Association of Plasma Concentration of Vitamin B12 With All-Cause Mortality in the General Population in the Netherlands | Complementary and Alternative Medicine | JAMA Network Open | JAMA Network
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Figure 1.  Cohort Recruitment Flowchart
Cohort Recruitment Flowchart

UAC indicates urinary albumin concentration.

Figure 2.  Association of Plasma Concentration of Vitamin B12 With Adjusted Risk of All-Cause Mortality
Association of Plasma Concentration of Vitamin B12 With Adjusted Risk of All-Cause Mortality

To convert plasma concentration of vitamin B12 to picomoles per liter, multiply by 0.7378. Shading indicates 95% CI.

Figure 3.  Kaplan-Maier Plot for Vitamin B12 Plasma Concentration and All-Cause Mortality
Kaplan-Maier Plot for Vitamin B12 Plasma Concentration and All-Cause Mortality

Quartile (Q) 1 indicates vitamin B12 plasma concentration less than 338.85 pg/mL (to convert to picomoles per liter, multiply by 0.7378); Q2 and 3, vitamin B12 plasma concentration 338.85 to 455.41 pg/mL; and Q4, vitamin B12 plasma concentration 455.41 pg/mL or higher.

Table 1.  Participant Characteristics According to Quartile of Plasma Concentration of Vitamin B12
Participant Characteristics According to Quartile of Plasma Concentration of Vitamin B12
Table 2.  Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality
Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality
Supplement.

eTable 1. Multivariable Linear Regression Analyses With Plasma Concentration of Vitamin B12 as the Dependent Variable

eTable 2. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality After Excluding Individuals With History of Cardiovascular Disease

eTable 3. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality After Excluding Individuals With History of Cancer

eTable 4. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality After Excluding Individuals With Low Plasma Concentrations Vitamin B12

eTable 5. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality After Excluding Individuals With High Plasma Concentrations Homocysteine

eTable 6. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality in All Individuals With Available Information

eTable 7. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality After Excluding Individuals With Mild to Moderated Loss of Kidney Function

eTable 8. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of All-Cause Mortality in a Design-Based Analysis

eTable 9. All-Cause Mortality Relative Risks for Each Stratum of Smoking Behavior

eTable 10. All-Cause Mortality Relative Risks for Each Stratum of Alcohol Consumption Behavior

eTable 11. All-Cause Mortality Relative Risks for Each Stratum of Age

eTable 12. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of Cancer Mortality

eTable 13. Prospective Associations of Plasma Concentration of Vitamin B12 With Risk of Cardiovascular Mortality

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    11 Comments for this article
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    B12 and mortality
    Tomoyuki Kawada, MD | Nippon Medcal School
    I have read the article by Flores-Guerrero et al. (1) with great interest. The authors conducted a prospective study to investigate the association between plasma vitamin B12 concentration and all-cause mortality. Median (interquartile range) plasma concentration of vitamin B12 was 394.42 (310.38-497.42) pg/mL, and 226 participants (4.1%) died during the median (interquartile range) of 8.2 (7.7-8.9) years of follow-up. Adjusted hazard ratio (HR) (95% confidence interval [CI]) of 1-SD increase of plasma vitamin B12 concentration for all-cause mortality was 1.25 (1.06-1.47). I have two concerns about their study.

    First, the authors cited a paper reporting the risk of higher
    plasma vitamin B12 concentrations on all-cause and cardiovascular mortality in the very old women (2). Adjusted HRs (95% CIs) of plasma vitamin B12 concentration with 100 pmol/L increase for all-cause and cardiovascular mortality were 1.10 (1.04-1.16) and 1.10 (1.02-1.18), respectively. Although a significant association between plasma vitamin B12 concentration and mortality was observed in older females in this study, both studies did not handle subjects with absolutely decreased levels of plasma vitamin B12 concentration. There is a need of further study to specify the depletion of plasma vitamin B12 concentration on subsequent mortality.

    Second, Hooshmand et al. investigated the association of plasma vitamin B12, red blood cell folate, and sulfur amino acids with the rate of total brain volume loss and the change in white matter hyper-intensity volume in older adults (3). By the multi-adjusted linear mixed models, higher baseline vitamin B12 and holo-transcobalamin levels were associated with a decreased rate of total brain volume loss. The aging was positively related to plasma vitamin B12 and holo-transcobalamin levels and negatively associated with brain volume. I suspect that aging of brain might gradually progress and neuropsychiatric information is also needed to understand the mechanism of the association.


    References

    1. Flores-Guerrero JL, Minovic I, Groothof D, et al. Association of plasma concentration of vitamin B12 with all-cause mortality in the general population in the Netherlands. JAMA Netw Open 2020;3(1):e1919274. doi: 10.1001/jamanetworkopen.2019.19274

    2. Mendonça N, Jagger C, Granic A, et al. Elevated total homocysteine in all participants and plasma vitamin B12 concentrations in women are associated with all-cause and cardiovascular mortality in the very old: The Newcastle 85+ Study. J Gerontol A Biol Sci Med Sci 2018;73(9):1258-1264. doi: 10.1093/gerona/gly035

    3. Hooshmand B, Mangialasche F, Kalpouzos G, et al. Association of vitamin B12, folate, and sulfur amino acids with brain magnetic resonance imaging measures in older adults: A longitudinal population-based study. JAMA Psychiatry 2016;73(6):606-613. doi: 10.1001/jamapsychiatry.2016.0274
    CONFLICT OF INTEREST: None Reported
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    Correlation consumption animal products and morality
    Frank Van Polanen Petel |
    Seems to me that you might have found (another) correlation between consumption of animal products and mortality? Since animal products are the only source and supplementation was excluded.
    CONFLICT OF INTEREST: None Reported
    High B12 is a known red flag for several common and fatal illnesses not corrected for in this analysis
    George Anstadt, MD | University of Rochester
    B12 physiology and metabolism is very complex. It plays an important role in every cell in the body e.g. as a cofactor in DNA replication and homocysteine metabolism. Microbiome perturbations influence levels. Several medications influence levels. Deficiency diseases are common in western society, ranging from fetal deaths, to anemias and cognitive and mood effects; these requiring supplementation. High levels are less common, but not rare, and are an important red flag for liver diseases, kidney disease, solid tumors and bone marrow dysplastic conditions. This article attempts to draw mortality conclusions from 10-year old single B12 level determinations without corrections for the often fatal diseases associated with high levels; high levels demand a careful work up, and should not be attributed to OTC supplementation. Further, the basic science of B12 elevation diseases so not suggest a casual relationship to the B12 elevation; rather the elevations reflect several passive mechanisms. B12 elevations do not cause of these fatal conditions and these findings should not change clinical practice.
    CONFLICT OF INTEREST: None Reported
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    Worth re-thinking and planning: over interpretation of the observed association between high B12 and mortality
    Rima Obeid, PhD | Department of Clinical Chemistry, Saarland University Hospital, Germany
    With great interest, I studied this paper and tried to figure out what is going on in this specific cohort. A long time ago, Waters et al. have shown that baseline vitamin B12 in community dwelling individuals is not associated with 10-y mortality PMID:5579454. Interestingly, vitamin B12 concentrations show low fluctuations upon repeated measurements over time (PMID: 24647388), thus generally making serum B12 an interesting potential predictor of future outcomes in a healthy population. In 2 large cohort studies (without previous selections), elevated B12 at baseline has been shown to be associated with prevalent albuminuria (OR 1.44 per 1 SD increase, 95% CI 1.10-1.87) and reduced kidney function PMID: 25644490, but not with incident (future) outcomes. The association with albuminuria was homocysteine-dependent .
    The association between plasma B12 and mortality in the PREVEND cohort is a post hoc analyses of data from 2 pooled cohorts that were “selected by” urinary albumin above 10mg/L (conditioning on renal function). The ratio of participants with/without urine albumin > 10 mg/L was 2.3 in the PREVEND (Figure 1), thus the study is dominated by people with microalbuminuria, suggesting possible selection bias and hence limited external validity or generalization to the general population - even if the present cohort is derived from the population. Intuitively, the reader would like to know how was the association in the two cohorts separated.
    Principally, to study an association between B12 and any future outcome we cannot select the participants based on factors causally related to the future Outcomes of interest. Moreover, co-variates to be adjusted for are those known to be associated with high B12 (e.g. pre-existing cancer, liver or renal dysfunction, etc) and factors that affect mortality in the same time (pre-existing diseases and age).
    Adjustments for covariates are expected to be judged from previous knowledge (not data-driven as in eTable 1). Moreover, to be able to adjust for 16-20 variables (including categories of any variable) as in the present work, there should be at least 300 events, which is not the case here. Table 1 shows that people in the upper quartile of serum B12 were more ill compared with the lowest or mid quartiles. In addition, the adjustments for renal function would better be according to albumin level in urine since this was a primary selection criteria for the majority of the participants and a previous report from PREVEND reported that, albuminuria, but not eGFR, was associated with CVD events and mortality PMID: 22835901.
    An additional main concern about the present study is: how would the association change if all subjects were free from albuminuria at baseline?
    eTable7 shows that when individuals with mild to moderate loss of renal function are excluded, and the model was further adjusted for preexisting mutual causes of high B12 and mortality (cancer and CVD), then the association is almost lost. Finally, tHcy is a possible mediator between B12 and the outcome (adjustment for tHcy is questionable). Taken together, it can be argued that previous diseases present in the cohort at baseline have caused high serum B12 and mortality in the same time and thus induced a seemingly meaningful association between high B12 and the study outcome [high B12 ← existing illnesses → future illnesses].
    CONFLICT OF INTEREST: None Reported
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    Harm from B12 is probalby due to cyanocobalamin
    J David Spence, M.D., FRCPC, FAHA | Robarts Research Institute, Western University, London, ON, Canada
    It is likely that harm from B12 in persons with impaired renal function is due to cyanocobalamin. Patients with renal failure have high plasma levels of thiocyanate[1], and cyanocobalamin requires decyanation to become active. In a trial in patients with diabetic nephropathy, B vitamins including 1000 mcg daily of cyanocobalamin accelerated the decline of renal function, and doubled cardiovascular events.[2] All the events occurred in participants with a glomerular filtration rate < 50 mL/ min/1. 73 m2. In the early studies of B vitamins for stroke prevention there was no benefit, but an analysis of the Vitamin Intervention for Stroke Prevention trial excluding patients with impaired renal function (eGFR in the lowest decile, 48 mL/ min/1. 73 m2, and those who received B12 supplements outside the randomized assignment, showed a 34% reduction of stroke/myocardial infarction/vascular death with B vitamins.[3] The Su.Fol.OM3 trial, in patients with the best renal function of all the trials and a much lower dose of cyanocobalamin (only 20 mcg daily) reported a 43% reduction of stroke with B vitamins. It is now becoming apparent that harm from cyanocobalamin among participants with impaired renal function obscured the benefit of B vitamins for lowering of homocysteine.[4]

    Koyama et al reported that methylcobalamin had the intended metabolic effects in patients with renal failure.[5] B vitamins to lower homocysteine do reduce the risk of stroke, but we should be using methylcobalamin or oxocobalamin rather than cyanocobalamin. For patients with the T allele of methylenetetrahydrofolate reductase we should probably also add folate supplementation beyond the dose achieved by folate fortification of the grain supply.[6]

    1. Koyama K, Yoshida A, Takeda A, Morozumi K, Fujinami T, Tanaka N. Abnormal cyanide metabolism in uraemic patients. Nephrol Dial Transplant. 1997;12(8):1622-8.
    2. House AA, Eliasziw M, Cattran DC, Churchill DN, Oliver MJ, Fine A, et al. Effect of B-vitamin therapy on progression of diabetic nephropathy: a randomized controlled trial. JAMA. 2010;303(16):1603-9.
    3. Spence JD, Bang H, Chambless LE, Stampfer MJ. Vitamin Intervention For Stroke Prevention trial: an efficacy analysis. Stroke. 2005;36(11):2404-9.
    4. Spence JD, Yi Q, Hankey GJ. B vitamins in stroke prevention: time to reconsider. Lancet Neurol. 2017;16(9):750-60.
    5. Koyama K, Ito A, Yamamoto J, Nishio T, Kajikuri J, Dohi Y, et al. Randomized controlled trial of the effect of short-term coadministration of methylcobalamin and folate on serum ADMA concentration in patients receiving long-term hemodialysis. Am J Kidney Dis. 2010;55(6):1069-78.
    6. Qin X, Spence JD, Li J, Zhang Y, Li Y, Sun N, et al. Interaction of serum vitamin B12 and folate with MTHFR genotypes on risk of ischemic stroke. Neurology. 2020.
    CONFLICT OF INTEREST: None Reported
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    Observations and musings about B-12
    William Liljequist, Esquire | The famous Staying out of the Hospital
    As a non-doctor, only commons sense is available to me.
    1) Hence, I figure that B-12 coming in the Cyanocobalamin variety might have more side effects than the Methylcobalamin variety. To be determined.

    2) While I have zero knowledge about how B-12 is absorbed, used, digested and excreted by the human body, I know that people who get B-12 prescribed might fall into two categories:

    a) Those that drink too much alcohol. If drinking alcohol is so prevalent in a patient that the doctor
    decides that a B-12 injection might be of use, then it stands to reason that both liver and kidney
    might already be overtaxed from both the liver trying its utmost to keep processing the alcohol,
    from the liver or kidney being strained by processing the acetaminophen or ibuprofen or other
    NSAID's that an alcoholic might consume, given the frequent headaches an alcoholic will experience.

    b) Those who are so old, that their liver and kidney no longer functions properly, such that any
    assimilation of B-12, whether of the Methyl - or Cyano variety, is so slowed down that more B-12
    remains in the plasma, instead of being used anywhere in the body, where it might be of use.

    3) I agree that Cyanocobalamin ought to be phased out, as I figure the less cyanide the body has to deal with, the better.

    All these musings are to be researched and the outcome determined. Let me know if I am wrong. I am just a tax accountant, so any doctor can prove me wrong, if they can. Occasionally I cut myself shaving
    with Occam's razor, but pretty rarely.
    CONFLICT OF INTEREST: None Reported
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    RESPONSE to Tomoyuki Kawada
    Jose L Flores-Guerrero | University Medical Center Groningen, Groningen, The Netherlands.
    Dear Tomoyuki Kawada
    We appreciate your interest on the article and your nice summary. Your insightful comment on the potential role of 1-Carbon metabolism on brain aging is also welcomed. Future investigation on the role of B-12 metabolism in the context of the brain aging deserves attention.


    On behalf of the authors,

    Jose L. Flores-Guerrero
    University Medical Center Groningen, Groningen, The Netherlands.
    CONFLICT OF INTEREST: None Reported
    RESPONSE to Rima Obeid,
    Jose L Flores-Guerrero | University Medical Center Groningen, Groningen, The Netherlands.
    Dear Rima Obeid,

    We appreciate your careful comment. Please note that adjustment for eGFR and urinary albumin excretion did not affect the association of vitamin B12 with all-cause mortality (Table 2, model 5). However, in a sensitivity analysis after excluding individuals with mild to moderate loss of kidney function (eGFR <60 mL/min/1.73m2), there was indeed no significant association of plasma concentration of vitamin B12 with all-cause mortality after adjustment for history of CVD (eTable 7 in the Supplement). This might be explained by the intrinsic relationship of CVD with CKD, and consequently the highly collinearity that such variables
    display in our data set. Importantly, in order to further investigate if our results could be applicable to the general population, we performed a Design-Based Analysis to weight the albuminuria enriched component in the PREVEND cohort. In this analysis, the association of plasma concentration of vitamin b12 with risk of all-cause mortality was significant after full adjustment.

    On behalf of the authors,

    Jose L. Flores-Guerrero
    University Medical Center Groningen, Groningen, The Netherlands.
    CONFLICT OF INTEREST: None Reported
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    RESPONSE to George Anstadt,
    Jose L Flores-Guerrero | University Medical Center Groningen, Groningen, The Netherlands. 
    Dear George Anstadt,

    We agree with your comment that 1-carbon metabolism is complex, and that the cause of high circulating vitamin B12 levels requires careful evaluation in individual patients. Please note that patients with active cancer did not participate in the second screening round of the PREVEND study, which was the starting point of our study. The results presented in the main text show that higher plasma concentrations of vitamin B12 were associated with all-cause mortality after adjustment for ferritin, hemoglobin and mean corpuscular volume (Table 2, model 3), as well as after additional adjustment for a history
    of cancer and cardiovascular disease (Table 2, model 4). In addition, excluding patients with a history of cancer, low plasma concentrations of vitamin B12, or high plasma concentrations of homocysteine did not materially change the results (supplemental tables). Further analysis showed that the association remained unchanged after excluding those participants who developed hematological or bone marrow dysplasias before, during and after the second screening round of the PREVEND. In such analysis, age- and sex-adjusted Cox regression that examined the vitamin B12 plasma concentration as a categorical variable with quartile 1 as the reference group, the fourth quartile of vitamin B12 plasma concentrations was associated with increased risk of mortality (HR, 1.63 [95% CI, 1.11-2.39]; P = .01). The association remained significant after full adjustment (adjusted HR, 1.75 [95% CI, 1.09- 2.80]; P = .02). Taken together, we believe it would be unlikely that the association of vitamin B12 levels with all-cause mortality was to a considerable extent attributable to undetected (hematological) malignancies or bone marrow dysplasias.
    We agree that our findings as such should not change clinical practice regarding vit B12 substitution therapy in patients diagnosed with vitamin B12 deficiency. However, our report may provide a rationale to re-evaluate the safety of vitamin B12 supplementation in the absence of vitamin B12 deficiency.

    On behalf of the authors,

    Jose L. Flores-Guerrero
    University Medical Center Groningen, Groningen, The Netherlands.
    CONFLICT OF INTEREST: None Reported
    READ MORE
    RESPONSE to J David Spence,
    Jose L Flores-Guerrero | University Medical Center Groningen, Groningen, The Netherlands.
    Dear J David Spence,

    We appreciate your interest on the article and your insightful comment. Future investigation on the role of thiocyanate and the activation of cyanocobalamin in renal function and mortality deserves attention.
    On behalf of the authors,
    Jose L. Flores-Guerrero
    CONFLICT OF INTEREST: None Reported
    RESPONSE to Frank Van Polanen Petel
    Jose L Flores-Guerrero | University Medical Center Groningen, Groningen, The Netherlands. 
    Dear Frank Van Polanen Petel,
    We appreciate your interest on the article and your insightful comment. As the only dietary source of vitamin B12 are animal products, it is possible that the present association could also be explained by the already reported association of higher animal protein intake with all-cause mortality. (JAMA Intern Med. 2016 November 1; 176(11): 1728., Am J Clin Nutr. 2019 May 1;109(5):1462-1471.). Unfortunately, the PREVEND cohort does not include data on dietary patterns.
    Our analysis were conducted after the exclusion of individuals who were prescribed with injectable vitamin B12 supplementation at the baseline measurements,
    nonetheless the Pharmacy records have no available information about the over-the-counter tablets of vitamin B12, and therefore our results cannot be adjusted for such variable.
    On behalf of the authors,
    Jose L. Flores-Guerrero
    University Medical Center Groningen, Groningen, The Netherlands.
    CONFLICT OF INTEREST: None Reported
    READ MORE
    Original Investigation
    Geriatrics
    January 15, 2020

    Association of Plasma Concentration of Vitamin B12 With All-Cause Mortality in the General Population in the Netherlands

    Author Affiliations
    • 1University Medical Center Groningen, Division of Nephrology, Department of Internal Medicine, University of Groningen, Groningen, the Netherlands
    • 2University Medical Center Groningen, Department of Laboratory Medicine, University of Groningen, Groningen, the Netherlands
    • 3Faculty of Science and Engineering, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, the Netherlands
    • 4University Medical Center Groningen, Division of Endocrinology, Department of Internal Medicine, University of Groningen, Groningen, the Netherlands
    JAMA Netw Open. 2020;3(1):e1919274. doi:10.1001/jamanetworkopen.2019.19274
    Key Points español 中文 (chinese)

    Question  Are plasma concentrations of vitamin B12 associated with risk of all-cause mortality among adults from the general population of the Netherlands?

    Findings  In this population-based cohort study including 5571 adults, higher plasma concentrations of vitamin B12 were associated with a 25% increased adjusted risk of all-cause mortality per 1-SD increase.

    Meaning  These findings suggest that higher plasma concentrations of vitamin B12 are associated with all-cause mortality, independent of traditional risk factors.

    Abstract

    Importance  Higher plasma concentrations of vitamin B12 have been associated with mortality in elderly and hospitalized populations, including patients with chronic kidney disease, but the association of plasma concentrations of vitamin B12 with mortality in the general population remains unclear.

    Objective  To investigate the association of plasma concentrations of vitamin B12 with all-cause mortality.

    Design, Setting, and Participants  This longitudinal cohort study used post hoc analysis to examine data from participants of the Prevention of Renal and Vascular End-stage Disease Study in Groningen, the Netherlands. Participants included individuals who completed the second screening visit beginning January 1, 2001, excluding those who were missing values of vitamin B12 plasma concentrations or used vitamin B12 supplementation. Follow-up time was defined between the beginning of the second screening round to end of follow-up on January 1, 2011. Data analysis was conducted from October 2, 2018, to February 22, 2019.

    Exposures  Plasma vitamin B12 concentration level.

    Main Outcomes and Measures  Death as recorded by the Central Bureau of Statistics of Groningen, the Netherlands.

    Results  A total of 5571 participants (mean [SD] age, 53.5 [12.0] years; 2830 [50.8%] men) were included in analyses. Median (interquartile range) plasma concentration of vitamin B12 was 394.42 (310.38-497.42) pg/mL. During the median (interquartile range) of 8.2 (7.7-8.9) years of follow-up, 226 participants (4.1%) died. According to quartiles of the distribution of plasma vitamin B12 concentration levels, mortality rates were 33.8 deaths per 10 000 person-years for the quartile with the lowest plasma concentration of vitamin B12 and 65.7 deaths per 10 000 person-years for the quartile with the highest plasma concentration of vitamin B12. After adjustment for multiple clinical and laboratory variables, Cox regression analyses found a significant association between higher vitamin B12 plasma concentration level and increased risk of all-cause mortality (hazard ratio per 1-SD increase, 1.25 [95% CI, 1.06-1.47]; P = .006).

    Conclusions and Relevance  These findings suggest that higher levels of plasma concentrations of vitamin B12 were associated with increased risk of all-cause mortality after adjusting for age, sex, renal function, and other clinical and laboratory variables. The mechanisms underlying this association remain to be established.

    Introduction

    Vitamin B12 is a hydrosoluble vitamin that plays a substantial role in 1-carbon metabolism. The 1-carbon pathway is involved in several biological functions beyond fetal development, such as mitochondrial metabolism, immune response, and nucleotide homeostasis in nonproliferative tissues.1

    While the deleterious effects of vitamin B12 deficiency, such as anemia, neuropsychiatric symptoms, and other clinical manifestations, are well established,2 the potential association of high plasma concentrations of vitamin B12 with adverse health outcomes has not been fully explored.3 Indeed, a potential association of high vitamin B12 plasma concentrations with excess mortality has been assessed in elderly3-9 and hospitalized10,11 populations, but it has not been explored in the general population, to our knowledge.

    An association of high plasma concentrations of vitamin B12 with increased risk of all-cause mortality has been reported among patients undergoing dialysis treatment.12 It has also been found that impaired renal function is associated with high plasma concentrations of vitamin B12.13,14 Furthermore, it has been found that combined supplementation of folic acid, vitamin B6, and vitamin B12 results in more rapid decline of renal function and an increase in occurrence of vascular events in patients with diabetic nephropathy.15 Taken together, these findings underscore the importance of further exploration of a possible role of chronic kidney disease (CKD) in the association of plasma concentrations of vitamin B12 with all-cause mortality.

    Therefore, this study aimed to assess the association of plasma concentrations of vitamin B12 with all-cause mortality in a population-based cohort study. In addition, we aimed to investigate whether findings were further associated with CKD or age, considering that approximately 35% of the elderly population has some degree of CKD.16 The Prevention of Renal and Vascular End-stage Disease (PREVEND) study is particularly suitable for such aims, since it has a wide age range and its design is enriched with a CKD component.

    Methods
    Study Cohort

    The PREVEND Study is a prospective population-based cohort study in the city of Groningen, the Netherlands. The design of the PREVEND Study has been described in detail elsewhere.17 Briefly, from 1997 to 1998, all residents from Groningen aged 28 to 75 years were invited to participate. A total of 40 856 individuals (47.8%) responded to the invitation to participate. From this group, 30 890 individuals had a urinary albumin concentration of less than 10 mg/L and 9966 individuals had a urinary albumin concentration of 10 mg/L or higher in their morning urine sample. After exclusion of individuals with type 1 diabetes and women who were pregnant, 7768 individuals with a urinary albumin concentration of 10 mg/L or higher and a randomly selected control group of 3395 individuals with a urinary albumin concentration of less than 10 mg/L were invited for further investigations in an outpatient clinic. A total of 8592 individuals completed an extensive examination. The PREVEND Study cohort was designed to include any people who met the inclusion criteria, regardless of race/ethnicity, which was recorded according to self-report and included in the analysis owing to potential racial/ethnic disparities in all-cause mortality.18

    We used data from 6894 participants who completed the second screening, starting January 1, 2001, excluding 1265 individuals with missing samples for assessment of vitamin B12 plasma concentrations and 58 individuals for use of injectable vitamin B12 supplementation, leaving a cohort of 5571 participants with complete information for the analysis (Figure 1). In this group, less than 1% of data on laboratory variables was missing. Participants lost to follow-up were considered as censored data. Educational level was categorized into low (ie, no, primary, basic vocational, and secondary education), medium (ie, senior secondary vocational and general senior secondary education), and high (higher professional and higher academic education) according to the International Standard Classification of Education.19 Drug dispensing data of injectable vitamin B12 supplementation was retrieved from the University Groningen Pharmacy dispensing database IADB.nl.20 This report follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline. The protocol for the PREVEND study was approved by the local ethics committee of the University Medical Center Groningen. All participants provided written informed consent, and all procedures were conducted according to the Declaration of Helsinki.21

    Laboratory Measurements

    Laboratory measurements were performed at the Central Laboratory of the University Medical Center Groningen, the Netherlands. Hematologic measurements, including hemoglobin, hematocrit, and mean corpuscular volume, were measured on a Coulter Counter STKS sample testing system (Coulter Corp) in fresh venous blood according to standard procedures. Plasma glucose level was measured directly after blood sampling. Ethylene diamine tetraacetic acid–anticoagulated plasma samples and sera were stored at −80 °C until analysis.

    Vitamin B12 plasma concentrations were measured on a Roche platform, using the validated Elecsys Vitamin B12 II assay (Roche Diagnostics). The performance of Elecsys Vitamin B12 II has been reported in greater detail elsewhere.22

    Total cholesterol (TC), triglyceride, serum creatinine, and serum cystatin C levels were measured using standard protocols, as described previously.23-25 Serum ferritin levels were measured using immunoassay, and serum transferrin levels were measured using immunoturbidimetric assay (Roche Diagnostics). Homocysteine concentrations were measured on a Roche Cobas analyzer (Roche Diagnostics). Urinary albumin excretion (UAE) rates were measured as described in two 24-hour urine collections and the results were calculated as a mean for analysis.25 The estimated glomerular filtration rates (eGFRs) were calculated using the Chronic Kidney Disease Epidemiology Collaboration combined creatinine–cystatin C equation.26

    Clinical Measurements

    Height and weight were measured with the participants standing without shoes or heavy outer garments. Body mass index (BMI) was calculated by dividing weight in kilograms by height in meters squared. Systolic and diastolic blood pressure values were recorded as the means of the last 2 recordings of the second visit.

    Ascertainment of End Point

    Participants were followed up from the date of the baseline visit until January 1, 2011. Data on mortality were obtained from the municipal register, and the cause of death was obtained by linking the number of the death certificate to the primary cause of death as coded by a physician from the Central Bureau of Statistics.

    Statistical Analysis

    Data are presented as the mean (SD) or median (interquartile range [IQR]) for continuous variables and percentages for categorical variables. Cross-sectional group differences among vitamin B12 plasma concentration groups at baseline were assessed by Welch 1-way test for normally distributed data, Kruskal-Wallis test for skewed distributed data, and χ2 test for categorical variables. Multivariable linear regression analyses were carried out to disclose the associations of vitamin B12 plasma concentrations with clinical covariates and laboratory parameters.

    For the prospective analysis, we plotted cumulative Kaplan-Meier curves for mortality during follow-up according to quartiles of vitamin B12 plasma concentrations. Quartile 1 was defined as vitamin B12 plasma concentration less than 338.85 pg/mL (to convert to picomoles per liter, multiply by 0.7378); quartile 2, 338.85 to 397.13 pg/mL; quartile 3, 397.14 to 455.41 pg/mL; and quartile 4, more than 455.41 pg/mL. Time-to-event Cox proportional hazards models were used to assess the hazard ratio (HR) and 95% CI of mortality among 5571 participants with full information at baseline. We calculated HRs in models adjusted for covariates selected on the basis of physiological plausibility and previous literature and organized in cumulative models of clinical significance. The cumulative models depict the influence of different physiological components on the association of vitamin B12 plasma concentrations with mortality. The choice for certain confounder adjustments (eg, adjust for systolic but not diastolic blood pressure) was made based on those variables that presented a stronger association on the cross-sectional logistic regression analysis (eTable 1 in the Supplement). The first model was adjusted for age and sex. The second model was further adjusted for relevant clinical variables: race/ethnicity, type 2 diabetes, smoking behavior, alcohol consumption, and education level, which were evaluated as dichotomous variables; BMI, systolic blood pressure, and homocysteine level were evaluated as continuous variables. Model 3 included variables in model 2 plus relevant variables involved in hematological homeostasis, such as ferritin and hemoglobin levels and mean corpuscular volume (continuous variables), which are also associated with plasma concentrations of vitamin B12. Model 4 was adjusted for the same variables as model 3 plus TC to high-density lipoprotein cholesterol ratio and glucose level (continuous variables), as a proxy for cardiometabolic risk. Model 5 was adjusted for the variables in model 4 plus history of cancer (dichotomous variable) and history of cardiovascular disease (CVD) (dichotomous variable), given the previous reports of the association of high plasma concentrations of vitamin B12 with cancer27 and the relevance of CVD in mortality risk. Model 6 was adjusted for the variables included in model 5 plus renal function (ie, eGFR and UAE rate as continuous variables), as this cohort was particularly enriched with participants with a component of CKD. Finally, model 7 was adjusted for the variables in model 6 plus aspartate aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyltransferase levels (continuous variables), as previous literature also suggests an important role of hepatic function on the metabolism of vitamin B12.1 Hazard ratios were computed per 1-SD increment of plasma concentration of vitamin B12. Given the right-skewed distribution, vitamin B12 plasma concentration data were loge transformed. Hazard ratios were also computed according to vitamin B12 plasma concentrations as a categorical variable, with the reference group as quartile 1. To improve the graphic presentation of HRs, quartile 2 and quartile 3 were combined into a single category. Proportionality of hazards assumptions were tested using weighted Schoenfeld residuals for each variable and for every model as a whole. Additionally, interactions with age, eGFR, and UAE rate were analyzed.

    Furthermore, we conducted sensitivity analysis consisting of (1) individuals without history of CVD; (2) individuals without history of cancer; (3) individuals without history of vitamin B12 deficiency, defined as plasma concentrations of vitamin B12 less than 200.60 pg/mL28; (4) individuals without history of vitamin B12 deficiency judged by plasma concentrations of homocysteine greater than 2.03 mg/L (to convert to micromoles per liter, multiply by 7.397)28; (5) all individuals with available information, including those with history of vitamin B12 supplementation; and (6) individuals without mild to moderate loss of kidney function (eGFR <60 mL/min/1.73 m2). By design, participants with an urinary albumin concentration of 10 mg/L or higher are overrepresented in the PREVEND Study cohort. Therefore, a design-based analysis was performed to take this overselection of participants with elevated UAE rates into account. This statistical weighting method allows conclusions to be generalized to the general population. In addition, all-cause mortality relative risks were estimated for each stratum of alcohol consumption, smoking behavior, and age. Finally, prospective associations of plasma concentrations of vitamin B12 with risk of cancer mortality and CVD mortality were assessed.

    All statistical tests were 2-sided, and a P value less than .05 was considered statistically significant. All statistical analyses were performed with R statistical software version 3.5.1 (R Project for Statistical Computing). Data analysis was conducted from October 2, 2018, to February 22, 2019.

    Results
    Baseline Characteristics

    Of 6894 PREVEND Study participants who completed the second round of screening, 5571 participants (mean [SD] age, 53.5 [12.0] years; 2830 [50.8%] men) were included in this study. Participant characteristics at baseline are shown in Table 1. The median (IQR) vitamin B12 plasma concentration was 394.42 (310.38-497.42) pg/mL (Table 1). A total of 195 participants (3.5%) had a low vitamin B12 plasma concentration (<220.60 pg/mL). After dividing participants by plasma concentration of vitamin B12, there were 1390 participants (mean [SD] age, 52.5 [12.3] years; 709 [51.0%] men) in quartile 1, the lowest concentration quartile; 2787 participants (mean [SD] age, 53.4 [11.9] years; 1444 [51.8%] men) in quartiles 2 and 3; and 1394 participants (mean [SD] age, 54.6 [11.6] years; 677 [48.5%] men) in quartile 4, the highest concentration quartile. Participants within the highest quartile of vitamin B12 plasma concentrations (>455.41 pg/mL) were more likely to be older and have higher BMI and blood pressure. Additionally, those participants also had higher concentrations of TC and glucose and higher UAE rates. Family history of CKD, history of cancer or CVD, and educational levels were similar across the quartiles of vitamin B12 plasma concentrations (Table 1).

    Associations at Baseline

    The associations of vitamin B12 plasma concentrations and other variables of interest were evaluated with univariable and multivariable regression analysis (eTable 1 in the Supplement). In a fully adjusted multivariable model, higher vitamin B12 plasma concentration remained positively associated with use of lipid-lowering medication (β = 0.04 [95% CI, 0.01-0.07]; P = .04), high-density lipoprotein cholesterol (β = 0.09 [95% CI, 0.05-0.13]; P < .001), ferritin (β = 0.05 [95% CI, 0.02-0.09]; P = .005), and hemoglobin (β = 0.15 [95% CI, 0.04-0.27]; P = .009) levels and inversely associated with mean corpuscular volume (β = −0.09 [95% CI, −0.12 to −0.05]; P < .001), homocysteine level (β = −0.34 [95% CI, −0.38 0.30]; P < .001), and eGFR (β = −0.11 [95% CI, −0.16, to −0.07]; P < .001).

    Longitudinal Analysis

    During median (IQR) follow-up of 8.2 (7.7-8.9) years, 226 participants (4.1%) died. Higher plasma concentrations of vitamin B12, when analyzed as HR per 1 loge SD increase, were associated with mortality after full adjustment (adjusted HR, 1.25 [95% CI, 1.06-1.47]; P = .006) (Table 2 and Figure 2). Kaplan-Meier curves for mortality according to quartiles of vitamin B12 plasma concentration are presented in Figure 3. There was an increased risk of all-cause mortality associated with the top quartile of vitamin B12 concentrations (P for log-rank test <.001). In age- and sex-adjusted Cox regression analysis that examined the vitamin B12 plasma concentration as a categorical variable with quartile 1 as the reference group, the fourth quartile of vitamin B12 plasma concentrations was associated with increased risk of mortality (HR, 1.73 [95% CI, 1.18-2.53]; P = .005) (Table 2). The association remained significant after full adjustment (adjusted HR, 1.85 [95% CI, 1.16-2.97]; P = .01) (Table 2). The proportional hazards assumptions were not violated for any of the variables in the full model. The interaction terms of vitamin B12 plasma concentration with age, eGFR, and UAE rate with all-cause mortality were not significant when included in either the crude or the sex- and age-adjusted models.

    Sensitivity Analyses

    Excluding patients with a history of CVD, cancer, low plasma concentrations of vitamin B12, or high plasma concentrations of homocysteine did not materially change the results (eTables 2, 3, 4, and 5 in the Supplement). Inclusion of participants with history of vitamin B12 supplementation also did not materially change the results (eTable 6 in the Supplement). In the analysis conducted after exclusion of individuals with mild to moderate loss of kidney function (eGFR <60 mL/min/1.73 m2), there was no association of plasma concentration of vitamin B12 with all-cause mortality after adjustment for history of CVD (eTable 7 in the Supplement). Nonetheless, the results of the design-based analysis are in line with the main results presented in Table 2 (eTable 8 in the Supplement). The relative risk of all-cause mortality across the different strata of smoking behavior (eTable 9 in the Supplement), alcohol consumption (eTable 10 in the Supplement), and age (eTable 11 in the Supplement) showed a consistent association of higher plasma concentration of vitamin B12 with increased risk of all-cause mortality. We found no independent associations of plasma concentration of vitamin B12 with cancer mortality (eTable 12 in the Supplement) or with CVD mortality (eTable 13 in the Supplement).

    Discussion

    In this prospective population-based cohort study, we investigated the associations of plasma concentration of vitamin B12 with all-cause mortality. Baseline characteristics, such as older age, high blood pressure, reduced eGFR, and elevated UAE rate, as well as increased concentrations of liver enzymes, were positively associated with higher plasma concentrations of vitamin B12, in agreement with findings from other studies.3,8,11 While the cross-sectional associations as found in this study do not provide an insight for a particular cause of death, we observed that high circulating vitamin B12 plasma concentrations were associated with significantly higher risk of all-cause mortality. The association remained significant after adjustment for established risk factors, including age, sex, BMI, type 2 diabetes, tobacco use, and alcohol consumption, as well as biomarkers associated with renal and liver function. We found no indication of change in this association by either renal function or age.

    In our study, the participants in the highest quartile of vitamin B12 had a mean age 1 year older than the total cohort’s mean age (53.5 years). However, the association we found of plasma concentration of vitamin B12 with all-cause mortality was independent of age. Previous studies have explored such an association only in elderly people. A study by Salles et al6 reported an association of high vitamin B12 plasma concentration with increased mortality risk among elderly individuals (mean age, 86 years). On the other hand, a study by Robinson et al5 reported that vitamin B12 plasma concentration levels were not associated with death risk in an elderly population. Moreover, in line with previous reports, we did not find a statistically significant difference between sexes on the association of vitamin B12 plasma concentration with all-cause mortality.

    Contrary to the findings reported in a study by Arendt et al,27 we did not find a significant association of vitamin B12 plasma concentration with risk of cancer mortality. Moreover, there was no cross-sectional association between concentrations of vitamin B12 plasma concentration and cancer history at baseline. In addition, the prospective association of vitamin B12 plasma concentration with all-cause mortality was not changed by adjustment for history of cancer at baseline. Also in sensitivity analyses, the association of plasma concentration of vitamin B12 with all-cause mortality persisted after exclusion of participants with history of cancer.

    Likewise, the association of all-cause mortality with high plasma concentrations of vitamin B12 could not be attributed to CVD. We demonstrated that adjustment for blood pressure, as well as lipid profile and type 2 diabetes, did not change the association. Furthermore, in sensitivity analyses conducted after exclusion of participants with history of CVD, the association remained. In addition, plasma concentration of vitamin B12 was not associated with risk of CVD mortality. Such results are in line with a 12-year follow-up study from 201929 that found that plasma concentrations of vitamin B12 were not associated with the incidence risk of atherosclerotic disease.

    Considering the universal role of the 1-carbon pathway in mammals, the explanation of the described association seems to rely in the role of vitamin B12 in the homeostasis of nonproliferative tissues6 rather than proliferative tissues,1 such as the bone marrow and other hematopoietic tissues, as we demonstrated that this association was independent of cancer history.

    To date, the underlying mechanism of the association of plasma concentration of vitamin B12 with mortality is incompletely understood, to our knowledge. The proposed mechanisms to explain the association are that high vitamin B12 plasma concentrations may represent a response to increased release of vitamin B12 from liver storage, decreased clearance, upregulation of haptocorrin and transcobalamin synthesis, or diminished affinity of vitamin B12 for transporter proteins.1,3 Those situations are often present as a consequence of liver damage or CKD, which could be represented by the baseline association of high plasma concentrations of vitamin B12 with elevated concentrations of hepatic enzymes. Nonetheless, a definite mechanism has not been described, to our knowledge.14,30

    The results of this study could also be clinically interpreted in the context of oral vitamin supplementation. Concern about the excess intake of vitamins, particularly vitamin B12, has gained attention. A 2010 study by Løland et al31 reported that vitamin B supplementation had no beneficial effect on the progression of coronary artery disease, as had been hypothesized previously. Moreover, in a prospective study with 75 864 women,32 vitamin B12 supplementation was associated with an increased risk of hip fracture. In that sense, our results may also suggest that caution should be taken when considering vitamin B12 supplementation in the absence of vitamin B12 deficiency.

    Strengths and Limitations

    This study has several strengths. To our knowledge, this is the first study reporting on the association of all-cause mortality with higher vitamin B12 plasma concentrations in the general population in which the vitamin B12 plasma concentration measurement was performed in unselected individuals, as we excluded participants with B12 supplementation, which is one source of bias in other studies.27 Moreover, we adjusted our results for several confounding variables, including liver function parameters, and found that the association remained, whereas other authors have reported loss of significance after such adjustment.11 Another strength of this study is the implementation of a robust method of plasma concentration of vitamin B12 quantification. The accuracy of the assay that we used has been evaluated using the vitamin B12 World Health Organization International Standard.33 Moreover, it has been reported that the Elecsys Vitamin B12 assay is not affected by anti–intrinsic factor antibodies, enhancing the reliability of this assay.34 Furthermore, this is the largest study to date reporting on such an association, to our knowledge, which enabled us to carry out sufficiently powered multivariable adjusted analyses and testing the robustness of the findings using several sensitivity analyses to provide solid evidence. Finally, the PREVEND cohort was enriched for increased albumin excretion. We therefore conducted design-based analyses, making our results valid for the general population.

    Several limitations of this study also need to be addressed. First, the PREVEND cohort study mainly comprises individuals of European ancestry, which could limit extrapolation of our findings to other races/ethnicities. Second, we did not have measurements of vitamin B12 plasma concentrations beyond baseline assessment, which limited us to evaluate the regression dilution of vitamin B12. Third, we only had access to pharmacy records on injectable vitamin B12 supplementation, but not for over-the-counter tablets, which could limit the implication of cobalamin supplementation; likewise, we had no data on reason for performing the vitamin B12 injections. Furthermore, it is worth noting that residual confounding is an important limitation in all observational studies. To further evaluate the associations of well-known mortality risk factors, we provided a supplementary analysis of all-cause mortality relative risks for each stratum of alcohol consumption as well as smoking behavior. Additionally, dietary patterns can also be influence plasma concentration of vitamin B12 and risk of mortality. In the PREVEND cohort, detailed dietary information was not available, which we consider a limitation. Therefore, the possibility of a noncausal association should not be discarded and deserves further investigation.

    Conclusions

    In this population-based cohort study, high plasma concentrations of vitamin B12 were associated with an increased risk of all-cause mortality. The prospective association was independent of age, renal function, and other comorbidities, such as history of cancer. Further investigation is needed to unravel the complexity of 1-carbon metabolism in different mortality causes, such as cardiometabolic disease and cancer.

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    Article Information

    Accepted for Publication: November 20, 2019.

    Published: January 15, 2020. doi:10.1001/jamanetworkopen.2019.19274

    Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2020 Flores-Guerrero JL et al. JAMA Network Open.

    Corresponding Author: Jose L. Flores-Guerrero, MD, MSc, University Medical Center Groningen, Division of Nephrology, Department of Internal Medicine, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands (j.l.flores.guerrero@umcg.nl).

    Author Contributions: Drs Dullaart and Bakker had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

    Concept and design: Flores-Guerrero, Navis, Dullaart, Bakker.

    Acquisition, analysis, or interpretation of data: Flores-Guerrero, Minović, Groothof, Gruppen, Riphagen, Kootstra-Ros, Muller Kobold, Hak, Gansevoort, de Borst, Dullaart, Bakker.

    Drafting of the manuscript: Flores-Guerrero, Hak, Dullaart.

    Critical revision of the manuscript for important intellectual content: Flores-Guerrero, Minović, Groothof, Gruppen, Riphagen, Kootstra-Ros, Muller Kobold, Hak, Navis, Gansevoort, de Borst, Bakker.

    Statistical analysis: Flores-Guerrero, Bakker.

    Obtained funding: Gansevoort, Bakker.

    Administrative, technical, or material support: Minović, Gruppen, Kootstra-Ros, Muller Kobold, Dullaart.

    Supervision: Navis, de Borst, Dullaart, Bakker.

    Conflict of Interest Disclosures: Dr de Borst reported receiving consultancy fees from Amgen, Bayer, Kyowa Hakko Kirin, Pharmacosmos, AstraZeneca, Sanofi Genzyme, and Vifor Fresenius Medical Care Renal Pharma (paid to the University of Groningen). No other disclosures were reported.

    Funding/Support: Dr Flores-Guerrero acknowledges support from the National Council of Science and Technology.

    Role of the Funder/Sponsor: The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

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