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Low Cobalamin Levels as Predictors of Cobalamin Deficiency: Importance of Comorbidities Associated with Increased Oxidative Stress
Accepted Manuscript Low Cobalamin (Vitamin B12) Levels as Predictors of Cobalamin Deficiency: Importance of Comorbidities Associated With Increased Oxidative Stress Lawrence R. Solomon, M.D. PII:
S0002-9343(15)00694-4
DOI:
10.1016/j.amjmed.2015.07.017
Reference:
AJM 13121
To appear in:
The American Journal of Medicine
Received Date: 12 May 2015 Revised Date:
30 June 2015
Accepted Date: 1 July 2015
Please cite this article as: Solomon LR, Low Cobalamin (Vitamin B12) Levels as Predictors of Cobalamin Deficiency: Importance of Comorbidities Associated With Increased Oxidative Stress, The American Journal of Medicine (2015), doi: 10.1016/j.amjmed.2015.07.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Low Cobalamin (Vitamin B12) Levels as Predictors of Cobalamin Deficiency: Importance of Comorbidities Associated With Increased Oxidative Stress
Author: Lawrence R. Solomon, M.D.
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Running Title: B12 and Oxidative Stress
Institutional Affiliation: Section of Palliative Care; Department of Medicine
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Yale University School of Medicine Corresponding Author:
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Lawrence R. Solomon, M.D.
Section of Palliative Care, Department of Medicine,
Yale University School of Medicine and Smilow Cancer Hospital 403 www; 333 Cedar Street
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P.O. Box 208021
New Haven, CT. 06520-8021
e-mail: [email protected]
Funding: None
Fax: 203-785-3712
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Telephone: 203-737-4353
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Conflicts of Interest: None
Role of Author: Lawrence R. Solomon was solely responsible for the design and performance of this study, analysis of the data and preparation of the manuscript.
Word Counts: Manuscript: 2,379 Abstract: 248 Figures: 3 Tables: 3 References: 34 Key Words: Oxidative stress; cobalamin; vitamin B12; methylmalonic acid; homocysteine
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ABSTRACT Background: Cobalamin (B12) deficiency can lead to irreversible neurocognitive changes if
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unrecognized. Screening involves measurement of serum cobalamin levels but the sensitive
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metabolic indicators of cobalamin deficiency, methylmalonic acid (MMA) and homocysteine
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(HCys), may be normal when cobalamin values are low and elevated when cobalamin values are
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normal. Since cobalamin is inactivated by oxidation, the relationship between these metabolites
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and comorbidities associated with increased oxidative stress (oxidant risks) in subjects with low
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and low-normal cobalamin levels was studied.
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Methods: A retrospective record-review was conducted of community-dwelling adults
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evaluated for cobalamin deficiency during a 12 year period with serum cobalamin values in the
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low (≤200 pg/ml; n=49) or low-normal (201-300 pg/ml; n=187) range and concurrent
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measurement of MMA.
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Results: When “No” oxidant risk was present, elevated MMA (>250 nmol/l) and HCys (>12.1
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µmol/l) values occurred in 50% and 30% of subjects respectively (p<0.01). In contrast, when
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“Three or More” oxidant risks were present, mean MMA and HCys values were significantly
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higher and elevated MMA and HCys values occurred in 84% and 78% of these subjects
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(p≤0.012). Pharmacologic doses of cyanocobalamin significantly decreased metabolite values in
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≥94% of treated subjects.
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Conclusion and Relevance: In subjects with low or low-normal cobalamin values, metabolic
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evidence of cobalamin deficiency is more frequent when three or more oxidant risks are present.
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Thus, defining a low serum cobalamin level to screen for cobalamin deficiency may be a
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“moving target” due to the variable presence and severity of often subtle, confounding clinical
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conditions in individual subjects.
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INTRODUCTION Cobalamin (i.e. vitamin B12) deficiency is common in vegetarians, the elderly and subjects using such frequently prescribed medications as proton pump inhibitors, H2-blockers and
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metformin1. If unrecognized, this disorder can lead to irreversible neurocognitive dysfunction.
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Measurement of serum cobalamin (Cbl) is initially performed to screen for Cbl deficiency.
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However, cut-off points suggested vary widely between 200 pg/ml and 500 pg/ml2,3. Moreover,
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even at Cbl levels ≤200 pg/ml, the lowest cut-off point commonly used to define Cbl deficiency,
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many subjects have neither clinical evidence of Cbl deficiency (i.e. megaloblastic anemia and/or
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neurocognitive disorders) nor elevated levels of the Cbl-dependent metabolites, methylmalonic
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acid (MMA) and homocysteine (HCys)(biochemical indicators of Cbl depletion)4-6. It is of note
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then that Cbl is readily inactivated by oxidation and that elevated MMA and HCys levels in
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subjects with normal Cbl values have been related to the presence of comorbidities associated
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with increased oxidative stress (oxidant risks)7. Since Cbl values between 201 and 300 pg/ml are
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often present in subjects with both metabolically and clinically significant Cbl deficiency, a
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retrospective study was performed to determine if metabolic changes consistent with Cbl
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deficiency were also related to the presence of oxidant risks in subjects with low (≤200 pg/ml)
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and low-normal (201-300 pg/ml) Cbl values9-13.
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METHODS
Laboratory Methods
Cbl, MMA and HCys were measured as previously described7. The laboratory reference range
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for Cbl was 201-1100 pg/ml (Quest Laboratory). Since Cbl values between 201 and 300 pg/ml
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are often present in subjects with both metabolically and clinically significant Cbl deficiency due
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to Cbl malabsorption, Cbl values ≤200 pg/ml were defined as “low” while Cbl values of 201-300
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pg/ml were defined as “low-normal” 9-13. The reference ranges for MMA and HCys were taken
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as 90-250 nmol/l and 5.4-12.1 µmol/l respectively8. When values were obtained on more than
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one occasion within a 6-week period, the lowest Cbl value and the highest metabolite values
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were used for analysis.
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Subjects
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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 August 1, 1993 and June 30,
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2005 with the measurement of both serum Cbl and MMA levels, was conducted as previously
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described8,14. Subjects were screened because of the presence of clinical findings consistent with
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Cbl deficiency or because of the presence of disorders known to lead to Cbl depletion1. Cbl
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values were “low” in 49 subjects and “low-normal” in 187 subjects. HCys values were obtained
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in 204 of these 236 individuals (86%).
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This study conforms to the principles of the Declaration of Helsinki of 1975 as revised in 2008
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and the institutional human investigation committee determined that further review was not
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required.
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Identification of Oxidant Risks
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All active medical conditions present in each subject were identified and a Medline search was performed to determine which disorders were associated with increased oxidative stress as
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defined by the presence of increased oxidative byproducts systemically including
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malondialdehyde and F2-isoprostanes (as indices of lipid oxidative damage); carbonylated
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proteins (as an index of protein oxidative damage); 8-hydroxy-2’-deoxyguanosine (as an index of
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DNA oxidative damage); and reduced and oxidized glutathione levels (as a general index of the
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redox state15. At least one oxidant risk was present in 152 subjects (64%) including advanced
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age (≥70 yrs) (n=61); hypertension (n=58); cigarette abuse (n=35); alcohol abuse (n=25);
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diabetes mellitus (n=24); malignancy (n=20); mild-moderate renal insufficiency (creatinine=1.4-
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2.4 mg/dl)(n=10); chronic infections (n=9); medication-dependent asthma (n=8); rheumatologic
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disorders (n=8); pregnancy (n=7); iron deficiency (n=7); hepatitis (n=4); neurodegenerative
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disorders (n=2); sickle cell disease (n=2); chronic pancreatitis (n=2); congestive heart failure
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(n=1); inflammatory bowel disease (n=1); recent myocardial infarction (n=1); hyperthyroidism
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(n=1); and an unexplained high sedimentation rate of 115 mm/hr (n=1). Subjects were then
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divided into 4 groups: “No” Oxidant Risks (n=84); “One” Oxidant Risk (n=70); “Two” Oxidant
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Risks (n=37) and “Three or More” (“Three+”) Oxidant Risks (n=45 - includes 35 subjects with
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three risks and 10 subjects with four risks).
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Cobalamin Treatment
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Patients were offered treatment if they had clinical findings known to be associated with Cbl
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deficiency or if MMA values were elevated. Since abnormal HCys values are less specific for
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Cbl deficiency, treatment was usually not offered to subjects with isolated elevated HCys
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values4. Patients were treated with cyanocobalamin 2 mg per day orally or 1 mg intramuscularly
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3 times a week for 2 weeks, weekly for 8 weeks and monthly thereafter. Metabolites were
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remeasured 1-3 months after beginning Cbl therapy. A response to Cbl therapy was defined as
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either a fall in the metabolite level to a value within the normal reference range (i.e. ≤250 nmol/l
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for MMA and ≤12.1 µmol/l for HCys) or a decrease in the metabolite by more than 1 standard
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deviation greater than its mean intra-individual variability as previously determined for this
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population (i.e >116 nmol/l for MMA and >3.6 µmol/l for HCys)8.
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Data Analysis
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Population studies suggest skewed distributions for Cbl, MMA, and HCys16. Thus, geometric means, 2-tailed Student t tests and paired t tests using log-transformed data, linear regression
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analyses and χ2 analyses were determined using StatPlus:mac (release 5.7, 2009, AnalystSoft,
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Vancouver, BC, Canada). A p value <0.05 was considered significant.
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RESULTS Patient Characteristics
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Age, gender, race and the distribution of oxidant risks were similar in the two Cbl groups
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(Table 1). Subjects with “low” Cbl values had higher mean MMA values than subjects in the
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“low-normal” group but the incidences of elevated MMA values were not significantly different
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in the two Cbl populations. Both mean HCys values and the incidences of elevated HCys values
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were the similar in subjects with “low” and “low-normal” Cbl levels..
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Relationship Between Oxidant Risks and MMA
When “No” oxidant risks were present, mean MMA values were not significantly different in
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subjects with “low” or “low-normal” Cbl levels (Figs. 1A). The presence of “One” or “Two”
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oxidant risks did not affect mean MMA values in either Cbl group. However, when “Three+”
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oxidant risks were present, mean MMA levels were significantly higher than when “No” or
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“One” oxidant risk was present within both Cbl populations. Mean MMA levels were also
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higher in subjects with “low” Cbl values than in subjects in the “low-normal” Cbl group when
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either “Two” or “Three+” oxidant risks were present. Overall, there was a significant inverse
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linear relationship between Cbl and MMA in each oxidant risk group but this relationship was
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more than 4 fold greater in subjects with “Three+” oxidant risks than in those with “No” oxidant
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risks (Fig. 2A).
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The pattern was the same when the incidences of high MMA values were considered except
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that values were similar in the “low” and “low-normal” Cbl populations even when “Two” and
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“Three+” oxidant risks were present (Table 2).
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Relationship Between Oxidant Risks and HCys
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Mean HCys values were not statistically different when subjects in the two Cbl populations
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with the same number of oxidant risks were compared (Fig. 1B). Mean HCys levels were also
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similar in subjects with “No” or “One” oxidant risk within both Cbl populations but were
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significantly higher when “Three+” oxidant risks were present. Overall, there was an inverse
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relationship between Cbl and HCys when “Three+” oxidant risks were present, but not when
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“No” oxidant risks were present (Fig. 2B).
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Similarly, the incidences of high HCys values were significantly greater in subjects with
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“Two” or “Three+” oxidant risks than in those with either “No’’ or “One” oxidant risk in both
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Cbl populations (Table 2). Moreover, when “No” oxidant risk was present in subjects “low” or
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“low-normal” Cbl values, the incidence of high HCys values (30%) was significantly lower than
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the incidence of high MMA values (50%)(p=0.0093)(Table 2). The incidence of high HCys
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values (22%) was also significantly lower than the incidence of high MMA values (43%) when
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only one oxidant risk was present (p=0.013). However, when “Two” or “Three+” oxidant risks
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were present, the incidences of high HCys (57% and 78%) and high MMA values (59% and
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84%) were similar (p≥0.48).
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Pattern of Increased Metabolite Values
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Both MMA and HCys were measured in 204 subjects in the two Cbl populations and at least
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one metabolite was elevated in 137 of them (67%). Isolated elevations in MMA values were
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significantly more frequent in subjects with “No” or “One” oxidant risk than in subjects with
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“Two” or “Three+” oxidant risks while combined elevations in both metabolites occurred
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significantly more frequently in subjects with “Three+” oxidant risks (Fig. 3). Thus, MMA
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values were elevated more frequently than HCys values when “No” or “One oxidant risk was
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present but the incidences of elevated values of both metabolites were similar when “Two” or
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“Three+” oxidant risks were present.
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Effect of Cbl Therapy on MMA and HCys Values
Overall, 81 of the 133 subjects with high MMA values were both treated with Cbl and had
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post-treatment values obtained (61%). Significant responses were noted in 78 of these 81
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subjects (96%). Similarly, 51 of the 84 subjects with high HCys values were evaluable for the
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effects of Cbl therapy (61%) and significant responses were noted in 48 of them (94%).
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Response rates and post-treatment values for both metabolites were not significantly different
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regardless of the number of oxidant risks present (Table 2).
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DISCUSSION
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Minimum optimum values suggested for serum Cbl have ranged widely from 200 pg/ml to 500
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pg/ml (150 to 360 pmol/l)2,3. However, even at the lowest cut-off value, most subjects have
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normal MMA and HCys values4-6. While genetic variants may alter the metabolic effectiveness
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of circulating Cbl by either decreasing the level of haptocorrin, the inactive binder of serum Cbl
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(i.e. “false” low Cbl levels) or decreasing the effective level of holotranscobalamin, the active
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transport form of Cbl (i.e. “false” normal Cbl levels), they are uncommon4. Moreover,
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metabolite values are often normal even when holotranscobalamin levels are low17,18.
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In the present study, it was of note that neither mean metabolite values nor the incidences of
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high metabolite values were significantly greater in subjects with “low” Cbl values than in those
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with “low-normal” Cbl values when “No” oxidant risks were present (Fig. 1; Table 2).
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However, MMA and HCys values were significantly higher when three or more oxidant risks
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were present in both Cbl populations (Table 2; Fig. 1). These findings are similar to those
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previously described in subjects with Cbl values well within the normal reference range7.
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Significantly, the incidences of elevated values for both metabolites were the same when Cbl
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values were in the “low-normal” range as when they were frankly “low”, even when adjusted for
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the number of oxidant risks present (Table 1, 2). Thus, Cbl deficiency should not be excluded as
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a cause for hematologic and/or neurocognitive abnormalities in subjects with Cbl values greater
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than the usual cut-off value of 200 pg/ml.
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MMA values were also more frequently increased than HCys values in subjects with “No” or
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“One” oxidant risk but not in those with “Three+” oxidant risks (Table 2, Fig. 3). Since MMA
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accumulation reflects decreased activity of a mitochondrial Cbl-dependent enzyme while HCys
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accumulation reflects decreased activity of a cytoplasmic Cbl-dependent enzyme, and since
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mitochondrial processes are more sensitive to oxidative damage, this observation is also
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consistent with a role for oxidative stress as a determinant of metabolic Cbl deficiency4,19.
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Many clinical disorders associated with increased oxidative stress are stable over time while others vary in severity depending on their natural history and the effectiveness of treatment.
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Thus, metabolic evidence of Cbl deficiency associated with oxidative inactivation of Cbl would
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also be expected to vary with the clinical status of the individual. Interestingly then, longitudinal
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follow-up of elevated MMA values in apparently healthy adults showed values to be stable in
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39%, decreased in 42% and increased in 16%20. Although high MMA values in this study did
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not predict for the development of clinical abnormalities, exposure of 7 subjects with low Cbl
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values (≤200 pg/ml) and 2 subjects with low-normal Cbl values (227 and 312 pg/ml) to nitrous
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oxide, a drug known to oxidize Cbl, resulted in acute precipitation of neurologic dysfunction21-28.
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Similarly, Cbl-responsive neurologic disorders have also been shown to be associated with the
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presence of prooxidant disorders and the increased oxidative stress associated with acute
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myocardial infarction has been linked to acute increases in urinary MMA excretion14,29,30. Thus,
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clinically overt Cbl deficiency may develop when susceptible individuals are exposed to
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additional oxidant stimuli. The findings in the current study also raise the possibility that, higher
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and more frequent doses of Cbl may be needed to correct Cbl deficiency due to increased
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oxidative stress than usually recommended and that fully reduced forms of Cbl (e.g.
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methylcobalamin) may be more effective than the partially reduced forms of Cbl currently
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prescribed (i.e. cyanocobalamin and hydroxocobalamin)4,31-33.
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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,
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since the severity of oxidative stress varies between and within the disorders identified as oxidant
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risks and since the presence of other oxidant risks may not have been recognized, this study was
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also limited by the absence of measurement of biomarkers of oxidative stress. Therefore,
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prospective studies with direct measures of oxidative stress are needed to confirm these
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observations. However, it is of note in this regard that a prospective study of 18 subjects with
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schizophrenia (a “prooxidant” disorder) and low-normal Cbl levels, found elevated levels of
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urinary MMA to be directly correlated with increased values of the oxidant marker,
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malondialdehyde in erythrocyte membranes34.
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It is concluded that, in subjects with low or low-normal Cbl values, metabolic evidence of Cbl
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deficiency is more frequent when disorders marked by increased oxidative stress are present and
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that HCys is a less reliable indicator of metabolic Cbl deficiency than MMA when the number of
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oxidant risks are limited. Thus, defining a low serum Cbl level to use as a cut-off value in
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screening for Cbl deficiency may be a “moving target” due to the variable presence and severity
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of often subtle, confounding clinical conditions both across different populations as well as
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within individual subjects. Since clinical manifestations of Cbl deficiency may develop or
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progress when subjects with metabolic Cbl deficiency are exposed to additional oxidant risks,
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measurement of MMA and HCys may be of value even when screening Cbl values are in the
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“normal” or “low-normal” range and correction of elevated metabolite values with Cbl therapy
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should be considered pending further studies.
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32. Xu G, Lv ZW, Feng Y, Tang WZ, Xu GX. A single-center randomized controlled trial of
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local methylcobalamin injection for subacute herpetic neuralgia. Pain Med 2013; 14:884-
306
894.
308 309
33. Kuwabara S, Nakazawa R, Azuma N et al. Intravenous methylcobalamin treatment of
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uremic and diabetic neuropathy in chronic hemodialysis patients. Int Med 1999; 38:472-5. 34. Ozcan O, Ipcioglu M, Gultepe M, Basoglu C. Altered red cell membrane compositions related to functional vitamin B12 deficiency manifested by elevated urine methylmalonic
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acid concentrations in patients with schizophrenia. Ann Clin Biochem 2008; 46:44-49.
313 314 315
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FIGURE LEGENDS Figure 1: Mean MMA and HCys Values Relative to Cbl Levels and Presence of Oxidant
320
Risk Factors
321
Values are geometric means for MMA (A) and HCys (B) for the number of subjects shown in
322
parentheses.
323
Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine
324
*p=0.012 and p=0.0054 vs MMA in subjects with Cbl≤200 pg/ml and “No” and “One” oxidant
325
risk respectively
326
†p=0.0010 and p<0.001 vs MMA in subjects with Cbl=201-300 pg/ml and “No” and “One”
327
oxidant risk respectively
328
§p=0.013 and 0.055 vs HCys in subjects with Cbl≤200 pg/ml and “No” and “One” oxidant risk
329
respectively
330
¶p<0.001 vs HCys in subjects with Cbl=201-300 pg/ml and either “No” or “One” oxidant
331
risk.
332
**p=0.0072 and p<0.001 vs HCys in subjects with Cbl=201-300 pg/ml and “No” and “One”
333
oxidant risk respectively..
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Figure 2: Linear Regression Analyses of the Relationships Between Cbl and MMA or
336
HCys Relative to the Number of Oxidant Risks Present
337
Linear regression analyses between Cbl and MMA (A) and HCys (B) were performed in subjects
338
with “low” and “low-normal” Cbl values and either “No” or “Three+” Oxidant Risks. Since only
339
2 subjects in the entire study group had Cbl values <100 pg/ml (both with “One” oxidant risk and
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340
Cbl values of 58 and 86 pg/ml respectively) regression lines were not extended below a Cbl
341
value of 100 pg/ml.
342
Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine
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Figure 3: Relationship of the Pattern of Increased MMA and HCys Values to the Presence
345
of Oxidant Risk Factors
346
Values plotted are the percent of those subjects in whom both metabolites were measured and in
347
whom an increased value of MMA and/or HCys was noted.
348
Abbreviations: MMA, methylmalonic acid; HCys, homocysteine
349
*p=0.025 and p<0.001 vs the incidences of isolated high MMA values in subjects with “Two” or
350
“Three+” oxidant risks respectively by χ2 analyses.
351
†p<0.001 vs the incidence of high MMA values in subjects with “No or One” oxidant risks;
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and p=0.025 and p<0.001 vs the incidences of high HCys values in subjects with “Two” and
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“Three+” oxidant risks respectively.
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Table 1: Patient Characteristics Cbl =201-300
(N=49)
(N=187)
57±19 (49)
56±17 (187)
[24-94]
[19-93]
14 (29%)
73 (39%)
Caucasian
37 (76%)
153 (82%)
Black
7 (19%)
Othera
5 (!0%)
Race (%):
Oxidant Risks (%): None
23 (12%) 11 (6%)
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Male (%)
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Age (yrs)
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Cbl ≤200
Characteristic
15 (31%)
69 (37%)
17 (35%)
53 (28%)
6 (12%)
31 (17%)
11 (22%)
34 (18%)
168 (49)
257 (187)
[58-199]
[201-300]
425 (49)b
278 (187)
[105-4911]
[101-1384]
MMA>250 nmol/l (%)
32 (65%)
101 (54%)
HCys (µmol/l)
12.3 (41)
11.2 (163)
[4.4-61.4]
[3.0-60.7]
18 (44%)
67 (41%)
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Cbl (pg/ml)
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MMA (nmol/l)
HCys >12.1 µmol/l (%)
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Values for Age are arithmetic means ± 1 SD while values for Cbl, MMA and HCys are geometric means for the number of subjects shown in parentheses with the range of values for each parameter shown in brackets.
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Abbreviations: Cbl; cobalamin; MMA, methylmalonic acid; HCys, homocysteine; yrs, years; SD, standard deviation a
Other racial groups include Indian (n=8); Phillipino (n=3); Arabic (n=3}; Asian (n=1); and
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Hispanic (n=1). b
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p<0.001 vs the mean MMA value in subjects with Cbl =201-300 pg/ml
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Table 2: Relationship of the Incidence of High Metabolite Values to the Number of Oxidant Risks Metabolite
Number of Oxidant Risks None
One
Two
Three+
MMA >250
≤200a
8/15 (55%)
8/17 (47%)
5/6 (63%)
11/11 (100%)b
nmol/l
201-300
34/69 (49%)
22/53 (42%)
18/31 (58%)
27/34 (79%)c
Hcys >12.1
≤200a
4/14 (29%)
3/13 (23%)
4/6 (67%)
7/8 (88%)d
µmol/l
201-300
19/63 (30%)
9/42 (21%)
16/29 (55%)e
22/29 (76%)f
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(pg/ml)
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a
Cbl
Incidences of high MMA and HCys values in subjects with Cbl levels ≤200 pg/ml were not
significantly greater than those in subjects with Cbl levels of 201-300 pg/ml and the same number of oxidant risks (p≥0.24). b
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p=0.0080 vs the incidence of high MMA values in subjects with Cbl levels ≤200 pg/ml and
“No” Oxidant Risks; and p=0.0034 vs the incidence of high MMA values in subjects with Cbl levels ≤200 pg/ml and “One” Oxidant Risk.
p=0.0034 vs the incidence of high MMA values in subjects with Cbl levels of 201-300 pg/ml
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c
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and “No” Oxidant Risks; and p<0.001 vs the incidence of high MMA values in subjects with Cbl levels of 201-300 pg/ml and “One” Oxidant Risk. d
p=0.0078 vs the incidence of high HCys values in subjects with Cbl levels ≤200 pg/ml and
“No” Oxidant Risks; and p=0.0041 vs the incidence of high HCys values in subjects with Cbl levels ≤200 pg/ml and “One” Oxidant Risk.
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e
p=0.022 vs the incidence of high HCys values in subjects with Cbl levels of 201-300 pg/ml and
“No” Oxidant Risks and p=0.0034 vs the incidence of high HCys values in subjects with Cbl levels of 201-300 pg/ml and “One” Oxidant Risk. f
either “No” Oxidant Risks or “One” Oxidant Risk. p values were obtained by χ2 analyses.
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p<0.001 vs the incidence of high HCys values in subjects with Cbl levels of 201-300 pg/ml and
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Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine
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Table 3: Effect of Cbl Therapy of Subjects With Elevated MMA and HCys Values Number
Treated
Risks
Elevated
(%)
MMA
None
42
30 (71%)
30 (100%)
484
173
<0.001
(nmol/l)
One
30
16 (53%)
14 (88%)
445
199
<0.001
Two
23
15 (65%)
14 (93%)
433
185
<0.001
Three+
38
20 (53%)
20 (100%)
516
174
<0.001
HCys
None
23
16 (70%)
16 (100%)
15.1
9.1
<0.001
(µmol/l)
One
12
6 (50%)
6 (100%)
16.1
10.1
0.0013
Two
20
11 (55%)
9 (82%)
15.6
10.6
<0.001
Three+
28
18 (64%)
17 (94%)
19.4
11.9
0.0037
Responders
Pre-
Post-
p
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Therapy Therapy
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Response criteria are defined in the Methods. Pre-therapy and Post-therapy metabolite values are geometric means for the number of treated subjects. “p” values were derived from paired ttests.
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Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine
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Figure 1: A) 900
(11)
800 700
MMA (nmol/l)
*
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Cbl ≤200 Cbl=201-300
p<0.001
600 500
(17)
300 (69)
None
(34)
(31)
One Two Number of Oxidant Risks
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p=0.021
(53)
200
B)
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(15)
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400
(6)
Three+
(8)
§
Cbl ≤200
19
Cbl=201-300
¶ (29)
13 11
p=0.17
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HCys (µ µ mol/l)
17
(14)
(63)
9 None
(29) (13)
**
(6)
(42)
One
Two
Number of Oxidant Risks
Three+
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Figure 2: A. 1500
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(r=-0.36; p=0.015)
1100 900
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MMA (nmol/l)
1300
Oxidant Risks: None Three+
700
(r=-0.22; p=0.049)
300 100 0
100
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500
200
300
400
B.
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Cbl (pg/ml)
35
Oxidant Risks: None Three+
(r=-0.32; p=0.050)
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HCys (µ µ mol/l)
30
20
15
(r=-0.012; p=0.92)
10 0
100
200 Cbl (pg/ml)
300
400
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100 p<0.001
None or One (N=75)
90 p<0.001
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Two (N=28)
80
Three+ (N=34)
70 p<0.001
60 50
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*
40
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30 20 10 0
Only MMA Increased
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Both MMA and HCys Increased
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% of Subjects With At Least One Metabolite Increased
Figure 3:
Only HCys Increased
All With MMA Increased
†
All With HCys Increased
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Highlights •
The incidences of elevated methylmalonic acid and homocysteine values, metabolic
and low-normal (201-300 pg/ml) cobalamin levels. •
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markers of cobalamin deficiency, were similar in subjects with low (≤200 pg/ml)
The incidences of high metabolite values were 1.5 to 3 fold higher in subjects with three or more comorbidities associated with increased oxidative stress than in
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Methylmalonic acid values were more frequently increased than homocysteine
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values.
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subjects with no oxidant risks.
S0002-9343(15)00694-4
DOI:
10.1016/j.amjmed.2015.07.017
Reference:
AJM 13121
To appear in:
The American Journal of Medicine
Received Date: 12 May 2015 Revised Date:
30 June 2015
Accepted Date: 1 July 2015
Please cite this article as: Solomon LR, Low Cobalamin (Vitamin B12) Levels as Predictors of Cobalamin Deficiency: Importance of Comorbidities Associated With Increased Oxidative Stress, The American Journal of Medicine (2015), doi: 10.1016/j.amjmed.2015.07.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT L SOLOMON
B12 AND OXIDATIVE STRESS
Title: Low Cobalamin (Vitamin B12) Levels as Predictors of Cobalamin Deficiency: Importance of Comorbidities Associated With Increased Oxidative Stress
Author: Lawrence R. Solomon, M.D.
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Running Title: B12 and Oxidative Stress
Institutional Affiliation: Section of Palliative Care; Department of Medicine
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Yale University School of Medicine Corresponding Author:
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Lawrence R. Solomon, M.D.
Section of Palliative Care, Department of Medicine,
Yale University School of Medicine and Smilow Cancer Hospital 403 www; 333 Cedar Street
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P.O. Box 208021
New Haven, CT. 06520-8021
e-mail: [email protected]
Funding: None
Fax: 203-785-3712
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Telephone: 203-737-4353
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Conflicts of Interest: None
Role of Author: Lawrence R. Solomon was solely responsible for the design and performance of this study, analysis of the data and preparation of the manuscript.
Word Counts: Manuscript: 2,379 Abstract: 248 Figures: 3 Tables: 3 References: 34 Key Words: Oxidative stress; cobalamin; vitamin B12; methylmalonic acid; homocysteine
1
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1
2
ABSTRACT Background: Cobalamin (B12) deficiency can lead to irreversible neurocognitive changes if
3
unrecognized. Screening involves measurement of serum cobalamin levels but the sensitive
4
metabolic indicators of cobalamin deficiency, methylmalonic acid (MMA) and homocysteine
5
(HCys), may be normal when cobalamin values are low and elevated when cobalamin values are
6
normal. Since cobalamin is inactivated by oxidation, the relationship between these metabolites
7
and comorbidities associated with increased oxidative stress (oxidant risks) in subjects with low
8
and low-normal cobalamin levels was studied.
9
Methods: A retrospective record-review was conducted of community-dwelling adults
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evaluated for cobalamin deficiency during a 12 year period with serum cobalamin values in the
11
low (≤200 pg/ml; n=49) or low-normal (201-300 pg/ml; n=187) range and concurrent
12
measurement of MMA.
13
Results: When “No” oxidant risk was present, elevated MMA (>250 nmol/l) and HCys (>12.1
14
µmol/l) values occurred in 50% and 30% of subjects respectively (p<0.01). In contrast, when
15
“Three or More” oxidant risks were present, mean MMA and HCys values were significantly
16
higher and elevated MMA and HCys values occurred in 84% and 78% of these subjects
17
(p≤0.012). Pharmacologic doses of cyanocobalamin significantly decreased metabolite values in
18
≥94% of treated subjects.
19
Conclusion and Relevance: In subjects with low or low-normal cobalamin values, metabolic
20
evidence of cobalamin deficiency is more frequent when three or more oxidant risks are present.
21
Thus, defining a low serum cobalamin level to screen for cobalamin deficiency may be a
22
“moving target” due to the variable presence and severity of often subtle, confounding clinical
23
conditions in individual subjects.
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3
INTRODUCTION Cobalamin (i.e. vitamin B12) deficiency is common in vegetarians, the elderly and subjects using such frequently prescribed medications as proton pump inhibitors, H2-blockers and
27
metformin1. If unrecognized, this disorder can lead to irreversible neurocognitive dysfunction.
28
Measurement of serum cobalamin (Cbl) is initially performed to screen for Cbl deficiency.
29
However, cut-off points suggested vary widely between 200 pg/ml and 500 pg/ml2,3. Moreover,
30
even at Cbl levels ≤200 pg/ml, the lowest cut-off point commonly used to define Cbl deficiency,
31
many subjects have neither clinical evidence of Cbl deficiency (i.e. megaloblastic anemia and/or
32
neurocognitive disorders) nor elevated levels of the Cbl-dependent metabolites, methylmalonic
33
acid (MMA) and homocysteine (HCys)(biochemical indicators of Cbl depletion)4-6. It is of note
34
then that Cbl is readily inactivated by oxidation and that elevated MMA and HCys levels in
35
subjects with normal Cbl values have been related to the presence of comorbidities associated
36
with increased oxidative stress (oxidant risks)7. Since Cbl values between 201 and 300 pg/ml are
37
often present in subjects with both metabolically and clinically significant Cbl deficiency, a
38
retrospective study was performed to determine if metabolic changes consistent with Cbl
39
deficiency were also related to the presence of oxidant risks in subjects with low (≤200 pg/ml)
40
and low-normal (201-300 pg/ml) Cbl values9-13.
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METHODS
Laboratory Methods
Cbl, MMA and HCys were measured as previously described7. The laboratory reference range
44
for Cbl was 201-1100 pg/ml (Quest Laboratory). Since Cbl values between 201 and 300 pg/ml
45
are often present in subjects with both metabolically and clinically significant Cbl deficiency due
46
to Cbl malabsorption, Cbl values ≤200 pg/ml were defined as “low” while Cbl values of 201-300
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pg/ml were defined as “low-normal” 9-13. The reference ranges for MMA and HCys were taken
48
as 90-250 nmol/l and 5.4-12.1 µmol/l respectively8. When values were obtained on more than
49
one occasion within a 6-week period, the lowest Cbl value and the highest metabolite values
50
were used for analysis.
51
Subjects
52
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4
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 August 1, 1993 and June 30,
54
2005 with the measurement of both serum Cbl and MMA levels, was conducted as previously
55
described8,14. Subjects were screened because of the presence of clinical findings consistent with
56
Cbl deficiency or because of the presence of disorders known to lead to Cbl depletion1. Cbl
57
values were “low” in 49 subjects and “low-normal” in 187 subjects. HCys values were obtained
58
in 204 of these 236 individuals (86%).
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This study conforms to the principles of the Declaration of Helsinki of 1975 as revised in 2008
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and the institutional human investigation committee determined that further review was not
61
required.
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Identification of Oxidant Risks
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All active medical conditions present in each subject were identified and a Medline search was performed to determine which disorders were associated with increased oxidative stress as
65
defined by the presence of increased oxidative byproducts systemically including
66
malondialdehyde and F2-isoprostanes (as indices of lipid oxidative damage); carbonylated
67
proteins (as an index of protein oxidative damage); 8-hydroxy-2’-deoxyguanosine (as an index of
68
DNA oxidative damage); and reduced and oxidized glutathione levels (as a general index of the
69
redox state15. At least one oxidant risk was present in 152 subjects (64%) including advanced
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age (≥70 yrs) (n=61); hypertension (n=58); cigarette abuse (n=35); alcohol abuse (n=25);
71
diabetes mellitus (n=24); malignancy (n=20); mild-moderate renal insufficiency (creatinine=1.4-
72
2.4 mg/dl)(n=10); chronic infections (n=9); medication-dependent asthma (n=8); rheumatologic
73
disorders (n=8); pregnancy (n=7); iron deficiency (n=7); hepatitis (n=4); neurodegenerative
74
disorders (n=2); sickle cell disease (n=2); chronic pancreatitis (n=2); congestive heart failure
75
(n=1); inflammatory bowel disease (n=1); recent myocardial infarction (n=1); hyperthyroidism
76
(n=1); and an unexplained high sedimentation rate of 115 mm/hr (n=1). Subjects were then
77
divided into 4 groups: “No” Oxidant Risks (n=84); “One” Oxidant Risk (n=70); “Two” Oxidant
78
Risks (n=37) and “Three or More” (“Three+”) Oxidant Risks (n=45 - includes 35 subjects with
79
three risks and 10 subjects with four risks).
80
Cobalamin Treatment
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Patients were offered treatment if they had clinical findings known to be associated with Cbl
82
deficiency or if MMA values were elevated. Since abnormal HCys values are less specific for
83
Cbl deficiency, treatment was usually not offered to subjects with isolated elevated HCys
84
values4. Patients were treated with cyanocobalamin 2 mg per day orally or 1 mg intramuscularly
85
3 times a week for 2 weeks, weekly for 8 weeks and monthly thereafter. Metabolites were
86
remeasured 1-3 months after beginning Cbl therapy. A response to Cbl therapy was defined as
87
either a fall in the metabolite level to a value within the normal reference range (i.e. ≤250 nmol/l
88
for MMA and ≤12.1 µmol/l for HCys) or a decrease in the metabolite by more than 1 standard
89
deviation greater than its mean intra-individual variability as previously determined for this
90
population (i.e >116 nmol/l for MMA and >3.6 µmol/l for HCys)8.
91
Data Analysis
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Population studies suggest skewed distributions for Cbl, MMA, and HCys16. Thus, geometric means, 2-tailed Student t tests and paired t tests using log-transformed data, linear regression
94
analyses and χ2 analyses were determined using StatPlus:mac (release 5.7, 2009, AnalystSoft,
95
Vancouver, BC, Canada). A p value <0.05 was considered significant.
96
RESULTS Patient Characteristics
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97
6
Age, gender, race and the distribution of oxidant risks were similar in the two Cbl groups
99
(Table 1). Subjects with “low” Cbl values had higher mean MMA values than subjects in the
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98
“low-normal” group but the incidences of elevated MMA values were not significantly different
101
in the two Cbl populations. Both mean HCys values and the incidences of elevated HCys values
102
were the similar in subjects with “low” and “low-normal” Cbl levels..
103
Relationship Between Oxidant Risks and MMA
When “No” oxidant risks were present, mean MMA values were not significantly different in
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subjects with “low” or “low-normal” Cbl levels (Figs. 1A). The presence of “One” or “Two”
106
oxidant risks did not affect mean MMA values in either Cbl group. However, when “Three+”
107
oxidant risks were present, mean MMA levels were significantly higher than when “No” or
108
“One” oxidant risk was present within both Cbl populations. Mean MMA levels were also
109
higher in subjects with “low” Cbl values than in subjects in the “low-normal” Cbl group when
110
either “Two” or “Three+” oxidant risks were present. Overall, there was a significant inverse
111
linear relationship between Cbl and MMA in each oxidant risk group but this relationship was
112
more than 4 fold greater in subjects with “Three+” oxidant risks than in those with “No” oxidant
113
risks (Fig. 2A).
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The pattern was the same when the incidences of high MMA values were considered except
115
that values were similar in the “low” and “low-normal” Cbl populations even when “Two” and
116
“Three+” oxidant risks were present (Table 2).
117
Relationship Between Oxidant Risks and HCys
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114
7
Mean HCys values were not statistically different when subjects in the two Cbl populations
119
with the same number of oxidant risks were compared (Fig. 1B). Mean HCys levels were also
120
similar in subjects with “No” or “One” oxidant risk within both Cbl populations but were
121
significantly higher when “Three+” oxidant risks were present. Overall, there was an inverse
122
relationship between Cbl and HCys when “Three+” oxidant risks were present, but not when
123
“No” oxidant risks were present (Fig. 2B).
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Similarly, the incidences of high HCys values were significantly greater in subjects with
125
“Two” or “Three+” oxidant risks than in those with either “No’’ or “One” oxidant risk in both
126
Cbl populations (Table 2). Moreover, when “No” oxidant risk was present in subjects “low” or
127
“low-normal” Cbl values, the incidence of high HCys values (30%) was significantly lower than
128
the incidence of high MMA values (50%)(p=0.0093)(Table 2). The incidence of high HCys
129
values (22%) was also significantly lower than the incidence of high MMA values (43%) when
130
only one oxidant risk was present (p=0.013). However, when “Two” or “Three+” oxidant risks
131
were present, the incidences of high HCys (57% and 78%) and high MMA values (59% and
132
84%) were similar (p≥0.48).
133
Pattern of Increased Metabolite Values
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Both MMA and HCys were measured in 204 subjects in the two Cbl populations and at least
135
one metabolite was elevated in 137 of them (67%). Isolated elevations in MMA values were
136
significantly more frequent in subjects with “No” or “One” oxidant risk than in subjects with
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“Two” or “Three+” oxidant risks while combined elevations in both metabolites occurred
138
significantly more frequently in subjects with “Three+” oxidant risks (Fig. 3). Thus, MMA
139
values were elevated more frequently than HCys values when “No” or “One oxidant risk was
140
present but the incidences of elevated values of both metabolites were similar when “Two” or
141
“Three+” oxidant risks were present.
142
Effect of Cbl Therapy on MMA and HCys Values
Overall, 81 of the 133 subjects with high MMA values were both treated with Cbl and had
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137
8
post-treatment values obtained (61%). Significant responses were noted in 78 of these 81
145
subjects (96%). Similarly, 51 of the 84 subjects with high HCys values were evaluable for the
146
effects of Cbl therapy (61%) and significant responses were noted in 48 of them (94%).
147
Response rates and post-treatment values for both metabolites were not significantly different
148
regardless of the number of oxidant risks present (Table 2).
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DISCUSSION
150
Minimum optimum values suggested for serum Cbl have ranged widely from 200 pg/ml to 500
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pg/ml (150 to 360 pmol/l)2,3. However, even at the lowest cut-off value, most subjects have
152
normal MMA and HCys values4-6. While genetic variants may alter the metabolic effectiveness
153
of circulating Cbl by either decreasing the level of haptocorrin, the inactive binder of serum Cbl
154
(i.e. “false” low Cbl levels) or decreasing the effective level of holotranscobalamin, the active
155
transport form of Cbl (i.e. “false” normal Cbl levels), they are uncommon4. Moreover,
156
metabolite values are often normal even when holotranscobalamin levels are low17,18.
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In the present study, it was of note that neither mean metabolite values nor the incidences of
158
high metabolite values were significantly greater in subjects with “low” Cbl values than in those
159
with “low-normal” Cbl values when “No” oxidant risks were present (Fig. 1; Table 2).
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However, MMA and HCys values were significantly higher when three or more oxidant risks
161
were present in both Cbl populations (Table 2; Fig. 1). These findings are similar to those
162
previously described in subjects with Cbl values well within the normal reference range7.
163
Significantly, the incidences of elevated values for both metabolites were the same when Cbl
164
values were in the “low-normal” range as when they were frankly “low”, even when adjusted for
165
the number of oxidant risks present (Table 1, 2). Thus, Cbl deficiency should not be excluded as
166
a cause for hematologic and/or neurocognitive abnormalities in subjects with Cbl values greater
167
than the usual cut-off value of 200 pg/ml.
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MMA values were also more frequently increased than HCys values in subjects with “No” or
169
“One” oxidant risk but not in those with “Three+” oxidant risks (Table 2, Fig. 3). Since MMA
170
accumulation reflects decreased activity of a mitochondrial Cbl-dependent enzyme while HCys
171
accumulation reflects decreased activity of a cytoplasmic Cbl-dependent enzyme, and since
172
mitochondrial processes are more sensitive to oxidative damage, this observation is also
173
consistent with a role for oxidative stress as a determinant of metabolic Cbl deficiency4,19.
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Many clinical disorders associated with increased oxidative stress are stable over time while others vary in severity depending on their natural history and the effectiveness of treatment.
176
Thus, metabolic evidence of Cbl deficiency associated with oxidative inactivation of Cbl would
177
also be expected to vary with the clinical status of the individual. Interestingly then, longitudinal
178
follow-up of elevated MMA values in apparently healthy adults showed values to be stable in
179
39%, decreased in 42% and increased in 16%20. Although high MMA values in this study did
180
not predict for the development of clinical abnormalities, exposure of 7 subjects with low Cbl
181
values (≤200 pg/ml) and 2 subjects with low-normal Cbl values (227 and 312 pg/ml) to nitrous
182
oxide, a drug known to oxidize Cbl, resulted in acute precipitation of neurologic dysfunction21-28.
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Similarly, Cbl-responsive neurologic disorders have also been shown to be associated with the
184
presence of prooxidant disorders and the increased oxidative stress associated with acute
185
myocardial infarction has been linked to acute increases in urinary MMA excretion14,29,30. Thus,
186
clinically overt Cbl deficiency may develop when susceptible individuals are exposed to
187
additional oxidant stimuli. The findings in the current study also raise the possibility that, higher
188
and more frequent doses of Cbl may be needed to correct Cbl deficiency due to increased
189
oxidative stress than usually recommended and that fully reduced forms of Cbl (e.g.
190
methylcobalamin) may be more effective than the partially reduced forms of Cbl currently
191
prescribed (i.e. cyanocobalamin and hydroxocobalamin)4,31-33.
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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,
194
since the severity of oxidative stress varies between and within the disorders identified as oxidant
195
risks and since the presence of other oxidant risks may not have been recognized, this study was
196
also limited by the absence of measurement of biomarkers of oxidative stress. Therefore,
197
prospective studies with direct measures of oxidative stress are needed to confirm these
198
observations. However, it is of note in this regard that a prospective study of 18 subjects with
199
schizophrenia (a “prooxidant” disorder) and low-normal Cbl levels, found elevated levels of
200
urinary MMA to be directly correlated with increased values of the oxidant marker,
201
malondialdehyde in erythrocyte membranes34.
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It is concluded that, in subjects with low or low-normal Cbl values, metabolic evidence of Cbl
203
deficiency is more frequent when disorders marked by increased oxidative stress are present and
204
that HCys is a less reliable indicator of metabolic Cbl deficiency than MMA when the number of
205
oxidant risks are limited. Thus, defining a low serum Cbl level to use as a cut-off value in
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screening for Cbl deficiency may be a “moving target” due to the variable presence and severity
207
of often subtle, confounding clinical conditions both across different populations as well as
208
within individual subjects. Since clinical manifestations of Cbl deficiency may develop or
209
progress when subjects with metabolic Cbl deficiency are exposed to additional oxidant risks,
210
measurement of MMA and HCys may be of value even when screening Cbl values are in the
211
“normal” or “low-normal” range and correction of elevated metabolite values with Cbl therapy
212
should be considered pending further studies.
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1. Stabler SP. Vitamin B12 deficiency. New Engl J Med 2013; 368: 149-160.
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2. Aparicio-Ugarriza R, Palacios G, Gonzalez-Cross M. A review of the cut-off points for the diagnosis of vitamin B12 deficiency in the general population. Clin Chem Lab Med 2015 doi
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3. Bailey RL, Carmel R, Green R et al. Monitoring of vitamin B12 nutritional status in the United States by using plasma MMA and serum vitamin B12. Am J Clin Nutr 2011; 94:552-
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4. Solomon LR. Disorders of cobalamin (vitamin B12) metabolism: emerging concepts in
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pathophysiology, diagnosis and treatment. Blood Reviews 2007; 21:113-130. 5. Holleland G, Schneede J, Ueland PM, Lund PK, Refsum H, Sandberg S. Cobalamin deficiency in general practice. Assessment of the diagnostic utility and cost-benefit analysis
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6. Fayet-Moore F, Petocz P, Samman S. Micronutrient status in female university students: iron, zinc, copper, selenium, vitamin B12 and folate. Nutrients 2014; 6:5103-5116.
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7. Solomon LR. Functional Cobalamin (Vitamin B12) Deficiency: Role of advanced age and disorders associated with increased oxidative stress. Europ J Clin Nutr 2015 69:687-692.
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8. Solomon LR. Cobalamin-responsive disorders in the ambulatory care setting: unreliability of
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cobalamin, methylmalonic acid and homocysteine testing. Blood 2005; 105:978-85. 9. Vogiatzoglu A, Oulhaj A, Smith DA et al. Determinants of plasma methylmalonic acid in a
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10. Smith AD, Refsum H. Do we need to reconsider the desirable blood level of vitamin B12? J Int Med 2011; 271:179-182. 11. Lindenbaum J, Savage DG, Stabler SP, Allen RN. Diagnosis of cobalamin deficiency: II.
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Relative sensitivities of serum cobalamin, methylmalonic acid and total homocysteine
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12. Magnus EM. Cobalamin and unsaturated transcobalamin values in pernicious anaemia: relation to treatment. Scand J Haematol 1986; 36:4576-65.
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13. Selhub J, Jacques PF, Dallal G, Choumenkovitch S, Rogers G. The use of blood concentrations of vitamins and theirs respective functional indicators to define folate and
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vitamin B12 status. Food Nutr Bull 2008; 29(suppl):567-73.
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14. Solomon LR. Diabetes mellitus as a cause of clinically significant functional cobalamin deficiency. Diabetes Care 2011; 34:1077-1080.
15. Dalle-Donne I, Rossi R, Colombo R, Guestarini D, Milzani A. Biomarkers of oxidative
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damage in human disease. Clin Chem 2006; 52:601-23. 16. Pfeiffer CM, Caudill SP, Gunter EW. Osterloh J, Sampson EJ. Biochemical indicators of B vitamin status in the US population after folic acid fortification: results from the National
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17. Sobczynska-Malefora A, Gorska R, Pelisser M, Ruwona P, Witchlow B, Harrington DJ. An
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audit of holotranscobalamin (“Active” B12) and methylmalonic acid assays for the
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18. Remacha AF, Sarda MP, Canals C et al. Role of serum holotranscobalamin (holoTC) in the
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diagnosis of patients with low serum cobalamin. Comparison with methylmalonic acid and
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homocysteine. Ann Hematol 2014; 93:565-569.
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19. Hansen JM, Go Y-M, Jones DP. Nuclear and mitochondrial compartmentation of oxidative
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stress and redox signaling. Ann Rev Pharmacol Toxicol 2006; 46:215-234.
20. Hvas A-M, Ellegard J, Nexo E. Increased plasma methylmalonic acid level does not predict clinical manifestations of vitamin B12 deficiency. Arch Int Med 2001; 161:1534-41.
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21. Schilling RF. Is nitrous oxide a dangerous anesthetic for vitamin B12-deficient subjects?
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patients with vitamin B12 deficiency. Arch Surg 1993; 128:1391-95. 23. Kinsella LJ, Green R. “Anesthesia paresthetica”: nitrous oxide induced cobalamin deficiency. Neurol 1995; 45:1608-10.
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22. Flippo TS, Holder WD. Neurologic degeneration associated with nitrous oxide anesthesia in
24. Rosener M, Dichgans J. Severe combined degeneration of the spinal cord after nitrous oxide anaesthesia in a vegetarian. J Neurol Neurosurg Psych 1996; 60:354. 25. Sesso RMCC, Iunes Y, Melo ACP. Myeloneuropathy following nitrous oxide anaesthesia in
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27. Renard D, Dutray A, Remy A, Castelnovo G, Labauge P. Subacute combined degeneration of the spinal cord caused by nitrous oxide anaesthesia. Neurol Sci 2009; 30:75-76.
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28. Alt RS, Morrissey RP, Gang MA, Hoffman RS, Schaumburg HH. Severe myeloneuropathy from acute high-dose nitrous oxide (N2O) abuse. J Emerg Med 2011; 41:378-80. 29. Solomon LR. Vitamin B12-responsive neuropathies: a case series. Nutr Neurosci 2015 Feb 24. [Epub ahead of print] PMID 25710280.
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30. Celik T, Kardesoglu E, Iyisoy A, Ozcan O, Kilic S, Yaman H. Urinary methylmalonic acid in patients with acute myocardial infarction. Med Princ Pract 2009; 18:217-222.
31. Solomon LR. Oral pharmacologic doses of cobalamin may not be as effective as parenteral
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cobalamin therapy in reversing hyperhomocysteinemia and methylmalonic acidemia in
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apparently normal subjects. Clin Lab Haematol 2006; 28:275-8.
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32. Xu G, Lv ZW, Feng Y, Tang WZ, Xu GX. A single-center randomized controlled trial of
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local methylcobalamin injection for subacute herpetic neuralgia. Pain Med 2013; 14:884-
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33. Kuwabara S, Nakazawa R, Azuma N et al. Intravenous methylcobalamin treatment of
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uremic and diabetic neuropathy in chronic hemodialysis patients. Int Med 1999; 38:472-5. 34. Ozcan O, Ipcioglu M, Gultepe M, Basoglu C. Altered red cell membrane compositions related to functional vitamin B12 deficiency manifested by elevated urine methylmalonic
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acid concentrations in patients with schizophrenia. Ann Clin Biochem 2008; 46:44-49.
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318
FIGURE LEGENDS Figure 1: Mean MMA and HCys Values Relative to Cbl Levels and Presence of Oxidant
320
Risk Factors
321
Values are geometric means for MMA (A) and HCys (B) for the number of subjects shown in
322
parentheses.
323
Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine
324
*p=0.012 and p=0.0054 vs MMA in subjects with Cbl≤200 pg/ml and “No” and “One” oxidant
325
risk respectively
326
†p=0.0010 and p<0.001 vs MMA in subjects with Cbl=201-300 pg/ml and “No” and “One”
327
oxidant risk respectively
328
§p=0.013 and 0.055 vs HCys in subjects with Cbl≤200 pg/ml and “No” and “One” oxidant risk
329
respectively
330
¶p<0.001 vs HCys in subjects with Cbl=201-300 pg/ml and either “No” or “One” oxidant
331
risk.
332
**p=0.0072 and p<0.001 vs HCys in subjects with Cbl=201-300 pg/ml and “No” and “One”
333
oxidant risk respectively..
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Figure 2: Linear Regression Analyses of the Relationships Between Cbl and MMA or
336
HCys Relative to the Number of Oxidant Risks Present
337
Linear regression analyses between Cbl and MMA (A) and HCys (B) were performed in subjects
338
with “low” and “low-normal” Cbl values and either “No” or “Three+” Oxidant Risks. Since only
339
2 subjects in the entire study group had Cbl values <100 pg/ml (both with “One” oxidant risk and
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340
Cbl values of 58 and 86 pg/ml respectively) regression lines were not extended below a Cbl
341
value of 100 pg/ml.
342
Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine
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Figure 3: Relationship of the Pattern of Increased MMA and HCys Values to the Presence
345
of Oxidant Risk Factors
346
Values plotted are the percent of those subjects in whom both metabolites were measured and in
347
whom an increased value of MMA and/or HCys was noted.
348
Abbreviations: MMA, methylmalonic acid; HCys, homocysteine
349
*p=0.025 and p<0.001 vs the incidences of isolated high MMA values in subjects with “Two” or
350
“Three+” oxidant risks respectively by χ2 analyses.
351
†p<0.001 vs the incidence of high MMA values in subjects with “No or One” oxidant risks;
352
and p=0.025 and p<0.001 vs the incidences of high HCys values in subjects with “Two” and
353
“Three+” oxidant risks respectively.
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Table 1: Patient Characteristics Cbl =201-300
(N=49)
(N=187)
57±19 (49)
56±17 (187)
[24-94]
[19-93]
14 (29%)
73 (39%)
Caucasian
37 (76%)
153 (82%)
Black
7 (19%)
Othera
5 (!0%)
Race (%):
Oxidant Risks (%): None
23 (12%) 11 (6%)
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Male (%)
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Age (yrs)
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Cbl ≤200
Characteristic
15 (31%)
69 (37%)
17 (35%)
53 (28%)
6 (12%)
31 (17%)
11 (22%)
34 (18%)
168 (49)
257 (187)
[58-199]
[201-300]
425 (49)b
278 (187)
[105-4911]
[101-1384]
MMA>250 nmol/l (%)
32 (65%)
101 (54%)
HCys (µmol/l)
12.3 (41)
11.2 (163)
[4.4-61.4]
[3.0-60.7]
18 (44%)
67 (41%)
One
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Two
Three+
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Cbl (pg/ml)
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MMA (nmol/l)
HCys >12.1 µmol/l (%)
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Values for Age are arithmetic means ± 1 SD while values for Cbl, MMA and HCys are geometric means for the number of subjects shown in parentheses with the range of values for each parameter shown in brackets.
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Abbreviations: Cbl; cobalamin; MMA, methylmalonic acid; HCys, homocysteine; yrs, years; SD, standard deviation a
Other racial groups include Indian (n=8); Phillipino (n=3); Arabic (n=3}; Asian (n=1); and
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Hispanic (n=1). b
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p<0.001 vs the mean MMA value in subjects with Cbl =201-300 pg/ml
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Table 2: Relationship of the Incidence of High Metabolite Values to the Number of Oxidant Risks Metabolite
Number of Oxidant Risks None
One
Two
Three+
MMA >250
≤200a
8/15 (55%)
8/17 (47%)
5/6 (63%)
11/11 (100%)b
nmol/l
201-300
34/69 (49%)
22/53 (42%)
18/31 (58%)
27/34 (79%)c
Hcys >12.1
≤200a
4/14 (29%)
3/13 (23%)
4/6 (67%)
7/8 (88%)d
µmol/l
201-300
19/63 (30%)
9/42 (21%)
16/29 (55%)e
22/29 (76%)f
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(pg/ml)
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a
Cbl
Incidences of high MMA and HCys values in subjects with Cbl levels ≤200 pg/ml were not
significantly greater than those in subjects with Cbl levels of 201-300 pg/ml and the same number of oxidant risks (p≥0.24). b
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p=0.0080 vs the incidence of high MMA values in subjects with Cbl levels ≤200 pg/ml and
“No” Oxidant Risks; and p=0.0034 vs the incidence of high MMA values in subjects with Cbl levels ≤200 pg/ml and “One” Oxidant Risk.
p=0.0034 vs the incidence of high MMA values in subjects with Cbl levels of 201-300 pg/ml
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c
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and “No” Oxidant Risks; and p<0.001 vs the incidence of high MMA values in subjects with Cbl levels of 201-300 pg/ml and “One” Oxidant Risk. d
p=0.0078 vs the incidence of high HCys values in subjects with Cbl levels ≤200 pg/ml and
“No” Oxidant Risks; and p=0.0041 vs the incidence of high HCys values in subjects with Cbl levels ≤200 pg/ml and “One” Oxidant Risk.
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e
p=0.022 vs the incidence of high HCys values in subjects with Cbl levels of 201-300 pg/ml and
“No” Oxidant Risks and p=0.0034 vs the incidence of high HCys values in subjects with Cbl levels of 201-300 pg/ml and “One” Oxidant Risk. f
either “No” Oxidant Risks or “One” Oxidant Risk. p values were obtained by χ2 analyses.
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p<0.001 vs the incidence of high HCys values in subjects with Cbl levels of 201-300 pg/ml and
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Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine
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Table 3: Effect of Cbl Therapy of Subjects With Elevated MMA and HCys Values Number
Treated
Risks
Elevated
(%)
MMA
None
42
30 (71%)
30 (100%)
484
173
<0.001
(nmol/l)
One
30
16 (53%)
14 (88%)
445
199
<0.001
Two
23
15 (65%)
14 (93%)
433
185
<0.001
Three+
38
20 (53%)
20 (100%)
516
174
<0.001
HCys
None
23
16 (70%)
16 (100%)
15.1
9.1
<0.001
(µmol/l)
One
12
6 (50%)
6 (100%)
16.1
10.1
0.0013
Two
20
11 (55%)
9 (82%)
15.6
10.6
<0.001
Three+
28
18 (64%)
17 (94%)
19.4
11.9
0.0037
Responders
Pre-
Post-
p
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Therapy Therapy
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Response criteria are defined in the Methods. Pre-therapy and Post-therapy metabolite values are geometric means for the number of treated subjects. “p” values were derived from paired ttests.
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Abbreviations: Cbl, cobalamin; MMA, methylmalonic acid; HCys, homocysteine
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Figure 1: A) 900
(11)
800 700
MMA (nmol/l)
*
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Cbl ≤200 Cbl=201-300
p<0.001
600 500
(17)
300 (69)
None
(34)
(31)
One Two Number of Oxidant Risks
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21
p=0.021
(53)
200
B)
SC
(15)
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400
(6)
Three+
(8)
§
Cbl ≤200
19
Cbl=201-300
¶ (29)
13 11
p=0.17
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HCys (µ µ mol/l)
17
(14)
(63)
9 None
(29) (13)
**
(6)
(42)
One
Two
Number of Oxidant Risks
Three+
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Figure 2: A. 1500
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(r=-0.36; p=0.015)
1100 900
SC
MMA (nmol/l)
1300
Oxidant Risks: None Three+
700
(r=-0.22; p=0.049)
300 100 0
100
M AN U
500
200
300
400
B.
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Cbl (pg/ml)
35
Oxidant Risks: None Three+
(r=-0.32; p=0.050)
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AC C
HCys (µ µ mol/l)
30
20
15
(r=-0.012; p=0.92)
10 0
100
200 Cbl (pg/ml)
300
400
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100 p<0.001
None or One (N=75)
90 p<0.001
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Two (N=28)
80
Three+ (N=34)
70 p<0.001
60 50
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*
40
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30 20 10 0
Only MMA Increased
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Both MMA and HCys Increased
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% of Subjects With At Least One Metabolite Increased
Figure 3:
Only HCys Increased
All With MMA Increased
†
All With HCys Increased
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Highlights •
The incidences of elevated methylmalonic acid and homocysteine values, metabolic
and low-normal (201-300 pg/ml) cobalamin levels. •
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markers of cobalamin deficiency, were similar in subjects with low (≤200 pg/ml)
The incidences of high metabolite values were 1.5 to 3 fold higher in subjects with three or more comorbidities associated with increased oxidative stress than in
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Methylmalonic acid values were more frequently increased than homocysteine
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values.
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•
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subjects with no oxidant risks.