Cobalamin absorption and metabolism

Cobalamin in the human diet is a bacterial product ingested and stored by animals and strict vegans are therefore liable to deficiency. The prevalence of cobalamin deficiency, as defined by serum vitamin B₁₂ <200 ng/l and methylmalonic acid >0.27 μmol/l, is 1.6% of subjects over 51 years of age in the 2001–2004 National Health and Nutrition Examination Survey¹ in the USA. Of this age group, 3.2% have a low serum B₁₂ <200 ng/l. Ingested cobalamin is released from food proteins by pepsin and acid and bound initially by transcobalamin I or haptocorrin (R binder). This binder also binds other cobinamides in the diet which are metabolically inert. Pancreatic enzymes release the cobalamin from transcobalamin I and permit binding by intrinsic factor. The intrinsic factor–cobalamin complex is attached to cubam, a multiligand receptor, which is a combination of cubilin² and amnionless and is taken up by endocytosis, into the ileal cell. The cobalamin is then released from the endosome and bound to transcobalamin II (holotranscobalamin) in the ileal cell and exported into the portal circulation. Cobalamin undergoes enterohepatic circulation via the liver and bile ducts with 1.4 μg/day excreted in the bile, of which 1 μg/day is reabsorbed in the ileum. Holotranscobalamin is the active form of cobalamin and is taken up by holotranscobalamin receptors (TCII receptors) on cells throughout the body, particularly liver, kidney and bone marrow cells. At the cellular level in the target tissue the holotranscobalamin undergoes endocytosis via the transmembrane TCII receptor. Holotranscobalamin then undergoes lysosomal degradation, releasing cobalamin for metabolic reactions.

Cobalamin is a cofactor in two important biochemical reactions. In the first, methylcobalamin acts as a cofactor for methionine synthase in the production of methionine from homocysteine. The remethylation of cobalamin requires the donation of the methyl group from methyltetrahydrofolate as it is converted to tetrahydrofolate, thus linking cobalamin to folate and 1-carbon metabolism. The second cobalamin reaction occurs in the mitochondrion. Cobalamin is converted to adenosylcobalamin, a cofactor for the enzyme methylmalonyl-CoA mutase, which converts methylmalonyl-CoA (the product of propionate metabolism) to succinyl-CoA. Methionine produced in the first reaction is converted to adenosylmethionine and is a vital source of methyl groups critical for a series of methylation reactions involving proteins, phospholipids, neurotransmitters, RNA and DNA. In cobalamin deficiency, methylmalonic acid and homocysteine levels are therefore elevated. Reduced methionine synthesis is thought to result in a decrease in methylation of myelin basic protein, resulting in the neuropathies associated with cobalamin deficiency which are irreversible once subacute combined degeneration³ of the cord has occurred.

Folate absorption and metabolism

Dietary folate polyglutamates are thermolabile, water-soluble vitamins found in leafy green vegetables. Folate deficiency arises from dietary deficiency, impaired absorption or increased requirements. Folate polyglutamates in the diet must be hydrolysed to monoglutamates by hydrolases, operating maximally at pH 5.5 in the presence of zinc and further converted to pteroylglutamate, before absorption can take place. Folate carriers transport pteroylpolyglutamate rapidly into the luminal cells where it is methylated using methyl cobalamin as cofactor and reduced to 5-methyltetrahydrofolate (5-methyl THF) in the enterocyte before entering the portal venous system. Unconverted pteroylglutamate remains in the luminal cells.

Just like cobalamin, there is significant enterohepatic recirculation of folate, amounting to 90 μg/day, and biliary drainage results in rapid fall in serum folate levels, whereas deprivation of dietary folate takes up to 3 weeks to cause serum levels to fall. Two-thirds of plasma folate is non-specifically bound to plasma folate binding proteins including albumen and one-third circulates as free folate.

There is sufficient retention of folate by the renal tubules to prevent urinary vitamin loss; this is achieved by megalin uptake of filtered folate-binding protein⁴ and bound folate. Cubam,² which binds intrinsic factor-cobalamin complex, is also important in the uptake of albumen from the renal tubules, which may also contribute to folate retention.

Folate transport into cells is dependent upon two mechanisms, reduced folate carrier (58 kDa), which is a low-affinity high-capacity system, and folate receptors (44 kDa), of which there are three isoforms – alpha and beta are attached to the cell surface through a glycosyl-phosphatidylinositol anchor and gamma is secreted by enteric mucosal cells. Methyltetrahydrofolate bound to the folate receptor undergoes endocytosis by clathrin-coated pits or caveolae. Antimetabolites such as methotrexate also bind to the reduced folate carrier and folate receptor. Passive diffusion is an alternative mechanism by which folate can enter cells. The relative contributions of the different mechanisms are not known. Folate receptors may be expressed on malignant cells and have become potential targets for delivery of cytotoxic agents linked to folate.

Folates participate in 1-C metabolism and thereby facilitate the essential cellular metabolism of methionine, serine, glycine, choline and histidine in the biosynthesis of purine and deoxythymidine monophosphate (dTMP) in the synthesis of pyrimidines and thus DNA (Fig. 10.1).

Figure 10.1 Metabolic diagram. Note: Cobalamin bound to transcobalamin II is in its most oxidized 3+ state and enters the cell by TCII receptor, it undergoes one reductase step to cobalamin (I) in the cytoplasm and a second reductase step to cobalamin (II) in the mitochondria. The methionine cycle takes place in the cytoplasm and the succinyl-CoA synthesis in the mitochondria.

Intracellular folates are compartmentalized between the cytosol and mitochondria and the major forms are tetrahydrofolate (THF), 5-methyl THF and 10-formyltetrahydrofolate (10-formyl THF). Homocysteine is converted to methionine by methionine synthetase using cobalamin as a cofactor and methyl THF as the methyl group donor. Cobalamin deficiency therefore results in inactivation of methionine synthase, resulting in accumulation of 5-methyl THF, which cannot be converted back to 5,10-methylene THF. Folate is then unavailable for pyrimidine and purine synthesis – the methyl-trap hypothesis, which was advanced to explain why cobalamin deficiency often results in a functional folate deficiency. Furthermore, methyl THF is a very poor substrate for the enzyme responsible for folate polyglutamation, folylpoly-γ-glutamate synthetase, which prefers THF and 10-formyl THF. Folate deficiency is thought to cause megaloblastic anaemia by inhibiting the production of 5,10-methylene THF polyglutamate form, which acts as a cofactor in the rate-limiting step in the production of DNA, the synthesis of deoxythymidine monophosphate (dTMP). Thus, in the absence of cobalamin, polyglutamate synthesis ceases and monoglutamate forms are not retained by cells. This explains why in cobalamin deficiency serum folate levels may be found to be elevated and red cell folate levels normal or low. These metabolic pathways explain the interrelationship of serum B₁₂, serum folate and red cell folate results seen in the clinical laboratory.

Folate deficiency is therefore associated with elevation of homocysteine levels, reduced methionine synthesis and defective purine and pyrimidine synthesis, the latter resulting in morphologically visible megaloblastic change in bone marrow cells. Cobalamin deficiency also results in a functional folate deficiency with indistinguishable megaloblastic change and/or the more insidious and dangerous potential for neurological damage.

Rationale for investigation of cobalamin or folate status

Investigation of the vitamin B₁₂ and folate status of individuals is not restricted to investigation of individuals with classical features of megaloblastic anaemia alone because neuropathy and neuro-psychiatric changes may occur in B₁₂ deficiency in the absence of macrocytosis or anaemia.^5–9 The finding that folate supplementation reduced the incidence of neural tube defects¹⁰ by 25–46% in the USA and Canada highlights the importance of defining optimum population folate levels. Increased plasma homocysteine and serum methylmalonic acid (MMA) levels have been advocated^11–14 as sensitive indicators of folate and cobalamin deficiency that may be subclinical. Introduction of metabolite testing to routine laboratory practice was limited in the past as a result of technical difficulty in measurement but is now becoming more available, though the clinical benefit of this approach has been questioned by some authors.^15,16 Elevated homocysteine levels are an independent vascular disease risk factor¹⁷ and are also associated with risk of idiopathic venous thrombosis.¹⁸ In the USA, dietary supplementation with folate was introduced in 1998 to reduce neural tube defects and by lowering homocysteine levels may achieve a further health gain in reduced myocardial infarction and stroke. Food supplementation with folate remains a controversial step in European countries because of a possible increased risk of cancers^19,20 (see Scientific Advisory Committee on Nutrition, at: www.sacn.gov.uk, for update). Increased plasma homocysteine levels occur in both B₁₂ and folate deficiency as a result of reduction in methionine synthesis (Fig. 10.1). Some laboratories have utilized homocysteine as an initial screening test for abnormalities of cobalamin and folate metabolism which may be particularly appropriate for investigation of suspected inherited cobalamin or folate disorders in children.^21,22 MMA measurement though not widely available in the UK, requiring gas chromatography-mass spectrometry (GC-MS), is available at a limited number of university departments. In contrast, plasma homocysteine measurement by high-performance liquid chromatography (HPLC) and commercial enzyme immunoassays is widely available. The limitations of total serum B₁₂ measurement have been highlighted by studies that showed poor positive predictive value (i.e. healthy persons with a low level or low cobalamin levels with no evidence of deficiency) and <100% negative predictive value of 95% (i.e. 5% clinically deficient with normal level).^15,23 In addition, some publications have highlighted the presence of severe cobalamin deficiency concurrent with a normal cobalamin level using some of the current commercial assays.^24,25 The introduction of holotranscobalamin assays,^26–28 now available on an Abbott automated immunoassay platform, provides a readily accessible method of assessing the physiologically active form of cobalamin rather than the less relevant total B₁₂. The advent of a commercial holotranscobalamin assay occurred over 20 years after depletion of serum holotranscobalamin II was identified as an early sign of cobalamin deficiency.²⁹ Adoption of new methodology in laboratory medicine in the UK requires careful evaluation in a clinical setting and this research appears inhibited by lack of training in research methodology and financial constraints in service laboratories. Serum methylmalonic acid is increased in renal impairment and 7.9% of subjects over 51 have levels in excess of 0.27 μmol/l.¹ MMA must therefore be used in conjunction with serum cobalamin and holotranscobalamin and must be interpreted in the light of renal function. Urinary MMA measurement can be used to compensate for the impact of renal impairment.

Haematological features of megaloblastic anaemia

Megaloblastic anaemia resulting from impaired DNA synthesis is characterized by the presence of megaloblastic red cell precursors in the bone marrow and occasionally also in the blood. Megaloblasts have a characteristic chromatin pattern (Fig. 10.2) and increased cytoplasm as a result of asynchrony of nuclear and cytoplasmic maturation with a relatively immature nucleus for the degree of cytoplasmic haemoglobinization. The delay in nuclear maturation caused by delay in DNA synthesis resulting from lack of vitamin B₁₂ or folate is also seen in all lineages, particularly granulocytic marrow precursors with giant metamyelocytes (Fig. 10.3) and hyperlobated neutrophils with increased lobe size as well as number of nuclear segments (see Chapter 5, Fig. 5.10). In severe pernicious anaemia, a progressive increase in mean red cell volume (MCV) up to 130 fl occurs, with oval macrocytes, poikilocytes and hypersegmentation of neutrophils (>5% with more than five nuclear lobes).³¹ The neutrophil hypersegmentation index is an equivalent automated parameter on some cell counters, although hypersegmentation does not always respond to a therapeutic trial. The mean platelet volume is decreased and there is increased platelet anisocytosis, as detected by the platelet distribution width (PDW). The MCV falls to 110–120 fl as megaloblastic change advances. Howell–Jolly bodies and basophilic stippling are seen in the red cells.

Figure 10.2 Photomicrographs of bone marrow films stained by May–Grünwald–Giemsa. (A) Megaloblasts. (B) Normoblasts for comparison.

Figure 10.3 Photomicrograph of bone marrow film stained by May–Grünwald–Giemsa showing giant metamyelocytes.

Testing strategy for suspected cobalamin or folate deficiency

Microbiological cobalamin and folate assays and competitive radiodilution binding assays for measurement of cobalamin and folate, which were often performed together, have largely been replaced by separate analysis by automated binding assays. The application of a suitable testing strategy for patients suspected of having cobalamin or folate deficiency is shown in Tables 10.1–10.3.

Table 10.1 Significance of clinical details

Table 10.3 Clinical and laboratory checklist for diagnosis of pernicious anaemia

a Reagents for Schilling tests currently unavailable. A non-isotopic B₁₂ absorption test utilizing recombinant intrinsic factor and holotranscobalamin measurement is under development.

Table 10.1 highlights the important clinical details that should be elicited by the clinician and submitted with the request to assist the laboratory in interpretation of the numeric results of cobalamin and folate assays. Ideally, test requests should not be accepted without this information, which could be incorporated into electronic order communication from user to laboratory.

Table 10.2 lists the important laboratory investigations that should be performed – results must not be reported in isolation from other laboratory results and clinical details. If investigations are performed in different laboratories, authorization and release of results requires access to all laboratory data on the individual patient. Laboratory information systems should facilitate this cross-disciplinary access. For example, intrinsic factor antibody results should be available to haematology or clinical chemistry laboratories undertaking cobalamin, MMA or homocysteine assays.

Table 10.3 provides a list of clinical and laboratory features for diagnosis of pernicious anaemia. These criteria avoid undue reliance on a single B₁₂ assay and should help clinicians to make a diagnosis even when some critical tests, e.g. Schilling tests, are not available.

In view of the lack of specificity and sensitivity of serum cobalamin assays and frequent lack of availability of other diagnostic tests, basing the diagnosis of pernicious anaemia, which requires lifelong parenteral B₁₂ therapy, solely on laboratory results, is not straightforward. A checklist of laboratory and clinical diagnostic criteria, as shown in Table 10.2, helps to achieve a greater degree of diagnostic certainty than any single diagnostic test and permits the diagnosis to be made even when a single diagnostic test is anomalous or unavailable. Clinical and other laboratory criteria thus provide additional supportive evidence of an autoimmune aetiology, even if the more demanding diagnostic laboratory criteria are not met.

Limitations of Cobalamin Assays

Sensitivity and Specificity of Cobalamin and Holotranscobalamin Assays

Utility of receiver operator characteristic curves

There has been little data on sensitivity and specificity of current B₁₂ assays, due to the difficulty in defining a truly deficient study population. Some authors have suggested B₁₂ assays and measurement of methylmalonic acid are no better than tossing a coin, to determine the presence or absence of deficiency. The study by Clarke et al.²⁸ provides data which permits calculation of the specificity and sensitivity of a current B₁₂ immunoassay in the detection of cobalamin deficiency in a community study of 1621 subjects over age 65 with normal renal function. Subjects were defined as cobalamin deficient if the methylmalonic acid was elevated above 0.75 μmol/l. Deficiency was found in 4.3% of subjects over 65 years of age with normal renal function. The mean B₁₂ level of these subjects was 151 pmol/l (202 ng/l) by Siemens Centaur assay (range 110–199 pmol/l). Table 10.4 illustrates the calculation to derive specificity and sensitivity for the Siemens Centaur B₁₂ assay using a cut-off point of 200 pmol/l (270 ng/l).

Table 10.4 The calculation to derive specificity and sensitivity for the Siemens Centaur B₁₂ assay

This study demonstrates that, while values over 270 ng/l have a high (98.4%) negative predictive value for the presence of disease, values below 270 ng/l include a high percentage (28.2%) of individuals with normal methylmalonic acid levels and presumably no evidence of cobalamin deficiency, resulting in a large grey area. This is reflected in the poor specificity (71.8%) of the assay using this cut-off point. The choice of the appropriateness of the cut-off point can be explored further using receiver operator characteristic (ROC) curves. Laboratories able to construct ROC curves will be able to select an appropriate cut-off point to identify those patients with possible or definite disease.

Selection of a cut-off point for cobalamin of below 150 ng/l, will identify subjects with higher probability of presence of cobalamin deficiency. Some authors¹³ have advocated choosing a cut-off point 25% below the reference range lower limit for any particular assay.

If, for example, a cut-off point of 125 pmol/l (168 ng/l) is chosen, the specificity of a value below this level, i.e. the number of normal individuals who fall below the cut-off point, will be markedly reduced (FP) and therefore the specificity of the test (TN/TN+FP) improves to 95%, although the detection of individuals with true deficiency who lie above the cut-off point, i.e. false negatives will be markedly increased, resulting in a sensitivity (TP/TP+FN) of 35%. The ROC curve therefore allows a laboratory to select a cut-off point that meets the objective of the laboratory – to have either a highly specific but low sensitivity test or to have a test of poor specificity but high sensitivity. Clearly cobalamin assays do not meet the criteria for an ideal test of high sensitivity and high specificity which would lie on the coordinates 0.1 to the left of the graph. The Axis-Shield/Abbott holotranscobalamin assay in this pivotal study is seen to have slightly superior ROC curves (see Fig. 10.4).

Figure 10.4 Receiver operator characteristic (ROC) curves (showing holotranscobalamin levels in μmol/l (abnormal <44 μmol/l) and B₁₂ levels in pmol/l (1 pmol/l = 1.344 ng/l). (A) The ROC curve for methylmalonic acid levels >0.75 μmol/l (definite metabolic B₁₂ deficiency). (B) Methylmalonic acid levels of >0.45 μmol/l (probable B₁₂ deficiency).

(Redrawn from Clarke et al. 2007).²⁸

Utility of holotranscobalamin, methylmalonic acid and homocysteine assays

Holotranscobalamin assays gave a greater area under the curve, 0.85 versus 0.76 in the above study, and superior sensitivity and specificity. Holotranscobalamin is the physiologically active fraction of total cobalamin.^27–29 An isolated abnormal result of serum B₁₂ should not be the sole criterion on which treatment decisions are based and a repeat assay and other confirmatory and clinical evaluation are necessary prior to a diagnostic conclusion. Additional secondary testing with metabolite levels, or holotranscobalamin and monitoring of treatment response is recommended. High B₁₂ levels have been described in subjects with no myeloproliferative neoplasm and who are not on cobalamin therapy or vitamin supplementation.³⁵ This is thought to be due to immunoglobulin-complexed B₁₂ resulting in assay interference. False normal B₁₂ levels^24,25 have been described in subjects with high titre intrinsic factor antibody²⁵ and may also occur due to presence of heterophile antibody interference.

Holotranscobalamin assays^27,28 may challenge total B₁₂ assays as a first-line test in cobalamin assessment. Holotranscobalamin has been shown to be unaffected by assay interference from high-titre intrinsic factor antibody levels.³⁶ In addition, holotranscobalamin is not subject to the 30% fall in total B₁₂ levels seen in normal pregnancy, which makes low total B₁₂ levels uninterpretable during pregnancy (Fig. 10.5).³⁰

Figure 10.5 Comparison of HoloTC, cobalamins and HoloHC in pregnant women at 18th, 32nd, 39th gestational week (gw) and at 8 weeks’ postpartum (8 pp) (n = 141). Means and 95% CI (calculated from the normal distributed log transformed data) for HoloTC (white), cobalamins (white + grey shaded) and measured HoloHC (grey + black shaded) are shown. The grey shaded area indicates HoloHC saturated with true cobalamins (cobalamins – HoloTC) and the black shaded area indicates HoloHC saturated with analogues (measured HoloHC + HoloTC – cobalamins). CI, confidence interval.

(Redrawn from Morkbak et al. (2007), Figure 1.³⁰)

Access to homocysteine, methylmalonic acid and holotranscobalamin assays facilitates more precise definition of cobalamin and folate status in patients in whom prolonged B₁₂ therapy may have been initiated inappropriately and continued unnecessarily or, conversely, discontinued inappropriately because of lack of confidence in the original assessment. The study by Gorringe et al.¹⁶ showed that only 27/49 patients were anaemic or macrocytic with a low total B₁₂ of <170 ng/l and elevated MMA. All of these patients treated with B₁₂ therapy corrected elevated MMA levels, suggesting the presence of a metabolic deficiency. However, only 15/27 showed a haematological response.

Homocysteine levels also fell by >25% in 47/49 patients after B₁₂ treatment. Many of these patients had no clinical evidence or symptoms of cobalamin deficiency and may reflect subjects with subclinical deficiency, which could have subtle cognitive impairment, or may just represent a compensated metabolic state of no clinical consequence. Elevation of homocysteine levels is seen in folate deficiency and is therefore less specific than MMA measurement. The causes of cobalamin deficiency are shown in Table 10.5.

Table 10.5 The causes of cobalamin deficiency

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		Supportive information/diagnostic tests
Reduced intake
	Strict vegetarian/vegan	Dietary history
	Dietary fad that excludes dairy products and meat	Ethnic origin/culture
	Breastfed babies of mothers who are vegetarian or cobalamin deficient
	Poor dietary intake in elderly
Malabsorption as a result of loss or inactivity of intrinsic factor
	Addisonian pernicious anaemia	Diagnostic criteria for pernicious anaemia (see Table 10.3)
	Gastrectomy (partial or total)	History of gastric surgery
	Bacterial overgrowth or parasitic infestation of small bowel	Radiolabelled lactose breath tests for bacterial overgrowth Repeat Schilling test post-antibiotic therapy
	Pancreatic dysfunction: failure of trypsin release of B12 from R binding proteins	Pancreatic function tests; exocrine pancreatic dysfunction results in abnormal Schilling test but clinical deficiency is rare
	Malabsorption as a result of failure of B12-intrinsic factor complex uptake in ileum – ileal resection