Myeloperoxidase-catalyzed chlorination of histamine by stimulated neutrophils.

To investigate neutrophil interactions with mediators released by mast cells at sites of inflammation, stimulated neutrophils were incubated with histamine. No accumulation of chlorinated histamine derivatives was detected in the medium. Instead, histamine inhibited the formation of chloramine derivatives of other amines. Incubation with radiolabeled histamine resulted in rapid uptake of label into the cells, and most of the label could be extracted and recovered as histamine. About 3% of the label taken up was incorporated into acid-precipitable forms. Uptake depended on myeloperoxidase (MPO)-catalyzed formation of chlorinating agents. Uptake was promoted by adding MPO and blocked by the MPO inhibitor dapsone, catalase, scavengers for hypochlorous acid and chloramines, or in a low-chloride medium, but not by histamine receptor antagonists. Incubation of histamine with MPO, hydrogen peroxide, and chloride resulted in formation of mono- and dichloramine derivatives of the primary amino group. Above pH 7.0, the chloramines were primarily in uncharged, lipophilic forms as indicated by partitioning into organic solvents. Histamine is a cation at neutral pH, but chlorination eliminated the charge on the amino group and shifted the pKa of the imidazole ring, resulting in formation of neutral histamine-chloramines. Incubation of neutrophils or other blood cells with radiolabeled histamine-chloramines resulted in rapid uptake of label, indicating membrane permeation by the uncharged, lipid-soluble forms. Incubation with labeled histamine-dichloramine also resulted in acid-precipitable incorporation. The results indicate that MPO-catalyzed chlorination of histamine could modulate histamine activity, tissue distribution, and metabolism at sites of inflammation.

Secretion and inactivation of myeloperoxidase by isolated neutrophils.

Neutrophils prevent infection by ingesting and killing microorganisms but oxidants and proteases released by neutrophils damage host tissues. Our aim was to identify factors that regulate oxidant production by the enzyme myeloperoxidase (MPO) following secretion of MPO into the medium. Cells stimulated with phorbol myristate acetate (PMA) or opsonized zymosan particles secreted MPO and released superoxide free radicals (.O2-). Dismutation of .O2- produced hydrogen peroxide (H2O2) and MPO catalyzed the oxidation of chloride ion by H2O2 to produce the toxic oxidant hypochlorous acid (HOCl). Adding the enzyme superoxide dismutase (SOD) to increase the rate of conversion of .O2- to H2O2 had pH-dependent effects on HOCl production. From pH 6.0 to 7.4, SOD promoted HOCl production by up to 500% but SOD had no effect at pH 7.6 and inhibited by 40 +/- 10% at pH 7.8. In further experiments at pH 7.0, MPO activity in the cells decreased by 25 +/- 2 and 44 +/- 4% during 1-h incubations with PMA and zymosan. Only 1 +/- 0 and 3 +/- 1% of the total activity was found in the medium, indicating that most of the secreted MPO was inactivated. Loss of activity was not accompanied by proteolytic destruction of the MPO protein, which was measured with anti-MPO antibodies. SOD raised the amount of active MPO in the medium two- to sevenfold, but adding deferoxamine to chelate iron or adding ferric ion had no effect. The ionophore A23187 was as effective as zymosan as a stimulus for MPO secretion but .O2- production by ionophore-stimulated cells was less than 4% of that of PMA- or zymosan-stimulated cells and most of the secreted MPO was found active in the medium. When PMA-stimulated cells were incubated with purified MPO, the added MPO activity was lost from the medium. Binding or proteolysis did not account for loss of activity as indicated by recovery of added radioiodinated MPO from the medium. The visible absorption spectrum of MPO was lost, indicating destruction of the iron-containing prosthetic group. Loss of activity and loss of the MPO spectrum were blocked by SOD but not by deferoxamine or catalase. The results indicate that, in the physiological pH range, inactivation of MPO in the medium suppressed HOCl production. Inactivation required O2- but not HOCl, H2O2, or free iron. Inactivation of secreted MPO may limit MPO-mediated damage to host tissues by stimulated neutrophils.

Oxidation of bromide by the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. Formation of bromamines.

Myeloperoxidase and eosinophil peroxidase catalyzed the oxidation of bromide ion by hydrogen peroxide (H2O2) and produced a brominating agent that reacted with amine compounds to form bromamines, which are long-lived oxidants containing covalent nitrogen-bromine bonds. Results were consistent with oxidation of bromide to an equilibrium mixture of hypobromous acid (HOBr) and hypobromite ion (OBr-). Up to 1 mol of bromamine was produced per mole of H2O2, indicating that bromamine formation prevented the reduction of HOBr/OBr- by H2O2 and the loss of oxidizing and brominating activity. Bromamines differed from HOBr/OBr- in that bromamines reacted slowly with H2O2, were not reduced by dimethyl sulfoxide, and had absorption spectra similar to those of chloramines, but shifted 36 nm toward higher wavelengths. Mono- and di-bromo derivatives (RNHBr and RNHBr2) of the beta-amino acid taurine were relatively stable with half-lives of 70 and 16 h at pH 7, 37 degrees C. The mono-bromamine was obtained with a 200-fold excess of amine over the amount of HOBr/OBr- and the di-bromamine at a 2:1 ratio of HOBr/OBr- to the amine. In the presence of physiologic levels of both bromide (0.1 mM) and chloride (0.1 M), myeloperoxidase and eosinophil peroxidase produced mixtures of bromamines and chloramines containing 6 +/- 4% and 88 +/- 4% bromamine. In contrast, only the mono-chloramine derivative (RNHCl) was formed when a mixture of hypochlorous acid (HOCl) and hypochlorite ion (OCl-) was added to solutions containing bromide and excess amine. The rapid formation of the chloramine prevented the oxidation of bromide by HOCl/OCl-, and the chloramine did not react with bromide within 1 h at 37 degrees C. The results indicate that when enzyme-catalyzed bromide or chloride oxidation took place in the presence of an amine compound at 10 mM or higher, bromamines were not produced in secondary reactions such as the oxidation of bromide by HOCl/OCl- and the exchange of bromide with chlorine atoms of chloramines. Therefore, the amount of bromamine produced by myeloperoxidase or eosinophil peroxidase was equal to the amount of bromide oxidized by the enzyme. Bromide was preferred over chloride as the substrate for both enzymes.

Leukocyte myeloperoxidase and salivary lactoperoxidase: identification and quantitation in human mixed saliva.

Human salivary lactoperoxidase (HS-LP) is synthesized and secreted by the salivary glands, whereas myeloperoxidase (MPO) is found in PMN leukocytes, which migrate into the oral cavity at gingival crevices. HS-LP levels vary with changes in salivary gland function, but increased numbers of MPO-containing leukocytes indicate infection or inflammation of oral tissues. To determine the contribution of each enzyme to the peroxidase activity of mixed-saliva samples, activity was assayed at pH 5.4 with tetramethylbenzidine as the substrate, with and without the inhibitor dapsone (4,4'-diaminodiphenylsulfone). Dapsone blocked the activity of HS-LP but not MPO. The enzymes were also separated and partially purified from the soluble portion of saliva samples and from detergent extracts of the saliva sediment. Chromatographic properties of the proteins were similar to those of LP from bovine milk (BM-LP) and MPO from human leukocytes. The identity and amounts of the enzymes were confirmed by the absorption spectra and by immunoblotting with antibodies to BM-LP and human MPO. Eosinophil peroxidase (EPO), a distinct enzyme found in eosinophilic leukocytes, was not detected by chromatography or with antibodies to human EPO. On average, 75% of the activity in samples from normal donors was due to MPO and 25% to HS-LP. When corrected for the lower specific activity of HS-LP in this assay, the average amount of MPO (3.6 micrograms/mL) was twice the amount of HS-LP (1.9 micrograms/mL). The amount of MPO corresponded to 1 x 10(6) PMN leukocytes/mL of saliva. The enzymes were distributed differently: Eighty-nine percent of the HS-LP was in the soluble portion of saliva, and 78% of the MPO was in the sediment, which contained 51% of the total activity. In contrast to results obtained with PMN leukocytes from blood, detergent was not required for MPO activity to be measured in saliva, indicating that the enzyme was accessible to peroxidase substrates. The results indicate that MPO is responsible for a large portion of peroxidase-catalyzed reactions in mixed saliva. The unique function of HS-LP may be carried out within the salivary glands, prior to secretion into the oral cavity.

Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci.

In secreted fluids, the enzyme lactoperoxidase (LP) catalyzes the oxidation of thiocyanate ion (SCN-) by hydrogen peroxide (H2O2), producing the weak oxidizing agent hypothiocyanite (OSCN-), which has bacteriostatic activity. However, H2O2 has antibacterial activity in the absence of LP and thiocyanate (SCN-). Therefore, LP may increase antibacterial activity by using H2O2 to produce a more effective inhibitor of bacterial metabolism and growth, or LP may protect bacteria against the toxicity of H2O2 by converting H2O2 to a less-potent oxidizing agent. To clarify the role of LP, the antibacterial activities of H2O2 and the LP-H2O2-SCN- system were compared by measuring loss of viability and inhibition of bacterial metabolism and growth. The relative toxicity of H2O2 and the LP system to oral streptococci was found to depend on the length of time that the bacteria were exposed to the agents. During incubations of up to 4 h, the LP system was from 10 to 500 times more effective than H2O2 as an inhibitor of glucose metabolism, lactic acid production, and growth. However, if no more H2O2 was added, the concentration of the inhibitor OSCN- fell because of slow decomposition of OSCN-, and when OSCN- fell below 0.01 mM, the bacteria resumed metabolism and growth. In contrast, the activity of H2O2 increased with time. H2O2 persisted in the medium for long periods of time because H2O2 reacted slowly with the bacteria and streptococci lack the enzyme catalase, which converts H2O2 to oxygen and water. After 24 h of exposure, H2O2 was as effective as the LP system as an inhibitor of metabolism. H2O2 also caused a time-dependent loss of viability, whereas the LP system had little bactericidal activity. The concentration of H2O2 required to kill half the bacteria within 15 s was 1.8 M (6%) but fell to 0.3 M (1%) at 2 min, to 10 mM (0.03%) at 1 h, and to 0.2 mM (0.0007%) with a 24-h exposure. The results indicate that if high levels of H2O2 can be sustained for long periods of time, H2O2 is an effective bactericidal agent, and the presence of LP and SCN- protects streptococci against killing by H2O2. Nevertheless, the combination of LP, H2O2, and SCN- is much more effective than H2O2 alone as an inhibitor of bacterial metabolism and growth.

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils.

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.

Mutagenic activity of chloramines.

Mutagenesis by chloramines and hypochlorous acid (HOCl) was studied to determine whether these agents could contribute to the mutagenic and potentially carcinogenic activity of stimulated leukocytes and whether environmental exposure to these agents is a cause for concern. Mutagenic activity was measured using the S. typhimurium TA97a, TA100 and TA102 tester strains. Because chloramines and HOCl are bactericidal, react rapidly with cell components, and can destroy the histidine and biotin required for the mutagenesis assay, activity can't be compared directly with that of less toxic or reactive agents. Nevertheless, chloramines were mutagenic when tested under appropriate conditions. TA100 was the most sensitive strain, and the most active mutagens were lipophilic dichloramines (RNCl2) including derivatives of histamine, ethanolamine and putrescine. Lipophilic monochloramines (RNHCl) such as histamine-monochloramine and NH2Cl were less active. Hydrophilic chloramines such as taurine-chloramines had low activity, and HOCl was inactive. The metabolic state of the bacteria was critical. Chloramines were mutagenic when added to bacteria with glucose at 37 degrees C, but killing predominated when chloramines were added at 4 degrees C or 25 degrees C, or at 37 degrees C without glucose. Production of chloramines and HOCl by leukocytes in vivo could contribute to the association of chronic inflammation and cancer as a result of: (1) the entry of membrane-permeable chloramines into normal cells followed by attack on intracellular components including DNA, and (2) the production of secondary mutagens such as compounds with carbonyl groups or carbon-chlorine bonds. On the other hand, chlorination of water supplies is perhaps more likely to destroy than create mutagens, and chloramines from the environment are unlikely to penetrate the skin and mucous membranes.

Evidence for a role of taurine in the in vitro oxidative toxicity of neutrophils toward erythrocytes.

Production of hydrogen peroxide and secretion of myeloperoxidase by stimulated neutrophils resulted in myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl), the reaction of HOCl with taurine to yield taurine monochloramine (TauNHCl), and accumulation of TauNHCl in the extracellular medium. When erythrocytes were present, the yield of TauNHCl was lower as the result of uptake of TauNHCl into erythrocytes. The zwitterion taurine was not taken up, but the anion TauNHCl and other anionic oxidants including taurine dichloramine (TauNCl2) and L-alanine chloramines were transported into erythrocytes by the anion-transport system. Oxidation of intracellular components such as glutathione (GSH) by taurine chloramines resulted in reduction of the chloramines and trapping of taurine within erythrocytes. At high oxidant:erythrocyte ratios, TauNHCl also oxidized hemoglobin (Hb) and depleted ATP, but caused little lysis. TauNCl2 was much more effective as a lytic agent. At low oxidant:erythrocyte ratios, the chloramines caused net loss of GSH when no glucose was provided, but Hb was not oxidized and GSH content returned to normal when glucose was added. Therefore, anionic chloramines may mediate oxidative toxicity when the neutrophil:erythrocyte ratio is high. Under more physiologic conditions, chlorination of taurine by neutrophils and the uptake and reduction of TauNHCl by erythrocytes prevents accumulation of oxidants and may protect blood cells, plasma components, and tissues against oxidative toxicity.

Chlorination of endogenous amines by isolated neutrophils. Ammonia-dependent bactericidal, cytotoxic, and cytolytic activities of the chloramines.

Isolated human neutrophilic leukocytes were stimulated to produce hydrogen peroxide (H2O2) and to secrete cytoplasmic granule components including myeloperoxidase into the medium. Myeloperoxidase catalyzed the oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), which reacted with endogenous nitrogen compounds to yield derivatives containing nitrogen-chlorine (N-Cl) bonds. Compounds available for reaction with HOCl were ammonia (NH+4), taurine, alpha-amino acids, and granule proteins and peptides that were released into the medium. A portion of the N-Cl derivatives formed under these conditions accumulated in the extracellular medium. These long lived oxidizing agents were characterized as hydrophilic, low molecular weight, mono-N-chloramine (RNHCl) derivatives based on their absorption spectrum, ability to oxidize 5-thio-2-nitrobenzoic acid and to chlorinate ammonia (NH+4), and behavior upon ultrafiltration, gel chromatography, and extraction with organic solvents. The RNHCl derivatives were of low toxicity, but reacted with NH+4 to yield the lipophilic oxidizing agent monochloramine (NH2Cl). Therefore, the addition of NH+4 conferred bactericidal, cytotoxic, and cytolytic activities on the RNHCl derivatives. The results indicate that taurine and other neutrophil amines protect neutrophils and other cells against oxidative attack by acting as a trap for HOCl and by competing with endogenous NH+4 for reaction with HOCl. However, the RNHCl derivatives act as a reserve of oxidizing equivalents that is converted to a toxic form when an increase in NH+4 concentration favors formation of NH2Cl.

Role of monochloramine in the oxidation of erythrocyte hemoglobin by stimulated neutrophils.

Stimulation of the oxygen (O2) metabolism of isolated human neutrophilic leukocytes resulted in oxidation of hemoglobin of autologous erythrocytes without erythrocyte lysis. Hb oxidation could be accounted for by reduction of O2 to superoxide (O-2) by the neutrophils, dismutation of O-2 to yield hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), the reaction of HOCl with endogenous ammonia (NH+4) to yield monochloramine ( NH2Cl ), and the oxidative attack of NH2Cl on erythrocytes. NH2Cl was detected when HOCl reacted with the NH+4 and other substances released into the medium by neutrophils. The amount of NH+4 released was sufficient to form the amount of NH2Cl required for the observed Hb oxidation. Oxidation was increased by adding myeloperoxidase or NH+4 to increase NH2Cl formation. Due to the volatility of NH2Cl , Hb was oxidized when neutrophils and erythrocytes were incubated separately in a closed container. Oxidation was decreased by adding catalase to eliminate H2O2, dithiothreitol to reduce HOCl and NH2Cl , or taurine to react with HOCl or NH2Cl to yield taurine monochloramine . NH2Cl was up to 50 times more effective than H2O2, HOCl, or taurine monochloramine as an oxidant for erythrocyte Hb, whereas HOCl was up to 10 times more effective than NH2Cl as a lytic agent. NH2Cl contributes to oxidation of erythrocyte components by stimulated neutrophils and may contribute to other forms of neutrophil oxidative cytotoxicity.

Myeloperoxidase-dependent effect of amines on functions of isolated neutrophils.

Isolated neutrophilic leukocytes were incubated with primary amines and related nitrogenous compounds. Stimulation of neutrophil oxygen (O2) metabolism with phorbol myristate acetate or opsonized zymosan resulted in production of hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (C1-) to hypochlorous acid (HOC1), and the reaction of HOC1 with the added compounds to yield nitrogen-chlorine (N-C1) derivatives. Formation of N-C1 derivatives of low lipid solubility resulted in accumulation of the derivatives in the extracellular medium. These oxidizing agents were identified and measured on the basis of their absorption spectra and their ability to oxidize 5-thio-2-nitrobenzoic acid to the disulfide form. The yield of N-Cl derivatives was in the order: taurine greater than Tris greater than spermidine greater than spermine greater than glucosamine greater than putrescine greater than guanidinoacetate. Accumulation of N-C1 derivatives was also observed in the absence of added amines, owing to the reaction of HOC1 with endogenous taurine and other amines that were released from the cells into the medium. In the presence of compounds that yield lipophilic N-C1 derivatives, little or no accumulation of oxidizing agents was observed. Instead, these compounds inhibited the accumulation of N-C1 derivatives that was obtained with taurine, and their effect was competitive with taurine. Inhibition was in the order: methylamine greater than ethanolamine greater than phenylethylamine greater than p-toluenesulfonamide greater than ammonia greater than guanidine. Formation of lipophilic N-C1 derivatives also resulted in inhibition of O2 uptake and glucose metabolism. Inhibition was prevented by adding catalase to eliminate H2O2, dapsone to inhibit myeloperoxidase, taurine to compete for reaction with HOC1, or compounds that are rapidly oxidized by HOC1 or N-C1 derivatives, to reduce these oxidizing agents. The results indicate that: (a) formation of N-C1 derivatives that do not penetrate biological membranes can protect leukocytes against the cytotoxicity of HOC1 and lipophilic N-C1 derivatives, and (b) formation of membrane-permeable N-C1 derivatives in the absence of target cells or readily oxidized substances results in oxidative attack by the N-C1 derivatives on leukocyte components and inhibition of leukocyte functions.

Myeloperoxidase-catalyzed incorporation of amines into proteins: role of hypochlorous acid and dichloramines.

Myeloperoxidase-catalyzed oxidation of chloride (Cl-) to hypochlorous acid (HOCl) resulted in formation of mono- and dichloramine derivatives (RNHCl and RNCl2) of primary amines. The RNCl2 derivatives could undergo a reaction that resulted in incorporation of the R moiety into proteins. The probable mechanism was attack of RNCl2 or an intermediate formed in the decomposition of RNCl2 on histidine, tyrosine, and cystine residues and on lysine residues at high pH. Incorporation of radioactivity from labeled amines into stable, high molecular weight derivatives of proteins was measured by acid or acetone precipitation and by gel chromatography and electrophoresis. Whereas formation of RNCl2 was favored at low pH, the subsequent incorporation reaction was favored at high pH. Up to several hours were required for the maximum amount of incorporation, which was less than 10% of the label in RNCl2. For the amines tested, incorporation was in the order histamine greater than 1,2-diaminoethane greater than putrescine greater than taurine greater than lysine greater than glucosamine greater than leucine greater than methylamine. Initiation of the reaction required HOCl, and oxidized forms of bromide, iodide, or thiocyanate did not substitute. Inhibitors of incorporation fell into three classes. First, ammonia or amines competed with the labeled amine for reaction with HOCl, so that larger amounts of HOCl were required. Second, readily oxidized substances such as sulfhydryl or diketo compounds or thioethers (methionine) reduced RNCl2. Third, certain compounds competed with protein as the acceptor for the incorporation reaction. The amount required to block incorporation into protein depended on protein concentration. Among these inhibitors were imidazole compounds (histidine), phenols (tyrosine), and disulfides (glutathione disulfide, GSSG). Low yields of derivatives of histidine, tyrosine, and GSSG were detected by thin-layer chromatography. Acid-precipitable derivatives were obtained by reacting RNCl2 with polyhistidine or polytyrosine, and to a lesser extent with polylysine at high pH, but not with other poly(amino acids). Precipitable derivatives were also obtained by incubating MPO-containing extracts from leukocyte granules with hydrogen peroxide, Cl-, and labeled amines. The extracts were found to have a high content of substances with primary amino groups, which competed for incorporation. The results account for oxidative incorporation of amines into proteins in leukocytes and provide evidence that HOCl and nitrogen-chlorine (N-Cl) derivatives are formed in these cells. The characteristics of the incorporation reaction suggest that it would not contribute significantly to the antimicrobial activity of myeloperoxidase (MPO). Nevertheless, the reaction may provide a sensitive method for studying MPO action in vivo.

Peroxidase antimicrobial system of human saliva: requirements for accumulation of hypothiocyanite.

Human saliva was fractionated to determine the components required for production and accumulation of the antimicrobial oxidizing agent, hypothiocyanite ion (OSCN-). The required components were: 1) peroxidase activity and thiocyanate ion (SCN-), 2) the saliva sediment, which produced hydrogen peroxide (H2O2) in the presence of oxygen and a divalent cation, and 3) heat-stable factors of the saliva supernatant. The supernatant factors were separated into high- and low-mol wt fractions. The high-mol wt fraction contained both peptide and carbohydrate, and its activity was partially inhibited by proteolytic treatment. The low-mol wt fraction contained carbohydrate and could be replaced by a number of mono- and di-saccharides. Glucosamine and N-acetyl glucosamine were the most effective, whereas neutral sugars such as sucrose were less effective. Sucrose competed with glucosamine, so that lower levels of OSCN- were obtained with increasing amounts of sucrose. The sugars stimulated production of H2O2 by the saliva sediment. Production of H2O2 was greater in the presence of glucosamine than of neutral sugars. Also, the ratio of OSCN- accumulation to H2O2 production was greater in the presence of glucosamine. The results suggest that peroxidase-mediated antimicrobial activity is modulated by the carbohydrate composition of whole saliva and by certain protein and glycoprotein components.

Hypothiocyanite ion: detection of the antimicrobial agent in human saliva.

The median concentration of hypothiocyanite (OSCN-) in freshly collected whole saliva was 10 microM. The OSCN- concentration increased to a median value of 36 microM during incubation for one h at 37 degrees in vitro. This increase was partially inhibited by adding certain sugars (especially sucrose). The results suggest that OSCN- is a naturally occurring component of human saliva. Also, dietary carbohydrate may inhibit OSCN- accumulation and antimicrobial action in saliva.