Haptocorrin

Food vitamin B 2 appears to bind to a saUvary transport protein referred to as the R-protein, R-binder, or haptocorrin. In the stomach, R-protein and the intrinsic factor competitively bind the vitamin. Release from the R-protein occurs in the small intestine by the action of pancreatic proteases, leading to specific binding to the intrinsic factor. The resultant complex is transported to the ileum where it is bound to a cell surface receptor and enters the intestinal cell. The vitamin is then freed from the intrinsic factor and bound to transcobalamin II in the enterocyte. The resulting complex enters the portal circulation. 

Mammalian intestinal absorption requires the presence of two receptors and two transporters, which is itself a unique feature. Specific transporters such as intrinsic factor, transcobalamin, and haptocorrin have been characterized,1113 as well as a number of receptors for passage across cell membranes. A number of biochemical studies on cell uptake1114 and receptors1115,1116 of cobala-mins have been reported. Genetic disorders that impair the synthesis, transport, or transmembrane passage of cobalamins and their consequences have been reviewed.1117,1118... 

The protein haptocorrin has been shovm in vitro to resist digestion by proteol3dic enzymes (Adkins and Lormerdal, 2003). Haptocorrin has been reported to inhibit the growth of enteropathogenic . coli, possibly by sequestering vitamin B12 that is required for microbial growth (Adkins and Lonnerdal, 2003). 

Adkins, Y., and Lonnerdal, B. (2003). Potential host-defense role of a human milk vitamin B-12-binding protein, haptocorrin, in the gastrointestinal tract of breastfed infants, as assessed with porcine haptocorrin in vitro. Am. ]. Clin. Nutr. 77,1234—1240. 

Figure 28-3. The absorption of vitamin B12 in humans.The sequence of transitions between protein-bound dietary vitamin B12 and circulating B12 bound to TCII. R, R proteins, haptocorrins IF, intrinsic factor TCII, transcobalamin II.

After the stomach s acidic environment facilitates the breakdown of vitamin B12 bound to food, the vitamin B12 binds to the intrinsic factor released by the stomach s parietal cells. The secretion of intrinsic factor generally corresponds to the release of hydrochloric acid and serves as a cell-directed carrier protein similar to transferrin for iron. This complex, resistant to degradation, forms in the duodenum and allows for subsequent absorption of vitamin B12 in the terminal ileum. The cobalamin-intrinsic factor complex is taken up into the ileal mucosal cell, the intrinsic factor is discarded, and the cobalamin is transferred to transcobalamin It, which serves as a transport protein. This complex is secreted into the circulation and is taken up by the fiver, bone marrow, and other cells. Transcobalamin 11 has a short half-fife of 1 hour and is rapidly cleared from the blood. Consequently, most circulating cobalamin is bound to serum haptocorrins (formerly transcobalamin I and transcobalamin IB) whose function is unknown. However, it should be noted that an alternate pathway for vitamin B12 absorption independent of intrinsic factor or an intact ter-... 

Haptocorrin—A group of carrier proteins which bind with vitamin Bi2 in the blood and aid in its transport. 

Ingested B12 can exist in two forms, either free or bound to dietary proteins. If free, the B12 binds to proteins known as R-binders (haptocorrins, also known as transcobalamin I), which are secreted by the salivary glands and the gastric mucosa, in either the saliva or the stomach. If the ingested B12 is bound to proteins, it must be released from the proteins by the action of digestive proteases both in the stomach... 

F. 40.7. Absorption, transport, and storage of vitamin B12. Dietary B12 binds to R-binders (haptocorrins) in the stomach and travels to the intestine, where the R-binders are destroyed by pancreatic proteases. The freed B12 then binds to intrinsic factor (IF). B12 is absorbed in the ileum and carried by called transcobalamins (TC) to the hver, where B12 is stored. 

Marchaj, A., Jacobsen, D. W., Savon, S. R., Brown, K. L. (1995). Kinetics and thermodynamics of the interaction of cyanocobalamin (vitamin Bjj) with haptocorrin Measurement of the highest protein-ligand binding constant yet reported, J. Am. Chem. Soc., 117 11640. 

An average supply of food with about 3-4 xg of vitamin B12 (1) is considered necessary for sustaining physical well being [148]. The proteins known to be involved in uptake and transport of cobalamin in humans are intrinsic factor (IF), transcobalamin (TC) and haptocorrin (HC) [148,259]. These three soluble proteins ensure that the needed amount of cobalamin reaches the two intracellular enzymes methionine synthase (in cytosol) and methylmalonyl-CoA mutase (in mitochondria) [148,260]. 

The total amount of cobalamin present in serum is in the magnitude of 400pmol/L. Circulating cobalamins are bound to two proteins. Transcobala-min (TC) carries approximately 20% of the cobalamins in serum and the protein haptocorrin (HC) carries the remaining 80% (Nexo and Andersen 1977). In contrast, the amount of unsaturated (apo) transcobalamin is high compared with that of haptocorrin (Figure 26.1). 

Only cobalamin present on transcobalamin is available for the cells (for a review, see Quadros 2010), while the function of cobalamin bound to haptocorrin remains to be clarified (for an overview, see Morkbak et al. 2007). Transcobalamin carries both of the two co-enzymes, 5 -deoxy adenosyl-coba-lamin and methyl-cobalamin, and also other forms of the vitamin that can be converted into the coenzymes such as cyanocobalamin and hydroxo-cobalamin... 

Haptocorrin carries both the active and inactive forms of the vitamin (Hardlei and Nexo 2009). Methyl-cobalamin is the most abundant active form of the cobalamins bound to haptocorrin (Nexo 1977). The nature of the inactive forms, the so-called analogues, remains to be clarified. Cobalamin-analogues cannot act as coenzymes in the cells. Therefore, care is usually taken to choose a method that measures the active forms of cobalamin without interference from the analogues. The distribution of cobalamins and analogues associated with the two binding proteins is shown in Figure 26.1. 

To our knowledge, all commercially available assays today employ intrinsic factor as the binding protein. The challenge with this is that the most commonly used source of intrinsic factor is the porcine stomach, a source that contains a substantial amount of haptocorrin in addition to intrinsic factor. Because of this, care is needed to remove haptocorrin prior to using porcine gastric intrinsic factor. It seems obvious that, in order to standardize assays for cobalamin across the analytical platforms, the use of recombinant intrinsic factor is relevant. Recombinant human intrinsic factor is available and has... 

Three Automated Platforms Reacts Differently Towards Changes in Sample Type. This section describes the design of assays for cobalamin from the three main players in automated cobalamin analysis on human serum (Abbott, Bayer Diagnostics and Roche) and demonstrates how these assays react if employed for the measurement of cobalamin in human milk. Human milk contains up to 200-fold more haptocorrin than serum and most of it is unsaturated with cobalamin. If haptocorrin is insufficiently denatured, it can interfere in the assays (Lildballe et al. 2009). 

In the Bayer Diagnostics analyser, cobalamin in the sample mixed with labelled cobalamin competes for binding with in-solubilized intrinsie faetor (Bayer Diagnostics 2008). The presence of active haptocorrin in the sample will give rise to a spurious result that is too high for cobalamin since part of the labelled cobalamin will bind to haptocorrin rather than to the intrinsic factor. 

In the Abbott analyser, solid-phase intrinsic factor and non-denatured haptocorrin in the sample to be analysed compete for sample cobalamin (Abbott 2007). Prior to the addition of labelled cobalamin, the sample is washed and haptocorrin—as well as the part of sample cobalamin bound to the haptocorrin—is removed. Consequently, the content of cobalamin present in the sample will be underestimated. 

In the Roche analyser, the label is on intrinsic factor and the solid-phase is linked to cobalamin (Roche 2008). Unsaturated sample haptocorrin and intrinsic factor compete for binding to sample cobalamin and thereby less samples cobalamin is bound to intrinsic factor. Following this, solid-phase cobalamin is added and the amount of solid-phase-cobalamin-intrinsic factor complex is measured. Provided that enough solid-phase cobalamin is present, the amount of cobalamin present in the sample will also be underestimated by this method. 

We discovered these analytical problems when seeking to analyse the content of cobalamin in human milk (Lildballe et al. 2009). However, we encountered the same problem for serum samples with a high content of unsaturated haptocorrin (Lildballe et al. 2011). Once such a problem is realized, it is possible to... 

The extracted cobalamins are mixed with excess cobalamin-binding protein of unknown origin and are allowed to bind. The reaction mixture is applied to a cobalamin-precoated microchip. The amount of unsaturated cobalamin-binding protein that binds to the chip is detected and is inversely proportional to the cobalamin concentration in the sample. The specificity of the method depends on the binding protein employed. If intrinsic factor is used as binding protein, only cobalamin will be quantified. If a haptocorrin-like protein is employed, the sum of cobalamin and analogues is measured. 

The detection system in HPLC is often based on ultraviolet (UV) light measurement of the effluent. Since the concentration of cobalamins in human samples is very low, our laboratory has developed a sophisticated method to determine low-level concentrations of HPLC-separated cobalamins (Hardlei and Nexo 2009). After immunoprecipitation of haptocorrin or transcobalamin followed by a proteolytic extraction and reversed-phase HPLC in darkness (to avoid conversion of methyl- and adenosyl-cobalamin into hydroxo-cobalamin), the fractions are vacuum-dried, re-dissolved in buffer and the cobalamin in the fractions is measured either by employing a sensitive cobalamin specific assay or an assay recognizing also the cobalamin analogues capable of measuring levels as low as 4pmol/L. 

Serum cobalamin consists of picomolar amounts of several forms of the vitamin bound to transcobalamin or haptocorrin. 

Measurement of serum cobalamin includes release of cobalamin from its binding proteins transcobalamin and haptocorrin, conversion of the various forms of cobalamin into one form of the vitamin, and quantification employing microbiological or protein binding assays. 

False levels of cobalamin may be encountered if the concentration of cobalamin unsaturated haptocorrin is high as observed in human milk. 

Measurement of cobalamin and analogues bound to haptocorrin is mainly used for research purposes. 

Cobalamin analogues Cobalamin analogues are cobalamin-related molecules not able to act as coenzymes for the cobalamin dependent human enzymes. In serum, both cobalamin and cobalamin analogues are present. The protein transcobalamin can only bind active forms of cobalamin, whereas the protein haptocorrin can bind both cobalamins and analogues. 

Hardlei, T.F., and Nexo, E., 2009. A new principle for measurement of cobalamin and corrinoids, used for studies of cobalamin analogs on serum haptocorrin. Clinical Chemistry. 55 1002-1010. 

Lildballe, D.L., Nguyen, K.Q., Poulsen, S.S., Nielsen, H.O., and Nexo, E., 2011. Haptocorrin as marker of disease progression in fibrolamellar hepatocellular carcinoma. European Journal of Surgical Oncology. 37 72-79. 

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