A new method of preparing radiolabeled cobalamin derivatives has been developed. The method involves the use of cobalamin-tri-n-butylstannyl hippurate conjugates as intermediates to obtain radioiodinated cobalamin-iodohippurate conjugates. The arylstannyl functionality was used as an exchangeable group to obtain high specific activity radioiodinations and to circumvent some deleterious side reactions common to cobalamins under electrophilic iodination conditions. The first step in the synthesis of tri-n--butylstannyl hippurate conjugates was to obtain free carboxylate groups on the cobalamin moiety. This was accomplished by mild acid hydrolysis of the b-, d-, or e-propionamide side chains on the corrin ring, followed by careful separation of the isomeric products. The second step was to couple a linking molecule (diaminododecane) to the carboxylate. The final step was to conjugate p-tri-n-butylstannyl hippurate to the cobalamin-diaminododecane adduct. All three isomeric cobalamin-p-tri-n-butylstannyl hippurate conjugates were prepared, as were the corresponding cobalamin-p-iodohippurate conjugates (HPLC standards). Radioiodination reactions were conducted with N-chlorosuccinimide and Na[*I]I in Me OH using conditions previously developed for arylstannylations. However, unlike the previous reactions, a key factor in obtaining the desired radioiodinated cobalamins was that the reaction be conducted under neutral conditions. Isolated yields of 40-65% were obtained for all three cobalamin isomers. Specific activities of 10-33% theoretical were obtained for the radioiodinated cobalamins. Evaluation of competitive binding of (nonradioactive) cobalamin-iodohippurate conjugates with recombinant human transcobalamin II showed that the e-isomer bound nearly as well as [57Co]cyanocobalamin (50%), whereas the b-isomer had decreased binding (6%) and the d-isomer was significantly decreased in its binding (0.7%). Two biodistributions of the radioiodinated e-isomer were conducted in athymic mice. One biodistribution investigated tissue localization in mice bearing a renal cell carcinoma xenograft, and the other biodistribution investigated tissue localization when the radioiodinated cyanocobalamin was mixed with 1% BSA prior to injection. A comparison of the results of the two biodistributions and a discussion of how they relate to previous [57/60Co]cyanocobalamin biodistributions are provided.

Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by receptor-mediated endocytosis. In genetic disorders that are characterized by congenital deficiency of TCII, intracellular Cbl deficiency occurs, resulting in an early onset of megaloblastic anemia that is sometimes accompanied by a neurologic disorder. To define the genetic basis for TCII deficiency, we have cloned and characterized the human gene that encodes this protein. The gene spans a minimum of 18 kbp and contains nine exons and eight introns, with a polyadenylation signal sequence located 509 bp downstream from the termination codon and a transcription initiation site beginning 158 bp upstream from the ATG translation start site. The 5′ flanking DNA does not have a TATA or CCAAT regulatory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5′-CCCC-3′ tetramers. This sequence is a motif for a trans-active transcription factor (ETF) that regulates expression of the epidermal growth factor receptor gene (EGFR), which also lacks TATA and CCAAT regulatory elements. A GC-rich sequence that binds the SP1 protein is located 356 nucleotides upstream from the first of the series of CCCC tetramers. Although this GC sequence is at an unusual location with respect to the CAP site, a 507-bp fragment containing this GC box drives the chloramphenicol acetyltransferase (CAT) reporter gene after transient transfection into NIH 3T3 cells. No CAT activity was observed when a 420-bp fragment lacking this GC box but containing the ETF-binding domains was similarly transfected into this cell line. One consensus and two atypical motifs for the c-myc ligand are located downstream and upstream, respectively, of the GC box, and this could explain the elevated plasma TCII observed in some patients with multiple myeloma, as the c-myc product is overexpressed in some myeloma cells. Restriction endonuclease digestion of genomic DNA from eight normal subjects with Taq I, Hinfl, Msp I, and Bgl I identified three patterns of restriction fragment length polymorphism (RFLP). A number of the exon/intron splice junctions of human TCII, TCI, and IF genes are located in homologous regions of these proteins, providing evidence that these genes have evolved by duplication of an ancestral gene. This characterization of the TCII gene and the RFLP should facilitate the identification of the mutation(s) responsible for the genetic abnormalities of TCII expression.

This report describes an investigation aimed at preparation of radioiodinated cyanocobalamin (CN-Cbl) monomers and dimers with improved water solubility and decreased nonspecific binding. In the investigation, synthesis and radioiodination reactions of one monomeric and two dimeric CN-Cbl derivatives were conducted. The initial step in the synthesis of the CN-Cbl derivatives was mild acid hydrolysis of CN-Cbl, 1, followed by separation of the resultant corrin ring b-, d-, and e-monocarboxylate isomers. The investigation was limited to preparation of conjugates of CN-Cbl-e-carboxylate, 2, as earlier studies had shown binding of that isomer with recombinant human transcobalamin II (rhTCII) was similar to CN-Cbl. In a second synthetic step, the hydrophilic linker moiety, 4,7,10-trioxa-1,13-tridecandiamine, 3, was conjugated with 2 to form the adduct, 4. The synthesis of a monomeric CN-Cbl derivative, 6a, which can be used for radioiodination, was accomplished by reaction of 4 with p-tri-n-butylstannylbenzoate tetrafluorophenyl (TFP) ester, 5a. Two CN-Cbl dimers containing the arylstannane radioiodination moiety were also synthesized. The first dimer, 8a, was synthesized by cross-linking 4 with a stannylbenzoyl-aminoisophthalate di-TFP ester, 7a. The second dimer, 11a, was synthesized by reacting benzene tricarboxylate tri-TFP ester, 10, in a stepwise manner with 1 equiv of the adduct of 5a and 3 (forming 9a), followed by 2 equiv of 4. Iodobenzoate HPLC standards, 6b, 8b, and 11b, used in the radioiodination studies, were prepared in a manner similar to that of the stannylbenzoate derivatives. Radioiodinations were performed by reacting 6a, 8a, or 11a with N-chlorosuccinimide and Na[(125)I]I in methanol under neutral conditions. Radiochemical yields of 17-42% were obtained. Evaluation of the binding properties of radiolabeled CN-Cbl conjugates with rhTCII showed that the dimer of CN-Cbl, 11b, bound more avidly than the monomer, 6b, and that the binding affinity of the dimer is essentially equivalent to that of unmodified CN-Cbl. Incubation of radioiodinated monomer, [(125)I]6b, and dimer, [(125)I]11b, with rhTCII followed by size-exclusion chromatographic analysis provided data that the monomer bound one rhTCII molecule whereas two rhTCII molecules were bound to approximately 30% of the dimer.

Transcobalamin II (TCII) is a cobalamin (Cbl, vitamin B12)-binding protein in mammalian plasma that facilitates the cellular uptake of the vitamin. To obtain human TCII in sufficient quantity for analytical studies, the complementary DNA (cDNA) encoding TCII was inserted into the plasmid PVL 1393, and the baculovirus expressing TCII was obtained by homologous recombination in Spodoptera frugiperda (SF9) insect cells by cotransfection with the wildtype virus. Under optimized conditions, SF9 cells infected with the recombinant virus secreted 2 to 4 micrograms of TCII per milliliter of culture medium. TCII did not accumulate in the SF9 cells and seemed to be constitutively secreted as observed previously in cultured human endothelial cells. The purified recombinant TCII has the same molecular weight by SDS-PAGE as purified human TCII. The recombinant TCII cross-reacts with an antiserum to native human TCII, binds Cbl and facilitates the uptake of Cbl in eukaryotic cells by binding to the receptor for TCII-Cbl on the plasma membrane of K562 cells. Amino acid sequence analysis of the purified recombinant TCII identified two polypeptides, one identical to the amino acid sequence deduced from the cDNA and a second lacking the first and second N-terminal residues. These sequences are identical to two TCII polypeptides purified from Cohn fraction III of pooled human plasma. The two forms of recombinant TCII have the same isoelectric points as the two predominant isoprotein forms of TCII in human serum. Since the baculovirus construct contains a single cDNA that can encode only one amino acid sequence, the two isoproteins in recombinant TCII must be generated by a mechanism other than allele specific expression. A plausible mechanism for generating isoproteins of nonglycosylated peptides, such as TCII, may be by splicing of the leader peptide at alternative sites.

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The uptake of 57Co-cyanocobalamin (CN-Cbl) and its conversion to 5- deoxyadenosylcobalamin (Ado-Cbl), methylcobalamin (Me-Cbl), and hydroxocobalamin (OH-Cbl) has been studied in phytohemagglutinin (PHA)- transformed lymphocytes from normal subjects and patients with patients with pernicious anemia. Uptake and conversion were much greater by PHA- stimulated lymphocytes than by mature non-transformed lymphocytes. In normal cells, uptake of 57Co-CN-Cbl and synthesis of the cobalamin coenzymes were approximately linear between 3 and 48 hr incubation. Ado- Cbl was the major cobalamin formed, and after 72 hr the cells contained about twice as much Ado-Cbl as Me-Cbl. Uptake by lymphocytes from patients with untreated pernicious anemia (PA) was greater than that by normal lymphocytes, but the proportions of Ado-Cbl and Me-Cbl synthesized by each were similar. Folic acid and methyltetrahydrofolate enhanced synthesis of Me-Cbl both in normal and in PA cells, while methotrexate and 5-fluorouracil depressed it. This depression was overcome by 5-formyltetrahydrofolate, suggesting that an uninterrupted folate cycle may play an important role in Me-Cbl synthesis.