A Locally-Produced Plant-Based Supplement Swathi Savi (SAVI) Enrichment Improves Body Growth, Bone Development, and Immune Functions in Protein Malnourished Mice: Implications for Strategies to Combat Child Malnutrition.

Child malnutrition is a critical global challenge. India alone is home to nearly 46 million stunted children, a third of the world's total. Supplementing locally-produced foods has been acknowledged as a sustainable strategy for combating child malnutrition. We used an established protein malnutrition (PM) model in young mice to evaluate the safety and efficacy of the SAVI-enriched diet as a food supplement to combat child malnutrition in India. Results indicate that feeding the SAVI-enriched diet improves body weight, lean muscle mass, bone, and immune health in PM young mice. Based on the results of our study in mice, we suggest future human trials to examine the supplement's potential benefits for humans.

Supplementation of Ocean-Based Advance Protein Powder (APP) for Restoration of Body Growth, Bone Development and Immune Functions in Protein Malnourished Mice: Implications for Preventing Child Malnutrition.

Child malnutrition is a global public health challenge. A protein malnutrition (PM) model in young mice was established in this study. The efficacy of an ocean-based protein (APP) extracted from by-catch fish as compared to casein and soy on restoring body weight, bone growth, and immunity of PM mice was evaluated. Results show that supplementation of APP increases body weight, lean muscle mass, bone area, mineral content and density. APP supplementation increases spleen, thymus weight, and interlukin-6 production. In conclusion, APP is an alternative source of protein to effectively restore body weight, bone growth and immune function of PM mice.

Growth, Bone Health, and Cognition: Nutritional Evaluation of a Sustainable Ocean-Based Advance Protein Powder (APP).

This study evaluated an alternative ocean-based fish protein, Advanced Protein Powder (APP) as a feasible, environmentally sustainable protein source to reduce childhood malnutrition. We completed a rodent feeding study to evaluate growth and development in young growing mice on a purified diet containing APP as compared to mice-fed diets using other common protein sources - casein, whey, and soy. Results suggested APP to be an effective and safe protein source and ensured normal body growth, bone development, and brain function in APP diet-fed mice. Evidence provided in this study supports considering the use of APP to reduce malnutrition among children worldwide.

Induction of regulatory T cells by green tea polyphenol EGCG.

Regulatory T cells (Treg) are critical in maintaining immune tolerance and suppressing autoimmunity. The transcription factor Foxp3 serves as a master switch that controls the development and function of Treg. Foxp3 expression is epigenetically regulated by DNA methylation, and DNA methyltransferase (DNMT) inhibitors can induce Foxp3 expression in naive CD4(+) T cells. We showed that EGCG, a major green tea polyphenol, could act as a dietary DNMT inhibitor, and induced Foxp3 and IL-10 expression in CD4(+) Jurkat T cells at physiologically relevant concentrations in vitro. We further showed that mice treated with EGCG in vivo had significantly increased Treg frequencies and numbers in spleen and lymph nodes and had inhibited T cell response. Induction of Foxp3 expression correlated with a concomitant reduction in DNMT expression and a decrease in global DNA methylation. Our data suggested that EGCG can induce Foxp3 expression and increase Treg frequency via a novel epigenetic mechanism. While the DNMT inhibitory effects of EGCG was not as potent as pharmacologic agents such as 5-aza-2'-deoxycytidine, the ability of dietary agents to target similar mechanisms offers opportunities for potentially sustained and longer-term exposures with lower toxicity. Our work provides the foundation for future studies to further examine and evaluate dietary strategies to modulate immune function.

Dietary soy and tea mitigate chronic inflammation and prostate cancer via NFκB pathway in the Noble rat model.

Chronic inflammation and nuclear factor-kappa B (NFκB) have been implicated in prostate cancer development; thus, dietary factors that inhibit NFκB may serve as effective chemo-preventative agents. Prostate cancer risk is significantly lower in Asian countries compared to the United States, which has prompted interest in the potential chemopreventative action of Asian dietary components such as soy and green tea. This study examined the effects of dietary soy and tea on NFκB activation and inflammation in vivo using a hormone-induced rat model for prostate cancer. Male Noble rats implanted with estradiol and testosterone were divided into 4 dietary groups: control, soy, tea, or soy+tea. NFκB activation and inflammatory cytokines were measured post implantation. The combination of soy and tea suppressed NFκB p50 binding activity and protein levels via induction of IκBα. Soy and tea also decreased prostate inflammatory infiltration, increased Bax/BcL2 ratio and decreased protein expression of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1ß compared to control. Soy and tea attenuated prostate malignancy by decreasing prostate hyperplasia. These effects were not apparent in groups treated with soy or tea alone. The ongoing in vivo studies thus far suggest that combination of foods, such as soy and tea, may inhibit hormone-induced proinflammatory NFκB signals that contribute to prostate cancer development.

Anti-inflammatory activity of soy and tea in prostate cancer prevention.

Prostate cancer is the leading cancer-related cause of death for men in the USA. Prostate cancer risk is significantly lower in Asian countries compared with the USA, which has prompted interest in the potential chemo-preventive action of soy and green tea that are more predominant in Asian diets. It has been proposed that chronic inflammation is a major risk factor of prostate cancer, acting as both an initiator and promoter. Specifically, the nuclear factor-kappa B (NF-kappaB) pathway has been implicated as an important mediator between chronic inflammation, cell proliferation and prostate cancer. Dietary factors that inhibit inflammation and NF-kappaB may serve as effective chemo-preventive agents. Recent studies have demonstrated that soy and green tea have anti-inflammatory properties, and may have the potential to block the inflammatory response during cancer progression. This minireview discusses the relationship between chronic inflammation and prostate cancer, emphasizing on the significance of NF-kappaB, and further explores the anti-inflammatory effects of soy and green tea. Finally, we propose that dietary strategies that incorporate these bioactive food components as whole foods may be a more effective means to target pathways that contribute to prostate cancer development.

Differential effects of whole soy extract and soy isoflavones on apoptosis in prostate cancer cells.

Previous studies have suggested that soy isoflavones exert anticarcinogenic effects against prostate cancer. We propose that soy extracts, containing a mixture of soy isoflavones and other bioactive components, would be a more potent chemo-preventive agent than individual soy isoflavones. We compared the apoptotic effects of whole soy extracts and individual soy isoflavones, genistein and daidzein, on prostate cancer cells. The soy extract contained 50% w/w of total isoflavones with approximately 1:5.5:3.5 ratios of genistin, daidzin and glycitin, respectively. Benign prostate hyperplasia (BPH-1), LnCap and PC3 cells were treated with varying concentrations of soy extract, genistein or daidzein and analyzed for cell cycle alterations and induction of apoptosis. At equal concentrations (25 micromol/L), soy extract induced a significantly higher percentage of cells undergoing apoptosis than genistein or daidzein (P < 0.001). No significant changes in cell cycle arrest or apoptosis were observed in non-cancerous BPH-1 cells treated with soy extract, suggesting that the effects of soy extract may be tumor cell specific. On the contrary, both genistein and daidzein induced apoptosis in BPH-1 cells, suggesting that individual isoflavones may have cytotoxicity in non-cancerous cells. Soy extracts also increased Bax expression in PC3 cells, but no significant changes in nuclear factor kappaB (NF kappaB) activation were detected, suggesting that the induction of apoptosis was independent of the NF kappaB pathway. Food products that bear a combination of active compounds may be more efficacious and safer as chemo-preventive agents than individual compounds. This 'whole-food'-based approach is significant for the development of public health recommendations for prostate cancer prevention.

Induction of proinflammatory response in prostate cancer epithelial cells by activated macrophages.

Emerging evidence indicates that chronic inflammation plays an important role in prostate carcinogenesis. Yet to date the precise molecular and cellular mechanisms linking inflammation to carcinogenesis remains unclear. The purpose of this study was to determine the local contribution of prostate epithelial cells to the inflammatory process. We characterized the inflammatory response elicited directly by prostate epithelial cells using an in vitro culture system in which androgen-dependent LNCaP prostate cancer epithelial cells were exposed to conditioned media from LPS-activated THP-1 macrophages. Upon exposure to activated macrophage conditioned media, LNCaP cells elicited a local proinflammatory response, as evidenced by NFkappaB activation, and the production of proinflammatory cytokines TNFalpha, IL-1beta, and IL-6. Furthermore, we observed a significant upregulation of the adhesion molecule VCAM-1 and nuclear estrogen receptor alpha (ERalpha) two biomarkers that correlate with tumor immune evasion and tumor progression. Our results suggest that prostate epithelial cells may play a significant role in sustaining and amplifying the inflammation process through NFkappaB activation and local production of proinflammatory cytokines that results in the recruitment and activation of additional immune cells in the prostate. At the same time, increased expression of VCAM-1 and ERalpha in prostate epithelial cells upon exposure to inflammatory conditions highlights the potential link between chronic inflammation and its involvement in promoting prostate cancer carcinogenesis.

Acute interstitial pneumonia in feedlot cattle: effects of feeding feather meal or vitamin E.

We evaluated the effects of feeding 1.5% cysteine-rich feather meal or 550 IU of vitamin E for 40 d before slaughter on the rates of death and emergency slaughter due to acute interstitial pneumonia (AIP) in commercial feedlots. Blood and lung tissue were collected at slaughter from 83 animals clinically diagnosed with AIP, 40 asymptomatic penmates, and 40 heifers receiving either feather meal (20) or vitamin E (20); the left lung was subsampled for histologic examination. Blood and lung tissue were analyzed for thiol adducts of 3-methyleneindolenine (3ME) and reduced glutathione. Supplementation with feather meal or vitamin E had no effect on the rates of death and emergency slaughter attributable to AIP and did not influence the levels of 3ME or reduced glutathione in blood or lung tissue. Although supplementation with greater amounts of feather meal or vitamin E may have been necessary to significantly affect factors related to feedlot AIP, increased supplementation would be uneconomical for commercial feedlots, given the relatively low incidence of AIP.

Xanthohumol, a prenylflavonoid derived from hops induces apoptosis and inhibits NF-kappaB activation in prostate epithelial cells.

There is increasing evidence that certain natural compounds found in plants may be useful as cancer chemopreventive or chemotherapeutic agents. Limited in vitro studies indicate that several prenylated flavonoids present in the hop plant (Humulus lupulus) possess anticarcinogenic properties. The purpose of this study was to investigate the anti-tumorigenic effects of xanthohumol (XN), the major prenylflavonoid in hops, on prostate cancer and benign prostate hyperplasia. BPH-1 and PC3 cell lines were used in our study to represent both non-tumorigenic hyperplasia and malignant prostate cancer. In both BPH-1 and PC3 cells, XN and its oxidation product, XAL, decreased cell viability in a dose dependent manner (2.5-20 microM) as determined by MTT assay and caused an increase in the formation of early and late apoptotic cells as determined by Annexin V staining and multicaspase assays. XN and its oxygenated derivative also induced cell cycle changes in both cells lines, seen in an elevated sub G1 peak at 48h treatment. Western blot analysis was performed to confirm the activation of proapoptotic proteins, Bax and p53. XN and its derivative caused decreased activation of NFkappaB. This work suggests that XN and its oxidation product, XAL, may be potentially useful as a chemopreventive agent during prostate hyperplasia and prostate carcinogenesis, acting via induction of apoptosis and down-regulation of NFkappaB activation in BPH-1 cells.

Effect of dietary melengestrol acetate on the incidence of acute interstitial pneumonia in feedlot heifers.

Over a 3-y period, 906,000 cattle were monitored in 23 feedlots in southern Alberta for symptoms of acute interstitial pneumonia (AIP). Plasma, urine, and lung tissue were collected at slaughter from 299 animals clinically diagnosed with AIP and from 156 healthy penmates and analyzed for 3-methylindole (3MI) derivatives and reduced glutathione concentration. From each animal, the left lung was subsampled for histologic examination. Concentrations of glutathione in lung tissue were reduced (P < 0.001) in animals showing clinical symptoms of AIP as compared with their asymptomatic penmates. Animals histologically confirmed as having AIP had higher levels of 3MI protein adducts in blood and lung tissue (P < 0.05) than did emergency-slaughtered animals without AIP. Within feedlots, where pens of heifers were fed either a standard dosage of melengestrol acetate (MGA) or none, the rate of death attributable to AIP was similar between treatment groups, but emergency slaughter after clinical diagnosis of AIP was done 3.2 times more often (P < 0.001) in the MGA-fed heifers than in the group not fed MGA. Use of MGA did not influence glutathione concentration. As growth performance of heifers given steroidal implants may not be improved by feeding MGA, the most cost-effective method of reducing the incidence of AIP-related emergency slaughter in feedlot heifers may be to eliminate MGA from the diet.

Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation.

Vitamin E disappearance is accelerated in cigarette smokers due to their increased oxidative stress and is inversely correlated with plasma vitamin C concentrations. Therefore, we hypothesized that ascorbic acid supplementation (500 mg, twice daily; 2 weeks) would normalize smokers' plasma alpha- and gamma-tocopherol disappearance rates and conducted a double-blind, placebo-controlled, randomized crossover investigation in smokers (n=11) and nonsmokers (n=13) given a single dose of deuterium-labeled alpha- and gamma-tocopherols (50 mg each d6-RRR-alpha and d2-RRR-gamma-tocopheryl acetate). During the placebo trial, smokers, compared with nonsmokers, had significantly (P<0.05) greater alpha- and gamma-tocopherol fractional disappearance rates and shorter half-lives. Ascorbic acid supplementation doubled (P<0.0001) plasma ascorbic acid concentrations in both groups and attenuated smokers', but not nonsmokers', plasma alpha- and gamma-tocopherol (P<0.05) fractional disappearance rates by 25% and 45%, respectively. Likewise, smokers' plasma deuterium-labeled alpha- and gamma-tocopherol concentrations were significantly higher (P<0.05) at 72 h during ascorbic acid supplementation compared with placebo. Ascorbic acid supplementation did not significantly change (P>0.05) time of maximal or maximal-labeled alpha- and gamma-tocopherol concentrations. Smokers' plasma F2alpha-isoprostanes were approximately 26% higher than nonsmokers (P>0.05) and were not affected by ascorbic acid supplementation in either group (P>0.05). In summary, cigarette smoking increased plasma alpha- and gamma-tocopherol fractional disappearance rates, suggesting that the oxidative stress from smoking oxidizes tocopherols and that plasma ascorbic acid reduces alpha- and gamma-tocopheroxyl radicals to nonoxidized forms, thereby decreasing vitamin E disappearance in humans.

Dietary supplementation of N-acetylcysteine enhances early inflammatory responses during cutaneous wound healing in protein malnourished mice.

Prolonged wound healing is a complication that contributes to the morbidity and mortality of protein malnutrition (PM). The molecular mechanisms that underlie impaired wound healing in PM may begin in the early inflammatory stage of the process. We hypothesized that the impaired wound healing observed in PM occurs as a consequence of excessive reactive oxygen species (ROS) production that impairs the wound healing process by depressing nuclear factor kappa B (NFkappaB) activation and the subsequent synthesis and release of proinflammatory cytokines that are critical mediators of the inflammatory response. In this study, we showed that the time to wound closure was significantly prolonged in PM mice. During the early wound healing in PM, inhibitory kappa B alpha (IkappaBalpha), interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) expression and neutrophil infiltration were significantly decreased in PM mice. The role of excess ROS in PM was demonstrated by using transgenic mice with overexpression of copper zinc superoxide dismutase and with dietary supplementation of N-acetylcysteine (NAC). Both interventions improved the extent of wound closure in PM mice. Moreover, NAC supplementation in PM mice restored the expression of IkappaBalpha, IL-1beta and TNF-alpha and infiltration of neutrophils to levels observed in control animals. These findings support the notion that wound healing defects in PM may result from dysregulation of ROS-mediated and NFkappaB-regulated signaling pathways.

Lower plasma alpha-carboxyethyl-hydroxychroman after deuterium-labeled alpha-tocopherol supplementation suggests decreased vitamin E metabolism in smokers.

BACKGROUND: Cigarette smoking increases the fractional disappearance rates of alpha-tocopherol and is associated with increased oxidative stress, but its effects on alpha-tocopherol metabolism are unknown. OBJECTIVE: We hypothesized that smokers would have less alpha-tocopherol available and consequently lower plasma alpha-carboxyethyl-hydroxychroman (alpha-CEHC), the alpha-tocopherol metabolite produced by a cytochrome P450-mediated process. DESIGN: Smokers and nonsmokers (n = 10 per group) were supplemented with deuterium-labeled alpha-tocopheryl acetates (75 mg each d3-RRR-alpha-tocopheryl and d6-all-rac-alpha-tocopheryl acetate) from day -6 to day -1, and plasma tocopherols and CEHCs were measured (day -6 through day 17). RESULTS: After 6 d of supplementation, plasma d3- and d6-alpha-tocopherol concentrations did not differ significantly between groups. Plasma d3- and d6-alpha-CEHCs were detectable only from day -5 to day 5. Before supplementation, unlabeled alpha- and gamma-CEHCs were approximately 60% and 40% lower, respectively, in smokers than in nonsmokers (P < or = 0.05). In addition, d0-, d3-, and d6-alpha-CEHC areas under the curves were approximately 50% lower in smokers (P < 0.05), and smokers had lower maximal d3-alpha-CEHC (P = 0.004) and d6-alpha-CEHC (P = 0.0006) concentrations. Notably, 2.9-4.7 times as much alpha-CEHC was produced from all-rac-alpha-tocopherol than from RRR-alpha-tocopherol. During supplementation, smokers had about one-half (P < 0.05) the plasma total, d6-, or d3-alpha-CEHC concentrations that nonsmokers did given similar alpha-tocopherol concentrations. CONCLUSIONS: Smoking did not increase alpha-tocopherol disappearance through P450-mediated tocopherol metabolism. Therefore, the mechanism of increased alpha-tocopherol disappearance in smokers likely operates through oxidation pathways, which is consistent with alpha-tocopherol's antioxidant function. Consequently, evaluating the molecular mechanism or mechanisms responsible for tocopherol metabolism under conditions of oxidative stress and the mechanisms that regulate alpha-tocopherol status is warranted.

{alpha}-Tocopherol disappearance is faster in cigarette smokers and is inversely related to their ascorbic acid status.

BACKGROUND: Cigarette smokers have enhanced oxidative stress from cigarette smoke exposure and from their increased inflammatory responses. OBJECTIVE: The objective of this study was to determine whether cigarette smoking increases plasma alpha-tocopherol disappearance in otherwise healthy humans. DESIGN: Smokers and nonsmokers (n = 10/group) were supplemented with deuterium-labeled alpha-tocopheryl acetates (75 mg each of d(3)-RRR-alpha-tocopheryl acetate and d(6)-all-rac-alpha-tocopherols acetate) for 6 evenings (days -6 to -1). Plasma alpha-tocopherols, ascorbic acid, uric acid, and F(2alpha)-isoprostanes were measured in blood samples collected on days -6 through 17. The urinary alpha-tocopherol metabolite, alpha-carboxy-ethyl-hydroxy-chroman (alpha-CEHC), was measured on days -6, 0, and 17 in 24-h urine samples. RESULTS: F(2alpha)-isoprostanes were, on average, approximately 40% higher in smokers than in nonsmokers. On day 0, plasma labeled and unlabeled alpha-tocopherol concentrations were not significantly different between groups. Smoking resulted in faster fractional disappearance of plasma alpha-tocopherol (0.215 +/- 0.011 compared with 0.191 +/- 0.009 pools/d; P < 0.05). Fractional disappearance rates of alpha-tocopherol correlated with plasma ascorbic acid concentrations in smokers (P = 0.021) but not in nonsmokers despite plasma ascorbic acid concentrations that were not significantly different between groups. By day 17, cigarette smoking resulted in lower plasma alpha-tocopherol concentrations and urinary excretion of labeled and unlabeled alpha-CEHC (P < 0.05). CONCLUSIONS: Cigarette smoking increased alpha-tocopherol disappearance. Greater rates of alpha-tocopherol disappearance in smokers appear to be related to increased oxidative stress accompanied by lower plasma ascorbic acid concentrations. Thus, smokers have an increased requirement for both alpha-tocopherol and ascorbic acid.

Effect of conjugated linoleic acid on Delta-5 desaturase activity in yeast transformed with fungal Delta-5 desaturase gene.

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers derived from linoleic acid (LA: delta9, 12-18:2), has been shown to exhibit various biological functions based on studies using cell culture and animal models. It was postulated that the beneficial effects of CLA were exerted through suppression of production of arachidonic acid (AA; delta5,8,11,14-20:4) and consequently, production of pro-inflammatory eicosanoids. In this study, we used the baker's yeast, Saccharomyces cerevisiae, transformed with fungal delta5-desaturase gene as a model, to study whether CLA affects the activity of delta5-desaturase, a rate-limiting step which converts dihomo-gamma-linolenic acid (DGLA; delta8,11, 14-20:3) to AA. The activity of delta5-desaturase was examined in the transformed yeast incubated in a medium supplemented with DGLA and one of four different CLA isomers (c9, t11-, t10, c12-, c9, c11- and t9, t11). Results show that all four isomers were taken up readily by the yeast, and all of them suppressed the conversion of DGLA to AA. The degree of suppression, which varied significantly among four isomers was modulated by the level of CLA isomers added in the medium. Since portions of these CLA isomers could be converted to form delta5-CLA metabolites (delta5, c9, t11-, delta5, t10, c12-, delta5, c9, c11- and delta5, t9, t11-18:3), it is suggested that CLA suppressed the delta5-desaturation of DGLA to AA through substrate competition between DGLA and CLA isomers.

Dietary influences on endocrine-inflammatory interactions in prostate cancer development.

Prostate cancer is the most frequently diagnosed non-cutaneous cancer and is the second leading cause of cancer death in American men. The focus of this review is to define the relationship between hormonal (testosterone/estrogens) stimulation of chronic inflammation, generation of reactive oxygen species (ROS), and uncontrolled prostate cell proliferation, and review putative dietary chemoprevention strategies that focus on these processes. It has been proposed that elevated estrogen in men who already have high blood testosterone are at high risk for prostate cancer. We hypothesized that elevated estrogen, in the presence of testosterone, causes prolonged activation of a redox-sensitive transcription factor, nuclear factor kappa B (NF kappa B), that initiates and amplifies an inflammatory cascade within the prostate and results in sustained oxidative and nitrative damage. The inflammatory cascade is proposed to link with uncontrolled proliferation through up-regulated Wnt signal and abnormal catenin accumulation in the prostate. Finally, a strategy that emphasizes a "whole food" based approach to cancer prevention by selecting food products that bear anti-inflammatory and anti-proliferative properties may be most promising as an effective dietary chemopreventive strategy.

Dietary zinc alters early inflammatory responses during cutaneous wound healing in weanling CD-1 mice.

Zinc deficiency is a well-known health problem associated with delayed wound healing, yet the precise mechanisms that underlie the delay remain unknown. We hypothesized that zinc deficiency delays wound healing as a result of decreased nuclear factor (NF)kappaB activation, reduced expression of proinflammatory cytokines [interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha], and a decrease in neutrophil infiltration during the early stage of cutaneous wound healing. We used a cutaneous, full-thickness excisional wound model in CD-1 mice to examine the rate of wound closure as well as mRNA levels of inhibitory (I)kappaBalpha, IL-1beta, and TNF-alpha and infiltration of neutrophils at the wound site of mice fed a diet containing <1 (deficient), 50 (control), 500, or 1000 microg zinc/g diet. Zinc deficiency reduced the rate of wound closure and mRNA levels of IL-1beta and TNF-alpha and attenuated infiltration of neutrophils at the wound site compared with controls. Interestingly, zinc supplementation at 1000 microg/g delayed the rate of wound closure and decreased mRNA levels of TNF-alpha and infiltration of neutrophils compared with mice fed the control diet. These findings demonstrate that zinc deficiency and high-dose zinc supplementation delay wound healing as a result of altered inflammatory responses and suggest that adequate zinc supplementation may have beneficial effects on the inflammatory responses to enhance cutaneous wound healing.

5-nitro-gamma-tocopherol increases in human plasma exposed to cigarette smoke in vitro and in vivo.

We hypothesized that the high concentrations of reactive nitrogen species in cigarette smoke and the known stimulatory effects of cigarette smoke on the inflammatory immune systems would lead to the formation of 5-nitro-gamma-tocopherol (NGT). In order to assess gamma-tocopherol nitration, human plasma was exposed in vitro to gas phase cigarette smoke (GPCS) or air for up to 6 h. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantitate NGT. Detector response was linear from 0.1 to 3 pmol NGT, with a detection limit of 20 fmol. After a 1 h lag time, 6 h plasma exposure to GPCS depleted approximately 75% of alpha-T, approximately 60% of gamma-T and increased NGT from 3 to 134 nmol/l. The increase in NGT accounted for approximately 20% of the gamma-T decrease. NGT also correlated (R2 = 0.9043) with nitrate concentrations in GPCS-exposed plasma. The physiologic relevance of NGT was evaluated in a group of healthy humans. Smokers (n = 15) had plasma NGT concentrations double those of nonsmokers (n = 19), regardless of corrections using lipids or gamma-T; plasma alpha-T and gamma-T concentrations were similar between the groups. Our results show that LC-MS can be successfully used for NGT quantitation in biologic samples. Importantly, NGT in smokers' plasma suggests that cigarette smoking causes increased nitrosative stress.