Background: Folate is important for DNA biosynthesis and many metabolic processes. Folate deficiency can cause macrocytic anemia, cardiovascular disease (CVD) and breast cancer. Several folate are commercially available, including synthetic versions such as folic acid and natural forms, such as that found in the alga chlorella. The enzyme 5, 10- methylenetetrahydrofolate reductase (MTHFR) polymorphisms C677T are associated with Alzheimer’s disease, neural tube defects and CVD.
Objective: To study the influence of Chlorella pyrenoidosa on individuals carrying the MTHFR C677T polymorphism.
Methods: Seven outpatients from Yoko Clinic (3 males, 4 females; 12–52 years) were enrolled. Three individuals carried the mutant homozygous genotype TT (TT), three were heterozygous genotype CT (CT). We conducted biochemical tests on peripheral blood taken before and during the 3 months when study subjects took 3 g of Chlorella pyrenoidosa daily.
Results: Overall, blood samples showed a tendency of negative correlation between folate and homocysteine levels before the study. The TT had a tendency toward low folate before taking chlorella (TT 5.53 ng/ml, CT 14.13 ng/ml) and the levels for the TT increased after taking chlorella (6.73 ng/ml). Two young female subjects who had started menstruating showed increased iron levels (before 58.5 mmg/dl, after 119 mmg/dl). One hyperlipidemic individual had a decrease in total cholesterol (before 305 mg/dl, after 256 mg/dl).
Conclusion: Cases in the TT group tended to have low serum concentrations of folate. Overall, the natural product chlorella might have beneficial influences for enhancing metabolism of folate and other nutrients by individuals with MTHFR polymorphisms.
Folate, Folic acid, MTHFR, Homocysteine
Folate is necessary for normal metabolic and developmental processes, as well as for DNA biosynthesis and methylation. During methionine synthesis, folate serves as a onecarbon donor. Several enzymes are involved in folate metabolism, including dihydrofolate reductase (DHFR), methylenetetrahydrofolate dehydrogenase (MTHFD1), 5, 10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and methionine synthase reductase (MSR).[1-3] MTHFR is a critical enzyme involved in homocysteine metabolism. Cases having a C to T mutation at the codon for amino acid 677 of MTHFR, and those who are homozygous TT at these residue, have MTHFR activity that is up to 70% lower than that of wild type.[4,5]
Folate deficiency is associated with infertility, macrocytic anemia, excess homocysteine, cardiovascular disease (CVD), developmental disorders, and breast cancer.[1,2,6,7] Meanwhile, the MTHFR C677T polymorphism is related to autism, Alzheimer’s disease, mental disorders (e.g., depression and schizophrenia), neural tube defects and CVD.[3-5,8,9,10]
A poor diet and/or metabolic dysfunction can create an environment that favors disease and metabolic disorders.[11,12] Folate fortification was initiated in the late 1990s and since then the impacts of folate on health and disease have been extensively studied.[1,2,13] There are many different folate supplements that are available, such as folic acid (FA) and L-5-MTHF and 5-formyltetrahydrofolate (5 formyl THF), as well as natural products that include multiple forms of folate, such as chlorella. Folate supplementation efforts appear to be successful as evidenced by a reduction in the rate of neural tube defects. However, some evidence suggests that intake of high amounts of synthetic FA may rsesult in excess FA in the blood, that could produce unexpected adverse effects.[1,6,7,13,14] High synthetic FA could also be a risk factor for autism and neural developmental disorders, increased body mass and insulin resistance, and colorectal cancer.
Chlorella pyrenoidosa is a unicellular green alga that grows in fresh water. Relative to other plants, chlorella is higher in protein and chlorophyll, and also has a high content of several vitamins including multiple forms of folate, vitamin B12 and vitamin D, minerals such as iron and magnesium, and dietary fiber. The proteins contained in chlorella include all the essential amino acids needed to maintain human growth and health.[15-18] Chlorella pyrenoidosa and chlorella extracts have been reported to exert a variety of beneficial effects, including reducing cholesterol, preventing stress-induced ulcers, enhancing resistance to infection and antineoplastic activity. In pregnant and lactating women, Chlorella pyrenoidosa was reported to decrease dioxin levels in breast milk and reduce the risk of anemia, proteinuria and edema. Chlorella is currently widely available as a nutritional supplement and health food.[15-18,19,20]
We previously confirmed that Chlorella pyrenoidosa includes many forms of folate such as 5-10-MTHF and 5-MTHF and 5 formyl MHF.
In this study, we investigated the influence of Chlorella pyrenoidosa on subjects carrying the MTHFR C677T polymorphism.
Population: Procedures in the present study were conducted between March and July 2017 and were in accordance with the guidelines of the Declaration of Helsinki (2000) for human experimentation. All study subjects provided informed consent. Seven outpatients from Yoko Clinic (Kitakyushu Japan) (3 males, 4 females; age range, 12–52 years) were enrolled in the current study. All study subjects reported their alcohol intake and kept daily record of their diet, which included two servings of uncooked leafy green vegetables.
Materials and study design: All study subjects were analyzed for MTHFR polymorphism C677T using commercial kits (GPL-SNP1000, The Great Plains Laboratory, Inc.; Lenexa, KY, USA and smartDNA Genomic Wellness, smart DNA, Pty Ltd.; Clayton, VIC, Australia). Three in seven subjects were mutant genotype TT (TT), three had a heterozygous genotype CT (CT) and one was normal CC type (CC). Table 1 shows the genotyping results and clinical characteristics for study subjects. We conducted blood biochemical tests (folate, homocysteine, vitamin B12, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH), hemoglobin, albumin, serum ferritin, serum iron level, total cholesterol) on peripheral blood samples taken before and each month of the 3 months of this study. The subjects took 3 g of Chlorella pyrenoidosa (Sun Chlorella A Tablets®, Sun Chlorella Corp. Kyoto Japan) twice daily for 3 months. Blood samples were taken from 4 of the 7 subjects for 14 days to examine changes that occur across the short term. One professional nutritionist provided direction of food intake such as plenty of green leaves and optimal protein and less processed food, sugar and oxidized oil. All subjects had already taken proper food intake for over six months.
|Case No.||Genotype||Age(years)||Sex||Past history||Disease|
|Case 2||TT||52||Male||Atrial Fibrillation (transient)||Hypothyroidism|
|Case 3||CC||46||Female||None||Reactive hypoglycemia|
|Case 4||CT||47||Male||Transient ischemic attack||Chronic fatigue syndrome|
|Case 5||TT||37||Male||none||High homocysteinemia|
|Case 6||CT||12||Female||Chronic sinusitis||Allergy for moth|
Table 1: Clinical characteristics and MTHFR genotype of study subjects.
Overall, the study subjects showed a tendency of negative correlation between folate and homocysteine blood levels before taking chlorella supplements (r=-0.43, p=0.33) in all subjects. Table 2 shows the data for the clinical parameters of the study subjects. All groups except for TT had normal concentrations of folate in the blood.
|Date||Case 1||Case 2||Case 3||Case 4|
Table 2(a): Clinical parameters of Folate level for short duration (ng /mL)
|Date||Case 1||Case 2||Case 3||Case 4||Case 5||Case 6||Case 7|
Table 2(b): Clinical parameters of Folate level for long duration (ng /mL)
|Date||Case 1||Case 2||Case 3||Case 4||Case 5||Case 6||Case 7|
Table 2(c): Clinical parameters of Homocystein level (nmol / mL)
|Date||Case 1||Case 2||Case 3||Case 4|
Table 2(d): Clinical parameters of TCHO (mg / dL)
Before Chlorella pyrenoidosa began, the TT group had a tendency of low folate concentration before this study (TT group 5.53 ng/ml, CT group 14.13 ng/ml). During chlorella intake, folate levels for the TT group increased to 6.73 ng/ml. One subject with high initial homocysteine levels showed a decrease in homocysteine after taking chlorella supplements (Figure 1). The high homocysteinemia case (TT) had taken Chlorella pyrenoidosa for 6 months before entry to this study. This case stopped taking 2 months before this study (Figure 2, 0 M to 2 M) and started taking again at 2 M. While not taking, there was no influence of tendency of a decrease in homocysteine (Figure 2). Two young females (aged 12 and 14) in the study were in an active growth period and had already started menstruating. The 12 year-old carried the CT genotype and the 14 year-old was homozygous TT. Both showed an albumin increase (average value before 4.05 g/dl, after 4.5 g/dl) (Figure 3a) as well as increased serum iron (before 58.5 microgram/dl, after 119 microgram/dl; Figure 3b) and ferritin levels (Figure 3c). One hyperlipidemia case (CT) had a decrease in total cholesterol with chlorella supplementation (before 305 mg/dl, after 256 mg/dl) (Figure 4). The cases of macrocytic anemia (MCV above normal range) showed improvement (data is not shown). There was no tendency about vitamin B12, hematocrit and hemoglobin.
Our results suggest that might have beneficial influence for folate metabolism in individuals with MTHFR C677T polymorphisms. The importance of folate and its metabolites has been extensively documented, and in the late 1990s fortification of foods with folate began, and the use of folate supplements, particularly for pregnant women, was encouraged. In contrast to folate present in foods, in supplements, synthetic FA, the monoglutamate form of folate, is typically present as pteroylmonoglutamic acid[1,21], which has a similar, but not identical structure as natural folate. The chemical structure of synthetic FA includes glutamate, an abundant neurotransmitter that is involved in activating ion flux in N-methyl-D-aspartate (NMDA) receptor channels. Thus, synthetic FA may directly compete for binding to NMDA receptor in neuronal synapses. Whether unmetabolized FA could affect the behavior of neurons in some neural circuits during development is currently being investigated. In humans, the activity of human DHFR, which converts folate to dihydrofolate and tetrahydrofolate is low compared to other animals.
Moreover, in humans synthetic FA is initially metabolized in the liver rather than in the intestine, which could result in high levels of unmetabolized FA in the blood.1 An increase in unmetabolized FA in the blood could in turn elicit autoantibodies against the folate receptor (FR) alpha and regulate DHFR activity to influence folate uptake in renal and intestinal epithelia.[1,25] Furthermore, excess synthetic FA intake is associated with a reduced cytotoxic activity of natural killer T cells in postmenopausal woman  and dysregulation of gene expression in lymphoblastoid cells. Other reports indicated that excess synthetic FA can have negative effects on mental health and increase the risk of colorectal cancer.[1,6] In pregnant women, excess eynthetic FA is associated with increased body fat mass and insulin resistance of offspring.
Chlorella is a natural substance that includes dihydrofolates and tetrahydrofolates such as 5-10-MTHF and 5-MTHF. Chlorella also contains higher levels of essential amino acids and chlorophyll compared to other plants, as well as large quantities of vitamins and minerals (e.g., iron and magnesium, vitamin B6, vitamin B12). Chlorella pyrenoidosa was previously reported to reduce the oxidation of erythrocyte membrane lipids18, and in pregnant and lactating women, improve anemia and reduce breast milk dioxin levels, respectively. Consistent with our results, an earlier study indicated that Chlorella pyenoidosa has cholesterol-lowering effects. The mechanisms associated with these effects are unclear, but the compounds present in chlorella could help chelate toxic substances such as dioxin, oxidative LDL, or heavy metals.[14-16] The beneficial nutrients contained in chlorella (e.g., essential amino acids, multiple forms of folate and vitamins B6, B12 and D, minerals) could enhance metabolism and tissue regeneration as well as promote DNA biosynthesis. The safety of chlorella has been well documented. [15-18]
In this study, the two girls (CT and TT genotype) showed improved iron metabolism and nutrition status after taking chlorella. Chlorella pyrenoidosa also reduced high levels of homocysteine but did not influence normal homocysteine levels. In terms of lipid metabolism, Chlorella pyrenoidosa could reduce total cholesterol, but did not have a negative effect on individuals with normal cholesterol levels.
Upon initiation of Chlorella pyrenoidosa, the concentration of folate in the blood gradually increased through day 4, and thereafter the levels of folate varied (6 day to 14 days) (Figure 4).
Individuals who carry the MTHFR polymorphism TT at residue 677 exhibit a tendency of low folate baseline levels, even if folate-rich diets containing foods such as uncooked leafy green vegetables are consumed daily. Appropriate food intake is necessary to keep in a healthy condition. A recent study suggested that the consumption of a mixed diet containing natural folate-rich foods can be almost as effective as synthetic FA supplements at improving folate status. Low folate levels are associated with several diseases, so individuals who carry MTHFR and other relevant polymorphisms are encouraged to take daily folate supplements. However, excess intake of folate or folate derivatives such as synthetic FA could lead to excess unmetabolized FA in the blood, which in turn may produce adverse health effects. Chlorella pyrenoidosa is a natural food that is a good source of many kinds of folate. This pilot case study with a limited number of subjects indicates that Chlorella pyrenoidosa might enhance folate metabolism in individuals who carry polymorphisms of MTHFR, particularly the TT genotype. Chlorella pyrenoidosa might also have beneficial influences for hyperlipidemic patients and reduce toxin accumulation in actively growing individuals. Chlorella pyrenoidosa also had influences for individuals with the CT genotype in terms of improved albumin and cholesterol levels. There was no side effect while study subjects were taking chlorella. Taken together, this initial study indicates that Chlorella pyrenoidosa can be a good source of natural folate that helps individuals who have MTHFR polymorphisms maintain healthy blood folate levels.
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