A simultaneous micro-determination of nicotinamide, N^1-methyl-2-pyridone-5-carboxamide (2-Py) and N^1-methyl-4-pyridone-3-carboxamide (4-Py), as well as an ultramicro-determination of N^1-methylnicotinamide (MNA) by HPLC are described. The method for measuring nicotinarnide, 2-Py and 4-Py employs a 7-ODS-L (4.6×250mm, particle size 7μm) column eluted with 10mM potassium dihydrogenphosphate-acetonitrile (96:4, v/v, pH adjusted to 3.0 by the addition of concentrated phosphoric acid) at a flow-rate of 1.0 ml/min. The UV detector was set at 260nm. The detection limits for nicotinamide, 2-Py and 4-Py were 10pmol (1.22ng), 2pmol (304pg) and 2pmol (304pg), respectively, at a signal-to-noise ratio 5:1. Isonicotinamide was used as an internal standard. MNA reacted with acetophenone in a strong alkali medium at 0℃ in the presence of a large amount of isonicotinamide. After being kept for 10min, formic acid was added and the mixture was kept for further 15min at 0℃. Then, the mixture was heated for 5 min. The reaction product, 1-methyl-7-phenyl-1, 5-dihydro-5-oxo-1; 6-naphthyridine was adsorbed on a 300-SCX (4.6×150mm, particle size 7μm) column, eluted with 25mM potassium dihydrogenphosphate containing 20% acetonitrile (pH 4.5) at a flow-rate of 1.0ml/min, and estimated at excitation wavelength of 382nm as well as emission wavelength of 440nm. The detection limit was 0.01pmol (1.36pg in terms of MNA). These techniques were applied to the analysis of biological materials.
Daily administration of 300mg/kg of latamoxef to rats fed a vitamin K deficient diet from 7 days to 9 days induced severe hypoprothrombinemia. The extrinsic and intrinsic coagulation activities in the latamoxef treated rats were decreased in higher degree than those in the rats fed a vitamin K deficient diet alone. Single oral administration of 0.1 or 1mg/kg of vitamin K_2 to the hypoprothrombinemic rats improved their coagulation activity. The coagulation activity was normalized completely by oral administration of 0.1mg/kg vitamin K_2 once a day for 2 days. These results suggest that latamoxef aggravates vitamin K deficiency and causes severe hypoprothrombinemia.
The presence of an enzyme which cleaves quinolinothiamin in a manner similar to the thiaminase II has been demonstrated in extracts of Euglena gracilis z, an unicellular eukaryote. The enzyme is considered to be localized in the mitochondrial membrane of this species. The pH optimum of the enzyme was shown to be around 9.5 and the optimum temperature was near 38℃. The Km value as calculated by the method of Lineweaver and Burk was found to be 1.7μM for quinolinothiamin. The splitting-activity towards quinolinothiamin was inhibited by 2-methyl-4-amino-5-hydroxymethyl pyrimidine (OMP), the pyrimidine moiety of thiamin. The enzyme activity in cells grown in a thiamin-limiting medium was higher than that in cells grown in a thiamin-rich medium. This might account for the fact that the requirement of this organism for OMP can be replaced by either pyrithiamine or quinolinothiamin. The enzyme appears to be involved in the production of OMP through the hydrolytic cleavage of these compounds.
Vitamin K_4 (Acetomenaphthone ; K_4) is orally administrated in the treatment or prevention of vitamin K deficiency. A High-performance liquid chromatographic (HPLC) method is studied for determination of K_4 in aqueous vitamin preparations for animal use. Samples were diluted with ethanol and followed by HPLC on a Nucleosil _5C_18 column. The mobile phase was water-acetonitrile (48:52) and the monitor wave length in ultraviolet detection was 226nm. The recovery rate. SD and CV of K_4 were 100%, 1.4 and 1.4% respectivery. The detection limit was 1.3×10^<-4>μg of K_4. The K_4 content in the aq. vitamin preparations decreased during storage due to hydrolysis of K_4 to menadiol monoacetate.