Ceptor (TC ), the TC 12/cobinamide(Cbi) complex is internalised into the

Ceptor (TC ), the TC 12/cobinamide(Cbi) complex is internalised into the lysosomes. TC is degraded and B12 is transported into the cytoplasm by the lysosomal membrane transporter 1 (LMBRD1). Intracellular, B12 serves as cofactor for the mitochondrial methylmalonylCoA mutase (MUT) and the cytosolic methionine synthase (MS) that acts in coordination with methylenetetrahydrofolate reductase (MTHFR). In plasma, TC circulates partly unsaturated with B12 (UB12BC). If the B12 supply to the cell is insufficient, methylmalonic acid (MMA) and homocysteine (tHcy) accumulates in the blood. The transcript level of the boxed components were analysed by quantitative Tunicamycin web reverse?transcript (q t? PCR and the circularised components were analysed biochemically in either tissue or blood (plasma) as indicated. doi:10.1371/journal.pone.0046657.gOrgan collectionImmediately after the mice were sacrificed, organs were collected and snap-frozen in 1326631 liquid nitrogen. The organs were stored at 280uC until further processing.Crude tissue extractionCrude aliquots of tissue 1485-00-3 manufacturer extracts were prepared in homogenisation buffer containing protease inhibitors as described previously [2]. The tissue-to-buffer ratios were: 231 g/L (kidney); 400 g/L (liver), and 140 g/L (salivary glands (submaxillary glands)).isoflurane anaesthesia, and pain after surgery was reduced as described below.Animals21 7-week-old female mice (strain 129.S6; Taconic, Denmark) were divided into three groups nd caged separately: control mice (C) n = 7 cobinamide-loaded mice (Cbi) n = 7; and vitamin B12loaded mice (B12) n = 7. The mice had free access to water and to standard chow (Altromin maintenance diet for rats and mice (1324) (19 pmol/g B12; 0.4 pmol/g Cbi) Altromin, Germany) except during 24-h urine collection once weekly in metabolic cages. After 1 week of acclimation (age 8 weeks), an osmotic minipump (Mini-Osmotic Pump Model 2004, Alzet Cupertino, CA, USA) was implanted into each animal. The mice were anesthetised with isoflurane (IsoFloH Vet), Abbott), and osmotic minipumps were inserted subcutaneously into the back by incision just below the neck region. After insertion, the wound was closed by absorbable suture. Prior to insertion, the pumps were equilibrated and filled following the manufacturer’s instructions. The pumps were filled with either saline (0.9 NaCl) (control mice), 17 mM dicyano-cobinamide (MW: 1042.12 g/mol SigmaAldrich, Saint Louis, MO, USA) in 0.9 NaCl (Cbi mice), or 7 mM cyanocobalamin (MW: 1355.37 g/mol; Sigma-Aldrich) in 0.9 NaCl (B12 mice). The latter represented the maximal amount of B12 that could be dissolved. According to the manufactures, the delivery rate of the pumps is 0.25 mL/hr equivalent to delivered rate of 4.25 nmol/h for Cbi mice and 1.75 nmol/h for B12 mice. To avoid wound biting between mice, the mice were housed in individual cages for 3 days after surgery. In addition, to ameliorate pain after surgery, analgesics was put into the drinking water (buprenorphine hydrochloride 0.06 mg/Analysis of haematological valuesWithin 2 hours after collection, 200 mL of EDTA blood from each mouse was analysed for haematological parameters on a Sysmex XE-2100 Automated Hematology System (Sysmex Corporation) [10].MMA, tHcy, cysteine, and methionine in plasma50 mL of heparinised plasma collected on day 27 was sent to BeVital (http://www.bevital.no/) for analysis of methylmalonic acid (MMA), total homocysteine (tHcy), total cysteine, and methionine levels using standardised.Ceptor (TC ), the TC 12/cobinamide(Cbi) complex is internalised into the lysosomes. TC is degraded and B12 is transported into the cytoplasm by the lysosomal membrane transporter 1 (LMBRD1). Intracellular, B12 serves as cofactor for the mitochondrial methylmalonylCoA mutase (MUT) and the cytosolic methionine synthase (MS) that acts in coordination with methylenetetrahydrofolate reductase (MTHFR). In plasma, TC circulates partly unsaturated with B12 (UB12BC). If the B12 supply to the cell is insufficient, methylmalonic acid (MMA) and homocysteine (tHcy) accumulates in the blood. The transcript level of the boxed components were analysed by quantitative reverse?transcript (q t? PCR and the circularised components were analysed biochemically in either tissue or blood (plasma) as indicated. doi:10.1371/journal.pone.0046657.gOrgan collectionImmediately after the mice were sacrificed, organs were collected and snap-frozen in 1326631 liquid nitrogen. The organs were stored at 280uC until further processing.Crude tissue extractionCrude aliquots of tissue extracts were prepared in homogenisation buffer containing protease inhibitors as described previously [2]. The tissue-to-buffer ratios were: 231 g/L (kidney); 400 g/L (liver), and 140 g/L (salivary glands (submaxillary glands)).isoflurane anaesthesia, and pain after surgery was reduced as described below.Animals21 7-week-old female mice (strain 129.S6; Taconic, Denmark) were divided into three groups nd caged separately: control mice (C) n = 7 cobinamide-loaded mice (Cbi) n = 7; and vitamin B12loaded mice (B12) n = 7. The mice had free access to water and to standard chow (Altromin maintenance diet for rats and mice (1324) (19 pmol/g B12; 0.4 pmol/g Cbi) Altromin, Germany) except during 24-h urine collection once weekly in metabolic cages. After 1 week of acclimation (age 8 weeks), an osmotic minipump (Mini-Osmotic Pump Model 2004, Alzet Cupertino, CA, USA) was implanted into each animal. The mice were anesthetised with isoflurane (IsoFloH Vet), Abbott), and osmotic minipumps were inserted subcutaneously into the back by incision just below the neck region. After insertion, the wound was closed by absorbable suture. Prior to insertion, the pumps were equilibrated and filled following the manufacturer’s instructions. The pumps were filled with either saline (0.9 NaCl) (control mice), 17 mM dicyano-cobinamide (MW: 1042.12 g/mol SigmaAldrich, Saint Louis, MO, USA) in 0.9 NaCl (Cbi mice), or 7 mM cyanocobalamin (MW: 1355.37 g/mol; Sigma-Aldrich) in 0.9 NaCl (B12 mice). The latter represented the maximal amount of B12 that could be dissolved. According to the manufactures, the delivery rate of the pumps is 0.25 mL/hr equivalent to delivered rate of 4.25 nmol/h for Cbi mice and 1.75 nmol/h for B12 mice. To avoid wound biting between mice, the mice were housed in individual cages for 3 days after surgery. In addition, to ameliorate pain after surgery, analgesics was put into the drinking water (buprenorphine hydrochloride 0.06 mg/Analysis of haematological valuesWithin 2 hours after collection, 200 mL of EDTA blood from each mouse was analysed for haematological parameters on a Sysmex XE-2100 Automated Hematology System (Sysmex Corporation) [10].MMA, tHcy, cysteine, and methionine in plasma50 mL of heparinised plasma collected on day 27 was sent to BeVital (http://www.bevital.no/) for analysis of methylmalonic acid (MMA), total homocysteine (tHcy), total cysteine, and methionine levels using standardised.

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