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Previously, our metabolomic, transcriptomic, and genomic studies characterized the ceramide/sphingomyelin pathway as a therapeutic target in Alzheimer's disease, and we demonstrated that FTY720, a sphingosine-1-phospahate receptor modulator approved for treatment of multiple sclerosis, recovers synaptic plasticity and memory in APP/PS1 mice. To further investigate how FTY720 rescues the pathology, we performed metabolomic analysis in brain, plasma, and liver of trained APP/PS1 and wild-type mice. APP/PS1 mice showed area-specific brain disturbances in polyamines, phospholipids, and sphingolipids. Most changes were completely or partially normalized in FTY720-treated subjects, indicating rebalancing the "sphingolipid rheostat", reactivating phosphatidylethanolamine synthesis via mitochondrial phosphatidylserine decarboxylase pathway, and normalizing polyamine levels that support mitochondrial activity. Synaptic plasticity and memory were rescued, with spermidine synthesis in temporal cortex best corresponding to hippocampal CA3-CA1 plasticity normalization. FTY720 effects, also reflected in other pathways, are consistent with promotion of mitochondrial function, synaptic plasticity, and anti-inflammatory environment, while reducing pro-apoptotic and pro-inflammatory signals., Competing Interests: Disclosures and competing interests Karel Kalecký and Teodoro Bottiglieri are authors of the Integrator software, which was used for quantification of chromatographic signal, and which implements an approach described in a patent application (Application # PCT/US24/51426, filed on October 15, 2024) that is currently pending. Dr. Kaddurah-Daouk is an inventor on a series of patents on use of metabolomics for the diagnosis and treatment of CNS diseases and holds equity in Metabolon Inc., Chymia LLC and PsyProtix.

Background: There is no clinically relevant serological marker for the early detection of oesophageal adenocarcinoma (EAC) and its precursor lesion, Barrett's oesophagus (BE)., Objective: To develop and test a blood-based assay for EAC and BE., Design: Oesophageal MicroRNAs of BaRRett, Adenocarcinoma and Dysplasia ( EMERALD ) was a large, international, multicentre biomarker cohort study involving 792 patient samples from 4 countries (NCT06381583) to develop and validate a circulating miRNA signature for the early detection of EAC and high-risk BE. Tissue-based miRNA sequencing and microarray datasets (n=134) were used to identify candidate miRNAs of diagnostic potential, followed by validation using 42 pairs of matched cancer and normal tissues. The usefulness of the candidate miRNAs was initially assessed using 108 sera (44 EAC, 34 EAC precursors and 30 non-disease controls). We finally trained a machine learning model (XGBoost+AdaBoost) on RT-qPCR results from circulating miRNAs from a training cohort (n=160) and independently tested it in an external cohort (n=295)., Results: After a strict process of biomarker discovery and selection, we identified six miRNAs that were overexpressed in all sera of patients compared with non-disease controls from three independent cohorts of different nationalities (miR-106b, miR-146a, miR-15a, miR-18a, miR-21 and miR-93). We established a six-miRNA diagnostic signature using the training cohort (area under the receiver operating characteristic curve (AUROC): 97.6%) and tested it in an independent cohort (AUROC: 91.9%). This assay could also identify patients with BE among patients with gastro-oesophageal reflux disease (AUROC: 94.8%, sensitivity: 92.8%, specificity: 85.1%)., Conclusion: Using a comprehensive approach integrating unbiased genome-wide biomarker discovery and several independent experimental validations, we have developed and validated a novel blood test that might complement screening options for BE/EAC., Trial Registration Number: NCT06381583., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2025. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Background/objectives: The MTHFR 677C>T gene variant results in a thermolabile MTHFR enzyme associated with elevated plasma homocysteine in TT individuals. Health risks associated with the TT genotype may be modified by dietary and supplemental folate intake. Supplementation with methyltetrahydrofolate (methylTHF) may be preferable to folic acid because it is the MTHFR product, and does not require reduction by DHFR to enter one-carbon folate metabolism. In the Mthfr 677C>T mouse model for this variant, female 677TT (TT) mice have an increased incidence of hepatic steatosis. The objective of this study was to compare the effects of methylTHF and folic acid supplementation on hepatic steatosis and one-carbon metabolism in this model., Methods: Male and female C57BL/6J 677CC (CC) and TT mice were fed control (CD), 5xmethylTHF-supplemented (MFSD), or 5xfolic-acid-supplemented (FASD) diets for 4 months. Liver sections were assessed for steatosis by Oil Red O staining. One-carbon metabolites were measured in the liver and plasma. MTHFR protein expression was evaluated in the liver., Results: MFSD had no significant effect on plasma homocysteine, liver SAM/SAH ratios, or hepatic steatosis in males or females as compared to CD. MTHFR protein increased in MFSD TT female liver, but remained <50% of the CC. FASD had no effect on plasma homocysteine but it decreased the liver MTHFR protein and SAM/SAH ratios, and increased hepatic steatosis in CC females., Conclusions: MethylTHF and folic acid supplementation had limited benefits for TT mice, while folic acid supplementation had negative effects on CC females. Further investigation is required to determine if these effects are relevant in humans., Competing Interests: The authors declare no conflicts of interest.

Scope: Human milk (HM) is rich in bioactive compounds and essential nutrients. While research has focused on lipids, minerals, immune markers, microbiota, and oligosaccharides, specific metabolites are less studied. This study uses targeted metabolomics to identify and quantify metabolites in HM and explores the impact of perinatal and dietary factors on the metabolomic profile., Methods and Results: In a cross-sectional study of 123 healthy lactating women, HM samples were collected up to 1 month postpartum and analyzed using the Biocrates MxP Quant 500 kit. Maternal and neonatal clinical, anthropometric, and nutritional data were collected. A total of 432 metabolites were quantified and categorized into 20 groups. The metabolomic profiles formed three distinct clusters, primarily driven by triglyceride concentration differences. Docosahexaenoic acid (DHA) levels were higher in HM from mothers with vaginal delivery compared to C-section births and differences in hexoses were found between exclusive and mixed-feeding practices. Maternal diets rich in lipids and animal proteins were associated with elevated amino acids, sphingolipids, and glycosyl-ceramides., Conclusion: The HM metabolome was grouped into three clusters influenced by delivery mode, lactation practices, and maternal diet. This comprehensive analysis opens new avenues to explore HM composition and offers valuable insights for future dietary interventions aimed at modulating HM., (© 2024 The Author(s). Molecular Nutrition & Food Research published by Wiley‐VCH GmbH.)

Metabolomics and lipidomics are pivotal in understanding phenotypic variations beyond genomics. However, quantification and comparability of mass spectrometry (MS)-derived data are challenging. Standardised assays can enhance data comparability, enabling applications in multi-center epidemiological and clinical studies. Here we evaluated the performance and reproducibility of the MxP ® Quant 500 kit across 14 laboratories. The kit allows quantification of 634 different metabolites from 26 compound classes using triple quadrupole MS. Each laboratory analysed twelve samples, including human plasma and serum, lipaemic plasma, NIST SRM 1950, and mouse and rat plasma, in triplicates. 505 out of the 634 metabolites were measurable above the limit of detection in all laboratories, while eight metabolites were undetectable in our study. Out of the 505 metabolites, 412 were observed in both human and rodent samples. Overall, the kit exhibited high reproducibility with a median coefficient of variation (CV) of 14.3 %. CVs in NIST SRM 1950 reference plasma were below 25 % and 10 % for 494 and 138 metabolites, respectively. To facilitate further inspection of reproducibility for any compound, we provide detailed results from the in-depth evaluation of reproducibility across concentration ranges using Deming regression. Interlaboratory reproducibility was similar across sample types, with some species-, matrix-, and phenotype-specific differences due to variations in concentration ranges. Comparisons with previous studies on the performance of MS-based kits (including the AbsoluteIDQ p180 and the Lipidyzer) revealed good concordance of reproducibility results and measured absolute concentrations in NIST SRM 1950 for most metabolites, making the MxP ® Quant 500 kit a relevant tool to apply metabolomics and lipidomics in multi-center studies., Competing Interests: DECLARATION OF INTERESTS G.K. is co-inventor (through Helmholtz Zentrum München) on patents regarding applications of metabolomics in diseases of the central nervous system and holds equity in Chymia LLC. A.L., T.H.P and T.K. were employed at biocrates life sciences ag at the time the experiment was conducted and/or at the time this manuscript was being written. K.C. is currently an AstraZeneca employee.

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is characterized by elevated plasma and tissue homocysteine levels. There is no cure, but HCU is typically managed by methionine/protein restriction and vitamin B 6 supplementation. Enzyme replacement therapy (ERT) based on human CBS has been developed and has shown significant efficacy correcting HCU phenotype in several mouse models by bringing plasma total homocysteine below the clinically relevant 100 μM threshold. As the reactive nature of homocysteine promotes disulfide formation and protein binding, and ERT is unable to normalize plasma total homocysteine levels, the mechanism of action of ERT in HCU remains to be further characterized. Here we showed that only a reduced homocysteine serves as a substrate for CBS and its availability restricts the homocysteine-degrading capacity of CBS. We also demonstrated that cells export homocysteine in its reduced form, which is efficiently metabolized by CBS in the culture medium. Availability of serine, a CBS co-substrate, was not a limiting factor in our cell-based model. Biological reductants, such as N-acetylcysteine, MESNA or cysteamine, increased the availability of the reduced homocysteine and thus promoted its subsequent CBS-based elimination. In a transgenic I278T mouse model of HCU, administration of biological reductants significantly increased the proportion of protein-unbound homocysteine in plasma, which improved the efficacy of the co-administered CBS-based ERT, as evidenced by significantly lower plasma total homocysteine levels. These results clarify the mechanism of action of CBS-based ERT and unveil novel pharmacological approaches to further increase its efficacy., Competing Interests: Declaration of competing interest TM is an inventor on patents related to pegtibatinase, provides ad-hoc consulting to Travere Therapeutics and receives research support from Travere Therapeutics. TB receives compensation for metabolomic analyses from Travere Therapeutics. WC, KL and SR are current or former employees and stockholders of Travere Therapeutics, which clinically develops pegtibatinase as an enzyme replacement therapy for classical homocystinuria. TMP and CS declare no conflicting interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO 2  + NO 3 ) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease., (© 2024 Federation of American Societies for Experimental Biology.)

Maternal nutrition during pregnancy and lactation plays an important role in the neurodevelopment of offspring. One-carbon (1C) metabolism, which centers around folic acid and choline, as well as other B vitamins, plays a key role during the closure of the neural tube of the developing fetus. However, the impact of these maternal nutritional deficiencies during pregnancy on offspring health outcomes after birth remains relatively undefined. Furthermore, maternal dietary deficiencies in folic acid or choline may impact other health outcomes in offspring - making this a valuable model. This protocol aims to outline the procedure for inducing a deficiency in 1C metabolism in female mice through dietary modifications. Females are placed on diets at weaning, up to 2 months of age, for 4-6 weeks prior to mating and remain on diet throughout pregnancy and lactation. Offspring from these females can be evaluated for health outcomes. Females can be used multiple times to generate offspring, and tissues from females can be collected to measure for 1C metabolite measurements. This protocol provides an overview of how to induce maternal dietary deficiencies in folic acid or choline to study offspring health outcomes.

Chronic kidney disease (CKD) impacts 14% of adults in the United States, and African American (AA) individuals are disproportionately affected, with more than 3 times higher risk of kidney failure as compared to White individuals. This study evaluated the effects of base-producing fruit and vegetables (FVs) on cardiorenal outcomes in AA persons with CKD and hypertension (HTN) in a low socioeconomic area. The "Cardiorenal Protective Diet" prospective randomized trial evaluated the effects of a 6-week, community-based FV intervention compared to a waitlist control (WL) in 91 AA adults (age = 58.3 ± 10.1 years, 66% female, 48% income ≤ USD 25K). Biometric and metabolomic variables were collected at baseline and 6 weeks post-intervention. The change in health outcomes for both groups was statistically insignificant ( p > 0.05), though small reductions in albumin to creatinine ratio, body mass index, total cholesterol, and systolic blood pressure were observed in the FV group. Metabolomic profiling identified key markers ( p < 0.05), including C3, C5, 1-Met-His, kynurenine, PC ae 38:5, and choline, indicating kidney function decline in the WL group. Overall, delivering a directed cardiorenal protective diet intervention improved cardiorenal outcomes in AA adults with CKD and HTN. Additionally, metabolomic profiling may serve as a prognostic technique for the early identification of biomarkers as indicators for worsening CKD and increased CVD risk.

Nutrition is a modifiable risk factor for ischemic stroke. As people age their ability to absorb some nutrients decreases, a primary example is vitamin B12. Older individuals with a vitamin B12 deficiency are at a higher risk for ischemic stroke and have worse stroke outcome. However, the mechanisms through which these occur remain unknown. The aim of the study was to investigate the role of vitamin B12 deficiency in ischemic stroke outcome and mechanistic changes in a mouse model. Ten-month-old male and female mice were put on control or vitamin B12 deficient diets for 4 weeks prior to and after ischemic stroke to the sensorimotor cortex. Motor function was measured, and tissues were collected to assess potential mechanisms. All deficient mice had increased levels of total homocysteine in plasma and liver tissues. After ischemic stroke, deficient mice had impaired motor function compared to control mice. There was no difference between groups in ischemic damage volume. However, within the ischemic damage region, there was an increase in total apoptosis of male deficient mice compared to controls. Furthermore, there was an increase in neuronal survival in ischemic brain tissue of the vitamin B12 deficient mice compared to controls. Additionally, there were changes in choline metabolites in ischemic brain tissue because of a vitamin B12 deficiency. The data presented in this study confirms that a vitamin B12 deficiency worsens stroke outcome in male and female mice. The mechanisms driving this change may be a result of neuronal survival and compensation in choline metabolism within the damaged brain tissue.

Scope: Disturbances in one-carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr 677C >T variant on NAFLD., Methods and Results: This study uses the new Mthfr 677C >T mouse model for the human MTHFR 677C >T variant. Mthfr 677CC and Mthfr 677TT mice were fed control diet (CD) or folate-deficient (FD) diets for 4 months. FD and Mthfr 677TT alter choline/methyl metabolites in liver and/or plasma (decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr 677TT , provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr 677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex-based differences in methylation potential, betaine, sphingomyelin, and trimethylamine-N-oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr 677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females., Conclusion: This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr 677TT , whereas Mthfr 677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD-related disorders., (© 2024 The Authors. Molecular Nutrition & Food Research published by Wiley‐VCH GmbH.)

Background and Aims: Participation in a healthy lifestyle intervention such as the Diabetes Prevention Program Group Lifestyle Balance-adapted for stroke (GLB-CVA) may reduce stroke burden. Identifying biomarkers associated with lifestyle changes may enhance an individualized approach to stroke recovery. We investigated metabolic biomarkers related to cardiovascular and neurological function in individuals with stroke in the GLB-CVA study and healthy (non-stroke) individuals., Methods and Results: Participants with chronic (>12 months) stroke were recruited to this wait-list randomized controlled trial if they were overweight (BMI ≥25 kg/m 2 ). Participants were randomized to (1) the GLB-CVA program to complete 22 educational sessions addressing behavioral principals of dietary and physical activity or (2) a 6 month wait-list control (WLC). Biomarkers [Plasma irisin, vascular endothelial growth factor, lipoprotein-associated phospholipase A2 (Lp-PLA2), insulin-like growth factor 1 and brain-derived neurotrophic factor (BDNF)] were collected at baseline, 3, and 6 months. Age-matched healthy individuals were recruited for biomarker assessment. Compared to healthy adults (n = 19), participants with stroke (GLB-CVA = 24; WLC = 24) at baseline had higher tHcy levels (p < 0.001) and lower PLA2 levels (p = 0.016). No statistically significant interactions were observed for any biomarkers between the GLB-CVA and WLC or between people who achieved 5% weight loss and those who did not., Conclusion: Participation in a 6-month healthy lifestyle program did not result in statistically significant changes to select metabolic biomarker levels for our participants with chronic stroke. However, participants with stroke demonstrated a unique biomarker profile compared to age-matched healthy individuals., Competing Interests: Declaration of competing interest No potential competing interest was reported by the authors., (Copyright © 2023 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Background: Folic acid (FA) is the oxidized form of folate found in supplements and FA-fortified foods. Most FA is reduced by dihydrofolate reductase to 5-methyltetrahydrofolate (5mTHF); the latter is the form of folate naturally found in foods. Ingestion of FA increases the plasma levels of both 5mTHF and unmetabolized FA (UMFA). Limited information is available on the downstream metabolic effects of FA supplementation, including potential effects associated with UMFA., Objective: We aimed to assess the metabolic effects of FA-supplementation, and the associations of plasma 5mTHF and UMFA with the metabolome in FA-naïve Bangladeshi adults., Methods: Sixty participants were selected from the Folic Acid and Creatine Trial; half received 800 μg FA/day for 12 weeks and half placebo. Plasma metabolome profiles were measured by high-resolution mass spectrometry, including 170 identified metabolites and 26,541 metabolic features. Penalized regression methods were used to assess the associations of targeted metabolites with FA-supplementation, plasma 5mTHF, and plasma UMFA. Pathway analyses were conducted using Mummichog., Results: In penalized models of identified metabolites, FA-supplementation was associated with higher choline. Changes in 5mTHF concentrations were positively associated with metabolites involved in amino acid metabolism (5-hydroxyindoleacetic acid, acetylmethionine, creatinine, guanidinoacetate, hydroxyproline/n-acetylalanine) and 2 fatty acids (docosahexaenoic acid and linoleic acid). Changes in 5mTHF concentrations were negatively associated with acetylglutamate, acetyllysine, carnitine, propionyl carnitine, cinnamic acid, homogentisate, arachidonic acid, and nicotine. UMFA concentrations were associated with lower levels of arachidonic acid. Together, metabolites selected across all models were related to lipids, aromatic amino acid metabolism, and the urea cycle. Analyses of nontargeted metabolic features identified additional pathways associated with FA supplementation., Conclusion: In addition to the recapitulation of several expected metabolic changes associated with 5mTHF, we observed additional metabolites/pathways associated with FA-supplementation and UMFA. Further studies are needed to confirm these associations and assess their potential implications for human health., Trial Registration Number: This trial was registered at https://clinicaltrials.gov as NCT01050556., (Copyright © 2023 American Society for Nutrition. All rights reserved.)

Atherosclerosis and resulting cardiovascular disease are the leading causes of death in the US. Hyperhomocysteinemia (HHcy), or the accumulation of the intermediate amino acid homocysteine, is an independent risk factor for atherosclerosis, but the intricate biological processes mediating this effect remain elusive. Several factors regulate homocysteine levels, including the activity of several enzymes and adequate levels of their coenzymes, including pyridoxal phosphate (vitamin B6), folate (vitamin B9), and methylcobalamin (vitamin B12). To better understand the biological influence of HHcy on the development and progression of atherosclerosis, apolipoprotein-E-deficient ( apoE -/- mice), a model for human atherosclerosis, were fed a hyperhomocysteinemic diet (low in methyl donors and B vitamins) (HHD) or a control diet (CD). After eight weeks, the plasma, aorta, and liver were collected to quantify methylation metabolites, while plasma was also used for a broad targeted metabolomic analysis. Aortic plaque burden in the brachiocephalic artery (BCA) was quantified via 14T magnetic resonance imaging (MRI). A severe accumulation of plasma and hepatic homocysteine and an increased BCA plaque burden were observed, thus confirming the atherogenic effect of the HHD. Moreover, a decreased methylation capacity in the plasma and aorta, indirectly assessed by the ratio of S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH) was detected in HHD mice together with a 172-fold increase in aortic cystathionine levels, indicating increased flux through the transsulfuration pathway. Betaine and its metabolic precursor, choline, were significantly decreased in the livers of HHD mice versus CD mice. Widespread changes in the plasma metabolome of HHD mice versus CD animals were detected, including alterations in acylcarnitines, amino acids, bile acids, ceramides, sphingomyelins, triacylglycerol levels, and several indicators of dysfunctional lipid metabolism. This study confirms the relevance of severe HHcy in the progression of vascular plaque and suggests novel metabolic pathways implicated in the pathophysiology of atherosclerosis.

Previous reports have provided evidence that insufficient or excessive maternal folic acid (FA) intake during pregnancy can alter neurodevelopment of the offspring by modulating prenatal neurogenesis. Furthermore, our earlier work in a mouse model confirmed long-term structural changes at the cellular level of either deficient or excessive FA supply by comparably reducing dendritic arborization of cortical projection neurons. Here, we report that excessive amounts of FA decrease arborization of deep layer projection neurons, but not upper layer neurons and that reduced complexity of deep layer neurons is not observed when folic acid is replaced by folinic acid, a stable reduced form of folate. In addition, deficiency of B 12 , a vitamin that critically regulates folate metabolism, causes even more marked decreases in neuronal arborization in both deep and upper layer neurons and particularly in combination with FA excess. Furthermore, both FA excess and B 12 deficiency affect synaptic density and morphology. Our findings point to neurodevelopmental risks associated with insufficient amounts of prenatal B 12 , particularly in association with high levels of FA intake, suggesting that the neurodevelopmental program is sensitive to an imbalance in the status of these interacting micronutrients., (© 2023. The Author(s).)

Epidemiological studies have reported discrepant findings on the relationship between folic acid intake during pregnancy and risk for gestational diabetes mellitus (GDM). To begin to understand how folic acid impacts metabolic health during pregnancy, we determined the effects of excess folic acid supplementation (5× recommendation) on maternal and fetal offspring metabolic health. Using a mouse (female C57BL/6J) model of diet-induced diabetes in pregnancy (western diet) and control mice, we show that folic acid supplementation improved insulin sensitivity in the female mice fed the western diet and worsened insulin sensitivity in control mice. We found no unmetabolized folic acid in liver from supplemented mice suggesting the metabolic effects of folic acid supplementation are not due to unmetabolized folic acid. Male fetal (gestational day 18.5) offspring from folic acid supplemented dams (western and control) had greater beta cell mass and density than those from unsupplemented dams; this was not observed in female offspring. Differential sex-specific hepatic gene expression profiles were observed in the fetal offspring from supplemented dams but this differed between western and controls. Our findings suggest that folic acid supplementation affects insulin sensitivity in female mice, but is dependent on their metabolic phenotype and has sex-specific effects on offspring pancreas and liver., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Maternal one-carbon metabolism plays an important role in early life programming. There is a well-established connection between the fetal environment and the health status of the offspring. However, there is a knowledge gap on how maternal nutrition impacts stroke outcomes in offspring. The aim of our study was to investigate the role of maternal dietary deficiencies in folic acid or choline on stroke outcomes in 3-month-old offspring. Adult female mice were fed a folic acid-deficient diet, choline-deficient diet, or control diet 4 weeks before pregnancy. They were continued on diets during pregnancy and lactation. Male and female offspring were weaned onto a control diet and at 2 months of age were subjected to ischemic stroke within the sensorimotor cortex via photothrombotic damage. Mothers maintained on either a folic acid-deficient diet or choline-deficient diet had reduced levels of S-adenosylmethionine in the liver and S-adenosylhomocysteine in the plasma. After ischemic stroke, motor function was impaired in 3-month-old offspring from mothers receiving either a folic acid-deficient diet or choline-deficient diet compared to the animals receiving a control diet. In brain tissue, there was no difference in ischemic damage volume. When protein levels were assessed in ischemic brain tissue, there were lower levels of active caspase-3 and hypoxia-inducible factor 1α in males compared to females and betaine levels were reduced in offspring from the mothers receiving a choline-deficient diet. Our results demonstrate that a deficient maternal diet at critical time points in neurodevelopment results in worse stroke outcomes. This study emphasizes the importance of maternal diet and the impact it can have on offspring health., Competing Interests: None

Background: Epidemiological data suggests statins could reduce the risk of dementia, and more specifically, Alzheimer's disease (AD). Pre-clinical data suggests statins reduce the risk of dementia through their pleiotropic effects rather than their cholesterol lowering effects. While AD is a leading cause of dementia, it is frequently found co-morbidly with cerebral small vessel disease and other vascular contributions to cognitive impairment and dementia (VCID), which are another leading cause of dementia. In this study, we determined if atorvastatin ameliorated hyperhomocysteinemia (HHcy)-induced VCID., Methods: Wild-type (C57Bl6/J) mice were placed on a diet to induce HHcy or a control diet each with or without atorvastatin for 14 weeks. Mice underwent novel object recognition testing before tissue collection. Plasma total cholesterol and total homocysteine as well as related metabolites were measured. Using qPCR and NanoString technology, we profiled glial cell-associated gene expression changes. Finally, microglial morphology, astrocyte end feet, and microhemorrhages were analyzed using histological methods., Results: Atorvastatin treatment of HHcy in mice led to no changes in total cholesterol but decreases in total homocysteine in plasma. While HHcy decreased expression of many glial genes, atorvastatin rescued these gene changes, which mostly occurred in oligodendrocytes and microglia. Microglia in HHcy mice with atorvastatin were trending towards fewer processes compared to control with atorvastatin, but there were no atorvastatin effects on astrocyte end feet. While atorvastatin treatment was trending towards increasing the area of microhemorrhages in HHcy mice in the frontal cortex, it only slightly (non-significantly) reduced the number of microhemorrhages. Finally, atorvastatin treatment in HHcy mice led to improved cognition on the novel object recognition task., Conclusions: These data suggest that atorvastatin rescued cognitive changes induced by HHcy most likely through lowering plasma total homocysteine and rescuing gene expression changes rather than impacts on vascular integrity or microglial changes., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Scope: Obesity and insulin resistance (IR) are associated with epigenetic changes of gene expression. However, the relationship between micronutrients, epigenetic regulation of gene expression, and IR during development of diet-induced obesity has yet to be defined. Our objective is to describe the effect of micronutrient addition to diets on IR and its related genes during obesity development., Methods and Results: Male C57BL/6J mice are fed a high-fat (HFD) or low-fat (LFD) diets with or without a multi-vitamin mineral mix (MVM) addition containing vitamins A, B1, B6, B12, and Zn, and Se for 9 weeks. Compared to LFD mice, HFD mice have higher body weight, IR, fasting glucose, insulin, C-peptide, leptin, and hepatic triglyceride concentrations, and dysregulated gene expression in liver, muscle, pancreas, and fat tissues (p < 0.05). The addition of MVM reduces these HFD-induced effects. HFD downregulates 27 genes associated with insulin regulation and adipose tissue function across all tissues by an average of 47% and upregulates five genes by 230% (p < 0.001). Adding MVM downregulates five genes and upregulates one in HFD-fed mice. Both HFD and MVM alter one-carbon metabolites., Conclusion: Addition of micronutrients to the HFD decreases IR and modifies associated gene expression in obese and lean mice., (© 2023 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

We performed liquid chromatography tandem mass spectrometry analysis with the targeted metabolomic kit Biocrates MxP Quant 500, in human brain cortex (Brodmann area 9) and putamen, to reveal metabolic changes characteristic of Parkinson's disease (PD) and PD-related cognitive decline. This case-control study involved 101 subjects (33 PD without dementia, 32 PD with dementia (cortex only), 36 controls). We found changes associated with PD, cognitive status, levodopa levels, and disease progression. The affected pathways include neurotransmitters, bile acids, homocysteine metabolism, amino acids, TCA cycle, polyamines, β-alanine metabolism, fatty acids, acylcarnitines, ceramides, phosphatidylcholines, and several microbiome-derived metabolites. Previously reported levodopa-related homocysteine accumulation in cortex still best explains the dementia status in PD, which can be modified by dietary supplementation. Further investigation is needed to reveal the exact mechanisms behind this pathological change., (© 2023. The Author(s).)

Folic acid (synthetic folate, FA) is consumed in excess in North America and may interact with common pathogenic variants in methylenetetrahydrofolate reductase (MTHFR); the most prevalent inborn error of folate metabolism with wide-ranging obesity-related comorbidities. While preclinical murine models have been valuable to inform on diet-gene interactions, a recent Folate Expert panel has encouraged validation of new animal models. In this study, we characterized a novel zebrafish model of mthfr deficiency and evaluated the effects of genetic loss of mthfr function and FA supplementation during embryonic development on energy homeostasis and metabolism. mthfr-deficient zebrafish were generated using CRISPR mutagenesis and supplemented with no FA (control, 0FA) or 100 μm FA (100FA) throughout embryonic development (0-5 days postfertilization). We show that the genetic loss of mthfr function in zebrafish recapitulates key biochemical hallmarks reported in MTHFR deficiency in humans and leads to greater lipid accumulation and aberrant cholesterol metabolism as reported in the Mthfr murine model. In mthfr-deficient zebrafish, energy homeostasis was also impaired as indicated by altered food intake, reduced metabolic rate and lower expression of central energy-regulatory genes. Microglia abundance, involved in healthy neuronal development, was also reduced. FA supplementation to control zebrafish mimicked many of the adverse effects of mthfr deficiency, some of which were also exacerbated in mthfr-deficient zebrafish. Together, these findings support the translatability of the mthfr-deficient zebrafish as a preclinical model in folate research., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Breastfeeding is the gold standard for early nutrition. Metabolites from the one-carbon metabolism pool are crucial for infant development. The aim of this study is to compare the breast-milk one-carbon metabolic profile to other biofluids where these metabolites are present, including cord and adult blood plasma as well as cerebrospinal fluid. Breast milk ( n = 142), cord blood plasma ( n = 23), maternal plasma ( n = 28), aging adult plasma ( n = 91), cerebrospinal fluid ( n = 92), and infant milk formula ( n = 11) samples were analyzed by LC-MS/MS to quantify choline, betaine, methionine, S-adenosylmethionine, S-adenosylhomocysteine, total homocysteine, and cystathionine. Differences between groups were visualized by principal component analysis and analyzed by Kruskal-Wallis test. Correlation analysis was performed between one-carbon metabolites in human breast milk. Principal component analysis based on these metabolites separated breast milk samples from other biofluids. The S-adenosylmethionine (SAM) concentration was significantly higher in breast milk compared to the other biofluids and was absent in infant milk formulas. Despite many significant correlations between metabolites in one-carbon metabolism, there were no significant correlations between SAM and methionine or total homocysteine. Together, our data indicate a high concentration of SAM in breast milk, which may suggest a strong demand for this metabolite during infant early growth while its absence in infant milk formulas may indicate the inadequacy of this vital metabolic nutrient., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Introduction: Vascular contributions to cognitive impairment and dementia (VCID) are a leading cause of dementia. An underappreciated, modifiable risk factor for VCID is hyperhomocysteinemia (HHcy), defined by elevated levels of plasma homocysteine, most often due to impaired B vitamin absorption in aged persons. Studies aimed at identifying neuropathologic features and gene expression profiles associated with HHcy have been lacking., Methods: A subset of research volunteers from the University of Kentucky Alzheimer's Disease Research Center longitudinal cohort came to autopsy and had ante mortem plasma homocysteine levels available. Brain tissue and blood plasma drawn closest to death were used to measure homocysteine and related metabolites in the current pilot study. Genetic expression profiles of inflammatory markers were evaluated using the Human Neuroinflammation NanoString panel. Further analyses included an evaluation of plasma homocysteine effects on amyloid beta, tau, ionized calcium-binding adaptor molecule 1, and glial fibrillary acidic protein immunohistochemistry in the frontal and occipital cortices. Analytes and other study outcomes were evaluated in relation to ante mortem HHcy status: We identified 13 persons with normal ante mortem plasma homocysteine levels (<14 µmol/L) and 18 who had high plasma homocysteine levels (≥14 µmol/L)., Results: Participants with HHcy demonstrated increased levels of several plasma homocysteine cycle metabolites such as total cysteine, S-adenosyl-homocysteine, cystathionine, and choline. Inflammatory gene expression profiles showed a general downregulation in the setting of elevated plasma homocysteine. HHcy was associated with more and longer microglial processes, but smaller and fewer astrocytes, especially in participants of older age at death. HHcy in older participants was also associated with occipital cortex microhemorrhages and increased severity of atherosclerosis throughout the cerebral vasculature., Conclusions: Increased plasma homocysteine and older age were associated with the downregulation of inflammatory gene expression markers in association with significant glial and vascular pathology changes. Impaired immune function is a plausible mechanism by which HHcy increases cerebrovascular damage leading to impaired cognitive function., Competing Interests: The authors declare they have no conflicts of interest. Author disclosures are available in the supporting information., (© 2022 The Authors. Alzheimer's & Dementia: Translational Research & Clinical Interventions published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Vascular contributions to cognitive impairment and dementia (VCID) particularly Alzheimer's disease and related dementias (ADRDs) are increasing; however, mechanisms driving cerebrovascular decline are poorly understood. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in the folate and methionine cycles. Variants in MTHFR, notably 677 C   > T, are associated with dementias, but no mouse model existed to identify mechanisms by which MTHFR 677C > T increases risk. Therefore, MODEL-AD created a novel knock-in (KI) strain carrying the Mthfr 677C > T allele on the C57BL/6J background ( Mthfr 677C > T ) to characterize morphology and function perturbed by the variant. Consistent with human clinical data, Mthfr 677C > T mice have reduced enzyme activity in the liver and elevated plasma homocysteine levels. MTHFR enzyme activity is also reduced in the Mthfr 677C > T brain. Mice showed reduced tissue perfusion in numerous brain regions by PET/CT as well as significantly reduced vascular density, pericyte number and increased GFAP-expressing astrocytes in frontal cortex. Electron microscopy revealed cerebrovascular damage including endothelial and pericyte apoptosis, reduced luminal size, and increased astrocyte and microglial presence in the microenvironment. Collectively, these data support a mechanism by which variations in MTHFR perturb cerebrovascular health laying the foundation to incorporate our new Mthfr 677C > T mouse model in studies examining genetic susceptibility for cerebrovascular dysfunction in ADRDs.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A and subsequent accumulation of glycosphingolipids with terminal α-D-galactosyl residues. The molecular process through which this abnormal metabolism of glycosphingolipids causes multisystem dysfunction in Fabry disease is not fully understood. We sought to determine whether dysregulated DNA methylation plays a role in the development of this disease. In the present study, using isogenic cellular models derived from Fabry patient endothelial cells, we tested whether manipulation of α-galactosidase A activity and glycosphingolipid metabolism affects DNA methylation. Bisulfite pyrosequencing revealed that changes in α-galactosidase A activity were associated with significantly altered DNA methylation in the androgen receptor promoter, and this effect was highly CpG loci-specific. Methylation array studies showed that α-galactosidase A activity and glycosphingolipid levels were associated with differential methylation of numerous CpG sites throughout the genome. We identified 15 signaling pathways that may be susceptible to methylation alterations in Fabry disease. By incorporating RNA sequencing data, we identified 21 genes that have both differential mRNA expression and methylation. Upregulated expression of collagen type IV alpha 1 and alpha 2 genes correlated with decreased methylation of these two genes. Methionine levels were elevated in Fabry patient cells and Fabry mouse tissues, suggesting that a perturbed methionine cycle contributes to the observed dysregulated methylation patterns. In conclusion, this study provides evidence that α-galactosidase A deficiency and glycosphingolipid storage may affect DNA methylation homeostasis and highlights the importance of epigenetics in the pathogenesis of Fabry disease and, possibly, of other lysosomal storage disorders., Competing Interests: None., (© 2022 Published by Elsevier Inc.)

Objectives: Acute kidney injury (AKI) remains a leading source of morbidity and mortality after cardiothoracic surgery. Insulin-like growth factor-binding protein 7 (IGFBP7), and tissue inhibitor of metalloproteinases-2 (TIMP-2), are novel early-phase renal biomarkers that have been validated as sensitive predictors of AKI. Here the authors studied the efficacy of these biomarkers for predicting AKI after left ventricular assist device (LVAD) implantation and cardiac transplantation., Design/setting/participants/interventions: This was a prospective study of 73 patients undergoing LVAD implantation (n = 37) or heart transplant (n = 36) from 2016 to 2017 at the authors' center. TIMP-2 and IGFBP7 were measured with the NephroCheck Test on urine samples before surgery and one-to-six hours after surgery. NephroCheck scores were assessed as predictors of moderate/severe AKI (Kidney Disease International Global Outcomes 2/3 creatinine criteria) within 48 hours of surgery, and the association with survival to one year was investigated., Measurements and Main Results: The LVAD and transplant cohorts overall were similar in demographics and baseline creatinine (p > 0.05), with the exception of having more African-American patients in the LVAD arm (p = 0.003). Eleven (30%) LVAD and 16 (44%) transplant patients developed moderate/severe AKI. Overall, AKI was associated with postsurgery NephroCheck (odds ratio [95% confidence interval] for 0.1 mg/dL increase: 1.36 [1.04-1.79]; p = 0.03), but not with baseline NephroCheck (p = 0.92). When analyzed by cohort, this effect remained for LVAD (1.68 [1.05-2.71]; p = 0.03) but not for transplant (p = 0.15). Receiver operating characteristic analysis showed postoperative NephroCheck to be superior to baseline creatinine in LVAD (p = 0.046). Furthermore, an increase of 0.1 mg/dL in postoperative NephroCheck was associated with a 10% increase in the risk of mortality (adjusted hazard ratio: 1.11 [1.01-1.21]; p = 0.04) independent of age and body mass index., Conclusion: Assessment of TIMP-2 and IGFBP7 within six hours after surgery appeared effective at predicting AKI in patients with LVADs. Larger studies are warranted to validate these findings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A vitamin B12 deficiency (vit. B12 def.) is common in the elderly, because of changes in metabolism. Clinical studies have reported that a vit. B12 def. results in worse outcome after stroke, and the mechanisms through which a vit. B12 def. changes the brain requires further investigation. This study investigated the role of vit. B12 def. on stroke outcome and mechanisms using aged female mice. Eighteen-month-old females were put on a control or vit. B12 def. diet for 4 weeks, after which an ischemic stroke was induced in the sensorimotor cortex. After damage, motor function was measured, the animals were euthanized, and tissues were collected for analysis. Vit. B12 def. animals had increased levels of total homocysteine in plasma and liver, and choline levels were also increased in the liver. Vit. B12 def. animals had larger damage volume in brain tissue and more apoptosis. The cecum tissue pathway analysis showed dysfunction in B12 transport. The analysis of mitochondrial metabolomics in brain tissue showed reduced levels of metabolites involved in the TCA cycle in vit. B12 def. animals. Motor function after stroke was impaired in vit. B12 def. animals. A dietary vit. B12 def. impairs motor function through increased apoptosis and changes in mitochondrial metabolism in brain tissue.

Food fortification and folic acid supplementation during pregnancy have been implemented as strategies to prevent fetal malformations during pregnancy. However, with the emergence of conditions where folate metabolism and transport are disrupted, such as folate receptor alpha autoantibody (FRαAb)-induced folate deficiency, it is critical to find a folate form that is effective and safe for pharmacologic dosing for prolonged periods. Therefore, in this study, we explored the absorption and tissue distribution of folic acid (PGA), 5-methyl-tetrahydrofolate (MTHF), l-folinic acid (levofolinate), and d,l-folinic acid (Leucovorin) in adult rats. During absorption, all forms are converted to MTHF while some unconverted folate form is transported into the blood, especially PGA. The study confirms the rapid distribution of absorbed folate to the placenta and fetus. FRαAb administered, also accumulates rapidly in the placenta and blocks folate transport to the fetus and high folate concentrations are needed to circumvent or overcome the blocking of FRα. In the presence of FRαAb, both Leucovorin and levofolinate are absorbed and distributed to tissues better than the other forms. However, only 50% of the leucovorin is metabolically active whereas levofolinate is fully active and generates higher tetrahydrofolate (THF). Because levofolinate can readily incorporate into the folate cycle without needing methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) in the first pass and is relatively stable, it should be the folate form of choice during pregnancy, other disorders where large daily doses of folate are needed, and food fortification.

Rationale & Objective: The proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor alirocumab is used in the general population to treat dyslipidemia, but little is known about the effects of alirocumab on lipid levels, biomarkers, the metabolome, and safety in individuals receiving maintenance dialysis., Study Design: Patients receiving maintenance dialysis for at least 3 months and with a low-density lipoprotein cholesterol level of >70 mg/dL were treated with alirocumab for 12 weeks. Laboratory measurements, drug levels, and safety assessments were obtained at baseline and every 4 weeks during the trial., Setting & Participants: In an outpatient setting, 14 patients completed the trial., Intervention: The patients were treated with alirocumab at a full dose of 150 mg every 2 weeks for 12 weeks. The patients were asked to report any adverse events every 2 weeks., Outcomes: There were no unexpected adverse events or laboratory abnormalities in this population receiving dialysis. The drug levels were the same as those for a population not receiving dialysis., Results: Alirocumab resulted in a 45% reduction in the low-density lipoprotein cholesterol level ( P  = 0.005) and a 35% reduction in the apolipoprotein B level ( P  = 0.06). There were no significant decreases in the levels of triglycerides, C-reactive protein, fibrinogen, or other inflammatory biomarkers tested. There were significant decreases in the levels of 7 ceramide, 5 sphingomyelin, and 5 cholesterol ester species., Limitations: This study was performed in only 14 patients who were administered alirocumab for only 12 weeks. This study did not address alirocumab treatment in patients with chronic kidney disease not receiving maintenance dialysis., Conclusions: Individuals receiving maintenance dialysis had a similar response to the PCSK9 inhibitor alirocumab as patients not receiving dialysis. The levels of inflammatory biomarkers were not clearly decreased by alirocumab, but the levels of ceramides, sphingomyelins, and cholesterol esters were significantly reduced., Trial Registration: Clinical Trials.gov as NCT03480568., (© 2022 The Authors.)

Vascular contributions to cognitive impairment and dementia (VCID) are the second leading cause of dementia behind Alzheimer's disease. Apolipoprotein E (ApoE) is a lipid transporting lipoprotein found within the brain and periphery. The APOE ε4 allele is the strongest genetic risk factor for late onset Alzheimer's disease and is a risk factor for VCID. Our lab has previously utilized a dietary model of hyperhomocysteinemia (HHcy) to induce VCID pathology and cognitive deficits in mice. This diet induces perivascular inflammation through cumulative oxidative damage leading to glial mediated inflammation and blood brain barrier breakdown. Here, we examine the impact of ApoE ε4 compared to ε3 alleles on the progression of VCID pathology and inflammation in our dietary model of HHcy. We report a significant resistance to HHcy induction in ε4 mice, accompanied by a number of related differences related to homocysteine (Hcy) metabolism and methylation cycle, or 1-C, metabolites. There were also significant differences in inflammatory profiles between ε3 and ε4 mice, as well as significant reduction in Serpina3n, a serine protease inhibitor associated with ApoE ε4, expression in ε4 HHcy mice relative to ε4 controls. Finally, we find evidence of pervasive sex differences within both genotypes in response to HHcy induction.