Gut, 1990, 31, 59-63 59 B12 Binding proteins Gut: first published as 10.1136/gut.31.1.59 on 1 January 1990. Downloaded from Graham Neale

This article is one of a series linked with the Festschrift for Christopher Booth. See Gut Festschrift 1989; 30.)

A reliable sensitive method for measuring within one to four years whereas in those taking a vitamin B12 in the circulation was first achieved vegan diet (which contains no B12) body stores at Hammersmith in 1950.' During the subse- persist for 10-15 years. Thus B12 status is in quent decade the vitamin B12 status was assessed part maintained by an effective enterohepatic of patients with a wide variety of conditions.2 circulation. This culminated in the determination ofa unique Vitamin B12 in food is bound to peptides and function for the ileum by Booth and Mollin: that other compounds. Its release from food may start of the absorption of vitamin B123. During the in the mouth but occurs primarily in the stomach course of these early studies greatly increased and duodenum. Studies in vitro suggest that a concentrations of vitamin B12 were found in low pH favours the release of vitamin B12. the serum of patients with very high leucocyte Curiously, however, in the gastrointestinal tract counts - for example, in chronic myeloid leu- B12 is not bound initially to IF. R proteins in kaemia and other myeloproliferative disorders saliva and gastric juice (vide infra) have a greater and in occasional patients with non-leukaemic affinity for the vitamin.7 R proteins transfer B12 leucocytosis.4 It was shown that circulating B12 is to IF only after their degradation by pancreatic nearly all protein bound and that raised values proteases (Figure). Failure to degrade the R were associated with an increase in the serum protein-IF complex seems to explain the malab- capacity for binding the vitamin. sorption shown in patients with pancreatic in- High values for circulating vitamin B12 were sufficiency. This defect is corrected by the also described in patients with liver cell damage administration of either pancreatic enzymes or including those with infective hepatitis, hepatic by cobinamide. Cobinamide is an analogue ofB12 tumours, congestive cardiac failure and absces- capable of displacing the vitamin from the R ses involving the liver or the subhepatic space. protein but not capable of binding to IF. It is Booth recognised the value of these observations suggested that normally unwanted and poten- http://gut.bmj.com/ in assessing the patients with pyrexias of un- tially harmful analogues ofcobalamin in ingested known cause and disturbed liver function.5 animal tissues and those produced by intestinal It had become clear by now that vitamin B12 bacteria are bound to R proteins. This prevents required specific transport proteins for absorp- their absorption from the upper gastrointestinal tion from the intestine and transport in the body tract. They are released by proteolysis in the (Figure). The proteins fall into three groups small intestine along with vitamin B12. The B12 (Tables I, II). binds specifically to IF rendering it available for on September 25, 2021 by guest. Protected copyright. absorption whereas cobalamin analogues in the gut and from bile are excreted in faeces. Never- Intrinsic factor and the intestinal passage theless the role of biliary and pancreatic secre- ofvitamin B12 tions in modulating the absorption ofvitamin B12 Intrinsic factor (IF) was first postulated in the remains poorly understood.89 Deficiencies of classical work of Castle (1930) and isolated and either bile or pancreatic enzymes are associated characterised by Grasbeck and his coworkers.6 with malabsorption ofcrystalline vitamin B12 but It is a glycoprotein of molecular weight around rarely ifever with clinical vitamin B12 deficiency. 60000, consisting of two polypeptide chains, Abnormalities of intrinsic factor (Table III) each binding one molecule ofIF. The complex is and the subsequent fate of B12-IF complex is very resistant to proteolytic digestion and quite discussed in this volume by Schjonsby.9 specific in its binding properties (much more so than the other B12 binding proteins). Intrinsic factor is essential for the absorption of B12 from Transcobalamins the gastrointestinal tract, and it is made and In plasma, B12 is bound to two main classes of secreted by gastric parietal cells in an amount proteins one with alpha-beta and the other with which greatly exceeds physiological require- ments (the mean concentration of 1,ug/ml pro- TABLE I Sources ofB,2 bindingproteins vides a binding capacity of more than 50 times requirements). The process of producing and Protein Alternative names Source secreting intrinsic factor is established early in Intrinsic Factors IF Gastric Juice Transcobalamin II TCII Liver prenatal life and in atrophic gastritis it persists Transcobalamin (Other tissues - long after the capacity to produce gastric acid has P-globulin binder probably many) R proteins R-binder Granulocytes been reduced to very low concentrations. TCI Myeloid cells A normal daily diet contains 5-15 ug vitamin TCI-III Salivary gland a Transcorrin (Other secretory cells Addenbrookes Hospital, B12 and the bile delivers further 5 ug per day Cobalophilin in the gastro- Cambridge into the duodenum. After total gastrectomy lack Leucocyte binder intestinal mucosa) Graham Neale of intrinsic factor leads to signs of B12 deficiency Salivary binder 60 Neale

Salivary 'fR't Gut: first published as 10.1136/gut.31.1.59 on 1 January 1990. Downloaded from

TC 11(10% Sat) TC 1l-B12

R Protein -IF (80% Sat) I I I I R-B12 analogues

Figure: Role ofB,2 binding proteins in the uptake and metabolism ofvitamin B,2. http://gut.bmj.com/

beta-electrophoretic mobility. Hall and Finkler TABLE iII Disorders ofthe binding protein associated with '° provided the further characterisation which is low levels ofcirculating B,2 the basis of the classification used today. They IF deficiency Congenital IF deficiency used ion-exchange chromatography on DEAE- Post gastrectomy Pernicious anaemia cellulose and with gradient elution they separ- Abnormal IF Decreased ileal binding

ated two transcobalamins TCI and TCII. TCI (Congenital disorder) on September 25, 2021 by guest. Protected copyright. Impaired transcellular Familial B12 malabsorption carries the bulk of endogenous B12 and TCII passage of B12 (Imerslund-Grasbeck) vitamin which has been recently absorbed. TCII Impaired transfer and Congenital TCII deficiency ('null' is sometimes referred to simply as transcobala- transport of B,2 allele or non-functioning TCII) min because it has a clearcut function in transfer- ring B12 to cell surface receptors and across cell tion. In this review the TCI proteins will be membranes. In contrast TCI, as originally des- called R binders (R is derived from the rapid cribed, is not a single protein. A family of electrophoretic mobility of these binding pro- proteins has emerged and the members of this teins in gastric juice compared with IF). The family are still far from being properly under- name is non-specific and so is suitable for a group stood with respect to either structure or func- of proteins which are closely related one to another but which have not been satisfactorily characterised (Table II). The function of R TABLE II Nature ofB,2 binding proteins proteins is still not clear. As indicated above they Specificity may serve as scavengers for useless and poten- Protein Nature Function of binding tially harmful cobalamin analogues and may also have an antibacterial action micro- IF MW 44 000* Promotes ileal uptake High by depriving Glycoprotein (specific receptor for organisms of vitamin B12-like substances." A dimer IF-B12) much information TC II MW 38 000* Essential for transfer, Inter- Despite these uncertainties Liver protein distribution, re- mediate has accumulated regarding the activities of TCII cycling and R proteins in the circulation even though In plasma 30 [sg/ml R proteins MW 60000* Binds endogenous B12 Low their concentrations are remarkably small (TCI/ Glycoproteins and analogues in 10 In clinical concentrations TCIII) From degrading circulation. (- ,mol/1). practice tissues In plasma 25 tg/ml are assessed indirectly by determining the equiv- From exocrine Possible antibacterial alent B12 binding capacity and expressing the glandst action results as pg vitamin B12 per ml. This is clearly *Representative weights based on amino acid and carbohydrate unsatisfactory and further progress is hampered composition and ultracentrifugation; tR proteins are found in saliva, gastric juice, bile, tears, CSF, breast milk, seminal fluid, by an inability to determine the holo- and apo- amniotic fluid as well as in plasma. proteins by direct assay. B12 Bindingproteins 61

Transcobalamin II (TCII) and the transport hypogammaglobulinaemia and possibly a killing of vitamin B12 defect of granulocytes.'7 Curiously the condition TCII is a plasma protein with P electrophorectic develops during infancy indicating that the fetus mobility which has a clearcut role in the physi- in utero has an alternative mechanism for deliver- Gut: first published as 10.1136/gut.31.1.59 on 1 January 1990. Downloaded from ology of vitamin B12. It picks up absorbed ing B12 to its tissues. Neurological defects occur vitamin B12, directs it to specific receptors on cell only if the diagnosis is long delayed (with the membranes and facilitates its transport into cells. anaemia corrected by the administration of folic Thus its role in the circulation is closely acid). Patients with TCII deficiency have low analogous to that of IF in the gut. It is probable normal values for the circulating vitamin (bound that all tissues require TCII for the adequate to R proteins) but respond to the administration uptake ofvitamin B12 and this has been shown in of large doses of Bl2. This is also true of patients several in vitro models (including reticulocytes, with apparently normal TCII binding but a lymphocytes, fibroblasts, homogenised brain failure of tissue uptake. and placenta). Nevertheless brain and liver may High values for circulating TCII may occur in have an alternative means of taking up vitamin active liver disease (alcoholism, acute viral hepa- B12 because although patients with congenital titis, metastatic cancer); in lymphoproliferative TCII deficiency have severe megaloblastic disorders (including lymphoma, multiple mye- anaemia they rarely develop neurological loma and autoimmune disease) and in disorders deficits; nor do they excrete abnormal amounts of the reticulo-endothelial system - for example, of methylmalonic acid or homocystine. Gaucher's disease (Table IV). The association of Normal serum contains 20-40 ,ug (0 7-1 5 increased values for circulating TCII with in- nM)/l TCII capable of binding 600-1300 ng flammatory and neoplastic disorders suggest that vitamin B12. Normally less than 10% of the the protein may be released from the liver as an binding capacity is used. TCII carries B12 both as acute phase reactant. adenosyl-cobalamin and as methyl-cobalamin The assessment of the patient with high values (B12 bound to circulating R proteins is primarily for TCII-B12 may be complicated by the findings in the methyl form). A reduction in the circulat- of immunoglobulin-TCII complexes. These are ing concentration of holo-TCII may be the found most commonly after treatment of perni- earliest sign of vitamin B12 deficiency.'2 This cious anaemia with B12 by injection. But com- potentially important observation awaits confir- plex formation may also occur spontaneously in mation. patients with hypergammaglobulinaemia and In animals TCII appears to be made primarily the amount formed appears to parallel the in the liver. In man reticulo-endothelial cells (as severity of the pathological process. in Gaucher's disease), lymphocytes (as in auto- immune disease) and intestinal cells, along with http://gut.bmj.com/ other tissues, may have the capacity to make R proteins (R binders; TC1-TC3; the protein. It has a molecular weight of about cobalophilin; haptocorrin) 40 000. Unlike the other binders it is not a glyco- R Binders ofvitamin B12 have alpha-beta electro- protein and it is immunologically quite distinct. phoretic mobility and are found in many body The NH2-terminal 19 amino acids have been fluids (Table II). Their function is still not determined but the elucidation of the molecular wholly clear. The binding occurs over a wide structure awaits the development of further range ofpH (1-12) and once bound the vitamin is on September 25, 2021 by guest. Protected copyright. monoclonal antibodies to key sites of the struc- unavailable to bacteria. This function may be ture.'3 analgous to that postulated for the iron binding Genetically TCII has been linked to the P protein lactoferrin.'8 In the gut, B12 bound to R- blood group system and assigned to chromosome proteins in ingested milk and in secreted bile 22. It is polymorphic with an autosomal codom- may be released from its binding by proteolysis inant pattern of inheritance. There are two and so made available for IF-mediated absorp- common codominant alleles, at least three less tion in the ileum. In the circulation R-proteins common forms and an allele which is silent. appear to have a specific transport function. Racial phenotypic differences are apparent and They provide a mechanism for the re-use of B12 these show some variation in B12 binding capa- released from tissues and they deliver unwanted city. '" These genetic differences are important in B12-analogues to the liver for clearance into bile. relating changes in the concentration of B12- Despite these seemingly important functions binding proteins to tissue pathology."' TCII delivers vitamin B12 to tissues where it TABLE IV Conditions with raised B,2-binding proteins binds to cell membranes. Adenosyl-Bl2 is bound (levels ofcirculating B,2 often high) more readily than other forms. With reticulo- cytes transfer into the cell occurs within minutes Rises in Common Other but in other tissues transfer may take somewhat R proteins longer. Receptor binding of TCII-B12 increases TCI Chronic myeloid Carcinomatosis leukaemia* Juvenile hepatoma as cells move from a resting state to active Eosinophilic leukaemia division.'6 In the liver TCII-B12 bound to the TCIII Polycvthaemiat Leukocytosis (Tnflammatory plasma membrane undergoes pinocytosis and is disease4) then transferred to lysosomes where it is released Transcobalamin Gaucher's disease Liver disease TCII Myeloma Lymphoma by proteolysis. The liver acts as the main store of Monocytic leukaemia (Cancer) vitamin B12 (Figure). Auto-immune Congenital deficiency of TCII is associated conditionsl with severe megaloblastic anaemia, mouth ulcer- *Good marker of early relapse; tNot raised in secondary ation, and recurrent infection associated with erythrocvtosis; tMav be useful in following progress of condition. 62 Neale

congenital R-protein deficiency appears not to necrotic debris. These are delivered to the liver carry important clinical implications.22 where they are taken up by a system for dealing The measured content of R-binders in the with asialoglycoproteins, rapidly cleared by hep-

blood depends on how the sample is obtained atocytes and excreted in bile (Figure). Gut: first published as 10.1136/gut.31.1.59 on 1 January 1990. Downloaded from and processed because of the in vitro release of Endogenous B12 analogues are not bound by IF TCIII from cells (vide infra). This can be mini- and so they escape reabsorption in the ileum. mised by immediate centrifugation and separa- Whether or not such analogues may be harmful tion ofEDTA-anticoagulated plasma in the cold. in man remains unresolved.2 About 80% of circulating vitamin B12 is carried Circulating R proteins are usually markedly on R-proteins and the measured concentration of raised in patients with chronic myeloid the vitamin is not affected by in vitro release of leukaemia, polycythaemia vera and some solid binding proteins. Normal plasma contains about tumours (especially hepatocellular carcinoma) 25 ,ug total R binders per litre most of which (Table IV). This is almost certainly mainly the are holo-proteins. The molecular weight is about result of increased synthesis of the protein but 60000 and each molecule is capable of binding because of changes in molecular structure the more than one molecule of B12 or analogue. half-life of cobalophilin is often much pro- Unlike TCII the molecule does not shrink when longed.'0 Be that as it may, it has become clear it binds B12. that in the plasma of normal subjects R proteins In the circulation R-proteins appear to be are derived primarily from granulocytes in which derived primarily from granulocytes. In body their subcellular localisation in specific granules fluids R-proteins come from glandular tissue is separate from the disposition of vitamin B12.23 especially lacrimal, salivary, mammary and The release of R proteins from granulocytes placental and exist largely as unbound apo- appear to explain the raised levels which are proteins. R-protein has also been demonstrated found in non-leukaemic leukocytosis (Table III). in the epithelial cells of the biliary tree, small intestine and colon.'9 Gastric juice contains the R proteins of swallowed saliva, some derived Intracellular cobalamin binding protein (ICB) from granulocytes and possibly some produced An intracellular binding protein exists which is locally. Bile is the richest source ofcobalamins in immunologically distinct from the R proteins body fluids. Nearly all is in the form of holo- and from TCII.24 It appears to be necessary for protein representing cobalamins being excreted the retention of B12 in cells and for the conver- by the liver bound to R protein. Despite the wide sion of B12 to metabolically active coenzyme range ofproteins produced by a variety oftissues forms such as methylcobalamin and 5'-deoxy they are determined by a single genetic locus adenosyl cobalamin. Most intra-cellular vitamin

with apparently only two alleles.20 Congenital B12 is bound to apo-enzymes and some appears to http://gut.bmj.com/ deficiency of R binder leads to low levels of exist free in the cytoplasm.25 circulating B12 but no clinical disturbance of B12 metabolism. The structure of R proteins have been exam- Clinical value of measuring the B12 binding ined in considerable detail. Those in the plasma proteins may be split into overlapping fractions by iso- The B12 binding proteins are not used in the

electric focussing.21 This observation has led to routine investigation of patients with haemato- on September 25, 2021 by guest. Protected copyright. considerable confusion. A separate entity called logical, inflammatory or neoplastic disorders. TCIII was characterised by what appeared to be The careful control needed in the taking of blood distinct physio-chemical properties. In contrast specimens and in laboratory methodology have with TCI, TCIII binds weakly to DEAE- inhibited development. Most reported studies cellulose, is iso-electric above pH 3 35, and is have been based on the capacity of plasma to cleared from the circulation very rapidly by the bind labelled cyanocobalamin which gives a liver. Nevertheless it shares immunological measure of unsaturated binding capacity identity, a common polypeptide backbone and (UBBC) and not of circulating holo-proteins. common amino acid sequences with the other R Nevertheless studies of B12 binding proteins are proteins. Differences are limited to the content necessary to evaluate patients with unusual of carbohydrate (fucose) and sialic acid, and forms of B12 responsive megaloblastosis especi- most authorities now agree that there are not two ally in those patients with congenital disorders; distinct classes of R proteins but a variable may be helpful in the elucidation of myeloprolif- spectrum depending on the source of the R erative pathology; and can be used as a marker of binder and the mode of separation. Nevertheless inflammation in patients with hepatic or intes- the variation in the R binding molecular struc- tinal disease. ture may have physiological significance. The Investigation is indicated in the megaloblastic more basic moiety (TCIII) carries little or no anaemias of infancy and in patients in whom endogenous B12, predominates within cells and there is a chance finding of raised values for secretions, and is rapidly cleared from the circulating B12 without obvious explanation. In circulation (which may be responsible for the patients with an 'acute phase' response TCII apparent lack of binding). The more acidic concentrations are often raised but B12 concen- binders (TCI) carry much endogenous B12, pre- trations are normal unless the liver is directly dominate in the plasma, and are cleared slowly involved. In this situation a raised concentration by the liver. Be that as it may R proteins bind for serum B12 may be a helpful diagnostic many B12-analogues and provide the body with pointer.4 a system for clearing B12 compounds released Low serum B12 concentrations without evi- from rapidly turning over tissues, pus cells and dence of B12 deficiency occur during pregnancy B,2Bindingproteins 63

and occasionally in patients with multiple mye- 8 Seetharam B, Alpers DH. Cellular uptake of cobalamin. Nutr Rev 1985; 43: 97-102. loma and associated disorders. In clinical prac- 9 Schionsby H. Vitamin B12 absorption and malabsorption. Gut tice, however, a means of determining the sig- 1989; 30: 1686-91. 10 Hall CA, Finkler AE. The dynamics of transcobalamins II. A

nificance ofa border line value for circulating B12 vitamin B 12-binding substance in plasma. J Lab Clin Med Gut: first published as 10.1136/gut.31.1.59 on 1 January 1990. Downloaded from is the most urgent issue especially in view of 1965; 65:459-68. 11 Carmel R. Cobalamin-binding proteins of man. In: Silber R, recent reports of B12-responsive neuropsychia- Gordon AS, Loboe J, Muggia FM, eds. Contemporary tric disorders with normal or near normal values haematologyloncology. New York: plenum 1981; 79-129. 12 Herzlich B, Herbert V. Depletion of serum holotranscobala- for serum B12 and without overt evidence of min II. An early sign of negative vitamin B12 balance. Lab megaloblastosis.2' The recent report of reduced Invest 1988; 58: 332-7. 13 Carmel R. Monoclonal antibodies to different sites on human holo-TCII as the earliest manifestation of B12 transcobalamin II. Proc Soc Exp Biol Med 1988; 188: 77-81. deficiency in the serum and thus a clinically 14 Porck HJ. Variant specific differences in human unsaturated transcobalamin II. Biochem Genet 1986; 24: 103-14. useful test is potentially important.'2 15 Kane SP, Hoffbrand AV, Allen RH, Neale G. A familial Thus it seems that Dr Beck's statement in a abnormality of circulating vitamin B12 binding proteins. Occurrence in a family with high serum concentrations of recent editorial is justified: 'To the stalwart little transcobalamin II. BrJ Haematol 1976; 33: 249-53. band of investigators of vitamin B12 there is 16 Hall CA, Colligan PD, Begley JA. Cyclic activity of receptors of cobalamin bound to TCII. J Cell Physiol 1987; 133: comfort in knowing that the stream ofimportant 187-91. scientific problems will never end'.26 Studies of 17 Hall CA. Congenital disorders of vitamin B12 transport and their contributions to concepts. Yale J Biol Med 1981; 54: these problems have already yielded two Nobel 485-95. prizes (Minot, Murphy and Whipple for the first 18 Bullen JJ, Rogers HJ, Leigh L. Iron-binding proteins in milk and resistance to Escherichia coli infection in infants. Br succesful treatment of pernicious anaemia and MedJ 1972; i: 69-75. Dorothy Hodgkin for unravelling the structure 19 Kudo H, Inada M, Ohshio G, Wakatsuki Y, Ogawa K, Hamashima Y, Myake T. Immuno-histochemical localisa- of vitamin B12) and B12 binding proteins may tion of vitamin B12 R-binder in the human digestive tract. well provide more surprises as we learn about the Gut 1987; 28: 399-45. 20 Azen EA, Denniston C. Genetic polymorphisms ofvitamin B12 evolutionary development ofB12 metabolism. binding (R) proteins ofhuman saliva detected by iso-electric focussing. Biochem Genet 1979; 17: 909-20. 1 Ross GIM. Vitamin B12 assay in body fluids. Nature (Lond) 21 Stenman U-H. Vitamin B12-binding proteins of R-type cobal- 1950; 166: 270-1. ophilin: characterisation and comparison of cobalophilin 2 Mollin DL, Ross GIM. The vitamin B12 concentrations of from different sources. ScandJ Haematol 1975; 14: 91-107. serum and urine of normals and of patients with megalo- 22 Carmel R. Plasma R binder deficiency. N EnglJ_ Med 1988; blastic and other diseases. J Clin Path 1952; 5: 129-39. 318: 1401-2. 3 Booth CC, Mollin DL. The site ofabsorption ofvitamin B12 in 23 Kane SP, Peters TJ. Analytical subcellular fractionation of man. Lancet 1959; i: 18-21. human granulocytes with reference to the localisation of 4 Mollin DL, Ross GIM. Serum vitamin B12 concentrations in vitamin B12 binding proteins. Clin Sci Mol Med 1975; 49: leukemia and in some other haematological conditions. BrJ 171-82. Haematol 1955; 1: 155-72. 24 Rosenberg LE, Patel L, Lilliequist AC. Absence of an 5 Neale G, Caughey DE, Mollin DL, Booth CC. Effects ofintra- intracellular cobalamin-binding protein in cultured fibro- hepatic and extra-hepatic infection on liver function. BrMed blasts from patients with defective synthesis of 5'-deoxy- J 1966; i: 382-7. adenosyl-cobalamin and methylcobalamin. Proc Natl Acad 6 Grasbeck R. Intrinsic factor and other B12 binding proteins. Sci USA 1975; 72: 4617-21.

Progr haematol 1%9; 6: 233-60. 25 Kolhouse JF, Allen RH. Recognition of two intracellular http://gut.bmj.com/ 7 Allen RH, Seetharam B, Allen NC, Podell E, Alpers DH. cobalamin binding proteins and their identification as Correction of cobalamin malabsorption in pancreatic in- methylamonyl-Co A mutase and methionine synthetase. sufficiency with a cobalamin analogue that binds with high Proc Natl Acad Sci USA 1977; 74: 921-5. affinity to R protein but not to intrinsic factor. J Clin Invest 26 Beck WS. Cobalamin and the nervous system. N Engl J Med 1978; 61: 1628-34. 1988; 318: 1752-4. on September 25, 2021 by guest. Protected copyright.