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Oral versus intramuscular administration of vitamin B12 for vitamin B12 deficiency in primary care: a pragmatic, randomized, noninferiority clinical trial (OB12) ForJournal: peerBMJ Open review only Manuscript ID bmjopen-2019-033687

Article Type: Research

Date Submitted by the 16-Aug-2019 Author:

Complete List of Authors: Sanz Cuesta, Teresa; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Research Unit; Instituto de Salud Carlos III, Health Services Research on Chronic Patients Network (REDISSEC) Escortell Mayor, Esperanza ; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Research Unit; Instituto de Salud Carlos III, Health Services Research on Chronic Patients Network (REDISSEC). Cura-Gonzalez, Isabel ; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Research Unit; Instituto de Salud Carlos III, Health Services Research on Chronic Patients Network (REDISSEC) Martin-Fernandez, Jesus; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Multiprofessional Teaching Unit of Primary and Community Care Oeste; Instituto de Salud Carlos III, Health Services Research on Chronic Patients Network (REDISSEC) Riesgo Fuertes, Rosario; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Multiprofessional Teaching Unit of Primary and Community Care Sur; Instituto de Salud Carlos III, Health Services Research on Chronic Patients Network (REDISSEC) Garrido-Elustondo, Sofía; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Multiprofessional Teaching Unit of Primary and Community Care Sureste; Instituto de Salud Carlos III, Health Services Research on Chronic Patients Network (REDISSEC) Mariño Suárez, Jose Enrique; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre El Greco Álvarez Villalba, Mar; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre María Jesús Hereza; Instituto de Salud Carlos III, Health Services Research on Chronic Patients Network (REDISSEC Gómez Gascón, Tomás; Comunidad de Madrid Servicio Madrileno de Salud, Fundación de Investigación e Innovación Biomédica de Atención Primaria; Instituto de Salud Carlos III, Health Services Research on Chronic Patients Network (REDISSEC) González García, Inmaculada; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Barajas González Escobar, Paloma; Comunidad de Madrid Servicio Madrileno de

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1 2 3 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Buenos 4 Aires 5 Vargas-Machuca Cabañero, Concepción; Comunidad de Madrid Servicio 6 Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare 7 Centre Guayaba 8 Noguerol Álvarez, Mar; Comunidad de Madrid Servicio Madrileno de 9 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Cuzco García de Blas González, Francisca; Comunidad de Madrid Servicio 10 Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare 11 Centre Mendiguchía Carriche; Instituto de Salud Carlos III, Health 12 Services Research on Chronic Patients Network (REDISSEC) 13 Baños Morras, Raquel; Comunidad de Madrid Servicio Madrileno de 14 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Buenos 15 Aires 16 Díaz Laso, Concepción; Comunidad de Madrid Servicio Madrileno de For peerSalud, Gerencia review Asistencial Atención only Primaria. Healthcare Centre 17 Fuentelarreina 18 Caballero Ramírez, Nuria; Comunidad de Madrid Servicio Madrileno de 19 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Juncal 20 Herrero de Dios, Alicia; Comunidad de Madrid Servicio Madrileno de 21 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Miguel 22 de Cervantes Fernández García, Rosa; Comunidad de Madrid Servicio Madrileno de 23 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Santa 24 Isabel 25 Herrero Hernández, Jesús; Comunidad de Madrid Servicio Madrileno de 26 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre 27 Lavapiés 28 Pose García, Belen; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Mendiguchía 29 Carriche 30 Sevillano Palmero, María Luisa; Comunidad de Madrid Servicio Madrileno 31 de Salud, Gerencia Asistencial Atención Primaria. Pharmacy Department 32 Mateo Ruiz, Carmen; Comunidad de Madrid Servicio Madrileno de Salud, 33 Gerencia Asistencial Atención Primaria. Pharmacy Department 34 Medina Bustillo, Beatriz; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Pharmacy Department 35 Aguilar Jiménez, Monica; Comunidad de Madrid Servicio Madrileno de 36 Salud, UICEC Hospital Ramón y Cajal, Plataforma SCReN;Unidad de 37 Farmacología Clínica, Hospital Ramón y Cajal; Instituto Ramón y Cajal de 38 Investigación Sanitaria IRYCIS 39 group, OB12; Comunidad de Madrid Servicio Madrileno de Salud 40 Vitamin B 12 Deficiency, Administration Oral, Equivalence Trial,, Keywords: 41 Injections Intramuscular, primary health care, Patient preference 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 2 of 38

1 2 3 4 5 6 7 8 9 I, the Submitting Author has the right to grant and does grant on behalf of all authors of the Work (as defined 10 in the below author licence), an exclusive licence and/or a non-exclusive licence for contributions from authors 11 who are: i) UK Crown employees; ii) where BMJ has agreed a CC-BY licence shall apply, and/or iii) in accordance 12 with the terms applicable for US Federal Government officers or employees acting as part of their official 13 duties; on a worldwide, perpetual, irrevocable, royalty-free basis to BMJ Publishing Group Ltd (“BMJ”) its 14 licensees and where the relevant Journal is co-owned by BMJ to the co-owners of the Journal, to publish the 15 Work in this journal and any other BMJ products and to exploit all rights, as set out in our licence. 16 17 The Submitting Author accepts and understands that any supply made under these terms is made by BMJ to 18 the Submitting Author Forunless you peer are acting as review an employee on behalf only of your employer or a postgraduate 19 student of an affiliated institution which is paying any applicable article publishing charge (“APC”) for Open 20 Access articles. Where the Submitting Author wishes to make the Work available on an Open Access basis (and 21 intends to pay the relevant APC), the terms of reuse of such Open Access shall be governed by a Creative 22 Commons licence – details of these licences and which Creative Commons licence will apply to this Work are set 23 out in our licence referred to above. 24 25 Other than as permitted in any relevant BMJ Author’s Self Archiving Policies, I confirm this Work has not been 26 accepted for publication elsewhere, is not being considered for publication elsewhere and does not duplicate 27 material already published. I confirm all authors consent to publication of this Work and authorise the granting 28 of this licence. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Oral versus intramuscular administration of vitamin B12 for vitamin B12 deficiency in 4 primary care: a pragmatic, randomized, noninferiority clinical trial (OB12) 5 6 7 8 Author names 9 10 Authors: Teresa Sanz-Cuesta, MD PhD1,2; Esperanza Escortell-Mayor, MD PhD1,2; Isabel del Cura- 11 González, MD PhD 1,2,3; Jesús Martín-Fernández, MD PhD2,3,4; Rosario Riesgo-Fuertes, MD2,5; Sofía 12 Garrido-Elustondo, MD2,6; José Enrique Mariño-Suárez, MD7; Mar Álvarez-Villalba, MD PhD2,8; Tomás 13 Gómez-Gascón, MD PhD2,9; Inmaculada González-García, MD10; Paloma González-Escobar, MD11; 14 Concepción Vargas-Machuca-Cabañero, MD PhD12; Mar Noguerol-Álvarez, MD13; Francisca García-de 15 Blas-González, MD PhD2,14; Raquel Baños-Morras, MD15; Concepción Díaz-Laso, MD PhD16; Nuria 16 For17 peer review18 only 19 17 Caballero-Ramírez, MD ; Alicia Herrero-de Dios, MD ; Rosa Fernández-García, MD ; Jesús Herrero- 18 Hernández, MD20; Belén Pose-García, RN14; María Luisa Sevillano-Palmero, Pharm21; Carmen Mateo- 19 Ruiz, Pharm21; Beatriz Medina-Bustillo, Pharm 21; Mónica Aguilar Jiménez, Pharm22 and OB12 Group23. 20 21 Author affiliations 22 1. Research Unit. Gerencia Asistencial de Atención Primaria (GAAP). Madrid. Spain. 23 2. Health Services Research on Chronic Patients Network (REDISSEC). Instituto Salud Carlos III. Madrid. Spain. 24 3. Preventive Medicine and Public Health Area. Health Sciences Faculty. Universidad Rey Juan Carlos, Alcorcón. 25 Madrid. Spain. 26 4. Multiprofessional Teaching Unit of Primary and Community Care Oeste. GAAP. Madrid. Spain. 27 5. Multiprofessional Teaching Unit of Primary and Community Care Sur. GAAP. Madrid. Spain. 28 6. Multiprofessional Teaching Unit of Primary and Community Care Sureste. GAAP.Madrid. Spain. 29 7. Healthcare Centre El Greco, Getafe. GAAP. Madrid. Spain. 30 8. Healthcare Centre Mª Jesús Hereza, Leganes. GAAP. Madrid. Spain. 31 9. Fundación de Investigación e Innovación Biomédica de Atención Primaria. Madrid. Spain. 32 10. Healthcare Centre Barajas. GAAP. Madrid. Spain. 33 11. Healthcare Centre Buenos Aires. GAAP. Madrid. Spain. 34 12. Healthcare Centre Guayaba. GAAP. Madrid. Spain. 35 13. Healthcare Centre Cuzco. Fuenlabrada. GAAP. Madrid. Spain. 36 14. Healthcare Centre Mendiguchía Carriche. Leganés. GAAP. Madrid, Spain. 37 15. Healthcare Centre Buenos Aires. GAAP. Madrid, Spain. 38 16. Healthcare Centre Fuentelarreina. GAAP. Madrid. Spain. 39 17. Healthcare Centre Juncal. Torrejón de Ardoz. GAAP. Madrid. Spain. 40 18. Healthcare Centre Miguel de Cervantes. Alcalá de Henares. GAAP. Madrid. Spain. 41 19. Healthcare Centre Santa Isabel. Leganés. GAAP. Madrid, Spain. 42 20. Healthcare Centre Lavapiés. GAAP. Madrid. Spain. 21. Pharmacy Department. GAAP. Madrid, Spain. 43 22. UICEC Hospital Ramón y Cajal, Plataforma SCReN; Unidad de Farmacología Clínica, Hospital Ramón y Cajal, 44 Madrid, España; Instituto Ramón y Cajal de Investigación Sanitaria, IRYCIS. 45 23. OB12 Group. 46 47 Corresponding author 48 Isabel del Cura-González 49 Head of the Primary Care Research Unit. Madrid Health Services. Spain 50 Associate professor. Department of Preventive Medicine and Public Health Rey Juan Carlos University 51 REDISSEC. Health Services Research on Chronic Patients Network. ISCIII 52 C/ San Martín de Porres 6, 28035 Madrid. Spain 53 e-mail: [email protected] 54 Phone number: +34913700697 55 56 Word count: 3056 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 38

1 2 3 Abstract 4 5 6 7 8 Objectives: To compare the effectiveness of oral versus intramuscular VB12 in patients aged ≥65 9 10 years with VB12 deficiency. 11 12 Design: Pragmatic, randomized, noninferiority, multicenter trial in patients ≥65 years in 22 13 14 primary healthcare centres in Madrid (Spain). Participants: 283 adults with VB12 deficiency were 15 16 For peer review only 17 randomly assigned to oral (n=140) or intramuscular (n=143) treatment arms. Interventions: The 18 19 intramuscular arm received 1mg VB12 on alternate days in weeks 1–2, 1mg/week in weeks 3–8, 20 21 and 1mg/month in weeks 9–52. The oral arm received 1mg/day in weeks 1–8 and 1 mg/week in 22 23 24 weeks 9–52. 25 26 Main outcomes: Serum VB12 concentration normalization at 8, 26, and 52 weeks. Non-inferiority 27 28 would be declared if the difference between arms is 10% or less. Secondary outcomes 29 30 31 included symptoms, adverse events, adherence to treatment, quality of life, patient preferences and 32 33 satisfaction. 34 35 Results: At week 8, the differences in the percentage of patients who normalized serum VB12 36 37 levels between the oral and intramuscular arm were -0.7% (95% CI: -3.2 to 1.8) analyzed per 38 39 40 protocol (PPT) and 4.8% (95% CI: -1.3 to 10.9) by intention-to-treat (ITT). At week 52, these 41 42 differences were -6.3% (95% CI: -11.9 to -0.1) and -6.8% (95% CI: -16.6 to 2.9), respectively. 43 44 Factors affecting the success rate at week 52 were age, OR=0.95 (95% CI: 0.91 to 0.99), and 45 46 47 having reached VB12 levels ≥281pg/mL at week 8, OR= 8.1 (95% CI: 2.4 to 27.3). Under a 48 49 Bayesian framework, noninferiority probabilities (Δ>-10%) at week 52 were 0.036 (PPT) and 50 51 0.060 (ITT). Quality of life and adverse effects were comparable across groups. 83.4% of patients 52 53 54 preferred the oral route. 55 56 57 58 2 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 5 of 38 BMJ Open

1 2 3 Conclusions: Oral administration was not less effective than intramuscular administration at 8 4 5 6 weeks. Although differences were found between administration routes at week 52, the probability 7 8 that the differences were below the noninferiority threshold was very low. 9 10 Trial registration: ClinicalTrials.gov (NCT 01476007) and EUDRACT (2010-024129-20). 11 12 Funding: Ministerio de Sanidad y Consumo Español. Instituto de Salud Carlos III (ISCIII). 13 14 15 European Regional Development Fund. 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 3 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 38

1 2 3 Strengths and limitations of this study 4 5 6  The present trial is the largest ever designed to compare the effectiveness of oral versus 7 8 intramuscular VB12 in patients aged ≥65 years with VB12 deficiency. It offers the longest 9 10 follow-up period and it is the first to evaluate, in addition to VB12 levels, clinical signs 11 12 13 and symptoms, health-related quality of life, and patient preferences. 14 15  The study design did not allow for masking the patients to the received treatment. However, 16 For peer review only 17 these limitations were compensated for by the objective measurement of the main outcome 18 19 20 variable. 21 22  The loss of patients was low at 8 and 26 weeks and higher at 52 weeks. This effect has 23 24 been observed in long follow-up pragmatic clinical trials. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 4 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 7 of 38 BMJ Open

1 2 3 INTRODUCTION 4 5 6 Vitamin B12 (VB12) is an essential nutrient for the synthesis of cellular DNA. Daily needs in 7 8 adults range from 1 to 2 μg/day. The Western diet is estimated to contain 7–30 μg/day of 9 10 cobalamin, of which 1–5 μg is absorbed and stored (estimated reserves of 2–5 mg), and therefore, 11 12 symptoms resulting from a VB12 deficit would not appear until 3–5 years after establishing a low- 13 14 15 ingestion or poor-absorption regimen.1 VB12 deficiency can lead to hematological and 16 For peer review only 17 neuropsychiatric disorders,2 as well as cardiovascular risk factors.3 The prevalence of VB12 18 19 deficiency in the elderly is highly variable across studies, which report values of 1.5% to 15%.4-7 20 21 22 In primary care, the most commonly observed causes of VB12 deficiency are related to 23 24 abnormalities in digestion or absorption 8 or the consequences of surgical resection.9 A deficiency 25 26 stemming solely from dietary habits is rare and usually affects strict vegans.10 In the elderly, 27 28 29 different alterations in the processes involved in VB12 absorption increase the prevalence of this 30 31 deficit, which can appear in the absence of specific symptoms, thereby hindering its diagnosis. 11 32 33 The traditional treatment for VB12 deficiency consists of intramuscular (IM) injection of 34 35 cyanocobalamin, generally 1 mg/day for one week, followed by 1 mg/week for one month, and 36 37 38 then 1 mg every 1 or 2 months ad perpetuum.9,12,13 The vitamin may, however, be administered 39 40 orally. Several studies have shown serum VB12 concentrations to normalize after taking large oral 41 42 doses.14,15 Studies taking into consideration the patients´ preferences have found differences in 43 44 16,17 45 favor of the oral route. Furthermore, oral treatment could avoid injection nuisances, reduce 46 47 unnecessary travel for the patients or nurses, and minimize treatment costs.18 48 49 Some authors have questioned the use of oral administration while others favor it, although 50 51 52 no firm conclusions can be drawn due to the methodological limitations of the evidence the authors 53 54 provide.9, 19,20,21 The 2018 Cochrane Review4 includes three randomized clinical trials comparing 55 56 57 58 5 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 8 of 38

1 2 3 the effectiveness of oral and IM administration. There are differences among the trials in terms of 4 5 6 treatment regimens and follow-up duration, ranging from 3 to 4 months, and average age of the 7 8 patients, as well as the frequency and VB12 daily dose for both routes. In terms of outcomes, 9 10 adverse events, and cost, the overall quality of the evidence was low due to the small number of 11 12 studies and limited sample sizes.22-24 In their conclusions, the authors state the need for trials with 13 14 15 improved methods for random allocation and masking, larger sample sizes, and information on 16 For peer review only 17 other relevant outcome variables that are preferably conducted in the primary care setting. 18 19 The aim of this study was to compare the effectiveness of oral- and IM-administered VB12 20 21 22 in the normalization of serum VB12 concentrations at 8, 26, and 52 weeks in patients aged ≥65 23 24 years with VB12 deficiency treated at primary healthcare centres (PHC). Secondary outcomes 25 26 included the safety (adverse events), quality of life, and adherence to treatment. Additional aims 27 28 29 were to describe patient preferences and satisfaction with treatment and to explore the immediate 30 31 response (8 weeks) as a normalization predictor of one-year outcomes to propose clinical 32 33 recommendations. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 6 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 9 of 38 BMJ Open

1 2 3 METHODS 4 5 6 Study Design and Participants 7 8 A pragmatic, randomized, multicenter, noninferiority clinical trial with a duration of 12 months 9 10 was conducted in a PHC. On ethical grounds, a placebo-controlled trial was not appropriate.25 11 12 Methodological issues of this trial have been published elsewhere (Supplement 1).26 13 14 15 Competitive recruitment was performed in 22 PHC in Madrid (Spain) from July 2014 to 16 For peer review only 17 November 2016. Eligible patients were 65 years of age or older, had been attending a PHC for 18 19 consultation on any medical matter, and had a serum VB12 concentration of <211 pg/mL. The 20 21 22 last criterion was modified before the start of recruitment due to the requirements of the 23 24 laboratory; the threshold selected in the protocol was <179 pg/mL. Written informed consent was 25 26 obtained from all participants. 27 28 29 Randomization and Masking 30 31 Physicians centrally randomized patients using an electronic online platform by simple 32 33 randomization at a 1:1 ratio. Neither the patients nor the health professionals were blinded. 34 35 Intervention 36 37 38 The pharmaceutical formulations used in the study are commercially available in Spain 39 40 (Optovite® vials). The treatment regimen was : a) IM route: 1 mg of cyanocobalamine on 41 42 alternate days during weeks 1–2, 1 mg/week during weeks 3–8 and 1 mg/month from weeks 9– 43 44 45 52; b) oral route: 1 mg/day of cyanocobalamin for 8 weeks and 1 mg/week from weeks 9–52. 46 47 The period between 1-8 weeks was considered as the charging period. 48 49 50 51 52 Outcomes 53 54 55 56 57 58 7 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 10 of 38

1 2 3 The main outcome was the normalization of serum VB12 concentrations (≥211 pg/mL) at 8, 26, 4 5 6 and 52 weeks. The secondary outcomes were the serum VB12 concentrations (pg/mL), adverse 7 8 events, adherence to treatment (good adherence was considered greater than 80%), quality of life 9 10 (EQ-5D-5L) and patient preferences and satisfaction were assessed. Anamnesis, demographic 11 12 and lifestyle information, clinical variables, analytical variables, and concomitant treatment were 13 14 15 recorded.26 16 For peer review only 17 Procedures 18 19 After signing the consent form, those who agreed to participate had serum VB12 concentrations 20 21 22 determined. If the VB12 value was <211 pg/mL, a hemogram, biochemical analysis, and anti- 23 24 intrinsic factor antibody levels were assessed.26 The patients also received a medication diary to 25 26 be filled out daily. Baseline data were collected by the family physician and/or a nurse. IM 27 28 29 treatments were administered by nurses in the health centres. The follow-up visits were 30 31 conducted during weeks 8, 26 and 52.26 32 33 Statistical Analysis 34 35 Sample size. Assuming that 70% of patients reach a serum VB12 concentration of ≥ 211 pg/mL 36 37 38 in both groups, for a threshold of noninferiority of 10%, statistical power of 60% with 39 40 significance set at p<0.05 and a 5% loss to follow-up, the final sample size was word 41 42 320 (160 in each arm). 43 44 45 As recommended for noninferiority studies, both PPT and ITT analyses were performed, 46 47 with the null hypothesis being a lack of differences between treatments at the three monitoring 48 49 points. Comparing both arms, we calculated the difference between the percentage of patients in 50 51 52 each treatment arm whose serum VB12 concentrations became normalized at 8, 26, and 52 53 54 weeks, with their 95% CI. If the confidence intervals do not fall outside the noninferiority limit 55 56 57 58 8 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 11 of 38 BMJ Open

1 2 3 (10%), it can be concluded that the oral treatment is not inferior to the intramuscular 4 5 27,28 6 treatment. In ITT analyses, missing values for the main outcome variable were added using 7 8 the 'last observation carried forward' (LOCF) method.29 9 10 To explore factors affecting the normalization of serum VB12 concentration at 52 weeks, 11 12 serum VB12 levels were studied at 8 weeks. A receiver operating characteristic (ROC) curve 13 14 15 was built to determine the likelihood ratios of each cutpoint after the charging period to “predict” 16 For peer review only 17 the normalization of levels at the end of the study.3 After this, a generalized linear model (GLM) 18 19 was built (function logit) 30,31. The normalization of serum VB12 levels at 52 weeks was the 20 21 22 dependent variable, and the treatment group was the independent variable. Variables considered 23 24 significant by the researchers from a clinical perspective were included in the model. To test the 25 26 noninferiority hypothesis, adding the information contained in these data to previous knowledge, 27 28 29 additional statistical analyses were performed using a Bayesian approach. Secondary outcome 30 31 variables were analyzed using the appropriate statistical tests, and their means or proportions 32 33 were used to estimate differences between groups. All analyses were performed using STATA 34 35 14 and EPIDAT 4.2 software. 36 37 38 Patient involvement 39 40 Patients were not involved in the development of plans for recruitment, design, outcome measures, 41 42 or implementation of the study conduct. No patients were asked to advise on the interpretation or 43 44 45 writing of the results. Patients have explained the experience of participating in the study on 46 47 the occasion of International Clinical Trial´s day in Radio Nacional de España (RNE). We will 48 49 pursue patient and public involvement in the development of an appropriate method for further 50 51 52 dissemination. 53 54 55 56 57 58 9 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 12 of 38

1 2 3 RESULTS 4 5 6 Characteristics of the Study Participants 7 8 A total of 2342 patients were offered participation and 2152 provided informed consent. A total 9 10 of 307 patients showed a VB12 deficit (14.3%), 283 of whom were allocated to receive VB12 11 12 treatment via the IM route (n=143) or orally (n=140). The follow-up period (52 weeks) was 13 14 15 completed by 229 patients (80.9%) (Figure 1). 16 For peer review only 17 The average age was 75.18 (6.34), and 58.3% of the patients were women. Table 1 18 19 describes the baseline characteristics of the patients included in the trial. No significant 20 21 22 differences were found between groups at baseline for demographic and medical characteristics 23 24 or for the study endpoints. 25 26 27 28 Table 1. Baseline Characteristics at Baseline by Group 29 30 Variable No. (%) 31 Oral route IM route Total 32 (n=140) (n=143) (n=283) 33 34 Sociodemographic data 35 Women 87 (62.1) 78 (54.5) 165 (58.3) 36 Age (years), mean (SD) 74.2 (5.8) 76.2 (6.7) 75.2 (6.3) 37 38 Educational level 39 Illiteracy-Incomplete-Primary education 110 (79.1) 116 (84.7) 226 (81.9) 40 Secondary – Higher education 29 (20.9) 21 (15.3) 50 (18.1) 41 Social occupational class a 42 43 Class I - IV 31 (27.7) 33 (27.3) 64 (27.5) 44 Class V - VI 81 (72.3) 88 (72.7) 169 (72.5) 45 Living alone 32 (21.4) 30 (22.2) 62 (21.9) 46 47 Clinical data 48 Tobacco habit 49 Ex-smoker 27 (19.7) 25 (18.4) 52 (19.0) 50 Smoker 9 (6.6) 10 (7.4) 19 (7.0) 51 52 Nonsmoker 101 (73.7) 101 (74.3) 202 (74.0) 53 Vegetarian 2 (1.4) 0 (0) 2 (0.7) 54 Having undergone gastrectomy 1 (0.7) 2 (1.4) 3 (1.1) 55 Symptoms 56 57 58 10 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 13 of 38 BMJ Open

1 2 3 Paresthesia 33 (23.6) 45 (31.5) 78 (27.6) 4 5 Asthenia 43 (30.7) 54 (37.8) 97 (34.3) 6 Loss of appetite 12 (8.6) 30 (21.0) 42 (14.8) 7 Sadness 37 (26.4) 53 (37.1) 90 (31.8) 8 Showing ≥1 symptoms 70 (50.0) 83 (58.0) 153 (54.1) 9 10 Signs 11 Glossitis 2 (1.4) 9 (6.3) 11 (3.9) 12 Position sensitivity 2 (1.4) 1 (0.7) 3 (1.1) 13 Vibration sensitivity 15 (10.7) 13 (9.1) 28 (9.9) 14 15 Showing ≥1 altered signs 16 (11.4) 21 (14.7) 37 (13.1) 16 Hemogram-ClinicalFor Biochemistry peer review only 17 Vitamin B12 (pg/mL), mean (SD) 173.1 (27.3) 166.4 (32.6) 169.7 (6.3) 18 19 Anemia 16 (11.4) 27 (18.9) 43 (15.2) 20 Hematocrit (%), mean (SD) 42.4 (4.0) 41.9 (4.2) 442.1 (4.1) 21 MCV (fL), mean (SD) 92.1 (6.7) 94.3 (7.4) 93.2 (7.1) 22 Anti-intrinsic factor antibody 15 (11.0) 15 (10.5) 30 (10.8) 23 24 Medication 25 Proton-pump inhibitors 57 (40.7) 64 (44.8) 121 (42.8) 26 Metformin 69 (49.3) 56 (39.2) 125 (44.2) 27 Scales 28 b 29 MMSE , mean (SD) 30.8 (4.6) 30.2 (4.8) 30.5 (4.7) 30 EQ-5D-5L-Utilities, mean (SD) 0.9 (0.1) 0.8 (0.2) 0.8 (0.2) 31 aNeoweberian occupational social class (CSO-SEE12). Gac Sanit. 2013;27(3):263–272. 32 bMini Mental State Examination. Maximum score= 35 points. Normal score= 30–35. Borderline score= 33 24–29 points. Scores < 24 points in patients aged >65 years and scores < 29 points in patients aged <65 34 years suggest cognitive impairment. 35 36 37 Primary Outcomes 38 39 40 At week 8, the difference in the success rate between the oral and IM routes was -0.7% (95%CI: 41 42 -3.2% to 1.8%) and 4.8% (95%CI: -1.3% to 10.9%) with the PPT and ITT analyses, respectively. 43 44 At week 26, these differences were -12.9% (95%CI: -17.9% to -6.1%) and -3.2% (95%CI: - 45 46 47 11.8% to 5.4%), respectively. At week 52, these differences were -6.3% (95%CI: -11.9% to - 48 49 0.07%) and -6.8% (95%CI: -16.6% to 2.9%), respectively (Figure 2). 50 51 In the PPT analysis under a Bayesian approach, the probabilities of differences in the 52 53 treatment effectiveness being >10% between the oral and IM groups were 0.001, 0.201, and 54 55 56 0.036 at weeks 8, 26, and 52, respectively. In the ITT analysis, these values were 0.000, 0.015, 57 58 11 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 14 of 38

1 2 3 and 0.060 at weeks 8, 26, and 52, respectively (Supplement 2). The result of the likelihood ratio 4 5 6 for the cutpoints at the main percentiles of the distribution of VB12 serum levels at week 8 to 7 8 predict normalization at the end of the study is shown in Supplement 3. The level at the 5th 9 10 percentile of the distribution was selected as the most useful value as it showed the best 11 12 classification ability. When patients did not reach this level at week 8, they were almost twelve 13 14 15 times more likely to not reach suitable VB12 levels at the end of the study than if they had 16 For peer review only 17 reached levels over 281 pg/mL (12~1/negative likelihood ratio). 18 19 In the ITT analysis, the factors affecting the success rate at week 52 were age, for each 20 21 22 year of increase in age the success rate decreased 5% and having attained VB12 levels of ≥281 23 24 pg/mL at week 8, which yielded a success rate 8.1 times higher (Table 2). 25 26 27 28 29 30 31 Table 2. Factors Associated with VB12 Concentration ≥ 211 pg/ml at Week 52 32 33 Variable Odds ratio Robust std. error P>z 95% CI 34 35 IM vs. oral route 1.10 0.370 0.776 (0.57 to 2.13) 36 Age 0.95 0.022 0.025 (0.91 to 0.99) 37 VB12 concentration 8.10 5.014 0.001 (2.41 to 27.25) 38 >281 pg/ml at week 8 39 Constant 0.78 0.622 0.755 (0.16 to 3.72) 40 41 GLM, N=265. Variance function: V(u) = u*(1-u/1) [Binomial]. Link function: g(u) = ln(u/(1-u)) 42 [Logit]. AIC= 0.89967. BIC = -1225.89. 43 44 45 46 The mean levels of VB12 for each follow-up visit were above the normalization 47 48 threshold in both groups, although these values were much greater in the IM group (Supplement 49 50 4). In 51 patients (36 IM and 5 oral), the levels of VB12 in week 8 were above the normal range 51 52 53 limit of the laboratory (≥911 pg/mL), so the treatment regimen was changed from the initial 54 55 planned pattern. 56 57 58 12 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 15 of 38 BMJ Open

1 2 3 Secondary Outcomes 4 5 6 In terms of quality of life and the presence of signs related to VB12 deficiency, no significant 7 8 differences were found between treatment arms at any of the follow-up visits (Table 3). 9 10 11 12 13 14 Table 3. Secondary Outcomes (Quality of Life and Exploratory Findings) at Weeks 8, 26 and 52 15 16 Visit Oral routeFor peerIM review route onlyMeans difference 17 18 N Mean (SD) N Mean (SD) (95% CI) 19 Quality of life (EQ-5D-5L Index) 20 Baseline 139 0.855 (0.139) 137 0.817 (0.197) 0.066 (-0.002 to 0.078) 21 Week 8 134 0.853 (0.158) 134 0.822 (0.204) 0.031 (-0.013 to 0.075) 22 Week 26 128 0.853 (0.153) 128 0.826 (0.191) 0.027 (-0.016 to 0.070) 23 Week 52 112 0.824 (0.179) 112 0.823 (0.194) 0.001 (-0.047 to 0.049) 24 At least one altered sign (glossitis and/or altered vibration sensitivity and/or altered position 25 sensitivity) 26 27 Visit N n (%) N n (%) Proportions difference (95% CI) 28 Baseline 140 16 (11.4%) 143 21 (14.7) -3.3 (-11.1% to 4.6) 29 Week 8 135 15 (11.1%) 130 13 (10.0) 1.1 (-6.3% to 8.5) 30 Week 26 131 14 (10.7%) 122 12 (9.8) 0.9 (-6.6% to 8.3) 31 Week 52 122 14 (12.5%) 117 9 (7.7) 3.8 (-3.7% to 11.2) 32 33 34 Eleven adverse events were reported and none of them were severe; five (3.57%) 35 36 occurred with patients in the oral arm and six (4.20%) with patients in the IM arm, yielding a 37 38 difference of -0.63% (95%CI: -5.12% to 3.87%). Three patients withdrew from the study: one 39 40 41 patient in the oral group due to urticaria, and two in the IM group due to reddening and pruritic 42 43 facial erythema and generalized itching (mainly in the cheeks with scarce urticariform lesions). 44 45 In three other cases, treatment for the adverse events was prescribed (constipation and erythema), 46 47 48 and in five cases, it was not necessary to take further measures. 49 50 At week 8, adherence to treatment was evaluated in 265 patients, of whom 95.5% were 51 52 adherent (97.8% oral and 93.8% IM); the difference between the groups was 3.9% (95%CI: -0.1 53 54 55 56 57 58 13 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 16 of 38

1 2 3 to 8.7). At week 52, adherence was evaluated in 229 patients, of whom 220 (96.1%) were 4 5 6 adherent (98.2% oral and 94.0% IM); the difference was 4.2% (95%CI: -0.7 to 9.1). 7 8 Overall, 89.5% of the patients reported being satisfied or very satisfied with the treatment 9 10 via the oral route (91.3%) and the IM route (87.6%). These differences were not statistically 11 12 significant. 13 14 15 83.4% of patients preferred the oral route (97.6% among the patients receiving VB12 16 For peer review only 17 orally vs. 68.6% of the patients in the IM group); the difference was 29.0% (95%CI: 20.3 to 18 19 37.7). 20 21 22 23 24 DISCUSSION 25 26 Main findings of the study 27 28 29 Supplementing VB12 in patients with VB12 deficiency, whether orally or 30 31 intramuscularly, achieves the normalization of VB12 levels in most cases. The oral route was not 32 33 inferior to the IM route during the charging period. Formally, the pre-established conditions for 34 35 determining the noninferiority of oral administration were not met for the complete follow-up 36 37 38 period, but these results merit a deeper analysis. 39 40 Differences between the administration routes were found at 26 and 52 weeks. The IM 41 42 maintenance treatment of 1 mg/month was effective in maintaining VB12 levels, while oral 43 44 45 administration of 1 mg/week had a probability of being inferior (by more than 10%) to the IM 46 47 route by 20% in the most unfavorable scenario (PPT). However, given that no strategy was 48 49 superior in the charging period, and in view of the model results showing that when VB12 levels 50 51 52 reached ≥ 281 pg/mL during the charging period, the success rate at 12 months was 8 times 53 54 higher, the probability of differences between groups to surpass Δ was very low, independent of 55 56 57 58 14 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 17 of 38 BMJ Open

1 2 3 the administration route. The most plausible explanation for the observed difference between 4 5 6 routes might be that in patients below this threshold, the maintenance oral dose should be higher 7 8 than the dose used in the present study. Some authors have recommended that an oral dose of 2 9 10 mg/week be administered as a maintenance dose.32 11 12 The incidence of adverse events was very low and similar for oral and intramuscular 13 14 15 administration, and nonserious adverse events were found. These findings were similar to other 16 For peer review only 17 studies.4 Patients’ preferences can be a decisive factor for determining the administration route. 18 19 In this trial, similar to previous studies,16 there was a clear preference for the oral route, 20 21 22 especially among the patients assigned to this group. 23 24 The effect of VB12 supplements on quality of life remains unclear,33,34 but the presents 25 26 results show that the treatment route does not improve patients´ perception of their health-related 27 28 29 quality of life or related symptoms. 30 31 We did not find significant differences in adherence. However, in usual practice, 32 33 adherence with the oral route could be more compromised than with the IM route, and this factor 34 35 should be taken into consideration to personalize prescription. 36 37 38 Comparison with other studies 39 40 The comparison with other studies is difficult, due to the treatment different doses used, 41 42 but especially because of the follow-up length had been inferior to 4 months and the number of 43 44 45 patients included were small. 46 47 As far as we know, the present trial is the largest clinical trial with the longest follow-up 48 49 period, and it is the first to evaluate, in addition to VB12 levels, clinical signs and symptoms, 50 51 22-24 52 health-related quality of life, and patient preferences. The 3 clinical trials described in the 53 54 2018 Cochrane Systematic Review4 had a duration between 3 and 4 months and included a total 55 56 57 58 15 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 18 of 38

1 2 3 of 153 patients. In the Saraswathy trial, patients in the oral route at 3 months normalised levels 4 5 24 6 20/30 (66.7%) vs 27/30 (90%) of the patients in the IM route. In Kuzminski's patients in the 7 8 oral route at 4 months normalised levels 18/18 (100%) vs 10/14 (71.4%) of the patients in the IM 9 10 route.22 These differences were statistically non-significant in both studies. 11 12 Strengths and limitations 13 14 15 Our study was pragmatic35 in both the inclusion and diagnostic methods criteria. The 16 For peer review only 17 majority of the patients with deficits included in this study did not present any symptomatology 18 19 or very low level symptoms, with no anemia, which is the common profile of most patients who 20 21 22 present with VB12 deficits in primary care. The study design did not allow for masking the 23 24 patients to the received treatment. However, these limitations were compensated for by the 25 26 objective measurement of the main outcome variable. 27 28 29 As occurs in all pragmatic clinical trials, patient recruitment was complicated, and the 30 31 sample size only reached 88.4% of the calculated necessary size, which predicts a loss of power 32 33 of the study. Hence, the analysis was complemented using Bayesian methods that allow for 34 35 studying a posteriori the likelihood of a difference between two outcomes to exceed a certain 36 37 38 limit.36 Under this approach, the a posteriori probability for differences to exceed the proposed 39 40 Δ=-10% was not significant during the charging period, and the probabilities were low but not 41 42 negligible in the PPT analysis and low in the ITT analysis over the complete follow-up period. 43 44 45 The loss of patients was low at 8 and 26 weeks and higher at 52 weeks. This effect has 46 47 been observed in long follow-up pragmatic clinical trials. Missing data were greater in the IM 48 49 arm, during the interval between randomization and initiation of treatment (6% IM vs 1% oral), 50 51 52 over 8 weeks (9% IM vs 4% oral) and over 26 weeks (15% IM vs 6%). These differences could 53 54 represent a lower acceptability of the IM route by patients, since the missing data were mostly 55 56 57 58 16 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 19 of 38 BMJ Open

1 2 3 due to patient abandonment. At 52 weeks, the number of losses in both arms was similar (20% 4 5 6 oral and 18% IM), and in the case of oral treatment, several of those losses corresponded to 7 8 withdrawals for not achieving particular levels of VB12. 9 10 Implications of the study findings 11 12 On the basis of our results and the available evidence, we propose the administration of 13 14 15 VB12 doses of 1 mg/day by the oral route during the charging period. Subsequently, the 16 For peer review only 17 recommended dose would vary as a function of the VB12 levels reached in the charging period. 18 19 For VB12 concentrations between the normal levels of 211 pg/mL (in our laboratory) and 281 20 21 th 22 pg/mL (the 5 percentile of the distribution in this trial), a dose of 2 mg/week is suggested. 23 24 When the levels reached in the charging period are between 281 and 380 pg/mL (the 20th 25 26 percentile of the distribution), it could be appropriate to perform an analysis between 8 and 26 27 28 29 weeks to confirm that normal levels are maintained. All the patients that reach levels of 380 30 31 pg/mL by week 8 could be maintained at the initial doses (1 mg/week) without subsequent 32 33 analyses during the year of follow-up. 34 35 If choosing the IM route, the proposed dose for the IM route during the first weeks may 36 37 38 be excessive for patients with VB12 deficiency. The scheduled IM dose should be reconsidered 39 40 in the first two weeks based on VB12 levels, and in light of this outcome, the scheduled dose 41 42 could be limited to 1 mg/week. Nevertheless, these recommendations must be assessed in further 43 44 45 research. 46 47 Oral administration of VB12 in patients older than 65 years is shown as a strategy that is 48 49 probably as effective as the intramuscular route, without adverse effects and preferred by 50 51 52 patients. We also must highlight the potential benefit of using the oral route in terms of safety for 53 54 patients with coagulant problems, for whom IM-administered medication is relatively 55 56 57 58 17 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 38

1 2 3 contraindicated. A small number of patients may require additional follow-up after the 8 weeks if 4 5 6 certain levels of VB12 in blood are not reached. 7 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 18 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 21 of 38 BMJ Open

1 2 3 4 5 6 Authors’ Contributions 7 8  Trial Management Committee: TSC; EEM; IdCG; JMF; RRF; SGE. 9 10  Technical Support Group: RRB; MRB; JCGM; MMM; PPG; GAC; EPC; SSD; MTRM; 11 12 13 SMI; AAL; MLSP; CMR; BMB; FRS; MGS; RGG; MAMS; MVH; FGBG; JEMS; TGG 14 15 RRG; IBL, MAJ; MAC, ASE, MSO, MAGM participated in different phases of the 16 For peer review only 17 design and the development of the research. 18 19 20  Healthcare Centres (managers)*: study coordination development in each healthcare 21 22 centre with principal investigator supervision. 23 24  Clinical Investigators: collected the data for the study, which included recruiting patients, 25 26 27 consenting, blood testing, applying an intervention, collecting data, and arranging and 28 29 performing follow-up for patients. 30 31  Statistical analysis: TSC; JMF; IDC; EEM; JGM; MMM. 32 33 34  Writing Committee: TSC; EEM; IDC; JMF, SGE RRF writing of the manuscript; MAV; 35 36 RRB, FGB reviewed the manuscript. All authors of the OB12 Group read and approved 37 38 the final manuscript. 39 40 41 Acknowledgments 42 43 To Margarita Blázquez Herranz, Instituto de Salud Carlos III, for making the impossible 44 45 possible. 46 47 To Juan Carlos Abánades Herranz for trusting us and his contribution to facilitate our work. 48 49 50 To Antonio Jesús Alemany López and Marta Sánchez-Celaya, Managing Director of Primary 51 52 Healthcare for accepting to sponsor this research after the dissolution of CAIBER. 53 54 55 56 57 58 19 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 22 of 38

1 2 3 To Fundación para la Investigación e Innovación Biomédica de Atención Primaria (FIIBAP) for 4 5 6 the grant awarded for the publishing of the research. 7 8 To Fernando Cava Valencia and Raquel Guillén Santos from the Clinical Laboratory 9 10 BRSALUD. 11 12 To Elena Tolosa Oropesa from the Pharmacy Department. Gerencia Asistencial de Atención 13 14 15 Primaria de Madrid. 16 For peer review only 17 To our colleagues from the Research Unit, including the assistants Marcial Caboblanco Muñoz, 18 19 Juan Carlos Gil and Isabel Morales Ropero for their unconditional support; researchers Juan A. 20 21 22 López Rodríguez, Jaime Barrio Cortes, Cristina Lozano Hernandez and Medical Residents 23 24 Daniel Toledo Bartolomé, Irene Wijers and Palmira Jurado Macías for their technical advice to 25 26 finalize this paper. 27 28 29 To Javier Zamora Romero, head of the Unit of Biostatistics, Hospital Ramón y Cajal of Madrid , 30 31 for the revision of the manuscript. 32 33 To all the professionals from the participating healthcare centers. 34 35 To all patients who contributed to this research. 36 37 38 39 40 Competing Interests 41 42 The authors declare that they have no competing interests. 43 44 45 46 47 Funding 48 49 The trial was financed by Ministerio de Sanidad y Consumo Español through their call for 50 51 52 independent clinical research, Orden Ministerial SAS/2377, 2010 (EC10-115, EC10-116, EC10- 53 54 117, EC10-119, EC10-122); CAIBER -Spanish Clinical Research Network, Instituto de Salud 55 56 57 58 20 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 38 BMJ Open

1 2 3 Carlos III (ISCIII) (CAI08/010044) and Gerencia Asistencial de Atención Primaria de Madrid. 4 5 6 This study is also supported by the Spanish Clinical Research Network (SCReN), funded by ISCIII- 7 8 Subdirección General de Evaluación y Fomento de la Investigación, project number 9 10 PT13/0002/0007, within the National Research Program I+D+I 2013-2016 and co-funded with 11 12 European Union ERDF funds (European Regional Development Fund). 13 14 15 The funders of the study had no role in the study design, data collection, onsite monitoring, data 16 For peer review only 17 analysis, data interpretation, or writing of the manuscript. 18 19 Ethics approval 20 21 Madrid Region Clinical Research Ethics Committee on February 8th, 2011. 22 23 24 Data sharing statement 25 26 Individual de-identified participant data will be shared at kindly request. This data will include 27 28 every variable used in the showed analysis and they will available for five years under request to 29 30 31 the correspondence author. 32 33 34 35 36 37 OB12 Group 38 Collaborating Investigators 39 40 Clinical Investigators 41 Healthcare Centre (HC) Guayaba: Tomás Gómez-Gascón* Concepción Vargas-Machuca Cabañero; Mª 42 ; Isabel Gutiérrez-Sánchez; Mª Ángeles Fernández-Abad; José Antonio Granados-Garrido; Javier Martínez- 43 Suberviola; Margarita Beltejar-Rodríguez; Carmen Coello-Alarcón; Susana Diez-Arjona. 44 HC El Greco: José Enrique Mariño-Suárez* Ana Ballarín-González; Ignacio Iscar-Valenzuela; José Luis 45 ; 46 Quintana-Gómez; José Antonio González-Posada-Delgado; Enrique Revilla-Pascual; Esther Gómez- Suarez; Yolanda Fernández-Fernández; Fernanda Morales-Ortiz; Isabel Ferrer-Zapata; Esperanza 47 Duralde-Rodríguez; Milagros Beamud-Lagos. 48 HC Barajas: Inmaculada González-García* Mª del Pilar Serrano-Simarro; Cristina Montero-García; María 49 ; 50 Domínguez-Paniagua; Sofía Causín-Serrano; Josefa Mª San Vicente-Rodríguez; Germán Reviriego-Jaén; Mª Margarita Camarero-Shelly; Rosa Mª Gómez-del-Forcallo. 51 52 HC Cuzco: Mar Noguerol-Álvarez*; María Ángeles Miguel-Abanto; Mª Lourdes Reyes-Martínez; Alejandro 53 Rabanal-Basalo; Carolina Torrijos-Bravo; Pilar Gutiérrez-Valentín; Jorge Gómez-Ciriano; Susana Parra 54 Román; Carolina Torrijos-Bravo; Judit León-González; Mª José Nebril-Manzaneque; Juana Caro-Berzal. 55 56 57 58 21 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 24 of 38

1 2 3 HC Mendiguchía Carriche: Francisca García-de Blas-González*; Belén Pose-García; Alberto López- 4 García-Franco; Mª Mar Álvarez-Villalba; Sonia Redondo-de-Pedro; Juan Carlos García-Álvarez; Elisa 5 Viñuela-Beneitez; Marisa López-Martín; Nuria Sanz-López. 6 HC Buenos Aires: Paloma González-Escobar*; Raquel Baños-Morras; Ana María Ibarra-Sánchez; Cecilio 7 Gómez-Almodóvar; Javier Muñoz-Gutiérrez; Carmen Molins-Santos; Cristina Cassinello-Espinosa. 8 HC Presentación Sabio Antonio Molina-Siguero* Rafael Sáez-Jiménez; Paloma Rodríguez-Almagro; 9 : ; Eva María Rey-Camacho; María Carmen Pérez-García. 10 11 HC Santa Isabel: Rosa Fernández-García*; Antonio Redondo-Horcajo; Beatriz Pajuelo-Márquez; 12 Encarnación Cidoncha-Calderón; Mª Jesús Galindo Rubio; Rosa Ana Escriva Ferrairo; José Francisco 13 Ávila-Tomas; Francisco De-Alba-Gómez; Mª Jesús Gómez-Martín; Alma María Fernández-Martínez. 14 HC Fuentelarreina: Concepción Díaz-Laso*; Rosa Feijoó-Fernández; José Vizcaíno-Sánchez-Rodrigo; 15 Victoria Díaz-Puente; Felisa Núñez-Sáez; Luisa Asensio-Ruiz; Agustín Sánchez-Sánchez; Orlando 16 Enríquez-Dueñas; SilviaFor Fidel-Jaimez; peer Rafael review Ruiz-Morote-Aragón; only Asunción Pacheco-Pascua; Belén 17 Soriano-Hernández; Eva Álvarez-Carranza; Carmen Siguero-Pérez. 18 HC Juncal: Nuria Caballero-Ramírez*; Ana Morán-Escudero; María Martín-Martín; Francisco Vivas-Rubio. 19 HC Miguel de Cervantes: Alicia Herrero-de-Dios*; Rafael Pérez-Quero; Mª Isabel Manzano-Martín; César 20 Redondo-Luciáñez. 21 HC San Martín de Valdeiglesias: Nuria Tomás-García*; Carlos Díaz-Gómez-Calcerrada; Julia Isabel 22 Mogollo-García; Inés Melero-Redondo; Ricardo González-Gascón. 23 HC Lavapiés: Jesús Herrero-Hernández*; María Carmen Álvarez-Orviz; María Veredas González- 24 Márquez; Teresa San Clemente-Pastor; Amparo Corral-Rubio. 25 HC General Ricardos: Asunción Prieto-Orzanco*; Cristina de la Cámara-Gonzalez; Mª Mercedes Parrilla- 26 Laso; Mercedes Canellas-Manrique; Maria Eloisa Rogero-Blanco 27 Paulino Cubero-González; Sara Sanchez-Barreiro; Mª Ángeles Aragoneses-Cañas; Ángela Auñón-Muelas; 28 Olga Álvarez-Montes 29 HC María Jesús Hereza: Mar Álvarez-Villalba*; Petra María Cortes-Duran; Pilar Tardaguila-Lobato; Mar 30 Escobar-Gallegos; Antonia Pérez-de-Colosia-Zuil; Jaime Inneraraty-Martínez; María Jesús Bedoya-Frutos; 31 María Teresa López-López; Nelly Álvarez-Fernández; Teresa Fontova-Cemeli; Josefa Marruedo-Mateo; 32 Josefa Díaz-Serrano; Beatriz Pérez-Vallejo. 33 HC Reyes Magos: Pilar Hombrados-Gonzalo*; Marta Quintanilla-Santamaría; Yolanda González-Pascual; 34 Luisa María Andrés-Arreaza; Soledad Escolar-Llamazares; Cristina Casado-Rodríguez; Luz Mª del Rey- 35 Moya; Mª Jesús Fernández-Valderrama; Alejandro Medrán-López; Julia Alonso-Arcas. 36 HC Barrio del Pilar: Alejandra Rabanal-Carrera*; Araceli Garrido-Barral; Milagros Velázquez-García; 37 Azucena Sáez-Berlanga; Mª Pilar Pérez-Egea; Rosario del Álamo-Gutiérrez; Pablo Astorga-Díaz; Carlos 38 Casanova-García; Ana Isabel Román-Ruiz; Mª Carmen Belinchón-Moya; Margarita Encinas-Sotillo; 39 Virtudes Enguita-Pérez. 40 HC Los Yébenes: Ester Valdés-Cruz*; Consuelo Mayoral-López; Alejandro Rabanal-Basalo; Teresa Gijón- 41 Seco; Francisca Martínez-Vallejo; Jesica Colorado-Valera. 42 HC María Ángeles López Gómez: Ana Sosa-Alonso*; Jeannet Sánchez-Yépez*; Dolores Serrano- 43 González; Beatriz López-Serrano; Inmaculada Santamaría-López; Paloma Morso-Peláez; Carolina López- 44 Olmeda; Almudena García-Uceda-Sevilla; Petra María Cortés-Durán; Mercedes del Pilar Fernández-Girón. 45 HC Arroyo de la Media Legua: Leonor González-Galán*; Mariano Rivera-Moreno; Luis Nistal Martín-de- 46 Serranos; Mª Jesús López-Barroso; Margarita Torres-Parras; María Verdugo-Rosado; Mª Reyes Delgado- 47 Pulpón; Elena Alcalá-Llorente 48 . 49 HC Federica Montseny: Sonsoles Muñoz-Moreno*; Isabel Vaquero-Turiño; Ana María Sánchez-Sempere; 50 Francisco Javier Martínez-Sanz; Clementa Sanz-Sanchez; Ana María Arias-Esteso. 51 HC Calesas: Diego Martín-Acicoya*; Pilar Kloppe-Villegas; Francisco Javier San-Andrés-Rebollo; 52 Magdalena Canals-Aracil; Isabel García-Amor; Nieves Calvo-Arrabal; María Milagros Jimeno-Galán. 53 HC Manuel Merino: Gloria de la Sierra-Ocaña*; María Mercedes Araujo-Calvo. 54 HC Doctor Cirajas: Julia Timoner-Aguilera*; María Santos Santander-Gutiérrez; Alicia Mateo-Madurga. 55 56 Technical Support Group 57 58 22 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 25 of 38 BMJ Open

1 2 3 Research Unit: Ricardo Rodríguez-Barrientos; Milagros Rico-Blázquez; Juan Carlos Gil-Moreno; Mariel 4 Morey-Montalvo; Pilar Pamplona-Gardeta. 5 Multiprofessional Teaching Units of Primary and Community Care: Gloria Ariza-Cardiel; Elena Polentinos- 6 Castro; Sonia Soto-Díaz; Mª Teresa Rodríguez-Monje. 7 Dirección Asistencial Sur: Susana Martín-Iglesias. 8 HC Pintores: Amaya Azcoaga-Lorenzo. 9 Pharmacy Department: María Luisa Sevillano-Palmero, Carmen Mateo-Ruiz, Beatriz Medina-Bustillo. 10 Agencia Pedro Laín Entralgo: Francisco Rodríguez-Salvanés; Marta García-Solano; Rocío González- 11 González; María Ángeles Martín-de la Sierra-San Agustín; María Vicente Herrero. 12 Hematology Department (Severo Ochoa): Ramón Rodríguez-González. 13 Endocrinology Department (HGCM): Irene Bretón-Lesmes. UICEC Hospital Ramón y Cajal, Plataforma 14 SCReN; Unidad de Farmacología Clínica, Hospital Ramón y Cajal, Madrid, España; Instituto Ramón y Cajal 15 de Investigación Sanitaria, IRYCIS: Mónica Aguilar Jiménez, Marta del Alamo Camuñas, Anabel Sánchez 16 Espadas, Marisa SerranoFor Olmeda peer y Mª Angeles review Gálvez Múgica. only 17 18 19 20 Principal Investigator: Teresa Sanz-Cuesta; Esperanza Escortell-Mayor; Isabel del Cura-González; Jesús 21 Martín-Fernández; Rosario Riesgo-Fuertes; Sofía Garrido-Elustondo. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 23 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 26 of 38

1 2 3 4 5 6 References 7 8 1. Hunt A, Harrington D, Robinson S. Vitamin B12 deficiency. BMJ. 2014;349:g5226. 9 10 2. Dharmarajan TS, Norkus EP. Approaches to vitamin B12 deficiency. Early treatment 11 12 may prevent devastating complications. Postgrad Med. 2001;110(1):99-105. 13 14 3. Pawlak R. Is vitamin B12 deficiency a risk factor for cardiovascular disease in 15 16 For peer review only 17 vegetarians? Am J Prev Med. 2015;48(6):e11-e26. 18 19 4. Wang H, Li L, Qin LL, Song Y, Vidal-Alaball J, Liu TH. Oral vitamin B12 versus 20 21 intramuscular vitamin B12 for vitamin B12 deficiency. Cochrane Database Syst Rev. 22 23 24 2018;3:CD004655. 25 26 5. Closas RG, Majem LS, Sales GS, Castellvell MO, Barba LR, Sanmartí LS. Distribución 27 28 de la concentración sérica de vitamina C, ácido fólico y vitamina B12 en una muestra 29 30 31 representativa de la población adulta de Cataluña. Med Clín. 2002;118(4):135-141. 32 33 6. Henriquez P, Doreste J, Deulofeu R, Fiuza MD, Serra-Majem L. Nutritional determinants 34 35 of plasma total homocysteine distribution in the Canary Islands. Eur J Clin Nutr. 36 37 2007;61(1):111-118. 38 39 40 7. Lindenbaum J, Rosenberg IH, Wilson PW, Stabler SP, Allen RH. Prevalence of 41 42 cobalamin deficiency in the Framingham elderly population. Am J Clin Nutr. 43 44 1994;60(1):2-11. 45 46 47 8. Niafar M, Hai F, Porhomayon J, Nader ND. The role of metformin on vitamin B12 48 49 deficiency: a meta-analysis review. Intern Emerg Med. 2015;10(1):93-102. 50 51 9. Andres E, Dali-Youcef N, Vogel T, Serraj K, Zimmer J. Oral cobalamin (vitamin B(12)) 52 53 54 treatment. An update. Int J Lab Hematol. 2009;31(1):1-8. 55 56 57 58 24 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 27 of 38 BMJ Open

1 2 3 10. Watanabe F, Yabuta Y, Tanioka Y, Bito T. Biologically active vitamin B12 compounds 4 5 6 in foods for preventing deficiency among vegetarians and elderly subjects. J Agric Food 7 8 Chem. 2013;61(28):6769-6775. 9 10 11. Langan RC, Zawistoski KJ. Update on vitamin B12 deficiency. Am Fam Physician. 11 12 2011;83(12):1425-1430. 13 14 15 12. Dali-Youcef N, Andres E. An update on cobalamin deficiency in adults. QJM. 16 For peer review only 17 2009;102(1):17-28. 18 19 13. Federici L, Loukili NH, Zimmer J, Affenberger S, Maloisel F, Andrès E. Manifestations 20 21 22 hématologiques de la carence en vitamine B12: données personnelles et revue 23 24 de la littérature. Rev Med Int. 2007;28(4):225-231. 25 26 14. Nilsson M, Norberg B, Hultdin J, Sandström H, Westman G, Lökk J. Medical 27 28 29 intelligence in Sweden. Vitamin B12: oral compared with parenteral? Postgrad Med J. 30 31 2005;81(953):191-193. 32 33 15. Vidal-Alaball J, Butler CC, Cannings-John R, et al. Oral vitamin B12 versus 34 35 intramuscular vitamin B12 for vitamin B12 deficiency. Cochrane Database Syst Rev. 36 37 38 2005(3):CD004655. 39 40 16. Metaxas C, Mathis D, Jeger C, Hersberger KE, Arnet I, Walter P. Early biomarker 41 42 response and patient preferences to oral and intramuscular vitamin B12 substitution in 43 44 45 primary care: a randomised parallel-group trial. Swiss Med Wkly. 2017;147(1314):1-9. 46 47 17. Kwong JC, Carr D, Dhalla IA, Tom-Kun D, Upshur RE. Oral vitamin B12 therapy in the 48 49 primary care setting: a qualitative and quantitative study of patient perspectives. BMC 50 51 52 Fam Pract. 2005;6(1):8. 53 54 55 56 57 58 25 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 28 of 38

1 2 3 18. Houle SK, Kolber MR, Chuck AW. Should vitamin B12 tablets be included in more 4 5 6 Canadian drug formularies? An economic model of the cost-saving potential from 7 8 increased utilisation of oral versus intramuscular vitamin B12 maintenance therapy for 9 10 Alberta seniors. BMJ Open. 2014;4(5):e004501. 11 12 19. Graham ID, Jette N, Tetroe J, Robinson N, Milne S, Mitchell SL. Oral cobalamin remains 13 14 15 medicine's best kept secret. Arch Gerontol Geriatr. 2007;44(1):49-59. 16 For peer review only 17 20. Shatsky M. Evidence for the use of intramuscular injections in outpatient practice. Am 18 19 Fam Physician. 2009;79(4):297-300. 20 21 22 21. Rey RR, Secades RM, Zemsch MP, Alvaredo MB, Gigirey AG. ¿Debemos utilizar la vía 23 24 oral como primera opción para el tratamiento del déficit de vitamina B12? Rev Clín 25 26 Español. 2007;207(4):179-182. 27 28 29 22. Kuzminski AM, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J. Effective treatment 30 31 of cobalamin deficiency with oral cobalamin. Blood. 1998;92(4):1191-1198. 32 33 23. Bolaman Z, Kadikoylu G, Yukselen V, Yavasoglu I, Barutca S, Senturk T. Oral versus 34 35 intramuscular cobalamin treatment in megaloblastic anemia: a single-center, prospective, 36 37 38 randomized, open-label study. Clin Ther. 2003;25(12):3124-3134. 39 40 24. Saraswathy AR, Dutta A, Simon EG, Chacko A. Randomized open label trial comparing 41 42 efficacy of oral versus intramuscular vitamin b12 supplementation for treatment of 43 44 45 vitamin b12 deficiency. Gastroenterology. 2012:142(1):S-216. 46 47 25. D'Agostino RB, Massaro JM, Sullivan LM. Non-inferiority trials: design concepts and 48 49 issues - the encounters of academic consultants in statistics. Stat Med. 2003;22(2):169- 50 51 52 186. 53 54 55 56 57 58 26 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 29 of 38 BMJ Open

1 2 3 26. Sanz-Cuesta T, Gonzalez-Escobar P, Riesgo-Fuertes R, et al. Oral versus intramuscular 4 5 6 administration of vitamin B12 for the treatment of patients with vitamin B12 deficiency: 7 8 a pragmatic, randomised, multicentre, non-inferiority clinical trial undertaken in the 9 10 primary healthcare setting (Project OB12). BMC Public Health. 2012;12:394. 11 12 27. Piaggio G, Elbourne DR, Pocock SJ, Evans SJ, Altman DG. Reporting of noninferiority 13 14 15 and equivalence randomized trials: extension of the CONSORT 2010 statement. JAMA. 16 For peer review only 17 2012;308(24):2594-2604. 18 19 28. Jones B, Jarvis P, Lewis JA, Ebbutt AF. Trials to assess equivalence: the importance of 20 21 22 rigorous methods. BMJ. 1996;313(7048):36-39. 23 24 29. Jakobsen JC, Gluud C, Wetterslev J, Winkel P. When and how should multiple 25 26 imputation be used for handling missing data in randomised clinical trials - a practical 27 28 29 guide with flowcharts. BMC Med Res Methodol. 2017;17(1):162. 30 31 30. Blough DK, Madden CW, Hornbrook MC. Modeling risk using generalized linear 32 33 models. J Health Econ. 1999;18(2):153-171. 34 35 31. Hardin JW, Hilbe JM. Generalized Linear Models and Extensions. 3rd ed. Texas: Taylor 36 37 38 & Francis; 2012. 39 40 32. Andres E, Vidal-Alaball J, Federici L, Loukili NH, Zimmer J, Kaltenbach G. Clinical 41 42 aspects of cobalamin deficiency in elderly patients. Epidemiology, causes, clinical 43 44 45 manifestations, and treatment with special focus on oral cobalamin therapy. Eur J Intern 46 47 Med. 2007;18(6):456-462. 48 49 33. van Uffelen JG, Chin APMJ, Hopman-Rock M, van Mechelen W. The effect of walking 50 51 52 and vitamin B supplementation on quality of life in community-dwelling adults with mild 53 54 55 56 57 58 27 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 30 of 38

1 2 3 cognitive impairment: a randomized, controlled trial. Qual Life Res. 2007;16(7):1137- 4 5 6 1146. 7 8 34. Hvas AM, Juul S, Nexo E, Ellegaard J. Vitamin B-12 treatment has limited effect on 9 10 health-related quality of life among individuals with elevated plasma methylmalonic acid: 11 12 a randomized placebo-controlled study. J Intern Med. 2003;253(2):146-152. 13 14 15 35. Loudon K, Treweek S, Sullivan F, Donnan P, Thorpe KE, Zwarenstein M. The PRECIS- 16 For peer review only 17 2 tool: designing trials that are fit for purpose. BMJ. 2015;350:h2147. 18 19 36. Bayarri MJ, Berger JO. The interplay of bayesian and frequentist analysis. Stat Sci. 20 21 22 2004;19(1):58-80. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 28 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 31 of 38 BMJ Open

1 2 3 Figure legends 4 5 6 Figure 1. Trial profile 7 8 Figure 2. Differences in proportions of patients recovering levels of VB12 (≥211 pg/ml) 9 10 between oral and intramuscular routes 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 29 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 2342BMJ assessed Open for eligibility Page 32 of 38

190 declined to participate • 44 lack of time 1 • 40 mistrust 2 • 35 declined to sign • 33 fear 3 • 21 not interested 4 • 17 others 5 6 2152 signed informed consent 7 8 171 declined to undergo blood test 9 1674 patients without B12 deficiency

10 Recruitment 11 For peer review only 12 307 VB12 deficiency 13 14 24 excluded: • 7 receiving anticoagulation treatment 15 • 4 receiving vitamin VB12 16 • 3 dropout • 2 folic ac. concentration < 2.3 ng/ml 17 • 1 severe disease 18 • 7 others 19 20 283 randomized 21 22 23 140 allocated to oral route: 143 allocated to IM route: • 139 received oral route • 135 received IM route 24 • 1 lost to follow-up • 6 lost to follow-up 25 • 1 withdrawn for disease Allocation 26 • 1 other 27 28 274 st 29 1 visit: start treatment 30 31 3 withdrawn for disease 1 withdrawn for adverse events 1 lost to follow-up 4 lost to follow-up 32 33 265 34 2nd visit: week 8 35 36 2 withdrawn for not recovering levels 1 withdrawn for not recovering levels 37 1 lost to follow-up 1 withdrawn for disease 38 6 lost to follow-up Follow-up 39 40 254 41 3rd Visit: week 26 42 1 withdrawn for disease 43 3 withdrawn for disease 17 withdrawn for not recovering levels 2 lost to follow-up 44 2 lost to follow-up 45 46 229 47 4th visit: week 52 48 49 112 analyzed Forper protocol peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml117 analyzed per protocol 50 • 28 excluded from analysis • 26 excluded from analysis 51 Analysis 140 analyzed by intention-to-treat 143 analyzed by intention-to-treat 52 53 54 55 56 57 58 59 60 Page 33 of 38 BMJ Open

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 174x93mm (600 x 600 DPI) 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 34 of 38

1 2 3 Supplement 2: Bayesian Analysis 4 5 6 7 Bayesian analysis is a highly appropriate analysis strategy when working with small 8 sample sizes. Previous knowledge about the studied item can be taken advantage of by means of 9 the assessment of the plausibility of a given hypothesis after incorporating the new observed data.1 10 The noninferiority hypothesis, formally Δ < -10%, was tested, taking into account the 11 2 12 observed results but also taking into account the results of the trials by Kuzminski et al. and 13 Saraswathy et al.3 14 P1 denotes the percentage of patients who responded to VB12 oral administration, and 15 P0 represents the percentage of those responding to VB12 intramuscular administration. Bayesian 16 analysis allows for calculating the probability of P1 being equal to or smaller than P0 by a 17 18 specified magnitude,For the noninferiority peer limit review (Δ < -10%). For only each of the parameters P1 and P0, 19 both measured at 8, 26 and 52 weeks, we selected a priori distributions from the family of beta 20 distributions with parameters a and b, which are related to the proportions of those responding in 21 each trial arm. The gamma distribution represents the a priori hypothesis of the distribution of 22 2 3 23 differences. According to the results of both trials by Kuzminski et al. and Saraswathy et al., 4 24 included in the review by Wang et al., 79.1% and 84.1% of patients normalized their VB12 levels 25 in the oral and IM treatment groups, respectively.4 The respective CIs associated with these prior 26 data were calculated, and parameters were chosen (a and b in the beta distribution) such that the 27 maximum density intervals of these distributions approximately coincided with the CI previously 28 29 obtained (see Figure 1). Beta distributions for the success rate in each arm of the trial were 30 obtained using binomial data. A total of 10000 simulations were made from these a posteriori 31 distributions, and the corresponding differences, P1-P0, were calculated yielding an a posteriori 32 distribution of differences. This distribution was used to derive simulation-based estimates of the 33 probability of relevant magnitudes concerning Δ: P1-P0>0.10 at weeks 8, 26, and 52. Both PPT 34 35 and ITT analyses were performed. EPIDAT 4.2 software was used for all computations. 36 Table 1 shows the a posteriori probability of differences in treatment effectiveness 37 between oral and IM routes at different weeks (8, 26 and 52). The probabilities of the differences 38 in treatment effectiveness being >10% between the oral and IM groups were 0.001, 0.201, and 39 0.036 at weeks 8, 26, and 52, respectively (per protocol analysis). In the intention-to-treat (ITT) 40 41 analysis, these values were 0.000, 0.015, and 0.060 at weeks 8, 26, and 52, respectively. 42 43 44 45 References 46 47 1. Berger JO, Bayarri MJ. The interplay of bayesian and frequentist analysis. Stat Sci. 48 2004;19(1):58-80. 49 2. Kuzminskia M, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J. Effective treatment of 50 cobalamin deficiency with oral cobalamin. Blood. 1998;92(4):1191-1198. 51 3. Saraswathy AR, Dutta A, Simon EG, Chacko A. Randomized open label trial comparing 52 efficacy of oral versus intramuscular vitamin b12 supplementation for treatment of vitamin b12 53 deficiency. Gastroenterology. 2012:142(1):S-216. 4. Wang H, Li L, Qin LL, Song Y, Vidal-Alaball J, Liu TH. Oral vitamin B12 versus intramuscular 54 vitamin B12 for vitamin B12 deficiency. Cochrane Database Syst Rev. 2018;3:CD004655. 55 56 57 58 59 60

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1 2 3 Figure 1. A priori distributions of the differences between oral and intramuscular treatment 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For peer review only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Table 1. A posteriori probability of differences in treatment effectiveness between 37 oral and IM routes at 8, 26, and 52 weeks. 38 39 A posteriori probability (Δ < -10%) Week 8 Week 26 Week 52 40 41 42 Per-protocol analysis 0.001 0.201 0.036 43 44 Intention-to-treat analysis 0.000 0.015 0.060 45 46 47 Δ: threshold of non-inferiority 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Supplement 3: Receiver Operating Characteristic (ROC) Curve 4 5 6 7 To explore factors affecting the normalization of serum VB12 concentration (yes/no) at 52 weeks, 8 serum VB12 levels were studied at 8 weeks (at the end of the “charging period”). An ROC curve was 9 built to determine the likelihood ratios for each cutpoint after the charging period to “predict” the 10 normalization of levels (serum VB12 levels ≥211 pg/mL) at the end of the study.1 11 12 13 Table 1 shows the results of the likelihood ratios for the cutpoints at the main percentiles of the 14 distribution of VB12 serum levels at week 8 (“charging period”) to predict normalized VB12 serum levels 15 at the end of the study. In Figure 1, the ROC curve is plotted. The level at the 5th percentile of the 16 distribution was selectedFor as the mostpeer useful value review as it showed best only classification ability and because 17 18 when patients did not reach this level at week 8, they were almost twelve times more likely to not reach 19 suitable VB12 levels at the end of the study than if they did reach levels over 281 pg at week 8 20 (12~1/negative likelihood ratio). 21 22 23 References 24 25 1. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) 26 curve. Radiology. 1982;143(1):29-36. 27 28 29 30 31 32 33 Table 1. Exploring the value of several cutpoints of OB12 serum levels at week 8 to “predict” 34 normalization of values of Vit B12 at the end of the study 35 36 Correctly 37 Cutpoint Sensitivity Specificity LR+ LR- Percentil 38 Classified 39 ≥ 281 0.977 0.273 94.30% 1.3435 0.0841 5 40 ≥ 328 0.963 0.546 94.30% 2.1193 0.0673 10 41 42 ≥ 353 0.931 0.636 91.70% 2.5608 0.1081 15 43 ≥ 389 0.895 0.818 89.10% 4.9197 0.129 20 44 ≥ 421 0.839 0.818 83.80% 4.617 0.1962 25 45 LR+: Positive Likelihood ratio. LR-: Negative Likelihood ratio 46 47 48 49 50 51 52 53 54 55 56 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 37 of 38 BMJ Open

1 2 3 Figure 1. ROC curve 4 5 6 7 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 2 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 38 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 452x329mm (72 x 72 DPI) 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 39 of 38 BMJ Open CONSORT Statement 2006 - Checklist for Non-inferiority and Equivalence Trials 1 2 Items to include when reporting a non-inferiority or equivalence randomized trial 3 4 PAPER SECTION Item Descriptor Reported on 5 And topic Page # 6 TITLE & 1 How participants were allocated to interventions (e.g., "random Page 1 and 2 ABSTRACT allocation", "randomized", or "randomly assigned"), 7 specifying that the trial is a non-inferiority or equivalence trial. 8 INTRODUCTION 2 Scientific background and explanation of rationale, Page 5 and 6 9 Background including the rationale for using a non-inferiority or equivalence design. 10 METHODS 3 Eligibility criteria for participants (detailing whether participants in the Page 7 11 Participants non-inferiority or equivalence trial are similar to those in any trial(s) that Supplement 1 12 established efficacy of the reference treatment) and the settings and 13 locations where the data were collected. 14 Interventions 4 Precise details of the interventions intended for each group detailing Page 7 15 whether the reference treatment in the non-inferiority or equivalence trial 16 Foris identical peer (or very reviewsimilar) to that in any onlytrial(s) that established 17 efficacy, and how and when they were actually administered. 18 Objectives 5 Specific objectives and hypotheses, including the hypothesis Page 6 19 concerning non-inferiority or equivalence. 20 Outcomes 6 Clearly defined primary and secondary outcome measures detailing Page 8 21 whether the outcomes in the non-inferiority or equivalence trial are 22 identical (or very similar) to those in any trial(s) that established efficacy 23 of the reference treatment and, when applicable, any methods used to 24 enhance the quality of measurements (e.g., multiple observations, 25 training of assessors). 26 Sample size 7 How sample size was determined detailing whether it was calculated Page 8 27 using a non-inferiority or equivalence criterion and specifying the margin 28 of equivalence with the rationale for its choice. When applicable, 29 explanation of any interim analyses and stopping rules (and whether 30 related to a non-inferiority or equivalence hypothesis). 31 Randomization -- 8 Method used to generate the random allocation sequence, including Page 7 32 Sequence details of any restrictions (e.g., blocking, stratification) Supplement 1 33 generation 34 Randomization -- 9 Method used to implement the random allocation sequence (e.g., Page 7 35 Allocation numbered containers or central telephone), clarifying whether the Supplement 1 36 concealment sequence was concealed until interventions were assigned. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 40 of 38

Randomization -- 10 Who generated the allocation sequence, who enrolled Page 7 and 8 1 Implementation participants, and who assigned participants to their groups. 2 Blinding (masking) 11 Whether or not participants, those administering the Not blinded 3 interventions, and those assessing the outcomes were blinded to 4 group assignment. If done, how the success of blinding was 5 evaluated. 6 Statistical methods 12 Statistical methods used to compare groups for primary Page 8 and 9 7 outcome(s), specifying whether a one or two-sided confidence interval Supplement 2 8 approach was used. Methods for additional analyses, such as Supplement 3 9 subgroup analyses and adjusted analyses. 10 RESULTS 13 Flow of participants through each stage (a diagram is strongly Figure 1 11 Participant flow recommended). Specifically, for each group report the numbers 12 of participants randomly assigned, receiving intended treatment, completing the study protocol, and analyzed for the primary 13 outcome. Describe protocol deviations from study as planned, 14 together with reasons. 15 Recruitment 14 Dates defining the periods of recruitment and follow-up. Page 7 and 8 16 For peer review only Figure 1 17 Baseline data 15 Baseline demographic and clinical characteristics of each group. Page 9 and 10 18 Table 1 19 Numbers analyzed 16 Number of participants (denominator) in each group included in Figure 1 and 20 each analysis and whether the analysis was “intention-to-treat” Figure 2 21 and/or alternative analyses were conducted. State the results in 22 absolute numbers when feasible (e.g., 10/20, not 50%). 23 Outcomes and 17 For each primary and secondary outcome, a summary of results Page 11 to 13 24 estimation for each group, and the estimated effect size and its precision Table 2 25 (e.g., 95% confidence interval). For the outcome(s) for which non- Table 3 26 inferiority or equivalence is hypothesized, a figure showing confidence Figure 2 27 intervals and margins of equivalence may be useful. Supplement 2 28 Ancillary analyses 18 Address multiplicity by reporting any other analyses performed, Page 12 29 including subgroup analyses and adjusted analyses, indicating 30 those pre-specified and those exploratory. 31 Adverse events 19 All important adverse events or side effects in each intervention Page 13 32 group. 33 DISCUSSION 20 Interpretation of the results, taking into account the non-inferiority Page 14 to16 34 Interpretation or equivalence hypothesis and any other study hypotheses, sources 35 of potential bias or imprecision and the dangers associated with 36 multiplicity of analyses and outcomes. 37 Generalizability 21 Generalizability (external validity) of the trial findings. Page 14 to 16 38 Overall evidence 22 General interpretation of the results in the context of current Page 16 and 39 evidence. 17 40 www.consort-statement.org 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open

Article Type: Research

Date Submitted by the 18-Nov-2019 Author:

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1 2 3 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Buenos 4 Aires 5 Vargas-Machuca Cabañero, Concepción; Comunidad de Madrid Servicio 6 Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare 7 Centre Guayaba 8 Noguerol Álvarez, Mar; Comunidad de Madrid Servicio Madrileno de 9 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Cuzco García de Blas González, Francisca; Comunidad de Madrid Servicio 10 Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare 11 Centre Mendiguchía Carriche; Instituto de Salud Carlos III, Health 12 Services Research on Chronic Patients Network (REDISSEC) 13 Baños Morras, Raquel; Comunidad de Madrid Servicio Madrileno de 14 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Buenos 15 Aires 16 Díaz Laso, Concepción; Comunidad de Madrid Servicio Madrileno de For peerSalud, Gerencia review Asistencial Atención only Primaria. Healthcare Centre 17 Fuentelarreina 18 Caballero Ramírez, Nuria; Comunidad de Madrid Servicio Madrileno de 19 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Juncal 20 Herrero de Dios, Alicia; Comunidad de Madrid Servicio Madrileno de 21 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Miguel 22 de Cervantes Fernández García, Rosa; Comunidad de Madrid Servicio Madrileno de 23 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Santa 24 Isabel 25 Herrero Hernández, Jesús; Comunidad de Madrid Servicio Madrileno de 26 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre 27 Lavapiés 28 Pose García, Belen; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Mendiguchía 29 Carriche 30 Sevillano Palmero, María Luisa; Comunidad de Madrid Servicio Madrileno 31 de Salud, Gerencia Asistencial Atención Primaria. Pharmacy Department 32 Mateo Ruiz, Carmen; Comunidad de Madrid Servicio Madrileno de Salud, 33 Gerencia Asistencial Atención Primaria. Pharmacy Department 34 Medina Bustillo, Beatriz; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Pharmacy Department 35 Aguilar Jiménez, Monica; Comunidad de Madrid Servicio Madrileno de 36 Salud, UICEC Hospital Ramón y Cajal, Plataforma SCReN;Unidad de 37 Farmacología Clínica, Hospital Ramón y Cajal; Instituto Ramón y Cajal de 38 Investigación Sanitaria IRYCIS 39 group, OB12; Comunidad de Madrid Servicio Madrileno de Salud 40 Primary Subject General practice / Family practice 41 Heading: 42 43 Secondary Subject Heading: Geriatric medicine, Evidence based practice, Health services research 44 Vitamin B 12 Deficiency, Administration Oral, Equivalence Trial,, Keywords: 45 Injections Intramuscular, primary health care, Patient preference 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 2 of 51

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1 2 3 Oral versus intramuscular administration of vitamin B12 for vitamin B12 deficiency in 4 primary care: a pragmatic, randomized, noninferiority clinical trial (OB12) 5 6 7 8 Author names 9 10 Authors: Teresa Sanz-Cuesta, MD PhD1,2; Esperanza Escortell-Mayor, MD PhD1,2; Isabel del Cura- 11 González, MD PhD 1,2,3; Jesús Martín-Fernández, MD PhD2,3,4; Rosario Riesgo-Fuertes, MD2,5; Sofía 12 Garrido-Elustondo, MD2,6; José Enrique Mariño-Suárez, MD7; Mar Álvarez-Villalba, MD PhD2,8; Tomás 13 Gómez-Gascón, MD PhD2,9; Inmaculada González-García, MD10; Paloma González-Escobar, MD11; 14 Concepción Vargas-Machuca-Cabañero, MD PhD12; Mar Noguerol-Álvarez, MD13; Francisca García-de 15 Blas-González, MD PhD2,14; Raquel Baños-Morras, MD15; Concepción Díaz-Laso, MD PhD16; Nuria 16 For17 peer review18 only 19 17 Caballero-Ramírez, MD ; Alicia Herrero-de Dios, MD ; Rosa Fernández-García, MD ; Jesús Herrero- 18 Hernández, MD20; Belén Pose-García, RN14; María Luisa Sevillano-Palmero, Pharm21; Carmen Mateo- 19 Ruiz, Pharm21; Beatriz Medina-Bustillo, Pharm 21; Mónica Aguilar Jiménez, Pharm22 and OB12 Group23. 20 21 Author affiliations 22 1. Research Unit, Gerencia Asistencial de Atención Primaria (GAAP), Madrid, Spain. 23 2. Health Services Research on Chronic Patients Network (REDISSEC), Instituto Salud Carlos III, Madrid, Spain. 24 3. Preventive Medicine and Public Health Area, Health Sciences Faculty, Universidad Rey Juan Carlos, Alcorcón, 25 Madrid, Spain. 26 4. Multiprofessional Teaching Unit of Primary and Community Care Oeste. GAAP, Madrid, Spain. 27 5. Multiprofessional Teaching Unit of Primary and Community Care Sur, GAAP, Madrid, Spain. 28 6. Multiprofessional Teaching Unit of Primary and Community Care Sureste, GAAP, Madrid, Spain. 29 7. Healthcare Centre El Greco, Getafe, GAAP, Madrid, Spain. 30 8. Healthcare Centre Mª Jesús Hereza, Leganés, GAAP, Madrid, Spain. 31 9. Fundación de Investigación e Innovación Biomédica de Atención Primaria, Madrid, Spain. 32 10. Healthcare Centre Barajas, GAAP, Madrid, Spain. 33 11. Healthcare Centre Buenos Aires, GAAP, Madrid, Spain. 34 12. Healthcare Centre Guayaba, GAAP, Madrid, Spain. 35 13. Healthcare Centre Cuzco, Fuenlabrada, GAAP, Madrid, Spain. 36 14. Healthcare Centre Mendiguchía Carriche, Leganés, GAAP, Madrid, Spain. 37 15. Healthcare Centre Buenos Aires, GAAP, Madrid, Spain. 38 16. Healthcare Centre Fuentelarreina, GAAP, Madrid, Spain. 39 17. Healthcare Centre Juncal, Torrejón de Ardoz, GAAP, Madrid, Spain. 40 18. Healthcare Centre Miguel de Cervantes, Alcalá de Henares, GAAP, Madrid, Spain. 41 19. Healthcare Centre Santa Isabel, Leganés, GAAP, Madrid, Spain. 42 20. Healthcare Centre Lavapiés, GAAP, Madrid, Spain. 21. Pharmacy Department, GAAP, Madrid, Spain. 43 22. UICEC Hospital Ramón y Cajal, Plataforma SCReN; Unidad de Farmacología Clínica, Hospital Ramón y Cajal, 44 Madrid, España; Instituto Ramón y Cajal de Investigación Sanitaria, IRYCIS. 45 23. OB12 Group. 46 47 Corresponding author 48 Isabel del Cura-González 49 Head of the Primary Care Research Unit, Madrid Health Services, Spain 50 Associate Professor, Department of Preventive Medicine and Public Health, Rey Juan Carlos University 51 REDISSEC (Health Services Research on Chronic Patients Network), ISCIII 52 C/ San Martín de Porres 6, 28035 Madrid, Spain 53 E-mail: [email protected] 54 Phone number: +34913700697 55 56 Word count: 3056 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 51

1 2 3 Abstract 4 5 6 7 8 Objectives: To compare the effectiveness of oral versus intramuscular vitamin B12 (VB12) in 9 10 patients aged ≥65 years with VB12 deficiency. 11 12 Design: Pragmatic, randomized, noninferiority, multicenter trial in patients ≥65 years in 22 13 14 primary healthcare centres in Madrid (Spain). Participants: 283 adults with VB12 deficiency were 15 16 For peer review only 17 randomly assigned to oral (n=140) or intramuscular (n=143) treatment arm. Interventions: The 18 19 intramuscular arm received 1mg VB12 on alternate days in weeks 1–2, 1mg/week in weeks 3–8, 20 21 and 1mg/month in weeks 9–52. The oral arm received 1mg/day in weeks 1–8 and 1 mg/week in 22 23 24 weeks 9–52. 25 26 Main outcomes: Serum VB12 concentration normalization (≥ 211 pg/mL) at 8, 26, and 52 weeks. 27 28 Noninferiority would be declared if the difference between arms is 10% or less. Secondary 29 30 31 outcomes included symptoms, adverse events, adherence to treatment, quality of life, patient 32 33 preferences and satisfaction. 34 35 Results: At week 8, the percentage of patients in each arm who achieved normal B12 levels was 36 37 well above 90%; the differences in this percentage between the oral and intramuscular arm were - 38 39 40 0.7% (95% CI: -3.2 to 1.8) by per-protocol (PPT) analysis and 4.8% (95% CI: -1.3 to 10.9) by 41 42 intention-to-treat (ITT) analysis. At week 52, the percentage of patients who achieved normal B12 43 44 levels was 73.6% in the oral arm and 80.4% in the intramuscular (IM) arm; these differences were 45 46 47 -6.3% (95% CI: -11.9 to -0.1) and -6.8% (95% CI: -16.6 to 2.9), respectively. Factors affecting the 48 49 success rate at week 52 were age, OR=0.95 (95% CI: 0.91 to 0.99), and having reached VB12 50 51 levels ≥281 pg/mL at week 8, OR= 8.1 (95% CI: 2.4 to 27.3). Under a Bayesian framework, 52 53 54 noninferiority probabilities (Δ>-10%) at week 52 were 0.036 (PPT) and 0.060 (ITT). Quality of 55 56 life and adverse effects were comparable across groups. 83.4% of patients preferred the oral route. 57 58 2 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 5 of 51 BMJ Open

1 2 3 Conclusions: Oral administration was no less effective than intramuscular administration at 8 4 5 6 weeks. Although differences were found between administration routes at week 52, the probability 7 8 that the differences were below the noninferiority threshold was very low. 9 10 Trial registration: ClinicalTrials.gov (NCT 01476007) and EUDRACT (2010-024129-20). 11 12 Funding: Ministerio de Sanidad y Consumo Español. Instituto de Salud Carlos III (ISCIII). 13 14 15 European Regional Development Fund. 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 3 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 51

1 2 3 Strengths and limitations of this study 4 5 6  This is the largest and longest follow-up randomized clinical trial in patients aged ≥65 years 7 8 with VB12 deficiency. 9 10  In addition to VB12 levels, this study incorporates patient-reported outcomes such as 11 12 13 symptoms, quality of life, and patient preferences. 14 15  The study design did not allow patient blinding; however, the main outcome measurement 16 For peer review only 17 was objective. 18 19 20  The rates of loss to follow-up were low at week 8 and week 26 and higher at week 52, 21 22 consistent with pragmatically designed clinical trials. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 4 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 7 of 51 BMJ Open

1 2 3 INTRODUCTION 4 5 6 Vitamin B12 (VB12) is an essential nutrient for the synthesis of cellular DNA. It is generally 7 8 accepted that daily needs in adults range from 1 to 2 μg/day, (1) but other standards recently 9 10 recommend 3-4 µg per day.(2) The Western diet is estimated to contain 7–30 μg/day of cobalamin, 11 12 of which 1–5 μg is absorbed and stored (estimated reserves of 2–5 mg); therefore, symptoms 13 14 15 resulting from a VB12 deficit would not appear until 3–5 years after establishing a low-ingestion 16 For peer review only 17 or poor-absorption regimen.(1) VB12 deficiency can lead to hematological and neuropsychiatric 18 19 disorders,(3) as well as cardiovascular risk factors.(4) The prevalence of VB12 deficiency in the 20 21 (5–8) 22 elderly is highly variable across studies, which report values of 1.5% to 15%. 23 24 In primary care, the most commonly observed causes of VB12 deficiency are related to 25 26 abnormalities in digestion (atrophic gastritis, achlorhydria) or absorption (autoimmune pernicious 27 28 29 anaemia, chronic pancreatitis, Crohn’s disease, the effect of medications that alter the mucosa of 30 31 the ileum such as metformin, antacids -proton-pump inhibitors and H2-receptor antagonists-, 32 33 antibiotics, and colchicine)(9) or the consequences of surgical resection.(10) A deficiency stemming 34 35 solely from dietary habits is rare and usually affects strict vegans.(11) In the elderly, different 36 37 38 alterations in the processes involved in VB12 absorption increase the prevalence of this deficit, 39 40 which can appear in the absence of specific symptoms, thereby hindering its diagnosis. (12) 41 42 The traditional treatment for VB12 deficiency consists of intramuscular (IM) injection of 43 44 45 cyanocobalamin, generally 1 mg/day for one week, followed by 1 mg/week for one month, and 46 47 then 1 mg every 1 or 2 months ad perpetuum.(10,13,14). The vitamin may, however, be administered 48 49 orally. Several studies have shown serum VB12 concentrations to normalize after taking large oral 50 51 (15,16) 52 doses. Studies taking into consideration the patients´ preferences have found differences in 53 54 55 56 57 58 5 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 8 of 51

1 2 3 favour of the oral route.(17,18) Furthermore, oral treatment could avoid injection nuisances, reduce 4 5 (19) 6 unnecessary travel for the patients or nurses, and minimize treatment costs. 7 8 Some authors have questioned the use of oral administration while others favour it, 9 10 although no firm conclusions can be drawn due to the methodological limitations of the evidence 11 12 the authors provide.(10,20–22) The 2018 Cochrane Review(5) includes three randomized clinical trials 13 14 15 comparing the effectiveness of oral and IM administration. There are differences among the trials 16 For peer review only 17 in terms of treatment regimens and follow-up duration, ranging from 3 to 4 months, and average 18 19 age of the patients, as well as the frequency and VB12 daily dose for both routes. In terms of 20 21 22 outcomes, adverse events, and cost, the overall quality of the evidence was low due to the small 23 24 number of studies and limited sample sizes.(23–25) In their conclusions, the authors state the need 25 26 for trials with improved methods for random allocation and masking, larger sample sizes, and 27 28 29 information on other relevant outcome variables that are preferably conducted in the primary care 30 31 setting. 32 33 The aim of this study was to compare the effectiveness of oral- and IM-administered VB12 34 35 in the normalization of serum VB12 concentrations at 8, 26, and 52 weeks in patients aged ≥65 36 37 38 years with VB12 deficiency treated at primary healthcare centres (PHC). Secondary outcomes 39 40 included safety (adverse events), quality of life, and adherence to treatment. Additional aims were 41 42 to describe patient preferences and satisfaction with treatment and to explore the immediate 43 44 45 response (8 weeks) as a normalization predictor of one-year outcomes to propose clinical 46 47 recommendations. 48 49 50 51 52 METHODS 53 54 Study design and participants 55 56 57 58 6 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 9 of 51 BMJ Open

1 2 3 A pragmatic, randomized, multicenter, noninferiority clinical trial with a duration of 12 months 4 5 (26) 6 was conducted in a PHC. On ethical grounds, a placebo-controlled trial was not appropriate. 7 8 Methodological issues of this trial have been published elsewhere (Supplement 1).(27) 9 10 Competitive recruitment was performed in 22 PHC in Madrid (Spain) from July 2014 to November 11 12 2016. Eligible patients were 65 years of age or older and had been attending a PHC for consultation 13 14 15 on any medical matter. Patients were assessed for eligibility and invited to participate 16 For peer review only 17 consecutively by their general practitioners. Written informed consent was obtained from all 18 19 participants. A blood test was performed, and in patients with a serum concentration of VB12 of 20 21 22 <211 pg/mL, the remaining inclusion and exclusion criteria were evaluated. The cut-off value 23 24 selected in the protocol was <179 pg/mL; this value was modified by the laboratory following the 25 26 recommendations of the provider. This change took place prior to the beginning of the recruitment. 27 28 29 Patient recruitment was always performed using the same methodology and cut-off point. The 30 31 procedures for measurement of the biomarkers were ADVIA Centaur XP (Siemens Diagnostics, 32 33 Tarrytown, NY, USA). 34 35 Randomization and masking 36 37 38 Patients were allocated by simple randomization at a 1:1 ratio to oral or intramuscular 39 40 administration of vitamin B12. The randomization system was incorporated into the electronic 41 42 data collection system to assure allocation concealment. Because of the nature of the intervention, 43 44 45 patients and general practitioners were aware of their treatment allocation. Analysis was performed 46 47 by the trial statistician, who was blinded to allocation. 48 49 50 51 52 Intervention 53 54 55 56 57 58 7 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 10 of 51

1 2 3 The pharmaceutical formulations used in the study are commercially available in Spain 4 5 6 (Optovite® vials). Its pharmaceutical presentation is in silk-screen-printed clear glass ampoules 7 8 that are presented in PVC blister support. The treatment regimen was : a) IM route: 1 mg of 9 10 cyanocobalamin on alternate days during weeks 1–2, 1 mg/week during weeks 3–8 and 1 11 12 mg/month during weeks 9–52; b) oral route: 1 mg/day of cyanocobalamin for 8 weeks and 1 13 14 15 mg/week during weeks 9–52. The period between 1-8 weeks was considered the charging period. 16 For peer review only 17 In the oral route, the medication was provided to the patient at the health centre, along with 18 19 instructions for self-administration at home. The information sheet explained to the patient the 20 21 22 procedure for oral administration, i.e., how to open the ampoule and dilute its contents in a glass, 23 24 then drink it. 25 26 In the IM route, the medication was administered by the nurse at the health centre. 27 28 29 30 31 Outcomes 32 33 The main outcome was the normalization of serum VB12 concentrations (≥211 pg/mL) at 8, 26, 34 35 and 52 weeks. The secondary outcomes were the serum VB12 concentrations (pg/mL), adverse 36 37 38 events, adherence to treatment (number of vials for the oral arm and the number of injections for 39 40 the IM arm during each visit; good adherence was considered greater than 80%), quality of life 41 42 (EQ-5D-3L) (28) and patient preferences and satisfaction were assessed. Anamnesis, demographic 43 44 45 and lifestyle information, clinical variables, analytical variables, and concomitant treatment were 46 47 recorded.(27) 48 49 Procedures 50 51 52 After signing the consent form, those who agreed to participate had serum VB12 concentrations 53 54 determined. If the VB12 value was <211 pg/mL, a hemogram, biochemical analysis, and anti- 55 56 57 58 8 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 11 of 51 BMJ Open

1 2 3 intrinsic factor antibody levels were assessed.(27) The patients also received a medication diary to 4 5 6 be filled out daily. Baseline data were collected by the family physician and/or a nurse. IM 7 8 treatments were administered by nurses in the health centres. The follow-up visits were 9 10 conducted during weeks 8, 26 and 52.(27) 11 12 Statistical analysis 13 14 15 Sample size. Assuming that 70% of patients reach a serum VB12 concentration of ≥ 211 pg/mL 16 For peer review only 17 in both groups, for a threshold of noninferiority of 10%, statistical power of 60% with 18 19 significance set at p<0.05 and a 5% loss to follow-up, the final sample size was word 20 21 22 320 (160 in each arm). 23 24 As recommended for noninferiority studies, both PPT and ITT analyses were performed, 25 26 with the null hypothesis being that there were differences between treatments at the three 27 28 29 monitoring points. Comparing both arms, we calculated the difference between the percentage of 30 31 patients in each treatment arm whose serum VB12 concentrations became normalized at 8, 26, 32 33 and 52 weeks, with their 95% CI. If the confidence intervals do not fall outside the noninferiority 34 35 limit (10%), it can be concluded that the oral treatment is not inferior to the intramuscular 36 37 38 treatment.(29,30) In ITT analyses, missing values for the main outcome variable were added using 39 40 the 'last observation carried forward' (LOCF) method.(31) 41 42 To explore factors affecting the normalization of serum VB12 concentration at 52 weeks, 43 44 45 serum VB12 levels were studied at 8 weeks. A receiver operating characteristic (ROC) curve 46 47 was built to determine the likelihood ratios of each cutpoint after the charging period to “predict” 48 49 the normalization of levels at the end of the study. After this, a generalized linear model (GLM) 50 51 (32,33) 52 was built (function logit). The normalization of serum VB12 levels at 52 weeks was the 53 54 dependent variable, and the treatment group was the independent variable. Variables considered 55 56 57 58 9 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 12 of 51

1 2 3 significant by the researchers from a clinical perspective were included in the model. To test the 4 5 6 noninferiority hypothesis, adding the information contained in these data to previous knowledge, 7 8 additional statistical analyses were performed using a Bayesian approach. Secondary outcome 9 10 variables were analyzed using the appropriate statistical tests, and their means or proportions 11 12 were used to estimate differences between groups. All analyses were performed using STATA 13 14 15 14 and EPIDAT 4.2 software. 16 For peer review only 17 Patient involvement 18 19 Patients were not involved in the development of plans for recruitment, design, outcome measures, 20 21 22 or implementation of the study conduct. No patients were asked to advise on the interpretation or 23 24 writing of the results. Patients explained the experience of participating in the study on 25 26 the occasion of International Clinical Trial´s day in Radio Nacional de España (RNE). We will 27 28 29 pursue patient and public involvement in the development of an appropriate method for further 30 31 dissemination. 32 33 34 35 36 RESULTS 37 38 Characteristics of the study participants 39 40 41 A total of 2342 patients were offered participation, and 2152 provided informed consent. A total 42 43 of 307 patients showed a VB12 deficit (14.3%), 283 of whom were allocated to receive VB12 44 45 treatment via the IM route (n=143) or orally (n=140). The follow-up period (52 weeks) was 46 47 48 completed by 229 patients (80.9%) (Figure 1). 49 50 The average age was 75.18 (6.34), and 58.3% of the patients were women. Table 1 51 52 describes the baseline characteristics of the patients included in the trial. No relevant differences 53 54 55 56 57 58 10 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 13 of 51 BMJ Open

1 2 3 were found between groups at baseline for demographic and medical characteristics or for the 4 5 6 study endpoints. 7 8 9 10 Table 1. Baseline characteristics at baseline by group 11 12 Variable No. (%) 13 Oral route IM route Total (n=283) 14 (n=140) (n=143) 15 Sociodemographic data 16 For peer review only 17 Women 87 (62.1) 78 (54.5) 165 (58.3) 18 Age (years), mean (SD) 74.2 (5.8) 76.2 (6.7) 75.2 (6.3) 19 Educational level 20 21 Illiteracy 4 (2.9) 7 (5.1) 11 (4.0) 22 Incomplete education 48 (34.5) 46 (33.6) 94 (34.1) 23 Primary education 58 (41.7) 63 (46.0) 121 (43.8) 24 25 Secondary education 16 (11.5) 10 (7.3) 26 (9.4) 26 Tertiary education 4 (2.9) 4 (2.9) 8 (2.9) 27 Higher education 9 (6.5) 7 (5.1) 16 (5.8) 28 a 29 Social occupational class 30 Class I - IV 31 (27.7) 33 (27.3) 64 (27.5) 31 Class V - VI 81 (72.3) 88 (72.7) 169 (72.5) 32 33 Living alone 32 (21.4) 30 (22.2) 62 (21.9) 34 Clinical data 35 Tobacco habit 36 37 Ex-smoker 27 (19.7) 25 (18.4) 52 (19.0) 38 Smoker 9 (6.6) 10 (7.4) 19 (7.0) 39 Nonsmoker 101 (73.7) 101 (74.3) 202 (74.0) 40 Vegetarian 2 (1.4) 0 (0) 2 (0.7) 41 42 Having undergone gastrectomy 1 (0.7) 2 (1.4) 3 (1.1) 43 Symptoms 44 Paresthesia 33 (23.6) 45 (31.5) 78 (27.6) 45 46 Asthenia 43 (30.7) 54 (37.8) 97 (34.3) 47 Loss of appetite 12 (8.6) 30 (21.0) 42 (14.8) 48 Sadness 37 (26.4) 53 (37.1) 90 (31.8) 49 50 Showing ≥1 symptom 70 (50.0) 83 (58.0) 153 (54.1) 51 Signs 52 Glossitis 2 (1.4) 9 (6.3) 11 (3.9) 53 54 Position sensitivity 2 (1.4) 1 (0.7) 3 (1.1) 55 Vibration sensitivity 15 (10.7) 13 (9.1) 28 (9.9) 56 57 58 11 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 14 of 51

1 2 3 Showing ≥1 altered sign 16 (11.4) 21 (14.7) 37 (13.1) 4 5 Hemogram-Clinical Biochemistry 6 Vitamin B12 (pg/mL), mean (SD) 173.1 (27.3) 166.4 (32.6) 169.7 (6.3) 7 Anemia b 16 (11.4) 27 (18.9) 43 (15.2) 8 9 Hematocrit (%), mean (SD) 42.4 (4.0) 41.9 (4.2) 442.1 (4.1) 10 MCV (fL), mean (SD) 92.1 (6.7) 94.3 (7.4) 93.2 (7.1) 11 Anti-intrinsic factor antibody 15 (11.0) 15 (10.5) 30 (10.8) 12 13 Medication 14 Proton-pump inhibitors (PPI) 57 (40.7) 64 (44.8) 121 (42.8) 15 Metformin 69 (49.3) 56 (39.2) 125 (44.2) 16 PPI and metforminFor peer review33 (23.6) 30only (21.0) 63 (22.3) 17 Scales 18 c 19 MMSE , mean (SD) 30.8 (4.6) 30.2 (4.8) 30.5 (4.7) 20 EQ-5D-Utilities, mean (SD) 0,817 (0,169) 0,855 (0,139) 0,836 (0,171) 21 22 aNeoweberian occupational social class (CSO-SEE12). Gac Sanit. 2013;27(3):263–272. 23 bAnaemia was defined by the World Health Organization criteria (haemoglobin <12 g/dL in women and 24 <13 g/dL in men). https://www.who.int/vmnis/indicators/haemoglobin 25 c 26 Mini Mental State Examination. Maximum score= 35 points. Normal score= 30–35. Borderline score= 27 24–29 points. Scores < 24 points in patients aged >65 years and scores < 29 points in patients aged <65 28 years suggest cognitive impairment. 29 30 31 Primary outcomes 32 33 At week 8, the difference in the success rate between the oral and IM routes was -0.7% (95%CI: 34 35 -3.2% to 1.8%) and 4.8% (95%CI: -1.3% to 10.9%) with the PPT and ITT analyses, respectively. 36 37 38 At week 26, these differences were -12.9% (95%CI: -17.9% to -6.1%) and -3.2% (95%CI: - 39 40 11.8% to 5.4%), respectively. At week 52, these differences were -6.3% (95%CI: -11.9% to - 41 42 0.07%) and -6.8% (95%CI: -16.6% to 2.9%), respectively (Figure 2). 43 44 45 In the PPT analysis under a Bayesian approach, the probabilities of differences in the 46 47 treatment effectiveness being >10% between the oral and IM groups were 0.001, 0.201, and 48 49 0.036 at weeks 8, 26, and 52, respectively. In the ITT analysis, these values were 0.000, 0.015, 50 51 and 0.060 at weeks 8, 26, and 52, respectively (Supplement 2). The result of the likelihood ratio 52 53 54 for the cutpoints at the main percentiles of the distribution of VB12 serum levels at week 8 to 55 56 predict normalization at the end of the study is shown in Supplement 3. The level at the 5th 57 58 12 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 15 of 51 BMJ Open

1 2 3 percentile of the distribution was selected as the most useful value because it showed the best 4 5 6 classification ability. When patients did not reach this level at week 8, they were almost twelve 7 8 times more likely to not reach suitable VB12 levels at the end of the study than if they had 9 10 reached levels over 281 pg/mL (12~1/negative likelihood ratio). 11 12 In the ITT analysis, the factors affecting the success rate at week 52 were age, for each 13 14 15 year of increase in age, the success rate decreased by 5%, and having attained VB12 levels of 16 For peer review only 17 ≥281 pg/mL at week 8, which yielded a success rate 8.1 times higher (Table 2). 18 19 20 21 22 23 24 Table 2. Factors associated with VB12 concentrations ≥ 211 pg/ml at week 52 25 26 Variable Odds ratio Robust std. error P>z 95% CI 27 28 IM vs. oral route 1.10 0.370 0.776 (0.57 to 2.13) 29 Age 0.95 0.022 0.025 (0.91 to 0.99) 30 VB12 concentration 8.10 5.014 0.001 (2.41 to 27.25) 31 >281 pg/ml at week 8 32 33 Constant 0.78 0.622 0.755 (0.16 to 3.72) 34 GLM, N=265. Variance function: V(u) = u*(1-u/1) [Binomial]. Link function: g(u) = ln(u/(1-u)) 35 [Logit]. AIC= 0.89967. BIC = -1225.89. 36 37 38 39 The mean levels of VB12 for each follow-up visit were above the normalization 40 41 threshold in both groups, although these values were much greater in the IM group (Supplement 42 43 4). In 51 patients (36 IM and 5 oral), the levels of VB12 in week 8 were above the normal range 44 45 46 limit of the laboratory (≥911 pg/mL), so the treatment regimen was changed from the initial 47 48 planned pattern. 49 50 Secondary outcomes 51 52 53 In terms of quality of life and the presence of signs related to VB12 deficiency, no significant 54 55 differences were found between treatment arms at any of the follow-up visits (Table 3). 56 57 58 13 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 16 of 51

1 2 3 Table 3. Secondary outcomes (Quality of life and exploratory findings) at weeks 8, 26 and 52 4 5 Visit Oral route IM route Mean difference 6 N Mean (SD) N Mean (SD) (95% CI) 7 8 Quality of life (EQ-5D-5L Index) 9 Baseline 139 0.855 (0.139) 137 0.817 (0.197) 0.066 (-0.002 to 0.078) 10 Week 8 134 0.853 (0.158) 134 0.822 (0.204) 0.031 (-0.013 to 0.075) 11 Week 26 128 0.853 (0.153) 128 0.826 (0.191) 0.027 (-0.016 to 0.070) 12 Week 52 112 0.824 (0.179) 112 0.823 (0.194) 0.001 (-0.047 to 0.049) 13 At least one altered sign (glossitis and/or altered vibration sensitivity and/or altered position 14 sensitivity) 15 Visit N n (%) N n (%) Proportion difference (95% CI) 16 For peer review only 17 Baseline 140 16 (11.4%) 143 21 (14.7) -3.3 (-11.1% to 4.6) 18 Week 8 135 15 (11.1%) 130 13 (10.0) 1.1 (-6.3% to 8.5) 19 Week 26 131 14 (10.7%) 122 12 (9.8) 0.9 (-6.6% to 8.3) 20 Week 52 122 14 (12.5%) 117 9 (7.7) 3.8 (-3.7% to 11.2) 21 22 23 Eleven adverse events were reported and none of them were severe; five (3.57%) 24 25 occurred with patients in the oral arm and six (4.20%) with patients in the IM arm, yielding a 26 27 difference of -0.63% (95%CI: -5.12% to 3.87%). Three patients withdrew from the study: one 28 29 30 patient in the oral group due to urticaria, and two in the IM group due to reddening and pruritic 31 32 facial erythema and generalized itching (mainly in the cheeks with scarce urticariform lesions). 33 34 In three other cases, treatment for the adverse events was prescribed (constipation and erythema), 35 36 37 and in five cases, it was not necessary to take further measures (Table 4). 38 39 Table 4. Description of adverse events by patient and route of administration 40 41 Route Adverse event Action 42 Constipation Administration of specific treatment 43 Generalized itching and hives on the cheeks Withdrawal 44 Dyspepsia Treatment not required 45 IM route 46 Constipation Administration of specific treatment 47 Redness and pruritic facial erythema Withdrawal 48 Erythema on forearms Administration of specific treatment 49 50 Urticaria on the neck and arms Treatment not required 51 Occasional postprandial dyspepsia Treatment not required 52 Oral route Occasional postprandial dyspepsia Treatment not required 53 Urticaria Withdrawal 54 55 Increased irritability and nervousness Treatment not required 56 57 58 14 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 17 of 51 BMJ Open

1 2 3 At week 8, adherence to treatment was evaluated in 265 patients, of whom 95.5% were 4 5 6 adherent (97.8% oral and 93.8% IM); the difference between the groups was 3.9% (95%CI: -0.1 7 8 to 8.7). At week 52, adherence was evaluated in 229 patients, of whom 220 (96.1%) were 9 10 adherent (98.2% oral and 94.0% IM); the difference was 4.2% (95%CI: -0.7 to 9.1). 11 12 Overall, 89.5% of the patients reported being satisfied or very satisfied with the treatment 13 14 15 via the oral route (91.3%) and the IM route (87.6%). The difference was 3.7% (95% CI: -4.0% to 16 For peer review only 17 11.3%). 18 19 A total of 83.4% of patients preferred the oral route (97.6% among the patients receiving 20 21 22 VB12 orally vs. 68.6% of the patients in the IM group); the difference was 29.0% (95%CI: 20.3 23 24 to 37.7). 25 26 27 28 29 DISCUSSION 30 31 Main findings of the study 32 33 Supplementing VB12 in patients with VB12 deficiency, whether orally or 34 35 intramuscularly, achieves the normalization of VB12 levels in most cases. The oral route was not 36 37 38 inferior to the IM route during the charging period. Formally, the pre-established conditions for 39 40 determining the noninferiority of oral administration were not met for the complete follow-up 41 42 period, but these results merit a deeper analysis. 43 44 45 Differences between the administration routes were found at 26 and 52 weeks. The IM 46 47 maintenance treatment of 1 mg/month was effective in maintaining VB12 levels, while oral 48 49 administration of 1 mg/week had a probability of being inferior (by more than 10%) to the IM 50 51 52 route by 20% in the most unfavourable scenario (PPT). However, given that no strategy was 53 54 superior in the charging period, and in view of the model results showing that when VB12 levels 55 56 57 58 15 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 18 of 51

1 2 3 reached ≥ 281 pg/mL during the charging period, the success rate at 12 months was 8 times 4 5 6 higher, the probability that the differences between groups would exceed Δ was very low, 7 8 independent of the administration route. The most plausible explanation for the observed 9 10 difference between routes might be that in patients below this threshold, the maintenance oral 11 12 dose should be higher than the dose used in the present study. Some authors have recommended 13 14 15 that an oral dose of 2 mg/week be administered as a maintenance dose.(34) 16 For peer review only 17 The incidence of adverse events was very low and similar for oral and intramuscular 18 19 administration, and nonserious adverse events were found. These findings were similar to other 20 21 (5) 22 studies. Patients’ preferences can be a decisive factor for determining the administration route. 23 24 In this trial, similar to previous studies,(17) there was a clear preference for the oral route, 25 26 especially among the patients assigned to this group. 27 28 (35,36) 29 The effect of VB12 supplements on quality of life remains unclear, but the present 30 31 results show that the treatment route does not improve patients´ perception of their health-related 32 33 quality of life or related symptoms. 34 35 We did not find significant differences in adherence. Adherence to the treatment via the 36 37 38 IM route was lower than expected. Although drug administration was assured once the patient 39 40 attended the consultation, the patient could choose not to attend appointments for various 41 42 reasons. However, in usual practice, adherence with the oral route could be more compromised 43 44 45 than with the IM route, and this factor should be taken into consideration to personalize 46 47 prescription. 48 49 Comparison with other studies 50 51 52 53 54 55 56 57 58 16 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 19 of 51 BMJ Open

1 2 3 The comparison with other studies is difficult, due to the treatment different doses used, 4 5 6 but especially because of the follow-up length had been inferior to 4 months and the number of 7 8 patients included was small. 9 10 As far as we know, the present trial is the largest clinical trial with the longest follow-up 11 12 period, and it is the first to evaluate, in addition to VB12 levels, clinical signs and symptoms, 13 14 15 health-related quality of life, and patient preferences. The 3 clinical trials(23–25) described in the 16 For peer review only 17 2018 Cochrane Systematic Review(5) had a duration between 3 and 4 months and included a total 18 19 of 153 patients. In the Saraswathy trial, patients in the oral route at 3 months normalised levels 20 21 (25) 22 20/30 (66.7%) vs 27/30 (90%) of the patients in the IM route. In Kuzminski's patients in the 23 24 oral route at 4 months normalised levels 18/18 (100%) vs 10/14 (71.4%) of the patients in the IM 25 26 route.(23) These differences were statistically non-significant in both studies. 27 28 29 Two studies have recently been published add evidence in favour of oral and sublingual 30 31 administration of VB12.(37,38) The follow-up of Molerio's study reached 24 months versus 12 32 33 months in our study. However, Molerio J performed a prospective uncontrolled study that included 34 35 26 patients submitted to total gastrectomy. All patients received oral VB12 supplementation (1 36 37 38 mg/day), and all of them maintained normalization V12 at 6, 12, 18, and 24 months. There was a 39 40 progressive increase in serum V12 levels within the first 12 months, which remained stable 41 42 thereafter.(37) The long-term effectiveness of the oral route in absorption-deficient people such as 43 44 45 gastrectomized patients would support the results of our study. 46 47 Bensky et al. compared the efficacy of sublingual vs. intramuscular administration of 48 49 vitamin B12 in a retrospective observational study from the computerized pharmacy records of 50 51 52 Maccabi Health Service (MHS). Among 4281 patients treated with VB12 supplements (830 53 54 (19.3%) with IM and 3451 (80.7%) with sublingual tablets, the IM group achieved a significant 55 56 57 58 17 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 51

1 2 3 increase in VB12 levels compared with the sublingual group, OR 1.85, CI 95% 1.5-2.3. (38) 4 5 6 Although this study has a large sample size, the important methodological limitations on its 7 8 effectiveness (retrospective design; reliance on clinical records; absence of epidemiological 9 10 information such as patient age and sex or the aetiology of the deficit) should be considered in the 11 12 interpretation of their results. 13 14 15 Strengths and limitations 16 For peer review only 17 Our study was pragmatic(39) in both the inclusion and diagnostic methods criteria. The 18 19 majority of the patients with deficits included in this study presented no symptomatology or very 20 21 22 low-level symptoms, with no anemia, which is the common profile of most patients who present 23 24 with VB12 deficits in primary care. The study design did not allow for masking the patients to 25 26 the received treatment. However, these limitations were compensated for by the objective 27 28 29 measurement of the main outcome variable. 30 31 As occurs in all pragmatic clinical trials, patient recruitment was complicated, and the 32 33 sample size reached only 88.4% of the calculated necessary size, which implies that the power of 34 35 the study was limited. Hence, the analysis was complemented using Bayesian methods that allow 36 37 38 for studying a posteriori the likelihood of a difference between two outcomes to exceed a certain 39 40 limit.(40) Under this approach, the a posteriori probability for differences to exceed the proposed 41 42 Δ=-10% was not significant during the charging period, and the probabilities were low but not 43 44 45 negligible in the PPT analysis and low in the ITT analysis over the complete follow-up period. 46 47 Loss to follow-up was low at 8 and 26 weeks and higher at 52 weeks. This effect has 48 49 been observed in pragmatic clinical trials with long follow-up periods. Missing data were greater 50 51 52 in the IM arm, during the interval between randomization and initiation of treatment (6% IM vs 53 54 1% oral), over 8 weeks (9% IM vs 4% oral) and over 26 weeks (15% IM vs 6%). These 55 56 57 58 18 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 21 of 51 BMJ Open

1 2 3 differences could represent a lower acceptability of the IM route by patients, since the missing 4 5 6 data were mostly due to patient dropout. At 52 weeks, the numbers of losses in the two arms 7 8 were similar (20% oral and 18% IM), and in the case of oral treatment, several of those losses 9 10 were withdrawals occasioned by not achieving particular levels of VB12. 11 12 Implications of the study findings 13 14 15 On the basis of our results and the available evidence, we propose the oral administration 16 For peer review only 17 of VB12 at 1 mg/day during the charging period. Subsequently, the recommended dose would 18 19 vary as a function of the VB12 levels reached during the charging period. For VB12 20 21 22 concentrations between the normal levels of 211 pg/mL (in our laboratory) and 281 pg/mL (the 23 24 5th percentile of the distribution in this trial), a dose of 2 mg/week is suggested. When the levels 25 26 reached in the charging period are between 281 and 380 pg/mL (the 20th percentile of the 27 28 29 distribution), it may be appropriate to perform an analysis between 8 and 26 weeks to confirm 30 31 that normal levels are maintained. All patients who reach a level of 380 pg/mL by week 8 could 32 33 be maintained at the initial dosage (1 mg/week) without subsequent analyses during the year of 34 35 follow-up. 36 37 38 If the IM route is chosen, the proposed dose for this route during the first few weeks may 39 40 be excessive for patients with VB12 deficiency. The scheduled IM dose should be reconsidered 41 42 in the first two weeks based on VB12 levels, and the scheduled dose could be limited to 1 43 44 45 mg/week if warranted by the outcome. Nevertheless, these recommendations must be assessed in 46 47 further research. 48 49 Oral administration of VB12 in patients older than 65 years is probably as effective as 50 51 52 intramuscular administration, and it also lacks adverse effects and is preferred by patients. We 53 54 must also highlight the potential benefit of the oral route in terms of safety for patients with 55 56 57 58 19 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 22 of 51

1 2 3 coagulation problems, for whom IM-administered medication is often contraindicated. A small 4 5 6 number of patients may require additional follow-up after 8 weeks if a certain concentration of 7 8 VB12 in blood is not reached. 9 10 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 20 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 51 BMJ Open

1 2 3 4 5 6 Authors’ Contributions 7 8  Trial Management Committee: TSC; EEM; IDC; JMF; RRF; SGE. 9 10  Healthcare Centres (managers)*: JEMS, TGG,MAV, IGG, PGE,CVMC,MNA, 11 12 13 FGBG,RBM,CDL,NCR,AHD, RFG,JHH,BPG study coordination development in each 14 15 healthcare centre with principal investigator supervision. 16 For peer review only 17  Technical Support Group**: participated in different phases of the design and 18 19 20 development of the research. MLSP; CMR; BMB; MAJ; coordinated the pharmaceutical 21 22 aspects. 23 24  Clinical Investigators: collected the data for the study, which included recruiting patients, 25 26 27 obtaining consent, performing blood tests, applying interventions, collecting data, and 28 29 arranging and performing follow-up for patients. 30 31  Statistical analysis: TSC; JMF; IDC; EEM with the collaboration of the Research Unit 32 33 34 (JGM and MMM). 35 36  Writing Committee: TSC; EEM; IDC; JMF, SGE, and RRF wrote the manuscript. All 37 38 authors in the OB12 Group read and approved the final manuscript. 39 40 41 Acknowledgements 42 43 To Margarita Blázquez Herranz, Instituto de Salud Carlos III, for making the impossible 44 45 possible. 46 47 To Juan Carlos Abánades Herranz for trusting us and his contribution to facilitate our work. 48 49 50 To Antonio Jesús Alemany López and Marta Sánchez-Celaya, Managing Director of Primary 51 52 Healthcare for accepting to sponsor this research after the dissolution of CAIBER. 53 54 55 56 57 58 21 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 24 of 51

1 2 3 To the Fundación para la Investigación e Innovación Biomédica de Atención Primaria (FIIBAP) 4 5 6 for the grant awarded for the publishing of the research. 7 8 To Fernando Cava Valencia and Raquel Guillén Santos from the Clinical Laboratory 9 10 BRSALUD. 11 12 To Elena Tolosa Oropesa from the Pharmacy Department, Gerencia Asistencial de Atención 13 14 15 Primaria de Madrid. 16 For peer review only 17 To our colleagues from the Research Unit, including the assistants Marcial Caboblanco Muñoz, 18 19 Juan Carlos Gil and Isabel Morales Ropero for their unconditional support; project manager Pilar 20 21 22 Pamplona-Gardeta, researchers Juan A. López Rodríguez, Jaime Barrio Cortes, Cristina Lozano 23 24 Hernandez and Medical Residents Daniel Toledo Bartolomé, Irene Wijers and Palmira Jurado 25 26 Macías for their technical advice in finalizing this paper. 27 28 29 To Javier Zamora Romero, head of the Unit of Biostatistics, Hospital Ramón y Cajal of Madrid, 30 31 for revising the manuscript. 32 33 To all the professionals from the participating healthcare centers. 34 35 To all patients who contributed to this research. 36 37 38 39 40 Competing Interests 41 42 The authors declare that they have no competing interests. 43 44 45 46 47 Funding 48 49 The trial was financed by Ministerio de Sanidad y Consumo Español through their call for 50 51 52 independent clinical research, Orden Ministerial SAS/2377, 2010 (EC10-115, EC10-116, EC10- 53 54 117, EC10-119, EC10-122); CAIBER – Spanish Clinical Research Network, Instituto de Salud 55 56 57 58 22 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 25 of 51 BMJ Open

1 2 3 Carlos III (ISCIII) (CAI08/010044); and Gerencia Asistencial de Atención Primaria de Madrid. 4 5 6 This study is also supported by the Spanish Clinical Research Network (SCReN), funded by 7 8 ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, project number 9 10 PT13/0002/0007, within the National Research Program I+D+I 2013-2016 and co-funded with 11 12 European Union ERDF funds (European Regional Development Fund). 13 14 15 The funders of the study had no role in the study design, data collection, onsite monitoring, data 16 For peer review only 17 analysis, data interpretation, or writing of the manuscript. 18 19 Ethics approval 20 21 th 22 Madrid Region Clinical Research Ethics Committee on February 8 , 2011. 23 24 Data sharing statement 25 26 Individual de-identified participant data will be shared upon reasonable request. These data will 27 28 29 include every variable used in the analysis shown in this report, and they will be available for 30 31 five years upon request to corresponding author. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 23 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 26 of 51

1 2 3 OB12 Group 4 Collaborating Investigators 5 6 Clinical Investigators 7 Healthcare Centre (HC) Guayaba: Tomás Gómez-Gascón*; Concepción Vargas-Machuca Cabañero; Mª 8 Isabel Gutiérrez-Sánchez; Mª Ángeles Fernández-Abad; José Antonio Granados-Garrido; Javier Martínez- 9 Suberviola; Margarita Beltejar-Rodríguez; Carmen Coello-Alarcón; Susana Diez-Arjona. 10 HC El Greco: José Enrique Mariño-Suárez*; Ana Ballarín-González; Ignacio Iscar-Valenzuela; José Luis 11 Quintana-Gómez; José Antonio González-Posada-Delgado; Enrique Revilla-Pascual; Esther Gómez- 12 Suarez; Yolanda Fernández-Fernández; Fernanda Morales-Ortiz; Isabel Ferrer-Zapata; Esperanza 13 Duralde-Rodríguez; Milagros Beamud-Lagos. 14 HC Barajas: Inmaculada González-García*; Mª del Pilar Serrano-Simarro; Cristina Montero-García; María 15 Domínguez-Paniagua; Sofía Causín-Serrano; Josefa Mª San Vicente-Rodríguez; Germán Reviriego-Jaén; 16 Mª Margarita Camarero-Shelly;For Rosapeer Mª Gómez-del-Forcallo. review only 17 HC Cuzco: Mar Noguerol-Álvarez*; María Ángeles Miguel-Abanto; Mª Lourdes Reyes-Martínez; Alejandro 18 Rabanal-Basalo; Carolina Torrijos-Bravo; Pilar Gutiérrez-Valentín; Jorge Gómez-Ciriano; Susana Parra 19 Román; Carolina Torrijos-Bravo; Judit León-González; Mª José Nebril-Manzaneque; Juana Caro-Berzal. 20 HC Mendiguchía Carriche: Francisca García-de Blas-González*; Belén Pose-García; Alberto López- 21 García-Franco; Mª Mar Álvarez-Villalba; Sonia Redondo-de-Pedro; Juan Carlos García-Álvarez; Elisa Viñuela-Beneitez; Marisa López-Martín; Nuria Sanz-López 22 . HC Buenos Aires: Paloma González-Escobar* Raquel Baños-Morras; Ana María Ibarra-Sánchez; Cecilio 23 ; Gómez-Almodóvar; Javier Muñoz-Gutiérrez; Carmen Molins-Santos; Cristina Cassinello-Espinosa. 24 HC Presentación Sabio: Antonio Molina-Siguero*; Rafael Sáez-Jiménez; Paloma Rodríguez-Almagro; Eva 25 María Rey-Camacho; María Carmen Pérez-García. 26 HC Santa Isabel: Rosa Fernández-García*; Antonio Redondo-Horcajo; Beatriz Pajuelo-Márquez; 27 Encarnación Cidoncha-Calderón; Mª Jesús Galindo Rubio; Rosa Ana Escriva Ferrairo; José Francisco 28 Ávila-Tomas; Francisco De-Alba-Gómez; Mª Jesús Gómez-Martín; Alma María Fernández-Martínez. 29 HC Fuentelarreina: Concepción Díaz-Laso*; Rosa Feijoó-Fernández; José Vizcaíno-Sánchez-Rodrigo; 30 Victoria Díaz-Puente; Felisa Núñez-Sáez; Luisa Asensio-Ruiz; Agustín Sánchez-Sánchez; Orlando 31 Enríquez-Dueñas; Silvia Fidel-Jaimez; Rafael Ruiz-Morote-Aragón; Asunción Pacheco-Pascua; Belén 32 Soriano-Hernández; Eva Álvarez-Carranza; Carmen Siguero-Pérez. 33 HC Juncal: Nuria Caballero-Ramírez*; Ana Morán-Escudero; María Martín-Martín; Francisco Vivas-Rubio. 34 HC Miguel de Cervantes: Alicia Herrero-de-Dios*; Rafael Pérez-Quero; Mª Isabel Manzano-Martín; César 35 Redondo-Luciáñez. 36 HC San Martín de Valdeiglesias: Nuria Tomás-García*; Carlos Díaz-Gómez-Calcerrada; Julia Isabel 37 Mogollo-García; Inés Melero-Redondo; Ricardo González-Gascón. 38 HC Lavapiés: Jesús Herrero-Hernández*; María Carmen Álvarez-Orviz; María Veredas González- 39 Márquez; Teresa San Clemente-Pastor; Amparo Corral-Rubio. 40 HC General Ricardos: Asunción Prieto-Orzanco*; Cristina de la Cámara-Gonzalez; Mª Mercedes Parrilla- 41 Laso; Mercedes Canellas-Manrique; Maria Eloisa Rogero-Blanco 42 Paulino Cubero-González; Sara Sanchez-Barreiro; Mª Ángeles Aragoneses-Cañas; Ángela Auñón-Muelas; 43 Olga Álvarez-Montes 44 HC María Jesús Hereza: Mar Álvarez-Villalba*; Petra María Cortes-Duran; Pilar Tardaguila-Lobato; Mar 45 Escobar-Gallegos; Antonia Pérez-de-Colosia-Zuil; Jaime Inneraraty-Martínez; María Jesús Bedoya-Frutos; María Teresa López-López; Nelly Álvarez-Fernández; Teresa Fontova-Cemeli; Josefa Marruedo-Mateo; 46 Josefa Díaz-Serrano; Beatriz Pérez-Vallejo. 47 HC Reyes Magos: Pilar Hombrados-Gonzalo*; Marta Quintanilla-Santamaría; Yolanda González-Pascual; 48 Luisa María Andrés-Arreaza; Soledad Escolar-Llamazares; Cristina Casado-Rodríguez; Luz Mª del Rey- 49 Moya; Mª Jesús Fernández-Valderrama; Alejandro Medrán-López; Julia Alonso-Arcas. 50 HC Barrio del Pilar: Alejandra Rabanal-Carrera*; Araceli Garrido-Barral; Milagros Velázquez-García; 51 Azucena Sáez-Berlanga; Mª Pilar Pérez-Egea; Rosario del Álamo-Gutiérrez; Pablo Astorga-Díaz; Carlos 52 Casanova-García; Ana Isabel Román-Ruiz; Mª Carmen Belinchón-Moya; Margarita Encinas-Sotillo; 53 Virtudes Enguita-Pérez. 54 HC Los Yébenes: Ester Valdés-Cruz*; Consuelo Mayoral-López; Alejandro Rabanal-Basalo; Teresa Gijón- 55 Seco; Francisca Martínez-Vallejo; Jesica Colorado-Valera. 56 57 58 24 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 27 of 51 BMJ Open

1 2 3 HC María Ángeles López Gómez: Ana Sosa-Alonso*; Jeannet Sánchez-Yépez*; Dolores Serrano- 4 González; Beatriz López-Serrano; Inmaculada Santamaría-López; Paloma Morso-Peláez; Carolina López- 5 Olmeda; Almudena García-Uceda-Sevilla; Petra María Cortés-Durán; Mercedes del Pilar Fernández-Girón. 6 HC Arroyo de la Media Legua: Leonor González-Galán*; Mariano Rivera-Moreno; Luis Nistal Martín-de- 7 Serranos; Mª Jesús López-Barroso; Margarita Torres-Parras; María Verdugo-Rosado; Mª Reyes Delgado- 8 Pulpón; Elena Alcalá-Llorente. 9 HC Federica Montseny: Sonsoles Muñoz-Moreno*; Isabel Vaquero-Turiño; Ana María Sánchez-Sempere; 10 Francisco Javier Martínez-Sanz; Clementa Sanz-Sanchez; Ana María Arias-Esteso. 11 HC Calesas: Diego Martín-Acicoya*; Pilar Kloppe-Villegas; Francisco Javier San-Andrés-Rebollo; 12 Magdalena Canals-Aracil; Isabel García-Amor; Nieves Calvo-Arrabal; María Milagros Jimeno-Galán. 13 HC Manuel Merino: Gloria de la Sierra-Ocaña*; María Mercedes Araujo-Calvo. 14 HC Doctor Cirajas: Julia Timoner-Aguilera*; María Santos Santander-Gutiérrez; Alicia Mateo-Madurga. 15 16 Technical Support GroupFor ** peer review only 17 Research Unit: Ricardo Rodríguez-Barrientos; Milagros Rico-Blázquez; Juan Carlos Gil-Moreno; Mariel Morey-Montalvo. Amaya Azcoaga Lorenzo. 18 Multiprofessional Teaching Units of Primary and Community Care: Gloria Ariza-Cardiel; Elena Polentinos- 19 Castro; Sonia Soto-Díaz; Mª Teresa Rodríguez-Monje. 20 Dirección Asistencial Sur: Susana Martín-Iglesias. 21 Pharmacy Department: María Luisa Sevillano-Palmero, Carmen Mateo-Ruiz, Beatriz Medina-Bustillo. 22 Agencia Pedro Laín Entralgo: Francisco Rodríguez-Salvanés; Marta García-Solano; Rocío González- 23 González; María Ángeles Martín-de la Sierra-San Agustín; María Vicente Herrero. 24 Hematology Department (Severo Ochoa): Ramón Rodríguez-González. 25 Endocrinology Department (HGCM): Irene Bretón-Lesmes. UICEC Hospital Ramón y Cajal, Plataforma 26 SCReN; Unidad de Farmacología Clínica, Hospital Ramón y Cajal, Madrid, España; Instituto Ramón y Cajal 27 de Investigación Sanitaria, IRYCIS: Mónica Aguilar Jiménez, Marta del Alamo Camuñas, Anabel Sánchez 28 Espadas, Marisa Serrano Olmeda, Mª Angeles Gálvez Múgica. 29 30 Principal Investigator: Teresa Sanz-Cuesta; Esperanza Escortell-Mayor; Isabel del Cura-González; Jesús 31 Martín-Fernández; Rosario Riesgo-Fuertes; Sofía Garrido-Elustondo. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 25 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 28 of 51

1 2 3 4 5 6 References 7 8 1. Hunt A, Harrington D, Robinson S. Vitamin B12 deficiency. Bmj. 2014;349(sep04 9 10 1):g5226–g5226. 11 12 2. Ströhle A, Richter M, González‐Gross M, Neuhäuser‐Berthold M, Wagner K, 13 14 Leschik‐Bonnet E, et al. The Revised D‐A‐CH‐Reference Values for the Intake of 15 16 For peer review only 17 Vitamin B 12 : Prevention of Deficiency and Beyond. Mol Nutr Food Res. 2019 Mar 18 19 28;63(6):1801178. 20 21 3. Dharmarajan TS, Norkus EP. Approaches to vitamin B12 deficiency. Early treatment may 22 23 24 prevent devastating complications. Postgrad Med. 2001;110(1):99–105; quiz 106. 25 26 4. Pawlak R. Is vitamin B12 deficiency a risk factor for cardiovascular disease in 27 28 vegetarians? Am J Prev Med. 2015;48(6):e11–26. 29 30 31 5. Wang H, Li L, Qin LL, Song Y, Vidal-Alaball J, Liu TH. Oral vitamin B 12 versus 32 33 intramuscular vitamin B 12 for vitamin B 12 deficiency. Cochrane Database Syst Rev. 34 35 2018 Mar 15;(3). 36 37 6. García Closas R, Serra Majem L, Sabater Sales G, Olmos Castellvell M, Ribas Barba L, 38 39 40 Salleras Sanmartí L. Distribución de la concentración sérica de vitamina C, ácido fólico y 41 42 vitamina B12 en una muestra representativa de la población adulta de Cataluña. Med Clin 43 44 (Barc). 2002;118(4):135–41. 45 46 47 7. Henríquez P, Doreste J, Deulofeu R, Fiuza MD, Serra-Majem L. Nutritional determinants 48 49 of plasma total homocysteine distribution in the Canary Islands. Eur J Clin Nutr. 50 51 2007;61(1):111–8. 52 53 54 8. Lindenbaum J, Rosenberg IH, Wilson PW, Stabler SP, Allen RH. Prevalence of 55 56 cobalamin deficiency in the Framingham elderly population. Am J Clin Nutr. 1994 57 58 26 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 29 of 51 BMJ Open

1 2 3 Jul;60(1):2–11. 4 5 6 9. Niafar M, Hai F, Porhomayon J, Nader ND. The role of metformin on vitamin B12 7 8 deficiency: a meta-analysis review. Intern Emerg Med. 2015 Feb;10(1):93–102. 9 10 10. Andrès E, Dali-Youcef N, Vogel T, Serraj K, Zimmer J. Oral cobalamin (vitamin B12) 11 12 treatment. An update. Int J Lab Hematol. 2009;31(1):1–8. 13 14 15 11. Watanabe F, Yabuta Y, Tanioka Y, Bito T. Biologically Active Vitamin B 12 Compounds 16 For peer review only 17 in Foods for Preventing Deficiency among Vegetarians and Elderly Subjects. J Agric 18 19 Food Chem. 2013 Jul;61(28):6769–75. 20 21 22 12. Langan RC, Zawistoski KJ. Update on vitamin B12deficiency. Am Fam Physician. 23 24 2011;83(12):1425–30. 25 26 13. Dali-Youcef N, Andrès E. An update on cobalamin deficiency in adults. QJM An Int J 27 28 29 Med. 2009;102(1):17–28. 30 31 14. Federici L, Loukili NH, Zimmer J, Affenberger S, Maloisel F, Andrès E. Manifestations 32 33 hématologiques de la carence en vitamine B12: données personnelles et revue de la 34 35 littérature. La Rev médecine interne. 2007;28(4):225–31. 36 37 38 15. Nilsson M, Norberg B, Hultdin J, Sandström H, Westman G, Lökk J. Medical intelligence 39 40 in Sweden. Vitamin B12: oral compared with parenteral? Postgrad Med J. 41 42 2005;81(953):191–3. 43 44 45 16. Vidal-Alaball, J, Butler, CC, Cannings-John, R, Goringe, A, Hood, K, McCaddon, A, 46 47 McDowell, I, Papaioannou A. Oral vitamin B12 versus intramuscular vitamin B12 for 48 49 vitamin B12 deficiency. Cochrane Database Syst Rev. 2005;(3). 50 51 52 17. Metaxas C, Mathis D, Jeger C, Hersberger KE, Arnet I, Walter P. Early biomarker 53 54 response and patient preferences to oral and intramuscular vitamin B12 substitution in 55 56 57 58 27 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 30 of 51

1 2 3 primary care: a randomised parallel-group trial. Swiss Med Wkly. 2017;147(1314):1–9. 4 5 6 18. Kwong JC, Carr D, Dhalla IA, Tom-Kun D, Upshur RE. Oral vitamin B12therapy in the 7 8 primary care setting: a qualitative and quantitative study of patient perspectives. BMC 9 10 Fam Pract. 2005 Dec 21;6(1):8. 11 12 19. Houle SKD, Kolber MR, Chuck AW. Should vitamin B12 tablets be included in more 13 14 15 Canadian drug formularies? An economic model of the cost-saving potential from 16 For peer review only 17 increased utilisation of oral versus intramuscular vitamin B12 maintenance therapy for 18 19 Alberta seniors. BMJ Open. 2014 May 2;4(5):e004501. 20 21 22 20. Graham ID, Jette N, Tetroe J, Robinson N, Milne S, Mitchell SL. Oral cobalamin remains 23 24 medicine’s best kept secret. Arch Gerontol Geriatr. 2007;44(1):49–59. 25 26 21. Shatsky M. Evidence for the use of intramuscular injections in outpatient practice. Am 27 28 29 Fam Physician. 2009;79(4):297–300. 30 31 22. Rabuñal Rey R, Monte Secades R, Peña Zemsch M, Bal Alvaredo M, Gómez Gigirey A. 32 33 ¿Debemos utilizar la vía oral como primera opción para el tratamiento del déficit de 34 35 vitamina B12? Rev Clínica Española. 2007;207(4):179–82. 36 37 38 23. Kuzminski a M, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J. Effective treatment 39 40 of cobalamin deficiency with oral cobalamin. Blood. 1998;92(4):1191–8. 41 42 24. Bolaman Z, Kadikoylu G, Yukselen V, Yavasoglu I, Barutca S, Senturk T. Oral Versus 43 44 45 Intramuscular Cobalamin Treatment in Megaloblastic Anemia: A Single-Center, 46 47 Prospective, Randomized, Open-Label Study. Clin Ther. 2003;25(12):3124–34. 48 49 25. Saraswathy AR, Dutta A, Simon EG, Chacko A. Randomized open label trial comparing 50 51 52 efficacy of oral versus intramuscular vitamin b12 supplementation for treatment of 53 54 vitamin b12 deficiency. In: Gastroenterology. AGA; 2012. p. S-216. 55 56 57 58 28 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 31 of 51 BMJ Open

1 2 3 26. D’Agostino RB, Massaro JM, Sullivan LM. Non-inferiority trials: design concepts and 4 5 6 issues - the encounters of academic consultants in statistics. Stat Med. 2002;22(2):169–86. 7 8 27. Sanz-Cuesta T, González-Escobar P, Riesgo-Fuertes R, Garrido-Elustondo S, del Cura- 9 10 González I, Martín-Fernández J, et al. Oral versus intramuscular administration of vitamin 11 12 B12 for the treatment of patients with vitamin B12 deficiency: a pragmatic, randomised, 13 14 15 multicentre, non-inferiority clinical trial undertaken in the primary healthcare setting 16 For peer review only 17 (Project OB12). BMC Public Health. 2012;12:394. 18 19 28. Herdman M, Badia X, Berra S. El EuroQol-5D: una alternativa sencilla para la medición 20 21 22 de la calidad de vida relacionada con la salud en atención primaria. Atención Primaria. 23 24 2001;28(6):425–9. 25 26 29. Piaggio G, Elbourne DR, Pocock SJ, Evans SJW, Altman DG, CONSORT Group. 27 28 29 Reporting of noninferiority and equivalence randomized trials: extension of the 30 31 CONSORT 2010 statement. JAMA. 2012 Dec;308(24):2594–604. 32 33 30. Jones B, Jarvis P, Lewis J a, Ebbutt a F. Trials to assess equivalence: the importance of 34 35 rigorous methods. BMJ. 1996;313(7048):36–9. 36 37 38 31. Jakobsen JC, Gluud C, Wetterslev J, Winkel P. When and how should multiple imputation 39 40 be used for handling missing data in randomised clinical trials – a practical guide with 41 42 flowcharts. BMC Med Res Methodol. 2017 Dec 6;17(1):162. 43 44 45 32. Blough DK, Madden CW, Hornbrook MC. Modeling risk using generalized linear models. 46 47 J Health Econ. 1999 Apr;18(2):153–71. 48 49 33. Hardin J, Hilbe J. Generalized Linear Models and Extensions. Third Edit. College Station: 50 51 52 Stata Press; 2012. 53 54 34. Andrès E, Vidal-Alaball J, Federici L, Loukili NH, Zimmer J, Kaltenbach G. Clinical 55 56 57 58 29 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 32 of 51

1 2 3 aspects of cobalamin deficiency in elderly patients. Epidemiology, causes, clinical 4 5 6 manifestations, and treatment with special focus on oral cobalamin therapy. Eur J Intern 7 8 Med. 2007 Oct;18(6):456–62. 9 10 35. van Uffele J, Chin A Paw M, Hopman-Rock M, W. van M. The effect of walking and 11 12 vitamin B supplementation on quality of life in community-dwelling adults with mild 13 14 15 cognitive impairment : a randomized , controlled trial. 2007;1137–46. 16 For peer review only 17 36. Hvas A, Juul S, Nexø E, Ellegaard J. Vitamin B-12 treatment has limited effect on health- 18 19 related quality of life among individuals with elevated plasma methylmalonic acid : a 20 21 22 randomized placebo-controlled study. 2003;c:146–52. 23 24 37. Moleiro J, Mão de Ferro S, Ferreira S, Serrano M, Silveira M, Dias Pereira A. Efficacy of 25 26 Long-Term Oral Vitamin B12 Supplementation after Total Gastrectomy: Results from a 27 28 29 Prospective Study. GE - Port J Gastroenterol. 2018;25(3):117–22. 30 31 38. Bensky MJ, Ayalon-Dangur I, Ayalon-Dangur R, Naamany E, Gafter-Gvili A, Koren G, 32 33 et al. Comparison of sublingual vs. intramuscular administration of vitamin B12 for the 34 35 treatment of patients with vitamin B12 deficiency. Drug Deliv Transl Res. 2019;9(3):625– 36 37 38 30. 39 40 39. Loudon K, Treweek S, Sullivan F, Donnan P, Thorpe KE, Zwarenstein M. The PRECIS-2 41 42 tool: designing trials that are fit for purpose. BMJ. 2015 May;350(04 May-10 43 44 45 May):h2147. 46 47 40. Berger JO, Bayarri MJ. The Interplay of Bayesian and Frequentist Analysis. Stat Sci. 48 49 2004;19(1):58–80. 50 51 52 53 54 55 56 57 58 30 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 33 of 51 BMJ Open

1 2 3 Figure legends 4 5 6 Figure 1. Trial profile 7 8 Figure 2. Difference between the oral and intramuscular routes in the proportion of patients 9 10 whose VB12 levels returned to normal (≥211 pg/ml) 11 12 13 14 15 16 For peer review only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 31 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 2342BMJ assessed Open for eligibility Page 34 of 51

Week 8 133/135 (98.5) 129/130 (99.2) -0.7 (-3.2 to 1.8) 1 For peer review only 2 Week 26 115/132 (87.1) 122/122 (100.0) -12.9 (-17.9 to -6.1)

3PPT 4 5 Week 52 103/112 (92.0) 115/117 (98.3) -6.3 (-11.9 to -0.07) 6 7 8 Week 8 133/140 (95.0) 129/143 (90.2) 4.8 (-1.3 to 10.9) 9

10AIT Week 26 115/140 (82.1) 122/143 (85.3) -3.2 (-11.8 to 5.4) 11 12 13 Week 52 103/140 (73.6) 115/143 (80.4) -6.8 (-16.6 to 2.9) 14 15 16 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml-20 -10 0 10 20 17 Diff proportions 18 Favors IM Favors oral 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 BMJ Open Page 36 of 51

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 5 STUDYPROTOCOL Open Access 6 7 8 Oral versus intramuscular administration of 9 10 vitamin B12 for the treatment of patients with 11 12 13 vitamin B12 deficiency: a pragmatic, randomised, 14 15 multicentre, non-inferiority clinical trial 16 For peer review only 17 undertaken in the primary healthcare setting 18 19 (Project OB12) 20 1* 2 3 4 21 Teresa Sanz-Cuesta , Paloma González-Escobar , Rosario Riesgo-Fuertes , Sofía Garrido-Elustondo , 22 Isabel del Cura-González5, Jesús Martín-Fernández6, Esperanza Escortell-Mayor7, Francisco Rodríguez-Salvanés8, 23 Marta García-Solano9, Rocío González-González10, María Ángeles Martín-de la Sierra-San Agustín11, 24 Carmen Olmedo-Lucerón12, María Luisa Sevillano Palmero13, Carmen Mateo-Ruiz14, Beatriz Medina-Bustillo15, 25 Antonio Valdivia-Pérez16, Francisca García-deBlas-González17, José Enrique Mariño-Suárez18, 26 19 20 21 22 27 Ricardo Rodríguez-Barrientos , Gloria Ariza-Cardiel , Luisa MaríaCabello-Ballesteros , Elena Polentinos-Castro , 23 24 25 26 28 Milagros Rico-Blázquez , Ma Teresa Rodríguez-Monje , Sonia Soto-Díaz , Susana Martín-Iglesias , 29 Ramón Rodríguez-González27, Irene Bretón-Lesmes28, María Vicente-Herrero29, Jesús Sánchez-Díaz30, 30 Tomás Gómez-Gascón31, Mercedes Drake-Canela32, Ángel Asúnsolo-del Barco33 and OB12 Group34 31 32 33 Abstract 34 35 Background: The oral administration of vitamin B12 offers a potentially simpler and cheaper alternative to 36 parenteral administration, but its effectiveness has not been definitively demonstrated. The following protocol was 37 designed to compare the effectiveness of orally and intramuscularly administered vitamin B12 in the treatment of 38 patients ≥65 years of age with vitamin B12 deficiency. 39 Methods/design: The proposed study involves a controlled, randomised, multicentre, parallel, non-inferiority clinical 40 trial lasting one year, involving 23 primary healthcare centres in the Madrid region (Spain), and patients ≥65 years of 41 age. The minimum number of patients required for the study was calculated as 320 (160 in each arm). Bearing in 42 mind an estimated 8-10% prevalence of vitamin B12 deficiency among the population of this age group, an initial 43 sample of 3556 patients will need to be recruited. 44 Eligible patients will be randomly assigned to one of the two treatment arms. In the intramuscular treatment arm, 45 vitamin B12 will be administered as follows: 1 mg on alternate days in weeks 1 and 2, 1 mg/week in weeks 3–8,and 46 1 mg/month in weeks 9–52. In the oral arm, the vitamin will be administered as: 1 mg/day in weeks 1–8 and 1 47 mg/week in weeks 9–52. The main outcome variable to be monitored in both treatment arms is the normalisation 48 of the serum vitamin B12 concentration at weeks 8, 26 and 52; the secondary outcome variables include the serum 49 concentration of vitamin B12 (in pg/ml), adherence to treatment, quality of life (EuroQoL-5D questionnaire), patient 50 51 52 * Correspondence: [email protected] 53 1Unidad de Apoyo a la Investigación. Gerencia Atención Primaria, Servicio 54 Madrileño de Salud, Calle Espronceda 24, Madrid 28003, Spain 55 Full list of author information is available at the end of the article 56 © 2012 Sanz-Cuesta et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the 57 Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 37 of 51 BMJ Open

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 2 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 5 3satisfaction and patient preferences. All statistical tests will be performed with intention to treat and per protocol. 6 Logistic regression with random effects will be used to adjust for prognostic factors. Confounding factors or factors 7 that might alter the effect recorded will be taken into account in analyses. 8 Discussion: The results of this study should help establish, taking quality of life into account, whether the oral 9 administration of vitamin B12 is an effective alternative to its intramuscular administration. If this administration 10 route is effective, it should provide a cheaper means of treating vitamin B12 deficiency while inducing fewer 11 adverse effects. Having such an alternative would also allow patient preferences to be taken into consideration at 12 the time of prescribing treatment. 13 Trial registration: This trial has been registered with ClinicalTrials.gov, number NCT 01476007, and under EUDRACT 14 number 2010-024129-20. 15 16 For peer review only 17 Background returned values of 1.9% and 3.4% respectively. What does 18 Vitamin B12 (cyanocobalamin), along with other deriva- appear to be constant in all studies reviewed for the 19 tives of folic acid, is a nutrient essential for the synthesis present work is that the prevalence of deficiency is 20 of DNA. Its deficiency is manifested through changes in greater among people aged 65–76 years. For example, 21 the number and morphology of erythrocytes, leucocytes the above Catalonian and Canary Island studies returned 22 and platelets, and by neurological alterations owed to the values of 3.8% and 8.5% for these age groups. Among 23 progressive demineralisation of the nervous system (a elderly patients belonging to the Framingham cohort, 24 consequence of defective myelin synthesis). Vitamin B12 Lidenbaun [9] observed a prevalence of over 5.3%. Other 25 is found mostly in food of animal origin. It is separated authors [10,11], however, report figures of 30-40% in eld- 26 from ingested food through the action of the gastric acid, erly people with degenerative neuropsychiatric disorders 27 and in the duodenum the vast majority binds to intrinsic and those receiving institutionalised care. 28 factor (IF). The vitamin B12/IF complex formed, which In the elderly, the symptoms of vitamin B12 deficiency 29 is very resistant to digestion, is then absorbed by endo- caused by deficient diets and/or digestive and/or absorp- 30 cytosis in the terminal ileum. Only 1-2% of vitamin B12 tion problems can be nonspecific, making a diagnosis of 31 absorption occurs independent of IF [1]. Daily vitamin deficiency more difficult. For example, up to 40% of eld- 32 B12 requirements vary between 1 and 2 μg/day in adults erly people show no haematological alterations. Further, 33 [2]. A balanced diet, however, provides somewhere be- neurological symptoms may appear before those of an- 34 tween 7 and 30 μg/day. Some of this excess can be stored aemia; indeed, only about 60% of elderly people with 35 (some 2–5 mg), meaning that deficiency symptoms may vitamin B12 deficiency are anaemic [12]. 36 not occur until 3–5 years after the diet fails to provide In primary healthcare in Spain, vitamin B12 deficiency 37 sufficient vitamin B12 or its absorption becomes inad- is diagnosed via the determination of the serum concen- 38 equate [3]. tration of the vitamin. Some studies [13-17] have 39 In the primary healthcare setting, the most commonly described the limitations of trying to diagnose vitamin 40 seen causes of vitamin B12 deficiency are related to ab- B12 deficiency exclusively via the measurement of this 41 normalities of digestion (atrophic gastritis, achlorhydria concentration, and report blood methylmalonic acid 42 or the consequences of gastrectomy) or absorption (MMA) and homocysteine concentrations to be more 43 (autoimmune pernicious anaemia, chronic pancreatitis, sensitive markers capable of detecting subclinical 44 ’ Crohn s disease, the effect of medications that alter the deficiency. 45 mucosa of the ileum, or the consequences of surgical re- The traditional treatment of vitamin B12 deficiency 46 section), and, to a lesser extent, a lack of exogenous sup- is the intramuscular injection of cyanocobalamin, 47 ply. The exact prevalence of vitamin B12 deficiency in generally 1 mg/day for one week, followed by 1 mg/ 48 industrialised countries is unknown; indeed, different week for one month, and then 1 mg every 1 or 49 studies using different definitions have reported it as be- 2monthsad perpetuum [4,18,19]. The vitamin may, 50 tween 5% and 60% [4]. Results have even differed widely however, be offered orally. In some circles this route 51 between similar studies using an identical definition of has been regarded as an effective alternative to par- 52 deficiency, and after stratifying by age [5]. In Spain, the enteral administration since the 1950s, during which 53 prevalence of vitamin B12 deficiency may reach 18% time several studies showed serum vitamin B12 con- 54 according to a meta-analysis of the studies undertaken centration to normalise after taking large oral doses. 55 up to 1999 [6]. However, population-based studies per- These results prompted the spread of oral adminis- 56 formed in Catalonia and the Canary Islands [7,8], both of tration in Sweden and Canada [3]. In the former 57 which used a serum vitamin B12 cut-off of 200 pg/ml, country, 13% of the population over 70 years of age 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 38 of 51

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 3 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 now receives treatment for vitamin B12 deficiency, include avoiding the displacement of patients to re- 5 with two of every three patients treated via the oral ceive injections, avoiding the discomfort of injection, 6 route [20]. However, in the rest of the world, the and a reduction in treatment costs [28,29]. 7 parenteral route remains the most used. Indeed, con- A further question still to be answered is that of the 8 troversy still surrounds the advantages and effective- optimum dose when using the oral route [3]. 9 ness of the oral route. Some authors question its use In summary, despite many studies indicating the oral 10 [21] while others favour it, although the methodo- administration of vitamin B12 to be easy, effective and 11 logical limitations of the evidence they provide less costly than intramuscular administration, their 12 means no firm conclusions can be drawn. In reviews designs, and in some cases their methodological limita- 13 of the literature published between 1999 and 2007, tions, mean that debate still surrounds the effectiveness 14 Daly-Youcef [4] and Andrés E [19] concluded that of the oral route. This may help explain why it is little 15 orally administered vitamin B12 provided effective used by health professionals [30]. 16 treatment for adult andFor elderly peer patients with review defi- Although someonly authors [31,32] recommend the use of 17 ciencies, although they highlighted that further stud- moderately high doses (which have obtained the best 18 ies were needed to determine its effectiveness in results), studies are still being performed to investigate this. 19 patients with severe neurological symptoms. Federicia In a randomised clinical trial involving five treatment arms 20 [22], who reviewed the treatment criteria followed in with doses of between 2.5 μg/day and 1000 μg/day, Eussen 21 different studies, concluded oral administration to be [33] concluded that a dose of at least 600 μg/day was 22 effective, but recommended further work to confirm required to obtain adequate results. However, in guidelines 23 this. Shatsky[23], who examined evidence derived published in 2012, the British Columbia Medical Associ- 24 from the use of oral and intramuscular administra- ation (Canadian Ministry of Health) recommended a dose 25 tion, indicated that high dose oral administration of 1000 μg/day for pernicious anemia or food-bound co- 26 appeared to be safe, effective and cost-effective, al- balamin malabsorption [34]. 27 though long term clinical trials were required to con- The proposed study examines the questions that, 28 firm this. In a prospective study performed in Spain according to the Cochrane review mentioned above [3], 29 involving commercially available multi-vitamin sup- are still to be answered, via a clinical trial (of ample dur- 30 plements, Rabuñal et al. [24] reported the effective- ation and with a large number of patients) in the primary 31 ness and tolerance of oral vitamin B12 to be healthcare setting. As recommended, one of the out- 32 excellent, but also indicated that the dosage to be comes examined is quality of life. The results obtained 33 used was yet to fully established. In 2005, a should provide high quality scientific evidence of use 34 Cochranereview[3]waspublishedthatexamined when taking treatment decisions in the primary health- 35 two randomised clinical trials - those reported by care centres, while allowing patient preference of admin- 36 Kuzminski [2] and Bolaman [25] - that studied the istration route to be taken into consideration. The 37 effectiveness of oral vs. intramuscular administration results may reveal oral treatment with vitamin B12 to be, 38 of vitamin B12 for the treatment of its deficiency. as Lederle [35] put it, “medicine’s best kept secret”. 39 The Kuzminski trial involved 33 patients (18 in the Aim 40 oral arm and 15 in the intramuscular arm), while the The aim of the proposed protocol is to compare the ef- 41 Bolaman trial involved 60 (26 in the oral arm and 15 fectiveness of orally and intramuscularly administered 42 in the intramuscular arm). The Cochrane concluded vitamin B12 in the normalisation of serum vitamin B12 43 that orally administered vitamin B12 appeared to be concentrations at 8, 26 and 52 weeks of treatment, in 44 as effective as the intramuscular route with respect patients aged ≥65 years with vitamin B12 deficiency trea- 45 to the short-term haematological and neurological ted at primary healthcare centres in the Madrid region, 46 responses observed in patients with deficiencies, but Spain. The secondary outcomes to be measured include 47 highlighted methodological limitations in both trials. the safety of both administration routes, quality of life 48 A large clinical trial was called for in the primary (measured using the EuroQoL-5D questionnaire) and ad- 49 healthcare setting, where a high percentage of herence to treatment. Patient preferences and satisfac- 50 patients with vitamin B12 deficiency is seen. The tion with treatment will also be recorded, along with 51 Cochrane review also underscored the need to in- 52 patient sociodemographic profiles, lifestyle habits, and clude a measurement of the quality of life as an out- 53 the clinical manifestation of each patient’s deficiency. come, and patient preference at the time of 54 prescribing treatment. Among other variables, three Methods/design 55 studies [24,26,27] have recorded patient views on the Study type 56 administration route, and record a high level of ac- This study takes the form a pragmatic, randomised, mul- 57 ceptance of the oral route, the advantages of which ticentre, non-inferiority clinical trial undertaken in the 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 39 of 51 BMJ Open

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 4 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 primary healthcare setting, with a duration of one year. severe systemic disease 5 For ethical reasons, a placebo controlled trial would not having been involved in any other trial involving 6 be appropriate [36]. the administration of any experimental treatment 7 The study involves 23 primary healthcare centres in in the 28 days prior to the start of the present 8 the Madrid region of Spain. The research team is com- study 9 posed of a clinical assistance group of 169 general practi- being treated for HIV, HVB or HVC infection 10 tioners and nurses, and a technical group of 22 health hypersensitivity to vitamin B12, or any of the 11 professionals including doctors of different specialities, vitamin preparation’s excipients 12 nurses and pharmacists. For the undertaking of field- receiving anticoagulation treatment 13 work, these 191 team members are divided into smaller being away from home and with no intention of 14 groups (with similar numbers of clinical and technical residing for the following year in the health 15 personnel), each in charge of one of five subprojects. district where consultation was made 16 Each subproject is led byFor a member peer of the technical review failing only to meet any inclusion criterion 17 personnel. Together, these five leaders form the coordin- limitations regarding oral treatment 18 ation group for the trial as a whole. 19 The trial protocol was approved by the Madrid Region Randomisation 20 Clinical Research Ethics Committee (Comité Ético de Inves- Participants will be enrolled consecutively by their gen- 21 tigación Clínica Regional de la Comunidad de Madrid)on eral practitioners when attending a primary healthcare 22 February 8th 2011, and has been registered with Clinical- centre in the study area (Figure 1). All patients without 23 Trials.gov number NCT 01476007, and under EUDRACT reason to be excluded will be invited to participate. 24 number 2010-024129-20 [Oral Versus Intramuscular Co- Those patients that accept this invitation will provide 25 balamin to treat Cobalamin Deficiency: Noninferiority ran- written, informed consent to be included. A blood sam- 26 domised controlled trial, pragmatic and multi-center in the ple will then be taken and part of this used to determine 27 primary healthcare setting (OB12 project)]. the serum vitamin B12 concentration (pg/ml). In those 28 returning a value of <179 pg/ml (defined as vitamin B12 29 Patients deficiency by the reference analytical laboratory analys- 30 ing the samples collected), the remaining fraction of the 31 1. Inclusion criteria: all participants must: sample will be analysed to provide a haemogram (reticu- 32 be ≥65 years of age locyte, erythrocyte, leucocyte and platelets counts), the 33 be attending a primary healthcare centre for values of biochemical variables (glucose, creatinine, 34 consultation on some medical matter GOT, GPT, GGT and ferritin), the folic acid concentra- 35 provide their informed consent to be included tion, and an anti-IF antibody count. Those who meet all 36 have a serum B12 concentration of <179 pg/ml. inclusion criteria, and no exclusion criteria, will then be 37 2. Exclusion criteria: patients meeting any of the randomly assigned to one arm of the treatment, i.e., oral 38 following conditions will be excluded: or intramuscular administration of vitamin B12. This will 39 having been treated (under medical prescription) be performed by means of a simple randomisation 40 in the last five years for vitamin B12 deficiency process performed by the electronic data collection sys- 41 serious neurological or psychiatric symptoms, tem. This guarantees that neither researcher nor patient 42 including psychotic problems has any choice with respect to the group to which the 43 dementia preventing the giving of informed latter is assigned. 44 consent to take part 45 atrophy of the optic nerve Sample size 46 serum folic acid concentration of <2.3 ng/ml The sample size required was determined bearing in 47 stage 4 kidney disease 4 (estimated glomerular mind the results of Kuzminski et al. [2]. In the latter 48 filtration rate [GFR] 15–29 ml/min) study the parenteral administration of vitamin B12 was 49 having received/suffering malabsorption-related: associated with an increase in serum concentrations of 50 ○ surgery or diseases affecting the jejunum-ileum the vitamin of >200 pg/ml at 4 months in over 70% of 51 ○ inflammatory-intestinal disease, e.g., Crohn’s patients. For the present trial, the level of non-inferiority 52 disease, ulcerative colitis of the oral treatment is set at a difference (delta) in re- 53 ○ celiac disease sponse compared to the parenteral treatment of ≤10%. 54 chronic pancreatitis This threshold was set given its importance from a clin- 55 myelodisplasia or malignant blood disease ical rather than a statistical viewpoint, and since it falls 56 haemophilia or other coagulation problems within the range normally accepted for this type of study 57 contraindicating parenteral administration [37]. 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 40 of 51

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 5 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 5 6 Patients ≥65 years of age assigned to No 7 the participating health centres and Informed consent EXCLUSION with no exclusión criteria given? No consent given 8 N= 3556 9 10 Yes 11 12 No 13 Vitamin B12<179pg/mL EXCLUSION 14 Inclusion criteria not met 15 16 For peer Yesreview only 17 18 19 Yes 20 Exclusion criteria EXCLUSION detected in analytical Exclusion criteria detected 21 results 22 23 24 No 25 26 27 Randomisation (n=320) 28 29 30 31 32 33 Oral group IM group 34 n=160 n=160 35 36 37 38 Follow-up 39 8, 26 and 52 40 weeks 41 42 Figure 1 Patient recruitment. 43 44 45 Assuming that the percentage of patients showing an in- the original number of patients to be enrolled so that 320 46 crease in serum vitamin B12 concentration to above with a vitamin B12 deficiency can be guaranteed is 3556. 47 179 pg/ml in both groups is 70%, means the study requires 48 at least 304 patients (152 in each arm) for a threshold of Blinding 49 non-inferiority of 10% and a statistical power of 60% with In studies with the present design it is impossible to 50 significance set at p < 0.05. Given the type of patients to blind the patient to the treatment received. However, 51 be studied, i.e., patients who have come to the health cen- this limitation is compensated for by the objective 52 tres for consultation, plus the fact that their own family measurement of the main outcome variable (the 53 doctors are members of the research team, a loss to fol- serum vitamin B12 concentration) and the random- 54 low-up of under 5% is expected. The minimum starting isation of the patients to the treatment groups. Fur- 55 sample size for each arm was therefore deemed to be ther, the persons charged with the statistical analysis 56 n = 160. With an expected prevalence of vitamin B12 defi- of the data will be blind to the identity of the 57 ciency of 8-10% (a figure of 9% was used in calculations), patients in each treatment arm. 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 41 of 51 BMJ Open

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 6 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 The intervention – Request analyses to be performed one week 5 The pharmaceutical formulations to be used in the study before next visit: haemogram and serum vitamin 6 are commercially available in Spain. The treatments will B12 7 involve: – Therapeutic plan: patient in oral arm – provision 8 of medication; patient in intramuscular arm – 9 Intramuscular route: 1 mg of vitamin B12 on provide appointments for injections 10 alternate days during weeks 1 and 2; 1 mg/week over 11 Visit 2 (week 8) weeks 3–8 (i.e., for 6 weeks); and 1 mg/month from 12 – Anamnesis: record lifestyle habits and use of weeks 9–52 13 alcohol Oral route: 1 mg/day of vitamin B12 for 8 weeks; 14 – Symptoms: if pathological at the first visit, record 1 mg/week from weeks 9–52 15 paresthesia, asthenia, loss or reduction of 16 Patients in both arms willFor undergo peer analytical monitor- reviewappetite, only sadness or change in level of happiness, 17 ing in weeks 8, 26 and 52. They will receive appoint- and concomitant pharmacological treatment 18 ments for the appropriate dates. The response to – Physical examination: if pathological at the first 19 treatment will be recorded alongside adherence to treat- visit examine for Hunter’s glositis, positional and 20 ment and the appearance of any adverse effects. vibrational sensitivity 21 – Record concomitant treatment 22 Work plan – Request analyses to be performed one week 23 Before work begins, the project will be presented to all before next visit: haemogram and serum vitamin 24 the research team members in a special meeting. Train- B12 25 ing sessions lasting 2–3 h will also be held at each par- – Questionnaires: EuroQoL-5D 26 ticipating health centre. These will involve a review of – Assessment of adverse effects 27 the inclusion and exclusion criteria, provide instructions – Therapeutic plan: patient in oral arm – provision 28 regarding the intervention, and examine the ethical of medication; patient in intramuscular arm – 29 requirements to be met for the trial to be held. provide appointments for injections – – 30 The procedures to be followed and information to be Assess adherence to treatment: oral route 31 recorded at each of a patient’s visits to a participating count number of vials used; intramuscular route: 32 health centre is as follows: count injections given 33 Selection Visit Visit 3 (week 26) 34 – – Signing of informed consent Anamnesis: record lifestyle habits and use of 35 – Assessment of inclusion/exclusion criteria alcohol 36 – – Recording of demographic data (age and sex) Symptoms: if pathological at the first visit, record 37 – Analysis: serum vitamin B12. If concentration is paresthesia, asthenia, loss or reduction of 38 <179 pg/ml the following analyses are to be appetite, sadness or change in level of happiness, 39 requested: haemogram, biochemical analysis and concomitant pharmacological treatment 40 – (glucose, creatinine, GOT/GPT/GGT), ferritin, Physical examination: if pathological at the first 41 ’ folic acid, anti-IF antibody level. If serum vitamin visit examine for Hunter s glositis, positional and 42 B12 concentration is >179 pg/ml: patient vibrational sensitivity 43 – preference questionnaire Record concomitant treatment 44 – – Randomisation of patients to treatment group Request analyses to be performed one week 45 Visit 1 (start of treatment) before next visit: haemogram and serum vitamin 46 – Anamnesis: record whether the patient lives alone B12 47 – or with others, lifestyle habits, use of alcohol, Questionnaire: EuroQoL-5D 48 – whether a vegan diet is followed, whether the Assessment of adverse effects 49 – – patient has undergone gastrectomy Therapeutic plan: patient in oral arm provision 50 – – Symptoms: record paresthesia, asthenia, loss or of medication; patient in intramuscular arm 51 reduction of appetite, sadness or change in state provide appointments for injections 52 – – of mind, concomitant pharmacological treatment Assess adherence to treatment: oral route 53 – Physical examination: for Hunter’s glositis, count number of vials used; intramuscular route: 54 positional and vibrational sensitivity count injections given 55 – Questionnaires: Lobo cognitive mini-exam, Visit 4 (week 52) 56 EuroQoL-5D – Anamnesis: record lifestyle habits and use of 57 – Record concomitant treatment alcohol 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 42 of 51

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 7 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 – Symptoms: record paresthesia, asthenia, loss or chloramphenicol, Fe, vitamin C and other vitamin 5 reduction of appetite, sadness or change in level supplements. 6 of happiness, and concomitant pharmacological 7 treatment Losses and withdrawals 8 – Physical examination: for Hunter’s glositis, Patients will be removed from the trial if any of the fol- 9 positional and vibrational sensitivity lowing conditions are met: 10 – Record concomitant treatment Serum vitamin B12 concentration still <179 pg/ml 11 – Questionnaires: EuroQoL-5D, satisfaction and after 8 weeks of treatment. Treatment will be 12 preferences deemed to have failed in these patients, and they will 13 – Assessment of haemogram and serum vitamin be further studied and treated outside the trial 14 B12 concentration according to normal clinical practice. 15 – Assessment of adverse effects Serious adverse events. 16 – Assess adherenceFor to treatment: peer oral route – review Voluntary only withdrawal or violation of the protocol. 17 count number of vials used; intramuscular route: At least two attempts will be made to contact by tele- 18 count injections given phone those patients who do not come for their sched- 19 uled visits. All patients will be informed that they can 20 abandon the study at any time without this affecting 21 Variables their future medical treatment in any way. 22 Outcome variables 23 The main outcome to be measured is the normalisation > Analysis 24 of the serum vitamin B12 concentration ( 179 pg/ml) at Descriptive analysis of the patients 25 8, 26 and 52 weeks. The secondary outcomes will be the The trial will involve a descriptive statistical analysis of 26 serum vitamin B12 concentration (pg/ml), adverse events the baseline characteristics of patients in both treatment 27 (description, moment of onset and resolution, intensity, arms. Quantitative variables will be described in terms of 28 cause, steps taken), adherence to treatment (measured at their measure of central tendency, mean or median (for 29 each patient visit via the number of vials used for those showing asymmetric distributions), and the corre- 30 patients in the oral arm, and the number of injections sponding dispersion, standard deviation or interquartile 31 given in the intramuscular arm), quality of life (measured range. Qualitative variables will be described in terms of 32 using the EuroQoL-5D questionnaire), and patient satis- proportions and their corresponding confidence 33 faction and preferences. intervals. 34 35 Anamnesis, demographic and lifestyle information Baseline comparisons 36 Including age, sex, whether the patient lives alone or The Student t test or Mann–Whitney U test (when the 37 with others, whether a vegan diet is followed, and the normal hypothesis is rejected) will be used to determine 38 use of alcohol (g/week). whether the two treatment arms are comparable based 39 on their quantitative baseline characteristics and known 40 Clinical variables prognostic factors. Comparisons on qualitative variables 41 Symptoms such as paresthesia, asthenia, loss or reduc- will be undertaken using the Pearson Chi-squared test or 42 tion of appetite, sadness or change in state of mind (an- Fisher’s Exact test as required. If cases of inequality are 43 amnesis), Hunter’s glositis, positional and vibrational detected, the confounding factors will be defined and ap- 44 sensitivity (all via physical examination), and cognitive propriate adjustments made. 45 decline (Lobo test). 46 Analysis of effectiveness of treatment (main outcome) at the 47 Analytical variables three monitoring points 48 Haemogram (complete blood cell and platelet count) Intention-to-treat and per-protocol analyses will both be 49 and biochemical analysis (folic acid, glucose, creatinine, performed, as is recommended for non-inferiority studies 50 GOT, GPT, GGT, ferritin, anti-IF antibodies). Blood ana- [38]. 51 lyses will be performed in plasma or serum as required The effectiveness of treatment will be analysed by 52 and under standard conditions. examining the therapeutic success achieved in each arm 53 at 8, 26 and 52 weeks, determining the 95% confidence 54 Concomitant treatment interval for the percentage of patients in each treatment 55 Recording of the taking of protein pump inhibitors, H2 arm whose serum vitamin B12 concentrations become 56 receptor antagonists, antacids, potassium, metformin, normalised. If the confidence intervals do not fall outside 57 colchicine, neomycin, p-aminosalicylic acid, parenteral the non-inferiority limit (10%), it can be concluded that 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 43 of 51 BMJ Open

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 8 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 the oral treatment is not inferior to the intramuscular Discussion 5 treatment. The within-patient percentage change in From a clinical point of view, the results obtained will 6 serum vitamin B12 concentration at each monitoring help establish whether the oral administration of vitamin 7 point will be determined, and the confidence intervals B12 is as effective as intramuscular treatment in the nor- 8 for the difference in the mean values for each arm malisation of serum vitamin B12 concentrations in 9 calculated. patients ≥65 years of age with a deficiency. Knowledge in 10 If the distribution of confounding factors differs in the this respect is important since oral administration should 11 two arms, explicative regression analysis will be per- provide these patients with greater autonomy, improve 12 formed in which the dependent variable will be the nor- patient satisfaction with treatment, and reduce treatment 13 malisation of the serum vitamin B12 concentration, and costs. Patients receiving anti-coagulation treatment, for 14 the independent variable will be the treatment group. whom intramuscular treatment may be contraindicated, 15 Repeated measures ANOVA will be used to examine should also benefit. The possibility of taking an oral 16 the change in serum vitaminFor B12 concentrationpeer in review each preparation only would also allow patient preferences to be 17 group at each monitoring point. taken into account when deciding on what treatment to 18 prescribe; indeed, patient preference is a factor of prime 19 Safety analysis importance in clinical decision-taking. The possibility of 20 The incidence of adverse events in the two arms will be providing treatment options in normal clinical practice 21 compared using the Pearson Chi-squared test or Fisher’s rests on two conditions being met: 1) that quality scien- 22 Exact test as required. tific information supports the effectiveness of the thera- 23 peutic options on offer, and 2) that heterogeneous 24 Quality of life analysis groups of patients have recorded their satisfaction with 25 The perception of quality of life by the patients of each these options. The present trial provides for information 26 arm will be assessed by comparing the EuroQol 5D in this respect to be gathered [39] and therefore treat- 27 scores (determined using a visual analogue scale) and the ment preferences to be taken into account at the time of 28 transformation of these scores into utility-based quality prescription. 29 of life values. The trial is also designed to provide information on 30 the effect of the normalisation of serum vitamin B12 31 Analysis of adherence to treatment concentrations by both treatments on patient-perceived 32 Adherence to treatment will be examined via the count- quality of life. Physicians commonly assume that taking 33 ing of oral doses taken in the oral arm, and the number oral supplements will be associated with a feeling of 34 of injections given in the intramuscular arm. An opera- greater well-being, although this has never been proven 35 tive indicator variable will then be defined to describe [40]. The present trial should also throw light on this. 36 the degree of adherence. The trial suffers from the practical limitation of having 37 to enrol a large number of patients to meet its sample size 38 Ethics requirements. However, a high degree of motivation is 39 The trial has been approved by the Madrid Region Clin- expected of the research team since its clinical assistance 40 ical Research Ethics Committee (February 8th 2011). It members are those involved in the enrolment process. 41 will be performed by qualified medical and scientific Further, the fact that the patients to be enrolled will be 42 staff. The rights and welfare of the patients will be seeking medical help (although not necessarily for vitamin 43 respected at all times. All patients will be adequately B12 deficiency) suggests few will be lost to follow-up. A 44 informed, both verbally and in writing, of the nature of further possible limitation is the low statistical power used 45 the trial, its aim, and its risks and possible benefits. in the calculation of the sample size. The 60% power con- 46 Given that the study is a non-inferiority trial, all patients templated requires a sample size of 304 patients (152 in 47 will be informed that the oral treatment is expected to each arm) – higher powers would increase the sample size 48 be as effective as the standard intramuscular treatment. required and the enrolment of such numbers cannot be 49 Signed, dated consent to be included will be required guaranteed. However, given the results reported in previ- 50 from each patient. ous studies (2,25,31-33) that used moderate/high doses of 51 Spanish law regarding the use of human subjects in clin- vitamin B12, it should be possible to demonstrate the non- 52 ical trials will be adhered to. The trial will respect all basic inferiority of the oral treatment with this power level. If 53 ethical principles of autonomy, justice, goodness of intent the 95% confidence interval were to cross the non-inferior- 54 and absence of malintent according to the standards of ity threshold, i.e., showing the results to be inconclusive, 55 good clinical practice enshrined in the Declaration of Hel- the intramuscular treatment would remain the treatment 56 sinki (Seoul, 2008) and the Oviedo Agreement (Convenio of choice. To determine the degree of adherence to treat- 57 de Oviedo) (1997). ment (and thus avoid outcome dilution effects) [41], the 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 44 of 51

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 9 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 number of doses taken orally and received by injection will 17Centro de Salud Centro de Salud Mendiguchia Carriche Gerencia de 5 be recorded. The characteristics of all the original 320 Atención Primaria. Servicio Madrileño de Salud, Calle Comunidad de Madrid 6 s/n, Leganés28912, Spain. 18Centro de Salud El Greco. Gerencia de Atención patients will be recorded to provide insight into the type 7 Primaria. Servicio Madrileño de Salud, Calle Avda. Reyes Católicos s/n, of patient left in the study after any withdrawals, as recom- Getafe28904, Spain. 19Unidad de Apoyo Técnico. Unidad de Apoyo a la 8 mended by the CONSORT group [41,42]. Basic informa- Investigación. Gerencia Atención Primaria. Servicio Madrileño de Salud, Calle 9 O’Donnell 55, Madrid28009, Spain. 20UDM Atención Familiar y Comunitaria tion (age, sex, etc.) on potentially eligible patients who 10 Oeste. Unidad de Apoyo a la Investigación. Gerencia Atención Primaria. decline to take part will also be recorded. This type of in- Servicio Madrileño de Salud, Calle Alonso Cano 8, Móstoles28933, Spain. 11 21 formation is of use when assessing the possible extrapola- Unidad Docente Multiprofesional Noroeste. Unidad de Apoyo a la 12 Investigación. Gerencia Atención Primaria. Servicio Madrileño de Salud, Avda. tion of the trial results to more general populations. 22 13 de España, 7 - 3 planta, Majadahonda28220, Spain. UDM Atención Familiar The decision not to take serum methylnalonic acid and y Comunitaria Norte. Unidad de Apoyo a la Investigación. Gerencia Atención 14 homocysteine concentrations into account as diagnostic Primaria. Servicio Madrileño de Salud, Calle Melchor Fernández Almagro, 1., 15 Madrid28029, Spain. 23Unidad de Apoyo a la Investigación. Gerencia markers and outcome variables was made bearing in 16 Atención Primaria, Servicio Madrileño de Salud, Calle Espronceda 24, mind that these are notFor normally determined,peer either review at Madrid28003, Spain. only24Centro de Salud M Ángeles López Gómez. Gerencia de 17 diagnosis or during follow-up, in patients with a vitamin Atención Primaria. Servicio Madrileño de Salud, Calle María Ángeles López 18 Gómez 2, Leganés28915, Spain. 25Unidad de Apoyo Técnico. Unidad de B12 deficiency. 19 Apoyo a la Investigación. Gerencia Atención Primaria. Servicio Madrileño de Finally, given the pragmatic nature of the proposed Salud, Calle O’Donnell 55, Madrid28009, Spain. 26Unidad de Apoyo a la 20 trial, the decision was taken to include consecutive Investigación. Unidad Docente Multiprofesional Sur. Gerencia Atención 21 Primaria, Servicio Madrileño de Salud, Avenida Juan de la Cierva s/n, patients seeking medical help at the participating centres, 27 22 Getafe28902, Spain. Servicio de Hematología. Hospital Severo Ochoa. thus ensuring the enrolment of subjects similar to those Servicio Madrileño de Salud, Avenida de Orellana s/n, Leganés28911, Spain. 23 28 that would be seen in normal clinical practice. Servicio de Endocrinología. Hospital Universitario Gregorio Marañón. 24 Servicio Madrileño de Salud, Calle Dr. Esquerdo 46, Madrid28007, Spain. 29Dirección General de Atención al Paciente. Servicio Madrileño de Salud, 25 Abbreviations Plaza Carlos Trías Bertrán 7, Madrid28020, Spain. 30Hospital Universitario Fe: Ferrum; g: Gram; GFR: Glomerular filtration rate; GGT: Gamma-glutamyl 26 clínico San Carlos. Servicio Madrileño de Salud, Calle Profesor Martín Lagos s/ transpeptidase; GOT: Glutamic oxaloacetic transaminase; GP: General n, Madrid28040, Spain. 31Profesor Asociado de Ciencias de la Salud. 27 practitioner; GPT: Glutamic-pyruvic transaminase; HIV: Human Departamento de Medicina. Facultad de Medicina. Universidad Complutense 28 immunodeficiency virus; HVB: Hepatitis B virus; HVC: Hepatitis C virus; de Madrid. Centro de Salud Guayaba. Dirección Asistencial Centro, Calle IF: Intrinsic factor; μg: Microgram; MMA: Methylmalonic acid; mg: Milligrams; 29 Antonia Rodríguez Sacristán 4, Madrid20044, Spain. 32Dirección Técnica de ng: Nanograms; pg: Picograms. 30 Procesos y Calidad. Gerencia Atención Primaria. Servicio Madrileño de Salud, 33 31 Calle Doctor Cirajas 20, Madrid28017, Spain. Universidad de Alcalá, Facultad Competing interests de Medicina, Campus Universitario, Ctra. Madrid-Barcelona Km 33,600., Alcalá 32 The authors declare that they have no competing interests. de Henares28871, Spain. 34Gerencia Atención Primaria, Servicio Madrileño de 33 Salud, Madrid, Spain. Author details 34 1Unidad de Apoyo a la Investigación. Gerencia Atención Primaria, Servicio ’ Madrileño de Salud, Calle Espronceda 24, Madrid 28003, Spain. 2Centro de Authors contributions 35 PGE y RRF conceived of the study and participated in its design. TSC; RRF; SGE; 36 Salud Buenos Aires. Dirección Asistencial Sureste. Gerencia Atención Primaria. Servicio Madrileño de Salud, Calle Pío Felipe s/n, Madrid28038, Spain. IdCG;JMF;EEM;participatedinthedesign and coordination of the study. FRS; 37 3Unidad de Apoyo a la Investigación. Unidad Docente Multiprofesional MGS; RGG; MAMS; COL; MLSP; CMR; BMB; AVP; FGBG; JEMS; RRB; GAC; LMCB; EPC; 38 (UDM) Atención Familiar y Comunitaria Sur. Gerencia Atención Primaria, MRB;MTRM;SSD;SMI;RRG;IBL;MVN;JSD;TGG;MDC;AABparticipatedindifferent Servicio Madrileño de Salud, Avenida Juan de la Cierva s/n, Getafe28902, phases of the design. TSC; RRF; SGE; IdCG; JMF; EEM directed the writing of the 39 Spain. 4Unidad de Apoyo a la Investigación. UDM Atención Familiar y manuscript. All authors OB12 Group read and approved the final manuscript. 40 Comunitaria Sureste. Gerencia Atención Primaria, Calle Hacienda de Pavones 41 271, Madrid28030, Spain. 5Unidad de Apoyo a la Investigación. Gerencia Funding Atención Primaria, Servicio Madrileño de Salud, Calle Espronceda 24, This study was funded by the Ministerio de Sanidad, Política Social e Igualdad, 42 Madrid28003, Spain. 6UDM Atención Familiar y Comunitaria Oeste. Unidad de Spain (EC10-115, EC10-116, EC10-117, EC10-119, EC10-122) and by CAIBER - 43 Apoyo a la Investigación. Gerencia Atención Primaria. Servicio Madrileño de Spanish Clinical Research Network. The authors thank the following persons 44 Salud, Calle Alonso Cano 8, Móstoles28933, Spain. 7Unidad de Apoyo a la for their contributions to this work: Dolores Otero-Criado, Carlos Carvajales Investigación. Gerencia Atención Primaria, Servicio Madrileño de Salud, Calle Fernández, Rosa Zurdo-Baz and Raisa González-Pérez. 45 Espronceda 24, Madrid28003, Spain. 8Hospital Universitario La Princesa. 46 Servicio Madrileño de Salud, Calle Diego de León 62, Madrid28006, Spain. The OB12 Group 47 9Dirección General de Sistemas de Información. Consejería de Sanidad, Healthcare Centre (HC) Barajasx: Germán Reviriego Jaén, Cristina Montero Comunidad de Madrid, Calle Julián Camarillo 4B 1, Madrid28037, Spain. García, Ana Isabel Sanz Lorente, Ma del Pilar Serrano Simarro, Julián Díaz 48 10CAIBER–Spanish Clinical Research Network. UCICEC Agencia Laín Entralgo, Sánchez, Irma Ma Ramos Gutiérrez, Josefa Ma San Vicente Rodríguez, Pilar 49 Calle Gran Vía 27, Madrid28013, Spain. 11CAIBER–Spanish Clinical Research Huelin Martín, Ma Inmaculada González García, Margarita Camarero Shelly, 50 Network. UCICEC Agencia Laín Entralgo, Calle Gran Vía 27, Madrid28013, Clarisa Reinares Martínez, Laura Villanova Cuadra, Rosa Ma Gómez del Spain. 12Hospital Universitario Gregorio Marañón. Servicio Madrileño de Forcallo. HC Doctor Cirajas: Francisco Endrino Gómez, Ma Rosario Ferreras 51 Salud, Calle Dr. Esquerdo 46, Madrid28007, Spain. 13Servicio de Farmacia. Eleta, Luis De Vicente Aymat, María Santos Santander Gutiérrez, Alicia Mateo 52 Dirección Asistencial Sureste. Gerencia Atención Primaria. Servicio Madrileño Madurga. HC Juncal: Nuria Caballero Ramírez, Ana Morán Escudero, 53 de Salud, Calle Hacienda de Pavones 271, Madrid28030, Spain. 14Servicio de Mercedes Rodríguez Franco, Mª Luz Meiriño Pérez, Mª Mar Zamora Gómez, Farmacia. Dirección Asistencial Sureste. Gerencia Atención Primaria. Servicio Francisco Vivas Rubio, María Martín Martín. HC Miguel de Cervantes: Rafael 54 Madrileño de Salud, Calle Hacienda de Pavones 271, Madrid28030, Spain. Pérez Quero, Mª Isabel Manzano Martín, Raimundo Pastor Sánchez, Alicia 55 15Servicio de Farmacia. Dirección Asistencial Sur. Gerencia Atención Primaria. Herrero de Dios, Cesar Redondo Luciáñez. HC Reyes Magos: Cristina Casado 56 Servicio Madrileño de Salud, Avenida Juan de la Cierva s/n, Getafe28902, Rodríguez, Luisa María Andrés Arreaza, Pilar Hombrados Gonzalo, Soledad Spain. 16Unidad de Medicina Preventiva, Hospital de Denia, Marina Salud, Escolar Llamazares, Francisco López Ortiz, Luz Mª del Rey Moya, Isabel 57 Agéncia Valenciana de Salut, Partida de Beniadlá, s/n, Dénia03700, Spain. Rodríguez López. HC Calesas: Diego Martín Acicoya, Pilar Kloppe Villegas, 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 45 of 51 BMJ Open

Sanz-Cuesta et al. BMC Public Health 2012, 12:394 Page 10 of 11 1 http://www.biomedcentral.com/1471-2458/12/394 2 3 4 Isabel García Amor, Magdalena Canals Aracil, José Javier Gómez Marco, 6. Ortega RM, Mena MC, Faci M, Santana JF, Serra-Majem L: Vitamin status in 5 Alberto González Álvaro, Fco Javier San Andrés Rebollo, Inés González López, different groups of the Spanish population: a meta-analysis of national studies 6 Isabel Herreros Hernanz, Antonio Revuelta Alonso, Nieves Calvo Arrabal, Mª performed between 1990 and 1999. Public Health Nutr 2001, 4(6A):1325–1329. 7 Milagros Jimeno Galán, Rosa García Hernández. HC Guayaba: Tomás Gómez 7. Garcia-Closas R, Serra-Majem L, Sabater-Sales G, Olmos-Castellvell M, Salleras- Gascón, Concepción Vargas-Machuca Cabañero, Mª Isabel Gutiérrez Sánchez, Sanmarti L: Distribución de la concentración sérica de vitamina C, ácido fólico y 8 Mª Angeles Fernández Abad, Margarita Beltejar Rodríguez, Javier Martínez vitamina B12 en una muestra representativa de la población adulta de 9 Suberviola, Miguel Angel Real Pérez, Carmen Coello Alarcón, Carlos San Cataluña. Med Clin (Barc) 2002, 118(4):135–141. 10 Andrés Pascua, José Antonio Granados Garrido. HC General Ricardos: 8. Henriquez P, Doroeste J, Deulofeu R, Fiuza M, Serra-Majem L: Nutritional Santiago Machín Hamalainen, Raquel Mateo Fernández, Cristina de la Cámara determinants of plasma total homocysteine distribution in the Canary Islands. 11 Gonzalez, José D.Garcés Ranz, Asunción Prieto Orzanco, Mª Teresa Marín Eur J Clin Nutr 2007, 61:111. 12 Becerra, Paulino Cubero González, Francisco R. Abellán López, Olga Álvarez 9. Lindenbaum J, Rosenberg IH, Wilson PW, Stabler SP, Allen RH: Prevalence of 13 Montes, Mercedes Canellas Manrique, Mª José San Telesforo Navarro, Mª cobalamin deficiency in the Framingham elderly population. Am J Clin Nutr Mercedes Parrilla Laso, Mª Ángeles Aragoneses Cañas, Angela Auñón Muelas 1994, 60(1):2–11. 14 HC Los Yébenes, Esther Valdés Cruz, Consuelo Mayoral Lopez, Teresa Gijon 10. Pautas E, Cherin P, De Jaeger C, Godeau P: Vitamin B 12 deficiency in the 15 Seco, Francisca Martinez Vallejo. HC Valle Inclán: Ana Isabel Menéndez aged. Presse Med 1999, 28(32):1767–1770. 16 Fernández, Mª del Mar De la Peña González, Mª Ángeles Maroto García, María 11. xvan Asselt DZ, Blom HJ, Zuiderent R, Wevers RA, Jakobs C, van den Broek Sánchez Cristóbal. HC Lavapiés: MªFor Carmen Álvarez peer Orviz, Jesús Herrero reviewWJ, et al: Clinical only significance of low cobalamin levels in older hospital 17 Hernández, Mª Veredas González Márquez, Mª Jesús López Rodríguez, Mª de patients. Neth J Med 2000, 57(2):41–49. 18 las Maravillas Almarza García, Mª Teresa San Clemente Pastor, Mª Ámparo 12. Smith DL: Anemia in the elderly. Am Fam Physician 2000, 62(7):1565–1572. 19 Corral Rubio. HC Colmenar Viejo Norte: Gonzalo Ruiz Zurita, Ángela Allue 13. Savage DG, Lindenbaum J, Stabler SP, Allen RH: Sensitivity of serum Bergua, Marta Cabrera Orozco, Mª del Puerto De Antonio García, Ana Isabel methylmalonic acid and total homocysteine determinations for 20 Cerezo Diviu, Inmaculada Solsons Roig, Pilar Gómez de Abia. HC diagnosing cobalamin and folate deficiencies. Am J Med 1994, 96(3): 21 Fuentelarreina: María Concepción Díaz Laso, Mª Luisa Asensio Ruiz, Carmen 239–246. 22 Siguero Pérez. HC Presentación Sabio: Antonio Molina Siguero, Inmaculada 14. Little DR: Ambulatory management of common forms of anemia. Am Cerrada Puri, Paloma Rodríguez Almagro, Rosa Rosanes González, Mª Carmen Fam Physician 1999, 59(6):1598–1604. 15. 23 Pérez García. HC Cuzco: Mar Noguerol Álvarez, Mª Ángeles de Miguel 15. Rasmussen K: P-methylmalonate and P-homocysteine: metabolic markers 24 Abanto, Mª Lourdes Reyes Martínez, Pilar Gutiérrez Valentín, Jorge Gómez of vitamin deficiencies. Background, validity and applications. Ugeskr – 25 Ciriano, Raquel Calzada Benito, Carolina Torrijos Bravo, David Ferreiro Laeger 1996, 158(27):3913 3918. González, Judit León González. HC San Martín de Valdeiglesias: Nuria Tomás 16. Herrmann W, Schorr H, Bodis M, Knapp JP, Muller A, Stein G, et al: Role of 26 García, Alberto Alcalá Faúndez, Eva Fernández López, Inés Melero Redondo, homocysteine, cystathionine and methylmalonic acid measurement for 27 Ricardo González Gascón. HC Pedroches: Jeannet Sánchez Yépez, Mercedes diagnosis of vitamin deficiency in high-aged subjects. Eur J Clin Invest – 28 del Pilar Fernández Girón, Beatriz López Serrano, Mª Teresa Rodríguez Monje, 2000, 30(12):1083 1089. Paloma Morso Pelaez, María Cortes Duran, Carolina López Olmeda, Almudena 17. Klee GG: Cobalamin and folate evaluation: measurement of 29 García- Uceda Sevilla, Dolores Serrano González, Inmaculada Santamaría methylmalonic acid and homocysteine vs vitamin B[12] and folate. Clin 30 López. HC Mendiguchía Carriche: Francisca García De Blas González, Alberto Chem 2000, 46(8 Pt 2):1277–1283. 31 López García-Franco, Amaya Azcoaga Lorenzo, Mar Álvarez Villalba, Belén 18. Andres E, Federici L, Affenberger S, Vidal-Alaball J, Loukili NH, Zimmer J, Pose García. HC Santa Isabel: Rosa Fernández García, Francisco de Alba et al: B12 deficiency: a look beyond pernicious anemia. J Fam Pract 2007, 32 Gómez, Antonio Redondo Horcajo, Beatriz Pajuelo Márquez, José Luis Gala 56(7):537–542. 33 Paniagua, Encarnación Cidoncha Calderón, Ángel Delgado Delgado, Mª Jesús 19. Andres E, Dali-Youcef N, Vogel T, Serraj K, Zimmer J: Oral cobalamin Gómez Martín, José Francisco Ávila Tomas. HC El Greco: José Enrique Mariño (vitamin B(12)) treatment An update. Int J Lab Hematol 2009, 31(1):1–8. 34 Suárez, José Luis Quintana Gómez, José Antonio González-Posada Delgado, 20. Nilsson M, Norberg B, Hultdin J, Sandstrom H, Westman G, Lokk J: Medical 35 Enrique Revilla Pascual, Esperanza Duralde Rodríguez, Milagros Beamud intelligence in Sweden. Vitamin B12: oral compared with parenteral? 36 Lagos. HC Arroyo de la Media Legua: Leonor González Galán, María Postgrad Med J 2005, 81(953):191–193. 37 Verdugo Rosado, Luis Nistal Martín de Serranos, Mª Jesús López Barroso, 21. Canales MA, de Paz R, Hernandez-Navarro F: Oral cobalamin therapy: a Mariano Rivera Moreno, Margarita Torres Parras, Mª Reyes Delgado Pulpon, note of caution. Med Clin (Barc) 2007, 128(19):757. 38 Elena Alcalá Llorente. HC Federica Montseny: Sonsoles Muñoz Moreno, Ana 22. 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Rabuñal R, Monte R, Peña M, Bal M, Gómez A: ¿Debemos utilizar la vía oral como primera opción para el tratamiento del déficit de vitamina B12? 44 Received: 4 May 2012 Accepted: 31 May 2012 Rev Clin Esp 2007, 207(4):179–182. 45 Published: 31 May 2012 25. Bolaman Z, Kadikoylu G, Yukselen V, Yavasoglu I, Barutca S, Senturk T: Oral 46 versus intramuscular cobalamin treatment in megaloblastic anemia: a References single-center, prospective, randomized, open-label study. Clin Ther 2003, 47 1. Dharmarajan TS, Norkus EP: Approaches to vitamin B12 deficiency. Early 25(12):3124–3134. 48 treatment may prevent devastating complications. Postgrad Med 2001, 26. Nyholm E, Turpin P, Swain D, Cunningham B, Daly S, Nightingale P, et al: – 49 110(1):99 105. quiz 106. Oral vitamin B12 can change our practice. Postgrad Med J 2003, 79 2. Kuzminski AM, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J: Effective (930):218–220. 50 treatment of cobalamin deficiency with oral cobalamin. Blood 1998, 92 27. 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1 2 3 Supplement 2: Bayesian Analysis 4 5 6 7 Bayesian analysis is a highly appropriate analysis strategy when working with small 8 sample sizes. Previous knowledge about the studied item can be taken advantage of by means of 9 the assessment of the plausibility of a given hypothesis after incorporating the new observed data.1 10 The noninferiority hypothesis, formally Δ < -10%, was tested, taking into account the 11 2 12 observed results but also taking into account the results of the trials by Kuzminski et al. and 13 Saraswathy et al.3 14 P1 denotes the percentage of patients who responded to VB12 oral administration, and 15 P0 represents the percentage of those responding to VB12 intramuscular administration. Bayesian 16 analysis allows for calculating the probability of P1 being equal to or smaller than P0 by a 17 18 specified magnitude,For the noninferiority peer limit review (Δ < -10%). For only each of the parameters P1 and P0, 19 both measured at 8, 26 and 52 weeks, we selected a priori distributions from the family of beta 20 distributions with parameters a and b, which are related to the proportions of those responding in 21 each trial arm. The gamma distribution represents the a priori hypothesis of the distribution of 22 differences. According to the results of both trials by Kuzminski et al.2 and Saraswathy et al.,3 23 4 24 included in the review by Wang et al., 79.1% and 84.1% of patients normalized their VB12 levels 25 in the oral and IM treatment groups, respectively.4 The respective CIs associated with these prior 26 data were calculated, and parameters were chosen (a and b in the beta distribution) such that the 27 maximum density intervals of these distributions approximately coincided with the CI previously 28 29 obtained (see Figure 1). Beta distributions for the success rate in each arm of the trial were 30 obtained using binomial data. A total of 10000 simulations were made from these a posteriori 31 distributions, and the corresponding differences, P1-P0, were calculated yielding an a posteriori 32 distribution of differences. This distribution was used to derive simulation-based estimates of the 33 probability of relevant magnitudes concerning Δ: P1-P0>0.10 at weeks 8, 26, and 52. Both PPT 34 35 and ITT analyses were performed. EPIDAT 4.2 software was used for all computations. 36 Table 1 shows the a posteriori probability of differences in treatment effectiveness 37 between oral and IM routes at different weeks (8, 26 and 52). The probabilities of the differences 38 in treatment effectiveness being >10% between the oral and IM groups were 0.001, 0.201, and 39 0.036 at weeks 8, 26, and 52, respectively (per protocol analysis). In the intention-to-treat (ITT) 40 41 analysis, these values were 0.000, 0.015, and 0.060 at weeks 8, 26, and 52, respectively. 42 43 44 45 References 46 47 1. Berger JO, Bayarri MJ. The interplay of bayesian and frequentist analysis. Stat Sci. 48 2004;19(1):58-80. 49 2. Kuzminskia M, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J. Effective treatment of 50 cobalamin deficiency with oral cobalamin. Blood. 1998;92(4):1191-1198. 51 3. Saraswathy AR, Dutta A, Simon EG, Chacko A. Randomized open label trial comparing 52 efficacy of oral versus intramuscular vitamin b12 supplementation for treatment of vitamin b12 53 deficiency. Gastroenterology. 2012:142(1):S-216. 4. Wang H, Li L, Qin LL, Song Y, Vidal-Alaball J, Liu TH. Oral vitamin B12 versus intramuscular 54 vitamin B12 for vitamin B12 deficiency. Cochrane Database Syst Rev. 2018;3:CD004655. 55 56 57 58 59 60

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1 2 3 4 5 6 Supplement 3: Receiver Operating Characteristic (ROC) Curve 7 8 9 To explore factors affecting the normalization of serum VB12 concentration (yes/no) at 52 weeks, 10 11 serum VB12 levels were studied at 8 weeks (at the end of the “charging period”). An ROC curve was 12 built to determine the likelihood ratios for each cutpoint after the charging period to “predict” the 13 normalization of levels (serum VB12 levels ≥211 pg/mL) at the end of the study.1 14 15 Table 1 shows the results of the likelihood ratios for the cutpoints at the main percentiles of the 16 distribution of VB12 serum levels at week 8 (“charging period”) to predict normalized VB12 serum levels 17 th 18 at the end of the study.For In Figure peer 1, the ROC curvereview is plotted. The onlylevel at the 5 percentile of the 19 distribution was selected as the most useful value as it showed best classification ability and because 20 when patients did not reach this level at week 8, they were almost twelve times more likely to not reach 21 suitable VB12 levels at the end of the study than if they did reach levels over 281 pg at week 8 22 (12~1/negative likelihood ratio). 23 24 25 26 References 27 28 1. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) 29 curve. Radiology. 1982;143(1):29-36. 30 31 32 33

34 35 Table 1. Exploring the value of several cutpoints of OB12 serum levels at week 8 to “predict” 36 normalization of values of Vit B12 at the end of the study 37 38 Correctly 39 Cutpoint Sensitivity Specificity LR+ LR- Percentil Classified 40 41 ≥ 281 0.977 0.273 94.30% 1.3435 0.0841 5 42 ≥ 328 0.963 0.546 94.30% 2.1193 0.0673 10 43 ≥ 353 0.931 0.636 91.70% 2.5608 0.1081 15 44 45 ≥ 389 0.895 0.818 89.10% 4.9197 0.129 20 46 ≥ 421 0.839 0.818 83.80% 4.617 0.1962 25 47 LR+: Positive Likelihood ratio. LR-: Negative Likelihood ratio 48 49 50 51 52 53 54 55 56 57 58 59 1 60

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1 2 3 4 5 6 Figure 1. ROC curve 7 8 9 10 11 12 13 14 15 16 17 18 For peer review only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 2 60

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1 2 3 4 5 6 7 8 9 10 11 12 For peer review only 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 BMJ Open Page 52 of 51

1 2 CONSORT Statement 2006 - Checklist for Non-inferiority and Equivalence Trials 3 4 Items to include when reporting a non-inferiority or equivalence randomized trial 5

6 PAPER SECTION Item Descriptor Reported on 7 And topic Page # 8 TITLE & 1 How participants were allocated to interventions (e.g., "random Page 1 and 2 9 ABSTRACT allocation", "randomized", or "randomly assigned"), 10 specifying that the trial is a non-inferiority or equivalence trial. 11 INTRODUCTION 2 Scientific background and explanation of rationale, Page 5 and 6 12 Background including the rationale for using a non-inferiority or equivalence design. 13 METHODS 3 Eligibility criteria for participants (detailing whether participants in the Page 7 14 Participants non-inferiority or equivalence trial are similar to those in any trial(s) that Supplement 1 15 established efficacy of the reference treatment) and the settings and 16 locations where the data were collected. 17 Interventions 4 Precise details of the interventions intended for each group detailing Page 7 18 Forwhether peer the reference review treatment in the non only-inferiority or equivalence trial 19 is identical (or very similar) to that in any trial(s) that established 20 efficacy, and how and when they were actually administered. 21 Objectives 5 Specific objectives and hypotheses, including the hypothesis Page 6 concerning non-inferiority or equivalence. 22 Outcomes 6 Clearly defined primary and secondary outcome measures detailing Page 8 23 whether the outcomes in the non-inferiority or equivalence trial are 24 identical (or very similar) to those in any trial(s) that established efficacy 25 of the reference treatment and, when applicable, any methods used to 26 enhance the quality of measurements (e.g., multiple observations, 27 training of assessors). 28 Sample size 7 How sample size was determined detailing whether it was calculated Page 8 29 using a non-inferiority or equivalence criterion and specifying the margin 30 of equivalence with the rationale for its choice. When applicable, 31 explanation of any interim analyses and stopping rules (and whether 32 related to a non-inferiority or equivalence hypothesis). 33 Randomization -- 8 Method used to generate the random allocation sequence, including Page 7 34 Sequence details of any restrictions (e.g., blocking, stratification) Supplement 1 35 generation 36 Randomization -- 9 Method used to implement the random allocation sequence (e.g., Page 7 37 Allocation numbered containers or central telephone), clarifying whether the Supplement 1 38 concealment sequence was concealed until interventions were assigned. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Randomization -- 10 Who generated the allocation sequence, who enrolled Page 7 and 8 4 Implementation participants, and who assigned participants to their groups. 5 Blinding (masking) 11 Whether or not participants, those administering the Not blinded 6 interventions, and those assessing the outcomes were blinded to 7 group assignment. If done, how the success of blinding was evaluated. 8 Statistical methods 12 Statistical methods used to compare groups for primary Page 8 and 9 9 outcome(s), specifying whether a one or two-sided confidence interval Supplement 2 10 approach was used. Methods for additional analyses, such as Supplement 3 11 subgroup analyses and adjusted analyses. 12 RESULTS 13 Flow of participants through each stage (a diagram is strongly Figure 1 13 recommended). Specifically, for each group report the numbers Participant flow 14 of participants randomly assigned, receiving intended treatment, 15 completing the study protocol, and analyzed for the primary 16 outcome. Describe protocol deviations from study as planned, 17 together with reasons. 18 Recruitment For14 Dates peer defining thereview periods of recruitment only and follow-up. Page 7 and 8 19 Figure 1 20 Baseline data 15 Baseline demographic and clinical characteristics of each group. Page 9 and 10 21 Table 1 22 Numbers analyzed 16 Number of participants (denominator) in each group included in Figure 1 and 23 each analysis and whether the analysis was “intention-to-treat” Figure 2 24 and/or alternative analyses were conducted. State the results in absolute numbers when feasible (e.g., 10/20, not 50%). 25 Outcomes and 17 For each primary and secondary outcome, a summary of results Page 11 to 13 26 estimation for each group, and the estimated effect size and its precision Table 2 27 (e.g., 95% confidence interval). For the outcome(s) for which non- Table 3 28 inferiority or equivalence is hypothesized, a figure showing confidence Figure 2 29 intervals and margins of equivalence may be useful. Supplement 2 30 Ancillary analyses 18 Address multiplicity by reporting any other analyses performed, Page 12 31 including subgroup analyses and adjusted analyses, indicating 32 those pre-specified and those exploratory. 33 Adverse events 19 All important adverse events or side effects in each intervention Page 13 34 group. 35 DISCUSSION 20 Interpretation of the results, taking into account the non-inferiority Page 14 to16 36 Interpretation or equivalence hypothesis and any other study hypotheses, sources 37 of potential bias or imprecision and the dangers associated with 38 multiplicity of analyses and outcomes. 39 Generalizability 21 Generalizability (external validity) of the trial findings. Page 14 to 16 40 Overall evidence 22 General interpretation of the results in the context of current Page 16 and 41 evidence. 17

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Article Type: Research

Date Submitted by the 28-Jan-2020 Author:

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1 2 3 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Buenos 4 Aires 5 Vargas-Machuca Cabañero, Concepción; Comunidad de Madrid Servicio 6 Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare 7 Centre Guayaba 8 Noguerol Álvarez, Mar; Comunidad de Madrid Servicio Madrileno de 9 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Cuzco García de Blas González, Francisca; Comunidad de Madrid Servicio 10 Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare 11 Centre Mendiguchía Carriche; Instituto de Salud Carlos III, Health 12 Services Research on Chronic Patients Network (REDISSEC) 13 Baños Morras, Raquel; Comunidad de Madrid Servicio Madrileno de 14 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Buenos 15 Aires 16 Díaz Laso, Concepción; Comunidad de Madrid Servicio Madrileno de For peerSalud, Gerencia review Asistencial Atención only Primaria. Healthcare Centre 17 Fuentelarreina 18 Caballero Ramírez, Nuria; Comunidad de Madrid Servicio Madrileno de 19 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Juncal 20 Herrero de Dios, Alicia; Comunidad de Madrid Servicio Madrileno de 21 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Miguel 22 de Cervantes Fernández García, Rosa; Comunidad de Madrid Servicio Madrileno de 23 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Santa 24 Isabel 25 Herrero Hernández, Jesús; Comunidad de Madrid Servicio Madrileno de 26 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre 27 Lavapiés 28 Pose García, Belen; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Mendiguchía 29 Carriche 30 Sevillano Palmero, María Luisa; Comunidad de Madrid Servicio Madrileno 31 de Salud, Gerencia Asistencial Atención Primaria. Pharmacy Department 32 Mateo Ruiz, Carmen; Comunidad de Madrid Servicio Madrileno de Salud, 33 Gerencia Asistencial Atención Primaria. Pharmacy Department 34 Medina Bustillo, Beatriz; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Pharmacy Department 35 Aguilar Jiménez, Monica; Comunidad de Madrid Servicio Madrileno de 36 Salud, UICEC Hospital Ramón y Cajal, Plataforma SCReN;Unidad de 37 Farmacología Clínica, Hospital Ramón y Cajal; Instituto Ramón y Cajal de 38 Investigación Sanitaria IRYCIS 39 group, OB12; Comunidad de Madrid Servicio Madrileno de Salud 40 Primary Subject General practice / Family practice 41 Heading: 42 43 Secondary Subject Heading: Geriatric medicine, Evidence based practice, Health services research 44 Vitamin B 12 Deficiency, Administration Oral, Equivalence Trial,, Keywords: 45 Injections Intramuscular, primary health care, Patient preference 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 2 of 52

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1 2 3 Oral versus intramuscular administration of vitamin B12 for vitamin B12 deficiency in 4 primary care: a pragmatic, randomized, noninferiority clinical trial (OB12) 5 6 7 8 Author names 9 10 Authors: Teresa Sanz-Cuesta, MD PhD1,2; Esperanza Escortell-Mayor, MD PhD1,2; Isabel del Cura- 11 González, MD PhD 1,2,3; Jesús Martín-Fernández, MD PhD2,3,4; Rosario Riesgo-Fuertes, MD2,5; Sofía 12 Garrido-Elustondo, MD2,6; José Enrique Mariño-Suárez, MD7; Mar Álvarez-Villalba, MD PhD2,8; Tomás 13 Gómez-Gascón, MD PhD2,9; Inmaculada González-García, MD10; Paloma González-Escobar, MD11; 14 Concepción Vargas-Machuca-Cabañero, MD PhD12; Mar Noguerol-Álvarez, MD13; Francisca García-de 15 Blas-González, MD PhD2,14; Raquel Baños-Morras, MD15; Concepción Díaz-Laso, MD PhD16; Nuria 16 For17 peer review18 only 19 17 Caballero-Ramírez, MD ; Alicia Herrero-de Dios, MD ; Rosa Fernández-García, MD ; Jesús Herrero- 18 Hernández, MD20; Belén Pose-García, RN14; María Luisa Sevillano-Palmero, Pharm21; Carmen Mateo- 19 Ruiz, Pharm21; Beatriz Medina-Bustillo, Pharm 21; Mónica Aguilar Jiménez, Pharm22 and OB12 Group23. 20 21 Author affiliations 22 1. Research Unit, Gerencia Asistencial de Atención Primaria (GAAP), Madrid, Spain. 23 2. Health Services Research on Chronic Patients Network (REDISSEC), Instituto Salud Carlos III, Madrid, Spain. 24 3. Preventive Medicine and Public Health Area, Health Sciences Faculty, Universidad Rey Juan Carlos, Alcorcón, 25 Madrid, Spain. 26 4. Multiprofessional Teaching Unit of Primary and Community Care Oeste. GAAP, Madrid, Spain. 27 5. Multiprofessional Teaching Unit of Primary and Community Care Sur, GAAP, Madrid, Spain. 28 6. Multiprofessional Teaching Unit of Primary and Community Care Sureste, GAAP, Madrid, Spain. 29 7. Healthcare Centre El Greco, Getafe, GAAP, Madrid, Spain. 30 8. Healthcare Centre Mª Jesús Hereza, Leganés, GAAP, Madrid, Spain. 31 9. Fundación de Investigación e Innovación Biomédica de Atención Primaria, Madrid, Spain. 32 10. Healthcare Centre Barajas, GAAP, Madrid, Spain. 33 11. Healthcare Centre Buenos Aires, GAAP, Madrid, Spain. 34 12. Healthcare Centre Guayaba, GAAP, Madrid, Spain. 35 13. Healthcare Centre Cuzco, Fuenlabrada, GAAP, Madrid, Spain. 36 14. Healthcare Centre Mendiguchía Carriche, Leganés, GAAP, Madrid, Spain. 37 15. Healthcare Centre Buenos Aires, GAAP, Madrid, Spain. 38 16. Healthcare Centre Fuentelarreina, GAAP, Madrid, Spain. 39 17. Healthcare Centre Juncal, Torrejón de Ardoz, GAAP, Madrid, Spain. 40 18. Healthcare Centre Miguel de Cervantes, Alcalá de Henares, GAAP, Madrid, Spain. 41 19. Healthcare Centre Santa Isabel, Leganés, GAAP, Madrid, Spain. 42 20. Healthcare Centre Lavapiés, GAAP, Madrid, Spain. 21. Pharmacy Department, GAAP, Madrid, Spain. 43 22. UICEC Hospital Ramón y Cajal, Plataforma SCReN; Unidad de Farmacología Clínica, Hospital Ramón y Cajal, 44 Madrid, España; Instituto Ramón y Cajal de Investigación Sanitaria, IRYCIS. 45 23. OB12 Group. 46 47 Corresponding author 48 Isabel del Cura-González 49 Head of the Primary Care Research Unit, Madrid Health Services, Spain 50 Associate Professor, Department of Preventive Medicine and Public Health, Rey Juan Carlos University 51 REDISSEC (Health Services Research on Chronic Patients Network), ISCIII 52 C/ San Martín de Porres 6, 28035 Madrid, Spain 53 E-mail: [email protected] 54 Phone number: +34913700697 55 56 Word count: 3056 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 52

1 2 3 A pragmatic, randomized, multicenter, noninferiority clinical trial with a duration of 12 months 4 5 (26) 6 was conducted in a PHC. On ethical grounds, a placebo-controlled trial was not appropriate. 7 8 Methodological issues of this trial have been published elsewhere (Supplement 1).(27) 9 10 Competitive recruitment was performed in 22 PHC in Madrid (Spain) from July 2014 to November 11 12 2016. Eligible patients were 65 years of age or older and had been attending a PHC for consultation 13 14 15 on any medical matter. Patients were assessed for eligibility and invited to participate 16 For peer review only 17 consecutively by their general practitioners. Written informed consent was obtained from all 18 19 participants. A blood test was performed, and in patients with a serum concentration of VB12 of 20 21 22 <211 pg/mL, the remaining inclusion and exclusion criteria were evaluated. The cut-off value 23 24 selected in the trial register/ trial protocol was <179 pg/mL; this value was modified by the 25 26 laboratory following the recommendations of the provider. This change took place prior to the 27 28 29 beginning of the recruitment. Patient recruitment was always performed using the same 30 31 methodology and cut-off point. The procedures for measurement of the biomarkers were ADVIA 32 33 Centaur XP (Siemens Diagnostics, Tarrytown, NY, USA). 34 35 Randomization and masking 36 37 38 Patients were allocated by simple randomization at a 1:1 ratio to oral or intramuscular 39 40 administration of vitamin B12. The randomization system was incorporated into the electronic 41 42 data collection system to assure allocation concealment. Because of the nature of the intervention, 43 44 45 patients and general practitioners were aware of their treatment allocation. Analysis was performed 46 47 by the trial statistician, who was blinded to allocation. 48 49 50 51 52 Intervention 53 54 55 56 57 58 7 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 10 of 52

1 2 3 significant by the researchers from a clinical perspective were included in the model. To test the 4 5 6 noninferiority hypothesis, adding the information contained in these data to previous knowledge, 7 8 additional statistical analyses were performed using a Bayesian approach. Secondary outcome 9 10 variables were analyzed using the appropriate statistical tests, and their means or proportions 11 12 were used to estimate differences between groups. All analyses were performed using STATA 13 14 15 14 and EPIDAT 4.2 software. 16 For peer review only 17 Patient involvement 18 19 Patients were not involved in the development of plans for recruitment, design, outcome measures, 20 21 22 or implementation of the study conduct. No patients were asked to advise on the interpretation or 23 24 writing of the results. Patients explained the experience of participating in the study on 25 26 the occasion of International Clinical Trial´s day in Radio Nacional de España (RNE). We will 27 28 29 pursue patient and public involvement in the development of an appropriate method for further 30 31 dissemination. 32 33 34 35 36 RESULTS 37 38 Characteristics of the study participants 39 40 41 A total of 2342 patients were offered participation, and 2152 provided informed consent. A total 42 43 of 307 patients showed a VB12 deficit (14.3%), 283 of whom were allocated to receive VB12 44 45 treatment via the IM route (n=143) or orally (n=140). The follow-up period (52 weeks) was 46 47 48 completed by 229 patients (80.9%) (Figure 1). 49 50 The average age was 75.2 (6.34), and 58.3% of the patients were women. Table 1 51 52 describes the baseline characteristics of the patients included in the trial. No relevant differences 53 54 55 56 57 58 10 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 13 of 52 BMJ Open

1 2 3 The comparison with other studies is difficult, due to the treatment different doses used, 4 5 6 but especially because of the follow-up length had been inferior to 4 months and the number of 7 8 patients included was small. 9 10 As far as we know, the present trial is the largest clinical trial with the longest follow-up 11 12 period, and it is the first to evaluate, in addition to VB12 levels, clinical signs and symptoms, 13 14 15 health-related quality of life, and patient preferences. The 3 clinical trials(23–25) described in the 16 For peer review only 17 2018 Cochrane Systematic Review(5) had a duration between 3 and 4 months and included a total 18 19 of 153 patients. In the Saraswathy trial, patients in the oral route at 3 months normalised levels 20 21 (25) 22 20/30 (66.7%) vs 27/30 (90%) of the patients in the IM route. In Kuzminski's patients in the 23 24 oral route at 4 months normalised levels 18/18 (100%) vs 10/14 (71.4%) of the patients in the IM 25 26 route.(23) These differences were statistically non-significant in both studies. 27 28 29 Two studies have recently been published and add evidence in favour of oral and sublingual 30 31 administration of VB12.(37,38) The follow-up of Moleiro's study reached 24 months versus 12 32 33 months in our study. However, Moleiro et al performed a prospective uncontrolled study that 34 35 included 26 patients submitted to total gastrectomy. All patients received oral VB12 36 37 38 supplementation (1 mg/day), and all of them maintained normalization V12 at 6, 12, 18, and 24 39 40 months. There was a progressive increase in serum V12 levels within the first 12 months, which 41 42 remained stable thereafter.(37) The long-term effectiveness of the oral route in absorption-deficient 43 44 45 people such as gastrectomized patients would support the results of our study. 46 47 Bensky et al. compared the efficacy of sublingual vs. intramuscular administration of 48 49 vitamin B12 in a retrospective observational study from the computerized pharmacy records of 50 51 52 Maccabi Health Service (MHS). Among 4281 patients treated with VB12 supplements (830 53 54 (19.3%) with IM and 3451 (80.7%) with sublingual tablets, the IM group achieved a significant 55 56 57 58 17 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 52

Week 8 133/135 (98.5) 129/130 (99.2) -0.7 (-3.2 to 1.8) 1 For peer review only 2 Week 26 115/132 (87.1) 122/122 (100.0) -12.9 (-17.9 to -6.1)

3PPT 4 5 Week 52 103/112 (92.0) 115/117 (98.3) -6.3 (-11.9 to -0.07) 6 7 8 Week 8 133/140 (95.0) 129/143 (90.2) 4.8 (-1.3 to 10.9) 9

Week 8 133/135 (98.5) 129/130 (99.2) -0.7 (-3.2 to 1.8) 1 For peer review only 2 Week 26 115/132 (87.1) 122/122 (100.0) -12.9 (-17.9 to -6.1)

3PPT 4 5 Week 52 103/112 (92.0) 115/117 (98.3) -6.3 (-11.9 to -0.07) 6 7 8 Week 8 133/140 (95.0) 129/143 (90.2) 4.8 (-1.3 to 10.9) 9

10ITT Week 26 115/140 (82.1) 122/143 (85.3) -3.2 (-11.8 to 5.4) 11 12 13 Week 52 103/140 (73.6) 115/143 (80.4) -6.8 (-16.6 to 2.9) 14 15 16 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml-20 -10 0 10 20 17 Diff proportions 18 Favors IM Favors oral 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 37 of 52 BMJ Open

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1 2 3 Supplement 2: Bayesian Analysis 4 5 6 7 Bayesian analysis is a highly appropriate analysis strategy when working with small 8 sample sizes. Previous knowledge about the studied item can be taken advantage of by means of 9 the assessment of the plausibility of a given hypothesis after incorporating the new observed data.1 10 The noninferiority hypothesis, formally Δ < -10%, was tested, taking into account the 11 2 12 observed results but also taking into account the results of the trials by Kuzminski et al. and 13 Saraswathy et al.3 14 P1 denotes the percentage of patients who responded to VB12 oral administration, and 15 P0 represents the percentage of those responding to VB12 intramuscular administration. Bayesian 16 analysis allows for calculating the probability of P1 being equal to or smaller than P0 by a 17 18 specified magnitude,For the noninferiority peer limit review (Δ < -10%). For only each of the parameters P1 and P0, 19 both measured at 8, 26 and 52 weeks, we selected a priori distributions from the family of beta 20 distributions with parameters a and b, which are related to the proportions of those responding in 21 each trial arm. The gamma distribution represents the a priori hypothesis of the distribution of 22 differences. According to the results of both trials by Kuzminski et al.2 and Saraswathy et al.,3 23 4 24 included in the review by Wang et al., 79.1% and 84.1% of patients normalized their VB12 levels 25 in the oral and IM treatment groups, respectively.4 The respective CIs associated with these prior 26 data were calculated, and parameters were chosen (a and b in the beta distribution) such that the 27 maximum density intervals of these distributions approximately coincided with the CI previously 28 29 obtained (see Figure 1). Beta distributions for the success rate in each arm of the trial were 30 obtained using binomial data. A total of 10000 simulations were made from these a posteriori 31 distributions, and the corresponding differences, P1-P0, were calculated yielding an a posteriori 32 distribution of differences. This distribution was used to derive simulation-based estimates of the 33 probability of relevant magnitudes concerning Δ: P1-P0>0.10 at weeks 8, 26, and 52. Both PPT 34 35 and ITT analyses were performed. EPIDAT 4.2 software was used for all computations. 36 Table 1 shows the a posteriori probability of differences in treatment effectiveness 37 between oral and IM routes at different weeks (8, 26 and 52). The probabilities of the differences 38 in treatment effectiveness being >10% between the oral and IM groups were 0.001, 0.201, and 39 0.036 at weeks 8, 26, and 52, respectively (per protocol analysis). In the intention-to-treat (ITT) 40 41 analysis, these values were 0.000, 0.015, and 0.060 at weeks 8, 26, and 52, respectively. 42 43 44 45 References 46 47 1. Berger JO, Bayarri MJ. The interplay of bayesian and frequentist analysis. Stat Sci. 48 2004;19(1):58-80. 49 2. Kuzminskia M, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J. Effective treatment of 50 cobalamin deficiency with oral cobalamin. Blood. 1998;92(4):1191-1198. 51 3. Saraswathy AR, Dutta A, Simon EG, Chacko A. Randomized open label trial comparing 52 efficacy of oral versus intramuscular vitamin b12 supplementation for treatment of vitamin b12 53 deficiency. Gastroenterology. 2012:142(1):S-216. 4. Wang H, Li L, Qin LL, Song Y, Vidal-Alaball J, Liu TH. Oral vitamin B12 versus intramuscular 54 vitamin B12 for vitamin B12 deficiency. Cochrane Database Syst Rev. 2018;3:CD004655. 55 56 57 58 59 60

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1 2 CONSORT Statement 2006 - Checklist for Non-inferiority and Equivalence Trials 3 4 Items to include when reporting a non-inferiority or equivalence randomized trial 5

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42 www.consort-statement.org 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Article Type: Research

Date Submitted by the 02-Apr-2020 Author:

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1 2 3 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Buenos 4 Aires 5 Vargas-Machuca Cabañero, Concepción; Comunidad de Madrid Servicio 6 Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare 7 Centre Guayaba 8 Noguerol Álvarez, Mar; Comunidad de Madrid Servicio Madrileno de 9 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Cuzco García de Blas González, Francisca; Comunidad de Madrid Servicio 10 Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare 11 Centre Mendiguchía Carriche; Instituto de Salud Carlos III, Health 12 Services Research on Chronic Patients Network (REDISSEC) 13 Baños Morras, Raquel; Comunidad de Madrid Servicio Madrileno de 14 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Buenos 15 Aires 16 Díaz Laso, Concepción; Comunidad de Madrid Servicio Madrileno de For peerSalud, Gerencia review Asistencial Atención only Primaria. Healthcare Centre 17 Fuentelarreina 18 Caballero Ramírez, Nuria; Comunidad de Madrid Servicio Madrileno de 19 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Juncal 20 Herrero de Dios, Alicia; Comunidad de Madrid Servicio Madrileno de 21 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Miguel 22 de Cervantes Fernández García, Rosa; Comunidad de Madrid Servicio Madrileno de 23 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Santa 24 Isabel 25 Herrero Hernández, Jesús; Comunidad de Madrid Servicio Madrileno de 26 Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre 27 Lavapiés 28 Pose García, Belen; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Healthcare Centre Mendiguchía 29 Carriche 30 Sevillano Palmero, María Luisa; Comunidad de Madrid Servicio Madrileno 31 de Salud, Gerencia Asistencial Atención Primaria. Pharmacy Department 32 Mateo Ruiz, Carmen; Comunidad de Madrid Servicio Madrileno de Salud, 33 Gerencia Asistencial Atención Primaria. Pharmacy Department 34 Medina Bustillo, Beatriz; Comunidad de Madrid Servicio Madrileno de Salud, Gerencia Asistencial Atención Primaria. Pharmacy Department 35 Aguilar Jiménez, Monica; Comunidad de Madrid Servicio Madrileno de 36 Salud, UICEC Hospital Ramón y Cajal, Plataforma SCReN;Unidad de 37 Farmacología Clínica, Hospital Ramón y Cajal; Instituto Ramón y Cajal de 38 Investigación Sanitaria IRYCIS 39 group, OB12; Comunidad de Madrid Servicio Madrileno de Salud 40 Primary Subject General practice / Family practice 41 Heading: 42 43 Secondary Subject Heading: Geriatric medicine, Evidence based practice, Health services research 44 Vitamin B 12 Deficiency, Administration Oral, Equivalence Trial,, Keywords: 45 Injections Intramuscular, primary health care, Patient preference 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 2 of 51

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1 2 3 Oral versus intramuscular administration of vitamin B12 for vitamin B12 deficiency in 4 primary care: a pragmatic, randomized, noninferiority clinical trial (OB12) 5 6 7 8 Author names 9 10 Authors: Teresa Sanz-Cuesta, MD PhD1,2; Esperanza Escortell-Mayor, MD PhD1,2; Isabel del Cura- 11 González, MD PhD 1,2,3; Jesús Martín-Fernández, MD PhD2,3,4; Rosario Riesgo-Fuertes, MD2,5; Sofía 12 Garrido-Elustondo, MD2,6; José Enrique Mariño-Suárez, MD7; Mar Álvarez-Villalba, MD PhD2,8; Tomás 13 Gómez-Gascón, MD PhD2,9; Inmaculada González-García, MD10; Paloma González-Escobar, MD11; 14 Concepción Vargas-Machuca-Cabañero, MD PhD12; Mar Noguerol-Álvarez, MD13; Francisca García-de 15 Blas-González, MD PhD2,14; Raquel Baños-Morras, MD15; Concepción Díaz-Laso, MD PhD16; Nuria 16 For17 peer review18 only 19 17 Caballero-Ramírez, MD ; Alicia Herrero-de Dios, MD ; Rosa Fernández-García, MD ; Jesús Herrero- 18 Hernández, MD20; Belén Pose-García, RN14; María Luisa Sevillano-Palmero, Pharm21; Carmen Mateo- 19 Ruiz, Pharm21; Beatriz Medina-Bustillo, Pharm 21; Mónica Aguilar Jiménez, Pharm22 and OB12 Group23. 20 21 Author affiliations 22 1. Research Unit, Gerencia Asistencial de Atención Primaria (GAAP), Madrid, Spain. 23 2. Health Services Research on Chronic Patients Network (REDISSEC), Instituto Salud Carlos III, Madrid, Spain. 24 3. Preventive Medicine and Public Health Area, Health Sciences Faculty, Universidad Rey Juan Carlos, Alcorcón, 25 Madrid, Spain. 26 4. Multiprofessional Teaching Unit of Primary and Community Care Oeste. GAAP, Madrid, Spain. 27 5. Multiprofessional Teaching Unit of Primary and Community Care Sur, GAAP, Madrid, Spain. 28 6. Multiprofessional Teaching Unit of Primary and Community Care Sureste, GAAP, Madrid, Spain. 29 7. Healthcare Centre El Greco, Getafe, GAAP, Madrid, Spain. 30 8. Healthcare Centre Mª Jesús Hereza, Leganés, GAAP, Madrid, Spain. 31 9. Fundación de Investigación e Innovación Biomédica de Atención Primaria, Madrid, Spain. 32 10. Healthcare Centre Barajas, GAAP, Madrid, Spain. 33 11. Healthcare Centre Buenos Aires, GAAP, Madrid, Spain. 34 12. Healthcare Centre Guayaba, GAAP, Madrid, Spain. 35 13. Healthcare Centre Cuzco, Fuenlabrada, GAAP, Madrid, Spain. 36 14. Healthcare Centre Mendiguchía Carriche, Leganés, GAAP, Madrid, Spain. 37 15. Healthcare Centre Buenos Aires, GAAP, Madrid, Spain. 38 16. Healthcare Centre Fuentelarreina, GAAP, Madrid, Spain. 39 17. Healthcare Centre Juncal, Torrejón de Ardoz, GAAP, Madrid, Spain. 40 18. Healthcare Centre Miguel de Cervantes, Alcalá de Henares, GAAP, Madrid, Spain. 41 19. Healthcare Centre Santa Isabel, Leganés, GAAP, Madrid, Spain. 42 20. Healthcare Centre Lavapiés, GAAP, Madrid, Spain. 21. Pharmacy Department, GAAP, Madrid, Spain. 43 22. UICEC Hospital Ramón y Cajal, Plataforma SCReN; Unidad de Farmacología Clínica, Hospital Ramón y Cajal, 44 Madrid, España; Instituto Ramón y Cajal de Investigación Sanitaria, IRYCIS. 45 23. OB12 Group. 46 47 Corresponding author 48 Isabel del Cura-González 49 Head of the Primary Care Research Unit, Madrid Health Services, Spain 50 Associate Professor, Department of Preventive Medicine and Public Health, Rey Juan Carlos University 51 REDISSEC (Health Services Research on Chronic Patients Network), ISCIII 52 C/ San Martín de Porres 6, 28035 Madrid, Spain 53 E-mail: [email protected] 54 Phone number: +34913700697 55 56 Word count: 3056 57 58 1 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 51

1 2 3 Abstract 4 5 6 7 8 Objectives: To compare the effectiveness of oral versus intramuscular vitamin B12 (VB12) in 9 10 patients aged ≥65 years with VB12 deficiency. 11 12 Design: Pragmatic, randomized, noninferiority, multicenter trial in patients ≥65 years in 22 13 14 primary healthcare centres in Madrid (Spain). Participants: 283 adults with VB12 deficiency were 15 16 For peer review only 17 randomly assigned to oral (n=140) or intramuscular (n=143) treatment arm. Interventions: The 18 19 intramuscular arm received 1mg VB12 on alternate days in weeks 1–2, 1mg/week in weeks 3–8, 20 21 and 1mg/month in weeks 9–52. The oral arm received 1mg/day in weeks 1–8 and 1 mg/week in 22 23 24 weeks 9–52. 25 26 Main outcomes: Serum VB12 concentration normalization (≥ 211 pg/mL) at 8, 26, and 52 weeks. 27 28 Noninferiority would be declared if the difference between arms is 10% or less. Secondary 29 30 31 outcomes included symptoms, adverse events, adherence to treatment, quality of life, patient 32 33 preferences and satisfaction. 34 35 Results: The follow-up period (52 weeks) was completed by 229 patients (80.9%). At week 8, the 36 37 percentage of patients in each arm who achieved normal B12 levels was well above 90%; the 38 39 40 differences in this percentage between the oral and intramuscular arm were -0.7% (133 out of 135 41 42 vs 129 out of 130; 95% CI: -3.2 to 1.8; p>0.999) by per-protocol (PPT) analysis and 4.8% (133 43 44 out of 140 vs 129 out of 143; 95% CI: -1.3 to 10.9; p=0.124) by intention-to-treat (ITT) analysis. 45 46 47 At week 52, the percentage of patients who achieved normal B12 levels was 73.6% in the oral arm 48 49 and 80.4% in the intramuscular (IM) arm; these differences were -6.3% (103 out of 112 vs 115 out 50 51 of 117; 95% CI: -11.9 to -0.1; p=0.025) and -6.8% (103 out of 140 vs 115 out of 143; 95% CI: - 52 53 54 16.6 to 2.9; p=0.171), respectively. Factors affecting the success rate at week 52 were age, 55 56 OR=0.95 (95% CI: 0.91 to 0.99), and having reached VB12 levels ≥281 pg/mL at week 8, OR= 57 58 2 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 5 of 51 BMJ Open

1 2 3 8.1 (95% CI: 2.4 to 27.3). Under a Bayesian framework, noninferiority probabilities (Δ>-10%) at 4 5 6 week 52 were 0.036 (PPT) and 0.060 (ITT). Quality of life and adverse effects were comparable 7 8 across groups. 83.4% of patients preferred the oral route. 9 10 Conclusions: Oral administration was no less effective than intramuscular administration at 8 11 12 weeks. Although differences were found between administration routes at week 52, the probability 13 14 15 that the differences were below the noninferiority threshold was very low. 16 For peer review only 17 Trial registration: ClinicalTrials.gov (NCT 01476007) and EUDRACT (2010-024129-20). 18 19 Funding: Ministerio de Sanidad y Consumo Español. Instituto de Salud Carlos III (ISCIII). 20 21 22 European Regional Development Fund. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 3 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 51

1 2 3 A pragmatic, randomized, multicenter, noninferiority clinical trial with a duration of 12 months 4 5 (26) 6 was conducted in a PHC. On ethical grounds, a placebo-controlled trial was not appropriate. 7 8 Methodological issues of this trial have been published elsewhere (Supplement 1).(27) 9 10 Competitive recruitment was performed in 22 PHC in Madrid (Spain) from July 2014 to November 11 12 2016. Eligible patients were 65 years of age or older and had been attending a PHC for consultation 13 14 15 on any medical matter. Patients were assessed for eligibility and invited to participate 16 For peer review only 17 consecutively by their general practitioners. Written informed consent was obtained from all 18 19 participants. A blood test was performed, and in patients with a serum concentration of VB12 of 20 21 22 <211 pg/mL, the remaining inclusion and exclusion criteria were evaluated. The cut-off value 23 24 selected in the trial register/ trial protocol was <179 pg/mL; this value was modified by the 25 26 laboratory following the recommendations of the provider. This change took place prior to the 27 28 29 beginning of the recruitment. Patient recruitment was always performed using the same 30 31 methodology and cut-off point. The procedures for measurement of the biomarkers were ADVIA 32 33 Centaur XP (Siemens Diagnostics, Tarrytown, NY, USA). 34 35 Randomization and masking 36 37 38 Patients were allocated by simple randomization at a 1:1 ratio to oral or intramuscular 39 40 administration of vitamin B12. The randomization system was incorporated into the electronic 41 42 data collection system to assure allocation concealment. Because of the nature of the intervention, 43 44 45 patients and general practitioners were aware of their treatment allocation. Analysis was performed 46 47 by the trial statistician, who was blinded to allocation. 48 49 50 51 52 Intervention 53 54 55 56 57 58 7 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 10 of 51

1 2 3 significant by the researchers from a clinical perspective were included in the model. To test the 4 5 6 noninferiority hypothesis, adding the information contained in these data to previous knowledge, 7 8 additional statistical analyses were performed using a Bayesian approach. Secondary outcome 9 10 variables were analyzed using the appropriate statistical tests, and their means or proportions 11 12 were used to estimate differences between groups. All analyses were performed using STATA 13 14 15 14 and EPIDAT 4.2 software. 16 For peer review only 17 Patient involvement 18 19 Patients were not involved in the development of plans for recruitment, design, outcome measures, 20 21 22 or implementation of the study conduct. No patients were asked to advise on the interpretation or 23 24 writing of the results. Patients explained the experience of participating in the study on 25 26 the occasion of International Clinical Trial´s day in Radio Nacional de España (RNE). We will 27 28 29 pursue patient and public involvement in the development of an appropriate method for further 30 31 dissemination. 32 33 34 35 36 RESULTS 37 38 Characteristics of the study participants 39 40 41 A total of 2342 patients were offered participation, and 2152 provided informed consent. A total 42 43 of 307 patients showed a VB12 deficit (14.3%), 283 of whom were allocated to receive VB12 44 45 treatment via the IM route (n=143) or orally (n=140). The follow-up period (52 weeks) was 46 47 48 completed by 229 patients (80.9%). Losses to follow-up were similar in both regimens, 28 out of 49 50 140 and 26 out of 143 losses in the oral and intramuscular arms respectively (p=0.697). (Figure 51 52 1). 53 54 55 56 57 58 10 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 13 of 51 BMJ Open

1 2 3 The average age was 75.2 (6.34), and 58.3% of the patients were women. Table 1 4 5 6 describes the baseline characteristics of the patients included in the trial. No relevant differences 7 8 were found between groups at baseline for demographic and medical characteristics or for the 9 10 study endpoints. 11 12 13 14 Table 1. Baseline characteristics at baseline by group 15 16 Variable For peer review onlyNo. (%) 17 18 Oral route IM route Total (n=283) 19 (n=140) (n=143) 20 Sociodemographic data 21 Women 87 (62.1) 78 (54.5) 165 (58.3) 22 23 Age (years), mean (SD) 74.2 (5.8) 76.2 (6.7) 75.2 (6.3) 24 Educational level 25 Illiteracy 4 (2.9) 7 (5.1) 11 (4.0) 26 27 Incomplete education 48 (34.5) 46 (33.6) 94 (34.1) 28 Primary education 58 (41.7) 63 (46.0) 121 (43.8) 29 Secondary education 16 (11.5) 10 (7.3) 26 (9.4) 30 31 Tertiary education 4 (2.9) 4 (2.9) 8 (2.9) 32 Higher education 9 (6.5) 7 (5.1) 16 (5.8) 33 Social occupational class a 34 35 Class I - IV 31 (27.7) 33 (27.3) 64 (27.5) 36 Class V - VI 81 (72.3) 88 (72.7) 169 (72.5) 37 Living alone 32 (21.4) 30 (22.2) 62 (21.9) 38 39 Clinical data 40 Tobacco habit 41 Ex-smoker 27 (19.7) 25 (18.4) 52 (19.0) 42 Smoker 9 (6.6) 10 (7.4) 19 (7.0) 43 44 Nonsmoker 101 (73.7) 101 (74.3) 202 (74.0) 45 Vegetarian 2 (1.4) 0 (0) 2 (0.7) 46 Having undergone gastrectomy 1 (0.7) 2 (1.4) 3 (1.1) 47 48 Symptoms 49 Paresthesia 33 (23.6) 45 (31.5) 78 (27.6) 50 Asthenia 43 (30.7) 54 (37.8) 97 (34.3) 51 52 Loss of appetite 12 (8.6) 30 (21.0) 42 (14.8) 53 Sadness 37 (26.4) 53 (37.1) 90 (31.8) 54 Showing ≥1 symptom 70 (50.0) 83 (58.0) 153 (54.1) 55 Signs 56 57 58 11 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 14 of 51

1 2 3 Glossitis 2 (1.4) 9 (6.3) 11 (3.9) 4 5 Position sensitivity 2 (1.4) 1 (0.7) 3 (1.1) 6 Vibration sensitivity 15 (10.7) 13 (9.1) 28 (9.9) 7 Showing ≥1 altered sign 16 (11.4) 21 (14.7) 37 (13.1) 8 9 Hemogram-Clinical Biochemistry 10 Vitamin B12 (pg/mL), mean (SD) 173.1 (27.3) 166.4 (32.6) 169.7 (6.3) 11 Anemia b 16 (11.4) 27 (18.9) 43 (15.2) 12 13 Hematocrit (%), mean (SD) 42.4 (4.0) 41.9 (4.2) 442.1 (4.1) 14 MCV (fL), mean (SD) 92.1 (6.7) 94.3 (7.4) 93.2 (7.1) 15 Anti-intrinsic factor antibody 15 (11.0) 15 (10.5) 30 (10.8) 16 Medication For peer review only 17 18 Proton-pump inhibitors (PPI) 57 (40.7) 64 (44.8) 121 (42.8) 19 Metformin 69 (49.3) 56 (39.2) 125 (44.2) 20 PPI and metformin 33 (23.6) 30 (21.0) 63 (22.3) 21 Scales 22 c 23 MMSE , mean (SD) 30.8 (4.6) 30.2 (4.8) 30.5 (4.7) 24 EQ-5D-Utilities, mean (SD) 0,817 (0,169) 0,855 (0,139) 0,836 (0,171) 25 26 aNeoweberian occupational social class (CSO-SEE12). Gac Sanit. 2013;27(3):263–272. 27 bAnaemia was defined by the World Health Organization criteria (haemoglobin <12 g/dL in women and 28 <13 g/dL in men). https://www.who.int/vmnis/indicators/haemoglobin 29 cMini Mental State Examination. Maximum score= 35 points. Normal score= 30–35. Borderline score= 30 31 24–29 points. Scores < 24 points in patients aged >65 years and scores < 29 points in patients aged <65 32 years suggest cognitive impairment. 33 34 35 Primary outcomes 36 37 At week 8, the difference in the success rate between the oral and IM routes was -0.7% (95%CI: 38 39 -3.2% to 1.8%; p>0,999) and 4.8% (95%CI: -1.3% to 10.9%; p=0.124) with the PPT and ITT 40 41 42 analyses, respectively. At week 26, these differences were -12.9% (95%CI: -17.9% to -6.1%; 43 44 p<0.001) and -3.2% (95%CI: -11.8% to 5.4%; p=0.470), respectively. At week 52, these 45 46 differences were -6.3% (95%CI: -11.9% to -0.07%; p=0.025) and -6.8% (95%CI: -16.6% to 47 48 2.9%; p=0.171), respectively (Figure 2). 49 50 51 In the PPT analysis under a Bayesian approach, the probabilities of differences in the 52 53 treatment effectiveness being >10% between the oral and IM groups were 0.001, 0.201, and 54 55 0.036 at weeks 8, 26, and 52, respectively. In the ITT analysis, these values were 0.000, 0.015, 56 57 58 12 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 15 of 51 BMJ Open

1 2 3 and 0.060 at weeks 8, 26, and 52, respectively (Supplement 2). The result of the likelihood ratio 4 5 6 for the cutpoints at the main percentiles of the distribution of VB12 serum levels at week 8 to 7 8 predict normalization at the end of the study is shown in Supplement 3. The level at the 5th 9 10 percentile of the distribution was selected as the most useful value because it showed the best 11 12 classification ability. When patients did not reach this level at week 8, they were almost twelve 13 14 15 times more likely to not reach suitable VB12 levels at the end of the study than if they had 16 For peer review only 17 reached levels over 281 pg/mL (12~1/negative likelihood ratio). 18 19 In the ITT analysis, the factors affecting the success rate at week 52 were age, for each 20 21 22 year of increase in age, the success rate decreased by 5%, and having attained VB12 levels of 23 24 ≥281 pg/mL at week 8, which yielded a success rate 8.1 times higher (Table 2). 25 26 27 28 29 30 31 Table 2. Factors associated with VB12 concentrations ≥ 211 pg/ml at week 52 32 33 Variable Odds ratio Robust std. error P>z 95% CI 34 35 IM vs. oral route 1.10 0.370 0.776 (0.57 to 2.13) 36 Age 0.95 0.022 0.025 (0.91 to 0.99) 37 VB12 concentration 8.10 5.014 0.001 (2.41 to 27.25) 38 >281 pg/ml at week 8 39 Constant 0.78 0.622 0.755 (0.16 to 3.72) 40 41 GLM, N=265. Variance function: V(u) = u*(1-u/1) [Binomial]. Link function: g(u) = ln(u/(1-u)) 42 [Logit]. AIC= 0.89967. BIC = -1225.89. 43 44 45 46 The mean levels of VB12 for each follow-up visit were above the normalization 47 48 threshold in both groups, although these values were much greater in the IM group (Supplement 49 50 4). In 51 patients (36 IM and 5 oral), the levels of VB12 in week 8 were above the normal range 51 52 53 limit of the laboratory (≥911 pg/mL), so the treatment regimen was changed from the initial 54 55 planned pattern. 56 57 58 13 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 16 of 51

1 2 3 Secondary outcomes 4 5 6 In terms of quality of life and the presence of signs related to VB12 deficiency, no significant 7 8 differences were found between treatment arms at any of the follow-up visits (Table 3). 9 10 Table 3. Secondary outcomes (Quality of life and exploratory findings) at weeks 8, 26 and 52 11 12 Visit Oral route IM route p Mean difference 13 N Mean (SD) N Mean (SD) (95% CI) 14 Quality of life (EQ-5D-5L Index) 15 16 Baseline 139 For0.855 peer137 0.817review (0.197) 0.066 only0.038 (-0.002 to 0.078) 17 (0.139) 18 Week 8 134 0.853 130 0.822 (0.204) 0.173 0.031 (-0.013 to 0.075) 19 (0.158) 20 Week 26 128 0.853 122 0.826 (0.191) 0.219 0.027 (-0.016 to 0.070) 21 (0.153) 22 Week 52 112 0.824 117 0.823 (0.194) 0.001 (-0.047 to 0.049) 23 (0.179) 0.958 24 25 At least one altered sign (glossitis and/or altered vibration sensitivity and/or altered position 26 sensitivity) 27 Visit N n (%) N n (%) p Proportion difference 28 (95% CI) 29 Baseline 140 16 (11.4%) 143 21 (14.7) 0.416 -3.3 (-11.1% to 4.6) 30 Week 8 135 15 (11.1%) 130 13 (10.0) 0.769 1.1 (-6.3% to 8.5) 31 Week 26 131 14 (10.7%) 122 12 (9.8) 0.824 0.9 (-6.6% to 8.3) 32 Week 52 122 14 (12.5%) 117 9 (7.7) 0.226 3.8 (-3.7% to 11.2) 33 34 35 Eleven adverse events were reported and none of them were severe; five (3.57%) 36 37 occurred with patients in the oral arm and six (4.20%) with patients in the IM arm, yielding a 38 39 40 difference of -0.63% (95%CI: -5.12% to 3.87%, p=0.786). Three patients withdrew from the 41 42 study: one patient in the oral group due to urticaria, and two in the IM group due to reddening 43 44 and pruritic facial erythema and generalized itching (mainly in the cheeks with scarce 45 46 47 urticariform lesions). In three other cases, treatment for the adverse events was prescribed 48 49 (constipation and erythema), and in five cases, it was not necessary to take further measures 50 51 (Table 4). 52 53 54 55 56 57 58 14 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 17 of 51 BMJ Open

1 2 3 Table 4. Description of adverse events by patient and route of administration 4 5 Route Adverse event Action 6 7 Constipation Administration of specific treatment 8 Generalized itching and hives on the cheeks Withdrawal 9 Dyspepsia Treatment not required IM route 10 Constipation Administration of specific treatment 11 12 Redness and pruritic facial erythema Withdrawal 13 Erythema on forearms Administration of specific treatment 14 Urticaria on the neck and arms Treatment not required 15 Occasional postprandial dyspepsia Treatment not required 16 For peer review only 17 Oral route Occasional postprandial dyspepsia Treatment not required 18 Urticaria Withdrawal 19 Increased irritability and nervousness Treatment not required 20 21 22 23 At week 8, adherence to treatment was evaluated in 265 patients, of whom 95.5% were 24 25 adherent (97.8% oral and 93.8% IM); the difference between the groups was 4% (95%CI: -0. 1% 26 27 to 8.7%; p=0.109). At week 52, adherence was evaluated in 229 patients, of whom 220 (96.1%) 28 29 30 were adherent (98.2% oral and 94.0% IM); the difference was 4.2% (95%CI: -0.7% to 9.1%; 31 32 p=0.172). 33 34 Overall, 89.5% of the patients reported being satisfied or very satisfied with the treatment 35 36 via the oral route (91.3%) and the IM route (87.6%). The difference was 3.7% (95% CI: -4.0% to 37 38 39 11.3%; p=0.348). 40 41 A total of 83.4% of patients preferred the oral route (97.6% among the patients receiving VB12 42 43 orally vs. 68.6% of the patients in the IM group); the difference was 29.0% (95%CI: 20.3 to 44 45 46 37.7;p<0.001). The preferences expressed by the patients referred to their potential choice 47 48 regardless of the arm to which they were assigned 49 50 DISCUSSION 51 52 53 Main findings of the study 54 55 56 57 58 15 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 18 of 51

1 2 3 Supplementing VB12 in patients with VB12 deficiency, whether orally or 4 5 6 intramuscularly, achieves the normalization of VB12 levels in most cases. The oral route was not 7 8 inferior to the IM route during the charging period. Formally, the pre-established conditions for 9 10 determining the noninferiority of oral administration were not met for the complete follow-up 11 12 period, but these results merit a deeper analysis. 13 14 15 Differences between the administration routes were found at 26 and 52 weeks. The IM 16 For peer review only 17 maintenance treatment of 1 mg/month was effective in maintaining VB12 levels, while oral 18 19 administration of 1 mg/week had a probability of being inferior (by more than 10%) to the IM 20 21 22 route by 20% in the most unfavourable scenario (PPT). However, given that no strategy was 23 24 superior in the charging period, and in view of the model results showing that when VB12 levels 25 26 reached ≥ 281 pg/mL during the charging period, the success rate at 12 months was 8 times 27 28 29 higher, the probability that the differences between groups would exceed Δ was very low, 30 31 independent of the administration route. The most plausible explanation for the observed 32 33 difference between routes might be that in patients below this threshold, the maintenance oral 34 35 dose should be higher than the dose used in the present study. Some authors have recommended 36 37 38 that an oral dose of 2 mg/week be administered as a maintenance dose.(34) 39 40 The incidence of adverse events was very low and similar for oral and intramuscular 41 42 administration, and nonserious adverse events were found. These findings were similar to other 43 44 (5) 45 studies. Patients’ preferences can be a decisive factor for determining the administration route. 46 47 In this trial, similar to previous studies,(17) there was a clear preference for the oral route, 48 49 especially among the patients assigned to this group. 50 51 52 53 54 55 56 57 58 16 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 19 of 51 BMJ Open

1 2 3 The effect of VB12 supplements on quality of life remains unclear,(35,36) but the present 4 5 6 results show that the treatment route does not improve patients´ perception of their health-related 7 8 quality of life or related symptoms. 9 10 We did not find significant differences in adherence. Adherence to the treatment via the 11 12 IM route was lower than expected. Although drug administration was assured once the patient 13 14 15 attended the consultation, the patient could choose not to attend appointments for various 16 For peer review only 17 reasons. However, in usual practice, adherence with the oral route could be more compromised 18 19 than with the IM route, and this factor should be taken into consideration to personalize 20 21 22 prescription. 23 24 Comparison with other studies 25 26 The comparison with other studies is difficult, due to the treatment different doses used, 27 28 29 but especially because of the follow-up length had been inferior to 4 months and the number of 30 31 patients included was small. 32 33 As far as we know, the present trial is the largest clinical trial with the longest follow-up 34 35 period, and it is the first to evaluate, in addition to VB12 levels, clinical signs and symptoms, 36 37 38 health-related quality of life, and patient preferences. The 3 clinical trials(23–25) described in the 39 40 2018 Cochrane Systematic Review(5) had a duration between 3 and 4 months and included a total 41 42 of 153 patients. In the Saraswathy trial, patients in the oral route at 3 months normalised levels 43 44 (25) 45 20/30 (66.7%) vs 27/30 (90%) of the patients in the IM route. In Kuzminski's patients in the 46 47 oral route at 4 months normalised levels 18/18 (100%) vs 10/14 (71.4%) of the patients in the IM 48 49 route.(23) These differences were statistically non-significant in both studies. 50 51 52 Two studies have recently been published and add evidence in favour of oral and sublingual 53 54 administration of VB12.(37,38) The follow-up of Moleiro's study reached 24 months versus 12 55 56 57 58 17 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 51

1 2 3 months in our study. However, Moleiro et al performed a prospective uncontrolled study that 4 5 6 included 26 patients submitted to total gastrectomy. All patients received oral VB12 7 8 supplementation (1 mg/day), and all of them maintained normalization V12 at 6, 12, 18, and 24 9 10 months. There was a progressive increase in serum V12 levels within the first 12 months, which 11 12 remained stable thereafter.(37) The long-term effectiveness of the oral route in absorption-deficient 13 14 15 people such as gastrectomized patients would support the results of our study. 16 For peer review only 17 Bensky et al. compared the efficacy of sublingual vs. intramuscular administration of 18 19 vitamin B12 in a retrospective observational study from the computerized pharmacy records of 20 21 22 Maccabi Health Service (MHS). Among 4281 patients treated with VB12 supplements (830 23 24 (19.3%) with IM and 3451 (80.7%) with sublingual tablets, the IM group achieved a significant 25 26 increase in VB12 levels compared with the sublingual group, OR 1.85, CI 95% 1.5-2.3. (38) 27 28 29 Although this study has a large sample size, the important methodological limitations on its 30 31 effectiveness (retrospective design; reliance on clinical records; absence of epidemiological 32 33 information such as patient age and sex or the aetiology of the deficit) should be considered in the 34 35 interpretation of their results. 36 37 38 Strengths and limitations 39 40 Our study was pragmatic(39) in both the inclusion and diagnostic methods criteria. The 41 42 majority of the patients with deficits included in this study presented no symptomatology or very 43 44 45 low-level symptoms, with no anemia, which is the common profile of most patients who present 46 47 with VB12 deficits in primary care. The study design did not allow for masking the patients to 48 49 the received treatment. However, these limitations were compensated for by the objective 50 51 52 measurement of the main outcome variable. 53 54 55 56 57 58 18 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 21 of 51 BMJ Open

1 2 3 As occurs in all pragmatic clinical trials, patient recruitment was complicated, and the 4 5 6 sample size reached only 88.4% of the calculated necessary size, which implies that the power of 7 8 the study was limited. Hence, the analysis was complemented using Bayesian methods that allow 9 10 for studying a posteriori the likelihood of a difference between two outcomes to exceed a certain 11 12 limit.(40) Under this approach, the a posteriori probability for differences to exceed the proposed 13 14 15 Δ=-10% was not significant during the charging period, and the probabilities were low but not 16 For peer review only 17 negligible in the PPT analysis and low in the ITT analysis over the complete follow-up period. 18 19 Loss to follow-up was low at 8 and 26 weeks and higher at 52 weeks. This effect has 20 21 22 been observed in pragmatic clinical trials with long follow-up periods. Missing data were greater 23 24 in the IM arm, during the interval between randomization and initiation of treatment (6% IM vs 25 26 1% oral), over 8 weeks (9% IM vs 4% oral) and over 26 weeks (15% IM vs 6%). These 27 28 29 differences could represent a lower acceptability of the IM route by patients, since the missing 30 31 data were mostly due to patient dropout. At 52 weeks, the numbers of losses in the two arms 32 33 were similar (20% oral and 18% IM), and in the case of oral treatment, several of those losses 34 35 were withdrawals occasioned by not achieving particular levels of VB12. 36 37 38 Implications of the study findings 39 40 On the basis of our results and the available evidence, we propose the oral administration 41 42 of VB12 at 1 mg/day during the charging period. Subsequently, the recommended dose would 43 44 45 vary as a function of the VB12 levels reached during the charging period. For VB12 46 47 concentrations between the normal levels of 211 pg/mL (in our laboratory) and 281 pg/mL (the 48 49 5th percentile of the distribution in this trial), a dose of 2 mg/week is suggested. When the levels 50 51 th 52 reached in the charging period are between 281 and 380 pg/mL (the 20 percentile of the 53 54 distribution), it may be appropriate to perform an analysis between 8 and 26 weeks to confirm 55 56 57 58 19 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 22 of 51

1 2 3 that normal levels are maintained. All patients who reach a level of 380 pg/mL by week 8 could 4 5 6 be maintained at the initial dosage (1 mg/week) without subsequent analyses during the year of 7 8 follow-up. 9 10 If the IM route is chosen, the proposed dose for this route during the first few weeks may 11 12 be excessive for patients with VB12 deficiency. The scheduled IM dose should be reconsidered 13 14 15 in the first two weeks based on VB12 levels, and the scheduled dose could be limited to 1 16 For peer review only 17 mg/week if warranted by the outcome. Nevertheless, these recommendations must be assessed in 18 19 further research. 20 21 22 Oral administration of VB12 in patients older than 65 years is probably as effective as 23 24 intramuscular administration, and it also lacks adverse effects and is preferred by patients. We 25 26 must also highlight the potential benefit of the oral route in terms of safety for patients with 27 28 29 coagulation problems, for whom IM-administered medication is often contraindicated. A small 30 31 number of patients may require additional follow-up after 8 weeks if a certain concentration of 32 33 VB12 in blood is not reached. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 20 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 51 BMJ Open