Biomedical subjects

T Y Aw

Publications and source records attributed to T Y Aw.

At least 55 records · Page 3Linked to original sources

PubMed1995

Microvascular responses to inhibition of nitric oxide production. Role of active oxidants.

The objective of this study was to assess the potential contribution of hydrogen peroxide (H2O2) to the leukocyte-endothelial cell adhesion and increased microvascular permeability observed in rat mesenteric venules after inhibition of nitric oxide synthesis with NG-nitro-L-arginine methyl ester (L-NAME). Leukocyte adherence and emigration and leakage of fluorescein isothiocyanate-labeled albumin were monitored in postcapillary venules before and after exposure of the tissue to L-NAME. H2O2 production in mesenteric tissue was monitored by using dihydrorhodamine 123 (DHR), the H2O2-sensitive fluorochrome. L-NAME elicited a rapid increase in both the rate of albumin extravasation and oxidation of DHR, which was followed by an increased adherence and emigration of leukocytes in postcapillary venules. Treatment with either catalase or dimethylthiourea attenuated the L-NAME-induced oxidative stress, albumin leakage, and leukocyte-endothelial cell adhesion. Oxidation of DHR was enhanced in animals treated with either 3-amino-1,2,4-triazole (ATZ), an inhibitor of endogenous catalase, or a combination of ATZ and maleic acid diethyl ester, which depletes intracellular glutathione. Animals receiving a CD11/CD18-specific antibody to prevent leukocyte adhesion/emigration exhibited a reduced oxidation of DHR in response to L-NAME. These findings indicate that most of the H2O2 (and secondarily derived oxidants) generated in mesenteric tissue exposed to an inhibitor of nitric oxide production is due to accumulation of activated leukocytes.

Amino Acid Oxidoreductases↗

PubMed1994

Omega 3-lipid peroxides injure CaCo-2 cells: relationship to the development of reduced glutathione antioxidant systems.

BACKGROUND/AIMS: Dietary polyunsaturated fats are significant sources of luminal lipid hydroperoxides whose accumulation can be injurious to the intestinal epithelium. The current study examines the cytotoxicity of peroxidized fish oil to CaCo-2 cells. METHODS: Chromate release from cells was used as an index of CaCo-2 injury, and day 1 and day 7 postconfluent monolayers were used to represent the immature and mature states, respectively. RESULTS: Air oxidation of fish oil yielded equimolar quantities of hydroperoxyeicosapentaenoic (20:5) and docosahexaenoic (22:6) acids. Their cytotoxicity were time- and concentration-dependent and were related to the developmental stages. A 100-mumol/L dose of hydroperoxides caused a 40% and a 15% 51Cr release from day 1 and day 7 cells, respectively. Cellular glutathione (GSH), GSH redox enzyme, and gamma-glutamyl cysteine synthetase activities were significantly lower in day 1 than in day 7 cells, indicating that hydroperoxide metabolism in immature cells is rate limited by reductant supply. GSH supplementation increased cell GSH in day 7 cells (twofold) but not in day 1 cells, suggesting a limited ability of immature cells to use exogenous GSH. CONCLUSIONS: These results show that nondifferentiated cells are more sensitive to oxidant-induced injury than mature cells. This enhanced susceptibility is associated with a lower GSH-dependent detoxication capacity of the immature cells.

Antioxidants↗

PubMed1994

Biliary glutathione promotes the mucosal metabolism of luminal peroxidized lipids by rat small intestine in vivo.

We previously found that exogenous GSH enhances mucosal GSH and promotes lipid hydroperoxide metabolism by rat small intestine (AW, T. Y., and M. W. WIlliams, 1992. Am. J. Physiol. 263:G665-G672). In this study, we have developed an in vivo bile and lymph fistula rat model to test the hypothesis that biliary GSH is an important luminal source of GSH. Peroxidized fish oil was infused into the proximal intestine, and hydroperoxide accumulation in lumen, mucosa, and lymph was determined. Diversion of bile decreased mucosal GSH and increased hydroperoxide accumulation in all fractions. Supplementation with GSH, but not with GSSG, increased tissue GSH and attenuated hydroperoxide accumulation (50-60%), consistent with enhancement of hydroperoxide removal by exogenous GSH. Addition of native bile deficient in GSH, but not cysteine, cystine, or GSSG, decreased luminal and lymph hydroperoxide levels by 20-30%. Amino acid supplementation concurrently attenuated hydroperoxide recoveries in these fractions by 30-40% and increased mucosal GSH by 40%, indicating a role for biliary amino acids in hydroperoxide elimination. The effect of amino acids was abolished by buthionine sulfoximine, confirming their role in GSH biosynthesis. Collectively, the results demonstrate that bile is a rich source of reductant for maintaining mucosal GSH to promote intestinal metabolism of luminal peroxidized lipids.

Analysis of Variance↗

PubMed1994

Development of ischemia/reperfusion tolerance in the rat small intestine. An epithelium-independent event.

In stable organ systems, such as the heart and kidneys, an oxidant stress induces an increase in endogenous antioxidant systems resulting in an increased resistance of the tissue to a subsequent oxidant challenge. The development of this oxidant tolerance requires 1.5-6 d. The aim of the present study was to determine whether oxidant tolerance can be induced in the small intestinal mucosa, a labile system whose epithelium turns over every 2-3 d. Ischemia/reperfusion-induced epithelial barrier dysfunction of the small intestinal mucosa was monitored in Sprague-Dawley rats whose intestines had been exposed to an ischemic insult 1, 24, or 72 h previously. At 24 h, but not 1 or 72 h after the initial ischemic insult, the mucosa was more resistant to ischemia/reperfusion-induced barrier dysfunction. The antioxidant status of the mucosa was enhanced at 24 h, but not at 1 or 72 h after the initial ischemic insult. This adaptation appears to be specific for oxidants, since an initial ischemic insult imposed 24 h earlier also protected against H2O2-induced, but not acid- or ethanol-induced, barrier dysfunction. Further studies indicated that the increase in antioxidant status of the mucosa observed 24 h after the initial ischemic insult was a result of adaptational changes in the lamina propria, rather than the epithelium. In vitro studies with isolated epithelial cells also indicated that epithelial cells do not develop oxidant tolerance. We conclude that the development of oxidant tolerance in the small intestinal mucosa does not involve an active participation of the epithelial lining.

Animals↗

PubMed1994

Glucose regulation of hydroperoxide metabolism in rat intestinal cells. Stimulation of reduced nicotinamide adenine dinucleotide phosphate supply.

The regulation of intestinal metabolism of t-butylhydroperoxide by glucose was examined in isolated enterocytes from proximal rat intestine. The basal rate of hydroperoxide elimination in control cells was 0.57 +/- 0.05 nmol/min per 10(6) cells, and was increased threefold by 10 mM exogenous glucose (1.74 +/- 0.14 nmol/min per 10(6) cells). Concurrently, cellular NADPH levels increased threefold (1.62 +/- 0.40 nmol/10(6) cells vs 0.57 +/- 0.14 nmol/10(6) cells in controls). The glucose effect was blocked by 6-aminonicotinamide and by 1,3-bis-(2-chloroethyl) 1-nitrosourea, consistent with glucose stimulation of NADPH production by the pentose phosphate shunt, and of NADPH utilization for glutathione disulfide reduction. The NADPH supply rate was quantified by controlled infusions of diamide, a thiol oxidant. At diamide infusion of 0.05 nmol/min per 10(6) cells, GSH and protein thiols in control cells were decreased significantly, consistent with a limited capacity for glutathione disulfide reduction. With glucose, cell GSH and protein thiols were preserved at a 10-fold higher diamide infusion which was reversed by 6-aminonicotinamide, supporting the view that glucose promotes glutathione disulfide reduction by increased NADPH supply. Collectively, the results demonstrate that intestinal metabolism of hydroperoxides subscribes to regulation by glucose availability. This responsiveness to glucose suggests that nutrient availability would be an important contributing factor in the detoxication of toxic hydroperoxides by the small intestine.

6-Aminonicotinamide↗

PubMed1992

Effect of chronic hypoxia on detoxication enzymes in rat liver.

Studies were performed to determine the effects of chronic hypoxia on enzymes that catalyze various detoxication reactions. Rats were exposed to room air or 10.5% O2 for 10 days, and microsomes and postmicrosomal supernatants were isolated from liver. Detoxication enzyme activities were measured by radiochemical and spectrophotometric assays, and immunoreactive protein amounts were measured by Western blot analysis. Total cytochrome P450, as measured by the CO-difference spectrum, and activities of superoxide dismutase (EC 1.15.1.1), epoxide hydrolase (EC 4.2.1.63), catalase (EC 1.11.1.6), glutathione disulfide reductase (EC 1.6.4.2), and glutathione (GSH) S-transferase (EC 2.5.1.18) were not affected by this extent of hypoxia. In contrast, 10 days of hypoxia decreased activities or immunoreactivities (% of aerobic) of GSH peroxidase (EC 1.11.1.9) (54%), cytochrome P450EtOH2 (42%), CYP3A1 (53%), sulfotransferase (EC 2.8.2.1) (77%) and UDP-glucuronosyltransferase (EC 2.4.1.17) (65%). Activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), an important enzyme in NADPH production was also decreased to 56% of the aerobic value, but Western blot analysis showed that the amount of protein reactive with antibodies to glucose-6-phosphate dehydrogenase was not affected by hypoxia. Thus, hypoxia may decrease activity of enzymes by regulatory mechanisms even though the amount of immuno-detectable enzyme is unchanged. Liver cells isolated from rats exposed to hypoxia also gave lower GSH synthetic rates than cells from normoxic rats. This result, together with the effect of hypoxia on glucose-6-phosphate dehydrogenase, indicates that the GSH supply for GSH-dependent detoxication reactions may be limited due to chronic hypoxia. To test directly whether chronic hypoxia increased sensitivity to a compound normally detoxified by a GSH-dependent reaction, sensitivity to tert-butyl hydroperoxide (t-BuOOH) of hepatocytes from rats exposed to in vivo hypoxia was compared to that from normoxic rats. The results showed that the cells from the hypoxic rats were much more sensitive to injury. Taken together, these results suggest that decreases in amounts and/or activities of detoxication enzymes during chronic hypoxia may result in increased susceptibility of cells to chemical injury.

Animals↗

PubMed1992

Postanoxic oxidative injury in rat hepatocytes: lactate-dependent protection against tert-butylhydroperoxide.

Previous studies in this laboratory showed that hypoxia and anoxia enhance the susceptibility of hepatocytes to tert-butylhydroperoxide (TBH)-induced oxidative injury. To determine whether preceding exposure to anoxia affects postanoxic sensitivity to oxidative injury, viability was studied in hepatocytes incubated under anoxic conditions followed by reoxygenation without or with tert-butylhydroperoxide addition. Results showed that a preceding exposure to 60 min of anoxia substantially increased the vulnerability of cells to injury by the oxidant. Because substantial tissue lactate can accumulate during anoxia, the effect of increased lactate on postanoxic injury due to TBH was determined. Results showed that added lactate protected in a concentration-dependent manner. The TBH elimination rate was stimulated by lactate, and the pyruvate production rate approached the rate of TBH elimination. Thus, lactate protects against postanoxic oxidative injury by supplying reducing equivalents for peroxide reduction. This suggests that lactate accumulation during ischemia may be beneficial and that supplementation with lactate could be considered as a means to protect against postischemic injury.

Animals↗

PubMed1992

Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH.

The absorption and lymphatic transport of peroxidized MaxEPA fish oil was studied using the lymph fistula rat to determine the role of mucosal glutathione (GSH) in intestinal metabolism of luminal lipid hydroperoxides. Decreasing intestinal GSH concentrations with buthionine sulfoximine (BSO, 1.15 +/- 0.20 nmol/g), diethyl maleate (DEM, 0.93 +/- 0.26 nmol/g), phorone (1.46 +/- 0.14 nmol/g), or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 1.54 +/- 0.18 nmol/g) compared with control (2.60 +/- 0.38 nmol/g) resulted in higher luminal recovery of the infused lipid hydroperoxide (% of infused dose): BSO (87.8 +/- 4.8%), DEM (86.1 +/- 1.3%), phorone (78.1 +/- 2.1%), and BCNU (71.7 +/- 4.8%) compared with control (52.8 +/- 4.3%). These results suggest that decreased elimination of luminal peroxidized lipids is associated with decreased tissue GSH. Treatment of rats with BSO, DEM, phorone, or BCNU resulted in dramatic increases in appearance of peroxidized lipids in lymph over 6-h lipid infusion (54.7 +/- 3.7, 57.7 +/- 4.6, 46.4 +/- 2.7, and 42.1 +/- 3.9 nmol, respectively) compared with control (20.5 +/- 3.4 nmol). The results are consistent with decreased intracellular metabolism of absorbed hydroperoxides and enhanced transport into lymph under GSH-deficient conditions. The current findings suggest that the function of the mucosal GSH peroxidase/oxidized glutathione (GSSG) reductase system may play an important role in intestinal handling of luminal lipid hydroperoxides. A compromised function of this detoxication mechanism in GSH-deficient states can significantly alter the metabolic fate of dietary peroxidized lipids.

Absorption↗

PubMed1992

Intestinal absorption and lymphatic transport of peroxidized lipids in rats: effect of exogenous GSH.

We previously found that mucosal glutathione (GSH) plays an important role in the intestinal metabolism of luminal peroxidized lipids [T. Y. Aw, M. W. Williams, and L. Gray. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G99-G106, 1992]. To determine the effects of exogenous GSH on lipid hydroperoxide elimination under conditions in which mucosal GSH was initially depleted with buthionine sulfoximine (BSO), we infused peroxidized lipid solutions without or with GSH into the proximal intestine of rats and monitored the steady-state output of hydroperoxides in lymph and recovery of luminal hydroperoxides. GSH supplementation in BSO-treated rats resulted in a concentration-dependent attenuation of lymphatic output of peroxidized lipids that was correlated with increases in mucosal GSH. Compared with BSO control, the luminal lipid hydroperoxide contents were significantly lower in GSH-supplemented rats, consistent with enhanced elimination of peroxidized lipids by exogenous GSH. The effect of GSH was ameliorated by the inhibitors of GSH uptake, suggesting that the uptake of GSH is required for promotion of intestinal removal of luminal hydroperoxides. Other thiols, either at comparable or higher concentrations than GSH, were without significant effects on lymphatic transport or luminal recovery of lipid hydroperoxides, indicating that these thiols are poor substitutes for GSH. Overall, the data are consistent with exogenous GSH being a source for cellular reduction of peroxidized lipids. Results from these studies could directly impact on future consideration of therapeutic means to increase cellular antioxidant systems to promote intestinal hydroperoxide detoxication.

Animals↗

PubMed1991

Effect of chronic hypoxia on acetaminophen metabolism in the rat.

The effect of chronic hypoxia (10.5% O2 for 8-9 days) on acetaminophen metabolism was studied in vivo or in isolated cell or microsomal systems. Results from in vivo studies with oral administration of acetaminophen showed that in hypoxic rats, the plasma appearance of the drug was delayed and the plasma half-life was increased. Analyses of the area under the curve (AUCoral) showed that this value was higher in hypoxic rats, whereas the rate constants for elimination (kelim) and absorption (kabs) were lower in these animals. Formation of the glucuronide and sulfate conjugates was decreased significantly (P less than 0.05) in hypoxic animals. The calculated volume of distribution (Vd) after an intravenous dose was not different in either group but total clearance (CL) was 35% lower in hypoxic rats. Studies with isolated hepatocytes from both groups revealed that glucuronidation and sulfation were inhibited markedly at low O2 concentrations. The O2 concentrations required for half-maximal production (P50 values) of glucuronide (2.3 microM O2) and sulfate (1.8 microM O2) conjugates in cells from hypoxic animals were lower than for control cells (5.3 microM and 3.9 microM O2 for glucuronide and sulfate conjugates, respectively). Maximal rates of conjugation in cells from hypoxic rats were 60-70% of control rates. Similar decreases in microsomal UDP-glucuronosyltransferase and cytosolic sulfotransferase activities were found in livers of animals exposed to chronic hypoxia. These lower P50 values are consistent with a lower P50 for oxidation of mitochondrial cytochromes in hypoxic cells. In comparison, the P50 for glutathione conjugation (4.1 microM O2) was not statistically different from control (4.6 microM O2), but the maximal rate was 65% higher. The results show that chronic hypoxia causes a change of absorptive processes and decreased glucuronidation and sulfation reactions which affects the disposition of acetaminophen and potentially the disposition of a variety of other exogenous and endogenous compounds.

Acetaminophen↗

PubMed1991

Oral glutathione increases tissue glutathione in vivo.

Mice were given an oral dose of glutathione (GSH) (100 mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min in blood plasma and after 1 h in liver, kidney, heart, lung, brain, small intestine and skin. In control mice, GSH concentrations in plasma increased from 30 microM to 75 microM within 30 min of oral GSH administration, consistent with a rapid flux of GSH from the intestinal lumen to plasma. Under these GSH-sufficient conditions, no increases over control values were obtained in GSH concentrations in most tissues except lung over the same time course. Mice pretreated for 5 days with the GSH synthesis inhibitor, L-buthionine-S,R-sulfoximine (BSO, 80 mumol/day) had substantially decreased tissue concentrations of GSH. Oral administration of GSH to these GSH-deficient animals gave statistically significant increases in GSH concentrations in kidney, heart, lung, brain, small intestine and skin but not in the liver. Administration of the equivalent amount of the constituent amino acids, glutamate, cysteine, and glycine, resulted in little change in GSH concentrations in all tissues in GSH-deficient animals. Thus, the results show that oral GSH can increase GSH concentrations in several tissues following GSH depletion, such as can occur in toxicological and pathological conditions in which GSH homeostasis is compromised.

Administration, Oral↗

PubMed1991

Postnatal changes in pyridine nucleotides in rat hepatocytes: composition and O2 dependence.

Postnatal changes in pyridine nucleotide concentration, composition, and oxidation-reduction characteristics were studied in liver cells from neonatal (newborn, d 4 and d 8) and adult rats to determine the development of hepatic pyridine nucleotide status and O2 dependence of oxidation of reducing equivalents. The results show that the total pyridine nucleotide concentrations in newborn and 4-d-old rat liver were low (30%) but increased to near adult values (80%) by d 8 postpartum. Analyses of the cellular distribution of NAD+, NADH, NADP+, and NADPH reveal that the reduced forms (NADH plus NADPH) accounted for over 50% of the total in the newborn and 4-d-old rats compared to 30% in adult animals. This relatively higher reductive capacity in hepatocytes of younger rats was largely the result of a significantly higher proportion of NADPH in these cells. Examination of the NADPH/NADP+ and NADH/NAD+ ratios show that they occur in an inverse relationship with postnatal age; the NADPH/NADP+ ratio was high at birth and decreased with age, whereas the reverse pattern was found for the NADH/NAD+ ratio. The result, that NADPH represents a significant percentage of the total pyridine nucleotides in neonatal cells in the early postnatal period, is consistent with a higher demand for NADPh for biosynthetic activities in association with tissue growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1990

Defining the resistance to oxygen transfer in tissue hypoxia.

Studies of O2 supply in freshly isolated adult mammalian cells provide new insight into the factors that limit mitochondrial oxygenation in vivo. Of particular importance, mitochondria are present at high densities and often in apparent clusters, both of which contribute to local O2 gradients under hypoxic conditions. Current evidence indicates that the mitochondrial distribution is a component of the differentiated phenotype of adult mammalian cells and that specific motors and anchoring mechanisms are present to allow redistribution in response to developmental, physiological and pathological challenges. To compare the importance of resistance to O2 transfer under different conditions and at different sites along the supply path in vivo, a simple mathematical expression of relative resistance to O2 supply is introduced. Under various pathophysiological conditions, this resistance increases in specific regions of the pulmonary, circulatory or cellular supply path and results in O2 deficiency in the mitochondria. Regardless of cause, the relative resistance increases dramatically in the vicinity of mitochondrial clusters during hypoxia.

Acute Disease↗

PubMed1990

Tributyltin stimulates apoptosis in rat thymocytes.

Treatment of rat thymocytes with micromolar concentrations of tributyltin caused a rapid increase in the cytosolic free Ca2+ concentration that was inhibited by Ni2+, which blocks Ca2+ influx through membrane channels. The elevation of cytosolic Ca2+ was associated with extensive DNA fragmentation, which was prevented by pretreatment of the cells with either of the intracellular Ca2+ chelators quin-2 or 1,2-bis(2-amino-phenoxy)ethane-N',N',N',N',-tetraacetic acid. Loss of thymocyte viability, which followed DNA fragmentation, was also prevented by the two Ca2+ chelators or by removing extracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The pattern of DNA fragmentation was characteristic of that produced by agents which activate a Ca2(+)- and Mg2(+)-dependent endogenous endonuclease during apoptosis or programmed cell death. Additional studies showed that other organotin compounds, including trimethyltin, triphenyltin, and dibutyltin had minimal effects on cytosolic Ca2+, DNA fragmentation, and cell viability. These results are consistent with a greater susceptibility of thymocytes to tributyltin and provide a basis for understanding its selective immunotoxicity in vivo.

Animals↗

PubMed1990

Glutathione-dependent protection against oxidative injury.

Functions of GSH in detoxication during radical-induced injury in specific pathological and toxicological conditions are discussed. GSH protects against oxidative damage in systems that scavenge radicals, eliminate lipid peroxidation products, preserve thiol-disulfide status of proteins, and repair oxidant damage. Several factors which affect cellular GSH homeostasis can affect these functions, including nutritional status, hypoxia and pharmacological intervention. Evidence from a variety of pathological and toxicological conditions, e.g. ischemia-reperfusion injury, chemically induced oxidative injury, radiation damage, aging, and degenerative diseases, indicate that GSH is a primary component of physiological systems to protect against oxidant and free-radical-mediated cell injury.

Animals↗

PubMed1990

Fate of dietary glutathione: disposition in the gastrointestinal tract.

Studies were performed in rats that had been fasted 24 h, fed a glutathione (GSH)-free semisynthetic diet (AIN-76), and fed the same diet supplemented with GSH. The results from the fasted rats and those fed GSH-free diet showed that the duodenum and jejunum contained 0.2-0.5 mumol of GSH/gram wet wt of luminal contents. The GSH contents of biliary juice was sufficient to maintain this amount of GSH in the intestinal lumen. Other analyses showed that cell sloughing, bacterial GSH content, and GSH secretion by epithelial cells of the jejunum were not sufficient to account for this content. GSH concentrations following consumption of a GSH-supplemented diet (5-50 mg/g AIN-76) showed a rapid increase in all regions of the small intestine and indicated that removal occurred primarily in the jejunum. However, the combined activities of brush-border gamma-glutamyltransferase and GSH uptake systems were not sufficient to remove all of the ingested GSH. Results from in situ vascular perfusions of small intestine showed that the upper jejunum is a principal site of GSH absorption. Measurements of the GSH-to-glutathione disulfide (GSSG) ratio in the lumen after ingestion of GSSG (5 mg/g diet) indicated that the upper small intestine also has a mechanism for reducing GSSG to GSH. The results therefore indicate that GSH is present in the lumen of the small intestine of rat under most if not all conditions. Although the physiological importance of luminal GSH remains unclear, it could potentially be used to detoxify reactive electrophiles in the diet or be absorbed for intracellular detoxication reactions.

Animals↗

PubMed1989

Heme catabolism in cultured hepatocytes: evidence that heme oxygenase is the predominant pathway and that a proportion of synthesized heme is converted rapidly to biliverdin.

Heme oxygenase has been considered to be involved in the predominant pathway of heme degradation in vivo. However, alternative pathways involving cytochrome P-450 reductase, and lipid peroxidation, have previously been demonstrated in vitro, and studies with cultured rat hepatocytes were interpreted to show a majority of endogenous hepatic heme breakdown by non-heme oxygenase pathways. To clarify the pathway of heme breakdown in hepatocytes and the role of heme oxygenase in this process, cultured hepatocytes were pre-labelled with 5-[5-14C]aminolevulinate [( 14C]ALA). Radioactivity in heme, carbon monoxide, and bile pigments was measured for 8-24 h after the removal of [14C]ALA. In cultured chick embryo hepatocytes, which lack biliverdin reductase, the rate of production of biliverdin IXa was closely similar to the rate of catabolism of exogenous heme and radioactivity in carbon monoxide and biliverdin IXa was similar to the loss of radioactivity from endogenous heme. These results support the conclusion that heme breakdown occurred predominantly, if not solely, by heme oxygenase. Also, no evidence of non-heme oxygenase pathways was found in the presence of tin protoporphyrin, an inhibitor of heme oxygenase or mephenytoin, an inducer of both cytochrome P-450 and heme oxygenase. Similarly, in untreated cultured rat hepatocytes, radioactivity in carbon monoxide corresponded with loss of radioactivity in endogenous heme. In other experiments with chick hepatocyte cultures, rates of heme synthesis and breakdown were measured, and data were fitted to various models of hepatic heme metabolism. The results observed were consistent only with models in which an appreciable fraction (control cells, 17%, mephenytoin treated cells, 41%) of the newly synthesized heme was degraded rapidly to biliverdin.

Animals↗

PubMed1989

Fractionation and analysis of mitochondria with polycarbonate membrane filters.

Polycarbonate membrane filters were used to fractionate mitochondrial populations depending on their aggregation or association with other subcellular structures. Isolated rat liver mitochondria penetrated through filters which have pore sizes larger than 1 micron. In contrast, mitochondria which were induced to aggregate in vitro by incubation at low pH were retained by the filters and thus could be separated from the single or small aggregates of mitochondria. Use of this membrane filtration method to analyze release of mitochondria from isolated hepatocytes showed that treatment with digitonin at concentrations only sufficient to lyse the plasma membrane did not release mitochondria. Homogenization or sonication following digitonin treatment released 25-50% of the mitochondria, but only a small fraction was intact. A high yield of intact mitochondria was released from digitonin-treated cells by a brief treatment with a low concentration of the proteolytic enzyme nagarse. Thus, this membrane filtration method provides a simple and rapid approach to analyze the extent of mitochondrial aggregation and association with other subcellular structures.

Animals↗