Model Studies Related to Vanadium Metabolism
- Model studies related to vanadium biochemistry: recent advances and perspectives
Although information about the metabolism of physiological amounts of vanadium in the higher forms of life remains scarce, an increasing amount of data has accumulated during recent years, mainly from animal studies. Some general aspects related to the absorption, transport, biological transformations, toxicity and excretion of vanadium become understandable.15,16
In Figure 1 we present a summary of this knowledge, briefly summarized, as follows: Dietary vanadium occurs mainly as H2VO4- and enters cells probably through the phosphate transport mechanism. Most of the ingested vanadium(V) undergoes a rapid one-electron reduction in the gastrointestinal tract, consistent with the fact that vanadium (V) is a rather strong oxidizing agent, especially at low pH. Most of the ingested and reduced vanadium remains unabsorbed and is excreted. Strong association between VO2+ and dietary fiber is postulated. In vivo, all the vanadium is converted to a common form. The organ distribution is essentially independent of the oxidation state and chemical nature of the form of the element. Evidently VO2+ undergoes autooxidation to vanadate in the presence of oxygen, whereas glutathione, ascorbate, cysteine and similar reducing agents can reduce vanadate. This means that endogenous reducing agents and dissolved oxygen ensure that both vanadium(V) and vanadium(IV) species are present in serum. Experimental evidence points to a relation between vanadium and iron metabolism. It has been suggested that the iron- transport protein transferrin may be also involved in vanadium transport. It remains to be determined if ferritin, the storage protein for iron, is also a useful storage system for vanadium. Interactions between vanadium species and serum albumin are probable, but very little is known about these processes and the possible nature of the complexes generated. Bone seems to be the major sink for vanadium. Final excretion of the small fraction of ingested and not retained vanadium occurs mainly through urine, as low-molecular-weight VO2+ complexes. Biliar excretion seems to be a secondary route.
We have performed a series of model studies related to different steps of the metabolic patterns in order to clarify some essential aspects.
2.1. Complexes of glutathione and related ligands
Reduced glutathione (GSH, Figure 2a), the tripeptide g-L-glutamyl-L-cysteinylglycine, the major non-protein thiol present in animal cells, is an extremely important biological reducing agent, involved in detoxification processes of exogenous materials17 and apparently plays a central role in vanadium metabolism.
Besides its reducing potential GSH can act as a ligand for the stabilization of the VO2+ oxocation.12,18,19 Consequently, we have repeatedly investigated the VO2+/GSH system. Our first studies, using electronic absorption spectroscopy, have shown that the interaction is strongly dependent on the initial metal-to-ligand ratios and the pH of the solution, and at least two different species were identified.20 More detailed speciation studies of this system have shown an even higher complexity.21,22 In the most recent and complete study, combining pH-potentiometry with EPR spectroscopy, visible absorption and circular dichroism measurements, it was shown that in the pH range between 5 and 7.5 and at any ligand-to-metal ratio between 10 and 140, the predominant complex, is a 2:1 species in which each GSH molecule coordinates through one O-atom of the deprotonated carboxylate group and the amino N-atom of the glutamyl residue.22 Four other species were identified in other pH-ranges.22 Previous EPR studies of this system also support VO2+ interaction through oxygens of the deprotonated carboxylate groups, and NH2 moieties.23
On the other hand, a series of model compounds with sulfhydryl-containing pseudopeptides, and investigated by a combination of numerous physicochemical techniques, also demonstrates the predominance of the cited 2:1 species in the pH range 5-7.24
The oxidation product of glutathione (GSSG, Figure 2b) can also interact with the VO2+ cation, and model speciation calculations reveal that in the pH range 6-7, GSSG is a more efficient oxovanadium(IV) binder than GSH.22 Two different 2:1 complexes, easily interconverted simply by changing the metal-to-ligand concentrations, are generated in the GSSG/VO2+ system. At low GSSG concentrations, coordination takes place through carboxylate groups, whereas at higher concentrations, N-donors appear to be principally involved in coordination.25 EPR studies at a 25 : 1 ligand-to-metal ratio suggest coordination through one or two monodentate carboxylate groups or through one or two a-amino acid moieties, i.e., COO_ + NH2 or 2COO- + 2 NH2.23
Most recently, Costa Pessoa et al.26 performed a detailed speciation study of this system. They concluded that in the pH range 6-8 and at a ligand-to-metal ratio = 10, a 1:1 species predominates. Coordination in this complex involves one carboxylate oxygen-atom and the NH2 group of the two terminal glutamyl moieties of GSSG.
These results confirm that both GSH and GSSG may participate in the stabilization and in the transport of VO2+ immediately after the GSH-mediated reduction of vanadate(V) in cellular systems.16,22
The amino acid L-cysteine is another potential reducing agent for vanadate in biological systems. Model studies in the VO3-/cysteine system show that vanadate is rapidly reduced, irrespective of the pH of the solution. At pH 6.8, reduction is followed by the formation of a purple complex. In this 2:1 ligand-to-metal species, the VO2+ cation interacts with the amino N-atom and the deprotonated -SH group of two amino acid molecules.27 This complex seems to be similar to the VO2+ complexes of cysteine esters of the same stoichiometry, which were isolated in the solid state.28,29 We also demonstrated that oxovanadium(IV) interacts with cystine, the oxidation product of L-cysteine. In this case, coordination apparently occurs through the carboxylate and amino groups.30 These results suggest that again both the excess of amino acid or its oxidation product was bind to the VO2+ cation.
2.2. Oxovanadium(IV) complexes of L-ascorbic acid and of its oxidation products
L-ascorbic acid (vitamin C, Figure 3) is another possible natural reducing agent of vanadates(V) to oxovanadium(IV). The reduced species can interact with the acid and with some of its oxidation products.12,31,32
A detailed study of the interaction of L-ascorbic acid with the VO2+ cation showed that different complexes are generated in solution at different pH-values. Some of them were isolated as powdered solids. As shown by spectroscopic studies, in these complexes the acid acts as a monodentate ligand through its deprotonated 3-hydroxo group, generating species of very low stability, consistent with the absence of chelation, suggesting that these VO2+/ascorbate complexes are probably not significant in the stabilization of the reduced vanadium.
On the other hand, the VO2+ cation might interact with some of the species generated after the oxidation of L-ascorbic acid.32 Dehydroascorbic acid, which is the primary oxidation product, is very unstable and undergoes a rapid series of transformations, first generating 2,3-diketogulonic acid, which can be further degraded to a mixture of oxalic and L-threonic acids.12,16,32 Dehydroascorbic acid interacts rapidly with VO2+. Solutions of these adduct are highly unstable towards the oxidation of the ligand. The VO2+/dehydroascorbate complex hydrolyzes irreversibly with opening of the lactone ring, generating a 2:1 ligand-to-metal complex, in which the enolized form of 2,3-diketogulonic acid is bidentate. A sodium salt of this complex anion, of composition Na2[VO(C6H6O7 )2].3H2O, has been isolated and characterized.32
Recently, we characterized some other VO2+ -complexes of this type, obtained as microcrystalline powders, by direct interaction of sodium metavanadate with ascorbic acid.33
2.3. Transferrin and serum albumin complexes
It is well known that in the oxidation states +3, +4 and +5, vanadium binds tightly to transferrin 3,13,34, forming vanadium-modified transferrins, which are believed to be involved in vanadium transport in higher organisms.35 Nevertheless, the coordination environment around vanadium in these systems is not yet totally known.
Vanadium(V) interaction with human serum transferrin has been investigated in detail, showing that two equivalents of vanadate are reversibly bound at the two metal-binding sites of the protein.36 The interaction was also modeled using the hexadentate ligand ethylenebis- (o-hydroxyphenlylglycine) showing that, at pH 9.5, the vanadium is bonded to phenolic residues as the VO2+ cation.36
In the case of the VO2+-complex of human lactoferrin, an octahedral structure with O3N equatorial coordination, involving one tyrosinate, one aspartate, one histidine and one monodentate carbonate, with another tyrosinate trans to the V=O bond has been proposed, on the basis of a computer simulation, using the atomic coordinates of the FeIII and CuII complexes.37 Vanadium(III), as V3+, and vanadium(V) as VO2+, can also be accommodated in a similar environment.37
Recently, Neves et al.38,39 have prepared some interesting model systems for the VO2+-transferrin complex, using N,O-donor ligands. With some of these models they were able to reproduce UV-Vis and EPR properties of oxovanadium(IV) complexes of human serum transferrin and ovotransferrin and they advanced some new proposals about the coordination sphere of vanadium in these systems. It has also been shown that some of these complexes can be oxidized chemically or electrochemically to the respective vanadium(V) complexes without changing the coordination sphere of the vanadium.39
Some aspects of the vanadium/albumin interactions are now understood.13,34 In one of the first EPR studies of the VO2+/bovine serum albumin system, it was found that the cation binds tightly probably at the specific sites for CuII, located at the N terminus of the polypeptide chain. There are also four or five additional weaker binding sites for VO2+ at carboxylate groups of the protein. However, the nature of the metal coordination sites could not be unambiguously established.40
The interaction of VOSO4 and NaVO3 with human serum albumin (HSA) was also investigated in aqueous solution at physiological pH, using gel and capillary electrophoresis and IR spectroscopic techniques. Gel electrophoresis results showed that a maximum of twenty VO2+ cations is bound per HSA molecule, at two sites with different affinities. Capillary electrophoresis confirmed the existence of two major binding sites for the oxovanadium(IV) cation, whereas VO3- has only a very weak binding affinity 41, consistent with previous studies.42
IR spectroscopic analysis showed that, as a consequence of the VO2+/HAS interaction, major structural changes are produced at the protein secondary structure.41
In a recent comparative study of the binding of vanadate to HSA, human fresh frozen plasma and human transferrin, it was demonstrated that the binding capacity of HSA is about one thousandth of those of the other two systems.43
2.4. Accumulation of vanadium in hard tissues and related systems
Bone seems to be the most active vanadium accumulator. The high skeletal retention of vanadate is probably related to its rapid exchange with bone phosphate, which must be favored by the strong similarities of PO43- and VO43-.
In order to investigate this exchange, calcium hydroxylapatite, Ca10(PO4)6(OH)2, was used as a model for the inorganic phase of bone.44 Under physiological conditions, the exchange was only observed with amorphous material. These model studies showed that the incorporation of small amount of vanadium into the phosphate sites only produces weak distortions at macroscopic (crystallographic parameters, crystal ordering) and microscopic (local distortions, weakening of chemical bonds) levels in the apatite lattice.44
Possible competition between VO2+ and CaII in the hydroxylapatite lattice has also been analyzed. Precipitation of Ca10(PO4)6(OH)2 in the presence of oxovanadium(IV)45 as well as interaction of apatite suspensions with the oxocation46 demonstrated that VO2+ is not incorporated into the apatite lattice, but that it is strongly adsorbed on the material surface.46,47
In addition, in vivo experiments, analyzing bone samples of rats treated with an interesting and promising antidiabetic drug,48 bis(maltolato)oxovanadium (IV), by means of electron spin-echo envelope modulation (ESEEM) spectroscopy, suggest that phosphate is involved in this surface interaction of VO2+ with bone.49
As it is known34,50 that VO2+ interacts with tropocollagen, it was useful to investigate whether it interacts with components of the organic matrix of bone. The interaction of VO2+ with chondroitin sulfate A (CSA), a well-known muchopolysaccharide present in connective tissues and other mineralized systems, was investigated in aqueous solutions by electron absorption spectroscopy and IR techniques. The generation of a complex species of stoichiometry VO(CSA)2, involving metal coordination to the carboxylate group and the glycosidic oxygen of the D-glucuronate units of CSA was demonstrated.51 It was also found that the two isolated components of CSA (D-glucuronic acid and N-acetylgalactosamine) behave towards VO2+ in a similar way as they do in the muchopolysaccharide.52
2.5. Vanadium excretion
As mentioned in the introduction, ingested vanadium is excreted most rapidly fecally. The postulated strong association of VO2+ withdietary fiber must be facilitated by the high affinity of the oxocation for a number of functional residues such as carboxylate, phosphate or hydroxo groups. However, very little information about the characteristics of these interactions in the gastrointestinal tract is available.
Concerning the final urinary excretion of the fraction of vanadium initially retained there exist a number of conflicting reports. Probably this excretion involves low-molecular-weight VO2+ complexes.15 However, other evidence also suggest the simultaneous presence of high molecular-weight complexes.53
In recent studies the low-molecular-weight vanadium species in urine was identified as a vanadium/ascorbate complex.54,55 But on the basis of our studies with this system, discussed above (section 2.2.), it is most likely that the ligand may be any of the oxidation products of ascorbic acid, perhaps 2,3-diketogulonic acid.
In the most recent study of this system it was shown that, after intraperitoneally 48V injection, vanadium in urine is found both as high-(protein-bound) and as low-molecular-weight species. The partition of these forms apparently depends on the time elapsed after vanadium administration. Different high- and low-molecular-weight forms were detected by chromatography depending of the elapsed times. But, after 48 h vanadium is largely excreted as a low-molecular-weight complex.56
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