Anal. Chem. 1998, 70, 2618-2622
In Vivo Voltammetric Detection of Rat Brain Lactate with Carbon Fiber Microelectrodes Coated with Lactate Oxidase
Anal. Chem. 1998.70:2618-2622. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 10/09/18. For personal use only.
Nataliya F. Shram,*,†,‡ Larissa I. Netchiporouk,‡,§ Claude Martelet,† Nicole Jaffrezic-Renault,† Chantal Bonnet,§ and Raymond Cespuglio§
Laboratory of Physicochemistry of Interfaces, UMR CNRS 5621 IFoS, Ecole Centrale de Lyon, BP 163, 69131 Ecully Cedex, France, Sector of Bioelectronics, Kiev University, P.O. Box 152, Kiev-1, 252001, Ukraine, and Department of Experimental Medicine, U INSERM 480, Claude Bernard University, 8 avenue Rockefeller, 69373 Lyon Cedex, France
To allow rat brain lactate measurement in vivo, a specific sensor based on a carbon fiber (O ) 30 µm) microelectrode coated with lactate oxidase was prepared. Combined with the differential normal pulse voltammetry measurement method, such a sensor, with a sensitivity of 9.15 ( 0.91 mA‚M-1‚cm-2, provided a lactate linear response in concentrations ranging from 0.1 to 2.0 mM. The measurements performed appeared to be essentially insensitive to usual interference caused by the electroactive compounds present in the brain (ascorbic acid and peptides). In vivo detection performed in the cortex of the anesthetized rat led to the determination of a lactate concentration of 0.41 ( 0.02 mM. Moreover, to validate the results obtained in vivo, an ex vivo determination of the lactate level was also performed in samples of brain tissue, plasma, and cerebrospinal fluid, using both voltammetry and a clinical analyzer with colorimetric-based detection. A good correlation was observed between the sets of data established by both methods.
detection.4-7 Regarding the sensors proposed, there are some general requirementssspecificity, stability, lack of interferences, and low cost. Besides the above aspects, in vivo application also implies some additional restrictions concerning the electrode material and configuration. In this sense, carbon fiber is now reported to be one of the most convenient materials due to its high biocompatibility and micrometric dimensions (8-30 µm). The suitability of such material is also reinforced by the possibility to improve the sensitivity of the measurements by chemical or electrochemical treatment of its surface.8-14 Concerning the measuring method, amperometry with constant potential remains the most commonly used,4,6,7,15-20 despite its drawbacks: insufficient specificity toward the substrate of interest, since the electroactive species with a lower oxidation potential can contribute to the sensor response, and electrode fouling by high molecular components in the physiological medium. From this point of view, differential normal pulse
* Address correspondence to this author, currently at Claude Bernard University. Phone: 33-4-78 77 71 26. Fax: 33-4-78 77 71 72. E-mail: cespugli@ univ-lyon1.fr. † Ecole Centrale de Lyon. ‡ Kiev University. § Claude Bernard University. (1) Siesjo, B. K. Brain Energy Metabolism; Wiley & Sons: New York, 1978. (2) Siesjo, B. K. Neurochem. Pathol. 1988, 9, 31-88. (3) Yao, H.; Sadoshima, S.; Fujii, K.; Ishitsuka, T.; Tamaki, K.; Fujishima, M. Eur. Neurol. 1987, 27, 182-187.
(4) Meyerhoff, C.; Bischof, F.; Mennel, F. J.; Sternberg, F.; Bican, J.; Pfeiffer, E. F. Biosens. Bioelectron. 1993, 8, 409-414. (5) Baker, D. A.; Gough, D. A. Anal. Chem. 1995, 67, 1536-1540. (6) Fray, A. E.; Forsyth, R. J.; Boutelle, M. G.; Fillenz, M. J. Physiol. 1996, 496, 49-57. (7) Hu, Y.; Wilson, G. S. J. Neurochem. 1997, 69, 1484-1490. (8) O’Neill, R. D. Analyst 1994, 119, 767-779. (9) Pantano, P.; Kuhr, W. G. Electroanalysis 1995, 7, 405-416. (10) Feng, J. X.; Brazell, M.; Renner, K.; Kasser, R.; Adams, R. N. Anal. Chem. 1987, 59, 1863-1867. (11) Cespuglio, R.; Faradji, H.; Ponchon, J. L.; Buda, M.; Riou, F.; Gonon, F.; Pujol, J. F.; Jouvet, M. Brain Res. 1981, 223, 287-298. (12) Netchiporouk, L. I.; Shul’ga, A. A.; Jaffrezic-Renault, N.; Martelet, C.; Olier, R; Cespuglio, R. Anal. Chim. Acta 1995, 303, 275-283. (13) Sakslund, H.; Wang, J.; Lu, F.; Hammerich, O. J. Electroanal. Chem. 1995, 397, 149-155. (14) Zhang, X.; Zhang, W.; Zhou, X.; Ogorevc, B. Anal. Chem. 1996, 68, 33383343. (15) Hendry, S. P.; Higgins I. J.; Bannister, J. V. J. Biotechnol. 1990, 15, 229238. (16) White, S. F.; Turner, A. P. F.; Bilitewski, U.; Schmid, R. D.; Bradley, J. Anal. Chim. Acta 1994, 295, 243-251. (17) Ito, N.; Matsumoto, T.; Fujiwara, H.; Matsumoto, Y.; Kayashima, S.; Arai, T.; Kikuchi, M.; Karube, I. Anal. Chim. Acta 1995, 312, 323-328. (18) Mizutani, F.; Yabuki, S.; Hirata, Y. Talanta 1996, 43, 1815-1820. (19) Sprules, S. D.; Hart, J. P.; Pittson R.; Wring, S. A. Electroanalysis 1996, 8, 539-543. (20) Palmisano, F.; De Benedetto, G. E.; Zambonin, C. G. Analyst 1997, 122, 365-369.
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S0003-2700(97)01299-7 CCC: $15.00
Direct in vivo measurements of lactate either in tissue or in biological fluids such as cerebrospinal fluid (CSF) or plasma are of great interest for studies related to energy production in anaerobic conditions.1 It is also reported that alterations in the processes of production and delivery of this compound may contribute to damage of the central nervous system in some pathological situations, such as hypoxia, ischemia, trauma, and seizures.2,3 Presently, reports on lactate sensors are relatively rare; this is particularly true for those dealing with direct in vivo
© 1998 American Chemical Society Published on Web 06/04/1998
voltammetry (DNPV) seems to be a more attractive method when used together with enzyme-modified electrodes.8,21-23 In this case, by recording the differential of the redox current as a function of the potential applied to a working electrode, one can obtain a voltammogram where separated peaks correspond to oxidation or reduction of different substances; a correct choice of the DNPV parameters yields in a better signal resolution for the substrate of interest. The present report concerns the measurement of lactate with a biosensor based on a carbon fiber microelectrode (CFME) coated with an enzymatic layer (lactate oxidase) and with an additional membrane of cellulose acetate. This sensor was used together with DNPV to record the oxidation current of the hydrogen peroxide generated by enzymatic conversion of lactate to pyruvate. The measurements were first performed in vitro to examine the sensor characteristics and then in vivo for lactate monitoring in the rat brain. Finally, to further validate the specificity and the sensitivity of the sensor prepared, ex vivo lactate determinations in samples of plasma, CSF, and brain tissue were performed with the method proposed and verified by using a clinical analyzer with a colorimetric principle of detection.
Figure 1. DNPV voltammograms obtained in vitro with the lactate sensor. The first two peaks are shown for each concentration of lactate. A peak appearance corresponds to the hydrogen peroxide oxidation at 0.8-1.0 V; the peak height increases proportionally to lactate concentrations. Abbreviations: DNPV, differential normal pulse voltammetry; h, peak height.
EXPERIMENTAL SECTION Enzymes and Chemicals. The lactate oxidase enzyme (LOx) (from Pediococcus sp., EC 1.1.3.2, activity 36 units/mg) was from Genzyme Diagnostics (Kent, England); L-(+)-lactic acid, cellulose acetate, phosphate-buffered saline (PBS) tablet), bovine serum albumin (BSA), L-ascorbic acid, and N-methyl-D-aspartic acid (NMDA) were from Sigma; hydroxyethylcellulose (HEC), medium viscosity, was from Fluka; and glutaraldehyde (25% aqueous solution) was from Merck. Peptides (posthypophyseal extract, 5 IU/mL) were obtained from Choay Laboratory (Paris, France). Preparation of the Electrodes. The working electrodes used in this study were prepared by a procedure similar to that described earlier for the glucose sensors.23 Briefly, a single carbon fiber (φ ) 30 µm, AVCO type, Lowell, MA) was inserted into a pulled glass capillary, and the glass was broken just at the end of the fiber. The fiber was then pushed out about 2-3 mm and stuck to the pipet with an epoxy resin. The emergent part of the fiber was cut at a length of 500 µm (active part of the sensor). Inside the pipet, electrical contact with the fiber was ensured by a silver wire inserted through the nonpulled tip of the pipet. To improve the selectivity and the sensitivity of the sensor, the active surface of the carbon fiber was electrochemically pretreated by applying successive runs of a triangular waveform potential (70 Hz, 2.4 V/10 s, 1.8 V/15 s, 1.4 V/3 s) through the electrode dipped in a PBS solution.11,12 Solution for the enzyme immobilization was prepared by dissolving 10 mg of LOx, 5 mg of BSA, and 5 µL of glycerol in 40 µL of a 3% HEC solution (in a 0.1 M phosphate buffer, pH 7.0). The active parts of the electrodes were dipped into 6 µL of this mixture, added with 1.2 µL of 5% glutaraldehyde solution. Afterward, the electrodes were dried for 1 h and then dip-coated with a cellulose acetate (CA) membrane (2% w/w solution in 1:1
w/w mixture of acetone and cyclohexanone) and left to dry for at least 2 h. The sensors prepared were stored at +4 °C. Instrumentation and Measurements. In vitro and in vivo lactate measurements as well as electrochemical pretreatment of the electrodes were carried out with a chlorinated silver wire as reference electrode (Ag|AgCl), a platinum wire as auxiliary electrode (Pt), and a carbon fiber lactate sensor as working electrode (WE). The electrodes were connected to a Biopulse pulse voltammetric unit (Tacussel, France) used in DNPV mode and to a chart recorder. The DNPV method, also described previously,21,23-25 consists of applying to a WE successive double pulsessprepulse, with a linearly increasing amplitude, and measuring pulse, with a constant amplitude. The signal recorded is the differential of the oxidation current, sampled just before and at the end of the measuring pulse, plotted as a function of the potential sweep. The DNPV voltammogram appears as peaks whose height (determined as indicated in Figure 1) is proportional to the concentrations of the substances oxidized at the WE surface. For in vitro experiments, the electrodes were placed in plastic cells containing 2 mL of PBS solutions with different lactate concentrations (except for the measurements where KH2PO4NaOH buffer of 10, 30, 50, and 100 mM at different pH values was used). In vivo studies were performed in the extracellular space of the rat brain cortex. OFA male rats (250-300 g, IFFA CREDO, Labresle, France) were anesthetized with sodium pentobarbital (60 mg/kg ip). After surgery, a lactate WE was inserted into the top 500 µm of the right cortex (bregma ) -2 mm, lateral ) 2 mm)26 by means of a microlift. Reference and auxiliary electrodes were afterward placed in contact with the dura close to the WE. The electrodes were then connected to the instrumental setup and the recordings started immediately. All the experiments involving animals were in accordance with the rules of the French Agriculture Ministry (Decree No. 87.848).
(21) Aoki, K.; Osteryoung, J.; Osteryoung, R. A. J. Electroanal. Chem. 1980, 110, 1-18. (22) Suaud-Chagny, M. F.; Gonon, F. G. Anal. Chem. 1986, 58, 412-415. (23) Netchiporouk, L. I.; Shram, N. F.; Jaffrezic-Renault, N.; Martelet, C.; Cespuglio, R. Anal. Chem. 1996, 68, 4358-4364.
(24) Suaud-Chagny, M. F.; Cespuglio, R.; Rivot, J. P.; Buda, M.; Gonon, F. G. J. Neurosci. Methods 1993, 48, 241-250. (25) Gonon, F. G.; Buda, M. J. Neurosci. 1985, 14, 765-774. (26) Paxinos G.; Watson, C. The Rat Brain in Stereotaxic Coordinates, 2nd ed.; Academic Press: New York, 1986.
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Ex vivo measurements of lactate were performed either with the lactate sensors or with a clinical analyzer (Corning 7200, Shimadzu, enzymatic-colorimetric method, 505 nm) in liquid samples containing homogenized cortical tissue biopsies, CSF drawn from the cisterna magna, and plasma. Experimental results are presented as mean ( SEM. RESULTS AND DISCUSSION DNPV Lactate Detection. The lactate sensor prepared detects the oxidation current of the hydrogen peroxide following the lactate enzymatic oxidation: LOx
lactate + O2 98 pyruvate + H2O2 +
H2O2 f O2 + 2H + 2e
-
(1) (2)
The DNPV measurements were carried out using the following parameters: potential was swept from 0.45 to 1.25 V with a scan rate of 10 mV/s (the potential step was 5 mV per 0.5 s); prepulse duration was 60-80 ms; the measuring pulse amplitude was 20 mV and it lasted 80 ms. The period of measurements was 2 min, so the time lapse between two successive scans was 40 s. Under these conditions, the response of the sensor resulted in a wellresolved DNPV peak appearing at 0.8-1.0 V vs Ag|AgCl. Its height was determined as the distance from its top to the line connecting the two minima. A typical response of the lactate sensor in different calibration solutions is shown in the Figure 1. To ensure that the sensor response was reproducible, at least three scans were registered for each in vitro measurement. Commonly, the linearity range of the sensors was between 0.1 and 2.0 mM of lactate, and their sensitivity in vitro was 9.15 ( 0.91 mA‚M-1‚cm-2 (n ) 35) (the sensitivity of the sensors reported was estimated to be about 1.08-6.03 nA‚mmol-1‚L; 1 mA‚M-1‚cm-2; 3.5 mA‚M-1‚cm-2; refs 4, 5, and 7 respectively). Oxygen Influence. The effect of the presence of oxygen on the sensor response was studied by calibrating the sensor in airsaturated solutions and in these solutions after bubbling with O2. The O2 dependence appears to be not significant up to about 1 mM lactate, and it becomes stronger for higher concentrations. In O2-saturated solutions, the linearity of the sensor response was extended up to 3-4 mM lactate. The partial pressure of oxygen determined in a hospital laboratory was 192 Torr for air-saturated and 706 Torr for oxygen-saturated calibration solutions. Calculation of the corresponding O2 concentrations (0.32 and 1.18 mM, respectively) shows that the observed linearity range of the sensor is at least 4 times higher than could be predicted from reaction 1. These estimations indicate that, despite the relatively low basal pO2 in cerebral medium (about 35-90 Torr1), the low levels of lactate can be detected using the sensor. Taking into account that the reported concentrations of lactate in the rat brain are in the range 0.35-1.12 mM,27,28 in vivo brain monitoring can be envisaged. Effect of pH and Buffer Concentrations. In this study, the response of the sensor to 1.5 mM lactate was examined for the phosphate buffer concentrations from 10 to 100 mM, in the pH range from 6 to 9 (Figure 2). The pH dependence of the sensor (27) Kuhr, W. G.; Korf, J. J. Cereb. Blood Flow Metab. 1988, 8, 130-137. (28) Demestre, M.; Boutelle, M.; Fillenz, M. J. Physiol. 1997, 499, 825-832.
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Figure 2. Influence of pH and buffer concentration on the sensor response obtained in a 1.5 mM lactate solution. Responses are given in relative values: 100% response is defined by the maximal response measured in a given buffer concentration; each point represents the mean of the measurements with five electrodes. Abbreviations are as in Figure 1.
response exhibits a large pH optimum between 7.5 and 8.5, which is sharper for lower buffer concentrations. The absolute current values (not shown in the figure) were about 2.5-fold lower in the 10 mM buffer than in the 30, 50, and 100 mM buffers at the same pH. This observation could be assigned to the local shift of pH resulting from the H2O2 oxidation (reaction). This decrease in pH might be attenuated in the highly buffered media. Thus, in physiological conditions (pH 7.4 and the buffer species concentration of about 40 mM), the sensor is expected to be in optimum working conditions. Effect of Interferences. During in vivo measurements, easily oxidizable species (like ascorbic acid, uric acid, etc.) can contribute to the biosensor response. To minimize this effect, (1) the electrodes were coated with a CA membrane (to block large molecules), and (2) the potential was scanned from 0.45V vs Ag|AgCl to exceed the oxidation potential of the interfering species. However, among the substances present in the cerebral medium, there is a great number of peptides which can be oxidized at a potential of about 0.7 V close to that of the H2O2. Even though the proper choice of the DNPV parameters allows one to separate the two oxidation peaks, some peptides were, nevertheless, tested as potential interferences. In this respect, the calibration solutions were added with 5% of posthypophyseal extract (containing mainly oxytocine and antidiuretic hormone), giving a final peptides concentration of 0.25 IU/mL. As shown in Figure 3, the addition of peptides or ascorbic acid (AA) at its physiological concentration changes the form of the signal for lactate but does not change its height significantly. The ratios of the DNPV peak, obtained in the lactate solution added with AA or peptides, to the peak produced in the interference-free solution were 0.95 ( 0.05 (n ) 5) and 0.92 ( 0.02 (n ) 7), respectively. Long-Term Stability. In this study, lactate sensors with different enzyme contents (10 mg as described above and 30 mg of LOx) were examined. The corresponding results are illustrated in Figure 4. A decrease in the response with time, likely due to enzyme loss or inactivation, was observed in both cases. We suppose to deal rather with the inactivation, considering the following aspects of the sensors behavior: (1) a reversible loss of their sensitivity after in vivo use, and (2) during the operational stability tests in vitro, the sensor response declined gradually after
Figure 3. DNPV peaks obtained in interference-free solutions of 1.5 mM lactate and in the presence of ascorbic acid (AA) or peptides. Addition of the interferences changes the form of the signal but not its height.
Figure 4. Long-term stability of the sensors with different enzyme loading: a sensor with 10 mg of lactate oxidase (LOx) membrane (O) and another one with 30 mg of LOx membrane (2) were used. Each point is a mean of three measurements performed in a 1.5 mM lactate solution.
some hours of working, while in the case of an enzyme loss, i.e., its leakage in the solution, some increment of the signal could be seen. The electrodes coated with the higher dose of enzyme appeared to be more stable and showed an improvement in their response after the first measurements. Such an effect can be explained by the structural modifications of the membrane becoming more porous. For the lower content of LOx, a competitive process of enzyme inactivation, more noticeable than for the higher dose, can prevail toward the changes of porosity and results in a decrease of the sensor response. However, since the sensors with the lower LOx loading (thus, less expensive) were satisfactory and sufficiently stable for in vivo acute measurements, they were used in all other experiments. Sensors with the greatest enzyme content should be recommended for chronic measurements in freely moving animals. In Vivo Lactate Monitoring. The electrodes were tested for a lactate level monitoring in a living rat brain. Usually, the stability of the basal signal was achieved in about 1 h, being slightly altered after anesthetic supply. According to the data obtained, it appears that the concentration of lactate in the extracellular compartment of the cortex is 0.41 ( 0.02 mM (n ) 10). It should be mentioned
Figure 5. In vivo changes induced in the extracellular brain lactate level by intraperitoneal injection of NMDA. Baseline level obtained before the substance administration is considered as 100%. Abbreviations: NMDA, N-methyl-D-aspartic acid; others as in Figure 1.
that the pentobarbital anesthesia (50 mg/kg ip) is reported to decrease the brain lactate level by 42%.29 It is known that the extracellular fluid lactate concentration can be increased by the stimulation of neuronal activity,30 and studies concerning such a stimulation by glutamate and NMDA were recently reported.28,31 In the present work, we have used NMDA, an ionotrophic glutamate receptors agonist. After we obtained a stable response of the sensor, a dose of NMDA (5 mg/1 mL NaCl, 0.9%) was administered ip to the animals. The ip route of administration was used only as a general test of responsivity since, in comparison with local injections, it allows a more homogeneous brain influx of the substances. Just after injection, the height of the voltammetric signal rose rapidly and reached its maximal value (+150%) 10 min later. Afterward, it decreased progressively and reached +25% of the baseline level 2 h later (Figure 5). This effect is in good agreement with data of the literature reporting an increased efflux of lactate in the striatum after NMDA local perfusion.31 Such an increase of the signal is most likely due to the stimulated (via NMDA receptors and lactate carriers) glycolysis, i.e., glucose uptake and lactate production. Validation of the Voltammetric Lactate Measurements. To validate our measurements, CSF, plasma, and brain tissue biopsies were sampled and lactate concentrations determined ex vivo either with the lactate sensor prepared or with the clinical analyzer. For DNPV detection, the samples were first diluted nine times by volume with a 50 mM phosphate buffer, and then the measurements were performed within each sample with at least three electrodes. An analysis of the results obtained is summarized in Table 1. For all the media studied, the concentrations determined with the sensors appear to be higher than those obtained with the standard procedure. Such a difference could be attributed to the technical difficulty in ensuring simultaneous measurements with both methods. So, just after sampling, the lactate concentration was determined with the clinical analyzer only, while the samples destined to the voltammetric detection were frozen and used 1 or 2 days later. Despite these conditions, there is a good (29) Miller, L. P.; Mayer, S.; Braun, L. D.; Geiger P.; Oldendorf, W. H. Neurochem. Res. 1988, 13, 377-382. (30) Tsacopoulos, M.; Magistretti, P. J. Neurosci. 1996, 16, 877-885. (31) Kuhr, W. G.; Korf, J. Eur. J. Pharmacol. 1988, 155, 145-149.
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Table 1. Lactate Measurements in Plasma, CSF, and Brain Tissue Performed by Using the Lactate Sensor Together with the DNPV and by a Standard Procedure (Colorimetric Principle)a
CFMEs analyzer
plasma, mmol L-1
CSF, mmol L-1
brain tissue, mmol kg-1
2.58 ( 0.29 (n ) 7) 2.18 ( 0.27 (n ) 13)
1.94 ( 0.27 (n ) 6) 1.70 ( 0.06 (n ) 13)
8.17 ( 0.57 (n ) 7) 7.27 ( 0.53 (n ) 7)
Mean values ( SEM are given, and the numbers of samples are indicated in parentheses; statistical comparison between both procedures did not reveal a significant difference (p < 0.05). Abbreviations: CFMEs, carbon fiber microelectrodes; CSF, cerebrospinal fluid. a
correlation between the voltammetric lactate detection and the standard method. Statistical tests did not reveal significant differences. Conclusions. The carbon fiber lactate sensor described here and used in combination with the DNPV method is suitable for specific lactate detection in vitro as well as in vivo. The results
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of our measurements correlate well with those obtained independently with a commercial instrument. Finally, the micrometric dimensions and high biocompatibility of the carbon fiber permit us to reach restricted brain areas with minimal tissue damage. Experimental perspectives are now opened for studying the cerebral metabolism by measuring simultaneously glucose and lactate in the animal, anesthetic-free and in chronic conditions. ACKNOWLEDGMENT This work was supported by French Foreign Ministry, CNRSERS5645, INSERM U480, and Rhoˆne-Alpes Region. We also thank P. Commerc¸ on and Prof. J. Pichot for their kind help in lactate determination by standard procedure, Prof. G. Annat for his help in the pO2 measurements, and C. Limoge for improving the English text.
Received for review December 2, 1997. Accepted April 7, 1998. AC971299F
