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Research Article
 

Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol



Fazlul Huq and Deena Ababneh
 
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ABSTRACT

Molecular modelling analyses based on molecular mechanics, semi-empirical (PM3) and DFT (at B3LYP/6-31G* level) calculations using the programs Spartan `02 and HyperChem 7.0 show that glycolic acid has high thermodynamic stability and low kinetic lability so that the reaction in which glycolic acid is converted to glyoxylic acid is indeed rate-determining. The metabolite glyoxal has the lowest LUMO-HOMO energy difference that makes it most kinetically labile. The high kinetic lability and the presence of electron-deficient region on the molecular surface may make glyoxal the most toxic metabolite as it can cause depletion of cellular glutathione, thus compromising antioxidant status of the cell.

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  How to cite this article:

Fazlul Huq and Deena Ababneh, 2007. Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol. Journal of Pharmacology and Toxicology, 2: 54-62.

DOI: 10.3923/jpt.2007.54.62

URL: https://scialert.net/abstract/?doi=jpt.2007.54.62

Introduction

Ethylene glycol is a widely used industrial chemical that is highly toxic that can cause both renal and developmental toxicity (Corley et al., 2005). When ingested in the form of antifreeze or other automobile products, ethylene glycol results in central nervous system depression, cardiopulmonary compromise and renal dysfunction (Eder et al., 1998). Ethylene glycol poisoning classically presents itself as a metabolic acidosis with an increased anion gap (Ammar and Heckering 1996). From a number of studies carried out to investigate the mechanism of toxicity of ethylene glycol (Mulinos et al., 1943; Coen and Weiss, 1966; Richardson, 1973; Marshall, 1982; Jacobsen et al. 1982, 1984) the metabolic degradation of ethylene glycol has been largely worked out. As shown in Fig. 1, it includes conversion to glycolaldehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and carbon dioxide and incorporation into glycine that occurs via a number of intermediates. It is believed that developmental toxicity is due to toxic metabolites rather than the parent compound, the most important one being glycolic acid (Carney, 1994; Carney et al., 1996; Pottenger et al., 2001; Jeffrey, 2001).

Following either oral or skin exposure to ethylene glycol, its metabolism involving alcohol dehydrogenase occurs mainly in the liver (Booth et al., 2004; Gessner et al., 1961). The rate-determining step in the metabolism of ethylene glycol is the step in which glycolic acid is converted to glyoxylic acid. In this study, molecular modelling analyses have been carried out to determine the relative stability of ethylene glycol and its metabolites with the aim of providing a better understanding on their relative toxicity in particular to find out whether any of the metabolites is likely to induce cellular toxicity by compromising the antioxidant status of the cell.

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 1: Metabolism of ethylene glycol. The major pathway is indicated by bold arrows. *Rate determining step. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase (Booth et al., 2004).

Computational Methods

The geometries of ethylene glycol, glycolaldehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and glycine have been optimised based on molecular mechanics, semi-empirical and DFT calculations, using the molecular modelling programs Spartan (2002) and HyperChem 7.0 (2002). Molecular mechanics calculations were carried out using MM+ force field. Semi-empirical calculations were carried out using the routine PM3. DFT calculations were carried using the program Spartan ’02 at B3LYP/6-31G* level. In optimization calculations, a RMS gradient of 0.001 was set as the terminating condition. For the optimised structures, single point calculations were carried to give heat of formation, enthalpy, entropy, free energy, dipole moment, solvation energy, energies for HOMO and LUMO. The order of calculations: molecular mechanics followed by semi-empirical followed by DFT minimized the chances of the structures being trapped in local minima rather than reaching global minima. To further check whether the global minimum was reached, some calculations were carried out with improvable structures. It was found that when the stated order was followed, structures corresponding to global minimum or close to that were reached in most cases. Although RMS gradient of 0.001 may not be sufficiently small for vibrational analysis, it is believed to be so for calculations associated with electronic energy levels. For the optimised structures, single point calculations were carried to give heat of formation, enthalpy, entropy, free energy, dipole moment and solvation energy, HOMO and LUMO. The study was carried out in the School of Biomedical Sciences, The University of Sydney during November 2005 to March 2006.

Results and Discussion

Table 1 gives the total energy, heat of formation as per PM3 calculation, enthalpy, entropy, free energy, dipole moment, energies of HOMO and LUMO as per both PM3 and DFT calculations for ethylene glycol and its metabolites glycolaldehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and glycine. Fig. 2-9 give the optimised structures of ethylene glycol and its metabolites glycolaldehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and glycine as per PM3 calculations using the program HyperChem 7.0.

Table 1: Calculated thermodynamic and other parameters for ethylene glycol and its metabolites
Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
*In atomic unit from DFT calculations

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 2: Structure of ethylene glycol giving the electrostatic potential and the HOMOs in (a) and surface electric charges in (b) where red indicates negative, blue indicates positive and green indicates neutral

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 3: Structure of glycolaldehyde giving the electrostatic potential and the HOMOs in (a) and surface electric charges in (b) where red indicates negative, blue indicates positive and green indicates neutral

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 4: Structure of glyoxal giving the electrostatic potential and the HOMOs in (a) and surface electric charges in (b) where red indicates negative, blue indicates positive and green indicates neutral

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 5: Structure of Glycolic acid giving the electrostatic potential and the HOMOs in (a) and surface electric charges in (b) where red indicates negative, blue indicates positive and green indicates neutral

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 6: Structure of glyoxylic acid giving the electrostatic potential and the HOMOs in (a) and surface electric charges in (b) where red indicates negative, blue indicates positive and green indicates neutral

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 7: Structure of oxalic acid giving the electrostatic potential and the HOMOs in (a) and surface electric charges in (b) where red indicates negative, blue indicates positive and green indicates neutral

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 8: Structure of formic acid giving the electrostatic potential and the HOMOs in (a) and surface electric charges in (b) where red indicates negative, blue indicates positive and green indicates neutral

Image for - Molecular Modelling Analysis of the Metabolic Activation of Ethylene Glycol
Fig. 9: Structure of glycine giving the electrostatic potential and the HOMOs in (a) and surface electric charges in (b) where red indicates negative, blue indicates positive and green indicates neutral

The structures also give regions of negative electrostatic potential (greyish-white envelopes), HOMOs (blue and red where red indicates HOMOs with high electron density) in (a) and surface charges in (b) where red indicates negative, blue indicates positive and green indicates neutral.

Preponderance of acids among the metabolites of ethylene glycol explains why ethylene glycol can cause metabolic acidosis.

The total energies of optimised structures of ethylene glycol, glycoladehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and glycine obtained from PM3 calculations in kcal mol-1 are -106.28, -92.31, -152.69, -72.27, -133.33, -188.55, -101.94 and -106.19, respectively. The corresponding values obtained from DFT calculations are -228.93, -229.03, -304.30, -227.81, -303.08, -378.32, -189.77 and -284.44 in atomic units. It can be seen that excepting oxalic acid (which is essentially one of the end-products in the metabolism of ethylene glycol), glycolic acid has the lowest value for the total energy from both PM3 and DFT calculations. The heats of formation in kcal mol-1 of ethylene glycol, glycoladehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and glycine from PM3 calculations are -97.78, -83.12, -138.47, -62.22, -116.83, -173.53, -90.12 and -92.51, respectively. Once again it can be seen that among ethylene glycol, glycoladehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and glycine, after oxalic acid, glycolic acid has the second lowest heat of formation. Since the calculated entropies of ethylene glycol, glycolaldehyde, glycolic acid, glyoxal and glyoxylic acid are found to be comparable, it can be seen that among glycoladehyde, glycolic acid, glyoxal and glyoxylic acid, glycolic acid has the highest thermodynamic stability. The difference in stability between glycolic acid and its metabolite glyoxylic acid is found to be the greatest except when we compare the total energies obtained from DFT calculations. The greater stability of glycolic acid than glyoxylic acid means that the activation energy for the conversion of glycolic acid to glyoxylic acid would be greater than the reverse reaction. Based on DFT calculations it is found that among glycolaldehyde, glycolic acid, glyoxal, glyoxylic acid and oxalic acid, the difference between HOMO and LUMO is greatest for glycolic acid (7.27 eV as against 5.96, 4.13, 4.86 and 5.77 eV for glycolaldehyde, glyoxal, glyoxylic acid and oxalic acid respectively). This means that the activation energy for the reaction: glycolic acid → glyoxylic acid would be very high. Thus it follows from both thermodynamic and kinetic considerations that the reaction: glycolic acid → glyoxylic acid would be the rate-determining step in the metabolism of ethylene glycol.

Glyoxal has the smallest LUMO-HOMO energy difference (4.13 eV from DFT calculations), indicating that the metabolite would be most labile kinetically. When density of electrostatic potential is considered, it is found that the metabolite has some electron-deficient regions on its molecular surface so that it can react with cellular glutathione. Thus the high reactivity of glyoxal would may makes it highly toxic as it would cause glutathione depletion, thus compromising the antioxidant status of the cell.

In the case of ethylene glycol, the electrostatic potential is found to be more negative around the two hydroxyl oxygen atoms, indicating that the positions may be subject to electrophilic attack. In the case of glycolaldehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid and formic acid, the electrostatic potential is found to be more negative around the carbonyl and/or the hydroxyl oxygen atoms, indicating that the positions may be subject to electrophilic attack. In the case of glycine, the electrostatic potential is found to be more negative around the carbonyl, hydroxyl oxygen atoms and the amino nitrogen atom, indicating that the positions may be subject to electrophilic attack.

In the case of ethylene glycol, the HOMOs with large electron density are found to be centred on the two hydroxyl oxygen atoms. In the case of glycolaldehyde, the HOMOs with large electron density are found to be centred on the carbonyl and hydroxyl oxygen atoms. In the case of glyoxal, the HOMOs with large electron density are found to be centred on the carbonyl and hydroxyl oxygen atoms. In the case of glyoxylic acid, the electrostatic potential is found to be more negative around the carbonyl and hydroxyl oxygen atoms, indicating that the positions may be subject to electrophilic attack. In the case of glyoxylic acid, the HOMOs with large electron density are found to be centred on the carbonyl and hydroxyl oxygen atoms. In the case of oxalic acid, the HOMOs with large electron density are found to be centred on the carbonyl and hydroxyl oxygen atoms. In the case of formic acid, the HOMOs with large electron density are found to be centred on the carbonyl and hydroxyl oxygen atoms. The overlap or close proximity of the positions high negative electrostatic potential with those of HOMO high electron density (e.g., carbonyl and hydroxyl oxygen atoms) give further support to the idea that the positions may be subject to electrophilic attack.

The abundance of red and yellow regions on the surface of ethylene glycol and metabolites glycolaldehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and glycine indicates that the surface predominates in negative charge so that the interaction of ethylene glycol, glycolaldehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid, formic acid and glycine with water and other biomolecules is more likely to be hydrophilic rather than hydrophobic. Hence the molecules would have high solubility in water and lower solubility in lipids. A similar conclusion was made earlier from solvation energy values.

Among metabolites of ethylene glycol, oxalic acid is found to have the highest solvation energy (-14.02 kcal mol-1 from PM3 calculations) and zero dipole moment. Glycine has larger dipole moment (4.40 from PM3 calculations) although its solvation energy is lower (-13.69 kcal mol-1 from PM3 calculations). The values show that a large dipole moment does not necessarily correspond to a high a solvation energy, pointing to the complexity of the process of dissolution in water.

Conclusions

Molecular modelling analysis using semi-empirical PM3 and DFT calculations were carried out to investigate the metabolism of ethylene glycol into glycoladehyde, glycolic acid, glyoxal, glyoxylic acid, oxalic acid and formic acid in which the rate-determining step is the conversion of glycolic acid into glyoxylic acid. The results of the calculation show that there are both thermodynamic and kinetic reasons why this is so. Glyoxal has the smallest LUMO-HOMO energy difference, indicating that the metabolite would be most labile kinetically and hence it may react most readily with biomolecules including glutathione as it has some electron-deficient regions on the molecular surface. Depletion of glutathione will induce cellular toxicity by compromising the antioxidant status of the cell.

Acknowledgment

Fazlul Huq is grateful to the School of Biomedical Sciences, The University of Sydney for the time release from teaching.

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