Serum β-Hydroxybutyrate decline: a common pathological factor for metabolic dysfunction-associated steatotic liver disease and cognitive impairment

Serum β-Hydroxybutyrate decline: a common pathological factor for metabolic dysfunction-associated steatotic liver disease and cognitive impairment | 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 Research Article Serum β-Hydroxybutyrate decline: a common pathological factor for metabolic dysfunction-associated steatotic liver disease and cognitive impairment Lijuan Nie, Xinyi Yang, Ying Wang, Jing Sun, Yaohuan Sun, Yujuan Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5403137/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 Background: Recently, cognitive impairment has emerged as a consequential aspect of metabolic dysfunction-associated steatotic liver disease (MASLD), prompting speculation that diminished β-Hydroxybutyrate (BHB) production might be intricately involved in its pathogenesis, along with that of cognitive decline. However, the intricate interplay between the circulating BHB levels and these conditions remains unclear. Methods: A BHB ELISA assay kit was used to quantify serum BHB concentrations in both healthy individuals and patients with MASLD or cognitive impairment. Pearson’s correlation analysis assessed associations between BHB levels and pertinent clinical parameters. The diagnostic utility of serum BHB levels in identifying MASLD and cognitive impairment was assessed using Receiver Operating Characteristic (ROC) curves. Results: The findings revealed a stark contrast: patients with MASLD and cognitive impairment exhibited notably lower serum BHB levels than healthy individuals. Furthermore, BHB levels were inversely associated with BMI (r = -0.407, P < 0.001), AST (r = -0.233, P = 0.044), TC (r = -0.266, P = 0.021), LDL-C (r = -0.236, P = 0.046), and HbA1c (r = -0.416, P < 0.001), while demonstrating a positively relationship with cognitive performance, as indicated by Mini-Mental State Examination (r = 0.434, P < 0.001) and Montreal Cognitive Assessment scores (r = 0.433, P < 0.001). Notably, ROC curve analysis underscored potential diagnostic significance of serum BHB in both MASLD and cognitive impairment. Intriguingly, supplementary research illuminated a negative correlation between serum BHB levels and age among healthy individuals (r = -0.775, P < 0.001). Conclusion: This work implies that a decrease on circulating BHB levels may lead to the occurrence of MASLD and cognitive impairment. Metabolic dysfunction-associated steatotic liver disease Cognitive impairment Age β-Hydroxybutyrate Biomarker Figures Figure 1 Figure 2 Figure 3 Background Amidst the rapid shifts in dietary patterns and lifestyles, the incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing annually. Currently, the global incidence of MASLD is 25% and is expected to reach 55.7% by 2040 [ 1 , 2 ]. The clinical burden of MASLD extends beyond liver-related morbidity or mortality and encompasses a range of extrahepatic complications [ 3 ]. Recently, cognitive impairment has been identified as a complication of MASLD [ 4 , 5 ]. Patients with MASLD experience memory impairment and decreased attention, which can significantly impede their daily functioning and overall quality of life [ 6 , 7 ]. Unfortunately, the precise mechanisms underlying MASLD-induced cognitive impairment remain elusive, and effective therapeutic interventions are scarce in clinical practice. β-Hydroxybutyrate (BHB), the predominant ketone body present in mammalian systems, arises from the fatty acids β-oxidation within liver during periods of fasting, extended physical exertion, and dietary carbohydrate restriction. This metabolic byproduct is readily taken up by energy-demanding tissues, where it undergoes a two-step conversion: initially to acetyl-CoA and subsequently to ATP. ATP, a vital energy currency, fuels essential tissues, such as the brain, and heart [ 8 ]. The intricate regulation of BHB synthesis is governed primarily by two key factors: the availability of fatty acids as precursors and the expression levels coupled with the enzymatic activity of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) [ 8 ]. Intriguing preclinical and clinical findings have revealed a decrease in blood ketone levels, paralleled by diminished HMGCS2 expression in the liver, among patients with metabolic abnormalities such as MASLD and in rodent models [ 9 – 11 ]. Furthermore, enhancing HMGCS2 expression or BHB supplement has been demonstrated to markedly improved hepatic steatosis and glucose homeostasis [ 12 – 14 ]. Remarkably, HMGCS2 fosters autophagy of Aβ precursor proteins and Tau/p-Tau, thereby mitigating cognitive decline associated with Alzheimer's disease (AD) through its modulation of ketone body production [ 15 ]. Additionally, BHB supplementation has demonstrated broad-spectrum benefits for cognitive function, inhibiting NLRP3 inflammasomes [ 16 ], bolstering BDNF expression in neurons [ 17 ], reducing Aβ accumulation in the brain [ 18 ], attenuating excessive microglia activation [ 18 ], and enhancing mitochondrial respiratory function in hippocampal neurons [ 18 ]. Collectively, these studies underscore the potential significance of ketone bodies, particularly BHB, in the pathophysiology and therapeutic strategies for MASLD and cognitive impairment. Nevertheless, the precise correlation linking circulating BHB levels to these conditions remains elusive, and the feasibility of BHB as a diagnostic biomarker for MASLD or cognitive decline has still to be conclusively finalized. This study aimed to explore the correlation between serum BHB levels, MASLD, and cognitive impairment. A reduction in circulating BHB levels may represent a common pathogenic factor contributing to MASLD and cognitive dysfunction. These discoveries pave the way for innovative pathways in clinical diagnosis and drug development for disorders such as MASLD and AD. Methods General information of patients with MASLD A retrospective analysis was conducted on a cohort of 43 individuals with MASLD, as per the 2020 International Expert Consensus Criteria, who were admitted to Jiangsu Province Hospital of Chinese Medicine between March 2022 and June 2023. The serum samples analyzed were pre-existing biochemical test sera that were meticulously preserved in the endocrinology department's biobank. Inclusion criteria were age between 18 and 80 years, duration of diabetes not exceeding 5 years, and a fasting C-peptide level of 2.0 ng/mL or above. The exclusion criteria were: cases with hyperglycemia attributed to alternative diabetes types, specific medications, or extraneous factors; individuals on drugs known to induce secondary MASLD (e.g., corticosteroids, estrogen, amiodarone, methotrexate); those with a history of acute metabolic complications, cardiovascular, or cerebrovascular events within the preceding 6 months; patients with severe primary systemic pathologies; liver dysfunction stemming from autoimmune liver disease, alcoholic fatty liver disease, viral hepatitis, or liver tumors; individuals with coexisting mental health disorders or severe cognitive impairments; and pregnant or lactating females. The biochemical test sera of the healthy control (HC) group were sourced from the health checkup center of Jiangsu Provincial Government Hospital, where HC individuals were confirmed to be devoid of lifelong mental or significant illnesses, and metabolic disorders. This study adhered to rigorous ethical standards, received approval from the Medical Ethics Committee of Jiangsu Province Hospital of Chinese Medicine (Approval No. 2022NL-071-02), and was conducted in compliance with standard biosecurity and institutional safety protocols. General information of patients with cognitive impairment Serum samples used for biochemical testing were sourced from the biobank of the Anhui Mental Health Center. Specifically, we collected 37 serum samples from patients diagnosed with mild cognitive impairment (MCI) and 28 from those diagnosed with AD. Notably, these patients did not receive any medication before serum collection. The diagnostic criteria for MCI encompassed subjective memory decline, preferably corroborated by knowledgeable individuals; objective testing indicating memory impairment or other cognitive domains inconsistent with age or educational background; preservation of daily functioning abilities; duration of symptoms exceeding 3 months; and the absence of meeting the diagnostic criteria for senile dementia. All patients fulfilled these diagnostic standards and were confirmed by doctors through a thorough examination of their medical history, consultations, and scale test results. The diagnostic criteria for AD adhered to the Diagnostic and Statistical Manual of Mental Disorders (fifth edition). For both MCI and AD, exclusion criteria were stringent and included the presence of other neurological or psychiatric disorders, severe primary systemic conditions, history of head injury or use of specific medications, alcohol or drug dependence within the past 6 months, and poor visual or auditory abilities. HC individuals were matched to patients with MCI and AD in terms of sex and age and were devoid of any mental or significant illnesses or metabolic disorders. This work obtained approval from the Medical Ethics Committee of Anhui Mental Health Center (Approval Number: HFSY-IRB-YJ- KYXM-KXM). Clinical assessment and measurement of serum BHB The clinical assessment included a comprehensive physical examination, including basic personal information and biochemical indices. Additionally, scores from Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination (MMSE) were obtained. Blood samples were collected from fasting individuals using standardized venous techniques in 5 mL capacity tubes. Following centrifugation, the sera were meticulously harvested and stored at -80 ℃ for future analysis. To quantify BHB levels in human serum, a dedicated BHB quantitative detection kit (RXJ99926; Quanzhou Ruixin Biotechnology Co., Ltd., Quanzhou, China) was used. Subsequently, a comparative analysis was conducted on the serum BHB levels of HC and patients with MCI and AD. Additionally, in-depth research has been conducted on the correlation between BHB levels and related indicators. The Receiver Operating Characteristic (ROC) curves were employed to assess the predictive capacity of BHB levels for MASLD and cognitive impairment severity. Statistical analysis Sex comparisons were assessed via Chi-square test. Normally distributed metrics were presented as mean ± SEM, with comparisons conducted using the independent sample t-test. For nonnormal variables, median and interquartile ranges were provided, with Mann-Whitney U test was utilized for comparisons. Pearson’s correlation tests evaluated relationships between BHB levels and other variables. Statistical evaluations were executed using SPSS version 26.0 software. A P -value of less than 0.05 is considered significant. Results Differences in BHB levels between HC individuals and patients with MASLD Thirty-eight HC individuals were matched to patients with MASLD based on age and sex. As illustrated in Table 1 , the HC group exhibited significantly lower levels of fasting blood glucose (FBG), hemoglobin A1c (HbA1c), triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and other biomarkers compared to the MASLD group. Using the BHB quantitative detection kit, the findings indicated that patients with MASLD (214.58 ± 2.04 µmol/L) had notably lower BHB levels than HC individuals (223.71 ± 1.88 µmol/L, P = 0.002; Fig. 1 A). Table 1 Comparison of characteristics between the MASLD and the HC groups. Variables HC MAFLD t/χ2/Z P -value Gender (female/male) 21/17 19/24 0.990 a 0.320 Age 42.29 ± 1.62 44.58 ± 1.58 1.011 0.315 BMI(kg/m 2 ) 21.89 ± 0.22 29.86 ± 0.47 15.334 < 0.001 FBG (mmol/L) 4.99(0.42) 7.57(2.70) 6.888 < 0.001 HbA1c (%) 4.83 ± 0.10 7.90 ± 0.21 13.132 < 0.001 ALT (U/L) 14.0(10.25) 31.0(27.5) 4.561 < 0.001 AST (U/L) 16.0(4.0) 21.5(13.5) 4.029 < 0.001 TC (mmol/L) 4.54 ± 0.09 4.90 ± 0.20 1.617 0.112 TG (mmol/L) 0.83(0.54) 1.33(1.26) 4.520 < 0.001 LDL-C (mmol/L) 2.57 ± 0.09 2.86 ± 0.12 1.854 0.068 HDL-C (mmol/L) 1.65 ± 0.05 1.23 ± 0.04 -6.506 < 0.001 Normal variables presented as mean ± SEM. Nonnormal variables were presented as medians and interquartile ranges. a Indicates χ 2 score. P < 0.05 was considered statistically significant. Further analyzing determined the correlation between serum BHB levels and a series of indicators, including Body mass index (BMI), age, AST, ALT, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and HbA1c. As illustrated in Fig. 1 B-G, BHB levels demonstrated significant negative correlations with age ( r= -0.251, P = 0.02), BMI (r = -0.407, P < 0.001), AST (r = -0.233, P = 0.044), TC (r = -0.266, P = 0.021), LDL-C (r = -0.23, P = 0.046), and HbA1c (r = -0.416, P < 0.001). ROC curve analysis revealed potential diagnostic significance of serum BHB levels (Fig. 1 H). Specifically, BHB had an area under the curve (AUC) of 0.719 ( P < 0.001). This maximum Youden index for BHB was 0.47, corresponding to a critical value of 218.30 µmol/L, yielding 84.2% sensitivity and 62.8% specificity in distinguishing between patients of MASLD and HC. Differences in BHB levels between HC individuals and patients with cognitive impairment Thirty-two HC matched for age, sex, and BMI to patients with MCI and AD were selected for analysis (Table 2 ). Cognitive function was evaluated using the MMSE and MoCA scales, where higher scores indicate superior cognitive performance. As presented in Table 2 , the MMSE and MoCA scores of the HC fell within the normal range and exhibited significant differences compared with those of patients with MCI and AD. The serum BHB levels were measured using a quantitative detection kit. Notably, BHB levels were significantly lower in patients with MCI (177.53 ± 2.36 µmol/L, P = 0.025) and patients with AD (176.41 ± 1.89 µmol/L, P = 0.003) compared to HC individuals (184.53 ± 1.84 µmol/L) (Fig. 2 A). Table 2 Comparison of characteristics between the AD, MCI, and HC groups. Variables HC MCI AD Test ( t or χ2) P -value Age 68.63 ± 9.32 71.70 ± 9.22 71.89 ± 9.17 1.258 0.289 Gender (female/male) 23/9 26/11 20/8 6.000 0.306 BMI (kg/m²) 22.72 ± 2.76 22.55 ± 2.81 21.23 ± 3.17 2.308 0.105 MMSE Score 28.88 ± 1.01 19.76 ± 4.46 2.75 ± 3.41 461.697 < 0.001 MOCA Score 26.81 ± 2.97 16.32 ± 6.25 1.46 ± 2.92 237.130 < 0.001 Normal variables presented as mean ± SEM. P < 0.05 was considered statistically significant. Additionally, correlation analyses were conducted between serum BHB levels and the MoCA and MMSE scores. As depicted in Fig. 2 B and 2 C, BHB levels had positive relationships with both MoCA (r = 0.433, P < 0.001) and MMSE (r = 0.434, P < 0.001) scores. Furthermore, ROC curve analysis demonstrated a potential usefulness of serum BHB levels for cognitive impairment (Fig. 2 D). The AUC for BHB was 0.696, confirming its diagnostic value. The maximum Youden index for BHB was 0.337, which corresponds to a critical BHB value of 174.13 µmol/L. At this threshold, it was 87.5% and 46.2% sensitive and specific for detecting cognitive impairment in patients with HC, respectively. BHB levels were significantly negatively correlated with age in HC individuals Age is closely associated with the incidence of both MASLD and cognitive impairment [ 19 , 20 ]. In current investigation, a relationship between BHB levels and age in HC individuals has been further investigated in depth. To achieve this, the HC individuals were stratified into two distinct age groups: individuals below 60 years and those aged 60 and above. The observations revealed that the serum BHB levels were markedly lower in the older group (187.27 ± 2.18 µmol/L) compared to the younger one (218.52 ± 2.97 µmol/L, Fig. 3 A). A significantly negative association of serum BHB levels with age was revealed through Pearson correlation analysis (r = -0.775, P < 0.001, Fig. 3 B). These results indicate a gradual decline in serum BHB levels as individuals age, in the absence of disease. Discussion In liver mitochondria, acetyl-CoA undergoes a sequence of reactions facilitated by HMGCS2, ultimately leading to the production of ketone bodies, including acetoacetic acid, acetone, and BHB [ 21 ]. These substances are subsequently oxidized and served as alternative energy sources for peripheral tissues. Notably, a remarkable negative relation existed between BHB levels and age in this work. Aging, accompanied by a myriad of conditions, such as dementia, cancer, cardiovascular disease, hypertension, arthritis, cataracts, osteoporosis, diabetes, MASLD, and AD, poses a significant health challenge [ 22 ]. The advantages of calorie restriction (CR) and ketogenic diet in prolonging lifespan and postponing age-related disease onset have been well documented across various species [ 23 , 24 ]. Circulating BHB is recognized as an anti-aging metabolite because its levels surge during CR and ketogenic diets [ 25 ]. Moreover, BHB supplementation has effectively prolonged lifespan of Caenorhabditis elegans , reduce senescence-related secretion phenotypes, and alleviate mammalian vascular cells’ senescence [ 26 , 27 ]. Recent research has shown a shortened maximum lifespan in HMGCS2 knockout mice, whereas administration of 3-butanediol, a precursor of ketone bodies, had a lifespan-extending effect [ 28 ]. Furthermore, exogenous BHB enhances stem cell homeostasis and intestinal stem cell functionality by activating the Notch signaling pathway, which is a pivotal axis for tissue regeneration [ 29 ]. Therefore, BHB has emerged as a promising regenerative mediator with the potential to mitigate age-related diseases [ 22 , 30 ]. However, the precise molecular mechanisms underlying BHB's ability to ameliorate age-related diseases remain unclear. The underlying pathophysiology of MASLD/MASH remains unclear, although latest preclinical and clinical researches have arisen interest in hepatic ketogenic impairment as a potential pathogenic factor. Research has demonstrated that ketogenesis impairment accelerates the progression of MASLD/MASH [ 31 , 32 ]. Notably, compared to MASLD, patients with MASH exhibit reduced HMGCS2 expression [ 33 ]. Additionally, blood ketone levels have been found to decrease in both patients with MASLD and in rodent models [ 9 , 10 ]. Consistent with these findings, this study revealed significantly lower serum BHB levels in patients with MASLD compared to HC individuals. Meta-analyses have confirmed the ketogenic diet was effective in reducing body weight, BMI, and waist circumference, improving HbA1c levels, and with improvement in blood lipid profiles [ 34 – 37 ]. Consistent with the research conducted by Lee et al. [ 38 ], BHB levels were found to be negatively correlated with age, body mass index, HbA1c, TC, LDL-C and AST levels in this research. All these findings indicate a role of BHB in metabolic disorders in patients with MASLD. As a potential insulin sensitizer [ 39 – 41 ], BHB is important in metabolic disease development, including MASLD and type 2 diabetes. A fatty liver can impair ketone production, and reduced BHB production may further disrupt glucose and lipid metabolism. AD is characterized as a progressively dementing disease for which there is no available effective treatment. A detailed study has implicated age-related metabolic changes as key drivers of AD pathology [ 22 ]. BHB, a water-soluble ketone, possesses the remarkable ability to effortlessly traverse this blood-brain barrier and energize brain in a low-glycemic state [ 42 ]. Prior research has established its role as a vital signaling molecule, capable of inhibiting the lipopolysaccharide-induced inflammatory response by modulating NF-κB’s activation [ 43 ]. Numerous studies have highlighted BHB's efficacy in ameliorating cognitive impairment through diverse mechanisms [ 16 – 18 ]. However, the relationship between endogenous BHB levels and AD progression remains unclear. To investigate this, a comparative analysis was conducted on serum BHB levels among patients with MCI, AD, and HC. This study showed a remarkable decline in serum BHB levels in both MCI and AD patients. Intriguingly, a linear correlation emerged between BHB levels and cognitive function, as evaluated by MoCA and MMSE scores. These observations indicate that circulating BHB levels are strongly associated with MCI and AD severity. Given that MASLD and cognitive impairment are closely associated with aging, accumulated clinical evidence underscores an independent link between MASLD and cognitive impairment. Animal studies further corroborated that a high-fat diet can adversely affect learning and memory in mice [ 44 , 45 ]. This study suggests a shared pathological denominator in the decline of circulating BHB in both MASLD and cognitive impairment, suggesting its potential role as a pathogenic factor in MASLD-related cognitive decline. To confirm this hypothesis, animal experiments were conducted. Study strengths and limitations This study is the first to elucidate the correlation between serum BHB levels, MASLD, and cognitive impairment, revealing the potential diagnostic significance of serum BHB levels in both MASLD and cognitive impairment. Notably, the serum BHB levels of healthy individuals is negatively correlated with age. Nevertheless, this study presented certain constraints. Conducted at a single institute with limited participant numbers, it primarily restricted the extent of the research findings' application. Also, a potential impact of external factors such as diet and exercise on BHB levels was overlooked. Furthermore, it did not investigate the effects of various medications on the BHB levels in patients with MASLD. Lastly, cognitive assessment is a complex process requiring the expertise of professional doctors, which necessitates the exclusion of direct cognitive function evaluation in patients with MASLD. Conclusion This study revealed that patients diagnosed with MASLD or cognitive impairment exhibited notably diminished serum levels of BHB compared to HC individuals. The observed decline in circulating BHB levels could serve as a shared underlying pathological mechanism in both MASLD and cognitive impairment. Consequently, measuring serum BHB levels is clinically important for diagnosing these diseases. Furthermore, the therapeutic approach of BHB supplementation may emerge as a focal area of clinical research for disorders such as MASLD and AD. Abbreviations AD Alzheimer's disease ALT Alanine aminotransferase AST Aspartate aminotransferase AUC Area under the curve BHB β-Hydroxybutyrate BMI Body mass index CR Calorie restriction FBG Fasting blood glucose HbA1c Hemoglobin A1c HC Healthy control HDL-C High-Density lipoprotein cholesterol HMGCS2 3-hydroxy-3-methylglutaryl CoA synthase 2 LDL-C Low-Density Lipoprotein Cholesterol MASLD Metabolic dysfunction-associated steatotic liver disease MCI Mild cognitive impairment MMSE Mini-Mental State Examination score MoCA Montreal Cognitive Assessment ROC Receiver operating characteristic TC Total cholesterol TG Triglyceride Declarations Acknowledgements Not applicable. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Lijuan Nie, Xinyi Yang, Ying Wang, Jing Sun, Yaohuan Sun, and Yujuan Zhang. The first draft of the manuscript was written by Lijuan Nie. All authors commented on previous versions of the manuscript. The manuscript was reviewed and edited by Xiqiao Zhou. All authors read and approved the final manuscript. Founding This study was supported by National Natural Science Foundation of China (No. 82474318), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, 035062005002-14), the Special Research Project of Jiangsu Administration of Traditional Chinese Medicine (ZT202105), and the Project Funded by Jiangsu Province Hospital of Chinese Medicine (KKZX01). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study protocols obtained approval from Ethics Committee of Jiangsu Province Hospital of Chinese Medicine (No. 2022NL-071-02; 02 June 2022), and Ethics Committee of Anhui Mental Health Center (No. HFSY-IRB-YJ-KYXM-KXM; 04 January 2024). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Wong VW, Ekstedt M, Wong GL, Hagstrom H. Changing epidemiology, global trends and implications for outcomes of NAFLD. J Hepatol. 2023;79(3):842-52. Le MH, Yeo YH, Zou B, Barnet S, Henry L, Cheung R, et al. Forecasted 2040 global prevalence of nonalcoholic fatty liver disease using hierarchical bayesian approach. Clin Mol Hepatol. 2022;28(4):841-50. Patel SS, Siddiqui MS. Current and Emerging Therapies for Non-alcoholic Fatty Liver Disease. Drugs. 2019;79(1):75-84. Colognesi M, Gabbia D, De Martin S. 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Bae HR, Kim DH, Park MH, Lee B, Kim MJ, Lee EK, et al. beta-Hydroxybutyrate suppresses inflammasome formation by ameliorating endoplasmic reticulum stress via AMPK activation. Oncotarget. 2016;7(41):66444-54. Gao Z, Yin J, Zhang J, Ward RE, Martin RJ, Lefevre M, et al. Butyrate improves insulin sensitivity and increases energy expenditure in mice. Diabetes. 2009;58(7):1509-17. Yang X, Wang R, Zhou H, Wang L, Wang R, Li H, et al. beta-hydroxybutyrate Alleviates Learning and Memory Impairment Through the SIRT1 Pathway in D-Galactose-Injured Mice. Front Pharmacol. 2021;12:751028. Fu SP, Li SN, Wang JF, Li Y, Xie SS, Xue WJ, et al. BHBA suppresses LPS-induced inflammation in BV-2 cells by inhibiting NF-kappaB activation. Mediators Inflamm. 2014;2014:983401. Kim JH, Seo HJ, Pang QQ, Kwon YR, Kim JH, Cho EJ. Protective effects of krill oil on high fat diet-induced cognitive impairment by regulation of oxidative stress. Free Radic Res. 2021;55(7):799-809. Kosari S, Badoer E, Nguyen JC, Killcross AS, Jenkins TA. Effect of western and high fat diets on memory and cholinergic measures in the rat. Behav Brain Res. 2012;235(1):98-103. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5403137","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":379568891,"identity":"1e91a1fd-e24a-46ea-8ed8-1f9e2369bfba","order_by":0,"name":"Lijuan Nie","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lijuan","middleName":"","lastName":"Nie","suffix":""},{"id":379568892,"identity":"a44a0f44-e3af-4ecd-be25-8f1d52fe7a49","order_by":1,"name":"Xinyi Yang","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Yang","suffix":""},{"id":379568893,"identity":"25283101-3090-407d-8622-6f8ead05a1d7","order_by":2,"name":"Ying Wang","email":"","orcid":"","institution":"Jiangsu Province Geriatric Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Wang","suffix":""},{"id":379568894,"identity":"300fcfbd-e00b-47e9-8004-0f2eafaf4cc9","order_by":3,"name":"Jing Sun","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Sun","suffix":""},{"id":379568895,"identity":"dcf73fd1-ddee-4b8d-bdca-d2ca9e1f2712","order_by":4,"name":"Yaohuan Sun","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yaohuan","middleName":"","lastName":"Sun","suffix":""},{"id":379568896,"identity":"abc2ad9d-c853-4f21-a754-c428557794a1","order_by":5,"name":"Yujuan Zhang","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yujuan","middleName":"","lastName":"Zhang","suffix":""},{"id":379568897,"identity":"dcef18cd-c823-4291-8a5c-febad239b64a","order_by":6,"name":"Xiqiao Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDACCRBhwMDDwMB8AMIBcYnUwpZAihYw4IGrxK9FfnbzMWmeAmsZ/vaebw8s2+yiGdibt0kw1NzBqYVxzrE0yRkG6TwSZ85uN5BsS85t4DlWJsFw7BlOLcwSOWYSHwwO8zDcyN0mIdl2ILcBJMLYcBinFjaQggSgFvkbOc8gWuTf4NfCA7PF4EYOG9QWHvxaJCTSki1BfjE8c8xMQuJccm4bT1qxRcIx3FrkZyQfvM3zx9pe7njzM2mJMrvcfvbDG298qMGtBRYK0LAA+Q7ESiCkAaaF8QNhlaNgFIyCUTACAQDaK0rFA77ZcAAAAABJRU5ErkJggg==","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Xiqiao","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-11-06 13:23:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5403137/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5403137/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70582221,"identity":"2b13b72d-39b1-43f5-aa0a-6d9320933d53","added_by":"auto","created_at":"2024-12-04 15:22:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1180628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations between serum BHB levels and MASLD.\u003c/strong\u003e (A). Decreased BHB levels in patients with MASLD compared to those in the HC group. (B). Correlation between BHB levels and age. (C). Correlation between BHB levels and BMI. (D). Correlation between BHB levels and HbA1c. (E). Correlation between BHB levels and TC. (F). Correlation between BHB levels and LDL-C. (G). Correlation between BHB levels and AST. (H). ROC curve for serum BHB in the identification of patients with MASLD. Data are mean ± SEM. \u003csup\u003e**\u003c/sup\u003e: \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, indicating statistically significant differences between HC and MASLD groups.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5403137/v1/ecfb88f2c2705fa24aa10f7a.png"},{"id":70582222,"identity":"41c740c7-f524-40d6-a3a6-cd666611fd1e","added_by":"auto","created_at":"2024-12-04 15:22:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":692330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations between serum BHB levels and cognitive impairment.\u003c/strong\u003e (A). Decreased BHB levels in patients with MCI and AD compared to those in the HC group. (B). Correlation between BHB levels and MoCA scores. (C). Correlation between BHB levels and MMSE scores. (D). ROC curve for serum BHB in identifying patients with cognitive impairment. Data are mean ± SEM. \u003csup\u003e*\u003c/sup\u003e: \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e: \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, indicating statistically significant differences between HC and MCI or AD groups.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5403137/v1/1ddceea8133b523fbda488c8.png"},{"id":70582223,"identity":"af81ee1d-1ce2-462c-8726-fd71ade0cd50","added_by":"auto","created_at":"2024-12-04 15:22:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":309233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations between serum BHB levels and age. \u003c/strong\u003e(A). Decreased BHB levels in individuals \u0026gt; 60 years of age compared to those aged \u0026lt; 60 years (B). Correlation between BHB levels and age in HC individuals. Data are mean ± SEM. ***: \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, indicating statistically significant differences between the two groups.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5403137/v1/258469eb20352b472033fbdf.png"},{"id":73833188,"identity":"ca4c874c-3fef-4ad2-b42a-f005b78c57f4","added_by":"auto","created_at":"2025-01-15 06:47:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3115563,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5403137/v1/7d7808d4-5a9e-4ffb-9bb3-0eaf53b3c566.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Serum β-Hydroxybutyrate decline: a common pathological factor for metabolic dysfunction-associated steatotic liver disease and cognitive impairment","fulltext":[{"header":"Background","content":"\u003cp\u003eAmidst the rapid shifts in dietary patterns and lifestyles, the incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing annually. Currently, the global incidence of MASLD is 25% and is expected to reach 55.7% by 2040 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The clinical burden of MASLD extends beyond liver-related morbidity or mortality and encompasses a range of extrahepatic complications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Recently, cognitive impairment has been identified as a complication of MASLD [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Patients with MASLD experience memory impairment and decreased attention, which can significantly impede their daily functioning and overall quality of life [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Unfortunately, the precise mechanisms underlying MASLD-induced cognitive impairment remain elusive, and effective therapeutic interventions are scarce in clinical practice.\u003c/p\u003e \u003cp\u003eβ-Hydroxybutyrate (BHB), the predominant ketone body present in mammalian systems, arises from the fatty acids β-oxidation within liver during periods of fasting, extended physical exertion, and dietary carbohydrate restriction. This metabolic byproduct is readily taken up by energy-demanding tissues, where it undergoes a two-step conversion: initially to acetyl-CoA and subsequently to ATP. ATP, a vital energy currency, fuels essential tissues, such as the brain, and heart [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The intricate regulation of BHB synthesis is governed primarily by two key factors: the availability of fatty acids as precursors and the expression levels coupled with the enzymatic activity of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Intriguing preclinical and clinical findings have revealed a decrease in blood ketone levels, paralleled by diminished HMGCS2 expression in the liver, among patients with metabolic abnormalities such as MASLD and in rodent models [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, enhancing HMGCS2 expression or BHB supplement has been demonstrated to markedly improved hepatic steatosis and glucose homeostasis [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Remarkably, HMGCS2 fosters autophagy of Aβ precursor proteins and Tau/p-Tau, thereby mitigating cognitive decline associated with Alzheimer's disease (AD) through its modulation of ketone body production [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, BHB supplementation has demonstrated broad-spectrum benefits for cognitive function, inhibiting NLRP3 inflammasomes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], bolstering BDNF expression in neurons [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], reducing Aβ accumulation in the brain [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], attenuating excessive microglia activation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and enhancing mitochondrial respiratory function in hippocampal neurons [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Collectively, these studies underscore the potential significance of ketone bodies, particularly BHB, in the pathophysiology and therapeutic strategies for MASLD and cognitive impairment. Nevertheless, the precise correlation linking circulating BHB levels to these conditions remains elusive, and the feasibility of BHB as a diagnostic biomarker for MASLD or cognitive decline has still to be conclusively finalized.\u003c/p\u003e \u003cp\u003eThis study aimed to explore the correlation between serum BHB levels, MASLD, and cognitive impairment. A reduction in circulating BHB levels may represent a common pathogenic factor contributing to MASLD and cognitive dysfunction. These discoveries pave the way for innovative pathways in clinical diagnosis and drug development for disorders such as MASLD and AD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneral information of patients with MASLD\u003c/h2\u003e \u003cp\u003eA retrospective analysis was conducted on a cohort of 43 individuals with MASLD, as per the 2020 International Expert Consensus Criteria, who were admitted to Jiangsu Province Hospital of Chinese Medicine between March 2022 and June 2023. The serum samples analyzed were pre-existing biochemical test sera that were meticulously preserved in the endocrinology department's biobank. Inclusion criteria were age between 18 and 80 years, duration of diabetes not exceeding 5 years, and a fasting C-peptide level of 2.0 ng/mL or above. The exclusion criteria were: cases with hyperglycemia attributed to alternative diabetes types, specific medications, or extraneous factors; individuals on drugs known to induce secondary MASLD (e.g., corticosteroids, estrogen, amiodarone, methotrexate); those with a history of acute metabolic complications, cardiovascular, or cerebrovascular events within the preceding 6 months; patients with severe primary systemic pathologies; liver dysfunction stemming from autoimmune liver disease, alcoholic fatty liver disease, viral hepatitis, or liver tumors; individuals with coexisting mental health disorders or severe cognitive impairments; and pregnant or lactating females. The biochemical test sera of the healthy control (HC) group were sourced from the health checkup center of Jiangsu Provincial Government Hospital, where HC individuals were confirmed to be devoid of lifelong mental or significant illnesses, and metabolic disorders. This study adhered to rigorous ethical standards, received approval from the Medical Ethics Committee of Jiangsu Province Hospital of Chinese Medicine (Approval No. 2022NL-071-02), and was conducted in compliance with standard biosecurity and institutional safety protocols.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneral information of patients with cognitive impairment\u003c/h3\u003e\n\u003cp\u003eSerum samples used for biochemical testing were sourced from the biobank of the Anhui Mental Health Center. Specifically, we collected 37 serum samples from patients diagnosed with mild cognitive impairment (MCI) and 28 from those diagnosed with AD. Notably, these patients did not receive any medication before serum collection. The diagnostic criteria for MCI encompassed subjective memory decline, preferably corroborated by knowledgeable individuals; objective testing indicating memory impairment or other cognitive domains inconsistent with age or educational background; preservation of daily functioning abilities; duration of symptoms exceeding 3 months; and the absence of meeting the diagnostic criteria for senile dementia. All patients fulfilled these diagnostic standards and were confirmed by doctors through a thorough examination of their medical history, consultations, and scale test results. The diagnostic criteria for AD adhered to the Diagnostic and Statistical Manual of Mental Disorders (fifth edition). For both MCI and AD, exclusion criteria were stringent and included the presence of other neurological or psychiatric disorders, severe primary systemic conditions, history of head injury or use of specific medications, alcohol or drug dependence within the past 6 months, and poor visual or auditory abilities. HC individuals were matched to patients with MCI and AD in terms of sex and age and were devoid of any mental or significant illnesses or metabolic disorders. This work obtained approval from the Medical Ethics Committee of Anhui Mental Health Center (Approval Number: HFSY-IRB-YJ- KYXM-KXM).\u003c/p\u003e\n\u003ch3\u003eClinical assessment and measurement of serum BHB\u003c/h3\u003e\n\u003cp\u003eThe clinical assessment included a comprehensive physical examination, including basic personal information and biochemical indices. Additionally, scores from Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination (MMSE) were obtained. Blood samples were collected from fasting individuals using standardized venous techniques in 5 mL capacity tubes. Following centrifugation, the sera were meticulously harvested and stored at -80 ℃ for future analysis. To quantify BHB levels in human serum, a dedicated BHB quantitative detection kit (RXJ99926; Quanzhou Ruixin Biotechnology Co., Ltd., Quanzhou, China) was used. Subsequently, a comparative analysis was conducted on the serum BHB levels of HC and patients with MCI and AD. Additionally, in-depth research has been conducted on the correlation between BHB levels and related indicators. The Receiver Operating Characteristic (ROC) curves were employed to assess the predictive capacity of BHB levels for MASLD and cognitive impairment severity.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSex comparisons were assessed via Chi-square test. Normally distributed metrics were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, with comparisons conducted using the independent sample t-test. For nonnormal variables, median and interquartile ranges were provided, with Mann-Whitney U test was utilized for comparisons. Pearson\u0026rsquo;s correlation tests evaluated relationships between BHB levels and other variables. Statistical evaluations were executed using SPSS version 26.0 software. A \u003cem\u003eP\u003c/em\u003e-value of less than 0.05 is considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDifferences in BHB levels between HC individuals and patients with MASLD\u003c/h2\u003e \u003cp\u003eThirty-eight HC individuals were matched to patients with MASLD based on age and sex. As illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the HC group exhibited significantly lower levels of fasting blood glucose (FBG), hemoglobin A1c (HbA1c), triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and other biomarkers compared to the MASLD group. Using the BHB quantitative detection kit, the findings indicated that patients with MASLD (214.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04 \u0026micro;mol/L) had notably lower BHB levels than HC individuals (223.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 \u0026micro;mol/L, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\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\u003eComparison of characteristics between the MASLD and the HC groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMAFLD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003et/χ2/Z\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender (female/male)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21/17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19/24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.990\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.320\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.315\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBMI(kg/m\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFBG (mmol/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.99(0.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.57(2.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.888\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHbA1c (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eALT (U/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.0(10.25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.0(27.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAST (U/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.0(4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.5(13.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTC (mmol/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTG (mmol/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.83(0.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33(1.26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLDL-C (mmol/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.068\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHDL-C (mmol/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNormal variables presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Nonnormal variables were presented as medians and interquartile ranges. a Indicates χ\u003csup\u003e2\u003c/sup\u003e score. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analyzing determined the correlation between serum BHB levels and a series of indicators, including Body mass index (BMI), age, AST, ALT, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and HbA1c. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-G, BHB levels demonstrated significant negative correlations with age ( r= -0.251, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), BMI (r = -0.407, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), AST (r = -0.233, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.044), TC (r = -0.266, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021), LDL-C (r = -0.23, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046), and HbA1c (r = -0.416, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). ROC curve analysis revealed potential diagnostic significance of serum BHB levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Specifically, BHB had an area under the curve (AUC) of 0.719 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This maximum Youden index for BHB was 0.47, corresponding to a critical value of 218.30 \u0026micro;mol/L, yielding 84.2% sensitivity and 62.8% specificity in distinguishing between patients of MASLD and HC.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDifferences in BHB levels between HC individuals and patients with cognitive impairment\u003c/h3\u003e\n\u003cp\u003eThirty-two HC matched for age, sex, and BMI to patients with MCI and AD were selected for analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Cognitive function was evaluated using the MMSE and MoCA scales, where higher scores indicate superior cognitive performance. As presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the MMSE and MoCA scores of the HC fell within the normal range and exhibited significant differences compared with those of patients with MCI and AD. The serum BHB levels were measured using a quantitative detection kit. Notably, BHB levels were significantly lower in patients with MCI (177.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36 \u0026micro;mol/L, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025) and patients with AD (176.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89 \u0026micro;mol/L, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) compared to HC individuals (184.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84 \u0026micro;mol/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\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\u003eComparison of characteristics between the AD, MCI, and HC groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMCI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTest\u0026nbsp;(\u003cem\u003et\u003c/em\u003e\u0026nbsp;or\u0026nbsp;χ2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.63\u0026thinsp;\u0026plusmn;\u0026thinsp;9.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.70\u0026thinsp;\u0026plusmn;\u0026thinsp;9.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.89\u0026thinsp;\u0026plusmn;\u0026thinsp;9.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.289\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u0026nbsp;(female/male)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.306\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBMI\u0026nbsp;(kg/m\u0026sup2;)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.23\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.308\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.105\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMMSE\u0026nbsp;Score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.76\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e461.697\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMOCA\u0026nbsp;Score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.32\u0026thinsp;\u0026plusmn;\u0026thinsp;6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e237.130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNormal variables presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, correlation analyses were conducted between serum BHB levels and the MoCA and MMSE scores. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, BHB levels had positive relationships with both MoCA (r\u0026thinsp;=\u0026thinsp;0.433, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and MMSE (r\u0026thinsp;=\u0026thinsp;0.434, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) scores. Furthermore, ROC curve analysis demonstrated a potential usefulness of serum BHB levels for cognitive impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The AUC for BHB was 0.696, confirming its diagnostic value. The maximum Youden index for BHB was 0.337, which corresponds to a critical BHB value of 174.13 \u0026micro;mol/L. At this threshold, it was 87.5% and 46.2% sensitive and specific for detecting cognitive impairment in patients with HC, respectively.\u003c/p\u003e\n\u003ch3\u003eBHB levels were significantly negatively correlated with age in HC individuals\u003c/h3\u003e\n\u003cp\u003eAge is closely associated with the incidence of both MASLD and cognitive impairment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In current investigation, a relationship between BHB levels and age in HC individuals has been further investigated in depth. To achieve this, the HC individuals were stratified into two distinct age groups: individuals below 60 years and those aged 60 and above. The observations revealed that the serum BHB levels were markedly lower in the older group (187.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18 \u0026micro;mol/L) compared to the younger one (218.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97 \u0026micro;mol/L, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A significantly negative association of serum BHB levels with age was revealed through Pearson correlation analysis (r = -0.775, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results indicate a gradual decline in serum BHB levels as individuals age, in the absence of disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn liver mitochondria, acetyl-CoA undergoes a sequence of reactions facilitated by HMGCS2, ultimately leading to the production of ketone bodies, including acetoacetic acid, acetone, and BHB [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These substances are subsequently oxidized and served as alternative energy sources for peripheral tissues. Notably, a remarkable negative relation existed between BHB levels and age in this work. Aging, accompanied by a myriad of conditions, such as dementia, cancer, cardiovascular disease, hypertension, arthritis, cataracts, osteoporosis, diabetes, MASLD, and AD, poses a significant health challenge [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The advantages of calorie restriction (CR) and ketogenic diet in prolonging lifespan and postponing age-related disease onset have been well documented across various species [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Circulating BHB is recognized as an anti-aging metabolite because its levels surge during CR and ketogenic diets [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, BHB supplementation has effectively prolonged lifespan of \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, reduce senescence-related secretion phenotypes, and alleviate mammalian vascular cells\u0026rsquo; senescence [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Recent research has shown a shortened maximum lifespan in HMGCS2 knockout mice, whereas administration of 3-butanediol, a precursor of ketone bodies, had a lifespan-extending effect [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, exogenous BHB enhances stem cell homeostasis and intestinal stem cell functionality by activating the Notch signaling pathway, which is a pivotal axis for tissue regeneration [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, BHB has emerged as a promising regenerative mediator with the potential to mitigate age-related diseases [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, the precise molecular mechanisms underlying BHB's ability to ameliorate age-related diseases remain unclear.\u003c/p\u003e \u003cp\u003eThe underlying pathophysiology of MASLD/MASH remains unclear, although latest preclinical and clinical researches have arisen interest in hepatic ketogenic impairment as a potential pathogenic factor. Research has demonstrated that ketogenesis impairment accelerates the progression of MASLD/MASH [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Notably, compared to MASLD, patients with MASH exhibit reduced HMGCS2 expression [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, blood ketone levels have been found to decrease in both patients with MASLD and in rodent models [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consistent with these findings, this study revealed significantly lower serum BHB levels in patients with MASLD compared to HC individuals. Meta-analyses have confirmed the ketogenic diet was effective in reducing body weight, BMI, and waist circumference, improving HbA1c levels, and with improvement in blood lipid profiles [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Consistent with the research conducted by Lee et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], BHB levels were found to be negatively correlated with age, body mass index, HbA1c, TC, LDL-C and AST levels in this research. All these findings indicate a role of BHB in metabolic disorders in patients with MASLD. As a potential insulin sensitizer [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], BHB is important in metabolic disease development, including MASLD and type 2 diabetes. A fatty liver can impair ketone production, and reduced BHB production may further disrupt glucose and lipid metabolism.\u003c/p\u003e \u003cp\u003eAD is characterized as a progressively dementing disease for which there is no available effective treatment. A detailed study has implicated age-related metabolic changes as key drivers of AD pathology [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. BHB, a water-soluble ketone, possesses the remarkable ability to effortlessly traverse this blood-brain barrier and energize brain in a low-glycemic state [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Prior research has established its role as a vital signaling molecule, capable of inhibiting the lipopolysaccharide-induced inflammatory response by modulating NF-κB\u0026rsquo;s activation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Numerous studies have highlighted BHB's efficacy in ameliorating cognitive impairment through diverse mechanisms [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the relationship between endogenous BHB levels and AD progression remains unclear. To investigate this, a comparative analysis was conducted on serum BHB levels among patients with MCI, AD, and HC. This study showed a remarkable decline in serum BHB levels in both MCI and AD patients. Intriguingly, a linear correlation emerged between BHB levels and cognitive function, as evaluated by MoCA and MMSE scores. These observations indicate that circulating BHB levels are strongly associated with MCI and AD severity.\u003c/p\u003e \u003cp\u003eGiven that MASLD and cognitive impairment are closely associated with aging, accumulated clinical evidence underscores an independent link between MASLD and cognitive impairment. Animal studies further corroborated that a high-fat diet can adversely affect learning and memory in mice [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This study suggests a shared pathological denominator in the decline of circulating BHB in both MASLD and cognitive impairment, suggesting its potential role as a pathogenic factor in MASLD-related cognitive decline. To confirm this hypothesis, animal experiments were conducted.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStudy strengths and limitations\u003c/h2\u003e \u003cp\u003eThis study is the first to elucidate the correlation between serum BHB levels, MASLD, and cognitive impairment, revealing the potential diagnostic significance of serum BHB levels in both MASLD and cognitive impairment. Notably, the serum BHB levels of healthy individuals is negatively correlated with age. Nevertheless, this study presented certain constraints. Conducted at a single institute with limited participant numbers, it primarily restricted the extent of the research findings' application. Also, a potential impact of external factors such as diet and exercise on BHB levels was overlooked. Furthermore, it did not investigate the effects of various medications on the BHB levels in patients with MASLD. Lastly, cognitive assessment is a complex process requiring the expertise of professional doctors, which necessitates the exclusion of direct cognitive function evaluation in patients with MASLD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed that patients diagnosed with MASLD or cognitive impairment exhibited notably diminished serum levels of BHB compared to HC individuals. The observed decline in circulating BHB levels could serve as a shared underlying pathological mechanism in both MASLD and cognitive impairment. Consequently, measuring serum BHB levels is clinically important for diagnosing these diseases. Furthermore, the therapeutic approach of BHB supplementation may emerge as a focal area of clinical research for disorders such as MASLD and AD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Alzheimer\u0026apos;s disease\u003c/p\u003e\n\u003cp\u003eALT \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Alanine aminotransferase\u003c/p\u003e\n\u003cp\u003eAST\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Aspartate aminotransferase\u003c/p\u003e\n\u003cp\u003eAUC \u0026nbsp; \u0026nbsp; \u0026nbsp;Area under the curve\u003c/p\u003e\n\u003cp\u003eBHB\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026beta;-Hydroxybutyrate\u003c/p\u003e\n\u003cp\u003eBMI \u0026nbsp; \u0026nbsp; \u0026nbsp; Body mass index\u003c/p\u003e\n\u003cp\u003eCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Calorie restriction\u003c/p\u003e\n\u003cp\u003eFBG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fasting blood glucose\u003c/p\u003e\n\u003cp\u003eHbA1c \u0026nbsp; \u0026nbsp; Hemoglobin A1c\u003c/p\u003e\n\u003cp\u003eHC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Healthy control\u003c/p\u003e\n\u003cp\u003eHDL-C \u0026nbsp; \u0026nbsp; High-Density lipoprotein cholesterol\u003c/p\u003e\n\u003cp\u003eHMGCS2\u0026nbsp; \u0026nbsp;3-hydroxy-3-methylglutaryl CoA synthase 2\u003c/p\u003e\n\u003cp\u003eLDL-C \u0026nbsp; \u0026nbsp; Low-Density Lipoprotein Cholesterol\u003c/p\u003e\n\u003cp\u003eMASLD\u0026nbsp; \u0026nbsp;\u0026nbsp;Metabolic dysfunction-associated steatotic liver disease\u003c/p\u003e\n\u003cp\u003eMCI \u0026nbsp; \u0026nbsp; \u0026nbsp; Mild cognitive impairment\u003c/p\u003e\n\u003cp\u003eMMSE \u0026nbsp; \u0026nbsp; Mini-Mental State Examination\u0026nbsp;score\u003c/p\u003e\n\u003cp\u003eMoCA \u0026nbsp; \u0026nbsp; Montreal Cognitive Assessment\u003c/p\u003e\n\u003cp\u003eROC \u0026nbsp; \u0026nbsp; \u0026nbsp; Receiver operating characteristic\u003c/p\u003e\n\u003cp\u003eTC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Total cholesterol\u003c/p\u003e\n\u003cp\u003eTG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Triglyceride\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by\u0026nbsp;Lijuan Nie, Xinyi Yang, Ying Wang, Jing Sun, Yaohuan Sun, and Yujuan Zhang. The first draft of the manuscript was written by Lijuan Nie. All authors commented on previous versions of the manuscript. The manuscript was reviewed and edited by Xiqiao Zhou. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFounding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by National Natural Science Foundation of China (No. 82474318), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, 035062005002-14), the Special Research Project of Jiangsu Administration of Traditional Chinese Medicine (ZT202105), and the Project Funded by Jiangsu Province Hospital of Chinese Medicine (KKZX01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis\u0026nbsp;study protocols obtained approval\u0026nbsp;from\u0026nbsp;Ethics Committee of Jiangsu Province Hospital of Chinese Medicine (No. 2022NL-071-02; 02 June 2022), and Ethics Committee of Anhui Mental\u0026nbsp;Health Center (No. HFSY-IRB-YJ-KYXM-KXM; 04 January 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWong VW, Ekstedt M, Wong GL, Hagstrom H. 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Behav Brain Res.\u003cem\u003e \u003c/em\u003e2012;235(1):98-103.\u003c/li\u003e\n\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":"Metabolic dysfunction-associated steatotic liver disease, Cognitive impairment, Age, β-Hydroxybutyrate, Biomarker","lastPublishedDoi":"10.21203/rs.3.rs-5403137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5403137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eRecently, cognitive impairment has emerged as a consequential aspect of metabolic dysfunction-associated steatotic liver disease (MASLD), prompting speculation that diminished β-Hydroxybutyrate (BHB) production might be intricately involved in its pathogenesis, along with that of cognitive decline. However, the intricate interplay between the circulating BHB levels and these conditions remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA BHB ELISA assay kit was used to quantify serum BHB concentrations in both healthy individuals and patients with MASLD or cognitive impairment. Pearson’s correlation analysis assessed associations between BHB levels and pertinent clinical parameters. The diagnostic utility of serum BHB levels in identifying MASLD and cognitive impairment was assessed using Receiver Operating Characteristic (ROC) curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The findings revealed a stark contrast: patients with MASLD and cognitive impairment exhibited notably lower serum BHB levels than healthy individuals. Furthermore, BHB levels were inversely associated with BMI (r = -0.407, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001), AST (r = -0.233, \u003cem\u003eP \u003c/em\u003e= 0.044), TC (r = -0.266, \u003cem\u003eP\u003c/em\u003e= 0.021), LDL-C (r = -0.236, \u003cem\u003eP\u003c/em\u003e = 0.046), and HbA1c (r = -0.416, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001), while demonstrating a positively relationship with cognitive performance, as indicated by Mini-Mental State Examination (r = 0.434, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001) and Montreal Cognitive Assessment scores (r = 0.433, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). Notably, ROC curve analysis underscored potential diagnostic significance of serum BHB in both MASLD and cognitive impairment. Intriguingly, supplementary research illuminated a negative correlation between serum BHB levels and age among healthy individuals (r = -0.775, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e This work implies that a decrease on circulating BHB levels may lead to the occurrence of MASLD and cognitive impairment.\u003c/p\u003e","manuscriptTitle":"Serum β-Hydroxybutyrate decline: a common pathological factor for metabolic dysfunction-associated steatotic liver disease and cognitive impairment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-04 15:22:41","doi":"10.21203/rs.3.rs-5403137/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":"8c7cd736-a0d6-410d-9a80-22633f1b39a5","owner":[],"postedDate":"December 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-15T06:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-04 15:22:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5403137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5403137","identity":"rs-5403137","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}