1. BIOCHEMISTRY OF FOLATE AND VITAMIN B12: October 15, 2014 Nutritional roles, and diagnosis of deficiencies

2. 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, SUCH AS DEVELOPING RBC

3. 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

4. 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 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.

5. WITHOUT VITAMIN B12, METHYL-THF CANNOT BE RECYCLED BACK TO TETRAHYDROFOLATE.

6. C CH 3 NADH+H+ NAD+ 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.

7. C CH 3 NADH+H+ NAD+ METHYLENE TETRAHYDROFOLATE METHY 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. VARIANTS OF MTHFR

8. 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

9. 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 BLOCKED, WITH NO B12 CYCLE #1 CYCLE #2 WHY WOULD ABUNDANT DIETARY FOLATE MAINTAIN CYCLE #1?

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. VITAMIN B12 DEFICIENCY CAUSES AND DIAGNOSIS

17. 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.

18. 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.

19. Diagnosis of B12 deficiency: There are several strategies

20. 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.

21. If B12 is adequate, propionyl-CoA is metabolized to succinate, for the TCA cycle. If B12 is deficient, methylmalonate appears in blood and urine.

22. 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 From some amino acids, and from odd-chain fatty acids

23. 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.

24. EPINEPHRINE BIOSYNTHESIS: What vitamin was needed to make the norepinephrine? Added methyl group

25. CREATINE SYNTHESIS Added methyl group

26. METHYLATION OF THE CYTOSINE BASE IN THE DNA STRAND: THIS IS VERY IMPORTANT IN GENE REGULATION!

27. 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.

28. 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.

29. 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.

30. 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. DETAILS OF THIS REACTION ROLE OF B6 IN REMOVING HOMOCYSTEINE

31. 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.

32. Folate supplements were strongly endorsed for prevention of CVD, in the period 1995-2010. WHAT IS THE EVIDENCE?

33. 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!

34. 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.

35. 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!

36. C CH 3 NADH+H+ NAD+ METHYLENE TETRAHYDROFOLATE METHYL TETRAHYDROFOLATE 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.

37. 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. WHAT IS MTHFR POLYMORPHISM?

38. THE POLYMORPHISM AT MTHFR IS A COMPLEX TOPIC. IT WILL BE ILLUSTRATED BY DISCUSSIONS USING THE BOARD, DURING CLASS.

39. Conversion of homocysteine to methionine depends on METHYL-tetrahydrofolate. The role of the enzyme MTHFR is very important to provide this form of folate

40. 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.

41. 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.