ARTICLE IN PRESS

Blood Reviews (2006) xxx, xxxxxx

www.elsevierhealth.com/journals/blre

REVIEW

Disorders of cobalamin (Vitamin B12) metabolism: Emerging concepts in pathophysiology, diagnosis and treatment
Lawrence R. Solomon
*

Section of Hematology, Department of Medicine, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520, United Kingdom

KEYWORDS
Cobalamin; Methylmalonic acid; Homocysteine; Vitamin B12; Diabetes mellitus

Summary Although cobalamin (vitamin B12) was isolated almost 60 years ago, its biochemical, physiologic and neurologic effects remain incompletely dened. New observations suggest renal regulation of cobalamin metabolism; actions of cobalamin on nucleic acid and protein function; and a role for cobalamin in cytokine and growth factor regulation. Clinically, no gold standard has emerged for the diagnosis of cobalamin deciency. Moreover, cobalamin resistance may occur in diabetes, renal insufciency and advanced age, leading to functional cobalamin deciency despite adequate cobalamin nutriture. Finally, high-dose cobalamin therapy may have salutary pharmacologic effects on neurologic function in a variety of disorders. Many studies lacked appropriate control groups. However, at this time, therapeutic trials with pharmacologic doses of cobalamin are suggested when ndings consistent with cobalamin deciency are present regardless of the results of diagnostic tests. While oral cobalamin immediate-release is adequate for many patients, its effectiveness in reversing neurologic abnormalities has yet to be established. c 2006 Elsevier Ltd. All rights reserved.

Introduction
Past reviews of cobalamin (Cbl)(Vitamin B12) deciency highlight diagnostic approaches to this disorder; the role of food Cbl malabsorption in Cbl depletion; and the presence of a subtle preclinical deciency state.14 Recent studies suggest unique biochemical and physiologic actions of Cbl; limitations in diagnostic testing; possible Cbl resistance; a role for Cbl as a pharmacologic agent; and

Abbreviations: Cbl, Cobalamin; MMA, Methylmalonic acid; HCys, Homocysteine; THF, Tetrahydrofolate; RDI, Recommended dietary intake; HIV, Human immunodeciency virus-1; TNF, Tumor necrosis factor a; EGF, Epidermal growth factor; IL6, Interleukin 6; OCS, Oral contraceptives; DM, Diabetes mellitus; TCA, Tricarboxylic acid. * Tel.: +203 785 6171; fax: +203 737 4244. E-mail address: [email protected].

0268-960X/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.blre.2006.05.001

ARTICLE IN PRESS
2 increasing use of oral Cbl therapy. These latter issues form the focus of this review. L.R. Solomon

Novel biochemical actions of cobalamin

Effects on nucleic acid metabolism and genetic regulation
Cbl coenzymes directly affect nucleic acid metabolism in vitro. Specically, methylcobalamin serves as a direct methyl donor in the DNA methylase reaction12 and Cbl binding by RNA receptors has been identied.1316 Whether these reactions occur in vivo remain to be determined. Similarly, growth of mammalian cells in Cblenriched media induces methionine synthase formation by modifying mRNA.1719 MethylCbl also stabilizes the methionine synthase enzyme protein in Cbl-decient rats.20 While enzyme induction by Cbl in vivo has not been studied, pharmacologic doses of B6 vitamers do induce synthesis of vitamin B6-related enzymes in red cells of normal and anemic human subjects.2123

Classic cobalamin biochemistry

Cbl participates in two enzymatic processes in mammalian cells. In the methionine synthase reaction, homocysteine (HCys) is converted to methionine allowing for the recycling of 5-methyl-tetrahydrofolate (THF) to N5,10 methylene-THF which is needed for the de novo synthesis of thymidylic acid and ultimately, for DNA formation (Fig. 1). Since conversion of N5,10-methylene-THF to N5-methylTHF is irreversible, Cbl deciency traps folic acid as N5-methyl-THF. Concurrently, HCys accumulates while methionine decreases, leading to a decrease in S-adenosylmethionine which further limits N5,10-methylene-THF formation by decreasing the synthesis of formyl-THF (formate starvation). Decreased methionine and S-adenosylmethionine may limit many methylation reactions including those involving DNA and myelin basic protein. In the methylmalonylCoA mutase reaction, methylmalonylCoA, derived from propionic acid synthesized by intestinal bacteria, is converted to succinylCoA, a precursor for fatty acid and heme synthesis (Fig. 2). Thus, Cbl deciency results in methylmalonic acid (MMA) accumulation. Impaired thymidylate synthesis, detected by the deoxyuridine suppression test, is the most sensitive marker of Cbl depletion, followed by elevation of MMA and HCys in peripheral blood.5 Similarly, decreased methylation of colonic DNA, increased uracil incorporation into DNA, and increased serum MMA and HCys levels antedate clinical changes in Cbl-depleted rats.6

Effects on nitric oxide and human immunodeciency virus-1 (HIV) metabolism

Cbl is a nitric oxide scavenger and inhibits nitric oxide synthase activity.2427 Since Cbl cream is effective treatment for atopic dermatitis, a disorder which responds to nitric oxide synthase inhibition, these observations may have clinical relevance.28 Similarly, Cbl decreases HIV infectivity by inhibiting integrase activity and low serum Cbl levels are associated with HIV progression in clinical studies.2931

Pathogenesis of clinical manifestations in Cbl deciency

Cbl deciency causes megaloblastic anemia and neurocognitive abnormalities but effects on immune function and bone formation have also been described.

Recommended dietary intake (RDI) of cobalamin

Based on studies of food Cbl absorption in normal subjects and of parenteral Cbl requirements in pernicious anemia, the RDI of Cbl was set at 23 lg/ day.7,8 However, micronucleus formation in peripheral blood lymphocytes, an index of chromosome breakage and loss, is minimized at plasma Cbl levels above 300 pmol/l (410 pg/ml), requiring a Cbl intake of 7 lg/day.9,10 Similarly, uracil misincorporation into leukocyte DNA in normal young women receiving folate supplements is lower at serum Cbl levels above 400 pg/ml (i.e. double the accepted lower limit of normal).11 Thus, the current RDI for Cbl may be inadequate to ensure genomic stability.

Pathogenesis of megaloblastic anemia

Megaloblastic anemia likely reects impaired thymidylic acid synthesis and misincorporation of uracil into DNA in hematopoietic precursors.32 Whether this results from trapping of N5-methyl THF and/or formate starvation remains uncertain.33 The roles of impaired DNA methylation and accelerated apoptosis are also unclear.34 Thus decreased methylation of colonic mucosa DNA occurs in Cbl-depleted rats, but methylation of DNA from

ARTICLE IN PRESS
Disorders of cobalamin (Vitamin B12) metabolism
dUR TdR

dUMP

TMP

DNA

N 5,10 Methylene THF PLP

DHF

PGA (Folic Acid)

Formyl THF

N 5-Methyl THF

THF

Formate B12 Methionine Methyl-B12 Homocysteine

(-)
S-Adenosyl Methionine S-Adenosyl Homocysteine

PLP

DNA

DNA Methylation

Cystathionine

Cysteine

Figure 1

Methionine synthase pathway.

peripheral blood leukocytes of patients with pernicious anemia is normal.6,35

Pathogenesis of neurocognitive defects

The spectrum of neurocognitive abnormalities in Cbl deciency is broad and the ndings on MRI and electrophysiologic examinations are diverse.3645 Moreover, neurologic changes often occur in the absence of hematologic abnormalities.37,41,45,46 Thus, establishing the biochemical basis for these clinical manifestations has proven elusive and suggested mechanisms include: impaired activity of methionine synthase and/or methylmalonylCoA mutase; accumulation of Cbl analogues; concurrent abnormalities in folate metabolism or nutriture; and alterations in cytokine and growth factor regulation.

While a role for both the methylmalonylCoA mutase and the methionine synthase pathways in the pathogenesis of neurocognitive dysfunction in Cbl deciency has been suggested, observations in experimental animals and in human subjects with inborn errors of Cbl metabolism do not consistently support either hypothesis.47,48 Similarly, a role for Cbl analogues, which inhibit Cbl-dependent enzymes, has not been conrmed in animal models.4952 Relatively increased serum folate, S-adenosylmethionine, cysteine and cysteine-glycine levels in patients with pernicious anemia and neurologic abnormalities suggest a role for folate-mediated inhibition of glycine N-methyltransferase in the pathogenesis of neuropathy.53 Thus, folate nutriture and genetic variations in folate or thiol metabolism may interact with impairment of Cbl-dependent

ARTICLE IN PRESS
4
METHYLMALONYL-CoA MUTASE PATHWAY
Propionic Acid
Intestinal Flora

L.R. Solomon

Oxaloacetic Acid 2-Methylcitric Acid AdoCbl Propionyl-CoA L-Methylmalonyl-CoA Succinyl CoA

MMA Odd-Chain Fatty Acids

CoA Heme

Branched-Chain Fatty Acids

+ Acetyl CoA

Even-Chain Fatty Acids

Figure 2

Methylmalonyl-CoA mutase pathway.

enzymes to produce nerve injury. In contrast, genetic polymorphisms of N-5,10-methylene tetrahydrofolate reductase do not correlate with the clinical manifestations of Cbl deciency.54 Polyamines are also decreased in the brains of Cbl-depleted rats but the signicance of this nding is unknown.55 Observations in Cbl-decient gastrectomized rats implicate cytokines and growth factors as mediators of neurologic damage.56,57 The myelinolytic cytokine, tumor necrosis factor a (TNF), is increased in the spinal uid of these animals while the neurotrophic cytokines, epidermal growth factor (EGF) and interleukin 6 (IL-6), are decreased. EGF mRNA is also absent in neurons and glial cells. Moreover, neurologic lesions are prevented in Cbldecient rats by intraventricular injections of TNF antibodies, EGF or IL-6 and induced in normal rats by intraventricular injections of either EGF antibodies or TNF. Similarly, Cbl-decient humans have increased TNF and decreased EGF levels in serum which improve with Cbl therapy.58 Regulation of TNF synthesis by S-adenosylmethionine links the classic Cbl biochemical pathways with the role of cytokines in neurologic dysfunction.57

abnormal lymphocyte subpopulations in Cbl-decient subjects with megaloblastic anemia suggest a role for Cbl in immune function.5964 Cbl-decient subjects have decreased total lymphocyte counts, decreased CD8+ cells and impaired natural killer cell activity which correct with high-dose methylCbl therapy.65,66 However, this regimen also increases total lymphocyte counts and CD8+ cell counts in normal subjects, suggesting a pharmacologic effect of Cbl.66

Pathogenesis of osteoporosis
Low serum Cbl levels increase the risk of osteoporosis.67,68 In vitro studies and the nding of low serum skeletal alkaline phosphatase and osteocalcin levels in Cbl-decient patients which correct with vitamin therapy suggest that Cbl is necessary for normal osteoblast activity.69,70 Signicantly, hip fractures are reduced in elderly subjects receiving 1500 lg/ day of mecoCbl in addition to 5 mg/day of folic acid orally.71 TNF may cause osteoporosis by stimulating osteoclast activity.72 Thus, the cytokine-related actions of Cbl may have a role in this setting as well.

Pathogenesis of immune dysfunction

Diagnosis of cobalamin deciency

An increased incidence of tuberculosis in vegetarians, impaired antibody responses to pneumococcal vaccine in elderly patients with low Cbl levels, and Tests for Cbl deciency include measurements of 1) total Cbl; 2) MMA and HCys, as indices of func-

ARTICLE IN PRESS
Disorders of cobalamin (Vitamin B12) metabolism tional Cbl deciency; and 3) holotranscobalamin as a measure of the metabolically active fraction of circulating Cbl. Each approach has signicant limitations. Moreover, since the pathogenesis of neurologic dysfunction in Cbl deciency remains unclear, these tests may not be reliable markers of neurocognitive impairment.73 5 also increase serum Cbl either by increasing release of hepatic Cbl stores or decreasing clearance of holohaptocorrin.79 In alcoholic liver disease, holohaptocorrin is increased but holotranscobalamin values are low and plasma levels of MMA and HCys are elevated, suggesting that tissue Cbl depletion may also occur in this setting.79,80

Total Cbl
Measurement of serum Cbl is commonly used to screen for Cbl deciency, but many patients with low Cbl levels are not Cbl-decient (i.e. false low values) while signicant clinical impairment may occur despite normal Cbl values (i.e. false high values)(Table 1).41,7375 Cbl levels may reect variations in the 2 major Cbl transport proteins, transcobalamin (formerly transcobalamin II) and haptocorrin (formerly transcobalamin I). Transcobalamin-bound Cbl represents less than 20% of circulating Cbl and readily enters tissues via specic receptors. Haptocorrinbound Cbl represents more than 80% of circulating Cbl but is metabolically inert.

Low Cbl levels

Haptocorrin values are lower in Caucasians than in African-Americans and genetic determinants of haptocorrin may explain 15% of false low serum Cbl values.81,82 Both serum Cbl and holohaptocorrin decrease progressively during pregnancy.83 Normal hematocrit, MCV and urine MMA values were noted in one study of 32 pregnant women with low serum Cbl values. However, other studies report increased urine MMA values; a signicant inverse relationship between serum Cbl and both hemoglobin and serum MMA levels; and a relationship between maternal serum Cbl and impaired Cbl status in the newborn.8387 Moreover, high serum MMA values were present in one-third of pregnant women regardless of the serum Cbl level and an association between high MMA values and neural tube defects has been reported.88,89 Thus, low serum Cbl levels in pregnant women warrant further evaluation. Oral contraceptives (OCS) also decrease Cbl and haptocorrin levels without increasing urine or serum MMA values.9094 However, many of these subjects were evaluated after only 34 months of OCS use which may have been insufcient time for tissue Cbl depletion.

High Cbl levels

Myeloproliferative disorders and renal failure increase both serum Cbl and haptocorrin levels.76,77 Although serum Cbl was not measured, the presence of Cbl-responsive elevations of MMA in 20% of patients with myeloproliferative disorders suggests that high Cbl values in these conditions can mask functional Cbl deciency.78 Hepatic disorders

Table 1

Determinants of serum cobalamin levels. FALSE HIGH/NORMAL VALUESb High Haptocorrin Levels: Myeloproliferative disorders Renal disease Increased tissue release of Cbl Liver disease Low/absent transcobalamin Low afnity transcobalamin polymorphisms Inherited Disorders of Cbl metabolism Recent Cbl therapy Cobalamin analoguesd: Nitrous oxide therapy High dose Vitamin C Intestinal bacterial overgrowth Assay methodological errors

FALSE LOW VALUESa Folate deciency Pregnancyc Oral contraceptivesc Multiple myelomac HIV infectionc Low haptocorrin levels

Abbreviations: Cbl, cobalamin; HIV, human immunodeciency virus-1. a Indicates low Cbl levels in the absence of impaired Cbl supply to tissues. b Indicates high or normal Cbl levels despite impaired Cbl supply to tissues. c See text. d Dependent on assay method.

ARTICLE IN PRESS
6 Moreover, women on OCS face different metabolic demands than those imposed by pregnancy. In contrast, hormone replacement therapy does not increase the risk of either low serum Cbl or elevated metabolite values.93,95 Multiple myeloma also decreases serum Cbl and haptocorrin levels. However, since increased uptake of Cbl by marrow myeloma cells has been suggested, further tests for functional Cbl deciency are warranted.96,97 Similarly, HIV infection decreases both Cbl and haptocorrin levels, but holotranscobalamin is also decreased and clinical abnormalities of gastrointestinal function associated with abnormal Schilling tests provide a mechanism for Cbl depletion in this patient population.98100 Nonetheless, metabolic abnormalities consistent with frank Cbl deciency are infrequent and Cbl-responsive hematologic abnormalities in HIV-infected patients are rare.98 While HCys was increased in 9 of 40 patients with serum Cbl levels <200 pmol/l, red cell folate values were also low and HCys values returned to normal after treatment with both Cbl and folic acid.100 Since MMA was not measured, the relative contributions of folate and Cbl deciencies to hyper-homocysteinemia is uncertain. However, an association between low serum Cbl levels and both peripheral neuropathy and myelopathy has been noted. Moreover, Cbl therapy improved the neuropathy but not the myelopathy in this uncontrolled study.101 Since the treatment regimen was not described and since improvement in myelopathy may require prolonged therapy, a role for Cbl deciency cannot be excluded.102 L.R. Solomon

MMA and HCys

Because of the limitations in serum Cbl, MMA and HCys are often measured as conrmatory indices of functional Cbl depletion.75,113,114 While increased levels of these metabolites were initially reported in almost all patients with clinically overt Cbl deciency, only those subjects with low serum Cbl levels were included in these studies.114116 Indeed when treatment was not restricted to patients with low serum Cbl levels, clinical responses to Cbl therapy were commonly seen even when Cbl, MMA and HCys values were normal, particularly in patients with neurologic impairment.73 Moreover, not all patients with signicant hematologic or neurologic abnormalities and elevated MMA and/or HCys values improve with Cbl therapy even when Non-Responders with confounding clinical disorders are excluded from analysis.73,75,114116 The intraindividual variation in MMA is small in normal subjects but much greater in patients with higher baseline MMA values and these uctuations may obscure the recognition of Cbl deciency.73,117 Renal dysfunction also increases serum MMA values, with a positive correlation noted between MMA and serum creatinine even for creatinine values within the normal range.118,119 However, an inverse relationship between MMA and Cbl persists in uremic subjects and MMA values decrease with Cbl therapy.120 Thus, high MMA values in renal failure may represent true tissue Cbl deciency (see Cbl Resistance). Conversely, MMA values may be normal in Cbl decient patients receiving antibiotics which can eradicate the intestinal ora needed to synthesize propionic acid (Fig. 2).113 Intraindividual variation in HCys is also low in healthy subjects but far greater in patients with high HCys values.73,120123 Moreover, many genetic, acquired and environmental factors elevate HCys levels.120 Thus, HCys is less specic than MMA for the diagnosis of Cbl deciency.

Normal Cbl levels

A common transcobalamin polymorphism (775G > C) with decreased afnity for Cbl and low holotranscobalamin levels has recently been identied.103 Increased MMA and HCys values, present in some but not all studies, suggest that homozygotes may be at risk for functional Cbl deciency at higher serum Cbl levels.103108 Similarly, partial transcobalamin deciency with autosomal dominant inheritance is associated with both megaloblastic anemia and neurologic abnormalities.109 Serum Cbl levels were normal in 12 of these 23 subjects. In contrast to familial recessive transcobalamin deciency which becomes clinically manifest in infancy, these patients may not become symptomatic until adulthood. Finally, current chemiluminescence and radioisotope dilution Cbl assay kits may give misleadingly normal values and the intraindividual variation in Cbl is greater than generally appreciated.73,110112

Holotranscobalamin
Holotranscobalamin levels are decreased in patients with elevated metabolite values as well as in subjects with clinically overt Cbl deciency.124127 However, 38% of patients with low holotranscobalamin values have normal deoxyuridine suppression tests, suggesting that this measurement is not specic for Cbl depletion.128 Moreover, increases in transcobalamin associated with inammation may also limit the diagnostic utility of this test.129,130

ARTICLE IN PRESS
Disorders of cobalamin (Vitamin B12) metabolism 7 served.146,147 Yet 730% of elderly subjects with normal serum Cbl levels have high metabolite values even when serum creatinine is normal.148162 Highdose Cbl therapy reduces MMA levels in most but not all elderly subjects with high MMA values.135,158,159,162 Moroever, maximum reduction of both mean MMA values and the incidence of elevated MMA levels requires oral Cbl doses of 5001000 lg/ day which are 167333 times higher than the RDI.163,164

Subtle cobalamin deciency

Subtle Cbl deciency, dened as elevated metabolite levels usually in asymptomatic patients with low or normal serum Cbl values, is prevalent in the elderly and has been associated with food Cbl malabsorption .4,131 The prognostic and clinical signicance of subtle Cbl deciency is uncertain. Anemia, neuropathy and impaired cognitive performance, as well as abnormalities in evoked potentials, somatosensory potentials and EEG recordings have all been reported in this disorder.132135 Moreover, in uncontrolled studies, Cbl therapy led to improvement in some, but not all, subjects.132,134 In contrast, other studies suggest that this disorder is not progressive and that treatment is without clinical benet.136138 However, since only 4 weekly Cbl injections were given with follow-up performed 2 months later and since MMA decreased to normal in only 69% of patients, therapy may have been inadequate.137,138 Importantly, subtle Cbl deciency may increase the risk of adverse events in other clinical settings. Thus, nitrous oxide anaesthesia, which oxidizes Cbl, can precipitate acute neurologic decompensation postoperatively in patients with unrecognized Cbl deciency while hematologic and gastrointestinal toxicity induced by the chemotheraeutic agent premetrexed is associated with high MMA and HCys values and ameliorated by folate and Cbl therapy.139141

Cobalamin resistance in renal disease

Increased MMA and HCys levels are present in hemodialysis patients despite high serum Cbl and holotranscobalamin levels. Moreover, pharmacologic doses of Cbl decrease both metabolites albeit usually not to normal values.165173 Signicantly, Cbl uptake by mononuclear cells from renal patients is decreased despite normal expression of holotranscobalamin receptors and elevated metabolite levels in these patients are inversely related to Cbl uptake.174 Since diminished vibration thresholds in hemodialysis patients improve with methylcobalamin therapy, these ndings may have clinical signicance.175

Cobalamin resistance in diabetes mellitus

Urinary MMA excretion increases in streptozotocindiabetic rats despite increased serum Cbl levels and high doses of methylcobalamin decrease urinary MMA to normal.176 While this study was not conrmed with a different MMA assay method, high-dose Cbl also improves nerve conduction and structure in these animals.177179 In human diabetics: MMA values are increased even when serum Cbl values are greater than 600 pg/ml;180 high metabolite values and neurologic abnormalities respond to Cbl therapy despite pretreatment Cbl levels >300 pg/ml;73 hyperhomocysteinemia is associated both with serum Cbl levels <350 pmol/ml (lower limit of normal = 150 pmol/l) and with the presence of diabetic neuropathy;181,182 Cbl therapy improves objective measures of cardiac autonomic dysfunction;183186 Cbl therapy improves symptoms of both peripheral neuropathy and autonomic dysfunction (randomized double-blind placebo-controlled study);187 and intrathecal highdose methylcobalamin can relieve symptoms of neuropathy.188 Nerve conduction velocities also improved in human diabetics treated with a high-dose combination of cyanocobalamin, lipid-soluble thiamine and pyridoxine.189 However, improvement also occurred in streptozotocin-diabetic rats treated only with lipid-soluble thiamine.190 Similarly, high-dose

Cobalamin resistance
Increased MMA levels despite normal serum Cbl values may be explained by insensitivity of serum Cbl to early Cbl depletion; abnormal plasma Cbl-binding proteins; or lack of specicity of MMA elevations for Cbl deciency (albeit not by clinically silent inborn errors of Cbl metabolism).142144 Alternatively, this picture may reect Cbl resistance, dened by the correction of abnormal metabolite levels in subjects with normal serum Cbl values by treatment with pharmacologic doses of Cbl. A role for the kidney in regulating Cbl metabolism has recently been proposed.145 Thus, cobalamin resistance in diabetes, renal insufciency and advanced age may reect the alteration in renal function common to these disorders.

Cobalamin resistance in the elderly

While decreased holotranscobalamin in elderly subjects has been reported, normal Cbl binding to serum transport proteins has recently been ob-

ARTICLE IN PRESS
8 Cbl in combination with high-dose folate improved endothelial function and insulin resistance in subjects with the metabolic syndrome.191 Since highdose Cbl therapy improves neurologic function in non-diabetic settings as well, these effects may result from nonspecic pharmacologic actions of Cbl rather than Cbl resistance (see Cbl AS A PHARMACOLOGIC AGENT).192195 L.R. Solomon disease; and the observation that some analogues may have a direct inhibitory effect on methionine synthase and methylmalonylCoA mutase activities.198,205,209,210 However, Cbl analogues are not associated with neurologic dysfunction in Cbl-decient bats and adequate trials of Cbl therapy in cognitive dysfunction have yet to be performed.50,52,211

AIDS-associated myelopathy Genetically determined causes of cobalamin resistance

Inborn errors of metabolism involving Cbl transport, intracellular metabolism, or interaction with the methionine synthase or methylmalonylCoA mutase apoenzymes are also characterized by elevation of MMA and/or HCys with normal serum Cbl levels.196 High dose Cbl therapy can improve both the metabolic and clinical manifestations of these rare disorders. Similar roles for other vitamins have been suggested in the setting of genetic polymorphisms which decrease coenzyme binding afnity.197 Decreased serum methionine and cerebrospinal uid S-adenosylmethionine levels have been reported in AIDS-associated myelopathy despite normal blood levels of Cbl, MMA and HCys.200 Moreover, central nerve conduction studies improved in this population after high-dose methionine therapy.212 However, high-dose Cbl therapy has yet to be evaluated.

Cobalamin as a pharmacologic agent

Since loading doses of Cbl far exceed physiologic requirements, clinical responses may result from pharmacologic effects on either Cbl-related processes or on cellular functions completely unrelated to the known biochemical actions of Cbl. Thus, neurologic responses to Cbl therapy were noted in 29 human subjects, 6 (21%) of whom had normal MMA and HCys values.73 Similarly, pharmacologic doses of methylcobalamin accelerate nerve regeneration in rats with acrylamide neuropathy;192 improve electrophysiologic parameters in the carpal tunnel syndrome;195 protect retinal cells from glutamateinduced injury in vitro;193 improve compound muscle action potentials in amyotrophic lateral sclerosis;194 and alter lymphocyte populations in normal human subjects.66 Pharmacologic doses of Cbl also improved neurologic function in hemodialysis patients as well as in diabetic rats and humans and transiently improved signs and symptoms of progressive vacuolar myelopathy following treatment of childhood leukemia.175,183189,213 The ability of Cbl to enter cells by a pathway independent of transcobalamin receptors provides a mechanism for the generation of high intracellular Cbl concentrations214 while the novel biochemical effects of Cbl and the pharmacologic actions of Cbl on cytokines and immune function provide possible explanations for these clinical observations.919,29,30,5659,66

Local cobalamin resistance in other neurocognitive disorders

Functional impairment in Cbl activity limited to the central nervous system has been proposed in multiple sclerosis, Alzheimers disease and AIDS-associated myelopathy.198200 Since serum Cbl, MMA and HCys levels would likely be normal in these settings, local Cbl resistance cannot be distinguished from nonspecic pharmacologic actions of Cbl on the nervous system.

Multiple sclerosis
In multiple sclerosis, high-dose Cbl therapy improved some objective and subjective measures of disease activity but these studies lacked adequate control groups.201,202 However, the combination of Cbl and interferon has signicantly more activity in a mouse model of demyelinating disease than either agent alone.203

Alzheimers disease
An increased risk of Alzheimers disease is associated with low serum Cbl levels in general and low holotranscobalamin levels in particular.204208 Moreover, the suggestion that increased Cbl oxidation in the central nervous system may play a role in this disorder is supported by the presence of Cbl analogues in human brain tissue; the relative increase in Cbl analogues in serum from patients with Alzheimers

Treatment of cobalamin deciency

Cbl therapy usually involves loading doses of daily to weekly parenteral injections over a period of

Disorders of cobalamin (Vitamin B12) metabolism

Table 2 Ref 226 228 229 227

Studies of oral cyanocobalamin therapy. N 18 30 10 26 Time (Mo) 4 1 3 3 Prior schilling No Yes (N = 10) Yes (N = 10) No Inclusion criteria Cbl MMA >376 ND ND ND HCys >17.9 >13.0 >13.0 ND Clinical None None None Hgb < 12 & MCV > 94 ND Megalo (Hgb 6 10.1 MCV P 106) <160 (x2) <200 (x2) <160 (x2) <160 Responses MMA 17/17 ND ND ND MCV 6/7 8/8 5/7 112 11 to 87 4 ND 7/8 Hgb 2/4 7/13 6/10 8.4 2.1 to 13.8 0.7 ND 7/7 Neuro 3/3 ND 2/4 4/6c M.S. 1/1a ND ?/2 3/3h

Oral regimen 2 mg/day 0.51.0 mg/d 35 mg/wk 1 mg/d 10 1 mg/wk 10 1 mg/mog 1 mg/db 1.5 mg/df

ARTICLE IN PRESS

164 230

20 8

1.5 1.5

No Yes (N = ?)

<300 <259

>271 (x2) NDe

>13.9 ND

20/20 NDe

ND ??d

ND ??d

All

104

>20

37/37 100%

26/30 87%

22/34 65%

9/13 69%

4/4 100%

Refs. [226,227] were randomized comparisons of parenteral and oral therapy. Inclusion criteria for Ref. [226] were a serum Cbl <160 pg/ml on 2 occasions and an elevated MMA or HCys value. Inclusion criteria for Refs. [228,229] were low serum Cbl on 2 occasions or a low serum Cbl on one occasion and an elevated HCys value. Units are: Cbl, pg/ml; MMA, nmol/ l; HCys, lmol/l. Abbreviations: Ref, reference number; N, number of subjects studied; Mo, months; Cbl, cobalamin; MMA, methylmalonic acid; Hgb, hemoglobin; MCV, mean corpuscular volume; Neuro, neuropathy; M.S., mental status; ND, not done; wk, weeks. a Method of testing not stated. b Treatment with 1 mg/day followed 6 weeks of treatment with 25 mcg/day and 6 weeks of treatment with 100 mcg/day. c Objective testing reported in 3 of 4 responders. d All 8 patients had mild neurologic or mental status changes which improved but details not provided. e 24 hour urine MMA excretion was elevated in 4 patients tested and returned to normal in the one patient with a post-treatment value. f Includes 3 patients on cyanocobalamin, 4 patients on hydroxocobalamin and 1 patient on methylcobalamin. g Used cyanocobalamin liquid for injection mixed with 20 ml fruit juice. h Determined by MMSE.

ARTICLE IN PRESS
10 13 months to replete body stores followed by a maintenance regimen. Specic recommendations depend on the form of Cbl used and the route of administration selected. L.R. Solomon high-dose oral cyanocobalamin can induce and maintain remissions in patients with megaloblastic anemia due to pernicious anemia.222224 More recently, a Cochrane Review based on 2 randomized trials concluded that daily oral therapy may be as effective as intramuscular administration in obtaining short term haematological and neurological responses in vitamin B12 decient patients.225227 However, of 104 patients in reported series, only 9 patients had pernicious anemia and 10 patients had atrophic gastritis (which causes both food Cbl malabsorption and intrinsic factor deciency) while at least 66 patients had food Cbl malabsorption or decreased dietary Cbl intake (Table 2).164,226230 More than 20 patients had antecedent Schilling tests involving a parenteral dose of Cbl. Moreover, most of the patients in these studies were selected only for the presence of low Cbl and high metabolite levels, some were selected for the presence of megaloblastic anemia and none were selected for the presence of neurologic abnormalities.164,226230 While neurologic responses were observed, these were often not objectively measured. Thus, since delay in effective therapy can lead to irreversible neurologic dysfunction, parenteral therapy should be strongly considered. Finally, while studies with

Form of cobalamin and frequency of administration

Intramuscular cyanocobalamin and hydroxocobalamin have distinct pharmacologic properties. Hydroxocobalamin has greater systemic retention and availability to cells than cyanocobalamin and cyanocobalamin therapy may be ineffective in some patients with inborn errors of metabolism.215218 Thus, while 1 mg maintenance doses of hydroxocobalamin may be given every 1.53 months, 1 mg doses of cyanocobalamin may be required as often as every 2 weeks.219 Importantly, individual patients require more frequent injections of either Cbl derivative to maintain normal serum Cbl levels and prevent clinical relapse.219221

Oral vs parenteral administration

Since Cbl is absorbed by intrinsic factor-independent passive diffusion in the small intestine, daily

Table 3

Classication of cobalamin-responsive disorders. MECHANISM Decreased intake Decreased absorption Increased destruction (e.g. Nitrous Oxide) Impaired utilization a) Hereditary - Inborn errors of Cbl metabolism b) Acquired i) Age P 70 yrs ii) Renal insufciency iii) Diabetes mellitus 2) Increased Requirements - Systemic a) Pregnancy ?? b) Abnormal plasma binders (TC; HC) c) Cbl analogues 3) Increased requirements - local i) Multiple Sclerosis ii) Alzheimers disease iii) AIDS-myelopathy 4) Unknown: (Nucleic Acid Binding; Binding of Metabolic Intermediates; Enzyme Induction; Enzyme Inhibition; Cytokine/ Growth factor regulation?) 1) 2) 3) 1) TESTS Cbl MMA/HCys High Low

CATEGORY DEFICIENCY

SYSTEMIC

Normal

High

FOCAL RESISTANCE

Normal

Normal

PHARMACOLOGIC

Normal

Normal

Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine; TC, transcobalamin; HC, haptocorrin.

ARTICLE IN PRESS
Disorders of cobalamin (Vitamin B12) metabolism oral Cbl involved immediate-release tablets or liquid formulations, many over-the-counter high dose Cbl tablets are timed release preparations which have not been adequately studied.231 11

Summary and conclusions

No single test has emerged as the gold standard for diagnosis of Cbl deciency.232 Moreover, Cbl requirements in normal subjects may be higher than previously appreciated; Cbl resistance may increase Cbl requirements in the setting of diabetes, renal insufciency and advanced age; and Cbl may have physiologic and pharmacologic effects which are as yet poorly understood. Thus, Cbl-responsive disorders may result from true Cbl deciency, systemic or local Cbl resistance; or nonspecic pharmacologic effects of high-dose Cbl therapy (Table 3). As a result, blood Cbl, MMA and HCys values often fail to predict whether or not a patient will respond to Cbl therapy. However, most clinical studies of high-dose Cbl therapy and neurologic function lacked appropriate control groups. Pending the completion of randomized, long-term, placebocontrolled trials of high-dose hydroxocobalamin, cyanocobalamin and methylcobalamin in neurologic disorders, prolonged (612 months)102 therapeutic trials with pharmacologic doses of parenteral Cbl (at least 1,000 lg intramuscularly 13 days/week) are warranted when clinical ndings consistent with Cbl deciency are present. Finally, the role of oral Cbl therapy in patients with neurologic abnormalities has not yet been established.

 High-dose cobalamin therapy may improve neurologic function even in the absence of cobalamin deciency.  Low serum cobalamin levels associated with pregnancy, oral contraceptive use, multiple myeloma or HIV infection warrant further evaluation.  High serum cobalamin levels in liver disease or myeloproliferative disorders may mask functional cobalamin deciency.  While the signicance of subtle cobalamin deciency is uncertain, therapy is suggested because of the presence of neurophysiologic abnormalities in some patients.  Individual patients with cobalamin deciency may require more frequent parenteral dosing to prevent relapse than is usually recommended.  While oral cobalamin is effective in many patients, its benet for neurocognitive dysfunction is uncertain and timed-release preparations should be avoided.

Research agenda
 Further dene the role of cobalamin in nucleic acid metabolism and genomic stability in normal subjects.  Determine biochemical effects of pharmacologic doses of cobalamin in vivo on metabolic processes in normal subjects and in subjects with neurologic disorders.  Determine the role of cobalamin in cytokine and growth factor regulation and on immune function.  Dene the mechanism of neurotoxicity in patients with cobalamin deciency.  Determine mechanisms of cobalamin resistance in diabetes, renal disease and the elderly.  Evaluate the role of the kidney in the regulation of cobalamin metabolism.  Explore pharmacologic role of cobalamin in neurologic disorders (including neuropathy associated with diabetes and HIV infection; multiple sclerosis; and Alzheimers disease) with particular attention to the form of Cbl (cyanocobalamin; hydroxocobalamin; or methylcobalamin), the dose of Cbl and the duration of therapy (i.e. 612 months).

Practice points
 The current RDI may underestimate cobalamin requirements in normal subjects.  Higher cobalamin levels may be needed in the elderly and in patients with diabetes or renal failure to prevent functional cobalamin deciency.  Common transcobalamin polymorphisms may also cause functional cobalamin deciency at normal serum cobalamin levels.  Since cobalamin, methylmalonic acid and homocysteine levels uctuate and neither predict nor preclude responses to cobalamin, cobalamin therapy is suggested for symptomatic patients regardless of the results of these diagnostic tests.

ARTICLE IN PRESS
12 L.R. Solomon
21. Solomon LR, Hillman RS. Vitamin B6 metabolism in human red cells: I. Variations in human red cells. Enzyme 1978;23:26273. 22. Solomon LR, Hillman RS. Regulation of vitamin B6 metabolism in human red cells. Am J Clin Nutr 1979;32:182431. 23. Solomon LR, Hillman RS. Vitamin B6 metabolism in idiopathic sideroblastic anaemia and related disorders. Brit J Haematol 1979;42:23953. 24. Brouwer M, Chamulitrat W, Ferruzzi G, Sauls DL, Weinberg JB. Nitric oxide interactions with cobalamins: biochemical and functional consequences. Blood 1996;88:185764. 25. Wolak M, Zahl A, Schneppensieper T, Stochel G, van Eldik R. Kinetics and mechanism of the reversible binding of nitric oxide to reduced cobalamin B(12r)(Cob(II)alamin. J Am Chem Soc 2001;123:978091. 26. Sharma VS, Pilz RB, Boss GR, Magde D. Reactions of nitric oxide with vitamin B12 and its precursor, cobinamide. Biochem 2003;42:89008. 27. Weinberg JB, Chen YC, Beasley BE, Ghosh DK. Cobalamins and cobinamides inhibit nitric oxide synthase enzymatic activity. Blood 2005;106:627a. Abstract. 28. Stucker M, Pieck C, Stoerb C, Niedner R, Hartung J, Altmeyer P. Topical vitamin B12 a new therapeutic approach in atopic dermatitis evaluation of efcacy and tolerability in a randomized placebo-controlled multicentre trial. Brit J Dermatol 2004;150:97783. 29. Weinberg JB, Shugars DC, Sherman PA, Sauls DL, Fyfe JA. Cobalamin inhibition of HIV-1 integrase and integration of HIV-DNA into cellular DNA. Biochem Biophys Res Commun 1998;246:3937. 30. Weinberg JB, Sauls DL, Misukonis MA, Shugars DC. Inhibition of productive human immunodeciency virus-1 infection by cobalamins. Blood 1995;86:12817. 31. Tang AM, Graham NMH, Chandra RK, Saah AJ. Low serum vitamin B12 concentrations are associated with faster human immunodeciency type 1 (HIV-1) disease progression. J Nutr 1997;127:34551. 32. Wickramasinghe SN, Fida S. Bone marrow cells from vitamin B12- and folate-decient patients misincorporate uracil into DNA. Blood 1994;83:165661. 33. Wickramasinghe SN. The wide spectrum of unresolved issues of megaloblastic anemia. 1999;36:318. 34. Ingram CF, Davidoff AN, Marais E, Sherman GG, Mendelow BV. Evaluation of DNA analysis for evidence of apoptosis in megalobalstic anemia. Brit J Haematol 1997;96:57683. 35. Perry J, Chanarin I, Deacon R, Lumb M. Methylation of DNA in megaloblastic anaemia. J Clin Pathol 1990;43:2112. 36. Healton EB, Savage DG, Brust JC, Garrett TJ, Lindenbaum J. Neurologic aspects of cobalamin deciency. Medicine 1991;70:22946. 37. Aaron S, Sudhir K, Vijayan J, Jacob J, Alexander M, Gnanamuthu C. Clinical and laboratory features and response to treatment in patients presenting with vitamin B12 deciency-related neurologic syndromes. Neurol India 2005;53:558. 38. Lee M, Chang H-S, Wu H-T, Weng H-H, Chen C-M. Intractable epilepsy as the presentation of vitamin B12 deciency in the absence of macrocytic anemia. Epilepsia 2005;46:11478. 39. Chatterjee A, Yapundich R, Palmer CA, Marson DC, Mitchell G. Leukoencephalopathy associated with cobalamin deciency. Neruology 1996;46:8324. 40. Ahn T-B, Cho J-W, Jeon BS. Unusual neurological presentations of vitamin B12 deciency. Europ J Neurol 2004;11:33941.

References
1. Green R, Kinsella L. Current concepts in the diagnosis of cobalamin deciency. Neurology 1995;45:143540. 2. Snow CF. Laboratory diagnosis of vitamin B12 and folate deciency. A guide for the primary care physician. Arch Intern Med 1999;159:128998. 3. Baik HW, Russell RM. Vitamin B12 Deciency in the Elderly. Annu Rev Nutr 1999;19:35777. 4. Carmel R. Current Concepts in Cobalamin Deciency. Annu Rev Med 2000;51:35775. 5. Carmel R, Rasmussen K, Jacobsen DW, Green R. Comparison of the deoxyuridine suppression test with serum levels of methylmalonic acid and homocysteine in mild cobalamin deciency. Brit J Haematol 1996;93:3118. 6. Choi S-W, Friso S, Ghandour H, Bagley PJ, Selhub J, Mason JB. Vitamin B12 deciency induces anomalies of base substitution and methylation in the DNA of rat colonic epithelium. J Nutr 2004;134:7505. 7. Herbert V. Recommended dietary intakes (RDI) of vitamin B12 in humans. Am J Clin Nutr 1987;45:6718. 8. Bailey LB. Folate and vitamin B12 recommended intakes and status in the United States. Nutr Rev 2004;62:S1420. 9. Fenech MF, Dresoti IE, Rinaldi JR. Folate, vitamin B12, homocysteine status and chromosome damage rate in lymphocytes of older men. Carcinogenesis 1997;18: 132936. 10. Fenech M, Aitken C, Rinaldi J. Folate, vitamin B12, homocysteine status and DNA damage in young Australian adults. Carcinogenesis 1998;19:116371. 11. Kapiszewska M, Kalemba M, Wojciech U, Milewicz T. Uracil misincorporation into DNA of leukocytes of young women with positive folate balance depends on plasma vitamin B12 concentrations and methylenetetrahydrofolate reductase polymorphisms. A pilot study. J Nutr Biochem 2005;16:46778. 12. Pfohl-Leszkowicz A, Keith G, Dirheimer G. Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: methylcobalamin can act as a methyl donor. Biochem 1991;30:804551. 13. Lorsch JR, Szostak JW. In vitro selection of RNA aptmers specic for cyanocobalamin. Biochem 1994;33:97382. 14. Lott WB, Takyar SS, Tuppen J, et al. Vitamin B12 and hepatitis C: molecular biology and human pathology. Proc Natl Acad Sci 2001;98:491621. 15. Takyar SS, Gowans EJ, Lott WB. Vitamin B12 stalls the 80 S ribosomal complex on the hepatitis C internal ribosome entry site. J Mol Biol 2002;319:18. 16. Li D, Lott WB, Martyn J, Haqshenas G, Gowans EJ. Differential effects on the hepatitis C virus (HCV) internal ribosome entry site by vitamin B12 and the HCV core protein. J Virol 2004;78:1207581. 17. Gulati S, Brody LC, Banerjee R. Posttranscriptional regulation of mammalian methionine synthase by B12. Biochem Biophys Res Commun 1999;259:43642. 18. Oltean S, Banerjee R. Nutritional modulation of gene expression and homocysteine utilization by vitamin B12. J Biol Chem 2003;278:2077884. 19. Oltean S, Banerjee R. A B12-responsive IRES element in human methionine synthase. J Biol Chem 2005;280: 326628. 20. Yamada K, Kawata T, Wada M, et al. Extremely low activity of methionine synthase in vitamin B12-decient rats may be related to effects on coenzyme stabilization rather than to changes in coenzyme function. J Nutr 2000;130:1894900.

ARTICLE IN PRESS
Disorders of cobalamin (Vitamin B12) metabolism
41. Saperstein DS, Wolfe GI, Gronseth GS, et al. Challenges in the identication of cobalamin-deciency polyneuropathy. Arch Neurol 2003;60:1296301. 42. Beitzke M, Pster P, Fortin J, Skrabal F. Autonomic dysfunction and hemodynamics in vitamin B12 deciency. Autonomic Neurosci: Basic and Clinical 2002;97:4554. 43. Al-Shubaili AF, Farah SA, Hussein JM, Trontelj JV, Khuraibet AJ. Axonal and demyelinating neuropathy with reversible proximal conduction block, an unusual feature of vitamin B12 deciency. Muscle Nerve 1998;21:13413. 44. Hemmer B, Glocker FX, Schumacher M, Deuschl G, Lucking CH. Subacute combined degeneration: clinical, electrophysiological, and magnetic resonance imaging ndings. J Neurol Neurosurg Psych 1998;65:8227. 45. Yu MK, Rodgers GM. Detection of occult cobalamin deciency by magnetic resonance imaging. Am J Hematol 2000;65:834. 46. Lindenbaum J, Healton EB, Savage DG, Brust JCM, Garrett ER, Podell ER, et al. Neuropsychiatric disorders caused by cobalamin deciency in the absence of anemia or macrocytosis. N Eng J Med 1988;318:17208. 47. Metz J. Cobalamin deciency and the pathogenesis of nervous system disease. Annu Rev Nutr 1992;12:5979. 48. Allen RH, Stabler SP, Savage DG, Lindenbaum J. Metabolic abnormalities in cobalamin (vitamin B12) and folate deciency. FASEB J 1993;7:134453. 49. Carmel R, Karnaze DS, Weiner JM. Neurologic abnormalities in cobalamin deciency are associated with higher cobalamin analogue values than are hematologic abnormalities. J Lab Clin Med 1988;111:5762. 50. Green R, Jacobsen DW. No discrepancy between vitamin B12 radioassays using puried intrinsic factor and R-binder in bats developing cobalamin deciency. Clin Res 1980;28:74A. 51. Kondo H, Osborne ML, Kolhouse JF, et al. Nitrous oxide has multiple deleterious effects on cobalamin metabolism and causes decreases in activities of both mammalian cobalamin-dependent enzymes in rats. J Clin Invest 1981;67:127083. 52. Van der Westhuyzen J, Fernandes-Costa F, Metz J, Kanazawa S, Drivas G, Herbert V. Cobalamin (vitamin B12) analogs are absent in plasma of fruit bats exposed to nitrous oxide. Proc Soc Exp Biol Med 1982;171:8891. 53. Carmel R, Melnyk S, James SJ. Cobalamin deciency with and without neurologic abnormalities: differences in homocysteine and methionine metabolism. Blood 2003;101:33028. 54. Carmel R, Pullarrkat V. Gene polymorphisms associated with diminished activity of 5,10-methylenetetrahydrofolate reductase do not explain the clinical manifestations of cobalamin deciency. Brit J Haematol 2003;12:9079. 55. Adachi K, Izumi M, Osano Y, et al. Polyamine concentrations in the brain of vitamin-B12 decient rats. Exp Biol Med 2003;228:106971. 56. Scalabrino G, Buccellato FR, Veber D, Mutti E. New basis for the neurotrophic action of vitamin B12. Clin Chem Lab Med 2003;41:14357. 57. Scalabrino G. Cobalamin (vitamin B12) in subacute combined degeneration and beyond: traditional interpretations and novel theories. Exp Neurol 2005;192:46379. 58. Peracchi M, Bamonti Catena F, Pomati M, De Franceschi M, Scalabrino G. Human cobalamin deciency: alterations in serum tumour necrosis factor-a and epidermal growth factor. Exp J Haematol 2001;67:1237. 59. Chanarin I, Stephenson E. Vegetarian diet and cobalamin deciency: their association with tuberculosis. J Clin Pathol 1988;41:75962.

13
60. Fata F, Herzlich BC, Schiffman G, Ast AL. Impaired antibody responses to pneumococcal polysaccharide in elderly patients with low serum vitamin B12 levels. Ann Int Med 1996;124:299304. 61. Imamura M, Fujimura K, Kuramoto A. Lymphocyte subpopulations in pernicious anemia. N Eng J Med 1984;311:56. 62. Wodzinski MA, Forrest MJ, Barnett D, et al. Lymphocyte subpopulations in patients with hydroxocobalamin responsive megaloblastic anaemia. J Clin Pathol 1985;38:5824. 63. Gogos CA, Kapatais-Zoumbos KN, Zoumbos NC. Lymphocyte subpopulations in megaloblastic anemia due to vitamin B12 deciency. Scand J Haematol 1986;37:3168. 64. Kubota K, Arai T, Tamura J, et al. Restoration of decreased suppressor cells by vitamin B12 therapy in patients with pernicious anemia. Am J Hematol 1987;24:2213. 65. Kubota K, Kurabayashi H, Kawada E, et al. Restoration of abnormally high CD4/CD8 ratio and low natural killer activity by vitamin B12 in a patient with post-gastrectomy megaloblastic anemia. Int Med 1992;31:1256. 66. Tamura J, Kubota K, Murakami H, et al. Immunomodulation by vitamin B12: augmentation of CD8+ T lymphocytes and natural killer (NK) cell activity in vitamin B12-decient patients by methyl-B12 treatment. Clin Exp Immunol 1999;116:2832. 67. Dhonukshe-Rutten RAM, Lips M, de Jong N, et al. Vitamin B12 status is associated with bone mineral content and bone mineral density in frail elderly women but not in men. J Nutr 2003;133:8017. 68. Tucker KL, Hannan MT, Qiao N, et al. Low plasma vitamin B12 is associated with lower BMD: the Framingham osteoporosis study. J Bone Miner Res 2005;20:1528. 69. Carmel R, Lau K-HW, Baylink DJ, Saxena S, Singer FR. Cobalamin and osteoblast-specic proteins. N Eng J Med 1988;319:705. 70. Kim GS, Kim C-H, Park JY, Lee K-U, Park CS. Effects of vitamin B12 on cell proliferation and cellular alkaline phosphatase activity in human bone marrow stromal osteoprogenitor cells and UMR106 osteoblastic cells. Metabolism 1996;45:14436. 71. Sato Y, Honda Y, Iwamoto J, Kanoko T, Satoh K. Effect of folate and mecobalamin on hip fractures in patients with stroke a randomized control trial. JAMA 2005;239: 10828. 72. Nanes MS. Tumor necrosis factor a: molecular and cellular mechanisms in skeletal pathology. Gene 2003;90:1 15. 73. Solomon LR. Cobalamin-responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid and homocysteine testing. Blood 2005;105: 97885. 74. Stabler SP, Allen RH, Savage DG, Lindenbaum J. Clinical spectrum and diagnosis of cobalamin deciency. Blood 1990;76:87181. 75. Matchar DB, McCrory DC, Millington DS, Feussner JR. Performance of the serum cobalamin assay for diagnosis of cobalamin deciency. Am J Med Sci 1994;308: 27683. 76. Carmel R, Vasireddy H, Aurangzeb I, George K. High serum cobalamin levels in the clinical setting clinical associations and holo-transcobalamin changes. Clin Lab Haem 2001;23:36571. 77. Ermens AAM, Vlasveld LT, Lindemans J. Signicance of elevated cobalamin (vitamin B12) levels in blood. Clin Biochem 2003;36:58590. 78. Faurschou M, Nielsen OJ, Jensen MK, Hasselbalch HC. High prevalence of hyperhomocysteinemia due to marginal

ARTICLE IN PRESS
14
deciency of cobalamin or folate in chronic myeloproliferative disorders. Am J Hematol 2000;65:13640. Baker H, Leevy CB, DeAngelis B, Frank O, Baker ER. Cobalamin (vitamin B12) and holotranscobalamin changes in plasma and liver tissue in alcoholics with liver disease. J Am Coll Nutr 1998;17:2358. Lambert D, Benhayoun S, Adjalla C, et al. Alcoholic cirrhosis and cobalamin metabolism. Digestion 1997;58: 6471. Carmel R, Brar S, Frouhar Z. Plasma total transcobalamin I: Ethnic/Racial patterns and comparison with lactoferrin. Am J Clin Pathol 2001;116:57680. Carmel R. Mild transcobalamin I (haptocorrin) deciency and low serum cobalamin concentrations. Clin Chem 2003;49:136774. Koebnick C, Hein UA, Dagnelie PC, et al. Longitudinal concentrations of vitamin B12 and vitamin B12-binding proteins during uncomplicated pregnancy. Clin Chem 2002;48:92833. Pardo J, Peled Y, Hod M, Sela BA, Rafael ZB, Orvieto R. Evaluation of low serum vitamin B12 in the nonanemic pregnant patient. Human Reproduction 2000;15:2246. Kalamegham R, Krishnaswamy K. Functional signicance of low serum vitamin B12 levels in pregnancy. Int J Vit Nutr Res 1977;47:526. McMullin MF, Young PB, Bailie KEM, Savage GA, Lappin TRJ, White R. Homocysteine and methylmalonic acid as indicators of folate and vitamin B12 deciency in pregnancy. Clin Lab Haem 2001;23:1615. Monsen A-LB, Ueland PM, Vollset SE, et al. Determinants of cobalamin staus in newborns. Pediatrics 2001;108:62430. Metz J, McGrath K, Bennett M, Hyland K, Bottiglieri T. Biochemical indices of vitamin B12 nutrition in pregnant patients with subnormal serum vitamin B12 levels. Am J Hematol 1995;48:2515. Adams MJ, Khoury MJ, Scanlon KS, Stevenson RE, et al. Elevated midtrimester serum methylmalonic acid levels as a risk factor for neural tube defects. Tetralogy 1995;51:3117. Gardyn J, Mittelman M, Zlotnik J, Sela BA, Cohen AM. Oral contraceptives can cause falsely low vitamin B12 levels. Acta Haematologica 2000;104:224. Shojania AM. Oral contraceptives: effects on folate and vitamin B12 metabolism. CMA J 1982;126:2447. Shojania AM, Wylie B. The effect of oral contraceptives on vitamin B12 metabolism. Am J Obst Gyn 1979;135:12934. Riedel B, Bjorke Monsen A-L, Ueland PM, Schneede J. Effects of oral contraceptives and hormone replacement therapy on markers of cobalamin status. Clin Chem 2005;51:77881. Sutterlin MW, Bussen SS, Rieger L, Dietl J, Steck T. Serum folate and vitamin B12 levels in women using modern oral contraceptives (OC) containing 20 lg ethinyl estradiol. Europ J Obstet Gyn Reproduc Biol 2003;107:5761. Carmel R, Howard JM, Green R, Jacobsen DW, Azen C. Hormone replacement therapy and cobalamin status in elderly women. Am J Clin Nutr 1996;64:8569. Hansen OP, Drivsholm A, Hippe E. Vitamin B12 metabolism in myelomatosis. Scand J Haematol 1977;18:395402. Ermens AA, Sonneveid P, Michiels JJ, Lindemans J. Increased uptake and accumulation of cobalamin by multiple myeloma bone marrow cells as a possible cause of low serum cobalamin. Europ J Haematol 1993;50:579. Remacha AF, Cadafalch J. Cobalamin deciency in patients infected with the human immunodeciency virus. Semin Hematol 1999;36:7587.

L.R. Solomon
99. Hansen M, Gimsing P, Ingeberg S, Jans H, Nexo E. Cobalamin binding in patients with HIV infection. Eur J Haematol 1992;48:22831. 100. Remacha AF, Cadafalch J, Sardar P, Barcelo M, Fuster M. Vitamin B12 metabolism in HIV-infected patients in the age of highly active retroviral therapy: role of homocysteine in assessing vitamin B12 status. Am J Clin Nutr 2003;77: 4204. 101. Kieburtz KD, Giang DW, Schiffer RB, Vakil N. Abnormal vitamin B12 metabolism in human immunodeciency virus infection: Association with neurologic dysfunction. Arch Neurol 1991;48:3124. 102. Puri V, Chaudry N, Goel S, Gulati P, Nehru R, Chowdhury D. Vitamin B12 deciency: a clinical and electrophysiological prole. Electromyogr Clin Neurophysiol:27384. 103. Namour F, Olivier J-L, Abdelmouttaleb A, et al. Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells in Caucasians: relation to transcobalamin and homocysteine concentration in blood. Blood 2001;97:10928. 104. Miller JW, Ramos MI, Garrod MG, Flynn MA, Green R. Transcobalamin II 755G > C polymorphism and indices of vitamin B12 status in healthy older adults. Blood 2002;100:71820. 105. Lievers KJA, Afman LA, Kluijtmans LAJ, et al. Polymorphisms in the transcobalamin gene: association with plasma homocysteine in healthy individuals and vascular disease patients. Clin Chem 2002;48:13839. 106. von Castel-Dunwoody KM, Kauwell GPA, Shelnutt KP, et al. Transcobalamin 776G C polymorphism negatively effects vitamin B12 metabolism. Am J Clin Nutr 2005;81:143641. 107. Gale DP, Cobbold JF, Chataway J. Steroid-responsive functional B12 deciency in association with functional transcobalamin II polymorphism 766 C G. Eur J Haematol 2006;76:758. 108. Wans S, Schutter K, Jacuicza S, Muller A, Luley C, Dierkea J. Analysis of the transcobalamin II 776G > C (259P > R) single nucleotide polymorphism by denaturing HPLC in healthy elderly: associations with cobalamin, homocysteine and holotranscobalamin II. Clin Chem Lab Med 2003;41:15326. 109. Teplitsky V, Huminer D, Zoldan J, Pitlik S, Shohat M, Mittelman M. Hereditary partial transcobalamin II deciency with neurologic mental and hematologic abnormalities in children and adults. Isr Med Assn J 2003;5:86872. 110. Zittoun J, Zittoun R. Modern clinical testing strategies in cobalamin and folate deciency. Semin Hematol 1999;36:3546. 111. Carmel R, Brar S, Agrawal A, Penha PD. Failure of assay to identify low cobalamin concentrations (letter). Clin Chem 2000;46:20178. 112. Vlasveld LT, vant Wout JW, Meeuwissen P, Castel A. High measured cobalamin (vitamin B12) concentration attributable to an analytical problem in testing serum from a patient with pernicious anemia. Clin Chem 2006;52:1578. 113. Lindenbaum J, Savage DG, Stabler SP, Allen RH. Diagnosis of cobalamin deciency: II. Relative sensitivities of serum cobalamin, methylmalonic acid and total homocysteine concentrations. Am J Hematol 1990;34:99107. 114. Stabler SP, Allen RH, Savage DG, Lindenbaum J. Clinical spectrum and diagnosis of cobalamin deciency. Blood 1990;76:87181. 115. Savage DG, Lindenbaum J, Stabler SP, Allen RH. Sensitivity of serum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deciencies. Am J Med 1994;96:23946.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91. 92. 93.

94.

95.

96. 97.

98.

ARTICLE IN PRESS
Disorders of cobalamin (Vitamin B12) metabolism
116. Moelby L, Rasmussen K, Jensen MK, Pedersen KO. The relationship between clinically conrmed cobalamin deciency and serum methylmalonic acid. J Intern Med 1990;228:37338. 117. Rasmussen K, Moller J, Ostergaard K, Ostergaard KM, Jensen J. Methylmalonic acid concentrations in serum of normal subjects: Biologic variability and effect of oral Lisoleucine loads before and after intramuscular administration of cobalamin. Clin Chem 1990;36:12959. 118. Hvas AM, Juul S, Gerdes LU, Nexo E. The marker of cobalamin deciency, plasma methylmalonic acid correlates to plasma creatinine. J Intern Med 2000;247:50712. 119. Moelby L, Rasmussen K, Ring T, Nielsen G. Relationship between methylmalonic acid and cobalamin in uremia. Kidney Intl 2000;57:26573. 120. Refsum H, Smith DA, Ueland PM, et al. Facts and recommendations about total homocysteine determinations: an expert opinion. Clin Chem 2004;50:332. 121. Garg UC, Zheng Z-J, Folsom AR, et al. Short-term and long-term variability of plasma homocysteine measurement. Clin Chem 1997;43:1415. 122. Thirup P, Ekelund S. Day-to-day, postprandial, and orthostatic variation of total plasma homocysteine. Clin Chem 1999;45:12803. 123. Santhosh-Kumar CR, Deutsch JC, Ryder JW, Kolhouse JF. Unpredictable intra-individual variations in serum homocysteine levels on folic acid supplementation. Europ J Clin Nutr 1997;51:18892. 124. Herzlich B, Herbert V. Depletion of holotranscobalamin II: an early sign of negative vitamin B12 balance. Lab Invest 1988;58:3327. 125. Hvas A-M, Nexo E. Holotranscobalamin as a predictor of vitamin B12 status. Clin Chem Lab Med 2003;41:148992. 126. Hvas A-M, Nexo E. Holotranscobalamin a rst choice assay for diagnosing early vitamin B12 deciency. J Int Med 2005;257:28998. 127. Chen X, Remacha AF, Pilar Sarda M, Carmel R. Inuence of cobalamin deciency compared with that of cobalamin absorption on serum holo-transcobalamin II. Am J Clin Nutr 2005;81:1104. 128. Wickramasinghe SN, Ratnayaka ID. Limited value of serum holo-transcobalamin II measurements in the differential diagnosis of macrocytosis. J Clin Pathol 1996;49:7558. 129. Molad Y, Rachmilewitz B, Sidi Y, Pinkhas J, Weinberger A. Serum cobalamin and transcobalamin levels in systemic lupus erythematosis. Am J Med 1990;88:1414. 130. Carmel R, Hollander D. Extreme elevation of transcobalamin II levels in multiple myeloma and other disorders. Blood 1978;51:105763. 131. Carmel R, Green R, Rosenblatt DS, Watkins D. Update on cobalamin, folate and homocysteine. Hematology:6281. 132. Karnaze DS, Carmel R. Neurologic and evoked potential abnormalities in subtle cobalamin deciency states including deciency without anemia and with normal absorption of free cobalamin. Arch Neurol 1990;47:100812. 133. Louwman MWJ, van Dusseldorp M, van de Vijver FJR, et al. Signs of impaired cognitive function in adolescents with marginal cobalamin status. Am J Clin Nutr 2000;72:7629. 134. Carmel R, Gott PS, Waters CH, et al. The frequently low cobalamin levels in dementia usually signify treatable metabolic, neurologic and electrophysiologic abnormalities. Eur J Haematol 1995;54:24553. 135. Andres E, Affenberger S, Vinzio S, et al. Food-cobalamin malabsorption in elderly patients: clinical manifestations and treatment. Am J Clin Nutr 2005;118:11549.

15
136. Hvas A-M, Ellegaard J, Nexo E. Increased plasma methylmalonic acid level does not predict clinical manifestations of vitamin B12 deciency. Arch Int Med 2001;161: 153441. 137. Hvas A-M, Ellegaard J, Nexo E. Vitamin B12 treatment normalizes metabolic markers but has limited clinical effect: A randomized placebo-controlled study. Clin Chem 2001;47:1396404. 138. Hvas A-M, Juul S, Nexo E, Ellegaard J. Vitamin B12 treatment has limited effect on health-related quality of life among individuals with elevated plasma methylmalonic acid: a randomized placebo-controlled study. J Int Med 2003;253:14652. 139. Marie RM, Le Biez E, Busson P, Schaeffer S. Nitrous oxide anesthesia-associated myelopathy. Arch Neurol 2000;57:3802. 140. Niyikza C, Baker SD, Seitz DE, Walling JM, et al. Homocysteine and methylmalonic acid: markers to predict and avoid toxicity from pemetrexed therapy. Molec Cancer Therapeutics 2002;1:54552. 141. Hazarika M, White RM, Johnson JR, Pazdur R. FDA drug approval summaries: premetrexed (Alimta). Oncologist 2004;9:4828. 142. Rasmussen K, Nathan E. The clinical evaluation of cobalamin determination of methylmalonic acid in serum or urine is not invalidated by the presence of heterozygous methylmalonic academia. J Clin Chem Clin Biochem 1990;28:41921. 143. Hvas AM, Ellegaard J, Lous J, Nexo E. Health technology assessment in clinical biochemistry. Methylmalonic acid: a Danish showcase. Scand J Clin Lab Invest 2003;63:31930. 144. Ledley FD, Levy HL, Shih VE, Benjamin R, Mahoney MJ. Benign methylmalonic aciduria. New Engl J Med 1984;311:10158. 145. Birn H, Nexo E, Christensen EI, Nielsen R. Diversity in rat tissue accumulation of vitamin B12 supports a distinct role for the kidney in vitamin B12 homeostasis. Nephrol Dial Transpl 2003;18:1095100. 146. Marcus DL, Shadick N, Crantz J, Gray J, Hernandez F, Freedman ML. Low serum B12 levels in a hematologically normal elderly subpopulation. J Am Geriatr Soc 1987;35:6358. 147. van Asselt DZB, Thomas CMG, Segers MFG, Blom HJ, Wevers RA, Hoefnagels WHL. Cobalamin-binding proteins in normal and cobalamin-decient older subjects. Ann Clin Biochem 2003;40:659. 148. Carmel R, Green R, Jacobsen DW, Rasmussen K, Florea M, Azen C. Serum cobalamin, homocysteine and methylmalonic acid concentrations in a multiethnic elderly population: ethnic and sex differences in cobalamin and metabolite abnormalities. Am J Clin Nutr 1999;70: 90410. 149. Clarke R, Refsum H, Birks J, et al. Screening for vitamin B12 and folate deciency in older patients. Am J Clin Nutr 2003;77:12417. 150. Campbell AK, Miller JW, Green R, Haan MN, Allen LH. Plasma vitamin B12 concentations in an elderly Latino population are predicted by serum gastrin concentrations and by crystalline vitamin B12 intake. J Nutr 2003;133: 27706. 151. Morris MS, Jacques PF, Rosenberg IH, Selhub J. Elevated serum methylmalonic acid concentrations are common among elderly Americans. J Nutr 2002;132:2799803. 152. Bates CJ, Schneede J, Mishra G, Prentice A, Mansoor MA. Relationship between methylmalinic acid, homocysteine, vitamin B12 intake and status and socio-economic indices in a subset of participants in the British National Diet and

ARTICLE IN PRESS
16
Nutrition Survey of people aged 65 years and older. Europ J Clin Nutr 2003;57:34959. Wolters M, Hermann S, Hahn A. B vitamin status and concentrations of homocysteine and methylmalonic acid in elderly German women. Am J Clin Nutr 2003;78:76572. Refsum H, Syajnik C, Gadkari M, et al. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deciency in Asian Indians. Am J Clin Nutr 2001;74:23341. Wolters M, Hermann S, Hahn A. Effect of multivitamin supplementation on homocysteine and methylmalonic acid blood concentrations in women over the age of 60. Eur J Nutr. Pennypacker LC, Allen RH, Kelly JP, et al. High prevalence of cobalamin deciency in elderly outpatients. J Am Geriatr Soc 1992;40:1197204. Lindenbaum J, Rosenberg IH, Wilson PWF, Stabler SP, Allen RH. Prevalence of cobalamin deciency in the Framingham elderly population. Am J Clin Nutr 1994;60:211. Bjorkegren K, Svardsudd K. Elevated serum levels of methlymalonic acid and homocysteine in elderly people: a population-based intervention study. J Int Med 1999;246:31724. Lewerin C, Nilsson-Ehle H, Matousek M, Steen B. Reduction of plasma homocysteine and serum methylmalonate concentrations in apparently healthy elderly subjects after treatement with folic acid, vitamin B12 and vitamin B6: a randomized trial. Europ J Clin Nutr 2003;57:142636. Holleland G, Schneede J, Ueland PM, Lund PK, Refsum H, Sandberg S. Cobalamin deciency in general practice. Assessment of the diagnostic utility and cost-benet analysis of methylmalonic acid determination in relation to current diagnostic strategies. Clin Chem 1999;45:18998. Joosten E, van de Berg A, Riezler R, et al. Metabolic evidence that deciences of vitamin B12 (cobalamin), folate and vitamin B6 occur commonly in elderly people. Am J Clin Nutr 1993;58:46876. Naurath HJ, Joosten E, Riezler R, Stabler SP, Allen RH, Lindenbaum J. Effects of vitamin B12, folate and vitamin B6 supplements in elderly people with normal serum vitamin concentrations. Lancet 1995;346:859. Eussen SJPM, de Groot LCPGM, Clarke R, et al. Oral cyanocobalamin supplementation in older people with vitamin B12 deciency: a dose-nding trial. Arch Intern Med 2005;165:116772. Rajan S, Wallace JI, Brodkin KI, Beresford SA, Allen RH, Stabler SP. Response of elevated methylmalonic acid to three doses levels of oral cobalamin in older adults. J Am Geriatr Soc 2002;50:178995. Herrmann W, Obeid R, Schorr H, Geisel J. Functional vitamin B12 deciency and determination of holotranscobalamin in populations at risk. Clin Chem Lab Med 2003;41:147888. Anwar W, Gerard P, Moutabarrek A, Namour F, Gueant J-L. End-stage renal disease increases plasma transcobalamin and neutralizes inuence of TCN 776C > G polymorphism. Kidney Intl:20923. letter. Kaplan LN, Mamer OA, Hoffer LJ. Parenteral vitamin B12 reduces hyperhomocysteinemia in end-stage renal disease. Clin Invest Med 2001;24:511. Mann B, Hyndman E, Burgess E, et al. Oral vitamin B12 and high-dose folic acid in hemodialysis patients with hyperhomocysteinemia. Kidney Intl 2001;59:11039. Elian KM, Hofer LJ. Hydroxocobalamin reduces hyperhomocysteinemia in end-stage renal disease. Metabolism 2002;51:8816.

L.R. Solomon
170. Hofer LJ, Elian KM. Parenteral vitamin B12 therapy of hyperhomocysteinemia in end-stage renal disease. Med Clin Exp 2004;27:103. 171. Nakhoul F, Abassi Z, Plawner M, et al. Comparative study of response to treatment with supraphysiologic doses of Bvitamins in hyperhomocysteinemic hemodialysis patients. IMAJ 2004;6:2137. 172. Hoffer LJ, Soboohi F, Golden M, Barre PE. Cobalamin dose regimen for maximum homocysteine reduction in end-stage renal disease. Metabolism 2005;54:83540. 173. Obeid R, Kuhlman MK, Kohler H, Herrmann W. Response of homocysteine, cystathionine and methylmalonic acid to vitamin treatment in dialysis patients. Clin Chem 2005;51:196201. 174. Obeid R, Kuhlmann M, Kirsch C-M, Herrmann W. Cellular uptake of vitamin B12 in patients with chronic renal failure. Nephron Clin Pract 2005;99:c428. 175. Koyama K, Yoshida A, Yakeda A, Morozumi K, Fujinami T. Efcacy of methylcobalamin in subclinical uraemic neuropathy as detected by measured vibration perception thresholds. Nephrology 1996;2:258. 176. Bhatt HR, Linnell JC, Matthews DM. Can faulty vitamin B12 (cobalamin) metabolism produce diabetic neuropathy? Lancet 1983;2:572. 177. Bailey LB, Molloy A, Scott J, Rice D. Streptozotocininduced diabetes is not a model for methylmalonic acidaemia. J Inherit Metab Dis 1989;12:42935. 178. Yagihashi S, Tokui A, Kashiwamura H, Takagi S, Imamura K. In vivo effect of methylcobalamin on the peripheral nerve structure in streptozotocin diabetic rats. Horm Metabol Res 1982;14:103. 179. Sonobe M, Yasuda H, Hatanaka I, et al. Methylcobalamin improves nerve conduction in streptozotocin-diabetic rats without affecting sorbitol or myo-inositol contents of sciatic nerve. Homr Metab Res 1988;20:7178. 180. Solomon LR. Elevated methylmalonic acid (MMA) and homocysteine (HCys) levels in patients with normal serum cobalamin (Cbl) values: diabetes mellitus (DM) and age as possible causes of Cbl resistance. Blood 2005;106:635a. abstract. 181. Smulders YM, Rakic M, Slaats EF, et al. Fasting and postmethionine homocysteine levels in NIDDM. Diabetes Care 1999;22:12532. 182. Ambrosch A, Dierkes I, Lobmann R, Kuhne W, Luley C, Lehnert H. Relation between homocysteinemia and diabetic neuropathy in patients with Type 2 diabetes mellitus. Diabet Med 2001;18:18592. 183. Beitzke M, Pster P, Fortin J, Skrabal F. Autonomic dysfunction and hemodynamics in vitamin B12 deciency. Autonomic Neurosci 2002;97:4554. 184. Yoshioka K, Tanaka K. Effect of methylcobalamin on diabetic autonomic neuropathy as assessed by power spectral analysis of heart rate variation. Horm Metab Res 1995;27:412. 185. Utsunomiya K, Narabayashi I, Tamura K, et al. Effects of aldose reductase inhibitor and vitamin B12 on myocardial uptake of iodine-123 metaiodobenzylguanidine in patients with non-insulin-dependent diabetes mellitus. Eur J Nucl Med 1998;25:16438. 186. Kuwabra S, Nakazawa R, Azuma N, et al. Intravenous methylcobalamin treatment for uremic and diabetic neuropathy in chronic hemodialysis patients. Intern Med 1999;38:4725. 187. Yaqub BA, Siddique A, Sulimani R. Effects of methylcobalamin on diabetic neuropathy. Clin Neurol Neurosurg 1992;94:10511.

153.

154.

155.

156.

157.

158.

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

ARTICLE IN PRESS
Disorders of cobalamin (Vitamin B12) metabolism
188. Ide H, Fujiya S, Asanuma Y, Tsuji M, Sakai H, Agishi Y. Clinical usefulness of intrathecal injection of methylcobalamin in patients with diabetic neuropathy. Clin Therapeutics 1987;9:18392. 189. Stracke H, Lindemann A, Federlin K. A benfotiaminevitamin B combination in treatment of diabetic polyneuropathy. Exp Clin Endcorinol Diabetes 1996;104:3116. 190. Stacke H, Hammes HP, Werkman D, et al. Efcacy of benfotiamine versus thiamine on function and glycation productis of peripheral nerves in diabetic rats. Exp Clin Endocrinol Diabetes 2001;109:3306. 191. Setola E, Monti LD, Galluccio E, et al. Insulin resistance and endothelial function are improved after folate and vitamin B12 therapy in patients with metabolic syndrome: relationship between homocysteine levels and hyperinsulinemia. Exp J Endocrinol 2004;151:4839. 192. Watanabe T, Kaji R, Oka N, Bara W, Kimura J. Ultra-high dose methylcobalamin promotes nerve regeneration in experimental acrylamide neuropathy. J Neurolog Sci 1994;122:1403. 193. Kikuchi M, Kahii S, Honda Y, Tamura Y, Kaneda K, Akaike A. Protective effects of methylcobalamin, a vitamin B12 analog, against glutamate-induced neurotoxicity in retinal cell culture. Invest Ophtalm Vis Sci 1997;38:84854. 194. Kaji R, Kodama M, Imamura A, et al. Effect of ultrahighdose methylcobalamin on compound muscle action potentials in amyotrophic lateral sclerosis: a double-blind controlled study. Muscle Nerve 1998;21:17758. 195. Sato Y, Honda Y, Iwamoto J, Kanoko T, Satoh K. Amelioration by mecocobalamin of sublcinical carpal tunnel syndrome involving unaffected limbs in stroke patients. J Neurol Sci 2005;231:138. 196. Fowler B. Genetic defects of folate and cobalamin metabolism. Eur J Pediatr 1998;15(Suppl2):S606. 197. Ames BN, Elson-Schwab I, Silver EA. High dose vitamin therapy stimulates variant enzymes with decreased coenzyme binding afnity (increased Km): relevance to genetic disease and polymorphisms. Am J Clin Nutr 2002;75:61658. 198. McCaddon A, Regland B, Hudson P, Davies G. Functional vitamin B12 deciency and Alzheimer disease. Neurology 2002;58:13959. 199. Miller A, Korem M, Almog R, Galboiz Y. Vitamin B12 demyelination, remyelination and repair in multiple sclerosis. J Neurol Sci 2005;233:937. 200. Di Rocco A, Bottiglieri T, Werner P, et al. Abnormal cobalamin dependent transmethylation in AIDS-associated myelopathy. Neurology 2002;58(march):2002. 201. Kira J, Tobimatsu S, Goto I. Vitamin B12 metabolism and massive-dose methyl vitamin B12 therapy in Japanese patients with multiple sclerosis. Intern Med 1994;33:826. 202. Wade DT, Young CA, Chaudhuri KR, Davidson DLW. A randomized placebo controlled exploratory study of vitamin B12, lofeperamine and L-phenylalanine (the CariLoser regime) in the treatment of multiple sclerosis. J Neurol Neurosurg Psych 2002;73:2469. 203. Mastonardi FG, Min W, Wang H, et al. Attenuation of experimental autoimmune encephalomyelitis and nonimmune demyelination by IFN-b plus vitamin B12: treatment to modify notch-1/sonic hedgehog balance. J Immunol 2004;172:641826. 204. Wang HX, Wahlin A, Basun H, Fastbom J, Winblad B, Fratiglioni L. Vitamin B12 and folate in relation to the development of Alzheimers disease. Neurol 2001;56: 118894. 205. McCaddon A, Hudson P, Abrahamsson L, Olofsson H, Regland B. Analogues, ageing and aberrant assimilation

17
of vitamin B12 in Alzheimers disease. Dementia Geriatr Cognitive Disorders 2001;12:1337. Zetterberg H, Nexo E, Regland B, et al. The transcobalamin (TC) codon 259 genetic polymorphism inuences holo-TC concentration in cerebrospinal uid from patients with Alzheimer Disease. Clin Chem 2003;49:11958. Refsum H, Smith AD. Low vitamin B12 status in conrmed Alzheimers disease as revealed by holotranscobalamin. J Neurol Neurosurg Psychiatry 2003;74:95961. McCaddon A, Blennow K, Hudson P, et al. Transcobalamin polymorphism and serum holo-transcobalamin in relation to Alzheimers disease. Dementia Geriatr Cognitive Disorders 2004;17:21521. Kanazawa S, Herbert V. Noncobalamin vitamin B12 analogues in human red cells, liver and brain. Am J Clin Nutr 1983;37:7747. Stabler SP, Brass EP, Marshall PD, Allen RH. Inhibition of cobalamin-dependent enzymes by cobalamin analogues in rats. J Clin Invest 1991;87:142230. Malouf R, Aranea SA. Vitamin B12 for cognition (review). Cochrane Library 2005;4:118. Di Rocco A, Werner P, Bottiglieri T, et al. Treatment of AIDS-associated myelopathy with L-methionine: a placebocontrolled study. Neurol 2004;63:12705. Olcay L, Irkkan C, Senbil N, Haci U, Basmaci M, Gurer YKY. Progressive vacuolar myelopathy and leukoencephalopathy in childhood acute lymphoblastic leukemia and transient improvement with vitamin B12. Pediatr Blood Cancer 2006; (in press). Berliner N, Rosenberg LE. Uptake and metabolism of free cyanocobalamin by cultured human broblasts from controls and a patient with transcobalamin II deciency. Metabolism: Clin Exper:2306. Chalmers JNM, Shinton NK. Comparison of hydroxocobalamin and cyanocobalamin in the treatment of pernicious anemia. Lancet 1965;2:13058. Boddy K, King P, Mervyn L, Macleod A, Adams JF. Retention of cyanocobalamin, hydroxocobalamin and coenzyme B12 after parenteral administration. Lancet 1968;2:7102. Hall CA, Begley JA, Green-Colligan PD. The availability of therapeutic hydroxocobalamin to cells. Blood 1984;63: 33541. Andersson HC, Shapira E. Biochemical and clinical response to hydroxocobalamin versus cyanocobalamin treatment in patients with methylmalonic academia and homocystinuria (cblC). J Pediatrics 1998;132:1214. Bastrup-Madsen P. Treatment of vitamin B12 deciency: evaluation of therapy with cyanocobalamin, hydroxocobalamin and depot cobalamin Betolvex. Scand J Gastroenterol(Suppl 29):8995. Tudhope GR, Swan HT, Spray GC. Patient variation in pernicious anaemia, as shown in a clinical trial of cyanocobalamin, hydroxocobalamin and cyanocobalamin-zinc tannate. Brit J Haematol 1967;13:21628. Magnus EM. Cobalamin and unsaturated transcobalamin values in pernicious anaemia: relation to treatment. Scand J Haematol 1986;36:45765. Chalmers JNM, Shinton NK. Absorption of orally administered vitamin B12 in pernicious anaemia. Lancet 1958;2:1298302. Hemsted EH, Mills J. Vitamin B12 in pernicious anaemia: intramuscular or oral? Lancet 1958;2:13023. Berlin R, Berlin H, Brante G, Pilbrant A. Vitamin B12 body stores during oral and parenteral treatment of pernicious anaemia. Acta Med Scand 1978;204:814. Vidal-Alaball J, Butler CC, Cannings-John R, et al. Oral vitamin B12 versus intramuscular B12 for vitamin B12

206.

207.

208.

209.

210.

211. 212.

213.

214.

215.

216.

217.

218.

219.

220.

221.

222.

223. 224.

225.

ARTICLE IN PRESS
18
deciency (Review). The Cochrane Database of Systematic Reviews 2005;3:121. 226. Kuzminski AM, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J. Effective treatment of cobalamin deciency with oral cobalamin. Blood 1998;92:11918. 227. Bolaman Z, Kadikoylu G, Yukselen V, Yavasoglu I, Barutca T, Senturk T. Oral versus intramuscular cobalamin treatment in megaloblastic anemia: a single-center, prospective, randomized open-label study. Clin Therapeutics 2003;25:312434. 228. Andres E, Kaltenback G, Noel E, et al. Efcacy of shortterm oral cobalamin therapy for the treatment of cobalamin deciencies related to food-cobalamin malabsorp-

L.R. Solomon
tion: a study of 30 patients. Clin Lab Haem 2003;25:1616. Andres E, Kurtz J-E, Perrin A-E, et al. Oral cobalamin therapy for the treatment of patients with food-cobalamin malabsorption. Am J Med 2001;111:1269. Kondo H. Haematological effects of oral cobalamin preparations on patients with megaloblastic anaemia. Acta Haematol 1998;99:2005. Solomon LR. Oral vitamin B12 therapy: a cautionary note. Blood 2004;103:2863. Green R. Unreliability of current assays to detect cobalamin deciency: nothing gold can stay. Blood 2005;105:910.

229.

230.

231. 232.