ORIGINAL RESEARCH

Local anesthetic failure associated with inflammation:

verification of the acidosis mechanism
and the hypothetic participation of inflammatory
peroxynitrite

Takahiro Ueno 1 Abstract: The presence of inflammation decreases local anesthetic efficacy, especially in
Hironori Tsuchiya 2 dental anesthesia. Although inflammatory acidosis is most frequently cited as the cause of such
Maki Mizogami 1 clinical phenomena, this has not been experimentally proved. We verified the acidosis mecha-
Ko Takakura 1 nism by studying the drug and membrane lipid interaction under acidic conditions together with
proposing an alternative hypothesis. Liposomes and nerve cell model membranes consisting
1
Department of Anesthesiology,
Asahi University School of Dentistry, of phospholipids and cholesterol were treated at different pH with lidocaine, prilocaine and
Mizuho, Gifu, Japan; 2 Department bupivacaine (0.05%–0.2%, w/v). Their membrane-interactive potencies were compared by
of Dental Basic Education, Asahi the induced-changes in membrane fluidity. Local anesthetics fluidized phosphatidylcholine
University School of Dentistry,
Mizuho, Gifu, Japan membranes with the potency being significantly lower at pH 6.4 than at pH 7.4 (p ⬍ 0.01),
supporting the acidosis theory. However, they greatly fluidized nerve cell model membranes
even at pH 6.4 corresponding to inflamed tissues, challenging the conventional mechanism.
Local anesthetics acted on phosphatidylserine liposomes, as well as nerve cell model mem-
branes, at pH 6.4 with almost the same potency as that at pH 7.4, but not on phosphatidylcholine,
phosphatidylethanolamine and sphingomyelin liposomes. Since the positively charged anesthetic
molecules are able to interact with nerve cell membranes by ion-paring with anionic components
like phosphatidylserine, tissue acidosis is not essentially responsible for the local anesthetic
failure associated with inflammation. The effects of local anesthetics on nerve cell model
membranes were inhibited by treating with peroxynitrite (50 μM), suggesting that inflammatory
cells producing peroxynitrite may affect local anesthesia.
Keywords: inflammatory acidosis, local anesthetic failure, membrane lipid interaction,
hypothetic mechanism, inflammatory peroxynitrite

Introduction
Inflammatory diseases alter the pharmacokinetics and pharmacodynamics of various
drugs, resulting in their decreased clinical effects and increased adverse effects (Slaviero
et al 2003; Sattari et al 2003; Aitken et al 2006). Such an inflammation-induced
alteration is well known especially in dental anesthesia. Local anesthetic failure or
difficulty to obtain satisfactory analgesia commonly occurs in the situations of pulpitis
and apical periodontitis (López and Diago 2006). The anesthetic efficacies of lidocaine
Correspondence: Hironori Tsuchiya and mepivacaine injections are remarkably affected in the teeth with irreversible pulpitis
Department of Dental Basic Education, (Dunbar et al 1996; Reisman et al 1997). The presence of pulpitis has been estimated
Building 3, Asahi University School
of Dentistry, 1851 Hozumi, Mizuho, to cause inferior alveolar nerve block to fail in approximately 30%–45% of cases
Gifu 501-0296, Japan (Cohen et al 1993; Potočnik and Bajrović 1999). Although a variety of mechanistic
Tel +81 58 329 1266
Fax +81 58 329 1266
hypotheses were proposed for explaining the decreased effects of local anesthetics in
Email hiro@[Link] the presence of inflammation, the most cited is the theory that the acidosis of inflamed

Journal of Inflammation Research 2008:1 41–48 41

© 2008 Ueno et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
which permits unrestricted noncommercial use, provided the original work is properly cited.
Ueno et al

tissues reduces the anesthetic potencies of administered drugs interactions of local anesthetics with membrane lipids by
(Meechan 1999; Becker and Reed 2006). lowering the reaction pH and varying the membrane lipid
Since clinically used local anesthetics are structurally the composition. Of particular interest were whether local
tertiary amines with aromatic rings, they exist as charged and anesthetics could change membrane fluidity at acidic pH
uncharged molecules with the relative amounts depending by interacting with lipid bilayers and whether any of lipid
on their medium pH and pKa values. While both species components were specifically responsible for the possible
are relevant to pharmacological activities, local anesthet- membrane interaction. For this purpose, we used the liposome
ics diffuse in uncharged form through nerve sheaths and system in which reaction conditions and membrane lipids
penetrate into cell membranes to reach the cytoplasmic were readily manipulated to examine the influence of pH
binding sites or receptors on transmembrane channels. In and the importance of individual lipids (Chatterjee and
addition to the blockade of voltage-gated sodium chan- Agarwal 1988). Based on the verification results, we propose
nels, the mode of action of anesthesia includes the drug an alternative hypothesis which was speculated from the
and membrane lipid interaction which induces the changes potential reactivity of peroxynitrite pathologically relevant
in membrane physicochemical property, fluidization or to inflammation.
disordering (Jastak et al 1995; Frangopol and Mihǎilescu
2001). These pharmacological features, together with a Materials and methods
correlation between anesthetic potency and lipid solubility Reagents
(Covino 1986), strongly suggest that the drugs interact with Lidocaine, prilocaine and bupivacaine, all in hydrochloride
membrane lipid bilayers to induce local anesthesia. Lactic salt form, were purchased from Sigma (St. Louis,
acid and acidic by-products are increasingly produced MO, USA), and 1,2-dipalmitoylphosphatidylcholine
and concentrated in and near inflamed tissues, causing the (DPPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC),
acidosis which lowers the tissue pH at least the order of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE),
0.5–1.0 pH unit (Punnia-Moorthy 1987; de Backer 2003). 1-palmitoyl-2-oleoylphosphatidylserine (POPS) and
The pKa values of almost local anesthetics in clinical use sphingomyelin (SM) from Avanti Polar Lipids (Alabaster,
are larger than 7.5 (Jastak et al 1995). According to the AL, USA). Cholesterol, 1,6-diphenyl-1,3,5-hexatriene (DPH)
Henderson-Hasselbalch equation (Log10 [uncharged drug]/ and peroxynitrite were obtained from Wako Pure Chemicals
[charged drug] = pH – pKa), a greater proportion of admin- (Osaka, Japan), Molecular Probes (Eugene, OR, USA) and
istered drugs should be the charged molecules with much Dojindo (Kumamoto, Japan), respectively. The concentra-
less membrane interactivity and permeability under acidic tion of peroxynitrite was spectrophotometrically determined
conditions corresponding to inflamed tissues. Therefore, according to manufacturer’s directions, and then diluted to
local anesthetic effects would be decreased in the presence be 10 mM with 0.1 M NaOH. The prepared peroxynitrite
of inflammation, leading to the hypothetic mechanism based solutions were stored at −80 ºC and consumed within three
on tissue acidosis. This hypothesis seems to be so theoretical weeks after preparation. Water of liquid chromatographic
and understandable that it has been conventionally accepted grade (Kishida, Osaka, Japan) and dimethyl sulfoxide of
despite being not experimentally confirmed. spectroscopic grade (Kishida) were used for preparing
The hydrophobic interaction underlies the effects of local reagent solutions. All other chemicals were of the highest
anesthetics on lipid bilayers (Ohki and Ohshima 1996; Fran- grade available commercially.
gopol and Mihǎilescu 2001; Tsuchiya et al 2005). However,
the drug action on biomembranes is not identical to that Liposome preparation
speculated from the organic: aqueous phase partition. Unlike DPH-labeled liposomes of the lipid bilayer structure (total lipids
experimental membrane specimens with the simple lipid of 0.14 mM) were prepared by hydrating the lipid dry film with
composition, nerve cell membranes consist of different phos- 20 mM sodium phosphate buffer of pH 7.4 and 6.4 (Wako
pholipid and sterol components (Svennerholm et al 1992). Pure Chemicals) containing 100 mM KCl as reported previ-
Their compositional variations greatly affect the membrane ously (Tsuchiya et al 2005). The molar ratio of DPH to mem-
interactivities of drugs (Tsuchiya et al 2007). brane lipids was adjusted to be 1 : 200. The lipid composition
Therefore, we attempted to verify the conventional of liposomal membranes was as follows: DPPC (100 mol%),
acidosis mechanism for the local anesthetic failure asso- POPC and cholesterol (55 : 45, mol%), POPE and cholesterol
ciated with inflammation. We comparatively studied the (55 : 45, mol%), POPS and cholesterol (55 : 45, mol%),

42 Journal of Inflammation Research 2008:1

 Anesthetic failure associated with inflammatory acidosis

and SM and cholesterol (55 : 45, mol%). Nerve cell model (3.93 mL) of pH 7.4 and 6.4 to be a final concentration of
membranes were prepared with POPC, POPE, POPS, SM 0.2% (w/v) for lidocaine, 0.2% (w/v) for prilocaine and
and cholesterol (11 : 16.5 : 11 : 16.5 : 45, mol%) according 0.05% (w/v) for bupivacaine. Immediately after that, per-
to the composition of major membrane lipids of peripheral oxynitrite in 0.1 M NaOH (50 μL) was added to be 50 μM
nerve cells (Svennerholm et al 1992). with vortex-mixing for 5 sec, followed by reaction for 15 min
at 37 ºC to induce the membrane effects of local anesthetics.
Membrane lipid interaction The corresponding volume of 0.1 M NaOH vehicle was added
Local anesthetics dissolved in dimethyl sulfoxide (20 μL) to controls. Thereafter, DPH polarization was measured as
were added to the liposome preparations (3.98 mL) of pH described above. The influence of peroxynitrite was evalu-
7.4 and 6.4 so that a final concentration was 0.2% (w/v) for ated by comparing the membrane-fluidizing effects of local
lidocaine, 0.2% (w/v) for prilocaine and 0.05% (w/v) for anesthetics treated with and without peroxynitrite.
bupivacaine. The concentration of dimethyl sulfoxide was
adjusted to be less than 0.5% (v/v) of the total volume so as Statistical analysis
not to influence the membrane fluidity of liposomes. After Results are expressed as mean ± SE (N = 6–8). Data were
reaction for 15 min at 37 ºC, DPH fluorescence polarization statistically analyzed by Student’s t-test (StatView 5.0; SAS
was measured by an RF-540 spectrofluorometer (Shimadzu, Institute, Cary, NC, USA). A P value ⬍0.05 was considered
Kyoto, Japan) equipped with a polarizer and a cuvette significant.
thermo-controller as reported previously (Tsuchiya 2001).
Compared with control values (dimethyl sulfoxide vehicle Results
added), the decrease of DPH polarization means the increase Lidocaine, prilocaine and bupivacaine acted on DPPC
of fluidity (membrane fluidization) in the hydrocarbon cores liposomal membranes at pH 7.4 to increase their fluidity
of lipid bilayers, reflecting the acting site of tested drugs as shown by DPH polarization decreases (Figure 1). At
(Mizogami et al 2002). The potencies to interact with liposo- pH 6.4, however, their membrane-fluidizing effects were
mal membranes were compared on the basis of the changing significantly weakened. Compared with pH 7.4, the decreas-
degree of polarization values relative to controls. ing values of polarization were reduced to 30.8 ± 1.2% for
lidocaine, 32.5 ± 0.9% for prilocaine and 32.5 ± 0.5% for
Peroxynitrite treatment bupivacaine.
Local anesthetics dissolved in dimethyl sulfoxide (20 μL) Even if the pH was lowered from 7.4 to 6.4, local
were added to the nerve cell model membrane preparations anesthetics more greatly fluidized nerve cell model

Lidocaine Prilocaine Bupivacaine

0

–0.005 ** **
**
Polarization change

–0.01

–0.015

pH 7.4
pH 6.4
–0.02

Figure 1 Effects of local anesthetics on DPPC liposomal membranes at different pH. Lidocaine (0.2%, w/v), prilocaine (0.2%, w/v) and bupivacaine (0.05%, w/v) were reacted
with DPPC liposomes for 15 min at 37 ºC at pH 7.4 and 6.4, followed by DPH fluorescence polarization measurements. Each result represents mean ± SE (N = 6–8). **p ⬍ 0.01,
compared with pH 7.4.

Journal of Inflammation Research 2008:1 43

Ueno et al

membranes compared with DPPC liposomal membranes by treating with peroxynitrite (Figure 4). The inhibition
(Figure 2). While the polarization decreasing values at pH 6.4 occurred at both pH 7.4 and pH 6.4, which showed no
relative to pH 7.4 were 69.6 ± 2.1% for lidocaine, 70.5 ± 1.9% differences in inhibition potency.
for prilocaine and 71.8 ± 2.9% for bupivacaine, their potencies
to interact with nerve cell model membranes at pH 6.4 were Discussion
2.2–2.3 times those with DPPC liposomal membranes. Representative anesthetics lidocaine, prilocaine and
The effects of pH lowering remarkably depended on bupivacaine were subjected to the membrane interactions
the kind of membrane component phospholipids. The at clinically used concentrations (Jastak et al 1995). All of
membrane-interactive potencies of lidocaine, prilocaine them fluidized DPPC liposomal membranes in 15 min, being
and bupivacaine were greatly diminished at pH 6.4 in consistent with their onset time (Uckan et al 1998; Sun et al
POPC, POPE and SM liposomal membranes (Figure 3). 1999). However, their membrane-interactive potencies were
However, all the tested anesthetics fluidized POPS liposo- significantly decreased at pH 6.4 which is almost comparable
mal membranes at pH 6.4 with almost the same potency to inflamed tissue conditions (Punnia-Moorthy 1987). Such
as that at pH 7.4. pH-dependence may provide experimental support for the
The relative potencies of local anesthetics to inter- conventional mechanism based on tissue acidosis.
act with liposomal membranes at pH 6.4 to pH 7.4 are Local anesthetics must traverse ensheathing perineu-
shown in Table 1, together with the relative concentrations ria which are hydrophobic (lipoid) barriers against the
of uncharged molecules which were calculated by the drugs to reach nerve fibers. They also must penetrate into
Henderson-Hasselbalch equation using the pKa values of or across neuronal cell membranes consisting of lipid
7.9 for lidocaine, 7.8 for prilocaine and 8.1 for bupivacaine bilayers to exert their pharmacological activities. When
(Courtney 1980). blocking voltage-gated sodium channels, local anesthet-
The fluidizing effects of lidocaine, prilocaine and ics also permeate cell membranes to bind to the receptors.
bupivacaine on nerve cell model membranes were inhibited Inflammation causes metabolic acidosis which lowers the

Lidocaine Prilocaine Bupivacaine

0

–0.005
Polarization change

–0.01
**
**
**

–0.015

pH 7.4
pH 6.4
–0.02

Figure 2 Effects of local anesthetics on nerve cell membranes at different pH. Lidocaine (0.2%, w/v), prilocaine (0.2%, w/v) and bupivacaine (0.05%, w/v) were reacted with
nerve cell model membranes for 15 min at 37 ºC at pH 7.4 and 6.4, followed by DPH fluorescence polarization measurements. Each result represents mean ± SE (N = 6–8).
**p ⬍ 0.01, compared with pH 7.4.

44 Journal of Inflammation Research 2008:1

 Anesthetic failure associated with inflammatory acidosis

pH 7.4 pH 6.4

PC

PC
PE

PS

PE

PS
SM

SM
PO

PO

PO

PO

PO

PO
0

∗∗ ∗∗
∗∗ ∗∗ ∗∗ ∗∗
−0.005 ∗∗
∗∗ ∗∗
Polarization change

−0.01

−0.015

−0.02
Lidocaine
Prilocaine
Bupivacaine
−0.025

Figure 3 Effects of local anesthetics on liposomal membranes consisting of different phospholipids. Lidocaine (0.2%, w/v), prilocaine (0.2%, w/v) and bupivacaine (0.05%, w/v)
were reacted with POPC, POPE, SM or POPS liposomes for 15 min at 37 ºC at pH 7.4 and 6.4, followed by DPH fluorescence polarization measurements. Each result represents
mean ± SE (N = 6–8). **p ⬍ 0.01, compared with pH 7.4.

pH of affected tissues (Punnia-Moorthy 1987; de Backer usually in an octanol:buffer system (Strichartz et al 1990).
2003). Such inflammatory conditions would alter the phar- While tertiary amine local anesthetics exist in uncharged
macokinetics and pharmacodynamics of local anesthetics (free base) or charged form depending on their pKa values
by reducing their interactivities with lipid bilayers and/or and medium pH, the uncharged molecules are much more
membrane lipids. membrane-interactive than the charged ones. As shown
There is a systematic relationship between anesthetic in Table 1, the relative potencies to interact with DPPC
potency and hydrophobicity to determine the membrane liposomal membranes decrease in all the tested drugs by
interactivity (Courtney 1980). The hydrophobicity of local lowering the pH from 7.4 to 6.4, which is correlated to the
anesthetics is characterized by their partition coefficients, decreasing relative concentrations of uncharged molecules.

Table 1 Relative concentrations of uncharged anesthetic molecules calculated by the Henderson-Hasselbalch equation and relative
potencies to interact with different liposomal membranes (mean ± SE, N = 6–8)
Relative potency to interact with
pH Relative concentration DPPC Nerve cell POPC and POPE and SM and POPS and
of uncharged anesthetic liposome model cholesterol cholesterol cholesterol cholesterol
molecules membrane liposome liposome liposome liposome
Lidocaine 7.4 100.0 100.0 ± 0.2 100.0 ± 1.5 100.0 ± 2.8 100.0 ± 1.8 100.0 ± 1.8 100.0 ± 1.0
6.4 12.8 29.8 ± 1.1** 70.2 ± 2.1** 36.7 ± 0.8** 34.1 ± 1.6** 27.5 ± 0.4** 98.2 ± 0.7
Prilocaine 7.4 100.0 100.0 ± 0.7 100.0 ± 1.8 100.0 ± 1.8 100.0 ± 2.4 100.0 ± 1.7 100.0 ± 0.6
6.4 13.5 31.5 ± 0.8** 71.1 ± 1.9** 38.9 ± 0.9** 35.5 ± 1.8** 27.6 ± 0.8** 94.1 ± 0.9**
Bupivacaine 7.4 100.0 100.0 ± 1.1 100.0 ± 1.4 100.0 ± 2.3 100.0 ± 1.3 100.0 ± 2.0 100.0 ± 0.6
6.4 11.8 31.4 ± 0.5** 72.4 ± 2.9** 42.1 ± 2.0** 37.5 ± 1.5** 27.4 ± 1.1** 99.5 ± 0.6
**p ⬍ 0.01 compared with pH 7.4.

Journal of Inflammation Research 2008:1 45

Ueno et al

Control
Treated with peroxynitrite

100
Relative polarization decrease (%)

∗∗
∗∗ ∗∗ ∗∗ ∗∗
50 ∗∗

0
Li

Pr

Bu

Li

Pr

Bu
do

do
ilo

ilo
pi

pi
ca

ca
ca

va

ca

va
in

in
in

ca

in

ca
e

e
e

e
in

in
e

e
pH 7.4 pH 6.4

Figure 4 Influence of peroxynitrite on local anesthetic membrane effects. Lidocaine (0.2%, w/v), prilocaine (0.2%, w/v) and bupivacaine (0.05%, w/v) were treated with 50 μM
peroxynitrite for 5 sec at pH 7.4 and 6.4. Their effects on nerve cell model membranes were determined after reaction for 15 min at 37 ºC by measuring DPH fluorescence
polarization. Each result represents mean ± SE (N = 6–8). **p ⬍ 0.01, compared with control (not treated with peroxynitrite).

The effects of lidocaine, prilocaine and bupivacaine membrane lipids under inflammatory acidic conditions. As
on nerve cell model membranes at pH 6.4 were much well as DPPC liposomes, liposomal membranes consisting
greater than presumed from their relative concentrations of POPC, POPE and SM showed much less fluidization
of uncharged molecules (Table 1). Although the rank order at pH 6.4 compared with pH 7.4. However, only POPS
is different between relative potency and concentration, liposomal membranes were significantly fluidized even at
this is interpretable by the difference in hydrophobicity of pH 6.4 by lidocaine, prilocaine and bupivacaine despite
local anesthetics. The hydrophobic interaction with phos- that their charged molecules predominated. With respect to
phatidylcholine membranes is estimated to be prilocaine structural difference of these phospholipids, DPPC, POPC,
⬍ lidocaine ⬍ bupivacaine in increasing order of intensity POPE and SM are zwitterionic, whereas POPS is acidic.
based on their chromatographic behaviors in the reversed- Positively charged (cationic) drugs show much higher affin-
phase partition system (Tsuchiya et al 2005). The partition ity to acidic (anionic) phospholipids like POPS compared
coefficients are greatest in the order of prilocaine ⬍ lidocaine with zwitterionic ones (de Wolf et al 1990; Tsuchiya et al
⬍⬍ bupivacaine for both uncharged and charged molecules 2007). Not only uncharged but also charged anesthetics are
(Strichartz et al 1990). The results of nerve cell model mem- able to interact with lipid bilayers, the former deeply in the
branes never support the acidosis mechanism, indicating hydrophobic core, but the latter in the head-group regions of
that the tissue acidification is not essentially responsible for phospholipids (Pasenkiewicz-Gierula et al 2003). Local anes-
the decreased effects of local anesthetics in the presence of thetics in charged form electrostatically interact with the neg-
inflammation. atively charged sites in membranes at pH lower than the pKa
Unlike DPPC liposomal membranes consisting of values and their adsorbed amounts depend on the membrane
phosphatidylcholine alone, nerve cell model membranes surface charge (Ohki and Ohshima 1996). In this study, local
were prepared with different phospholipids (POPC, POPE, anesthetics with pKa over 7.8 showed less fluidizing effects
POPS and SM) and cholesterol to resemble peripheral nerve on DPPC, POPC, POPE and SM liposomal membranes
cells (Svennerholm et al 1992). Such lipid composition at pH 6.4 as the uncharged molecules decreased. On the
may explain why local anesthetics are able to interact with other hand, their effects on POPS liposomal membranes

46 Journal of Inflammation Research 2008:1

 Anesthetic failure associated with inflammatory acidosis

were significantly great even at pH 6.4, contradicting to the the activities of sensory nerves and ion channels to possibly
relative decrease of uncharged molecules, but correlating induce afferent hyperexcitability and hyperalgesia (Flake
to the relative increase of charged molecules. As known in and Gold 2005; Wang et al 2007). In addition to these patho-
certain amphiphilic drugs (Štolc et al 1989), the cationic physiological changes, it is hypothesized that inflammatory
moieties of local anesthetics possibly pair with the anionic peroxynitrite could participate in the decrease of local
head-groups of acidic phospholipids to form the seemingly anesthetic effects.
uncharged ion-pairs, which could penetrate into lipid bilayers In conclusion, inflammatory acidosis is not as important
and modify membrane fluidity. Acidic phosphatidylserine is for the decreased efficacy of local anesthesia as recognized
referred to as a counter-ion to pair with positively charged conventionally. As a novel mechanistic hypothesis,
anesthetics. peroxynitrite relevant to inflammation may interact with local
Local anesthetics have been verified to interact anesthetics to modify their pharmacological activities.
with nerve cell membranes at the pH corresponding to
inflammatory acidosis. Tissues can buffer the excess acidity Acknowledgments
(Wennberg et al 1982) and such buffering is more potent in This study was partly supported by a Grant-in-Aid for Sci-
inflamed tissues (Punnia-Moorthy 1988). These pathophysi- entific Research 20592381 (to H.T.) from the Japan Society
ological features are also unfavorable to the conventional for the Promotion of Science.
mechanism based on tissue acidosis, requiring an alternative
theory for the local anesthetic failure associated with Disclosures
inflammation. None of the authors disclose conflicts of interest.
Inflammatory cells produce peroxynitrite (Ródenas et al
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48 Journal of Inflammation Research 2008:1