Distribution of Vitamin B12 Absorption in a Community and Factors Associated with the Response to Long-term Oral Supplementation

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OBJECTIVE In a secondary data analysis of a B-12 absorption (CobaSorb) study and a B-12 supplementation trial in a B-12 insufficient Indian rural community, we examined i) the distribution of plasma holo-transcobalamin (holoTC) response during the CobaSorb test, and ii) determinants of plasma B-12 response to long-term, oral supplementation with ‘physiological doses’ of B-12. METHODS The participants in the Pune Maternal Nutrition Study first underwent a B-12 absorption study (CobaSorb protocol). Subsequently, they participated in a 12-month-long, double-blind RCT of daily oral B-12 (0, 2, or 10 µg) and folic acid (0 or 200 µg). We studied distribution of plasma holoTC response in the CobaSorb test, and used linear mixed-effect model to determine predictors of B-12 absorption and response to supplementation. RESULTS Participants (313) included children (n = 109, 57 boys, mean age 9y, weight 21.9 kg, BMI 13.6 kg/m 2 ), mothers (n = 108, 30y, 47.7 kg, 19.3 kg/m 2 ), and fathers (n = 96, 37y, 59.3 kg, 21.4 kg/m 2 ). The plasma holoTC response during the absorption test was continuously distributed and was negatively associated with weight and positively with the dose of B-12 (10 or 2 µg). Response to long-term B-12 supplementation was positively predicted by absorption test response, dose of B-12, length of supplementation, and compliance. CONCLUSIONS The continuous distribution of plasma holoTC response during the absorption test supports physiological absorption of B-12 in a community. CobaSorb protocol could be used to unravel the intricacies of B-12 absorption. Health sciences/Medical research/Epidemiology Health sciences/Health care/Nutrition Figures Figure 1 Figure 2 INTRODUCTION Vitamin B12, a water-soluble vitamin, is an essential component of many metabolic pathways but cannot be synthesized in humans. It is available almost exclusively from animal products in the diet and possibly from the intestinal microbiota. Worldwide, B-12 insufficiency and deficiency are increasingly driven by reduced intake of animal products, particularly red meat, aging populations, and by food-B12 (cobalamin) malabsorption [ 1 , 2 ]. The interpretation of biomarkers of B-12 status requires an understanding of the process of B-12 absorption and metabolism. Orally consumed B-12 attaches to salivary carrier proteins and is transferred to gastric intrinsic factor (IF), which mediates intestinal absorption. Up to 70% of absorbed vitamin is transported by haptocorrin for storage in the liver. The remaining 30% is transported by transcobalamin II and is delivered to peripheral tissues for cellular metabolism [ 1 , 3 ]. The transcobalamin -B-12 complex is called holo-transcobalamin (holoTC) or ‘active B-12’ [ 4 ]. Vitamin B-12 promotes cytoplasmic methylation reactions, which generate methionine from homocysteine and provide S-adenosyl methionine (SAM), a universal methyl donor. Vitamin B12 is also a coenzyme for a mutase enzyme, which converts methylmalonic acid (MMA) to propionic acid, a source of energy in mitochondria. Elevated circulating total homocysteine (tHcy) and MMA concentrations indicate reduced tissue availability of B-12. Elevated tHcy may also reflect deficiencies of other ‘methyl’ vitamins (folate, pyridoxine, riboflavin) while elevated MMA is usually considered a specific biomarker of B-12 deficiency [ 1 – 4 ]. Conventional ideas about B-12 absorption are based on the Schilling test, used to help diagnose pernicious anaemia, and resulted in binary reports of ‘absorber’/ ‘non-absorber’ [ 5 ]. Schilling’s test used a radioactive isotope of cobalt and is not available now. Stable isotope labelled vitamin is being used to investigate B-12 absorption in specialized research laboratories [ 6 ]. The CobaSorb test investigates the absorption of B-12 from the gut and its bioavailability by measuring the rise in circulating levels of holoTC after oral administration of ‘physiological’ doses of B-12. It may be used in routine clinical practice. There are only a few reports of this measure with healthy controls and little information on the distribution of absorption characteristics in B12-deficient populations [ 7 ]. India has a high prevalence of low plasma B-12 status, attributed to the low intake of animal-origin foods. In our cross-sectional and longitudinal cohort studies, we have found a high prevalence of low B-12 status (< 150 pmol/L coupled with hyperhomocysteinemia in 21 to 70%) throughout the life course [ 8 – 11 ]. We used the CobaSorb protocol of Bor et al [ 12 ] to investigate B-12 absorption in 313 participants of the Pune Maternal Nutrition Study (parents and children) by measuring the rise in circulating holoTC concentration using physiological doses of oral B-12 (10 and 2 µg x 3 doses, 6 hours apart) [ 13 ]. This study [ 13 ] showed that the 24-hour rise in holoTC after an oral challenge with B-12 was normal in > 90% of participants based on Bor’s criteria. The same parents and children subsequently participated in a randomized controlled trial of physiological doses of B-12 and folic acid (daily 2 or 10 µg B-12, with or without 200 µg folic acid), for 12 months [ 14 ]. Circulating B-12 concentrations were measured at baseline, and 4 and 12 months after starting the supplementation. These datasets [ 13 , 14 ] allowed a secondary analysis to be undertaken to study the distribution of the increase in holoTC in the CobaSorb test and to examine the relationships between the increase in holoTC during the absorption test and the response of circulating B-12 concentrations after 4 and 12 months of daily supplementation. SUBJECTS AND METHODS Study Setting and Participants Briefly, the Pune Maternal Nutrition Study (PMNS) is a community-based, prospective birth cohort established during 1993-94 in six villages near Pune [ 15 ]. The original PMNS enrolled 814 pregnant women for the study of maternal nutrition and fetal growth. An additional 153 women from the same population were enrolled (extended PMNS) after the completion of the original study to standardize ultrasonic measurements of early fetal growth in this undernourished population. Parents and children in the original and the extended PMNS have been followed up regularly to study physical growth and the evolution of type 2 diabetes and cardiovascular disease. One of the important nutritional findings in the PMNS was the high rates of low B-12 concentrations and hyperhomocysteinemia in this largely vegetarian population [ 11 ]. We performed a B-12 absorption study (CobaSorb test: rise in circulating concentrations of holoTC in response to 3 physiological oral doses of B-12, 6 h apart) to rule out absorption defects in this population [ 13 ]. This was followed by a pilot trial of physiological dose B-12 and folic acid supplementation to improve one-carbon metabolism [ 14 ], in anticipation of our intergenerational trial [ 16 ] to reduce the primordial risk of diabesity in the population. The KEM Hospital Ethics Committee approved both studies (KEMHRC/VSP/Dir Off/EC/065; Project No. 067). The timeline of the B-12 absorption protocol [ 13 ] and B-12 supplementation trial [ 14 ] is summarized in Fig. 1 . Absorption study protocol One hundred and nineteen families, from the follow-up in the extended PMNS, were invited to participate (May to November 2006); 109 families participated [ 13 ]. The participants were admitted overnight for the absorption study in the Diabetes Unit, KEM Hospital & Research Centre, Pune. Standardised food and facilities were provided. Demographic details and anthropometric measurements were recorded. A fasting blood sample was collected (baseline), and a 10 µg (64 families) or 2 µg (44 families) B-12 (cyanocobalamin) capsules were administered under supervision every 6 h x 3 times. A fasting blood sample was collected the following morning (post-dose sample) to measure the rise in circulating holoTC concentration. Supplementation study The same participants were enrolled in this randomized, stratified, placebo-controlled longitudinal trial with a factorial design (April 2007 – March 2008; ISRCTN59289820, Protocol No. 079877/Z/06/Z) [ 14 ]. The participants were randomized to receive one of three daily doses of B-12 (0, 2, or 10 µg) and two doses of folic acid (0 or 200 µg), forming six groups (B0F0, B2F0, B10F0, B0F200, B2F200, and B10F200). Randomization was computer-based. The unit of randomization was the family, making it a cluster-randomized trial. Stratification was by the children’s baseline plasma B12 concentrations (below and above the median value of 188 pmol/L). The duration of supplementation was 12 months, and a non-fasting blood sample was collected at 4 and 12 months after starting supplementation to measure circulating B-12 concentrations. Compliance with B-12 supplements was assessed monthly by counting the unused capsules and as a percentage of the capsules consumed. Anthropometry and Laboratory Measurements Height was measured to the nearest 0.1cm using a wall-mounted stadiometer (CMS Instruments, London, UK), and body weight to the nearest 0.05 kg (Conveigh, Electronic Instruments Ltd, Mumbai, India). Plasma B-12 and folate were measured by microbiological assays using a colistin sulfate-resistant strain of Lactobacillus leishmannii [ 17 , 18 ] and a chloramphenicol-resistant strain of Lactobacillus casei I [ 19 , 20 ], with inter-batch CVs of < 8% and < 7%, respectively. Plasma holoTC was measured using magnetic beads (microspheres) with an immobilized monoclonal antibody specific for human transcobalamin, followed by the conventional microbiological assay developed for cobalamin estimation [ 21 , 22 ]. The combined B-12 marker (cB12) was estimated, using plasma B-12, total homocysteine, and folate concentrations, using Fedosov’s method [ 23 ]. B-12 adequacy status at baseline, 4, and 12 months was estimated using the cut points of cB12 defined for epidemiological purposes, viz. B-12 adequacy: cB12 >-0.5, transitional B-12 status: cB12 ≥ -2.5 and ≤ -0.5, low B-12 status: cB12 < -2.5 [ 23 ]. Statistical methods Data are presented as mean ± SD, or as median and 25th–75th percentile when not normally distributed. The Shapiro-Wilk test was used to test normality. Variables not normally distributed were transformed to the natural logarithm before analysis (weight, plasma B-12, folate, and holoTC). Correlation and linear mixed effects regression analyses were used to explore associations and predictors. B-12 Absorption Study We studied the distribution of the rise in holoTC concentration during the absorption study by plotting it graphically. We investigated the contribution of the following predictors to the rise in plasma holoTC: body weight, sex, and dose of B-12 (2 or 10 µg), using linear mixed-effects regression analysis. B-12 Supplementation trial Predictors of circulating B-12 concentrations at 4 and 12 months included: absorption performance (the change in holoTC during the absorption test, adjusted for the dose of B-12), the dose of B-12 (0, 2, or 10 µg), and folic acid (0 or 200 µg) in the supplementation trial, and the compliance and length of supplementation. We used linear mixed-effects regression analyses for this purpose. All statistical analyses were carried out using R software (R ver. 4.4). RESULTS Recruitment and participant flow The consort diagram (Supplementary Fig. 1) shows the flow of participants in the two studies and exclusions or drop-outs. A total of 313 individuals participated in the B-12 absorption study; 311 had all measurements. Participants (300) were randomized in the supplementation trial; 291 had all measurements at 4 months and 287 at 12 months. Baseline characteristics Children (n = 109; 57 boys, 52 girls) were 9 years old, mothers (n = 108) were 30 years old, and fathers (n = 96) were 37 years old. Children weighed 21.9 kg, mothers 47.7 kg, and fathers 59.3 kg. Low plasma B-12 (< 150 pmol/L) concentrations were present in 27% of children, 48% of mothers, and 72% of fathers. Absorption Study Plasma B-12 and holoTC concentrations The median rise in holoTC concentrations was 42.0 pmol/L from baseline for the 10 µg dose (55.0 in children, 39.0 in mothers, 39.0 in fathers), and 23.0 pmol/L for the 2 µg dose (39.0 in children, 19.0 in mothers, 10.0 in fathers) (Table 1). Overall, plasma holoTC increased 4.8-fold with the 10 µg dose and 2.2-fold with the 2 µg dose; the rise was greater in children than in the parents (Table 1 and Fig. 2 ). Using Bor et al.’s 2004 criterion (rise in plasma holoTC < 15% and < 15 pmol/L after 3 doses of B-12) [ 12 ], 10% of participants (10 fathers, 5 mothers, and 4 children) in the 10 µg group were classified as poor absorbers. In a subsequent study by Bor et al., including patients with inherited B-12 malabsorption (Imerslund-Grasbeck syndrome or intrinsic factor deficiency), a rise of ≥ 6 pmol/L of holoTC was proposed as a cut point to define ‘adequate’ B-12 absorption (100% sensitivity and 72% specificity) [ 24 ]. Using this cut point, 3.7% of participants in our study (3 fathers, 2 mothers, and 2 children) could be classified as inadequate absorbers. Figure 2 shows the results of the CobaSorb Absorption Study for children, fathers, and mothers. Panel A presents a box plot of baseline and 24-hour holoTC concentrations in the 2 and 10 µg groups, while panel B illustrates a box plot of the rise in holoTC concentrations for the same dosage groups. Panel C displays density plots of the increase in plasma holoTC at 24 hours following three doses of 2 and 10 µg. The distributions are nearly bell-shaped, with a slight right skew due to a few outliers (Shapiro-Wilk test, p < 0.001 for plasma holoTC at baseline, 24 hours, and the change). These distributions were normalized using a log transformation for further analysis. The density plots can be interpreted in terms of the peak (average), width (variability), and shifts (comparing the effects of 2 and 10 µg B-12 doses). The peaks shift further to the right in children compared to parents, indicating a higher average rise for both doses. The width is smaller for the 2-µg dose than for the 10-µg dose, suggesting less variability, and the curves for the 10-µg dose are shifted to the right compared to the 2-µg dose, demonstrating a greater rise. The predominant characteristic of the density plots shows that the distribution is continuous and not binary or bimodal. In multiple regression analysis, the increase in holoTC concentration exhibited a small and inverse association with body weight [β (SE): -0.565 (0.060), P < 0.0001] and a strong and direct association with the doses of B-12 (10 vs. 2 µg) [β (SE): 19.881 (2.680), P < 0.0001] (Supplementary Table 1). Supplementation Study Plasma B-12 concentrations increased clinically significantly in those who received B-12 supplements, and plasma folate concentrations increased in those who received folic acid (Supplementary Table 2, Supplementary Fig. 2). At 12 months, the median B-12 rise was 1.9-fold (95% CI: 1.8–2.2) from baseline in the 10 µg group, compared to 1.5-fold (95% CI: 1.3–1.7) in the 2 µg group. Using the combined B-12 marker (cB12), the B-12 adequacy status remained stable in the placebo group (baseline 46.0% and 12 months 50.0%), while it improved from 41.5 to 73.3% in the 2 µg group, and from 35.7 to 85.7% in the 10 µg group ( P < 0.001 for the B-12 groups) (Table 2). The median compliance with supplements at 12 months was 86%. Compliance was ≥ 80% in 71% and only 3% were < 50% compliant. Predictors of response to supplementation Participants' 4- and 12-month responses were examined in cross-sectional and longitudinal models (Table 3). The response was inversely associated with age and weight (colinear so results are shown only for weight) and directly associated with B-12 dose, duration of supplementation, and compliance. In addition, the response was directly associated with the rise in holoTC measured in the absorption study (‘absorption performance’). At 12 months, the largest effect was of B-12 dose (for 2 µg: β (SE): 0.166 (0.057), P = 0.004; for 10 µg: 0.411 (0.056), P < 0.0001. The effect size of absorption performance (rise in plasma holoTC during CobaSorb test) was 0.002 (0.001), P = 0.012 (Table 3). DISCUSSION This secondary analysis of two linked community-based studies of B-12 metabolism [ 13 , 14 ] with largely vegetarian Indian people resident in Pune, India, expands our understanding of the absorption of B-12 and how it relates to response during long-term supplementation. We believe that ours is perhaps the largest study of B-12 absorption in a free-living community. Based on the criteria of Bor [ 24 ] the Cobasorb test demonstrated that 95% of participants could effectively absorb B-12. Our secondary analysis adds new knowledge in that i) the rise in plasma holoTC concentration (a measure of absorption after an oral challenge with ‘physiological’ dose B-12) is continuously distributed and ii) that it follows the expected physiological rules i.e. the concentration achieved was proportional to the dose and inversely to the weight of the participant. This conflicts with the common belief among clinicians and researchers to define B-12 absorption as Yes or No based on the obsolete Schilling test. Furthermore, the width (spread) of the distribution of rise in holoTC concentration was larger for the 10 µg compared to the 2 µg dose. This suggests that the physiological (carrier-mediated) absorption at near the recommended dietary allowance of 2 µg dose is more tightly regulated and therefore less variable than the additional contribution of passive absorption at the higher 10 µg dose. This understanding could inform a more ‘personalized’ and physiological approach towards dose in clinical practice and public health interventions. Recent exciting discoveries using stable isotopes of B-12 are expected to throw further light on this amazingly complex physiological process [ 6 , 25 – 27 ] and define a better model of absorption. We extended our secondary analysis to investigate if the performance during the CobaSorb test would predict the response to long-term supplementation. Allowing for body weight, compliance, and dose of B-12, the ‘absorption performance’ was a significant direct predictor, though admittedly the size of the effect was rather small. This finding leads us to believe that within physiological limits, the individual’s position within a population ‘tracks’ after an intervention and will inform personalized nutrition. The Schilling test, now obsolete because of restrictions on using radioactive isotopes, did not measure circulating B-12 concentrations and was performed mostly in ‘patient’ populations, with few controls or volunteers [ 5 ]. Henze et al [ 28 ] used a new assay for B-12 and described the distribution of serum B-12 concentration after an oral, supraphysiological dose of 1 mg B-12 in 32 normal participants (age range 22–75 y) as “Gaussian”. Much of this response would have been by passive absorption. A better understanding of the physiology of active B-12 absorption and binding with transport proteins led to the use of holoTC measurement in B-12 absorption protocols [ 4 , 12 , 24 ]. Bor et al stressed that the dose of B-12 (9 µg x 3 doses) in the CobaSorb test is still near-physiological [ 12 , 24 ]. The use of an even more physiological dose (2 µg x 3 doses) in a proportion of our subjects further supports the use of a rise in circulating holoTC concentration to study B-12 absorption [ 13 ] in different settings. Importantly, it uses routine laboratory facilities without radioactive markers (14C-labeled B12) or expensive and rare isotopes (13C-labeled B12), [ 6 , 25 – 27 , 29 , 30 ]. Strengths and limitations This analysis was possible only because the same cohort participated in both trials, there were only a few drop-outs and high compliance. The participants were representative of a community where high B-12 insufficiency and poor dietary B-12 intake are prevalent [ 8 , 10 ]. The studies were population-based, involving otherwise healthy individuals, and included both children and adults with a balanced sex ratio. The sample size was large, and the doses of B-12 used were ‘physiological’. These contribute to the strength of the statistical interpretations. On the other hand, this was a specific population with long term vegetarian practices, which may limit application to other populations. We could not find any published studies investigating similar predictors of response to supplementation to compare our results. Based on the outcome of this trial, a primordial trial [ 16 ] in the PMNS cohort was designed with a 2 µg dose. In summary, the absorption study revealed that the distribution of the absorption characteristics was continuous and not binary and that it followed physiological expectations. Long-term supplementation study showed that the absorption performance of the participant was a predictor of response to supplementation. Our findings expand the knowledge about B-12 absorption and provide a basis for further studies. Declarations Data Availability: The raw data supporting the conclusions of this article will be made available upon reasonable request and adequate institutional permissions. Acknowledgements We thank K. J. Coyaji, Director of the KEM Hospital, Pune, for providing the required facilities. We are grateful to the participants of the Pune Maternal Nutrition Study for taking part in these studies. RW is supported by a senior research fellowship from the Council of Scientific and Industrial Research, India Author Contributions CY conceived the idea of this secondary data analysis. UD, ER, and LP wrote the first draft of the manuscript. DB was involved in performing the laboratory analysis. SB, RW, OD, ER, CF, HR, CY, and UD were involved in the data analysis and interpretation of the results. All authors contributed to the article and approved the submitted version. Funding: The original research projects were supported by the Wellcome Trust, London, UK. Ethical Approval KEM Hospital Ethics Committee approved both the studies (KEMHRC/VSP/Dir Off/EC/065; Project No. 067). The B12 supplementation RCT was registered at the ISRCTN. Clinical Trial Registry number and website where it was obtained: ISRCTN59289820. DOI: https://doi.org/10.1186/ISRCTN59289820 https://www.isrctn.com/ Competing Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Allen LH, Miller JW, de Groot L, Rosenberg IH, Smith AD, Refsum H, et al. Biomarkers of nutrition for development (BOND): Vitamin B-12 review. J Nutr. 2018;148:1995S–2027S. Carmel R. 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Tables Tables are available in the Supplementary Files section. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Table1B12paper.xlsx Table 1 Table2B12paper.xlsx Table 2 Table3B12paper.xlsx Table 3 B12paperSupplementaryFile06092025.pdf Electronic Supplemental Material Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 13 Oct, 2025 Review # 2 received at journal 12 Oct, 2025 Reviewer # 2 agreed at journal 29 Sep, 2025 Review # 1 received at journal 15 Sep, 2025 Reviewer # 1 agreed at journal 14 Sep, 2025 Reviewers invited by journal 14 Sep, 2025 Editor assigned by journal 10 Sep, 2025 Submission checks completed at journal 10 Sep, 2025 First submitted to journal 09 Sep, 2025 Unknown event 08 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7551002","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":514739845,"identity":"0ee9dd8e-f15f-4841-b359-8705ec42eb09","order_by":0,"name":"Chittaranjan Yajnik","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYLACCQYJA34QI6GAFC2SDSAtBiRYZGBwAEwRoZS//4zhB8sdFsbG51cnfnhgwCDPL3aAgJtu5BhLSJ6RMDO78XazBNBhhjNnJxBwkASPgYRkm4SN2Y2zG0BaEgxuE9LCf8b4B0iL8Yyzm38Qp4Uhxwxki5kBf+824myRuJFWZgHUYixxg3ebRYKBBGG/8Pcf3nxbsq3OsL//7OabPyps5PmlCWhhYOAwYJYA2wdWKUFIOQiwP2D8ALbvADGqR8EoGAWjYCQCALQxPwDJN6fQAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2911-2378","institution":"King Edward Memorial Hospital and Research Centre","correspondingAuthor":true,"prefix":"","firstName":"Chittaranjan","middleName":"","lastName":"Yajnik","suffix":""},{"id":514739846,"identity":"ac814434-0348-4b89-bf8a-83b367eb8caa","order_by":1,"name":"Elaine Rush","email":"","orcid":"https://orcid.org/0000-0001-7713-6203","institution":"Auckland University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Elaine","middleName":"","lastName":"Rush","suffix":""},{"id":514739847,"identity":"3858a162-e1b5-44a9-a9c6-74ce255562fd","order_by":2,"name":"Dattatray Bhat","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dattatray","middleName":"","lastName":"Bhat","suffix":""},{"id":514739848,"identity":"7e8512aa-01df-4303-90cf-b5b0e9c5a901","order_by":3,"name":"Rucha Wagh","email":"","orcid":"","institution":"King Edward Memorial Hospital and Research Centre, Pune","correspondingAuthor":false,"prefix":"","firstName":"Rucha","middleName":"","lastName":"Wagh","suffix":""},{"id":514739849,"identity":"bc3e0756-84a3-4422-9914-56019af05726","order_by":4,"name":"Onkar Deshmukh","email":"","orcid":"","institution":"King Edward Memorial Hospital and Research Centre, Pune","correspondingAuthor":false,"prefix":"","firstName":"Onkar","middleName":"","lastName":"Deshmukh","suffix":""},{"id":514739850,"identity":"16622e79-ef48-4859-b2da-c2032a7bf2da","order_by":5,"name":"Rasika Ladkat","email":"","orcid":"","institution":"King Edward Memorial Hospital and Research Centre, Pune","correspondingAuthor":false,"prefix":"","firstName":"Rasika","middleName":"","lastName":"Ladkat","suffix":""},{"id":514739851,"identity":"4b2b51ad-a609-42e5-a303-3f689c39e013","order_by":6,"name":"Lindsay Plank","email":"","orcid":"https://orcid.org/0000-0003-2737-0151","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Lindsay","middleName":"","lastName":"Plank","suffix":""},{"id":514739852,"identity":"52de973d-14b2-498d-b632-4fc53126122b","order_by":7,"name":"Souvik Bandopadhyay","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Souvik","middleName":"","lastName":"Bandopadhyay","suffix":""},{"id":514739853,"identity":"c4e609c3-f64f-4b94-bb86-4d4f64631466","order_by":8,"name":"C. Fall","email":"","orcid":"","institution":"Southampton General Hospital","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"","lastName":"Fall","suffix":""},{"id":514739854,"identity":"04cf9c4b-b140-4c48-b4b9-7cfa25576ae5","order_by":9,"name":"Helga Refsum","email":"","orcid":"","institution":"Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.","correspondingAuthor":false,"prefix":"","firstName":"Helga","middleName":"","lastName":"Refsum","suffix":""},{"id":514739855,"identity":"8d9c6f18-b0a0-4240-9528-d60dda84503f","order_by":10,"name":"Urmila Deshmukh","email":"","orcid":"https://orcid.org/0000-0002-3372-5628","institution":"King Edward Memorial Hospital Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Urmila","middleName":"","lastName":"Deshmukh","suffix":""}],"badges":[],"createdAt":"2025-09-06 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1","display":"","copyAsset":false,"role":"figure","size":107891,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic presentation of B-12 Absorption and Supplementation studies.\u003c/p\u003e\n\u003cp\u003efoot note\u003cstrong\u003e: \u003c/strong\u003e[FA, folic acid; holoTC, holo-transcobalamin; RCT, randomized controlled trial]\u003c/p\u003e","description":"","filename":"Figure1B12paper.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7551002/v1/4139d103eb596e2bcb8b5123.jpg"},{"id":91954300,"identity":"aa569368-e263-4f70-8e3c-5c66d1077cde","added_by":"auto","created_at":"2025-09-23 07:07:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83723,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the CobaSorb Absorption Study in children, fathers, and mothers after 3 doses of 2 and 10 µg B-12. A: Box plots of baseline and 24 hr plasma holoTC. B: Rise in plasma holoTC from the baseline. C: Density plots of the rise in plasma holoTC.\u003c/p\u003e","description":"","filename":"Figure2B12paper.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7551002/v1/81f7dc43eef99121f3cd4e3f.jpg"},{"id":91958033,"identity":"def6aeeb-e710-4dfd-aff2-99fd2584a409","added_by":"auto","created_at":"2025-09-23 07:31:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3650795,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7551002/v1/d761637f-050e-4e13-b6be-d3c0a3b5cb3a.pdf"},{"id":91954298,"identity":"d9ee889f-8c53-4286-9771-e80724cd0ebd","added_by":"auto","created_at":"2025-09-23 07:07:02","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9967,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"Table1B12paper.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7551002/v1/f837c43a59b4a8477df2a588.xlsx"},{"id":91956452,"identity":"90c95428-2e7e-4d03-9db1-2575da8dc447","added_by":"auto","created_at":"2025-09-23 07:15:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9888,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"Table2B12paper.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7551002/v1/16757c015048bc54266bf7b9.xlsx"},{"id":91954301,"identity":"128bf6ec-2f13-40b0-b7c7-ebc41327555d","added_by":"auto","created_at":"2025-09-23 07:07:02","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10721,"visible":true,"origin":"","legend":"Table 3","description":"","filename":"Table3B12paper.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7551002/v1/ec82f86f9862b246d1c1d9be.xlsx"},{"id":91954306,"identity":"ef1d79ae-a2fa-4a58-ab2c-5f311e8b307f","added_by":"auto","created_at":"2025-09-23 07:07:02","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":366626,"visible":true,"origin":"","legend":"Electronic Supplemental Material","description":"","filename":"B12paperSupplementaryFile06092025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7551002/v1/3cc0fbe643db9441d82b2ee8.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Distribution of Vitamin B12 Absorption in a Community and Factors Associated with the Response to Long-term Oral Supplementation","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eVitamin B12, a water-soluble vitamin, is an essential component of many metabolic pathways but cannot be synthesized in humans. It is available almost exclusively from animal products in the diet and possibly from the intestinal microbiota. Worldwide, B-12 insufficiency and deficiency are increasingly driven by reduced intake of animal products, particularly red meat, aging populations, and by food-B12 (cobalamin) malabsorption [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The interpretation of biomarkers of B-12 status requires an understanding of the process of B-12 absorption and metabolism.\u003c/p\u003e\u003cp\u003eOrally consumed B-12 attaches to salivary carrier proteins and is transferred to gastric intrinsic factor (IF), which mediates intestinal absorption. Up to 70% of absorbed vitamin is transported by haptocorrin for storage in the liver. The remaining 30% is transported by transcobalamin II and is delivered to peripheral tissues for cellular metabolism [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The transcobalamin -B-12 complex is called holo-transcobalamin (holoTC) or \u0026lsquo;active B-12\u0026rsquo; [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Vitamin B-12 promotes cytoplasmic methylation reactions, which generate methionine from homocysteine and provide S-adenosyl methionine (SAM), a universal methyl donor. Vitamin B12 is also a coenzyme for a mutase enzyme, which converts methylmalonic acid (MMA) to propionic acid, a source of energy in mitochondria. Elevated circulating total homocysteine (tHcy) and MMA concentrations indicate reduced tissue availability of B-12. Elevated tHcy may also reflect deficiencies of other \u0026lsquo;methyl\u0026rsquo; vitamins (folate, pyridoxine, riboflavin) while elevated MMA is usually considered a specific biomarker of B-12 deficiency [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConventional ideas about B-12 absorption are based on the Schilling test, used to help diagnose pernicious anaemia, and resulted in binary reports of \u0026lsquo;absorber\u0026rsquo;/ \u0026lsquo;non-absorber\u0026rsquo; [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Schilling\u0026rsquo;s test used a radioactive isotope of cobalt and is not available now. Stable isotope labelled vitamin is being used to investigate B-12 absorption in specialized research laboratories [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The CobaSorb test investigates the absorption of B-12 from the gut and its bioavailability by measuring the rise in circulating levels of holoTC after oral administration of \u0026lsquo;physiological\u0026rsquo; doses of B-12. It may be used in routine clinical practice. There are only a few reports of this measure with healthy controls and little information on the distribution of absorption characteristics in B12-deficient populations [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIndia has a high prevalence of low plasma B-12 status, attributed to the low intake of animal-origin foods. In our cross-sectional and longitudinal cohort studies, we have found a high prevalence of low B-12 status (\u0026lt;\u0026thinsp;150 pmol/L coupled with hyperhomocysteinemia in 21 to 70%) throughout the life course [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe used the CobaSorb protocol of Bor et al [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] to investigate B-12 absorption in 313 participants of the Pune Maternal Nutrition Study (parents and children) by measuring the rise in circulating holoTC concentration using physiological doses of oral B-12 (10 and 2 \u0026micro;g x 3 doses, 6 hours apart) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] showed that the 24-hour rise in holoTC after an oral challenge with B-12 was normal in \u0026gt;\u0026thinsp;90% of participants based on Bor\u0026rsquo;s criteria. The same parents and children subsequently participated in a randomized controlled trial of physiological doses of B-12 and folic acid (daily 2 or 10 \u0026micro;g B-12, with or without 200 \u0026micro;g folic acid), for 12 months [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Circulating B-12 concentrations were measured at baseline, and 4 and 12 months after starting the supplementation.\u003c/p\u003e\u003cp\u003eThese datasets [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] allowed a secondary analysis to be undertaken to study the distribution of the increase in holoTC in the CobaSorb test and to examine the relationships between the increase in holoTC during the absorption test and the response of circulating B-12 concentrations after 4 and 12 months of daily supplementation.\u003c/p\u003e"},{"header":"SUBJECTS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Setting and Participants\u003c/h2\u003e\u003cp\u003eBriefly, the Pune Maternal Nutrition Study (PMNS) is a community-based, prospective birth cohort established during 1993-94 in six villages near Pune [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The original PMNS enrolled 814 pregnant women for the study of maternal nutrition and fetal growth. An additional 153 women from the same population were enrolled (extended PMNS) after the completion of the original study to standardize ultrasonic measurements of early fetal growth in this undernourished population. Parents and children in the original and the extended PMNS have been followed up regularly to study physical growth and the evolution of type 2 diabetes and cardiovascular disease. One of the important nutritional findings in the PMNS was the high rates of low B-12 concentrations and hyperhomocysteinemia in this largely vegetarian population [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We performed a B-12 absorption study (CobaSorb test: rise in circulating concentrations of holoTC in response to 3 physiological oral doses of B-12, 6 h apart) to rule out absorption defects in this population [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This was followed by a pilot trial of physiological dose B-12 and folic acid supplementation to improve one-carbon metabolism [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], in anticipation of our intergenerational trial [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] to reduce the primordial risk of diabesity in the population. The KEM Hospital Ethics Committee approved both studies (KEMHRC/VSP/Dir Off/EC/065; Project No. 067).\u003c/p\u003e\u003cp\u003eThe timeline of the B-12 absorption protocol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and B-12 supplementation trial [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAbsorption study protocol\u003c/h3\u003e\n\u003cp\u003eOne hundred and nineteen families, from the follow-up in the extended PMNS, were invited to participate (May to November 2006); 109 families participated [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The participants were admitted overnight for the absorption study in the Diabetes Unit, KEM Hospital \u0026amp; Research Centre, Pune. Standardised food and facilities were provided. Demographic details and anthropometric measurements were recorded. A fasting blood sample was collected (baseline), and a 10 \u0026micro;g (64 families) or 2 \u0026micro;g (44 families) B-12 (cyanocobalamin) capsules were administered under supervision every 6 h x 3 times. A fasting blood sample was collected the following morning (post-dose sample) to measure the rise in circulating holoTC concentration.\u003c/p\u003e\n\u003ch3\u003eSupplementation study\u003c/h3\u003e\n\u003cp\u003eThe same participants were enrolled in this randomized, stratified, placebo-controlled longitudinal trial with a factorial design (April 2007 \u0026ndash; March 2008; ISRCTN59289820, Protocol No. 079877/Z/06/Z) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The participants were randomized to receive one of three daily doses of B-12 (0, 2, or 10 \u0026micro;g) and two doses of folic acid (0 or 200 \u0026micro;g), forming six groups (B0F0, B2F0, B10F0, B0F200, B2F200, and B10F200). Randomization was computer-based. The unit of randomization was the family, making it a cluster-randomized trial. Stratification was by the children\u0026rsquo;s baseline plasma B12 concentrations (below and above the median value of 188 pmol/L). The duration of supplementation was 12 months, and a non-fasting blood sample was collected at 4 and 12 months after starting supplementation to measure circulating B-12 concentrations. Compliance with B-12 supplements was assessed monthly by counting the unused capsules and as a percentage of the capsules consumed.\u003c/p\u003e\n\u003ch3\u003eAnthropometry and Laboratory Measurements\u003c/h3\u003e\n\u003cp\u003eHeight was measured to the nearest 0.1cm using a wall-mounted stadiometer (CMS Instruments, London, UK), and body weight to the nearest 0.05 kg (Conveigh, Electronic Instruments Ltd, Mumbai, India). Plasma B-12 and folate were measured by microbiological assays using a colistin sulfate-resistant strain of Lactobacillus leishmannii [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and a chloramphenicol-resistant strain of Lactobacillus casei I [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], with inter-batch CVs of \u0026lt;\u0026thinsp;8% and \u0026lt;\u0026thinsp;7%, respectively. Plasma holoTC was measured using magnetic beads (microspheres) with an immobilized monoclonal antibody specific for human transcobalamin, followed by the conventional microbiological assay developed for cobalamin estimation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe combined B-12 marker (cB12) was estimated, using plasma B-12, total homocysteine, and folate concentrations, using Fedosov\u0026rsquo;s method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. B-12 adequacy status at baseline, 4, and 12 months was estimated using the cut points of cB12 defined for epidemiological purposes, viz. B-12 adequacy: cB12 \u0026gt;-0.5, transitional B-12 status: cB12 \u0026ge; -2.5 and \u0026le; -0.5, low B-12 status: cB12 \u0026lt; -2.5 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStatistical methods\u003c/h3\u003e\n\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, or as median and 25th\u0026ndash;75th percentile when not normally distributed. The Shapiro-Wilk test was used to test normality. Variables not normally distributed were transformed to the natural logarithm before analysis (weight, plasma B-12, folate, and holoTC). Correlation and linear mixed effects regression analyses were used to explore associations and predictors.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eB-12 Absorption Study\u003c/h2\u003e\u003cp\u003eWe studied the distribution of the rise in holoTC concentration during the absorption study by plotting it graphically. We investigated the contribution of the following predictors to the rise in plasma holoTC: body weight, sex, and dose of B-12 (2 or 10 \u0026micro;g), using linear mixed-effects regression analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eB-12 Supplementation trial\u003c/h3\u003e\n\u003cp\u003ePredictors of circulating B-12 concentrations at 4 and 12 months included: absorption performance (the change in holoTC during the absorption test, adjusted for the dose of B-12), the dose of B-12 (0, 2, or 10 \u0026micro;g), and folic acid (0 or 200 \u0026micro;g) in the supplementation trial, and the compliance and length of supplementation. We used linear mixed-effects regression analyses for this purpose. All statistical analyses were carried out using R software (R ver. 4.4).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRecruitment and participant flow\u003c/h2\u003e\u003cp\u003eThe consort diagram (Supplementary Fig.\u0026nbsp;1) shows the flow of participants in the two studies and exclusions or drop-outs. A total of 313 individuals participated in the B-12 absorption study; 311 had all measurements. Participants (300) were randomized in the supplementation trial; 291 had all measurements at 4 months and 287 at 12 months.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBaseline characteristics\u003c/h2\u003e\u003cp\u003eChildren (n\u0026thinsp;=\u0026thinsp;109; 57 boys, 52 girls) were 9 years old, mothers (n\u0026thinsp;=\u0026thinsp;108) were 30 years old, and fathers (n\u0026thinsp;=\u0026thinsp;96) were 37 years old. Children weighed 21.9 kg, mothers 47.7 kg, and fathers 59.3 kg. Low plasma B-12 (\u0026lt;\u0026thinsp;150 pmol/L) concentrations were present in 27% of children, 48% of mothers, and 72% of fathers.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAbsorption Study\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003ePlasma B-12 and holoTC concentrations\u003c/h2\u003e\u003cp\u003eThe median rise in holoTC concentrations was 42.0 pmol/L from baseline for the 10 \u0026micro;g dose (55.0 in children, 39.0 in mothers, 39.0 in fathers), and 23.0 pmol/L for the 2 \u0026micro;g dose (39.0 in children, 19.0 in mothers, 10.0 in fathers) (Table\u0026nbsp;1). Overall, plasma holoTC increased 4.8-fold with the 10 \u0026micro;g dose and 2.2-fold with the 2 \u0026micro;g dose; the rise was greater in children than in the parents (Table\u0026nbsp;1 and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUsing Bor et al.\u0026rsquo;s 2004 criterion (rise in plasma holoTC\u0026thinsp;\u0026lt;\u0026thinsp;15% and \u0026lt;\u0026thinsp;15 pmol/L after 3 doses of B-12) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], 10% of participants (10 fathers, 5 mothers, and 4 children) in the 10 \u0026micro;g group were classified as poor absorbers. In a subsequent study by Bor et al., including patients with inherited B-12 malabsorption (Imerslund-Grasbeck syndrome or intrinsic factor deficiency), a rise of \u0026ge;\u0026thinsp;6 pmol/L of holoTC was proposed as a cut point to define \u0026lsquo;adequate\u0026rsquo; B-12 absorption (100% sensitivity and 72% specificity) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Using this cut point, 3.7% of participants in our study (3 fathers, 2 mothers, and 2 children) could be classified as inadequate absorbers.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results of the CobaSorb Absorption Study for children, fathers, and mothers. Panel A presents a box plot of baseline and 24-hour holoTC concentrations in the 2 and 10 \u0026micro;g groups, while panel B illustrates a box plot of the rise in holoTC concentrations for the same dosage groups. Panel C displays density plots of the increase in plasma holoTC at 24 hours following three doses of 2 and 10 \u0026micro;g. The distributions are nearly bell-shaped, with a slight right skew due to a few outliers (Shapiro-Wilk test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for plasma holoTC at baseline, 24 hours, and the change). These distributions were normalized using a log transformation for further analysis. The density plots can be interpreted in terms of the peak (average), width (variability), and shifts (comparing the effects of 2 and 10 \u0026micro;g B-12 doses). The peaks shift further to the right in children compared to parents, indicating a higher average rise for both doses. The width is smaller for the 2-\u0026micro;g dose than for the 10-\u0026micro;g dose, suggesting less variability, and the curves for the 10-\u0026micro;g dose are shifted to the right compared to the 2-\u0026micro;g dose, demonstrating a greater rise. The predominant characteristic of the density plots shows that the distribution is continuous and not binary or bimodal.\u003c/p\u003e\u003cp\u003eIn multiple regression analysis, the increase in holoTC concentration exhibited a small and inverse association with body weight [β (SE): -0.565 (0.060), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] and a strong and direct association with the doses of B-12 (10 vs. 2 \u0026micro;g) [β (SE): 19.881 (2.680), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eSupplementation Study\u003c/h2\u003e\u003cp\u003ePlasma B-12 concentrations increased clinically significantly in those who received B-12 supplements, and plasma folate concentrations increased in those who received folic acid (Supplementary Table\u0026nbsp;2, Supplementary Fig.\u0026nbsp;2). At 12 months, the median B-12 rise was 1.9-fold (95% CI: 1.8\u0026ndash;2.2) from baseline in the 10 \u0026micro;g group, compared to 1.5-fold (95% CI: 1.3\u0026ndash;1.7) in the 2 \u0026micro;g group. Using the combined B-12 marker (cB12), the B-12 adequacy status remained stable in the placebo group (baseline 46.0% and 12 months 50.0%), while it improved from 41.5 to 73.3% in the 2 \u0026micro;g group, and from 35.7 to 85.7% in the 10 \u0026micro;g group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for the B-12 groups) (Table\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eThe median compliance with supplements at 12 months was 86%. Compliance was \u0026ge;\u0026thinsp;80% in 71% and only 3% were \u0026lt;\u0026thinsp;50% compliant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePredictors of response to supplementation\u003c/h2\u003e\u003cp\u003eParticipants' 4- and 12-month responses were examined in cross-sectional and longitudinal models (Table\u0026nbsp;3). The response was inversely associated with age and weight (colinear so results are shown only for weight) and directly associated with B-12 dose, duration of supplementation, and compliance. In addition, the response was directly associated with the rise in holoTC measured in the absorption study (\u0026lsquo;absorption performance\u0026rsquo;). At 12 months, the largest effect was of B-12 dose (for 2 \u0026micro;g: β (SE): 0.166 (0.057), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004; for 10 \u0026micro;g: 0.411 (0.056), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. The effect size of absorption performance (rise in plasma holoTC during CobaSorb test) was 0.002 (0.001), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012 (Table\u0026nbsp;3).\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis secondary analysis of two linked community-based studies of B-12 metabolism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] with largely vegetarian Indian people resident in Pune, India, expands our understanding of the absorption of B-12 and how it relates to response during long-term supplementation. We believe that ours is perhaps the largest study of B-12 absorption in a free-living community. Based on the criteria of Bor [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] the Cobasorb test demonstrated that 95% of participants could effectively absorb B-12. Our secondary analysis adds new knowledge in that i) the rise in plasma holoTC concentration (a measure of absorption after an oral challenge with \u0026lsquo;physiological\u0026rsquo; dose B-12) is continuously distributed and ii) that it follows the expected physiological rules i.e. the concentration achieved was proportional to the dose and inversely to the weight of the participant. This conflicts with the common belief among clinicians and researchers to define B-12 absorption as Yes or No based on the obsolete Schilling test. Furthermore, the width (spread) of the distribution of rise in holoTC concentration was larger for the 10 \u0026micro;g compared to the 2 \u0026micro;g dose. This suggests that the physiological (carrier-mediated) absorption at near the recommended dietary allowance of 2 \u0026micro;g dose is more tightly regulated and therefore less variable than the additional contribution of passive absorption at the higher 10 \u0026micro;g dose. This understanding could inform a more \u0026lsquo;personalized\u0026rsquo; and physiological approach towards dose in clinical practice and public health interventions.\u003c/p\u003e\u003cp\u003eRecent exciting discoveries using stable isotopes of B-12 are expected to throw further light on this amazingly complex physiological process [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and define a better model of absorption.\u003c/p\u003e\u003cp\u003eWe extended our secondary analysis to investigate if the performance during the CobaSorb test would predict the response to long-term supplementation. Allowing for body weight, compliance, and dose of B-12, the \u0026lsquo;absorption performance\u0026rsquo; was a significant direct predictor, though admittedly the size of the effect was rather small. This finding leads us to believe that within physiological limits, the individual\u0026rsquo;s position within a population \u0026lsquo;tracks\u0026rsquo; after an intervention and will inform personalized nutrition.\u003c/p\u003e\u003cp\u003eThe Schilling test, now obsolete because of restrictions on using radioactive isotopes, did not measure circulating B-12 concentrations and was performed mostly in \u0026lsquo;patient\u0026rsquo; populations, with few controls or volunteers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Henze et al [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] used a new assay for B-12 and described the distribution of serum B-12 concentration after an oral, supraphysiological dose of 1 mg B-12 in 32 normal participants (age range 22\u0026ndash;75 y) as \u0026ldquo;Gaussian\u0026rdquo;. Much of this response would have been by passive absorption. A better understanding of the physiology of active B-12 absorption and binding with transport proteins led to the use of holoTC measurement in B-12 absorption protocols [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Bor et al stressed that the dose of B-12 (9 \u0026micro;g x 3 doses) in the CobaSorb test is still near-physiological [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The use of an even more physiological dose (2 \u0026micro;g x 3 doses) in a proportion of our subjects further supports the use of a rise in circulating holoTC concentration to study B-12 absorption [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] in different settings. Importantly, it uses routine laboratory facilities without radioactive markers (14C-labeled B12) or expensive and rare isotopes (13C-labeled B12), [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStrengths and limitations\u003c/h2\u003e\u003cp\u003eThis analysis was possible only because the same cohort participated in both trials, there were only a few drop-outs and high compliance. The participants were representative of a community where high B-12 insufficiency and poor dietary B-12 intake are prevalent [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The studies were population-based, involving otherwise healthy individuals, and included both children and adults with a balanced sex ratio. The sample size was large, and the doses of B-12 used were \u0026lsquo;physiological\u0026rsquo;. These contribute to the strength of the statistical interpretations. On the other hand, this was a specific population with long term vegetarian practices, which may limit application to other populations.\u003c/p\u003e\u003cp\u003eWe could not find any published studies investigating similar predictors of response to supplementation to compare our results. Based on the outcome of this trial, a primordial trial [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] in the PMNS cohort was designed with a 2 \u0026micro;g dose.\u003c/p\u003e\u003cp\u003e In summary, the absorption study revealed that the distribution of the absorption characteristics was continuous and not binary and that it followed physiological expectations. Long-term supplementation study showed that the absorption performance of the participant was a predictor of response to supplementation. Our findings expand the knowledge about B-12 absorption and provide a basis for further studies.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability: \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article will be made available upon reasonable request and adequate institutional permissions.\u003cstrong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank K. J. Coyaji, Director of the KEM Hospital, Pune, for providing the required facilities. We are grateful to the participants of the Pune Maternal Nutrition Study for taking part in these studies. RW is supported by a senior research fellowship from the Council of Scientific and Industrial Research, India\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCY conceived the idea of this secondary data analysis. UD, ER, and LP wrote the first draft of the manuscript. DB was involved in performing the laboratory analysis. SB, RW, OD, ER, CF, HR, CY, and UD were involved in the data analysis and interpretation of the results. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The original research projects were supported by the Wellcome Trust, London, UK.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKEM Hospital Ethics Committee approved both the studies (KEMHRC/VSP/Dir Off/EC/065; Project No. 067). The B12 supplementation RCT was registered at the ISRCTN. Clinical Trial Registry number and website where it was obtained: ISRCTN59289820.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDOI: https://doi.org/10.1186/ISRCTN59289820 \u0026nbsp;https://www.isrctn.com/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllen LH, Miller JW, de Groot L, Rosenberg IH, Smith AD, Refsum H, et al. Biomarkers of nutrition for development (BOND): Vitamin B-12 review. J Nutr. 2018;148:1995S\u0026ndash;2027S.\u003c/li\u003e\n\u003cli\u003eCarmel R. Malabsorption of food cobalamin. Baillieres Clin Haematol. 1995;8:639\u0026ndash;55.\u003c/li\u003e\n\u003cli\u003eCarmel R. Biomarkers of cobalamin (vitamin B-12) status in the epidemiologic setting: a critical overview of context, applications, and performance characteristics of cobalamin, methylmalonic acid, and holotranscobalamin II. Am J Clin Nutr. 2011;94:348S\u0026ndash;58S.\u003c/li\u003e\n\u003cli\u003eNexo E, Hoffmann-L\u0026uuml;cke E. Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility. Am J Clin Nutr. 2011;94:359S\u0026ndash;65S.\u003c/li\u003e\n\u003cli\u003eSchilling RF. The absorption and utilization of vitamin B12. Am J Clin Nutr. 1955;3:45\u0026ndash;51.\u003c/li\u003e\n\u003cli\u003eDevi S, Pasanna RM, Shamshuddin Z, Bhat K, Sivadas A, Mandal AK, et al. Measuring vitamin B-12 bioavailability with [13C]-cyanocobalamin in humans. Am J Clin Nutr. 2020;112:1504\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eB\u0026auml;rebring L, Lamberg-Allardt C, Thorisdottir B, Ramel A, S\u0026ouml;derlund F, Arnesen EK, et al. Intake of vitamin B12 in relation to vitamin B12 status in groups susceptible to deficiency: a systematic review. Food Nutr Res. 2023;67:8626.\u003c/li\u003e\n\u003cli\u003eRefsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, Orning L, et al. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am J Clin Nutr. 2001;74:233\u0026ndash;41.\u003c/li\u003e\n\u003cli\u003eYajnik C, Deshmukh U, Katre P, Limaye T. Vitamin B12 deficiency in developing and newly industrialising countries. In: Vitamin B12- Advances and Insights. Edited by Rima Obeid. 1\u003csup\u003est\u003c/sup\u003e ed. CRC Press; 2017. pp. 131\u0026ndash;50. \u003c/li\u003e\n\u003cli\u003eYajnik C, Deshpande SS, Lubree HG, Naik S, Bhat D, Uradey BS, et al. Vitamin B12 deficiency and hyperhomocysteinemia in rural and urban Indians. J Assoc Physicians India. 2006;54:82.\u003c/li\u003e\n\u003cli\u003eYajnik CS, Deshpande SS, Jackson AA, Refsum H, Rao S, Fisher DJ, et al. Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: the Pune Maternal Nutrition Study. Diabetologia. 2008;51:29\u0026ndash;38.\u003c/li\u003e\n\u003cli\u003eBor MV, Nexo E, Hvas AM. Holo-transcobalamin concentration and transcobalamin saturation reflect recent vitamin B12 absorption better than does serum vitamin B12. Clin Chem. 2004;50:1043\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eBhat DS, Thuse NV, Lubree HG, Joglekar CV. Increases in plasma holotranscobalamin can be used to assess vitamin B12 absorption in individuals with low plasma vitamin B12. J Nutr. 2009;139:2119\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eDeshmukh US, Joglekar CV, Lubree HG, Ramdas LV, Bhat DS, Naik SS, et al. Effect of physiological doses of oral vitamin B12 on plasma homocysteine: a randomized, placebo-controlled, double-blind trial in India. Eur J Clin Nutr. 2010;64:495\u0026ndash;502.\u003c/li\u003e\n\u003cli\u003eRao S, Yajnik CS, Kanade A, Fall CH, Margetts BM, Jackson AA, et al. Intake of micronutrient-rich foods in rural Indian mothers is associated with the size of their babies at birth: Pune Maternal Nutrition Study. J Nutr. 2001;131:1217\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eKumaran K, Yajnik P, Lubree H, Joglekar C, Bhat D, Katre P, et al. The Pune Rural Intervention in Young Adolescents (PRIYA) study: design and methods of a randomised controlled trial. BMC Nutr. 2017;3:1\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eKelleher B, Broin S. Microbiological assay for vitamin B12 performed in 96-well microtitre plates. J Clin Pathol. 1991;44:592\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eKelleher B, Walshe K, Scott J, O\u0026apos;Broin S. Microbiological assay for vitamin B12 with use of a colistin-sulfate-resistant organism. Clin Chem. 1987;33:52\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eHorne D, Patterson D. Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates. Clin Chem. 1988;34:2357\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eTamura T, Freeberg L, Cornwell P. Inhibition by EDTA of growth of Lactobacillus casei in the folate microbiological assay and its reversal by added manganese or iron. Clin Chem. 1990;36:1993.\u003c/li\u003e\n\u003cli\u003eRefsum H, Johnston C, Guttormsen AB, Nexo E. Holotranscobalamin and total transcobalamin in human plasma: determination, determinants, and reference values in healthy adults. Clin Chem. 2006;52:129\u0026ndash;37.\u003c/li\u003e\n\u003cli\u003eUlleland M, Eilertsen I, Quadros EV, Rothenberg SP, Fedosov SN, Sundrehagen E, et al. Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum. Clin Chem. 2002;48:526\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eFedosov SN, Brito A, Miller JW, Green R, Allen LH. Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points. Clin Chem Lab Med. 2015;53:1215\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003eBor MV, Cetin M, Aytac S, Altay C, Nexo E. Nonradioactive vitamin B12 absorption test evaluated in controls and in patients with inherited malabsorption of vitamin B12. Clin Chem. 2005;51:2151\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eMiller JW, Green R. Assessing vitamin B-12 absorption and bioavailability: read the label. Am J Clin Nutr. 2020;112:1420\u0026ndash;1.\u003c/li\u003e\n\u003cli\u003eKurpad AV, Pasanna RM, Hegde SG, Patil M, Mukhopadhyay A, Sachdev HS, et al. Bioavailability and daily requirement of vitamin B12 in adult humans: an observational study of its colonic absorption and daily excretion as measured by [13C]-cyanocobalamin kinetics. Am J Clin Nutr. 2023;118:1214\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eFedosov SN. New insights into mechanisms of vitamin B12 uptake and conversion. Am J Clin Nutr. 2023;118:1073\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eHenze E, M\u0026auml;nner S, Clausen M, Malfertheiner P, Hellwig D, Ditschuneit H, et al. The Schilling test cannot be replaced by an absorption test with unlabeled vitamin B12. Klin Wochenschr. 1988;66:332\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eCarkeet C, Dueker SR, Lango J, Buchholz BA, Miller JW, Green R, et al. Human vitamin B12 absorption measurement by accelerator mass spectrometry using specifically labeled 14C-cobalamin. Proc Natl Acad Sci U S A. 2006;103:5694\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eGarrod MG, Rossow HA, Calvert CC, Miller JW, Green R, Buchholz BA, et al. 14C-Cobalamin absorption from endogenously labeled chicken eggs assessed in humans using accelerator mass spectrometry. Nutrients. 2019;11:2148.\u003cstrong\u003e\u003cbr\u003e \u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-nutrition","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejcn","sideBox":"Learn more about [European Journal of Clinical Nutrition](http://www.nature.com/ejcn/)","snPcode":"41430","submissionUrl":"https://mts-ejcn.nature.com/cgi-bin/main.plex","title":"European Journal of Clinical Nutrition","twitterHandle":"@ejcneditor","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7551002/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7551002/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBACKGROUND\u003c/h2\u003e\u003cp\u003eInsufficient dietary intake and/or reduced gastrointestinal absorption lead to low vitamin B12 (B-12) status.\u003c/p\u003e\u003ch2\u003eOBJECTIVE\u003c/h2\u003e\u003cp\u003eIn a secondary data analysis of a B-12 absorption (CobaSorb) study and a B-12 supplementation trial in a B-12 insufficient Indian rural community, we examined i) the distribution of plasma holo-transcobalamin (holoTC) response during the CobaSorb test, and ii) determinants of plasma B-12 response to long-term, oral supplementation with \u0026lsquo;physiological doses\u0026rsquo; of B-12.\u003c/p\u003e\u003ch2\u003eMETHODS\u003c/h2\u003e\u003cp\u003eThe participants in the Pune Maternal Nutrition Study first underwent a B-12 absorption study (CobaSorb protocol). Subsequently, they participated in a 12-month-long, double-blind RCT of daily oral B-12 (0, 2, or 10 \u0026micro;g) and folic acid (0 or 200 \u0026micro;g). We studied distribution of plasma holoTC response in the CobaSorb test, and used linear mixed-effect model to determine predictors of B-12 absorption and response to supplementation.\u003c/p\u003e\u003ch2\u003eRESULTS\u003c/h2\u003e\u003cp\u003eParticipants (313) included children (n\u0026thinsp;=\u0026thinsp;109, 57 boys, mean age 9y, weight 21.9 kg, BMI 13.6 kg/m\u003csup\u003e2\u003c/sup\u003e), mothers (n\u0026thinsp;=\u0026thinsp;108, 30y, 47.7 kg, 19.3 kg/m\u003csup\u003e2\u003c/sup\u003e), and fathers (n\u0026thinsp;=\u0026thinsp;96, 37y, 59.3 kg, 21.4 kg/m\u003csup\u003e2\u003c/sup\u003e). The plasma holoTC response during the absorption test was continuously distributed and was negatively associated with weight and positively with the dose of B-12 (10 or 2 \u0026micro;g). Response to long-term B-12 supplementation was positively predicted by absorption test response, dose of B-12, length of supplementation, and compliance.\u003c/p\u003e\u003ch2\u003eCONCLUSIONS\u003c/h2\u003e\u003cp\u003eThe continuous distribution of plasma holoTC response during the absorption test supports physiological absorption of B-12 in a community. CobaSorb protocol could be used to unravel the intricacies of B-12 absorption.\u003c/p\u003e","manuscriptTitle":"Distribution of Vitamin B12 Absorption in a Community and Factors Associated with the Response to Long-term Oral Supplementation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 07:06:57","doi":"10.21203/rs.3.rs-7551002/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-13T15:34:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-12T21:24:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-29T06:55:16+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-15T13:02:59+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-14T08:50:36+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-09-14T06:09:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-10T14:33:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-10T14:25:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Clinical Nutrition","date":"2025-09-09T18:22:01+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-09-08T15:16:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-nutrition","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejcn","sideBox":"Learn more about [European Journal of Clinical Nutrition](http://www.nature.com/ejcn/)","snPcode":"41430","submissionUrl":"https://mts-ejcn.nature.com/cgi-bin/main.plex","title":"European Journal of Clinical Nutrition","twitterHandle":"@ejcneditor","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"52cf9605-e5a0-43c3-b11d-594b44177a67","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54679621,"name":"Health sciences/Medical research/Epidemiology"},{"id":54679622,"name":"Health sciences/Health care/Nutrition"}],"tags":[],"updatedAt":"2026-03-31T19:05:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 07:06:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7551002","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7551002","identity":"rs-7551002","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}