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Chem. Res. Toxicol. 2006, 19, 1066-1073
Thiocyanate Cannot Inhibit the Formation of Reactive Nitrogen Species in the Human Oral Cavity in the Presence of High Concentrations of Nitrite: Detection of Reactive Nitrogen Species with 4,5-Diaminofluorescein Umeo Takahama,*,† Sachiko Hirota,‡ and Takayuki Oniki† Department of Bioscience, Kyushu Dental College, Kitakyushu 803-8580, Japan, and Department of Nutritional Science, Kyushu Women’s UniVersity, Kitakyushu 807-8586, Japan ReceiVed February 20, 2006
In the human oral cavity, nitrite is reduced to nitric oxide (NO) by certain bacteria. 4,5Diaminofluorescein (DAF-2) was transformed to a fluorescent component triazolfluorescein (DAF-2T) in a bacterial fraction of saliva in the presence of nitrite. No detectable consumption of DAF-2 and formation of DAF-2T were observed in bacterial fraction in the absence of nitrite. The nitrite-dependent transformation of DAF-2 to DAF-2T was inhibited by catalase, SCN-, and CN- suggesting the participation of peroxidases in saliva in the transformation. The formation of DAF-2T, which was observed by the addition of an NO generating reagent (()-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR 3) to bacterial fraction, was also inhibited by catalase, SCN-, and CN-. The degree of the inhibition by SCN- decreased as the concentration of nitrite or NOR 3 was increased. Superoxide dismutase (SOD) enhanced nitrite- and NOR 3-induced fluorescence increase in the bacterial fraction, and the degree of the enhancement decreased as the concentrations of nitrite and NOR 3 were increased. In whole saliva filtrate, the inhibitory effects of SCN- on the fluorescence increase decreased as the concentration of nitrite was increased, but the enhancement by SOD was not significantly affected by the increase in the concentration of nitrite. As salivary bacteria produce O2-, H2O2, and NO and as peroxidase/H2O2/nitrite systems in saliva produce NO2, the effects of SCN- are discussed taking SCN--dependent inhibition of NO2 formation by peroxidases in saliva into consideration and the effects of SOD are discussed taking O2--dependent consumption of NO into consideration. It is concluded that when the rate of the formation of NO is high, SCN- is not effective enough to inhibit the formation of N2O3 in the oral cavity. Introduction Nitrate, which is ingested as a food component, is secreted into the oral cavity as a salivary component. The nitrate secreted is reduced to nitrite, and the nitrite formed is reduced further to nitric oxide (NO) by certain bacteria in the oral cavity (1-3). The NO formed has been suggested to have an antibacterial function (1, 3-5) but is autooxidized producing reactive nitrogen species such as NO2 and N2O3 as shown below:
2NO + O2 f 2NO2 (k ) 2.8 ∼ 11.6 × 106 M-2 s-1) NO + NO2 f N2O3 (k ) 1.1 × 109 M-1 s-1)
(1) (2)
It has been reported that the transformation of 4,5-diaminofluorescein (DAF-2)1 to a fluorescent component triazolfluorescein (DAF-2T) is a convenient reaction to detect the formation of NO in various biological systems (6-9). In a previous paper, we also reported that NO formed in whole human saliva could be detected by the transformation of DAF-2 to DAF-2T (10). * To whom correspondence should be addressed. Fax: (81)93-581-3202. E-mail:
[email protected]. † Kyushu Dental College. ‡ Kyushu Women’s University. 1 Abbreviations: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium; ABTS+•, ABTS cation radical; DAF-2, 4,5-diaminofluorescein; DAF-2T, triazolfluorescein; HRP, horseradish peroxidase; DHR, dihydrorhodamine; NOR 3, (()-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro3-hexenamide; SOD, superoxide dismutase.
However, the mechanism of the transformation remained to be elucidated in the whole saliva. Two reactions are possible for the transformation of DAF-2 to DAF-2T. One is a reaction between DAF-2 and N2O3 or its equivalents (11), and the other is a reaction between DAF-2 radical or its derivatives and NO (12, 13). It is known that salivary peroxidase and myeloperoxidase, which are derived from salivary glands and leukocytes, respectively, are present in the human oral cavity and that in addition to NO, O2- and H2O2 are formed by bacteria in the cavity (14). It is also known that NO rapidly reacts with O2- producing an oxidant ONOO- (pKa ) 6.8). Therefore, peroxidase-catalyzed and ONOO-/ONOOH-dependent oxidation of DAF-2 to DAF-2 radical are possible in the saliva. Horseradish peroxidase (HRP)catalyzed and ONOO-/ONOOH-dependent oxidation of DAF-2 have been reported (12, 13). On the other hand, if NO2 is produced by reactions other than reaction 1 in NO generating systems, the formation of DAF-2T by a N2O3-dependent reaction may be enhanced as N2O3 is formed by reaction 2. The production of NO2 by reactions other than reaction 1 is possible by peroxidase-catalyzed oxidation of nitrite in the presence of H2O2 (15-20). In addition to the oxidation of nitrite, peroxidasecatalyzed oxidation of NO may also contribute to the transformation of DAF-2 to DAF-2T. The oxidation of NO by peroxidases in saliva is deduced from the reports that hemeproteins including HRP can oxidize NO to NO+ (21-23). If oxidation products of NO are formed by peroxidases in saliva, they may be included in the transformation of DAF-2 to DAF2T.
10.1021/tx060038a CCC: $33.50 © 2006 American Chemical Society Published on Web 08/01/2006
ReactiVe Nitrogen Species in the Human Oral CaVity
Figure 1. Procedure of fractionation of mixed whole saliva.
Thiocyanate is a normal salivary component and is a substrate for salivary peroxidase. It has been reported that physiological concentrations of SCN- (about 1 mM) can inhibit salivary peroxidase-catalyzed oxidation of nitrite to NO2 in the presence of physiological concentrations of nitrite (around 0.2 mM) (15, 16). The concentration of nitrite increases to 1-2 mM when nitrate-rich foods are ingested (24). An objective of the present study is to elucidate how DAF-2 is transformed to DAF-2T in the human saliva. The other objective is to elucidate whether SCN- can or cannot inhibit the formation of reactive nitrogen species in the human oral cavity when the concentration of nitrite is high. The results obtained in this study suggest (i) that DAF-2 was mainly transformed to DAF-2T by N2O3-dependent reaction and (ii) that SCN- was not effective enough to inhibit the formation of N2O3 when the concentration of nitrite was high. In addition, effects of superoxide dismutase (SOD) on the formation of DAF-2T were also studied to discuss the contribution of ONOO-/ONOOH to the formation of DAF-2T.
Experimental Procedures Reagents. DAF-2 was obtained from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). (()-(E)-4-Ethyl-2-[(E)-hydroxyimino]5-nitro-3-hexenamide (NOR 3) was obtained from Dojindo (Kumamoto, Japan). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium (ABTS) was from Wako Pure Chem. Ind. (Osaka, Japan). Catalase from beef liver was from Roche Diagnostics GmbH (Mannheim, Germany). SOD from bovine erythrocytes, HRP (type II), and dihydrorhodamine (DHR) 123 were from Sigma Japan (Tokyo). Inactivated SOD was prepared by incubating the solution (1 mg/mL) in boiling water for 5 min. Preparation of Saliva. Mixed whole saliva (5 mL) was collected at 9:00-10:00 a.m. from five healthy volunteers when required by chewing Parafilm after their informed consent had been obtained. The saliva collected was fractionated as shown in Figure 1. The collected saliva was passed through two layers of nylon filter nets [380 mesh (32 µm) net, Sansho, Tokyo, Japan] to remove epithelial cells and other particles. The filtrate was used as whole saliva filtrate. Whole saliva filtrate was centrifuged at 20000g for 5 min, and the sediment was suspended in 5 mL of 50 mM sodium phosphate (pH 7.0). This fraction mainly contained bacteria with some contamination of leukocytes when observed using a microscope and was used as a bacterial fraction. The supernatant (5 mL) obtained after the centrifugation was dialyzed against 1 L of 10 mM sodium phosphate (pH 7.0) for about 24 h at 4 °C. After centrifugation of the dialyzed saliva at 20000g for 5 min, the supernatant was used as a salivary peroxidase fraction. This fraction was a mixture of salivary peroxidase and myeloperoxidase. The above fractions were kept on ice until use for experiments. Measurements of Fluorescence. The transformation of DAF-2 to a fluorescence component DAF-2T was measured at about 25 °C using a spectrofluorometer (RF-550, Shimadzu, Kyoto, Japan). The reaction mixture (0.5 mL) contained 10 µM DAF-2 in 0.5 mL of bacterial fraction when bacterial fraction was used. When whole
Chem. Res. Toxicol., Vol. 19, No. 8, 2006 1067 saliva filtrate was used, the reaction mixture (0.5 mL) contained 0.25 mL of whole saliva filtrate and 0.25 mL of 0.1 M sodium phosphate (pH 7.0). The reason that whole saliva filtrate was mixed with the buffer solution was to prevent the increase in pH due to the decrease in concentration of carbon dioxide in whole saliva filtrate and to decrease the viscosity of the saliva. The excitation and emission wavelengths were 485 and 538 nm, respectively, as reported previously (10, 11). The excitation light was passed through two glass filters ND-13 (11% transmission at 485 nm) and B-440 (18% transmission at 485 nm) from Hoya (Tokyo, Japan). Various reagents were added to the above reaction mixtures. Fluorescence increase due to the transformation of DAF-2 to DAF-2T was also studied using an NO generating reagent NOR 3 or FK 409 (2528), which decomposed with a half-life of about 3 h at pH 7.0 in this study. The half-life was determined by measuring the change in the concentration of NOR 3 by HPLC as described below. Anaerobic experiments were carried out under a stream of nitrogen gas. Oxidation of DHR 123 to rhodamine 123 was also measured fluorometrically using the spectrofluorometer and filters described above. The excitation and emission wavelengths were 500 and 535 nm, respectively, as reported previously (29). The reaction mixture (0.5 mL) contained 10 µM DHR 123 in bacterial fraction or whole saliva filtrate. Spectrophotometric Measurements. Oxidation of ABTS and quercetin was measured using a 557 spectrophotometer (Hitachi, Tokyo, Japan). Oxidation of ABTS to ABTS+• was recorded by double-beam mode at 734 nm (30). The reaction mixture (1 mL) contained 0.05 mL of salivary peroxidase fraction and 0.1 mM ABTS in 50 mM sodium phosphate (pH 7.0). Oxidation of quercetin was recorded by dual-wavelength mode (∆A330-390) (31) and the absorbance change was due to the formation of a stable component 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (15). The reaction mixture (1 mL) contained 10 µM quercetin in the bacterial fraction. The absorbance change of 0.011 (path length of the measuring beam ) 4 mm) was equivalent to the formation of 1 µM H2O2. The relation was determined from H2O2-induced absorbance changes observed in the mixture that contained 10 µM quercetin and 25 µg/mL of HRP in 50 mM sodium phosphate (pH 7.0). HPLC. DAF-2T formed by the transformation of DAF-2 was identified by HPLC using a Shim-Pack VP-ODS column (15 cm × 4.6 mm i.d.) (Shimadzu) as reported previously (10). The mobile phase was a mixture of acetonitrile and 10 mM sodium phosphate (pH 7.2) (6:94, v/v), and the flow rate was 1 mL/min. DAF-2T separated by the column was detected with a spectrofluometric detector (RF-550) and a spectrophotometric detector with a photodiode array (SPD-M10Avp, Shimadzu). The excitation and emission wavelengths for detection of DAF-2 were 485 and 538 nm, respectively. The wavelengths used for spectrophotometric detection of DAF-2 and DAF-2T were 486 and 491 nm, respectively, and the retention times of DAF-2 and DAF-2T were 8.1 and 11.5 min, respectively. As the molar extinction coefficient of DAF-2 (7.1 × 104 M-1 cm-1 at 486 nm) was similar to that of DAF-2T (7.3 × 104 M-1 cm-1 at 491 nm) (32), the amount of DAF-2T formed was estimated from known concentrations of DAF-2 by comparing peak areas. NOR 3 was separated using a Shim-pack CLC-ODS column (15 cm × 6 mm i.d.) (Shimadzu). The mobile phase was a mixture of methanol and 25 mM KH2PO4 (1:2, v/v), and the flow rate was 1 mL/min. NOR 3 was detected at 280 nm using a spectrophotometric detector with a photodiode array (SPD-M10Avp). The retention time was 10.5 min. Nitrite and SCN- in Saliva. The salivary concentration of nitrite was measured using a Griess-Romijn nitrite reagent, and the concentration of SCN- in saliva was measured using Fe3+ under acidic conditions as reported previously (33).
Results Nitrite-Induced Fluorescence Increase. It has been reported that nitrite is reduced to NO in bacterial fractions of human
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Figure 3. Fluorescence increase under anaerobic conditions. The reaction mixture (0.5 mL) contained 10 µM DAF-2 in 0.5 mL of bacterial fraction. Where indicated by downward arrows, 0.2 mM NaNO2 was added. Upward arrows show the addition of 2 or 10 µM H2O2. Trace a, under aerobic conditions; and traces b-d, under anaerobic conditions. Traces a and b, no addition; trace c, 2 mM NaSCN; and trace d, catalase (1300 units/mL).
Figure 2. Time courses of fluorescence increase and effects of concentration of nitrite and some reagents on fluorescence increase. The reaction mixture (0.5 mL) contained 10 µM DAF-2 in 0.5 mL of bacterial fraction. Upper panel: Time courses of fluorescence increase. Where indicated by an arrow, 0.2 mM NaNO2 was added. Trace 1, SOD (34 units/mL); trace 2, KCN (0.2 mM); trace 3, NaSCN (1 mM); and trace 4, catalase (1300 units/mL). These reagents were added where indicated by a white arrow. A dashed line is fluorescence increase without addition of any reagents. Lower panel: Effects of concentration of nitrite. The rate of fluorescence increase was estimated from the slope 10 min after the addition of NaNO2. Effects of reagents were calculated from rates of fluorescence increase before and after the addition of reagents: O, rate of fluorescence increase before the addition of SOD and SCN- (control); b, fluorescence increase after the addition of 34 units/mL of SOD relative to control; and 4, fluorescence increase after the addition of 1 mM NaSCN relative to control. Each data point represents the mean ( SD (n ) 4).
saliva (1, 10) and that DAF-2 is transformed to a fluorescent component DAF-2T during the formation of NO under aerobic conditions (10). DAF-2T formed has been identified from its retention time, absorption spectrum, excitation and emission spectra, and mass spectra (10). Figure 2A shows a typical time course of fluorescence increase by the addition of 0.2 mM sodium nitrite to a bacterial fraction. The fluorescence intensity started increasing on the addition, and the rate was accelerated during incubation attaining to a constant rate. Although the rate of the fluorescence increase was different from that of Figure 2, essentially the same results were obtained using other bacterial fractions. Effects of concentration of nitrite on the fluorescence increase are shown in Figure 2B. As the concentration of nitrite was increased, the rate of the fluorescence increase increased. The result suggests that the normal salivary concentration of nitrite (around 0.2 mM) (33, 34) can participate in the formation
of DAF-2T. The nitrite-induced fluorescence increase was enhanced by SOD (34 units/mL) (Figure 1) but not by inactivated SOD (not shown). SCN- (1 mM), CN- (0.2 mM), and catalase (1300 units/mL) inhibited the fluorescence increase (Figure 2). Maximal inhibition by SCN- was about 80% in the presence of 0.2 mM nitrite, and the half-maximal inhibition was observed at about 0.4 mM SCN- (data not shown). Degrees of the SOD-induced enhancement and the SCN--dependent inhibition decreased as the concentration of nitrite was increased (Figure 2B). No significant increase in fluorescence and no detectable decrease in concentration of DAF-2 were observed when 10 µM DAF-2 was incubated in bacterial fraction for 30 min in the absence of nitrite. By the addition of 0.2 mM nitrite, the decrease in the concentration of DAF-2 was observed and the concentration decreased during 30 min of incubation, which ranged from 0.7 to 3.1 µM depending on bacterial fraction [average ) 1.81 µM (n ) 9)]. DAF-2T was formed during the decrease in concentration of DAF-2 and the concentration of DAF-2T increased during 30 min of incubation ranged from 0.5 to 2.1 µM [(average ) 1.25 µM (n ) 9)]. There were nearly linear relations between the fluorescence increase and the decrease in concentration of DAF-2 and between the decrease in concentration of DAF-2 and the increase in concentration of DAF-2T. The relation between the fluorescence increase and the increase in concentration of DAF-2T was also nearly linear. One micromolar DAF-2T corresponded to the fluorescence increase of about 20 (arbitrary unit). Figure 3 shows a nitrite-induced fluorescence increase in bacterial fraction under anaerobic conditions. The fluorescence increase was nearly completely inhibited by replacing air with nitrogen gas (trace b). By the addition of H2O2 under anaerobic conditions, fluorescence increased and the fluorescence increase was dependent on the concentration of H2O2 added. The H2O2-
ReactiVe Nitrogen Species in the Human Oral CaVity
induced fluorescence increase was suppressed by SCN- and catalase (traces c and d). The H2O2-induced fluorescence increase in the presence of nitrite was also observed under aerobic conditions (data not shown). Rates of the formation of NO in bacterial fractions were estimated using a spin trapping reagent, a complex of Fe2+, and N-(dithiocarboxy)sarcosine. A nitrite-induced increase in ESR signal due to formation of Fe2+/ N-(dithiocarboxy)sarcosine/NO complex has been reported in the bacterial fraction of human saliva (10). In this study, an increase in the ESR signal was also observed after the addition of 0.2 mM nitrite to a bacterial fraction in the presence of 5 mM N-(dithiocarboxy)sarcosine and 1.5 mM Fe2+ in the bacterial fraction. The increase in the ESR signal was about 9-fold faster under anaerobic than aerobic conditions. This may be due to competition between the O2 and the radical trapping reagent for NO. NOR 3-Induced Fluorescence Increase. Time courses of fluorescence increase induced by 20 µM NOR 3 are shown in Figure 4. In 50 mM sodium phosphate (pH 7.0), SOD (34 units/ mL) enhanced the fluorescence increase about 10% (traces in A). Denatured SOD (equivalent to 34 units/mL) did not affect the fluorescence increase. SCN- (2 mM), CN- (1 mM), and catalase (1300 units/mL) did not affect the fluorescence increase either. In the bacterial fraction, native SOD enhanced the fluorescence increase about 2-fold (traces in B) but inactivated SOD did not (data not shown). Catalase (1300 units/mL), SCN(2 mM), and CN- (1 mM) inhibited the fluorescence increase in bacterial fraction, and the degree of the inhibition was about 30%. In the buffer solution, 20 µM NOR 3 decomposed at a rate of about 0.054 µM/min. Figure 4C shows effects of SOD and SCN- on the fluorescence increase in bacterial fraction in the presence of various concentrations of NOR 3. The inhibition by SCN- and the enhancement by SOD decreased as the concentration of NOR 3 was increased. Oxidation of ABTS by Salivary Peroxidase/H2O2 Systems. ABTS is known to be oxidized to ABTS+• by peroxidases including salivary peroxidase (14) and by reactive nitrogen species (30). To confirm that peroxidases in saliva could oxidize nitrite to NO2, effects of nitrite on H2O2-dependent formation of ABTS+• were studied in the presence of salivary peroxidase preparation [Figure 5, traces in (A)]. The formation of ABTS+• was nearly linear as a function of time in the absence of nitrite and attained to a maximal value (trace A). The initial rate of the formation of ABTS+• was enhanced by nitrite, but the amount of ABTS+• formed decreased as the concentration of nitrite was increased (traces B-F). By the second addition of 10 µM H2O2 to trace f as indicated by a white arrow, ABTS+• was formed again, and the rate of ABTS+• formation and the amount of ABTS+• formed were similar to those observed by the first addition of 10 µM H2O2. This result indicates that a nitrite-dependent decrease in the formation of ABTS+• was due to the consumption of H2O2 but not due to the inactivation of peroxidases in salivary peroxidase fraction by nitrite/H2O2 systems. Figure 5 (traces in B) shows effects of concentration of H2O2 on peroxidase-catalyzed formation of ABTS+• in the presence (solid curves) and absence (dashed curves) of 0.2 mM nitrite. As the concentration of H2O2 was increased, the rate of ABTS+• formation decreased in the absence of nitrite, whereas the rate increased in the presence of nitrite. Thiocyanate (1 mM) completely inhibited H2O2-induced oxidation of ABTS in the presence and absence of 0.2-1 mM nitrite (data not shown). Production of H2O2 by Bacterial Fraction. Bacteria in the oral cavity produce O2-, which transforms to H2O2 (14). Then,
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Figure 4. Effects of SOD and SCN- on NOR 3-induced fluorescence increase. (A,B) Time courses of fluorescence increase. The reaction mixture (0.5 mL) contained 10 µM DAF-2 in 0.5 mL of 50 mM sodium phosphate (pH 7.0) or bacterial fraction. (A) Buffer solution and (B) bacterial fraction. Where indicated by arrows, 20 µM NOR 3 was added. White arrows indicate the addition of reagents other than NOR 3. SOD, 17 units; catalase, 650 units; NaSCN, 1 mM; and KCN, 1 mM. (C) Effects of concentration of NOR 3 on the fluorescence increase. The reaction mixture (0.5 mL) contained 10 µM DAF-2 in 0.5 mL of bacterial fraction. Effects of SCN- and SOD were calculated from rates of fluorescence increase before and after the addition of reagents: O, fluorescence increase before the addition of SCN- and SOD (control); 4, rate of fluorescence increase after the addition of 1 mM NaSCN relative to control; and 2, rate of fluorescence increase after the addition of 34 units/mL of SOD relative to control. Each data point represents the mean ( SD (n ) 4-5).
the rate of H2O2 production of bacterial fraction was studied. No detectable oxidation of 0.1 mM ABTS was observed in bacterial fraction in the presence and absence of nitrite. This may be due to low affinity of ABTS to peroxidases in the bacterial fraction. On the other hand, the oxidation of quercetin (10 µM) was observed in the bacterial fraction. Figure 6 shows typical data. By the addition of quercetin, an increase in ∆A330-390, which was due to the difference in absorbance between 330 and 390 nm, was observed, and then ∆A330-390 decreased nearly linearly. From the absorbance change, the rate of H2O2 formation was estimated to be 0.11 ( 0.04 µM/min (mean ( SD; n ) 13). The oxidation was enhanced by HRP (5 µg/mL) by about 30% and inhibited by catalase (1300 unit/ mL) by 50-75%. Nitrite (1 mM) and SCN- (1 mM) enhanced and suppressed, respectively, the oxidation of quercetin. The oxidation of quercetin in the presence of catalase and SCNwas enhanced by HRP. It has been reported that SCN- is a
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Figure 6. Oxidation of quercetin in bacterial fraction. The reaction mixture (1 mL) contained 10 µM quercetin in 1 mL of 50 mM sodium phosphate (pH 7.0) or bacterial fraction. Where indicated by white upward arrows, quercetin was added. (A) A 50 mM concentration of sodium phosphate (pH 7.0). (B) Bacterial fraction. An upward arrow indicates the addition of 1 mM NaNO2, 25 µg/mL of HRP, 1300 units/ mL of catalase, or 1 mM NaSCN. A downward arrow shows the addition of 25 µg/mL of HRP. Figure 5. Effects of concentration of nitrite on oxidation of ABTS by salivary peroxidase/H2O2 system. The reaction mixture (1.0 mL) contained 0.1 mM ABTS and 0.05 mL of salivary peroxidase preparation in 1 mL of 50 mM sodium phosphate (pH 7.0). (A) Effects of concentration of sodium nitrite. H2O2 (10 µM) was added where indicated by an arrow: A, no addition; B, 0.02 mM; C, 0.1 mM; D, 0.2 mM; E, 0.4 mM; and F, 0.8 mM NaNO2. Where indicated by a white arrow (trace F), 10 µM H2O2 was added again. (B) Effects of concentration of H2O2. Dashed curves, without NaNO2; and solid curves, with 0.2 mM NaNO2. Where indicated by an arrow, H2O2 was added as follows: A, 2.5 µM; B, 5 µM; C, 10 µM; and D, 20 µM.
poor substrate for HRP (35) and quercetin is known to be a good substrate for HRP (36) as well as salivary peroxidase (37). Effects of SCN- and SOD on Fluorescence Increase in Whole Saliva Filtrate. Figure 7 shows effects of the concentration of nitrite on the fluorescence increase in whole saliva filtrate. Rates of the fluorescence increase in whole saliva filtrate increased as a function of the concentration of nitrite although deviations were large. The fluorescence increase was inhibited by 5 mM SCN- in the presence of low concentrations of nitrite (
