1. BIOCHEMISTRY OF FOLATE AND VITAMIN B12: Oct 1, 2014 BASIC BIOCHEMICAL PATHWAYS

2. IMPORTANCE OF FOLATE AND B12 IN NUTRITION: KEY FUNCTIONS Folate is essential for DNA replication, because it’s needed for a critical nucleic acid In addition, folate is needed for most of the activities of B12: synthesis of neurotransmitters, and lipids in the central nervous system. Their biochemistry is closely interrelated.

3. RESOURCE: THE MEDICAL BIOCHEMISTRY PAGE http://themedicalbiochemistrypage.org/home.html

4. KEY POINTS ABOUT FOLATE AND B12: They both donate METHYL groups to other molecules. -METHYLENE-THF: donates a CH 2 group, that is needed for DNA synthesis -METHYL-THF:is a CH 3 donor for many biochemical steps that use vitamin B12. These slides outline important pathways that involve folate and B12.

5. DIETARY FORMS, TRANSPORT FORMS, AND FOLATE INSIDE THE CELL

6. TETRAHYDROFOLATE, WITH 3 GLUTAMIC ACID SIDE CHAINS. THIS IS TYPICAL OF FOLATE IN FOODS SUCH AS VEGETABLES. Only the monoglutamate can be absorbed by humans. If a person has a digestive disorder, the monoglutamate is provided, since it’s absorbed more easily than the form available naturally in foods. Vitamin pills, flour, Wheaties, etc only have the monoglutamate. CLEAVAGE SITE, FOR ENYZME IN DUODENUM

7. Product of enzyme cleavage: Folate-monoglutamate This form is absorbed through the small intestine, carried in plasma to the tissues, and then taken up into cells. After it enters cells, more glutamate residues are added, to remake the POLYGLUTAMATE

8. POLYGLUTAMATE: RESYNTHESIZED IN CELLS. It does not cross the cell membrane, so it remains inside the cells as part of the folate pool.

9. REDUCTIONS AND CARBON TRANSFERS: The formation of the active metabolites of folate

10. FOLIC ACID, WITH A SINGLE GLUTAMATE: Typical of what’s present in vitamins and supplements. H H The first double bond is removed by NADPH: this is DIHYDROFOLATE. NADPH + H + NADP+

11. DIHYDROFOLATE (DHF): FIRST ACTIVE FORM IN PATHWAYS IN THE CELL TETRAHYDROFOLATE (THF): SECOND ACTIVE FORM IN CELL METABOLISM

12. + -CH 2 - C ADDITION OF METHYLENE (CH 2 ): TO MAKE METHYLENE-TETRAHYDOFOLATE OH-CH 2 -C-COOH N H2H2 SERINE H-C-COOH N H2H2 H H GLYCINE THE METHYLENE GROUP (CH 2 ) COMES FROM SERINE. THE REACTION IS B 6 -DEPENDENT (PLP)

13. C CH 3 NADH+H+ NAD+ METHYLENE TETRAHYDROFOLATE METHYL TETRAHYDROFOLATE The molecule on the left donates CH 2 for DNA synthesis: the molecule on the donates CH 3 in many reactions. The reaction also needs riboflavin (FAD). THE REDUCTION OF METHYLENE-THF TO METHYL-THF IS NOT REVERSIBLE!

14. The previous reaction, the conversion of METHYLENE-THF to METHYL-THF, is done by an important enzyme called Methylene-tetrahydrofolate-reductase (MTHFR). There are major genetic variants (polymorphisms) in humans, across different populations, and these variants have significant effects. We will discuss this extensively on Oct 2.

15. The previous slides illustrate all the forms of folate that will be shown in the next slides. These slides show how folate pathways are inter-related, with transfer of CH 2 (for DNA synthesis) and for CH 3 (methylation pathways using B12)

16. dUMP DIETARY FOLATE REDUCTION BY NAPDH DIHYDROFOLATE TETRAHYDROFOLATE REDUCTION BY NAPDH METHYLENE TETRAHYDROFOLATE SERINE GLYCINE METHYL GROUP ADDITION METHYL TETRAHYDROFOLATE REDUCTION BY NADH HCys Met SUBSTRATE S-adenosyl- Met S-adenosyl- HCys METHYLATED SUBSTRATE dTMP (methylated) DNA synthesis REQUIRES Vit B12 CYCLE #1 CYCLE #2 FOLATE PROVIDES METHYL GROUPS BY TWO DIFFERENT CYCLES

17. METHYLENE-THF: DONATES [CH 2 ] FOR DNA SYNTHESIS DEOXYTHYMIDINE (dTMP): REQUIRED FOR DNA SYNTHESIS, AND FOR CELL DIVISON THIS PATHWAY IS VERY ACTIVE IN RAPIDLY-DIVIDING CELLS

18. METHYL-THF: Transfers a CH 3 group (which requires B12) to make S-adenosyl-methionine (SAM)

19. S-adenosyl-methionine (often called SAM) is a very important CH 3 donor. It participates in hundreds of pathways. The function of the methyl-THF/B12 pathway is to make SAM. The intermediates were shown on a previous slide. This methyl came from methyl-THF, and will next be donated to a recipient molecule.

20. From S-adenosyl methionine There are many reactions like this!

21. This is a VERY complicated slide. It outlines the REQUIREMENT for B12, as intermediate to make SAM. Study of B12 function is a very advanced topic.

22. The complex stucture of vitamin B12. There is an atom of cobalt in each B12 molecule.

23. An additional form of folate, FORMINOTETRAHYDROFOLATE, is necessary for the catabolism of the amino acid HISTIDINE. The folate functions as the ACCEPTOR for the amino group, and the histidine carbon skeleton is finally converted to alpha-keto-glutarate. The NH came from the amino acid Folate deficiency causes a change in histidine metabolism. This is the basis of a useful test that examine metabolites in urine. This will be examined in the lecture on October 2.

24. SUMMARY OF BASIC FOLATE/B12 PATHWAYS: -Dietary folate is absorbed, transported to cells, and converted back to a polyglutamate -A complex series of reductions and methyl transfers occurs, which involves serine -One form (methylene-THF) transfers CH 2 to make deoxythymidine, needed for DNA synthesis and cell division -Another form (methyl-THF) transfers CH 3, to make S-adenosyl-methionine (SAM), a step that requires B12. The SAM molecule is a key methyl donor for many pathways in the cell

25. Megadose supplements and health benefits In the last 50 years, 3 separate vitamins have been promoted as having “miraculous” benefits, when consumed high levels: Vitamin C: 1-5 grams/day, for cold prevention Vitamin E: 0.5-1 gram/day, to prevent CHD Folate (5 mgs/day)/B12 (100 ugs/day), also to decrease CHD Benefits are questionable, but folate is protective against birth defects, a highly reliable observation.

26. Ten 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. Why was this decision taken?

27. SPINA BIFIDA: Can occur early in the first trimester. Incidence reduced 50-75% by folate supplementation! The risk may be higher if the embryo has the thermolabile variant of MTHFR (to be discussed shortly).

28. 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.

29. There is evidence now that 1 mg/day is protective. We will discuss optimal doses of folate on Oct 2.