Daniel A. von Deutsch

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.

Cross-linking density alters early metabolic activities in chondrocytes encapsulated in poly(ethylene glycol) hydrogels and cultured in the rotating wall vessel.

In designing a tissue engineering strategy for cartilage repair, selection of both the bioreactor, and scaffold is important to the development of a mechanically functional tissue. The hydrodynamic environment associated with many bioreactors enhances nutrient transport, but also introduces fluid shear stress, which may influence cellular response. This study examined the combined effects of hydrogel cross‐linking and the hydrodynamic environment on early chondrocyte response. Specifically, chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels having two different cross‐linked structures, corresponding to a low and high cross‐linking density. Both cross‐linked gels yielded high water contents (92% and 79%, respectively) and mesh sizes of 150 and 60 Å respectively. Cell‐laden PEG hydrogels were cultured in rotating wall vessels (RWV) or under static cultures for up to 5 days. Rotating cultures yielded low fluid shear stresses (≤0.11 Pa) at the hydrogel periphery indicating a laminar hydrodynamic environment. Chondrocyte response was measured through total DNA content, total nitric oxide (NO) production, and matrix deposition for glycosaminoglycans (GAG). In static cultures, gel cross‐linking had no effect on DNA content, NO production, or GAG production; although GAG production increased with culture time for both cross‐linked gels. In rotating cultures, DNA content increased, NO production decreased, and overall GAG production decreased when compared to static controls for the low cross‐linked gels. For the high cross‐linked gels, the hydrodynamic environment had no effect on DNA content, but exhibited similar results to the low cross‐linked gel for NO production, and matrix production. Our findings demonstrated that at early culture times, when there is limited matrix production, the hydrodynamic environment dramatically influences cell response in a manner dependent on the gel cross‐linking, which may impact long‐term tissue development. Biotechnol. Bioeng. 2009;102: 1242–1250.

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 hindlimb suspension and clenbuterol treatment on polyamine levels in skeletal muscle.

Polyamines are unbiquitous, naturally occurring small aliphatic, polycationic, endogenous compounds. They are involved in many cellular processes and may serve as secondary or tertiary messengers to hormonal regulation. The relationship of polyamines and skeletal muscle mass of adductor longus, extensor digitorum longus, and gastrocnemius under unloading (hindlimb suspension) conditions was investigated. Unloading significantly affected skeletal muscle polyamine levels in a fiber-type-specific fashion. Under loading conditions, clenbuterol treatment increased all polyamine levels, whereas under unloading conditions, only the spermidine levels were consistently increased. Unloading attenuated the anabolic effects of clenbuterol in predominately slow-twitch muscles (adductor longus), but had little impact on clenbuterol’s action as a countermeasure in fast- twitch muscles such as the extensor digitorum longus. Spermidine appeared to be the primary polyamine involved in skeletal muscle atrophy/hypertrophy.

VALIDATION OF A SOLILD PHASE EXTRACTION PROCEDURE FOR THE GC-MS IDENTIFICATION AND QUANTITATION OF COCAINE AND THREE METABOLITES IN BLOOD, URINE, AND MILK

A simple and widely used solid-phase extraction procedure (United Chemical Technologies Method Handbook) was applied for the GC-MS identification and quantitation of cocaine (COC), benzoylecgonine (BE), cocaethylene (COCE), and m-hydroxy- benzoylecgonine (HBE) in blood, urine, and milk. The method which utilizes BSTFA as a derivatizing agent yielded abundant diagnostic ions with high m/z values. Linear quantitative response curves were generated for the analytes of interest over a concentration range of 5–1000 ng/mL. Linear regression analyses of the standard curve in the three specimen types exhibited correlation coefficients ranging from 0.997 to 1.000. The LOD values for COC, COCE, and derivatives of BE, and HBE in the three specimen types ranged from 2.5 to 5.0 ng/mL. The LOQ values, however, ranged from 5.0 to 10.0 ng/mL. 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 detection and quantitation of COC, BE, COCE, and HBE was demonstrated successfully in human blood and urine samples, as well as blood samples obtained from cocaine-treated (subcutaneously) rats.

Toward Understanding the Influence of Soil Metals and Sulfate Content on Plant Thiols

Plants respond to increased concentrations of metals by a number of mechanisms, including chelation with phytochelatins (PCs). Soil specimens and plants (Veronica anagalis-aquatica, Typha domingensis, Cynodon dactylon, Chenopodium album, Rumex dentatus, Amaranthus gracilis, Chenopodium murale, Inula viscosa) leaves were collected from two sites in northern Jordan and subsequently metals (cadmium, copper, and lead), sulfate, and PC (from leaves) levels were determined. One of these sites was contaminated with metals and the other served as a control site. The contaminated site had elevated cadmium, copper, lead, and sulfate levels. This increase of metal and sulfate levels in the soil at the contaminated site correlated with a rise in plant total glutathione (GSHT) and cysteine (CYST). These increases were not attributed to an elevation in total phytochelatin levels. However, a significant increase in the ratio of short-chain phytochelatins to the total phytochelatin stores was observed. The individual effects of metals and sulfate on glutathione, short-chain PCs and long-chain PCs levels were dissimilar.

Anabolic Doping Agents

This chapter discusses the different types of doping agents that have been used since the pre-Christian era, such as hallucinogenic mushrooms and alcohol, up to those currently used by athletes today. Today, articles 2.1 through 2.8 of the World Anti-Doping Code, define doping as the violation of one or more of these articles through the use of prohibited substances or methods. In the present discussion on anabolic doping agents, the classes of banned substances covered include stimulants, anabolic agents, peptide hormones, and β2-adrenoceptor agonists, as well as masking and antiestrogenic agents. The pharmacokinetics, pharmacodynamics, and toxicology of these substances will be discussed along with some potential historical consequences of the use of certain doping agents by both axis and allied forces during World War II, and the terrible consequences that might be attributed to their use.