• YAKHAK HOEJI
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• v.39 no.4
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• pp.411-416
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• 1995

The single-pass perfusion experiments were performed in anesthetized rats to investigate the effects of perfusates and their osmolality on the water transport and to determine the correlation between the extent of water transport and the volume change of perfusate. Phenol red was used as a nonabsorbable marker. In normal rats, when perfused at a flow rate of 0.5 ml/min, 2-(N-rnorpholino) ethanesulfonic acid (MES) and S$\phi$rensen's phosphate buffers showed minimal net water transport as 0.125 and 0.173 %/cm of intestinal length, respectively. Hypotonic perfusate of 200 mOsm/kg of water and hypertonic perfusate of 400 mOsm/kg of water generated significant water transport compared with isotonic perfusate of 300 mOsm/kg of water. There was a linear correlation between the extent of water transport and the volume change of perfusate, suggesting that the volume change can be used as a measure of water transport.

• Archives of Pharmacal Research
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• v.23 no.1
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• pp.22-25
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• 2000

Formation of glutathione (GSH) conjugates with 2- or 6-(1-hydroxymethyl)- and 2-(1-hydroxyethyl)-DMNQ derivatives (DMNQ, 5,8-dimethoxy-1,4-naphthoquone was carried out in phosphate buffer (pH 7.4), in the presence of glutathione-S-transferase (GST), in rat liver S-9 fraction and by perfusion, and the rates of conjugates formation were compared and correlated to cytotoxicity. The GSH conjugates of 6-(1-hydroxyalkyl)-DMNQ derivatives were formed faster than 2-(1-hydroxyalkyl)-DMNQ derivatives under all of the media, implying that steric hindrance was the cause of lowering the rate of conjugate formation of 2-substituted derivatives. For both isomers, addition of GST did not improve the reaction rate, compared with that in buffer, while the reaction in the S-9 fraction and the perfusate was accelerated to a great extent. The catalytic effect of the S-9 fraction and the perfusate contain an effective system relaxing the steric hindrance of 2-(1-hydroxyalkyl)-DMNQ derivatives. Furthermore, a good correlation between the formation of the GSH conjugates and the cytotoxic activity of both naphthazarin isomers suggests that the steric hindrance is a cause of lowering the cytotoxicity of 2-isomers.

• YAKHAK HOEJI
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• v.44 no.3
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• pp.237-244
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• 2000

Liver isolated from 18 hours fasted rats was subjected to $N_2$hypoxia (for 45 min) followed by reoxygenation (for 30 min). The perfusion medium used was Krebs-Henseleit bicarbonate buffer (pH 7.4, $37^{\circ}C$). Vitamin C (0.5 mM) and trolox C (0.5 mM), soluble vitamin E analog, were added to perfusate. Lactate dehydrogenase (LDH), total glutathione, oxidized glutathione, lipid peroxide and drug-metabolizing enzymes were measured. After hypoxia LDH significantly increased but this increase was attenuated by vitamin C and combination of vitamin C and E. Total glutathione and oxidized glutathione in perfusate markedly increased during hypoxia and this increase was inhibited by vitamins C, E and its combination. Similarly; oxidized glutathione and lipid peroxide in liver tissue increased after hypoxia and reoxygenation and this increase was inhibited by vitamin I and combination of vitamin C and E. Hepatic drug metabolizing function (phase I, II) were suppressed during hypoxia but improved during reoxygenation. While vitamins C and E only increased glucuronidation, the combination of vitamin C and E increased the oxidation, glucuronidation and sulfation. Our findings suggest that vitamins C and E synergistically ameliorates hepatocellular damage as indicated by abnormalities in drug metabolizing function during hypoxia/reoxygenation and that this protection is in major part, caused by decreased oxidative stress.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.6 no.1
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• pp.28-37
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• 1996

Benzidine, an aromatic amine used primarily in the manufacture of azo dyes, is recognized as a urinary bladder carcinogen in humans. In rats, mice, and hamsters, chronic exposure to benzidine resulted in tumors of the liver. The present study was undertaken to suggest analyzing the metabolites of benzidine with the optimal condition, identify the metabolites of benzidine, and observe time variance of the metabolites in the isolated perfusated rat liver. N-acetylbenzidine was synthesized by acetylation of benzidine with acetic anhydride and separated by thin layer chromatography(TLC) and high performance liquid chromatography(HPLC). To analysis benzidine and the metabolites of benzidine, HPLC operating condition has been optimized by means of preliminary experiment. The mobile phase consisted of acetonitrile(37%) in phosphate buffer, flow rate maintained at 1.0 ml/min. Optimal detective conditions were electrochemicaldetector(ECD) at 0.75 V for benzidine and N-acetylbenzidine and ultravioletdetector(UVD) at 287 nm for N,N'-diacetylbenzidine. The separation system was composed of a guard column and a separation column(Polymer C18, $4.6{\times}250cm$) at a temparature of $40^{\circ}C$. The perfusion system was equilibrated for 30 minutes before addition of benzidine to the perfusate. Samples of the perfusate were collected at time intervals(0, 10, 20, 30, 60, 90, 120 min) during the 2 hour perfusion. Before analyzing samples by HPLC/ECD/UVD, samples had been treated with sep-pak. Samples of perfusate analyzed by HPLC/ECD/UVD and the metabolites of benzidine in the isolated perfused rat liver were N-acetylbenzidine and N,N'-diacetylbenzidine. Benzidine metabolized over 60% during the initial 30 minutes of perfusion, extensively by 1 hour, and was undetectable in the perfusate. N-acetylbenzidine increased by 30 minutes of perfusion, declined. N,N'-diacetylbenzidine increased the 0-90 minutes period, remained constant during the 90-120 minutes period.

• Journal of Chest Surgery
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• v.13 no.4
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• pp.338-345
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• 1980

We have modified an isolated perfusion rat heart model of cardiopulmonary bypass, with which we are able to screen the effects of various cardioplegic solutions and hypothermia upon the ability of the heart to survivie during and recover from period of ischemic arrest. The modified experimental model was differed from the original as follow : a heat coil chamber of atrial and aortic reservoir provided temperature control, and the perfusate was gassed with each pure oxygen and pure carbon dioxide in 95:5 ratio. The Langendorff perfusion was initiated for a 10 minute period by introducing perfusate at $37^{\circ}C.$ into the aorta from the aortic reservoir located 100 cm above the heart. The isolated perfused working rat heart model was a left heart preparation in which oxygenated perfusion medium (at $37^{\circ}C.$) entered the cannulated left atrium at a pressure of 20 cm $H_{2}O$ and was passed to the ventricle, from which it was sponeously elected(no electrical pacing) via an aortic cannula, against a hydrostatic pressure of 100cm $H_{2}O$. during this working period various indices of cardiac functin were measured. The cardiac functions were stable for over 3 hour with perfusion of Krebs-Henseleit bicarbonate buffer solution containing only glucose (11.1 mM/L). The percentage of cardiac functins were maintained about 94% on heart rate, 80.6% on peak aortic pressure, 87.7% on coronary flow and 76.3% on aortic flow rate after 3 hour of working heart perfusion at a pressure of 20 cm $H_{2}O$. We believe this preparation to be a good biochemical model for the human heart which offers many advantages including economic, speed of preparation, reproducibility, and the ability to handle large numbers.

• Biomolecules & Therapeutics
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• v.5 no.1
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• pp.1-7
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• 1997

This study was done to investigate the effect of vitamin C on hypoxia/reoxygenation-induced hepatic injury ul isolated perfused rat liver. Isolated livers from rats fasted 18 hours were subjected to 45 min of hypoxia followed by reoxygenation for 45 min. The perfusion medium used was Krebs-Henseleit bicarbonate buffer (pH 7.4) and 0.5 mmol/L of vitamin C was added to the perfusate. Alanine aminotransferase (ALI) and lactate dehydrogenase (LDH) levels were significantly increased by hypoxia/reoxygenation. These increases were augmented by vitamin C. Glucose output and bile flow were markedly decreased by hypoxia/reoxygenation. Vitamin C aggavated the decrease of glucose output but had little effect on bile flow. Our findings suggest that hypoxia/reoxygenation diminishes hepatic metabolic and secretory functions, and vitamin C significantly aggravates these changes.

• Archives of Pharmacal Research
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• v.20 no.5
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• pp.471-475
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• 1997

Livers isolated from 18 hours fasted rats were subjected to N$_{2}$ hypoxia (for 45 min) followed by reoxygenation (for 45 min). The perfusion medium used was Krebs-Henseleit bicarbonate buffer (KHBB, pH 7.4). Lactate and alanine were added as gluconeogenic and ureagenic substrates and Trolox C was also added to perfusate. Oxygen consumption, lactate dehydrogenase (LDH), alanine transaminase (ALT), total glutathione, oxidized glutathione, bile flow, glucose and urea were measured. After hypoxia oxygen consumption significantly dropped but Trolox C had no influence on this decrease. ALT and LDH were significantly increased by hypoxia/reoxygenation. This increase was markedly attenuated in the presence of Trolox C. The total glutathione and oxidized glutathione efflux increased following hypoxia, which were prevented by the treatment of Trolox C. Bile flow rate decreased following hypoxia/reoxygenation but did not continue to decrease in the reoxygenation phase by Trolox C. Following hypoxia/reoxygenation glucose and urea releases decreased. Trolox C had no influence on inhibition of glucose and urea production. These results suggest that Trolox C protected the liver cells against hypoxia/reoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model.

• Journal of Chest Surgery
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• v.20 no.4
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• pp.643-654
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• 1987

The effect of prostacyclin[PGI, ] on myocardial preservation during global ischemia was studied in the isolating working rabbit heart model. Forty hearts underwent a 15 minute period of retrograde nonworking perfusion with Krebs-Henseleit buffer solution [37*C] and were switched over to the working mode for 15 minutes. After baseline measurement of heart rate, peak aortic pressure, aortic flow, and coronary flow, all hearts were subjected to 60 minutes of ischemic arrest at 10*C induced with St. Thomas Hospital cardioplegic solution: Group I had single dose cardioplegia, Croup II double dose, Croup III oxygenated double dose, and Group IV single dose with PCI, infusion [10ng/min./gm heart weight]. Hearts were then revived with 15 minute period of nonworking reperfusion at normothermia, followed by 30 minutes of working perfusion. Repeat measurements of cardiac function were obtained and expressed as a percent of the preischemic baseline values. Oxygen content of arterial perfusate and coronary effluent was measured by designed time interval. Leakage of creatine kinase was determined during post-ischemic reperfusion period. Finally wet hearts were weighed and placed in 120*C oven for 36 hours for measurement of dry weight. In the PGI, treated group [IV], heart rate increased consistently throughout the period of reperfusion from 100*5.0% [p<0.001] to 107*6.2% [p<0.001]. The percent recovery of aortic flow showed 95*5.7% [p<0.001] at the first 3 minute and full recovery through the subsequent time. Coronary flow was augmented significantly in the 3 minute [96*6.2%, p<0.001] and then sustained above baseline values. Among the Croup I, II, and III, all hemodynamic values were significantly below preischemic levels. PGI2 relatively increased oxygen delivery [1.22*0.19ml/min, p<0.001] and myocardial oxygen consumption [0.90*0.13ml/min, p<0.001] during reperfusion period. Leakage of creatine kinase in the PGI2 group was 9.3*1.58IU/15min [p<0.001]. This was significantly lower than Group I [33.0*2.68 IU/15min]. The water content of PCI2 treated hearts [81*0.9%, p<0.001] was also lower than the other groups.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1998.11a
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• pp.191-191
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• 1998

It has been hypothesized that formation of oxygen-derived free radicals may play an important part in ischemically induced tissue injury. In this study, the effects of vitamin C treatment on hepatic reperfusion model were investigated. Livers isolated from 18 hrs fasted rats were subjected to low flow hypoxia (1 $m\ell$/g liver/min, for 45min) followed by reoxygenation (for 30min). The perfusion medium used was Krebs-Henseleit bicarbonate buffer (KHBB, pH 7.4) and vitamin C (0.25, 0.5, 1.0 and 2.0 mM) was added to perfusate. 7-Ethoxycoumarin was used as substrate of phase and metbolism. After hypoxia oxygen consumption significantly dropped but vitamin C 0.25, 0.5 and 1.0 mM treatments restored oxygen consumption to the level of control group. LDH and lipid peroxidation were not changed in all experimental groups. Oxidation, phase metabolism, significantly decreased following hypoxia but improved during reoxygenation. Vitamin C 0.25 mM treatment significantly improved the oxidation of 7-ethoxycoumarin during hypoxia and reoxygenation, but the oxidation significantly decreased by vitamin C 2.0 mM treatment. Similarly, sulfate conjugation decreased in hypoxic group, but this decrease was inhibited by vitamin C 0.25, 0.5 and 1.0 mM treatments. Our findings suggest that hypoxia/reoxygenation diminishes hepatic drug metabolizing function, vitamin C at concentration of 0.25-1.0 mM ameliorates but at higher concentration aggravates these hypoxia/reoxygenation-induced changes.

• Korean Journal of Food Science and Technology
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• v.26 no.3
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• pp.213-220
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• 1994

Gastrodia (G) Rhizoma has been used clinically as an oriental herbal medicine with sedative, anticonvulsive, and depressor effects. The present study tested effects of G. Rhizoma extracts on the coronary circulation and myocardial oxygen consumption in perfused rat hearts. Sprague Dawley rats (SD) and spontaneously hypertensive rats (SHR) were employed as experimental animals and nonworking Langendorff heart perfusion technique introduced for heart experiments. G. Rhizoma extracts were prepared from grinding G. Rhizoma into powder, extracting in water and 50% ethanol for 4 or 16 hr and diluting with Krebs-Henseleit bicarbonate perfusion buffer to be 70%. Hearts were perfused with bicarbonate buffer oxygenated with 95% $O_{2}:$ 5% $CO_{2}$ at constant coronary perfusion pressure of $90cmH_{2}O$. The diluted extracts were infused into coronary arteries in a concentration of $1{\sim}5\;{\mu}M$ for $7{\sim}8 min. While in SD water- or ethanol-extracts of G. Rhizoma extracted for 16 hr increased coronary perfusate flow (CPF) and decreased coronary vascular resistance (CVR), ethanol-extracts in SHR produced coronary vasoconstriction associated with enhanced CVR. G. Rhizoma extracts-induced increase in CPF reduced myocardial oxygen extraction, and thus myocardial oxygen consumption ($MVO_{2}$) remained at that observed prior to infusion of extracts. In SD and SHR 16 hr-water-extracts markedly altered coronary venous effluent pH and $Pco_{2}$ and evoked metabolic acidosis, which could be a coronary vasodilator mechanism decreasing CVR. In this study, the extracts decreasing CVR in SD and SHR did not augment the lactate production. Therefore, although the effects of the extracts on cardiac function and coronary circulation depended on solvents and duration for extraction, the 16hr-water-extracts, at least, exhibited coronary vasodilation in SD and SHR. Conversely, ethanol-extracts constricted coronary arteries in SHR. G. Rhizoma extracts-induced vasodilation might be due to the metabolic acidosis rather than due to the increased lactate production. The results indicate that G. Rhizoma extracts obtained from proper extracting procedures can be used as a safe and clinically applicable herbal medicine in the cardiovascular diseases such as coronary artery disease and hypertension for vasodilatory and antihypertensive actions.