Abstract
Coβ-4-ethylphenyl-cob(III) alamin (EtPhCbl) is an organometallic analogue of vitamin B12 (CNCbl) which binds to transcobalamin (TC), a plasma protein that facilitates the cellular uptake of cobalamin (Cbl). In vitro assays with key enzymes do not convert EtPhCbl to the active coenzyme forms of Cbl suggesting that administration of EtPhCbl may cause cellular Cbl deficiency. Here, we investigate the in vivo effect of EtPhCbl in mice and its ability, if any, to induce Cbl deficiency. We show that EtPhCbl binds to mouse TC and we examined mice that received 3.5 nmol/24h EtPhCbl (n=6), 3.5 nmol/24h CNCbl (n=7) or NaCl (control group) (n=5) through osmotic mini-pumps for four weeks. We analyzed plasma, urine, liver, spleen, submaxillary glands and spinal cord for Cbl and markers of Cbl deficiency including methylmalonic acid (MMA) and homocysteine (tHcy). Plasma MMA (mean±SEM) was elevated in animals treated with EtPhCbl (1.01±0.12 µmol/L) compared to controls (0.30±0.02 µmol/L) and CNCbl (0.29±0.01 µmol/L) treated animals. The same pattern was observed for tHcy. Plasma total Cbl concentration was higher in animals treated with EtPhCbl (128.82±1.87 nmol/L) than in CNCbl treated animals (87.64±0.93 nmol/L). However, the organ levels of total Cbl were significantly lower in animals treated with EtPhCbl compared to CNCbl treated animals or controls, notably in the liver (157.07±8.56 pmol/g vs. 603.85±20.02 pmol/g, and 443.09±12.32 pmol/g, respectively). Differences between the three groups was analysed using one-way ANOVA and, Bonferroni post-hoc test. EtPhCbl was present in all tissues, except the spinal cord, accounting for 35-90% of total Cbl. In conclusion, treatment with EtPhCbl induces biochemical evidence of Cbl deficiency. This may in part be caused by a compromised tissue accumulation of Cbl.
Highlights
Cobalamins (Cbls) are delivered in several molecular forms differing by the axial ligand attached to the cobalt atom bound by the corrin macro ring (Figure 1)
We explored the binding of ethylphenyl-cob(III) alamin (EtPhCbl) to mouse TC and report binding characteristics identical to those of H2OCbl (EtPhCbl converted to its aquoform by exposure to light) and comparable to the binding characteristics of CNCbl (Figure 2)
We report that mice treated with EtPhCbl for four weeks show signs of an impaired metabolism of Cbl characterized by tissue depletion of Cbl and an increased level of the metabolic markers of Cbl deficiency, methylmalonic acid (MMA) and tHCy
Summary
Cobalamins (Cbls) are delivered in several molecular forms differing by the axial ligand attached to the cobalt atom bound by the corrin macro ring (Figure 1). The various forms of Cbl are reduced by the enzyme methylmalonic aciduria and homocystinuria type C protein (CblC), and, subsequently, 5′-deoxyadenosyl-cobalamin (AdoCbl) and methylcobalamin (CH 3Cbl) are formed. These are coenzymes for the two known mammalian Cbl dependent enzymes. A Cbl analogue not reduced by CblC is expected to inhibit Cbl metabolism resulting in the symptoms and findings of Cbl deficiency. Severe Cbl deficiency may induce megaloblastic anemia and a neuropathy known as subacute combined degeneration and, characterized by irregular demyelination of the white matter and astrogliosis [3]
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