PurposeHydroxocobalamin (HOCbl) is the dominating Cbl form in food, whereas cyanocobalamin (CNCbl) is common in vitamin pills and oral supplements. This study compares single-dose absorption and distribution of oral HO[57Co]Cbl and CN[57Co]Cbl in Cbl-deficient and normal rats.MethodsMale Wistar rats (7 weeks) were fed a 14-day diet with (n = 15) or without (n = 15) Cbl. We compared the uptakes of HO[57Co]Cbl (free or bound to bovine transcobalamin) and free CN[57Co]Cbl administered by gastric gavage (n = 5 in each diet group). Rats were sacrificed after 24 h. Blood, liver, kidney, brain, heart, spleen, intestines, skeletal muscle, 24-h urine and faeces were collected, and the content of [57Co]Cbl was measured. Endogenous Cbl in tissues and plasma was analysed by routine methods.ResultsMean endogenous plasma-Cbl was sevenfold lower in deficient vs. normal rats (190 vs. 1330 pmol/L, p < 0.0001). Cbl depletion increased endogenous Cbl ratios (tissue/plasma = k in/k out) in all organs except for the kidney, where the ratio decreased considerably. Twenty-four-hour accumulation of labelled Cbl showed that HOCbl > CNCbl (liver) and CNCbl > HOCbl (brain, muscle and plasma).ConclusionsThe Cbl status of rats and the administered Cbl form influence 24-h Cbl accumulation in tissues and plasma.Electronic supplementary materialThe online version of this article (doi:10.1007/s00394-017-1424-0) contains supplementary material, which is available to authorized users.
Cbl added to milk (spiked with rbTC) has high bioavailability matching that of free Cbl. OHCbl and CNCbl are absorbed equally well, but much more OHCbl accumulated in the liver. Benefits of oral supplementation with OHCbl compared to CNCbl should be investigated.
Recent rat studies show different tissue distributions of vitamin B 12 (B 12 ), administered orally as hydroxo-B 12 ) (predominant in food) and cyano-B 12 ) (common in supplements). Here we examine male Wistar rats kept on a low-B 12 diet for 4 weeks followed by a 2-week period on diets with HO-B 12 (n 9) or CN-B 12 (n 9), or maintained on a low-B 12 diet (n 9). Plasma B 12 was analysed before, during and after the study. The content of B 12 and its variants (HO-B 12 , glutathionyl-B 12 , CN-B 12 , 5'-deoxyadenosyl-B 12 (ADO-B 12 ), and methyl-B 12 (CH 3 -B 12 )) were assessed in the tissues at the end of the study. A period of 4 weeks on the low-B 12 diet reduced plasma B 12 by 58 % (from median 1323 (range 602-1791) to 562 (range 267-865) pmol/l, n 27). After 2 weeks on a high-B 12 diet (week 6 v. week 4), plasma B 12 increased by 68 % (HO-B 12 ) and 131 % (CN-B 12 ). Total B 12 in the tissues accumulated differently: HO-B 12 > CN-B 12 (liver, spleen), HO-B 12 < CN-B 12 (kidneys), and HO-B 12 ≈CN-B 12 (brain, heart). Notably, more than half of the administered CN-B 12 remained in this form in the kidneys, whereas HO-B 12 was largely converted to the bioactive ADO-B 12 . Only <10 % of the other cofactor, CH 3 -B 12 , were found in the tissues. In conclusion, dietary CN-B 12 caused a higher increase in plasma and total kidney B 12 but provided less than half of the active coenzymes in comparison to dietary HO-B 12 . These data argue that HO-B 12 may provide a better tissue supply of B 12 than CN-B 12 , thereby underscoring the lack of a direct relation between plasma B 12 and tissue B 12 . ]-coordinating anions. These reactions occur irrespectively of light (6) . All forms of B 12 are metabolically transformed into and CH 3 -B 12 in the cell (3) . Here, ADO-B 12 acts as a cofactor for methylmalonyl-CoA mutase in the conversion of methylmalonyl-CoA to succinyl-CoA in the mitochondria. CH 3 -B 12 acts as a cofactor for methionine synthase in the folatedependent methylation of homocysteine to methionine in the cytoplasm (1,2) .Both human and animal studies have demonstrated that synthetic and natural forms of B 12 are absorbed equally (7)(8)(9)(10)(11) . However, our recent data show that HO-B 12 accumulates in the liver to a higher degree than CN-B 12 , but that the patterns are opposite in the brain and plasma. These observations were based on the administration of acute doses of radiolabelled HO-B 12 and CN-B 12 to rats (10,11) . In accordance with this, acute human studies showed CN-B 12 to cause a 2-3-fold higher increase in the active circulating B 12 , holotranscobalamin, relative to HO-B 12 upon oral administration (12) . These findings Abbreviations: ADO-B 12 ,
We suggest a novel kinetic approach to quantifying receptor–ligand interactions via the cellular transport and/or accumulation of the ligand. The system of cobalamin (Cbl, vitamin B12) transport was used as a model, because Cbl is an obligatory cofactor, taken up by animal cells with the help of a transport protein and a membrane receptor. Bovine transcobalamin (bTC) stimulated the cellular accumulation and transcytosis of radioactive [57Co]Cbl in polarized monolayers of Caco-2 cells. The bovine protein was much more efficient than human TC. The transport was inhibited in a dose-dependent manner by the unlabeled bTC-Cbl complex, the ligand-free bTC, and the receptor-associated protein (RAP). This inhibition pattern implied the presence of a megalin-like receptor. Quantitative assessment of kinetic records by the suggested method revealed the apparent concentration of receptors in vitro (≈15 nM), as well as the dissociation constants of bTC–Cbl ( Kd = 13 nM) and RAP ( Kd = 1.3 nM). The data were used to estimate the effective luminal concentrations of TC-specific receptors in kidneys (3.8 µM) and intestine (50 nM), the tissues resembling polarized Caco-2 cells.
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