Total Cobalamin Research Articles

Response of the methionine synthase system to short‐term culture with homocysteine and nitrous oxide and its relation to methionine dependence

We compared the metabolic response of a methionine(Met)-dependent (P60) human glioma cell line with that of a Met-independent variant (P60H) when cultured in a homocysteine (Hcy) medium and exposed to N2O. In Hcy medium (without Met), remethylation of Hcy in P60H cells was enhanced and supported growth, whereas remethylation was low in P60 cells, which failed to thrive under these conditions. Both cell types seemed to contain adequate amounts of folates and total cobalamin (Cbl). P60 cells showed increased total and methylcobalamin (CH3Cbl) content after the shift to a Hcy medium, but the high, stable level of CH3Cbl detected in P60H cells was not attained. Further metabolic differences were induced by N2O exposure, which markedly reduced Met-synthase activity in cell-free extracts in both cell lines and completely blocked intact-cell Hcy remethylation in P60, whereas Hcy remethylation was only partly inhibited in P60H cells cultured in Met medium. The residual Hcy remethylation in P60H cells may be related to only a moderate depletion of CH3Cbl. The resulting high CH3Cbl level relative to Met-synthase activity during N2O exposure was even higher in Hcy medium. These findings in P60H cells probably reflect increased provision of Cbl to support Hcy remethylation under metabolic strain. The inability of P60 to furnish CH3Cbl to the enzyme may explain both the Met-dependent phenotype and the increased sensitivity of Hcy remethylation to N2O exposure in these cells. Int. J. Cancer 72:301–306, 1997. © 1997 Wiley-Liss, Inc.

Atherogenesis and the homocysteine-folate-cobalamin triad: do we need standardized analyses?

Bioscientists, physicians and nutritionists are newly interested in the homocysteine-folate-cobalamin triad, in part because homocysteine may be important both in atherogenesis and thrombogenesis. Homocysteine imbalance may be an early marker for cobalamin disorders because cobalamin is a cofactor in remethylation of homocysteine to methionine.In 139 men and 32 women of similar mean age of 65 years, we measured markers which have been cited as risk for atherosclerosis: serum homocysteine, folate, total cobalamin, holotranscobalamin I and II, (TCI and TCII), total serum cholesterol (SCHOL), high density lipoprotein cholesterol (HDLC), triglycerides (STG) as well as red blood cell (RBC) folate, food records and body composition by whole body counting of potassium-forty (40K).Statistical relationships among the data showed healthy women had lower mean serum homocysteine and their mean RBC folate and TCI and TCII were higher than men. Eighty-three subjects had TCII much lower than 60 pg/ml (subnormal), yet only 11 of these men and two women had total cobalamin < 200 pg/ml (abnormal). Fifty-two subjects with serum homocysteine greater than 17.5 nmol/ml had TCII less than 60 pg/ml, suggesting serum homocysteine may be a marker for early cobalamin negative balance. None of the subjects in the study had serum folate below abnormal values, i.e., less than 1.6 mg/ml. All subjects had RBC folate within normal range. Serum homocysteine showed inverse relationship with RBC folate and serum total cobalamin, TCI and TCII.1) importance of using serum holotranscobalamin TCI and TCII as markers of cobalamin deficiency, 2) necessity to use documented quantitative components of dietary intake if strong comparisons are to be made among quantitative values of serum or plasma homocysteine, folate, cobalamin, and nutrients in food intake.

Cobalamin forms and analogues in plasma and myeloid cells during chronic myelogenous leukaemia related to clinical condition.

Plasma and buffy coat specimens of CML patients with untreated disease, in chronic and accelerated phase, and in overt blastic crises were analysed for the cobalamin patterns using non-polar extraction, thin-layer chromatography and bioautography. Splenic tissue specimens from four splenectomized patients in accelerated phase were analysed similarly. Cellular extracts were separated into two compartments by adsorption to haptocorrin antibodies. Plasma concentrations of all cobalamin forms were increased in CML. The proportion of methylcobalamin was significantly lower than in a reference population, and a low plasma proportion of methylcobalamin was associated with a poor prognosis. Buffy coat cells and splenic tissue had a higher proportion of 5'-deoxyadenosylcobalamin and a lower proportion of methylcobalamin than plasma; still, there was relatively more methylcobalamin than in normal tissue. The haptocorrin-bound compartment differed from the non-haptocorrin compartment in untreated or chronic phase patients by binding less methylcobalamin. An estimate of cobalamin analogues in plasma was achieved by comparing two different isotope dilution assays employing a cobalamin-specific binder, intrinsic factor, and a nonspecific binder, hog non-intrinsic factor. Values for total cobalamin and analogues were increased to the same degree in CML plasma.

Effets de la transfusion sanguine sur les dosages sériques du fer, de la capacité totale de fixation de la transferrine, de la ferritine, de la vitamine B12 et des folates

To evaluate the influence of blood transfusion on the serum levels of different nutrients, the levels of total iron binding capacity, ferritin, cobalamin and folate were determined before and 24 hours after 2 or 3 packed red cell transfusions, by forty patients with anemia of various causes. No significant change was found in these levels before and after blood transfusion, except for iron and folate.

On the Role of two Different Cobalt(II) Species in Coenzyme B12-Dependent 2-Methyleneglutarate Mutase fromClostridium barkeri

Purified 2-methyleneglutarate mutase from Clostridium barkeri contains adenosylcobalamin (coenzyme B12) and varying amounts of oxygen-stable cob(II)alamin. The content of the latter was estimated by EPR spectroscopy at 6-11% of the total cobalamin (2-4 mol/mol enzyme). Tryptic digestion of the enzyme liberated the prosthetic groups, cob(II)alamin being oxidized by air to aquocobalamin. HPLC analysis of the released cobamides from several preparations revealed > 90% adenosylcobalamin and < 10% aquocobalamin. Treatment of active 2-methyleneglutarate mutase with 8M urea followed by gelfiltration yielded an inactive enzyme from which 50% of the adenosylcobalamin and up to 70% of the cob(II)alamin was removed. Addition of adenosylcobalamin to the urea-treated enzyme resulted in complete reactivation, but the content of cob(II)alamin was not increased. These data suggest that the oxygen-stable cob(II)alamin is not involved in catalysis. In the presence of the competitive inhibitor itaconate (methylenesuccinate, Ki = 0.7mM), an alteration of the UV/visible spectrum at 470 nm as well as a new line in the EPR spectrum of the enzyme (around g = 2.1) was observed. The results indicate the formation of an unusual, oxygen sensitive Co(II) species during catalysis. The EPR signal of the oxygen-stable cob(II)alamin (gx,y = 2.24) remained unchanged under those conditions.

Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri.

The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively. Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa). Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin. This preparation could be reactivated to 95-100% by addition of adenosylcobalamin. The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity. 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin. Upon incubation of the mutase with [5'-3H]adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity. During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin. EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme. Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose. The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.

INCREASED APOTRANSCOBALAMIN II LEVELS IN RHEUMATOID ARTHRITIS

The distribution of endogenous cobalamin among serum cobalamin-binding proteins was studied in 30 patients with active rheumatoid arthritis (RA) and 27 in clinical remission. The mean total serum cobalamin concentration (holo-transcobalamin I and II) was similar in both groups of patients, whereas mean apotranscobalamin II was significantly increased in patients with active RA. The clinical significance of this finding is not yet established but it might be a useful parameter for the evaluation of disease activity in RA.

1] Determination of cobalamins in biological material

This chapter discusses the determination of cobalamins in biological material, and mainly concerns with “true cobalamins.” In animal tissues, five different cobalamins have been identified in cells and extracellular fluids: MeCbl, AdoCbl, OH-Cbl, CN-Cbl, and HSO 3 -Cbl. In addition, several cobalamin-like substances (i.e., other corrinoids and cobalamin analogs) are found in animal and bacterial materials under various conditions. The major aims of a cobalamin determination are (1) the accurate estimation of total cobalamin content, and (2) estimation of the relative amount of each cobalamin form. The determination of cobalamins from certain compartments and speculatively extrapolate these principles to other compartments are discussed in the chapter. The assay depends on a preliminary extraction of cobalamins from tissue or fluid, further isolation and/or purification of the cobalamins before resolution of the different forms, and quantification of each form. Two methods are presented in the chapter. One, using thin-layer chromatography (TLC), is a refinement of earlier methods. The other employs high performance liquid chromatography (HPLC). The recovery of each cobalamin form from each compartment should be complete in the ideal assay. Since unknown biological materials may contain cobalamin-binding substances of varying stability in the extraction procedure, some compartments may be overestimated at the expense of other compartments. For example, cobalamins bound to transcobalamin are liberated easily by acidification, while their liberation from haptocorrin requires boiling in addition. Thus recovery studies are essential when examining biological materials that might contain uncharacterized cobalamin-binding proteins.

A sex difference in serum cobalamin and transcobalamin levels

In healthy young adults, serum levels of cobalamin, unsaturated and total cobalamin binding capacity, and transcobalamin (TC) II are significantly higher in females, while TC III is higher in males. These findings have relevance to the reference values in normals for these tests.

Cobalamin and folate binding proteins in human tumour tissue.

The serum of an 84 year old man with disseminated carcinoma was found to contain extremely high concentrations of cobalamin and of a cobalamin binding protein with trans-cobalamin I characteristics. Tumour tissue samples obtained at necropsy contained considerably higher concentrations of cobalamin binding protein (R-binder) than normal tissues. Tumour tissues also contained increased concentrations of specific folate binding protein. In all tissues studied a close correlation existed between unsaturated cobalamin and unsaturated folate binding and between total cobalamin and total folate binding. These results suggest related mechanisms for the synthesis of cobalamin binding proteins of the R-binder class and folate binding proteins by tumour tissue.

Determination of cobalamins in biological material: I. Improvement in the unequal recovery of cobalamins by preincubation with cadmium acetate

Ethanol extraction of 28 specimens of human plasma in the dark showed that the total cobalamin value is increased up to 2 1/2 times by adding 400 micrometers of potassium cyanide. The addition of cyanide significantly changed the substance concentration (fractional content times substance concentration of total cobalamins) for all the cobalamin forms except 5'-deoxyadenosylcobalamin. The ethanol extraction was also studied with 57Co-labeled cobalamin forms. The recovery of [57Co]hydroxo- and [57Co]sulfitocobalamin from both plasma and hemolysate was inadequate. The recovery of [57Co]5'-deoxyadenosylcobalamin was only studied from liver tissue; the extraction recovery of this form was equal to that of 57Co]cyano- and [57Co]methylcobalamin. Preincubation with 0.25 M of cadmium acetate or extraction with 400 micrometers of potassium cyanide almost equalized the recovery of all five cobalamins. It is concluded that (i) the substance concentrations of the individual cobalamins presented hitherto are erroneous, owing to inadequate ethanol extraction of hydroxo- and sulfitocobalamin; (ii) about one-third of the cobalamins in plasma consists of hydroxocobalamin, which is not extractable by the methods used hitherto; (iii) studies of the cobalamin forms in body fluids and tissue cells need to be repeated by methods ensuring equal extraction of all the cobalamins; and (iv) the extraction of the cobalamins must be uniform for determination of the substance concentration of total cobalamins as well as the fractional amounts of all cobalamins.

Tissue Distribution of Methylcobalamin in Rats Fed Amino Acid-Defined, Methyl-Deficient Diets

Total cobalamin and methylcobalamin levels were determined in tissues of male F344 rats fed a complete, amino acid-defined diet or a diet deficient in methionine, choline and/or cyanocobalamin. Total cobalamin levels in rats fed the complete diet were (picograms/milligram tissue ± SEM): liver, 67 ± 13; kidneys, 738 ± 133; spleen, 23 ± 2; and adrenals, 268 ± 36. Corresponding methylcobalamin levels were: liver, 1.6 ± 0.5; kidneys, 107.6 ± 22.2; spleen, 0.3 ± 0.1; and adrenals, 26.9 ± 5.3; these values represent 2.4, 14.5, 1.4 and 9.7%, respectively, of the total cobalamin levels. Total cobalamin levels of all tissues studied were altered by cobalamin deprivation alone or in conjunction with methionine and/or choline deprivation. Methylcobalamin levels were more resistant to dietary alteration. Regardless of the presence or absence of methionine and cobalamin in the diet, choline deprivation always decreased the proportion of methylcobalamin in the liver. Kidney levels of methylcobalamin, like those of total cobalamin, were decreased by removal of cobalamin from the complete or the methyl-deficient diets. The results demonstrate that cobalamin, methionine and choline exert quite different effects on tissue levels of the cobalamins in rats.

Internal Transport and Metabolism of Cobalamin in Spirometra mansonoides (Cestoda) Spargana

The anterior 2 mm of the sparganum of Spirometra mansonoides contained 35 to 48% of the total endogenous cobalamin (Cbl) with only 2 to 4% in the terminal 1 cm. [57Co]cobalamin taken up in the posterior region of spargana was transported directionally to the anterior region and concentrated there resulting in a uniform depletion of [57Co]Cbl along the remaining body length. The anterior 2 mm contained 24% of the sparganum's holo-MMCoAM activity with only 5% in the terminal 1 cm. When head and body preparations were chromatographed on Sephadex G-150 columns eluates from both regions exhibited single peaks of MMCoAM activity at an approximate molecular weight of 150,000 and two peaks of endogenous Cbl. The first Cbl peak cochromatographed with MMCoAM activity and the second Cbl peak eluted at the position of free Cbl. Spargana rapidly converted CN-[57Co]Cbl to hydroxocobalamin (OH-Cbl) and adenosylcobalamin (Ado-Cbl) in vitro. In the head region significant amounts of Ado-Cbl were present (25%) although OH-Cbl was the predominant form of Cbl (45%) after 264 hr in a Cbl-free medium. In the body Ado-Cbl was the predominant form of Cbl (44%) although significant amounts of OH-Cbl were present (27%) after 264 hr in a Cbl-free medium. No methylcobalamin (Me-Cbl) was detected, but an unidentified cobalamin-containing entity (Fraction 4) was present. Based on these results we propose that the sparganum takes up cobalamin, concentrates it in the anterior, "head" region, sequentially metabolizes it to the hydroxyl then the adenosyl forms, and that the adenosylated form is used in this region of high, anaerobic metabolism as an obligatory coenzyme for MMCoAM, in coordination with the differentiation of the sparganum to the adult cestode.

Effects of low cobalamin diet and chronic cyanide toxicity on cobalamin distribution in baboons

This paper reports the bodily distribution of total cobalamin and individual dual cobalamins at the termination of an experiment on the effects of a low cobalamin diet and chronic cyanide of thiocyanate administration in baboons. The results show that the distribution of cobalamins in the tissues of the baboon can be altered by a low cobalamin diet and also by chronic intoxication with cyanide, whether or not the animals are on a low cobalamin diet. All animals on the low cobalamin diet showed a reduction in total and individual cobalamins. In blood plasma and erythrocytes, kidney, spleen, testis and brain, the proportion of methylcobalamin tended to be disproportionately reduced in cobalamin-depleted animals. This reduction was lessened or prevented by the administration of cyanide. Neither cyanide not thiocyanide produced a significant increase in the proportion of cyanocobalamin in plasma, though thiocyanate produced a large increase in cyanocobalamin in erythrocytes. In liver, cyanocobalamin was more than doubled by the administration of cyanide to cobalamin-depleted animals.

Distribution of endogenous cobalamin between the transcobalamins in various mammals.

1. Plasma samples from ten mammals were chromatographed on Sephadex G-200 columns and the total cobalamin content of each fraction was determined. 2. Unlike the situation in man, the bulk of the endogenous plasma cobalamin was found attached to a transcobalamin II-like protein in all ten animals. Transcobalamin 0 carried between 3 and 20% and this proportion appeared to be inversely related to the plasma total cobalamin. No endogenous cobalamin peak corresponding to transcobalamin I was detected in any species, though in the rabbit 5.3% of the plasma total cobalamin was attached to a protein of apparent molecular weight 176 000.

Interrelationships between Vitamin B12 and Folic Acid in Myelomatosis: Cobalamin Coenzyme and Tetrahydrofolic Acid Function

Cobalamin and folate metabolism was investigated in 43 patients with myelomatosis, in 8 control subjects of similar age and 22 younger controls. Plasma total cobalamin was lower in myeloma patients than in either of the control groups and methylcobalamin (Me-Cbl) was disproportionately reduced. Erythrocyte levels of total cobalamin were very similar in patients and elderly controls but were half the levels in younger controls. Erythrocyte levels of Me-Cbl were slightly higher in patients than in the dlderly controls. FIGLU excretion after L-histidine was elevated in 53% of the patients but values did not correlate with serum or erythrocyte folate or with plasma total cobalamin. FIGLU excretion decreased after DL-methionine or Me-Cbl only in patients whose FIGLU excretion was initially high. The results are discussed in the light of the 'methylfolate trap hypothesis' and suggest that some patients with myelomatosis have insufficient activity of methionine synthetase to meet the additional metabolic demand for one carbon compounds.

Isolation of Coenzyme B12 from Liver

The identity of the major cobamide in liver has not been satisfactorily established. Cyanocobalamin (vitamin B12, 5,6dimethylbenzimidazolylcobamide cyanide), which has been obtained in crystalline form with liver as a source material (1, 2), is certainly a very minor component of the cobalamins in liver. Analyses of liver concentrates, both by a paper chromatographicbioautographic method (3) and by a chemical method (4), have shown that cyanocobalamin is usually not present in detectable amounts and seldom constitutes as much as 10% of the total cobalamin. The preparation of cyanocobalamin from liver probably was made possible by the presence of cyanide in the charcoal used in the isolation process. Examination of a liver extract by the chromatographic-bioautographic method (3) indicated that hydroxocobalamin (vitamin B12b, 5,6-dimethylbenzimidazolylcobamide hydroxide) was the most abundant cobalamin. This compound was also obtained in pure form from liver (1, 5). However, the recent discovery of the cobamide coenzymes in bacteria (6,7) and the demonstration that they constitute a large part of the total cobamides in these organisms (8,9) raised the possibility that coenzyme Bit (5,6-dimethylbenzimidazolylcobamide coenzyme, DMBC coenzyme, DBC coenzyme) may be a constituent of liver. Since coenzyme B12, like the adenylcobamide coenzyme (lo), is relatively unstable and is readily converted to hydroxocobalamin or cyanocobalamin by treatment with light or cyanide,’ respectively, the inadvertent decomposition of the coenzyme, if present in liver extracts, could account for previous observations on the occurrence of the two cobalamin vitamins. The present paper reports some observations on the occurrence and abundance of a cobamide coenzyme, which has been identified as coenzyme Blz, in livers of rabbit, chicken, sheep, and man. A brief report of the occurrence of coenzyme Blz in rabbit liver has already appeared (7).

Stability of injected vitamin B12 content of dog liver.

An investigation was made of livers from 5 dogs receiving 3.6 to 14 μg (specific activity = 1 mc/mg) of vitamin B12-Co60 intravenously. The vitamin B12-Co60, was retain ed by the livers as an intact cobalamin, probably as cyanocobalamin. This was demonstrated by the method of reverse isotope dilution. Direct isotope dilution indicated an average total cobalamin concentration of 0.36 μg/g of dog liver. The authenticity of retained vitamin B12-Co60, introduced by injection, as an isotope dilution tracer was demonstrated by simultaneous analysis with vitamin B12 Co57. Degradation of vitamin B12 to ionic cobalt was negligible.

The Vitamin B12 Content of Certain Fishery Materials

Irregularities occurring in the microbiological assay of herring (Clupea pallasi) materials for vitamin B12 (cobalamin) are described and the probable reasons for the differences discussed. With the employment of a Lactobacillus leichmannii assay procedure and conditions under which vitamin B12 (cyano-cobalamin) and vitamin B13a (hydroxo-cobalamin) caused identical growth response, it was found that normal commercial flame-dried herring meals had about the same total cobalamin content as specially prepared meals dried at 38° to 43.5 °C. Chromatographic separation, elution and assay of the cobalamins in herring-meal samples indicated that substantially all of the activity found by direct assays of the meals was due to the vitamin itself. Hexane extraction had little effect on the cobalamin content, but heating the meals reduced it.