ENDOTHELIAL NITRIC OXIDE CONCENTRATIONS IN THE SALIVA OF JIU-JITSU ATHLETES

ABSTRACT Introduction This article discusses the production of nitric oxide under the influence of sport-specific physical training, measured by the salivary nitrite of Jiu-Jitsu athletes. Objectives To verify the potential of the sport to produce optimal levels of nitric oxide stimulated by exertion, and to quantify training-related nitric oxide concentrations. Method The study participants were 14 volunteer athletes from the Tatame project (extension project), who were monitored for nine months in their training routine, providing samples of unstimulated saliva. Samples were collected each month, in three periods of the day: in the morning upon waking, immediately before training, and immediately after training. Salivary nitrite was quantified by the colorimetric Griess assay. Training heart rates were also monitored in order to establish training intensity. Results Mean monthly salivary nitrite levels showed a significant correlation with mean monthly heart rates, suggesting that salivary nitrite responds to training. However, salivary nitrite concentrations measured immediately after training were always lower than in the pre-training period. Conclusion The post-training reduction in concentrations was due to the nature of the sport studied, since because it involves a fight, the intense sympathetic stimulation inhibited salivary gland activity and irrigation, preventing salivary nitrite from producing an increase in circulating nitric oxide. Level of evidence IV; Case series.

A Comparison of Different Approaches to Quantify Nitric Oxide Release from NO-Releasing Materials in Relevant Biological Media

The development of solid materials that deliver nitric oxide (NO) are of interest for several therapeutic applications. Nevertheless, due to NO’s reactive nature, rapid diffusion and short half-life, reporting their NO delivery characteristics is rather complex. The full knowledge of this parameter is fundamental to discuss the therapeutic utility of these materials, and thus, the NO quantification strategy must be carefully considered according to the NO-releasing scaffold type, to the expected NO-releasing amounts and to the medium of quantification. In this work, we explore and discuss three different ways of quantifying the release of NO in different biological fluids: haemoglobin assay, Griess assay and NO electrochemical detection. For these measurements, different porous materials, namely zeolites and titanosilicates were used as models for NO-releasing platforms. The oxyhaemoglobin assay offers great sensitivity (nanomolar levels), but it is only possible to monitor the NO release while oxyhaemoglobin is not fully converted. On the other hand, Griess assay has low sensitivity in complex biological media, namely in blood, and interferences with media make NO measurements questionable. Nevertheless, this method can measure micromolar amounts of NO and may be useful for an initial screening for long-term release performance. The electrochemical sensor enabled real-time measurements in a variety of biological settings. However, measured NO is critically low in oxygenated and complex media, giving transient signals, which makes long-term quantification impossible. Despite the disadvantages of each method, the combination of all the results provided a more comprehensive NO release profile for these materials, which will help to determine which formulations are most promising for specific therapeutic applications. This study highlights the importance of using appropriate NO quantification tools to provide accurate reports.

Methyl 3,4,5-trimethoxycinnamate suppresses inflammation in RAW264.7 macrophages and blocks macrophage–adipocyte interaction

Methyl 3,4,5-trimethoxycinnamate (MTC) is a bioactive natural phenylpropanoid. We evaluated anti-inflammatory effects of synthetic MTC in RAW264.7 macrophages and RAW264.7–3T3-L1 adipocytes co-culture. Levels of cytokines and chemokines, as well as NO and PGE2 in cell supernatants were analysed using ELISAs, Griess assay and enzyme immunoassays, respectively. In-cell cytoblot was used to assess levels of proteins; while DNA binding and reporter gene assays were used to measure transcription factor DNA binding and transcriptional activities, respectively. Glucose uptake in adipocytes was evaluated with 2‐deoxy‐2‐[(7‐nitro‐2, 1, 3‐benzoxadiazol‐4‐yl) amino]‐d‐glucose uptake. MTC (5–20 µM) suppressed LPS + IFNγ-induced release of TNFα, IL-6 and IL-1β, as well as NO/iNOS and PGE2/COX-2 levels in RAW264.7 cells. Furthermore, there was a reduction in phospho-IκB and phospho-p65 proteins, accompanied by a reduction in total IκB in RAW264.7 cells. Further studies showed that MTC also produced a reduction in NF-κB DNA binding and luciferase activity. Treatment of RAW264.7 cells with MTC (5–20 µM) resulted in enhanced DNA binding of Nrf2 and an increase in ARE-luciferase activity. In a macrophage–adipocyte co-culture, the compound reduced the release of TNFα, IL-6, IL-1β, MCP-1 and RANTES, while enhancing glucose uptake and activation of AMPKα. Our results suggest that MTC produced anti-inflammatory and antioxidant activities in macrophages. MTC also prevented inflammation in macrophage–adipocyte co-culture. The effect of MTC on glucose uptake in adipocytes is proposed to be linked to activation of AMPK.

Phenosafranin-Based Colorimetric-Sensing Platform for Nitrite Detection Enabled by Griess Assay

A facile and effective colorimetric-sensing platform based on the diazotization of phenosafranin for the detection of NO 2 − under acidic conditions using the Griess assay is presented. Diazotization of commercial phenosafranin produces a color change from purplish to blue, which enables colorimetric quantitative detection of NO 2 − . Optimal detection conditions were obtained at a phenosafranin concentration of 0.25 mM, HCl concentration of 0.4 M, and reaction time of 20 min. Under the optimized detection conditions, an excellent linearity range from 0 to 20 μM was obtained with a detection limit of 0.22 μM. Favorable reproducibility and selectivity of the colorimetric sensing platform toward NO 2 − were also verified. In addition, testing spiked ham sausage, bacon, and sprouts samples demonstrated its excellent practicability. The presented colorimetric sensing platform is a promising candidate for the detection of NO 2 − in real applications.

Dithiothreitol activity by particulate oxidizers of SOA produced from photooxidation of hydrocarbons under varied NO_<i>x</i> levels

When hydrocarbons (HCs) are atmospherically oxidized, they form particulate oxidizers, including quinones, organic hydroperoxides, and peroxyacyl nitrates (PANs). These particulate oxidizers can modify cellular materials (e.g., proteins and enzymes) and adversely modulate cell functions. In this study, the contribution of particulate oxidizers in secondary organic aerosols (SOAs) to the oxidative potential was investigated. SOAs were generated from the photooxidation of toluene, 1,3,5-trimethylbenzene, isoprene, and α-pinene under varied NOx levels. Oxidative potential was determined from the typical mass-normalized consumption rate (reaction time t = 30 min) of dithiothreitol (DTTt), a surrogate for biological reducing agents. Under high-NOx conditions, the DTTt of toluene SOA was 2–5 times higher than that of the other types of SOA. Isoprene DTTt significantly decreased with increasing NOx (up to 69 % reduction by changing the HC ∕ NOx ratio from 30 to 5). The DTTt of 1,3,5-trimethylbenzene and α-pinene SOA was insensitive to NOx under the experimental conditions of this study. The significance of quinones to the oxidative potential of SOA was tested through the enhancement of DTT consumption in the presence of 2,4-dimethylimidazole, a co-catalyst for the redox cycling of quinones; however, no significant effect of 2,4-dimethylimidazole on modulation of DTT consumption was observed for all SOA, suggesting that a negligible amount of quinones was present in the SOA of this study. For toluene and isoprene, mass-normalized DTT consumption (DTTm) was determined over an extended period of reaction time (t = 2 h) to quantify their maximum capacity to consume DTT. The total quantities of PANs and organic hydroperoxides in toluene SOA and isoprene SOA were also measured using the Griess assay and the 4-nitrophenylboronic acid assay, respectively. Under the NOx conditions (HC ∕ NOx ratio: 5–36 ppbC ppb−1) applied in this study, the amount of organic hydroperoxides was substantial, while PANs were found to be insignificant for both SOAs. Isoprene DTTm was almost exclusively attributable to organic hydroperoxides, while toluene DTTm was partially attributable to organic hydroperoxides. The DTT assay results of the model compound study suggested that electron-deficient alkenes, which are abundant in toluene SOA, could also modulate DTTm.

Dithiothreitol Activity by Particulate Oxidizers of SOA Produced from Photooxidation of Hydrocarbons under Varied NO_<i>x</i> Levels

When hydrocarbons are atmospherically oxidized, they form particulate oxidizers, including quinones, organic hydroperoxides, and peroxyacyl nitrates (PANs). These particulate oxidizers can modify cellular materials (e.g., proteins and enzymes), and adversely modulate cell functions. In this study, the contribution of particulate oxidizers in secondary organic aerosols (SOA) to the oxidative potential was investigated. SOA were generated from the photooxidation of toluene, 1,3,5-trimethylbenzene, isoprene, and α-pinene under varied NOx levels. Oxidative potential was determined from the typical mass-normalized consumption rate (reaction time t = 30 min) of dithiothreitol (DTTt), a surrogate for biological reducing agents. At high NOx conditions, the DTTt of toluene SOA was 2–5 times higher than that of other types of SOA. Isoprene DTTt significantly decreased with increasing NOx (up to 69 % reduction by changing the hydrocarbon / NOx ratio from 30 to 5). The DTTt of 1,3,5-trimethylbenzene and α-pinene SOA was insensitive to NOx under the experimental conditions of this study. The significance of quinones to the oxidative potential of SOA was tested through the enhancement of DTT consumption in the presence of 2,4-dimethylimidazole, a co-catalyst for the redox cycling of quinones; however, no significant effect of 2,4-dimethylimidazole on modulation of DTT consumption was observed for all SOA, suggesting that a negligible amount of quinones was present in SOA of this study. For toluene and isoprene, mass-normalized DTT consumption (DTTm) was determined over an extended period of reaction time (t = 2 h) to quantify their maximum capacity to consume DTT. The total quantities of PANs and organic hydroperoxides in toluene SOA and isoprene SOA were also measured using the Griess assay and the 4-nitrophenylboronic acid assay, respectively. The amount of organic hydroperoxides was substantial, while PANs were found to be insignificant for both SOA. Isoprene DTTm was almost exclusively attributable to organic hydroperoxides, while toluene DTTm was partially attributable to organic hydroperoxides. The results of the model compound study suggest that electron-deficient alkenes, which are abundant in toluene SOA, could also modulate DTTm.