Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.

Cystathionine ß-synthase-deficient homocystinuria (HCU) is a poorly understood, life-threatening inborn error of sulfur metabolism. Analysis of hepatic glutathione (GSH) metabolism in a mouse model of HCU demonstrated significant depletion of cysteine, GSH, and GSH disulfide independent of the block in trans-sulfuration compared with wild-type controls. HCU induced the expression of the catalytic and regulatory subunits of γ-glutamyl ligase, GSH synthase (GS), γ-glutamyl transpeptidase 1, 5-oxoprolinase (OPLAH), and the GSH-dependent methylglyoxal detoxification enzyme, glyoxalase-1. Multiple components of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant-response regulatory axis were induced without any detectable activation of Nrf2. Metabolomic analysis revealed the accumulation of multiple γ-glutamyl amino acids and that plasma ophthalmate levels could serve as a noninvasive marker for hepatic redox stress. Neither cysteine, nor betaine treatment was able to reverse the observed enzyme inductions. Taurine treatment normalized the expression levels of γ-glutamyl ligase C/M, GS, OPLAH, and glyoxalase-1, and reversed HCU-induced deficits in protein glutathionylation by acting to double GSH levels relative to controls. Collectively, our data indicate that the perturbation of the γ-glutamyl cycle could contribute to multiple sequelae in HCU and that taurine has significant therapeutic potential for both HCU and other diseases for which GSH depletion is a critical pathogenic factor.-Maclean, K. N., Jiang, H., Aivazidis, S., Kim, E., Shearn, C. T., Harris, P. S., Petersen, D. R., Allen, R. H., Stabler, S. P., Roede, J. R. Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.

Cystathionine beta-synthase deficiency alters hepatic phospholipid and choline metabolism: Post-translational repression of phosphatidylethanolamine N-methyltransferase is a consequence rather than a cause of liver injury in homocystinuria.

Classical homocystinuria (HCU) due to inactivating mutation of cystathionine ß-synthase (CBS) is a poorly understood life-threatening inborn error of sulfur metabolism. A previously described cbs-/- mouse model exhibits a semi-lethal phenotype due to neonatal liver failure. The transgenic HO mouse model of HCU exhibits only mild liver injury and recapitulates multiple aspects of the disease as it occurs in humans. Disruption of the methionine cycle in HCU has the potential to impact multiple aspect of phospholipid (PL) metabolism by disruption of both the Kennedy pathway and phosphatidylethanolamine N-methyltransferase (PEMT) mediated synthesis of phosphatidylcholine (PC). Comparative metabolomic analysis of HO mouse liver revealed decreased levels of choline, and choline phosphate indicating disruption of the Kennedy pathway. Alterations in the relative levels of multiple species of PL included significant increases in PL degradation products consistent with enhanced membrane PL turnover. A significant decrease in PC containing 20:4n6 which primarily formed by the methylation of phosphatidylethanolamine to PC was consistent with decreased flux through PEMT. Hepatic expression of PEMT in both the cbs-/- and HO models is post-translationally repressed with decreased levels of PEMT protein and activity that inversely-correlates with the scale of liver injury. Failure to induce further repression of PEMT in HO mice by increased homocysteine, methionine and S-adenosylhomocysteine or depletion of glutathione combined with examination of multiple homocysteine-independent models of liver injury indicated that repression of PEMT in HCU is a consequence rather than a cause of liver injury. Collectively, our data show significant alteration of a broad range of hepatic PL and choline metabolism in HCU with the potential to contribute to multiple aspects of pathogenesis in this disease.

Identification of ABC transporters acting in vitamin B12 metabolism in Caenorhabditis elegans.

Vitamin B12 (cobalamin, Cbl) is a micronutrient essential to human health. Cbl is not utilized as is but must go through complex subcellular and metabolic processing to generate two cofactor forms: methyl-Cbl for methionine synthase, a cytosolic enzyme; and adenosyl-Cbl for methylmalonyl-CoA mutase, a mitochondrial enzyme. Some 10-12 human genes have been identified responsible for the intracellular conversion of Cbl to cofactor forms, including genes that code for ATP-binding cassette (ABC) transporters acting at the lysosomal and plasma membranes. However, the gene for mitochondrial uptake is not known. We hypothesized that ABC transporters should be candidates for other uptake and efflux functions, including mitochondrial transport, and set out to screen ABC transporter mutants for blocks in Cbl utilization using the nematode roundworm Caenorhabditis elegans. Thirty-seven mutant ABC transporters were screened for the excretion of methylmalonic acid (MMA), which should result from loss of Cbl transport into the mitochondria. One mutant, wht-6, showed elevated MMA excretion and reduced [14C]-propionate incorporation, pointing to a functional block in methylmalonyl-CoA mutase. In contrast, the wht-6 mutant appeared to have a normal cytosolic pathway based on analysis of cystathionine excretion, suggesting that cytosolic methionine synthase was functioning properly. Further, the MMA excretion in wht-6 could be partially reversed by including vitamin B12 in the assay medium. The human ortholog of wht-6 is a member of the G family of ABC transporters. We propose wht-6 as a candidate for the transport of Cbl into mitochondria and suggest that a member of the corresponding ABCG family of ABC transporters has this role in humans. Our ABC transporter screen also revealed that mrp-1 and mrp-2 mutants excreted lower MMA than wild type, suggesting they were concentrating intracellular Cbl, consistent with the cellular efflux defect proposed for the mammalian MRP1 ABC transporter.

Molecular and biochemical investigations of patients with intermediate or severe hyperhomocysteinemia.

A discrepancy has been identified between numbers of expected and identified patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. Patients homozygous for the frequent c.833T>C (p.I278T) are most often responsive to vitamin B6, and can present with a total-homocysteine (tHcy) <100 µM on a normal diet. In Denmark, patients with tHcy <100 µM are not routinely sequenced for CBS(2) mutations. This study investigated the prevalence of CBS mutations and the common methylenetetrahydrofolate reductase (MTHFR) c.677C>T polymorphism in patients with tHcy ≥ 50 µM and the association with clinical manifestations. We studied a cohort of patients with intermediate and severe hyperhomocysteinemia (tHcy ≥ 50 µM) determined between 1996 and 2011. Among the 413 eligible patients, 184 (45%) patients agreed to participate in the present follow-up study. A MTHFR(3)c.677TT genotype was found in 49% of the patients. Eight patients were found to have mutations in CBS(2). Of those, two were homozygous for c.833T>C (p.I278T), and four were compound heterozygous for c.833T>C. One c.833T>C (p.I278T) compound heterozygote was identified by lowering the threshold for sequencing from tHcy at 100 µM to 50 µM. The most prominent clinical presentation among patients with a CBS(2) mutation was thrombosis presenting at a median age of 25 years. In case of arterial or venous thrombosis without any explanation in individuals below 40 years, tHcy should be part of the thrombophilia screening. When tHcy is between 50 and 100 µM genotyping for the MTHFR(3) c.677TT is relevant, and when tHcy >100 µM CBS should be genotyped.

Identification of specific corrinoids reveals corrinoid modification in dechlorinating microbial communities.

Cobalamin and other corrinoids are essential cofactors for many organisms. The majority of microbes with corrinoid-dependent enzymes do not produce corrinoids de novo, and instead must acquire corrinoids produced by other organisms in their environment. However, the profile of corrinoids produced in corrinoid-dependent microbial communities, as well as the exchange and modification of corrinoids among community members have not been well studied. In this study, we applied a newly developed liquid chromatography tandem mass spectrometry-based corrinoid detection method to examine relationships among corrinoids, their lower ligand bases and specific microbial groups in microbial communities containing Dehalococcoides mccartyi that has an obligate requirement for benzimidazole-containing corrinoids for trichloroethene respiration. We found that p-cresolylcobamide ([p-Cre]Cba) and cobalamin were the most abundant corrinoids in the communities. It suggests that members of the family Veillonellaceae are associated with the production of [p-Cre]Cba. The decrease of supernatant-associated [p-Cre]Cba and the increase of biomass-associated cobalamin were correlated with the growth of D. mccartyi by dechlorination. This supports the hypothesis that D. mccartyi is capable of fulfilling its corrinoid requirements in a community through corrinoid remodelling, in this case, by importing extracellular [p-Cre]Cba and 5,6-dimethylbenzimidazole (DMB) (the lower ligand of cobalamin), to produce cobalamin as a cofactor for dechlorination. This study also highlights the role of DMB, the lower ligand produced in all of the studied communities, in corrinoid remodelling. These findings provide novel insights on roles played by different phylogenetic groups in corrinoid production and corrinoid exchange within microbial communities. This study may also have implications for optimizing chlorinated solvent bioremediation.

Vitamin B-12 Status Differs among Pregnant, Lactating, and Control Women with Equivalent Nutrient Intakes.

BACKGROUND: Limited data are available from controlled studies on biomarkers of maternal vitamin B-12 status. OBJECTIVE: We sought to quantify the effects of pregnancy and lactation on the vitamin B-12 status response to a known and highly controlled vitamin B-12 intake. METHODS: As part of a 10-12 wk feeding trial, pregnant (26-29 wk gestation; n = 26), lactating (5 wk postpartum; n = 28), and control (nonpregnant, nonlactating; n = 21) women consumed vitamin B-12 amounts of ∼8.6 µg/d [mixed diet (∼6 µg/d) plus a prenatal multivitamin supplement (2.6 µg/d)]. Serum vitamin B-12, holotranscobalamin (bioactive form of vitamin B-12), methylmalonic acid (MMA), and homocysteine were measured at baseline and study-end. RESULTS: All participants achieved adequate vitamin B-12 status in response to the study dose. Compared with control women, pregnant women had lower serum vitamin B-12 (-21%; P = 0.02) at study-end, whereas lactating women had higher (P = 0.04) serum vitamin B-12 throughout the study (+26% at study-end). Consumption of the study vitamin B-12 dose increased serum holotranscobalamin in all reproductive groups (+16-42%; P ≤ 0.009). At study-end, pregnant (vs. control) women had a higher holotranscobalamin-to-vitamin B-12 ratio (P = 0.04) with ∼30% (vs. 20%) of total vitamin B-12 in the bioactive form. Serum MMA increased during pregnancy (+50%; P < 0.001) but did not differ by reproductive state at study-end. Serum homocysteine increased in pregnant women (+15%; P = 0.009) but decreased in control and lactating women (-16-17%; P < 0.001). Despite these changes, pregnant women had ∼20% lower serum homocysteine than the other 2 groups at study-end (P ≤ 0.02). CONCLUSION: Pregnancy and lactation alter vitamin B-12 status in a manner consistent with enhanced vitamin B-12 supply to the child. Consumption of the study vitamin B-12 dose (∼3 times the RDA) increased the bioactive form of vitamin B-12, suggesting that women in these reproductive states may benefit from vitamin B-12 intakes exceeding current recommendations. This trial was registered at clinicaltrials.gov as NCT01127022.

Altered hepatic sulfur metabolism in cystathionine ß-synthase-deficient homocystinuria: regulatory role of taurine on competing cysteine oxidation pathways.

Cystathionine ß-synthase-deficient homocystinuria (HCU) is a serious life-threatening inborn error of sulfur metabolism with poorly understood pathogenic mechanisms. We investigated the effect of HCU on hepatic cysteine oxidation in a transgenic mouse model of the disease. Cysteine dioxygenase (CDO) protein levels were 90% repressed without any change in mRNA levels. Cysteinesulfinic acid decarboxylase (CSAD) was induced at both the mRNA (8-fold) and protein (15-fold) levels. Cysteine supplementation normalized CDO protein levels without reversing the induction of CSAD. Regulatory changes in CDO and CSAD expression were proportional to homocysteine elevation, indicating a possible threshold effect. Hepatic and blood taurine levels in HCU animals were decreased by 21 and 35%, respectively, and normalized by cysteine supplementation. Expression of the cytoplasmic (GOT1) and mitochondrial (GOT2) isoforms of glutamic-oxaloacetic transaminase were repressed in HCU animals by 86 and 30%, respectively. HCU induced regulatory changes in CSAD, CDO, and GOT1 expression were normalized by taurine supplementation, indicating that cysteine is not the only sulfur compound that regulates hepatic cysteine oxidation. Collectively, our results indicate that HCU induces significant alterations of sulfur metabolism with the potential to contribute to pathogenesis and that cysteine and taurine have the potential to serve as adjunctive treatments in this disease.

Assessment of upper airway patency in spontaneously breathing non-intubated neonates and infants undergoing conventional MRI of head and neck.

PURPOSE: Neonates and infants frequently undergo MRI examinations of the brain or head and neck in spontaneous respiration. This study aims to evaluate the patency of the upper airway and associated risk factors in spontaneously breathing neonates and infants undergoing MRI of head and neck. METHODS: Airway patency was assessed on sagittal and axial MRI images of the head and neck region for neonates and infants retrospectively. Anteroposterior diameters were measured at the soft palate and tongue levels as well as a lateral diameter at the tongue level for the patent airway. Chart review for risk factors was carried out. RESULTS: A total of 831 children between 0 and 12 months of age had an MRI. Eighty-two children with spontaneous ventilation were included. The airway was occluded in 29/82 (35%) of children. Twenty-four out of 29 (83%) children with airway occlusion had a depressed level of consciousness, 7/24 (29%) of whom were sedated with a single dose of benzodiazepine and 17/24 (71%) were on anti-epileptic therapy for an underlying seizure disorder and/or hypoxic ischemic encephalopathy. Forty-three out of 82 (65%) of children had an open airway. The airway diameters (mean ± SD) were 5.9 ± 2 mm (anteroposterior (AP) at soft palate), 7.4 ± 2.9 mm (lateral at soft palate), and 6.3 mm ± 1.6 (AP at dorsum of tongue). CONCLUSION: A significant proportion of spontaneously breathing neonates and infants with hypoxic ischemic encephalopathy or sedation show evidence of airway obstruction during MRI. Careful pre-MRI screening for decision of spontaneous breathing versus artificial airway support during MRI and robust airway monitoring during MRI are required for these vulnerable children.

Sustainable growth of Dehalococcoides mccartyi 195 by corrinoid salvaging and remodeling in defined lactate-fermenting consortia.

Corrinoids are essential cofactors of reductive dehalogenases in Dehalococcoides mccartyi, an important bacterium in bioremediation, yet sequenced D. mccartyi strains do not possess the complete pathway for de novo corrinoid biosynthesis. Pelosinus sp. and Desulfovibrio sp. have been detected in dechlorinating communities enriched from contaminated groundwater without exogenous cobalamin corrinoid. To investigate the corrinoid-related interactions among key members of these communities, we constructed consortia by growing D. mccartyi strain 195 (Dhc195) in cobalamin-free, trichloroethene (TCE)- and lactate-amended medium in cocultures with Desulfovibrio vulgaris Hildenborough (DvH) or Pelosinus fermentans R7 (PfR7) and with both in tricultures. Only the triculture exhibited sustainable dechlorination and cell growth when a physiological level of 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, was provided. In the triculture, DvH provided hydrogen while PfR7 provided corrinoids to Dhc195, and the initiation of dechlorination and Dhc195 cell growth was highly dependent on the growth of PfR7. Corrinoid analysis indicated that Dhc195 imported and remodeled the phenolic corrinoids produced by PfR7 into cobalamin in the presence of DMB. Transcriptomic analyses of Dhc195 showed the induction of the CbiZ-dependent corrinoid-remodeling pathway and BtuFCD corrinoid ABC transporter genes during corrinoid salvaging and remodeling. In contrast, another operon annotated to encode a putative iron/cobalamin ABC transporter (DET1174-DET1176) was induced when cobalamin was exogenously provided. Interestingly, a global upregulation of phage-related genes was observed when PfR7 was present. These findings provide insights into both the gene regulation of corrinoid salvaging and remodeling in Dhc195 when it is grown without exogenous cobalamin and microbe-to-microbe interactions in dechlorinating microbial communities.

Cystathionine protects against endoplasmic reticulum stress-induced lipid accumulation, tissue injury, and apoptotic cell death.

Cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine) is a non-proteinogenic thioether containing amino acid. In mammals, cystathionine is formed as an intermediate of the transsulfuration pathway by the condensation of serine and homocysteine (Hcy) in a reaction catalyzed by cystathionine ß-synthase (CBS). Cystathionine is subsequently converted to cysteine plus ammonia and α-ketobutyrate by the action of cystathionine γ-lyase (CGL). Pathogenic mutations in CBS result in CBS-deficient homocystinuria (HCU) which, if untreated, results in mental retardation, thromboembolic complications and connective tissue disorders. Currently there is no known function for cystathionine other than serving as an intermediate in transsulfuration and to date, the possible contribution of the abolition of cystathionine synthesis to pathogenesis in HCU has not been investigated. Using both mouse and cell-culture models, we have found that cystathionine is capable of blocking the induction of hepatic steatosis and kidney injury, acute tubular necrosis, and apoptotic cell death by the endoplasmic reticulum stress inducing agent tunicamycin. Northern and Western blotting analysis indicate that the protective effects of cystathionine occur without any obvious alteration of the induction of the unfolded protein response. Our data constitute the first experimental evidence that the abolition of cystathionine synthesis may contribute to the pathology of HCU and that this compound has therapeutic potential for disease states where ER stress is implicated as a primary initiating pathogenic factor.

Disruption of shmt1 impairs hippocampal neurogenesis and mnemonic function in mice.

Impaired folate-mediated one-carbon metabolism (OCM) has emerged as a risk factor for several diseases associated with age-related cognitive decline, but the underlying mechanisms remain unknown and thus hinder the identification of subpopulations most vulnerable to OCM disruption. Here we investigated the role of serine hydroxymethyltransferase 1 (SHMT1), a folate-dependent enzyme regulating de novo thymidylate biosynthesis, in influencing neuronal and cognitive function in the adult mouse. We observed Shmt1 expression in the hippocampus, including the granule cell layer of the dentate gyrus (DG), and examined hippocampal neurogenesis and hippocampal-dependent fear conditioning in mice deficient for Shmt1. We used a 3 × 3 design in which adult male Shmt1(+/+), Shmt1(+/-), and Shmt1(-/-) mice were fed folic acid control (2 mg/kg), folic acid-deficient (0 mg/kg), or folic acid-supplemented (8 mg/kg) diets from weaning through the duration of the study. Proliferation within the DG was elevated by 70% in Shmt1(+/-) mice, yet the number of newborn mature neurons was reduced by 98% compared with that in Shmt1(+/+) mice. Concomitant with these alterations, Shmt1(+/-) mice showed a 45% reduction in mnemonic recall during trace fear conditioning. Dietary folate manipulations alone did not influence neural outcomes. Together, these data identify SHMT1 as one of the first enzymes within the OCM pathway to regulate neuronal and cognitive profiles and implicate impaired thymidylate biosynthesis in the etiology of folate-related neuropathogenesis.

Reduced MTHFD1 activity in male mice perturbs folate- and choline-dependent one-carbon metabolism as well as transsulfuration.

Impaired utilization of folate is caused by insufficient dietary intake and/or genetic variation and has been shown to prompt changes in related pathways, including choline and methionine metabolism. These pathways have been shown to be sensitive to variation within the Mthfd1 gene, which codes for a folate-metabolizing enzyme responsible for generating 1-carbon (1-C)-substituted folate derivatives. The Mthfd1(gt/+) mouse serves as a potential model of human Mthfd1 loss-of-function genetic variants that impair MTHFD1 function. This study investigated the effects of the Mthfd1(gt/+) genotype and folate intake on markers of choline, folate, methionine, and transsulfuration metabolism. Male Mthfd1(gt/+) and Mthfd1(+/+) mice were randomly assigned at weaning (3 wk of age) to either a control (2 mg/kg folic acid) or folate-deficient (0 mg/kg folic acid) diet for 5 wk. Mice were killed at 8 wk of age following 12 h of food deprivation; blood and liver samples were analyzed for choline, methionine, and transsulfuration biomarkers. Independent of folate intake, mice with the Mthfd1(gt/+) genotype had higher hepatic concentrations of choline (P = 0.005), betaine (P = 0.013), and dimethylglycine (P = 0.004) and lower hepatic concentrations of glycerophosphocholine (P = 0.002) relative to Mthfd1(+/+) mice. Mthfd1(gt/+) mice also had higher plasma concentrations of homocysteine (P = 0.0016) and cysteine (P < 0.001) as well as lower plasma concentrations of methionine (P = 0.0003) and cystathionine (P = 0.011). The metabolic alterations observed in Mthfd1(gt/+) mice indicate perturbed choline and folate-dependent 1-C metabolism and support the future use of Mthfd1(gt/+) mice as a tool to investigate the impact of impaired 1-C metabolism on disease outcomes.

Profiles of cognitive functioning in a population-based sample of centenarians using factor mixture analysis.

UNLABELLED: BACKGROUND/STUDY CONTEXT: The goal of the study was to identify and characterize latent profiles (clusters) of cognitive functioning in centenarians and the psychometric properties of cognitive measures within them. METHODS: Data were collected from cross-sectional, population-based sample of 244 centenarians (aged 98 to 108, 15.8% men, 20.5% African American, 38.0% community-dwelling) from 44 counties in northern Georgia participating in the Georgia Centenarian Study (2001-2008). Measures included the Mini-Mental State Examination (MMSE), Severe Impairment Battery (SIB), Wechsler Adult Intelligence Scale-III Similarities subtest (WAIS), Hand Tapping, Behavioral Dyscontrol Scale (BDS), Controlled Oral Word Association Test (COWAT), and Fuld Object Memory Evaluation (FOME). The Global Deterioration Rating Scale (GDRS) was used to independently evaluate criterion-related validity for distinguishing cognitively normal and impaired groups. Relevant covariates included directly assessed functional status for basic and instrumental activities of daily living (DAFS), race, gender, educational attainment, Geriatric Depression Scale Short Form (GDS), and vision and hearing problems. RESULTS: Results suggest two distinct classes of cognitive performance in this centenarian sample. Approximately one third of the centenarians show a pattern of markedly lower cognitive performance on most measures. Group membership is independently well predicted (area under the curve [AUC] = .83) by GDRS scores (sensitivity 67.7%, specificity 82.4%). Membership in the lower cognitive performance group was more likely for individuals who were older, African Americans, had more depressive symptoms, lower plasma folate, carriers of the apolipoprotein E (APOE) ε4 allele, facility residents, and individuals who died in the 2 years following interview. CONCLUSIONS: In a population expected to have high prevalence of dementia, latent subtypes can be distinguished via factor mixture analysis that provide normative values for cognitive functioning. The present study allows estimates for normative cognitive performance in this age group.

Nuclear localization of de novo thymidylate biosynthesis pathway is required to prevent uracil accumulation in DNA.

Uracil accumulates in DNA as a result of impaired folate-dependent de novo thymidylate biosynthesis, a pathway composed of the enzymes serine hydroxymethyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase. In G1, this pathway is present in the cytoplasm and at S phase undergoes small ubiquitin-like modifier-dependent translocation to the nucleus. It is not known whether this pathway functions in the cytoplasm, nucleus, or both in vivo. SHMT1 generates 5,10-methylenetetrahydrofolate for de novo thymidylate biosynthesis, a limiting step in the pathway, but also tightly binds 5-methyltetrahydrofolate in the cytoplasm, a required cofactor for homocysteine remethylation. Overexpression of SHMT1 in cell cultures inhibits folate-dependent homocysteine remethylation and enhances thymidylate biosynthesis. In this study, the impact of increased Shmt1 expression on folate-mediated one-carbon metabolism was determined in mice that overexpress the Shmt1 cDNA (Shmt1tg+ mice). Compared with wild type mice, Shmt1tg+ mice exhibited elevated SHMT1 and TYMS protein levels in tissues and evidence for impaired homocysteine remethylation but surprisingly exhibited depressed levels of nuclear SHMT1 and TYMS, lower rates of nuclear de novo thymidylate biosynthesis, and a nearly 10-fold increase in uracil content in hepatic nuclear DNA when fed a folate- and choline-deficient diet. These results demonstrate that SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in nuclear DNA and indicate that SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity.

Altered expression of apoA-I, apoA-IV and PON-1 activity in CBS deficient homocystinuria in the presence and absence of treatment: possible implications for cardiovascular outcomes.

Classical homocystinuria (HCU) is caused by mutations in cystathionine beta-synthase (CBS) which, if untreated, typically results in cognitive impairment, thromboembolic complications and connective tissue disturbances. Paraoxonase-1 (PON1) and apolipoprotein apoA-I are both synthesized in the liver and contribute to much of the cardioprotective effects of high density lipoprotein. Additionally, apoA-I exerts significant neuro-protective effects that act to preserve cognition. Previous work in a Cbs null mouse model that incurs significant liver injury, reported that HCU dramatically decreases PON1 expression. Conflicting reports exist in the literature concerning the relative influence of homocysteine and cysteine upon apoA-I expression. We investigated expression of PON1 and apoA-I in the presence and absence of homocysteine lowering therapy, in both the HO mouse model of HCU and human subjects with this disorder. We observed no significant change in plasma PON1 paraoxonase activity in either mice or humans with HCU indicating that this enzyme is unlikely to contribute to the cardiovascular sequelae of HCU. Plasma levels of apoA-I were unchanged in mice with mildly elevated homocysteine due to CBS deficiency but were significantly diminished in both mice and humans with HCU. Subsequent experiments revealed that HCU acts to dramatically decrease apoA-I levels in the brain. Cysteine supplementation in HO mice had no discernible effect on plasma levels of apoA-I while treatment to lower homocysteine normalized plasma levels of this lipoprotein in both HO mice and humans with HCU. Our results indicate that plasma apoA-I levels in HCU are inversely related to homocysteine and are consistent with a plausible role for decreased expression of apoA-I as a contributory factor for both cardiovascular disease and cognitive impairment in HCU.

Long-term betaine therapy in a murine model of cystathionine beta-synthase deficient homocystinuria: decreased efficacy over time reveals a significant threshold effect between elevated homocysteine and thrombotic risk.

Classical homocystinuria (HCU) is caused by deficiency of cystathionine ß-synthase and is characterized by connective tissue disturbances, mental retardation and cardiovascular disease. Treatment for pyridoxine non-responsive HCU typically involves lowering homocysteine levels with a methionine-restricted diet and dietary supplementation with betaine. Compliance with the methionine-restricted diet is difficult and often poor. Investigating optimization of the efficacy of long-term betaine treatment in isolation from a methionine-restricted diet is precluded by ethical considerations regarding patient risk. The HO mouse model of HCU developed in our laboratory, exhibits constitutive expression of multiple pro-inflammatory cytokines and a hypercoagulative phenotype both of which respond to short-term betaine treatment. Investigation of the effects of long-term betaine treatment in the absence of methionine-restriction in HO HCU mice revealed that the ability of betaine treatment to lower homocysteine diminished significantly over time. Plasma metabolite analysis indicated that this effect was due at least in part, to reduced betaine-homocysteine S-methyltransferase (BHMT) mediated remethylation of homocysteine. Western blotting analysis revealed that BHMT protein levels are significantly repressed in untreated HCU mice but are significantly induced in the presence of betaine treatment. The observed increase in plasma homocysteine during prolonged betaine treatment was accompanied by a significant increase in the plasma levels of TNF-alpha and IL-1beta and reversion to a hypercoagulative phenotype. Our findings are consistent with a relatively sharp threshold effect between severely elevated plasma homocysteine and thrombotic risk in HCU and indicate that the HO mouse model can serve as a useful tool for both testing novel treatment strategies and examining the optimal timing and dosing of betaine treatment with a view toward optimizing clinical outcome.

Two patients with hepatic mtDNA depletion syndromes and marked elevations of S-adenosylmethionine and methionine.

This paper reports studies of two patients proven by a variety of studies to have mitochondrial depletion syndromes due to mutations in either their MPV17 or DGUOK genes. Each was initially investigated metabolically because of plasma methionine concentrations as high as 15-21-fold above the upper limit of the reference range, then found also to have plasma levels of S-adenosylmethionine (AdoMet) 4.4-8.6-fold above the upper limit of the reference range. Assays of S-adenosylhomocysteine, total homocysteine, cystathionine, sarcosine, and other relevant metabolites and studies of their gene encoding glycine N-methyltransferase produced evidence suggesting they had none of the known causes of elevated methionine with or without elevated AdoMet. Patient 1 grew slowly and intermittently, but was cognitively normal. At age 7 years he was found to have hepatocellular carcinoma, underwent a liver transplant and died of progressive liver and renal failure at age almost 9 years. Patient 2 had a clinical course typical of DGUOK deficiency and died at age 8 ½ months. Although each patient had liver abnormalities, evidence is presented that such abnormalities are very unlikely to explain their elevations of AdoMet or the extent of their hypermethioninemias. A working hypothesis is presented suggesting that with mitochondrial depletion the normal usage of AdoMet by mitochondria is impaired, AdoMet accumulates in the cytoplasm of affected cells poor in glycine N-methyltransferase activity, the accumulated AdoMet causes methionine to accumulate by inhibiting activity of methionine adenosyltransferase II, and that both AdoMet and methionine consequently leak abnormally into the plasma.

Versatility in corrinoid salvaging and remodeling pathways supports corrinoid-dependent metabolism in Dehalococcoides mccartyi.

Corrinoids are cobalt-containing molecules that function as enzyme cofactors in a wide variety of organisms but are produced solely by a subset of prokaryotes. Specific corrinoids are identified by the structure of their axial ligands. The lower axial ligand of a corrinoid can be a benzimidazole, purine, or phenolic compound. Though it is known that many organisms obtain corrinoids from the environment, the variety of corrinoids that can serve as cofactors for any one organism is largely unstudied. Here, we examine the range of corrinoids that function as cofactors for corrinoid-dependent metabolism in Dehalococcoides mccartyi strain 195. Dehalococcoides bacteria play an important role in the bioremediation of chlorinated solvents in the environment because of their unique ability to convert the common groundwater contaminants perchloroethene and trichloroethene to the innocuous end product ethene. All isolated D. mccartyi strains require exogenous corrinoids such as vitamin B(12) for growth. However, like many other corrinoid-dependent bacteria, none of the well-characterized D. mccartyi strains has been shown to be capable of synthesizing corrinoids de novo. In this study, we investigate the ability of D. mccartyi strain 195 to use specific corrinoids, as well as its ability to modify imported corrinoids to a functional form. We show that strain 195 can use only specific corrinoids containing benzimidazole lower ligands but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base. This study of corrinoid utilization and modification by D. mccartyi provides insight into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment.