ascorbic-acid and thiocyanate

Catalytic consumption of nitric oxide (NO) by myeloperoxidase and related peroxidases is implicated as playing a key role in impairing NO bioavailability during inflammatory conditions. However, there are major gaps in our understanding of how peroxidases consume NO in physiological fluids, in which multiple reactive enzyme substrates and antioxidants are present. Notably, ascorbate has been proposed to enhance myeloperoxidase-catalyzed NO consumption by forming NO-consuming substrate radicals. However, we show that in complex biological fluids ascorbate instead plays a critical role in inhibiting NO consumption by myeloperoxidase and related peroxidases (lactoperoxidase, horseradish peroxidase) by acting as a competitive substrate for protein-bound redox intermediates and by efficiently scavenging peroxidase-derived radicals (e.g., urate radicals), yielding ascorbyl radicals that fail to consume NO. These data identify a novel mechanistic basis for how ascorbate preserves NO bioavailability during inflammation. We show that NO consumption by myeloperoxidase Compound I is significant in substrate-rich fluids and is resistant to competitive inhibition by ascorbate. However, thiocyanate effectively inhibits this process and yields hypothiocyanite at the expense of NO consumption. Hypothiocyanite can in turn form NO-consuming radicals, but thiols (albumin, glutathione) readily prevent this. Conversely, where ascorbate is absent, glutathione enhances NO consumption by urate radicals via pathways that yield S-nitrosoglutathione. Theoretical kinetic analyses provide detailed insights into the mechanisms by which ascorbate and thiocyanate exert their protective actions. We conclude that the local depletion of ascorbate and thiocyanate in inflammatory microenvironments (e.g., due to increased metabolism or dysregulated transport) will impair NO bioavailability by exacerbating peroxidase-catalyzed NO consumption.

Topics: Animals; Antioxidants; Ascorbic Acid; Biocatalysis; Cattle; Humans; Models, Biological; Nitric Oxide; Peroxidase; Plasma; Thiocyanates

β(2)-agonists are the most effective bronchodilators for the rapid relief of asthma symptoms, but for unclear reasons, their effectiveness may be decreased during severe exacerbations. Because peroxidase activity and nitrogen oxides are increased in the asthmatic airway, we examined whether salbutamol, a clinically important β(2)-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)), both absorption spectroscopy and mass spectrometry indicated formation of a new metabolite with features expected for the nitrated drug. The new metabolites showed an absorption maximum at 410 nm and pK(a) of 6.6 of the phenolic hydroxyl group. In addition to nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, formed by elimination of the formaldehyde group, was detected (m/z 255.13) by mass spectrometry. It is noteworthy that the latter metabolite was detected in exhaled breath condensates of asthma patients receiving salbutamol but not in unexposed control subjects, indicating the potential for β(2)-agonist nitration to occur in the inflamed airway in vivo. Salbutamol nitration was inhibited in vitro by ascorbate, thiocyanate, and the pharmacological agents methimazole and dapsone. The efficacy of inhibition depended on the nitrating system, with the lactoperoxidase/H(2)O(2)/NO(2)(-) being the most affected. Functionally, nitrated salbutamol showed decreased affinity for β(2)-adrenergic receptors and impaired cAMP synthesis in airway smooth muscle cells compared with the native drug. These results suggest that under inflammatory conditions associated with asthma, phenolic β(2)-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy.

Topics: Adrenergic beta-2 Receptor Agonists; Albuterol; Ascorbic Acid; Asthma; Breath Tests; Bronchi; Catalysis; Child; Cyclic AMP; Dapsone; Humans; Hydrogen Peroxide; Mass Spectrometry; Methimazole; Nitrites; Peroxidases; Receptors, Adrenergic, beta-2; Thiocyanates

Myeloperoxidase (MPO) is a heme enzyme, released by activated leukocytes at sites of inflammation, which catalyzes the formation of the potent oxidant, hypochlorous acid (HOCl), from H2O2. HOCl is a key component of the inflammatory response and is bactericidal but has been linked with several human pathologies as a result of damage to host tissue. Elevated plasma MPO levels are a strong independent risk factor, and predictor of outcomes, for cardiovascular disease. Rate constants for reaction of HOCl with individual biological targets and the products of these reactions have been determined, but the targets of HOCl in complex biological fluids such as plasma are incompletely defined. In this study, rate constants (M(-1) s(-1)) for the reactions of ascorbate with HOCl (ca. 6 x 10(6)) and imidazole chloramine (7.7 x 10(4)) have been determined to supplement known kinetic parameters. HOCl-mediated oxidation of the major plasma protein, albumin, was investigated both experimentally and computationally; these approaches provide consistent data. The computational studies were extended to examine the fate of HOCl in plasma. The model predicts that plasma proteins consume the majority of HOCl with limited damage to other materials. Ascorbate or alpha-tocopherol, even at the levels achieved in human supplementation studies, do not attenuate these reactions. In contrast, elevated levels of thiocyanate ions (SCN(-)), as detected in heavy smokers, can modulate HOCl-mediated reactions as a result of the formation of the highly specific oxidant hypothiocyanous acid (HOSCN). These observations support the hypothesis that MPO-generated HOSCN is a key agent in smoking-enhanced atherosclerosis.

Topics: alpha-Tocopherol; Ascorbic Acid; Humans; Hypochlorous Acid; Kinetics; Models, Theoretical; Oxidants; Oxidation-Reduction; Peroxidase; Serum Albumin; Smoking; Thiocyanates

4-Hydroxyphenylacetic acid (HPA) and nitrite are present in human mixed whole saliva, and HPA can be nitrated by peroxidase/hydrogen peroxide (H(2)O(2))/nitrite systems in the oral cavity. Thus, the objectives of the present study were to estimate the concentrations of HPA, nitrated HPA [4-hydroxy-3-nitrophenylacetic acid (NO(2)HPA)], nitrite, and thiocyanate (SCN(-)) in saliva from 73 patients with periodontal diseases and to elucidate the conditions necessary to induce nitration of HPA. High concentrations of HPA, nitrite, and SCN(-) were found in the saliva of patients older than 50 yr of age. NO(2)HPA was detected in seven patients who were older than 60 yr of age. Nitrite-dependent formation of NO(2)HPA by a bacterial fraction prepared from mixed whole saliva was faster at pH 5.3 than at pH 7, and increased as the rate of H(2)O(2) formation increased. The formation of NO(2)HPA was inhibited by SCN(-) and by salivary antioxidants such as uric acid, ascorbic acid, and glutathione. These results suggest that nitration can proceed at an acidic site in the oral cavity where H(2)O(2) is produced under conditions of decreased concentrations of SCN(-) and of antioxidants.

Topics: Acids; Adult; Age Factors; Aged; Aged, 80 and over; Antioxidants; Ascorbic Acid; Bacteria; Female; Gingival Hemorrhage; Gingivitis; Glucose; Glucose Oxidase; Glutathione; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Male; Middle Aged; Mouth; Nitrites; Periodontal Diseases; Peroxidases; Phenylacetates; Saliva; Smoking; Thiocyanates; Uric Acid; Young Adult

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H(2)O(2) with concurrent evolution of O(2). This pseudocatalatic degradation of H(2)O(2) to O(2) is solely dependent on ascorbate and is blocked by a spin trap, alpha-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H(2)O with subsequent regeneration of ascorbate by the reduction of MDA with H(2)O(2) evolving O(2) through the intermediate formation of O(2)(-). Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN(-) in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN(-). Further kinetic studies indicate that SCN(-) itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN(-) blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H(2)O(2) to O(2). Binding studies by optical difference spectroscopy indicate that SCN(-) binds LmAPX (Kd = 100 +/- 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN(-) acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.

Topics: Animals; Ascorbate Peroxidases; Ascorbic Acid; Catalase; Cloning, Molecular; Guaiacol; Hydrogen Peroxide; Kinetics; Leishmania major; Ligands; Oxidation-Reduction; Oxygen; Peroxidases; Protozoan Proteins; Recombinant Proteins; Thiocyanates

Extraction spectrophotometric method has been developed for the determination of tricyclic drugs such as trazodone (TZH), amineptine (APH) and amitriptyline (ATPH) hydrochlorides in pure form and in the dosage forms coming from different Egyptian markets. The method based on the formation of ion-pairs between these drugs under investigation and inorganic complex of Mo(V)-thiocyanate followed by its extraction with methylene chloride. The optimum conditions for the ion-pairs formation are established. The method permits the determination of TZH, APH and ATPH over the concentration range of 2-28, 2-32 and 1-30 microg ml(-1), respectively. The Sandell sensitivity (S) is found to be 0.105, 0.138 and 0.118 g cm(-2) for TZH, APH and ATPH, respectively. The SD is found to be 0.16-0.377, 0.12-0.259 and 0.091-0.286 and the R.S.D. are 0.14-0.55, 0.12-0.399 and 0.095-0.485 for TZH, APH and ATPH, respectively. The method is applicable for the assay of the investigated drugs in different dosage forms and the results are in good agreement with those obtained by the official method.

Topics: Absorption; Amitriptyline; Antidepressive Agents, Tricyclic; Ascorbic Acid; Dibenzocycloheptenes; Dose-Response Relationship, Drug; Hydrochloric Acid; Hydrogen-Ion Concentration; Models, Biological; Molybdenum; Solvents; Spectrophotometry; Temperature; Thiocyanates; Time Factors; Trazodone

When saliva, with its high nitrite content derived from the enterosalivary recirculation of dietary nitrate, meets acidic gastric juice, the nitrite is converted to nitrous acid, nitrosative species, and nitric oxide. In healthy volunteers this potentially mutagenic chemistry is focused at the gastric cardia. We have studied the location of this luminal chemistry in Barrett's patients during acid reflux.. Ten Barrett's patients were studied before and after administration of 2 mmol nitrate. Using microdialysis probes we measured nitrite, ascorbic acid, total vitamin C, and thiocyanate concentrations and pH simultaneously in the proximal oesophagus, Barrett's segment, hiatal sac, proximal stomach, and distal stomach. In a subgroup, real time nitric oxide concentrations were also measured.. During acid reflux, Barrett's segment was the anatomical site with maximal potential for acid catalysed nitrosation, with its median concentration of nitrite exceeding that of ascorbic acid in two of 10 subjects before nitrate and in four of nine after nitrate. Thiocyanate, which catalyses acid nitrosation, was abundant at all anatomical sites. On entering the acidic Barrett's segment, there was a substantial fall in nitrite and the lowest ascorbic acid to total vitamin C ratio, indicative of reduction of salivary nitrite to nitric oxide at this anatomical site. Episodes of acid reflux were observed to generate nitric oxide concentrations of up to 60 muM within the Barrett's segment.. The interaction between acidic gastric refluxate and nitrite rich saliva activates potentially mutagenic luminal nitrosative chemistry within Barrett's oesophagus.

Topics: Aged; Ascorbic Acid; Barrett Esophagus; Esophageal Neoplasms; Esophagoscopy; Female; Gastroesophageal Reflux; Humans; Hydrogen-Ion Concentration; Male; Microdialysis; Middle Aged; Nitrates; Nitric Oxide; Nitrites; Nitrosation; Precancerous Conditions; Saliva; Thiocyanates

A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H(2)O(2). The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.

Topics: Amino Acids; Ascorbic Acid; Azides; Blotting, Western; Catalase; Cyclic N-Oxides; Cysteine; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Free Radicals; Glutathione; Heme; Hydrogen Peroxide; Hydrogen-Ion Concentration; Immunochemistry; Iodides; Lactoperoxidase; Models, Chemical; Nitrites; Nitrogen Oxides; Oxygen; Oxygen Consumption; Phenol; Spin Labels; Thiocyanates; Time Factors; Tyrosine

It has been hypothesized that dietary nitrite augments the antimicrobial activity of gastric acid after conversion to nitric oxide and other reactive nitrogen intermediates, thus resulting in increased resistance against gastrointestinal infection. In this study, we showed that the reducing agents ascorbic acid and glutathione reduced the activity of acidified nitrite against Yersinia enterocolitica (P < 0.001). In contrast, iodide and thiocyanate increased the antimicrobial activity (P < 0.001), whereas hydroxyacids (citrate, lactate, and tartarate) had no measurable effects.

Topics: Acids; Anti-Infective Agents; Antioxidants; Ascorbic Acid; Citric Acid; Glutathione; Humans; Iodides; Lactic Acid; Nitrites; Tartrates; Thiocyanates; Yersinia enterocolitica; Yersinia Infections

There is evidence that LDL oxidation may render the lipoprotein atherogenic. The myeloperoxidase-hydrogen peroxide (MPO/H2O2) system of activated phagocytes may be involved in this process. Chloride is supposed to be the major substrate for MPO, generating reactive hypochlorous acid (HOCl), modifying LDL. The pseudo-halide thiocyanate (SCN-) has been shown to be a suitable substrate for MPO, forming reactive HOSCN/SCN*. As relatively abundant levels of SCN- are found in plasma of smokers--a well-known risk group for cardiovascular disease--the ability of SCN- to act as a catalyst of LDL atherogenic modification by MPO/H2O2 was tested. Measurement of conjugated diene and lipid hydroperoxide formation in LDL preparations exposed to MPO/H2O2 revealed that SCN- catalyzed lipid oxidation in LDL. Chloride did not diminish the effect of SCN- on lipid oxidation. Surprisingly, SCN inhibited the HOCl-mediated apoprotein modification in LDL. Nitrite--recently found to be a substrate for MPO--showed some competing properties. MPO-mediated lipid oxidation was inhibited by heme poisons (azide, cyanide) and catalase. Ascorbic acid was the most effective compound in inhibiting the SCN- -catalyzed reaction. Bilirubin showed some action, whereas tocopherol was ineffective. When LDL oxidation was performed with activated human neutrophils, which employ the MPO pathway, SCN- catalyzed the cell-mediated LDL oxidation. The MPO/H2O2/SCN- system may have the potential to play a significant role in the oxidative modification of LDL--an observation further pointing to the link between the long-recognized risk factors of atherosclerosis: elevated levels of LDL and smoking.

Topics: Antioxidants; Apoproteins; Ascorbic Acid; Azoles; Catalase; Catalysis; Chlorides; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Female; Free Radicals; Heme; Humans; Hydrogen Peroxide; Hypochlorous Acid; Isoindoles; Lipid Metabolism; Lipid Peroxidation; Lipids; Lipoproteins, LDL; Male; Neutrophils; Nitrites; Organoselenium Compounds; Oxygen; Peroxidase; Phagocytes; Phagocytosis; Risk Factors; Smoking; Thiocyanates; Time Factors; Ultracentrifugation

Formation of OH radicals in the stomach is possible by Fenton-type reactions, as gastric juice contains ascorbic acid (AA), iron ions and H2O2. An objective of the present study is to elucidate the effects of salivary SCN- and NO2- on the hydroxylation of salicylic acid which was induced by H2O2/Fe(II) and AA/H2O2/Fe(II) systems. Thiocyanate ion inhibited the hydroxylation of salicylic acid by the above systems in acidic buffer solutions and in acidified saliva. The inhibition by SCN- was deduced to be due to SCN- -dependent scavenging of OH radicals. Nitrite ion could enhance the SCN- -dependent inhibition of the hydroxylation induced by AA/H2O2/Fe(II) systems. The enhancement was suggested to be due to scavenging of OH radicals by NO which was formed by the reactions among AA, HNO2 and SCN- contained in the reaction mixture. The concentrations of SCN- and NO2-, which were effective for the inhibition, were in ranges of their normal salivary concentrations. These results suggest that salivary SCN- can cooperate with NO2- to protect stomach from OH radicals formed by AA/H2O2/Fe(II) systems under acidic conditions.

Topics: Acids; Ascorbic Acid; Ferrous Compounds; Free Radical Scavengers; Gastric Mucosa; Humans; Hydrogen Peroxide; Hydroxyl Radical; Hydroxylation; Nitrites; Oxidation-Reduction; Salicylic Acid; Saliva; Thiocyanates

Saliva contains substantial concentrations of nitrite derived from the enterosalivary recirculation of dietary nitrate.. We have investigated factors in gastric juice influencing the fate of nitrite in swallowed saliva. When nitrite (100 microM) is added to human gastric juice pH 1.5 or pH 2.5 at 37 degrees C containing physiological concentrations of thiocyanate (1 mM) and ascorbic acid (200 microM), it is converted to nitric oxide within a few seconds.. The reduction of nitrite to nitric oxide is slower at pH 3.5 and very little is generated at pH 4.5. The rate of nitric oxide generation at acid pH increases with increasing thiocyanate concentration. The concentration of nitric oxide generated in the above way is maintained until the ascorbic acid is depleted by the recycling of nitric oxide to nitrite. In gastric juice depleted of ascorbic acid, very little nitrite is reduced to nitric oxide at any pH.. These studies indicate that in the healthy acid-secreting stomach most salivary nitrite will be reduced to nitric oxide at the gastro-oesophageal junction and gastric cardia where it first encounters gastric juice.

Topics: Ascorbic Acid; Gastric Juice; Humans; Hydrogen-Ion Concentration; Nitric Oxide; Nitrites; Oxygen; Saliva; Thiocyanates; Time Factors

Saliva has a high nitrite concentration, derived from the enterosalivary recirculation of dietary nitrate, and is the main source of nitrite entering the acidic stomach. Acidification of nitrite in the presence of secondary amines or amides generates potentially carcinogenic N-nitroso compounds. The reaction is inhibited by ascorbic acid and catalysed by thiocyanate.. To determine whether there is intragastric regional variation in the chemical conditions promoting luminal nitrosation following nitrate ingestion.. Using microdialysis probes, we measured concentrations of nitrite, ascorbic acid, total vitamin C, and thiocyanate simultaneously in saliva, the distal oesophagus, cardia, and the proximal and distal stomach of 17 healthy volunteers before and following intragastric nitrate (2 mmol) administration.. The median pH in the distal oesophagus, cardia, and proximal and distal stomach were 7, 2.6, 1.9, and 1.7, respectively, before, and were similar following nitrate administration. Mean nitrite concentration in the distal oesophagus was similar to that of saliva, being 29.1 micro M and 36.7 micro M, respectively, before nitrate and increasing to 181.6 micro M and 203.3 micro M after nitrate ingestion. Within the stomach, mean (SEM) nitrite concentration following nitrate was higher in the cardia (45.5 (12.7) micro M) than in the mid (7.8 (3.1)) (p<0.01) or distal (0.8 (0.6)) (p<0.1) stomach, and ascorbic acid concentration was lower at the cardia (13.0 (6.1)) than in the mid (51 (19.2)) (p<0.02) or distal (86 (29)) (p<0.01) stomach. Consequently, the median ascorbic acid to nitrite ratio was lowest at the cardia (0.3) (p<0.01) versus the mid (7.8) or distal (40) stomach. Thiocyanate concentration was similar throughout the stomach.. The conditions favouring luminal generation of N-nitroso compounds from dietary nitrate are maximal at the most proximal cardia region of the acidic stomach and may contribute to the high incidence of mutagenesis at this site.

Topics: Adolescent; Adult; Ascorbic Acid; Cardia; Dialysis; Esophagus; Female; Humans; Hydrogen-Ion Concentration; Male; Middle Aged; Nitrates; Nitrites; Nitrosation; Saliva; Stomach; Thiocyanates

We have examined the suitability of microdialysis probes for examining nitrosative chemistry within localized regions of the lumen of the gastrointestinal tract. Chemical nitrosation occurs maximally at pH 2.5 in the presence of nitrite and thiocyanate and absence of ascorbic acid. Nitrite and thiocyanate are delivered into the stomach in saliva and ascorbic acid is secreted in gastric juice.. We used a benchtop model to reproduce the nitrosative chemistry occurring in the human upper GI tract and assessed the ability of the microdialysis probes to measure it.. The microdialysis probes were reliable at measuring nitrite ascorbic acid, total vitamin C and thiocyanate in both aqueous solutions and human gastric juice over the full range of intragastric pH, i.e. 1.5, 2.5, 3.5, 5.0 and 7.0. The probes were also reliable at measuring these chemicals under conditions simulating the active interaction between nitrite and ascorbic acid. Under such conditions with ascorbic acid in excess the probes gave a more accurate assessment of the nitrite level than that obtained by directly sampling the gastric juice. This was due to the probes not being subject to artefactual measurement of nitric oxide as nitrite. This was prevented by the rapid diffusion of nitric oxide through the probe collecting tube.. Microdialysis probes provide a reliable means of examining nitrosative chemistry within the lumen of the upper GI tract. In addition, they have the advantage of measuring this chemistry in very local regions and of simultaneously comparing the chemistry in different regions of the upper GI tract.

Topics: Ascorbic Acid; Esophagogastric Junction; Gastric Juice; Gastric Mucosa; Humans; Microdialysis; Models, Biological; Nitrites; Nitrosation; Reproducibility of Results; Thiocyanates

The uptake of L-ascorbate (vitamin C) and its oxidized form, dehydro-L-ascorbic acid (DHAA), was evaluated in brush border membrane vesicles isolated from adult human duodenum, jejunum and ileum. Ascorbate was taken up along the entire length of the small intestine with a threefold higher initial uptake rate in distal than proximal segments. Ascorbate uptake was Na(+)-dependent, potential-sensitive and saturable (K(m), 200 micromol/L), whereas DHAA transport involved facilitated diffusion (K(m), 800 micromol/L). Pharmacologic experiments were conducted to characterize further these transport mechanisms. DHAA uptake was not mediated by the fructose carrier GLUT5, the uridine transporter or the 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive anion exchanger of the apical membrane. DIDS and sulfinpyrazone, an inhibitor of the urate/lactate exchanger, both significantly reduced the initial rate of ascorbate uptake. Acidic pH inhibited ascorbate uptake, and this effect was not due to a transmembrane proton gradient. Increasing concentrations of glucose in the transport media also significantly inhibited ascorbate uptake, but no effect of glucose was seen when glucose internalization was blocked by phlorizin. Preloading the vesicles with glucose inhibited ascorbate uptake similarly, indicating that glucose interferes with the ascorbate transporter from the internal side of the membrane. The results of this study suggest that DHAA crosses the apical membrane by facilitated diffusion, whereas ascorbate transport is a Na(+)-dependent, electrogenic process modulated by glucose.

Topics: Adult; Ascorbic Acid; Azides; Biological Transport; Dehydroascorbic Acid; Glucose; Humans; Intestinal Absorption; Intestine, Small; Microvilli; Thiocyanates; Tissue Distribution

Erythrocyte and plasma antioxidant enzyme activities and antioxidants as well as concentrations of total sulfate and thiocyanate were estimated in a group of healthy subjects and three groups of smokers (cigarette smokers, mixed tobacco smokers, and miscellaneous tobacco smokers). Plasma vitamin E, uric acid, ascorbic acid, ceruloplasmin, and urinary total sulfate concentrations were decreased, whereas dehydroascorbate and urinary thiocyanate concentrations were elevated in the three groups of smokers in comparison to the corresponding levels of the control subjects. On the other hand, erythrocyte superoxide dismutase and catalase as well as plasma superoxide dismutase activities were elevated in subjects of the three groups of smokers compared with the corresponding activity in subjects of the control group. Plasma catalase activity is statistically unaffected by smoking, but blood glutathione peroxidase activities were decreased in the three groups of smokers in comparison with the corresponding levels of the control group. There were also statistically meaningful differences between mean values of the antioxidant concentrations and the activities of the antioxidant enzymes in most of the smokers groups.

Topics: Adult; Antioxidants; Ascorbic Acid; Catalase; Ceruloplasmin; Dehydroascorbic Acid; Erythrocytes; Glutathione Peroxidase; Humans; Male; Smoking; Sulfates; Superoxide Dismutase; Thiocyanates; Uric Acid; Vitamin E

We have modified an automated measurement system of urinary iodine (UI) and established a sensitive UI assay system by using ultraviolet (UV) digestion. The automated system is sensitive enough to detect concentrations of UI < 0.78 mumol/L (< 10 micrograms/dL) in a small volume of urine (500 microL). Sample throughput is > 30/h, including a water washing. The within-assay imprecision (CV) was < or = 10% in the UI range of 0.10-3.00 mumol/L; the between-assay CV was usually < or = 15% in the same range. Analytical recovery of iodine added to urine samples was consistently > 90%. The theoretical values were recovered when UV irradiation was used but not in its absence. High (supraphysiological) doses of thiocyanate or ascorbic acid, which are major interfering substances to the ceric-arsenious acid reaction, did not interfere with this system. The correlation between UI determined by this method and by the acid digestion method was linear (r = 0.994). For samples containing iodine at < 1.00 mumol/L, the correlation between values by both methods was still significant (r = 0.937). UI in an iodine-deficient area in Ukraine, measured by this system, ranged from 0.06 to 1.83 mumol/L (median 0.44 mumol/L, n = 95), significantly lower than in Japan (range 0.23-50.70 mumol/L, median 4.70 mumol/L, n = 84) and consistent with mild iodine deficiency. This modified automated assay system, therefore, is useful and applicable for screening UI in inhabitants of iodine-deficient areas.

Topics: Arsenates; Ascorbic Acid; Autoanalysis; Cerium; Humans; Iodine; Japan; Quality Control; Reference Values; Sensitivity and Specificity; Sulfates; Thiocyanates; Ukraine; Ultraviolet Rays

A mathematical model has been developed to estimate the rates of formation of nitrosamines and nitrosamides in the human stomach, under a variety of physiological and environmental conditions. The model combines a detailed description of the kinetics of N-nitrosation with mass balance equations which account for gastric emptying, dilution and absorption. The simulations were based on a typical schedule of dietary inputs, and included variations in gastric pH and in the volume of the stomach contents over a 24-h period. Consideration of these transient phenomena allowed a distinction to be made between amines or amides present in the diet and in gastric or salivary secretions. A comparison of the theoretical results with available data on the nitrosation of proline suggests that the model accurately predicts gastric rates of nitrosamine formation under control conditions, and correctly represents the strong catalytic effects of thiocyanate and the inhibitory effects of ascorbic acid or ascorbate ion. The results further suggest that nitrosoproline (NPro) excretion is not an accurate index of gastric nitrosation under physiological (low-dose) conditions, even when corrections are made for dietary intake of NPro. The predicted rates of formation of N-nitrosodimethylamine (NDMA), even for a diet high in dimethylamine, were found to be a factor of approximately 10(2) to 10(3) lower than published estimates of the dietary exposure to preformed NDMA. Thus, these findings do not support the hypothesis that gastric formation of NDMA from dietary dimethylamine poses a serious additional health risk. The results are presented in a graphical and tabular form which makes it possible to readily estimate the rates of formation of other nitrosamines or nitrosamides in the stomach, under various assumed conditions.

Topics: Ascorbic Acid; Gastric Mucosa; Gastrointestinal Contents; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Theoretical; Nitrates; Nitrites; Nitrosamines; Nitroso Compounds; Thiocyanates

To elucidate the factors governing the formation of N-nitrosamines in the stomach, the formation of N-nitrosoproline (NPro) was studied under controlled conditions, using a dog equipped with a Thomas cannula. Solutions containing nitrite, proline and in some cases ascorbic acid and/or SCN-, were infused into the stomach and samples taken to determine gastric [nitrite], [NPro], [ASC], [SCN-] and pH as functions of time. (Brackets indicate molar concentrations; ascorbic acid and ascorbate ion are denoted together by ASC.) Previous work showed that the rapid decline of [nitrite] in the stomach was due primarily to absorption. Additional experiments here in which ASC, proline or NPro were infused together with a non-absorbable marker, in the absence of nitrite, demonstrated that there was negligible absorption or secretion of these substances in the stomach. Thus, changes in [ASC] and [NPro] with time could be interpreted quantitatively in terms of rates of chemical reaction and dilution of the stomach contents. A mathematical model, based on mass balance equations for the various chemical species and chemical kinetic data obtained previously from in vitro studies, was developed for this purpose. The ability of ASC to inhibit nitrosation (by reaction with nitrite) was shown to be highly dependent on initial [ASC] and on the rate of entry of O2 into the stomach from blood. The rate of NPro formation in the absence of ASC and SCN-, the inhibitory effects on nitrosation of ASC and the catalytic effects of SCN-, were all accurately predicted by the mathematical model. This suggests that similar models may prove useful in estimating rates of intragastric N-nitrosation reactions in humans, under various assumed conditions.

Topics: Animals; Ascorbic Acid; Dogs; Female; Gastric Mucosa; Nitrites; Nitrogen Oxides; Nitrosamines; Oxygen; Proline; Thiocyanates

Plasma levels of vitamins A, C, and E, selenium, and carotenes were determined in 125 male cigarette smokers and 125 age- and race-matched nonsmokers. The smokers had a mean daily consumption of 30.6 cigarettes and a cumulative consumption of 22.8 pack years. Plasma levels of vitamin C and total carotenes were significantly (p less than 0.05) lower in smokers than those of nonsmokers, while levels of vitamin A, selenium, and vitamin E were not significantly different between these two groups. Similar results were found when only those subjects not taking any form of dietary supplements were included for analysis. Except for negative correlation between vitamin A and pack-year, no significant correlates were observed between plasma levels of these micronutrients and indices of smoking status or cigarette consumption in smokers. These data suggest that chronic cigarette smoking is associated with depressed levels of plasma vitamin C and carotenes; however, the relationship between smoking and these plasma micronutrients is still unclear.

Topics: Adult; Ascorbic Acid; Blood Cell Count; Carboxyhemoglobin; Carotenoids; Humans; Male; Selenium; Smoking; Thiocyanates; Vitamin A; Vitamin E

Dehydroascorbic acid (DHA), the reversibly oxidized form of vitamin C, was taken up much more rapidly than L-glucose into membrane vesicles prepared from the maternal face of the human placental syncytiotrophoblast. DHA uptake was sensitive to inhibition by cytochalasin B and was independent of a sodium concentration gradient. At equilibrium, the concentration of DHA in the vesicles did not exceed that of the medium. DHA and the D-glucose analogue, 3-O-methyl-D-glucose (3-O-MG) appeared to compete with one another for the transporter. The 3-O-MG and DHA inhibitory constants were indistinguishable. Vesicles loaded with a high concentration of 3-O-MG and suspended in low 3-O-MG displayed a marked, transitory enhancement of DHA, but not L-glucose uptake. These findings suggest that DHA is taken into the first cellular boundary of the placenta between maternal and fetal circulations by the sodium-independent monosaccharide transporter. In contrast to DHA, L-ascorbic acid, the reversibly reduced form of vitamin C, was taken into these vesicles much more slowly. This uptake was not affected by cytochalasin B nor by a sodium concentration gradient; it appeared to occur by simple diffusion.

Topics: 3-O-Methylglucose; Ascorbic Acid; Chlorides; Dehydroascorbic Acid; Female; Humans; Methylglucosides; Monosaccharide Transport Proteins; Placenta; Pregnancy; Sodium; Thiocyanates

A method for the determination of cyanide and thiocyanate in blood plasma and red cells of humans was established. It involved high-performance liquid chromatography and fluorometric detection by the König reaction. Calibration curves for cyanide and thiocyanate were linear in the range 1-200 pmol and 2-300 pmol, respectively. Clean-up methods for the determination of cyanide and thiocyanate in red cells were also developed. These methods were applied for the determination of cyanide and thiocyanate in the blood of smokers and non-smokers.

Topics: Ascorbic Acid; Barbiturates; Chemical Phenomena; Chemistry; Chloramines; Chromatography, High Pressure Liquid; Cyanides; Erythrocytes; Humans; Kinetics; Pyridines; Smoking; Spectrometry, Fluorescence; Thiocyanates; Tosyl Compounds