Efficacy of different routes of vitamin B12 supplementation for the treatment of patients with vitamin B12 deficiency: A systematic review and network meta-analysis

Background This systematic review and network meta-analysis aimed to evaluate the three different administration routes of vitamin B12: oral, intramuscular (IM), and sublingual (SL) routes. Methods We searched four electronic databases (PubMed, Scopus, Web of Science, and Cochrane CENTRAL Register of Controlled Trials). We included only comparative studies. We performed a frequentist network meta-analysis to measure network estimates for the relative outcomes. Moreover, we conducted a pairwise meta-analysis using a random effect model to obtain direct estimates for outcomes. All outcomes were continuous, and the relative treatment effects were pooled as mean difference (MD) with 95% confidence intervals. Results Thirteen studies were included in the meta-analysis, with a total of 4275 patients. Regarding increasing vitamin B12 levels, the IM route ranked first, followed by the SL route (MD = 94.09 and 43.31 pg/mL, respectively) compared to the oral route. However, these differences did not reach statistical significance owing to the limited number of studies. Regarding the hemoglobin level, the pooled effect sizes showed no difference between all routes of administration that could reach statistical significance. However, the top two ranked administration routes were the oral route (78.3) and the IM route (49.6). Conclusion All IM, oral, and SL routes of administration of vitamin B12 can effectively increase the level of vitamin B12 without significant differences between them, as thought previously. However, the IM route was the top-ranked statistically but without clinical significance. We found no significant difference among studied administrated routes in all other CBC parameters and homocysteine levels. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s11845-023-03602-4.


Introduction
Vitamin B12 is a water-soluble vitamin that may be derived from foods, including fish, meat, dairy products, and cereals that have been fortified.It can also be taken in supplement form.After being extracted by gastric acid, it travels to the terminal ileum, where it is co-absorbed along with the intrinsic factor, an enzyme from the stomach's parietal cells (Fig. 1) [1,2].Vitamin B12 is essential for neurologic function, red blood cell creation, and DNA synthesis and is a coenzyme for three primary biochemical conversions: homocysteine to methionine, 5-methyltetrahydrofolate to tetrahydrofolate, and methylmalonic acid to succinyl coenzyme A [1,2].
There are different routes for the administration of any vitamin.Oral and intramuscular (IM) vitamin B12 are the most common routes for treating vitamin B12 deficiency, and several studies have evaluated their efficacy [9,10].However, in the case of vitamin B12 malabsorption, irrespective of the cause of malabsorption, the intramuscular route is preferred [11,12].According to the British Society for Hematology recommendations, for individuals with severe insufficiency and malabsorption syndromes, IM vitamin B12 delivery is preferable; however, oral replacement therapy may be recommended for patients with asymptomatic, moderate illness who have no concerns about absorption or compliance [13].
The sublingual (SL) route of vitamin B12 administration has been evaluated as an alternative route to oral and IM routes, especially in the case of vitamin B12 malabsorption [14][15][16], but there is no sufficient data to consider this route in the guidelines.This systematic review and network metaanalysis aimed to assess the efficacy of those three different routes of administration of vitamin B12.

Methods
For this research, we used the same format that the PRISMA statement recommends for systematic reviews and metaanalyses [17].Every stage was carried out in a manner entirely consistent with the Cochrane Handbook of Systematic Reviews and Meta-analyses of Interventions [18].

Eligibility criteria
Studies were included in our review if they satisfied the following criteria: Population: studies on patients with vitamin B12 deficiency Intervention and comparator: studies where the experimental and control groups received vitamin B12 through any of these routes of administration: SL, IM, and oral Outcome: studies reporting at least any of the following serum levels: vitamin B12, folate, hemoglobin, hematocrit, mean corpuscular volume, plasma homocysteine, urine methylmalonic acid, leukocyte, and platelets Study design: comparative studies whose design was controlled trials with patients allocated to receive SL, IM, or oral vitamin B12 in a random or non-random allocation manner.We considered both blinded and open-label studies.We also included controlled prospective and retrospective observational studies with extreme caution; these studies were separated from randomized controlled trials in subgroups and were only considered in calculating the pooled effect size if their results were consistent with randomized controlled trials.In case of discrepancy between randomized controlled trials and observational studies, the results highlighted this, and the outcomes of randomized controlled trials were prioritized Studies for which complete full texts were not accessible, studies not published in English, studies whose data were unsuitable for extraction and analysis, and studies that were described as abstracts only or thesis were all excluded.

Selection process
Five authors (S.D., A.K., R.A.D., T.E., and T.F.) separately screened the titles and abstracts of all identified articles to determine their relevance to this meta-analysis.The second step involved screening the full-text articles of the included abstracts to determine the final eligibility for metaanalysis.Duplicates were excluded using Endnote (Clarivate Analytics, PA, USA).The discussion was used to settle any disagreements.

Data collection process and data items
Five reviewers (S.D., A.K., R.A.D., T.E., and T.F.) collected data onto a standardized data extraction sheet.Extracted information focused on four primary areas: (1) summary of the included studies (including study ID, title, study design, country, inclusion criteria, exclusion criteria, interventions, dose, sample size, treatment duration, and the main findings); (2) study population characteristics (including age, sex body mass index, serum level of vitamin B12, hematocrit, hemoglobin, and mean corpuscular volume (MCV) levels); (3) risk of bias domains according to the study design; and (4) outcome measures (the primary outcomes were the serum levels of vitamin B12 and hemoglobin and the secondary outcomes includes the levels of MCV, homocysteine, platelets count, and white blood cells count).The discussion was used to settle any disagreements.

Assessing the risk of bias in the individual studies
We independently evaluated the quality of each included study by two authors (S.D. and T.E.).The Cochrane assessment tool was used for randomized clinical trials (ROB2) [19].Newcastle Ottawa scale (NOS) was used to assess the risk of bias for observational studies [20].For non-randomized controlled trials, we used the Cochrane ROBINS-I tool [21].A third author (O.A.A.) solved any disagreements.

Statistical analysis
We measured network estimates for the related outcomes using a frequentist network meta-analysis of aggregate data.The evident heterogeneity in the intervention comparison effects across studies was accommodated using the random effect model as a framework.Moreover, we conducted a pairwise meta-analysis using a random effect model to obtain direct estimates for outcomes.The transitivity assumption was examined to determine if patient and research characteristics were sufficiently comparable across comparisons.Additionally, using a loop-specific methodology, we assessed the consistency assumption locally in a closed loop [22].The surface under the cumulative ranking (SUCRA) was used to rank the intervention's hierarchy in the network model; then, we estimated the mean ranks [23].A comparison-adjusted funnel plot was used to explore the potential publication bias [23].
We used a three-level hierarchical network meta-analysis to incorporate the exchangeability between different study designs to predict an effect estimate for each study design individually [24].Thus, this model allows strength to be borrowed within the different classes of study designs, strengthening interference and potentially reducing the uncertainty around each study design and consequently increasing the ability to inform decision-making frameworks.All data of this approach were pooled as Cohen's d with 95% confidence intervals.
All outcomes of interest were continuous, and the relative treatment effects were pooled as mean difference (MD) with 95% confidence intervals.All analyses were done in STATA version 17 using the network command.

Literature search results
Our literature search process retrieved 22,262 records.Five thousand two hundred ten duplicates were removed using Endnote, and 17,052 were screened for title and abstract.One hundred forty-nine articles were qualified for full-text screening after being subjected to title and abstract screening.The meta-analysis comprised 13 of these investigations.No further papers were included after manually searching the references of the listed studies.Figure 2 illustrates the PRISMA flow diagram of the study recruitment process.

Characteristics of the included studies
Thirteen studies were included in the meta-analysis, with a total of 4275 patients.In all studies, patients were assigned to receive either oral, SL, or IM vitamin B12.A summary of the included studies and the characteristics of the population in each study are provided in Tables 1 and 2, respectively.According to the Cochrane ROB 2 tool, four studies had some concerns, two had high, and one had a low risk of bias.According to ROBINS-I checklists, one study had a high risk of bias, and one had a moderate risk of bias.Two studies had good quality, and one had fair quality, according to the NOS tool (Supplementary 2).

Vitamin B12
The network of treatment comparisons for vitamin B12 included three active individual nodes (Fig. S1; top panel).Each node represents a different administration route; the oral administration route was the most well-connected intervention with all other interventions directly linked to it; therefore, it has been used as the reference for comparison.Figure 3A shows network estimates of treatment effect on vitamin B12 levels for different route administrations compared with the oral route.Network meta-analysis showed that either the IM route (MD 94.09 pg/mL, 95% CI [− 93.36 to 281.54]) or the SL route (MD 43.31 pg/mL, 95% CI [− 228.92 to 315.54]) compared to the oral route did not reach a significant difference to increase vitamin B12 levels.
According to SUCRA values, the top-ranked intervention for increasing levels of vitamin B12 was the IM route (74.2), followed by the SL route (48.4) (Table 3(A)).We performed a three-level hierarchical model to investigate the pooled effect sizes according to the study design in which there was no significant difference between the IM and the oral routes compared to the SL route (P = 0.31, 0.16, respectively).There was a significant difference between the oral and the IM routes (Cohen's d − 0.74, 95% CI [− 1.06 to − 0.43]; P < 0.001).The pooled effect sizes were not homogenous (I 2 > 50%); Fig. S2.

Hb
Five studies comprising 3730 patients reported Hb.The network diagram included three individual nodes, (Fig. S1; bottom panel).Each node represents a different drug intervention, in which the oral route was the well-connected route of administration with all other routes directly linked to it.
Figure 3B shows network estimates of treatment effect on Hb levels for different administration routes compared to the oral route.Pooled effect sizes showed no difference between all routes of administration to increase Hb without substantial statistical evidence (Fig. 3B).According to SUCRA values, the top two ranked administration routes were the oral route (78.3) followed by the IM route (49.6) (Table 3

(B)).
The three-level hierarchical model showed no significant difference among all comparisons of administration routes; Cohen's d was 0.07 for IM vs SL routes, 0.06 for oral vs IM routes, and 0.22 for oral vs SL routes.The pooled effect size was homogenous (I 2 = 0%) (Fig. S3).

Secondary outcomes
Five included studies comprising 3605 patients reported change in MCV, four studies comprising 140 patients reported  3C-F summarize the results for secondary outcomes.Network meta-analysis showed no evidence of differences among all possible comparisons for secondary outcomes (MCV, homocysteine levels, platelet counts, and WBC counts).Supplementary Table 5 presents SUCRA values.However, none of the three-level hierarchical models showed any significance among all possible comparisons of administration routes (Figs.S6-S9).

Significance of the study
To our knowledge, this is the most comprehensive network meta-analysis comparing the efficacy of SL, IM, and oral routes of administration of vitamin B12 in patients with vitamin B12 deficiency.The significance of this paper is not only to compare the three routes of administration but also to evaluate them and determine which route is the best to administrate the drug.The study also opens the door for more future research about the administration routes of vitamin B12 and other vitamins.

Summary of the findings
The current article evaluated 13 studies, of which eight were randomized clinical trials comparing different administration routes of vitamin B12 in patients with vitamin B12 deficiency.A total of 4275 patients with vitamin B12 deficiency were included in the final analysis.We found that irrespective of the route of vitamin B12 administration, serum vitamin B12 levels were increased.When comparing the different routes, the top-ranked route for increasing levels of vitamin B12 was the IM route, followed by the SL route.However, this difference has no clinical significance.
Interestingly, we found no significant difference among studied administrated routes in all other CBC parameters such as Hb, MCV, platelets count, WBC count, and homocysteine level.Given the fact that vitamin B12 levels were increased insignificantly among all routes, the preference of the administrated route should be referenced to the advantages and disadvantages of each route as well as the patient situation, which will be decided according to the physician's opinion.

Sublingual vs intramuscular and oral routes of administration
A previous systematic review by Cochrane compared the effectiveness of oral versus IM routes of vitamin B12 administration.It showed that both effectively improve serum vitamin B12 levels for people suffering from vitamin B12 deficiency.However, the dosage of oral vitamin B12 might be a clinical factor that affects this outcome [9].This contributes to the absorption mechanism of oral vitamin B12, which does not depend only on intrinsic factors, but the absorption can be done through passive diffusion.Passive diffusion accounts for 1.2% of overall absorption, with little effect on bioavailability in patients with pernicious anemia or gastroduodenal resection [49,50].
As our results showed, there is no difference between them in terms of efficacy, but in terms of patients' tolerance, advantages, and disadvantages of each route, the sublingual route is preferred over IM and oral routes.SL vitamin B12 is as adequate as IM and oral vitamin B12.However, the SL route of administration has more advantages and fewer disadvantages than IM and oral routes.So, we recommend using SL vitamin B12 rather than oral and IM, especially in patients who do not tolerate IM injection and patients who need prolonged vitamin B12 supplementation, such as patients with long-term metformin use and patients with pernicious anemia or with gastro-duodenal resection [51][52][53][54].In terms of cost, the IM route is higher in cost than the SL and oral routes [2,55].
We recommend future research directions to determine when to start with SL, IM, or oral vitamin B12 in different situations.

Implications in practice
As there is no apparent clinical significance, the treatment should depend on the patient's condition.We advise physicians to examine every patient carefully to exclude every cause that may lead to malabsorption and then choose the most appropriate route.SL route could be used in patients with pernicious anemia or malabsorption causes.However, the oral route cannot be excluded as it did not depend on intrinsic factors only, as the simple diffusion (without the need for intrinsic factors) is now considered another mechanism for the absorption of vitamin B12 [49,50].
Before this meta-analysis, the IM route was generally the preferred route, but our results showed that no route is usually preferred, and SL and oral routes should be considered comparable alternative routes.

Strength points and limitations
Based on our knowledge, this is the most updated metaanalysis on this topic, including all available evidence based on our inclusion criteria of including only controlled studies, either RCTs or controlled observational studies, to reach the highest accessible quality of evidence from the available evidence found in the literature.We are the first meta-analysis comparing the different administration routes of vitamin B12, and the first meta-analysis includes the SL route of administration in the analysis.
The limitations of this work are that we included RCTs, non-RCTs, and observational studies, which may lower the overall quality of evidence of the included studies.We Fig. 4 Advantages and disadvantages of each route of administration cannot find the full of one study which seems to be included.The head-by-head comparison between the three interventions was made only in one paper of the included studies.Additionally, there was variability between the included studies in the follow-up duration, which may cause heterogeneity in the results obtained.

Conclusion
All IM, oral, and SL routes of administration of vitamin B12 can effectively increase the level of vitamin B12 without significant differences between them, as thought previously.However, the IM route was the top-ranked statistically, followed by the SL and then the oral routes, but without clinical significance.We found no significant difference among studied administrated routes in all other CBC parameters such as Hb, MCV, platelets count, WBC count, and homocysteine level.

Fig. 2
Fig. 2 PRISMA flow diagram of studies' screening and selection

Fig. 3
Fig. 3 Network estimates of treatment effect on each outcome

Table 1
Summary of included studies

Table 2 (
IMchange in homocysteine levels, four studies comprising 3588 patients reported change in platelets count, and only three studies with 3430 patients reported change in WBC count.FiguresS4 and S5show networks of routes of administration for secondary outcomes.Figure