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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.

Several cobalamin (Cbl) dimers have been prepared for evaluation as potential antiproliferative agents in the treatment of AIDS-related lymphoma. The Cbl dimers were synthesized by cross-linking Cbl carboxylates, produced by acid hydrolysis of the b-, d-, and e-propionamide side chains of cyanocobalamin (CN-Cbl), through an isophthalate molecule. Linking molecules were used between the Cbl carboxylates and the isophthalate moiety. The linkers were incorporated to provide a distance between the two Cbl molecules such that the dimeric Cbls might bind two molecules of transcobalamin II (TCII), the Cbl transport protein in plasma. Initially, the linking moiety used was 1,12-diaminododecane, but the resulting dimers had low aqueous solubility. To improve the solubility of the dimers, 4,7,10-trioxa-1,13-tridecanediamine was employed as the linking moiety. This improved the water solubility of the dimers considerably, while retaining the distance between the Cbl molecules at 41-42 A (fully extended). To introduce additional substitution on Cbl dimers, 5-aminoisophthalic acid was used as the cross-linking reagent. p-Iodobenzoyl and p-(tri-n-butylstannyl)benzoyl conjugates of 5-aminoisophthalate were synthesized and used to prepare Cbl dimers. The stannylbenzoyl-conjugated Cbl dimers were prepared as precursors to be used in radioiodination reactions, and the iodobenzoyl-conjugated Cbl dimers were prepared as HPLC standards for the radioiodinated product. Attempts to iodinate/radioiodinate the stannylbenzoyl Cbl dimers were unsuccessful. Although an explanation for this is not readily apparent, the failure to react may be due to the lipophilicity of the linker used and the steric environment of the two Cbl moieties. A biotinylated derivative of 5-aminoisophthalate was also synthesized and used to prepare biotinylated-Cbl dimers. In a competitive rhTCII binding assay with [57Co]CN-Cbl, Cbl dimers containing the lipophilic diaminododecane linking moiety had decreased binding avidities compared to those of Cbl monomers substituted at the same corrin ring carboxylate. However, Cbl dimers containing the water-solubilizing trioxadiamine linker appeared to have avidities similar to those of the Cbl monomers.

Several cobalamin (Cbl) dimers have been prepared for evaluation as potential antiproliferative agents in the treatment of AIDS-related lymphoma. The Cbl dimers were synthesized by cross-linking Cbl carboxylates, produced by acid hydrolysis of the b-, d-, and e-propionamide side chains of cyanocobalamin (CN-Cbl), through an isophthalate molecule. Linking molecules were used between the Cbl carboxylates and the isophthalate moiety. The linkers were incorporated to provide a distance between the two Cbl molecules such that the dimeric Cbls might bind two molecules of transcobalamin II (TCII), the Cbl transport protein in plasma. Initially, the linking moiety used was 1,12-diaminododecane, but the resulting dimers had low aqueous solubility. To improve the solubility of the dimers, 4,7,10-trioxa-1,13-tridecanediamine was employed as the linking moiety. This improved the water solubility of the dimers considerably, while retaining the distance between the Cbl molecules at 41-42 A (fully extended). To introduce additional substitution on Cbl dimers, 5-aminoisophthalic acid was used as the cross-linking reagent. p-Iodobenzoyl and p-(tri-n-butylstannyl)benzoyl conjugates of 5-aminoisophthalate were synthesized and used to prepare Cbl dimers. The stannylbenzoyl-conjugated Cbl dimers were prepared as precursors to be used in radioiodination reactions, and the iodobenzoyl-conjugated Cbl dimers were prepared as HPLC standards for the radioiodinated product. Attempts to iodinate/radioiodinate the stannylbenzoyl Cbl dimers were unsuccessful. Although an explanation for this is not readily apparent, the failure to react may be due to the lipophilicity of the linker used and the steric environment of the two Cbl moieties. A biotinylated derivative of 5-aminoisophthalate was also synthesized and used to prepare biotinylated-Cbl dimers. In a competitive rhTCII binding assay with [57Co]CN-Cbl, Cbl dimers containing the lipophilic diaminododecane linking moiety had decreased binding avidities compared to those of Cbl monomers substituted at the same corrin ring carboxylate. However, Cbl dimers containing the water-solubilizing trioxadiamine linker appeared to have avidities similar to those of the Cbl monomers.

The plasma protein transcobalamin II (TCII) binds and delivers cobalamin (Cbl; vitamin B12) to all cells, which internalize the TCII/Cbl complex by receptor-mediated endocytosis. Congenital deficiency of TCII results in intracellular Cbl deficiency, one effect of which is to disrupt DNA synthesis, leading to megaloblastic anemia. We report here an in vitro culture system in which cell growth is dependent on delivery of Cbl to cells by TCII. Recombinant human holo-TCII was shown to support in dose-dependent manner the growth of the human erythroleukemic cell line K562 and the murine lymphoma cell line BW5147. Free Cbl also supported cell growth; however, at 100- to 1,000-fold higher concentrations than those effective in the presence of apo-TCII. To determine if cellular depletion of Cbl could be achieved by interfering with interactions between TCII/Cbl and its cell-surface receptor, several monoclonal antibodies raised against human TCII were studied. Three antibodies, found to compete for the same binding site on TCII, proved to be effective inhibitors of TCII/Cbl-dependent cell growth. Our results suggest that monoclonal anti-TCII antibodies that block the function of this protein may prove useful in antitumor therapies.

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 is a plasma protein that binds vitamin B12 (cobalamin) as it is absorbed in the terminal ileum and distributes it to tissues. The circulating transcobalamin II-cobalamin complex binds to receptors on the plasma membrane of tissue cells and is then internalized by receptor-mediated endocytosis. A number of genetic abnormalities are characterized either by a failure to express transcobalamin II or by synthesis of an abnormal protein. These disorders result in cellular cobalamin deficiency and megaloblastic anaemia. In this chapter we review the structural and functional properties of transcobalamin II, the receptor for the transcobalamin-cobalamin complex and the clinical disorders that are associated with perturbation of circulating transcobalamin II. In addition, we provide emerging data about the molecular genetics of transcobalamin II which has emanated from our own and other laboratories.

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.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene.

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.

1. The B-vitamin status of fifty-nine patients, mainly from the lower socio-economic classes in Bombay, with a history of chronic malnutrition, and of alcoholism of 1·5–20 years’ duration, was studied before and during treatment, and in relation to their clinical, especially neurological, condition. These patients were divided into two neurological categories: (1) those with peripheral neuropathy (mainly sensory and distal) alone, (2) those with mental changes (mainly confusion and disorientation) also. Both categories frequently showed pellagrous pigmentation and mucocutaneous signs of B-vitamin deficiency.2. Thiamin and erythrocyte transketolase (EC2.2.1.1) activity, riboflavin, nicotinic acid, pantothenic acid, total and pyridoxal fraction of vitamin B6, folate and total vitamin B12were estimated in the blood and the cerebrospinal fluid (CSF) of these patients, and also in the blood of sixty-nine control subjects and in the CSF of some of them. The concentrations of all the vitamins, except vitamin B12, were highly significantly lower in the blood of patients of category 1 compared to the controls, and erythrocyte transketolase activity and pyridoxal concentration in patients of category 2 were significantly lower than those of category 1 patients. Blood pantothenic acid and folate concentrations were reduced less consistently.3. Serum vitamin B12concentration was significantly increased (though within normal range) in the patients compared to the control group, probably because of the moderate hepatic insufficiency (as assessed by liver function tests) in the former.4. The concentrations of thiamin, riboflavin, nicotinic acid and total vitamin B6were also highly significantly lower in the CSF in patients of category 1 compared with controls. Furthermore, thiamin, nicotinic acid and total vitamin B6concentrations were significantly lower in patients of category 2 than those of category 1 patients, indicating that CSF levels reflect better the neurological status of these patients.5. There was a moderate increase in the blood concentration of all the vitamins tested, after a relatively poor hospital diet alone. There was a concurrent increase in the blood levels of thiamin, riboflavin, nicotinic acid and pantothenic acid after parenteral treatment with either thiamin or nicotinic acid. The administration of pyridoxine resulted in a significant increase in the blood levels of riboflavin and the pyridoxal fraction of vitamin B6.

Transcobalamin II (TCII) has been purified from Cohn fraction III of human plasma by batchwise binding to and then elution from carboxymethyl-Sephadex, affinity chromatography using photo-labile aminopropyl cobalamin coupled to activated Sephacryl S-200, and finally chromatography through carboxymethyl cellulose. The yield was approximately 80%. The addition of protease inhibitors in all steps of the purification procedure and extensive washing of the carboxymethyl-Sephadex prior to eluting the TCII minimized degradation of the protein and the final preparation of holo-TCII contained 1 mol of cobalamin/mol of protein. A single polypeptide of 43,000 daltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 19 amino acids have been determined for human TCII. 12 of the amino acids are homologous with rabbit TCII and six are homologous with human R-binder, but there is no homology with human intrinsic factor.

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.

Transcobalamin II (TCII) is a cobalamin (Cbl) binding protein in the plasma that mediates the cellular uptake of Cbl. Although the synthesis of TCII by a variety of cultured mammalian cells and by some isolated perfused organs has been reported, no single tissue has been identified as the source of TCII in vivo. In this study, we demonstrate that cultured human umbilical vein endothelial cells secrete a protein that binds CN[57Co]Cbl, elutes from a Sephacryl S-200 column in the same position as TCII, and precipitates with an antiserum to purified human TCII. The biosynthesis of TCII by these cells was confirmed by demonstrating the incorporation of [35S]methionine into a nascent protein that immunoprecipitated with anti-TCII and which, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) had an Mr of 43,000, the same as human TCII. This secreted protein also had the functional properties of TCII because it facilitated the uptake of CN[57Co]Cbl by the same endothelial cells that secreted it as well as other cell lines that express the membrane receptor for TCII. We also present evidence that the venous endothelium could be the source of TCII in vivo by showing that an intact umbilical vein in an isolated umbilical cord, when perfused with medium containing [35S]methionine, secretes a radiolabeled nascent protein with the same immunoreactive and electrophoretic properties as human TCII. These studies demonstrate that the endothelial cell, which has been shown to secrete a number of plasma proteins, also synthesizes and secretes TCII both in vitro and as an intact endothelium in situ, and therefore, could be the source of circulating TCII in vivo.

The distribution of cobalamin cofactors was investigated in the livers and tumors of rats bearing transplanted Morris 7777 or 7800 hepatomas, in the livers of rats treated with the hepatocarcinogen diethylnitrosamine, and in normal rats. There was a significant increase in the proportion of methylcobalamin both in livers and tumors from rat bearing the hepatomas 7777 and 7800 compared to the proportion of methylcobalamin in the livers of normal rats. The total cobalamin content of the hepatomas was significantly lower than that of host or control livers. Similarly, the total cobalamin content of the livers from the tumor-bearing rats was less than that in control animals. The administration to rats of an acute dose of diethylnitrosamine led to an 84% increase in the hepatic concentration of methylcobalamin. Chronic administration of diethylnitrosamine slightly increased the hepatic methylcobalamin concentration, but this was not statistically significant. Liver weight was reduced, and the hepatic content of total cobalamin fell to 55% of that in control animals.

Transcobalamin II (TCII) has been purified from Cohn fraction III of human plasma by batchwise binding to and then elution from carboxymethyl-Sephadex, affinity chromatography using photo-labile aminopropyl cobalamin coupled to activated Sephacryl S-200, and finally chromatography through carboxymethyl cellulose. The yield was approximately 80%. The addition of protease inhibitors in all steps of the purification procedure and extensive washing of the carboxymethyl-Sephadex prior to eluting the TCII minimized degradation of the protein and the final preparation of holo-TCII contained 1 mol of cobalamin/mol of protein. A single polypeptide of 43,000 daltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 19 amino acids have been determined for human TCII. 12 of the amino acids are homologous with rabbit TCII and six are homologous with human R-binder, but there is no homology with human intrinsic factor.

Cobalamin coenzymes and a series of related corrinoid analogs have been analyzed by high-performance liquid chromatography on reverse-phase C8 and C18 columns using both isocratic and gradient elution systems and 254-nm absorbance detection. In the isocratic mode, retention times for sulfitocobalamin, cyanocobalamin, methylcobalamin, adenosylcobalamin, and aquacobalamin on a LiChrosorb C8 column were 1.1, 1.6, 2.2, 2.9, and 4.7 min, respectively. In the gradient mode, corresponding retention times were 9.7, 10.2, 12.8, 11.4, and 9.0 min. Closely related structural analogs of adenosylcobalamin such as 1,N6-ethenoadenosylcobalamin, formycinylcobalamin, and 2,6-diaminonebularinylcobalamin were clearly resolved from naturally occurring cobalamins by isocratic elution. In the gradient system, the order of elution of cobalamins was related to the hydrophobicity of the upper-axial ligand. This was demonstrated by determining the retention times of aminoalkylcobalamin homologs (C2, 9.8; C3, 10.5; C5, 11.2; C8, 12.8; and C11, 14.9 min). The usefulness of this method was demonstrated by analyzing 57Co-labeled cobalamins present in extracts of Lactobacillus leichmannii and murine leukemia L1210 cells.

Five patients presenting clinically with a form B12-deficiency neuromyelopathy, with cord involvement in all and proximal muscle weakness in two of them, were investigated for their neurologic, hematologic and vitamin status. Megaloblastosis and achlorhydria were present in all, and impaired absorption of 57Co vitamin B12 and of D-xylose was detected in four. Total cyanide extracted vitamin B12 (A) was lowered in all cases and noncyanide extractable (B) in four of the five, being zero in three. All five responded to injections of hydroxocobalamin. In two patients sequential estimations showed that both A and B, especially the latter, rose steeply initially, normalizing at 50% of A after some weeks. Moiety B is suggested to be physiologically the more active and dissociable form of vitamin B12. Markedly elevated initial serum folate levels, and their subsequent fall under treatment with B12, indicated the operation of the "methyltetrahydrofolate trap". Blood levels of thiamin, nicotinic acid and pantothenic acid were within normal limits. However, serum riboflavin (B2) total vitamin B6 and pyridoxal were reduced in all where tested. Vitamin B6 deficiency could have resulted from its own malabsorption and have contributed to be B12 deficiency. Vitamin B2 and B6 levels also corrected themselves on B12 therapy. The B-vitamin deficiencies in our patients probably resulted from intestinal malabsorption, with a possible factor of malnutrition consequent to their strictly vegetarian diet.

1. The B-vitamin status of fifty-nine patients, mainly from the lower socio-economic classes in Bombay, with a history of chronic malnutrition, and of alcoholism of 1-5-20 years' duration, was studied before and during treatment, and in relation to their clinical, especially neurological, condition. These patients were divided into two neurological categories: (I) those with peripheral neuropathy (mainly sensory and distal) alone, (2) those with mental changes (mainly confusion and disorientation) also. Both categories frequently showed pellagrous pigmentation and mucocutaneous signs of B-vitamin deficiency. 2. Thiamin and erythrocyte transketolase (EC 2.2.1.1) activity, riboflavin, nicotinic acid, pantothenic acid, total and pyridoxal fraction of vitamin B6, folate and total vitamin B12 were estimated in the blood and the cerebrospinal fluid (CSF) of these patients, and also in the blood of sixty-nine control subjects and in the CSF of some of them. The concentrations of all the vitamins, except vitamin B12, were highly significantly lower in the blood of patients of category I compared to the controls, and erythrocyte transketolase activity and pyridoxal concentration in patients of category 2 were significantly lower than those of category I patients. Blood pantothenic acid and folate concentrations were reduced less consistently. 3. Serum vitamin B12 concentration was significantly increased (though within normal range) in the patients compared to the control group, probably because of the moderate hepatic insufficiency (as assessed by liver function tests) in the former. 4. The concentrations of thiamin, riboflavin, nicotinic acid and total vitamin B6 were also highly significantly lower in the CSF in patients of category I compared with controls. Furthermore, thiamin, nicotinic acid and total vitamin B6 concentrations were significantly lower in patients of category 2 than those of category I patients, indicating that CSF levels reflect better the neurological status of these patients. 5. There was a moderate increase in the blood concentration of all the vitamins tested, after a relatively poor hospital diet alone. There was a concurrent increase in the blood levels of thiamin, riboflavin, nicotinic acid and pantothenic acid after parenteral treatment with either thiamin or nicotinic acid. The administration of pyridoxine resulted in a significant increase in the blood levels of riboflavin and the pyridoxal fraction of vitamin B6.

Cobalamin metabolism has been investigated in a new case of transcobalamin II (TC II) deficiency. Using the chromatobioautographic technique, an abnormal distribution of cobalamins was detected in the child's erythrocytes and reduced synthesis of adenosylcobalamin but not of methylcobalamin in cultured fibroblasts. These results suggest that there may be a close link between TC II-mediated cobalamin transport and intracellular synthesis of adenosylcobalamin (Ado-Cbl).

Only 20 patients with optic neuritis of unknown aetiology could be collected in Bombay over a period of two and a half years. Only six of them were smokers and only three (one a non-smoker) had bilateral centrocaecal scotomata similar to those found in tobacco amblyopia. In 11 untreated patients the serum vitamin B(12) and plasma thiocyanate levels were estimated and found to show no significant differences from those in normal control subjects. Treatment with hydroxocobalamin in the majority of cases and with cyanocobalamin, corticotrophin, or prednisolone in the rest showed equally good results; spontaneous improvement was seen in one case.No significant role could be assigned to smoking (cyanide) or to vegetarianism in the production of optic neuritis in these patients; nor was there any evidence of depletion of total (cyanide-extracted) B(12) or of an increase in the proportion of non-cyanide-extracted B(12) in the serum.

Vitamin B(12), thiocyanate, and folate levels in the blood were estimated in 69 apparently normal subjects, of whom 26 were non-vegetarian non-smokers, 19 non-vegetarian smokers, 15 vegetarian non-smokers, and nine vegetarian smokers. The serum total (cyanide-extracted) B(12) level (value A) ranged from 105 to 728 pg/ml, with a mean of 292 pg/ml. The highest values were found in non-vegetarian non-smokers and the lowest in vegetarian smokers. There was no significant difference in value A between smokers as a group and non-smokers as a group. On the other hand, in vegetarians value A was very significantly lower than in non-vegetarians regardless of their smoking habits.It is suggested that A may represent both the protein-bound and free forms of vitamin B(12) in the blood, and B mainly the free B(12), which may be the physiologically active form. The plasma thiocyanate level varied from 1.0 to 15 mumol/100 ml, being, as expected, much higher in smokers (mean 8.20 mumol/100 ml) than in non-smokers (mean 2.02 mumol/100 ml). There was a rough correlation between falling B(12) levels and rising thiocyanate levels. The serum folate level ranged from 2.75 to 15.75 ng/ml, and was slightly but significantly higher in vegetarians (mean 6.60 ng/ml) than in non-vegetarians (mean 4.79 ng/ml), reflecting the greater content of folate in a vegetarian diet.

Studies of cobalamin metabolism in case of juvenile pernicious anaemia during haematological remission showed that while plasma cobalamins were normal there was some depletion of erythrocyte cobalamins. In addition, the plasma distribution of apo- and holo-transcobalamins was markedly abnormal, holo-TCII being increased, yet the patient's plasma promoted less cellular synthesis of adenosylcobalamin by cultured lymphocytes than did plasma from a control subject. The results suggest that holo-TCII may in this case have been ineffective and provide further evidence for the importance of TCII in intracellular synthesis of adenosylcobalamin.

1. The B-vitamin status of fifty-nine patients, mainly from the lower socio-economic classes in Bombay, with a history of chronic malnutrition, and of alcoholism of 1-5-20 years' duration, was studied before and during treatment, and in relation to their clinical, especially neurological, condition. These patients were divided into two neurological categories: (I) those with peripheral neuropathy (mainly sensory and distal) alone, (2) those with mental changes (mainly confusion and disorientation) also. Both categories frequently showed pellagrous pigmentation and mucocutaneous signs of B-vitamin deficiency. 2. Thiamin and erythrocyte transketolase (EC 2.2.1.1) activity, riboflavin, nicotinic acid, pantothenic acid, total and pyridoxal fraction of vitamin B6, folate and total vitamin B12 were estimated in the blood and the cerebrospinal fluid (CSF) of these patients, and also in the blood of sixty-nine control subjects and in the CSF of some of them. The concentrations of all the vitamins, except vitamin B12, were highly significantly lower in the blood of patients of category I compared to the controls, and erythrocyte transketolase activity and pyridoxal concentration in patients of category 2 were significantly lower than those of category I patients. Blood pantothenic acid and folate concentrations were reduced less consistently. 3. Serum vitamin B12 concentration was significantly increased (though within normal range) in the patients compared to the control group, probably because of the moderate hepatic insufficiency (as assessed by liver function tests) in the former. 4. The concentrations of thiamin, riboflavin, nicotinic acid and total vitamin B6 were also highly significantly lower in the CSF in patients of category I compared with controls. Furthermore, thiamin, nicotinic acid and total vitamin B6 concentrations were significantly lower in patients of category 2 than those of category I patients, indicating that CSF levels reflect better the neurological status of these patients. 5. There was a moderate increase in the blood concentration of all the vitamins tested, after a relatively poor hospital diet alone. There was a concurrent increase in the blood levels of thiamin, riboflavin, nicotinic acid and pantothenic acid after parenteral treatment with either thiamin or nicotinic acid. The administration of pyridoxine resulted in a significant increase in the blood levels of riboflavin and the pyridoxal fraction of vitamin B6.