The author: Professor Yasser Metwally

http://yassermetwally.com

INTRODUCTION

December 1, 2009 — Even though vitamin B12 (B12) refers specifically to cyanocobalamin, the terms cobalamin (Cbl), B12, and vitamin B12 are generally used interchangeably. Cbl is required as a cofactor in several enzymatic reactions. The 2 active forms of Cbl are methylCbl and adenosylCbl (Fig. 1). [1] Fig. 1 shows the biochemical pathways involved in Cbl metabolism and Fig. 2 shows the steps involved in the gastrointestinal processing and absorption of Cbl. Cbl is transferred across the intestinal mucosa into portal blood where it binds to transCbl II (TCII). The liver takes up approximately 50% of the Cbl and the rest is transported to other tissues. TCII-bound Cbl is taken up by cells through TCII receptor-mediated endocytosis. Cbl bound to transcobalamin II (holoTC) is the fraction of total vitamin B12 available for tissue uptake. Intracellular lysosomal degradation releases Cbl for conversion to methylCbl or adenosylCbl. Most of the Cbl secreted in the bile is reabsorbed. The estimated daily losses of Cbl are minute compared with body stores. Hence, even in the presence of severe malabsorption, 2 to 5 years may pass before Cbl deficiency develops. [2] Similarly, a clinical relapse in pernicious anemia after interrupting Cbl therapy takes approximately 5 years before it is recognized.

Figure 1. Biochemistry of cobalamin and folate deficiency. MethylCbl is a cofactor for a cytosolic enzyme, methionine synthase, in a methyl-transfer reaction that converts homocysteine (Hcy) to methionine. Methionine is adenosylated to SAM, a methyl group donor required for biologic methylation reactions involving proteins, neurotransmitters, and phospholipids. Decreased SAM production leads to reduced myelin basic protein methylation and white matter vacuolization in Cbl deficiency. Methionine also facilitates the formation of formyltetrahydrofolate (THF), which is involved in purine synthesis. During the process of methionine formation methylTHF donates the methyl group and is converted into THF, a precursor for purine and pyrimidine synthesis. Impaired DNA synthesis could interfere with oligodendrocyte growth and myelin production. AdenosylCbl is a cofactor for l-methylmalonyl CoA (CoA) mutase, which catalyzes the conversion of l-methylmalonyl CoA to succinyl CoA in an isomerization reaction. Accumulation of methylmalonate and propionate may provide abnormal substrates for fatty acid synthesis. The branched-chain and abnormal odd-number carbon fatty acids may be incorporated into the myelin sheath. The biologically active folates are in the THF form. MethylTHF is the predominant folate and is required for the Cbl-dependent remethylation of Hcy to methionine. Methylation of deoxyuridylate to thymidylate is mediated by methyleneTHF. Impairment of this reaction results in accumulation of uracil, which replaces the decreased thymine in nucleoprotein synthesis and initiates the process that leads to megaloblastic anemia. CH3, methyl group; Cbl, cobalamin; THF1 and THFn, monoglutamated and polyglutamated forms of tetrahydrofolate. (Click to enlarge table)

Figure 2. Physiology of cobalamin absorption. In the stomach, Cbl bound to food is dissociated from proteins in the presence of acid and pepsin. The released Cbl binds to R proteins secreted by salivary glands and gastric mucosa. In the small intestine, pancreatic proteases partially degrade the R protein-Cbl complex at neutral pH and release Cbl, which then binds with IF. IF is a Cbl-binding protein secreted by gastric parietal cells. The IF-Cbl complex binds to specific receptors in the ileal mucosa and is internalized. In addition to the IF-mediated absorption of ingested Cbl, there is a nonspecific absorption of Cbl that occurs by passive diffusion at all mucosal sites. This is a relatively inefficient process by which 1–2% of the ingested amount is absorbed. Cbl, cobalamin; H+, acidic, OH-, alkaline; IF, intrinsic factor.  (Click to enlarge table)

• Causes of deficiency

Not infrequently the cause of Cbl deficiency is unknown. Most patients with clinically expressed Cbl deficiency have malabsorption related to intrinsic factor such as that seen in pernicious anemia. Cbl deficiency is particularly common in the elderly and is most likely caused by the high incidence of atrophic gastritis and achlorhydria-induced food-Cbl malabsorption. [3, 4] Food-bound Cbl malabsorption is rarely associated with clinically significant deficiency. Cbl deficiency is commonly seen following gastric surgery. Partial gastrectomy and bariatric surgery cause food-bound Cbl malabsorption; partial gastrectomy may also be associated with loss of intrinsic factor. [5, 6, 7, 8] Acid reduction therapy such as with H2 blockers and prolonged use of drugs like metformin can also cause Cbl deficiency. [9, 10] Other causes of Cbl deficiency include conditions associated with malabsorption such as ileal disease or resection, bacterial overgrowth, pancreatic disease, and tropical sprue. Helicobacter pylori infection of the stomach may be associated with mucosal atrophy, hypochlorhydria, and impaired splitting of bound Cbl from food proteins. Competition for Cbl secondary to parasitic infestation by the fish tapeworm Diphyllobothrium latum may cause Cbl deficiency. Certain hereditary enzymatic defects can also manifest as disorders of Cbl metabolism. [11] Increased prevalence of B12 deficiency has been recognized in patients infected with human immunodeficiency virus (HIV) with neurologic symptoms but the precise clinical significance of this is unclear. [12, 13] In AIDS-associated myelopathy the Cbl- and folate-dependent transmethylation pathway is depressed and cerebrospinal fluid and serum levels of S-adenosyl methionine are reduced. [14] Despite low B12 levels in many patients with AIDS, serum homocysteine (Hcy) and methylmalonic acid (MMA) levels are normal and Cbl supplementation fails to improve clinical manifestations.

Nitrous oxide (N2O, “laughing gas”) is a commonly used inhalational anesthetic that has been abused because of its euphoriant properties. N2O irreversibly oxidizes the cobalt core of Cbl and renders methylCbl inactive.[15] Clinical manifestations of Cbl deficiency appear relatively rapidly with N2O toxicity because the metabolism is blocked at the cellular level, but they may be delayed up to 8 weeks. [16, 17] Postoperative neurologic dysfunction can be seen with N2O exposure during routine anesthesia if subclinical Cbl deficiency is present. [6, 18] N2O toxicity caused by inhalant abuse has been reported among dentists, other medical personnel, and university students. [19, 20, 21]

Vitamin B12 deficiency is only rarely the consequence of diminished dietary intake. Strict vegetarians may rarely develop mild Cbl deficiency after years. The low vitamin B12 level noted in vegetarians is often without clinical consequences. Clinical manifestations are more likely when poor intake begins in childhood wherein limited stores and growth requirements act as additional confounders.

• Clinical significance

Neurologic manifestations may be the earliest and often the only manifestation of Cbl deficiency. [22, 23, 24, 25, 26, 27, 28] The severity of the hematologic and neurologic manifestations may be inversely related in a particular patient. Relapses are generally associated with the same neurologic phenotype. The commonly recognized neurologic manifestations may include a myelopathy with or without an associated neuropathy, cognitive impairment, optic neuropathy (impaired vision, optic atrophy, centrocecal scotomas), and paresthesias without abnormal signs.

The best characterized neurologic manifestation of Cbl deficiency is a myelopathy that has commonly been referred to as subacute combined degeneration. The neurologic features typically include a spastic paraparesis, extensor plantar response, and impaired perception of position and vibration. Accompanying optic nerve involvement may be present. The most severely involved regions are the cervical and upper thoracic posterior columns. Changes are also seen in the lateral columns. Involvement of the anterior columns is rare. Spongiform changes and foci of myelin and axon destruction are seen in the spinal cord white matter. There is myelin loss followed by axonal degeneration and gliosis. Neuropsychiatric manifestations include decreased memory, personality change, psychosis, and rarely delirium. [24, 26] High total vitamin B12 intake or higher vitamin B12 concentrations and MMA levels have been associated with slower cognitive decline among the very elderly. [29, 30] A recent study of Cbl status and rate of brain volume loss in community-dwelling elderly individuals noted that decrease in brain volume was greater among those with lower Cbl and holoTC levels and higher plasma total Hcy and MMA levels. [31]

Rarely reported neurologic manifestations related to Cbl deficiency include cerebellar ataxia, orthostatic tremors, ophthalmoplegia, vocal cord paralysis, a syringomyelia-like distribution of motor and sensory deficits, and autonomic dysfunction. [32, 33, 34, 35, 36, 37, 38, 39]

Clinical, electrophysiologic, and pathologic involvement of the peripheral nervous system has been described with Cbl deficiency. In a one study Cbl deficiency was detected in 27 of 324 patients with a polyneuropathy. [40] Clues to possible B12 deficiency in a patient with polyneuropathy included a relatively sudden onset of symptoms, findings suggestive of an associated myelopathy, onset of symptoms in the hands, macrocytic red blood cells, and the presence of a risk factor for Cbl deficiency.

Serum Cbl can be normal in some patients with Cbl deficiency and serum MMA and total Hcy levels are useful in diagnosing patients with Cbl deficiency. [2, 41, 42, 43, 44, 45] The sensitivity of the available metabolic tests for Cbl deficiency has facilitated the development of the concept of subclinical Cbl deficiency. [46, 47] This refers to biochemical evidence of Cbl deficiency in the absence of hematologic or neurologic manifestations. These biochemical findings should respond to Cbl therapy if Cbl deficiency is their true cause. The frequency of subclinical Cbl deficiency is estimated to be at least 10 times that of clinical Cbl deficiency and its incidence increases with age. [47, 48, 49] The cause of subclinical Cbl deficiency includes food-bound Cbl malabsorption but is frequently unknown; the course is often stationary. [50, 51] Some of these individuals may have subtle neurologic and neurophysiologic abnormalities of uncertain significance that may respond to Cbl therapy. [52, 53] The presence of a low Cbl level in association with neurologic manifestations does not imply cause and effect or indicate the presence of metabolic Cbl deficiency. The incidence of cryptogenic polyneuropathy, cognitive impairment, and Cbl deficiency increases with age and Cbl deficiency may be a chance occurrence rather than causative. Although Cbl levels are frequently low in the elderly, up to one-third are falsely low by clinical and metabolic criteria, and many of the remainder are clinically innocent. [3, 48, 54, 55] The clinical impact of subclinical Cbl deficiency and its appropriate management are uncertain. If it is unclear whether an elevated MMA or Hcy level is caused by Cbl deficiency, the response to empirical parenteral B12 replacement can be assessed.

• Investigations

Serum Cbl determination has been the mainstay for evaluating Cbl status.12, 65, 66 The older microbiologic and radioisotopic assays have been replaced by immunologically based chemiluminescence assays. Although it is a widely used screening test, serum Cbl measurement has technical and interpretive problems and lacks sensitivity and specificity for the diagnosis of Cbl deficiency (Table 1). [2, 42, 43, 44, 47, 48, 56, 57, 58, 59, 60, 61] Levels of serum MMA and plasma total Hcy are useful as ancillary diagnostic tests. [2, 41, 42, 43, 44, 45] The specificity of MMA is superior to that of plasma Hcy. Although plasma total Hcy is a very sensitive indicator of Cbl deficiency, its major limitation is its poor specificity. Table 1 indicates causes other than Cbl deficiency that can result in abnormal levels of Cbl, MMA, and Hcy. Hcy should be measured either fasting or after an oral methionine load. Immediate refrigeration of the blood sample is important because levels of Hcy increase if whole blood is left at room temperature for hours. Measuring MMA and Hcy is also useful in patients with N2O toxicity and those with inherited metabolic disorders. In these conditions vitamin B12-dependent pathways are impaired despite normal vitamin B12 levels. Vitamin B12 bound to holoTC is the fraction of total vitamin B12 available for tissue uptake and therefore has been proposed by some as a potentially useful alternative indicator of vitamin B12 status. [62, 63, 64, 65, 66, 67, 68, 69, 70]

Table 1.  Common causes, other than Cbl deficiency, for abnormal Cbl, MMA, and Hcy levels [46, 47, 56]

An increase in the mean corpuscular volume may precede development of anemia. [71] The presence of neutrophil hypersegmentation may be a sensitive marker for Cbl deficiency and may be seen in the absence of anemia or macrocytosis. Megaloblastic bone marrow changes may be seen.

To determine the cause of Cbl deficiency, tests directed at determining the cause of malabsorption are undertaken. Concerns regarding cost, accuracy, and radiation exposure have led to a significant decrease in the availability of the Schilling test. [82] Elevated serum gastrin and decreased pepsinogen I levels are seen in 80% to 90% of patients with pernicious anemia but the specificity of these tests is limited. [73] Elevated gastrin levels are a marker for hypochlorhydria or achlorhydria, which are invariably seen with pernicious anemia. Elevated gastrin levels may be seen in up to 30% of the elderly. [4] Elevated serum gastrin levels are approximately 70% specific and sensitive for pernicious anemia. [74] Anti-intrinsic factor antibodies are specific (>95%) but lack sensitivity and are found in approximately 50% to 70% of patients with pernicious anemia. [75, 76, 77] Studies suggest that antiparietal cell antibodies may not be seen as commonly as was earlier believed and therefore have limited usefulness. [77] False-positive results for the gastric parietal cell antibody are common. They may be seen in 10% of people more than 70 years old and are also present in other autoimmune endocrinopathies. A common approach is to combine the specific but insensitive intrinsic factor antibody test with the sensitive but nonspecific serum gastrin or pepsinogen level in patients with Cbl deficiency. [55]

Electrophysiologic abnormalities include nerve conduction studies suggestive of a sensorimotor axonopathy, and abnormalities on somatosensory evoked potentials, visual evoked potentials, and motor evoked potentials. [78, 79]

• Neuroimaging of Vitamin B 12 deficiency

Magnetic resonance imaging (MRI) abnormalities include a signal change in the subcortical white matter and posterior and lateral columns. (Fig. 3) [79, 80, 81, 82] Similar spinal cord MRI findings are seen with nitrous oxide toxicity. [31] Brain T2-hyperintensities seen in Cbl deficiency may show significant improvement with vitamin B12 replacement [93] Contrast enhancement involving the dorsal or lateral columns may be present. [94, 95] The dorsal column may show a decreased signal on T1-weighted images. [95] Other reported findings include cord atrophy and anterior column involvement. [86, 87] Treatment may be accompanied by reversal of cord swelling, contrast enhancement, and signal change. [79, 80, 82, 85, 87] Increased T2 signals involving the cerebellum are also reported. [37, 78] Rarely striking diffuse white matter abnormalities suggestive of a leukoencephalopathy may be seen. [37, 89]  {Click for more details)

Figure 3. TSET2W sagittal images show symmetric well defined hyperintensity involving bilateral posterior columns.

Figure 1. Subacute combined degeneration-vitamin B12 deficiency. (A) Sagittal T2-weighted image with signal abnormality in posterior columns (arrows). (B) Sagittal T1- weighted image shows no significant intramedullary signal abnormality. (C) Axial gradient-echo sequence confirms posterior column signal abnormality (arrow). (Click to enlarge figure)

• Management

The goals of treatment are to reverse the signs and symptoms of deficiency, replete body stores, ascertain the cause of deficiency, and monitor response to therapy. With normal Cbl absorption, oral administration of 3 to 5 µg of cyanoCbl may suffice. In patients with food-bound Cbl malabsorption caused by achlorhydria, 50 to 100 µg of cyanoCbl given orally is often adequate. [90] More recent studies have shown blunted metabolic responses in elderly persons with subclinical deficiency until oral doses reached 500 µg or more. [91, 92, 93] Patients with Cbl deficiency caused by achlorhydria-induced food-bound Cbl malabsorption show normal absorption of crystalline B12 but are unable to digest and absorb Cbl in food because of achlorhydria. The more common situation is one of impaired absorption where parenteral therapy is required. A short course of daily or weekly therapy is often followed by monthly maintenance therapy (see Table 1). If the oral dose is large enough, even patients with an absorption defect may respond to oral Cbl. [94] The response to oral therapy is less predictable and relapse occurs following cessation of oral therapy sooner than with parenteral regimens. [23, 95]

Patients with pernicious anemia have a higher risk of gastric cancer and carcinoids, and therefore should undergo endoscopy. [96] Patients with pernicious anemia also have a higher frequency of thyroid disease and iron deficiency and should be screened for these conditions. [97, 98]

Patients with B12 deficiency are prone to develop neurologic deterioration following N2O anesthesia. This situation is preventable by prophylactic vitamin B12 given weeks before surgery in individuals with a borderline B12 level who are expected to receive N2O anesthesia. Intramuscular B12 should be given to patients with acute N2O poisoning. Methionine supplementation has also been proposed as a first-line therapy. [99] With chronic exposure, immediate cessation of exposure should be ensured. In AIDS-associated myelopathy, a possible benefit of administration of the S-adenosyl methionine precursor, l-methionine, was suggested by a pilot study but not confirmed in a subsequent double-blind study. [100, 101]

Response to treatment may relate to the extent of involvement and delay in starting treatment.36 Remission correlates inversely with the time lapsed between symptom onset and initiation of therapy. Response of the neurologic manifestations is variable, may be incomplete, often starts in the first week, and is complete in 6 months. [55] Response of the hematologic derangements is prompt and complete. Reticulocyte count begins to increase within 3 days and peaks around 7 days. Red blood cell count begins to increase by 7 days and is followed by a decline in mean corpuscular volume with normalization by 8 weeks. MMA and Hcy levels normalize by 10 to 14 days. Cbl and holoTC levels increase after injection regardless of the benefit. Hence, MMA and Hcy are more reliable for monitoring response to therapy. In patients with severe Cbl deficiency, replacement therapy may be accompanied by hypokalemia as a result of proliferation of bone marrow cells that use potassium. The clinical significance of this hypokalemia is unproven. [55, 102]

HydroxoCbl is commonly used in parts of Europe. It is more allergenic but has superior retention and may permit injections every 2 to 3 months. [103] Compared with hydroxoCbl, cyanoCbl binds to serum proteins less well and is excreted more rapidly. [104] Intranasal administration of hydroxoCbl has been associated with fast absorption and normalization of Cbl levels. [105] Advantages of delivering Cbl by the nasal or sublingual route are unproven. Oral preparations of intrinsic factor are available but not reliable. Antibodies to intrinsic factor may nullify its effectiveness in the intestinal lumen.

Neurologically affected patients may have high folate levels. [28] Inappropriate folate therapy may delay recognition of the Cbl deficiency and cause neurologic deterioration. Anemia caused by Cbl deficiency often responds to folate therapy but the response is incomplete and transient. It is unclear if routine folate supplementation may compromise the early diagnosis of the hematologic manifestations or worsen the neurologic consequences

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