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The Many Faces of Cobalamin (Vitamin B12) Deficiency

      Abstract

      Although cobalamin (vitamin B12) deficiency was described over a century ago, it is still difficult to establish the correct diagnosis and prescribe the right treatment. Symptoms related to vitamin B12 deficiency may be diverse and vary from neurologic to psychiatric. A number of individuals with vitamin B12 deficiency may present with the classic megaloblastic anemia.
      In clinical practice, many cases of vitamin B12 deficiency are overlooked or sometimes even misdiagnosed. In this review, we describe the heterogeneous disease spectrum of patients with vitamin B12 deficiency in whom the diagnosis was either based on low serum B12 levels, elevated biomarkers like methylmalonic acid and/or homocysteine, or the improvement of clinical symptoms after the institution of parenteral vitamin B12 therapy. We discuss the possible clinical signs and symptoms of patients with B12 deficiency and the various pitfalls of diagnosis and treatment.

      Abbreviations and Acronyms:

      CoA (coenzyme A), holoTC (holotranscobalamin), IF (intrinsic factor), IM (intramuscularly), MMA (methylmalonic acid)
      Several scientific articles and textbooks have described the clinical presentation of patients with cobalamin (vitamin B12) deficiency.
      • Healton E.B.
      • Savage D.G.
      • Brust J.C.
      • Garrett T.J.
      • Lindenbaum J.
      Neurologic aspects of cobalamin deficiency.
      • Stabler S.P.
      Vitamin B12 deficiency.
      After the classic presentation of Addison-Biermer disease with megaloblastic anemia, many generations of doctors have been educated with the view that vitamin B12 deficiency exclusively presents itself with this type of anemia. Additional cases have been reported in which neurologic abnormalities were the main presenting symptom, with subacute combined degeneration of the spinal cord as one of the most feared manifestations,
      • Stone R.E.
      • Spies T.D.
      Vitamin B12 and subacute combined degeneration of the spinal cord.
      often leading to permanent disability. Lindenbaum et al
      • Lindenbaum J.
      • Healton E.B.
      • Savage D.G.
      • et al.
      Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis.
      reported a large series of 40 patients who had neurologic symptoms or psychiatric disorders caused by vitamin B12 deficiency but who had no anemia or macrocytosis. Psychiatric symptoms may vary from depression to mania, psychosis, and occasionally suicidal thoughts (Supplemental Table 1, available online at http://mcpiqojournal.org).
      • Zucker D.K.
      • Livingston R.L.
      • Nakra R.
      • Clayton P.J.
      B12 deficiency and psychiatric disorders: case report and literature review.
      The reason why some patients mainly present with megaloblastic anemia and others with neurologic symptoms remains unknown.
      Laboratory investigations with the establishment of low serum B12 levels and elevated levels of methylmalonic acid (MMA) are the cornerstone of diagnostics, but normal levels of serum B12 and MMA do not exclude symptomatic B12 deficiency. In clinical practice, many cases of B12 deficiency are overlooked or sometimes even misdiagnosed because of misconceptions and misbeliefs among health care professionals. We have summarized the most frequently encountered misconceptions and misbeliefs regarding vitamin B12 deficiency in Table 1.
      Table 1Frequently Reported Misconceptions and Misbeliefs Regarding Vitamin B12 Deficiency
      Diagnosis: You do not have vitamin B12 deficiency because
      • You have no anemia
      • You have no macrocytic anemia
      • Your serum vitamin B12 level is within the reference range of 140 to 450 pmol/L
      • Your serum vitamin B12 level is only moderately low
      • Your serum vitamin B12 level may be low, but your plasma level of methylmalonic acid is completely normal
      • Vitamin B12 deficiency only occurs in elderly people
      • Vitamin B12 deficiency never occurs in children
      Treatment
      • Oral therapy is as good as or even better than painful injections to alleviate neurologic symptoms
      • You may stop treatment because your serum vitamin B12 level has normalized
      • You must stop treatment because your serum vitamin B12 level is elevated
      • You must stop after 5 injections because vitamin B12 can have serious adverse effects
      • You must stop the injections because your symptoms have worsened
      • We must measure your serum vitamin B12 level after 3 injections to see whether the treatment is successful
      • We must stop vitamin B12 injections now that you are pregnant
      In this review, we discuss a number of typical patients who were seen at our outpatient clinic, and we summarize the possible clinical signs and symptoms of patients with vitamin B12 deficiency and the pitfalls of diagnosis and treatment.

      Case Descriptions

      Patient A

      Patient A is a 55-year-old woman admitted to the psychiatry department of our hospital because of depression. In addition to symptoms related to her depression, she reported pain in the lower legs, paresthesia and numbness in the feet, and difficulty walking. Her history included Graves disease, for which she had undergone thyroidectomy several years earlier and was subsequently prescribed levothyroxine. We were invited for consultation because of slightly elevated free thyroxine levels. The patient had no symptoms suggestive of thyrotoxicosis. On examination, she had signs of severe peripheral neuropathy of the lower legs (absent reflexes, superficial and deep sensation) and several skin lesions compatible with vitiligo. Laboratory studies (also summarized in Supplemental Table 2, available online at http://mcpiqojournal.org) (reference ranges provided parenthetically) revealed a hemoglobin level of 7.0 mmol/L (>7.5 mmol/L) and mean corpuscular volume of 105 fl (85-98 fl). Her serum vitamin B12 level was 51 pmol/L (145-450 pmol/L). These findings prompted the diagnosis of pernicious anemia caused by vitamin B12 deficiency, and hydroxocobalamin injections were initiated immediately. Within 3 to 4 weeks, the patient reported that her symptoms gradually lessened, and her walking capacity improved. Additional laboratory tests revealed the presence of antibodies against parietal cells and intrinsic factor (IF). Six weeks later, the attending psychiatric resident ordered an additional serum vitamin B12 measurement. Because the serum vitamin B12 level was greater than 1476 pmol/L, hydroxocobalamin injections were stopped after consultation with an internal medicine resident. The patient was discharged home 3 months later, but no follow-up appointment at our outpatient clinic was made. The discharge letter did not mention the diagnosis of pernicious anemia, nor the treatment with hydroxocobalamin injections. This exclusion went unnoticed until 8 months later, when we realized she was lost to follow-up and invited her for a follow-up visit. She presented at our outpatient clinic shortly thereafter and reported a severe increase of her symptoms. One month earlier, her primary care physician had ordered measurement of her serum vitamin B12 level, which was 251 pmol/L; he did not restart treatment because he was unaware of the earlier findings. We strongly advised the patient to recommence hydroxocobalamin injections, which she continued twice weekly for the following 2 years. Gradually, her symptoms abated. Two years later, there were still bouts of pain, numbness, and paresthesia, but she was able to walk supported only by a walking stick. This case is a clear example of pernicious anemia in the constellation of a polyglandular autoimmune syndrome. Unwarranted cessation of therapy may lead to severe worsening of neurologic abnormalities and irreversible damage.

      Patient B

      Patient B is a 17-year-old girl who had a 2-year history of fatigue, sleepiness, numbness in her hands, dizziness, exertional dyspnea, and problems with concentrating and finding the correct words while in conversations. Table 2 summarizes her most important symptoms. Because her mother had presented with symptomatic vitamin B12 deficiency 6 months earlier, she was tested as well. Clinical examination did not reveal major abnormalities. Laboratory examination yielded a total serum vitamin B12 level of 112 pmol/L, with an intermediate level of holotranscobalamin (holoTC) of 54 pmol/L (40-125 pmol/L) and a low total haptocorrin concentration of 162 pmol/L (203-596 pmol/L). Her MMA value was 178 nmol/L (<300 pmol/L), homocysteine level was 11 pmol/L (<10 pmol/L), and folate concentration was 19 nmol/L (10-36 nmol/L). Antibodies against parietal cells, IF, and transglutaminase/gliadin were absent. Because of our clinical suspicion of vitamin B12 deficiency, she was treated with hydroxocobalamin, 1000-μg injections twice weekly, with a beneficial effect on her symptoms (Table 2). As can be appraised from her symptom list, many improved considerably or resolved completely. This case illustrates the clear clinical syndrome of vitamin B12 deficiency despite intermediate holoTC and normal MMA levels.
      Table 2Self-Reported Symptoms of Patient B Before and 1 and 6 Months After Initiation of Hydroxocobalamin Therapy
      Symptoms rated on a scale of 0 to 10, in which 0 = no symptoms and 10 = worst symptoms.
      VariableBefore treatmentAfter treatment
      1 mo6 mo
      Numbness in hands541
      Difficulties focusing543
      Not being able to find the right words640
      Mood changes760
      Pain in mouth and tongue664
      Fatigue, weakness990
      Nausea, reduced appetite860
      Menstrual pains998
      Pain in joints663
      Dyspnea on exertion681
      Dizzy spells750
      Pale skin554
      Feeling cold880
      Muscle cramps664
      Stomach complaints, acidity640
      a Symptoms rated on a scale of 0 to 10, in which 0 = no symptoms and 10 = worst symptoms.

      Patient C

      Patient C is a 15-year-old girl. She had no specific symptoms. Her exercise capacity was normal, and her school grades were fine. Because her brother was diagnosed as having symptomatic vitamin B12 deficiency at age 17 years and was subsequently successfully treated, she was referred for evaluation. Physical examination yielded no abnormalities. Laboratory investigation revealed a serum vitamin B12 level of 94 pmol/L initially and 114 pmol/L on repeated testing, MMA value of 218 nmol/L, homocysteine concentration of 78.4 μmol/L, and folate level of 6.7 nmol/L. Extensive evaluation showed no additional abnormalities, especially no signs of celiac disease, or antibodies against parietal cells or IF. Whole-exome sequencing of genes related to metabolic disorders yielded only the well-known homozygous c.677C>T mutation in the gene coding for methylenetetrahydrofolate reductase. It is known that people with this mutation have increased risk of mild hyperhomocysteinemia with vitamin B12 and/or folate deficiency.
      • Kim J.
      • Kim H.
      • Roh H.
      • Kwon Y.
      Causes of hyperhomocysteinemia and its pathological significance.
      This patient had no clinical symptoms related to vitamin B12 deficiency and a severely elevated homocysteine level, but the genetic mutation is not considered to be the sole cause of this patient's vitamin B12 deficiency and marked elevation of homocysteine.

      Patient D

      Patient D is a 33-year-old woman with a history of fatigue, inertia, indolence, paresthesia in her hands and feet, difficulties concentrating, problems with remembering things, and word finding disturbances. An acquaintance mentioned to her the possibility of vitamin B12 deficiency. Her primary care physician ordered a serum vitamin B12 measurement, which was 190 pmol/L, and subsequently advised her to start hydroxocobalamin injections. Her MMA and homocysteine levels were unfortunately not measured. After 5 weeks of treatment, her symptoms had decreased considerably. Her physician then ordered repeated serum vitamin B12 measurement, which revealed a value of more than 1476 pmol/L. Subsequently, the physician became concerned about possible vitamin B12 intoxication and asked the patient to stop treatment. Five to 6 weeks later, her symptoms had increased substantially. She was referred to our clinic for evaluation. Her main question was why this successful treatment was stopped. Hydroxocobalamin injections were restarted, in a frequency of twice weekly, which resulted in a gradual improvement of her symptoms. This case illustrates the difficulties of an incomplete diagnostic work-up and also the dangers of measuring serum vitamin B12 levels during parenteral administration. Hydroxocobalamin is not toxic, and successful treatment should not be stopped. Usually, the period between injections can be prolonged when all symptoms have disappeared.

      Patient E

      Patient E is a 68-year-old woman who was treated by a hematologist in another hospital for a period of 3 years because of macrocytic anemia (hemoglobin, 7.5 pmol/L; mean corpuscular volume, 110 fl). There was no history of alcohol abuse. Extensive evaluation revealed no other abnormalities, and her serum vitamin B12 value was 301 pmol/L. She had already been treated for several years with levothyroxine because of primary hypothyroidism. Myelodysplastic syndrome was diagnosed by bone marrow aspiration, and she received blood transfusions twice. In 2017, she experienced progressive pains in her legs and almost completely lost the feeling in her feet; she was not able to walk outside her house anymore. Even walking in her home was extremely difficult and painful. Additional laboratory examinations did not reveal any new abnormalities. She was admitted to the neurology department in another hospital and found to have severe polyneuropathy. Magnetic resonance imaging of the spinal cord showed symmetric bilateral high signal within the dorsal columns, suggestive of subacute combined degeneration of the cord; magnetic resonance imaging of the brain revealed minimal white matter lesions. Again, her serum vitamin B12 level was normal, as were thiamine, pyridoxine, and folate values. No signs or laboratory abnormalities consistent with connective tissue disease or other causes of polyneuropathy were found. Biochemical and microbiological evaluation of cerebrospinal fluid revealed no abnormalities. After 2 weeks of observation, her doctors decided to initiate hydroxocobalamin treatment. Two days later, results of additional testing were returned: her MMA level was 37,000 nmol/L, and her homocysteine value was 165 μmol/L. These findings confirmed the existence of severe vitamin B12 deficiency. Additional testing yielded a high titer of antibodies against IF and parietal cells. Because of uncertainty about the diagnosis, she was referred to our outpatient clinic 2 months later. No additional clues arose from history or physical examination. Repeated examination of the bone marrow specimen did not confirm a diagnosis of myelodysplastic syndrome and showed only a slight increase of erythropoiesis. The most likely diagnosis was Addison-Biermer disease and interference in the vitamin B12 assay by the IF autoantibodies. No serum samples were left to test this hypothesis. Six months after diagnosis and initiation of high-dose hydroxocobalamin treatment, the patient still had considerable neurologic damage, loss of sensations in her feet and legs, and inability to walk without the use of a rollator walker. The anemia has resolved completely. This case is a classic example of how the assay for serum vitamin B12 can be wrong because of interference of the assay by IF autoantibodies.

      Patient F

      Patient F is a 62-year-old woman with a body mass index of 36.6 kg/m2. She underwent a Roux-en-Y gastric bypass procedure 8 months earlier, which resulted in a 40-kg weight loss. During a routine visit to the outpatient clinic, she reported fatigue and difficulties in concentrating and executing difficult tasks. She took multivitamins that were prescribed by the physician in the bariatric surgery center. Before the operation, she underwent a laboratory evaluation that revealed the following: serum vitamin B12, 303 pmol/L; folate, 15.4 nmol/L; and vitamin D, 59 nmol/L. At reassessment, her serum vitamin B12 concentration was 249 pmol/L, and her MMA level was 1380 nmol/L. Because of the suspicion of vitamin B12 deficiency, treatment with hydroxocobalamin injections, 1000 μg twice weekly, was initiated, after which most of her symptoms disappeared within 6 to 8 weeks. This case illustrates that vitamin B12 levels within the reference range do not exclude symptomatic vitamin B12 deficiency.

      Discussion

      We have described the disease spectrum of a number of patients who attended our outpatient clinic with possible vitamin B12 deficiency in whom the diagnosis was either based on low serum vitamin B12 levels, elevated biomarkers like MMA and/or homocysteine, or the improvement of clinical symptoms after the institution of parenteral vitamin B12 therapy. Altogether, these cases show the considerable heterogeneity of vitamin B12 deficiency. Not all cases were easy to recognize or diagnose. The most prevalent symptoms of vitamin B12 deficiency are neurologic, such as paresthesia in hands and feet, muscle cramps, dizziness, cognitive disturbances, ataxia, and erectile dysfunction, as well as fatigue, psychiatric symptoms like depression, and macrocytic anemia.
      • Healton E.B.
      • Savage D.G.
      • Brust J.C.
      • Garrett T.J.
      • Lindenbaum J.
      Neurologic aspects of cobalamin deficiency.
      • Stabler S.P.
      Vitamin B12 deficiency.
      • Kim J.M.
      • Stewart R.
      • Kim S.W.
      • Yang S.J.
      • Shin I.S.
      • Yoon J.S.
      Predictive value of folate, vitamin B12 and homocysteine levels in late-life depression.
      • Oberlin B.S.
      • Tangney C.C.
      • Gustashaw K.A.
      • Rasmussen H.E.
      Vitamin B12 deficiency in relation to functional disabilities.
      • Hunt A.
      • Harrington D.
      • Robinson S.
      Vitamin B12 deficiency.
      • Green R.
      • Allen L.H.
      • Bjørke-Monsen A.L.
      • et al.
      Vitamin B12 deficiency (published correction appears in Nat Rev Dis Primers. 2017;3:17054).
      However, there is evidence that in the Western world as well as China, less than 20% of people with demonstrable low serum vitamin B12 levels have macrocytic anemia.
      • Metz J.
      A high prevalence of biochemical evidence of vitamin B12 or folate deficiency does not translate into a comparable prevalence of anemia.
      • den Elzen W.P.
      • Westendorp R.G.
      • Frölich M.
      • de Ruijter W.
      • Assendelft W.J.
      • Gussekloo J.
      Vitamin B12 and folate and the risk of anemia in old age: the Leiden 85-Plus Study.
      • Yildirim T.
      • Yalcin A.
      • Atmis V.
      • et al.
      The prevalence of anemia, iron, vitamin B12, and folic acid deficiencies in community dwelling elderly in Ankara, Turkey.
      • Wong C.W.
      • Ip C.Y.
      • Leung C.P.
      • Leung C.S.
      • Cheng J.N.
      • Siu C.Y.
      Vitamin B12 deficiency in the institutionalized elderly: a regional study.
      Although the demonstration of low serum vitamin B12 levels is considered diagnostic, there is a poor correlation between these levels and symptoms,
      • Oberlin B.S.
      • Tangney C.C.
      • Gustashaw K.A.
      • Rasmussen H.E.
      Vitamin B12 deficiency in relation to functional disabilities.
      • Hunt A.
      • Harrington D.
      • Robinson S.
      Vitamin B12 deficiency.
      • Solomon L.R.
      Low cobalamin levels as predictors of cobalamin deficiency: importance of comorbidities associated with increased oxidative stress.
      and even people with vitamin B12 levels below 140 pmol/L may not have symptoms.
      • Martin S.S.
      • Daya N.
      • Lutsey P.L.
      • et al.
      Thyroid function, cardiovascular risk factors, and incident atherosclerotic cardiovascular disease: the Atherosclerosis Risk in Communities (ARIC) Study.
      This factor sheds a different light on the discussion regarding appropriate cutoff levels for serum vitamin B12 and related parameters. Some investigators suggest that it is necessary to establish different reference cutoffs according to age and the applied analytic method.
      • Aparicio-Ugarriza R.
      • Palacios G.
      • Alder M.
      • Gonzaléz-Gross M.
      A review of the cut-off points for the diagnosis of vitamin B12 deficiency in the general population.
      However, serum vitamin B12 tests also may fail because many people with symptoms related to cobalamin deficiency may have serum vitamin B12 levels above the lower reference level of 140 pmol/L.
      • Solomon L.R.
      Cobalamin-responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid, and homocysteine testing.
      • Roessler F.C.
      • Wolff S.
      Rapid healing of a patient with dramatic subacute combined degeneration of spinal cord: a case report.
      Although several factors may be of influence, in a considerable number of cases this issue can be caused by the earlier use of oral supplementation with multivitamins or high-dose oral vitamin B12 preparations.
      • Dobson R.
      • Alvares D.
      The difficulties with vitamin B12.
      It has been reported that even a dose of 10 μg/d can increase vitamin B12 levels to more than 200 pmol/L in elderly individuals (>65 years).
      • Hill M.H.
      • Flatley J.E.
      • Barker M.E.
      • et al.
      A vitamin B-12 supplement of 500 μg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.
      Oral supplementation may increase the serum vitamin B12 level but often not enough to replenish the vitamin B12 levels in the tissues
      • Hill M.H.
      • Flatley J.E.
      • Barker M.E.
      • et al.
      A vitamin B-12 supplement of 500 μg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.
      unless very high doses (1000-2000 μg/d) are used.

      Biochemistry and Diagnosis

      Vitamin B12 deficiency may be easily overlooked in patients when only total serum vitamin B12 is used as a status marker.
      • Solomon L.R.
      Cobalamin-responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid, and homocysteine testing.
      • Vugteveen I.
      • Hoeksma M.
      • Monsen A.L.
      • et al.
      Serum vitamin B12 concentrations within reference values do not exclude functional vitamin B12 deficiency in PKU patients of various ages.
      • Schrempf W.
      • Eulitz M.
      • Neumeister V.
      • et al.
      Utility of measuring vitamin B12 and its active fraction, holotranscobalamin, in neurological vitamin B12 deficiency syndromes.
      • Herrmann W.
      • Obeid R.
      Cobalamin deficiency.
      • Vashi P.
      • Edwin P.
      • Popiel B.
      • Lammersfeld C.
      • Gupta D.
      Methylmalonic acid and homocysteine as indicators of vitamin B-12 deficiency in cancer.
      Many investigators therefore advocate measuring one or both of the additional functional biomarkers MMA and homocysteine to establish vitamin B12 deficiency. Especially in people with so-called borderline vitamin B12 levels, ie, those between 140 and 300 pmol/L, elevated MMA and/or homocysteine values may aid in establishing a possible diagnosis of deficiency.
      • Solomon L.R.
      Cobalamin-responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid, and homocysteine testing.
      • Spence J.D.
      Metabolic vitamin B12 deficiency: a missed opportunity to prevent dementia and stroke.
      • Harrington D.J.
      Laboratory assessment of vitamin B12 status.
      The mechanism by which these biomarkers may demonstrate cobalamin deficiency can be derived from the role of vitamin B12 in our body. Vitamin B12 is a pivotal cofactor in 2 enzymatic reactions. Its deficiency will impair the proper function of these enzymes and lead to accumulation of the substrate. Methylmalonyl–coenzyme A (CoA) mutase, which catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA, is one of the vitamin B12–dependent enzymes, and impaired function will cause elevations of methylmalonyl-CoA and thereby MMA after cleavage of CoA. The other vitamin B12–dependent enzyme is methionine synthase, which regenerates methionine from homocysteine, and impaired activity as the consequence of vitamin B12 deficiency will lead to accumulation of homocysteine.
      • Stabler S.P.
      • Marcell P.D.
      • Podell E.R.
      • Allen R.H.
      • Savage D.G.
      • Lindenbaum J.
      Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry.
      • Bailey R.L.
      • Durazo-Arvizu R.A.
      • Carmel R.
      • et al.
      Modeling a methylmalonic acid-derived change point for serum vitamin B-12 for adults in NHANES.
      From this process, it can be understood how low tissue levels of cobalamin will increase MMA and homocysteine, and a clear relationship between serum vitamin B12, MMA, and homocysteine has been described in the National Health and Nutrition Examination Survey population.
      • Bailey R.L.
      • Durazo-Arvizu R.A.
      • Carmel R.
      • et al.
      Modeling a methylmalonic acid-derived change point for serum vitamin B-12 for adults in NHANES.
      Nevertheless, the sensitivity and specificity of elevated MMA and/or homocysteine levels in patients with symptoms associated with vitamin B12 deficiency are unknown. Also, it has been documented that MMA levels are elevated in people with severely impaired renal function.
      • Iqbal N.
      • Azar D.
      • Yun Y.M.
      • Ghausi O.
      • Ix J.
      • Fitzgerald R.L.
      Serum methylmalonic acid and holotranscobalamin-II as markers for vitamin B12 deficiency in end-stage renal disease patients.
      Similarly, elevated homocysteine values can also be the consequence of folate or vitamin B6 deficiency, as well as impaired renal function, hypothyroidism, and certain medications.
      • Kim J.
      • Kim H.
      • Roh H.
      • Kwon Y.
      Causes of hyperhomocysteinemia and its pathological significance.
      In a separate study, we calculated from the National Health and Nutrition Examination Survey and Lifelines epidemiological studies that MMA and/or homocysteine levels are elevated above current reference values (>300 nmol/L and 10 μmol/L, respectively) in only 73% of people with low serum vitamin B12 levels of less than 140 pmol/L and in 28% of patients with serum vitamin B12 levels between 140 and 300 pmol/L (B.H.R.W., H.J.C.M.W., J.E. Kootstra-Ros, et al, unpublished data, 2019). Other studies have confirmed that normal levels of MMA may be measured even in situations of very low vitamin B12 levels.
      • Ispir E.
      • Serdar M.A.
      • Ozgurtas T.
      • et al.
      Comparison of four automated serum vitamin B12 assays.
      In addition, there are isolated reports that serum vitamin B12, homocysteine, and MMA levels are unreliable predictors of vitamin B12–responsive neurologic disorders.
      • Graber J.J.
      • Sherman F.T.
      • Kaufmann H.
      • Kolodny E.H.
      • Sathe S.
      Vitamin B12-responsive severe leukoencephalopathy and autonomic dysfunction in a patient with “normal” serum B12 levels.
      Genetic studies have found that in addition to mutations in the MMUT gene (for expansion of gene symbols, use search tool at www.genenames.org) associated with methylmalonylaciduria, single-nucleotide polymorphisms in HIBCH and ACSF3 have been associated with MMA levels.
      • Sloan J.L.
      • Johnston J.J.
      • Manoli I.
      • et al.
      Exome sequencing identifies ACSF3 as a cause of combined malonic and methylmalonic aciduria.
      • Molloy A.M.
      • Pangilinan F.
      • Mills J.L.
      • et al.
      A common polymorphism in HIBCH influences methylmalonic acid concentrations in blood independently of cobalamin.
      Similarly, serum vitamin B12 levels may also be influenced by specific polymorphisms or mutations.
      • Carmel R.
      • Parker J.
      • Kelman Z.
      Genomic mutations associated with mild and severe deficiencies of transcobalamin I (haptocorrin) that cause mildly and severely low serum cobalamin levels.
      • Oussalah A.
      • Besseau C.
      • Chery C.
      • et al.
      Helicobacter pylori serologic status has no influence on the association between fucosyltransferase 2 polymorphism (FUT2 461 G→A) and vitamin B-12 in Europe and West Africa.
      • Surendran S.
      • Adaikalakoteswari A.
      • Saravanan P.
      • Shatwaan I.A.
      • Lovegrove J.A.
      • Vimaleswaran K.S.
      An update on vitamin B12-related gene polymorphisms and B12 status.
      One of these factors is the gene FUT2, in which the FUT2 secretor variant genotype AA is associated with a 10% to 25% higher total and haptocorrine-bound vitamin B12 level but not holoTC/active vitamin B12.
      • Velkova A.
      • Diaz J.E.L.
      • Pangilinan F.
      • et al.
      The FUT2 secretor variant p.Trp154Ter influences serum vitamin B12 concentration via holo-haptocorrin, but not holo-transcobalamin, and is associated with haptocorrin glycosylation.
      During recent years, the clinical usefulness of measuring holoTC as a screening for vitamin B12 status has received attention. HoloTc is the biologically active form of vitamin B12 in plasma. Some studies have suggested that holoTC has a better diagnostic accuracy than total serum vitamin B12 level.
      • Nexo E.
      • Hoffmann-Lücke E.
      Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility.
      • Heil S.G.
      • de Jonge R.
      • de Rotte M.C.
      • et al.
      Screening for metabolic vitamin B12 deficiency by holotranscobalamin in patients suspected of vitamin B12 deficiency: a multicentre study.
      However, holoTc also has a large window with indeterminate levels,
      • Harrington D.J.
      Laboratory assessment of vitamin B12 status.
      and the reference values strongly depend on the assay method used.
      • Nexo E.
      • Hoffmann-Lücke E.
      Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility.
      It has been suggested that patients with holoTC levels between 23 and 75 pmol/L require further testing of MMA levels to document, or rule out, true vitamin B12 deficiency.
      • Herrmann W.
      • Obeid R.
      Utility and limitations of biochemical markers of vitamin B12 deficiency.
      Also, approximately 63% of people with low holoTc levels (<27 pmol/L, indicative of true deficiency) have normal levels of MMA, while 9% of patients with holoTC values above 63 pmol/L have elevated MMA levels (defined as >300 nmol/L). This issue raises questions about whether holoTC measurement is really superior to measurement of total serum vitamin B12 plus MMA for determining vitamin B12 deficiency
      • Herrmann W.
      • Obeid R.
      Utility and limitations of biochemical markers of vitamin B12 deficiency.
      but also indicates that MMA is a poor indicator of vitamin B12 deficiency. Indeed, in another study, both serum vitamin B12 and holoTC levels were weak predictors of abnormal MMA levels.
      • Schrempf W.
      • Eulitz M.
      • Neumeister V.
      • et al.
      Utility of measuring vitamin B12 and its active fraction, holotranscobalamin, in neurological vitamin B12 deficiency syndromes.
      A more mathematical approach toward establishing vitamin B12 deficiency was proposed by Fedosov et al,
      • Fedosov S.N.
      Biochemical markers of vitamin B12 deficiency combined in one diagnostic parameter: the age-dependence and association with cognitive function and blood hemoglobin.
      • Fedosov S.N.
      • Brito A.
      • Miller J.W.
      • Green R.
      • Allen L.H.
      Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points.
      who calculated a single combined indicator of vitamin B12 status in which levels of total serum vitamin B12, holoTC, MMA, and homocysteine are taken into account. This is an elegant approach to making a proper diagnosis, but its validation in the context of both functional vitamin B12 deficiency and systematically evaluated response to vitamin B12 supplementation therapy (see subsequent discussion) is required.

      Assay Interference

      Several published case reports have shown functional vitamin B12 deficiency in patients with apparently normal serum vitamin B12 levels. In some of these patients, interference of serum vitamin B12 assays by IF antibodies has been demonstrated,
      • Hamilton M.S.
      • Blackmore S.
      • Lee A.
      Possible cause of false normal B-12 assays.
      • Yang D.T.
      • Cook R.J.
      Spurious elevations of vitamin B12 with pernicious anemia.
      • Scarpa E.
      • Candiotto L.
      • Sartori R.
      • Radossi P.
      • Maschio N.
      • Tagariello G.
      Undetected vitamin B12 deficiency due to false normal assay results.
      • Wainwright P.
      • Narayanan S.
      • Cook P.
      False-normal vitamin B12 results in a patient with pernicious anaemia.
      and it has been reported that assays fail to measure low total vitamin B12 concentrations in some samples because of an unknown artifact.
      • Carmel R.
      • Agrawal Y.P.
      Failures of cobalamin assays in pernicious anemia [letter] [published correction appears in N Engl J Med. 2012;367(10):976].
      Carmel and Agrawal
      • Carmel R.
      • Agrawal Y.P.
      Failures of cobalamin assays in pernicious anemia [letter] [published correction appears in N Engl J Med. 2012;367(10):976].
      reported that in 25% of patients with pernicious anemia, the assay may have produced false-normal values. We strongly believe that our patient E fits the description of interfering anti-IF antibodies. She had overt macrocytic anemia, initially diagnosed as myelodysplastic syndrome. Repeated serum vitamin B12 measurements yielded normal results, and in the following 3 years, she had development of symptoms compatible with severe polyneuropathy and nerve damage, signs of subacute combined degeneration of the spinal cord. After both MMA and homocysteine levels were found to be grossly elevated, it was realized that she had severe vitamin B12 deficiency. An additional factor that may have added to her protracted course was that she had been treated with high doses of folate. It is well known that folate therapy may mask anemia, and not treating with cobalamin may accelerate neurologic damage in people with vitamin B12 deficiency.
      • Dickinson C.J.
      Does folic acid harm people with vitamin B12 deficiency?.

      Possible Causes of Vitamin B12 Deficiency

      The polyglandular autoimmune syndrome is easily recognizable as a cause of deficiency in patients A and E. Polyglandular autoimmune syndromes are characterized by a number of (endocrine) diseases in which autoantibodies directed against certain organs or cell constituents can be demonstrated in the blood.
      • Eisenbarth G.S.
      • Gottlieb P.A.
      Autoimmune polyendocrine syndromes.
      • Van den Driessche A.
      • Eenkhoorn V.
      • Van Gaal L.
      • De Block C.
      Type 1 diabetes and autoimmune polyglandular syndrome: a clinical review.
      • Michels A.W.
      • Gottlieb P.A.
      Autoimmune polyglandular syndromes.
      • Vrkljan A.M.
      • Pašalić A.
      • Strinović M.
      • Perić B.
      • Kruljac I.
      • Miroševć G.
      Coexistence of Addison's disease and pernicious anemia: is the new classification of autoimmune polyglandular syndrome appropriate?.
      Table 3 summarizes the most prevalent disorders, varying from autoimmune thyroid disease (Hashimoto or Graves disease) to type 1 diabetes, Addison disease, and vitiligo. In the situation of vitamin B12 deficiency, both antibodies against IF and antibodies against parietal cells can be found, although it must be realized that the sensitivity of these measurements is low. One study found that only 55% of people of Western European descent with documented pernicious anemia had anti-IF antibodies.
      • Carmel R.
      Reassessment of the relative prevalences of antibodies to gastric parietal cell and to intrinsic factor in patients with pernicious anaemia: influence of patient age and race.
      However, this was not a very recent study, and methodology to demonstrate antibodies may have changed. Studies that assessed different assays to measure anti-IF antibodies have yielded discrepant results.
      • Carmel R.
      Reassessment of the relative prevalences of antibodies to gastric parietal cell and to intrinsic factor in patients with pernicious anaemia: influence of patient age and race.
      • Corcuff J.B.
      • Morel S.
      • Georges A.
      • de Bosredon K.
      • Bordenave L.
      Do intrinsic factor antibodies assays provide univocal answers in Biermer's disease?.
      • Berth M.
      • Bonroy C.
      • Guerti K.
      • Uyttenbroeck W.
      • Uytterhoeven M.
      Comparison of five commercially available ELISA kits for the determination of intrinsic factor antibodies in a vitamin B12 deficient adult population.
      Conversely, some investigators have argued that testing for gastric parietal cell antibodies is an appropriate screening test for pernicious anemia.
      • Khan S.
      • Del-Duca C.
      • Fenton E.
      • et al.
      Limited value of testing for intrinsic factor antibodies with negative gastric parietal cell antibodies in pernicious anaemia.
      Table 3Spectrum of Polyglandular Autoimmune Syndrome
      OrganDiseaseAntigen
      ThyroidHashimoto diseaseThyroid peroxidase
      Graves diseaseThyrotropin receptor, thyroid-stimulating immunoglobulins
      PancreasType 1 diabetesGlutamic acid decarboxylase 65, Islet antigen 2, zinc transporter 8
      Adrenal glandsAddison disease21-Hydroxylase
      GonadsAutoimmune oophoritis17α-Hydroxylase, side-chain cleavage enzyme
      Premature menopauseAldehyde (retinal) dehydrogenases, selenium-binding protein 1
      Autoimmune orchitisVarious antigens of sperm cell or testicular basement membrane
      Pituitary glandLymphocytic hypophysitisVarious antigens suggested
      Parathyroid glandsHypoparathyroidism
      Often in combination with mucocutaneous candidiasis.
      NACHT leucine-rich repeat protein 5
      IntestineCeliac diseaseGluten
      StomachAtrophic gastritisParietal cells, intrinsic factor
      LiverAutoimmune hepatitisCytochrome P450 1A2, 2A6
      Sclerosing cholangitisUnknown
      Primary biliary cirrhosisMitochondrial antigens, like subunits (E2) of the pyruvate dehydrogenase complex
      BloodThrombocytopeniaPlatelet surface glycoproteins
      Hemolytic anemiaVarious antigens
      SkinVitiligoTyrosinase
      Hair folliclesBaldness/alopeciaTyrosinase
      a Often in combination with mucocutaneous candidiasis.
      Other causes that can lead to vitamin B12 deficiency are summarized in Supplemental Table 3 (available online at http://mcpiqojournal.org). One important cause is celiac disease, in which gluten hypersensitivity will cause atrophy of the intestinal villi and consequently malabsorption, not only for vitamin B12 but also for a variety of other vitamins. Celiac disease is one of the disorders in which vitamin B12 absorption may be restored after dietary adjustment with complete avoidance of gluten. Other factors that may contribute to vitamin B12 malabsorption, hence deficiency, are certain medications like metformin and proton pump inhibitors,
      • Nehra A.K.
      • Alexander J.A.
      • Loftus C.G.
      • Nehra V.
      Proton pump inhibitors: review of emerging concerns.
      • Miller J.W.
      Proton pump inhibitors, H2-receptor antagonists, metformin, and vitamin B-12 deficiency: clinical implications.
      H2 receptor blockers, and antacids. Metformin use was associated with an increase in MMA and worsening neuropathy score in patients with type 2 diabetes.
      • Out M.
      • Kooy A.
      • Lehert P.
      • Schalkwijk C.A.
      • Stehouwer C.D.A.
      Long-term treatment with metformin in type 2 diabetes and methylmalonic acid: post hoc analysis of a randomized controlled 4.3year trial.
      In addition, vitamin B12 malabsorption may be more prominent in older individuals, rendering these people at increased risk of symptomatic vitamin B12 deficiency.
      • Pennypacker L.C.
      • Allen R.H.
      • Kelly J.P.
      • et al.
      High prevalence of cobalamin deficiency in elderly outpatients.
      • Andrès E.
      • Affenberger S.
      • Vinzio S.
      • et al.
      Food-cobalamin malabsorption in elderly patients: clinical manifestations and treatment.
      Inadequate dietary intake is another important cause of vitamin B12 deficiency. Because vitamin B12 is mainly present in food products from animal origin, vegetarians and vegans are at especially high risk for development of vitamin B12 deficiency.
      • Rizzo G.
      • Laganà A.S.
      • Rapisarda A.M.
      • et al.
      Vitamin B12 among vegetarians: status, assessment and supplementation.
      Finally, a new group of vitamin B12–deficient individuals is rapidly developing as the number of people undergoing bariatric surgery worldwide is increasing.
      • Parrott J.
      • Frank L.
      • Rabena R.
      • Craggs-Dino L.
      • Isom K.A.
      • Greiman L.
      American Society for Metabolic and Bariatric Surgery Integrated Health Nutritional Guidelines for the Surgical Weight Loss Patient 2016 Update: Micronutrients.
      • Kornerup L.S.
      • Hvas C.L.
      • Abild C.B.
      • Richelsen B.
      • Nexo E.
      Early changes in vitamin B12 uptake and biomarker status following Roux-en-Y gastric bypass and sleeve gastrectomy.
      Bariatric surgery has become one of the major treatments for (morbid) obesity, and reduced production of both IF and gastric acid, as well as reduced intake of vitamin B12–containing foods, contribute to the development of vitamin B12 deficiency (patient F). A recent study from The Netherlands reported a 60% incidence of post–bariatric surgery vitamin B12 deficiency, defined as a serum MMA level greater than 300 pmol/L.
      • Smelt H.J.
      • Smulders J.F.
      • Said M.
      • Nienhuijs S.W.
      • Boer A.K.
      Improving bariatric patient aftercare outcome by improved detection of a functional vitamin B12 deficiency.
      Interestingly, these investigators also reported that positive results of parenteral vitamin B12 administration were reported by patients without functional vitamin B12 deficiency (MMA <300 nmol/L), suggesting that supplementation itself, regardless of the actual vitamin B12 status, improves clinical symptoms.
      • Smelt H.J.
      • Smulders J.F.
      • Said M.
      • Nienhuijs S.W.
      • Boer A.K.
      Improving bariatric patient aftercare outcome by improved detection of a functional vitamin B12 deficiency.
      Finally, severe cases of vitamin B12 deficiency have been reported in young individuals using nitrous oxide as a recreational drug.
      • Morris N.
      • Lynch K.
      • Greenberg S.A.
      Severe motor neuropathy or neuronopathy due to nitrous oxide toxicity after correction of vitamin B12 deficiency.
      • Massey T.H.
      • Pickersgill T.T.
      • Peall J.K.
      Nitrous oxide misuse and vitamin B12 deficiency.
      • Buizert A.
      • Sharma R.
      • Koppen H.
      When the laughing stops: subacute combined spinal cord degeneration caused by laughing gas use.

      Familial/Genetic Causes of Vitamin B12 Deficiency

      The literature on familial cases of vitamin B12 deficiency is limited. Classically, the Imerslund-Gräsbeck syndrome is mentioned as an example of a genetic cause of vitamin B12 deficiency due to selective malabsorption, but its prevalence is very low. Other causes may be mutations in the gene for intrinsic factor,
      • Ferrand A.
      • Siu V.M.
      • Rupar C.A.
      • et al.
      Biochemical and hematologic manifestations of gastric intrinsic factor (GIF) deficiency: a treatable cause of B12 deficiency in the Old Order Mennonite population of southwestern Ontario.
      genes encoding the vitamin B12 transporting transcobalamins,
      • Nashabat M.
      • Maegawa G.
      • Nissen P.H.
      • et al.
      Long-term outcome of 4 patients with transcobalamin deficiency caused by 2 novel TCN2 mutations.
      • Yildirim Z.K.
      • Nexo E.
      • Rupar T.
      • Buyukavci M.
      Seven patients with transcobalamin deficiency diagnosed between 2010 and 2014: a single-center experience.
      and genes involving intracellular vitamin B12 metabolism. The list of single-nucleotide polymorphisms associated with serum vitamin B12 concentration is gradually increasing, and a recent review reported 59 gene polymorphisms found in a wide variety of populations, although the majority were reported in people of Western European descent.
      • Surendran S.
      • Adaikalakoteswari A.
      • Saravanan P.
      • Shatwaan I.A.
      • Lovegrove J.A.
      • Vimaleswaran K.S.
      An update on vitamin B12-related gene polymorphisms and B12 status.
      Recent studies have found an association between homozygosity of the TT methylenetetrahydrofolate reductase C677T genotype and vitamin B12 deficiency.
      • Zittan E.
      • Preis M.
      • Asmir I.
      • et al.
      High frequency of vitamin B12 deficiency in asymptomatic individuals homozygous to MTHFR C677T mutation is associated with endothelial dysfunction and homocysteinemia.
      • Shiran A.
      • Remer E.
      • Asmer I.
      • et al.
      Association of vitamin B12 deficiency with homozygosity of the TT MTHFR C677T genotype, hyperhomocysteinemia, and endothelial cell dysfunction.
      • Al-Batayneh K.M.
      • Zoubi M.S.A.
      • Shehab M.
      • et al.
      Association between MTHFR 677C>T polymorphism and vitamin B12 deficiency: a case-control study.
      In the near future, dedicated evaluation of genetic mutations in genes that are associated with vitamin B12 metabolism, for instance with whole-exome sequencing, may shed light on familial cases.

      Consequences of Maternal and Infant Low B12 Status

      Maternal vitamin B12 deficiency during pregnancy may be associated with an increased incidence of neural tube defects and brain development retardation,
      • Molloy A.M.
      Should vitamin B12 status be considered in assessing risk of neural tube defects?.
      as well as preterm birth and low birth weight.
      • Rogne T.
      • Tielemans M.J.
      • Chong M.F.
      • et al.
      Associations of maternal vitamin B12 concentration in pregnancy with the risks of preterm birth and low birth weight: a systematic review and meta-analysis of individual participant data.
      Wnt signaling has been reported to be disrupted in the developing cerebellum of the offspring of vitamin B12– and folate-deficient female rats
      • Willekens J.
      • Hergalant S.
      • Pourié G.
      • et al.
      Wnt signaling pathways are dysregulated in rat female cerebellum following early methyl donor deficiency.
      and is associated with long-term disabilities of behavior and memory.
      • Guéant J.L.
      • Caillerez-Fofou M.
      • Battaglia-Hsu S.
      • et al.
      Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase.
      Ethnic differences in vitamin B12 levels have been observed in pregnancy, possibly related to differences in intake of animal-derived foods.
      • Schroder T.H.
      • Sinclair G.
      • Mattman A.
      • et al.
      Pregnant women of South Asian ethnicity in Canada have substantially lower vitamin B12 status compared with pregnant women of European ethnicity.
      Children born to mothers with normal to high folate levels and low serum vitamin B12 values have higher truncal adiposity and insulin resistance,
      • Yajnik C.S.
      • Deshpande S.S.
      • Jackson A.A.
      • et al.
      Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: the Pune Maternal Nutrition Study.
      which may influence long-term risk of development of type 2 diabetes and cardiovascular disease.
      • Krishnaveni G.V.
      • Hill J.C.
      • Veena S.R.
      • et al.
      Low plasma vitamin B12 in pregnancy is associated with gestational 'diabesity' and later diabetes.
      • Rush E.C.
      • Katre P.
      • Yajnik C.S.
      Vitamin B12: one carbon metabolism, fetal growth and programming for chronic disease.
      Insufficient vitamin B12 status has an important negative influence on children's development and cognitive functioning.
      • Obeid R.
      • Murphy M.
      • Solé-Navais P.
      • Yajnik C.
      Cobalamin status from pregnancy to early childhood: lessons from global experience.
      • Kvestad I.
      • Hysing M.
      • Shrestha M.
      • et al.
      Vitamin B-12 status in infancy is positively associated with development and cognitive functioning 5 y later in Nepalese children.
      Nutritional vitamin B12 deficiency in children, and a late diagnosis of this condition, may lead to irreversible neurologic damage such as growth and motor retardation and even convulsions,
      • Demir N.
      • Koc A.
      • Üstyol L.
      • Peker E.
      • Abuhandan M.
      Clinical and neurological findings of severe vitamin B12 deficiency in infancy and importance of early diagnosis and treatment.
      and recent studies indicate a relationship between maternal vitamin B12 status and bone mass in the offspring.
      • Roman-Garcia P.
      • Quiros-Gonzalez I.
      • Mottram L.
      • et al.
      Vitamin B12-dependent taurine synthesis regulates growth and bone mass.
      Disturbed vitamin B12 status as well as (maternal) genetic factors may influence DNA methylation patterns in the newborn and thereby predispose individuals to specific disease later in life.
      • McKay J.A.
      • Groom A.
      • Potter C.
      • et al.
      Genetic and non-genetic influences during pregnancy on infant global and site specific DNA methylation: role for folate gene variants and vitamin B12.

      Natural Course of Vitamin B12 Deficiency

      Importantly, the natural course of vitamin B12 deficiency is not clearly understood. In typical patients, in whom clear signs and symptoms associated with (poly)neuropathy and/or megaloblastic anemia are explained by the unequivocal finding of low serum vitamin B12 levels, like in patient A, it is not particularly difficult to make a diagnosis and institute treatment. However, more widespread screening for vitamin B12 deficiency may result in situations in which low serum vitamin B12 levels are found but without clear symptomatology.
      • Green R.
      • Allen L.H.
      • Bjørke-Monsen A.L.
      • et al.
      Vitamin B12 deficiency (published correction appears in Nat Rev Dis Primers. 2017;3:17054).
      Parenteral vitamin B12 administration is also used—according to the package insert—for the prevention of vitamin B12–associated problems. In some individuals without symptoms, low levels of serum vitamin B12 may be associated with reductions in the binding protein haptocorrin.
      • Carmel R.
      Mild transcobalamin I (haptocorrin) deficiency and low serum cobalamin concentrations.
      However, this condition may be difficult to discriminate from true vitamin B12 deficiency in the presence of symptoms, as is evidenced by patient B.

      Just a Vitamin, or More?

      Some of the cases reported in this article, as well as the study by Smelt et al
      • Smelt H.J.
      • Smulders J.F.
      • Said M.
      • Nienhuijs S.W.
      • Boer A.K.
      Improving bariatric patient aftercare outcome by improved detection of a functional vitamin B12 deficiency.
      mentioned previously, suggest that supplementation of vitamin B12 itself, regardless of the actual vitamin B12 status, improves clinical symptoms. This hypothesis may put our thinking on the pathophysiology of vitamin B12 deficiency in a different perspective. Maybe we should not regard vitamin B12 as a vitamin that needs to be restored to normal levels to ensure proper functioning of metabolic pathways but rather as a general nerve-protecting and nerve-regenerating compound. There are some clues in the medical literature that support this concept. Cobalamin may regulate the balance between neurotoxic and neurotrophic agents.
      • Scalabrino G.
      • Buccellato F.R.
      • Veber D.
      • Mutti E.
      New basis of the neurotrophic action of vitamin B12.
      • Scalabrino G.
      The multi-faceted basis of vitamin B12 (cobalamin) neurotrophism in adult central nervous system: lessons learned from its deficiency.
      Micronutrients including vitamin B12 might improve the neuropathy score in patients with type 2 diabetes,
      • Farvid M.S.
      • Homayouni F.
      • Amiri Z.
      • Adelmanesh F.
      Improving neuropathy scores in type 2 diabetic patients using micronutrients supplementation.
      but in this study, a mixture of nutrients was used, including multivitamins and vitamin B12 orally. Another study reported that parenteral vitamin B12 administration was more effective than nortriptyline in the treatment of painful diabetic neuropathy.
      • Talaei A.
      • Siavash M.
      • Majidi H.
      • Chehrei A.
      Vitamin B12 may be more effective than nortriptyline in improving painful diabetic neuropathy.
      A 2005 meta-analysis
      • Sun Y.
      • Lai M.S.
      • Lu C.J.
      Effectiveness of vitamin B12 on diabetic neuropathy: systematic review of clinical controlled trials.
      reported that a high-dose oral cyanocobalamin or methylcobalamin had beneficial effects on symptoms of diabetic neuropathy, such as pain and paresthesia. In 3 studies, methylcobalamin therapy also improved autonomic symptoms. Effects on vibration perception and electrophysiologic measures were not consistent. However, one review did not find any evidence that the use of oral vitamin B12 supplements is associated with improvement in the clinical symptoms of diabetic neuropathy.
      • Jayabalan B.
      • Low L.L.
      Vitamin B supplementation for diabetic peripheral neuropathy.
      Low central nervous system cobalamin levels may play a role in the development of multiple sclerosis.
      • Scalabrino G.
      • Veber D.
      • Tredici G.
      Relationships between cobalamin, epidermal growth factor, and normal prions in the myelin maintenance of central nervous system.
      • Scalabrino G.
      • Veber D.
      • De Giuseppe R.
      • Roncaroli F.
      Low levels of cobalamin, epidermal growth factor, and normal prions in multiple sclerosis spinal cord.
      Similar observations, and even reports on beneficial effects of vitamin B12 injection therapy, have been reported in patients with myalgic encephalomyelitis, with and without fibromyalgia.
      • Regland B.
      • Andersson M.
      • Abrahamsson L.
      • Bagby J.
      • Dyrehag L.E.
      • Gottfries C.G.
      Increased concentrations of homocysteine in the cerebrospinal fluid in patients with fibromyalgia and chronic fatigue syndrome.
      • Regland B.
      • Forsmark S.
      • Halaouate L.
      • et al.
      Response to vitamin B12 and folic acid in myalgic encephalomyelitis and fibromyalgia.
      Also, there are clues that low vitamin B12 status is related to the development of chemotherapy-induced peripheral neuropathy.
      • Schloss J.M.
      • Colosimo M.
      • Airey C.
      • Vitetta L.
      Chemotherapy-induced peripheral neuropathy (CIPN) and vitamin B12 deficiency.
      Prospective studies to assess whether vitamin B12 supplementation may prevent chemotherapy-induced peripheral neuropathy are planned or ongoing.
      • Schloss J.
      • Colosimo M.
      B vitamin complex and chemotherapy-induced peripheral neuropathy.
      A recent intriguing study comes from US investigators, who demonstrated low vitamin B12 levels in the brain tissue of aged individuals, especially those older than 60 years, and of people with autism and schizophrenia.
      • Zhang Y.
      • Hodgson N.W.
      • Trivedi M.S.
      • et al.
      Decreased brain levels of vitamin B12 in aging, autism and schizophrenia.
      They speculate that on one hand vitamin B12 status in the brain compartment is distinctly regulated during aging from the rest of the body, while on the other hand may contribute to impaired brain function and in the etiology of neurologic disorders. These observations are supported by a recent study that reported improvement of symptoms in children with autism spectrum disorder treated with frequent hydroxocobalamin injections.
      • Hendren R.L.
      • James S.J.
      • Widjaja F.
      • Lawton B.
      • Rosenblatt A.
      • Bent S.
      Randomized, placebo-controlled trial of methyl B12 for children with autism.
      Finally, in a prospective study, Brito et al
      • Brito A.
      • Grapov D.
      • Fahrmann J.
      • et al.
      The human serum metabolome of vitamin B-12 deficiency and repletion, and associations with neurological function in elderly adults.
      found that one injection of high-dose (10 mg) cyanocobalamin, pyridoxine, and thiamine increased several metabolic markers of mitochondrial function oxidative stress, nerve function, and myelin integrity such as acylcarnitines, plasmalogens, phospholipids, and sphingomyelins.

      How Should Treatment Be Given?

      Traditionally, vitamin B12 is administered by intramuscular (IM) injection because of the low degree of resorption after oral administration as a consequence of the underlying disease. For parenteral therapy in cases of megaloblastic anemia, it has been advised to administer 1000 μg hydroxocobalamin IM twice weekly for a period of 5 weeks, and the dose is reduced to 1000 μg IM every 2 months thereafter. In the United States, often cyanocobalamin is used.
      • Green R.
      • Allen L.H.
      • Bjørke-Monsen A.L.
      • et al.
      Vitamin B12 deficiency (published correction appears in Nat Rev Dis Primers. 2017;3:17054).
      • Solomon L.R.
      Low cobalamin levels as predictors of cobalamin deficiency: importance of comorbidities associated with increased oxidative stress.
      In case of neurologic symptoms or abnormalities, it is suggested to administer hydroxocobalamin, 1000 μg once or twice weekly for a period of up to 2 years, and the package insert for hydroxocobalamin has included these particular instructions for several decades. However, it is ill defined which neurologic symptoms or abnormalities require such intensive treatment. Clinical practice has shown that in a substantial number of patients seen in a tertiary care setting, injection frequency cannot be reduced after the initial loading regimen. This topic has gained much interest recently, and studies evaluating this phenomenon are ongoing.
      • Pernicious Anaemia Society
      More good news! Another step forward in B12 research. Pernicious Anaemia Society website.
      Since the 1990s, interest in the possibilities of oral instead of parenteral administration has emerged. In 2014, a Dutch Viewpoint on Vitamin B12 Treatment was presented by the Dutch Organization of General Practitioners.
      NHG-Standpunt Diagnostiek van vitamine-B12-deficiëntie.
      Motivation to publish this Viewpoint was the observed increase in serum vitamin B12 testing and findings of abnormal test results. In this Viewpoint, it was stated that oral vitamin B12 administration was preferred over parenteral supplementation. This advice has been based on a very limited number of clinical trials. A 2005 Cochrane review evaluated 2 randomized controlled trials comprising a total of 108 participants and suggested that oral vitamin B12 may be as effective as IM administration in obtaining short-term hematologic and neurologic responses in vitamin B12–deficient patients.
      • Vidal-Alaball J.
      • Butler C.C.
      • Cannings-John R.
      • et al.
      Oral vitamin B12 versus intramuscular vitamin B12 for vitamin B12 deficiency.
      The first study by Kuzminski et al
      • Kuzminski A.M.
      • Del Giacco E.J.
      • Allen R.H.
      • Stabler S.P.
      • Lindenbaum J.
      Effective treatment of cobalamin deficiency with oral cobalamin.
      was performed in 33 vitamin B12–deficient patients, the majority with atrophic gastritic or pernicious anemia, and reported that serum vitamin B12 levels were significantly higher and MMA levels significantly lower in the oral group (n=18) compared to the IM group (n=15). Similar numbers of patients reported improvement (n=2) or clearance of symptoms (n=2) in both groups. Typically in patients using vitamin B12 injections, serum vitamin B12 levels exceed the upper limit of normal (1476 pmol/L), and this finding suggests that there may be underdosing in the injection group. Patients were randomized to receive 2000 μg of oral cyanocobalamin administered with breakfast daily for 4 months or 1000 μg of cyanocobalamin administered IM with increasing intervals. Evaluation was at 4 months, when the oral group was still using supplementation but the IM group had received the most recent injection 1 month earlier. In addition, parenteral administration consisted of cyanocobalamin, not hydroxocobalamin, and it has been reported that hydroxocobalamin has a longer retention in plasma and a lower excretion in urine than an equivalent dose of cyanocobalamin.
      • Hall C.A.
      • Begley J.A.
      • Green-Colligan P.D.
      The availability of therapeutic hydroxocobalamin to cells.
      A second study performed in Turkey
      • Bolaman Z.
      • Kadikoylu G.
      • Yukselen V.
      • Yavasoglu I.
      • Barutca S.
      • Senturk T.
      Oral versus intramuscular cobalamin treatment in megaloblastic anemia: a single-center, prospective, randomized, open-label study.
      included 70 patients with megaloblastic anemia. In 8 of 24 participants in the oral group, vitamin B12 deficiency was caused by poor nutrition. Ten patients were excluded because they did not appear for follow-up, and it should be noted that these patients were mainly randomized to the oral supplementation group. Research into the etiology of vitamin B12 deficiency was rather poor. Only 19 patients had anti–parietal cell antibodies assessed, which was due to costs as stated by the authors. Finally, the prevalence of neurologic symptoms was low. Only 7 of 60 participants reported altered cognitive function. Taken together, the articles provide very weak support for oral rather than parenteral vitamin B12 administration, and the large number of participants in the study by Bolaman et al
      • Bolaman Z.
      • Kadikoylu G.
      • Yukselen V.
      • Yavasoglu I.
      • Barutca S.
      • Senturk T.
      Oral versus intramuscular cobalamin treatment in megaloblastic anemia: a single-center, prospective, randomized, open-label study.
      with an etiology of poor nutrition (and not malabsorption) strongly biases these results in favor of simple oral vitamin B12 administration, which is the default in any nutritional insufficiency of vitamin B12 including vegetarianism. However, dosage is important in this respect. A study by Hill et al
      • Hill M.H.
      • Flatley J.E.
      • Barker M.E.
      • et al.
      A vitamin B-12 supplement of 500 μg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.
      found that supplementation with 500 μg of cyanocobalamin did not normalize moderately elevated MMA levels in healthy elderly people (aged 65 years or older). A recent Cochrane review confirmed that there is only very low-quality evidence that oral vitamin B12 appears as safe as IM vitamin B12.
      • Wang H.
      • Li L.
      • Qin L.L.
      • Song Y.
      • Vidal-Alaball J.
      • Liu T.H.
      Oral vitamin B12 versus intramuscular vitamin B12 for vitamin B12 deficiency.
      The authors concluded that further trials should conduct better randomization and blinding procedures, recruit more participants, provide adequate reporting, and measure important outcomes such as the clinical signs and symptoms of vitamin B12 deficiency, health-related quality of life, socioeconomic effects, and adverse events adequately.
      • Wang H.
      • Li L.
      • Qin L.L.
      • Song Y.
      • Vidal-Alaball J.
      • Liu T.H.
      Oral vitamin B12 versus intramuscular vitamin B12 for vitamin B12 deficiency.
      Serious adverse effects, even with high doses of hydroxocobalamin, have never been reported.
      • Zerbe N.F.
      • Wagner B.K.
      Use of vitamin B12 in the treatment and prevention of nitroprusside-induced cyanide toxicity.
      • Izumi Y.
      • Kaji R.
      Clinical trials of ultra-high-dose methylcobalamin in ALS.
      However, some patients do report acneiform eruptions or rosacea.
      • Jansen T.
      • Romiti R.
      • Kreuter A.
      • Altmeyer P.
      Rosacea fulminans triggered by high-dose vitamins B6 and B12.

      Possible Placebo Effects Unlikely

      In discussions on the effect of hydroxocobalamin treatment, often the suggestion is made that part of the effect is that of a placebo. Indeed, there is evidence that placebo treatments can have large and sustained effects on clinical outcomes in multiple disorders, as reviewed by Ashar et al,
      • Ashar Y.K.
      • Chang L.J.
      • Wager T.D.
      Brain mechanisms of the placebo effect: an affective appraisal account.
      who suggested that this effect may be particularly prominent in disorders in which emotion and motivation play a central role. In vitamin B12 deficiency, there is no evidence that recovery of megaloblastic anemia or normalization of elevated MMA levels as a consequence of hydroxocobalamin injections can be regarded as a placebo effect. Yet, when patients' neurologic or cognitive symptoms improve by injection therapy, it is often ascribed to a placebo effect. Several studies have assessed the effects of oral vitamin B12 supplementation in a variety of disorders, usually in people with normal serum vitamin B12 levels,
      • Eussen S.J.
      • de Groot L.C.
      • Joosten L.W.
      • et al.
      Effect of oral vitamin B-12 with or without folic acid on cognitive function in older people with mild vitamin B-12 deficiency: a randomized, placebo-controlled trial.
      • Almeida O.P.
      • Ford A.H.
      • Flicker L.
      Systematic review and meta-analysis of randomized placebo-controlled trials of folate and vitamin B12 for depression.
      with a surprising benefit in, for example, aphthous stomatitis.
      • Volkov I.
      • Rudoy I.
      • Freud T.
      • et al.
      Effectiveness of vitamin B12 in treating recurrent aphthous stomatitis: a randomized, double-blind, placebo-controlled trial.
      We have not found many well-designed studies of hydroxocobalamin injections in patients with neurologic symptoms caused by vitamin B12 deficiency. One study reported favorable effects of hydroxocobalamin injections in patients with mechanical or irritative lumbago,
      • Mauro G.L.
      • Martorana U.
      • Cataldo P.
      • Brancato G.
      • Letizia G.
      Vitamin B12 in low back pain: a randomised, double-blind, placebo-controlled study.
      whereas another well-designed study reported improvement of symptoms in children with autism spectrum disorder.
      • Hendren R.L.
      • James S.J.
      • Widjaja F.
      • Lawton B.
      • Rosenblatt A.
      • Bent S.
      Randomized, placebo-controlled trial of methyl B12 for children with autism.
      Clearly, there is a need for well-conducted double-blind, randomized studies in patients with vitamin B12 deficiency who report the benefit of continued frequent (twice weekly) hydroxocobalamin injections vs oral supplementation.

      Conclusion

      The spectrum of symptoms and signs suggestive of vitamin B12 deficiency is reasonably well defined. Nevertheless, many of the symptoms are nonspecific and may occur as a consequence of other diseases. Currently, no research has documented the positive and negative predictive values of specific symptoms or symptom scores for the presence of vitamin B12 deficiency.
      Patients with low serum vitamin B12 levels may have no symptoms (yet). Nevertheless, they are at high risk for development of symptoms. There is a tendency among physicians to consider a serum vitamin B12 level higher than 140 pmol/L as normal, but many symptomatic patients may present with such levels, for instance because of taking oral vitamin supplementation. This does not mean that their tissue vitamin B12 levels are normal as well. Methylmalonic acid and homocysteine are not very sensitive biomarkers, but there is currently no good alternative, although systematic evaluation of more advanced metabolic factors may lead to the application of better biomarkers. When serum total vitamin B12 levels are low or questionable, the combination of total vitamin B12, active vitamin B12, MMA, and homocysteine may be the best strategy, but the validity of this combined biomarker approach
      • Fedosov S.N.
      • Brito A.
      • Miller J.W.
      • Green R.
      • Allen L.H.
      Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points.
      needs to be validated in larger prospective studies and especially validated against objective markers of treatment response. In case of doubt, when results of biomarker measurements are equivocal, a trial with parenteral hydroxocobalamin injections may be considered, as was done in patients B and D. Because symptom improvement in long-standing (subclinical) vitamin B12 deficiency may take some time, we usually advise a treatment regimen of twice weekly hydroxocobalamin injections for 3 months, after which a thorough reevaluation is performed with systematic evaluation of symptom score as demonstrated in patient B (Table 2). There is no proof in large prospective, double-blind studies that oral supplementation is as effective in reducing symptoms associated with vitamin B12 deficiency as parenteral treatment.

      Supplemental Online Material

      Supplemental material can be found online at http://mcpiqojournal.org. Supplemental material attached to journal articles has not been edited, and the authors take responsibility for the accuracy of all data.

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        • Pernicious Anaemia Society
        More good news! Another step forward in B12 research. Pernicious Anaemia Society website.
        (Published June 29, 2018. Accessed December 28, 2018)
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