Low methylcobalamin in liver tissues is an artifact as shown by a revised extraction procedure

Fedosov, Sergey N.; Nexø, Ebba; Heegaard, Christian W.

doi:10.1016/j.bbagen.2023.130315

Cited by 3 publications

(6 citation statements)

References 48 publications

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“…This procedure preserved the original composition of Cbls in an artificial mixture, containing GSCbl, CNCbl, HOCbl, AdoCbl, and MeCbl in blood plasma with 10 mM glutathione (GSH), see Supplementary Section S4. GSH is capable of converting MeCbl, CNCbl, and HOCbl to GSCbl, if all heated together [28,30]. Our tests showed that GSH did not enter the phenol-chloroform phase and could not disturb the original balance of Cbls.…”

Section: Intracellular Cbls and The Extraction And Analysis Of Cbl Fo...mentioning

confidence: 60%

“…A puzzling dispersion in the proportions between MeCbl and AdoCbl was reported for cell cultures [11,[19][20][21]23,24], while animal and human tissues seemingly exposed a great prevalence of endogenous AdoCbl above MeCbl [25][26][27]. Noteworthy, we have recently demonstrated that several widely used methods for the tissue extraction of Cbl can convert MeCbl to HOCbl, thereby masking the true balance of intracellular Cbl coenzymes [28].…”

Section: Introductionmentioning

confidence: 86%

“…The possible reasons for such conspicuous discrepancies were thoroughly discussed earlier [11] and cover the incubation time of cultured cells, cell-type specificity, and the composition of the culture medium. Here, we would like to attract attention to a recent observation that the conditions of extraction can critically affect the balance between AdoCbl and MeCbl due to the conversion of MeCbl → HOCbl, which takes place during a harsh heating in the presence of endogenous reductants (e.g., thiols like GSH) [28]. Importantly, "a cold extraction" should not be accompanied by "a hot evaporation" of the liquid phase, because 16 h at 45 • C would match approximately 1 h at 85 • C. Therefore, no heating is advised during lyophilization in a centrifugal evaporator.…”

Section: General Observationsmentioning

confidence: 99%

“…Therefore, the enzymatic pair of MS + CblE effectively intercepts CblD••Cbl, resulting in a 10-fold higher probability of MeCbl synthesis vs. AdoCbl (k +7 and k +8 in Table 2). We have earlier speculated [28] that a lower intracellular content of MS in comparison to MMCM [36] guarantees an early equilibration of MS with Cbl, whereupon the rest of the "excessive" Cbl is delivered to MMCM. Only at this point does the synthesis of AdoCbl start gaining momentum, while the production of MeCbl slows down.…”

Section: Kinetic Model: Steps 7 and 8 The Synthesis Of Mecbl And Adocblmentioning

confidence: 99%

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

Fedosov,

Nexo,

Heegaard

2024

Nutrients

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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.

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Section: Intracellular Cbls and The Extraction And Analysis Of Cbl Fo...mentioning

confidence: 60%

Section: Introductionmentioning

confidence: 86%

Section: General Observationsmentioning

confidence: 99%

Section: Kinetic Model: Steps 7 and 8 The Synthesis Of Mecbl And Adocblmentioning

confidence: 99%

See 2 more Smart Citations

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

Fedosov,

Nexo,

Heegaard

2024

Nutrients

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“…The samples were centrifuged on a benchtop centrifuge (20 000 g, 10 min) and stored frozen at −20 °C. This procedure is based on a previously established efficient method for extraction of total Cbl tested in an artificial mixture with a very sturdy Cbl-binder HC from buffalo milk ( Fedosov, Nexo & Heegaard, 2023 ).…”

Section: Methodsmentioning

confidence: 99%