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Nutrition and Disease

Very Low Oral Doses of Vitamin B-12 Increase Serum Concentrations in Elderly Subjects with Food-Bound Vitamin B-12 Malabsorption¹

² INSERM U 258, Hôpital Paul Brousse, Villejuif, France; ³ Biochimie, hôpital Necker, Paris; ⁴ Biochimie, hôpital Emile Roux, Limeil-Brévannes, France; ⁵ Hématologie Biologique, hôpital Henri Mondor, Créteil, France; ⁶ Agence Générale des Equipements et Produits de Santé, Paris, France; ⁷ Médecine Gériatrique, hôpital Emile Roux, Limeil-Brévannes, France; ⁸ Pharmacie, hôpital Emile Roux, Limeil-Brévannes, France; and ⁹ Santé Publique, hôpital Georges Pompidou, Paris, France

^* To whom correspondence should be addressed. E-mail: jacques.blacher{at}htd.aphp.fr.

	ABSTRACT

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
The BOSSANOVA study, a randomized double-blind trial, was designed to test the ability of very low oral doses of vitamin B-12 to increase the serum vitamin B-12 concentration in elderly subjects with food-bound vitamin B-12 malabsorption, and to determine whether there was a dose response. We also aimed to quantitatively assess the most efficient dose to be added to flour in addition to folic acid (flour cofortification with vitamin B-12 and folic acid). Sixty-seven patients were randomly assigned to 1 of 6 groups receiving various daily oral doses of vitamin B-12 (i.e., 2.5, 5, 10, 20, 40, or 80 µg/d) for 30 d. The dose-response was tested for different biological variables using a mixed model, taking into account the variable's initial value (between-subject effect), a linear log-dose effect, and a linear log (dose x time) interaction, where time was d 15 or d 30. We planned to determine the amount of oral vitamin B-12 that would increase the serum vitamin B-12 concentration by 37 pmol/L (50 ng/L). Significant between-subject effects were found for serum vitamin B-12, plasma homocysteine, and methylmalonic acid concentrations, but a log-dose effect was found only for vitamin B-12 (P < 0.001). The slope of the line tended to be higher (P = 0.07) at d 30 than at d 15. For a mean serum vitamin B-12 increase of 37 pmol/L, a dose of 5.9 (95% CI, 0.9–12.1) µg/d was needed. We concluded that very low oral doses of vitamin B-12 increased serum vitamin B-12 concentrations in elderly subjects with subclinical vitamin B-12 deficiency, following a log-dose pattern. Our results could be beneficial in the design of a public health program for safe flour cofortification with folic acid.

	Introduction

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

More recently, during the 1990s, the concept of food-bound vitamin B-12 malabsorption emerged (2). Approximately 30–40% of patients with subclinical deficiency were suspected to have food-bound vitamin B-12 malabsorption, with normal free vitamin B-12 absorption (2). Food-bound vitamin B-12 malabsorption is a frequent condition in the elderly (2,3). The prevalence of vitamin B-12 deficiency was 12% in 548 free-living elderly Americans, surviving members of the original Framingham Study cohort (3).

Pernicious anemia is usually treated with parenteral vitamin B-12, but food-bound vitamin B-12 malabsorption can be treated with oral vitamin B-12 (4,5). Although high doses were first tested (1000 µg/d), recent reports show that lower doses, ranging from 10 to 1000 µg/d, can be effective (6–11). Although the vitamin B-12 recommended daily allowance (RDA) for adults >51 y is 2.4 µg/d (12), to our knowledge, very low doses (<10 µg/d) have rarely been tested. A recent study tested the effects of 2.5, 100, 250, 500, and 1000 µg oral doses of vitamin B-12 on sensitive markers of vitamin B-12 status (11).

Flour fortification with low doses of folic acid has been performed since 1998 in the US and in Canada on the basis of individual trial results, which showed a reduction in the incidence of neural tube defects (NTD) (13–18). Indeed, a striking decreased incidence of NTD-affected births has been observed over the last 5 y (19–21). However, health effects are difficult to ascertain in the absence of a public health program evaluation (22). The main concern with a high daily consumption of folic acid (>1 mg/d) is a possible masking of the anemia and macrocytosis due to vitamin B-12 deficiency, which consequently, could lead to neurological disease (12,23). Flour fortification with both folic acid and vitamin B-12 has been recommended to avoid this risk (22,24). However, an essential question remains unanswered concerning this combined fortification: namely, which dose of vitamin B-12 should be added to flour?

We therefore designed the BOSSANOVA study (B-12 per OS chez les Sujets Agés carencés par NOn absorption de la Vitamine B-12 Alimentaire) to test the ability of a very low oral dose to increase serum vitamin B-12 in subjects with subclinical deficiency (primary goal) and to assess whether there is a dose response. The final objective was to obtain quantitative data for determining the optimal level for vitamin B-12 flour fortification.

	Methods

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
    Population. The BOSSANOVA study took place between June 2003 and March 2004 in Emile Roux Hospital, Limeil-Brévannes, France. Emile Roux hospital is a geriatric hospital in the suburb of Paris with 884 beds in 4 different departments. All 4 departments participated in the BOSSANOVA study. Every patient entering Emile Roux hospital during the study period was screened for vitamin B-12 status. In subjects with low serum vitamin B-12 (<162 pmol/L), further inclusion criteria were: >70 y of age, absence of fatal disease and life expectancy <1 mo, planned hospitalization duration >1 mo, lack of relevant signs of vitamin B-12 deficiency (i.e., absence of severe cognitive impairment with a Mini Mental Status Examination (MMSE) 23 of 30 points (25); hemoglobin >100 g/L; absence of clinical peripheral neuropathy; absence of clinical signs of subacute combined degeneration of the spinal cord), absence of known vitamin B-12 hypersensitivity, willingness to give a written informed consent to participate in this study, and food-bound vitamin B-12 malabsorption according to Carmel's criteria, excluding the Schilling test (2).

The Carmel criteria includes low serum vitamin B-12 level, normal results of Schilling's test, no dietary vitamin B-12 deficiencies, and at least one of the following conditions: 1) gastric disease, including atrophic gastritis, type A atrophic gastritis, gastric disease associated with Helicobacter Pylori infection, partial gastrectomy, by-pass gastric surgery (obesity), vagotomy, and Zollinger-Ellison syndrome; 2) pancreatic insufficiency resulting from alcohol abuse or cystic fibrosis; 3) gastric or intestinal bacterial overgrowth, including achlorhydria, sprue tropical, Ogilvie syndrome; 4) chronic drug treatment such as ingestion of acid-suppressing drugs (cimetidine, ranitidine, omeprazole), or biguanides (metformine); 5) alcohol abuse; and 6) aging.

Screening for vitamin B-12 deficiency in Emile Roux hospital was performed using the Bayer Diagnostics ACS:180SE cobalamin competition assay with detection by chemiluminescence (26).

The BOSSANOVA study was approved by the Committee for the Protection of Human Subjects in Biomedical Research of Pitié-Salpétrière Hospital, Paris VI University. Written informed consent was obtained from all participants.

    Design. The BOSSANOVA study was a randomized, double-blind trial evaluating the biological effects of daily oral vitamin B-12 supplementation at low dosages for 30 d. We chose 30 d for 3 reasons. First, in the absence of clinical manifestations of vitamin B-12 malabsorption, we considered it ethical to postpone pharmacological supplementation, but not by >1 mo. Second, it would have been difficult to further extend the follow-up period in this frail geriatric population. Finally, several previous studies have already tested vitamin B-12 supplementation for 30 d (6–8). Patients were randomly assigned to 1 of 6 groups of vitamin B-12 (2.5, 5, 10, 20, 40, or 80 µg/d) and treated with this daily oral dose for 30 d. Daily doses were administered to the patients in the morning, after breakfast, under the supervision of medical staff. Randomization was centrally performed by la Délégation Régionale à la Recherche Clinique (AP-HP) de Paris – Ile de France and transmitted by fax to the medical staff (M. R., C. R.). Both participants and medical staff who administered the interventions (M. R., C. R.) and performed the dosages (G. M.) were unaware of group assignment.

The pharmaceutical preparations (all containing 10 mL liquid) containing different amounts of vitamin B-12 were obtained from the dilution of a 1-mg ampoule (Renaudin Pharmaceutics) supplied by Emile Roux Pharmacy. Validation of the pharmaceutical preparations was performed by cyanocobalamin quantitation on 30 samples of 2.5 and 80 µg in 10 mL solutions (6 samples/dosage each day for 5 d) by reversed phase HPLC with spectrophotometric detection. The chromatographic analysis was performed on a Thermo Separation HPLC system, consisting of a P1000 pump and AS1000 auto sampler (Thermo Quest). The HPLC signal processing was performed using the Acquisition system logiciel PC1000. The analytical column used to achieve chromatographic analysis was an Hypersil Elite C₁₈ column (5 µm, 250 mm x 4.6 mm, Thermo Quest). The detection was achieved by spectrometry UV at 363 nm with a UV 6000 detector. The flow rate was fixed at 0.8 mL/min). Results met the defined specifications (theory ± 5%). Pharmaceutical preparations were prepared twice each week.

The nutritional status of subjects was assessed on d 1 by body weight, BMI (kg/m²), and bicipital and tricipital skinfold thicknesses. Nutrient intakes were recorded once weekly with a specific dietary records. Intakes of energy, folate, and vitamin B-12 were calculated by a dietician using the French computerized food composition table (CIQUAL) (27). For each intake, the mean of all dietary records available was calculated and used in the analyses.

Intrinsic factor antibody was measured on d 1. Fasting serum vitamin B-12 and plasma homocysteine were measured on d 1, 15, and 30 to assess the evolution of the serum vitamin B-12 concentration. Blood cell counts, plasma folic acid, and plasma methylmalonic acid (MMA) were measured at d 1 and 30.

    Biological measurements. For biological testing, blood was drawn early in the morning from fasting patients before they took medication. Serum samples obtained at d 1, 15, and 30 were frozen at –20°; vitamin B-12 concentrations were then simultaneously determined using the referent microbiologic and radioisotopic method (28). The between-days CV was 5.8% (n = 48) at 265 pmol/L. The plasma MMA concentration was measured using stable isotope dilution capillary GC-MS (29). Normal values for plasma MMA are <2.25 µmol/L (29); the between-days CV was 6.23% at 1.17 µmol/L The plasma total homocysteine concentration was measured using an immunological method and IMX (Abbott Laboratory) equipment (30). Normal values are <14.6 µmol/L (30); the between-days CV was 3.1% (n = 20) at 12.5 µmol/L. Plasma and erythrocyte folate concentrations were measured by an immunological method and Architect (Abbott Laboratory) equipment (31). Blood cell counts were determined (Argos 3 ABX) and plasma creatinine concentration (Hitachi 911 analyzer with Roche reagents) was measured.

    Statistical analysis. To calculate the number of patients to be included in the BOSSANOVA study, we estimated a dose-response model, using all published studies relating short-term biological effects of low oral doses of vitamin B-12 in food-bound vitamin B-12 malabsorption patients. The model took into account 3 studies (6–8) that tested the effects of different oral dosages (10, 50, 100, 250, 500, and 1000 µg/d for 30 d) on serum vitamin B-12. The dose-response relation between the oral dose of vitamin B-12 and the relative mean increase in serum vitamin B-12 concentration was almost log linear from 10 to 1000 µg. The model showed that serum vitamin B-12 concentrations increased by 11% for each double-dose of vitamin B-12 (Fig. 1). For 6 different doses (2.5, 5, 10, 20, 40, and 80 µg/d), with = 0.05 and with ß = 0.20, 10 subjects were needed for each dosage. Because some randomized subjects could have intrinsic factor antibodies and would therefore be excluded from the final analysis, we included 10% more participants than needed, according to the sample size calculation. Ultimately 67 patients completed the study.

View larger version (13K): [in this window] [in a new window]   Figure 1  Relation between the logarithm of the oral dose of vitamin B-12 (10–1000 µg/d) and the relative serum vitamin B-12 increase (% of the initial concentration), obtained from aggregated published data.

	Results

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
From June 2003 to March 2004, 1277 inpatients were screened for vitamin B-12 status; 190 (15%) had a serum vitamin B-12 concentrations <162 pmol/L. Among these, 89 had vitamin B-12 serum concentrations of 136 ± 18 pmol/L, fulfilled all inclusion criteria, and were randomized. Sixty-seven patients completed the BOSSANOVA study; the 22 remaining randomized subjects who did not complete the study were excluded for one of the following reasons: hospitalization <1 mo (n = 9), transfer to another hospital for urgent surgery or intensive care (n = 7), consent withdrawal (n = 4), or death (n = 2) (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Figure 2  Flow of participants throughout the phases of the BOSSANOVA randomized trial.

At baseline, the plasma homocysteine and MMA concentrations were elevated but the plasma and RBC folate concentrations were within normal ranges (Table 1). Importantly, 76% (51/67) of the study population were folate deficient (<6.8 nmol/L plasma). This was not reflected by the mean, which was within the normal range, because 10 patients had very high levels due to folic acid supplementation.

View larger version (6K): [in this window] [in a new window]   Figure 3  Relation between the serum vitamin B-12 concentration increase at d 30 and the dose of oral vitamin B-12 after 30 d of supplementation in the BOSSANOVA study. Values are means + SEM, n = 67. Dotted lines: for an increase of 37 pmol/L, a log dose of 1.7766 is needed, corresponding to a dose of 5.9 µg/d (95% CI, 0.9–12.1).

	Discussion

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

In this study, only 2 patients exhibited positive intrinsic factor antibody, confirming that in elderly with low serum vitamin B-12 concentrations, food-bound vitamin B-12 malabsorption was more frequent than true pernicious anemia (2). Furthermore, because these 2 patients had significantly increased serum vitamin B-12 levels after oral supplementation, the causes of vitamin B-12 deficiency in these 2 patients are uncertain. It is important to note that the vitamin B-12 assays performed for the screening were not used for analysis. Part of the patients included in the BOSSANOVA study, on the basis of a low screening dosage, had a normal serum vitamin B-12 concentrations at the 2nd determination several days later using a more precise method (28). Therefore, our study population should be described as older patients with low to low-normal serum vitamin B-12 concentrations.

There is undoubtedly an oral alternative to parenteral treatment for vitamin B-12 deficiency, regardless of its etiology (1–11). Our study showed that very low doses of crystalline cyanocobalamin can normalize serum vitamin B-12 levels in the studied population but with substantial variability among subjects at low doses: at d 30, the mean vitamin B-12 level was in the low-normal range (179 ± 61 pmol/L), but 29 patients (43%) still exhibited serum vitamin B-12 <162 pmol/L. However, in the BOSSANOVA study, because the mean serum vitamin B-12 concentration was higher at d 30 than at d 15, and, because a previous study reported an increase in serum vitamin B-12 levels between the 1st and the 4th mo of high-dose oral vitamin B-12 treatment (32), the maximal biological effect was probably not obtained at d 30 in our study population. Indeed, 1 mo of vitamin B-12 supplementation at these low doses did not result in significant decreases in plasma homocysteine and MMA concentrations. The absence of normalization of these 2 biological variables could be related to insufficient doses, insufficient treatment time, or both. Previous studies have shown that different oral vitamin B-12 doses could be associated with different homocysteine and MMA responses. Seal et al. (6) reported that a treatment with oral vitamin B-12 at low doses (10 and 50 µg/d for 1 mo) increased mean serum vitamin B-12, but homocysteine was not significantly decreased. Rajan et al. (33) performed an intervention study in 23 older adults with food-bound vitamin B-12 malabsorption who received sequential daily oral treatment with 25 µg followed by 100 µg and 1000 µg/d crystalline vitamin B-12, each dose for 6 wk. Although serum MMA concentrations were significantly decreased after the first 2 study periods (to a similar extent), a much more dramatic decrease occurred after 6 wk with 1000 µg vitamin B-12/d. Kuzminski et al. (32) performed a randomized trial comparing the effects of oral vs. parenteral vitamin B-12 treatments; the greatest homocysteine and methylmalonic decreases were obtained after 30 d, compared with 2 or 4 mo, because of the very high treatment dose (2000 µg vitamin B-12/d). Finally, in the BOSSANOVA study, the absence of a decrease in plasma homocysteine and MMA was probably related to the low doses. The long-term biological effect of low doses, as would be used in flour fortification, needs further investigation.

To assess from previously published data the dose of vitamin B-12 that should be added to flour to improve low to normal concentrations of serum vitamin B-12, as observed in subjects with protein-bound malabsorption, we evaluated 3 studies (6–8). The first was a randomized trial that assessed the efficacy of oral cyanocobalamin, 10 and 50 µg/d for 1 mo, in patients with serum vitamin B-12 concentrations ranging from 100 to 150 pmol/L (6). The second was an open trial measuring the biological effects of a daily dose of 100 µg of vitamin B-12 in patients with a serum vitamin B-12 concentration <162 pmol/L (7). The third was an open trial assessing the effects of 250, 500, or 1000 µg daily doses of vitamin B-12 in patients with a serum vitamin B-12 concentration <162 pmol/L (8). Using these data, the increment from a low to a normal serum B-12 concentration (e.g., 111 pmol/L to 148 pmol/L) might be achieved with a daily dose of 10 µg of vitamin B-12. In our experiment, the mean required dose was 5.9 µg/d, with a relatively large 95% CI (0.9–12.1 µg/d), which did not exclude the 10 µg/d dose. Bor et al. (34) reported that a daily vitamin B-12 intake of 6 µg appeared to be sufficient to correct all the vitamin B-12-related variables measured in a study of 98 postmenopausal Danish women.

The nutrient intakes measured in this study revealed that our patients had relatively low folate and vitamin B-12 intakes compared with the RDA. Those patients would have then benefitted from a combined flour fortification with both folic acid and vitamin B-12. Folic acid flour fortification is the best way to prevent neural tube defect-affected pregnancies in the population (35). With respect to folic acid flour fortification, one method of preventing the potential adverse outcome of vitamin B-12 deficiency masking would be to combine flour fortification with both folic acid and vitamin B-12 (22,36). In Europe, Hungary is the only country that has started fortification with both vitamins, but results have not yet been published (37). Preliminary results from folic acid fortification in Chile demonstrate an efficient and substantial increase in folate status, without change after 6 mo, in vitamin B-12 levels, both in women of reproductive age (38), and in a sample of elderly subjects with vitamin B-12 deficiency (39). Our results could be used in the design of a public health program for safe flour fortification with folic acid plus vitamin B-12 (40). Further research is needed to determine the feasibility and effects of such a combined fortification (41,42).

	ACKNOWLEDGMENTS

 
The authors sincerely thank Lucie Alche, Christian Aussel, Nicolas Best, and Saliha Djane. The BOSSANOVA study would not have been possible without their contributions.

	FOOTNOTES

 
¹ The BOSSANOVA study was publically funded (Contrat de Recherche Clinique) and managed by la Délégation Régionale à la Recherche Clinique (AP-HP) de Paris, Ile de France.

Manuscript received 23 February 2006. Initial review completed 14 May 2006. Revision accepted 13 November 2006.

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