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Biomedical subjects

W E Smith

Publications and source records attributed to W E Smith.

At least 55 records · Page 3Linked to original sources
PubMed1995

Quantitative determination of functional thiol groups on intact cell surfaces by resonance Raman spectroscopy.

The sensitivity and selectivity of resonance Raman spectroscopy, combined with electronic spectroscopy, has been used to develop a method to quantify the membrane thiol population in situ in viable erythrocytes. This technique is based on the thiol-disulfide reaction of Ellman's reagent (5,5'-dithio-bis-2-nitrobenzoic acid). It has the advantage that continuous monitoring of lysis is simple and a correction can be made for any interference resulting from lysis. In addition, the extent of reaction can be expressed as a ratio of the reagent signal, providing an internal calibrant.

Calibration↗
PubMed1994

Alteration of the erythrocyte glutathione redox balance by N-acetylcysteine, captopril and exogenous glutathione.

The effect of the thiol containing compounds N-acetylcysteine and captopril on glutathione metabolism in human erythrocytes has been investigated non-invasively using 1H spin echo NMR. N-Acetylcysteine was found to increase the reduced form of glutathione while captopril increased the oxidized form, but no changes in the total glutathione concentration were observed. Incubation of the cells with buthionine sulphoximine to inhibit de novo glutathione synthesis did not affect the response. The results show that these compounds act by altering the redox balance of glutathione rather than by stimulating its synthesis, and that their mechanisms of action are different, and not simply due to the presence of the thiol group.

Acetylcysteine↗
PubMed1994

The interaction of colloidal metals with erythrocytes.

The interactions of citrate reduced colloids (Ag, Au, and Bi) with intact erythrocytes and erythrocyte lysate have been studied by 1H spin echo NMR. Silver colloid is observed to induce cellular depletion of cytosolic glutathione and bismuth colloid induces cytosolic glutathione oxidation in the intact cell. In comparison, there is no detectable effect with gold colloid. With red cell lysate the three colloids all remove glutathione from the spectrum. The metal salts AgNO3 and NaAuCl4 both oxidize intracellular glutathione to diglutathione whereas BiO(NO3) has no effect. Thus colloidal preparations have a different reactivity to their parent metal salts. The differences observed between the three types of colloids (silver, gold, and bismuth) are unique to the colloids studied. None of the colloids studied were biologically inert in the erythrocyte model used.

Bismuth↗
PubMed1994

Increased susceptibility to oxidation of vitamin E in mitochondrial fractions compared with synaptosomal fractions from rat brains.

The in vitro oxidation of vitamin E (alpha tocopherol) in rat brain synaptosomes and mitochondria by 2,2'-azo-bis-(2'-amidinopropane) dihydrochloride (ABAPH), a free radical generator, was studied. Subcellular fractions (300 micrograms total protein) were suspended in different buffers at pH 7.4 and incubated at 37 degrees C. In the presence of 0.5 mM ABAPH the mitochondrial alpha tocopherol began to get oxidized after a lag time or induction time of 15 min compared with a lag time of 30 min for the synaptosomal fraction. Thus the reserve of reducing compounds that are responsible for delaying tocopherol oxidation is less in mitochondria than in synaptosomes. More tocopherolquinone was produced during incubations without ABAPH compared with incubations in the presence of ABAPH suggesting that the mechanism of oxidation of tocopherol differs under these two conditions. When mitochondria were incubated in buffer without oxidants the production of tocopherolquinone preceded that of thiobarbituric acid reactive substances, an indicator of peroxidation of fatty acids. Therefore, alpha tocopherol is active as an anti-oxidant in mitochondrial membranes and the production of alpha tocopherolquinone could be a monitor of mild membrane oxidation under in vitro conditions. The ease of oxidation of mitochondrial tocopherol suggests a general vulnerability of the mitochondrial membranes to oxidation. Adding vitamin E or its water soluble analogs during in vitro experiments may improve the stability and viability of mitochondria. Furthermore, antioxidant protection by vitamin E may be crucial for the maintenance of tissues, such as brain, whose function is critically dependent upon the availability of high energy phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidines↗
PubMed1994

The assessment of the HO. scavenging action of therapeutic agents.

A new method is reported for the assessment of the HO. scavenging action of therapeutic agents. It is based on the photolysis of zinc oxide and has a detection limit of 3.3%. The scavenging order of the compounds tested was penicillamine > rentiapril > ascorbic acid > cysteine > glutathione > thiomalic acid > N-acetylcysteine > myocrysin > methionine. None were as effective as DMSO. It is argued that these compounds can have an in vivo protective effect where HO. is produced from oxidant producing cells, thus limiting radical induced damage.

Azo Compounds↗
PubMed1994

Intracellular and extracellular antioxidant buffering levels in erythrocytes from pregnancy-induced hypertension.

Both intracellular (lysate thiol, lysate glutathione and lysate superoxide dismutase) and extracellular (plasma thiol, plasma glutathione and membrane thiol) antioxidant buffering levels were measured in red blood cells from patients with pregnancy-induced hypertension (PIH). We found the following. (1) The levels of plasma thiol and plasma glutathione in PIH women with proteinuria were markedly lower than that in the normal pregnancy. (2) The concentrations of lysate glutathione and superoxide dismutase in PIH women with proteinuria were significantly decreased compared with that in the normal pregnancy. (3) With the exception of plasma and lysate glutathione, all the tested antioxidant markers were not significantly different between PIH women without proteinuria and normal pregnancy. (4) There was no statistical correlation between antioxidant buffering level and BP in patients with PIH. We concluded that both extracellular and intracellular antioxidant buffering levels were decreased in patients with PIH, especially in those with proteinuria. The reduction of the antioxidant buffering level could account for several important pathophysiological features of PIH, such as the elevation of intracellular calcium, decreased red blood cell deformability and endothelial damage.

Adult↗
PubMed1993

A liquid chromatographic method for the simultaneous determination of alpha-tocopherol and tocopherolquinone in human red blood cells and other biological samples where tocopherol is easily oxidized during sample treatment.

A liquid chromatographic method for the simultaneous determination of alpha-tocopherol and tocopherolquinone in human red blood cells is described. Tocopherols in the red cell membrane are very susceptible to oxidation during sample processing. Red cell samples are saponified in the presence of a mixture of butylated hydroxytoluene, ascorbic acid, and pyrogallol and then extracted with hexane. The tocopherol compounds are separated on a C-18 column using a mobile phase containing 12% acetonitrile, 83% methanol, and 5% buffer (NaH2PO4.H2O, 7.5 mM final concentration) and are detected electrochemically. The mixture of antioxidants is essential to avoid loss of the tocopherol compounds during processing of samples. The use of acetonitrile in the mobile phase results in the separation of tocopherolquinone from delta-tocopherol. The proposed method may be generally suitable for the analysis of biological samples where tocopherols are especially vulnerable to oxidation. The levels of tocopherolquinone and delta-tocopherol in normal red cells are quite small (less than 1% of alpha-tocopherol). The ratio of tocopherol and tocopherolquinone concentrations might serve as a useful index of the redox status of red cell membranes, particularly under in vitro conditions.

Biological Assay↗
PubMed1993

The interaction of sodium nitroprusside with peripheral white blood cells in vitro: a rationale for cyanide release in vivo.

Isolated monocytes and polymorphonuclear leukocytes release the cyanide anion from the hypotensive agent, sodium nitroprusside. The proposed mechanism involves the production of hydrogen peroxide which oxidizes the substitution inert d6 iron(II) transition metal complex to a labile d5 iron(III) complex. Consequently, the release of cyanide becomes more favoured. This mechanism is supported by the chemical release of the cyanide anion from sodium nitroprusside in vitro by hydrogen peroxide and the demonstrated ability of monocytes and polymorphonuclear leukocytes to generate hydrogen peroxide ex vivo when chemically stimulated by the nitroprusside anion. The results suggest that within a clinical environment white cell counts and cell activation induced as a consequence of disease processes may be important for predicting the toxicity of sodium nitroprusside.

Cells, Cultured↗
PubMed1993

Site of action for the inhibition by gold sodium thiomalate of rabbit platelet activation.

The inhibitory action of the gold-based drug gold sodium thiomalate was investigated in rabbit platelets. Gold sodium thiomalate at concentrations of 0.25-13 x 10(-4) M inhibits collagen-, ADP-, and 9,11,dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F2 alpha (U46619)-induced aggregation as well as collagen- and U46619-induced serotonin release. This inhibition occurs in both Tyrodes-albumin or Tyrodes-gelatin buffer systems. Preincubation of gold sodium thiomalate with platelets resulted in less inhibition as the time of preincubation increased. The inhibitory effect of gold sodium thiomalate could be removed by washing the platelets. Other sulfhydryl-reacting compounds, such as D-penicillamine, thiomalic acid, 5,5'-dithiobis-2-nitrobenzoic acid, and 6,6'-dithiodinicotinic acid, were all capable of inhibiting collagen-induced aggregation and serotonin release. Evidence is presented that gold sodium thiomalate interferes with the activation of rabbit platelets by several activators, that this action of gold sodium thiomalate is similar to the action of other sulfhydryl-reacting agents, that this inhibition is likely occurring at the membrane, and that the action of the drug is not dependent on the presence of albumin.

Animals↗
PubMed1993

Flow compensation in MRI using a phase-corrected real reconstruction.

Flow- and motion-related artifacts are problematic in clinical MR imaging. In this paper we discuss the utility of a phase-corrected real reconstruction to reduce flow artifacts. This technique is particularly useful when flow-compensation pulse sequences may not be possible, such as when a very short echo delay or small field-of-view is desired. We will demonstrate that the phase-corrected real reconstruction provides superior results to the magnitude reconstruction either used alone or in conjunction with existing flow-compensation techniques.

Artifacts↗
PubMed1993

Prostacyclin, thromboxane and antioxidant levels in pregnancy-induced hypertension.

1. Plasma 6-keto-prostaglandin F1 alpha (6-keto-PGF 1 alpha, a major metabolite of prostacyclin), plasma thromboxane B2 (TXB2, a major metabolite of thromboxane A2) and five antioxidants (indirect markers of reactive oxygen species) namely, plasma thiol, erythrocyte lysate thiol, erythrocyte superoxide dismutase, plasma total glutathione and erythrocyte membrane thiol, were measured in 25 healthy non-pregnant women, 36 normotensive pregnant women and 35 women with pregnancy-induced hypertension (PIH). 2. The levels of TXB2 were significantly increased in normal pregnant women and PIH women with or without proteinuria compared with non-pregnant women. The concentrations of TXB2 in PIH women with proteinuria were higher than those without proteinuria (P < 0.05). 3. The levels of 6-keto-PGF1 alpha in healthy non-pregnant women and PIH women with or without proteinuria were significantly lower than that in normotensive pregnant women (all of three P < 0.01). There were no significant differences between healthy non-pregnant women and PIH women with and without proteinuria. 4. The ratio of TXB2 to 6-keto-PGF1 alpha was markedly elevated in PIH women with or without proteinuria compared with normotensive pregnant women and healthy non-pregnant women. The difference between PIH women with proteinuria and those without proteinuria was not significant (P > 0.05). 5. The levels of plasma thiol, superoxide dismutase and glutathione were significantly decreased in PIH women compared with normotensive pregnant women. 6. There were significant positive correlations between the levels of prostaglandins and antioxidant activity.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Evidence of oxidative stress in chronic heart failure in humans.

Chronic heart failure (CHF) due to coronary artery disease (CAD) has been shown to be associated with increased plasma thiobarbituric reactive substances (TBARS) and reduced plasma thiol (PSH) concentrations, suggesting oxidative stress (OS). The aims of the present studies were (a) to determine whether OS is due to CAD or CHF per se and (b) to determine if a wider range of more specific markers of OS are abnormal in CHF. In the first study, two groups of patients (n = 15 each) were compared. Group 1 (11 male, mean age 56 years) had CHF due to CAD and group 2 (12 male, mean age 53 years) had non-CAD CHF. Median plasma TBARS in controls was 7.6 nmol.ml-1, 10.0 nmol.ml-1 in group 1 and 9.3 nmol.ml-1 in group 2 (P < 0.01 both groups vs control). Median PSH was 505 384 and 364 nmol.ml-1 (P < 0.05 and P < 0.01 vs control) respectively. Fifty-three patients with CHF were recruited in the second study. Malondialdehyde and PSH were 10.3 and 409 nmol.ml-1 respectively, compared to control values of 7.9 and 560 nmol.ml-1 (both P < 0.001). The median values for the following additional measures of OS in controls and patients were: erythrocyte superoxide dismutase 131 vs 114 U.l-1 (P = 0.005); caeruloplasmin oxidase 97 vs 197 U.l-1 (P < 0.01); erythrocyte glutathione 1.56 nmol.ml-1 vs 1.77 nmol.ml-1 (P < 0.02); plasma conjugated dienes 0.28 vs 0.33 optical density units (P = ns).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Studies of oxidative stress in cellular systems. The interaction of monocytes and erythrocytes.

1H spin echo NMR spectroscopy is used to follow the interaction of intact and viable erythrocytes and monocytes obtained from different sources in mixed cultures. After a lag time (270 min) erythrocyte glutathione is observed to become more oxidised. This result is believed to occur as a consequence of monocyte activation generating hydrogen peroxide or hypochlorous acid, which is targeted at the erythrocyte. The red cell in turn employs its sulphydryl system as an anti-oxidant defence.

Erythrocytes↗
PubMed1992

Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization.

1. The gene CYP102 encoding cytochrome P-450 BM-3 and subgenes encoding the cytochrome P-450 and cytochrome P-450 reductase domains have been cloned in Escherichia coli. 2. The protein products of these genes have been overexpressed and purified to homogeneity. 3. The cytochrome P-450 domain is purified in the ferric low-spin state, but is readily converted into the high-spin state by addition of the substrate palmitate (Ks = 1 microM). The cytochrome P-450 reductase domain readily reduces cytochrome c. Mixing the two domains reconstitutes only about one-thousandth of the fatty acid hydroxylase activity associated with the intact cytochrome P-450 BM-3. 4. The X-band e.p.r. spectra of both the cytochrome P-450 domain and intact cytochrome P-450 BM-3 give g-values indicating low-spin ferric haem. The spectra are virtually identical with those of the equivalent form of cytochrome P-450 cam indicating that the haem ligation in cytochrome P-450 BM-3 is identical with that of cytochrome P-450 cam. 5. Resonance Raman spectra of the substrate-free and substrate-bound forms of the cytochrome P-450 domain are given. Spectral differences in comparison with cytochrome P-450 cam may reflect subtle electronic differences between the respective haem environments.

Bacillus megaterium↗
PubMed1992

Clinical analysis by 1H spin-echo NMR. 2. Oxidation of intracellular glutathione as a consequence of penicillamine therapy in rheumatoid arthritis.

Spin echo NMR analysis is used to monitor the effect of penicillamine on intact erythrocytes obtained from patients suffering from rheumatoid arthritis during a 12-week period of therapy. The results are compared to the previously reported in vitro effects of the compound (McKay, C. N. N.; et al. Biochim. Biophys. Acta 1986, 888, 30-35). At clinical assessment at week 12, the 20 patients were divided into responder and nonresponder groups. The intracellular glutathione in the responder group is more oxidized (P less than 0.01) than in the nonresponder group. A retrospective analysis of the two patient groups at the initial assessment following the commencement of therapy indicated that in the nonresponder group intracellular glutathione was significantly more reduced (P less than 0.02) than in the responder group. It is postulated that penicillamine stimulates cellular defense against the oxidation of the cell membrane at the expense of cytosolic glutathione. This initial study suggests that spin-echo NMR analysis of erythrocyte glutathione can act as an early indicator of a clinical response to penicillamine therapy.

Arthritis, Rheumatoid↗