Oxidative Damage and Mitochondrial Dysfunction in Cystathionine Beta-synthase Deficiency

Oxidative Damage and Mitochondrial Dysfunction in Cystathionine Beta-synthase Deficiency | 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 Oxidative Damage and Mitochondrial Dysfunction in Cystathionine Beta-synthase Deficiency Mehmet Cihan Balci, Asuman Gedikbasi, Sukru Anil Dogan, Sevde Kahraman, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4732434/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Cystathionine beta-synthase deficiency (CBSD) is the most prevalent inherited disorder of homocysteine metabolism in the transsulphuration pathway. Research have suggested oxidative stress and inflammation as candidate pathogenic mechanisms in CBSD. This study aims to evaluate mitochondrial dysfunction and oxidative stress biomarkers in cystathionine beta-synthase deficiency (CBSD) patients, which may aid in understanding the pathogenesis of CBSD and improving treatment. The study group comprised 23 patients with a diagnosis of CBSD and healthy controls. We analysed serum levels of NAD + and NADH by fluorometric assay, FGF-21 and GDF-15 by ELISA, mitochondrial DAMPs by real time qRT-PCR, total homocysteine levels in plasma by enzymatic test and compared the results in CBSD group with healthy controls. In patient group, a positive correlation was found between the total homocysteine level and both GDF-15 and NAD + /NADH levels. Furthermore, there was a negative correlation between total homocysteine levels and both total NAD + +NADH and NADH levels. The alterations in NAD + , FGF-21, GDF-15 levels, and NAD + /NADH ratio in patients suggest that oxidative damage coexists with mitochondrial dysfunction in CBSD. Assessment of oxidative damage and addition of anti-oxidant therapy together with mitochondrial support may have additional benefits in reducing long-term morbidity in CBSD patients. Cystathionine Beta Synthase Deficiency Fibroblast Growth Factor 21 Growth Differentiation Factor 15 Mitochondria Oxidative Stress Introduction Cystathionine beta-synthase deficiency (CBSD) (OMIM 236200) is the most prevalent inherited disorder of homocysteine (Hcy) metabolism in the transsulphuration pathway, characterised by increased levels of homocysteine, methionine, S-adenosylmethionine, S-adenosylhomocysteine, and impaired synthesis of endogenous cystathionine and cysteine. Untreated CBSD patients are at high risk of developing osteoporosis, thromboembolism, ectopia lentis, mental retardation, marfanoid habitus, skeletal abnormalities, seizures, psychiatric disturbances, and cognitive dysfunction 1 . The current treatment for CBSD involves a restricted methionine diet and the use of betaine along with folate, vitamin B 12 and pyridoxine 2,3 . Oxidative stress is the result of an imbalance between the production of reactive species and the tissue's antioxidant defence. This can occur due to an increase in reactive species generation or a decrease in antioxidant levels 4 . Research has demonstrated that oxidative stress plays a role in the pathophysiology of several inborn errors of metabolism 5,6 . The available data suggest that the accumulation of toxic metabolites leads to an increase in free radicals and a decrease in antioxidant defences in these diseases 6 . The repair systems involve distinct actors, including antioxidant enzymes, DNA repair enzymes, chaperones, and proteases which require energy and mitochondrial products, like NAD + , to function properly 7 . NAD + may affect anti-oxidation and generation of oxidative stress via various pathways 8 . In this respect, NAD + deficiency has been associated with mitochondrial dysfunction 9,10 . Some studies suggest that NADH also has direct anti-oxidative effects 11,12 . Fibroblast growth factor 21 (FGF-21) modulates lipid and glucose homeostasis 13 . Growth differentiation factor 15 (GDF-15) is a TGF-β family protein produced in response to inflamm ation and oxidative stress to ensure tissue homeostasis 14 . FGF-21 and GDF-15 have emerged as biomarkers of mitochondrial dysfunction 15–17 . Although Molema et al. reported that the levels of these two biomarkers are increased in different inherited metabolic diseases 18 and are not diagnostic for mitochondrial diseases, there are several findings that FGF-21 and GDF-15 are associated with mitochondrial dysfunction 19–21 . Mitochondrial damage-associated molecular patterns (DAMPs), such as mitochondrial DNA and N-formyl peptides, are released after severe traumatic mitochondrial injury, affecting the respiratory chain and increasing oxidative stress 22 . The release of mitochondrial DAMPs correlates with altered mitochondrial metabolism and increased levels of reactive oxygen species (ROS), which can cause oxidative modification of DAMPs 23 . Research have suggested oxidative stress 4 and inflammation 24 as candidate pathogenic mechanisms in CBSD. This study aims to evaluate mitochondrial dysfunction and oxidative stress biomarkers in CBSD patients, which may aid in understanding the pathogenesis of CBSD and improving treatment. Results The study group comprised 23 patients (13 females) with CBSD and 23 healthy controls (15 females). Patients with CBSD had a mean age of 19.8 ± 8.2 (median: 16; range: 11–46) years, while the control group had a mean age of 16.7 ± 5.2 (median: 17; range: 2–27) years (Table 1). There was no statistically significant difference in age and sex distribution between the groups. The sum of NAD + and NADH levels was lower in the CBSD group (median: 42.1 pmol/µL; range: 40.5–45.8) compared to healthy controls (median: 44.6 pmol/µL; range: 42.1–45.5) (p = 0.000). The NAD + levels were reduced (mean: 16 ± 1.2 pmol/µL) and the NADH levels were increased (mean: 25.5 ± 2.2 pmol/µL) in CBSD group, relative to controls (29.0 ± 0.6 and 15 ± 0.6 pmol/µL) (p = 0.000/p = 0.000). The NAD + /NADH ratio was lower in the CBSD group (mean: 0.66 ± 0.10) than in the healthy group (mean: 1.89 ± 0.06) (p = 0.000) (Table 1). FGF-21 levels were increased in individuals with CBSD (median: 446 pg/ml; range: 96-2984) compared to healthy controls (median: 30 pg/ml; range: 17–150) (p = 0.000). Likewise, GDF-15 levels were higher in patients with CBSD (median: 153 pg/ml; range: 88–375) in respect to the control group (median: 78 pg/ml; range: 14–138) (p = 0.000) (Table 1). The mean mitochondrial DAMPs (MT-COX1/GAPDH; MT-ND1/RNaseP) levels in the CBSD group were found to be significantly elevated in the CBSD group (2.4 ± 0.4; 2.2 ± 0.3) in comparison to the control group (1.0 ± 0.1; 1.1 ± 0.07) (p = 0.000) (Table 1). In the CBSD group, total homocysteine levels (mean: 104.5 ± 66 µmol/L, range: 14–250) were significantly higher than homocysteine levels those in control group (mean: 8.9 ± 1.7 µmol/L) (p = 0.000) (Table 1). In patients with cystathionine beta synthase deficiency, a positive correlation was found between the total homocysteine level and both GDF-15 and NAD + /NADH levels. Furthermore, there was a negative correlation between total homocysteine levels and both total NAD + +NADH and NADH levels. There was a negative correlation between NAD + levels and NADH levels. Mitochondrial DAMPs levels were positively correlated with each other (Table 2 ). Table 1. Comparison of age and biochemical data between patient and control groups CBDS Group Control Group p Mean±SD Median Mean±SD Median Age (year) 19.8 ± 8.2 16 (11-46) 16.7 ± 5.2 17 (2-27) 0.415 m Gender n (%) Male 10 (43%) 8 (34%) 0.546 X² Female 13 (57%) 15 (66%) Total NAD + +NADH (pmol/μL) 42 ± 1.5 42.1 (40.5-45.8) 44 ± 1 44.6 (42.1-45.5) 0.000* m NAD + (pmol/μL) 16 ± 1.2 16.5 (14-19) 29 ± 0.6 29.2 (27-29.9) 0.000* t NADH (pmol/μL) 25.5 ± 2.2 25.6 (20-29) 15 ± 0.6 15.3 (14-16.3) 0.000* t NAD + /NADH 0.66 ± 0.1 0.63 (0.51-0.96) 1.89 ± 0.06 1.89 (1.74-1.98) 0.000* t FGF-21 (pg/ml) 891 ± 826 446 (96-2984) 52 ± 42 30 (17-150) 0.000* m GDF-15 (pg/ml) 178 ± 68 153 (88-375) 81 ± 35 78 (14-138) 0.000* m MT-COX1/GAPDH 2.4 ± 0.4 2.5 (1.7-3.1) 1 ± 0.1 1 (0.9-1.2) 0.000* t MT-ND1/RNaseP 2.2 ± 0.3 2 (1.5-2.9) 1.1 ± 0.07 1.1 (0.9-1.2) 0.000* t Total Homocysteine (µmol/L) 104.5 ± 66 84 (14-250) 8.9 ± 1.7 8.7 (6.2-13.7) 0.000* t m Mann-whitney u test / t test/ X² Chi-square test CBSD: Cystathionine beta-synthase deficiency, FGF-21: Fibroblast growth factor-21, GDF-15: Growth differantiation factor-15, NAD: Nicotinamide adenine dinucleotide, NADH: Nicotinamide adenine dinucleotide + hydrogen, SD: Standart deviation Table 2. Correlation coefficients of variables in Cystathionine β-synthase deficiency patients 1 2 3 4 5 6 7 8 9 10 1 Age 1 2 FGF-21 -0.315 1 3 GDF-15 0.322 -0.085 1 4 Total NAD+NADH -0.195 0.139 -0.326 1 5 NAD -0.007 -0.129 0.386 -0.253 1 6 NADH -0.129 0.169 -0.446* 0.829** -0.750** 1 7 NAD/NADH 0.069 -0.144 0.464* -0.569** 0.934** -0.928** 1 8 MT-COX1/ GAPDH -0.162 -0.196 0.042 -0.225 0.262 -0.305 0.273 1 9 MT-ND1/ RNaseP -0.111 -0.055 -0.044 0.060 0.155 -0.048 0.087 0.722** 1 10 Total Homocysteine 0.184 -0.030 0.514* -0.449* 0.371 -0.521* 0.490* 0.147 -0.029 1 *. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed). FGF-21:Fibroblast growth factor-21, GADPH: Glyceraldehyde-3-phosphate dehydrogenase, GDF-15: Growth differantiation factor-15, MT-COX1: Nicotinamide adenine dinucleotide + hydrogen, MT-ND1: Mitochondrially encoded NADH-ubiquinone oxidoreductase chain 1, NAD: Nicotinamide adenine dinucleotide, NADH: Nicotinamide adenine dinucleotide + hydrogen, RNaseP: Ribonuclease P Discussion Though the pathophysiology of CBSD is not well understood, it has been proposed that Hcy excess or cysteine deficiency, rather than methionine accumulation, is more likely to be involved in the pathogenesis of the disease 1 . On the other hand, oxidative stress has been suggested to play an important role in the pathophysiology of homocystinuria 5,25–27 . This was also rooted to researches that reported an association between Hcy and oxidative stress 28,29 . NAD + and its reduced form NADH are key components in redox reactions 30 . The intracellular NAD + concentration is directly proportional to the redox potential. A decreased amount of mitochondrial signalling molecules, particularly NAD + , has been postulated to be responsible for compromised mitochondrial function and repair mechanisms in chronic disease states 9,10 . In our study, the NAD + level and NAD + /NADH ratio were found to be decreased in CBSD patients. In contrast, healthy controls showed normal NAD + and NAD + /NADH levels. These findings suggest the presence of oxidative stress in CBSD patients. Another study similarly showed that Hcy induces oxidative stress by upregulating protease-activated receptor-4 (PAR-4) and enhancing iNOS and NAD(P)H oxidase expression 31 . FGF-21 and GDF-15 have been identified as reliable tools for the diagnosis of mitochondrial diseases 20 . The fact that FGF-21 and GDF-15 levels were statistically significantly different in patients with CBSD compared to healthy controls suggests that these patients suffered oxidative damage and mitochondrial dysfunction similar to mitochondrial diseases. The levels of these two biomarkers have been documented to be increased in various inherited metabolic diseases 18,19,32 . Dysfunctional mitochondria may release DAMPs, which can trigger inflammasomes and a systemic inflammatory response 33,34 . Levels of mitochondrial DAMPs were found to be higher in CBSD patients than in healthy subjects. Also, levels of FGF-21, GDF-15, NAD + , NADH and NAD + /NADH, which are markers of oxidative stress and mitochondrial dysfunction, differed between CBSD patients and healthy subjects, suggesting the presence of oxidative stress and mitochondrial dysfunction in CBSD patients. Previous studies have reported that oxidative damage can occur in hepatocytes of CBSD patients and in animal experiments with induced hyperhomocystinemia 29,35 . Our study suggests that patients with CBSD experience mitochondrial dysfunction besides endoplasmic reticulum damage due to oxidative stress. Accumulation of the pro-oxidant Hcy 29 and decreased hepatic antioxidant capacity due to reduced activity of antioxidant enzymes, including glutathione peroxidase, are possible causes of increased mitochondrial and oxidative damage in homocystinuria 36 . Vanzin et al discovered a significant negative correlation between antioxidant content and Hcy levels 37 . They also observed a positive correlation between malondialdehyde levels and Hcy levels, implying a possible mechanistic role for Hcy in the oxidative damage observed in homocystinuria. The positive correlation between total homocysteine level and GDF-15, as well NAD + /NADH levels and negative correlation with total NAD + +NADH and NADH levels of patients with CBSD in our study, were consistent with these previous findings. Biomarkers of CBSD patients shows an increase in oxidative damage and a reduction in antioxidant defences, likely due to the generation of reactive species induced by Hcy 27 . The alterations in NAD + , FGF-21, GDF-15 levels, and NAD + /NADH ratio in patients with homocystinuria suggest that oxidative damage coexists with mitochondrial dysfunction in CBSD patients. Vanzin et al. demonstrated that patients with homocystinuria experience protein and lipid oxidative damage, which can be ameliorated through therapy 27,38 . In this respect, assessment of oxidative damage and addition of anti-oxidant therapy together with mitochondrial support may have additional benefits in reducing long-term morbidity in CBSD patients. Reproducibility is important, especially in rare diseases, and more studies on this subject are needed to support these results. Methods The study group comprised 23 patients with a diagnosis of CBSD who were in follow-up, between 1986 and 2023. Patients with CBSD were initially diagnosed by enzyme and/or molecular tests. All patients with a diagnosis of CBSD who were in follow-up, were involved in the study. The control group was composed of age- and gender-matched 23 healthy volunteers. An ethical approval (file number; 2023/1637) was obtained from Istanbul Faculty of Medicine Ethics Committee before the study and written consent was obtained from each patient or guardian. This study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. We analysed serum levels of NAD + and NADH by fluorometric assay, FGF-21 and GDF-15 by ELISA, mitochondrial DAMPs by real time qRT-PCR, total homocysteine levels in plasma by enzymatic test and compared the results in CBSD group with healthy controls. Descriptive statistics included mean, standard deviation, median, minimum and maximum. Shapiro-Wilk test was used to analyse whether or not the variables were normally distributed. The independent non-parametric data were analysed by Mann-Whitney U test. Independent Samples t-test was performed to analyse independent parametric data. Chi-square test was used to analyse qualitative variables. Correlation analyses were made by Pearson and Spearman’s tests. A p < 0.05 is considered to be statistically significant. IBM SPSS Statistics (Version 26.0. Armonk, NY: IBM Corp.) was used for the statistical analysis. Declarations Author Contributions: Mehmet Cihan Balcı: Conceptualization, Methodology, Data Curation, Formal analysis, Writing - Original Draft Asuman Gedikbasi: Resources, Visualization Sevde Kahraman: Resources, Data Curation Suzin Tatoryan: Investigation Sebnem Tekin Neijmann: Resources, Investigation, Meryem Karaca: Conceptualization, Methodology Fatmahan Atalar: Visualization Gulden Gokcay: Conceptualization, Methodology, Writing - Review & Editing, Visualization Data availability: The data that support the findings of this study are available from the corresponding author upon reasonable request. Division of Nutrition and Metabolism, Istanbul Faculty of Medicine, Children’s Hospital, Istanbul University, Istanbul, Türkiye. Ethical approval: An ethical approval (file number; 2023/1637) was obtained from Istanbul Faculty of Medicine Ethics Committee before the study and written informed consent was obtained from each patient or guardian. The authors declare no competing interests. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors. This research was funded by Istanbul University Scientific Research Projects Unit (Project number:TSA-2022-39178). The funding source had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. References Mudd, S. H. et al. The natural history of homocystinuria due to cystathionine beta-synthase deficiency. Am J Hum Genet 37 , 1-31 (1985). Morris, A. A. et al. Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. J Inherit Metab Dis 40 , 49-74 (2017). https://doi.org:10.1007/s10545-016-9979-0 Rao, T. N., Radhakrishna, K., Mohana Rao, T. S., Guruprasad, P. & Ahmed, K. Homocystinuria due to cystathionine beta synthase deficiency. 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Cite Share Download PDF Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 14 Oct, 2024 Reviews received at journal 11 Oct, 2024 Reviewers agreed at journal 27 Sep, 2024 Reviews received at journal 19 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviewers invited by journal 26 Jul, 2024 Editor assigned by journal 26 Jul, 2024 Editor invited by journal 16 Jul, 2024 Submission checks completed at journal 15 Jul, 2024 First submitted to journal 12 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4732434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":336876057,"identity":"b7da0cba-debe-4f1a-ae2f-4ab5ab05c97b","order_by":0,"name":"Mehmet Cihan Balci","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYFACHjCZwMDOwHDgYwOIzdh4gLCWBCBkZmA4OLOBQQKopYF4Lcy8YC1A6/Bp0G3vPbrh4w+7PP5m5oeHbXfY1Om2HwbaUmMTjUuL2ZlzaTdnJCQXSxxmMziceyZNwuxMIlDLsbTcBlxabuSY3eZJOJDYcJiH4XBu22EJMyD7AGPDYdxa7r8xu/0HqGU+SIslSMv5hwS03OAxu80A1LIBpIURpOUGIVvO5Jjd7ElLLjYE+uVgb1ua5LYbQFsS8Pnl+BmzGz9s7PLkjjc//vCzzYbf7Hz6wwcfamxwasEBEkhTPgpGwSgYBaMADQAAuLFpqleizTcAAAAASUVORK5CYII=","orcid":"","institution":"Istanbul University","correspondingAuthor":true,"prefix":"","firstName":"Mehmet","middleName":"Cihan","lastName":"Balci","suffix":""},{"id":336876058,"identity":"37878ee1-de66-4f61-ad5e-3fbed73e4256","order_by":1,"name":"Asuman Gedikbasi","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Asuman","middleName":"","lastName":"Gedikbasi","suffix":""},{"id":336876059,"identity":"f4b513a0-e332-4ba2-ad75-9de683cc079f","order_by":2,"name":"Sukru Anil Dogan","email":"","orcid":"","institution":"Boğaziçi University","correspondingAuthor":false,"prefix":"","firstName":"Sukru","middleName":"Anil","lastName":"Dogan","suffix":""},{"id":336876060,"identity":"c799fde3-269c-48fb-9d6a-767b943784b2","order_by":3,"name":"Sevde Kahraman","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Sevde","middleName":"","lastName":"Kahraman","suffix":""},{"id":336876061,"identity":"cd0faa5e-2fef-4b67-a4d5-0bf618fffa00","order_by":4,"name":"Suzin Tatoryan","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Suzin","middleName":"","lastName":"Tatoryan","suffix":""},{"id":336876062,"identity":"db2b53de-a8b6-485a-9ee8-d72c13ab80e0","order_by":5,"name":"Sebnem Tekin Neijmann","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Sebnem","middleName":"Tekin","lastName":"Neijmann","suffix":""},{"id":336876063,"identity":"5440e096-6553-4270-b81f-37c617ea2743","order_by":6,"name":"Meryem Karaca","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Meryem","middleName":"","lastName":"Karaca","suffix":""},{"id":336876064,"identity":"3e3894a2-e365-4cb7-ad15-cd103aa42206","order_by":7,"name":"Fatmahan Atalar","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Fatmahan","middleName":"","lastName":"Atalar","suffix":""},{"id":336876065,"identity":"8168cd47-51dd-4fbd-99b9-8e4df9a82cef","order_by":8,"name":"Gulden Gokcay","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Gulden","middleName":"","lastName":"Gokcay","suffix":""}],"badges":[],"createdAt":"2024-07-12 19:55:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4732434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4732434/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-80273-w","type":"published","date":"2024-11-21T15:58:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69835543,"identity":"2e92d7a4-9a05-4ce2-9981-3bf698ae9063","added_by":"auto","created_at":"2024-11-25 16:13:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":519709,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4732434/v1/5fa6e35e-6ae5-4d6b-90dc-6d78e0843f17.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOxidative Damage and Mitochondrial Dysfunction in Cystathionine Beta-synthase Deficiency \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCystathionine beta-synthase deficiency (CBSD) (OMIM 236200) is the most prevalent inherited disorder of homocysteine (Hcy) metabolism in the transsulphuration pathway, characterised by increased levels of homocysteine, methionine, S-adenosylmethionine, S-adenosylhomocysteine, and impaired synthesis of endogenous cystathionine and cysteine. Untreated CBSD patients are at high risk of developing osteoporosis, thromboembolism, ectopia lentis, mental retardation, marfanoid habitus, skeletal abnormalities, seizures, psychiatric disturbances, and cognitive dysfunction \u003csup\u003e1\u003c/sup\u003e. The current treatment for CBSD involves a restricted methionine diet and the use of betaine along with folate, vitamin B\u003csub\u003e12\u003c/sub\u003e and pyridoxine \u003csup\u003e2,3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOxidative stress is the result of an imbalance between the production of reactive species and the tissue's antioxidant defence. This can occur due to an increase in reactive species generation or a decrease in antioxidant levels \u003csup\u003e4\u003c/sup\u003e. Research has demonstrated that oxidative stress plays a role in the pathophysiology of several inborn errors of metabolism \u003csup\u003e5,6\u003c/sup\u003e. The available data suggest that the accumulation of toxic metabolites leads to an increase in free radicals and a decrease in antioxidant defences in these diseases \u003csup\u003e6\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe repair systems involve distinct actors, including antioxidant enzymes, DNA repair enzymes, chaperones, and proteases which require energy and mitochondrial products, like NAD\u003csup\u003e+\u003c/sup\u003e, to function properly \u003csup\u003e7\u003c/sup\u003e. NAD\u003csup\u003e+\u003c/sup\u003e may affect anti-oxidation and generation of oxidative stress via various pathways \u003csup\u003e8\u003c/sup\u003e. In this respect, NAD\u003csup\u003e+\u003c/sup\u003e deficiency has been associated with mitochondrial dysfunction \u003csup\u003e9,10\u003c/sup\u003e. Some studies suggest that NADH also has direct anti-oxidative effects \u003csup\u003e11,12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFibroblast growth factor 21 (FGF-21) modulates lipid and glucose homeostasis \u003csup\u003e13\u003c/sup\u003e. Growth differentiation factor 15 (GDF-15) is a TGF-β family protein produced in response to inflamm\u003c/p\u003e \u003cp\u003eation and oxidative stress to ensure tissue homeostasis \u003csup\u003e14\u003c/sup\u003e. FGF-21 and GDF-15 have emerged as biomarkers of mitochondrial dysfunction \u003csup\u003e15\u0026ndash;17\u003c/sup\u003e. Although Molema et al. reported that the levels of these two biomarkers are increased in different inherited metabolic diseases \u003csup\u003e18\u003c/sup\u003e and are not diagnostic for mitochondrial diseases, there are several findings that FGF-21 and GDF-15 are associated with mitochondrial dysfunction \u003csup\u003e19\u0026ndash;21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMitochondrial damage-associated molecular patterns (DAMPs), such as mitochondrial DNA and N-formyl peptides, are released after severe traumatic mitochondrial injury, affecting the respiratory chain and increasing oxidative stress \u003csup\u003e22\u003c/sup\u003e. The release of mitochondrial DAMPs correlates with altered mitochondrial metabolism and increased levels of reactive oxygen species (ROS), which can cause oxidative modification of DAMPs \u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eResearch have suggested oxidative stress \u003csup\u003e4\u003c/sup\u003e and inflammation \u003csup\u003e24\u003c/sup\u003e as candidate pathogenic mechanisms in CBSD. This study aims to evaluate mitochondrial dysfunction and oxidative stress biomarkers in CBSD patients, which may aid in understanding the pathogenesis of CBSD and improving treatment.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe study group comprised 23 patients (13 females) with CBSD and 23 healthy controls (15 females). Patients with CBSD had a mean age of 19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2 (median: 16; range: 11\u0026ndash;46) years, while the control group had a mean age of 16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 (median: 17; range: 2\u0026ndash;27) years (Table\u0026nbsp;1). There was no statistically significant difference in age and sex distribution between the groups.\u003c/p\u003e\n\u003cp\u003eThe sum of NAD\u003csup\u003e+\u003c/sup\u003e and NADH levels was lower in the CBSD group (median: 42.1 pmol/\u0026micro;L; range: 40.5\u0026ndash;45.8) compared to healthy controls (median: 44.6 pmol/\u0026micro;L; range: 42.1\u0026ndash;45.5) (p\u0026thinsp;=\u0026thinsp;0.000). The NAD\u003csup\u003e+\u003c/sup\u003e levels were reduced (mean: 16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 pmol/\u0026micro;L) and the NADH levels were increased (mean: 25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 pmol/\u0026micro;L) in CBSD group, relative to controls (29.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 and 15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 pmol/\u0026micro;L) (p\u0026thinsp;=\u0026thinsp;0.000/p\u0026thinsp;=\u0026thinsp;0.000). The NAD\u003csup\u003e+\u003c/sup\u003e/NADH ratio was lower in the CBSD group (mean: 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10) than in the healthy group (mean: 1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06) (p\u0026thinsp;=\u0026thinsp;0.000) (Table\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eFGF-21 levels were increased in individuals with CBSD (median: 446 pg/ml; range: 96-2984) compared to healthy controls (median: 30 pg/ml; range: 17\u0026ndash;150) (p\u0026thinsp;=\u0026thinsp;0.000). Likewise, GDF-15 levels were higher in patients with CBSD (median: 153 pg/ml; range: 88\u0026ndash;375) in respect to the control group (median: 78 pg/ml; range: 14\u0026ndash;138) (p\u0026thinsp;=\u0026thinsp;0.000) (Table\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eThe mean mitochondrial DAMPs (MT-COX1/GAPDH; MT-ND1/RNaseP) levels in the CBSD group were found to be significantly elevated in the CBSD group (2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4; 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3) in comparison to the control group (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1; 1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) (p\u0026thinsp;=\u0026thinsp;0.000) (Table\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eIn the CBSD group, total homocysteine levels (mean: 104.5\u0026thinsp;\u0026plusmn;\u0026thinsp;66 \u0026micro;mol/L, range: 14\u0026ndash;250) were significantly higher than homocysteine levels those in control group (mean: 8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 \u0026micro;mol/L) (p\u0026thinsp;=\u0026thinsp;0.000) (Table\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eIn patients with cystathionine beta synthase deficiency, a positive correlation was found between the total homocysteine level and both GDF-15 and NAD\u003csup\u003e+\u003c/sup\u003e/NADH levels. Furthermore, there was a negative correlation between total homocysteine levels and both total NAD\u003csup\u003e+\u003c/sup\u003e+NADH and NADH levels. There was a negative correlation between NAD\u003csup\u003e+\u003c/sup\u003e levels and NADH levels. Mitochondrial DAMPs levels were positively correlated with each other (Table\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"640\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"89.375%\" colspan=\"10\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eComparison of age and biochemical data between patient and control groups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.65625%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"2.96875%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.65625%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.90625%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"31.71875%\" colspan=\"4\" valign=\"bottom\"\u003e\n \u003cp\u003eCBDS Group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.09375%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003eControl Group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.625%\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.532286212914485%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"15.18324607329843%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eMean\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.418848167539267%\" valign=\"top\"\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.31064572425829%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eMean\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.418848167539267%\" valign=\"top\"\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\"\u003e\n \u003cp\u003eAge (year)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e19.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e8.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e16 (11-46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e16.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e17 (2-27)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.415\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.636505460218409%\" rowspan=\"2\"\u003e\n \u003cp\u003eGender \u0026nbsp;n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.88455538221529%\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.57254290171607%\" colspan=\"3\" valign=\"bottom\"\u003e\n \u003cp\u003e10 (43%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.792511700468019%\" colspan=\"3\"\u003e\n \u003cp\u003e8 (34%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.546\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003csup\u003eX\u0026sup2;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.089430894308943%\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.682926829268293%\" colspan=\"3\" valign=\"bottom\"\u003e\n \u003cp\u003e13 (57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.78048780487805%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" colspan=\"3\"\u003e\n \u003cp\u003e15 (66%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.78048780487805%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\"\u003e\n \u003cp\u003eTotal NAD\u003csup\u003e+\u003c/sup\u003e+NADH (pmol/\u0026mu;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e42.1 (40.5-45.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e44.6 (42.1-45.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\"\u003e\n \u003cp\u003eNAD\u003csup\u003e+\u0026nbsp;\u003c/sup\u003e(pmol/\u0026mu;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e16.5 (14-19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e29.2 (27-29.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003et\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\"\u003e\n \u003cp\u003eNADH (pmol/\u0026mu;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e25.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e25.6 (20-29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e15.3 (14-16.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003et\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e/NADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e0.63 (0.51-0.96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e1.89 (1.74-1.98)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003et\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\"\u003e\n \u003cp\u003eFGF-21 (pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\" valign=\"bottom\"\u003e\n \u003cp\u003e891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e826\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e446 (96-2984)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\" valign=\"bottom\"\u003e\n \u003cp\u003e30 (17-150)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\"\u003e\n \u003cp\u003eGDF-15 (pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e153 (88-375)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e78 (14-138)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eMT-COX1/GAPDH\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e2.5 (1.7-3.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e1 (0.9-1.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003et\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eMT-ND1/RNaseP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e2 (1.5-2.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e1.1 (0.9-1.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003et\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.521060842433698%\" colspan=\"2\"\u003e\n \u003cp\u003eTotal Homocysteine (\u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.5522620904836195%\"\u003e\n \u003cp\u003e104.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.5241809672386895%\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e84 (14-250)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.49609984399376%\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.14820592823713%\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.252730109204368%\"\u003e\n \u003cp\u003e8.7 (6.2-13.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.644305772230889%\" valign=\"top\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.9641185647425896%\"\u003e\n \u003cp\u003e\u003csup\u003et\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"12\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003csup\u003em\u0026nbsp;\u003c/sup\u003eMann-whitney u test / \u003csup\u003et\u003c/sup\u003etest/\u003csup\u003eX\u0026sup2;\u0026nbsp;\u003c/sup\u003eChi-square test\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCBSD: Cystathionine beta-synthase deficiency, FGF-21: Fibroblast growth factor-21, GDF-15: Growth differantiation factor-15, NAD: Nicotinamide adenine dinucleotide, NADH: Nicotinamide adenine dinucleotide + hydrogen, SD: Standart deviation \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"644\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"13\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eCorrelation coefficients of variables in Cystathionine \u0026beta;-synthase deficiency patients\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eFGF-21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.315\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eGDF-15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eTotal NAD+NADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.386\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e-0.253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eNADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.446*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e0.829**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e-0.750**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eNAD/NADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.464*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e-0.569**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e0.934**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\n \u003cp\u003e-0.928**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eMT-COX1/ \u0026nbsp;GAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e-0.225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e0.262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\n \u003cp\u003e-0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eMT-ND1/ RNaseP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e0.155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\n \u003cp\u003e-0.048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.087\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\n \u003cp\u003e0.722**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"2.950310559006211%\" valign=\"bottom\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.012422360248447%\"\u003e\n \u003cp\u003eTotal Homocysteine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.514*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e-0.449*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.850931677018634%\"\u003e\n \u003cp\u003e0.371\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.006211180124224%\"\u003e\n \u003cp\u003e-0.521*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e0.490*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\"\u003e\n \u003cp\u003e0.147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e-0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.298136645962733%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"1.0869565217391304%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"13\"\u003e\n \u003cp\u003e*. Correlation is significant at the 0.05 level (2-tailed).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"13\"\u003e\n \u003cp\u003e**. Correlation is significant at the 0.01 level (2-tailed).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"13\"\u003e\n \u003cp\u003eFGF-21:Fibroblast growth factor-21, GADPH: Glyceraldehyde-3-phosphate dehydrogenase, GDF-15: Growth differantiation factor-15, MT-COX1: Nicotinamide adenine dinucleotide + hydrogen, MT-ND1: Mitochondrially encoded NADH-ubiquinone oxidoreductase chain 1, NAD: Nicotinamide adenine dinucleotide, NADH: Nicotinamide adenine dinucleotide + hydrogen, RNaseP: Ribonuclease P \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThough the pathophysiology of CBSD is not well understood, it has been proposed that Hcy excess or cysteine deficiency, rather than methionine accumulation, is more likely to be involved in the pathogenesis of the disease \u003csup\u003e1\u003c/sup\u003e. On the other hand, oxidative stress has been suggested to play an important role in the pathophysiology of homocystinuria \u003csup\u003e5,25\u0026ndash;27\u003c/sup\u003e. This was also rooted to researches that reported an association between Hcy and oxidative stress \u003csup\u003e28,29\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e and its reduced form NADH are key components in redox reactions \u003csup\u003e30\u003c/sup\u003e. The intracellular NAD\u003csup\u003e+\u003c/sup\u003e concentration is directly proportional to the redox potential. A decreased amount of mitochondrial signalling molecules, particularly NAD\u003csup\u003e+\u003c/sup\u003e, has been postulated to be responsible for compromised mitochondrial function and repair mechanisms in chronic disease states \u003csup\u003e9,10\u003c/sup\u003e. In our study, the NAD\u003csup\u003e+\u003c/sup\u003e level and NAD\u003csup\u003e+\u003c/sup\u003e/NADH ratio were found to be decreased in CBSD patients. In contrast, healthy controls showed normal NAD\u003csup\u003e+\u003c/sup\u003e and NAD\u003csup\u003e+\u003c/sup\u003e/NADH levels. These findings suggest the presence of oxidative stress in CBSD patients. Another study similarly showed that Hcy induces oxidative stress by upregulating protease-activated receptor-4 (PAR-4) and enhancing iNOS and NAD(P)H oxidase expression \u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFGF-21 and GDF-15 have been identified as reliable tools for the diagnosis of mitochondrial diseases \u003csup\u003e20\u003c/sup\u003e. The fact that FGF-21 and GDF-15 levels were statistically significantly different in patients with CBSD compared to healthy controls suggests that these patients suffered oxidative damage and mitochondrial dysfunction similar to mitochondrial diseases. The levels of these two biomarkers have been documented to be increased in various inherited metabolic diseases \u003csup\u003e18,19,32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDysfunctional mitochondria may release DAMPs, which can trigger inflammasomes and a systemic inflammatory response \u003csup\u003e33,34\u003c/sup\u003e. Levels of mitochondrial DAMPs were found to be higher in CBSD patients than in healthy subjects. Also, levels of FGF-21, GDF-15, NAD\u003csup\u003e+\u003c/sup\u003e, NADH and NAD\u003csup\u003e+\u003c/sup\u003e/NADH, which are markers of oxidative stress and mitochondrial dysfunction, differed between CBSD patients and healthy subjects, suggesting the presence of oxidative stress and mitochondrial dysfunction in CBSD patients.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that oxidative damage can occur in hepatocytes of CBSD patients and in animal experiments with induced hyperhomocystinemia \u003csup\u003e29,35\u003c/sup\u003e. Our study suggests that patients with CBSD experience mitochondrial dysfunction besides endoplasmic reticulum damage due to oxidative stress.\u003c/p\u003e \u003cp\u003eAccumulation of the pro-oxidant Hcy \u003csup\u003e29\u003c/sup\u003e and decreased hepatic antioxidant capacity due to reduced activity of antioxidant enzymes, including glutathione peroxidase, are possible causes of increased mitochondrial and oxidative damage in homocystinuria \u003csup\u003e36\u003c/sup\u003e. Vanzin et al discovered a significant negative correlation between antioxidant content and Hcy levels \u003csup\u003e37\u003c/sup\u003e. They also observed a positive correlation between malondialdehyde levels and Hcy levels, implying a possible mechanistic role for Hcy in the oxidative damage observed in homocystinuria. The positive correlation between total homocysteine level and GDF-15, as well NAD\u003csup\u003e+\u003c/sup\u003e/NADH levels and negative correlation with total NAD\u003csup\u003e+\u003c/sup\u003e+NADH and NADH levels of patients with CBSD in our study, were consistent with these previous findings.\u003c/p\u003e \u003cp\u003eBiomarkers of CBSD patients shows an increase in oxidative damage and a reduction in antioxidant defences, likely due to the generation of reactive species induced by Hcy \u003csup\u003e27\u003c/sup\u003e. The alterations in NAD\u003csup\u003e+\u003c/sup\u003e, FGF-21, GDF-15 levels, and NAD\u003csup\u003e+\u003c/sup\u003e/NADH ratio in patients with homocystinuria suggest that oxidative damage coexists with mitochondrial dysfunction in CBSD patients. Vanzin et al. demonstrated that patients with homocystinuria experience protein and lipid oxidative damage, which can be ameliorated through therapy \u003csup\u003e27,38\u003c/sup\u003e. In this respect, assessment of oxidative damage and addition of anti-oxidant therapy together with mitochondrial support may have additional benefits in reducing long-term morbidity in CBSD patients. Reproducibility is important, especially in rare diseases, and more studies on this subject are needed to support these results.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe study group comprised 23 patients with a diagnosis of CBSD who were in follow-up, between 1986 and 2023. Patients with CBSD were initially diagnosed by enzyme and/or molecular tests. All patients with a diagnosis of CBSD who were in follow-up, were involved in the study. The control group was composed of age- and gender-matched 23 healthy volunteers. An ethical approval (file number; 2023/1637) was obtained from Istanbul Faculty of Medicine Ethics Committee before the study and written consent was obtained from each patient or guardian. This study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.\u003c/p\u003e \u003cp\u003eWe analysed serum levels of NAD\u003csup\u003e+\u003c/sup\u003e and NADH by fluorometric assay, FGF-21 and GDF-15 by ELISA, mitochondrial DAMPs by real time qRT-PCR, total homocysteine levels in plasma by enzymatic test and compared the results in CBSD group with healthy controls.\u003c/p\u003e \u003cp\u003eDescriptive statistics included mean, standard deviation, median, minimum and maximum. Shapiro-Wilk test was used to analyse whether or not the variables were normally distributed. The independent non-parametric data were analysed by Mann-Whitney U test. Independent Samples t-test was performed to analyse independent parametric data. Chi-square test was used to analyse qualitative variables. Correlation analyses were made by Pearson and Spearman\u0026rsquo;s tests. A p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered to be statistically significant. IBM SPSS Statistics (Version 26.0. Armonk, NY: IBM Corp.) was used for the statistical analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMehmet Cihan Balcı:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Data Curation, Formal analysis, Writing - Original Draft \u003cstrong\u003eAsuman Gedikbasi:\u003c/strong\u003e Resources, Visualization \u003cstrong\u003eSevde Kahraman:\u003c/strong\u003e Resources, Data Curation \u003cstrong\u003eSuzin Tatoryan:\u003c/strong\u003e Investigation \u003cstrong\u003eSebnem Tekin Neijmann:\u003c/strong\u003e Resources, Investigation, \u003cstrong\u003eMeryem Karaca:\u003c/strong\u003e Conceptualization, Methodology \u003cstrong\u003eFatmahan Atalar:\u003c/strong\u003e Visualization \u003cstrong\u003eGulden Gokcay:\u003c/strong\u003e Conceptualization, Methodology, Writing - Review \u0026amp; Editing, Visualization\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability:\u003c/strong\u003e The data that support the findings of this study are available from the corresponding author upon reasonable request. Division of Nutrition and Metabolism, Istanbul Faculty of Medicine, Children\u0026rsquo;s Hospital, Istanbul University, Istanbul, T\u0026uuml;rkiye.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthical approval:\u003c/strong\u003e An ethical approval (file number; 2023/1637) was obtained from Istanbul Faculty of Medicine Ethics Committee before the study and written informed consent was obtained from each patient or guardian.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare no competing interests. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors. This research was funded by Istanbul University Scientific Research Projects Unit (Project number:TSA-2022-39178). The funding source had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMudd, S. H.\u003cem\u003e et al.\u003c/em\u003e The natural history of homocystinuria due to cystathionine beta-synthase deficiency. \u003cem\u003eAm J Hum Genet\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 1-31 (1985).\u003c/li\u003e\n\u003cli\u003eMorris, A. A.\u003cem\u003e et al.\u003c/em\u003e Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. \u003cem\u003eJ Inherit Metab Dis\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 49-74 (2017). https://doi.org:10.1007/s10545-016-9979-0\u003c/li\u003e\n\u003cli\u003eRao, T. N., Radhakrishna, K., Mohana Rao, T. S., Guruprasad, P. \u0026amp; Ahmed, K. 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S.\u003cem\u003e et al.\u003c/em\u003e Lipid, Oxidative and Inflammatory Profile and Alterations in the Enzymes Paraoxonase and Butyrylcholinesterase in Plasma of Patients with Homocystinuria Due CBS Deficiency: The Vitamin B12 and Folic Acid Importance. \u003cem\u003eCell Mol Neurobiol\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 899-911 (2015). https://doi.org:10.1007/s10571-015-0185-7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cystathionine Beta Synthase Deficiency, Fibroblast Growth Factor 21, Growth Differentiation Factor 15, Mitochondria, Oxidative Stress","lastPublishedDoi":"10.21203/rs.3.rs-4732434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4732434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCystathionine beta-synthase deficiency (CBSD) is the most prevalent inherited disorder of homocysteine metabolism in the transsulphuration pathway. Research have suggested oxidative stress and inflammation as candidate pathogenic mechanisms in CBSD. This study aims to evaluate mitochondrial dysfunction and oxidative stress biomarkers in cystathionine beta-synthase deficiency (CBSD) patients, which may aid in understanding the pathogenesis of CBSD and improving treatment. The study group comprised 23 patients with a diagnosis of CBSD and healthy controls. We analysed serum levels of NAD\u003csup\u003e+\u003c/sup\u003e and NADH by fluorometric assay, FGF-21 and GDF-15 by ELISA, mitochondrial DAMPs by real time qRT-PCR, total homocysteine levels in plasma by enzymatic test and compared the results in CBSD group with healthy controls. In patient group, a positive correlation was found between the total homocysteine level and both GDF-15 and NAD\u003csup\u003e+\u003c/sup\u003e/NADH levels. Furthermore, there was a negative correlation between total homocysteine levels and both total NAD\u003csup\u003e+\u003c/sup\u003e+NADH and NADH levels. The alterations in NAD\u003csup\u003e+\u003c/sup\u003e, FGF-21, GDF-15 levels, and NAD\u003csup\u003e+\u003c/sup\u003e/NADH ratio in patients suggest that oxidative damage coexists with mitochondrial dysfunction in CBSD. Assessment of oxidative damage and addition of anti-oxidant therapy together with mitochondrial support may have additional benefits in reducing long-term morbidity in CBSD patients.\u003c/p\u003e","manuscriptTitle":"Oxidative Damage and Mitochondrial Dysfunction in Cystathionine Beta-synthase Deficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-07 15:26:04","doi":"10.21203/rs.3.rs-4732434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-14T04:42:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-11T10:16:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107868787885675599953526113042008104516","date":"2024-09-27T15:27:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-19T19:17:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51859146409766669848945034386446842414","date":"2024-08-08T05:51:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80499072038020439164545463359150476152","date":"2024-07-31T18:56:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-26T14:50:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-26T14:49:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-16T05:45:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-15T04:15:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-12T19:54:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52c79567-c497-412f-885b-2e3de4c0a11b","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:08:21+00:00","versionOfRecord":{"articleIdentity":"rs-4732434","link":"https://doi.org/10.1038/s41598-024-80273-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-21 15:58:12","publishedOnDateReadable":"November 21st, 2024"},"versionCreatedAt":"2024-08-07 15:26:04","video":"","vorDoi":"10.1038/s41598-024-80273-w","vorDoiUrl":"https://doi.org/10.1038/s41598-024-80273-w","workflowStages":[]},"version":"v1","identity":"rs-4732434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4732434","identity":"rs-4732434","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}