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Detection of Vitamin B₁₂ Deficiency in Older People by Measuring Vitamin B₁₂ or the Active Fraction of Vitamin B₁₂, Holotranscobalamin

¹ Clinical Trial Service Unit, University of Oxford, Oxford, United Kingdom.
² Hightown Surgery, Hightown Gardens, Banbury, United Kingdom.
³ Department of Clinical Biochemistry, AS, Aarhus University Hospital, Aarhus, Denmark.
⁴ Department of Clinical Chemistry, Umeå University Hospital, Umeå, Sweden.
⁵ Division of Clinical Geratology, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom.
⁶ Locus for Homocysteine and Related Vitamins, University of Bergen, Bergen, Norway.
⁷ School of Biochemistry and Immunology, Trinity College, Dublin, Republic of Ireland.

^aAddress correspondence to this author at: Clinical Trial Service Unit, Richard Doll Building, Old Road Campus, Roosevelt Drive, Oxford, OX3 7LF, United Kingdom. Fax 00-44-1865-743985; e-mail robert.clarke{at}ctsu.ox.ac.uk.

	Abstract

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Background: Impaired vitamin B₁₂ function and decreased vitamin B₁₂ status have been associated with neurological and cognitive impairment. Current assays analyze total vitamin B₁₂ concentration, only a small percentage of which is metabolically active. Concentrations of this active component, carried on holotranscobalamin (holoTC), may be of greater relevance than total vitamin B₁₂.

Methods: We compared the utility of serum holoTC with conventional vitamin B₁₂ for detection of vitamin B₁₂ deficiency in a population-based study of older people, using increased methylmalonic acid (MMA) concentrations as a marker of metabolic vitamin B₁₂ deficiency in the overall population (n = 2403) and in subsets with normal (n = 1651) and abnormal (n = 752) renal function.

Results: Among all participants, 6% had definite (MMA >0.75 µmol/L) and 16% had probable (MMA >0.45 µmol/L) metabolic vitamin B₁₂ deficiency. In receiver operating characteristic curves for detection of definite vitamin B₁₂ deficiency, holoTC had a greater area under the curve (AUC) compared with vitamin B₁₂ in all participants (0.85 vs 0.76; P <0.001) and in subsets with normal (AUC: 0.87 vs 0.79; P <0.001) and abnormal (AUC: 0.85 vs 0.74; P = 0.002) renal function. Similar findings were observed for detection of moderate vitamin B₁₂ deficiency. Whereas the positive predictive value for both holoTC and vitamin B₁₂ was greater for detection of probable than definite vitamin B₁₂ deficiency, both tests were associated with more false-positive than true-positive test results.

Conclusions: HoloTC has a modestly superior diagnostic accuracy compared with conventional vitamin B₁₂ for the detection of vitamin B₁₂ deficiency, but neither test can be recommended to screen asymptomatic populations.

	Introduction

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Concerns that older individuals with undiagnosed vitamin B₁₂ deficiency may be put at risk by mandatory folic acid fortification have prompted interest in screening older people for undiagnosed vitamin B₁₂ deficiency (1). Approximately 5%–20% of older people may have undiagnosed vitamin B₁₂ deficiency, defined by low serum concentrations of vitamin B₁₂ together with increased concentrations of methylmalonic acid (MMA)¹ or homocysteine (tHcy) (2)(3)(4). Population-based studies that screened for anemia, depression, cognitive impairment, and neuropathy in relation to vitamin B₁₂ status in older people have indicated that most older individuals with undiagnosed vitamin B₁₂ deficiency did not have anemia or neurological disease (4); however, the proportion that may subsequently develop signs of vitamin B₁₂ deficiency is not known. In clinical practice, vitamin B₁₂ deficiency commonly presents as macrocytic anemia, and the neurological signs may occur without anemia in 20% of cases (5). The administration of folic acid to individuals with vitamin B₁₂ deficiency can correct the anemia but may worsen the neurological dysfunction (6). The possibility that mandatory folic acid fortification could result in an insidious progression of neurological disease in individuals with vitamin B₁₂ deficiency prompted some to advocate universal screening for vitamin B₁₂ deficiency in older people (2).

The diagnosis of vitamin B₁₂ deficiency is complicated by poor sensitivity and specificity of the conventional vitamin B₁₂ assays (7)(8)(9). The advent of novel assays for holotranscobalamin (holoTC), the active fraction of vitamin B₁₂, may improve the detection of vitamin B₁₂ deficiency, but the results of previous studies that addressed this question were conflicting (10)(11)(12)(13). Approximately 80% of circulating vitamin B₁₂ is biologically unavailable, and the rest comprises holoTC, which is the part of vitamin B₁₂ bound to transcobalamin that delivers vitamin B₁₂ to all cells in the body (10). We compared the diagnostic utility of holoTC with conventional vitamin B₁₂ testing to detect definite metabolic vitamin B₁₂ deficiency (defined as MMA >0.75 µmol/L) or probable metabolic vitamin B₁₂ deficiency (defined as MMA >0.45 µmol/L) in the overall population and in subsets with normal or abnormal renal function in a population-based study of older people living in the UK.

	Materials and Methods

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study population
The study population included all participants with data on vitamin status in population-based studies of older people living in Oxford city (Oxford Healthy Aging Project, OHAP) and Oxfordshire (Banbury B₁₂ study) (2). OHAP is a longitudinal cohort study of 2741 randomly selected people ages 65 years that is a component part of the Medical Research Council Cognitive Function and Aging Study (14). In 1993, we randomly selected the population sample from general practice registers for people living in Oxford city to provide equal numbers of individuals ages 65 to 74 years and 75 years. Research nurses visited study participants in their homes and carried out a structured interview. The collected data included medical history, smoking habits, education, and use of medication (including multivitamin supplements or vitamin B₁₂ injections). All surviving participants who had not previously refused to be interviewed were invited to provide a blood sample in 1995. Nonfasting blood samples were collected into vacutainers that were allowed to clot at room temperature, and the serum was separated within 2 h and stored at –80 °C until it was shipped on dry ice or thawed for analysis.

Participants in the Banbury B₁₂ study were recruited from a random sample of people ages 75 years living in their own homes and registered with 3 general practices in Banbury, Oxfordshire (4). Individuals who were known to have a terminal illness or were living in institutions were excluded. Eligible participants (n = 1934) were invited to participate in the study, and those who agreed (n = 1000) were asked to provide written informed consent. Participants were visited in their homes by a research nurse between March 2003 and April 2004, and the data collected included medical history and use of medication. Nonfasting venous blood samples were collected and kept chilled (using a cooling box to ensure that the temperature was maintained <4 °C) until the serum was separated at the local hospital laboratory within 2 h of blood collection and stored at –40 °C until analysis. Participants in both studies provided signed consent, and the protocols (in accordance with the current version of the Helsinki Declaration) were approved by research ethics committees of the participating institutions.

laboratory methods
Frozen blood samples were thawed for measurements of serum concentrations of creatinine, holoTC, tHcy, MMA, and vitamin B₁₂. We measured serum holoTC concentrations in the OHAP study at Aarhus University Hospital, Aarhus, Denmark, using an ELISA modified for use on an automated analyzer (13). We measured serum holoTC concentrations in the Banbury B₁₂ study at the Clinical Trial Service Unit using a RIA (AXIS-Shield ASA) that has been shown to have very good agreement with the ELISA assay (13)(15). We measured serum tHcy concentrations on an Abbott IMx automated analyzer by means of a fluorescence polarization immunoassay in OHAP (2) and by gas chromatography-mass spectrometry in the Banbury study (4). We measured serum vitamin B₁₂ concentrations on an ACS Centaur with an automated chemiluminescence system (Bayer A/S), using a competitive protein-binding assay at Aarhus University Hospital in both studies. In the Banbury B₁₂ study, we also assessed vitamin B₁₂ concentrations using a Beckman assay. We measured serum MMA concentrations at the University of Bergen, Bergen, Norway, using stable isotope–dilution capillary gas chromatography-mass spectrometry in both studies (16).

statistical methods
We summarized continuous variables as medians and interquartile ranges. Individuals with extreme increases of vitamin B₁₂ (>1000 pmol/L) or holoTC (>400 pmol/L) or who reported use of vitamin B₁₂ injections were excluded. We used Spearman correlation coefficients to examine linear associations between the different biochemical markers. We defined metabolic vitamin B₁₂ deficiency as definite if serum MMA was >0.75 µmol/L and probable if serum MMA was >0.45 µmol/L. We defined renal function as normal if serum creatinine concentration was <97 µmol/L in women and <124 µmol/L in men. We assessed the performance of specific cutoff points for either test to detect vitamin B₁₂ deficiency using the odds of being affected given a positive result (OAPR), the likelihood ratio, and positive predictive value in addition to the sensitivity and specificity. We used ROC plots to display the sensitivity and specificity of serum concentrations of holoTC and of vitamin B₁₂ for detection of definite and probable vitamin B₁₂ deficiency. The area under the curve (AUC) of the ROC plots for either test to detect vitamin B₁₂ deficiency was estimated in the overall population and in subsets without and with abnormal renal function. Because holoTC was measured using a slightly different method in the OHAP study (2) compared with the Banbury study (4), we carried out a sensitivity analysis to compare holoTC with vitamin B₁₂ separately in the OHAP and Banbury studies. Because vitamin B₁₂ had been measured using both a Centaur (Bayer) method and a Beckman method in the Banbury study, we performed comparisons of holoTC with vitamin B₁₂ using both assays for vitamin B₁₂ in the Banbury study (4).

	Results

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characteristics of the study population
Among the 2559 individuals with data on vitamin status in both studies, 70 who reported current use of vitamin B₁₂ injections were excluded, as were 13 other individuals with extreme values of vitamin B₁₂ (>1000 pmol/L) or holoTC (>400 pmol/L) (indicating likely vitamin B₁₂ treatment, but failure to report it), leaving 2476 untreated individuals for analysis. HoloTC or creatinine concentrations were missing for some individuals; complete data were available on 2403 individuals. The mean (SD) age of all participants was 79.2 (6.2) years, and 41% were men. The main analyses were carried out in all 2403 individuals with complete data and in subsets of 1651 individuals with normal renal function and 752 with abnormal renal function.

distribution of vitamin b₁₂ status
Table 1 shows the distribution of median (interquartile range) for age and vitamin B₁₂, holoTC, tHcy, MMA, and creatinine concentrations in all participants and in subsets with normal or abnormal renal function. As concentrations of vitamin B₁₂ or holoTC in the overall population declined, MMA concentrations increased. Individuals with holoTC concentrations <45 pmol/L or vitamin B₁₂ <200 pmol/L tended to have increased MMA concentrations, but most did not have MMA >0.75 µmol/L. Among individuals with normal renal function, those identified with definite metabolic vitamin B₁₂ deficiency showed highly significant differences in holoTC, vitamin B₁₂, and tHcy concentrations compared with participants without vitamin B₁₂ deficiency, demonstrating the importance of assessing associations before and after stratification for level of renal function (Table 1 ).

Table 2 shows a shift in the percentage with low concentrations of serum vitamin B₁₂ and holoTC with increasing age, with a corresponding shift in the proportion with increased concentrations of MMA with increasing age. Serum holoTC concentrations positively correlated with vitamin B₁₂ (correlation coefficient: r = 0.61, P <0.001) and inversely correlated with MMA (r = –0.44, P <0.001) and tHcy (r = –0.38, P <0.001). In contrast, the associations of vitamin B₁₂ with both metabolites were less extreme, with inverse correlations with MMA (r = –0.34, P <0.001) and tHcy (r = –0.32, P <0.001) in the subset with normal renal function. Serum creatinine concentrations positively correlated with MMA (r = 0.16, P <0.001) and tHcy (r = 0.31, P <0.001) but not with holoTC (r = 0.002, P = 0.92).

comparison of holotc with vitamin b₁₂
There was a tradeoff between the sensitivity and specificity of any particular level of holoTC or vitamin B₁₂ concentrations to detect vitamin B₁₂ deficiency in all participants and in the subsets with different levels of renal function. Among those with normal renal function, the interval of cutoff points for holoTC concentrations was between 32 pmol/L (90th percentile specificity) and 58 pmol/L (90th percentile sensitivity). The corresponding interval for vitamin B₁₂ was between 157 pmol/L (90th percentile specificity) and 272 pmol/L (90th percentile sensitivity). A sensitivity that equaled the specificity was observed for holoTC at 44 pmol/L and for vitamin B₁₂ at 199 pmol/L. The value obtained when the sensitivity equaled the specificity was higher for holoTC (77%) than for vitamin B₁₂ (73%) for definite vitamin B₁₂ deficiency in those with normal renal function. Hence, cutoff points of 45 pmol/L for holoTC and 200 pmol/L for vitamin B₁₂ were used to compare the utility of both tests for diagnosis of vitamin B₁₂ deficiency. Fig. 1A shows a comparison of the ROC plots for holoTC and vitamin B₁₂ to detect individuals with definite metabolic vitamin B₁₂ deficiency in individuals with normal renal function. Fig. 1B shows a comparison of the ROC plots for holoTC and vitamin B₁₂ to detect individuals with probable metabolic vitamin B₁₂ deficiency in individuals with normal renal function. Analysis of ROC curves indicated that holoTC had a greater AUC [0.87 (SE 0.02)] compared with vitamin B₁₂ [0.79 (0.03)] for the diagnosis of definite vitamin B₁₂ deficiency (P = 0.01) in individuals with normal renal function.

View larger version (25K): [in this window] [in a new window]   Figure 1. ROC curves displaying the sensitivity and specificity of holoTC and vitamin B12 for diagnosis of definite vitamin B12 deficiency (MMA >0.75 µmol/L; A) and probable vitamin B12 deficiency (MMA >0.45 µmol/L; B) in individuals with normal renal function (n = 1651).

replication of the findings in independent populations
The AUCs for holoTC and vitamin B₁₂ to detect definite or probable metabolic vitamin B₁₂ deficiency in all participants and in the subset with normal renal function are shown in Table 3 . Using both definitions of either definite or probable vitamin B₁₂ deficiency in each individual population and all participants, holoTC had a modestly superior diagnostic utility for detection of vitamin B₁₂ deficiency compared with conventional vitamin B₁₂ testing. Although there was some attenuation in the AUC for detection of vitamin B₁₂ deficiency in the overall population compared with the subset with normal renal function, the superior diagnostic utility of holoTC compared with vitamin B₁₂ was unaffected by the level of renal function. Among the 752 individuals with renal impairment in both studies, the modestly superior diagnostic utility of holoTC compared with vitamin B₁₂ for detection of definite metabolic vitamin B₁₂ deficiency [AUC (SE): 0.84 (0.03) vs 0.74 (0.03); P = 0.0002] was also observed, with a similar finding for probable metabolic vitamin B₁₂ deficiency [0.77 (0.02) vs 0.71 (0.02); P = 0.001], respectively.

Table 3 also shows comparisons of holoTC with vitamin B₁₂ in the individual studies. Despite holoTC being measured using a slightly different method in the OHAP compared with the Banbury study, the modestly superior diagnostic utility of holoTC compared with vitamin B₁₂ was observed in both studies. In the Banbury study, vitamin B₁₂ was measured using both a Centaur [mean (SD) 246 (93) pmol/L, n = 868] and Beckman method [224 (92) pmol/L], and the AUCs for the 2 vitamin B₁₂ assays to detect metabolic vitamin B₁₂ deficiency were similar (Table 3 ). Hence, irrespective of which assay was used to measure vitamin B₁₂, there was a modestly superior diagnostic utility for holoTC compared with vitamin B₁₂ to detect metabolic vitamin B₁₂ deficiency in the Banbury study, except for the subset with normal renal function using the more extreme cutoff point for metabolic vitamin B₁₂ deficiency, where the difference was not statistically significant (Table 3 ).

utility of population screening for vitamin b₁₂ deficiency
Table 4 compares the utility of holoTC with conventional vitamin B₁₂ testing to detect individuals with definite (MMA >0.75 µmol/L) or probable (MMA >0.45 µmol/L) vitamin B₁₂ deficiency using cutoff points that provided equal sensitivity and specificity for each assay. A cutoff point of 45 pmol/L for holoTC that had an equal sensitivity and specificity of 79% had a positive predictive value of definite vitamin B₁₂ deficiency of 12.5% and an OAPR of 1.14. A cutoff point of 200 pmol/L for vitamin B₁₂ that had an equal sensitivity and specificity of 74% had a positive predictive value of 10.8% and an OAPR of 1.12. As expected, the positive predictive value and the OAPR were higher for detection of metabolic vitamin B₁₂ deficiency than those for definite vitamin B₁₂ deficiency because of the higher prevalence of the outcomes. Nevertheless, use of these cutoff points for both holoTC and vitamin B₁₂ was associated with more false positives than true positives for both definite and probable vitamin B₁₂ deficiency. Additional analysis using holoTC and vitamin B₁₂ simultaneously (using cutoff points of equal sensitivity and specificity for each test) or the ratio of holoTC/vitamin B₁₂ did not decrease the excess proportion of false positives to true positives identified using either test alone (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study involving 2403 randomly selected older people demonstrated a modest superior diagnostic utility of holoTC compared with conventional vitamin B₁₂ testing for the detection of both definite and probable metabolic vitamin B₁₂ deficiency. The superior diagnostic utility was confirmed not only in the overall population (AUC 0.85 vs 0.76; P <0.001) and in those with normal renal function (AUC 0.87 vs 0.79; P <0.001), but also in those with abnormal renal function (AUC 0.85 vs 0.74; P = 0.002). Using cutoff points of equal sensitivity and specificity at 45 pmol/L for holoTC and 200 pmol/L for vitamin B₁₂, holoTC had a better diagnostic accuracy than vitamin B₁₂ (77% vs 73%) for detection of definite metabolic vitamin B₁₂ deficiency in individuals with normal renal function.

The results of this study differ from a population-based study of 607 individuals in North America (12) that reported no difference in the diagnostic utility of holoTC compared with vitamin B₁₂ to detect vitamin B₁₂ deficiency. Our study, involving an older population, detected 141 individuals with definite and 390 with probable vitamin B₁₂ deficiency and hence had greater statistical power to detect modest differences in the AUC for ROC plots of vitamin B₁₂ deficiency than the 37 individuals detected with vitamin B₁₂ deficiency in the North American study (12). The results of the present study confirm the superior diagnostic utility of holoTC compared with vitamin B₁₂ previously reported in highly selected populations, either people with increased MMA concentrations (10) or vegetarians (17)(18).

The superior diagnostic accuracy of holoTC compared with vitamin B₁₂ was confirmed in 2 independent populations in Oxfordshire using slightly different assay methods to measure holoTC concentrations (13)(15). Moreover, irrespective of whether a Beckman or Centaur assay was used to measure vitamin B₁₂ in the Banbury study, there was a modestly superior diagnostic utility for holoTC compared with vitamin B₁₂ to detect metabolic vitamin B₁₂ deficiency (with the exception of detection of definite metabolic vitamin B₁₂ deficiency among the subset with normal renal function). Whereas the nonsignificant difference (P = 0.07) between the diagnostic utility of the 2 assays in the latter subset may have arisen because of random error (chance), an alternative explanation that the superior diagnostic utility of holoTC resulted from limitations of the Centaur vitamin B₁₂ assay cannot be completely excluded.

It had been originally suggested that holoTC may be the earliest metabolic change when an individual enters a negative vitamin B₁₂ balance, and consequently holoTC should have superior diagnostic accuracy compared with conventional vitamin B₁₂ testing for detection of vitamin B₁₂ deficiency (19). The advent of several different methods to measure holoTC provided an opportunity to test these predictions (13)(15). However, some experts have advocated caution in the interpretation of low holoTC test results and about the use of holoTC as a surrogate for a Schilling test (11). Clearly, a low holoTC concentration cannot distinguish general cobalamin insufficiency (deficient intake) from malabsorption (idiopathic or drug-induced) (11). Nevertheless, because holoTC is more sensitive than conventional vitamin B₁₂ testing in response to low doses of oral vitamin B₁₂, use of holoTC before and after oral ingestion of vitamin B₁₂ could still be developed into a test that might be suitable as an alternative to the Schilling test (20).

implications for public health
Impaired vitamin B₁₂ function and decreased vitamin B₁₂ status have been associated with neurological and cognitive impairment (4)(5)(6), but whether these associations are causal is uncertain. Nevertheless, detection of impaired vitamin B₁₂ status and the choice of the optimum test to achieve this may be relevant to prevent and treat neurological dysfunction and prevent cognitive impairment.

In view of the high prevalence of low vitamin B₁₂ status in older people (2)(3)(4) and the uncertainty about the proportion of such individuals who may be put at risk because of folic acid fortification, universal screening to detect vitamin B₁₂ deficiency in the elderly has been advocated (2). If the utility of screening is solely to detect affected individuals with metabolic vitamin B₁₂ deficiency, then the present study suggests that neither test is suitable for this purpose. Clinical vigilance for vitamin B₁₂ deficiency will continue to be important to minimize the risk of disability associated with vitamin B₁₂ deficiency in older people, as will public health and clinical strategies to avoid an excessive intake of folic acid (>1000 µg/day folic acid where the risk of aggravation of neurological function in people with vitamin B₁₂ deficiency is greatest). This study demonstrated that holoTC had a modest superior diagnostic accuracy compared with vitamin B₁₂ for the detection of vitamin B₁₂ deficiency, but neither test could be recommended to screen asymptomatic populations for vitamin B₁₂ deficiency.

	Acknowledgments

 
Grant/funding support: The MRC CFAS program was funded by the Medical Research Council and the Department of Health. Blood collection and analysis was supported by grants from the Medical Research Council, European Union (QLK3-CT-2002-01775), Health Foundation, London (554/1236), Food Standards Agency, Clothworkers Foundation, and the Joan Dawkins Foundation of the British Medical Association. All the analyses were carried out independently of the sources of support.

Financial disclosures: None declared. The Clinical Trial Service Unit has a policy of not accepting honoraria or other payments from the pharmaceutical industry, except for reimbursement of costs for participation in scientific meetings.

Acknowledgements: We are grateful to all the participants in the Oxford Healthy Aging Project and the Banbury B₁₂ project. The MRC CFAS provided administrative support for the main interview data in the OHAP study.

	Footnotes

 
¹ Nonstandard abbreviations: MMA, methylmalonic acid; tHcy, homocysteine; holoTC, holotranscobalamin; OHAP, Oxford Healthy Aging Project; OAPR, odds of being affected given a positive result; and AUC, area under the curve.

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