Photodegradation of cobalamins in aqueous solutions and in human blood
Introduction
Cobalamin, vitamin B12, is a micronutrient synthesized only by certain bacteria. It is obtained from animal food (meat, milk, fish, shellfish, poultry, eggs) and is essential to human health, as it is necessary for DNA synthesis, formation and maintenance of myelin sheaths, synthesis of neurotransmitters, and for erythropoiesis [1], [2].
Human plasma contains a variety of cobalamins (Fig. 1) including methylcobalamin (MeCbl, 64%), adenosylcobalamin (AdCbl, 15%), hydroxocobalamin (OHCbl, 12%) and cyanocobalamin (CNCbl (sometimes called vitamin B12), 9%) [3]. AdCbl (or coenzyme B12) and MeCbl are biochemically active forms of vitamin B12 in the human body. OHCbl and CNCbl are used as therapeutical agents and as supplements, but in cells they have to be converted either to AdCbl or MeCbl. AdCbl serves as a radical repository for a group of enzymes that catalyze unusual isomerizations, whereby a hydrogen atom is interchanged with an electron-withdrawing group on a neighboring carbon atom [4], [5]. AdCbl can be enzymatically produced in the human mitochondrion by adenosyltransferase, and is an essential cofactor for the enzyme methylmalonyl-coenzyme A mutase, which converts l-methylmalonyl coenzyme A to succinyl coenzyme A, which then enters the Krebs cycle [6]. In the cytoplasm methionine synthase requires MeCbl and catalyzes the conversion of homocysteine to methionine by transfer of a methyl group from methyltetrahydrofolate [7]. Intracellular deficiency of cobalamin or defects in cobalamin metabolism results in accumulation of methylmalonic acid and homocysteine in blood and urine [1], [8].
A typical Western diet gives 3–12 μg of cobalamin per day can be compared with the estimated daily requirement intake of 2.4 μg (World Health Organization (WHO) recommendation), and healthy humans have large vitamin B12 stores (about 2–5 mg) mostly in the liver [9], [10], [11]. Therefore, vitamin B12 deficiency does not occur under normal circumstances for 3–5 years [1], [10]. Vitamin B12 deficiency develops when people do not consume enough vitamin or when the body does not absorb or store enough of the vitamin. Vitamin B12 deficiency (<150 pmol/L) is common throughout the world, with a prevalence increasing with age [10], [12], [13], [14], [15].
Osmancevic et al. [16] reported that the plasma concentration of vitamin B12 decreased by 15% (P = 0.003), and the plasma concentration of homocysteine increased by 21% (P = 0.000) in psoriasis patients from Norway during 15 days of climate therapy (heliotherapy) at Gran Canaria. The patients followed a strict sun exposure schedule, and received a mean ultraviolet B (UVB, 280–315 nm) dose of 11.5 J/cm2 and a mean ultraviolet A (UVA, 315–400 nm) dose of 452 J/cm2 during 15 days. The decrease in serum vitamin B12 during heliotherapy cannot be explained by insufficient intake of this vitamin. Some forms of vitamin B12 are photolabile in solutions [17], [18], [19], [20], [21], [22], [23], [24], [25], and based on this the authors suggested that serum vitamin B12 is photodegradated during exposure to sunlight [16]. However, this hypothesis has yet neither been proven nor rejected. The photostability of cobalamins in vitro (cells) or in vivo (animal, humans) has not been studied so far. Contradictory studies have been published regarding the photostability of vitamin B12 in blood samples exposed to fluorescent light [26], [27], [28], [29]. Modern fluorescent lamps used for laboratory lighting emit mostly visible light and almost no ultraviolet (UV) radiation, where cobalamins also absorb (Fig. 2). In all studies blood was stored in plastic tubes. Plastic partially absorbs UV radiation.
Lamps with different emission spectra and intensities have been used to study the photostability of cobalamins in aqueous solutions [17], [18], [19], [20], [21], [22], [23], [24], [25], but no comprehensive study has been done to compare the photostability of cobalamins. In addition, the studies did not evaluate a possible influence of UVA on photostability of cobalamins. It is known that intracellular cobalamins react rapidly with nitric oxide and superoxide [21], [30], [31], [32], which can be generated in skin or blood during exposure to UVA [33], [34]. Additionally, skin and blood contain many endogenous photosensitizers (porphyrins, flavins) absorbing in the UVA region, and exposure to UVA leads to the generation of reactive oxygen species [35], [36]. Singlet oxygen, superoxide anions, hydrogen peroxide and triplet-state riboflavin radicals are generated under exposure of riboflavin (RF) to UVA [37]. Reactive oxygen species are quenched by antioxidants in human skin which may, due to this, indirectly be degraded. Cobalamins may also quench singlet oxygen and hydrogen peroxide, and it may photodegrade indirectly. However, the indirect photodegradation of cobalamins due to their antioxidant properties have not been studied.
In the present study we have investigated the photodegradation of four major forms of vitamin B12 (MeCbl, AdCbl, OHCbl and CNCbl) under UVA exposure in aqueous solutions at physiological pH by absorption spectroscopy. The degradation of OHCbl exposed to UVA in the absence and presence of the endogenous photosensitizer RF was investigated. Serum vitamin B12 concentrations before and after summer were measured in four patients with psoriasis.
Section snippets
Chemicals
Hydroxocobalamin (OHCbl), cyanocobalamin (CNCbl), adenosylcobalamin (AdCbl), methylcobalamin (MeCbl), riboflavin (RF) and Dulbecco’s phosphate buffered saline (PBS) were purchased from Sigma–Aldrich Norway AS (Oslo, Norway).
OHCbl, CNCbl, AdCbl and RF were dissolved in PBS, while MeCbl was dissolved in ethanol (50 mg/ml). The final stock solutions of all vitamin B12 forms were made in PBS (pH = 7.4) at a concentration of 0.1 mM. The solutions of OHCbl and CNCbl were stored at 3–4 °C, while AdCbl and
Absorption measurements
All studied vitamin B12 derivatives have larger absorbance in the UVA region than in the visible (Fig. 2). The inserts represent the absorbance at the main peak wavelength in the UVA region as a function of the cobalamin concentration (Fig. 2). Linear regressions indicate that at these concentrations the absorbance follows the Beer–Lambert–Bouguer law. The absorbance intensities were normalized to unity at the optical absorption maxima in the UVA region: OHCbl at 351 nm, CNCbl at 361 nm, AdCbl at
Discussion
The active forms of cobalamin, AdCbl and MeCbl, which participate in important biological reactions in humans, were found to be extremely sensitive to UVA radiation in aqueous solutions (Fig. 4, Fig. 5C–D). Under UVA exposure they degrade very fast and are converted to OHCbl (Fig. 4, Fig. 5, Fig. 6), which was found to be the most photostable cobalamin under direct exposure to UVA radiation. However, OHCbl is an antioxidant, and may degrade indirectly in the presence of reactive oxygen and
Conclusions
Four major forms of cobalamin, OHCbl, CNCbl, AdCbl and MeCbl, in aqueous solutions under physiological pH, are sensitive to UVA radiation. OHCbl is the most stable cobalamin. AdCbl and MeCbl, the biologically active forms of vitamin B12, are converted to OHCbl within seconds during UVA exposure. Our pilot study on humans demonstrates that serum vitamin B12 concentrations are not significantly affected during summertime in the Scandinavia. There is no consensus on whether vitamin B12 is degraded
Abbreviations
AdCbl, adenosylcobalamin; CNCbl, cyanocobalamin; MeCbl, methylcobalamin; OHCbl, hydroxocobalamin; UV, ultraviolet radiation; UVA, ultraviolet A radiation (315–400 nm), UVB, ultraviolet B radiation (280–315 nm).
Acknowledgements
The present work was supported by the South-Eastern Norway Regional Health Authority and by Oslo University Hospital. We appreciate the help of Dr. Gunnar Volden (Ski, Norway) in taking blood samples. We also appreciate the help of the personnel at Fürst Medical Laboratory (Oslo, Norway).
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