Clinical Pathobiochemistry of Vitamin B12 Deficiency: Improving Our Understanding by Exploring Novel Mechanisms with a Focus on Diabetic Neuropathy

Vitamin B12 (B12) is an essential cofactor of two important biochemical pathways, the degradation of methylmalonic acid and the synthesis of methionine from homocysteine. Methionine is an important donor of methyl groups for numerous biochemical reactions, including DNA synthesis and gene regulation. Besides hematological abnormalities (megaloblastic anemia or even pancytopenia), a deficiency in B12 may cause neurological symptoms, including symptoms resembling diabetic neuropathy. Although extensively studied, the underlining molecular mechanism for the development of diabetic peripheral neuropathy (DPN) is still unclear. Most studies have found a contribution of oxidative stress in the development of DPN. Detailed immunohistochemical investigations in sural nerve biopsies obtained from diabetic patients with DPN point to an activation of inflammatory pathways induced via elevated advanced glycation end products (AGE), ultimately resulting in increased oxidative stress. Similar results have been found in patients with B12 deficiency, indicating that the observed neural changes in patients with DPN might be caused by cellular B12 deficiency. Since novel results show that B12 exerts intrinsic antioxidative activity in vitro and in vivo, B12 may act as an intracellular, particularly as an intramitochondrial, antioxidant, independent from its classical, well-known cofactor function. These novel findings may provide a rationale for the use of B12 for the treatment of DPN, even in subclinical early states.


Introduction
Vitamin B 12 (B 12 ) is an essential cofactor for two enzymes in human metabolism: methylmalonyl-CoA mutase (catalyzing the conversion of methylmalonyl-CoA to succinyl-CoA), and methionine synthase (catalyzing the synthesis of methionine from homocysteine). While an inherited defect of methylmalonyl-CoA mutase causes methylmalonic aciduria, severe acquired B 12 deficiency, mostly due to reduced uptake of B 12 , causes classical pernicious anemia. It may also cause neurological symptoms, most commonly sensory, but also motoric or painful neuropathy, symptoms that are also common in DPN.
The precise molecular mechanism leading to DPN is under debate [1][2][3][4]. Previous reviews implicate a variety of hyperglycemia-dependent pathways, including the sorbitol, glucosamine and the AGE/RAGE pathways, possibly leading to oxidative stress and induction of inflammatory pathways [5]. However, none of these pathways have been unequivocally shown to be causally responsible for the development of DPN. To date, other than glycemic control, there is no evidence-based therapy available which may prevent DPN [5][6][7][8]. Numerous reports indicate beneficial effects of B 12 supplementation, while other studies did not find significant effects [9,10]. In a recent extensive review, the therapeutic effects of B 12 on DPN were evaluated, and significant effects of B 12 on DPN could be demonstrated [10]. However, no molecular mechanism has been hitherto established for the beneficial effects of B 12 in the prevention or treatment of DPN. Furthermore, it is not known which B 12 levels should be considered as indicating B 12 deficiency (and thus needing B 12 supplementation). Indeed, B 12 serum concentrations observed in different states of DPN may be only marginally reduced or even unchanged. In addition, elevated homocysteine and methylmalonic acid levels, both biomarkers of cellular B 12 deficiency, are common in the elderly and are associated with neurological abnormalities [11]. It was suggested that a functional B 12 deficiency is present in these patients, despite normal B 12 serum levels. This would mean that intracellular B 12 does not always reflect serum levels. Notably, patients with DPN do not always show signs of classical B 12 deficiency, e.g., pernicious anemia, which suggests that DPN may develop at lower cellular (and not necessarily lower serum) levels of B 12 . It should be underlined that subclinical B 12 deficiency may develop very slowly because the liver contains large amounts of B 12 ; thus, clinical signs develop slowly and may be overlooked.
Accordingly, in this review, we first summarize the current knowledge of the possible involvement of B 12 deficiency in the development of DPN and provide novel data introducing the property of B 12 working as redox system. Second, we discuss the issue and the implications of "functional" or "subclinical" B 12 deficiencies. For the detection of such subclinical B 12 deficiencies, cut-off values need to be defined. Such cut-off values should include cut-off values for overt B 12 deficiency, ranges for inadequate B 12 supply and values for adequate supply.

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Chemistry; • Vitamin B 12 sources, physiological uptake and causes of deficiency; • Intracellular processing and reduction/oxidation function of B 12 ; • Physiological functions of Vitamin B 12 ; • Clinical pathophysiology of B 12 deficiency.

Chemistry
Vitamin B 12 or cobalamin is a water-soluble vitamin with a complex structure ( Figure 1). The unique characteristic of B 12 is that a single cobalt atom is bound in the center of a ring of four pyrroles (corrin) similar to the Fe atom bound in the center of the heme ring of hemoglobin. Accordingly, similar to the heme system of hemoglobin where the upper axial ligand O 2 can be easily exchanged, the upper axial ligand of B 12 is also exchangeable. Physiological ligands of B 12 are a methyl (-CH 3 ), hydroxy (-OH) or a 5'-desoxyadenosyl unit [12]. Between them, hydroxy-B 12 is the more stable form, and is used, as well as cyano-B 12 , for pharmacological administration of B 12 in Europe and the US, respectively ( Figure 1).

Figure 1.
Structure and modifications of Vitamin B12 (cobalamin). Cobalamin (Cbl) is the only coba containing vitamin. The central cobalt atom (magenta) is coordinated by a corrin ring (blue) with residues (R = acetyl or propionylamide) (red). Only the upper axial position (L) (green) can b exchanged for different ligands: the physiological ligands 5′-desoxyadenosyl-Cbl or methyl-Cb which are needed for two different biochemical reactions, or the pharmacologically administere forms of B12 cyano-Cbl (more in USA) or hydroxy-Cbl (more in Europe). The unique characteristi of B12 is that a single cobalt atom is bound in the center of a ring of four pyrroles (corrin) similar t the Fe atom bound in the center of the heme ring of hemoglobin The only difference between th tetrapyrrolic ring of corrins and heme is that the A and D pyrroles are directly bound in corrin while in heme, the four rings are all bound via a carbon bridge.

Vitamin B12 Sources, Physiological Uptake and Causes of Deficiency
B12 is not present in foods obtained from plants, and since it cannot be produced b humans, it needs to be obtained from a diet containing products derived from animals, suc as meat, dairy products and particularly liver, which is the most important B12 storage orga (80% of total storage) in mammals. The recommended daily requirement depends o gender. Adult women require 2.4 µg of B12 per day. This requirement increases to 2.8 µg o B12 per day during pregnancy and breastfeeding. Adult men can meet their needs b consuming 2.6 µg of B12 per day. Lesser amounts are recommended for young infants [13 The daily turnover of B12 is less than 0.1%. Deficiencies occur when supplies are reduced t 300 µg of B12 per day. As mentioned above, B12 deficiency may be easily overlooked: th clinical signs of deficiency may develop slowly because in humans, B12 storage ranging from 2 to 5 mg may last for more than a year.
The sequential stages of uptake of B12 (Table 1, left column) [3,14] are as follows: (1 The first step is dietary intake of free or protein-bound B12. (2) In the stomach gastric cell release proteases, e.g., pepsin and HCl, and in this acidic milieu, protein-bound B12 i released by protein digestion and free B12 is bound by haptocorrin. This complex and intrinsic factor (IF) secreted by parietal cells are transferred to the duodenum. (3) In th duodenum, haptocorrin-bound B12 (also named "transcobalamin I") is released from haptocorrin by proteolytic digestion, and the released B12 binds to IF with high affinity. (4 The B12-IF complex is bound and taken up by specific mucosal receptors in the dista ileum. (5) After internalization, B12 is released and free B12 is secreted into the blood, bound to transcobalamin and transported to peripheral organs/targets. (6) B12 bound t transcobalamin II (HoloTC) is taken up via the ubiquitous receptor CD 320, and B12 i liberated in the lysosomes. Note that only about 10-20% of B12 is bound to transcobalami II (HoloTC) in the blood, while the majority of B12 (ca. 80%) is bound to transcobalamin . Cobalamin (Cbl) is the only cobalt containing vitamin. The central cobalt atom (magenta) is coordinated by a corrin ring (blue) with 7 residues (R = acetyl or propionylamide) (red). Only the upper axial position (L) (green) can be exchanged for different ligands: the physiological ligands 5 -desoxyadenosyl-Cbl or methyl-Cbl, which are needed for two different biochemical reactions, or the pharmacologically administered forms of B 12 cyano-Cbl (more in USA) or hydroxy-Cbl (more in Europe). The unique characteristic of B 12 is that a single cobalt atom is bound in the center of a ring of four pyrroles (corrin) similar to the Fe atom bound in the center of the heme ring of hemoglobin The only difference between the tetrapyrrolic ring of corrins and heme is that the A and D pyrroles are directly bound in corrins, while in heme, the four rings are all bound via a carbon bridge.

Vitamin B 12 Sources, Physiological Uptake and Causes of Deficiency
B 12 is not present in foods obtained from plants, and since it cannot be produced by humans, it needs to be obtained from a diet containing products derived from animals, such as meat, dairy products and particularly liver, which is the most important B 12 storage organ (80% of total storage) in mammals. The recommended daily requirement depends on gender. Adult women require 2.4 µg of B 12 per day. This requirement increases to 2.8 µg of B 12 per day during pregnancy and breastfeeding. Adult men can meet their needs by consuming 2.6 µg of B 12 per day. Lesser amounts are recommended for young infants [13]. The daily turnover of B 12 is less than 0.1%. Deficiencies occur when supplies are reduced to 300 µg of B 12 per day. As mentioned above, B 12 deficiency may be easily overlooked: the clinical signs of deficiency may develop slowly because in humans, B 12 storage ranging from 2 to 5 mg may last for more than a year.
The sequential stages of uptake of B 12 (Table 1, left column) [3,14] are as follows: (1) The first step is dietary intake of free or protein-bound B 12 . (2) In the stomach gastric cells release proteases, e.g., pepsin and HCl, and in this acidic milieu, protein-bound B 12 is released by protein digestion and free B 12 is bound by haptocorrin. This complex and intrinsic factor (IF) secreted by parietal cells are transferred to the duodenum. (3) In the duodenum, haptocorrin-bound B 12 (also named "transcobalamin I") is released from haptocorrin by proteolytic digestion, and the released B 12 binds to IF with high affinity. (4) The B 12 -IF complex is bound and taken up by specific mucosal receptors in the distal ileum. (5) After internalization, B 12 is released and free B 12 is secreted into the blood, bound to transcobalamin and transported to peripheral organs/targets. (6) B 12 bound to transcobalamin II (HoloTC) is taken up via the ubiquitous receptor CD 320, and B 12 is liberated in the lysosomes. Note that only about 10-20% of B 12 is bound to transcobalamin II (HoloTC) in the blood, while the majority of B 12 (ca. 80%) is bound to transcobalamin I. The latter complex cannot be taken up by peripheral cells. Therefore, determination of HoloTC is superior to the traditional determination of total B 12 , i.e., B 12 bound to both transcobalamin II and transcobalamin I (see Section 4.1). Table 1. Stages of B 12 absorption and metabolism and possible defects leading to B 12 deficiency.

Stages of B 12 Metabolism
Defects/Causes of B12 Deficiency 1. Dietary uptake: B 12 present in free or protein-bound form is taken up in the diet or as a drug.

Binding of B 12 by IF in the duodenum:
In the duodenum, the B 12 -haptocorrin complex is digested by proteases, and the released B 12 is bound by IF with high affinity.

Absorption in ileum:
The B 12 -IF complex is bound to and taken up by specific mucosal receptors in the terminal ileum.

Blood-borne transport by transcobalamins:
After internalization by the enterocytes, B 12 is exported into the blood and subsequently bound to transcobalamins (TC). The complex with holotranscobalamin (HoloTC) carries 10-20% of total B 12 . HoloTC is the circulating form of B 12 which can be taken up by the target cell via the ubiquitous receptor CD 320. The majority of B 12 is transported by TC I, but this B 12 complex cannot be taken up by peripheral cells.
Congenital defects (very rare in adults)

Intracellular transport and lysosomal metabolism:
The internalized complex is transported to the lysosomes and degraded, thereby liberating B 12 . Free intracellular B 12 can be metabolized into methylcobalamin or adenosylcobalamin, both being cofactors, the first for methionine synthase and the second for methylmalonyl-CoA mutase.

Congenital defects (very rare in adults)
Possible defects that may lead to B 12 deficiency are listed in the right column of Table 1 [1][2][3]. (1) Inadequate B 12 supply, either due to a strict vegan diet or eating disorders, may cause B 12 deficiency. (2) Reduced acidification in the stomach, due to aging or drugs (Proton Pump Inhibitors or H 2 -Receptor antagonists) may lead to reduced release of B 12 . The term "Food cobalamin malabsorption" is used to describe these cases of B 12 deficiency when normal amounts of B 12 are ingested with food but the vitamin cannot be released from dietary proteins. These individuals can absorb B 12 from supplements in which the vitamin is not protein-bound or when B 12 is administered as a drug. Food cobalamin malabsorption is common among older people, in whom the resulting B 12 deficiency is, in many cases, "subclinical", i.e., featuring no clinical signs, such as megaloblastic anemia. (3) One of the most common causes is an insufficiency of the parietal cells, which may have been destroyed by autoimmune mechanisms or resected by gastrectomy. This autoimmunity can be diagnosed by detecting autoantibodies against parietal cell and/or IF. For the detection of such atrophic gastritis, anti-IF antibodies are more specific than anti-parietal cell antibodies (100% vs. 90%), but their sensitivity is much lower (37% vs. 81%) [15]. (4) An important cause of reduced resorption of B 12 are forms of malabsorption which may be caused by surgery or inflammatory bowel diseases (M. Crohn or Colitis ulcerosa) involving the distal ileum. Furthermore, numerous observational and interventional studies, including meta-analyses, indicate that the use of metformin may reduce B 12 bioavailability [9,[16][17][18][19][20][21][22]. The strongest evidence comes from a randomized clinical trial reported by De Jager et al. [23]. Although there are indications that metformin reduces the B 12 uptake in the terminal ileum [16,20,22], the exact molecular mechanism by which chronic metformin treatment may cause B 12 deficiency remains unclear [9,16,17,24,25]. Different mechanisms have been suggested: (i) Metformin interferes with the absorption of B 12 by impairing the calcium-dependent binding of the IF-B 12 complex to the cubilin receptor on enterocytes. (ii) Metformin might enhance hepatic B 12 accumulation, thereby altering B 12 tissue distribution and metabolism. (iii) Metformin may interfere with the reabsorption of bile acids in the enterohepatic circulation because some B 12 is excreted in bile and may not be reabsorbed under this condition [26]. Considering that nearly all diabetic patients take metformin and many also proton pump inhibitors (mostly for gastric protection because of concomitant use of aspirin or other antiplatelets for cardiovascular complications of diabetes), it is not surprising that B 12 deficiency is common in diabetic patients. (5) Congenital defects of transport proteins (transcobalamins) or (6) congenital defects of intracellular transport or processing may cause B 12 deficiency with the respective clinical signs.

Intracellular Processing and Reduction/Oxidation Function of B 12
The sophisticated intracellular transport and processing of B 12 deserves detailed attention. Not only because multiple (but rare) inherited defects have been found causing various forms of B 12 deficiency, as extensively reviewed by Froese DS et al. [27], but also because these results have elucidated the properties of B 12 acting as redox system: B 12 bound to HoloTC is taken up by receptor mediated endocytosis. This complex is then directed to the lysosomes, degraded, and free B 12 is then transported into the cytosol ( Figure 2) [28]. Within the cytosol, B 12 is processed by several proteins and the central cobalt is reduced to Co 2+ to facilitate the exchange of the axial ligand, e.g., hydroxy-or methyl-B 12 , for the appropriate ligands needed [27]. The final B 12 -protein complex is then targeted to the cytosolic methionine synthase and the active form, methyl-B 12 , is formed by reduction of the central cobalt of B 12 to Co 1+ . Methyl-B 12 presents the active cofactor of methionine synthase. The physiological function of B 12 in methionine synthase is discussed below.
If bound to other protein(s) containing a mitochondrial leader sequence, B 12 is targeted to the mitochondria where the methylmalonyl-CoA mutase resides [29]. In the mitochondria, B 12 is further processed by adenosyl transferase (ATR), an enzyme catalyzing the ATP-dependent synthesis of adenosyl-B 12 (Co 1+ ), which is then transferred to methylmalonyl-CoA mutase (MUT), yielding the active enzyme ( Figure 2) [30,31]. Together, these data show that B 12 can be processed and cycled to different oxidation states, i.e., with Co 1+ , Co 2+ or Co 3+ , in the cytosol and mitochondria as well. A comprehensive overview of the clinical characteristics, treatments and outcomes of nutritional and acquired B 12 deficiencies, impairments in B 12 absorption and intracellular trafficking has been previously published [32]. After degradation of the transport protein, free Cbl is transported into the cytosol where Cbl x+ is "denuded" from the ligand by the β-ligand transferase activity, thus being converted to Cbl 2+ and entering the branching point: (i) either it remains in the cytosol and enters the methionine cycle for the synthesis of methionine, as outlined in the figure, undergoing reduction and oxidation reactions from 3+ to 1+ and vice versa, or (ii) B12 is transported into the mitochondria by a still unknown way to serve as cofactor of methylmalonyl mutase (MUT). After binding to adenosyl transferase (ATR) (blue rhombus), Cbl 2+ is reduced to Cbl 1+ (step 1, yellow stars). In a second step (yellow star 2), by using ATP, Cbl is adenosylated (Ado-Co 3+ ) and triphosphate (PPPi) is liberated. This complex can transfer adenosylated Cbl to apo MUT (grey pentagon) (step 3). The MUT loaded by adenosylated Cbl 3+ can catalyze the conversion of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA, which may be metabolized further via the tricarbonic acid cycle. The Cbl off-loaded ATR binds ATP and is regenerated by taking up Cbl 2+ from inactive MUT (step 4). The reaction schemes indicate the reduction/oxidation versatility of B12. Several auxiliary steps are omi ed for clarity.

Physiological Functions of Vitamin B12
The first strictly B12-dependent enzyme is methionine synthase, which is essential for the formation of methionine. This enzyme catalyzes the transfer of the methyl unit from methyl-tetrahydrofolate (methyl-THF) to homocysteine, yielding methionine via a two-step reaction ( Figure 3). In the first step, the methyl group from methyl-THF is transferred to B12, thus forming methyl-B12 and releasing THF. In the second step, the methyl group from methyl-B12 is transferred to homocysteine, yielding methionine. In turn, methionine transfers its methyl-unit via the S-adenosylmethionine/S-adenosylhomocysteine cycle to numerous metabolites and macromolecules for methylation of, e.g., lipids, proteins and DNA. Methylation of DNA is important for epigenetic regulation of gene expression. Methyl-B12 receives its methyl-unit from methyl-THF provided from methylene-THF in the folate cycle, indicating that both the cobalamin and the folate metabolism are tightly interrelated. The folate cycle provides methylene-THF for the synthesis of thymidine. Since B12 deficiency leads to impaired conversion of homocysteine to methionine, elevated plasma homocysteine levels may indicate functional B12 deficiency. Since the oxidation state of B 12 /Cbl is dependent on the ligand, -CN, -OH or -CH 3 , the oxidation state is indicated by (x+). After degradation of the transport protein, free Cbl is transported into the cytosol where Cbl x+ is "denuded" from the ligand by the β-ligand transferase activity, thus being converted to Cbl 2+ and entering the branching point: (i) either it remains in the cytosol and enters the methionine cycle for the synthesis of methionine, as outlined in the figure, undergoing reduction and oxidation reactions from 3+ to 1+ and vice versa, or (ii) B 12 is transported into the mitochondria by a still unknown way to serve as cofactor of methylmalonyl mutase (MUT). After binding to adenosyl transferase (ATR) (blue rhombus), Cbl 2+ is reduced to Cbl 1+ (step 1, yellow stars). In a second step (yellow star 2), by using ATP, Cbl is adenosylated (Ado-Co 3+ ) and triphosphate (PPPi) is liberated. This complex can transfer adenosylated Cbl to apo MUT (grey pentagon) (step 3). The MUT loaded by adenosylated Cbl 3+ can catalyze the conversion of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA, which may be metabolized further via the tricarbonic acid cycle. The Cbl off-loaded ATR binds ATP and is regenerated by taking up Cbl 2+ from inactive MUT (step 4). The reaction schemes indicate the reduction/oxidation versatility of B 12 . Several auxiliary steps are omitted for clarity.

Physiological Functions of Vitamin B 12
The first strictly B 12 -dependent enzyme is methionine synthase, which is essential for the formation of methionine. This enzyme catalyzes the transfer of the methyl unit from methyl-tetrahydrofolate (methyl-THF) to homocysteine, yielding methionine via a two-step reaction ( Figure 3). In the first step, the methyl group from methyl-THF is transferred to B 12 , thus forming methyl-B 12 and releasing THF. In the second step, the methyl group from methyl-B 12 is transferred to homocysteine, yielding methionine. In turn, methionine transfers its methyl-unit via the S-adenosylmethionine/S-adenosylhomocysteine cycle to numerous metabolites and macromolecules for methylation of, e.g., lipids, proteins and DNA. Methylation of DNA is important for epigenetic regulation of gene expression. Methyl-B 12 receives its methyl-unit from methyl-THF provided from methylene-THF in the folate cycle, indicating that both the cobalamin and the folate metabolism are tightly interrelated. The folate cycle provides methylene-THF for the synthesis of thymidine. Since B 12 deficiency leads to impaired conversion of homocysteine to methionine, elevated plasma homocysteine levels may indicate functional B 12 deficiency. In the first step catalyzed by serine hydroxymethyltransferase (SHMT), a one-carbon unit is transferred from serine to tetrahydrofolate (THF), yielding 5,10-methylentetrahydrofolate which is subsequently converted to methyl-THF by methylentetrahydrofolate-reductase (MTHFR). This reaction is irreversible. The one-carbon (methylen-or methyl-) units are highlighted in green, enzymes in red and cobalamin (Cbl) in magenta. Starting from methyl-THF to the left, the methyl unit is transferred to Cbl to form methyl-Cbl, which is further transferred to homocysteine, yielding methionine. Both steps are catalyzed by methionine synthase. Together, the methyl-group is transferred from methyl-THF to methionine via a methyl transfer chain which is strictly dependent on B12. Methionine serves as an important and universal donor for methyl units via the S-adenosylmethionine (SAMet)/adenosylhomocysteine (SAHcys) cycle for various acceptors (e.g., lipids, DNA etc.). On the other side, methylene-THF serves as a one-carbon donor for the synthesis of deoxythymidine-monophosphate (dTMP) from deoxyuracil-monophosphate (dUMP) catalyzed by thymidylate synthase. Thymidine is an absolute essential building block for the synthesis of DNA. In case of severe B12 deficiency, methyl-THF accumulates and the folate cycle is blocked. Thus, B12 deficiency may lead to a secondary and functional folate deficiency ("folate trap"). DHF indicates dihydrofolate.
The second strictly B12-dependent human enzyme is methylmalonyl-CoA mutase, which is essential for the conversion of methylmalonyl-CoA to succinyl-CoA and subsequently to succinate, a common intermediate of the tricarboxylic acid cycle. This enzyme is at the end of an important biochemical degradation chain of propionyl-CoA arising during the catabolism of the amino acids methionine, isoleucine, threonine and valine, and odd chain fatty acids. After carboxylation by propionyl-CoA carboxylase, methylmalonyl-CoA is formed, which is the substrate for methylmalonyl-CoA mutase. This strictly B12-dependent enzyme contains one mol of adenosyl-B12 per subunit [12] and is located in the mitochondrial matrix. If the activity of methylmalonyl-CoA mutase is absent or decreased, e.g., due to hereditary defects of the apoprotein or to B12 deficiency, toxic methylmalonic acid (MMA) is formed and the excess of methylmalonic acid is excreted in the urine. The full clinical expression of methylmalonyl-CoA mutase deficiency is seen in the "inherited methylmalonic aciduria" of newborns. In any case, elevated urinary excretion or elevated serum levels of methylmalonic acid serve as specific laboratory biomarkers for functional B12 deficiency (see Section 4.1).

Clinical Pathophysiology of B12 Deficiency
B12 deficiency may manifest with hematological abnormalities and neurological symptoms. The classic hematological abnormality in B12 deficiency is megaloblastic anemia, possibly accompanied by leukocytopenia and/or thrombocytopenia. All of them can be fully explained from the physiologic role of B12 as a cofactor of methionine synthetase. In the absence of sufficient B12, methyl-THF accumulates (Figure 3) in the cytosol (where methionine In the first step catalyzed by serine hydroxymethyltransferase (SHMT), a one-carbon unit is transferred from serine to tetrahydrofolate (THF), yielding 5,10-methylentetrahydrofolate which is subsequently converted to methyl-THF by methylentetrahydrofolate-reductase (MTHFR). This reaction is irreversible. The one-carbon (methylen-or methyl-) units are highlighted in green, enzymes in red and cobalamin (Cbl) in magenta. Starting from methyl-THF to the left, the methyl unit is transferred to Cbl to form methyl-Cbl, which is further transferred to homocysteine, yielding methionine. Both steps are catalyzed by methionine synthase. Together, the methyl-group is transferred from methyl-THF to methionine via a methyl transfer chain which is strictly dependent on B 12 . Methionine serves as an important and universal donor for methyl units via the S-adenosylmethionine (SAMet)/adenosylhomocysteine (SAHcys) cycle for various acceptors (e.g., lipids, DNA etc.). On the other side, methylene-THF serves as a one-carbon donor for the synthesis of deoxythymidine-monophosphate (dTMP) from deoxyuracil-monophosphate (dUMP) catalyzed by thymidylate synthase. Thymidine is an absolute essential building block for the synthesis of DNA. In case of severe B 12 deficiency, methyl-THF accumulates and the folate cycle is blocked. Thus, B 12 deficiency may lead to a secondary and functional folate deficiency ("folate trap"). DHF indicates dihydrofolate.
The second strictly B 12 -dependent human enzyme is methylmalonyl-CoA mutase, which is essential for the conversion of methylmalonyl-CoA to succinyl-CoA and subsequently to succinate, a common intermediate of the tricarboxylic acid cycle. This enzyme is at the end of an important biochemical degradation chain of propionyl-CoA arising during the catabolism of the amino acids methionine, isoleucine, threonine and valine, and odd chain fatty acids. After carboxylation by propionyl-CoA carboxylase, methylmalonyl-CoA is formed, which is the substrate for methylmalonyl-CoA mutase. This strictly B 12dependent enzyme contains one mol of adenosyl-B 12 per subunit [12] and is located in the mitochondrial matrix. If the activity of methylmalonyl-CoA mutase is absent or decreased, e.g., due to hereditary defects of the apoprotein or to B 12 deficiency, toxic methylmalonic acid (MMA) is formed and the excess of methylmalonic acid is excreted in the urine. The full clinical expression of methylmalonyl-CoA mutase deficiency is seen in the "inherited methylmalonic aciduria" of newborns. In any case, elevated urinary excretion or elevated serum levels of methylmalonic acid serve as specific laboratory biomarkers for functional B 12 deficiency (see Section 4.1).

Clinical Pathophysiology of B 12 Deficiency
B 12 deficiency may manifest with hematological abnormalities and neurological symptoms. The classic hematological abnormality in B 12 deficiency is megaloblastic anemia, possibly accompanied by leukocytopenia and/or thrombocytopenia. All of them can be fully explained from the physiologic role of B 12 as a cofactor of methionine synthetase. In the absence of sufficient B 12 , methyl-THF accumulates (Figure 3) in the cytosol (where methionine synthetase resides) and subsequently in the nucleus. Palmer et al. [33] showed that cytosolic B 12 depletion causes nuclear 5-methyl-THF accumulation. Methyl-THF cannot be converted back to methylene-THF because this reaction is not reversible (Figure 3). In this way, the folate cycle is blocked and a functional folate deficiency develops ("folate trap") [3,34]. The reduced methylene-THF leads to impaired thymidine biosynthesis. Thymidine is an absolute essential building block for the synthesis of DNA. A shortage of thymidine leads to genome instability and a slow-down or even blockade in cell proliferation, which is most obvious in fast dividing cells such as bone marrow cells. This mechanism represents the background for the evolution of both megaloblastic/pernicious anemia and leukocytopenia and thrombocytopenia.
In contrast to the hematological abnormalities, the neuropsychiatric symptoms of B 12 deficiency cannot be fully explained by the function of B 12 as a cofactor of either of the two above-mentioned enzymes. Previous reports suggest that B 12 deficiency-associated dysfunction of methionine synthase reduces the availability of methionine and subsequently S-adenosyl-methionine, which is an important methyl group donor ( Figure 2). Transfer of methyl groups to various metabolites such as lipids, proteins (e.g., histones) and nucleic acids (e.g., DNA) has been described for more than 100 biochemical reactions [35]. The neurological symptoms would then result from reduced methylation of neuronal lipids and neuronal proteins, such as myelin basic protein, which makes up approximately one-third of the myelin of peripheral nerves and the spinal cord [36][37][38]. However, this assumption is actually far from proven [39]. Reduced genomic DNA methylation leading to defects in gene regulation was found in diabetic neuropathy (see Section 3.2). An even less likely theory suggests dysfunction of methylmalonyl-CoA mutase, which as an important detoxifying enzyme, is highly expressed in most tissues, including the nervous system [40]. Accumulation of MMA in the CNS would then be responsible for some of the neurological or psychiatric symptoms of B 12 deficiency. In this respect, the new findings about the intracellular redox cycle of B 12 (see Section 2.2) and its possible action as a cellular antioxidant (see Section 3.3) may provide a better explanation for the neurological symptoms of B 12 deficiency than its role as cofactor of the two enzymes [41,42].

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Biochemical pathways leading to diabetic neuropathy; • Common pathophysiological pathways of B12 deficiency-induced neuropathy and diabetic neuropathy; • B12 may act as an intracellular antioxidant.
As mentioned before, the neurological symptoms of B 12 deficiency are similar to those arising in the course of diabetic neuropathy. If any, the only difference is that sole sensory neuropathy occurs more frequently in B 12 deficiency. However, B 12 deficiency may also cause motoric or painful neuropathy, symptoms that are common in DPN. The most prevalent forms of diabetic neuropathy are peripheral, autonomic and painful neuropathy. Notably, in 10-18% of patients, nerve damage is already present at the time of diagnosis of diabetes, and neuropathy has been shown to occur even in the prediabetic state [5,43,44], which suggests that there may also be other factors contributing to neuropathy besides glucose levels or the diabetes state itself. Since B 12 deficiency is quite common in type 2 diabetic patients (see Section 2.1) and since B 12 deficiency causes neurological symptoms similar to DPN, B 12 deficiency may cause the neuropathy occurring during the course of diabetes or just accelerate the progression of DPN [9]. In this case, B 12 supplementation may help to prevent or reduce the development of DPN.

Biochemical Pathways Leading to Diabetic Neuropathy
Numerous molecular mechanisms leading to diabetic autonomous and sensory peripheral neuropathy have been suggested. The respective literature was recently re-viewed [4,5,45]. The proposed pathomechanisms include the hyperglycemia-induced elevated sorbitol and glucosamine pathway, dysregulated glycolysis, and mitochondrial oxidative phosphorylation in diabetic nerve tissues leading to mitochondrial dysfunction and oxidative stress [46]. Numerous reports indicate an increased presence of reactive oxygen species (ROS) in DPN [47][48][49][50].
Similar to the formation of HbA1c, chronic hyperglycemia also increases the glycation of other body proteins. Long-living proteins, however, undergo further "aging", yielding numerous protein-bound products called AGE. These elevated AGE lead to an increased binding to and stimulation of the specific receptor for AGE (RAGE), thereby activating inflammatory pathways. Earlier studies showed that deletion of RAGE improved pain perception in diabetic mice [51]. This group and others have shown that the High Mobility Globulin Box 1 Protein (HMGB1) is involved in the activation of RAGE in the RAGE pathway [1,2,52]. Recent studies illustrated roles for HMGB1 in painful DPN as treatment of diabetic mice or rats with the HMGB1 inhibitor glycyrrhizin improved mechanical and thermal pain thresholds [53,54]. Together, the suggested elevated glucose-induced mechanisms finally enhance inflammatory signal pathways and elevate oxidative stress mediated by ROS in neural cells [47].

B 12 Deficiency-Induced Neuropathy and Diabetic Neuropathy Share Common Pathophysiological Pathways
While these glucocentric pathways are all based on elevated glucose levels and enhanced glucose metabolism, they do not fully explain why neuropathy may occur very early in the course of diabetes or in the prediabetic stage. There are some data suggesting that B 12 deficiency may be a significant contributor. Straightforward evidence for a causal involvement of insufficient B 12 supply in DPN comes from experimental animals [55]. This group has generated a mouse lacking the Cd320 receptor, which is responsible for the cellular uptake of HoloTC. These mice develop B 12 deficiency in the nervous system and mild anemia. The structural pathology in the spinal cord presented as loss of myelin in the axonal tracts with inflammation. The sciatic nerve showed increased nonuniform, internodal segments, suggesting demyelination and remyelination in progress. Consistent with these changes, the Cd320 −/− mouse showed an increased latency to thermal nociception. Further, a lower amplitude of compound action potential in the sural nerve suggested that the functional capacity of the heavily myelinated axons was preferentially compromised, leading to loss of peripheral sensation. These changes resemble the clinical phenotype seen in human DPN, thus providing a strong argument that neural tissue B 12 deficiency may be causally involved in the development of DPN.
There are also human data indicating a significant contribution of B 12 in DPN. First, treatment with B 12 showed positive effects on DPN in several clinical trials [10,[56][57][58][59][60][61][62][63][64][65][66][67], but not in all. For instance, one recent review found no evidence that the use of oral B 12 supplements is associated with improvement in the clinical symptoms or in the electrophysiological markers of diabetic neuropathy [63]. However, this review comprised only 4 studies with 363 patients. Second, recent studies point to common pathology/pathophysiological mechanisms. For instance, a recent report shows a low level of genomic DNA methylation in DPN of type 2 diabetic patients but not in other complications of diabetes, such as diabetic retinopathy or nephropathy, indicating that a reduced level of genomic DNA methylation is a relatively specific risk factor for DPN [68]. Similarly, when investigating DNA methylation and gene expression in human sural nerve biopsies, Guo et al. found that DNA methylation is a mechanism in the regulation of gene expression in DPN [69,70]. Considering that reduced DNA-methylation can be one of the consequences of B12-deficiency (see Section 2.4), these data provide a mechanistic explanation of how B12-deficiency may be involved in the pathogenesis of DPN. However, to date, a precise molecular mechanism causally linking impaired methylation to DPN has not been shown. In addition, the influence of B12 treatment on the DNA methylation pattern and subsequent biological alterations has not been studied in humans yet.
Even stronger support for an important role of B 12 deficiency in neuropathy is provided by a series of immunohistochemical studies. Haslbeck and coworkers investigated the presence of ROS and inflammatory markers in sural nerve biopsies obtained from patients with diabetic or other causes of neuropathy. They found elevated ROS biomarkers and an AGE-mediated activation of proinflammatory pathways, as indicated by an activation of the AGE-RAGE-NFκBp65 pathway in DPN [71]. In most cases, staining of proinflammatory and ROS biomarkers were colocalized in epineural vessels, perineural cells and in Schwann cells. In patients with impaired glucose tolerance and DPN, the staining patterns of sural nerves were similar but less intense compared with those found in patients with overt diabetes, indicating that ROS production and proinflammatory pathways are activated even before chronic hyperglycemia occurs [42]. To evaluate the specificity of the effects, different forms of peripheral neuropathies were studied. The authors observed a striking similarity between diabetic and B 12 deficiency-induced neuropathy, while the staining patterns were different or absent in sural nerve biopsies from patients with other neuropathies (monoclonal gammopathy of unknown significance, Charcot-Marie-Tooth disease, idiopathic peripheral neuropathy and alcohol abuse-induced neuropathy), indicating that the pathogenesis of these neuropathies may be different [41]. Further evidence that B 12 deficiency may lead to mitochondrial dysfunction and oxidative stress is provided by an extensive investigation by Luciani et al. [72]. They found that reduced methylmalonyl-CoA mutase activity and subsequently elevated cellular MMA levels cause extensive mitochondrial dysfunction, including abnormal mitochondrial networks, dysfunctional bioenergetics and increased oxidative stress, as shown by elevated ROS production. Treatment with two different mitochondria-targeted antioxidants normalized mitochondrial ROS production, dysfunction and structural alterations. This finding suggests that reduced B 12 bioavailability may lead to reduced mitochondrial methylmalonyl-CoA mutase activity and increased oxidative stress and that specific treatment may rescue mitochondrial integrity. Together, the present data suggest that the development of DPN is due to mitochondrial dysfunction leading to increased local ROS production and that this derangement can possibly be prevented with suitable drug treatment.

Evidence for a Role of B 12 Acting as an Intracellular Antioxidant
While the molecular mechanisms of the essential need of B 12 for the activity of methylmalonyl-CoA mutase and methionine synthase are broadly documented, there is little information on the molecular properties allowing B 12 to serve as an antioxidant. As previously reviewed, the cobalt oxidation state in the center of B 12 can vary substantially during B 12 trafficking and processing in the cytosol and the mitochondria (Figure 2) [12]. In vitro studies showed that B 12 exerts superoxide dismutase activity, i.e., B 12 can react with superoxide (O 2 − ), producing H 2 O 2 at high rates approaching the reaction rates of the physiological defense enzyme superoxide dismutase [73]. In this reaction, superoxide oxidates Co 1+ to Co 3+ , which can be reduced back to Co 2+ in the cellular environment; thus, a catalytic cycle is formed. In human aortic endothelial cells, this group showed that B 12 protects against superoxide-induced cell injury [74]. Recent in vitro and in vivo studies indicate that B 12 may function as an endogenous antioxidant in neuronal cells [75], thus supporting a novel role for B 12 acting as an intracellular antioxidant [76].
In a previous report, the current knowledge of human studies supporting the antioxidative potential of B 12 was systematically reviewed [77]. Six studies found evidence for antioxidative properties of B 12 , the results of four studies remained unclear, and the results of one study did not support any antioxidative properties of B 12 . The potential antioxidant properties may act at several stages: (i) by directly scavenging ROS, (ii) indirectly by preserving high glutathione levels, (iii) by reducing oxidative stress induced by metabolic pathways, and (iv) by modulation of cytokine production. Possibly, the suggested pathways may act, at least in part, in common. In a diabetic rat model for DPN, Mizukami et al. found that treatment with methyl-B 12 prevented activation of protein kinase C in nerve tissue and reduced the diabetes-induced increased number of 8-oxo guanosine positive cells in the endoneurium [78]. Since 8-oxo guanosine is an integrative marker for oxidative stress, these results indicate that administered pharmacological B 12 preparations may help to prevent the diabetes-induced increase in ROS in peripheral nerves.
Taken together, these findings suggest a causal role for B 12 deficiency in different forms of neuropathies including DPN, very likely through mitochondrial dysfunction and oxidative stress/ROS formation, and support the possibility that B 12 acts as a cellular antioxidant in subclinical forms of functional B 12 deficiency, independent of the classical mechanisms of B 12 serving as a cofactor. This notion is supported by the fact that B 12 supplementation may improve clinical symptoms regardless of the actual serum B 12 status [79]. Wolffenbuttel et al. suggest that "B 12 may not be only a vitamin in the general sense, but rather a general nerve protecting and nerve regenerating factor particularly in DPN" [79]. However, although numerous aspects indicate the beneficial action of B 12 for the prevention or treatment of (sub-)clinical DPN, there are no reports in the literature unequivocally linking B 12 treatment with its beneficial effects on DPN on a molecular basis. Therefore, concrete evidence, particularly in humans, is still lacking.

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Biomarkers of B 12 deficiency; • Laboratory biomarkers and decision limits for B 12 deficiency in the general population; • B 12 deficiency in the elderly population: special considerations and decision limits.

Biomarkers of B 12 Deficiency
Because, as mentioned above, clinical symptoms and signs may be subtle or absent in cases of subclinical B 12 deficiency, the diagnosis has to be supported by determination of laboratory biomarkers [14,79]. The following parameters are available for assessing B 12 adequacy or deficiency: serum (total) B 12 , HoloTC, homocysteine (HCys) (Figure 3), and methylmalonic acid (MMA) (Figure 2). Serum B 12 and HoloTC concentrations are direct biomarkers for the estimation of the body's B 12 supply. Among them, the determination of B 12 serum concentration is most widely used in routine clinical practice to document the presence of B 12 deficiency. However, concentrations of total serum B 12 do not adequately mirror the actual supply of B 12 in the human body. Rather HoloTC, which reflects the biological form of the body's B 12 supply, is the most sensitive biomarker for early B 12 deficiency [80]. One study using receiver operator curves (ROC) for the comparison of the two biomarkers determined in 2403 individuals found that that HoloTC was superior compared with conventional B 12 measurements for diagnosing B 12 deficiency [81]. HoloTC had a greater area under the ROC compared with serum B 12 concentrations (0.85 vs. 0.76) in all participants. Similarly, Gwathmay and Grogan [82] report that HoloTC is a more sensitive biomarker for B 12 deficiency. In another study, Herrmann et al. [83] found values of 0.879 vs. 0.836 for HoloTC and total B 12 , respectively, supporting the notion that HoloTC is modestly superior compared with serum B 12 concentrations. However, for the sake of completeness, we also note that there is also a single reference that HoloTC is not superior to total B 12 [84].
An increase in the metabolites homocysteine (HCys) (Figure 3) and methylmalonic acid (MMA) (Figure 2) is considered to mirror cellular deficiency of B 12 , also termed "functional B 12 deficiency". Nevertheless, they seem not to be valuable as screening tests. For instance, in the aforementioned study of Herrmann et al. [83], which included 111 vegetarian subjects, HoloTC was found to be the earliest biomarker for B 12 deficiency followed by MMA, and serum B 12 was the latest biomarker. Most importantly, however, mainly MMA and, to a lesser degree, HCys concentrations were influenced by renal function. In a cohort of 1143 apparently healthy elderly Swiss participants >60 years, HCys and MMA levels were influenced by renal function as estimated by eGFR [85]. In this extensive study, the authors found an age-dependent increase in HCys and MMA concentrations, which were inversely related to eGFR, indicating that not age per se, but rather the age-dependent decrease in GFR is responsible for the increase in MMA and HCys when GFR is below 36 mL/min. Notably, both B12 and HoloTC were unrelated to eGFR. Taken together, the present data suggest that HoloTC is a more sensitive biomarker for the detection of insufficient B 12 supply than any other biomarker and that modestly decreased HoloTC may also indicate the presence of early cellular or functional B 12 deficiency. MMA and HCys levels can also be useful for differentiation of B 12 from folate deficiency: normal MMA and increased homocysteine levels are consistent with folate deficiency, and conversely, increased MMA levels without elevated folate levels indicate B 12 deficiency.

Laboratory Biomarkers for B 12 Deficiency in the General Population and Decision Limits
From the previous discussion on the available biomarkers (Section 4.1) it becomes apparent that no single biomarker is capable of covering all possible aspects of B 12 deficiency (screening, subclinical/cellular deficiency, clinically overt deficiency). Furthermore, there is no consensus in the literature with regard to the cut-offs for each of the abovementioned biomarkers. Reviewing 69 publications on cut-off points for the diagnosis of B 12 deficiency in the general population [86], the authors report on broad ranges of decision limits: 100-350 pmol/L for serum total B 12 ; 20-50 pmol/L for HoloTC; 0.21-0.47 µmol/L for methylmalonic acid; and 10-21.6 µmol/L for homocysteine. These authors conclude that it is necessary to establish different reference cut-offs according to age and the analytical method used. It is, therefore, not surprising that for the screening of B 12 deficiency, numerous algorithms have been developed [1,87].
We suggest here an algorithm for practical application based on a suggestion of Herrmann et al. (modified from [88], Figure 4). Inevitably, the diagnosis of B 12 deficiency is probabilistic, i.e., "(very) likely", "possible" or "unlikely". Because HoloTC is the most specific and most sensitive laboratory biomarker for the detection of B 12 deficiency, HoloTC should be used for screening. If serum levels of HoloTC are above 50 pmol/L, B 12 deficiency is very unlikely. If serum levels of HoloTC are below 35 pmol/L and MMA levels are above 271 nmol/L, B 12 deficiency is likely. If serum levels of HoloTC are below 35 pmol/L and MMA levels are below 271 nmol/L, a negative B 12 balance is likely, i.e., there is no cellular B 12 deficiency but an insufficient B 12 supply. If serum levels of HoloTC fall into the range of 35-49 pmol/L, MMA values above 271 nmol/L suggest B 12 deficiency, and if they are below 271 nmol/L B 12 , deficiency is unlikely. In any case renal dysfunction should be excluded.
responsible for the increase in MMA and HCys when GFR is below 36 mL/min. Notably, both B12 and HoloTC were unrelated to eGFR. Taken together, the present data suggest that HoloTC is a more sensitive biomarker for the detection of insufficient B12 supply than any other biomarker and that modestly decreased HoloTC may also indicate the presence of early cellular or functional B12 deficiency. MMA and HCys levels can also be useful for differentiation of B12 from folate deficiency: normal MMA and increased homocysteine levels are consistent with folate deficiency, and conversely, increased MMA levels without elevated folate levels indicate B12 deficiency.

Laboratory Biomarkers for B12 Deficiency in the General Population and Decision Limits
From the previous discussion on the available biomarkers (Section 4.1) it becomes apparent that no single biomarker is capable of covering all possible aspects of B12 deficiency (screening, subclinical/cellular deficiency, clinically overt deficiency). Furthermore, there is no consensus in the literature with regard to the cut-offs for each of the above-mentioned biomarkers. Reviewing 69 publications on cut-off points for the diagnosis of B12 deficiency in the general population [86], the authors report on broad ranges of decision limits: 100-350 pmol/L for serum total B12; 20-50 pmol/L for HoloTC; 0.21-0.47 µmol/L for methylmalonic acid; and 10-21.6 µmol/L for homocysteine. These authors conclude that it is necessary to establish different reference cut-offs according to age and the analytical method used. It is, therefore, not surprising that for the screening of B12 deficiency, numerous algorithms have been developed [1,87].
We suggest here an algorithm for practical application based on a suggestion of Herrmann et al. (modified from [88], Figure 4). Inevitably, the diagnosis of B12 deficiency is probabilistic, i.e., "(very) likely", "possible" or "unlikely". Because HoloTC is the most specific and most sensitive laboratory biomarker for the detection of B12 deficiency, HoloTC should be used for screening. If serum levels of HoloTC are above 50 pmol/L, B12 deficiency is very unlikely. If serum levels of HoloTC are below 35 pmol/L and MMA levels are above 271 nmol/L, B12 deficiency is likely. If serum levels of HoloTC are below 35 pmol/L and MMA levels are below 271 nmol/L, a negative B12 balance is likely, i.e., there is no cellular B12 deficiency but an insufficient B12 supply. If serum levels of HoloTC fall into the range of 35-49 pmol/L, MMA values above 271 nmol/L suggest B12 deficiency, and if they are below 271 nmol/L B12, deficiency is unlikely. In any case renal dysfunction should be excluded. . Algorithm for the detection of B12 deficiency. An algorithm for the detection of B12 deficiency is shown (adapted and modified from Herrmann and Obeid [88]). This algorithm suggests that HoloTC be used for screening of B12 deficiency. For further differentiation, determination of serum MMA is used, provided that there is no severe renal insufficiency. For details see text (Sections 4.1 and 4.2). deficiency is shown (adapted and modified from Herrmann and Obeid [88]). This algorithm suggests that HoloTC be used for screening of B 12 deficiency. For further differentiation, determination of serum MMA is used, provided that there is no severe renal insufficiency. For details see text (Sections 4.1 and 4.2).

B 12 Deficiency in the Elderly Population
The situation in elderly people deserves particular consideration [1,2,14,87,89,90]. From a clinical point of view, some disorders occur in the elderly more frequently than in younger patients. These include the so-called "Food cobalamin malabsorption" (see Section 2.1) [91] and the classic autoimmune atrophic gastritis, as well as diabetes and cardiovascular disease, the latter often being treated with medications potentially leading to B 12 malabsorption (Section 2.1). In these cases, B 12 deficiency is mostly only moderate. Furthermore, clinical symptoms are usually mild or even absent in the elderly. It follows that B 12 deficiency is in many cases subclinical or "functional", and there is an imperative need for laboratory biomarkers to set or support the diagnosis as early as possible. However, laboratory diagnosis faces two issues. First, in most studies, age appears to be an influence factor for laboratory biomarkers of B 12 deficiency [81,83,86]. However, as mentioned before, several other studies show that not age per se but rather age-related renal insufficiency may increase serum levels of HCys and particularly MMA since GFR declines with age [83]. Thus, the usefulness of increased HCys and MMA, generally considered to be markers of "functional" B 12 deficiency, is limited in the elderly. In a comprehensive study (n = 1143), HoloTC performed better (AUC = 0.923) than B 12 (AUC = 0.884) and appeared to be the superior biomarker for screening of B 12 deficiency, also in elderly people above 60 years (and up to slightly more than 80 years) [85]. Second, since specific symptoms of B 12 deficiency are usually very mild or absent, the question arises whether decision limits for treating (suspected) B 12 deficiency should be set higher in the elderly than in younger patients in order to enhance the sensitivity of each biomarker. Based on their results, the authors of the aforementioned study [85] provided a suggestion for decision limits, as shown in Table 2. It is important to note that the B 12 values suggesting sufficient B 12 supply are much higher than those suggested by other authors and also higher than the values suggested by the WHO (which are shown in the table for comparison). For instance, the respective cut-offs for total serum B 12 levels are 316 vs. 221 pmol/L ( Table 2). These data suggest that B 12 decision levels need to be higher in elderly people to ensure sufficient B 12 supply and to avoid cellular B 12 deficiency.

Summary of Laboratory Assessment of B 12 Deficiency
Although a gold standard for diagnosing B 12 deficiency is lacking [92], laboratory biomarkers are valuable for screening, for the diagnosis of B 12 deficiency and for therapeutic monitoring of B 12 deficiency. While B 12 levels are widely used, HoloTC levels appear to react earlier and appear more sensitive for documenting B 12 deficiency. Although the biomarkers MMA and HCys may indicate functional B 12 deficiency in tissues, they are not valuable as screening tests since they are less sensitive and are influenced by renal function (Table 2). MMA can also be useful for differentiation of B 12 from folate deficiency. Subclinical and overt B 12 deficiency may be easily overlooked in the elderly population since clinical signs are absent or nonspecific, laboratory biomarkers may be influenced by reduced kidney function, and B 12 absorption is impaired by reduced gastric acidity, e.g., due to proton-pump inhibitors or other drugs, e.g., metformin.

Conclusions
B 12 is an essential cofactor for two important enzymes in mammals. Besides its function serving as a cofactor, recent findings suggest that the central Co atom of B 12 undergoes several reduction/oxidation steps ranging from 1+ to 3+, both in the cytosol and in mitochondria. The recognition that B 12 exerts intrinsic antioxidative activity indicates a new role of B 12 in cellular metabolism beyond its classical, well-known cofactor function.
Since several studies point to an involvement of oxidative stress in the development of DPN, the antioxidative potential of B 12 suggests that B 12 administration may be justified, not only for the therapy of overt DPN, but also in early stages of DPN, or even for prevention of DPN when cellular oxidative stress is present but symptoms are mostly absent. The absence of a clinical improvement after B 12 administration in some studies does not exclude a possible favorable effect of B 12 because oxidative stress is probably only one of the multiple mechanisms leading to DPN. Of course, further studies are needed to elucidate in more detail the complex physiological action of B 12 and to prove the putative favorable action of B 12 administration, particularly in the early stages of DPN.