Anal. Chem. 1093, 65, 857-059
A Radioisotope Dilution Method for Quantltatlon of Total Vitamin 8 1 2 In Biological Samples Using Isolated Euglena Pellicle Fragments as a Solid-Phase Vitamin Bl2-Blndlng Material Fumio Watanabe,'l+ Yoshihisa NakanoJ Erhard Stupperieh,s Kenji Ushikoshi,l Setuo Ushikoshi,l Ikuo Ushikoshi,l and Shozaburo Kitaoka' Laboratory of Nutrition and Food Science, Hagoromo-gakuen College, 1-89-1 Hamadera-minamimachi, Sakai, Osaka 592, Japan, Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 591, Japan, Abteilung Angewandte Mikrobiologei, Universitat Ulm, Albert-Einstein-Allee 11, 0-7900,F.R.G., and Ushikoshi Institute for Physiology, Sakura, Chiba 285, Japan
INTRODUCTION Euglenagracilis z is one of the most suitable organisms for microbiological assay of vitamin B12, cobalamin (Cbl), and found the widest application,1p2but the microbiological assay has been replaced by radioisotope dilution assay (RIDA) because the microbiological assay is technically difficult and several kits for the RIDA are commercially available. All the commercial assay kits use hog intrinsic factor (IF) because IF is the most specific mammalian Cbl-binding protein. In all the commercial assay kits, IF that was purified from hog gastric juice by Cbl-affinity chromatography and then covalently coupled t o polymer beads is used. Thus, the commercial assay kits are fairly expensive. The Euglena pellicle (a cell membrane complex) fragments isolated by a stepwise sucrosedensity gradient centrifugation have Cbl-binding activity,since Cbl-binding protein involved in the Cbl uptake system is embedded in the pellicle fragments.3 In this paper, we have devised a RIDA method for quantitation of total Cbl using isolated Euglena pellicle fragments aa a solid-phase Cbl-binding material and also discuss the availability of this method in assaying human serum Cbl content.
EXPERIMENTAL SECTION Preparation of Euglena Pellicle Fragments. E. gracilis z was cultured for 5 days at 27 "C with illumination (2000 lux) in Koren-Hutner m e d i ~ m .We ~ used a Cbl-deficient (50 ng/L of medium) medium for preparation of Euglena pellicle fragments. Euglena pelliclefragmentswere prepared by the modified method of Nakano et al.5 The Euglena cells grown for 5 days in the Cbl-deficient medium were collected by centrifugation at 2000g for 10 min, washed twice with water, and suspended with 10 volumesof 0.2 M potassiumphosphate buffer, pH 7.2, containing 0.3 M sucrose. The suspended cells were disrupted by sonic oscillation in an ice bath and then centrifuged at 2000g for 30 min, a pellet containing paramylon and pellicle fragments being obtained. The pellet was suspended with 10volumes of the above buffer, applied to a stepwise density gradient of 1.9,1.4, and 1.0 M sucrose in the same buffer, and then centrifuged at loooOg for 30 min. The fraction banding between 1.9 and 1.4 M sucrose, which contained most of the pellicle,was collected and suspended with the same buffer. The size of the pellicle fragments obtained by this method has been reported to be 0.5-2.0 pm.5 The fraction of the pellicle fragments was stored at -20 "C for experiments. RIDA for Total Cbl in the Biological Sample. Total Cbl was extracted from human serum by the method of boiling with
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* Corresponding author. +
KCN in acid P H . ~The Cbl assay with the Euglena pellicle fragments was done as follows: the assay mixture (1.0 mL) contained 10 mM sodium phosphate buffer, pH 7.2, 1 nM 57Co-CN-Cbl(15 Ci/mmol; Amersham International PLC, England), the isolated pellicle fragments (36 pg of protein), and a sample or standard cyanocobalamin (CN-Cbl). Cbl content was calibrated with various amounts (100-3000 pg/mL) of standard CN-Cbl which was added to the mixture. The mixture, after incubation for 2 min at 25 "C in the dark, was immediatelyfiltered through a cellulose acetate membrane filter (0.45 pm, 25-mm diameter) and washed three times with 5 mL of 10 mM sodium phosphate buffer, pH 7.2, containing 0.1 M NaC1. The dried membrane filter was cut into small pieces and then placed in a sample tube (12 X 75 mm). Radioactivity on the membrane filter was counted for 5 min with a y radiation counter (Packard Model No. B5003, Cobra Auto-gamma;the detector consists of a thallium-activated, sodium iodide crystal that is optically coupled to a photomultiplier and is surrounded by a minimum of 3 in. of lead shielding). The radioactivity was expressed as counts per minute. The amount of nonspecific binding of Cbl to the cellulose acetate membrane filter (blank) was estimated to be
