[Show abstract][Hide abstract] ABSTRACT: Mechanisms underlying gender difference of atherogenesis were investigated focusing on direct effects of estrogen on the artery.
First, male and female apoE(-/-) mice were fed an atherogenic diet for 16 weeks from 10 weeks of age. Second, female apoE(-/-) mice were ovariectomized (ovx) or sham operated at 8 weeks of age, and 2-weeks afterwards, one-third of each ovx-group received conjugated equine estrogens (CEE) (0, 2.5 or 5.0 μg/day) for 16 weeks. Atherosclerotic lesions were examined after experimental periods. To clarify anti-atherogenic effect of 17β-estradiol (E2) on artery, neutral cholesteryl ester hydrolase (N-CEase) activity in aorta and peritoneal macrophages, and E2-treated J774A.1 cells were measured.
First, atherosclerotic lesion in female mice was significantly less than male mice without any changes in serum lipids and lipoprotein profile. N-CEase activity in aorta and peritoneal macrophages in female mice was significantly higher than male mice. Second, atherosclerotic lesion in ovx-group was significantly greater than sham-group. CEE-replacement to ovx-group decreased atherosclerotic lesion in a dose-dependent manner. N-CEase activity in aorta and peritoneal macrophages was decreased in ovx-group compared to sham-group, and restored by CEE-replacement in macrophages. To study detailed mechanisms, J774A.1 cells were treated with E2. E2 significantly increased N-CEase activity, and cAMP-dependent protein kinase (A-kinase) type II activity and the protein in cytosol fraction without any changes of total protein of A-kinase type II.
These results suggest that gender difference of atherogenesis is partly accounted for activation of N-CEase through estrogen-dependent translocation of A-kinase type II in macrophages.
[Show abstract][Hide abstract] ABSTRACT: We compared ascorbic acid (AA) levels in the blood and TPA- and fMLP-stimulated superoxide (O(2)(•-)) production in neutrophils of pre-, early, and late hypertensive stroke-prone spontaneously hypertensive rats (SHRSP) with those of age-matched Wistar Kyoto rats (WKY), or two other normotensive strains of rats. Plasma and lymphocyte AA levels were about two-fold higher in SHRSP as early as 4 weeks old compared to WKY, and also higher than those of Wistar and Sprague-Dawley (SD) rats. Levels of AA were high in the liver and adrenal glands of SHRSP, indicating congenitally high AA levels. The production of O(2)(•-) in neutrophils was about two-fold higher in SHRSP than in WKY even at 4 weeks of age, and increased with age in both strains. Among SHRSP, AA levels in lymphocytes decreased at the late hypertensive stages with a decrease in hepatic l-gulono-γ-lactone oxidase (GLO) activities. These data suggest that bi-phasic AA levels in the blood of SHRSP comprise congenitally high levels and a decrease after persistent hypertension due to enhanced O(2)(•-) production and a decrease in de novo AA synthesis through GLO.
[Show abstract][Hide abstract] ABSTRACT: In the present study, we examined the antiatherosclerotic effects of 3 edible mushrooms, Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Hypsizygus marmoreus (Bunashimeji), in atherosclerosis-susceptible C57BL/6J, apolipoprotein E-deficient (apoE(-/-)) mice. Male apoE(-/-) mice (6 weeks of age) were fed a normal diet (cholesterol concentration <66 mg/100 g) or a normal diet containing 3% dried Eringi, Maitake, or Bunashimeji mushroom powder for 10 weeks. Food intake, body weight, serum total cholesterol (TC), and serum triacylglycerols (TG) were measured periodically during the experimental period. At the end of the experiment (at 16 weeks of age), the atherosclerotic lesion area was measured in cross-sections of the aortic root. Serum TC concentrations in the Bunashimeji group were significantly lower than that in the control group at 8, 10, 12, 14, and 16 weeks of age. Serum TC concentrations in the Eringi, and Maitake groups were significantly lower than that in the control group only at 12 weeks of age. There was no significant difference in the serum TG concentrations in all groups during the experimental period. The atherosclerotic lesions were significantly decreased in the Eringi, Maitake, and Bunashimeji groups than that in the control group at the end of the experiment. Dietary supplementation with the Bunashimeji mushroom powder had the strongest antiatherosclerotic effect among 3 mushrooms. In conclusion, supplementation of the 3 edible mushrooms prevents the development of atherosclerosis, even normal diet. Antiatherosclerotic effect is partly via lowering of serum TC concentrations; further mechanisms should be investigated.
Nutrition research 05/2008; 28(5):335-42. DOI:10.1016/j.nutres.2008.03.010 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Green tea catechins possess potent antioxidative properties and protect against various oxidative diseases. Malignant stroke-prone spontaneously hypertensive rats (M-SHRSP) develop severe hypertension and spontaneous stroke at early ages. We previously reported that ingestion of green tea catechins prevents cerebral ischemic damage in a middle cerebral artery occlusion and reperfusion rat stroke model, in association with increased plasma epigallocatechin-gallate (EGCG) concentrations. In this study, we examined whether tea catechin intake decreases the incidence of spontaneous stroke in M-SHRSP.
Male M-SHRSP ingested 0.5% green tea catechin extract (Polyphenon E) in their drinking water beginning at 5 weeks of age, and blood pressure, heart rate, and locomotor activity were continuously monitored from 8 weeks using a telemetry system. Stroke onset was assessed by the appearance of neurologic symptoms, body weight loss, and circadian rhythm disturbances in heart rate, blood pressure, and locomotor activity.
Tea catechin ingestion significantly delayed stroke onset by 10 days compared to the control group. Although there was no difference in blood pressure at 10 weeks, the rate of in blood pressure increase in the tea catechin group was significantly smaller than that in the control group. Plasma NO2- and NO3- concentrations increased after stroke in both groups without significant difference between the two groups. Plasma EGCG concentration significantly decreased at post-stroke compared with that of pre-stroke.
Continuous ingestion of green tea catechins from an early age prevented the development of spontaneous stroke in M-SHRSP, probably by inhibiting the further development of high blood pressure at later ages.
Medical science monitor: international medical journal of experimental and clinical research 03/2007; 13(2):BR40-5. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the reproductive system of male rat offspring (F1) and the sex ratio of the subsequent generation (F2) were examined. Female Holtzman rats were gavaged with an initial loading dose of 400 ng/kg TCDD prior to mating, followed by weekly maintenance doses of 80 ng/kg during mating, pregnancy, and the lactation period. Maternal exposure to TCDD had no significant effects on fetus/pup (F1) mortality, litter size, or sex ratio on gestation day (GD) 20 or postnatal day (PND) 2. The TCDD concentration in maternal livers and adipose tissue on GD20 was 1.21 and 1.81 ng/kg, respectively, and decreased at weaning to 0.72 in the liver and 0.84 in the adipose tissue. In contrast, the TCDD concentration in pup livers was 1.32 ng/kg on PND2 and increased to 1.80 ng/kg at weaning. Ventral prostate weight of male offspring was significantly decreased by TCDD exposure on PND28 and 120 compared with that of controls. Weight of the testes, cauda epididymides, and seminal vesicle, and sperm number in the cauda epididymis were not changed by TCDD exposure at PND120. TCDD- or vehicle-exposed male offspring were mated with unexposed females. The sex ratio (percentage of male pups) of F2 offspring was significantly reduced in the TCDD-exposed group compared with controls. These results suggest that in utero and lactational TCDD exposures affect the development of male gonads in offspring (F1), leading to changes in the sex ratio of the subsequent generation (F2).
[Show abstract][Hide abstract] ABSTRACT: The effects of in utero and lactational exposure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on brain sexual differentiation were investigated. TCDD was orally administered to pregnant Holtzman rats on gestation day (GD) 15, and the activity of brain aromatase, a key enzyme for sexual differentiation, was measured in offspring on postnatal day (PND) 2. Changes in sexual dimorphisms of saccharin preference and the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) were examined in adult offspring. In controls, litter means of brain aromatase activity were higher in males than in females. In utero exposure to 200 ng/kg TCDD significantly decreased the sex ratio of aromatase activity (male/female) on PND 2. Offspring were weaned on PND28 and the saccharin test was started on PND84. In controls, saccharin (0.25%) intake (g/kg body weight) was significantly higher in female offspring than in males. In utero exposure to 200 ng/kg TCDD significantly increased saccharin intake in male offspring compared with control males, whereas 800 ng/kg TCDD had no effect. Neither dose of TCDD influenced saccharin intake of female offspring. In controls, SDN-POA volume was significantly greater in males than in females at 14 weeks of age. Exposure to 200 ng/kg TCDD significantly decreased SDN-POA volume in males, whereas 800 ng/kg TCDD had no effect. Neither doses of TCDD influenced the SDN-POA volume in female offspring. These results suggest that in utero and lactational TCDD exposure dose-dependently induces demasculinization in male offspring by inhibiting brain aromatase activity in the hypothalamus-preoptic area during central nervous system development.
[Show abstract][Hide abstract] ABSTRACT: To characterize the role of nitric oxide (NO) in stroke, NO was measured using an in vivo microdialysis technique and electron spin resonance spectrometry in malignant stroke-prone spontaneously hypertensive rats (M-SHRSP), stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). The brain dialysate NO level was higher in SHRSP than in WKY. NO was not detected in M-SHRSP hippocampus microdialysate after stroke except after the administration of N-tert-butyl-alpha-phenylnitrone (PBN). In addition, very little NO was generated in M-SHRSP brain tissue with hemorrhage. These data demonstrate an association between NO and stroke in M-SHRSP. Further, PBN administration results in maintenance of NO levels after stroke in M-SHRSP.
[Show abstract][Hide abstract] ABSTRACT: Green tea catechins possess potent antioxidative properties, and the preventive effects against various oxidative diseases have been reported. The purpose of this study is to examine preventive and protective effects of green tea catechins on various deteriorative processes following stroke.
Male Wistar rats were given ad libitum water with or without 0.25 and 0.5% tea catechin extract for 5 days prior to the operation and during the experiment. Right middle cerebral artery was occluded for 2 h, then reperfused for 22 h. Brain slices were stained with triphenyltetrazolim chloride to assess infarct area. Concentrations of plasma EGCg, and serum NOx were analyzed by HPLC. Detection of iNOS expression, neutrophil infiltration and peroxynitrite formation in the penumbra was performed by immunostain. Neurologic deficit was scored by posture reflex.
Tea catechins dose-dependently reduced the brain infarct area and volume. Infarct volume was inversely correlated with plasma EGCg concentration. Dark staining for iNOS, neutrophils and peroxynitrite were observed in vessel wall of small arteries in control ischemic hemisphere, while in catechins (0.5%)-treated rats iNOS was detected slightly, and staining for neutrophils and peroxynitrite was not seen. Catechin ingestion blocked a 3-fold increase in serum NOx concentration in the jugular vein, and also reduced by 35% a 2-fold increase of plasma lipid peroxide level seen in control rats after reperfusion. Neurologic deficits were significantly alleviated by 0.5% catechin ingestion.
Daily intake of green tea catechins efficiently protects the penumbra from irreversible damage due to cerebral ischemia, and consequent neurologic deficits.
Medical science monitor: international medical journal of experimental and clinical research 07/2004; 10(6):BR166-74. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously reported that intracellular free cholesterol at physiological concentrations regulates the activity of neutral cholesterol esterase (N-CEase) in macrophages. The objective of the present study is to investigate whether the regulation of N-CEase by cholesterol is generally observed in other types of cells such as adipocytes with high activity of hormone-sensitive lipase (HSL), the same gene product as N-CEase. 3T3-L1 adipocytes were cultured with and without cholesterol (1-30 microg/mL) or 25-hydroxycholesterol (0.1-10 microg/mL), and changes in the N-CEase activity, expression of HSL mRNA, and protein were examined. Incubation (24 h) of cells with cholesterol did not change N-CEase activity, but incubation with 25-hydroxycholesterol decreased the activity in a concentration-dependent manner by 24 (24 h) and 54% (36 h). Quantitative reverse transcription-PCR indicated that 25-hydroxycholesterol (10 microg/mL) did not influence expression of HSL mRNA. However, Western blot analysis showed that this sterol reduced HSL protein by 72 (24 h) and by 93% (36 h), respectively. It was concluded that sterol-mediated regulation of HSL/N-CEase occurs not only in macrophages but also in adipocytes, and regulation appears to occur not at a transcriptional level but by a post-transcriptional process. Sterol-mediated proteolysis may be involved in the loss of HSL protein.
[Show abstract][Hide abstract] ABSTRACT: Peroxynitrite is assumed to play a crucial role in brain damage associated with the overproduction of nitric oxide (NO). The purpose of this study is to examine time-dependent changes of nitrite and nitrate (NOx) concentration in the circulation, and peroxynitrite formation as well as the expression of inducible nitric oxide synthase (iNOS) in the penumbra of rat brains during transient middle cerebral artery occlusion (MCAO) of Wistar rat for 2 h and reperfusion for 4-70 h. NOx concentration in the circulation was continuously monitored at the right jugular vein by microdialysis. The expression of iNOS was detected at 22-70 h after reperfusion in vascular walls and the cortex. Nitrotyrosine, a marker of peroxynitrite, appeared 4 h after reperfusion in the cortex, increasing substantially at 22-46 h in vascular walls. NOx level in dialysate increased immediately after MCAO. After a gradual decrease, the level increased again 4 h after reperfusion, reaching a maximum at 46 h. Brain myeloperoxidase activity, a marker of neutrophil infiltration, was not detected 4 h after reperfusion, but greatly increased at 22 h and then decreased. These results suggest that a marked increase of NOx level in the circulation might reflect the expression of iNOS, while neuronal NOS may contribute to peroxynitrite formation in the cortex observed at an earlier phase of reperfusion. This study indicates that monitoring NOx level in the circulation serves to assess the progress of stroke, and to determine appropriate therapeutic measures.
Brain Research 10/2002; 951(1):113-20. DOI:10.1016/S0006-8993(02)03145-1 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malignant stroke-prone spontaneously hypertensive rats (M-SHRSP) develop hypertension and stroke at earlier ages than do nonmalignant SHRSP. Our previous findings suggested that reactive oxygen species were involved in the development of stroke in this strain. Nitric oxide (NO) which is more released at ischemia, might play a crucial role in stroke development by producing peroxynitrite, a neurotoxic substance. This study investigated whether the development of cerebrovascular lesion in M-SHRSP could be assessed by the fluctuation of serum NO(x) concentration, and whether peroxynitrite is associated with brain damage. Serum NO(x) levels were examined using an automated NO detector. Stroke-onset was temporally assessed according to a known method: changes in body weight, water intake, and neurologic symptoms. Cerebral lesions were confirmed by magnetic resonance imaging (MRI), and Evans blue extravasation at autopsy. MRI taken just after estimated stroke onset disclosed brain lesions. The baseline serum NO(x) level remained at 15-18 micromol/l, but the level gradually increased prior to stroke, and significantly at stroke onset. A marked rise in serum NO(x) occurred subsequently at poststroke. Immunohistochemical staining of nitrotyrosine, a peroxynitrite marker, was detected around vessels, neuronal cells and parenchyma in cerebral lesions. Stroke occurred in 50% of male M-SHRSP at 80 days of age. In conclusion, this study provides the first evidence for fluctuation of serum NO(x) at the onset of spontaneous stroke accompanying the appearance of peroxynitrite in brain lesions. Monitoring serum NO(x) would serve to assess the development of brain lesions at least in spontaneous stroke model.
Brain Research 10/2002; 949(1-2):147-56. DOI:10.1016/S0006-8993(02)02975-X · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aromatase catalyzes intraneuronal conversion of androgens to estrogens in the hypothalamus-preoptic area at a specific time during development. This local estrogen formation has a central role in sexual differentiation. To investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on sexual differentiation, TCDD (800 and 1600 ng/kg) was orally administered to pregnant Holtzman rats on gestation day (GD) 15, and the change in brain aromatase activity of fetuses (GD20) and pups (postnatal day 2) was examined. Litter means of brain aromatase activity in the hypo-preoptic area of control fetuses were higher in males than in females (female/male ratio was 0.7). This ratio of aromatase activity increased in a TCDD-concentration dependent manner, reaching 1 at the higher dose. There was a similar change in aromatase activity in brains of pups (PND 2). These results suggest that in utero TCDD exposure induces demasculinization in male offspring by inhibiting the aromatase activity in the brain during central nervous system development.
[Show abstract][Hide abstract] ABSTRACT: Malignant stroke-prone spontaneously hypertensive rats (M-SHRSP), separated from SHRSP, develop severe hypertension and spontaneously develop stroke at early ages. Using this model of cerebrovascular stroke, influence of stroke-onset on the autonomic nervous system was investigated. Heart rate (HR), systolic and diastolic blood pressures (SBP and DBP) and locomotive activity were monitored during development of stroke using a telemetry system. Stroke-onset was assessed by neurologic symptoms, changes in body weight, fluid intake and serum NOx level. The rat displayed a nocturnal pattern of circadian rhythms. At stroke-onset, mean HR over 24 h increased by 20 to 30 bpm and rapidly increased at post stroke, approximately 100 bpm higher than that at pre stroke. Circadian variation in HR, which was normally 50 bpm higher during night than during day, attenuated at stroke-onset, and it was blunted or reversed at post stroke. BP variation, which was approximately 7 mmHg higher at night than at day, decreased one or two days before stroke-onset and reversed at post stroke, especially in DBP. Insufficient falls in HR and BP during the day mainly accounted for the disturbed circadian variations. Variation of locomotive activity also decreased. These changes serve as reliable and accurate markers for stroke-onset in evaluation of drugs for the prevention and outcome predictions of stroke.
The Japanese Journal of Pharmacology 03/2001; 85(2):197-202. DOI:10.1254/jjp.85.197
[Show abstract][Hide abstract] ABSTRACT: Green tea contains various antioxidative flavan-3ols (tea catechins), such as (-)-epigallocatechin gallate (EGCg, the major catechin), which exert potent inhibitory effects on LDL oxidation in vitro and ex vivo in humans. In this study, the antiatherogenic effects of tea catechins were examined in atherosclerosis-susceptible C57BL/6J, apoprotein (apo)E-deficient mice. Male apoE-deficient mice (10 wk old) were fed an atherogenic diet for 14 wk; during that time, one group (tea) was supplied drinking water supplemented with green tea extract (0.8 g/L), and another group (control) was offered the vehicle only. The tea extract consisted of the following (g/100 g): EGCg, 58.4; (-)-epigallocatechin (EGC), 11.7; (-)-epicatechin (EC), 6.6; (-)-gallocatechingallate (GCg), 1.6; (-)-epicatechin gallate (ECg), 0.5; and caffeine, 0.4. The estimated actual intake of tea catechin was 1.7 mg/(d. mouse). Tea ingestion did not influence plasma cholesterol or triglyceride concentrations. Plasma lipid peroxides were reduced in the tea group at wk 8, suggesting that the in vivo oxidative state is improved by tea ingestion. Atheromatous areas in the aorta from the arch to the femoral bifurcation and aortic weights were both significantly attenuated by 23% in the tea group compared with the control group. Aortic cholesterol and triglyceride contents were 27 and 50% lower, respectively, in the tea group than in the control group. These results suggest that chronic ingestion of tea extract prevents the development of atherosclerosis without changing the plasma lipid level in apoE-deficient mice, probably through the potent antioxidative activity of the tea.
Journal of Nutrition 02/2001; 131(1):27-32. · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diesel exhaust particles (DEP) are assumed to be a causal substance for pulmonary inflammation. As peroxynitrite is recently implicated in inflammation and cytotoxity, the hypothesis was tested that instillation of DEP induces formation of peroxynitrite in cells migrated in lung. Rats were intratracheally instilled with DEP suspension (2 mg/0.5 ml/kg) and killed 24 h later. Alveolar cells were collected by broncho-alveolar lavage. Population of alveolar cells increased more than twice by DEP exposure, mainly due to a large increase of neutrophils. Peroxynitrite formation (NG-nitro-l-arginine methylester and superoxide dismutase inhibitable chemiluminescence) was detected in alveolar cells from treated rats, and 12-O-tetradecanoylphorbol 13-acetate-stimulation enhanced it. In addition, DEP induced expression of inducible NO synthase mRNA in these cells. But peroxynitrite was not detectable in cells from control. These results indicate that DEP exposure results in peroxynitrite formation in migrated cells, which leads to pulmonary inflammation.
[Show abstract][Hide abstract] ABSTRACT: Oxidation of low density lipoprotein (LDL) plays crucial roles in atherogenesis. We previously reported that green tea polyphenols (flavan 3-ols), especially epigallocatechingallate (EGCg) and epicatechingallate, exerted potent inhibitory effects on LDL oxidation in vitro. To examine whether intake of green tea polyphenols renders LDL resistant to ex vivo oxidation in humans, 22 male volunteers aged between 22 and 32 years were recruited and assigned the same dietary regimen for 2 weeks. After a 1-week baseline period, they were equally divided into two groups: control and tea. The tea group ingested 300 mg of green tea polyphenol extract twice daily for 1 week. Plasma EGCg concentration at the end of the experiment was 56 nmol/L on average (56% in free form) in the tea group; no EGCg was detected before the experiment. Plasma concentrations of lipids, ascorbate, alpha-tocopherol, and lipid peroxides did not change before and after the experiment in either group, but beta-carotene was higher in the tea group (P< 0.01 by paired Student'st-test). LDL (0.1 mg/mL) was incubated with 5 microM Cu(2+) and the oxidation was measured by absorbance at 234 nm. The lag time was significantly prolonged by 13.7 min in the tea group (P < 0.05 by paired Student'st-test, before versus after), whereas such a change was not observed in the control group. These results suggest that daily consumption of seven to eight cups (approximately 100 mL each cup) of green tea may increase resistance of LDL to in vivo oxidation, leading to reduction in the risk of cardiovascular diseases.
The Journal of Nutritional Biochemistry 04/2000; 11(4):216-22. DOI:10.1016/S0955-2863(00)00068-1 · 3.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Contractile response to angiotensin II (AngII) of vascular smooth muscle was compared between 12-week-old, stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY). AngII induced phasic and tonic contraction in denuded aortic ring preparation. The phasic contraction was concentration-dependent (AngII 10(-9) to 10(-6) mol/L) and similar in both strains. However, the relaxation after phasic contraction was significantly attenuated in SHRSP compared with that in WKY. To examine the recovery of contractile responses to the repeated stimulation, AngII was applied three times at 20- and 60-min intervals. The first maximal contraction was similar in both strains, but the response to the second stimulation was significantly reduced in SHRSP, compared with all three responses in WKY. These results suggested that Ca2+-sequestration into the Ca2+ store is delayed in SHRSP. Cyclopiazonic acid (10(-5) mol/L), an intracellular Ca2+-pump inhibitor, decreased spontaneous relaxation and increased the sustained contraction in WKY, whereas it did not affect the contraction in SHRSP. Insulin, which modulates tonic contraction by facilitating Ca2+-extrusion, was applied at peak contraction by AngII. It enhanced relaxation after phasic contraction in a concentration-dependent manner in SHRSP, but it did not affect the relaxation in WKY. These results suggest that increased sustained contraction observed in SHRSP reflects at least partly the impaired Ca2+-pump activity leading to hypertension.
American Journal of Hypertension 07/1999; 12(6):590-5. DOI:10.1016/S0895-7061(99)00019-9 · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of phospholipid composition in cholesteryl ester (CE)-micellar substrates on neutral cholesterol esterase (N-CEase) activity was examined. N-CEase preparation was incubated with micelles composed of cholesteryl-[1-14C]-oleate, sodium taurocholate, and phosphatidylcholine (PC)/phosphatidylethanolamine (PE) at varying ratios (%PE:0 = PC only, 17, 33, 50, 66, 83). The activity increased dependently with the increase in PE content; the activity with the micelles containing the highest ratio of PE was 2.5-fold compared with the micelles consisting of PC only. Vmax with the micelles of 83, 66, and 50% PE was 3.1-, 2.7-, and 1.9-fold, respectively, compared with the micelles of PC only. Each micellar preparation was chromatographed through a Superose 6 column by the FPLC system. In 66 and 83% PE-containing micelles, PC, PE, CE, and part of sodium taurocholate eluted completely together in a single peak, whereas in micelles with 33 and 50% PE they eluted loosely together. The micelles with PC only or 17% PE formed PC-micelles without including CE and PE. It is concluded that PE plays a critical role in the formation of CE micelles with PC, and in the interaction with N-CEase. The CE-micelles with 66-83% PE serve as substrates for sensitive and reproducible N-CEase assay.