BIOCHEMISTRY OF FOLATEAND VITAMIN B12:

October 15, 2014

Nutritional roles, and diagnosisof deficiencies

METHYLENE-THF: DONATES [CH2] FOR DNA SYNTHESIS

DEOXYTHYMIDINE (dTMP): REQUIRED FOR DNA SYNTHESIS, AND FOR CELL DIVISON

THIS PATHWAY IS VERY ACTIVE IN RAPIDLY-DIVIDING CELLS,

SUCH AS DEVELOPING RBC

WITHOUT NEW DNASYNTHESIS, CELLSWILL NOT DIVIDE, BUTCAN GET LARGER.

THIS IS CALLED:

MEGALOBLASTICANEMIA

FOLATE AND B12DEFICIENCY PRODUCEA SIMILAR ANEMIA.

THIS IS EXPLAINED BYTHE METHY-FOLATE TRAP.

LARGE RBC:Megaloblastic anemia

dUMP

DIETARYFOLATE

REDUCTION BYNAPDH

DIHYDROFOLATE

TETRAHYDROFOLATE

REDUCTION BYNAPDH

METHYLENETETRAHYDROFOLATE

SERINE

GLYCINE

METHYL GROUPADDITION

METHYLTETRAHYDROFOLATE

REDUCTIONBY NADH

HCys Met

SUBSTRATE

S-adenosyl-Met

S-adenosyl-HCys

METHYLATEDSUBSTRATE

dTMP(methylated)

DNAsynthesis

REQUIRESVit B12

CYCLE#1

CYCLE#2

FOLATE PROVIDES METHYL GROUPSBY TWO DIFFERENT CYCLES

This step is not reversible.

If B12 is deficient, folateaccumulates in this form,

(called the folate trap).

Result: folate is not availablefor cycle #1.

WITHOUT VITAMIN B12, METHYL-THF CANNOTBE RECYCLED BACK TO TETRAHYDROFOLATE.

C CH3

NADH+H+ NAD+

METHYLENETETRAHYDROFOLATE

METHYLTETRAHYDROFOLATE

THE REDUCTION OF METHYLENE-THF TOMETHYL-THF IS NOT REVERSIBLE.

IF METHYL-THF IS FORMED, IT CANNOT BECONVERTED BACK TO METHYLENE-THF.

C CH3

NADH+H+ NAD+

METHYLENETETRAHYDROFOLATE

METHYTETRAHYDROFOLATE

The enzyme methylene-tetrahydrofolate-reductase (MHTFR)catalyzes this reaction. That enzyme has common variants, which is the topic of your reading assignment. Different formsof MFTHR also influence Hcy levels.

VARIANTS OF MTHFR

During lecture, I will put specific details aboutvariants of MTHFR on the board.

We will discuss:

-variations in gene sequences

-how that affects the enzyme protein

-the interaction with plasma Hcy levels

dUMP

DIETARYFOLATE

REDUCTION BYNAPDH

DIHYDROFOLATE

TETRAHYDROFOLATE

REDUCTION BYNAPDH

METHYLENETETRAHYDROFOLATE

SERINE

GLYCINE

METHYL GROUPADDITION

METHYLTETRAHYDROFOLATE

REDUCTIONBY NADH

HCys Met

SUBSTRATE

S-adenosyl-Met

S-adenosyl-HCys

METHYLATEDSUBSTRATE

dTMP(methylated)

DNAsynthesis

BLOCKED, WITHNO B12

CYCLE#1

CYCLE#2

WHY WOULD ABUNDANT DIETARY FOLATEMAINTAIN CYCLE #1?

VITAMIN B12 DEFICIENCY

-Vitamin B12 deficiency develops veryslowly. It’s common to store 2 mg,and the body only uses about 1 microgram/day, so deficiency takes several years.

-HOWEVER: long-term deficiency causesirreversible damage to the CNS.

-Conclusion: it’s IMPORTANT that deficiencynot be masked, and be readily diagnosed.

BOTH: B12 deficiency and folate deficiency cause megaloblastic anemia.

BUT: high-dose folate MASKS the anemia,and delays diagnosis of B12 deficiency.

MEANWHILE: the CNS damage continuesfrom the B12 deficiency.

PUBLIC HEALTH CONCLUSION: Don’t consume so much folate, that the earlyanemia of B12 deficiency is prevented.

About years ago, the decision was made to add folateto flour, breakfast cereal, bakery products, andmany other consumer goods.

As a result, folate deficiency is now very rarein many parts of the world.

This decision was made to decrease theincidence of birth defects, especially spina bifida.

Summary of a study done between 1985-1990

The dose was 4 mg/day, given to women who hadpreviously had a child with an NTD.

ASSIGNMENT: Review the study (posted)that describes prevention of NTD with supplements of dietary folate.

BUT IT WAS IMPORTANT TO DEFINE THESMALLEST EFFECTIVE DOSE.

Us of 4 mg/day would certainlymask presence of B12 deficiency.

Assignment: the Lancet paper (1997), that seeks todefine the minimal dose needed.

FOLATE INTAKE FROM FORTIFICATION HAS EXCEEDED INITIAL PROJECTIONS

When the U.S. Food and Drug Administration set the folic acid fortification regulation in 1996, the projected increase in folic acid intake was 100 µg/day. Data from a study with 1480 subjects showed that folic acid intake increased by 190 µg/day.

Folic acid intake above the upper tolerable intake level (1000 µg folic acid/day) increased only among those individuals consuming folic acid supplements as well as folic acid found in fortified grain products.

Taken together, folic acid fortification has led to a bigger increase in folic acid intake than first projected.

VITAMIN B12 DEFICIENCY

CAUSES AND DIAGNOSIS

B12 requires INTRINSIC FACTOR (IF) to be absorbed.

In some autoimmune diseases, Intrinsic Factor is not made, and B12 deficiency can slowly develop.

This disorder is calledPERNICIOUS ANEMIA.

WHERE IS B12 DEFICIENCY A PROBLEM?

-Deficiency of intrinsic factor ( a protein in the stomach) results of lack of proper complex formation, and Vit B12 absorption is very poor. This occurs in some autoimmune diseases (linkage to arthritis,etc).

-The whole process is favored by gastric acidity, so lack of HCl production (called ACHLORHYDRIA) aggravates this problem

-STRICT vegetarians do not get enough B12

(HOWEVER, WE STORE AT LEAST 2 mg of B12,ENOUGH FOR SEVERAL YEARS. There is a long lag time, after conversion to strict vegan diet, beforedeficiency is a problem.

Diagnosis of B12 deficiency: There are several strategies

If EITHER folate or B12 are lacking, Hcy accumulates. This is useful, but elevated Hcy is NOT SPECIFIC.

Elevated Hcy has become a standard procotocol.

If B12 is adequate, propionyl-CoA is metabolizedto succinate, for the TCA cycle.

If B12 is deficient, methylmalonate appearsin blood and urine.

Without B12, this accumulates and enters the bloodstream, and is measured. This does NOT accumulate with folate deficiency.

Vit B12-dependent step:the methyl group isremoved to make succinate

From some amino acids, andfrom odd-chain fatty acids

EXAMPLES OF BIOCHEMICAL PATHWAYS WHERES-ADENOSYLMETHIONINE (SAM) TRANSFERS

A METHYL GROUP TO A SUBSTRATE

All these functions require B12.

Of course, they also require folate.

There may be 100 reactions in human biochemistrythat require SAM for methylation.

EPINEPHRINE BIOSYNTHESIS: What vitaminwas needed to make the norepinephrine?

Addedmethylgroup

CREATINE SYNTHESIS

Addedmethylgroup

METHYLATION OF THE CYTOSINE BASEIN THE DNA STRAND: THIS IS VERYIMPORTANT IN GENE REGULATION!

Specific diagnosis of folate deficiency:

-measurement of RBC folate is common.Folate <140 microgram/L indicates deficiency.

-elevated Hcy can indicate either folateor B12 deficiency, and more specificdiagnosis is needed. Variants of MTHFRare also a factor.

-the catabolism of the amino acid HISTIDINErequires folate. In folate deficiencyN-formino-glutamate will appear in the urine,and that’s a reasonable test.

With lack of folate,this compound willappear in the urine.

Folate is the ACCEPTORof the nitrogen group inred, and histidine isconverted to glutamate.

If EITHER folate or B12 are lacking, Hcy accumulates. This is useful, but elevated Hcy is NOT SPECIFIC.

Elevated Hcy has become a standard procotocol.

Homocysteine is condensed with Serine to make Cystathionine, which is non-toxic. THIS REQUIRES VITAMIN B (as PLP). Many studies use a supplement that provides folate/B12/B6.

DETAILSOF THIS

REACTION

ROLE OF B6 INREMOVING

HOMOCYSTEINE

Measurement of homocysteine (Hcy) may become a standard procedure in clinical diagnostics. As a screening procedure,it identifies:-folate deficiency-B12 deficency

In addition, high Hcy indicates:-declining renal function-B6 deficiency

-To be used effectively, it needs to be followed by specific diagnosis of theCAUSE for the increased Hcy.

Folate supplements were strongly endorsed for prevention of CVD,

in the period 1995-2010.

WHAT IS THE EVIDENCE?

INTERVENTIONS TO LOWER HCY AND PREVENTIONOF CARDIOVASCULAR DISEASE

• It has been know since about 1990 that high Hcy inplasma was ASSOCIATED with increased CVD.

• Therefore, it was hypothesized that lowering Hcywith vitamins would decrease CVD risk

• Several studies have been published that used folate/B12/B6• to decrease cardiovascular risk.

• The general conclusion: this therapy does not have muchbenefit for CVD reduction.

• The connection is with the kidney. Hcy increases duringrenal failure..AND renal failure very often leads toheart disease!

More recent work has focused on folate/B12/B6 and related nutrients, and cognitive decline.

DOUAD ET AL, PROC NAT ACAD SCIENCES, 110: 9532-9528, 2013

In an initial, randomized controlled study on elderly subjects with increased dementia risk (mild cognitive impairment according to 2004 Petersen criteria),we showed that high-dose B-vitamin treatment (folic acid 0.8 mg, vitamin B6 20 mg, vitamin B12 0.5 mg) slowed shrinkage of the whole brain volume over 2 y.

We additionally show that the beneficial effect of B vitamins is confinedto participants with high homocysteine (above the median, 11 μmol/L) and that, in these participants, a causal Bayesian network analysis indicates the following chain of events: B vitamins lower homocysteine, which directly leads to a decrease in GM atrophy, thereby slowing cognitive decline.

Our results show that B-vitamin supplementation can slow the atrophy of specific brain regions that are a key component of the AD process and that areassociated with cognitive decline. Further B-vitamin supplementationtrials focusing on elderly subjets with high homocysteine levelsare warranted to see if progression to dementia can be prevented.

The paper by Douad et al on Alzheimers provides evidence the these vitamins

can prevent cerebral atrophy(if Hcy levels are high).

WE NEED EVIDENCE FROM CONTROLLED TRIALS WITH

FOLATE/B12/B6 SUPPLEMENTS,AND EFFECTS ON COGNITION!

C CH3

NADH+H+ NAD+

METHYLENETETRAHYDROFOLATE

METHYLTETRAHYDROFOLATE

In addition to intake of B12 and folate, there is research on variants of a key enzyme in the folate pathway, MTHFR,which carries out the conversion shown here.

Some variants of the enzyme (linked to the “TT” allele in the gene) don’t function as well as other variants. This will be discussed in class.

The MTHFR DNA base at position 677 in the gene has two possibilities: C (cytosine) or T (thymine). C at position 677 (leading to an alanine at amino acid 222) is the normal allele. The 677T allele (leading to a valine substitution at amino acid 222) encodes a thermolabile enzyme with reduced activity.

THIS LEADS TO A PROTEIN WITH A DIFFERENTAMINO ACID.

MANY PEOPLE HAVE THE VARIANT DNASEQUENCE THAT LEADS TO THE CHANGEIN THE PROTEIN STRUCTURE.

WHAT IS MTHFR POLYMORPHISM?

THE POLYMORPHISM AT MTHFRIS A COMPLEX TOPIC.

IT WILL BE ILLUSTRATED BYDISCUSSIONS USING THE BOARD,DURING CLASS.

Conversion of homocysteine to methionine dependson METHYL-tetrahydrofolate. The role of the enzymeMTHFR is very important to provide this form of folate

In a population from Northern China (Crider et al, AJCN, 2011), it tooklarge daily doses of folate (4 mg/day) to achieve normal levels of homocysteinein the group with the T allele on both genes. The CC group only needed100 µg/day, and there was no effect of added dietary folate.

Why is this important? Consider thepublication on use of folate/B12/B6

to minimize loss of gray matterin old people with AD.

It was most effective for subjectsthat had high levels of Hcy.