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Biomedical subjects

M Ashraf

Publications and source records attributed to M Ashraf.

At least 91 records · Page 5Linked to original sources
PubMed1993

Demonstration of hydroxyl radical and its role in hydrogen peroxide-induced myocardial injury: hydroxyl radical dependent and independent mechanisms.

We investigated the mechanism of hydrogen peroxide (H2O2) action on myocardial injury in relation to hydroxyl radical (.OH) formation. Isolated rat hearts were perfused with a concentration of H2O2 (300 microM) known to produce cardiac injury. Perfusion of H2O2 for 15 min caused severe myocardial dysfunction, morphological damage, ATP depletion, and lipid peroxidation. Hydrogen peroxide concentration in the coronary effluent was reduced approximately 40% reflecting a myocardial H2O2 consumption of 12.7 +/- 0.9 mumol/15 min/g wet tissue (n = 12). One of the .OH-generated derivatives, 2,3-dihydroxybenzoic acid (2,3-DHBA), formed from reaction with salicylic acid, was detected in the coronary effluent by high-performance liquid chromatography at 23.16 +/- 4.05 nmol/15 min/g wet tissue. Catalase (200 U/ml, n = 6) added to the perfusate attenuated all parameters of myocardial injury by eliminating H2O2 from the perfusate, and thus .OH was not detected in the effluent. Deferoxamine (5 mM, n = 7) added to the perfusate reduced morphological damage and lipid peroxidation, but not dysfunction or ATP depletion. Deferoxamine significantly reduced .OH production; 2,3-DHBA was 5.22 +/- 3.56 nmol/15 min/g wet tissue. The present study provides evidence that .OH is produced in the H2O2-perfused heart. The adverse H2O2-mediated myocardial outcomes documented in this study appear to arise from both .OH-dependent and .OH-independent mechanisms.

Adenosine Triphosphate↗
PubMed1993

Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation. Is the hydroxyl radical really the most damaging radical species?

Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (.OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H2O2 as a possible precursor of .OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of .OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83 +/- 0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of .OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094 +/- 0.021 units/mg protein) and increased fivefold after 120 minutes (0.428 +/- 0.069 units/mg protein). Similarly, MDA increased from 0.78 +/- 0.14 nmol/mg protein at 15 minutes to 1.65 +/- 0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the .OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 microM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate .OH endogenously; 2) maximum .OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in .OH generation; 4) there was no significant production of .OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional .OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than .OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.

Animals↗
PubMed1993

Role of magnesium in the management of hypertension.

Twenty-two patients receiving long term diuretic treatment for arterial hypertension (19) and congestive heart failure (3) received magnesium chloride 10 mmol/day for four months. Both systolic and diastolic pressures decreased significantly, by a mean of 13 +/- 9 mmHg. No significant changes were recorded in serum or urinary electrolytes except for magnesium.

Adult↗
PubMed1993

Subcellular distribution of xanthine oxidase during cardiac ischemia and reperfusion: an immunocytochemical study.

Oxygen-derived free radicals are known to take part in cardiac injury during post-ischemic reperfusion (I/R). Xanthine oxidase (XO) is closely associated with the generation of superoxide radicals. We have determined the distribution of XO in rat myocardium after ischemia (I) and I/R by immunocytochemical method using murine monoclonal antibody against XO (bovine milk) and by enzyme histochemistry (EHC) in situ. Frozen sections of periodate-lysine-paraformaldehyde (PLP) fixed myocardium after 15, 60 and 90 min ischemia and 15 min ischemia and 30 min reperfusion were processed for immunocytochemistry and EHC. In other experiments, rats were treated with allopurinol, an inhibitor of XO, and hearts were processed for immunocytochemistry. By immunoperoxidase and immunofluorescence methods, a deep staining of interstitial cells, capillary and small blood vessels was observed, but the staining intensity of these cells was increased after reperfusion, in comparison to the normal and ischemic heart tissue. In the electron microscope, an immunoperoxidase reaction product was seen in the cytoplasm of interstitial, endothelial and smooth muscle cells. Similarly, EHC studies by nitroblue tetrazolium staining showed an increase in enzymatic activity in the tissue after reperfusion. The allopurinol-treated I/R tissue exhibited reduced staining. The data suggest that XO activity increases during ischemia but intensifies after reperfusion. The enzyme is localized in interstitial cells, coronary vessel endothelium and smooth muscle cells. XO is constantly present in the interstitial cells of the myocardium and it is a new finding not previously reported. It is further suggested that myocardial interstitium may be one of the major sites where oxygen derived radicals are generated during ischemia.

Allopurinol↗
PubMed1992

Production of hydroxyl radicals and their disassociation from myocardial cell injury during calcium paradox.

The production of hydroxyl radicals during calcium paradox injury was investigated by measuring the production of 2,5-dihydroxybenzoic acid (2,5-DHBA) from salicylate. Four groups of rats were analyzed. In the first group, isolated hearts were perfused with calcium-free medium for 10 minutes followed by perfusion with medium containing Ca++ for 10 minutes. In the other groups, 0.25 microM N,N'-diphenyl-1,3-phenylenediamine (DPPD), 80 microM cytochrome c, or 450 U/ml catalase was added. Coronary effluent was analyzed for the presence of 2,5-DHBA, and tissue sections were examined using light microscopy. In the first group, 2,5-DHBA production began during the calcium-free period, peaked tenfold 60-90 sec. into the Ca repletion period, and declined thereafter. The increase in 2,5-DHBA was accompanied by severe cell damage. Cytochrome c reduced 2,5-DHBA production, and catalase almost completely inhibited 2,5-DHBA production, while DPPD had no effect on 2,5-DHBA production. None of the three additives provided any complete morphological protection. The data provide evidence for the production of hydroxyl radicals during calcium-paradox injury, that their production is dependent upon the presence of hydrogen peroxide, and that cell damage in the calcium paradox is not primarily mediated by the extracellular hydroxyl radicals.

Animals↗
PubMed1992

Quantification of hydroxyl radical and its lack of relevance to myocardial injury during early reperfusion after graded ischemia in rat hearts.

To elucidate the pathophysiological role of the hydroxyl radical (.OH) during the postischemic reperfusion of the heart, we measured the .OH product in the coronary effluent from isolated perfused rat heart during a 30-minute reperfusion period after various ischemic intervals of 5, 10, 15, 20, 30, and 60 minutes. Salicylic acid was used as the probe for .OH, and its derivative, 2,5-dihydroxybenzoic acid (2,5-DHBA), was quantified using high-performance liquid chromatography with ultraviolet detection. 2,5-DHBA was negligible in the effluent from nonischemic hearts, but a significant amount was detected from the hearts rendered ischemic for 10 minutes or longer. The peak of 2,5-DHBA was seen within 90 seconds after the onset of reperfusion in every group. The accumulated amount of 2,5-DHBA was maximal in the group with 15-minute ischemia (6.73 +/- 1.04 nmol/g wet heart wt after 30 minutes of reperfusion); it decreased as the ischemic time was prolonged and was 2.38 +/- 0.84 nmol/g wet wt after 30 minutes of reperfusion in the group with 60-minute ischemia. In the model of 15-minute ischemia/30-minute reperfusion, there was no correlation between the accumulated amount of 2,5-DHBA and functional recovery (+/- dP/dt, heart rate, and coronary flow), lactate dehydrogenase release, and morphological damage. Although treatment with 0.5 mM deferoxamine, an iron chelator, significantly decreased 2,5-DHBA (from 6.73 +/- 1.04 to 2.29 +/- 0.80 nmol/g wet wt after 30 minutes of reperfusion, p less than 0.01), it failed to reduce the postischemic myocardial injury in the group with 15-minute ischemia. The results suggest that .OH production is influenced by the preceding ischemic interval and that .OH does not exert an immediate direct effect on postischemic damage during early reperfusion in the isolated perfused rat heart, although a possibility remains that the small portion of .OH trapped by salicylic acid may not be intimately associated with myocardial injury.

Animals↗
PubMed1992

Hydroxyl radical production during early reperfusion after different periods of ischemia in rat hearts and its effect on myocardial function. .OH in postischemic heart.

To elucidate the significance of hydroxyl radical (.OH) in postischemic reperfusion injury, we measured the .OH production in the coronary effluent collected from isolated perfused rat hearts during reperfusion period of 15 minutes after various ischemic intervals ranging from 5 to 60 minutes. Salicylic acid was used as a probe for .OH formation, and its derivative, 2,5-dihydroxybenzoic acid (2,5-DHBA), was quantified using high performance liquid chromatography. A significant amount of 2,5-DHBA was detected from the hearts rendered ischemic for 10 minutes and longer. The peak of 2,5-DHBA was seen within 90 seconds after the onset of reperfusion in every group, and the accumulated amount of 2,5-DHBA was maximal in 15 minutes ischemia group (3.97 +/- 0.49 nmol/g/15 minutes reperfusion) in contrast to 1.22 +/- 0.30 nmol/g/15 minute in 60 minutes ischemia. This study demonstrated an ischemic time-dependent .OH production during reperfusion, and no direct effect of .OH was observed on the post-ischemic injury related to myocardial function.

Analysis of Variance↗
PubMed1992

Maximal .OH production is seen upon reoxygenation of viable anoxic cultured cardiomyocytes but not of compromised cells.

Hydroxyl radicals (.OH) in isolated cultured cardiomyocytes upon reoxygenation (reoxy) were measured after graded anoxia (A) using high performance liquid chromatography (HPLC). Isolated myocytes were subjected to A for 15, 30, 60, 90 and 120 minutes and reoxy for 120 seconds. Supernatant was collected after reoxy, extracted with ether and injected into HPLC for measuring hydroxylation products of salicylic acid (2,5-DHBA) as an indicator of .OH formation. 2,5-DHBA was detected maximally after 15 minutes of A and 120 seconds of reoxygenation (34.2 +/- 3 pmol/mg protein), at which time 80% of cells had maintained their rod shape and 99% of cells excluded both trypan blue (TB) and horseradish peroxidase (HP). There was significantly less (P < 0.05) 2,5-DHBA in the group subjected to 15 minutes of A only without reoxy (7.95 +/- 1.2 pmol/mg protein). 2,5-DHBA decreased to 13.1 +/- 2 pmol/mg protein at 120 minutes of A/120 seconds reoxy. With increasing anoxic time, the number of rod-shaped cells decreased from 80% at 15 minutes to 30% at 120 minutes, while the number of TB/HP positive cells increased from 0% at 15 minutes to 100% at 120 minutes. The cell membrane blebs were nonexistent at 15 minutes, but at 120 minutes A intense bleb formation was observed. These data suggest that .OH is produced upon reoxygenation of anoxic cultured cardiomyocytes and their production is maximum when majority of myocytes are viable.

Animals↗
PubMed1992

Ultrastructural effects of hydrogen peroxide on the sarcolemma of rat heart.

Ultrastructural effects of hydrogen peroxide (H2O2) on the sarcolemma of the isolated rat heart were investigated with transmission electron microscopy combined with biochemical, enzyme histochemical, and freeze fracture techniques. Three hundred microM H2O2 were continuously administered to the Langendorff perfused isolated rat hearts. A significant amount of lipid peroxidation associated with depressed Na-K-ATPase activity was observed after 15 minutes of H2O2 perfusion (Group I), and consequently the cell membrane permeability was greatly increased. When 2.5 mM,N'-diphenyl-1,4-phenylenediamine (DPPD), a potent antioxidant, was added to the perfusate, the lipid peroxidation was totally inhibited (Group II). DPPD prevented an increase in the cell membrane permeability. However, Na-K-ATPase activity was not restored by DPPD. Decreased cytochemical staining of Na-K-ATPase was associated with an increase in cell membrane permeability. H2O2 appears to affect, not only lipids but also, intramembranous proteins embedded in the cell membrane. The combined effects of H2O2 on the membrane lipid and proteins result in the formation of membranous blebs.

Animals↗
PubMed1992

Effect of exogenous hydrogen peroxide on myocardial function and structure in isolated rat heart.

A time- and dose-dependent effect of exogenous hydrogen peroxide was determined on myocardial function, structure, high energy phosphate and lipid peroxidation in the isolated perfused rat heart. Hydrogen peroxide induced a dose-dependent decrease in cardiac function whereas 200 microM hydrogen peroxide reduced +dP/dt to 50% of control value after 10 mins. The effect of 300 microM hydrogen peroxide was more severe after 15 mins; changes observed with this dose were reversible within 10 mins of perfusion, becoming irreversible after 15 mins. Lipid peroxidation and severe morphological damage were observed after 10 mins of perfusion with 300 microM hydrogen peroxide. When 16 mEq potassium ions were added in the perfusion buffer during hydrogen peroxide perfusion, the degree of tissue damage and loss of ATP were attenuated. However, lipid peroxidation was not inhibited by high potassium ions. When 0.25 microM N,N'-diphenyl-1,4-phenylenediamine, a potent antioxidant, was added to the perfusate, lipid peroxidation was totally inhibited and the degree of tissue damage was decreased. However, depletion of tissue ATP and functional deterioration were not influenced. These results suggest that hydrogen peroxide-mediated ATP loss was independent of lipid peroxidation.

Adenosine Triphosphate↗
PubMed1992

Changes in fibroblast-derived trypomastigotes of Trypanosoma cruzi during long-term culture.

Fibroblast-derived trypomastigotes (FDTs) of Trypanosoma cruzi that had been in culture for extended periods of time were found to differ in their ability to proliferate in culture when compared to blood-form trypomastigotes (BFTs) and FDTs that had been recently established from blood-forms. "Old" FDTs transform into amastigotes/spheromastigotes and epimastigotes and readily incorporate [3H]thymidine in medium alone or in the presence of mouse spleen cells, whereas "new" FDTs and BFTs did not incorporate [3H]thymidine although they did transform in culture. These differences should be considered when FDTs are used for physiologic and immunologic studies of T. cruzi.

Animals↗
PubMed1991

Detection of hydroxyl radicals in the post-ischemic reperfused heart using salicylate as a trapping agent.

The formation of hydroxyl radical in the post-ischemic reperfused heart was measured with high performance liquid chromatography and ultraviolet detection using salicylic acid. Hydroxyl radicals react with salicylic acid yielding 2,3- and 2,5-dihydroxybenzoic acid, which can be separated by the liquid chromatography. Isolated rat hearts were perfused with 1 mM salicylic acid and were subjected to 30 mins of global ischemia followed by aerobic or anaerobic reperfusion at 37 degrees C. The effluent from the hearts was collected at various intervals, extracted with ether, and injected into the high performance liquid chromatography unit. 2,5-dihydroxybenzoic acid was present only after aerobic reperfusion and was not detected before ischemia. The liquid chromatography peak of 2,3-dihydroxybenzoic acid was too small for quantitation. The concentration of 2,5-dihydroxybenzoic acid was the highest within 300 s of reperfusion. 2,5-dihydroxybenzoic acid was not detected in the ischemic hearts during anaerobic reperfusion. In ischemic hearts perfused with mannitol, the amount of 2,5-dihydroxybenzoic acid after reperfusion was reduced. These data suggest that hydroxyl radicals are produced in the post-ischemic reperfused heart and that the present method is useful and reliable for the measurement of hydroxyl radicals in the heart.

Animals↗
PubMed1991

Development of an intracutaneous depot for drugs. Binding, drug accumulation and retention studies, and mechanism of depot.

In previous studies we reported that the permeation of dexamethasone and hydrocortisone across the skin was decreased and penetration into the skin was increased in the presence of Transcutol (TC) when compared to water as a solvent. The objective of this investigation was to study the effect of TC on the binding of dexamethasone and hydrocortisone to the skin, and on the accumulation and retention in the skin. Adsorption and desorption studies were conducted for dexamethasone and hydrocortisone with full thickness skin (FTS), and epidermis. The amount of dexamethasone and hydrocortisone adsorbed and desorbed with FTS and epidermis was essentially the same. In the presence of TC the amount of dexamethasone and hydrocortisone adsorbed was increased by 100% with both FTS and epidermis, whereas there was no difference in the amount of dexamethasone and hydrocortisone desorbed. A topical delivery system was developed with and without TC and was evaluated in vivo using the rat as an animal model for hydrocortisone accumulation after multiple dosing. The systemic body burden was reduced by 70% and skin retention of hydrocortisone was increased by 100% in all the layers of the skin. The detection of hydrocortisone accumulation and retention were studied by autoradiography and electron microscopy, and the results support the hydrocortisone depot in the skin due to TC.

Administration, Cutaneous↗
PubMed1991

Immunocytochemical localization of xanthine oxidase in rat myocardium.

Monoclonal antibodies (MAb's) to xanthine oxidase (XO) (from bovine milk) were produced by hybridoma technique. Culture supernatants were initially screened using enzyme-linked immunoabsorbant assay (ELISA). Forty four positive clones were subcloned and further characterized by ELISA and immunoblot analysis. Out of fifteen clones, which were positive in immunoblot analysis, one clone N2-26 was also positive in immunocytochemical studies. Indirect immunoperoxidase and enzyme histochemistry staining showed that XO activity is present in endothelial cells of capillaries, small blood vessels and also in interstitial cells. Electron microscopy revealed that diaminobenzidine reaction product was distributed in the cytoplasm of interstitial cells and endothelial cells of capillaries and small blood vessels. This is the first report of the presence of XO in interstitial cells and endothelial cells of small blood vessels. Allopurinol, which inhibits the xanthine oxidase activity, did not have any effect on the immunocytochemical staining. Our results in normal rat heart suggest that XO activity is confined to interstitial cells, endothelial cells of capillaries and small blood vessels.

Animals↗
PubMed1990

Prospective study of nerve conduction parameters and serum magnesium following cisplatin therapy.

This study evaluated the effects of cisplatin on 18 nerve conduction parameters of median, ulnar, peroneal, and sural nerves in relation to age, magnesium nadir, average magnesium, and magnesium replacement in gynecologic oncology patients. The 37 patients in this study received cisplatin (70 mg/m2) at 4-week intervals either as a single agent (17 patients) or in combination with doxorubicin and cyclophosphamide (20 patients) following inpatient hydration. The patients were placed randomly on either magnesium supplementation (intravenous plus oral) or placebo. For all patients, nerve conduction studies were performed before and after cisplatin therapy in the same EMG laboratory. Statistical analysis revealed that postcisplatin nerve conduction parameters were significantly predictable based upon pretherapy nerve conduction parameters, magnesium supplementation, total cisplatin, age, magnesium nadir, and average magnesium. Total cisplatin, age, and serum magnesium level were significant predictors in 8, 2, and 2 of the 18 nerve conduction parameters, respectively. Following cisplatin therapy, there was a significant decrease in sensory nerve action potential amplitude of median, ulnar, and sural nerves, whereas sensory latency of median and ulnar nerves was significantly increased. Cisplatin therapy had no effect on motor nerve conduction parameters of median, ulnar, and peroneal nerves. Factors responsible for the decrease in sural sensory action potential amplitude were not identified. Sensory nerve action potential amplitude and sensory latency of ulnar nerve are the two best objective parameters that can be utilized to monitor patients for adverse nerve conduction side effects of cisplatin.

Action Potentials↗
PubMed1990

Evaluation of high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA) and particle concentration fluorescence immunoassay (PCFIA) methods for the screening, quantitation and pharmacokinetic study of furosemide in horses.

Equine plasma and urine samples were analyzed by using a high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA) and particle concentration fluorescence assay (PCFIA). Although ELISA and PCFIA were rapid, simple and sensitive for the screening of furosemide, they did not give reproducible quantitative results. The HPLC method, which required relatively longer analysis time, provided simple and reproducible quantitative analysis of furosemide in plasma and urine. The performance of the three methods was compared for the quantitation of furosemide in plasma obtained from thoroughbred mares dosed intravenously with furosemide (500 micrograms/kg (n = 7) and 1.0 mg/kg (n = 5)). Although the plasma furosemide profiles determined by ELISA, PCFIA and HPLC were similar, ELISA and PCFIA methods exhibited considerable variation in values. At high furosemide concentrations, the PCFIA method gave better quantitative values than ELISA. However, at trace furosemide concentrations the PCFIA method gave false positive values which were not confirmed by HPLC or ELISA. The pharmacokinetic values obtained from the HPLC data and the pharmacokinetic values obtained previously from the gas chromatographic data were comparable. The data obtained by ELISA and PCFIA were not suitable for the pharmacokinetic calculations.

Animals↗
PubMed1990

Needle knife papillotomy: how safe and how effective?

Between January 1986 and July 1988 needle knife papillotomy was attempted in 103 patients after failure of conventional access for endoscopic sphincterotomy (96 cases) or diagnostic cholangiography (seven cases). Access was obtained at the same session in 36 cases and at a subsequent attempt within 2 to 5 days in a further 43, an overall success rate of 77%. The procedure related morbidity and mortality in the therapeutic group were 5.2% and 2.0% respectively. There were no deaths or complications in the diagnostic group. Needle knife papillotomy is a valuable adjunct to conventional techniques of biliary access.

Aged↗