Emi Miyamoto

Feeding dried purple laver (nori) to vitamin B12-deficient rats significantly improves vitamin B12 status.

To clarify the bioavailability of vitamin B12 in lyophylized purple laver (nori; Porphyra yezoensis), total vitamin B12 and vitamin B12 analogue contents in the laver were determined, and the effects of feeding the laver to vitamin B12-deficient rats were investigated. The amount of total vitamin B12 in the dried purple laver was estimated to be 54.5 and 58.6 (se 5.3 and 7.5 respectively) microg/100 g dry weight by Lactobacillus bioassay and chemiluminescent assay with hog intrinsic factor respectively. The purple laver contained five types of biologically active vitamin B12 compounds (cyano-, hydroxo-, sulfito-, adenosyl- and methylcobalamin), in which the vitamin B12 coezymes (adenosyl- and methylcobalamin) comprised about 60 % of the total vitamin B12. When 9-week-old vitamin B12-deficient rats, which excreted substantial amounts of methylmalonic acid (71.7(se 20.2) micromol/d) in urine, were fed the diet supplemented with dried purple laver (10 microg/kg diet) for 20 d, urinary methylmalonic acid excretion (as an index of vitamin B12 deficiency) became undetectable and hepatic vitamin B12 (especially adenosylcobalamin) levels were significantly increased. These results indicate that vitamin B12 in dried purple laver is bioavailable to rats.

Analysis of Vitamin B12 in Food by Silica Gel 60 TLC and Bioautography with Vitamin B12-Dependent Escherichia coli 215

Abstract To evaluate whether certain foods contain vitamin B12 or inactive corrinoids, a simple technique, bioautography with vitamin B12-dependent Escherichia coli mutant after separation of the sample by silica gel 60 thin-layer chromatography, is available. By using the method, vitamin B12-compounds found in some edible cyanobacteria are readily identified. This bioautography has great advantages (simplicity, speed, and inexpensiveness) for the analysis of vitamin B12-compounds in food.

Characterization of vitamin B12 compounds from Korean purple laver (Porphyra sp.) products.

Vitamin B(12) contents of various Korean purple laver products were determined with the microbiological vitamin B(12) assay method. Although a substantial amount (133.8 microg/100 g) of vitamin B(12) was found in dried purple laver, seasoned and toasted laver products contained lesser vitamin B(12) contents (about 51.7 microg/100 g). The decreased vitamin B(12) contents in the seasoned and toasted laver products, however, were not due to loss or destruction of vitamin B(12) during the toasting process. Silica gel 60 thin layer chromatography-bioautogram analysis indicated that all Korean laver products tested contain true vitamin B(12), but not inactive corrinoid compounds. In vitro gastrointestinal digestion experiments indicated that digestion rate of vitamin B(12) from the dried Korean purple laver was estimated to be 50% under pH 2.0 conditions (as a model of normal gastric function). These results suggest that Korean purple laver products would be excellent vitamin B(12) sources for humans, especially vegetarians.

Characterization of a Corrinoid Compound in the Edible (Blue-Green) Alga, Suizenji-nori

The edible blue-green alga (cyanobacterium), Suizenji-nori, contained 143.8±22.4 μg of vitamin B12 per 100 g dry weight of the alga (mean±SE, n=4). A corrinoid compound was purified from the dried Suizenji-nori, and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified corrinoid compound were not identical to those of true vitamin B12, but to those of pseudovitamin B12 which is inactive for humans.

Methyladeninylcobamide functions as the cofactor of methionine synthase in a Cyanobacterium, Spirulina platensis NIES-39

To clarify the physiological function of pseudovitamin B12 (or adeninylcobamide; AdeCba) in Spirulina platensis NIES‐39, cobalamin‐dependent methionine synthase (MS) was characterized. We cloned the full‐length Spirulina MS. The clone contained an open reading frame encoding a protein of 1183 amino acids with a molecular mass of 132 kDa. Deduced amino acid sequences of the Spirulina MS contained critical residues identical to cobalamin‐, zinc‐, S‐adenosylmethionine‐, and homocysteine‐binding motifs. The recombinant Spirulina enzyme showed higher affinity for methyladeninylcobamide than methylcobalamin as a cofactor. These results indicate that Spirulina cells can utilize AdeCba synthesized as the cofactor for MS.

Effect of Vitamin B12-Enriched Thraustochytrids on the Population Growth of Rotifers

Newly isolated thraustochytrids showed uptake of vitamin B12 from the culture into the cells. Cultivation of thraustochytrids in a medium containing 1 μg/ml of vitamin B12 greatly increased the contents of vitamin B12 in the cells. Similarly to Schizochytrium limacinum, odd numbered fatty acids decreased in the cells of new isolates cultivated with vitamin B12. Vitamin B12-enriched thraustochytrids, strain mh0186, enhanced the population growth of rotifers fed on the cells as sole feed.

TLC Analysis of a Corrinoid Compound from Dark Muscle of the Yellowfin Tuna (Thunnus albacares)

Abstract A significant amount of vitamin B12 (52.9±8.9 µg/100 g) was found in the dark muscle of the yellowfin tuna (Thunnus albacares), in comparison to that of the light muscle. A corrinoid compound was purified to homogeneity from the dark muscle and partially characterized. TLC and HPLC patterns of the purified corrinoid compound were identical to those of authentic vitamin B12. These results indicate that dark muscle of the yellowfin tuna would be an excellent source of vitamin B12 for humans and aminals.

Uptake and physiological function of vitamin B12 in a photosynthetic unicellular coccolithophorid alga, Pleurochrysis carterae.

The photosynthetic coccolithophoid alga, Pleurochrysis (Hymenomonas) carterae, could take up and accumulate exogenous vitamin B12, most of which was converted into the coenzyme forms of vitamin B12. Two vitamin B12-dependent enzyme activities (methylmalonyl-CoA mutase, 2.6±0.4 nmol/min/mg protein and methionine synthase, 85.1±38.9 pmol/ min/mg protein) could be found in a cell homogenate of the vitamin B12-supplemented alga. Most of the methylmalonyl-CoA mutase activity and 19.2% of the vitamin B12 accumulated by the algal cells were recovered in the macromolecular fractions with Mr of 150 kDa, although the remaining vitamin B12 was found only in free vitamin B12 fractions.

Purification and Characterization of Corrinoid Compounds from a Japanese Fish Sauce

Abstract A Japanese fish sauce “Ishiru,” which was made from squid by a traditional food manufacturing process, contained the highest amounts (5.5 ± 2.3 µg/100 g) of B12 among various fish sauces tested. Two corrinoid compounds were purified from the fish sauce Ishiru and partially characterized. TLC and HPLC patterns of the main red‐colored compound, purified from the fish sauce, were identical to those of authentic vitamin B12, but minor compounds could not be identified. Fish sauce may not be suitable for use as a good vitamin B12 source, judging from the low daily intake of the sauce and occurrence of the unknown corrinoid‐compound.

TLC SEPARATION AND ANALYSIS OF VITAMIN B12 AND RELATED COMPOUNDS IN FOOD

ABSTRACT To evaluate why differences between the vitamin B12 contents determined by both microbiological and intrinsic factor-chemi-luminescence B12 assay methods occur in some edible shellfish and algal foods, or how much loss of B12 occurs in food during microwave heating, some B12-compounds and their degradation products formed during microwave heating were purified and characterized using silica gel 60 thin layer chromatography. Although dried green and purple lavers (nori), some algal health foods, and most shellfish contained considerable amounts of true B12, pseudovitamin B12, an inactive B12-compound, predominated in spirulina tablets. Significant loss of B12 occurred in foods during microwave heating due to the conversion of B12 to inactive B12 degradation products. These results indicate that thin-layer chromatography has great advantages (simplicity, flexibility, speed, and relative inexpensiveness) for the separation and analysis of B12 compounds in foods.