B Vitamin Intake and the Risk of Colorectal Cancer Development: A Systematic Review and Meta-Analysis of Observational Studies

Methylenetetrahydrofolate Reductase; Newcastle-Ottawa Preferred Reporting for Systematic Reviews and Meta-Analyses; Quality of Life; Relative S-Adenosyl-homocysteine; S-Adenosylmethio-nine; Background: Cancer research has increased interest in lifestyle factors. These can affect colorectal cancer burden, which is the second common cause of cancer death worldwide, although 40% of the cases would be preventable. Aim: We aimed to conduct a systematic review and meta-analysis on relationship between B vitamin intake and colorectal cancer where 14 publications were analysed. Methods: We carried out a systematic search of scientific literature based on PRISMA statements. Combined effect size (CES), confident interval (CI), prediction interval (PI), I2 and publication bias were calculated during statistical analysis of selected studies. Results: Combined effect sizes showed inverse association between higher intake of vitamin B2 (CES = 0.90; CI95% 0.83 - 0.97; I2 = 0.00%, p = 0.910), B6 (CES = 0.80; CI95% 0.68-0.92; I2 = 9.17%, p = 0.359) and CRC. We could not confirm the higher dietary intake of vitamin B12 reducing the risk of CRC. Vitamin B2 and B6 could compensate the effect of MTHFR C677T polymorphism (CES = 0.81; CI95% 0.64 - 0.98; I2 = 0.00%, p = 0.515) as well. Conclusion: Our results suggest that optimal intake of vitamin B2 and B6 could be important dietary factors in prevention of CRC. The association with vitamin B12 is inconsistent as its bioavailability is affected by other lifestyle factors. Vitamin B2 and B6 could influence MTHFR enzyme activity, therefore these vitamins might be incorporated into screening process of CRC with recommendations for specific diet.

Introduction most frequent cancer type and the second most common cause of cancer death worldwide, although around 40% of the cases would be preventable [4]. Countries with better and careful cancer prevention programs have more chance to fight against CRC [5].
Dietary intake of methyl donors (such as folate, choline, betaine, methionine and vitamin B2, B6 and B12) could have important role in cancer prevention by reducing the risk of cancer and could contribute to the success of cancer therapies and to reach better quality of life (QoL) of the patients [6][7][8]. Dietary methyl donors are food components, which provide methyl groups for the one-carbon metabolism, which consists of two main metabolic cycles: the folate cycle and the methionine cycle [9]. Methionine has a universal methyl group and can be added to several molecules; thus, its sufficient amount supports the normal DNA methylation [10]. It is also well known that inadequate DNA methylation may lead to development of cancer [6].
The optimal function of one-carbon metabolism requires specific vitamins as well as minerals. B vitamins are catalytic co-enzymes in these processes; therefore, they can influence the availability of methyl groups [10]. Moreover, B vitamins are important in energy-yielding metabolism, oxygen transport and neuronal functions. They play essential roles in basic metabolic pathways and fundamental cellular functions consequently have an impact on cognitive and psychological processes, including mental and physical fatigue [7,11]. Besides nutritional and other lifestyle factors, genetically determined components influence the development of CRC as well. One of these is the single nucleotide polymorphism (SNP) of the methylenetetrahydrofolate reductase (MTHFR) gene. MTHFR is involved in the one-carbon metabolism, where this enzyme activates folic acid. It has a common SNP at the position of 677 (MTHFR C677T). The heterozygous mutation (CT) results in a reduced enzyme activity around 65% of the normal level, while the homozygous (TT) mutation causes only 30% enzyme activity, and both reduce the level of DNA methylation [12][13][14].In this meta-analysis our aim was to systematically collect publicly available data, and summarize and update the scientific knowledge about the associations between dietary B2, B6 and B12 vitamin intake and the risk of CRC in adult patients, which has already published until 15th March 2021. Moreover, we aimed to highlight the importance of the need for standardization of the way how to explain the result of a meta-analysis as well.

Study Characteristics
Our systematic review and meta-analysis based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statements [15] (Table S1) focused on vitamin B2, B6 and B12 intake and the polymorphisms of MTHFR (where data were collected from cohort and case-control studies, respectively), and their effects on colorectal cancer risk in adults.

Literature Search
We carried out a systematic scientific literature search in

Study Selection and Quality Assessment
Identified records were screened by titles and abstracts and after removal of duplicated studies, publications were reviewed based on inclusion and exclusion criteria. Inclusion criteria were:

1.
Publications had to be written in English.

2.
Papers had to be original articles.

3.
Patients had to be adults.

4.
The exposure of interest was vitamin B2, B6 and B12.

5.
The outcome of interest was the diagnosis of colorectal cancer. All studies with only animal or in vitro experiments were excluded. After screening process, the remained 35 studies were assessed by eligibility criteria, which were: 1. odds ratio (OR), relative risk (RR) or hazard ratio (HR) with 95% confidence interval (CI) had to be calculated in the article; 2. the studies had to be cohort or case-control studies (these only were accepted if they discussed the association between B2, B6 and B12 vitamin intake and MTHFR polymorphism in CRC. Articles, which met all the criteria were reviewed again and these publications formed the basis of our quantitative analysis. We applied the Newcastle-Ottawa Scale (NOS) for assessing the quality of included publications in our meta-analysis [16].
the studies using random effects model [17][18][19]. Overall ORs (combined effect size, CES) and the corresponding 95% CIs and 95% prediction intervals (PIs) were calculated. The studies were tested using I2 statistic and Cochran's Q test. In order to identify possible sources of heterogeneity, we explored studies with outlier effect sizes using funnel plot and Galbraith plot [20]. We also used the "Trim and fill" method within funnel plot to estimate true effect size and the dispersion of the combined effect size (heterogeneity) [19]. In this process both observed and adjusted combined effects size (CES) were calculated with related CI and PI, respectively [21,22]. We carried out Egger's regression test [23] and Begg & Mazumdar's rank correlation test to inspect possible publication bias [24]. Publication and other biases of the individual studies were evaluated according to the information found in the original articles. All statistical analysis were implemented by the tools of Meta-Essentials [25].

Study Characteristics
In the first analysis consisting of 9 selected articles, we   (Tables 1 & 2); therefore, we compared the highest versus lowest intake and related ORs in all cases.   method also showed significant heterogeneity (p = 0.002) as well.
We visualized effect sizes of vitamin B12 intake to select outliers but neither funnel plot nor Galbraith plot ( Figure 3B

Association between B Vitamin Intake and MTHFR Polymorphism
According to random effects model we found that higher dietary intake of vitamin B2 and B6 could decrease the risk of CRC in patients with MTHFR C667T polymorphism. The calculated CES was 0.81 with CI95% 0.64 -0.98 (PI95% value was the same).
Heterogeneity was not detected among the included studies (I 2 = 0.00%; p = 0.515) ( Figure 3A). There was also no evidence for heterogeneity by "Trim and fill" method as well. We assessed

publication bias in which Egger's regression test and Begg &
Mazumdar's rank correlation test did not show publication bias with levels of significance 0.759 and 0.340, respectively.

Discussion
The importance of nutritional vitamin and mineral intake has Therefore, there is an expectation and necessity to measure and evaluate the effects of these vitamins, compounds and products [6,7,10,38]. Several studies suggested that dietary methyl-donors and related vitamins can contribute to cancer prevention [8,[39][40][41].
Dietary methyl-donors, such as folate, betaine, choline,methionine and B vitamins provide methyl groups for the one-carbon metabolism of which vitamin B2, B6 and B12 can influence the availability of methyl groups [38,7,10]. B vitamins, additionally, take part in energy-yielding metabolism, oxygen transport and neuronal functions thus they affect the cognitive and psychological processes, including mental and physical fatigue [7,11].
We performed a systematic review and meta-analysis to collect Some publication has already been written that patients in the higher quartile of vitamin B12 intake had more chance to smoking and drinking alcohol, and because of this the utilization of vitamin B12 is decreased in their case [42,43]. As stated by Ishihara et al., there is possibility for positive association between vitamin B12 intake and the risk of CRC, written in their study, which remained after the adjustment of smoking habits and alcohol intake. Therefore, their result represents more likely the effect of smoking and alcohol consumption on the risk of CRC, which is a well-known positive association, rather than the dietary intake of vitamin B12 [33]. All the smoking habits, alcohol consumption and gastrointestinal disorders should be considered if we examine the effect of vitamin B12 intake on the risk of CRC as these factors make it difficult to involve patients properly into any study group based only on their known vitamin B12 intake [33,44]. This information led us to exclude vitamin B12 intake from the further analysis. After the exclusion of the study of Ishihara et al. the group of the studies became homogeneous, which is essential criterion for calculating publication bias.
Genetic polymorphisms also can influence the risk of CRC. which then similarly can arrest DNA methylation [6,38].
In the second part of our analysis, we investigated the association between MTHFR C667T polymorphism and intake of vitamin B2 and B6. We could confirm that appropriate intake of vitamin B2 and B6 could be possibly protective in diminishing or even eliminating the negative effect of the reduced enzyme activity in the folate cycle in case of homozygote TT patients. Additionally, vitamin B2 intake have already been reported as a protective factor for breast and cervical cancer as well, highlighting its potential protective role in cancer prevention [47][48][49]. There was no evidence for publication bias, indicating that the pooled results may be unbiased. We excluded the group of vitamins B12 from this analysis as well, because the effect of vitamin B12 is influenced by numerous factors as we have already described above. In contrast to the first analysis, in the second, the effect of B vitamins was handled altogether as both vitamin B2 and B6 play role in the onecarbon cycle, which is regulated by MTHFR.
The limitations of our study are similar to other meta-analysis, where several confounding factors (e.g., inadequate controls, obtained at baseline may have changed over the long follow-up period, high intake of vitamins may have been at lower risk due to other healthy habits and behaviors, adjusted variables differed in the studies) could affect the pooled result. Additionally, nutrients which was not measured in the studies could influence the risk of CRC even after an adjustment process. Details of other possible biases were described in the original papers. We used more searching engines to increase the chance for achieve the highest amount of searching terms related to our analysis as possible. We used additional metrics from Borenstein, which gives additional, valid and meaningful interpretation of the results.
In conclusion, we found that vitamin B2 and B6 may be an effective dietary component to decrease CRC risk, and they can be an important part of a dietary intervention, or a special diet during/ after cancer treatment. We found that an adequate intake of vitamin B2 and B6 -and probably B12 -could compensate the consequence of the reduced enzyme activity of MTHFR in CRC development.
Therefore, it may give the opportunity to incorporate a genetic test of the MTHFR polymorphism into the screening process of CRC with recommendations for specific diet for those in need.

Ethical Statement -Not Applicable
Ethical approval was not sought for this study because of its design (systematic review & meta-analysis).

Data Availability Statements
The data that support the findings of this study are openly available in the references number 13-14, 26-37.