Serum homocysteine concentration in clinically healthy Thoroughbreds

Serum homocysteine concentration in clinically healthy Thoroughbreds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Serum homocysteine concentration in clinically healthy Thoroughbreds Marcin Gołyński, Santiago Alonso Sousa, Adrian Farringdon, Anna Cywińska, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7600643/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Homocysteine (HCY) is a sulfur-containing amino acid that is metabolically linked to folic acid (FA) and is recognised as a risk marker for coagulation, vascular, and neurodegenerative disorders in humans and small animals. However, the use of serum HCY in equine medicine is limited due to paucity of research concerning its diagnostic utility in this species. This study aimed to evaluate serum HCY concentrations in relation to age, thyroid status (total thyroxine, TT4), and folic acid (FA) in a uniform group of trained, clinically healthy Thoroughbred geldings. Initially, eighty-eight horses were recruited, with 61 deemed clinically healthy following examination. Older animals (≥ 10 years of age) exhibited significantly higher HCY and FA concentrations, while their TT4 levels were lower. A weak positive correlation between HCY and age was observed. This significant age-related increase in HCY was also confirmed in the subset of 19 horses that had all haematological and biochemical parameters within the reference range. Interestingly, in older horses within this healthy subset, a strong negative correlation was found between HCY and the total number of races (TNR), as well as between FA and cumulative fast work distance (CFWD), suggesting that greater historical training loads are linked to a more favourable HCY/FA profile. This study indicates that age significantly influences HCY metabolism in horses, with serum concentrations increasing in older animals. Additionally, it provides new evidence that an individual's athletic history may affect these metabolic pathways. These findings underscore the importance of considering age and athletic activity when interpreting HCY levels and represent a crucial step towards establishing a comprehensive reference range for HCY in horses. Biological sciences/Biochemistry Health sciences/Biomarkers Health sciences/Medical research Biological sciences/Physiology Biological sciences/Zoology homocysteine HCY folic acid FA thyroxine TT4 equine Thoroughbred biomarkers age-related metabolism exercise physiology Introduction Homocysteine (HCY) is an endogenous, sulfur-containing amino acid that serves as an intermediate metabolite in the process of transmethylating methionine into cysteine [ 1 , 2 ]. Its metabolism includes the transsulfuration of HCY to cysteine and remethylation to methionine with the participation of the active form of folic acid (FA) as the “important sweeper” of HCY [ 3 , 4 ]. Hyperhomocysteinemia is widely recognised as both a marker and a risk factor for certain human diseases, as it plays a role in the development of conditions associated with coagulation disorders, vascular health, and neurodegeneration [ 5 , 6 , 7 , 8 ]. Similarly, in small animals, it has been the subject of research in cardiac, enteric and kidney diseases [ 10 , 11 , 12 ]. It has been shown that hyperhomocysteinemia, akin to the condition observed in humans, occurs in canine hypothyroidism, with the onset of the disease showing a positive correlation with HCY levels [ 9 ]. The available literature on HCY concentration in horses is limited. The role of this amino acid was evaluated in the context of embryonic resorption, laminitis, arrhythmia, parasitemia, oxidative stress and acute exercise [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Similarly, only a few studies assessed serum homocysteine concentration in healthy horses [ 14 , 15 , 16 , 17 ]. Studies examining homocysteine levels in horses should consider training loads, as physical activity—particularly the frequency, intensity, and duration of training—can lead to biochemical changes that influence HCY metabolic pathways, both through mechanisms dependent on oxidative stress and those independent of it [ 16 , 20 , 21 ]. As previously indicated, serum HCY concentration may be useful in assessing the health status of individual horses. The key issue, however, is to determine the normal HCY concentration, which includes the concentrations in the serum of healthy horses of various breeds and activities [ 1 ]. Thus, the aim of the present study was to assess the HCY concentration in trained, clinically healthy Thoroughbred horses. Materials and methods The present study was carried out in accordance with relevant guidelines and regulations and the methods are reported in accordance with ARRIVE guidelines. The study was approved by the City University of Hong Kong Animal Ethics Committee (AN-STA-00000246) and the Hong Kong Jockey Club (HKJC) Animal Ethics Committee (ERC/051/2023). Eighty-eight retired, still working under the saddle, Thoroughbred geldings, 4–23 years old (Median = 7), fed balanced feed, with salt and water ad libitum , kept under the same housing conditions as one herd, owned by HKJC Beas River Equestrian Centre (Hong Kong, SAR China) were initially recruited into the study. Inclusion criteria were: 1) being kept under the same housing conditions and 2) being of a healthy clinical status determined on physical examination carried out by one of the equine clinicians (SAS, AF). Exclusion criteria were: 1) animals that received medication in the last 7 days or, 2) animals with a history of health issues in the last 14 days. Subsequently, based on this criteria, 61 clinically healthy horses were qualified for further observation. All animals participated in regular training based on individualised schedules, primarily determined by their age. The horses, based on their age and prior racing activity, were divided into two groups: 1) the horses up to 10 years of age (n = 49), and 2) the horses above 10 years of age (n = 12). No control group has been used, because the study was conducted in clinically healthy animals. The following parameters were used to assess the condition of horses: 1) total number of races (TNR) as the total number of races in career, 2) cumulative fast work distance (CFWD) as the total distance a horse covers during high-intensity exercise sessions, such as galloping or race-pace workouts, over a defined period - this typically includes distances covered at speeds above 13.3–14.3 m/s (approximately 47.9–65.5 km/h; 29.7–40.7 mph), which corresponds to gallop or near-race conditions. To minimise potential confounders, blood samples were collected directly after clinical examination at 6:00–8:00 AM before feeding and training from the cephalic vein into K2EDTA tubes for haematology analysis and into plain tubes to obtain serum after centrifugation at 1,500 x g for 15 min. Haematological analysis was performed with Sysmex XN-1000 analyser (Sysmex Corporation, Japan). Creatine kinase (CK), Triglycerides (TRIG), Gamma-glutamyltransferase (GGT), Lactate dehydrogenase (LDH), Cholesterol (CHOL), Albumin (ALB), Total protein (TP), Phosphate (P), Bicarbonate (CO2), Alkaline phosphatase (ALP), Glucose (GLU), Urea, Creatinine (CREA), Aspartate aminotransferase (AST), Bilirubin (BIL), Calcium (Ca), and Magnesium (Mg) were measured using photometric method by Cobas 6000/c501 (Roche Diagnostics, Switzerland). Sodium (Na), Potassium (K) and Chloride (Cl) were determined using ion-selective electrodes. The haematological and serum biochemical profiles were used to confirm the good health of the horses. Based on the knowledge of HCY metabolism [ 1 ], the HCY serum concentration of healthy horses was analysed in the context of total serum thyroxine (TT4) and serum FA levels. Homocysteine (HCY), total T4 (TT4), and Folic acid (FA) were measured in serum using chemiluminescent immunoassay by Immulite 2000 XPi (Siemens, Germany). Serum Amyloid A (SAA) was measured in serum using an immunoturbidimetric method by Cobas C303 (Roche Diagnostics, Switzerland). Laboratory analyses were performed by the City University of Hong Kong Veterinary Diagnostic Laboratory and the Hong Kong Jockey Club Veterinary Services Department. Statistical analysis The significance of the differences between the concentrations of selected parameters (HCY, TT4, FA) in the age groups (the horses < 10 years old and the horses ≥ 10 years old) was assessed by the Mann-Whitney U test. The same test was applied to evaluate the significance of the differences in the age groups of horses selected on the basis of lack of alterations in haematological and blood biochemical values. P < 0.05 was considered significant. Spearman's R correlations were calculated to assess the relationships between the parameters (HCY, TT4, FA) and factors such as age and activity (TNR, CFWD). Data are presented as the median and interquartile range (IQR). Statistical analysis was performed in Statistica (Version13.3.0, TIBCO Software). The statistical analysis was conducted by a person (AC) blinded to the inclusion and exclusion criteria. Results Routine haematological and serum biochemical parameters varied widely among individuals (Table 1 ). Only 19 horses had all haematological and blood biochemical values within the reference ranges or within 10% below or above the normal reference interval established by the testing laboratory. Table 1 Median and interquartile range (IQR) of routine haematological and serum biochemical parameters of clinically healthy horses (n = 61). Parameter Median (IQR) Range Lab. reference range WBC [x10 9 /L] 6.2 (1.53) 3.49–11.67 5–11 NEU [x10 9 /L] 3.4 (0.85) 2.81–7.46 2.8–7.5 LYM [x10 9 /L] 2.37 (0.46) 1.4–5.66 1.2–3.7 MON [x10 9 /L] 0.38 (0.11) 0.2–0.58 0.2–0.7 EOS [x10 9 /L] 0.06 (0.04) 0.0-0.58 0-0.2 BAS [x10 9 /L] 0.02 (0.02) 0.0-0.05 0-0.05 PLT [x10 9 /L] 102.0 (26.0) 33.0-148.0 60–210 RBC [x10 12 /L] 8.46 (0.91) 7.2–10.0 7–10 HGB [g/dL] 13.6 (1.3) 12.0-16.6 12–17 PCV [%] 39.5 (3.9) 34.2–46.9 33–50 MCV [fl] 46.6 (2.6) 43.9–51.1 44–54 MCH [pg] 16.0 (0.9) 14.8–17.4 15–18 MCHC [g/dL] 34.3 (0.9) 32.0-35.5 32–36 CK [IU/L] 182.2 (90.0) 59.0-373.0 60–330 TRIG [mmol/L] 0.23 (0.12) 0.08–0.62 0.12–0.58 GGT [IU/L] 11.9 (6.2) 3.5–22.5 6–32 LDH [IU/L] 269.3 (94.3) 114.7-461.8 112–456 CHOL [mmol/L] 2.03 (0.78) 0.71–3.29 1.32–2.8 ALB [g/L] 26.88 (6.7) 10.6-39.43 26–41 TP [g/L] 45.86 (11.08) 15.43–62.88 56–76 P [mmol/L] 0.76 (0.23) 0.4–1.15 0.5–1.5 CO2 [mmol/L] 21.27 (3.97) 11.19–29.39 24–30 ALP [IU/L] 84.0 (24.5) 26.3-126.8 109–315 GLU [mmol/L] 3.0 (0.94) 1.07–4.58 3.4–7.4 UREA [mmol/L] 5.12 (1.48) 2.17–7.2 3.9–9.6 CREA [mmol/L] 108.9 (22.9) 42.5–186.0 35–194 AST [IU/L] 217.5 (74.9) 70.0-406.2 160–412 BIL [mmol/L] 28.04 (12.15) 9.73–87.51 0-54.7 Ca [mmol/L] 2.44 (0.44) 0.94–3.09 2.5–3.3 Mg [mmol/L] 0.71 (0.12) 0.28–0.94 0.65–1.02 Na [mmol/L] 128.0 (20.0) 65.0-149.0 128–142 K [mmol/L] 4.15 (1.3) 2.7-10.14 2.9–4.6 Cl [mmol/L] 91.1 (15.4) 44.2-104.9 98–109 SAA [mg/L] 2.0 (0.3) 0.00-68.4 0–20 Abbreviations: WBC – White blood cells, NEU - Neutrophils, LYM - Lymphocytes, MON - Monocytes, EOS - Eosinophils, BAS - Basophils, PLT - Platelet, RBC – Red blood cells, HGB - Haemoglobin, PCV – Packed cell volume, MCV – Mean corpuscular volume, MCH - Mean corpuscular haemoglobin, MCHC - Mean corpuscular haemoglobin concentration, CK - Creatine kinase, TRIG - Triglycerides, GGT - Gamma-glutamyltransferase, LDH Lactate dehydrogenase, CHOL - Cholesterol, ALB - Albumin, TP - Total Protein, P - Phosphate, CO2 - Bicarbonate, ALP - Alkaline phosphatase, GLU - Glucose, CREA - Creatinine, AST - Aspartate aminotransferase, BIL - Bilirubin, Ca - Calcium, Mg – Magnesium, Na - Sodium, K – Potassium, Cl – Chloride, SAA – Serum Amyloid A. In the whole group of 61 horses, the concentrations of HCY and TT4 (Table 2 ) weakly but significantly correlated with the age (r = 0.29, p < 0.05 and r=-0.37, p < 0.05, respectively). The concentrations of TT4 were significantly (p = 0.007) lower in the horses ≥ 10 years old, and the concentrations of HCY and FA were significantly (p = 0.003 and p = 0.03, respectively) higher in this group (Table 3 ). In the age group ≥ 10 years, there was a moderately significant (r = 0.58, p < 0.05) correlation between the concentrations of HCY and TT4. Table 2 Median and interquartile range (IQR) of Homocysteine (HCY), Total Thyroxine (TT4) and Folic Acid (FA) in the serum of clinically healthy horses (n = 61). Parameter Median (IQR) Range Lab reference range HCY [µmol/L] 5.5 (4.88) 2.0-18.7 Not defined TT4 [nmol/L] 16.0 (9.4) 6.4–32.3 7–27 FA [nmol/L] 13.3 (5.4) 2.3–26.1 Not defined Table 3 The differences between the concentrations of Homocysteine (HCY), Total Thyroxine (TT4) and Folic Acid (FA) in the group of clinically healthy horses (n = 61), divided by age. Parameter Median (IQR) P value Horses < 10 years (n = 49) Horses ≥ 10 years (n = 12) HCY [µmol/L] 5.0 (4.27) 8.72 (4.52) 0.003 TT4 [nmol/L] 16.3 (8.8) 11.6 (8.3) 0.007 FA [nmol/L] 12.8 (3.3) 17.8 (8.5) 0.030 In the horses with no alterations in haematological and serum biochemical parameters (n = 19), the concentration of HCY was significantly (p = 0.046) higher in the horses ≥ 10 years of age (Table 4 ). Table 4 The differences between the concentrations of Homocysteine (HCY), Total Thyroxine (TT4), and Folic Acid (FA) in the age groups of horses with no alterations in haematological and serum biochemical values. Parameter Median (IQR) P value Horses < 10 years (n = 13) Horses ≥ 10 years (n = 6) HCY[µmol/L] 5.7 (3.89) 9.98 (3.72) 0.046 TT4 [nmol/L] 15.2 (9.2) 13.15 (7.5) 0.365 FA [nmol/L] 12.2 (2.6) 17.8 (7.3) 0.244 In a group of older horses (≥ 10 years) with no alterations in haematological and serum biochemical values, the concentration of HCY strongly, significantly correlated with the TNR (r=-0.83, p < 0.05) and the concentration of FA significantly and very strongly correlated with the CFWD (r=-0.9, p < 0.05) (Table 5 ). Table 5 Total Number of Races (TNR) and Cumulative Fast Work Distance (CFWD) in the age groups of horses with no alterations in haematological and blood biochemical values. Parameter Median (IQR) P value Horses < 10 years (n = 13) Horses ≥ 10 years (n = 6) TNR 11 (14) 21 (17) 0.087 CFWD 73 (53) 42 (52) 0.323 Discussion The Hong Kong Jockey Club (HKJC), one of the largest charitable donors and taxpayers in the city, primarily manages Thoroughbred horse races at the Happy Valley and Sha Tin Racecourses and provides care to horses retired from racing [ 22 ]. Consequently, this unique arrangement enables the recruitment of a homogeneous group of horses for analysis in various contexts. In this study, 88 Thoroughbred geldings from HKJC Beas River Equestrian Centre were randomly selected, and 61 horses subjected to regular training according to the schedule did not show any clinical issues. In this group, classified as clinically healthy animals, only 19 individuals had all haematological and routine biochemical parameters within reference ranges, or not deviating from the reference range by more than 10%, which did not indicate the underlying disease. The concentration of HCY in the blood of 61 clinically healthy horses positively correlated with the age of the animals and was significantly higher in older horses ≥ 10 years. This tendency was confirmed also in the subgroup of clinically healthy horses with no alterations in haematological and serum biochemical parameters; the concentration of HCY was significantly higher in older animals. Similar results were observed in humans; HCY concentration was lowest in individuals aged 30 to 50, but showed a significant increase in both males and females aged 50 and above. In males over the age of 60, HCY levels rose significantly, surpassing the reference range of 5–15 µmol/L [ 23 , 27 ]. Other researchers have noted that HCY concentrations in humans tend to rise with age during adulthood, and that average HCY levels also vary based on sex and ethnicity [ 24 ]. This could be connected to the development of age-related disorders, as elevated homocysteine levels have been observed in cardiovascular diseases with thrombotic origins, as well as in neurodegenerative diseases and dementia [ 25 ]. In Berhane et al.’s research [ 14 ], the normal range of serum HCY in ponies was determined at 1.3–14.7 µmol/L, which is close to the above-mentioned range found in human serum. Although there were no differences in HCY serum levels between winter and spring, there were no data about age, gender, reproductive status and the sport training of animals in this study. As such, the established HCY normal range may not be an accurate reference for all horses. Evaluated concentration of HCY in rest mares seems to depend on sport discipline or breed – mean value in 6–11 years old jumping Sella Italiana was lower (3.16 ± 0.60µmol/L) than in 5–9 years old Thoroughbred racehorses (5.88 ± 0.78µmol/L) [ 16 ]. In another study, the circadian rhythm of HCY in mares was determined, and the obtained results were similar [ 26 ]. In research conducted on resting horses of different breeds by Mitchell et all [ 17 ], the mean of HCY was 4.65µmol/L. A higher mean value (6.16 µmol/L) was observed by another study, but these were conducted in Thoroughbred mares [ 15 ]. Although the reproductive cycle significantly impacts HCY levels in females, and HCY influences fertility and pregnancy establishment [ 29 ], the aforementioned values are comparable to our findings in geldings. Our study is the first to assess the serum homocysteine concentration in a uniform group of thoroughbred geldings. Our study revealed that in all clinically healthy older horses, HCY concentration increased with TT4 concentration. We have previously confirmed the inverse relationship between these parameters in dogs [ 9 ], but such a relation occurred in canine hypothyroidism, which leads to hyperhomocysteinemia. However, in healthy animals, when thyroid function is normal, there are no disturbances in HCY metabolism directly related to thyroid hormone deficiency, including reduced FA concentration and its effect on HCY remethylation to methionine [ 1 , 4 , 9 ]. Therefore, individuals with abnormal serum total thyroxine concentration were excluded in the present study. The serum concentration of FA (vitamin B9) in all healthy horses was higher in older animals, over 10 years of age. This may indicate a decline in the efficiency of HCY scavenging mechanisms by FA in older animals, although we did not find a correlation between FA and HCY concentrations, which is reversed in dogs diagnosed with hypothyroidism [ 9 ]. However, the present study indicated the correlation between both parameters and the performance indicators in older, clinically healthy horses with no alterations in haematological and blood biochemical values. HCY concentrations were lower and FA higher in individuals at higher training levels – such relationships have been demonstrated for the first time in horses. Likewise, baseline HCY levels are lower in humans who engage in regular training, whereas in amateur runners, homocysteine-dependent oxidative stress poses a significant risk factor for vascular diseases [ 20 , 28 ]. The main limitation of our study, which prevents the use of the obtained results as a reference range in clinical practice, is the relatively small number of individuals with normal values of all blood parameters. A similar problem applies to other studies on HCY in horses [ 14 , 15 , 16 , 17 , 26 ]. To develop a reference range for HCY in horses, it is necessary to examine the concentration of this amino acid in a significantly larger number of clinically and laboratory-healthy individuals. However, the data presented in this study serve as another step towards defining a reference range for HCY in horses. Declarations Conflicts of Interest: The authors declare no conflict of interest. Funding: This work is supported by the late Madam CHONG Kin Wo, a City University of Hong Kong donor. Author Contribution Conceptualization, M.G. and P.M.B.; blood sample collection and animal health and welfare oversight, S.A.S and A.F.; data analysis, M.G. and A.C.; writing—original draft preparation, M.G., M.C. and P.M.B.; writing—review and editing, M.G., S.A.S., M.C. and P.M.B.; project administration and funding acquisition, P.M.B. Acknowledgement Authors would like to thank the late Madam CHONG KinWo for supporting research into the role of homocysteine in horses. Data Availability Data supporting the findings of this study are available within the article. The raw data from the diagnostic laboratory are available from the corresponding author [P.M.B.] on request. References Gołyński, M. et al. Homocysteine— Potential. Novel Diagn. Indicator Health Disease Horses Anim. 13 , 1311. (2023). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7600643","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":530238683,"identity":"811aeea4-ec46-41fd-875d-095aa6191178","order_by":0,"name":"Marcin Gołyński","email":"","orcid":"","institution":"Nicolaus Copernicus University in Toruń","correspondingAuthor":false,"prefix":"","firstName":"Marcin","middleName":"","lastName":"Gołyński","suffix":""},{"id":530238684,"identity":"920cc72a-f329-4ef6-a18b-91f0f480fb31","order_by":1,"name":"Santiago Alonso Sousa","email":"","orcid":"","institution":"City University of Hong 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Bęczkowski","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACCRBxgIGBn4GxASJygIENxsSvRbKBsbGBNC0GB2DWENJiLt188OOPMzaJm48fbn/M28Ygx3cjge3hDDxaLOccS5aQuJGWuO1MYmMzUIux5I0EdsMNeLQY3MgxYzD4cDh32w1GsJbEDUBbJB/g1ZL/jSEBqGXzDIiWeiK05LAxHLhxOHeDBERLggFICz6HAf1iLNlwJq1+BtAvM+eckzCceeZhmyQ+7wND7OHHH8dsjPnbjz/48KbMRp7vePIxyR58DkPmMPGAowl/RKJqYfyBV+0oGAWjYBSMVAAAgnlZEQQ6E+sAAAAASUVORK5CYII=","orcid":"","institution":"City University of Hong Kong","correspondingAuthor":true,"prefix":"","firstName":"Paweł","middleName":"Marek","lastName":"Bęczkowski","suffix":""}],"badges":[],"createdAt":"2025-09-12 12:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7600643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7600643/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93648235,"identity":"37ce4afe-5965-4856-b41f-8a87401195a8","added_by":"auto","created_at":"2025-10-16 04:55:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46904,"visible":true,"origin":"","legend":"","description":"","filename":"Homocysteinerevisedclean190925DOC.docx","url":"https://assets-eu.researchsquare.com/files/rs-7600643/v1/e31713884ef081ef12bd6883.docx"},{"id":93648392,"identity":"b2b002a0-00f4-4dcd-ba97-8a3ef3a6343d","added_by":"auto","created_at":"2025-10-16 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07:54:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":672814,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7600643/v1/2b384c87-f4e2-451c-85aa-0e6458ce2a68.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Serum homocysteine concentration in clinically healthy Thoroughbreds","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHomocysteine (HCY) is an endogenous, sulfur-containing amino acid that serves as an intermediate metabolite in the process of transmethylating methionine into cysteine [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Its metabolism includes the transsulfuration of HCY to cysteine and remethylation to methionine with the participation of the active form of folic acid (FA) as the \u0026ldquo;important sweeper\u0026rdquo; of HCY [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Hyperhomocysteinemia is widely recognised as both a marker and a risk factor for certain human diseases, as it plays a role in the development of conditions associated with coagulation disorders, vascular health, and neurodegeneration [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, in small animals, it has been the subject of research in cardiac, enteric and kidney diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been shown that hyperhomocysteinemia, akin to the condition observed in humans, occurs in canine hypothyroidism, with the onset of the disease showing a positive correlation with HCY levels [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe available literature on HCY concentration in horses is limited. The role of this amino acid was evaluated in the context of embryonic resorption, laminitis, arrhythmia, parasitemia, oxidative stress and acute exercise [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Similarly, only a few studies assessed serum homocysteine concentration in healthy horses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Studies examining homocysteine levels in horses should consider training loads, as physical activity\u0026mdash;particularly the frequency, intensity, and duration of training\u0026mdash;can lead to biochemical changes that influence HCY metabolic pathways, both through mechanisms dependent on oxidative stress and those independent of it [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs previously indicated, serum HCY concentration may be useful in assessing the health status of individual horses. The key issue, however, is to determine the normal HCY concentration, which includes the concentrations in the serum of healthy horses of various breeds and activities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Thus, the aim of the present study was to assess the HCY concentration in trained, clinically healthy Thoroughbred horses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe present study was carried out in accordance with relevant guidelines and regulations and the methods are reported in accordance with ARRIVE guidelines. The study was approved by the City University of Hong Kong Animal Ethics Committee (AN-STA-00000246) and the Hong Kong Jockey Club (HKJC) Animal Ethics Committee (ERC/051/2023).\u003c/p\u003e\u003cp\u003eEighty-eight retired, still working under the saddle, Thoroughbred geldings, 4\u0026ndash;23 years old (Median\u0026thinsp;=\u0026thinsp;7), fed balanced feed, with salt and water \u003cem\u003ead libitum\u003c/em\u003e, kept under the same housing conditions as one herd, owned by HKJC Beas River Equestrian Centre (Hong Kong, SAR China) were initially recruited into the study. Inclusion criteria were: 1) being kept under the same housing conditions and 2) being of a healthy clinical status determined on physical examination carried out by one of the equine clinicians (SAS, AF). Exclusion criteria were: 1) animals that received medication in the last 7 days or, 2) animals with a history of health issues in the last 14 days. Subsequently, based on this criteria, 61 clinically healthy horses were qualified for further observation. All animals participated in regular training based on individualised schedules, primarily determined by their age. The horses, based on their age and prior racing activity, were divided into two groups: 1) the horses up to 10 years of age (n\u0026thinsp;=\u0026thinsp;49), and 2) the horses above 10 years of age (n\u0026thinsp;=\u0026thinsp;12). No control group has been used, because the study was conducted in clinically healthy animals.\u003c/p\u003e\u003cp\u003eThe following parameters were used to assess the condition of horses: 1) total number of races (TNR) as the total number of races in career, 2) cumulative fast work distance (CFWD) as the total distance a horse covers during high-intensity exercise sessions, such as galloping or race-pace workouts, over a defined period - this typically includes distances covered at speeds above 13.3\u0026ndash;14.3 m/s (approximately 47.9\u0026ndash;65.5 km/h; 29.7\u0026ndash;40.7 mph), which corresponds to gallop or near-race conditions.\u003c/p\u003e\u003cp\u003eTo minimise potential confounders, blood samples were collected directly after clinical examination at 6:00\u0026ndash;8:00 AM before feeding and training from the cephalic vein into K2EDTA tubes for haematology analysis and into plain tubes to obtain serum after centrifugation at 1,500 x g for 15 min. Haematological analysis was performed with Sysmex XN-1000 analyser (Sysmex Corporation, Japan). Creatine kinase (CK), Triglycerides (TRIG), Gamma-glutamyltransferase (GGT), Lactate dehydrogenase (LDH), Cholesterol (CHOL), Albumin (ALB), Total protein (TP), Phosphate (P), Bicarbonate (CO2), Alkaline phosphatase (ALP), Glucose (GLU), Urea, Creatinine (CREA), Aspartate aminotransferase (AST), Bilirubin (BIL), Calcium (Ca), and Magnesium (Mg) were measured using photometric method by Cobas 6000/c501 (Roche Diagnostics, Switzerland). Sodium (Na), Potassium (K) and Chloride (Cl) were determined using ion-selective electrodes. The haematological and serum biochemical profiles were used to confirm the good health of the horses.\u003c/p\u003e\u003cp\u003eBased on the knowledge of HCY metabolism [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], the HCY serum concentration of healthy horses was analysed in the context of total serum thyroxine (TT4) and serum FA levels. Homocysteine (HCY), total T4 (TT4), and Folic acid (FA) were measured in serum using chemiluminescent immunoassay by Immulite 2000 XPi (Siemens, Germany). Serum Amyloid A (SAA) was measured in serum using an immunoturbidimetric method by Cobas C303 (Roche Diagnostics, Switzerland). Laboratory analyses were performed by the City University of Hong Kong Veterinary Diagnostic Laboratory and the Hong Kong Jockey Club Veterinary Services Department.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe significance of the differences between the concentrations of selected parameters (HCY, TT4, FA) in the age groups (the horses\u0026thinsp;\u0026lt;\u0026thinsp;10 years old and the horses\u0026thinsp;\u0026ge;\u0026thinsp;10 years old) was assessed by the Mann-Whitney U test. The same test was applied to evaluate the significance of the differences in the age groups of horses selected on the basis of lack of alterations in haematological and blood biochemical values. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. Spearman's R correlations were calculated to assess the relationships between the parameters (HCY, TT4, FA) and factors such as age and activity (TNR, CFWD). Data are presented as the median and interquartile range (IQR). Statistical analysis was performed in Statistica (Version13.3.0, TIBCO Software). The statistical analysis was conducted by a person (AC) blinded to the inclusion and exclusion criteria.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eRoutine haematological and serum biochemical parameters varied widely among individuals (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Only 19 horses had all haematological and blood biochemical values within the reference ranges or within 10% below or above the normal reference interval established by the testing laboratory.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMedian and interquartile range (IQR) of routine haematological and serum biochemical parameters of clinically healthy horses (n\u0026thinsp;=\u0026thinsp;61).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (IQR)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRange\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLab. reference range\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWBC [x10\u003csup\u003e9\u003c/sup\u003e/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6.2 (1.53)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.49\u0026ndash;11.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u0026ndash;11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNEU [x10\u003csup\u003e9\u003c/sup\u003e/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.4 (0.85)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.81\u0026ndash;7.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.8\u0026ndash;7.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLYM [x10\u003csup\u003e9\u003c/sup\u003e/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.37 (0.46)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.4\u0026ndash;5.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c4\"\u003e\u003cp\u003e0-0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBAS [x10\u003csup\u003e9\u003c/sup\u003e/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.02 (0.02)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0-0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0-0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePLT [x10\u003csup\u003e9\u003c/sup\u003e/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e102.0 (26.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e33.0-148.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60\u0026ndash;210\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRBC [x10\u003csup\u003e12\u003c/sup\u003e/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.46 (0.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.2\u0026ndash;10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u0026ndash;10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHGB [g/dL]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.6 (1.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.0-16.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12\u0026ndash;17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePCV [%]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e39.5 (3.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e34.2\u0026ndash;46.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33\u0026ndash;50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCV [fl]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e46.6 (2.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e43.9\u0026ndash;51.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44\u0026ndash;54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCH [pg]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16.0 (0.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14.8\u0026ndash;17.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15\u0026ndash;18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCHC [g/dL]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e34.3 (0.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32.0-35.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u0026ndash;36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCK [IU/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e182.2 (90.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.0-373.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60\u0026ndash;330\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTRIG [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.23 (0.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.08\u0026ndash;0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.12\u0026ndash;0.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGGT [IU/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11.9 (6.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.5\u0026ndash;22.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLDH [IU/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e269.3 (94.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e114.7-461.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e112\u0026ndash;456\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCHOL [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.03 (0.78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.71\u0026ndash;3.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.32\u0026ndash;2.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALB [g/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26.88 (6.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10.6-39.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26\u0026ndash;41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTP [g/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e45.86 (11.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15.43\u0026ndash;62.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56\u0026ndash;76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.76 (0.23)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.4\u0026ndash;1.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u0026ndash;1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCO2 [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21.27 (3.97)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11.19\u0026ndash;29.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u0026ndash;30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALP [IU/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e84.0 (24.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26.3-126.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e109\u0026ndash;315\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGLU [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.0 (0.94)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.07\u0026ndash;4.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.4\u0026ndash;7.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUREA [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.12 (1.48)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.17\u0026ndash;7.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.9\u0026ndash;9.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCREA [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e108.9 (22.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e42.5\u0026ndash;186.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35\u0026ndash;194\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAST [IU/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e217.5 (74.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70.0-406.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e160\u0026ndash;412\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBIL [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28.04 (12.15)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.73\u0026ndash;87.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0-54.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCa [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.44 (0.44)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.94\u0026ndash;3.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.5\u0026ndash;3.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMg [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.71 (0.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.28\u0026ndash;0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.65\u0026ndash;1.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNa [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e128.0 (20.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e65.0-149.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e128\u0026ndash;142\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.15 (1.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.7-10.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.9\u0026ndash;4.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCl [mmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e91.1 (15.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e44.2-104.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98\u0026ndash;109\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSAA [mg/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.0 (0.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00-68.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u0026ndash;20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: WBC \u0026ndash; White blood cells, NEU - Neutrophils, LYM - Lymphocytes, MON - Monocytes, EOS - Eosinophils, BAS - Basophils, PLT - Platelet, RBC \u0026ndash; Red blood cells, HGB - Haemoglobin, PCV \u0026ndash; Packed cell volume, MCV \u0026ndash; Mean corpuscular volume, MCH - Mean corpuscular haemoglobin, MCHC - Mean corpuscular haemoglobin concentration, CK - Creatine kinase, TRIG - Triglycerides, GGT - Gamma-glutamyltransferase, LDH Lactate dehydrogenase, CHOL - Cholesterol, ALB - Albumin, TP - Total Protein, P - Phosphate, CO2 - Bicarbonate, ALP - Alkaline phosphatase, GLU - Glucose, CREA - Creatinine, AST - Aspartate aminotransferase, BIL - Bilirubin, Ca - Calcium, Mg \u0026ndash; Magnesium, Na - Sodium, K \u0026ndash; Potassium, Cl \u0026ndash; Chloride, SAA \u0026ndash; Serum Amyloid A.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the whole group of 61 horses, the concentrations of HCY and TT4 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) weakly but significantly correlated with the age (r\u0026thinsp;=\u0026thinsp;0.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and r=-0.37, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively). The concentrations of TT4 were significantly (p\u0026thinsp;=\u0026thinsp;0.007) lower in the horses\u0026thinsp;\u0026ge;\u0026thinsp;10 years old, and the concentrations of HCY and FA were significantly (p\u0026thinsp;=\u0026thinsp;0.003 and p\u0026thinsp;=\u0026thinsp;0.03, respectively) higher in this group (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the age group\u0026thinsp;\u0026ge;\u0026thinsp;10 years, there was a moderately significant (r\u0026thinsp;=\u0026thinsp;0.58, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) correlation between the concentrations of HCY and TT4.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMedian and interquartile range (IQR) of Homocysteine (HCY), Total Thyroxine (TT4) and Folic Acid (FA) in the serum of clinically healthy horses (n\u0026thinsp;=\u0026thinsp;61).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (IQR)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRange\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLab reference range\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHCY [\u0026micro;mol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.5 (4.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.0-18.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNot defined\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTT4 [nmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16.0 (9.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.4\u0026ndash;32.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u0026ndash;27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFA [nmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.3 (5.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.3\u0026ndash;26.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNot defined\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe differences between the concentrations of Homocysteine (HCY), Total Thyroxine (TT4) and Folic Acid (FA) in the group of clinically healthy horses (n\u0026thinsp;=\u0026thinsp;61), divided by age.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eMedian (IQR)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHorses\u0026thinsp;\u0026lt;\u0026thinsp;10 years (n\u0026thinsp;=\u0026thinsp;49)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHorses\u0026thinsp;\u0026ge;\u0026thinsp;10 years (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHCY [\u0026micro;mol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.0 (4.27)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.72 (4.52)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTT4 [nmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16.3 (8.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11.6 (8.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFA [nmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.8 (3.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17.8 (8.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.030\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the horses with no alterations in haematological and serum biochemical parameters (n\u0026thinsp;=\u0026thinsp;19), the concentration of HCY was significantly (p\u0026thinsp;=\u0026thinsp;0.046) higher in the horses\u0026thinsp;\u0026ge;\u0026thinsp;10 years of age (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe differences between the concentrations of Homocysteine (HCY), Total Thyroxine (TT4), and Folic Acid (FA) in the age groups of horses with no alterations in haematological and serum biochemical values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eMedian (IQR)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHorses\u0026thinsp;\u0026lt;\u0026thinsp;10 years (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHorses\u0026thinsp;\u0026ge;\u0026thinsp;10 years (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHCY[\u0026micro;mol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.7 (3.89)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.98 (3.72)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.046\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTT4 [nmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.2 (9.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13.15 (7.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.365\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFA [nmol/L]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.2 (2.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17.8 (7.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.244\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn a group of older horses (\u0026ge;\u0026thinsp;10 years) with no alterations in haematological and serum biochemical values, the concentration of HCY strongly, significantly correlated with the TNR (r=-0.83, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the concentration of FA significantly and very strongly correlated with the CFWD (r=-0.9, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTotal Number of Races (TNR) and Cumulative Fast Work Distance (CFWD) in the age groups of horses with no alterations in haematological and blood biochemical values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eMedian (IQR)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHorses\u0026thinsp;\u0026lt;\u0026thinsp;10 years (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHorses\u0026thinsp;\u0026ge;\u0026thinsp;10 years (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11 (14)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21 (17)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.087\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCFWD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e73 (53)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42 (52)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.323\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Hong Kong Jockey Club (HKJC), one of the largest charitable donors and taxpayers in the city, primarily manages Thoroughbred horse races at the Happy Valley and Sha Tin Racecourses and provides care to horses retired from racing [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Consequently, this unique arrangement enables the recruitment of a homogeneous group of horses for analysis in various contexts.\u003c/p\u003e\u003cp\u003eIn this study, 88 Thoroughbred geldings from HKJC Beas River Equestrian Centre were randomly selected, and 61 horses subjected to regular training according to the schedule did not show any clinical issues. In this group, classified as clinically healthy animals, only 19 individuals had all haematological and routine biochemical parameters within reference ranges, or not deviating from the reference range by more than 10%, which did not indicate the underlying disease.\u003c/p\u003e\u003cp\u003eThe concentration of HCY in the blood of 61 clinically healthy horses positively correlated with the age of the animals and was significantly higher in older horses\u0026thinsp;\u0026ge;\u0026thinsp;10 years. This tendency was confirmed also in the subgroup of clinically healthy horses with no alterations in haematological and serum biochemical parameters; the concentration of HCY was significantly higher in older animals. Similar results were observed in humans; HCY concentration was lowest in individuals aged 30 to 50, but showed a significant increase in both males and females aged 50 and above. In males over the age of 60, HCY levels rose significantly, surpassing the reference range of 5\u0026ndash;15 \u0026micro;mol/L [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Other researchers have noted that HCY concentrations in humans tend to rise with age during adulthood, and that average HCY levels also vary based on sex and ethnicity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This could be connected to the development of age-related disorders, as elevated homocysteine levels have been observed in cardiovascular diseases with thrombotic origins, as well as in neurodegenerative diseases and dementia [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn Berhane et al.\u0026rsquo;s research [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the normal range of serum HCY in ponies was determined at 1.3\u0026ndash;14.7 \u0026micro;mol/L, which is close to the above-mentioned range found in human serum. Although there were no differences in HCY serum levels between winter and spring, there were no data about age, gender, reproductive status and the sport training of animals in this study. As such, the established HCY normal range may not be an accurate reference for all horses. Evaluated concentration of HCY in rest mares seems to depend on sport discipline or breed \u0026ndash; mean value in 6\u0026ndash;11 years old jumping Sella Italiana was lower (3.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u0026micro;mol/L) than in 5\u0026ndash;9 years old Thoroughbred racehorses (5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u0026micro;mol/L) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In another study, the circadian rhythm of HCY in mares was determined, and the obtained results were similar [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In research conducted on resting horses of different breeds by Mitchell et all [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the mean of HCY was 4.65\u0026micro;mol/L. A higher mean value (6.16 \u0026micro;mol/L) was observed by another study, but these were conducted in Thoroughbred mares [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although the reproductive cycle significantly impacts HCY levels in females, and HCY influences fertility and pregnancy establishment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the aforementioned values are comparable to our findings in geldings. Our study is the first to assess the serum homocysteine concentration in a uniform group of thoroughbred geldings.\u003c/p\u003e\u003cp\u003eOur study revealed that in all clinically healthy older horses, HCY concentration increased with TT4 concentration. We have previously confirmed the inverse relationship between these parameters in dogs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], but such a relation occurred in canine hypothyroidism, which leads to hyperhomocysteinemia. However, in healthy animals, when thyroid function is normal, there are no disturbances in HCY metabolism directly related to thyroid hormone deficiency, including reduced FA concentration and its effect on HCY remethylation to methionine [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, individuals with abnormal serum total thyroxine concentration were excluded in the present study.\u003c/p\u003e\u003cp\u003eThe serum concentration of FA (vitamin B9) in all healthy horses was higher in older animals, over 10 years of age. This may indicate a decline in the efficiency of HCY scavenging mechanisms by FA in older animals, although we did not find a correlation between FA and HCY concentrations, which is reversed in dogs diagnosed with hypothyroidism [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the present study indicated the correlation between both parameters and the performance indicators in older, clinically healthy horses with no alterations in haematological and blood biochemical values. HCY concentrations were lower and FA higher in individuals at higher training levels \u0026ndash; such relationships have been demonstrated for the first time in horses. Likewise, baseline HCY levels are lower in humans who engage in regular training, whereas in amateur runners, homocysteine-dependent oxidative stress poses a significant risk factor for vascular diseases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe main limitation of our study, which prevents the use of the obtained results as a reference range in clinical practice, is the relatively small number of individuals with normal values of all blood parameters. A similar problem applies to other studies on HCY in horses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To develop a reference range for HCY in horses, it is necessary to examine the concentration of this amino acid in a significantly larger number of clinically and laboratory-healthy individuals. However, the data presented in this study serve as another step towards defining a reference range for HCY in horses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work is supported by the late Madam CHONG Kin Wo, a City University of Hong Kong donor.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, M.G. and P.M.B.; blood sample collection and animal health and welfare oversight, S.A.S and A.F.; data analysis, M.G. and A.C.; writing\u0026mdash;original draft preparation, M.G., M.C. and P.M.B.; writing\u0026mdash;review and editing, M.G., S.A.S., M.C. and P.M.B.; project administration and funding acquisition, P.M.B.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthors would like to thank the late Madam CHONG KinWo for supporting research into the role of homocysteine in horses.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData supporting the findings of this study are available within the article. The raw data from the diagnostic laboratory are available from the corresponding author [P.M.B.] on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGołyński, M. et al. \u003cem\u003eHomocysteine\u0026mdash; Potential. Novel Diagn. Indicator Health Disease Horses Anim.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1311. (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrosnan, J. T. \u0026amp; Brosnan, M. E. The sulfur-containing amino acids: An overview. \u003cem\u003eJ. Nutr.\u003c/em\u003e \u003cb\u003e136\u003c/b\u003e, 1636S\u0026ndash;1640S (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFroese, D. S., Fowler, B. \u0026amp; Baumgartner, M. R. Vitamin B12, folate, and the methionine remethylation cycle\u0026mdash;Biochemistry, pathways, and regulation. \u003cem\u003eJ. Inherit. Metab. 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The influence of exercise on the daily rhythm of serum homocysteine in horses. \u003cem\u003eJ. Physiol. Sci.\u003c/em\u003e \u003cb\u003e56\u003c/b\u003e (6), 455\u0026ndash;458 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUeland, P. M. et al. Total homocysteine in plasma or serum: Methods and clinical applications. \u003cem\u003eClin. Chem.\u003c/em\u003e \u003cb\u003e39\u003c/b\u003e, 1764\u0026ndash;1779 (1993).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoubert, L. M. \u0026amp; Manore, M. M. The role of physical activity level and B-vitamin status on blood homocysteine levels. \u003cem\u003eMed. Sci. Sport Exerc.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 1923\u0026ndash;1931 (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRizzo, A. \u0026amp; Sciorsci, R. L. Role of homocysteine metabolism in animal reproduction: A review. \u003cem\u003eRes. Vet. Sci.\u003c/em\u003e \u003cb\u003e122\u003c/b\u003e, 29\u0026ndash;35 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"homocysteine, HCY, folic acid, FA, thyroxine, TT4, equine, Thoroughbred, biomarkers, age-related metabolism, exercise physiology","lastPublishedDoi":"10.21203/rs.3.rs-7600643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7600643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHomocysteine (HCY) is a sulfur-containing amino acid that is metabolically linked to folic acid (FA) and is recognised as a risk marker for coagulation, vascular, and neurodegenerative disorders in humans and small animals. However, the use of serum HCY in equine medicine is limited due to paucity of research concerning its diagnostic utility in this species. This study aimed to evaluate serum HCY concentrations in relation to age, thyroid status (total thyroxine, TT4), and folic acid (FA) in a uniform group of trained, clinically healthy Thoroughbred geldings.\u003c/p\u003e\u003cp\u003eInitially, eighty-eight horses were recruited, with 61 deemed clinically healthy following examination. Older animals (\u0026ge;\u0026thinsp;10 years of age) exhibited significantly higher HCY and FA concentrations, while their TT4 levels were lower. A weak positive correlation between HCY and age was observed. This significant age-related increase in HCY was also confirmed in the subset of 19 horses that had all haematological and biochemical parameters within the reference range. Interestingly, in older horses within this healthy subset, a strong negative correlation was found between HCY and the total number of races (TNR), as well as between FA and cumulative fast work distance (CFWD), suggesting that greater historical training loads are linked to a more favourable HCY/FA profile.\u003c/p\u003e\u003cp\u003eThis study indicates that age significantly influences HCY metabolism in horses, with serum concentrations increasing in older animals. Additionally, it provides new evidence that an individual's athletic history may affect these metabolic pathways. These findings underscore the importance of considering age and athletic activity when interpreting HCY levels and represent a crucial step towards establishing a comprehensive reference range for HCY in horses.\u003c/p\u003e","manuscriptTitle":"Serum homocysteine concentration in clinically healthy Thoroughbreds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 04:55:44","doi":"10.21203/rs.3.rs-7600643/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82aa24e2-a736-4372-bde8-67189e3f96c1","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56367111,"name":"Biological sciences/Biochemistry"},{"id":56367112,"name":"Health sciences/Biomarkers"},{"id":56367113,"name":"Health sciences/Medical research"},{"id":56367114,"name":"Biological sciences/Physiology"},{"id":56367115,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2025-11-10T07:54:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-16 04:55:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7600643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7600643","identity":"rs-7600643","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}