Imad K. Abukhalaf

Acute inhibition of glutathione biosynthesis alters endothelial function and blood pressure in rats

The cardiovascular and biochemical responses during acute oxidative stress induced by D,L-buthionine-(S,R)-sulfoximine (BSO) were investigated in Sprague-Dawley rats. Mean arterial pressure, heart rate and vascular reactivity were measured after subcutaneous injection of BSO (4 mmol/kg). Control rats received saline. Levels of GSH and GSSG in blood and tissues as well as renal superoxide were determined. Nitric oxide, prostacyclin and thromboxane A(2) in plasma and aorta, and isoprostane in plasma were also measured. Blood pressure was elevated at 24 h (121+/-2 vs. 104+/-2 mm Hg), with increased reactivity to phenylephrine (by a 59+/-4 vs. 45+/-2 mm Hg change), and impaired response to sodium nitroprusside (by a -35+/-2 vs. -63+/-2 mm Hg change), P<0.05. The GSH:GSSG ratio was reduced at 8 and 24 h in blood (4.1+/-0.6 and 5.1+/-0.3, respectively, vs. 8.5+/-0.2), and at 8 h in the aorta (1.0+/-0.2 vs. 2.9+/-0.5), heart (1.6+/-0.3 vs. 2.3+/-0.1) and kidney (2.1+/-0.2 vs. 3.7+/-0.4), P<0.05. Superoxide fluorescence was increased at 24 h via NADH (4131+/-194 vs. 2853+/-199), NADPH (2874+/-272 vs.1479+/-257) and succinate (2475+/-133 vs. 1594+/-2150), P<0.05. Plasma prostacyclin was reduced at 8 and 24 h (36+/-4 and 52+/-13, respectively, vs. 310+/-44 pg/ml), P<0.001, whereas nitric oxide was reduced at 24 h (6.4+/-1 vs. 22+/-2 microM), P<0.01. Also at 24 h, thromboxane A(2) was increased both in plasma (374+/-154 vs. 61+/-10 pg/ml) and the aorta (174.4+/-38 vs. 27+/-3.4 pg/mg), P<0.05. Thus, acute BSO-induced oxidative stress alters blood pressure and endothelial function by mechanisms involving increased plasma levels and aortic release of thromboxane A(2) and reduced nitric oxide and prostacyclin.

β‐Agonist‐induced alterations in organ weights and protein content: Comparison of racemic clenbuterol and its enantiomers

Clenbuterol is a relatively selective beta2-adrenergic partial agonist that has bronchodilator activity. This drug has been investigated as a potential countermeasure to microgravity- or disuse-induced skeletal muscle atrophy because of presumed anabolic effects. The purpose of this study was to: 1) analyze the anabolic effect of clenbuterols (-)-R and (+)-S enantiomers (0.2 mg/kg) on muscles (cardiac and skeletal) and other organs; and 2) compare responses of enantiomers to the racemate (0.4 mg/kg and 1.0 mg/kg). Male Sprague Dawley rats were treated with: a) racemic clenbuterol (rac-clenbuterol, 0.4 or 1.0 mg/kg); b) enantiomers [clenbuterol (-)-R or (+)-S]; or c) vehicle (1.0 mL/kg buffered saline). Anabolic activity was determined by measuring tissue mass and protein content. HPLC teicoplanin chiral stationary phase was used to directly resolve racemic clenbuterol to its individual enantiomers. In skeletal muscle, both enantiomers had equal anabolic activity, and the effects were muscle- and anatomic region-specific in magnitude. Although the enantiomers did not affect the ventricular mass to body weight ratio, clenbuterol (+)-S induced a small but significant increase in ventricular mass. Both clenbuterol enantiomers produced significant increases in skeletal muscle mass, while being less active in producing cardiac ventricular muscle hypertrophy than the racemic mixture.

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Muscle-Specific Effects of Hindlimb Suspension and Clenbuterol in Mature Male Rats

Anabolic agents are useful tools for probing the mechanisms by which muscle fibers perceive and respond to disuse. β2-Adrenergic agonists exert protective, and/or reparative, effects on atrophying muscle tissue. The effects of one such agent, clenbuterol (Cb), were examined on muscle mass, total protein content, and myofibrillar protein content in selected hindlimb muscles [adductor longus (ADL), extensor digitorum longus (EDL), plantaris (PLAN), soleus (SOL)] of mature male rats, under different loading conditions. Pair-fed rats were divided into four experimental groups: vehicle- and Cb-treated nonsuspended, vehicle- and Cb-treated hindlimb suspended (HLS). Experiments lasted 14 days, during which the rats received subcutaneous injections of 1 mg/kg Cb or 1 ml/kg vehicle. HLS induced significant atrophy in all muscles, except the EDL, in a generally fiber type-related pattern. However, myofibrillar protein content was affected in a more regional pattern. Cb treatment of nonsuspended rats induced hypertrophy in all muscles, in a generally uniform pattern. However, myofibrillar protein content was affected in a more fiber type-related pattern. Cb treatment of HLS rats reduced or eliminated HLS-induced atrophy in all muscles, in a muscle-specific pattern. Overall, the ADL and SOL were most susceptible to HLS-induced atrophy. The PLAN had the greatest magnitude of Cb-induced sparing of atrophy. The results show that, in mature male rats, Cb exerts anabolic effects that are load-dependent and muscle-specific. Responses to this drug cannot be reliably predicted by fiber-type composition alone.

Comparative analytical quantitation of clenbuterol in biological matrices using GC-MS and EIA.

A simple and sensitive procedure utilizing GC-MS for the identification and quantitation of clenbuterol in biofluids and tissues is described. This improved method utilizes trimethylboroxine for the derivatization of clenbuterol, requires only 1 mL/g of biological sample, and most importantly does not require an extra cleaning step for urine specimens prior to extraction. Linear quantitative response curves have been generated for derivatized clenbuterol over a concentration range of 5-200 ng/mL. The extraction efficiency at four representative points of the standard curve exceeded 90% in both specimen types (plasma and urine). Linear regression analyses of the standard curve in both specimen types exhibited correlation coefficients ranging from 0.997 to 1.000. The Limit of detection (LOD) and Limit of quantitation (LOQ) values for plasma specimens were determined to be 0.5 and 1.5 ng/mL respectively. For urine specimens, LOD and LOQ values were 0.2 and 0.7 ng/microL respectively. Percentage recoveries ranged from 91 to 95% for urine and 89 to 101% for plasma. Precision and accuracy (within-run and between-run) studies reflected a high level of reliability and reproducibility of the method. In addition to its reliability, sensitivity and simplicity, this modified procedure is more efficient and cost effective, requiring less time, only 1 mL of sample, and minimal amounts of extraction solvents. The applicability of the method for the detection and quantitation of clenbuterol in biological tissues of rats treated with the drug was demonstrated successfully. For comparative analysis of clenbuterol in plasma and liver samples, both GC-MS and enzyme immunoassay (EIA) methods are found to be suitable. Due to potential antibody-cross reactivity with EIA, the GC-MS method is the method of choice for most samples because of its specificity. However, the EIA method is considered the method of choice for analysis of clenbuterol found in concentrations below the limits of quantitation by GC-MS due to its sensitivity.

Distribution and Muscle-Sparing Effects of Clenbuterol in Hindlimb-Suspended Rats

Based on their anabolic properties in skeletal muscles, β-adrenergic agonists are of interest as potential countermeasures to microgravity-induced skeletal muscle atrophy. The levels of clenbuterol (Cb), a β2-adrenergic agonist, in both plasma and skeletal muscle were higher in hindlimb-suspended rats than in their nonsuspended Cb-treated controls. Cb treatment was shown to help maintain the body weight in suspended rats, while reducing the amount of mesenteric fat. However, hindlimb suspension attenuated Cb’s lipolytic effects. In skeletal muscle, the magnitude of response to unloading and Cb treatment followed a general regional pattern and was muscle and type specific. The highest magnitude of response to unloading was in predominantly slow-twitch muscles, and the least responsive were the predominately fast-twitch muscles.

Effect of UV on the susceptibility of acid-soluble Skh-1 hairless mouse collagen to collagenase

Background/Purpose: Photoaging of the skin is a result of chronic exposure to environmental ultraviolet radiation (UV). The milieu provided by the extracellular matrix, which significantly influences the behaviour of resident fibroblasts, depends critically on the supermolecular collagen structure. We ask whether direct photochemical treatment of type I collagen with solar wavelengths capable of reaching the dermis can modify the substrates susceptibility to collagenase in a model in vitro system.

Rapid HPLC Procedure for the Quantitation of Phytochelatins in Plant Tissue Extracts

Abstract A simple HPLC procedure for the identification and quantitation of phytochelatins (PCs) in plant tissue extracts is described. The method, which does not require a derivatization step utilizes only 20 µL of sample volume. Linear quantitative response curve was generated for phytochelatin 3 (PC3) over a concentration range of 1.33 µmol/L–6.66 mmol/L. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.996. Limit of detection (LOD) and limit of quantitation (LOQ) values were 0.1 and 0.5 µmol, respectively. Phytochelatin 3 recovery using this method was relatively high (above 85%). Intra‐assay and inter‐assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of a wide range of PC concentrations in plant tissue extracts was demonstrated successfully.

Fluorescence of putative chromophores in Skh-1 and citrate-soluble calf skin collagens

Background/purpose: Fluorescence of Skh‐1 hairless mouse and calf skin acid‐soluble type I collagens are envelopes of several bands putatively due to tyrosine (excitation/emission peak at 275/300 nm), dihydroxyphenylalanine (dopa; 280/325 nm), tyrosine aggregate (285/360 nm), dityrosine 325/400 nm), and advanced glycation end (AGE) product (370/450 nm), respectively. As these fluorophores can be markers of pathological conditions, we wish to present further evidence for or against these assignments.

Influences of prostanoids and nitric oxide on post-suspension hypotension in female Sprague–Dawley rats

Impairment in cardiovascular functions sometimes manifested in astronauts during standing postflight, may be related to the diminished autonomic function and/or excessive production of endothelium-dependent relaxing factors. In the present study, using the 30 degrees head-down tilt (HDT) model, we compared the cardiovascular and biochemical effects of 7 days of suspension and a subsequent 6-h post-suspension period between suspended and non-suspended conscious female Sprague-Dawley rats. Mean arterial pressure (MAP) and heart rate were measured prior to suspension (basal), daily thereafter, and every 2h post-suspension. Following 7 days of suspension, MAP was not different from their basal values, however, upon release from suspension, MAP was significantly reduced compared to the non-suspended rats. Nitric oxide levels were elevated while thromboxane A(2) levels declined significantly in both plasma and tissue samples following post-suspension. The levels of prostacyclin following post-suspension remained unaltered in plasma and aortic rings but was significantly elevated in carotid arterial rings. Therefore, the post-suspension reduction in mean arterial pressure is due mostly to overproduction of nitric oxide and to a lesser extent prostacyclin.