Vitamin B12 Coenzyme Functions

by Jack Norris, RD

Contents

• Introduction
• B12 Functions
• Homocysteine Clearance
• Folate, B12, and the Methylfolate Trap
  • Table 1: Upper Limit for Supplemental Folate
  • MTHFR Variant and Supplementation
  • Folate Forms and their Effects
    • Table 2. MTHFR Variant and Supplementation
• Lack of Anemia Does Not Mean B12 Status Is Healthy
• Table 3: B12 Levels in Neurological Patients without Macrocytic Anemia
• Methylmalonic Acid (MMA)
• References

Introduction

Eussen et al. summarize vitamin B12’s functions in humans:

Vitamin B-12 is involved in one-carbon metabolism, during which it plays a role in the transfer of methyl groups and methylation reactions that are important for the synthesis and metabolism of neurotransmitters and phospholipids in the central nervous system. Moreover, vitamin B-12 is required for nucleic acid synthesis and hematopoiesis and for the metabolism of fatty acids and amino acids in the mitochondrial citric acid cycle. In addition to causing anemia, vitamin B-12 deficiency has been linked with several neurologic disorders, such as neuropathy, myelopathy, dementia, depression, memory impairment, and cerebrovascular disease. Although prolonged vitamin B-12 deficiency may eventually result in irreversible neurologic damage and cognitive impairment, early stages of vitamin B-12 deficiency—detected by elevated concentrations of plasma total homocysteine and methylmalonic acid and decreased concentrations of holotranscobalamin—may result in milder forms of cognitive impairment in the absence of anemia (Eussen et al., 2006).

B12 Functions

In the cells of mammals, there are two different co-enzyme forms of vitamin B12 (Scalabrino, 2001; Seetharam & Li, 2000):

• Methylcobalamin
  • Used by the enzyme methionine synthase to turn homocysteine into methionine. Methionine is further converted to the important methyl donor, S-adenosylmethionine (SAM, aka SAMe)
• 5′-deoxyadenosylcobalamin
  • Used by the enzyme methylmalonyl-CoA mutase to convert methylmalonyl-CoA to succinyl-CoA

Homocysteine Clearance

Methionine is an essential amino acid provided by the diet. Some methionine is turned into homocysteine and without adequate vitamin B12, homocysteine builds up in the blood. Although it’s not clearly a causative factor, elevated homocysteine is associated with early death, cardiovascular disease, and dementia. For more information, see Homocysteine and Mild B12 Deficiency in Vegans.

Folate, B12, and the Methylfolate Trap

Author’s note: I used AI in the literature search, analysis, and editing for this section. (Last reviewed: April 2026.)

Vitamin B12 deficiency, which most commonly results from impaired B12 absorption rather than inadequate dietary intake, has typically presented as fatigue due to red blood cells not forming correctly. This type of anemia goes by two related names:

• Macrocytic anemia – above normal mean corpuscular volume (MCV), meaning the red blood cells are larger than normal on average
• Megaloblastic anemia – a specific subtype of macrocytic anemia in which abnormally large, immature red blood cell precursors (megaloblasts) are observed in the bone marrow, and the red blood cells show characteristic changes under a microscope

Folate is needed for DNA synthesis because it provides the one-carbon units required to produce thymidine, a building block of DNA. Folate from food is converted to a form called 5-MTHF during digestion and liver metabolism, and it is in this form that folate circulates in the bloodstream (EFSA, 2023, Figure 4). To be used for DNA synthesis, however, 5-MTHF first needs to be converted into a different form with the help of B12. When B12 is deficient, folate gets stuck in its unusable circulating form, DNA synthesis slows, and red blood cell precursors can’t divide properly. This bottleneck is called the methylfolate trap.

Because DNA is required for cell division, rapidly dividing cells, such as red blood cells, are impacted first. Red blood cell precursors in the bone marrow divide continuously, and when DNA synthesis slows, they divide more slowly than normal. However, the hemoglobin they produce continues to be made at a relatively normal rate. The result is cells that grow large but divide slowly, producing the oversized red blood cells (macrocytes) characteristic of this deficiency. When enough of these accumulate in circulation, the result is macrocytic anemia.

Folic acid, the synthetic form of folate found in supplements and fortified foods, can bypass the methylfolate trap. Unlike 5-MTHF, folic acid is converted to THF through a different enzyme pathway that doesn’t require B12. This means that a high intake of folic acid can restore DNA synthesis in red blood cell precursors even when B12 is severely deficient, correcting the anemia and returning red blood cell size to normal. By resolving the most visible symptom of B12 deficiency, folic acid may delay diagnosis and potentially allow neurological damage to progress undetected. However, doses of folic acid up to 1,000 µg per day, well above the 400 µg found in a typical multivitamin, are considered unlikely to mask B12 deficiency (EFSA, 2023, Section 3.4.1.2).

Table 1 provides the amounts of folic acid considered by the European Food Safety Authority to be unlikely to mask B12 deficiency for different age groups.

Table 1. Upper Limit (UL) for supplemental folate
Age group	UL males and females (µg/day)
4–6 months	200
7–11 months	200
1–3 years	200
4–6 years	300
7–10 years	400
11–14 years	600
15–17 years	800
Adults	1,000
Pregnant women	1,000
Lactating women	1,000
Supplemental folate includes folic acid from fortified foods and supplements, and supplements of 5-MTHF-glucosamine and l-5-MTHF-Ca. Source: EFSA, 2023, Section 4

MTHFR Variant and Supplementation

A common genetic variant called the MTHFR 677C→T polymorphism reduces the activity of the MTHFR enzyme, which converts 5,10-methyleneTHF into 5-MTHF primarily in the liver and intestinal cells. This reduces the amount of 5-MTHF released into the bloodstream and available to target cells. People who are homozygous for this variant (carrying two copies of the T-allele) tend to have lower folate status and elevated homocysteine levels, and may have folate requirements up to 20% higher than average (EFSA, 2023, Section 3.2.5.3).

Because their MTHFR enzyme is impaired, these individuals are often advised to supplement with 5-MTHF directly, in the forms of calcium-L-methylfolate (L-5-MTHF-Ca) or 5-MTHF-glucosamine, rather than folic acid. Although folic acid can enter the DNA synthesis pathway via a route that initially bypasses MTHFR, producing 5-MTHF from folic acid ultimately still requires the MTHFR enzyme and, therefore, folic acid is no more effective than food folate at correcting the shortfall in 5-MTHF available for homocysteine clearance via the methylation cycle (see EFSA, 2023, Figure 4). Supplementing with pre-formed 5-MTHF bypasses the impaired enzyme entirely.

However, the EFSA applies the same tolerable upper intake level of 1,000 µg/day to these 5-MTHF forms as to folic acid, noting that natural reduced folates have also been reported to correct megaloblastic anemia in B12-deficient individuals. The EFSA acknowledges that the evidence base for 5-MTHF specifically is limited and that additional research is needed (EFSA, 2023, Section 4).

Folate Forms and their Effects

Table 2 summarizes the effects of different forms of folate on the folate cycle depending on vitamin B12 status and MTHFR genetic variant. Folate that synthesizes DNA when B12 is deficient can mask a B12 deficiency.

Table 2. Folate Form, B12 Status, and MTHFR 677C→T Variant
B12 Status	MTHFR 677C→T	Synthesizes DNA			Clears Homocysteine
		Food Folate	Folic Acid	5-MTHF	Food Folate	Folic Acid	5-MTHF
Sufficient	No	Y	Y	Y	Y	Y	Y
Sufficient	Yes	Y	Y	Y	Ns	Ns	Y
Deficient	No	N	Y	?	Nt	Nt	Nt
Deficient	Yes	N	Y	?	Nt	Ns,t	Nt
Y – yes  •  N – no  •  Ns – no, due to conversion to 5-MTHF being slowed  •  Nt – no, due to being in the methylfolate trap  •  ? – theoretically, DNA should not be synthesized when B12 is deficient, but there is some evidence it is (EFSA, 2023, Section 4).

Lack of Anemia Does Not Mean B12 Status Is Healthy

Traditionally, the existence of macrocytic anemia was relied on to indicate a B12 deficiency. However, neurological disorders due to B12 deficiency commonly occur in the absence of a macrocytic anemia.

Lindenbaum et al. (Lindenbaum et al., 1988, USA) examined 141 cases of neurological problems due to B12 deficiency. 40 (28%) had no macrocytic anemia (iron deficiency may have contributed to a lack in 6 patients, and folate therapy could account for 2 others). These 40 had very high serum MMA levels (range: .76-187 µmol/l, 78% > 2 µmol/l) and homocysteine levels (23-289 µmol/l, 45% > 100 µmol/l). Characteristic features of patients with B12 deficiency but without macrocytic anemia included: sensory loss, inability to move muscles smoothly (ataxia), dementia, and psychiatric disorders. They also had borderline (and sometimes normal) B12 levels (see Table 3). One patient died during the first week of treatment, but the other 39 benefited from B12 therapy. Some patients had residual abnormalities after years of treatment.

Table 3. B12 Levels in Neurological Patients Without Macrocytic Anemia (pg/ml)
Number of Patients	Serum B12
2	> 200
16	100-200
22	< 100

In a 2011 study from Korea, among 35 patients with vitamin B12 deficiency, most of whom had neurological symptoms, none had anemia (Kim et al., 2011).

Methylmalonic Acid (MMA)

The second coenzyme form of B12, adenosylcobalamin, takes part in the conversion of methylmalonyl-CoA to succinyl-CoA. When B12 is not available, methylmalonyl-CoA levels increase. Methylmalonyl-CoA is then converted to methylmalonic acid (MMA) which then accumulates in the blood and urine. Since B12 is the only coenzyme required in this pathway, MMA levels are the best indicators of a B12 deficiency.

Elevated MMA levels can also be caused by genetic defects, kidney failure, low blood volume, gut bacteria changes, pregnancy, and thyroid disease (Minet et al., 2000).

For more information on elevated methylmalonic acid, see Minimizing Methylmalonic Acid Levels.

References

European Food Safety Authority (EFSA) Panel on Nutrition, Novel Foods and Food Allergens (NDA Panel); Turck D, Bohn T, Castenmiller J, et al. Scientific opinion on the tolerable upper intake level for folate. EFSA J. 2023 Nov 13;21(11):e08353.

Eussen SJ, de Groot LC, Joosten LW, et al. Effect of oral vitamin B-12 with or without folic acid on cognitive function in older people with mild vitamin B-12 deficiency: a randomized, placebo-controlled trial. Am J Clin Nutr. 2006;84(2):361-370.

Kim HI, Hyung WJ, Song KJ, Choi SH, Kim CB, Noh SH. Oral vitamin B12 replacement: an effective treatment for vitamin B12 deficiency after total gastrectomy in gastric cancer patients. Ann Surg Oncol. 2011 Dec;18(13):3711-7.

Lindenbaum J, Healton EB, Savage DG, Brust JC, Garrett TJ, Podell ER, Marcell PD, Stabler SP, Allen RH. Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis. N Engl J Med. 1988 Jun 30;318(26):1720-8.

Minet JC, Bisse E, Aebischer CP, Beil A, Wieland H, Lutschg J. Assessment of vitamin B-12, folate, and vitamin B-6 status and relation to sulfur amino acid metabolism in neonates. Am J Clin Nutr. 2000 Sep;72(3):751-7.

Scalabrino G. Subacute combined degeneration one century later. The neurotrophic action of cobalamin (vitamin B12) revisited. J Neuropathol Exp Neurol. 2001 Feb;60(2):109-20.

Seetharam B, Li N. Transcobalamin II and its cell surface receptor. Vitam Horm. 2000;59:337-66.