Every medical student learns about the vitamin B12 (cyanocobalamin) deficit, but there is much less awareness on the elevated plasma levels of B12, although this abnormality is not so uncommon and could be the expression of a severe disease. The prevalence of high plasma B12 level varies between 10-18% in different patient populations.^1,2 The interest for understanding the pathogeny of the high vitamin B12 levels becomes even more obvious, as from the total samples referred to the laboratory for a presumed vitamin B12 deficit, 15% had, in fact, a higher than normal vitamin B12 value.³

Through this article, we will decode this paradox, by presenting the clinical significance of high plasma levels of vitamin B12 and by reviewing the literature regarding the pathogenic mechanisms that lead to it. We will also propose a pathophysiological classification, in order to facilitate the clinical approach of a patient with high B12 vitamin levels.

Terminology

Vitamin B12 is a corrinoid that specifically contains a cobalt atom inside the corrin ring. Due to the cobalt atom, vitamin B12 is called cyanocobalamin (Cbl). There are several forms of Cbl in the human body, taken from the food as hydroxy-, methyl and adenosyl cobalamin. The last two are the active forms at cellular level.⁴

In clinical practice, the most common determination is the total serum level of vitamin B12 by chemiluminescence immunoassay. The test measures the total Cbl, no matter the plasma transporter. Normal values are considered between the range of 240-680 pmol/L. A higher than the reference value is defined as hypercobalaminemia or hypervitaminemia B12.

The determination of the "active B12”, meaning by that the Cbl transported by the transcobalamin II (TCII), had been recently standardized.⁵ Normal values are 40-150 pmol/L and a higher than normal value is referred to as hyperholotranscobalaminemia. The utilization of an undifferentiated terminology creates confusion when interpreting results and in comparing different studies.

Review

Absorption, transport and B12 metabolism

Corrinoids are synthetized only by some prokaryote species. Because the human body is not able to produce vitamin B12, the requirements for B12 are entirely dependent on the intake. The intake of vitamin B12 is either as free form or as vitamin bound to proteins; besides Cbl, food also contains corrinoids.⁶ The free form of Cbl binds to the salivary haptocorrin (HC, also known as protein R or transcobalamin I). Inside the stomach and the duodenum, after proteolysis of the bound Cbl, vitamin B12 is captured (and protected from further degradation) by the HC secreted locally. Pancreatic enzymes, in the duodenum, degrade HC and release Cbl. At this point inside the digestive tract, Cbl is taken by the intrinsic factor (IF) secreted by the parietal gastric cells.⁷ The Cbl-IF complex reaches the terminal ileum where the absorption of vitamin B12 finally takes place, through a mechanism which is both active and saturable, involving a specific receptor for IF, cubam.⁸ The paracellular absorption, independent of the IF, is very limited in normal individuals, with a balanced diet; it represents only 2-5% of the total Cbl absorbed.⁹ When the exogenous intake is very high (mainly from food supplements or from fortified foods), this mechanism might become a significant absorption route.

Inside the enterocytes, the lysosomal cathepsin unbinds the Cbl-IF complex. Cbl is transported in the basal pole, from where it is released in plasma through the ABC transporter MRP1.¹⁰MRP1 (mutidrug resistance protein 1) is a protein from the ATP-binding cassette family, present in polarized cells. MRP1 was initially described as membrane transporter for the efflux of anticancer agents. Afterwards, MRP1 was found to be a multitasking transmembrane transporter for a variety of molecules, such as organic acids, xenobiotics, glutathione, cystenil leukotriene 4, estrogen and free Cbl.¹¹ As any ATP-binding cassette transporter, MRP1 contains a nucleotide binding domain (NBD) that binds ATP and transmembrane domains. The efflux of molecules through MRP1 is energy consuming, activated by the ATP lysis after attaching to the NBD. Under physiological circumstances, a couple of hours after the ileal absorption, Cbl is present in the portal circulation bound to the TC II.¹²

In plasma, vitamin B12 is not only captured by TCII. Both vitamin B12 and its related compounds are also transported inside the circulatory system by HC, TCIII (an isoform of HC).

TC I and TC III are glycosylated proteins with MW of about 60-70 kDa belonging to the haptocorrin superfamily; these represent the plasma forms of the HC. TC I and TC III are secreted from granulocyte derived cells and considered as markers of the secondary granules of the neutrophils. This explains why the TCI and TCIII increase in myeloproliferative disorders.¹. Distinct from TCII, HC has the capacity to bind not only Cbl, but also the analogs of Cbl (the corrinoids). Corrinoids are present in foods, but endogenous production has been also postulated: a possible mechanism is the colonic absorption of the corrinoids released by the microbiota. Some indirect evidences of the transformation, inside the human body, of vitamin B12 in corrinoids were found. At least for the fetal stage, the identification of corrinoids in the amniotic fluid, considering that only Cbl (and not the analogs) is transferred via the placenta,¹³ was considered as proof. The biological role of the corrinoids in humans is unknown. The inevitable presence of the corrinoids leads to the assignment of a scavenger role for analogs of vitamin B12 to HC, that leverages the Cbl binding to TCII;¹⁴ related to a possible antibacterial defense role, the initial experimental results have been invalidated.¹⁵ Uptake of Cbl bound to HC is not possible, except for the hepatocytes.¹⁶

Only Cbl bound to TCII, a protein with a molecular weight of 42-47 kDa, is transferred to the cells. TCII that incorporates Cbl becomes holotranscobalamin (holoTC).¹⁰ TC II is synthetized by the liver, but also by the endothelium and monocytes. The cellular uptake is mediated by the CD320, a membrane receptor able to bind TCII. This receptor is part of the low-density lipoprotein receptor (LDLR) family; binding to CD320, it initiates the endocytosis of holoTC. The number of CD320 receptors on the surface of the cells apparently depends on the cellular requirement for Cbl;¹⁷ the expression of CD320 is present in all human cells and is upregulated in cells with high proliferation rate. This might explain the rapid uptake of B12 in neoplastic cells, for their intense mitotic activity.¹⁸

Roughly 80% of the circulating vitamin B12 is transported by HC and TCIII and only around 20% is bound to TCII. The presence of several transport proteins and the preferential binding of Cbl to HC explain the major differences between the hypervitaminemia and hyperholotranscobalaminemia. This difference also explains the coexistence of a normal (or even high) level of serum vitamin B12 with the typical clinical signs of vitamin B12 deficit, megaloblastic anemia or neuropathy. Therefore, the B12 deficit confirmation should rely on the holoTC measurement or the determination of the active fraction of the B12 vitamin (holoTC/total B12 vitaminemia).¹⁹ Another possibility is to identify high homocysteine (Hcy) and methylmalonic acid plasma levels, molecules that accumulate when cellular deficit of B12 is present.

Subsequently to the cellular uptake, the more acidic intracellular milieu favors the dissociation of holoTC and releases Cbl. The intracellular traffic (lysosomes-cytoplasm-mitochondria) is assured by Cbl D and F proteins. The adenosylation of Cbl in mitochondria needs cobalamin B protein, while the cytoplasmic methylation needs cobalamin G protein.¹⁰Methylcobalamin is the cofactor of the methionine-synthase, an important enzyme involved in the folic acid dependent purine and pyrimidine synthesis.²⁰Adenosylcobalamin plays a role in the fatty acids degradation by methylmalonyl-CoA mutase and in the cellular respiration.²¹ If Cbl is neither used in the cellular metabolism, nor stored, it could be exported though the same ABC transporter as in the enterocyte. The intimate mechanism of the set up equilibrium and the regulation factors of the balance between intracellular Cbl and plasma Cbl is not yet known; therefore, the specific conditions when Cbl efflux from cells takes place, others than those related to cell destruction, are not defined.

The enterohepatic cycle (5-7 µg of Cbl/day) and the proximal tubular reabsorption of Cbl maintain the reserves and meet the cellular requirements for many years (about 5 years, in general) after the intake stops.²² If the enterohepatic circuit is interrupted (e.g., by bariatric surgery), the deficit becomes manifest more rapidly, in about 6 months.²³ In the experimental short-bowel syndrome, (after an extended intestinal resection) on animals, the readjustment process and the remodeling of the remaining intestine do not include the apical expression of cubam and the possibility to compensate the B12 deficit.²⁴

The majority of the absorbed vitamin B12 from the distal ileum is stored in the liver, directly or by uptake through the endothelial cells, as CD320 seems to be scarcely represented on the hepatocytes membranes.²⁵ Cbl and its analogs are secreted in the bile. Due to the high selectivity of Cbl for IF, up to 90% of the Cbl that reaches the duodenum is reabsorbed.²⁶ In biliary tract obstructions, the absorption and the entero-hepatic circuit is impaired.²⁷

HoloTC is filtered by the glomeruli and is reabsorbed in the proximal tubes, by a saturable mechanism, mediated by 2 transporters: cubam and megalin. After the internalization, the Cbl-TCII complex is stored in the lysosomes, the TC is metabolized and Cbl is probably released in the basal pole by a different transporter than MRP1.¹⁰In renal failure, the alteration of the transcobalamin renal metabolism leads to the plasma accumulation and to the identification of a high total serum value of vitamin B12 in the same patients.²⁸ There is normally an inverse relation between the serum value of Cbl and Hcy, but this relation is lost in renal failure, and above normal levels of vitamin B12 are requested in order to maintain the normal Hcy values.²⁹ The preferential accumulation of HC might explain this phenomenon.

Clinical implications

In clinical practice, a comprehensive evaluation of the vitamin B12 status involves the B12 vitaminemia, the holoTC and the metabolic deficit markers (methylmalonic acid and Hcy) measurement. Based on these parameters, we propose the following pathophysiological classification of a high serum B12 level.

Disproportionate hypercobalaminemia

Disproportionate hypercobalaminemia refers to a high total level of B12, with normal or low level of holoTC. This type of hypervitaminemia might be encountered in a physiological context, as an adaptative process to a temporarily increased cellular requirement or by excessive synthesis of the corrinoid transporters, in pathological situations.

Transient (self-limiting) hypervitaminemia.After sustained effort demanded by some physical activities (e.g., cycling) the level of B12 increases;³⁰ a possible mobilization from the stores was considered. But this increase is always present. Another study tested the level of vitamin B12 during the recovery period after endurance exercises, and found it to be decreased.³¹ The authors explained this finding to be related with an increased utilization. Indeed, Cbl is an important co-factor for the methyl transfer for the synthesis of several molecules involved in the adaptation to effort (acetylcholine, creatine, DNA) or in the energy production (through the participation to the succinyl CoA synthesis for the Krebs cycle functioning); therefore, a biological role in organism adaptation to exercise makes sense. These human studies are also supported by animal experiments³² but a conclusive demonstration of the role and the kinetics of Cbl in exercise is not available.

Excessive synthesis of HC develops in certain forms of neoplasia. It has been described in hematological malignancies (chronic myeloid leukemia, polycythemia vera, primary myelofibrosis, primary hypereosinophilic syndrome, acute leukemia or primary thrombocytosis)²²but also in some solid tumors. Concerning the hematological neoplasia, the relationship seems to be direct, as these cells are able to produce HC and an increase of their number will be reflected in a higher plasma HC.

The association between hypercobalaminemia and solid tumors was documented for the first time in 1975 by Carmel et al,³³most frequently in the hepatocellular carcinoma and in hepatic metastasis, but also in breast, colon, stomach and pancreatic cancer.³⁴Hypervitaminemia is related to a direct synthesis of TC by the tumor itself or secondary to the leukocytosis induced by the tumor.^2,12 In the majority of the tumors the level of the holoTC is under the normal range, but the HC is significantly high.³

Hypervitaminemia has also been considered as a prognostic factor. The correlation between the size of the tumor, particularly of the hepatic ones and the serum value of vitamin B12 was proposed as a significant marker for worse prognosis. The vitamin B12 - C reactive protein index (BCI), obtained by multiplying the serum level of B12 (pmol/L) with the serum level of C reactive protein (mg/L) has high predictive value for neoplasm mortality. A BCI > 40,000 was associated with a 3 months mortality of 90%.³⁵The physiopathological model of this predictive value is the increase in TC secretion in the cells from the inflammatory infiltrate and in the tumor cells, together with the high release of vitamin B12 from subclinical hepatic metastasis.

Impaired transport of B12 (functional deficit of vitamin B12) in hypervitaminemia or normovitaminemia. The "functional deficit of vitamin B12” needs a special comment. In a strict sense, there is no functional deficit, as vitamin B12 has normal structure and function. The proper term should be "impaired transport of B12”, as there is a reduction of the vitamin bound by TCII and therefore of the Cbl available for cellular uptake. The presence of a surplus of HC and TCIII could even contribute to this, because the vitamin B12 fixed by these transporters reduces the availability of Cbl for the TCII. This pathological mechanism is more prominent in chronic alcoholism. In patients with severe alcoholic hepatic disease, both an increase of the plasmatic levels of TC I and TC III, and a decrease in TC II have been demonstrated. The raise in HC reduces the excretion of Cbl, leading to hypervitaminemia and the low TC II impairs the cellular uptake.³⁶In this context, the hypervitaminemia masks hypoholotrans-cobalaminemia and the subsequent cellular depletion of B12. The metabolic signs of deficit (increase in serum methylmalonic acid and in Hcy) and even the clinical manifestations (megaloblastic anemia and neuropathy) become evident.

Proportionate hypercobalaminemia

Proportionate hypercobalaminemia refers to hypercobalaminemia with hyperholotrans-cobalaminemia, namely a high level of vitamin B12 and a high level of holoTC. Because the fraction of the vitamin B12 bound to HC is largely predominant, the sole increase of the holoTC, although theoretically possible, is unlikely to determine hypervitaminemia.

The pathogenic mechanisms are represented by an excessive exogenous intake, increase of the TCII levels, insufficient cellular uptake, high release from deposits, and insufficient renal excretion.

Excessive exogenous intake. Excessiveoral consumption is easily identified by the anamnesis (although, in general, it is not spontaneously specified by the patient). Both transcellular and paracellular mechanisms of absorption are present. The most frequent cause is the self-administration of multivitamins containing B12. The long-term parenteral administration of Cbl can associate the development of antibodies against TCII, resulting a reduction of the TCII clearance.²

Insufficient cellular uptake. In newborns with methylmalonic acidemia mutations of CD320 from homozygote deletion of a codon, c.262_264GAG (p.E88del), of the CD320 gene have been identified.¹⁸ The abnormal CD320 protein is not able to perform the appropriate endocytosis of the Cbl-TCII complex, with deficit symptoms if vitamin B12 is not substituted in sufficient amount.

Excessive release from deposits is a mechanism reserved for the severe liver diseases. The release from the destructed liver cells converges with the reduced hepatic and general cellular uptake to increase the plasma level of the Cbl. The fact that the liver is an important source of TCII contributes to the low tissue capture. Acute hepatitis is associated with high vitamin B12 in 25-40% of the cases; this increase was attributed to the release of Cbl from the hepatocytes and to the reduction in the TCII synthesis.¹²

In liver cirrhosis, plasma Cbl might reach 5 times the upper normal level; a parallelism between the level of B12 and the severity of cirrhosis has been suggested.³⁷ The main mechanism is the reduction of the liver uptake of holoTC and of tissue uptake of B12, confirmed in biopsies from cirrhotic patients.¹² The same pathological process seems to be responsible for the hypercobalaminemia in children with cystic fibrosis.³⁸ In hepatic tumors, the total increase in vitamin B12 is frequently associated with the increase in holoTC; the incidence raises up to 50% in hepatocellular carcinoma.²²Several mechanisms contribute to this increase: on the one hand, the diminished hepatic clearance of HC-cobalamin; on the other hand, the release of Cbl from the necrotic hepatocytes.² The low hepatic clearance could be attributed to the precarious vascularization of the remaining liver and to a reduction of the number of HC receptors from the surface of the tumor cells.

As a paradox, in anorexia nervosa, despite the extremely low nutritional intake, the plasma level of B12 increases, in correlation with the transaminases level, most probably from the hepatic distruction.³⁹

Decreased excretion of the B12 and of the Cbl transporters. As far as Cbl is concerned, kidney failure is a very peculiar clinical situation. In the initial stages, an increase in HC and TCII is expected, from a diminished renal clearance. Therefore, we included this stage in the second category, the balanced hypervitaminemia.

As renal dysfunction evolves and regular dialysis is initiated, the Cbl levels decrease, as direct effect of the dialysis, if not properly substituted.⁴⁰ "Resistance to vitamin B12” described in these patients could be, in fact, a preferential increase of HC compared to TCII, with a reduction of the cellular availability of Cbl relative to the vitamin B12 in plasma. Patients with this biological pattern should be included in the unbalanced hypervitaminemia category.

Increase of the TC II. Occasionally, high levels of HC and TCIII are present in chronic inflammatory syndromes, as a consequence of the proliferation of the immune cells and because TCII is also an acute phase protein. Hypercobalaminemia has been reported in Gaucher disease, in systemic lupus erythematosus, rheumatic polyarthritis or Still disease.^2a

Even if B12 hypervitaminemia is not frequent, it often reflects a severe disorder. Therefore, the accurate diagnosis is very important. The clinical signs reflect the underlying disease and might be followed by the intracellular metabolic markers and even by megaloblastic anemia and neuropathy.

In contrast to the high serum value, treatment with B12 vitamin might improve part of the symptoms, particularly those derived from the impaired transport of B12. This emphasizes the importance of the compartmentalization of the biological compounds inside the human body even if they are usually measured in the accessible biological fluids (plasma or urine). Understanding the absorption, transport and metabolic implications of B12 vitamin allows for the interpretation of the current tests used for the exploration of Cbl: the global determination of vitamin B12, the measurement of the holoTC, the methylmalonic acid and the Hcy and for the integration of the results in the systemic role of B12 vitamin.

Significant progress has been made in terms of understanding how this micronutrient interferes with human biology, but there are many questions that need to be answered, such as the source of the corrinoids in human body, the relation between the analogs and the Cbl, the role, if any, played by the corrinoids, the renal handling of the Cbl and of the transcobalamins, the cellular uptake and the equilibrium between the cellular and the plasma levels. The comprehensive understanding of this equilibrium is very important to elucidate the contradictory results obtained by now regarding the vitamin B12 circuit during and after exercise, the relation between the stores, the cellular turnover and the tumor growth, or when the liver deposit function is lost. The increase in the ratio between holoTC and total B12 vitaminemia in different pathological situations needs further clarification.

The specific effects of hypervitaminosis B12 (others than masking a deficit) or of the hyperholotranscobalaminemia are not known. In this respect, the systematization proposed by us could also serve to a better consolidation of the clinical data reported in different studies and for the characterization of the biological picture of the excess B12 vitamin.

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