WO2021043196A1 - 5-甲基四氢叶酸的用途 - Google Patents

5-甲基四氢叶酸的用途 Download PDF

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WO2021043196A1
WO2021043196A1 PCT/CN2020/113157 CN2020113157W WO2021043196A1 WO 2021043196 A1 WO2021043196 A1 WO 2021043196A1 CN 2020113157 W CN2020113157 W CN 2020113157W WO 2021043196 A1 WO2021043196 A1 WO 2021043196A1
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methyltetrahydrofolate
folic acid
acid
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zebrafish
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成永之
连增林
顾睿
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LIANYUNGANG JINKANG HEXIN PHARMACEUTICAL CO Ltd
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LIANYUNGANG JINKANG HEXIN PHARMACEUTICAL CO Ltd
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Priority to EP20860261.5A priority Critical patent/EP4026548A4/en
Priority to US17/639,993 priority patent/US20230043678A1/en
Priority to JP2022514615A priority patent/JP7591299B2/ja
Priority to CN202080062025.8A priority patent/CN114340635B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention belongs to the field of medicine, and relates to a new use of a composition containing 5-methyltetrahydrofolate in the preparation of a medicine or health food for peri-pregnancy and/or pregnancy women to prevent neonatal congenital heart disease, abortion, and stillbirth.
  • Congenital heart disease (abbreviated as congenital heart disease) has become the main disease of birth defects and the most common heart disease in childhood. Congenital heart disease has seriously endangered the lives and quality of life of children, and is an important cause of death in children aged 0 to 5 years.
  • VSD ventricular septal defect
  • ASD atrial septal defect
  • PDA patent ductus arteriosus
  • TOF tetralogy of Fallot
  • TGA great artery displacement
  • TGA tricuspid valve Atresia or stenosis and pulmonary artery stenosis
  • PS pulmonary artery stenosis
  • Folic acid refers to pteroyl glutamate, synthesized folic acid, also called vitamin B9, vitamin M, and its English name is folic acid.
  • folic acid In medical applications, folic acid is used to prevent neonatal neural tube defects, prevent and treat megaloblastic anemia.
  • the World Health Organization recommends that the daily intake of lactating mothers and pregnant women should be 400 ⁇ g/day. High intake of folic acid can cause unmetabolized folic acid and reduced folic acid (5-methyltetrahydrofolate and its derivatives) in the blood circulation.
  • Folic acid is a synthetic oxidized form, which is not found in natural foods. Folic acid needs to be converted into tetrahydrofolate by human dihydrofolate reductase (DHFR) before it can be used by the human body, and this process is very slow in the human body .
  • DHFR human dihydrofolate reductase
  • Some scholars have reported that a few hours after taking more than 200 ⁇ g of folic acid, the blood plasma Can detect unmetabolized folic acid [Bailey SW, Ayling JE.
  • the Hungarian research team pointed out in a randomized controlled study of folic acid supplementation to prevent neural tube defects from 1984 to 1991.
  • the experimental group began to take a multivitamin containing 0.8mg of folic acid from 1 month before pregnancy until 3 months after pregnancy was confirmed.
  • the incidence of neural tube defects in the control group was greatly reduced, and it was also found that the incidence of congenital heart disease was also reduced by 40% [Czeizel,Andrew E.Prevention of congenital abnormalities by periconceptional multivitamin supplementation.[J].Bmj,1993,306(6893 ):1645-1648.].
  • the above-mentioned studies have flaws.
  • OR value is 1.28 (95% CI 0.33–4.95) [van Beynum IM, Kapusta L, Bakker MK, den Heijer M, Blom HJ, walle HE. Protective effect of periodical folic acid supplements on the risk of defect: a Registry-based case-control study in the northern Netherlands. Eur Heart J. 2010; 31(4):464-471.].
  • the birth rate related to ASD rose rapidly after 1980.
  • the inventor found through research that the incidence of congenital heart disease in the urban population in China is much higher than that in the rural population, and it has a tendency to gradually increase.
  • the global incidence of birth defects is 6.42% in low-income countries, 5.57% in middle-income countries, and 4.72% in high-income developed countries.
  • China’s economy is developing rapidly, the standard of living and medical care has improved rapidly, especially It is the eastern developed cities, such as Shanghai, and the southern Jiangsu area as a whole has reached the level of developed countries, but the infant birth defect rate has not shown a corresponding level of control.
  • the birth defect rate of the urban population is higher than that of the rural population.
  • Formaldehyde is a colorless and irritating gas, easily soluble in water and ethanol, and is widely used in human industrial production, including chemical industry, wood industry, textile industry, and anti-corrosion engineering.
  • human industrial production including chemical industry, wood industry, textile industry, and anti-corrosion engineering.
  • formaldehyde With the continuous improvement of health requirements, people pay more and more attention to the harm of formaldehyde in the environment. It is inevitable that formaldehyde will be produced in the process of house decoration, and it is difficult to completely remove the formaldehyde. Therefore, the content of formaldehyde in new houses is often higher than the safe value. This also increases the risk of miscarriage of pregnant women and impaired fetal health.
  • 5-methyltetrahydrofolate can rescue zebrafish embryos affected by formaldehyde, and at the same time, high-concentration 5-methyltetrahydrofolate is non-toxic and non-teratogenic.
  • the present invention is based on animal experiments. Studies have found that if folic acid is taken during pregnancy, unmetabolized folic acid will have teratogenicity on embryos, and its long-term or excessive supplementation can cause the occurrence of congenital heart disease. However, 5-methyltetrahydrofolate is not teratogenic, and it can prevent the occurrence of congenital heart disease when taken during pregnancy.
  • the present invention provides a medicine or health food containing 5-methyltetrahydrofolate, and the medicine or health food is used for pregnant women to prevent neonatal congenital heart disease.
  • the medicine or health food according to the present invention is used for pregnant women to prevent the occurrence of neonatal congenital heart disease.
  • the congenital heart disease refers to the most common type of congenital malformations, which refers to anatomical structure abnormalities caused by heart and large blood vessel formation disorders or developmental abnormalities during embryonic development, or birth Circumstances where the channel that should be automatically closed fails to close afterwards.
  • the congenital heart disease includes three subgroups of diseases: 1. Congenital aortic malformations, including unclosed arterial ducts, aortic stenosis, pulmonary artery stenosis, pulmonary atresia and other congenital malformations of the aorta 2. Congenital heart septal malformations, including atrial-ventricular septal defect (AVSD s ), ventricular septal defect (VSD s ), atrial septal defect (ASD s ), tetralogy of Fallot, main pulmonary artery septal defect and other congenitals Diaphragm deformities; 3. Other congenital heart diseases. Congenital heart diseases, including congenital malformations of the heart cavity, congenital malformations of the aorta and mitral valve.
  • AVSD s atrial-ventricular septal defect
  • VSD s ventricular septal defect
  • ASD s atrial septal defect
  • tetralogy of Fallot main pulmonary
  • the medicine or health food of the present invention contains an effective amount of 5-methyltetrahydrofolate, and it also contains pharmaceutically acceptable auxiliary materials or adjuvants.
  • enteral dosage forms such as oral, sublingual or rectal administration
  • oral dosage forms may be tablets, capsules, oral liquids, dripping pills, pills, powders, granules
  • injections The type can be powder injection, solution, emulsion, suspension.
  • the dosage of the 5-methyltetrahydrofolate is 0.05-50 mg/day, preferably 0.2-0.8 mg/day.
  • Another aspect of the present invention is to provide a medicine or health food for preventing abortion of pregnant women due to formaldehyde and protecting the fetus.
  • the medicine or health food for preventing pregnant women from aborting due to formaldehyde and protecting fetuses it can prevent pregnant women from aborting due to long-term exposure to formaldehyde.
  • the medicine or health food for preventing abortion of pregnant women due to formaldehyde and protecting fetuses it can be applied to treat pregnant women's abortion symptoms of fetuses caused by formaldehyde.
  • the medicine or health food for preventing abortion of pregnant women due to formaldehyde and protecting the fetus can be in various dosage forms known in the art.
  • enteral dosage forms such as oral, sublingual or rectal administration
  • oral dosage forms may be tablets, capsules, oral liquids, dripping pills, pills, powders, granules
  • injections The type can be powder injection, solution, emulsion, suspension.
  • the dosage of 5-methyltetrahydrofolate is 0.05-50 mg/day, preferably 0.2-0.8 mg/day, to treat abortion
  • the aura dosage is 0.8-200mg/day, preferably 20mg/day.
  • dietary supplements in the United States can claim related functions (FDA agrees that dietary supplements can describe the relationship between a food or dietary ingredient and a health state or reduce the risk of a disease) Therefore, the related 5-methyltetrahydrofolate can be used not only to prepare medicines or health foods for the prevention of congenital heart disease, abortion and stillbirth, but also to prepare dietary supplements and formula foods for special medical purposes with the above-mentioned functions.
  • Exemplary such as multivitamin tablets containing 5-methyltetrahydrofolate, formula milk powder for pregnant women and so on.
  • the said cardiac malformations are preferably those caused by ethanol, lead nitrate and aristolochic acid.
  • the 5-methyltetrahydrofolate includes 5-methyltetrahydrofolate or a pharmaceutically acceptable salt thereof.
  • the 5-methyltetrahydrofolate is selected from 5-methyl-(6S)tetrahydrofolate, 5-methyl-(6R)tetrahydrofolate, or 5-methyl(6R,S)tetrahydrofolate, namely Including 5-methyltetrahydrofolate optical isomers, mixtures of optical isomers, especially pure optical natural isomers.
  • the pharmaceutically acceptable salt includes the conversion of the basic group in 5-methyltetrahydrofolate into the corresponding acid addition salt, and the conversion of the acid group in 5-methyltetrahydrofolate into the corresponding alkali addition salt; Preference is given to hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, sodium, potassium, magnesium, calcium and ammonium salts, substituted ammonium salts, or salts with arginine or lysine.
  • the term "5-methyltetrahydrofolate” includes 5-methyl-(6S)tetrahydrofolate, 5-methyl-(6R)tetrahydrofolate, 5-methyl(6R,S)tetrahydrofolate, Folic acid includes optical isomers of 5-methyltetrahydrofolate, especially pure optical natural isomers, mixtures of optical isomers, such as racemic mixtures, and their physiologically acceptable salts. Among them, 5-(methyl)-(6S)tetrahydrofolate is particularly preferred.
  • the physiologically acceptable salt refers to the conversion of the basic group in 5-methyltetrahydrofolate into the corresponding acid addition salt
  • the acid may be an inorganic acid, such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; Organic acids, such as formic acid, acetic acid, propionic acid, diethyl acetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid or niacin, etc. .
  • the physiologically acceptable salt can also refer to the conversion of the acidic group in 5-methyltetrahydrofolate into the corresponding base addition salt.
  • Suitable salts are, for example, sodium, potassium, magnesium, calcium and ammonium salts, Substituted ammonium salts, and salts formed with arginine or lysine.
  • the folic acid in the present invention refers to pteroyl glutamate, which is synthesized folic acid, also called vitamin B9 and vitamin M, and the English name is folic acid.
  • the research paper [Baohong M, Jie Q, Nan Z, et al. Maternal folic acid supplementation and dietary intake and general heart defects[J]. PLOS ONE, 2017, 12(11): e0187996-.] shows that before pregnancy In January, pregnant women with daily intake of folic acid more than 266.5 ⁇ g had a higher incidence of neonatal atrioventricular septal defect (ASD) than the pregnant group with daily intake of 115.97 ⁇ 265.5 ⁇ g.
  • ASSD neonatal atrioventricular septal defect
  • Folic acid supplementation in pregnant women can also cause unmetabolized folic acid in the blood circulation.
  • American scholars are studying a study of folic acid supplementation in pregnant women [Obeid R, Kasoha M, Kirsch S H. Concentrations of unmetabolized folic acid and primary folate formswomen at delivery and in umbilical cord blood[J].American Journal of Clinical Nutrition,2010,92(6):1416-1422.]
  • 25 pregnant women who supplemented with folic acid during pregnancy detected 0.19nmol/L in serum.
  • Metabolizing folic acid the concentration of folic acid in umbilical cord blood is higher than 0.27nmol/L, which suggests that unmetabolized folic acid may accumulate in the fetus at a higher concentration than the mother.
  • folic acid has teratogenic effects on zebrafish embryos.
  • the intervention of folic acid can cause zebrafish embryo pericardium edema, zebrafish embryo heart rate slow, and body length shortening.
  • folic acid can cause zebrafish.
  • folic acid may also have teratogenic effects on human embryonic development.
  • the critical period for human embryonic development and heart formation is 8 weeks before the formation of early fetal cardiovascular circulation at 6-8 weeks of pregnancy.
  • Vascular development and pericardial development have teratogenic effects, which can cause neonatal cardiovascular abnormalities or diaphragm abnormalities, which can lead to congenital heart disease.
  • the inventors further observed through zebrafish that 5-methyltetrahydrofolate has no teratogenic effect on zebrafish embryos. There is no difference between the normal embryo group and the 5-methyltetrahydrofolate intervention group. Under the intervention of 5-methyltetrahydrofolate , The zebrafish embryonic heart looks normal, and the blood vessels in the intestinal reticulum develop normally.
  • the inventor collected zebrafish embryos under the intervention of folic acid at different times (24h, 48h), and used quantitative PCR technology to detect the expression of different transcription factors in the zebrafish embryos.
  • the results showed that the expression of transcription factors NKx2.5, amhc, vmhc, hand2, has2, mef2a, mef2c, bmp2b, ephrinB2, and ephB4 in zebrafish embryos under the intervention of folic acid were all affected, among which NKx2.5, hand2, has2, mef2c Both ephB4 and ephB4 have been proven to be important transcription factors during heart development.
  • the inventors found that folic acid has a greater impact on mef2c, bmp2b, vmhc, has2, and ephb4.
  • the four members of the human mef2 family include mef2a, mef2b, mef2c and mef2d.
  • mef2b and mef2c are first activated in the heart embryo on the 7th day of the embryo, and mef2c is the key to the formation of primitive heart tube mesoderm cells during embryonic development.
  • the absence of mef2c can cause severe heart structural abnormalities, especially atrial septal defect or ventricular septal defect [Qiao XH, Wang F, Zhang XL, et al. MEF2C loss-of-function mutation contributions to congenital heart defects. Int J Med Sci. 2017; 14(11):1143-1153.].
  • Folic acid affects the expression of mef2c, which is also a possible reason for the increase in the birth rate of ASD.
  • hyaluronic acid is highly expressed in heart development and plays an important role in cell migration and transformation. Human has gene expression regulates the synthesis of hyaluronic acid. It has been reported that has2 gene mutations may affect the formation of heart compartments in embryos. [Zhu X, Deng X, Huang G, et al. A novel mutation of Hyaluronan synthesis 2 gene in Chinese children with ventricular septal defect.PLoS One.2014; 9(2):e87437.].
  • the inventors simultaneously collected zebrafish embryos under the intervention of 5-methyltetrahydrofolate at different times (24h, 48h), and used quantitative PCR technology to detect the expression of different transcription factors in zebrafish embryos.
  • the results showed that under the intervention of 5-methyltetrahydrofolate, no effect on the expression of transcription factors was found, and the expression of related transcription factors was not different from that of the normal control group.
  • the inventors found that 5-methyltetrahydrofolate cannot save zebrafish embryos from deformity of pericardial edema caused by synthetic folic acid, but it can save zebrafish’s abnormal heart rate and shortened body length. .
  • Example 9 the inventors investigated the effect of different doses of folic acid on the heart of fetal rats. The results showed that under a certain dose, the ventricular wall was too thin in the heart of fetal rats, and the ventricular wall was visible in some cases. Comparing the fetuses born from female mice with folic acid and 5-methyltetrahydrofolate, the incidence of congenital heart disease in fetal mice in the low-dose folic acid group was 5.95%, and the incidence of congenital heart disease in the low-dose folic acid group was 5.95%. The incidence of congenital heart disease in fetal rats was 22.2%, while the 5-methyltetrahydrofolate group had no congenital heart disease, which further proved the effect of folic acid on embryonic heart development.
  • 5-methyltetrahydrofolate can prevent the teratogenic effects of alcohol, lead nitrate, and aristolochic acid A on the embryonic heart induced by alcohol, and the above substances represent alcoholism, heavy metals, drugs and other environmental factors caused by different environmental factors. Heart malformations in newborns, so 5-methyltetrahydrofolate has a wide range of prevention of congenital heart disease.
  • the inventors found that 5-methyltetrahydrofolate can prevent heart malformations caused by folic acid deficiency.
  • the medicine or health food of the present invention contains 0.1mg-200mg of 5-methyltetrahydrofolate per dose. In preventive medication, it is preferable to use 5-methyltetrahydrofolate containing 0.1mg-1mg per dose.
  • the specific dosage depends on various factors, such as the patient's age, weight, time and route of administration, individual conditions, etc.
  • the optimal dosage is preferably 0.1-1 mg/day, for example 0.8 mg/day.
  • the medicine or health food contains various carriers, excipients and/or auxiliary functional agents, such as water, oil, benzyl alcohol, polyethylene glycol, triacetin, gelatin, lecithin, Carbohydrates such as cyclodextrin, lactobiose or starch, magnesium stearate, talc, silica gel or cellulose.
  • auxiliary functional agents are, for example, stabilizers, antioxidants, buffers, bacteriostatic agents and the like.
  • the excipient or carrier is microcrystalline cellulose, or a combination of microcrystalline cellulose and croscarmellose sodium, or micronized silica gel.
  • the present invention finds for the first time that after pregnant women take folic acid, the unmetabolized folic acid that enters the blood circulation can cause congenital heart disease.
  • Folic acid is a synthetic oxidized form that is not found in natural foods. Folic acid needs to pass through human dihydrofolate reductase. (DHFR) converts it into tetrahydrofolate before it can be used by the human body. This process is very slow in humans. A few hours after taking more than 200 ⁇ g of folic acid, unmetabolized folic acid can be detected in the plasma, which is also folic acid and congenital heart disease. The clinical statistical results are confused and the possible reasons for the inconsistent conclusions. The present invention also explains the contradictory reasons of previous studies.
  • DHFR human dihydrofolate reductase
  • the present invention also found that 5-methyltetrahydrofolate has the effect of preventing congenital heart disease.
  • the preventive dosage of 5-methyltetrahydrofolate can exceed 1mg.
  • synthetic folic acid it has no teratogenic effect when used in large doses. Realizing the prevention of birth defects at large doses, especially the prevention of neonatal congenital heart disease, has significant progress.
  • Figure 1 The birth rates of different subtypes of congenital heart disease in China from 1980 to 2019 and the birth rates of atrial septal defect deformities in different regions of the world from 1970 to 2017.
  • Figure 2a The effect of 10mM L-5-MTHF-Ca on the survival rate of zebrafish embryos in Experimental Example 1; b. The effect of L-5-MTHF-Ca on the development of zebrafish embryos in Experimental Example 1.
  • Fig. 3 The effect of L-5-MTHF-Ca on the heart rate and body length of zebrafish at 48hpf and 72hpf in Experimental Example 1.
  • Figure 4a The survival rate of zebrafish embryos of MTX and MTX+L-5-MTHF-Ca in experimental example 2; ns, not significantly different from the control group; ****, p ⁇ 0.005; b: MTX model The effect of rescue group with L-5-MTHF-Ca on zebrafish embryo development.
  • Fig. 5 The effect of MTX and MTX+L-5-MTHF-Ca on zebrafish embryo pericardial swelling rate, heart rate and zebrafish embryo body length in Experimental Example 2; ns, the difference is not significant compared with the control group;*** *, p ⁇ 0.005; ***, p ⁇ 0.01; *, p ⁇ 0.1.
  • Figure 6a The effect of different concentrations of folic acid on the survival rate of zebrafish embryos in Experimental Example 3; b: the effect of different concentrations of folic acid on the heart rate of zebrafish embryos.
  • Fig. 7 The effect of different concentrations of folic acid at 8hpf and 24hpf on the morphology of zebrafish embryos in Experimental Example 3.
  • Figure 8 The effect of 10mM L-5-MTHF-Ca, 8mM FA, 6mM FA and 4mM FA on zebrafish heart in experimental example 3 at 72hpf; left image: 72hpf microscope observation of the heart embryo development under the influence of different concentrations of folic acid, found Pericardial edema malformation ⁇ atrium and ventricle elongated, intestinal reticulum blood vessel development disorder; right picture: the effect of folic acid at 72hpf on zebrafish body length.
  • Fig. 9 The effect of folic acid in experimental example 4 on the expression of different transcription factors in zebrafish embryos.
  • Figure 10 The effect of 5-methyltetrahydrofolate in Experimental Example 4 on the expression of different transcription factors in zebrafish embryos.
  • Figure 11a The effect of FA and FA+L-5-MTHF-Ca on the survival rate of zebrafish embryos in Experimental Example 5; b: The effect of FA and FA+L-5-MTHF-Ca on the development of zebrafish embryos in Experimental Example 5 Impact.
  • Figure 13a The effect of formaldehyde and formaldehyde + L-5-MTHF-Ca on the survival rate of zebrafish embryos in Experimental Example 6; b: the effect of formaldehyde and formaldehyde + L-5-MTHF-Ca on the development of zebrafish embryos.
  • Fig. 14 The influence of 48hpf zebrafish embryo heart rate and 72hpf zebrafish embryo body length in Experimental Example 6.
  • Figure 15a The effect of low-dose homocysteine on myocardial trabecula in experimental example 7; b: low-dose homocysteine caused a small amount of lymphocyte infiltration in the intercellular space of myocardium; c: low-dose homocysteine caused A pale pink serous substance can be seen in the right ventricle; d: high-dose homocysteine causes fetal mouse myocyte plasma to stain red.
  • Fig. 18 The effect of high-dose (36.44mg/kg) folic acid in experimental example 8 on the heart of fetal rats.
  • Fig. 19 The effect of high-dose (18.22mg/kg) folic acid in experimental example 8 on the heart of fetal rats.
  • Figure 20 The effect of high dose (9.11 mg/kg) folic acid in experimental example 8 on the heart of fetal rats.
  • Fig. 21 is a typical heart slice of the fetus in the 2.2775 mg/kg folic acid group in Experimental Example 9.
  • Figure 22 shows a typical heart slice of a fetal rat in the 0.911 mg/kg folic acid group in Experimental Example 9.
  • Figure 23 Ultrasonography of the heart of newborn mice on the 6th day in Experimental Example 10.
  • Control group (A) low-dose folic acid group (B), high-dose folic acid group (C), low-dose methyltetrahydrofolate group (D) methylene High-dose group of tetrahydrofolate (E).
  • Figure 24 Echocardiographic data of six-day-old newborn mice in Experimental Example 10, control group (A), low-dose folic acid group (B), high-dose folic acid group (C), low-dose methyltetrahydrofolate group (D) Methylenetetrahydrofolate high-dose group (E).
  • FIG. 25 Typical heart slices of fetal rats in each group in Experimental Example 10.
  • Fig. 26 In experimental example 11 a: Pb(NO3)2 and Pb(NO3)2+L-5-MTHF-Ca survival rate test on zebrafish embryos; b: Pb(NO3)2 model and L-5 The effect of -MTHF-Ca rescue group on zebrafish embryo development.
  • FIG. 28 In experimental example 11, A: ethanol and ethanol+L-5-MTHF-Ca on the survival rate of zebrafish; B: the effect of ethanol and L-5-MTHF-Ca on the development of zebrafish.
  • Fig. 30 Survival rate test of aristolochic acid A and aristolochic acid A+L-5-MTHF-Ca on zebrafish embryos in Experimental Example 11.
  • Fig. 31 The effect of aristolochic acid A model and L-5-MTHF-Ca+ aristolochic acid A group on zebrafish embryo development in experimental example 11.
  • 5-methyltetrahydrofolate calcium salt 1g is added to 200g of micro-powdered silica gel, mixed well, and then compressed into a tablet machine to form a tablet to obtain 5-methyltetrahydrofolate tablets each containing 2mg.
  • the transgenic zebrafish came from the Institute of Model Animals, Nanjing University.
  • the fish used in the experiment was an adult zebrafish younger than 1.
  • the pH value of the rearing water body was 7 ⁇ 0.2, the temperature was about 28°C, and the time ratio of light to darkness was 14h:10h, and Artemia eggs were fed twice a day.
  • One female, two males and three zebrafish were placed in the spawning box the night before, and the eggs were collected the next morning.
  • the appearance of the hearts of each group was normal, and the blood vessels of the intestinal reticulum were normal (see Figure 2).
  • the heart rate of zebrafish embryos was measured at 48hpf and the body length of zebrafish embryos at 72hpf. It can be found that there is no difference in heart rate and body length between the L-5-MTHF-Ca of 10mML and the control group (see Figure 3).
  • the transgenic zebrafish came from the Institute of Model Animals, Nanjing University.
  • the fish used in the experiment was an adult zebrafish younger than 1.
  • the pH value of the rearing water body was 7 ⁇ 0.2
  • the temperature was about 28°C
  • the time ratio of light and darkness was 14h:10h
  • Artemia eggs were fed twice a day.
  • One female, two males and three zebrafish were placed in the spawning box the night before, and the eggs were collected the next morning.
  • the eggs are placed in embryo culture medium (prepared with 0.2g/L of sea salt) and cultured at 28°C.
  • the zebrafish embryos were placed in a 24-well plate at 6hpf and cultured with drugs, 10 per well, and 1mL of 1.5mM MTX solution was added, until the 10hpf was terminated, and then transferred to the egg fluid to continue the culture to 72hpf. If the embryo is found dead, clean up in time. Observe the phenotype of the deformity, record the number of all zebrafish with abnormal deformity, and calculate the deformity rate and rescue rate of each group. The morphological defects, heart rate, heart morphology and other indicators were evaluated by SMZ745T stereo inverted microscope.
  • Both 1.5mM MTX and 1.5mM MTX+10mM L-5-MTHF-Ca did not have obvious embryonic developmental delay; at 24hpf, 1.5mM MTX caused zebra Fish are underdeveloped, with malformations such as small heads and short tails; at 48hpf, 1.5mM MTX causes zebrafish torso curvature, intersegmental vascular hypoplasia, and pericardial edema; at 72hpf, 1.5mM MTX causes zebrafish body length changes Short, hypoplasia of the intestinal reticulum. And 10mM L-5-MTHF-Ca can rescue the embryonic developmental abnormalities caused by 1.5mM MTX.
  • the deformity rate in the MTX group was 100%, while the deformity rate was only 10% after adding L-5-MTHF-Ca.
  • the transgenic zebrafish came from the Institute of Model Animals, Nanjing University.
  • the fish used in the experiment was an adult zebrafish younger than 1.
  • the pH value of the rearing water body was 7 ⁇ 0.2, the temperature was about 28°C, and the time ratio of light to darkness was 14h:10h, and Artemia eggs were fed twice a day.
  • One female, two males and three zebrafish were placed in the spawning box the night before, and the eggs were collected the next morning. Fish eggs are placed in embryo culture medium (prepared with 0.2g/L sea salt) and cultured at 28°C.
  • solvent 20Mm NaHCO 3 1mL6mM FA solution (solvent 20mM NaHCO 3), 1mL4mM FA solution (solvent 20mM NaHCO 3), 1mL2mM FA (solvent 20mM NaHCO 3), 1mL 10mM L -5-MTHF-Ca
  • the solvent is 20 mM NaHCO 3 ), and it acts until 72hpf is terminated.
  • the embryo If the embryo is found dead, it will be cleaned up in time. Each test was repeated 3 times. If the embryo is found dead, it will be cleaned up in time. Observe the phenotype of the deformity, record the number of all zebrafish with abnormal deformity, and calculate the deformity rate and rescue rate of each group. The morphological defects, heart rate, heart morphology and other indicators were evaluated by SMZ745T stereo inverted microscope.
  • zebrafish embryos at different developmental stages were collected for testing.
  • the transgenic zebrafish came from the Institute of Model Animals, Nanjing University.
  • the fish used in the experiment was an adult zebrafish younger than 1.
  • the pH value of the rearing water body was 7 ⁇ 0.2, the temperature was about 28°C, and the time ratio of light to darkness was 14h:10h, and Artemia eggs were fed twice a day.
  • One female, two males and three zebrafish were placed in the spawning box the night before, and the eggs were collected the next morning.
  • the eggs are placed in embryo culture medium (prepared with 0.2g/L of sea salt) and cultured at 28°C.
  • -Ca solvent is 20mM NaHCO 3 , folic acid
  • RNA of each group of zebrafish was extracted with Trizol according to the instructions, and the embryos at 2 time points of 24 and 48hpf were extracted for follow-up experiments. .
  • This qRT-PCR experiment uses the fluorescent dye SYBR Green dye. qRT-PCR was operated in accordance with the instructions of the SYBR Green qPCR Master Mix kit, and finally tested on the ABI (HT 7900) PCR machine.
  • the gene primers used were synthesized by Jinweizhi Biotechnology Co., Ltd., and the specific information is shown in Table 1.
  • the ⁇ Ct method was used to calculate the relative expression levels of other genes.
  • the results are as follows.
  • the zebrafish eggs form a complete heart 48 hours after fertilization, which is composed of atria and ventricles, and there are valves between the atrio-ventricles.
  • the cardiomyogenic genes of the slab cells are expressed. These cardiomyogenic genes include Nkx-2 family genes, Mef2 family genes and so on.
  • folic acid interferes with the expression of various genes.
  • the expression of various genes is affected to different degrees (see Figure 9), while 5-methyltetrahydrofolate (folate) is compared with the normal control group. There was no difference in the control group (see Figure 10).
  • folic acid deficiency is related to the formation of congenital heart disease, but folic acid itself is also related to the formation of congenital heart disease.
  • folic acid increased the expression of mef2c gene and decreased the expression of bmp2b gene at 24hpf, while folic acid inhibited the expression of has2, mef2c, and ephb4 genes at 48hpf, and increased the expression of vmhc.
  • mef2c is a key member of the primordial heart tube germ layer during embryonic development, has2 also affects the formation of the embryonic heart compartment, which is also the possible reason for the significant increase in the birth rate of ASD.
  • the transgenic zebrafish came from the Institute of Model Animals, Nanjing University.
  • the fish used in the experiment was an adult zebrafish younger than 1.
  • the pH value of the rearing water body was 7 ⁇ 0.2, the temperature was about 28°C, and the time ratio of light to darkness was 14h:10h, and Artemia eggs were fed twice a day.
  • One female, two males and three zebrafish were placed in the spawning box the night before, and the eggs were collected the next morning.
  • the eggs are placed in embryo culture medium (prepared with 0.2g/L of sea salt) and cultured at 28°C.
  • the zebrafish embryos were cultured on a 24-well plate at 2hpf, 10 strips per well, 1mL of 6mM FA solution (solvent 20mM NaHCO 3 ) was added, and the effect was continued until 72hpf was terminated. If the embryo is found dead, it will be cleaned up in time. Each test was repeated 3 times. Choose 6mM FA to make the model, and use 10mM L-5-MTHF-Ca to carry out the rescue experiment, and set the 20mM NaHCO 3 solvent control group. Observe the phenotype of the deformity, record the number of all zebrafish with abnormal deformity, and calculate the deformity rate and rescue rate of each group. The morphological defects, heart rate, heart morphology and other indicators were evaluated by SMZ745T stereo inverted microscope.
  • 10mM L-5-MTHF-Ca can save the survival rate, bradycardia and shortened body length of zebrafish embryos caused by 6mM FA, but it cannot save pericardial edema and pigmentation disorders.
  • the transgenic zebrafish came from the Institute of Model Animals, Nanjing University.
  • the fish used in the experiment was an adult zebrafish younger than 1.
  • the pH value of the rearing water body was 7 ⁇ 0.2, the temperature was about 28°C, and the time ratio of light to darkness was 14h:10h, and Artemia eggs were fed twice a day.
  • One female, two males and three zebrafish were placed in the spawning box the night before, and the eggs were collected the next morning.
  • the eggs are placed in embryo culture medium (prepared with 0.2g/L of sea salt) and cultured at 28°C.
  • Zebrafish embryos were cultured on a 24-well plate with drugs (10mM L-5-MTHF-Ca) starting at 2hpf, 10 per well, and 1mL of 30mM HCHO solution was added. If the embryo is found dead, clean up in time. Each test was repeated 3 times. Observe the phenotype of the deformity, record the number of all zebrafish with abnormal deformity, and calculate the deformity rate and rescue rate of each group. The morphological defects, heart rate, heart morphology and other indicators were evaluated by SMZ745T stereo inverted microscope.
  • 30mM HCHO and 30mM HCHO+10mM L-5-MTHF-Ca have similar phenotypes to the zebrafish in the control group.
  • brightfield and fluorescence microscopes were used to observe and photograph zebrafish embryos.
  • the normal embryos were similar to the 30mM HCHO and 30mM HCHO+10mM L-5-MTHF-Ca groups.
  • the head and tail were well developed, the whole body was pigmented, and the heart beats. Obviously, the blood vessels between the somites develop normally, and blood can be seen flowing throughout the body.
  • normal embryos and 30mM HCHO and 30mM HCHO+10mM L-5-MTHF-Ca affect the appearance of the heart, and the blood vessels of the intestinal reticulum develop normally.
  • the fetus was taken by cesarean section under anesthesia with chloral hydrate.
  • the high-dose HCY model pregnant rats were investigated in the same way as above, the dose was (200mg/kg/d), and the other conditions were the same.
  • the heart of the fetus was dissected and sliced for observation. The results are as follows.
  • HCY low-dose HCY caused fetal rat myocardial trabeculae to widen and the atria were filled with red blood cells (see Figure 15, a). A small amount of lymphocyte infiltration in the myocardial space (see Figure 15, b). A small amount of lymphocyte infiltration in the right myocardial space (see Figure 15, c). High doses of HCY caused the cytoplasm of fetal mouse muscle to stain red, as shown by the black tip (see Figure 15, d). The ventricles and atria are filled with red blood cells, and the myocardial trabecula is widened (see Figure 16). The above experiment shows that HCY can affect the heart of fetal mice, and it is speculated that high levels of human homocysteine may also cause congenital heart disease.
  • the 16 pregnant rats were randomly divided into 4 groups according to their body weights, 4 rats in each group: normal group, high-dose folic acid group, medium-dose folic acid group, and low-dose folic acid group.
  • the groups were divided into high-dose folic acid group 36.44mg/kg, medium-dose folic acid group 18.22mg/kg, low-dose folic acid group 9.11mg/kg and blank control group.
  • the administration method was intragastric administration and the administration route was 1ml/100g. Solution. Except that the normal group was given pure water, the other groups started to be administered on the second day after the administration, once a day, for 21 consecutive days.
  • the pregnant rats in each group had free access to food and water.
  • Sample preparation preparation of paraffin sections of conventional tissue samples, 1 paraffin block for each sample; section: 20 sections of each numbered sample. The starting point of the mounting is when the wax block shows the mounting of the ventricles during the sectioning. On average, there are 4 consecutive tissue points mounted on each slide.
  • HE staining 20 slides for each sample, and 1 slide for HE staining, a total of 10 slides.
  • Observation and analysis Leica panoramic image acquisition and comparative analysis. The fetal rat heart was dissected and sectioned for observation. The results are as follows.
  • the atria and ventricles were well differentiated, and the pericardium, endocardium and epicardium were intact.
  • the longitudinal section, oblique section or cross section of myocardial fibers can be observed at the same time.
  • the myocardial fibers are arranged neatly and regularly, the texture is clear, and the muscle fibers are branched and anastomosed into a net.
  • Myocardial fibers are divided into fiber bundles of varying sizes by loose connective tissue, and there are abundant blood vessels in the bundles. Myocardial trabeculae are densely developed in the atria and ventricles.
  • the cardiomyocytes are normal, the nuclei are neatly arranged, oval or spherical, and light blue. There are a few round or oval red-stained blood cells between the myocardial fibers. No obvious pathological changes. Due to development or cutting angle problems, some animals did not see the aortic structure connected to the ventricle (see Figure 17).
  • the experiment was divided into folic acid group (6 groups) and blank control group.
  • the folic acid control group was folic acid 4.555mg/kg/d (group 1) and folic acid 2.2775mg/kg/ d (2 groups), folic acid 0.911 mg/kg/d (3 groups), folic acid 0.4555 mg/kg/d (4 groups), folic acid 0.22775 mg/kg/d (5 groups), folic acid 0.113875 mg/kg (6 groups) ).
  • Each of the folic acid group and the blank control group consisted of 4 pregnant mice, 4.555 mg/kg/d (group 1), folic acid 2.2775 mg/kg/d (group 2), and folic acid 0.911 mg/kg/d (group 3).
  • the method is intraperitoneal injection, folic acid 0.4555mg/kg/d (4 groups), folic acid 0.22775mg/kg/d (5 groups), folic acid 0.113875mg/kg (6 groups) by gavage, and blank control group
  • the vaginal plug was checked the next morning. The day when the vaginal plug was seen was set as the 0th day of pregnancy.
  • the number of fetuses in each group is shown in Figure 30.
  • the fetal mouse heart was taken for pathological examination, and the results are as follows.
  • 4.555mg/kg (group 1) has basically differentiated into atria and ventricles, and there are no obvious pathological changes in the pericardium, endocardium, endocardium, and myocardium, as shown in the figure.
  • the development of the left and right atria is basically normal.
  • the cut surface of the right atrium is generally slightly larger than that of the left atrium.
  • Abundant myocardial trabeculae can be seen in the cavity wall.
  • the atrial cavity of individual individuals is small.
  • the right ventricle of individual individuals also showed lacuna stenosis. Individual individuals can also see that the right ventricular wall is too thin.
  • the large blood vessel lumen connected to the ventricle can be seen in the slices, and the valves in the lumen can be seen in some sections.
  • group 2 has basically differentiated into ventricles and atria, pericardium is intact, myocardium, epicardium and endocardium are intact, and there is no necrosis or inflammatory cell infiltration. Abundant myocardial trabeculae can be seen in the left and right atria, and only a small section of the left atrium can be seen in individual individuals ( Figure 21, d). Atrial septal defect can be seen in individual individuals ( Figure 21, d). The left and right ventricles are basically developed and formed, the ventricle is rich in myocardial trabeculae, connected to the vascular lumens, and individual intravascular valves ( Figure 21, b, e).
  • the atrial and ventricular openings and the mitral valve can be seen in individual sections ( Figure 21, d).
  • the right ventricular wall is too thin ( Figure 21, b, d).
  • 0.911mg/kg (group 3) has basically differentiated into ventricles and atria, pericardium is intact, myocardium, epicardium and endocardium are intact, and no necrosis or inflammatory cell infiltration is seen.
  • the left and right atria are relatively full, and the myocardial trabecula is abundant in the atria. Only a small part of the left atrium was seen in some individual slices ( Figure 22, c, e).
  • the left and right ventricles are basically developed and formed, and individual individuals have thicker left ventricle walls and narrow cavities ( Figure 22, d).
  • the ventricular wall is rich in blood vessels, and abundant myocardial trabeculae can be seen in the ventricle.
  • the myocardial trabecula was abundant in the left and right atria, and the atrium section was small.
  • the left and right ventricular walls are thick, the cavities are narrow, the myocardial trabeculae are closely arranged, and the ventricular walls are rich in blood vessels.
  • the ventricular section of individual individuals is smaller and the staining is darker.
  • the myocardial trabecula was abundant in the left and right atria at 0.22775mg/kg (group 5).
  • the large blood vessels connected to the ventricle and the valves in the lumen can be seen on most of the cut surfaces, but only a few are not seen.
  • 0.113875mg/kg (group 6) has basically differentiated into ventricles and atria, pericardium is intact, only a few sections do not see pericardium, myocardium, epicardium and endocardium intact, no necrosis and inflammatory cell infiltration .
  • the left and right atria are relatively full, and the myocardial trabecula is abundant in the atria.
  • mice Female C57BL/6J mice, 7 weeks old, 75 mice, male C57BL/6J mice, 8 weeks old, 25 mice, provided by Shanghai Lingchang Biotechnology Co., Ltd., certificate number: SCXK (Shanghai) 2018-0003. After the animals were purchased, they were bred adaptively for about 10 days, until the weight of female mice reached about 20g and the weight of male mice reached about 25g, and the experiment started after the body matured.
  • mice Female mice were randomly divided into 5 groups according to body weight, namely: vehicle control group, folic acid 151.66 ⁇ g/kg, folic acid 303.32 ⁇ g/kg, 5-methyltetrahydrofolate calcium (folic acid equivalent) 151.66 ⁇ g/kg, 5-methyl Calcium tetrahydrofolate (equivalent to folic acid) 303.32 ⁇ g/kg.
  • a unit mass of 1.1275 calcium 5-methyltetrahydrofolate is equal to a unit mass of 1 folate.
  • mice There are 15 animals in each group, and they are numbered by cutting toe method.
  • the rats were given intragastric administration once a day.
  • the male and female mice were caged at a ratio of 3:1, and the vaginal suppository was observed daily from the next day, and 0.5 days of pregnancy was recorded from the day the vaginal suppository was seen.
  • the female mice stopped the administration, the male mice were removed, and the pregnant female mice were reared alone until littering.
  • mice were randomly selected from each group, and when they grew to 6 days of age, small animal cardiac ultrasound observations (Vevo 2100 Imaging System, VisualSonics, Toronto, ON, Canada) were performed.
  • mice After the test, the mouse’s thoracic cavity and abdominal cavity were opened, and the heart morphology and heart beating were observed under a body-field microscope (SMZ168, Motic, Xiamen, Fujian, China), and photographed and recorded video (Motic Image Plus 3.0, Motic, Xiamen) ,Fujian,China). He was then executed, and the heart, liver, kidneys and lungs were collected. The hearts and livers of newborn rats were selected from each group, fixed with 4% paraformaldehyde, paraffin-embedded sections, and H&E stained. Half of the other tissues are immersed and fixed in RNA later, and the other half is directly immersed in liquid nitrogen and frozen for subsequent experiments.
  • SMZ168 Motic, Xiamen, Fujian, China
  • the electrodes of newborn mice are not inserted under the skin like adult mice, but only in contact with the skin, so the measured signal is not very stable and has not been statistically summarized.
  • the transgenic zebrafish came from the Institute of Model Animals, Nanjing University.
  • the fish used in the experiment was adult zebrafish less than 1 year old.
  • the pH value of the rearing water body was 7 ⁇ 0.2
  • the temperature was about 28°C
  • the time ratio of light and darkness was 14h:10h
  • Artemia eggs were fed twice a day.
  • One female, two males and three zebrafish were placed in the spawning box the night before, and the eggs were collected the next morning. Fish eggs are placed in embryo culture medium (prepared with 0.2g/L sea salt) and cultured at 28°C.
  • the zebrafish embryos were placed in a 24-well plate at 2hpf and cultured with drugs, 10 per well, and the drug solution of the concentration in the above table was added respectively until the 8hpf was terminated. After the embryos were washed, they were transferred to the egg fluid and continued to be cultured to 72hpf. If the embryo is found dead, clean up in time. Each test was repeated 3 times.
  • the concentration selected in the experiment of 5-methyltetrahydrofolate preventing malformation is recorded in the table. Observe the phenotype of the deformity, record the number of all zebrafish with abnormal deformity, and calculate the deformity rate and rescue rate of each group.
  • the morphological defects, heart rate, heart morphology and other indicators were evaluated by SMZ745T stereo inverted microscope.
  • 2,4mM Pb(NO3)2 can reduce the survival rate of embryos, but the addition of 10mM L-5-MTHF-Ca group cannot save the impact of 2,4mM Pb(NO3)2 on embryo survival, compared with the control group .
  • 2,4mM Pb(NO3)2 and 2,4mM Pb(NO3)2+10mM L-5-MTHF-Ca did not have obvious embryonic developmental delay; 24hpf At this time, 2,4mM Pb(NO3)2 did not significantly affect the development and growth of zebrafish.
  • the zebrafish embryo has formed the development of the head and tail, and the tail will swing; at 48hpf, 2,4mM Pb(NO3)2 It did not affect the development of zebrafish intersegmental blood vessels, and there was no pericardial edema; at 72hpf, 2,4mM Pb(NO3)2 caused zebrafish body length to shorten, and a large number of embryos appeared deformities, mainly manifested as a curved trunk , The tail is short, but there is no significant difference in the development of blood vessels in the intestinal reticulum. And 10mM L-5LMTHF-Ca can rescue the embryonic developmental abnormalities caused by 2,4mM Pb(NO3)2 (see Figure 26).
  • 10mM L-5-MTHF-Ca can save all the abnormalities caused by 2,4mM Pb(NO3)2 in zebrafish embryo development.
  • the 0.9% EtOH group and 1.2% EtOH group caused the malformed phenotype of zebrafish embryos with bradycardia, and had no effect on pigment growth and intersegmental vascular development; 0.9% and 1.2% EtOH plus 10mM L-5-MTHF- Ca can relieve slow heart rate.
  • various concentrations of EtOH lead to the abnormal phenotype of zebrafish embryo pericardial edema. Adding 10mM L-5-MTHF-Ca at a concentration of 0.6% can reduce the rate of pericardial edema; 1.2% EtOH plus 10mM L-5- The MTHF-Ca group had no effect on alleviating pericardial edema (see Figure 29).
  • ethanol causes early embryonic developmental delay, pericardial edema, slow heart rate, shortened body length, and malformed phenotypes of intestinal reticulum vascular development disorders on zebrafish embryos, and has an effect on pigment growth and intersegmental vascular development. no effect.
  • Adding 10mM L-5-MTHF-Ca can alleviate pericardial edema under the condition of low concentration (0.6%) EtOH, alleviate the shortening of body length under the condition of high concentration (1.2%) EtOH, and improve the survival rate of embryos. Under the conditions of medium and high concentration (0.9%, 1.2%) EtOH, L-5-MTHF-Ca relieves bradycardia more obviously, and has a certain effect on preventing deformities.
  • 10mM L-5-MTHF-Ca can save the developmental malformations and death of zebrafish embryos caused by 1 ⁇ 5 ⁇ M aristolochic acid A to a certain extent.

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Abstract

5-甲基四氢叶酸用于制备围孕期和/或妊娠期妇女预防新生儿先心病缺陷的药物或保健食品的用途。5‐甲基四氢叶酸具有预防先天性心脏病的作用，由有关5‐甲基四氢叶酸的预防用量可以超过1mg，与合成叶酸相比大剂量使用没有致畸作用，可以实现大剂量下预防出生缺陷，用于预防新生儿先天性心脏病。

Description

5-甲基四氢叶酸的用途 技术领域
本发明属于医药领域，涉及含5-甲基四氢叶酸的组合物的在制备用于围孕期和/或妊娠期妇女预防新生儿先心病、流产、死胎的药物或保健食品新用途。
背景技术
先天性心脏病(简称先心病)已经成为出生缺陷的主要病种，也是儿童时期最常见的心脏病。先心病已经严重危害患儿生命和生活质量，是0～5岁儿童死亡的重要原因。
先心病分为多种亚型，其中室间隔缺损(VSD)，房间隔缺损(ASD)，动脉导管未闭(PDA)，法洛四联症(TOF)，大动脉位移(TGA)，三尖瓣闭锁或狭窄和肺动脉狭窄(PS)为前7种最常见的亚型。
目前，使用叶酸预防神经管缺陷的效果已经得到证实，在使用叶酸预防先心病方面，各国专家却存在争议。目前，大部分学者认为补充叶酸有助于预防先心病，甚至有人提出叶酸应使用更高的剂量来预防先心病[Huhta J C,Linask K.When should we prescribe high-dose folic acid to prevent congenital heart defects？[J].Current Opinion in Cardiology,2015,30(1):125-131.]。叶酸，是指喋酰谷氨酸，合成叶酸，又叫维生素B9、维生素M，英文名为folic acid。在医药应用中，叶酸应用于预防新生儿神经管畸形，预防和治疗巨幼细胞性贫血，世界卫生组织推荐乳母、孕妇每日摄入量应为400μg/天。高摄入量的叶酸会导致血液循环中存在未代谢叶酸以及还原叶酸(5-甲基四氢叶酸及其衍生物)。
叶酸是一种合成的氧化形式，在天然食品中没有发现，叶酸需要通过人的二氢叶酸还原酶(DHFR)将其转化为四氢叶酸，才能被人体利用，而这个过程在人体中很慢。有研究表明人肝脏代谢叶酸的速率在生理pH下是大鼠的1.79％，因此服用叶酸后，血液中可能会检测到未代谢的叶酸，有学者报道服用超过200μg的叶酸后数小时，血浆中能够检测到未代谢的叶酸[Bailey SW,Ayling JE.The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake.Proc Natl Acad Sci U S A.2009；106(36):15424-15429.]。此外，未代谢叶酸在细胞内蓄积有可能会导致DHFR活性降低。
回顾已有的叶酸和先心病关联性的研究，早期的北美地区的几项研究值得一提。其中一项研究，通过电话采访在1987年至1998年对加利福尼亚的207例出生有心脏锥干异常的母亲和481例随机选择的无畸形婴儿母亲，显示多种维生素或叶酸强化谷物的母亲的婴儿患有锥状动 脉性心脏病的风险降低了[Shaw GM,O'Malley CD,Wasserman CR,Tolarova MM and Lammer EJ.Maternal periconeptional use of multivitamins and reduced risk for conotruncal heart defects and limb deficiencies among offspring.American Journal of Medical Genetics.1995；59:536-545.]，但是该研究有关估算值不显著。之后在1999年至2004年加利福利亚的研究中，未发现叶酸复合维生素对心脏锥干异常有预防作用[Shaw GM,Carmichael SL,Yang W and Lammer EJ.Periconceptional nutrient intakes and risks of conotruncal heart defects.Birth Defects Res A Clin Mol Teratol.2010；88:144-51.]。1993-1996年，在加拿大波士顿、美国费城、加拿大多伦多的医院开展的一项对照研究表明，在产前使用含叶酸的维生素与心脏缺陷的类型之间未发现显著的关联[Werler MM,Hayes C,Louik C,Shapiro S and Mitchell AA.Multivitamin supplementaion and risk of birth defects.American Journal of Epidemiology.1999；150:675-682.]。在巴尔的摩-华盛顿的一项研究中，没有发现怀孕前补充叶酸400mg/天的预防作用[Scanlon K S,Ferencz C,Loffredo C A,et al.Preconceptional folate intake and malformations of the cardiac outflow tract.Baltimore-Washington Infant Study Group.[J].Epidemiology,1998,9(1):95-98.]。在1997年至2004年美国国家出生缺陷预防研究报告中指出，一项多中心研究母体糖尿病和含叶酸的多种维生素对多种类型的心脏缺陷的联合作用，得出未调整OR值为0.95(95％CI 0.85-1.06)，二者关系并不大[Correa A,Gilboa SM,Botto LD,Moore CA,Hobbs CA,Cleves MA,Riehle-Colarusso TJ,Waller DK,Reece EA and National Birth Defects Prevention S.Lack of periconceptional vitamins or supplements that contain folic acid and diabetes mellitus-associated birth defects.Am J Obstet Gynecol.2012；206:218e1-13.]。
匈牙利研究团队在1984-1991年一项补充叶酸预防神经管畸形的随机对照研究中指出，实验组人群从怀孕前1个月开始服用含有0.8mg叶酸的复合维生素直至确认怀孕后3个月停止，对照组的神经管畸形发生率极大降低，同时也发现先心病发生率也降低了40％[Czeizel,Andrew E.Prevention of congenital abnormalities by periconceptional multivitamin supplementation.[J].Bmj,1993,306(6893):1645-1648.]。然而上述研究存在缺陷，纳入的先心病例太少了，仅有10例暴露的心脏缺陷病例和17例未暴露的心脏缺陷病例。随后的评估研究发现，流感和普通感冒并伴有并发症的孕妇与锥周缺损风险较高相关，而大剂量的叶酸能够降低锥周缺损的风险[Csáky-Szunyogh,Melinda,Vereczkey A,
Figure PCTCN2020113157-appb-000001
Zsolt,et al.Risk and protective factors in the origin of conotruncal defects of heart--a population-based case-control study.[J].American Journal of Medical Genetics Part A,2013,161(10):2444-2452.]。
澳大利亚的1997至1998年的一项研究表明，叶酸与后代心脏缺陷的风险之间没有关联 [Bower C,Miller M,Payne J,Serna P.Folate intake and the primary prevention of non-neural birth defects.Aust N Z J Public Health.2006；30(3):258-261.]。在荷兰北部的病例对照研究中，作者表示使用叶酸的妇女患心脏缺陷的婴儿风险降低约20％，然而报告中对心脏病亚型的分析中，房室间隔缺损的风险却因为补充叶酸而提高，OR值为1.28(95％CI 0.33–4.95)[van Beynum IM,Kapusta L,Bakker MK,den Heijer M,Blom HJ,de Walle HE.Protective effect of periconceptional folic acid supplements on the risk of congenital heart defects:a registry-based case-control study in the northern Netherlands.Eur Heart J.2010；31(4):464-471.]。
挪威的研究团队[Leirgul E,Gildestad T,Nilsen R M,et al.Periconceptional Folic Acid Supplementation and Infant Risk of Congenital Heart Defects in Norway 1999-2009[J].Paediatric and Perinatal Epidemiology,2015,29(5):391-400.]指出，未能发现围孕期补充叶酸与重症先心病发生率的降低相关。同时，却发现0.4mg叶酸补充后，间隔缺损型先心病的发生风险增加20％。进一步的，2000年至2009年挪威和1996年至2003年丹麦的共197123例婴儿病例分析研究中，部分数据可能包含前述的研究，得出结论：叶酸与后代先心病出生缺陷的风险无关，包括严重缺损，圆锥缺陷或室间隔缺陷[
Figure PCTCN2020113157-appb-000002
N,Olsen SF,Basit S,et al.Association Between Maternal Folic Acid Supplementation and Congenital Heart Defects in Offspring in Birth Cohorts From Denmark and Norway.J Am Heart Assoc.2019；8(6):e011615.]。
加拿大也有学者[Bedard T,Lowry R B,Sibbald B,et al.Folic acid fortification and the birth prevalence of congenital heart defect cases in Alberta,Canada[J].Birth Defects Research Part A:Clinical and Molecular Teratology,2013,97(8):564-570.]统计了亚伯达省1995-2002年20万左右的出生例，发现1998年叶酸强化政策实行前后，总的先心病的发生率并未发生变化。
在中国，同样开展了几项叶酸对先心病的预防研究，在广东，湖北，福建和山西的医院病例对照研究表示，叶酸补充剂可将先心病心脏缺陷风险降低65％[Li X,Li S,Mu D,et al.The association between periconceptional folic acid supplementation and congenital heart defects:a case-control study in China.Prev Med.2013；56(6):385-389.]。中国学者[Baohong M,Jie Q,Nan Z,et al.Maternal folic acid supplementation and dietary folate intake and congenital heart defects[J].PLOS ONE,2017,12(11):e0187996-.]。2010年至2012年在甘肃省的一项出生列队研究中指出，叶酸具有降低新生儿先心病的概率，低摄入量的孕妇产下先心病患儿的风险与补充叶酸组比几乎增加了2倍。需要注意的是上述研究报告也指出，大于221.03μg/天的叶酸的摄入会导致房室间隔缺损风险增加，OR值1.20(95％CI 0.58–2.51)。2004年至2016年广东的一项研究，以约8379例先心病病例和6918例对照例为基础，作者认为孕早期孕妇使用叶酸与冠心病的低 风险有关[Qu Y,Lin S,Zhuang J,et al.First-Trimester Maternal Folic Acid Supplementation Reduced Risks of Severe and Most Congenital Heart Diseases in Offspring:A Large Case-Control Study.J Am Heart Assoc.2020；9(13):e015652.]。该数据结果与前人不一致的解释可能是在中国2010年免费叶酸补充计划之前，很少有孕妇使用叶酸，其叶酸水平本来就较低所导致的，并且该研究未调查孕妇的饮食中叶酸的水平以及孕妇本身的叶酸水平，未讨论剂量等有关因素，此外，文中作者承认统计的病例中仅有少数体现了先心病的亚型，可能并不具有代表性。
讨论因果的先决条件是设计有关具有叶酸补充剂使用和婴儿患心脏缺陷风险的具体信息的研究，但是由于不同的设计，偏见或混淆导致上述的一些研究结果相互矛盾，同时也可以通过区域的饮食习惯中叶酸的水平差异来解释。
综上所述，叶酸对先心病的作用依然存在质疑，上述的十五项叶酸与先心病有关的临床研究中，明确支持叶酸有助于先心病预防的有八项，其中匈牙利的研究病例数较少，而荷兰、挪威、中国的研究报告虽然支持叶酸有助于降低先心病风险，但是也在报告的细节中体现了叶酸补充有可能会导致房室间隔缺损的风险提高。上述研究仅是临床的数据统计或电话随访研究并不是双盲对照试验，由于研究作者对现有技术的认识偏见，先心病是否与叶酸相关，需要进一步确认。
目前，国际医界大部分认同围孕期妇女的叶酸补充有助于降低先心病的发生风险，在中国，孕妇补充叶酸也已经是共识，但是有关先心病发病率依然在增加。统计世界各国的先心病发病率，特别是房间隔缺损(ASD)的有关数据(见说明书附图1)，结果令人惊奇，1998年之后全球北美地区和亚洲有关ASD的出生率显著上升，而欧洲和南美地区则一定幅度下降[Liu Y,Chen S,Zühlke L,et al.Global birth prevalence of congenital heart defects 1970-2017:updated systematic review and meta-analysis of 260 studies.Int J Epidemiol.2019；48(2):455-463.]。值得注意的是，在1998年美国颁布了强制在谷物、面粉、面包等主食中添加叶酸的要求，加拿大也推出了类似的要求。而欧洲却没有强制性的要求，此外欧洲国家对叶酸补充剂的依从性很低，根据2009年欧洲先天异常监测发表的报告表明，叶酸补充剂的依从性介于5％至40％之间。
在中国，1980年之后有关ASD的出生率快速上升。发明人通过研究发现，中国的先心病的城市人口发病率远高于农村人口，并且有逐渐拉大的趋势。据世界卫生组织估计，全球低收入国家出生缺陷发生率为6.42％，中等收入国家为5.57％，而高收入发达国家为4.72％，虽然中国经济发展迅速，生活水平和医疗保健水平快速提高，尤其是东部发达城市，比如上海，苏南地区整体达到了发达国家的水平，但是婴儿出生缺陷率呈却并未呈现出相应水平的控制，反而城市人口比农村人口的出生缺陷率更高，从文献报道来看，先心病近年来，中国先心病CHD 缺陷发生率大幅度上升，从1980年到2004年缓慢上升，到2005年至2019年大幅上升。根据研究报道[Zhao L,Chen L,Yang T,et al.Birth prevalence of congenital heart disease in China,1980-2019:a systematic review and meta-analysis of 617 studies.Eur J Epidemiol.2020；35(7):631-642.]中国的先心病出生率，从1980-1984年的每千名婴儿0.201到2015-2019年的每前面婴儿4.905。值得注意的是在中国，1980年至2019年，随着时间的推移ASD的出生率显著增加，从1980年约千分之0.25，到2000年的千分之0.5，2000年之后迅速上升至千分之3，ASD几乎占据了先心病的半壁江山。
尽管在此期间，中国开始实施全国性孕妇叶酸增补，部分地区提供免费叶酸的政策，神经管畸形的发生率通过叶酸的补充显著的降低了而先心病发生率却反而大大增加，但结合流行病统计和当地叶酸使用情况，先心病发明人谨慎怀疑，叶酸的补充与先心病发生率提高是否有一定相关性并在本发明所公开的研究中证实了叶酸(未代谢叶酸)会导致先心病发病率提高。
甲醛是一种无色有刺激性气体，易溶于水和乙醇中，在人类工业生产中广泛应用，包括化学工业、木材工业、纺织工业、防腐工程中。随着对健康要求的不断提高，人们越来越重视环境中甲醛的危害。房屋装修过程中不可避免的会产生甲醛，而这些甲醛很难完全去除。因此新房中甲醛的含量往往高于安全值。这也增加了孕妇流产、胎儿健康受损的危险。发明人在研究先心病过程中意外发现5-甲基四氢叶酸能够挽救受甲醛影响的斑马鱼胚胎，同时高浓度5-甲基四氢叶酸无毒性、无致畸性。
发明内容
本发明基于动物实验，研究发现在孕期服用叶酸，未代谢叶酸会对胚胎具有致畸性，其长期或过度的补充可引发先心病的发生。而5-甲基四氢叶酸却无致畸性，在孕期服用具有预防先心病发生的作用。
本发明提供一种药物或保健食品，其含有5-甲基四氢叶酸，所述药物或保健食品用于孕妇预防新生儿先心病。
根据本发明的药品或保健食品，其用于孕妇预防新生儿先心病的发生。
根据本发明的药品或保健食品，所述先心病先心病是指先天畸形中最常见的一类，指在胚胎发育时期由于心脏及大血管形成障碍或发育异常而引起的解剖结构异常，或出生后应自动关闭的通道未能闭合的情况。
根据本发明的药品或保健食品，所述先心病先心病包括三个亚组疾病：一、先天性大动脉畸形，包括动脉导管未闭合，主动脉狭窄，肺动脉狭窄，肺动脉闭锁和其他大动脉先天性畸形； 二、先天性心脏隔膜畸形，包括心房-室间隔缺损(AVSD s)，室间隔缺损(VSD s)，房间隔缺损(ASD s)，法洛四联症，主肺动脉间隔缺损和其他先天性隔膜畸形；三、其他先心病先心病，包括心腔的先天性连接畸形，先天性主动脉和二尖瓣畸形。
根据本发明的药品或保健食品，所述药品或保健食品包含有效量的5-甲基四氢叶酸，其还含有药学上可接受的辅料或助剂。
根据本发明所述的药品或保健食品，其可以是本领域已知的各种剂型。例如经肠给药剂型，例如口服、舌下含服或直肠给药；示例性的，口服剂型可以是片剂、胶囊、口服液、滴丸、丸剂、粉剂、颗粒剂；示例性的，注射剂型可以是粉针剂、溶液剂、乳剂、混悬剂。
根据本发明的药品或保健食品，所述5-甲基四氢叶酸的使用剂量为0.05～50mg/日，优选的为0.2～0.8mg/日。
本发明的另一方面是提供一种防止孕妇因甲醛流产、保护胎儿的药物或保健食品。
根据所述防止孕妇因甲醛流产、保护胎儿的药物或保健食品，其能够预防孕妇因长期在甲醛环境中的流产。
根据所述防止孕妇因甲醛流产、保护胎儿的药物或保健食品，其能够应用于治疗孕妇因甲醛导致胎儿的流产先兆症状。
根据本发明所述防止孕妇因甲醛流产、保护胎儿的药品或保健食品，其可以是本领域已知的各种剂型。例如经肠给药剂型，例如口服、舌下含服或直肠给药；示例性的，口服剂型可以是片剂、胶囊、口服液、滴丸、丸剂、粉剂、颗粒剂；示例性的，注射剂型可以是粉针剂、溶液剂、乳剂、混悬剂。
根据本发明所述防止孕妇因甲醛流产、保护胎儿的药品或保健食品，所述5-甲基四氢叶酸的使用剂量为0.05～50mg/日，优选的为0.2～0.8mg/日，治疗流产先兆用量为0.8～200mg/日，优选20mg/日。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本方面上述基本技术思想前提下，还可以做出其他多种形式的修改、替换或变更。
特别指出的是，由于各国法律法规的不同，比如美国的膳食营养补充剂可以宣称有关功能(FDA同意膳食营养补充剂可以描述一种食物或膳食成分与健康状态或降低一种疾病风险的关系)，因此有关5-甲基四氢叶酸不仅可以应用于制备预防先心病、流产及死胎药品或保健食品，还可以应用于制备具有上述功能的膳食补充剂、特殊医学用途配方食品。示例性的，比如含有5-甲基四氢叶酸的复合维生素片，孕妇专用的配方奶粉等等。
5-甲基四氢叶酸钙在制备预防胎儿心脏畸形的药物或保健食品中的应用。
所述的心脏畸形优选为乙醇、硝酸铅、马兜铃酸导致的心脏畸形。
所述5-甲基四氢叶酸包括5-甲基四氢叶酸或其药学可接受的盐。
所述5-甲基四氢叶酸选自5-甲基-(6S)四氢叶酸，5-甲基-(6R)四氢叶酸，或5-甲基(6R，S)四氢叶酸，即包括5-甲基四氢叶酸的旋光异构体，旋光异构体的混合物，特别是纯旋光天然异构体。
所述药学可接受的盐包括5-甲基四氢叶酸中的碱性基团转化为相应的酸加成盐，5-甲基四氢叶酸中酸性基团转化为相应的碱加成盐；优选盐酸、硫酸、硝酸、磷酸、钠、钾、镁、钙和铵盐，取代的铵盐，或与精氨酸或赖氨酸形成的盐。
5-甲基四氢叶酸在制备预防重金属，酒精，药物导致的先心病的药物中的应用。
发明详述
本发明中，术语“5-甲基四氢叶酸”包括5-甲基-(6S)四氢叶酸，5-甲基-(6R)四氢叶酸，5-甲基(6R，S)四氢叶酸，即包括5-甲基四氢叶酸的旋光异构体、特别是纯旋光天然异构体，旋光异构体的混合物、例如是外消旋混合物，以及它们生理学上可接受的盐。其中5-(甲基)-(6S)四氢叶酸是特别优选的。
所述生理上可接受的盐，是指5-甲基四氢叶酸中的碱性基团转化为相应的酸加成盐，所述酸可以是无机酸，例如盐酸、硫酸、硝酸、磷酸；有机酸，例如甲酸、乙酸、丙酸、二乙基乙酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸，酒石酸、苹果酸、柠檬酸、葡糖酸、抗坏血酸或烟酸等。
所述生理上可接受的盐，也可指5-甲基四氢叶酸中的酸性基团转化为相应的碱加成盐，合适的盐，例如是钠、钾、镁、钙和铵盐，取代的铵盐，以及与精氨酸或赖氨酸形成的盐。
本发明所述叶酸，是指喋酰谷氨酸，合成叶酸，又叫维生素B9、维生素M，英文名为folic acid。研究论文[Baohong M,Jie Q,Nan Z,et al.Maternal folic acid supplementation and dietary folate intake and congenital heart defects[J].PLOS ONE,2017,12(11):e0187996-.]中表明，怀孕前一月，每日摄入叶酸超过266.5μg的孕妇，其新生儿房室隔膜缺损(ASD)的发生率高于每日摄入在115.97～265.5μg的孕妇组，而作者忽视了这个现象，依然认为叶酸的补充有利于新生儿先心病发生率降低。265.5μg/天依然低于联合国推荐摄入量，这个现象作者未进行进一步的讨论。补充叶酸会提高新生儿先心病发生率这个论点是颠覆性的论点，未被人们所发现，主要原因可能为新生儿缺陷发病例的绝对值本身就很低，先心病出生缺陷虽然是主要的类型，但是也仅在5‰以下，直接通过临床对照来验证则需要纳入上万名孕妇，这是不现实的，而之前 的研究报告由于都是对病历的统计分析无法控制纳入病例的本身的叶酸水平以及饮食影响，难以排除其影响。
孕妇补充叶酸也会导致血液循环中存在未代谢的叶酸，美国学者在研究孕妇补充叶酸的一项研究[Obeid R,Kasoha M,Kirsch S H.Concentrations of unmetabolized folic acid and primary folate forms in pregnant women at delivery and in umbilical cord blood[J].American Journal of Clinical Nutrition,2010,92(6):1416-1422.]中记载，25名怀孕期间补充叶酸的孕妇在血清中检测到0.19nmol/L的未代谢叶酸，脐带血中叶酸的浓度更高为0.27nmol/L，这提示未代谢叶酸可能在胎儿中积累的浓度大于母体。
本发明的发明人发现叶酸对斑马鱼胚胎具有致畸作用，叶酸介入下会导致斑马鱼胚胎心包膜水肿，斑马鱼胚胎心率过缓，体长变短，通过显微镜观察发现，叶酸会导致斑马鱼心包膜水肿、色素形成障碍、体节间血管发育不全。由此可得出，叶酸同样可能会对人体胚胎发育产生致畸作用，人体胚胎发育，心脏形成的关键时期为8周之前，在妊娠6-8周形成早期胎儿的心血管循环，而叶酸对血管发育、心包膜发育均有致畸的作用，会导致新生儿心血管异常或隔膜畸形，从而导致先心病。
发明人进一步通过斑马鱼观察到5-甲基四氢叶酸对斑马鱼胚胎无致畸作用，正常胚胎组与5-甲基四氢叶酸介入组无差异，在5-甲基四氢叶酸干预下，斑马鱼胚胎心脏外形正常，肠下网血管发育正常。
在一个实施例中，发明人收集不同时间(24h，48h)的叶酸干预下的斑马鱼胚胎，采用定量PCR技术检测斑马鱼胚胎中不同的转录因子的表达。结果发现，叶酸干预下斑马鱼胚胎中的转录因子NKx2.5、amhc、vmhc、hand2、has2、mef2a、mef2c、bmp2b、ephrinB2、ephB4的表达均受到影响，其中NKx2.5、hand2、has2、mef2c、ephB4均已经被证明是对心脏发育过程中重要的转录因子，发明人发现叶酸对mef2c、bmp2b、vmhc、has2、ephb4影响较大。
人类的mef2家族的四个成员，包括mef2a，mef2b，mef2c和mef2d，其中mef2b和mef2c首先在胚胎第7天在心脏胚胎中被激活，而mef2c是胚胎发育过程中形成原始心管中胚层细胞关键成员，mef2c的缺失会导致严重的心脏结构异常，特别是会导致房间隔缺损或室间隔缺损[Qiao XH,Wang F,Zhang XL,et al.MEF2C loss-of-function mutation contributes to congenital heart defects.Int J Med Sci.2017；14(11):1143-1153.]。叶酸影响mef2c的表达，这也是导致ASD出生率增加的可能原因。此外，透明质酸在心脏发育中高度表达，在细胞迁移和转化中起重要作用，人类的has基因表达调控透明质酸的合成，已经有报道显示has2基因突变可能会影响胚胎中心脏的间隔形成[Zhu X,Deng X,Huang G,et al.A novel mutation of Hyaluronan synthase  2 gene in Chinese children with ventricular septal defect.PLoS One.2014；9(2):e87437.]。
发明人同时收集了不同时间(24h，48h)的5-甲基四氢叶酸干预下的斑马鱼胚胎，采用定量PCR技术检测斑马鱼胚胎中不同的转录因子的表达。结果发现，在5-甲基四氢叶酸干预下，未发现其对转录因子的表达的影响，有关转录因子的表达与正常对照组相比无差异。
在一个实施例中，发明人发现5-甲基四氢叶酸并不能挽救斑马鱼胚胎由于合成叶酸导致的心包膜水肿的畸形，但可以挽救斑马鱼心率过缓、体长变短的畸形表现。
为了进一步确定叶酸对胚胎心脏发育的影响，在实施例9中发明人考察了不同剂量的叶酸对胎鼠心脏的影响，结果表明在一定剂量下，胎鼠心脏可见心室壁过薄，个别可见室间隔缺损，在实施例10中对摄入叶酸的母鼠以及5-甲基四氢叶酸的母鼠所生胎儿进行比较发现，低剂量叶酸组胎鼠先心病发病率为5.95％，高剂量叶酸组胎鼠先心病发病率为22.2％，而5-甲基四氢叶酸组则无先心病的胎鼠，进一步证明了叶酸对胚胎心脏发育的影响。
进一步的,发明人发现5-甲基四氢叶酸能够预防酒精、硝酸铅、马兜铃酸A诱导的胚胎心脏致畸的作用，而上述物质分别代表了酗酒、重金属、药物等不同环境因素导致的新生儿心脏畸形，因此5-甲基四氢叶酸具有广泛的预防先心病发生的作用。
在本发明一个实施例中，发明人发现5-甲基四氢叶酸能够预防因缺乏叶酸导致的心脏畸形。
本发明的药物或保健食品，每剂量中含有0.1mg-200mg的5-甲基四氢叶酸，在预防用药时，优选使用每剂量含有0.1mg-1mg的5-甲基四氢叶酸。其具体的使用剂量取决于各种因素，例如患者的年龄、体重、给药时间和途径、个体情况等等，优选最佳剂量为0.1-1mg/日，例如是0.8mg/天。
本发明中，所述药物或保健食品，含有各种载体、赋形剂和/或辅助功能剂，例如水、油、苯甲醇、聚乙二醇、甘油三乙酸酯、明胶、卵磷脂、环糊精、乳二糖或淀粉等糖类，硬脂酸镁、滑石、硅胶或纤维素。所述辅助功能剂，例如是稳定剂、抗氧剂、缓冲剂、抑菌剂等等。在本发明的一个实施方式中，所述赋形剂或载体是微晶纤维素，或者微晶纤维素与交联羧甲基纤维素钠的组合，或者微粉硅胶。
本发明首次发现，孕妇服用叶酸后，进入血液循环的未代谢叶酸会导致先天性心脏病，叶酸是一种合成的氧化形式，在天然食品中没有发现，叶酸需要通过人的二氢叶酸还原酶(DHFR)将其转化为四氢叶酸，才能被人体利用，而这个过程在人类中很慢，服用超过200μg的叶酸后数小时，血浆中能够检测到未代谢的叶酸，这也是叶酸与先心病临床统计结果混淆，结论不一致的可能原因。本发明也解释了前人研究相互矛盾的原因，上世纪80‐90年代由 于营养、和叶酸补充的政策未推出，孕妇本身的叶酸水平可能比较低，容易做出正面的结论。而随着含有营养补充的叶酸食品的越来越多，大剂量下的叶酸难以做出叶酸与先心病的相关性。此外由于2000年以前间隔缺陷发病率非常低，尽管统计上发现叶酸有可能与该亚型有一定相关性，研究作者在认知缺陷和偏见下可能忽略了这个现象，或认为是统计偏差。
虽然叶酸已经大规模应用于预防胎儿的先天畸形，但是本发明通过分析流行病学资料与该地区叶酸补充情况的却发现补充叶酸之后先心病发病率提高的矛盾性的结果，谨慎的提出叶酸补充会提高先心病发病率，本发明的动物实验和表观遗传学的结果也证实了该结论。
本发明也发现5‐甲基四氢叶酸具有预防先天性心脏病的作用，由有关5‐甲基四氢叶酸的预防用量可以超过1mg，与合成叶酸相比大剂量使用没有致畸作用，可以实现大剂量下预防出生缺陷，特别是预防新生儿先天性心脏病方面具有显著的进步性。
附图说明
图1 1980-2019年中国不同亚型的先心病出生率及1970-2017年世界不同地区房间隔缺损畸形的出生率。
图2a:实验例1中10mM L-5-MTHF-Ca对斑马鱼胚胎存活率的影响；b实验例1中L-5-MTHF-Ca对斑马鱼胚胎发育的影响。
图3实验例1中L-5-MTHF-Ca在48hpf及72hpf时对斑马鱼心率及体长的影响。
图4a:实验例2中MTX和MTX+L-5-MTHF-Ca对斑马鱼胚胎存活率；ns，与对照组比差异不显著；****，p＜0.005；b:MTX造模组与L-5-MTHF-Ca拯救组对斑马鱼胚胎发育的影响。
图5实验例2中MTX及MTX+L-5-MTHF-Ca对斑马鱼胚胎心包膜肿率、心率及斑马鱼胚胎体长的影响；ns，与对照组比差异不显著；****，p＜0.005；***，p＜0.01；*，p＜0.1。
图6a:实验例3中不同浓度叶酸对斑马鱼胚胎存活率的影响；b:不同浓度叶酸对斑马鱼胚胎心率的影响。
图7实验例3中不同浓度叶酸在8hpf和24hpf对斑马鱼胚胎形态的影响。
图8实验例3中10mM L-5-MTHF-Ca、8mM FA、6mM FA及4mM FA在72hpf对斑马鱼心脏的影响；左图:72hpf显微镜观察不同浓度叶酸影响下的心脏胚胎发育情况,发现心包膜水肿畸形\心房和心室拉长,肠下网血管发育障碍；右图：叶酸在72hpf对斑马鱼体长的影响。
图9实验例4中叶酸对斑马鱼胚胎不同转录因子表达的影响。
图10实验例4中5-甲基四氢叶酸对斑马鱼胚胎不同转录因子表达的影响。
图11a:实验例5中FA和FA+L-5-MTHF-Ca对斑马鱼胚胎的成活率的影响；b:实验例5中FA与FA+L-5-MTHF-Ca对斑马鱼胚胎发育的影响。
图12实验例5中48hpf斑马鱼胚胎心包膜水肿率、心率和72hpf斑马鱼胚胎体长。
图13a:实验例6中甲醛和甲醛+L-5-MTHF-Ca对斑马鱼胚胎成活率的影响；b:甲醛和甲醛+L-5-MTHF-Ca对斑马鱼胚胎发育的影响。
图14实验例6中48hpf斑马鱼胚胎心率和72hpf斑马鱼胚胎体长的影响。
图15a:实验例7中低剂量同型半胱氨酸对心肌小梁的影响；b:低剂量同型半胱氨酸导致心肌细胞间隙小量淋巴细胞浸润；c:低剂量同型半胱氨酸导致右心室内可见淡粉色浆液性物质；d:高剂量同型半胱氨酸导致胎鼠肌细胞浆染红。
图16实验例7中高高剂量同型半胱氨酸对胎鼠心脏影响。
图17实验例8正常组大鼠心脏切片图。
图18实验例8中高剂量(36.44mg/kg)叶酸下，对胎鼠心脏的影响。
图19实验例8中高剂量(18.22mg/kg)叶酸下，对胎鼠心脏的影响。
图20实验例8中高剂量(9.11mg/kg)叶酸下，对胎鼠心脏的影响。
图21实验例9中2.2775mg/kg叶酸组中胎鼠典型的心脏切片图。
图22实验例9中0.911mg/kg叶酸组中胎鼠的典型心脏切片图。
图23实验例10中6日新生小鼠心脏超声图，对照组(A)，叶酸低剂量组(B)，叶酸高剂量组(C)，甲基四氢叶酸低剂量组(D)亚甲基四氢叶酸高剂量组(E)。
图24实验例10中六日龄新生小鼠超声心动图数据，对照组(A)，叶酸低剂量组(B)，叶酸高剂量组(C)，甲基四氢叶酸低剂量组(D)亚甲基四氢叶酸高剂量组(E)。
图25实验例10中各组胎鼠典型的心脏切片图。
图26实验例11中a：Pb(NO3)2和Pb(NO3)2+L-5-MTHF-Ca对斑马鱼胚胎的成活率试验；b：Pb(NO3)2造模组与L-5-MTHF-Ca拯救组对斑马鱼胚胎发育的影响。
图27实验例11中醋酸铅与甲基四氢叶酸影响下48hpf斑马鱼胚胎心率和72hpf斑马鱼胚胎体长及畸形率。
图28实验例11中A:乙醇和乙醇+L-5-MTHF-Ca对斑马鱼成活率；B:乙醇与L-5-MTHF-Ca对斑马鱼发育的影响。
图29实验例11中乙醇和甲基四氢叶酸影响下48hpf对斑马鱼胚胎心率和斑马鱼胚胎体长与畸形率。
图30实验例11中马兜铃酸A和马兜铃酸A+L-5-MTHF-Ca对斑马鱼胚胎的存活率试验。
图31实验例11中马兜铃酸A造模组与L-5-MTHF-Ca+马兜铃酸A组对斑马鱼胚胎发育的影响。
图32实验例11中马兜铃酸A与5-甲基四氢叶酸影响下的48hpf斑马鱼胚胎心包膜水肿率、心率和72hpf斑马鱼胚胎体长。
具体实施方式
实施例1
0.4g的5-甲基四氢叶酸钙盐，与700g的微晶纤维素混合，干法制粒后，灌1000粒胶囊，制成每粒含0.4mg的5-甲基四氢叶酸钙胶囊制剂。
实施例2
1g的5-甲基四氢叶酸钙盐，加入200g的微粉硅胶，混匀，后于压片机压片成形，制得每粒含有2mg的5-甲基四氢叶酸片。
实验例1 5-甲基四氢叶酸钙(L-5-MTHF-Ca)对斑马鱼胚胎存活率和心血管发育的影响
转基因型斑马鱼(fli-1:EGFP)来自于南京大学模式动物研究所。实验所用鱼是年龄小于1的成年斑马鱼，饲养水体的pH值为7±0.2，温度在28℃左右，光照和黑暗的时间比是14h:10h，每天喂食两次丰年虫卵。前一天晚上把一雌两雄三条斑马鱼放在产卵盒中，第二天早上收集鱼卵。鱼卵放于胚胎培养液(0.2g/L的海盐配置而成)中，28℃培养。放于24孔板培养，每孔10条(n＝10)，已预先加入1mL的胚胎培养液。每组试验重复3次。斑马鱼胚胎在受精后2h(2hpf)开始加药，一直作用到72hpf终止。于8、24、48、72hpf记录每组死亡数。加药期间不喂食，发现胚胎死亡及时清理。观察所致畸形的表型，记录畸形存在的异常的所有斑马鱼数目。成活率、形态缺陷、心率、心脏形态等指标通过SMZ745T体视倒置显微镜观察评估。
试验结果如下，我们发现10mML的L-5-MTHF-Ca并没有对胚胎存活率产生影响，与正常组对比8hpf时，正常胚胎和10mML的L-5-MTHF-Ca胚胎表型相似，已近完成外包。24hpf时，正常胚胎和10mML的L-5-MTHF-Ca相似，已经形成头部和尾部的发育，并且尾部会有摆动现象。48hpf后，各组头部尾部发育完善，全身色素形成，心脏搏动明显，体节间血管发育正常，可见血液在全身流动。72hpf时，各组心脏外形正常，肠下网血管发育正常(见图2)。在48hpf测量了斑马鱼胚胎的心率，72hpf测量斑马鱼胚胎的体长，可以发现10mML的L-5-MTHF-Ca与对照组在心率和体长均无差别(见图3)。
实验例2 L 5-MTHF-Ca对甲氨蝶呤(MTX)致叶酸缺乏斑马鱼胚胎模型心血管发育系统的影响
转基因型斑马鱼(fli-1:EGFP)来自于南京大学模式动物研究所。实验所用鱼是年龄小于1 的成年斑马鱼，饲养水体的pH值为7±0.2，温度在28℃左右，光照和黑暗的时间比是14h:10h，每天喂食两次丰年虫卵。前一天晚上把一雌两雄三条斑马鱼放在产卵盒中，第二天早上收集鱼卵。鱼卵放于胚胎培养液(0.2g/L的海盐配置而成)中，28℃培养。斑马鱼胚胎在6hpf开始放于24孔板加药培养，每孔10条，加入1mL的1.5mM MTX溶液，一直作用到10hpf终止，后转入到卵液继续培养至72hpf。发现胚胎死亡及时清理。观察畸形的表型，记录畸形存在异常的所有斑马鱼数目，计算各组的畸形率和挽救率。形态缺陷、心率、心脏形态等指标通过SMZ745T体视倒置显微镜观察评估。
结果如下(见图4)，我们发现1.5mM MTX会严重导致胚胎存活率低(存活率低于0.1)，加10mM L 5-MTHF-Ca组可以挽救1.5mM MTX对胚胎存活率产生的影响，与对照组差异不显著。与正常对照组比，评价不同时间点斑马鱼胚胎的表型，1.5mM MTX和1.5mM MTX+10mM L-5-MTHF-Ca均没有出现明显的胚胎发育迟缓；24hpf时，1.5mM MTX导致斑马鱼发育不全，出现小头、短尾等畸形现；48hpf时，1.5mM MTX致斑马鱼躯干弯曲、体节间血管发育不全、心包膜水肿；72hpf时，1.5mM MTX致斑马鱼体长变短，肠下网血管发育不全。而10mM L-5-MTHF-Ca可以拯救1.5mM MTX所致的胚胎发育畸形。MTX组畸形率为100％，而加入L-5-MTHF-Ca之后畸形率仅为10％。
为进一步量化评估10mM L-5-MTHF-Ca对MTX的挽救效果，48hpf时评估了斑马鱼胚胎心包膜水肿、10s心率和72hpf各组间斑马鱼的体长。由图5可以看出，1.5mM MTX严重导致斑马鱼心包膜水肿、心率过缓、体长变短，而加10mM L-5-MTHF-Ca组则可以挽救斑马鱼心包膜水肿、心率过缓、体长变短的畸形表型。
实验例3叶酸(FA)对斑马鱼胚胎的致畸作用
转基因型斑马鱼(fli-1:EGFP)来自于南京大学模式动物研究所。实验所用鱼是年龄小于1的成年斑马鱼，饲养水体的pH值为7±0.2，温度在28℃左右，光照和黑暗的时间比是14h:10h，每天喂食两次丰年虫卵。前一天晚上把一雌两雄三条斑马鱼放在产卵盒中，第二天早上收集鱼卵。鱼卵放于胚胎培养液(0.2g/L的海盐配置而成)中，28℃培养。斑马鱼胚胎在2hpf开始放于24孔板加药培养，每孔5条(n＝5)，各孔分别加入1mL20mM NaHCO 3溶液、1mL 10Mm FA溶液(溶剂为20mM NaHCO 3)、1mL 8mM FA溶液(溶剂为20Mm NaHCO 3)、1mL6mM FA溶液(溶剂为20mM NaHCO 3)、1mL4mM FA溶液(溶剂为20mM NaHCO 3)、1mL2mM FA(溶剂为20mM NaHCO 3)、1mL 10mM L-5-MTHF-Ca(溶剂为20mM NaHCO 3)，一直作用到72hpf终止。发现胚胎死亡及时清理。每组试验重复3次。发现胚胎死亡及时清理。观察畸形的表型，记录畸形存在异常的所有斑马鱼数目，计算各组的畸形率和挽救率。形态缺陷、心 率、心脏形态等指标通过SMZ745T体视倒置显微镜观察评估。
结果如下，我们发现20mM NaHCO 3不影响斑马鱼的存活率，10mM L-5-MTHF-Ca也不影响斑马鱼的存活率，而所有FA组均显示胚胎存活率低，随着剂量增多斑马鱼胚胎死亡率也不断提高(见图6)，同时叶酸也影响了胚胎的心率，特别是对48hpf时的胚胎心率影响最大。我们也观察到叶酸也影响斑马鱼胚胎的体长，特别是在72hpf时，8mM浓度的叶酸显著缩短了胚胎的体长。
与正常对照组对比，评价不同时间点各个对照组斑马鱼胚胎的表型，8hpf正常对照组，L-5-MTHF-Ca组、NaHCO 3组、2mM FA均已外包完全，而其他浓度的叶酸试验组的斑马鱼胚胎均显示外包不完全的现象，24hpf时发现叶酸能够导致胚胎的头和尾部发育畸形(见图7)。72hpf，正常斑马鱼、L-5-MTHF-Ca组、NaHCO 3组、2mM FA胚胎心脏外形较正常，肠下网血管发育正常,而4mM及以上浓度的FA组均发现斑马鱼胚胎的心包膜水肿畸形、心房和心室拉长、肠下网血管发育障碍(见图8)。
实验例4叶酸(FA)对斑马鱼胚胎发育过程中不同转录因子表达的影响
为了探讨叶酸在斑马鱼胚胎发育过程中对其表观遗传的影响，收集了不同发育时期的斑马鱼胚胎进行检测。转基因型斑马鱼(fli-1:EGFP)来自于南京大学模式动物研究所。实验所用鱼是年龄小于1的成年斑马鱼，饲养水体的pH值为7±0.2，温度在28℃左右，光照和黑暗的时间比是14h:10h，每天喂食两次丰年虫卵。前一天晚上把一雌两雄三条斑马鱼放在产卵盒中，第二天早上收集鱼卵。鱼卵放于胚胎培养液(0.2g/L的海盐配置而成)中，28℃培养。斑马鱼胚胎在2hpf开始放于24孔板加药培养，每孔10条(n＝10)，各孔分别加入1mL的8mM FA溶液(溶剂为20mM NaHCO 3)、1ml的8mM L-5-MTHF-Ca(溶剂为20mM NaHCO 3，叶源酸)，分别于24h、48hpf，用4％多聚甲醛溶液固定过夜，保存在甲醇溶液中，置-20℃备用。斑马鱼胚胎在2hpf开始给药，每24h收集每组表型明显的10条斑马鱼，根据说明书用Trizol提取每组斑马鱼的RNA，每组提取24、48hpf 2个时间点的胚胎进行后续实验。
本qRT-PCR实验用的是荧光染料SYBR Green染料。qRT-PCR按照SYBR Green qPCR Master Mix试剂盒的说明书操作，最后在ABI(HT 7900)PCR仪上检测。所用基因引物由金唯智生物科技有限公司合成，具体信息见表1。以β-actin的表达量为内参，采用ΔΔCt方法计算其他基因的相对表达水平。
表1目标和参考基因的引物序列
Figure PCTCN2020113157-appb-000003
Figure PCTCN2020113157-appb-000004
结果如下，斑马鱼卵在受精后48h形成完整的心脏，由心房和心室组成，房室之间存在瓣膜。在各种信号分子作用下，生心板细胞的心肌形成基因进行表达，这些心肌形成基因包括Nkx-2家族基因、Mef2家族基因等等。我们发现叶酸会干扰各种基因的表达，与正常对照组相比，各种基因表达均受到不同程度的影响(见图9)，而5-甲基四氢叶酸(叶源酸)则与正常对照组无差异(见图10)。我们认为叶酸缺乏与先心病形成有关，但是叶酸(folic acid)本身也与先心病形成有关。我们发现叶酸在24hpf时提高了mef2c基因的表达，降低了bmp2b基因的表达，而在48hpf时叶酸抑制了has2、mef2c、ephb4基因的表达，提高了vmhc的表达。而mef2c是胚胎发育过程中形成原始心管胚层细胞的关键成员，has2也影响胚胎心脏间隔的形成，这也是ASD出生率显著增加的可能原因。
实验例5 L-5-MTHF-Ca挽救FA对斑马鱼胚胎致畸、存活率、心率及体长的影响
为了考察5-甲基四氢叶酸是否能够降低FA的致畸性，开展了以下实验。转基因型斑马鱼(fli-1:EGFP)来自于南京大学模式动物研究所。实验所用鱼是年龄小于1的成年斑马鱼，饲养水体的pH值为7±0.2，温度在28℃左右，光照和黑暗的时间比是14h:10h，每天喂食两次丰年虫卵。前一天晚上把一雌两雄三条斑马鱼放在产卵盒中，第二天早上收集鱼卵。鱼卵放于胚胎培养液(0.2g/L的海盐配置而成)中，28℃培养。斑马鱼胚胎在2hpf开始放于24孔板加 药培养，每孔10条，加入1mL的6mM FA溶液(溶剂为20mM NaHCO 3)，一直作用到72hpf终止。发现胚胎死亡及时清理。每组试验重复3次。选取6mM FA造模，同时用10mM L-5-MTHF-Ca进行挽救试验，并设置20mM NaHCO 3的溶剂对照组。观察畸形的表型，记录畸形存在异常的所有斑马鱼数目，计算各组的畸形率和挽救率。形态缺陷、心率、心脏形态等指标通过SMZ745T体视倒置显微镜观察评估。
结果如下，选取6mM FA造模，同时用10mM L-5-MTHF-Ca进行挽救试验，并设置20mM NaHCO3的溶剂对照组，我们发现20mM NaHCO3不影响斑马鱼的存活率，而6mM FA会严重导致胚胎存活率低，但是加10mML-5-MTHF-Ca组可以挽救6mM FA对胚胎存活率产生影响。与正常对照组对比，评价不同时间点斑马鱼胚胎的表型(见图11)，8hpf时，正常胚胎已近完成外包，6mM FA和6mM FA+10mM L-5-MTHF-Ca组均会使斑马鱼胚胎发育延迟。24hpf，正常斑马鱼胚胎已经形成头部和尾部的发育，并且尾部会有摆动现象。6mM FA会使斑马鱼出现小头短尾，6mM FA+10mM L-5-MTHF-Ca则会缓解这种畸形。48hpf时，6mM FA和6mM FA+10mM L-5-MTHF-Ca均会致斑马鱼心包膜水肿、色素形成障碍、体节间血管发育不全。72hpf时，6mM FA和6mM FA+10mM L-5-MTHF-Ca组均导致斑马鱼肠下网血管发育障碍。但20mM NaHCO3(溶剂对照组)在各个时间点观察，其表型与正常组相似。
为进一步量化评估10mM L-5-MTHF-Ca对6mM FA的挽救效果，48hpf时评估了斑马鱼胚胎心包膜水肿、10s心率和72hpf各组间斑马鱼的体长。由图12可以看出，6mM FA严重导致斑马鱼心包膜水肿，但10mM L-5-MTHF-Ca并不能挽救心包膜水肿的畸形；6mM FA严重导致斑马心率过缓、体长变短，而加10mM L-5-MTHF-Ca组则可以挽救斑马鱼心率过缓、体长变短的畸形表型。20mM NaHCO 3(溶剂对照组)对斑马鱼胚胎心脏、心率和体长均无影响。
综上所述，10mM L-5-MTHF-Ca可以挽救6mM FA导致斑马鱼胚胎降低的存活率、心率过缓和体长缩短，但无法挽救心包膜水肿和色素发育障碍。
实验例6 L-5-MTHF-Ca挽救甲醛HCHO对斑马鱼胚胎存活率的影响
转基因型斑马鱼(fli-1:EGFP)来自于南京大学模式动物研究所。实验所用鱼是年龄小于1的成年斑马鱼，饲养水体的pH值为7±0.2，温度在28℃左右，光照和黑暗的时间比是14h:10h，每天喂食两次丰年虫卵。前一天晚上把一雌两雄三条斑马鱼放在产卵盒中，第二天早上收集鱼卵。鱼卵放于胚胎培养液(0.2g/L的海盐配置而成)中，28℃培养。斑马鱼胚胎在2hpf开始放于24孔板加药(10mM L-5-MTHF-Ca)培养，每孔10条，加入1mL的30mM HCHO溶液。发现胚胎死亡及时清理。每组试验重复3次。观察畸形的表型，记录畸形存在异常的所有斑马 鱼数目，计算各组的畸形率和挽救率。形态缺陷、心率、心脏形态等指标通过SMZ745T体视倒置显微镜观察评估。
结果如下，选取30mM HCHO造模，同时用10mM L-5-MTHF-Ca进行挽救，发现30mM HCHO会严重导致胚胎存活率低，但是加10mM L-5-MTHF-Ca组可以挽救30mM HCHO对胚胎存活率产生影响，与对照组对比，评价不同时间点斑马鱼胚胎的表型(见图13)，8hpf时，正常胚胎已近完成外包，30mM HCHO和30mM HCHO+10mM L-5-MTHF-Ca组均会使斑马鱼胚胎发育延迟。24hpf，正常斑马鱼胚胎已经形成头部和尾部的发育，并且尾部会有摆动现象，30mM HCHO和30mM HCHO+10mM L-5-MTHF-Ca与control组斑马鱼表型相似。48hpf时，用明场和荧光显微镜对斑马鱼胚胎进行观察拍照，正常胚胎和30mM HCHO和30mM HCHO+10mM L-5-MTHF-Ca作用组相似，头部尾部发育完善，全身色素形成，心脏搏动明显，体节间血管发育正常，可见血液在全身流动。72hpf时，正常胚胎和30mM HCHO和30mM HCHO+10mM L-5-MTHF-Ca作用心脏外形正常，肠下网血管发育正常。
进一步量化评估10mM L-5-MTHF-Ca对30mM HCHO的挽救效果，48hpf时评估了斑马鱼胚胎心包膜水肿、10s心率和72hpf各组间斑马鱼的体长。由图14可以看出，30mM HCHO和30mM HCHO+10mM L-5-MTHF-Ca对斑马鱼心包膜、心率、体长方面均无影响。
综上所述，30mM HCHO对斑马鱼致死但不致畸，10mM L-5-MTHF-Ca可以挽救30mM HCHO导致斑马鱼胚胎降低的存活率。该发现说明L-5-MTHF-Ca能够挽救因长期在甲醛环境下孕妇的胚胎的高死亡率。
实验例7同型半胱氨酸对胎鼠发育影响
为了考察同型半胱氨酸对胎儿心脏的影响，选取SD大鼠，雌雄大鼠按1：1比例合笼交配过夜，次日清晨检查阴栓，见阴栓之日即定未妊娠第0天，孕鼠单独饲养。用1％同型半胱氨酸溶液(HCY)，按1ml/100g注射体积计算注射量，剂量为(100mg/kg/d)，孕鼠妊娠第7天，每日腹腔注射1次，直至孕17天，孕第19天在注射一次，每次注射时回抽无液体，孕第20天水合氯醛麻醉下，剖宫取胎。按上述同样方法考察高剂量HCY模型孕鼠，剂量为(200mg/kg/d)，其余条件一致。胎鼠解剖取心脏，切片观察，结果如下。
低剂量的HCY导致胎鼠心肌小梁增宽，心房内充满红细胞(见图15，a)。心肌间隙少量淋巴细胞浸润(见图15,b)。右心肌间隙少量淋巴细胞浸润(见图15,c)。高剂量的HCY导致胎鼠肌细胞浆染红，如黑色尖头所示(见图15,d)。心室、心房充满红细胞，心肌小梁增宽(见图16)。上述实验说明HCY会影响胎鼠心脏，推测人类的同型半胱氨酸的高水平同样也会可 能导致先心病发生。
实验例8高剂量叶酸对胎鼠心脏的影响(预实验)
SD成年健康雌、雄大鼠各28只，重约200～250g，购买于斯贝福(北京)生物技术有限公司。所有动物购入后，观察动物的一般生理指标、体重和进食情况。适应性喂养一周。标准颗粒饲料饲养，自由饮水。自然昼夜光线照明，室温18～26℃，相对湿度40％～70％。
将获取的16只孕鼠按体重随机分为4个组每组4只：正常组、高剂量叶酸组、中剂量叶酸组、低剂量叶酸组。分组分别为高剂量叶酸组36.44mg/kg，中剂量叶酸组18.22mg/kg，低剂量叶酸组9.11mg/kg及空白对照组，给药方式为灌胃给药，给药途径为1ml/100g溶液。除正常组给予纯水外，其他各组分组后第2d开始给药，每日一次，连续给药21天，各组孕鼠均自由进食饮水。雌鼠给药7天后，雌雄大鼠按1：1比例合笼交配过夜，次日清晨检查阴栓，见阴栓之日即定为妊娠第0天，孕鼠单独饲养，再继续给药21天，剖宫取胎。
常规组织病理学石蜡样本制备、连续切片(厚度5um)、HE染色。样本制备：常规组织样本石蜡切片制备，每个样本1个石蜡块；切片：每个编号样本切片20张。裱片起始点为切片时蜡块可见有心室起裱片，平均每张玻片上裱有4个连续组织点。HE染色：每个样本20张玻片，隔张取1张做HE染色，共10张。观察分析：Leica全景图像采集，并进行对比分析。解剖胎鼠心脏，进行切片观察，结果如下。
正常组心房与心室分化良好，心包膜、心内外膜完整。可同时观察到心肌纤维的纵切、斜切或横切面。心肌纤维排列整齐规则，纹理清晰，肌纤维分支并吻合成网。心肌纤维被疏松结缔组织分成大小不等的纤维束，束内见有丰富的血管。心房与心室内心肌小梁密集发达。心肌细胞正常，细胞核排列整齐，呈卵圆形或者是圆球形，蓝色淡染。心肌纤维之间有少量圆形或椭圆形红染的血细胞。无明显病理变化。由于发育或切割角度问题，部分动物未见与心室相连的大动脉结构(见图17)。
高剂量叶酸组中，多数动物心脏发育延迟(n＝3)，共同特点是心脏体积较小，心壁较薄(C9-1，C9-4，C9-5)。部分动物室间隔消失(n＝1)，右心室缺损(n＝4)，为实性肌肉组织，或只有很小的腔体，几乎看不到心肌小梁；左心室腔体内壁心肌小梁缺失，或只有少量短粗的心肌小梁；左右心房基本正常(C9-2，C9-3，C9-4，C9-5)。个别动物心脏出现不同的镜下特征，表现为左右心房腔体明显扩张，左右心室腔体大小相似，左心室有较稀疏的心肌小梁，右心室内壁光滑，几乎看不到心肌小梁(C9-1)。由于发育或切割角度问题，部分动物未见与心室相连的大动脉结构(C9-3，C9-4)(见图18)。
中剂量叶酸组中，分化形成心室与心房，可见完整心包膜与心内膜、心外膜。心肌纤维的纵切、斜切或横切面均可见。心肌纤维发育较发达，排列规则，纹理清晰。未见组织细胞变性坏死或炎性细胞浸润。但个别动物心室与心房表现出一定程度发育迟缓(n＝1)，心房心室体积较小(D1-3)。部分动物可见心室腔狭窄，分化迟缓，心室内壁光滑，甚至心室缺损(n＝2)，心室腔几乎不可见，为实性肌肉组织，心肌小梁短小稀疏(D1-3，D1-5)，心房内心肌小梁稀少(D1-3)(见图19)。
低剂量叶酸组中，心房与心室分化完成，心肌纤维呈束状整齐排列，可见心肌纤维的不同切面，纹理清晰，心肌纤维之间有较丰富的毛细血管。但部分动物心室分化延迟，共同特点是右心室缺损(n＝3)，只有很小的腔体；左右心房腔体稍微扩张，基本正常(E3-3，E3-4)。个别动物心脏明显发育延迟(n＝1)，心脏体积较小，室间隔消失，右心室缺损，为实性肌肉组织，看不到心肌小梁；左心室腔体变小，内壁只有少量的心肌小梁；左右心房萎缩变小(E3-5)(见图20)。
统计数据见下表2，由图可知，叶酸同时也可能影响大鼠的产仔率，高剂量下会降低产仔率。
表2叶酸对大鼠胎鼠发育影响汇总
Figure PCTCN2020113157-appb-000005
实验例9药理剂量叶酸对胎鼠心脏影响
为了考察药理剂量下叶酸对胎鼠心脏的影响，实验分为叶酸组(6组)及空白对照组，叶 酸对照组分别为叶酸4.555mg/kg/d(1组)、叶酸2.2775mg/kg/d(2组)、叶酸0.911mg/kg/d(3组)、叶酸0.4555mg/kg/d(4组)、叶酸0.22775mg/kg/d(5组)、叶酸0.113875mg/kg(6组)。叶酸组及空白对照组每组为4只孕鼠，4.555mg/kg/d(1组)、叶酸2.2775mg/kg/d(2组)、叶酸0.911mg/kg/d(3组)给药方式为腹腔注射，叶酸0.4555mg/kg/d(4组)、叶酸0.22775mg/kg/d(5组)、叶酸0.113875mg/kg(6组)灌胃给药、空白对照组的给药方式为自然喂养。雌鼠给药7天后，雌雄大鼠按1：1比例合笼交配过夜，次日清晨检查阴栓，见阴栓之日即定为妊娠第0天，孕鼠单独饲养，再继续给药21天，剖宫取胎，各组胎鼠数量见图30。取胎鼠心脏进行病理检查，结果如下。
4.555mg/kg(1组)基本已分化形成心房和心室，心包膜及心内膜、心内膜、心肌层未见明显病理变化见图。左右心房发育基本正常，右心房切面普遍略大于左心房，腔体内壁可见丰富的心肌小梁。个别个体心房腔体较小。左右心室基本形成，心室壁血管丰富，常见左心壁较厚，腔隙狭窄，甚至未见明显的左心室腔隙(n＝3)，或仅见紧密排布的心肌小梁。个别个体右心室也表现出腔隙狭窄。个别个体还可见右心室壁过薄。切片中均可见与心室相连的大血管管腔，部分切面可见管腔内的瓣膜。
2.2775mg/kg(2组)基本已分化形成心室与心房，心包膜完整，心肌层，心外膜与心内膜完整，未见坏死与炎性细胞浸润。左右心房内可见丰富的心肌小梁，个别个体左心房仅见小部分切面(图21，d)。个别个体可见房间隔缺损(图21，d)。左右心室基本发育形成，心室内心肌小梁丰富，可见相连的血管管腔，个别可见血管内瓣膜(图21，b，e)。个别切面可见心房心室开口及二尖瓣瓣膜(图21，d)。部分个体左心室缺损(n＝2)，或仅见排列疏松的心肌小梁而不见明显腔隙(图21，b，c)。部分个体还可见右心室壁过薄(图21，b，d)。
0.911mg/kg(3组)基本已分化形成心室与心房，心包膜完整，心肌层，心外膜与心内膜完整，未见坏死与炎性细胞浸润。左右心房均较为充盈，心房内心肌小梁丰富。部分个体切片内仅见小部分左心房(图22，c，e)。左右心室基本发育形成，个别个体左心室心壁较厚，腔隙狭窄(图22，d)。心室壁血管丰富，心室内可见丰富的心肌小梁。大部分切片中可见与心室连接的大血管及管腔内的瓣膜结构(图22，a，b，d，e)。个别切片中可见二尖瓣及右心房室开口(图22，c)。个别个体还可见右心室壁过薄(图22，c，e)。个别心房或心室壁有缺损(n＝1)。
0.4555mg/kg(4组)左右心房内心肌小梁丰富，心房切面较小。左右心室壁较厚，腔隙狭窄，心肌小梁排列紧密，心室壁血管丰富。个别个体心室切面较小，染色较深。0.22775mg/kg(5组)左右心房内心肌小梁丰富。左右心室壁较厚，腔隙狭窄，心肌小梁排列紧密，心室壁 血管丰富，部分个体左心室腔隙不可见(n＝5)。大部分切面可见与心室相连的大血管，及管腔内瓣膜，仅个别未见。
0.113875mg/kg(6组)基本已分化形成心室与心房，心包膜完整，仅个别切片未见心包膜，心肌层，心外膜与心内膜完整，未见坏死与炎性细胞浸润。左右心房均较为充盈，心房内心肌小梁丰富。部分个体心房壁有破损(n＝1)，心包腔内有积血，由于没有相应的病理变化，可能与采样有关。个别个体心脏较小。左右心室内心肌小梁丰富，部分个体左心室壁较厚，左心室腔隙狭窄。个别个体可见室间隔缺损(n＝3)，右心室狭窄，或可见右心室壁过薄。
将全部叶酸组进行统计，如下
表3叶酸对大鼠胎鼠心脏发育的影响统计
Figure PCTCN2020113157-appb-000006
由于样本量还是比较少，未做统计学归纳。但从病理结果来看，叶酸容易造成心室缺陷或间隔缺损。
实验例10叶酸与5-甲基四氢叶酸对小鼠胎鼠的发育影响
雌性C57BL/6J小鼠，7周龄，75只，雄性C57BL/6J小鼠，8周龄，25只，由上海灵畅生物科技有限公司提供，合格证号：SCXK(沪)2018-0003。动物购入后适应性饲养10天左右，至雌性小鼠体重达到约20g，雄性小鼠体重达到约25g，体成熟后开始实验。雌性小鼠按体重随机分为5组，分别为：溶媒对照组、叶酸151.66μg/kg、叶酸303.32μg/kg、5-甲基四氢叶酸钙(折合叶酸)151.66μg/kg、5-甲基四氢叶酸钙(折合叶酸)303.32μg/kg。单位质量1.1275的5-甲基四氢叶酸钙等于单位质量1的叶酸。
每组15只，采用剪趾法编号。分组第二天开始每天一次灌胃给药。预给药1周后，雌雄小鼠以3:1比例合笼，并于次日起每日观察阴栓，见阴栓之日起记为怀孕0.5天。合笼14天后，母鼠停止给药，移出雄鼠，怀孕雌鼠单独饲养直至产仔。
取出当日新生鼠，称量体重。每组随机选取部分新生小鼠，出生当日进行心电图检测(NeoNatal Mouse，iWorx,Dover,NH,USA)。
另外每组随机选取4-5只小鼠，待其生长到6日龄进行小动物心脏超声观察(Vevo 2100 Imaging System,VisualSonics,Toronto,ON,Canada)。
检测结束后剖开小鼠胸腔及腹腔，在体视野显微镜(SMZ168,Motic,Xiamen,Fujian,China)下观察其心脏形态及心脏跳动情况，拍照并拍摄视频记录(Motic Image Plus 3.0,Motic,Xiamen,Fujian,China)。随后处死，采集心脏，肝脏，肾脏及肺脏。每组选取新生鼠心脏及肝脏，4％多聚甲醛固定，石蜡包埋切片并进行H&E染色。其它组织一半采取RNA later浸泡固定，另外一半直接液氮浸泡冻存，用于后续实验。
结果如下：
叶酸对新生小鼠心脏超声的影响，对照组(A)，叶酸低剂量组(B)，叶酸高剂量组(C)，甲基四氢叶酸低剂量组(D)及甲基四氢叶酸高剂量组(E),从动力学基本知识来看，B和C动力流相与对照组有明显差别，而D和E组与对照组比较相似。见图23，六日龄新生小鼠超声心动图。
室间隔舒张末厚度及收缩末厚度(毫米)无明显差异(A-B)；叶酸高剂量给药组左室舒张末及收缩末内径(毫米)明显低于其它组，且左室舒张末内径与其它组的差异具有统计学意义(C，D)；各组之间射血分数和短轴缩短分数(％)无明显统计学差异(E，F)。见图24，六日龄新生小鼠超声心动图数据。
1日龄新生小鼠扑杀后取得心脏，4％多聚甲醛固定，石蜡包埋后切片并进行H&E染色，普通显微镜下拍照。对照组，甲基四氢叶酸低剂量给药组，甲基四氢叶酸高剂量给药组新生小鼠心肌完整。叶酸高低剂量给药组新生小鼠左心房及心室之间的部分可见一小块心壁有缺陷(B，C)。见图25。
表4叶酸对大鼠胎鼠心脏发育的影响统计
Figure PCTCN2020113157-appb-000007
Figure PCTCN2020113157-appb-000008
新生小鼠电极不像成年小鼠一样插入皮下，只是和皮肤接触，所以测得的信号不是很稳定，未统计归纳。
上述结果表明，5-甲基四氢叶酸钙没有直接的致心脏发育畸形的能力，而叶酸则可能导致先天性心脏病。
实验例11 5-甲基四氢叶酸钙预防心脏畸形的研究
转基因型斑马鱼(fli-1:EGFP)来自于南京大学模式动物研究所。实验所用鱼是年龄小于1年的成年斑马鱼，饲养水体的pH值为7±0.2，温度在28℃左右，光照和黑暗的时间比是14h:10h，每天喂食两次丰年虫卵。前一天晚上把一雌两雄三条斑马鱼放在产卵盒中，第二天早上收集鱼卵。鱼卵放于胚胎培养液(0.2g/L的海盐配制而成)中，28℃培养。
根据文献调研，选取三种不同类型的，据文献报道有导致先心病的物质，分别为乙醇、硝酸铅、马兜铃酸。见下表。
表4:发育毒性实验中的挽救剂和药物浓度
Figure PCTCN2020113157-appb-000009
斑马鱼胚胎在2hpf开始放于24孔板加药培养，每孔10条，分别加入上表中浓度的药物溶液，一直作用到8hpf终止，清洗胚胎后转入到卵液继续培养至72hpf。发现胚胎死亡及时清理。每组试验重复3次。
5-甲基四氢叶酸预防畸形的实验中选择的浓度记录在表中。观察畸形的表型，记录畸形存在异常的所有斑马鱼数目，计算各组的畸形率和挽救率。形态缺陷、心率、心脏形态等指标通过SMZ745T体视倒置显微镜观察评估。
1、5-甲基四氢叶酸对硝酸铅预防心脏畸形的结果
我们发现2,4mM Pb(NO3)2能降低胚胎的存活率，但是加10mM L-5-MTHF-Ca组并不能挽救2,4mM Pb(NO3)2对胚胎存活率产生影响，与control组对比，评价不同时间点斑马鱼胚胎的表型，8hpf时，2,4mM Pb(NO3)2和2,4mM Pb(NO3)2+10mM L-5-MTHF-Ca均没有出现明显胚胎发育迟缓；24hpf时，2,4mM Pb(NO3)2并未明显影响斑马鱼的发育生长，斑马鱼胚胎已经形成头部和尾部的发育，并且尾部会有摆动现象；48hpf时，2,4mM Pb(NO3)2并未影响斑马鱼体节间血管发育，也未有心包膜水肿的现象；72hpf时，2,4mM Pb(NO3)2致斑马鱼体长变短，大量胚胎出现畸形现象，主要表现为躯干弯曲，尾巴短小，但是肠下网血管发育没有明显差异。而10mM L-5LMTHF-Ca可以拯救2,4mM Pb(NO3)2所致的胚胎发育畸形(见图26)。
进一步量化评估10mM L-5-MTHF-Ca对Pb(NO3)2的挽救效果，由于未出现胚胎心包膜水肿的现象，所以48hpf时只评估了斑马鱼胚胎10s心率和72hpf各组间斑马鱼的体长。图27可以看出，Pb(NO3)2严重导致斑马鱼心率过缓、体长变短，胚胎畸形率增高，并且表现出了显著的浓度依赖性，而加10mM L-5-MTHF-Ca组则可以挽救斑马鱼心率过缓、体长变短及躯干异常弯曲的畸形表型。
综上所述，10mM L-5-MTHF-Ca可以挽救2,4mM Pb(NO3)2导致斑马鱼胚胎发育的所有畸形。
2、5-甲基四氢叶酸对酒精预防心脏畸形的结果
我们发现1.2％EtOH组存活率与对照组间差异明显，但1.2％EtOH+10mM L-5-MTHF-Ca组可以挽救存活率，且其存活率与control组之间无差异(图28，A)。72hpf时，0.6％和0.9％EtOH组斑马鱼形态与对照组相似，1.2％EtOH导致斑马鱼肠下网血管发育障碍，而1.2％EtOH+10mM L-5-MTHF-Ca组无法改善该缺陷(图28，B)。
48hpf时，0.9％EtOH组和1.2％EtOH组导致斑马鱼胚胎心率过缓的畸形表型，对色素生长和体节间血管发育无影响；0.9％和1.2％EtOH加10mM L-5-MTHF-Ca可以缓解心率过缓。48hpf时，各浓度EtOH导致斑马鱼胚胎心包膜水肿畸形表型，在0.6％的浓度下加10mM L-5-MTHF-Ca可以降低心包膜水肿率；1.2％EtOH加10mM L-5-MTHF-Ca组对缓解心包膜水肿没有作用(见图29)。
综上所述，乙醇对斑马鱼胚胎造成早期胚胎发育迟缓、心包膜水肿、心率过缓、体长变短、肠下网血管发育障碍的畸形表型，对色素生长、体节间血管发育无影响。加入10mM L-5-MTHF-Ca可以在低浓度(0.6％)EtOH的条件下缓解心包膜水肿，在高浓度(1.2％)EtOH的 条件下缓解体长变短，提高胚胎的存活率，在中高浓度(0.9％、1.2％)EtOH的条件下，L-5-MTHF-Ca缓解心率过缓较为明显，有一定预防畸形作用。
3、5-甲基四氢叶酸对马兜铃酸A致畸形的预防作用
选取1～5μM马兜铃酸A造模，同时用10mM L-5-MTHF-Ca进行挽救试验，我们发现2～5μM马兜铃酸A会严重导致胚胎存活率降低，加10mM L-5-MTHF-Ca能够挽救马兜铃酸A对胚胎存活率的影响(图30)，但对于高浓度马兜铃酸A组(5μM)，L-5-MTHF-Ca挽救效果不显著。
与control组对比，评价不同时间点斑马鱼胚胎的表型(见图31)，8hpf和24hpf时，1～5μM马兜铃酸A和1～5μM马兜铃酸A+10mM L-5-MTHF-Ca均没有出现明显胚胎发育迟缓、畸形及死亡现象；48hpf时，马兜铃酸A处理组胚胎随着马兜铃酸A浓度升高，逐渐出现心包膜水肿、心率变缓、躯干弯曲、全身血流消失、积血、卵黄囊不透明等现象，并随马兜铃酸A浓度升高而加重；同时，高浓度组出现体节间血管发育不良现象；而10mM L-5-MTHF-Ca可以挽救马兜铃酸A导致的心包膜水肿、心率过缓及体节间血管发育不良等畸形；72hpf时，马兜铃酸A处理组斑马鱼肠下网血管发育出现完全缺失现象，体长也变短，而10mM L-5-MTHF-Ca一定程度上可以挽救马兜铃酸A导致的斑马鱼体长变短、肠下网发育缺失现象。
为进一步量化评估10mM L-5-MTHF-Ca对马兜铃酸A的挽救效果，48hpf时评估了斑马鱼胚胎心包膜水肿率、10s心率和72hpf各组间斑马鱼的体长。由图32可以看出，1～5μM马兜铃酸A严重导致斑马鱼心包膜水肿、心率过缓、体长变短，而加10mM L-5-MTHF-Ca则可以一定程度上挽救斑马鱼心包膜水肿、心率过缓、体长变短的畸形表型。
综上所述，10mM L-5-MTHF-Ca在一定程度上可以挽救1～5μM马兜铃酸A导致的斑马鱼胚胎发育畸形及死亡。

Claims (20)

  1. 5-甲基四氢叶酸用于制备围孕期和/或妊娠期妇女预防新生儿先心病的药物或保健食品的用途。
  2. 根据权利要求1的用途，其特征在于所述先心病选自以下任意一种亚组疾病：一、先天性大动脉畸形，包括动脉导管未闭合，主动脉狭窄，肺动脉狭窄，肺动脉闭锁或其他大动脉先天性畸形中的任意一种；二、先天性心脏隔膜畸形，包括心房-室间隔缺损(AVSDs)，室间隔缺损(VSDs)，房间隔缺损(ASDs)，法洛四联症，主肺动脉间隔缺损或其他先天性隔膜畸形中的任意一种；三、其他先心病，包括心腔的先天性连接畸形，先天性主动脉或二尖瓣畸形中的任意一种。
  3. 根据权利要求1的用途，所述先心病为重金属或酒精或药物导致的先天性心脏病。
  4. 根据权利要求1的用途，所述5-甲基四氢叶酸包括5-甲基四氢叶酸或其药学可接受的盐。
  5. 根据权利要求4的用途，所述5-甲基四氢叶酸选自5-甲基-(6S)四氢叶酸，5-甲基-(6R)四氢叶酸，或5-甲基(6R，S)四氢叶酸，即包括5-甲基四氢叶酸的旋光异构体，旋光异构体的混合物，特别是纯旋光天然异构体。
  6. 根据权利要求4的用途，其特征在于所述药学可接受的盐包括5-甲基四氢叶酸中的碱性基团转化为相应的酸加成盐，5-甲基四氢叶酸中酸性基团转化为相应的碱加成盐；优选盐酸、硫酸、硝酸、磷酸、钠、钾、镁、钙和铵盐，取代的铵盐，或与精氨酸或赖氨酸形成的盐。
  7. 根据权利要求1-6任一项的用途，所述药品或保健食品的剂型包括注射剂、片剂、胶囊剂、丸剂、口服液、颗粒剂或散剂。
  8. 5-甲基四氢叶酸用于制备围孕期和/或妊娠期妇女预防流产或死胎的药品或保健食品的用途。
  9. 根据权利要求8的用途，其特征在于所述流产或死胎是由胎儿心脏发育畸形所导致的。
  10. 根据权利要求8的用途，其特征在于所述流产或死胎是由围孕期和/或妊娠期妇女在含有甲醛环境中所造成的。
  11. 根据权利要求8的用途，所述5-甲基四氢叶酸包括5-甲基四氢叶酸或其药学可接受的盐。
  12. 根据权利要求11所述的用途，所述5-甲基四氢叶酸包括5-甲基所述5-甲基四氢叶酸包括5-甲基-(6S)四氢叶酸，5-甲基-(6R)四氢叶酸，5-甲基(6R，S)四氢叶酸，即包括5-甲基四氢叶酸的旋光异构体，旋光异构体的混合物、特别是纯旋光天然异构体。
  13. 根据权利要求11所述的用途，其特征在于所述药学可接受的盐包括5-甲基四氢叶酸中的碱性基团转化为相应的酸加成盐，5-甲基四氢叶酸中酸性基团转化为相应的碱加成盐；优选盐酸、硫酸、硝酸、磷酸、钠、钾、镁、钙和铵盐，取代的铵盐，或者与精氨酸或赖氨酸形成的盐。
  14. 根据权利要求8-13任一项所述的用途，其特征在于所述药品或保健食品的剂型包括注射剂、片剂、胶囊剂、丸剂、口服液、颗粒剂或散剂。
  15. 5-甲基四氢叶酸在制备预防胎儿心脏畸形的药物或保健食品中的应用。
  16. 根据权利要求15所述的应用，其特征在于所述心脏病为重金属或酒精或药物导致的先天性心脏病。
  17. 根据权利要求16所述的应用，其特征在于所述的心脏畸形为乙醇、硝酸铅、马兜铃酸导致的心脏畸形。
  18. 根据权利要求15的用途，所述5-甲基四氢叶酸包括5-甲基四氢叶酸或其药学可接受的盐。
  19. 根据权利要求18的用途，所述5-甲基四氢叶酸选自5-甲基-(6S)四氢叶酸，5-甲基-(6R)四氢叶酸，或5-甲基(6R，S)四氢叶酸，即包括5-甲基四氢叶酸的旋光异构体，旋光异构体的混合物，特别是纯旋光天然异构体。
  20. 根据权利要求15的用途，其特征在于所述药学可接受的盐包括5-甲基四氢叶酸中的碱性基团转化为相应的酸加成盐，5-甲基四氢叶酸中酸性基团转化为相应的碱加成盐；优选盐酸、硫酸、硝酸、磷酸、钠、钾、镁、钙和铵盐，取代的铵盐，或与精氨酸或赖氨酸形成的盐。
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