Serum albumins and detoxication of anti-cholinesterase agents

Article abstract of DOI:10.1016/j.cbi.2010.03.001

Serum albumin displays an esterase activity that is capable of hydrolysing the anti-cholinesterase compounds carbaryl, paraoxon, chlorpyrifos-oxon, diazoxon and O-hexyl, O-2,5-dichlorphenyl phosphoramidate. The detoxication of all these anti-cholinesterase compounds takes place at significant rates with substrate concentrations in the same order of magnitude as expected during in vivo exposures, even when these substrate concentrations are between 15 and 1300 times lower than the recorded K_m constants. Our data suggest that the efficacy of this detoxication system is based on the high concentration of albumin in plasma (and in the rest of the body), and not on the catalytic efficacy itself, which is low for albumin. We conclude the need for a structure-activity relationship study into the albumin-associated esterase activities because this protein is universally present in vertebrates and could compensate for reduced levels of other esterases, i.e., lipoprotein paraoxonase, in some species. It is also remarkable that the biotransformation of xenobiotics can be reliably studied in vitro, although conditions as similar as possible to in vivo situations are necessary.

Full text of DOI:10.1016/j.cbi.2010.03.001

Chemico-Biological Interactions 187 (2010) 325–329
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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Serum albumins and detoxication of anti-cholinesterase agentsଝ
Miguel A. Sogorb∗, Eugenio Vilanova
Instituto de Bioingeniería, Unidad de Toxicología y Seguridad Química, Universidad Miguel Hernández de Elche, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 March 2010
Serum albumin displays an esterase activity that is capable of hydrolysing the anti-cholinesterase
compounds carbaryl, paraoxon, chlorpyrifos-oxon, diazoxon and O-hexyl, O-2,5-dichlorphenyl phos-
phoramidate. The detoxication of all these anti-cholinesterase compounds takes place at significant rates
with substrate concentrations in the same order of magnitude as expected during in vivo exposures, even
when these substrate concentrations are between 15 and 1300 times lower than the recorded Km con-
stants. Our data suggest that the efficacy of this detoxication system is based on the high concentration
of albumin in plasma (and in the rest of the body), and not on the catalytic efficacy itself, which is low for
albumin. We conclude the need for a structure–activity relationship study into the albumin-associated
esterase activities because this protein is universally present in vertebrates and could compensate for
reduced levels of other esterases, i.e., lipoprotein paraoxonase, in some species. It is also remarkable that
the biotransformation of xenobiotics can be reliably studied in vitro, although conditions as similar as
possible to in vivo situations are necessary.
Keywords:
Detoxication
Albumin
Hydrolysis
Paraoxonase
Organophosphorus
Carbamate
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
paraoxon and other OPs, such as diazoxon or chlorpyrifos-oxon,
in a calcium-dependent way [5]. Paraoxonase plays a critical role
Organophosphorus compounds (OPs)1 and carbamates are
mainly used as pesticides. OPs are chiefly used as insecticides, while
carbamates are also used as fungicides, nematocides and herbicides
[1,2]. Both types of compounds exert their pesticides effect through
the inhibition of acetylcholinesterase (AChE) by either phospho-
rylation [1] or carbamylation [2] of a serine residue of the active
centre of the enzyme. The study of the pesticide routes of biotrans-
formation and detoxication is needed to perform appropriate risk
assessments. In this sense, it is well-known that the main enzymes
involved in the detoxication of OPs and carbamates are phospho-
triesterases and carboxylesterases [3,4].
in the in vivo detoxication of certain OPs, such as diazoxon and
chlorpyrifos-oxon [6–7], but surprisingly not in the detoxication
of paraoxon since paraoxonase knock-out mice are not more sen-
sitive to the toxic effects of paraoxon than wild-type animals [7].
This raises the question about the real role of paraoxonase in the
detoxication of paraoxon.
Carboxylesterases are widely distributed enzymes in certain tis-
sues, especially in the liver, brain and gastrointestinal tract [8].
These carboxylesterases can be stoichiometrically phosphorylated
or carbamylated by OPs and carbamates, respectively, without
detectable adverse effects. These reactions between non-essential
carboxylesterases and OPs and carbamates are indeed a detox-
ication route since each scavenged molecule of pesticide is not
available to reach the nervous system and to cause the toxic effects
by inhibiting AChE [4].
The most studied phosphotriesterase is paraoxonase (EC
3.1.8.1), which is a lipoprotein that is capable of hydrolysing
Serum albumin displays an EDTA-resistant esterase activity
which is capable of hydrolysing certain anti-cholinesterase com-
pounds such as the OPs paraoxon, O-hexyl, O-2,5-dichlorophenyl
phosphoramidate (HDCP) [9,10] and soman [11], and the carba-
mate carbaryl [12,13]. The interactions between OPs and albumin
have recently drawn attention since the phosphorylated adducts of
albumin might be used as biomarkers of exposure to OPs [14,15].
The enzymes involved in the detoxication of xenobiotics are
usually studied in vitro under conditions that are far from the tox-
icologically relevant conditions in vivo. For human paraoxonase,
the enzyme hydrolyses paraoxon, chlorpyrifos-oxon or diazoxon
with Km ranging (depending on the isoform) between 0.5 and 3 mM
ଝ
This work was presented as a poster at the 10th International Meeting on
ˇ
Cholinesterases (Sibenik, Croatia).
∗
Corresponding author at: Instituto de Bioingeniería, Unidad de Toxicología y
Seguridad Química, Universidad Miguel Hernández de Elche; Avenida de la Univer-
sidad s/n, Alicante, 03202 Elche, Spain. Tel.: +34 966658506; fax: +34 965222033.
E-mail address: msogorb@umh.es (M.A. Sogorb).
1
Abbreviations and organophosphorus compound common names:
AChE, acetylcholinesterase; BSA, bovine serum albumin; BuChE, butyryl-
cholinesterase; carbaryl, 1-Naphthalenylmethyl carbamate; chlorpyrifos,
O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) thiophosphate; chlorpyrifos-oxon,
O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate; diazoxon, phosphoric
acid, diethyl 6-methyl-2-(1-methylethyl)-4-pyrimidinyl ester; HDCP, O-hexyl,
O-2,5-dichlorphenyl phosphoramidate; HSA, human serum albumin; OPs,
organophosphorus compounds; paraoxon, O,O-diethyl 4-nitrophenyl phosphate.
0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.03.001

326
M.A. Sogorb, E. Vilanova / Chemico-Biological Interactions 187 (2010) 325–329
[7]. Thus, the hydrolysing activities associated with paraoxonase
are studied in vitro using substrate concentrations of mM units,
while the internal OPs concentrations during in vivo poisonings are
several orders of magnitude lower than these figures. A similar con-
clusion may be drawn when we compare enzyme concentrations
because the activities are assayed in vitro using highly diluted sam-
ples (a few ␮l of plasma/ml solution), while detoxication under
physiological conditions takes place in 100% plasma.
The enzyme concentration may also prove a critical point to
consider in order to gain understanding of the role of albumin
in the detoxication of OPs and carbamate pesticides. The paraox-
onase concentration in human plasma is around 50 mg/l [16]
(approximately 1.3 ␮M when considering a molecular weight of
40 kDa [17]), while the albumin concentration is around 46 g/l [18]
(670 ␮M when considering a molecular weight of 69 kDa [17]). This
means that, in vivo, paraoxonase and OPs react at concentrations in
Fig. 1. Detoxication of OPs by human serum albumin. The hydrolyses of paraoxon (panel A), HDCP (panel B), diazoxon (panel C) and chlorpyrifos-oxon (panel D) were
assayed by incubating solutions of 1 ␮M paraoxon, 20 ␮M HDCP, 80 ␮M diazoxon and 2 ␮M chlorpyrifos-oxon with 9 mg HSA/ml (131 ␮M HSA) (for paraoxon and HDCP) or
13 mg HSA/ml (189 ␮M HSA) (for diazoxon and chlorpyrifos-oxon), or with buffer (50 mM TRIS pH 7.4). After degradation, the inhibitory power against chicken serum BuChE
of the resulting solutions was assayed. For this purpose, samples were incubated for 3 min at 37 ◦C with chicken serum, and the residual BuChE was assayed according to
Ellman’s procedure [27] and expressed as a percentage against a sample assayed in the absence of OP. The differences in the percentages of BuChE activity in the presence
and absence of HSA without incubation (0 h of exposure) can be explained on the basis of the OPs quickly bound to albumin without turnover. In all cases, the mean SD
of three independent samples is provided (*statistically different from the equivalent sample incubated for 0 h, p < 0.05; **statistically different from the equivalent sample
incubated for 0 h, p < 0.01).

M.A. Sogorb, E. Vilanova / Chemico-Biological Interactions 187 (2010) 325–329
327
the same order of magnitude, while the albumin concentration in
vivo is several orders of magnitude higher than their substrates.
In this work we compare the role of serum albumins in the
detoxication of paraoxon, chlorpyrifos-oxon, diazoxon, HDCP and
carbaryl under conditions that approach physiologically relevant
conditions. For this purpose, we employed enzyme concentrations
higher than the substrate, and substrate concentrations of the same
order of magnitude as expected during moderate or acute poi-
soning. We concluded that serum albumin plays a critical role in
the detoxication of paraoxon and carbaryl, and probably in that of
HDCP, and is of minor (although not negligible) relevance in the
detoxication of chlorpyrifos-oxon and diazoxon.
2. Materials and methods
2.1. Chemicals
Paraoxon, chlorpyrifos-oxon and diazoxon, with purities rang-
ing between 94.5% and 96.0%, were obtained from Sigma Chemicals
(Madrid, Spain), Dr. Ehrenstorfer (Augsburg, Germany), and Chem
Service (West Chester, USA), respectively. HDCP (95% purity) was
supplied by Dr. Nauman (Bayer Chemical Co., Germany). All other
chemicals (always of analytical grade) were obtained from either
Sigma Chemicals (Madrid, Spain) or local suppliers.
Fig. 2. Time course of paraoxon hydrolysis by human serum albumin. A solution of
0.33 ␮M paraoxon was incubated at 37 ◦C in the presence of either buffer or 9.5 mg
HSA/ml (138 ␮M HSA). After incubation, an aliquot of each sample was incubated for
3 min at 37 ◦C with chicken serum. The residual BuChE was assayed according to Ref.
[27], and expressed as, a percentage against a sample assayed in the absence of OP.
In all cases, the mean SD of three independent samples is provided (*statistically
different from the equivalent sample incubated with buffer for at least p < 0.05 or
lower).
2.2. Biological materials
(Fig. 1A). However, the residual BuChE inhibitory power after the
incubation of the same 1 ␮M paraoxon solution with 9 mg HSA/ml
(131 ␮M HSA) was 39% lower than before incubation (Fig. 1A).
A 4 h incubation of a 20 ␮M HDCP solution with buffer or HSA at
37 ◦C led to significant reductions in the BuChE inhibitory potency
in both cases, although this reduction was greater in the sample
incubated with HSA (41%) than in that incubated with buffer (20%)
(Fig. 1B).
Human serum albumin (HSA) and bovine serum albumin (BSA),
both essentially fatty acid-free and with 99% purity, were all
obtained from Sigma Chemicals (Madrid, Spain). Chicken serum
was also purchased from Sigma Chemicals.
2.3. Hydrolysis of paraoxon, chlorpyrifos-oxon, HDCP and
diazoxon
Incubation of 80 ␮M diazoxon and 2 ␮M chlorpyrifos-oxon solu-
tions with buffer did not alter the residual BuChE inhibitory potency
of these solutions, although incubation with HSA lowered the
BuChE inhibition potency by 24% and 80% in the cases of diazoxon
(Fig. 1C) and chlorpyrifos-oxon (Fig. 1D), respectively. It is remark-
able that chlorpyrifos-oxon was totally detoxified since the solution
displayed no capability to inhibit chicken serum BuChE after 1 h of
exposure to HSA.
The hydrolysis of paraoxon by HSA was time-dependent. The
incubation of a solution of 0.33 ␮M paraoxon with buffer main-
tained the BuChE inhibition power of the solution (at around 75%)
with no significant changes (Fig. 2). However, the incubation of this
same paraoxon solution with 9.5 mg HSA/ml (138 ␮M HSA) caused
a time-dependent reduction in the BuChE inhibition power. This
reduction was statistically significant after 2 h (Fig. 2). The total
reduction in the BuChE inhibition power reached 27% after 6 h of
incubation with HSA (Fig. 2).
In order to gain knowledge of the physiological relevance of
albumin-associated esterase activities, we determined the kinetics
for the hydrolysis of paraoxon and chlorpyrifos-oxon. HSA hydrol-
ysed chlorpyrifos-oxon with a higher Vmax and Km than paraoxon
(Table 1). The catalytic efficiency (Vmax/Km) of HSA for the hydrol-
ysis of chlorpyrifos-oxon was almost 4 times higher than for the
hydrolysis of paraoxon (Table 1).
The hydrolysis of paraoxon, chlorpyrifos-oxon, HDCP and dia-
zoxon was mainly assayed by the loss of the inhibitory potency
against chicken serum butyrylcholinesterase (BuChE) as a biosen-
sor of OP degradation. For this purpose, the BuChE inhibitory
potency of the solutions before and after incubation with HSA was
compared. If incubation with HSA significantly lowered the OP
concentration, then the solution’s capability to inhibit the enzyme
would also be lower. Similar approaches have been previously used
successfully by other authors [19]. Chicken serum was chosen as a
source of BuChE since it is well-known that this tissue does not
express significant enzymatic activities involved in the hydroly-
sis of OPs [3]. In addition, the exposure time of BuChE to the tested
solution was 3 min, a very short time compared with that employed
for degradation (between 1 and 6 h, depending on the assayed
OP).
In some cases (for determining kinetic constants), the hydroly-
sis of paraoxon, chlorpyrifos-oxon and HDCP was assayed using
well-established spectrometric procedures based on monitoring
the released hydrolysis product [9,20].
The hydrolysis of carbaryl was assayed by monitoring the
1-naphthol released during the carbaryl hydrolysis by gas chro-
matography coupled with mass spectrometry [21].
3. Results
3.2. Hydrolysis of carbaryl by bovine serum albumin
3.1. Hydrolysis of organophosphorus compounds by human
serum albumin
The hydrolysis of carbaryl by BSA exhibited two well differen-
tiated behaviours. BSA (7 mg/ml, 102 ␮M) was able to hydrolyse
more than 75% of the carbaryl present in the solutions whose con-
centrations were higher than 50 ␮M in 3 h (Fig. 3). However, the
A 4 h incubation of 1 ␮M paraoxon with buffer at 37 ◦C brought
no significant changes in the BuChE inhibition power of the solution

328
M.A. Sogorb, E. Vilanova / Chemico-Biological Interactions 187 (2010) 325–329
Table 1
Kinetic constants for the hydrolysis of several substrates by albumin. The hydrolysis of paraoxon and chlorpyrifos-oxon was assayed with HSA by spectrometric methods,
while the hydrolysis of carbaryl was assayed with BSA by gas chromatography coupled with mass spectrometry [21]. Vmax and Km were obtained using the Lineweaver–Burk
equation. The turnover numbers (kcat) were calculated using a molecular weight of 69 kDa for both albumins [17] and by assuming that only one active centre in each molecule
of enzyme is involved in the hydrolysis of the substrates.
Enzyme
Substrate
kcat (min−1
)
Km (␮M)
Catalytic efficiency (kcat/Km) (min−1 M−1
)
HSA
HSA
BSA
Paraoxon
Chlorpyrifos-oxon
Carbaryl
1.5 × 10−3
4.0 × 10−2
9.9 × 10−3
377
2640
380
3.9
15
26
have attempted to study in vitro the hydrolysing activities in situ-
ations that mimic in vivo mild to severe poisonings.
There are no data available about the toxic concentration
reached in the central nervous system during OP poisonings. Thus
in each case, we chose the OP concentration that caused between
60% and 80% of the BuChE inhibition for 3 min of exposure. Under
such conditions, HSA caused significant detoxication of paraoxon,
HDCP, chlorpyrifos-oxon and diazoxon (Fig. 1) at concentrations of
between 3.5 and 5.1 times lower than the physiological albumin
concentration. This suggests that at 46 mg HSA/ml, that is, the real
albumin concentration circulating in plasma [18], similar percent-
ages of hydrolysis would be reached in one-third or one-fifth of the
time employed for the assay.
Knocked-out mice for the paraoxonase gene were more sus-
ceptible to the toxic effect of chlorpyrifos-oxon than wild-type
mice [6], although the sensitivity of these animals to paraoxon
was similar to that of wild-type animals [7]. Li et al. [7] studied
the catalytic efficiencies (Vmax/Km ratios) for the hydrolysis of
paraoxon and chlorpyrifos-oxon by paraoxonase. They concluded
that the isoenzyme R192 hydrolysed chlorpyrifos-oxon with
an efficacy 41 times higher than paraoxon, while this record
increased to 214 in the case of the isoenzyme Q192. These figures,
together with the findings reported in this work, might explain
the surprising results obtained with transgenic mice. Indeed, the
Km for paraoxon hydrolysis was lower than for chlorpyrifos-oxon
hydrolysis (Table 1), thus enabling albumin to hydrolyse paraoxon
more efficiently than chlorpyrifos-oxon at the lower substrate
concentrations found during in vivo exposures. In conclusion:
(1) differences in the catalytic efficiencies determine that the
absence of paraoxonase is more relevant for chlorpyrifos-oxon
detoxication than for paraoxon detoxication and (2) differences in
the Km determine that transgenic animals can overcome the lack
of paraoxonase with albumin for the detoxication of paraoxon,
but not for the detoxication of chlorpyrifos-oxon. Previous studies
in which we studied the capability of human serum to detox-
ify paraoxon and chlorpyrifos-oxon by calcium-dependant
and EDTA-resistant routes corroborate these conclusions
[22].
Fig. 3. Hydrolysis of carbaryl by bovine serum albumin. Carbaryl samples were incu-
bated for 3 h with 7 mg HSA/ml (102 ␮M HSA) at 37 ◦C. The released 1-naphthol was
extracted with an organic solvent and quantified by gas chromatography coupled to
mass spectrometry [21], and was expressed as a percentage in relation to the initial
carbaryl concentration.
total hydrolysed carbaryl was lower than 25% when the carbaryl
concentration was 30 ␮M or lower (Fig. 3).
The kinetics for the hydrolysis of carbaryl by BSA showed a Km in
the same order of magnitude as the recorded Km for the hydrolysis
of paraoxon by HSA (Table 1), although the hydrolysis of carbaryl by
BSA occurred with a Vmax 8.5 times higher than the hydrolysis of
paraoxon by HSA. The catalytic efficiency of BSA for the hydrol-
ysis of carbaryl was 6.6 and 1.7 times higher than the catalytic
efficiency of HSA for hydrolysing paraoxon and chlorpyrifos-oxon,
respectively (Table 1).
4. Discussion
This work demonstrates that serum albumins are able to detox-
ify a variety of OPs as well as carbaryl. Hydrolysis takes place at
significant rates at much lower substrate concentrations than, or in
the same order of magnitude as, the recorded Km. We consider that
the most probable mechanism for detoxication of these substrates
is its catalytic hydrolysis. Nevertheless, we cannot refuse the possi-
bility of an alternative or simultaneous mechanism of detoxication
in which certain side-chain of albumin might be phosphorylated or
carbamylated without turnover.
4.2. Hydrolysing activities of carbaryl associated with human
serum albumin under physiological conditions
As with OPs, there is no information available about the internal
carbaryl concentration reached during carbaryl poisonings. Gor-
don and co-workers [23] orally dosed rats with 175 mg carbaryl/kg
bw to cause around 50% of brain cholinesterase inhibition 4 h after
exposure. It is well-established that carbaryl is totally absorbed by
the gastrointestinal route and does not accumulate in the organ-
ism [24]. Therefore, this dose might cause an internal exposure
of 870 ␮M, which is around twofold higher than the recorded Km
(Table 1). By extrapolating the data shown in Fig. 3, we will be able
to estimate that this carbaryl could be hydrolysed after a few hours
of exposure to 46 mg HSA/ml.
4.1. Hydrolysing activities of organophosphorus compounds
associated with human serum albumin under physiological
conditions
In vitro assays for the biochemical and toxicological characteri-
sation of biotransformation enzymes are usually performed under
conditions that are far from physiologically relevant situations. We
Li et al. [16] reported the presence of BuChE, paraoxonase
and albumin esterase, but not carboxylesterase, in human plasma.

M.A. Sogorb, E. Vilanova / Chemico-Biological Interactions 187 (2010) 325–329
329
Paraoxonase is unable to hydrolyse carbaryl, thus the detoxication
of this compound must necessarily be assigned to either albumin or
BuChE. Albumin concentration is around 46 mg/ml (670 ␮M) while
BuChE concentration is 5 mg/l (60 nM with a molecular weight
of 85 kDa). Therefore if we consider the above estimated inter-
nal carbaryl concentrations, we may conclude that most of the
carbaryl will be detoxified by albumin. In mouse plasma, a third
esterase (a carboxylesterase), which is capable of hydrolysing car-
baryl, can be found at 80 mg/l [16] (1.3 ␮M with a molecular weight
of 62 kDa [17]). Therefore, this carboxylesterase might contribute
to carbaryl detoxication to a greater extent than BuChE, although
the main relevance must still be assigned to albumin. For all
these enzymes (albumin, BuChE and carboxylesterase), the carbaryl
detoxication mechanism may include a spontaneous reactivation of
the carbamylated active centres. Indeed, it is well-known that the
carbamylation of Ser residues (and probably that of the Tyr residues
as well) is not as stable as phosphorylation because the carbamy-
lated enzyme can be reactivated by nucleophilic molecules (usually
water molecules) [4].
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4.3. Final remarks and conclusions
Our data suggest that albumin plays a highly relevant role
in the detoxication of paraoxon and carbaryl (and probably that
of HDCP as well), but plays a minor role in the detoxication of
chlorpyrifos-oxon and diazoxon. The capability of serum albu-
min to hydrolyse paraoxon and carbaryl has long since been
known [25,26]. However, the role of these hydrolysing activi-
ties has been underestimated given the low catalytic efficacy of
albumin, especially when compared with other enzymes such
as paraoxonase. Yet low catalytic efficiency is compensated by
the extremely high enzyme concentration (46 mg/ml, 670 ␮M),
similarly to insect strains that over-express non-essential car-
boxylesterases for detoxifying OPs and carbamates for developing
resistances [4]. The traditional underestimation of the role of
albumin in the detoxication of paraoxon has been due to the per-
formance of in vitro assays in conditions far from physiological
situations, and the subsequent extrapolation of the conclusions to
in vivo exposures.
This work demonstrates that: (1) biotransformation (activation
and detoxication) might occur by unexpected routes and (2) the
biotransformation of xenobiotics can be studied in vitro, although
it is mandatory to use conditions that mimic the in vivo situations
as much as possible.
[21] M.A. Sogorb, C. Álvarez-Escalante, V. Carrera, E. Vilanova, An in vitro approach
for demonstrating the critical role of serum albumin in the detoxication of
the carbamate carbaryl at in vivo toxicologically relevant concentrations, Arch.
Toxicol. 81 (2007) 113–119.
Finally, we conclude the need for a structure–activity relation-
ship study into the esterase activities associated with albumin
because this protein is universally present in vertebrates and could
confer resistance to those individuals or species with reduced levels
of other esterases.
[22] M.A. Sogorb, S. García-Argüelles, V. Carrera, E. Vilanova, Serum albumin is
as efficient as paraxonase in the detoxication of paraoxon at toxicologically
relevant concentrations, Chem. Res. Toxicol. 21 (2008) 1524–1529.
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Conflicts of interest
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The authors declare that there are no conflicts of interest.
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This study has been supported by Spanish Ministry of the Envi-
ronment (Grant A051/2007/3-14.4).
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