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Interfering with Nitric Oxide Measurements

2002; Elsevier BV; Volume: 277; Issue: 50 Linguagem: Inglês

10.1074/jbc.m209130200

1083-351X

Xin Zhang, Won Suk Kim, Nathan G. Hatcher, Kurt Potgieter, Leonid L. Moroz, Rhanor Gillette, Jonathan V. Sweedler,

Electrochemical sensors and biosensors

4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N2O3 from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry. 4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N2O3 from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry. NO is involved in a variety of important biological functions in the cardiovascular system, the central and peripheral nervous system, the reproductive system, and the immune system (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Google Scholar, 2Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Google Scholar, 3Szabó C. Brain Res. Bull. 1996; 41: 131-141Google Scholar, 4Dawson V.L. Dawson T.M. Prog. Brain Res. 1998; 118: 215-229Google Scholar, 5Bogdan C. Nat. Immunol. 2001; 2: 907-916Google Scholar, 6Singh V.K. Mehrotra S. Narayan P. Pandey C.M. Agarwal S.S. Immunol. Res. 2000; 22: 1-19Google Scholar). NO is normally generated via an enzymatically regulated pathway with the conversion ofl-arginine to citrulline by a family of at least three distinct nitric-oxide synthase (NOS) 1The abbreviations used are: NOS, nitric-oxide synthase; AA, ascorbic acid; ASW, artificial seawater; CE, capillary electrophoresis; DAF, diaminofluorescein; DAF-2, 4,5-diaminofluorescein; DAF-2T, DAF-2 triazole; DEA/NONOate, diethylamine NONOate; DHA, dehydroascorbic acid; ESI, electrospray ionization; LIF, laser-induced fluorescence; MCC, metacerebral cell; MS, mass spectrometry. enzymes, the neuronal, inducible, and endothelial NOS forms (or NOS-I, -II, and -III) (7Bredt D.S. Hwang P.M. Snyder S.H. Nature. 1990; 347: 768-770Google Scholar, 8Shaul P.W. Annu. Rev. Physiol. 2002; 64: 749-774Google Scholar, 9Reutov V.P. Biokhimiya. 2002; 67: 293-311Google Scholar). Endothelial and neuronal NOS, also named constitutive NOS, are calcium-dependent. Although cells expressing constitutive NOS generally produce small amounts of NO (submicromolar at the cellular level), inducible NOS, which is synthesized in immune-competent cells when stimulated by cytokines, endotoxins, and other biologically active compounds, is calcium-independent and produces NO at higher levels (1–10 μm in microphages) (10Nagano T. Yoshimura T. Chem. Rev. 2002; 102: 1235-1269Google Scholar). Although in situ and immunohistochemical techniques allow one to determine whether NOS is present in a particular tissue, whether the NOS is actively producing NO under specific conditions and the amount of NO produced are important questions to answer to understand the physiological roles of NOS. Direct detection (or imaging) of NO production in a biological system was unsatisfactory until the development of a series of fluorescent indicators for NO, the diaminofluoresceins (DAFs) in 1998 by the Nagano group (11Kojima H. Nakatsubo N. Kikuchi K. Kawahara S. Kirino Y. Nagoshi H. Hirata Y. Nagano T. Anal. Chem. 1998; 70: 2446-2453Google Scholar). In the presence of oxygen, NO nitrosates DAFs to produce the highly fluorescent triazofluoresceins. DAFs provide the advantages of sensitivity (detection limits of 5 nm), simple protocols, and noncytotoxicity, and they are also believed to offer high specificity to NO. Since then, an increasing number of researchers have used them for NO detection and NO imaging (10Nagano T. Yoshimura T. Chem. Rev. 2002; 102: 1235-1269Google Scholar,12Nagano T. Luminescence. 1999; 14: 283-290Google Scholar). The specificity of 4,5-diaminofluorescein (DAF-2), a commonly used form of DAF, has been examined. Broillet et al. (13Broillet M.C. Randin O. Chatton J.Y. FEBS Lett. 2001; 491: 227-232Google Scholar) reported that Ca2+, Mg2+, or incident light promoted the production of DAF-2T, the reaction product of DAF-2 and NO, by using NO donors in Ringer solution. It was later reported (14Suzuki N. Kojima H. Urano Y. Kikuchi K. Hirata Y. Nagano T. J. Biol. Chem. 2002; 277: 47-49Google Scholar) that the enhancement of fluorescence intensity was not caused by increasing the reaction rate between DAF-2 and NO but by the potentiation of the release rate of NO from NO donors, and it was concluded that DAF-2 could provide reliable information on NO production in a biological system. Recently, Roychowdhury et al. (15Roychowdhury S. Luthe A. Keilhoff G. Wolf G. Horn T.F.W. Glia. 2002; 38: 103-114Google Scholar) evaluated the efficiency and specificity of DAF-2 for the detection of endogenously produced NO in mouse glia cell culture and reported that DAF-2 was more sensitive for peroxynitrite. Jourd'heuil (16Jourd'heuil D. Free Radical Biol. Med. 2002; 33: 676-684Google Scholar) described that DAF-2 may be oxidized by either peroxynitrite or a peroxidase to form an intermediate that directly combines with NO to bypass NO/O2reaction and suggested that the results from DAF-2 fluorometric assays are quantitatively difficult to interpret in cells when oxidants and NO are cogenerated. In addition, Nagata et al. (17Nagata N. Momose K. Ishida Y. J. Biochem. (Tokyo). 1999; 125: 658-661Google Scholar) investigated the effects of reducers on the NO-induced nitrosation of DAF-2 and reported that catecholamines attenuated the fluorescence induced by NO donor, whereas ascorbic acid (AA, ascorbate, or vitamin C) and other reducers abolished the fluorescence. The authors suggested that the attenuation was mainly through anti-oxidative action of the reducers and oxidized NO and N2O3 or possibly through a direct interaction of reducers with DAF-2. We demonstrate here that DAF-2 directly reacts with dehydroascorbic acid (DHA) and AA to form fluorescent compounds with emission spectra similar to that of DAF-2T. AA is the primary water-soluble antioxidant in biological systems, donating reducing hydrogen when being oxidized to DHA (18Carr A. Frei B. Faseb J. 1999; 13: 1007-1024Google Scholar, 19May J.M. Front. Biosci. 1998; 2: d1-d10Google Scholar). Uptake of AA into cells has been suggested to be via DHA, which is preferentially transported through the cell membrane (20Agus D.B. Gambhir S.S. Pardridge W.M. Spielholz C. Baselga J. Vera J.C. Golde D.W. J. Clin. Invest. 1997; 100: 2842-2848Google Scholar). Once inside the cell, DHA is rapidly reduced to AA through enzymatic processes that involve GSH, NADH, or NADPH reductants (21Deutsch J.C. J. Chromatogr. A. 2000; 881: 299-307Google Scholar). Tissues vary widely in AA content with a range of 1–10 mm (22Grunewald R.A. Brain Res. Rev. 1993; 18: 123-133Google Scholar, 23Washko P.W. Wang Y.H. Levine M. J. Biol. Chem. 1993; 268: 15531-15535Google Scholar, 24Ek A. Strom K. Cotgreave I.A. Biochem. Pharmacol. 1995; 50: 1339-1346Google Scholar, 25Rice M.E. Russo-Menna I. Neuroscience. 1998; 82: 1213-1223Google Scholar), but the highest concentrations occur in the pituitary and adrenal glands and in the brain (22Grunewald R.A. Brain Res. Rev. 1993; 18: 123-133Google Scholar). For example, catecholamines require ascorbic acid for synthesis and protection from oxidation. Large quantities of AA are required during conception (26Luck M.R. Jeyaseelan I. Scholes R.A. Biol. Reprod. 1995; 52: 262-266Google Scholar). Leukocytes, particularly neutrophils, possess high concentrations of AA and may be the primary vehicle for its distribution throughout the body. It has been suggested that AA accumulates in activated human neutrophils to 14 mmconcentrations (23Washko P.W. Wang Y.H. Levine M. J. Biol. Chem. 1993; 268: 15531-15535Google Scholar). This study reports a cross-reaction of DHA/AA with DAF-2, confounding detection and measurements of NO levels in biological systems. We investigated the effect of this newly described reaction using capillary electrophoresis (CE) with wavelength-resolved fluorescence detection and chemically characterized the new products using electrospray ionization-mass spectrometry (ESI-MS). Because of the extremely high physiological concentrations of AA and DHA colocalized with nanomolar levels of NO, even a poor reactivity of DAF-2 to DHA/AA can generate significant amounts of DAF-2-DHA/AA products. This is a potentially serious complication for direct NO measurements using DAF-2 because the same tissues that contain NOS enzymes have high levels of AA and DHA. All of the chemicals used were of the highest available purity and were obtained from Sigma unless otherwise noted. Three different buffers were used in this work. The 0.1 m phosphate buffer (pH 7.4 ± 0.1) was prepared using 0.54 g of monobasic sodium phosphate (NaH2PO4·H2O; Fisher) and 1.64 g of dibasic sodium phosphate (Na2HPO4·7H2O; Fisher) in 100 ml of ultrapure water (Millipore, Bedford, MA). The 0.1 macetate buffer (pH 5.0 ± 0.1) was prepared using 0.96 g of sodium acetate (CH3COONa·3H2O) and 0.17 ml of glacial acetic acid dissolved in a final volume of 100 ml of ultrapure water. The borate buffer (pH 8.8 ± 0.1) was prepared using 3.0 g of boric acid (H3BO3) and 9.2 g of sodium borate (Na2B4O7·10H2O) in 1.00 liters of milli-Q water. DAF-2 was obtained either in solid form or in Me2SO solution. If the solid was used, DAF-2 was first dissolved in Me2SO (Fisher) to make a high concentration of stock solution and then diluted into the desired buffer. The final amount of Me2SO in the DAF-2 solution was less than 1%. The l-ascorbic acid was purchased from Fisher. DHA exists as a dimer in the crystalline form, but it spontaneously converts to a hydrated monomer in aqueous solution. The NO donor, diethylamine NONOate diethylammonium salt (DEA/NONOate), decomposes to release free NO into solution with a half-life of 2.5 min at neutral pH and 37 °C (27Hrabie J.A. Klose J.R. Wink D.A. Keefer L.K. J. Org. Chem. 1993; 58: 1472-1476Google Scholar). NO gas with 99% purity was obtained from Matheson Gas (through S. J. Smith Welding Supply, Urbana, IL). To prepare the NO-saturated solution, NO gas, after passing through 0.5 mm sodium hydroxide, was bubbled into either ultrapure water or desired buffer solutions, which had been boiled and flushed with nitrogen gas for 20 min. The saturated solution should contain ∼1.9 mm NO at room temperature (28Beckman J.S.W.D.A. Crow J.P. Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. John Wiley & Sons, Inc., New York1996: 61-70Google Scholar). A laboratory-assembled CE system with 257-nm excitation and a charge-coupled device/spectrograph for wavelength-resolved fluorescence emission spectra was employed for monitoring the reaction products of DAF-2 with other compounds. This CE system, described in detail elsewhere (29Fuller R.R. Moroz L.L. Gillette R. Sweedler J.V. Neuron. 1998; 20: 173-181Google Scholar, 30Park Y.H. Zhang X. Rubakhin S.S. Sweedler J.V. Anal. Chem. 1999; 71: 4997-5002Google Scholar, 31Zhang X. Fuller R.R. Dahlgren R.L. Potgieter K. Gillette R. Sweedler J.V. Fresenius J. Anal. Chem. 2001; 369: 206-211Google Scholar), allows sensitive detection and spectral identification of species that fluoresce with 257-nm excitation. Because the outlet end of the capillary is located in a sheath flow assembly, the same detection conditions (pH 8.8) are assured for all analytes regardless of the separation conditions (such as samples prepared at different pHs). Sample injection was performed electrokinetically at 2.5 kV (current, ∼2.8 μA) for 10.0 s from a 360-nl stainless steel nanovial, and an approximately 4-nl sample was injected into the capillary. The separation voltage was maintained at 25 kV (current, ∼30 μA). All of the experiments were performed at room temperature. The fluorescence measurements were performed with a spectrophotometer (F-3010, Hitachi, Tokyo, Japan). Emission fluorescence (scan range, 500–600 nm) of a 0.2 ml of reaction mixture was monitored upon excitation at 495 nm. Excitation spectra (scan range, 410–500 nm) were measured when the emission wavelength was set at 515 nm. Both the excitation and emission bandpass were 5 nm. Scan speed and response time were 60 nm/min and 2 s, respectively. The fluorescence counts were calculated with Microsoft Excel software. Electrospray mass spectral data were recorded with a Micromass quadrupole/time-of-flight mass spectrometer (Ultima API, Micromass UK, Wythenshawe, Manchester, UK) fitted with a Z-spray ion source. The operating conditions were as follows: ESI mode, positive; needle voltage, 3 KV; ion source temperature, 80 °C; and collision energy, 30 eV. The data were acquired over the mass rangem/z 100–1000. Because of the short lifetime of NONOate, the measured NO donor solid was kept on dry ice before usage. Immediately after dissolution and dilution to desired concentrations in buffers at 4 °C, the NO donor solutions were rapidly transferred to the reaction mixture at room temperature as described below. For the purpose of evaluation and calibration, a series of known concentrations of NO donor solution was added to and mixed with DAF-2. If the effects of other chemicals (such as DHA, AA, or GSH) were monitored, NONOate solution was combined with the compound just prior to the addition of DAF-2 solution. Unless otherwise stated, all of the reactions were at room temperature for 30 or 40 min (30-min reaction for CE assays and 40-min reaction for fluorimetric measurements). The reaction products were subjected to subsequent CE or fluorometric analysis or transferred to dry ice for storage if immediate analysis was not available. Because of the instability of DHA, AA, and GSH, all of the solutions were freshly made from solids just before the experiment and dissolved in the nitrogen-purged buffer solution containing 3 mm EDTA. If any stock solution was used for more than one protocol, it was kept at 4 °C and used within 30 min. Both pH 7.4 and 5.0 media were tested. Furthermore, NO bubbling solution at both pH levels were also used as an alternative NO source for the above experimental protocols. Aplysia californica (80–120 g) were obtained from the Aplysia Research Facility (Miami, FL). Larger animals (120–200 g) were collected off the seacoast in Santa Barbara, CA. The animals were kept in artificial seawater (ASW) continuously aerated at 14–15 °C. For dissections, the animals were anesthetized by injecting a volume of isotonic MgCl2 equal to half the body weight of the animal. The cerebral ganglia were treated with 1% protease IX (Sigma, P-6141) in ASW at +34 °C for 1–2 h. Individual metacerebral cells (MCCs) were isolated with tungsten needles and transferred into stainless-steel nanovials by a homemade micropipette. Either two or four MCCs were placed into each individual vial. For cell incubation, ASW was gently aspirated from the sample nanovial, and 300 nl of the incubation solution (10 μm DAF-2 solution in pH 7.4 phosphate buffer) was added. Nanovials with samples were immediately placed into a humidified chamber for incubation times of ∼2 h. The samples were directly injected into the CE system for analysis or stored frozen for a few hours before the analysis. Pleurobranchaea californica (200–500 g) were obtained from Sea-life Supply (Sand City, CA) and kept in ASW at 12–14 °C until use. The procedure for Pleurobranchaea cell isolation has been described previously (29Fuller R.R. Moroz L.L. Gillette R. Sweedler J.V. Neuron. 1998; 20: 173-181Google Scholar). MCCs and surrounding cells were isolated together from individual animal and transferred to the bottom tip of 150-μl plastic vials (Beem, Bronx, NY) after removal of the excess ASW. Approximately 0.9 μl of DAF-2 solution (10 μm, pH 7.4) was added to the vial. The cells were homogenized using a homemade capillary homogenizer and incubated at room temperature for 45 min. Immediately before CE analysis, 360 nl of the incubation solution was transferred to the nanovials for injection. Various solutions were assayed using CE-LIF detection to determine the specificity of the DAF-2 reaction with NO. To provide the detection of the greatest number of potential products, we chose to use 257-nm excitation instead of the more conventional 488-nm excitation because most aromatic compounds are detectable using this wavelength. CE can separate unreacted DAF-2 and the reaction products so that each migrates past the detector at a different time, allowing the fluorescence of each DAF-2 product to be measured individually (assuming that the different reaction products have different electrophoretic migration times). In this case, a homemade CE system that also provides simultaneous wavelength-resolved detection was used to obtain fluorescence emission profiles for all of the fluorescent analytes (29Fuller R.R. Moroz L.L. Gillette R. Sweedler J.V. Neuron. 1998; 20: 173-181Google Scholar, 30Park Y.H. Zhang X. Rubakhin S.S. Sweedler J.V. Anal. Chem. 1999; 71: 4997-5002Google Scholar, 31Zhang X. Fuller R.R. Dahlgren R.L. Potgieter K. Gillette R. Sweedler J.V. Fresenius J. Anal. Chem. 2001; 369: 206-211Google Scholar). As shown in Fig. 1A, DAF-2 and DAF-2T were separated using CE and detected by LIF with a 257-nm excitation. In all DAF-2 standards, we found an impurity whose level directly correlated with DAF-2 concentration. Because this impurity did not react with any compound under any conditions tested in this study, we used its peak as an internal standard to correct injection bias. Integrated fluorescence (over 520–600 nm) of both DAF-2T and DAF-2 responded linearly (r 2 = 0.9995 and 0.9808, respectively) to the NONOate concentration within the tested 3–100 μm range. Similar linear results were observed for pH 5.0 solutions. The results at both pH levels showed stoichiometric conversion of DAF-2 to DAF-2T. DAF-2 cross-reacted with DHA to produce fluorescent compounds, which are called the DAF-2-DHAs. Combining DHA and DAF-2 in solution (pH 7.4) for 30 min produced two new major compounds detected with CE-LIF, DAF-2-DHA1 and DAF-2-DHA2 (Fig. 1B). DAF-2 in the presence of both DHA and NO generated DAF-2-DHAs as well as DAF-2T (Fig. 1C). Comparison of these three reaction protocols (Fig. 1D) demonstrated that more DAF-2 was used by 1 mm DHA than 30 μm NO donor (Fig. 1D, left panel). Moreover, the signal of DAF-2-DHAs was almost unchanged with the presence of NO (Fig. 1D, middle panel), whereas the amount of DAF-2T generated (from 30 μm NO donor) decreased by about 45% with the inclusion of 1 mm DHA (Fig. 1D,right panel). DAF-2 reacted with AA to generate the same fluorescent compounds as DAF-2 with DHA. Although stronger DAF-2-DHA peak signals were observed for samples with longer incubation times of 7–24 h (data not shown), only a small amount was detected after an incubation period of 30 min using CE-LIF (Fig. 2). Concomitantly, AA attenuated the formation of DAF-2T to a greater extent (∼98%) than DHA (45%). Besides using an NO donor, authentic NO bubbled in buffer solutions (both pH 5 and pH 7.4) were tested. In all cases, similar results were obtained. Time course (5 min to 24 h) examinations of the reaction of DAF-2 with DHA/AA at both pH 5 and 7.4 demonstrated ever-increasing DAF-2-DHA2 signals as well as complex patterns of a series of unresolved peaks between DAF-2-DHA1 and DAF-2-DHA2 (data not shown). The major reaction products were those observed at pH 7.4 with a 30-min incubation time, DAF-2-DHA1 and DAF-2-DHA2 (Figs. 1 and 2). A reaction time longer than 24 h produced only DAF-2-DHA2 for DAF-2 + DHA/AA at both pHs. The reaction of DAF-2 + NO, DAF-2 + DHA/AA and the effect of DHA and AA on the fluorescence of DAF-2 in response to NO were monitored using a fluorimeter with 495-nm excitation. This scheme measured the total fluorescence from all fluorescent compounds in the solution. Fluorescence spectra of DAF-2 with NONOate at different concentrations are shown in Fig. 3. Fluorescence intensity was linear with the NONOate concentration (r 2 = 0.9974) within the tested range of 10 nm to 10 μm. Appreciable fluorescence signal was observed from DAF-2 + DHA mixtures. As shown in Fig. 3, the fluorescence intensity of the reaction mixture of DAF-2 with 1 mm DHA was in between the fluorescence produced by 100 nm and 1 μm NONOate. A semi-quantitative calculation based on the calibration curve of DAF-2T fluorescence versus NO donor concentration revealed that 1 mm DHA produces a fluorescence signal indistinguishable from ∼300 nm NONOate, 10 μm DHA + 10 nm NONOate, and 1 mm AA + 20 nmNONOate after a incubation period of 40 min with DAF-2. Furthermore, the fluorescence emission profiles of DAF-2-DHAs and DAF-2T showed a high degree of similarity (both had emission maxima at 515 nm). The excitation maxima differed slightly between DAF-2-DHAs (485 nm) and DAF-2T (495 nm). Consistent with the CE-LIF assays, similar attenuation was observed on the total fluorescence of DAF-2 + NO when DHA or AA was present (Fig. 4). The total fluorescence from DAF-2T generated from 1 μm NONOate was attenuated by about 57 and 75% with 1 mm DHA and AA, respectively. The DAF-2-DHA products were characterized with ESI-MS. The solvent dependence of the reaction products aided in this analysis. In an ammonium acetate media (pH 4) where CE-LIF mainly showed DAF-2-DHA2, a molecular ion with m/z 519.1 was observed from the DAF-2 + DHA reaction mixture. An additional weak peak at m/z 501.1 was detected from a reaction mixture prepared in water, which has a weak DAF-2-DHA1 peak and a strong DAF-DHA2 signal in the CE-LIF analysis. These two ions withm/z 519.1 and 501.1 (M+H+) were therefore assigned to DAF-2-DHA2 and DAF-2-DHA1, respectively. We name these compounds DAF-2-DHA-518 and DAF-2-DHA-500. Analysis of the MS-MS fragmentation pattern and the proposed structures of the two compounds are summarized in Table I.Table ISummary of MS-MS analysis of ion m/z of 519.1 and 501.1 and proposed structures for the corresponding DAF-2-DHA-518 and DAF-2-DHA-500, respectively Open table in a new tab The reaction of DAF-2 with DHA/AA was also observed in the cellular samples using CE-LIF. Fig. 5 demonstrates typical wavelength-resolved electropherograms of molluscan MCCs and a few small adjacent cells homogenated and incubated with DAF-2 solution. Besides the expected cellular compounds (such as the neurotransmitter, serotonin; amino acids, tryptophan and tyrosine; NOS cofactor, tetrahydrobiopterin) that are natively fluorescent with 257-nm excitation, we also detected DAF-2-DHAs generated from DAF-2 in reaction with endogenous DHA/AA. In the control samples without DAF-2 incubation, no DAF-2-DHAs were observed. Unresolved peaks eluting between DAF-2-DHA1 and DAF-2-DHA2, as in the time course of DAF-2 + DHA/AA reaction, were also observed. CE-LIF results indicate that AA and DHA react with DAF-2 under physiologically relevant conditions to form a unique series of fluorescent products, from which we identified two major compounds, DAF-2-DHA-518 and DAF-2-DHA-500. We propose a reaction mechanism where the two primary amines from DAF-2, acting as electron-donating groups, react with the 1,2-carbonyl groups from DHA/AA to form a conjugated system containing -C=N moieties. Reactions between ascorbate and diaminobenzene, the end functional group of DAF-2, have been documented (32Parish H.A. Gilliom R.D. Carbohydr. Res. 1982; 102: 302-307Google Scholar, 33Awad L.F. Carbohydr. Res. 2000; 326: 34-42Google Scholar, 34Somogyi L. Liebigs Ann. Org. Bioorg. Chem. 1995; 4: 721-724Google Scholar). The reaction products between these two reactants have been previously suggested to contain either a quinoxaline structure (see Table I footnote) with the five-member ring of ascorbate remaining closed (32Parish H.A. Gilliom R.D. Carbohydr. Res. 1982; 102: 302-307Google Scholar) or a 1H-quinoxaline-2-one structure (see Table I footnote) with the ascorbate ring opened (33Awad L.F. Carbohydr. Res. 2000; 326: 34-42Google Scholar, 34Somogyi L. Liebigs Ann. Org. Bioorg. Chem. 1995; 4: 721-724Google Scholar). From these reports, we expect a similar reaction scheme resulting in the two predicted structures in Table I. These structures have been partially confirmed by ESI-MS and MS-MS analysis, although complete MS-MS fragmentation was not obtained. The proposed reaction and reaction products are further supported by an earlier study that reported the use of 2,3-diaminonaphthalene to detect AA (35Yang J. Tong C. Jie N. Zhang G. Ren X. Hu J. Talanta. 1997; 44: 855-858Google Scholar). Yang et al. (35Yang J. Tong C. Jie N. Zhang G. Ren X. Hu J. Talanta. 1997; 44: 855-858Google Scholar) proposed a similar reaction scheme in which the two primary amine groups from 2,3-diaminonaphthalene reacted with the carbonyl moiety on ascorbate to form a quinoxaline structure. Interestingly, 2,3-diaminonaphthalene, a previously defined fluorescent indicator for NO, reacts with NO in the presence of oxygen to generate a triazole structure; this is the reaction upon which the development of DAFs and diamino-rhodamines as fluorescent NO indicators was based (12Nagano T. Luminescence. 1999; 14: 283-290Google Scholar). It is reasonable to predict that other structurally similar members of the DAF family such as DAF-FM and diamino-rhodamine 4M-AM also react with DHA/AA to form fluorescent products similar to those we have described here. The reactivity of DAF-FM and diamino-rhodamine 4M-AM with DHA/AA and the fluorescence properties of the products (if any) will be the focus of future investigations. Here, we report the formation of fluorescent products of both DHA and AA combined with DAF-2. AA autooxidizes to form DHA (36Deutsch J.C. Anal. Biochem. 1998; 265: 238-245Google Scholar). Thus it is likely that DAF-2 + AA fluorescent products may be acting through DHA, given that only small amount of same products, DAF-2-DHA-500 and DAF-2-DHA-518, were observed for the DAF-2 + AA mixture (Fig. 2). To ensure the presence of AA alone in solution, it is necessary to carefully control the reaction conditions. To suppress the AA autooxidation, we could eliminate oxygen from the solution, but the reaction of DAF-2 with NO requires the presence of oxygen. A second alternative would be the use of GSH at pH 7.4 to prevent AA oxidation (37Winkler B.S. Biochim. Biophys. Acta. 1992; 1117: 287-290Google Scholar); however, we found no significant difference on the reaction products of DAF-2 + AA with or without GSH. Rather, GSH itself was found to attenuate the DAF-2T signal, consistent with the observation by Nagata et al. (17Nagata N. Momose K. Ishida Y. J. Biochem. (Tokyo). 1999; 125: 658-661Google Scholar). Thus this method is not practical in our study for the preservation of AA in solution. It is unclear from our data whether or not AA reacts directly with DAF-2. The unresolved peaks between DAF-2-DHA1 and DAF-2-DHA2 observed in the cellular analysis (Fig. 5) and in the time course studies may be attributed to the ascorbate cascade resulting from AA autooxidation to DHA and the subsequent DHA degradation (38Kimoto E. Tanaka H. Ohmoto T. Choami M. Anal. Biochem. 1993; 214: 38-44Google Scholar, 39Hughes D.E. Van Deusen S. Analyst. 1989; 114: 169-172Google Scholar). Both DHA and AA attenuate the formation of DAF-2T. The effect by DHA is mainly due to the competition for DAF-2. Physiologically relevant levels of NO may be low enough so that the reaction of DHA with DAF-2 interferes with NO measurements. AA seems to inhibit the DAF-2 + NO reaction to a greater extent. This can be attributed to additional redox activity between AA and N2O3 to form NO, which decreases the available amount of N2O3 to react with DAF-2 to form DAF-2T (17Nagata N. Momose K. Ishida Y. J. Biochem. (Tokyo). 1999; 125: 658-661Google Scholar). As we have shown, DAF-2 reacts with DHA/AA to form fluorescent compounds, DAF-2-DHAs. Unfortunately, standard fluorescence spectroscopy is not able to distinguish DAF-2-DHAs from DAF-2T because of the similarity in the emission profiles. Although the fluorescence properties (extinction coefficient and fluorescence quantum yield) of the DAF-2-DHAs are much lower than those of DAF-2T, the concentration of ascorbate in biological systems (0.1 to 10 mm) is often 3 orders of magnitude higher than the expected NO level (10 nm to 10 μm). As an example, the measured AA and DHA concentrations in the Aplysia MCC neuron are ∼1 mm and ∼300 μmrespectively. 2Kim, W. S., Dahlgren, R. L., Moroz, L. L., and Sweedler, J. V. (2000) Anal. Chem. 74,5614–5620. Our observation of DAF-2-DHA peaks in the MCC neurons incubated with DAF-2 clearly indicates that this reaction occurs under endogenous physiological conditions. Thus we suggest that the fluorescence observed with spectrofluorometry upon DAF-2 application may contain a significant DAF-2-DHAs signal, in addition to the expected DAF-2T fluorescence. NO research has expanded of the last 5 years. There have been more than 100 studies published using variants of DAF fluorescence indicators to detect NO in biological systems. However we demonstrate here novel reactions of DAF-2 with DHA/AA. Without chromatographic or electrophoretic separation, fluorescence spectroscopy detection lacks the ability to differentiate compounds with similar emission profiles. Several studies have used high performance liquid chromatography to separate weakly fluorescent DAF-2 and highly fluorescent DAF-2T but have not reported other fluorescent compounds from the direct reaction with DAF-2 (10Nagano T. Yoshimura T. Chem. Rev. 2002; 102: 1235-1269Google Scholar, 41Nakatsubo N. Kojima H. Kikuchi K. Nagoshi H. Hirata Y. Maeda D. Imai Y. Irimura T. Nagano T. FEBS Lett. 1998; 427: 263-266Google Scholar, 42Itoh Y. Ma F.H. Hoshi H. Oka M. Noda K. Ukai Y. Kojima H. Nagano T. Toda N. Anal. Biochem. 2000; 287: 203-209Google Scholar). Our work appears to be the first report that DAF-2 reacts with physiologically relevant compounds other than NO-related compounds. NO is an important messenger molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in biological systems. AA plays an important antioxidant role in many of the same biological activities, including endothelial function, infection and inflammation, and fertilization (26Luck M.R. Jeyaseelan I. Scholes R.A. Biol. Reprod. 1995; 52: 262-266Google Scholar, 43May J.M. Free Radical Biol. Med. 2000; 28: 1421-1429Google Scholar, 44Calabrese V. Bates T.E. Stella A.M.G. Neurochem. Res. 2000; 25: 1315-1341Google Scholar, 45Knight J.A. Ann. Clin. Lab. Sci. 2000; 30: 145-158Google Scholar, 46Foissner I. Wendehenne D. Langebartels C. Durner J. Plant J. 2000; 23: 817-824Google Scholar). It has also been suggested that AA potentiates NO synthesis in endothelial cells and preserves the endothelial NO activity (47Heller R. Munscher-Paulig F. Grabner R. Till U. J. Biol. Chem. 1999; 274: 8254-8260Google Scholar, 48Carr A. Frei B. Free Radical Biol. Med. 2000; 28: 1806-1814Google Scholar). Based on these findings, our experimental results demonstrate a significant potential bias in the methodology of NO assays using DAF-2. It has been suggested that the major advantage of DAF-2 fluorescence spectroscopy over other NO sensitive imaging/detection techniques (such as electron paramagnetic resonance or EPR with NO spin trapping) is the ability to detect the basal NO production from endothelial and neuronal cells, with submicromolar NO concentrations (41Nakatsubo N. Kojima H. Kikuchi K. Nagoshi H. Hirata Y. Maeda D. Imai Y. Irimura T. Nagano T. FEBS Lett. 1998; 427: 263-266Google Scholar, 49López-Figueroa M.O. Caamano C. Marin R. Guerra B. Alonso R. Morano M.I. Akil H. Watson S.J. Biochim. Biophys. Acta. 2001; 1540: 253-264Google Scholar, 50Leikert J.F. Rathel T.R. Muller C. Vollmar A.M. Dirsch V.M. FEBS Lett. 2001; 506: 131-134Google Scholar). The intracellular level of ascorbate is as high as 9 mm in human endothelial cells (24Ek A. Strom K. Cotgreave I.A. Biochem. Pharmacol. 1995; 50: 1339-1346Google Scholar) and is in the range of 1–10 mm in neurons (25Rice M.E. Russo-Menna I. Neuroscience. 1998; 82: 1213-1223Google Scholar). Our data indicate that these high concentrations of AA can attenuate the DAF-2T level to below its detection limits or even prevent the formation of DAF-2T. Concomitantly, fluorescence of DAF-2-DHAs generated from the DAF-2 + AA/DHA reactions is expected that is indistinguishable from DAF-2T fluorescence. To exclude this possibility, further work using specific constitutive NOS inhibitors should be performed. We have observed a constant increase of DAF-2-DHAs signal with longer DAF incubation periods that may be misinterpreted as inducible NO production. As one example, DAF-2 has been used together with NOS-IIin situ hybridization to simultaneously monitor NO production and inducible NOS mRNA expression in an experimental model of central nervous system inflammation (51López-Figueroa M.O. Day H.E.W. Lee S. Rivier C. Akil H. Watson S.J. Brain Res. 2000; 852: 239-246Google Scholar). NO production estimated from DAF fluorescence was observed in a much broader area than inducible NOS mRNA expression and remained at a high level for more than 24 h even though the inducible NOS mRNA signal returned to base line. The authors hypothesized that the interleukin-1β (used to induce inflammation) also activated remote cells that, together with other immune-related molecules, penetrated the blood-brain barrier and progressively increased NO production. Our study suggests the possibility that significant amounts of DHA/AA in these tissues are in fact producing the fluorescence signal because AA is one of the major cytoprotective agents to biotic and abiotic stress. A growing body of evidence suggests that animals and plants increase their capacity to scavenge reactive oxygen species and reactive nitrogen species in response to such stresses generated in inflammation (52Grace S.C. Logan B.A. Philos. Trans. R. Soc. Lond-Biol. Sci. 2000; 355: 1499-1510Google Scholar, 53Floyd R.A. Proc. Soc. Exp. Biol. Med. 1999; 222: 236-245Google Scholar). It has even been reported that extracellular DHA levels increase in the disease state (23Washko P.W. Wang Y.H. Levine M. J. Biol. Chem. 1993; 268: 15531-15535Google Scholar). Although our results indicate the potential interference of measuring NO production using DAF-2, many of the prior reports have used elegantly designed controls such as NOS inhibitors and NO scavengers (40Kuo R.C. Baxter G.T. Thompson S.H. Stricker S.A. Patton C. Bonaventura J. Epel D. Nature. 2000; 406: 633-636Google Scholar, 41Nakatsubo N. Kojima H. Kikuchi K. Nagoshi H. Hirata Y. Maeda D. Imai Y. Irimura T. Nagano T. FEBS Lett. 1998; 427: 263-266Google Scholar, 42Itoh Y. Ma F.H. Hoshi H. Oka M. Noda K. Ukai Y. Kojima H. Nagano T. Toda N. Anal. Biochem. 2000; 287: 203-209Google Scholar, 46Foissner I. Wendehenne D. Langebartels C. Durner J. Plant J. 2000; 23: 817-824Google Scholar, 49López-Figueroa M.O. Caamano C. Marin R. Guerra B. Alonso R. Morano M.I. Akil H. Watson S.J. Biochim. Biophys. Acta. 2001; 1540: 253-264Google Scholar, 50Leikert J.F. Rathel T.R. Muller C. Vollmar A.M. Dirsch V.M. FEBS Lett. 2001; 506: 131-134Google Scholar, 54Kojima H. Nakatsubo N. Kikuchi K. Urano Y. Higuchi T. Tanaka J. Kudo Y. Nagano T. Neuroreport. 1998; 9: 3345-3348Google Scholar), so that the results are, at least qualitatively, unchanged with this unwanted reaction pathway. Furthermore, the work reported here also indicates the possibility of using DAF-2 to image ascorbate distributions in the central nervous system and other tissues. The use of DAFs and other related compounds as fluorescent probes for ascorbate is certainly an intriguing possibility. We gratefully thank Dr. Shao-Ching Hung for the help with fluorimeter measurements; Elena Romanova for help with the dissection of animals and preparation of cellular samples; the Mass Spectrometry Facility, especially Furong Sun, for the technical support; and Sarah Sheeley for the use of the NO bubbling system. We also thank Jiping Fu for the helpful discussion of organic reaction mechanisms.