Kunihiko Shindo

Measurement of leukotriene B4 in arterial blood of asthmatic patients during wheezing attacks

Abstract. To investigate whether leukotriene B4 is present in the arterial blood of asthmatic patients during wheezing attacks. 20 ml of arterial blood was drawn from the inguinal artery of five patients. Leukotriene B4 was detected in all five individuals, and its identity was confirmed by a combination of high pressure liquid chromatography and radioimmunoassay techniques. The concentration of leukotriene B4 was 48.34 ± 16.27 pg ml−1 (mean value ± SE). However, in five control subjects the leukotriene B4 concentration was found to be 9.43 ± 5.44 pg ml−1. Thus there was a significant difference between the two groups (P < 0.05). These results suggest that leukotriene B4 may be important in elucidation of the pathogenesis of bronchial asthma.

Plasma levels of leukotriene E4 during clinical course of chronic obstructive pulmonary disease

We investigated the relationship between circulating leukotriene E4 (LTE4) and chronic obstructive pulmonary disease (COPD) by measuring plasma levels of leukotriene E4 in patients with COPD and 10 normal controls. We also investigated the relationship between LTE4 levels and FEV1 and PaO2. Leukotriene E4 was measured by high performance liquid chromatography (HPLC) and radioimmunoassay. The mean leukotriene E4 level in patients with COPD during remission, during acute exacerbation before and after prednisolone treatment were 16.8[4.02], 41.7[21.9], and 19.5[3.78] pg/ml (mean[SD]), respectively. In contrast, the mean leukotriene E4 level of 10 normal controls was 11.8[4.49] pg/ml. Thus, the mean LTE4 level during an acute exacerbation of COPD was significantly lower in patients after prednisolone treatment than in patients before prednisolone treatment. The mean LTE4 level in patients after prednisolone treatment did not significantly differ from that in patients during remission and in normal controls (Scheffe F-test, P < 0.05) (Fig. 1). Mean FEV1 (% predict) values were 51.4[9.02] (mean[SD]), 38.0[4.82], and 44.2[4.48] on the three occasions, respectively; corresponding mean PaO2 values (mmHg) were 84.0[5.01] (mean[SD]), 61.3[1.66], and 80.6[5.30], respectively. Leukotriene E4 levels were significantly correlated with PaO2 and relatively with FEV1 in the patients during acute exacerbation before prednisolone treatment. Thus, we suggest that leukotriene E4 levels in arterial blood reflect the severity of COPD lung and oral prednisolone reduces the plasma levels of leukotriene E4 in patients with COPD.

The deconjugation ability of bacteria isolated from the jejunal fluids in the blind loop syndrome with high 14CO2 excretion using the breath analysis technique and thin-layer chromatography

Five patients with blind loop syndrome (Billroth II) were examined by measuring 14CO2 specific activity of expired breath samples taken at intervals after a meal containing glycine-1-14C cholate. The 5 patients tested showed a marked increase of 14CO2 specific activity. Furthermore, the ability of deconjugation of bacteria isolated from the jejunal fluids in the efferent loop of these patients was tested by thin layer chromatography. The bacterial species identified from the samples were as follows: enterococcus, Lactobacillus (L) buchneri, L. bifidus, L. brevis, Eubacterium (E) lentum, Bacteroides (B) vulgaricus, B. filamentosum, Corynebacterium (C) granulosum, Escherichia (E) coli, Staphylococcus (S) epidermidis, and Aerobacter (A) aerogenes. These species of bacteria, except E. coli and A. aerogenes, showed the deconjugation ability by which conjugated bile acids in ox gall was hydrolyzed. Administration of chloramphenicol (1g per day for 14 days orally divided doses) to the 5 patients reduced 14CO2 specific activity significantly. On the other hand, 9 healthy men (control subjects) who were tested showed a flat curve, and 8 of the 9 had no growth of bacteria isolated from the jejunal fluids. The remaining healthy man showed an overgrowth of E. coli and Pseudomonas (P) aeruginosa, but the species did not have the ability of deconjugation. Thus, we concluded that the patients with blind loop syndrome(Billroth II) had the bacterial overgrowth in the efferent loop that contained species with deconjugation ability, and, as a result the bacterial overgrowth contributed to causing abnormalities (increased deconjugation) in the metabolism of bile acids in the small intestine. When the concentration of conjugated bile acids in the small intestine was reduced to levels below the critical micellar concentration by several factors, fat malabsorption and subsequent steatorrhea were induced (1,-4). Furthermore, H. Fromm and A. F. Hofmann presented in vivo that the patients with blind loop syndrome had fat malabsorption and the patients who had a high 14CO2 output after oral administration of glycine-1-14C cholate showed a low 14CO2 output after oral administration of antibiotic drug (5,6). However, there has been no report on the deconjugation ability of bile acids of bacteria isolated from the jejunal fluids in the efferent loop of patients with Billroth II who had positive breath tests.(ABSTRACT TRUNCATED AT 400 WORDS)

Deconjugation of bile acids by human intestinal bacteria

SummaryThe purpose of this report is to present the deconjugation of bile acids by numbers of strains of bacteria in the small intestine and feces. The small intestinal juice was aseptically aspirated by a double lumen tube with a rubber cover on the tip devised by us (“Fukushima Type 1”). Bile acids were analyzed with thin layer chromatography. The results: 1) Among aerobic bacteria, species of which all of the strains split conjugated bile acids was enterococcus, and most of the strains split were Staphylococcus (S.) epidermidis and Lactobacillus (L.) bifidus. Species of which none of the strains split were Escherichia (E.) coli, E. communior, E. freundii, L. plantarum, L. acidophilus, L. buchneri, L. cellobiosus, L. bulgaricus, S. aureus, Aerobacter aerogenes, Pseudomonas aeruginosa, Candida, proteus, serratia, and almost none of the species split was Intermediate coliform bacilli. 2) Among anaerobic bacteria, species of which all of the strains split were Bacteroides (B.) vulgatus, B. thetaiotaomicron, B. uniformis, Corynebacterium (C.) granulosum, C. avidum, Peptostreptococcus (Peptostrept.) putridus, Eubacterium (Eubact.) lentum, Peptococcus (Pept.) grigoroffii, Pept. anaerobius, Veillonella (V.) orbiculus, and most of the strains split were Coryne. diphtheroides, Eubact. parvum, Peptostrept. intermedius. Species of which none of the strains split were Coryne. parvum, Peptostrept. micros, V. alcalescens, V. parvula, Catenabacterium (Catena.) catenaforme, and Catena, filamentosum. 3) All or none, or almost all or none, of the strains of each species tested split conjugated bile acids, and it seems probable that the presence or absence of this ability would be a proper character of each species.

Effect of doxazosin therapy on glucose tolerance and lipid metabolism in hypertensive patients with impaired glucose tolerance

The effects of long-term monotherapy with doxazosin, an alpha 1-blocker, or placebo on blood pressure (BP), glucose tolerance, and serum lipid levels were investigated prospectively in 43 hypertensive patients with impaired glucose tolerance. The levels of plasma glucose, serum lipids, fructosamine, and glycated hemoglobin A1c (Hb A1c) were determined before and during long-term (mean treatment period, 6.7 months) therapy with doxazosin (n = 23) or placebo (n = 20). A 75-g oral glucose tolerance test was performed before and during therapy. Significant decreases in both systolic and diastolic BP were maintained during doxazosin therapy; BP did not change in the placebo group. Neither fasting nor post-glucose-load venous plasma glucose levels were altered, and there was no significant change in the insulinogenic index in either group. Glucose intolerance was slightly improved with significant reductions in Hb A1c and fructosamine levels during doxazosin therapy. Serum total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol levels were significantly decreased, and high-density lipoprotein cholesterol levels were significantly increased in patients treated with doxazosin. Moreover, TC, LDL cholesterol, and apolipoprotein B levels were significantly decreased in patients with hypercholesterolemia (TC > or = 5.69 mmol/L). In contrast, there were no significant changes in Hb A1c, fructosamine, and lipid levels in the placebo group. These results suggest that long-term doxazosin therapy may improve glucose and lipid metabolism in hypertensive patients. Doxazosin appears useful as an antihypertensive agent for hypertensive patients with either impaired glucose metabolism or dyslipidemia.

Effect of cilazapril therapy on glucose and lipid metabolism in patients with hypertension

The effects of long-term monotherapy with cilazapril, an angiotensin-converting enzyme inhibitor, on blood pressure, glucose tolerance, and serum lipid profiles were prospectively investigated in 66 patients with hypertension: 23 with normal glucose tolerance and 43 with glucose intolerance (including 9 patients with non-insulin-dependent diabetes mellitus). The levels of plasma glucose, serum insulin, serum lipids, glycated hemoglobin A(lc) (Hb A(lc)), and fructosamine were determined before and during long-term (mean +/- SD, 26.2 +/- 1.2 weeks) therapy with cilazapril. A 75-g oral glucose tolerance test was performed before and during treatment. Significant reductions in both systolic and diastolic blood pressures in both patient groups were maintained during the study. Neither fasting nor post-glucose load venous plasma glucose levels were altered in either group of patients, and no patient with normal glucose tolerance developed diabetes mellitus during the study. There was no significant change in the insulinogenic index (delta serum insulin/delta venous plasma glucose at 30 minutes post-glucose load) in either group, and glucose intolerance was slightly improved with significant reductions (P < 0.01) in Hb A(lc) and fructosamine in the patient group with impaired glucose tolerance. Serum total cholesterol (TC), low-density lipoprotein cholesterol, and triglyceride levels were significantly (P < 0.01) decreased and high-density lipoprotein cholesterol levels increased in patients with hypercholesterolemia (TC levels > or = 5.69 mmol/L). These results suggest that long-term cilazapril therapy may improve glucose and lipid metabolism in hypertensive patients with impaired glucose tolerance. Cilazapril also appears to be useful as an antihypertensive agent for hypertensive patients with either impaired glucose tolerance or hypercholesterolemia.

Cough‐Challenge Trial with a New Angiotensin‐Converting Enzyme Inhibitor, Imidapril

This study was conducted to examine whether imidaprilat, an active diacid of the angiotensin‐converting enzyme (ACE) inhibitor imidapril, preferentially inhibits angiotensin I degradation rather than bradykinin degradation, and whether imidapril is less active than other ACE inhibitors in inducing cough in patients with hypertension. The effect of imidaprilat on the inhibition of pressor response to angiotensin I and augmentation of depressor response to bradykinin was compared with that of enalaprilat and captopril in anesthetized rats. To determine the incidence of cough associated with imidapril, patients with a history of ACE inhibitor‐induced dry cough were enrolled in a randomized, open‐labeled, crossover trial with two 6‐week periods to be treated with imidapril or amlodipine, a calcium‐channel blocker. The recurrence of cough was assessed during both treatments. In the animal study, there were no significant differences in the ratio of inhibition of pressor response to angiotensin I and the augmentation of depressor response to bradykinin among the ACE inhibitors. In the cough‐challenge trial, a total of 60 patients with hypertension were enrolled in the study. Cough and cough related symptoms recurred in 98.3% of the patients (59/60) during imidapril therapy. In contrast, only two patients reported cough during treatment with amlodipine. These results indicate that imidapril has no selectivity in inhibiting angiotensin I‐ and bradykinin‐degradation in rats, and that clinically it is not different from other ACE inhibitors in inducing cough in patients with hypertension.

Azelastine hydrochloride inhibits platelet activating factor-like activity in human eosinophils.

We investigated the inhibitory effect of azelastine hydrochloride (azelastine), an anti-asthmatic drug, on platelet-activating factor (PAF)-like activity in eosinophils obtained from asthmatic and non-asthmatic patients. Eosinophils were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 mumol) for 15 min. PAF-like activity was detected by aggregation of washed guinea-pig platelets. PAF-like activity released from asthmatic eosinophils without preincubation of azelastine was 2.36 [1.02] (mean [SD], ng/10(7) cells) in supernatants and 13.87 [4.77] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M azelastine, PAF-like activity reduced to 1.85 [0.46] (mean [SD], ng/10(7) cells), 1.11 [0.14], and 0.88 [0.09] (n = 15) in the supernatants, and 11.83 [2.93], 8.32 [1.41], and 6.27 [1.25] (n = 15) in the cell pellets, respectively. PAF-like activity in non-asthmatic eosinophils without preincubation of azelastine was 2.01 [0.86] (mean [SD], ng/10(7) cells) in supernatants and 7.44 [0.99] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M azelastine, PAF-like activity reduced to 1.73 [0.64] (mean [SD], ng/10(7) cells), 1.12 [0.23], and 0.84 [0.17] (n = 20) in the supernatants, and 6.26 [2.08], 4.65 [0.88], and 3.02 [0.43] (n = 20) in the cell pellets, respectively. Our results showed that preincubation with azelastine caused a dose-dependent inhibition of intra and extracellular PAF-like activity from asthmatic and non-asthmatic eosinophils in the same manner.

Effect of azelastine hydrochloride on release and production of platelet activating factor in human neutrophils

Effect of azelastine hydrochloride (azelastine) on release and production of platelet-activating factor (PAF) in neutrophils obtained from asthmatic and non-asthmatic patients was investigated. Neutrophils were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 microM) for 15 min. PAF-like activity was detected by aggregation of washed guinea pig platelets. PAF-like activity released from asthmatic neutrophils without preincubation of azelastine was 5.67[0.89] (mean[SD], ng/10(7) cells) in supernatants and 21.8[0.76] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M of azelastine, PAF-like activity reduced to 5.96[0.97] (mean[SD], ng/10(7) cells), 3.49[0.63], and 1.89[0.09] (n = 15) in the supernatants, and 20.7[0.97], 13.9[0.29], and 8.91 [0.99] (n = 15) in the cell pellets, respectively. PAF-like activity in non-asthmatic neutrophils without preincubation of azelastine was 4.67[0.19] (mean[SD], ng/10(7) cells) in supernatants and 18.5[0.34] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M of azelastine, PAF-like activity reduced to 4.39[0.51] (mean[SD], ng/10(7) cells), 2.77[0.22], and 1.75[0.07] (n = 15) in the supernatants, and 17.9[0.54], 10.8[0.25], and 5.97 [0.59] (n = 15) in the cell pellets, respectively. Our results showed that preincubation with azelastine caused a dose-dependent inhibition of intra and extracellular PAF-like activity from asthmatic and non-asthmatic neutrophils in the same manner.

Inhibitory effect of amphotericin B on leukotriene B4 synthesis in human neutrophils in vitro

Our objective was to determine the effect of amphotericin B on leukotriene (LT) A4 hydrolase in human neutrophil cytosol and to examine its effect on intact neutrophils in vitro. Cytosolic fractions were assayed for LTA4 hydrolase and 5-lipoxygenase activity in the presence or absence of amphotericin B. The IC50 of amphotericin B for LTA4 hydrolase activity was 0.72 microM. No inhibition of 5-lipoxygenase activity in the cytosolic fraction was detected. The IC50 of amphotericin B for leukotriene B4 synthesis in intact neutrophils was 0.43 microM. The 5-hydro(per) oxy-eicosatetraenoic acid (5-H(P)ETE) synthesis was diminished in intact cells by 66.8 [3.4]% (mean[SEM]) in the presence of 0.01 mM amphotericin B. Thus, amphotericin B inhibited the synthesis of LTB4 and 5-H(P)ETE in neutrophils in vitro. Differences between the results of studies on cytosol and on intact cells suggest that amphotericin B is involved in a complex interaction in the intact cell.