Methylenetetrahydrofolate reductase deficiency and high-dose FA supplementation disrupt embryonic development of energy balance and metabolic homeostasis in zebrafish.

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.

Dysregulation of hepatic one-carbon metabolism in classical homocystinuria: Implications of redox-sensitive DHFR repression and tetrahydrofolate depletion for pathogenesis and treatment.

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 (NO2 + NO3) 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.

Metabolomic Effects of Folic Acid Supplementation in Adults: Evidence from the FACT Trial.

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.

Folic acid supplementation in a mouse model of diabetes in pregnancy alters insulin sensitivity in female mice and beta cell mass in offspring.

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.

Dietary vitamin B12 deficiency impairs motor function and changes neuronal survival and choline metabolism after ischemic stroke in middle-aged male and female mice.

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.

Linking biomarkers with healthy lifestyle outcomes after stroke: Supplementary results of a 12-month randomized controlled trial.

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/m2). 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.

Atorvastatin rescues hyperhomocysteinemia-induced cognitive deficits and neuroinflammatory gene changes.

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.

Analysis of differential neonatal lethality in cystathionine ß-synthase deficient mouse models using metabolic profiling.

Cystathionine beta-synthase (CBS) is a key enzyme of the trans-sulfuration pathway that converts homocysteine to cystathionine. Loss of CBS activity due to mutation results in CBS deficiency, an inborn error of metabolism characterized by extreme elevation of plasma total homocysteine (tHcy). C57BL6 mice containing either a homozygous null mutation in the cystathionine ß-synthase (Cbs-/- ) gene or an inactive human CBS protein (Tg-G307S Cbs-/- ) are born in mendelian numbers, but the vast majority die between 18 and 21 days of age due to liver failure. However, adult Cbs null mice that express a hypomorphic allele of human CBS as a transgene (Tg-I278T Cbs-/- ) show almost no neonatal lethality despite having serum tHcy levels similar to mice with no CBS activity. Here, we characterize liver and serum metabolites in neonatal Cbs+/- , Tg-G307S Cbs-/- , and Tg-I278T Cbs-/- mice at 6, 10, and 17 days of age to understand this difference. In serum, we observe similar elevations in tHcy in both Tg-G307S Cbs-/- and Tg-I278T Cbs-/- compared to control animals, but methionine is much more severely elevated in Tg-G307S Cbs-/- mice. Large scale metabolomic analysis of liver tissue confirms that both methionine and methionine-sulfoxide are significantly more elevated in Tg-G307S Cbs-/- animals, along with significant differences in several other metabolites including hexoses, amino acids, other amines, lipids, and carboxylic acids. Our data are consistent with a model that the neonatal lethality observed in CBS-null mice is driven by excess methionine resulting in increased stress on a variety of related pathways including the urea cycle, TCA cycle, gluconeogenesis, and phosphatidylcholine biosynthesis.

Longitudinal profiling of plasma and urine metabolites during liver regeneration in living liver donors.

BACKGROUND: Knowledge of metabolic processes affected by major hepatectomy (MHx), and the metabolic pathways involved in liver regeneration and recovery of function, is limited and mainly derived from animal models. Assessment of restoration of hepatic function is essential in human living liver donors (LD). METHODS: We used a targeted metabolomic approach to longitudinally quantify changes in plasma and urine biomarkers from healthy LD. The biomarkers were analyzed before MHx and at scheduled intervals up to 12 months thereafter. RESULTS: Marked changes were found in the concentration of 15 primary and secondary plasma bile acids. Most significant changes occurred 2 days after MHx and persisted for up to 3 months. In addition, there were significant changes in acylcarnitine, phospholipid, and amino acid metabolism. The sum of aromatic amino acids and the Fischer ratio, both metabolic markers of liver damage, and the symmetrically demethylated arginine to arginine ratio, a marker of kidney function, were affected. CONCLUSIONS: This is the first comprehensive longitudinal study investigating metabolic processes during recovery of liver function after MHx in LD. It provides further evidence of full restoration of metabolic processes 3 months after MHx and supports future investigation to understand how metabolic changes affect donors' hepatic function.

Urinary Cell-Cycle Arrest Biomarkers as Early Predictors of Acute Kidney Injury After Ventricular Assist Device Implantation or Cardiac Transplantation.

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.

Choline and Folic Acid in Diets Consumed during Pregnancy Interact to Program Food Intake and Metabolic Regulation of Male Wistar Rat Offspring.

BACKGROUND: North American women consume high folic acid (FA), but most are not meeting the adequate intakes for choline. High-FA gestational diets induce an obesogenic phenotype in rat offspring. It is unclear if imbalances between FA and other methyl-nutrients (i.e., choline) account for these effects. OBJECTIVE: This study investigated the interaction of choline and FA in gestational diets on food intake, body weight, one-carbon metabolism, and hypothalamic gene expression in male Wistar rat offspring. METHODS: Pregnant Wistar rats were fed an AIN-93G diet with recommended choline and FA [RCRF; 1-fold, control] or high (5-fold) FA with choline at 0.5-fold [low choline and high folic acid (LCHF)], 1-fold [recommended choline and high folic acid (RCHF)], or 2.5-fold [high choline and high folic acid (HCHF)]. Male offspring were weaned to an RCRF diet for 20 wk. Food intake, weight gain, plasma energy-regulatory hormones, brain and plasma one-carbon metabolites, and RNA sequencing (RNA-seq) in pup hypothalamuses were assessed. RESULTS: Adult offspring from LCHF and RCHF, but not HCHF, gestational diets had 10% higher food intake and weight gain than controls (P < 0.01). HCHF newborn pups had lower plasma insulin and leptin compared with LCHF and RCHF pups (P < 0.05), respectively. Pup brain choline (P < 0.05) and betaine (P < 0.01) were 22-33% higher in HCHF pups compared with LCHF pups; methionine was ∼23% lower after all high FA diets compared with RCRF (P < 0.01). LCHF adult offspring had lower brain choline (P < 0.05) than all groups and lower plasma 5-methyltetrahydrofolate (P < 0.05) than RCRF and RCHF groups. HCHF adult offspring had lower plasma cystathionine (P < 0.05) than LCHF adult offspring and lower homocysteine (P < 0.01) than RCHF and RCRF adult offspring. RNA-seq identified 144 differentially expressed genes in the hypothalamus of HCHF newborns compared with controls. CONCLUSIONS: Increased choline in gestational diets modified the programming effects of high FA on long-term food intake regulation, plasma energy-regulatory hormones, one-carbon metabolism, and hypothalamic gene expression in male Wistar rat offspring, emphasizing a need for more attention to the choline and FA balance in maternal diets.

Metabolomic studies identify changes in transmethylation and polyamine metabolism in a brain-specific mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and the quintessential disorder of mechanistic Target of Rapamycin Complex 1 (mTORC1) dysregulation. Loss of either causative gene, TSC1 or TSC2, leads to constitutive mTORC1 kinase activation and a pathologically anabolic state of macromolecular biosynthesis. Little is known about the organ-specific metabolic reprogramming that occurs in TSC-affected organs. Using a mouse model of TSC in which Tsc2 is disrupted in radial glial precursors and their neuronal and glial descendants, we performed an unbiased metabolomic analysis of hippocampi to identify Tsc2-dependent metabolic changes. Significant metabolic reprogramming was found in well-established pathways associated with mTORC1 activation, including redox homeostasis, glutamine/tricarboxylic acid cycle, pentose and nucleotide metabolism. Changes in two novel pathways were identified: transmethylation and polyamine metabolism. Changes in transmethylation included reduced methionine, cystathionine, S-adenosylmethionine (SAM-the major methyl donor), reduced SAM/S-adenosylhomocysteine ratio (cellular methylation potential), and elevated betaine, an alternative methyl donor. These changes were associated with alterations in SAM-dependent methylation pathways and expression of the enzymes methionine adenosyltransferase 2A and cystathionine beta synthase. We also found increased levels of the polyamine putrescine due to increased activity of ornithine decarboxylase, the rate-determining enzyme in polyamine synthesis. Treatment of Tsc2+/- mice with the ornithine decarboxylase inhibitor α-difluoromethylornithine, to reduce putrescine synthesis dose-dependently reduced hippocampal astrogliosis. These data establish roles for SAM-dependent methylation reactions and polyamine metabolism in TSC neuropathology. Importantly, both pathways are amenable to nutritional or pharmacologic therapy.

Relationship of Cerebrospinal Fluid Vitamin B12 Status Markers With Parkinson's Disease Progression.

BACKGROUND: Using blood specimens from untreated early Parkinson's disease (PD) patients from the DATATOP trial, we found that subjects in the low serum vitamin B12 tertile experienced greater annualized change in ambulatory capacity score, whereas those with moderately elevated (>15 µmol/L) total homocysteine had greater annualized declines in the Mini-Mental State Exam. METHODS: In this this study we sought to determine whether levels of cerebrospinal fluid (CSF) B12 markers were also associated with progression of PD. RESULTS: The annualized change in the UPDRS "walking" item, a component of the ambulatory capacity score, was worse in the low B12 tertile. No association with change in the Mini-Mental State Exam was seen for those 7% with the highest baseline CSF total homocysteine. CONCLUSIONS: In these untreated early-PD subjects, low CSF B12 predicted greater worsening of the UPDRS "walking" item, whereas CSF total homocysteine was not associated with progression of cognitive impairment. These findings extend and partially support our findings in serum. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.

Methyl-donor deficiency is a risk factor for neurodegenerative diseases. Dietary deficiency of the methyl-donors methionine and choline [methionine-choline-deficient (MCD) diet] is a well-established model of nonalcoholic steatohepatitis (NASH), yet brain metabolism has not been studied in this model. We hypothesized that supplemental betaine would protect both the liver and brain in this model and that any benefit to the brain would be due to improved liver metabolism because betaine is a methyl-donor in liver methylation but is not metabolically active in the brain. We fed male Sprague-Dawley rats a control diet, MCD diet, or betaine-supplemented MCD (MCD+B) diet for 8 wk and collected blood and tissue. As expected, betaine prevented MCD diet-induced NASH. However, contrary to our prediction, it did not appear to do so by stimulating methylation; the MCD+B diet worsened hyperhomocysteinemia and depressed liver methylation potential 8-fold compared with the MCD diet. Instead, it significantly increased the expression of genes involved in ß-oxidation: fibroblast growth factor 21 and peroxisome proliferator-activated receptor α. In contrast to that of the liver, brain methylation potential was unaffected by diet. Nevertheless, several phospholipid (PL) subclasses involved in stabilizing brain membranes were decreased by the MCD diet, and these improved modestly with betaine. The protective effect of betaine is likely due to the stimulation of ß-oxidation in liver and the effects on PL metabolism in brain.-Abu Ahmad, N., Raizman, M., Weizmann, N., Wasek, B., Arning, E., Bottiglieri, T., Tirosh, O., Troen, A. M. Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.

Neuropathology of vitamin B12 deficiency in the Cd320-/- mouse.

In humans, vitamin B12 deficiency causes peripheral and CNS manifestations. Loss of myelin in the peripheral nerves and the spinal cord (SC) contributes to peripheral neuropathy and motor deficits. The metabolic basis for the demyelination and brain disorder is unknown. The transcobalamin receptor-knockout mouse ( Cd320-/-) develops cobalamin (Cbl) deficiency in the nervous system, with mild anemia. A decreased S-adenosylmethionine: S-adenosylhomocysteine ratio and increased methionine were seen in the brain with no significant changes in neurotransmitter metabolites. The structural pathology in the SC presented as loss of myelin in the axonal tracts with inflammation. The sciatic nerve (SN) showed increased nonuniform, internodal segments suggesting demyelination, and remyelination in progress. Consistent with these changes, the Cd320-/- mouse showed an increased latency to thermal nociception. Further, lower amplitude of compound action potential in the SN suggested that the functional capacity of the heavily myelinated axons were preferentially compromised, leading to loss of peripheral sensation. Although the metabolic basis for the demyelination and the structural and functional alterations of the nervous system in Cbl deficiency remain unresolved, the Cd320-/- mouse provides a unique model to investigate the pathologic consequences of vitamin B12 deficiency. -Arora, K., Sequeira, J. M., Alarcon, J. M., Wasek, B., Arning, E., Bottiglieri, T., Quadros, E. V. Neuropathology of vitamin B12 deficiency in the Cd320-/- mouse.

Correction to: Post-mortem tissue analyses in a patient with succinic semialdehyde dehydrogenase deficiency (SSADHD). I. Metabolomic outcomes.

Upon publication, it was noted that five of the on-line supplementary figures had incorrect figure: figure legend associations. These were supplementary Figs. 6, 7, 14, 15, and 23.

Post-mortem tissue analyses in a patient with succinic semialdehyde dehydrogenase deficiency (SSADHD). I. Metabolomic outcomes.

Metabolomic characterization of post-mortem tissues (frontal and parietal cortices, pons, cerebellum, hippocampus, cerebral cortex, liver and kidney) derived from a 37 y.o. male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) was performed in conjunction with four parallel series of control tissues. Amino acids, acylcarnitines, guanidino- species (guanidinoacetic acid, creatine, creatinine) and GABA-related intermediates were quantified using UPLC and mass spectrometric methods that included isotopically labeled internal standards. Amino acid analyses revealed significant elevation of aspartic acid and depletion of glutamine in patient tissues. Evidence for disruption of short-chain fatty acid metabolism, manifest as altered C4OH, C5, C5:1, C5DC (dicarboxylic) and C12OH carnitines, was observed. Creatine and guanidinoacetic acids were decreased and elevated, respectively. GABA-associated metabolites (total GABA, γ-hydroxybutyric acid, succinic semialdehyde, 4-guanidinobutyrate, 4,5-dihydroxyhexanoic acid and homocarnosine) were significantly increased in patient tissues, including liver and kidney. The data support disruption of fat, creatine and amino acid metabolism as a component of the pathophysiology of SSADHD, and underscore the observation that metabolites measured in patient physiological fluids provide an unreliable reflection of brain metabolism.

One-carbon metabolism supplementation improves outcome after stroke in aged male MTHFR-deficient mice.

The prevalence of stroke increases with age and the ability to absorb all nutrients from our diets decreases with age. Nutrition is a modifiable risk factor for stroke, which is a leading cause of death and disability in world-wide. Deficiencies in one­carbon metabolism, including in methyltetrahydrofolate reductase (MTHFR), have been linked to increased risk of stroke. The Mthfr+/- mice mouse model mimic the phenotype of the MTHFR677C➔T polymorphism, such as elevated levels of homocystine. Using this mouse model, the aim of this study was to investigate the impact of dietary supplementation with 5-methylTHF, vitamin B12, and choline after ischemic stroke. Male Mthfr+/- and wildtype littermate control mice were aged (~1.5-year-old) and were placed on control diet (CD) 4-weeks prior to sensorimotor cortex damage using photothrombosis (PT), a model for ischemic stroke. Post-operatively, one group of Mthfr+/- and wildtype littermate mice were placed on 5-methylTHF, vitamin B12, and choline supplemented diet (SD). Four weeks after PT and SD motor function was assessed using the accelerating rotarod, forepaw asymmetry, and ladder beam walking tasks. Total homocysteine and cysteine levels were measured in blood. Brain tissue was processed to assess lesion volume and investigate biochemical and molecular changes. After PT and SD, Mthfr+/- mice were able to stay on the accelerating rotarod longer and used their impaired forepaw to explore more when compared to CD animals. Furthermore, total homocysteine levels in plasma and lesion volume were reduced in Mthfr+/+ and Mthfr+/- SD mice. Within the damage site, there were reduced levels of apoptotic cell death and increased neuroprotective cellular response in the brains of SD treated Mthfr+/- mice. This study reveals a critical role for one­carbon supplementation, with 5-methylTHF, vitamin B12, and choline, in supporting improvement after ischemic stroke damage.

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

Gamma-Hydroxybutyrate content in dried bloodspots facilitates newborn detection of succinic semialdehyde dehydrogenase deficiency.

Increased gamma-hydroxybutyric acid in urine and blood are metabolic hallmarks of succinic semialdehyde dehydrogenase deficiency, a defect of 4-aminobutyric acid metabolism. Here, we examined the hypothesis that succinic semialdehyde dehydrogenase deficiency could be identified via measurement of gamma-hydroxybutyric acid in newborn and post-newborn dried bloodspots. Quantitation of gamma-hydroxybutyric acid using liquid chromatography-tandem mass spectrometry in twelve archival newborn patient dried bloodspots was 360 ±â€¯57 µM (mean, standard error; range 111-767), all values exceeding the previously established cutoff for newborn detection of 78 µΜ established from 2831 dried bloodspots derived from newborns, neonates and children. Gamma-hydroxybutyric acid in post-newborn dried bloodspots (n = 19; ages 0.8-38 years) was 191 ±â€¯65 µM (mean, standard error; range 20-1218), exceeding the aforementioned GHB cutoff for patients approximately 10 years of age or younger. Further, gamma-hydroxybutyric acid in post-newborn dried bloodspots displayed a significant (p < .0001) inverse correlation with age. This preliminary study suggests that succinic semialdehyde dehydrogenase deficiency may be identified in newborn and post-newborn dried bloodspots via quantitation of gamma-hydroxybutyric acid, while forming the platform for more extensive studies in affected and unaffected dried bloodspots.