center">374.2</td>
<td valign="top" align="center">392.4</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">24</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Etiocholanolone</td>
<td valign="top" align="center">405.5</td>
<td valign="top" align="center">346.2</td>
<td valign="top" align="center">91.1</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">72</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Etiocholanolone-d5</td>
<td valign="top" align="center">410.3</td>
<td valign="top" align="center">351.2</td>
<td valign="top" align="center">105.0</td>
<td valign="top" align="center">16</td>
<td valign="top" align="center">72</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Dehydroepiandrosterone</td>
<td valign="top" align="center">403.4</td>
<td valign="top" align="center">344.2</td>
<td valign="top" align="center">105.1</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">68</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Dehydroepiandrosterone-d5</td>
<td valign="top" align="center">408.0</td>
<td valign="top" align="center">349.2</td>
<td valign="top" align="center">162.2</td>
<td valign="top" align="center">28</td>
<td valign="top" align="center">48</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Dihydrotestosterone</td>
<td valign="top" align="center">405.4</td>
<td valign="top" align="center">346.2</td>
<td valign="top" align="center">91.1</td>
<td valign="top" align="center">32</td>
<td valign="top" align="center">80</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Dihydrotestosterone-d3</td>
<td valign="top" align="center">408.5</td>
<td valign="top" align="center">349.2</td>
<td valign="top" align="center">81.1</td>
<td valign="top" align="center">24</td>
<td valign="top" align="center">60</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Tetrahydrocortisol</td>
<td valign="top" align="center">481.0</td>
<td valign="top" align="center">118.1</td>
<td valign="top" align="center">116.0</td>
<td valign="top" align="center">56</td>
<td valign="top" align="center">44</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Tetrahydrocortisol-d5</td>
<td valign="top" align="center">486.0</td>
<td valign="top" align="center">118.2</td>
<td valign="top" align="center">60.1</td>
<td valign="top" align="center">48</td>
<td valign="top" align="center">68</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Tetrahydrocortisone</td>
<td valign="top" align="center">479.0</td>
<td valign="top" align="center">118.0</td>
<td valign="top" align="center">59.1</td>
<td valign="top" align="center">40</td>
<td valign="top" align="center">64</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Tetrahydrocortisone-d5</td>
<td valign="top" align="center">484.0</td>
<td valign="top" align="center">118.0</td>
<td valign="top" align="center">116.1</td>
<td valign="top" align="center">52</td>
<td valign="top" align="center">40</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Tetrahydro-11-deoxycortisol</td>
<td valign="top" align="center">465.0</td>
<td valign="top" align="center">118.1</td>
<td valign="top" align="center">59.1</td>
<td valign="top" align="center">48</td>
<td valign="top" align="center">80</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
<tr>
<td valign="top" align="left">Tetrahydro-11-deoxycortisol-d5</td>
<td valign="top" align="center">470.0</td>
<td valign="top" align="center">118.1</td>
<td valign="top" align="center">115.9</td>
<td valign="top" align="center">44</td>
<td valign="top" align="center">44</td>
<td valign="top" align="center">&#x0002B;</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap position="float" id="T6">
<label>Table 4</label>
<caption><p>Assay performance of the non-derivatization and keto-derivatization methods.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th valign="top" align="left"><bold>Detection method</bold></th>
<th valign="top" align="left"><bold>Steroid hormones</bold></th>
<th valign="top" align="center"><bold>IDL (pg)</bold></th>
<th valign="top" align="center"><bold>MDL (ng/ mL)</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">Non-derivatization method</td>
<td valign="top" align="left">Cortisol</td>
<td valign="top" align="center">0.02</td>
<td valign="top" align="center">0.13</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Cortisone</td>
<td valign="top" align="center">0.02</td>
<td valign="top" align="center">0.18</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">11-deoxycortisol</td>
<td valign="top" align="center">0.01</td>
<td valign="top" align="center">0.06</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">11-deoxycorticosterone</td>
<td valign="top" align="center">0.02</td>
<td valign="top" align="center">0.02</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Corticosterone</td>
<td valign="top" align="center">0.02</td>
<td valign="top" align="center">0.04</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Progesterone</td>
<td valign="top" align="center">0.01</td>
<td valign="top" align="center">0.01</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">17&#x003B1;-OH progesterone</td>
<td valign="top" align="center">0.01</td>
<td valign="top" align="center">0.09</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Androstenedione</td>
<td valign="top" align="center">0.03</td>
<td valign="top" align="center">0.09</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Testosterone</td>
<td valign="top" align="center">0.01</td>
<td valign="top" align="center">0.19</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">21-deoxycortisol</td>
<td valign="top" align="center">0.01</td>
<td valign="top" align="center">0.03</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Aldosterone</td>
<td valign="top" align="center">0.06</td>
<td valign="top" align="center">0.06</td>
</tr>
<tr>
<td valign="top" align="left">Keto-derivatization method</td>
<td valign="top" align="center">Pregnenolone</td>
<td valign="top" align="center">0.02</td>
<td valign="top" align="center">0.03</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">17&#x003B1;-OH pregnenolone</td>
<td valign="top" align="center">0.02</td>
<td valign="top" align="center">0.02</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Tetrahydrocortisol</td>
<td valign="top" align="center">0.02</td>
<td valign="top" align="center">0.03</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Tetrahydrocortisone</td>
<td valign="top" align="center">0.04</td>
<td valign="top" align="center">0.02</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Tetrahydro-11-deoxycortisol</td>
<td valign="top" align="center">0.01</td>
<td valign="top" align="center">0.02</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Dehydroepiandrosterone</td>
<td valign="top" align="center">0.03</td>
<td valign="top" align="center">0.04</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Dihydrotestosterone</td>
<td valign="top" align="center">0.04</td>
<td valign="top" align="center">0.01</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Etiocholanolone</td>
<td valign="top" align="center">0.02</td>
<td valign="top" align="center">0.06</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>The detection rate of steroid hormones in the non-derivatization and keto derivatization methods in dogs with HCC, HAC, and both diseases are described in <xref ref-type="table" rid="T7">Table 5</xref>. 21-Deoxycortisol and aldosterone were detected in one-fourth of the total samples. Androstenedione and dehydroepiandrosterone were hardly detected around 10%. Etiocholanolone was detected only in one HAC dog while testosterone and dihydrotestosterone were detected in each dog of the HCC with the HAC group. The detection rate of 19 steroids was not significantly different between groups (<italic>p</italic> &#x0003E; 0.05). Steroid hormones with a detection rate of higher than 50% were selected for further statistical analysis.</p>
<table-wrap position="float" id="T7">
<label>Table 5</label>
<caption><p>Detection rate (%) of steroid hormones in the non-derivatization and keto-derivatization methods.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th valign="top" align="left"><bold>Detection method</bold></th>
<th valign="top" align="left"><bold>Steroid hormones</bold></th>
<th valign="top" align="center"><bold>Overall dogs (<italic>N =</italic> 40)</bold></th>
<th valign="top" align="center"><bold>HCC</bold><break/> <bold>(<italic>N =</italic> 15)</bold></th>
<th valign="top" align="center"><bold>HAC</bold><break/> <bold>(<italic>N =</italic> 15)</bold></th>
<th valign="top" align="center"><bold>Both diseases (<italic>N =</italic> 10)</bold></th>
<th valign="top" align="center"><bold>Chi Square (<italic>p</italic> &#x0003C; 0.05)</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">Non-derivatization method</td>
<td valign="top" align="left">Progesterone (%)</td>
<td valign="top" align="center">95 (38/40)</td>
<td valign="top" align="center">93 (14/15)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">90 (9/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">17&#x003B1;-OH progesterone (%)</td>
<td valign="top" align="center">68 (27/40)</td>
<td valign="top" align="center">67 (10/15)</td>
<td valign="top" align="center">67 (10/15)</td>
<td valign="top" align="center">70 (7/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">11-deoxycorticosterone (%)</td>
<td valign="top" align="center">86 (34/40)</td>
<td valign="top" align="center">80 (12/15)</td>
<td valign="top" align="center">93 (14/15)</td>
<td valign="top" align="center">80 (8/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">11-deoxyortisol (%)</td>
<td valign="top" align="center">100 (40/40)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (10/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Corticosterone (%)</td>
<td valign="top" align="center">100 (40/40)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (10/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Cortisol (%)</td>
<td valign="top" align="center">100 (40/40)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (10/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Cortisone (%)</td>
<td valign="top" align="center">100 (40/40)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (10/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Aldosterone (%)</td>
<td valign="top" align="center">25 (10/40)</td>
<td valign="top" align="center">27 (4/15)</td>
<td valign="top" align="center">27 (4/15)</td>
<td valign="top" align="center">20 (2/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">21-deoxycortisol (%)</td>
<td valign="top" align="center">35 (14/40)</td>
<td valign="top" align="center">20 (3/15)</td>
<td valign="top" align="center">47 (7/15)</td>
<td valign="top" align="center">40 (4/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Androstenedione (%)</td>
<td valign="top" align="center">13 (5/40)</td>
<td valign="top" align="center">13 (2/15)</td>
<td valign="top" align="center">20 (3/15)</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x02013;</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Testosterone (%)</td>
<td valign="top" align="center">5 (2/40)</td>
<td valign="top" align="center">7 (1/15)</td>
<td valign="top" align="center">7 (1/15)</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x02013;</td>
</tr>
<tr>
<td valign="top" align="left">Keto-derivatization method</td>
<td valign="top" align="left">Pregnenolone (%)</td>
<td valign="top" align="center">100 (40/40)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (10/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">17&#x003B1;-OH pregnenolone (%)</td>
<td valign="top" align="center">78 (31/40)</td>
<td valign="top" align="center">80 (12/15)</td>
<td valign="top" align="center">73 (11/15)</td>
<td valign="top" align="center">80 (8/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Tetrahydrocortisol (%)</td>
<td valign="top" align="center">93 (37/40)</td>
<td valign="top" align="center">87 (13/15)</td>
<td valign="top" align="center">93 (14/15)</td>
<td valign="top" align="center">100 (10/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Tetrahydrocortisone (%)</td>
<td valign="top" align="center">100 (40/40)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (15/15)</td>
<td valign="top" align="center">100 (10/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Tetrahydro-11-deoxycortisol (%)</td>
<td valign="top" align="center">48 (19/40)</td>
<td valign="top" align="center">33 (5/15)</td>
<td valign="top" align="center">40 (6/15)</td>
<td valign="top" align="center">80 (8/10)</td>
<td valign="top" align="center">NS</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Dehydroepiandrosterone (%)</td>
<td valign="top" align="center">10 (4/40)</td>
<td valign="top" align="center">13 (2/15)</td>
<td valign="top" align="center">13 (2/15)</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x02013;</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Dihydrotestosterone (%)</td>
<td valign="top" align="center">5 (2/40)</td>
<td valign="top" align="center">7 (1/15)</td>
<td valign="top" align="center">7 (1/15)</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x02013;</td>
</tr>
<tr>
<td/>
<td valign="top" align="left">Etiocholanolone (%)</td>
<td valign="top" align="center">3 (1/40)</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">7 (1/15)</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x02013;</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>Principle component analysis explains no steroid pattern difference among the groups (<xref ref-type="fig" rid="F2">Figure 2</xref>). <xref ref-type="fig" rid="F3">Figures 3</xref>, <xref ref-type="fig" rid="F4">4</xref> compare the median concentrations of 9 steroids and 2 metabolites, respectively. None of the 11 steroid concentrations significantly differed between dogs with HCC, HAC, and both diseases (<italic>p</italic> &#x0003E; 0.05).</p>
<fig id="F2" position="float">
<label>Figure 2</label>
<caption><p>Principal component analysis (PCA) of the concentration of 11 steroids. Points labeled in white circles are dogs with HCC, in gray circles are dogs with HAC, and in asterisk are dogs with both diseases.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-1014792-g0002.tif"/>
</fig>
<fig id="F3" position="float">
<label>Figure 3</label>
<caption><p>Comparison of 9 steroids concentration <bold>(A&#x02013;I)</bold> in baseline serum of HCC (<italic>n</italic> = 15), HAC (<italic>n</italic> = 15), and HCC with HAC dogs (<italic>n</italic> = 10). Bars; the median concentration of steroids in each group.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-1014792-g0003.tif"/>
</fig>
<fig id="F4" position="float">
<label>Figure 4</label>
<caption><p>Comparison of 2 metabolites concentration <bold>(A,B)</bold> in baseline serum of HCC (<italic>n</italic> = 15), HAC (<italic>n</italic> = 15), and HCC with HAC dogs (<italic>n</italic> = 10). Bars; the median concentration of steroids in each group.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-1014792-g0004.tif"/>
</fig>
</sec>
<sec sec-type="discussion" id="s4">
<title>Discussion</title>
<p>In this study, steroid profiling of the baseline serum was performed to clarify the relationship between HCC and HAC. The co-occurrence of massive HCC and HAC was comparable, 32% (10/31) in this study and 23% (10/44) in a previous study (<xref ref-type="bibr" rid="B9">9</xref>). However, the baseline serum steroid profiling at the time of diagnosis was failed to find the association between HCC and HAC.</p>
<p>Liver cirrhosis and fibrosis caused by viral infections are the major causes of HCC development in humans but are rarely reported in dogs. Moreover, NAFLD occurs in 20% of people with Cushing&#x00027;s disease and precedes HCC development because steroid hormones cause multi-hit theories in Cushing patients with NAFLD (<xref ref-type="bibr" rid="B32">32</xref>). Excess glucocorticoids stimulate gluconeogenesis in the liver and consequently cause hepatic steatosis, steatohepatitis, and fibrosis in humans (<xref ref-type="bibr" rid="B33">33</xref>). Although hepatopathy with glycogen accumulation was found in dogs with HAC, steroid-induced hepatic inflammation, progressive findings of liver cirrhosis, or fibrosis, such as human HCC, seemed not to be preceded in dogs (<xref ref-type="bibr" rid="B34">34</xref>). Other confounding factors, such as changes in expression of steroidogenic factors, genetic mutation or epigenetic changes, and inflammatory cytokines, are thought to influence the occurrence of HAC in dogs with HCC. To our knowledge, there is only one online dissertation report on TP 53 gene mutation in canine HCC (<xref ref-type="bibr" rid="B35">35</xref>).</p>
<p>Chronic exposure to exogenous or endogenous glucocorticoid hormones is considered to cause vacuolar hepatopathy, which is a potential risk of developing HCC (<xref ref-type="bibr" rid="B33">33</xref>). Furthermore, adrenocortical hormone, concentrations are expected to be high in dogs with HAC because of hyperfunctions of the adrenal glands (<xref ref-type="bibr" rid="B31">31</xref>). However, a comparison of 11 steroid hormones (<xref ref-type="fig" rid="F3">Figures 3</xref>, <xref ref-type="fig" rid="F4">4</xref>) did not show a significant difference between dogs with HCC, HAC, and both diseases. Our findings indicate that steroid profile may not be involved in HCC development, although there was a noticeable concurrence of HAC and HCC. Sepesy et al. support our findings that 45% of vacuolar hepatopathy occurred without endogenous or exogenous glucocorticoid exposure in dogs (<xref ref-type="bibr" rid="B14">14</xref>). In Scottish terriers, vacuolar hepatopathy dogs show HAC suggestive signs with changes in non-cortisol hormones. However, some vacuolar hepatopathy dogs developed HCC with or without HAC suggestive signs and non-cortisol changes (<xref ref-type="bibr" rid="B8">8</xref>). Even in Scottish terriers, the exact mechanism of steroidogenesis disorders in the progression of vacuolar hepatopathy to HCC has been unclear.</p>
<p>Aldosterone and 21-deoxycortisol were detected in one-third of the samples with low concentration. In the canine mineralocorticoid pathway, zonal expression of aldosterone synthase is limited to a single gene (CYP11B1), with no expression of a zone-specific enzyme (CYP11B2) when compared with humans (<xref ref-type="bibr" rid="B16">16</xref>). Androgen concentration in many dogs was measured under the MDL value. The current study included only 7 intact dogs; the remaining 33 were neutered or spayed. Even though androgens can activate the oncoprotein transcription in human HCC and neuter status might be a risk factor for some malignancies in dogs, current findings suggest that androgens may not be involved in the development of dog HCC (<xref ref-type="bibr" rid="B36">36</xref>&#x02013;<xref ref-type="bibr" rid="B38">38</xref>).</p>
<p>Additionally, even though our results do not support the idea that steroidogenesis changes may impact on the development of hepatocarcinogenesis, the coincidence of HAC in HCC is a concern. HCC may also play a role in the steroid imbalance as an alternative to our hypothesis. Hepatocarcinogenesis may disturb steroidogenesis or steroid metabolism in the liver. Steroids and metabolites in liver tissues and 24-h urine samples may be useful to confirm metabolic changes inside the liver in future analysis. This study only evaluated serum steroids at a single time point and was unable to determine when HCC or HAC first started in dogs. Therefore, follow-up case studies were warranted to prove the alternative theory.</p>
<p>The reliable measurement of steroid hormones is a powerful technique to investigate their hormonal status in endocrine-related diseases. By combining the elution effect and a biphenyl column, the technique provided sufficient column separation and symmetric peaks for isobaric steroids, such as 21-deoxycortisol, 11-deoxycortisol, and corticosterone (<xref ref-type="bibr" rid="B39">39</xref>). Following the non-derivatization preparation steps, derivatization with the keto-reagent is simple, fast, and sensitive when compared with other different derivatization strategies (<xref ref-type="bibr" rid="B40">40</xref>&#x02013;<xref ref-type="bibr" rid="B42">42</xref>). The MDL values were acceptable, and the present LC/MS/MS method could be used to measure multi-steroid hormones (<xref ref-type="table" rid="T6">Table 4</xref>).</p>
<p>To conclude our study, we developed the LC/MS/MS method for measuring multiple steroid hormones in a small amount of serum. The concentration of 19 serum steroids was not different in dogs with HCC, HAC, and both diseases. We concluded that HAC is not associated with the development of HCC, and steroidogenesis may not contribute to hepatocarcinogenesis. Additional measurement of steroids inside the liver and metabolites from the liver should be used to conduct more in-depth research on steroid imbalance in hepatocarcinogenesis.</p>
</sec>
<sec sec-type="data-availability" id="s5">
<title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="supplementary-material" rid="SM1">Supplementary material</xref>, further inquiries can be directed to the corresponding author.</p>
</sec>
<sec id="s6">
<title>Ethics statement</title>
<p>The animal study was reviewed and approved by Laboratory Animal Experimentation Committee of the Graduate School of Veterinary Medicine, Hokkaido University (Approval No. 18-0142). Written informed consent was obtained from the owners for the participation of their animals in this study.</p>
</sec>
<sec id="s7">
<title>Author contributions</title>
<p>TO: conceptualization, formal analysis, investigation, data curation, visualization, writing-original draft, and project administration. NS: conceptualization, writing-original draft, review, and editing. YI: methodology, validation, and funding acquisition. TI: methodology and validation. NN: investigation. NY, KS, and KM: resources. KN: supervision. MT: conceptualization, writing-review and editing, supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.</p>
</sec>
<sec sec-type="funding-information" id="s8">
<title>Funding</title>
<p>This study was supported by the Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (YI; JP18H04132, JP22K18425, JP22K05980 and MT; JP20H03139) and the MEXT project for promoting public utilization of advanced research infrastructure (program for supporting the introduction of the new sharing system, JPMXS0420100619). TO was supported by the World Leading Innovative and Smart Education (WISE) program (1801) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.</p>
</sec>
<sec sec-type="COI-statement" id="conf1">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec sec-type="disclaimer" id="s9">
<title>Publisher&#x00027;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
</body>
<back>
<ack><p>The authors are grateful for the funding provided by the Japanese Society for the Promotion of Science, the MEXT project, and WISE program. The authors would like to thank all staff at HUVTH for their kind support in the sample collection of this research work.</p>
</ack>
<sec sec-type="supplementary-material" id="s10">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fvets.2022.1014792/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fvets.2022.1014792/full#supplementary-material</ext-link></p>
<supplementary-material xlink:href="Data_Sheet_1.xlsx" id="SM1" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet" xmlns:xlink="http://www.w3.org/1999/xlink"/>
</sec>
<ref-list>
<title>References</title>
<ref id="B1">
<label>1.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Patnaik</surname> <given-names>AK</given-names></name> <name><surname>Hurvitz</surname> <given-names>AI</given-names></name> <name><surname>Lieberman</surname> <given-names>PH</given-names></name></person-group>. <article-title>Canine hepatic neoplasms: a clinicopathologic study</article-title>. <source>Vet Pathol.</source> (<year>1980</year>) <volume>17</volume>:<fpage>553</fpage>&#x02013;<lpage>64</lpage>. <pub-id pub-id-type="doi">10.1177/030098588001700504</pub-id><pub-id pub-id-type="pmid">7404966</pub-id></citation></ref>
<ref id="B2">
<label>2.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Patnaik</surname> <given-names>AK</given-names></name> <name><surname>Hurvitz</surname> <given-names>AI</given-names></name> <name><surname>Lieberman</surname> <given-names>PH</given-names></name> <name><surname>Johnson</surname> <given-names>GF</given-names></name></person-group>. <article-title>Canine hepatocellular carcinoma</article-title>. <source>Vet Pathol.</source> (<year>1981</year>) <volume>18</volume>:<fpage>427</fpage>&#x02013;<lpage>38</lpage>. <pub-id pub-id-type="doi">10.1177/030098588101800402</pub-id><pub-id pub-id-type="pmid">6266116</pub-id></citation></ref>
<ref id="B3">
<label>3.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>van Sprundel</surname> <given-names>RGHM</given-names></name> <name><surname>van den Ingh</surname> <given-names>TSGAM</given-names></name> <name><surname>Guscetti</surname> <given-names>F</given-names></name> <name><surname>Kershaw</surname> <given-names>O</given-names></name> <name><surname>Kanemoto</surname> <given-names>H</given-names></name> <name><surname>van Gils</surname> <given-names>HM</given-names></name> <etal/></person-group>. <article-title>Classification of primary hepatic tumours in the dog</article-title>. <source>Vet J.</source> (<year>2013</year>) <volume>197</volume>:<fpage>596</fpage>&#x02013;<lpage>606</lpage>. <pub-id pub-id-type="doi">10.1016/j.tvjl.2013.05.027</pub-id><pub-id pub-id-type="pmid">24011584</pub-id></citation></ref>
<ref id="B4">
<label>4.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Llovet</surname> <given-names>JM</given-names></name> <name><surname>Kelley</surname> <given-names>RK</given-names></name> <name><surname>Villanueva</surname> <given-names>A</given-names></name> <name><surname>Singal</surname> <given-names>AG</given-names></name> <name><surname>Pikarsky</surname> <given-names>E</given-names></name> <name><surname>Roayaie</surname> <given-names>S</given-names></name> <etal/></person-group>. <article-title>Hepatocellular carcinoma</article-title>. <source>Nat Rev Dis Primers.</source> (<year>2021</year>) <volume>7</volume>:<fpage>6</fpage>. <pub-id pub-id-type="doi">10.1038/s41572-020-00240-3</pub-id><pub-id pub-id-type="pmid">33479224</pub-id></citation></ref>
<ref id="B5">
<label>5.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Samant</surname> <given-names>H</given-names></name> <name><surname>Amiri</surname> <given-names>HS</given-names></name> <name><surname>Zibari</surname> <given-names>GB</given-names></name></person-group>. <article-title>Addressing the worldwide hepatocellular carcinoma: epidemiology, prevention and management</article-title>. <source>J Gastrointest Oncol.</source> (<year>2021</year>) <volume>12</volume>:<fpage>361</fpage>&#x02013;<lpage>73</lpage>. <pub-id pub-id-type="doi">10.21037/jgo.2020.02.08</pub-id><pub-id pub-id-type="pmid">34422400</pub-id></citation></ref>
<ref id="B6">
<label>6.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Selmic</surname> <given-names>LE</given-names></name></person-group>. <article-title>Hepatobiliary neoplasia</article-title>. <source>Vet Clin N Am Small Anim Pract.</source> (<year>2017</year>) <volume>47</volume>:<fpage>725</fpage>&#x02013;<lpage>35</lpage>. <pub-id pub-id-type="doi">10.1016/j.cvsm.2016.11.016</pub-id><pub-id pub-id-type="pmid">28063743</pub-id></citation></ref>
<ref id="B7">
<label>7.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Lapsley</surname> <given-names>JM</given-names></name> <name><surname>Wavreille</surname> <given-names>V</given-names></name> <name><surname>Barry</surname> <given-names>S</given-names></name> <name><surname>Dornbusch</surname> <given-names>JA</given-names></name> <name><surname>Chen</surname> <given-names>C</given-names></name> <name><surname>Leeper</surname> <given-names>H</given-names></name> <etal/></person-group>. <article-title>Risk factors and outcome in dogs with recurrent massive hepatocellular carcinoma: a Veterinary Society of Surgical Oncology case-control study</article-title>. <source>Vet Comp Oncol</source>. (<year>2022</year>) <volume>20</volume>:<fpage>697</fpage>&#x02013;<lpage>709</lpage>. <pub-id pub-id-type="doi">10.1111/vco.12824</pub-id><pub-id pub-id-type="pmid">35488436</pub-id></citation></ref>
<ref id="B8">
<label>8.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Cortright</surname> <given-names>CC</given-names></name> <name><surname>Center</surname> <given-names>SA</given-names></name> <name><surname>Randolph</surname> <given-names>JF</given-names></name> <name><surname>McDonough</surname> <given-names>SP</given-names></name> <name><surname>Fecteau</surname> <given-names>KA</given-names></name> <name><surname>Warner</surname> <given-names>KL</given-names></name> <etal/></person-group>. <article-title>Clinical features of progressive vacuolar hepatopathy in Scottish terriers with and without hepatocellular carcinoma: 114 cases (1980-2013)</article-title>. <source>J Am Vet Med Assoc.</source> (<year>2014</year>) <volume>245</volume>:<fpage>797</fpage>&#x02013;<lpage>808</lpage>. <pub-id pub-id-type="doi">10.2460/javma.245.7.797</pub-id><pub-id pub-id-type="pmid">25229531</pub-id></citation></ref>
<ref id="B9">
<label>9.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Leela-Arporn</surname> <given-names>R</given-names></name> <name><surname>Ohta</surname> <given-names>H</given-names></name> <name><surname>Nagata</surname> <given-names>N</given-names></name> <name><surname>Sasaoka</surname> <given-names>K</given-names></name> <name><surname>Tamura</surname> <given-names>M</given-names></name> <name><surname>Dermlim</surname> <given-names>A</given-names></name> <etal/></person-group>. <article-title>Epidemiology of massive hepatocellular carcinoma in dogs: a 4-year retrospective study</article-title>. <source>Vet J.</source> (<year>2019</year>) <volume>248</volume>:<fpage>74</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1016/j.tvjl.2019.04.011</pub-id><pub-id pub-id-type="pmid">31113567</pub-id></citation></ref>
<ref id="B10">
<label>10.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hirose</surname> <given-names>N</given-names></name> <name><surname>Uchida</surname> <given-names>K</given-names></name> <name><surname>Kanemoto</surname> <given-names>H</given-names></name> <name><surname>Ohno</surname> <given-names>K</given-names></name> <name><surname>Chambers</surname> <given-names>JK</given-names></name> <name><surname>Nakayama</surname> <given-names>H</given-names></name></person-group>. <article-title>A retrospective histopathological survey on canine and feline liver diseases at the University of Tokyo between 2006 and 2012</article-title>. <source>J Vet Med Sci.</source> (<year>2014</year>) <volume>76</volume>:<fpage>1015</fpage>&#x02013;<lpage>20</lpage>. <pub-id pub-id-type="doi">10.1292/jvms.14-0083</pub-id><pub-id pub-id-type="pmid">24717415</pub-id></citation></ref>
<ref id="B11">
<label>11.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Liptak</surname> <given-names>JM</given-names></name> <name><surname>Dernell</surname> <given-names>WS</given-names></name> <name><surname>Monnet</surname> <given-names>E</given-names></name> <name><surname>Powers</surname> <given-names>BE</given-names></name> <name><surname>Bachand</surname> <given-names>AM</given-names></name> <name><surname>Kenney</surname> <given-names>JG</given-names></name> <etal/></person-group>. <article-title>Massive hepatocellular carcinoma in dogs: 48 cases (1992&#x02013;2002)</article-title>. <source>J Am Vet Med Assoc.</source> (<year>2004</year>) <volume>225</volume>:<fpage>1225</fpage>&#x02013;<lpage>230</lpage>. <pub-id pub-id-type="doi">10.2460/javma.2004.225.1225</pub-id><pub-id pub-id-type="pmid">15521445</pub-id></citation></ref>
<ref id="B12">
<label>12.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Zimmerman</surname> <given-names>KL</given-names></name> <name><surname>Pandera</surname> <given-names>DL</given-names></name> <name><surname>Panciera</surname> <given-names>RJ</given-names></name> <name><surname>Oliver</surname> <given-names>JW</given-names></name> <name><surname>Hoffmann</surname> <given-names>WE</given-names></name> <name><surname>Binder</surname> <given-names>EM</given-names></name> <etal/></person-group>. <article-title>Hyperphosphatasemia and concurrent adrenal gland dysfunction in apparently healthy Scottish Terriers</article-title>. <source>J Am Vet Med Assoc.</source> (<year>2010</year>) <volume>237</volume>:<fpage>178</fpage>&#x02013;<lpage>86</lpage>. <pub-id pub-id-type="doi">10.2460/javma.237.2.178</pub-id><pub-id pub-id-type="pmid">20632791</pub-id></citation></ref>
<ref id="B13">
<label>13.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Nestor</surname> <given-names>DD</given-names></name> <name><surname>Holan</surname> <given-names>KM</given-names></name> <name><surname>Johnson</surname> <given-names>CA</given-names></name> <name><surname>Schall</surname> <given-names>W</given-names></name> <name><surname>Kaneene</surname> <given-names>JB</given-names></name></person-group>. <article-title>Serum alkaline phosphatase activity in Scottish Terriers versus dogs of other breeds</article-title>. <source>J Am Vet Med Assoc.</source> (<year>2006</year>) <volume>228</volume>:<fpage>222</fpage>&#x02013;<lpage>4</lpage>. <pub-id pub-id-type="doi">10.2460/javma.228.2.222</pub-id><pub-id pub-id-type="pmid">16426191</pub-id></citation></ref>
<ref id="B14">
<label>14.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Sepesy</surname> <given-names>LM</given-names></name> <name><surname>Center</surname> <given-names>SA</given-names></name> <name><surname>Randolph</surname> <given-names>JF</given-names></name> <name><surname>Warner</surname> <given-names>KL</given-names></name> <name><surname>Erb</surname> <given-names>HN</given-names></name></person-group>. <article-title>Vacuolar hepatopathy in dogs: 336 cases (1993-2005)</article-title>. <source>J Am Vet Med Assoc</source>. (<year>2006</year>) <volume>229</volume>:<fpage>246</fpage>&#x02013;<lpage>52</lpage>. <pub-id pub-id-type="doi">10.2460/javma.229.2.246</pub-id><pub-id pub-id-type="pmid">16842046</pub-id></citation></ref>
<ref id="B15">
<label>15.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Zimmerman</surname> <given-names>KL</given-names></name> <name><surname>Panciera</surname> <given-names>DL</given-names></name> <name><surname>Hoeschele</surname> <given-names>I</given-names></name> <name><surname>Monroe</surname> <given-names>WE</given-names></name> <name><surname>Todd</surname> <given-names>SM</given-names></name> <name><surname>Werre</surname> <given-names>SR</given-names></name> <etal/></person-group>. <article-title>Adrenocortical challenge response and genomic analyses in Scottish Terriers with increased alkaline phosphate activity</article-title>. <source>Front Vet Sci.</source> (<year>2018</year>) <volume>5</volume>:<fpage>231</fpage>. <pub-id pub-id-type="doi">10.3389/fvets.2018.00231</pub-id><pub-id pub-id-type="pmid">30356827</pub-id></citation></ref>
<ref id="B16">
<label>16.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Sanders</surname> <given-names>K</given-names></name> <name><surname>Mol</surname> <given-names>JA</given-names></name> <name><surname>Kooistra</surname> <given-names>HS</given-names></name> <name><surname>Slob</surname> <given-names>A</given-names></name> <name><surname>Galac</surname> <given-names>S</given-names></name></person-group>. <article-title>New insights in the functional zonation of the canine adrenal cortex</article-title>. <source>J Vet Intern Med.</source> (<year>2016</year>) <volume>30</volume>:<fpage>741</fpage>&#x02013;<lpage>50</lpage>. <pub-id pub-id-type="doi">10.1111/jvim.13946</pub-id><pub-id pub-id-type="pmid">27108660</pub-id></citation></ref>
<ref id="B17">
<label>17.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bennaim</surname> <given-names>M</given-names></name> <name><surname>Shiel</surname> <given-names>RE</given-names></name> <name><surname>Mooney</surname> <given-names>CT</given-names></name></person-group>. <article-title>Diagnosis of spontaneous hyperadrenocorticism in dogs. Part 1: pathophysiology, aetiology, clinical and clinicopathological features</article-title>. <source>Vet J.</source> (<year>2019</year>) <volume>252</volume>:<fpage>105343</fpage>. <pub-id pub-id-type="doi">10.1016/j.tvjl.2019.105342</pub-id><pub-id pub-id-type="pmid">31554593</pub-id></citation></ref>
<ref id="B18">
<label>18.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Michelotti</surname> <given-names>A</given-names></name> <name><surname>de Scordilli</surname> <given-names>M</given-names></name> <name><surname>Palmero</surname> <given-names>L</given-names></name> <name><surname>Guardascione</surname> <given-names>M</given-names></name> <name><surname>Masala</surname> <given-names>M</given-names></name> <name><surname>Roncato</surname> <given-names>R</given-names></name> <etal/></person-group>. <article-title>NAFLD-related hepatocarcinoma: the malignant side of metabolic syndrome</article-title>. <source>Cells</source>. (<year>2021</year>) <volume>10</volume>:<fpage>2034</fpage>. <pub-id pub-id-type="doi">10.3390/cells10082034</pub-id><pub-id pub-id-type="pmid">34440803</pub-id></citation></ref>
<ref id="B19">
<label>19.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Sasaki</surname> <given-names>N</given-names></name> <name><surname>Oo</surname> <given-names>T</given-names></name> <name><surname>Yasuda</surname> <given-names>Y</given-names></name> <name><surname>Ichise</surname> <given-names>T</given-names></name> <name><surname>Nagata</surname> <given-names>N</given-names></name> <name><surname>Yokoyama</surname> <given-names>N</given-names></name> <etal/></person-group>. <article-title>Simultaneous steroids measurement in dogs with hyperadrenocorticism using a column-switching liquid chromatography-tandem mass spectrometry method</article-title>. <source>J Vet Med Sci.</source> (<year>2021</year>) <volume>83</volume>:<fpage>1634</fpage>&#x02013;<lpage>42</lpage>. <pub-id pub-id-type="doi">10.1292/jvms.21-0406</pub-id><pub-id pub-id-type="pmid">34544959</pub-id></citation></ref>
<ref id="B20">
<label>20.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Behrend</surname> <given-names>EN</given-names></name> <name><surname>Kemppainen</surname> <given-names>RJ</given-names></name> <name><surname>Boozer</surname> <given-names>AL</given-names></name> <name><surname>Whitley</surname> <given-names>EM</given-names></name> <name><surname>Smith</surname> <given-names>AN</given-names></name> <name><surname>Busch</surname> <given-names>KA</given-names></name></person-group>. <article-title>Serum 17-&#x003B1;-hydroxyprogesterone and corticosterone concentrations in dogs with nonadrenal neoplasia and dogs with suspected hyperadrenocorticism</article-title>. <source>J Am Vet Med Assoc.</source> (<year>2005</year>) <volume>227</volume>:<fpage>1762</fpage>&#x02013;<lpage>7</lpage>. <pub-id pub-id-type="doi">10.2460/javma.2005.227.1762</pub-id><pub-id pub-id-type="pmid">16342524</pub-id></citation></ref>
<ref id="B21">
<label>21.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Chapman</surname> <given-names>PS</given-names></name> <name><surname>Mooney</surname> <given-names>CT</given-names></name> <name><surname>Ede</surname> <given-names>J</given-names></name> <name><surname>Evans</surname> <given-names>H</given-names></name> <name><surname>O&#x00027;Connor</surname> <given-names>J</given-names></name> <name><surname>Pfeiffer</surname> <given-names>DU</given-names></name> <etal/></person-group>. <article-title>Evaluation of the basal and post-adrenocorticotrophic hormone serum concentrations of 17-hydroxyprogesterone for the diagnosis of hyperadrenocorticism in dogs</article-title>. <source>Vet Rec.</source> (<year>2003</year>) <volume>153</volume>:<fpage>771</fpage>&#x02013;<lpage>5</lpage>. <pub-id pub-id-type="doi">10.2460/javma.2005.227.1095</pub-id><pub-id pub-id-type="pmid">14735992</pub-id></citation></ref>
<ref id="B22">
<label>22.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Stanczyk</surname> <given-names>FZ</given-names></name> <name><surname>Clarke</surname> <given-names>NJ</given-names></name></person-group>. <article-title>Advantages and challenges of mass spectrometry assays for steroid hormones</article-title>. <source>J Steroid Biochem Mol Biol.</source> (<year>2010</year>) <volume>121</volume>:<fpage>491</fpage>&#x02013;<lpage>5</lpage>. <pub-id pub-id-type="doi">10.1016/j.jsbmb.2010.05.001</pub-id><pub-id pub-id-type="pmid">20470886</pub-id></citation></ref>
<ref id="B23">
<label>23.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Lane</surname> <given-names>MB</given-names></name> <name><surname>Flatland</surname> <given-names>B</given-names></name> <name><surname>Olin</surname> <given-names>SJ</given-names></name> <name><surname>Fecteau</surname> <given-names>KA</given-names></name> <name><surname>Rick</surname> <given-names>M</given-names></name> <name><surname>Giori</surname> <given-names>L</given-names></name></person-group>. <article-title>Analytic performance evaluation of a veterinary-specific ELISA for measurement of serum cortisol concentrations of dogs</article-title>. <source>J Am Vet Med Assoc.</source> (<year>2018</year>) <volume>253</volume>:<fpage>1580</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.2460/javma.253.12.1580</pub-id><pub-id pub-id-type="pmid">30668258</pub-id></citation></ref>
<ref id="B24">
<label>24.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Lurye</surname> <given-names>JC</given-names></name> <name><surname>Behrend</surname> <given-names>EN</given-names></name> <name><surname>Kemppainen</surname> <given-names>RJ</given-names></name></person-group>. <article-title>Evaluation of an in-house enzyme-linked immunosorbent assay for quantitative measurement of serum total thyroxine concentration in dogs and cats</article-title>. <source>J Am Vet Med Assoc.</source> (<year>2002</year>) <volume>221</volume>:<fpage>243</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.2460/javma.2002.221.243</pub-id><pub-id pub-id-type="pmid">12118587</pub-id></citation></ref>
<ref id="B25">
<label>25.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Stanczyk</surname> <given-names>FZ</given-names></name> <name><surname>Clarke</surname> <given-names>NJ</given-names></name></person-group>. <article-title>Advantages and challenges of mass spectrometry assays for steroid hormones</article-title>. <source>J Steroid Biochem Mol Biol.</source> (<year>2010</year>) <volume>121</volume>:<fpage>491</fpage>&#x02013;<lpage>5</lpage>.<pub-id pub-id-type="pmid">20470886</pub-id></citation></ref>
<ref id="B26">
<label>26.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kushnir</surname> <given-names>MM</given-names></name> <name><surname>Rockwood</surname> <given-names>AL</given-names></name> <name><surname>Roberts</surname> <given-names>WL</given-names></name> <name><surname>Yue</surname> <given-names>B</given-names></name> <name><surname>Bergquist</surname> <given-names>J</given-names></name> <name><surname>Meikle</surname> <given-names>AW</given-names></name></person-group>. <article-title>Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories</article-title>. <source>Clin Biochem.</source> (<year>2011</year>) <volume>44</volume>:<fpage>77</fpage>&#x02013;<lpage>88</lpage>. <pub-id pub-id-type="doi">10.1016/j.clinbiochem.2010.07.008</pub-id><pub-id pub-id-type="pmid">20627096</pub-id></citation></ref>
<ref id="B27">
<label>27.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Pitt</surname> <given-names>JJ</given-names></name></person-group>. <article-title>Principles and applications of liquid chromatography-mass spectrometry in clinical biochemistry</article-title>. <source>Clin Biochem Rev.</source> (<year>2009</year>) <volume>30</volume>:<fpage>19</fpage>&#x02013;<lpage>34</lpage>.<pub-id pub-id-type="pmid">19224008</pub-id></citation></ref>
<ref id="B28">
<label>28.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Santa</surname> <given-names>T</given-names></name></person-group>. <article-title>Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry</article-title>. <source>Biomed Chromatogr.</source> (<year>2011</year>) <volume>25</volume>:<fpage>1</fpage>&#x02013;<lpage>10</lpage>. <pub-id pub-id-type="doi">10.1002/bmc.1548</pub-id><pub-id pub-id-type="pmid">21058414</pub-id></citation></ref>
<ref id="B29">
<label>29.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Santa</surname> <given-names>T</given-names></name></person-group>. <article-title>Derivatization in liquid chromatography for mass spectrometric detection</article-title>. <source>Drug Discov Ther.</source> (<year>2013</year>) <volume>7</volume>:<fpage>9</fpage>&#x02013;<lpage>17</lpage>. <pub-id pub-id-type="doi">10.5582/ddt.2013.v7.1.9</pub-id><pub-id pub-id-type="pmid">23524938</pub-id></citation></ref>
<ref id="B30">
<label>30.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Cullen</surname> <given-names>JM</given-names></name></person-group>. <article-title>Summary of the World Small Animal Veterinary Association Standardization Committee guide to classification of liver disease in dogs and cats</article-title>. <source>Vet Clin N Am Small Anim Pract</source>. (<year>2009</year>) <volume>39</volume>:<fpage>395</fpage>&#x02013;<lpage>418</lpage>. <pub-id pub-id-type="doi">10.1016/j.cvsm.2009.02.003</pub-id><pub-id pub-id-type="pmid">19524786</pub-id></citation></ref>
<ref id="B31">
<label>31.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Behrend</surname> <given-names>EN</given-names></name> <name><surname>Kooistra</surname> <given-names>HS</given-names></name> <name><surname>Nelson</surname> <given-names>R</given-names></name> <name><surname>Reusch</surname> <given-names>CE</given-names></name> <name><surname>Scott-Moncrieff</surname> <given-names>JC</given-names></name></person-group>. <article-title>Diagnosis of spontaneous canine hyperadrenocorticism: 2012 acvim consensus statement (small animal)</article-title>. <source>J Vet Intern Medicine.</source> (<year>2013</year>) <volume>27</volume>:<fpage>1292</fpage>&#x02013;<lpage>304</lpage>. <pub-id pub-id-type="doi">10.1111/jvim.12192</pub-id><pub-id pub-id-type="pmid">24112317</pub-id></citation></ref>
<ref id="B32">
<label>32.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname> <given-names>M</given-names></name> <name><surname>Ma</surname> <given-names>F</given-names></name> <name><surname>Guan</surname> <given-names>M</given-names></name></person-group>. <article-title>Role of steroid hormones in the pathogenesis of nonalcoholic fatty liver disease</article-title>. <source>Metabolites.</source> (<year>2021</year>) <volume>11</volume>:<fpage>320</fpage>. <pub-id pub-id-type="doi">10.3390/metabo11050320</pub-id><pub-id pub-id-type="pmid">34067649</pub-id></citation></ref>
<ref id="B33">
<label>33.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tarantino</surname> <given-names>G</given-names></name> <name><surname>Finelli</surname> <given-names>C</given-names></name></person-group>. <article-title>Pathogenesis of hepatic steatosis: The link between hypercortisolism and nonalcoholic fatty liver disease</article-title>. <source>World J Gastroenterol.</source> (<year>2013</year>) <volume>19</volume>:<fpage>6735</fpage>&#x02013;<lpage>43</lpage>. <pub-id pub-id-type="doi">10.3748/wjg.v19.i40.6735</pub-id><pub-id pub-id-type="pmid">24187449</pub-id></citation></ref>
<ref id="B34">
<label>34.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Rogers</surname> <given-names>WA Ruebner BH</given-names></name></person-group>. <article-title>A retrospective study of probable glucocorticoid-induced hepatopathy in dogs</article-title>. <source>J Am Vet Med Assoc.</source> (<year>1977</year>) <volume>170</volume>:<fpage>603</fpage>&#x02013;<lpage>6</lpage>.<pub-id pub-id-type="pmid">849910</pub-id></citation></ref>
<ref id="B35">
<label>35.</label>
<citation citation-type="book"><person-group person-group-type="author"><name><surname>Gomez</surname> <given-names>DS</given-names></name></person-group>. <source>Mutational evaluation of TP53 gene in the canine hepatocellular carcinoma: a comparative approach</source> [Dissertation/master&#x00027;s thesis]. <publisher-name>University of S&#x00027;ao Paulo, Brazil</publisher-name> (<year>2019</year>).</citation>
</ref>
<ref id="B36">
<label>36.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Aicher</surname> <given-names>KM</given-names></name> <name><surname>Cullen</surname> <given-names>JM</given-names></name> <name><surname>Seiler</surname> <given-names>GS</given-names></name> <name><surname>Lunn</surname> <given-names>KF</given-names></name> <name><surname>Mathews</surname> <given-names>KG</given-names></name> <name><surname>Gookin</surname> <given-names>JL</given-names></name></person-group>. <article-title>Investigation of adrenal and thyroid gland dysfunction in dogs with ultrasonographic diagnosis of gallbladder mucocele formation</article-title>. <source>PLoS ONE.</source> (<year>2019</year>) <volume>14</volume>:<fpage>e0212638</fpage>. <pub-id pub-id-type="doi">10.1371/journal.pone.0212638</pub-id><pub-id pub-id-type="pmid">30811473</pub-id></citation></ref>
<ref id="B37">
<label>37.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Giannitrapani</surname> <given-names>L</given-names></name> <name><surname>Soresi</surname> <given-names>M</given-names></name> <name><surname>la Spada</surname> <given-names>E</given-names></name> <name><surname>Cervello</surname> <given-names>M</given-names></name> <name><surname>D&#x00027;Alessandro</surname> <given-names>N</given-names></name> <name><surname>Montalto</surname> <given-names>G</given-names></name></person-group>. <article-title>Sex hormones and risk of liver tumor</article-title>. <source>Ann N Y Acad Sci.</source> (<year>2006</year>) <volume>1089</volume>:<fpage>228</fpage>&#x02013;<lpage>36</lpage>. <pub-id pub-id-type="doi">10.1196/annals.1386.044</pub-id><pub-id pub-id-type="pmid">17261770</pub-id></citation></ref>
<ref id="B38">
<label>38.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Smith</surname> <given-names>AN</given-names></name></person-group>. <article-title>The role of neutering in cancer development</article-title>. <source>Vet Clin N Am Small Anim Pract.</source> (<year>2014</year>) <volume>44</volume>:<fpage>965</fpage>&#x02013;<lpage>75</lpage>. <pub-id pub-id-type="doi">10.1016/j.cvsm.2014.06.003</pub-id><pub-id pub-id-type="pmid">25174910</pub-id></citation></ref>
<ref id="B39">
<label>39.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Keevil</surname> <given-names>BG</given-names></name></person-group>. <article-title>Novel liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for measuring steroids</article-title>. <source>Best Pract Res Clin Endocrinol Metab.</source> (<year>2013</year>) <volume>27</volume>:<fpage>663</fpage>&#x02013;<lpage>74</lpage>. <pub-id pub-id-type="doi">10.1016/j.beem.2013.05.015</pub-id><pub-id pub-id-type="pmid">24094637</pub-id></citation></ref>
<ref id="B40">
<label>40.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Shiraishi</surname> <given-names>S</given-names></name> <name><surname>Lee</surname> <given-names>PWN</given-names></name> <name><surname>Leung</surname> <given-names>A</given-names></name> <name><surname>Goh</surname> <given-names>VHH</given-names></name> <name><surname>Swerdloff</surname> <given-names>RS</given-names></name> <name><surname>Wang</surname> <given-names>C</given-names></name></person-group>. <article-title>Simultaneous measurement of serum testosterone and dihydrotestosterone by liquid chromatography-tandem mass spectrometry</article-title>. <source>Clin Chem.</source> (<year>2008</year>) <volume>54</volume>:<fpage>1855</fpage>&#x02013;<lpage>63</lpage>. <pub-id pub-id-type="doi">10.1373/clinchem.2008.103846</pub-id><pub-id pub-id-type="pmid">18801940</pub-id></citation></ref>
<ref id="B41">
<label>41.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Qin</surname> <given-names>Q</given-names></name> <name><surname>Feng</surname> <given-names>D</given-names></name> <name><surname>Hu</surname> <given-names>C</given-names></name> <name><surname>Wang</surname> <given-names>B</given-names></name> <name><surname>Chang</surname> <given-names>M</given-names></name> <name><surname>Liu</surname> <given-names>X</given-names></name> <etal/></person-group>. <article-title>Parallel derivatization strategy coupled with liquid chromatography-mass spectrometry for broad coverage of steroid hormones</article-title>. <source>J Chromatogr.</source> (<year>2020</year>) <volume>1614</volume>:<fpage>460709</fpage>. <pub-id pub-id-type="doi">10.1016/j.chroma.2019.460709</pub-id><pub-id pub-id-type="pmid">31784081</pub-id></citation></ref>
<ref id="B42">
<label>42.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Keski-Rahkonen</surname> <given-names>P</given-names></name> <name><surname>Huhtinen</surname> <given-names>K</given-names></name> <name><surname>Poutanen</surname> <given-names>M</given-names></name> <name><surname>Auriola</surname> <given-names>S</given-names></name></person-group>. <article-title>Fast and sensitive liquid chromatography-mass spectrometry assay for seven androgenic and progestagenic steroids in human serum</article-title>. <source>J Steroid Biochem Mol Biol.</source> (<year>2011</year>) <volume>127</volume>:<fpage>396</fpage>&#x02013;<lpage>404</lpage>. <pub-id pub-id-type="doi">10.1016/j.jsbmb.2011.06.006</pub-id><pub-id pub-id-type="pmid">21684334</pub-id></citation></ref>
</ref-list> 
</back>
</article> 