Vitamin B12 Deficiency Induces Imbalance in Melanocytes Homeostasis—A Cellular Basis of Hypocobalaminemia Pigmentary Manifestations

Abstract

Vitamin B12 deficiency causes significant changes in cellular metabolism leading to various clinical symptoms, such as hematological, psychiatric, and neurological disorders. We hypothesize that skin pigmentation disorders may be a diagnostically important manifestation of vitamin B12 deficiency, however the cellular and molecular mechanisms underlying these effects remain unknown. The aim of this study was to examine the effect of vitamin B12 deficiency on melanocytes homeostasis. Hypocobalaminemia in vitro model was developed by treating epidermal melanocytes with synthesized vitamin B12 antagonist—hydroxycobalamin(c-lactam). The cells were examined using immunoenzymatic, spectrophotometric, and fluorimetric assays as well as image cytometry. Significant melanogenesis stimulation—the increase of relative melanin content and tyrosinase activity up to 131% and 135%, respectively—has been indicated. Cobalamin-deficient cells displayed the elevation (by 120%) in reactive oxygen species level. Moreover, the redox status imbalance was stated. The study provided a scientific evidence for melanocytes homeostasis disturbance under hypocobalaminemia, thus indicating a significant element of the hyperpigmentation mechanism due to vitamin B12 deficiency. Furthermore, the implication between pigmentary and hematological and/or neuropsychiatric symptoms in cobalamin-deficient patients may be an important issue.

Cobalamin Deficiency: Effect on Homeostasis of Cultured Human Astrocytes

“…To verify whether the astrocytes cultured with (OH)Cbl(c-lactam) were cobalamin deficient, concentration of Hcy was measured in the medium samples collected at days 7, 13, 20, and 27. For this purpose quantitative sandwich enzyme-linked immuno-sorbent assay (ELISA) was performed using commercially available Human Hcy ELISA Kit (Abbexa), according to the previously described method [13].…”

“…Intracellular thiols, by their ability to be reversible oxidized, determine cellular redox homeostasis, and thus the decrease of reduced thiol level is recognized as a marker of oxidative stress [25]. Analysis of thiol status in astrocytes after the 27 day culture in medium with/without (OH)Cbl(c-lactam) was performed using the NucleoCounter NC-3000 system according to a previously reported method [13]. In brief, the assay is based on staining with a VitaBright 48 (VB-48) probe, which selectively reacts with free thiol groups forming a fluorescent product [26].…”

“…As demonstrated in Figure 1a, at day 20 and 27 of the treatment, the number of cells was significantly lower than in the control culture. method [13]. In brief, the assay is based on staining with a VitaBright 48 (VB-48) probe, which selectively reacts with free thiol groups forming a fluorescent product [26].…”

“…The state of hypocobalaminemia in cellulo can be detected by measuring extracellular homocysteine level [13]. To determine a time after which astrocytes cultured with (OH)Cbl(c-lactam) were cobalamin deficient, we measured the concentration of homocysteine in the medium samples collected during cell subculturing.…”

“…Hydroxycobalamin(c-lactam) was synthesized by Prof. Dorota Gryko and Dr. Keith ó Proinsias (Institute of Organic Chemistry, Polish Academy of Science, Warsaw, Poland). The synthesis and identification of the compound were performed as described previously [13].…”