For spinal or epidural anesthesia, a larger concentration of lidocaine than of bupivacaine is required (1). Anesthetic potency depends on lipid solubility (2). Lipid solubility of bupivacaine is eight to nine times larger than that of lidocaine (3). Local anesthetics block the sodium channels when they reach the axoplasm (4). The increase in the intracellular concentration of local anesthetics augments the anesthetic potency (5). The greater potency of bupivacaine is thought to be related to its higher lipid solubility, tissue permeability, and affinity for sodium channels than that of lidocaine (3). However, details of the intracellular concentrations and anesthetic potency of local anesthetics that are required to inhibit nerve conduction are currently unavailable.
The objective of this study was to determine the axoplasmic (intracellular) concentration of lidocaine and bupivacaine and to compare its permeability based on the ratio of the intra- to extracellular concentration when nerve conduction is blocked.
All animal experiments were approved by the Animal Care Committee of the University of Miyazaki. The giant axon of the crayfish, Procambarus clarkii, was enzymatically prepared as described previously (5). In brief, the abdominal nerve bundles from a crayfish (weight, 38–48 g; n = 40) were isolated under hypothermic narcosis by soaking a crayfish in ice water for 10 min. The four giant axons in each bundle were exposed by removal of the nerve sheath between the second and third ganglia in HEPES-van Harrevelds solution (195 mM of NaCl, 5.4 mM of KCl, 13.5 mM of CaCl2, 2.6 mM of MgCl2, 7.5 mM of NaHCO3, and 5 mM of HEPES) adjusted to a pH value of 7.60 by adding 2 N of NaOH. The nerve bundle was treated for 30 min in 1 mg/mL of collagenase (Wako Pure Chemical, Osaka, Japan) to ease microelectrode insertion.
The recording chamber had two compartments and was made using acrylic materials (Fig. 1). The chamber had stimulus and perfusion pools (1.5 mL in volume) separated by Vaseline. The nerve bundle was fixed on a silicon sheet stuck on a basal plane of the perfusion pool and perfused at 1.5 mL/min with HEPES-van Harrevelds solution. The portion of the nerve in the stimulus pool was fully covered with liquid paraffin to prevent the nerve from drying and the leaking drugs from perfusing. One of four axons in a new nerve bundle was used for each measurement. All experiments were performed at room temperature (25°C–27°C).
We used a continuous stirred tank reactor (CSTR) to gradually increase the local anesthetic concentration from zero to the final concentration in the perfusion solution. The concentration of local anesthetic in the recording chamber was fixed to slowly increase linearly to facilitate the accurate measurement of the nerve blocking time and the intracellular local anesthetic concentration. Under the assumption of perfect mixing, the concentration of local anesthetic in the recording chamber, C(t), was predicted using the following equation: C(t) = C0(1 − e−Qt/V)(1 − e−Qt/V′), where Q is the flow rate through the tank, C0 is the concentration of local anesthetics in the syringe, V is the stirring tank volume, V′ is the recording chamber volume, and t is the elapsed time (6). It was assumed that there was no drug reaction in the CSTR, and the local anesthetic concentration in the recording chamber was 0 at t = 0; C(t=0) = 0. The equation was confirmed to measure the lidocaine concentrations of the recording chamber by the fluorescence polarization immunoassay method (SRL, Tokyo, Japan) during perfusion.
A conventional glass microelectrode was filled with 4 M of KCl and inserted into one of the two median giant axons at the region between the second and third ganglia. The membrane potential (Em) was measured using a high-impedance amplifier (FD-233; WPI, Sarasota, FL) and continuously monitored using a storage oscilloscope (VC-11; Nihonkoden, Tokyo, Japan). The action potentials were evoked by a square-wave pulse of supramaximal intensity (double the threshold voltage for 0.2 ms) using an electrical stimulator (SEN-3301, Nihonkoden). The stimulation, 20 stimulus pulses with 20 Hz bursts repeated 60 times with a 60-s interval, was started for the measurements of tonic (0.016 Hz = 1/min) and phasic (20 Hz) blocks. The action potentials and Em were continuously recorded. Perfusion was started to gradually increase the local anesthetic concentration from zero over a period of 60 min. Before the experiment, the concentrations of lidocaine and bupivacaine required to block the action potentials at approximately 50 min after the start of the experiment were determined. Nerve blocks in the absence of action potentials were evaluated; one or more (0 to 19) disappearances of action potentials within 20 pulses were determined to be a phasic block, and complete (all 20) disappearance was defined as a tonic block. The time elapsed from the start of the perfusion of local anesthetics until the occurrence of a phasic or a tonic block was recorded. In each experiment, the local anesthetic concentration of the perfusion solution was predicted using the CSTR equation. The action potentials and intracellular local anesthetic concentration were not measured simultaneously because of the difficulty of measuring the intracellular local anesthetic concentration because of the significant amount of noise in our setting when electrical burst stimulation was applied. The electrical stimulation did not alter the intracellular local anesthetic concentration (7).
Local anesthetic-sensitive microelectrodes were constructed according to previously described methods (5,7). Each microelectrode contained 98.2 wt/% of o-nitrophenol octyl ether, 1.8 wt/% of polyvinylchloride, 2 mM of tungstophosphoric acid hydrate, and 2 mM of lidocaine HCl or bupivacaine HCl. The microelectrode was back filled to 4 M of KCl. Local anesthetic sensors were calibrated with each anesthetic dissolved in an intracellular-like solution containing 100 mM of KCl, 10 mM of NaCl, and 5 mM of HEPES at a pH value of 7.2 within the concentration range of 0.01–10 mM (5 mM in bupivacaine). The local anesthetic sensor response was linear over a concentration range of 0.08–10 mM. Typical calibration graphs for the electrodes are shown in Figure 2. The slope of the electrode responses was determined as 71 ± 4.3 mV/log [lidocaine] (mean ± sd) and 98 ± 8.7 mV/log [bupivacaine] by regression analysis of the linear portion of the calibration curves. The electrode potentials were measured repeatedly with different concentrations of local anesthetics (8), and the coefficient of variance ranged from 0.63% to 1.12% (mean, 0.81%). The influence of pH values on the response of the local anesthetic sensor was determined using previously described methods (5,8). A change in pH value did not affect the response of the local anesthetic sensor between pH values of 6.5 and 8.0. Selectivity coefficients, which express the ability of the local anesthetic-sensitive microelectrode to distinguish different ions, were evaluated by the fixed-interference method (5,9). The selectivity coefficient values for Na+, K+, and Ca2+ were 1.06 × 10−4, 1.56 × 10−5, and 8.75 × 10−5, respectively.
A local anesthetic-sensitive microelectrode and a conventional glass microelectrode were inserted into the same axon (Fig. 1). The Em and the potential of the local anesthetic-sensitive microelectrode (ELA, where ELA = Elid or Ebup) were monitored continuously using a storage oscilloscope. Because ELA is the sum of Em and the potential produced by each local anesthetic cation, the Em measurement must be subtracted from ELA. The average increases in the CSTR concentrations during perfusion of a local anesthetic were 0.20 mM/min for lidocaine and 0.03 mM/min for bupivacaine. We confirmed that the local anesthetic-sensitive microelectrode was able to respond to the change in local anesthetics before this experiment. The intracellular local anesthetic concentration was calculated by the following equation (5): [LAi] = 10(E − C)/S, where [LAi] is the intracellular local anesthetic concentration expressed in millimoles, E is the difference between ELA and Em during exposure to a local anesthetic, C is the relative ELA at 1 mM of the local anesthetic from 0 mM, and S is the slope of the linear portion of the calibration curve.
All values were expressed as mean ± sd. Study groups were compared using the Student’s t-test. A value of P < 0.05 was considered statistically significant.
The mean elapsed times until the occurrence of a tonic block produced by lidocaine and bupivacaine were similar (Table 1). The mean elapsed times until the onset of a phasic block were significantly shorter than those until the onset of a tonic block for both lidocaine and bupivacaine. Furthermore, the mean elapsed time until the onset of a phasic block was significantly shorter for bupivacaine than for lidocaine.
Typical tracings of the predicted and intracellular lidocaine and bupivacaine concentrations and the lidocaine concentration obtained in a recording chamber by a fluorescence polarization immunoassay are shown in Figure 3. The predicted (extracellular) concentrations of lidocaine and bupivacaine obtained using the CSTR equation were 10.93 ± 0.60 mM and 1.84 ± 0.13 mM in the tonic block and 10.33 ± 0.65 mM and 1.23 ± 0.13 mM in the phasic block, respectively. The intracellular concentrations at the tonic and phasic blocks were 3.71 ± 0.95 mM and 3.42 ± 0.90 mM in the lidocaine perfusion and 0.34 ± 0.09 mM and 0.21 ± 0.06 mM in the bupivacaine perfusion, respectively (Table 1). The ratio of the intra- to extracellular concentrations at the tonic and phasic blocks was significantly larger in the lidocaine (34.0% ± 8.9% and 33.1% ± 8.6%) than in the bupivacaine perfusion (18.5% ± 5.0% and 17.1% ± 4.5%) (Fig. 4).
The present results demonstrated that an 11- to 16-fold larger intracellular concentration of lidocaine than bupivacaine was required to block a single nerve and that the ratio of the intra- to extracellular concentration was significantly larger for lidocaine than for bupivacaine. The results also demonstrated that a use-dependent (phasic) block was predominantly achieved with bupivacaine when the extracellular concentration was gradually increased, and a single nerve was blocked by tonic or phasic stimulation.
The difference in the intra- to extracellular concentration between lidocaine and bupivacaine could have resulted from the difference in the extracellular perfusion concentration between lidocaine and bupivacaine. Furthermore, the difference could have been affected by the intracellular pH value change during perfusion. The administration of 10 mM of procaine at an extracellular pH value of 7.5 decreases the intracellular pH value from 7.36 to 6.97 in full-grown Xenopus oocytes (10). When the intracellular pH value is decreased, the increase in the base traversing into the axoplasm and the conversion from the base to the cation in the axoplasm accelerate the amount of cation in the axoplasm (5). If intracellular acidification is induced by local anesthetics, especially by lidocaine, the change in the intracellular pH value may contribute to the difference in the ratio of the intra- to extracellular concentration. In contrast to the acidification, Willoughby et al. (11) have shown that 0.3 U of intracellular alkalinization are induced by the administration of 3 mM of procaine to snail neurones; however, intracellular alkalinization could not explain the marked increase in the intracellular lidocaine concentration. In this study, the intracellular pH value was not measured because of the marked interference of local anesthetics with the H+-sensitive microelectrode. The change in the intracellular pH value and other details could not be clarified.
The physicochemical properties of lidocaine and bupivacaine are important for the explanation of the differences in the intra- to extracellular concentration. Although the pKa values for lidocaine (7.77) and bupivacaine (8.10) are different at 36°C (12), they change with various experimental conditions, such as the temperature, pH value, and buffer (13). At 25°C, the pKa values of lidocaine and bupivacaine were 8.19 and 8.21, respectively (12). Then, the ionized forms of lidocaine and bupivacaine at 25°C were approximately 80% at a pH value of 7.6. The physicochemical properties were not closely related to the marked increase in intracellular lidocaine concentration.
Kanai et al. (7) have shown that the ratio of the intra- to extracellular concentrations is 25% each in the giant axon of the crayfish when 1 mM of S(-) bupivacaine, R(+) bupivacaine, or ropivacaine is perfused for 15 minutes. In our study, we used a continuous increase of the extracellular concentration and achieved a large concentration of more than 1 mM. Furthermore, we determined the intracellular concentrations to produce tonic and phasic blocks. The difference in the ratio of the intra- to extracellular concentration between their results and ours seems to be related to the experimental conditions.
The relative potency valence for lidocaine has previously been considered to be four- to eightfold less than that for bupivacaine in vitro (14). Bupivacaine produces a phasic block at one eighth of the concentration of lidocaine or less (15). In the present study, the relative potency of lidocaine was found to be six- to eightfold that of bupivacaine, based on the extracellular concentration. When the intracellular concentration required to produce anesthetic potency was compared, lidocaine required a concentration 16-fold larger than bupivacaine to achieve a phasic block in a single giant axon of the crayfish. The large difference in the potency valence between lidocaine and bupivacaine may be related to their affinity for sodium channels.
In conclusion, the ratio of the intra- to extracellular concentration of lidocaine was larger than that of bupivacaine when the nerve was blocked, and a use-dependent (phasic) block was induced with bupivacaine at a significantly smaller concentration than lidocaine in the giant axon of a crayfish in vitro.
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© 2006 International Anethesia Research Society
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