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An estimated 500,000 individuals in the US, mostly steroid-dependent asthmatics, suffer severe adverse reactions to sulfites in foods, beverages, and pharmaceutical products. In an attempt to understand the pathogenesis of sulfite hypersensitivity, we have developed an assay for the determination of total serum sulfite by utilizing: (a) reductive release of serum protein-bound sulfite; (b) derivatization of free sulfite with monobromobimane; (c) separation of sulfite-bimane from thiol-bimanes by reversed-phase HPLC; and (d) quantitation of sulfite-bimane by fluorescence detection. The detection limit of this assay was 0.44 mumol/L serum sulfite. The intra- and interassay CVs for total serum sulfite at 5.4 mumol/L were 8.1% and 22.0%, respectively. The standard addition method was used to determine total serum sulfite in normal subjects. More than 70 samples were prepared in 2-3 h, followed by automated overnight analysis. The mean concentrations (+/- SD) of total serum sulfite in female (n = 41) and male (n = 35) donors were 4.63 +/- 2.33 and 5.16 +/- 2.68 mumol/L, respectively (not statistically significant: P = 0.368). The combined mean concentration of total sulfite in both sexes was 4.87 +/- 2.49 mumol/L. There was no correlation between total serum sulfite and total serum cysteine, cysteinylglycine, homocysteine, subject age, serum cobalamin, or serum folic acid. The reference range (mean +/- 2 SD) for total serum sulfite in normal subjects is 0-9.85 mumol/L.

A rapid solid-phase extraction procedure utilizing C18 reverse-phase mini-columns has been established for dopamine, 3,4-dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindoleacetic acid in tissue perfusate samples. The method rapidly separates the analytes from protein in albumin-containing tissue perfusates. The analytes which are recovered in high yield are then determined by high-performance liquid chromatography with electrochemical detection. The mean interassay coefficient of variation for the recovery of dopamine, 3, 4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid was 12, 7, 13 and 8 percent, respectively.

High plasma homocysteine (tHcy) is a risk factor for cardiovascular disease and stroke and Alzheimer's disease (AD). An inverse relationship has been reported between tHcy and plasma B12 and folate levels. Seventy-nine AD patients and 156 controls from three Arab villages in northern Israel participated. Plasma tHcy, B12 and folate levels were determined. Data were analyzed using univariate statistical tests and logistical regression with confounders. tHcy was significantly higher in AD patients (20.6+/-8.7 micromol/l) than in controls (16.4+/-6.5 micromol/l) (p=0.03) after correction for year of birth, gender and smoking status. Plasma B12 (322.9+/-136.0/350.5+/-175.3 pmol/l) and plasma folate (4.5+/-3.8/4.9+/-2.6 nmol/l) levels did not differ significantly between AD patients and controls. Subjects in the highest tHcy tertile or in the lowest B12 and folate tertiles did not have greater risk to develop AD. In this population residing in Arab villages in northern Israel, tHcy levels were significantly higher among AD patients than in controls. Plasma B12 and folate levels were lower among cases but were not significant. There was not a significant association between plasma tHcy, B12 and folate levels in controls or AD patients. High levels of tHcy may suggest the need for folate and vitamin B12 supplementation in this population.

To study the association between Alzheimer s disease (AD) and plasma total homocysteine (tHcy), dietary folate and vitamin B6.64 AD patients were matched by gender, age, and smoking status to 64 healthy controls. tHcy was determined using an automated immunoassay. Dietary patterns for three age periods (20-39, 40-59, and 60 + yrs) were assessed using a questionnaire adapted from the Block Health Habits and History Questionnaire. Respondents (cases by proxy) reported food frequencies, which were translated into estimated daily nutrient intakes. APOE genotype, cognitive performance (CDR, MMSE), blood lipids, and albumin were obtained for patients and controls.tHcy did not differ significantly between controls (11.5 +/- 3.7 mmol/L) and AD patients (12.3 +/- 4.3 mmol/L)(p=0.25). tHcy levels were not related in AD patients or controls to education, CDR, MMSE, blood lipids, albumin or ApoE genotype (p0.15). There was a negative correlation between plasma tHcy and triglyceride levels in AD patients (p=0.023), but not in controls. AD patients consumed significantly less dietary vitamin B6 (p=0.05) and folate (p=0.001) after age 60 than controls.Although plasma tHcy levels were higher in cases than controls, this difference was not significant. tHcy levels were not related to cognitive status. Plasma tHcy was inversely correlated with triglyceride levels in AD patients but not in controls.

CONTEXT: It has been suggested that total blood homocysteine concentrations are associated with the risk of ischemic heart disease (IHD) and stroke. OBJECTIVE: To assess the relationship of homocysteine concentrations with vascular disease risk. DATA SOURCES: MEDLINE was searched for articles published from January 1966 to January 1999. Relevant studies were identified by systematic searches of the literature for all reported observational studies of associations between IHD or stroke risk and homocysteine concentrations. Additional studies were identified by a hand search of references of original articles or review articles and by personal communication with relevant investigators. STUDY SELECTION: Studies were included if they had data available by January 1999 on total blood homocysteine concentrations, sex, and age at event. Studies were excluded if they measured only blood concentrations of free homocysteine or of homocysteine after a methionine-loading test or if relevant clinical data were unavailable or incomplete. DATA EXTRACTION: Data from 30 prospective or retrospective studies involving a total of 5073 IHD events and 1113 stroke events were included in a meta-analysis of individual participant data, with allowance made for differences between studies, for confounding by known cardiovascular risk factors, and for regression dilution bias. Combined odds ratios (ORs) for the association of IHD and stroke with blood homocysteine concentrations were obtained by using conditional logistic regression. DATA SYNTHESIS: Stronger associations were observed in retrospective studies of homocysteine measured in blood collected after the onset of disease than in prospective studies among individuals who had no history of cardiovascular disease when blood was collected. After adjustment for known cardiovascular risk factors and regression dilution bias in the prospective studies, a 25% lower usual (corrected for regression dilution bias) homocysteine level (about 3 micromol/L [0.41 mg/L]) was associated with an 11% (OR, 0.89; 95% confidence interval [CI], 0.83-0.96) lower IHD risk and 19% (OR, 0.81; 95% CI, 0.69-0.95) lower stroke risk. CONCLUSIONS: This meta-analysis of observational studies suggests that elevated homocysteine is at most a modest independent predictor of IHD and stroke risk in healthy populations. Studies of the impact on disease risk of genetic variants that affect blood homocysteine concentrations will help determine whether homocysteine is causally related to vascular disease, as may large randomized trials of the effects on IHD and stroke of vitamin supplementation to lower blood homocysteine concentrations.

Mild hyperhomocysteinemia has been established as a new independent risk factor for atherosclerosis and thrombosis. The metabolic syndrome of insulin resistance is associated with a high risk of coronary heart disease. Our objective was to determine if any relationship exists between the metabolic syndrome of insulin resistance in non-diabetic subjects and total serum homocysteine levels. Sixty-six healthy volunteers (33 males and 33 females) were selected from the population of Pilsen. Insulin resistance was measured by the Insulin Suppression Test using Octreotide. Steady-state plasma glucose concentrations at the end of the test period provided a quantitative measure of insulin resistance. Serum homocysteine level was estimated by high-pressure liquid chromatography. Serum folate and vitamin B12 were estimated using commercial kits on an Abbott IMx analyzer. All other laboratory tests were performed by standard methods in a routine biochemical laboratory. Subjects with the highest tertile of steady-state plasma glucose showed a significantly higher body mass index, blood pressure, fasting plasma triglyceride levels, plasminogen activator inhibitor-1 and lower HDL-cholesterol, i.e. an insulin resistance pattern. These subjects had significantly lower serum homocysteine levels compared with non-insulin resistant subjects. The negative association of insulin resistance and serum homocysteine was unexpected. The contribution of plasma folate levels to serum homocysteine levels and serum creatinine was significantly negative and positive, respectively.

In banked human erythrocytes (RBCs), biochemical and functional changes are accompanied with vesiculation and reduced in vivo survival. We hypothesized that some of these changes might have resulted from oxidative modification of membrane lipids, proteins, or both as a result of atrophy of the antioxidant defense system(s). In banked RBCs, we observed a time-dependent increase in protein clustering, especially band 3; carbonyl modification of band 4.1; and malondialdehyde, a lipid peroxidation product. Examination of the antioxidative defense system showed a time-dependent decline in glutathione (GSH) concentration and glutathione-peroxidase (GSH-PX) activity, with a concomitant increase in extracellular GSH, cysteine, and homocysteine, and unchanged catalase activity. When subjected to acute oxidant stress by exposure to ferric/ascorbic acid or tert-butylhydroperoxide (tert-BHT), catalase activity showed a steeper decline compared with GSH-PX. The results demonstrate that GSH and GSH-PX appear to provide the primary antioxidant defense in stored RBCs, and their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.

Static light scattering measurements have been used to determine the molecular mass (65.3 kDa) and second virial coefficient (3.66 x 10(-4) mol mL g-2) for the complex between chicken serum haptocorrin (HC) and aquacobalamin (H2OCbl). Dynamic light scattering measurements have also been used to determine the translational diffusion coefficient of the H2OCbl-HC complex as a function of concentration. From the diffusion coefficient at infinite dilution (4.71 x 10(-7) cm2 s-1), the hydrodynamic radius (45.5 A) and rotational correlation time (85.4 ns) have been calculated. Using the latter, and measured values of the 31P NMR linewidths of the H2OCbl-HC complex at several field strengths, a detailed analysis of the 31P nuclear relaxation is possible. The chemical shift anisotropy term from the transverse relaxation equation is determined to be 95.7 ppm, and the average phosphorus-proton internuclear distance is 2.05 A. For comparison to protein-free H2OCbl, the chemical shift anisotropy of the phosphorus atom was studied by solid state NMR spectroscopy and the 31P relaxation by solution T1 measurements. These studies give values of 110.3 ppm for the chemical shift anisotropy term and 2.45 A for the average phosphorus-proton internuclear distance. The results are consistent with a significant change in the conformation of the H2OCbl phosphodiester upon binding to haptocorrin which could be due to a shortening of the axial Co-N bond.

On the basis of recent retrospective and prospective studies, it is now widely accepted that increased total plasma homocysteine is a risk factor for cardiovascular disease. Impaired enzyme function as a result of genetic mutation or deficiency of the essential B vitamins folic acid, B12, and B6 can lead to hyperhomocysteinemia. Oxidized forms of homocysteine account for 98-99% of total plasma homocysteine. Although there is uncertainty as to whether increased homocysteine is causal or merely a proxy for cardiovascular disease, several lines of evidence suggest that it may play a role in atherothrombotic disease. Homocysteine appears to alter the anticoagulant properties of endothelial cells to a procoagulant phenotype. Mildly increased homocysteine causes dysfunction of the vascular endothelium. Folic acid effectively lowers homocysteine concentration in the plasma. Intervention studies are urgently needed to determine if lowering homocysteine is effective in decreasing the morbidity and mortality of cardiovascular disease.

An estimated 500,000 individuals in the US, mostly steroid-dependent asthmatics, suffer severe adverse reactions to sulfites in foods, beverages, and pharmaceutical products. In an attempt to understand the pathogenesis of sulfite hypersensitivity, we have developed an assay for the determination of total serum sulfite by utilizing: (a) reductive release of serum protein-bound sulfite; (b) derivatization of free sulfite with monobromobimane; (c) separation of sulfite-bimane from thiol-bimanes by reversed-phase HPLC; and (d) quantitation of sulfite-bimane by fluorescence detection. The detection limit of this assay was 0.44 mumol/L serum sulfite. The intra- and interassay CVs for total serum sulfite at 5.4 mumol/L were 8.1% and 22.0%, respectively. The standard addition method was used to determine total serum sulfite in normal subjects. More than 70 samples were prepared in 2-3 h, followed by automated overnight analysis. The mean concentrations (+/- SD) of total serum sulfite in female (n = 41) and male (n = 35) donors were 4.63 +/- 2.33 and 5.16 +/- 2.68 mumol/L, respectively (not statistically significant: P = 0.368). The combined mean concentration of total sulfite in both sexes was 4.87 +/- 2.49 mumol/L. There was no correlation between total serum sulfite and total serum cysteine, cysteinylglycine, homocysteine, subject age, serum cobalamin, or serum folic acid. The reference range (mean +/- 2 SD) for total serum sulfite in normal subjects is 0-9.85 mumol/L.

High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;178:208), which used monobromobimane to specifically derivatize thiols, has been extensively modified to allow for rapid processing of samples. As a result,80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limit of detectionor = 4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 mumol/L, respectively; P0.001) and healthy female donors (7.85 and 10.34 mumol/L, respectively; P0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.

Glutathionylcobalamin (GSCbl), the complex formed between glutathione (GSH, gamma-glutamylcysteinylglycine) and aquacobalamin (H2OCbl), has been implicated as an intermediate in the pathway for the formation of the cobalamin coenzymes. In chemical model studies, GSCbl has been shown to be a substrate for methylcobalamin formation in the presence of S-adenosylmethionine and a thiol reductant. Although GSCbl was first described in 1964, the structure of this compound, particularly the site of GSH coordination, has been unknown. GSCbl was prepared by reacting GSH (5-fold molar excess) with H2OCbl in 0.1 M sodium phosphate (pH 6.5) and was purified by gel-permeation chromatography on a Bio-Gel P2 polyacrylamide column. By use of a combination of homonuclear [homonuclear J-correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and absorption-mode nuclear Overhauser effect spectroscopy (NOESY)] and inverse detected heteronuclear [1H-detected heteronuclear multiple-quantum coherence (HMQC) and 1H-detected multiple-bond heteronuclear multiple-quantum coherence (HMBC) spectroscopies] two-dimensional NMR methods at 600 MHz, the complete 1H and 13C NMR spectra of GSCbl have now been assigned. Comparison of the 1H and 13C NMR chemical shifts of the GS moiety of GSCbl to those of free GSH and GS- shows that by far the largest differences occur at the cysteine alpha and beta positions. This result strongly suggests that GSH is coordinated to the cobalt atom in GSCbl via the cysteine sulfur atom.

Binding of neopentylcobalamin and benzylcobalamin to the apoprotein of a haptocorrin from chicken serum has been demonstrated spectrophotometrically. The spectra of the protein-bound cobalamins strongly resemble those of base-on alkylcobalamins and show that when unbound these sterically hindered alkylcobalamins are only approximately 75% (benzyl) and 40% (neopentyl) base-on, at neutral pH and at 5 degrees C. The haptocorrin was found to stabilize the spontaneous thermal decomposition of the neutral species of benzylcobalamin and neopentylcobalamin by 470-fold (3.6 kcal) and 166-fold (3.0 kcal), respectively, relative to the protein-free species. After correction of the activation parameters for the thermal decomposition of the protein-free, neutral alkylcobalamins for the relative proportions of base-on and base-off species, the haptocorrin was found to stabilize the base-on species of both alkylcobalamins by 275- to 1400-fold (approximately 3.3 to 4.3 kcal). From the temperature dependence of the decomposition reactions, the enthalpies of activation are found to be essentially identical for the protein-free and protein-bound species of either cobalamin. Thus, stabilization of the thermal decomposition of these sterically hindered alkylcobalamins by haptocorrin is entirely due to entropic factors.

Plasma membrane receptors for the serum cobalamin-binding protein transcobalamin II (TCII) were identified on human leukemia K562 and HL-60 cells using immunoaffinity-purified human TCII labeled with [57Co]cyanocobalamin. The Bmax values for TCII receptors on proliferating K562 and HL-60 cells were 4,500 and 2,700 per cell, respectively. Corresponding dissociation constants (kd) were 8.0 x 10(-11) mol/L and 9.0 x 10(-11) mol/L. Rabbit TCII also bound to K562 and HL-60 cells but with slightly reduced affinities. Calcium was required for the binding of transcobalamin II to K562 cells. Brief treatment of these cells with trypsin resulted in almost total loss of surface binding activity. After removal of trypsin, surface receptors for TCII slowly reappeared, reaching pretrypsin treatment densities only after 24 hours. Reappearance of receptors was blocked by cycloheximide. TCII receptor densities on K562 and HL-60 cells correlated inversely with the concentration of cobalamin in the culture medium. This suggests that intracellular stores of cobalamin may affect the expression of transcobalamin receptors. Nonproliferating stationary-phase K562 cells had low TCII receptor densities (less than 1,200 receptors/cell). However, the density of TCII receptors increased substantially when cells were subcultured in fresh medium. Up-regulation of receptor expression coincided with increased 3H-thymidine incorporation, which preceded the resumption of cellular proliferation as measured by cell density. In the presence of cytosine arabinoside, which induces erythroid differentiation, K562 cells down-regulated expression of TCII receptors. When HL-60 cells were subcultured in fresh medium containing dimethysulfoxide to induce granulocytic differentiation, the up-regulation of TCII receptors was suppressed. This event occurred well before a diminution of 3H-thymidine incorporation and cessation of proliferation. Thus, changes in the regulation of expression of TCII receptors correlate with both the proliferative and differentiation status of cells.

A vitamin B12-binding protein (haptocorrin) from chicken serum has been purified to homogeneity by photodissociative affinity chromatography and characterized by gel electrophoresis and UV-visible spectrophotometry of its aquocobalamin, hydroxocobalamin, and cyanocobalamin complexes. The haptocorrin is a glycoprotein with a molecular mass of about 70 kDa and a protein moiety of about 40 kDa. 31P NMR resonances of the haptocorrin-cobalamin complexes are relatively broad singlets (with or without proton decoupling) shifted downfield by 0.7-1.0 ppm from the position of the free cobalamin resonances. From the line width data, the relaxation of the phosphorus nucleus is found to be dominated by chemical shift anisotropy with a very minor (13%) component from dipolar interaction with the two nearest neighbor protons. The rotational correlation time of the haptocorrin at 25 degrees C is estimated to be 85 ns and the activation energy for rotational correlation 3.9 +/- 0.3 kcal mol-1. The downfield shift of the 31P resonances of cobalamins upon binding to the haptocorrin cannot be due to hydrogen bonding phosphodiester moiety or displacement of the axial base by a group on the protein. Calculations also show that the downfield shift is very unlikely to be due to dipolar deshielding of the phosphorus nucleus by the ring current of an aromatic residue of the protein. It is concluded that the downfield shift of the 31P resonance must be due to sterically induced changes in phosphodiester conformation which may, or may not, involve steric compression of the axial Co-N bond.

Folate and B12 coenzymes are involved in the biosynthesis of purines, pyrimidines and amino acids and thus play an important role in cell replication. Regulation of folate- and B12-dependent metabolism, and the possible exploitation of such regulation in cancer chemotherapy, can be achieved at several different levels: (a) transport of the vitamins into cells; (b) conversion of these vitamins to their coenzyme forms; and (c) reactions catalyzed by coenzyme-dependent systems. Transport of folate compounds into mammalian cells is an energy-dependent, carrier-mediated process. Three separate lines of evidence (substrate competition, mutant sublines and sulfhydryl inhibitors) indicate that L1210 cells utilize two separate carrier systems for folate compounds. One is specific for folate, while the other is specific for 5-methyl tetrahydrofolate; the latter system also transports tetrahydrofolate, 5-formyl tetrahydrofolate (folinate) and amethopterin (Methotrexate). In contrast, Lactobacillus casei utilizes a single carrier system for all of the folate compounds. The intracellular conversion of folate to the coenzyme, tetrahydrofolate, is mediated by the TPNH-dependent enzyme, dihydrofolate reductase. This enzyme is the target for folate antagonists such as amethopterin. Interaction with the drug, however, is markedly dependent upon prior binding of TPNH to the enzyme. Treatment of cells with amethopterin generally causes the level of dihydrofolate reductase to rise in subsequent generations. This has been attributed to a decreased rate of degradation of the enzyme (stabilized by complex formation with amethopterin) rather than to an increased rate of synthesis. Polyacrylamide gel electrophoresis and absorbance spectra have been used to study the interaction of dihydrofolate reductases from L1210 cells and L. casei with various substrates and inhibitors. Transport of vitamin B12 is an energy-dependent, carrier-mediated process. In mammalian cells transport requires, in addition to the membrane carrier system, an external protein, transcobalamin-II. Analogs of adenosyl cobalamin inhibit this process. Conversion of B12 to its coenzyme forms (adenosyl- and methyl cobalamin) is accomplished by the two-step reduction of B12a (CoIII) to B12r (CoII) to B12s (CoI), followed by reaction of the latter with ATP or adenosyl methionine. A variant of this system has been encountered in the activation of the B12-containing methionine synthetase by TPNH and two flavoproteins.

Sephacryl beads containing an immobilized aminopropylcobalamin-transcobalamin-II complex serve as foci for the adherence of L1210 murine leukemia cells. Bead-cell interaction does not occur when (A) nonderivatized beads are used; (B) transcobalamin-II is omitted or presaturated with cyanocobalamin in the preparation of the bead complex; (C) intrinsic factor replaces transcobalamin-II; and (D) the complex is removed from beads by photolysis. These observations suggest that adherence results from the ability of transcobalamin-II to form a bridge between immobilized cobalamin on the bead and receptors in the plasma membrane of the cell.

The kinetics of the reaction of cyanide with free aquocobalamin (H2OCbl) and with aquocobalamin bound to a vitamin B12-binding protein (haptocorrin) from chicken serum (HC-H2OCbl) have been investigated as a function of temperature and pH. The mechanism of replacement of H2O from protein-bound and free H2OCbl is apparently the same and involves attack of both CN- (k1) and HCN (k2). The reactions with HC-H2OCbl are somewhat slower than those with free H2OCbl, k1 being 22-fold smaller and k2 7-fold smaller at 25 degrees C. The relatively small effect of the protein on the rate constants supports the view that the metal in HC-H2OCbl is readily accessible to solvent. Activation parameters suggest that the transition states for ligand substitution are stabilized by nucleophilic participation (at least by CN-) and that ligand substitution on the protein-bound cobalamin proceeds through more ordered, concerted transition states. The latter effect suggests that the Co-O bond of H2OCbl is strengthened upon binding to the haptocorrin.

Absorption of radiolabeled cobalamin in baboons was assessed by whole body counting. Retention of biliary cobalamin and an aqueous solution of cyanocobalamin was measured in normal baboons and in baboons after total gastrectomy by using 57Co-labeled biliary cobalamin and 58C0-cyanocobalamin, with and without baboon gastric juice containing intrinsic factor. Radiolabeled biliary cobalamin was obtained by intravenous injection of 57Co-cyanocobalamin in baboons and collection of bile through a cannula placed in the common bile duct. Cobalamin absorption was not completely abolished by gastrectomy and biliary cobalamin was better retained than cyanocobalamin; intrinsic factor enhanced absorption of both forms. After gastrectomy there was steady depletion of liver and serum cobalamin levels, which ceased after a new equilibrium was reached between a progressively diminishing cobalamin loss and the impaired but significant residual level of absorption. These studies in the nonhuman primate provide further information concerning the enterohepatic circulation of cobalamin and suggest that the form of cobalamin in bile may be more readily absorbed than is cyanocobalamin or that bile itself may have an enhancing effect on cobalamin absorption. The data also suggest that physiologically significant amounts of cobalamin may be absorbed in the absence of a gastric source of intrinsic factor.

A vitamin B12-binding protein (haptocorrin) from chicken serum has been purified to homogeneity by photodissociative affinity chromatography and characterized by gel electrophoresis and UV-visible spectrophotometry of its aquocobalamin, hydroxocobalamin, and cyanocobalamin complexes. The haptocorrin is a glycoprotein with a molecular mass of about 70 kDa and a protein moiety of about 40 kDa. 31P NMR resonances of the haptocorrin-cobalamin complexes are relatively broad singlets (with or without proton decoupling) shifted downfield by 0.7-1.0 ppm from the position of the free cobalamin resonances. From the line width data, the relaxation of the phosphorus nucleus is found to be dominated by chemical shift anisotropy with a very minor (13%) component from dipolar interaction with the two nearest neighbor protons. The rotational correlation time of the haptocorrin at 25 degrees C is estimated to be 85 ns and the activation energy for rotational correlation 3.9 +/- 0.3 kcal mol-1. The downfield shift of the 31P resonances of cobalamins upon binding to the haptocorrin cannot be due to hydrogen bonding phosphodiester moiety or displacement of the axial base by a group on the protein. Calculations also show that the downfield shift is very unlikely to be due to dipolar deshielding of the phosphorus nucleus by the ring current of an aromatic residue of the protein. It is concluded that the downfield shift of the 31P resonance must be due to sterically induced changes in phosphodiester conformation which may, or may not, involve steric compression of the axial Co-N bond.

Biliary excretion of cobalamin was studied in baboons after intravenous injection of 57Co-radiolabeled cyanocobalamin. Radioactivity appeared in bile after 20 min, and up to 4.4% of the dose was recovered in 6 h. It was estimated that 4 microgram cobalamin traverses the baboon biliary tract each day. These studies in the nonhuman primate confirm that there is considerable biliary excretion of cobalamin and that enterohepatic circulation of the vitamin is important for maintenance of normal cobalamin balance.

The kinetics of the reaction of cyanide with free aquocobalamin (H2OCbl) and with aquocobalamin bound to a vitamin B12-binding protein (haptocorrin) from chicken serum (HC-H2OCbl) have been investigated as a function of temperature and pH. The mechanism of replacement of H2O from protein-bound and free H2OCbl is apparently the same and involves attack of both CN- (k1) and HCN (k2). The reactions with HC-H2OCbl are somewhat slower than those with free H2OCbl, k1 being 22-fold smaller and k2 7-fold smaller at 25 degrees C. The relatively small effect of the protein on the rate constants supports the view that the metal in HC-H2OCbl is readily accessible to solvent. Activation parameters suggest that the transition states for ligand substitution are stabilized by nucleophilic participation (at least by CN-) and that ligand substitution on the protein-bound cobalamin proceeds through more ordered, concerted transition states. The latter effect suggests that the Co-O bond of H2OCbl is strengthened upon binding to the haptocorrin.

The heptapeptide angiotensin-(1-7) is a circulating biologically active product of the renin-angiotensin system. In this study, we evaluated the role of the vascular endothelium in the formation of angiotensin-(1-7). Metabolism of 125I-angiotensin I was investigated using confluent cultured bovine and human aortic and umbilical vein endothelial cells. The fetal calf serum-supplemented medium was replaced by serum-free medium containing 0.2% bovine serum albumin. One hour later, this medium was replaced by serum-free medium containing 125I-angiotensin I. After incubation of 125I-angiotensin I for various intervals at 37 degrees C, the medium was collected and analyzed for formed products by high-performance liquid chromatography. Products of angiotensin I metabolism were identified by comparison of their retention times with those of radiolabeled standards. The contribution of proteases released into the medium was evaluated by incubation of 125I-angiotensin I with medium previously incubated for 1 hour with endothelial cells. Incubation of 125I-angiotensin I with bovine and human endothelial cells produced a time-dependent generation of 125I-angiotensin-(1-7) greater than 125I-angiotensin II greater than 125I-angiotensin-(1-4). Generation of angiotensin peptides was not due to the presence of proteases in the medium. When human umbilical endothelial cells were incubated in the presence of the angiotensin converting enzyme inhibitor enalaprilat (1 microM), generation of angiotensin II was undetectable. In contrast, angiotensin-(1-7) production increased by an average of 30%.(ABSTRACT TRUNCATED AT 250 WORDS)