Determination of trace concentrations of dissolved nitric oxide in a biological buffer.

A new real-time method for measuring a trace concentration of nitric oxide (NO) in a complex matrix routinely used in pharmacological studies of its bioactivity is described. NO was quantified as a gas by chemiluminescence after extraction from a continuous liquid sample flow with a limit of detection of 0.042 nmol dm(-3) at a signal to noise ratio of 3. Theories to calculate the concentration of NO in the liquid sample flow from a direct measurement of NO in the extraction carrier gas are presented. The efficiency of extraction is determined by a stopflow experiment. An example is presented of the measurement of the steady-state concentrations of NO in Krebs-bicarbonate buffer at pH 7.4 and 37 degrees C when its liquid surface is sequentially exposed to gases containing various concentrations of NO in O2 plus CO2.

Controlled prospective randomised trial on the effects on pulmonary haemodynamics of the ambulatory long term use of nitric oxide and oxygen in patients with severe COPD.

BACKGROUND: Pulmonary hypertension is a frequent complication of severe chronic obstructive pulmonary disease (COPD) and a major cause of morbidity and mortality in this condition. Based on the improved survival of these patients due to long term oxygen therapy and the potent and selective pulmonary vasodilation by inhaled nitric oxide, the safety and effectiveness of the combined inhalation of these two gases over a 3 month period was assessed. METHODS: Forty patients with secondary pulmonary hypertension due to COPD were randomly assigned to receive either oxygen alone or "pulsed" inhalation of nitric oxide with oxygen over a period of 3 months. "Pulsed" inhalation of nitric oxide was used to reduce pulmonary ventilation-perfusion mismatch and formation of toxic reaction products of nitric oxide and oxygen. RESULTS: Compared with oxygen alone, the combined inhalation of nitric oxide and oxygen caused a significant decrease in mean (SE) pulmonary artery pressure (from 27.6 (4.4) mm Hg to 20.6 (4.9) mm Hg, p<0.001) and pulmonary vascular resistance index (from 569.7 (208.1) to 351.3 (159.9) dyne x s(-1) x cm(-5) x m(-2), p<0.001) without decreasing arterial oxygenation. Cardiac output increased by 0.5 litres (from 5.6 (1.3) l/min to 6.1 (1.0) l/min, p=0.025). Systemic haemodynamics and left heart function remained unchanged during this period and no increase in toxic reaction products of nitric oxide was observed. CONCLUSIONS: This is the first controlled trial indicating that the "pulsed" inhalation of nitric oxide together with oxygen may be safely and effectively used for the long term treatment of severe COPD.

Circulating nitrite anions are a directly acting vasodilator and are donors for nitric oxide.

Inhaled nitric oxide (NO) is a pulmonary vasodilator, but also acts systemically, causing negative cardiac inotropic effects and a fall in systemic vascular resistance. Circulating metabolites of NO are presumed to be responsible. We questioned the role of nitrite anions and the manner in which they might contribute to these effects. Nitrite and nitrate anions coexist in blood, while circulating levels of dissolved NO are very low. Nitrate anions are not biologically active, but nitrite anions may have a biological role through the release of NO. In vitro, at 37 degrees C and in aerated Krebs bicarbonate solution, the steady-state concentration of dissolved NO was proportional to the concentration of NO in the gas. Nanomolar concentrations of dissolved NO coexisted with micromolar concentrations of nitrite anions. The idea of an equilibrium between the two in solution was also supported by the observed release of NO from nitrite anions in the absence of gas. With rings of precontracted pig pulmonary arteries (prostaglandin F(2alpha); 10 micromol/l), the steady-state concentration of dissolved NO causing 50% relaxation (EC(50)) was 0.84+/-0.25 nmol/l, corresponding to a gaseous concentration of 2.2 p.p.m. The EC(50) of nitrite was 4.5+/-0.7 micromol/l, a concentration normally found in plasma. The estimated concentration of dissolved NO derived from this nitrite was 4.5 pmol/l, some 100 times lower than would be needed to cause relaxation. The rate of exhalation of NO was increased and pulmonary vascular resistance was reduced by the addition of nitrite solution to the perfusate of isolated perfused and ventilated pig lungs, but only when millimolar concentrations were achieved. Thus circulating nitrite anions are a direct vasodilator, only being a carrier of effective amounts of "free" NO at higher than physiological concentrations.

Exhaled nitric oxide is reduced in infants with cystic fibrosis.

BACKGROUND: Exhaled nitric oxide levels are low in patients with cystic fibrosis (CF), despite the chronic inflammation present in the airways. This study aimed to determine whether levels of exhaled nitric oxide were reduced prior to the onset of respiratory symptoms in infants with CF. METHODS: The levels of exhaled nitric oxide were measured using a chemiluminescence analyser in five infants with CF and 11 healthy control subjects, both groups having a mean age of 48.6 days. RESULTS: Mean levels of exhaled nitric oxide were significantly lower in infants with CF than in the control group (4.9 ppb v 12.1 ppb; p=0.01). CONCLUSIONS: This finding may be the key to understanding the inflammatory processes in early cystic fibrosis and may lead to novel treatment approaches.

Eradication of house dust mite from homes of atopic asthmatic subjects: a double-blind trial.

BACKGROUND: House dust mite (HDM) allergens can accumulate to very high levels in homes. From the observed sensitivity of HDMs to heat and their allergens to steam, a novel treatment of furnishings has been developed. OBJECTIVE: We sought to determine whether combined steam and heat treatment of home furnishings reduced asthmatic patients' bronchial hyperreactivity (BHR) and lowered HDM antigen loads. METHODS: The homes of 30 asthmatic subjects aged 18 to 45 years were randomly allocated into 3 groups. In groups 1 and 2 mattresses and duvets were treated with hot air (110 degrees C), followed by steam and then heat again. All their carpets were steam cleaned. Group 2 also had a special ventilation system installed above each patient's bedroom. The homes of subjects in group 3 were sham treated. Neither patient nor laboratory staff was aware of the types of treatment. Der p 1 and 2 levels in the household dust from the lounge, bedroom carpet, and beds were determined before and after treatment and then at 6 and 12 months. BHR, measured by using histamine PD(20) values, was recorded during the 4-week run-in period and at 3, 6, 9, 12 months after treatment. RESULTS: Active heat-steam treatment of homes caused a sustained reduction of Der p 1 (P =.003) and Der p 2 (P =.001) compared with no change in sham-treated group 3 homes. Patients whose homes were treated showed a 4-fold reduction in BHR at 9 months in group 1 and throughout the posttreatment period in group 2. No change was observed in the asthmatic subjects whose homes were not treated. These improvements were sustained for 12 months in the homes with bedroom ventilation units. CONCLUSIONS: A single treatment of home furnishings reduced mite allergen load to below the risk level for sensitization and improved the asthmatic patients' BHR by 4-fold.

A randomized trial of dehumidification in the control of house dust mite.

BACKGROUND: House dust mites (HDM) are sensitive to humidity. Few studies have adequately examined the potential of dehumidification in reducing HDM numbers. OBJECTIVE: The study examined the effect of portable domestic dehumidifiers, and a behavioural programme to reduce humidity, on HDM counts and HDM allergen levels. METHODS: A randomized controlled trial was undertaken. A total of 76 homes were allocated to three groups that received a portable domestic dehumidifier, a behavioural programme, or no intervention. Humidity, temperature, HDM counts (trap and vacuum samples), HDM allergen, and other details of the home environment were measured on four occasions over a period of 1 year. Interventions and measurements were concerned predominantly with one bedroom. RESULTS: There was a reduction in relative humidity in the dehumidifier group, but not the behavioural group. A decline in HDM trap counts was observed for all three groups. Change scores did not indicate that the dehumidifier group had a greater decline than the other groups. A secondary analysis examining presence or absence of HDM showed a shift from households having HDM at baseline to households not having HDM in the final round for some trap measures. Change score analysis indicated that this shift was greater in the dehumidifier group compared with other groups. The dehumidifier group did not show a greater decline in HDM allergen than that seen in the other two groups. CONCLUSION: Neither the dehumidifier nor the behavioural intervention had a major effect on HDM counts or allergen levels. However, the study did have a number of limitations relating to size, timing of intervention, and running of the dehumidifiers. The secondary data analysis may indicate some effect of dehumidification, but clearly this effect was small. There is a need for more information on the effects of reducing ambient humidity on the distribution of HDM within their habitats.

Hemodynamic effects of basal and stimulated release of endogenous nitric oxide in isolated human lungs.

Background-We compared the hemodynamic responses to inhibition or stimulation of endothelial nitric oxide (NO) release of isolated explanted lungs from transplantation recipients with pulmonary hypertension and in normotensive unallocated donor lungs. Methods and Results-Lungs from 10 patients with severe pulmonary hypertension (SPH) and from 16 patients with severe chronic obstructive lung disease (COLD) were studied. Fourteen normotensive lungs were studied as controls. The lungs were perfused at a constant flow. In protocol 1 N(G)-nitro-L-arginine methyl ester caused a similar rise in baseline pulmonary artery pressure (PAP) that was similar in SPH (+17.1+/-4.2 mm Hg; n=5), COLD (+15.5+/-4.8 mm Hg; n=8), and control lungs (+14.5+/-1.5 mm Hg; n=7). Arterial occlusion demonstrated that most of the changes with N(G)-nitro-L-arginine methyl ester were precapillary. The response to sodium nitroprusside (10(-8) to 10(-4) mol/L) was similar in all groups. In protocol 2, the lungs were preconstricted, and acetylcholine (10(-9) to 10(-5) mol/L) caused a lesser fall in PAP in both COLD and SPH lungs compared with control (-41.9+/-8.6%, -55. 7+/-7.6%, and -73.2+/-2.5%, respectively; P<0.05), whereas sodium nitroprusside (10(-5) mol/L) decreased PAP to initial levels in all lungs. Conclusions-Stimulated release of NO is impaired in arteries of lungs with plexogenic or hypoxemic pulmonary hypertension. In contrast, basal release of NO appears to be maintained.

Minimizing the inhaled dose of NO with breath-by-breath delivery of spikes of concentrated gas.

BACKGROUND: Pulmonary vasodilatation with a 100 ppm concentration of NO given as a short burst of a few milliliters at the beginning of each breath (NOmin) was compared with conventionally inhaled NO, in which a full breath of 40 ppm of NO was inhaled (NOCD). METHODS AND RESULTS: NOmin was studied in 16 patients with severe pulmonary hypertension and in 16 isolated porcine lungs with experimentally induced pulmonary hypertension. We compared volumes of 8 to 38 mL of 100 ppm NO in N2 injected at the beginning of each breath with conventional inhalation of 40 ppm NO in air. NOCD and NOmin were studied in 4 pigs after inhibition of NO synthase with NG-nitro-L-arginine methyl ester (1 to 2 mg/kg IV) had raised the pulmonary vascular resistance index (PVRI) from 4.4+/-0.8 to 10. 0+/-1.6 mm Hg. L-1. min-1. kg-1. A similar comparison was made in 7 isolated porcine lungs after the thromboxane analogue U46619 (10 pmol. L-1. min-1) increased the mean PVRI from 4.6+/-0.8 to 12.2+/-1. 3 mm Hg. L-1. min-1. kg-1. Patients' mean PVRI was reduced from 29. 2+/-3.7 to 24.0+/-3.1 with NOmin and 24.5+/-3.3 mm Hg. L-1. min-1. m-2 (mean+/-SEM) with NOCD. In isolated porcine lungs, there was the same reduction of PVRI for NOmin and NOCD between 12.7% and 34.8%. CONCLUSIONS: A small volume of NO inhaled at the beginning of the breath was equally effective as NOCD but reduced the dose of NO per breath by 40-fold, which ranged from 1.2x10(-8) (0.4 microg) to 1. 6x10(-7) mol/L (4.8 microg) compared with 5.3x10(-7) (16 microg) to 1.2x10(-6) mol/L (36 microg) per breath with NOCD.

The pulmonary hypertensive fawn-hooded rat has a normal serotonin transporter coding sequence.

The coding sequence of the serotonin transporter gene was compared in two strains of rat-the Wistar and the fawn-hooded rat (FHR). The FHR has an inherited platelet storage-pool deficiency and a widespread impairment of serotonin storage. It is also susceptible to systemic and pulmonary hypertension. The FHR provides a model to study the genetics in human systemic and pulmonary hypertension. We measured platelet function in these two strains by measuring incorporation of radiolabeled serotonin into a platelet suspension and found significant differences in serotonin uptake and release. The coding sequence for the serotonin transporter in the FHR has yet to be reported. No differences were found in the predicted amino acid sequence between these two strains of rat, either in the platelet or the lung samples or when compared with the published sequence of the brown rat. We conclude that differences in the primary structure of the serotonin transporter gene do not account for the altered serotonin storage in the FHR strain.

Treatment of pulmonary hypertension with the continuous infusion of a prostacyclin analogue, iloprost.

OBJECTIVE: To compare prostacyclin with an analogue, iloprost, in treatment of severe pulmonary hypertension. PATIENTS: Eight patients with severe pulmonary hypertension: primary in five, thromboembolic pulmonary hypertension in three. METHODS: All patients underwent right heart catheterisation. Mean (SEM) right atrial pressure was 9.9 (2.2) mm Hg, mean pulmonary artery pressure 67.4 (3.0) mm Hg, cardiac index 1.75 (0.13) l/min/m2 and mixed venous oxygen saturation 59.1(3.1)%. Continuous intravenous epoprostenol (prostacyclin, PGI2) or iloprost was given for phase I (three to six weeks); the patients were then crossed over to receive the alternate drug in an equivalent phase II. MAIN OUTCOME MEASURES: Exercise tolerance was measured at baseline and at the end of phase I and II with a 12 minute walk; distance covered, rest period, percentage drop in arterial oxygen saturation (delta Sao2%) and percentage rise in heart rate (delta HR%). RESULTS: Walking distance covered rose from (mean (SEM)) 407.5 (73) to 591 (46) m with PGI2 (p = 0.004) and to 602.5 (60) m while on iloprost (p = 0.008). Rest period decreased from 192 (73) seconds at baseline to 16 (16) seconds with PGI2 (p = 0.01) and to 58 (34) seconds with iloprost (p = 0.008). Delta HR% was 37.5(6)% at baseline, 35(3)% on PGI2, and 24(6)% on iloprost (p = 0.04). CONCLUSIONS: Both intravenous PGI2 and iloprost caused significant improvement in exercise tolerance. Iloprost offers an alternative to PGI2 treatment of severe pulmonary hypertension.

Induction of nitric oxide synthase II does not account for excess vascular nitric oxide production in experimental cirrhosis.

BACKGROUND/AIMS: Excess production of nitric oxide (NO) reduces vasoconstriction of cirrhotic rat aorta. Expression of the inducible nitric oxide synthase (NOS II) in endothelial cells or vascular smooth muscle could account for this, as described with endotoxin or lipopolysaccharide (LPS) treatment. Alternatively, the endothelial NOS enzyme (NOS III) could be activated by an as-yet undescribed mechanism. Here we describe a combined study of the basal release of NO and quantitative measurement of the mRNA for NOS II and NOS III from thoracic aorta of an animal model of cirrhosis. METHODS: Thoracic aortas of six normal, six cirrhotic (secondary biliary cirrhosis) and six intra-peritoneal LPS-treated (15 mg/kg) rats were removed. Dissected aortic rings were precontracted with norepinephrine (NE; 10(-6) M) and relaxed with acetylcholine (Ach; 10(-6) M) with or without pre-incubation with the specific NOS inhibitor (L-NNA, 10(-5) M). Total RNA was extracted from aorta, reverse transcribed (RT) and used in polymerase chain reaction (PCR) amplification with primers specific for NOS II, NOS III and beta-actin. PCR products were hybridized with fluorescein labelled cDNA probes and the relative intensity on film was analyzed by densitometry. RESULTS: Compared to normal, NE caused 32% less contraction in cirrhotic and 43% less contraction in LPS-treated aortic rings. This response was corrected to normal by L-NNA pre-incubation. All the contracted rings relaxed with Ach. NOS II mRNA, was expressed only in LPS-treated aorta and not in aorta from normal and cirrhotic rats. NOS III mRNA levels were the same in normal, cirrhotic and LPS-treated rats: 1176 +/- 170, 1233 +/- 626 and 979 +/- 423, in arbitrary densitometry units, respectively. CONCLUSIONS: NO is overproduced by aorta from cirrhotic and LPS-treated rats. In LPS-treated rats this could result from expression of NOS II, but this was not the case in the cirrhotic rat aorta. Comparable amounts of NOS III mRNA suggest that, in cirrhosis, increased activity of this enzyme and not increased NOS III expression is responsible for the overproduction of NO.

Bronchiolitis obliterans syndrome in heart-lung transplantation: surveillance biopsies.

Transbronchial biopsies (TBBs) are useful to diagnose acute rejection and infection in patients with lung transplants. The value of routine surveillance biopsies (S-TBBs) is not known, and such biopsies with a clinical indication are not without risk and are expensive. One hundred twenty-six 6-mo survivors of heart-lung transplantation (HLT) were studied to determine the effect of stopping S-TBBs on the development of bronchiolitis obliterans syndrome (BOS) and subsequent survival. Fifty-one received transplants while S-TBB was part of routine care (group A), and 75 received transplants after this practice was stopped (group B). There was no difference in patient characteristics. Group A had shorter graft ischemia (p < 0.01) and longer postoperative ventilation (p < 0.01). Maintenance immunosuppression was similar, but group A had more steroid pulses in the second 6 mo after HLT (p < 0.01). The number of patients free from any functional deterioration at 49 to 60 mo after HLT declined to 39% in group A and 64% in group B. The risk of developing BOS grade 1 in group A relative to group B was 1.63 (95% confidence intervals: 0.96-2.79, p = 0.07). Patient survival was similar in the two groups. A total of 86 TBBs were taken in the absence of any signs or symptoms and had low diagnostic yield. In summary, there was no increased incidence of BOS after stopping S-TBBs.

Inhaled nitric oxide and arterial oxygen tension in patients with chronic obstructive pulmonary disease and severe pulmonary hypertension.

BACKGROUND: Inhaled nitric oxide (NO) is a selective pulmonary vasodilator which can improve gas exchange in acute lung injury. However, it is uncertain that this effect on arterial oxygenation can be generalised to all lung diseases. METHODS: The effects of inhaled NO on gas exchange were studied in nine patients with chronic obstructive pulmonary disease (COPD), 11 patients with severe pulmonary hypertension, and 14 healthy volunteers. A randomized sequence of 40 ppm of NO or air was inhaled for 20 minutes through an orofacial mask. RESULTS: Inhaled NO reduced mean (SE) transcutaneous arterial oxygen tension (TcPO2) from 9.6 (0.3) to 8.9 (0.4) kPa in healthy volunteers and from 7.4 (0.6) to 7.0 (0.5) kPa in patients with COPD. There was no change in TcPO2 in patients with severe pulmonary hypertension. During inhalation of NO and air no change occurred in transcutaneous arterial carbon dioxide tension (TcPCO2), arterial oxygen saturation (SaO2) measured by pulse oximeter, or cardiac output determined by the transthoracic impedance method. CONCLUSIONS: Inhaled NO does not improve TcPO2 nor increase cardiac output in normal subjects and patients with COPD, suggesting that inhaled NO worsens gas exchange. This could represent inhaled NO overriding hypoxic pulmonary vasoconstriction in COPD. The finding that TcPO2 also fell when normal subjects inhaled NO suggests that a similar mechanism normally contributes to optimal gas exchange. Whilst inhaled NO can improve oxygenation, this effect should not be considered to be a general response but is dependent on the type of lung disease.

Measurement of health-related quality of life before and after heart-lung transplantation.

BACKGROUND: The measurement of the quality of the outcome of treatment as viewed by patients is becoming increasingly recognized as an important aspect of decision making in all health services. This major study, which set out to measure the health-related quality of life outcomes of heart and lung transplantation, developed from the experience gained in the United Kingdom and United States studies of the cost and benefits of heart transplantation in the 1980s. METHODS: The design was prospective with a cohort of patients completing a variety of generic and specific health-related quality of life questionnaires at intervals before and after heart and lung transplantation. The sample size was not prescribed; one of the aims of the project was to test the feasibility of introducing routine monitoring of health-related quality of life outcomes as an integral part of a developing transplant service. RESULTS: Before the operation, there was evidence of deterioration over time in all dimensions of the Nottingham Health Profile. In comparing Profile scores at less than 3 months before with those at 3 to 6 months after transplantation, statistically significant improvements were evident (p < 0.001). Mean scores at intervals up to 2 years after transplantation showed little change over time and compared well with those from a general population sample. Hospital Anxiety and Depression Scale scores were reduced significantly (p < 0.01) by 1 year after heart and lung transplantation. In the pretransplantation period, at least 90% of 101 patients had some level of restriction in home and leisure activities, reducing to between 2% and 24% at 1 year after transplantation. Similarly, 79 patients (78%) had chest pain, and 101 (100%) were breathless before transplantation, reducing to 54% and 39%, respectively, at 1 year after surgery; by which time, for 8 of 10 patients, the problem was occasional. CONCLUSIONS: Highly significant improvements were observed in the physical, social, and emotional dimensions of health-related quality of life of patients after heart-lung transplantation. The advantages and feasibility of combining generic and condition-specific questionnaires are shown together with the need to develop new measures with greater sensitivity to the smaller peaks and troughs of recovery.

Heart-lung-liver transplantation.

We report three patients with cystic fibrosis and one patient with primary biliary cirrhosis and plexogenic pulmonary hypertension who have undergone heart-lung-liver transplantation as a combined procedure. Liver transplantation was necessary in the three patients with cystic fibrosis because of portal hypertension secondary to either hepatic fibrosis or established cirrhosis in addition to their advanced lung disease. Three of the four patients were alive at 20, 50, and 100 months after transplantation (one patient with cystic fibrosis died on day 16 of pneumonia) with well-preserved pulmonary function (forced expiratory volume in 1 second 110%, 49%, and 100% predicted, respectively), normal hepatic function and New York Heart Association class 1 performance status. Heart-lung and concurrent liver transplantation is a feasible and successful procedure with a satisfactory long-term outcome in selected patients with advanced pulmonary and hepatic disease.