Importance and uses of uric acid (urate) precursors in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures
Uric acid is not a direct additive to any of the classical cell culture media. However, it is present in animal sera. Consequently, uric acid is typically present in complete serum-supplemented classical cell culture systems. Urate has not been generally recognized as a useful additive during the development of serum-free cell culture media. However, several defined and serum-free media formulations do contain the direct precursors of uric acid, xanthine and hypoxanthine. Under appropriate conditions, xanthine and hypoxanthine may represent precursor pools for uric acid.
Ames' Medium contains hypoxanthine (6.02 µM) and Medium 199 (an early attempt to formulate a defined medium) contains both hypoxanthine (2.2 µM) and xanthine (2.2 µM).
Uric acid has unique and important antioxidant and metal chelating activities and may contribute to the improved performance of serum-free media that are supplemented with xanthine and/or hypoxanthine. Nutrient Mixtures, Ham's F-10, Ham's F-12 Coon's (F12C) and Kaighn's (F12K) modifications and Serum-Free/Protein Free Hybridoma Medium all contain 30 µM of hypoxanthine. Many of the precursor media used to develop proprietary media for CHO cells also contain the urate precursor, hypoxantine. These CHO related media include Nutrient Mixture, Ham's F-12; MCDB medium 302 with 30 µM hypoxanthine. DMEM/Ham's Nutrient Mixture F-12 (50:50), a basal medium used for the development of various proprietary CHO media, contains 12.4 µM of hypoxanthine.
Waymouth's Medium MB has the highest level of hypoxanthine at 184 µM. This media was designed as an early serum-free medium to grow L292 cells and various tumorigenic cell lines.
To better understand how urate derived from xanthine and hypoxanthine may contribute to the performance of serum-free media used to culture cells such as CHO cells see below.
Urate is not generally recognized as a component of cell culture. However, it is found in human and bovine sera in relatively high concentrations ranging from 10 to 50 µg/mL. It is a fortuitous component of all serum-supplemented media. Uric acid has a unique and important role as a multifaceted aqueous extracellular antioxidant. It is important to understand the chemistry and functions of uric acid in the context of its possible use in serum- and protein-free media.
Urate is the conjugate acid of uric acid. At pH =7.4, it exists predominantly as monobasic salts, probably NaHC5H2N4O3 and KHC5H2N4O3 or as metal complexes. It is a very weak diprotic organic acid with limited solubility in water. Solubility of the acid is approximately 70 µg/mL. However, the salts are somewhat more soluble. The sodium salt has a solubility limit of 800 µg/mL. The solubility limit of urate in cell culture media is probably somewhere between these values.
Urate Chemistry: Urate forms stable coordination complexes with iron (both ferrous and ferric forms), and manganese ions. Urate’s and ascorbate’s stability constants for ferric iron are 1011 and 102, respectively. When urate is present very little iron binds to ascorbate.
Ascorbate can reduce
In the process, ascorbate is oxidized and lost as an effective antioxidant. These reduced metals can catalyze the formation of hydroxyl free radicals through Fenton chemistry and the formation of organic alkoxyl and peroxyl radicals from lipid hydroperoxides.. The tight binding of iron by urate might prevent their reduction by, and concomitant oxidation of ascorbate. Removal of iron from the redox cycle by urate has the indirect impact of protecting lipids from peroxidation.
Urate shares the anti-oxidant quality of ascorbate and alpha-tocopherol of breaking lipid peroxidation chain reactions by providing the protons and electrons required to form lipid hydroperoxides. Under certain conditions urate can function as a pro-oxidant. When copper present in vitro is able to undergo redox cycling, lipid hydroperoxides can have mixed fates. Copper (II) reacts with lipid hydroperoxides to form lipid peroxide radicals and reduced copper. These lipid peroxides radicals can be converted back to hydroperoxides by agents such as urate, ascorbate or alpha-tocopherol. Copper (I) reacts with lipid hydroperoxides to form lipid alkoxyl radicals and copper (II). Lipid alkoxyl radicals can decay to a number of oxidation products and radicals including conjugated dienes and organic aldehydes. The fate of lipid hydroperoxides in vitro depends upon the concentrations and ratio of copper (II) to copper (I). Kaur, H., et. al., (1990) reported that uric acid can be oxidized by free Cu (II) to various oxidized products, including anion radical and allantoin. In the process Cu (II) is reduced to Cu (I). The reduction of copper by urate did not occur when the copper was bound by albumin or histidine. Bagnati has suggested that urate may promote the propagation of lipid peroxidation by accelerating the reduction of copper (II) to copper (I). This suggests that urate may not promote Cu(I) mediated in vitro oxidation of hydroperoxides when albumin or histidine is present.
Nitric Oxide is a small gaseous molecule that may form in cell culture from L-arginine as a product of cellular nitric oxide synthetase (EC 18.104.22.168) activity, and by non-enzymatic processes. Ferrous:ascorbate complexes may react non-enzymatically with nitrite (NO2) and generate nitric oxide that may subsequently bind with the ferrous:ascorbate complex to form a three member complex, nitrosyl:ferrous:ascorbate.
Nitric oxide is a free radical that reacts rapidly with other free radicals, including superoxide radicals found in cell culture to form peroxynitrite anion (ONOO-) and peroxynitrous acid. This acid:base pair exist in equilibrium with a pKa = 6.56.8. The peroxynitrite anion is fairly stable, but peroxynitrous acid is a very reactive, strongly oxidizing molecule that spontaneously decays by O-O bond homolysis. The predominant initial homolytic products are nitrite and hydroxyl free radicals. The hydroxyl free radical is extremely reactive. The nitrite free radical appears to attack protein bound tyrosine. The production of the peroxynitrite is often detected by measuring the presence of 3-nitrotyrosine. Urate binds to and blocks peroxynitrite mediated tyrosine nitration and apoptosis.
When peroxynitrite is formed in physiological solution where carbon dioxide and bicarbonate are present, the early products of homolysis are nitrite and carbonate radicals.
Urate is a peroxynitrite scavenger because it appears to reduce damage caused by this molecule. Urate is most likely exerting its protective effect by reacting with the products of peroxynitrite, the nitrite and carbonate free radicals, rather than the peroxynitrite itself.
Excess purines are degraded in vivo by xanthine dehydrogenase, XDH (EC 22.214.171.124). XDH normally functions as a dehydrogenase, but low oxygen conditions and calcium can promote its conversion to an oxidase (XO) (EC 126.96.36.199). XDH oxidizes hypoxanthine and xanthine to uric acid and NADH. XO also metabolizes hypoxanthine and xanthine to uric acid. However, instead of donating electron equivalents to NAD it donates them to oxygen resulting in the formation of superoxide and hydrogen peroxide. When XDH is induced by low oxygen concentrations into a form that produces urate and peroxides, urate:ferrous and urate:cuprous complexes may form and participate in Fenton chemistry. However, the hydroxyl radicals formed are likely to attack the urate molecule directly. In this way urate may act as a sacrificial antioxidant, i.e. a free radical