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ACTIVATION OF CARBOXYLIC ACID IN CELLS

1. Introduction
Carboxylic acids constitute a large and heterogeneous class of both endogenous and
xenobiotic compounds, comprising, among others, pesticides, monomers for plastics, and
food constituents such as preservatives and flavorus. Even if a compound is not a
carboxylic acid it may, if having the suitable structure, be metabolized to a carboxylic acid
through phase I metabolism. Carboxylic acids also account for a very large number of
endogenous metabolites in biological systems such as fatty acids, keto-acids, bile acids,
messenger molecules and breakdown products form hormones and other endogenous
molecules. The physicochemical properties of all these carboxylic acids cover a very wide
span that ranges from very hydrophilic small-chains fatty acids (formic and acetic acid
being the smallest) to hydrophobic long-chain fatty acids, prostanoids, bide acids, etc.
2. General metabolism of carboxylic acid-containing compounds
The metabolic activation of the carboxylic acid moiety proceeds through two metabolic
pathways, which both result in chemically reactive metabolites that are capable of
participating in nucleophilic-type reactions leading to the formation of covalently protein
adducts.
2.1. Glucuronidation
The quantitatively most important route of metabolism of carboxylic acids is
glucuronidation, yielding the corresponding -1-O-acyl glucuronides. Which are more
polar than the parent compound due to the hydrophilic nature of the linked glucuronic acid
moiety. Glucuronidation is carried out by the family of UDP-glucuronosyl transferases
(UGTs) that require uridine-diphosphate glucuronic acid (UDPGA) as a co-factor. The
UGTs are membrane-bound enzymes found in the endoplasmic reticulum, and consist of
a number of more or less substrate-specific isoforms, displaying a number of
polymorphisms, similar to those of the cytochrome P450 family (Gibson, Skett, 2001).
The largest enzyme activity of UGTs is found in the liver, but significant catalytic activity
is also present in the kidneys and wall of the small intestines (Gibson, Skett, 2001). Acyl
glucuronides are largely excreted from cells and are either actively transported into the
systemic circulation or excreted into the small intestine via the bile. In the bile and
intestine, acyl glucuronides may undergo hydrolysis to yield the parent/unconjugated
compound (aglycone), thus giving rise to potential entero-hepatic circulation, with
reabsorption of the aglycone from the gut back into the portal circulation. The hydrolysis
reactions may be enzymemediated by -glucuronidase enzymes or nonspecific
esterases and chemically by hydroxide ions (alkaline environment). Similar hydrolysis
reactions may take place in plasma, if the acyl glucuronide is released into the systemic
circulation (Skonberg et al., 2008).
2.2. Acyl-CoA thioester formation

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Carboxylic acid can also be bioactivated to acyl-coenzyme A thioesters derivatives (acyl-
CoAs). The biochemistry of acyl-CoA thioester formation is well known from lipid
metabolism, where fatty acids are known to be metabolized by acyl-CoA ligases to acyl-
CoAs before into mitochondria. The prior formation of acyl-CoA is necessary in the
metabolism of fatty acids and biosynthetic reactions such as fatty acid elongation and
lipid biosynthetic reactions such as the formation of triglycerides. Acyl-CoA ligases are
defined according to cellular location and substrate specificity and can be divided into
three families based on the substrate fatty acid chain length: short-chain (C2 - C5),
medium-chain (C4 C12) and long-chain (C10 C22) (Knights, 1998; Knights et al., 2007).
Activation of carboxylic acid by formation of an acyl-CoA derivative is of special
importance because of its widespread use by all organism. The basic reaction occurs by
two steps. The first is a displacement reaction on the phosphorus atom of ATP to form an
acyl adenylate, a mixed anhydride of the carboxylic acid and a substituted phosphoric
acid. Such mixed a anhydride intermediates, or acyl phosphates, are central to much of
cellular energy metabolism because they preserve the high group-transfer potential of a
phosphor group of ATP while imparting a high group-transfer potential to the acyl group
(Metzler & Metzler, 2001). This allows the acyl group to be transferred onto the sulfur
atom of coenzyme A to form an acyl-coenzyme A in which the high group transfer
potential of the acyl group is conserved. Two additional steps are linked to this sequence.
The inorganic pyrophosphate that is displaced in the first step is hydrolyzed to two
molecules of HPO42- and the ADP formed is phosphorylated by ATP to form ADP. Figure
1 shows the activation of carboxylic acid by formation of acyl-CoA.
2.3. Acyl-glutathione thioester formation
Glutathione (GSH), an endogenous tripeptide, is the main nonprotein thiol in mammalian
cells, where it functions as an antioxidant and nucleophilic scavenger of varied types of
chemically reactive electrophilic metabolites. Formation of acyl-glutathione thioester
conjugates (acyl-SGs) may result from spontaneous reactions of electrophilic ester or
thioester metabolites with intracellular glutathione; or it can be catalyzed by glutathione-
S-transferases (Grillo, Benet, 2002; Shore et al., 1995). In general, due to the nucleophilic
scavenger property of GSH, formation of glutathione adducts are considered a
detoxification pathway. However, acyl-SGs themselves may be as reactive as acyl-CoAs
towards nucleophilic functional in proteins (Grillo, Benet, 2002).
The routes for metabolism of carboxylic acid-containing compounds to chemically
reactive intermediates, as well as nontoxic products, are shown in Figure 2.

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Figure 1 Activation of Carboxylic Acid by formation of acyl-CoA (Metzler & Metzler, 2001)

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Figure 2 Overview of the metabolism of carboxylic acids and some of the processes in which the metabolites may be
involved (Skonberg et al., 2008).

3. Mechanism of acetylcholine
The transmission of messages over the juncture between a nerve cell and another cell
requires that electrical signals be converted to chemical signals. The chemical messenger
is termed a neurotransmitter.
Acetylcholine (ACh) is synthesized in certain neurons by the enzyme choline
acetyltransferase from the compounds choline and acetyl-CoA. Organic mercurial
compounds have a high affinity for sulfhydryl groups, which causes dysfunction of the
enzyme choline acetyl transferase. This inhibition may lead to acetylcholine deficiency,
and can have consequences on motor function (Hasselmo, 1995).
ACh is stored at the ending of the neurons in membrane-enclosed vesicles (synaptic
vesicles), and released in response to a nerve impulse traveling down the neuron. ACh
is released in the automatic nervous system: pre- and post-ganglionic parasympathetic
neurons; preganglionic sympathetic neurons (and also postganglionic sudomotor
neurons). The release mechanism is constituted for several steps (Sudhof, 1995), first,
the vesicles carry the neurotransmitter to its interior by a transporter protein with 12
transmembrane domains, using an electrochemical gradient generated by a proton (H +)
pump (ATPase) (Trends, 1998). Most synaptic vesicles (~ 90%) containing the
neurotransmitter are not free in the cytoplasm but are bound to the presynaptic terminal
cytoskeleton by the interaction of proteins present in the vesicle membrane (synapsins I
and II) with cytoskeletal proteins.
There are two main classes of acetylcholine receptors (AChR) in the membranes of cells:
Nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors
(mAChR). They are named for the ligands used to discover the receptors. Nicotine mimics

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the action of acetylcholine at nicotinic receptors and muscarine (an alkaloid from the
mushroom Amanita muscaria) mimics acetylcholine at the muscarinic receptors
(Blakemore & Jennett 2001). Skeletal muscle has nicotinic receptors, while muscarinic
recpetors are found in smooth muscle, glands, and the heart.
Nicotinic AChRs are ionotropic receptors permeable to sodium, potassium, and chloride
ions. They are stimulated by nicotine and acetylcholine and blocked by curare. Most
peripheral AChRs are nicotinic, such as those on the heart and blood vessels or at the
neuromuscular junction. They are also found in wide distribution through the brain, but in
relatively low numbers (Flores & Segura, 2005).
Muscarinic receptors are metabotropic and affect neurons over a longer time frame. They
are stimulated by muscarine and acetylcholine, and blocked by atropine. Muscarinic
receptors are found in both the central nervous system and the peripheral nervous
system, in heart, lungs, upper GI tract, and sweat glands (Flores & Segura, 2005).
Drugs that inhibit ACh breakdown are effective in altering cholinergic neurotransmission.
In fact, the irreversible inhibition of AChE by isopropylfluoroesters are so toxic that they
can be incompatible with lifeinhibiting the muscles for respiration. This inhibition is
produced because ACh molecules accumulate in the synaptic space, keep the receptors
occupied, and cause paralysis. Two notable examples are insecticides and the gases
used in biological warfare. The mechanism of action of these irreversible inhibitors of
AChE is that they carbamylate the AChE, rendering it inactive. The carbamylation
inactivates both the acetyl and choline binding domains. A recently developed antidote to
these inhibitors cleaves the nerve gas so that it will dissociate from the AChE. In contrast
to the irreversible inhibitors, the reversible AChE inhibitors are effective in transiently
increasing the ACh level and are effective in diseases and conditions where an increased
ACh level is desired. The clinically important compound, eserine (physostigmine),
reversibly inhibits AChE (Skonberg et al., 2008).

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