- Email: [email protected]
Microelectrode for In Vivo Real‐Time Detection of NO
584
[50]
cell biology and physiology
whole tissues, such as brain, using fMRI technology. The availability of more potent magnetic fields could benefit this type of study for other organs in which apoptosis is a major pathological issue, such as the heart under myocardial infarction. References Dawson, D. A. (1994). Nitric oxide and focal cerebral ischemia: Multiplicity of actions and diverse outcome. Cerebrovasc. Brain Metab. Rev. 6, 299–324. Green, D. R. (1998). Apoptotic pathways: The roads to ruin. Cell 94, 695–698. Mattson, M. P., Culmsee, C., and Yu, Z. F. (2000). Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue Res. 301, 173–187. Meldrum, B., and Garthwaite, J. (1990). Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11, 379–387. Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355–365. Shi, Y. (2002). Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell 9, 459–470. Takuma, H., Tomiyama, T., Kuida, K., and Mori, H. (2004). Amyloid beta peptide‐induced cerebral neuronal loss is mediated by caspase‐3 in vivo. J. Neuropathol. Exp. Neurol. 63, 255–261. Thompson, C. B. (1995). Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1462. Zhang, J., and Steiner, J. P. (1995). Nitric oxide synthase, immunophilins and poly(ADP‐ ribose) synthetase: Novel targets for the development of neuroprotective drugs. Neurol. Res. 17, 285–288.
[50] Microelectrode for In Vivo Real‐Time Detection of NO By TAYFUN DALBASTI and EMRAH KILINC Abstract
Nitric oxide (NO) is gaining importance with its diverse spectrum of clinic effects. However, there is still a need for an ideal sensor to monitor its concentration in tissue. An ideal sensor should not interfere with the ongoing physiological process, while making fast, reliable, and repeatable measurements. We have designed a microelectrode for electrochemical NO measurement from tissue with relatively low interference and reliable results upon calibration. Details of electrode preparation and calibration procedure are explained along with an experiment to monitor effects of photodynamic therapy.
METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96050-3
[50]
microelectrode for
IN VIVO DETECTION OF
NO
585
Nitric Oxide
Nitric oxide, an endogenously synthesized free radical of great physiological and pathophysiological importance, displays various biological functions after the induction of the corresponding NO synthase (NOS). Neuronal NOS (nNOS)–derived NO acts as a neuronal signal in the central nervous system; inducible NOS (iNOS)–derived NO contributes to the pathology of many clinical conditions, and endothelial‐derived NOS (eNOS)–originated NO regulates vascular tone in the endothelial cell systems. It was biologically studied in detail and first characterized in vivo as an endothelial‐derived relaxing factor (EDRF) in 1983 by Furchgott (1983) and in 1987 by Palmer et al. The biosynthesis of NO has been reviewed by various authors (Butler and Williams, 1993; Moncada et al., 1991; Stamler, 1994). Background of Detection Measuring NO in biological media is extremely hard because of its very low concentration values (nanomolar levels) and supershort lifetime (nanoseconds) (Dalbasti et al., 2002). Most of the techniques detecting NO release use indirect methods based on the determination of secondary species or adducts (e.g., nitrites and L‐citrulline). Current techniques for direct measurements of NO include some spectroscopic methods (such as chemiluminescence, UV–vis spectroscopy, etc.) and electrochemical methods. Several types of electrochemical electrodes for NO detection have been reported in the literature (Pallini et al., 1998). Electrochemical Detection Principle NO is usually oxidized at the surface of the working electrode at a positive potential of approximately 800–900 mV (vs. Ag/AgCl reference), resulting in the generation of a small redox current. This oxidation redox potential may vary in accordance to the electrode material used or the catalytic compound used for surface modification. The overall electrode reaction for aqueous NO oxidation may be summarized as follows: NO e
!
þ900 mV
NOþ
Because NOþ is a relatively strong Lewis acid, in the presence of OH, it is converted into nitrite (NO 2 ): NOþ þ OH ! HNO2
586
cell biology and physiology
[50]
SCHEME 1. A typical three‐electrode potentiostat.
The oxidation potential of NO 2 is very close to that of NO in aqueous solution; therefore it can be further oxidized into nitrate (NO 3 ). The oxidation current flowing between the working and reference electrodes is directly proportional to the concentration of NO oxidized as described earlier. This technique of applying such a constant redox potential and sampling the response current versus time is known as amperometry (chronoamperometry as well). Thus, the redox current proportional to NO concentration is measured amperometrically using a NO meter or a conventional potentiostat (Zhang and Broderick, 2000). NO meters and potentiostats use three electrode systems for sampling response current, which is illustrated and simplified in Scheme 1. A Sample Experiment: Electrode Construction and Application for In Vivo Real‐Time Detection
There is a demand for an ideal NO detection method that is fast, durable, repeatable, specific, sensitive, and biocompatible, and that should not interfere with the tissue and physiological processes. So far, no electrode design fulfills these requirements. Electrochemical electrode designs are most close to these specifications. Following the instructions below,
[50]
microelectrode for
IN VIVO DETECTION OF
NO
587
constructing a working NO measurement electrode is the main aim of this chapter. Please keep in mind that this electrode design is only for approved experimental animal studies, and none of the procedures described hereafter are tested for human subjects. Chemicals and Materials Required Pt–Ir Alloy Wire Teflon‐insulated, multistranded Pt–Ir alloy wire (total diameter 130 m) (10% Ir–90% Pt), Medwire (http://www.sigmundcohn.com/ english/medwire.html) Nafion A synthetic perfluorinated polymer, containing sulfonic and carboxylic acid functions, solution containing 5% in a mixture of lower aliphatic alcohols and water; highly toxic and flammable; Fluka 70160 o‐Phenylenediamine (OPD) CAS [95‐54‐5], brownish‐yellow crystals; slightly soluble in water, freely soluble in alcohol, chloroform, ether; may darken in storage; FW 108.14 g/mol, Sigma P 2903 ‐Aminolevulinic acid hydrochloride (ALA) CAS [5451‐09‐2], freely soluble in water, FW 167.6 g/mol, Sigma A 3785 Potassium phosphate monobasic (KH2PO4) (potassium dihydrogenphosphate) CAS [7778‐77‐0], FW 136.1 g/mol, Sigma Sigma P 5379 Potassium phosphate dibasic (K2HPO4) (dipotassium hydrogen phosphate) CAS [7758‐11‐4], FW 174.2 g/mol, Sigma P 8281 Sulfuric acid (H2SO4) CAS [7664‐93‐9], 95–98%, FW 98.08 g/mol, Aldrich 32,050‐1 Potassium nitrite (KNO2) CAS [7758‐09‐0], FW 85.10 g/mol, Sigma P 7391 Nitrogen gas (N2) (Extra Pure) Local source: Habas sinai ve Tibbi Gazlar Istihsal Endu¨ strisi A.S. Istanbul/Turkey [Phone: þ90 (216) 452 56 00 Fax: þ90 (216) 452 25 70, www.habas.com.tr] Bunsen burner Preparation of Solutions NO stock solution may easily be prepared by bubbling NO gas through deoxygenated 0.05 M phosphate buffer (1.36 g/L KH2PO4 and 6.96 g/L K2HPO4, pH ¼ 7.4) for 30 min. Such an NO‐bubbled buffer will have a
588
cell biology and physiology
[50]
concentration of about 2 mM (1.95 mmol/L) NO at saturation (Mesaros et al., 1997; Young, 1981). The buffer to be bubbled with NO should be freshly prepared and heated to boiling just before cooling in an ice bath immediately. This boiling and cooling procedure is necessary to de‐gas the solution (oxygen free). Boiling may preferably be performed in an ultrasonic cleaner, which will help increase the de‐gas performance. The O2‐free buffer may then be bubbled with pure N2 for 30 min to help it remain O2 free. The NO gas used to saturate the buffer may either be supplied in commercial tanks (Matheson Tri‐Gas, www.matheson‐trigas.com/mathportal) or be prepared simultaneously in the lab. The production of NO gas in the lab is simpler and cheaper than the commercial choice, but maximum attention should be paid during the process, as NO immediately on contact with air is converted to the extremely poisonous nitrogen dioxide (NO2)!! (Budavari, 2001). Under adequate ventilation, NO gas may be produced in leak‐free glassware or solution wash bottles. For this procedure in a leak‐free glass chamber, 125 mol of 6 M H2SO4 is added in portions onto 50 g of KNO2. In a highly acidic media, KNO2 is decomposed, and equivocal NO is formed, which is simultaneously transferred through a leak‐free connection into the buffer solution sitting in an ice bath, kept under N2 atmosphere. When this bubbling procedure is performed for 30 min, the buffer is saturated with NO in a concentration of 1.95 mM, as indicated earlier, which is stable for 48 h at 4 . Electrode Fabrication Working electrodes (microelectrodes) may be prepared from Teflon‐ insulated Pt–Ir alloy wire (Pt 90%–Ir 10%, 130 m total diameter) by removing 3 mm of the Teflon coating (from the tip of the wire) using ordinary flame (Bunsen burner). Pt–Ir alloy may be preferred because of the reported electrocatalytic effect of iridium in redox reactions (Wang et al., 1996), and pure Pt is more fragile than the Pt–Ir alloy, which makes it harder to apply to real tissue samples. Electrodes should be preconditioned in 0.5 M H2SO4 solution by applying a constant potential of 1.9 V for 30 s, followed by a cyclic voltametry between the ranges 0.25 and þ1.1 V with a scan rate of 100 mV/s for 10 min. Preconditioning is necessary to remove any impurity left on the wire after the ordinary flame removal of the Teflon layer. Electrode fouling is another problem faced with non‐pretreated electrodes, in which response current decays rapidly and is lost in seconds.
[50]
microelectrode for
IN VIVO DETECTION OF
NO
589
Prepared electrodes should be dip‐coated four times (four layers) with 1/10th diluted Nafion solution, and each dried at 200 for 4 min as indicated (Friedemann et al., 1996; Kilinc et al., 2002). Nafion layers formed on the wire surface are responsible for repelling the negatively charged species and ions that might interfere during in vivo applications. Such interferences may include highly electroactive species, such as ascorbic acid, uric acid, and various drug metabolites. Finally, electropolymerization of 50 mM OPD (in buffer) should be performed on the Nafion‐coated electrodes by applying 0.650 V (vs. Ag/ AgCl reference) for 10 min. This additional layer of OPD on Nafion will improve the conductance of the electrode and help the Nafion layer in repelling the species of interference. Electrodes should be calibrated individually before use. Electrode Calibration A series of standard NO solutions may be prepared by diluting this NO‐saturated stock for use in electrode calibration. Increasing the final concentration of NO with 10 nm increments in a 10 mL standard electrochemical cell may help in obtaining a calibration plot linear enough to work in a range of in vivo NO levels (Dalbasti et al., 2002; Friedemann et al., 1996; Kilinc et al., 2002). NO Detection Albino rats weighing around 250 g may be used for in vivo recordings. A midline skin incision over the scalp may be performed to expose bregma and lambdoid sutures (under anesthesia). An approximately 4.0‐mm diameter burr hole is necessary over the cerebellar hemisphere (taking great care to keep the dura intact). The dura may be opened under microscopic control. The reference and auxiliary electrodes have to be placed under the skin close to the working electrode. In the control group of recordings, the Pt–Ir working electrode should carefully be placed in the cortex, and steady state recordings must be obtained. In the study group, 50 l, 5 mg/ml ALA solution in 0.05 M phosphate buffer (pH 7.4) should be applied topically with a syringe (100 l total volume) attached to a 26‐gauge needle (Fig. 1). After waiting 10 min for ALA diffusion in tissue, the intracortical electrode was placed, and simultaneous current‐time recordings were obtained (0.90 V vs. Ag/AgCl) until a steady state current recording was observed. This level must be recorded as a baseline, and then
590
cell biology and physiology
[50]
FIG. 1. Typical amperogram showing the in vivo nitric oxide (NO) release in rat cerebellum in the presence (5 mg/ml) (A) and absence (B) of ALA during ALA‐mediated PDT. Arrows indicate the time of application of the 150‐W excitation light.
broad‐spectrum light (150‐W halogen lamp) was applied from a 5‐cm distance to the surface while recording was continued to show the generation of NO during ALA‐mediated PDT. In the study group, in contrast to the control group (where ALA is not administered), an amperogram similar to Fig. 1A should be obtained. In the control group, because NO is not released in the tissue, no increase in the response current should be observed (Fig. 1B). Commercial Electrochemical NO Measurement Systems
Although alternative direct and indirect measurement techniques are present in literature for NO detection, for commercially available systems, only a few electrochemical choices are present. However, with some basic electronics knowledge, one could design a simple measurement system to be used with NO electrodes. As mentioned earlier, the idea is to apply constant voltage to the electrode and measure the current flowing through. Because electrode impedance is high (several mega ohms) and measured current is very low (pico‐nano amperes), special precautions should be taken in component selection and circuit design. The details of the companies providing commercial hardware and software for NO detection are summarized in Table I.
[50]
TABLE I COMPANIES PROVIDING SYSTEMS FOR NO DETECTION Address
Web www.wpiinc.com
Innovative Instruments, Inc.
Innovative Instruments, Inc., 8533 Queen Brooks Ct., Tampa, FL 33637
www.2in.com/
Diamond General Development Corporation
333 Parkland Plaza, Ann Arbor, MI 48103‐6202
www.diamondgeneral.com
Inter Medical Co., Ltd.
40‐4, 3‐chome, Imaike, Chikusa, NAGOYA, 464‐0850, Japan
www1.sphere.ne.jp/intermed
Free Radical Detection Nitric Oxide Detection Oxygen Detection Neurotransmitter Detection pH Meters and Electrodes Nitric Oxide Detection Oxygen Detection Reference Electrodes pH Sensors Nitrate Reductor Nitric Oxide Detection Oxygen Detection Amplifier/Display Meters Calibration Cells Nitric Oxide Detection Oxygen Detection Electrode for Spine Functions
Phone: (941) 371‐1003 Fax: (941) 377‐5428 [email protected]
Phone: (813) 727‐0676 Fax: (813) 914‐8686 [email protected]
Phone: (734) 332‐0200 Toll Free: (800) 678‐9856 Fax: (734) 332‐4775 [email protected] Phone: þ81 52 731 8000 Fax: þ81 52 731 5050 intermed‐2@mbs. sphere.ne.jp
NO
175 Sarasota Center Boulevard, Sarasota, FL 34240
Contact
IN VIVO DETECTION OF
World Precision Instruments, Inc.
Products
microelectrode for
Company
591
592
[51]
cell biology and physiology
References Budavari, S. (2001). ‘‘The Merck Index,’’ 13th Ed. Merck Research Laboratories, Whitehouse Station, NJ. Butler, A. R., and Williams, D. L. H. (1993). The physiological role of nitric oxide. Chem. Soc. Rev. 22(4), 233–241. Dalbasti, T., Cagli, S., Kilinc, E., Oktar, N., and Ozsoz, M. (2002). Online electrochemical monitoring of nitric oxide during photodynamic therapy. Nitric Oxide 7, 301–305. Friedemann, M. N., Robinson, S. W., and Gerhardt, G. A. (1996). o‐Phenylenediamine‐ modified carbon fiber electrodes for the detection of nitric oxide. Anal. Chem. 68, 2621–2628. Furchgott, R. (1983). Role of endothelium in response of vascular smooth muscle. Circulation Res. 53(5), 557–573. Kilinc, E., Yetik, G., Dalbasti, T., and Ozsoz, M. (2002). Comparison of electrochemical detection of acetylcholine‐induced nitric oxide release (NO) and contractile force measurement of rabbit isolated carotid artery endothelium. J. Pharmaceut. Biomed. Analysis 28, 345–354. Mesaros, S., Grunfeld, S., Mesarosova, A., Bustin, D., and Malinski, T. (1997). Determination of nitric oxide saturated (stock) solution by chronoamperometry on a porphyrin microelectrode. Anal. Chim. Acta 339, 265–270. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991). Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142. Pallini, M., Curulli, A., Amine, A., and Palleschi, G. (1998). Amperometric nitric oxide sensors: A comparative study. Electroanalysis 10(15), 1010–1016. Palmer, R. M. J., Ferrige, A. G., and Moncada, S. (1987). Nitric oxide release accounts for the biological activity of endothelium‐derived relaxing factor. Nature 327, 524–526. Stamler, J. S. (1994). Redox signaling: Nitrosylation and related target interactions of nitric oxide. Cell 78, 927–936. Wang, J., Rivas, G., and Chicharro, M. (1996). Iridium‐dispersed carbon paste enzyme electrodes. Electroanalysis 8(5), 434–437. Young, C. L. (ed.) (1981). ‘‘Oxides of Nitrogen,’’ Solubility Data Series, Vol. 8. IUPAC, Pergamon Press, Oxford. Zhang, X., and Broderick, M. (2000). Amperometric detection of nitric oxide. Mod. Asp. Immunobiol. 1(4), 160–165.
[51] Real‐Time Detection of Nitric Oxide Isotopes in Lung Function Tests By H. HELLER, R. GA¨ BLER , and K.‐D. SCHUSTER Abstract
In lung function tests, the determination of the pulmonary diffusing capacity (D) using the single‐breath method is a commonly applied technique. The calculation of D is performed on the basis of accurate measurements of indicator gas concentrations. In this chapter, we demonstrate the METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96051-5
[50]
cell biology and physiology
whole tissues, such as brain, using fMRI technology. The availability of more potent magnetic fields could benefit this type of study for other organs in which apoptosis is a major pathological issue, such as the heart under myocardial infarction. References Dawson, D. A. (1994). Nitric oxide and focal cerebral ischemia: Multiplicity of actions and diverse outcome. Cerebrovasc. Brain Metab. Rev. 6, 299–324. Green, D. R. (1998). Apoptotic pathways: The roads to ruin. Cell 94, 695–698. Mattson, M. P., Culmsee, C., and Yu, Z. F. (2000). Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue Res. 301, 173–187. Meldrum, B., and Garthwaite, J. (1990). Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11, 379–387. Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355–365. Shi, Y. (2002). Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell 9, 459–470. Takuma, H., Tomiyama, T., Kuida, K., and Mori, H. (2004). Amyloid beta peptide‐induced cerebral neuronal loss is mediated by caspase‐3 in vivo. J. Neuropathol. Exp. Neurol. 63, 255–261. Thompson, C. B. (1995). Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1462. Zhang, J., and Steiner, J. P. (1995). Nitric oxide synthase, immunophilins and poly(ADP‐ ribose) synthetase: Novel targets for the development of neuroprotective drugs. Neurol. Res. 17, 285–288.
[50] Microelectrode for In Vivo Real‐Time Detection of NO By TAYFUN DALBASTI and EMRAH KILINC Abstract
Nitric oxide (NO) is gaining importance with its diverse spectrum of clinic effects. However, there is still a need for an ideal sensor to monitor its concentration in tissue. An ideal sensor should not interfere with the ongoing physiological process, while making fast, reliable, and repeatable measurements. We have designed a microelectrode for electrochemical NO measurement from tissue with relatively low interference and reliable results upon calibration. Details of electrode preparation and calibration procedure are explained along with an experiment to monitor effects of photodynamic therapy.
METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96050-3
[50]
microelectrode for
IN VIVO DETECTION OF
NO
585
Nitric Oxide
Nitric oxide, an endogenously synthesized free radical of great physiological and pathophysiological importance, displays various biological functions after the induction of the corresponding NO synthase (NOS). Neuronal NOS (nNOS)–derived NO acts as a neuronal signal in the central nervous system; inducible NOS (iNOS)–derived NO contributes to the pathology of many clinical conditions, and endothelial‐derived NOS (eNOS)–originated NO regulates vascular tone in the endothelial cell systems. It was biologically studied in detail and first characterized in vivo as an endothelial‐derived relaxing factor (EDRF) in 1983 by Furchgott (1983) and in 1987 by Palmer et al. The biosynthesis of NO has been reviewed by various authors (Butler and Williams, 1993; Moncada et al., 1991; Stamler, 1994). Background of Detection Measuring NO in biological media is extremely hard because of its very low concentration values (nanomolar levels) and supershort lifetime (nanoseconds) (Dalbasti et al., 2002). Most of the techniques detecting NO release use indirect methods based on the determination of secondary species or adducts (e.g., nitrites and L‐citrulline). Current techniques for direct measurements of NO include some spectroscopic methods (such as chemiluminescence, UV–vis spectroscopy, etc.) and electrochemical methods. Several types of electrochemical electrodes for NO detection have been reported in the literature (Pallini et al., 1998). Electrochemical Detection Principle NO is usually oxidized at the surface of the working electrode at a positive potential of approximately 800–900 mV (vs. Ag/AgCl reference), resulting in the generation of a small redox current. This oxidation redox potential may vary in accordance to the electrode material used or the catalytic compound used for surface modification. The overall electrode reaction for aqueous NO oxidation may be summarized as follows: NO e
!
þ900 mV
NOþ
Because NOþ is a relatively strong Lewis acid, in the presence of OH, it is converted into nitrite (NO 2 ): NOþ þ OH ! HNO2
586
cell biology and physiology
[50]
SCHEME 1. A typical three‐electrode potentiostat.
The oxidation potential of NO 2 is very close to that of NO in aqueous solution; therefore it can be further oxidized into nitrate (NO 3 ). The oxidation current flowing between the working and reference electrodes is directly proportional to the concentration of NO oxidized as described earlier. This technique of applying such a constant redox potential and sampling the response current versus time is known as amperometry (chronoamperometry as well). Thus, the redox current proportional to NO concentration is measured amperometrically using a NO meter or a conventional potentiostat (Zhang and Broderick, 2000). NO meters and potentiostats use three electrode systems for sampling response current, which is illustrated and simplified in Scheme 1. A Sample Experiment: Electrode Construction and Application for In Vivo Real‐Time Detection
There is a demand for an ideal NO detection method that is fast, durable, repeatable, specific, sensitive, and biocompatible, and that should not interfere with the tissue and physiological processes. So far, no electrode design fulfills these requirements. Electrochemical electrode designs are most close to these specifications. Following the instructions below,
[50]
microelectrode for
IN VIVO DETECTION OF
NO
587
constructing a working NO measurement electrode is the main aim of this chapter. Please keep in mind that this electrode design is only for approved experimental animal studies, and none of the procedures described hereafter are tested for human subjects. Chemicals and Materials Required Pt–Ir Alloy Wire Teflon‐insulated, multistranded Pt–Ir alloy wire (total diameter 130 m) (10% Ir–90% Pt), Medwire (http://www.sigmundcohn.com/ english/medwire.html) Nafion A synthetic perfluorinated polymer, containing sulfonic and carboxylic acid functions, solution containing 5% in a mixture of lower aliphatic alcohols and water; highly toxic and flammable; Fluka 70160 o‐Phenylenediamine (OPD) CAS [95‐54‐5], brownish‐yellow crystals; slightly soluble in water, freely soluble in alcohol, chloroform, ether; may darken in storage; FW 108.14 g/mol, Sigma P 2903 ‐Aminolevulinic acid hydrochloride (ALA) CAS [5451‐09‐2], freely soluble in water, FW 167.6 g/mol, Sigma A 3785 Potassium phosphate monobasic (KH2PO4) (potassium dihydrogenphosphate) CAS [7778‐77‐0], FW 136.1 g/mol, Sigma Sigma P 5379 Potassium phosphate dibasic (K2HPO4) (dipotassium hydrogen phosphate) CAS [7758‐11‐4], FW 174.2 g/mol, Sigma P 8281 Sulfuric acid (H2SO4) CAS [7664‐93‐9], 95–98%, FW 98.08 g/mol, Aldrich 32,050‐1 Potassium nitrite (KNO2) CAS [7758‐09‐0], FW 85.10 g/mol, Sigma P 7391 Nitrogen gas (N2) (Extra Pure) Local source: Habas sinai ve Tibbi Gazlar Istihsal Endu¨ strisi A.S. Istanbul/Turkey [Phone: þ90 (216) 452 56 00 Fax: þ90 (216) 452 25 70, www.habas.com.tr] Bunsen burner Preparation of Solutions NO stock solution may easily be prepared by bubbling NO gas through deoxygenated 0.05 M phosphate buffer (1.36 g/L KH2PO4 and 6.96 g/L K2HPO4, pH ¼ 7.4) for 30 min. Such an NO‐bubbled buffer will have a
588
cell biology and physiology
[50]
concentration of about 2 mM (1.95 mmol/L) NO at saturation (Mesaros et al., 1997; Young, 1981). The buffer to be bubbled with NO should be freshly prepared and heated to boiling just before cooling in an ice bath immediately. This boiling and cooling procedure is necessary to de‐gas the solution (oxygen free). Boiling may preferably be performed in an ultrasonic cleaner, which will help increase the de‐gas performance. The O2‐free buffer may then be bubbled with pure N2 for 30 min to help it remain O2 free. The NO gas used to saturate the buffer may either be supplied in commercial tanks (Matheson Tri‐Gas, www.matheson‐trigas.com/mathportal) or be prepared simultaneously in the lab. The production of NO gas in the lab is simpler and cheaper than the commercial choice, but maximum attention should be paid during the process, as NO immediately on contact with air is converted to the extremely poisonous nitrogen dioxide (NO2)!! (Budavari, 2001). Under adequate ventilation, NO gas may be produced in leak‐free glassware or solution wash bottles. For this procedure in a leak‐free glass chamber, 125 mol of 6 M H2SO4 is added in portions onto 50 g of KNO2. In a highly acidic media, KNO2 is decomposed, and equivocal NO is formed, which is simultaneously transferred through a leak‐free connection into the buffer solution sitting in an ice bath, kept under N2 atmosphere. When this bubbling procedure is performed for 30 min, the buffer is saturated with NO in a concentration of 1.95 mM, as indicated earlier, which is stable for 48 h at 4 . Electrode Fabrication Working electrodes (microelectrodes) may be prepared from Teflon‐ insulated Pt–Ir alloy wire (Pt 90%–Ir 10%, 130 m total diameter) by removing 3 mm of the Teflon coating (from the tip of the wire) using ordinary flame (Bunsen burner). Pt–Ir alloy may be preferred because of the reported electrocatalytic effect of iridium in redox reactions (Wang et al., 1996), and pure Pt is more fragile than the Pt–Ir alloy, which makes it harder to apply to real tissue samples. Electrodes should be preconditioned in 0.5 M H2SO4 solution by applying a constant potential of 1.9 V for 30 s, followed by a cyclic voltametry between the ranges 0.25 and þ1.1 V with a scan rate of 100 mV/s for 10 min. Preconditioning is necessary to remove any impurity left on the wire after the ordinary flame removal of the Teflon layer. Electrode fouling is another problem faced with non‐pretreated electrodes, in which response current decays rapidly and is lost in seconds.
[50]
microelectrode for
IN VIVO DETECTION OF
NO
589
Prepared electrodes should be dip‐coated four times (four layers) with 1/10th diluted Nafion solution, and each dried at 200 for 4 min as indicated (Friedemann et al., 1996; Kilinc et al., 2002). Nafion layers formed on the wire surface are responsible for repelling the negatively charged species and ions that might interfere during in vivo applications. Such interferences may include highly electroactive species, such as ascorbic acid, uric acid, and various drug metabolites. Finally, electropolymerization of 50 mM OPD (in buffer) should be performed on the Nafion‐coated electrodes by applying 0.650 V (vs. Ag/ AgCl reference) for 10 min. This additional layer of OPD on Nafion will improve the conductance of the electrode and help the Nafion layer in repelling the species of interference. Electrodes should be calibrated individually before use. Electrode Calibration A series of standard NO solutions may be prepared by diluting this NO‐saturated stock for use in electrode calibration. Increasing the final concentration of NO with 10 nm increments in a 10 mL standard electrochemical cell may help in obtaining a calibration plot linear enough to work in a range of in vivo NO levels (Dalbasti et al., 2002; Friedemann et al., 1996; Kilinc et al., 2002). NO Detection Albino rats weighing around 250 g may be used for in vivo recordings. A midline skin incision over the scalp may be performed to expose bregma and lambdoid sutures (under anesthesia). An approximately 4.0‐mm diameter burr hole is necessary over the cerebellar hemisphere (taking great care to keep the dura intact). The dura may be opened under microscopic control. The reference and auxiliary electrodes have to be placed under the skin close to the working electrode. In the control group of recordings, the Pt–Ir working electrode should carefully be placed in the cortex, and steady state recordings must be obtained. In the study group, 50 l, 5 mg/ml ALA solution in 0.05 M phosphate buffer (pH 7.4) should be applied topically with a syringe (100 l total volume) attached to a 26‐gauge needle (Fig. 1). After waiting 10 min for ALA diffusion in tissue, the intracortical electrode was placed, and simultaneous current‐time recordings were obtained (0.90 V vs. Ag/AgCl) until a steady state current recording was observed. This level must be recorded as a baseline, and then
590
cell biology and physiology
[50]
FIG. 1. Typical amperogram showing the in vivo nitric oxide (NO) release in rat cerebellum in the presence (5 mg/ml) (A) and absence (B) of ALA during ALA‐mediated PDT. Arrows indicate the time of application of the 150‐W excitation light.
broad‐spectrum light (150‐W halogen lamp) was applied from a 5‐cm distance to the surface while recording was continued to show the generation of NO during ALA‐mediated PDT. In the study group, in contrast to the control group (where ALA is not administered), an amperogram similar to Fig. 1A should be obtained. In the control group, because NO is not released in the tissue, no increase in the response current should be observed (Fig. 1B). Commercial Electrochemical NO Measurement Systems
Although alternative direct and indirect measurement techniques are present in literature for NO detection, for commercially available systems, only a few electrochemical choices are present. However, with some basic electronics knowledge, one could design a simple measurement system to be used with NO electrodes. As mentioned earlier, the idea is to apply constant voltage to the electrode and measure the current flowing through. Because electrode impedance is high (several mega ohms) and measured current is very low (pico‐nano amperes), special precautions should be taken in component selection and circuit design. The details of the companies providing commercial hardware and software for NO detection are summarized in Table I.
[50]
TABLE I COMPANIES PROVIDING SYSTEMS FOR NO DETECTION Address
Web www.wpiinc.com
Innovative Instruments, Inc.
Innovative Instruments, Inc., 8533 Queen Brooks Ct., Tampa, FL 33637
www.2in.com/
Diamond General Development Corporation
333 Parkland Plaza, Ann Arbor, MI 48103‐6202
www.diamondgeneral.com
Inter Medical Co., Ltd.
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Free Radical Detection Nitric Oxide Detection Oxygen Detection Neurotransmitter Detection pH Meters and Electrodes Nitric Oxide Detection Oxygen Detection Reference Electrodes pH Sensors Nitrate Reductor Nitric Oxide Detection Oxygen Detection Amplifier/Display Meters Calibration Cells Nitric Oxide Detection Oxygen Detection Electrode for Spine Functions
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[51]
cell biology and physiology
References Budavari, S. (2001). ‘‘The Merck Index,’’ 13th Ed. Merck Research Laboratories, Whitehouse Station, NJ. Butler, A. R., and Williams, D. L. H. (1993). The physiological role of nitric oxide. Chem. Soc. Rev. 22(4), 233–241. Dalbasti, T., Cagli, S., Kilinc, E., Oktar, N., and Ozsoz, M. (2002). Online electrochemical monitoring of nitric oxide during photodynamic therapy. Nitric Oxide 7, 301–305. Friedemann, M. N., Robinson, S. W., and Gerhardt, G. A. (1996). o‐Phenylenediamine‐ modified carbon fiber electrodes for the detection of nitric oxide. Anal. Chem. 68, 2621–2628. Furchgott, R. (1983). Role of endothelium in response of vascular smooth muscle. Circulation Res. 53(5), 557–573. Kilinc, E., Yetik, G., Dalbasti, T., and Ozsoz, M. (2002). Comparison of electrochemical detection of acetylcholine‐induced nitric oxide release (NO) and contractile force measurement of rabbit isolated carotid artery endothelium. J. Pharmaceut. Biomed. Analysis 28, 345–354. Mesaros, S., Grunfeld, S., Mesarosova, A., Bustin, D., and Malinski, T. (1997). Determination of nitric oxide saturated (stock) solution by chronoamperometry on a porphyrin microelectrode. Anal. Chim. Acta 339, 265–270. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991). Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142. Pallini, M., Curulli, A., Amine, A., and Palleschi, G. (1998). Amperometric nitric oxide sensors: A comparative study. Electroanalysis 10(15), 1010–1016. Palmer, R. M. J., Ferrige, A. G., and Moncada, S. (1987). Nitric oxide release accounts for the biological activity of endothelium‐derived relaxing factor. Nature 327, 524–526. Stamler, J. S. (1994). Redox signaling: Nitrosylation and related target interactions of nitric oxide. Cell 78, 927–936. Wang, J., Rivas, G., and Chicharro, M. (1996). Iridium‐dispersed carbon paste enzyme electrodes. Electroanalysis 8(5), 434–437. Young, C. L. (ed.) (1981). ‘‘Oxides of Nitrogen,’’ Solubility Data Series, Vol. 8. IUPAC, Pergamon Press, Oxford. Zhang, X., and Broderick, M. (2000). Amperometric detection of nitric oxide. Mod. Asp. Immunobiol. 1(4), 160–165.
[51] Real‐Time Detection of Nitric Oxide Isotopes in Lung Function Tests By H. HELLER, R. GA¨ BLER , and K.‐D. SCHUSTER Abstract
In lung function tests, the determination of the pulmonary diffusing capacity (D) using the single‐breath method is a commonly applied technique. The calculation of D is performed on the basis of accurate measurements of indicator gas concentrations. In this chapter, we demonstrate the METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96051-5