Introduction

The intrinsic factor (IF) is a glycoprotein produced by the parietal cells (oxyntic cells) located at the gastric body and fundus. It plays a crucial role in the transportation and absorption of the vital micronutrient vitamin B12 (cobalamin, Cbl) by the terminal ileum. Insufficiency of intrinsic factor could lead to devastating consequences on body homeostasis. The effect ranges from hematological to neurological disorders and, in unfortunate cases, fatal cardiovascular disease. 

Cbl binds the first Cbl-binding protein haptocorrin (HC) in the stomach, following Cbl separation from the protein of food, to form the Cbl-HC complex. In the duodenum, pancreatic proteases break the HC-Cbl complex. Therefore, Cbl binds IF to form the IF-Cbl complex the ligand of the cubam receptor localized in the distal ileum. Subsequently, receptor-mediated endocytosis into enterocyte accomplished. Finally, lysosomal enzymes liberate the Cbl molecule from the complex to travel directly to the target tissue through the plasma riding transcobalamin II (TC II).

Cellular Level

Cobalamin, the passenger

Cbl is the most complex water-soluble vitamin, made by microorganisms, mostly anaerobes, present only in animal products, plus negligible amounts produced by colonic flora. Therefore, strict vegetarians are at a high risk of Cbl deficiency. The recommended dietary allowance (RDA) of Cbl is 2.4 micrograms/day for adults.[1] Vitamin B12 is essential for DNA synthesis and fatty acid metabolism. Cbl is stored in the liver, with bile execration of 1 to 10 micrograms/day.

The organometallic compound Cbl consists of a cobalt (Co) atom positioned in the center of six coordination sites. Four of the coordination sites are formed by corrin rings (four reduced pyrrole rings), resembling the structure of heme (iron atom in the center of a porphyrin ring). The fifth site (lower ligand) forms from the alpha 5,6-dimethylbenzimidazole (DMB) covalently attached to the corrin rings through a ribo-phosphate group. In addition to a variable region (R-group) at the sixth site (upper ligand). The upper ligand is characteristic of each form of Cbl. For example, a hydroxyl group in (OH-Cbl) and cyanide in (Cyano-Cbl). 

Two forms of cobalamin are biologically active in the human body. 5-deoxyadenosylcobalamin (Ado-Cbl) and methylcobalamin (Met-Cbl), sharing in fatty acid catabolism and methionine synthesis, respectively. Cyanocobalamin (CN-Cbl) is convenient for medical use based on its chemical stability and will undergo a conversion reaction to be active inside the body.[2] Most Cbl forms are photosensitive and converted to OH-Cbl when exposed to UV light.[3] It is worth mentioning that OH-Cbl is used as an antidote for cyanide toxicity; cyanide replaces the OH group in the upper ligand of the cobalt atom. Therefore, cyanide excretion is facilitated through the urine in the form of CN-Cbl.[4]

The molecular bases of Cbl-binding proteins

In its journey, three binding proteins transport Cbl until it reaches the final destination, haptocorrin, intrinsic factor, and transcobalamin II (TC-II). Haptocorrin is encoded in the TCN1 gene located on chromosome 11 with the intrinsic factor gene (GIF).[5] TC-II is phylogenetically the oldest, located on chromosome 22.[6] All three genes show homology, indicating total or partial gene duplication of an ancestral gene.

All three Cbl-binding proteins share the primary protein structure. The N-terminal domain (alpha-domain) consists of alpha-helices linked by a single flexible linker, to the C-terminal domain (beta domain) consists of beta-helices. The beta domain side chains composition is a determinant of the specificity of ligand binding.[6] In terms of Cbl specificity, intrinsic factor is the highest, and haptocorrin is the least specific. Glycosylation is characteristic of haptocorrin and, to a lesser extent, IF and is absent in TC-II. Three significant steps explain the binding model: (1) ligand attachment to the beta domain, (2) primary assembly of alpha-domain and beta domain, and (3) fixation of the ligand ''sandwiched'' in between the two domains.[7]

Intrinsic factor structure:

Intrinsic factor present in gastric juice is highly specific for genuine Cbl. Intrinsic factor has two binding sites, one for binding Cbl and the other for the ileal cubilin receptor. The glycoprotein is organized as a 30 kDa N-terminal peptide fragment (alpha-domain) and a 20 kDa C-terminal glycopeptide fragment (beta domain), linked by a protease-sensitive linker.[8] The beta domain must bind the lower ligand (DMB) in Cbl via hydrogen bond. Later, conformational changes force the Cbl molecule to become sandwiched in the middle of the two domains.[6]

Haptocorrin structure:

Haptocorrin glycoprotein has a molecular mass of 60 to 70 kDa attributed mainly to glycans.[9] Unlike other Cbl–binding proteins, HC has a high affinity for Cbl and a group of cobalamin analogs called nicotinamide, the Cbl derivatives missing the nucleotide-side chain (the lower ligand).[10] The high affinity for cobinamide explained by the composition of the C-terminal domain, which has three large amino acid side chains compensate for the missing nucleotide by forming a hydrophobic bond with the Cbl apolar side. Asialoglycoprotein receptor (ASGR1) in the liver uptakes only HC that lacks sialic acid in its terminus.[9]

Functionally, haptocorrin plays an essential role in Cbl transport within the GI tract and through the circulation, in addition to the excretion of Cbl analogs that compete with Cbl binding sites in the enzymes. 

Trascobalamin structure:

TC-II is a 45 kDa non-glycosylated protein[11], responsible for 10% to 30% of Cbl transportation in the bloodstream and the remaining Cbl plus other cobinamides bound to haptocorrin. Cellular uptake of the saturated TC-II occurs via TCblR (CD320) receptors in the tissues.[12]

Development

The process of cobalamin transportation and absorption begins early in life and utilizes a variety of mechanisms with fetal development.

The first of these mechanisms is the intrinsic factor-dependent system of absorption. During embryo-fetal development, cobalamin and intrinsic factor are as a meal via the ingestion of amniotic fluid. The Cbl and Cbl-binding proteins contained within the amniotic fluid parallel to maternal blood concertation.[13] Also, the intrinsic factor receptor cubam is present in the whole length of the gastrointestinal tract, not only the distal portion, during 10 to 19 weeks of gestation.[14] Further studies show that the fetal stomach is capable of secreting active intrinsic factor resembles isoprotein patterns of IF found in amniotic fluid but differs from that of adults. Within the fetus, amniotic fluid is continuously circulating in and out. Therefore, fetal parietal cells could contribute part of the IF in the amniotic fluid.[15]

In neonates, the digestive tract is not well developed compared to the adult GI tract. Intrinsic factor secretion is low during this period, and there is a gradual increase in IF secretion during the first four months. Therefore, the presence of intrinsic factor independent system of absorption proposed. Cbl binds haptocorrin found in the mother's milk, and the Cbl-HC complex is absorbed via putative intestinal receptors until the IF-dependent system develops well.[16]

Function

Met-Cbl is a cofactor for the methionine synthase enzyme. In this reaction, the methyl group of methyltetrahydrofolate transferred to homocysteine to form methionine and tetrahydrofolate. Methionine is converted to s-adenosyl methionine (SAM), a methyl donor for phospholipid biosynthesis, especially that of the myelin sheath. Tetrahydrofolate is the free form of folate essential for purine synthesis. Dysfunction of this reaction leads to the following:

1. Folate traps in methyltetrahydrofolate form; therefore, less tetrahydrofolate is available for DNA synthesis, resulting in megaloblastic anemia.
2. Less methionine for phospholipid synthesis leads to demyelination of neurons.
3. Homocysteine accumulates, leading to endothelial dysfunction predisposing to cardiovascular events plus homocystinuria.

Ado-Cbl is a cofactor for methylmalonyl CoA mutase enzyme. In this reaction, the methylmalonyl-CoA (a product from odd number fatty acid catabolism) converted to succinyl-CoA. The latter makes its way into two pathways: the citric acid cycle and heme synthesis. Accumulation of methylmalonyl-CoA responsible for:

1.  Neurological disorders due to the incorporation of methylmalonyl-CoA into the myelin sheath, and the result is neuron demyelination. 
2.  Methylmalonic aciduria

Mechanism

Cbl is bound to dietary proteins upon food preparation, and because of the heat effect, Cbl is liberated partially from food. When exposed to light, all forms of free Cbl undergo conversion to OH-Cbl. Another source of Cbl is bound to HC, available within the bile, and delivered to the GI tract via the enterohepatic circulation.

Intra-gastric Events

Within the stomach, the proteolytic effect of pepsin and the low pH liberate the remaining Cbl and Cbl analogs encapsulated in the food. In some food sources including liver, Cbl readily released even when the stomach has natural pH.[17]

Substances that stimulate the release of acids contribute to intrinsic factor release; these include gastrin, histamine, and insulin, in addition to the vagal stimulation. The stomach secretes intrinsic factor more than the required for Cbl absorption. Thus, only the considerable loss in intrinsic factor secretion capacity results in Cbl deficiency.[18]

Deity Cbl binds salivary HC with affinities of 50- and 3-fold that of intrinsic factor in pH 2 and pH 8, respectively.[19] Salivary HC binds Cbl in the stomach (where some amount of HC is produced also), and it hardly does so in the mouth because Cbl binds to food components. Since HC is resistant to acid and pepsin, Cbl binds HC to for protection from acid deformation. In addition, Cbl binding to HC will prevent its uptake by bacteria.[20]

Intra-duodenal Events

By entering the small intestine, Cbl remains bound to HC even in the relatively neutral environment of the digest. HC is sensitive to pancreatic proteases trypsin and chymotrypsin, which will degrade HC moiety. Subsequently, intrinsic factor will exclusively bind the free Cbl. Intrinsic factor binds only active Cbl from diet and bile, ensuring that inactive analogs are excreted.

Ileal Events

The receptor complex cubam (an abbreviation for its two subunits) is located at the ileal apical membrane at the terminal ileum. Cubam is also present in the small intestine and the proximal tubules of the kidney, where it mediates the IF-Cbl complex and filtered protein endocytosis, respectively. Cubilin (CUBL) is the ligand-binding subunit that recognizes the IF-Cbl complex and amnionless (AMN) subunit for membrane anchorage plus endocytic capacitation.

Cubilin is a multiligand peripheral membrane protein that interacts directly with the IF-Cbl complex and consists of a short N-terminus without a membrane-spanning motif, eight epidermal growth factor (EGF) repeats, and a cluster of 27 CUB domains.[21] CUB of cubilin binds with high affinity to the two domains of intrinsic factor in Ca-dependent binding in Cbl presence. CUB interacts with the alpha domain of intrinsic factor whereas CUB interacts with the beta domain. CUB and CUB assist in positioning the two CUBs; the remaining CUBs form binding sites to other ligands rather than Cbl, such as filtered protein in the kidney.[22] 

Amnionless is an integral membrane protein that binds the N-terminal residues of cubilin to assist its fixation to the cell membrane. 

After its recognition by cubilin, the IF-Cbl complex gets endocytosed into the enterocyte bound to the cubam complex. Inside the endosome, the IF-Cbl complex gets liberated, and cubam gets recycled to the apical membrane. Within the lysosome, Cbl is released from IF by the action of lysosomal cathepsin L.[23] Cbl will be exported from the lysosome to the cytoplasm via a transporter called LMBD1.[24] The transport of Cbl to the bloodstream is mediated by ABC transporter multidrug resistance protein 1 (MRP1) located in the basolateral membrane of ileal cells.[25]

Enterohepatic circulation (EHC)

Most of the Cbl is transported in the bloodstream in the form of HC-Cbl, which is taken by the liver via the ASGR1 receptor to be stored. EHC is useful in retaining the Cbl but will lead to major storage depletion in the conditions of suboptimal absorption of Cbl.[26]

Related Testing

Tests for intrinsic factor insufficiency: 

• Anti-intrinsic factor antibody (Specific to pernicious anemia).
• Anti-parietal cells antibody.
• Schilling test: This is less commonly used. If the antibodies are negative, it is performed to assist Cbl uptake. It is done by injecting free Cbl intramuscularly and oral intake of radiolabeled Cbl. If the intrinsic factor is sufficient and there is no issue in its function, then the urine will show the radiolabeled Cbl. Otherwise, it will not.  

Tests for autoimmune atrophic gastritis: 

• Gastroscopy with histopathology: shows lymphocytes and plasma cells in lamina propria and pseudohypertrophy of parietal cells. 
• High fasting gastrin.
• Low pepsinogen I

Others tests: 

• Complete blood count (CBC):

Megaloblastic anemia: the CBC will demonstrate low hematocrit and low hemoglobin (less than 13 g/dL for men, under 12 g/dL for women), with mean corpuscular volume (MCV) greater than 100 fL.

• Peripheral blood smear:

Shows pancytopenia, oval macrocytosis, and hypersegmented neutrophils (at least five lobes) in case of megaloblastic anemia.

• Bone marrow examination:

Megaloblastic hyperplasia in megaloblastic anemia.

• Serum B12 level:

A level below 150 pg per mL is diagnostic for Cbl.[27]

Pathophysiology

IF insufficiency:

1) Autoimmune atrophic gastritis:

Autoimmune gastritis (type A gastritis) accounts for 10% of chronic gastritis cases. Type A gastritis is associated with loss of the gastric body and fundal parietal cells (which secretes acids and IF) caused by a cell-mediated autoimmune reaction characterized by the following:

• Achlorhydria: defective gastric acid secretion.
• Low pepsinogen I
• Hypergastrenemia: Defective acid secretion forces antral G cells to secrete the hormone gastrin, which is responsible for parietal cell stimulation. Prolonged stimulation of antral G cells forces them to undergo hyperplasia.
• IF insufficiency: will lead to Cbl deficiency and pernicious anemia.
• Autoantibody against parietal cells and IF:

Parietal cell antibody (PCA) is an IgA or IgG immunoglobulin that targets H/K ATPase, the gastric proton pump for acid secretion. Studies show that high homology exists between the beta subunit of PCA and that of H. pylori urease, suggesting that the autoimmune trigger is mediated by molecular mimicry due to long-standing H. pylori infection.[28] Another possibility is that the H/K ATPase released by cell turnover is recognized by the antigen-presenting cells in the adjacent lymph nodes and subsequent activation of CD4+ T lymphocytes. 

Intrinsic factor antibody (IFA) is less predominant than PCA, yet it is the specific marker for pernicious anemia. IFA is an IgG immunoglobulin classified into two types. Type 1 IFA operates against the Cbl binding site, while type 2 IFA targets the ileal receptor-binding site.[29]

2) Medical interventions: 

Some drugs, like proton pump inhibitors, induce gastric atrophy and impair gastric function, causing IF deficiency. Also, the diminished secretion of acids impairs Cbl absorption because acid is needed to liberate Cbl from the food component. Some bariatric procedures, such as sleeve gastrectomy, decrease the mucosal surface area, leading to IF deficiency.

Impaired Cbl transfer to IF:

The pancreatic proteases are needed to liberate Cbl from HC. In a disease like pancreatic insufficiency, Cbl is trapped in Cbl-HC form and, therefore, not ready to bind intrinsic factor.

Impaired attachment to ileal receptors:

Imerslund–Grasbeck syndrome (IGS) is a rare autosomal recessive disorder caused by a mutation in one of the two subunits of the cubam receptors preventing the IF-Cbl complex endocytosis. Cubilin is encoded by the CUBN gene located on chromosome 10, and amnionless is encoded by the AMN gene on chromosome 14.[30] The disease is characterized by Cbl deficiency and mild proteinuria.

Certain conditions (eg, celiac disease and inflammatory bowel disease) affect the distal ileum and harm its ability to bind intrinsic factor. Especially patients with Crohn disease undergoing ileal resection are at higher risk.

Clinical Significance

Subacute Combined Degeneration (SCD)

Demyelination of the spinal cord caused by Cbl deficiency:

• Dorsal column demyelination: loss of sense of vibration, proprioception (ataxia), and stereognosis
• Lateral corticospinal tract demyelination: motor deficit and hyperreflexia

Other Clinical Manifestations of Cbl Deficiency

Hematological manifestations:

• Megaloblastic anemia: light-headedness, palpitations, weakness, and anginal pain
• Thrombocytopenia: purpura

Neuropsychiatric manifestations:

• Dementia
• Polyneuritis
• Positive Babinski reflex

GIT manifestations:

• Hunter glossitis is a shiny, smooth tongue caused by the atrophy of the lingual papillae.

Cardiovascular manifestation:

• Congestive heart failure caused by severe anemia
• Coronary artery disease, limb ischemia, or cerebrovascular events secondary to hyperhomocysteinemia

Cbl deficiency in newborns:

• Neural tube defects
• Failure to thrive
• Hypotonia
• Anemia