Synthetic vs. organic B12 metabolism—is cyanocobalamin inferior?

Cyanocobalamin is a common synthetic form of vitamin B₁₂ used in supplements and fortified foods—how does it compare to natural forms?

Vitamin B₁₂ (cobalamin, Cbl) has the most complex structure of all vitamins, which consists of a central cobalt atom bound to a corrin ring, a displaceable lower (a) ligand (5,6-dimethylbenzimidazole, DMBI) and a variable upper (b) ligand (e.g. cyano-, methyl-, 5’-deoxyadenosyl-, etc.) ¹ (see).

Cbl was originally isolated as cyanocobalamin (CNCbl), which was later recognised as an artefact arising from extraction methods ². Further advances led to identification of natural forms in microbes, animals and humans ^2–5, where methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) serve as vital coenzymes for methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), respectively.

In the diet, animal foods mainly contain protein-bound Ado-, Me- and hydroxycobalamin (HOCbl) ^6,7, while supplements can contain free-form CNCbl or natural forms. CNCbl is still widely used owing to its low cost and stability. Cbl stability in water is affected by various factors (e.g. light, heat, pH, ROS, other vitamins, polyphenols, etc.), where MeCbl is often least and CNCbl most stable ^8–11. However, Me-, Ado- and CNCbl can all undergo photoaquation to HOCbl, which was the most stable in saline ¹¹ and main form in natural seawater ¹².

While CNCbl has favourable stability, as an unnatural form the burden of proof surely lies with bioactivity—does it work as well? The efficacy of different B₁₂ forms has been the topic of recent reviews ^13,14, which I will add to.

Bioavailability

On consumption, Cbl is initially bound by haptocorrin (HC) in the oral–gastric phase and then by intrinsic factor in the small intestine, which enables active (receptor-mediated) absorption in the ileum; while a minor fraction of free Cbl can also be absorbed passively ¹⁵. In blood, Cbl is transported by HC and transcobalamin (TC), the latter of which mediates transport to tissues and so better reflects ‘active B₁₂’.

Early human studies with radiolabelled Cbl (synthetic and natural forms) and whole-body monitoring generally reported similar oral absorption (refs in ¹⁶), or lower for low-dose AdoCbl ^17,18; while injection of high-dose HOCbl had greater retention and non-specific binding to blood plasma proteins ^18,19. In recent trials, low-dose oral CNCbl elevated plasma B₁₂ and/or holoTC more than HOCbl over 2 days ¹⁶ and 8 weeks ^20,21. In 1 trial (2x 2.8ug CNCbl vs dietary B₁₂) this was related to CNCbl accumulation on HC, although both forms similarly lowered functional biomarkers (i.e. homocysteine and MMA) ²⁰. Whereas in another trial, with more equivalent 3ug supplements, HOCbl seemed to lower MMA slightly better at 8 weeks ²¹.

Recent animal studies also show similar absorption, but different metabolic efficacy ^22,23. In particular, while CNCbl (human equiv. 60ug/day) increased B₁₂ more in blood and kidney, HOCbl increased B₁₂ more in liver, with a similar trend in other organs, and was better converted to other forms ²³. Several earlier in vitro studies also suggested greater efficacy of natural Cbl in cells and mitochondria (refs in ^13,19). For instance, in human cells, both HO- and MeCbl had greater cellular uptake (2x after 4hrs) and conversion to coenzymes (over 24hr), while CNCbl seemed to have more reversal of attachment and internalisation ^19,24; although all forms supported similar methionine synthesis (after 1hr ²⁵) and cell division (over days ²⁴).

Cell processing

Cbl metabolism is highly redox-dependent, where the cobalt coordination (a/b ligands) and oxidation states (I–III) determine reactivity ¹. Inside cells, the cytosolic chaperone MMACHC binds Cbl in the ‘base-off’ state (i.e. a-axial displacement of DMBI) and mediates reductive removal of upper ligands (e.g. decyanation, dealkylation or denitration), generating Cbl(I/II) products which are oxidised to Cbl(II) or aquacob(III)alamin (OH₂Cbl) ^26–30. Cbl processing and trafficking may be facilitated by multiprotein complexes which include MMACHC, MMADHC, MS and MSR ³¹. MMADHC acts as branch point for Cbl delivery to the enzymes MS (cytosol) and MCM (mitochondria), which mediate conversion to coenzymes ³². MSR stabilises MS, via reduction of OH₂Cbl, and maintains MS activity, via reduction of Cbl(II) ³³.

Regarding reaction rates, dealkylation proceeds quicker with MeCbl than AdoCbl ²⁹. HO- and CNCbl go through similar redox-dependent processing ^26,30,33, although the latter is slower (e.g. MMACHC ³⁰, MS ^25,34 and in vivo ^21,23); perhaps due to Co-CN binding affinity and a lower redox potential ^1,9,35,36.

Cbl processing is promoted by glutathione ^5,34,37,38 and related Nrf2 activity ^39,40. Accordingly, decyanation of CNCbl requires NADPH ²⁶ and is supported by glutathione ^25,37 (i.e. aerobic binding and anaerobic metabolism ³⁰), while removal of natural ligands requires glutathione ^27–30,41. Also, on cellular uptake of the TC-Cbl complex into lysosomes and degradation, some Cbl may be released in free form ^42,43. Free HO/OH₂Cbl can readily complex with glutathione to form glutathionylcobalamin (GSCbl) ^25,30,36; a putative protector ^44,45, antioxidant ⁴⁶ and precursor to coenzymes ^25,36–38. In particular, GSCbl had quicker coenzyme activity than HO-, Me- or CNCbl ²⁵ (although see ³⁴). Subsequently, bovine MMACHC was shown to mediate deglutathionylation of GSCbl, via a glutathione-dependent reaction analogous to dealkylation of coenzymes ⁴¹. The reaction with GSCbl is also rapid, being an order of magnitude higher than with coenzymes ⁴¹.

Host factors

Genetic, environmental and disease-related factors can affect B₁₂ metabolism, where differences between forms may become more relevant. Foremost of which, rare inborn errors affecting MMACHC (i.e. CblC disorders), where HOCbl ^14,26,35 or CNCbl ²⁷ may be advantageous. More prevalently, various SNPs may slow Cbl uptake or processing (e.g. TCN2 and MTRR) ^5,13,47.

Some other factors may specifically affect CNCbl. For instance, since the 1960s, elevated CNCbl has been reported in several populations, including smokers, Leber’s and kidney disease ^2,48. Further, a meta-analysis of RCTs linked high-dose CNCbl to adverse CVD outcomes in people with impaired renal function, obscuring benefit in those with good function ⁴⁹; potentially also of relevance in elderly ⁵⁰. A human brain autopsy study also found very elevated CNCbl levels (15-fold; 1:1 ratio with MeCbl) in fetal samples, which may be a result of prenatal supplements and/or decreased metabolism ⁵. Note, in animal models, CNCbl also accumulated in kidneys and brain ²³, suggesting organ-specific metabolism.

Host cyanide metabolism may be important. Intracellular processing of CNCbl releases cyanide ²⁶, which is mainly metabolised via sulfur-dependent pathways ^51,52; including MST (cytosol and mitochondria) and TST (aka. rhodanese; mitochondria) catalysis to thiocyanate (SCN^–), and non-enzymatic reactions ^53,54. Even 1mg of CNCbl provides little cyanide compared to background dietary levels ⁵⁵ (see). Conversely however, increased basal cyanide levels, due to exposure (e.g. smoking, diet or SNP) or impaired metabolism (e.g. Leber’s and kidney disease), may affect Cbl status ^2,48. Indeed, cyanide has extremely high affinity for HOCbl (> glutathione), resulting in conversion to CNCbl ³⁶, and also reacts with coenzymes ^56,57. Note, treatment of neuro-symptoms in 5 HD patients with intravenous MeCbl was associated with higher CNCbl (4x) and slightly lower thiocyanate ⁴⁸. Ultimately, saturation of cyanide detox pathways may favour auxiliary formation of CNCbl, sequestering cyanide and Cbl, at the expense of coenzymes ⁴⁸. Importantly, cyanide metabolism is organ-dependent, being notably lower in brain—a major target of toxicity—and more so in early and later life ^51,58. As noted above, even the normal brain can accumulate CNCbl, where concern was raised as to whether it could act as a competitive antagonist ^5,23.

B₁₂ metabolism may be even more generally impaired in ageing and chronic disease. For instance, early observations suggested MeCbl markedly declined during ageing in plasma, liver and brain ². More recently, a marked decline in total, GS-, Ado- and MeCbl was reported in the ageing human brain (i.e. 61–80yrs), which occurred prematurely in autism and schizophrenia ⁵. In the periphery, functional B₁₂ deficiency increases with age in relation to amnionless ⁵⁹ and ‘oxidant risks’ ⁶⁰, the presence of which impaired response to high-dose CNCbl ⁶⁰. Note, in other trials on older people, very high doses of CNCbl were required to lower functional biomarkers ^61,62, as compared to younger people (trials above) ^20,21.

Host redox biology may be important. A recent systematic review found some evidence of a relation between B₁₂ and redox status ⁶³. Reciprocally, an oxidised redox status in ageing and disease may affect B₁₂ status ^5,39,40,60. In particular, depletion of glutathione may impair brain transport and conversion to coenzymes ^5,34,37–40. In ageing neurons, alternate splicing of MS (via deletion of exons 19 and 20) may also increase Cbl(I) susceptibility to oxidation ³⁸. Moreover, many diseases involve chronic inflammation, neutrophil activation and production of hypochlorous acid (HOCl) ⁶⁴, a potent oxidant which can destroy Cbl to release toxic products, including redox-active cobalt, cyanogen chloride and cyanide ^64,65.

Intriguingly, in healthy adults, a positive correlation was found between cerebrospinal fluid B₁₂ and 8-OHdG—a marker of DNA oxidation ⁶⁶. The authors speculated as to whether this may involve CNCbl supplements and reduced brain cyanide detoxification capacity ⁶⁶. Note, other aspects of multivitamin-mineral supplements can also promote oxidation ⁶⁷.

Gut microbiome?

Cbl is synthesised exclusively by microbes, yet potential effects on the gut microbiome seem rarely considered. Human faecal samples contain a variety of corrinoids, less than 2% of which is Cbl ⁶⁸. Over 80% of sequenced human gut bacteria are predicted to use corrinoids, where they serve diverse functions as enzyme cofactors and genetic regulators (e.g. corrinoid riboswitches). However, since less than 25% possess synthetic capacity, they may be a precious resource for commensals ⁶⁸ and pathogens ⁶⁹. Further competition between host and microbes may occur under conditions of SIBO ^68,70, and influence bacterial pathogenesis at various body sites ^69,70.

B₁₂ is often supplemented at high doses (e.g. >100x RDA), due to poor absorption (relative to body stores), which exceed active transport (via intrinsic factor) and so are absorbed by passive diffusion at a rate of ~1% ¹⁵. Consequently, most will end up in the gut ⁶⁸. In mice, high-dose CNCbl (human equiv. 5mg for 16 days) greatly increased gut Cbl, but lowered microbial corrinoid analogues ⁷¹. This treatment had no effect on microbial diversity, SCFAs or IBD, but did selectively deplete Bacteroides ⁷¹. A couple of studies have compared CN- and MeCbl. In a human colonic simulation, they differently shifted the microbiome and metabolism, with MeCbl appearing more favourable ⁷². While in mice, only CNCbl (human equiv. 1.5mg for 3 days) aggravated IBD, in relation to increased Enterobacteriaceae (e.g. E. coli), modulation of bacterial enzymes and riboswitches ⁷³.

In sum, while synthetic CNCbl has favourable stability, perhaps this comes at the cost of slower metabolism and some other potentially unfavourable characteristics. Could this hinder its efficacy in chronic disease and ageing? Many authors have long favoured or suggested natural B₁₂ forms (e.g. HO- or MeCbl) to circumvent the conditions and issues discussed above ^{2,5,13,48,50,60,72,74}. With natural form supplements the burden of proof perhaps lies more on stability.

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