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BIOCHEMISTRY OF FOLATE AND VITAMIN B12: October 15, 2014 Nutritional roles, and diagnosis PowerPoint Presentation
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BIOCHEMISTRY OF FOLATE AND VITAMIN B12: October 15, 2014 Nutritional roles, and diagnosis of deficiencies. METHYLENE-THF : DONATES [CH 2 ] FOR DNA SYNTHESIS. DEOXYTHYMIDINE (dTMP): REQUIRED FOR DNA SYNTHESIS, AND FOR CELL DIVISON.

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slide1

BIOCHEMISTRY OF FOLATE

AND VITAMIN B12:

October 15, 2014

Nutritional roles, and diagnosis

of deficiencies

slide2

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

slide3

WITHOUT NEW DNA

SYNTHESIS, CELLS

WILL NOT DIVIDE, BUT

CAN GET LARGER.

THIS IS CALLED:

MEGALOBLASTIC

ANEMIA

FOLATE AND B12

DEFICIENCY PRODUCE

A SIMILAR ANEMIA.

THIS IS EXPLAINED BY

THE METHY-FOLATE TRAP.

LARGE RBC:

Megaloblastic

anemia

slide4

FOLATE PROVIDES METHYL GROUPS

BY TWO DIFFERENT CYCLES

DNA

synthesis

DIETARY

FOLATE

REDUCTION BY

NAPDH

DIHYDROFOLATE

dTMP

(methylated)

REDUCTION BY

NAPDH

CYCLE

#1

TETRAHYDROFOLATE

SERINE

dUMP

METHYL GROUP

ADDITION

CYCLE

#2

GLYCINE

METHYLENE

TETRAHYDROFOLATE

REQUIRES

Vit B12

REDUCTION

BY NADH

This step is not reversible.

If B12 is deficient, folate

accumulates in this form,

(called the folate trap).

Result: folate is not available

for cycle #1.

METHYL

TETRAHYDROFOLATE

HCys

Met

S-adenosyl-

HCys

S-adenosyl-

Met

SUBSTRATE

METHYLATED

SUBSTRATE

slide5

WITHOUT VITAMIN B12, METHYL-THF CANNOT

BE RECYCLED BACK TO TETRAHYDROFOLATE.

slide6

C

NADH+H+ NAD+

CH3

METHYLENE

TETRAHYDROFOLATE

METHYL

TETRAHYDROFOLATE

THE REDUCTION OF METHYLENE-THF TO

METHYL-THF IS NOT REVERSIBLE.

IF METHYL-THF IS FORMED, IT CANNOT BE

CONVERTED BACK TO METHYLENE-THF.

slide7

C

VARIANTS OF MTHFR

NADH+H+ NAD+

CH3

METHY

TETRAHYDROFOLATE

METHYLENE

TETRAHYDROFOLATE

The enzyme methylene-tetrahydrofolate-reductase (MHTFR)

catalyzes this reaction. That enzyme has common variants,

which is the topic of your reading assignment. Different forms

of MFTHR also influence Hcy levels.

slide8

During lecture, I will put specific details about

variants of MTHFR on the board.

We will discuss:

-variations in gene sequences

-how that affects the enzyme protein

-the interaction with plasma Hcy levels

slide9

WHY WOULD ABUNDANT DIETARY FOLATE

MAINTAIN CYCLE #1?

DNA

synthesis

DIETARY

FOLATE

REDUCTION BY

NAPDH

DIHYDROFOLATE

dTMP

(methylated)

REDUCTION BY

NAPDH

CYCLE

#1

TETRAHYDROFOLATE

SERINE

dUMP

METHYL GROUP

ADDITION

CYCLE

#2

GLYCINE

METHYLENE

TETRAHYDROFOLATE

BLOCKED, WITH

NO B12

REDUCTION

BY NADH

METHYL

TETRAHYDROFOLATE

HCys

Met

S-adenosyl-

HCys

S-adenosyl-

Met

SUBSTRATE

METHYLATED

SUBSTRATE

slide10

VITAMIN B12 DEFICIENCY

-Vitamin B12 deficiency develops very

slowly. 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 causes

irreversible damage to the CNS.

-Conclusion: it’s IMPORTANT that deficiency

not be masked, and be readily diagnosed.

slide11

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 continues

from the B12 deficiency.

PUBLIC HEALTH CONCLUSION: Don’t consume so much folate, that the early

anemia of B12 deficiency is prevented.

slide12

About years ago, the decision was made to add folate

to flour, breakfast cereal, bakery products, and

many other consumer goods.

As a result, folate deficiency is now very rare

in many parts of the world.

This decision was made to decrease the

incidence of birth defects, especially spina bifida.

slide13

Summary of a study done between 1985-1990

The dose was 4 mg/day, given to women who had

previously had a child with an NTD.

ASSIGNMENT: Review the study (posted)

that describes prevention of NTD with

supplements of dietary folate.

slide14

BUT IT WAS IMPORTANT TO DEFINE THE

SMALLEST EFFECTIVE DOSE.

Us of 4 mg/day would certainly

mask presence of B12 deficiency.

Assignment: the Lancet paper (1997), that seeks to

define the minimal dose needed.

slide15

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.

slide16

VITAMIN B12 DEFICIENCY

CAUSES AND DIAGNOSIS

slide17

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 called

PERNICIOUS ANEMIA.

slide18

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, before

deficiency is a problem.

slide19

Diagnosis of B12 deficiency:

There are several strategies

slide20

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.

slide21

If B12 is adequate, propionyl-CoA is metabolized

to succinate, for the TCA cycle.

If B12 is deficient, methylmalonate appears

in blood and urine.

slide22

From some amino acids, and

from odd-chain fatty acids

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 is

removed to make succinate

slide23

EXAMPLES OF BIOCHEMICAL PATHWAYS WHERE

S-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 biochemistry

that require SAM for methylation.

slide24

Added

methyl

group

EPINEPHRINE BIOSYNTHESIS: What vitamin

was needed to make the norepinephrine?

slide25

Added

methyl

group

CREATINE SYNTHESIS

slide26

METHYLATION OF THE CYTOSINE BASE

IN THE DNA STRAND: THIS IS VERY

IMPORTANT IN GENE REGULATION!

slide27

Specific diagnosis of folate deficiency:

-measurement of RBC folate is common.

Folate <140 microgram/L indicates deficiency.

-elevated Hcy can indicate either folate

or B12 deficiency, and more specific

diagnosis is needed. Variants of MTHFR

are also a factor.

-the catabolism of the amino acid HISTIDINE

requires folate. In folate deficiency

N-formino-glutamate will appear in the urine,

and that’s a reasonable test.

slide28

With lack of folate,

this compound will

appear in the urine.

Folate is the ACCEPTOR

of the nitrogen group in

red, and histidine is

converted to glutamate.

slide29

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.

slide30

ROLE OF B6 IN

REMOVING

HOMOCYSTEINE

DETAILS

OF THIS

REACTION

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.

slide31

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 the

CAUSE for the increased Hcy.

slide32

Folate supplements were strongly

endorsed for prevention of CVD,

in the period 1995-2010.

WHAT IS THE EVIDENCE?

slide33

INTERVENTIONS TO LOWER HCY AND PREVENTION

  • OF CARDIOVASCULAR DISEASE
  • It has been know since about 1990 that high Hcy in
  • plasma was ASSOCIATED with increased CVD.
  • Therefore, it was hypothesized that lowering Hcy
  • with 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 much
  • benefit for CVD reduction.
  • The connection is with the kidney. Hcy increases during
  • renal failure..AND renal failure very often leads to
  • heart disease!
slide34

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 confined

to 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 are

associated with cognitive decline. Further B-vitamin supplementation

trials focusing on elderly subjets with high homocysteine levels

are warranted to see if progression to dementia can be prevented.

slide35

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!

slide36

C

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.

NADH+H+ NAD+

CH3

METHYLENE

TETRAHYDROFOLATE

METHYL

TETRAHYDROFOLATE

slide37

WHAT IS MTHFR POLYMORPHISM?

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 DIFFERENT

AMINO ACID.

MANY PEOPLE HAVE THE VARIANT DNA

SEQUENCE THAT LEADS TO THE CHANGE

IN THE PROTEIN STRUCTURE.

slide38

THE POLYMORPHISM AT MTHFR

IS A COMPLEX TOPIC.

IT WILL BE ILLUSTRATED BY

DISCUSSIONS USING THE BOARD,

DURING CLASS.

slide39

Conversion of homocysteine to methionine depends

on METHYL-tetrahydrofolate. The role of the enzyme

MTHFR is very important to provide this form of folate

slide40

In a population from Northern China (Crider et al, AJCN, 2011), it took

large daily doses of folate (4 mg/day) to achieve normal levels of homocysteine

in the group with the T allele on both genes. The CC group only needed

100 µg/day, and there was no effect of added dietary folate.

slide41

Why is this important? Consider the

publication on use of folate/B12/B6

to minimize loss of gray matter

in old people with AD.

It was most effective for subjects

that had high levels of Hcy.