Graduate Thesis Or Dissertation


Ascorbylation- the biological relevance of covalently bound ascorbic acid Public Deposited

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  • Ascorbic acid, or vitamin C, is well known as a co-factor for proline-hydroxylase and as an anti-oxidant. However, it is also capable of forming covalent bonds, particularly in the role of a nucleophile, henceforth termed 'ascorbylation'. The ascorbylation of electrophiles can occur under physiological conditions. Furthermore, there are a number of ascorbylated natural products found in plants and these compounds are reviewed here. Many of these natural products have biological activity. For example, the ascorbylated indole derivative ascorbigen, found in Brassica vegetables, can induce quinone reductase and thus may have a role in cancer chemoprevention. Thus, we hypothesize that ascorbic acid has important biological relevance beyond the role of antioxidant and in the hydroxylation of proline. If ascorbic acid, which is present in cells and very high concentrations, ascorbylates electrophiles, this present a new pathway in the detoxification of toxic compounds. For example, acrolein is a toxic compound which alkylates DNA and protein, leading to cancer among other health issues. Acrolein also reacts rapidly with ascorbic acid. Once ascorbylated, acrolein is no longer electrophilic and furthermore, the ascorbyl-acrolein (AscACR) molecule is more hydrophilic and this could lead to improved elimination of the molecule. Essentially, ascorbylation could be a previously unrecognized form of phase II metabolism, analogous to glutathione conjugation. The first major goal of this research is to detect ascorbylated metabolites of electrophiles (i.e. AscACR) from biological samples. Additionally, because ascorbylated compounds may have dramatically altered ADME characteristics compared to the non-ascorbylated compound, synthetically ascorbylated compounds may function as prodrugs. That is, a drug with poor pharmacokinetic parameters may be improved with the addition of an ascorbyl moiety, and that ascorbyl moiety may be cleaved via a retro-Michael reaction upon deliver to the site of action. The second goal of this work is to produce an ascorbylated prodrug. The candidate of choice was xanthohumol. Xanthohumol is a biologically active Michael acceptor which has very poor bioavailability due to low hydrophilicity- a characteristic which can be improved via ascorbylation. AscACR is easily synthesized. However, it was exceedingly difficult to detect in any biological samples using LC-MS/MS. It was hypothesized originally that AscACR may be a product of oxidative stress but it could not be found in samples of human subjects exposed to oxidative stress. Samples of rats exposed to CCl₄, a well known inducer of oxidative stress, similarly contained no detectable amounts of AscACR. Even rat or cell cultures exposed to acrolein directly did not produce samples with detectable amounts of AscACR. In retrospect, this was likely due to the instability of AscACR in biological media. However, a degradation product of AscACR was detected by LC-MS/MS in THP-1 cells exposed to acrolein diacetate (which was used to deliver acrolein intracellularly). This degradation product is produced via the hydrolysis of the lactone of AscACR, followed by decarboxylation and racemization to two stereoisomers, the mixture of which we term 5,6,7,8-tetrahydroxy-4-oxooctanal (THO). This same reaction scheme occurs with the natural product ascorbigen under certain conditions, and indeed is involved in the biosynthetic pathway of dactylose A and B. Vitamin C adequate THP-1 cells which are exposed to acrolein (via acrolein diacetate), produce THO. Thus, biologically, ascorbylation is involved in the detoxification of electrophiles; it is a new sort of phase II metabolism (albeit without an enzyme). The ascorbylation of acrolein itself is chemical; THO is also detected in the incubation of ascorbic acid and acrolein-diacetate no cell control. However, AscACR, the precursor of THO, was only detected in the no cell control. This is strong evidence that an enzyme, or some related biocatalyst, is involved in the conversion of AscACR to THO. In the prodrug work, the ascorbylated form of xanthohumol (AscXN) failed to serve as a prodrug. The synthesis of AscXN was poor. AscXN itself was unstable. In a caco-2 cell study modeling intestinal absorption, AscXN failed to show any transport across the caco-2 monolayer. While AscXN failed to function as a prodrug, this may have been due to the choice in candidate, rather than failure of the hypothesis itself.
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