Study of the compatibility of oral magnesium oxide preparations sold in Japan with the ICH-Q3D guideline for elemental impurities.

Magnesium oxide has been widely used as an antacid and constipation remedy. Currently in Japan, magnesium oxide preparations manufactured by five medical companies are marketed as prescribed generic drugs. In this study, we focused on metal elemental impurities present in 330 mg magnesium oxide tablets manufactured by each of these companies. The content of such impurities was determined by atomic absorption spectrometry and inductively coupled plasma mass spectrometry. We confirmed whether the content conformed to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Guideline for Elemental Impurities (ICH-Q3D) based on the 30% control threshold. The content of these impurities varied among the five products (preparations A-E), but in all cases met the oral permitted daily exposure (PDE) criteria stipulated in ICH-Q3D. In 5 lots of preparation C and all lots of preparation D, the equivalent cadmium (Cd) intake for a daily maximum dosage of 2 g was higher than the 30% control threshold of 1.5 µg/day. By cluster analysis, preparations A-E were classified into preparations A + B and C + D + E and/or preparations A + B, C + D and E. The present study showed that all 5 preparations sold in Japan meet the PDE value standard of ICH-Q3D, and that preparations A and B meet the 30% control threshold. It is important that for preparations failing to meet the criteria, further improvements need to be sought, and impurities in magnesium oxide preparations need to be monitored to ensure their safety.

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ-AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)(1).

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ-aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the (13) C-enrichment ratios ((13) C-ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l-[3-(13) C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%-67%, compared with that in our previous d-[1-(13) C]glucose feeding experiment (Iida et al. 2002). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C-13(2) of chl a was labeled with the (13) C-carbon of l-[methyl-(13) C]methionine derived from l-[3-(13) C]alanine via [2-(13) C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l-[3-(13) C]serine. The phytyl moiety of chl a was also labeled on C-P2, C-P3(1) , C-P4, C-P6, C-P7(1) , C-P8, C-P10, C-P11(1) , C-P12, C-P14, C-P15(1) , and C-P16 from (13) C-isoprene (2-[1,2-methyl,3-(13) C3 ]methyl-1,3-butadiene) generated from l-[3-(13) C]alanine via [2-(13) C]acetyl CoA.

Metabolic pathways leading from amino acids to vitamin B12 in Propionibacterium shermanii, and the sources of the seven methyl carbons.

The metabolic pathways leading from l-[2-13C]aspartic acid, [2-13C]glycine and l-[methyl-13C]methionine to vitamin B12 were investigated, focusing on the biosynthetic pathways leading to the aminopropanol moiety of vitamin B12 and on the role of the Shemin pathway leading to delta-aminolevulinic acid (a biosynthetic intermediate of tetrapyrrole), by means of feeding experiments with Propionibacterium shermanii in combination with 13C-NMR spectroscopy. The 13C-methylene carbons of l-[2-(13)C]aspartic acid, which is transformed to [2-13C]glycine via l-[2-13C]threonine, and [2-13C]glycine added to the culture medium served mainly to enrich the seven methyl carbons of the corrin ring through C-methylation by S-adenosyl-l-[methyl-13C]methionine derived from catabolically generated l-[methyl-13C]methionine in the presence of tetrahydrofolic acid. The results indicate that the catabolism of these amino acids predominates over pathways leading to (2R)-1-amino-2-propanol or delta-aminolevulinic acid in P. shermanii. Feeding of l-[methyl-13C]methionine efficiently enriched all seven methyl carbons. In the cases of [2-13C]glycine and l-[methyl-13C]methionine, the 13C-enrichment ratio of the methyl carbon at C-25 (the site of the first C-methylation) was less than those of the other six methyl carbons, probably due to the influence of endogenous d-glucose in P. shermanii. The almost identical 13C-enrichment ratios of the other six methyl carbons indicated that these C-methylations during vitamin B12 biosynthesis were completed before the amino acids were completely consumed. However, in the case of l-[2-13C]aspartic acid, the 13C-enrichment ratios of five methyl carbons were low and similar, whereas the last two sites of C-methylation (C-53 and C-35) were not labeled, presumably because of complete consumption of the smaller amount of added label. The ratios of 13C-incorporation into the seven methyl carbons are influenced by the conditions of amino acid feeding experiments in a manner that is dependent upon the order of C-methylation in the corrin ring of vitamin B12.

Identification of tetrapyrrole compounds excreted by Rhodobacter sphaeroides and sources of the methyl hydrogens of bacteriochlorophyll a biosynthesized by R. sphaeroides, based on 13C-NMR spectral analysis of coproporphyrin III tetramethyl ester.

Red-fluorescent tetrapyrrole compounds excreted by Rhodobacter sphaeroides into the culture broth were concluded to be coproporphyrinogen (Copro'gen) III and uroporphyrinogen (Uro'gen) I, based on the (13)C-NMR spectral identification of coproporphyrin (Copro) III tetramethyl ester and uroproporphyrin (Uro) I octamethyl ester. The sources of the methyl hydrogens of bacteriochlorophyll a were established by analysis of the (13)C-NMR spectra of (2)H,(13)C-Copro III tetramethyl ester chemically derived from (2)H,(13)C-Copro'gen III biosynthesized through the feeding of delta-amino[2-(13)C]levulinic acid (ALA) to R. sphaeroides in medium containing 50% (2)H(2)O. We confirmed the previous finding that one of the methyl hydrogens was derived from water in the medium during decarboxylation of four acetyl side chains of Uro'gen III to generate Copro'gen III. It was further shown that the other hydrogen atoms, previously reported to be derived from methylene hydrogens at C-2 of ALA, had been exchanged with hydrogen of water in the medium in the biosynthetic pathways leading from ALA to Copro'gen III.

Mechanism of the ring contraction process in vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii under aerobic conditions.

The mechanism of the ring contraction process during vitamin B(12) biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with delta-amino[1-(13)C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and delta-amino[1-(13)C,1,1,4-(18)O(3)]levulinic acid in combination with (13)C-NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin-3x from formation of the gamma-lactone from the ring A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ring D by cleavage of the carbon-oxygen bond at C1 of precorrin-3x) in the aerobe Pseudomonas denitrificans was not seen in P. shermanii under aerobic conditions, and the mechanism of the ring contraction process in P. shermanii was the same irrespective of the presence or absence of oxygen.

Evaluation of two biosynthetic pathways to delta-aminolevulinic acid in Euglena gracilis.

delta-Aminolevulinic acid (ALA), which is an intermediate in the biosynthesis of chlorophyll a, can be biosynthesized via the C5 pathway and the Shemin pathway in Euglena gracilis. Analysis of the (13)C-NMR spectrum of (13)C-labeled methyl pheophorbide a, derived from 13C-labeled chlorophyll a biosynthesized from d-[1-(13)C]glucose by E. gracilis, provided evidence suggesting that ALA incorporated in the (13)C-labeled chlorophyll a was synthesized via both the C5 pathway and the Shemin pathway in a ratio of between 1.5 and 1.7 to one. The methoxyl carbon of the methoxycarbonyl group at C-132 of chlorophyll a was labeled with (13)C. The phytyl moiety of chlorophyll a was labeled on C-P2, C-P3(1), C-P4, C-P6, C-P7(1), C-P8, C-P10, C-P11(1), C-P12, C-P14, C-P15(1) and C-P16.