Concentrations Of Cyanocobalamin Research Articles

Study of the protective effects of cyanocobalamin on methotrexate induced nephrotoxicity in rats.

Background: Methotrexate (MTX) is a chemotherapeutic drug, used mainly in many cancerous stages, inflammatory and auto-immune diseases, but its use has been limited by its nephrotoxicity. Cyanocobalamin is a water-soluble vitamin possessing nephro-protective properties. The aim of this study was to investigate the effect of cyanocobalamin on the nephrotoxicity of methotrexate. Methods: In this study 42 albino adult female rats were used, divided into six groups each containing seven rats (n=7). First group: Control group (Negative control), 7 rats were injected intraperitoneally with 0.5ml/kg/day NS. Second group: 7 rats were injected intraperitoneal with a single dose of methotrexate (20 mg/kg) for 4 days. Third Group: 7 rats were given intraperitoneal cyanocobalamin at a dose (1.5 mg/kg/day) for two weeks, fourth, fifth, sixth group: 7 rats from each group were injected intraperitoneal with different concentrations of cyanocobalamin (0.5, 1, 1.5 mg/kg /day) respectively for two weeks and MTX (20 mg/kg) which was injected only on day 11. On day 15, rats from all groups were euthanized, and blood samples were taken for biochemical tests, including evaluating serum urea and creatinine. The kidneys were extracted for histological investigation and evaluation of antioxidant (GSH) and oxidative stress (MDA) by using kidney tissue homogenates. Results: This study revealed that kidney damage produced by the MTX (group II) is manifested by significantly elevated (P<0.05) urea and creatinine. On the contrary, the cyanocobalamin groups (IV, V, VI) significantly (P<0.05) reduced urea and creatinine. Renal antioxidant defense systems, such as reduced glutathione depleted by MTX therapy, were restored to normal levels by cyanocobalamin. Furthermore, cyanocobalamin reduced oxidative stress (MDA) and histologically reduced renal tissue injury induced by MTX. Conclusions: In conclusion, the study revealed that cyanocobalamin has a nephroprotective action upon MTX-induced renal damage in rats; cyanocobalamin may offer a protective effect, such as antioxidant action.

Study of the protective effects of cyanocobalamin on methotrexate induced nephrotoxicity in rats.

Background: Methotrexate (MTX) is a chemotherapeutic drug, used mainly in many cancerous stages, inflammatory and auto-immune diseases, but its use has been limited by its nephrotoxicity. Cyanocobalamin is a water-soluble vitamin possessing nephro-protective properties. The aim of this study was to investigate the effect of cyanocobalamin on the nephrotoxicity of methotrexate. Methods: In the study 42 albino adult female rats were used, divided into six groups each containing seven rats (n=7). 1 st group: Control group (Negative control), 7 rats were injected intraperitoneally with 0.5ml/kg/day NS. Second group: 7 rats were injected intraperitoneally with a single dose of methotrexate (20 mg/kg) for 4 days. Third Group: 7 rats were given intraperitoneally cyanocobalamin at a dose (1.5 mg/kg/day) for two weeks, fourth, fifth, sixth group: 7 rats from each group were injected intraperitoneally with different concentrations of cyanocobalamin (0.5, 1, 1.5 mg/kg /day), respectively, for two weeks and MTX (20 mg/kg), which was injected only on day 11. On day 15, rats from all groups were euthanized, and blood samples were taken for biochemical tests, including evaluating serum urea and creatinine. The kidneys were extracted for histological investigation and evaluation of antioxidant (GSH) and oxidative stress (MDA) by using kidney tissue homogenates. Results: This study revealed that kidney damage, produced by the MTX (group II), is manifested by significantly elevated (P<0.05) urea and creatinine. On the contrary, the cyanocobalamin groups (IV, V, VI) significantly (P<0.05) reduced urea and creatinine. Renal antioxidant defense systems, such as reduced glutathione depleted by MTX therapy, were restored to normal levels by cyanocobalamin. Furthermore, cyanocobalamin reduced oxidative stress (MDA) and histologically reduced renal tissue injury induced by MTX. Conclusions: In conclusion, the study revealed that cyanocobalamin has a nephroprotective action upon MTX-induced renal damage in rats; cyanocobalamin may offer a protective effect, such as antioxidant action.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus-oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

Effects of dietary cyanocobalamin on growth performance, non‐specific immune response, antioxidant capacity, haematological parameters, body composition and liver cyanocobalamin concentration of fingerling major carp, Catla catla (Hamilton)

Effects of dietary cyanocobalamin on growth performance, non‐specific immune response, antioxidant capacity, haematological parameters, body composition and liver cyanocobalamin concentration of fingerling major carp, <i>Catla catla</i> (Hamilton)

Effect of chemically defined growth medium components on characteristics of bacterial inclusion bodies

AbstractBACKGROUNDInactive aggregated protein deposits formed during fermentation of recombinant bacteria – inclusion bodies (IBs) – continue to be a significant source of active proteins. Understanding how the microenvironment, in particular growth medium, affects the quality and quantity of the IBs would be desirable for optimal production of proteins. In this study, we explore the effect of various chemically defined media components on IBs using a variety of analytical techniques. Expression of a biotherapeutic, ranibizumab, has been chosen as a case study.RESULTSA repertoire of nutrients, including but not limited to carbon and nitrogen source, vitamins, cofactors, buffering agents and minerals, were examined. Sucrose, biotin, pantothenate, glutathione, K2HPO4, K2SO4, cyanocobalamin and CaSO4 were shortlisted based on experimentation. A design of experiments (DOE) study was performed to identify optimal concentrations of and interactions between these nutrients. Higher concentrations of sucrose, biotin and pantothenate, and lower concentrations of cyanocobalamin, CaSO4 and glutathione, resulted in larger IBs and greater product expression. Further, K2HPO4 and K2SO4 concentrations did not seem to have a meaningful impact on product formation.CONCLUSIONSMedium composition affects size and quality of IBs. The size of IBs exhibits a positive correlation with the quantity of product expressed, as well as the purity of the product, but does not correlate with the total amount of host cell proteins produced. IB size could be used as a key process attribute during bioreactor optimization, and the otherwise difficult task of measuring product concentration in the fermentation broth could be avoided. © 2020 Society of Chemical Industry

Effects of different levels of dietary cyanocobalamin on growth, liver cyanocobalamin concentration, antioxidant capacity, intestinal enzymes and non-specific immune response for optimum inclusion in the commercial feeds of fingerling Channa punctatus (Bloch)

For increasing the aquaculture production, it is crucial to develop quality feeds with all the essential nutrients including vitamins. Since vitamins are essential nutrient required in small amount to perform structural and functional roles, information on the dietary vitamin requirements is essential. This study was conducted to investigate the effects of varying levels of dietary cyanocobalamin on growth, liver cyanocobalamin concentration, antioxidant capacity, intestinal enzymes and non-specific immune response and optimize dietary requirement for developing feeds for intensive culture of fingerling Channa punctatus. Seven purified diets (450 g/kg CP, 18.71 kJ/g GE) with 0, 0.02, 0.04, 0.08, 0.16, 0.32 and 0.64 mg/kg of cyanocobalamin were fed to fish (4.27 ± 0.18 g) until satiation for 12-week. Maximum live weight gain (845%), protein efficiency ratio (1.50), best feed conversion ratio (1.48), protein deposition (25.98%) and protein gain (6.24 g/fish) were recorded in fish fed 0.08 mg cyanocobalamin per kg diet. Hematocrit, red blood cells, hemoglobin, mean cell volume and mean cell hemoglobin enhanced significantly with the increase of cyanocobalamin up to 0.08 mg/kg diet and then leveled off. However, intestinal protease, amylase and lipase activities increased (P < .05) as the amounts of dietary cyanocobalamin increased up to 0.16 mg/kg. Dietary cyanocobalamin levels significantly (P < .05) affected liver glutathione peroxidase, superoxide dismutase, catalase, total serum protein, lysozyme and alkaline phosphatase activities which were found to be highest in fish fed 0.16 mg cyanocobalamin per kg diet. Liver cyanocobalamin concentrations increased linearly (P < .05) with the incremental doses of dietary cyanocobalamin. However, biometric indices including HSI and VSI all showed little difference (P < .05) among all the treatments while CF exhibited upward trend up to 0.08 mg/kg dietary cyanocobalamin. Live weight gain, feed conversion ratio, protein deposition, hematocrit, hemoglobin, superoxide dismutase, catalase, glutathione peroxidase, alkaline phosphatase, protease and lipase activities were regressed against increasing concentrations of dietary cyanocobalamin using broken-line model which yielded the requirement in the range of 0.067–0.15 mg/kg diet. The information generated in this feeding trial will enable feed manufacturers to develop cyanocobalamin-balanced feeds for intensive culture of C. punctatus.

Production and characterization of cyanocobalamin-enriched lettuce (Lactuca sativa L.) grown using hydroponics.

When lettuces (Lactuca sativa L.) grown for 30 days in hydroponic culture were treated with various concentrations of cyanocobalamin for 24 h, its content in their leaves increased significantly from nondetectable to 164.6 ± 74.7 ng/g fresh weight. This finding indicated that consumption of only two or three of these fresh leaves is sufficient to meet the Recommended Dietary Allowance for adults of 2.4 μg/day. Analyses using a cobalamin-dependent Escherichia coli 215 bioautogram and LC/ESI-MS/MS demonstrated that the cyanocobalamin absorbed from the nutrient solutions by the leaves did not alter any other compounds such as coenzymes and inactive corrinoids. Gel filtration indicated that most (86%) of the cyanocobalamin in the leaves was recovered in the free cyanocobalamin fractions. These results indicated that cyanocobalamin-enriched lettuce leaves would be an excellent source of free cyanocobalamin, particularly for strict vegetarians or elderly people with food-bound cobalamin malabsorption.

Liquid chromatographic mass spectrometric (LC/MS/MS) determination of plasma hydroxocobalamin and cyanocobalamin concentrations after hydroxocobalamin antidote treatment for cyanide poisoning

Cyanide poisoning occurs in individuals after fire smoke inhalation and after oral ingestion of cyanide. Hydroxocobalamin (HOCbl), a hydroxylated form of vitamin B12, is often used as an antidote to treat cyanide toxicity. It has a high affinity for cyanide and rapidly removes cyanide from tissue by forming cyanocobalamin (CNCbl). Little information is available on the pharmacokinetics of HOCbl and CNCbl largely because of the lack of analytical methods for analyzing HOCbl and CNCbl. In this study, we developed a new liquid chromatographic mass spectrometric (LC/MS/MS) method for the quantitative analysis of plasma HOCbl and CNCbl in the porcine (Sus scrofa) model. The method uses on-column extraction, reversed phase gradient chromatography, and multiple reaction monitoring (MRM) for quantitation. MRM transitions monitored were 664.7→147.3 and 664.7→359.2 for HOCbl and 678.8→147.3, 678.8→359.1 678.8→457.1 for CNCbl. The limit of detection (LOD) and the lower limit of quantitation (LLOQ) were 1.0 and 1.0μmole/L, respectively, for plasma HOCbl and 0.1 and 0.5μmole/L for plasma CNCbl. The within-day and between-day CVs were 4.3 and 6.4% for plasma HOCbl at 500.0μmole/L and 5.5 and 5.7% for CNCbl at 100.0μmole/L (n=6). The plasma HOCbl and CNCbl calibrations curves were linear from 100.0 to 2000.0 and 50.0 to 500.0μmole/L, respectively. Based on 6 separate calibration curves the average linear regression coefficient (R2) for both HOCbl and CNCbl was 0.992. The LC/M/MS method was found to be accurate and precise and has been validated by determining the plasma HOCbl and CNCbl concentrations in 11 pigs that were treated with HOCbl for cyanide poisoning.

Vitamin B12 protects against superoxide-induced cell injury in human aortic endothelial cells

Superoxide (O2•−) is implicated in inflammatory states including arteriosclerosis and ischemia–reperfusion injury. Cobalamin (Cbl) supplementation is beneficial for treating many inflammatory diseases and also provides protection in oxidative-stress-associated pathologies. Reduced Cbl reacts with O2•− at rates approaching that of superoxide dismutase (SOD), suggesting a plausible mechanism for its anti-inflammatory properties. Elevated homocysteine (Hcy) is an independent risk factor for cardiovascular disease and endothelial dysfunction. Hcy increases O2•− levels in human aortic endothelial cells (HAEC). Here, we explore the protective effects of Cbl in HAEC exposed to various O2•− sources, including increased Hcy levels. Hcy increased O2•− levels (1.6-fold) in HAEC, concomitant with a 20% reduction in cell viability and a 1.5-fold increase in apoptotic death. Pretreatment of HAEC with physiologically relevant concentrations of cyanocobalamin (CNCbl) (10–50nM) prevented Hcy-induced increases in O2•− and cell death. CNCbl inhibited both Hcy and rotenone-induced mitochondrial O2•− production. Similarly, HAEC challenged with paraquat showed a 1.5-fold increase in O2•− levels and a 30% decrease in cell viability, both of which were prevented with CNCbl pretreatment. CNCbl also attenuated elevated O2•− levels after exposure of cells to a Cu/Zn-SOD inhibitor. Our data suggest that Cbl acts as an efficient intracellular O2•− scavenger.

Effect of multiple intravenous injections of butaphosphan and cyanocobalamin on the metabolism of periparturient dairy cows

Numerous adjunct therapeutic agents have been investigated for the treatment or control of fat mobilization syndrome in periparturient dairy cows. The aim of this study was to determine the effects of multiple i.v. injections of 10% butaphosphan and 0.005% cyanocobalamin combination (Catosal, Bayer Animal Health, Leverkusen, Germany) between 1 and 2 wk antepartum (a.p.) on the metabolism and health of dairy cows. Forty-five late-gestation Holstein-Friesian cows (second pregnancy) were allocated randomly to 1 of 3 groups with 15 cows/group: group C6 (6 daily i.v. injections of butaphosphan at 10mg/kg of body weight (BW) and cyanocobalamin at 5μg/kg of BW in the last 2 wk of gestation); group C3 (3 daily i.v. injections of butaphosphan at 10mg/kg of BW and cyanocobalamin at 5μg/kg of BW in the last week of gestation); and group C0 (equivolume daily i.v. injections of 0.9% NaCl solution). Serum biochemical analysis was performed on jugular venous blood samples that were periodically obtained a.p. and postpartum (p.p.). Health status and milk production were monitored p.p. Serum cyanocobalamin concentration increased in groups C6 and C3 p.p. Multiple daily i.v. injections of Catosal before parturition increased p.p. glucose availability, as evaluated by p.p. serum glucose concentration, and decreased peripheral fat mobilization and ketone body formation, as evaluated by p.p. serum nonesterified fatty acid and β-OH butyrate concentrations. The number of puerperal infections in the first 5 d after calving was decreased in group C6, relative to group C0. We conclude that multiple injections of Catosal during the close-up period have a beneficial effect on the metabolism of periparturient dairy cows. Our results are consistent with the hypothesis that high-producing dairy cows in early lactation may have a relative or actual deficiency of cyanocobalamin.

Effects of concentrations of cyanocobalamin in the gestation diet on some criteria of vitamin B12 metabolism in first-parity sows1,2

In swine nutrition, little is known about the role of vitamin B(12) in the reproductive processes. The current study was undertaken to obtain information on the dose-response pattern of different metabolic criteria related to the homeostasis of vitamin B(12) and homocysteine in gestating sows receiving various concentrations of dietary vitamin B(12) (cyanocobalamin). Homocysteine is a detrimental intermediate metabolite of the vitamin B(12)-dependent remethylation pathway of Met. Forty nulliparous (Large White x Landrace) sows were randomly assigned during gestation to dietary treatments containing 5 concentrations of cyanocobalamin (0, 20, 100, 200, or 400 microg/kg). During lactation, a diet containing 25 microg of cyanocobalamin/kg (as-fed) was given to all sows. During gestation, plasma vitamin B(12) increased as concentrations of dietary cyanocobalamin increased (linear and quadratic, P < 0.01) and the effect persisted during lactation (21 d postpartum) both in plasma (linear and quadratic, P < 0.05) and the liver (linear and quadratic, P < 0.04). Plasma homocysteine decreased with concentrations of cyanocobalamin provided to sows during gestation (linear, quadratic, and cubic, P < 0.01). At parturition, vitamin B(12) in colostrum increased as concentrations of cyanocobalamin increased (linear and quadratic, P < 0.01), but the treatment effect persisted (linear, P = 0.01) only up to 1 d postfarrowing. However, in piglets there was no treatment effect (P = 0.59) on plasma vitamin B(12) before colostrum intake, but a linear effect of concentrations of cyanocobalamin (P = 0.04) was observed 1 d later. Plasma homocysteine in piglets during lactation decreased with increasing concentrations of cyanocobalamin given to sows in gestation (linear and quadratic, P < 0.01). Based on a broken-line regression model, the concentrations of dietary cyanocobalamin that maximized plasma vitamin B(12) and minimized plasma homocysteine of sows during gestation were estimated to be 164 and 93 microg/kg, respectively. The maximal residual responses in sows and piglets during lactation were observed with treatments of 100 or 200 microg of cyanocobalamin/kg. The dietary cyanocobalamin concentration necessary to optimize the response of these metabolic criteria remains to be refined within lower and narrower ranges of cyanocobalamin concentrations (i.e., <200 mg/kg). Moreover, the biological significance of such concentrations of cyanocobalamin needs to be validated with performance criteria by using greater numbers of animals during several parities.

Non-enzymatic degradation of chlorofluorocarbon 113 using cyanocobalamin under anaerobic conditions

Chlorofluorocarbon 113 (CFC113) was rapidly and completely degraded non-enzymatically in systems containing cyanocobalamin with titanium (III) citrate as the reductant. Hydrochlorofluorocarbon 123a (HCFC123a) accounted for up to 25% of the degraded CFC113. Chlorotrifluoroethene was also detected. Increasing concentrations of cyanocobalamin increased CFC113 removal rates asymptotically and also decreased the fraction of HCFC123a remaining. Key words: chlorofluorocarbon 113, cyanocobalamin, anaerobic degradation, non-enzymatic.

Relation of blood cyanide to plasma cyanocobalamin concentration after a fixed dose of hydroxocobalamine in cyanide poisoning: Houeto P, Hoffman JR, Imbert M, et al. Lancet. 1995;346:923–926

Relation of blood cyanide to plasma cyanocobalamin concentration after a fixed dose of hydroxocobalamine in cyanide poisoning: Houeto P, Hoffman JR, Imbert M, et al. Lancet. 1995;346:923–926

Relation of blood cyanide to plasma cyanocobalamin concentration after a fixed dose of hydroxocobalamin in cyanide poisoning

Hydroxocobalamin combines with cyanide to form cyanocobalamin. We hypothesised that the amount of cyanocobalamin formed after a fixed dose of hydroxocobalamin given for cyanide poisoning would correlate with initial blood cyanide concentration. We determined blood cyanide concentration in 12 patients exposed to residential fires, and compared this with markers of the amount of cyanocobalamin formed after treatment with 5 g intravenous hydroxocobalamin. All relationships were highly correlated ( r 2 0·79-0·95), for the whole group, and there appeared to be an almost linear relationship for the 9 patients with initial cyanide concentration below 40 μmol/L. Above this concentration, no further cyanocobalamin was formed from a single 5 g dose of hydroxocobalamin. In one patient with initial blood cyanide concentration of 96 μmol/L, however, plasma cyanocobalamin concentration approximately doubled after a second 5 g dose of hydroxocobalamin. 5 g of hydroxocobalamin appears capable of binding all available cyanide ions for blood cyanide concentrations up to about 40 μmol/L. Beyond this, more hydroxocobalamin must be given for remaining cyanide ions to be bound. This information will allow clinicians to use rapidly measurable plasma cyanocobalamin concentrations to gauge severity of exposure and evaluate adequacy of treatment.

Determination of hydroxocobalamin and cyanocobalamin by derivative spectrophotometry in cyanide poisoning.

Hydroxocobalamin (OHCo) and cyanocobalamin (CNCo) are determined directly in biological media, without extraction, by using first derivative spectrophotometry. We diluted 200 mL of plasma, urine, or standards with 1.8 mL of pH 6 buffer (boric acid, potassium dihydrogen orthophosphate, and potassium hydroxide). The first derivative spectra of the dilutions were plotted between 320 and 400 nm. At the exact zero-crossing point for hydroxocobalamin, the derivative values of cyanocobalamin concentration were determined. The same procedure was followed for hydroxocobalamin at the zero-crossing point for cyanocobalamin. The derivative values of the concentration curves are linear in the range 5-100 microM. The minimum detection limit is approximately 5 microM for hydroxocobalamin of cyanocobalamin on the determination of hydroxocobalamin or vice versa, although the spectra strongly overlap. The method is fast and simple to use, thus making it easy to assess the in vivo transformation of hydroxocobalamin into cyanocobalamin after the administration of high doses of hydrocobalamin in cyanide poisoning.

Ultramicrodetermination of cyanocobalamin in elemental diet by solid-phase extraction and high-performance liquid chromatography with visible detection

The ultramicrodetermination of cyanocobalamin (9 ng/g) in elemental diet containing 46 kinds of compounds, with concentrations at least 50–10 6 times higher than that of cyanocobalamin, was performed by Sep-Pak C 18 purification and concentration of cyanocobalamin followed by HPLC with detection at 550 nm. The method is simple, rapid, sensitive and reproducible. The calibration graph was linear in the range of 0-0.2 μg. The recovery of cyanocobalamin was over 95% by the standard addition method. There was good agreement between the cyanocobalamin concentrations indicated and found.

Vitamin nutritional status in elderly Chinese subjects living in chronic care institutions

Dietary intake and blood levels of some vitamins were assessed in 208 elderly subjects (mean age 78.4±8.6 years) living in four chronic care institutions in Hong Kong. Compared to elderly free-living subjects, both men and women had lower intakes of thiamine, nicotinic and ascorbic acid, while in addition men had lower intakes of riboflavin and retinol equivalent. Plasma folate and 25 hydroxyvitamin D (25 OHD) concentrations were lower among both men and women in institutions. Men also had lower plasma retinol concentration and poorer thiamine status, while women had lower plasma ascorbic acid and cyanocobalamin concentrations. Vitamin supplementation and ability to self feed did not greatly affect vitamin nutritional status in this group of institutionalized subjects. Compared to elderly Caucasians living in chronic care institutions, elderly Chinese had poorer riboflavin, pyridoxine, nicotinic acid, vitamin A, and ascorbic acid status, but better cyanocobalamin, folic acid, and vitamin D status. Different dietary habits and quantity of sunshine probably contribute to these differences. Improvement in the vitamin nutritional status of elderly Chinese in chronic care institutions is desirable.

Diffusion permeability of cyanocobalamin in human placenta.

The molecular weight of cyanocobalamin is well suited to distinguish the two patterns of size discrimination found in the hemochorial and epitheliochorial placentas. The concentrations of cyanocobalamin that were used ensured that nondiffusional transport was negligible, and experiments on guinea pigs confirmed that the diffusion permeabilities measured with cyanocobalamin were the same as those expected for inert hydrophilic substances of similar molecular weight. All human studies were performed on volunteers who were scheduled for elective cesarean section under spinal or epidural anesthesia. Endogenous plasma concentrations of cobalamin were measured in 10 pregnant volunteers and their newborns. Ten additional volunteers were given 1 mg of cyanocobalamin by intramuscular injection 44 min before delivery. Placental permeability, calculated as the ratio of fetal uptake and the concentration-time integral between maternal and fetal plasmas, was 14.3 microliter X min-1 X g placental wt-1. The permeability of the human placenta parallels that of the histologically similar guinea pig placenta, at a slightly higher level, up to molecular weights of 1,355.

Activation of the methylreductase system from Methanobacterium bryantii by corrins.

Corrins activated the methylreductase system from Methanobacterium bryantii three- to fivefold in extracts resolved from low-molecular-weight factors. Corrins did not substitute for ATP and component B, which were also required for maximal activity. The concentration of diaquacobinamides required for one-half maximal activity was 1 microM. The concentrations of cyanocobalamin, methylcobalamin, Co alpha-(5-hydroxybenzimidazoyl)-Co beta-cyanocobamide, and 5'-deoxyadenosylcobinamide required for one-half maximal activity were between 4 and 7 microM. Deoxyadenosylcobalamin was nearly inactive. Activation was independent of thiols, coenzyme M, and ATP. Activation was also observed after partial purification of the methylreductase system by agarose column chromatography. Corrins were required in catalytic concentrations, methylcobalamin was not required, and methanogenesis was enzymatic. Corrin activation of the methylreductase is a novel effect on methanogenesis. However, the physiological significance of the corrin activation is uncertain.

Kinetics of the Attachment of Intrinsic Factor-Bound Cobamides to Ileal Receptors

To determine whether the molecular configuration of vitamin B(12) influences the attachment of intrinsic factor-vitamin B(12) complex to ileal microvillous membrane receptor sites, we have examined the kinetics of uptake of intrinsic factor-bound cyanocobalamin by brush borders and microvillous membranes isolated from guinea pig ileum, and have compared this uptake with that of intrinsic factor alone and with that of intrinsic factor complexed with various analogs of cyanocobalamin. We first studied the kinetics of binding of cyanocobalamin and other cobamides to human gastric intrinsic factor. The binding of cyanocobalamin showed saturation kinetics and, at relatively high concentrations of cyanocobalamin, a Scatchard plot of binding was linear. The dissociation constant for the intrinsic factor-cyanocobalamin complex was 0.066 nM. When the binding of various vitamin B(12) analogs to intrinsic factor was determined by competition experiments, the analogs could be separated into two categories: those with affinities similar to that of cyanocobalamin and those with affinities much lower than that of cyanocobalamin. The affinity of cyanocobalamin for intrinsic factor was not altered by various substitutions at the -CN position, while removal of a single amido group on the corrin ring of substitution of the dimethylbenzimidazole base greatly reduced affinity. Removal of the base totally abolished binding. These findings, confirming those reported by others, are consistent with the concept that the cyanocobalamin molecule fits into a "pocket" in the intrinsic factor molecule, with the nucleotide base facing inward and the -CN side of the planar corrin ring facing outward. We then investigated the attachment of intrinsic factor-bound cyanocobalamin to ileal receptor. Attachment to microvillous membranes showed saturation kinetics with a dissociation constant of 0.25 nM. Attachment was rapid and was 70% complete within 5 min; the second-order rate constant for attachment was 1.3 x 10(6) M(-1) s(-1). The half-time for dissociation of intrinsic factor-bound cyanocobalamin from the ileal receptor was approximately 35 min. Free intrinsic factor inhibited the attachment of intrinsic factor-bound cyanocobalamin, but the rate of attachment of free intrinsic factor was slower than that of intrinsic factor bound to cyanocobalamin. When intrinsic factor was complexed with various analogs of cyanocobalamin, the affinities of these complexes for ileal microvillous membranes were similar to that of intrinsic factor-bound cyanocobalamin. These findings suggest that the molecular configuration of vitamin B(12) is not a major determinant in the interaction between intrinsic factor-bound vitamin B(12) and its ileal receptor site.