Alpha lipoic acid (ALA) protects proteins against the hydroxyl free radical-induced alterations: rationale for its geriatric topical application

Abstract

The well known OH free radical scavenging properties of α-lipoic acid (ALA) cannot be easily utilized for biological experiments, because the compound is practically insoluble in water. We elaborated a simple method of preparing its Na-salt (Na-ALA) which proved to be water soluble. It has been demonstrated by ESR spin trapping experiments with DMPO, using the Fenton reaction as the source of OH free radicals that Na-ALA maintains its OH free radical scavenging ability: it reacts nearly an order of magnitude faster with these radicals than the spin trap itself. It was tested in two different systems to determine whether Na-ALA was able to protect bovine serum albumin (BSA) against the OH free radical-induced polymerization and protein oxidation. (i) OH free radicals were generated by Fenton reaction in the presence of BSA. This protein is polymerized by these radicals shown by the loss of its water solubility; Na-ALA exerted a considerable protective effect against this type of protein damage. (ii) BSA oxidation was induced by Co-gamma irradiation of 80 krad, resulting in a strong increase in the protein carbonyl content. Na-ALA inhibited this carbonyl formation very efficiently. The data suggest that the interaction of the OH radical with Na-ALA takes place on the disulfide group, yielding thiosulfinate or thiosulfonate. The results indicate that the geriatric topical application of Na-ALA may have an established rationale.

Introduction

The compound α-lipoic acid (ALA) has been used in both animal experiments and as a therapy for various diseases in humans (Suzuki et al., 1992, Kahler et al., 1993, Guillausseau, 1994, Jacob et al., 1995, Ou et al., 1995), and further data reviewed by Packer et al. (1995). From a gerontological point of view, it is particularly important to note that ALA displayed a protective effect in various models against age-dependent cognitive deficits (Choi, 1988, Altenkirch et al., 1993, Stoll et al., 1993, Greenamyre et al., 1994). In addition, ALA proved to be useful also in topical applications against aging signs of the skin (Perricone, 1997, Perricone, 1999).

The investigation of ALA began during the 1930s, originally identified as a bacterial growth factor which was designated ‘acetate-replacing factor’ or ‘pyruvate oxidation factor’ (Reed, 1957). The name ALA was given by Reed et al. (1951) who isolated a crystalline organic acid which proved identical to the acetate-replacing and pyruvate oxidation factors. In the same year, Patterson et al. (1951) isolated a derivative of ALA, and called it β-lipoic acid (BLA). The chemical structure of ALA was identified: it is 1,2-dithiolane-3-valeric acid, or 1,2-dithiolane-3-pentanoic acid, or dl-thioctic acid, or 6,8-dithiooctanoic acid, whereas BLA is an oxidized form of ALA, being a thiosulfinate version of it (Fig. 1) (Reed, 1957, Biewenga and Bast, 1995).

ALA is an ubiquitous natural compound: it forms the prostethic group of the Coenzyme-A (CoA) in the mitochondrial (e.g. pyruvateor- or α-ketoglutarate-) dehydrogenase systems (Reed, 1957, Lehninger, 1970). ALA is a relatively small molecule (mwt: 206). Its heterocyclic 1,2-dithiolane ring contains an -S-S- group, and the C-atom of α-position to either of the S atoms is asymmetric, giving rise to two possible d- and two possible l-enantiomers of the molecule. In CoA only the d-AI A may be incorporated. ALA can be synthesized relatively easily in racemic form.

ALA is a partially oxidized variant of the molecule (Fig. 1a). This can easily be reduced to a compound called dihydrolipoic acid (DHLA) shown by Fig. 1b. As a matter of fact, this reduced form performs the temporary binding of the acetyl group, in the CoA, and it is reoxidized again at the end of the process, i.e. becomes ready for repeating the transacetylating function (Lehninger, 1970). It has been observed rather early that ALA prevented the consequences of vitamin C deficiency in guinea pigs, and of vitamin E deficiency in rats (Rosenberg and Culik, 1959), i.e. it acted as an antioxidant.

More recent studies have revealed that ALA and DHLA are potent quenchers of various reactive oxygen species, as reviewed by Packer et al. (1995). A number of studies in various model systems have demonstrated these molecules effectively neutralizing free radicals such as the OH free radical, hypochlorous acid, singlet oxygen, but not hydrogen peroxide (Suzuki et al., 1991, Scott et al., 1994, Passwater, 1996). Based on their properties, both the ALA and DHLA have been characterized as ‘ideal antioxidants’ (Packer et al., 1995), although DHLA has been shown to have both antioxidant and prooxidant effects (Suzuki et al., 1991, Scott et al., 1994.

The mechanism of the free radical scavenging processes performed by these compounds remains somewhat controversial. Most authors assume that DHLA is chiefly responsible for the antioxidant effects (Armstrong and Webb, 1966, Peinado et al., 1989, Muller and Menzel, 1990, Handelman et al., 1994) etc. This assumption is supported by the fact that when ALA is used in vitro or in vivo systems, a reduction to DHLA results. However, other experiments show that ALA is directly able to scavenge hypochlorous acid (HOCl) (Haenen and Bast, 1991, Biewenga and Bast, 1995) as well as the OH free radical (Scott et al., 1994, Passwater, 1996). This apparent contradiction is explained by the formation of BLA (Fig. 1c). One of the S-atoms in the dithiolane ring of BLA becomes tetravalent and the same S-atom may take up further oxygen, and thus becomes a thiosulfonate (Fig. 1d) in which the S-atom is hexavalent (Biewenga and Bast, 1995). These valence changes fully explain the ability of ALA to directly scavenge the OH free radical without reduction to DHLA.

Although the popularity of ALA as a possible therapeutic agent is growing, some basic problems still persist. Namely, because ALA is almost completely insoluble in water, the possibilities of experimental explorations of its effect in aqueous systems are limited. On the other hand, it seemed to be important to reveal whether the OH free radical scavenger activity of this compound may really be exploited as a protective effect on proteins against the OH free radical induced oxidations, conformational alterations or cross linking.