Kinetics of Cellular Cobalamin Uptake and Conversion: Comparison of Aquo/Hydroxocobalamin to Cyanocobalamin

Abstract

Cyanocobalamin (CNCbl) and aquo/hydroxocobalamin (HOCbl) are the forms of vitamin B₁₂ that are most commonly used for supplementation. They are both converted to methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl), which metabolize homocysteine and methylmalonic acid, respectively. Here, we compare the kinetics of uptake and the intracellular transformations of radiolabeled CNCbl vs. HOCbl in HeLa cells. More HOCbl was accumulated over 4-48 h, but further extrapolation indicated similar uptake (>90%) for both vitamin forms. The initially synthesized coenzyme was MeCbl, which noticeably exceeded AdoCbl during 48 h. Yet, the synthesis of AdoCbl accelerated, and the predicted final levels of Cbls were MeCbl ≈ AdoCbl ≈ 40% and HOCbl ≈ 20%. The designed kinetic model revealed the same patterns of the uptake and turnover for CNCbl and HOCbl, apart from two steps. First, the "activating" intracellular processing of the internalized HOCbl was six-fold faster. Second, the detachment rates from the cell surface (when the "excessive" Cbl-molecules were refluxed into the external medium) related as 4:1 for CNCbl vs. HOCbl. This gave a two-fold faster cellular accumulation and processing of HOCbl vs. CNCbl. In medical terms, our data suggest (i) an earlier response to the treatment of Cbl-deficiency with HOCbl, and (ii) the manifestation of a successful treatment initially as a decrease in homocysteine.

Keywords: HeLa cells; cobalamin; kinetics; uptake; vitamin B12.

Figure 5

The kinetic scheme of the final model. The following steps were considered: (1) the export of receptor R (specific for TC-XCbl) to the cell surface; (2) the clearance of “empty” R from the surface; (3) the R-mediated uptake of TC-XCbl followed by the degradation of the protein moieties and the appearance of XCbl in the cytoplasm; (4) the recirculation of the unprocessed XCbl to the cell surface; (5) the dissociation of XCbl from the cells and its binding to surplus TC; (6) the intracellular reduction of absorbed XCbl to [Co²⁺]Cbl; (7) the synthesis of MeCbl in the cytoplasm; (8) the synthesis of AdoCbl in the mitochondria. Steps 7 and 8 describe the equilibriums between MeCbl/AdoCbl and [Co²⁺]Cbl. All parameters (Table 2) for CNCbl and HOCbl schemes are identical except for steps 5 and 6, as highlighted by yellow squares. See the main text for further details.

Figure 1

The intracellular processing of Cbl (notation of steps generally follows ref. [12]). Different Cbl forms (XCbl) circulate in blood, bound to an excess of the specific carrier transcobalamin (TC). The TC–XCbl complex enters the cell with the help of its specific receptor (R). Both protein moieties (TC and R) are endocytosed and degrade in lysosomes, whereupon free XCbl enters the cytoplasm with the help of a lysosomal membrane transporter (omitted in this figure). The liberated XCbl is reduced in the cytoplasm by the enzyme chaperon CblC and thus loses its original X-group. The superfluous XCbl leaves the cell unprocessed via the MRP1/ABCC1 transporter (ABC). The processed [Co²⁺]Cbl is delivered with the help of the adapter protein CblD to either cytoplasmic methionine synthase (MS) and MS reductase (CblE), or to the mitochondrial methylmalonyl-CoA mutase (MMCM) and adenosyltrasferase (CblB). The first pair of enzymes produces and utilizes MeCbl, while the second pair synthesizes and uses AdoCbl.

Figure 2

Time-dependent records for the cellular uptake of radioactive Cbls, normalized to 10⁷ cells (open symbols). Closed symbols notate the data of exploratory experiments (24 h), where the number of cells was assessed as 10⁷. (a) Intracellular Cbl. The experimental measurements are shown as the individual points for CN[⁵⁷Co]Cbl (Δ, green) and HO[⁵⁷Co]Cbl (○, red). The experimental dispersion was estimated as σ = 4.15%, see Supplementary Section S3. The solid lines (tagged as “CN” and “HO” for the respective Cbl-ligands) depict kinetic simulations according to the final kinetic model of Cbl uptake (see Section 3.5). Here, 100% of Cbl units % corresponds to 1.1 pmol of ⁵⁷Cbl (bound to 2.2 pmol of TC) in 5 mL of the medium. (b) Tightly associated surface Cbl. The points correspond to measured surface radioactivity released after trypsinolysis of the cells. The experimental dispersion was estimated as σ = 2.44%. The solid lines show simulations based on the final kinetic model. The lower dashed curve (shown in red) depicts the turnovers of surface HO[⁵⁷Co]Cbl if assuming k_5HO = k_5CN, see Section 3.5 and Table 2. The turnover of the surface receptor R (dotted lines at the top) was theoretically simulated but not measured, see more in Section 4.3 of the Discussion. Abbreviations R_CN and R_OH reflect simulations of R-turnover in the experiments with CN[⁵⁷Co]Cbl and HO[⁵⁷Co]Cbl, respectively. The other notation is explained in the legend to panel (a).

Figure 3

The HPLC elution profiles of the intracellular Cbls. Experiments with (a) CN[⁵⁷Co]Cbl or (b) HO[⁵⁷Co]Cbl. The records are presented as a percentage of radioactivity in each HPLC fraction, normalized to the total radioactivity in the profile. The time of incubation (cells + ⁵⁷Cbl) is shown in the upper left corner of each panel. The tags above the peaks correspond to NH₃Cbl (generated from HOCbl and its substituted variants), CNCbl, AdoCbl, and MeCbl. Solid arrows schematically depict transitions of the peaks during incubation of cells with ⁵⁷Cbl (4–48 h). The dotted arrow in panel (b) indicates that a part of the breakthrough peak (1.5 min for 4 h of incubation) was assumed to be HOCbl, unspecifically bound to some peptides via cobalt coordination, see the main text for further details.

Figure 4

Intracellular conversions of the accumulated Cbls. Experiments with (a) CN[⁵⁷Co]Cbl; and (b) HO[⁵⁷Co]Cbl. Percent units % reflect fractions of each individual Cbl in the pool of total radioactivity added to the cells. Thus, 10% corresponds to ≈4.4 nM of the intracellular concentration (assuming 25 μL of intracellular volume for 10⁷ cells). Experimental measurements are shown as the individual points. The experimental dispersion of data was assessed as σ = 1.42% for panel a and σ = 2.15% for panel (b), see Supplementary Section S3. The curves depict our computer simulations based on the designed kinetic model (Figure 5). The tags “CN”, “Ado”, and “Me” correspond to the respective Cbl forms. The tag “Co²⁺” assembles all different reduction grades of Cbl in one category, isolated as HOCbl. The tag “HO_in” in panel B represents the absorbed but unprocessed intracellular HO[⁵⁷Co]Cbl, which cannot be experimentally differentiated from the pool of processed [Co²⁺]-forms. The representation of HO_in was assessed from the model simulations.