Objectives::Folates are B vitamins that are essential for several molecular, cellular, and biological processes, including nucleotide synthesis, methylation, and methionine cycling. The physiological impacts of these processes on health also extend to cell proliferation, folate deficiency anemia, and reduction of the risk of birth defects during pregnancy. The primary objective of this study was to characterize the binding affinities of different folate forms, folic acid (FA), 5-methyltetrahydrofolate (5MTHF), and folinic acid, to the folate receptors α and β, and to the bovine milk folate binding protein. These three dietary forms of folate are found in enriched grains (FA), various fruits and leafy vegetables (folinic acid), and red blood cells (5MTHF).Methods::The half maximal inhibitory concentration values and binding curves of each of these folates for each receptor were determined.Results::Our results indicated that FA had the highest affinity for all folate receptors, followed by 5MTHF, and lastly, by folinic acid, examined by several orders of magnitudes.Conclusion::These data are expected to provide new insights into the therapeutic applications of the different forms of folate in a variety of diseases.

Objective::The purpose of this study was to examine the role of rare variants in the one-carbon metabolic pathway in the etiology of the cerebral folate deficiency (CFD) syndrome. The CFD syndrome is a neurometabolic syndrome identified by low concentrations of 5-methyltetrahydrofolate (5-MTHF) in the cerebrospinal fluid (CSF) in spite of near-normal peripheral folate levels resulting in neurodevelopmental disorders.Methods::The localized folate metabolism impairments in CFD are thought to be either the result of mutations in genes responsible for folate transport or folate turnover through degradation. Genes that have been previously implicated in the etiology of CFD include folate receptor alpha-1 (FOLR1), dihydrofolate reductase, proton-coupled folate transporter, and capicua. We performed whole-exome sequencing (WES) analysis of a CFD patient that revealed 99 novel missense mutations, of which 21 were classified as damaging mutations through the Poly-Phen2 prediction algorithm. In vitro functional studies were conducted by transient transfection of wild-type and mutant MTHFS into HEK293T cells to determine the impact of the variants on enzyme activity. Results::Of the damaging variants identified in the WES studies, we focused on the gene coding for the enzyme 5,10-methenyl-tetrahydrofolate synthetase (MTHFS). This enzyme catalyzes the production of methenyl THF which is subsequently converted to 5-MTHF. The CFD patient described within was found to carry a homozygous mutation, c.101G>T (p.R34L, rs200058464) in MTHFS, while the parents of the proband are heterozygotes for the MTHFS gene, and the healthy sibling is not a carrier. Conclusion::The mutant allele displayed a 50% reduction in luciferase activity ( P < 0.05), suggesting that homozygous loss of the MTHFS gene may play a significant role in the development of CFD.

目的·研究普通叶酸与活性叶酸补充对亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)677TT型不明原因反复流产(unexplained recurrent pregnancy loss,URPL)患者红细胞叶酸水平的影响.方法·选取2021年1-12月于北京大学第三医院生殖医学中心就诊的MTHFR 677TT型URPL患者45例.按照叶酸补充方式将其分为3组,包括A组16例(研究开始前尚未接受任何形式的叶酸补充,研究开始后进行活性叶酸补充),B组15例(研究开始前进行过普通叶酸的补充,研究开始后进行活性叶酸补充),以及C组14例(研究开始前进行过普通叶酸的补充,研究开始后进行普通叶酸与活性叶酸联合补充).分别于入组时(第一次)、入组补充后(第二次)对3组患者的红细胞5-甲基四氢叶酸(5-methyltetrahydrofolate,5-MTHF)浓度进行检测,并开展比较.结果·在3组患者中,任意2组的第一次红细胞5-MTHF浓度间差异均无统计学意义.与第一次红细胞5-MTHF浓度相比,3组患者的第二次红细胞5-MTHF浓度均有提高(均P=0.000),且B组患者的红细胞5-MTHF浓度的增幅高于A组(t=2.373,P=0.049),但与C组间差异无统计学意义.结论·与补充普通叶酸相比,补充活性叶酸可以更好地在短时间内提高MTHFR 677TT型URPL患者的红细胞叶酸水平.

目的 探讨二仙汤治疗迟发性抑郁症(LOD)的效果及可能作用机制.方法 40只雄性7～8周龄Wistar大鼠分为正常组和青年抑郁组各20只.60只20月龄雄性Wistar大鼠分为衰老组、迟发抑郁组、二仙汤组各20只.青年抑郁组大鼠采用慢性不可预计应激刺激(CUMS)6周建立抑郁模型,迟发抑郁组、二仙汤组衰老大鼠采用CUMS6周建立LOD模型,正常组和衰老组不进行任何干预.造模的同时进行灌胃给药,二仙汤组大鼠给予二仙汤8 g/(kg·d)灌胃,正常组、青年抑郁组、衰老组、迟发抑郁组给予4 ml/(kg·d)纯净水灌胃,各组均给药6周.采用糖水偏爱实验、强迫游泳实验评估大鼠抑郁样行为,旷场实验评价大鼠自发活动能力,T迷宫实验评估大鼠认知功能;Western Blot法检测海马组织神经元核心抗原(NeuN)、巢蛋白、双皮质素(DCX)及脉络丛组织中闭锁小带蛋白1(ZO-1)、叶酸α受体(FRα)、还原型叶酸转运体(RFC)、质子耦合叶酸转运体(PCFT)蛋白表达;采用免疫荧光法检测海马齿状回Ki-67/巢蛋白双阳性细胞数、5-溴脱氧尿嘧啶核苷(BrdU)/DCX双阳性细胞数及脉络丛组织中ZO-1、FRα、RFC、PCFT蛋白表达;ELISA法检测血浆、脑脊液、海马组织中5-甲基四氢叶酸(5-MTHF)含量;采用Pearson相关分析对各组大鼠脑脊液、海马组织中5-MTHF含量与海马组织中巢蛋白、DCX、NeuN蛋白表达进行相关性分析.结果 与正常组比较,青年抑郁组及迟发抑郁组大鼠糖水偏爱度降低,旷场总路程、穿格次数减少,强迫游泳不动时间延长,海马组织NeuN、巢蛋白、DCX蛋白表达均降低,海马齿状回Ki-67/巢蛋白双阳性细胞数、BrdU/DCX双阳性细胞数减少,ZO-1、FRα蛋白表达及荧光强度减少(P＜0.05或P＜0.01);青年抑郁组大鼠第3、4天及迟发抑郁组大鼠第2～4天T迷宫正确率均明显降低,迟发抑郁组大鼠脑脊液、海马组织中5-MTHF含量明显降低(P＜0.05或P＜0.01).与青年抑郁组相比,迟发抑郁组大鼠旷场总路程、穿格次数减少,第1～4天T迷宫正确率均显著降低,海马组织NeuN、巢蛋白、DCX蛋白表达及海马齿状回Ki-67/巢蛋白双阳性细胞数、BrdU/DCX双阳性细胞数减少(P＜0.05或P＜0.01).与迟发抑郁组比较,二仙汤组大鼠糖水偏爱度及旷场总路程、穿格次数均升高,强迫游泳不动时间缩短,第3～4天T迷宫正确率明显升高,海马组织NeuN、巢蛋白、DCX蛋白表达升高,海马齿状回Ki-67/巢蛋白双阳性细胞数、BrdU/DCX双阳性细胞数增加,脑脊液、海马组织中5-MTHF含量明显升高,脉络丛中ZO-1、FRα、RFC、PCFT蛋白表达及荧光强度升高(P＜0.05或P＜0.01).相关性分析结果显示,脑脊液(r=0.466)和海马组织(r=0.522)中5-MTHF含量与海马组织中巢蛋白表达存在正相关(P＜0.05).结论 二仙汤可改善LOD模型大鼠抑郁样行为及认知障碍,其机制可能通过减轻脑组织脉络丛结构损伤,改善脉络丛叶酸转运障碍,从而促进海马神经发生.

目的 探讨孤独症谱系障碍(autism spectrum disorder,ASD)患儿是否存在叶酸代谢异常,为建立ASD的早期预警信号和开展ASD儿童的靶向营养干预提供依据.方法 收集2015年10月-2016年9月期间在哈尔滨医科大学儿童发育行为研究中心就诊和接受康复训练,以及在本地某特教学校上学的典型ASD患者110名作为病例组,另选110名性别、年龄匹配的正常儿童青少年作为对照组.采用化学发光微粒子免疫技术(CMIA)检测2组儿童青少年血清中叶酸、维生素B12及同型半胱氨酸(Hcy)的浓度;应用酶联免疫吸附试验技术(ELISA)测定2组儿童青少年血清中叶酸代谢产物四氢叶酸(THFA)、5-甲基四氢叶酸(5-MTHF)、叶酸受体α(FRα)、叶酸受体自身抗体(FRAA)的含量,进行统计学分析.结果 ASD组血清叶酸和维生素B12的水平低于对照组,Hcy的水平高于对照组,差异均有统计学意义(t值分别为-3.12,-4.63,2.86,P值均＜0.05);ASD组血清叶酸代谢产物5-MTHF的含量低于对照组,FRAA的含量高于对照组,差异均有统计学意义(t值分别为-2.28,2.88,P值均＜0.05);而THFA,FRα 2组间差异均无统计学意义(P值均＞0.05).结论 ASD患者体内存在明显的叶酸代谢异常,叶酸、维生素B12、5-MTHF水平降低,而Hcy、FRAA含量升高.ASD患者叶酸代谢异常可能成为ASD的预警信号,应及时开展靶向营养干预.

叶酸在人体内作为重要的辅因子参与一碳代谢,同时在嘌呤核苷酸和嘧啶核苷酸的合成和转化过程中起着重要作用.孤独症谱系障碍(autism spectrum disorder,ASD)是一类病因未明的神经系统发育障碍,发病机制复杂,患者主要表现为社交障碍和重复、刻板行为.叶酸代谢过程中相应酶的功能缺失、叶酸转运障碍以及相关的遗传变异都与ASD的发病风险增加有关.多项研究表明,ASD患者人群中可能存在一种亚型,因患者体内存在α型叶酸受体自身抗体而导致脑脊液中5-甲基四氢叶酸含量低于正常水平,使患者表现出相应的症状.在这种情况下,患者可以通过口服5-甲酰四氢叶酸(又称亚叶酸)使得疾病症状明显改善.本文主要就叶酸代谢过程中与ASD发病机制及治疗相关的研究进行综述.

目的 检测甲氨蝶呤(MTX)诱导叶酸代谢障碍神经管畸形(NTDs)小鼠胚胎神经组织的基因组拷贝数变异(CNVs),探讨叶酸代谢障碍与NTDs发生的分子遗传学机制.方法 采用本实验室已建立的MTX诱导的叶酸代谢障碍NTDs C57BL/6J小鼠模型,于孕第7.5天采用MTX干预,收集孕鼠血清及NTDs小鼠胚胎神经组织;用NimbleGen高分辨率微阵列比较基因组杂交(array-CGH)芯片对NTDs小鼠胚胎神经组织进行全基因组CNVs分析,反转录(RT)-PCR验证新发现的CNVs;用液相色谱串联质谱法(LC-MS/MS)与酶学方法检测孕鼠血清中叶酸与其相关代谢产物水平及二氢叶酸还原酶(DHFR)活性.结果 array-CGH分析发现,NTD小鼠胚胎神经组织全基因组存在3个高可信度CNVs,分别位于XqE3、XqA1.1-qA2和XqA1.1染色体,RT-PCR验证了这3个CNVs的存在.与对照组相比,MTX干预后,NTDs孕鼠血清中5-甲基四氢叶酸、5-甲酰四氢叶酸、S-腺苷甲硫氨酸水平及DHFR活性明显降低,差异均有统计学意义(P均＜0.05).结论 MTX诱导的NTDs小鼠胚胎神经组织中存在明显的CNVs,叶酸代谢紊乱可通过CNVs导致小鼠胚胎神经管发育障碍.

Objective This study aimed to investigate the effects of N,N-dimethylglycine (DMG) on the concentration and metabolism of plasma homocysteine (pHcy) in folate-sufficient and folate-deficient rats. Methods In this study, 0.1％ DMG was supplemented in 20％ casein diets that were either folate-sufficient (20C) or folate-deficient (20CFD). Blood and liver of rats were subjected to assays of Hcy and its metabolites. Hcy and its related metabolite concentrations were determined using a liquid chromatographic system. Results Folate deprivation significantly increased pHcy concentration in rats fed 20C diet (from 14.19 ± 0.39 μmol/L to 28.49 ± 0.50 μmol/L; P < 0.05). When supplemented with DMG, pHcy concentration was significantly decreased (12.23 ± 0.18 μmol/L) in rats fed 20C diet but significantly increased (31.56 ± 0.59 μmol/L) in rats fed 20CFD. The hepatic methionine synthase activity in the 20CFD group was significantly lower than that in the 20C group; enzyme activity was unaffected by DMG supplementation regardless of folate sufficiency. The activity of hepatic cystathionine β-synthase (CBS) in the 20CFD group was decreased but not in the 20C group; DMG supplementation enhanced hepatic CBS activity in both groups, in which the effect was significant in the 20C group but not in the other group. Conclusion DMG supplementation exhibited hypohomocysteinemic effects under folate-sufficient conditions. By contrast, the combination of folate deficiency and DMG supplementation has deleterious effect on pHcy concentration.

Human methylenetetrahydrofolate reductase (MTHFR), an essential enzyme in folate metabolism, catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.