Vitamin B₁₂ Carboxylic Acid Characterization in the Viscera Edible Portions of Lobster, Crayfish, Crab, and Shrimp

Abstract

The vitamin B 12 content of lobster, crayfish, crab, and shrimp viscera was determined using a microbiological assay based on Lactobacillus delbrueckii subsp. lactis ATCC7830. These viscera contained high vitamin B 12 levels (7.2−118.6 μg/100 g of wet weight). We purified vitamin B 12 compounds from the extracts of these viscera using an immunoaffinity column. Corrinoid compounds were identified using liquid chromatography-electrospray ionization tandem mass spectrometry. The viscera tested contained vitamin B 12 -b-, -d-, and -e-monocarboxylic acids and vitamin B 12 -be-dicarboxylic acid, modified with peripheral propionamide side chains of the corrin ring. These vitamin B 12 carboxylic acids are specifically located in the viscera edible portions of lobster, crayfish, crab, and shrimp.

Characterization of Vitamin B12 Compounds in Commercially Available Livestock Livers Used as Foods

“…The MS/MS spectrum of peak d indicated that the singly charged ion of m / z 359.10 was predominantly formed due to the B 12 nucleotide moiety (Figure C). These results were identical to MS and MS/MS data of authentic B 12 as described previously . Minor peaks with retention times of 4.5 min (peak 1), 4.7 min (peak 2), 5.0 min (peak 3), and 6.8 min (peak 5) corresponded to ion peaks at m / z 672.3 (peak a, factor III), m / z 679.8 (peak b, factor A), m / z 672.8 (peak c, pseudo-B 12 ), and m / z 695.8 (peak e, factor S), respectively; the remaining absorbance peaks with retention times of 7.4 min (peak 6), 8.6 min (peak 7), 10.0 min (peak 8), and 11.6 min (peak 9) corresponded to the retention times of ion peaks f-1, f-2, f-3, and f-4 at m / z 678.8.…”

“…Similarly, a considerable proportion of putative B 12 monocarboxylic acids, including B 12 - b -monocarboxylic acid (approximately 2%), B 12 - d -monocarboxylic acid (approximately 3%), and unknown B 12 monocarboxylic acids (approximately 1%), were found in chicken and cow livers. It was reported that shrimp viscera contained a substantial amount of B 12 monocarboxylic acids, which might be an intermediate compound of B 12 biosynthesis in bacteria living in the environment in which shrimps live . However, we do not have further information on why various B 12 monocarboxylic acids occur in such livestock livers.…”

“…Thus, to clarify these minor B 12 compounds, liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) analysis was conducted. Figure A shows the total ion (TIC) and selected ion monitoring chromatogram (factor III m / z 672.3; factor A m / z 679.8; pseudo-B 12 m / z 672.8; B 12 m / z 678.3; factor S m / z 695.8; B 12 monocarboxylic acids m / z 678.8) of naturally occurring corrinoid compounds − of B 12 compounds purified from cow liver extracts. The B 12 compounds were eluted as nine peaks: one major peak (approximately 90% the sum of the detected nine peak areas) with a retention time of 5.8 min (peak 4) and eight minor peaks with retention times of 4.5 min (peak 1), 4.7 min (peak 2), 5.0 min (peak 3), 6.8 min (peak 5), 7.4 min (peak 6), 8.6 min (peak 7), 10.0 min (peak 8), and 11.6 min (peak 9), during monitoring of the absorbance at 361 nm.…”

“…B 12 compounds were purified from the solution using an immunoaffinity column (EASI-EXTRACT B 12 P80; φ 8.0 mm × 60 mm, R-Biopharm, Darmstadt, Germany), according to the manufacturer’s protocol. The eluate was evaporated to dryness under reduced pressure, dissolved in 100 μL of Milli-Q water, and used as an HPLC sample, as described previously . The HPLC apparatus (SPD-10AV UV–vis detector, SCL-10A VP system controller, DGU-20A 3 degasser, LC-10Ai liquid chromatograph, CTO-20A column oven) and a reversed-phase HPLC column (Wakosil-II 5C18RS, φ 4.6 mm × 150 mm; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) were used.…”

“…The eluate was evaporated to dryness under reduced pressure, dissolved in 100 μL of Milli-Q water, and used as an HPLC sample, as described previously. 15 The HPLC apparatus (SPD-10AV UV−vis detector, SCL-10A VP system controller, DGU-20A 3 degasser, LC-10Ai liquid chromatograph, CTO-20A column oven) and a reversed-phase HPLC column (Wakosil-II 5C18RS, φ 4.6 mm × 150 mm; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) were used. The B 12 compounds were isocratically eluted with 20% (v/v) methanol containing 1% (v/v) ■ RESULTS AND DISCUSSION Determination of the B 12 Content in Cow, Chicken, and Pig Livers Using HPLC.…”