The aim of this study was to clarify the role of free radicals generated in the postischemic myocardium in a canine coronary occlusion (90min)-reperfusion (5h) model of myocardial infarction. Myocardial samples were taken from the central infarcted region (infarct), the noninfarcted region within the area at risk (risk), and the endocardial-to-epicardial border between the infarct region and the area at risk (border). Free-radical generation was assessed by electron paramagnetic resonance (EPR) spectroscopy using a spin trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and a luminol-enhanced chemiluminescence assay. DMPO-adducts detected by EPR spectroscopy consisted of DMPO-OH, which markedly increased after reperfusion in the ischemic myocardium, especially in the border, and showed a peak after 3h of reperfusion (p<0.01). Similarly, a marked increase in the chemiluminescence of the border was observed after reperfusion, which showed a peak after 3h of reperfusion (p<0.01) and thereafter declined. The generating activity of free radicals in the border increased significantly (p<0.01) after reperfusion and its peak was also observed at 3h after reperfusion. However, the activity in the infarct was nearly zero after 1h of reperfusion. Administration of a free-radical scavenger, CV-3611 (5mg/kg, i.v.), before reperfusion reduced the free radical generation in the border. These results indicate that the increased generation of free radicals at the peri-infarcted site may relate to the extension of reperfusion-associated myocardial injury.
The direct role of albumin in L-tryptophan transport into hepatocytes was studied by examining the effect of bovine serum albumin (BSA) on L-tryptophan uptake by isolated rat hepatocytes in comparison with that on the uptake of L-phenylalanine and L-leucine, which are known to be taken up into hepatocytes via the transport systems common to L-tryptophan. When the uptake rates of these amino acids were determined in an incubation medium containing BSA and at the same free-form substrate concentration, the rate of L-tryptophan uptake increased, while the rates of L-phenylalanine and L-leucine uptakes remained unchanged. L-Tryptophan was well bound to BSA, while L-phenylalanine and L-leucine were scarcely bound to the protein. BSA was clearly bound to the incubated cells. From the kinetic analysis of L-tryptophan uptake by hepatocytes with and without BSA, BSA was found to enhance the affinity between L-tryptophan and its transport site on the surface of liver cells. In hepatocytes pretreated with BSA, only L-tryptophan uptake was stimulated, in which the affinity between L-tryptophan and its transport site was enhanced. A significant amount of BSA was bound to the cells pretreated with the protein. When the effects of BSA co-addition and its pretreatment on the binding of L-tryptophan to plasma membranes prepared from rat livers were examined, the protein was found to promote the binding of L-tryptophan to the membranes. The present results suggest that albumin can facilitate L-tryptophan transport into hepatocytes by enhancing the affinity between the amino acid and its transport site present on the cell surface through its direct interaction with both the amino acid and the plasma membrane.
This study attempted to elucidate the pathological role of peripheral blood polymorphonuclear leukocytes (PMNs) in the damage to hepatic sinusoidal endothelial cells (HSECs) in hepatic injury. Wistar male rats weighing about 200g received a single injection of 1g/kg body weight of galactosamine (GalN) intraperitoneally for the induction of hepatitis. The level of serum glutamic pyruvate transaminase activity increased time-dependently concomitantly with serum endotoxin level and hepatic lipid peroxide content. Histological studies using light and electron microscopy showed that a large number of PMNs infiltrated the midzonal area of the liver and that HSECs in contact with the PMNs were injured 6h after GalN injection. Superoxide anion production by PMNs isolated from the peripheral blood of rats treated with GalN was increased significantly compared with that from control rats, as estimated by the reduction of exogenously added cytochrome c in the presence of phorbol myristate acetate. A cytotoxicity study using the 51Cr release assay revealed that PMNs isolated from GalN-treated rats caused more serious injury to the HSECs than those from control rats. These results suggest that the oxygen-derived free radicals released from the activated PMNs directly injure HSECs and lead to a disturbance of sinusoidal microcirculation, causing an extended liver cell necrosis in GalN hepatitis.
In order to elucidate the mechanisms pertaining to the interrelation between atherosclerotic changes and lipid peroxides in the aorta, we examined the effects of increased lipid peroxides in plasma and in the aorta on cholesteryl ester-hydrolyzing enzymes by feeding rats a small quantity of autoxidized linoleic acid hydroperoxide (LHPO). In LHPO-fed rats, plasma and hepatic lipid peroxides were elevated in an inverse relation to vitamin E levels. Aortic cholesteryl ester content was increased by 42% in the treated rats along with a 65% increase in the lipid peroxide content. The activities of acid (AL) and neutral (NL) cholesteryl ester hydrolases of the LHPO group were reduced in aorta, mononuclear leukocytes, hepatic lysosomes, and microsomes compared with those of the control group. The addition of purified linoleate hydroperoxide and serum lipid peroxides, and low-density lipoprotein (LDL) isolated from LHPO-fed rats also showed a dose-dependent inhibition of both AL and NL activities of mononuclear leukocytes in vitro. The LDL showed high lipid peroxide values that were about 6 times over the normal LDL value and exhibited characteristics typical of oxidatively modified LDL; that is to say, the net negative charges increased along with a degradation of the tertiary structure of the apolipoprotein. The results suggest that an increase in blood lipid peroxides may alter LDL into an oxidatively modified form that is then incorporated into cells via scavenger receptors, thus inhibiting the intracellular cholesteryl ester hydrolases and allowing an increase in cellular cholesteryl esters. Oxidatively modified LDL thereby becomes one of the factors that initiates and causes the development of atherosclerotic changes in the aorta.
Metabolic changes in folic acid and their polyglutamyl derivatives were studied in streptozotocin-induced diabetic rats. Polyglutamylfolates, which constituted about 70% of the folates (determined by the Lactobacillus casei microbiological assay) in normal rat liver, were observed to be hydrolyzed to a greater extent to simple monoglutamyl forms as the diabetes progressed. Only 4-5% conjugated folates were observed 25 days after the injection of streptozotocin. Total methylfolates, which represented 70-75% of total folates, were lowered by 60% and methylpolyglutamylfolates by 95% in the diabetic rat. An imbalance in the two folate-metabolizing enzymes, folyl conjugase and folylpolyglutamate synthetase, were observed in diabetes, the former being 2-3-fold higher and the latter being 7-8-fold lower. [14C]Folate uptake studies indicated that folates accumulated for up to the first 24h in diabetic rat liver, and then were depleted over the next 48h period, indicating an impaired polyglutamylation and storage of folates.
The plasma levels of atrial natriuretic factor (ANF), renin activity and aldosterone level in a lean control group (group I, n=20), obese normotensive individuals (group II, n=46), and obese hypertensives (group III, n=14) were estimated along with echocardiographic examination. According to findings obtained from echocardiography, normotensive and hypertensive obese subjects with impaired left ventricular diastolic function were subclassified as groups IIa and IIIa, respectively. ANF was found to be increased in obese normotensive and obese hypertensive groups compared with the control group level. Obese normotensive and obese hypertensive subjects with impaired left ventricular function were found to have higher plasma ANF than their unimpaired counterparts. An inverse relation was found between plasma ANF and plasma renin activity or aldosterone level. There were a significant decrease in fractional shortening and increase in left atrial diameter in groups IIa and IIIa compared with control group. We conclude that impairment of the left ventricular diastolic function caused by hypertrophy of the left ventricle in obese normotensives and obese hypertensives produces elevation of pressure in the left atrium and an increase in the secretion of ANF. Cardiac chamber in pressure is a reflection of this increased circulating ANF.
Anti-human pancreatic elastase 1 autoantibodies occurring in sera of patients with pancreatic diseases specifically bound elastase 1 but showed little binding with proelastase 1. Binding of proelastase 1 to the autoantibodies was effectively elevated by tryptic digestion of the proelastase 1 for activation, suggesting that the autoantibodies recognized some conformational change from proelastase 1 to elastase 1. Gel filtration analysis of the 125I-labeled elastase 1 and proelastase 1 added to sera revealed that both elastase 1 and proelastase 1 existed as conjugates with α1-antitrypsin or α2-macroglobulin in autoantibody-negative sera; but in autoantibody-positive sera, more than 80% of elastase 1 was assumed to form an immune complex with the autoantibodies, while proelastase 1 did not seem to form an immune complex, but existed as conjugates with the serum protease inhibitors as was the case of autoantibody-negative serum. Therefore, conversion of proelastase 1 into the active form does not seem to occur in serum. These results indicate that anti-elastase 1 autoantibodies are produced as a consequence of the leakage of unusually large amounts of elastase 1 into the circulation after an attack of pancreatitis.
The relationship of dietary fat to atherosclerosis is an area of continuing research. In order to establish if there is a seasonal variation in lipid status, we determined the dietary intake of lipids as well as their serum levels in 65 (33 males and 32 females) free-living subjects (67-75 years) in winter and summer. Both fat and cholesterol intakes were generally higher for men than women, but no seasonal differences were found for either gender. Nor in either sex was there any significant association of dietary fat intake (polyunsaturated/saturated fatty acid ratio) with either triglycerides or cholesterol levels in serum. Dietary unsaturated fatty acid but not the cholesterol intake appeared to have a marginal effect on serum cholesterol. According to the Canadian Society of Clinical Chemists reference standards for total serum cholesterol, 18% of the men and 41% of the women in this study population would be classified as high-risk (>5.2mmol/liter). HDL cholesterol, however, was consistently higher in the women. Total fat intake contributed 34% of total energy for both genders regardless of the season.