Biomedical subjects
Publications and source records attributed to N Benjamin.
The use of acidified nitrite as a potential antifungal agent was investigated by exposing clinical isolates of Candida albicans, Candida glabrata, Candida tropicalis, Cryptococcus neoformans, Aspergillus fumigatus and Rhodotorula to varying concentrations of acidified nitrite at different pH levels to determine the minimal fungicidal concentration after 10 min exposure.
To test the hypothesis that a combination of high salivary nitrate and high nitrate-reducing capacity are protective against dental caries, 209 children attending the Dental Institute, Barts and The London NHS Trust were examined. Salivary nitrate and nitrite levels, counts of Streptococcus mutans and Lactobacillus spp., and caries experience were recorded. Compared with control subjects, a significant reduction in caries experience was found in patients with high salivary nitrate and high nitrate-reducing ability. Production of nitrite from salivary nitrate by commensal nitrate-reducing bacteria may limit the growth of cariogenic bacteria as a result of the production of antimicrobial oxides of nitrogen, including nitric oxide.
Mycobacterium ulcerans disease (Buruli ulcer) is a serious ulcerating skin disease which is common in many tropical countries. Standard treatment, by extensive excision and skin grafting, is not available in rural communities where the disease is common. We evaluated the efficacy and safety of treatment with topical nitrogen oxides. Thirty-seven patients with a clinical diagnosis of Buruli ulcer caused by M. ulcerans disease were randomly assigned to one of two groups. In one group, two creams containing sodium nitrite (6%, wt/wt) or citric acid monohydrate (9%, wt/wt) were applied daily for 6 weeks, while the other group received a placebo. In the second 6 weeks, both groups received the nitrogen oxide-generating combination of creams. Treatment was continued for another 4 weeks for patients whose ulcers were not healed after 12 weeks. The ulcer surface area was monitored by weekly tracings made by assessors blinded to the treatment. In the first 6 weeks, patients on sodium nitrite and citric acid monohydrate (group I, active treatment) showed a rapid decrease in ulcer size from 28.6 +/- 5.6 cm2 (mean +/- standard error) to 12.6 +/- 3.2 cm2, a decrease significantly greater than that in group II (from 15.3 +/- 3.1 to 11.7 +/- 3.7 cm2; P = 0.03). Five ulcers in the placebo group enlarged during this period, compared with one in the active group. In the second 6 weeks (both groups on active treatment), the rates of healing were similar for the two groups and there was a significant reduction in ulcer size in group II (previously on placebo) compared to the first 6 weeks. Yellow pigmentation of the skin, which disappeared 3 days after treatment was stopped, was the only side effect to date. We conclude that creams releasing nitrogen oxides increase the healing rate of ulcers caused by M. ulcerans infection with minimal adverse events. This is the first controlled trial of any form of therapy which demonstrates efficacy in treating this disease.
Mycobacterium ulcerans, which causes Buruli ulcer, was exposed to acidified nitrite or to acid alone for 10 or 20 min. Killing was rapid, and viable counts were reduced below detectable limits within 10 min of exposure to 40 mM acidified nitrite. M. ulcerans is highly susceptible to acidified nitrite in vitro.
Platelets play an important role in the development of vascular disease, while vegetarian diets, which are rich in inorganic nitrate, protect against it. This study was performed to assess the effect of potassium nitrate (KNO(3)) ingestion on platelet function in humans. Oral KNO(3) (2 mmol) was given to healthy volunteers and its effect on platelet function assessed by measuring the aggregant effect of collagen. Blood samples were taken for measurement of plasma S-nitrosothiols (RSNO) and platelet cyclic GMP and nitrotyrosine levels. Gastric juice samples were taken for measurement of RSNO. In a separate study, the effect of oral KNO(3) on portal RSNO levels in patients with intrahepatic porto-systemic shunts was assessed. KNO(3) caused a significant increase in gastric RSNO levels, from 0.46 +/- 0.06 to 3.62 +/- 2.82 microM (t(max) 45 min; P < 0.001), and significantly inhibited platelet function (t(max) 60 min; P < 0.001). There was no effect on systemic or portal RSNO, platelet cGMP or platelet nitrotyrosine levels. Oral KNO(3) inhibits platelet aggregation. The time course suggests that gastric RSNO production may be involved in this effect. The protection against vascular events associated with a high intake of vegetables may be due to their high nitrate content.
Nitric oxide (NO) may be generated and delivered into the skin via a novel system of sodium nitrite and ascorbic acid. This placebo-controlled, double-blind trial compared the analgesic properties of this system alone and when supplemented with lidocaine. The pain of dorsal hand vein cannulation was assessed in 100 volunteers. The NO-generating system was prepared by mixing two gels, the first KY jelly and sodium nitrite (10% w/v), the second KY jelly and ascorbic acid (10% w/v). NO-generating gel was the placebo treatment, and when combined with lidocaine (final concentration 5%), formed the active treatment. The gels were applied to the dorsum of the hands bilaterally and simultaneously for 10 min. Following cannulation, pain perception was measured with a verbal rating score (VRS) and a visual analogue score (VAS). The active formulation significantly decreased the VRS (p < 0.0001) and also reduced the mean VAS by > 40% compared with placebo (p < 0.001). This investigation suggests a 10-min topical application of anaesthetic combined with the NO-generation system may provide effective analgesia for venous cannulation in adults.
The S-nitrosothiols (RSNOs) are thought to represent a circulating endogenous reservoir of nitric oxide (NO), and may have potential as donors of NO, distinct from currently used agents. They have the general formula RSNO, and naturally occurring examples include S-nitrosocysteine, S-nitrosoglutathione and S-nitrosoalbumin, in which R is an amino acid, polypeptide and protein respectively. RSNOs have anti-platelet properties, a theoretical role in the treatment of asthma and the potential to be used as agents to treat infectious diseases ranging from the common cold to AIDS. RSNOs are relatively unstable, being degraded to release NO and the corresponding disulphide. Their stability is influenced by the properties of the R group, heat, light, the presence of transition metal ions (in particular copper) and the presence of other thiols. RSNOs participate in transnitrosation reactions in which the -NO group is transferred to another thiol to form a more stable RSNO. Thus the stability of RSNOs in vivo is difficult to predict, and this has led to the development of more stable analogues, in which the properties of R are modified. Potential interactions of RSNOs include that with ascorbic acid (vitamin C), which enhances the ability of copper to catalyse their degradation. Transnitrosation reactions with thiol-containing enzymes can influence protein function, and the intracellular thiol glutathione, levels of which are influenced by many disease states, can also influence stability. Thus, although RSNOs have many theoretically useful properties, there are also many aspects of their biochemistry that need to be addressed before their therapeutic potential can be fully realized.
The antimicrobial agent nitric oxide (NO) is formed in the mouth and its concentration is directly related to salivary nitrite, which in turn is related to dietary nitrate intake. The aim of this study was to determine whether nitrite under acidic conditions will have an inhibitory effect, possibly occurring through NO production, on the periodontal disease pathogens Fusobacterium nucleatum, Eikenella corrodens and Porphyromonas gingivalis. Whereas the growth of these organisms was inhibited by a more acid pH, the addition of nitrite caused a marked, further dose-dependent reduction in bacterial numbers after exposure. The ability of these bacteria to recover from nitrite exposure was also affected by pH and nitrite concentration. At acidity levels below pH 5.0, low concentrations of nitrite (0.2 mM) caused effective complete killing of the periodontal bacteria. Addition of sodium thiocyanate did not increase the bacteriostatic or bacteriocidal activity of acidified nitrite against any of the 3 bacteria. These results demonstrate the possibility that nitrite in saliva, under appropriate conditions, may have an effect on the growth and survival of the bacteria implicated in periodontal disease.
INTRODUCTION: Glucocorticoid-remediable aldosteronism (GRA) is a rare inherited cause for hypertension associated with a significant morbidity and mortality at an early age. Individuals with this abnormality frequently present with severe hypertension which is resistant to standard antihypertensive therapy, a strong family history of hypertension, intracranial haemorrhage, and sporadic hypokalaemia. However many affected individuals may appear phenotypically indistinguishable from normal essential hypertensives but remain at high risk of morbidity and mortality. OBJECTIVE: To determine how effective random or targeted screening of hypertensive patients is for the detection of GRA. DESIGN: A prospective study involving the screening of 300 hypertensive patients chosen at random attending the Aberdeen Hypertension Clinic and, during the same period, the targeted screening of patients with a medical and family history suggestive of GRA. SETTING: A University hospital with a primary catchment of 500,000 inhabitants and a hypertension clinic population of over 8500 patients. RESULTS: Random screening failed to identify any GRA mutation-positive individuals. Targeted screening of selected individuals revealed two index families and four further families containing 40 mutation-positive individuals. CONCLUSION: Targeted screening of hypertensive individuals with a family history of hypertension, cerebral haemorrhage, a history of hypertension from an early age, resistant hypertension which has proven difficult to control and hypokalaemia revealed two index cases and four further individuals and 30 hypertensive and 10 normotensive members of their families with GRA.
AIMS: Nitric oxide is generated from sweat nitrite in the acidic environment of the skin surface and is thought to contribute to protection against infection. This study examined the sensitivity of Trichophyton mentagrophytes, T. rubrum, Candida albicans, Streptococcus pyogenes, Staphylococcus aureus and Propionibacterium acnes to acidified nitrite. METHODS AND RESULTS: Organisms were cultured in varying concentrations of nitrite and pH for different lengths of time, before being transferred to recovery medium. With the exception of Strep. pyogenes, addition of nitrite increased the antimicrobial activity of acid solutions against all organisms tested. The rank order of sensitivity was: C. albicans < T. rubrum < T. mentagrophytes < Staph. aureus < P. acnes, with P. acnes being most sensitive. CONCLUSION: This work has shown that acidified nitrite is microbiocidal to common cutaneous pathogens. The concentrations of nitrite required to kill pathogenic fungi and bacteria in in vitro assays were higher than the concentrations of nitrite measured in sweat. However, additional co-factors in vivo and in sweat may potentiate the effect of acidified nitrite. SIGNIFICANCE AND IMPACT OF THE STUDY: Pharmacological preparations of acidified nitrite are novel antimicrobial agents. These data suggest skin organisms which may be sensitive to this treatment.
BACKGROUND: In patients with intact renal function and low dietary nitrate intake, plasma nitrate concentrations reflect endogenous nitric oxide production and are shown to be increased during inflammatory processes. The aim of this study was to compare plasma nitrate concentrations and hence endogenous nitric oxide production in children with infectious and noninfectious diarrhea and to determine whether plasma nitrate concentrations could serve as a discriminant test between acute and chronic diarrhea in children. METHODS: Three groups of patients were identified: 14 patients with acute gastroenteritis, 13 patients with chronic noninfectious diarrhea, and 14 patients with no evidence of gastrointestinal pathology and no underlying infectious process, who served as control subjects. Plasma nitrate concentrations were determined spectrophotometrically using the Greiss reaction before reduction to nitrite with a copper-coated cadmium column. RESULTS: Mean plasma nitrate concentrations were 405.3 micromol/L +/- 281.6 micromol/L (standard deviation) in patients with infectious diarrhea, 134.7 micromol/L +/- 77.0 micromol/L in patients with chronic diarrhea, and 54.1 micromol/L +/- 20.1 micromol/L in control subjects (F = 42.6, P < 0.0001; analysis of variance). Plasma nitrate concentrations were significantly higher in the infectious diarrhea group compared with the noninfectious diarrhea and control groups (Student-Newman-Keuls test, P < 0.5). CONCLUSIONS: Although an optimal cutoff concentration cannot be defined, plasma nitrate concentrations in excess of 300 micromol/L are suggestive of an infectious process whereas values less than 100 micromol/L are indicative of noninfectious diarrhea.
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Nitric oxide (NO) is a mediator of essential biological processes, including vasodilatation, anti-microbial activity and wound healing. A chemical system using sodium nitrite and ascorbic acid has been developed which generates significant amounts of NO. The originally described system was messy and impractical, and the high acidity may cause pain and further tissue damage in ulcerated skin. To overcome this, a selectively permeable, hydrophilic polyester co-polymer membrane system (Sympatex ) has been identified that can be placed between the NO-generating chemicals and the skin. The aim of the present study was to determine whether NO derived from this chemical system was able to diffuse through the membrane and have a measurable vasodilatory effect on forearm skin in healthy volunteers. The Sympatex 10 microm membrane was found to be highly permeable to NO, while preventing passage of the constituents of the NO-generation gel to the skin. The transmembrane NO-generation system had a vasodilatory effect comparable with that resulting from direct topical application. Additionally, the NO generated was effective in killing Staphylococcus aureus and Escherichia coli at doses lower than those required to increase skin blood flow. The vasodilatory and anti-microbial effects of this system may be useful as a patch-based topical therapy for skin ulceration, particularly when there is concomitant ischaemia and infection.
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The role of nitric oxide (NO) and free radicals in the development of microvascular disease in type 1 diabetes remains unclear. We have measured NO and isoprostane (a stable marker of in vivo lipid peroxidation) production in 13 type 1 diabetic subjects with normal urinary albumin excretion and 13 healthy volunteers. Whole-body NO synthesis was quantified by measuring the urinary excretion of 15N-nitrate after the intravenous administration of L-[15N]2-arginine. The urinary excretion of the major urinary metabolite of 15-F2t-isoprostane (8-iso-prostaglandin-F2alpha), 2,3-dinor-5,6-dihydro-F2t-IsoP, was quantified as a marker of in vivo lipid peroxidation. Whole-body NO synthesis was significantly higher in diabetic subjects compared with control subjects (342 vs. 216 nmol 15N-nitrate/mmol creatinine [95% CI of the difference 45-207], P = 0.005). This increase was not explained by a difference in renal function between the 2 groups. There was no difference in 2,3-dinor-5,6-dihydro-F2t-IsoP excretion between diabetic subjects and control subjects (44.8+/-7.8 vs. 41.4+/-10.0 ng/mmol creatinine, mean +/- 95% CI). However, there was an inverse correlation between NO synthesis and free radical activity in subjects with diabetes (r = -0.62, P = 0.012) that was not observed in control subjects (r = 0.37, P = 0.107). We conclude that whole-body NO synthesis is higher in type 1 diabetic subjects with normal urinary albumin excretion than in control subjects. The inverse correlation between isoprostane production and NO synthesis in diabetic subjects is consistent with the hypothesis that NO is being inactivated by reactive oxygen species.
Xanthine oxidoreductase (XOR) is a mammalian enzyme that possesses a series of redox centers, which use either NAD(+) or molecular oxygen for oxidation of the purines xanthine and hypoxanthine to uric acid. The ability of XOR to act as an NADH oxidase is a less well recognized function of the enzyme, and it is this function that we used to explore the metabolism of glyceryl trinitrate. The antiplatelet effect of nitric oxide (NO) on platelet aggregation was used as a bioassay to assess the bioconversion of glyceryl trinitrate to NO by XOR. The thromboxane mimetic U46619, 2 microM, was used to stimulate platelet aggregation in platelet-rich plasma prepared from healthy drug-free human volunteers. All incubations were carried out at 37 degrees C for 2 min after the addition of U46619. XOR produced a dose-dependent antiaggregant effect when incubated with glyceryl trinitrate (GTN), 220 microM. This did not occur when GTN or XOR was incubated with platelet-rich plasma independently. The antiaggregant effect of XOR plus GTN was dose dependently inhibited by allopurinol, with an IC(50) of 100 microM. The addition of superoxide dismutase (SOD), 100 U/ml produced a shift to the left in the antiaggregant dose-response curve for XOR. The IC(50) for XOR at 200 U/l without SOD was decreased to 80 U/l with SOD. Oxyhemoglobin, an extracellular NO scavenger, produced a dose-dependent, noncompetitive inhibition of the antiaggregant effect of XOR plus GTN. These findings suggest that GTN may be reduced to NO in vitro by the enzyme XOR in sufficient amounts to inhibit platelet aggregation.
BACKGROUND: Patients with Raynaud's syndrome have abnormal digital vasoconstriction, which may be secondary to impaired synthesis of, or impaired sensitivity to, nitric oxide. We studied the effect on microcirculation of a nitric-oxide-generating system applied topically to the finger and forearm of healthy volunteers and patients with primary Raynaud's syndrome. METHODS: We did a single-blind, randomised, placebo controlled, cross-over study of the microcirculatory response to topical application of a nitric-oxidegenerating gel in 20 patients with severe Raynaud's syndrome, and ten healthy volunteers. We prepared the nitric-oxide-generating system by mixing a solution of KY jelly and sodium nitrite (5% weight/volume), with a solution of KY jelly and ascorbic acid (5% weight/volume). About 0.5 mL of each solution was separately applied to the skin of the forearm (3 cm2), and then mixed with a sterile cotton bud. A similar procedure was done simultaneously on the other arm with KY jelly only (placebo). The procedure was then repeated on the finger pulps. Changes in skin microcirculatory volume and flux were measured bilaterally by infrared photoplethysmography and laser doppler fluxmetry, respectively. FINDINGS: In the forearm, blood flow increased significantly after application of the active gel both in patients with Raynaud's syndrome (microcirculatory volume from mean area under the curve 98 [SE 14] to 1024 [130]; microcirculatory flux from 5060 [462] to 74,800 [3940]) and in healthy controls (volume from 85 [19] to 1020 [60]; flux from 4420 [435] to 84,500 [7000]). In the fingers, although baseline blood flow was lower in patients than in controls, both groups showed increases with application of active gel (volume from 1100 [194] to 3280 [672] and 2380 [441] to 6160 [1160], respectively; flux from 33,400 [4200] to 108,000 [13,600] and 52,000 [8950] to 185,000 [19,500]). Increases in blood flow with placebo gel were not significant. No adverse effects were reported. INTERPRETATION: In primary Raynaud's syndrome, topical application of a nitric-oxide-generating system can stimulate an increase in both microcirculatory volume and flux.