Of tissue B12 levels to 33?0 of those observed in control mice. However, this study only lasted 4 weeks. Prolonged treatment with an inactive B12 analogue such as Cbi could further deplete B12 from the cells and, in time, cause B12 deficiency.Figure 3. Changes in transcript levels after treatment of mice with Cbi or B12. Kidney, liver and salivary glands were removed from mice treated for 27 days with saline (control, n = 7), 4.25 nmol/h Cobinamide (Cbi, n = 7) or 1.75 nmol/h vitamin B12 (B12, n = 7). We analysed the transcript levels by q t CR. The results (Mean +SEM) for each sample are relative to the mean value for the corresponding tissue from control mice. Non arametric Wilcoxon tests were employed to calculate significant differences, p,0.05 are indicated with * for comparison to control mice. No significant difference was observed between Cbi and controls or Cbi and B12 mice. LMBRD1: lysosomal B12 transporter. 1326631 MS: methionine synthase. MTHFR: methylenetetrahydrofolate reductase. MUT: methylmalonyl oA mutase. TC: transcobalamin. TC : transcobalamin Rubusoside site receptor (CD320). doi:10.1371/journal.pone.0046657.gHigh-dose B12 increases tissue B12 and influences markers of B12 metabolismHigh doses of B12 increased plasma B12 and decreased the level of MMA. Unexpectedly, the other metabolic marker of B12 deficiency, tHCY, increased and so did cysteine, another metabolite of the homocysteine pathway. To our knowledge no previous studies have shown that B12 load may lead to an increased level of tHCY, thus suggesting that in relation to the metabolism of homocysteine, both too little B12 and too much may be disadvantageous. Our results show that the major part of surplus B12, and therefore possibly also B12 conjugates, will end up in the kidney. The liver is able to double the amount of B12 internalised. High dose B12 resulted in previously undescribed changes in the transcript levels of genes involved in the transport and metabolism of the vitamin. Firstly, we found a reduced transcript level of B12-dependent enzyme MTHFR in the kidneys. This reduction was not related to an accumulation of methionine in this group of mice. This could be expected, as MTHFR is involved in the conversion of methionine into methyl-tetrahydrofolate, see Figure 1. but apparently, the reduction in MTHFR mRNA wasrespectively, accumulation in the kidney far exceeded a 
factor of 2. This is explained by the kidney’s role in rodent B12 metabolism. The kidney acts as a reservoir for 
B12 and accumulates the vitamin during loading, whereas the vitamin is liberated from the kidney during deficiency [18]. The results for the Cbi-loaded animals show cellular depletion of B12 and a PD-1/PD-L1 inhibitor 1 substantial accumulation of Cbi, notably in the liverOverload of the B12 Transport System in Micenot enough to cause accumulation of methionine. Secondly, we found that TC and TC-R are reduced in salivary gland. We have no explanation to offer for this observation. However, a downregulation of the TC-R fits well with our observation of a relatively low accumulation of salivary gland B12 in the B12-treated mice.transport of B12 into the cells. Thus, if the conjugate is inactive with respect to B12, this may lead to changes in B12 status.ConclusionsTaken together, our study shows that mice are a suitable model for studies of the transport and effect of B12 conjugates. Furthermore, our study emphasises the importance of monitoring B12 metabolism during treatment with B12 conjugates or analogues.Implicatio.Of tissue B12 levels to 33?0 of those observed in control mice. However, this study only lasted 4 weeks. Prolonged treatment with an inactive B12 analogue such as Cbi could further deplete B12 from the cells and, in time, cause B12 deficiency.Figure 3. Changes in transcript levels after treatment of mice with Cbi or B12. Kidney, liver and salivary glands were removed from mice treated for 27 days with saline (control, n = 7), 4.25 nmol/h Cobinamide (Cbi, n = 7) or 1.75 nmol/h vitamin B12 (B12, n = 7). We analysed the transcript levels by q t CR. The results (Mean +SEM) for each sample are relative to the mean value for the corresponding tissue from control mice. Non arametric Wilcoxon tests were employed to calculate significant differences, p,0.05 are indicated with * for comparison to control mice. No significant difference was observed between Cbi and controls or Cbi and B12 mice. LMBRD1: lysosomal B12 transporter. 1326631 MS: methionine synthase. MTHFR: methylenetetrahydrofolate reductase. MUT: methylmalonyl oA mutase. TC: transcobalamin. TC : transcobalamin receptor (CD320). doi:10.1371/journal.pone.0046657.gHigh-dose B12 increases tissue B12 and influences markers of B12 metabolismHigh doses of B12 increased plasma B12 and decreased the level of MMA. Unexpectedly, the other metabolic marker of B12 deficiency, tHCY, increased and so did cysteine, another metabolite of the homocysteine pathway. To our knowledge no previous studies have shown that B12 load may lead to an increased level of tHCY, thus suggesting that in relation to the metabolism of homocysteine, both too little B12 and too much may be disadvantageous. Our results show that the major part of surplus B12, and therefore possibly also B12 conjugates, will end up in the kidney. The liver is able to double the amount of B12 internalised. High dose B12 resulted in previously undescribed changes in the transcript levels of genes involved in the transport and metabolism of the vitamin. Firstly, we found a reduced transcript level of B12-dependent enzyme MTHFR in the kidneys. This reduction was not related to an accumulation of methionine in this group of mice. This could be expected, as MTHFR is involved in the conversion of methionine into methyl-tetrahydrofolate, see Figure 1. but apparently, the reduction in MTHFR mRNA wasrespectively, accumulation in the kidney far exceeded a factor of 2. This is explained by the kidney’s role in rodent B12 metabolism. The kidney acts as a reservoir for B12 and accumulates the vitamin during loading, whereas the vitamin is liberated from the kidney during deficiency [18]. The results for the Cbi-loaded animals show cellular depletion of B12 and a substantial accumulation of Cbi, notably in the liverOverload of the B12 Transport System in Micenot enough to cause accumulation of methionine. Secondly, we found that TC and TC-R are reduced in salivary gland. We have no explanation to offer for this observation. However, a downregulation of the TC-R fits well with our observation of a relatively low accumulation of salivary gland B12 in the B12-treated mice.transport of B12 into the cells. Thus, if the conjugate is inactive with respect to B12, this may lead to changes in B12 status.ConclusionsTaken together, our study shows that mice are a suitable model for studies of the transport and effect of B12 conjugates. Furthermore, our study emphasises the importance of monitoring B12 metabolism during treatment with B12 conjugates or analogues.Implicatio.