Active secretion and protective effect of salivary nitrate against stress in human volunteers and rats

Up to 25% of the circulating nitrate in blood is actively taken up, concentrated, and secreted into saliva by the salivary glands. Salivary nitrate can be reduced to nitrite by the commensal bacteria in the oral cavity or stomach and then further converted to nitric oxide (NO) in vivo, which may play a role in gastric protection. However, whether salivary nitrate is actively secreted in human beings has not yet been determined. This study was designed to determine whether salivary nitrate is actively secreted in human beings as an acute stress response and what role salivary nitrate plays in stress-induced gastric injury. To observe salivary nitrate function under stress conditions, alteration of salivary nitrate and nitrite was analyzed among 22 healthy volunteers before and after a strong stress activity, jumping down from a platform at the height of 68 m. A series of stress indexes was analyzed to monitor the stress situation. We found that both the concentration and the total amount of nitrate in mixed saliva were significantly increased in the human volunteers immediately after the jump, with an additional increase 1 h later (p<0.01). Saliva nitrite reached a maximum immediately after the jump and was maintained 1 h later. To study the biological functions of salivary nitrate and nitrite in stress protection, we further carried out a water-immersion-restraint stress (WIRS) assay in male adult rats with bilateral parotid and submandibular duct ligature (BPSDL). Intragastric nitrate, nitrite, and NO; gastric mucosal blood flow; and gastric ulcer index (UI) were monitored and nitrate was administrated in drinking water to compensate for nitrate secretion in BPSDL animals. Significantly decreased levels of intragastric nitrate, nitrite, and NO and gastric mucosal blood flow were measured in BPSDL rats during the WIRS assay compared to sham control rats (p<0.05). Recovery was observed in the BPSDL rats upon nitrate administration. The WIRS-induced UI was significantly higher in the BPSDL animals compared to controls, and nitrate administration rescued the WIRS-induced gastric injury in BPSDL rats. In conclusion, this study suggests that stress promotes salivary nitrate secretion and nitrite formation, which may play important roles in gastric protection against stress-induced injury via the nitrate-dependent NO pathway.     

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N^ω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.     

The gastric mucus layers: constituents and regulation of accumulation

The mucus layer continuously covering the gastric mucosa consists of a loosely adherent layer that can be easily removed by suction, leaving a firmly adherent mucus layer attached to the epithelium. These two layers exhibit different gastroprotective roles; therefore, individual regulation of thickness and mucin composition were studied. Mucus thickness was measured in vivo with micropipettes in anesthetized mice [isoflurane; C57BL/6, Muc1-/-, inducible nitric oxide synthase (iNOS)-/-, and neuronal NOS (nNOS)-/-] and rats (inactin) after surgical exposure of the gastric mucosa. The two mucus layers covering the gastric mucosa were differently regulated. Luminal administration of PGE(2) increased the thickness of both layers, whereas luminal NO stimulated only firmly adherent mucus accumulation. A new gastroprotective role for iNOS was indicated since iNOS-deficient mice had thinner firmly adherent mucus layers and a lower mucus accumulation rate, whereas nNOS did not appear to be involved in mucus secretion. Downregulation of gastric mucus accumulation was observed in Muc1-/- mice. Both the firmly and loosely adherent mucus layers consisted of Muc5ac mucins. In conclusion, this study showed that, even though both the two mucus layers covering the gastric mucosa consist of Muc5ac, they are differently regulated by luminal PGE(2) and NO. A new gastroprotective role for iNOS was indicated since iNOS-/- mice had a thinner firmly adherent mucus layer. In addition, a regulatory role of Muc1 was demonstrated since downregulation of gastric mucus accumulation was observed in Muc1-/- mice.     

Protection from nonsteroidal anti-inflammatory drug (NSAID)-induced gastric ulcers by dietary nitrate

Nitrate is abundant in our diet with particularly high levels in many vegetables. Ingested nitrate is concentrated in saliva and reduced to nitrite by bacteria in the oral cavity. We recently reported that application of nitrite-containing saliva to the gastric mucosa increases superficial blood flow and mucus generation via acid-catalyzed formation of bioactive nitrogen oxides including nitric oxide. Here we studied if dietary supplementation with nitrate would protect against gastric damage caused by a nonsteroidal anti-inflammatory drug. Rats received sodium nitrate in the drinking water for 1 week in daily doses of 0.1 or 1 mmol kg(-1). Control rats received 1 mmol kg(-1) sodium chloride. Diclofenac (30 mg kg(-1)) was then given orally and the animals were examined 4 h later. In separate experiments we studied the effects of dietary nitrate on intragastric NO levels and mucus formation. Luminal levels of NO gas were greatly increased in nitrate-fed animals. The thickness of the mucus layer increased after nitrate supplementation and gene expression of MUC6 was upregulated in the gastric mucosa. Nitrate pretreatment dose dependently and potently reduced diclofenac-induced gastric lesions. Inflammatory activity was reduced in the rats receiving nitrate as indicated by lower mucosal myeloperoxidase activity and expression of inducible NO synthase. We conclude that dietary nitrate protects against diclofenac-induced gastric ulcers likely via enhanced nitrite-dependent intragastric NO formation and concomitant stimulation of mucus formation. Future studies will reveal if a diet rich in nitrate can offer an additional nutritional approach to preventing and treating peptic ulcer disease.     

Aqueous humor nitric oxide in patients with central retinal vein occlusion

PurposeIt has been suggested that nitric oxide (NO) has a role in ischemic retinopathies. Since retinal ischemia may develop in retinal vein occlusion, we investigated the presence of nitric oxide in the pathogenesis of central retinal vein occlusion (CRVO).MethodsEighteen consecutive patients with CRVO were included in this study. Aqueous humor specimens were obtained within 21 days of diagnosis. Samples of aqueous humor were also collected from 20 control patients undergoing cataract surgery. For each sample after reduction of nitrate to nitrite with vanadium chloride (VCl₃), we used spectrophotometric method for simultaneous detection of nitrate and nitrite (NO_x).ResultsMean level of aqueous humor NO_x in CRVO and control group was 94.1 ± 23.2 μmol/l and 55.6 ± 11.0 μmol/l, respectively. The difference between two groups was statistically significant (p < 0.0001).ConclusionsOur results may support involvement of nitric oxide in the pathogenesis of CRVO.     

Dietary phenolic acids and ascorbic acid: Influence on acid-catalyzed nitrosative chemistry in the presence and absence of lipids

Acid-catalyzed nitrosation and production of potentially carcinogenic nitrosative species is focused at the gastroesophageal junction, where salivary nitrite, derived from dietary nitrate, encounters the gastric juice. Ascorbic acid provides protection by converting nitrosative species to nitric oxide (NO). However, NO may diffuse into adjacent lipid, where it reacts with O₂ to re-form nitrosative species and N-nitrosocompounds (NOC). In this way, ascorbic acid promotes acid nitrosation. Using a novel benchtop model representing the gastroesophageal junction, this study aimed to clarify the action of a range of water-soluble antioxidants on the nitrosative mechanisms in the presence or absence of lipids. Caffeic, ferulic, gallic, or chlorogenic and ascorbic acids were added individually to simulated gastric juice containing secondary amines, with or without lipid. NO and O₂ levels were monitored by electrochemical detection. NOC were measured in both aqueous and lipid phases by gas chromatography–tandem mass spectrometry. In the absence of lipids, all antioxidants tested inhibited nitrosation, ranging from 35.9 ± 7.4% with gallic acid to 93 ± 0.6% with ferulic acid. In the presence of lipids, the impact of each antioxidant on nitrosation was inversely correlated with the levels of NO they generated (R² = 0.95, p < 0.01): gallic, chlorogenic, and ascorbic acid promoted nitrosation, whereas ferulic and caffeic acids markedly inhibited nitrosation.     

Positive correlation between plasma nitrite and performance during high-intensive exercise but not oxidative stress in healthy men

Several studies suggest that exercise is associated with elevated oxidative stress which diminishes NO bioavailability. The aim of the present study was to investigate a potential link between NO synthesis and bioavailability and oxidative stress in the circulation of subjects performing high-intensive endurance exercise. Twenty-two male healthy subjects cycled at 80% of their maximal workload. Cubital venous blood was taken before, during and after exercise, and heparinized plasma was generated. Plasma concentrations of nitrite and nitrate were quantified by GC–MS and of the oxidative stress biomarker 15(S)-8-iso-PGF_2α by GC–MS/MS. pH and pCO₂ fell and HbO₂ increased upon exercise. The duration of the 80% phase (d80) was 740 ± 210 s. Subjects cycled at 89.2 ± 3.3% of their peak oxygen uptake. Plasma concentration of nitrite (P < 0.01) and 15(S)-8-iso-PGF_2α (P < 0.05) decreased significantly during exercise. At the end of exercise, plasma nitrite concentration correlated positively with d80 and performed work (w80) (each P < 0.05). Changes in nitrate concentration also correlated positively with d80 (P < 0.05) and w80/kg (P < 0.01). These findings provide evidence of a favorable effect of nitrite on high-intensive endurance exercise. The lack of association between 15(S)-8-iso-PGF_2α and NO bioavailability (nitrite concentration) and NO biosynthesis (nitrate concentration) suggest that oxidative stress, notably lipid peroxidation, is not linked to the l-arginine/NO pathway in healthy male subjects being on endurance exercise.     

A critical evaluation of salivary testosterone as a method for the assessment of serum testosterone

Although salivary testosterone (T) is often used in clinical studies accuracy is mostly questionable. State of the art data for men is sparse and for women absent. Our objective was to perform a critical evaluation of salivary T (Sal-T) as a method for indirect assessment of serum T using state of the art methods. Saliva was collected via ‘Salivette’ and ‘passive drooling’ methods. Sal-T and free T in serum after equilibrium dialysis were measured by LC-MS/MSResultsEvaluation of Sal-T results versus free T by equilibrium dialysis (ED-T) for men gave: ‘Salivette’ Sal-T = 0.05 + 0.88x ED-T, r = 0.43; ‘passive drooling’ Sal-T = 0.17 + 0.91x ED-T r = 0.71. In women, correlation was comparable but values are higher than free T: ‘passive drooling’ Sal-T = 0.12 + 2.32x ED-T, r = 0.70. The higher than expected T values in saliva, appear to be explained by T binding to salivary proteins. Iso-electric focusing of saliva proteins, followed by fractionation and LC–MS/MS assay of T showed marked testosterone peaks at pH 5.3 and 8.4, providing evidence for T binding in saliva to proteins such as albumin and proline rich protein (PRP).ConclusionsPassive drooling is the collection method of choice for testosterone in saliva. Sal-T is not directly comparable to serum free T due to T binding to saliva proteins, which substantially affects the low Sal-T in women but not the higher Sal-T in healthy adult men.     

Combined effect of bone marrow derived mesenchymal stem cells and nitric oxide inducer on injured gastric mucosa in a rat model

AimTo study the effect of intravenous injection of bone marrow mesenchymal stem cells (BMMSCs), alone and combined with NO inducer in gastric ulcer healing in a rat model.MethodsRats were divided into controls, gastric ulcer, gastric ulcer receiving mesenchymal stem cells (MSCs), gastric ulcer receiving NO inducer (l-Arginine), gastric ulcer receiving MSCs plus NO inducer (l-Arginine) groups. MSCs were given in a dose of (10⁶cells) by intravenous injection. l-Arginine was given 300 mg/kg body weight intraperitoneally. 24 h and 7 days after BMMSCs and NO inducer injection, VEGF, PGE, TNF-α were assessed by ELISA. Gene expression of HGF, caspase-3, eNOS and BAX/Bcl-2 in gastric tissues were studied by real time PCR. Histopathology staining of gastric tissues was performed.ResultsInjection of MSCs or NO inducer or both to the gastric ulcer group significantly decreased caspase-3 and BAX genes expression (apoptotic factors) and increased Bcl-2 gene expression (anti-apoptotic factor) compared to that of the gastric ulcer group after both 24 h and 7 days with more significant results in the gastric group received both MSCs and NO inducer. HGF gene expression was significantly increased in the groups injected with MSCs or NO inducer or both compared with the corresponding gastric ulcer group (p < 0.05, p < 0.05 & p < 0.001 respectively). There was a significant decrease in the mean PGE2 and TNF-α levels in the gastric ulcer group receiving MSCs, the gastric ulcer group receiving NO and the gastric ulcer group receiving both MSCs and  NO compared to the gastric ulcer group after both 24 h and 7 days. Histopathological examination of gastric tissue of groups that received stem cells or NO alone, showed mucosal regenerative changes with increased thickness together with reduced inflammatory cellular infiltrate in the submucosa and decreased congestion. There was complete restoration in gastric mucosa in the group that received both stem cells and NO.ConclusionAdministration of MSCs, NO, or MSCs plus NO may exert a therapeutic effect on the mucosal lesion in gastric ulcer through their anti-inflammatory, angiogenic and antiapoptotic actions.     

Protective effect of anacardic acids from cashew (Anacardium occidentale) on ethanol-induced gastric damage in mice

Cashew nut-shell liquid and the contained anacardic acids (AAs) have been shown to possess antioxidant, lipoxygenase inhibitory, anti-Helicobacter pylori and antitumor properties. Despite these known effects, hitherto there were no published reports on their likely gastroprotective effects. The present study was designed to verify whether AAs afford gastroprotection against the ethanol-induced gastric damage and to examine the underlying mechanism(s). Gastric damage was induced by intragastric administration of 0.2 mL of ethanol (96%). Mice in groups were pretreated orally with AAs (10, 30 and 100 mg/kg), misoprostol (50 μg/kg), or vehicle (2% Tween 80 in saline, 10 mL/kg), 45 min before ethanol administration. They were sacrificed 30 min later, the stomachs excised, and the mucosal lesion area (mm²) measured by planimetry. Gastroprotection was assessed in relation to inhibition of gastric lesion area. To study the gastroprotective mechanism(s), its relations to capsaicin-sensitive fibers, endogenous prostaglandins, nitric oxide and ATP-sensitive potassium channels were analysed. Treatments effects on ethanol-associated oxidative stress markers GSH, MDA, catalase, SOD, and total nitrate/nitrite levels as an index of NO were measured in gastric tissue. Besides, the effects of AAs on gastric secretory volume and total acidity were analysed in 4-h pylorus-ligated rat. AAs afforded a dose-related gastroprotection against the ethanol damage and further prevented the ethanol-induced changes in the levels of GSH, MDA, catalase, SOD and nitrate/nitrite. However, they failed to modify the gastric secretion or the total acidity. It was observed that the gastroprotection by AAs was greatly reduced in animals pretreated with capsazepine, indomethacin, l-NAME or glibenclamide. These results suggest that AAs afford gastroprotection principally through an antioxidant mechanism. Other complementary mechanisms include the activation of capsaicin-sensitive gastric afferents, stimulation of endogenous prostaglandins and nitric oxide, and opening of K⁺_ATP channels. These combined effects are likely to be accompanied by an increase in gastric microcirculation.     

Nitrite correlates with 3-nitrotyrosine but not with the F2-isoprostane 15(S)-8-iso-PGF2α in urine of rheumatic patients

In vitro studies suggested that nitrite may play a cytoprotective role in inflammation. The aim of the present clinical study was to investigate the relationship between the NO metabolites nitrite and nitrate and the biomarkers of oxidative stress 3-nitrotyrosine (3-NT) and 15(S)-iso-PGF_2α in patients suffering from chronic inflammatory rheumatic diseases. In morning urine from 28 patients with different chronic inflammatory rheumatic diseases (23–82 years of age) and from 41 healthy persons of both genders, nitrite and nitrate were quantitated by GC-MS, and 3-NT and 15(S)-iso-PGF_2α by GC-MS/MS. Mean creatinine-corrected urinary excretion rates of nitrite (1.1 versus 0.19 μmol/mmol, P = 0.00012) and 3-NT (1.2 versus 0.39 nmol/mmol, P = 0.01629), but not of nitrate (105 versus 106 μmol/mmol), were significantly elevated in rheumatism as compared to health. Urinary excretion rate of 15(S)-iso-PGF_2α did not differ between patients and healthy subjects (65 versus 69 pmol/mmol creatinine, P = 0.48). In rheumatism, urinary 3-NT correlated closely with nitrite (R = 0.788, P < 0.0001) and moderately with nitrate (R = 0.45, P < 0.016), but did not correlate with 15(S)-iso-PGF_2α (R = ?0.083, P = 0.68). In healthy persons there was no correlation between urinary 3-NT and nitrite or nitrate. Our study suggests that urinary nitrite may represent a novel specific biomarker of nitrative stress in chronic inflammatory rheumatic disease. In another eight patients with chronic inflammatory rheumatic diseases we found higher nitrite concentrations in synovial fluid as compared to serum (1.30 versus 0.35 μM). We hypothesize that in chronic inflammatory rheumatic diseases nitrite concentration is elevated in the inflamed joint and contributes to the inactivation of myeloperoxidase-catalyzed production of hypochloric acid by forming nitryl chloride which eventually nitrates tyrosine to form 3-NT.     

Salivary detection of periodontopathic bacteria and periodontal health status in dental students

ObjectiveSaliva may become a potential source of contamination through vertical and horizontal transmissions as well as cross-infections. This study aims to use saliva as a screening tool to detect putative periodontal pathogens in a young population with fairly good oral hygiene.Materials and methodsStimulated saliva samples were obtained from 134 dental students (20.5 ± 1 years, range 18–22 years). Among those, 77 subjects also completed a periodontal examination including attachment loss, modified dental, gingival and plaque indices (AL, mDI, GI and PI). The test bacteria were identified using a 16S rRNA-based PCR detection method.ResultsOne or more of the test bacteria was found in 67% of the subjects. Prevotella nigrescens was detected as single bacterium in 16% of the subjects followed by Treponema denticola (4%), Porphyromonas gingivalis (2%), Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans (1%) and Tannerella forsythia (1%). Two or more pathogens were detected in 42% of the subjects. Clinical examination revealed health with no attachment loss (AL) in 84% of the students. In no AL group, 38% of the students were pathogen free while this was 25% for students in localized AL group (p > 0.05). There was a statistically significant association between the detection of salivary periodontal pathogen in general and higher PI (p = 0.018) and GI (p = 0.043).ConclusionWithin the limits of this study, it is possible to detect all six periodontal pathogens in the saliva of dental students. Although a correlation can be observed between the presence of salivary periodontal pathogen and clinical signs of inflammation such as plaque accumulation and gingival bleeding, detection of specific bacteria in saliva is not related to the presence of localized AL based on the presented study population.     

Dietary nitrite induces occludin nitration in the stomach

The clinical implications of the nitrate–nitrite–nitric oxide pathway have been extensively studied in recent years. However, the physiological impact of bioactive nitrogen oxides produced from dietary nitrate has remained largely elusive. Here, we report a hitherto unrecognized nitrite-dependent nitrating pathway that targets tight junction proteins in the stomach. Inorganic nitrate, nitrite or saliva obtained after the consumption of lettuce were administered by oral gavage to Wistar rats. The enterosalivary circulation of nitrate was allowed to occur for 4?h after which the animals were euthanized and the stomach collected. Nitrated occludin was detected by immunoprecipitation in the gastric epithelium upon inorganic nitrite administration (p??NO production rates from inorganic and salivary nitrite under simulated gastric conditions, suggests that competing reactions at acidic pH determine the production of nitrating agents (^?NO₂) or other, more stable, oxides. Accordingly, it is shown in vitro that salivary nitrite yields higher steady state concentrations of ^?NO (0.37?±?0.01?μM) than sodium nitrite (0.12?±?0.03?μM). Dietary-dependent reactions involving the production of nitrogen oxides should be further investigated as, in the context of occludin nitration, the consumption of green leafy vegetables (with high nitrate content), if able to modulate gut barrier function, may have important implications in the context of leaky gut disorders.     

Dietary nitrate increases gastric mucosal blood flow and mucosal defense

Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria. In the acidic stomach, nitrite is further reduced to bioactive nitrogen oxides, including nitric oxide (NO). In this study, we investigated the gastroprotective role of nitrate intake and of luminally applied nitrite against provocation with diclofenac and taurocholate. Mucosal permeability ((51)Cr-EDTA clearance) and gastric mucosal blood flow (laser-Doppler flowmetry) were measured in anesthetized rats, either pretreated with nitrate in the drinking water or given acidified nitrite luminally. Diclofenac was given intravenously and taurocholate luminally to challenge the gastric mucosa. Luminal NO content and nitrite content in the gastric mucus were determined by chemiluminescence. The effect of luminal administration of acidified nitrite on the mucosal blood flow was also investigated in endothelial nitric oxide synthase-deficient mice. Rats pretreated with nitrate or given nitrite luminally had higher gastric mucosal blood flow than controls. Permeability increased more during the provocation in the controls than in the nitrate- and nitrite-treated animals. Dietary nitrate increased luminal NO levels 50 times compared with controls. Nitrate intake also resulted in nitrite accumulation in the loosely adherent mucous layer; after removal of this mucous layer, blood flow was reduced. Nitrite administrated luminally in endothelial nitric oxide synthase-deficient mice increased mucosal blood flow. We conclude that dietary nitrate and direct luminal application of acidified nitrite decrease diclofenac- and taurocholate-induced mucosal damage. The gastroprotective effect likely involves a higher mucosal blood flow caused by nonenzymatic NO production. These data suggest an important physiological role of nitrate in the diet.     

Assessment of salivary cotinine concentration among general non-smokers population: Before and after Spanish smoking legislations

BackgroundIn Spain, two smoke-free laws have been passed (Law 28/2005 and Law 42/2010).This study evaluates the association between Spanish smoking legislations and the second-hand smoke (SHS) exposure in an adult non-smoking population cohort in Barcelona (Spain).MethodsThis is a longitudinal study, before and after the implementation of two national smoking bans, in a representative sample of adults (≥16 years old) from Barcelona (Spain) surveyed in 2004–2005 and followed up in 2013–2014 (n = 736). We only analyzed non-smokers (n = 397). We obtained 9 ml of saliva sample for analysis of cotinine, a biomarker of recent tobacco exposure. We calculated geometric means of salivary cotinine concentration and their geometric standard deviation. We used linear mixed effect models, with individuals as random effects, to model the percentage change in salivary cotinine concentration and their 95% confidence intervals.ResultsThe percentage of participants with saliva samples with measurable concentrations of cotinine fell from 92.4% to 64.2% after both Spanish smoking legislations. The geometric mean of salivary cotinine concentration significantly decreased 88% (from 0.98 ng/mL to 0.12 ng/mL, p < 0.001) after the implementation of the two Spanish smoke-free legislations. The decrease of the GM salivary cotinine concentration was statistically significant independently of the sociodemographic variables.ConclusionThere was a large reduction in the salivary cotinine concentration among adult non-smokers and higher cotinine concentrations among those declaring exposure to SHS at home after both legislations. Moreover, after both Spanish smoke-free laws salivary cotinine concentration was homogenized according to sociodemographic variables.     

Dietary nitrate reduces maximal oxygen consumption while maintaining work performance in maximal exercise

The anion nitrate—abundant in our diet—has recently emerged as a major pool of nitric oxide (NO) synthase-independent NO production. Nitrate is reduced stepwise in vivo to nitrite and then NO and possibly other bioactive nitrogen oxides. This reductive pathway is enhanced during low oxygen tension and acidosis. A recent study shows a reduction in oxygen consumption during submaximal exercise attributable to dietary nitrate. We went on to study the effects of dietary nitrate on various physiological and biochemical parameters during maximal exercise. Nine healthy, nonsmoking volunteers (age 30 ± 2.3 years, VO_2max 3.72 ± 0.33 L/min) participated in this study, which had a randomized, double-blind crossover design. Subjects received dietary supplementation with sodium nitrate (0.1 mmol/kg/day) or placebo (NaCl) for 2 days before the test. This dose corresponds to the amount found in 100–300 g of a nitrate-rich vegetable such as spinach or beetroot. The maximal exercise tests consisted of an incremental exercise to exhaustion with combined arm and leg cranking on two separate ergometers. Dietary nitrate reduced VO_2max from 3.72 ± 0.33 to 3.62 ± 0.31 L/min, P < 0.05. Despite the reduction in VO_2max the time to exhaustion trended to an increase after nitrate supplementation (524 ± 31 vs 563 ± 30 s, P = 0.13). There was a correlation between the change in time to exhaustion and the change in VO_2max (R² = 0.47, P = 0.04). A moderate dietary dose of nitrate significantly reduces VO_2max during maximal exercise using a large active muscle mass. This reduction occurred with a trend toward increased time to exhaustion implying that two separate mechanisms are involved: one that reduces VO_2max and another that improves the energetic function of the working muscles.     

Saliva as a medium for aldosterone measurement in repeated sampling studies

BackgroundSaliva is a readily available biological fluid, making it convenient in diagnosis of diseases and in multi-sampling protocols. Several salivary steroids give a useful index of free plasma levels. Increased incidence of primary aldosteronism (PA) in approximately 10% of the hypertensive population has increased interest in the mineralocorticoid aldosterone.MethodsA biotinylated-aldosterone tracer and a commercially available antibody are used in a time-resolved fluorescence immunoassay (TR-FIA) to measure salivary aldosterone (SA). Saliva was collected in various multi-sampling protocols: Investigation of diurnal rhythm in healthy and PA patients, ACTH stimulation test and posture test in healthy subjects.ResultsMethod validation showed a sensitivity of 19 ng/L and intra-/inter-assay precision between 7.2–10.1% and 8.7–15.7%, respectively. SA correlated significantly (y = 0.2995x ± 0.01, r² = 0.60) to plasma aldosterone measured by a commercial radioimmunoassay. SA (median; 95%CI) was at 111 (95–127) ng/L in PA (n = 84) and 50 (44–56) ng/L in healthy subjects (n = 60). After change in posture, aldosterone increased in both, saliva (57 (47–63) ng/L to 95 (84–117) ng/L) and plasma (26 (26–41) ng/L to 135 (110–181) ng/L). Peak levels were reached after 1 h, and were higher in females than in males.ConclusionsSA correlates well to plasma aldosterone and mirrors responses during conditions of stress. SA is significantly higher in PA, and the diurnal rhythm seen in the healthy is blunted in PA. We additionally found gender-dependent differential responses to posture, with higher increases in females. Measurement of aldosterone in saliva presents a useful and convenient method for application in multi-sampling studies.     

Counteraction of early circulatory derangement by administration of low dose steroid treatment at the onset of established endotoxemic shock is not directly mediated by TNF-α and IL-6

BackgroundOnce a septic condition is progressing, administration of steroids in the pro-inflammatory phase of septic shock ought to yield maximal effect on the subsequent, devastating inflammatory response. Recently, a retrospective study showed that early initiation of corticosteroid therapy improved survival in septic shock. We aimed to prospectively evaluate effects of early administrated hydrocortisone therapy on physiologic variables in a porcine model of septic shock.ExperimentEight anesthetized pigs were given a continuous infusion of endotoxin during this 6 h prospective, randomized, parallel-grouped placebo-controlled experimental study. At the onset of endotoxemic shock, defined as the moment when the mean pulmonary arterial pressure reached the double baseline value, the pigs were either given a single intravenous dose of hydrocortisone (5 mg kg^?1) or the corresponding volume of saline.ResultsMean arterial pressure and systemic vascular resistance index were significantly higher (both p < 0.05), and heart rate was significantly lower (p < 0.05), in the endotoxin + hydrocortisone group as compared to the endotoxin + saline group. Body temperature and blood hemoglobin levels increased significantly in the endotoxin + saline group (both p < 0.05). Urinary hydrocortisone increased significantly in both groups (p < 0.05). There were no significant differences in the plasma levels of TNF-alpha, IL-6 or nitrite/nitrate between the groups.ConclusionEarly treatment with hydrocortisone ameliorates some endotoxin mediated circulatory derangements, fever response and microvascular outflow. Our results suggest that these effects are not directly mediated by the pro-inflammatory cytokines TNF-alpha or IL-6, nor by NO.     

An effective technique for the processing of saliva for the analysis of leptin and adiponectin

The recovery of protein from saliva has been extensively investigated as a method to monitor health. The aim of this study was to compare filtration and centrifugation as two methods of saliva processing necessary for determining the levels of salivary leptin and adiponectin. Thirty-seven healthy patients (median age of 45 years; range 35–73) participated in the study. Unstimulated whole saliva was collected by a drooling technique. An aliquot was filtered using a Millex-Millipore^® (0.45 μm PVDF Dura Pore membrane) syringe and a second aliquot was centrifuged at 15 000 × g for 15 min at 4 °C. Leptin and adiponectin levels were analyzed using an ELISA kit for serum (RayBio^®, GA, USA) with minor modifications. Leptin and adiponectin levels following the filtration technique yielded comparable results with those after centrifugation. Correlation was observed between filtered and centrifuged salivary leptin levels ((r = 0.9155; 95% CI 0.8362–0.9573; p < 0.0001) with concordance correlation coefficient k 0.9114 (95% CI 0.8332–0.9539)). Less correlation was observed for adiponectin ((r = 0.5718; 95% CI 0.3041–0.7558; p = 0.0002) with concordance correlation coefficient k 0.5586 (95% CI 0.2977–0.7419)). Using a Bland–Altman plot, similar measurements for both adipocytokines were observed with mean difference within a 95% CI, and interpreted as no systematic differences between the two processing techniques. This study showed that filtration is an alternative saliva processing technique to retrieve supernatant for protein analysis. Filtered saliva yielded leptin and adiponectin concentrations comparable with those obtained from centrifuged saliva.