Akihiro Matsumoto

Increased Nitric Oxide in the Exhaled Air of Patients with Decompensated Liver Cirrhosis

Patients with liver cirrhosis often present with several systemic hemodynamic disturbances, including hypotension, low systemic vascular resistance, and a reduced sensitivity to vasoconstrictors [1]. As cirrhosis progresses, vascular resistance continues to decrease, and the low arterial pressure may lead to secondary disturbances in renal and hepatic blood flow and to ascites [1]. The precise mechanisms of these hemodynamic disorders have not yet been clearly elucidated. Excessive production of vasodilators, such as prostacyclin, bradykinin, substance P, and atrial natriuretic peptide, has been proposed, but there is no clear evidence to show that vasodilators are involved. Vallance and Moncada [2] hypothesized that nitric oxide, originally discovered as an endothelium-derived relaxing factor [3], may be a causative factor in hemodynamic disorders in patients with liver cirrhosis. High concentrations of circulating endotoxin are frequently found in patients with cirrhosis who have no clinical evidence of infection [4]. Thus, the endotoxemia of liver cirrhosis may induce nitric oxide synthase directly in blood vessels or indirectly through cytokines, leading to an increased synthesis and release of nitric oxide that may account for the hemodynamic abnormalities. Recent studies show that nitric oxide concentration in exhaled air can be measured [5-7] and that it is increased in patients with bronchial asthma [5, 6]. To test the hypothesis that an increased synthesis and release of nitric oxide accounts for hemodynamic abnormalities in patients with liver cirrhosis, we investigated whether nitric oxide output in exhaled air is increased in these patients. Methods Patients Fifty-six patients were consecutively selected from those hospitalized in our department. All had biopsy-proven chronic hepatitis or liver cirrhosis; none had primary lung disease, hypertension, or infection. They could walk in the ward unaided and did not need intensive care. Physical examination findings and blood data were analyzed to classify hepatocellular function in liver cirrhosis according to the Child criteria. The clinical background of these patients is summarized in Table 1. Healthy volunteers served as controls (15 men; 34 2 years of age; body surface area, 1.84 0.03 m2). All medications were discontinued 24 hours before each study began. No antihypertensives or vasodilators, including nitrates and angiotensin-converting enzyme inhibitors, were used in these patients. The study was approved by the hospital ethics committee, and informed consent was obtained from each study participant. Table 1. Clinical Background of Patients with Chronic Hepatitis and Liver Cirrhosis Nitric Oxide Measurement The nitric oxide concentration in exhaled air was determined at least 3 hours after meals while each participant was at rest in the sitting position, as previously described [7]. Each participant was asked to inhale synthetic air (Taiyo Sanso Co., Osaka, Japan) free of nitric oxide (< 3 parts per billion [ppb]) through a mask and a T-valve, and to exhale the air into a wide-bore Teflon tube (internal diameter, 25 mm; length, 600 mm). Exhaled air was continuously drawn from this tube with a vacuum pump and was introduced into a chemiluminescence analyzer (APNE-350E, Horiba Co., Kyoto, Japan). Measurement of nitric oxide concentration was based on the reaction of nitric oxide with ozone. The sensitivity of the analyzer to nitric oxide ranged from 2 to 1000 ppb. The system was calibrated with dilutions of certified nitric oxide gas (450 ppb in nitrogen; Taiyo Sanso Co.) using mass flowmeters (Estec Co., Kyoto, Japan). Expired volume was measured with a hot-wire flow meter connected to the T-valve on the expiratory side, and minute ventilation was calculated using a breath-by-breath respirometer (RM-280, Minato Medical Science Co., Tokyo, Japan). The nitric oxide concentration and minute ventilation were recorded with a computer-assisted data recorder (DS1100, Fukuda Denshi Co., Tokyo, Japan), and the output of nitric oxide was calculated as follows: nitric oxide output = (nitric oxideex nitric oxidein) x minute ventilation/body surface area, where nitric oxideex was the nitric oxide concentration in exhaled air, and nitric oxidein was the nitric oxide concentration in inhaled air. Nitric oxide concentration and minute ventilation were monitored simultaneously for 10 minutes, and the data obtained during the last 3 minutes was averaged. During the study period, the ambient levels of nitric oxide concentration were less than 5 ppb. Nitric oxide output was reproducible in patients with cirrhosis and in controls (coefficient of variation, 10.8% [n = 5] for patients and 9.3% [n = 5] for controls) on separate days, and there was no significant time-course change in nitric oxide output at rest. Echocardiographic Measurement To examine the relation between systemic hemodynamics and nitric oxide production, we measured cardiac output using transthoracic two-dimensional echocardiography (SSD-2200, Aloka Co., Tokyo, Japan) in 19 patients with liver cirrhosis and in 6 controls. This was done on the same day that nitric oxide concentrations were measured. A physician, who was blinded to the patient characteristics and the exhaled nitric oxide output values, obtained echocardiographic views and recorded them on videotape. Another physician, who was also blinded to these data, measured cardiac output using the echocardiographic images. Left ventricular dimension was measured in the long-axis view of the left ventricle while the patient was in the left lateral decubitus position. Left ventricular volume and cardiac index were obtained by the following formulae according to the Teichholz equation [8]: left ventricular volume = 7.0 x dimension3/(2.4 + dimension); cardiac index = (left ventricular end-diastolic volume -end-systolic volume) x heart rate/body surface area. Blood pressure was measured with a sphygmomanometer. Total peripheral resistance index was calculated as mean blood pressure 80/cardiac index. The cardiac index obtained by this method on separate days was reproducible in patients with cirrhosis and in controls (coefficient of variation, 9.1% [n = 6] in patients and 8.6% [n = 5] in controls). Statistical Analysis Values are expressed as the mean SE. Differences between patients and controls were compared using one-way analysis of variance (ANOVA) followed by the Fisher test. The correlation coefficient was calculated using the least-squares method. Statistical significance was set at P < 0.05. Results Patients with decompensated liver cirrhosis had markedly depressed liver function but normal serum creatinine levels (Table 1). There were no intergroup differences in minute ventilation per m2 body surface area (patients with chronic hepatitis, 5.1 0.3 L/min; Child A patients, 5.6 0.3 L/min; Child B patients, 5.4 0.2 L/min; Child C patients, 6.2 0.3 L/min; and controls, 5.4 0.2 L/min; P = 0.12). The level of exhaled nitric oxide output per m2 body surface area was significantly greater in patients with Child C (190 11 nL/min; P < 0.001) or Child B liver cirrhosis (166 12 nL/min; P < 0.001) than in controls (97 8 nL/min) (Figure 1). In patients with Child A liver cirrhosis (119 10 nL/min; P = 0.17) or chronic hepatitis (129 19 nL/min; P = 0.13), the level of nitric oxide output per m2 body surface area was similar to that in controls. Figure 1. Nitric oxide (NO) output in exhaled air in controls, patients with chronic hepatitis (CH), and patients with liver cirrhosis. The results of hemodynamic measurements showed that patients with Child C liver cirrhosis had a greater cardiac index per m2 body surface area (4.3 0.3 L/min compared with 2.9 0.2 L/min; P < 0.001) and a smaller total peripheral resistance per m2 body surface area (1732 125 dyne/s x cm5 compared with 2680 235 dyne/s x cm5; P = 0.004) than controls. There was a positive correlation between the level of nitric oxide output and cardiac index (r = 0.621; P < 0.001) (Figure 2). Figure 2. Relation between nitric oxide (NO) output and cardiac index in patients with liver cirrhosis and controls. Discussion We have shown that nitric oxide output is increased in the air exhaled by patients with cirrhosis, especially patients with decompensated cirrhosis. Although we did not identify the origin of the increased synthesis of nitric oxide, several potential sources can be considered. Patients with liver cirrhosis often have endotoxemia even when they have no signs of infection [4], and elevated concentrations of cytokines, such as tumor necrosis factor-, have been shown in patients with liver diseases [9, 10]. The liver may produce large amounts of nitric oxide in these patients: Hepatocytes and Kupffer cells are known to produce nitric oxide in vitro in response to lipopolysaccharide and several cytokines [11, 12]. The plasma levels of cytokines, including tumor necrosis factor, are much lower in patients with liver cirrhosis than in these in vitro studies [10-15]. However, in vitro studies have also shown that endotoxin and cytokines also induce nitric oxide synthase in other tissues, including vascular endothelium, smooth muscle, and bronchial epithelium [13-15]. Thus, it is possible that vascular and bronchial tissues in the lungs of patients with liver cirrhosis produce nitric oxide as a result of continuous stimulation by the lower concentrations of cytokines, because the plasma levels of cytokines in patients with cirrhosis are similar to those in normal persons who have become hypotensive through the administration of endotoxin [10, 16]. Because most nitric oxide is inactivated by hemoglobin or rapidly metabolized to nitrite and nitrate [3], nitric oxide in exhaled air may be the residual of excessive local production of nitric oxide by the lung rather than a product of the liver. Because plasma nitrite and nitrate levels reflect the sum of nitric oxide production in the entire body, includ

End-tidal CO2 pressure decreases during exercise in cardiac patients: association with severity of heart failure and cardiac output reserve.

OBJECTIVES We measured end-tidal CO2 pressure (PETCO2) during exercise and investigated the relationship between PETCO2 and exercise capacity, ventilatory parameters and cardiac output to determine the mechanism(s) of changes in this parameter. BACKGROUND It is unclear whether PETCO2 is abnormal at rest and during exercise in cardiac patients. METHODS Cardiac patients (n = 112) and normal individuals (n = 29) performed exercise tests with breath-by-breath gas analysis, and measurement of cardiac output and arterial blood gases. RESULTS PETCO2 was lower in patients than in normal subjects at rest and decreased as the New York Heart Association class increased, whereas the partial pressure of arterial CO2 did not differ among groups. Although PETCO2 increased during exercise in patients, it remained lower than in normal subjects. PETCO2 in relation to cardiac output was similar in patients and normal subjects. PETCO2 at the respiratory compensation point was positively correlated with the O2 uptake (r = 0.583, p < 0.0001) and the cardiac index at peak exercise (r = 0.582, p < 0.0001), and was negatively correlated with the ratio of physiological dead space to the tidal volume. The sensitivity and specificity of PETCO2 to predict an inadequate cardiac output were 76.6% and 75%, respectively, when PETCO2 at respiratory compensation point and a cardiac index at peak exercise that were less than the respective control mean-2 SD values were considered to be abnormal. CONCLUSIONS PETCO2 was below normal in cardiac patients at rest and during exercise. PETCO2 was correlated with exercise capacity and cardiac output during exercise, and the sensitivity and specificity of PETCO2 regarding decreased cardiac output were good. PETCO2 may be a new ventilatory abnormality marker that reflects impaired cardiac output response to exercise in cardiac patients diagnosed with heart failure.

Measurement of plasma brain natriuretic peptide level as a guide for cardiac overload.

OBJECTIVES We examined whether measurement of the plasma BNP concentrations might be useful for the early diagnosis of the existence and severity of disease in patients with heart disease in daily clinical practice. METHODS AND RESULTS The plasma BNP and ANP concentrations in 415 patients with heart disease and hypertension and 65 control subjects were measured. Patients with heart disease had higher plasma BNP and ANP concentrations than did those with hypertension or control subjects. Among the etiology of cardiac diseases, specifically dilated cardiomyopathy and hypertrophic cardiomyopathy, was associated with the highest plasma BNP concentrations, whereas dilated cardiomyopathy was associated with the highest plasma ANP concentrations. Plasma BNP concentrations showed an increase as the severity of the heart disease, as graded according to the NYHA classification of cardiac function, increased. In both patients with heart disease and hypertension, the plasma BNP values were higher in those who had abnormalities in their echocardiogram and electrocardiogram as compared to those without any abnormalities. The plasma BNP levels also showed a significant correlation with left ventricular wall thickness and left ventricular mass. On the other hand, the plasma ANP levels showed significant correlations with left ventricular dimension. Receiver operative characteristic analysis revealed that plasma BNP levels showed substantially high sensitivity and specificity to detect the existence of heart diseases. CONCLUSION Measurements of the plasma BNP concentrations is useful to detect the existence of the diseases, and abnormalities of left ventricular function and hypertrophy in patients with heart disease in daily clinical practice.

Effects of exercise on plasma level of brain natriuretic peptide in congestive heart failure with and without left ventricular dysfunction

This study was designed to determine whether plasma brain natriuretic peptide (BNP) increases in response to exercise in patients with congestive heart failure and to show what kind of hemodynamic abnormalities induce increased secretion of BNP during exercise. Plasma levels of atrial natriuretic peptide (ANP) and BNP and hemodynamic parameters were measured during upright bicycle exercise tests in seven patients with dilated cardiomyopathy and nine with mitral stenosis. At rest, there were no intergroup differences in cardiac output or pulmonary capillary wedge pressure; however, the group with dilated cardiomyopathy had higher left ventricular end-diastolic pressures and lower left ventricular ejection fractions than did the group with mitral stenosis. Plasma ANP levels were comparable between the dilated cardiomyopathy group (170 +/- 77 [SE] pg/ml) and the mitral stenosis group (106 +/- 33 pg/ml) (p, not significant), whereas BNP was significantly higher in the dilated cardiomyopathy group (221 +/- 80 pg/ml) than in the other group (37 +/- 10 pg/ml) (p < 0.05). The plasma concentration of BNP but not of ANP significantly correlated with left ventricular end-diastolic pressure and volume. Exercise increased plasma ANP and BNP in the two groups. The dilated cardiomyopathy group had a larger increment in BNP (+157 +/- 79 pg/ml) than did the mitral stenosis group (+17 +/- 5 pg/ml) (p < 0.05), although the increase in pulmonary capillary wedge pressure was greater in the mitral stenosis group. Thus exercise increases plasma levels of BNP, and impaired left ventricular function may be a main factor in the greater increment in BNP during exercise in patients with congestive heart failure.

Bile acids increase intracellular Ca2+ concentration and nitric oxide production in vascular endothelial cells

The effects of bile acids on intracellular Ca2+ concentration [Ca2+]i and nitric oxide production were investigated in vascular endothelial cells. Whole‐cell patch clamp techniques and fluorescence measurements of [Ca2+]i were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO2−. Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca2+]i concentration‐dependently, while cholic acid showed no significant effect. These effects resulted from the first mobilization of Ca2+ from an inositol 1,4,5‐triphosphate (IP3)‐sensitive store, which was released by ATP, then followed by Ca2+ influx. Both bile acids and ATP induced the activation of Ca2+‐dependent K+ current. Oscillations of [Ca2+]i were occasionally monitored with the Ca2+‐dependent K+ current in voltage‐clamped cells and Ca2+ measurements of single cells. The intracellular perfusion of heparin completely abolished the ATP effect, but failed to inhibit the bile acid effect. Deoxycholic acid and chenodeoxycholic acid enhanced NO2− production concentration‐dependently, while cholic acid did not enhance it. The bile acids‐induced nitric oxide production was suppressed by NG‐nitro‐L‐arginine methyl ester, exclusion of extracellular Ca2+ or N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesulphonamide hydrochloride (W‐7) and calmidazolium, calmodulin inhibitors. These results provide novel evidence showing that bile acids increase [Ca2+]i and subsequently nitric oxide production in vascular endothelial cells. The nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in liver diseases including cirrhosis.

Increased excretion of nitric oxide in exhaled air of patients with chronic renal failure

Nitric oxide exerts multiple effects on renal function. It remains unclear whether endogenous nitric oxide production is increased or decreased in patients with chronic renal failure. To evaluate endogenous nitric oxide production in these patients we studied exhaled nitric oxide output by an ozone chemiluminescence method and plasma NO2(-)/NO3(-) levels by the Griess method in 40 patients with end-stage chronic renal failure who underwent regular continuous ambulatory peritoneal dialysis (n=30) or haemodialysis (n=10), and in 28 healthy subjects. Patients with chronic renal failure had a higher exhaled nitric oxide concentration [39+/-3 versus 19+/-1 parts per billion, (mean+/-S.E.M.), P<0.0001], a greater nitric oxide output (177+/-11 versus 96+/-7 nl.min-1.m-2, P<0.001) and a higher plasma NO2(-)/NO3(-) concentration (96+/-14 versus 33+/-4 micromol, P<0.01) than controls. These values did not differ between patients on haemodialysis and those on continuous ambulatory peritoneal dialysis. Patients with chronic renal failure had significantly higher plasma concentrations of both interleukin-1beta and interferon-gamma than controls. The exhaled nitric oxide output did not correlate with plasma NO2(-)/NO3(-) or with peritoneal dialysate NO2(-)/NO3(-), but plasma NO2(-)/NO3(-) correlated with dialysate NO2(-)/NO3(-) in patients who underwent continuous ambulatory peritoneal dialysis (r=0.77, P<0.01). Haemodialysis for 4 h acutely decreased plasma NO2(-)/NO3(-) (92+/-17 versus 50+/-8 micromol, P<0.05) and cGMP concentration (16.5+/-4.3 versus 5.1+/-1. 7 pmol/ml, P<0.01), but did not decrease exhaled nitric oxide output. The increase in exhaled nitric oxide with the simultaneous increase in circulating cytokines suggests that nitric oxide synthase seems to be induced significantly in patients with chronic renal failure. Increased endogenous nitric oxide production may have a pathophysiological role in patients with uraemia.

Role of insulin resistance in heart and skeletal muscle F-18 fluorodeoxyglucose uptake in patients with non-insulin-dependent diabetes mellitus.

BackgroundAltered heart and skeletal glucose usage has been reported in patients with non-insulin-dependent diabetes mellitus (NIDDM). Although elevations in plasma free fatty acid (FFA) concentrations have been implicated in reduced myocardial 18fluorine-fluoro-2-deoxy-d-glucose uptake (MFU), the specific role of whole-body insulin resistance in MFU in patients with NIDDM compared with skeletal muscle metabolism remains controversial.PurposeMFU and skeletal muscle 18fluorine-fluoro-2-deoxy-d-glucose uptake (SMFU) were compared with positron emission tomography and the whole-body glucose disposal rate (GDR) during hyperinsulinemic euglycemic clamping in 26 normotensive asymptomatic patients with NIDDM who were not taking medication. These factors were also compared in 12 age-matched control subjects to increase the knowledge of the influence of whole-body insulin resistance on MFU. In addition, independent factors for both SMFU and MFU were investigated.ResultsGDR in control subjects (10.0±2.97 mg/min per kilogram) was significantly higher than in patients with NIDDM (4.05±2.37 mg/min per kilogram, P<.01). SMFU in patients with NIDDM (0.826 +-0.604 mg/min per 100g) was significantly lower than that in control subjects (1.86±1.06 mg/min per 100g, P<.01). MFU in patients with NIDDM (5.35±2.10 mg/min per 100 g) was also significantly lower than that of control subjects (7.05±1.66 mg/min per 100 g, P =.0182). SMFU significantly correlated with GDR (r=.727, P<.01) and FFA (r=-.52, P <.01) in patients with NIDDM. MFU also correlated with GDR (r=.778, P<.01) and FFA (r=-.72, P<.01) in patients with NIDDM. Multivariate stepwise regression analysis showed that GDR (F=36.8) was independently related to MFU (r=.85, P<.01) whereas FFA was not (F=1.763), where F is the value for statistical analysis of multivariate stepwise regression analysis.ConclusionInsulin resistance is the most essential factor for both heart and skeletal muscle FDG uptake in patients with NIDDM.

Kinetics of oxygen uptake at onset of exercise related to cardiac output, but not to arteriovenous oxygen difference in patients with chronic heart failure ☆

Patients with chronic heart failure performed exercise tests to evaluate the relation of kinetics of oxygen uptake to cardiac output and arteriovenous oxygen difference at the onset of exercise. The kinetics of oxygen uptake are primarily determined by cardiac output; these kinetics are useful in evaluating exercise intolerance and cardiac output response during exercise.

Inhibition of carnitine synthesis modulates protein contents of the cardiac sarcoplasmic reticulum Ca2+-ATPase and hexokinase type I in rat hearts with myocardial infarction

Abstract It was previously reported than inhibition of carnitine synthesis by 3-(2,2,2-trimethyl-hydrazinium) propionate (MET-88) restores left ventricular (LV) systolic and diastolic function in rats with myocardial infarction (MI). Preservation of the calcium uptake function of sarcoplasmic reticulum Ca2+-ATPase (SERCA2) is one of the possible mechanisms by which MET-88 alleviates hemodynamic dysfunction. To test this hypothesis, the effects of MET-88 on protein content of SERCA2 were evaluated using the same rat model of heart failure. Myocardial protein content of hexokinase, which is one of the key enzymes of glucose utilization, was also measured. Either MET-88 (MET-88 group) or a placebo (MI group) was administered for 20 days to rats with MI induced by coronary artery ligation. The control group underwent sham surgery (no ligation) and received placebo. In LV myocardial homogenates, the myocardial SERCA2 protein content was 32% lower (p<0.05) in the MI group than in the control group. However, in the MET-88 group myocardial SERCA2 content was the same as in the control group. Hexokinase I protein content was 29% lower (p<0.05) in the MI group compared with the control. In contrast, hexokinase II protein content did not differ significantly among the three groups. Consequently, inhibition of carnitine synthesis ameliorates depression of SERCA2 and hexokinase I protein content which may reduce tissue damage caused by MI.

Inhaled nitric oxide and exercise capacity in congestive heart failure

Vol 349 • April 5, 1997 999 most closely related of the previously characterised hantaviruses. The nucleotide sequence of the Rio Mamore virus, a South American virus previously identified in O microtis from Bolivia by Hjelle et al, is not yet available for comparisons from the GeneBank. Our data shows the co-existence of four phylogenetically different new and hantaviruses in central Argentina, two associated with HPS. O flavescens, commonly found along water courses in central Argentina, has been identified as a reservoir for Lechiguanas virus which is associated with HPS in this region.