Development of a method for the separation of corrinoids in ovine tissues by HPLC

A method has been developed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to routinely estimate the distribution of corrinoids (the cobalamins hydroxocobalamin, methylcobalamin and 5'-deoxyadenosylcobalamin, and cobalamin analogues) in liver, plasma, milk, intestinal fluid and faeces. Corrinoids were extracted with a sodium acetate buffer, separated by HPLC and quantified by RIDA. Recoveries of corrinoids were 29% for hydroxocobalamin, 50% for 5'-deoxyadenosylcobalamin and 64% for methylcobalamin. The method allows the routine analysis of many samples and maintains good standards of precision.     

Cobalamins and the spectrochemical series

UV-visible-NIR spectra of a variety of cobalamins were run in water and methanol. A broad absorption band (band A) with extinction coefficients of about an order of magnitude less than those of the alphabeta bands was found in the red and NIR regions for Cl-cobalamin (Cl-cbl), Br-cbl, I-cbl, SC(NH(2))(2)-cbl(+) and SeCN-cbl. OCrO(3)-cbl(-), which also has a broad absorption band in the NIR was prepared for the first time. After deconvolution, similar broad bands were seen in the visible region for many other cobalamins. The wavelengths for band A placed the cobalamins in an order similar to the spectrochemical series but different from that of the alphabeta and gamma bands (pi-pi* transitions), which follow the nephelauxetic series. Band A was ascribed to a ligand-to-metal charge transfer (LMCT) transition from a pi orbital in the corrin ring to Co(iii). This is the first systematic study of LMCT bands in cobalamins.     

Determination of cobalamins using capillary electrophoresis inductively coupled plasma mass spectrometry

The determination of cobalamins using capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) was investigated. Both capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) modes of operation were studied. The optimal separation of four cobalamin species (cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5′-deoxyadenosylcobalamin) and a potentially harmful corrinoid analogue (cobinamide dicyanide) was obtained using CZE at a pH of 2.5. Both 20 mM phosphate and 20 mM formate buffers were used with success, although the formate buffer provided improved resolution. The CZE-ICP-MS method was used to quantify cyanocobalamin in a vitamin supplement and the analytical results were in good agreement (±5%) with values obtained by ICP-MS for total Co levels. The solution detection limits for cobalamins using CZE-ICP-MS were approximately 50 ng/ml. MEKC was found to be useful for the screening of vitamin preparations because it provided a rapid means of distinguishing cyanocobalamin (the form most commonly used in vitamin preparations) from free cobalt. The separation of free cobalt and cyanocobalamin using MEKC was achieved in less than 10 min.     

Heteronuclear NMR Studies of Cobalt Corrinoids. 18. Correlation of Structure and Magnetic Resonance Parameters in Base-On Cobalamins(1)

Recent X-ray crystal structure determinations (including a new X-ray determination of the structure of cyano-13-epicobalamin reported herein) create a series of seven base-on cobalamins structurally characterized by modern crystallographic techniques in which the intramolecular equilibrium constant for coordination of the axial benzimidazole ligand (Bzm) varies from 76.6 to 4.90 x 10(7). For the five normal, unepimerized cobalamins, the free energy change for this equilibrium correlates linearly with the axial Co-N bond length (r(2) = 0.99). Absolute assignment of the (1)H and (13)C NMR spectra of two of these structurally characterized cobalamins (CH(3)Cbl and CN-13-epiCbl) together with literature assignments for the other complexes now provides reliable (13)C NMR assignments and chemical shifts for all seven complexes. The magnetic anisotropies of the central cobalt atom of all seven complexes, estimated by a method described earlier, are well correlated with the axial Co-N bond distance (r(2) = 0.97) and the free energy of coordination of the Bzm ligand (r(2) = 0.95). The (31)P NMR chemical shift of the phosphodiester moiety of the nucleotide loop is excellently correlated to the axial Co-N bond length (r(2) = 0.996) of the unepimerized cobalamins and provides a reliable method of estimating this bond length. The (15)N chemical shifts of the axially coordinated Bzm nitrogen vary strongly with the axial Co-N bond distance and correlate linearly with this structural parameter (r(2) = 0.991) except for the case of H(2)OCbl(+), which deviates substantially. However, there is a good linear correlation (r(2) = 0.98) of this (15)N chemical shift with the free energy of Bzm coordination for the five unepimerized cobalamins. Attempts to correlate (13)C NMR chemical shifts with structural, thermodynamic, and corrin ring conformational parameters are discussed.     

Similarities and differences between cobalamins and cobaloximes. Accurate structural determination of methylcobalamin and of LiCl- and KCl-containing cyanocobalamins by synchrotron radiation

The accurate crystal structure determinations of MeCbl (1), CNCbl.2LiCl (2), and CNCbl.KCl (3), based on synchrotron diffraction data collected at 100 K and using high-quality single crystals, are reported. Refinements gave R1 indices of 0.0834 (1), 0.0434 (2), and 0.0773 (3). The influence of the water of crystallization and ion content on the crystal packing of these and other cobalamins (XCbl) is discussed, and a relationship between the crystal packing and the corrin side chain conformations is presented. An analysis of the bond lengths within the corrin moiety, based on 13 accurate structures with several X groups, shows that the trend of the C-C and C-N distances can be interpreted in terms of electronic and steric factors. The variation in structural, NMR and IR spectroscopic, and electrochemical properties are compared with those of cobaloximes, the B12 model, when X is varied. This comparison indicates that the pi-back-donation from metal to the CN axial ligand and the transmission of the trans influence of the X ligand are more effective in cobalamins than in cobaloximes. These findings are consistent with a significantly greater availability of electron charge on Co in cobalamins, and, hence, a semiquantitative evaluation of the electronic difference between the cobalt centers in the two systems is allowed.     

Antivitamins B12—A Structure‐ and Reactivity‐Based Concept

B₁₂‐antimetabolites are compounds that counteract the physiological effects of vitamin B₁₂ and related natural cobalamins. Presented here is a structure‐ and reactivity‐based concept of the specific ′antivitamins B₁₂′: it refers to analogues of vitamin B₁₂ that display high structural similarity to the vitamin and are ′locked chemically′ to prevent their metabolic conversion into the crucial organometallic B₁₂‐cofactors. Application of antivitamins B₁₂ to healthy laboratory animals is, thus, expected to induce symptoms of B₁₂‐deficiency. Antivitamins B₁₂ may, hence, be helpful in elucidating still largely puzzling pathophysiological phenomena associated with B₁₂‐deficiency, and also in recognizing physiological roles of B₁₂ that probably still remain to be discovered.     

Combined spectroscopic/computational studies of vitamin B12 precursors: geometric and electronic structures of cobinamides

Vitamin B(12) (cyanocobalamin) and its biologically active derivatives, methylcobalamin and adenosylcobalamin, are members of the family of corrinoids, which also includes cobinamides. As biological precursors to cobalamins, cobinamides possess the same structural core, consisting of a low-spin Co(3+) ion that is ligated equatorially by the four nitrogens of a highly substituted tetrapyrrole macrocycle (the corrin ring), but differ with respect to the lower axial ligation. Specifically, cobinamides possess a water molecule instead of the nucleotide loop that coordinates axially to Co(3+)cobalamins via its dimethylbenzimidazole (DMB) base. Compared to the cobalamin species, cobinamides have proven much more difficult to study experimentally, thus far eluding characterization by X-ray crystallography. In this study, we have utilized combined quantum mechanics/molecular mechanics (QM/MM) computations to generate complete structural models of a representative set of cobinamide species with varying upper axial ligands. To validate the use of this approach, analogous QM/MM geometry optimizations were carried out on entire models of the cobalamin counterparts for which high-resolution X-ray structural data are available. The accuracy of the cobinamide structures was assessed further by comparing electronic absorption spectra computed using time-dependent density functional theory to those obtained experimentally. Collectively, the results obtained in this study indicate that the DMB → H(2)O lower axial ligand switch primarily affects the energies of the Co 3d(z(2))-based molecular orbital (MO) and, to a lesser extent, the other Co 3d-based MOs as well as the corrin π-based highest energy MO. Thus, while the energy of the lowest-energy electronic transition of cobalamins changes considerably as a function of the upper axial ligand, it is nearly invariant for the cobinamides.     

碱退式辅酶B~1~2类似物的合成和光谱性质

本工作用改进的方法合成了一类新的辅酶B~1~2碱退式类似物-含2'-脱氧核苷钴啉醇酰胺。它们分别是2',5'-二脱氧尿苷钴啉醇酰胺(2'dUrdCbi), 2',5'-二脱氧腺苷钴啉醇酰胺(2'dAdoCbi), 2',5'-二脱氧胞苷钴啉醇酰胺(2'dCytCbi)以及5'-脱氧胸苷钴啉醇酰胺(ThyCbi)。研究了它们的UV-Vis和^1H NMR光谱性质。并且和相应钴胺素的有关性质作了比较。     

The profound influence of a single metal-carbon bond on the reactivity of bio-relevant cobalt(III) complexes

This Perspective reports the development of mechanistic insight over the past 6 years, on the substitution behaviour of cobalamins that contain a single Co-C bond. The effect of the alkyl group, located in the trans position, on the thermodynamic, kinetic and ground state trans effect, was studied in detail. The substitution reactions of different alkylcobalamins with CN- were investigated, the apparent mechanistic discrepancy reported for the co-enzyme B12 was resolved and a logical explanation could be offered. In addition, a complete picture of the effect of pressure on the UV-Vis spectra of different base-on and base-off cobalamins is presented, which clearly shows the role of the alkyl group in controlling the equilibrium between five- and six-coordinate species, and the possible participation of such species in the studied ligand substitution reactions. The kinetics of the base-on/base-off equilibration was studied for the first time using a pH-jump technique. All in all the novel mechanistic information adds to the understanding of the profound effect that a single metal-carbon bond can have on the reactivity of such Co(III) complexes.     

Quantitative determination of alpha-, beta-, gamma- and delta-tocopherols in human serum by high-performance liquid chromatography and gas chromatography-mass spectrometry as trimethylsilyl derivatives with a two-step sample preparation

Using a two-step sample preparation with Extrelut and silica gel extraction in Pasteur pipettes it is possible to quantify all tocopherols in human serum samples by means of normal-phase HPLC with fluorescence detection (lambda(ex) 295 nm, lambda(em) 330 nm) or by GC-MS of their trimethylsilyl (TMS) derivatives. The method has been used in pharmacoepidemiological studies concerning the exposition with vitamin E-containing drugs in Germany. The recovery for all tocopherols is 98% and the limit of detection is 50 pg for alpha-tocopherol in the HPLC and 40 pg for all TMS-tocopherols in the GC-MS method using the selected ion monitoring mode with a well-tuned GCQ system. Linearity of calibration is excellent for both methods over the full physiological relevant range. Due to the low sample amount needed, the method is suitable for epidemiological and paediatric research.     

Accurate redetermination of the X-ray structure and electronic bonding in adenosylcobalamin

The electronic structure of adenosylcobalamin (B12 coenzyme, AdoCbl) has been calculated by a density functional method, using the orthogonalized linear combination of the atomic orbital method (OLCAO). Since a fixed accurately determined geometry was needed in such calculations, the crystal structure of adenosylcobalamin has been redone and refined to R = 0.065, using synchrotron diffraction data. Comparison with the recently reported electronic structures of cyano- (CNCbl) and methylcobalamin (MeCbl) shows that the net charges and bond orders vary only on the axial donors. The values in the three cobalamins suggest that the Co-C bond in MeCbl has a strength similar to that in AdoCbl, but it is significantly weaker that that in CNCbl. Present results are compared with those previously reported for the analogous corrin derivatives; i.e., simplified cobalamins with the side chains a-f replaced by H atoms. Despite a qualitative agreement, a discrepancy in the calculated HOMO-LUMO gap is found.     

Multi-walled carbon nanotubes as solid-phase extraction adsorbents for the speciation of cobalamins in seafoods by liquid chromatography

Multi-walled carbon nanotubes (MWCNTs) were evaluated as potential adsorbents for miniaturized solid-phase extraction coupled to liquid chromatography. The adsorption capacity of this sorbent was applied to assess the speciation of four cobalamins representing the various forms of vitamin B₁₂. The preconcentration on the MWCNTs was based on the retention of analytes by introducing the sample online into the mini-column system. Dimethyl sulfoxide was used to elute the retained vitamins for liquid chromatographic analysis. The experimental conditions of the continuous flow device, which affect the enrichment procedure, such as the type and amount of nanotubes, the volume, pH and flow rate of the sample solution, and the eluent and its volume, were optimized. For detection purposes, a diode array device was used and good resolution was obtained with a mobile-phase acetonitrile–phosphate buffer and gradient elution. Specificity was demonstrated by the retention characteristics and UV spectra and by comparing the peak purity index with commercial standards. Linearity, precision, recovery, and sensitivity were satisfactory. Detection limits ranged from 0.35 to 30 ng mL⁻¹. The method was successfully applied to the determination of cobalamins in seafoods, which were extracted from the sample with a buffer solution using an ultrasonic probe. The reliability of the procedure was checked by analyzing a certified reference material.     

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog

Nitrosylcobalamin (NO-Cbl), a novel vitamin B₁₂ analog and anti-tumor agent, functions as a biologic ‘Trojan horse’, utilizing the vitamin B₁₂ transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. A stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an Ascentis^® RP-Amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min^?1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. Additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B₁₂ therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.     

The effect of on-column structural changes of proteins on their HPLC behavior

Whenever proteins are found in environments different from those provided by physiological conditions, structural alterations can occur which can dramatically affect their adsorption and chromatographic behavior. The resultant behavior is often kinetically controlled and thus dependent on such factors as contact time of the protein with the adsorbent surface. Examples are given of the appearance of multiple peaks from seemingly pure species, as a result of these structural changes. In one case (papain in reversed-phase LC), multiple peaks are shown to arise from different conformational states. In a second case (beta-lactoglobulin A in hydrophobic interaction chromatography), a series of three peaks is a result of self-association or aggregation. Finally, recent work on an examination of structural changes of proteins on chromatographic supports, by means of intrinsic fluorescence and HPLC, is presented. The value of these studies for the elucidation of the retention mechanism in HPLC and the assessment of purity of proteins is demonstrated.     

Regio- and stereoselective glycosylation: synthesis of 5-haloimidazole alpha-ribonucleosides

We describe the synthesis of novel 5-haloimidazole ribonucleosides as precursors of modified cobalamins. A regio- and stereoselective glycosylation of protected ribose with silylated 4(5)-haloimidazoles produces 5-haloimidazole ribonucleosides predominantly in the alpha-configuration (60-75%) without any 4-substituted imidazole ribonucleoside. The structure of the 5-fluoroimidazole ribonucleoside was confirmed by X-ray crystallography and 2D NMR spectroscopy.     

Artifactual-free analysis of S-nitrosoglutathione and S-nitroglutathione by neutral-pH, anion-pairing, high-performance liquid chromatography. Study on peroxynitrite-mediated S-nitration of glutathione to S-nitroglutathione under physiological conditions

The endogenous potent vasodilators and inhibitors of platelet aggregation S-nitrosoglutathione (GSNO) and S-nitroglutathione (GSNO2) are frequently analyzed by high-performance liquid chromatography (HPLC) using mobile phases of acidic pH. These systems are associated with problems stemming from rapid and considerable artifactual formation of GSNO from glutathione (GSH) and ubiquitous nitrite. We describe a novel ion-pairing HPLC method with UV absorbance detection at 334 nm for the highly specific and interference-free analysis of GSNO and GSNO2 in the presence of high GSH and nitrite concentrations. Complete avoidance of artifactual formation of GSNO was accomplished by using the anion-pairing agent tetrabutylammoniumhydrogen sulphate in the mobile phase that enables analysis of GSNO at neutral pH, at which GSH and nitrite do not react to form GSNO. This HPLC system was used to study formation of GSNO2 from GSH and peroxynitrite under physiological conditions. We found by this HPLC system that peroxynitrite (0-300 microM) reacts with GSH (0-5 mM) to form GSNO2 at a mean yield of 2%. Analysis of the same samples by a cation-pairing HPLC system with acidic mobile phase (pH 2.0) revealed, however, GSNO plus GSNO2 formation of the order of 20% due to on column reaction of GSH with peroxynitrite-derived nitrite to form GSNO. Ammonium sulfamate is frequently used to remove nitrite from thiol-containing solutions under acidic conditions. By means of the anion-pairing HPLC system it is demonstrated that nitrite removal by this method is incomplete even when ammonium sulfamate is used at high concentrations. These findings underscore the absolute requirement of neutral pH conditions for the analysis of GSNO. The novel anion-pairing HPLC method should be useful to provide reliable data on formation, reaction and metabolism of GSNO and GSNO2 in biological fluids using various detectors including mass spectrometers.     

Direct zonal liquid chromatographic method for the kinetic study of actinomycin-DNA binding

The binding of an anticancer drug (actinomycin D or ACTD) to double-stranded DNA (dsDNA) was studied by means of high-performance liquid chromatography (HPLC). ACTD is an antitumor antibiotic containing one chromophore group and two pentapeptidic lactone cycles that binds dsDNA. Incubations of ACTD with DNA were performed at physiological pH. The complexed and free ligand concentrations of the mixture were quantified at 440 nm from their separation on a size-exclusion chromatographic (SEC) column using the same buffer for the elution and the sample incubation. The DNA and the ACTD-DNA complexes were eluted at the column exclusion volume while the ligand was retained on the support. An apparent binding curve was obtained by plotting the amount emerging at the exclusion column volume against that eluted at free ACTD retention volume. A dissociating effect was evidenced and the binding parameters were significantly different from those obtained at equilibrium by visible absorbance titration. The equilibrium binding parameters determined by absorption spectroscopy were used as starting data in the numerical simulations of the chromatographic process. The results showed a strong dependency of the apparent binding parameters on the reaction kinetics. Finally the comparison of the apparent binding curve obtained from the HPLC experiments and from the numerical simulations permitted an evaluation of the dissociation rate constant (kd = 0.004 s(-1)).     

Analysis of corrinoids in ovine tissues

Corrinoids from various ovine tissue samples (liver, blood, small intestinal fluid and faeces) were analysed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to estimate the distribution of corrinoids--the cobalamins hydroxocobalamin (OH-cbl), methylcobalamin (me-cbl) and 5'-deoxyadenosylcobalamin (ado-cbl), and cobalamin analogues--in these tissues. Samples were taken from either cobalt-deficient or cobalt-replete ewes, and ruminant and pre-ruminant lambs. In liver, ado-cbl predominated, followed by analogues, OH-cbl and me-cbl. Supplementation with either cobalt (ruminant) or vitamin B12 injections (pre-ruminant) increased the amount of ado-cbl and decreased analogues. In blood, OH-cbl predominated, followed by ado-cbl, analogues and me-cbl, respectively. In small intestinal fluid, the distribution from largest to smallest percentage was analogues, ado-cbl, OH-cbl and me-cbl. In faeces, analogues constituted the greatest proportion, followed by OH-cbl, ado-cbl and me-cbl, respectively. Owing to the small sample sizes only cautionary interpretations can be made. In contrast to humans, where me-cbl constitutes the highest proportion of corrinoids in plasma and ado-cbl in the liver, in sheep the amount of ado-cbl was consistently higher than me-cbl in all tissues. This may be due to the higher metabolic need of sheep for ado-cbl due to gluconeogenesis. Analogues and OH-cbl were found in each tissue, contrary to previous postulations. The much higher amount of vitamin B12 in small intestinal fluid compared with faeces indicates that a large proportion of the vitamin is absorbed by the gastro-intestinal tract.     

X-ray structural characterization of imidazolylcobalamin and histidinylcobalamin: cobalamin models for aquacobalamin bound to the B12 transporter protein transcobalamin

The X-ray structures of imidazolylcobalamin (ImCbl) and histidinylcobalamin (HisCbl) are reported. These structures are of interest given that the recent structures of human and bovine transcobalamin prepared in their holo forms from aquacobalamin show a histidine residue of the metalloprotein bound at the beta-axial site of the cobalamin (Wuerges, J. et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4386-4391). The beta-axial Co-N bond distances for ImCbl and HisCbl are 1.94(1) and 1.951(7) A, respectively. The alpha-axial Co-N bond distances to the 5,6-dimethylbenzimidazole are 2.01(1) and 1.979(8) A for ImCbl and HisCbl, respectively, and are typical for cobalamins with weak sigma-donor ligands at the beta-axial site. The corrin fold angles of 11.8(3) degrees (ImCbl) and 12.0(3) degrees (HisCbl) are smaller than those typically observed for cobalamins.     

Derivatization,Separation and Determination of Steroids and Triterpenoids by Reverse-Phase HPLC and UV Detection

The steroids were derivatised with aroyl chloride to yield the corresponding benzoates. Among them, their 4-methoxybenzoates were obtained in greatest yield and were analyzed by HPLC with the smallest detection limit (10 ng) by means of a UV detector (254 nm). This method of derivatization can be applied for analysis of steroids and triterpenoids from crude products with the HPLC technique.