Malondialdehyde modification of proteins in vitro is enhanced in the presence of acetaldehyde.

To test the hypothesis that the cardioprotective effect of alcohol is related to the inhibition of malondialdehyde (MDA) modification of proteins by acetaldehyde (AA), we studied the effect of AA on MDA modification of bovine serum albumin (BSA) in vitro. BSA was incubated simultaneously with a fixed concentration of MDA (70 mM) and different concentrations of AA (120, 60, 30, 10, or 0 mM) for 24 h at 37 degrees C. The MDA-modified or AA-modified BSA was quantitated with immunoblotting by using specific anti-MDA and specific anti-AA protein antisera, respectively. In another set of experiments, BSA was incubated sequentially, first with different concentrations of AA and then with 70 mM of MDA. In both incubation protocols, the presence of AA and AA modification of BSA enhanced MDA binding. These in vitro observations suggest that the putative cardioprotective effects of alcohol or wine cannot be ascribed to AA-mediated reduction in MDA protein formation, a possible biochemical pathway of accelerated atherosclerosis.

Effect of select antioxidants on malondialdehyde modification of proteins.

To determine whether commonly used antioxidants alter malondialdehyde (MDA) modification of proteins, a known mechanism of free radical-related tissue injury, we studied the effect of adding 1 mg/mL of pycnogenol, 5 mM of alpha-tocopherol, 5 mM of ascorbate, and 0.2 mg/mL of an ethanol equivalent of red and white wine on MDA-protein content of endothelial cells in culture. The addition of pycnogenol but not of the other antioxidants was associated with significant reduction in MDA-protein content compared with controls (0.521 +/- 0.041 in arbritrary units versus 1.011 +/- 0.021, P < 0. 001). To determine whether the observed effect occurs distal to MDA generation, the effect of these antioxidants on the modification of bovine serum albumin with MDA generated in a cell-free system was studied. In this cell-free assay, pycnogenol but not the other antioxidants reduced MDA-BSA generation by approximately 50%. It is concluded that pycnogenol may reduce MDA modification of proteins at a step distal to MDA generation. This may be an additional mechanism of protective effects of pycnogenol against oxidative stress.

Age-related changes in the thyroid hormone effects on malondialdehyde-modified proteins in the rat heart.

To determine the age-related changes in thyroid hormone (TH) effects on malondialdehyde (MDA)-modified proteins in cardiac tissue, rats at 4, 12, and 25 months of age were studied. Hyperthyroidism was induced with daily injection of L-triiodothyronine (15 microg/100 g) intraperitoneally for 10 days. Hypothyroidism was induced with 0. 025% methimazole in the drinking water for 4 weeks. MDA proteins were measured with immunoblots using a specific anti-MDA antiserum. MDA was measured as thiobarbituric acid reactive substance. Hypothyroidism in 4-month-old rats was associated with significant reduction in MDA proteins compared to euthyroid rats (13.4 +/- 5.9% vs. 99.8 +/- 10.4% of controls P < 0.001). Hyperthyroidism did not result in a significant change of MDA proteins. In aged rats, neither hypothyroidism nor hyperthyroidism was associated with significant changes in cardiac MDA proteins. The changes in MDA proteins did not correlate with cardiac MDA concentrations. In young rats, the MDA concentrations (nmol/mg) were significantly reduced in hypothyroidism (2.71 +/- 0.21) and were increased in hyperthyroidism (8.19 +/- 0.78) compared to euthyroid values (5.06 +/- 0.71) P < 0. 01. In aged rats, cardiac MDA content was significantly increased during both hyperthyroidism and hypothyroidism. We conclude that alterations in MDA protein content is yet another potential biochemical effect of TH in cardiac tissue. This particular effect is significantly blunted with age.

A six-month, placebo-controlled comparison of the safety and efficacy of salmeterol or beclomethasone for persistent asthma.

BACKGROUND: There is a paucity of data comparing the long-term safety and efficacy of long-acting inhaled beta2-agonists versus low-dose inhaled corticosteroids in the treatment of asthma. OBJECTIVE: To compare the safety and efficacy of salmeterol xinafoate, beclomethasone dipropionate (BDP), and placebo over a 6-month treatment period in patients with persistent asthma. METHODS: Salmeterol (42 microg twice daily), BDP (84 microg four times daily), or placebo was administered via metered-dose inhaler to 386 adolescent and adult inhaled corticosteroid-naive patients in a randomized, double-blind, double-dummy, parallel-group study. Eligible patients demonstrated a forced expiratory volume in 1 second (FEV1) from 65% to 90% of predicted values. Pulmonary function, symptom control, frequency of asthma exacerbations, bronchial hyperresponsiveness (BHR) to methacholine challenge, and adverse events were assessed. RESULTS: There were few statistically significant differences between the two active treatments over 6 months of therapy. Asthma symptoms and lung function were significantly improved with both salmeterol and BDP compared with placebo (changes from baseline in FEV1 of 0.28 L (SE = 0.04) and 0.23 L (SE = 0.04), respectively, compared with 0.08 L (SE = 0.04); P < or = .014). There were no significant differences among the treatment groups with respect to the distribution of asthma exacerbations over time. Both salmeterol and BDP significantly reduced BHR compared with placebo (P < or = .033; changes from baseline of 1.29 (SE = 0.26) and 1.42 (SE = 0.24) doubling doses at 6 months, respectively, compared with 0.24 (SE = 0.29) doubling dose for placebo). No rebound effect in BHR was seen upon cessation of any of the three treatment regimens. There were no clinically important differences in the safety profiles among the three treatments. CONCLUSIONS: Both salmeterol and BDP are effective and well-tolerated when administered for 6 months to inhaled corticosteroid-naive patients with persistent asthma.

Malondialdehyde binding of rat cerebral proteins is reduced in experimental hypothyroidism.

To determine the effect of thyroid hormones (TH) on cerebral tissue malondialdehyde (MDA) content and accumulation of MDA-bound proteins, hyperthyroid rats and hypothyroid rats were compared to euthyroid controls. Hyperthyroidism was induced by daily injection of l-3,5, 3'-triiodothyronine (15 ug (100 g)-1) intraperitoneally daily for 10 days. Hypothyroidism was induced with 0.025% methimazole in the drinking water for 4 weeks. Immunoblot analysis of cerebral and plasma proteins was carried out using a specific anti-MDA-protein antiserum. MDA was measured as thiobarbituric acid reactive substance. Hypothyroidism was associated with significant reduction in MDA-proteins of plasma (59.3+/-9.5% vs. 99.8+/-23.0% of control p<0.05) and cerebral tissue (17.6+/-19.9% vs. 100.2+/-29.0% of control p<0.001). Hyperthyroidism did not significantly alter MDA-protein distribution. These changes did not correlate with cerebral tissue or plasma MDA concentration. It is concluded that hypothyroidism in rats is associated with significant decrease in MDA-bound proteins. This may have some clinical and biological implications.

Antibodies against malondialdehyde (MDA) in MRL/lpr/lpr mice: evidence for an autoimmune mechanism involving lipid peroxidation.

Previous studies have shown that lipid peroxidative processes may play a role in disease pathogenesis in lupus-prone MRL/lpr mice. Studies were thus performed to determine if an immune response against malondialdehyde (MDA), a highly reactive byproduct of lipid peroxidation, was present in these mice. By using MDA-modified mouse serum albumin (MSA) as antigens in ELISA, we found that these mice produce high levels of MDA-specific antibodies in the complement-fixing IgG2a and IgG2b subclasses. Anti-MDA antibodies were also found in MRL/+ mice but in significantly lower levels. The specificity of these antibodies was verified by inhibition ELISA. MDA may contribute to disease pathogenesis in these mice by altering the immunogenicity of self molecules, eliciting an immune response and forming immune complexes that may deposit in tissues.

Glycosylation enhances malondialdehyde binding to proteins.

To determine whether glycosylation of proteins increases their susceptibility to modification with malondialdehyde (MDA), bovine serum albumin, which was pretreated with 500 mg/dl dextrose at 37 degrees C for 0, 1, 2, and 4 weeks, were incubated with 100 mM MDA at 37 degrees C for 24 h. The MDA content of the protein samples were determined after dialysis using thiobarbituric acid (TBA) assay. In addition, a specific anti-MDA protein antiserum was used to demonstrate MDA proteins with immunoblotting technique. The MDA content of BSA preincubated with dextrose for 4 weeks and reincubated with MDA (0.0649 +/- 0.0019 microgram MDA/mg protein) was significantly higher (p < .001) than the MDA content of BSA preincubated with dextrose for only one (0.0227 +/- 0.0031 microgram/mg) or two (0.0347 +/- 0.0034 microgram/mg) weeks or the MDA content of nonglysolated BSA incubated with MDA at the same experimental conditions (0.0201 +/- 0.0029 microgram/mg). These differences could also be found in the immunoblots. However, the correlation of TBA assay with the estimates on immunoblots was poor. It is likely that the immunoblotting assay is more of an estimate of the number of BSA molecules modified with MDA, rather than MDA content of each BSA molecule. It is concluded that in vitro glycosylation of proteins increases their susceptibility to MDA-modification. This may well be an additional pathway of diabetes-related modification of proteins.

Cholesterol enriched diet enhances malondialdehyde modification of proteins in cerebral microvessels of rabbits.

To determine the role of dietary cholesterol on malondialdehyde (MDA) modification of proteins, the cerebral microvessels of rabbits fed a cholesterol enriched diet for 8 weeks were compared to control rabbits. The MDA proteins were estimated with immunoblotting using a specific polyclonal antiserum against MDA proteins. Cholesterol-fed rabbits had a significantly increased MDA protein band at 175 kDa compared to control rabbits (65.6 +/- 6.8 OD versus 16.5 +/- 2.5 OD, P < 0.01). An increase in MDA protein was also found in rabbits intravenously injected with 2 mg of MDA-modified low density lipoproteins or MDA-modified rabbit serum albumin (RSA) at 0, 2, and 4 weeks of observation. The MDA proteins were not increased in cerebral tissue of cholesterol-fed rabbits. It is concluded that a high cholesterol diet is associated with increased MDA modification of proteins in cerebral microvessels. The mechanism could be related to increased circulatory MDA proteins since exogenously administered MDA-LDL or MDA-RSA resulted in similar increases in MDA proteins in cerebral microvessels.

Antiallergic activity of tylogenin, a novel steroidal compound from Tylophora sylvatica.

Tylogenin, a steroidal aglycone generated by acid hydrolysis from two seasonal glycosides occurring in Tylophora sylvatica, inhibits IgE-induced basophil mediator release for allergic reactions. In the rabbit basophil-dependent serotonin release (BDSR) assay system, the inhibitory activity of tylogenin (geom mean IC50 = 39 microM) was significantly (P < 0.05) greater than that of its parent glycosides, tylophoroside (geom mean IC50 263 microM) and acetyltylophoroside (geom mean IC50 331 microM), and that of dexamethasone (geom mean IC50 = 912 microM). The activity of tylogenin was found to increase with the incubation time. In the human leukocyte-dependent histamine release (LDHR) test model, the glycosides had only a minimal activity. In contrast, tylogenin, with a geom mean IC50 = 49 microM, exerted a significantly (P < 0.05) greater potency than dexamethasone (IC50 = 257 microM). These results suggest that tylogenin could represent a new class of antiallergic agents.

Diabetes-related changes in the protein composition of rat cerebral microvessels.

To determine the effect of diabetes mellitus on cerebral microvessel protein composition, post translational modification of proteins with glucose and malondialdehyde (MDA) was determined and the abundant protein species found in cerebral microvessels isolated from control and streptozotocin-induced diabetic rats were studied. Two dimensional gel electrophoresis and computer assisted densitometry revealed that only one out of 25 quantitated proteins was significantly altered in diabetic rats after 5 weeks of uncontrolled hyperglycemia. The level of glycosylation of cerebral microvessel protein mixture was significantly increased in diabetic rats compared to control rats (168.8 +/- 25 vs 109.5 +/- 4.8 nmol/mg) (p < 0.05). Western blot analysis of cerebral microvessel proteins from diabetic rats using a specific antibody against MDA-modified proteins revealed three protein spots with molecular weights of approximately 60,000 Kd. These were shown not to be contaminants from cerebral tissue or plasma proteins modified with MDA. It is concluded that short duration of streptozotocin-induced diabetes mellitus in rats is associated with some qualitative changes in protein composition of cerebral microvessels. These changes may contribute to the diabetes-related alterations in the blood-brain barrier.

Tissue-specific distribution of malondialdehyde modified proteins in diabetes mellitus.

A potential mechanism of diabetes-related tissue damage is modification of various proteins by lipid peroxidation by-products such as malondialdehyde (MDA). To determine the extent of MDA derivatization of various proteins in diabetes mellitus, Western blots were carried out using a specific anti-MDA antiserum to study proteins in plasma and various tissues of control and streptozotocin (STZ)-induced diabetic rats. The concentration of MDA-proteins was highest in plasma compared to other tissues tested. Diabetes was associated with a reduction in MDA-protein content of plasma, lung and liver while in the heart, testicle, cerebrum and kidney the MDA-protein concentration was not altered. Insulin treatment of diabetic rats normalized MDA-protein content of plasma but not in the lung or liver. A large interindividual variability in various protein species was observed within a group. This was partly attributed to polymerization of MDA-proteins. It is concluded that although diabetes is associated with increased lipid peroxidation the content of MDA-proteins in plasma and in some tissues is decreased.

Age-related changes in tissue content of malondialdehyde-modified proteins.

One of the possible mechanisms of the age-related modifications of proteins is the result of reaction with malondialdehyde (MDA), a lipid peroxidation byproduct. To determine the effect of age on the extent of MDA derivatization of proteins in plasma and various tissues, male Fisher 344 rats at 4, 12 and 26 months of age were studied. Protein electrophoresis and immunoblotting was carried out using a specific antiserum against MDA-protein complexes. The concentration of MDA-proteins in plasma (mean +/- SD of optical density in 50 micrograms protein) was 201.6 +/- 47.7 in 4 month old rats, 197.4 +/- 67 in 12 month old rats and 101.4 +/- 22.7 in 26 month old rats (P < 0.01). The MDA protein content of testicle, liver and heart was increased in 12 month old rats compared to 4 and 26 months old rats. There were no age-related differences in MDA-protein content of lung, brain, and kidney. Because of the interindividual variability of MDA-protein profiles within an age group distinct age-related changes in the distribution of various MDA protein bands could not be documented.

Pollen changes during five decades of urbanization in Tucson, Arizona.

Five decades of daily pollen measurements have catalogued a growing number of imported and native plants in Tucson, Arizona, a city that has a high prevalence of allergic rhinitis and asthma. These plants have contributed a large proportion of the overall pollen rain and include mulberry, olive, juniper, cypress, privet, mesquite, Bermuda grass, ragweed, Palmer's amaranth, and Russian thistle. Factors contributing to these increases in pollen include population growth as well as changing architectural and landscape preferences. The institution of pollen control laws, inner city development, and nonreplacement of allergenic trees along with the growing popularity of desert landscaping suggests that trend reversal may be occurring.

Tylophora compounds as Na+/K(+)-ATPase inhibitors.

1. Acetyltylophoroside (AcT) and tylogenin inhibit Na+/K(+)-ATPase in spite of having structures very different from cardiac glycosides (CGs). 2. Calculation of the lowest energy conformations of AcT and tylogenin and superpositions of these with the X-ray conformations of CGs and chlormadinone acetate led to a model for the interaction of these different types of Na+/K(+)-ATPase inhibitors with the receptor.

Malondialdehyde modified proteins and their antibodies in the plasma of control and streptozotocin induced diabetic rats.

One of the possible mechanisms of diabetes-related tissue damage is modification of various proteins via lipid peroxidation byproducts such as malondialdehyde (MDA). To determine the extent of MDA derivatization of plasma proteins, Western blots were carried out using anti-MDA antisera to study plasma proteins in control and streptozotocin (STZ)-induced diabetic rats. Since MDA can modify proteins and may alter or enhance their antigenicity, we screened plasma samples for anti-MDA antibodies using enzyme-linked immunosorbent assay (ELISA), and confirmed antibody specificity by inhibition ELISA. Circulating immune complexes containing MDA were also assayed. This study is the first demonstration of the existence in plasma of MDA-modified proteins with a molecular weight of approximately 100 Kd. Both control and diabetic rats have similar concentrations of plasma anti-MDA antibodies and circulating immune complexes. These results do not support the notion that diabetes alters the immune response to MDA modified proteins. Whether MDA modification of proteins participate in immunological processes that lead to tissue injury remains to be demonstrated.

Adrenal function in adult asthmatics during long-term daily treatment with 800, 1,200, and 1,600 micrograms triamcinolone acetonide. Multicenter study.

A study to assess the effect of the long-term use of triamcinolone acetonide (TA) on adrenal function was conducted with 143 male and female patients with asthma who were randomly assigned to receive 800, 1200, or 1,600 micrograms of TA daily for six months. Adrenal function was assessed prior to treatment and after two weeks and one, three, and six months of TA use. The effect of TA was evaluated by measuring plasma cortisol levels just prior to and 30 min after a bolus IV injection of 0.25 mg cosyntropin. Adrenal suppression was assumed if the plasma concentration of cortisol did not increase by at least 7 micrograms/dl from the prestimulation value, and remained below 18 micrograms/dl 30 min after the cosyntropin injection. Urine collected for 24 h prior to each cosyntropin stimulation was assayed for free cortisol and related metabolites to confirm suppression. Although all treatment regimens caused some reduction in the 24-h excretion of corticosteroid products, none of the mean values was below the normal ranges. The mean data indicate that TA had no significant effect on adrenal function at any dose or at any time for the patients overall. Individually, three patients exhibited some reduction in adrenal function.

Antigen enhances neuronally induced contraction of intrapulmonary bronchi from IgE-producing rabbits.

The effect of in vitro antigen exposure on contraction induced by electrical field stimulation (EFS) was examined in bronchial rings isolated from rabbits producing specific IgE antibodies. After exposure to antigen, tissues showed an enhanced isometric contractile response to EFS especially at low frequencies, leading to a significant change in the mean slope factor (p less than 0.05) derived from modeling the log frequency response curve using a 4-parameter logistic function. Also, the mean log EF20 +/- SEM decreased from 1.03 +/- 0.05 to 0.88 +/- 0.07 Hz (p less than 0.02). This antigen-induced effect was blocked by pretreatment with 3 microM chlorpheniramine and not observed in unsensitized tissues. Antigen challenge of tissues passively sensitized with IgE (but not IgG) antibodies led to a similar EFS-enhancing effect, significantly reducing the mean slope factor (p less than 0.025). Substituting EFS with exogenous acetylcholine resulted in no antigen-induced enhancement of contraction. The data suggest that antigen-IgE interaction leads to local histamine release sufficient to enhance the function of excitatory airway neurons.

Inhaled epinephrine and oral theophylline-ephedrine in the treatment of asthma.

Inhaled and oral over-the-counter bronchodilators are used for self-therapy by asthmatic patients. To evaluate their safety and efficacy, we compared epinephrine and theophylline combined with ephedrine with inhaled metaproterenol and the placebo. Twelve asthmatic patients were studied in a randomized, double-blind, placebo-controlled, crossover trial comparing forced expiratory volume in 1 second (FEV1) after two inhalations of epinephrine (0.2 mg/inh), 1 minute apart, followed in 15 minutes by theophylline (130 mg) with ephedrine (24 mg) versus two inhalations of metaproterenol (0.65 mg/inh), 1 minute apart, versus placebo inhaler and tablets. Onset of FEV1 greater than 15% above baseline values occurred within 15 seconds after inhalations for 100% of epinephrine-treated patients, 92% of metaproterenol-treated patients, and 33% of placebo-treated patients. FEV1 responses were significantly greater (P less than .05) for epinephrine at 0.66 to 1.66 minutes compared with the responses of metaproterenol, and epinephrine and theophylline that was combined with ephedrine compared with metaproterenol beginning at 2 hours. Mean duration of activity was 5.7 hours for the epinephrine- and theophylline with ephedrine-treated patients, 4.9 hours for metaproterenol-treated patients, and 2 hours for the placebo group. There were statistically significant differences for patients receiving epinephrine and theophylline with ephedrine versus the placebo group (P less than .001), metaproterenol patients versus the placebo group (P = .02), and patients receiving epinephrine and theophylline with ephedrine versus metaproterenol-treated patients (P less than .05). Compared with inhaled metaproterenol, inhaled epinephrine followed in 15 minutes by a theophylline-ephedrine tablet had a significantly earlier onset, longer duration of action, numerically greater peak effect, and patient preference.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical properties of malondialdehyde-protein adducts.

Malondialdehyde (MDA), a product of lipid peroxidation, can bind to and modify proteins by adduct formation. To determine whether MDA adducts were immunogenic, MDA was added to rabbit serum albumin (RSA) in order to characterize MDA-proteins and to immunize rabbits. Bound MDA was proportional to the concentration of MDA added in the range of 2.5-20 mM as measured by thiobarbituric acid reactivity. MDA adducts of RSA migrated further toward the anode than native serum protein in zone and immunoelectrophoresis indicating increased negative charge. Rabbits immunized with MDA-RSA produced high titers of IgG antibodies to MDA-RSA as measured by enzyme-linked immunosorbent assay (ELISA). Hapten specificity of the antibody was demonstrated by antisera reactivity with MDA-RSA but not with unaltered RSA. Our findings support the possibility that MDA may serve as a hapten to form neoantigens which may represent a pathway by which lipid peroxidation could produce tissue damage via an immunologic mechanism.

SCH 434: a new antihistamine/decongestant for seasonal allergic rhinitis.

In a double-blind, multicenter study, we compared the effects of SCH 434 (Claritin-D; Schering Corp., Kenilworth, N.J.), a new sustained-release, combination antihistamine/decongestant medication, with the effects of its individual components and placebo in 435 patients with seasonal allergic rhinitis. SCH 434 contains 5 mg of loratadine, a nonsedating antihistamine, and 120 mg of pseudoephedrine as the decongestant component. Administered twice daily in this study, SCH 434 effected a 50% decrease in total symptom scores at day 4 and was significantly (p less than or equal to 0.03) more effective than the components alone or the placebo. Loratadine or pseudoephedrine alone, with 43% and 33% decline in symptom scores, respectively, also was more effective than placebo (p less than 0.05). As expected, pseudoephedrine alone was more effective than loratadine (p less than 0.01) in relieving nasal stuffiness; SCH 434 was more effective (p less than or equal to 0.01) than placebo and loratadine in relieving nasal stuffiness. All treatments were safe and well tolerated, although insomnia and dry mouth were noted in a significant number of patients who received either SCH 434 or pseudoephedrine. No serious side effects were noted. The incidence of sedation did not differ significantly among the four treatment groups. We conclude that SCH 434 is a safe and effective treatment for symptoms of seasonal allergic rhinitis. The combination drug (SCH 434) was better than its components for some, but not all, symptoms.