Lipoic (Thioctic) Acid, in Cell Culture

Importance and uses of lipoic acid in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures

Lipoic Acid (aka Thioctic acid), a Serum-Free Medium Supplement, Useful In Biomanufacturing; Tissue Engineering and Specialty Media

Non-protein bound lipoic acid (aka thioctic acid), is a water and lipid soluble disulfide fatty acid that can move across the cell membrane of lymphocytes and fibroblasts. The activity of lipoic acid is mediated by its sulfur atoms as they participate in intra- and extra- cellular oxidation reduction reactions and in transition metal chelation.

Lipoic acid was introduced into media basal formulae in Ham's Nutrient Mixtures originally developed for the clonal growth of Chinese Hamster Ovary cells (CHO). It is found in Nutrient Mixtures, Ham's F-10, Ham's F-12; Nutrient Mixture, Ham's F-12 Kaighn's Modification (F12K); Dulbecco's Modified Eagle's Medium (DMEM)/Ham's Nutrient Mixture F-12 (50:50); all MCDB Media formulations, and Serum-Free/Protein Free Hybridoma Medium. It is likely that lipoic acid is also present in proprietary media developed from Ham's nutrient basal media. Consequently, lipoic acid may be important in heterologous protein biomanufacturing involving CHO cells and tissue engineering.

Lipoic acid participates in numerous chemical processes both inside and outside of the cell. This combined with its ability to cross the cell membrane make lipoic acid an especially important component of serum- and protein- free media.

Primary Functions of Lipoic acid in Cell Culture Systems

Lipoic acid (LA) is synthesized by eukaryotic cells and is not considered a vitamin. The in vivo synthesis of alpha-lipoic acid in mammalian systems is poorly understood, but is reported to involve octanoyl-ACP, which is produced during fatty acid synthesis and a lipoyl synthetase that adds the sulfur atoms. Lipoic acid and its reduced form, dihydrolipoic acid (DHLA), are involved in several aspects of cell energy and amino acid metabolism, as well as in defense against oxidative stress and apoptosis.

Energy and Amino Acid Metabolism

Lipoic acid is required for the metabolism of pyruvate; the TCA cycle intermediate, alpha-ketoglutarate, and the amino acids glycine, leucine, isoleucine and valine. Bound lipoic acid, lipoamide, is a coenzyme of unique E2 enzymes of four mitochondrial enzyme complexes: dihydrolipoamide S-acetyltransferase (EC 2.3.1.12) of the pyruvate dehydrogenase; dihydrolipoamide S-succinyltransferase (2.3.1.61) of the alpha-ketoglutarate complex; dihydrolipoamide branched-chain transacylase (no EC number) of the branched-chain keto-acid dehydrogenase complex; and aminomethytransferase (EC 2.1.2.1), a component of the glycine cleavage system.

Lipoamide is also a coenzyme of component X found in the dehydrogenase complexes.

Glucose Mimetic

Lipoic acid helps cells overcome oxidative stress-induced insulin-resistance. The uptake of glucose into cells is regulated by a family of hexose-specific GLUT transporters. GLUT-4 is a very important member of the GLUT transporters family because its activity is regulated by insulin. Insulin is frequently added to low-serum and serum-free cell cultures. Oxidative stress can induce cells to become insulin resistant by interfering with the insulin signaling pathway that regulates the activation of GLUT-4. Lipoic acid has been shown to overcome oxidative stress-induced insulin-resistance in vitro. Under conditions of oxidative stress, lipoic acid may replace insulin as an agent that supports increased glucose uptake mediated by the GLUT-4 transporter.

Antioxidant Functions

Lipoic acid and dihydrolipoic acid exert their antioxidant effects by several mechanisms. They regenerate endogenous antioxidants, remove transition metals from redox reactions by chelation and react non-enzymatically (scavenging) with reactive oxygen species.

The antioxidant and anti-apoptotic functions of LA and DHLA are inter-related and dependent upon the ability of non protein bound LA/DHLA to cross cell membranes and to undergo both enzymatic and non-enzymatic oxidation and reduction. Handelman, G. J., et.al. (1994) reported that lymphocytes and fibroblasts absorb lipoic acid from the medium, reduce it to DHLA and efficiently release it into the culture medium. Jones, W., et.al. (2002) reported that cultured human endothelial cells absorb LA, reduce it and release DHLA into the cell culture medium. Peinado, J., et.al. (1989) reported that the uptake of lipoic acid by hepatocytes was largely carrier-mediated and may involve the medium-chain fatty acids uptake system. The reduction of lipoic acid inside cells is mediated by at least three enzymes that couple either NADH or NADPH oxidation. These three enzymes are mitochondrial NADH-dependent dihydrolipoamide dehydrogenase (EC 1.8.1.4), and the cytoplasmic NADPH-dependent thioredoxin (EC 1.8.1.9) and glutathione (EC 1.6.4.2) reductases . Ames, E. S., et.al. (1996) and Jones, W., et.al. (2002) suggest that thioredoxin reductase may be more important for reducing lipoic acid in mammalian cells than dihydrolipoamide dehydrogenase. Lipoamide dehydrogenase reduces the R-enantiomer of lipoic acid more efficiently than the S-enantiomer, Biewenga, G. P., et.al. (1996) and Haramake, N., et.al. (1997). Whether the S or R enantiomer of lipoic acid is reduced more efficiently, the reduction occurs in the cytoplasm or mitochondria and whether the co-reductant is NADH or NADPH depends upon the specific cell type and status.

The ability of cells to recycle DHLA to the extracellular milieu is an important determinant of their ability to stimulate glutathione synthesis, resist glutamate toxicity and protect themselves from lipid peroxidation.

Glutathione Synthesis

Lipoic acid, is able to increase glutathione in cells. The beneficial effects of lipoic acid on GSH levels are seen at low doses, between 25 and 100 µM. Concentrations over 2 mM may cause apoptosis. Dihydrolipoic acid stimulates glutathione synthesis by improving cystine utilization. When lipoic acid is added to medium it is absorbed, reduced and released back into the culture medium as dihydrolipoic acid, DHLA. DHLA is able to reduce cystine to cysteine. For a number of cell types, the availability of cysteine from the extracellular milieu is a rate-limiting step in glutathione synthesis.

Glutamate Toxicity

Lipoic acid, protects cells from glutamate toxicity. L-cysteine is the rate limiting amino acid for the synthesis of glutathione. L-cysteine can be taken up from the extracellular milieu both as L-cysteine and L-cystine. These two forms are transferred into the cell by different uptake mechanisms. L-cystine is transported primarily by the Xc- system and L-cysteine is transported primarily by the ASC system. Different cell types have different relative dependence on these two channels. The sensitivity of a cell to glutamate toxicity can be correlated to its dependency on the Xc- system. L-glutamate is a competitive inhibitor for uptake of L-cystine by the Xc- system and for cells that primarily utilize the Xc- system high glutamate concentrations can reduce the rate of glutathione synthesis. If the production of glutathione is suppressed apoptosis can occur. Lipoic acid helps protect the cell from glutamate induced apoptosis by reducing extracellular L-cystine to L-cysteine. This increased ratio of L-cysteine to L-cystine reduces dependency upon the Xc- system and increases the effectiveness of the ASC system.

Lipid Peroxidation

Dihydrolipoic acid, protects cells membranes from lipid peroxidation. Once lipid peroxidation is initiated, it is self-propagating and if not stopped it will destabilize cell membranes. Membrane peroxyl radicals are converted to stable lipid hydroperoxides by vitamin E. The reduction of the lipid peroxyl radical generates the chromanoxyl radical of vitamin E. Ascorbate reduces the chromanoxyl radical to regenerate vitamin E and is itself oxidized to the ascorbate radical. Dihydrolipoic acid can reduce the ascorbate radical to ascorbate. The recycling of lipoic acid back to dihydrolipoic acid by glutathione and thioredoxin reductase links this antioxidation cycle back to the major reducing resources of the cell, glutathione and NADPH. Glutathione can also reduce the ascorbate radical directly.

Protein Repair

Methionine sulfoxide reductase (EC 1.8.4.6) reduces protein-methionine S-oxides to methionine. The reducing equivalents can come from reduced thioredoxin or dihydrolipoic acid. This facilitates the repair of proteins such alpha-1 antiprotease that are damaged by oxidation.

Chemical Attributes of Lipoic acid that make it a Useful Serum-Free Medium Supplement

Acid: C₈H₁₄O₂S₂

Molecular Weight: 206.3

Sodium Salt: C₈H₁₃NaO₂S₂

Molecular Weight: 228.3

Lipoic acid (LA) is an eight-carbon fatty acid containing sulfurs attached at carbons 6 and 8. Carbon atom 6 is asymmetric and lipoic acid exists as two enantiomers designated R (d) and S (l). The naturally occurring form is R. Most, but not all, commercially available lipoic acids are mixtures of R and S enantiomers. When lipoic acid is oxidized, the sulfide atoms form a five member-ring, dithiolane, composed of two sulfur and three carbon atoms. Its sulfur atoms and stereochemical form confer most of the characteristics that give lipoic acid its chemical and biochemical activities. LA sulfur atoms are easily reduced and oxidized. The reduced form of lipoic acid is a vicinal dithiol called dihydrolipoic acid, DHLA. The LA/DHLA redox couple can interact with a wide range of reactive oxygen species, other antioxidants and thiol compounds.

Non protein bound lipoic acid, is a water and lipid soluble fatty acid. Its solubility in water is controlled by its carboxylate moiety. The octanoic backbone is hydrophobic. Lipoic acids limited water solubility and hydrophobicity affect its stability and utility in cell culture systems. Factors that affect the availability of lipoic acid for use by cells in culture include its possible loss upon storage at low temperatures, its filterability, and the complexes it forms.

Iron and Copper

The in vitro interactions of LA and DHLA with iron and copper mediate the transition metal based prooxidant and antioxidant effects of lipoic acid. Cu (I) and Fe (II) promote the formation of hydroxyl radicals via Fenton reactions. Dihydrolipoic acid (DHLA) chelates Cu (II) and forms a stable complex below pH 6. At physiological pH, this complex is destabilized. In this destabilized form, Cu (II) can be reduced to Cu (I) by DHLA. DHLA also promotes hydroxyl radical formation by chelating Fe (III) and reducing it to Fe (II). Consequently, DHLA is a pro-oxidant in vitro when chelatable copper or iron is present. Lipoic acid can chelate Cu (II) and Fe (II) and keep these metals from catalyzing the formation of hydroxyl radicals.

When iron or copper are present in cell culture, the effect that lipoic acid has on protecting cells depends to some degree on the cells. Since healthy cells tend to keep lipoic acid in the reduced form, the potential exists that copper and iron will have prooxidant effects when they are accessible.

Radical Scavenger Activities

Lipoic acid and dihydrolipoic acid scavenge a wide range of radicals and oxidants in vitro. Both lipoic acid and DHLA can scavenge hydroxyl radicals; hypochlorous acid; peroxynitrous acid, and singlet oxygen. In addition, DHLA can scavenge superoxide and aqueous peroxyl radials. Hydrogen peroxide is not scavenged by either form of lipoic acid.

Lipoic Acid Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eukaryotic Cells in Serum-free Cultures.