[A modified method for labelling proteins with 125I and chloramine-T as oxidative agents. Advantages of a low chloramine-T: protein ratio (author's transl)].
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Chloramines↗
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[Kinetics of bacteriophage OX 174 disinfection by chloramin B, activated chloramin B, and Dikonit (author's transl)].
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Ammonium Chloride↗
Microtiter plate assay for phagocyte-derived taurine-chloramines.
Despite their potential importance, the role of phagocyte-derived chloramines ("long-lived oxidants") has not yet been investigated in inflammatory or infectious diseases. We have developed a sensitive spectrophotometric microtiter plate assay for chloramines based on their capacity to oxidize potassium iodide (KI). Consistent levels of endogenous chloramines were detected in normal human polymorphonuclear neutrophil (PMN) supernatants after stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Exogenous taurine strongly enhanced chloramine secretion and was used to quantify the chlorinating potential of PMN. Taurine-chloramines were also detectable in monocyte supernatants, although in smaller amounts. The specificity of the KI assay was assessed both in terms of effect of compounds inhibiting (KBr) or interacting with (sodium azide and catalase) chloramine formation and by showing that PMN from patients with chronic granulomatous disease, due to a hereditary lack of oxidative response capacity, were unable to produce chloramines. Taurine-chloramine levels secreted by PMA (but not zymosan)-stimulated PMN were closely related to the cellular luminol-amplified chemiluminescence (CL) responses although the CL assay failed to detect chloramines in PMN supernatants. We consider that this KI assay should be of use in studying the role of long-lived phagocyte-derived oxidants in clinical medicine.
Chloramines↗
Abolition with chloramine-T of inactivation in barnacle muscle fibers results in stimulation of the ouabain-insensitive sodium efflux.
The hypothesis that chloramine-T stimulates the basal Na+ efflux in barnacle fibers as the result of the entry of trigger Ca2+ into the myoplasm from the bathing medium was examined in this study. Two reasons for doing so can be given. One is that the oxidant is known to abolish inactivation in sodium and potassium channels. The other is that L-type Ca2+ channels are present in barnacle fibers, and an increase in internal free Ca2+ in these fibers is known to stimulate the Na+ efflux, particularly in ouabain-poisoned fibers. The results of the experiments are as follows: (i) Chloramine-T exerts a biphasic effect on the Na+ efflux: inhibition is followed by stimulation, the threshold concentration being 10(-5) M. This is also found to be the threshold concentration for shortening of these fibers. (ii) The kinetics of the inhibitory effect resemble those of ouabain. (iii) Ouabain is without effect on the stimulatory phase caused by chloramine-T. (iv) Application of chloramine-T after the full effect of 10(-4) M-ouabain is reached elicits solely a stimulatory response. (v) The dose-response curves for the stimulatory action of chloramine-T in unpoisoned and ouabain-poisoned fibers are alike except that the threshold concentration is less than 10(-5) M in poisoned fibers. (vi) Basal light emission from unpoisoned and ouabain-poisoned fibers loaded with the photoprotein, aequorin, some 60 min beforehand increases as soon as they are exposed to 10(-4) M chloramine-T. The response recorded in unpoisoned fibers is monophasic and usually transitory, whereas it is multiphasic and usually sustained in ouabain-poisoned fibers. (vii) The dose-response curve for chloramine-T shows a shift to the left in poisoned fibers. (viii) The magnitude of the rise in light emission depends on the external Ca2+ concentration. A rise fails to take place in the nominal absence of external Ca2+. Taken together, these results support the above hypothesis that chloramine-T causes the entry of trigger Ca2+ into the myoplasm from the outside and provide evidence that stimulation of the Na+ efflux is associated not only with this event but also with a reduced Na+ gradient resulting from inhibition of the membrane Na+/K(+)-ATPase system by the oxidant. Thus, the suggestion put forward is that this oxidant promotes reverse Na+/Ca2+ exchange and is able to exert multiple effects on membrane transport.
Animals↗
Pholasin: a new chemiluminescent probe for the detection of chloramines derived from human phagocytes.
We have previously reported that normal human polymorphonuclear neutrophils (PMN) release taurine-chloramine, a long-lived oxidant, in response to stimulation by phorbol myristate acetate (PMA) or opsonized zymosan in the presence of exogenous taurine. We now describe a new, simple, and highly sensitive method for the detection of chloramines, including taurine-chloramine, using the chemiluminescent probe Pholasin, the luciferin of the mollusc Pholas dactylus. Taurine-chloramine (N-chlorotaurine) detection was assessed with both a colorimetric method (based on the oxidation of potassium iodide) and with the Pholasin-dependent chemiluminescence (CL) method. The taurine-chloramine concentration in PMN supernatants determined using the potassium iodide (KI) method correlated closely with Pholasin-dependent CL. This CL was also assessed in nonoxidative conditions. No taurine-chloramine was detected in supernatants of lymphocytes and PMN from patients with an oxidative burst defect (chronic granulomatous disease, CGD) with the KI method, but Pholasin-dependent CL was consistently observed. The use of methionine, a specific chloramine scavenger in our incubation conditions, allowed us to define a methionine-inhibitable fraction of Pholasin-dependent CL (i.e., chloramine-induced CL).
Child↗
Effect of an exposure to chloramine-T on the immunoreactivity of glucagon.
Various preparations of glucagon treated with chloramine-T under different conditions have been studied with respect to their immunoreactivity toward two different glucagon antisera; one specific for pancreatic glucagon and the other capable of reacting with enteroglucagon as well. The glucagon preparations exposed to chloramine-T for different periods reacted almost identically with the nonspecific antibody whether they were used as tracer or standard. On the contrary, treatment with chloramine-T under severe conditions led to reduced immunoreactivity toward the specific antibody. Inclusion of dimethyl sulfoxide (DMSO) in the chloramine-T reaction resulted in preservation of the immunoreactivity of the treated preparations. The cyanogen bromide cleaved-glucagon, (1-26) homoserine lactone, showed little cross-reactivity with the specific antibody whereas it reacted to a similar extent with the nonspecific antibody as natural glucagon did. Amino acid analysis of the hormone exposed to chloramine-T demonstrated that the methionine residue at position 27 in the glucagon molecule had been oxidized to methionine sulfoxide. In addition, tryptophan had also been affected. DMSO protected methionine and tryptophan from the oxidative action of chloramine-T. We postulate from these results that the change in the immunoreactivity toward the specific antibody of glucagon exposed to chloramine-T is mainly due to oxidation of the methionine residue at position 27 in the molecule. The usefulness of DMSO in the iodination process is also discussed.
Animals↗
Illness in hemodialysis patients after exposure to chloramine contaminated dialysate.
In September 1987, patients at an outpatient dialysis center were exposed to chloramine contaminated dialysate when the carbon filter in a recently modified water treatment system failed. Forty-one patients required transfusion to treat the resultant hemolytic anemia. Epidemiologic investigation demonstrated that the mortality rate among dialysis center patients increased during the 5 months after chloramine exposure when compared with the 12 months before chloramine exposure, but no deaths could be attributed to the exposure. Chloramine is commonly used as a disinfectant in municipal water supplies, and has previously been reported to cause hemolytic anemia in patients undergoing dialysis. Hemodialysis centers in cities that use chloramine in water supplies must design water treatment systems with adequate means for removing chloramine and must monitor processed water closely to ensure that chloramine contamination does not occur. Dialysis centers that make changes in their water processing systems should evaluate all components of the system before changes are made, and must ensure that after modifications are made, processed water meets the standards set by the Association for Advancement of Medical Instrumentation.
Ambulatory Care Facilities↗
Immunological and biophysical properties of hepatitis B antigen labeled by the chloramine-T and by the lactoperoxidase methods.
Optimal conditions were sought for the radiolabeling of microgram quantities of hepatitis B surface antigen (HBs Ag) employing the chloramine-T or lactoperoxidase iodination procedures. Preparations of HBsAg labeled by these procedures are referred to as chloramine-T preparations and lactoperoxidase preparations, respectively. Labeled HBsAg having specific activities between 10-20 muCi/mug were found to display the greatest degree of sensitivity for unlabeled HBsAg and for anti-HBs using a double-antibody radioimmunoassay (RIA-DA). Increasing the specific activity above this level redulted in a decreased affinity of labeled 1251-HBs Ag for anti-HBs, indicating that soluble antigenic alterations had developed. At equivalent specific activities, chloramine-T preparations competed less effectively for unlabeled HBs Ag than lactoperoxidase preparations, and anti-HBs endpoint titers were slightly reduced, especially among preparations of high specific activity (greater than or equal to 65 muCi/mug). Chloramine-T preparations of HBs Ag (sp. act. 15--30 muCi/mug) showed essentially no antigenic deterioration over a 2-month period at minus 196 degrees C or minus 70 degrees C. Utilization of optimally labeled 1251-HBs Ag has increased the sensitivity of the RIA-DA for unlabeled HBs Ag 30-fold to a level below 1 ng/ml and enhanced antiamine-T method revealed that only the most acidic population was labeled (pH 3.75+/-0.5). In contrast, six antigenic components with distinct pI values ranging from 3.7 to 5.2 were detected by RIA-DA in both unlabeled HBs ag and in the chloramine-T preparation. This indicated that the chloramine-T method did not radically change the relative number or charge of each of the pI populations present in purified preparations of HBs Ag. Analysis of HBs Ag iodinated by the lactoperoxidase procedure revealed the presence of three of four populations of particles with pI values ranging from 3.9 to 4.5, suggesting that this procedure labels HBs Ag more uniformly.
Chloramines↗
Asthma due to industrial use of chloramine.
Seven brewery workers developed asthmatic symptoms after using chloramine (chloramine-T) powder as a sterilising agent. They gave positive weal and flare reactions to skin-prick tests with solutions of chloramine at strengths that caused no reactions in unexposed controls. The symptoms did not recur once the men had been removed from areas in which chloramine was handled. As well as causing irritant effects, inhaling dry or liquid aerosols of chloramine may cause sensitisation, with workers being prone to allergic asthma on reexposure. In view of this, measures should be taken to ensure that chloramine is not inhaled.
Adult↗
High pressure liquid chromatographic determination of chloramine-T in food.
A high pressure liquid chromatographic (HPLC) method for determining chloramine-T (N-chloro-N-sodium-p-toluenesulfonadmie) in foods such as ice cream, minced meat, and shrimp is described. Deproteinized samples are treated with sulfite to convert chloramine-T to p-toluenesulfonamide (p-TSA) and extracted, and HPLC analysis is performed on concentrated extracts. Sample extracts are chromatographed on a reverse phase 10 micrometers Lichrosorb RP-18 column with acetonitrile-water (190), and quantitated by an ultraviolet detector (220 nm) and digital integrator. The relationship between recorded peak area and concentration was linear to 0.600 micrograms p-TSA/microL. The detection limit was 2.5 ng pTSA/microL, corresponding to a chloramine-T concentration of 0.8 mg/kg in samples. Recoveries of added chloramine-T were 88% for ice cream, 73% for minced meat, and 51% for shrimp. Precision data indicate a relative standard deviation of 1.54 and 2.14% for the complete analysis of ice cream with levels of 15 and 63 mg chloramine-T/kg (n = 5 determinations), respectively. The HPLC method was applied to chloramine-T determinations in 62 ice cream, 25 minced meat, and 25 shrimp samples.
Animals↗
Prevention of chloramine-induced hemolysis in dialyzed patients.
Chloramines, compounds made up of chlorine and ammonia, when present in tap water used for dialysis cause methemoglobinemia and hemolysis. Ascorbic acid addition has been reported to effectively neutralize chloramines in vitro and in patients dialyzed with the single batch dialysis delivery system. We extended these observations to patients dialyzed with the proportioning dialysis delivery system where exposure time of ascorbic acid to chloramines is shorter. This may be important since we found that the half time of the reaction between ascorbic acid and chloramines is 4 minutes. Red cell oxidant sensitivity in 15 patients was assessed by incubating red cells with ascorbate-cyanide and measuring methemoglobin which averaged 2.17 +/- 0.42 g/100 ml (SEM) before dialysis and 2.87 +/- 0.52 g/100 ml after dialysis (NS). Reduced glutathione (GSH) levels were also measured as an index of red cell oxidant damage. GSH decreased from a mean of 7.40 +/- 0.59 micromoles/g Hb before dialysis to 6.98 +/- 0.52 micronmoles/g Hb after dialysis (P less than 0.01). In 2 patients there was no change in 51Cr red cell survival when dialyzed on either the proportioning system or other chloramine free systems. We conclude that addition of ascorbic acid to neutralize chloramines in tap water is also effective when using the proportioning dialysis delivery system.
Adult↗
Atopic allergy to chloramine-T and the demonstration of specific IgE antibodies by the radioallergosorbent test.
Chloramine-T is a small molecular oxidizing agent that has been widely used as a disinfectant since the beginning of this century. It is generally used in a 5% solution but it is also supplied in powder form. Sporadic case reports of immediate-type sensitization to this agent associated with symptoms of asthma, rhinitis and urticaria have appeared during recent decades. In one of the reports, specific IgE antibodies in sera of four patients who developed asthmatic symptoms after exposure to chloramine-T were demonstrated using a radioimmuno-assay. Three cases of bronchial asthma in workers who had handled chloramine-T powder are described in the present report. Positive skin-prick test reactions to chloramine-T were observed and specific IgE antibodies to human serum albumin treated with chloramine-T were detected using the classic radioallergosorbent (RAST) technique in all three patients.
Adult↗
Physiological studies of chloramine resistance developed by Klebsiella pneumoniae under low-nutrient growth conditions.
This study investigated the physiological mechanisms of resistance to chloramines developed by Klebsiella pneumoniae grown in a nutrient-limited environment. Growth under these conditions resulted in cells that were smaller than cells grown under high-nutrient conditions and extensively aggregated. Cellular aggregates ranged from 10 to more than 10,000 cells per aggregate, with a mean population aggregate size of 90 cells. This aggregation may have been facilitated by the presence of extracellular polymer material. By using glucose as a reference of capsule content, it was determined that growth under low-nutrient conditions produced cells with 8 x 10(-14) to 41 x 10(-14) g of carbohydrate per cell, with a mean +/- standard deviation of 27 x 10(-14) +/- 16 x 10(-14) g of carbohydrate per cell. In comparison, growth under high-nutrient conditions resulted in 2.7 x 10(-14) to 5.9 x 10(-14) g of carbohydrate per cell, with a mean and standard deviation of 4.3 x 10(-14) +/- 1.2 x 10(-14) g of carbohydrate per cell. Cell wall and cell membrane lipids also varied with growth conditions. The ratio of saturated to unsaturated fatty acids in cells grown under low-nutrient conditions was approximately five times greater than that in cells grown under high-nutrient conditions, suggesting possible differences in membrane permeability. An analysis of sulfhydryl (-SH) groups revealed no quantitative difference with respect to growth conditions. However, upon exposure to chloramines, only 33% of the -SH groups of cells grown under low-nutrient conditions were oxidized, compared with 80% oxidization of -SH groups in cells grown under high-nutrient conditions. The reduced effectiveness of chloramine oxidization of -SH groups in cells grown under low-nutrient conditions may be due to restricted penetration of chloramines into the cells, conformational changes of enzymes, or a combination of both factors. The results of this study suggest that chloramine resistance developed under low-nutrient growth conditions may be a function of multiple physiological factors, including cellular aggregation and protection of sulfhydryl groups within the cell.
Bacterial Adhesion↗
Effects of chloramine on Bacillus subtilis deoxyribonucleic acid.
The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically. Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments. Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks. DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high. Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases. The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation. Both treatments introduced a small fraction of alkali-sensitive lesions in DNA. DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity.
Bacillus subtilis↗
[Biochemical activity of opportunistic antibiotic and chloramine-sensitive and resistant microorganisms isolated from healthy and sick people].
Activity of oxidation-reduction enzymes such as succinate dehydrogenase, peroxidase and catalase was studied in staphylococci isolated from healthy persons and patients as well as from the air and implements of medical institutions. The isolates were resistant either to antibiotics or to chloramine B or to the both. The results showed that development of resistance to antibiotics and chloramine B in the staphylococci was accompanied by a decrease in the activity of succinate dehydrogenase, peroxidase and catalase. In the strains resistant only to chloramine B the activity of the enzymes was practically at the same level as in the strains resistant only to antibiotics. In the strains resistant to both antibiotics and chloramine B, the activity of succinate dehydrogenase, peroxidase and catalase did not practically differ from that in the strains resistant either to antibiotics or to chloramine B.
Anti-Bacterial Agents↗
Spectrodensitometric determination of chloramine-T in ice cream.
A spectrodensitometric method has been developed for the quantitative determination of chloramine-T (N-chloro-N-sodium-p-toluenesulfonamide) in ice cream. Chloramine-T is extracted and converted into p-toluenesulfonamide (p-TSA) followed by thin layer chromatographic separation of concentrated extracts on silica gel and quantitation of the p-TSA spots from standards and samples by direct scanning with a reflectance densitometer at 228 nm. A linear relationship was obtained between recorded peak area and concentration for 0.5--7.0 microgram p-TSA/spot. The reproducibility of the complete method was 2.87% (n = 9 determinations). The detection limit of the scanning procedure was 0.5 microgram p-TSA/spot, corresponding to a concentration of 4 mg chloramine-T/kg sample. The average recovery was 88 +/- 3% (P = 95%) for 10 ice cream samples spiked with chloramine-T at levels ranging from 10 to 55 mg/kg. The described method was used to assay 146 commercial ice cream and whipped cream samples for chloramine-T.
Chloramines↗
Chloramine-T effect on sodium conductance of neuroblastoma cells as studied by whole-cell clamp and single-channel analysis.
Patch-clamp experiments were done on sodium channels of neuroblastoma cells (N1E-115) in the presence of tetraethylammonium ions to block potassium channels. In Ringer solution whole-cell records revealed a diphasic INa inactivation with the fast (tau 0) component. being clearly larger than the slow (tau 1 approximately 3 tau 0) component. In single-channel studies on inside-out patches the mean open time, to, turned out to be only a fraction of tau 0 and almost independent of membrane potential. After external application of chloramine-T INa inactivation of whole cells was delayed with both tau 0 and tau 1 increased, and incomplete, i.e. a persistent current component emerged. The latter was maximal at a more positive membrane potential than the peak current. Also, after chloramine-T treatment the peak INa increased, particularly at weak depolarizations. In inside-out patches the equally effective internal application of chloramine-T led to bursting channel openings with mean burst times (tb) approximately 6 ms, and gap times (tg) approximately 20 ms, where gap is defined as a closure of greater than or equal to 1.5 ms. Within the bursts to was approximately 2 ms, again clearly shorter than tau 0; the mean close time, tc was approximately 0.5 ms. The single-channel conductance was approximately 13 pS and unaffected by chloramine-T. Diphasic INa inactivation and the fact that to less than tau 0 led to an extension of the model of Aldrich and Stevens [J Neurosci 7:418-431 (1987)], in which overall kinetics is determined by the openings rather than closures of the sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Chloramines↗
Chloramine mutagenesis in Bacillus subtilis.
Chloramine (which occurs widely as a by-product of sanitary chlorination of water supplies) is shown to be a weak mutagen, when reversion of trpC to trpC in Bacillus subtilis is used as an assay. Some DNA-repair mutants appear to be more sensitive to chloramine, suggesting the involvement of DNA targets in bactericide. The influence of plating media on survival of cells treated with chloramine suggests a bacterial repair system acting upon potentially lethal lesions induced by chloramine.
Bacillus subtilis↗