Functional cobalamin (Cbl; vitamin B12) deficiency (that is, high levels of the Cbl-dependent metabolites, methylmalonic acid (MMA) and homocysteine (HCys), despite normal serum Cbl values) is common in the elderly and is associated with neurocognitive abnormalities, but its cause is unknown. As only reduced Cbls are metabolically active, the possibility that functional Cbl deficiency is associated with disorders having biomarkers indicative of increased oxidative stress (oxidant risks) was considered.
A retrospective record review of community-dwelling adults evaluated over a 12-year period for Cbl deficiency in a primary care setting who had serum Cbl values ⩾400 pg/ml (n=170).
When no oxidant risks were present, older subjects (⩾70 years) had higher metabolite values than younger individuals (<70 years). MMA values were even higher in the elderly when one oxidant risk was present and in younger subjects when two or more oxidant risks were present. Even at Cbl levels ⩾800 pg/ml, MMA values were increased in 73% of elderly subjects with at least one oxidant risk. HCys values were also higher in both age groups when at least two oxidant risks were present. Cyanocobalamin therapy decreased MMA and HCys values in 86 and 76% of subjects, respectively, with nonresponders more likely to have two or more oxidant risks.
Functional Cbl deficiency is associated with disorders marked by increased oxidative stress particularly in the elderly; it occurs even when Cbl levels are high and is not consistently corrected with high-dose cyanocobalamin therapy. Thus, current approaches to recognizing and managing this disorder may be inadequate.
Presence of elevated levels of the cobalamin (Cbl; vitamin B12)-dependent metabolites, methylmalonic acid (MMA) and homocysteine (HCys), in asymptomatic patients with low serum Cbl levels has been termed ‘subtle’ or ‘subclinical’ Cbl deficiency, suggesting a continuum of Cbl depletion resulting first in a fall in serum Cbl levels, progressing next to metabolite accumulation and resulting finally in hematologic and/or neurocognitive disorders, which evolve over several years.1 However, 7–30% of elderly subjects have ‘functional’ Cbl deficiency, defined as the presence of elevated metabolite values despite Cbl levels well within the normal reference range, and this disorder has also been linked to neurocognitive abnormalities.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13
The pathophysiology of functional Cbl deficiency is unknown and <17% of the variation in MMA can be explained by the determinants of age, Cbl level and renal function.14 It is noteworthy then that Cbl is an active superoxide scavenger;15, 16 reduced forms of Cbl are required for its coenzyme activity;2 cellular uptake of Cbl may be impaired by oxidative byproducts;17 and functional Cbl deficiency occurs after exercise and in many other pro-oxidant disorders.18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 Significantly, diabetes and folate excess, both of which are associated with increased oxidative stress, have been linked to functional Cbl deficiency in the elderly but not in younger individuals.10, 18, 21 These observations suggest that functional Cbl deficiency may be associated with oxidative stress and that this relationship may be particularly prevalent in the elderly. Thus, a retrospective study was performed to test this hypothesis by determining the relationship of functional Cbl deficiency to the number of oxidant-stress-related disorders (oxidant risks) present in elderly and younger individuals evaluated for the presence of Cbl deficiency.
Patients and methods
Serum Cbl, serum MMA and plasma HCys were measured as previously described.9 The reference range for Cbl was 201–1100 pg/ml. However, Cbl values between 201 and 400 pg/ml have often been found in subjects with both metabolically and clinically significant Cbl deficiency due to frank depletion of Cbl stores related to decreased dietary intake or malabsorption.14, 34, 35, 36, 37 Thus, subjects with Cbl values ⩾400 pg/ml were considered to have adequate Cbl stores and ‘functional’ Cb; deficiency was defined as the presence of high metabolite values when Cbl values were ⩾400 pg/ml. The reference ranges for MMA and HCys were taken as 90–250 nmol/l and 5.4–12.1 μmol/l, respectively.9 When values were obtained on more than one occasion within a 4-week period, the lowest Cbl value and the highest metabolite values were used for analysis. Tests for intrinsic factor antibodies (IF Abs) were performed in 59 subjects (35%), including 46 of the 78 subjects with increased values for MMA and/or HCys (59%).
A retrospective review of the medical records of all community-dwelling subjects evaluated by the author for Cbl deficiency in a primary care setting between 1 August 1993 and 30 June 2005 was conducted as previously described.9, 10 Subjects were initially tested because of the presence of clinical findings consistent with Cbl deficiency or because of the presence of disorders known to lead to Cbl depletion.
Of the 473 subjects evaluated with the measurement of MMA, 175 had Cbl values ⩾400 pg/ml (37%). Two patients on chronic hemodialysis and 3 patients taking high-dose vitamin B12 supplements were excluded, leaving 170 patients evaluable for analysis of the relationship of age, Cbl level and oxidant risk factors to MMA. Hcys was measured in 161 of these subjects (95%).
This study conforms to the principles of the Declaration of Helsinki of 1975 as revised in 2008, and the institutional human investigation committee determined that further review was not required.
Identification of oxidant risks
All active medical conditions present in each subject were identified, and a Medline search was performed to determine which of these disorders were associated with increased oxidative stress (oxidant risks) as defined by the presence of increased oxidative byproducts systemically.38 At least one oxidant risk was present in 88 subjects (52%), including diabetes mellitus (DM; n=36), cigarette abuse (n=18), mild–moderate renal insufficiency (creatinine=1.4–2.2 mg/dl; n=19), alcohol abuse (n=11), malignancy (n=14), neurodegenerative disorders (n=5), chronic infections (n=6), congestive heart failure (n=5), rheumatologic disorders (n=5), medication-dependent asthma (n=4), pregnancy (n=3), inflammatory bowel disease (n=2), iron overload (n=2), unexplained high sedimentation rates of 85 and 115 mm/h (n=2), hyperthyroidism (n=1) and cirrhosis (n=1). Based on the presence of these disorders, subjects were divided into three groups: ‘No’ oxidant risks; ‘One’ oxidant risk; and ‘Two or More’ (‘Two+’) oxidant risks. In a preliminary analysis, MMA and HCys values relative to the number of oxidant risks were similar in subjects <60 years old and in those 60–69 years old (data not shown). Thus, subjects were further divided into a younger age group (<70 years old; n=100) and an older population (⩾70 years old; n=70).
Patients were considered for treatment if they had clinical findings known to be associated with Cbl deficiency or if MMA values were elevated. As abnormal HCys values are less specific for Cbl deficiency, treatment was not offered to subjects with isolated high HCys values.2 Patients were treated with cyanocobalamin 2 mg per day orally or 1 mg intramuscularly 3 times a week for 2 weeks, weekly for 8 weeks and monthly thereafter. Metabolites were remeasured 1–3 months after beginning Cbl therapy. A response to Cbl therapy was defined as either a fall in the metabolite level to a value within the normal reference range (that is, ⩽250 nmol/l for MMA and ⩽12.1 μmol/l for HCys) or a decrease in the metabolite by more than 1 s.d. greater than its mean intraindividual variability as previously determined for this population (that is, >116 nmol/l for MMA and >3.6 μmol/l for HCys).9
Population studies suggest skewed distributions for Cbl, MMA and HCys.39 Thus, geometric means, two-tailed Student's t-tests and paired t-tests using log-transformed data, and χ2 analyses were determined using StatPlus:mac (release 5.7, 2009, AnalystSoft, Vancouver, BC, Canada).
In the younger population, Cbl values were not significantly different in the three oxidant risk categories but both mean age and the incidence of male sex were higher in subjects with ‘Two+’ oxidant risks than in those with ‘No’ or ‘One’ oxidant risk (Table 1). In the older population, age, sex and Cbl values were similar in all three oxidant risk groups. However, Cbl values were consistently higher in older subjects than in younger individuals with the same number of oxidant risks, and this difference was statistically significant in subjects with ‘Two+’ oxidant risks. The incidences of DM, renal disease and smoking, the three most prevalent oxidant risk factors identified, were similar in the two age groups when only ‘One’ oxidant risk was present.
Relationship between oxidant risks and MMA values relative to age
In the elderly, mean MMA values were significantly higher when ‘One’ or ‘Two+’ oxidant risks were present than when ‘No’ oxidant risk was identified (Figure 1a). In contrast, in the younger population, mean MMA values were the same when ‘No’ or ‘One’ oxidant risk was present but significantly higher when ‘Two+’ oxidant risks were identified. MMA values were consistently higher in the elderly than in younger subjects in all oxidant risk categories and these differences were statistically significant when ‘One’ or ‘Two+’ oxidant risks were present.
The pattern was the same when the incidences of elevated MMA values were considered (Figure 2a). Even when Cbl values were ⩾800 pg/ml in elderly subjects with at least one oxidant risk, MMA values were elevated in 8 of 11 individuals (73%).
A further analysis was performed to determine whether the effect of single oxidant risks on MMA values in the older population was solely due to the relatively high prevalence of DM in these subjects. In the elderly, the presence of either DM or any non-DM oxidant risk factor was associated with higher mean MMA values than when ‘No’ oxidant risk was identified and mean MMA values were not significantly different in elderly DM and non-DM subjects (P=0.35; Table 2). MMA values >250 nmol/l were also significantly more frequent in elderly subjects with either DM (83%) or any non-DM oxidant risk (63%) than in elderly subjects with ‘No’ oxidant risk (22%; P=0.0041 and P=0.0083, respectively).
Relationship between oxidant risks and HCys values relative to age
When ‘No’ or ‘One’ oxidant risk was present, mean HCys values were significantly higher in older subjects than in younger individuals (Figure 1b). The presence of ‘One’ oxidant risk was not associated with higher mean HCys values in either age group, but the presence of ‘Two+’ oxidant risks was associated with a 36% higher mean HCys value in the elderly (P<0.001) and with a 52% higher mean HCys value in the younger age group (P<0.001). Mean HCys values were similar in the two age groups when ‘Two+’ oxidant risks were present (P=0.30).
The pattern was the same when the incidences of elevated HCys values were considered (Figure 2b). Even when Cbl values were ⩾800 pg/ml in elderly subjects with at least one oxidant risk, HCys values were elevated in 6 of 10 individuals (60%).
Pattern of increased metabolite values in subjects with functional Cbl deficiency
At least one metabolite was increased in 73 of the subjects in whom both MMA and HCys were measured. Increased MMA values were significantly more frequent than increased HCys values, with increases in both metabolites present in 41% of subjects, with isolated increases in MMA present in 40% of subjects and with isolated increases in HCys present in 19% of subjects (Figure 3).
IF Abs in subjects with functional Cbl deficiency
IF Abs were measured in 46 of the 64 subjects with increased MMA values (72%) and were negative in 43 (93%), equivocal in 2 (4%) and positive in 1 (2%). Overall, Cbl values in subjects with negative tests for IF Abs (565 pg/ml; n=56) were not significantly different from those in untested subjects (582 pg/ml; n=111; P=0.54).
Functional Cbl deficiency in vegetarian subjects
Fourteen subjects on vegetarian diets were identified, all of whom were <70 years old: 11 with no oxidant risks and 3 with one oxidant risk. MMA values were increased in three subjects; both MMA and HCys values were increased in one subject; and an isolated increase in HCys was present in one subject.
Effect of Cbl therapy on MMA and HCys values
Fifty of the 64 subjects with high MMA values were evaluable for the effects of Cbl therapy (78%) and significant responses were noted in 43 of them (86%; Table 3). Similarly, 25 of the 44 subjects with high HCys values were evaluable for the effects of Cbl therapy (57%) and significant responses were noted in 19 of them (76%). Even when pre-therapy Cbl values were ⩾800 pg/ml, significant responses were noted in seven of eight treated subjects with pre-therapy elevations in MMA (88%) and in three of five treated subjects with pre-therapy elevations in HCys (60%). In the subjects with negative tests for IF Abs treated with Cbl, significant responses occurred in 33 of the 37 with high MMA values (89%) and in 15 of the 18 with high HCys values (83%). In 23 treated subjects who had elevated values for both MMA and HCys, responses were noted in both metabolites in 16 (70%), only in MMA in 3 (13%), only in HCys in 1 (4%) and in neither metabolite in 3 (13%).
Decreases in metabolite values occurred in both age groups and in all oxidant risk categories. However, ‘Two+’ oxidant risks were present in five of the seven MMA nonresponders (71%) and in 5 of the 6 HCys nonresponders (83%). Moreover, mean post-treatment values for MMA (242 nmol/l) and HCys (12.7 μmol/l) in all treated subjects with ‘Two+’ oxidant risks were significantly higher than the corresponding values in all treated subjects with ‘No’ or ‘One’ oxidant risk (200 nmol/l and 9.6 μmol/l, respectively; P=0.047 and 0.0034).
In the present study, the incidence of functional Cbl deficiency was found to be increased in subjects with disorders associated with increased oxidative stress and this relationship was more marked in the elderly even though Cbl levels were consistently higher in older subjects than in younger individuals with the same number of oxidant risks (Figures 1 and 2; Table 1). Functional Cbl deficiency occurred in elderly subjects even when Cbl values were ⩾800 pg/ml. Moreover, cyanocobalamin therapy significantly decreased MMA and HCys values in most but not all subjects, with nonresponders more likely to have a ‘Two+’ oxidant risk (Table 3; text). As tests for IF Abs were positive in only 1 of the 46 subjects with an increase in MMA tested (2%), these results cannot be explained by a Cbl assay artefact with ‘false normal’ Cbl values masking frank Cbl deficiency.40 Taken together, these findings suggest a relationship between functional Cbl deficiency and both advanced age and disorders associated with increased oxidative stress. Significantly, increased levels of biomarkers indicative of increased oxidative stress are also associated with ageing per se.
It was also noteworthy that MMA values were more frequently increased than HCys values (Figure 3). As MMA accumulation results from decreased activity of a mitochondrial Cbl-dependent enzyme, whereas HCys accumulation results from decreased activity of a cytoplasmic Cbl-dependent enzyme, and as oxidative damage is more likely to impair mitochondrial processes, this observation also suggests a role for oxidative stress in this disorder.2, 41
The optimum lower levels suggested for serum Cbl have been variably based on the distribution of Cbl values in the general population, on the point below which clinical signs or symptoms of deficiency begin to appear, or the point below which MMA and HCys values begin to increase. Using these criteria, minimum optimum values suggested for serum Cbl have ranged from 200 to 550 pg/ml (150–400 pmol/l).14, 34, 35, 36, 37 Nonetheless, the relationships of MMA and HCys values to age and oxidant risks in the current study population were the same regardless of the criteria used to define the lower limit of normal for serum Cbl (see Supplementary Table 1).
As functional Cbl deficiency associated with oxidative inactivation of Cbl does not require depletion of vitamin stores, this disorder could develop more rapidly than classic Cbl deficiency. This has indeed been shown to be the case when Cbl oxidation is induced by nitrous oxide exposure.42 Moreover, higher and more frequent doses of Cbl may be required to correct functional Cbl deficiency than are needed for the treatment of classic Cbl deficiency.2, 43 Finally, a fully reduced form of Cbl (for example, methylcobalamin) may be more effective in this setting than the partially reduced forms of Cbl usually administered (that is, cyanocobalamin and hydroxocobalamin).44
As vegetarian diets increase blood levels of antioxidants and decrease markers of oxidative stress, it is possible that they might prevent functional Cbl deficiency.45, 46, 47, 48 However, functional Cbl deficiency was identified in 5 of the 14 vegetarian subjects (36%) identified in the present study. Previous reports also suggest that functional Cbl deficiency occurs in vegetarians, as the incidences of high MMA and HCys values were consistently greater than the incidence of low Cbl values in these populations.49, 50, 51, 52 However, relatively low cutoff values for Cbl of 213–221 pg/ml were used to define Cbl deficiency in these reports, and confounding disorders associated with increased oxidative stress were not identified. Thus, further studies are needed to determine whether vegetarian diets can protect against functional Cbl deficiency.
This study was limited in that it was a retrospective analysis with patients selected because of a suspicion of Cbl deficiency based on clinical findings or predisposing risk factors. Moreover, as the severity of oxidative stress varies between and within the disorders identified as oxidant risks and as the presence of other oxidant risks may not have been recognized, this study was also limited by the absence of measurement of biomarkers of oxidative stress. Therefore, prospective controlled trials with direct measures of oxidative stress are needed to confirm these observations. These trials could focus on subjects with specific disorders strongly associated with increased oxidative stress (for example, DM, cancer) or on unselected populations of different ages and should include biomarkers of oxidative stress, including malondialdehyde and F2-isoprostanes (as indices of lipid oxidative damage), carbonylated proteins (as an index of protein oxidative damage), 8-hydroxy-2’-deoxyguanosine (as an index of DNA oxidative damage) and reduced and oxidized glutathione levels (as a general index of the redox state).38 In fact, a small controlled prospective study of 18 subjects with schizophrenia, a disorder in which increased oxidative stress has been described, found elevated levels of urinary MMA to be directly correlated with increased values of malondialdehyde in erythrocyte membranes.53 As Cbl levels in these subjects were in the low to normal range and not significantly different than that in the control group, whereas plasma HCys values were normal, this pattern was considered consistent with functional Cbl deficiency.
It is concluded that functional Cbl deficiency occurs in elderly subjects with multiple oxidant risks even when high to normal serum Cbl levels are present and that treatment requirements may be different than previously suggested. As functional Cbl deficiency has been linked to neurocognitive disorders, current approaches to recognizing and treating Cbl deficiency in the ill-elderly need to be reassessed.2, 3, 4, 5, 6, 7, 8, 9, 10, 11
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I thank D Bonke (Merck, Darmstadt, Germany) for his suggestion regarding the possible effect of oxidative stress on cobalamin metabolism and R Green (Department of Medical Pathology and Laboratory Medicine, University of California Davis Medical Center, Sacramento, CA, USA) for his suggestion regarding the possible therapeutic role of methylcobalamin.
The author declares no conflict of interest.
Supplementary Information accompanies this paper on European Journal of Clinical Nutrition website
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Solomon, L. Functional cobalamin (vitamin B12) deficiency: role of advanced age and disorders associated with increased oxidative stress. Eur J Clin Nutr 69, 687–692 (2015). https://doi.org/10.1038/ejcn.2014.272
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