Folate and B12 Flashcards
What vitamin is folate?
vitamin B9
How was folate initially investigated?
(vitamin B9) was initially investigated as a dietary factor that prevented anemia of pregenancy and as a growth factor present in green leafy vegetables (Foliage)
* folate = “folium” (leaf)
Metabolic functions of folate
Intracellular folate is important for many cellular process:
* Methylation reactions (Lipids, proteins, DNA, RNA)
* Nucleic acid synthesis (synthesis of DNA precursors)
* Amino acid metabolism (Eg. methionine, cysteine, histidine, serine/glycine)
What are the two common forms of folate?
- folate found naturally in foods
- folic acid which is the form found in supplements and fortifie foods
Structure of folate vs. folic acid
The double bond on N5 and N8 is folic acid but with a single bond it is folate. Can also add on more glutamate which give more negative charges
* folate: single bond at N3, N5, N8, 4 Hs
* folic acid double bonds at N3, N5, N8
Where is the active site of folate?
N-5 and N-10
* where substances are added or donated. So methyl groups can be added or donated from here
What are the folate derivatives?
Essentially acts as a substrate for these reactions; so not bound to enzymes like other B vitamins
* most common is THF - tetrahydrofolate?
What role does folate have with histidine?
histidine catabolism to glutamate which is a gluconeogenic precursor
* there is a transfer of the formimino group from formiminoglutamate to THF whereby formimino-THF is converted to 5,10-methenyl-THF
What happens to histidine catabolism with folate deficiency?
buildup of formiminoglutamate in urine
Role of folate in synthesis of DNA precursors
- Pyrimidine synthesis
- purine biosynthesis
Role of folate in pyrimidine synthesis
Thymidylate
* A methyl group on serine is added to uracil to make thymine via THF.
* Converts THF to DHF so need to covert back to THF before it can be used again. This uses NADPH (niacin) to bring back folate. Maintaining active form of THF is important.
What other vitamin is important in the pyrimidine synthesis?
Conversion of DHF back to THF uses niacin (NADPH).
What is Methotrexate?
anti-cancer drug & folate antagonist
* traps DHF in cell, which impairs folate metabolism and reduces cell division when this recycling pattern gets out of hand with cancer
Folate role in purine biosynthesis
Adenine & Guanine
* both require folate
What base pairs require folate?
- thymine
- adenine
- guanine
What is the idea behind folates role in ‘one-carbon metabolism’?
Should be able to draw diagram
Methionine synthase: Folate plays a role in maintaining the essential AA methionine by adding a methyl group to homocysteine via methionine synthase, this cycle is important for many processes.
* methionine is required for cell proliferation, protein synthesis, RNA synthesis and transamination
* methionine is converted to S-adenosyl-Met (AdoMet or SAM) which is important for transmethylation reactions
* AdoMet → AdoHcy → homocysteine
* homocystein can be converted to cystein but ultimately the body wants to maintain as much methionine as possible so homocysteine can be converted back to methionine and the methyl group comes from folate
What are other important vitamins that a play a role in the folate dependant methionine resynthesis cycle?
- Niacin & Riboflavin play a role in making methyl-THF via MTHFR which then donates the methyl group to homocysteine to form methionine
- B12 play a role in methionine synthase
- Pyrodixine plays a role in proteins synthesis (glycine, serine, cystein) within the folate cycle
How might B12 deficiency effect folate metabolism?
B12 is neccessary for methionine synthase in assisting the methyl THF to donate its methyl group to homocysteine. This is the only thing that methyl-THF is used for so there can be a build up of methyl-THF and of homocysteine without B12
* MS (methionine synthase) only way for methyl-THF to be demethylated in tissues to yield free THF
What are some transmethylation reactions which folate is important for?
folate plays a role by ensuring methionine production in order to make SAM which is used in the transmethylation reactions
* epigenetic methylations (DNA/ RNA)
* sterol methylation (catecholamines)
* phospholipid methylation
* creatine synthesis
* protein synthesis
Folate role in epigenetics
- DNA methylation
- histone methylation
Folate in DNA methylation?
Epigenetic pattern during developement whereby some genes are turned off when methylated or activated without the methylation. So without folate can have disproportionate genes turned on or off.
Folate in histone methylation?
Genes wrapped around histones and in order to turn on they have to be unwrapped. Methylating turns off the genes associated so that genes wrapped around cannot be activated but with no methylation they can then unwrap.
folate role in sterol methylation
synthesis of catacholamines include dopamine, epinephrine (adrenaline), and norepinephrine (noradrenaline)
* important for stress responses
* Difference is a methyl group so methylate nor to epi
folate role in PL methylation
In PL metabolism, phosphocholine is the main head group which has 3 methyl groups and all of these use SAM for synthesis
* PC is important for membranes and transport of the lipids;
What would happen in Phospholipid Methylation with folate deficiency?
folate deficiency would inhibit the reaction for synthesis of PC via SAM so would get huge fatty livers because would not be able to move VLDL out of the liver
What would happen with sterol methylation if folate deficient?
Would not be able to form epinephrin
Folate role in creatine synthesis
transmethylation reaction whereby SAM donate methyl group to Guanidinoacetate (starts from arginine) to form creatine
How much of SAM (AdoMet) goes towards creatine synthesis?
This reaction actually consumes most of AdoMet ~30-50%
Folate metabolism overview; draw diagram
How is the RDI of folate determined?
The RDI, is the amount of dietary folate sufficient to maintain red cell folate levels.
How is folate requirements expressed?
We dont use ug, because bioavailability of supplement is different from naturaI sources so it is expressed in Dietary Folate Equivalents (DFE) to take into account the different availabilities of food, supplements, and fortification.
* Synthetic folic acid taken in as supplements and fortified cereal gain foods are 1.7 times more bioavailable as most food folate.
* μg DFE = μg food folate + (1.7 x μg folic acid)
What are the folate requirements?
Folate requirements rise with age.
* 400µg M/F
* greater in pregnancy and lactation
folate upper limit
UL = 1 mg/day folic acid
* high doses folate can mask development of B12 deficiency - Anemia is first sign of deficiency of both folate and B12. But if folic acid is is really high and B12 deficient it does not cause anemia and never know and are then at risk for neuropathy
Dietary sources of folate
found in plant & animal tissues
* Dietary sources include beans, nuts, meat (especially organ meat), dairy products, fruits, grains, and cereals.
principle sources of folate
liver, dark green leafy veg, legumes
How is folate typically present in food naturally?
mostly found in polyglutamate form which is how it is metabolically trapped
* mostly bound to proteins & storage polysaccharides
* folic acid only found when foods are fortified & when dietary folates are oxidized
Bioavailability of food folate
<50% for folate but folic acid is high
* depends on form (poly- or mono-Glu)
* organic acids inhibit conjugase activity
* trapping of folate in plant material
* losses during preparation
How is folate status determined?
- serum folate = short-term balance & recent intake (What we have recently eaten)
- RBC folate reflects tissue depletion = longer-term balance; more reliable
How is folate absorbed?
dietary folate as poly- and mono- Glu folates must first be converted to THF which can then be absorbed to enterocyte. folic acid can be transported in enterocyte as is, where it is converted to THF before being absorbed for further circulation. In circulation goes to liver first where THF may become bile (goes to gut), or becomes poly-Glu (metabolically trapped) and used for function or sent to other tissues for use
How do monoglutamate and
polyglutamate forms of THF differ?
- monoglutamates = circulatory; membrane- transportable
- polyglutamates = intracellular; activity & storage
Folate deficiency and related disease/conditions
- Main is megaloblastic anemia
- Cancer (Dont have enough folate to make DNA more likely to get mutations ans may be cause of cancer)
- Vascular Disease
- Birth Defects (most common defects)
- Folate deficiency may impair memory/learning, and lipoprotein metabolism (If give elderly people supplement s may reduce conversion to dementia)
Causes of Folate Deficiency
- Main cause is Dietary Inadequacy
- Malabsorption
- Metabolic Disorders, Alcohol and Drugs
- Demographic factors: Age, sex, poor
Important of folate in the folate dependant methionine cycle?
to maintain methylation cycle
How do genetics effect folate requirements?
polymorphisms in methylene tetrahydrofolate reductase (MTHFR) which converts 5,10-methylene-THF to 5-methyl THF (which is what is used on MS)
What other vitamins are required for MTHFR?
Reaction requires Riboflavin (B2) and Niacin (B3)
What are the different MTHFR polymorphisms and prevalence?
- 9% have TT - homozygous affect polymorphism (mutation in both)
- 44% have CT - heterozygous (one mutation)
- 47% have CC - normal
Importance of neural tube during development
The development and closure of the neural tube, which forms into the brain and spinal cord, is normally completed within 28 days after conception.
What are NTDs?
Neural Tube Defects
* thought to be caused by failure of the neural tube to close
Folate Deficiency and NTDs
Folate status is associated with the risk of NTDs in an inverse dose-response relationship
* Polymorphism in MTHFR increases the risk of NTDs. The homozygous (TT) mutation doubles the risk of NTDs
* Mutations in other genes, i.e. human folate carrier and methionine synthase increases susceptibility to NTD
What food requires mandatory fortification of folate and when did this happen?
In 1997, US and Canada instituted mandatory folate fortification of flour and grains.
How did mandatory folate fortification change the rate of NTDs?
Rate of NTDs was reduced but not as good as was projected
What was the set amount of folic acid fortification?
final FDA decision, in March 1996,
to mandate the fortification of all enriched cereal grain products at the level of 140 ug folic acid per 100 g of grain by January, 1998.
* In the early 1990s, despite the mounting evidence indicating that folic acid reduced the incidence of NTDs, two U.S. Health agencies initially came to opposite conclusions.
* In 1993, the FDA approved, the health claim that folic acid reduces the risk of NTDs.
How did folic acid intake change after fortification?
Average intake of folic acid from fortified foods post-fortification: ~ 200 ug/d 1 (manufacturers put in way more)
* Expected intake of folic acid following mandatory fortification: 100 ug/d
* <3% of adults have folate intake <RDA 2
What are the cut offs for folate status and what is the prevalence of this in Canada?
- 305 mol/L Deficiency cut-off; so very few in Canada are deficient
- 1360 mol/L High concentration cut-off; so a lot of people have excess
What are potential concerns of excess folate?
No Toxicity of high doses of folate has been reported (not widely studied) but…
* Masking of Vitamin B12 deficiency.
* [Very Recent] A potential increase in the risk of colorectal (and other) cancer.
How might folate mask B12 deficiency
pre- vs. post- folic acid fortification has seen an increase in women who show B12 defiency but excess folate
* fortification essentially created this group
How might folate deficiency and excess contribute to cancer?
essential if folate is taken earlier on when condition are still normal it will likely decrease risk but if taken in adenoma (benign tumour) stage it may increase risk
* Folate deficiency limits the synthesis of thymidine and leads to misincorporation of uracil or thymide into DNA leaving breaks the predispose the DNA to mutation and initiation of cancer
* However…Folate plays a central role in DNA methylation since it provides methyl groups for the synthesis of AdoMe so excess may cause hypermethylation of promotor regions with global DNA hypomethylation which is a common observation in tumors.
Other research aroud folate excess
- High folate in the presence of B2 deficiency in pregnant women may increase the risk of developing T2DM in offspring
- Maternal folate excess during pregnancy increased serum glucose and insulin in offspring of rats
- other effects of chronic exposure to unmetabolized folic acid?
- Reduced natural killer cell cytotoxicity
- increased lipid storage
What is the name for B12
Cobalamin
Structure of Cobalamin
Large molecule with a central cobalt
What is the active site of B12?
The cobalt
What is the metabolic role of B12
Involved in only two enzymes
* methionine synthesis
* methylmalonyl CoA Mutase
B12 role in MS
accepts the methyl from methyl-THF and donates it to homocysteine to form methionine
Methyl Trap Hypothesis
- lack of B12
- lack of MS activity
- ↓ synthesis of methionine & THF
- ↓ AdoMet
- ↑ conversion of methylene-THF to CH3-THF
CH3-THF may be trapped because:
- reductase rxn irreversible
- MS rendered inactive by lack of coenzyme (methylcobalamin)
Role of B12 in Methylmalonyl CoA Mutase
Methylmalonyl CoA Mutase enzyme is dependant on B12 for isomerization of methylmalonyl CoA to succinyl CoA in order for assocaited proteins to enter the CAC
What happens with Methylmalonyl CoA Mutase in B12 deficiency?
methylmalonyl CoA converted to
methylmalonic acid
* Most sensitive marker of B12 deficiency
Historical view in B12
In the19th century, an anemia was described in association with disease of the stomach and was called pernicious anemia (Fatal)
* In the 1920s Minot and Murphy described the first treatment (1 pound of raw liver a day). They received the Nobel Prize for this discovery
Absorption of B12
Absorption of vitamin B12 requires the action of an Intrinsic Factor (IF) and transcobalamin–II (TC-II)
* B12 in ingested and IF is released from stomach parietal cells. IN the distal iluem IF binds B12 and IF receptor then recognized it and carries it into enterocyte. A lysosome in the enterocyte releases B12 where it then binds with TC-II and leaves the enterocyte TC-II binds to the TC-II receptor of peripheral tissue and is transported into cells
Best dietary sources of B12
- meats (beef liver, beef products, chicken products)
- dairy products
- fish and seafood
- eggs
Sources of fruits and vegetables with B12
none contain B12
non-meat sources of B12
- mushrooms
- tempeh
- saurkraut
B12 DRIs
- 2.4 ug/d M/F
- increased need in pregnancy and lactation
- ↑ in elderly due to malabsorption
- no UL set
What are tests to determine B12 status?
- schilling test
- Microbiological assay for total plasma B12
- Competitive radioassay for plasma B12 using IF
- elevated levels of methylmalonic acid and homocysteine
What is the schilling test for determining vitamin B12 status?
Schilling test = tests for B12 malabsorption
* stage I - 57Co+B12 load: if 57Co excretion is low the problem is absorption
* stage II - 57Co+IF: if 57Co excretion normalized the problem is IF; if not – may be bacterial overgrowth
* stage III - Itreatment with antibiotics & retest
What else is important to test with B12?
important that both B12 and folate test
* Often comes with folate deficiency with anemia
What are signs and symptoms of B12 defiency?
- tissues with high (than normal?) turnover rates (e.g. RBC, gut) - due to ↓ availability of THF involved in DNA synthesis
- megaloblastic anemia
- neuropathy - due to impaired myelin formation related to depletion of AdoMet
factors relating to B12 deficiency
dietary deficiency is uncommon and we have large B12 stores which are depleted slowly (turnover = months)
* malabsorption more likely
Populations at risk
- Elderly = “tea & toast” diets (not as much meat)
- Chronic alcoholics
- Strict vegans
Vitamin B12 deficiency in vegans/ vegetarians
Risk of developing deficiency over many year and most don’t show deficiency because
* small amounts are available through contamination of plant food
* high reabsorption efficiency
* slow turnover
pernicious anemia
same as megaloblastic anemia
* identical to anemia in folate deficiency
* inability to absorb B12 due to impaired IF
What are the 2 types of pernicious anemia?
- Addisonian = genetic; can be due to IF antibodies – IF is ABSENT
- Non-Addisonian = gastrectomy & age = ↓ IF synthesis – IF is INADEQUATE
Treatment for B12 deficiency
oral dose or intramuscular injections (suggests alternate transport system in SI)
Best indicator for B12 deficiency due to dietary intake
methylmalonic acid is most sensitive
* comparing status of methylmalonic acid od control vs. vegans
Should we have mandatory vitamin B12 fortification?
- It might avoid the potential problem of masking B12 deficiency in those with high folate levels.
- Irrespective of folate status, it may be good for those populations that are know to have low B12 status (i.e. Elderly, vegetarians, and breastfed infants).
- It may prevent NTDs that are suspected to be due to poor cobalamin status.
What are the concerns of B12 fortification?
No adverse effects have been associated with excess B12 intake from food or supplements in healthy individuals but governments need to be assured that it will be safe as well as effective.
* biggest concern is increase cost to companies and consumers (cost vs. benefits)
What is a possible reason for the low toxicity of B12?
One possible reason for the low toxicity- small % of B12 is absorbed in intestine.
What we need to know before fortifying with B12
- Dose-response studies
- Evidence of benefit
- Cobalamin is pink - pink flour? Is that okay
- Evidence of a lack of adverse effects
- Stability studies (unlike folic acid, cyanocobalamin is unstable)
- Need to plan follow-up studies to document effects on NTDs, cognition and anemia
