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Biomedical subjects

K G Allen

Publications and source records attributed to K G Allen.

At least 37 records · Page 2Linked to original sources
PubMed1993

Thromboxane production in copper-deficient and marginal platelets: influence of superoxide dismutase and lipid hydroperoxides.

Platelet thromboxane (TX) production was examined in response to dietary copper. Groups of eight rats were fed copper-deficient, -marginal, and -adequate diets providing 0.5, 1.7, and 7.5 micrograms Cu/g, respectively, with controlled dietary Se and vitamin E. Platelets were purified and washed by centrifugation. Separate platelet samples from each rat were challenged with 10 micrograms/ml of collagen and 1 unit/ml (27.3 nM) of thrombin in Tyrode's buffer, 2.0 mM Ca2+. Platelet copper-dependent superoxide dismutase (CuSOD) activity showed a significant depression with reduced diet copper, but platelet glutathione peroxidase activity was unaffected. Challenged platelet TX production showed a significant 1.5- to 2.5-fold increase in response to both dietary copper deficiency and marginality, with highly significant negative correlations between challenged platelet TX production and platelet CuSOD activity and between TX production and copper status (liver copper). Endogenous (unchallenged) platelet lipid hydroperoxide concentrations, measured as free fatty acid hydroperoxides by a glutathione-disulfide-specific glutathione reductase recycling assay, showed a nonsignificant 47-67% increase in copper deficiency. Pooled data showed a significant 71% increase in platelet lipid hydroperoxides in copper deficiency. Platelet TX production showed a significant correlation with endogenous lipid hydroperoxides. The results suggest that dietary copper insufficiency increases platelet TX synthesis through changes in CuSOD in a dose-responsive (diet copper and platelet CuSOD activity) manner, and that platelet TX synthesis is influenced by lipid hydroperoxides (peroxide tone).

Animals↗
PubMed1992

Iron-catalyzed reactions cause lipid peroxidation in the intact heart.

The chemical targets and mechanisms of iron-catalyzed oxidative injury in myocardium are poorly understood. Oxygen metabolites, in the presence of iron, can initiate free-radical chain reactions in unsaturated membrane lipids, generating lipid peroxides and causing membrane injury. We examined whether exposure to iron-catalyzed oxidative injury would increase myocardial lipid peroxide levels as injury evolved in the intact heart. Isolated, buffer perfused rabbit hearts were exposed for 30 min to 100 uM Fe2+/500 uM ADP and 10 uM H2O2 (IRON group, n = 5), saline vehicle (CON group, n = 6) or 500 uM ADP and 10 uM H2O2 without iron (ADP, n = 5). Lipid peroxides were measured in cytosol and membrane fractions by a new method, using the lipid peroxide-induced oxidation of exogenous GSH to GSSG, catalyzed by the enzyme glutathione peroxidase. The results indicated that iron-catalyzed lipid peroxidation occurs in the intact heart during chemically-mediated oxidative injury.

Animals↗
PubMed1992

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.

Animals↗
PubMed1992

Copper-marginal and copper-deficient diets decrease aortic prostacyclin production and copper-dependent superoxide dismutase activity, and increase aortic lipid peroxidation in rats.

Agonist challenged aortic prostacyclin production was examined in copper-adequate, -marginal and -deficient rats fed AIN-based diets providing 6.7, 1.7 and 0.8 micrograms Cu/g, respectively. Aortic rings were incubated in Krebs-Henseleit salts, 10 mmol/L HEPES buffer, pH 7.4, 95%:5% O2:CO2, 37 degrees C, and equilibrated for 1 h. Equilibrated rings were challenged with buffer (basal), 273.0 nmol/L thrombin and angiotensin II at 84.6 pmol/L and 846.0 pmol/L. Prostacyclin production, determined at 10 minutes by RIA as 6-keto prostaglandin F1 alpha, in basal and 84.6 pmol/L angiotensin II ring incubations was significantly reduced by 28 to 48% in copper-deficient rats. With thrombin or 846.0 pmol/L angiotensin II prostacyclin production was significantly reduced by 18 to 55% in copper-marginal and copper-deficient rats. Copper-dependent superoxide dismutase activity was significantly depressed by 30 and 57% in aortae of copper-marginal and copper-deficient rats. Lipid peroxidation, estimated by the thiobarbituric acid test, was significantly increased by 85% in copper-deficient rats, with a nonsignificant 40% increase in aortae from copper-marginal rats. The results suggest that the decreases in aortic prostacyclin production in aortae from both copper-deficient and copper-marginal rats are associated, in a dose-dependent manner, with copper-dependent superoxide dismutase depression and increases in aortic lipid peroxidation.

Analysis of Variance↗
PubMed1992

Inhibition of elevated hepatic glutathione abolishes copper deficiency cholesterolemia.

Dietary copper deficiency causes hypercholesterolemia and increased hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (MHG-CoA) reductase activity and increased hepatic glutathione (GSH) in rats. We hypothesized that inhibition of GSH production by L-buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor, would abolish the cholesterolemia and increased HMG-CoA reductase activity of copper deficiency. In two experiments, two groups of 20 weanling male rats were fed diets providing 0.4 and 5.8 micrograms Cu/g, copper-deficient (Cu-D) and copper-adequate (Cu-A), respectively. At 35 days plasma cholesterol was significantly elevated by 30 to 43% in Cu-D and 10 animals in each of the Cu-D and Cu-A groups were randomly assigned to receive 10 mM BSO solution in place of drinking water and continued on the same diets for another 2 wk. At necropsy Cu-D animals had a significant 52 to 58% increase in plasma cholesterol. BSO administration abolished the cholesterolemia in Cu-D rats, but had no influence on plasma cholesterol of Cu-A rats. Hepatic GSH was increased 39 to 82% in Cu-D rats and BSO abolished this increase. BSO was without effect on cardiac hypertrophy, plasma and liver copper, and hematocrit indices of copper status. Liver microsome HMG-CoA reductase activity was significantly increased 85 to 288% in Cu-D rats and BSO administration abolished this increase in activity in Cu-D rats. The results suggest that copper deficiency cholesterolemia and elevated HMG-CoA reductase activity are a consequence of elevated hepatic GSH, and provide evidence for GSH regulation of cholesterol metabolism in intact animals.

Animals↗
PubMed1990

Determination of picomole quantities of hydroperoxides by a coupled glutathione peroxidase and glutathione disulfide specific glutathione reductase assay.

A procedure to quantitate picomole amounts of hydroperoxides based on GSSG formation is described. Hydroperoxides are incubated with GSH and glutathione peroxidase, and the GSSG formed is measured by a GSSG-specific glutathione reductase recycling assay. Prior to analysis the remaining GSH is removed with N-ethylmaleimide. N-Ethylmaleimide inhibition of the recycling assay is prevented by alkaline hydrolysis of the N-ethylmaleimide, without GSSG hydrolysis, at pH 11. The method is rapid, 30 min, with a limit of detection of 142 pmol calculated by linear regression analysis. Stoichiometric amounts of GSSG are produced in response to hydroperoxides. An application of the method is shown with air oxidation of arachidonic acid solutions over 4 h at room temperature. The method is sufficiently sensitive to quantitate the low amounts of hydroperoxides present in fresh arachidonic acid samples.

Arachidonic Acid↗
PubMed1988

Use of 2,3,2-tetramine as a hepatic copper chelating agent for treatment of copper hepatotoxicosis in Bedlington terriers.

Five Bedlington Terriers with inherited copper (Cu) hepatotoxicosis and with hepatic Cu concentrations ranging from 3,000 to 11,000 micrograms/g of dry weight (normal, less than 350 micrograms/g of dry weight) were treated daily for up to 200 days with 2,3,2-tetramine tetrahydrochloride. During treatment, no change was made in the dietary Cu intake, which ranged from 12 to 16 micrograms/g of dry diet. Concentrations of hepatic and serum Cu, iron, and zinc were determined before and at the conclusion of the treatment period. In one dog, 24-hour urinary Cu concentration was measured before and during treatment. A liver biopsy specimen obtained after treatment had significantly (P less than 0.05) reduced hepatic Cu concentration (3,282 micrograms/g of dry weight; a 54.9% reduction), compared with the pretreatment value (7,281 micrograms/g of dry weight). After treatment, there was an overall general lessening of the extent of hepatic morphologic damage. Cytochemical examination for Cu in rhodanine-stained biopsy specimens revealed decreased numbers of Cu-laden hepatic lysosomes. The mean daily urinary Cu concentration increased as much as 25-fold during 2,3,2-tetramine treatment. Hepatic iron and zinc concentrations and serum Cu concentrations remained within normal ranges after treatment. Clinical or laboratory evidence of 2,3,2-tetramine toxicosis was not detected during treatment. These findings indicated that in affected Bedlington Terriers, 2,3,2-tetramine was a safe and rapid chelating agent of hepatic Cu.

Animals↗
PubMed1988

Copper deficiency depresses rat aortae superoxide dismutase activity and prostacyclin synthesis.

Prostaglandin synthesis shows dependence on lipid hydroperoxides and resultant oxygen derived radical formation. In view of the importance of dietary copper in cytosolic copper dependent superoxide dismutase (Cu/Zn SOD) activity and the role of SOD in oxygen radical formation, the influence of dietary copper on prostacylin (PGI2) synthesis and SOD activity in rat aorta was examined. Copper deficient (0.5 micrograms Cu/g diet) rats showed a significant 47% reduction in PGI2 synthesis rates by aortic ring incubations in comparison to copper adequate (6.0 micrograms Cu/g diet) animals. Aortic SOD activity was reduced by 46% in copper deficiency in comparison to copper adequate animals. Marginal dietary copper (1.6 micrograms Cu/g diet) significantly reduced aortic SOD activity by 32% but was without effect on aortic ring incubation PGI2 synthesis. These results indicate that dietary copper deficiency, and the resultant decrease in SOD activity, depresses aortic PGI2 synthesis.

Animals↗
PubMed1988

Copper deficiency and tissue glutathione concentration in the rat.

Copper deficiency in rats increased renal vein and arterial (heart) plasma GSH concentration by approximately 50%. There was no change in plasma GSSG concentration. Renal vein plasma GSSG/GSH ratio was decreased in copper deficiency, which is consistent with previous reports showing a copper-dependent thiol oxidase activity in the renal basement membrane. No change occurred in arterial plasma GSSG/GSH ratio. Hepatic GSH concentrations were also elevated by 50% in copper deficiency, GSSG concentrations were unaffected, but GSSG/GSH ratio was depressed. Renal and cardiac tissue GSH and GSSG were unaffected by copper deficiency. The decreased SOD activity and GSH-Px activity observed in copper deficiency may contribute to increased hepatic and plasma GSH concentrations.

Animals↗
PubMed1987

Tetramine cupruretic agents: a comparison in dogs.

The cupruretic effects of 2 tetramine (N,N'-bis (2-aminoethyl) alkane diamine) chelators were examined in healthy dogs fed a commercial dog food containing 12.2 micrograms of copper/g of dry diet. Two groups of 3 dogs each were given either 300 mg of 2,2,2-tetramine tetrahydrochloride or 2,3,2-tetramine tetrahydrochloride for 23 consecutive days. Serum and 24-hour urine samples obtained before drug administration and during therapy were analyzed for copper, zinc, and iron concentrations. Both tetramines produced a significant cupruresis without significant changes in serum copper or in serum or urine zinc and iron concentrations. The 2,3,2-tetramine tetrahydrochloride produced a 4- to 9-fold greater cupruresis than did 2,2,2-tetramine tetrahydrochloride and resulted in a daily loss of more than 2 mg of copper in the urine. The dogs had no laboratory or clinical evidence of toxic side effects to either cupruretic agent during the treatment period. The results of the present study indicate that 2,3,2-tetramine should be an effective decoppering drug for use in dogs.

Animals↗
PubMed1984

A new procedure to analyze free fatty acids. Application to 20-mg brain tissue samples.

Fatty acids were analyzed by a new method which involved their isolation from hexane extracts of serum or brain tissue in aqueous potassium hydroxide (10 microliter) and methylation directly in this solution with methyl iodide. The resulting fatty acid methyl esters were partitioned into ethylene chloride (25 microliter) and were quantitated by gas-liquid chromatography. The procedure was documented by comparison with conventional methylation reactions on serum fatty acids. This method, which avoids thin-layer chromatography and which measures individual free fatty acid concentrations in 20-mg brain tissue samples, should be of particular value for examining regional free fatty acids in brain following ischemia and trauma.

Animals↗
PubMed1984

Marginal copper deficiency in rats. Aortal morphology of elastin and cholesterol values in first-generation adult males.

A marginal, 2 parts per million (ppm) copper diet (experimental) was fed to female rats for 4 months prior to breeding, through gestation/lactation, and to the weaned offspring to determine the consequences in adult, male offspring on cholesterol values and aortal morphology. Liver copper concentrations of the dams and pups at day 21 of lactation and of the 117-day-old offspring who consumed the experimental diet were lower (P less than 0.0001) than corresponding rats fed a 10 ppm copper diet (control). However, statistically significant differences due to dietary treatments were not evident in pre- or post-weaning gain in body weight, litter size, cannibalism of pups, or total cholesterol concentrations of the serum and aorta. Ultrastructural examination of experimental offspring aortas revealed focally abnormal features of endothelial cells, the subendothelial space, collagen fibers, smooth muscle cells, and particularly elastin. The ultrastructural irregularities of elastin included discontinuous regions of the internal elastic lamina comprised of stained clumps of elastin of irregular size and shape. The results of this study suggest that a marginal copper nutriture begun in utero will elicit morphologic abnormalities of the aorta in rats that are otherwise without overt signs of copper deficiency.

Animals↗
PubMed1981

Decreased plasma lecithin: cholesterol acyltransferase activity in copper-deficient rats.

The effect of dietary copper deficiency on rat plasma lecithin: cholesterol acyltransferase (LCAT) activity was examined. Three groups of rats were used: ad libitum-fed copper-deficient group (0.6 microgram Cu/g diet); pair-fed control group (5.9 microgram Cu/g diet); and an ad libitum-fed control group (5.9 microgram Cu/g diet). Copper deficiency produced a significant hypercholesterolemia (57% increase, P less than 0.001) in 63 days with a significant increase in plasma free cholesterol (44%, P less than 0.001). Despite the significant increase in plasma free cholesterol, fractional and molar LCAT activity were markedly decreased (74.2%, P less than 0.001 and 63.7%, P less than 0.005, respectively) in copper-deficient rats. The results of this study suggest that dietary copper deficiency affects LCAT activity.

Animals↗
PubMed1981

Dietary L-histidine-induced hypercholesterolemia and hypocupremia in the rat.

The effect of histidine-supplemented stock diets on plasma cholesterol and on the amelioration of the hypercholesterolemia by simultaneous copper supplementation was examined in rats. Weanling male Sprague-Dawley rats, five groups of 10 animals each, were fed ground stock diets unsupplemented or supplemented with 8% L-histidine, 8% L-histidine plus 222.8 micrograms Zn/g and 8% L-histidine plus 56.6 micrograms Cu/g. On day 46 plasma and liver cholesterol, copper and zinc concentrations were determined by colorimetry and atomic absorption spectrophotometry. Histidine supplementation produced hepatomegaly, depressed growth, a 131.8% (P less than 0.001) increase in plasma cholesterol and significant reductions in plasma copper and zinc and liver copper concentrations. The simultaneous feeding of histidine and zinc-supplemented stock diet failed to alter the hypercholesterolemia despite elevation in plasma zinc to values in excess of control animals. The simultaneous feeding of histidine and copper-supplemented stock diets abolished the hypercholesterolemia, reduced the hepatomegaly observed with histidine alone and elevated tissue copper concentrations to values in excess of control animals. Tissue copper and cholesterol concentrations showed significant negative correlations. No correlations between zinc and cholesterol were observed. The results suggest that histidine-induced hypercholesterolemia is due to changes in copper status, and is abolished by simultaneous copper supplementation.

Animals↗
PubMed1978

Identification of the functional ionic groups of papain by pH/rate profile analysis.

The pH dependence of papain catalysis was analyzed by a scheme which evaluates the kinetic contribution of both protonated and unprotonated species of functional groups involved in catalysis. Kinetic measurements were made at constant pH, without buffers, by automatic titration. The rate-determining step for papain-catalyzed hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester, determined by nucleophile competition, changed from acylation below pH 6.5 to mixed acylation-deacylation above pH 6.5. Kinetic analysis indicated that three prototropic groups governed the pH-specificity of alpha-N-benzoyl-L-arginine ethyl ester hydrolysis. These prototropic groups had pKa values of 4.8, 6.5 to 6.7, and 8.7. Theoretical treatment of the kinetics provided an excellent fit with the experimentally found profile when the contribution of all three prototropic groups was considered. Analysis showed that, in acid, the pathways of papain catalysis were functional with either two or three active-site protons. In base, a single functional ionic pathway is associated with an active site with only one proton. Pathways involving an unprotonated active site are catalytically inoperative in both acid and base. These results indicate that papain exhibits several catalytically functional ionic pathways. The results are discussed in terms of pKa assignments, and the mechanism of papain catalysis.

Arginine↗