Edwin L. Thomas

Preparation and Characterization of Chloramines

Publisher Summary This chapter discusses the preparation and characterization of chloramines. Chloramines represent an important class of leukocyte oxidants and contribute to oxidative microbicidal, cytotoxic and cytolytic activities, the chemical modification of regulatory substances, and the uptake and metabolism of nitrogen compounds. These are products of the reaction of hypochlorous acid (HOCl) or other chlorinating agents with primary and secondary amines. The production of HOCl by leukocytes is the result of peroxidase-catalyzed oxidation of chloride (Cl – ) by hydrogen peroxide (H 2 O 2 ). HOCl is in equilibrium with hypochlorite (OCl – ) and chlorine (Cl 2 ). The most abundant N-moieties available for reaction with HOCl in biological systems are primary amino groups, such as taurine, polyamines, amino sugars, lysine residues, and protein amino termini. Leukocyte peroxidases catalyze the oxidation of bromide (Br – ), iodide (I – ), thiocyanate (SCN – ), and Cl – . N–Cl derivatives are used within minutes of preparation to avoid decomposition or rearrangement. However, most are stable at 4° for hours or days at the appropriate pH.

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.

Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase

Conditions were optimized for measuring the activity of myeloperoxidase (MPO) and the eosinophil peroxidase (EPO) with tetramethylbenzidine (TMB) as the substrate. Detergents caused a small increase in the measured activity of the purified enzymes and were required when isolated neutrophils or eosinophils were assayed. Sharp concentration optima were observed with both ionic and non-ionic detergents. Activity was also influenced by halide ions. Bromide or iodide caused up to a 7-fold increase in EPO activity and a 1.5-fold increase in MPO activity. The effect of bromide is notable because the bromide-containing detergent CETAB is often used to extract the enzymes for assay and purification. Stimulation by bromide or iodide was consistent with peroxidase-catalyzed oxidation of the halides to hypohalous acids (HOBr and HOI), which oxidized TMB. MPO catalyzes the oxidation of chloride to hypochlorus acid (HOCl), which also oxidized TMB, but chloride up to 20 mM had little effect on the assay. Both MPO and EPO catalyze thiocyanate oxidation, but the product (HOSCN) was a poor oxidant for TMB, and thiocyanate inhibited the measured activities. Stimulation by bromide or iodide could be used to facilitate detection of EPO and to distinguish between MPO and EPO. Activities could also be distinguished based on the greater sensitivity of EPO to inhibition by thiocyanate, azide, aminotriazole, and dapsone. Methods reported here may prove useful for measuring leukocyte influx into inflamed tissues, detecting MPO or EPO deficiencies, and measuring enzyme synthesis and secretion.

Taurine and hypotaurine content of human leukocytes

Taurine (T) was reported to be at high concentrations in human leukocytes. It was proposed that T is a scavenger for chlorinated oxidants produced by the myeloperoxidase system of monocytes and neutrophils. Hypotaurine (HT) would be a more effective scavenger, and HT could also detoxify products of bromide or iodide oxidation produced by the eosinophil peroxidase system. Methods previously used to measure T in leukocytes might oxidize HT to T or fail to separate T and HT. Therefore, we examined T and HT content, uptake, and biosynthesis in isolated blood cells and cultured tumor cells derived from hematopoietic/lymphoid cells. Platelets and all leukocytes including monocytes, lymphocytes, neutrophils, and eosinophils had high T levels (10–20 mM), and all except eosinophils had substantial HT levels (0.3–1 mM). Intracellular levels were 500‐times higher than in plasma. Erythrocytes were the only blood cells with low levels of both T and HT. Tumor cells from lymphoid (CCRF‐CEM) and myeloid (HL‐60, K‐562, RWLeu4, HEL) lineages took up and concentrated T and HT from the bovine calf serum in the culture medium, and intracellular levels were similar to those in leukocytes. When cells were cultured in HT‐supplemented media, HT almost completely replaced T, and HT was not converted to T. Levels of T were not raised by culturing cells with possible precursors, but HT levels were raised when cysteine sulfinic acid was present. Washed tumor cells took up T and HT by way of a β‐amino acid transport system, but uptake by leukocytes was very low. Therefore, leukocytes may acquire T and HT by active uptake rather than biosynthesis, and uptake may be completed during differentiation in the bone marrow. Though HT is low relative to T, HT levels may be sufficient to protect leukocytes from toxic oxidants.

Cytotoxicity of Chloramines

Publisher Summary This chapter describes the cytotoxicity of chloramines. The binding of ligands to the membrane-surface receptors of neutrophilic leukocytes stimulates the reduction of oxygen (O 2 ) to superoxide (O 2 – ) and the secretion of cytoplasmic granule components including myeloperoxidase into the intracellular phagolysosome compartment and the extracellular medium. When neutrophils are incubated with target cells, nitrogen–chlorine (N–Cl) derivatives react with both types of cells so that results are influenced by the neutrophil:target cell ratio and the reactivity of N–Cl derivatives toward neutrophil and target cell components. Methods for evaluating the cytotoxicity of N–Cl derivatives consist of measuring oxidation, chlorination, or other chemical modifications of cell components and measuring lysis or the failure of specific structural or functional components of cells. Stimulated neutrophils undergo a progressive myeloperoxidase-dependent inhibition of O 2 and glucose metabolism. This inhibition results from the reaction of hypochlorous acid (HOCl) with ammonium (NH 4 + ) produced by the cells. Monochloramine (NH 2 Cl) is produced as a result and inactivates neutrophil components.

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. J. Leukoc. Biol. 61: 293–302; 1997.

Hypothiocyanite Ion: Detection of the Antimicrobial Agent in Human Saliva

The median concentration of hypothiocyanite (OSCN-) in freshly collected whole saliva was 10 μM. The OSCN- concentration increased to a median value of 36 μM during incubation for one h at 37° 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.

Peroxidase Antimicrobial System of Human Saliva: Hypothiocyanite Levels in Resting and Stimulated Saliva

The antimicrobial oxidizing agent hypothiocyanite ion (OSCN-) was measured in resting (drooling) and stimulated (expectorated) whole saliva. Stimulation of the saliva flow rate resulted in a rapid decrease in OSCN- concentration, whereas the thiocyanate ion (SCN—) concentration and peroxidase activity were increased. The decrease in OSCN — levels was greater than could be accounted for by dilution of the whole saliva volume. Assuming that the antimicrobial activity of the salivary peroxidase system is proportional to OSCN- concentration, this system may be more effective in resting saliva than in stimulated saliva.

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 (H 2O2) in the presence of oxygen and a divalent cation, and 3) heatstable 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 H202 by the saliva sediment. Production of H202 was greater in the presence of glucosamine than of neutral sugars. Also, the ratio of OSCN- accumulation to H202 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.

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

Abstract The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [ 3 H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of 32 P i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μ m and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.