VITAMINS

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Cannabinoids are psychoactive components in marijuana, and their specific membrane-bound receptors are present in the brain and other animal tissues. Arachidonoylethanolamide was found as an endogenous ligand for the cannabinoid receptors, and referred to as anandamide. Anandamide is enzymatically hydrolyzed to arachidonic acid and ethanolamine resulting in the loss of its biological activity. We partially purified "anandamide amidohydrolase" from microsomes of porcine brain. The purified enzyme also catalyzed the reverse reaction, namely, the condensation of arachidonic acid and ethanolamine to form anandamide. This reversibility was confirmed with the recombinant anandamide amidohydrolase of rat liver which was overexpressed in COS-7 cells. The recombinant enzyme showed a wide substrate specificity hydrolyzing primary amides and esters of unsaturated fatty acids as well as anandamide. 2-Arachidonoylglycerol, which was recently identified as another endogenous ligand for cannabinoid receptors, was also hydrolyzed efficiently by the same enzyme. The enzyme was distributed in various organs such as liver, brain, alimentary tract, and eye tissues. The enzyme activity of small intestine was underestimated by the presence of endogenous lipid inhibitors, and was increased by the acetone precipitation of the enzyme to remove lipids. In addition to the hydrolysis, anandamide was oxygenated at C-12 or C-15 of the arachidonate moiety by mammalian 12- and 15-1ipoxygenases. These results suggest the in vivo functions of these enzymes to regulate the biological activity of anandamide.

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We studied the origin of the methyl group attached to C-2 of the pyrimidine moiety of thiamin (Pm) in Escherichia coil [3-^<13>C]glucose and [4-^<13>C]glucose were used as tracers and were incorporated into the ribose moiety of 5-aminoimidazole ribonucleotide(AIR), an intermediate of Pm via pentose phosphate pathway in E. coil The incorporation of these labels into Pm was examined, together with the measurment of the rate of the incorporation into histidine in E. coli. The label from [4-^<13>C]glucose was not incorporated into Pm, while the label from [3-^<13>C]glucose was effectively incorporated into it, indicating the incorporation of C-2 of ribose moiety of AIR into Pm. The biosynthetic conversion process from AIR to Pm in E. coli is proposed based on the results.

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Recently, two radioimmunoassay systems with ^<125>I-labelled tracer for 1α,25-dihydroxyvitamin D(1,25(OH)_2D) have been developed: IDS-RIA and INCSTAR-RIA. These assay systems provide simple and rapid methods that do not require sample pre-purification by HPLC before the binding assay, and are shown to be useful especially in individuals who do not receive vitamin D treatment. However, its clinical use has been still limited because of the limited number of studies, which demonstrate the cross-reactivities of vitamin D metabolites and the bias from the administered vitamin D related medicine. In this study, we investigated the cross-reactivities of various vitamin D metabolites by adding those compound to normal pool serum and compared the assays with an accepted radioreceptor assay (RRA) in patients with end-stage renal diseases under 1α-OH-D_3 therapy. Regarding the cross-reactivity of 1,25(OH)_2D_3 as 100 %, the indexes of 23(S)25(R)-1,25(OH)_2D_3-26,23-lactone, 1,24,25(OH)_3D_3, 1,24(OH)_2D_3 and 1,25(OH)_2D_3-24-oxo-D_3 were 33.5%, 169%, 81.7% and 150% in IDS-RIA and 0%, 28.1%, 44.3% and 24.0% in INCSTAR-RIA, respectively. In patients with end-stage renal diseases, the values from each RIA were well correlated with those from RRA. In conclusion, these new RIA assay systems for serum 1,25(OH)_2D are useful methods in patients under the vitamin D treatment.

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